NMR characterization and conformational analysis of a potent papain-family cathepsin L-like cysteine protease inhibitor with different behaviour in polar and apolar media

Article abstract of DOI:10.1016/j.molstruc.2014.07.046

We recently reported the synthesis, of a potent papain-family cathepsin L-like cysteine protease inhibitor, as new lead compound for the development of new drugs that can be used as antiprotozoal agents. The investigation of its conformational profile is

Full text of DOI:10.1016/j.molstruc.2014.07.046

Journal of Molecular Structure 1076 (2014) 337–343
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Journal of Molecular Structure
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NMR characterization and conformational analysis of a potent
papain-family cathepsin L-like cysteine protease inhibitor with
different behaviour in polar and apolar media
b
c
b
a
Archimede Rotondo a, , Roberta Ettari , Maria Zappalà , Carlo De Micheli , Enrico Rotondo
⇑
a Dipartimento di Scienze Chimiche, University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy
b Dipartimento di Scienze Farmaceutiche, University of Milan, Via Mangiagalli, 25, 20133 Milano, Italy
c Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, University of Messina, Viale Annunziata, 98168 Messina, Italy
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
ꢀ
We chose the ester as interesting case
study because of its biological
properties.
ꢀ
The retention of the compound in
apolar media focused our curiosity on
this system.
ꢀ
ꢀ
NMR data prove different structural
features in methanol and chloroform.
Intramolecular interactions play a key
role in the detected conformational
switches.
ꢀ
Only the dynamic conformation
typical of polar media can arise the
biological activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2014
Received in revised form 16 July 2014
Accepted 17 July 2014
Available online 23 July 2014
We recently reported the synthesis, of a potent papain-family cathepsin L-like cysteine protease inhibi-
tor, as new lead compound for the development of new drugs that can be used as antiprotozoal agents.
The investigation of its conformational profile is crucial for the in-depth understanding of its biological
behaviour. Our careful NMR analysis has been based on the complete and total assignment of 1H, 13C,
15N and 19F signals of the molecule in both CDCl3 and CD3OH, which could reproduce in some way a sce-
nario of polar and not polar phases into the biological environment. In this way it has been unveiled a
different behaviour of the molecule in polar and apolar media. In CDCl3 it is possible to define stable con-
formational arrangements on the basis of the detected through space contacts, whereas, in CD3OH a
greater conformational freedom is envisaged: (a) by the overlap of any of the CH2 diastereotopic reso-
nances (unable to distinguish asymmetric molecular sides because of the free rotation about the single
bonded chains), (b) by the less definite measured vicinities not consistent with just one conformation
and (c) by the evident loss or switching of key intramolecular hydrogen interactions.
Keywords:
Cysteine protease inhibitors
1H
13C
15N NMR
Conformational analysis
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
relevant diseases are malaria and Human African Trypanosomiasis
(HAT) [1]. Malaria is the most widespread and severe tropical dis-
Neglected tropical diseases (NTDs) are a group of chronic dis-
abling infections affecting over one billion people, two of the most
ease, it is caused in humans by several species of the Plasmodium
genus, being P. falciparum together with P. vivax responsible of
more than 95% cases of malaria [2]. On the other hand, HAT is
caused by protozoa of Trypanosoma genus: T. brucei gambiense
and T. b. rhodesiense which are responsible for the transmission
⇑
Corresponding author. Tel.: +39 090 6765714; fax: +39 090 393756.
E-mail address: arotondo@unime.it (A. Rotondo).
http://dx.doi.org/10.1016/j.molstruc.2014.07.046
0022-2860/Ó 2014 Elsevier B.V. All rights reserved.

338
A. Rotondo et al. / Journal of Molecular Structure 1076 (2014) 337–343
of the disease to humans, causing chronic and acute forms of the
pathology respectively [3]. The currently available therapeutic pro-
tocols are characterized by many drawbacks such as poor efficacy,
toxicity, parasites resistance to drugs, cost and route of administra-
tion [4–6]. This situation urges to identify new targets for malaria
and HAT treatment. With this aim we focused our attention on the
cathepsin L-like cysteine proteases, falcipain-2 (FP-2) and rhodes-
ain, which have been validated as promising targets for the treat-
ment of protozoan infections [7,8].
In particular FP-2 of P. falciparum, takes part to the cleavage of
the cytoskeletal elements of the erythrocyte membrane and it is
involved in the haemoglobin degradation. On the other hand, rho-
desain of T. b. rhodesiense, is responsible for the degradation of the
blood–brain barrier (BBB) and for the immune evasion.
Consequently, we evaluated its apparent permeability (Papp) across
the Caco-2 monolayer with an obtained medium permeability of 1
due to a low mass balance (sum of amount in the apical and baso-
lateral chambers) which has been calculated to amount to 45%.
This poor mass balance could be attributed to the strong interac-
tions of 1 with the components of cell membranes, which are sup-
posed to be strong because of the high lipophilicity of the
molecule. In order to account for its behaviour in the physiological
conditions, herein we report the NMR structural characterization
and conformational analysis of 1 in both, polar and apolar media.
Experimental
Synthesis of 1
In this context, our research group has been actively involved in
the synthesis and development of papain-family cysteine protease
inhibitors [9,10]. Among all the synthesized compounds the most
The synthesis of compound1 has been previously reported by our
group, [12] and it is briefly shown in Scheme 2. The R enantiomer of
the Garner’s ester 2, was firstly converted into key intermediate 3
which, at reflux under acidic conditions, afforded the 3-hydroxy-
methyl substituted 1,4-benzodiazepine 4 through Boc deprotection
of the nitrogen, cleavage of the oxazolidine ring and intramolecular
condensation to form an imino bond. Intermediate 5, obtained from
compound 4 via a series of standard reactions (Scheme 2), was then
condensed with commercially available allylamine to afford unsat-
urated amide 7. Such a derivative was desilylated and the free pri-
mary alcohol reacted with 4-chloro-2-trifluoromethylphenyl
isocyanate to afford carbamate 7, a fully functionalized scaffold at
the P1–P3 region. The terminal olefin was used to insert the required
‘‘warhead’’ via the cross metathesis methodology, using the Hov-
eyda–Grubbs 2nd generation catalyst (a phosphine-free N-hetero-
cyclic carbene ruthenium complex), which actually represents the
catalyst of choice for these kind of reactions [13].
promising inhibitor was proven to be the
a,b-unsaturated ester 1
(Scheme 1), characterized by the presence of a 1,4-benzodiazepine
(BDZ) scaffold, introduced internally to a peptide backbone, in
which the fused aromatic ring mimics a phenylalanine residue at
the P2 site, strongly preferred in this position from both proteases,
while the hydroxyl group of the scaffold has been employed to tie
substituents able to establish additional interactions with the S3
site of the target enzymes. At the C-terminal moiety an a,b-unsat-
urated ester has been introduced as warhead, able to act as a
Michael acceptor, thus leading to an irreversible alkylation of the
enzyme targets.
The use of an appropriate scaffold, incorporated into a character-
istic peptide sequence, has been selected because this strategy could
be particularly advantageous in terms of potency and selectivity, as
described also in our recent studies on other proteases [11].
The a,b-unsaturated ester 1 was proven to possess an extraor-
dinary potency towards FP-2 (k2nd = 3,571,000 Mꢁ1 minꢁ1) and a
binding affinity (Ki) of 17 nM [12]. Compound 1 was screened also
against rhodesain towards which it showed a moderate potency
Analytical methods
(k2nd = 34,600 Mꢁ1 minꢁ1) and a Ki value of 0.81
lM. Overall an
1H, 13C{1H} and 15N{1H} NMR spectra of 1 were recorded on
Bruker Avance 300 MHz NMR spectrometer equipped with a BBI
probe and operating at frequencies of 300.13, 75.47, 30.42 MHz;
many experiments were double checked on a Varian 500 MHz
spectrometer equipped with a ONE_NMR probe and operating at
499.74, 125.73, 50.65 MHz respectively; moreover it was necessary
to record 19F spectra at 469.65 MHz. Compound 1 was dissolved in
excellent selectivity has been detected towards the protozoan cys-
teine proteases with respect to the papain-family human cysteine
proteases cathepsin B (k2nd = 6000 Mꢁ1 minꢁ1 and Ki = 7.3
l
M) and
M). Additionally, when tested on P. falciparum, ester 1
showed a good profile with an IC50 = 12 M. However the discrep-
L (Ki = 8.4
l
l
ancy between the activity of 1 against the enzyme and the parasite
(nanomolar versus micromolar) might be explained by the
difficulty for this inhibitor to cross the parasite membranes.
500
lL of CDCl3 (10 mg) and in 500 lL of CD3OH (saturated
solution, about 3 mg). Calibration was attained using as internal
Scheme 1. Molecular representation and atom labelling scheme for 1.

A. Rotondo et al. / Journal of Molecular Structure 1076 (2014) 337–343
339
Ph
NBoc
O
c
a,b
O
NH
(R) COOMe
(R)
O
O
N
2
3
Boc
Ph
O
N
d,e,f
N
g,h,i
NH
N
TBSO
+
H2N
OH
HO
O
O
4
6
5
O
CF3
N
O
CF3
N
H
N
l
H
N
O
N
N
O
N
H
N
H
COOMe
O
O
O
O
Cl
Cl
7
1
Scheme 2. Reagents and conditions: (a) LiOH, MeOH, 0 °C-rt, 6 h; (b) i-BuOCOCl, NMM, CH2Cl2, 0 °C-rt, 30 min, then 2-aminobenzophenone, reflux, 20 min, rt, 12 h; (c) HCl/
MeOH, reflux, 5 h, then NaHCO3, MeOH, rt, 12 h; (d) TBS-Cl, imidazole, CH2Cl2, 0 °C-rt, 12 h; (e) NaH, 0 °C, 1 h then BrCH2COOEt, rt, 12 h; (f) LiOH, MeOH, 0 °C-rt, 6 h; (g) HATU,
CH2Cl2, rt, 12 h; (h) TBAF, THF, rt, 5 h; (i) 4-Chloro-2-(trifluoromethyl)phenyl isocyanate, rt, 12 h; (l) methyl acrylate, Hoveyda–Grubbs II generation catalyst, CH2Cl2, 100 °C, 2 h,
MW.
standard residual proton signal of the solvent ([D3] methanol:
d = 3.31 ppm; CDCl3 d = 7.26 ppm) and the 13C solvent septuplet
d = 49.0 ppm and triplet d = 79.5 respectively [14]. 15N calibration
was referred to the CH3NO2 as external standard (90% CH3NO2 in
CD3OH d = 380.5 ppm).
times [16] are then evaluated and reported for both studied envi-
ronments (Table 2).
Theoretical models
The model of inhibitor 1 was built in the MOLEFACTURE routine
of the VMD program operating with AMBERTOOLS PM3 minimiza-
tion methods. Several models were treated by GAUSSIAN03 soft-
ware package [17] with semi-empirical methods at the MP3MM
level [18]. Some of the meaningful models are herein reported to
better understand the NMR findings.
Complete and unambiguous assignment was pursued by several
1D and 2D homo- and hetero-nuclear NMR experiments such as 2D-
TOCSY, 2D-NOESY, 13C-HSQC, 13C-HMBC and 15N-HSQC. Table 1
reports the assigned resonances in both solvents; this is at the basis
of any further consideration. According to the many data available
we can assess that the potent cysteine protease inhibitor is more sol-
uble in apolar solvents such as chloroform. In the raw solvent
though, it is not possible to study the conformational features
because the residual DCl molecules involve amidic protons (espe-
cially 5-NH) in some kind of dynamic prototropic equilibrium. This
is the reason why, after many assays, we decided to add small
amounts of insoluble KOH to the sample in order to get rid of the
unwanted line-broadening effects (because of the slight shifts
caused by the base, H2O would be the best choice but it does not sep-
arate from the chloroform phase giving a very nasty signals around
4.8 ppm). On the KOH saturated sample we performed the exact
measurement of the NMR constants with a complete and total
assignment of the 1H and H-bound 13C resonances. It was also pos-
sible to distinguish the different geminal protons on the basis of NOE
dipolar couplings (Fig. 1, Tables 1 and 2 and supplementary data).
As an alternative solvent we have chosen the non-conventional
CD3OH for the following reasons: (a) because of its capability to
dissolve enough of the molecule to record 1H, 13C and 15N chemical
shifts (cs); and (b) to detect also the NAH amidic resonances in a
polar protic solvent somewhat reminding a biological environment
(Fig. 1, Tables 1 and 2 and supplementary data).
Results and discussion
Preliminary remarks
Conformational analysis of BDZ derivatives have attracted the
attention of many researchers [19] because of the known pharma-
cological activities of these constructs which are related to their
structure according to the known ‘‘structure–activity relationship’’
(SAR) [20]. The seven-membered ring might theoretically assume
many conformations [21], but, because of specific substituents
[20] or coordination modes [22], only few of the conformers keep
a thermodynamic relevance. This is demonstrated by both theoret-
ical [23] and experimental evidences [24]. The molecule 1 can
assume just two BDZ boat conformations with the benzo-fused
ring upward (facing the 3A-CH2) or downward (facing the 3D-
CH). We think these conformations are both populated in solution,
but NOE confirms always the prevalent presence of the latter. This
was also the biologically active conformation [10c,10d]. Our chal-
lenge is to evaluate the conformational behaviour of the whole 1
molecular scaffold in different solvents since it may shed light on
the biological behaviour of this inhibitor.
Selective DPFGSE 1D NOE [15] spectra, which were cleaner and
more sensitive, have been integrated by the Bruker Topspin soft-
ware package (version 2.5) and Mestrenova (version 6.0.2-5475).
These integration data were then converted into proton-proton
average distances using the two-spin approximation and the
geminal 3A-CH2 NOE integration as reference (1.78 Å). The average
distances extracted by many experiments at different mixing
Spectroscopical discussion
Conformational analyses of biologically interesting compounds,
by itself, is a fundamental step towards the whole knowledge of

340
A. Rotondo et al. / Journal of Molecular Structure 1076 (2014) 337–343
Table 1
1H cs of compound 1 in chloroform and in methanol at 298 K.
Code
1H CDCl3
1H CD3OH
1H & 19F Delta
Parent 13C CDCl3
Parent 13C CD3OH
13C & 15N delta
CH3
2-CH
3-CH
4-CH2
5-NH
3.69
5.84
6.82
4.00
6.59
4.31
4.67
7.74
7.62
7.28
7.36
7.61
7.39
7.47
4.05
4.94
5.06
7.05
7.55
7.49
8.06
ꢁ65a
3.70
5.97
6.90
4.01
8.62
4.61
+0.01
+0.13
+0.08
+0.01
+2.03
+0.3
51.9
121.7
143.6
40.4
52.0
121.7
145.9
41.2
+0.2
0.0
+2.3
+0.8
+0.4
ꢁ1.1
109.9b
53.0
110.3b
51.9
7-CH2
70-CH2
4B-CH
3B-CH
2B-CH
1B-CH
OP-CH
MP-CH
PP-CH
3D-CH
2A-CH2
2A0-CH2
1A-NH
3C-CH
5C-CH
6C-CH
CF3
ꢁ0.06
ꢁ0.14
+0.04
+0.03
ꢁ0.08
ꢁ0.02
+0.04
+0.04
+0.05
ꢁ0.09
ꢁ0.21
+1.85
+0.12
+0.13
ꢁ0.37
+3.3
7.58
7.66
7.31
7.28
7.58
7.43
7.51
4.108
4.85
123.0
133.0
125.5
131.0
130.2
128.6
131.6
61.9
123.4
133.4
126.0
131.5
130.9
129.2
131.9
63.0
+0.4
+0.4
+0.5
+0.5
+0.7
+0.8
+0.3
+1.1
+0.9
65.3
66.2
8.90
7.67
7.62
95.7b
126.3
133.0
124.7
93.8b
127.1
133.9
130.8
ꢁ1.9
+0.8
+0.9
+6.1
7.70
ꢁ62.70
ꢁ62.71
In bold character there are the most relevant cs variations.
a
Concerns 19F resonances instead of 1H.
b
Refers to 15N resonances instead of 13C.
Table 2
1H average distances detected by 1d NOE integrations at 298 K.
Contact in CDCl3
CDCl3 (Å)
Contact in CD3OH
CD3OH (Å)
2-CH
2-CH
2-CH
3-CH
3-4CH
3-CH
4-CH2
5-NH
5-NH
5-NH
CH3
5-NH
4-CH2
5-NH
CH3
4.85
3.20
3.71
4.85
n.d
2-CH
2-CH
2-CH
3-CH
3-4CH
3-CH
4-CH2
5-NH
CH3
5.21
3.55
4.18
4.70
5.38
3.45
3.05
2.98
5-NH
4-CH2
5-NH
CH3
4-CH2
5-NH
7-CH2
4-CH2
5-NH
2.94
2.94
3.60
2.54
3.95
4.04
1.78
2.62
2.54
4.32
4.04
4.32
4.04
2.51
2.62
1.76
3.85
4.32
4.85
4.32
nd
70-CH2
700-CH2
4B-CH
3B-CH
700-CH2
4B-CH
4B-CH
3B-CH
3B-CH
1A-NH
4B-CH
3A0-CH2
3A00-CH2
3A00-CH2
1A-NH
OP-CH
OP-CH
6C-CH
3B-CH
OP-CH
5-NH
5-NH
–
4B-CH
3B-CH
–
4.47
nd
–
5-NH
70-CH2
700-CH2
70-CH2
70-CH2
700-CH2
3D-CH
3D-CH
3D-CH
3D-CH
3A0-CH2
3A0-CH2
3A0-CH2
3A00-CH2
3A00-CH2
2B-CH
1B-CH
7-CH2
4B-CH
2.78
7-CH2
3B-CH
nd
3D-CH
3D-CH
3D-CH
1A-NH
4B-CH
3A-CH2
nd
3.98
2.66
Fig. 1. 1H NMR spectra at 500 MHz: (a) in KOH saturated CDCl3 at 25 °C; (b) in
CD3OH at 5 °C with the OAH suppression. The complete 1H assignment is possible
showing the crucial differences.
–
–
–
nd
4.23
3A-CH2
3A-CH2
1A-NH
OP-CH
the interaction mechanisms occurring in the chemistry of life [25].
Moreover, many biological molecules travelling within biological
systems pass through many different environments; this change
of media often triggers conformational mutations. Being the title
compound soluble in chloroform, it was straightforward to per-
form the first NMR analysis in this solvent. From the 1H NMR spec-
trum it was immediately clear that two over three of the CH2
methylene signals look split according to diasterotopic behaviour
of these nuclei. Usually this effect is remarkable in the case of
rather rigid conformations; consistently, after the complete and
total assignment of the 1H and 13C signals, it was possible to
observe different dipolar couplings (‘‘through the space’’ NOE
information) for the mentioned geminal nuclei. The many defined
1D NOE data allowed the evaluation of the average distances
(Table 2): provided there is a certain conformational freedom, it
is possible to find a PM3MM minimised conformation which suits
the main detected contacts and some other experimental data
(Fig. 2). First we would like to point out the long range contact
3A-CH2
2B-CH
1B-CH
6C-CH
3B-CH
OP-CH
nd
2.50
3.0
nd
Entries in bold were used as reference to determine all the other average distances.
3D-CH/4BCH & 3D-CH/1BCH assessing the boat conformation of
the BDZ with the benzo-fused ring on the same side of the 3D-
CH group; on the other side the close contact 5-NH/2-CH witnesses
that there is a certain stiffness of the warhead conformation. BDZ
conformation, retained also in CD3OH (Table 2), actually does not
diverge too much from the supposed active conformation inside
the macromolecular model of the complex [10c], however the war-
head and the carbamoyl pendant chain look quite over folded in
CDCl3.

A. Rotondo et al. / Journal of Molecular Structure 1076 (2014) 337–343
341
The 19F spectrum, showed two different signals with about 1:2
integration ratio in CD3OH, shielded respect to the single resonance
observed in CDCl3; on another hand, in strong dipolar and protic
solvents chemical shift is well deshielded (Fig. 2). This suggest
CF3 group is deep inside an intramolecular network of dipolar
interactions in CDCl3 whereas, in CD3OH a permanent intramolec-
ular 1A-NHꢂ ꢂ ꢂCF3 hydrogen bond has to be invoked; moreover this
is known to play a very important role in respect to the biological
activity of many substrates [26], including the present one [10c]; in
strong polar media this interaction is demonstrated to fade out
because of the competing solvent interactions (Fig. 2 and supple-
mentary data).
The permanent 1A-NHꢂ ꢂ ꢂCF3 interaction in CD3OH is confirmed
by the lack of any 1A-NH through the space contacts despite the
CDCl3 detection of few long-range couplings. According to our
knowledge it would be pretty unusual that intra-molecular H-
bonds are favoured in polar solvents, however, the overall data
together with few minimizations run by semi-empirical methods
at PM3MM level, revealed the competing and/or alternative pres-
ence of the 1A-NHꢂ ꢂ ꢂO@C intra-molecular interaction which is rea-
sonably prevailing in chloroform (reduction of the dipolar
moment) accounting also for the evident rigid conformation
around the 3A-CH2 torsional angles (Fig. 3) which easily gains flex-
ibility in presence of possible H-donors (protic solvents).
Fig. 2. 19F Spectrum in progressively more polar environments: according to our
knowledge [26] the 1A-NHꢂ ꢂ ꢂCF3 interaction gets more and more permanent in
pure CD3OH eventually fading out in strong polar environments.
In CD3OH, the most remarkable NMR spectral changes concern
the CH2 signals which are not any more split. This is per se the evi-
dence that dihedral angles around CH2 groups enhanced their
degrees of freedom. Specifically, by adding just few microliters of
CD3OH in the CDCl3 solution, 3A-CH2 signals quickly coalesce into
one resonance whereas the other geminal 7-CH2 protons begin to
get closer keeping though their own identity. This experiment
reveals that dihedral angles around 3A-CH2, which are in some
way constrained in chloroform, seem right away to free in
polar-protic solvents.
Generally speaking, the folded conformation presents
a
crowded network of loose intramolecular dipolar interactions
Fig. 3. Sound conformation in chloroform as minimized with semi-empirical
methods PM3MM: most of the NOE contacts are consistent with this conformation
and the entangled intramolecular interactions match the other NMR evidences. This
is the most populated conformation in chloroform but it is not the only one.
Fig. 4. (a) Representation of a stable conformation (PM3MM global minimum) still
presenting one of the intramolecular interactions involving the endocyclic carbonyl
group and (b) free conformation detected in pure CD3OH, the most dihedral angles
show free rotation.

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A. Rotondo et al. / Journal of Molecular Structure 1076 (2014) 337–343
perfectly matching the main detected CDCl3 NOE. It is easy to
understand that the structure opens up when many possible sol-
vent-dipolar interactions occur (Fig. 4). Interestingly, beyond the
amide protons, the most relevant 1H chemical shift differences
concern 6C-CH and 4B-CH (Table 1) and the most shifted 13C is
the 6C-CH, these changes well match the intramolecular
3A-NHꢂ ꢂ ꢂ3D-C@O switching. The presence of 3A-NH intramolecu-
lar hydrogen interactions in both solvents is also confirmed by
the 15N NMR resonance of 1A-NH which is at much lower frequen-
cies from what expected; it is more than 15 ppm downshift respect
to the 5-NH which should be theoretically more shielded (Table 1).
Also the comparison of many 1H NMR profiles and line-broadening
measurements highlights 1A-NH resonance is always the less
involved in chemical proton exchange testifying this site is pretty
protected by the molecule itself (this is not the case of strongly
polar solvents, see supplementary material and Fig. 2). Also the
other amidic 5-NH, in CDCl3, is involved in a weaker intramolecular
H-bond with the endocyclic carbonyl group (3D-C@O), this is keep-
ing the 7-CH2 resonance splitting.
fades out leaving room to many possible solvent-intermolecular
interactions.
Acknowledgements
This work was financially supported by the Ministero dell’Ist-
ruzione, dell’Universita‘ e della Ricerca Scientifica e Tecnologica
[MIUR-PRIN2010-2011, Grant 2010W7YRLZ_004 (M.Z.)].
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2014.
07.046. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References
In summary we can talk about a whole intramolecular bonding
network which is gradually switching from a folded structure,
overwhelming in CDCl3 (Fig. 3), to a much more dynamically
opened structure in pure CD3OH where solvent-intermolecular
interactions prevail but the local 1A-NHꢂ ꢂ ꢂCF3 turns out favoured
(Fig. 4). Experiments with the mixture of solvents show that, once
the aromatic branch gains freedom, dihedral angles on the ali-
phatic warhead branch are still a little locked as also witnessed
by the global minimum found by theoretical calculations
(Fig. 4a). Another important difference in the two solvents has to
be underlined is related to the warhead fragment: in MeOH, the
simultaneous presence of the couples 5-NH/2-CH & 5NH/3-CH;
4-CH2/2-CH & 4-CH2/3-CH; CH3/2-CH & CH3/3-CH dipolar cou-
plings, accounts for a certain flexibility of the terminal warhead
in respect to the more embedded CDCl3 arrangement (Fig. 4b).
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Conclusions
We carried out a thorough conformational analysis of the inhib-
itor 1 in apolar and polar media, assuming that these can simulate
specific biological environments. Although the presented ester is
less soluble in polar and protic solvents, these media are necessary
for its biological activity; indeed, whereas the aryl carbamoyl moi-
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mainly retained in apolar media, is not suitable to fit the catalytic
sites of the protease under investigation. Intermediate conditions,
generated by mixtures of solvents, also revealed that the carbam-
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1A-NH—2D-C@O interaction, while in a polar and protic environ-
ment (pure CD3OH) also the weaker interaction 5-NH—2D-C@O
fades out leaving room to interactions with the solvent. These last
ones appear to be of utmost importance to impart to the unsatu-
rated ester a conformational freedom necessary to induce a pro-
ductive fit with the catalytic sites which produces the observed
biological activity. To complete this analysis we have to report that,
in very strong polar conditions (in d6-DMSO and 20% of water),
even the last amphiphilic 1A-NH—CF3 interaction eventually
ˇ
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