Synthesis and conformational analysis of benzimidazole-based reverse turn mimics

Article abstract of DOI:10.1016/j.tetlet.2007.12.110

New benzimidazole-based tetrapeptide mimics were synthesized and their conformational features were studied by NMR and molecular modeling techniques. All the analyses led to the conclusion that a β-turn is stabilized in both 2 and 3. Since values of the t

Full text of DOI:10.1016/j.tetlet.2007.12.110

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Tetrahedron Letters 49 (2008) 1293–1296
Synthesis and conformational analysis of benzimidazole-based
reverse turn mimics
Giordano Lesma, Alessandro Sacchetti *, Alessandra Silvani
`
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via G. Venezian 21, 20133 Milano, Italy
Received 21 November 2007; revised 12 December 2007; accepted 19 December 2007
Available online 25 December 2007
Abstract
New benzimidazole-based tetrapeptide mimics were synthesized and their conformational features were studied by NMR and mole-
cular modeling techniques. All the analyses led to the conclusion that a b-turn is stabilized in both 2 and 3. Since values of the torsion
angles do not allow an univocal definition of the b-turn type, structures 2 and 3 could be assigned to the generic type IV classification.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
of peptidic bond. In our strategy, the N-substituted benz-
imidazole 1 (Fig. 1) is designed as the dipeptide mimic of
the i + 1ꢀi + 2 residues core of b-turn motif. Herein, we
report the synthesis of new benzimidazole-based b-turn
mimics 2 and 3 and their conformational analysis by
The design, synthesis and application of peptidomimetic
compounds have been for many years a focal point of a
research aimed to develop new therapeutics with peptide-
like activity and enhanced resistance towards proteases.1
Among the different strategies, of particular interest and
success has been the replacement of a dipeptide substruc-
ture in a natural substrate with a constrained rigid ana-
logue, capable to induce a folding in the peptide chain.2
Reverse turns are structural motifs commonly found in
bioactive peptides that, besides being fundamental in pro-
tein folding, play a central role as molecular recognition
elements.3 During the past decade, a large array of highly
functionalized nitrogen heterocycles, mainly fused bicyclic
lactams, have been synthesized and employed as reverse
turn mimics.4 In search of new, small peptidomimetics that
combine the structural rigidity with the ease of synthetic
access, we focused on the benzimidazole moiety. Benzimid-
azole is a very common aromatic pharmacophore, which is
present as a rigid scaffold in many different biologically
active molecules.5 We envisaged that the amidine group
of the benzimidazole ring could provide a rigid bioisostere
1
molecular modeling calculations6 and H NMR7 spectro-
scopy. These structures have been prepared through a
straightforward synthetic path, relying on the condensation
of ortho-phenylenediamine with an aminoacid, thus mak-
ing in principle the easy preparation of a library of deriva-
tives possible. In this study, we employed L-alanine and
L-proline as the amino acid counterpart of condensation
with ortho-phenylenediamine, realizing the synthesis of 2
(Ac–AlaBidGly–NHMe, Bid = 1H-benzo[d]imidazole) and
3 (Ac–ProBidGly–NHMe) derivatives. These compounds
were selected after preliminary investigation by computa-
tional methods (vide infra) for their good b-turn mimic
O
O
O
NMe
H
NMe
H
AA2
N
N
N
H
O
O
N
N
N
N
N
AA1
N
R
H
1
2
3
*
Corresponding author. Tel.: +39 250314081; fax: +39 250314078.
Fig. 1.
E-mail address: alessandro.sacchetti@unimi.it (A. Sacchetti).
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.12.110

1294
G. Lesma et al. / Tetrahedron Letters 49 (2008) 1293–1296
propensity. Compound 3 was chosen to investigate the
effect on the reverse turn propensity of an increased steric
hindrance and conformational rigidity with respect to 2.
O
i + 2
C
α
C
i + 3
α
C
i
+ 2
N + 3
i
H
Ni + 2
i + 1
α
d
= dC
α
α
α
i + 3
α i - C
C
N
β
(Ci-C i +1-C i +2-N
)
O
2. Results and discussion
i +3
αi
C
+ 1
Ci
R
Ni +1
αi
C
The synthesis of tetrapeptide mimics is outlined in
Scheme 1. Reaction of ortho-phenylenediamine with N-
acetyl-L-alanine produced intermediate 5 which was cycli-
zed in acetic acid to yield the benzimidazole derivative 6.
Alkylation with bromoacetic acid methylester followed
by treatment with methylamine allowed to obtain the
desired product 2.8 The same synthetic strategy was used
for the preparation of 3. After condensation of N-acetyl-
L-proline with ortho-phenylenediamine (DCC/HOBt),
amide 7 was cyclized to compound 8. Alkylation of 8 with
bromoacetic acid methylester and subsequent treatment
with methylamine afforded 3.9
Fig. 2. Definition of parameters to characterize b-turn propensity of BID
systems.
Table 1
MC/EM conformational analysis for 2 and 3
No. of
conf.
<6 kcal/
mol
%
%
%
% H bond
(10
membered
ring)
˚
da < 7 A
jbj < 30°
jbj < 60°
Compound 2
In vacuo
In water
11
12
27 (3)
42 (5)
27 (3)
33 (4)
36 (4)
42 (5)
27 (3)
27 (3)
3. Conformational analysis: computational studies
Compound 3
In vacuo
7
7
57 (4)
57 (4)
43 (3)
43 (3)
43 (3)
43 (3)
43 (3)
43 (3)
Evaluation of the ability of 2 and 3 to induce reverse
turn conformations was made by means of computational
tools,10 by computing and analyzing characteristic geomet-
ric parameters3a (Fig. 2).
In water
Results are reported as percentage of conformers which meet the
requirements (the occurrence numbers are given in parentheses).
Two main geometric features are used to characterize a
˚
b-turn: a value of the interatomic distance da (Cai–Cai+3
)
distance da < 7 A and a torsion angle jbj < 30°. The first
˚
less than 7 A and the measure of the virtual torsion angle
b, defined by Ci–Cai+1–Cai+2–Ni+3, for which jbj < 30° is
associated with a tight reverse turn, while jbj < 60° is usu-
ally referred to an open reverse turn.11 Another feature of
b-turns is the presence12 of a 10-membered ring hydrogen
bond between the carbonyl oxygen at Ci and the hydrogen
on Ni+3. The computational procedure consisted of an
unconstrained Monte Carlo/energy minimization confor-
mational search using the molecular mechanics MMFF94
force field13 both in water and in vacuo. Only conforma-
tions within 6 kcal/mol of the global minimum were kept.
Results are reported in Table 1 as percentage of conformers
which meet the cited requirements. Compound 2 shows
moderate percentage of conformers satisfying the b-turn
geometry. In vacuo, only three conformers (27%) have a
two lowest energy conformers are b-turn mimics and the
next conformer (conf. no. 3), which is not predicted to be
a b-turn, is 1.08 Kcal/mol above the minimum. In water,
a more favourable situation is revealed: 42% of conformers
˚
have da < 7 A and jbj < 60°. In this case, the two first con-
formers are b-turn mimics as well. Conformer no. 3 is not a
b-turn mimic and its energy is 1.56 Kcal/mol above the
minimum. A better situation is observed for compound 3.
In this case, no difference between in vacuo and in water
simulations is observed. A good percentage (57%) of con-
˚
formers have a distance da < 7 A and 43% have a torsion
angle jbj < 60. Again, the first two lowest energy conform-
ers are good b-turn mimics and the next conformer (conf.
no. 3, DE = 2.88 Kcal/mol in vacuo, DE = 2.57 Kcal/mol
in water) is not a b-turn.
1. BrCH2CO2Me,NaH,
N-acetyl-L-alanine,
DMF, 80 °C,2 h.
NH2
O
isobutylchloroformate,
NMM,DMF
AcOH,
H
2. MeNH2, methanol
86%
65 °C,1 h
NHAc
N
N
2
NHAc
N
57%
98%
NH2
H
5
6
NH2
1.BrCH2CO2Me,NaH,
DMF, 80 °C,2 h.
NH2
O
N-acetyl-L-proline,
DCC,HOBt,DCM
AcOH,
Ac
N
H
65 °C,1 h
2. MeNH2, methanol
Ac
N
N
N
3
N
H
94%
51%
79%
7
8
Scheme 1. Synthesis of tetrapeptide mimics 2 and 3.

G. Lesma et al. / Tetrahedron Letters 49 (2008) 1293–1296
1295
Table 2
Characteristics of low-energy conformers calculated for 2 and 3
Table 4
Spectroscopic data of tetrapeptide mimics 2 and 3
a
b
c
d
˚
da (A)
b (°)
u2 (°)
w2 (°)
u3 (°)
w3 (°)
d DMSO-d6 (ppm)
Dd/DT (ppb/K)
2
3
5.38
5.57
11.04
ꢀ8.72
ꢀ84.40
123.65
113.98
ꢀ115.26
ꢀ127.66
81.00
63.79
NHMe
NHAc
NHMe
8.14
7.40
8.35, 8.24
ꢀ3.2
ꢀ6.3
ꢀ4.0
2
3
ꢀ68.32
a
b
c
Ci–Ni+1–Cai+1–Ci+1 torsion angle.
i+1–Cai+1–Ci+1–Ni+2 torsion angle.
i+1–Ni+2–Cai+2–Ci+2 torsion angle.
i+2–Cai+2–Ci+2–Ni+3 torsion angle.
N
C
N
d
of a b-turn conformation is to investigate the presence of
the intramolecular hydrogen bond (CiO–H–Ni+3) by means
1
of variable temperature H NMR experiments. The tem-
perature dependence (Dd/DT) of the H NMR chemical
1
Computational studies support the prediction that mol-
ecules 2 and 3 would easily achieve a b-turn conformation,
with the observation that the presence of a proline residue
in 3 is a favourable condition. The calculated backbone
geometries for 2 and 3 are reported in Table 2. The u
and w backbone torsion angles in residue i+1 and i+2
define the specific b-turn type.3a Since analysis of the tor-
sion angles does not allow an univocal classification of
the b-turn type, structures 2 and 3 are assigned to the gen-
eric type IV14 classification.
An analysis to evaluate and quantify the similarity of 2
and 3 to standard type b-turns was performed by superim-
posing the atoms of the amide backbone. Results are
reported as scores,15 for which a value of 1 means a perfect
similarity (Table 3). This analysis confirms the impossibil-
ity to unambiguously assign a b-turn type, even if a slight
preference for a type II b-turn geometry is revealed for
both 2 (score 0.87) and 3 (score 0.89). The lowest energy
conformers for 2 and 3 are shown in Figure 3.
shifts of the amide protons for 2 and 3 was evaluated in
DMSO-d6. The use of this solvent has been previously
reported in the literature.16 In this solvent, intermolecular
hydrogen bonds and those to the solvent are readily
cleaved by increasing temperature. In general, for the
chemical shift of the NH amide proton, a value of Dd/DT
below ꢀ4.0 ppb/K is considered as an evidence of the
absence of intramolecular hydrogen bonding.6b All analy-
ses were performed on 3.0 mM solutions to assure the
absence of significant molecular aggregation. Variable tem-
perature NMR coefficients were measured by recording
four 1H NMR spectra between 298 and 343 K. Results
are summarized in Table 4. For compound 2 the NHMe
amide hydrogen appeared downfield at 8.14 ppm, with a
coefficient of ꢀ3.2 ppb/K, according to an H-bonded con-
formation. As expected, the NHAc hydrogen is not
involved in H-bond (Dd/DT = ꢀ6.3 ppb/K). The 1H
NMR spectrum of 3 revealed the presence of two sets of
signals in a 3:1 ratio (coalescence was observed at 410 K)
for the presence of two conformers. The temperature
coefficient values for the NHMe hydrogen of both con-
formers of compound 3 (Dd/DT = ꢀ4.0 for both conform-
ers) suggested that these protons were involved in an
equilibrium between a hydrogen-bonded and a non-hydro-
gen-bonded state.
1
4. Conformational analysis: H NMR studies
Complete characterization of compounds 2 and 3 was
realized through mono- and bidimensional NMR spectro-
scopy. A well established procedure to confirm the presence
In summary, in this Letter, we have described the syn-
thesis of new tetrapeptide mimics. Both MM calculations
and spectroscopic NMR investigations support the conclu-
sion that the Bid-based scaffolds 2 and 3 are good reverse
turns. In particular, VT NMR studies indicated the pre-
sence of an equilibrium between a hydrogen-bonded state
and a non-hydrogen-bonded state. A type IV b-turn can
be postulated for both 2 and 3 in their lowest energy state,
based on the analysis of the torsion angles.
Table 3
Similarity analysis of 2 and 3 with standard type b-turns
I
I0
II
II0
III
III0
V
V0
2
3
0.80
0.84
0.83
0.80
0.87
0.89
0.80 0.79
0.78 0.82
0.80 0.65
0.78 0.67
0.53
0.50
Results are reported as scores, see Ref. 15.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2007.12.110.
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Fig. 3. Lowest energy conformers obtained by MM/MC calculations.

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˚
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