Design, Synthesis, and Conformational Analysis of Proposed β-Turn Mimics from Isoxazoline-Cyclopentane Aminols

Article abstract of DOI:10.1002/chem.201503062

Constrained aminols from oxazanorbornene derivatives have the geometrical features to be used as β-turn inducers. Four different stereoisomers were prepared and spectroscopically characterized (MD calculations, NMR-titration and VT-NMR experiments). Temperature coefficients in DMSO are indicative for the existence of an intramolecular hydrogen bond. Chirooptical properties revealed a β-turn arrangement of all the synthesized compounds, where, depending on the absolute configuration of the cyclopentane spacer, they can be labeled as left- or right-handed turns.

Full text of DOI:10.1002/chem.201503062

DOI: 10.1002/chem.201503062
Communication
&
Biomimetic Synthesis
Design, Synthesis, and Conformational Analysis of Proposed
b-Turn Mimics from Isoxazoline-Cyclopentane Aminols
Misal Giuseppe Memeo, Mariella Mella, Valentina Montagna, and Paolo Quadrelli*[a]
Dedicated to Irene Lucia Novara and Peppino Quadrelli on the occasion of their 85th birthday
Abstract: Constrained aminols from oxazanorbornene de-
rivatives have the geometrical features to be used as b-
turn inducers. Four different stereoisomers were prepared
and spectroscopically characterized (MD calculations,
NMR-titration and VT-NMR experiments). Temperature co-
efficients in DMSO are indicative for the existence of an in-
tramolecular hydrogen bond. Chirooptical properties re-
vealed a b-turn arrangement of all the synthesized com-
Figure 1. Compounds and products for turn induction. In the inset: criteria
for the identification of b-turns.
pounds, where, depending on the absolute configuration
of the cyclopentane spacer, they can be labeled as left- or
right-handed turns.
Critical distances are defined between the Ca carbon atoms,
which must be <7 Š. A second requirement is the distance be-
tween the carbonyl oxygen of the first amino acid and the
In the regulation of nearly every biological process, a pivotal
role is dictated by peptide ligands and protein receptors.[1] Be-
sides the understanding of the bioactivity, three-dimensional
interactions are essential for the design of analogues during
the drug discovery process. The determination of the spatial
orientation deeply involves the conformational analysis on the
basis of spectroscopic techniques and molecular modelling.[2]
As far as flexible molecules are concerned, the key point is the
correspondence among conformation found in solution, real
physical meaning, and in-silico determined conformations.[3]
Conformationally constrained molecules are the probes to get
information about the three-dimensional interplay between
ligand and receptor. Many efforts have been made in this
area[4] to mimic secondary structural features and b-turns are
the most common observed in small peptides and proteins.[2]
Turns are essential for protein structure and also occur within
protein binding sites, at protein–protein interfaces and in small
bioactive peptides, playing a crucial role in recognition.[5] Most
b-turns contain an intramolecular hydrogen bond between the
carbonyl oxygen of the first residue (i) and the amide NH
proton of the fourth residue (i+3), forming a pseudo-ten-
membered ring (Figure 1).[6]
amide hydrogen of the fourth one, which must be <4 Š. In
general, the use of cage amino acids allows for the induction
of a b-turn by the complete replacement of the three amino
acids required normally.[7–9]
The synthesis of analogues designed to stabilize a peptide
chain is constantly pursued and examples of cyclic and bicyclic
modifications have been recently reported in literature,[9a] for
example, spirolactam-bicyclic and tricyclic proline based sys-
tems,[10] medium-ring heterocyclic compounds[11] and confor-
mationally constrained b-amino acids.[12]
Cage a-amino acids of type 1 incorporate a bulky hydropho-
bic disubstituted cage while b-amino acids of type 2 find in
the norbornene skeleton the best residue to generate a turn-
inducer.[7] Non-racemic amides 3 were the key step for a suc-
cessful application of this type of chemistry towards metallo-
protease inhibitors. The use of peptidic reverse turns is often
limited because natural peptides are not resistant to enzymatic
degradation. Nonpeptidic (S)-aminobicyclo[2.2.2] octane-2-car-
boxylic acid 4 (H-(S)-ABOC-OH) were reported as reverse turns
into peptides.[5]
Recently we have proposed the synthesis of a new class of
cyclopentane aminols starting from cyclopentadiene using the
nitrosocarbonyl intermediates (RCONO, 5) chemistry.[13]
These intermediates, generated by the mild oxidation of ni-
trile oxides with tertiary amine N-oxides or by oxidation of
hydroxamic acids, are efficiently trapped by cyclopentadiene
(Scheme 1) to afford the hetero Diels–Alder (HDA) cycload-
ducts 6, highly reactive dipolarophiles employed to
synthesize the conformationally restricted carbocyclic aminols
8 through amide hydrolysis and NÀO bond cleavage of the
cycloadducts 7.[7,14]
[a] Dr. M. G. Memeo, Prof. M. Mella, Dr. V. Montagna, Prof. P. Quadrelli
Department of Chemistry
University of Pavia
Viale Taramelli 12
27100 - Pavia (Italy)
E-mail: paolo.quadrelli@unipv.it
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201503062.
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Scheme 1. Synthetic pathway of isoxazoline-cyclopentane aminols through
nitrosocarbonyl chemistry. Boat- and chair-like conformations of the aminol
8. Side chain structures are shown as hydrogen-bonded (b) and non-hydro-
gen-bonded (n).
We wish to present here our conceptual approach towards
the preliminary design of original turn mimics. Boat- and chair-
like conformations are possible for the aminolic structures (cal-
culated in the gas phase at the DFT B3LYP/6-31+G(d,p)
level);[15] the aim is to ascertain whether adequate elongation
from the hydroxy and amino groups with side chains incorpo-
rating aminoacidic residues can provide a stabilized turn struc-
ture (Scheme 1) of type hydrogen-bonded (b) or non-hydro-
gen-bonded (n).[16]
The regioisomeric aminols 8a,b, prepared according to a pre-
viously reported methodology,[7,14] nicely resemble the struc-
tures of external mimetics because of the reduced conforma-
tional flexibility determined by the isoxazoline ring fused to
the cyclopentane moiety.[17] To develop our concept, the syn-
thetic strategy for the preparation of depsipeptide-like struc-
tures is outlined in Scheme 2.
The protection of the hydroxy group was performed under
standard procedure affording the amines 9a,b in 85% each.
Compounds 9a,b were coupled with the commercially avail-
able l-Alalnine N-Boc protected. Diastereoisomers 10a and
11 a were isolated in good yields and similarly diastereoisomers
10b and 11 b obtained from 9b. The deprotection of the hy-
droxy functionalities was secured by standard nBu4NF treat-
ment. The alcohols of type 12a,b/13a,b were obtained in very
good yields.
Scheme 2. Synthesis of the turn mimic compounds: a) TBDMSiCl, Imidazole,
CH2Cl2, RT, 18 h. b) N-Boc-l-Ala, HBTU, DIEA, CH2Cl2, RT, 48 h. c) nBu4NF, THF,
RT, 3 h. d) HOOC-CH2-COOtBu, DIC, DMAP, CH2Cl2, RT, 48 h. Temperature coef-
ficients of amide and carbamate protons of 14a,b and 15a,b in CDCl3
(298.15–318.15 K) and DMSO (298.15–348.15 K). Yields and [a]D (C=1,
MeOH) are also reported.
dure in DCM at room temperature for 48 h. The ester deriva-
tives 14a/15a and 14b/15b were obtained from good to opti-
mum yields.
The alcoholic groups of the compounds 12 and 13 were
then derivatized as esters of a desimmetrized malonic acid
easily prepared upon treatment with malonic acid with anhy-
drous pyridine in ter-butanol and adding dropwise an equimo-
lecular amount of methanesulfonyl chloride.[18] This choice was
done to avoid the presence of a further stereogenic center
and to terminate the elongation with a carboxylic moiety
facing the amino group on the opposite chain. The esterifica-
tion was conducted with a typical DIC/DMAP coupling proce-
The conformational analysis[19] relies upon a series of CD
analyses coupled with MD and DFT calculations as well as NMR
experiments.[20,21] Collection of the CD spectra was obtained
from MeOH solutions (10À5 m solutions) of diastereoisomers
14a,b within a range of wavelength between 200–300 nm
(Figure 2). The CD profile of compound 14a has a positive ab-
sorption band at 212.9 nm and a second negative band cen-
tered at 226.5 nm. This profile can be addressed to a left b-
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The extent of H-bonding in diastereoisomers 14,15 in CDCl3
and DMSO was evaluated through temperature coefficients of
the amide and carbamate protons at 298.15 and 318.15 K for
CDCl3 and up to 348.15 K for DMSO as well as DMSO-titration
experiments.[22] It can be assumed that lower temperature co-
efficients values in CDCl3 ꢀ2.4 ppb/K are related not only to
shielded protons but also to accessible ones; hence, only
values significantly larger than 2.4 ppb/K in CDCl3 can be un-
ambiguously assigned to NH protons initially shielded, which
become exposed to the solvent upon increasing tempera-
ture.[5,23]
On the other hand, low temperature coefficients in DMSO
(<5 ppb/K) are related to inaccessible protons to the solvent.
Diluted solutions (10À3 m) of the compounds 14,15 in the deu-
terated solvents of choice were used to record the 1H NMR
spectra. Scheme 2 reports the temperature coefficients values
expressed in Dppb/DK highlighted in red for CDCl3 and in blue
for DMSO. The results of the determinations indicate that in
CDCl3 the four diastereoisomers display somewhat a borderline
situation since the values of the amide protons are in the
range 2.5–5.0 Dppb/DK and similarly happens for the carba-
mate protons. The temperature coefficients in DMSO indicate
that the amide protons, ranging at 3.2–4.2 Dppb/DK for all the
four substrates, are inaccessible to the solvent, while carba-
mate protons have Dppb/DK values higher than the threshold
values (8.6–9.2 Dppb/DK) indicating the accessibility of those
protons to the solvent.
We also performed DMSO-titration experiments with gradual
addition of DMSO to CDCl3 solutions (10À3 m) of the four dia-
stereoisomers 14,15. Figure 3 shows the plots of the four com-
pounds. The results clearly indicated that chemical shifts of the
amide protons (solid lines with solid symbols) remain almost
unchanged over the addition of increasing amounts of DMSO
to the CDCl3 solutions; the range of variation DNH is 0.09-
0.18 ppm indicating the inaccessibility of the amide proton,
engaged in H-bonding with the oxygen atom of the carbonyl
group.
Figure 2. CD spectra of diastereoisomers 14a,b and 15a,b experimental
(top), calculated (bottom)
turn in accordance with those reported in literature.[22] On the
other side, the CD spectrum of 15a shows a comparable trend
with a negative absorption band at 213.0 nm and a second
positive band centered at 227.6 nm, consistent for a right b-
turn.
Similarly, the CD profile of compound 14b has a negative
absorption band at 212.9 nm and a second positive band cen-
tered at 229.6 nm. This profile can be addressed to a right b-
turn in accordance with those reported in literature.[19]
On the other side, the CD spectrum of 15b shows a positive
absorption band at 212.8 nm and a second negative band cen-
tered at 227.7 nm, consistent for a left b-turn. Simulation of
the CD absorption profile has been conducted by TD-DFT cal-
culation at the B3LYP/6-31 g(d,p) level simulating a methanolic
solution.[15]
The calculated CD spectra nicely fit with the experimental
profiles indicating that inversion of the configurations at the
isoxazoline-norbornane moieties correspond to an inversion of
the chirooptical properties of the products at hand. As the cal-
culated CD spectrum of the conformer 14a nicely fit the exper-
imental one obtained the configuration can be assigned
beyond any reasonable doubt. The same approach was fol-
lowed for all the other diastereoisomers (see the Supporting
Information).
Figure 3. DMSO-titration of compounds 14,15. Solid lines (solid symbols) in-
dicate amide protons and dashed lines (open symbols) the carbamate pro-
tons.
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A larger chemical shift variation is however observed for the
carbamate NH proton (dashed lines with open symbols) with
DNH values ranging from 1.23 to 1.43 ppm. The carbamate NH
protons are prone to be attached by the DMSO molecule
through a hydrogen bond, being more accessible. To properly
rationalize all the data obtained from DMSO titration and VT-
NMR experiments, and to better understand the dynamic be-
havior of 14,15, unrestrained MD simulations were performed
both in DMSO and water to compare theoretical calculations
and NMR experiments as well as to probe their behavior in
biological systems.[15]
lated CD spectra allowed for the identification of the absolute
configuration of every single diastereoisomer.
The driving force that makes so efficient these compounds
in inducing b-turns can be related to the presence of a fused
isoxazolinic ring or similar rings to the cyclopentane moiety.
This latter usually adopts an envelope conformation with the
flap directed toward the isoxazoline ring thus giving a boat-
like appearance to the bicyclic system. The chair-like conforma-
tion was found higher in energy (from B3LYP/6-31 g(d) opti-
mized structures). The boat-like conformation allows for the
relief of non-bonded interactions between the heterocyclic
ring and the substituents on the adjacent cyclopentane car-
bons and causes the dihedral angles between the isoxazoline
protons and the adjacent trans cyclopentane protons to be
near 908. This results in a flattening of the cyclopentane enve-
lope pushing the amino and hydroxy groups close each
other.[14b]
Through the calculated trajectories it is possible to analyze
the conformations assumed by the cyclopentane ring, which
plays the pivotal role in the side chains pairing and then what
kind of interactions occurs between these. In Figure 4, the di-
The reported diastereoisomeric compounds represents
a “proof of concept” for the use of simple and easily prepared
aminols for the synthesis of non-peptidic turn-inducers. It is
worthwhile to note that the aminols themselves display the
crucial role in the formation of the b-turn arrangement. Further
experiments will aim to ascertain the influence of the nature of
the two side chains on the type of turn for a better under-
standing of applicability/reliability of these scaffolds as b-turn
inducers.
Experimental Section
Figure 4. Dihedral angles (H4isox. and bridged methylene) values along 10 ns
simulations in DMSO as solvent.
Syntheses, characterization, and detailed procedures for all the
compounds can be found in the Supporting Information.
hedral angle values assumed by every compound along the
simulation are reported in DMSO solutions (see the Supporting
Information for simulation in water).
Acknowledgements
Dihedral angle values within 808 and 1108 are consistent
with boat-like conformations while higher values with chair-
like conformations. Three out of four diastereoisomers spend
more time with the cyclopentane methylene upward with the
exception of 15b showing a preference for a chair-like confor-
mation. In the boat-like conformations all the geometrical re-
quirements imposed by the definition of b-turn are respected
(see the Supporting Information).
Financial support: University of Pavia, MIUR (PRIN 2011, CUP:
F11J12000210001) and COST Action CM1004.
Keywords: amino alcohols · b-turn inducers · cycloadditions ·
Diels–Alder reactions · nitrosocarbonyl compounds
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Published online on September 29, 2015
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