Sugar furanoid trans-vicinal diacid as a γ-turn inducer: Synthesis and conformational study

Article abstract of DOI:10.1039/c3ob41462k

A simple method for the synthesis of a sugar furanoid trans vicinal diacid and its incorporation into the N-terminal tetrapeptide sequence (H-Phe-Trp-Lys-Thr-OH) to get glycopeptide 8 has been described. 2D NMR and MD simulation studies of 8 clearly show that the sugar diacid adopts a γ-turn conformation towards the N-terminus.

Full text of DOI:10.1039/c3ob41462k

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Sugar furanoid trans-vicinal diacid as a γ-turn inducer:
synthesis and conformational study†
Cite this: Org. Biomol. Chem., 2013, 11,
6874
Madhuri Vangala,a Snehal A. Dhokale,b Rupesh L. Gawade,c
Received 16th July 2013,
Accepted 5th August 2013
Rajamohanan R. Pattuparambil,b Vedavati G. Puranikc and Dilip D. Dhavale*a
DOI: 10.1039/c3ob41462k
www.rsc.org/obc
A simple method for the synthesis of a sugar furanoid trans
vicinal diacid and its incorporation into the N-terminal tetra-
peptide sequence (H-Phe-Trp-Lys-Thr-OH) to get glycopeptide 8
has been described. 2D NMR and MD simulation studies of 8
clearly show that the sugar diacid adopts a γ-turn conformation
towards the N-terminus.
Biological receptors recognise peptides in a well defined,
compact and predictable three-dimensional structure which is
often composed of secondary structures such as β-turns1 and
γ-turns.2 The β-turns consist of a tetrapeptide sequence indu-
cing a reversal in chain direction in which the distance
between the Cα(i) and the Cα(i + 3) is <7 Å, while the γ-turns
consist of a tripeptide sequence. In general, β/γ-turns are
stabilized by a hydrogen bond between CvO of residue i and
NH of residue (i + 3)/(i + 2), forming a pseudo ten-3/seven-
membered ring.4 These turn structures in peptides are impor-
tant for understanding the molecular mechanism of peptide–
protein or protein–protein interactions that enforce a specific
backbone conformation.5 In this direction, various peptidic
and non-peptidic scaffolds were designed to assess their
potential as β-turn6 or γ-turn mimetics7 although the latter are
relatively rare. Among various peptide mimetics, furanoid
sugars8 have gained much acclaim in DNA/RNA due to the
puckered conformation of the furanose ring. Moreover, sugar
furanoid frameworks are found to be inducers of secondary
structures in oligomers9 as well as in short peptide
sequences.10 In this respect, a number of constrained amino
Fig. 1 Known furanoid α,β-sugar amino acids and diacids.
acid analogues11 as well as α-/β-sugar amino acids,12 I/II
(Fig. 1), are known to be stabilizers of β-turns13 or helix indu-
cers in homooligomers.14 In addition, segments containing
diacid functionalities, suitable for C-terminus linkages, have
been explored and some of them showed parallel sheet folding
between attached peptide strands. For example Gellmann et al.
reported the synthesis of cis-1,2-cyclohexane dicarboxylic acid
III as a linker that promotes parallel β-sheet secondary struc-
ture in water.15 Chakraborty et al. have demonstrated the bi-
directional elongation of sugar diacid16 IV or its carbasugar/
pyrrolidine analogue with identical peptide strands that led to
the formation of a C2-symmetric reverse-turn mimetic or favor
a pseudo β-turn.17 Although a number of linkages containing
diacids or equivalents have been explored in organic/aqueous
solvents,18 no report on the use of a sugar vicinal diacid as a
promoter of secondary structures, to the best of our knowl-
edge, is known so far. As a part of our continuous interest in
sugar amino acids19/iminosugars20 we report here a new sugar
furanoid trans vicinal type diacid V (Fig. 1) and demonstrate
its first applicability by incorporating it into the tetrapeptide
(H-Phe-Trp-Lys-Thr-OH) N-terminus that induces γ-turns. Our
efforts in this direction are depicted below.
aDepartment of Chemistry, Garware Research Centre, University of Pune, Pune,
411 007, India. E-mail: ddd@chem.unipune.ac.in
bCentral NMR Facility, National Chemical Laboratory, Dr Homi Bhabha Road,
Pune 411 008, India
cCentre for Materials Characterization, National Chemical Laboratory,
Dr Homi Bhabha Road, Pune 411 008, India
†Electronic supplementary information (ESI) available: Copies of 1H and 13C
NMR, HRMS spectra of compounds 2–5; IR of 7; HPLC, HRMS of 8; 2DNMR data
of 8; crystallographic data (CIF) of compounds 2 and 3. CCDC 936034 and
936035. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3ob41462k
The required V was synthesized starting from the diaceto-
nide D-glucose (Scheme 1). Thus, the PCC oxidation of
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Scheme 1 Synthesis of a trans vicinal sugar furanoid acid 5.
diacetone D-glucose gave ketone 1. Reaction of ketone 1 with
trichloromethyl anion, generated using CHCl3/LHMDS,
afforded trichloromethyl alcohol 2 in 48% yield as the only dia-
stereomer. The ‘R’ configuration at the C-3 centre of 2 was con-
firmed by X-ray crystallography‡ (see data files in ESI†) and by
comparing the analytical data with those reported.21 Treat-
ment of 2 with NaN3 and NaOH (4 eq.) in dioxane–water
Fig. 2 ORTEP diagram of compound 3.
trans β-sugar amino acid as a structure-inducing template to
make cyclic somatostatin analogues.25 Inspired by this obser-
vation, we incorporated the sugar acid 5 into a tetrapeptide
sequence (H-Phe-Trp-Lys-Thr-OH) using 2-chlorotrityl chloride
1
afforded α-azido acid as is evident from the IR and H NMR of
crude product. The formation of azido acid proceeded by
in situ formation of epoxychloride followed by the attack of
NaN3 to give azido acid chloride that was hydrolyzed to azido
acid.22,23 At this stage, crude α-azido acid was directly sub-
jected to condensation with benzylamine using EDC–HOBt, to
give amide 3. The ‘S’ absolute configuration at the newly gen-
erated stereocentre C-3 in 3 was confirmed by X-ray crystallo-
graphy‡ (Fig. 2) (see data files in ESI†). The protection of C-3
acid group with benzylamine not only allowed us to determine
the absolute configuration at C-3 but also gave the option of
easy manipulation of the 5,6-isopropylidene group to get C-4 acid
functionality that could be incorporated into the tetrapeptide
so as to determine possible H-bonding and turn mimetics.
Thus, the 5,6-isopropylidene group in 3 was deprotected
with 85% AcOH to give diol 4 in 90% yield. The diol 4 on oxi-
dative cleavage using NaIO4 in THF–H2O gave an aldehyde
which on oxidation with oxone24 gave sugar acid 5. The unique
features of compound 5 are
resin (0.8 mmol g−1
) by manual solid phase synthesis
(Scheme 2). The attachment of the first amino acid, i.e., Fmoc-
Thr(But) (2 eq.), to a solid polymer was done using DIPEA.
Fmoc deprotection was done using 20% piperidine in DMF
and the coupling of succeeding amino acids, i.e., Fmoc-Lys-
(Boc), Fmoc-Trp and Fmoc-Phe, was done using HBTU, HOBt
and DIPEA. Subsequently, sugar acid 5 was incorporated into
the N-terminus of the tetrapeptide under the same coupling
conditions to give resin bound glycopeptide 7. In the next step,
(i) Vicinal diacid functionality offering selective protection
of either of the carboxyl groups.
(ii) Presence of cis β-azido acid as a precursor for β-amino
acid with a sugar framework.
(iii) Presence of α-azido acid functionality with a cyclic qua-
ternary centre at C-3 as a synthon for sugar α-amino acid.
Having sugar acid 5 in hand, we planned the introduction
of 5 into the N-terminus of the peptide sequence by keeping
azide functionality intact to avoid the possibility of additional
secondary structures. Kessler et al. replaced the Phe-Pro bridge
of the Veberpeptide (cyclo[-Phe-Pro-Phe-D-Trp-Lys-Thr-]) with
Scheme 2 Solid phase synthesis of a glycopeptide.
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7 was treated with 0.2% TFA in DCM to yield glycopeptide 8, showed very little changes in shift Δδ ∼ 0.16 ppm suggesting
with all the protecting groups intact. The crude compound was its involvement in intramolecular hydrogen bonding between
then purified using RP-HPLC and analyzed by HRMS (S18–S20 NHV and 31CO, in accordance with the observed strong NOE
of ESI†), yielding the glycopeptide 8 in more than 95% purity correlation between NHV and 32CH (Fig. 4).
and in 30–41% yield.
This fact is further substantiated by the molecular
In order to understand the influence of vicinal diacid dynamics (MD) simulations for 8 that were carried out at
linkage, conformational analysis of glycopeptide 8 was per- 295 K using Schrodinger software on macromodel 9.9 imple-
formed. We determined the solution structure of 8 by 2D NMR menting the OPLS_2005 force field in CD3CN (dielectric con-
spectroscopy. Complete proton and carbon signal assignments stant 37.5). Monte Carlo conformational analyses were
of 8 were done on a 4 mM solution in CD3CN using a performed by generating 3000 starting structures, followed by
combination of 2D COSY, HMBC, HSQC, NOESY and TOCSY minimization of these structures to a gradient of 0.05 kJ Å−1
(see data in ESI†).
mol−1 with an RMS deviation of 0.09 kcal mol−1. For minimiz-
The backbone has been well defined, suggesting a predomi- ation, ten distance restraints obtained from the NOESY experi-
nantly single conformation. Assignment of all the amide ment (S40 of ESI†) were used that generated 35 conformers out
protons was done using 15N HSQC spectroscopy and was of which 15 conformations within 1 kcal mol−1 of the global
found to be in the range of 5.2–9.5 ppm. The NOESY spectrum minimum energy were considered. Superimposition of 15
was characterized by numerous strong and medium cross lower energy structures showed the existence of 7-membered
peaks (Fig. 3). Sequential NOEs were observed between NHI– hydrogen bonding, forming an intramolecular C7 turn around
6CH, NHII–12CH, NHIII–23CH, NHIII–24CH2 (weak NOE) and NHV and 31CO at a distance of 1.9 Å (Fig. 5).
NHIV–32CH. In the sugar residue NOEs were observed between
NHV–32CH, 35CH–38CH, 34CH–38CH, and 32CH–37CH. In
addition, we observed some interresidual NOEs between NHV–
t
19CH, 17CH–6CH, and equivalent Bu 53/54/55CH3–14/17/18CH,
suggesting a possible secondary structure. The NOE cross peak
between NHV–32CH indicated the possibility of 7-membered
H-bonding formation leading to a γ-turn between NHV–31CO. In
order to confirm this fact, we studied the effect of solvent on the
changes in chemical shift of NH protons.
Thus, a solvent titration study of compound 8 in CD3CN
was performed using 5 µL of DMSO-d6 for each addition. The
chemical shift changes of all the amide protons are presented
in a graph (S39 of ESI†). It is evident from the graph that the
chemical shifts of NHI–IV, VI, and VII showed changes in shift
Δδ in the range of 0.49–0.73 ppm suggesting their involvement
in intermolecular hydrogen bonding. In contrast, the NHV
Fig. 4 DMSO titration studies.
Fig. 3 NOE cross peaks in glycopeptide 8 (thick line – strong NOE; dotted line
– medium NOE).
Fig. 5 Superimposed 15 lowest energy conformations forming a γ-turn.
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In conclusion, we have synthesized a sugar furanoid trans
vicinal acid 5 from diacetone D-glucose in overall 28.6% yield.
In the sugar acid 5, the C-3 acid was converted to benzylamide
as a continuation of the peptide sequence and C-4 acid was
incorporated into a tetrapeptide sequence (H-Phe-Trp-Lys-Thr-
OH) for conformational analysis. The 2D NMR and MD simu-
lation studies of glycopeptide 8 clearly demonstrated that con-
strained sugar acid 5 can serve as an efficient inducer of
γ-turns. Further application of this highly functionalized sugar
acid 5 as a mixed α, β-sugar azido/amino acid will be exploited
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‡Crystallographic data of compounds 2 and 3 have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication no.
CCDC 936034 and 936035 respectively.
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