Synthesis and structural studies of peptides containing a mannose-derived furanoid sugar amino acid

Article abstract of DOI:10.1039/b712365p

A mannose-derived furanoid sugar amino acid (Maa) induced helical turns in peptides having repeat units of Maa(Bn₂)-Phe-Leu, which aggregated into head-to-tail duplexes in the longer oligomers. The Royal Society of Chemistry.

Full text of DOI:10.1039/b712365p

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and structural studies of peptides containing a mannose-derived
furanoid sugar amino acid†‡
Tushar Kanti Chakraborty,* Dipankar Koley, Rapolu Ravi and Ajit Chand Kunwar*
Received 10th August 2007, Accepted 19th September 2007
First published as an Advance Article on the web 3rd October 2007
DOI: 10.1039/b712365p
A mannose-derived furanoid sugar amino acid (Maa) induced helical turns in peptides having
repeat units of Maa(Bn2)-Phe-Leu, which aggregated into head-to-tail duplexes in the longer oligomers.
Introduction
Results and discussion
Since they were first reported as useful peptide building blocks,
the pyranoid1 and furanoid2 sugar amino acids have been used
extensively by many research groups world wide as conforma-
tionally constrained scaffolds in peptidomimetic studies.3 They
have also emerged as an important class of synthetic monomers
leading to many de novo oligomeric libraries with interesting
structures and properties.4 For example, the linear homooligomers
of a glucose-derived furanoid sugar amino acid (Gaa) had a
well-defined structure in CDCl3 with repeating b-turns, each
Compounds 1–4 were synthesized using standard solution phase
peptide coupling methods and the final products were purified
by silica gel column chromatography and fully characterized by
spectroscopic methods before the conformational studies.
Detailed NMR studies of 1–4 were carried out in CDCl3.
For 1 and 2, only one set of peaks was observed, even when
the temperature was lowered to 228 K. Amide proton chemical
shifts and solvent titration studies support the involvement of the
PheNH in hydrogen bonding.5 The presence of NOE connectivities
between PheNH and the preceding MaaC5H support PheNH–
CO(Boc) H-bonds in 1 and 2 and PheNH(5)-Maa(4)CO H-bonds
in 2, which result in a 10-membered pseudo b-turn in 1 and
two such turns in 2, like those observed earlier.4c Evidence for
these sheet-like structures is further provided by the 3JNH–CaH being
>8 Hz.
NMR spectra of both 3 and 4 in CDCl3 displayed two sets
of peaks. The major to minor populations are in the ratio of
about 3 : 2 for 3, whereas for 4 we could not obtain the exact
population of the minor isomer; however it was believed to be
<5%. The exchange peaks in the ROESY spectra between these
species show that they are in equilibrium with each other. The
rate of exchange between the two species was slow in the NMR
time scale, being 0.9 Hz and 0.1 Hz for 3 and 4, respectively.5,6
The first indications of the major isomer having a different and
compact structure in 3 and 4, compared to 1 and 2, reside in the
very large (∼8 ppm) chemical shifts of all the amide protons (dNH),
excluding the BocNH. Solvent titration studies were carried out
by adding DMSO-d6 to the CDCl3 solutions of the peptides 3 and
4. Insignificant changes were observed in the chemical shifts of the
amide protons on addition of up to 6% DMSO-d6. This indicates
that most of the amides of the major species of 3 and 4 were H-
bonded. Fig. 1 shows the solvent titration plots for 4. When more
DMSO-d6 was added to the CDCl3 solution, a gradual growth of
the minor species at the expense of the major one was observed.
In pure DMSO-d6 only the peaks of the minor species were seen,
that belonged to structures similar to those observed for 1 and 2.
The NMR data for the major species in 3 and 4 strongly
suggest the presence of dimeric structures in CDCl3 solution.
The extensive network of H-bonds seen in the NMR spectra
probably arises due to the formation of this dimer-like association
of the individual strands. These observations, along with the
changes in the isomer populations during variable temperature
involving a 10-membered ring structure with intramolecular
4g
=
hydrogen bonds between NHi → C Oi − 2
.
A very similar
structure was also seen in the corresponding heterooligomers
with repeating Gaa-Leu-Val units which displayed well-defined
turn structures with repetitive 10/10/9-H-bonding patterns.4e In
contrast, the octameric chain of C-glycosyl a-D-lyxofuranose
configured tetrahydrofuran amino acid, having the C-2 and C-
5 substituents on the tetrahydrofuran ring trans to each other,
adopted a well-defined helical secondary structure stabilized by
16-membered (i, i − 3) inter-residue hydrogen bonds, similar
to a p-helix, in a nonpolar solvent like CDCl3.4d However,
homooligomers of a mannose-derived sugar amino acid (Maa),
having a similar 2,5-trans configuration, failed to exhibit any
well-defined structure in polar solvents.4n In the present study,
we investigate the conformational biases imposed by the Maa
residues in the linear oligomeric peptides 1–4 having repeat units
of Maa(Bn2)-Phe-Leu.
Indian Institute of Chemical Technology, Hyderabad, 500 007, India. E-mail:
chakraborty@iict.res.in, kunwar@iict.res.in
† IICT Communication No. 060304
‡ Electronic supplementary information (ESI) available: Characterisa-
tion data for 1–4 and MD simulation details for 3 and 4. See DOI:
10.1039/b712365p
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3
also have twisted strands. The JNH–CaH of >10.5 Hz for Phe was
followed by <9.3 Hz for Leu throughout the strand implying the
presence of alternating φ magnitudes, a requirement for twisted
b-sheets.8
The dimer-like association of the individual strands is evident
from the large number of inter-residue NOEs shown schemat-
ically in Fig. 2. The observed NOEs between the protons
at two termini, like Boc–OMe, Maa(1)NH–OMe, Maa(1)NH–
Leu(12)CaH and Phe(2)NH–OMe (Fig. 3 shows some of
them), provide strong evidence for the proposed anti-parallel
dimeric structure. NOE connectivities, Leu(3)CaH–Leu(9)CaH,
Leu(3)CaH–Maa(10)NH, Leu(3)NH–Leu(12)NH, Maa(1)C2H–
Phe(11)CaH, Maa(4)NH–Leu(9)CaH, Phe(2)CaH–Maa(10)C2H
and Leu(6)NH–Leu(9)NH, are consistent with a distinctly com-
plex duplex structure consisting of two anti-parallel twisted b-
strands, with all the above-mentioned NOEs being inter-strand
Fig. 1 Solvent titration plots of 4 (500 MHz, 303 K, CDCl3 with 0–33.3%
DMSO-d6): (a) NMR spectrum of 4 in pure CDCl3; (b) to (i) NMR spectra
of 4 on addition of 20 lL, 40 lL, 60 lL, 80 lL, 120 lL, 160 lL, 200 lL
and 300 lL of DMSO-d6 to 600 lL CDCl3 solution.
3
in nature.9 All the NMR observations, e.g. JNH–CaH, H-bonding
information and NOEs across the strands, support the formation
of a duplex structure from twisted strands.
and dilution studies, imply a possible equilibrium between a single-
(minor) and a double-stranded (major) species. The dimerisation
constants (Kdim), for 3 with 40% and 4 with 5% populations of the
monomer, were 4.7 × 102 M−1 and 3.2 × 104 M−1, respectively.5,7
ESI mass spectral studies of compounds 3 and 4 showed the
presence of doubly-charged dimeric species, in addition to the
singly-charged monomer, further supporting the dimerisation of
two monomeric units. The experimental peak shapes were matched
with the calculated isotopic distribution patterns and the mass
differences between the isotopic peaks arising from the doubly-
charged dimeric species were 0.5 Da and those from the singly
charged monomeric species showed differences of 1.0 Da.5
Fig.
2
The characteristic NOEs in the ROESY spectrum of 4:
1, Phe(2)NH–Leu(3)NH; 2, Phe(5)NH–Leu(6)NH; 3, Phe(8)NH–
Leu(9)NH; 4, Phe(11)NH–Leu(12)NH; 5, Leu(3)NH–Leu(12)NH;
6, Leu(6)NH–Leu(9)NH; 7, Maa(4)NH–Leu(9)CaH; 8, Leu(3)CaH–
Maa(10)NH; 9, Phe(2)CaH–Maa(10)C2H; 10, Leu(3)CaH–Leu(9)CaH;
11, Maa(1)C2H–Phe(11)CaH; 12, Maa(4)C2H–Phe(8)CaH and
Phe(5)CaH–Maa(7)C2H; 13, Maa(1)NH–Leu(12)CaH.
The structure of 4 is discussed here in detail as it existed almost
3
exclusively as a single species. The JNH–CaH being >9 Hz for
all but the Maa(1) residue strongly supported an antiperiplanar
arrangement of the amide and the Ca protons, corresponding to φ
∼ −120◦ in the b-region of the Ramachandran map, suggesting a
basic b-stranded structure. Additional evidence for this structure
was provided by the CaH chemical shifts being >5 ppm for both
Phe and Leu residues. Since the proteins containing a b-sheet
structure have been shown to have the b-strands with a natural
right handed twist, it was envisaged that the structure in 4 could
The b-sheets and strands, especially in globular proteins, display
right handed twists10 and the extent of curvature depends on many
factors like the entropic considerations, the number and types of
residues, the torsional angles of the peptide chains, intra- and
Fig. 2 Schematic representation of the intra- and inter-residue NOEs seen in the ROESY spectrum of 4.
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inter-strand non-covalent interactions, etc.11–13 Theoretical calcu-
lations have shown that periodicity of the twists depends on the
nature of the b-sheets.10,11 For an anti-parallel arrangement, the
periodicity is about 15 residues per turn. The mannose-derived
furanoid sugar amino acid with 2,5-trans geometry permitted
significant changes in the direction of the chain propagation,
thereby introducing enhanced twists in the strands, which might
have resulted in the nucleation of novel structures deduced from
the NMR data.
Incorporating all the above information, molecular dynamics
calculations were carried out using the distance constraints from
the volume integrals from the ROESY correlations and two-spin
approximation. Fig. 4 shows the stereo-view of one of the lowest
energy MD structures. The structures were revealing. The presence
of anti-parallel twisted dimeric b-strands, as envisaged, resulted
in an unprecedented right-handed double helical structure. The
average values of the dihedral angles, φ and w, of the backbone
from these structures are −127 9◦ and −59 8◦ for Phe and
−96 12◦ and 138 14◦ for Leu, respectively.
Leu(12)CO with Phe(2ꢀ)NH and Leu(3ꢀ)NH. The strong NOE
correlations between all the adjacent PheNH and LeuNH are
consistent with these observations. The observed H-bonding
patterns are also supported by the IR spectrum of 4 in CHCl3,
where three amide bands were seen, both in the NH stretch region
with values of 3376, 3331 and 3280 cm−1 and the C O stretch
=
region with values of 1676, 1653, and 1639 cm−1. The presence
of these bands in the IR spectrum is attributed to all the amides
with no H-bonds, single H-bonds as well as three centre H-bonds,
based on earlier studies.14
All the NMR observations, like 3JNH-CaH, H-bonding infor-
mation and NOEs across the strands, suggest a similar duplex
structure for 3. The structures for the minor species in both 3 and
4 could not be obtained in detail. However, some of the NMR
spectral parameters and NOEs which could be attributed to the
minor conformers suggest that their structures were like those
proposed for 1 and 2.
Conclusion
In conclusion, detailed NMR and mass spectral studies carried
out here indicate strong aggregation of 3 and 4 into dimeric
structures. Furthermore, the inter-strand NOEs seen across the
length of these oligomers strongly suggest that the strands are
intertwined. The double helical structure described here is unique,
very similar to those observed earlier for gramicidins and their
structural analogs.15 It is likely that studies in other solvents and
on peptides with different sequences can lead to other interesting
structures. Incorporation of charged residues in lieu of Phe and
Leu can also provide scope for additional interactions, though at
the cost of deviation from a hydrophobic peptide core important
for generation of the twisted b-strands.
Experimental section
The peptides 1–4 were synthesized following standard so-
lution phase peptide coupling methods16 using 1-ethyl-3-[3-
(dimethylamino)propyl]-carbodiimide hydrochloride (EDCI) and
1-hydroxybenzotriazole (HOBt) as coupling agents and dry DMF
and/or CH2Cl2 as solvents. In the racemization free fragment
condensation strategy that was followed, the Boc-Maa(Bn2)-OH
was first coupled with the dipeptide H-Phe-Leu-OMe as efficiently
as with any normal amino acid using the reagents mentioned
above to give the tripeptide Boc-Maa(Bn2)-Phe-Leu-OMe 1. While
saponification of one-half of 1 using LiOH in THF–MeOH–H2O
gave the acid, Boc-deprotection of the other half using TFA–
CH2Cl2 freed the N-terminus, which was then coupled with the
acid to furnish 2. Coupling of 1 and 2 under similar conditions
gave 3 and ‘2 + 2’ provided the linear tetramer 4.
Fig. 3 Stereo-view of one of the energy-minimized MD structures of 4
(top) and its top view (bottom). The benzyl groups and the amino acid
side-chains are not shown for clarity.
The MD structures clearly show that the Maa residues impart
˚
significant twists in the individual strands with a pitch of ∼18 A
and a periodicity of about 6 residues per turn. Two of these
strands intertwine in an anti-parallel double helical structure with
˚
an inner pore of ∼1 A in diameter (Fig. 4). All the observed
NOEs can now be rationalized based on the findings from the
MD calculations. We have, for further discussion, differentiated
the two strands by marking the residues as natural and primed
numbers. Leu carbonyls are involved in three centre H-bonds with
Phe and Leu amide protons in the other chain, e.g. Leu(3)CO
with Phe(11ꢀ)NH and Leu(12ꢀ)NH, Leu(6)CO with Phe(8ꢀ)NH
and Leu(9ꢀ)NH, Leu(9)CO with Phe(5ꢀ)NH and Leu(6ꢀ)NH and
Spectral data of peptide 1
1H NMR (CDCl3, 500 MHz): see Table S1 in the support-
ing information‡; HRMS (ESI) m/z calculated for [M + H]+
C41H54N3O9: 732.3860, found: 732.3850.
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Spectral data of peptide 2
D. D. Long, C. M. Baker, B. Odell, G. H. Grant, A. A. Edwards,
G. E. Tranter, G. W. J. Fleet and M. D. Smith, J. Org. Chem., 2005,
70, 2082; (e) T. K. Chakraborty, S. Roy, S. K. Kumar and A. C.
Kunwar, Tetrahedron Lett., 2005, 46, 3065; (f) L. F. Bornaghi, B. L.
Wilkinson, M. J. Kiefel and S.-A. Poulsen, Tetrahedron Lett., 2004, 45,
9281; (g) T. K. Chakraborty, P. Srinivasu, S. S. Madhavendra, S. K.
Kumar and A. C. Kunwar, Tetrahedron Lett., 2004, 45, 3573; (h) T. Q.
Gregar and J. Gervay-Hague, J. Org. Chem., 2004, 69, 1001; (i) D. F. A.
Hunter and G. W. J. Fleet, Tetrahedron: Asymmetry, 2003, 14, 3831;
(j) M. D. Smith, T. D. W. Claridge and M. S. P. Sansom, Org. Biomol.
Chem., 2003, 1, 3647; (k) A. Vescovi, A. Knoll and U. Koert, Org.
Biomol. Chem., 2003, 1, 2983; (l) S. A. W. Gruner, V. Truffault, G.
Voll, E. Locardi, M. Sto¨ckle and H. Kessler, Chem.–Eur. J., 2002, 8,
4365; (m) Y. Suhara, Y. Yamaguchi, B. Collins, R. L. Schnaar, M.
Yanagishita, J. E. K. Hildreth, I. Shimada and Y. Ichikawa, Bioorg.
Med. Chem., 2002, 10, 1999; (n) T. K. Chakraborty, S. Jayaprakash, P.
Srinivasu, M. G. Chary, P. V. Diwan, R. Nagaraj, A. R. Sankar and
A. C. Kunwar, Tetrahedron Lett., 2000, 41, 8167; (o) D. E. A. Brittain,
M. P. Watterson, T. D. W. Claridge, M. D. Smith and G. W. J. Fleet,
J. Chem. Soc., Perkin Trans. 1, 2000, 3655; (p) N. L. H. Hungerford,
T. D. W. Claridge, M. P. Watterson, R. T. Aplin, A. Moreno and G. W. J.
Fleet, J. Chem. Soc., Perkin Trans. 1, 2000, 3666; (q) N. Hungerford and
G. W. J. Fleet, J. Chem. Soc., Perkin Trans. 1, 2000, 3680.
1H NMR (CDCl3, 500 MHz): see Table S2 in the supporting
information‡; HRMS (ESI) m/z calculated for [M + NH4]+
C76H98N7O15: 1348.7120, found: 1348.7154.
Spectral data of peptide 3
1H NMR (CDCl3, 600 MHz): see Table S3 in the supporting
information‡; HRMS (ESI) m/z calculated for [M + NH4]+
C111H139N10O21: 1948.0116, found: 1948.1011.
Spectral data of peptide 4
1H NMR (CDCl3, 750 MHz): see Table S4 in Supporting
Information‡; HRMS (ESI) m/z calculated for [M + Na]+
C146H176N12O27Na: 2552.2665, found: 2552.2766.
Acknowledgements
5 See Supporting Information for details‡.
6 B. Reif, V. Wittmann, H. Schwalbe, C. Griesinger, K. Worner, K. Jahn-
Hofmann and J. W. Engels, Helv. Chim. Acta, 1997, 80, 1952.
7 C. S. Wilcox, in Frontiers in Supramolecular Organic Chemistry and
Photochemistry, ed. H.-J. Schneider, H. Durr, VCH, New York, 1991;
p. 123.
8 A. G. Cochran, N. J. Skelton and M. A. Starovasnik, Proc. Natl. Acad.
Sci. U. S. A., 2001, 98, 5578.
9 The other likely NOEs, Maa(4)C2H–Phe(8)CaH and Phe(5)CaH–
Maa(7)C2H overlapped (cross-peak 12 in Fig. 1) and could not be
distinguished. However, in analogy with the data for 3, we believe that
these two long-range NOEs are also present in 4, although they were
not used in the MD calculations.
Authors wish to thank DST, New Delhi for financial support. D.K.
and R.R. are thankful to CSIR, New Delhi for research fellow-
ships. A.C.K. thanks Profs. M. Goerlach and R. Ramachandran
for 750 MHz NMR spectra and INSA-DFG for supporting the
exchange program. Authors thank the Mass Spectrometry Centres
at IICT and CCMB for their help.
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©