An Unusual Reverse Turn Structure Adopted by a Furanoid Sugar Amino Acid Incorporated in Gramicidin S

Article abstract of DOI:10.1021/ja0397254

A new reverse turn, replacing one of the native type II′ β-turns in the cyclic peptide antibiotic gramicidin S, induced by a furanoid sugar amino acid is revealed. The C₃-hydroxyl function plays a pivotal role by acting as a H-bond acceptor, co

Full text of DOI:10.1021/ja0397254

Published on Web 02/25/2004
An Unusual Reverse Turn Structure Adopted by a Furanoid Sugar Amino Acid
Incorporated in Gramicidin S
Gijsbert M. Grotenbreg,† Mattie S. M. Timmer,† Antonio L. Llamas-Saiz,‡ Martijn Verdoes,†
Gijsbert A. van der Marel,† Mark J. van Raaij,‡ Herman S. Overkleeft,† and Mark Overhand*,†
Leiden Institute of Chemistry, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands, and
Unidad de Rayos X (RIAIDT), Laboratorio Integral de Dina´mica y Estructura de Biomole´culas “Jose´ R.
Carracido”, Edificio CACTUS, Campus Sur, UniVersidad de Santiago, 15782 Santiago de Compostela, Spain
Received November 21, 2003; E-mail: overhand@chem.leidenuniv.nl
Peptides and proteins display an extraordinary structural diversity
and are instrumental in numerous biological events. Correct folding
of these biomolecules is imperative for their functioning. Key
components contributing to the overall folding are secondary
structure elements such as helices, sheets, and turns.1 Adoption of
nonproteinogenic residues or sequences has appreciably contributed
to further our understanding of the factors that are at the basis of
secondary structure. Besides mimicking spatial arrangements found
in polypeptides, peptide-like molecules have been designed with
the aim to enhance resistance to proteolytic activity, to attain
structural stabilization, and to introduce additional functionalization
sites.2 Synthetic peptide analogues are now widely recognized as
important lead compounds, both in the development of new
materials3 and in the generation of therapeutic agents.4
Increasing research efforts have been devoted to the study of
reverse turns. In this common motif, the polypeptide chain reverses
its overall direction. The γ- and â-turns describe three and four
consecutive residues, respectively, in which the CdO of the first
residue i is H-bonded to the NH of residue i + 2 or i + 3. Further
classification of the turn motifs can be made on the basis of their
peptide backbone geometry with specific angular and torsional
parameters.5 Factors influencing turn motifs include hydrophobic
interactions, conformational bias, side chain participation, and intra-
and interresidue interactions.
peptide sequences does not lead to regular â-turn structures.10
Instead, one of the hydroxyl functionalities (C3-OH) on the
furanoid SAA proved to be actively involved in stabilizing the
observed secondary structure by acting as hydrogen acceptor.
Our focus in the area of peptidomimetics is directed at the
determination of the structural consequences of incorporating SAA
building blocks in selected oligopeptides. Ultimately, we aim to
attain tailor-made peptidomimetic building blocks able to induce
the desired secondary structure combined with the opportunity to
introduce extra functionalities on the turn region. To this end, we
have selected gramicidin S (GS), a cyclic decapeptide antibiotic
with the primary sequence cyclo-(Pro-Val-Orn-Leu-DPhe)2, as
a suitable model peptide. GS adopts a C2-symmetric amphiphilic
antiparallel â-sheet structure11 with two type II′ â-turns with DPhe
and Pro at positions i + 1 and i + 2, respectively, and is widely
recognized as a good system to study the effect of potential artificial
reverse turn inducers.12 We here report the in-depth study, through
NMR and X-ray analysis, of synthetic GS analogue 7, with SAA
1 as a replacement of one of the DPhe-Pro dipeptides in GS. NMR
and crystallographic analysis of 7 revealed the involvement of C3-
OH in the final overall secondary structure by inducing an
unprecedented turn motif. The implications of this secondary
structure element on the overall structure, as well as oligomeric
assemblies of 7, are disclosed.
Recently, sugar amino acids (SAAs), carbohydrate derivatives
featuring an amine and a carboxylic acid, have emerged as a
promising new class of peptidomimetics.6 Oligopeptides containing
SAA building blocks have been assembled with the aim to improve
their biostability. Furthermore, examples of these structurally and
functionally diverse molecular scaffolds have been found to induce
well-defined secondary structures in oligomeric constructs, including
reverse turns.7,8 An attractive feature of the use of carbohydrate-
based peptidomimetics as turn motifs is the presence of additional
functionalities on the furanose or pyranose core stemming from
the parent sugar, enabling further functionalization. For instance,
Smith et al. demonstrated the incorporation of a pyranoid SAA as
â-turn inducer in a heptapeptide corresponding to the C-terminus
of the R2 subunit of mammalian ribonucleotide reductase. The
remaining hydroxyl functions were equipped with methylene
carboxylate (mimicking aspartic acid) and isobutyl (mimicking
leucine) functionalities, resulting in an artificial ensemble that
closely resembles the native peptide sequence.9 Notably, the residual
functionalities present at the parent core of SAAs may also prohibit
the formation of the targeted secondary structural motif. The latter
is exemplified by the finding of Chakraborty and co-workers that
the incorporation of furanoid SAA 1 (Scheme 1) in short linear
The synthesis of cyclic peptide 7 was accomplished as follows
(Scheme 1). Fmoc-Leu-OH was condensed with the 4-(4-
hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB)-func-
tionalized 4-methylbenzhydrylamine (MBHA)-resin under the
agency of N,N′-diisopropylcarbodiimide (DIC) and a catalytic
amount of 4-(dimethylamino)pyridine (DMAP) to furnish 3.
Standard Fmoc-based solid-phase peptide synthesis (condensating
agents; Castro’s reagent,13 N-hydroxybenzotriazole (HOBt) and
N,N′-diisopropylethylamine (DiPEA), Fmoc cleavage; 20% piperi-
dine in NMP) using the appropriate amino acid building blocks
(Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-DPhe-OH, Fmoc-
Pro-OH, and Fmoc-Leu-OH) followed by analogous condensa-
tion of azido acid 2 furnished immobilized nonapeptide 4. At this
stage, the azide functionality was converted to the corresponding
amine employing Staudinger reduction conditions (PMe3, 1,4-
dioxane, and H2O). Mild acidic cleavage from the resin (1% TFA
in CH2Cl2) afforded the partially protected linear peptide which
was directly cyclized using Castro’s reagent, HOBt, and DiPEA
under highly dilute conditions. The thus obtained cyclic peptide
was purified by size-exclusion chromatography (Sephadex LH-20)
to furnish the homogeneous target compound 5 in 96% overall yield,
based on 3. Removal of the pivaloyl- and Boc-protecting groups
(1% NaOMe in MeOH and 50% TFA in CH2Cl2, respectively) and
† Leiden Institute of Chemistry.
‡ Universidad de Santiago.
9
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J. AM. CHEM. SOC. 2004, 126, 3444-3446
10.1021/ja0397254 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Synthesis of GS Analogue 7a
a (a) Sequential coupling (Xaa or 2, BOP, HOBt, DiPEA) and deprotection (piperidine/NMP 1/4 v/v) steps; (b) PMe3, 1,4-dioxane, H2O; (c) TFA/DCM
(1/99 v/v); (d) BOP, HOBt, DiPEA; (e) NaOMe, MeOH; (f) TFA/DCM (1/1 v/v).
Figure 2. Pleated sheet structure of 7 with the intramolecular H-bonds
depicted in green. Water molecules, Leu-, Val-, and Orn-side chains as
Figure 1. (A) GS; (B) observed long-range NOEs for 7.
well as hydrogens were omitted for clarity.
subsequent reversed-phase HPLC purification finally gave GS
analogue 7 in 59% yield.
The 1H NMR resonance assignment of peptide 7 was ac-
complished using a combination of COSY, TOCSY, and ROESY
data sets. Subsequently, we compared the thus obtained structural
information with the antiparallel â-sheet structure adopted by GS.11
The structure of GS is characterized, apart from the two DPhe-
Pro turn regions, by four H-bonds, two shared between the Leu4
and Val2′ residues and two between the Leu4′ and Val2 residues
(Figure 1A). Besides numerous short-range NOEs, the observation
of interstrand NH-NH (Val2-Leu9 and Val7-Leu4), NH-HR
(Val7-DPhe5 and Leu9-Orn3), and HR-HR (Orn3-Orn8) NOEs
in 7 confirms the preservation of the overall â-sheet structure and
indicates the presence of three H-bonds (Figure 1B).14 However, a
strong NH-NH NOE between SAA1-NH and Val2-NH was
observed, indicating their close proximity. The latter observation
strongly suggests that residue SAA1 does not induce a regular â-turn
conformation.
With the aim to create a better understanding of the overall
structure and the implications of the introduction of SAA 1 in one
of the turn regions of GS, we obtained and analyzed crystallographic
data of 7. To this end, a solution of 7 in a 1:1 mixture of MeOH
and H2O in the presence of spermidine tri-HCl (or 1,5-diamino-
pentane di-HCl) was allowed to evaporate slowly under oil. The
resulting needle-shaped crystals were subjected to X-ray diffraction
analysis. The structure was solved and refined to 1.2 Å resolution
(the diffraction limit of the crystals).
Figure 3. (A) Turn region of GS; (B) turn region of 7; (C) crystal structure
of turn region of 7. Side chains and hydrogens were omitted for clarity.
by the furanose moiety, allows it to function as a H-bond acceptor.
The structure that results from formation of a H-bond with SAA1-
NH is in full agreement with the data obtained from the NMR
studies. As a consequence, the amide bond linking residues Leu9
and SAA1 is flipped, causing the SAA1-NH to extend into the
turn region leading to a novel secondary structure with a H-bond
between a side chain functionality and the amide NH of the
synthetic dipeptide isostere incorporated (Figure 3). The structure
adopted by SAA 1 in compound 7 constitutes, to our knowledge,
an unprecedented turn structure.
Perusal of the molecular packing of 7 reveals the presence of
cyclic assemblies of six crystallographically equivalent molecules,
with the hydrophilic Orn side chain residues extending into the
core and the Val, Leu, and DPhe residues forming a hydrophobic
periphery (Figure 4A). The structure is stabilized by intermolecular
H-bonds between SAA1-CdO and Orn3-NH of one â-sheet with
Orn8-NH and Pro6-CdO of the next, respectively (Figure 4B).
This results in a novel hexameric â-barrel-like structure corre-
sponding to a 12-stranded â-barrel of approximately 13 Å height
(the length of the unit cell c-axis).
As can be seen in Figure 2, peptide 7 adopts a pleated sheet
structure with two H-bonds shared between the Leu4 and Val7
residues and one between Leu9-NH and Val2-carbonyl similar to
that reported for GS, but with a larger right-handed twist15 in the
overall â-sheet structure. Interestingly, the SAA residue induces
an unusual turn structure with the C3-OH in close proximity to
the SAA1-NH (Figure 3). The protrusion of this hydroxyl function
into the turn region, enabled by the C3-endo conformation16 adopted
It has been reported that the parent cyclodecapeptide GS itself
adopts oligomeric structures of a different nature. X-ray analysis
of a crystal structure of a GS-urea-water complex revealed
channel-like structures composed of six crystallographically equiva-
lent GS molecules assembled in a double spiral of two left-handed
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Figure 4. (A) Top view of the hexameric assembly of 7 with the SAA
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helices.17 Changes in the turn region, while of relatively small
consequence on the secondary structure of the cyclic peptide itself
(both GS and 7 adopt a pleated â-sheet), may therefore have a
profound effect on oligomeric assemblies thereof, at least in their
crystal structures. Interestingly, â-barrels are found to be at the
basis of the mode of action of many pore-forming proteins,
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R-hemolysin.18 The results presented here may therefore be of use
for the future development of novel transmembrane channels and
may contribute in the design of artificial â-barrel-like molecules
based on cyclic peptides with applications such as bactericidal
agents.19,20
Acknowledgment. This work was financially supported by the
Council for Chemical Sciences of the Netherlands Organization
for Scientific Research (CW-NWO), the Netherlands Technology
Foundation (STW), DSM Research, and the Spanish Ministry of
Science and Technology (Ramo´n y Cajal fellowship and research
grant BMC2002-2436). We thank Nico Meeuwenoord and Hans
van der Elst for their technical assistance. Kees Erkelens and Fons
Lefeber are gratefully acknowledged for their assistance with NMR
experiments. X-ray data were acquired on apparatus partially funded
by an EU FEDER infrastructure grant.
Supporting Information Available: Experimental procedures
and spectral data for all new compounds (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
CCDC-216610 contains the crystallographic data for this paper. These
data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge CB21EZ, UK; fax: (+44) 1223-336-033;
or deposit@ccdc.cam.ac.uk).
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