β-peptides that adopt stable cyclic hairpin-like conformations

Tetrahedron 68 (2012) 2391e2400

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Design, synthesis and structural analysis of mixed a/b-peptides that adopt stable cyclic hairpin-like conformations
M. Otero b, Antonio Llamas-Saiz c, Mark J. van Raaij d, Lianne I. Lageveen e, Matthijs van der Knaap a, Jose Henk J. Busscher e, Gijsbert M. Grotenbreg f, Gijsbert A. van der Marel a, Herman S. Overkleeft a, Mark Overhand a, * a

Bio-Organic Synthesis, Leiden Institute Of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands Departamento de Bioquímica y Biología Molecular, Universidad de Santiago, Santiago de Compostela, Spain Unidad de Rayos X (RIAIDT), Edificio CACTUS, Universidad de Santiago de Compostela, Santiago de Compostela, Spain d Laboratorio M-4, Dpto de Estructura de Macromoleculas, Centro Nacional de Biotecnologia, c/Darwin 3, Campus Cantoblanco, E-28049 Madrid, Spain e Department of Biomedical Engineering, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands f Department of Microbiology, National University of Singapore, CeLs, 28 Medical Drive, Singapore 117456, Singapore b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 September 2011 Received in revised form 23 December 2011 Accepted 9 January 2012 Available online 18 January 2012

The strategic replacement of four a-amino acid residues of a cyclo-(aaaaa)2 peptide by b-, b2- or b3amino acids residues provided a series of novel 2:1 a/b-mixed peptides that were designed to adopt cyclic hairpin-like structures. It was shown that conformationally stable cyclo-(ababa)2 isomers can be obtained using both enantiomers of the central two basic a-amino acid residues, a known a-amino acid turn sequence and several combinations of facing b-amino acid residues with no side chain or a hydrophobic side chain having specific regio- and stereochemistry. The X-ray analysis of two derivatives provides molecular details of the intra-molecular hydrogen bonding interaction, dihedral angles of the backbone and side chain positioning of the novel cyclic hairpin-like structures. One of these isomers forms an unprecedented hexagon-shaped nano-channel assembly in the crystal structure. Well-defined cyclic hairpin-like structures as described here and derivatives that can be readily designed based on this research can be used as scaffolds onto which functional groups can be grafted in a spatially controlled manner and as b-hairpin mimics with specific biological properties. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Cyclic mixed a/b-peptides H-bonding pattern Hairpin-like structure Nano-channels

1. Introduction Enormous progress has been made in the design of linear and cyclic oligomers with predetermined structures by making use of the properties of b- and g-amino acids.1e9 In particular hydrogen bond formation between specific homologue residues govern the formation of turn-, helix-, and sheet-like structures, as well as molecular assemblies. In recent years, it has been shown that even greater structural diversity can be obtained by combining different classes of amino acids homologues with each other.10e15 Though many mixed a/b-peptides adopt helical structures,13,14 several variants of hairpin structures were obtained as well.16e25 For instance, the peptide sequences bbaabb,23 aabaabaa,24 aabaaaa baa,22 and aabaaaaaabaa,16 all form hairpin-like structures, each with a distinct intra-molecular hydrogen bonding pattern. Besides the turn-forming propensity of the central two a-amino acids, both

* Corresponding author. Fax: þ31 71 5274307; e-mail addresses: overhand@ chem.leidenuniv.nl, [email protected] (M. Overhand). 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2012.01.015

the position in these sequences, as well as the regio- and stereochemistry of the side chains of the built-in b-amino acid residues strongly influence the stability of the secondary structures. Guided by these results and our own structural work on modified cyclic peptides26e32 we realized that related rigid hairpin structures containing mixed a-/b-amino acids can be designed having a cyclic backbone. To attain well-defined hairpin-like structures a cyclic seamless peptide backbone must comply with specific geometrical requirements to allow a regular pattern of intra-molecular hydrogen bonding interactions. The requirements for the formation of conformationally stable cyclic hairpin structures for apeptides are well established,33e37 as exemplified by the natural product gramicidin S (GS, cyclo-(Pro-Val-Orn-Leu-(R)-Phe)2). GS is a C2 symmetric cyclic decapeptide (Fig. 1a) containing four intra-molecular hydrogen bonding interactions (blue arrows Fig. 1a),38e41 resulting in a central 14-membered H-bonding ring, flanked by two 10-membered H-bonding rings (Fig. 1b). In addition to the alignment of the interstrand hydrogen bond forming amides, the stereochemistry of the amino acid side

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a

b

c

Fig. 1. (a) The structure of the antibiotic peptide gramicidin S (GS). Blue arrows indicate intra-molecular hydrogen bonds, in green are the two (R)-a-amino acid residues. (b) Schematic representation of the cyclo-(aaaaa)2 motif in which the central 14-membered H-bonding ring segment is flanked by two 10-membered H-bonded ring segments and two two-residue turns. (c) Schematic representation of the designed cyclo-(ababa)2 motif, indicating is the alternative hydrogen bonding pattern.

chains is of importance for the formation of a stable secondary structure. The presence of an (R)-a-amino acid residue (corresponding to a D-amino acid in the Fisher notation)42 in each turn region stabilizes the cyclic hairpin structure in solution. The side chains of the strand residues in the cyclic hairpin structure adopt well-defined positions with the hydrophobic side chains (i-Pr and i-Bu) on one face of the molecule and the hydrophilic groups (aminopropyl) of the central residues on the opposing face.38e41 GS does not readily aggregate and is soluble in aqueous media. Using GS as a basis we set out to replace a-amino acid residues in the strand regions by b-amino acids, in order to obtain mixed a/b-sequences with hairpin characteristics. It was decided to retain the two turn a-amino acid residues as well as the two central residues in the strand regions. Thus, the two a-amino acid residues neighboring the central ornithine residue in each strand of the regular cyclic hairpin structure of GS were replaced by unsubstituted b-amino acids (1). An alternative pattern of stabilizing intra-molecular hydrogen bonding16e25 interactions would ensue to accommodate the four additional methylene groups in the peptide backbone, namely a central 10-membered H-bonding ring, flanked by two 14-membered H-bonding rings (Fig. 1c). iso-Propyl and iso-butyl side chains were introduced, analogously to the stereochemistry and the relative position of the side chains in the strand regions of GS, via the dual incorporation of (R)-b2hVal and (S)-b3hLeu residues (peptides 2e4, Fig. 2). To probe the stability of the secondary structure the stereochemistry of the side chains of the b-amino acid residues were varied (peptides 5e7) and a second series of compounds were prepared (8e11, Fig. 2) containing two (R)-Orn residues in which the b2- and b3-amino acid residues have swapped their positions in the sequence. Here we present the synthesis of a series of symmetric and asymmetric cyclo-(ababa)2 peptides (1e11) and the study of their structural properties using spectroscopic and diffraction techniques. In addition, as it is known and well documented that b-amino acid containing peptides may display antibacterial activity (for examples of antibacterial bpeptides see Refs. 43e45, for examples of antibacterial mixed a/ b-peptides see Refs. 46e48), we also report the antibacterial activity of peptides 1e11.

2. Results and discussion 2.1. Synthesis The linear precursors of the cyclo-(ababa)2 peptides 1e11 were prepared using suitably protected a- and b-amino acid building blocks following a solid-phase Fmoc peptide chemistry strategy. The required (S)- or (R)-configured Fmoc-a-amino acids of which the Orn residues have their side chains protected with a Boc-group, FmocebAlaeOH and both enantiomers of the hydrophobic b3amino acids are commercially available. Both enantiomers of Fmoceb2hVal were synthesized following adaptation of the literature procedures.49e51 The hyper-acid labile HMPB-MBHA resin52 was functionalized with either FmocebAlaeOH or Fmoce(R)-PheeOH, in the presence of the coupling reagent DIC and catalytic DMAP and elongated using standard conditions. The assembled decameric partially protected peptides were cleaved from the resin by repetitive mild acid treatment and cyclization of the C/N-terminus realized in DMF under high dilution (0.01 M) under the agency of PyBOP/HOBt/ DiPEA. Removal of reagents was affected by LH-20 gel filtration, the Boc-groups of the Orn residues of the cyclic peptides were removed using TFA and the fully deprotected peptides were purified using preparative HPLC purification (20e45% overall yield). 2.2. Crystallographic analysis The cyclo-(ababa)2 peptides 1e11 were subjected to controlled evaporation conditions using a homemade set of crystallization buffers and solvents. cyclo-(ab2ab3a)2 6 and cyclo-(ab3ab2a)2 11 provided single crystals that were suitable for X-ray diffraction and their high resolution structures are compared with those of GS38 in Fig. 3. Based on the orientation and distances of the amides of the opposing strands (Table 1), the crystal structures of compounds 6 and 11 have four well-defined intra-molecular hydrogen bonding interactions. These results confirm the anticipated four stabilizing intra-molecular hydrogen bonding pattern of cyclo-(ababa)2 peptides. The distances and angles of the two H-bonds of the turn regions and the dihedral angles of the (R)-Phe-Pro sequences of all three peptides have comparable values (Table 2). The turn regions of GS and peptides 6 and 11 are thus highly similar. The side chains of the two facing strands of all three peptides position themselves perpendicular with respect to the hydrogen

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Fig. 2. The structures of the symmetric and asymmetric cyclo-(ababa)2 peptides 1e11. Table 2 Dihedral angles of the amino acid residues of GS, 6 and 11 based on the X-ray data. In brackets the deviation of the dihedral angles of the turn residues from an ideal type II0 b-turn are indicated (angleobservedangleideal) Peptide

Residue

4(D4) [ ]

q [ ]

j(Dj) [ ]

GS

Pro(1) Val(2) Orn(3) Leu(4) (R)-Phe(5) Pro(6) Val(7) Orn(8) Leu(9) (R)-Phe(10)

84(4) 87 135 137 52(8) 80(0) 108 113 141 54(6)

d d d d d d d d d d

2(2) 139 134 107 126(6) 5(5) 126 135 103 129(9)

6

Pro(1) (R)-b2hVal(2) Orn(3) (R)-b3hLeu(4) (R)-Phe(5) Pro(6) (R)-b2hVal(7) Orn(8) (R)-b3hLeu(9) (R)-Phe(10)

76(4) 127 96 96 53(7) 92(12) 126 128 78 55(5)

d 178 d 162 d d 177 d 161 d

10(10) 120 162 154 130(10) 15 (15) 149 124 125 127(7)

11

Pro(1) (S)-b3hLeu(2) (R)-Orn(3) (R)-b2hVal(4) (R)-Phe(5) Pro(6) (S)-b3hLeu(7) (R)-Orn(8) (R)-b2hVal(9) (R)-Phe(10)

81(9) 79 144 147 51(9) 71(19) 85 142 145 56(4)

d 160 d 177 d d 156 d 175 d

7(7) 116 145 109 133(13) 15(15) 130 144 118 130(10)

Fig. 3. X-ray crystal structures of GS (top) and the peptides 6 (middle) and 11 (bottom), with their intra-molecular hydrogen bonding patterns (left, side chains of the aaa and the bab-strand regions are omitted for clarity) and their side chain positions (right).

Table 1 Intra-molecular hydrogen bonding distances and angles of facing strand residues of the X-ray structures of GS,6a 6 and 11 Peptide

Donor

Acceptor

d(D/A) [ A]

d(H/A) [ A]

D/O]C [ ]

GS

N(2) N(4) N(7) N(9)

O(9) O(7) O(4) O(2)

2.94 2.91 3.04 2.90

2.11 2.06 2.19 2.03

134 159 134 150

6

N(2) N(3) N(7) N(8)

O(9) O(8) O(4) O(3)

3.03 2.92 2.92 2.84

2.19 2.09 2.08 1.98

132 155 143 145

N(2) N(3) N(7) N(8)

O(9) O(8) O(4) O(3)

3.00 2.98 3.07 2.94

2.16 2.11 2.25 2.07

132 164 134 171

11

bond pattern. As a result of the regio- and stereochemistry of the facing bab strands of peptide 6, that is (R)-b2-(S)-a-(R)-b3, both isopropyl groups are oriented on the concave face of the molecule while the iso-butyl and the aminopropyl groups are located on the convex face (Fig. 3 right middle). The (S)-b3-(R)-a-(R)-b2 regio- and stereochemistry of the facing bab strands of peptide 11 provides

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a cyclic hairpin-like structure in which both hydrophobic iso-butyl and iso-propyl side chains are located on the concave face with the two hydrophilic aminopropyl side chains on the opposite face (Fig. 3 right bottom). This side chain positioning introduces amphiphilic characteristics to the molecule and in this respect, peptide 11 resembles GS. Peptides 6 and 11 adopt a bent hairpin-like structure, rather than a pleated sheet, and the observed values of the dihedral angles53 4, q and j are listed in Table 2. The hydrogen bonding network between the peptide subunits as observed in the crystal packing of GS and peptides 6 and 11 is depicted in Fig. 4. In our previously published crystal structure of GS38 not all amides in the strand regions are involved in intersubunit hydrogen bonding interactions. The GS monomers in the crystal structure form closely packed double-helical tubes with a hydrophobic exterior and a hydrophilic interior (Fig. 4, top).38 Peptide 6 forms a hydrogen bonding network with no discrete channels in its crystal (Fig. 4, middle). Apart from the four intramolecular hydrogen bonds present per monomer 11, each babstrand interacts with a neighboring molecule via inter-subunit hydrogen bonds (Fig. 4, bottom). The turn regions of each subunit

of 11 interact with two other subunits in the lateral direction via hydrophobic and p-stacking interactions. Six lateral subunits come full circle, forming a hexagon-shaped nano-channel structure with a cationic interior diameter of w26  A. The hydrophobic exterior (w45  A) of the hexagon-shaped molecular assembly interacts with its neighbors with each of its six molecular walls. 2.3. NMR analysis The structures of the peptides 6 and 11 in solution were evaluated using NMR and compared with the well studied cyclic bhairpin structure of GS using this technique.41,54e61 Compounds 6 and 11 dissolve readily in polar solvents and as judged by the dispersion of the amide proton signals of their NMR spectra there are no multiple conformations in CD3OH (Fig. 5, left). Using a combination of COSY and TOCSY spectra the NMR signals of 6 and 11 could be assigned. The 3JNHeHa coupling constant values of the Orn and (R)-Orn residues are >7 Hz, while the 3JNHeHa values of the (R)-Phe residues are <4 Hz (Fig. 5 middle). The chemical shift perturbation26,30,62e64 of the Orn and (R)-Orn residues have a DdHa

Fig. 4. Molecular assembly of GS, peptide 6 and 11 in their crystals. (Top): GS subunits assemble in a helical fashion with inter-subunit hydrogen bonds (blue dashed lines) in the A).6a (Middle): Part of the hydrogen bonding network observed in the crystal X-ray structure of peptide 6, each monomers pattern (3,3,2,3,3)n forming a double-helical channel (14  has four hydrogen bonds with four other subunits (blue dashed lines), no channels are present. (Bottom): Each monomer 11 forms inter-subunit hydrogen bonding interactions (blue dashed lines) with its neighbors, the turn regions interact via p-stacking interactions with one lateral subunit, six subunits come full circle forming a hexagon-shaped nanochannels (26  A).

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Fig. 5. The amide regions of the 600 MHz 1D NMR spectra in CD3OH of GS (blue) and peptides 6 (green) and 11 (red) (left). 3JNHeHa coupling constant values of the Orn and (R)-Orn and (R)-Phe residues (middle). The chemical shift perturbation of the a-protons of the a-amino acid residues of GS, 6, and 11 (right).

>0.1 ppm and the Pro and (R)-Phe residues a DdHa <0 ppm (Fig. 5 right). Both the magnitude of the 3JNHeHa coupling constant values and the chemical shift perturbation of the a-protons, of the aamino acid residues of GS, 6 and 11 concord that the Orn and (R)-

Orn residues are part of an extended conformation of a strand region and that the (R)-Phe, Pro residues are part of a turn region.65,66 The interpretation of the coupling constants of peptides 6 and 11 corresponding to the b-amino acid residues,53 although not directly

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comparable with GS, is of interest. For instance the amide signal of the (R)-b2hVal residues of peptide 11 appears as a doublet with a large coupling constant 3JNHeHba/b of 10 Hz. Using the Karplus equation for 4 dihedral angles of amino acid residues,67,68 this large coupling constant corresponds to 4 w120 . The value of this dihedral angle is in agreement with the measured dihedral angle 4 as determined from the X-ray crystal structure (Table 2). The NOESY spectra of 6 and 11 displayed several signals characteristic of a hairpin-like structure. Indicative of an extended conformation of an amino acid residue as part of a strand region is the presence of a strong sequential NOE interaction between Ha in the i position and the NH of the amino acid in the iþ1 position, together with a weak or absence NHeNH NOE between these residues.65,66 As can be seen in Fig. 6 in both peptide 6 and 11 the strong NOE signal between the Ha of the (S)-Orn/(R)-Orn residues with the NH of the sequential b-amino acid, while the NHeNH interaction is absent. Furthermore, both peptides 6 and 11 have a characteristic sequential NHeNH interaction of the b-amino acid residue at the i1 position with the NH of the Orn (6) or (R)-Orn with the amino acid in the iþ1 position (11) (Fig. 6). This data strongly indicates the presence of a bab-strand. Peptide 11 also displays a long range NOE interaction between Ha of (R)-Phe and the NH (R)-b3hLeu, which supports the presence of a b-turn. In addition to a NOE signal between Ha (R)-Phe and NH (R)-b2hVal, peptide 6 also shows a weak interstrand NOE between NH (R)b2hVal and Hb (R)-b2hLeu, also in agreement with the crystal structure. More details concerning the NMR data can be found in the experimental section (Tables S1e3).

Fig. 6. Enlargement of the NOESY spectra of peptide 6 (top) and 11 (bottom), displaying the NHeHa interactions (NHeHb for the b-amino acids).

In conclusion, the NMR data is in full agreement with the structures of peptides 6 and 11 as determined by X-ray. Based on the magnitude of the 3JNHeHa coupling constant values and the chemical shift perturbation of the a-protons of the a-amino acid residues of peptides 1e4, and 8e11 (Fig. 7), as well as the dispersion of the amide signals (Table S1) it is concluded that they all adopt distinct, but generally similar, cyclic hairpin-like structures as

peptides 6 and 11. More structural data is required, however, to determine the exact conformation of these peptides. In contrast, the NMR spectra of peptides 5 and 7, containing (S)-b2hVal and (S)b3hLeu residues and (S/R)-b2hVal and (S)-b3hLeu, respectively, appear as broad signals. As peptides 5 and 7 readily dissolve, the broad NMR signals are most likely due to the absence of a welldefined secondary structure in solution, though it is possible that these peptides are actually not monomeric in methanol solution.

2.4. Biological activity Finally peptides 1e11 were evaluated for their biological activity (Table 3). As can be seen, none of the tested peptides, with the exception of 8 displayed any antibacterial activity comparable to the all-a-peptide starting point, gramicidin S.

3. Conclusion The strategic replacement of four a-amino acid residues of a cyclo-(aaaaa)2 peptide by their hydrophobic b-amino acid counterparts provided a series of novel 2:1 a/b-mixed peptides (1e11) that were designed to adopt cyclic hairpin-like structures. It was shown that conformationally stable cyclo-(ababa)2 isomers can be obtained using several stereochemical combinations of a, b, b2and b3-amino acids in the facing bab strand regions while keeping the two turn regions constant. Two of the here reported thirteen peptides, that is, 6 and 11, provided X-ray crystal structures. The Xray data showed the anticipated stabilizing intra-molecular hydrogen bonding pattern, specific side chains positioning and an unaltered structure of the two turn regions as compared with the parent compound (GS). The regio- and stereochemistry of the amino acids of the two facing bab strands determine the side chain positioning. Peptides 6 and 11 adopt a bent conformation, rather than a pleated sheet structure. The bent conformation is intrinsic to the cyclo-(ababa)2 structure and is probably enhanced by the presence of the four unsubstituted sp3-hybridized carbon atoms in the macrocyclic backbone. By using NMR techniques it was shown that peptides 1e4 and 8e11 adopt similar stable cyclic hairpin-like structures in solution. However, not every a/b-amino acid combination provided a structurally stable cyclo-(ababa)2 structure, as peptides 5 and 7 appear not to adopt a well-defined conformation in solution. At this moment we do not have a plausible explanation for this latter finding. Deeper understanding of stabilizing/destabilizing factors of this type of cyclic hairpin-like peptides may be obtained by synthesizing derivatives containing disubstituted b2,3amino acids,1e9,69,70 turn modified analogues,19 or the design of related cyclic hairpin-like structures combining a-,b-, and g-amino acids with a predetermined intra-molecular hydrogen bonding pattern. The here described cyclo-(ababa)2 molecules may be applicable in various ways. Well-defined cyclic hairpin-like structures can be used as scaffolds onto which functional groups can be grafted in a spatially controlled manner71,72 and as b-hairpin mimics with specific biological properties.73e78 With respect to the latter, we aim for the development of membrane disrupting amphiphilic cationic peptides antibiotics that kill antibiotic resistant strains. The hexagon-shaped cationic nano-channel assemblies as observed in the crystal packing of peptide 11 are related yet distinct from our earlier reported hexameric b-barrel-like pore X-ray structure of a GS turn modified cyclo-(aaaadaaaa) derivative31 and selfassembling peptide nano-tubes with remarkable material and biological properties.11,79e81 Currently, we are preparing a series of cyclo-(ab3ab2a)2 derivatives of 11 to explore the possibility of inducing their self-assembly under chemically controlled conditions.

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Fig. 7. 3JNHeHa coupling constant values of the (S)-Orn, (R)-Orn and (R)-Phe residues of peptides 1e4 and 8e10 (left), and the chemical shift perturbation of the a-protons of the aamino acid residues of GS (1e4 and 8e10) (right).

Table 3 Antibacterial activity of the cyclic mixed a/b-peptides 1e11 in mg/mL (MIC) Strain

GS 1

S. aureus 7323 8 S. aureus 7388 8 CNS 5277 4 CNS 5115 16 CNS 7368 8 E. faecalis 1131 8 E. coli ATCC 25922 32 P. aeruginosa AK1 16 8 P. aeruginosa ATCC19582 S. mitis BMS 8 S. mitis ATCC 4 33399

32 32 32 >64 32 16 >64 >64 >64

2

3

4

5

6

>64 >64 64 64 64 >64 >64 >64 >64

>64 >64 64 64 64 >64 >64 >64 >64

>64 16 32 64 16 >64 >64 >64 >64

>64 >64 >64 >64 >64 64 >64 >64 >64

>64 32 >64 >64 32 >64 >64 32 >64 >64 8 >64 >64 8 >64 >64 8 >64 >64 64 >64 >64 >64 >64 >64 >64 >64

>64 >64 >64 >64 >64 >64

16 >64 >64 16 64 >64

8

10

11 >64 64 >64 >64 >64 >64 >64 >64 >64

32 >64 >64 64 >64 >64

4. Experimental 4.1. General (S)-Fmoceb3hLeueOH and (R)-Fmoceb3hLeueOH were obtained from Senn Chemicals AG. Solvents and chemicals were used as received from their supplier. Solvents were stored over 4 A molecular sieves. Solvents for column chromatography and extractions were of technical grade and distilled prior to use. THF was distilled over LiAlH4, CH2Cl2 over CaH2 and MeOH over NaBH4 prior to use. All reactions were performed at room temperature under inert atmosphere. Flash chromatography was performed on Screening Devices silica gel 60 (0.04e0.063 mm). TLC analysis was conducted on DC-alufolien (Merck, Kieselgel 60, F254) with detection by UV-absorption at 254 nm or by spraying with a solution of ninhydrin (3 g/L) in EtOH/AcOH (20:1 v/v), or KMnO4 solution (20 g in 1% aq K2CO3), followed by charring at 150  C. 1H and 13C NMR spectra were recorded with a Bruker DMX-600 spectrometer (600/150 MHz) or with a Bruker AV400liq spectrometer (400/100 MHz). Chemical shifts d are given in parts per million relative to TMS (0 ppm) or CD3OH (3.31 ppm) as internal standard. High resolution mass spectra were recorded by direct injection (2 mL of a 2 mM solution in water/acetonitrile; 50:50 v/v and 0.1% formic acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electro spray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 250  C) with resolution R¼60,000 at m/z 400 (mass range m/z¼150e2000) and dioctylphthalate (m/ z¼391.28428) as a ‘lock mass’. The high resolution mass spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). LC/MS analyses were performed on an LCQ Adventage Max (Thermo Finnigan) equipped with a Gemini C18 column (Phenomenex). The applied buffers were

A: H2O, B: MeCN, and C: 1.0% aq TFA. HPLC purifications were performed with a Gilson GX-281 automated HPLC system, equipped with a preparative Gemini C18 column (15021.20 mm, 5 m). The applied buffers were: A: 0.2% aq TFA, B: MeCN. The analysis of coupling constants, temperature coefficients, and chemical shift perturbations were performed with GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. 4.2. General peptide synthesis Loading of the HMPB-MBHA-resin: The HMPB-MBHA-resin (theoretical loading¼1.2 mmol/g, 2 mmol, 1.67 g) was suspended in 1,2-dichloroethane (10 mL) and concentrated thrice. Then a solution of the first amino acid (5 equiv, 10 mmol), DIC (5 equiv, 10 mmol, 1.54 mL), and DMAP (0.01 equiv, 20 mmol, 3 mg) in dry DCM/DMF (50 mL, 10:1 v/v) was added. The mixture was shaken for 3 h and then drained, washed subsequently with DCM, NMP, DCM, and Et2O. The resin was dried before determination of the loading. The loading procedure was repeated when the loading of the resin was found to be too low. Stepwise elongation: FmoceDPheeHMPB-MBHA-resin (loading of the resin was 0.50 mmol/g, 100 mmol, 200 mg) was submitted to nine cycles of Fmoc solid-phase synthesis with the appropriate commercial amino acid building blocks, or Fmoceb2hValeOH. The side chain of ornithine is protected with a Boc-group. Fmoc removal was effected by treatment with 20% piperidine in NMP for 210 min. The resin was subsequently washed with NMP, DCM, MeOH, and finally NMP. FmoceAAeOH (2.5 equiv, 250 mmol), HCTU (2.5 equiv, 250 mmol, 103 mg) in NMP was pre-activated for 1 min after the addition of DiPEA (3 equiv, 300 mmol, 53 mL) and then added to the resin. The suspension was shaken for 1.5 h. The resin was washed with NMP, DCM, MeOH, and NMP. Cleavage from the resin: After the final Fmoc deprotection the resin was washed with NMP and DCM and treated with 5 mL 1% TFA in DCM (610 min). The filtrates were collected, diluted with toluene (15 mL), and concentrated under reduced pressure. The residue was coevaporated with toluene (250 mL). Cyclization: In DMF (80 mL) were dissolved PyBOP (5 equiv, 500 mmol, 260 mg), HOBt (5 equiv, 500 mmol, 77 mg), and DiPEA (15 equiv, 1.5 mmol, 262 mL). The linear decapeptide was dissolved in DMF (5 mL) and added dropwise over 1 h to the reaction mixture. After addition the mixture was stirred for 16 h. The reaction mixture was concentrated in vacuo and the crude mixture was subjected to LH-20 size exclusion chromatography. Boc deprotection: The peptide was dissolved in DCM (2 mL) and TFA (2 mL) was added. The mixture was stirred for 2 h, concentrated, and coevaporated with toluene (210 mL). The obtained crude product was applied to preparative HPLC purification. Using

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gradients of aqueous TFA and acetonitrile the cyclic peptides 1e13 were obtained in the yield range 20e45%. 4.2.1. cyclo-(Pro-bAla1-Orn-bAla2-(R)-Phe)2 (1). 1H NMR (600 MHz, CD3OH) d 8.47 (NH Phe, d, J¼3.7 Hz, 2H), 8.37 (NH bAla2, t, J¼6.0 Hz, 2H), 8.23 (NH Orn, d, J¼8.2 Hz, 2H), 7.85 (H3 Orn, 4H), 7.83 (NH bAla1, dd, J¼5.7, 6.4 Hz, 2H), 7.34e7.26 (Ar. Phe, 10H), 4.66 (Ha Orn, 2H), 4.40 (Ha Phe, 2H), 4.31 (Ha Pro, 2H), 3.67 (Hd Pro, 2H), 3.62 (Hb bAla1, 2H), 3.51 (Hb bAla2, 2H), 3.31 (Hb bAla1, 2H), 3.19 (Hb bAla2, 2H), 3.00 (Hb Phe, 2H), 2.95 (Hd Orn, 4H), 2.94 (Hb Phe, 2H), 2.51 (Ha bAla2, 2H), 2.47 (Hd Pro, 2H), 2.41 (Ha bAla1, 4H), 2.35 (Ha bAla2, 2H), 2.00 (Hg Orn, 2H), 1.97 (Hb Pro, 2H), 1.87 (Hb Orn, 2H), 1.76 (Hg Pro, 2H), 1.66 (Hb Orn, 2H), 1.66 (Hb Pro, 2H), 1.51 (Hg Pro, 2H); 13C (150 MHz, CD3OH) d 174.9, 173.8, 173.9, 173.5, 173.2, 137.0, 130.5, 129.6, 128.4, 61.7, 55.7, 53.6, 49.9, 47.9, 40.4, 37.9, 37.2, 37.1, 36.6, 35.0, 30.1, 24.9, 24.5; LC/MS: tR¼4.76 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1001.67 [MþH]þ; HRMS: calculated for [C50H73N12O10]þ: m/z 1001.55671; found: m/z 1001.55734. (2). 1H NMR 4.2.2. cyclo-(Pro-(S)-b3hLeu-Orn-bAla-(R)-Phe)2 (600 MHz, CD3OH) d 8.63 (NH Phe, d, J¼4.1 Hz, 2H), 8.46 (NH Orn, d, J¼8.3 Hz, 2H), 8.17 (NH b3hLeu, d, J¼9.0 Hz, 2H), 7.74 (NH bAla, t, J¼5.1 Hz, 2H), 7.31e7.23 (Ar. Phe, 10H), 4.48 (Ha Phe, 2H), 4.42 (Ha, Orn, 2H), 4.32 (Ha Pro, 2H), 4.29 (Hb b3hLeu, 2H), 3.56 (Hd Pro, 2H), 3.51 (Ha bAla, 2H), 3.30 (Ha bAla, 2H), 2.99 (Hb Phe, 4H), 2.95 (Hd Orn, 4H), 2.62 (Ha bAla, 4H), 2.56 (Hd Pro, 2H), 2.42 (Ha b3hLeu, 2H), 2.28 (Ha b3hLeu, 2H), 1.94 (Hb Pro, 4H), 1.84 (Hg Orn, 4H), 1.70 (Hg Pro, 2H), 1.69 (Hb Orn, 4H), 1.55 (Hd b3hLeu, 2H), 1.52 (Hg Pro, 2H), 1.44 (Hg b3hLeu, 4H), 0.91 (H3 b3hLeu, 6H), 0.82 (H3 b3hLeu, 6H); 13C (150 MHz, CD3OH) d 173.8, 173.5, 173.1, 172.9, 172.2, 137.3, 130.4, 129.6, 128.2, 61.6, 55.5, 53.6, 47.9, 47.8, 46.7, 44.1, 42.7, 40.4, 37.9, 37.6, 36.2, 31.1, 30.3, 25.9, 24.9, 24.6, 23.6; LC/MS: tR¼5.55 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1113.67 [MþH]þ; HRMS: calculated for [C58H89N12O10]þ: m/z 1113.68191; found: m/z 1113.68273. (3). 1H NMR 4.2.3. cyclo-(Pro-(R)-b2hVal-Orn-bAla-(R)-Phe)2 (600 MHz, CD3OH) d 8.64 (NH Phe, d, J¼3.8 Hz, 2H), 8.35 (NH bAla, t, J¼5.4 Hz, 2H), 8.25 (NH Orn, d, J¼8.3 Hz, 2H), 7.88 (H3 Orn, 4H), 7.50 (NH b2hVal, t, J¼5.4 Hz, 2H), 7.32e7.19 (Ar. Phe, 10H), 4.50 (Ha Phe, 2H), 4.50 (Ha Orn, 2H), 4.30 (Ha Pro, 2H), 3.85 (Hb bAla, 2H), 3.60 (Hd Pro, 2H), 3.54 (Hb b2hVal, 2H), 3.40 (Hb bAla, 2H), 3.26 (Hb b2hVal, 2H), 3.04 (Hb Phe, 2H), 3.01 (Hd Orn 4H), 2.93 (Hb Phe, 2H), 2.57 (Ha bAla, 4H), 2.57 (Hd Pro, 2H), 2.50 (Ha b2hVal, 2H), 1.98 (Hg 0 Pro, 4H), 1.84 (Hb b2hVal, 2H), 1.73 (Hg Orn, 4H), 1.66 (Hb Orn, 4H), 0 0 b 1.66 (H Pro, 2H), 1.52 (Hb Pro, 2H), 0.93 (Hg b2hVal, 6H), 0.89 (Hg 2 13 b hVal, 6H); C (150 MHz, CD3OH) d 175.5, 173.8, 173.7, 173.4, 173.1, 137.1, 130.4, 129.6, 128.3, 61.7, 55.5, 53.3, 52.8, 48.6, 47.9, 41.0, 40.5, 38.0, 36.9, 35.8, 30.8, 30.1, 29.6, 24.6, 21.1, 20.1; LC/MS: tR¼5.28 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1085.40 [MþH]þ; HRMS: calculated for [C56H85N12O10]þ: m/z 1085.65061; found: m/z 1085.65151. 4.2.4. cyclo-(Pro-(R)-b2hVal-Orn-(S)-b3hLeu-(R)-Phe)2 (4). 1H NMR (600 MHz, CD3OH) d 8.51 (NH Phe, d, J¼3.5 Hz, 2H), 8.42 (NH Orn, d, J¼7.2 Hz, 2H), 8.11 (NH b3hLeu, d, J¼8.9 Hz, 2H), 7.89 (H3 Orn, 4H), 7.65 (NH b2hVal, br s, 2H), 7.31e7.23 (Ar. Phe, 10H), 4.44 (Ha Phe, 2H), 4.41 (Ha Orn, 2H), 4.31 (Ha Pro, 2H), 4.29 (Hb b3hLeu, 2H), 3.65 (Hb b2hVal, 2H), 3.52 (Hd Pro, 2H), 3.15 (Hb b2hVal, 2H), 3.04 (Hd Orn, 2H), 2.98 (Hb Phe, 4H), 2.98 (Hd Orn, 2H), 2.74 (Hd Pro, 2H), 2.65 (Ha b2hVal, 2H), 2.44 (Ha b3hLeu, 2H), 1.93 (Ha b3hLeu, 2H), 1.91 (Hb Pro, 2H), 1.85 (Hb’ b2hVal, 2H), 1.81 (Hb Orn, 2H), 1.73 (Hb Pro, 2H), 1.69 (Hg b3hLeu, 2H), 1.69 (Hg Pro, 2H), 1.68 (Hg Orn, 4H), 1.60 (Hg b3hLeu, 2H), 1.59 (Hg Pro, 2H), 1.47 (Hb Orn, 2H), 1.32 (Hd b3hLeu, 0 0 2H), 0.99 (Hg b2hVal, 6H), 0.99 (H3 b3hLeu, 6H), 0.93 (Hg b2hVal, 3 3 13 6H), 0.93 (H b hLeu, 6H); C NMR (150 MHz, CD3OH) d 173.3,

173.0, 172.7, 172.5, 172.5, 137.4, 130.3, 129.6, 128.2, 74.8, 68.8, 64.0, 61.6, 58.4, 30.2, 25.9, 23.7, 22.6; LC/MS: tR¼6.61 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1197.40 [MþH]þ; HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z 1197.77691. (5). LC/MS: 4.2.5. cyclo-(Pro-(S)-b2hVal-Orn-(S)-b3hLeu-(R)-Phe) tR¼6.10 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1197.67 [MþH]þ; HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z 1197.77705. 4.2.6. cyclo-(Pro-(R)-b2hVal-Orn-(R)-b3hLeu-(R)-Phe)2 (6). 1H NMR (600 MHz, CD3OH) d 8.78 (NH Phe, d, J¼2.5 Hz, 2H), 8.58 (NH Orn, d, J¼9.3 Hz, 2H), 8.26 (NH b3hLeu, d, J¼9.3 Hz, 2H), 7.87 (H3 Orn, 4H), 7.45 (NH b2hVal, d, J¼10.0 Hz, 2H), 7.32e7.24 (Ar. Phe, 10H), 4.60 (Ha Orn, 2H), 4.40 (Ha Phe, 2H), 4.44 (Hb b2hVal, 2H), 4.34 (Hb b3hLeu, 2H), 4.30 (Ha Pro, 2H), 3.74 (Hd Pro, 2H), 3.05 (Hb Phe, 2H), 3.02 (Hg Orn, 2H), 2.92 (Hg Orn, 2H), 2.94 (Hb Phe, 2H), 2.86 (Ha b2hVal, 2H), 2.77 (Ha b3hLeu, 2H), 2.57 (Ha b3hLeu, 2H), 2.54 (Hd 0 Pro, 2H), 2.05 (Hb Pro, 4H), 1.87 (Hb b2hVal, 2H), 1.77 (Hg Orn, 4H), g d 3 1.74 (H Pro, 2H), 1.68 (H b hLeu, 2H), 1.67 (Hb Orn, 4H), 1.64 (Hg b3hLeu, 2H), 1.58 (Hg Pro, 2H), 1.22 (Hg b3hLeu, 2H), 0.94 (H3 b3hLeu, 0 3H), 0.85 (H3 b3hLeu, 3H), 0.83 (Hg b2hVal, 6H); 13C (150 MHz, CD3OH) d 174.4, 173.4, 173.3, 173.2, 172.6, 136.9, 130.4, 129.6, 128.3, 61.7, 55.7, 52.6, 51.3, 49.9, 47.9, 46.4, 42.9, 42.3, 40.3, 39.4, 37.5, 31.6, 30.3, 27.6, 26.0, 25.0, 24.4, 23.8, 22.9, 21.4; LC/MS: tR¼6.32 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1197.67 [MþH]þ; HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z 1197.77698. 4.2.7. cyclo-(Pro-(S)- b 2 hVal-Orn-(S)- b 3 hLeu-(R)-Phe-Pro-(R)b2hVal-Orn-(S)-b3hLeu-(R)-Phe) (7). LC/MS: tR¼6.28 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1197.60 [MþH]þ; HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z 1197.77714. (8). 1H NMR 4.2.8. cyclo-(Pro-bAla1-(R)-Orn-bAla2-(R)-Phe)2 (600 MHz, CD3OH) d 8.64 (NH Phe, d, J¼3.8 Hz, 2H), 8.30 (NH Orn, d, J¼7.6 Hz, 2H), 8.24 (NH bAla2, t, J¼5.4 Hz, 2H), 7.90 (H3 Orn, 4H), 7.69 (NH bAla1, t, J¼5.1 Hz, 2H), 7.31e7.17 (Ar Phe, 10H), 4.50 (Ha Phe, 2H), 4.39 (Ha Orn, 2H), 4.26 (Ha Pro, 2H), 3.61 (Hd Pro, 2H), 3.59 (Hb bAla2, 2H), 3.46 (Hb bAla1, 2H), 3.38 (Hb bAla1, 2H), 3.35 (Hb bAla2, 2H), 3.03 (Hd Orn, 2H), 3.02 (Hb Phe, 2H), 2.96 (Hd Orn, 2H), 2.96 (Hb Phe, 2H), 2.56 (Hd Pro, 2H), 2.53 (Ha bAla1, 4H), 2.51 (Ha bAla2, 2H), 2.43 (Ha bAla2, 2H), 1.89 (Hb Pro, 2H), 1.85 (Hb Orn, 2H), 1.73 (Hb Pro, 2H), 1.72 (Hg Orn, 2H), 1.71 (Hb Orn, 2H), 1.68 (Hg Pro, 2H), 1.65 (Hg Orn, 2H), 1.51 (Hg Pro, 2H); 13C NMR (150 MHz, CD3OH) d 173.8, 173.7, 173.4, 173.4, 173.1, 137.1, 130.3, 129.5, 128.3, 61.8, 55.5, 53.8, 48.0, 40.8, 40.4, 37.9, 37.3, 36.8, 36.2, 35.5, 30.4, 24.9, 24.6; LC/MS: tR¼4.84 min (10/90 min MeCN, 15 min run); ESI-MS: m/z 1001.60 [MþH]þ; HRMS: calculated for [C50H72N12O10]þ: m/z 1001.55671; found: m/z 1001.55734. 4.2.9. cyclo-(Pro-(S)-b3hLeu-(R)-Orn-bAla-(R)-Phe)2 (9). 1H NMR (600 MHz, CD3OH) d 8.64 (NH Phe, d, J¼2.9 Hz, 2H), 8.44 (NH bAla, t, J¼2.9 Hz, 2H), 8.42 (NH Orn, J¼8.6 Hz, 2H), 7.92 (H3 Orn, 4H), 7.37 (NH b3hLeu, d, J¼9.5 Hz, 2H), 7.30e7.23 (Ar Phe, 10H), 4.52 (Ha Orn, 2H), 4.44 (Ha Phe, 2H), 4.43 (Hb b3hLeu, 2H), 4.30 (Ha Pro, 2H), 3.79 (Hb bAla, 2H), 3.60 (Hd Pro, 2H), 3.23 (Hb bAla, 2H), 3.05 (Hb Phe, 2H), 3.02 (Hd Orn, 4H), 2.92 (Hb Phe, 2H), 2.56 (Ha bAla, 2H), 2.46 (Ha bAla, 2H), 2.45 (Hd Pro, 2H), 2.43 (Ha b3hLeu, 4H), 1.94 (Hb Pro, 2H), 1.86 (Hb Orn, 2H), 1.73 (Hb Orn, 2H), 1.71 (Hg Orn, 2H), 1.68 (Hb Pro, 2H), 1.67 (Hg Orn, 2H), 1.62 (Hg Pro, 2H), 1.60 (Hg b3hLeu, 2H), 1.53 (Hg Pro, 2H), 1.52 (Hd b3hLeu, 2H), 1.19 (Hg b3hLeu, 2H), 0.87 (H3 b3hLeu, 6H), 0.83 (H3 b3hLeu, 6H); 13C NMR (150 MHz, CD3OH) d 173.7, 173.3, 173.1, 173.0, 172.7, 137.0, 130.4, 129.6, 128.3, 61.9, 55.7, 53.0, 47.8, 46.2, 44.3, 43.0, 40.5, 37.7, 37.0, 35.4, 31.1, 31.1, 30.4, 26.2, 24.7, 24.5, 23.8; LC/MS: tR¼6.82 min (10/90 min MeCN, 15 min

M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400

run); ESI-MS: m/z 1113.47 [MþH]þ; HRMS: calculated for [C58H89N12O10]þ: m/z 1113.68191; found: m/z 1113.68281. 4.2.10. cyclo-(Pro-bAla-(R)-Orn-(R)-b2hVal-(R)-Phe)2 (10). 1H NMR (600 MHz, CD3OH) d 8.49 (NH Phe, d, J¼5.3 Hz, 2H), 8.30 (NH Orn, d, J¼7.7 Hz, 2H), 7.92 (NH bAla, t, J¼5.2 Hz, 2H), 7.86 (H3 Orn, 4H), 7.81 (NH b2hVal, t, J¼5.4 Hz, 2H), 7.33e7.25 (Ar. Phe, 10H), 4.72 (Ha Phe, 2H), 4.36 (Ha Orn, 2H), 4.25 (Ha Pro, 2H), 3.73 (Hd Pro, 2H), 3.43 (Hb b2hVal, 2H), 3.42 (Hb bAla, 2H), 3.33 (Hb b2hVal, 2H), 3.33 (Hb bAla, 2H), 2.98 (Hb Phe, 4H), 2.98 (Hd Orn, 2H), 2.94 (Hd Orn, 2H), 2.78 (Hd Pro, 2H), 2.61 (Ha bAla, 2H), 2.46 (Ha bAla, 2H), 1.86 (Hb Pro, 2H), 0 1.85 (Ha b2hVal, 2H), 1.85 (Hb Orn, 2H), 1.80 (Hb Pro, 2H), 1.76 (Hb 2 g b g b hVal, 2H), 1.75 (H Pro, 2H), 1.72 (H Orn, 2H), 1.65 (H Orn, 4H), 0 0 1.59 (Hg Pro, 2H), 0.94 (Hg b2hVal, 6H), 0.85 (Hg b2hVal, 6H); 13C NMR (150 MHz, CD3OH) d 176.5, 174.1, 173.8, 173.5, 172.6, 137.3, 130.4, 129.5, 128.2, 61.6, 54.6, 54.0, 53.4, 48.2, 40.6, 40.5, 38.5, 37.3, 36.1, 30.5, 30.3, 30.0, 24.9, 24.9, 21.0, 20.3; LC/MS: tR¼5.84 min (10/90 min MeCN, 15 min run); ESI-MS: m/z 1085.33 [MþH]þ; HRMS: calculated for [C56H85N12O10]þ: m/z 1085.65061; found: m/z 1085.65166. 4.2.11. cyclo-(Pro-(S)-b3hLeu-(R)-Orn-(R)-b2hVal-(R)-Phe)2 (11). 1H NMR (600 MHz, CD3OH) d 8.54 (NH Orn, d, J¼7.7 Hz, 2H), 8.47 (NH Phe, s, 2H), 8.12 (NH b2hVal, s, 2H), 7.94 (H3 Orn, 4H), 7.57 (NH b3hLeu, d, J¼8.8 Hz, 2H), 7.32e7.24 (Ar. Phe, 10H), 4.50 (Ha Orn, 2H), 4.41 (Ha Phe, 2H), 4.34 (Ha Pro, 2H), 4.30 (Hb b3hLeu, 2H), 3.76 (Hb b2hVal, 2H), 3.58 (Hd Pro, 2H), 3.15 (Hb b2hVal, 2H), 3.08 (Hb Phe, 2H), 3.01 (Hd Orn, 4H), 2.91 (Hb Phe, 2H), 2.51 (Ha b3hLeu, 2H), 2.50 (Hd Pro, 2H), 2.40 (Ha b2hVal, 2H), 2.34 (Ha b3hLeu, 2H), 2.00 (Hb Pro, 2H), 1.94 (Hb Orn, 2H), 1.87 (Hb’ b2hVal, 2H), 1.75 (Hb Orn, 2H), 1.75 (Hg Orn, 2H), 1.73 (Hg b3hLeu, 2H), 1.68 (Hg Orn, 2H), 1.61 (Hg Pro, 2H), 1.60 (Hb Pro, 2H), 1.57 (Hd b3hLeu, 2H), 1.53 (Hg Pro, 2H), 0 0 1.22 (Hg b3hLeu, 2H), 1.00 (Hg b2hVal, 6H), 0.96 (Hg b2hVal, 6H), 3 3 3 3 13 0.88 (H b hLeu, 6H), 0.82 (H b hLeu, 6H); C NMR (150 MHz, CD3OH) d 176.3, 173.3, 173.0, 172.9, 172.8, 136.9, 130.4, 129.6, 128.3, 61.7, 55.6, 53.3, 52.6, 47.9, 46.8, 43.7, 42.9, 40.7, 40.6, 38.0, 31.3, 30.7, 30.3, 26.1, 24.6, 24.4, 23.8, 21.4, 20.5, 20.3; LC/MS: tR¼7.83 min (10/90 min MeCN, 15 min run); ESI-MS: m/z 1097.53 [MþH]þ; HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/ z 1197.77631.

2399

performed at beamline BM30A and high and low resolution intensity data were collected at beamline ID14-1 at the ESRF (Grenoble, France) by performing one 4 scan (1 oscillation per image). A Images were collected with DNA software84 and processed to 1.10  with MOSFLM.85 Low and high resolution datasets were scaled and merged with POINTLESS and SCALA.86 4.3.3. Structure determination of compounds 6 and 11. Initial phases for solving the structure of compound 11 were obtained with ACORN,87 using a mixed molecular replacement/ab initio procedure with the Leu-DPhe-Pro-Val b-turn of native GS38 used as a starting search fragment. ACORN produced suitable electronic density maps and by means of COOT88 the complete structure of compound 11 could be traced. The structure of compound 6 was solved by direct methods using the program SHELXD.89 Both structures were refined with no intensity cutoff using the fullmatrix least-squares methods on F2 refinement implemented in SHELXL89 included in the WinGX package.90 Throughout the refinement, bond-length, bond-angle, and planarity restraints were imposed. All non-H atoms were refined anisotropically with suitable rigid-bond and similarity restraints. All hydrogen positions were calculated and refined using a riding atom model. Both structures have one crystallographically independent molecule per asymmetric unit with several disordered side chains. Selected crystallographic data is reported in Tables S4 and S5. Final figures were created using PyMOL (Delano Scientific, Palo Alto CA, USA). CCDC 773292 and CCDC 805623 contain the supplementary crystallographic data for structures 6 and 11, respectively. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Acknowledgements We thank STW for financing this research, Spanish Ministry of
Science and Innovation for grant BFU2008-01588 and for a Jose Castillejo fellowship to J.M.O. and the Xunta de Galicia for an ~ o fellowship to J.M.O. We thank the ESRF for the Angeles Alvarin provision of beam time on beamlines ID14-1 and BM30A. We thank Kees Erkelens, Fons Lefeber, and Hans van den Elst for their technical assistance with the analysis and purification of the peptides.

4.3. Crystallographic data Supplementary data 4.3.1. Crystallization and crystallographic data collection of compound 6. Colorless plate-shaped crystals were obtained after slow evaporation of 2 mL droplets of 16 mg/mL peptide in 50% solution of MeOH in H2O plus 2 mL of 1.0 M NaOH in MeOH under paraffin oil in a Terasaki plate. A plate-shaped crystal was mounted in air and then rapidly placed under a 100 K dry nitrogen stream. Intensity data were collected using a BrukereNonius FR591 Kappa CCD2000 X-ray diffractometer with Cu Ka radiation and multilayer confocal optics by performing one 4 and five u scans (2 oscillations per image) at different k and 2q angle settings. The exposure time used was 300 s per degree at generator settings (45 kV, 90 mA). Raw images were integrated using DENZO.83 The resulting intensities were scaled, corrected for absorption effects and the crystal unitcell parameters calculated by global refinement using SCALEPACK.83 4.3.2. Crystallization and crystallographic data collection of compound 11. Colorless crystals were obtained by slow evaporation of 2 mL droplets of 17 mg/mL peptide in 50% (vol/vol) solution of MeOH in H2O plus 2 mL of 20% (vol/vol) of glycerol and 0.1 M Nacacodylate in MeOH under paraffin oil in a Terasaki plate. A prism-shaped crystal was mounted in a cryoloop and directly flashfrozen in liquid nitrogen. Preliminary diffraction tests were

Supplementary data related to this article can be found online at doi:10.1016/j.tet.2012.01.015. References and notes 1. Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodivers. 2004, 1, 1111e1239. 2. Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev. 2001, 101, 3219e3232. 3. Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101, 3893e4011. 4. Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr. Med. Chem. 1999, 6, 905e925. 5. DeGrado, W. F.; Schneider, J. P.; Hamuro, Y. J. Pept. Res. 1999, 54, 206e217. 6. Gellman, S. H. Acc. Chem. Res. 1998, 31, 173e180. 7. Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015e2022. 8. Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 13071e13072. 9. Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913e941. 10. Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Chem. Rev. 2011, 111, 657e687. 11. Brea, R. J.; Reiriz, C.; Granja, J. R. Chem. Soc. Rev. 2010, 39, 1448e1456. 12. Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Acc. Chem. Res. 2009, 42, 1628e1639. 13. Horne, W. S.; Gellman, S. H. Acc. Chem. Res. 2008, 41, 1399e1408. 14. Chatterjee, S.; Roy, R. S.; Balaram, P. J. R. Soc. Interface 2007, 4, 587e606. 15. Seebach, D.; Hook, D. F.; Gl€ attli, A. Biopolymers 2006, 84, 23e37. 16. Lengyel, G. A.; Frank, R. C.; Horne, W. S. J. Am. Chem. Soc. 2011, 133, 4246e4249.

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