Site-selective incorporation of alpha- and beta-amino acid derivatives : towards new gramicidin S-based bactericides

towards new gramicidin S-based bactericides

Knaap, M. van der

Citation

Knaap, M. van der. (2010, September 8). Site-selective incorporation of alpha- and beta- amino acid derivatives : towards new gramicidin S-based bactericides. Retrieved from https://hdl.handle.net/1887/15935

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Mixing Į- and β-Amino Acids in the Strand of Gramicidin S – Towards a New Class of Cyclic β-Hairpins

6.1 I

Gramicidin S (GS, 1) is built up from eight L- and two D-α-amino acids. This allows for the unique β-hairpin structure of this cyclic peptide, and is coupled to its special biological properties. The D-Phe-Pro segments form type II´ β-turns, interconnected by β-sheets of Val- Orn-Leu tripeptides. GS has an amphiphilic structure, with the leucine and valine side chains on one side and the positively charged ornithine side chains on the other.^[1-3] In Part because of the amphiphilicity, GS is rendered as a strong antibiotic. The antibacterial activity, coupled with strong toxicity against mammalian cells, has led to considerable attention from organic chemists, biochemists and medicinal chemists. In the hunt for broadly applicable antibiotics, a lot of research has been devoted to the preparation and biological evaluation of derivatives of

gramicidin S. Numerous publications have appeared on the influence of mutations of one or more amino acid residues on the biological activity, leading to more insight into the structure- activity-relationships.

A plethora of natural and non-natural amino acids with countless functionalities and structural properties are available, so a virtually unlimited number of GS derivatives can be made. Although α-amino acids are at the basis of the unique structure of gramicidin S, confining to these building blocks limits the design of secondary structures, and therefore activity profiles, of the gramicidin S derivatives. Incorporating building blocks other than α-amino acids may broaden the scope of design considerably and this is currently a field of intense research.^[4,5] For example, the advent of β-amino acids^[6-8] has expanded the scope of new peptide structures,^[9-12] as β-peptides (oligomers of β-amino acids) have displayed structures resembling helices,^[7,13-17]

turns^[18-21] and sheets.^[19,22] It has been shown as well that combinations of α- and β-amino acids may lead to specific structures.^[23,24] Depending on the number and position of the specific amino acids the oligomers will display a different structure, though most of them are helical.^[25-29] Only few designs towards pleated sheets built up by both α- and β-amino acids have been published over the years. In 2001 Karle and co-workers^[30] reported on a β-turn consisting of α-amino acids, with on both sides an α/β-dipeptide. In effect, two β³-homophenylalanine residues in the strand faced each other. CD measurements and X-ray analysis confirmed the β-hairpin structure and the stabilisation of the strand by hydrogen-bonding interactions.

It was reasoned that incorporation of β-amino acids in the strand of gramicidin S (1) would lead to a structurally new β-hairpin/β-sheet hybrid (2). Additionally, incorporation of β-amino acids allows for the addition of more functionalities, making fine-tuning of the structure and hydrophobicity/hydrophilicity ratio possible. As shown in Figure 1 (Bottom left), a βαβ- tripeptide would lead to a β-sheet pattern, stabilised by four hydrogen bonds. Assuming that the hydrogen bonds lie in a plane, and that sp³-carbons lead to a kink in the plane of the sheet, a simplified three dimensional model of gramicidin S and its derivatives can be made. The viability

of this simplification is justified when compared to the crystal structure of GS. The model (2) for the βαβ-sheet leads to two additional bends in the sheet, as both the Į- and β-carbons are sp³- hybridised. It also indicates the positioning of the side chains in order to create an amphiphilic structure. When the L-ornithine residue are retained in the middle, the L-valine should be replaced by (S)-β²-homovaline and ^L-leucine by (S)-β³-homoleucine. To probe these hypotheses a series of GS analogues containing β-amino acids was prepared. Besides the fully substituted derivative (6), the analogues lacking hydrophobic side chains (3) and the semi-substituted homologues (4 and 5) were made. The influence of stereochemistry of the side chains on the structure of β-GS was analysed by the preparation of peptides with one (8) or two (7 and 9) stereocenters inverted. Various NMR techniques and X-ray crystallography were used to elucidate the three dimensional structure. If the peptides were to adopt the pleated sheet

Figure 1 Top panel: Chemical structure of gramicidin S with hydrogen bonds indicated and the simplified representation of the three dimensional structure on the right. The ornithines are represented by the +-signs, the hydrophobic side chains by the black dots. Bottom panel: The hydrogen bond-pattern of the GS derivatives containing β-amino acids. Also the amino acid numbering is indicated. On the right the simplified three dimensional representation is shown.

NH N

H

HN H

N O

O O

O HN HN

NH NH

O

O O

O N

N O

O

I II III IV V

NH HN

NH O

HN NH HN NH

O N

O

O

O O

O HN

H2N NH2

O

O N

O

1, Gramicidin S

2

structure they would be amphiphilic and likely to be active against bacteria. Therefore peptides 3- 9 in Figure 2 were tested for their activity against bacteria.

6.2 R

D

6.2.1 S

Peptide synthesis was performed as described earlier (Scheme 1).^[31] The synthesis started with the hyper-acid labile HMPB-MBHA resin 10.^[32] The alcohol was esterified with either Fmoc- βAla-OH or Fmoc-^DPhe-OH, in combination with DIC and catalytic DMAP to produce 11 and 12 respectively. With the appropriately loaded resins in hand, step-wise solid phase peptide

syntheses were carried out by making use of the Fmoc-based strategy. Once fully assembled, the decameric peptides were cleaved from the resin by mild acid treatment. The crude linear peptides

Figure 2 

NH N

H

HN H

N O

O O

O HN HN

NH NH

O

O O

O

N N O

O

R₂ R₁

R2 R1

3: R1= R2= H

4: R1=ⁱBu; R2= H

5: R1= H; R2=ⁱPr

6: R1=ⁱBu; R2=ⁱPr

NH N

H

HN H

N O

O O

O HN HN

NH NH

O

O O

O

N N O

O

NH N

H

HN H

N O

O O

O HN HN

NH NH

O

O O

O

N N O

O

R₁ R2

7

8: R1=ⁱPr; R2= H

9: R₁= H; R₂=ⁱPr NH₂

H2N

NH2

H2N

NH2

H2N

Scheme 1 Reagents and conditions i) Fmoc-βAla-OH (5 eq), DIC (5 eq), DMAP (0.01 eq), DCM; ii) Fmoc-^DPhe-OH (5 eq), DIC (5 eq), DMAP (0.01 eq), DCM; iii) a) 20% pip/NMP (2 × 10 min); b) Fmoc-aa-OH (2.5 eq), HCTU (2.5 eq), DiPEA (3 eq), NMP; iv) 1% TFA/DCM (6 × 10 min); v) PyBOP (5 eq), HOBt (5 eq), DiPEA (15 eq), DMF; vi) 50% TFA/DCM.

were cyclised in DMF under dilute (0.01 M) conditions under the agency of PyBOP/HOBt/DiPEA. Removal of the coupling reagents was affected by LH-20 gel filtration, followed by Boc-removal and preparative HPLC purification.

6.2.2 NMR

The obtained peptides were all subjected to 1D and 2D NMR analyses. Using a combination of COSY and TOCSY spectra all signals could be unambiguously assigned. From the 1D ¹H NMR spectra it appeared that, with the exception of peptides 8 and 9, all peptides adopted a single, defined structure. Peptides 8 and 9 did not give

clearly resolved signals. The spectra of the other peptides (3-7) were interpreted in more detail.

Coupling constants. A first indication of the

structure is given by examining the ³JNH-Hα values.

A small coupling constant (J < 4) indicates involvement of that residue in a turn structure,

whereas a large value (J > 7) indicates ^{Figure 3}

3JNH-Hα of peptides 2-6. Couplings indicated with * are triplets.

-AA (II) E Orn (III)

-AA (IV )

E Phe

(V)

D

0 2 4 6 8 10

3 4 5

* 6

* *

7

* * *

Residue 3 JHND

involvement in a sheet.^[33] Figure 3 shows the ³JNH-Hα values of peptides 3-7. Interestingly the amide of the β²hVal residue in peptide 6 appeared as a broad singlet and in peptide 7 as a doublet with large coupling constant (10 Hz), instead of the expected triplets. The signals that resulted in triplets (for βAla and β²hVal) had coupling constants of 5.10-5.97 Hz, indicating an extended structure for these residues. In general it can be seen that the D-phenylalanine residues give small couplings (2.52-4.08 Hz, which corresponds to a turn structure) while the coupling constants for the ornithine and β³-homoleucine residues are large (7.19-9.33 Hz, which correlates with a sheet), thus confirming a cyclic β-hairpin structure.

Temperature coefficients. Another indication that 3-7 adopted the expected structure was

found by examining the effect of temperature on the chemical shift of the amide proton signals.^[34,35] Towards this end, the peptides were dissolved in DMSO and ¹H NMR spectra were

recorded at different temperatures. From the data the ppb/K were calculated. Large changes in chemical shift indicate interaction with solvent and therefore lack of involvement in hydrogen bonding. Protons involved in interstrand hydrogen bonding are less influenced by temperature changes. From Figure 1 it may be anticiptated that the amide protons of residues 2/2 (βAla or β²hVal) and 3/3 (Orn) are involved in hydrogen bonding. The data in Table 1 agrees with this hypothesis, but the trend is less pronounced for the amide protons of the ornithine residues, indicating that these hydrogen bonds are less strong than the hydrogen bonds of the amide protons of the βAla/β²hVal residues.

Chemical shift perturbation is an empirical method to probe the secondary structure of

peptides and proteins and it is based on the observation that the chemical shift of the Į-proton of a certain amino acid residue is influenced by the secondary structure in which it is involved.^[36]

 3 4 5 6 7

AA(II) 3.23 2.30 0.70 2.46 0.37 Orn(III) 4.45 4.95 4.43 3.19 5.31

AA(IV) 5.33 3.33 7.74 4.46 2.56

DPhe(V) 4.94 4.71 5.39 4.56 3.88

Table 1 Temperature coefficients of the amide proton signals in ppb/K. In brackets the amino acid numbering is given.

The chemical shift perturbation is defined as the difference between the observed chemical shift and the literature value of the chemical shift of the residue in a random coil.^[36] A positive value indicates involvement in a sheet, a negative value involvement in a turn and zero points at a random coil structure. As the random coil values have not been determined for any β-amino acid, this method can only be applied to the α-amino acids. Figure 4 shows the chemical shift perturbation values for residues I (Pro), III (Orn) and V (^DPhe) in peptides 3-7.

As is obvious proline and ^D-phenylalanine are involved in turns and ornithine is part of the sheet in all instances. Though this technique is quite limited in this case, as β-amino acids can not be analysed, it indeed confirms the secondary structure of peptides 3-7.

Nuclear Overhauser Effects. CROESY spectra were recorded for peptides 3-7. NOE crosspeaks

similar to gramicidin S were observed; all peptides showed sequential NH-H^α crosspeaks (or NH- H^β in the case of the amide of ornithine and the adjacent β-amino acid residue) and in none of the peptides interaction between two α-protons (or β-protons for the β-amino acids) were observed, confirming the strand structure in these peptides. In the turn, nuclear Overhauser effects were observed between the amides of β²hVal/βAla and the H^α of proline and between the NH of D-phenylalanine and the H^δ of proline. More data concerning the NOE signals are presented in the experimental section.

Figure 4 Chemical shift perturbations of the natural amino acids in 3-7. Literature values are from [36]. The value for Lys is used to calculate the perturbation of H^α of Orn.

Chemical Shift Perturbation

Pro ( I)

Orn (III)

Phe (V)

D

-0.2 0.0 0.2

3 4 5 6 7

amino acid residue

'GD (ppm)

Figure 5 Crystal structure of 7 with the top view (left panel) and the side view (right panel). In the top view the side chains are omitted for clarity.

6.2.3 C

One of the peptides yielded crystals suitable for Röntgen diffraction analysis. The structure of 7 was solved and the top and side views are displayed in Figure 5. As can be seen in the top view,

the hydrogen bonds as proposed in Figure 1 are indeed formed in the peptide, thus leading to an anti-parallel β-sheet. What can also be observed from the top view are the two type II´ β-turns that impose the change in directionality of the sheets. Also the side view shows this sheet structure with accompanying hydrogen bonds and β-turns. What is interesting in this view is the bend in the sheet. The sheet in gramicidin S also posses this bend,^[37] but this is not as pronounced as in GS derivative 7.

6.4 B

6.4.1 A

A

In Table 2 the antibacterial activities of peptides 3-9 are presented and compared to the parent compound gramicidin S. It is clear that none of the new peptides are as effective in lysing bacterial cells as GS. For peptides 3-5 this may be explained by the fact that they lack two or four of the hydrophobic side chains, and therefore lack the necessary hydrophobicty to disrupt the

integrity of the bacterial cell membrane. Peptide 6 has the same side chains as GS, but still is only very slightly active against some selected bacterial strains. Most interesting is the activity of 8.

This molecule appears to be the most active antibiotic from the series tested, but it is epimeric in one of the valine side chains and is therefore not thought to be amphiphilic, a prerequisite for these kinds of antibiotic peptides. The literature, however, has shown more nonamphiphilic gramicidin analogues with antibacterial activity.^[38-43]

6.5 C

In this Chapter the synthesis of cyclic, mixed α/β-peptides has been described. Based on a simplified model described in the introduction a series of gramicidin S derivatives with β-amino acids in the strand have been synthesised. These peptides were subjected to a variety of NMR techniques. These showed that in all cases, except 8 and 9, the peptides adopted a cyclic β-hairpin structure. Also when de stereochemistry of the β³hLeu was inverted a peptide (7) with well defined secondary structure was obtained. However, inversion of the stereochemistry of one or two of the β²hVal residues led to distortion of the structure (i.e. 8 and 9). The crystal structure

Strain GS 3 4 5 6 7 8 9

S.aureus7323 8 32 >64 >64 >64 >64 32 >64

S.aureus7328 32 >64 >64 16 >64 32 >64

CNS5277 4 32 64 64 32 >64 32 >64

CNS5115 >64 64 64 64 >64 8 >64

CNS7368 32 64 64 16 >64 8 >64

E.faecalis1131 8 16 >64 >64 >64 >64 8 64

E.coliATCC25922 >64 >64 >64 >64 >64 64 >64

P.aeruginosaAK1 16 >64 >64 >64 >64 >64 >64 >64

P.aeruginosaATCC19582 >64 >64 >64 >64 >64 >64 >64

S.mitis BMS >64 >64 >64 16 >64 32 >64

S.mitis ATCC33399 4 >64 >64 >64 16 >64 64 64

Table 2 Antibaterial activity of GS and 3-9 in μg/mL.

obtained from peptide 7 confirmed the observation that at the β³hLeu-position variation in the stereochemistry of the side chains are allowed. Unfortunately the antibacterial assay did not support the amphiphilicity of 3-6, but the reason for this is at the moment unclear.

6.6 E

S

General

Solvents and chemicals were used as received from their supplier. Solvents were stored over 4 Å molecular sieves (or 3 Å MS for MeOH). Fmoc-β³hLeu-OH and Fmoc-D-β³hLeu-OH were obtained from Senn Chemicals AG. Fmoc- β²hVal-OH was synthesised as described in Chapter 5. ¹H and ¹³C NMR spectra were recorded with a Bruker DMX- 600 spectrometer (600/150 MHz). Chemical shifts δ are given in ppm relative to MeOD (3.31 ppm) as internal standard. High resolution mass spectra were recorded by direct injection (2 μL of a 2 μM 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 R = 60000 at m/z 400 (mass range m/z = 150-2000) and dioctylphthalate (m/z = 391.28428) 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 a 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 (150 u 21.20 mm, 5μ). 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.

Antibacterial screening

Bacteria were stored at -70°C and grown at 30°C on Columbia Agar with sheep blood (Oxoid, Wesel, Germany) suspended in physiological saline until an optical density of 0.1 AU (at 595 nm, 1 cm cuvette). The suspension was diluted (10 x) with physiological saline, and 2 PL of this inoculum was added to 100 PL growth medium, Nutrient Broth from Difco (ref. nr. 234000, lot nr. 6194895) with yeast extract (Oxoid LP 0021, lot nr. 900711, 2 g/400 mL broth) in Microtiter plates (96 wells). The peptides GS and 3-9 were dissolved in ethanol (4 g/L) and diluted in distilled water (1000 mg/L), and two-fold diluted in the broth (64, 32, 16, 8, 4 and 1 mg/L). After incubation at 30 °C (24-96 h) the MIC was determined as the lowest concentration inhibiting bacterial growth.

General Peptide Synthesis

Loading of the HMPB-MBHA-resin 11 and 12: The resin 10 (theoretical loading = 1.2 mmol/g, 2 mmol, 1.67 g) was suspended in dichloroethane and concentrated thrice. Then a solution of the first amino acid (5 eq, 10 mmol), DIC (5 eq, 10 mmol, 1.54 mL) and DMAP (0.01 eq, 20 μmol, 3 mg) in dry DCM/DMF (50 mL, 10:1 v/v) was added. The mixture was shaken for 3 hours and then drained, washed subsequently with DCM, NMP, DCM and Et2O. The resin was dried before determination of the loading. The procedure was repeated when necessary.

Stepwise Elongation: Fmoc-^DPhe-HMPB-MBHA-resin 12 (Loading of the resin was 0.50 mmol/g, 100 μmol, 200 mg) or Fmoc-βAla-HMPB-MBHA-resin 11 (L = 0.55 mmol/g, 100 μmol, 182 mg) was submitted to nine cycles of Fmoc solid-phase synthesis with the appropriate commercial amino acid building blocks, or Fmoc-β²hVal-OH. The amino

group on the side chain of ornithine was protected with a Boc-group. Fmoc removal was effected by treatment with 20% piperidine in NMP for 2 x 10 min. The resin was subsequently washed with NMP, DCM, MeOH, and finally NMP. The Fmoc-AA-OH (2.5 eq, 250 μmol), HCTU (2.5 eq, 250 μmol, 103 mg) in NMP was pre-activated for 1 min after the addition of DiPEA (3 eq, 300 μmol, 53 μL) 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 (6 x 10 min). The filtrates were collected and coevaporated with toluene (3 x 50 mL).

Cyclisation: In DMF (80 mL) were dissolved PyBOP (5 eq, 500 μmol, 260 mg), HOBt (5 eq, 500 μmol, 77 mg), and DiPEA (15 eq, 1.5 mmol, 262 μL). The linear decapeptide was dissolved in DMF (5 mL) and added dropwise over 1 h

to the coupling cocktail. 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 (2 × 10 mL). The obtained crude product was applied to

Peptide  NHamide H^ H^ H^ H^ H^

3 ^DPhe 8.47(3.68) 4.40 3.00/2.94 7.347.26 

 Ala 8.37(t,5.97) 2.51/2.35 3.51/3.19 

 Orn 8.23(8.18) 4.66 1.87/1.66 2.00 2.95 7.85

 Ala 7.83(5.74/6.38) 2.41 3.62/3.31 

 Pro  4.31 1.97/1.66 1.76/1.51 3.67/2.47 

4 ^DPhe 8.63(4.08) 4.48 2.99 7.317.23 

 Orn 8.46(8.26) 4.42 1.69 1.84 2.95 

 ³hLeu 8.17(9.03) 2.42/2.28 4.29 1.44 1.55 0.91/0.82

 Ala 7.74(t,5.10) 2.62 3.51/3.30 

 Pro  4.32 1.94 1.70/1.52 3.56/2.56 

5 ^DPhe 8.64(3.83) 4.50 3.04/2.93 7.327.19 

 Ala 8.35(t,5.43) 2.57 3.58/3.40 

 Orn 8.25(8.25) 4.50 1.66 1.73 3.01 7.88

 ²hVal 7.50(t,5.43) 2.50 3.54/3.26 1.84(=') 0.93/0.89(=') 

 Pro  4.30 1.66/1.52 1.98 3.60/2.57 

6 ^DPhe 8.51(3.45) 4.44 2.98 7.317.23 

 Orn 8.42(7.19) 4.41 1.81/1.47 1.68 2.98/3.04 7.89

 ³hLeu 8.11(8.85) 2.44/1.93 4.29 1.69/1.60 1.47/1.32 0.99/0.93

 ²hVal 7.65(brs) 2.65 3.65/3.15 1.85(=') 0.99/0.93(=') 

 Pro  4.31 1.91/1.73 1.69/1.59 3.52/2.74 

7 ^DPhe 8.78(2.52) 4.40 3.05/2.94 7.327.24 

 ^D³hLeu 8.26(9.33) 2.57/2.77 4.34 1.64/1.22 1.68 0.94/0.85

 Orn 8.58(9.33) 4.60 1.67 1.77 3.02/2.92 7.87

 ²hVal 7.45(10.00) 2.86/2.71 4.44 1.87(=') 0.83(=') 

 Pro  4.30 2.05 1.74/1.58 3.74/2.54 

Table 3 ¹H NMR Data for Peptides 3-7 (600 MHz, CD3OH). In brackets the ³JNH-Hα are given.

preparative HPLC purification.

Peptide 3

13C (150 MHz, CD3OH) δ 174.68, 173.82, 173.68, 173.49, 173.23, 137.03, 130.45, 129.63, 128.40, 61.69, 55.66, 53.61, 49.85, 49.43, 48.58, 47.93, 40.42, 37.93, 37.20, 37.06, 36.56, 35.01, 30.63, 30.12, 24.89, 24.54; LC/MS: Rt = 4.76 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1001.67 [M + H]⁺; HRMS: calculated for [C50H73N12O10]⁺: m/z 1001.55671; found: m/z 1001.55734.

Peptide 4

13C (150 MHz, CD3OH) δ 173.81, 173.49, 173.12, 172.86, 172.22, 137.32, 130.38, 129.56, 128.24, 61.63, 55.54, 53.64, 49.43, 48.57, 47.90, 47.83, 46.71, 44.06, 42.69, 40.37, 37.94, 37.63, 36.16, 31.05, 30.66, 30.25, 25.88, 24.90, 24.60, 23.61, 22.15; LC/MS: Rt = 5.55 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1113.67 [M + H]⁺; HRMS: calculated for [C58H89N12O10]⁺: m/z 1113.68191; found: m/z 1113.68273.

Peptide 5

13C (150 MHz, CD3OH) δ 175.54, 173.82, 173.71, 173.44, 173.14, 137.10, 130.37, 129.60, 128.34, 61.69, 55.47, 53.26, 52.84, 49.43, 48.57, 47.94, 40.96, 40.47, 37.95, 36.86, 35.82, 30.76, 30.05, 29.61, 24.60, 21.11, 20.13; LC/MS: Rt = 5.28 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1085.40 [M + H]⁺; HRMS: calculated for [C56H85N12O10]⁺: m/z 1085.65061; found: m/z 1085.65151.

3 ^DPheNH H^Ala(m)

 ^DPheH^ H^Pro(s),H^Ala⁴(s)

 Ala⁴NH H^Orn(s),H^Ala⁴(w)

 OrnNH H^Ala(s)

 Ala²NH H^Pro(m),H^{ D}Phe(w),H^Pro(m)

4 ^DPheNH H^³hLeu(m),H^³hLeu(s/m),H^³hLeu(m)

 ^DPheH^ H^Pro(s)

 ³hLeuNH H^Orn(s),H^Pro(w)

 ³hLeuH^ H^Pro(m)

 OrnNH H^³hLeu(m),H^Ala(s)

 AlaNH H^Pro(m),H^Orn(w),H^Pro(m) 5 ^DPheNH H^Ala(m)

 ^DPheH^ H^Pro(s)

 AlaNH H^Orn(m)

 OrnNH H^²hVal(m)

 ²hValNH H^Pro(w/m)

6 ^DPheNH H^³hLeu(w),H^³hLeu(s)

 ³hLeuNH H^Orn(s)

 OrnNH H^²hVal(m)

 OrnH^ H^Pro(w)

 ²hValNH H^³hLeu(s),H^Pro(w)

7 ^DPheNH NH^D³hLeu(w),H^{ D}³hLeu(w/s)

 ^D³hLeuNH H^Orn(s)

 OrnNH H^^D³hLeu(w),H^²hVal(s),H^²hVal(vw),H^²hVal(vw)

 ²hValNH NHOrn(s),H^Pro(m),H^Pro(m)

Table 4 NOE Data for peptides 3-7. CROE spectra were recorded at 600 MHz in CD3OH, mixing time 200 ms.

Peptide 6

13C NMR (150 MHz, CD3OH) δ 173.26, 172.99, 172.72, 172.49, 172.46, 137.42, 130.33, 129.58, 128.24, 74.77, 68.82, 63.96, 61.64, 58.37, 30.22, 25.88, 23.65, 22.58; LC/MS: Rt = 6.61 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1197.40 [M + H]⁺; HRMS: calculated for [C64H101N12O10]⁺: m/z 1197.77581; found: m/z 1197.77691.

Peptide 7

13C (150 MHz, CD3OH) δ 174.42, 173.36, 173.32, 173.22, 172.64, 136.91, 130.41, 129.56, 128.34, 61.73, 55.68, 52.64, 51.27, 49.85, 49.43, 48.57, 47.93, 46.36, 42.90, 42.26, 40.31, 39.39, 37.50, 31.55, 30.26, 27.63, 26.01, 25.00, 24.38, 23.83,

Formula (C₆₄H₁₀₀N₁₂O₁₀) H₂O Formulaweight 1213.56

Wavelenght[Å] 1.54178 Crystalsystem monoclinic

Spacegroup P2₁

a[Å] 17.3105(10)

b[Å] 10.7558(7)

c[Å] 18.4678(12)

[º] 90

[º] 97.839(3)

[º] 90

Cellvolume[ų] 3406.4(4) Ucalc[g/cm³] 1.183 Nº.form.unitsZ 2 P^[mm1] 0.66

F(000) 1312

crystalsize[mm³] 0.08x0.07x0.01

T[K] 100(2)

Trange[º] 0.98247.27 Measuredreflections 33462 Uniquereflections 3294 Completeness[%] 99.1

Redundancy 5.9

R(int) 0.071

Datainrefinement 3289 DatawithFo>4sig(Fo) 2554

Av.I/sig(I) 9.25

H,KandLmin 0,0,17 H,KandLmax 16,10,17

Nºparameters 926

Extinctioncoef. 0.0028(4)

wR2 0.2012

R1(obsdata) 0.0700

R1(alldata) 0.0937

Goof=S 1.045

'Fpeak/hole[eÅ^3] 0.36/0.24 Table 4 Selected crystallographic data obtained for compound 7.

22.85, 21.38, 17.89; LC/MS: Rt = 6.32 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1197.67 [M +H]⁺; HRMS:

calculated for [C64H101N12O10]⁺: m/z 1197.77581; found: m/z 1197.77698.

Peptide 8

LC/MS: Rt = 6.28 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1197.60 [M + H]⁺; HRMS: calculated for [C64H101N12O10]⁺: m/z 1197.77581; found: m/z 1197.77714.

Peptide 9

LC/MS: Rt = 6.10 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1197.67 [M + H]⁺; HRMS: calculated for [C64H101N12O10]⁺: m/z 1197.77581; found: m/z 1197.77705.

Crystallisation and crystal structure determination of compound 2:

Colourless plate-shaped crystals were obtained after slow evaporation of 2 μL droplets of 16 mg/mL peptide in 50%

solution of MeOH in H2O plus 2 μL of 1.0 M NaOH in MeOH under paraffin oil in Terasaki plates. A 0.08 x 0.07 x 0.01 mm plate crystal was mounted in air and then rapidly placed under a 100 K dry nitrogen stream. Intensity data were collected using a Bruker–Nonius FR591 Kappa CCD2000 X-ray diffractometer with Cu Kα radiation and multilayer confocal optics by performing one φ and five ω scans (2° oscillations per image) at different κ and 2θ angle settings. The exposure time used was 300 s per degree at generator settings (45 kV, 90 mA). Raw images were integrated using DENZO.^[44] The resulting intensities were scaled, corrected for absorption effects and the crystal unit-cell parameters calculated by global refinement using SCALEPACK.^[44] The structure was solved by direct methods using the program SHELXD^[45] and refined with no intensity cutoff using the full-matrix least-squares methods on F² refinement implemented in SHELXL^[45] included in the WinGX package^[46]. 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. CCDC 773292 contains the supplementary crystallographic data. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/

data_request/cif.

6.7 R

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