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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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

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Synthesis and Evaluation of Smaller Ring Analogues of Peptide Antibiotic Gratisin

4.1INTRODUCTION

Of the cyclic cationic amphiphilic peptides, gramicidin S^[1,2] (1, GS, Figure 1) has enjoyed a great deal of attention from various research fields, not in the least because of its unique biological profile. GS is highly active against a wide range of bacteria, including pathogens that have built up resistance against a broad range of antibiotics, though it is less well able to kill Gram-negative bacteria.^[3] Because it is thought that the antibacterial activity lies in its ability to lyse bacterial cell membranes, it is not very likely that bacteria will easily build up resistance against gramicidin S.^[4-7] Broad medical application has been hampered however, by the toxicity of the cyclic peptide towards mammalian cells.^[8] Developing highly active cationic amphiphilic peptides active against pathogens like bacteria and fungi, but that leave mammalian cell membranes untouched is therefore a valuable approach to combat the expanding number of multiply resistant pathogens.

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Gratisin (2, GR), a related cyclic peptide, also isolated from Bacillus Brevis,^[9,10] is built up from twelve amino acids. Compared to GS, GR also contains a Val-Orn-Leu tripeptide, but displays two slightly altered turns. Instead of type II β-turns constituted by ^DPhe-Pro, gratisin has two ^DPhe-Pro-^DTyr sequences. As no crystal structures or high resolution NMR data are available, the exact structure of gratisin is unknown. It is likely however, that Val-Orn-Leu sequences form antiparallel β-sheets, similar to gramicidin S, whereas the ^DPhe-Pro-^DTyr tripeptides on the other hand may adopt α-turn configurations.^[11-13] These assumptions direct to a three dimensional structure that is amphiphilic, with the ornithines projecting to one side of the β-sheet and the valine and leucine side chains to the other side. Like GS, gratisin is known to be a potent antibiotic as well, but it is slightly less hemolytic,^[14] making it attractive research target. Likely, knowledge of the structure will give insight into the exact mode of action. If the structure and biological profiles are compared, less hemolytic, but strongly antibiotic analogues of gratisin and gramicidin S come into view. Towards the same goal, besides gramicidin S and gratisin, a hybrid structure (3) between GS and GR was synthesised,

Figure 1

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in which one of the two D-tyrosine residues is lacking, rendering an undecameric cyclic peptide.

A known artificial cyclic antibacterial peptide is glycosylated octapeptide 4.^[15] This peptide contains alternating D- and L-amino acids, which causes the peptide to be disk shaped, with the side chains pointing outwards. The design of the peptide was based on the complex cyclic glycopeptide mannopeptimycin,^[16] which displays its antibacterial activity by binding lipid A and thereby inhibiting the transglycosylation step in bacterial cell wall biosynthesis. However the designed peptide 4 also showed membrane disrupting ability and hemolysis. On the basis of peptide 4 octameric peptide 5 with alternating D- and L-amino acids is designed. Peptide 5 is derived from GS by removal of two proline residues and inversion of the stereochemistry of the two ornithine residues, thereby mimicking the stereochemistry of 4. Three hybrid structures between peptide 5 and GS (1) were synthesised as well: two decameric peptides with either one (6) or two D-ornithines (7) and a cyclic 9-mer (8) with an additional proline compared to 5. All peptides were assessed for their structure by NMR spectroscopic techniques, and for their activity against bacterial- and erythrocytic cell membranes.

4.2RESULTS AND DISCUSSION

In order to synthesise peptides 1-3 and 5-8, an approach was used in which the side chain of ornithine was anchored to the solid support, which allowed for on resin cyclisation of the peptides. By performing the cyclisations on solid support, the likeliness that two different peptides will get in each other proximity and produce dimers or trimers, is very low (pseudo

Scheme 1 Reagents and conditions i) allyl bromide, DiPEA, MeCN; ii) 50% TFA in DCM

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dilution). For this approach a suitably protected ornithine residue was necessary. Following the approach by Albericio and co-workers,^[17] the side chain of ornithine was used as attachment point for the resin. Standard Fmoc/^tBu chemistry was used and the carboxylic acid of the Orn residue was protected as the allyl ester during the peptide elongation. The Allyl ester can be mildly deprotected on resin using palladium(0) species. After liberation of the acid functionality the cyclisation can be performed on resin.

The Fmoc-Orn-OAll 11a and Fmoc-D-Orn-OAll 11b were synthesised from Fmoc- Orn(Boc)-OH 9a and Fmoc-D-Orn(Boc)-OH 9b respectively (Scheme 1).^[17] Esterification was performed using allyl bromide and DiPEA to yield 10ab, after which the Boc group could be removed by treatment with strong acid to yield the trifluoroacetic acid salt of allyl esters 11a and 11b. Reaction of this amino acid building block with o-chlorotrityl chloride resin 12 in the presence of excess DiPEA yielded loaded resins 13a and 13b (Scheme 2), which were used as the starting materials in the automated stepwise peptide synthesis. Repeated cycles of Fmoc-deprotection with piperidine in NMP and couplings with five equivalents of the amino acids and HCTU as the coupling reagent in the presence of DiPEA yielded the linear octa-, nona-, deca-, undeca-, and dodecapeptides, protected with Fmoc carbamate and allyl ester (14). Deallylation was performed in degassed chloroform, with catalytic Pd(PPh3)4 and

Scheme 2 Reagents and conditions i) 11a or 11b, DiPEA (5 eq), DCM; ii) 20% pip/NMP; iii) Fmoc-aa-OH (5 eq), HCTU (5 eq), DiPEA (10 eq) (1.5 h); iv) Pd(PPh3)4, sodium p-toluenesulfinate, CHCl3; v) PyAOP (5 eq), HOAt (5 eq), DiPEA (10 eq), 0.8 M LiCl/DMF; vi) 1% TFA/DCM; v) 50%

TFA/DCM

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sodium p-toluenesulfinate as the scavenger. After ester deprotection, the Fmoc group was removed to give the amino acids 15. As monitored by LC/MS analysis, cyclisations worked best with PyAOP/HOAt as coupling reagent and with DiPEA and LiCl in DMF. In case a cyclisation was not complete the coupling procedure was repeated until all of the linear peptide was converted to the cyclic product 16. Using mild acid cleavage the peptides were liberated from the resin and 50% TFA in DCM was subsequently used to acidolyse the remaining Boc protective group. This furnished the peptides 1-3 and 5-8 in good to reasonable purities. The crude products were subjected to preparative HPLC purification to give the homogeneous products. Unfortunately, peptide 5 proved to be insoluble in all solvents tested and it was therefore impossible to purify the crude cyclisation product. The insolubility is presumably caused by extensive hydrogen bonding between the disk-like peptides, thereby forming aggregates.^[18-20]

Important information may be obtained from the ³JNH-αH values, as these correlate with the dihedral angle. Small coupling (J < 4 Hz) constants indicate involvement in turns structures.

Large coupling constant (J > 5 Hz), on the other hand, point towards a β-sheet.^[21] Figure 2 depicts the coupling constants ³JNH-αH for peptides 1 (GS), 3, 6 and 8. The structure of peptide 1 (GS) is known in detail, as high resolution crystal structures are available.^[22] The ³JNH-αH

match those for the β-hairpin structure (see Experimental Section for detailed NMR data). For peptide 3 one of the ^DPhe residues gives a ³JNH-αH of 3.30 Hz, the other of ~ 0 Hz. The first is clearly involved in a type II β-turn, but the latter has a dihedral angle (-30°) that is smaller than normal (-60°) for an i + 1 residue in a II β-turn. This hints at an α-turn,^[23,24] although detailed structural information is not available at the moment. The coupling constant of the

D-tyrosine residue in the i + 3 position is 8.56 Hz (dihedral angle of about -80°), which could imply a I´αLS-turn, although the dihedral angle of ^DPhe shows an appreciable distortion (-48°

versus -30° found). The sheet appears to be maintained in this molecule. The data for peptide 6 also indicates that the molecule adopts a structure with at least one β-turn (the signal of the NH of the second ^DPhe residue appeared as a singlet). The other residues seem to form β-sheet despite the incorporation of a D-amino acid in the strand. The significantly high coupling

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constant for one of the D-phenylalanine residues in 8 indicate that it does not adopt a β-turn structure, while data for the other ^DPhe residue could not be obtained due to overlap with other signals. The chemical shift for the amide proton of this residue is unexpectedly low (7.89 versus 8.35), indicating that the structure deviates significantly from a β-turn. One of the

valine residues shows a rather low coupling constant indicating that this residue adopts a conformation that deviates slightly from that for a β-sheet. Probably it is being forced into a pseudo-turn conformation, although this is unfavourable for a β-branched residue.

The chemical shifts of the α-protons also give information about the structure. The observed chemical shifts may be compared to the chemical shifts of the same residue in a random coil.^[25] A negative value indicates involvement in a turn, a positive value indicates at involvement in a sheet, a value close to zero points at an unstructured segment. The chemical

Figure 2 ³JNH-αH in Hertz for pepides 1, 3, 6 and 8.

1

Val (II)

Orn (III )

Leu (IV)

Phe (V)

D

0 2 4 6 8 10

3 JHNH-HD

3

Val (II) Val (VIII)

Orn (III)

Orn (IX)

Leu (IV)Leu (X) Phe (V)

D Phe (XI)

D Tyr (VII)

D

0 2 4 6 8 10

3 JHNH-HD

6

Val (II) Val (VII)

Orn (III)

L Orn (VIII)

D Leu (IV

) Leu (IX

) Phe (V)

D DPhe (X)

0 2 4 6 8 10

3 JHNH-HD

8

Val (II) Val (VI)

Orn (III)

D Orn (VI

I)

D Leu (IV)

Leu (VII I)

Phe (V)

D Phe (IX)

D

0 2 4 6 8 10

3 JHNH-HD

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shift perturbations are depicted in Figure 3. These data support the findings from the ³JNH-Hα

values. ^DPhe, Pro and ^DTyr all have negative chemical shift perturbation, showing that peptide 3 contains two turns, a β- and an α-turn, interconnected by two β-strands. Peptide 6 adopts a

β-hairpin structure, despite the fact that 6 contains a D-amino acid in the strand, but valine seems to compensate for the steric hindrance caused by inversion of the stereochemistry of ornithine. Peptide 8 adopts mainly a random coil. Interestingly proline seems to be involved in a turn, while ^DPhe does not, suggesting that proline forms a γ-turn.

The CROESY spectra show crosspeaks that confirm the data presented above. Peptide 3 showed the same interstrand NOEs as peptide 1, indicating the antiparallel β-sheet. Also the NOE between H^α of ^DPhe-11 and H^δ of Pro-1 was observed, confirming a type II´ β-turn. In the other turn, interestingly, showed NOE a interaction between NH-Val-8 and the α-proton

Figure 3 Chemical shift perturbations of the α-protons in peptides 1, 3, 6 and 8.

1

Pro (I) Val (II)

Orn (III)

Leu (IV)

Phe (V)

D

0.0 0.5

Perturbation (ppm)

3

Pro (I) Pro (VI)Val (VIII

) Val (II)

Orn (IX)Orn (II I) Leu (IV)Leu (X)

Phe (XI)

D DPhe (V)Tyr (VII )

D

0.0 0.5

Perturbation (ppm)

6

Pro (I) Pro (VI)

Val (II) Val (VII)

Orn (III)

L Orn (VIII)

D Leu (IV) Leu (IX)

Phe (V)

D Phe (X)

D

0.0 0.5

Perturbation (ppm)

8

Pro ( I)

Val (II) Val (VI)

Orn (III)

D Orn (

VII)

D Leu (IV)

Leu (VI II)

Phe ( V)

D Phe (IX)

D

0.0 0.5

Perturbation (ppm)

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of ^DPhe, the H^α, H^β and NH of the tyrosine residue. In addition the NH of ^DTyr interacted with the H^δ of Pro-6 and the H^α of Phe-5. All this data indicate the formation of a sheet-turn structure. Peptide 6 shows, besides the sequential couplings, interstrand NOEs between the NHs of leucine and valine, suggesting that it forms an antiparallel β-sheet. The turn structures are confirmed by interactions between the α-proton of D-phenylalanine and H^δ of proline.

Finally, peptide 8 only showed sequential NOE interactions indicating that it does not form a β-sheet with hydrogen bonds between the strands. This is also supported by a NOE between the amides of ornithine-7 and leucine-8, showing that the amides of ornithine and leucine both directing inside the ring, or pointing outwards, which is impossible to determine without additional high resolution data.

Gratisin (2) did not yield spectra with clearly dispersed amide signals, indicating that this peptide did not adopt a singly well-defined structure in methanol, DMSO or at elevated temperatures.^[26,27] This peptides was therefore not analysed in further detail by NMR spectroscopy.

Nor peptide 7 yielded high resolution NMR data, suitable for structural analysis.

Fortunately though 7 yielded crystals suitable for Röntgen diffraction and the structure was solved (Figure 4). Compound 7 is an analogue of gramicidin S, in which the two ornithine

Figure 4 Crystal structure of 7 with side (left) and top view (right)

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residues have been replaced with D-ornithines. The two D-Phe-Pro sequences seem to retain mainly the β-turn comformation. The pleated sheet structure is completely lost, however and a twist is introduced, caused by a steric clash between the ornithine side chains and the surrounding aliphatic side chains. Curiously the four hydrogen bonds are retained, resulting in a horse saddle shape.

4.3BIOLOGICAL RESULTS

Table 1 presents the biological activity of the purified peptides 1-3 and 6-8, with respect to antibacterial activity. None of the presented peptides compares in activity to gramicidin S.

Interesting is the absence of activity of gratisin (2), which is in contrast to what has been reported in the literature^[14] and the cause of this discrepancy is not clear. The hybrid peptide 3 shows moderate activity. Another surprising aspect is that the GS peptide with one D- ornithine (6) shows no activity, while the one with two D-ornithines (7) regains some of its activity, which is comparable to the cyclic nonapeptide 8. Although 7 does not show a segregation between the ornithine and the hydrophobic side chains, it does adopt a amphiphilic structure with the ornithine side chains on one side of the molecule and the hydrophobic proline and D-phenylalanine side chains on the other. This would explain the activity of 7.

Strain 1 2 3 6 7 8

S.aureus7323 4 >64 16 >64 32 16

S.aureus7388 4 >64 4 >64 4 4

CNS5277 4 >64 16 >64 16 8

CNS5115 8 >64 4 >64 32 64

CNS7368 4 >64 16 >64 4 4

E.faecalis1131 16 >64 32 >64 32 >64

E.coliATCC25922 16 >64 64 >64 >64 >64

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

P.aeruginosaATCC19582 32 >64 >64 >64 >64 >64

S.mitisBMS 4 >64 32 >64 4 4

S.mitisATCC33399 16 >64 32 >64 >64 64

Table 1 MIC values for peptides 1-3 and 6-8 in μg/mL

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4.4CONCLUSIONS

Most of the peptides described in this thesis were synthesised by a solution phase cyclisation protocol, which works very well for peptides with a preference for the cyclic β- hairpin conformation. In the current Chapter an alternative approach was used, as not all peptides were expected to have a high cyclisation propensity. The on-resin cyclisation conditions presented in this Chapter take advantage of a condition known as pseudodilution, which prevents the formation of dimeric and other oligomeric species, instead of the desired cyclic product. In this manner cyclic octa-, nona-, deca-, undeca and dodecamers 1-3 and 5-8 were prepared and subsequently analysed by NMR spectroscopy. Peptide 5, however, proved to be insoluble, attributable to its disk shaped conformation, indirectly proving the expected structure. Two additional peptides (2 and 7) gave poorly dispersed ¹H NMR spectra, indicating their lack of preference for a single, specific structure in methanol. Peptide 3 gave NMR spectra that indicated a hybrid structure between gramicidin S and gratisin, with two β- strands and two different turns.

Peptide 6 also adopted a β-hairpin conformation with a slightly distorted strand due to the

D-ornithine it contains. The cyclic nonamer 8 did not adopt the β-strand/β-turn structure, but instead mainly a random coil-like conformation. The ^DPhe-Pro dipeptide did not adopt a β- turn, but the proline apparently adopted a γ-turn conformation.

Unfortunately, none of the peptides showed antibacterial activity that approached that of GS, but interesting are the absence of activity of gratisin (2) and epimeric peptide 6. Recently Tamaki and co-workers^[28] described the antimicrobial properties of several cyclic undecameric peptides derived from gramicidin S. These peptides displayed high antibacterial activity, comparable to GS, but the hemolytic activity of the new molecules was significantly diminished. Although the peptides presented in this Chapter did not display high activity against bacteria, these newly designed peptides are interesting starting point for the development of antimicrobial peptides.

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4.5EXPERIMENTAL SECTION

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). Peptides were synthesised on an Applied Biosystems 433A Peptide Synthesizer. 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 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 analyses of coupling constants, 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 1a-m 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). Incubation at 30 °C (24-96 h) and the MIC was determined as the lowest concentration inhibiting bacterial growth.

General Peptide Synthesis

Resin Loading Resin 12 was swollen in dry CH2Cl2 for 20 min and drained. Fmoc-L-Orn-OAll 11a or Fmoc-D- Orn-OAll 11b (4 eq) in DCM and DiPEA (5 eq) were added to the resin. After shaking overnight the resin was drained and washed with DCM/MeOH/DiPEA (80:10:10 v/v/v) thrice and DCM (3 ×) and dried in order to determine the loading by Fmoc analysis.

Peptide chain elongation The loaded resins 13a or 13b were subjected to repeated cycles of Fmoc deprotection and amino acid coupling in an automated fashion. Fmoc deprotections were done with 20% piperidine in NMP for (5 × 3 min, or until UV indicated complete deprotection) and subsequent washings with NMP. Fort the couplings 5 eq of the appropriate Fmoc-aa-OH, 5 eq HCTU and 10 eq DiPEA were added to the resin and agitated for 1 hour before draining and NMP wash steps. The N^δ of ornithine residues were protected with a Boc group and the phenol of tyrosine was protected as the tert-butyl ether.

Allyl Removal The resin 14 (100 μmol) was swollen in CH2Cl2 for 20 minutes, drained and, Pd(PPh3)4 (0.5 eq, 50 μmol, 58 mg) in degassed N-methylmorpholine/CHCl3 (1:9 v/v) were added. This mixture was shaken for 5 h, drained and washed with 1% p-toluenesulfinic acid sodium salt in DMF (3 × 2 min), 1% DiPEA (3 × 2 min) and CH2Cl2. The procedure was repeated in case of incomplete deprotection.

Fmoc deprotection The N-terminal Fmoc-group was removed by treatment with 20% piperidine in NMP (2 × 10 min). The resin was filtered and washed with NMP and DCM.

Cyclisation The resin 15 (100 μmol) was swollen in DMF for 20 min and filtered off. Subsequently the resin was washed with DMF (3 × 3 min), 10% DiPEA in DMF (3 × 3 min) and 0.8 M LiCl in DMF (3 × 3 min). The immobilised peptide was then treated with PyAOP (5 eq, 500 μmol, 261 mg), HOAt (5 eq, 500 μmol, 68 mg) and DiPEA (20 eq, 2mmol, 345 μL) in 0.8 M LiCl in DMF and shaken overnight. The resin was washed with DMF and CH2Cl2. The procedure was repeated in case the cyclisation was not complete.

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Cleavage from resin The peptides were cleaved from the resin by treatment with 1% TFA/CH2Cl2 (6 × 5 mL × 10 min). The fractions were collected in toluene (50 mL) and concentrated in vacuo and halfway another 50 mL ol toluene was added to remove traces of TFA.

Boc deprotection The remaining protecting group was removed by treatment of the peptide with 50% TFA in CH2Cl2 (2 mL) for 2 h. The resulting solution was concentrated and coevaporated with toluene (3 × 50 mL)

  NH H^ H^ H^ H^ H^

1 Pro(I)  4.34 1.99/1.68 1.69/1.57 3.73/2.46 

 Val(II) 7.70(8.80) 4.15 2.26 0.95/0.87 

 Orn(III) 8.70(9.10) 4.98 2.04/1.61 1.76 3.04/2.87 

 Leu(IV) 8.74(9.39) 4.66 1.53/1.40 1.50 0.89 

 ^DPhe(V) 8.94(2.35) 4.50 3.09/2.94 7.337.24 

3 Pro(I)  4.36 1.99/1.69 1.70/1.57 3.71/2.45 

 Pro(VI)  4.31 1.88/1.44 1.58/1.52 3.51/2.61 

 Val(II) 7.71(8.68) 4.09 2.27 0.96/0.88 

 Val(VIII) 8.28(8.07) 4.03 2.11 0.94 

 Orn(III) 8.36(9.05) 4.83 1.49 1.35 2.42/2.37 

 Orn(IX) 8.70(8.56) 4.96 2.05/1.71 1.79 3.03/2.98 7.92

 Leu(IV) 8.98(9.05) 4.82 1.59 1.56 0.90 

 Leu(X) 8.53(9.54) 4.64 1.60/1.39 1.54 0.89 

 ^DTyr(VII) 8.03(8.56) 4.48 2.86/2.58 7.05/6.73 6.70(OH)

 ^DPhe(V) 8.73(s) 4.50 3.12/3.05 

 ^DPhe(XI) 8.88(3.30) 4.52 3.08/2.94 7.30/7.23 

6 Pro(I)  4.33 1.97/1.71 1.57 3.67/2.47 

 Pro(VI)  4.37 2.01/1.62 1.72 3.67/2.46 

 Val(II) 7.72(9.67) 4.03 2.35 0.93 

 Val(VII) 7.48(9.39) 4.39 2.25 0.97 

 Orn(III) 7.74(9.70) 5.11 1.87/1.57 1.57 2.98 

 ^DOrn(VIII) 8.38(8.51) 5.28 1.81/1.74 1.71 3.10/2.89 7.74

 Leu(IV) 8.48(9.39) 4.59 1.63/1.39 1.54 0.94 

 Leu(IX) 8.31(7.63) 4.56 1.66/1.51 1.54 0.95 

 ^DPhe(V) 9.01(3.23) 4.49 3.07/2.95 

 ^DPhe(X) 9.09(s) 4.44 3.09/2.91 7.337.24 

8 Pro(I)  4.28 2.02/1.90 1.87/1.71 3.74/3.10 

 Val(II) 7.88(5.86) 3.94 2.16 0.95 

 Val(VI) 8.09(7.34) 4.03 2.08 0.89 

 ^DOrn(III) 7.61(8.44) 4.43 1.88 1.70 2.96 

 ^DOrn(VII) 8.29(8.44) 4.40 1.94 1.69/1.73 2.94/3.07 7.85

 Leu(IV) 7.82(6.52) 4.45 1.54 1.35 0.85 

 Leu(VIII) 8.07(7.75) 4.29 1.44 1.37 0.82 

 ^DPhe(V) 8.35(7.14) 4.83 3.06/2.95 7.297.19 

 ^DPhe(IX) 7.89() 4.68 3.13/2.92 

Table 2 NMR Data for peptides 1, 3, 6 and 8. In brackets the ³JNH-Hαare given in Hz

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Peptide 1

13C NMR (150 MHz, CD3OH) δ 173.55, 173.52, 173.42, 172.74, 172.39, 136.83, 130.35, 129.65, 128.46, 61.95, 60.36, 55.92, 52.42, 51.42, 47.89, 41.95, 40.55, 37.25, 31.94, 30.74, 30.60, 25.61, 24.59, 24.40, 23.16, 22.99, 19.60, 19.41; LC/MS: Rt = 7.47 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1142.2 [M + H]⁺; HRMS: calculated for [C60H93N12O10]⁺: m/z 1141.71321; found: m/z 1141.71429.

Peptide 2

LC/MS: Rt = 6.41 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1469.1 [M + H]⁺; HRMS: calculated for [C78H111N14O14]⁺: m/z 1467.83987; found: m/z 1467.841333.

Peptide 3

13C NMR (150 MHz, CD3OH) δ 175.30, 174.67, 173.65, 173.61, 173.56, 173.49, 173.08, 173.04, 172.89, 172.85, 172.63, 157.76, 136.89, 136.60, 131.43, 130.59, 130.35, 129.64, 129.58, 128.44, 128.17, 116.43, 116.28, 61.84, 61.17,

1 NHVal(II)  H^^DPhe(V)(w) H^Pro(I)(m) H^Pro(I)(s)

 NHOrn(III)  H^Val(II)(s) H^Val (II) (m) 

 NHLeu(IV)  H^Orn(III) (m) 

 NH^DPhe(V)  H^Leu(IV)(s) 

 H^^DPhe(V)  H^Pro(I)(s) 

3 NHVal(II)  NHLeu(X)(w) H^Pro (I) (w) 

 NHOrn(III)  H^Val(II)(s) H^Val (II) (w) 

 NHLeu(IV)  NHVal(VIII) (w) H^Orn (III) (s) H^{ D}Tyr (VII) (w)

 NH^DPhe(V)  H^Leu(IV)(m) 

 NH^DTyr(VII)  H^Pro(VI)(s) H^Pro (VI) (s) H^{ D}Phe (V)

 NHVal(VIII)  NH^DTyr(VII) (m) H^{ D}Phe (V) (m) H^{ D}Tyr (VII) (m) H^^DTyr(VII)(w)

 NHOrn(IX)  H^Val(VIII) (s) H^Val (VIII) (s) 

 NHLeu(X)  H^Orn(IX) (s) 

 NH^DPhe(XI)  H^Leu(X)(s) 

6 NHVal(II)  H^Pro(I) H^Pro (I) 

 NHOrn(III)  H^Val(II)(s) 

 NHLeu(IV)  NHVal(VII) (w) H^Orn (III) (s) 

 NH^DPhe(V)  H^Leu(IV)(s) 

 NHVal(VII)  H^Pro(VI) H^Pro (VI) 

 NHDorn(VIII)  HVal(VII) (s) H^Val (VII) (w) 

 NHLeu(IX)  NHVal(II)(w) H^{ D}Orn (VIII) (s) 

 NH^DPhe(X) H^Leu(IX)(s) 

 H^^DPhe(X)  H^Pro(I) 

 H^^DPhe(V)  H^Pro(VI) 

8 NH^DOrn(III)  H^Val(II)(w) 

 NHLeu(IV)  H^Dorn(III) (w) 

 NH^DPhe(V)  H^Leu(IV)(s) 

 NHVal(VI)  H^^DPhe(V) (m) 

 NH^DOrn(VII)  H^Val(VI)(s) 

 NHLeu(VIII)  NH^DOrn(VII) (w) H^{ D}Orn (VII) (s) 

 NH^DPhe(IX)  H^Pro(I)(s) 

Table 3 NOE interactions for peptides 1, 3, 6 and 8 (600 MHz, CD3OH, mixing time: 200 ms).

[78]

61.07, 60.67, 57.89, 55.94, 55.52, 53.21, 52.98, 52.13, 51.62, 48.01, 47.70, 43.88, 41.76, 40.60, 40.50, 40.32, 40.14, 37.68, 37.22, 31.89, 31.19, 30.59, 30.32, 28.63, 25.78, 25.52, 24.69, 24.59, 24.35, 24.10, 23.11, 22.90, 22.32, 19.84, 19.54, 19.46; LC/MS: Rt = 6.90 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1305.2 [M + H]⁺; HRMS:

calculated for [C69H102N13O12]⁺: m/z 1304.77654; found: m/z 1304.77773.

Peptide 6

13C NMR (150 MHz, CD3OH) δ 174.04, 173.87, 173.86, 173.55, 173.36, 173.34, 173.26, 172.80, 172.54, 171.81, 136.99, 136.54, 130.37, 129.64, 129.61, 128.53, 128.43, 61.86, 61.77, 60.48, 59.69, 55.79, 55.67, 52.77, 52.74, 52.56, 52.19, 47.99, 47.51, 43.93, 42.03, 40.86, 37.44, 37.24, 32.55, 32.47, 32.38, 30.57, 30.43, 28.86, 26.08, 26.02, 24.65, 24.58, 24.33, 24.03, 23.60, 23.20, 22.56, 22.38, 20.04, 19.84, 19.05, 18.71; LC/MS: Rt = 5.72 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1142.2 [M + H]⁺; HRMS: calculated for [C60H93N12O10]⁺: m/z 1141.71321; found: m/z 1141.713999.

Peptide 7

13C NMR (150 MHz, CD3OH) δ 174.31, 173.46, 173.43, 173.07, 171.89, 137.12, 130.47, 129.61, 128.35, 101.10, 62.49, 60.80, 55.08, 54.70, 52.69, 52.39, 48.17, 43.99, 40.61, 40.51, 30.57, 30.55, 30.38, 26.19, 25.10, 24.58, 24.53, 24.10, 23.83, 22.64, 20.06, 19.29; LC/MS: Rt = 5.34 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1142.2 [M + H]⁺; HRMS: calculated for [C60H93N12O10]⁺: m/z 1141.71321; found: m/z 1141.71454.

Peptide 8

LC/MS: Rt = 6.48 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1044.60 [M + H]⁺; HRMS calculated for [C55H85N11O9]⁺: m/z 1044.66045; found: m/z 1044.66150.

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