Synthetic modifications of the antibiotic peptide gramicidin S : conformational and biological aspects Knijnenburg, A.D.

Synthetic modifications of the antibiotic peptide gramicidin S : conformational and biological aspects

Knijnenburg, A.D.

Citation

Knijnenburg, A. D. (2011, September 29). Synthetic modifications of the antibiotic peptide gramicidin S : conformational and biological aspects.

Retrieved from https://hdl.handle.net/1887/17882

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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/17882

Note: To cite this publication please use the final published version (if

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* Knijnenburg, A. D.; Spalburg, E.; de Neeling, A. J.; Mars-Groenendijk, R. H.; Noort, D.; Grotenbreg, G.

M.; van der Marel, G. A.; Overkleeft, H. S.; Overhand, M., ChemMedChem 2009, 4, 1976.

Introduction

The decapeptide gramicidin S (GS, cyclo -(Pro-Val-Orn-Leu-

Phe)

, Figure 1) is a prominent member of the Cationic Antimicrobial Peptide (CAP) family.

GS kills several Gram-positive bacteria by disrupting the bacterial lipid bilayer, but is less effective against Gram-negative strains. In combination with the antibiotic agent framycetin, which targets Gram-negative bacteria, GS has proven to be an effective topical antibiotic to treat external ear infections.

GS cannot be used to treat systemic infections because of its toxicity towards human erythrocytes (hemolysis).

The membrane-disrupting ability of GS is attributed

to its amphipathic characteristics caused by the opposite position of the hydrophilic side chains of the Orn residues with respect to the hydrophobic side chains of the Leu and Val residues. The β-strand sequences (Val-Orn-Leu) adopt an antiparallel β-sheet. The resulting rigid cyclic β-hairpin structure is stabilized by four intramolecular hydrogen bonding interactions and the presence of two

-amino acids in the two type II’ β-turns.

Both the β-strand and the β-turn regions of GS have been

Ring-extended derivatives of gramicidin S with furanoid

sugar amino acids in the turn region ^*

modified by amino acid substitution as a strategy to obtain structurally related molecules with an improved biological profile.

β-strand modification of derivatives of GS has been achieved by extension of the macrocyclic ring via the incorporation of four additional amino acid residues.

Some of these ring- extended derivatives have an improved biological profile.

The tetradecameric GS14

(Figure 2), one of the previously described ring-extended derivatives containing

-Tyr and Lys rather than

-Phe and Orn,

adopts an amphiphilic cyclic β-hairpin structure with extended β-strand regions. Also several GS derivatives with a modified β-turn region were reported.

It was found that GS derivative 2 (Figure 1), containing a furanoid sugar amino acid (SAA) dipeptide isoster 1 (R

= R

= OH), adopts a twisted cyclic β-hairpin structure, resembling the secondary structure of GS with three of the four intra-molecular hydrogen bonding interactions intact. The presence of the conformationally restricted SAA causes one of the amides of the turn region to flip outwards (as indicated in Figure 1). GS analog 2 is largely biologically inactive. Substitution of one of the hydroxyl functionalities of the furanoid core with a benzyloxy group (3) restores the biological activity to a certain extent, without changing the overall twisted cyclic β-hairpin secondary structure.

In this chapter the synthesis and conformational evaluation is presented of a series of extended GS derivatives containing furanoid SAAs in the turn region (5- 7, Figure 2).

To investigate the influence of hydrophobic characteristics of the SAA moieties, hydrophobic benzyl groups were introduced at both the 2' and 3' hydroxyl functionalities of the sugar core. The antimicrobial and hemolytic activity of compounds 5-7 were compared with those of 4, having

-Phe and Orn residues rather than

-Tyr and Lys, as well as GS14

and GS.

Figure 1: The primary structures of gramicidin S (GS) and of GS analogs 2 and 3 modified

by the introduction of furanoid sugar amino acid SAA 1.

55

Results and discussion

All compounds were synthesized employing a slight modification of a standard step-wise solid phase protocol (see Experimental section) using the highly acid- sensitive HMPB-BHA resin.

After mild acidic cleavage from the solid support, the partially protected linear peptides containing four Orn(Boc) residues were cyclized under highly dilute conditions. The cyclized peptides were purified using gel filtration, followed by Boc deprotection with strong acid. The peptides were purified with preparative HPLC and lyophilized resulting in the TFA salts of GS14

and compounds 4-7 in a overall yield of 10-40 %.

The solution structures of GS14 and 4-7 were evaluated by NMR and circular dichroism (CD) (Figure 3 A-C). The general structural characteristics of peptides 5-7 were established with NMR and resemble those of GS14 and 4. The

J

coupling constants of the Orn, Leu and Val residues are all between 7 and 9 Hz (Figure 3A), which is a strong indication that they are part of a β-strand (extended) conformation. The

J

values of the

-Phe residues were in the range of 2-4 Hz, which is typical for an amino acid as part of a β-turn.

NH NH

HN NH

HN

HN NH

HN NH O

N

O

O

O O O

O

NH O

O O

O H

N O

O N HN

O

GS14^[12], R = OH, n = 2 4, R = H, n = 1

R

R

NH₃⁺

n NH₃⁺

+H₃N ⁺H₃N

NH NH

HN NH

HN

HN NH

HN NH O

N

O

O

O O O

O

NH NH₃⁺

+H₃N

O

O +H₃N

NH₃⁺ O

O H

N

O R₁ R₂ O

O HN

5, R1=OH, R2=OH 6, R₁=OH, R₂=OBn 7, R₁=OBn, R₂=OBn

n

n n

Figure 2: Ring-extended gramicidin S analogs GS14 and 4 and β-turn modified analogs 5-7.

The chemical shift perturbations (∆ δ H

, Figure 3B) of oligopeptides give an indication of the structural environment of a particular residue.

The Val-Orn- Leu residues all show chemical shift perturbations higher than 0.1 ppm (Figure 3B), indicating that these residues are part of an extended β-strand conformation.

The negative values of the Pro and

-Phe residues imply the presence of a turn motif. Based on related GS derivatives

it is justified to conclude that the peptides adopt cyclic β-hairpin-like conformations.

However, it is obvious, both from the significantly higher coupling constants and the larger chemical shift perturbations, that compounds GS14 and 4 adopt a more stable cyclic β-hairpin than the SAA modified derivatives 5-7. This observation is corroborated by the CD spectra (Figure 3C). CD indicates that the tetradecameric peptides (GS14 and 4) have the typical GS-like excitation spectrum with two negative minima around 205 and 222 nm. Analogs 5-7 have a weaker minimum around 220 nm, because Figure 3 A-C: Secondary structure analysis of GS analogs GS14 and 4-7: [A]

J

in CD

OH at 298 K; [B] ∆δH

(= δH

- δH

random coil) in CD

OH at 298 K; [C] CD in MeOH at 298 K.

200 220 240

-250 -200 -150 -100 -50 0 50

C

GS14 4 5 6 7

 (nm)

52-1 [] x 10(deg cmdmol)

GS14 4 5 6 7

Val 1 Orn

2 Leu

3 Orn 4Val 5

Phe 6

D Pro

7 Leu

8 Orn

9 Val

10 Orn 11

Leu 12 phe 13

D Pro 14 -0.2

0.0 0.2 0.4 0.6 0.8

B

Residue

H

 in ppm

Val1 Orn2

Leu3 Orn

4Val5 Phe

6

D Leu

8 Orn

9 Val1

0 Orn11

Leu12 Phe13

D

0 2 4 6 8 10

A

Residue

3JHN (Hz)

57

the cyclic β-hairpin structure is less stable due to the presence of the SAA moieties.

GS14 and compounds 4-7 were screened against a small series of Gram- positive and Gram-negative bacteria and their hemolytic properties were determined (Table 1, Figure 4). As a control GS was included in the assays.

Compound 4 is not only more bactericidal, but also less hemolytic than compound GS14

. Interestingly, analogs 5-7 are more antibacterially active and less hemolytic than compound 4. The most important finding is that all SAA-modified analogs (5-7) exhibit more activity against the two Gram-negative bacterial strains included in our assay. Analog 7 even shows a slightly better antimicrobial activity than GS.

Table 1. Cytotoxic (Hemolytic) and Antimicrobial Activity (MIC) of GS, GS14 and GS analogs 2-7

Analog Erythrocytesa S. aureusb S. epidermidisb E. faecalisb B. cereusb P. aeruginosac E. colic

GS 19.0 4 4 8 4 64 32

GS14 0.4 >64 64 32 32 >64 >64

2

> 300 64 16-32 >64 16-32 >64 >64

3

≈25 2 2 16 4 64 >64

4 1.7 16 8 8 16 64 32

5 10.9 8 4 8 4 16 8

6 6.1 4 4 8 4 16 16

7 4.7 4 4 8 2 16 16

[a]

Hemolytic activity HC

(the concentration at 50% lysis of the erythrocytes) in μM;

[b]

Gram-positive bacteria, MIC in mg/L;

Gram-negative bacteria, MIC in mg/L Molecular weight of analogs: GS: 1369,49; GS14: 2126.23; 2: 1284.34; 3:1374.47; 4:

2038.12; 5: 1952.97; 6: 2043.09; 7: 2133.22.

0 1 2 3

0 20 40 60 80 100

4 GS GS14

7 5 6

log [c] inM

% Hemolysis

Figure 4: Hemolytic activity of GS, GS14 and GS analogs 4-7.

Conclusion

In conclusion, the substitution of two amino acids (

-Phe-Pro) in one of the turn regions in compound 4 (a ring-extended derivative of GS) by a series of hydrophilic and hydrophobic SAAs dipeptide isosteres induces a slight distortion of the cyclic β-hairpin secondary structure. Although peptides 5-7 have a less well-defined secondary structure than template 4, compounds 6, 7 and especially 5 have an improved biological profile, namely, increased antibacterial activity and reduced hemolytic activity. The results presented in this chapter indicate that a decreased hydrophobicity in the β-turn and slight distortion of the cyclic β- hairpin structure is of importance for an improved therapeutic profile of ring- extended derivatives of GS.

Experimental section

General:

Light petroleum ether with a boiling range of 40-60 ºC was used. All other solvents used under anhydrous conditions were stored over 4Å molecular sieves except for methanol which was stored over 3Å molecular sieves. Solvents used for work-up and silica gel column chromatography were of technical grade and distilled before use. All other solvents were used without further purification.

Reactions were monitored by TLC-analysis. Peptides were synthesized on an Applied Biosystems 433A Peptide Synthesizer. The linear peptides were cleaved from resin, cyclized and purified by RP- HPLC (Gilson GX-281) with a preparative Gemini C18 column (Phenomenex 21.2 mmϕ x 150 mmL, 5μm particle size). The applied eluents were A: 0.1 % aq. TFA, B: MeCN. The linear peptides and cyclized peptides were analyzed with LC/MS (detection simultaneously at 214 and 254 nm) equipped with an analytical C18 column (4.6 mmϕ x 250 mmL, 5μm particle size). The applied buffers were A:

H2O, B: MeCN and C: 1.0 % aq. TFA. High resolution mass spectra were recorded by direct injection (2 μL of a 2 μM solution in H2O/MeCN; 50/50: v/v and 0.1% formic acid) on a mass spectrometer Thermo Finnigan LTQ Orbitrap equipped with an electrospray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 523 K) with resolution R = 60000 at m/z 400 (mass range m/z = 150-2000 ) and dioctylphthalate (m/z = 391.28428) as lock mass. Optical rotations were measured on a Propol automatic polarimeter (sodium D-line, λ = 589 nm). Specific rotations [α]D are given in degrees per centimeter and the concentration c is given in mg/ml in the specific solvent. IR spectra were recorded on a Perkin Elmer Paragon 1000 FT-IR Spectrometer. CD and hemolytic curves were analyzed with Graphpad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA. ¹H-, ¹³C-APT, Standard DQF-COSY (512c x 2084c) and TOCSY (400c x 2048c) NMR spectra were recorded on a Bruker DMX 600 equipped with a pulsed field gradient accessory and a cryo-probe.

Antibacterial assays:

The following bacterial strains were used: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853).

Bacteria were stored at –70 ºC and grown at 30 ºC on Columbia Agar with sheep blood (Oxoid, Wesel, Germany) suspended in physiological saline solution until an optical density of 0.1 AU (at 595 nm, 1 cm cuvette). The suspension was diluted (10 x) with physiological saline solution, and 2

59 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, GS14 and 4-7 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 ºC (24-96 h) the MIC was determined as the lowest concentration inhibiting bacterial growth.

Hemolytic assays:

Freshly drawn heparinized blood was centrifuged for 10 minutes at 1000g at 10 ºC. Subsequently, the erythrocyte pellet was washed three times with 0.85% saline solution and diluted with saline solution to a 1/25 packed volume of red blood cells. The peptides to be evaluated were dissolved in a 30% DMSO/0.5 mM saline solution to give a 1.5 mM solution of peptide. If a suspension was formed, the suspension was sonicated for a few seconds. A 1% Triton-X solution was prepared. Subsequently, 100 μl of saline solution was dispensed in columns 1-11 of a microtiter plate, and 100 μl of 1% Triton solution was dispensed in column 12. To wells A1-C1, 100 μl of the peptide was added and mixed properly. 100 μl of wells A1-C1 was dispensed into wells A2-C2. This process was repeated until wells A10-C10, followed by discarding 100 μl of wells A10-C10. These steps were repeated for the other peptides. The red blood cell solution (50 μl) was added to the wells and the plates were incubated at 37 ºC for 4 hours. After incubation, the plates were centrifuged at 1000g at 10 ºC for 4 min. In a new microtiter plate, 50 μl of the supernatant of each well was dispensed into a corresponding well. The absorbance at 405 nm was measured and the percentage of hemolysis was determined.

General synthetic procedure (Figure 5):

(a) Stepwise elongation: Resin (HMPB-BHA, 100-200 mesh, Novabiochem) preloaded with Fmoc- Leu (2.8 g, 0.53 mmol/g, 1.5 mmol) was subjected to four cycles of Fmoc solid-phase synthesis using commercially available building blocks in the order: Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc- Orn(Boc)-OH, Fmoc-Leu-OH. (a) deprotection with 20% piperidine in NMP (3x 50 mL, 15 min); (b) washing with NMP (2 x 50 ml) and DCM (2 x 50 mL); (c) pre-activation of the following building block (3 eq) in 50 mL NMP, 0.2 M HBTU in NMP and DiPEA (6 eq) and subsequent coupling with the resin and shaken for 3h; (d) washing with NMP (2 x 50 mL) and DCM (2 x 50 mL). Couplings were monitored for completion by the Kaiser test and LC/MS after mini-cleavage (1 mg in TFA, filtrate dissolved in 1:1:1 H2O/MeCN/t-BuOH). (e) The resin was capped 10 min. with 0.45 M Ac2O in NMP (5 eq) and DiPEA (1 eq); (f) The resin was washed with MeOH (2 x 50 mL), NMP (2 x 50 mL), DCM (2 x 50 mL). Dry resin 10: 3.73 g, 0.4 mmol/g. LC/MS: Rt5.61min, linear gradient 10→90% B in 13.5 min.; m/z = 794.7 [M+H]⁺.

(b) Incorporation of SAAs 8 and 9: SAA 8 (1.75 eq., 0.7 mmol) was coupled to the dried resin 10 (995 mg, 0.4 mmol); SAA 9 (1.5 eq, 0.3 mmol) was coupled to the resin 10 (498 mg, 0.2 mmol): (a) The resin was washed with MeOH (2 x 50 mL), NMP (2 x 50 mL), DCM (2 x 50 mL); (b) deprotection with 20% piperidine in NMP (3 x 10 mL); (c) pre-activation of 8 and 9 with HBTU (3 eq, 0.2 M HBTU in NMP), DiPEA (6 eq) in 10 mL NMP and subsequent coupling with the resin and shaken for 4 h; (d) washing with NMP (2 x 50 mL) and DCM (2 x 50 mL); LC/MS 11: Rt 5.69 min, linear gradient 10→90% B in 13.5 min.; m/z = 847.0 [M+H]⁺. LC/MS 12: Rt 7.05 min, linear gradient 10→90% B in 13.5 min.; m/z = 937.4 [M+H]⁺.

(c) Azide reduction: resin 11 and 12 were washed with 1,4-dioxane (3 x 10 mL), and taken up in 1,4- dioxane (10 mL) to which trimethylphosphine (16 eq, 1M in THF) pre-mixed with H2O (0.6 eq) was added. The resin was shaken for 24 hours and complete reduction was determined by LC/MS.

(d) Automated SPPS elongation: resin 11 (0.2 mmol) and resin 12 (0.1 mmol) were subjected to 7 cycles of SPPS with the use of commercially available building blocks in the following order: Fmoc- Val-OH, Fmoc-Orn(Boc)-OH, Fmoc-Leu-OH, Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-D-Phe-

OH, Fmoc-Pro-OH and subsequent Fmoc deprotection. LC/MS 13: Rt 4.97 min, linear gradient 10→90% B in 13.5 min.; m/z = 1605.9 [M+H]⁺. LC/MS 14: Rt 5.05 min, linear gradient 10→90% B in 13.5 min.; m/z = 1695.3 [M+H]⁺.

(e) Cleavage from resin: The peptides were released from the resin by mild acidic cleavage (4 times 10 min, 10 mL 1% TFA in DCM). The fractions were collected and coevaporated with toluene (3 times 50mL) to give the crude linear peptide which was immediately cyclized without further purification.

Cyclization: The crude partially protected peptide in DMF (20 mL) was dropwise added over 16h to a solution of HOBt (5 eq), pyBOP (5 eq) and DiPEA (15 eq) in DMF (160 mL). The solvent was removed under diminished pressure and the residue applied to a Sephadex® size exclusion column (50.0 mmϕ x 1500 mmL) and eluted with MeOH. The protected peptides (15 and 17) were LC/MS and HRMS analyzed.

(f) Hydrogenation: Protected peptide 15 (121 mg, 60.9 μmol) was hydrogenated with 10%

Pd(OH)2/C (40 mg) in MeOH (30 mL). The mixture was filtered over Celite® yielding peptide 16 (96 mg, 50.6 μmol); LC/MS Rt 5.52 min, linear gradient 10→90% B in 13.5 min.; m/z = 1497.1 [M+H]⁺. Deprotection: The Boc-protection groups of peptides 15-17 were removed by addition of TFA/TIS/H2O mixture (10 mL, 95/2.5/2.5) and subsequently the peptide was purified by preparative RP-HPLC. The yield of peptides 5-7 after HPLC is based on purified protected state.

Fmoc-L ii Fmoc-L-O-V-O-L

iii

iv, v

N₃-SAAx-L-O-V-O-L 11, x= 8 12, x= 9 V-O-L-O-V-SAAx-L-O-V-O-L

13, x = 8 14, x = 9

vi,vii

15, R₁= OH, R₂= OBn, R₃= Boc 16, R₁= OH, R₂= OH, R₃= Boc 17, R₁= OBn, R₂= OBn, R₃= Boc

NH NH

HN NH

HN

HN NH

HN NH O

N

O

O

O O O

O

NH NHR₃

R₃HN

O

O R₃HN

NHR₃ O

O H

N

O OR₁ OR₂

viii ix

10

O

O HN O

BnO N₃

OH OH O

i

O

BnO N₃

OBn OH O

8 9

5, R₁= OH, R₂= OH, R₃= H^.TFA 6, R₁= OH, R₂= OBn, R₃= H^.TFA 7, R₁= OBn, R₂= OBn, R₃= H^.TFA

Figure 5: Reagents and conditions: (i) NaH, BnBr, DMF, 0ºC, 5h, 71% r.o.s.; (ii) SPPS; (iii)

SAAx (1.5 eq), HBTU, DiPEA, DMF, 16h;(iv) PMe

(1M in 9:1 THF/H

O, 25 eq), 16h (v)

61

2,5-Anhydro-6-azido-4-O-Benzyl-6-deoxy-

-gluconic acid (8)

Rf: 0.1 (1:1 EtOAc/PE, 1% AcOH); HRMS (ESI) m/z 316.09044 [M+Na]⁺, calcd.

316.09039 for C13H15N3O5;[α]D20

+ 247.0 (c = 1, in CHCl3); IR 3427 (s), 2923 (m), 2362 (s), 2338 (s), 2103 (s), 1737 (s), 1495 (m), 1454 (s), 1211 (s), 1092 (s); ¹H NMR (400 MHz, CDCl3) δ 7.41 – 7.27 (m, 5H, CH Benzyl), 6.29 (br. s, 1H, OH), 4.68 (d, J = 3.9, 1H, H1), 4.60 (dd, J = 11.8, 31.6, 2H, CH2 Benzyl), 4.56 – 4.52 (m, 1H, H2), 4.14 (td, J = 2.7, 4.9, 1H, H4), 3.88 (d, J = 1.6, 1H, H3), 3.60 – 3.53 (m, 2H, H5, H5^’);¹³C NMR (101 MHz, CDCl3) δ 172.1 (C=O), 136.9 (C^q Benzyl), 128.6-127.8 (CH Benzyl), 85.5 (C3), 83.1 (C4), 81.5 (C1), 75.9 (C2), 72.1 (CH2 Benzyl), 52.2 (C5).

2,5-Anhydro-6-azido-3,4-O-di-Benzyl-6-deoxy-

-gluconic acid (9)

Compound 8^[17] (427 mg, 1.46 mmol) was coevaporated with toluene and dissolved in DMF (20 mL). The solution was cooled (0 ºC), sodium hydride as a 60% suspension in mineral oil (122 mg, 3.05 mmol) was added, followed by addition of Benzylbromide (190 μL, 1.6 mmol). Stirring was continued and the reaction mixture was allowed to warm to room temperature during 5 h. The reaction mixture was quenched with H2O (10 mL), diluted with EtOAc (50 mL) and treated with 1M HCl solution (25 mL). The organic layer was dried (Na2SO4), concentrated under reduced pressure and the residue purified using silica gel column chromatography using 1:8 EtOAc/PE containing 1% AcOH as the eluent. Compound 9 was obtained as a colorless oil (85 mg, 0.22 mmol, 71% yield based on recovered starting material). HRMS (ESI) m/z 406.13715 [M+Na]⁺ (calcd. 406.13734 for C20H21N3O5). [α]D20

+49.4 (c = 1, in CHCl3). IR: 2928 (m), 2363 (s), 2338 (s), 2103 (s), 1737 (s), 1496 (m), 1455 (s), 1210 (s), 1094 (s). ¹H NMR (400 MHz, CDCl3) δ 8.33 (br. s, 1H, OH), 7.42 – 7.16 (m, 10H, CH Benzyl), 4.78 (d, J = 4.6, 1H, H1), 4.54 (dd, J = 11.9 29.0, 2H, CH2 Benzyl), 4.44 (dd, J = 11.8, 20.0, 2H, CH2 Benzyl), 4.29 (dd, J = 1.4, 4.6, 1H, H2), 4.18 (td, J = 2.5, 6.4, 1H, H4), 3.88 – 3.87 (m, 1H, H3), 3.48 (ddd, J = 6.4, 12.7, 66.1, 2H, H5, H5^’); ¹³C NMR (101 MHz, CDCl3) δ 172.1 (C=O), 136.9, 136.7 (2xC^q Benzyl), 128.6-127.7 (CH Benzyl), 83.6 (C4), 82.8 (C3), 82.6 (C2), 80.6 (C1), 72.8, 71.9 (2xCH2 Benzyl), 52.09 (C5).

cyclo-[Leu-Lys-Val- Lys -Leu-

Tyr-Pro-Val- Lys-Leu-Lys-Val-

Tyr-Pro]

4TFA (GS14)

cyclized; washed; deprotected; purified twice by semi-preparative RP-HPLC (linear gradient of 30- 39%, 3 CV) and lyophilization of the combined fractions furnished the peptide (11.5 mg, 5.4 μmol, 5.4%); LC/MS Rt 7.26 min, linear gradient 10→90%

B in 13.5 min.; m/z = 1670.1 [M+H]⁺; HRMS (ESI) m/z 1670.07529 [M+H]⁺, calcd. 1670.07675 for C85H140N18O16; IR 3278 (m), 2962 (s), 1668 (s), 1634 (s), 1539 (m), 1456 (m), 1201 (s), 1134 (s); ¹H NMR (600 MHz, CD3OH) δ 8.84 (d, J = 3.8, NH^DTyr14), 8.83 (d, J = 8.3, 1H, NH Leu11), 8.78 (d, J = 3.2, 1H, NH^DTyr7), 8.77 (d, J = 9.6, NH Val4), 8.76 (d, J = 9.4, 1H, NH Leu6), 8.71 (d, J = 9.3, 1H, NH Val13), 8.56 (d, J = 8.5, 1H, NH Lys5), 8.52 (d, J = 9.4, 1H, NH Lys12), 8.37 (d, J = 9.4, 1H, NH Lys3), 8.34 (d, J = 9.4, 1H, NH Lys10), 7.88 (d, J = 8.5, 1H, NH Leu2), 7.72 (d, J = 8.8, 1H, NH Val9), 7.03 (d, J

= 7.3, 4H, H2,6^DTyr), 6.71 (d, J = 7.9, 4H, H3,5^DTyr), 5.09-5.02 (m, 4H, Hα Lys), 4.67 – 4.64 (m, 1H, Hα

Leu11), 4.62 – 4.58 (m, 1H, Hα Leu6), 4.51 – 4.33 (m, 6H, Hα Pro, Leu2, ^DTyr, Val4), 4.16 (t, J = 10.2, Hα

Val13), 4.14 (t, J = 9.1, Hα Val9), 3.72 – 3.69 (m, 1H, Hδd Pro8), 3.66 – 3.64 (m, 1H, Hδd Pro1), 3.01 – 2.98 (m, 4H, H^εd,u Lys5,12), 2.96 – 2.90 (m, 2H, Hβd^DTyr), 2.89 – 2.85 (m, 4H, H^εd,u Lys3,10), 2.83 – 2.79 (m, 2H, Hβu^DTyr), 2.52 (dd, J = 8.6, 17.8, 1H, Hδu Pro8), 2.46 (dd, J = 8.9, 17.8, 1H, Hδu Pro1), 2.26 (dd,

O BnO N₃

OBn OH O

NH NH

HN NH

HN

HN N H

HN N H O

N O

O O O O

O

NH O

O O

O H N O O

N HN

O OH

HO

NH3+ NH3+

NH3+ NH3+

O BnO N₃

OH OH O

J = 6.8, 14.2, 1H, Hβ Val9), 1.99-1.87 (m, 8H, Hβ Val4,13, Hβd Pro, Hβd Lys), 1.79 – 1.25 (m, 32H, Hβd,u,γd

Leu, Hβu,γd,uδd,u Lys, Hβu,γd,u Pro), 1.01 – 0.68 (m, 36H, CH3 Val, CH3 Leu).

cyclo-[Leu-Orn-Val-Orn-Leu-

Phe-Pro-Val-Orn-Leu-Orn-Val-

Phe-Pro]

4TFA (4)

HMPB-BHA-Fmoc-Orn(Boc) preloaded resin was subjected to 13 cycles of SPPS; cleaved from resin;

cyclized; washed; deprotected; purified by semi- preparative RP-HPLC (linear gradient of 32-41%, 3 CV) and lyophilization of the combined fractions furnished the peptide (63.4 mg, 31.1 μmol, 31.1%); LC/MS Rt 8.00 min, linear gradient 10→90% B in 13.5 min.; m/z = 1581.7 [M+H]⁺; HRMS (ESI) m/z 1582.02251 [M+H]⁺, calcd.1582.02432 for C81H132N18O14; IR 3278 (m), 2962 (m), 1635 (s), 1539 (m), 1456 (m), 1202 (s), 1133 (s); ¹H NMR (600 MHz, CD3OH) δ 9.00 (d, J = 2.6, 1H, NH^DPhe14), 8.95 (d, J = 2.5, 1H, NH^DPhe7), 8.83 (d, J = 9.0, 1H, NH Leu11), 8.73 (d, J = 9.5 Hz, 1H, NH Val4), 8.70 (d, J = 9.4, 1H, NH Orn5), 8.69 (d, J = 9.4, 1H, NH Orn12), 8.67 (d, J = 9.4, 1H, NH Leu6), 8.53 (d, J = 9.6, 1H, NH Val13), 8.50 (d, J = 9.4 Hz, 1H, NH Orn3), 8.46 (d, J = 9.6, 1H, NH, Orn10), 7.93 (d, J = 8.3, 1H, NH Leu2), 7.78 (d, J = 8.5, 1H, NH Val9), 7.37-7.21 (m, 10H, CH Ar), 5.07-5.01 (m, 4H, Hα Orn3,5,10,12), 4.72 – 4.63 (m, 1H, Hα Leu6), 4.56 – 4.51 (m, 2H, Hα ^DPhe), 4.42 – 4.34 (m, 5H, Hα Leu2,11, Val4, Pro1,8), 4.21 (t, J = 9.8, 1H, Hα Val13), 4.10 (t, J = 8.6, 1H, HαVal9), 3.75 – 3.72 (m, 1H, Hδd Pro8), 3.70 - 3.67 (m, 1H, Hδd

Pro1), 3.12 - 3.04 (m, 4H, Hβd^DPhe, Hδd Orn5,12), 2.95-2.86 (m, 8H, Hβu^DPhe, Hδd,u Orn), 2.47 (dd, J = 8.3, 16.4 1H, Hδ Pro8), 2.41 (dd, J = 9.2, 17.4, 1H, Hδu Pro1), 2.31 (dd, J = 7.3, 14.5, 1H, Hβ Val9), 2.11 – 1.89 (m, 8H, Hβ Val4,13, Hβd Orn, Hβd Pro1,8), 1.85 – 1.21 (m, 27H, Hβd,u,γ Leu, Hβu,γd,u Orn, Hβu,γd,u Pro), 0.99-0.77 (m, 36H, CH3 Val, CH3 Leu). ¹³C NMR (151 MHz, CD3OH) δ 174.00, 173.85, 173.77, 173.75, 173.47, 172.77, 162.48, 162.25, 136.93, 134.54, 130.51, 129.45, 128.82, 128.78, 127.67, 61.01, 59.80, 58.55, 58.23, 54.97, 52.19, 51.59, 51.26, 50.74, 46.96, 46.91, 46.89, 46.87, 43.60, 41.17, 41.11, 40.91, 39.79, 39.59, 39.53, 36.37, 36.30, 35.98, 33.02, 30.85, 30.78, 29.73, 29.66, 29.53, 24.99, 24.66, 24.07, 24.00, 23.61, 23.41, 22.56, 22.23, 21.64, 20.65, 20.19, 18.88, 18.61, 18.34, 18.08.

cyclo-[SAA-Leu-Orn-Val-Orn-Leu-

Phe-Pro-Val-Orn-Leu-Orn-Val]

4TFA (5)

The peptide (96 mg, 50.6 μmol) was deprotected and purified by preparative RP-HPLC (linear gradient of 35- 44%, 3 CV) and yielded 32.3 mg, 16.5 μmol, 32.7%; LC/MS Rt 5.70 min, linear gradient 10→90% B in 13.5 min.; m/z = 1497.1 [M+H]⁺; HRMS (ESI) m/z 1496.95631 [M+H]⁺, calcd.1496.95630 for C73H125N17O16; IR 3269 (m), 2962 (m), 1622 (s), 1539 (m), 1456 (m), 1201 (s), 1132 (s); ¹H NMR (600 MHz, CD3OH) δ 8.82 (d, J = 3.5, 1H, NH^DPHe7), 8.49 (d, J = 7.0, 2H, NH Leu6,11), 8.45 (d, J = 8.0, 4H, NH Orn3,5,10,12), 8.35 (d, J = 7.4, 1H, NH Val13), 8.31 (br. s, 1H, NH SAA), 8.17 (d, J = 7.5, 1H, NH Val4), 8.07 (d, J = 7.7, 1H, NH Leu2), 7.99 – 7.86 (m, 2H, NH2 Orn), 7.84 (d, J = 8.0, 1H, NH Val9), 7.37 – 7.18 (m, 5H, CH Ar), 4.82 - 4.76 (m, 4H, Hα Orn3,5,10,12), 4.63 - 4.56 (m, 3H, Hα Leu6,11, Hα^DPhe7), 4.54 (d, J = 4.7, 1H, H1 SAA), 4.50 – 4.47 (m, 1H, HαLeu2), 4.37 (d, J = 5.9, 1H, HαPro8), 4.32 (t, J = 8.0, 1H, HαVal13), 4.29 – 4.28 (m, 1H, H2 SAA), 4.18 (t, J = 8.4, 1H, Hα Val4), 4.07 (t, J = 8.2, 1H, Hα Val9), 4.08 – 4.04 (m, 1H, H4 SAA), 3.95 (s, 1H, H3 SAA), 3.73 (t, J = 7.6, 1H, Hδd Pro8), 3.62 - 3.60 (m, 1H, H5d SAA), 3.49 – 3.47 (m, 1H, H5u

SAA), 3.16 – 2.88 (m, 10H, Hδd,δu Orn, Hβd,βu^DPhe7), 2.64 – 2.57 (m, 1H, Hδu Pro8), 2.26 (dd, J = 7.0, 14.2, 1H, Hβ Val9), 2.11 (dd, J = 6.9, 14.0, 1H, HβVal4), 2.08 – 1.85 (m, 6H, Hβd Orn, Hβd Pro, Hβ

Val13), 1.86 – 1.44 (m, 20H, Hβd,u,γ Leu, Hβu,γd,u Orn), 1.39 (t, J = 7.5, 1H, Hγ Leu6), 1.14 – 0.73 (m, 36H, CH3 Val, CH3 Leu). ¹³C NMR (151 MHz, CD3OH) δ 175.06, 174.37, 174.04, 173.97, 173.69,

63 128.38, 116.81, 86.36, 82.65, 79.48, 78.92, 61.87, 60.98, 60.98, 60.57, 59.63, 55.69, 54.23, 53.62, 53.62, 53.57, 52.96, 52.89, 52.63, 51.98, 47.95, 43.57, 42.11, 41.58, 40.70, 40.59, 40.53, 40.44, 37.53, 33.31, 31.80, 31.49, 30.44, 30.38, 30.10, 29.64, 25.76, 25.66, 25.19, 25.03, 24.76, 24.62, 23.84, 23.48, 23.09, 22.79, 22.22, 21.60, 19.83, 19.71, 19.63, 19.50, 19.10, 18.85.

cyclo-[SAA-Leu-Orn-Val-Orn-Leu-

Phe-Pro-Val-Orn-Leu-Orn-Val]

4TFA (6)

The peptide (126 mg, 63.4 μmol) was deprotected and subsequently the peptide was purified by preparative RP-HPLC (linear gradient of 36-45%, 3 CV and yielded 31.6 mg, 15.5 μmol, 24.4 %; LC/MS Rt 6.28 min, linear gradient 10→90% B in 13.5 min.;

m/z = 1588.2 [M+H]⁺; HRMS (ESI) m/z 1587.00271 [M+H]⁺, calcd.1587.00325 for C80H131N17O16; IR 3271 (m), 2962 (m), 1622 (s), 1539 (m), 1456 (m), 1201 (s), 1131 (s); ¹H NMR (600 MHz, CD3OH) δ 8.81 (d, J = 4.2 Hz, 1H, NH^DPhe7), 8.47 (d, J = 8.0, 2H, NH Leu6,11), 8.43 (d, J = 8.2, 3H, NH Orn3,10,12), 8.37 (d, J = 8.4, 1H, NH Orn5), 8.33 (d, J = 7.4, 1H, NH Val13), 8.27 (t, J = 4.6, 1H, NH SAA), 8.11 (d, J = 7.5, 1H, NH Val4), 8.03 (d, J = 7.7, 1H, NH Leu2), 7.94 (br. s, NH2 Orn), 7.85 (d, J = 7.9, 1H, NH Val9), 7.39 – 7.22 (m, 10H, CH Ar), 4.80 - 4.77 (m, 3H, Hα Orn3,10,12), 4.61 (dd, J = 11.7, 31.2, 1H, CH2

Benzyl), 4.59 – 4.57 (m, 4H, Hα Orn5,Leu6,11,^DPhe7), 4.53 (d, J = 3.9, 1H, H1 SAA), 4.49 – 4.46 (m, 2H, Hα Leu2, H2 SAA), 4.37 – 4.36 (m, 1H, Hα Pro8), 4.33 (t, J = 7.9, 1H, Hα Val13), 4.21 (s, 1H, H4 SAA), 4.16 (t, J = 8.2, 1H, Hα Val4), 4.05 (t, J = 8.1, 1H, Hα Val9), 3.88 (s, 1H, H3 SAA), 3.73 – 3.71 (m, 1H, Hδd Pro8), 3.62 – 3.60 (m, 1H, H5d SAA), 3.46 – 3.43 (m, 1H, H5u SAA), 3.09 – 2.91 (m, 10H, Hδd,u

Orn, Hβd,u^DPhe7), 2.63 - 2.61 (m, 1H, Hδu Pro8), 2.26 (dd, J = 6.9, 14.2, 1H, Hβ Val9), 2.10 (dd, J = 6.9, 13.8, 1H, Hβ Val4), 2.08 – 1.85 (m, 6H, Hβd Orn, Hβd Pro8, Hβ Val13), 1.85 – 1.41 (m, 20H, Hβd,u,γ Leu, Hβu,γd,u Orn), 1.42 – 1.37 (m, 1H, Hγ Leu6), 1.14 – 0.73 (m, 36H, CH3 Val, CH3 Leu); ¹³C NMR (151 MHz, CD3OH) δ 175.01, 174.43, 174.05, 173.97, 173.71, 173.68, 173.61, 173.51, 173.40, 173.00, 172.86, 172.74, 171.95, 138.97, 137.03, 130.38, 129.63, 129.40, 128.85, 128.72, 128.39, 101.28, 87.23, 85.51, 83.72, 77.10, 72.81, 61.86, 61.06, 60.66, 59.63, 55.64, 54.33, 53.67, 53.59, 52.93, 52.75, 52.00, 47.98, 43.44, 42.63, 42.11, 41.67, 40.72, 40.64, 40.56, 40.46, 37.58, 33.24, 31.96, 31.46, 30.45, 30.38, 30.02, 29.61, 29.51, 25.76, 25.67, 25.08, 25.04, 24.76, 24.66, 23.86, 23.50, 23.10, 22.75, 22.75, 22.17, 21.59, 19.85, 19.66, 19.51, 19.05, 18.89.

cyclo-[SAA-Leu-Orn-Val-Orn-Leu-

Phe-Pro-Val-Orn-Leu-Orn-Val]

4TFA (7)

Cyclic peptide (147 mg, 70.8 μmol) was deprotected and subsequently the peptide was purified by preparative RP-HPLC (linear gradient of 35-44, 3 CV) and yielded 13.5 mg, 6.34 μmol, 9.0 %; LC/MS Rt

6.32 min, linear gradient 10→90% B in 13.5 min.;

m/z = 1678.6 [M+H]⁺; HRMS (ESI) m/z 1677.04439 [M+H]⁺, calcd.1677.05020 for C87H137N17O16 IR 3278 (m), 2962 (m), 1634 (s), 1539 (m), 1456 (m), 1202 (s), 1131 (s); ¹H NMR (600 MHz, CD3OH) δ 8.71 (d, J = 3.0, 1H, NH^DPhe7), 8.39 (d, J = 7.5, 3H, NH Orn5,12, Leu2), 8.36 (d, J = 8.0, 3H, NH Leu6,11, Orn10), 8.24 (d, J = 7.5, 4H, NH Orn3, Val4,13), 8.23 – 8.19 (m, 1H, NH SAA), 7.90 (d, J = 7.5, 1H, NH Val9), 7.808 (br. s. NH2 Orn), 7.42 – 7.21 (m, 15H, CH Ar), 4.64 – 4.60 (m, 5H, Hα Orn3,5,10,12, ^DPhe7), 4.59 (d, J = 4.2, 1H, H1 SAA), 4.54 – 4.52 (m, 4H, CH2 Benzyl), 4.51 – 4.47 (m, 3H, Hα Leu2,6,11), 4.35 – 4.33 (m, 1H, Hα Pro8), 4.29 (t, J = 8.0, 1H, Hα

Val4), 4.25 (d, J = 4.1, 1H, H2 SAA), 4.17 (d, J = 7.6, 1H, H4 SAA), 4.05 (t, J = 6.5, 1H, Hα Val13), 3.97 (t, J = 7.7, 1H, Hα Val9), 3.92 (s, 1H, H3 SAA), 3.73 – 3.69 (m, 1H, Hδd Pro8), 3.55 – 3.51 (m, 1H, H5d

SAA), 3.42 – 3.40 (m, 1H, H5u SAA), 3.04 – 2.94 (m, 10H, Hδd,u Orn, Hβd,u^DPhe7), 2.75 – 2.73 (m, 1H, Hδu Pro8), 2.24 (dd, J = 5.0, 13.0, 1H, Hβ Val9), 2.07 – 2.05 (m, 2H, Hβ Val4,13), 1.99 – 1.96 (m, 1H, Hβd

Pro8), 1.90 – 1.82 (m, 3H, Hβd Orn,5,10,12), 1.80-1.48 (m, 20H, Hβd,u,γ Leu, Hβu,γd,u Orn), 1.45 – 1.38 (m, 1H, Hγ Leu6) 1.05–0.79 (m, 36H, CH3 Val, CH3 Leu); ¹³C NMR (151 MHz, CD3OH) δ 175.01, 174.43, 174.05, 173.97, 173.71, 173.68, 173.61, 173.51, 173.40, 173.00, 172.87, 172.74, 171.95, 162.99, 162.76, 138.97, 137.03, 130.38, 129.63, 129.40, 128.85, 128.72, 128.38, 87.23, 85.51, 83.72, 77.10, 72.81, 61.86, 61.05, 60.66, 59.62, 55.64, 54.33, 53.67, 53.59, 52.93, 52.75, 52.00, 47.98, 43.44, 42.63, 42.11, 41.66, 40.72, 40.64, 40.56, 40.46, 37.58, 33.25, 31.96, 31.46, 30.45, 30.38, 30.02, 29.61, 29.50, 25.76, 25.67, 25.08, 25.04, 24.76, 24.66, 23.86, 23.50, 23.10, 22.74, 22.17, 21.59, 19.84, 19.66, 19.51, 19.05, 18.89.

References and notes

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[2] Hancock, R. E. W.; Chapple, D. S. Antimicrobial Agents Chemother. 1999, 43, 1317.

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[5] Wadsten, C. J.; Bertilsson, C. A.; Sieradzki, H.; Edstrom, S. Archiv. Otorhino-Laryngol. 1985, 242, 135.

[6] (a) Kaprelyants, A. S.; Nikiforov, V. V.; Miroshnikov, A. I.; Snezhkova, L. G.; Eremin, V. A.;

Ostrovskii, D. N. Biochemistry-Moscow 1977, 42, 252; (b) Hull, S. E.; Karlsson, R.; Main, P.;

Woolfson, M. M.; Dodson, E. J. Nature 1978, 275, 206; (c) Katsu, T.; Kuroko, M.; Morikawa, T.;

Sanchika, K.; Fujita, Y.; Yamamura, H.; Uda, M. Biochim. Biophys. Acta 1989, 983, 135; (d) Prenner, E. J.; Lewis, R. N. A. H.; McElhaney, R. N. Biochim. Biophys. Acta Biomembr. 1999, 1462, 201; (e) Wu, M.; Maier, E.; Benz, R.; Hancock, R. E. W. Biochemistry 1999, 38, 7235; (f) Salgado, J.; Grage, S. L.; Kondejewski, L. H.; Hodges, R. S.; McElhaney, R. N.; Ulrich, A. S. J.

Biomol. NMR 2001, 21, 191; (g) Grotenbreg, G. M.; Witte, M. D.; van Hooft, P. A. V.; Spalburg, E.; Reiss, P.; Noort, D.;. de Neeling, A. J.; Koert, U.; van der Marel, G. A.; Overkleeft, H. S.;

Overhand, M. Org. Biomol. Chem. 2005, 3, 233; (h) Llamas-Saiz, A. L.; Grotenbreg, G. M.;

Overhand, M.; van Raaij, M. J. Acta Crystallogr. 2007, D63, 401.

[7] Schmidt, G. M. J.; Hodgkin, D. C.; Oughton, B. M.; Biochem. J. 1957, 65, 744.

[8] Hodgkin, D. C.; Oughton, B. M. Biochem. J. 1957, 65, 752.

[9] Izumiya, N.; Kato, T.; Aoyagi, H.; Waki, M.; Kondo, M. Synthetic aspects of biologically active cyclic peptides-gramicidin S and tyrocidines, Halstead (Wiley), New York, 1979.

[10] Ovchinnikov, Y. A.; Ivanov, V.T. The proteins (editor: H. Neurath and R. Hill), Academic Press, New York, 1979, 5, 391 – 398

[11] Wishart, D. S.; Kondejewski, L. H.; Semchik, P. D.; Sykes, B. D.; Hodges, R. S. Lett. Pep. Sci., 1996, 3, 53.

[12] Kondejewski, L. H.; Farmer, S. W.; Wishart, D. S.; Kay, C. M.; Hancock, R. E. W.; Hodges, R. S.

J. Biol. Chem. 1996, 271, 25261.

[13] Kondejewski, L. H.; Jelokhani-Niaraki, M.; Farmer, S. W.; Lix, B.; Kay, C. M.; Sykes, B. D.;

Hancock, R. E. W.; Hodges, R. S. J. Biol. Chem. 1999, 274, 13181.

[14] Gibbs, A. C.; Kondejewski, L. H.; Gronwald, W.; Nip, A. M.; Hodges, R. S.; Sykes, B. D.;

Wishart, D. S. Nat. Struct. Biol. 1998, 5, 284.

[15] Jelokhani-Niaraki, M.; Kondejewski, L. H.; Wheaton, L. C.; Hodges, R. S. J. Med. Chem. 2009, 52, 2090.

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van Raaij, M. J.; Overkleeft, H. S.; Overhand, M. J. Am. Chem. Soc. 2004, 126, 3444.

65 [17] Grotenbreg, G. M.; Buizert, A. E. M.; Llamas-Saiz, A. L.; Spalburg, E.; van Hooft, P. A. V.; de

Neeling, A. J.; Noort, D.; van Raaij, M. J.; van der Marel, G. A.; Overkleeft, H. S.; Overhand, M.

J. Am. Chem. Soc. 2006, 128, 7559.

[18] See for relevant examples: (a) Jelokhani-Niaraki, M.; Hodges, R. S.; Meissner, J. E.; Hassenstein, U. E.; Wheaton, L. Biophys. J. 2008, 95, 3306; (b) Sato, K.; Nagai, U. J. Chem. Soc. Perk. Trans.

1 1986, 1231; (c) Ripka, W. C.; De Lucca, G. V.; Bach, A. C.; Pottorf, R. S.; Blaney, J. M.

Tetrahedron 1993, 49, 3609; (d) Andreu, D.; Ruiz, S.; Carreño, C.; Alsina, J.; Albericio, F.;

Jiménez, M. A.; de la Figuera, N.; Herranz, R.; García-López, M. T.; González-Muñiz, R. J. Am.

Chem. Soc. 1997, 119, 10579; (e) Roy, S.; Lombart, H. G.; Lubell, W. D.; Hancock, R. E. W.;

Farmer, S. W. J. Pep. Res. 2002, 60, 198; (f) Xiao, J.; Weisblum, B.; Wipf, P. Org. Lett. 2006, 8, 4731.

[19] Timmer, M. S. M.; Verdoes, M.; Sliedregt, L.; van der Marel, G. A.; van Boom, J. H.; Overkleeft, H. S. J. Org. Chem. 2003, 68, 9406.

[20] Chakraborty, T. K.; Jayaprakash, S.; Diwan, P. V.; Nagaraj, R.; Jampani S. R. B.; Kunwar, A. C.

J. Am. Chem. Soc. 1998, 120, 12962.

[21] Grotenbreg, G. M.; Kronemeijer, M.; Timmer, M. S. M.; El Oualid, F.; van Well, R. M.;

Verdoes, M.; Spalburg, E.; van Hooft, P. A. V.; de Neeling, A. J.; Noort, D.; van Boom, J. H.; van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. J. Org. Chem. 2004, 69, 7851.

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[23] Wüthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons, New York, 1986.

[24] Maynard, A. J.; Sharman, G. J.; Searle, M. S. J. Am. Chem. Soc. 1998, 120, 1996.

[25] Griffiths-Jones, S. R.; Maynard, A. J ; Searle, M. S. J. Mol. Biol. 1999, 292, 1051.

[26] (a) The δHα of the amino acid residues in GS are not significantly affected when using methanol instead of water as solvent system. For the Orn residues the random coil value of Lys is taken. (b) Kraus, E. M.; Chan, S. I. J. Am. Chem. Soc. 1982, 104, 6953.

[27] Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992, 31, 1647.

[28] Wishart, D. S.; Kondejewski, L.H.; Semchuk, P. D.; Kay, C. M.; Hodges, R. S.; Sykes, B. D.

Design, Synthesis and Characterization of a Water-soluble ^β-sheet Peptide, in Techniques in Protein Chemistry VI (editor: J.W. Crabb) Academic Press, Orlando, 1995, 451-458.