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

: 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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* Knijnenburg, A. D.; Spalburg, E.; de Neeling, A. J.; Mars-Groenendijk, R. H.; Noort, D.; van der Marel, G. A.; Overkleeft, H. S.; Overhand M. Manuscript in preparation

 

₈₅

         

Introduction

The amphipathicity and hydrophobicity of cationic antimicrobial peptides (CAPs) are important factors that contribute to their ability to kill micro–

organisms.

^[1]

The antimicrobial peptide gramicidin S (GS, cyclo –VOLP

^D

F)

2

) is a member of the CAP family and active against Gram–positive and certain Gram–

negative bacteria.

^[2]

Unfortunately GS is toxic and for this reason has been the subject of many studies aiming to improve the therapeutic profile.

^[3]

GS has an amphipathic nature in that the cyclic β–hairpin structure has an apolar side comprising Leu and Val amino acid residues and a polar side comprising ornithine as side chains.

^[4]

By increasing the ring–size of GS with four additional amino acids the amphipathicity can be influenced. This is exemplified by tetradecameric

Ring-extended gramicidin S analogs with strand modification and sugar amino acids that vary in

ring-size in the turn region ^*

analog 1 (Figure 1), which follows the rules of Schwyzer/Gibbs

^[5]

since it has 2(2n+1) amino acid residues (n = 3), and thus forms a stable cyclic β–hairpin in solution. Compound 1 has four cationic charges and six apolar residues in the strand regions and was found to be very hemolytic.

^[6]

The replacement of an

L

– ornithine by the corresponding

D

–amino acid leads to a molecule with an altered amphipathicity in that the cationic charge of the

D

–ornithine is situated at the apolar side of the molecule (8, Figure 1, see also Chapter 4). As a result, the therapeutic profile of 8, that is potent antimicrobial activity with reduced hemolytic activity, is significantly improved when compared to compound 1.

^[6]

Hydrophobicity of GS analogs can be further down–tuned by using sugar amino acids (SAAs) as dipeptides isosteres of varying ring–size replacing one of the turn regions, as was shown in Chapter 2.

^[7]

In addition, the hydrophobicity of SAAs can also be altered by the introduction of benzyl groups on the hydroxyl group(s) of the carbohydrate derived core, as shown in Chapter 3.

^[8]

This chapter describes an approach to modify both the amphipathicity and hydrophobicity of tetradecameric GS analogs by combining the strategies described in Chapters 2 and 3. SAAs of varied ring–size, that is an oxetane, a furanoid and a pyranoid, are used to replace one of the turns of the tetradecameric templates 1, having solely

L

–ornithines, and template 8, having three

L

–ornthines and one

D

–ornithine. To further influence the hydrophobicity of the turn region the SAAs were incorporated as either mono–hydroxyl or mono–benzyloxy

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

O

2, R = H 3, R = Bn

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

8

OR HN

O NH3+ NH3+

NH₃⁺ NH₃⁺

1

4, R = H 5, R = Bn

6, R = H 7, R = Bn HN

O

HN

O O

OR O OR

NH₃⁺ NH₃⁺

NH3+ NH3+

O

9, R = H 10, R = Bn

OR HN

O 11, R = H 12, R = Bn

13, R = H 14, R = Bn HN

O

HN

O O

OR O OR

Figure 1: Tetradecameric GS analogs with strand and turn modifications 1-14.

87

species. The resulting peptides 1–14 were subjected to a conformational analysis by NMR. Furthermore, the antimicrobial activity and toxicity (hemolytic activity) were investigated.

Results and discussion

Synthesis. In order to construct the tetradecameric analogs 2–7 and 9–14 the appropriately functionalized SAAs were synthesized (23–27, Scheme 2). The oxetane and pyranoid SAAs (25, 26 and 27) were synthesized according to a previous described procedure (Chapter 2).

^[6]

A difficult and lengthy synthetic route is known for the furanoid SAA building block 24.

^[9]

Pedersen and Watterson, however, described independently a short synthetic route on large scale with simple work–up procedures towards intermediates 20 and 22, respectively.

^[10]

It was decided to modify this synthetic route to obtain the desired furanoid SAAs 23 and 24.

D

–glucono–1,5–lactone (15) was first acetylated under acidic conditions. Subsequent elimination of the acetate under basic conditions resulted in the exclusive formation of the 2,3–unsaturated lactone 16.

Hydrogenation of the double bond in the presence of catalytic amounts of palladium on carbon under high hydrogen pressure gave lactone 17. Deacetylation under basic conditions and neutralization resulted in the formation of a 1,4–

lactone, which was protected with an isopropylidene acetal to give 18. Mesylation and ring opening under acidic conditions led to the conversion into 2,5–anhydride 20. The azide was then introduced via tosylation of the primary alcohol and treatment with sodium azide resulting in compound 22. Hydrolysis of the methyl ester gave SAA 23 and subsequent benzylation gave SAA 24.

O O O

OR O O

OAc AcO

AcO O

O OH HO

HO O

OH

i O

OAc AcO

AcO O

ii

iii

O v RO

HO

OMe O

16 17

vii

20, R = H 21, R = Ts N₃ O

R₂O

OR₁ O

22, R₁= Me, R₂= H 23, R₁= H, R₂= H 24, R₁= OH, R₂= Bn

viii vi 18, R = H

19, R = Ms iv

15

ix

Scheme 1: Reagents and conditions: (i) 1. 60% aq. HClO

4

, Ac

2

O, 2. Et

3

N, DCM, 0

º

C,

quant.; (ii) 10% Pd/C, 160 Bar H

2,

EtOAc, 92%; (iii) 1. MeONa, MeOH, 2. DOWEX H

⁺

, 50

º

C, 3. H

2

SO

4

, MgSO

4

, acetone, 76%; (iv) MsCl, pyridine, 0

º

C, 76%; (v) 1. TFA, H

2

O, 100

º

C,

2. DOWEX H

⁺

, MeOH, 59%; (vi) TsCl, DMAP, DCM, 0

º

C, 72%; (vii) NaN

3

, DMF, 85, 0

º

C,

85%; (viii) LiOH, THF/H

2

O, 95%; (ix) Compound 23, NaH, benzyl bromide, DMF, 36%.

Peptides 1–14 were synthesized according to a silimar procedure described in Chapter 3 and 4 (Scheme 2).

^[7,8]

Acid–labile HMPB–BHA resin was loaded with the pentapeptide 28. Coupling of SAAs 23–27 was accomplished with the coupling reagent HCTU and base DiPEA and was followed by azide reduction on resin using a solution of aqueous trimethylphosphine. The peptides were subsequently elongated by automated Fmoc–based solid phase chemistry resulting in peptides 34–38 containing either

L

–ornithine or

D

–ornithine at the fourth position of the sequence (Scheme 2). The peptides were released from the resin under mild acidic conditions and cyclized under highly dilute conditions. Final deprotection using strong acid resulted in peptides 3–7 and 10–14. Peptides 3 and 10 were hydrogenated under acidic conditions to obtain peptides 2 and 9.

NMR analysis. The general secondary structure of a peptide can be derived from the coupling constants of amide protons (

³

J

HNα

) and chemical shift perturbations of the alpha protons (∆δH

α

).

^[11]

These values were extracted from the

1

H NMR spectra of all peptides (1–14). Only the

³

J

HNα

and ∆δH

α

of the non–

benzylated analogs (2, 4, 6, 9, 11 and 13) and templates 1 and 8 are given in Figures 2 and 3, because of the similarity between the non–benzylated and the benzylated peptides. All peptides show well–resolved

¹

H spectra. Peptide residues were assigned using recorded 2D–TOCSY and 2D–cROESY

^[12]

spectra. The

³

J

HNα

of the Val, Orn and Leu residues of peptide 1–14 are all above 7 Hz, which indicates

Scheme 2: Reagents and conditions: (i) coupling of SAA 23-27, HCTU, DiPEA, DMF, 16h;

(ii) PMe

3

(1M in 9:1 THF/H

2

O, 16 eq), 16h; (iii) standard sequential Fmoc SPPS; (iv) 1%

TFA in DCM; (v) pyBOP, HOBt, DiPEA, DMF; (vi) 95/2.5/2.5 TFA/TIS/H

2

O; (vii) H

2

, Pd-

black in 1,4-dioxane/aq. HCl. Numbering of amino acids N→C: cyclo-(Val1-Orn2-Leu3-D-

Orn4-Val5-D-Phe6-Pro7/SAA-Leu8-Orn9-Val10-Orn11-Leu12-D-Phe13-Pro14)

89

Figure 2: [A]

³

J

HNα

of peptides 1, 2, 4 and 6 containing

L

-ornithines; [B]

³

J

HNα

of peptides 8, 9, 11 and 13 having one

D

-ornithine.

Figure 3: [A] ∆δH

α

of peptides 1, 2, 4 and 6 having

L

-ornithines.

Val1

Orn2 Leu3 Orn4 Val5 Phe6/SAA D

Leu8 Orn9 Val10

Orn11 Leu12 Phe13 D 0

2 4 6 8 10

A

1 2 4 6

Residue 3JHN (Hz)

Val1

Orn2 Leu 3

Orn4 Val5 Phe

6/SAA D

Leu8 Orn9 Val10

Orn11 Leu12 Phe13 D 0

2 4 6 8 10

B

8 9 11 13

Residue 3JHN (Hz)

Val 1

Orn 2 Leu 3

Orn 4 Val 5 Phe

6 / SAA D

Pro 7

Leu 8 Orn 9 Val 10

Orn 11 Leu 12 Phe 13

D Pro 1

-0.2 4 0.0 0.2 0.4 0.6 0.8

A

1 2 6 4

Residue

H (ppm)

that these residues are part of an extended β–sheet conformation (Figure 2A and 2B). The turn residue

D

–Phe has a coupling constant around 3 Hz, which is a typical value for a turn residue, thus confirming that all peptides adopt cyclic β–

hairpin structure in methanol.

^[11]

The chemical shift perturbation of peptides 1–14 also corroborated the β–sheet character of the Val, Orn and Leu residues since these values were all above 0.1 ppm (Figure 3A and 3B). The turn residues (

D

–Phe and Pro) show negative perturbations which is a characteristic of a β–turn region.

^[11]

Physical and biological properties. The antimicrobial activity of peptides 1–14 was tested on Gram–positive and Gram–negative bacteria (Table 1). The toxicity of the peptides was evaluated by performing an hemolytic assay (Figure 4).

Peptides 9–14, having a

D

–ornithine substitution and a SAA in the β–turn, did not show broad spectrum activity against Gram–positive and Gram–negative bacteria.

They only displayed significant activity against the Gram–positive strain S.

epidermidis . Additionally, peptides 9–14 have low hemolytic activity. In contrast, analogs 2–7, only having

L

–ornithines and a SAA in the turn, display antimicrobial activity against all tested strains. Notable is the higher activity of 2–7 against Gram–negative bacteria compared to GS and template 1. Unfotunately, these peptides (2–7) were equally or more toxic than GS.

The retention time on a reverse phase column was used as an indication of peptide hydrophobicity

^[13]

(Table 1). The benzylation of the hydroxyl functionality of the SAAs generally results in a more hydrophobic peptide. These retention time data also show that the introduction of a

D

–ornithine leads to a peptide series (9–

14) that is more hydrophilic than the series with only

L

–ornithines (2–7).

Figure 3: [B] ∆δH

α

of the peptides 8, 9, 11 and 13 having one

D

-ornithine. ∆δH

α = δHα

- δH

α

random coil.

Val 1 Orn 2

Leu 3 Orn 4

Val 5 Phe 6 / SAA 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 1.0

B

8 9 11 13

Residue

H (ppm)

91

Figure 4: Hemolytic curves of GS and compounds 1-14.

0 1 2 3

0 20 40 60 80 100

1 2 3 4

5 6 7 GS

Concentration in log [M]

% Hemolysis

0 1 2 3

0 20 40 60 80 100

8 9 10 11

12 13 14 GS

Concentration in log [M]

% Hemolysis

Table 1. Antimicrobial activity (MIC) and retention times of GS and 1-14.

Analogs Retention timea S. aureusb S. epidermidisb E. faecalisb B. cereusb P. aeruginosac E. colic

GS 8.38 4 2 8 4 64 32

1 7.67 32 2 32 32 64 32

2 6.63 8 1 4 2 4 2

3 6.67 8 2 16 16 16 8

4 6.71 16 8 16 16 16 16

5 7.30 4 4 8 8 8 8

6 6.67 32 8 32 32 32 16

7 7.42 4 2 8 8 8 16

8 6.25 64 4 64 16 >64 64

9 5.76 >64 16 >64 64 64 64

10 6.34 32 4 64 16 32 16

11 5.79 >64 16 >64 >64 >64 64

12 6.31 32 4 64 16 64 16

13 5.56 >64 64 >64 >64 >64 >64

14 6.15 >64 8 >64 32 >64 32

[a]

Retention times from RP-HPLC in minutes (see experimental section);

^[b]

Gram-positive bacteria, MIC in mg/L;

^[c]

Gram-negative bacteria, MIC in mg/L. For detailed experimental set up: see Experimental section. Molecular weight GS: 1369.49; 1,8: 2038.12; 2,9: 1922.95;

3,10: 2013.07; 4,11: 1936.97; 5,12: 2027.09; 6,13:1951.00; 7,14: 2041.12.

Conclusion

In this chapter a study was presented in which subtle changes in hydrophobicity and amphipathicity were introduced in tetradecameric GS analogs by incorporating varying ring–size SAAs and by

D

–ornithine substitution. To this end peptides 1–14 were prepared, ranging from hydrophilic peptides to hydrophobic peptides. The peptides (1–14) were all able to form cyclic β–hairpins in the solvent methanol. Unfortunately, the peptides (9–14) having a

D

–ornithine substitution in the strand were biologically inactive. Based on their retention times on a HPLC column under controlled conditions,

^[13]

it was found that these peptides are too hydrophilic and these properties are in agreement with the general trend that hydrophilic peptides of this type are not antimicrobially active.

^[14]

Peptides 2–7 showed higher antimicrobial activity against Gram–positive and Gram–negative bacteria compared to template 1 and GS. However, no improvement of the biological profile was achieved since the peptide 2–7 were equally toxic or even more toxic than 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. The linear peptides were cleaved from resin, cyclized and purified by RP–HPLC (Gilson GX–281) with a preparative Gemini C18 column (Phenomenex 21.2mmϕ x 150 mmL, 5μm particle size) or semi–preparative (250 x 21.2 mm, 5μg 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 eluents 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. Hemolytic curves were analyzed with Graphpad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA. 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.

NMR spectroscopy:

1H and ¹³C–APT NMR for all intermediates (16–24) were recorded on a Bruker AV–400 (400/100 MHz) or a Bruker DMX 600. The spectra of the peptides (3–7, 9–14) were recorded on a Bruker DMX 600 equipped with a pulsed field gradient accessory and a cryo–probe. For the 2D cROESY spectra (200 msec mixing time) the peptides were dissolved in CD3OH. Standard DQF–COSY (512c x

93 2084c) and TOCSY (400c x 2048c) spectra were recorded using presaturation for solvent suppression.

cROESY^[22] spectra (400c x 2048c, τ^mix = 180 ms) were recorded using the presat solvent suppression.

All spectra were recorded in phase–sensitive mode, using either the TPPI or states–TPPI for quadrature detection in the indirect dimension. Homonuclear coupling constants were determined from the corresponding ¹H spectra.

2,4,6–Tri–O–acetyl–3–deoxy–

^D

–erythro–hex–2–enono–1,5–lactone (16)

D–Glucono–1,5–lactone (40 gram, 225 mmol) was added in portions over 10–15 min to Ac2O (100 mL, 1 mol) containing a few drops of 60% aq. HClO4. The mixture was kept for 1 h at room temperature and the clear solution was then concentrated in vacuo. The crude product was dissolved in CH2Cl2 (300 mL) and cooled to 0ºC, and Et3N (48 ml, 1,5 mol) was added in one portion. The yellow solution was kept for 15 min at 0ºC. It was then washed with 2M HCl and with water, dried (Na2SO4) and concentrated in vauco. The residue was purified by column chromatography (1:3 EtOAc/PE) leaving 16 as a colorless syrup (65 gram, 225 mmol, 100%). ¹H NMR (400 MHz, CDCl3) δ 6.45 (d, J = 4.2 Hz, 1H, H3), 5.63 (dd, J = 5.8, 4.2 Hz, 1H, H4), 4.77 (dd, J = 10.3, 4.6 Hz, 1H, H5), 4.36 (ddd, J = 34.8, 12.3, 4.6 Hz, 2H, H6,6^’), 2.27 (s, 3H, acetyl), 2.14 (s, 3H, acetyl), 2.11 (s, 3H, acetyl).¹³C NMR (101 MHz, CDCl3) δ 170.39, 169.66, 168.14 (3x C=O), 157.18 (C1), 139.61 (C2), 126.13 (C3), 77.83 (C5), 64.16 (C4), 61.97 (C6), 20.68, 20.61, 20.31 (3xCH3 acetyl).

2,4,6–Tri–O–acetyl–3–deoxy–

^D

–arabino–hexnono–1,5–lactone (17)

The unsaturated lactone 16 (57.2 gram, 200 mmol) was dissolved in EtOAc (200 mL) and hydrogenated overnight at 65 Bar in the presence of 10% Pd/C. Filtration and concentration gave 17 (52.7 gram, 183 mmol, 92%) as a colorless syrup. ¹H NMR (400 MHz, CDCl3) δ 5.58 (dd, J = 12.4, 7.1 Hz, 1H, H2), 5.19 (td, J = 6.1, 3.2 Hz, 1H, H4), 4.65 – 4.60 (m, 1H, H5), 4.36 (dd, J = 12.3, 3.6 Hz, 1H, H6), 4.27 (dd, J = 12.3, 5.0 Hz, 1H, H6^’), 2.44 (ddd, J = 14.3, 12.4, 6.2 Hz, 1H, H3), 2.38 – 2.26 (m, 1H, H3^’), 2.19 (s, 3H, Acetyl), 2.14 (s, 3H, Acetyl), 2.11 (s, 3H, Acetyl). ¹³C NMR (101 MHz, CDCl3) δ 170.30, 169.68, 167.31 (3x C=O), 77.32 (C5), 65.70 (C4), 63.99 (C2), 62.70 (C6), 30.49 (C3), 20.90, 20.66, 20.63 (3x CH3 acetyl).

3–Deoxy–5,6–O–isopropylidene–

^D

–arabino–hexono–1,4–lactone (18)

Acetate 17 (18.48 gram, 64.11 mmol) was deacetylated in methanol (100 mL) with MeONa (0.02 N of 5.56M MeONa in MeOH). The reaction was stirred for 3 h. after which the mixture was acidified with Dowex H⁺(pH = 5). The mixture was heated overnight to 50 ºC. The resin was filtered off and the mixture was concentrated in vacuo. The crude residue was dissolved in aceton and H2SO4 (1 mL) and MgSO4 were added (10 gram). After completion (R^f 0.6, EtOAc) the mixture was neutralized with TEA and filtered over Celite®. The mixture was concentrated and subjected to a flash column (1:1 EtOAc/PE) yielding 18 (9.816 gram, 48.55 mmol, 76%). ¹H NMR (400 MHz, MeOD) δ 4.56 (dd, J = 10.7, 8.7 Hz, 1H, H2), 4.44 – 4.39 (m, 1H, H4), 4.26 (dd, J = 11.9, 5.6 Hz, 1H, H5), 4.13 (dd, J = 8.6, 6.7 Hz, 1H, H6), 3.84 (dd, J = 8.7, 5.4 Hz, 1H, H6^’), 2.69–2.63 (m, 1H, H3), 1.98 (q, J = 10.4, 1H, H3^’), 1.42 (s, 3H, CH3 isoprop), 1.35 (s, 3H, CH3 isoprop).¹³C NMR (101 MHz, MeOD) δ 178.51 (C=O), 111.11 (C^q isoprop), 77.65 (C5), 77.30 (C4), 68.80 (C2), 66.78 (C6), 34.13 (C3), 26.63 (CH3), 25.22 (CH3).

3–Deoxy–5,6–O–isopropylidene–

^D

–arabino–hexono–1,4–lactone (19)

Lactone 18 (9.78 gram, 48.37 mmol) was coevaporated with toluene and dissolved in pyridine (50 mL). The mixture was cooled to 0ºC. Mesylchloride (2eq 7.49 ml) was added dropwise. After 1 h. the reaction was completed (R^f 0.8, EtOAc). Ice and water were added, keeping the temperature at 0ºC which cause the product to precipitate. The solids were filtered off, washed with water and ether and dried to give mesylate 19

O OAc AcO

AcO O

O OAc AcO

AcO O

O O O

OH O

O O O

OMs O

(7.02 gram, 25.03 mmol, 76%). ¹H NMR (600 MHz, CDCl3, T = 303K) δ 5.38 (t, J = 9.5 Hz, 1H, H2), 4.39 (dt, J = 9.2, 6.3 Hz, 1H, H4), 4.21 (dd, J = 11.2, 6.3 Hz, 1H, H5), 4.14 (dd, J = 8.8, 6.6 Hz, 1H, H6), 3.90 (dd, J = 9.0, 4.6 Hz, 1H, H6^’), 3.27 (s, 3H, CH3 mesyl), 2.90 (ddd, J = 14.5, 8.9, 5.9 Hz, 1H, H3), 2.39 (dt, J = 13.2, 9.8 Hz, 1H, H3^’), 1.45 (s, 3H, CH3 isoprop), 1.36 (s, 3H, CH3 isoprop).

Methyl–2,5–anhydro–3–deoxy–

D

–ribo–hexonate (20)

A suspension of mesyl 19 (7.02 gram, 25.03 mmol) in water (50 mL) and TFA (0.5 mL) was boiled for 3 h. The solution was concentrated and the residue was dissolved in MeOH (100 mL) and Amberlite IR120 H⁺ was added.The solution was stirred for 3 h. The Amberlite H⁺ was filtered off and the mixture was neutralized with Et3N and concentrated. The methylester was purified with column chromatography (100% EtOAc) to yield 20 as colorless oil (2.46 gram, 13.94 mmol 59 %).

1H NMR (400 MHz, MeOD) δ 4.68 – 4.61 (m, 1H, H1), 4.27 (dt, J = 5.7, 2.8 Hz, 1H, H3), 3.91 (td, J = 5.0, 2.9 Hz, 1H, H4), 3.75 (s, 3H, CH3), 3.58 – 3.56 (m, 2H, H5, H5^’), 2.29 – 2.09 (m, 2H, H2, H2^’).¹³C NMR (101 MHz, MeOD) δ 175.30 (C=O), 89.45 (C4), 77.33 (C1), 73.15 (C3), 63.51 (C5), 52.90 (CH3), 39.68 (C2).

Methyl–6–O–p–toluenesulfonyl–2,5–anhydro–3–deoxy–

^D

–ribo–hexonate (21)

The residue (419 mg, 2.38 mmol) was coevaporated with toluene (3 x 50 mL) and dissolved in DCM (100 mL). Et3N (1.2 eq, 397 μL), p–TsCl (1.1 eq, 499 mg) and DMAP (10 mg) were added and the mixture was stirred till completion (R^f 0.50, 1:1 EtOAc/PE). The mixture was washed with 1M HCl, sat. aq. NaHCO3

and brine. The organic layer was dried (Na2SO4) and concentrated. The residue was subjected to a column chromatography (1:4 EtOAc/PE) to yield tosyl 21 as a slightly yellow oil (566 mg, 1.71 mmol, 72%). ¹H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.3 Hz, 2H, CH tosyl), 7.36 (d, J = 8.2 Hz, 2H, CH tosyl), 4.67 – 4.64 (m, 1H, H1), 4.42 (dt, J = 5.6, 2.8 Hz, 1H, H3), 4.16 – 4.07 (m, 2H, H4, H5), 4.00 (dd, J = 9.5, 5.4 Hz, 1H, H5^’), 3.73 (s, 3H, CH3 ester), 2.68 (br. s, 1H, OH), 2.45 (s, 3H, CH3 tosyl), 2.27 (ddd, J = 13.3, 7.1, 3.1 Hz, 1H, H2), 2.24 – 2.10 (m, 1H, H2^’).¹³C NMR (101 MHz, CDCl3) δ 172.29 (C=O), 145.12, (C^q), 132.18 (C^q), 129.90, 127.91 (CH tosyl), 84.25 (C4), 76.52 (C1), 72.68 (C3), 69.13 (C5), 52.21 (CH3), 38.07 (C2), 21.58 (CH3).

Methyl 6–azido–2,5–anhydro–3–deoxy–

D

–ribo–hexonate (22)

Tosyl 21 (1.65 gram, 1.55 mmol) was dissolved in DMF (50 mL) and NaN3 was added (5 eq, 1.62 gram). The reaction was heated overnight at 85ºC. TLC analysis showed complete conversion of starting material (R^f 0.51, 1:1 EtOAc/PE). DMF was evaporated. The residue was subjected to column chromatography (1:4 EtOAc/PE) and yielded 22 a colorless oil (264 mg, 1.33 mmol, 85%).

1H NMR (400 MHz, CDCl3) δ 4.71 (t, J = 7.7 Hz, 1H, H1), 4.35 (dd, J = 8.1, 4.7 Hz, 1H, H3), 4.05 (td, J

= 5.4, 3.2 Hz, 1H, H4), 3.77 (s, 3H, CH3), 3.45 (qd, J = 12.8, 5.4 Hz, 2H, H5, H5^’), 2.29 (dd, J = 7.8, 4.8 Hz, 2H, H2,H2^’), 1.86 (br. s, 1H, OH).¹³C NMR (101 MHz, CDCl3) δ 172.31 (C=O), 85.69 (C4), 76.58 (C1), 73.47 (C3), 52.62 (C5), 52.33 (CH3), 38.68 (C2).

6–Azido–2,5–anhydro–3–deoxy–

D

–ribo–hexonic acid (23)

Methylester 22 (1.31 mmol, was dissolved in a mixture of THF and H2O (7:1, 16 mL) and stirred for 3 h. The solvent was evaporated and the residue was dissolved in H2O (50 mL) and Amberlyte H⁺. The mixture was stirred for 2 h. and the resin was filtered off. The filtrate was evaporated in vacuo yielding compound 23 (1.24 mmol, 232 mg, 95%, Rf 0.1 1:1 EtOAc/PE, 1% AcOH); ¹H NMR (400 MHz, CDCl3) δ 4.73 (t, J = 7.6 Hz, 1H, H1), 4.37 (dd, J = 9.7, 5.4 Hz, 1H, H3), 4.08 (q, J = 4.3 Hz, 1H, H4), 3.61 (qd, J = 13.0, 4.3 Hz, 2H, H5,H5^’), 2.38 (dd, J = 7.6, 5.5 Hz, 2H, H2,H2^’).¹³C NMR (101 MHz, CDCl3) δ 173.21 (C=O), 85.02

N3 O HO

OMe O HO O

HO

OMe O

95 (C4), 76.16 (C1), 72.48 (C3), 51.94 (C5), 38.78 (C2). [α] ^{D 20} + 39.6, c = 0.5 in 1–propanol; HRMS (ESI) m/z 188.06645 [M+H]⁺, 188.06658 calcd. for C6H10N3O4.

6–Azido–4–O–benzyl–2,5–anhydro–3–deoxy–

^D

–ribo–hexonic acid (24)

Compound 23 (133 mg, 0.71 mmol) was dissolved in DMF and cooled to 0 ºC and NaH was added (2.5 eq, 71 mg). Subsequently benzyl bromide was added (1.1 eq, 92 μL). The solution was stirred for 3 h. at 0 ºC and product formation was observed by TLC (R^f 0.57, EtOAc, 1% AcOH). The mixture was quenched with water and the volatiles were evaporated. The mixture was stirred with DOWEX H⁺ for 1 h. in H2O.

The resin was filtered off and the solvent was evaporated. The crude product was subjected to a column (1:4 EtOAc/PE, 1% AcOH) and the product was collected as a colorless oil (71 mg, 0.25 mmol, 36%). Note: this reaction is prone for racemisation and keeping the solution at cold temperature is essential.

Alternative procedure: Compound 6 (78 mg, 0.39 mmol) was coevaporated with toluene trice and subsequently dissolved in DCM/cyclo–hexane (1:3, 10 mL). Benzylimidate (2.5 eq 181 μL) and triflic acid (cat. 1 droplet) were added to the solution and the mixture was heated overnight to 35 ºC.

Product formation was seen (Rf 0.8, 1:1 EtOAc/PE), although starting material was still present. The solution was filtered, neutralized with Et3N and the volatiles were removed in vacuo. The crude product was subjected to a flash column to remove the excess of benzylimidate. The product was dissolved in a THF/H2O mixture (7:1, 14 mL) and LiOH was added (2 eq, 18 mg). The mixture was stirred for 2 h. and TLC analysis showed full conversion (R^f 0.57, EtOAc, 1% AcOH). The solvent was removed in vacuo and the crude product was stirred with DOWEX H⁺ in H2O for 1 h. The resin was filtered off and the solvent was evaporated. The crude product was subjected to a column (1:4 EtOAc/PE, 1% AcOH) and the product was collected as colorless oil (10 mg, 0.04 mmol, 9%). ¹H NMR (400 MHz, CDCl3) δ 7.48 – 7.28 (m, 5H), 4.71 (t, J = 7.8 Hz, 1H, H1), 4.57 (d, J = 11.7 Hz, 1H, CH2 benzyl), 4.49 (d, J = 11.7 Hz, 1H, CH2 benzyl), 4.20 (dd, J = 7.9, 4.1 Hz, 1H, H4), 4.05 (dd, J = 5.8, 3.1 Hz, 1H, H3), 3.52–3.42 (m, 2H, H5,5^’), 2.58 – 2.35 (m, 1H, H2), 2.35 – 2.10 (m, 1H, H2^’). ¹³C NMR (101 MHz, CDCl3) δ 175.71 (C=O), 137.16 (C^q), 128.57, 128.08, 127.71 (CH benzyl), 83.63 (C4), 79.46 (C3), 76.46 (C1), 71.73 (CH2 benzyl), 52.33 (C5), 35.96 (C2).

General Peptide Synthesis

(a) Stepwise elongation for LOVOL: Preloaded HMPB–BHA resin with Fmoc–Leucine (1.24 g, 0.81 mmol/g, 1 mmol) was submitted to 4 cycles of automated solid–phase synthesis using the Fmoc based solid phase peptide synthesis protocols with the building blocks in the order: Fmoc–Orn(Boc)–OH, Fmoc–Val–OH, Fmoc–Orn(Boc)–OH, Fmoc–Leu–OH. The amino acids were coupled with 3 eq HCTU and 3 eq of amino acid in 30 minutes. The resin was air–dried (1.57 g; loading 0.64 mmol/g).

(b) General incorporation of SAA: (a) The resin was washed with MeOH (2 x 10 mL), NMP (2 x 10 mL), DCM (2 x 10 mL); (b) coupling of SAA to resin (for conditions: see experimental procedure analogs 2–7, 9–14) (c) washing with NMP (2 x 10 mL) and DCM (2 x 10 mL).

(c) Azide reduction: First the resins (29–33) were capped with Ac2O (5 eq), DiPEA (5 eq) in 4 mL NMP for 10 minutes. The resins were subsequently washed with 1,4–dioxane (3 x 10 mL), and taken up in 1,4–dioxane (10 mL) to which trimethylphosphine (16 eq, 1 M in THF) pre–mixed with H2O (0.6 eq) was added. The resin was shaken for 24 h.; the reduction of the azide functionality was monitored with the Kaiser test.

(d) Automated SPPS elongation: Azide reduced resins (29–33) were subjected to 7 cycles of SPPS with the use of commercially available building blocks in the following order: Fmoc–Val–OH, Fmoc–L–Orn(Boc)–OH/Fmoc–D–Orn(Boc)–OH, Fmoc–Leu–OH, Fmoc–Orn(Boc)–OH, Fmoc–Leu–

OH, Fmoc–Pro–OH, Fmoc–d–Phe–OH and subsequent Fmoc deprotection resulting in peptides 34–

43.

N₃ O BnO

OH O

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

(f) Cyclization: To a solution of HOBt (5 eq), pyBOP (5 eq) and DIPEA (15 eq) in DMF (160 mL) were dropwise added the crude peptides in DMF (20 mL) over a periode of 16 h using the syringepump. The solvent was removed under reduced pressure and the resulting mixture was applied to a Sephadex® size exclusion column (50.0 mmϕ x 1500 mmL) and eluted with MeOH.

(g) Deprotection: The Boc–protection groups were removed by addition of a TFA/TIS/H2O mixture (10 mL, 95/2.5/2.5) and subsequently the peptides (2–7 and 8–14) were purified by preparative and semi–preparative RP–HPLC.

(h) Hydrogenation: peptides 3 and 10 (3: 20.65 mg, 10.26 μmol; 10: 18.51 mg, 9.19 μmol) were hydrogenated under a hydrogen atmosphere in 1,4–dioxane (1 mL) and aq. HCl (0.7 M, 1 mL) with Pd–black (20 mg). The mixture was filtered over Celite® yielding the crude peptide 2 and 9.

cyclo–[SAA

4

–OH–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–Orn–Val]

^.

4TFA (2)

The hydrogenated peptide was RP–HPLC purified (linear gradient of 38–47%, 3 CV) and yielded 2 as white powder (15.71 mg, 8.17 μmol, 80%). HRMS (ESI) m/z 1466.94742 [M+H]⁺, 1466.94373 calcd. for C72H124N17O15; ¹H NMR (600 MHz, CD3OH) δ 8.98 (d, J = 9.4 Hz, 1H, NH Val5), 8.93 (br. s, 1H, NHD–Phe13), 8.73 (d, J = 9.1 Hz, 1H, NH Leu3), 8.65 (d, J = 9.0 Hz, 3H, NH Leu8,12, Orn9), 8.62 (d, J = 8.9 Hz, 1H, NH Orn4), 8.53 (d, J = 8.8 Hz, 1H, NH Orn11), 8.44 (d, J = 9.2 Hz, 1H, NH Orn2), 8.01 (t, J = 5.6 Hz, 1H, NH SAA), 7.97 (br. s., NH2

Orn), 7.93 (d, J = 5.7 Hz, 1H, NH Val10), 7.75 (d, J = 8.6 Hz, 1H, NH Val1), 7.36, – 7.22 (m, 5H), 5.10 (d, J = 7.3 Hz, 1H, H^α Orn11), 5.03–5.01 (m, 2H, H^α Orn2,9), 4.97 – 4.91 (m, 1H, H^α Orn4), 4.80 – 4.74 (m, 1H), 4.72 (d, J = 6.4 Hz, 1H), 4.70 – 4.61 (m, 2H, H^α Leu3,12), 4.52 (dt, J = 10.9, 4.4 Hz, 1H, H^αD– Phe13), 4.41 (dd, J = 9.3, 7.2 Hz, 1H, H^α Leu8), 4.37 (d, J = 6.7 Hz, 1H, H^α Pro14), 4.25–4.19 (m, 2H, H^α Val5,10), 4.16 (br. s, 1H), 4.12 (t, J = 8.6 Hz, 1H, H^α Val1), 3.72 (dd, J = 12.8, 5.3 Hz, 1H), 3.57 – 3.50 (m, 2H), 3.13 – 3.00 (m, 2H), 3.00 – 2.86 (m, 8H), 2.49 (q, J = 8.9 Hz, 1H), 2.30 (dd, J = 14.8, 6.9 Hz, 1H), 2.13 – 2.03 (m, 1H), 2.03 – 1.92 (m, 3H), 1.88 – 1.41 (m, 23H), 1.42 – 1.33 (m, 1H), 1.05 – 0.75 (m, 36H).¹³C NMR (151 MHz, CD3OH) δ 174.61, 174.08, 173.97, 173.92, 173.69, 173.53, 173.42, 173.40, 172.85, 172.74, 172.58, 137.02, 130.51, 129.80, 128.61, 88.74, 85.37, 70.70, 62.05, 60.81, 59.09, 56.07, 53.68, 53.59, 53.55, 53.36, 52.74, 52.41, 51.78, 49.72, 47.95, 44.34, 42.20, 41.49, 41.43, 40.95, 40.84, 40.69, 37.43, 34.21, 31.87, 30.81, 30.73, 30.35, 30.25, 25.90, 25.87, 25.72, 25.66, 25.27, 25.11, 24.81, 24.56, 24.05, 23.29, 23.20, 23.15, 22.61, 21.92, 20.16, 19.92, 19.72, 19.61, 19.44, 19.04.

LC/MS: R^t 4.75 min, linear gradient 10→90% B in 13.5 min.; m/z = 1467.3 [M+H]⁺.

cyclo–[SAA

4

–OBn–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–Orn–Val]

^.

4TFA (3)

SAA 25 (105.3 mg, 2 eq) was pre–activated with HBTU (3 eq, 3 mL of 0.2 M HBTU in NMP), DIPEA (6 eq, 199 μL) in 10 mL NMP and subsequently coupled to the resin 28 (498 mg, 0.2 mmol, 0.402 mmol/g) for 4 h.; 29: LC/MS Rf 6.21 min, linear gradient 10→90% B in 13.5 min.; m/z = 817.33 [M+H]⁺. Resin 29 (0.1 mmol) was subjected to steps c–g and deprotected peptide was RP–HPLC purified (linear gradient of 36–45%, 3 CV) and yielded 3 as white powder (40.6 mg, 20.2 μmol, 20%). HRMS (ESI) m/z 1556.99245 [M+H]⁺, 1556.99268 calcd. for C79H130N17O15; ¹H NMR (600 MHz, CD3OH) δ 8.85 (d, J = 3.3 Hz, 1H, NHD– Phe13), 8.66 (d, J = 8.7 Hz, 1H, NH Val5), 8.55 (d, J = 10.0 Hz, 1H, NH Leu12), 8.53 (d, J = 8.8 Hz, 1H, NH Leu5), 8.53 (d, J = 8.9 Hz, 1H, NH Orn11), 8.49 (d, J = 8.2 Hz, 1H, NH Orn9), 8.47 (d, J = 10.0 Hz,

97 1H, NH Val10), 8.40 (d, J = 8.3 Hz, 2H, NH Orn2,4), 8.13 (t, J = 5.5 Hz, 1H, NH SAA4), 8.06 (d, J = 6.2 Hz, 1H, NH Leu8), 7.93 (br. s, NH2 Orn), 7.83 (br. s, NH2 Orn), 7.78 (d, J = 8.2 Hz, 1H, NH Val1), 7.40 – 7.21 (m, 10H), 4.93–4.83 (m, 4H, H^α Orn2,9,11), 4.79– 4.77 (m, 2H, H^α Orn4), 4.62–4.54 (m, 4H, H^α Leu3,12, CH benzyl, H^αD–Phe13), 4.43 (d, J = 11.6 Hz, 1H, CH benzyl), 4.36–4.34 (m, 2H, H^α Pro14, H^α Val10), 4.32–4.28 (m, 1H, H^α Leu8), 4.25–4.20 (m, 2H, H^α Val5), 4.05 (t, J = 8.4 Hz, 1H, H^α Val1), 3.72–

3.69 (m, 1H), 3.50–3.47 (m, 1H), 3.41–3.39 (m, 1H), 3.07–3.02 (m, 2H), 2.99 – 2.89 (m, 8H), 2.78–

2.76 (m, 1H), 2.55 (q, J = 8.3 Hz, 1H), 2.29–2.24 (m, 1H), 2.10 – 1.87 (m, 3H), 1.87 – 1.45 (m, 23H), 1.39–1.35 (m, 1H), 0.98 (d, J = 4.9 Hz, 3H), 0.97 (d, J = 5.2 Hz, 3H), 0.94 – 0.85 (m, 27H), 0.83 (d, J = 6.1 Hz, 3H). ¹³C NMR (151 MHz, CD3OH) δ 174.72, 174.51, 174.07, 173.76, 173.62, 173.53, 173.07, 172.92, 172.88, 172.70, 138.53, 137.08, 130.52, 129.78, 129.66, 129.26, 128.92, 128.56, 86.31, 83.17, 77.41, 72.36, 62.02, 61.09, 60.92, 59.57, 55.88, 54.20, 53.78, 53.60, 53.34, 52.91, 51.94, 48.05, 43.57, 42.30, 42.22, 41.62, 40.90, 40.86, 40.74, 40.58, 37.62, 33.71, 31.62, 30.86, 30.79, 30.66, 30.55, 30.41, 29.97, 25.93, 25.90, 25.78, 25.32, 25.15, 24.83, 24.70, 24.05, 23.46, 23.18, 23.03, 22.44, 21.78, 20.03, 19.98, 19.83, 19.69, 19.52, 19.10. LC/MS: R^t 6.13 min, linear gradient 10→90% B in 13.5 min.; m/z = 1557.3 [M+H]⁺.

cyclo–[SAA

5

–OH–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–Orn–Val]

^.

4TFA (4)

SAA 23 (28. 1 mg, 1.5 eq) was pre–activated with HCTU (1.4 eq, 57.9 mg), DIPEA (2.8 eq, 46 μL) in 10 mL NMP and subsequently coupled to the resin 28 (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h.; 30: LC/MS R^t 5.14 min, linear gradient 10→90% B in 13.5 min.; m/z = 741.33 [M+H]⁺. Resin 30 (0.1 mmol) was subjected to steps c–g and deprotected peptide was RP–HPLC purified (linear gradient of 38–47%, 3 CV) and yielded 4 as white powder (12.0 mg, 6.2 μmol, 6.2%). HRMS (ESI) m/z 1480.96331 [M+H]⁺, 1480.96138 calcd. for C73H126N17O15; ¹H NMR (600 MHz, CD3OH) δ 9.05 (d, J = 9.6 Hz, 1H, NH Val5), 8.94 (d, J = 3.1 Hz, 1H, NHD–Phe13), 8.78 (d, J = 9.1 Hz, 1H, NH Leu3), 8.66 (d, J = 8.4 Hz, 1H, NH Orn9), 8.65 (d, J = 7.5 Hz, 1H, NH Leu12), 8.62 (d, J = 9.6 Hz, 1H, NH Leu8), 8.54 (d, J = 8.4 Hz, 1H, NH Orn11), 8.48 (br. s, 1H, NH2 Orn), 8.45 (d, J = 8.5 Hz, 1H, NH Orn4), 8.43 (d, J = 8.0 Hz, 1H, NH Orn2), 8.28 (t, J = 6.2 Hz, 1H, NH SAA5), 8.01 (d, J = 6.4 Hz, 1H, NH Val10), 7.92 (br. s, NH2 Orn), 7.76 (d, J = 8.6 Hz, 1H, NH Val1), 7.37 – 7.18 (m, 5H), 5.12 – 5.08 (m, 2H, Hα Orn4,11), 5.08 – 5.00 (m, 2H, Hα Orn2,9), 4.68 – 4.65 (m, 2H, Hα Leu3,12), 4.57 – 4.48 (m, 1H, HαD–Phe13), 4.42 (dd, J = 10.8, 5.4 Hz, 1H), 4.38 – 4.35 (m, 2H, Hα Pro14, Hα Leu8), 4.27 – 4.15 (m, 2H, Hα Val10), 4.11 – 4.09 (m, 2H, Hα Val1,5), 4.07 – 4.05 (m, 1H), 3.91 (dd, J = 13.9, 7.0 Hz, 1H), 3.73 (dd, J = 12.5, 5.4 Hz, 1H), 3.10 – 3.05 (m, 3H), 3.00 – 2.83 (m, 8H), 2.48 (d, J = 8.1 Hz, 1H), 2.30 (dd, J = 15.0, 6.9 Hz, 1H), 2.09 (dd, J = 11.8, 5.6 Hz, 1H), 2.05 – 1.28 (m, 32H), 1.08 – 0.70 (m, 36H). ¹³C NMR (151 MHz, CD3OH) δ 175.21, 175.18, 174.27, 173.94, 173.73, 173.68, 173.65, 173.55, 173.49, 173.43, 172.85, 172.80, 172.60, 163.23, 163.01, 137.02, 130.51, 129.82, 128.63, 89.45, 79.08, 73.59, 62.05, 60.92, 60.58, 59.36, 56.09, 54.07, 53.37, 53.20, 52.69, 52.44, 51.77, 49.72, 47.94, 44.63, 42.23, 42.13, 41.86, 41.40, 40.70, 37.42, 34.26, 31.84, 31.67, 30.73, 30.51, 29.63, 25.91, 25.70, 25.39, 25.15, 24.83, 24.55, 24.27, 23.45, 23.23, 23.11, 22.45, 21.81, 19.94, 19.71, 19.69, 19.34, 19.27. LC/MS: R^t 5.81 min, linear gradient 10→90% B in 13.5 min.; m/z = 1482.3 [M+H]⁺.

cyclo–[SAA

5

–OBn–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–Orn–Val]

^.

4TFA (5)

SAA 24 (33.2 mg, 1.2 eq) was pre–activated with HCTU (1.2 eq, 50 mg), DIPEA (3 eq, 60 μL) in 10 mL NMP and subsequently coupled to the resin 28 (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h.; 31: LC/MS R^t 6.31 min, linear gradient 10→90% B in 13.5 min.; m/z = 831.53 [M+H]⁺. Resin 31 (0.05 mmol) was subjected to steps c–g and deprotected peptide was RP–HPLC purified (linear gradient of 43–52%, 3 CV) and yielded 5 as white powder (11.84 mg, 5.84 μmol, 12%). HRMS (ESI) m/z 11571.01082 [M+H]⁺, 1571.00833 calcd. for C80H132N17O15; ¹H NMR (600 MHz, CD3OH) δ 9.04 (d, J = 9.6 Hz, 1H, NH Val5), 8.94 (s, 1H, NHD– Phe13), 8.76 (d, J = 9.0 Hz, 1H, NH Leu3), 8.65 (d, J = 7.6 Hz, 1H, NH Orn11), 8.64 (d, J = 7.7 Hz, 1H, NH Leu12), 8.63 (d, J = 9.3 Hz, 1H, NH Val10), 8.55 (d, J = 8.1 Hz, 1H, NH Orn9), 8.43 (d, J = 9.2 Hz, 2H, NH Orn2,4), 8.33 (t, J = 5.9 Hz, 1H, NH SAA), 8.01 (d, J = 6.3 Hz, 1H, NH Leu8), 7.92 (br s. NH2

Orn), 7.77 (d, J = 8.6 Hz, 1H, NH Val1), 7.41 – 7.20 (m, 10H), 5.14 – 5.12 (m, 1H, Hα Orn4), 5.09 – 4.99 (m, 2H, Hα Orn2,11), 4.88 (m 1H, Hα Orn9), 4.72 – 4.61 (m, 2H, Hα Leu3,12), 4.57 – 4.50 (m, 3H, Hα D–Phe13), 4.39 – 4.37 (m, 3H, Hα Pro, Hα Val10), 4.28 (s, 1H), 4.19 (dt, J = 10.9, 5.5 Hz, 1H, Hα Leu8), 4.15 – 4.05 (m, 3H, Hα Val1,5), 3.94 (dd, J = 13.8, 7.0 Hz, 1H), 3.73 (dd, J = 12.8, 5.3 Hz, 1H), 3.15 – 3.01 (m, 3H), 3.00 – 2.78 (m, 8H), 2.49 (q, J = 8.9 Hz, 1H), 2.37 – 2.24 (m, 2H), 2.05 – 1.25 (m, 32H), 1.04 – 0.71 (m, 36H). ¹³C NMR (151 MHz, CD3OH) δ 175.17, 174.86, 174.29, 173.95, 173.72, 173.67, 173.64, 173.59, 173.48, 173.43, 172.84, 172.80, 172.58, 163.25, 163.02, 139.28, 137.01, 130.50, 129.81, 129.56, 128.95, 128.85, 128.62, 87.15, 81.77, 79.41, 72.21, 62.04, 60.93, 60.60, 59.37, 56.08, 54.15, 53.34, 53.14, 52.68, 52.44, 51.76, 49.75, 47.93, 44.65, 42.57, 42.23, 41.43, 40.96, 40.81, 40.75, 40.70, 39.34, 37.41, 34.26, 31.81, 31.79, 30.72, 30.68, 30.49, 29.54, 25.90, 25.70, 25.35, 25.25, 25.14, 24.82, 24.55, 24.30, 23.46, 23.22, 23.11, 22.44, 21.79, 19.95, 19.72, 19.65, 19.64, 19.34, 19.28. LC/MS: R^t 6.39 min, linear gradient 10→90% B in 13.5 min.; m/z = 1571.5 [M+H]⁺.

cyclo–[SAA

6

–OH–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–Orn–Val]

^.

4TFA (6)

SAA 26 (62.4 mg, 1.55 eq) was pre–activated with HCTU (1.45 eq, 120 mg), DIPEA (2.9 eq, 96 μL) in 10 mL NMP and subsequently coupled to the resin 28 (400 mg, 0.2 mmol, 0.5 mmol/g) for 4 h.; 32: LC/MS Rt 5.28 min, linear gradient 10→90% B in 13.5 min.; m/z = 755.33 [M+H]⁺. Resin 32 (0.1 mmol) was subjected to steps c–g and deprotected peptide was RP–HPLC purified (linear gradient of 37–46%, 3 CV) and yielded 5 as white powder (1.8 mg, 0.9 μmol, 1%). HRMS (ESI) m/z 747.99199 [M+H]²⁺, 747.99215 calcd. for C74H129N17O15; ¹H NMR (600 MHz, CD3OH) δ 8.92 (d, J = 3.2 Hz, 1H, NHD–Phe13), 8.74 (d, J = 8.9 Hz, 1H, NH Leu8), 8.63 (d, J = 8.3 Hz, 1H, NH Orn11), 8.62 (d, J = 8.5 Hz, 1H, NH Leu12), 8.61 (d, J = 9.3 Hz, 2H, NH Val5,10), 8.53 (d, J = 8.3 Hz, 1H, NH Orn4), 8.42 (t, J = 9.0 Hz, 2H, NH Orn2,9), 8.09 (d, J = 8.5 Hz, 2H, NH Leu3, NH SAA6), 7.77 (d, J = 8.5 Hz, 1H, NH Val1), 7.38 – 7.21 (m, 5H), 5.11– 5.10 (m, 1H, Hα Orn9), 5.04 – 5.01 (m, 2H, Hα Orn2,11), 4.87 – 4.85 (m, 1H, Hα Orn4), 4.67 – 4.64 (m, 2H, Hα Leu8,12), 4.53 (dd, J = 10.7, 4.6 Hz, 1H, HαD–Phe13), 4.50 – 4.40 (m, 2H, Hα Leu3), 4.39 – 4.32 (m, 3H, Hα Pro14 Hα Val10), 4.15 (t, J = 9.4 Hz, 1H, Hα Val5), 4.11 (t, J = 8.4 Hz, 1H, Hα Val1), 3.94 – 3.90 (m, 2H), 3.73 (t, J = 9.0 Hz, 1H), 3.37 – 3.18 (m, 3H), 3.12–3.01 (m, 2H), 3.01 – 2.78 (m, 8H), 2.50 (dd, J = 17.2, 8.2 Hz, 1H), 2.30 (dd, J = 14.6, 7.0 Hz, 1H), 2.14 – 1.90 (m, 6H), 1.86 – 1.44 (m, 26H), 1.43 – 1.30 (m, 1H), 1.18–1.12 (m, 1H), 1.06 – 0.71 (m, 36H). ¹³C NMR (151 MHz, CD3OH) δ 175.15, 174.28, 174.15, 174.10, 173.97, 173.71, 173.47, 172.85, 172.62, 163.23, 137.01, 130.49, 129.80, 128.61, 81.67, 75.82, 66.12, 62.04, 60.89, 59.39, 56.05, 54.10, 53.39, 52.73, 52.55, 52.42, 51.79, 47.95, 42.25, 42.13, 40.96, 40.81, 40.69, 40.05, 37.44, 34.16, 32.48, 31.86, 31.77, 30.93, 30.71, 29.77, 29.56, 25.91, 25.87, 25.71, 25.29, 24.85, 24.58, 24.28, 23.44, 23.21, 23.13,

NH NH

HN N H

HN

HN N H

HN N H O

N O

O O O O

O

NH NH3+

+H3N O

O +H3N

NH3+ O

O H N O O HN

O OH

99 22.54, 21.93, 19.97, 19.75, 19.65, 19.60, 19.28. LC/MS: R^t 5.77 min, linear gradient 10→90% B in 13.5 min.; m/z = 1495.3 [M+H]⁺.

cyclo–[SAA

6

–OBn–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–Orn–Val]

^.

4TFA (7)

SAA 27 (44 mg, 1.5 eq) was pre–activated with HCTU (1.

5 eq, 63 mg), DIPEA (3 eq, 50 μL) in 10 mL NMP and subsequently coupled to the resin 28 (200 mg, 0.1 mmol, 0.5 mmol/g) for 4 h.; 33: LC/MS Rt 6.68 min, linear gradient 10→90% B in 13.5 min.; m/z = 845.33 [M+H]⁺. Resin 32 (0.1 mmol) was subjected to steps c–g and deprotected peptide was RP–HPLC purified (linear gradient of 43–52%, 3 CV) and yielded 7 as white powder (14.3 mg, 7.0 μmol, 7%). HRMS (ESI) m/z 1585.02578 [M+H]⁺, 1585.02398 calcd. for C81H134N17O15; ¹H NMR (600 MHz, CD3OH) δ 8.92 (d, J = 3.3 Hz, 1H, NHD–Phe13), 8.73 (d, J = 8.9 Hz, 1H, NH Leu3), 8.63 (d, J = 8.9 Hz, 2H, NH Val10, Leu12), 8.61 (d, J = 9.2 Hz, 2H, NH Orn11, Val5), 8.53 (d, J = 8.2 Hz, 1H, NH Orn9), 8.42 (d, J = 9.2 Hz, 2H, NH Orn2,4), 8.11 (d, J = 8.4 Hz, 1H, NH Leu8), 7.92 (t, J = 6.1 Hz, 1H, NH SAA), 7.77 (d, J = 8.5 Hz, 1H, NH Val1), 7.40 – 7.22 (m, 10H), 5.09 (t, J = 7.0 Hz, 1H, Hα Orn4), 5.04 – 5.02 (m, 2H, Hα Orn2,11), 4.87 – 4.83 (m, 1H, Hα Orn9), 4.73 – 4.60 (m, 3H, Hα Leu3,12), 4.58 – 4.49 (m, 2H, HαD–Phe13), 4.44 – 4.42 (m, 1H, Hα Leu8), 4.37 – 4.36 (m, 2H, Hα Pro14, Hα Val10), 4.15 (t, J = 9.4 Hz, 1H, Hα Val5), 4.11 (t, J = 8.5 Hz, 1H, Hα Val1), 3.96 – 3.87 (m, 2H), 3.73 (dd, J = 12.7, 5.3 Hz, 1H), 3.46 (d, J = 9.3 Hz, 1H), 3.27 – 3.16 (m, 2H), 3.16 – 3.01 (m, 3H), 3.01 – 2.89 (m, 6H), 2.84 (s, 2H), 2.49 (t, J = 8.5 Hz, 1H), 2.38 – 2.24 (m, 2H), 2.10 (dd, J = 13.3, 2.7 Hz, 1H), 2.08 – 1.90 (m, 4H), 1.86 – 1.43 (m, 26H), 1.41 – 1.25 (m, 1H), 1.12 (d, J = 14.7 Hz, 1H), 1.03 – 0.74 (m, 36H). ¹³C NMR (151 MHz, CD3OH) δ 175.16, 174.30, 174.13, 174.01, 173.98, 173.70, 173.68, 173.51, 173.44, 172.86, 172.81, 172.61, 139.88, 137.01, 130.50, 129.81, 129.53, 128.99, 128.86, 128.61, 80.05, 75.93, 73.54, 71.75, 62.04, 60.91, 60.89, 59.39, 56.05, 54.17, 53.39, 53.37, 52.72, 52.57, 52.45, 51.81, 49.72, 47.96, 42.25, 42.12, 40.96, 40.79, 40.16, 37.44, 34.14, 31.85, 31.81, 30.94, 30.81, 30.72, 29.56, 29.26, 29.01, 25.91, 25.87, 25.71, 25.23, 24.84, 24.58, 24.31, 23.44, 23.19, 23.13, 22.56, 21.91, 19.98, 19.76, 19.66, 19.62, 19.30. LC/MS: R^t 6.51 min, linear gradient 10→90% B in 13.5 min.;

m/z = 1585.4 [M+H]⁺.

cyclo–[SAA

4

–OH–Leu–Orn–Val–Orn–Leu–

^D

Phe–Pro–Val–Orn–Leu–

^D

Orn–Val]

^.

4TFA (9)

The hydrogenated peptide was RP–HPLC purified (linear gradient of 31–40%, 3 CV) and yielded 9 as white powder (5.74 mg, 2.98 μmol, 32%). HRMS (ESI) m/z 11466.94689 [M+H]⁺, 1466.94573 calcd. for C72H124N17O15; ¹H NMR (600 MHz, CD3OH) δ 8.81 (d, J = 3.9 Hz, 1H. NH D– Phe13), 8.55 (d, J = 8.4 Hz, 1H, NH Orn2), 8.54 (d, J = 7.7 Hz, 1H, NH Orn9), 8.45 (d, J = 8.3 Hz, 1H, NH Orn11), 8.42 (d, J = 8.8 Hz, 1H, NH Leu12), 8.33 (d, J = 8.3 Hz, 2H, NH Leu8, NH SAA4), 8.30 (d, J = 8.0 Hz, 1H, NH Leu3), 8.24 (d, J = 8.2 Hz, 1H, NH Val5), 7.98 (d, J = 8.4 Hz, 1H, NH Val10), 7.94 (d, J = 7.8 Hz, 1H, NH Orn4), 7.86 (d, J = 8.0 Hz, 1H, NH Val1) 7.85 (br. s. NH2 Orn), 7.32–7.24 (m, 5H), 4.82 – 4.78 (m, 1H, Hα Orn11), 4.72 (d, J = 4.6 Hz, 1H), 4.63 – 4.46 (m, 8H, HαD–Phe13, Hα Orn2,4,9, Hα Leu3,8,12), 4.41 – 4.34 (m, 1H, Hα Pro14), 4.32 (t, J = 7.4 Hz, 1H, Hα Val5), 4.26 (t, J = 7.9 Hz, 1H, Hα Val10), 4.21 (t, J = 4.3 Hz, 1H), 4.06 – 3.95 (m, 2H, Hα Val1), 3.72–3.69 (m, 1H), 3.65 (s, 1H), 3.21 – 3.14 (m, 2H), 3.06 (dd, J = 12.8, 5.7 Hz, 1H), 3.03 – 2.90 (m, 9H), 2.63 (dd, J = 17.9, 9.4 Hz, 1H), 2.26 (td, J = 13.6, 6.8 Hz, 1H), 1.95 – 1.49 (m, 29H), 1.44 – 1.35 (m, 2H), 1.06 – 0.82 (m, 36H). ¹³C NMR (151 MHz, CD3OH) δ 175.34, 174.57, 174.30, 174.17, 173.99, 173.81, 173.55, 173.51, 173.48, 173.37, 172.74, 163.12, 162.90, 137.20, 130.53, 129.77, 128.53, 89.10, 86.08, 72.83, 64.59, 61.94, 61.29, 60.27, 55.73, 54.44, 54.10, 53.66, 53.42, 53.39, 52.90, 52.29, 49.72, 48.06, 43.22, 42.32, 41.89, 41.81, 40.85, 40.67, 40.51, 37.71, 32.34, 32.21, 31.51, 31.12, 30.65, 29.75,

NH NH

HN N H

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 O HN

O OH

NH NH

HN N H

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 O HN O

OBn