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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[51]

Application of the Triazole Moiety in the Turn-Region of Gramicidin S to Mimic the Phenyl-Ring

3.1 I

The cyclic decapeptide gramicidin S (GS, 1)

is a highly potent biocidal natural product, active against a broad range of bacteria, both Gram-positive and Gram-negative.

Although the exact mode of action is not known, it is thought that the basis of its activity is the rigid conformation of the peptide: GS is comprised of two type II' β-turns, and two β-strands, stabilized by four interstrand hydrogen bonds (Figure 1).

In this way the hydrophobic and hydrophilic residues are segregated on different faces of the molecule, rendering the peptide amphiphilic. This amphiphilicity appears to be a prerequisite for the antibacterial activity of the cationic peptide.

Unfortunately, the membrane lytic properties of GS are not limited to bacteria or other

life-threatening pathogens, confining its use to topical applications. However, because GS

targets the cell membrane and not a gene product, it is a very attractive starting point for the

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development of a new antibiotic with broad medical applications. Towards this goal countless derivatives of GS have been designed and synthesised, with the hope to obtain a peptide with high antibacterial activity and low toxicity towards mammalian cells.

The turns of GS, consisting of the

Phe-Pro motif, can be modified by changing the amino acids or by incorporation of dipeptide isosteres, like sugar amino acids (SAAs).

For example, replacement of the

-phenylalanine by

-tyrosine (2) led to a decrease in antibacterial activity compared to the parent compound, while benzylation of the phenolic position (3) restored the activity.

Grotenbreg et al. also replaced the

Phe-Pro segment of GS by a sugar derived ε-amino acid (4).

The crystal structure of the SAA-modified GS showed a slightly altered turn, in which one of the free hydroxyls was involved in an intra- residual hydrogen-bond. Also the antibacterial activity of the peptide was lost.

Arylmethylation of the hydroxyl not involved in hydrogen-bonding (5) did not result in a major change in structure,

however, the activity was restored. These findings led to the conclusions that alterations in the β-turn are allowed, hydrophobicity in the turn is vital for the antibacterial activity and aromaticity in the turn is an additional benefit to the membrane lytic activity of GS.

In the previous chapter it was shown that the

-phenylalanine residue in GS is a feasible position for modifications. A popular and synthetically easily accessible aromatic functionality is the 1,2,3-triazole. The past years a lot of attention has been devoted to the preparation of triazoles (8 and 9) by means of the 1,3-dipolar Huisgen cycloaddition reaction between an azide 6 and a terminal alkyne 7 (Figure 2).

The question addressed in this chapter is

Figure 1

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whether the phenyl ring in GS may be substituted by a triazole, without loss of antibacterial activity. An asymmetric GS derivative with the unsubstituted triazole instead of the phenyl ring was prepared, as well as the corresponding imidazole analogue. As shown by Yamada et al.

a basic residue ((Z)-(β-3-pyridyl)-α,β-dehydroalanine) at this position has a lowering effect on the hemolytic activity of GS. The copper(I)-catalysed Huisgen cycloaddition reaction has been widely used in conjugation reactions. Therefore, incorporation of

-propargylglycine instead of

-phenylalanine into GS will render the molecule easily modifiable or conjugatable

at this position. This was investigated by the synthesis of a series of 1,4-substituted triazoles, with increasing aromatic bulk. Besides the copper(I)-catalysed triazole synthesis, leading exclusively to the 1,4-product (8), the Ru(II)-catalysed conjugation between azides and alkynes is known to lead to the predominant formation of the 1,5-product (9, Figure 2).

This Ru(II)-catalysed reaction was also used to sample the space available around the i +1 position of the β-turn for modifications.

3.2 R

D

The required azides 6a-e were accessed from their corresponding alcohols 10a-e by reaction with diphenyl phosphoryl azide (DPPA) and DBU (Scheme 1),

with the exception

Figure 2

Scheme 1 Reagents and conditions i) DPPA, DBU, toluene, 0 °C, (6b: 100%, 6c: 100%, 6d: 97%, 6e:

80%; ii) NaN3, DMSO (100%)

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of benzyl azide 6a, which was prepared by azidolysis of benzyl bromide 10a. For the synthesis of alkyne-containing GS stepwise Fmoc-based solid-phase synthesis was applied. This started with the hyper-acid labile HMPB-MBHA resin, loaded with Fmoc-Leu-OH 11, which was exposed to a repeated cycle of Fmoc-deprotection with 20% piperidine in NMP, followed by coupling with the appropriate Fmoc-aa-OH, HCTU and DiPEA. As the last amino acid the commercial N-Fmoc-

-propargylglycine was incorporated. Once the decamer 12 was assembled, the N-terminal Fmoc-group was removed and the peptide liberated from the solid support under mild acidic conditions, leaving the Boc-groups in place. Cyclisation was performed under dilute conditions in DMF, followed by LH-20 size-exclusion column chromatography, to give protected 7. The protected di-Boc-

-histidyl-GS derivative 14 was synthesised in a similar fashion, and then deprotected to give the free peptide 15.

With both peptide 7 and a variety of azides (6a-e) in hand, further synthetic steps were taken. Cycloadditions were performed under the agency of in situ generated copper(I) in DMF to give GS derivatives containing a 1,4-triazole, still containing the Boc groups on the ornithine side chains (Scheme 3). This method appeared to work well for all azides involved.

Scheme 2 Reagents and conditions i) 20% pip/NMP (2 x 10 min); ii) Fmoc-aa-OH (2.5 eq), HCTU (2.5 eq), DiPEA (3 eq), NMP (1.5 h); iii) 1% TFA/DCM (6 x 10 min); iv) PyBOP (5 eq), HOBt (5 eq), DiPEA (15 eq), 0.01M DMF; v) 50% TFA/DCM.

[55]

To obtain the unsubstituted triazole, 2,4-dimethoxybenzyl azide (6e) was employed in the click reaction and the obtained triazole 8e was deprotected with 50% TFA in DCM under the influence of brief microwave irradiation to yield 8f. Towards the synthesis of the 1,5-triazoles 9a-d the procedure of Pradere et al.

was followed. This implied the reaction of the alkyne and azide under the agency of catalytic Cp*RuCl(PPh

)

and microwave irradiation. It was found that with increasing steric hindrance of the azide, the reaction time increased (phenyl ≤ naphtyl < phenantryl < pyrenyl). All the obtained peptides were deprotected with TFA in DCM and purified by preparative HPLC (8a-d,f and 9a-d). Due to difficult removal of catalyst impurities the yields were invariably low for the 1,5-triazole series (9a-d).

The obtained peptides were subjected to NMR studies. The signals were unambiguously assigned by a combination of

H NMR, COSY and TOCSY spectra (Data can be found in experimental section). The spectra of the 1,4-triazoles 8a-d, the ‘bare’ triazole 8f and the histidine analogue 15 showed the characteristics of two type II' β-turns connected by two β- sheets. That is, small coupling constants (2.5 < J

< 3.5 Hz) for the amide protons of

- phenylalanine,

-histidine and

-triazolalanine were observed. The J

of the strand amino

Scheme 3 Reagents and conditions i) CuSO4, sodium ascorbate, 6a-e, DMF; ii) TFA/DCM; iii) Cp*RuCl(PPh3)2, 6a-d, THF, μW; iv) 50% TFA/DCM, μW.

Scheme 4 Reagents and conditions i) Cp*RuCl(PPh3)2, 6a, THF, μW; ii) LiOH, dioxane/H2O; iii) TFA/DCM; iv) FmocOSu, NaHCO3, dioxane/H2O

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acid residues, on the other hand, showed large coupling constants (J

> 8 Hz). Both values support the cyclic β-hairpin structure.

Also the CROESY spectra showed characteristic cross-peaks. Surprisingly, the

H- and

C NMR spectra of the 1,4-substituted triazoles 8 were identical to the spectra of the 1,5-triazoles 9. Also co-injection of compounds 8a and 9a on an analytical C

HPLC column strongly indicated that the products from both reactions were identical. Apparently, both the copper(I) and the ruthenium(II) catalysed reaction led to the same product, presumably the 1,4-substituted triazoles. To verify this hypothesis the Cp*RuCl(PPh

)

-catalysed reaction was applied to the Boc-

-propargylglycine methyl ester 16

and benzyl azide (Scheme 4). HPLC analysis indicated that the Ru(II)-catalysed cycloaddition reaction at the monomer level gave an unseparable mixture, presumably of 1,4- and 1,5-triazoles (17). NMR analysis was hampered by the presence of two regioisomers, split up in two rotamers. Further synthesis was performed on this regioisomeric mixture. A deprotection/reprotection sequence led to the regioisomeric Fmoc-building block 18, which was used in solid-phase peptide synthesis similar to the synthesis of 15. Before Boc- deprotection a mixture of the two products with identical mass was observed by HPLC analysis, presumably the two regioisomers. Curiously, this difference was not observed after treatment with 50% TFA in DCM. Also the NMR of the HPLC-purified product showed a single product, matching 8a obtained before. Closely examining the NOE signals in the CROESY spectrum did not indicate a NOE crosspeak between the benzylic protons and the β- protons of the amino acid, which would likely arise in the 1,5-substituted product. From this it can be concluded that somehow a regioisomerisation reaction occurs during the deprotection step, although the exact mechanism is yet unknown.

3.1.2 B

The antibacterial activities of triazoles 8a-d,f and imidazole 15 are listed in Table 1.

Substituted 1,4-triazoles 8a-d are at least equally potent to GS. Histidine analogue 15 appears

[57]

to be more potent in killing Gram-negative bacteria than GS. It is surprising therefore that non-methylarylated 8f eliminates bacteria only at higher concentrations.

Also the hemolytic activities of triazoles 8a-8d, 8f and histidine 15 were determined and the results are presented in Figure 3. The most active compounds (8b-d) are all more toxic than gramicidin S. The benzyl substituted derivative 8a and 8f both show lower hemolytic activity, which may be correlated with a lower diminished ability to eliminate bacteria. The highly antibacterial histidine analogue 15 displays the lowest hemolytic activity of all peptides tested.

3.3 C

A series of triazole- and imidazole derivatives of GS was prepared. Key molecule in the synthetic scheme was GS-derivative 7 in which one

-phenylalanine was substituted by a

- propargylglycine. The triazoles were synthesized using a copper(I) catalysed cycloaddition reaction between 7 and aromatic azides (6a-d). The acid-labile dimethoxybenzyl group was used to prepare the 1H-triazole (8f). An alternative cycloaddition reaction, catalysed by Cp*RuCl(PPh

)

and known to lead to the 1,5-substituted triazoles 9a-d was applied as well.

The obtained peptides, however, appeared to be identical to the 1,4-products 8a-d, as judged by NMR spectroscopy and HPLC analysis. In order to explain this result, the 1,5-substituted triazolalanine building block (18) was synthesised by the Ru(II)-catalysed cycloaddition reaction. This yielded a regioisomeric mixture of 1,4- and 1,5-triazoles, which was

Bacterialstrain: GS 8a 8b 8c 8d 8f 15

S.aureus7323 8 8 4 4 8 16 4

S.aureus7388 4 4 4 4 32 4

CNS5277 4 8 4 4 4 16 4

CNS5115 8 4 4 8 32 4

CNS7368 8 4 4 4 8 4

E.faecalis1131 8 16 4 4 16 64 16

E.coliATCC25922 32 64 64 >64 32 8

P.aeruginosaAK1 16 64 >64 64 8 64 4

P.aeruginosaATCC19582 64 >64 64 >64 >64 16

S.mitisBMS 16 4 4 4 32 4

S.mitisATCC33399 4 32 4 8 16 64 16

Table 1 Antibacterial activities in μg/mL of 8a-d,f and 15 compared to GS.

[58]

incorporated in the cyclic peptide. Acid treatment of the Boc-protected regioisomeric mixture gave a single GS derivative 8a. These observations suggest that during the acid deprotection a regioisomerisation occured. However, further experimentation is required to verify this hypothesis and if it is possible to synthesise the deprotected cyclic peptides 14a-d via a different route.

The methylarylated 1,4-triazoles 8a-d appeared to be highly potent in killing bacteria, showing that substituted triazoles are good mimics for the phenyl-ring in the turn region of GS. Also histidine analogue 15 showed strong activity against both Gram-positive and Gram- negative bacteria, making it even more potent than GS, which is only weakly active against Gram-negative strains. The ‘bare’ triazole 8f on the other hand, was only moderately active against Gram-positive bacteria. In general the hemolytic activity could be correlated with the antibacterial activity, i.e. the higher the antibacterial activity, the higher the toxicity towards blood cells. The positive exception to this is the highly antibacterial peptide 15. This histidine analogue of GS was considerably less hemolytic than the mother compound.

3.4 E

S

3.4.1 General Procedures

Solvents and chemicals were used as received from their supplier. Solvents were stored over 4 Å molecular sieves (or 3 Å MS for MeOH). Solvents for extractions and silica gel chromatography were of technical grade and distilled before use. ¹H and ¹³C NMR spectra were recorded with a Bruker AV-400 (400/100 MHz), or Bruker DMX-600 spectrometer (600/150 MHz). Chemical shifts δ are given in ppm relative to tetramethylsilane (0 ppm) or CD3OH (3.31 ppm) as internal standard. High resolution mass spectra were recorded by direct injection (2 μL

0 1 2 3

0 50 100

8a 8b 8c 8d 8f 15 GS DMSO log [c] in uM

% Hemolysis

Figure 3 Hemolytic activity of peptides 8a-d, 8f and 15.

[59]

of a 2 μM solution in water/acetonitrile; 50:50 v/v and 0.1% formic acid) on a mass spectrometer (Termo Finnigan LTQ Orbitrap) equipped with an electrospray 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% aq TFA. HPLC purifications were performed on 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.

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 solution 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 8a-d, 8f and 15were dissolved in ethanol (4 g/L) and diluted in distilled water (1000 mg/L), and dilution in the broth to achieve the right concentration (64, 32, 16, 8, 4 and 1 mg/L). The bacteria were incubated at 30 °C (24-96 h) and the MIC was determined as the lowest concentration inhibiting bacterial growth.

Hemolytic assay

Freshly drawn heparinised blood was centrifuged for 10 minutes at 1000g at 10°C. Subsequently, the erythrocyte pellet was washed three times with 0.85% saline solution and diluted with saline 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. Subsequently, 50 μL of the red blood cell solution was added to the wells and the plates were incubated at 37°C for 4 hours. After incubation, the plates were centrifuged at 1000g at 10°C 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.

Benzyl azide (6a)^[26]

Benzyl bromide (20 mmol, 2.38 mL) was dissolved in 0.5 M NaN3 (1.1 eq, 44 mL) in DMSO. The mixture was stirred for 1 h and quenched with H2O (150 mL). The product was extracted with Et2O (3 u 50 mL).

The ethereal layer was dried (MgSO4), filtered and evaporated to yield the pure compound in quantitative yield.

1H NMR (400 MHz, CDCl3) δ 7.43-7.32 (m, 5H), 4.35 (s, 2H); ¹³C NMR (100 MHz, CDCl3) δ 135.31, 128.78, 128.25, 128.16, 65.80.

Preparation of azides 6b-e from their corresponding alcohols.

The alcohol (5 mmol) was dissolved in toluene (dry, 9 mL) and cooled to 0 °C under argon atmosphere. Then DPPA (1.2 eq, 6 mmol, 1.32 mL) and DBU (1.2 eq, 6 mmol, 0.92 mL) were subsequently added. When TLC indicated completion of the reaction, the mixture was diluted with Et2O and washed with water. The organic fractions were dried over MgSO4, filtered and evaporated. The pure compound was obtained by silica gel column chromatography (10% toluene in PE).

1-(azidomethyl)naphthalene (6b)^[27]

Following the general procedure and column chromatography the title compound was obtained in 99% yield (4.95 mmol).

[60]

1H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz, 1H), 7.82-7.76 (m, 2H), 7.52-7.44 (m, 4H), 4.63 (s, 2H); ¹³C NMR (100 MHz, CDCl3) δ 133.8, 131.2, 130.9, 129.3, 128.7, 127.1, 126.6, 126.0, 125.1, 123.3, 52.8.

9-(azidomethyl)phenanthrene (6c)^[28]

Column chromatography yielded 4c in 99% (4.95 mmol).

1H NMR (400 MHz, CDCl3) δ 8.74 (d, J = 7.6 Hz, 1H), 8.67 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.71-7.61 (m, 4H), 4.80 (s, 2H); ¹³C NMR (100 MHz, CDCl3) δ 131.04, 130.89, 130.76, 130.00, 129.36, 128.79, 128.27, 127.27, 127.07, 126.95, 126.89, 124.25, 123.30, 122.59, 53.61.

1-(azidomethyl)pyrene (6d)

The title compound was obtained in 97% yield (4.85 mmol).

1H NMR (400 MHz, CDCl3) δ 8.13-7.92 (m, 7H), 7.83 (d, J = 8 Hz, 2H), 4.89 (s, 2H); ¹³C NMR (100 MHz, CDCl3) δ 131.6, 131.1, 130.6, 129.1, 128.2, 128.1, 127.3, 127.2, 126.1, 125.5, 125.4, 124.9, 124.5, 122.5, 53.0; IR neat (cm^-1) 3042.0, 2099.7, 1593.6, 1249.8, 1225.8, 894.6, 818.5, 752.0, 699.0, 663.7.

2,4-dimethoxybenzyl azide (6e)^[29]

Column chromatography (5% Et2O/PE + 0.1% Et3N) was applied to obtain the title compound in 80% yield (4 mmol).

1H NMR (400 MHz, CDCl3) δ 7.14 (d, J = 10.4 Hz, 1H), 6.49-6.44 (m, 2H), 4.27 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H);

13C NMR (100 MHz, CDCl3) δ 161.2, 158.8, 131.0, 116.2, 104.0, 98.6, 55.3, 49.8. IR neat (cm^-1): 2090.6, 1611.6, 1587.7, 1507.9, 1288.2, 1265.9, 1156.9, 1128.6, 1032.3

General peptide synthesis

Stepwise Elongation: Fmoc-Leu-HMPB-MBHA resin 11 (Loading of the resin was 0.56 mmol/g, 1.5 mmol) was submitted to nine cycles of Fmoc solid-phase synthesis with the appropriate commercial amino acid building blocks. The amino group on the side chain of ornithine was protected with a Boc-group. Fmoc removal was effected by treatment with 20% piperidine in NMP for 2 u 10 min. The resin was subsequently washed with NMP, DCM, MeOH, and finally NMP. The Fmoc-aa-OH (3.75 mmol, 2.5 eq), HCTU (3.75 mmol, 2.5 eq) in NMP was pre-activated for 1 min after the addition of DiPEA (7.5 mmol, 5 eq) and then added to the resin. The suspension was shaken for 1.5 h. The resin was washed with NMP, DCM, MeOH and NMP.

Cleavage from the resin: After the final Fmoc deprotection the resin was washed with NMP and DCM and treated with 50 mL 1% TFA in DCM (6 u 10 min). The filtrates were collected and coevaporated with toluene (3 u 100 mL).

Cyclisation: In DMF (1.2 L) were dissolved PyBOP (7.5 mmol, 5 eq), HOBt (7.5 mmol, 5 eq), and DiPEA (22.5 mmol, 15 eq). The linear decapeptide (1.5 mmol) was dissolved in DMF (15 mL) and added dropwise over 1 h to the coupling cocktail. After addition the mixture was stirred for 16 h. The reaction mixture was concentrated in vacuo and the crude mixture was subjected to LH-20 size-exclusion chromatography.

cyclo-(^DPhe-Pro-Val-Orn(Boc)-Leu-^DPrg-Pro-Val-Orn(Boc)-Leu) (7)

LC/MS: Rt = 4.75 min (50 → 90% MeCN, 15 min run); ESI-MS: m/z 1289.67 [M + H]⁺. HRMS: calculated for [C66H105N12O14]⁺: m/z 1289.78677; found: m/z 1289.78814

General procedure for Cu(I)-catalysed click reactions

Alkyne 7 (50 mg, 39 μmol) and the appropriate azide 6a-e (3 eq) were dissolved in DMF (750 μL) and an aqueous solution of CuSO4 was added (1 M, 0.1 eq, 39 μL), followed by an aqueous solution of sodium ascorbate (1 M, 0.15 eq, 59 μL). When TLC (CHCl3/MeOH 9:1 v/v) indicated completion, DMF was evaporated and the residue was redissolved in DCM and washed with 1N HCl and NaHCO3-solution. The organics wre dried (MgSO4), filtered and evaporated.

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General procedure for Cp*RuCl(PPh3)2-catalysed reactions

7 (50 mg, 39 μmol) and the appropriate azide 6a-d were dissolved in 750 μL THF. The solution was purged with argon and kept under an inert atmosphere during the reaction. Cp*RuCl(PPh3)2 (0.1 eq, 4 μmol) was added and the reaction mixture was exposed to microwave irradiation for 15 min (or 30 min if the reaction was incomplete). The crude product was then applied to short silicagel chromatography (5% MeOH/DCM).

General procedure for Boc-doprotection.

The appropriate peptide was dissolved in DCM (2 mL) and TFA (2 mL) was added. The mixture was left for 4 hours, before it was concentrated in vacuo and coevaporated with toluene (3 u 10 mL). The crude products were purified by preparative RP-HPLC purification.

Peptide 8a

1H NMR (600 MHz, CD3OH) δ 8.93 (NH ^DPhe, d, J = 2.99 Hz, 1H), 8.87 (NH ^DTrA, d, J = 3.05 Hz, 1H), 8.74 (NH Leu, 1H), 8.72 (NH Leu, 1H), 8.70 (NH Orn, 2H), 7.93 (triazole, s, 1H), 7.83 (NH2 Orn, brs, 2H), 7.73 (NH2 Orn, brs, 2H), 7.70 (NH Val, d, J = 8.99 Hz, 1H), 7.64 (NH Val, d, J = 8.92 Hz, 1H), 7.39-7.35 (Ar Bn, m, 5H), 7.33- 7.24 (Ar Phe, m, 5H), 5.57 (CH2Ph, s, 2H), 4.99 (H^α Orn, m, 2H), 4.67 (H^α Leu, m, 2H), 4.51 (H^α Phe, Hα TrA, m, 2H), 4.33 (H^α Pro, m, 2H), 4.13 (H^α Val,1H), 4.11 (H^α Val,1H), 3.87 (H^δ Pro, m, 1H), 3.73 (H^δ Pro, m, 1H), 3.08 (H^β TrA, m, 2H), 3.03 (H^δ Orn, 1H), 2.99 (H^δ Orn, 1H), 2.95 (H^β Phe, m, 2H), 2.86 (H^δ Orn, 1H), 2.81 (H^δ Orn, 1H), 2,67 (H^δ Pro, 1H), 2.46 (H^δ, 1H), 2.26 (H^β Val, 2H) 2.04 (H^β Pro, 1H), 2.03 (H^β Orn, 4H), 1.98 (H^β Pro, 1H), 1.75 (H^γ Orn, 2H), 1.69 (H^γ Pro, 2H), 1.68 (H^β Pro, 1H), 1.59 (H^β Pro, 2H), 1.56 (H^γ Orn, 2H), 1.55 (H^γ Pro, 2xH), 1.50 (H^β Leu, 4H), 1.40 (H^γ Leu, 2H), 0.96 (H^γ Val, 6H), 0.89 (H^δ Leu, 12H), 0.87 H^γ Val, 6H); ¹³C NMR (150 MHz, CD3OH) δ 173.53, 172.39, 172.36, 136.83, 136.81, 130.35, 130.04, 129.72, 129.65, 129.19, 128.47, 124.47, 62.07, 61.95, 60.41, 60.38, 55.92, 54.92, 54.10, 52.37, 51.39, 51.31, 47.88, 40.56, 31.92, 31.86, 25.60, 25.57, 24.58, 23.20, 23.16, 22.98, 22.92, 19.57, 19.48; LC/MS: Rt = 8.06 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1222.73 [M + H]⁺; HRMS: calculated for [C63H96N15O10]⁺: m/z 1222.74591; found: m/z 1222.74704

Peptide 8b

1H NMR (600 MHz, CD3OH) δ 8.92 (NH Phe, d, J = 3.30 Hz, 1H), 8.82 (NH TrA, d, J = 3.32 Hz, 1H), 8.71 (NH Leu, d, J = 9.37 Hz, 1H), 8.70 (NH Leu, d, J = 9.43, 1H), 8.67 (NH Orn, d, J = 9.42 Hz, 2H), 8.25-7.51 (naph, m, 7H), 7.85 (triazole, s, 1H), 7.82 (NH2 Orn, brs, 2H), 7.70 (NH2 Orn, brs, 2H), 7.69 (NH Val, d, J = 8.97 Hz, 1H), 7.54 (NH Val, 1H), 7.33-7.23 (Ar Phe, m, 5H), 6.07 (CH2Naph, dd, J = 14.4 Hz, J = 41.4 Hz, 2H), 4.95 (H^α Orn, 2H), 4.64 (H^α Leu, 1H), 4.59 (H^α Leu, 1H), 4.49 (H^α Phe, 1H), 4.41 (H^α TrA, 1H), 4.33 (H^α Pro, 1H), 4.15 (H^α Pro, 1H), 4.13 (H^α Val, 1H), 4.07 (H^α Val, 1H), 3.72 (H^δ Pro, 1H), 3.61 (H^δ Pro, 1H), 3.09 (H^β TrA, 1H), 3.08 (H^β Phe, 1H), 3.02 (H^δ Orn, 1H), 3.01 (H^β TrA, 1H), 2.96 (H^δ Orn, 1H), 2.92 (H^β Phe, 1H), 2.84 (H^δ Orn, 1H), 2.79 (H^δ Orn, 1H), 2.46 (H^δ Pro, 1H), 2.32 (H^δ Pro, 1H), 2.24 (H^β Val, 1H), 2.20 (H^β Val, 1H), 2.03 (H^β Orn, 2H), 2.01 (H^β Orn, 2H), 1.99 (H^β Pro, 1H), 1.75 (H^γ Orn, 1H), 1.73 (H^β Pro, 1H), 1.70 (H^γ Orn, 1H), 1.68 (H^γ Pro, 1H), 1.68 (H^β Pro, 1H), 1.57 (H^γ Pro, 1H), 1.56 (H^γ Orn, 1H), 1.54 (H^γ Orn, 1H), 1.52 (H^β Leu, 2H), 1.47 (H^γ Pro, 1H), 1.46 (H^β Leu, 2H), 1.37 (H^γ Leu, 1H), 1.36 (H^γ Leu, 1H), 1.09 (H^β Pro, 1H), 1.07 (H^γ Pro, 1H), 0.94 (H^γ Val, 3H), 0.88 (H^δ Leu, 12H), 0.86 (H^γ Val, 6H), 0.85 (H^γ Val, 3H); ¹³C NMR (150 MHz, CD3OH) δ 173.59, 173.53, 173.41, 173.32, 172.81, 172.70, 172.37, 172.33, 161.23, 143.30, 136.83, 135.45, 132.41, 131.97, 130.98, 130.35, 130.01, 129.65, 129.44, 128.46, 128.00, 127.37, 126.55, 124.42, 124.09, 105.40, 67.89, 61.92, 60.37, 55.92, 54.04, 52.89, 52.35, 51.38, 51.26, 47.88, 41.93, 41.70, 40.54, 40.45, 37.24, 31.91, 31.81, 30.81, 30.70, 30.59, 30.41, 27.01, 25.59, 25.54, 24.56, 24.39, 24.17, 23.19, 23.15, 22.97, 22.89; LC/MS: Rt = 7.66 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1273.1 [M + H]⁺; HRMS: calculated for [C67H98N15O10]⁺: m/z 1272.76156; found: m/z 1272.76242

Peptide 8c

1H NMR (600 MHz, CD3OH) δ 8.92 (NH Phe, d, J = 3.30 Hz, 1H), 8.85-7.64 (phen, m, 9H), 8.82 (NH TrA, d, J = 3.32 Hz, 1H), 8.71 (NH Leu, d, J = 9.37 Hz, 1H), 8.70 (NH Leu, d, J = 9.43 Hz, 1H), 8.67 (NH Orn, d, J = 9.42 Hz, 2H), 7.89 (triazole, s, 1H), 7.82 (NH2 Orn, brs, 2H), 7.70 (NH2 Orn, brs, 2H), 7.69 (NH Val, d, J = 8.97 Hz, 1H), 7.54 (NH Val, 1H), 7.33-7.23 (Ar Phe, m, 5H), 6.12 (CH2Phen, dd, J = 14.77 Hz, J = 38.75 Hz, 2H), 4.95 (H^α Orn,

[62]

2H), 4.64 (H^α Leu, 1H), 4.59 (H^α Leu, 1H), 4.49 (H^α Phe, 1H), 4.41 (H^α TrA, 1H), 4.33 (H^α Pro, 1H), 4.15 (H^α Pro, 1H), 4.13 (H^α Val, 1H), 4.07 (H^α Val, 1H), 3.72 (H^δ Pro, 1H), 3.61 (H^δ Pro, 1H), 3.09 (H^β Phe, 1H), 3.08 (H^β Phe, 1H), 3.02 (H^δ Orn, 1H), 3.01 (H^β Phe, 1H), 2.92 (H^β Phe, 1H), 2.96 (H^δ Orn, 1H), 2.84 (H^δ Orn, 1H), 2.79 (H^δ Orn, 1H), 2.46 (H^δ Pro, 1H), 2.32 (H^δ Pro, 1H), 2.24 (H^β Val, 1H), 2.20 (H^β Val, 1H), 2.03 (H^β Orn, 2H), 2.01 (H^β Orn, 2H), 1.99 (H^β Pro, 1H), 1.75 (H^γ Orn, 1H), 1.73 (H^β Pro, 1H), 1.70 (H^γ Orn, 1H), 1.68 (H^β Pro, 1H), 1.68 (H^γ Pro, 1H), 1.57 (H^γ Pro, 1H), 1.56 (H^γ Orn, 1H), 1.54 (H^γ Orn, 1H), 1.52 (H^β Leu, 2H), 1.47 (H^γ Pro, 1H), 1.46 (H^β Leu, 2H), 1.37 (H^γ Leu, 1H), 1.36 (H^γ Leu, 1H), 1.09 (H^β Pro, 1H), 1.07 (H^γ Pro, 1H), 0.94 (H^γ Val, 3H), 0.88 (H^δ Leu, 12H) 0.86 (H^γ Val, 6H), 0.85 (H^γ Val, 3H); ¹³C NMR (150 MHz, CD3OH) δ 173.51, 173.40, 172.67, 172.36, 172.32, 143.36, 136.82, 132.36, 132.30, 132.06, 130.96, 130.89, 130.34, 129.91, 129.64, 128.90, 128.46, 128.30, 128.25, 128.22, 124.97, 124.63, 124.38, 123.69, 61.93, 61.87, 60.35, 55.91, 53.98, 53.48, 52.34, 51.37, 51.24, 47.87, 47.83, 41.92, 40.52, 31.90, 31.77, 30.58, 25.58, 25.51, 24.54, 23.17, 23.14, 22.97, 22.86, 19.57, 19.51, 19.42; LC/MS:

Rt = 8.36 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1323.1 [M + H]⁺; HRMS: calculated for [C71H100N15O10]⁺: m/z 1322.77721; found: m/z 1322.77827

Peptide 8d

1H NMR (750 MHz, CD3OH) δ 8.92-8.07 (Ar pyr, m, 9H), 8.88 (NH Phe, d, J = 2.53 Hz, 1H), 8.77 (NH TrA, d, J

= 2.40 Hz, 1H), 8.67 (NH Leu, d, J = 9.17 Hz, 1H), 8.66 (NH Leu, d, J = 9.72 Hz, 1H), 8.65 (NH Orn, d, J = 9.82 Hz, 1H), 8.62 (NH Orn, d, J = 9.29 Hz, 1H), 7.90 (triazole, s, 1H), 7.80 (NH2 Orn, brs, 2H), 7.68 (NH Val, d, J = 8.90 Hz, 1H), 7.64 (NH2 Orn, brs, 2H), 7.44 (NH Val, d, J = 8.86 Hz, 1H), 7.31-7.23 (Ar Phe, m, 5H), 6.36 (CH2pyr, dd, J = 14.89 Hz, J = 72.78 Hz, 2H), 4.90 (H^α Orn, 1H), 4.88 (H^α Orn, 1H), 4.62 (H^α Leu, 1H), 4.55 (H^α Leu, 1H), 4.48 (H^α Phe, 1H), 4.36 (H^α TrA, 1H), 4.32 (H^α Pro, 1H), 4.11 (H^α Val, 1H), 4.01 (H^α Pro, 1H), 3.98 (H^α Val, 1H), 3.72 (H^δ Pro, 1H), 3.47 (H^δ Pro, 1H), 3.10 (H^β Phe, 1H), 3.08 (H^β Phe, 1H), 3.00 (H^β Phe, 1H), 2.99 (H^δ Orn, 1H), 2.92 (H^δ Orn, 1H), 2.92 (H^β Phe, 1H), 2.81 (H^δ Orn, 1H), 2.76 (H^δ Orn, 1H), 2.47 (H^δ Pro, 1H), 2.23 (H^β Val, 1H), 2.17 (H^δ Pro, 1H), 2.13 (H^β Val, 1H), 2.00 (H^β Orn, 1H), 1.98 (H^β Orn, 1H), 1.97 (H^β Pro, 1H), 1.72 (H^γ Orn, 2H), 1.68 (H^γ Pro, 1H), 1.68 (H^γ Orn, 2H), 1.66 (H^β Pro, 1H), 1.56 (H^γ Pro, 1H), 1.53 (H^β Orn, 1H), 1.52 (H^β Orn, 1H), 1.50 (H^β Leu, 2H), 1.42 (H^γ Leu, 1H), 1.35 (H^β Leu, 2H), 1.31 (H^γ Leu, 1H), 1.30 (H^γ Pro, 1H), 1.15 (H^γ Pro, 1H), 0.93 (H^γ Val, 3H), 0.85 (H^δ Leu, 6H), 0.84 (H^γ Val, 3H), 0.80 (H^δ Leu, 6H), 0.80 (H^γ Val, 6H), 0.72 (H^β Pro, 1H), 0.59 (H^β Pro, 1H); ¹³C NMR (187 MHz, CD3OH) δ 173.52, 173.44, 173.38, 172.70, 172.68, 172.28, 143.36, 136.81, 133.45, 132.50, 131.86, 130.34, 129.73, 129.64, 129.35, 129.23, 128.46, 128.20, 127.61, 127.03, 126.78, 126.10, 125.49, 124.32, 123.38, 61.93, 61.73, 60.35, 60.30, 55.90, 53.98, 53.01, 52.31, 51.35, 51.21, 49.85, 47.87, 47.67, 41.91, 41.64, 40.51, 31.90, 31.73, 30.59, 25.56, 25.49, 24.53, 23.13, 22.95, 22.83, 19.47, 19.41, 19.37;

LC/MS: Rt = 9.21 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1346.80 [M + H]⁺; HRMS: calculated for [C73H100N15O10]⁺: m/z 1346.77721; found: m/z 1346.77851

cyclo-(^DPhe-Pro-Val-Orn-Leu-^DTrA-Pro-Val-Orn-Leu)∙2TFA (8f)

The Boc-protected peptide was dissolved in DCM (2 mL) and TFA was added (2 mL). The mixture was heated under microwave irradiation for 5 min. The crude product was concentrated in vacuo and coevaporated with toluene (3 × 10 mL) to remove traces of TFA. The pure product was obtained by preparative RP-HPLC purification.

1H NMR (600 MHz, CD3OH) δ 8.93 (NH Phe, d, J = 2.98 Hz, 1H), 8.87 (NH TrA, d, J = 2.86 Hz, 1H), 8.77 (NH Leu, d, J = 9.43 Hz, 1H), 8.73 (NH Leu, d, J = 9.40 Hz, 1H), 8.73 (NH Orn, d, J = 9.52 Hz, 1H), 8.72 (triazole, s, 1H), 8.71 (NH Orn, d, J = 10.04, 1H), 7.71 (NH Val, d, J = 9.01 Hz, 2H), 7.33-7.24 (Ar Phe, m, 5H), 4.97 (H^α Orn, 2H), 4.67 (H^α Leu, 1H), 4.66 (H^α Leu, 1H), 4.61 (H^α TrA, 1H), 4.57 (H^α Pro, 2H), 4.49 (H^α Phe, 1H), 4.15 (H^α Val, 2H), 3.73 (H^δ Pro, 2H), 3.14 (H^β TrA, 2H), 3.09 (H^β Phe, 1H), 3.03 (H^δ Orn, 2H), 2.93 (H^β Phe, 1H), 2.84 (H^δ Orn, 2H), 2.47 (H^δ Pro, 2H), 2.27 (H^β Val, 2H), 2.05 (H^β Orn, 2H), 2.00 (H^β Pro, 2H), 1.77 (H^γ Orn, 4H), 1.69 (H^β Pro, 2H), 1.69 (H^γ Pro, 2H), 1.59 (H^β Orn, 2H), 1.58 (H^γ Pro, 2H), 1.54 (H^β Leu, 2H), 1.51 (H^γ Leu, 2H), 1.41 (H^β Leu, 2H), 0.97 (H^γ Val, 6H), 0.89 (H^γ Val, 6H), 0.89 (H^δ Leu, 12H); ¹³C NMR (150 MHz, CD3OH) δ 173.56, 173.54, 173.42, 172.76, 172.73, 172.39, 136.84, 130.35, 129.65, 128.46, 62.18, 61.95, 60.41, 60.37, 55.92, 52.40, 51.40, 51.34, 48.15, 47.88, 41.95, 41.47, 40.53, 31.93, 30.79, 30.59, 25.61, 25.59, 24.57, 23.20, 23.16, 22.99, 22.91, 19.59, 19.48, 19.43; LC/MS: Rt = 7.55 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1132.60 [M + H]⁺; HRMS: calculated for [C56H90N15O10]⁺: m/z 1132.69896; found: m/z 1132.69989

[63]

cyclo-(^DPhe-Pro-Val-Orn-Leu-^DHis-Pro-Val-Orn-Leu) (15g)

1H NMR (600 MHz, CD3OH) δ 8.94 (NH, Phe, d, J = 3.12 Hz, 1H), 8.86 (NH His, d, J = 3.60 Hz, 1H), 8.82 (2-CH im, s, 1H), 8.74 (NH Leu, d, J = 8.77 Hz, 2H), 8.74 (NH Orn, d, J = 8.88 Hz, 2H), 7.70 (NH Val, d, J = 8.97 Hz, 1H), 7.63 (NH Val, d, J = 8.97 Hz, 1H), 7.45 (5-CH im, s, 1H), 7.32-7.24 (Ar Phe, m, 5H), 4.98 (H^α Orn, 2H), 4.71 (H^α His, 1H), 4.63 (H^α Leu, 2H), 4.51 (H^α Pro, 1H), 4.49 (H^α Phe, 1H), 4.34 (H^α Pro, 1H), 4.19 (H^α Val, 1H), 4.14 (H^α Val, 1H), 4.09 (H^δ Pro, 1H), 3.74 (H^δ Pro, 1H), 3.37 (H^δ Pro, 1H), 3.18 (H^β His, 2H), 3.10 (H^β Phe, 1H), 3.02 (H^δ Orn, 2H), 2.95 (H^β Phe, 1H), 2.88 (H^δ Orn, 2H), 2.48 (H^δ Pro, 1H), 2.29 (H^β Val, 1H), 2.26 (H^β Val, 1H), 2.17 (H^β Pro, 1H), 2.06 (H^β Pro, 1H), 2.06 (H^β Orn, 2H), 2.01 (H^β Pro, 1H), 2.00 (H^γ Pro, 1H), 1.92 (H^γ Pro, 1H), 1.73 (H^γ Orn, 4H), 1.69 (H^γ Pro, 1H), 1.69 (H^β Pro, 1H), 1.63 (H^β Orn, 2H), 1.57 (H^γ Pro, 1H), 1.53 (H^β Leu, 2H), 1.49 (H^γ Leu, 2H), 1.42 (H^β Leu, 2H), 0.99 (H^γ Val, 3H), 0.95 (H^γ Val, 3H), 0.91 (H^γ Val, 3H), 0.88 (H^δ Leu, 12H), 0.86 (H^γ Val, 3H); ¹³C NMR (150 MHz, CD3OH) δ 173.49, 173.45, 173.43, 172.72, 172.45, 172.39, 172.25, 136.85, 130.36, 130.14, 129.63, 128.43, 118.85, 62.31, 61.92, 60.41, 60.38, 55.92, 53.17, 52.44, 51.44, 51.33, 48.38, 47.86, 41.96, 41.70, 40.53, 37.24, 31.92, 31.87, 30.87, 30.74, 30.71, 30.56, 25.73, 25.62, 25.54, 24.65, 24.57, 24.39, 23.22, 23.14, 23.01, 22.86, 19.59; LC/MS: Rt = 6.81 min (10 → 90% MeCN, 15 min run); ESI-MS: m/z 1131.53 [M + H]⁺; HRMS: calculated for [C57H91N14O10]⁺: m/z 1131.70371; found: m/z 1131.70509

(R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(1-benzyl-1H-1,2,3-triazol-5-yl)propanoic acid (18)

Methyl ester 16 (115 mg, 0.51 mmol) was dissolved in THF (2 mL), together with benzyl azide 6a (2eq, 1.01 mmol, 130 μL). The solution was purged with argon. Cp*RuCl(PPh3)2 (0.1 eq) was added and heated in the microwave for 15 min. The crude product was applied directly to silca gel chromatography (10 → 50 % EtOAc in PE).

The regioisomeric mixture 17 (0.14 mmol) was dissolved in dioxane/water (3 mL, 2:1 v/v) and LiOH (3 eq, 0.42 mmol, 10 mg) was added. The mixture was stirred until TLC indicated full consumption of the starting material.

The mixture was acidified with 1N HCl, and extracted with EtOAc. The organics were washed with brine, dried over MgSO4, filtered and concentrated.

LC/MS: Rt = 6.47 min (1,4-product), 6.60 (1,5-product) (10 → 90% MeCN, 15 min run); ESI-MS: m/z 346.93 [M + H]⁺.

The residue was dissolved in DCM (1 mL) and TFA (1 mL) was added. After 5 h the solvents were evaporated and coevaporated with toluene (3 × 10 mL). The free amino acid was suspended in dioxane (2 mL) and saturated NaHCO3 (2 mL) and FmocOSu (1 eq, 0.14 mmol, 47mg) were added. After 4 h the mixture was acidified with conc. HCl and extracted with EtOAc. The organics were washed with water, brine, dried (MgSO4), filetered and evaporated. The crude mixture was applied to silica gel chromatography (50% EtOAc in PE) to yield the title compound in 73% combined yield.

3.5 R

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