Total Synthesis and Configurational Revision of Mozamide A, a

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Total Synthesis and Configurational Revision of Mozamide A – a Hydroxy-Brunsvicamide Lukas Junk, and Uli Kazmaier J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02836 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 12, 2019

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The Journal of Organic Chemistry

Total Synthesis and Configurational Revision of Mozamide A – a Hydroxy-Brunsvicamide Lukas Junk and Uli Kazmaier*[a] Saarland University, Organic Chemistry I, Campus, Bldg. C4.2, D-66123 Saarbrücken, Germany. Supporting Information Placeholder HO

HO

NH

NH O

O O

N O

NH

HN

NH

O

O

N Ph NH

O Structure revision via total synthesis

NH

HN

NH

O

Ph NH

O

O HN

HN

O

O

HN

HN

COOH

COOH Mozamide A (proposed)

Mozamide A

ABSTRACT: The marine cyclopeptide mozamide A, a member of the class of anabaenopeptin-type peptides, was synthesized for the first time via a convergent and flexible route. The installation of the substituted tryptophan moieties was accomplished at the very end of the synthesis and thus allows easy modifications at this position. Comparison of the nmr data of the synthesized cyclopeptide with the natural product clearly indicates, that the originally proposed structure of mozamide A can not be correct. The synthesis of two other diastereomers allowed the correction of the configuration of three amino acid building blocks. Mozamide A contains L-Val, D-Lys and LIle (instead of D-Val, L-Lys and L-allo-Ile) and is a hydroxylated brunsvicamide.

INTRODUCTION With an age of ~600 million years, marine sponges are considered to be the oldest multicellular animals on earth.1 During this period, the sessile animals had to adapt to a variety of environmental conditions. This might be one of the reasons why sponges are among the best sources of marine natural products.2 Furthermore, sponges are host to diverse symbiotic microorganisms.3 In fact, it could be shown that many of the natural products isolated from sponges are actually produced by symbiotic bacteria.4 Due to their diverse biological properties, these molecules play a major role in pharmaceutical research and development5 which makes them interesting and rewarding targets for total synthesis. On the one hand, the total synthesis of natural products serves as a tool to provide sufficient quantities of the molecules, thus enabling detailed biological studies. On the other hand, the synthetic construction of a proposed structure can be seen as its ultimate proof. Despite modern spectroscopic and chromatographical methods, the total synthesis of numerous natural products showed that their structures were elucidated incorrectly and their structures had to be revised.6 The cyclic peptides mozamide A (1a) and B (2a), (Figure 1) were isolated in 1997 by Faulkner and co-workers from a sponge of the genus Theonella, which was collected in Mozambique.7 Their structures were elucidated via NMR and MS experiments and the configurations of the constituting amino acids were determined by degradation of the peptides followed by derivatization and chiral GC analysis. Faulkner et al. found D-Val and

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five L-configured amino acids, including L-allo-Ile in the sidechain. However, due to a lack of material, no biological properties for 1a or 2a were reported. Br

HO

NH

NH

O N O

R

NH

O O

4

NH

HN

NH

O

O

N O

Ph NH

O

R HN

O

O

NH

HN

NH

O

Ph NH

O O

HN COOH

HN

NH

O

Ph NH

HN

O

HN COOH

1a Mozamide A: R = H 2a Mozamide B: R = CH3

NH

O HN

HN

O

N O

Brunsvicamide A: R = H Brunsvicamide B: R = CH3

COOH Psymbamide A

Figure 1. Structures of (proposed) mozamides, brunsvicamides and psymbamide A.

The mozamides 1a and 2a belong to the family of anabaenopeptin-type peptides, which consists of at least 94 congeners with a similar peptidic structure.8 These molecules consist of a pentapeptide, which is cyclized via the ε-nitrogen of the N-terminal D-lysine and usually, an N-methylated amino acid can be found in position 4. The α-nitrogen of the lysine is linked to another amino acid via an ureido bond. All amino acids except of lysine are usually L-configured. The mozamides (and konbamide,9 whose correct configuration has yet to be determined) seem to be the only exception with an L-Lys next to a Dconfigured amino acid. Anabaenopeptin-type peptides show a wide range of biological properties. The most prominent among these is the inhibition of Zn-containing peptidases like carboxypeptidase A.10 Furthermore, some anabaenopeptins show the inhibition of clinically relevant carboxypeptidase U or carboxypeptidase TAFIa.11 For some congeners, inhibition of serine proteases like elastase, trypsin and chymotrypsin was demonstrated.12 Additionally, a few anabaenopeptin-type peptides showed inhibition of protein phosphatases.13 For example, brunsvicamides A – C (Fig. 1), which were isolated from Tychonema cyanobacteria, were described as potent inhibitors of protein phosphatase B (PPB) in mycobacterium tuberculosis (Mtb).14 Initially, an L-Lys unit was also proposed for the brunsvicamides. However, the total synthesis of brunsvicamides A and B by Arndt and Waldmann et al. showed that the molecules actually contain a D-Lys-unit.15 However, the synthetic compounds showed no inhibition of PPB in Mtb. Furthermore, Arndt and Waldmann et al. synthesized a small library of brunsvicamide analogs and tested them for inhibition of carboxypeptidase A and B.10c While brunsvicamide A and B were strong inhibitors of the peptidases, molecules bearing an L-Lys showed almost no activity. Additionally, the inhibition via a different mode of action of human leukocyte elastase was described for brunsvicamides A – C.16 Brunsvicamides and mozamides show a very similar molecular constitution, which only differs in a hydroxylation of the indole moiety. However, the proposed configurations of Val, Lys and Ile are different in brunsvicamides and mozamides. Our working group has a long-standing interest in the synthesis of peptidic natural products,17 especially with the goal to develop routes which allow the synthesis of derivatives in a straight-forward manner.18 We recently described the total synthesis and configurational revision of keramamides A and L, two other tryptophan-containing anabaenopeptin-type peptides.19 The late-stage installation of the indole moieties ACS Paragon Plus Environment

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allowed the synthesis of both natural products from a common precursor. In order to proof the utility and flexibility of our methodology, we chose mozamide A (1a) as a synthetic target. This route should also provide access to synthetic derivatives, and therefore enable the determination of structure-activity relationships. For example, an uncommon 5-bromotryptophan, which can be found in psymbamide A (Figure 1) would be easily accessible using our strategy. Thus, the retrosynthesis of 1a is based on the late-stage installation of the 5hydroxyindole on a stannylated precursor A,20 which should be accessible via hydrostannation of a propargyl glycine unit in B (Scheme 1). 3+3-Disconnection of cyclic precursor B leads to tripeptides C,19 which is the same peptide we used in our synthesis of keramamides A and L, and D, which had to be prepared from the amino acids. SnBu3 O

O O

N NH

O

HN

NH

HN

NH

O

O

NH

NH

O

O HN

HN

O

O

HN

HN

COOR

COOR

B

A

NH2

O BocHN

Ph

Ph

1a NH

O

N O

H N

N O

COOH Ph

O

+ MeOOC

N H

H N

N H

C

COOtBu

O D

Scheme 1. Retrosynthetic approach to (proposed) mozamide A (1a).

RESULTS AND DISCUSSION Our synthetic endeavors towards 1a, the proposed structure of mozamide A, thus began with the synthesis of tripeptide D from Alloc-protected L-Lys (3a) (Scheme 2). Coupling with H-D-Val-OtBu·HCl followed by N-deprotection and reaction with L-allo-Ile derived isocyanate 5a yielded the Nε-Cbz-protected tripeptide 6a. The hydrogenolysis of the Cbzgroup had to be carried out under slightly acidic conditions to avoid undesired cyclizations. The deprotected tripeptide was then coupled to tripeptide C,19 which was activated as a mixed anhydride, to give linear hexapeptide 7a. Deprotection of C- and Ntermini followed by cyclization with TBTU and DIPEA gave rise to alkyne-bearing cyclic peptide 8a in 73% yield. The Ru-catalyzed hydrostannation could be realized using a method by Fürstner and co-workers21 in excellent yield. Stannylated peptide 9a was then subjected to our sequence for indole synthesis.20 Stille-coupling with O-benzyl protected 4-amino-3-iodophenol E provided the corresponding aniline derivative (10a). Azidation with tBuONO and TMSN3 gave rise to azide 11a, which could be transformed to protected indole 12a by photochemical nitrene C–H-insertion. Hydrogenolysis of the benzyl ether (to 13a) and cleavage of the methyl ester both required prolonged reaction times. Finally, 1a, exhibiting the proposed structure of mozamide A, could be isolated in 44% yield over two steps.



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Scheme 2. Synthesis of the proposed structure of mozamide A (1a) NHCbz OH

AllocHN

NHCbz

H-D-Val-OtBu·HCl ClCOOiBu, NMM

THF, 20 °C to rt, 16 h 82%

O

H N

AllocHN

COOtBu

MeOOC

N H

O O

1) H2, Pd/C, 1 M HCl MeOH, rt, 2 h 2) C,ClCOOiBu, NMM THF, 20 °C to rt, 43 h 52%

O

6a

Ph

O

O

NH

NH

HN

NH

O

1) TFA/DCM, 2 h 2) TBTU, DIPEA DCM, rt, 41 h 73%

O HN

Bu3SnH [Cp*Ru(MeCN)3]PF6

Ph NH

DCM, rt, 20 min 87%

O HN

NH

O

HN

COOMe

7a

O

N

tBuOOC NH

8a

BnO

SnBu3

COOMe R 1O

N3

O

NH

O O

N HN

NH

O

NH

Ph NH

O HN

COOtBu

O

O

H N

N

H N

N H

4a BocHN

O

O

2) 5a, DCM, rt, 66 h 86%

O

3a

NHCbz

1) Pd(PPh3)4, DMBA DCM, rt, 1 h

O

O O

N

1) E, Pd(PPh3)4, CuTC Ph2PO2NBu4 DMF, RT, 2 h

O

NH

O

NH

2) TMSN3, tBuONO MeCN, 4.5 h 0 °C to rt 59%

Ph NH

O MeCN,rt, 12 h

O

COOMe 5a

BnO

I

Ph NH

O

COOR2 1 2 12a (R = Bn, R = Me): 36%

11a

OCN

O

HN COOMe

NH2

NH HN

HN

9a

HN

O HN

COOMe

NH

h (365 nm)

O

HN

O

N

HN

1) H2, Pd/C, Pd(OH2)/C MeOH, 52 h 2) Me3SnOH, C2H4Cl2, 80 °C, 62 h

E

1 2 1a (R = R = H): 44%

NMM: N-methylmorpholine; DMBA: N,N’-dimethylbarbituric acid; TBTU: N,N,N’,N’-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate; Cp*: η5-C5Me5; CuTC: copper (I) thiophene-2-carboxylate.

However, comparison of the NMR data of 1a and natural 1 showed no good accordance. We thus hypothesized that 1a is actually a diastereomer of 1. Based on the structures of other anabaenopeptin-type peptides, especially brunsvicamide A, we found it reasonable that the lysine had to be D-configured. The incorporation of two D amino acids into the cyclic peptide however seemed very unlikely. Based on the structures of the brunsvicamides, we assumed that 1 contains D-Lys next to L-Val. The configuration of the -branched sidechain should be determined by the preparation of the two diastereomers, either bearing L-allo-Ile or L-Ile. The synthesis of the diastereomers 1b (L-allo-Ile) and 1c (L-Ile) thus started from NαAlloc-Nε-Cbz-D-Lys (3b) and could be completed in a straight-forward manner using the same strategy we used before (Scheme 3). Only the very last step caused some problems in case of 1c. The reaction had to be heated for 5 d in order to achieve 90% conversion. However, the product had to be purified several times by flash chromatography and preparative HPLC, resulting in a final yield of only 9%. Luckily, the amount was sufficient to obtain well resolved NMR spectra, so that the spectra of the three isomers 1a – c could be compared to the natural product. ACS Paragon Plus Environment

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Scheme 3. Synthesis of mozamide stereoisomers 1b and 1c SnBu3

3b

O

1) H-D-Val-OtBu·HCl ClCOOiBu, NMM THF, 20 °C to rt, 16 h 2) Pd(PPh3)4, DMBA DCM, rt, 1 h 3) 5a/5b, DCM, rt, 17 h

BocHN

O

H N

N O

O

Ph NH

tBuOOC

4) H2, Pd/C, 1 M HCl MeOH, rt, 2 h 5) C,ClCOOiBu, NMM THF, 20 °C to rt, 44 h

NH

O HN

O

3) Bu3SnH [Cp*Ru(MeCN)3]PF6 DCM, rt, 20 min

NH

HN

NH

O

HN

NH

COOMe

): 60% ): 46%

9b ( 9c (

3) h (365 nm) MeCN,rt, 12 h

NH N HN

NH

O

): 35% ): 47%

Ph

NH

O

1) H2, Pd/C, Pd(OH2)/C MeOH, 52 h

O

NH

O

HO

O O

NH

HN

BnO 1) E, Pd(PPh3)4, CuTC Ph2PO2NBu4 DMF, RT, 2 h 2) TMSN3, tBuONO MeCN, 4 h/3 h 0 °C to rt

Ph

O

COOMe 7b ( 7c (

O

N O

1) TFA/DCM, 2 h 2) TBTU, DIPEA DCM, rt, 41 h

2) Me3SnOH, C2H4Cl2, 80 °C, 62 h

NH

O

O

N O

NH

HN

NH

O

Ph NH

O HN

O

HN

O

HN COOMe

12b ( 12c (

HN

): 32% ): 35%

COOH 1b ( ): 53% 1c ( ): 19% (crude) (after prep. HPLC: 5%)

NMM: N-methylmorpholine; DMBA: N,N’-dimethylbarbituric acid; TBTU: N,N,N’,N’-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate; Cp*: η5-C5Me5; CuTC: copper (I) thiophene-2-carboxylate.

Figure 2 shows the Δδ(1H) values of all six α-protons of the amino acids in 1a – c compared to natural 1. 1a (blue bars) shows no good accordance to the natural product, whereas 1b (red bars) deviates only in the tryptophan and isoleucine units. For 1c (green bars) the Δδ(1H) values are below 0.1 ppm in the whole range of the spectrum (see Supporting Information for complete NMR data). Some values (e.g. Val N-H and Lys N-H) could not be exactly reproduced (Δδ = 0.07 ppm) which is possibly caused by small differences in the protonation state or intramolecular hydrogen bonding in the molecule. Apart from these discrepancies the 1H-NMR spectrum of 1c shows an almost perfect accordance to the spectrum reported for mozamide A. The 13C-NMR spectra of 1a – c showed larger deviances from the spectrum of the natural product of up to 2.2 ppm for Lys-α and 1.1 ppm for Val-α. However, these differences were observed in all three diastereomers. We suppose that the reported values for Lys-α and Val-α might be reported incorrectly (both were reported to resonate at a chemical shift of 56.7 ppm, which could be a copy-paste error). The same might be true for Phe-β (Δδ = 1.3 – 1.4 ppm). Because of the lack of original data or an authentic sample of the natural product, we can however not confirm these assumptions. Since the NMR spectra of 1c show a good accordance to those of mozamide A, we propose the actual structure of mozamide A to be 1c.



Figure 2. Δδ(1H) values of α-protons of all constituting six amino acids in 1a, 1b and 1c; Δδ = δ(1) – δ(1a–c); all spectra were recorded in DMSO-d6 at 500 MHz; Me5HT: N-Methyl-5-hydroxytryptophan.

Obviously, 1 contains a D-Lys unit next to L-Val and L-Ile in the side chain. This is not only in perfect accordance to most other anabaenopeptin-type peptides, but is the exact configuration of these amino acids in brunsvicamide A, making 1 a hydroxylated version of brunsvicamide A. At first, this seems surprising since 1 was isolated from a marine sponge whereas brunsvicamide A was isolated from terrestrial cyanobacteria. However, this finding indicates that mozamide A might in fact be produced by symbiotic cyanobacteria living inside of Theonella sponges. CONCLUSIONS In conclusion, we synthesized mozamide A (1c) for the first time in a convergent manner. We could show that the originally proposed structure of 1 is not consistent with the NMR data. After synthesizing two diastereomers and comparing their spectral data to that of natural 1, we propose that the natural product contains a D-Lys-unit next to L-Val and LIle. Therefore, 1a is a hydroxylated version of brunsvicamide A. The synthetic installation of the indole moiety on a late stage should give access to derivatives of the natural product, which might enable the evaluation of structure-activity relationships. We will report on the synthesis of further mozamide derivatives as well as their biological evaluation in due course.

For conditions, experimental dures, 1H and C 13 the porting The aprocedure tative rNaepresenSupMR nd example. analytical Information. spectra, serves following procedetailed general data, see as EXPERIMENTAL SECTION

All air- or moisture-sensitive reactions were carried out in dried glassware (>100 °C) under an atmosphere of nitrogen or argon. THF was distilled over Na/benzophenone prior to use. Ethyl acetate (EtOAc) and petroleum ether (PE) were distilled prior to use. Reactions were monitored by analytical TLC, which was performed on precoated silica gel on TLC PET-foils. Visualization was accomplished with UV light (254 nm) and KMnO4 solution. The products were purified by flash chromatography on silica gel columns (0.04–0.063 mm) or by automated flash chromatography. Mixtures of EtOAc and PE were generally used as eluents. Melting points were determined with a melting point apparatus and are uncorrected. 1H and 13C{1H} NMR spectra were recorded with a 400 MHz (101 MHz) or 500 MHz (126 MHz) spectrometer in CDCl3 or DMSO-d6. Chemical shifts are reported in ppm relative to Si(CH3)4, and CHCl3 was used as the internal standard. ACS Paragon Plus Environment

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Multiplicities are reported as br (broad signal), s (singlet), d (doublet), t (triplet), q (quartet), sept (septet) and m (multiplet). Mass spectra were recorded with a highresolution sector field spectrometer (CI) or a UHR-TOF spectrometer using the ESI technique. Optical rotations were measured in a thermostated (20 ± 1 °C) cuvette. The radiation source used was a sodium vapor lamp (λ = 589 nm). The concentrations are given in g/100 mL. Starting Materials Unless otherwise stated, all starting materials were commercially available and were used without further purification. 4-Amino-3-iodophenol,22 Ph2PO2NBu4,23 and H-D-Leu-Ot-Bu24 were prepared by literature procedures. General Procedure A for the Peptide Bond Formation via Mixed Anhydrides.25 The carboxylic acid (1.0 eq.) was dissolved in THF (10 mL/mmol) in a round bottom flask and the resulting solution was cooled to –20 °C. 4–methylmorpholine (NMM) (1.1 eq) was added in one portion and ibutyl chloroformate (IBCF) (1.05 eq) was added dropwise with a syringe. After stirring for 15 min at –20 °C, the amine component (1.0 eq) was added. In case of hydrochloride salts, 1.0 eq. NMM was additionally added. This mixture was slowly warmed to room temperature in the cooling bath during the specified time. The reaction mixture was diluted with EtOAc and subsequently washed with 1 M HCl, H2O, sat. NaHCO3 solution and brine. The organic phase was dried (MgSO4), filtered and concentrated. The residue was purified by flash chromatography. General Procedure B for Alloc Cleavage and Formation of Urea Tripeptides. General Procedure B.1: The N-Alloc protected dipeptide was dissolved in DCM (10 mL/mmol). N,N’-dimethyl barbituric acid (3.0 eq) and Pd(PPh3)4 (3 mol-%) were added at rt. After 1 h, TLC control usually showed full conversion. The solvent was evaporated, the residue was taken up in EtOAc and washed with sat. NaHCO3 solution three times. The combined aqueous phases were extracted once with EtOAc. The combined organic layers were dried (Na2SO4) and concentrated. General Procedure B.2: The crude deprotected dipeptide was taken up in DCM (5 mL/mmol) and added dropwise to a solution of freshly prepared isocyanate in DCM (5 mL/mmol) at rt. After stirring for the specified time, the solvent was evaporated and the residue was purified by flash chromatography. General Procedure C for N-Boc and tert-Butyl Ester Cleavage and Cyclization of hexapeptides. The linear hexapeptide was dissolved in DCM (5 mL/mmol) and TFA (5 mL/mmol) was added at rt. After 2 – 3 h LC/MS usually showed full conversion. The solvent was evaporated and the residue was taken up in CHCl3 and concentrated three times. The deprotected peptide was dried in high vacuum for >1 h. Subsequently, the peptide was dissolved in DCM (10 – 20 mL) and added dropwise to a solution of TBTU (3.0 eq) and DIPEA (3.0 eq) in DCM (1000 mL/mmol rel. to the peptide) over the course of 10 min at rt. The solution was stirred for the specified time until LC/MS showed complete conversion. The solvent was then evaporated in vacuo. The residue was taken up in EtOAc and washed with 1 M HCl, H2O, sat. NaHCO3 solution and brine. The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography.



General Procedure D for Stille Cross Coupling of Vinyl Stannanes with o-Iodoanilines.26 DMF was degassed by bubbling with Ar for 10 min prior to use. Ph2PO2NBu4 (2.0 eq) was placed in a Schlenk tube and carefully melted in high vacuum. Before it solidified, 0.5 mL of DMF were added. Then, Pd(PPh3)4 (5 mol-%), CuTC (2.0 eq) and the o-iodoaniline (1.1 eq) were added followed by a solution of the vinyl stannane (1.0 eq) in DMF (final concentration 0.05 M). The solution was stirred until full conversion was observed by TLC. Subsequently, H2O and EtOAc were added and the mixture was shaken vigorously in the closed Schlenk tube. The mixture was filtered over Celite, the layers were separated and the organic phase was washed twice with H2O (10x the amount of DMF used) and once with brine. The combined aqueous phases were extracted once with EtOAc. The combined EtOAc phases were dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography using SiO2:K2CO3 9:1 (w/w) as the stationary phase. tert-Butyl Να-allyloxycarbonyl-Νε-benzyloxycarbonyl-L-lysyl-D-valinate (4a) According to general procedure A, Alloc-L-Lys(Cbz)-OH 3a (747 mg, 1.91 mmol) was reacted with HCl·H-D-Val-Ot-Bu (400 mg, 1.91 mmol), IBCF (0.26 mL, 2.01 mmol) and NMM (0.44 mL, 4.01 mmol) in THF (20 mL). The reaction was worked up after 16 h. Flash chromatography (PE/EtOAc 6:4) yielded 4a (816 mg, 1.57 mmol, 82%) as a colorless 1 solid; mp 97 – 99 °C; Rf = 0.20 (PE/EtOAc 6:4); [α]20 D = −19.5 (c = 1.0, CHCl3). H NMR (400 MHz, CDCl3): δ = 0.88 (d, J = 6.9 Hz, 3 H), 0.92 (d, J = 6.9 Hz, 3 H), 1.40 (m, 2 H), 1.46 (s, 9 H), 1.53 (m, 2 H), 1.66 (m, 1 H), 1.88 (m, 1 H), 2.15 (septd, J = 6.9, 4.6 Hz, 1 H), 3.19 (m, 2 H), 4.18 (m, 1 H), 4.41 (dd, J = 8.6, 4.5 Hz, 1 H), 4.56 (m, 2 H), 4.84 (m, 1 H), 5.09 (s, 2 H), 5.20 (d, J = 10.4 Hz, 1 H), 5.29 (d, J = 17.2 Hz, 1 H), 5.44 (bs, 1 H), 5.89 (ddt, J = 17.0, 10.6, 5.6 Hz, 1 H, 2-H), 6.50 (d, J = 8.2 Hz, 1 H), 7.29–7.37 (m, 5 H). 13C{1H} NMR (101 MHz, CDCl3): δ = 17.6, 18.9, 22.3, 28.0, 29.4, 31.4, 32.1, 40.3, 54.8, 57.3, 65.9, 66.6, 82.1, 117.9, 128.0, 128.1, 128.5, 132.5, 136.6, 156.1, 156.6, 170.7, 171.4. HRMS (CI): calcd for C27H42N3O7+ [M+H]+: 520.3017, found: 520.3026. tert-Butyl Nα-allyloxycarbonyl-Νε-benzyloxycarbonyl-D-Lysyl-L-valinate (4b) According to general procedure A, Alloc-D-Lys(Cbz)-OH 3b (1.13 g, 2.82 mmol) was reacted with HCl·H-L-Val-OtBu (568 mg, 2.71 mmol), IBCF (0.37 mL, 2.85 mmol) and NMM (0.59 mL, 5.69 mmol) in THF (27 mL). The reaction was worked up after 17 h. Automated flash chromatography (PE/EtOAc 100:0–7:3) afforded 4b (1.64 g, 2.05 mmol, 76%) as a 1 colorless resin; [α]20 D = +20.8 (c = 1.0, CHCl3); Rf = 0.19 (PE/EtOAc 7:3). H NMR (400 MHz, CDCl3): δ = 0.88 (d, J = 6.9 Hz, 3 H), 0.91 (d, J = 6.9 Hz, 3 H), 1.40 (m, 2 H), 1.45 (s, 9 H), 1.53 (m, 2 H), 1.66 (m, 1 H), 1.87 (m, 1 H), 2.15 (septd, J = 6.9, 4.5 Hz, 1 H), 3.18 (m, 2 H), 4.19 (m, 1 H), 4.41 (dd, J = 8.7, 4.5 Hz, 1 H), 4.56 (m, 2 H), 4.90 (m, 1 H), 5.09 (s, 2 H), 5.19 (d, J = 10.4 Hz, 1 H), 5.29 (ddt, J = 17.2, 1.4, 1.4 Hz, 1 H), 5.49 (bs, 1 H), 5.89 (ddt, J = 17.2, 10.5, 5.6 Hz, 1 H), 6.55 (d, J= 8.2 Hz, 1 H), 7.28–7.37 (m, 5 H). 13C{1H} NMR (101 MHz, CDCl3): δ = 17.6, 18.9, 22.3, 28.0, 29.4, 31.4, 32.1, 40.3, 54.8, 57.3, 65.9, 66.6, 82.1, 117.9, 128.0, 128.1, 128.5, 132.5, 136.6, 156.1, 156.6, 170.7, 171.4. HRMS (CI): calcd for C27H42N3O7+ [M+H]+: 520.3017, found: 520.3014. Methyl (((S)-6-(((benzyloxy)carbonyl)amino)-1-(((R)-1-(tert-butoxy)-3-methyl-1oxobutan-2-yl)amino)-1-oxohexan-2-yl)carbamoyl)-L-allo-isoleucinate (6a) According to general procedure B, 4a (352 mg, 0.678 mmol) was reacted with DMBA (318 mg, 2.03 mmol), Pd(PPh3)4 (23 mg, 20 µmol) and freshly prepared 5a27 (116 mg, 0.678 ACS Paragon Plus Environment

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mmol). The reaction was worked up after 66 h. Flash chromatography (PE/EtOAc 1:1, 4:6) afforded 6a (355 mg, 0.585 mmol, 86%) as a colorless solid; mp = 115–117 °C; [α]20 D = −12.7 (c = 1.0, CHCl3); Rf = 0.23 (PE/EtOAc 4:6). Major rotamer: 1H NMR (400 MHz, CDCl3): δ = 0.80 (d, J = 6.9 Hz, 3 H), 0.85–0.93 (m, 9 H), 1.18 (m, 1 H), 1.33–1.41 (m, 3 H), 1.44 (s, 9 H), 1.50 (m, 2 H), 1.60 (m, 1 H), 1.76–1.92 (m, 2 H), 2.15 (m, 1 H), 3.14 (m, 1 H), 3.20 (m, 1 H), 3.68 (s, 3 H), 4.34 (m, 1 H), 4.39 (dd, J = 8.7, 4.4 Hz, 1 H), 4.58 (dd, J = 9.1, 3.8 Hz, 1 H), 5.10 (m, 2 H), 5.36 (m, 1 H), 5.59 (d, J = 9.1 Hz, 1 H), 5.82 (d, J = 7.1 Hz, 1 H), 6.90 (d, J = 8.7 Hz, 1 H), 7.28–7.37 (m, 5 H). 13C{1H} NMR (101 MHz, CDCl3): δ = 11.8, 14.6, 17.5, 18.9, 22.4, 26.2, 28.0, 29.4, 31.3, 32.2, 37.5, 40.3, 52.1, 54.0, 56.0, 57.3, 66.6, 81.7, 128.0, 128.1, 128.4, 136.6, 156.9, 157.9, 170.6, 172.9, 174.4. Minor rotamer (selected signals): 1H NMR (400 MHz, CDCl3): δ = 2.96 (m, 1 H), 4.21 (m, 1 H), 5.48 (bs, 1 H), 6.29 (bs, 1 H), 6.37 (bs, 1 H). Rotameric ratio: 8:2. HRMS (CI): calcd for C31H50N4O8+ [M+H]+: 607.3701, found: 607.3678. Methyl (((R)-6-(((benzyloxy)carbonyl)amino)-1-(((S)-1-(tert-butoxy)-3-methyl-1oxobutan-2-yl)amino)-1-oxohexan-2-yl)carbamoyl)-L-allo-isoleucinate (6b) According to general procedure B, 4b (509 mg, 0.980 mmol) was reacted with DMBA (459 mg, 2.94 mmol), Pd(PPh3)4 (34 mg, 30 µmol) and freshly prepared 5a27 (169 mg, 0.987 mmol). The reaction was worked up after 17 h. Automated flash chromatography (PE/EtOAc 100:0–55:45) afforded 6b (520 mg, 0.857 mmol, 87%) as a pale yellow resin; 1 [α]20 D = +21.8 (c = 1.0, CHCl3); Rf = 0.16 (PE/EtOAc 1:1). H NMR (400 MHz, CDCl3): δ = 0.81 (d, J = 6.9 Hz, 3 H), 0.87 (d, J = 6.9 Hz, 3 H), 0.89–0.92 (m, 6 H), 1.16 (m, 1 H), 1.34–1.41 (m, 3 H), 1.44 (s, 9 H), 1.47–1.54 (m, 2 H), 1.68 (m, 1 H), 1.79–1.89 (m, 2 H), 2.13 (septd, J= 6.8, 4.9 Hz, 1 H), 3.16 (m, 2 H), 3.68 (s, 3 H), 4.28 (m, 1 H), 4.40 (dd, J = 8.7, 4.7 Hz, 1 H), 4.54 (dd, J = 9.2, 4.3 Hz, 1 H), 5.08 (m, 2 H), 5.13 (m, 1 H), 5.58 (d, J = 9.2 Hz, 1 H), 5.84 (d, J = 7.1 Hz, 1 H), 6.91 (d, J = 8.8 Hz, 1 H), 7.28–7.36 (m, 5 H). 13C{1H} NMR (101 MHz, CDCl3): δ = 11.7, 14.6, 17.6, 18.9, 22.3, 26.2, 28.0, 29.4, 31.3, 32.2, 37.7, 40.4, 52.0, 54.0, 56.3, 57.4, 66.6, 81.8, 128.0, 128.4, 136.5, 156.8, 157.7, 170.8, 172.5, 174.0. HRMS (CI): calcd for C31H51N4O8+ [M+H]+: 607.3701, found: 607.3735. Methyl (((R)-6-(((benzyloxy)carbonyl)amino)-1-(((S)-1-(tert-butoxy)-3-methyl-1oxobutan-2-yl)amino)-1-oxohexan-2-yl)carbamoyl)-L-isoleucinate (6c) According to general procedure B, 4b (513 mg, 0.987 mmol) was reacted with DMBA (462 mg, 2.96 mmol), Pd(PPh3)4 (34 mg, 30 µmol) and freshly prepared 5b27 (179 mg, 1.05 mmol). The reaction was worked up after 17 h. Automated flash chromatography (PE/EtOAc 100:0–1:1) afforded 6c (538 mg, 0.887 mmol, 90%) as a pale yellow resin; [α]20 D = +21.6 (c = 1.0, CHCl3); Rf = 0.19 (PE/EtOAc 1:1). Mixture of rotamers (ratio: 6:4). 1H NMR (400 MHz, CDCl ): δ = 0.84–0.92 (m, 12 H), 1.14 (m, 1 H), 1.34–1.41 (m, 3 H), 1.44 3 (s, 9 H), 1.51 (m, 2 H), 1.67 (m, 1 H), 1.77–1.87 (m, 2 H), 2.13 (septd, J = 6.8, 4.8 Hz, 1 H), 3.17 (m, 2 H), 3.68 (s, 3 H), 4.28 (m, 1 H), 4.40 (dd, J = 8.6, 4.7 Hz, 1 H), 4.43 (dd, J = 8.9, 5.2 Hz, 1 H), 5.09 (m, 2 H), 5.11 (m, 1 H), 5.61 (d, J = 8.9 Hz, 1 H), 5.81 (d, J = 7.0 Hz, 1 H), 6.89 (d, J = 8.7 Hz, 1 H), 7.28–7.36 (m, 5 H). 13C{1H} NMR (101 MHz, CDCl3): δ = 11.6, 15.4, 17.6, 18.9, 22.3, 25.1, 28.0, 29.4, 31.3, 32.2, 38.0, 40.4, 51.9, 54.0, 57.4, 66.6, 81.8, 128.0, 128.4, 136.5, 156.8, 157.5, 170.8, 172.5, 173.6. HRMS (CI): calcd for C31H51N4O8+ [M+H]+: 607.3701, found: 607.3705. Methyl (((S)-1-(((R)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-6-((S)-2((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-N,4-dimethylpentanamido)pent-4ynamido)-3-phenylpropanamido)-1-oxohexan-2-yl)carbamoyl)-L-alloisoleucinate (7a) ACS Paragon Plus Environment


To a solution of 6a (594 mg, 0.979 mmol) in MeOH (10 mL) 1 M aq. HCl (1.1 mL, 1.1 mmol) and Pd/C (10 wt%, 59 mg) were added. The mixture was stirred under H2 (1 bar) for 2 h and was then filtered over celite. The filtrate was concentrated and the residue dried by lyophilization, which afforded the deprotected tripeptide (600 mg, 83% purity). This tripeptide (583 mg, 0.951 mmol) was reacted according to general procedure A with C (473 mg, 0.951 mmol), which was activated with IBCF (0.13 mL, 1.00 mmol) and NMM (0.22 mL, 2.00 mmol) in THF (10 mL). The reaction was worked up after 43 h. Flash chromatography (DCM/MeOH 97:3, 96:4) afforded 7a (467 mg, 0.496 mmol, 52%) as a colorless solid; mp = 95–96 °C; [α]20 D = −95.6 (c = 1.0, CHCl3); Rf = 0.25 (DCM/MeOH 95:5). Mixture of rotamers (ratio: 6:4). Major rotamer: 1H NMR (500 MHz, CDCl3): δ = 0.60 (d, J = 6.9 Hz, 3 H), 0.86–0.89 (m, 6 H), 0.92 (d, J = 6.9 Hz, 3 H), 0.93 (d, J = 7.0 Hz, 3 H), 0.97 (d, J = 6.4 Hz, 3 H), 1.20 (m, 1 H), 1.29–1.36 (m, 3 H), 1.44 (s, 9 H), 1.47 (m, 2 H), 1.49 (s, 9 H), 1.58 (m, 1 H), 1.65 (m, 2 H), 1.74 (m, 1 H), 1.85–1.93 (m, 2 H), 2.04 (t, J = 2.6 Hz, 1 H), 2.14 (m, 1 H), 2.28 (s, 3 H), 2.53 (ddd, J = 17.5, 9.2, 2.7 Hz, 1 H), 2.92 (ddd, J = 17.5, 5.9, 2.7 Hz, 1 H, 10-Hb), 2.95–3.02 (m, 2 H), 3.15 (m, 1 H), 3.53 (m, 1 H), 3.75 (s, 3 H), 4.33 (m, 1 H), 4.40 (dd, J = 8.9, 4.5 Hz, 1 H), 4.49 (m, 1 H), 4.55 (m, 1 H), 4.60 (dd, J = 9.7, 4.0 Hz, 1 H), 4.89 (dd, J = 9.0, 6.1 Hz, 1 H), 5.18 (d, J = 6.4 Hz, 1 H), 5.62 (d, J = 7.5 Hz, 1 H), 6.40 (d, J = 9.7 Hz, 1 H), 6.97–7.01 (m, 2 H), 7.15 (m, 2 H), 7.20 (m, 1 H), 7.24 (m, 2 H), 8.48 (d, J = 7.9 Hz, 1 H). 13C{1H} NMR (126 MHz, CDCl3): δ = 11.7, 14.8, 17.4, 18.9, 18.9, 20.9, 21.6, 23.5, 24.9, 26.4, 28.0, 28.0, 28.3, 28.5, 29.5, 31.3, 36.9, 37.3, 38.1, 40.8, 48.5, 52.2, 52.8, 55.9, 56.8, 57.2, 58.6, 71.9, 80.0, 81.0, 81.5, 126.7, 128.6, 129.1, 137.3, 156.8, 158.8, 169.5, 170.8, 171.5, 172.7, 173.6, 175.4. Minor rotamer (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.77 (d, J = 6.9 Hz, 3 H), 0.98 (d, J = 6.4 Hz, 3 H), 1.39 (s, 9 H), 1.44 (s, 9 H), 1.96 (t, J = 2.6 Hz, 1 H), 2.58 (m, 1 H), 2.69 (m, 1 H), 3.46 (m, 1 H), 3.73 (s, 3 H), 4.27 (m, 1 H), 4.36 (dd, J = 8.9, 4.5 Hz, 1 H), 4.66 (dd, J = 9.7, 4.0 Hz, 1 H), 4.99 (m, 1 H,), 5.46 (d, J = 8.6 Hz, 1 H), 5.96 (d, J = 6.9 Hz, 1 H), 6.26 (d, J = 9.1 Hz, 1 H), 6.93 (d, J = 8.9 Hz, 1 H). 13C{1H} NMR (126 MHz, CDCl3): δ = 11.7, 14.8, 17.4, 18.9, 18.9, 20.9, 21.6, 23.5, 24.9, 26.4, 28.0, 28.3, 28.5, 29.5, 31.3, 36.9, 37.3, 38.1, 40.8, 48.5, 52.2, 52.8, 55.9, 56.8, 57.2, 58.6, 71.9, 80.0, 81.0, 81.5, 126.7, 128.6, 129.1, 137.3, 156.8, 158.8, 169.5, 170.8, 171.5, 172.7, 173.6, 175.4. HRMS (ESI): calcd for C49H80N7O11+ [M+H]+: 942.5910, found: 942.5933. Methyl (((R)-1-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-6-((S)-2((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-N,4-dimethylpentanamido)pent-4ynamido)-3-phenylpropanamido)-1-oxohexan-2-yl)carbamoyl)-L-alloisoleucinacte (7b) To a solution of 6b (489 mg, 0.806 mmol) in MeOH (8 mL) 1 M HCl (aq.) (0.89 mL, 0.89 mmol) and Pd/C (10 wt%, 49 mg) were added. The mixture was stirred under H2 (1 bar) for 3 h and was then filtered over celite. The filtrate was concentrated and the residue dried by dissolving in DCM and evaporation in vacuo three times. Drying in high vacuum afforded the deprotected tripeptide (413 mg, purity 99%, 0.806 mmol), which was reacted according to general procedure A with C (423 mg, 0.806 mmol), which was activated with IBCF (0.11 mL, 0.846 mmol) and NMM (175 µL, 1.69 mmol) in a mixture of THF (10 mL) and DCM (5 mL). After 19 h, another 0.2 eq of activated tripeptide C in THF (2 mL) and NMM (83.2 µL, 0.806 mmol) were added. The reaction was worked up after 44 h. Automated flash chromatography (DCM/MeOH 100:0–97:3) afforded 7b (688 mg, 0.730 mmol, 91%) as a colorless solid; mp = 86–87 °C; [α]20 D = −32.6 (c = 1.0, CHCl3); Rf = 0.30 (DCM/MeOH 95:5). Mixture of rotamers (ratio: 1:1). Rotamer 1:28 1H NMR (500 MHz, CDCl3): δ = 0.82 (d, J = 6.9 Hz, 3 H), 0.88 (d, J = 6.9 Hz, 3 H), 0.89–0.95 (m, 9 H), 0.97 (d, J = 6.5 Hz, 3 H), 1.18 (m, 1 H), 1.33 (m, 2 H), 1.40 (m, 2 H), 1.44 (s, 9 H), 1.45 (m, 1 H), 1.47 (s, 9 H), 1.64 (m, 2 H), 1.71–1.83 (m, 3 H), 1.89 (m, 1 H), 1.98 (t, J = 2.6 Hz, 1 H), 2.14 (m, 1 H), ACS Paragon Plus Environment

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2.23 (s, 3 H), 2.55 (ddd, J = 17.7, 10.2, 2.5 Hz, 1 H), 2.89–2.93 (m, 2 H), 3.04 (m, 1 H), 3.29 (m, 1 H), 3.35 (m, 1 H), 3.69 (s, 3 H), 4.11 (m, 1 H), 4.38 (dd, J = 6.7, 4.7 Hz, 1 H), 4.51 (m, 1 H), 4.55 (dd, J = 9.3, 4.4 Hz, 1 H), 4.58 (m, 1 H), 4.78 (dd, J = 10.2, 5.1 Hz, 1 H), 5.37 (d, J = 7.0 Hz, 1 H), 5.78 (d, J = 9.5 Hz, 1 H), 5.91 (d, J = 6.9 Hz, 1 H), 6.85 (m, 1 H), 7.13 (m, 2 H), 7.21 (m, 1 H), 7.25 (m, 2 H), 8.07 (d, J = 8.2 Hz, 1 H). 13C{1H} NMR (126 MHz, CDCl3): δ = 11.7, 14.7, 17.5, 18.5, 18.9, 21.4, 22.3, 23.5, 24.6, 26.2, 28.0, 28.5, 28.8, 30.9, 31.3, 37.5, 37.8, 38.7, 40.5, 48.6, 52.0, 54.9, 56.2, 56.3, 57.4, 58.7, 71.7, 79.9, 81.0, 81.6, 126.8, 128.7, 129.0, 137.1, 157.1, 157.9, 168.7, 170.8, 171.5, 172.8, 173.7, 174.0. Rotamer 2 (selected signals): 1H NMR (500 MHz, CDCl3): δ = 0.80 (d, J = 6.9 Hz, 3 H), 0.87 (d, J = 6.9 Hz, 3 H), 1.38 (s, 9 H), 1.43 (s, 9 H), 1.95 (t, J = 2.5 Hz, 1 H), 2.81 (m, 2 H), 2.93 (s, 3 H), 3.11–3.17 (m, 2 H), 3.26–3.31 (m, 2 H), 4.22 (m, 1 H), 4.36 (dd, J = 6.5, 4.7 Hz, 1 H), 4.48 (m, 1 H), 5.60 (d, J = 7.9 Hz, 1 H), 5.80 (d, J = 9.7 Hz, 1 H), 6.02 (d, J = 6.9 Hz, 1 H), 6.97 (d, J = 7.4 Hz, 1 H), 7.17 (m, 2 H). 13C{1H} NMR (126 MHz, CDCl3): δ = 14.7, 17.6, 18.0, 19.0, 21.6, 21.9, 23.4, 24.8, 26.2, 28.3, 31.1, 31.2, 34.5, 36.3, 37.7, 38.6, 41.0, 49.2, 54.5, 57.4, 59.7, 70.8, 79.7, 80.0, 81.6, 126.7, 128.5, 129.0, 137.6, 156.0, 158.0, 169.3, 170.6, 171.0, 172.8, 174.1, 174.8. HRMS (ESI): calcd for C49H80N7O11+ [M+H]+: 942.5910, found: 942.5926. Methyl (((R)-1-(((S)-1-(tert-butoxy)-3-methyl-1-oxobutan-2-yl)amino)-6-((S)-2((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-N,4-dimethylpentanamido)pent-4ynamido)-3-phenylpropanamido)-1-oxohexan-2-yl)carbamoyl)-L-isoleucinate (7c) To a solution of 6c (511 mg, 0.842 mmol) in MeOH (8.5 mL) 1 M aq. HCl (0.93 mL, 0.93 mmol) and Pd/C (10 wt%, 51 mg) were added. The mixture was stirred under H2 (1 bar) for 3 h and was then filtered over celite. The filtrate was concentrated and the residue dried by dissolving in DCM and evaporation in vacuo three times. Drying in high vacuum afforded the deprotected tripeptide (433 mg, purity 99%, 0.842 mmol), which was reacted according to General procedure A with C (441 mg, 0.842 mmol) which was activated with IBCF (0.12 mL, 0.884 mmol) and NMM (183 µL, 1.77 mmol) in a mixture of THF (10 mL) and DCM (5 mL). After 19 h, another 0.2 eq of activated tripeptide C in THF (2 mL) and NMM (86.9 µL, 0.842 mmol) were added. The reaction was worked up after 44 h. Automated flash chromatography (DCM/MeOH 100:0–97:3) afforded 7c (530 mg, 0.563 mmol, 67%) as a colorless solid; mp = 92–94 °C; [α]20 D = −25.1 (c = 1.0, CHCl3); Rf = 0.16 (DCM/MeOH 95:5). .Mixture of rotamers (ratio: 1:1). Rotamer 1: 1H NMR (500 MHz, CDCl3): δ = 0.86–0.96 (m, 15 H, 8-H), 0.98 (d, J = 6.5 Hz, 3 H), 1.16 (m, 1 H), 1.38 (m, 2 H), 1.42 (m, 1 H), 1.45 (s, 9 H), 1.54 (m, 2 H), 1.66 (m, 2 H), 1.69–1.80 (m, 2 H, 7-H), 1.81–1.89 (m, 2 H), 1.99 (t, J = 2.6 Hz, 1 H), 2.15 (m, 1 H), 2.25 (s, 3 H), 2.58 (ddd, J = 17.7, 10.3, 2.4 Hz, 1 H), 2.87–2.93 (m, 2 H), 3.07 (m, 1 H), 3.29–3.37 (m, 2 H), 3.70 (s, 3 H), 4.25 (m, 1 H), 4.39 (m, 1 H), 4.45 (m, 1 H), 4.52 (m, 1 H), 4.61 (m, 1 H), 4.79 (dd, J = 10.3, 5.0 Hz, 1 H), 5.42 (d, J = 6.9 Hz, 1 H), 5.83 (d, J = 8.8 Hz, 1 H), 6.03 (d, J = 6.9 Hz, 1 H), 6.84 (m, 1 H), 7.10 (d, J = 8.8 Hz, 1 H), 7.15 (m, 2 H), 7.22 (m, 1 H), 7.29 (m, 2 H), 8.06 (d, J = 8.2 Hz, 1 H). 13C{1H} NMR (126 MHz, CDCl ): δ = 11.6, 15.5, 17.5, 18.5, 18.9, 21.4, 22.3, 23.5, 24.6, 25.1, 3 28.0, 28.0, 28.3, 28.8, 31.1, 31.3, 37.5, 37.9, 38.5, 40.5, 49.2, 51.9, 54.3, 56.1, 57.4, 57.4, 58.7, 71.7, 79.9, 80.9, 81.6, 126.8, 128.5, 129.0, 137.1, 155.9, 157.7, 168.6, 170.8, 171.4, 172.7, 170.9, 173.6, 173.6. Rotamer 2 (selected signals): 1H NMR (500 MHz, CDCl3): δ = 1.32 (m, 1 H), 1.38 (s, 9 H), 1.45 (s, 9 H), 1.53 (m, 1 H), 1.96 (t, J = 2.6 Hz, 1 H), 2.80 (m, 2 H), 2.94 (s, 3 H), 3.13–3.21 (m, 3 H), 3.27 (m, 1 H), 4.15 (m, 1 H), 4.38–4.42 (m, 2 H), 5.60 (d, J = 7.9 Hz, 1 H), 5.88 (d, J = 8.0 Hz, 1 H), 5.91 (d, J = 6.9 Hz, 1 H), 7.18 (m, 2 H). 13C{1H} NMR (126 MHz, CDCl3): δ = 15.5, 17.6, 18.0, 21.6, 21.9, 23.4, 24.8, 25.2, 28.5, 31.2 31.2, 34.4, 36.3, 38.6, 41.0, 48.6, 54.7, 55.8, 59.6, 70.8, 79.7, 80.0, 81.6, 126.7, 128.7, 129.0,



137.6, 157.1, 157.8, 169.2, 170.6, 170.9, 172.7, 173.7, 174.8. HRMS (ESI): calcd for C49H80N7O11+ [M+H]+: 942.5910, found: 942.5912. Methyl (((3S,6S,9S,12R,15S)-3-benzyl-9-isobutyl-12-isopropyl-7-methyl2,5,8,11,14-pentaoxo-6-(prop-2-yn-1-yl)-1,4,7,10,13-pentaazacyclononadecan-15yl)carbamoyl)-L-allo-isoleucinate (8a) According to general procedure C, 7a (200 mg, 0.212 mmol) was deprotected and reacted with TBTU (204 mg, 0.636 mmol) and DIPEA (111 µL, 0.636 mmol) in DCM (212 mL). After 13 h further TBTU (34 mg, 0.106 mmol) and DIPEA (18.5 µL, 0.160 mmol) were added. The reaction was worked up after 41 h. Flash chromatography (DCM/MeOH 96:4) afforded cyclic peptide 8a (119 mg, 0.155 mmol, 73%) as a colorless solid; mp = 149–149 °C; Rf = 0.12 (DCM/MeOH 95:5). Mixture of rotamers (ratio: 8:2). Major rotamer: 1H NMR (400 MHz, DMSO-d6): δ = 0.76 (d, J = 6.9 Hz, 3 H), 0.79 (d, J = 6.9 Hz, 3 H), 0.81–0.86 (m, 9 H), 0.88 (d, J = 6.3 Hz, 3 H), 1.15 (m, 1 H), 1.25–1.36 (m, 3 H, 11-H), 1.40–1.52 (m, 3 H), 1.56–1.72 (m, 4 H), 1.80 (m, 1 H), 2.12 (s, 3 H), 2.45 (m, 1 H), 2.68 (m, 1 H), 2.85 (dd, J = 13.8, 10.5 Hz, 1 H), 2.90–2.95 (m, 2 H), 3.17 (dd, J = 13.9, 4.5 Hz, 1 H), 3.28 (m, 1 H), 4.06 (m, 1 H), 4.23 (dd, J = 8.1, 4.4 Hz, 1 H), 4.29 (dd, J = 9.0, 4.1 Hz, 1 H), 4.51 (ddd, 1 H), 4.81– 4.90 (m, 2 H), 6.42 (d, J = 9.0 Hz, 1 H), 6.49 (d, J = 7.2 Hz, 1 H), 7.12 (m, 2 H), 7.16–7.28 (m, 4 H), 7.65–7.71 (m, 2 H), 8.54 (d, J = 7.9 Hz, 1 H). 13C{1H} NMR (101 MHz, DMSO-d6): δ = 11.6, 14.7, 16.6, 18.4, 19.1, 21.2, 21.3, 23.5, 24.0, 25.8, 27.2, 28.1, 30.3, 31.0, 36.8, 37.3, 38.0, 41.0, 46.3, 51.7, 53.3, 54.3, 55.1, 57.0, 58.1, 74.0, 80.6, 126.2, 128.2, 129.0, 137.7, 157.3, 167.7, 170.1, 170.7, 172.0, 172.5, 173.4. Minor rotamer (selected signals): 1H NMR (400 MHz, DMSO-d6): δ = 3.91 (m, 1 H), 3.98 (m, 1 H), 4.69 (m, 1 H), 6.58 (d, J = 6.3 Hz, 1 H), 6.99 (d, J = 9.0 Hz, 1 H), 8.36 (d, J = 7.3 Hz, 1 H). HRMS (CI): calcd for C40H61N7O8+ [M]+: 767.4576, found: 767.4582. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-9-isobutyl-12-isopropyl-7-methyl2,5,8,11,14-pentaoxo-6-(prop-2-yn-1-yl)-1,4,7,10,13-pentaazacyclononadecan-15yl)carbamoyl)-L-allo-isoleucinate (8b) According to general procedure C, 7b (400 mg, 0.425 mmol) was deprotected and reacted with TBTU (409 mg, 1.28 mmol) and DIPEA (0.30 mL, 1.70 mmol) in DCM (425 mL). The reaction was worked up after 40.5 h. Flash chromatography (DCM/MeOH 96:4) afforded cyclic peptide 8b (182 mg, 0.237 mmol, 56%) as a colorless solid; mp = 159–162 °C; [α]20 D = −117.2 (c = 1.0, CHCl3); Rf = 0.19 (DCM/MeOH 95:5). 1H NMR (400 MHz, DMSO-d6): δ = 0.79 (d, J = 6.9 Hz, 3 H, 19-H), 0.85 (t, J = 7.4 Hz, 3 H, 18-H), 0.90–0.94 (m, 9 H), 1.01 (d, J = 6.6 Hz, 3 H), 1.06–1.18 (m, 2 H), 1.28 (m, 1 H), 1.33–1.47 (m, 3 H), 1.55–1.66 (m, 4 H), 1.78 (m, 1 H), 1.80 (s, 3 H), 1.89 (m, 1 H), 1.95 (m, 1 H), 2.52 (m, 2 H), 2.73 (dd, J = 13.3, 13.3 Hz, 1 H), 2.81 (m, 1 H), 2.92 (m, J = 2.5 Hz, 1 H), 3.42 (m, 1 H), 3.59 (s, 3 H), 3.64 (m, 1 H), 3.86–3.93 (m, 2 H), 4.26 (dd, J = 9.2, 4.5 Hz, 1 H), 4.44 (m, 1 H), 4.67 (m, 1 H), 5.07 (dd, J = 9.6, 5.4 Hz, 1 H), 6.41 (d, J = 9.2 Hz, 1 H), 6.46 (d, J = 6.6 Hz, 1 H), 6.74 (d, J = 7.1 Hz, 1 H), 7.03 (d, J = 6.9 Hz, 2 H), 7.14 (m, 1 H), 7.18 (m, 2 H), 7.49 (m, 1 H), 8.77 (d, J = 4.7 Hz, 1 H), 9.01 (d, J = 9.1 Hz, 1 H). 13C{1H} NMR (101 MHz, DMSO-d6): δ = 11.5, 14.8, 17.3, 18.9, 18.9, 20.1, 21.3, 23.5, 24.3, 25.8, 26.8, 28.5, 30.0, 31.7, 36.6, 37.5, 38.2, 39.5, 47.6, 51.7, 54.4, 54.5, 55.4, 58.0, 58.3, 73.7, 81.2, 126.0, 128.3, 128.9, 138.4, 157.4, 168.5, 170.4, 172.0, 172.3, 172.5, 173.4. HRMS (ESI): calcd for C40H62N7O8+ [M+H]+: 768.4654, found: 768.4647. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-9-isobutyl-12-isopropyl-7-methyl2,5,8,11,14-pentaoxo-6-(prop-2-yn-1-yl)-1,4,7,10,13-pentaazacyclononadecan-15yl)carbamoyl)-L-isoleucinate (8c) ACS Paragon Plus Environment

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Page 13 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


According to general procedure C, 7c (492 mg, 0.522 mmol) was deprotected and reacted with TBTU (504 mg, 1.57 mmol) and DIPEA (0.36 mL, 2.09 mmol) in DCM (520 mL). The reaction was worked up after 41 h. Flash chromatography (DCM/MeOH 97:3, 96:4) afforded cyclic peptide 8c (272 mg, 0.354 mmol, 68%) as a colorless solid; mp = 155–157 1 °C; [α]20 D = −99.8 (c = 1.0, CHCl3); Rf = 0.17 (DCM/MeOH 95:5). H NMR (400 MHz, DMSOd6): δ = 0.80–0.86 (m, 6 H), 0.88–0.95 (m, 9 H), 1.01 (d, J = 6.7 Hz, 3 H), 1.07–1.15 (m, 2 H), 1.29–1.47 (m, 4 H), 1.55–1.65 (m, 4 H), 1.70 (m, 1 H), 1.80 (s, 3 H), 1.89 (m, 1 H), 1.96 (m, 1 H), 2.52 (m, 2 H), 2.74 (dd, J = 13.3, 13.3 Hz, 1 H), 2.80 (m, 1 H), 2.92 (t, J = 2.5 Hz, 1 H), 3.42 (m, 1 H), 3.58 (s, 3 H), 3.63 (m, 1 H), 3.86–3.92 (m, 2 H), 4.10 (dd, J = 8.7, 5.8 Hz, 1 H), 4.43 (m, 1 H), 4.67 (m, 1 H), 5.07 (dd, J = 9.8, 5.2 Hz, 1 H), 6.42 (d, J = 6.7 Hz, 1 H), 6.47 (d, J = 8.8 Hz, 1 H), 6.78 (d, J = 7.0 Hz, 1 H), 7.03 (d, J = 6.9 Hz, 2 H), 7.14 (m, 1 H), 7.19 (m, 2 H), 7.48 (m, 1 H), 8.77 (d, J = 4.6 Hz, 1 H), 9.02 (d, J = 9.1 Hz, 1 H). 13C{1H} NMR (101 MHz, DMSO-d6): δ = 11.3, 15.6, 17.3, 18.9, 18.9, 20.1, 21.3, 23.5, 24.3, 24.7, 26.8, 28.5, 29.9, 31.7, 36.9, 37.5, 38.2, 39.4, 47.6, 51.5, 54.5, 54.5, 56.8), 58.0, 58.3, 73.7, 81.2, 126.0, 128.3, 128.9, 138.4, 157.2, 168.5, 170.4, 172.0, 172.3, 172.5, 173.0. HRMS (ESI): calcd for C40H62N7O8+ [M+H]+: 768.4654, found: 768.4642. Methyl (((3S,6S,9S,12R,15S)-3-benzyl-9-isobutyl-12-isopropyl-7-methyl2,5,8,11,14-pentaoxo-6-(2-(tributylstannyl)allyl)-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (9a) Peptide 8a (88 mg, 0.115 mmol) was dissolved in DCM (5 mL) under an atmosphere of Ar. [Cp*Ru(MeCN)3] PF6 (11.6 mg, 23.0 µmol) was added in one portion, and immediately, a solution of Bu3SnH (90 µL, 0.344 mmol) in DCM (6 mL) was added via syringe over the course of 30 seconds. A change of color from deep purple to light brown occurred after ca. 1 eq. of Bu3SnH was added. The mixture was stirred for 20 min before the solvent was removed in vacuo. Automated flash chromatography (DCM/MeOH 100:0–97:3) afforded 9a (106 mg, 0.100 mmol, 87%) as a pale brown solid; mp = 103–106 °C; Rf = 0.14 (DCM/MeOH 95:5). Mixture of rotamers (ratio: 6:4). Major rotamer: 1H NMR (500 MHz, DMSO-d6): δ = 0.71 (d, J = 7.1 Hz), 0.77–0.79 (m, 9 H), 0.82–0.89 (m, 21 H), 1.15 (m, 1 H), 1.21–1.33 (m, 11 H), 1.43 (m, 6 H), 1.55 (m, 1 H), 1.58–1.65 (m, 2 H), 1.72–1.82 (m, 2 H), 2.16 (m, 1 H), 2.21 (m, 1 H), 2.23 (s, 3 H), 2.76–2.85 (m, 2 H), 3.00 (m, 1 H), 3.10 (m, 1 H), 3.18 (m, 1 H), 3.63 (s, 3 H), 4.08 (m, 1 H), 4.29 (dd, J = 9.1, 4.2 Hz, 1 H), 4.34 (m, 1 H), 4.55 (m, 1 H), 4.63 (m, 1 H), 4.76 (m, 1 H), 5.25 (m, 1 H), 5.70 (m, 1 H), 6.41 (d, J = 9.1 Hz, 1 H), 6.53 (d, J = 7.3 Hz, 1 H), 7.13 (m, 2 H), 7.17 (m, 1 H), 7.21–7.26 (m, 3 H), 7.59 (m, 1 H), 7.83 (d, J = 8.0 Hz, 1 H), 8.64 (d, J = 8.2 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 9.5, 11.6, 13.5, 14.7, 16.4, 19.0, 21.3, 21.6, 23.2, 24.1, 25.8, 26.8, 27.6, 28.6, 29.3, 30.9, 31.2, 36.8, 37.8, 38.2, 38.3, 41.2, 46.3, 51.7, 53.2, 54.1, 55.0, 56.3, 60.2, 126.2, 127.9, 128.1, 129.1, 137.4, 151.3, 157.3, 168.4, 170.1, 170.6, 171.7, 172.4, 173.4. 119Sn NMR (149 MHz, DMSOd6): δ = −43.6. Minor rotamer (selected signals): 1H NMR (500 MHz, DMSO-d6): δ = 2.66 (m, 1 H), 2.93 (m, J = 13.7, 9.6 Hz, 1 H), 3.59 (m, 1 H), 3.99 (m, 1 H), 4.17 (m, 1 H), 5.17 (m, 1 H), 5.58 (m, 1 H), 6.45 (d, J = 9.4 Hz, 1 H), 6.58 (d, J = 6.5 Hz, 1 H), 7.02 (d, J = 8.9 Hz, 1 H), 7.33 (m, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 8.30 (d, J = 7.5 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 9.3, 11.5, 17.4, 18.9, 20.9, 23.2, 28.5, 51.8, 54.9, 56.4, 126.1, 128.0, 129.1, 138.6, 157.3, 169.3, 171.8, 172.7, 173.3. HRMS (ESI): calcd for C52H90N7O8Sn+ [M+H]+: 1060.5867, found: 1060.5904. Methyl (((3S,6S,9S,12S,15R)-3-Benzyl-9-isobutyl-12-isopropyl-7-methyl2,5,8,11,14-pentaoxo-6-(2-(tributylstannyl)allyl)-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (9b)



8b (157 mg, 0.204 mmol) was reacted with [Cp*Ru(MeCN)3]PF6 (20.6 mg, 40.9 µmol) and Bu3SnH (0.11 mL, 0.408 mmol) in DCM (5 mL) according to the procedure for 9a. The reaction was worked up after 1 h. Flash chromatography (DCM/MeOH 97:3) afforded 9b (134 mg, 0.127 mmol, 62%) as a colorless solid; mp = 103–106 °C; [α]20 D = −108 (c = 0.1, MeOH); Rf = 0.24 (DCM/MeOH 95:5). 1H NMR (400 MHz, DMSO-d6): δ = 0.78 (d, J = 6.9 Hz, 3 H), 0.80–0.87 (m, 21 H, 18-H, 30-H), 0.88–0.93 (m, 6 H, 25-H), 1.01 (d, J = 6.6 Hz, 3 H), 1.09–1.18 (m, 2 H), 1.23 (m, 6 H), 1.28–1.35 (m, 3 H), 1.36–1.49 (m, 8 H), 1.54–1.67 (m, 3 H), 1.73–1.79 (m, 2 H), 1.79 (s, 3 H), 1.98 (m, 1 H), 2.19 (dd, J = 14.8, 6.4 Hz, 1 H), 2.74– 2.87 (m, 3 H), 3.34 (m, 1 H), 3.55 (m, 1 H), 3.59 (s, 3 H), 3.87–3.93 (m, 2 H), 4.25 (dd, J = 9.0, 4.4 Hz, 1 H), 4.49 (m, 1 H), 4.68 (m, 1 H), 4.78 (t, J = 6.5 Hz, 1 H), 5.29 (d, J = 63.5 Hz, 1 H), 5.82 (d, J = 138.6 Hz, 1 H), 6.38 (d, J = 9.1 Hz, 1 H), 6.45 (d, J = 6.6 Hz, 1 H), 6.87 (d, J = 7.1 Hz, 1 H), 7.06 (d, J = 7.0 Hz, 2 H), 7.13–7.23 (m, 3 H), 7.33 (m, 1 H), 8.75 (d, J = 4.7 Hz, 1 H), 8.85 (d, J = 9.0 Hz, 1 H). 13C{1H} NMR (101 MHz, DMSO-d6): δ = 9.4, 11.5, 13.6, 14.8, 18.8, 19.0, 20.4, 21.4, 23.4, 24.2, 25.8, 26.7, 27.9, 28.2, 28.6, 30.0, 31.5, 36.6, 36.8, 37.7, 38.3, 39.6, 47.6, 51.7, 54.7, 54.7, 55.4, 57.9, 60.6, 126.1, 128.3, 128.8, 128.9, 138.2, 151.2, 157.4, 169.1, 170.8, 171.5, 172.0, 172.5, 173.3. 119Sn NMR (149 MHz, DMSO-d6): δ = −42.2. HRMS (ESI): calcd for C52H90N7O8Sn+ [M+H]+: 1060.5867, found: 1060.5914. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-9-isobutyl-12-isopropyl-7-methyl2,5,8,11,14-pentaoxo-6-(2-(tributylstannyl)allyl)-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-isoleucinate (9c) 8c (247 mg, 0.322 mmol) was reacted with [Cp*Ru(MeCN)3]PF6 (32.4 mg, 64.3 µmol) and Bu3SnH (0.17 mL, 0.644 mmol) in DCM (6 mL) according to the procedure for 9a. The reaction was worked up after 1 h. Flash chromatography (DCM/MeOH 97:3) afforded 9c (234 mg, 0.221 mmol, 69%) as a colorless solid; mp = 117–119 °C; [α]20 D = −120 (c = 0.1, MeOH); Rf = 0.26 (DCM/MeOH 95:5). 1H NMR (400 MHz, DMSO-d6): δ = 0.79–0.86 (m, 24 H), 0.89–0.92 (m, 6 H), 1.01 (d, J = 6.7 Hz, 3 H), 1.08–1.15 (m, 2 H), 1.23 (m, 6 H), 1.28– 1.36 (m, 3 H), 1.37–1.50 (m, 9 H), 1.53–1.64 (m, 3 H), 1.70 (m, 1 H), 1.78 (m, 1 H), 1.79 (s, 3 H), 1.98 (m, 1 H), 2.19 (dd, J = 14.8, 6.7 Hz, 1 H), 2.75–2.86 (m, 3 H), 3.34 (m, 1 H), 3.54 (m, 1 H), 3.58 (s, 3 H), 3.87–3.93 (m, 2 H), 4.10 (dd, J = 8.7, 5.9 Hz, 1 H), 4.49 (m, 1 H), 4.68 (m, 1 H), 4.78 (t, J = 6.6 Hz, 1 H), 5.29 (d, J = 63.2 Hz, 1 H), 5.82 (d, J = 139.4 Hz, 1 H), 6.41 (d, J = 6.8 Hz, 1 H), 6.44 (d, J = 8.8 Hz, 1 H), 6.91 (d, J = 7.1 Hz, 1 H), 7.06 (m, 2 H), 7.14 (m, 1 H), 7.20 (m, 2 H), 7.32 (m, 1 H), 8.74 (d, J = 4.9 Hz, 1 H), 8.85 (d, J = 9.2 Hz, 1 H). 13C{1H} NMR (101 MHz, DMSO-d6): δ = 9.4, 11.2, 13.6, 15.6, 18.8, 19.0, 20.4, 21.4, 23.4, 24.2, 24.7, 26.7, 27.9, 28.2, 28.6, 30.0, 31.5, 36.8, 36.9, 37.7, 38.3, 39.6, 47.6, 51.5, 54.7, 54.8, 56.8, 57.8, 60.5, 126.1, 128.3, 128.8, 128.9, 138.2, 151.2, 157.2, 169.1, 170.8, 171.5, 172.0, 172.5, 172.9. 119Sn NMR (149 MHz, DMSO-d6): δ = −42.3. HRMS (ESI): calcd for C52H90N7O8Sn+ [M+H]+: 1060.5867, found: 1060.5913. 4-(Benzyloxy)-2-iodoaniline (E) 4-Hydroxy-2-iodoaniline (200 mg, 0.851 mmol) was dissolved in DMF (1.6 mL) under an atmosphere of N2 and NaH (60% dispersion, 41 mg, 1.02 mmol) were added portionwise. After 10 min, benzyl chloride (0.12 mL, 1.02 mmol) was added and the reaction mixture was warmed to rt over night. After 16 h, H2O was added and the aqueous phase was extracted twice with EtOAc. The organic phase was washed with H2O and saturated NaCl solution, dried (Na2SO4) and concentrated. Automated flash chromatography (PE/EtOAc 100:0–9:1) afforded 194 mg (0.597 mmol, 70%) of benzlyated aminophenol E as an orange coloured solid; mp = 64–65 °C; Rf = 0.26 (PE/EtOAc 8:2). 1H NMR (400 MHz, CDCl3): δ = 3.63 (bs, 2 H), 4.96 (s, 2 H), 6.69 (d, J = 8.7 Hz, 1 H), 6.84 (dd, J = 8.7, 2.8 Hz, 1 H), 7.31 (d, J = 2.8 Hz, 1 H), 7.33 (m, 1 H), 7.36–7.43 (m, 4 H). 13C{1H} NMR (101 MHz, ACS Paragon Plus Environment

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Page 15 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CDCl3): δ = 71.0, 84.2, 115.3, 117.0, 124.9, 127.5, 127.9, 128.5, 136.9, 141.1, 151.8. HRMS (CI): calculated for C13H13INO+ [M+H]+: 326.0036, found: 326.0028. Methyl (((3S,6S,9S,12R,15S)-6-(2-(2-amino-5-(benzyloxy)phenyl)allyl)-3-benzyl9-isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucin (10a) According to general procedure D, 9a (122 mg, 115 µmol) was reacted with E (41.2 mg, 127 µmol), CuTC (44 mg, 230 µmol), Ph2PO2NBu4 (106 mg, 230 µmol) and Pd(PPh3)4 (6.6 mg, 5.8 µmol) in DMF (1 mL) for 2 h. Flash chromatography (SiO2/K2CO3 9:1 w/w, DCM/MeOH 97:3, 96:4) afforded 10a (65.1 mg, 67.3 µmol, 59%) as a pale brown solid; mp = 131–134 °C; Rf = 0.07 (DCM/MeOH 95:5). 1H NMR (400 MHz, CDCl3): δ = 0.78 (d, J = 6.8 Hz, 3 H), 0.79 (d, J = 6.7 Hz, 3 H), 0.88–0.95 (m, 12 H), 1.13–1.29 (m, 3 H), 1.34–1.47 (m, 3 H), 1.50–1.62 (m, 3 H), 1.69 (m, 1 H), 1.91 (m, 1 H), 2.07 (m, 1 H), 2.41 (m, 1 H), 2.77 (s, 3 H), 2.98–3.09 (m, 2 H), 3.14–3.20 (m, 2 H), 3.21–3.36 (m, 2 H), 3.44 (bs, 2 H), 3.63 (m, 1 H), 3.72 (s, 3 H), 4.43–4.50 (m, 2 H), 4.56–4.62 (m, 2 H), 4.76 (m, 1 H), 4.97 (s, 2 H), 5.13 (s, 1 H), 5.22 (s, 1 H), 5.80 (d, J = 7.2 Hz, 1 H), 6.02 (d, J = 9.1 Hz, 1 H), 6.36 (d, J = 9.1 Hz, 1 H), 6.53 (d, J = 2.8 Hz, 1 H), 6.62 (d, J = 8.6 Hz, 1 H), 6.64 (m, 1 H), 6.76–6.82 (m, 3 H), 7.20 (m, 2 H), 7.25 (m, 1 H), 7.28–7.33 (m, 2 H), 7.34–7.41 (m, 4 H). 13C{1H} NMR (101 MHz, CDCl3): δ = 11.8, 14.8, 16.7, 19.4, 19.9, 22.3, 22.8, 24.8, 26.4, 26.9, 28.3, 29.5, 36.6, 37.3, 37.5, 37.5, 38.9, 40.9, 47.7, 52.1, 53.1, 55.8, 55.9, 57.3, 63.9, 70.7, 115.5, 116.0, 117.7, 118.7, 127.1, 127.3, 127.4, 128.0, 128.5, 128.9, 129.1, 136.4, 137.0, 137.1, 144.0, 151.9, 157.9, 170.6, 171.0, 171.6, 172.1, 173.6, 174.2. HRMS (ESI): calcd for C53H75N8O9+ [M+H]+: 967.5652, found: 967.5666. Methyl (((3S,6S,9S,12S,15R)-6-(2-(2-amino-5-(benzyloxy)phenyl)allyl)-3-benzyl9-isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (10b) According to general procedure D, 9b (107 mg, 101 µmol) were reacted with E (36.1 mg, 111 µmol), CuTC (38.5 mg, 202 µmol), Ph2PO2NBu4 (92.8 mg, 202 µmol) and Pd(PPh3)4 (5.8 mg, 5.1 µmol) in DMF (2 mL) for 2 h. Flash chromatography (SiO2/K2CO3 9:1 w/w, DCM/MeOH 97.5:2.5, 96:4) afforded 10b (69.8 mg, 72.2 µmol, 71%) as a pale brown solid; 1 mp = 141–144 °C; [α]20 D = −127 (c = 0.1, MeOH); Rf = 0.13 (DCM/MeOH 95:5). H NMR (500 MHz, DMSO-d6): δ = 0.75 (d, J = 6.5 Hz, 3 H), 0.78 (d, J = 6.9 Hz, 3 H), 0.84 (t, J = 7.4 Hz, 3 H), 0.88–0.90 (m, 6 H), 0.99 (d, J = 6.8 Hz, 3 H), 1.09–1.17 (m, 2 H), 1.21–1.32 (m, 3 H), 1.40 (m, 2 H), 1.53–1.63 (m, 3 H), 1.59 (s, 3 H), 1.69–1.81 (m, 2 H), 1.95 (m, 1 H), 2.41 (dd, J = 15.4, 7.7 Hz, 1 H), 2.84 (dd, J = 13.3, 13.3 Hz, 1 H), 2.86 (m, 1 H), 2.97 (dd, J= 15.4, 6.7 Hz, 1 H), 3.37 (m, 1 H), 3.55–3.62 (m, 4 H), 3.87–3.92 (m, 2 H), 4.25 (dd, J = 9.1, 4.4 Hz, 1 H), 4.41 (s, 2 H), 4.51 (ddd, J = 12.4, 9.0, 3.3 Hz, 1 H), 4.63 (m, 1 H), 4.81 (t, J = 7.7 Hz, 1 H), 4.89 (s, 2 H), 5.02 (s, 1 H), 5.30 (s, 1 H), 6.39 (d, J = 9.2 Hz, 1 H), 6.42 (d, J = 2.9 Hz, 1 H), 6.45 (d, J = 6.5 Hz, 1 H), 6.54 (d, J = 8.7 Hz, 1 H), 6.66 (dd, J = 8.7, 2.9 Hz, 1 H), 6.83 (d, J = 7.4 Hz, 1 H), 7.06 (m, 2 H), 7.11 (m, 1 H), 7.18 (m, 2 H), 7.30 (m, 1 H), 7.34–7.39 (m, 4 H), 7.41 (m, 1 H), 8.76 (d, J = 4.9 Hz, 1 H), 8.95 (d, J = 8.9 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.5, 14.8, 18.8, 18.9, 20.3, 21.1, 23.3, 24.0, 25.8, 27.7, 28.5, 30.0, 31.6, 33.4, 36.6, 37.4, 38.2, 39.2, 47.9, 51.7, 54.6, 54.6, 55.4, 57.8, 58.9, 69.7, 114.7, 115.0, 115.9, 117.3, 126.0, 127.5, 127.6, 127.8, 128.2, 128.3, 128.9, 137.6, 138.4, 138.8, 144.8, 149.7, 157.4, 169.4, 170.7, 171.6, 172.0, 172.3, 173.4. HRMS (ESI): calcd for C53H75N8O9+ [M+H]+: 967.5652, found: 967.5662.



Methyl (((3S,6S,9S,12S,15R)-6-(2-(2-amino-5-(benzyloxy)phenyl)allyl)-3-benzyl9-isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-isoleucinate (10c) According to general procedure D, 9c (204 mg, 193 µmol) were reacted with E (68.9 mg, 212 µmol), CuTC (73.6 mg, 386 µmol), Ph2PO2NBu4 (177 mg, 386 µmol) and Pd(PPh3)4 (11.2 mg, 9.7 µmol) in DMF (4 mL) for 1.5 h. Flash chromatography (SiO2/K2CO3 9:1 w/w, DCM/MeOH 97.5:2.5, 96:4) afforded 10c (134 mg, 139 µmol, 72%) as a pale brown solid; 1 mp = 138–140 °C; [α]20 D = −127 (c = 0.1, MeOH); Rf = 0.17 (DCM/MeOH 95:5). H NMR (500 MHz, DMSO-d6): δ = 0.74 (d, J = 6.4 Hz, 3 H), 0.82 (d, J = 7.1 Hz, 3 H), 0.84 (t, J = 7.6 Hz, 3 H), 0.88 (d, J = 6.3 Hz, 3 H), 0.89 (d, J = 6.0 Hz, 3 H), 0.99 (d, J = 6.6 Hz, 3 H,), 1.07–1.17 (m, 2 H), 1.24 (m, 1 H), 1.28–1.44 (m, 4 H), 1.52–1.64 (m, 3 H), 1.61 (s, 3 H), 1.67–1.76 (m, 2 H), 1.96 (m, 1 H), 2.40 (dd, J = 15.3, 7.7 Hz, 1 H), 2.80–2.90 (m, 2 H), 2.98 (dd, J = 15.5, 6.9 Hz, 1 H), 3.37 (m, 1 H), 3.53–3.63 (m, 4 H), 3.86–3.94 (m, 2 H), 4.09 (dd, J = 8.6, 5.9 Hz, 1 H), 4.40 (s, 2 H), 4.51 (m, 1 H), 4.62 (m, 1 H), 4.81 (t, J = 7.3 Hz, 1 H), 4.89 (s, 2 H), 5.02 (s, 1 H), 5.30 (s, 1 H), 6.39–6.47 (m, 3 H), 6.54 (d, J = 8.7 Hz, 1 H), 6.66 (dd, J = 8.7, 2.7 Hz, 1 H), 6.86 (d, J = 7.4 Hz, 1 H), 7.06 (m, 2 H), 7.12 (m, 1 H), 7.18 (m, 2 H), 7.30 (m, 1 H), 7.33– 7.42 (m, 5 H), 8.74 (d, J = 4.6 Hz, 1 Hu), 8.94 (d, J = 8.9 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.3, 15.6, 18.8, 18.9, 20.3, 21.1, 23.3, 24.0, 24.7, 27.7, 28.5, 30.0, 31.6, 33.4, 36.9, 37.4, 38.2, 38.9, 47.9, 51.5, 54.6, 54.6, 56.8, 57.8, 58.9, 69.7, 114.7, 115.0, 115.9, 117.3, 126.0, 127.5, 127.6, 127.8, 128.2, 128.3, 128.9, 137.6, 138.4, 138.8, 144.8, 149.7, 157.2, 169.4, 170.7, 171.6, 172.0, 172.3, 173.0. HRMS (ESI): calcd for C53H75N8O9+ [M+H]+: 967.5652, found: 967.5672. Methyl (((3S,6S,9S,12R,15S)-6-(2-(2-azido-5-(benzyloxy)phenyl)allyl)-3-benzyl-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (11a) 10a (64 mg, 66.2 µmol) was dissolved in MeCN (2 mL) and tBuONO (43.7 µL, 331 µmol, 5.0 eq.) followed by TMSN3 (46.3 µL, 331 µmol, 5.0 eq) were added at 0 °C. After 4 h, further tBuONO (43.7 µL, 331 µmol, 5.0 eq.) and TMSN3 (46.3 µL, 331 µmol, 5.0 eq.) were added at rt, resulting in gas development. After 4.5 h, the mixture was diluted with EtOAc and extracted with H2O three times. The organic phase was dried (Na2SO4) and concentrated in vacuo. Automated flash chromatography (DCM/MeOH 100:0–97:3) afforded 11a (48.0 mg, 48.3 µmol, 73%) as a yellow solid; mp = 125–128 °C; Rf = 0.15 (DCM/MeOH 95:5). 1H NMR (500 MHz, CDCl3): δ = 0.78–0.82 (m, 6 H), 0.87–0.94 (m, 12 H), 1.13–1.26 (m, 3 H), 1.34–1.45 (m, 3 H), 1.50–1.67 (m, 4 H), 1.92 (m, 1 H), 2.08 (m, 1 H), 2.44 (m, 1 H), 2.85 (m, 3 H), 2.91–3.03 (m, 2 H), 3.19 (m, 2 H), 3.23–3.32 (m, 2 H), 3.71 (m, 1 H), 3.73 (s, 3 H), 4.44 (m, 1 H), 4.48 (dd, J = 9.8, 4.6 Hz, 1 H), 4.59 (dd, J = 8.8, 3.3 Hz, 1 H), 4.62 (m, 1 H), 4.76 (m, 1 H), 5.04 (s, 2 H), 5.07 (s, 1 H), 5.09 (s, 1 H), 5.85 (d, J = 6.5 Hz, 1 H), 5.97 (d, J = 9.0 Hz, 1 H), 6.36 (d, J = 8.6 Hz, 1 H), 6.58 (d, J = 5.9 Hz, 1 H), 6.66 (d, J = 1.9 Hz, 1 H), 6.79–6.85 (m, 2 H), 6.98 (m, 1 H), 7.09 (d, J = 8.7 Hz, 1 H), 7.19–7.24 (m, 2 H), 7.25–7.30 (m, 2 H), 7.31–7.37 (m, 2 H), 7.38–7.44 (m, 4 H). 13C{1H} NMR (126 MHz, CDCl3): δ = 11.8, 14.7, 16.6, 19.3, 19.8, 22.3, 22.8, 24.8, 26.4, 26.8, 28.5, 29.5, 35.8, 37.2, 37.6, 37.6, 39.2, 40.8, 47.6, 52.1, 53.3, 55.8, 55.8, 57.3, 63.9, 70.4, 115.3, 117.2, 119.4, 119.7, 127.3, 127.4, 128.3, 128.7, 128.8, 129.1, 129.3, 133.4, 136.3, 136.3, 142.9, 156.0, 157.8, 170.5, 170.7, 171.6, 172.1, 173.3, 174.0. HRMS (ESI): calcd for C53H73N10O9+ [M+H]+: 993.5557, found: 993.5573. Methyl (((3S,6S,9S,12S,15R)-6-(2-(2-azido-5-(benzyloxy)phenyl)allyl)-3-benzyl-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinacte (11b) ACS Paragon Plus Environment

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Page 17 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10b (68 mg, 70.3 µmol) was dissolved in DMF (1.5 mL) and tBuONO (46.5 µL, 353 µmol) and TMSN3 (49.0 µL, 352 µmol) were added at 0 °C. After 3 h, further tBuONO (46.5 µL, 352 µmol) and TMSN3 (49.0 µL, 352 µmol) were added at rt. After 4 h, the mixture was diluted with EtOAc and extracted with H2O three times. The organic phase was dried (Na2SO4) and concentrated in vacuo. Flash chromatography (DCM/MeOH 97:3) afforded 11b (55.2 mg, 55.6 µmol, 79%) as a pale yellow solid; [α]20 D = −71 (c = 0.1, MeOH); Rf = 0.24 (DCM/MeOH 95:5). 1H NMR (500 MHz, DMSO-d6): δ = 0.74 (d, J = 6.5 Hz, 3 H), 0.77 (d, J = 6.9 Hz, 3 H), 0.84 (t, J = 7.4 Hz, 3 H), 0.86 (d, J = 6.6 Hz, 3 H), 0.89 (d, J = 6.6 Hz, 3 H), 0.98 (d, J = 6.7 Hz, 3 H), 1.06–1.17 (m, 3 H), 1.23–1.31 (m, 2 H), 1.34–1.41 (m, 2 H), 1.48 (s, 3 H), 1.54–1.61 (m, 3 H), 1.78 (m, 1 H), 1.83 (m, 1 H), 1.95 (m, 1 H), 2.62 (dd, J = 15.0, 10.3 Hz, 1 H), 2.81–2.87 (m, 2 H), 3.02 (dd, J = 15.1, 4.6 Hz, 1 H), 3.37 (m, 1 H), 3.56 (m, 1 H), 3.58 (s, 3 H), 3.86–3.91 (m, 2 H), 4.24 (dd, J = 9.2, 4.4 Hz, 1 H), 4.50 (ddd, J = 12.4, 8.9, 3.6 Hz, 1 H), 4.55 (m, 1 H), 4.72 (dd, J = 10.1, 5.3 Hz, 1 H), 5.04 (s, 2 H), 5.13 (s, 1 H), 5.29 (s, 1 H), 6.38 (d, J = 9.2 Hz, 1 H), 6.46 (d, J = 6.6 Hz, 1 H), 6.66 (d, J = 2.9 Hz, 1 H), 6.78 (d, J = 7.3 Hz, 1 H), 7.00 (dd, J = 8.8, 2.9 Hz, 1 H), 7.04 (m, 2 H), 7.10 (m, 1 H), 7.15–7.19 (m, 3 H), 7.32 (m, 1 H), 7.35–7.42 (m, 5 H), 8.79 (d, J = 5.3 Hz, 1 H), 8.85 (d, J = 8.8 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.5, 14.8, 18.8, 18.9, 20.2, 20.5, 23.5, 23.8, 25.8, 27.8, 28.5, 29.9, 31.5, 34.0, 36.6, 37.2, 38.2, 38.5, 48.1, 51.7, 54.5, 54.6, 55.4, 57.9, 59.9, 69.4, 115.4, 115.7, 119.9, 120.3, 126.0, 127.5, 127.9, 128.2, 128.4, 128.7, 128.8, 134.8, 136.7, 138.4, 143.1, 155.4, 157.4, 169.3, 170.7, 171.9, 172.0, 172.4, 173.4. HRMS (ESI): calcd for C53H73N10O9+ [M+H]+: 993.5557, found: 993.5586. Methyl (((3S,6S,9S,12S,15R)-6-(2-(2-azido-5-(benzyloxy)phenyl)allyl)-3-benzyl-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-isoleucinate (11c) 10c (112 mg, 116 µmol) was dissolved in DMF (2 mL) and tBuONO (80.7 µL, 579 µmol) and TMSN3 (80.7 µL, 579 µmol) were added at 0 °C. After 2 h, further tBuONO (76.5 µL, 579 µmol) and TMSN3 (80.7 µL, 579 µmol) were added at rt. After 3 h, the reaction mixture was diluted with EtOAc and extracted with H2O three times. The organic phase was dried (Na2SO4) and concentrated in vacuo. Flash chromatography (DCM/MeOH 97:3) afforded 11c (95.4 mg, 96.1 µmol, 83%) as a pale yellow solid; mp = 135–135 °C (decomp.); [α]20 D = −76 (c = 0.1, MeOH); Rf = 0.21 (DCM/MeOH 95:5). 1H NMR (500 MHz, DMSO-d6): δ = 0.74 (d, J = 6.5 Hz, 3 H), 0.82 (d, J = 6.9 Hz, 3 H), 0.84 (t, J = 7.6 Hz, 3 H), 0.86 (d, J = 6.7 Hz, 3 H), 0.88 (d, J = 6.7 Hz, 3 H), 0.98 (d, J = 6.7 Hz, 3 H), 1.06–1.16 (m, 3 H), 1.26 (m, 1 H), 1.32– 1.41 (m, 3 H), 1.48 (s, 3 H), 1.53–1.60 (m, 3 H), 1.70 (m, 1 H), 1.82 (m, 1 H), 1.95 (m, 1 H), 2.61 (dd, J = 15.0, 10.3 Hz, 1 H), 2.80–2.89 (m, 2 H), 3.02 (dd, J = 15.0, 4.8 Hz, 1 H), 3.37 (m, 1 H), 3.55 (m, 1 H), 3.57 (s, 3 H), 3.85–3.89 (m, 2 H), 4.09 (dd, J = 8.7, 5.8 Hz, 1 H), 4.49 (ddd, J = 12.4, 9.1, 3.5 Hz, 1 H), 4.55 (m, 1 H), 4.72 (dd, J = 10.0, 5.4 Hz, 1 H), 5.04 (s, 2 H), 5.13 (s, 1 H), 5.29 (s, 1 H), 6.42 (d, J = 6.7 Hz, 1 H), 6.44 (d, J = 8.8 Hz, 1 H), 6.66 (d, J = 2.8 Hz, 1 H), 6.82 (d, J = 7.4 Hz, 1 H), 7.00 (dd, J = 8.8, 2.9 Hz, 1 H), 7.04 (m, 2 H), 7.10 (m, 1 H), 7.15–7.18 (m, 3 H), 7.32 (m, 1 H), 7.36–7.43 (m, 5 H), 8.78 (d, J = 5.2 Hz, 1 H), 8.85 (d, J = 8.7 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.3, 15.6, 18.8, 18.9, 20.2, 20.5, 23.5, 23.8, 24.7, 27.8, 28.5, 29.9, 31.6, 34.0, 36.9, 37.2, 38.2, 38.5, 48.1, 51.5, 54.5, 54.6, 56.8, 57.9, 59.9, 69.4, 115.4, 115.7, 119.9, 120.3, 126.0, 127.5, 127.9, 128.2, 128.4, 128.7, 128.8, 134.8, 136.7, 138.4, 143.1, 155.4, 157.2, 169.3, 170.7, 171.9, 172.0, 172.4, 173.0. HRMS (ESI): cald for C53H73N10O9+ [M+H]+: 993.5557, found: 993.5564. Methyl (((3S,6S,9S,12R,15S)-3-benzyl-6-((5-(benzyloxy)-1H-indol-3-yl)methyl)-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (12a) ACS Paragon Plus Environment


A solution of 11a (47.5 mg, 47.8 µmol) in MeCN (5 mL) was irradiated with a UV-LED (365 nm) for 15 h. Subsequently, the solvent was evaporated in vacuo. Purification of the residue by automated flash chromatography (twice, DCM/MeOH 100:0–96:4) afforded 12a (16.5 mg, 17.1 µmol, 36%) as a yellow solid; mp = 146–148 °C; [α]20 D = −110 (c = 0.1, MeOH); Rf = 0.05 (DCM/MeOH 95:5). 1H NMR (400 MHz, DMSO-d6): δ = −0.41 (m, 1 H), 0.07 (d, J = 6.5 Hz, 3 H), 0.32 (d, J = 6.5 Hz, 3 H), 0.63 (d, J = 6.9 Hz, 3 H), 0.72 (d, J = 6.8 Hz, 3 H), 0.70–0.88 (m, 6 H), 1.05–1.17 (m, 3 H), 1.25–1.33 (m, 3 H), 1.51 (m, 2 H), 1.62 (m, 1 H), 1.68–1.83 (m, 2 H), 2.16 (m, 1 H), 2.41 (s, 3 H), 2.76 (dd, J = 14.7, 10.9 Hz, 1 H), 2.89 (dd, J = 13.8, 9.5 Hz, 1 H), 2.97 (m, 1 H), 3.11–3.21 (m, 1 H), 3.28 (dd, J = 15.0, 2.9 Hz, 1 H), 3.62 (s, 3 H), 4.11 (m, 1 H), 4.21 (dd, J = 8.4, 4.1 Hz, 1 H), 4.26 (dd, J = 9.1, 4.1 Hz, 1 H), 4.31 (m, 1 H), 4.60 (ddd, J = 8.7, 8.7, 5.4 Hz, 1 H), 4.68 (dd, J = 10.8, 3.3 Hz, 1 H), 5.05 (s, 2 H), 6.39 (d, J = 9.0 Hz, 1 H), 6.48 (d, J = 7.5 Hz, 1 H), 6.79 (dd, J = 8.8, 2.3 Hz, 1 H), 6.99 (d, J = 2.3 Hz, 1 H), 7.10 (d, J = 2.2 Hz, 1 H), 7.17 (m, 2 H), 7.20–7.24 (m, 2 H), 7.25–7.32 (m, 3 H), 7.38 (m, 2 H), 7.45 (m, 2 H), 7.60 (m, 1 H), 7.82 (d, J = 8.1 Hz, 1 H), 8.35 (d, J = 8.2 Hz, 1 H), 10.76 (d, J = 2.0 Hz, 1 H). 13C{1H} NMR (101 MHz, DMSO-d6): δ = 11.5, 14.7, 16.4, 18.9, 19.2, 21.5, 22.7, 23.0, 23.5, 25.8, 27.3, 29.1, 30.7, 31.0, 36.8, 37.8, 38.3, 38.8, 45.6, 51.7, 52.9, 54.3, 55.0, 56.5, 61.2, 70.0, 101.5, 109.2, 111.8, 112.2, 124.8, 126.3, 127.3, 127.5, 127.6, 128.2, 128.3, 129.1, 131.4, 137.5, 137.8, 152.5, 157.3, 168.9, 170.3, 170.3, 172.3, 172.4, 173.4. HRMS (ESI): calcd for C53H73N8O9+ [M+H]+: 965.5495, found: 965.5500. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-6-((5-(benzyloxy)-1H-indol-3-yl)methyl)-9-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (12b) A solution of 11b (53 mg, 53.4 µmol) in MeCN (5.5 mL) was irradiated with a UV-LED (365 nm) for 12 h. Subsequently, the solvent was evaporated in vacuo. Purification of the residue by flash chromatography (DCM/MeOH 97:3, 96:4) afforded 12b (29.3 mg, 30.4 µmol, 57%) as a pale yellow solid; mp = 159–162 °C; [α]20 D = −146 (c = 0.1, MeOH); Rf = 0.14 (DCM/MeOH 95:5). 1H NMR (500 MHz, DMSO-d6): δ = −0.51 (m, 1 H), 0.26 (d, J = 6.5 Hz, 3 H), 0.33 (d, J = 6.6 Hz, 3 H), 0.78 (d, J = 6.9 Hz, 3 H), 0.84 (t, J = 7.4 Hz, 3 H), 0.87 (d, J = 6.7 Hz, 3 H), 0.90 (m, 1 H), 0.94 (d, J = 6.6 Hz, 3 H), 1.07–1.16 (m, 2 H), 1.22–1.34 (m, 2 H), 1.35–1.47 (m, 3 H), 1.56 (m, 2 H), 1.78 (m, 1 H), 1.92 (m, 1 H), 1.92 (s, 3 H), 2.74–2.83 (m, 2 H), 2.88 (m, 1 H), 3.14 (dd, J = 14.9, 2.4 Hz, 1 H), 3.44 (dd, J = 13.9, 2.8 Hz, 1 H), 3.58 (s, 3 H), 3.61 (m, 1 H), 3.81 (dd, J = 7.9, 7.9 Hz, 1 H), 3.85 (m, 1 H), 4.24 (dd, J = 9.1, 4.4 Hz, 1 H), 4.27 (m, 1 H), 4.61 (ddd, J = 12.4, 9.4, 3.2 Hz, 1 H), 4.75 (dd, J = 11.2, 3.1 Hz, 1 H), 5.01 (d, J = 11.8 Hz, 1 H), 5.05 (d, J = 11.8 Hz, 1 H), 6.39 (d, J = 9.2 Hz, 1 H), 6.45 (d, J = 6.5 Hz, 1 H), 6.72 (d, J = 7.1 Hz, 1 H), 6.78 (dd, J = 8.7, 2.2 Hz, 1 H), 6.93 (d, J = 2.1 Hz, 1 H), 7.08 (d, J = 7.3 Hz, 2 H), 7.19 (m, 2 H), 7.20–7.25 (m, 3 H), 7.33 (m, 1 H), 7.40 (m, 2 H), 7.40–7.45 (m, 3 H), 8.49 (d, J = 5.0 Hz, 1 H), 8.86 (d, J = 9.0 Hz, 1 H), 10.73 (s, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.5, 14.8, 18.7, 19.0, 19.3, 20.2, 22.5, 22.6, 23.0, 25.8, 27.6, 28.4, 29.9, 31.5, 36.6, 37.0, 37.7, 38.3, 47.2, 51.7, 54.3, 54.5, 55.3, 57.7, 60.7, 70.2, 102.3, 109.3, 111.4, 111.9, 124.7, 126.1, 127.6, 127.7, 127.7, 128.3, 128.3, 128.9, 131.3, 137.7, 138.4, 152.3, 157.4, 169.9, 170.8, 171.9, 172.1, 172.4, 173.4. HRMS (ESI): calcd for C53H73N8O9+ [M+H]+: 965.5495, found: 965.5518. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-6-((5-(benzyloxy)-1H-indol-3-yl)methyl)-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-isoleucinate (12c) A solution of 11c (90.7 mg, 91.3 µmol) in MeCN (9 mL) was irradiated with a UV-LED (365 nm, 50% intensity) for 13 h. Subsequently, the solvent was evaporated in vacuo. Purification of the residue by flash chromatography (DCM/MeOH 97:3, 96:4) afforded 12c ACS Paragon Plus Environment

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(51.0 mg, 52.8 µmol, 58%) as a pale yellow solid; [α]20 D = −133 (c = 0.1, MeOH); Rf = 0.09 1 (DCM/MeOH 95:5). H NMR (500 MHz, DMSO-d6): δ = −0.51 (m, 1 H), 0.26 (d, J = 6.5 Hz, 3 H), 0.34 (d, J = 6.6 Hz, 3 H), 0.82 (d, J = 6.8 Hz, 3 H), 0.84 (t, J = 7.4 Hz, 3 H), 0.86 (d, J = 6.6 Hz, 3 H), 0.88 (m, 1 H), 0.94 (d, J = 6.7 Hz, 3 H), 1.07–1.14 (m, 2 H), 1.28–1.46 (m, 5 H), 1.56 (m, 2 H), 1.70 (m, 1 H), 1.92 (s, 3 H), 1.94 (m, 1 H), 2.74–2.82 (m, 2 H), 2.87 (m, 1 H), 3.15 (dd, J = 14.9, 2.5 Hz, 1 H), 3.43 (dd, J = 14.0, 2.9 Hz, 1 H), 3.57 (s, 3 H), 3.60 (m, 1 H), 3.80 (dd, J = 7.9, 7.9 Hz, 1 H), 3.85 (m, 1 H), 4.08 (dd, J = 8.7 Hz, 5.8 Hz, 1 H), 4.26 (m, 1 H), 4.61 (ddd, J = 12.4, 9.2, 3.3 Hz, 1 H), 4.75 (dd, J = 11.2 Hz, 3.3 Hz, 1 H), 5.01 (d, J = 11.8 Hz, 1 H), 5.05 (d, J = 11.8 Hz, 1 H), 6.41 (d, J = 6.7 Hz, 1 H), 6.45 (d, J = 8.8 Hz, 1 H), 6.76 (d, J = 7.2 Hz, 1 H), 6.79 (dd, J = 8.7, 2.3 Hz, 1 H), 6.93 (d, J = 2.2 Hz, 1 H), 7.08 (d, J = 7.2 Hz, 2 H), 7.19 (m, 2 H), 7.21–7.25 (m, 3 H), 7.32 (m, 1 H), 7.38 (m, 2 H), 7.40 (m, 1 H), 7.44 (m, 2 H), 8.48 (d, J = 5.1 Hz, 1 H), 8.87 (d, J = 9.0 Hz, 1 H), 10.73 (d, J = 1.9 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.3, 15.6, 18.7, 19.0, 19.3, 20.2, 22.5, 22.6, 23.0, 24.7, 27.6, 28.4, 29.9, 31.5, 36.9, 37.0, 37.7, 38.3, 47.2, 51.5, 54.4, 54.5, 56.8, 57.7, 60.7, 70.2, 102.3, 109.3, 111.4, 111.9, 124.7, 126.1, 127.5, 127.6, 127.7, 128.3, 128.3, 128.9, 131.3, 137.7, 138.4, 152.3, 157.2, 169.9, 170.8, 171.9, 172.1, 172.4, 173.0. HRMS (ESI): calcd for C53H73N8O9+ [M+H]+: 965.5495, found: 965.5508. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-6-((5-hydroxy-1H-indol-3-yl)methyl)-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (13b) To a solution of 12b (27 mg, 30.9 µmol) in MeOH (5 mL) Pd/C (5.4 mg) and Pd(OH)2/C (5.4 mg) were added. The reaction mixture was stirred under H2 (3.1 bar) for 17 h. It was filtered over Celite and the filtrate was concentrated in vacuo. Flash chromategraphy (DCM/MeOH 95:5, 94:6) afforded 13b (19.8 mg, 22.6 µmol, 73%) as a colorless solid; mp 1 = 191–193 °C; [α]20 D = −172 (c = 0.1, MeOH); Rf = 0.24 (DCM/MeOH 92:8). H NMR (500 MHz, DMSO-d6): δ = −0.49 (m, 1 H), 0.24 (d, J = 6.6 Hz, 3 H), 0.37 (d, J = 6.7 Hz, 3 H), 0.77 (d, J = 6.9 Hz, 3 H), 0.84 (t, J = 7.4 Hz, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 0.88 (m, 1 H), 0.94 (d, J = 6.8 Hz, 3 H), 1.09–1.16 (m, 2 H), 1.23–1.32 (m, 2 H), 1.37–1.44 (m, 3 H), 1.57 (m, 2 H), 1.77 (m, 1 H), 1.90 (s, 3 H), 1.93 (m, 1 H), 2.70–2.78 (m, 2 H), 2.87 (m, 1 H), 3.08 (dd, J = 14.9, 2.9 Hz, 1 H), 3.43 (dd, J = 14.0, 3.0 Hz, 1 H), 3.58 (s, 3 H), 3.61 (m, 1 H), 3.82 (t, J = 7.7 Hz, 1 H), 3.85 (m, 1 H), 4.22 (m, 1 H), 4.24 (dd, J = 9.2, 4.4 Hz, 1 H), 4.59 (ddd, J = 12.4, 9.0, 3.4 Hz, 1 H), 4.69 (dd, J = 11.3, 3.5 Hz, 1 H), 6.40 (d, J = 9.2 Hz, 1 H), 6.47 (d, J = 6.6 Hz, 1 H), 6.58 (dd, J = 8.6, 2.3 Hz, 1 H), 6.72 (d, J = 7.2 Hz, 1 H), 6.81 (d, J = 2.4 Hz, 1 H), 6.84 (d, J = 2.3 Hz, 1 H), 7.09 (m, 2 H), 7.09 (d, J = 8.6 Hz, 1 H), 7.17 (m, 1 H), 7.23 (m, 2 H), 7.47 (dd, J = 8.2, 2.6 Hz, 1 H), 8.43 (d, J = 5.1 Hz, 1 H), 8.53 (s, 1 H), 8.81 (d, J = 8.9 Hz, 1 H), 10.55 (d, J = 2.2 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.5, 14.8, 18.6, 18.9, 19.6, 20.2, 22.3, 22.6, 23.1, 25.8, 27.6, 28.4, 30.0, 31.5, 36.6, 37.1, 37.8, 38.3, 47.3, 51.7, 54.4, 54.5, 55.3, 57.6, 60.8, 102.2, 108.6, 111.3, 111.6, 124.2, 126.1, 127.9, 128.3, 128.9, 130.5, 138.4, 150.4, 157.4, 169.9, 170.8, 171.9, 172.2, 172.3, 173.4. HRMS (ESI): calcd for C46H67N8O9+ [M+H]+: 875.5026, found: 875.5043. Methyl (((3S,6S,9S,12S,15R)-3-benzyl-6-((5-hydroxy-1H-indol-3-yl)methyl)-9isobutyl-12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucinate (13c) To a solution of 12c (50.4 mg, 52.2 µmol) in MeOH (5 mL) Pd/C (10 mg) and Pd(OH)2/C (10 mg) were added. The reaction mixture was stirred under H2 (3.1 bar) for 15.5 h. It was filtered over Celite and the filtrate was concentrated in vacuo. Flash chromategraphy (DCM/MeOH 95:5, 94:6) afforded 13c (26.7 mg, 30.5 µmol, 58%) as a colorless solid; mp 1 = 186–188 °C; [α]20 D = −125 (c = 0.1, MeOH); Rf = 0.21 (DCM/MeOH 92:8). H NMR (500 ACS Paragon Plus Environment


MHz, DMSO-d6): δ = −0.49 (m, 1 H), 0.24 (d, J = 6.5 Hz, 3 H), 0.37 (d, J = 6.6 Hz, 3 H), 0.81 (d, J = 6.8 Hz, 3 H), 0.83 (t, J = 7.5 Hz, 3 H), 0.86 (d, J = 6.6 Hz, 3 H), 0.94 (d, J = 6.7 Hz, 3 H), 1.06–1.14 (m, 2 H), 1.27–1.36 (m, 2 H), 1.37–1.44 (m, 3 H), 1.56 (m, 2 H), 1.69 (m, 1 H), 1.90 (s, 3 H), 1.94 (m, 1 H), 2.73 (dd, J = 15.2 Hz, 11.2 Hz, 1 H), 2.76 (m, 1 H), 2.87 (m, 1 H), 3.08 (dd, J = 14.9, 2.3 Hz, 1 H), 3.43 (m, 1 H), 3.57 (s, 3 H), 3.60 (m, 1 H), 3.82 (t, J = 7.8 Hz, 1 H), 3.85 (m, 1 H), 4.08 (dd, J = 8.7, 5.9 Hz, 1 H), 4.22 (m, 1 H), 4.58 (ddd, J = 12.2, 9.0, 3.3 Hz, 1 H), 4.69 (dd, J = 11.3, 3.3 Hz, 1 H), 6.43 (d, J = 6.6 Hz, 1 H), 6.46 (d, J = 8.8 Hz, 1 H), 6.58 (dd, J = 8.6, 2.2 Hz, 1 H), 6.75 (d, J = 7.1 Hz, 1 H), 6.81 (d, J = 2.2 Hz, 1 H), 6.83 (d, J = 2.0 Hz, 1 H), 7.08–7.11 (m, 3 H), 7.18 (m, 1 H), 7.23 (m, 2 H), 7.47 (dd, J = 8.2, 2.3 Hz, 1 H), 8.43 (d, J = 4.9 Hz, 1 H), 8.52 (s, 1 H), 8.81 (d, J = 8.9 Hz, 1 H), 10.55 (d, J = 1.9 Hz, 1 H). 13C{1H} NMR (126 MHz, DMSO-d ): δ = 11.3, 15.6, 18.6, 18.9, 19.6, 20.2, 22.3, 22.6, 23.1, 6 24.7, 27.6, 28.3, 30.0, 31.5, 36.9, 37.1, 37.8, 38.3, 47.3, 51.5, 54.5, 54.5, 56.8, 57.6, 60.8, 102.2, 108.6, 111.3, 111.6, 124.2, 126.1, 127.9, 128.3, 128.9, 130.4, 138.4, 150.4, 157.2, 169.9, 170.8, 171.9, 172.2, 172.3, 173.0. HRMS (ESI): calcd for C46H67N8O9 [M+H]+: 875.5026, found: 875.5056. (((3S,6S,9S,12R,15S)-3-Benzyl-6-((5-hydroxy-1H-indol-3-yl)methyl)-9-isobutyl12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucine (1a) To a solution of 12a (15.2 mg, 15.7 µmol) in MeOH (2 mL) were added Pd/C (7.5 mg) and Pd(OH)2/C (4.5 mg) and the mixture was stirred under an atmosphere of H2 (1 bar) for 52 h. It was then filtered over Celite and concentrated in vacuo. The residue was dissolved in 1,2-dichloroethane (1.5 mL), Me3SnOH (28.4 mg, 0.157 mmol) was added and the mixture was heated to 80 °C for 62 h. The mixture was then diluted with EtOAc, washed with 1 M HCl and H2O. The organic phase was dried (Na2SO4) and concentrated in vacuo. Automated RP flash chromatography (H2O/MeCN 90:10–0:100) afforded 1a (6.0 mg, 7.0 1 µmol, 44%) as a colorless solid; [α]20 D = −35 (c = 0.024, MeOH). H NMR (500 MHz, DMSOd6): δ = −0.47 (m, 1 H), 0.09 (d, J = 6.3 Hz, 3 H), 0.31 (d, J = 6.4 Hz, 3 H,), 0.61 (d, J = 6.7 Hz, 3 H), 0.72 (d, J = 6.6 Hz, 3 H), 0.75 (d, J = 6.5 Hz, 3 H), 0.81 (t, J = 7.3 Hz, 3 H), 1.01 (m, 1 H), 1.09 (m, 1 H), 1.15 (m, 1 H), 1.26–1.37 (m, 4 H), 1.48 (m, 1 H), 1.65 (m, 2 H), 1.78 (m, 1 H), 2.15 (m, 1 H), 2.37 (s, 3 H), 2.65 (m, 1 H), 2.86 (dd, J = 13.6, 10.4 Hz, 1 H), 2.91 (m, 1 H), 3.16 (m, 1 H), 3.21–3.27 (m, 2 H), 4.03 (m, 1 H), 4.11 (m, 1 H), 4.24 (dd, J = 8.8, 3.3 Hz, 1 H), 4.35 (m, 1 H), 4.52 (m, 1 H), 4.60 (m, 1 H), 6.20 (bs, 1 H), 6.57 (dd, J = 8.6, 1.9 Hz, 1 H), 6.61 (m, 1 H), 6.79 (d, J = 1.4 Hz, 1 H), 6.91 (d, J = 1.6 Hz, 1 H), 7.03 (bs, 1 H), 7.10 (d, J = 8.6 Hz, 1 H), 7.16 (m, 2 H), 7.20 (m, 1 H), 7.28 (m, 2 H), 7.44 (m, 1 H), 7.84 (d, J = 8.1 Hz, 1 H), 8.31 (m, 1 H), 8.59 (bs, 1 H), 10.58 (s, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.8, 14.7, 16.0, 19.0, 19.0, 21.4, 22.7, 23.0, 23.6, 25.9, 27.7, 29.0, 30.8, 30.8, 37.1, 37.6, 38.6, 39.1, 45.5, 53.3, 54.4, 55.3, 56.1, 61.4, 101.6, 108.5, 111.5, 111.9, 124.2, 126.3, 127.6, 128.2, 129.1, 130.6, 137.7, 150.7, 157.4, 169.0, 170.3, 170.4, 172.4, 172.6, 174.8. HRMS (ESI): calcd for C45H65N8O9+ [M+H]+: 861.4869, found: 861.4893. (((3S,6S,9S,12S,15R)-3-Benzyl-6-((5-hydroxy-1H-indol-3-yl)methyl)-9-isobutyl12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-allo-isoleucine (1b) 13b (19.2 mg, 21.9 µmol) was dissolved in 1,2-dichloroethane (2 mL), Me3SnOH (39.7 mg, 0.219 mmol) was added and the mixture was heated to 80 °C. After 57 h, further Me3SnOH (19.8 mg, 0.110 mmol) was added. After 82 h, the mixture was diluted with EtOAc, washed with 1 M HCl and H2O. The organic phase was dried (Na2SO4) and concentrated in vacuo. Automated RP flash chromatography (H2O/MeCN 100:0–20:80) afforded 1b (13.5 mg, 1 15.7 µmol, 72%) as an off-white solid; [α]20 D = −87 (c = 0.024, MeOH). H NMR (500 MHz, ACS Paragon Plus Environment

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DMSO-d6): δ = −0.49 (m, 1 H), 0.24 (d, J = 6.5 Hz, 3 H), 0.37 (d, J = 6.6 Hz, 3 H), 0.76 (d, J = 6.9 Hz, 3 H), 0.84 (t, J = 7.4 Hz, 3 H), 0.87 (d, J = 6.6 Hz, 3 H), 0.89 (m, 1 H), 0.94 (d, J = 6.7 Hz, 3 H), 1.08–1.14 (m, 2 H), 1.23–1.33 (m, 2 H), 1.34–1.45 (m, 2 H), 1.56 (m, 2 H), 1.80 (m, 1 H), 1.89 (s, 3 H), 1.91 (m, 1 H), 2.70–2.77 (m, 2 H), 2.87 (m, 1 H), 3.07 (dd, J = 14.8, 2.5 Hz, 1 H), 3.42 (m, 1 H), 3.60 (m, 1 H), 3.78 (t, J = 7.7 Hz, 1 H), 3.86 (m, 1 H), 4.15 (dd, J = 9.1, 3.7 Hz, 1 H), 4.22 (m, 1 H), 4.58 (ddd, J = 12.4, 9.2, 3.4 Hz, 1 H), 4.69 (dd, J = 11.2, 3.2 Hz, 1 H), 6.24 (d, J = 9.1 Hz, 1 H), 6.51 (d, J = 6.5 Hz, 1 H), 6.58 (dd, J = 8.6, 2.2 Hz, 1 H), 6.74 (d, J = 6.3 Hz, 1 H), 6.81 (d, J = 2.2 Hz, 1 H), 6.84 (d, J = 2.0 Hz, 1 H), 7.06–7.10 (m, 3 H), 7.17 (m, 1 H), 7.23 (m, 2 H), 7.46 (m, 1 H), 8.43 (d, J = 5.0 Hz, 1 H), 8.52 (bs, 1 H), 8.81 (d, J = 9.0 Hz, 1 H), 10.55 (d, J = 1.7 Hz, 1 H), 12.49 (bs, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.7, 14.8, 18.9, 19.0, 19.5, 20.2, 22.3, 22.6, 23.1, 26.0, 27.6, 28.4, 30.0, 31.6, 36.7, 37.1, 37.7, 38.4, 47.2, 54.4, 54.5, 55.2, 57.8, 60.7, 102.2, 108.6, 111.3, 111.6, 124.2, 126.1, 127.9, 128.3, 128.9, 130.4, 138.4, 150.4, 157.6, 169.9, 170.8, 172.1, 172.2, 172.3, 174.5. HRMS (ESI): calcd for C45H65N8O9+ [M+H]+: 861.4869, found: 861.4894. (((3S,6S,9S,12S,15R)-3-Benzyl-6-((5-hydroxy-1H-indol-3-yl)methyl)-9-isobutyl12-isopropyl-7-methyl-2,5,8,11,14-pentaoxo-1,4,7,10,13pentaazacyclononadecan-15-yl)carbamoyl)-L-isoleucine (1c) 13c (25.1 mg, 28.7 µmol) was dissolved in 1,2-dichloroethane (5 mL), Me3SnOH (51.9 mg, 0.287 mmol) was added and the mixture was heated to 80 °C. After 39 h and 82 h, further Me3SnOH (25.9 mg, 0.144 mmol) was added. After 5 d, the mixture was diluted with EtOAc, washed with 1 M HCl and H2O. The organic phase was dried (Na2SO4) and concentrated in vacuo.. Automated RP flash chromatography (H2O/MeCN 100:0–20:80) followed by preparative HPLC afforded 1c (2.3 mg, 2.7 µmol, 9%) as a colorless solid; [α]20 D 1 = −107 (c = 0.024, MeOH). H NMR (500 MHz, DMSO-d6): δ = −0.49 (m, 1 H), 0.24 (d, J = 6.5 Hz, 3 H), 0.37 (d, J = 6.6 Hz, 3 H), 0.82–0.85 (m, 6 H), 0.86 (d, J = 6.6 Hz, 3 H), 0.88 (m, 1 H), 0.94 (d, J = 6.7 Hz, 3 H), 1.07–1.12 (m, 2 H), 1.29–1.35 (m, 2 H), 1.37–1.45 (m, 2 H), 1.56 (m, 2 H), 1.72 (m, 1 H), 1.89 (s, 3 H), 1.92 (m, 1 H), 2.70–2.78 (m, 2 H), 2.87 (m, 1 H), 3.08 (m, 1 H), 3.42 (m, 1 H), 3.60 (m, 1 H), 3.79 (t, J = 7.7 Hz, 1 H), 3.86 (m, 1 H), 4.04 (dd, J = 8.5, 4.9 Hz, 1 H), 4.22 (m, 1 H), 4.58 (m, 1 H), 4.69 (dd, J = 11.1, 3.3 Hz, 1 H), 6.31 (bs, 1 H), 6.45 (d, J = 6.3 Hz, 1 H), 6.58 (dd, J = 8.6, 2.2 Hz, 1 H), 6.74 (m, 1 H), 6.81 (d, J = 2.3 Hz, 1 H), 6.84 (d, J = 2.1 Hz, 1 H), 7.08 (m, 2 H), 7.09 (d, J = 8.5 Hz, 1 H), 7.17 (m, 1 H), 7.23 (m, 2 H), 7.46 (m, 1 H), 8.43 (d, J = 5.0 Hz, 1 H), 8.52 (s, 1 H), 8.81 (d, J = 8.9 Hz, 1 H), 10.55 (d, J = 1.9 Hz, 1 H), 12.49 (bs, 1 H). 13C{1H} NMR (126 MHz, DMSO-d6): δ = 11.5, 15.8, 18.9, 19.0, 19.5, 20.2, 22.3, 22.6, 23.1, 24.6, 27.6, 28.4, 30.0, 31.6, 36.9, 37.1, 37.7, 38.4, 47.3, 54.5, 54.5, 57.1, 57.8, 60.8, 102.2, 108.6, 111.3, 111.6, 124.2, 126.1, 127.9, 128.3, 128.9, 130.5, 138.4, 150.4, 157.3, 169.9, 170.8, 172.0, 172.2, 172.3, 173.9. HRMS (ESI): calcd for C45H65N8O9+ [M+H]+: 861.4869, found: 861.4861. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Comparisons of NMR data, NMR spectra (pdf) AUTHOR INFORMATION Corresponding Author ACS Paragon Plus Environment


*Email: [email protected]

Notes The authors declare no competing financial interest. Acknowledgements This work was supported by Saarland University. We also thank Fabian Panter for recording high resolution mass spectra and performing preparative HPLC separation.

References 1 Li, C. W.; Chen, J. Y.; Hua, T. E. Precambrian sponges with cellular structures. Science 1998, 279, 879–882. 2 Mehbub, M. F.; Lei, J.; Franco, C.; Zhang, W. Marine sponge derived natural products between 2001 and 2010: trends and opportunities for discovery of bioactives. Mar. Drugs 2014, 12, 4539– 4577. 3 Lee, Y.; Lee, J.; Lee, H. K. Microbial symbiosis in marine sponges. J. Microbiol. 2001, 39, 254– 264. 4 Piel, J. Metabolites from symbiotic bacteria. Nat. Prod. Rep. 2009, 26, 338–362. 5 Martins, A.; Vieira, H.; Gaspar, H.; Santos, S. Marketed marine natural products in the pharmaceutical and cosmeceutical industries: tips for success. Mar. Drugs 2014, 12, 1066–1101. 6 a) Nicolaou, K. C. Snyder, S. A. Chasing molecules that were never there: misassigned natural products and the role of chemical synthesis in modern structure elucidation. Angew. Chem. Int. Ed. 2005, 44, 1012–1044; b) Maier, M. E. Structural revisions of natural products by total synthesis. Nat. Prod. Rep. 2009, 26, 1105–1124. 7 Schmidt, E. W.; Harper, M. K.; Faulkner, D. Mozamides A and B, cyclic peptides from a Theonellid sponge from Mozambique. J. Nat. Prod. 1997, 60, 779–782. 8 Spoof, L.; Błaszczyk, A.; Meriluoto, J.; Cegłowska, M., Mazur-Marzec, H. Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria. Mar. Drugs 2016, 14, 1–14. 9 a) Kobayashi, J.; Sato, M.; Murayama, T.; Ishibashi, M.; Wälchi, M. R.; Kanai, M.; Shoji, J.; Ohizumi, Y. Konbamide, a novel peptide with calmoduiin antagonistic activity from the Okinawan marine sponge Theonella sp. J. Chem. Soc., Chem. Commun. 1991, 1050–1052; b) Ishibashi, M.; Li, Y.; Sato, M.; Kobayashi, J. Stereochemistry of the modified tryptophan residues contained in konbamide and keramamide a isolated from marine sponges of the genus Theonella. Nat. Prod. Lett. 1994, 4, 293–296; c) Schmidt, U.; Weinbrenner, S. What is the Structure of the Calmodulin Antagonist Konbamide fromTheonella sp.? Synthesis of Two Isomers by Direct Biomimetic Introduction of Bromine in Hydroxytryptophan-Containing Cyclic Peptides. Angew. Chem. Int. Ed. 1996, 35, 1336– 1338. 10 a) Itou, Y., Suzuki, S.; Ishida, K.; Murakami, M. Anabaenopeptins G and H, potent carboxypeptidase A inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-595). Bioorg. Med. Chem. Lett. 1999, 9, 1243–1246; b) Murakami, M.; Suzuki, S.; Itou, Y.; Kodani, S.; Ishida, K. New anabaenopeptins, potent carboxypeptidase-A inhibitors from the cyanobacterium Aphanizomenon flos-aquae..J. Nat. Prod. 2000, 63, 1280–1282. c) Walther, T.; Renner, S.; Waldmann, H.; Arndt, H.-D. Synthesis and structure-activity correlation of a brunsvicamideinspired cyclopeptide collection. ChemBioChem 2009, 10, 1153–1162; d) Harms, H.; Kurita, K. L.; Pan, L.; Wahome, P. G.; He, H.; Kinghorn, A. D.; Carter, G. T.; Linington, R. G. Discovery of anabaenopeptin 679 from freshwater algal bloom material: Insights into the structure-activity ACS Paragon Plus Environment

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Total Synthesis and Configurational Revision of Mozamide A, a

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