Direct Peptide Cyclization and One-Pot Modification Using the MeDbz Linker

Article abstract of DOI:10.1021/acs.joc.8b01237

The one-pot synthesis and modification of cyclic peptides through a self-cleaving on-resin protocol is described. We apply Dawson's MeDbz linker to achieve direct intramolecular peptide cyclization by thioesterification followed by S → N acyl shift. This native chemical ligation approach requires no activating additive and allows direct modification of the crude cyclic peptides in one-pot. The strategy was applied to synthesize 5 cyclic peptide natural products of varying ring size. Finally, one-pot modifications include desulfurization, fluorophore conjugation, and intramolecular disulfide formation.

Full text of DOI:10.1021/acs.joc.8b01237

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Note
Direct Peptide Cyclization and One-Pot Modification Using the MeDbz Linker
Bengt Herbert Gless, and Christian Adam Olsen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01237 • Publication Date (Web): 06 Aug 2018
Downloaded from http://pubs.acs.org on August 7, 2018
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Direct Peptide Cyclization and One-Pot Modification Using the MeDbz
Linker
Bengt H. Gless and Christian A. Olsen*
Center for Biopharmaceuticals and Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Universitetsparken 2, DKꢀ2100, Copenhagen, Denmark.
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*Correspondence: cao@sund.ku.dk
Abstract
The oneꢀpot synthesis and modification of cyclic peptides through a selfꢀcleaving onꢀresin protocol is
described. We apply Dawson’s MeDbz linker to achieve direct intramolecular peptide cyclization by
thioesterification followed by S→N acyl shift. This native chemical ligation approach requires no
activating additive and allows direct modification of the crude cyclic peptides in oneꢀpot. The strategy
was applied to synthesize 5 cyclic peptide natural products of varying ring size. Finally, oneꢀpot
modifications include desulfurization, fluorophore conjugation, and intramolecular disulfide formation.
Cyclic peptides are receiving increasing interest in drug discovery to fill the lack of promising medium
1
sized therapeutics (500–5000 Da). Their constrained nature may result in predetermined conformations
that bind a target protein with high affinity, presumably in part due to lower entropic penalty upon
binding. For these reasons, macrocyclic peptides have found interest for development of ligands that
2
,3
target protein–protein interactions. Furthermore, improved stability with respect to proteolytic
degradation is often achieved by cyclization. Numerous ways have been developed to synthesize
macrocyclic peptides, including the commonly applied headꢀtoꢀtail cyclization of side chain protected
4
linear peptides under high dilution conditions. Alternatively, chemoselective approaches using
5
ꢀ7
unprotected peptides for macrocyclization have been developed. In particular, native chemical
8
9ꢀ16
ligation (NCL) and its recent advances
have enabled the development of diverse strategies based on
17ꢀ21
22
23ꢀ26
either Boc solidꢀphase peptide synthesis (SPPS),
O→S and N→S
shift thioester formation,
2
7,28
29ꢀ31
32
safetyꢀcatch linkers,
Cꢀterminal hydrazides,
Cꢀterminal oꢀaminoanilides,
Nꢀacylurea
3
3ꢀ35
36
surrogates
and Cꢀterminal βꢀthiolactones. Taking advantage of the pseudoꢀdilution effect on solid
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38,39
40ꢀ48
support, onꢀresin cyclizations
and cleavage inducing cyclization approaches
have been
3
developed for various chemistries. We and others have recently exploited Dawson’s N ꢀFmocꢀ3,4ꢀ
34
methylꢀdiaminobenzoic acid (FmocꢀMeDbz) linker using onꢀresin protocols to synthesize cyclic
4
9,50
51ꢀ53
thiodepsipeptides,
Cꢀterminally modified peptides
and homodetic cyclopeptides via attack of the
54
Nꢀterminal amine. Here, we apply this linker for the cleavageꢀinducing oneꢀpot cyclization and
modification of headꢀtoꢀtail cyclized homodetic peptides by NCL (Figure 1).
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In our previously reported protocol, we applied a MeDbzꢀGlyꢀresin, which allowed for the global
deprotection of the peptides on solid support after formation of the activated Nꢀacylꢀbenzimidazolinone
(Nbz) moiety and the triggering of a cleavageꢀinducing cyclization, upon the addition of pHꢀcontrolled
50
buffer to the resin. We envisioned that subjecting Nꢀterminal cysteineꢀcontaining peptides to these
conditions would lead to a chemoselective cyclization–cleavage event with subsequent S→N shift
yielding homodetic peptides 2 (Figure 1). For our initial attempts, we chose a model hexapeptide (3) to
test our hypothesis (Figure S1A and Table 1).
Figure 1 Envisioned strategy for the synthesis of cyclic peptides.
First, the cyclization–cleavage reaction was performed in 0.2 M phosphate buffer–MeCN (1:1) for 2 h
at 50 °C on MeDbzꢀChemMatrix resin 4. The resulting crude HPLC chromatogram showed several
products, which furnished two peaks upon reduction with TCEP. The major peak corresponded to
monomer 3 and the minor peak was isolated and identified as cyclic homodetic peptide dimer 5 by mass
1
spectrometry, H NMR spectroscopy, and chemical reactivity tests (Figure S1A–B and Table 1). Since
50
we did not encounter dimerization using the protocol for the synthesis of thiodepsipeptides, we
synthesized an Nꢀacetylated version of the hexamer to investigate the dimer formation further (Figure
S1C). Here, we only detected the expected thiodepsipeptide, which may suggest a mechanism for the
dimer formation involving site–site nucleophilic attack on the resin (Figure S2A) as also previously
39,43,45,48,55
reported for resinꢀbound peptides.
However, it could also be imagined that monomer 3
cleaves another peptide to form an adduct that may cause dimer formation through different
mechanisms (Figure S2B and C).
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We first envisioned that temporary protection of the Nꢀterminal amine could prevent dimer formation in
the cyclization–cleavage step and release a thiodepsipeptide. Removal of the protecting group in buffer
would then lead to the desired S→N shift to furnish the desired cyclic peptide. Such a protecting group
would need to be stable towards TFA, aqueous buffer at 50 °C, as well as nucleophiles. The
propargyloxycarbonyl group (Proc) fulfills these requirements and can be removed using palladium(II)
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complexes in aqueous media. Thus, we synthesized the Procꢀprotected hexapeptide and performed the
cyclization (Figure S3A). Subsequent LCꢀMS analysis of the crude material showed excellent purity
and confirmed the identity of the Procꢀprotected thiodepsipeptide. However, we were not able to
2
remove the protecting group using PdCl under the reported conditions. We therefore examined the
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photolabile 6ꢀnitroveratryloxycarbonyl (Nvoc) group, which exhibited stability in both TFA and
buffer. Cyclization gave a single peak with the correct m/z for the Nvocꢀprotected thiodepsipeptide, but
subsequent UV irradiation led to decomposition (Figure S3B). Instead, we decided to investigate
different resins with varying loading and swelling properties to limit dimer formation. We performed
test reactions applying MeDbz on PEGA, TentaGel, and TentaGel XV resins and all resulted in
improved monomer–dimer ratios compared to the ChemMatrix resin (Table 1). The highꢀswelling
TentaGel XV resin proved superior with an 11% reduction in the dimer formation (Table 1, entry 4).
Applying these conditions, we observed satisfying isolated yield of 3 (30%) with only 4% of 5 based on
the resin loading.
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Table 1: Dimer ratios for different resins
b
resin
resin loading
mmol/g)
ratio of 3–5
(
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c
ChemMatrix
0.50
0.34
0.22
0.21
73:27
80:20
78:22
84:16
d
PEGA
c
TentaGel
c
TentaGel XV
a
b
The resin loadings were determined by Fmoc deprotection and subsequent UV measurement. The ratios of 3 to 5 were
determined by analytical HPLC (230 nm). Cyclization reactions were performed in 0.2 M phosphate buffer–MeCN (1:1) for
h at 50 °C unless otherwise noted. This reaction was performed in 0.1 M phosphate bufferꢀDMF (7:3) to increase the
c
d
2
swelling of the resin
NCL protocols without the use of thiol additives have been used for oneꢀpot ligation–
3
6,58ꢀ60
61
desulfurization
and oneꢀpot ligation–iodine oxidation reactions. We therefore envisioned that
the absence of activating additives in our cyclization–cleavage protocol would allow direct
modifications of the cysteine residue, including oneꢀpot radical desulfurization to give cyclic alanineꢀ
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containing peptides. In addition, the use of penicillamine (Pen) instead of Cys would give rise to
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valineꢀcontaining macrocycles. We also expected that our protocol could be used for direct
intramolecular disulfide formation by iodine oxidation, subsequent to the cyclization–cleavage step,
providing cyclic disulfide containing peptides. Functionalizing cyclic peptides received recent
6
4,65
interest
and we therefore envisioned that the absence of nucleophilic additives would allow us to
2 reaction via the cysteine residue and furnish labeled cyclic peptides,
install functionalities by S
N
requiring just a single purification step. In order to probe the scope, we chose several cyclic peptide
natural compounds with varying ring size that contain alanine, valine, or cysteine (Figure 2). We
66
prepared the Cys (6) and Ala (7) mutants of the hexameric natural peptide destoamide B (8) to
compare the yields for the oneꢀpot desulfurization. The synthesis of destoamide B V1C (6) proceeded in
a good isolated yield (23%) and the direct desulfurization using VAꢀ044 and TCEP to provide
destoamide B V1A (7) proceeded within 2 h under ambient atmosphere to give a similar yield (18%).
When performing this oneꢀpot modification for the Penꢀcontaining peptide to give destoamide B (8)
itself, a considerable amount of hydrolyzed linear peptide was detected (m/z +18) together with a
peptide with the correct m/z (Figure S4A). Analysis of this product by NMR spectroscopy revealed a
linear peptide with a Cꢀterminal succinimide moeity (Figure S4B). The steric congestion of the
penicillamine side chain presumably slows down the S→N shift, leaving the thioester exposed for
hydrolysis or intramolecular nucleophilic attack of the Asn side chain. We then prepared several
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alanineꢀcontaining natural compounds including stellarin G (5ꢀmer) (9), stylostatin 1 (7ꢀmer) (10),
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cyclonellin (8ꢀmer) (11), and crotogossamide (9ꢀmer) (12) in yields between 13–31% based on the
resin loading (Figure 2).
1
) buffer/MeCN
50 °C, 2 h
SH
H
N
O
1
1
1
1
1
1
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1
2
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2) VA-044, TCEP,
red. glutathione,
H2N peptide
MeNbz
37 °C, 2-3 h
O
H
N
O
H
O
HO
HN
HN
N
O
HN
O
O
NH
HN
NH
O
NH2
N
O
O
H
HN
O
O
NH
Stellarin G (
yield = 31% (5-mer)
9)
N
H
R
2
O
R
1
Destoamide B V1C (
6
), R1 = SH, R2 = H
N
O
O
yield = 23% (6-mer
)
O
NH
NH
O
O
1 2
Destoamide B V1A (7), R , R = H
yield = 18% (6-mer
)
NH
O
NH
H
N
Destoamide B ( ), R1 R2 = Me
8
,
O
succinimdeformation
O
NH
NH2
NH
OH
OH
H2N
NH
Stylostatin 1 (10
yield = 16% (7-mer
reported yield: 7%
)
)
a
O
NH2
O
O
H
N
O
HN
NH
OO
HN
N
O
N
H
N
O
NH
H
N
NH
O
O
NH
O
NH
O
O
O
O
NH
NH
NH OH
O
HO
HN
OH
H
N
Cyclonellin (11
)
yield = 20% (8-mer
)
O
O
O
O
HO
NH2
H
NH
N
O
Crotogossamide (12
yield = 13% (9-mer
reported yield: 22–28%
)
)
HN
O
b
H2N
O
R1 HN
O
R1
O
NH
NH
HN
N
O
NH
2
O
O
H
N
O
O
2
H N
O
N
H
Tyrocidine A V1A (13); R1 = H
yield = 24% (10-mer
)
Tyrocidine A (13a); = Me
R
1
a
42 b
Figure 2 Scope of cyclization–oneꢀpot desulfurization. Yield as stated in the manuscript.
Yield as stated in the
62
manuscript.
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Finally, we attempted the syntheses of the antibiotic tyrocidine A (13a), a 10ꢀmer valineꢀcontaining
peptide, and its alanine mutant tyrocidine A V1A (13). The latter was obtained in good yield but when
employing Pen as the Nꢀterminal amino acid, we again encountered hydrolysis and were not able to
synthesize tyrocidine A. Thus, applying penicillamine to obtain valineꢀcontaining peptides appears to be
a limitation of the protocol based on the two examples attempted here. The cyclization of tetrameric
peptides is challenging due to conformational constraints and could not be achieved using our
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protocol.
Next, we tested our protocol for the synthesis of a peptide with an internal disulfide bond by
7
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synthesizing sunflower trypsin inhibitor 1 (SFTIꢀ1, 14) (Figure 3A). In contrast to the previously
applied conditions, TCEP was added to the cyclization reaction to prevent disulfide formation while the
peptide is still attached to the resin. The presence of an internal cysteine can lead to the formation of a
thiolactone intermediate in the cyclization–cleavage step, which has been shown to effectively promote
77
thiolꢀadditive free NCL and should therefore lead to the final peptide. Indeed, the cyclization reaction
gave a major peak, which corresponded to the reduced form of SFTIꢀ1. Addition of an iodine solution
quickly oxidized the peptide and gave bicyclic form of SFTIꢀ1 (14) in a yield of 11% after HPLC
purification.
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A. One-pot disulfide formation
SH
H
O
1
) buffer/MeCN,
TCEP, 50 °C, 2 h
N
S
H2N peptide
2
) iodine, rt, 10 min
MeNbz
SH
S
O
H
N
O
N
N
NH
H
O
HN
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
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3
3
4
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5
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5
5
5
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5
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0
1
2
3
4
5
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7
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9
0
1
2
3
4
5
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0
1
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0
1
2
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0
1
2
3
4
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8
9
0
O
N
H
NH
2
OH
O
NH
O
O
NH
S
S
O
HN
SFTI-1 (14
yield = 11% (14-mer
)
NH
O
)
HO
HN
HN
O
O
reported yields:
O
a
b
50% , 34%
N
O
c
d
7
% , 42%
N
NH
O
N
H
HO
O
NH
2
B. One-pot cysteine labeling
SH
1) buffer/MeCN
0 °C, 2 h
R
H
N
O
S
5
H2N peptide
2) TCEP, Br-
R
or I-R
rt, 0.5-1 h
MeNbz
H
H
N
N
S
O
S
O
O
“
Crotogossamide-alkyne” (15
)
HO C
2
O
yield = 13% (9-mer
)
H
N
S
OH
“Destoamide B-fluorescein” (17
yield = 12% (6-mer
O
N3
)
“Stylostatin 1-azide” (16
)
)
yield = 15% (7-mer
)
a
33 b
30
Figure 3 Oneꢀpot, postcyclization modification. Yield based on resin loading. Yield based on purified linear precursor.
Yield based on resin loading. Yield as stated in the manuscript.
c
23 d
47
Lastly, we prepared cyclic peptides containing handles for copperꢀcatalyzed azide–alkyne click
7
8,79
chemistry
or a fluorophore (Figure 3B). We chose haloacetamides as electrophiles due the ease of
preparation. First, we introduced an alkyne handle into crotogossamide (12) by addition of N-propargylꢀ
iodoacetamide directly to the reaction mixture after the cyclization. The reaction went to completion in
30 min, yielding the modified peptide 15 in a similar yield (13%) to natural product 12. Next, we
prepared an azideꢀmodified version of stylostatin 1 (10) using a Nꢀ(4ꢀazidophenyl)ꢀbromoacetamide.
The alkylation proceeded in 1 h and provided the modified peptide 16 in a yield of 15%. Encouraged by
these results, we subjected destoamide B V1C (6) to labeling with bromoacetamideꢀfunctionalized
fluorescein under the same conditions. The fluorescent peptide 17 was obtained within 1 h in only
slightly decreased yield (12%) compared to destoamide B V1A (7) (Figure 3B). The ease of synthesis
and the variety of commercial haloacetamides offer access to a vast number of possible modifications in
a single step that does not require additional purification steps.
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In conclusion, we have developed a procedure for the cleavage, cyclization, and modification of
homodetic cyclopeptides using the MeDbz linker in oneꢀpot fashion. The absence of activating
additives allowed us to perform direct desulfurization subsequent to the cyclization, enabling efficient
preparation of 4 natural cyclic peptides together with 3 analogues thereof. Further, we demonstrated
oneꢀpot intramolecular disulfide formation in the preparation of SFTIꢀ1 (14) as well as installation of
various handles for bioconjugation using haloacetamide reagents. The developed chemistry offers rapid
access to cyclic peptides of varying ring size and, importantly, enables a variety of postꢀcyclization
modifications without requiring additional purification steps.
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Experimental Section
General Methods and Materials
All reagents and solvents were obtained from commercial suppliers. Flash column chromatography was
performed on silica gel 60 (particle size 35–70 ꢀm). Analytical highꢀperformance liquid
chromatography (HPLC) was performed on a C18 column (2.6 ꢀm, 100 Å, 150 × 4.60 mm) using a
diode array ultraviolet detector. A gradient with eluent A (water–MeCN–TFA, 95:5:0.1) and eluent B
(0.1% TFA in MeCN) rising linearly from 0 to 95% of B over 15.0 min was applied at a flow rate of
ꢀ1
1 mL min to monitor reactions and a gradient over 30 min was applied to determine the purity of
peptides ( = 210 nm). Ultraꢀhighꢀperformance liquid chromatography (UPLC) mass spectrometry
MS) analyses were performed on a C18 column (1.7 ꢀm, 100 Å, 50 × 2.10 mm). A gradient with eluent
C (0.1% HCOOH in water) and eluent D (0.1% HCOOH in MeCN) rising linearly from 0 to 95% of D
λ
(
ꢀ1
over 5.20 min at a flow rate of 0.6 mL min was applied. Preparative HPLC purification was performed
on a C18 column (5 ꢀm, 100 Å, 250 × 21.2 mm) or a C8 column (5 ꢀm, 100 Å, 250 × 21.2 mm) using a
diode array ultraviolet detector. Fractions containing the purified target peptide were identified using
UPLCꢀMS or matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDIꢀ
TOF MS). Selected fractions were pooled and lyophilized. MALDIꢀTOF mass spectra were recorded
using a matrix of alphaꢀcyanoꢀ4ꢀhydroxycinnamic acid or 2,5ꢀdihydroxybenzoic acid in water/MeCN
(1:1) containing 0.1% TFA. Highꢀresolution mass spectra (HRMS) were recorded using either MALDI,
1
or electrospray ionization (ESI). H NMR spectra were recorded at 400 MHz or 600 MHz at 298 K.
1
3
C NMR spectra were recorded at 101 MHz or 150 MHz at 298 K. Chemical shifts are reported in parts
per million (ppm) relative to the deuterated solvent peak of DMSOꢀd = 2.50 ppm; = 39.52 ppm)
or CDCl = 7.26 ppm; = 77.16 ppm) as internal standard.
General procedure for preparation of MeDbz resins
6
(δ
H
δ
C
3
(δ
H
δ
C
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Aminomethyl resin (0.30 mmol) was placed in a polypropylene syringe equipped with a fritted disk,
swelled in DMF for 30 min and washed with DMF (5 × 5 mL).
Coupling of the first residue. FmocꢀGlyꢀOH or FmocꢀRink amide linker (1.20 mmol, 4.0 equiv), HATU
(445 mg, 1.17 mmol, 3.9 equiv), and i-Pr NEt (417 ꢀL, 2.40 mmol, 8.0 equiv) were preꢀincubated in
2
DMF (6.0 mL, 0.2 M) for 2 min and then added to the resin. After 2 h, the resin was washed with DMF
1
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(
3 × 5 mL), MeOH (3 × 5 mL), and CH
Pr NEt–CH Cl , 2:2:6, v/v/v, 5.0 mL). After 2 h, the resin was washed with DMF (3 × 5 mL), MeOH
3 × 5 mL), and CH Cl (3 × 5 mL).
Fmoc deprotection. The resin was treated with piperidine in DMF (1:4, v/v, 5.0 mL) (1 × 2 min,
× 20 min) and washed with DMF (3 × 5 mL), MeOH (3 × 5 mL), and CH Cl (3 × 5 mL).
Coupling of second residue. FmocꢀMeDbzꢀOH (S14) (466 mg, 1.20 mmol, 4.0 equiv), HATU (445 mg,
.17 mmol, 3.9 equiv), and i-Pr NEt (417 ꢀL, 2.40 mmol, 8.0 equiv) were preꢀincubated in DMF
6.0 mL, 0.2 M) for 2 min and then added to the resin. After 2 h, the resin was washed with DMF
(3 × 5 mL), MeOH (3 × 5 mL), and CH Cl (3 × 5 mL). After coupling of the second residue, the resin
2
Cl
2
(3 × 5 mL) and treated with a capping solution (Ac
2
O–i-
2
2
2
(
2
2
1
2
2
1
2
(
2
2
was dried under high vacuum for 16 h and the loading was determined using the outlined procedure.
The following resins were prepared using the general procedure:
MeDbzꢀRinkꢀChemMatrix resin (0.43 mmol/g)
MeDbzꢀGlyꢀChemMatrix resin (0.50 mmol/g)
MeDbzꢀGlyꢀTentaGel resin (0.22 mmol/g)
MeDbzꢀGlyꢀTentaGel XV resin (0.21 mmol/g)
MeDbzꢀGlyꢀPEGA resin (0.33 mmol/g)
Resin-loading determination
Vacuumꢀdried resin (10 mg) was agitated in DMF (2.0 mL) containing 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀ
ene (DBU) (40.0 ꢀL). After 30 min, the resin was allowed to settle and 16.0 ꢀL of the solution were
diluted with MeCN (984 ꢀL) and the absorption at 294 nm or 304 nm was determined. The loading was
calculated using the following equations:
ꢂꢂꢃꢄ
ꢅ
ꢇꢈ.ꢉꢀ×ꢀꢊꢉꢋꢈꢀꢌꢂ
ꢂꢂꢃꢄ ꢇꢗ.ꢗꢀ×ꢀꢊꢗꢘꢈꢀꢌꢂ
ꢆ =
ꢅ ꢍꢎꢏꢅꢐꢑꢀꢃꢒꢀꢓꢎꢔꢏꢌꢀꢕꢂꢅꢖ
loadingꢀ ꢁ
ꢆ =
or ꢀloadingꢀ ꢁ
ꢍꢎꢏꢅꢐꢑꢀꢃꢒꢀꢓꢎꢔꢏꢌꢀꢕꢂꢅꢖ
General protocol for automated peptide synthesis
Automated peptide synthesis was carried out on an automated peptide synthesizer using standard Fmoc
SPPS chemistry. The following Fmocꢀprotected amino acids with side chain protecting groups were
used: FmocꢀAlaꢀOH, FmocꢀArg(Pbf)ꢀOH, FmocꢀAsn(Trt)ꢀOH, FmocꢀAsp(Ot-Bu)ꢀOH, FmocꢀCys(Trt)ꢀ
OH, FmocꢀGln(Trt)ꢀOH, FmocꢀGlyꢀOH, FmocꢀGly(Dmb)ꢀOH, FmocꢀIleꢀOH, FmocꢀLeuꢀOH, FmocꢀDꢀ
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LeuꢀOH, FmocꢀLys(Boc)ꢀOH, FmocꢀOrn(Boc)ꢀOH, FmocꢀPheꢀOH, FmocꢀDꢀPheꢀOH, FmocꢀProꢀOH,
FmocꢀSer(t-Bu)ꢀOH, FmocꢀThr(t-Bu)ꢀOH, FmocꢀTrp(Boc)ꢀOH, FmocꢀTyr(t-Bu)ꢀOH, and FmocꢀValꢀ
OH. The following amino acids were used as Nꢀterminal amino acids: BocꢀCys(Trt)ꢀOH, Procꢀ
Cys(Trt)ꢀOH (S10), NvocꢀCys(Trt)ꢀOH (S11) and BocꢀPen(Trt)ꢀOH. SPPS was performed on
0.02 mmol scale using MeDbzꢀGlyꢀresins resin containing additional 5% MeDbzꢀRinkꢀChemMatrix.
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Fmoc deprotection was performed in two stages: 1) piperidine in DMF (2:3, v/v) for 3 min and 2)
piperidine in DMF (1:4, v/v) for 12 min. The deprotection was followed by washing with DMF
(
5 × 45 s). The first coupling reaction was performed as double coupling using FmocꢀXaaꢀOH
(6.0 equiv to the resin loading), HATU (5.9 equiv) and i-Pr NEt in NMP (12 equiv, 2.0 M) in DMF
final concentration = 0.15 M) for 90 min for each coupling. The remaining coupling reactions were
performed as double couplings with FmocꢀXaaꢀOH (6.0 equiv to the resin loading), HBTU (5.9 equiv)
and i-Pr NEt in NMP (12 equiv, 2.0 M) in DMF (final concentration = 0.15 M) for 40 min for each
2
(
2
coupling. Couplings reactions for FmocꢀGly(Dmb)ꢀOH, ProcꢀCys(Trt)ꢀOH (S10), NvocꢀCys(Trt)ꢀOH
(S11) and BocꢀPen(Trt)ꢀOH were performed manually in a polypropylene syringe equipped with a
2
fritted disk using 2.5 equiv of amino acid, HATU (2.5 equiv) and i-Pr NEt (5 equiv) in DMF (final
concentration = 0.10 M) for 120 min.
General procedure for N-acyl-benzimidazolinone (Nbz) formation
After automated peptide elongation, the resin (0.02 mmol, 1.0 equiv) was transferred into a
polypropylene syringe equipped with a fritted disk using CH
2
Cl
2
and the resin was washed with CH
2
Cl
2
2
(5 × 1 min). A solution of 4ꢀnitrophenylꢀchloroformate (20.1 mg, 0.10 mmol, 5.0 equiv) in CH
2
Cl
(
1.0 mL) was added to the resin and the suspension was agitated for 30 min. The resin was then washed
with CH Cl (2 × 1 min) and the procedure was repeated. The resin was then washed with CH Cl
(3 × 1 min) and DMF (3 × 1 min) and a solution of i-Pr NEt (87 ꢀL, 0.50 mmol, 25.0 equiv) in DMF
2
2
2
2
2
(1.0 mL) was added to the resin. After 15 min, the resin was washed with DMF (3 × 1 min) and the
procedure was repeated. The resin was then washed with DMF (3 × 1 min), i-Pr NEt in DMF (5%, v/v)
2
(
3 × 1 min), DMF (3 × 1 min), MeOH (3 × 1 min), and CH Cl (3 × 1 min) and dried under high
2
2
vacuum.
General procedures for one-pot peptide cyclization
Dried peptidyl MeNbzꢀGlyꢀTentaGel XV resin (0.02 mmol, 1.0 equiv) containing additional 5%
MeNbzꢀRinkꢀChemMatrix was placed in a polypropylene syringe equipped with a fritted disk and
treated with a deprotection/cleavage cocktail (2.0 mL, TFA–i-Pr SiH–water, 94:3:3, v/v/v). After 1 h
3
[2 h for peptides containing Arg(Pbf)], the cleavage solution was collected for assessment of the peptide
synthesis and the resin was washed with CH Cl (3 × 1 min), DMF (3 × 1 min), and CH Cl (3 × 1 min)
2
2
2
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and dried under suction for several minutes. The cyclization buffer [5.0 mL, phosphate buffer (0.2 M,
pH = 6.8)–MeCN, 1:1, v/v; for procedure D, a TCEP solution (0.5 M, pH = 6.8, 0.2 mL, 0.10 mmol,
5.0 equiv) was added to the buffer] was added to the resin (final concentration = 4.0 mM) and the
suspension was agitated at 50 °C.
Procedure A: For peptides containing a cysteine. After 2 h, a TCEP solution (0.5 M, pH = 6.8) (0.2 mL,
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0.10 mmol, 5.0 equiv) was added and the syringe was agitated for 15 min at room temperature. The
solution was removed from the resin and the resin was rinsed with fresh cyclization buffer. The
combined peptideꢀcontaining washings were pooled and purified by preparative HPLC. Fractions
containing pure peptide were lyophilized to afford the desired cyclized peptide.
Procedure B: For thiodepsipeptides. After 2 h, the solution was removed from the resin and the resin
was rinsed with fresh cyclization buffer. The combined peptideꢀcontaining washings were pooled and
purified by preparative HPLC. Fractions containing pure peptide were lyophilized to afford the desired
cyclized peptide.
Procedure C: For desulfurization of peptides. After 2 h, the solution was removed from the resin into a
50 mL centrifugal tube containing a magnetic stir bar and the resin was rinsed with fresh cyclization
buffer. To the solution was added a TCEP solution (0.5 M, pH = 6.8, 3.6 mL, final
concentration = 0.2 M), reduced glutathione (123 mg, 0.40 mmol, 20.0 equiv) and VAꢀ044·HCl
(64.6 mg, 0.20 mmol, 10.0 equiv) and the reaction mixture was stirred for 2ꢀ3 h at 37 °C. The reaction
mixture was monitored by analytic HPLC and upon complete desulfurization purified by preparative
HPLC. Fractions containing pure peptide were lyophilized to afford the desired cyclized peptide.
Procedure D: For peptides containing an internal disulfide bond. After 2 h, the solution was removed
from the resin into a 50 mL centrifugal tube containing a magnetic stir bar and the resin was rinsed with
fresh cyclization buffer. An iodine solution in MeCN (0.2 M, ca. 0.40 mL, 4.0 equiv) was added
dropwise to the solution until the decoloring of the iodine solution stopped. Excess iodine was quenched
2 2 3
with a drop of saturated Na S O solution and the reaction mixture was purified by preparative HPLC.
Fractions containing pure peptide were lyophilized to afford the desired cyclized peptide.
Procedure E: For cysteine labeling of peptides using bromo/iodoacetamides. After 2 h, a TCEP
solution (0.5 M, pH = 6.8, 0.2 mL, 0.10 mmol, 5.0 equiv) was added and the syringe was agitated for
15 min at room temperature. The solution was removed from the resin into a 50 mL centrifugal tube
containing a magnetic stir bar and the resin was rinsed with fresh cyclization buffer. A solution of
bromo/iodoacetamides (3.0 equiv) in DMF (0.1 mL) was added to the solution and the reaction mixture
was stirred for 0.5–1.0 h at room temperature. The reaction mixture was monitored by analytic HPLC
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and upon complete alkylation purified by preparative HPLC. Fractions containing pure peptide were
lyophilized to afford the desired cyclized peptide.
Model peptide (3). The peptide was synthesized on a 10 ꢀmol scale using procedure A. Preparative RPꢀ
HPLC (C18, 05–95% B in 30 min) afforded model peptide (3) as a fluffy white solid after
lyophilization (2.0 mg, 30%). Analytical HPLC t
R
= 19.1 min, purity 95% (
λ
= 210 nm). HRMS
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+
+
1
(
ESI/QꢀTOF) m/z: [M+H] Calcd for C31
DMSOꢀd ):
m, 1H), 1.67 (dp, J = 13.4, 6.6 Hz, 1H), 1.90–2.00 (m, 1H), 2.30 (t, J = 8.6 Hz, 1H), 2.51–2.55 (m,
H
45
N
8
O
7
S 673.3126; found 673.3123. H NMR (600 MHz,
6
δ 0.81–0.87 (m, 9H), 0.91 (d, J = 6.5 Hz, 3H), 1.42 (dt, J = 13.7, 7.1 Hz, 1H), 1.46–1.52
(
4H), 2.66 (dt, J = 13.8, 8.9 Hz, 1H), 2.89–2.98 (m, 1H), 3.04 (dd, J = 14.5, 6.2 Hz, 1H), 3.21–3.28 (m,
2H), 3.80 (dd, J = 14.8, 5.7 Hz, 1H), 4.14 (dt, J = 8.9, 6.2 Hz, 1H), 4.18–4.21 (m, 1H), 4.21–4.26 (m,
1H), 4.38 (td, J = 7.3, 5.3 Hz, 1H), 4.58 (q, J = 6.8 Hz, 1H), 6.95 (s, 1H), 6.97 (t, J = 7.5 Hz, 1H), 7.03–
7.08 (m, 3H), 7.28 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.35 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H),
.72 (d, J = 7.4 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.44 (d, J = 5.6 Hz, 1H), 8.53 (t, J = 5.7 Hz, 1H), 8.58
d, J = 8.0 Hz, 1H), 10.80 (d, J = 2.4 Hz, 1H).
7
(
Peptide dimer (5). The peptide was isolated in the synthesis of model peptide (3) on a 10 ꢀmol scale
using procedure A. Preparative RPꢀHPLC (C18, 05–95% B in 30 min) afforded peptide dimer (5) as a
fluffy white solid after lyophilization (0.5 mg, 8%). Analytical HPLC t = 22.4 min, purity 88%
R
+
+
1
(
(
(
λ
= 210 nm). UPLCꢀMS m/z: [M+H] Calcd for C62
600 MHz, DMSOꢀd ): 0.79–0.87 (m, 18H), 0.87 (d, J = 6.6 Hz, 6H), 1.43–1.54 (m, 4H), 1.56–1.63
m, 2H), 1.99–2.06 (m, 2H), 2.14 (t, J = 8.6 Hz, 2H), 2.44–2.50 (m, 2H) 2.52–2.60 (m, 2H), 2.60–2.69
89 16 14 2
H N O S 1345.62; found 1345.70. H NMR
6
δ
(m, 2H), 2.75–2.81 (m, 2H), 3.01 (dd, J = 14.9, 9.3 Hz, 2H), 3.14–3.18 (m, 2H), 3.69 (dd, J = 16.6, 5.2
Hz, 2H), 3.79 (dd, J = 16.6, 5.9 Hz, 2H), 4.11–4.17 (m, 2H), 4.19–4.26 (m, 2H), 4.34–4.40 (m, 2H),
4
.50–4.57 (m, 2H), 4.60 (q, J = 7.0 Hz, 2H), 6.92 (s, 2H), 6.95 (t, J = 7.5 Hz, 2H), 7.04 (t, J = 7.3 Hz,
H), 7.15–7.21 (m, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.35 (s, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.77 (d, J = 8.1
2
Hz, 2H), 8.02 (d, J = 7.4 Hz, 2H), 8.07 (d, J = 7.4 Hz, 2H), 8.15 (d, J = 7.4 Hz, 2H), 8.23 (d, J = 7.1 Hz,
2H), 8.25–8.30 (m, 2H), 10.79 (d, J = 2.3 Hz, 2H).
Destoamide B V1C (6). The peptide was synthesized using procedure A. Preparative RPꢀHPLC (C8,
05–95% B in 30 min) afforded destoamide B V1C (6) as a fluffy white solid after lyophilization
(3.2 mg, 23%). Analytical HPLC t = 20.0 min, purity 96% ( = 210 nm). HRMS (ESI/QꢀTOF) m/z:
λ
R
+
+
1
[
M+H] Calcd for C H N O S 687.3283; found 687.3278. H NMR (600 MHz, DMSOꢀd ):
δ
0.77
32 47
8
7
6
(d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.6 Hz, 3H), 0.84–0.90 (m, 6H), 1.35–1.62 (m, 6H), 2.10 (t, J = 8.4 Hz,
1H), 2.55 (d, J = 6.2 Hz, 2H), 2.69–2.78 (m, 2H), 3.06 (dd, J = 14.7, 5.9 Hz, 1H), 3.27 (dd, J = 14.7, 5.9
Hz, 1H), 3.33 (dd, J = 15.1, 5.4 Hz, 1H), 3.78 (dd, J = 15.1, 5.4 Hz, 1H), 4.00 (ddd, J = 11.4, 7.5, 3.9
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Hz, 1H), 4.28 (q, J = 7.4 Hz, 1H), 4.34–4.40 (m, 2H), 4.55 (q, J = 6.1 Hz, 1H), 6.90 (s, 1H), 6.96 (ddd,
J = 7.9, 6.9, 1.0 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H), 7.05 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.28–7.33 (m,
3
H), 7.35 (d, J = 7.1 Hz, 1H), 7.47 (d, J = 7.9 Hz, 1H), 8.31 (d, J = 6.8 Hz, 1H), 8.49 (d, J = 7.6 Hz,
H), 8.65 (d, J = 7.5 Hz, 1H), 8.67 (t, J = 5.5 Hz, 1H), 10.82 (d, J = 2.4 Hz, 1H).
1
Destoamide B V1A (7). The peptide was synthesized using procedure C. Preparative RPꢀHPLC (C18,
05–95% B in 30 min) afforded destoamide B V1A (7) as a fluffy white solid after lyophilization
1
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2.4 mg, 18%). Analytical HPLC t
R
= 19.2 min, purity 96% (
λ
= 210 nm). HRMS (ESI/QꢀTOF) m/z:
): 0.78 (d,
+
+
1
[
M+H] Calcd for C32
H
47
N
8
O
7
655.3562; found 655.3558. H NMR (600 MHz, DMSOꢀd
6
δ
J = 6.3 Hz, 3H), 0.81 (d, J = 6.5 Hz, 3H), 0.84–0.90 (m, 6H), 1.18 (d, J = 6.7 Hz, 3H), 1.36–1.42 (m,
2
H), 1.43–1.49 (m, 2H), 1.50–1.62 (m, 2H), 2.45–2.50 (m, 1H), 2.51–2.57 (m, 1H), 3.07 (dd, J = 14.6,
.6 Hz, 1H), 3.28 (dd, J = 14.6, 6.1 Hz, 1H), 3.32 (dd, J = 15.0, 5.8 Hz, 1H), 3.77 (dd, J = 15.0, 5.3 Hz,
5
1H), 4.01 (ddd, J = 11.3, 7.5, 3.8 Hz, 1H), 4.22 (q, J = 7.5 Hz, 1H), 4.27 (p, J = 6.7 Hz, 1H), 4.32–4.37
m, 1H), 4.53 (q, J = 6.1 Hz, 1H), 6.89 (s, 1H), 6.96 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 6.98 (d, J = 2.4 Hz,
H), 7.05 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.22 (d, J = 6.9 Hz, 1H), 7.28 (s, 1H), 7.29–7.32 (m, 2H), 7.48
d, J = 7.9 Hz, 1H), 8.09 (d, J = 6.8 Hz, 1H), 8.53 (d, J = 7.5 Hz, 1H), 8.60 (d, J = 7.5 Hz, 1H), 8.69 (t,
(
1
(
J = 5.6 Hz, 1H), 10.82 (d, J = 2.4 Hz, 1H).
Stellarin G (9). The peptide was synthesized using procedure C. Preparative RPꢀHPLC (C18, 05–
50% B in 30 min) afforded stellarin G (9) as a fluffy white solid after lyophilization (2.9 mg, 31%).
+
Analytical HPLC t
R
= 15.7 min, purity 99% (
λ
= 210 nm). UPLCꢀMS m/z: [M+H] Calcd for
+
1
C
23
H
34
N
5
O
6
476.25; found 476.23. H NMR (600 MHz, DMSOꢀd
6
):
δ
0.78 (d, J = 6.5 Hz, 3H), 0.85
(
d, J = 6.5 Hz, 3H), 1.17 (d, J = 7.3 Hz, 3H), 1.21 (d, J = 6.9 Hz, 3H), 1.31–1.40 (m, 1H), 1.39–1.47 (m,
H), 1.71 (ddd, J = 13.1, 10.1, 5.0 Hz, 1H), 2.80–2.89 (m, 2H), 3.34 (dd, J = 14.5, 5.1 Hz, 1H), 3.89–
3.97 (m, 2H), 4.06 (p, J = 7.3 Hz, 1H), 4.17 (p, J = 7.1 Hz, 1H), 4.27 (q, J = 8.1 Hz, 1H), 6.64 (d, J =
.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.6 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 7.3
Hz, 1H), 8.20 (d, J = 7.9 Hz, 1H), 8.37 (t, J = 5.6 Hz, 1H), 9.17 (s, 1H). The spectral data is in
1
8
6
7
accordance with the literature.
Stylostatin 1 (10). The peptide was synthesized using procedure C. Preparative RPꢀHPLC (C18, 05–
95% B in 30 min) afforded stylostatin 1 (10) as a fluffy white solid after lyophilization (2.4 mg, 16%).
+
Analytical HPLC t
R
= 18.0 min, purity 95% (
λ
= 210 nm). UPLCꢀMS m/z: [M+H] Calcd for
+
1
C
36
H
55
N
8
O
9
743.40; found 743.32. H NMR (600 MHz, DMSOꢀd
6
):
δ
0.71–0.76 (m, 1H) 0.77–0.83
(m, 9H), 0.85 (d, J = 6.1 Hz, 3H), 1.14 (d, J = 6.5 Hz, 3H), 1.21–1.32 (m, 1H), 1.50–1.57 (m, 4H),
1.57–1.63 (m, 2H), 1.68–1.76 (m, 1H), 2.17 (dd, J = 11.8, 6.5 Hz, 1H), 2.59–2.63 (m, 1H), 3.01–3.11
(m, 3H), 3.15 (dd, J = 13.8, 4.2 Hz, 1H), 3.20–3.28 (m, 1H), 3.68–3.70 (m, 2H), 3.84–3.89 (m, 1H),
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2
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2
2
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3
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3
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6
4.03 (dd, J = 8.6, 4.9 Hz, 1H), 4.16 (ddd, J = 12.1, 7.7, 4.3 Hz, 1H), 4.28–4.33 (m, 2H), 4.43 (d, J = 7.4
Hz, 1H), 4.44–4.50 (m, 1H), 7.12–7.16 (m, 2H), 7.19–7.23 (m, 1H), 7.26–7.31 (m, 3H), 7.34 (d, J = 7.4
Hz, 1H), 7.76 (d, J = 5.4 Hz, 1H), 7.78 (s, 1H), 8.04 (d, J = 9.6 Hz, 1H), 8.47 (d, J = 3.8 Hz, 1H), 8.59
6
8
(d, J = 7.7 Hz, 1H), 8.65 (d, J = 4.8 Hz, 1H). The spectral data is in accordance with the literature.
Cyclonellin (11). The peptide was synthesized using procedure C. Preparative RPꢀHPLC (C18, 05–
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50% B in 30 min) afforded cyclonellin (11) as a fluffy white solid after lyophilization (3.8 mg, 20%).
+
Analytical HPLC t
R
= 13.9 min, purity 95% (
λ
= 210 nm). UPLCꢀMS m/z: [M+H] Calcd for
+
1
C
45
H
63
N
12
O
12 963.47; found 963.44. H NMR (600 MHz, DMSOꢀd
6
):
δ
1.05 (d, J = 6.4 Hz, 3H), 1.25–
1.30 (m, 1H), 1.31 (d, J = 7.4 Hz, 3H), 1.34–1.41 (m, 1H), 1.47–1.54 (m, 1H), 1.61–1.70 (m, 1H), 1.70–
.78 (m, 1H), 1.78–1.85 (m, 2H), 1.85–1.92 (m, 2H), 1.92–2.00 (m, 1H), 2.17–2.24 (m, 1H), 2.48–2.53
m, 1H), 2.65–2.72 (m, 1H), 2.75 (dd, J = 14.0, 4.6 Hz, 1H), 2.95–3.01 (m, 3H), 3.15 (dd, J = 13.9, 4.2
Hz, 1H), 3.22–3.28 (m, 1H), 3.52–3.61 (m, 2H), 3.62–3.72 (m, 2H), 3.83 (dt, J = 9.9, 6.9 Hz, 1H),
1
(
3
.86–3.90 (m, 1H), 4.05–4.10 (m, 2H), 4.11–4.14 (m, 1H), 4.36–4.41 (m, 1H), 4.71 (dd, J = 9.9, 3.7 Hz,
H), 4.79 (ddd, J = 12.6, 9.1, 3.8 Hz, 1H), 4.94 (td, J = 9.5, 4.7 Hz, 1H), 5.49 (d, J = 12.4 Hz, 1H), 6.61
1
(
d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 6.89–6.96 (m, 4H), 7.35 (s, 1H), 7.37 (d, J = 9.9 Hz, 1H),
.50 (t, J = 5.5 Hz, 1H), 7.74 (d, J = 9.6 Hz, 1H), 7.98 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 8.34 (d, J = 6.6
Hz, 1H), 8.52 (d, J = 8.9 Hz, 1H), 8.62 (d, J = 6.8 Hz, 1H), 9.13 (br s, 2H). The spectral data is in
7
6
9
accordance with the literature.
Crotogossamide (12). The peptide was synthesized using procedure C. Preparative RPꢀHPLC (C8, 05–
50% B in 30 min) afforded crotogossamide (12) as a fluffy white solid after lyophilization (2.1 mg,
+
13%). Analytical HPLC t
R
= 17.3 min, purity 96% (
λ
= 210 nm). UPLCꢀMS m/z: [M+H] Calcd for
+
1
C
37
H
57
N
10
O
11 817.42; found 817.36. H NMR (600 MHz, DMSOꢀd
6
):
δ
0.59 (d, J = 6.8 Hz, 3H), 0.73
(t, J = 7.4 Hz, 3H), 0.82 (d, J = 5.9 Hz, 3H), 0.87 (d, J = 6.0 Hz, 3H), 0.98–1.06 (m, 1H), 1.27 (d, J =
.1 Hz, 3H), 1.29–1.33 (m, 1H), 1.48–1.55 (m, 2H), 1.47–1.62 (m, 1H), 1.70–1.79 (m, 1H), 2.48–2.53
m, 1H), 2.72 (dd, J = 15.6, 5.2 Hz, 1H), 2.97 (dd, J = 14.2, 9.8 Hz, 1H), 3.17 (dd, J = 14.2, 5.5 Hz,
7
(
1H), 3.37–3.43 (m, 1H), 3.62–3.69 (m, 3H), 3.75 (dd, J = 16.8, 6.2 Hz, 1H), 3.85–3.95 (m, 3H), 4.04 (q,
J = 5.8 Hz, 1H), 4.24–4.34 (m, 3H), 4.38 (p, J = 7.2 Hz, 1H), 4.99–5.07 (m, 1H), 6.85 (s, 1H), 7.16–
7.21 (m, 3H), 7.22–7.28 (m, 2H), 7.36 (d, J = 7.2 Hz, 1H), 7.39 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.72
(d, J = 7.4 Hz, 1H), 8.06 (t, J = 6.1 Hz, 1H), 8.16 (t, J = 6.1 Hz, 1H), 8.23 (d, J = 7.2 Hz, 1H), 8.31 (s,
70
1H), 8.35 (s, 1H), 8.51 (s, 1H). The spectral data is in accordance with the literature.
Tyrocidine A V1A (13). The peptide was synthesized using procedure C. Preparative RPꢀHPLC (C8,
5–95% B in 30 min) afforded tyrocidine A V1A (13) as a fluffy white solid after lyophilization
0
(
5.9 mg, 24%). Analytical HPLC t = 25.4 min, purity 97% (
λ
= 210 nm). HRMS (ESI/QꢀTOF) m/z:
R
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8
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+
+
1
[M+H] Calcd for C64
H
84
N
13
O
13 1242.6306; found 1242.6302. H NMR (600 MHz, DMSOꢀd
.35–0.47 (m, 1H), 0.92 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H), 1.00–1.06 (m, 1H), 1.21–1.28 (m,
H), 1.34–1.40 (m, 1H), 1.41–1.50 (m, 2H), 1.61–1.70 (m, 5H), 1.74–1.81 (m, 1H), 1.85–1.91 (m, 1H),
6
): δ
0
5
1.95–2.04 (m, 1H), 2.16 (q, J = 9.0 Hz, 1H), 2.25 (t, J = 12.9 Hz, 1H), 2.37–2.41 (m, 1H), 2.67–2.89
m, 5H), 2.91–2.96 (m, 2H), 2.98–3.03 (m, 1H), 3.05–3.12 (m, 1H), 3.27–3.35 (m, 2H), 3.79–3.84 (m,
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
7
H), 4.07 (d, J = 8.0 Hz, 1H), 4.26–4.35 (m, 2H), 4.47–4.55 (m, 3H), 4.65 (p, J = 6.7 Hz, 1H), 5.19–
.25 (m, 1H), 5.51–5.57 (m, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.89 (s, 1H), 6.92 (d, J = 8.5 Hz, 2H), 7.03–
.16 (m, 6H), 7.17–7.27 (m, 11H), 7.35 (d, J = 7.1 Hz, 1H), 7.45 (m, 3H), 7.54 (s, 1H), 7.88 (d, J = 8.5
Hz, 1H), 7.98 (s, 1H), 8.31 (d, J = 9.5 Hz, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.91–8.97 (m, 2H), 9.06 (d, J =
.8 Hz, 1H), 9.18 (br s, 1H), 9.26 (d, J = 3.7 Hz, 1H).
9
SFTI-1 (14). FmocꢀGly(Dmb)ꢀOH was used to prevent aspartimide formation during SPPS. The peptide
was synthesized using procedure D. Preparative RPꢀHPLC (C18, 20–50% B in 30 min) afforded SFTIꢀ1
(
14) as a fluffy white solid after lyophilization (3.2 mg, 11%). Analytical HPLC t
R
= 17.7 min, purity
1513.73; found 1513.55;
757.37; found 757.53. H NMR (600 MHz, DMSOꢀd ): 0.54
d, J = 6.7 Hz, 3H), 0.69 (t, J = 7.4 Hz, 3H), 0.76–0.86 (m, 6H), 0.91–1.05 (m, 2H), 1.21–1.34 (m, 7H)
+
+
9
105 18 18 2
5% (λ = 210 nm). UPLCꢀMS m/z: [M+H] Calcd for C67H N O S
2
+
2+
2
1
[M+2H] Calcd for C67
H
106
N
18
O
18
S
6
δ
(
1
1
2
.35–1.40 (m, 1H), 1.41–1.50 (m, 1H), 1.51–1.57 (m, 2H), 1.62–1.79 (m, 6H), 1.80–1.90 (m, 4H),
.91–2.03 (m, 3H), 2.04–2.09 (m, 1H), 2.17–2.23 (m, 1H), 2.23–2.29 (m, 1H), 2.59–2.66 (m, 1H),
.70–2.84 (m, 5H), 2.90–3.01 (m, 2H), 3.02–3.12 (m, 2H), 3.22 (d, J = 13.4 Hz, 1H), 3.44–3.55 (m,
5H), 3.65 (dd, J = 17.0, 5.8 Hz, 1H), 3.71–3.84 (m, 4H), 3.81 (d, J = 7.7 Hz, 1H), 3.87 (dd, J = 17.0, 6.4
Hz, 1H), 4.13 (d, J = 6.4 Hz 1H), 4.16–4.25 (m, 5H), 4.25–4.34 (m, 4H), 4.54–4.64 (m, 2H), 4.95 (d, J
=
8.5 Hz, 1H), 5.20 (d, J = 6.4 Hz, 1H), 5.26–5.35 (m, 2H), 5.72–5.75 (m, 1H), 7.10–7.14 (m, 1H), 7.19
(t, J = 7.6 Hz, 2H), 7.22 –7.27 (m, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.35 (d, J = 7.6 Hz, 2H), 7.49–7.52 (m,
H), 7.63–7.72 (m, 3H), 7.80–7.85 (m, 1H), 8.18 (t, J = 6.3 Hz, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.47 (br
s, 1H), 8.64 (d, J = 9.6 Hz, 1H), 8.70 (d, J = 7.0 Hz, 1H), 8.81 (d, J = 9.4 Hz, 1H), 12.40 (br s, 1H). The
2
7
6
spectral data is in accordance with the literature.
Crotogossamide-alkyne (15). The peptide was synthesized using procedure E and 2ꢀiodoꢀNꢀ(propꢀ2ꢀynꢀ
1ꢀyl)acetamide (S16) (13.3 mg, 0.06 mmol, 3.0 equiv) as alkylating reagent. Preparative RPꢀHPLC
(
C18, 05–95% B in 30 min) afforded crotogossamideꢀalkyne (15) as a fluffy offꢀwhite solid after
lyophilization (2.4 mg, 13%). Analytical HPLC t
R
= 18.9 min, purity 95% (
λ
= 210 nm). HRMS
+
+
1
(ESI/QꢀTOF) m/z: [M+H] Calcd for C H N O S 944.4295; found 944.4282. H NMR (600 MHz,
4
2
62 11 12
6
DMSOꢀd ): δ 0.51 (d, J = 6.8 Hz, 3H), 0.74 (t, J = 7.4 Hz, 3H), 0.82 (d, J = 5.9 Hz, 3H), 0.88 (d, J = 6.0
Hz, 3H), 0.99–1.08 (m, 1H), 1.31–1.38 (m, 1H), 1.48–1.64 (m, 3H), 1.70–1.78 (m, 1H), 2.48–2.53 (1H
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5
5
6
signal not fully visible due to DMSOꢀd
6
signal), 2.79–2.84 (m, 2H), 3.02–3.10 (m, 2H), 3.10 (t, J = 2.5
Hz, 1H), 3.22–3.29 (m, 3H), 3.36 (d, J = 14.4 Hz, 1H), 3.43 (dd, J = 16.6, 4.9 Hz, 1H), 3.66–3.76 (m,
4H), 3.83–3.86 (m, 1H), 3.88 (dd, J = 5.6, 2.5 Hz, 1H), 3.90 (dd, J = 5.6, 2.5 Hz, 1H), 3.91–3.94 (m,
1H), 3.94–3.97 (m, 1H) 4.02 (q, J = 5.6 Hz, 1H), 4.22–4.36 (m, 3H), 4.62 (q, J = 7.8 Hz, 1H), 6.85 (s,
1H), 7.15–7.20 (m, 3H), 7.22–7.26 (m, 2H), 7.30 (d, J = 6.6 Hz, 1H), 7.37–7.44 (m, 2H), 7.67–7.74 (m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H), 7.99 (t, J = 6.3 Hz, 1H), 8.13–8.22 (m, 2H), 8.32 (t, J = 5.4 Hz, 1H), 8.50 (t, J = 5.5 Hz, 1H), 8.68
br s, 1H), 8.77 (br s, 1H).
(
Stylostatin 1-azide (16). The peptide was synthesized using procedure E and Nꢀ(4ꢀazidophenyl)ꢀ2ꢀ
bromoacetamide (S17) (15.3 mg, 0.06 mmol, 3.0 equiv) as alkylating reagent. Preparative RPꢀHPLC
(C18, 20–60% B in 30 min) afforded stylostatin 1ꢀazide (16) as a fluffy offꢀwhite solid after
lyophilization (2.9 mg, 15%). Analytical HPLC t
R
= 21.8 min, purity 98% (
λ
= 210 nm). HRMS
+
+
1
(
ESI/QꢀTOF) m/z: [M+H] Calcd for C44
H
61
N
12
O
10S 949.4349; found 949.4334. H NMR (600 MHz,
DMSOꢀd6):
δ
0.67–0.74 (m, 1H), 0.77–0.82 (m, 9H), 0.82–0.85 (m, 3H), 0.85–0.91 (m, 1H), 1.26–1.33
(m, 1H), 1.50–1.57 (m, 5H), 1.58–1.65 (m, 1H), 1.73–1.81 (m, 1H), 2.17 (dd, J = 11.8, 6.4 Hz, 1H),
2
.58–2.63 (m, 1H), 2.75 (dd, J = 13.3, 4.3 Hz, 1H), 2.95–3.00 (m, 2H), 3.03 (dd, J = 15.5, 3.2 Hz, 1H),
.09 (dd, J = 15.5, 4.6 Hz, 1H), 3.15 (dd, J = 13.8, 4.0 Hz, 1H), 3.23–3.29 (m, 1H), 3.36 (s, 2H), 3.68–
3
3.70 (m, 2H), 3.84–3.87 (m, 1H), 4.07 (dd, J = 8.7, 5.1 Hz, 1H), 4.13 (ddd, J = 12.0, 7.6, 4.1 Hz, 1H),
4
.30–4.36 (m, 2H), 4.44 (d, J = 7.2 Hz, 1H), 4.64 (ddd, J = 9.8, 7.7, 4.5 Hz, 1H), 7.04 (d, J = 8.9 Hz,
H), 7.14–7.20 (m, 3H), 7.21–7.25 (m, 2H), 7.26 (s, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 8.9 Hz,
2
2H), 7.79 (d, J = 5.5 Hz, 1H), 7.82 (s, 1H), 7.99 (d, J = 9.7 Hz, 1H), 8.41 (d, J = 7.5 Hz, 1H), 8.52 (d, J
3.8 Hz, 1H), 8.87 (d, J = 5.0 Hz, 1H), 10.05 (s, 1H).
Destoamide B-fluorescein (17). The peptide was synthesized using procedure E and 5ꢀ
bromoacetamido)fluorescein (S18) (28.1 mg, 0.06 mmol, 3.0 equiv) as alkylating reagent. Preparative
RPꢀHPLC (C18, 05–95% B in 30 min) afforded destoamide Bꢀfluorescein (17) as a fluffy yellow solid
=
(
after lyophilization (2.5 mg, 12%). Analytical HPLC t
R
= 21.3 min, purity 96% (
λ
= 210 nm). HRMS
+
+
1
(ESI/QꢀTOF) m/z: [M+Na] Calcd for C54
H
59
N
9
NaO13S 1096.3845; found 1096.3840. H NMR (600
MHz, DMSOꢀd6):
J = 6.4 Hz, 3H), 1.32–1.39 (m, 1H), 1.41–1.49 (m, 2H), 1.50–1.61 (m, 3H), 2.52–2.59 (m, 2H), 2.86–
.96 (m, 2H), 3.06 (dd, J = 14.7, 5.9 Hz, 1H), 3.27 (dd, J = 14.7, 5.9 Hz, 1H), 3.33 (dd, J = 14.9, 5.8
δ 0.77 (d, J = 6.4 Hz, 3H), 0.80 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H), 0.86 (d,
2
Hz, 1H), 3.41 (s, 2H), 3.78 (dd, J = 14.9, 5.1 Hz, 1H), 3.99 (ddd, J = 11.3, 7.4, 3.8 Hz, 1H), 4.25–4.31
(m, 1H), 4.43 (td, J = 8.1, 4.1 Hz, 1H), 4.50–4.57 (m, 2H), 6.51–6.55 (m, 2H), 6.58 (dd, J = 8.7, 2.3 Hz,
2
H), 6.67 (d, J = 2.3 Hz, 2H), 6.89 (s, 1H), 6.95 (ddd, J = 7.9, 6.9, 1.0 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H),
.04 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.28–7.32 (m, 2H), 7.33–7.39 (m, 2H),
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7.47 (d, J = 7.9 Hz, 1H), 7.80 (dd, J = 8.3, 2.0 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 8.41 (d, J = 6.8 Hz,
1
1
H), 8.51 (d, J = 7.7 Hz, 1H), 8.63–8.69 (m, 2H), 10.11 (br s, 2H), 10.49 (s, 1H), 10.83 (d, J = 2.4 Hz,
H).
Model thiodepsipeptide (S1). The peptide was synthesized on a 10 ꢀmol scale using procedure B.
Preparative RPꢀHPLC (C18, 05–95% B in 30 min) afforded model thiodepsipeptide (S1) as a fluffy
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
white solid after lyophilization (3.6 mg, 50%). Analytical HPLC t
R
= 18.1 min, purity 98%
+
+
(λ
= 210 nm). HRMS (ESI/QꢀTOF) m/z: [M+H] Calcd for C H N O S 715.3232; found 715.3229.
33 47
8
8
1
H NMR (600 MHz, DMSOꢀd6):
δ 0.77 (d, J = 6.5 Hz, 3H), 0.85 (d, J = 6.5 Hz, 3H), 0.88–0.93 (m,
6
2
1
H), 1.48 (t, J = 11.9 Hz, 1H), 1.52–1.63 (m, 2H), 1.79 (s, 3H), 2.12 (dq, J = 13.7, 6.8 Hz, 1H), 2.54–
.57 (m, 1H), 2.59–2.63 (m, 1H), 2.88 (dd, J = 12.7, 3.5 Hz, 1H), 2.90–2.98 (m, 1H), 2.97–3.03 (m,
H), 3.13 (dd, J = 14.8, 5.0 Hz, 1H), 3.62 (dd, J = 16.7, 5.9 Hz, 1H), 3.76 (dd, J = 16.7, 4.8 Hz, 1H),
4.01–4.06 (m, 1H), 4.28 (ddd, J = 8.3, 6.5, 4.5 Hz, 1H), 4.34–4.39 (m, 1H), 4.39–4.44 (m, 1H), 4.46–
4
.51 (m, 1H), 6.92–6.97 (m, 2H), 7.04 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.25 (d, J = 8.3 Hz,
H), 7.31 (d, J = 8.1 Hz, 1H), 7.41 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.93 (d, J = 8.3 Hz, 1H), 8.10 (d, J
1
= 7.3 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.24–8.31 (m, 2H), 10.83 (d, J = 2.3 Hz, 1H).
N-((prop-2-yn-1-yloxy)carbonyl)-S-trityl-L-cysteine (Proc-Cys(Trt)-OH) (S10). The compound was
56
prepared using a procedure adapted from the literature. HꢀCys(Trt)ꢀOH (0.50 g, 1.38 mmol, 1.0 equiv)
was suspended in NaOH (2 M, 25 mL) and the pH was adjusted to 8.5. Propargyl chloroformate
(0.68 mL, 7.00 mmol, 5.0 equiv) was added dropwise to the suspension and the reaction mixture was
stirred for 4 h. The reaction mixture was then diluted with EtOAc (50 mL) and acidified with saturated
citric acid (10 mL). The acidic phase was extracted with EtOAc (3 × 50 mL) and the combined organic
layers were dried over Na
2 4
SO and concentrated under reduced pressure. Purification of the crude
product by flash column chromatography (SiO
2
, CH
2
Cl
2
/MeOH/AcOH = 95/5/0.5) afforded the title
ꢀ
compound S10 as white solid (487 mg, 1.09 mmol, 79%). UPLCꢀMS m/z: [MꢀH] Calcd for
ꢀ
1
C
26
H22NO
4
S 444.13; found 444.30. H NMR (400 MHz, CDCl
3
): δ 2.46–2.50 (m, 1H), 2.64–2.76 (m,
2H), 4.28 (dt, J = 8.1, 5.6 Hz, 1H), 4.58–4.73 (m, 2H), 5.22 (d, J = 8.1 Hz, 1H), 7.17–7.46 (m, 15H).
1
3
C NMR (101 MHz, CDCl
3
):
δ
33.7, 52.9, 53.2, 67.5, 75.2, 77.9, 127.1 (3C), 128.2 (6C), 129.6 (6C),
56
144.3 (3C), 155.0, 175.0. The spectral data is in accordance with the literature.
N-(((4,5-dimethoxy-2-nitrobenzyl)oxy)carbonyl)-S-trityl-L-cysteine (Nvoc-Cys(Trt)-OH) (S11).
solution of 6ꢀnitroveratryloxycarbonyl chloride (NvocꢀCl, 152 mg, 0.55 mmol, 1.1 equiv) in dioxane
1.0 mL) was added dropwise to a solution of HꢀCys(Trt)ꢀOH (182 mg, 0.50 mmol, 1.0 equiv) and
Na CO (117 mg, 1.10 mmol, 2.2 equiv) in dioxane/water (1:1, 10 mL, v/v) at 0 °C. After 30 min was
A
(
2
3
the reaction mixture allowed to warm to room temperature and stirred for 20 h. The reaction mixture
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was then evaporated to dryness, redissolved in water (10 mL) and washed with EtOAc (3 × 10 mL). The
aqueous phase was acidified with saturated citric acid (5 mL), extracted with EtOAc (3 × 10 mL) and
the combined organic layers dried over Na
of the crude product by flash column chromatography (SiO
afforded the title compound S11 as yellow solid (169 mg, 0.28 mmol, 56%). HRMS (ESI/QꢀTOF) m/z:
2
SO
4
and concentrated under reduced pressure. Purification
2
, CH Cl /MeOH/AcOH = 90/10/0.5)
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
1
[
M+Na] Calcd for C32
29 2 8 6
H N NaO S 625.1615; found 625.1610. H NMR (400 MHz, DMSOꢀd ): δ
2.41–2.47 (m, 1H), 2.56–2.60 (m, 1H), 3.80–3.92 (m, 7H), 5.37 (s, 1H) 7.17 (s, 1H), 7.20–7.38 (m,
13
1
6H), 7.71 (s, 1H), 7.77 (br s, 1H). C NMR (101 MHz, DMSOꢀd
6
): δ 33.5, 48.6, 56.1, 56.2, 62.3,
66.0, 108.1, 109.7, 126.7 (3C), 128.0 (6C), 128.4, 129.1 (6C), 138.9, 144.3 (3C), 147.6, 153.5, 155.3.
4-(Methylamino)-3-nitrobenzoic acid (S12). The compound was prepared using a procedure adapted
50
from the literature. A solution of methylamine (30 mL, 33% in MeOH, 320 mmol, 12.0 equiv) was
added to a solution of 4ꢀfluoroꢀ3ꢀnitrobenzoic acid (5.00 g, 27.0 mmol, 1.0 equiv) in MeOH (66 mL)
and the reaction mixture was stirred at room temperature. After 18 h, the reaction mixture was poured
into water (75 mL), acidified with HCl (35%, ~18 mL) to reach pH = 5, and the resulting yellow
precipitate was collected by filtration and washed with water. Drying for 24 h under high vacuum gave
1
the title compound S12 (5.20 g, 26.5 mmol, 98%) as a yellow solid. H NMR (600 MHz, DMSOꢀd ):
δ
6
3
.00 (d, J = 4.9 Hz, 3H), 7.03 (d, J = 9.1 Hz, 1H), 7.97 (dd, J = 9.1 Hz, 2.0 Hz, 1H), 8.50−8.56 (m, 1H),
13
8.60 (d, J = 2.0 Hz, 1H), 12.8 (s, 1H). C NMR (151 MHz, DMSOꢀd
6
):
δ
29.9, 114.4, 116.8, 128.3,
5
0
130.4, 136.0, 147.9, 166.0. The spectral data is in accordance with the literature.
3-Amino-4-(methylamino)benzoic acid (S13). The compound was prepared using a procedure adapted
5
0
from the literature. 4ꢀ(Methylamino)ꢀ3ꢀnitrobenzoic acid (S12) (5.20 g, 26.5 mmol, 1.0 equiv) was
hydrogenated over Pd/C (250 mg, 10% wt) at atmospheric pressure in MeOH (250 mL) at room
temperature. After 16 h, the catalyst was removed by filtration and evaporation of the solvent under
1
reduced pressure gave the title compound S13 (4.31 g, 25.9 mmol 98%) as a dark solid. H NMR
(
600 MHz, DMSOꢀd
H), 7.22 (dd, J = 8.3 Hz, 2.0 Hz, 1H). C NMR (151 MHz, DMSOꢀd
120.9, 133.9, 141.2, 168.4. The spectral data is in accordance with the literature.
Fmoc-3-amino-4-(methylamino)benzoic acid (Fmoc-MeDbz-OH) (S14). The compound was prepared
6
): δ 2.76 (s, 3H), 5.26 (br s, 1H), 6.37 (d, J = 8.3 Hz, 1H), 7.15 (d, J = 2.0 Hz,
1
3
1
6
): δ 29.8, 107.3, 114.3, 118.1,
5
0
50
using a procedure adapted from the literature. A solution of Fmocꢀchloride (6.00 g, 23.2 mmol,
.9 equiv) in MeCN (48 mL) was added dropwise to a solution of 3ꢀaminoꢀ4ꢀ(methylamino)ꢀbenzoic
acid (S13) (4.31 g, 25.9 mmol, 1.0 equiv) and i-Pr NEt (4.27 mL, 24.5 mmol, 0.95 equiv) in a mixture
0
2
of MeCN in water (96 mL, 1:1, v/v) until all starting material was consumed. The solvent was
evaporated under reduced pressure and the precipitate collected by filtration and washed with MeCN.
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Drying for 24 h under high vacuum gave the title compound S14 (6.16 g, 15.9 mmol, 61%) as a grey
+
+
1
solid. UPLCꢀMS m/z: [M+H] Calcd for C H N O 389.15; found 389.17. H NMR (600 MHz,
23
21
2
4
DMSOꢀd
6
): δ
2.78 (d, J = 3.6 Hz, 3H), 4.30−4.40 (m, 3H), 5.87 (br s, 1H), 6.62 (d, J = 8.5 Hz, 1H),
7.28−7.38 (m, 2H), 7.45 (t, J = 7.4 Hz, 2H), 7.61−7.80 (m, 4H), 7.92 (d, J = 7.4 Hz, 2H), 8.71 (br s,
1
3
1
H), 12.15 (br s, 1H). C NMR (151 MHz, DMSOꢀd
6
): δ 29.5, 46.7, 66.0 (2C), 108.8, 116.7, 120.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2C), 122.0, 125.3 (2C), 127.1 (2C), 127.6 (2C), 140.7 (2C), 143.8 (2C), 154.7, 167.3. The spectral data
5
0
is in agreement with the literature.
2-Chloro-N-(prop-2-yn-1-yl)acetamide (S15). The compound was prepared using a procedure adapted
8
0
from the literature. A solution of chloroacetyl chloride (0.38 mL, 4.8 mmol, 1.2 equiv) in CH
5 mL) was added dropwise to a solution of propargyl amine (0.26  L, 4.00 mmol, 1.0 equiv) and i-
Pr NEt (0.84 mL, 4.8 mmol, 1.2 equiv) in CH Cl (27 mL) at 0 °C. The iceꢀbath was removed and the
reaction stirred for 30 min. The reaction mixture was then diluted with CH Cl (20 mL), washed with
2 2
Cl
(
2
2
2
2
2
saturated NaHCO
3
(3 × 20 mL), 2 M HCl (3 × 20 mL) and brine (1 × 20 mL) and dried over Na
2 4
SO .
Evaporation of the solvent under reduced pressure gave the title compound S15 (421 mg, 3.2 mmol,
+
+
1
80%) as a brown oil. UPLCꢀMS m/z: [M+H] Calcd for C
5
H
7
ClNO 132.02; found 132.12. H NMR
2.28 (t, J = 2.6 Hz, 1H), 4.07 (s, 2H), 4.10 (dd, J = 5.4, 2.6 Hz, 2H), 6.78 (br s,
): 29.7, 42.5, 72.3, 78.7, 165.8. The spectral data is in accordance
3
(400 MHz, CDCl ): δ
13
1
H). C NMR (101 MHz, CDCl
3
δ
8
1
with the literature.
2-Iodo-N-(prop-2-yn-1-yl)acetamide (S16). The compound was prepared using a procedure adapted
80
from the literature. To a solution of chloroacetamide S15 (180 mg, 1.37 mmol, 1.0 equiv) in acetone
(15 mL) was added NaI (614 mg, 4.10 mmol, 3.0 equiv) and the reaction mixture was refluxed for 18 h.
The solvent was removed under reduced pressure and the remaining residue was partitioned between
EtOAc (20 mL) and water (20 mL). The organic layer was separated, washed with saturated Na
3 × 20 mL), water (1 × 20 mL) and brine (1 × 20 mL) and dried over Na SO . Evaporation of the
solvent under reduced pressure gave the title compound S16 (151 mg, 0.68 mmol, 50%) as a brown
2 2 3
S O
(
2
4
+
+
1
solid. UPLCꢀMS m/z: [M+H] Calcd for C
2.28 (t, J = 2.6 Hz, 1H), 3.71 (s, 2H), 4.07 (dd, J = 5.3, 2.6 Hz, 2H), 6.22 (br s, 1H). C NMR (101
MHz, CDCl ): ꢀ1.3, 30.4, 72.4, 78.7, 166.5.
N-(4-azidophenyl)-2-bromoacetamide (S17). A solution of bromoacetyl bromide (0.13 mL, 1.49 mmol,
.0 equiv) in anhydrous CH Cl (1 mL) was added dropwise to a solution of 4ꢀazidoaniline
hydrochloride (253 mg, 1.49 mmol, 1.0 equiv) and i-Pr NEt (0.52 mL, 2.98 mmol, 2.0 equiv) in
anhydrous CH Cl (3.7 mL) at 0 °C for 10 min. The iceꢀbath was removed and the reaction stirred for
5 7 3
H INO 223.96; found 223.95. H NMR (400 MHz, CDCl ):
13
δ
3
δ
1
2
2
2
2
2
2 2 3
30 min. The reaction mixture was then diluted with CH Cl (20 mL), washed with saturated NaHCO
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2 4
(3 × 20 mL), 2 M HCl (3 × 20 mL) and brine (1 × 20 mL) and dried over Na SO . Evaporation of the
solvent under reduced pressure gave the title compound S17 (353 mg, 1.38 mmol, 93%) as a brown
+
+
1
solid. HRMS (ESI/QꢀTOF) m/z: [M+H] Calcd for C
(400 MHz, CDCl3): 4.02 (s, 2H), 7.02 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 8.12 (s, 1H).
C NMR (101 MHz, CDCl3): 29.5, 119.8 (2C), 121.7 (2C), 134.0, 137.0, 163.4.
-(Bromoacetamido)fluorescein (S18). To a suspension of 5ꢀaminofluorescein (isomer I, 100 mg,
0.29 mmol, 1.0 equiv) and NaHCO (73.0 mg, 0.87 mmol, 3.0 equiv) in anhydrous THF (10 mL) at
8 8 4
H BrN O 254.9876; found 254.9877. H NMR
δ
1
3
δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
3
0
°C was added dropwise a solution of bromoacetyl bromide (30.2 ꢀL, 0.35 mmol, 1.2 equiv) in
anhydrous THF (1 mL) over 10 min. The iceꢀbath was removed and the reaction stirred for 2 h. The
reaction mixture was then partitioned between with EtOAc (20 mL) and water (10 mL). The aqueous
phase was acidified with saturated citric acid (5 mL) and extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine (20 mL), dried over Na SO and concentrated under
2
4
reduced pressure. Purification of the crude product by flash column chromatography (SiO
CH Cl /MeOH/ = 9/1) afforded the title compound S18 as an orange solid (70 mg, 0.15 mmol, 52%).
HRMS (ESI/QꢀTOF) m/z: [M+H] Calcd for C H BrNO 468.0077; found 468.0074. H NMR
2
,
2
2
+
+
1
22
15
6
(400 MHz, DMSOꢀd
6
): δ 4.10 (s, 2H), 6.53–6.62 (m, 4H), 6.67 (d, J = 2.3 Hz, 2H), 7.24 (d, J = 8.3 Hz,
1H), 7.82 (dd, J = 8.3, 2.0 Hz, 1H), 8.25–8.33 (m, 1H), 10.10 (s, 2H), 10.85 (s, 1H). (Signals reported
13
for the open acid conformation) C NMR (101 MHz, DMSOꢀd
6
): δ 30.1, 83.1, 102.2 (2C), 109.6 (2C),
112.5 (2C), 113.6, 124.6, 126.4, 127.0, 129.1 (2C), 140.1, 147.4, 151.9 (2C), 159.4 (2C), 165.5, 168.4.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website. Supporting
1
13
figures, copies of HPLC traces as well as H and C NMR spectra.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: cao@sund.ku.dk
ORCID
Bengt H. Gless: 0000ꢀ0003ꢀ0935ꢀ3278
Christian A. Olsen: 0000ꢀ0002ꢀ2953ꢀ8942
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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This work was supported by the Carlsberg Foundation (2013ꢀ01ꢀ0333 and CF15ꢀ011; C.A.O.) and the
University of Copenhagen (partial Ph.D. fellowship for B.H.G.).
References
(
1) White, A. M.; Craik, D. J. Discovery and Optimization of Peptide Macrocycles. Expert Opin. Drug
Discovery 2016, 11, 1151ꢀ1163.
2) Hill, T. A.; Shepherd, N. E.; Diness, F.; Fairlie, D. P. Constraining Cyclic Peptides to Mimic
Protein Structure Motifs. Angew. Chem. Int. Ed. 2014, 53, 13020ꢀ13041.
3) Hosseinzadeh, P.; Bhardwaj, G.; Mulligan, V. K.; Shortridge, M. D.; Craven, T. W.; PardoꢀAvila,
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
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4
4
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5
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5
5
6
0
1
2
3
4
5
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7
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0
1
2
3
4
5
6
7
8
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0
1
2
3
4
5
6
7
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0
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2
3
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8
9
0
1
2
3
4
5
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7
8
9
0
(
(
F.; Rettie, S. A.; Kim, D. E.; Silva, D.ꢀA.; Ibrahim, Y. M.; Webb, I. K.; Cort, J. R.; Adkins, J. N.;
Varani, G.; Baker, D. Comprehensive Computational Design of Ordered Peptide Macrocycles. Science
2017, 358, 1461ꢀ1466.
(4) White, C. J.; Yudin, A. K. Contemporary Strategies for Peptide Macrocyclization. Nat. Chem.
2
011, 3, 509ꢀ524.
5) Kleineweischede, R.; Hackenberger, C. P. R. Chemoselective Peptide Cyclization by Traceless
Staudinger Ligation. Angew. Chem. Int. Ed. 2008, 47, 5984ꢀ5988.
6) Hili, R.; Rai, V.; Yudin, A. K. Macrocyclization of Linear Peptides Enabled by Amphoteric
Molecules. J. Am. Chem. Soc. 2010, 132, 2889ꢀ2891.
7) Rohrbacher, F.; Deniau, G.; Luther, A.; Bode, J. W. Spontaneous HeadꢀtoꢀTail Cyclization of
Unprotected Linear Peptides with the KAHA Ligation. Chem. Sci. 2015, 6, 4889ꢀ4896.
8) Dawson, P. E.; Muir, T. W.; ClarkꢀLewis, I.; Kent, S. B. H. Synthesis of Proteins by Native
Chemical Ligation. Science 1994, 266, 776ꢀ779.
9) BlancoꢀCanosa, J. B.; Dawson, P. E. An Efficient FmocꢀSPPS Approach for the Generation of
(
(
(
(
(
Thioester Peptide Precursors for Use in Native Chemical Ligation. Angew. Chem. Int. Ed. 2008, 47,
6851ꢀ6855.
(10) Ollivier, N.; Dheur, J.; Mhidia, R.; Blanpain, A.; Melnyk, O. Bis(2ꢀsulfanylethyl)amino Native
Peptide Ligation. Org. Lett. 2010, 12, 5238ꢀ5241.
(
11) Fang, G.ꢀM.; Li, Y.ꢀM.; Shen, F.; Huang, Y.ꢀC.; Li, J.ꢀB.; Lin, Y.; Cui, H.ꢀK.; Liu, L. Protein
Chemical Synthesis by Ligation of Peptide Hydrazides. Angew. Chem. Int. Ed. 2011, 50, 7645ꢀ7649.
12) Wang, P.; Dong, S.; Shieh, J.ꢀH.; Peguero, E.; Hendrickson, R.; Moore, M. A. S.; Danishefsky, S.
J. Erythropoietin Derived by Chemical Synthesis. Science 2013, 342, 1357ꢀ1360.
13) Zheng, J.ꢀS.; Tang, S.; Huang, Y.ꢀC.; Liu, L. Development of New Thioester Equivalents for
Protein Chemical Synthesis. Acc. Chem. Res. 2013, 46, 2475ꢀ2484.
14) Bondalapati, S.; Jbara, M.; Brik, A. Expanding the Chemical Toolbox for the Synthesis of Large
(
(
(
and Uniquely Modified Proteins. Nat. Chem. 2016, 8, 407ꢀ418.
(15) Burke, H. M.; McSweeney, L.; Scanlan, E. M. Exploring Chemoselective SꢀtoꢀN Acyl Transfer
Reactions in Synthesis and Chemical Biology. Nat. Commun. 2017, 8, 15655.
(16) Kulkarni, S. S.; Sayers, J.; Premdjee, B.; Payne, R. J. Rapid and Efficient Protein Synthesis
through Expansion of the Native Chemical Ligation Concept. Nat. Rev. Chem. 2018, 2, 0122.
(
17) Zhang, L.; Tam, J. P. Synthesis and Application of Unprotected Cyclic Peptides as Building
Blocks for Peptide Dendrimers. J. Am. Chem. Soc. 1997, 119, 2363ꢀ2370.
18) Shao, Y.; Lu, W.; Kent, S. B. H. A Novel Method to Synthesize Cyclic Peptides. Tetrahedron
Lett. 1998, 39, 3911ꢀ3914.
19) Daly, N. L.; Love, S.; Alewood, P. F.; Craik, D. J. Chemical Synthesis and Folding Pathways of
Large Cyclic Polypeptides: Studies of the Cystine Knot Polypeptide Kalata B1. Biochemistry 1999, 38,
0606ꢀ10614.
20) Yan, L. Z.; Dawson, P. E. Synthesis of Peptides and Proteins without Cysteine Residues by Native
(
(
1
(
Chemical Ligation Combined with Desulfurization. J. Am. Chem. Soc. 2001, 123, 526ꢀ533.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(21) Raz, R.; Burlina, F.; Ismail, M.; Downward, J.; Li, J.; Smerdon S. J.; Quibell, M.; White P. D.;
Offer, J. HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization. Angew. Chem.
Int. Ed. 2016, 55, 13174ꢀ13179.
(
22) Chen, J.; Warren, J. D.; Wu, B.; Chen, G.; Wan, Q.; Danishefsky, S. J. A Route to Cyclic Peptides
and Glycopeptides by Native Chemical Ligation Using in situ Derived Thioesters. Tetrahedron Lett.
006, 47, 1969ꢀ1972.
23) Hemu, X.; Taichi, M.; Qiu, Y.; Liu, D.ꢀX.; Tam, J. P. Biomimetic Synthesis of Cyclic Peptides
Using Novel Thioester Surrogates. Biopolymers 2013, 100, 492ꢀ501.
24) Boll, E.; Drobecq, H.; Lissy, E.; Cantrelle, F.ꢀX.; Melnyk, O. Kinetically Controlled
Chemoselective Cyclization Simplifies the Access to Cyclic and Branched Peptides. Org. Lett. 2016,
8, 3842ꢀ3845.
2
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1
(25) Shelton, P. M. M.; Weller, C. E.; Chatterjee, C. A Facile NꢀMercaptoethoxyglycinamide (MEGA)
Linker Approach to Peptide Thioesterification and Cyclization. J. Am. Chem. Soc. 2017, 139, 3946ꢀ
3949.
(26) Terrier, V. P.; Delmas, A. F.; Aucagne, V. Efficient Synthesis of Cysteineꢀrich Cyclic Peptides
through Intramolecular Native Chemical Ligation of NꢀHnbꢀCys Peptide CryptoꢀThioesters. Org.
Biomol. Chem. 2017, 15, 316ꢀ319.
(
27) Ueda, S.; Fujita, M.; Tamamura, H.; Fujii, N.; Otaka, A. Photolabile Protection for Oneꢀpot
Sequential Native Chemical Ligation. Chembiochem 2005, 6, 1983ꢀ1986.
28) Aboye, T. L.; Li, Y.; Majumder, S.; Hao, J.; Shekhtman, A.; Camarero, J. A. Efficient Oneꢀpot
Cyclization/Folding of Rhesus ThetaꢀDefensinꢀ1 (RTDꢀ1). Bioorg. Med. Chem. Lett. 2012, 22, 2823ꢀ
826.
29) Zheng, J.ꢀS.; Tang, S.; Guo, Y.; Chang, H.ꢀN.; Liu, L. Synthesis of Cyclic Peptides and Cyclic
(
2
(
Proteins via Ligation of Peptide Hydrazides. ChemBioChem 2012, 13, 542ꢀ546.
(30) Chen, Y.ꢀQ.; Chen, C.ꢀC.; He, Y.; Yu, M.; Xu, L.; Tian, C.ꢀl.; Guo, Q.ꢀX.; Shi, J.; Zhang, M.; Li,
Y.ꢀM. Efficient Synthesis of Trypsin Inhibitor SFTIꢀ1 via Intramolecular Ligation of Peptide
Hydrazide. Tetrahedron Lett. 2014, 55, 2883ꢀ2886.
(
31) Jin, K.; Li, T.; Chow H. Y.; Liu, H.; Li, X. P−B Desulfurization: An Enabling Method for Protein
Chemical Synthesis and SiteꢀSpecific Deuteration. Angew. Chem. Int. Ed. 2017, 56, 14607ꢀ14611.
32) Wang, J.ꢀX.; Fang, G.ꢀM.; He, Y.; Qu, D.ꢀL.; Yu, M.; Hong, Z.ꢀY.; Liu, L. Peptide oꢀ
Aminoanilides as CryptoꢀThioesters for Protein Chemical Synthesis. Angew. Chem. Int. Ed. 2015, 54,
194ꢀ2198.
33) Gunasekera, S.; Aboye, T. L.; Madian, W. A.; ElꢀSeedi, H. R.; Göransson, U. Making Ends Meet:
(
2
(
MicrowaveꢀAccelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification
and Native Chemical Ligation. Int. J. Pept. Res. Ther. 2013, 19, 43ꢀ54.
(34) BlancoꢀCanosa, J. B.; Nardone, B.; Albericio, F.; Dawson, P. E. Chemical Protein Synthesis Using
a SecondꢀGeneration NꢀAcylurea Linker for the Preparation of PeptideꢀThioester Precursors. J. Am.
Chem. Soc. 2015, 137, 7197ꢀ7209.
(35) Thell, K.; Hellinger, R.; Sahin, E.; Michenthaler, P.; GoldꢀBinder, M.; Haider, T.; Kuttke, M.;
Liutkeviciute, Z.; Göransson, U.; Gründemann, C.; Schabbauer, G.; Gruber, C. W. Oral Activity of a
NatureꢀDerived Cyclic Peptide for the Treatment of Multiple Sclerosis. Proc. Natl. Acad. Sci. U. S. A.
2
016, 113, 3960ꢀ3965.
36) Chen, H.; Xiao, Y.; Yuan, N.; Weng, J.; Gao, P.; Breindel, L.; Shekhtman, A.; Zhang, Q. Coupling
of Sterically Demanding Peptides by βꢀThiolactoneꢀMediated Native Chemical Ligation Chem. Sci.
018, 9, 1982ꢀ1988.
37) Vaino, A. R.; Janda, K. D. SolidꢀPhase Organic Synthesis:ꢁ A Critical Understanding of the Resin.
(
2
(
J. Comb. Chem. 2000, 2, 579ꢀ596.
(38) Kates, S. A.; Solé, N. A.; Johnson, C. R.; Hudson, D.; Barany, G.; Albericio, F. A Novel,
Convenient, ThreeꢀDimensional Orthogonal Strategy for SolidꢀPhase Synthesis of Cyclic Peptides.
Tetrahedron Lett. 1993, 34, 1549ꢀ1552.
ACS Paragon Plus Environment

Page 23 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(39) TullaꢀPuche, J.; Barany, G. OnꢀResin Native Chemical Ligation for Cyclic Peptide Synthesis. J.
Org. Chem. 2004, 69, 4101ꢀ4107.
(40) Camarero, J. A.; Cotton, G. J.; Adeva, A.; Muir, T. W. Chemical Ligation of Unprotected Peptides
Directly from a Solid Support. J. Pept. Res. 1998, 51, 303ꢀ316.
(
41) Sun, Y.; Lu, G.; Tam, J. P. A Thioester Ligation Approach to Amphipathic Bicyclic Peptide
Library. Org. Lett. 2001, 3, 1681ꢀ1684.
42) Rosenbaum, C.; Waldmann, H. Solid Phase Synthesis of Cyclic Peptides by Oxidative Cyclative
Cleavage of an Aryl Hydrazide Linker—Synthesis of Stylostatin 1. Tetrahedron Lett. 2001, 42, 5677ꢀ
680.
43) Bourne, G. T.; Golding, S. W.; McGeary, R. P.; Meutermans, W. D. F.; Jones, A.; Marshall, G. R.;
(
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
(
Alewood, P. F.; Smythe, M. L. The Development and Application of a Novel SafetyꢀCatch Linker for
BOCꢀBased Assembly of Libraries of Cyclic Peptides. J. Org. Chem. 2001, 66, 7706ꢀ7713.
(44) Ravn, J.; Bourne, G. T.; Smythe, M. L. A Safety Catch Linker for FmocꢀBased Assembly of
Constrained Cyclic Peptides. J. Pept. Sci. 2005, 11, 572ꢀ578.
(45) Ahsanullah; Rademann, J. Cyclative Cleavage through Dipolar Cycloaddition: PolymerꢀBound
Azidopeptidylphosphoranes Deliver Locked cisꢀTriazolylcyclopeptides as Privileged Protein Binders.
Angew. Chem. Int. Ed. 2010, 49, 5378ꢀ5382.
(
46) Kumarn, S.; Chimnoi, N.; Ruchirawat, S. Synthesis of Integerrimide A by an OnꢀResin Tandem
FmocꢀDeprotectionꢀMacrocyclisation Approach. Org. Biomol. Chem. 2013, 11, 7760ꢀ7767.
47) Selvaraj, A.; Chen, H.ꢀT.; YaꢀTing Huang, A.; Kao, C.ꢀL. Expedient OnꢀResin Modification of a
Peptide CꢀTerminus through a Benzotriazole Linker. Chem. Sci. 2018, 9, 345ꢀ349.
48) Ohara, T.; Kaneda, M.; Saito, T.; Fujii, N.; Ohno, H.; Oishi, S. HeadꢀtoꢀTail Macrocyclization of
CysteineꢀFree Peptides Using an oꢀAminoanilide Linker. Bioorg. Med. Chem. Lett. 2018, 28, 1283ꢀ
286.
49) Acosta, G. A.; Royo, M.; de la Torre, B. G.; Albericio, F. Facile SolidꢀPhase Synthesis of Headꢀ
(
(
1
(
side Chain Cyclothiodepsipeptides through a Cyclative Cleavage from MeDbzꢀResin. Tetrahedron Lett.
2017, 58, 2788ꢀ2791.
(50) Gless, B. H.; Peng, P.; Pedersen, K. D.; Gotfredsen, C. H.; Ingmer, H.; Olsen, C. A. Structure–
Activity Relationship Study Based on Autoinducing Peptide (AIP) from Dog Pathogen S. schleiferi.
Org. Lett. 2017, 19, 5276ꢀ5279.
(
51) Arbour, C. A.; Saraha, H. Y.; McMillan, T. F.; Stockdill, J. L. Exploiting the MeDbz Linker To
Generate Protected or Unprotected CꢀTerminally Modified Peptides. Chem. Eur. J. 2017, 23, 12484ꢀ
2488.
52) Arbour, C. A.; Kondasinghe, T. D.; Saraha, H. Y.; Vorlicek, Teanna L.; Stockdill, J. L.
1
(
EpimerizationꢀFree Access to CꢀTerminal Cysteine Peptide Acids, Carboxamides, Secondary Amides,
and Esters via Complimentary Strategies. Chem. Sci. 2018, 9, 350ꢀ355.
(53) Arbour, C. A.; Stamatin, R. E.; Stockdill, J. L. Sequence Diversification by Divergent CꢀTerminal
Elongation of Peptides. J. Org. Chem. 2018, 83, 1797ꢀ1803.
(54) Abdel Monaim, S. A. H.; Acosta, G. A.; Royo, M.; ElꢀFaham, A.; de la Torre, B. G.; Albericio, F.
SolidꢀPhase Synthesis of Homodetic Cyclic Peptides from FmocꢀMeDbzꢀResin. Tetrahedron Lett.
2
(
018, 59, 1779ꢀ1782.
55) Punna, S.; Kuzelka, J.; Wang, Q.; Finn, M. G. HeadꢀtoꢀTail Peptide Cyclodimerization by Copperꢀ
Catalyzed Azide–Alkyne Cycloaddition. Angew. Chem. Int. Ed. 2005, 44, 2215ꢀ2220.
56) Jbara, M.; Maity, S. K.; Seenaiah, M.; Brik, A. Palladium Mediated Rapid Deprotection of Nꢀ
(
Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of
Synthetically Challenging Proteins. J. Am. Chem. Soc. 2016, 138, 5069ꢀ5075.
(
1
(
57) Patchornik, A.; Amit, B.; Woodward, R. B. Photosensitive Protecting Groups. J. Am. Chem. Soc.
970, 92, 6333ꢀ6335.
58) Schmalisch, J.; Seitz, O. Acceleration of Thiol AdditiveꢀFree Native Chemical Ligation by
Intramolecular S → S Acyl Transfer. Chem. Commun. 2015, 51, 7554ꢀ7557.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 25
1
2
3
4
5
6
7
8
9
1
1
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1
1
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4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
(59) Tsuda, S.; Mochizuki, M.; Nishio, H.; Yoshiya, T. Combination of ThiolꢀAdditiveꢀFree Native
Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine.
ChemBioChem 2016, 17, 2133ꢀ2136.
(
60) Wang, Y.; Han, L.; Yuan, N.; Wang, H.; Li, H.; Liu, J.; Chen, H.; Zhang, Q.; Dong, S. Traceless
βꢀMercaptanꢀAssisted Activation of Valinyl Benzimidazolinones in Peptide Ligations Chem. Sci. 2018,
, 1940ꢀ1946.
61) Sakamoto, K.; Tsuda, S.; Nishio, H.; Yoshiya, T. 1,2,4ꢀTriazoleꢀAided Native Chemical Ligation
between PeptideꢀNꢀAcylꢀN'ꢀMethylꢀBenzimidazolinone and Cysteinyl Peptide. Chem. Commun. 2017,
3, 12236ꢀ12239.
62) Wan, Q.; Danishefsky, S. J. FreeꢀRadicalꢀBased, Specific Desulfurization of Cysteine: a Powerful
Advance in the Synthesis of Polypeptides and Glycopolypeptides. Angew. Chem. Int. Ed. 2007, 46,
248ꢀ9252.
9
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
(
9
(63) Haase, C.; Rohde, H.; Seitz, O. Native Chemical Ligation at Valine. Angew. Chem. Int. Ed. 2008,
47, 6807ꢀ6810.
(64) White, C. J.; Hickey, J. L.; Scully, C. C. G.; Yudin, A. K. SiteꢀSpecific Integration of Amino Acid
Fragments into Cyclic Peptides. J. Am. Chem. Soc. 2014, 136, 3728ꢀ3731.
(65) Malins, L. R.; deGruyter, J. N.; Robbins, K. J.; Scola, P. M.; Eastgate, M. D.; Ghadiri, M. R.;
Baran, P. S. Peptide Macrocyclization Inspired by NonꢀRibosomal Imine Natural Products. J. Am.
Chem. Soc. 2017, 139, 5233ꢀ5241.
(66) Song, Y.; Li, Q.; Liu, X.; Chen, Y.; Zhang, Y.; Sun, A.; Zhang, W.; Zhang, J.; Ju, J. Cyclic
Hexapeptides from the Deep South China SeaꢀDerived Streptomyces scopuliridis SCSIO ZJ46 Active
Against Pathogenic GramꢀPositive Bacteria. J. Nat. Prod. 2014, 77, 1937ꢀ1941.
(67) Zhao, Y.ꢀR.; Wang, X.ꢀK.; Zhou, J.; Cheng, C.ꢀX.; Huang, X.ꢀL.; Wu, H.ꢀM. Stellarins F and G,
Two New Cyclopeptides from Stellaria yunnanensis. Chin. J. Chem. 1995, 13, 552ꢀ557.
(68) Pettit, G. R.; Srirangam, J. K.; Herald, D. L.; Erickson, K. L.; Doubek, D. L.; Schmidt, J. M.;
Tackett, L. P.; Bakus, G. J. Isolation and Structure of Stylostatin 1 from the Papua New Guinea Marine
Sponge Stylotella aurantium. J. Org. Chem. 1992, 57, 7217ꢀ7220.
(69) Milanowski, D. J.; Rashid, M. A.; Gustafson, K. R.; O'Keefe, B. R.; Nawrocki, J. P.; Pannell, L.
K.; Boyd, M. R. Cyclonellin, a New Cyclic Octapeptide from the Marine Sponge Axinella carteri. J.
Nat. Prod. 2004, 67, 441ꢀ444.
(
70) QuintyneꢀWalcott, S.; Maxwell, A. R.; Reynolds, W. F. Crotogossamide, a Cyclic Nonapeptide
from the Latex of Croton gossypifolius. J. Nat. Prod. 2007, 70, 1374ꢀ1376.
71) Roskoski Jr., R.; Kleinkauf, H.; Gevers, W.; Lipmann, F. Isolation of EnzymeꢀBound Peptide
Intermediates in Tyrocidine Biosynthesis. Biochemistry 1970, 9, 4846ꢀ4851.
72) Shute, R. E.; Kawai, M.; Rich, D. H. Conformationally Constrained Biologically Active Peptides:
(
(
Tentative Identification of the Antimitogenic Bioactive Confomer of the Naturally Occurring Cyclic
Tetrapeptides. Tetrahedron 1988, 44, 685ꢀ695.
(73) Singh, S. B.; Zink, D. L.; Liesch, J. M.; Mosley, R. T.; Dombrowski, A. W.; Bills, G. F.; Darkinꢀ
Rattray, S. J.; Schmatz, D. M.; Goetz, M. A. Structure and Chemistry of Apicidins, a Class of Novel
Cyclic Tetrapeptides without a Terminal αꢀKeto Epoxide as Inhibitors of Histone Deacetylase with
Potent Antiprotozoal Activities. J. Org. Chem. 2002, 67, 815ꢀ825.
(
74) Glenn, M. P.; Kelso, M. J.; Tyndall, J. D. A.; Fairlie, D. P. Conformationally Homogeneous
Cyclic Tetrapeptides:ꢁ Useful New ThreeꢀDimensional Scaffolds. J. Am. Chem. Soc. 2003, 125, 640ꢀ
41.
75) Kitir, B.; Maolanon, A. R.; Ohm, R. G.; Colaço, A. R.; Fristrup, P.; Madsen, A. S.; Olsen, C. A.
6
(
Chemical Editing of Macrocyclic Natural Products and Kinetic Profiling Reveal Slow, TightꢀBinding
Histone Deacetylase Inhibitors with Picomolar Affinities. Biochemistry 2017, 56, 5134ꢀ5146.
(76) Luckett, S.; Garcia, R. S.; Barker, J. J.; Konarev, A. V.; Shewry, P. R.; Clarke, A. R.; Brady, R. L.
HighꢀResolution Structure of a Potent, Cyclic Proteinase Inhibitor from Sunflower Seeds. J. Mol. Biol.
1999, 290, 525ꢀ533.
ACS Paragon Plus Environment

Page 25 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
(77) Tsuda, S.; Yoshiya, T.; Mochizuki, M.; Nishiuchi, Y. Synthesis of CysteineꢀRich Peptides by
Native Chemical Ligation without Use of Exogenous Thiols. Org. Lett. 2015, 17, 1806ꢀ1809.
(78) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on Solid Phase:ꢁ [1,2,3]ꢀTriazoles by
Regiospecific Copper(I)ꢀCatalyzed 1,3ꢀDipolar Cycloadditions of Terminal Alkynes to Azides. J. Org.
Chem. 2002, 67, 3057ꢀ3064.
(79) Rostovtsev V. V.; Green L. G.; Fokin V. V.; Sharpless, K. B. A Stepwise Huisgen Cycloaddition
Process: Copper(I)ꢀCatalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew.
Chem. Int. Ed. 2002, 41, 2596.
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
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2
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2
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1
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3
4
5
6
7
8
9
0
(80) Herner, A.; Marjanovic, J.; Lewandowski, T. M.; Marin, V.; Patterson, M.; Miesbauer, L.; Ready,
D.; Williams, J.; Vasudevan, A.; Lin, Q. 2ꢀArylꢀ5ꢀcarboxytetrazole as a New Photoaffinity Label for
Drug Target Identification. J. Am. Chem. Soc. 2016, 138, 14609ꢀ14615.
(81) Hanna, R. J.; Allan, C.; Lawrence, C.; Meyer, O.; Wilson, D. N.; Hulme, N. A. Optimizing the
Readout of LanthanideꢀDOTA Complexes for the Detection of LigandꢀBound Copper(I). Molecules
2017, 22, 802.
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