Applications of Convertible Isonitriles in the Ligation and Macrocyclization of Multicomponent Reaction-Derived Peptides and Depsipeptides

Article abstract of DOI:10.1021/acs.joc.6b01150

Peptide ligation and macrocyclization are among the most relevant approaches in the field of peptide chemistry. Whereas a variety of strategies relying on coupling reagents and native chemical ligation are available, there is a continuous need for efficient peptide ligation and cyclization methods. Herein we report on the utilization of convertible isonitriles as effective synthetic tools for the ligation and macrocyclization of peptides arising from isocyanide-based multicomponent reactions. The strategy relies on the use of convertible isonitriles - derived from Fukuyama amines - and peptide carboxylic acids in Ugi and Passerini reactions to afford N-alkylated peptides and depsipeptides, respectively, followed by conversion of the C-terminal amide onto either N-peptidoacyl indoles or pyrroles. Such activated peptides proved efficient in the ligation to peptidic, lipidic and fluorescently labeled amines and in macrocyclization protocols. As a result, a wide set of N-substituted peptides (with methyl, glycosyl and amino acids as N-substituents), cyclic N-methylated peptides and a depsipeptide were produced in good yields using conditions that involve either classical heating or microwave irradiation. This report improves the repertoire of peptide covalent modification methods by exploiting the synthetic potential of multicomponent reactions and convertible isonitriles.

Full text of DOI:10.1021/acs.joc.6b01150

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Article
Applications of Convertible Isonitriles in the Ligation and Macrocyclization
of Multicomponent Reaction-Derived Peptides and Depsipeptides
Ludger A. Wessjohann, Micjel C Morejón, Gerardo Manuel Ojeda
Carralero, Cristiano R. Bohn-Rhoden, and Daniel G. Rivera
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01150 • Publication Date (Web): 08 Jul 2016
Downloaded from http://pubs.acs.org on July 11, 2016
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Applications of Convertible Isonitriles in the Ligation and Macrocyclization of
Multicomponent Reaction-Derived Peptides and Depsipeptides
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Ludger A. Wessjohann†,* Micjel C. Morejón,† Gerardo M. Ojeda,‡ Cristiano R. B. Rhoden,†,§ and Daniel
G. Rivera,†,*
†Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120,
Halle/Saale, Germany. Tel: +49 345 5582 1301.
‡Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, 10400,
La Habana, Cuba. Tel: +53 78792331.
Corresponding Authors: *Eꢀmail: wessjohann@ipbꢀhalle.de; Eꢀmail: dgr@fq.uh.cu
§Present Address: (C. R. B. Rhoden) Centro Universitário Franciscano, UNIFRA, Santa Maria, Brasil.
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Graphical Abstract
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Abstract
Peptide ligation and macrocyclization are among the most relevant approaches in the field of peptide
chemistry. Whereas a variety of strategies relying on coupling reagents and native chemical ligation are
available, there is a continuous need for efficient peptide ligation and cyclization methods. Herein we
report on the utilization of convertible isonitriles as effective synthetic tools for the ligation and
macrocyclization of peptides arising from isocyanideꢀbased multicomponent reactions. The strategy
relies on the use of convertible isonitriles – derived from Fukuyama amines – and peptide carboxylic
acids in Ugi and Passerini reactions to afford Nꢀalkylated peptides and depsipeptides, respectively,
followed by conversion of the Cꢀterminal amide onto either Nꢀpeptidoacyl indoles or pyrroles. Such
activated peptides proved efficient in the ligation to peptidic, lipidic and fluorescently labeled amines
and in macrocyclization protocols. As a result, a wide set of Nꢀsubstituted peptides (with methyl,
glycosyl and amino acids as Nꢀsubstituents), cyclic Nꢀmethylated peptides and a depsipeptide were
produced in good yields using conditions that involve either classical heating or microwave irradiation.
This report improves the repertoire of peptide covalent modification methods by exploiting the synthetic
potential of multicomponent reactions and convertible isonitriles.
Keywords
Peptide ligation, Nꢀmethyl peptides, multicomponent reactions, convertible isonitriles, fluorescent
labeling, macrocyclization
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Introduction
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Isonitrileꢀbased multicomponent reactions (IꢀMCRs) have proven to be powerful tools for the synthesis
and derivatization of peptides and peptidomimetics.1 Among the IꢀMCRs, the Ugi fourꢀcomponent
reaction2 (Ugiꢀ4CR) has the greatest applicability on this field,1 not only because it utilizes amino and
carboxylic groups but also due to its high chemical efficiency and diversityꢀgenerating character. A
common application of the Ugiꢀ4CR has been the ligation of – at least – two amino acids or peptide
fragments to assemble Nꢀsubstituted oligopeptidic skeletons. This Ugiꢀligation strategy has enabled the
preparation of naturally occurring peptides3 as well as synthetic ones of medicinal,4 catalytic5 and
biomimetic6 importance. Another emerging application of this type of IꢀMCR is its utilization in the
synthesis of cyclic peptides7 and peptidomimetics,8 by means of approaches using the multicomponent
process for assembling the acyclic scaffold, for the ring closure, or both.9
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In peptide and medicinal chemistry, a strategy comprising an Ugiꢀ4CR followed by either an efficient
macrolactamization or peptide ligation can be a useful tool for the construction of Nꢀfunctionalized
(e.g., methyl, dyeꢀlabeled) (cyclo)peptides. However, a drawback limiting the implementation of this
strategy is the poor reactivity in acylation processes of the Cꢀterminal secondary amide. A solution for
this can be found in the development of convertible isonitriles,10,11 reagents that upon participation in
the Ugiꢀ4CR – and eventual activation – generate reactive amides suitable for followꢀup derivatization.
The utilization of these reagents has enabled effective applications of IꢀMCRs in the synthesis of
naturally occurring compounds and analogues.3b,12
The derivatizations reported so far for convertible isonitrileꢀderived amides include hydrolysis,
methanolysis, intramolecular acylations to five and sixꢀmembered lactones and lactams, as well as
acylation of aliphatic amines.10,11,12 However, to our knowledge, applications in crucial approaches of
peptide chemistry such as ligation of two peptide fragments and macrolactamization have remained
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elusive so far. Herein we report on the utilization of convertible isonitriles for the derivatization of
peptides by IꢀMCRs and their subsequent activation to enable either ligation to a second peptide or
macrolactamization under dilution conditions. Of course, such a ligation is not limited to inter and
intramolecular couplings, but can be extended to attaching lipids, labels and glycosidic moieties. To
develop this strategy, we focused on the utilization of the structurally related and versatile convertible
isocyanides 1 and 2 (scheme 1A). 1ꢀIsocyanoꢀ2ꢀ(2,2ꢀdimethoxyethyl)benzene (1) was developed
independently by the groups of Wessjohann11b and Kobayashi11c in 2007, while 4ꢀ
isocyanopermethylbutaneꢀ1,1,3ꢀtriol (IPB, 2) was reported by Wessjohann and coꢀworkers in 2012.11a
Both isonitriles show excellent reactivity in IꢀMCRs and are available in multigram scale from amines
previously introduced by Fukuyama et. al.13
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Scheme 1. Strategy for the utilization of convertible isonitriles 1 and 2 in peptide ligation and
macrocyclization.
Results and Discussion
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As depicted in scheme 1, the approach devised to exploit the potential of convertible isonitriles on
peptide chemistry comprises their utilization in the Ugiꢀ4CR by reaction with a peptidic carboxylic acid,
a primary amine and an oxoꢀcompound to produce a larger peptide incorporating an Nꢀalkylated amino
acid, in this case preferentially Gly by the use of paraformaldehyde. The internal Nꢀalkylation of
peptides, and especially Nꢀmethylation,14 has proven to be a successful way to improve pharmacological
properties such as metabolic stability, membrane permeability and pharmacokinetics, as compared with
their nonꢀNꢀalkylated congeners.14 As mentioned before, the reactivity of the Cꢀterminal secondary
amides derived from other convertible isocyanides has enabled their conversion to terminal carboxylic
acids and esters, but not their utilization either in the direct ligation to another peptide fragment or in
macrolactamization processes. On the other hand, the activation mode of terminal amides derived from
isonitriles 1 and 2 comprises the conversion – upon mild acidic treatment – to Nꢀacyl indoles and
pyrroles,13 respectively. Both types of activated acyl groups are known to react readily with primary and
secondary amines,11a,b,13 thus paving the way for the development of both peptide ligation and
intramolecular acylation of a peptidic amine, i.e., Nꢀterminus or side chain.
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As shown in scheme 1B, our strategy encompasses the implementation of the Ugiꢀ4CR with Bocꢀ
protected peptides as the carboxylic acid component and either isonitrile 1 or 2, thus enabling the
simultaneous activation of the Cꢀterminus – by formation of either Nꢀpeptidoacyl indole or pyrrole –
and the deprotection of the Nꢀterminal residue upon mild acidic treatment (also known as UDAC, Ugiꢀ
DeprotectionꢀActivationꢀCyclization/Condensation). Consequently, headꢀtoꢀtail macrocyclization can
be straightforward by setting up dilution conditions typically required to cyclize oligopeptides, while
ligation to another peptide fragment ideally requires previous capping of the Nꢀterminus, e.g., by
acetylation or a Nꢀterminal protection not cleaved upon activation. Thus, the Fmoc protecting group
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may be installed at the Nꢀterminus, while Lys side chains could be orthogonally protected with e.g.,
Cbz, if concomitant deprotection is not desired during acidꢀmediated Cꢀterminal activation.
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To prove the scope of this strategy, we decided to implement a ligation process wherein peptides having
various carboxylic groups could be selectively ligated by the one taking part in the Ugiꢀ4CR with a
convertible isonitrile and being subsequently activated. Peptide synthesis was carried out either in
solution using the Boc tactic or by a stepwise solidꢀphase Fmoc strategy on the AmꢀMBHA resin.15
Oligopeptides used as substrates of the IꢀMCRs and of the ligation processes are either known
compounds reported by our group or were prepared as described in the Supporting Information.
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Scheme 2. Ligation of Ugiꢀmodified peptides via Nꢀpeptidoacyl indoles and pyrroles.
As depicted in scheme 2, we initially carry out a comparison of the ligation efficiency of an Nꢀacyl
indole and Nꢀacyl pyrrole having identical peptide sequences and reacting them with the same
nucleophilic aminopeptide. For this, tripeptide 3 was reacted in parallel with glycine tꢀbutyl ester,
paraformaldehyde and isonitriles 1 and 2 to furnish the branched Nꢀsubstituted peptides 4 and 5,
respectively, in excellent yield after 24 h. Both intermediates were subjected to Cꢀterminal activation
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and simultaneous Boc and tꢀbutyl ester removal by treatment with 20% TFA in CH2Cl2, followed by
acetylation of the N-terminus to render Nꢀpeptidoacyl indole 6 and pyrrole 7, which were used without
further purification. A variety of protocols were studied to assess the best conditions to ligate the Cꢀ
activated peptides to the model tripeptide PheꢀLeuꢀAlaꢀOMe. Tested conditions include stirring a
solution of both peptides in either DMF or THF and either at room temperature, 50 °C or 70 °C during
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several hours. The best conversion was found with DMF as solvent at 70 °C for 24 h. A solution to
shorten the ligation time was the use of microwave irradiation at 70 °C for 30 min, which proved
effective in the ligation of the two peptide fragments similar to the traditional heating for 24 h. Several
parallel experiments demonstrated that peptides functionalized at the Cꢀterminus as Nꢀacyl indole are
more activated than those having the Nꢀacyl pyrrole. This was consistent with the synthesis of peptide 8,
which was obtained as isolated pure product in 72% yield from 6 and only in 51% from 7. HPLC
monitoring of routes A and B shown in scheme 2 confirmed higher conversion into peptide 8 in the
ligation process based on intermediate 6 than that involving 7 (see the Supporting Information).
In an endeavour to expand the scope of the peptide ligation, we turned to ligate peptides having
unprotected Glu and Asp side chains. Peptide 9 was reacted with isonitrile 1, methylamine and
paraformaldehyde in the Ugiꢀ4CR to produce Nꢀmethylated peptide 10 in excellent yield. The Cꢀ
terminal activation/deprotection procedure followed by Nꢀterminal capping rendered Nꢀpeptidoacyl
indole 11 bearing an unprotected Glu side chain and activated Cꢀterminus. As before, the MWꢀassisted
ligation process proved success in the conjugation to tetrapeptide LeuꢀAlaꢀPheꢀIleꢀNH2 yielding the Nꢀ
methylated octapeptide 12 with 74% of conversion after 30 min, as indicated by analytical RPꢀHPLC
analysis. As shown in scheme 2, a third example of ligation was implemented with an Nꢀpeptidoacyl
pyrrole as intermediate. For this, tripeptide 13 was combined with isonitrile 2 to render the Nꢀ
methylated peptide 14, which was subjected to deprotection of the Asp side chain by saponification
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followed by Cꢀterminal activation upon acidic treatment. Final ligation to tetrapeptide PheꢀValꢀGluꢀAlaꢀ
OH was also undertaken under MW irradiation, albeit it required longer reaction time to achieve peptide
16 with a 67% conversion. With the last two examples, we demonstrated the success of the ligation of
unprotected Nꢀpeptidoacyl indoles and pyrroles to aminopeptides bearing Cꢀterminal methyl esters,
carboxamides and free carboxylic acid groups. Finally, since peptide 13 was initially protected at the Nꢀ
terminus with an acid stable benzoyl group, Nꢀterminal capping between the Ugiꢀ4CR and the Cꢀ
terminal activation can be avoided, saving this extra step altogether.
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Scheme 3. Ligation strategy to glycosylated, lipidated and fluorescently tagged peptides.
Next we turned to assess the scope of the ligation process to produce glycoꢀ, lipoꢀ and fluorescently
labelled peptides. As depicted in scheme 3, an initial strategy relied on employing glycosyl amine 18 –
derived from the biologically relevant Nꢀacetyl glucosamine – in the Ugiꢀ4CR with peptide 17 and
isonitrile 1 to afford Nꢀglycosylated peptide 19 in good yield. After acidic treatment to enable the Cꢀ
terminal activation and Boc removal, followed by Nꢀterminal acetylation to yield Nꢀacyl indole 20, this
latter intermediate was ligated to tetrapeptide HꢀPheꢀLys(Cbz)ꢀGlyꢀPheꢀOMe to afford the remarkably
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complex Nꢀglycosylated nonapeptide 21. HPLC monitoring of this reaction showed a moderate
conversion of 56%, while a substantial amount of intermediate 20 remained unreacted after 45 min
under MW irradiation. However, a longer reaction time was not considered, as decomposition of both
compounds 20 and 21 becomes competitive after 1h.
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Thus far, ligation at the Cꢀtermini of peptides was the focus, while such a type of derivatization is also
possible at the side chains of peptides endowed with Asp and Glu. Scheme 3 illustrates the Ugiꢀ4CRs of
hexapeptide 22 – having an unprotected Asp side chain and protected N and Cꢀtermini – with isonitrile
2 and the methylamine/paraformaldehyde combination to afford the side chainꢀfunctionalized peptide
23 in excellent yield. Classic activation by acidic treatment rendered Nꢀpeptidoacyl pyrrole 24 in
quantitative yield, which was subsequently used in the ligation protocol without further purification.
Thus, 24 was reacted with the 7ꢀnitrobenzꢀ2ꢀoxaꢀ1,3ꢀdiazolꢀ4ꢀyl (NBD)ꢀderived fluorescent amine 25[16]
and nꢀdocecylamine to furnish the sideꢀchain fluorescentlyꢀtagged peptide 26 and the lipidated version
27 in 59% and 67% yield, respectively, after column chromatography. It is worthꢀmentioning that first
attempts to produce sideꢀchain Nꢀglycosides by reaction of activated peptide 24 with a glycosyl amine
were thwarted by poor conversion and significant decomposition upon conjugation conditions with
either MW or traditional heating.
After demonstrating the scope of convertible isonitriles in Ugiꢀ4CR/activation/ligation protocols, we
turned to demonstrate applications in the synthesis of both cyclic depsipeptides and Nꢀmethylated
peptides. Cyclic depsipeptides are natural products composed by amino acids (or bioisosters) and at
least one hydroxy acid, thus forming a lactone bond in the cyclopeptidic skeleton. Previously, IꢀMCRs
have been employed to produce cyclic depsipeptide mimics,17 though convertible isocyanides have not
been used for such purposes yet. As shown in scheme 4, our strategy to cyclic depsipeptides comprised
the implementation of the Passerini 3ꢀcomponent reaction (Passeriniꢀ3CR) between Nꢀprotected peptide
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28, isonitrile 1 and aqueous formaldehyde in chloroform under MW irradiation to furnish depsipeptide
29 in excellent yield. Treatment of 29 with TFA in CH2Cl2 enabled the simultaneous Nꢀterminal
deprotection and the Cꢀterminal activation (UDAC) by formation of the Nꢀacyl indole, thus paving the
way for the macrolactamization step. A variety of macrocyclization conditions were studied for this and
other Cꢀactivated peptides (not including heating by MW irradiation), resulting in the selection of a 5
mM concentration in DMF under basic conditions at 70 °C as the most suitable one. The reaction time
for such macrolactamizations was set to 48 hours, but in some cases longer reaction times might be
required for higher yields. Under these conditions, the cyclic depsipeptide 30 was obtained in 57% yield
of isolated pure product, while no byproducts derived from the feared cleavage of the lactone bond were
detected by HPLC and ESIꢀMS analysis of the crude product.
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Scheme 4. Macrocyclization strategy to a cyclic depsipeptide and Nꢀmethylated peptides.
A similar approach was utilized for the synthesis of Nꢀmethylated peptides endowed with different
sequences and macrocyclic ring sizes, but relying on the Ugiꢀ4CR for the installation of the Nꢀ
methylated amide bond. Initially, Nꢀprotected tetrapeptide 31 was reacted with isonitrile 1, methylamine
and paraformaldehyde in the Ugiꢀ4CR to produce Nꢀmethylated peptide 32 in excellent yield. The Cꢀ
terminal activation/deprotection procedure rendered the corresponding Nꢀpeptidoacyl indole, which was
subjected to the macrocyclization protocol to furnish cyclic pentapeptide 33 in good yield over two
steps. The same UDAC sequence was employed for the synthesis of Nꢀmethylated cyclic hexapeptide
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36 and heptapeptide 39 in 69% and 63% yield, respectively, over three steps. As shown in scheme 4, the
experience gained in the previous protocols led us to implement the synthesis of 36 in 39 without
chromatographic purification of any intermediate, which was possible mainly due the high reactivity of
isonitrile 1 in combination with methylamine, paraformaldehyde and peptide carboxylic acids, enabling
the Ugiꢀ4CRs to proceed quantitatively in 24 h.
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Besides of the headꢀtoꢀtail cyclization, the method should also enable the side chainꢀtoꢀside chain
cyclization of peptides. For this, peptide 40, having both termini capped and an unprotected Glu side
chain, was submitted to the Ugiꢀ4CR with methylamine, paraformaldehyde and isonitrile 2 to produce
the side chainꢀfunctionalized peptide 41 in excellent yield. As compound 41 is protected at the Nꢀ
terminus with Boc, camphor sulphonic acid (CSA, 0.1 equiv) and quinoline (0.1 equiv) in toluene were
chosen for the Cꢀterminal activation, conditions upon which the Boc group remained unaffected.[13a]
Subsequently, the Cbz group of the Lys side chain was orthogonally cleaved by hydrogenation, and the
resulting intermediate was cyclized under previously described conditions to furnish the side chain
crossꢀlinked tetrapeptide 42 in 58% yield over three steps. With this final example, we have proven that
convertible isonitriles 1 and 2 are suitable reagents for the incorporation of either Nꢀalkylated or
depsipeptide moieties by means of Ugiꢀ4CR and Passeriniꢀ3CR, respectively, while enabling the mild
activation of either the peptide Cꢀterminus or side chain for ligation and macrocyclization purposes.
Conclusions
We have demonstrated the feasibility of using convertible isonitriles – derived from Fukuyama amines –
as synthetic means to enable peptide ligation and macrocyclization. The strategy comprises their
utilization in IꢀMCRs such as the Ugiꢀ4CR and the Passeriniꢀ3CR for the assembly of Nꢀalkylated
peptides and depsipeptides, respectively, followed by either side chain or Cꢀterminal activation by
acidic treatment to afford Nꢀpeptidoacyl indoles or pyrroles. The latter intermediates proved to be
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properly activated to enable either the ligation to nucleophilic aminopeptides or macrolactamization
under diluted conditions. Both ligation and macrocyclization protocols required either microwave
irradiation or heating at 70 °C to proceed n reasonable time, thus providing a variety of Nꢀsubstituted
(cyclo)peptides and a cyclic depsipetide. These results provide further evidence of the potential of
convertible isonitriles and IꢀMCRs as powerful synthetic tools in peptide chemistry.
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Experimental Section
Convertible isonitriles 1 and 2 were synthesized according to references 11a and 11b. Peptides 3, 9, 17,
28, 31, 34, and 37 are known compounds and were produced according to references 6b and 18. Peptide
synthesis grade DMF, CH2Cl2, iPr2EtN, TFA, and HPLCꢀgrade acetonitrile were used. HPLC analysis
was performed in a reverseꢀphase (RP) C18 column (4.6× 150 mm, 5µm). A linear gradient from 5% to
60% of solvent B in solvent A over 35 min at a flow rate of 0.8 mL/min was used (solvent A: 0.1% (v/v)
TFA in water, solvent B: 0.05% (v/v) TFA in acetonitrile). Detection was accomplished at 226 or 254
nm. Flash column chromatography was performed on silica gel 60 (>230 mesh) and analytical thin layer
chromatography (TLC) was performed using silica gel aluminum sheets. For peptides that were not
purified to >95% by column chromatography on silica, purity was assessed by RPꢀHPLC and
characterization of an analytical sample was made by electrospray ionization mass spectrometry (ESIꢀ
MS). The high resolution ESI mass spectra were obtained either from a 70e Fourier transform ion
cyclotron resonance (FTꢀICR) mass spectrometer equipped with an Infinity cell, a 7.0 Tesla
superconducting magnet or from an Orbitrap Elite mass spectrometer equipped with an HESI
electrospray ion source. Reactions involving microwave irradiation were performed in a Robotic
1
Microwave Synthesizer (Biotage Emrys Personal Chemistry Optimizer Microwave Synthesizer). H
13
NMR and C NMR spectra were recorded on a 400 spectrometer at 399.94 MHz and 100.57 MHz,
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respectively. Chemical shifts (δ) are reported in ppm relative to TMS (1H NMR) and to the solvent
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signal (13C NMR).
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General procedure for the Ugi-4CR: A solution of the amine (0.6 mmol, 1.2 equiv.) and
paraformaldehyde (0.6 mmol, 1.2 equiv.) in MeOH/CH2Cl2 (5 mL, 5:1, v/v) is stirred for 1 h at room
temperature. NEt3 (0.6 mmol) is added when amine hydrochlorides were employed as amino
components. The peptide carboxylic acid (0.5 mmol, 1 equiv.) and the convertible isocyanide (0.5
mmol, 1 equiv.) are then added and the reaction mixture is stirred at room temperature for 24 h. The
volatiles are then concentrated under reduced pressure and the resulting crude product is dissolved in 50
mL of CHCl3. The organic phase is washed sequentially with an aqueous saturated solution of citric
acid (30 mL), aqueous 10% NaHCO3 (30 mL), and brine (30 mL), and then dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The crude product is purified by flash
column chromatography (CH2Cl2/MeOH) on silica to afford the corresponding Nꢀalkylated peptide.
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General procedure for the simultaneous Boc/tBu removal and C-terminal activation by
conversion of Ugi-4CR-derived amides into N-peptidoacyl indoles and pyrroles: The Ugiꢀ4CRꢀ
derived peptide (0.5 mmol) is dissolved in CH2Cl2 (5 mL) and treated with trifluoroacetic acid (1 mL) at
0 °C. The reaction mixture is allowed to reach room temperature and stirred for 2 h, then concentrated
under reduced pressure. TFA is removed completely by repetitive addition and evaporation of CH2Cl2 to
furnish the TFA salt of the Cꢀactivated peptide, which is used without further purification.
General acetylation procedure of the free N-terminal peptide: The peptide (0.5 mmol) is dissolved
in 5 mL of CH2Cl2 and treated with iPr2EtN (0.52 mL, 3 mmol) and Ac2O (0.28 ꢀL, 3 mmol). The
reaction mixture is stirred at room temperature for 2 h and then the volatiles are evaporated under
reduced pressure. The product is dissolved in EtOAc (25 mL) and washed vigorously with aq. 10% HCl
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(2×15 mL) and brine (15 mL). The organic phase is dried over anhydrous Na2SO4 and concentrated
under reduced pressure to afford the Nꢀacetyl peptide.
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General peptide ligation procedure under MW irradiation: The peptidyl or alkyl amine (0.25 mmol,
1 equiv.) and the Nꢀpeptidoacyl indole or pyrrole (0.25 mmol, 1 equiv.) are dissolved in 5 mL of DMF
in a 10 mL glass tube. iPr2EtN (0.17 mL, 1 mmol, 4 equiv.) is added and the glass tube is sealed and
introduced in the microwave reactor. The flask is irradiated for 30 min (150 W) under highꢀspeed
magnetic stirring at 70 °C, while the reaction course is monitored by TLC. Additional cycles of 15 min
are applied in cases of poor consumption of the starting material. The volatiles are removed under
reduced pressure and the reaction product is washed several times with frozen diethyl ether, then taken
up in ca. 20ꢀ30 mL of MeOH and filtered through a pad of silica gel C18 to partially remove the indole
or pyrrole derivatives. The resulting solution is concentrated to dryness and the crude product is purified
either by flash column chromatography or analyzed by RPꢀHPLC and ESIꢀMS. In the latter case, the
crude peptide is taken up in 2:1 acetonitrile/water and lyophilized prior to HPLC analysis and
purification.
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Peptide 4: HCl⋅GlyꢀOtBu (100 mg, 0.6 mmol), Et3N (83ꢀL, 0.6 mmol), paraformaldehyde (18 mg, 0.6
mmol), peptide 3 (225 mg, 0.5 mmol) and isonitrile 1 (95.5 mg, 0.5 mmol) were reacted in
MeOH/CH2Cl2 (5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure. Flash column
chromatography purification (CH2Cl2/MeOH 12:1) afforded peptide 4 (360 mg, 92%) as a white
amorphous solid. 1H NMR (400 MHz, CDCl3): δ = 8.88 (s, 1H); 7.33 (m, 1H); 7.27 (m, 1H); 7.26ꢀ7.05
(m, 9H); 6.97 (m, 1H); 5.22 (m, 1H); 4.62 (t, 1H, J=5.7 Hz); 4.57 (m, 1H) 4.54ꢀ4.50 (m, 2H); 4.38ꢀ4.31
(m, 2H); 4.17ꢀ4.06 (m, 2H); 3.38 (s, 3H); 3.37 (s, 3H); 3.06 (dd, 1H, J=13.8/5.2 Hz); 3.01 (m, 1H); 2.91
(m, 2H); 1.80ꢀ1.76 (m, 1H); 1.67ꢀ1.61 (m, 2H); 1.40 (s, 9H); 1.38 (s, 9H); 1.36 (d, 3H, J=7.0 Hz); 0.89
13
(d, 3H, J=6.4 Hz), 0.87 (d, 6H, J=6.4 Hz). C NMR (100 MHz, CDCl3): δ = 17.7, 22.6, 22.9, 25.1,
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28.2, 28.4, 38.3, 40.8, 41.1, 47.7, 49.9, 51.6, 52.6, 55.3, 55.5, 80.5, 81.4, 104.5, 122.3, 126.6, 126.8,
128.6, 129.1, 129.3, 130.5, 131.2, 136.3, 136.5, 155.9, 166.7, 167.3, 171.5, 171.6, 171.9, 173.0. HRMS
(ESIꢀFTꢀICR) m/z: 806.4322 [M+Na]+, calcd. for C41H61O10NaN5: 806.4316.
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Peptide 5: HCl⋅GluꢀOtBu (100 mg, 0.6 mmol), Et3N (83ꢀL, 0.6 mmol), paraformaldehyde (18 mg, 0.6
mmol), peptide 3 (225 mg, 0.5 mmol) and isonitrile 2 (87 mg, 0.5 mmol) were reacted in MeOH/CH2Cl2
(5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure. Flash column chromatography
1
purification (CH2Cl2/MeOH 10:1) afforded peptide 5 (364 mg, 95%) as a white amorphous solid. H
NMR (400 MHz, CDCl3): δ = 7.36 (d, 1H, J=9.2 Hz); 7.26ꢀ7.21 (m, 3H); 7.15 (m, 2H); 7.11 (m, 1H);
7.07 (m, 1H); 6.99 (m, 1H); 5.24 (m, 1H); 4.66 (m, 1H); 4.60ꢀ4.51 (m, 3H); 4.32ꢀ4.27 (m, 3H); 4.25
(dd, 1H, J=17.2/4.5 Hz); 4.12 (dd, 1H, J=17.1/4.6 Hz); 3.53 (m, 1H); 3.38 (s, 3H); 3.32 (s, 6H); 3.27
(m, 1H); 3.06 (dd, 1H, J=13.8/5.2 Hz); 2.96 (dd, 1H, J=13.9/7.9 Hz); 1.81ꢀ1.72 (m, 3H); 1.66ꢀ1.60 (m,
2H); 1.41 (s, 9H); 1.30 (s, 9H); 1.35 (d, 3H, J=7.2 Hz); 0.88 (d, 3H, J=6.4 Hz), 0.87 (d, 6H, J=6.4 Hz).
13C NMR (100 MHz, CDCl3): δ = 17.9, 22.0, 22.3, 25.1, 28.2, 38.2, 40.7, 42.6, 41.3, 47.6, 49.1, 49.9,
51.4, 55.3, 55.4, 56.2, 76.2, 80.3, 81.0, 102.5, 126.9, 128.8, 129.2, 136.3, 155.9, 166.2, 170.0, 171.6,
171.9, 172.6, 173.3. HRMS (ESIꢀFTꢀICR) m/z: 788.4427 [M+Na]+, calcd. for
C38H63O11NaN5:788.4422.
Peptide 8: Peptide 4 (235 mg, 0.3 mmol) was subjected to the general procedure for the simultaneous
Boc/tBu removal and Cꢀterminal activation, followed by the general acetylation procedure to yield
quantitatively the Nꢀpeptidoacyl indole 6, which was used forward without previous purification. Cꢀ
activated peptide 6 (151 mg, 0.25 mmol) was reacted with the hydrochloride salt of tripeptide HꢀPheꢀ
LeuꢀAlaꢀOMe (100 mg, 0.25 mmol) for 30 minutes in the presence of iPr2EtN according to the general
ligation procedure. Flash column chromatography purification (CH2Cl2/MeOH 10:1) afforded peptide 8
(153 mg, 72%) as a white amorphous solid. In parallel, peptide 5 was subjected to the general procedure
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for the simultaneous Boc/tBu removal and Cꢀterminal activation, followed by acetylation to give 7,
which was equally submitted to the ligation procedures to furnish 8 (108 mg, 51%) after column
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chromatography. Mixture of two conformers. H NMR (400 MHz, CD3OD): δ = 7.27ꢀ7.16 (m, 10H);
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5.13, 4.97 (2×m, 1H); 4.67ꢀ4.48 (m, 3H); 4.30ꢀ4.24 (m, 2H); 4.19 (d, J=18.8 Hz); 4.17 (d, J=17.2 Hz);
3.93 (d, J=17.2 Hz); 3.91 (d, J=17.2 Hz); 3.79 (d, J=17.6 Hz); 3.74 (s, 3H); 3.19ꢀ3.09 (m, 2H); 2.95ꢀ
2.85 (m, 2H); 1.90 (s, 3H); 1.75ꢀ1.42 (m, 6H); 1.36 (d, 3H, J=6.8 Hz); 1.31 (d, 3H, J=6.6 Hz); 0.99 (d,
3H, J=6.6 Hz); 0.94 (d, 3H, J=6.2 Hz); 0.90 (d, 6H, J=6.4 Hz); 0.86 (d, 3H, J=6.4 Hz); 0.85 (d, 3H,
J=6.4 Hz). 13C NMR (100 MHz, CD3OD): δ = 17.7, 17.8, 22.0, 22.4, 22.8, 22.9, 23.0, 23.2, 23.3, 23.4,
25.6, 5.7, 25.8, 38.5, 39.0, 39.7, 41.6, 41.8, 42.2, 46.1, 46.2, 46.8, 47.0, 50.6, 52.7, 53.0, 53.4, 53.5,
55.1, 55.3, 56.0, 56.5, 59.3, 127.6, 127.8, 129.4, 129.5, 130.2, 130.4, 130.6, 138.2, 138.4, 171.0, 171.4,
172.0, 172.1, 172.6, 172.9, 173.1, 173.4, 173.5, 173.8, 174.4, 174.7, 174.8, 175.4. HRMS (ESIꢀFTꢀICR)
m/z: 874.4331 [M+Na]+, calcd. for C43H61O11NaN7: 874.4327.
Peptide 10: HCl⋅MeNH2 (40 mg, 0.6 mmol), Et3N (83ꢀL, 0.6 mmol), paraformaldehyde (18 mg, 0.6
mmol), peptide 9 (334 mg, 0.5 mmol) and isonitrile 1 (95.5 mg, 0.5 mmol) were reacted in
MeOH/CH2Cl2 (5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure. Flash column
chromatography purification (CH2Cl2/MeOH 15:1) afforded peptide 10 (378 mg, 91%) as a pale yellow
amorphous solid. 1H NMR (400 MHz, CDCl3): δ = 9.07 (s, 1H); 7.30ꢀ7.07 (m, 14H); 6.79 (d, 1H, J=8.6
Hz); 6.76 (d, 1H, J=8.8 Hz); 5.73 (m, 1H); 5.07 (m, 1H); 4.94 (m, 1H); 4.69 (t, 1H, J=5.8 Hz); 4.42 (m,
1H); 4.37 (d, 1H, J=15.8 Hz); 4.08 (d, 1H, J=15.8 Hz); 3.38 (s, 3H); 3.37 (s, 3H); 3.24 (s, 3H); 3.21ꢀ
3.19 (m, 2H); 3.03 (m, 1H); 3.01 (m, 1H); 2.90 (m, 2H); 2.38 (m, 2H); 2.19 (m, 2H); 1.41 (s, 9H); 1.39
(s, 9H). 13C NMR (100 MHz, CDCl3): δ = 26.9, 28.2, 28.4, 30.6, 35.3, 38.3, 38.5, 40.8, 51.6, 53.2, 53.6,
55.2, 55.9, 80.8, 81.3, 104.5, 122.3, 126.6, 127.7, 126.8, 127.0, 128.5, 128.6, 129.0, 129.1, 129.3, 130.8,
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131.3, 132.8, 136.1, 136.3, 136.4, 157.9, 167.0, 170.7, 171.9, 172.1, 173.0. HRMS (ESIꢀFTꢀICR) m/z:
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854.4321 [M+Na]+, calcd. for C45H61O10NaN5:854.4316.
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Peptide 12: Peptide 10 (249 mg, 0.3 mmol) was subjected to the general procedure for the simultaneous
Boc/tBu removal and Cꢀterminal activation, followed by the acetylation procedure to yield
quantitatively the Nꢀpeptidoacyl indole 11, which was used forward without previous purification. Cꢀ
activated peptide 11 (163 mg, 0.25 mmol) was reacted with the trifluoroacetate salt of tetrapeptide Hꢀ
LeuꢀAlaꢀPheꢀIleꢀNH2 (144 mg, 0.25 mmol) for 30 minutes in the presence of iPr2EtN according to the
general ligation procedure to afford peptide 12 (239 mg) as a pale yellow amorphous solid. RPꢀHPLC
analysis of the crude product showed 74% of conversion. An analytical sample was purified by RPꢀ
HPLC to >95% purity for ESIꢀMS characterization. Rt = 20.7 min. HRMS (ESIꢀFTꢀICR) m/z: 996.5190
[MꢀH]ꢀ, calcd. for C52H70O11N9: 996.5200.
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General peptide coupling procedure. The Bocꢀprotected
L
ꢀaminoacid (1.0 mmol, 1.0 equiv.), HOBt
ꢀaminoacid methyl ester
(168 mg, 1.1 mmol, 1.1 equiv.), EDC (210 mg, 1.1 mmol, 1.1 equiv.) and the
L
hydrochloride are suspended in dry CH2Cl2 (15 mL). Et3N (0.15 mL, 1.1 mmol, 1.1 equiv.) is syringed
in one portion and the resulting solution is stirred at room temperature overnight (~12 h). The reaction
mixture is then diluted with 100 mL EtOAc, transferred to a separatory funnel and sequentially washed
with 0.5 M aqueous solution of citric acid (2×50 mL) and saturated aqueous suspension NaHCO3 (2×50
mL). The organic phase is dried over MgSO4, filtered and concentrated under reduced pressure.
General Boc removal procedure: The peptide is dissolved in a 4 M HCl solution in dioxane (2 mL)
and the solution is stirred at room temperature. As the material dissolved, gas evolution could be
detected and the pressure that built up inside the reaction flask is regularly relieved by opening the
reaction flask. After 30 min, usually no starting material is detected by thin layer chromatography and
the reaction is concentrated under a stream of dry N2. The volatiles are then fully removed by
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concentrating the resulting thick oily residue under reduced pressure in the rotary evaporator and then
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placing the flask under high vacuum for 2 h. The resulting salt was used forward assuming quantitative
yield.
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Peptide 13: NꢀBocꢀValꢀOH (217 mg, 1.0 mmol) was coupled to HCl·LeuꢀOBzl (257 mg, 1.0 mmol)
according to the peptide coupling procedure, following by deprotection of the Nꢀterminus by Boc
removal. The same protocol was employed for the coupling of NꢀAsp(Me)ꢀOH (247 mg, 1.0 mmol).
Flash column chromatography purification (CH2Cl2/MeOH 15:1) furnished peptide BocꢀAsp(OMe)ꢀ
ValꢀLeuꢀOBzl (433 mg, 79%) as a white amorphous solid. This latter tripeptide (412 mg, 0.75 mmol)
was subjected to deprotection of the Nꢀterminus by the Boc removal procedure. The resulting rude
peptide is dissolved in 10 mL of CH2Cl2 and treated with iPr2EtN (0.52 mL, 3 mmol) and BzCl (0.28
ꢀL, 3 mmol). The reaction mixture was stirred at room temperature for 8 h and then the volatiles
evaporated under reduced pressure. The crude product was dissolved in EtOAc (25 mL) and washed
with aq. 10% HCl (2×10 mL) and brine (2×10 mL). The organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure to afford the Nꢀbenzoyl peptide. The resulting product
was dissolved in MeOH (30 mL) and 10% Pd/C (80 mg) was added. The mixture was subjected
successively to hydrogen atmosphere and vacuum and finally stirred under hydrogen atmosphere for 24
h. The catalyst was removed by filtration over a pad of Celite and the filtrate was evaporated under
reduced pressure. Flash column chromatography purification (CH2Cl2/MeOH 15:1) furnished peptide
1
13 (268 mg, 77%) as a white amorphous solid. H NMR (400 MHz, CDCl3): δ = 7.95 (d, 1H, J = 8.1
Hz), 7.82 (m, 2H), 7.56 (m, 1H), 7.53 – 7.47 (m, 1H), 7.41 (m, 2H), 7.17 (d, 1H, J = 8.1 Hz), 5.09 (m,
1H), 4.61 – 4.50 (m, 1H), 4.31 (m, 1H), 3.66 (s, 3H), 2.94 (d, 2H, J = 6.1 Hz), 2.24 – 2.11 (m, 1H), 1.66
(m, 3H), 0.90 (m, 12H). 13C NMR (100 MHz, CDCl3): δ = 17.9, 19.2, 21.8, 23.0, 25.0, 30.5, 35.6, 40.8,
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50.2, 51.2, 52.3, 59.3, 125.6, 127.5, 128.8, 132.2, 133.3, 167.8, 171.3, 171.6, 172.5, 175.6. HRMS (ESIꢀ
FTꢀICR) m/z: 462.2245 [MꢀH]ꢀ, calcd. for C23H32O7N3: 462.2246.
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Peptide 14: HCl⋅MeNH2 (40 mg, 0.6 mmol), Et3N (83ꢀL, 0.6 mmol), paraformaldehyde (18 mg, 0.6
mmol), peptide 13 (232 mg, 0.5 mmol) and isonitrile 2 (87 mg, 0.5 mmol) were reacted in
MeOH/CH2Cl2 (5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure. Flash column
chromatography purification (CH2Cl2/MeOH 18:1) afforded peptide 14 (292 mg, 86%) as a white
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amorphous solid. H NMR (400 MHz, CD3OD): δ = 7.83 (m, 2H); 7.58ꢀ7.51 (m, 1H); 7.46 (m, 2H);
5.04 (t, 1H, J=7.1 Hz); 4.87 (m, 1H); 4.52 (m, 1H); 4.28 (m, 1H); 4.11ꢀ3.97 (m, 2H), 3.69 (s, 3H); 3.45ꢀ
3.39 (m, 1H); 3.37 (s, 3H); 3.34ꢀ3.30 (m, 6H); 3.27ꢀ3.14 (m, 3H); 3.02 (m, 1H); 2.92 (s, 1H); 2.86 (dd,
1H, J=16.6/7.7 Hz); 2.11 (m, 1H); 1.79ꢀ1.62 (m, 4H); 1.55 (m, 1H); 1.00ꢀ0.86 (m, 12H). 13C NMR (100
MHz, CD3OD): δ = 18.3, 19.8, 22.0, 23.7, 25.8, 32.1, 36.3, 36.7, 37.3, 41.3, 42.7, 51.7, 52.3, 52.5, 53.5,
53.7, 57.6, 57.7, 59.7, 77.6, 103.5, 128.5, 129.6, 133.0, 135.0, 170.2, 170.8, 172.8, 173.1, 173.3, 174.7.
HRMS (ESI) m/z: 678.3715 [MꢀH]ꢀ, calcd. for C33H52O10N5: 678.3720.
Peptide 16: Peptide 14 (204 mg, 0.3 mmol) was dissolved in THF/H2O (2:1, 15 mL) and LiOH (50 mg,
1.2 mmol) is added at 0 °C. The mixture was stirred at 0 °C for 3 h and then acidified with aqueous 10%
NaHSO4 to pH 3. The resulting phases were separated and the aqueous phase was additionally extracted
with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The resulting crude was subjected to the general procedure for the Cꢀterminal
activation to yield quantitatively the Nꢀpeptidoacyl pyrrole 15, which was used forward without
previous purification. Cꢀactivated peptide 15 (146 mg, 0.25 mmol) was reacted with the trifluoroacetate
salt of tetrapeptide HꢀPheꢀValꢀGluꢀAlaꢀOH (145 mg, 0.25 mmol) for 45 minutes in the presence of
iPr2EtN according to the general ligation procedure to afford peptide 16 (212 mg) as a white amorphous
solid. RPꢀHPLC analysis of the crude product showed 67% purity. A sample was purified by RPꢀHPLC
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to >95% purity for characterization. Rt = 9.72 min. H NMR (600 MHz, CD3OD): δ = 7.89 – 7.80 (m,
2H), 7.52 (m, 1H), 7.48 – 7.40 (m, 2H), 7.30 – 7.14 (m, 5H), 5.00 (t, 1H, J = 7.3 Hz), 4.77 – 4.69 (m,
1H), 4.65 (m, 1H), 4.49 – 4.39 (m, 1H), 4.32 (m, 1H), 4.27 (m, 1H), 4.24 – 4.15 (m, 1H), 4.08 (m, 1H),
4.04 – 3.89 (m, 2H), 3.21 – 3.15 (m, 1H), 3.11 – 3.03 (m, 2H), 3.01 – 2.88 (m, 3H), 2.86 – 2.79 (m, 1H),
2.58 – 2.47 (m, 1H), 2.46 – 2.36 (m, 2H), 2.15 – 2.02 (m, 3H), 1.98 – 1.89 (m, 1H), 1.71 – 1.59 (m, 2H),
1.59 – 1.48 (m, 1H), 1.45 – 1.40 (m, 1H), 1.40 – 1.34 (m, 3H), 1.30 (dd, 1H, J = 14.1, 6.6 Hz), 1.02 –
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0.75 (m, 18H). C NMR (151 MHz, CD3OD): δ = 18.9, 19.8, 19.9, 22.1, 23.6, 25.8, 28.5, 30.2, 31.2,
36.3, 37.3, 38.7, 41.5, 52.1, 53.8, 56.1, 57.5, 57.6, 59.2, 60.2, 61.0, 127.7, 128.4, 128.5, 128.6, 129.3,
129.5, 129.6, 130.3, 130.4, 131.2, 134.1, 138.5, 166.7, 168.4, 171.4, 172.0, 172.2, 172.6, 173.2, 173.5,
174.5, 175.3, 180.4. HRMS (ESI) m/z: 965.4619 [MꢀH]ꢀ, calcd. for C47H65O14N8: 965.4626.
Peptide 19: Glucosyl amine 18 (208 mg, 0.6 mmol), paraformaldehyde (18 mg, 0.6 mmol), peptide 17
(257 mg, 0.5 mmol) and isonitrile 1 (95.5 mg, 0.5 mmol) were reacted in MeOH/CH2Cl2 (10 mL, 5:1,
v/v) for 36 h according to the general Ugiꢀ4CR procedure. Flash column chromatography purification
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(CH2Cl2/MeOH 12:1) afforded glycopeptide 19 (383 mg, 72%) as a pale yellow amorphous solid. H
NMR (400 MHz, CDCl3): δ = 9.12 (s, 1H); 7.62 (d, 1H, J=7.2 Hz); 7.48 (m, 1H); 7.24ꢀ7ꢀ08 (m, 4H);
5.62 (d, 1H, J=8.2 Hz); 5.50 (d, 1H, J=8.2 Hz); 5.39 (t, 1H, J=9.4 Hz); 5.00 (m, 1H); 4.87 (m, 1H);
4.75ꢀ4.64 (m, 3H); 4.58 (t, 1H, J=5.6 Hz); 4.44ꢀ4.40 (m, 1H); 4.36ꢀ4.30 (m, 3H); 4.14 (m, 1H); 4.06 (d,
1H, J=15.8 Hz); 3.87 (m, 1H); 3.39 (s, 3H); 3.37 (s, 3H); 2.90 (m, 2H); 2.01, 2.04, 2.05, 2.08 (4×s,
4×3H); 1.81 (m, 1H); 1.73ꢀ1.62 (m, 2H); 1.56ꢀ1.46 (m, 2H); 1.43 (s, 9H); 1.36 (d, 3H, J=7.0 Hz); 0.96
13
(d, 3H, J=6.0 Hz); 0.93ꢀ0.86 (m, 12H). C NMR (100 MHz, CDCl3): δ = 15.9, 17.4, 18.9, 20.7, 21.3,
21.4, 22.2, 22.7, 22.8, 24.6, 24.9, 28.3, 28.4, 30.5, 37.5, 40.0, 41.2, 43.5, 48.6, 51.4, 51.9, 52.5, 53.6,
57.2, 59.3, 62.8, 68.6, 69.7, 73.2, 79.7, 84.4, 104.5, 122.3, 126.9, 129.3, 130.5, 131.1, 136.1, 158.3,
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1710.2, 171.4, 171.7, 171.9, 172.3, 172.5, 172.8. HRMS (ESIꢀFTꢀICR) m/z: 1086.5590 [M+Na]+, calcd.
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for C51H81O17NaN7: 1086.5587.
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Peptide 21: Peptide 19 (320 mg, 0.3 mmol) was subjected to the general procedure for the simultaneous
Boc/tBu removal and Cꢀterminal activation, followed by the acetylation procedure to yield
quantitatively the Nꢀpeptidoacyl indole 20, which was used forward without previous purification. Cꢀ
activated peptide 20 (235 mg, 0.25 mmol) was reacted with the hydrochloride salt of tetrapeptide Hꢀ
PheꢀLys(Cbz)ꢀGlyꢀPheꢀOMe (170 mg, 0.25 mmol) for 30 minutes in the presence of iPr2EtN according
to the general ligation procedure to afford peptide 21 (257 mg) as a pale yellow amorphous solid. RPꢀ
HPLC analysis of the crude product showed 56% purity. An analytical sample was purified by RPꢀ
HPLC to >95% purity for ESIꢀMS characterization. Rt = 17.3 min. HRMS (ESIꢀFTꢀICR) m/z:
1492.7234 [M+Na]+, calcd. for C73H103O21N11Na: 1492.7228.
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Bz-Ala-Leu-Phe-Val-Glu-Ala-OMe (22): NꢀBocꢀGlu(OBzl)ꢀOH (337 mg, 1.0 mmol) was coupled to
HCl·AlaꢀOMe (139 mg, 1.0 mmol) according to the peptide coupling procedure, following by
deprotection of the Nꢀterminus by Boc removal. The same protocol was employed for the sequential
coupling of NꢀBocꢀValꢀOH (217 mg, 1.0 mmol) and NꢀBocꢀPheꢀOH (265 mg, 1.0 mmol). Flash column
chromatography purification (CH2Cl2/MeOH 15:1) furnished peptide BocꢀPheꢀValꢀGlu(OBzl)ꢀAlaꢀ
OMe (411 mg, 72%) as a white amorphous solid. This latter tetrapeptide (400 mg, 0.6 mmol) was
subjected to deprotection of the Nꢀterminus by the Boc removal procedure, followed by sequential
coupling of NꢀBocꢀLeuꢀOH (139 mg, 0.6 mmol) and NꢀBocꢀAlaꢀOH (113 mg, 0.6 mmol). Flash column
chromatography purification (CH2Cl2/MeOH 12:1) furnished hexapeptide BocꢀAlaꢀLeuꢀPheꢀValꢀ
Glu(OBzl)ꢀAlaꢀOMe (414 mg). This latter peptide was subjected to Nꢀterminal deprotection by the Boc
removal procedure, followed by benzoylation of the Nꢀterminus and removal of the benzyl protecting
group of the Glu side chain according to the procedures described for peptide 13. Flash column
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white amorphous solid. H NMR (400 MHz, DMSOꢀd6): δ = 8.54 – 8.40 (m, 2H), 8.35 – 8.21 (m, 1H),
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8.08 – 7.99 (m, 1H), 7.98 – 7.90 (m, 1H), 7.90 – 7.82 (m, 2H), 7.52 (m, 1H), 7.45 (m, 2H), 7.26 – 7.07
(m, 5H), 6.39 (m, 2H), 4.61 – 4.51 (m, 1H), 4.45 (m, 1H), 4.36 – 4.29 (m, 1H), 4.25 (m, 2H), 4.15 (m,
1H), 3.96 – 3.84 (m, 2H), 3.63 – 3.56 (m, 3H), 3.45 (s, 3H), 3.26 (s, 1H), 3.19 (m, 2H), 3.04 (dd, 1H, J
= 14.0, 4.0 Hz), 2.95 (m, 2H), 2.83 – 2.74 (m, 2H), 2.45 – 2.40 (m, 2H), 2.39 – 2.32 (m, 1H), 2.01 –
1.91 (m, 1H), 1.84 (m, 4H), 1.66 (p, 1H, J = 5.4 Hz), 1.55 (m, 3H), 1.37 (t, 1H, J = 7.3 Hz), 1.27 (m,
6H), 1.21 – 1.13 (m, 1H), 0.76 (m, 9H), 0.71 (dd, 3H, J = 6.5, 3.6 Hz). 13C NMR (100 MHz, DMSOꢀd6)
δ = 16.7, 17.6, 18.1, 19.1, 21.5, 21.7, 22.9, 23.0, 24.7, 28.4, 29.2, 36.1, 36.2, 36.5, 37.1, 41.2, 44.9,
47.51, 48.5, 48.9, 51.8, 52.8, 53.3, 57.8, 99.2, 102.0, 126.1, 127.4, 127.5, 127.9, 128.2, 129.1, 129.3,
131.3, 134.0, 137.6, 137.7, 137.9, 144.6, 145.2, 166.0, 166.2, 168.4, 171.1, 171.0, 172.1, 172.2, 172.9.
HRMS (ESIꢀFTꢀICR) m/z: 765.3823 [MꢀH]ꢀ, calcd. for C39H53O10N6: 765.3829.
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Peptide 23: HCl⋅MeNH2 (40 mg, 0.6 mmol), Et3N (83ꢀL, 0.6 mmol), paraformaldehyde (18 mg, 0.6
mmol), peptide 22 (383 mg, 0.5 mmol) and isonitrile 2 (87 mg, 0.5 mmol) were reacted in
MeOH/CH2Cl2 (8 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure. Flash column
chromatography purification (CH2Cl2/MeOH 15:1) afforded peptide 23 (452 mg, 92%) as a white
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amorphous solid. H NMR (400 MHz, CD3OD): δ = 7.88 (d, 2H, J=7.5 Hz); 7.59ꢀ7.50 (m, 1H); 7.46
(m, 2H); 7.24ꢀ7.10 (m, 5H); 6.92 (s, 1H); 4.68 (dd, 1H, J=9.9/4.8 Hz); 4.55ꢀ4.46 (m, 2H); 4.45ꢀ4.35 (m,
2H); 4.34ꢀ4.26 (m, 1H); 4.20 (dd, 1H, J=11.7/7.6 Hz); 4.16ꢀ4.06 (m, 1H); 4.02 (m, 1H); 3.70 (s, 3H);
3.39 (m, 1H); 3.38ꢀ3.34 (m, 3H); 3.31 (m, 9H); 3.20 (dd, 1H, J = 13.4/4.7 Hz); 3.05 (m, 2H); 3.02ꢀ2.94
(m, 1H); 2.93ꢀ2.89 (m, 1H); 2.58 (m, 1H); 2.48 (m, 1H); 2.15ꢀ2.07 (m, 2H); 2.01ꢀ1.92 (m, 1H); 1.72 (m,
2H); 1.60 (m, 1H); 1.45 (m, 4H); 1.40 (m, 6H); 0.98ꢀ0.79 (m, 12H). 13C NMR (101 MHz, CD3OD): δ =
17.3, 17.7, 19.8, 22.1, 23.4, 25.8, 28.7, 28.0, 30.9, 31.9, 35.4, 36.8, 37.4, 38.4, 42.8, 51.7, 52.2, 52.7,
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53.1, 53.6, 53.7, 55.9, 56.5, 57.5, 57.7, 60.4, 77.7, 103.6, 126.1, 127.7, 128.6, 129.4, 129.5, 130.2,
130.3, 132.9, 135.0, 138.4, 138.6, 139.2, 170.1, 170.4, 171.3, 173.3, 173.7, 174.5, 175.2, 175.3, 175.5.
HRMS (ESI) m/z: 981.5287 [MꢀH]ꢀ, calcd. for C49H73O13N8: 981.5303.
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Peptide 26: Peptide 23 (392 mg, 0.4 mmol) was subjected to the general procedure for the Cꢀterminal
activation to yield quantitatively the Nꢀpeptidoacyl pyrrole 24, which was used forward without
previous purification. Cꢀactivated peptide 24 (133 mg, 0.15 mmol) was reacted with the NBDꢀderived
fluorescent amine 25 (48 mg, 0.20 mmol) for 30 minutes in the presence of iPr2EtN according to the
general ligation procedure. Flash column chromatography purification (CH2Cl2/MeOH 15:1) afforded
pure peptide 26 (94 mg, 59%) as a white amorphous solid. Rt = 11.7 min. 1H NMR (400 MHz, DMSOꢀ
d6): δ = 8.54ꢀ8.40 (m, 2H); 8.35ꢀ8.21 (m, 1H); 8.08ꢀ7.99 (m, 1H); 7.98ꢀ7.90 (m, 1H); 7.90ꢀ7.82 (m,
2H); 7.52 (m, 1H); 7.45 (m, 2H); 7.26ꢀ7.07 (m, 5H); 6.39 (m, 2H); 4.61ꢀ4.51 (m, 1H); 4.45 (m, 1H);
4.36ꢀ4.29 (m, 1H); 4.25 (m, 2H); 4.15 (m, 1H); 3.96ꢀ3.84 (m, 2H); 3.63ꢀ3.56 (m, 3H); 3.45 (s, 3H); 3.26
(s, 1H); 3.19 (m, 2H); 3.04 (dd, 1H, J=14.0/ 4.0 Hz); 2.95 (m, 2H); 2.83ꢀ2.74 (m, 2H); 2.45ꢀ2.40 (m,
2H); 2.39ꢀ2.32 (m, 1H); 2.01ꢀ1.91 (m, 1H); 1.84 (m, 4H); 1.66 (d, 1H, J=5.4 Hz); 1.55 (m, 3H); 1.37 (t,
13
1H, J=7.3 Hz); 1.27 (m, 6H); 1.21ꢀ1.13 (m, 1H); 0.76 (m, 9H); 0.71 (m, 3H). C NMR (100 MHz,
DMSOꢀd6): δ = 16.7, 17.6, 18.1, 19.1, 21.5, 21.7, 22.9, 23.0, 24.1, 28.4, 29.2, 36.1, 36.2, 36.5, 37.1,
41.2, 45.0, 47.5, 48.5, 48.9, 51.9, 52.8, 53.3, 57.8, 99.2, 102.0, 126.1, 127.4, 127.5, 127.9, 128.2, 129.1,
129.3, 131.3, 134.0, 137.6, 137.7, 137.90, 144.6, 145.2, 166.0, 166.2, 168.4, 171.0, 171.0, 172.1, 172.2,
172.9, 174.9. HRMS (ESI) m/z: 1055.4952 [MꢀH]ꢀ, calcd. for C51H67O13N12: 1055.4956.
Peptide 27: Nꢀpeptidoacyl pyrrole 24 (133 mg, 0.15 mmol), obtained as describe above, was reacted
with nꢀdodecylamine (100 mg, 0.20 mmol) for 30 minutes in the presence of iPr2EtN according to the
general ligation procedure. Flash column chromatography purification (CH2Cl2/MeOH 20:1) afforded
pure peptide 27 (102 mg, 67%) as a white amorphous solid. Rt = 16.0 min. H NMR (400 MHz, DMSOꢀ
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d6): δ = 8.07ꢀ7.94 (m, 1H); 7.93ꢀ7.84 (m, 2H); 7.56ꢀ7.50 (m, 1H); 7.49ꢀ7.40 (m, 2H); 7.29ꢀ7.09 (m,
5H); 4.57 (m, 1H); 4.47 (m, 1H); 4.31 (m, 1H); 4.25 (m, 2H); 4.20ꢀ4.08 (m, 1H); 3.93ꢀ3.80 (m, 2H);
3.63ꢀ3.56 (m, 3H); 3.35 (m, 6H); 3.07ꢀ2.99 (m, 3H); 2.95ꢀ2.90 (m, 2H); 2.79ꢀ2.72 (m, 2H); 2.42ꢀ2.32
(m, 1H); 2.07 (s, 1H); 2.03ꢀ1.92 (m, 1H); 1.88 (m, 1H); 1.78 (m, 1H); 1.60ꢀ1.49 (m, 1H); 1.41ꢀ1.33 (m,
2H); 1.30 (m, 2H); 1.28 (m, 2H); 1.27ꢀ1.25 (m, 2H); 1.25ꢀ1.20 (m, 18H); 0.87ꢀ0.69 (m, 15H). 13C NMR
(100 MHz, DMSOꢀd6): δ = 14.0, 16.8, 17.0, 17.6, 18.1, 18.2, 19.2, 21.6, 21.7, 22.1, 22.9, 23.0, 24.1,
26.4, 27.7, 28.7, 29.0, 30.4, 31.3, 34.0, 34.1, 36.1, 37.1, 38.4, 41.0, 47.5, 49.1, 50.2, 50.7, 51.1, 51.9,
53.7, 57.8, 126.1, 126.2, 127.4, 127.5, 127.9, 127.9, 128.2, 129.2, 129.3, 131.3, 134.0, 138.0, 166.0,
167.6, 167.9, 170.6, 171.0, 171.9, 172.1, 172.7, 173.0. HRMS (ESI) m/z: 1003.6232 [MꢀH]ꢀ, calcd. for
C54H83O10N9: 1003.6238.
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General macrocyclization procedure: The Cꢀactivated peptide (0.25 mmol, 1 equiv.) is dissolved in
DMF (50 mL) and treated with iPr2EtN (0.17 mL, 1 mmol, 4 equiv.). The reaction mixture is stirred for
48 h at 70 °C and then concentrated under reduced pressure. The reaction product is taken up in ca. 20ꢀ
30 mL of MeOH and filtered through a pad of silica gel C18 to partially remove the indole or pyrrole
derivatives and the resulting solution is concentrated to dryness. The resulting crude cyclic peptide is
purified by flash column chromatography (CH2Cl2/ MeOH).
Depsipeptide 29: Peptide 28 (222 mg, 0.3 mmol) was dissolved in 5 mL of THF a 10 mL glass tube and
treated with 0.1 mL of 40% aqueous formaldehyde and isonitrile 1 (57 mg, 0.3 mmol). The flask was
irradiated for 30 min (300 W) under highꢀspeed magnetic stirring 60 °C and then the volatiles were
evaporated under reduced pressure. The crude product was purified by flash column chromatography
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(CH2Cl2/MeOH 20:1) to furnish depsipeptide 29 (256 mg, 89%) as a white amorphous solid. H NMR
(400 MHz, CDCl3): δ = 9.18 (s, 1H); 7.74 (d, 1H, J=7.8 Hz); 7.29 (d, 1H, J=7.2 Hz); 7.34ꢀ7.18 (m,
10H); 7.25ꢀ7.20 (m, 2H); 7.14ꢀ7.08 (m, 2H); 6.84 (d, 1H, J=8.2 Hz); 5.60 (m, 1H); 5.15 (m, 1H); 5.08
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(s, 2H); 4.85 (d, 1H, J=15.4 Hz); 4.67 (d, 1H, J=15.4 Hz); 4.61 (m, 1H); 4.43 (m, 2H); 4.22 (m, 1H);
3.40 (s, 3H); 3.38 (s, 3H, CH3O); 3.24ꢀ3.14 (m, 2H); 3.04 (dd, 1H, J=14.0/5.2 Hz); 2.96ꢀ2.88 (m, 3H);
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2.14 (m, 1H); 1.96 (m, 2H); 1.85 (m, 1H); 1.56ꢀ1.44 (m, 4H); 1.37 (s, 9H); 0.93ꢀ0.81 (m, 12H). C
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NMR (100 MHz, CDCl3): δ = 11.3, 15.4, 17.4, 19.2, 23.4, 22.7, 24.6, 28.1, 29.2, 30.2, 30.9, 36.2, 36.7,
37.9, 40.4, 42.1, 52.3, 54.8, 55.3, 56.0, 58.0, 59.0, 63.6, 66.4, 80.7, 107.0, 124.8, 125.6, 127.0, 127.5,
127.9, 128.4, 128.7, 129.1, 131.0, 135.5, 135.9, 136.6, 155.9, 156.9 165.4, 170.4, 170.9, 171.5, 172.1.
HRMS (ESIꢀFTꢀICR) m/z: 983.5105 [M+Na]+, calcd. for C51H72O12NaN6: 983.5106.
Cyclic Depsipeptide 30: Depsipeptide 29 (240 mg, 0.25 mmol) was subjected to the procedure for the
simultaneous Boc/tBu removal and Cꢀterminal activation to yield quantitatively the intermediate Nꢀ
peptidoacyl indole, which was next submitted to the general macrocyclization procedure. Flash column
chromatography purification (CH2Cl2/MeOH 18:1) afforded the pure cyclic depsipeptide 30 (98 mg,
57%) as a white amorphous solid. 1H NMR (400 MHz, CDCl3): δ = 8.21 (d, 1H, J=7.8 Hz); 7.64 (d, 1H,
J=7.4 Hz); 7.59 (d, 1H, J=7.0 Hz); 7.51 (d, 1H, J=7.1 Hz); 7.35ꢀ7.31 (m, 5H); 5.11 (s, 2H); 4.92 (d, 1H,
J=15.8 Hz); 4.72 (d, 1H, J=15.7 Hz); 4.50 (m, 1H); 4.37 (m, 1H); 4.28 (m, 2H); 3.18ꢀ3.13 (m, 3H); 3.08
(dd, 1H, J=14.0/5.2 Hz); 2.09 (m, 1H); 1.86 (m, 2H); 1.72 (m, 1H); 1.52ꢀ1.49 (m, 4H); 0.93ꢀ0.84 (m,
13
12H). C NMR (100 MHz, CDCl3): δ = 11.1, 15.3, 17.6, 19.0, 24.2, 29.4, 30.3, 31.4, 36.5, 36.7, 39.6,
40.8, 53.4, 56.6, 58.7, 59.9, 63.4, 66.5, 126.9, 127.7, 127.9, 128.1, 128.5, 128.8, 129.1, 129.3, 136.3,
136.7, 157.8, 169.8, 170.9, 171.5, 172.2, 172.8. HRMS (ESIꢀFTꢀICR) m/z: 702.3477 [M+Na]+, calcd.
for C36H49O8NaN5: 702.3479.
Peptide 32: HCl⋅MeNH2 (20 mg, 0.3 mmol), Et3N (44ꢀL, 0.3 mmol), paraformaldehyde (9 mg, 0.3
mmol), peptide 31 (137 mg, 0.25 mmol) and isonitrile 1 (48 mg, 0.25 mmol) were reacted in
MeOH/CH2Cl2 (5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure. Flash column
chromatography purification (CH2Cl2/MeOH 15:1) afforded peptide 32 (180 mg, 92%) as a pale yellow
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amorphous solid. 1H NMR (400 MHz, CDCl3): δ = 8.91 (s, 1H); 7.31 (m, 1H); 7.27 (m, 1H); 7.26ꢀ7.07
(m, 9H); 6.96 (m, 1H); 5.78 (m, 1H); 5.24 (m, 1H); 4.63 (t, 1H, J=5.6 Hz); 4.59ꢀ4.52 (m, 4H); 4.36 (m,
1H, J=15.8 Hz); 4.08 (m, 1H, J=15.9 Hz); 3.39 (s, 3H); 3.37 (s, 3H); 3.25 (s, 3H); 3.08 (dd, 1H,
J=13.9/5.0 Hz); 3.08 (dd, 1H J=13.9/5.0 Hz); 2.96 (dd, 1H J=13.9/8.0 Hz); 2.90 (m, 2H); 1.81ꢀ1.74 (m,
1H); 1.66ꢀ1.60 (m, 1H); 1.55ꢀ1.42 (m, 2H); 1.38 (s, 9H); 1.37 (d, 3H, J=7.1 Hz); 0.96 (d, 3H, J=6.4
Hz); 0.94 (d, 3H, J=6.4 Hz); 0.87 (d, 6H, J=6.4 Hz). 13C NMR (100 MHz, CDCl3): δ = 17.9, 18.8, 19.0,
21.9, 22.1, 25.1, 28.2, 38.3, 40.4, 40.7, 41.3, 47.6, 51.4, 51.7, 51.9, 55.3, 55.7, 80.4, 104.5, 122.3, 126.6,
126.9, 128.6, 129.2, 129.3, 130.5, 131.2, 136.2, 136.6, 155.8, 166.7, 171.5, 170.8, 171.9, 172.9. HRMS
(ESIꢀFTꢀICR) m/z: 805.4472 [M+Na]+, calcd. for C41H62O9NaN6: 805.4476.
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32
33
34
35
36
37
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39
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Cyclic peptide 33: Peptide 32 (156 mg, 0.2 mmol) was subjected to the procedure for the simultaneous
Boc/tBu removal and Cꢀterminal activation to yield quantitatively the intermediate Nꢀpeptidoacyl
indole, which was next submitted to the general macrocyclization procedure. Flash column
chromatography purification (CH2Cl2/MeOH 16:1) afforded the pure cyclic peptide 33 (101 mg, 61%)
1
as a white amorphous solid. H NMR (400 MHz, CD3OD): δ = 7.28ꢀ7.21 (m, 5H); 4.84 (dd, 1H,
J=9.9/4.6 Hz); 4.55 (dd, 1H, J=11.0/3.8 Hz); 4.45 (q, 1H, J=6.9 Hz); 4.38 (d, 1H, J=14.1 Hz); 3.44 (d,
1H, J=10.1 Hz); 3.34 (m, 1H); 3.31 (m, 1H); 3.24 (d, 1H, J=14.1 Hz); 3.17 (s, 3H); 2.82 (dd, 1H,
J=11.0/14.1 Hz); 2.51ꢀ2.43 (m, 1H); 1.73ꢀ1.62 (m, 1H); 1.53ꢀ1.46 (m, 2H); 1.39 (d, 3H, J=6.9 Hz);
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1.01ꢀ0.95 (m, 12H). C NMR (100 MHz, CD3OD): δ = 17.0, 20.0, 21.7, 23.6, 26.2, 29.7, 37.8, 38.3,
41.9, 49.3, 51.0, 54.6, 56.6, 66.2, 127.8, 129.5, 129.9, 138.9, 172.1, 173.1, 174.5, 174.5, 175.2. HRMS
(ESIꢀFTꢀICR) m/z: 783.5354 [M+H]+, calcd. for C40H73O10N5: 783.5357.
Cyclic peptide 36: HCl⋅MeNH2 (20 mg, 0.3 mmol), Et3N (44ꢀL, 0.3 mmol), paraformaldehyde (9 mg,
0.3 mmol), peptide 34 (165 mg, 0.25 mmol) and isonitrile 1 (48 mg, 0.25 mmol) were reacted in
MeOH/CH2Cl2 (5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure to afford
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quantitatively peptide 35. This latter peptide was subjected without further purification to the procedure
for the simultaneous Boc removal and Cꢀterminal activation to yield quantitatively the intermediate Nꢀ
peptidoacyl indole, which was next submitted to the general macrocyclization procedure. Flash column
chromatography purification (CH2Cl2/MeOH 18:1) afforded the pure cyclic peptide 36 (106 mg, 69%)
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18
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36
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44
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as a white amorphous solid. H NMR (400 MHz, DMSOꢀd6): δ = 7.54 (d, 1H, J=7.2 Hz); 7.27 (br. m,
5H); 4.84ꢀ4.74 (m, 4H); 4.62ꢀ4.58 (m, 2H); 4.46 (m, 1H); 4.14 (m, 1H); 4.04 (d, 1H, J=5.7 Hz); 3.97 (d,
1H, J=7.2 Hz); 3.42 (d, 1H, J=15.2 Hz); 3.40 (dd, 1H, J=14.0/4.4 Hz); 3.18 (s, 3H); 3.13 (m, 1H); 2.27
(m, 1H); 1.85ꢀ1.79 (m, 2H); 1.63 (m, 4H); 1.48 (d, 3H, J=7.2); 1.36ꢀ1.28 (m, 4H); 1.01ꢀ0.89 (m, 12H);
0.74 (d, 3H, J=8.4 Hz); 0.72 (d, 3H, J=9.0 Hz). 13C NMR (100 MHz, CDCl3): δ = 11.0, 15.4, 15.9, 18.3,
19.6, 22.4, 22.9, 24.6, 25.0, 28.5, 29.7, 34.0, 36.3, 38.5, 40.7, 48.3, 54.6, 55.8, 55.9, 56.0, 56.1, 126.9,
128.6, 129.0, 137.0, 170.3, 172.0, 172.8, 173.0, 174.1, 174.3. HRMS (ESIꢀFTꢀICR) m/z: 637.3685
[M+Na]+, calcd. for C32H50O6NaN6: 637.3689.
Cyclic peptide 39: HCl⋅MeNH2 (20 mg, 0.3 mmol), Et3N (44ꢀL, 0.3 mmol), paraformaldehyde (9 mg,
0.3 mmol), peptide 37 (183 mg, 0.25 mmol) and isonitrile 1 (48 mg, 0.25 mmol) were reacted in
MeOH/CH2Cl2 (5 mL, 5:1, v/v) for 24 h according to the general Ugiꢀ4CR procedure to afford
quantitatively peptide 38. This latter peptide was subjected without further purification to the procedure
for the simultaneous Boc removal and Cꢀterminal activation to yield quantitatively the intermediate Nꢀ
peptidoacyl indole, which was next submitted to the general macrocyclization procedure. Flash column
chromatography purification (CH2Cl2/MeOH 16:1) afforded the pure cyclic peptide 39 (108 mg, 63%)
as a white amorphous solid. Mixture of the S-cis and S-trans isomers of the Nꢀmethylated amide bond.
1H NMR (400 MHz, CDCl3): δ = 8.81, 8.45 (2×d, 1H, J=8.8 Hz); 8.41, 8.34 (2×d, 1H, J=6.2 Hz); 7.71
(d, 1H, J=9.5 Hz); 7.67, 7.47 (2×d, 1H, J=5.4 Hz); 7.55, 7.41 (2×br. s, 1H); 7.27 (br. m, 5H); 6.73, 6.60
(2×d, 1H, J=7.8 Hz); 4.70 (td, 1H, J=10.2/4.5 Hz); 4.60 (m, 1H); 4.54ꢀ4.45 (m, 2H); 4.27ꢀ4.12 (m, 4H);
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3.30 (m, 1H); 3.29 (s, 3H, CH3N); 3.20 (dd, 1H, J=14.0/4.4 Hz); 3.03 (dd, 1H, J=14.1/10.0 Hz); 1.91
(m, 2H); 1.69 (m, 2H); 1.62 (m, 1H); 1.42 (d, 3H, J=7.3, CH3); 1.23 (d, 3H, J=7.0, CH3); 1.01ꢀ0.84 (m,
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18H, 6×CH3). C NMR (100 MHz, CDCl3): δ = 18.1, 18.9, 20.9, 21.4, 22.3, 22.9, 23.1, 23.4, 24.6,
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36
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24.8, 32.4, 33.9, 36.8, 40.2, 40.2, 49.1, 49.7, 51.4, 52.2, 52.6, 55.8, 57.7, 127.0, 128.5, 128.6, 128.7,
129.0, 137.0, 169.3, 171.3, 171.8, 172.3, 172.6, 173.0, 173.2. HRMS (ESIꢀFTꢀICR) m/z: 708.4063
[M+Na]+, calcd. for C35H55O7NaN7: 708.4060.
Boc-Glu-Leu-Phe-Lys(Cbz)-NHCH2CH2pF-C6H4 (40): NαꢀBocꢀLys(Cbz)ꢀOH (380 mg, 1.0 mmol)
was coupled to 2ꢀ(4ꢀfluorophenyl)ethanꢀ1ꢀamine (139 mg, 1.0 mmol) according to the peptide coupling
procedure, following by deprotection of the Nꢀterminus by Boc removal subsequent coupling of NꢀBocꢀ
PheꢀOH (265 mg, 1.0 mmol). Flash column chromatography purification (CH2Cl2/MeOH 18:1)
furnished peptide BocꢀPheꢀLys(Cbz)ꢀNHCH2CH2pFꢀC6H4 (544 mg, 84%) as a white amorphous solid.
This latter peptide (389 mg, 0.6 mmol) was subjected to deprotection of the Nꢀterminus by the Boc
removal procedure, followed by sequential coupling of NꢀBocꢀLeuꢀOH (139 mg, 0.6 mmol) and NꢀBocꢀ
Glu(OMe)ꢀOH (157 mg, 0.6 mmol). The resulting cure peptide was dissolved in THF/H2O (2:1, 10 mL)
and LiOH (105 mg, 2.5 mmol) was added at 0 °C. The mixture was stirred at 0 °C for 3 h and then
acidified with aquouse 10% NaHSO4 to pH 3. The resulting phases are separated and the aqueous phase
is additionally extracted with EtOAc (2×30 mL). The combined organic phases are dried over
anhydrous Na2SO4 and concentrated under reduced pressure to yield the crude deprotected peptide.
Flash column chromatography purification (CH2Cl2/MeOH 15:1) furnished the peptide 40 (368 mg,
69%) as a white amorphous solid. 1H NMR (400 MHz, CD3OD): δ = 7.32 (d, 4H, J = 4.4 Hz), 7.28 –
7.16 (m, 8H), 6.98 (t, 2H, J = 8.8 Hz), 5.05 (m, 2H), 4.58 (dt, 1H, J = 14.4/7.1 Hz), 4.24 (m, 2H), 4.11 –
3.99 (m, 1H), 3.36 (m, 2H), 3.11 (m, 3H), 2.97 (dd, 1H, J = 14.1/8.7 Hz), 2.75 (t, 2H, J = 7.3 Hz), 2.37
(t, 2H J = 7.5 Hz), 1.99 (m, 1H), 1.85 (m, 1H), 1.73 (m, 1H), 1.66 – 1.56 (m, 2H), 1.54 – 1.46 (m, 3H),
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