On the scope of the double Ugi multicomponent stapling to produce helical peptides

Article abstract of DOI:10.1016/j.bioorg.2021.104987

The stabilization of helical structures by peptide stapling approaches is now a mature technology capable to provide a variety of biomedical applications. Recently, it was shown that multicomponent macrocyclization is not only an effective way to introduc

Full text of DOI:10.1016/j.bioorg.2021.104987

Bioorganic Chemistry 113 (2021) 104987
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
On the scope of the double Ugi multicomponent stapling to produce
helical peptides
,
b
a
,b,*
a
Manuel G. Ricardoa , Yadiel Vazquez-Mena , Yuleidys Iglesias-Morales ,
´
´
Ludger A. Wessjohannb *, Daniel G. Riveraa
,
a Laboratory of Synthetic and Biomolecular Chemistry, Faculty of Chemistry, University of Havana, Zapata y G, Havana 10400, Cuba
b Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
The stabilization of helical structures by peptide stapling approaches is now a mature technology capable to
provide a variety of biomedical applications. Recently, it was shown that multicomponent macrocyclization is
not only an effective way to introduce conformational constraints but it also allows to incorporate additional
functionalities to the staple moiety in a one-pot process. This work investigates the scope of the double Ugi
multicomponent stapling approach in its capacity to produce helical peptides from unstructured sequences. For
this, three different stapling combinations were implemented and the CD spectra of the cyclic peptides were
measured to determine the effect of the multicomponent macrocyclization on the resulting secondary structure.
A new insight into some structural factors influencing the helicity type and content is provided, along with new
prospects on the utilization of this methodology to diversify the molecular tethers linking the amino acid side
chains.
Peptide secondary structures
Macrocyclization
Cyclic peptides
Multicomponent reactions
Peptide stapling
Conformation control
1. Introduction
A special class of side chain-to-side chain tethering is the two-
component peptide stapling [17,18], which comprises the utilization
Multicomponent reactions (MCRs) are currently considered powerful
peptide macrocyclization tools that introduce conformational con-
straints in short and medium-size peptide sequences and – at the same
time – allow the generation of skeletal diversity [1]. Examples of MCRs
used in peptide cyclization are the Strecker [2], the Passerini [3], the A3-
coupling [4] and the Petasis [5,6] three-component reactions, as well as
various variants of Ugi-type four-component reactions [7–11]. In most
cases, these macrocyclization processes consist of a single MCR that
connects the two reactive ends of the peptide backbone or side chains.
Conceptually, this type of multicomponent cyclization approach is
similar to the classic lactamization [12,13], ring-closing metathesis
[14,15] and other metal-catalyzed peptide cyclization processes [16],
but with the advantage of adding one or two additional components
during the ring closure if desired. Linking of two amino acid side chains
separated along the sequence is referred to as peptide stapling, a term
of a bifunctional linker that connects a pair of reactive side chains via
two consecutive reactions in one pot, a first one forming an acyclic in-
termediate and a second one for the ring closure. This type of stapling
has been implemented by double Cu(I)-catalyzed azide-alkyne cyclo-
addition [17,18], double thiol-ene coupling [19], bis-alkylation [20]
and bis-arylation [21] of Cys thiols, etc.
Fig. 1 compares a multicomponent stapling based on a single MCR
with the variant based on a double MCR [22]. This latter approach
comprises the reaction of two equally-functionalized side chains (e.g.,
two amines, carboxylic acids, thiols, etc.) with a bifunctional linker in
the presence of – at least – two equivalents of the other components. This
methodology was originally designed for the synthesis of steroidal [23]
and natural product-like macrocycles [24] and it has more recently been
adapted to peptide stapling using Ugi [25,26] and Petasis [5] MCRs. An
important application of the multicomponent stapling approach has
originally coined for the synthesis of all-hydrocarbon bridged
α-helical
been the synthesis of functionalized α-helical peptides with proven
peptides [14,15] but currently employed for a variety of cyclization
methods connecting two side chains to enforce helicity [17].
success as inhibitors of protein-protein interactions [26] and as anti-
microbial agents [27]. Herein, we investigate the scope of the double
* Corresponding authors at: Laboratory of Synthetic and Biomolecular Chemistry, Faculty of Chemistry, University of Havana, Zapata y G, Havana 10400, Cuba (D.
G. Rivera) and Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
E-mail addresses: wessjohann@ipb-halle.de (L.A. Wessjohann), dgr@fq.uh.cu (D.G. Rivera).
https://doi.org/10.1016/j.bioorg.2021.104987
Received 19 January 2021; Received in revised form 19 April 2021; Accepted 8 May 2021
Available online 12 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M.G. Ricardo et al.
Bioorganic Chemistry 113 (2021) 104987
Ugi multicomponent stapling approach in its capacity to produce helices
using peptide sequences that do not show helical propensity in an un-
constrained form. For this, we implemented various stapling combina-
tions that allow tuning the length, flexibility and functionality of the side
chain linkers derived from the double Ugi macrocyclization. The helical
character of the Ugi-stapled peptides is assessed using circular dichroism
(CD), thus providing new insights into the structural factors of the Ugi-
derived moieties influencing the helicity of the cyclic peptides.
diisocyanobutane to render macrocyclic peptide 2, 3 and 4, respec-
tively. The overall yields of the stapled peptides (ca. 30%) can be
considered as good, taking into account the complex SPPS and macro-
cyclization protocol used to produce them with the latter comprising the
formation of 8 covalent bonds in one pot. In parallel, peptide 5 was
macrocyclized with the longer 4,4′-biarylether diisocyanide to furnish
stapled peptide 6 with the Glu’s in positions 2 and 13 (i → i + 11)
tethered.
Scheme 1B compares the CD spectra of the unconstrained and stapled
peptides in 20% trifluoroethanol (TFE)/10 mM phosphate buffer (PB), a
solvent mixture that, besides helping stabilize a helical conformation,
allows solubilizing these hydrophobic peptides that are insoluble in pure
water or PB solution. The almost identical CD spectra of peptides 1–4
(left panel) suggest that a short peptide bridging with the i → i + 7
diacid/diisocyanide combination did not lead to the desired stabiliza-
tion of a helical structure in, regardless of the linkers used, i.e. more
flexible or rigid or kinked ones. It seems that the extended tether linking
the two Glu side chains – including the aromatic or aliphatic linker and
the two peptidic moieties formed in the Ugi reactions – in this combi-
nation of building blocks (v.i. for other ones) is not capable to impose
sufficient conformational constraints into this sequence to force it into a
helical conformation. Accordingly, the CD pattern of these peptides
corresponds to either an unstructured conformation (i.e., random coil)
or extended structure [30]. This is in contrast to previous results in
which we proved that 14-mer peptides with double Ugi stapling at Glu
residues placed at i and i + 7 positions showed a significant increase in
2. Results and discussion
To stabilize helical peptides via stapling approaches, the side chain-
to-side chain tethering is typically conducted at amino acids whose side
chains are pointing towards the same face in an ideal helix, i.e., in res-
idues i → i + 4, i → i + 7, or i → i + 11 along the peptide sequence.
Typical lactam-bridged helical peptides produced either by lactamiza-
tion [12,13] or Ugi reaction [11,27,28] have been linked at the side
chains of Glu/Asp and Lysusually placed at i, i + 4 positions (see
Fig. 1A). On the other hand, the use of a bifunctional linker allows
connecting residues of the same type and more separated in the
sequence, that is, i → i + 7 and even i → i + 11. In this work, we sought to
implement a variety of double Ugi multicomponent macrocyclizations to
staple peptides at amino acids placed at these latter positions, and thus
evaluate the effect of the Ugi combinations on the helical content of the
resulting cyclic peptides.
Scheme 1A depicts the initial double Ugi multicomponent macro-
cyclization featuring the diacid/diisocyanide combination, in which not
only the nature and flexibility of the linker is varied but also the distance
of the two linked residues. The classic Ugi four-component reaction
comprises the condensation of a carboxylic acid, a primary amine, an
oxo-compound and an isocyanide to form an N-substituted dipeptide
moiety [29]. Methylamine and paraformaldehyde were chosen as the
simplest amine and oxo components, while the peptides were employed
as dicarboxylic acid building blocks and aromatic or aliphatic diiso-
cyanides as bifunctional linkers. Peptides 1 and 5 were produced by
solid phase peptide synthesis (SPPS), then released from the resin (see
the Supporting Information, SI) in deprotected form and used as crude
products in the Ugi stapling protocol. Peptide 1 bears two Glu residues at
positions 2 and 9 (i, i + 7), while 5 has the two Glu at positions 2 and 13
(i, i + 11). Both acyclic peptides include only very few residues with
propensity to favor helical conformations, and accordingly, their CD
spectra prove a random coil conformation. Therefore, they were
considered suitable to study the effect of the Ugi stapling on the sec-
ondary structure of thus constrained macrocyclic derivatives. The
multicomponent macrocyclization procedure consisted in the slow
addition with syringe pumps of two solutions, one of the peptide and
another one of the diisocyanide, to a reaction mixture containing the
imine derived from the condensation of methyl amine and
paraformaldehyde.
α
-helicity [26]. Nevertheless, it is worth-mentioning that those 14-mer
peptides were based on a helical binding fragment of the P53 protein
showing per se a higher helical propensity even in the acyclic form [26].
Considering that the diacid/diisocyanide combination of the double
Ugi stapling at i → i + 7 was unable to impose a helical conformation in
the 10-mer sequence, we looked at the comparison of the CD spectra of
the 14-mer linear peptide 5 and its stapled analog 6 (scheme 1B, right
panel). In this case, there is a significant conformational change derived
from the double Ugi stapling with the biarylether diisocyanide [23],
which results in stapled peptide 6 showing a significant
α-helicity as
evidenced by CD. The -helical content of peptides can be estimated by
α
the mean residue helicity at 222 nm, a value commonly downshifted for
short peptides to 215–220 nm [13,31,32]. By means of the equation
developed by Baldwin [33], the percent of helicity is correlated with the
ratio [θ]222/[θ]max, where [θ]max = (ꢀ 44 000 + 250 T)(1 ꢀ k/n), T is
temperature in degrees Celsius, n is the number of peptide units and k is
an finite factor related to the peptide length. Following the consider-
ation of Fairlie [13] and Baldwin [34] for carboxyamidated peptides, a
suitable value for k is 3.0. The [θ]max at T = 25 ◦C for this 14-mer
peptide 6 (n = 14) was found to be ꢀ 29 700. Using the corresponding
[θ]max, the previous equation results in 81% helicity for stapled peptide
6. As shown in the helical wheel in scheme 1C, an ideal α-helix can place
the tethered Glu side chains pointing towards the same face, thus fa-
voring the stapling process that stabilizes this conformation if a suitable
linker is employed. Therefore, the success of this design rests on the
capacity of the rather long tether – including the biaryl ether fragment
and the Ugi-derived peptidic moieties – to expand the distance between
the two Glu residues placed as far as in positions i and i + 11. To our
knowledge, this is the first time that an Ugi multicomponent macro-
cyclization is employed to staple peptides at residues separated by ten
Such pseudo-dilution condition has proven success in previous dou-
ble Ugi multicomponent macrocyclizations with a variety of substrates
[23–25], as it reduces formation of undesired oligomers and large re-
action volumes. After the slow addition is completed (ca. 24 h), the
reaction mixture is stirred for a total of 72 h to ensure high macro-
cyclization conversion. In this way, peptide 1 was subjected to the Ugi
stapling procedure with p- and m-xylylene diisocyanides [24] and 1,4-
Fig. 1. Comparison of the multicomponent peptide stapling using A) a single MCR and B) a double MCR approach.
2

M.G. Ricardo et al.
Bioorganic Chemistry 113 (2021) 104987
Scheme 1. (A) Double Ugi multicomponent macrocyclization featuring the diacid/diisocyanide combination for the synthesis of i → i + 7 and i → i + 11 stapled
peptides. Overall yields of peptides resulting from the complete synthetic route including the SPPS and multicomponent macrocyclization. (B) CD spectra of the
acyclic and stapled peptides in 20% TFE/phosphate buffer. (C) Wheel representation of an ideal helical peptide with the sequence of stapled peptide 6. Yields refer to
the overall sequence including SPPS and Ugi macrocyclization.
amino acids.
After the failure of the diacid/diisocyanide combination to produce
also lipids or fluorescent tags or any other function provided by the
isocyanide component [11].
helical conformations in the 10-mer sequence of peptide 1, we sought to
implement another combination capable to shorten the tether connect-
ing the residues i and i + 7. For this, we turned to the diacid/diamine
combination comprising the double Ugi reaction of a peptide dicar-
boxylic acid with a diamine building block in presence of para-
formaldehyde and an isocyanide. Scheme 2 shows the double Ugi
multicomponent stapling of peptide 1 with m-xylylene-diamine and 1,5-
pentane-diamine and cyclohexyl and benzyl isocyanides to render sta-
pled peptides 8 and 9, respectively. In this case, the diimine preforma-
tion was accomplished by first mixing the diamine with
paraformaldehyde and then slowly adding – with syringe pumps – the
solutions of the diimine and the peptide to a stirring solution of the
isocyanide. The overall yields of the cyclic peptides can be also
considered very good, being in the same range of the diacid/diisocya-
nide combination. In addition, this diacid/diamine variant of the Ugi
stapling variant may be regarded as similar to a double lactamization of
a peptide dicarboxylic acid with a diamine linker [35]. However, it of-
fers a synthetic advantage over that classic stapling technique in the
sense that allows the incorporation of exocyclic functionalities as N-
substituents of the tertiary amides formed. As depicted in scheme 2,
these functionalities can be either of aliphatic or aromatic nature, but
Analysis of the CD spectra of stapled peptides 8 and 9 – in compar-
ison to peptide 1 (scheme 2B, left panel) – reveals that the nature of the
linker, and thus the combination of Ugi-components chosen, definitely
influence the secondary structure stabilized in these peptides. The two
constrained peptides show a maximum at 190 nm and a minimum near
205 nm but a different ellipticity around 222 nm. Thus, the CD pattern of
peptide 8 tethered with the m-xylylene-diamine shows a positive ab-
sorption around 220 nm, which suggests an extended helical structure
resembling a polyproline-II helix [29]. In contrast, the double Ugi sta-
pling with the more flexible 1,5-pentane-diamine rendered peptide 9.
This shows a clear
α
-helical character as evidenced by CD. The
maximum at 193 nm and the minima at 202 and 222 nm confirm the
α
-helical structure with a relative percent of α-helicity of 68%, calcu-
lated as explained before ([θ]max of ꢀ 26 400, T = 25 ◦C, n = 10).
Because of this result, we also sought to prepare an additional cyclic
peptide using the same combination of the Ugi stapling but starting from
linear peptide 7 having two Asp residues replacing the Glu at positions 2
and 9. The double Ugi macrocyclization furnished stapled peptide 10,
which has only 2 carbons less in the macrocyclic ring than 9, but even so
shows another helical conformation. Scheme 2B (central panel) depicts
the CD spectrum of 10 corroborating that the shorter side chain tether of
3

M.G. Ricardo et al.
Bioorganic Chemistry 113 (2021) 104987
Scheme 2. (A) Double Ugi multicomponent macrocyclization featuring the diacid/diamine combination for the synthesis of i → i + 7 stapled peptides. Overall yields
of peptides resulting from the complete synthetic route including the SPPS and multicomponent macrocyclization. (B) CD spectra of the linear and stapled peptides in
20% TFE/phosphate buffer. Yields refer to the overall sequence including SPPS and Ugi macrocyclization.
peptide 10 leads to a 310-helix, as evidenced by a [θ]220/203 ratio of 0.4
that is characteristic of this type of helical conformation [13].
after the multicomponent stapling. Once more, it was proven that even
small changes in the side chain linker – derived either from variation of
the Ugi-derived connectors or the side chain tether – provokes different
types of conformational constraints defining the secondary structure. In
addition, comparison of the CD spectra of stapled peptides 8, 9 and 10
with those of the unconstrained precursors 1 and 7 – which show
random coil conformations – ratifies that the diacid/diamine combina-
tion (i → i + 7) is well suited to constrain 10-mer peptides into helical
While both peptides 9 and 10 include the same 1,5-pentane linkers, it
seems that the shorter side chain tether (Asp compared to Glu) of the
latter leads to a more compact 310-helix, while the longer tether of 9
tends to fix a α-helical conformation. Nevertheless, in both cases the 1,5-
pentane linker proved capable of introducing sufficient conformational
constraints to transform unstructured acyclic peptides into helical ones
Scheme 3. (A) Double Ugi multicomponent macrocyclization featuring the diamine/diisocyanide combination for the synthesis of i → i + 7 stapled peptides. Overall
yields of peptides resulting from the complete synthetic route including the SPPS and multicomponent macrocyclization. (B) CD spectra of the linear and stapled
peptides in 20% TFE/phosphate buffer. Yields refer to the overall sequence including SPPS and Ugi macrocyclization.
4

M.G. Ricardo et al.
Bioorganic Chemistry 113 (2021) 104987
conformations. Many more diamines with more or less rigidity, length,
and functionality (e.g., heterocyclic, polyether, fluorescent, etc.) are
commercially available and can be used to span the scope of this variant
of double Ugi multicomponent stapling.
the diamine/diisocyanide combination, always using the i → i + 7
linking strategy. We proved that both the type of bifunctional combi-
nation and the nature and flexibility of the linker influence the resulting
helicity, or the absence of any structured conformation. Although 20%
TFE had to be used as co-solvent in the CD spectra measurements – a
solvent known to favor the helical content – it was proven that the
double Ugi stapling is responsible for the stabilization of helical struc-
tures in several peptide-internal linker combinations, since the acyclic
peptides show a typical random coil pattern under the same conditions,
as do some tethered ones. Thus, the peptide diacid/diisocyanide com-
bination – in these examples – was unable to fix a helical conformation
in 10-mer peptides cross-linked at i, i + 7 residues, but the same com-
By considering Ugi-reactive functional groups naturally present in
amino acid side chains, we envisioned other additional variants of the
double Ugi macrocyclization approach that involve amino groups of
peptides. Hence, we devised the implementation of a variant that has
never been employed in multicomponent peptide stapling, that is, the
diamine/diisocyanide combination with a peptide diamine. Scheme 3
shows the implementation of this variant using a liner 10-mer peptide
featuring the same sequence of peptides 1 and 7 but with ornithine (Orn)
instead of Glu or Asp at positions 2 and 9. We realized that by using Orn
and not the proteinogenic Lys, we could access the same macrocyclic
ring sizes in the new stapled peptides 12, 13 and 14 than for those
peptides already shown in scheme 1, of course also using the same dii-
socyanides. Therefore, direct comparision is possible, to see if this new
diamine/diisocyanide combination would render a different result than
the inverse diacid/diisocyanide one, which did not provide helical
peptides for the 10-mer sequence. Cyclic peptides 12, 13, and 14 were
produced in reasonably good overall yield via a macrocyclization pro-
tocol previously optimized. This required the preformation of the dii-
mine by reaction of 11 with paraformaldehyde, followed by the slow
parallel addition of this diimine solution and the diisocyanide to a stir-
red solution of acetic acid.
bination did lead to very high
+ 11 residues. Unfortunately, a precise prediction, which components
will give an -helix, a 310-, polyproline- or other helix for varied peptide
α-helicity in a 14-mer peptide linked at i, i
α
sequences and i → i + n distances is yet impossible, but the ease and
variability of Ugi-stapling allows rapid experimental selection of the best
linker for a chosen purpose. While in this basic study very simple
monofunctional components were employed, i.e., methyl amine, acetic
acid, cyclohexyl and benzyl isocyanides, this multicomponent stapling
approach also has the capacity to incorporate more complex, function-
alized appendages for a variety of purposes, including fluorescent labels,
lipids, PEGs, and cationic or anionic groups, as shown by us before in
other contexts[11]. The only requirement is to have the primary amines,
carboxylic acids and isocyanides properly functionalized, for which
commercially available reactants provide numerous alternatives. In
principle, also different aldehydes or ketones can be use, but the
preferred use of paraformaldehyde is due to the poor stereoselectivity of
the Ugi reaction in non-symmetric oxo components. This is also the
reason for not using Ugi stapling combinations with dialdehyde linkers,
as they would lead to the formation of diastereomeric cyclic peptides of
difficult characterization and – depending on the purpose – perhaps poor
applicability. Nevertheless, the three easy combinations of bifunctional
components explored in this work already offer countless opportunities
for stabilizing helical peptides via variation of 2 of the 3 linker compo-
nents and the nature and positions of the amino acids to be tethered. We
are convinced that many biomedical applications can profit from the
exploitation of this powerful concomitant stapling/functionalization
technology.
In all the macrocyclization combinations included here, a key step is
the formation of the imine by condensation of the amine – either a Lys/
Orn side chain or an added component – with formaldehyde. Whereas
such imines should typically participate in the Ugi reactions, previous
reports describe the reaction of Arg and Tyr side chains with
formaldehyde-derived imines [36,37]. Since all peptide sequences used
in our examples incorporate Arg, it is expected the formation of by-
products derived from the reaction of the Arg guanidinium group and
the imine. Analysis of HPLC traces of crude products conveys that such
side reactions might take place (see the Supporting Information), but the
Ugi-stapled peptides are the major products of this procedure.
Comparison of the CD spectra of the unconstrained peptide 11 with
the three cyclic ones 12, 13, and 14 reveals that this variant of double
Ugi stapling can also lead to helical structures. As shown in scheme 3B,
there are differences in the CD spectra of the stapled peptides, indicating
that – in this case – the dissimilar linkers have influence on the resulting
conformations. Thus, while the macrocyclization with m-xylylene-dii-
socyanide led to unstructured peptide 12 (CD spectrum similar to the
acyclic precursor 11), the CD spectrum of peptide 14 suggests the sta-
bilization of a 310-helix after the stapling with the p-xylylene linker. In
contrast, the Ugi stapling with the 1,4-diisocyanobutane-having a
4. Experimental section
4.1. General
All starting materials were purchased from commercial sources and
used without further purification. 1H NMR and 13C NMR spectra were
recorded on a Varian Mercury 400 spectrometer at 399.94 MHz and
100.57 MHz, respectively. Chemical shifts (δ) are reported in ppm
relative to the TMS (1H NMR) and to the solvent signal (13C NMR). High
resolution mass spectra were obtained in an Orbitrap Elite mass spec-
trometer (Thermo Fisher Scientific, Germany) equipped with an ESI
electrospray ion source (positive spray voltage 4.5 kV, negative spray
voltage 3.5 kV, capillary temperature 275 ◦C, source heater temperature
250 ◦C, FTMS resolution 30000). A TripleToF 6600–1 mass spectrometer
(Sciex) was also used for high-resolution mass spectrometry, equipped
with an ESI-DuoSpray-Ion-Source and controlled by Analyst 1.7.1 TF
software (Sciex). The ESI source operation parameters were as follows:
ion spray voltage: 5,500 V, nebulizing gas: 60p.s.i., source temperature:
deeper minimum at 222 nm seems to favor some α-helicity in peptide
13, corroborating once more that small changes in both the shape and
flexibility of the linker – even using the same stapling combination –
determine the type of helicity or the absence of a structured confor-
mation. It is worth-noticing that not only the macrocycle ring size is
important in this type of double Ugi stapling (see that peptides 2, 3 and 4
have the same ring size than 14, 12 and 13, respectively), but the
presence of endo- or exo-cyclic amides and substituents is also relevant
as they can ultimately define the conformation of the Ugi-derived in-
ternal linker. This is especially evident for tertiary amides as they usu-
ally occur as a mixture of s-cis and s-trans rotamers, either of which can
impose dissimilar constraints and thus affect the secondary structure.
◦
450 C, drying gas: 70p.s.i., curtain gas: 35p.s.i. Data acquisition was
3. Conclusions
performed in the MS1-ToF mode, scanned from 100 to 1500 Da with an
accumulation time of 50 ms. Analytical RP-HPLC analysis was per-
formed on an Agilent 1100 system with a reverse-phase C18 column
We have provided novel insights into the effect of different combi-
nations of the double Ugi stapling on the secondary structure of the
resulting cyclic peptides. A generic 10-mer peptide sequence was chosen
to implement three different variants of this multicomponent macro-
cyclization. These are the diacid/diisocyanide, the diacid/diamine and
(4.6 × 150 mm, 5 μm) and a PDA detector. A linear gradient from 5% to
100% of solvent B in solvent A over 20 min at a flow rate of 0.8 mL
minꢀ 1 was used. Preparative RP-HPLC was performed on a Knauer 1001
system with UV detector K-2501. Separation was achieved using a C18
5

M.G. Ricardo et al.
Bioorganic Chemistry 113 (2021) 104987
column (25 × 250 mm, 25
μ
m). A linear gradient from 15% to 45% of
4.3.2. Stapled peptide 2
solvent B in solvent A over 30 min at a flow rate of 5 mL minꢀ 1 was used.
Detection was accomplished at 210 and 254 nm. Solvent A: 0.1% (v/v)
formic acid (FA) in water. Solvent B: 0.1% (v/v) FA in acetonitrile.
Circular dichroism spectra were collected on a Jasco J-815 spec-
tropolarimeter equipped with a temperature controller at 25 ◦C in 1 mm
path length cuvettes. The wavelength range used was from 260 to 185
nm, using the standard measurement parameters: 50 nm/sec speed and
16 accumulations.
The title compound (10.8 mg, 35%) was obtained by combining the
linear peptide 1 (26 mg, 0.02 mmol) with p-xylylene diisocyanide (3.1
mg, 0.02 mmol), paraformaldehyde and methylamine hydrochloride
according to the multicomponent stapling procedure described above.
Rt
=
14.0 min. HR-MS m/z: 773.4169 [M+2H]2+
,
calcd. for
2+
C
75H108N20
O
: 773.4205.
16
4.3.3. Stapled peptide 3
The title compound (12.1 mg, 39%) was obtained by combining the
linear peptide 1 (26 mg, 0.02 mmol) with m-xylylene diisocyanide (3.1
mg, 0.02 mmol), paraformaldehyde and methylamine hydrochloride
according to the multicomponent stapling procedure described above.
4.2. Synthesis of linear peptides
Rt
=
14.2 min. HR-MS m/z: 773.4242 [M+2H]2+
,
calcd. for
4.2.1. Solid phase peptide synthesis
2+
C
75H108N20
O
: 773.4205.
Peptide synthesis was carried out automatically on an INTAVIS
ResPepSL peptide synthesizer by a stepwise Fmoc/tBu strategy using
MBHA S RAM resin (Irich Biotech) at 0.1 mmol scale. Coupling reactions
were accomplished in DMF using 5-fold excess of the amino acid residue
and PyBOP and 10-fold excess of NMM. Fmoc removal was achieved
with a solution of 20% piperidine in DMF. N-terminus acetylation was
performed manually using a mixture of acetic anhydride (10 equiv) and
DIPEA (10 equiv) in DMF for 30 min at room temperature, then washing
with DMF (3 × 1 min) and DCM (2 × 1 min). Final cleavage is performed
mixing the resin with the cocktail TFA/TIS/H2O (95:2.5:2.5). The crude
peptides were precipitated from diethyl ether (-20 ◦C), then taken up in
1:2 acetonitrile/water and lyophilized.
16
4.3.4. Stapled peptide 4
The title compound (9.9 mg, 33%) was obtained by combining the
linear peptide 1 (26 mg, 0.02 mmol) with 1,4-diisocyanibutane (2.2 mg,
0.02 mmol), paraformaldehyde and methylamine hydrochloride ac-
cording to the multicomponent stapling procedure described above. Rt
=
14.3 min. HR-MS m/z: 749.4211 [M+2H]2+
,
calcd. for
2+
C71H110N20
O
: 749.4205.
16
4.3.5. Stapled peptide 6
The title compound (11.7 mg, 29%) was obtained by combining the
linear peptide 5 (34 mg, 0.02 mmol) with biarylether diisocyanide (4.4
mg, 0.02 mmol), paraformaldehyde and methylamine hydrochloride
according to the multicomponent stapling procedure described above.
4.2.2. Linear peptide 1
Ac-Phe-Glu-Ala-Leu-Gln-Trp-Arg-Ala-Glu-Leu-NH2 (117 mg, 91%
crude yield, 94% purity) was synthesized according to the protocol
described above. Rt = 13.2 min. HR-MS m/z: 1303.6699 [M+H]+, calcd.
for C61H91N16O+16: 1303.6799.
Rt = 17.6 min. HR-MS m/z: 1014.5352 [M+2H]2+, calcd. for
2+
C71H110N20
O
: 1014.5287.
16
4.3.6. Peptide diacid/diamine combination
Paraformaldehyde (1.8 mg, 0.06 mmol) and the diamine (0.03
mmol) are stirred in 2 mL of MeOH/DCM 2:1 (v/v) for 1 h at room
temperature. The resulting solution and another one of the peptide
diacid (0.02 mmol in 2 mL of MeOH) are simultaneously added using
syringe pumps (flow rate: 80 µL × hꢀ 1) to the isocyanide component
prevously disolved in a 16 mL mixture of MeOH/DCM 2:1 (v/v). Once
the addition is completed, the reaction mixture is stirred for additional
48 h and concentrated under reduced pressure. The resulting mixture is
dissolved in TFA (2 mL), then precipitated from diethyl ether (ꢀ 20 ◦C),
taken up in 1:2 acetonitrile/water, lyophilized and purified by RP-HPLC.
4.2.3. Linear peptide 5
Ac-Leu-Glu-Phe-Leu-Ala-Leu-Ser-Trp-Ala-Gln-Arg-Ala-Glu-Phe-NH2
(158 mg, 92% crude yield, 77% purity) was synthesized according to the
protocol described above. Rt = 15.8 min. HR-MS m/z: 861.4670
[M+2H]2 +, calcd. for C82H121N20
O
: 861.4547.
2+
21
4.2.4. Linear peptide 7
Ac-Phe-Asp-Ala-Leu-Gln-Trp-Arg-Ala-Asp-Leu-NH2 (114 mg, 90%
crude yield, 96% purity) was synthesized according to the protocol
described. Rt = 13.6 min. HR-MS m/z: 1275.6399 [M+H] +, calcd. for
C
59H87N16O+16: 1275.6486.
4.3.7. Stapled peptide 8
The title compound (10.8 mg, 32%) was obtained by combining the
linear peptide 1 (26 mg, 0.02 mmol) with m-xylylene diamine (4.1 mg,
0.03 mmol), paraformaldehyde and cyclohexyl isocyanide (7.4 µL, 0.06
mmol) according to the multicomponent stapling procedure described
4.2.5. Linear peptide 11
Ac-Phe-Orn-Ala-Leu-Gln-Trp-Arg-Ala-Orn-Leu-NH2 (142 mg, 95%
crude yield, 97% purity) was synthesized according to the protocol
+
described above. Rt = 10.1 min. HR-MS m/z: 1273.7514 [M+H]
,
above. Rt = 17.9 min. HR-MS m/z: 841.4843 [M+2H]2+, calcd. for
calcd. for C61H97N18O+12: 1273.7533.
2+
C85H126N20
O
: 841.4831.
16
4.3.8. Stapled peptide 9
4.3. Double Ugi multicomponent stapling
The title compound (12.3 mg, 37%) was obtained by combining the
linear peptide 1 (26 mg, 0.02 mmol) with 1,5-diaminobutane (3.1 mg,
0.03 mmol), paraformaldehyde and benzyl isocyanide (7.3 µL, 0.06
mmol) according to the multicomponent stapling procedure described
4.3.1. Peptide diacid/diisocyanide combination
Paraformaldehyde (1.8 mg, 0.06 mmol), methylamine hydrochloride
(4.0 mg, 0.06 mmol) and DIPEA (10.5 µL, 0.06 mmol) are stirred in dry
MeOH (2 mL) for 1 h at room temperature and next diluted with 14 mL
of MeOH/DCM 2:1 (v/v). Two solutions, one of the peptide diacid (0.02
mmol in MeOH, 2 mL) and another of the diisocyanide (0.02 mmol in
MeOH, 2 mL), are simultaneously added to the reaction mixture using
syringe pumps (flow rate: 80 µL × hꢀ 1). Once the addition is completed
after ca. 24 h, the mixture is stirred for an additional 48 h and then
concentrated under reduced pressure. The resulting mixture is dissolved
in TFA (2 mL), then precipitated from diethyl ether (ꢀ 20 ◦C), taken up in
1:2 acetonitrile/water, lyophilized and purified by RP-HPLC.
above. Rt = 16.9 min. HR-MS m/z: 832.4572 [M+2H]2+, calcd. for
2+
C84H120N20
O
: 832.4596.
16
4.3.9. Stapled peptide 10
The title compound (9.5 mg, 29%) was obtained by combining the
linear peptide 7 (25.5 mg, 0.02 mmol) with 1,5-diaminobutane (3.1 mg,
0.03 mmol), paraformaldehyde and benzyl isocyanide (7.3 µL, 0.06
mmol) according to the multicomponent stapling procedure described
above. Rt = 17.0 min. HR-MS m/z: 818.4459 [M+2H]2+, calcd. for
6

M.G. Ricardo et al.
Bioorganic Chemistry 113 (2021) 104987
2+
[5] M.G. Ricardo, D. LLanes, L.A. Wessjohann, D.G. Rivera, Introducing the Petasis
reaction for the late-stage multicomponent diversification, labeling and stapling of
peptides, Angew. Chem. Int. Ed. 58 (2019) 2700–2704.
C82H116N20
O
: 818.4439.
16
4.3.10. Peptide diamine/diisocyanide combination
[6] A. Yamaguchi, S.J. Kaldas, S.D. Appavoo, D.B. Diaz, A.K. Yudin, Conformationally
stable peptide macrocycles assembled using the Petasis borono-Mannichreactio,
Chem. Commun. 55 (2019) 10567–10570.
Paraformaldehyde (1.8 mg, 0.06 mmol), DIPEA (10.5 µL, 0.06 mmol)
and the linear peptide 11 (30 mg, 0.02 mmol) are stirred in dry MeOH
(2 mL) for 1 h at room temperature. The resulting solution and the
diisocyanide (0.02 mmol in 2 mL of MeOH) are separately and simul-
taneously added using syringe pumps (flow rate: 80 µL.hꢀ 1) to a solution
of acetic acid (3.4 µL, 0.06 mol) in 16 mL of MeOH/DCM 2:1 (v/v). Once
the addition is completed, the mixture is stirred additional 48 h and
concentrated under reduced pressure. The resulting residue is dissolved
in TFA (2 mL), then precipitated from diethyl ether (ꢀ 20 ◦C), taken up in
1:2 acetonitrile/water, lyophilized and purified by RP-HPLC.
¨
[7] A. Failli, H. Immer, M. Gotz, Can. J. Chem. 57 (1979) 3257–3261.
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amphoteric molecules, J. Am. Chem. Soc. 132 (2010) 2889–2891.
´
[9] A.V. Vasco, C.S. Perez, F.E. Morales, H.E. Garay, D. Vasilev, J.A. Gavín, L.
A. Wessjohann, D.G. Rivera, Macrocyclization of peptide side chains by the Ugi
reaction: achieving peptide folding and exocyclic N-functionalization in one shot,
J. Org. Chem. 80 (2015) 6697–6707.
[10] J.R. Frost, C.C.G. Scully, A.K. Yudin, Oxadiazole grafts in peptide macrocycles, Nat.
Chem. 8 (2016) 1105–1111.
´
[11] A.V. Vasco, Y. Mendez, A. Porzel, J. Balbach, L.A. Wessjohann, D.G. Rivera,
A Multicomponent stapling approach to exocyclic functionalized helical peptides:
adding lipids, sugars, PEGs, labels, and handles to the lactam bridge, Bioconjugate
Chem. 30 (2019) 253–259.
4.3.11. Stapled peptide 12
[12] M.E. Houston, C.L. Gannon, C.M. Kay, R.S. Hodges, Lactam bridge stabilization of
The title compound (10.0 mg, 32%) was obtained by combining the
linear peptide 11 (30 mg, 0.02 mmol) with m-xylylene diisocyanide (3.1
mg, 0.02 mmol), paraformaldehyde and acetic acid according to the
α
-helical peptides: Ring size, orientation and positional effects, J. Pept. Sci. 1
(1995) 274–282.
[13] N.E. Shepherd, H.N. Hoang, G. Abbenante, D.P. Fairlie, Single turn peptide alpha
helices with exceptional stability in water, J. Am. Chem. Soc. 127 (2005)
2974–2983.
multicomponent stapling procedure described above. Rt = 11.0 min.
HR-MS m/z: 787.4352 [M+2H]2+, calcd. for C77H114N20
O
: 787.4361.
2+
[14] C.E. Schafmeister, J. Po, G.L. Verdine, An all-hydrocarbon cross-linking system for
enhancing the helicity and metabolic stability of peptides, J. Am. Chem. Soc. 122
(2000) 5891–5892.
16
4.3.12. Stapled peptide 13
[15] Y.W. Kim, T.N. Grossmann, G.L. Verdine, Synthesis of all-hydrocarbon stapled
±-helical peptides by ring-closing olefin metathesis, Nat. Protoc. 6 (2011)
761–771.
The title compound (6.7 mg, 22%) was obtained by combining the
linear peptide 11 (30 mg, 0.02 mmol) with 1,4-diisocyanobutane (2.2
mg, 0.02 mmol), paraformaldehyde and acetic acid according to the
[16] D.G. Rivera, G.M. Ojeda-Carralero, L. Reguera, E.V. Van der Eycken, Peptide
Macrocyclization by Transition Metal Catalysis, Chem. Soc. Rev. 49 (2020)
2039–2059.
multicomponent stapling procedure described above. Rt = 13.6 min.
HR-MS m/z: 763.4366 [M+2H]2+, calcd. for C73H114N20
O
: 763.4361.
2+
[17] Y.H. Lau, P. de Andrade, Y. Wu, D.R. Spring, Peptide stapling techniques based on
different macrocyclisation chemistries, Chem. Soc. Rev. 44 (2015) 91–102.
[18] K. Sharma, D.L. Kunciw, W. Xu, M.M. Wiedmann, Y. Wu, H.F. Sore, W.R.J.
D. Galloway, Y.H. Lau, L.S. Itzhaki, D.R. Spring, Double-click Stapled Peptides for
Inhibiting Protein-Protein Interactions (chapter 8), in: Cyclic Peptides: From
Bioorganic Synthesis to Applications, The Royal Society of Chemistry, London,
2017, pp. 164–187.
16
4.3.13. Stapled peptide 14
The title compound (7.8 mg, 25%) was obtained by combining the
linear peptide 11 (30 mg, 0.02 mmol) with p-xylylene diisocyanide (3.1
mg, 0.02 mmol), paraformaldehyde and acetic acid according to the
[19] Y. Wang, D.H.-C. Chou, A Thiol–ene coupling approach to native peptide stapling
and macrocyclization, Angew. Chem. Int. Ed. 54 (2015) 10931–10934.
[20] A.M. Spokoyny, Y. Zou, J.J. Ling, H. Yu, Y.S. Lin, B.L. Pentelute, A perfluoroaryl-
cysteine SNAr chemistry approach to unprotected peptide stapling, J. Am. Chem.
Soc. 135 (2013) 5946–5949.
multicomponent stapling procedure described above. Rt = 13.5 min.
HR-MS m/z: 787.4349 [M+2H]2+, calcd. for C77H114N20
O
: 787.4361.
2+
16
[21] H. Jo, N. Meinhardt, Y. Wu, S. Kulkarni, X. Hu, K.E. Low, P.L. Davies, W.
Declaration of Competing Interest
F. Degrado, D.C. Greenbaum, Development of α-helical calpain probes by
mimicking a natural protein-protein interaction, J. Am. Chem. Soc. 134 (2012)
17704–17713.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[22] L.A. Wessjohann, D.G. Rivera, O.E. Vercillo, Multiple Multicomponent
Macrocyclization including Bifunctional Building Blocks (MiBs): A Strategic
Development Towards Macrocycle Diversity, Chem. Rev. 109 (2009) 796–814.
[23] L.A. Wessjohann, D.G. Rivera, F. Coll, Synthesis of steroidꢀ biaryl ether hybrid
macrocycles with high skeletal and side chain variability by multiple
multicomponent macrocyclization including bifunctional building Blocks, J. Org.
Chem. 71 (2006) 7521–7526.
Acknowledgments
We acknowledge financial support from the Alexander von Hum-
boldt Foundation (AvH) and the German Academic Exchange Service
(DAAD).
[24] D.G. Rivera, O.E. Vercillo, L.A. Wessjohann, Rapid generation of macrocycles with
natural-product-like side chains by multiple multicomponent macrocyclizations
(MiBs), Org. Biomol. Chem. 6 (2008) 1787–1795.
[25] M.G. Ricardo, F.M. Vicente, H. Garay, O. Reyes, L.A. Wessjohann, D.G. Rivera,
Bidirectional macrocyclization of peptides by double multicomponent reactions,
Org. Biomol. Chem. 13 (2015) 438–446.
Appendix A. Supplementary material
[26] M.G. Ricardo, A.M. Ali, J. Plewka, E. Surmiak, B. Labuzek, C.G. Neochoritis,
¨
J. Atmaj, L. Skalniak, R. Zhang, T.A. Holak, M. Groves, D.G. Rivera, A. Domling,
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104987.
Multicomponent Stapling as a Driven Tool for the Development of Inhibitors of
Protein-Protein Interactions, Angew. Chem Int. Ed. 59 (2020) 5235–5241.
´
[27] A.V. Vasco, M. Brode, Y. Mendez, O. Valdez, D.G. Rivera, L.A. Wessjohann,
Synthesis of Lactam-Bridged and Lipidated Cyclo-Peptides as Promising Anti-
Phytopathogenic Agents, Molecules 25 (2020) 811.
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8