Macrocyclization of Peptide Side Chains by the Ugi Reaction: Achieving Peptide Folding and Exocyclic N-Functionalization in One Shot

Article abstract of DOI:10.1021/acs.joc.5b00858

The cyclization of peptide side chains has been traditionally used to either induce or stabilize secondary structures (β-strands, helices, reverse turns) in short peptide sequences. So far, classic peptide coupling, nucleophilic substitution, olefin metat

Full text of DOI:10.1021/acs.joc.5b00858

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Article
Macrocyclization of Peptide Side Chains by Ugi Reaction: Achieving
Peptide Folding and Exocyclic N-Functionalization in One Shot
Aldrin V Vasco, Carlos S. Pérez, Fidel E Morales, Hilda E Garay, Dimitar
Vasilev, Jose A Gavin, Ludger A. Wessjohann, and Daniel García Rivera
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2015
Downloaded from http://pubs.acs.org on June 4, 2015
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Macrocyclization of Peptide Side Chains by Ugi Reaction: Achieving
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Peptide Folding and Exocyclic N-Functionalization in One Shot
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Aldrin V. Vasco,†,‡ Carlos S. Pérez,† Fidel E. Morales,† Hilda E. Garay,ǁ Dimitar Vasilev,§
José A. Gavín,ǂ Ludger A. Wessjohann,§,* and Daniel G. Rivera,†,*
†Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y
G, 10400, La Habana, Cuba.
‡Facultad de Ingeniería Química. Instituto Superior Politécnico José Antonio Echeverría,
CUJAE, Calle 114 # 11901, 11500, La Habana, Cuba.
ǁSynthetic Peptides Group, Center for Genetic Engineering and Biotechnology, P.O. Box
6162, La Habana, Cuba.
ǂInstituto Universitario de Bioorgánica “Antonio González and Departamento de Química
Orgánica, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain.
§Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3,
Dꢀ06120, Halle/Saale, Germany.
Corresponding Authors: *Emails: dgr@fq.uh.cu; wessjohann@ipbꢀhalle.de
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Keywords
Cyclic peptides, macrocyclization, multicomponent reactions, secondary structures, NMR
Abstract
The cyclization of peptide side chains has been traditionally used to either induce or stabilize
secondary structures (βꢀstrands, helices, reverse turns) in short peptide sequences. So far,
classic peptide coupling, nucleophilic substitution, olefin metathesis and click reactions have
been the methods of choice to fold synthetic peptides by means of macrocyclization. This
article describes the utilization of the Ugi reaction for the side chainꢀtoꢀside chain and side
chainꢀtoꢀtermini macrocyclization of peptides, thus enabling access not only to stable folded
structures but also the incorporation of exocyclic functionalities as Nꢀsubstituents. Analysis of
the NMRꢀderived structures revealed the formation of helical turns, βꢀbulges, and αꢀturns in
cyclic peptides crossꢀlinked at i, i + 3 and i, i + 4 positions, proving the folding effect of the
multicomponent Ugi macrocyclization. Molecular dynamics simulation provided further
insights on the stability and molecular motion of the side chain crossꢀlinked peptides.
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Introduction
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Constraining short peptides into proteinꢀlike bioactive conformations remains as a synthetic
challenge with growing implications in the development of peptideꢀbased therapeutic agents.1
Cyclization2,3 and conjugation to topological templates4 are the most successful methods to
introduce conformational constraints in peptides designed either as protein ligands or
mimetics of protein epitopes. However, cyclization stands as the most effective, and
consequently employed, way to access folded peptide sequences mimicking biologically
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relevant secondary structures (i.e.,
βꢀhairpins, βꢀstrands, α
ꢀhelix and turns).3 In addition,
cyclic peptides usually exhibit an enhanced binding affinity to the biological target5 as well as
improved pharmacological properties compared to their acyclic analogues.6 Consequently,
there has been a resurgence of chemical2,6 and enzymatic7 approaches aiming at the efficient
cyclization of peptides either by their termini or side chains.
While headꢀtoꢀtail and side chainꢀtoꢀmain chain cyclizations are of wide incidence in natural
nonꢀribosomal peptides, the disulfide bridge is nature’s solution for the structural
preorganization of peptides and proteins by means of side chainꢀtoꢀside chain linkage.
Synthetic chemists have developed a variety of side chainꢀtoꢀside chain cyclization methods
to constrain peptides to strand, helix, turn and loop conformations.2,3 Such approaches include
the lactam bridge formation between the side chains of Lys and Asp/Glu residues,8 the CuIꢀ
catalyzed alkyneꢀazide 1,3ꢀdipolar cycloaddition,9 olefin metathesis,10 nucleophilic
substitution11 and arylꢀaryl coupling,12 among others.
In the last years, isocyanideꢀbased multicomponent reactions (IꢀMCRs) have emerged as
powerful macrocyclization procedures enabling the rapid access to new macrocyclic
chemotypes,13 including pseudoꢀpeptidic ones.14 In particular, the Ugi fourꢀcomponent
reaction was the first one being employed for the headꢀtoꢀtail cyclization of peptides as far as
in 1979.15 However, the interest on utilizing this reaction for peptide cyclization dropped over
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the following 30 years,13b mainly because of the longer reaction times and lower yields
compared to the classic peptide coupling protocols. Despite the drawbacks related to the Ugiꢀ
cyclization by the peptide termini, there are other synthetic options involving the utilization of
peptide side chains that may lack such problems, and yet remain unexplored.
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Figure 1. Peptide macrocyclization strategy using the Ugi reaction.
Figure 1 illustrates the four possibilities of peptide macrocyclization by means of the Ugi
reaction, including the headꢀtoꢀtail cyclization (A) previously reported.15 As this reaction is
the condensation of an amino and a carboxylic group with an oxo component and an
isocyanide, cyclization strategies B, C and D rely on the presence of Glu/Asp and Lys/Orn
along the peptide sequence. Perhaps the most important feature of this strategy – and the one
that distinguishes it more from the classic peptide coupling – is the generation of an exocyclic
functionality as part of the
Nꢀsubstituted amide formed during the ring closing step. We
envisioned that the exploitation of such a characteristic may provide a variety of applications
not so easily available for conventional cyclization methods based on coupling reagents.
Herein we demonstrate that the Ugiꢀ4CR is a suitable procedure for the macrocyclization of
peptide side chains, thus leading to cyclic peptides bearing a tertiary lactam bridge instead of
a secondary one, i.e.,
N
ꢀsubstituted cyclic peptides. Our aim is to prove the potential of this
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approach to fulfil two objectives in only one synthetic operation; i.e., the incorporation of an
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exocyclic functionality as part of the
constraints leading to folded peptides. To get deep insights into the threeꢀdimensional
structures of the ꢀsubstituted cyclic peptides in solution, a molecular modeling study based
Nꢀalkylation and the introduction of conformational
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N
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on NMR restraints and molecular dynamics was performed.
Results and Discussion
Macrocyclization of peptide side chains by Ugi reaction
Several reports have shown the scope and limitations of the Ugi reaction in the
macrocyclization of dissimilar substrates,16 including peptidic ones.17,18 According to the
mechanism of the Ugi reaction (Fig. 1B) – in which the initially formed
αꢀadduct evolves
through an intramolecular acylation (Mumm rearrangement) to the final product – the
migration capacity of the amino component is crucial on the reaction length and efficiency. In
this sense, Yudin et. al. have proposed that the slow kinetics of the Ugi macrocyclization in
short peptides is due to the slow transannular attack of the amine to the mixed anhydride.19 As
an alternative to the conventional Ugi reaction, Yudin’s group introduced the use of an
amphoteric aziridine aldehyde in a new multicomponent peptide macrocyclization deviating
from the Mumm rearrangement.19 Such a modified Ugi macrocyclization allowed the efficient
and highly stereoselective headꢀtoꢀtail cyclization of short and mediumꢀsize peptides.19,20 In
contrast to linear peptides, small pentapeptoids (i.e.,
Nꢀalkylated pentaglycines) have been
successfully cyclized by the Ugi reaction,17 confirming that a greater flexibility of the amine
moiety facilitates the macrocyclic Ugiꢀbased ring closure.
Recently, we described the development of a bidirectional macrocyclization strategy based on
the execution of two Ugi reactions between peptide diacids and diisocyanide crossꢀlinkers.18
In that study, macrocyclizations based on side chain carboxylic groups were much more
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efficient than those relying on the more conformationally constrained backbone carboxylic
groups. In a similar way, we anticipated that unidirectional Ugi macrocyclizations using the
flexible Glu/Asp and Lys side chains would be less problematic than the headꢀtoꢀtail version,
as they may skip the difficulties associated with the slow Mumm rearrangement.
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Scheme 1 depicts three examples of side chainꢀtoꢀside chain peptide macrocyclization by
means of the Ugi reaction between Glu/Asp and Lys side chains. Our aim lies at utilizing the
cyclization to fold peptide scaffolds onto turn structures, while introducing an
Nꢀsubstitution
as an exocyclic functionality. As mentioned above, several peptide cyclization methods have
been employed to stabilize turns,3 though none of them allows for the simultaneous exocyclic
N
ꢀfunctionalization of the cyclization site. The potential of accomplishing cyclization and Nꢀ
substitution in one step is remarkable, as three crucial goals can be achieved in one shot: a)
ꢀalkylation,21 b) accessing specific
improving pharmacological properties through the
N
secondary structures of biological relevance and c) employing the resulting exocyclic
functionality, e.g., for conjugation/immobilization strategies.
Nꢀ
As shown in scheme 1, three different oligopeptides were subjected to side chainꢀtoꢀside
chain Ugi macrocyclization in solution. Such linear peptides were produced either by a
stepwise solutionꢀphase synthesis or by a standard Fmoc solidꢀphase protocol.22 The strategy
was the introduction of amino acids with Ugiꢀreactive side chains located at
i, i + 3 and i, i + 4
positions, as these combinations are known to stabilize both turns and helical structures.3a,23
Thus, peptides 1 and 3 bear the Lys/Glu and Glu/Lys pairs located at the
running from the to ꢀterminus direction, while peptide 5 has the Lys and Asp residues
located at and + 4 positions. It must be noticed that amino acid sequences were not
i and i + 3 positions
N
C
i
i
designed to favor a periodic secondary structure (e.g., αꢀhelix and βꢀsheet), but the goal is to
assess the effect of the Ugi macrocyclization on the peptide folding.
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Scheme 1. Side chainꢀtoꢀside chain peptide macrocyclizations by Ugi reaction.
Macrocyclizations were carried out in methanol at 2 mM concentration, always using
paraformaldehyde as the oxo component in order to form only one diastereomer. Besides of
using commercially available isocyanides, synthesis of cyclic peptide 6 demonstrates the
possibility of incorporating an exocyclic reactive functionality suitable for further
derivatization. In all cases, preꢀformation of the imine was initially accomplished at higher
concentration (10 mM), followed by dilution to the final concentration and addition of the
isocyanide. HPLC monitoring of Ugi macrocyclization affording 2 proved high conversion (
≥
80%) after 72 h of reaction. Thus, the general reaction time was fixed to 72 h for all
macrocyclizations to enable comparison of efficiency among the different approaches. Cyclic
peptides 2, 4 and 6 were obtained in good yield (ca. 60%) and high purity (> 95%) after
preparative HPLC purification, which renders enough material for assessing the solution
threeꢀdimensional structure through NMR analysis.
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Scheme 2. Side chainꢀtoꢀtermini peptide macrocyclizations by Ugi reaction.
To expand the scope of the Ugi macrocyclization, we sought to assess the efficacy of this
method for tethering peptide side chains to each one of the termini, thus showing the two
remaining Ugiꢀcyclizing combinations. Scheme 2 depicts the implementation of headꢀtoꢀside
chain and side chainꢀtoꢀtail macrocyclizations leading to peptides 8 and 10, respectively. As
shown, tetra and pentapeptides were cyclized by side chains of amino acids separated two
residues from one of the termini. Interestingly, the yield of isolated cyclic peptide 8 was the
lowest among all macrocyclizations, but there were no great differences between the
procedure yielding 10 and those giving, for example, 2 and 4. It must be noticed that the more
efficient Ugi macrocyclizations are those employing the amino group of the flexible Lys side
chain, despite of the fact that macrocycles 2, 4, 6 and 10 feature different ring sizes.
Alternatively, Ugi macrocyclization to cyclic peptide 8 comprises the reaction of a backbone
terminal amino group, which seems to share with the headꢀtoꢀtail approach the mechanistic
complications derived from a slow transannular acylation. Two additional cyclizations (not
shown) featuring the same headꢀtoꢀGlu side chain cyclization, but with different tetrapeptide
sequences, were also studied. In those cases, HPLC/ESIꢀMS monitoring showed conversion
no greater than 50% after 72 h, confirming the difficulties associated with the Ugi
macrocyclization of short peptides utilizing the
Nꢀterminus as amino component. So far, we
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can summarise that the Ugi reaction is an effective method for side chainꢀtoꢀside chain and
side chainꢀtoꢀtail cyclizations, enabling a straightforward access to macrocyclic peptides
6
7
8
bearing an exocyclic Nꢀsubstitution. Albeit yields seem to be lower than those of peptide side
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chain cyclization with coupling agents, it must be considered that 4 covalent bonds are
formed in the multicomponent reaction, while only one is formed in the peptide coupling.
Three-dimensional structure of side chain-to-side chain cross-linked peptides
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To assess the effect of the Ugi macrocyclization on the threeꢀdimensional solutionꢀphase
structures of the side chain crossꢀlinked peptides, we chose compounds 2, 4 and 6 to be
studied by means of NMR and molecular dynamics simulation. Similarly to other Lysꢀ
Asp/Glu crossꢀlinked peptides made by classic lactamization, it is likely that the Ugi
macrocyclization does not only introduce significant conformational constraints onto small
peptides, but also leads to folded structures.3a Due to low solubility in water, structural
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characterization was accomplished based on 1D and 2D H NMR spectroscopy in DMSOꢀd6
at 300 K. Full NMR assignment of these compounds is reported in tables 1 and 2. Final
structures were derived from coupling constants (3J NHCHα) and ROE constraints and obtained
by simulated annealing and refinement protocols in XplorꢀNIH 24
.
We expected that cyclic peptides 2 and 4 – crossꢀlinked with Lys and Glu separated by two
residues – might have a Cα ꢀCαi+3 distance lower than 7 Å, typical of reverse and helical turns.
i
For cyclic peptide 2, coupling constants 3J
were higher than 8 Hz for all residues,
NHCHα
except for Cꢀterminal Ala (see supporting information, Table S1). Such values are indicative
of a wellꢀstructured peptide skeleton of low conformational freedom, and are commonly
ꢀstrand structures. Intriguingly, only three signals were observed in the CαH
region of the 1H NMR spectrum. The HSQCꢀedit spectrum of 2 (Fig. 2) shows an overlapping
associated to
β
of H
α resonances of Lys2, Val4 and Glu5 at 4.30 ppm, which makes impossible the
unequivocal assignment of ROE crossꢀpeaks involving these hydrogens and, consequently, its
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use as experimental constrain in structure refinement. A total of 37 distance constraints (5
strong, 18 medium and 14 weak; 22 interꢀresidual: 10 of them sequential and 12 nonꢀ
sequential) resulted from analysis of ROE intensities. Six dihedral restraints were used in the
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MD simulation, 5
three rounds of simulation, we could stereospecifically assign the methylene hydrogens of
Phe3, the H of Lys3, and methyl groups of Val4.
φ
angles from the 3JNHCHα data and one χ1 angle from the Phe residue. After
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ε
Figure 2. Expanded CαH region of HSQCꢀedit spectrum of peptide 2.
Figure 3A (top) depicts the superimposition of the 20 lowest energy structures of cyclic
peptide 2, featuring 0.14 Å average RMSD to the mean structure for the peptide backbone
3
that confirms the great conformational rigidity suggested by the JNHCHα values. As shown,
the NMRꢀderived solution structures of 2 comprise a reverse turn with propensity to helicity,
i.e., the
whether such a behavior remains in time, we performed a further MD simulation during 80 ns
from the average NMRꢀderived structure. Figures 3B (top) shows the distribution in time
φ and ψ angles of Phe3, Val4 and Glu5 lay within the helical region. To determine
φ/ψ
of the four central amino acids obtained from MD, confirming that the most populated states
are confined to the helical space of Ramachandran plots. Also shown are three of the most
representative structures obtained during simulation time, which show that the greater
mobility lies at the first two residues Leu1 and Lys2. This comprises that the two Nꢀterminal
residues may pass through a pseudoꢀplanar conformation, while residues Phe3, Val4, and
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Glu5 mostly remain in an helical conformation. In addition, various relevant hydrogen bonds
4
5
6
were detected during MD simulation, including COLys2···NHAla6 (6% in time;
i, i+4
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characteristic of ꢀhelix), COLys2···NHGlu5 (2% in time) and COPhe3···NHAla6 (3% in time).
α
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Table 1. 1H NMR assignment of cyclic peptides 2, 4 and 6.
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5
6
7
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Cyclic peptide 2
Residue
Leu
NH (m, J (Hz))
7.36 (d, 8.0)
7.87 (d, 8.2)
Hα
Hβ
Hγ
Others
4.02
4.30
1.20, 1.38
1.69, 1.42
1.56
0.81 , 0.85 (2×CH3δ)
1.52, 1.41 (Hδ); 2.90, 3.45 (Hε)
Lys
1.25, 1.35
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7.17 (Hδ); 7.20 (Hε) ; 7.13 (Hζ)
Phe
Val
Glu
Ala
8.73 (d, 8.9 )
6.72 (d, 8.7)
8.32 (d, 9.1)
8.23 (d, 3.9)
4.42
4.30
4.30
4.03
2.77, 3.15
2.00
ꢀ
ꢀ
0.83, 0.90
2.35, 2.40
ꢀ
ꢀ
1.57, 2.21
1.21
3.64 (OCH3)
Cbz group
Exocyclic Nꢀsubstituent produced by cyclization
3.72, 3.90 (H1); 7.54 (H3, J=7.8
Hz); 3.50 (H4); 1.65, 1.63, 1.21,
1.11, 1.19, 1.17, 1.15 (not assigned)
5.01 (H2); 7.34 (H4); 7.35 (H5);
7.30 (H6)
Cyclic peptide 4
Hβ
Residue
NH (m, J (Hz))
Hα
Others
Hγ
Val
Ile
7.92 (d, 9.0)
7.84 (d, 8.8)
7.96 (d, 7.1)
8.15 (d, 6.4)
7.82 (d, 6.7)
7.94 (d, 9.4)
8.03 (d, 8.1)
8.31 (t, 6.0)
4.18
4.15
1.93
1.69
0.79; 0.81
1.85 (acetyl CH3)
0.79 (CH3 δ)
1.41,1.05 (CH2); 0.79 (CH3)
Glu
Ala
Gln
Lys
Phe
Gly
4.26
1.75, 1.84
1.26
2.26, 2.61
ꢀ
4.07
ꢀ
2.07
1.16
ꢀ
ꢀ
4.02
1.76
7.31; 6.80 (NH2)
1.48, 1.36 (Hδ); 3.21 (Hε)
7.22 (Hδ); 7.24 (Hε); 7.17 (Hζ)
7.18, 7.08 (terminal CONH2)
4.25
1.55, 1.35
3.03, 2.80
ꢀ
4.49
3.56, 3.65
ꢀ
Exocyclic Nꢀsubstituent produced by cyclization
3.70, 3.85 (H1); 7.40 (H3); 1.23 (H4)
Cyclic peptide 6
Hα
Hβ
Others
Hγ
ꢀ
Residue
Phe
NH (m, J (Hz))
7.23 (Hδ); 7.25 (Hε); 7.18 (Hζ)
8.03 (d, 8.5)
4.52
2.93, 2.68
1.73 (acetyl CH3)
Lys
Gly
8.05 (d, 8.4)
8.44 ( m )
ꢀ
4.34
4.38, 3.72
4.16
1.68, 1.44
ꢀ
1.30
1.69, 1.42 (Hδ); 3.06, 3.42 (Hε)
ꢀ
ꢀ
Pro
2.19, 1.82
1.30
1.93; 1.89
3.73; 3.51 (δ)
Ala I
Asp
8.24 (d, 6.3)
7.53 (d, 7.6)
7.60 (d, 7.5)
4.14
ꢀ
ꢀ
ꢀ
ꢀ
4.59
3.12, 2.54
1.23
ꢀ
Ala II
4.09
7.01; 7.04 (terminal CONH2)
Exocyclic Nꢀsubstituent produced by cyclization
4.08, 3.60 (H1); 7.65 (H3); 3.11, 3.02 (H4);
1.63 (H5); 2.20 (H6); 3.58 (H8)
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Table 2. 13C NMR assignment for cyclic peptides 2, 4 and 6.
4
5
6
7
8
9
Cyclic peptide 2
Residue
Leu
CO
Cα
Cβ
Cγ
24.2
Others
21.3; 23.2 (2×CH3 δ)
28.1 (Cδ ), 48.4 (Cε)
128.8 (Cδ); 128.2 (Cε), 126.2 (Cζ)
ꢀ
171.8
171.4
170.2
172.4
170.4
172.5
52.9
52.4
54.3
56.0
50.5
49.0
40.9
32. 9
37.3
31.4
27.1
17.0
Lys
22.6
Phe
137.9
17.4; 19.2
27.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Val
Glu
171.4 (Cε)
Ala
ꢀ
52.0 (OCH3)
Cbz group
Exocyclic Nꢀsubstituent produced by cyclization
48.7 (C1); 167.3 (C2); 47.5 (C4);
32.3; 32.4; 25.2; 24.7; 24.7 (not
assigned)
155.9 (C1); 65.3 (C2); 137,2 (C3);
127.6 (C4); 128.4 (C5); 127.8 (C6)
Cyclic peptide 4
Residue
CO
Cα
Cβ
Others
Cγ
171.1
57.7
30.4
18.2; 19.3
22.5 (acetyl CH3); 165.9 (acetyl CO)
Val
Ile
170.4
170.3
171.9
171.2
171.7
171.3
170.7
56.7
51.9
48.9
53.1
51.7
54.0
41.9
36.6
27.7
17.8
27.1
31.9
37.4
ꢀ
24.4 (CH2); 15.2 (CH3)
11.07 (Cδ)
Glu
Ala
Gln
Lys
Phe
Gly
28.3
ꢀ
171.8 (Cδ)
ꢀ
31.3
21.9
137.6
ꢀ
173.8 (Cδ)
27.4 (Cδ); 47.9 (Cε)
129.2 (Cδ); 128.1 (Cε); 126.3 (Cζ)
ꢀ
Exocyclic Nꢀsubstituent produced by cyclization
47.5 (C1); 167.7 (C2); 50.1 (C4); 28.6 (C5)
Cyclic peptide 6
Residue
Phe
NH (m, J (Hz))
Hα
Hβ
Others
Hγ
129.2 (Cδ); 128.0 (Cε); 126.2 (Cζ);
169.1 (acetyl CO); 22.5 (acetyl CH3)
171.0
53.8
37.7
138.0
Lys
Gly
171.6
169.6
172.7
172.6
170.9
173.8
52.1
41.1
61.4
49.7
50.6
48.4
31.5
ꢀ
22.2
27.4 (Cδ) ; 48.6(Cε)
ꢀ
24.5
ꢀ
ꢀ
Pro
29.1
16.2
34.0
17.9
46.6 (Cδ)
Ala I
Asp
ꢀ
ꢀ
ꢀ
170.4
ꢀ
Ala II
Exocyclic Nꢀsubstituent produced by cyclization
48.5 (C1); 168.0 (C2); 38.0 (C4); 24.4 (C5);
30.1 (C6); 173.2 (C7); 51.3 (C8)
Cyclic peptide 4 also contains a
Nꢀsubstituted lactam bridge of Glu and Lys side chains at i
and
i
+3 positions, but with the opposed directionality with respect to peptide 2. Previously, it
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5
6
7
8
has been reported that the position of the amide group in the linker is crucial for stabilization
of helical structures in peptide crossꢀlinked at i, i+4 posistions.8e As a result, we sought to
evaluate whether the change in the amide position might be important also in the folding of
9
peptides crossꢀlinked at i, i+3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. A) Superimposition of 20 lowest energy structures of cyclic peptides 2 and 4. B)
Distribution in time of and angles (Ramachandran plots) for central amino acids of
φ
ψ
peptides 2 and 4, and some representative structures as obtained from MD simulation.
For peptide 4, 3JNHCHα coupling constants of residues Val1, Ile2, Lys6 and Phe7 were > 8Hz,
3
while JNHCHα values of central residues Glu3, Ala4 and Gln5 were between 6 Hz and 7 Hz.
Analysis of the ROESY spectrum of 4 resulted in a total of 43 distance constraints (13 strong,
16 medium and 14 weak; 28 interꢀresidual: 22 of them sequential and only 6 nonꢀsequential).
An important characteristic is the abundance of intense dαN(i, i+1) ROEs, which is indicative
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of
β
ꢀsheet structures, but the lack of long range dαN(i, j) and dNN (i, j) ROEs. Along with the
3
5
high JNHCHα values of residues Val1, Ile2, Lys6 and Phe7, ROESYꢀderived information
6
7
8
suggests
β
ꢀstrand conformations for sequences flanking the central amino acids Ala4 and
9
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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52
53
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55
56
57
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60
Gln5. During molecular modeling, five dihedral restraints from JNHCHα coupling constants
were considered. The stereospecific assignment of relevant methylene hydrogens was
possible after three rounds of simulation. Figure 3A (bottom) depicts the superimposition of
the final 20 lowest energy structures of 4 (0.68 Å RMSD difference to the mean structure for
the backbone), which comprise a
βꢀstrand with a change in the directionality due to the
presence of a reverse turn at the central residues. Further MD analysis carried out during 80
ns of simulation revealed that the reverse turn transits from a tighter (only 17% in time) to a
looser disposition (most populated), as shown in the two representative conformations
illustrated in Figure 3B (bottom). MD simulation also confirmed that the average distance
CαGlu3ꢀCαLys6 is around 7 Å (6.77
peptide 4, the irregularity in the
±
0.51 Å). In the looser conformations of the central turn of
β
ꢀstrand chain caused by residues Ala4 and Gln5 resembles a
β
ꢀbulge, i.e., a protuberance that affects the directionality of ꢀstrands but in a less drastic
β
manner than
β
ꢀturns.25 This is supported by the fact that
φ and ψ angles of residue Ala4 lay
within the rightꢀhanded helical region of Ramachandran plot, while the remaining residues
mostly populate the
bulges.25
βꢀsheet space (Figure 3B), which is a common characteristic of classic βꢀ
Combined NMR/MD analysis was also performed for peptide 6, which is crossꢀlinked at
i, i +
4 residues by a ꢀsubstituted lactam bridge. Previously, it has been reported that
N
lactamization of Lys and Asp residues located at
i, i + 4 positions is a very effective way to
stabilize an
α
ꢀhelix in small peptides.8a In our case, we chose to introduce the turnꢀinducing
amino acids Pro and Gly at the middle of the peptide sequence instead of using other
sequences favoring helicity (e.g., polyꢀAla or polyꢀLeu). Accordingly, we anticipated that
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6
cyclic peptide 6 could fold onto a pseudoꢀplanar
conformation.
αꢀturn rather than in a αꢀhelical
7
8
For peptide 6, 3JNHCHα coupling constants higher than 8 Hz were only found for residues Phe1
9
3
and Lys2, while JNHCHα values of the remaining residues were between 6 and 8 Hz. As a
10
11
12
13
14
15
16
17
18
19
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22
23
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42
43
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46
47
48
49
50
51
52
53
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56
57
58
59
60
result, there is no clear inclination either to helical or to βꢀstrand structures. A total of 42
distance constraints (6 strong, 7 medium and 29 weak; 19 interꢀresidual: 16 of them
sequential and only 3 nonꢀsequential) resulted from ROESY spectrum. As most interꢀresidual
ROE contacts were between sequential amino acids, these evidences indicate low propensity
to either helical or tight turn conformations.
Figure 4. A) Superimposition of 20 lowest energy structures of cyclic peptide 6. B) Plot of
CαLys2ꢀCαAsp6 distance vs time as obtained from MD simulation.
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5
6
7
8
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Figure 4A shows the superimposition of the 20 lowest energy NMRꢀderived structures of 6
(0.86 Å RMSD difference to the mean structure for the backbone), illustrating that this
peptide occurs in a rather loose reverse turn including five residues (αꢀturn). Analysis of the
distance CαLys2ꢀCαAsp6 reveals that, in fifteen of the twenty lowest energy structures, the value
is lower than 7 Å, whilst five structures had a distance greater than 7 Å. To get deeper insight
into the loose character of the αꢀturn, we performed a MD simulation during 60 ns starting
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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43
44
45
46
47
48
49
50
51
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53
54
55
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57
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60
from the NMRꢀderived lowest energy structure. Figure 4B depicts the plot of CαLys2ꢀCα
Asp6
distances versus time, indicating that such distances varies from 6.5 Å to around 9 Å.
However, most populated states lay within a distance of 7ꢀ7.5 Å, confirming the relatively
loose character of the reverse turn.
Conclusions
We have proven that the Ugi reaction is a suitable approach for the macrocyclization of
peptide side chains, enabling the simultaneous access to folded peptide conformations and
exocyclic
higher for side chainꢀtoꢀside chain combinations based on Glu/Asp and Lys residues, while
the cyclization of a carboxylic side chain with an ꢀterminal amine provided poorer results.
Nꢀfunctionalization at the resulting lactam bridge. Macrocyclization efficiency was
N
Side chainꢀtoꢀside chain crossꢀlinked peptides were analyzed by NMR and MD simulation to
assess the folding effect of the Ugi macrocyclization. Thus, cyclization at i→i+3 positions led
to folded structures such as an helical turn and a loose βꢀturn resembling a βꢀbulge, while the
i→i+4 combination resulted in formation of a loose αꢀturn. The most populated states arising
from molecular dynamics simulation are consisted with the NMRꢀderived structures of the
three modelled compounds, proving the preference for the folded conformations of the
peptide backbone skeletons. Ongoing studies are directed to assess the effect of the
Nꢀ
substituent nature on the stability of specific secondary structures as well as the utilization of
such exocyclic appendages as derivatization sites, e.g., for lipidation or glycosylation.
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Experimental Section
General
3
4
5
6
7
8
9
1H NMR and 13C NMR spectra were recorded at 400/600 MHz for 1H, 100/150 MHz for 13C,
at 300K. Chemical shifts (δ) are reported in parts per million relative to TMS using the
10
11
12
13
14
15
16
17
18
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20
21
22
23
24
25
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32
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35
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
residual solvent signals as secondary standards. Coupling constants (J) are reported in Hertz.
Splitting patterns that could not be easily interpreted are designated as multiplet (m) or broad
singlet (br. s). Carbon resonances were assigned using additional information provided by
DEPT spectra recorded with phase angles of 135º. For compounds subjected to structure
determination, NMR peak assignments were accomplished by analysis of standard TOCSY
(mixing time 60 ms), gꢀCOSY, gꢀHSQC, HMBC and trꢀROESY (500 ms spinꢀlock) spectra.
Peptides were characterized by electrospray ionization mass spectrometry (ESIꢀMS) in a
hybrid quadrupoleꢀ timeꢀofꢀflight instrument (QTOF1, Waters, USA) fitted with a nanospray
ion source. All reagents and solvents were used as received, with the exception of CH2Cl2,
DMF and DIPEA that were dried by distillation from CaH2 over argon prior to use as reaction
solvent, and DMF was stored over 4Å molecular sieves. Flash column chromatography was
carried out using silica gel 60 (230ꢀ400 mesh) and analytical thin layer chromatography
(TLC) was performed using silica gel aluminum sheets.
General solution-phase peptide synthesis: The
Nꢀprotected amino acid (1.0 mmol, 1.0
equiv.), HOBt (149 mg, 1.1 mmol, 1.1 equiv.), EDC (241 mg, 1.1 mmol, 1.1 equiv.) and the
amino acid methyl ester (or peptide methyl ester) hydrochloride are suspended in dry CH2Cl2
(10 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 of 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
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4
5
mL). The organic phase is dried over MgSO4, filtered and concentrated under reduced
pressure to dryness.
6
7
8
General methyl ester removal procedure: The peptide (1.0 mmol) is dissolved in THF/H2O
9
(2:1, 30 mL) and LiOH (105 mg, 2.5 mmol) is added at 0
for 3 h and then acidified with aqueous 10% NaHSO4 to pH 3. The resulting phases are
separated and the aqueous phase is additionally extracted with EtOAc (2 50 mL). The
organic phases are combined and dried over anhydrous Na2SO4 and concentrated under
reduced pressure to yield the ꢀdeprotected peptide.
°C. The mixture is stirred at 0 °C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
×
C
General Cbz removal procedure: The product is dissolved in absolute EtOH (30 mL) and
10% Pd/C (80 mg) is added. The mixture is subjected successively to hydrogen atmosphere
and vacuum and finally stirred under hydrogen atmosphere (1 atm.) for 24 h. The catalyst is
removed by filtration over a pad of Celite and the filtrate is evaporated under reduced
pressure.
General Boc and tert-butyl ester removal procedure: The crude peptide is exposed to high
vacuum for 1 hour before dissolving it in a 4 M HCl solution in dioxane (2 mL) for
simultaneous Boc and tBu removal. 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 for about 30 min.
The volatiles are then fully removed by concentrating the resulting thick oily residue under
reduced pressure in the rotary evaporator and then placing the flask under high vacuum. If
required, the hydrochloride salt can be crystallized from frozen diethyl ether.
CbzꢀLeuꢀLys(Boc)ꢀPheꢀValꢀGlu(tBu)ꢀAlaꢀOMe (1): CbzꢀGlu(tBu)ꢀOH (337 mg, 1.0 mmol)
was coupled to HCl·AlaꢀOMe (140 mg, 1.0 mmol) according to the general solutionꢀphase
peptide synthesis procedure, following by deprotection of the
Nꢀterminus by Cbz removal.
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The same protocol was employed for the coupling of CbzꢀValꢀOH (251 mg, 1.0 mmol), and
4
5
6
7
8
CbzꢀPheꢀOH (270 mg, 0.9 mmol) to obtain the tetrapeptide CbzꢀPheꢀValꢀGlu(
with 54% yield (361 mg, 0.54 mmol) as a white amorphous solid after liquid chromatography
purification in silica ( ꢀhexane/AcOEt 5:1). After ꢀCbz deprotection according to the
tBu)ꢀAlaꢀOMe
9
n
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
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34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
general procedure, the tetrapeptide was coupled with CbzꢀLys(Boc)ꢀOH (204 mg, 0.54
mmol), and CbzꢀLeuꢀOH (125 mg, 0.47 mmol) respectively. Flash column chromatography
purification (CH2Cl2/MeOH 25:1) furnished hexapeptide CbzꢀLeuꢀLys(Boc)ꢀPheꢀValꢀ
Glu(
(600 MHz, DMSOꢀd6):
= 7.8 Hz, 1H, N ), 7.83 (d,
8.3 Hz, 1H, N ), 7.36–7.27 (m, 5H, Ar), 7.23–7.13 (m, 5H, Ar), 6.69 (t,
(s, 2H), 4.56 (m, 1H), 4.29 (dd, = 15.2, 7.8 Hz, 1H), 4.27 (dd, = 13.3, 8.8 Hz, 1H), 4.21–
4.17 (m, 1H), 4.17 (dd, = 8.5, 7.0 Hz, 1H), 4.01 (m, 1H), 3.61 (s, 3H), 3.00 (dd, = 13.9, 4.6
Hz, 1H), 2.84 (dd, = 12.1, 5.9 Hz, 2H), 2.79 (dd, = 14.2, 9.1 Hz, 1H), 2.27–2.23 (m, 2H),
1.97–1.90 (m, 2H), 1.79–1.71 (m, 1H), 1.61–1.51 (m, 2H), 1.37 (s, 9H), 1.35 (s, 9H,
t
Bu)ꢀAlaꢀOMe (334 mg, 0.33 mmol, 33% overall) as a white amorphous solid. 1H NMR
8.20 (d, = 7.6 Hz, 1H, N ), 8.06 (d, = 7.2 Hz, 1H, N ), 7.98 (d,
= 11.1 Hz, 1H, N ), 7.83 (d, = 5.3 Hz, 1H, N ), 7.40 (d,
= 5.4 Hz, 1H), 5.01
δ
J
H
J
H
J
H
J
H
J
H
J =
H
J
J
J
J
J
J
J
C(C
H
3)3), 1.48 – 1.08 (m, 13H), 1.21 (d,
J
= 7.1 Hz, 3H), 0.86–0.78 (m, 12H), 0.85–0.78 (m,
172.5, 172.2, 172.2, 171.5, 171.5, 170.7, 170.4,
1H).13C NMR (150 MHz, DMSOꢀd6):
δ
156.0, 155.7 (
77.5( ); 65.5 (
37.3, 32.0, 30.9 (
C
O); 137.7, 137.1 (
C
); 129.2, 128.5, 128.1, 127.9, 127.7, 126.3 (
CH); 80.0,
C
C
H2); 57.5, 53.6, 53.2, 52.5 (
C
H); 52.1 ( H3); 51.1, 48.0 ( H); 40.7; 39.5,
C
C
C
H2); 30.9 (
CH); 29.3 (CH2); 28.4, 27.8 (
CH3); 26.3 (CH2); 24.3(CH); 23.2
(CH2); 22.6, 21.5, 19.2, 18.2, 18.1(C
H3). ESIꢀMS m/z: 1032.58 [M+Na]+, calcd. for
C52H79O13NaN7: 1032.56. This product (334 mg, 0.33mmol) was subjected to simultaneous
Boc and tertꢀbutylester removal according to the general procedure to afford peptide 1 (256
mg, 0.3 mmol, 91%). The final mixture was recrystalized from Et2O/AcOEt (10 mL, 5:1) and
used forward without further purification.
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General solid-phase peptide synthesis: Peptides were synthesized manually on AmꢀMBHA
4
5
6
7
8
resin (250 mg, 0.45 mmol/mg) by a stepwise Fmoc/
using DIC/HOBt activation, and completion of the coupling reaction was monitored by the
ninhydrin test. Fmocꢀdeprotection was carried out using 20% piperidine solution in DMF.
tBu strategy. Amino acids were coupled
9
N
ꢀ
10
11
12
13
14
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16
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terminal acetylation was accomplished with 20% Ac2O in DMF and DIEA for 30 min.
Peptides were cleaved from the resin with the cocktail TFA/TIPS/water (95:2.5:2.5),
precipitated from frozen diethyl ether, then taken up in 1:1 acetonitrile/water and lyophilized.
Linear peptides were analyzed by HPLC in a reverseꢀphase (RP) C18 column (Vydac, 4.6 ×
150 mm, 5
ꢁm) to prove purity > 90%, and characterized by ESIꢀMS.
AcꢀValꢀIleꢀGluꢀAlaꢀAsnꢀLysꢀPheꢀGlyꢀNH2 (3): The peptide (101 mg) was obtained in 94.5 %
purity by the general solidꢀphase peptide synthesis.
[M+H]+, calcd. for C43H70O11N10: 932.52.
Rt= 13.8 min. ESIꢀMS m/z: 932.54
AcꢀPheꢀLysꢀGlyꢀProꢀAlaꢀAspꢀAlaꢀNH2 (5): The peptide (95 mg) was obtained in 91.3%
purity by the general solidꢀphase peptide synthesis.
[M+H]+, calcd. for C39H59O14N10: 746.38.
Rt= 10.3 min. ESIꢀMS m/z: 746.41
BocꢀPheꢀValꢀIleꢀGlu(
to CbzꢀIleꢀOH (265 mg, 1.0 mmol) according to the general peptide coupling procedure,
following by deprotection of the ꢀterminus by Cbz removal. The same protocol was
tBu)ꢀOMe (7): HꢀGlu(tBu)ꢀOMe·HCl (254 mg, 1.0 mmol) was coupled
N
employed for the coupling of CbzꢀValꢀOH (241 mg, 0.96 mmol). After Cbz removal, a final
coupling with BocꢀPheꢀOH (210 mg, 0.79 mmol) was performed. Flash column
chromatography purification (CH2Cl2/MeOH 25:1) furnished pure tetrapeptide BocꢀPheꢀValꢀ
1
IleꢀGlu(
CDCl3):
1H), 4.38 (d,
t
Bu)ꢀOMe (284 mg, 42% yield) as a white amorphous solid. H NMR (400 MHz,
δ
7.29–7.07 (m, 5H), 4.71 (t,
J
= 6.7 Hz, 1H), 4.56–4.49 (m, 1H), 4.47 (d,
J = 7.1 Hz,
J
= 5.6 Hz, 1H), 3.53 (s, 3H), 3.14 (dd,
J
= 12.3, 10.1 Hz, 1H), 2.94 (dd, J =
12.3, 4.2 Hz, 1H), 2.68–2.51 (m, 2H), 2.26–1.84 (m, 4H), 1.41 (s, 9H), 1.35 (s, 9H), 1.31ꢀ1.12
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(m, 2H), 1.08–0.73 (m, 12H). 13CꢀNMR (100 MHz, CDCl3):
171.8, 158.6 ( O); 137.3 ( ); 129.4, 129.2, 127.1 ( H); 81.9; 80.5 (
H3); 51.7 ( H); 38.3 (
δ
173.3, 173.0, 172.2, 172.1,
C
C
C
C
); 57.5, 56.7, 55.2 ( H);
C
52.4 (
C
C
C
C
H2); 35.5 (
C
H); 31.1 (
C
H2); 30.4 (
C
H); 28.6, 28.1 (
C
H3); 26.1,
24.8 (
H2); 20.2, 19.5, 16.3, 11.6 (
C
H3). ESIꢀMS (ESIꢀFTꢀICR) m/z: 699.42 [M+Na]+, calcd.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
for C35H56O9NaN4: 699.39. This product was subjected to Boc and tertꢀbutyl ester
simultaneously removal according to the general procedure to afford peptide 7 (198 mg, 0.38
mmol). The final mixture was recrystalized from Et2O/AcOEt (10 mL, 5:1) and used forward
without further purification.
CbzꢀValꢀLys(Boc)ꢀLeuꢀPheꢀAlaꢀOMe (9): HꢀAlaꢀOMe·HCl 140 mg, 1.0 mmol) was coupled
to BocꢀPheꢀOH (265 mg, 1.0 mmol) according to the general solutionꢀphase peptide synthesis
procedure, following by deprotection of the
Nꢀterminus by Boc removal. The same protocol
was employed for the coupling of BocꢀLeuꢀOH (231 mg, 1.0 mmol), and CbzꢀLys(Boc)ꢀOH
(327 mg, 0.86 mmol) to obtain the tetrapeptide CbzꢀLys(Boc)ꢀLeuꢀPheꢀAlaꢀOMe. After Cbz
removal, a final coupling with CbzꢀValꢀOH (196 mg, 0.78 mmol) was performed. Flash
column chromatography purification (CH2Cl2/MeOH 25:1) furnished pure pentapeptide Cbzꢀ
1
ValꢀLys(Boc)ꢀLeuꢀPheꢀAlaꢀOMe (322 mg, 39% overall) as a white amorphous solid. H
NMR (400 MHz, CDCl3/CD3OD 95:5):
1H), 4.52 (t, = 5.7 Hz, 1H), 4.40–4.32 (m, 2H), 4.16 (q,
(dd, = 13.4, 10.8 Hz, 1H), 3.21–3.08 (m, 2H), 2.92 (dd,
δ
7.35–7.11 (m, 10H), 5.63 (s, 2H), 4.65–4.56 (m,
= 5.3 Hz ,1H), 3.65 (s, 3H), 3.25
= 13.3, 4.8 Hz, 1H), 2.06–1.99 (m,
= 5.2 Hz, 3H), 1.08 – 0.79
J
J
J
J
1H), 1.84–1.68 (m, 2H), 1.54–1.16 (m, 7H), 1.34 (s, 9H), 1.30 (d,
(m, 12H). 13C NMR (100 MHz, CDCl3/CD3OD 95:5):
J
δ
173.1, 172.5, 172.2, 171.1, 168.1,
H); 79.7 ( );
158.3, 157.8 (
66.9 ( H2); 57.6, 54.4, 53.8 (
30.5 ( H); 29.8 ( H2); 28.0 (
C
O); 136.5, 135.4 (
H); 52.1(
H3); 25.3 (
C
); 129.6, 129.4, 128.4, 128.3, 127.9, 127.0 (
H3); 51.4, 48.3( H); 42.5, 40.9, 38.6, 33.2 (
H); 24.0 ( H2); 20.7, 19,4, 18.2, 18.2, 17.4 (
C
C
C
C
C
C
C
H2);
H3).
C
C
C
C
C
C
ESIꢀMS (ESIꢀFTꢀICR) m/z: 847.50 [M+Na]+, calcd. for C43H64O10NaN6: 847.46. This
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product was subjected to Boc and methyl ester removal according to the general procedures to
afford peptide 9 (234 mg, 0.33 mmol). The final mixture was recrystalized from Et2O/AcOEt
(10 mL, 5:1) and used forward without further purification.
9
10
11
12
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14
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General procedure for the Ugi reaction-based macrocyclization: A solution of
paraformaldehyde (0.13 mmol, 1.3 equiv.), Et3N (0.1 mmol, 1 equiv) and the peptide (0.1
mmol, 1 equiv.) in MeOH (5 mL) was stirred for 1 h at room temperature and next diluted to
50 mL of MeOH. The isocyanide (0.2 mmol, 2 equiv.) was added and the reaction mixture
was stirred for 96 h and then concentrated under reduced pressure. The crude peptide was
dissolved in the minimum amount of TFA, precipitated from frozen diethyl ether, then taken
up in 1:1 acetonitrile/water and lyophilized. Peptides were purified by preparative RPꢀHPLC
on the HPLC system LaChrom (Merck Hitachi, Germany). Separation was achieved by RP
C18 column (Vydac, 25 × 250 mm, 25 ꢁm). A linear gradient from 15% to 45% of solvent B
over 50 min at a flow rate of 5 mL/min was used. Detection was accomplished at 226 nm.
Solvent A: 0.1% (v/v) TFA in water. Solvent B: 0.05% (v/v) TFA in acetonitrile.
N-Substituted cyclic peptide 2: Peptide 1 (85 mg, 0.1 mmol), cyclohexylisocyanide (20 ꢁL,
0.15 mmol), paraformaldehyde (4 mg, 0.13 mmol) were reacted according to the general
cyclization procedure. Purification by preparative RPꢀHPLC afforded the cyclic peptide 2 (55
mg, 56%) as a white amorphous solid. Purity of 98% as determined by analytical RPꢀHPLC.
R
t = 14.96 min. ESIꢀMS m/z: 997.59 [M+H]+, calcd. for C51H74O11N8Na: 997.54.
N-Substituted cyclic peptide 4: Peptide 3 (93 mg, 0.1 mmol), tertꢀbutylisocyanide (17 ꢁL,
0.15 mmol), paraformaldehyde (4 mg, 0.13 mmol) were reacted according to the general
cyclization procedure. Purification by preparative RPꢀHPLC afforded the pure cyclic peptide
4 (58 mg, 56%) as a white amorphous solid. Purity of 97% as determined by analytical RPꢀ
HPLC.
R
t= 15.53 min. ESIꢀMS m/z: 1027.59 [M+H]+, calcd. for C49H79O12N12: 1027.59.
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N-Substituted cyclic peptide 6: Peptide 5 (89 mg, 0.1 mmol), methyl 4ꢀisocyanobutanoate
4
5
6
7
8
(21 ꢁL, 0.15 mmol), paraformaldehyde (4 mg, 0.13 mmol) were reacted according to the
general cyclization procedure. Purification by preparative RPꢀHPLC afforded the pure cyclic
peptide 6 (32 mg, 36%) as a white amorphous solid. Purity of 95% as determined by
9
10
11
12
13
14
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analytical RPꢀHPLC.
885.45.
R
t= 14.65 min. ESIꢀMS m/z: 885.44 [M+H]+, calcd. for C41H61O12N10:
N-substituted cyclic peptide 8: Peptide 7 (56 mg, 0.1 mmol), tertꢀbutylisocyanide (17 ꢁL,
0.15 mmol), paraformaldehyde (6 mg, 0.2 mmol) were reacted according to the general
cyclization procedure. Purification by Flash column chromatography afforded the pure cyclic
peptide 8 (27 mg, 44%) as a white amorphous solid. 1H NMR (600 MHz, acetoneꢀd6):
δ 7.73
(bs, 1H), 7.51 (bs, 1H), 7.38 (bs, 1H), 7.36 – 6.98 (m, 6H), 5.10ꢀ5.01 (m, 1H), 4.84ꢀ4.76 (m,
1H), 4.38ꢀ4.30 (m, 1H), 4.23ꢀ4.17 (m, 1H), 3.90 – 3.79 (m, 2H), 3.66 (s, 3H), 3.50ꢀ
3.41(m,1H), 3.15 – 3.05 (m, 1H), 2.63 (m, 1H), 2.54 – 1.88 (m, 4H), 1.70ꢀ1.60 (m, 1H), 1.34
(s, 9H), 1.41 – 1.14 (m, 2H), 1.04 – 0.81 (m, 12H). 13C NMR (150 MHz, acetoneꢀd6):
176.0, 173.8, 173.1, 172.0, 171.5, 170.6 ( O); 138.9 ( ); 129.7, 129.3, 127.7 ( H); 63.5,
63.2, 62.6 ( H); 53.3 ( ), 51.9 ( H); 50.7 ( H3); 48.0 ( H2); 36.8 ( H2), 36.6 ( H); 34.6
H3). ESIꢀMS (ESIꢀ
δ
C
C
C
C
C
C
C
C
C
C
(C
H2); 31.5 (
CH); 28.7 (CH3); 25.7, 25.3 (CH2), 19.9, 19.8, 16.1, 12.3 (C
FTꢀICR) m/z: 638.36 [M+Na]+, calcd. for C32H49O7NaN5: 638.35.
N-Substituted cyclic peptide 10: Peptide 9 (71 mg, 0.1 mmol), cyclohexylisocyanide (20 ꢁL,
0.15 mmol), paraformaldehyde (6 mg, 0.2 mmol) were reacted according to the general
cyclization procedure. Purification by flash column chromatography afforded the pure cyclic
1
peptide 10 (42 mg, 51%) as a white amorphous solid. H NMR (600 MHz, CDCl3/CD3OD
95:5):
= 10.2, 3.7 Hz, 1H), 4.69 (dd,
4.21 (q, = 5.7 Hz, 1H), 4.12 (d,
δ
7.51 – 7.29 (m, 10H), 5.26 (d,
J = 12.4 Hz, 1H), 5.22 (d, J = 12.4 Hz, 1H), 4.77 (dd, J
J
= 9.5, 3.1 Hz, 1H), 4.47 (d,
J
= 6.0 Hz, 1H), 4.35 (m, 1H),
J
J
= 16.0 Hz, 1H), 3.97 (d, J = 16.0 Hz, 1H), 3.84ꢀ3.77 (m,
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1H), 3.59 – 3.50 (m, 1H), 3.33 – 3.23 (m, 1H), 3.07 (dd,
J = 14.3, 10.4 Hz, 1H), 2.08 – 2.00
4
5
6
(m, 1H), 1.98ꢀ1.90 (m, 2H), 1.89 – 1.69 (m, 7H), 1.69ꢀ 1.24 (m, 10H), 1.52 (d,
J = 7.2 Hz,
3H), 1.12 (d,
J = 6.7 Hz, 3H), 1.08 (d, J = 6.4 Hz, 6H), 1.05 (d, J
= 6.5 Hz, 3H). 13C NMR
7
8
9
(150 MHz, CDCl3/CD3OD 95:5):
136.6, 135.9 ( ); 128.3, 127.9, 127.9, 127.5, 127.1, 126.2 (
50.9, 49.3 ( H); 49.2 ( H2), 48.9 ( H), 48.3, 40.3, 36.4, 32.0, 31.9 (
26.4 ( H2), 24.9 ( H), 24.4, 24.1 ( H2), 21.9, 20.7, 18.4, 17.2, 16.2 (C
δ
173.1, 172.8, 171.6, 171.4, 171.1, 168.3, 156.8 (
H), 66.3 ( H2), 57.5, 55.1, 53.5,
H2); 30.9 ( H); 27.4,
H3). ESIꢀMS (ESIꢀFTꢀ
CO);
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
26
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31
32
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43
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C
C
C
C
C
C
C
C
C
C
C
ICR) m/z: 854.48 [M+Na]+, calcd. for C45H65O8NaN7: 854.48.
NMR structure determination
Crossꢀpeaks in tꢀROESY spectra were assigned and integrated in Sparky NMR 26
Distance
.
constraints from ROE intensities were generated using pseudoꢀatoms corrections where
needed, and placed into three groups: strong (2.8 Å upper limit), medium (3.5 Å upper limit)
and weak (5.0 Å upper limit). The lower limit for NOE restraints was always maintained at
3
1.8 Å. Backbone dihedral angle restraints were inferred from JNHCHa coupling constants in
3
1D spectrum at 300 K,
φ
was restrained to ꢀ120
±
30° for JNHCHa ≥ 8 Hz as reported by
Fairlie et. al..8e Peptide bond
ω
angles were all set trans. Structure calculations were carried
out using XplorꢀNIH 2.33 package.24 The calculations were performed using the standard
force field parameter set (PARALLHDG.PRO) and topology file (TOPALLHDG.PRO)
within XplorꢀNIH with inꢀhouse modifications to ensure peptide cyclization and amide
substitution. Structures were visualized and analyzed using VMDꢀXplor
Nꢀ
.
NMR structure determination was performed through simulated annealing regularization and
refinement in torsion angle space, using experimental data as interꢀproton distances and
dihedral angles restraints. For simulated annealing regularization 200 starting structures were
randomly generated. A 100 ps molecular dynamics simulation at 3500 K was performed with
a timeꢀstep of 3 fs. The system was cooled from 3500 to 25 K, with a temperature step of 12.5
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K. At each temperature step, 0.2 ps of molecular dynamics simulation was performed. A 500
steps torsion angle minimization was performed and finally the system was optimized by
means of 500 steps conjugated gradient Powell Cartesian minimization.
9
10
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The refinement protocol consisted in a slow cooling simulated annealing from the regularized
structures. A 10 ps molecular dynamics simulation at 3000 K was achieved with a timeꢀstep
of 3 fs. The system was cooled with a temperature step of 12.5 K and a simulation time of 0.2
ps at each temperature. A 500 torsion angle minimization was performed afterwards a second
500 steps minimization was achieved in Cartesian coordinates. A finally 1000 steps Powell
minimization with an energy function nonꢀdependent of experimental restraints was executed.
(for more details, see Supporting Information)
Molecular Dynamics Simulations
Molecular dynamics simulation was performed with the NAMD27 software taking as starting
coordinates the corresponding NMR structure. The CHARMMꢀ3628 force field was used with
convenient parameterizations for nonꢀstandard residues, i.e. peptide
Nꢀfuncionalized
cyclization, with the help of Paratool plugin within VMD29 molecular package. Before each
simulation 60 ps MD equilibration was performed. All simulations were performed using
periodic boundary conditions in a cubic box with explicit solvent. The model proposed by
Laaksonen30 was employed for the treatment of DMSO molecules. Simulation temperature
was always set to 310 K, using Langevin dynamics for temperature control (damping
coefficient of 1/ps). Langevin piston was utilized for maintaining a pressure of 1 atm. A 12 Å
cutoff with a switching function was employed for nonꢀbonded Van der Waals and
electrostatics interactions. A 2 fs timeꢀstep was always used with the SHAKE algorithm for
freeze hydrogenꢀheavy atom interatomic distances. Final trajectories were analyzed and
visualized in VMD29 and Vega ZZ 31 softwares.
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Supporting Information: ESIꢀMS and HPLC chromatograms of linear peptides produced by
solidꢀphase peptide synthesis. HPLC chromatograms, ESIꢀMS and NMR spectra of all final
cyclic peptides. This material is available free of charge via the Internet at http://pubs.acs.org/.
References
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11
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1. (a) Robinson, J. A. J. Pept. Sci. 2013, 19, 127. (b) Craik, D. J.; Fairlie, D. P.; Liras, S.;
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K.; Taneja, V.; Satyanarayanajois, S. D. J. Med. Chem. 2011, 54, 5307. (c) Bodé, C. A.;
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L.; Daugherty, M. D.; Franke, A. D.; Guy, K. J. Am. Chem. Soc. 2006, 128, 3496. (d)
Woolley, G. A.; Jaikaran, A. S. I.; Berezovski, M.; Calarco, J. P.; Krylov, S. N.; Smart O. S.;
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Sci.) 2002, 66, 49. (g) Condon, S. M.; Morize, I.; Darnbrough, S.; Burns, C. J.; Miller, B. E.;
Uhl, J.; Burke, K.; Jariwala, N.; Locke, K.; Krolikowski, P. H.; Kumar, N. V.; Labaudiniere,
R. F. J. Am. Chem. Soc. 2000, 122, 3007.
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Maekawa, H.; Ge, N.ꢀH.; Görbitz, C. H.; Rongved, P.; Ottersen, O. P.; AmiryꢀMoghaddam,
M.; Klaveness, J. J. Org. Chem. 2011, 76, 1228. (c) Cantel, S.; Isaad, A. L. C.; Scrima, M.;
Levy, J. J.; DiMarchi, R. D.; Rovero, P.; Halperin, J. A.; D’Ursi, A. M.; Papini, A. M.;
Chorev, M. J. Org. Chem. 2008, 73, 5663.
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