Design and stereoselective synthesis of ProM-2: A spirocyclic diproline mimetic with polyproline type II (PPII) helix conformation

Article abstract of DOI:10.1002/chem.201406493

With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Full text of DOI:10.1002/chem.201406493

DOI: 10.1002/chem.201406493
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&
Peptidomimetics
Design and Stereoselective Synthesis of ProM-2: A Spirocyclic
Diproline Mimetic with Polyproline Type II (PPII) Helix
Conformation
CØdric Reuter,[a] Robert Opitz,[b] Arne Soicke,[a] Stephan Dohmen,[a] Matthias Barone,[b]
Slim Chiha,[a] Marco Tobias Klein,[a] Jçrg-Martin Neudçrfl,[a] Ronald Kühne,*[b] and Hans-
Günther Schmalz*[a]
Abstract: With the aim of developing polyproline type II
helix (PPII) secondary-structure mimetics for the modulation
of prolin-rich-mediated protein–protein interactions, the
novel diproline mimetic ProM-2 was designed by bridging
the two pyrrolidine rings of a diproline (Pro–Pro) unit
through a Z-vinylidene moiety. This scaffold, which closely
resembles a section of a PPII helix, was then stereoselectively
synthesized by exploiting a ruthenium-catalyzed ring-closing
metathesis (RCM) as a late key step. The required vinylpro-
line building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=
tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester,
were prepared on a gram scale as pure stereoisomers. The
difficult peptide coupling of the sterically demanding build-
ing blocks was achieved in good yield and without epimeri-
zation by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate (HATU)/N,N-diisopropyl-
ethylamine (DIPEA). The RCM proceeded smoothly in the
presence of the Grubbs II catalyst. Stereostructural assign-
ments for several intermediates were secured by X-ray crys-
tallography. As a proof of concept, it was shown that certain
peptides containing ProM-2 exhibited improved (canonical)
binding towards the Ena/VASP homology 1 (EVH1) domain
as a relevant protein interaction target.
Introduction
challenging targets for the development of small molecules
that act as specific inhibitors/modulators of such interactions
by mimicking the PRM in the active conformation.
The search for small molecules that allow the selective modu-
lation of pharmacologically relevant protein–protein interac-
tions has become an important challenge in chemical biology
and medicinal chemistry.[1] Once the active conformation of
short peptide sequences, which usually take over this function
in nature, has been determined, it is, in principle, possible to
design and synthesize geometrically defined peptide mimet-
ics[2] to target such interactions.[3] As an important example,
proline-rich recognizing domains (PRDs) are able to selectively
bind to proline-rich motifs (PRMs) in a structurally well-under-
stood fashion.[4] These protein–protein interactions play an im-
portant role in a variety of relevant cellular processes, such as
tyrosine kinase receptor signaling,[5] endocytosis,[6] cytoskeletal
rearrangements,[7] transcription,[8] and splicing,[9] and represent
A constitutive property of all PRMs is that their core motif
needs to adopt a left-handed polyproline type II (PPII) helix
conformation to be recognized by the respective PRD. PPII
helices are characterized by a complete lack of main-chain hy-
drogen bonding and exhibit a helical pitch of 9.3 Š (three resi-
dues per turn) with F and Y angles of about À75 and 1458,
respectively.[10] Based on this information, we recently intro-
duced the “rationally designed” scaffold ProM-1 as a rigid di-
proline analogue that displayed a virtually perfect PPII confor-
mation (Figure 1).[11,12] Herein, we now report the design and
synthesis of a second PPII-structured dipeptide mimetic, that
is, the spirocyclic scaffold ProM-2, as an alternative module
(Pro–Pro substitute) for the development of geometrically de-
fined PRD-binding small molecules.
[a] Dr. C. Reuter, Dr. A. Soicke, S. Dohmen, S. Chiha, M. T. Klein,
Dr. J.-M. Neudçrfl, Prof. Dr. H.-G. Schmalz
Department of Chemistry, University of Cologne
Greinstrasse 4, 50939 Cologne (Germany)
E-mail: schmalz@uni-koeln.de
Results and Discussion
Design
[b] Dr. R. Opitz, M. Barone, Dr. R. Kühne
Leibniz-Institut für Molekulare Pharmakologie (FMP)
Campus Berlin-Buch, Robert-Rçssle-Str. 10
13125 Berlin (Germany)
Taking a model of a Pro–Pro unit in a PPII helix conformation
as a starting point, we recognized that the geometry could be
locked by stereoselectively introducing a stiff vinylidene bridge
between the two pyrrolidine rings. Initially, this led to the
design of the scaffold ProM-1 (Figure 1),[12] which was subse-
E-mail: kuehne@fmp-berlin.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201406493.
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domain by means of molecular modeling.[16] As depicted in
Figure 2, the model suggested that the decreased binding af-
finity of the peptide with the F[ProM-1]PP core resulted from
an unfavorable steric interaction between the vinylidene
bridge of ProM-1 and the protein surface, leading to a partial
displacement of the ligand (see Figure 2a and b). To avoid this
unfavorable interaction, a different Pro–Pro mimetic (still rigidi-
fied in a PPII conformation, but with a different backbone ge-
ometry) was required. After re-analyzing the model of the
parent PPII diproline structure, we found that rigidification
could also be achieved by introducing a vinylidene bridge be-
tween the two pyrrolidine rings in a different manner. Thus,
the spirocyclic scaffold ProM-2 was conceived (Figure 1). The
computational model suggested that the introduction of
ProM-2 as a substitute for the first two proline units of the
core motif of the ActA-derived ligand (i.e., F-[ProM-2]-PP)
should lead to a better fit (i.e., a closer contact) to the binding
site, and therefore, to a higher binding affinity (Figure 2c and
d).
Figure 1. Design of the PPII Pro–Pro mimetics ProM-1 (previous work)[11,12]
and ProM-2 (this work). In both structures, the diproline unit is locked in
a virtually perfect PPII helix conformation.
quently incorporated as a Pro–Pro substitute into the core
motif of a PRM ligand binding to the SH3 domain.[12a] Indeed,
the resulting ProM-1-containing peptides exhibited pro-
nounced binding affinity to the PRD target (SH3 domain).
Thus, we could demonstrate (for the first time) that it was pos-
sible to modify the core motif of a proline-rich ligand without
complete loss of affinity.[12a]
More recently, we turned our attention to the Ena/VASP (vas-
odilator-stimulated phosphoprotein) proteins as a relevant
target for the design of PRD-binding ligands. Ena/VASP protein
family members are multifunctional regulators involved in the
remodeling of the actin cytoskeleton,[13] that is, fundamental
cellular processes, including axon guidance and cell migration.
All Ena/VASP proteins contain an Ena/VASP homology 1 (EVH1)
domain specialized in recognizing PRMs as natural interaction
partners.[4a,5c] A pathological ligand is the protein ActA of the
intracellular pathogen Listeria monocytogenes, which is known
for causing listeriosis in mammals.[14] In this particular case, the
Ena/VASP-EVH1 domain recognizes four sequence sections of
ActA that all contain a FPxxP motif and satisfy the consensus
motif (F/W)PxfP of the Ena/VASP-EVH1 domains, in which
x represents any amino acid and f is any aliphatic amino
acid.[5a,c] Notably, the first and last proline residues are essential
for binding, and therefore, are highly conserved in the partner
proteins of Ena/VASP-EVH1 domains. Starting with the ActA-
derived peptide Ac-332SFEFPPPPTEDEL-NH2, we tried to replace
pairs of prolines in the ActA-derived model ligand by ProM-1.
We found that Ac-SFE-FPP[ProM-1]-TEDEL-NH2 showed im-
proved binding to the EVH1 domain of VASP and all four pro-
lines could even be substituted for ProM-1 (i.e., Ac-SFE-
F[ProM-1][ProM-1]-TEDEL-NH2) without loss of affinity relative
to the wild-type peptide. However, substitution of the first two
prolines by ProM-1 (i.e., Ac-SFE-F[ProM-1]PP-TEDEL-NH2) re-
sulted in a decrease of the binding affinity to the VASP-EVH1
domain.[15]
Figure 2. Calculated superimposed molecular models (in two perspectives)
of the core regions of PRM ligands binding to the Ena/VASP-EVH1 domain.
Wild-type ligand FPPPP (a; gray) and the synthetic ligand F[ProM-1]PP (b;
yellow with ProM-1 shown in green); wild-type ligand FPPPP (c; gray) and
the synthetic ligand F[ProM-2]PP (d; yellow with ProM-2 shown in magen-
ta). The models are based on the crystal structure 1evh[17] of the EnaH-EVH1
domain and on crystal structures of ProM-1 and ProM-2.
Synthesis
To synthesize the ProM-2 scaffold (as its fluorenylmethyloxycar-
bonyl (Fmoc)-protected derivative 1), we decided to follow
a metathesis-based strategy related to that previously used for
the synthesis of other ProMs.[12] As shown in Scheme 1, retro-
synthetic analysis leads to a dipeptidic precursor of type 2,
which, in turn, is derived from two vinylproline building blocks
of types 3 and 4 (Scheme 1).
The synthesis of a trans-5-propenylproline building block of
type 3 (Scheme 2) started from l-proline (5, >99% ee), which
was first double-protected by treatment with an excess of
methyl chloroformate in the presence of NEt3 to give the car-
bamate/ester 6 in 88% yield.[12b] Functionalization at C-5 was
To understand these observations, we investigated the inter-
action of the ProM-1-containing peptides with the EVH1
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Scheme 3. Synthesis of 12. a) Cl3CCHO (2 equiv), MeCN, RT, 15 h; b) LiCl,
LDA, HCO2Me, THF, À788C, 30 min; c) MePPh3Br, KOtBu, toluene, 808C, 2 h,
then 10, RT, 1 h; d) AcCl (10 equiv), MeOH, RT, 7 days; e) Boc2O, DIPEA,
CH2Cl2, RT, 2.5 days; f) LiOH, THF/MeOH/H2O, 508C, 5 h.
Scheme 1. Strategy (retrosynthetic analysis) for the synthesis of 1 based on
ring-closing metathesis (RCM); PG=protecting group.
then initiated by electrochemical oxidation of 6 using graphite
plate electrodes (48”28 mm, 600 mA, 5 mm, 08C) in a 0.05m
solution of Bu4NBF4 in methanol.[12b,18] Without any purification,
the resulting N,O-acetal 7, which was obtained in nearly quan-
titative yield as a 1:1 mixture (GC-MS) of diastereomers, was
further converted into the 5-propenylproline derivative 8 in
66% yield through a BF3-mediated reaction with a cuprate pre-
pared in situ from the corresponding Grignard reagent.[19] One
can assume that the reaction proceeds through diastereoselec-
tive attack of the cuprate at an intermediate N-acyliminium
ion. Notably, product 8 was obtained with virtually perfect
trans selectivity (as a 9:1 mixture of the Z/E isomers) by using
the Grignard reagent freshly prepared from (Z)-1-bromopro-
pene.[20]
Figure 3. Structure of the 2-formylproline derivative 10 (left) and of N-Boc-2-
vinylproline (12; right) in the crystalline state.
system in a cisoid configuration with the CCl3 substituent in
the exo (pseudoequatorial) position.
The conversion of aldehyde 10 into vinyl derivative 11
through a Wittig reaction was achieved in 91% yield by using
thoroughly dried MePPh3Br (1408C, high vacuum, 48 h) (lit.:[22]
74%). The final conversion of 11 into the N-Boc-protected 2-vi-
nylproline 12 was initiated by treatment with an excess of
AcCl in MeOH. The resulting crude 2-vinylproline methyl ester
was redissolved in dichloromethane and treated with Boc2O in
the presence of Hünig’s base. After purification, the N-Boc-pro-
tected methyl ester intermediate was hydrolyzed with LiOH in
a solvent mixture of MeOH/THF/H2O to afford 12 in good over-
all yield (33% over 6 steps). The structure of 12 was secured
by X-ray analysis (Figure 3, right) and showed that the pyrroli-
dine ring adopted an envelope conformation with the central
methylene unit in an out-of-plane position (to minimize Pitzer
strain).
Scheme 2. Synthesis of 9. a) ClCO2Me (3.5 equiv), NEt3 (3.0 equiv), MeOH, RT,
15 h; b) À2eÀ (600 mA, 5 mm), Bu4NBF4, MeOH, 08C; c) BrMgCH=CHCH3,
CuBr·SMe2, Et2O, À408C, then addition of BF3·Et2O and 7 at À788C to 108C;
d) TMSI (2 equiv), CH2Cl2, reflux, 3 h.
Finally, selective monodeprotection of 8 (cleavage of the
methyl carbamate functionality) was achieved with iodotrime-
thylsilane (TMSI) in dichloromethane[21] to afford the trans-5-
propenylproline ester 9 (Z/E=9:1) in 38% yield over four steps
on a gram scale. The relative (and absolute) configuration of
(Z)-9 was unequivocally proven by means of X-ray crystal-struc-
ture analysis of its hydro iodide salt (see the Supporting Infor-
mation).
We next attempted to conclude the planned synthesis of
the spirocyclic scaffold 1 (Scheme 1) by peptide coupling of
the two building blocks 9 and 12 and subsequent RCM
(Scheme 4).
However, we recognized that the coupling of sterically hin-
dered acid 12 with the (bulky) secondary amine 9 represented
Preparation of the second building block, that is, the 2-vinyl-
proline derivative 12, was achieved as shown in Scheme 3 by
slightly modifying the synthesis of Bittermann and Gmeiner.[22]
First, l-proline (5) was protected by treatment with chloral[23]
and the resulting cyclic N,O-acetal was formylated (lithium dii-
sopropylamide (LDA), HCO2Me) to diastereoselectively[24] give
the aldehyde 10 in 54% yield over two steps.[22] The configura-
tion of 10 was again secured by X-ray crystallography
(Figure 3, left). The structure nicely shows the bicyclic ring
Scheme 4. Unexpected epimerization during the coupling of 9 and 12. a) 2-
(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU), N,N-diisopropylethylamine (DIPEA), NMP, 858C, 24 h; b) Grubbs II cat-
alyst (30 mol%), CH2Cl2, microwave (300 W), 558C, 7 h.
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a rather difficult task. By using HATU[25] as a coupling agent in
the presence of Hünig’s base (DIPEA) in N-methyl-2-pyrrolidone
(NMP),[26] and heating the mixture for 24 h to 858C, we suc-
ceeded in obtaining a dipetidic product (13) in at least 31%
yield. To our surprise, epimerization at the ester-substituted
stereocenter occurred during peptide coupling, as unequivo-
cally proven by X-ray crystal-structure analysis at the stage of
the metathesis product 14 (Figure 4, left).
Scheme 6. Coupling of the building blocks 17 (2 equiv) and 12. a) HATU
(1.1 equiv), DIPEA (1.98 equiv), NMP, 858C, 24 h.
The importance of minimizing the amount of base in the
(difficult) peptide-coupling step is reflected by the fact that
coupling product 18 epimerizes under the reaction conditions
in the presence of base. Thus, when a solution of pure 18 in
NMP was heated in the presence of two equivalents of DIPEA
(in the absence of the coupling reagent) to 858C for 20 h, vir-
tually complete epimerization to epi-18 was observed
(Figure 5). Clearly, the cis-2,5-disubstituted pyrrolidine deriva-
tive epi-18 is the thermodynamically more stable diastereomer.
Figure 4. Structures of 14 (left) and 18 (right) in the crystalline state.
The metathesis of 13 was also difficult. Nevertheless, by
using 30 mol% of Grubbs II catalyst under microwave condi-
tions[27] (300 W, closed vessel), we could obtain product 14 in
69% yield (in addition to 30% of unreacted 13).
To optimize the peptide-coupling step, we decided to
switch to tert-butyl ester 17 (instead of 9), with the hope that
additional steric hindrance would reduce the kinetic acidity of
the a-ester position to suppress epimerization. This building
block (17) was available in gram amounts, starting from the
easily accessible methoxylated proline derivative 15,[12b,18,19a]
which was reacted with a vinyl cuprate reagent, according to
a procedure reported by Nagaike et al.,[19c] to give 16 as a pure
trans diastereomer. Cleavage of the N-Boc group with trime-
thylsilyl trifluoromethanesulfonate (TMSOTf) in CH2Cl2 then
smoothly afforded the trans-2,5-disubstituted pyrrolidine 17 in
67% overall yield (Scheme 5).
Figure 5. Epimerization of 18 to epi-18. The signals depicted in the NMR
spectrum correspond to the CH group of the vinyl moiety at the (“left”) 2,5-
disubstituted pyrrolidine ring.
Although epimerization can occur (as shown) at the stage of
the coupling products (to give 13 or epi-18, respectively), we
cannot exclude that the amine building block 9 epimerizes
under the coupling conditions, followed by a (kinetically
preferred)[26] reaction of the less hindered cis-2,5-disubstituted
pyrrolidine, to directly yield the epimerized product 13
(Scheme 4).
The remaining major task in the synthesis of 1 was metathe-
sis cyclization of 18 to 19 (Scheme 7). This was initially ach-
ieved in 68% yield by employing either 30 mol% of Grubbs II
catalyst (15 h) or 5 mol% of the nitro-Grela catalyst (72 h). No-
tably, the yield of 19 could be further improved by performing
the reaction in Et2O with 5 mol% of Grubbs II catalyst in the
presence of 7.5 mol% of CuI.[30] Global deprotection and subse-
quent Fmoc protection under the established conditions[12] fi-
nally delivered 1, that is, the ProM-2-related target structure, in
97% yield.
Scheme 5. Synthesis of the trans-5-vinylproline 17: a) CuBr·SMe2, MgBrCH=
CH2, À408C, À788C, BF3·Et2O, À788C, 2 h, RT, 2 h; b) TMSOTf, CH2Cl2, 08C,
5 min.
Coupling of the building blocks 12 and 17 under the estab-
lished conditions (HATU, DIPEA) gave rise to a product (18)
that contained only minor amounts of the undesired isomer
epi-18 (18/epi-18=6:1). Epimerization could even be com-
pletely suppressed by reducing the amount of DIPEA to
1.98 equivalents. The optimized protocol delivered stereo-
chemically pure dipeptide 18 in a notable yield of 72%, even
on a gram scale (Scheme 6). The configuration of 18 was se-
cured by X-ray crystallography (Figure 4).
The configuration of 19 was secured by X-ray crystal-struc-
ture analysis (Figure 6). A crystal structure was also obtained
from the epimeric compound epi-19 (prepared by metathesis
of a 6:1 mixture of 19 and epi-19 and subsequent chromato-
graphic separation of the product diastereomers). The struc-
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Scheme 7. Synthesis of 1. a) Grubbs II catalyst (30 mol%), toluene, reflux,
15 h; b) TFA/CH2Cl2 1:1, RT, 1 h; c) Fmoc-Cl, NaHCO3, H2O/THF, RT, 15 h.
Figure 7. Superimposed structures of 14. Left: polymorph 1 (black) superim-
posed with polymorph 3 (red). Right: polymorph 2 (black) superimposed
with polymorph 3 (red). This figure was created by using the program
PLATON.[32]
ally the same conformation in all three structures. Thus, the
polymorphism of compound 14 seems to result just from dif-
ferent packing of the molecules within the crystal.[33]
Binding studies
Figure 6. Structure of the tricyclic compounds 19 (left) and epi-19 (right) in
the crystalline state. The conformation of 19 almost perfectly matches that
of the PPII-helix structure.
As mentioned previously, the main motivation of this work was
the search for PPII secondary-structure mimetics that bind to
EVH1 domains of the Ena/VASP proteins.[34,35] Herein, we fo-
cused on the EVH1 domain of EnaH, which is the human ver-
sion of the protein Ena, because modeling was performed by
using the crystal structure of this domain.
ture nicely shows that the diproline unit of 19 is locked in a vir-
tually perfect PPII-type conformation, as predicted. In contrast,
compound epi-19 can be regarded as a b-turn mimetic.[22]
By using standard methods of solid-phase peptide synthesis,
the ActA-derived model ligand (Ac-SFE-FPPPP-TEDEL-NH2) and
two derivatives, in which the first two proline residues were
substituted by either ProM-1 or ProM-2, were prepared and
their binding affinity towards EnaH-EVH1 was determined by
using tryptophan-based fluorescence titration (FT) and isother-
mal titration calorimetry (ITC), respectively. As the results
shown in Table 2 reveal, exchange of the Pro–Pro unit within
the model ligand for either ProM-1 or ProM-2 did not lead to
a loss of affinity.
Polymorphism of 14
An interesting observation made during this study concerned
the occurrence of polymorphs[31] of compound 14 (Scheme 4),
which we obtained in crystalline form. As mentioned previous-
ly, X-ray crystal-structure analysis of a crystal picked from the
sample revealed the formation of unexpected (epimerized)
product 14. To rule out the possibility that we might have acci-
dentally picked a crystal of the minor diastereomer present in
the mixture, we investigated a second crystal (with different
cell parameters); however, this also corresponded to product
14. We then even found a third polymorph of 14 in the same
sample. All three polymorphs of 14 crystallized in the same
space group (P212121). The cell constants are given in Table 1.
The very similar affinities may reflect the contribution of the
flanking epitopes (SFE and TEDEL) to compensate for the less
favorable position of the ProM-1 vinylidene bridge in this case
(see the Design section). Notably, we could confirm through
1H–15N HSQC NMR spectroscopy[36] that the ProM-1 and ProM-2
[a]
Table 2. Binding data for peptides of type Ac-SFE-F[XX]PP-TEDEL-NH2
against EnaH-EVH1 determined either by using tryptophan-based FT or
ITC at 258C.
Table 1. Cell constants of the three polymorphs of 14.
Parameter
Polymorph 1
Polymorph 2
Polymorph 3
Pro–Pro
ProM-1
ProM-2
a [Š]
b [Š]
c [Š]
7.007
15.229
17.490
8.944
13.539
15.249
9.983
11.188
16.360
FT
KD [mm]
DG8 [kJmolÀ1
13.1Æ0.6
8.0Æ1.0
12.0Æ1.0
]
À27.9Æ0.1
À29.1Æ0.3
À28.1Æ0.3
ITC
KD [mm]
21Æ1
24Æ3
21Æ5
Because no solvent molecules were incorporated in any of
the three structures, it was of interest to investigate whether
the polymorphism originated from conformational or packing
effects. A comparison of the three different polymorphs of 14
(Figure 7)[32] clearly revealed that the molecules adopted virtu-
DG8 [kJmolÀ1
DH8 [kJmolÀ1
]
]
À26.7Æ0.1
À21.2Æ0.3
À5.5Æ0.4
À26.4Æ0.4
À21.8Æ0.6
À5.0Æ1.0
À26.7Æ0.8
À17.0Æ2.0
À10.0Æ2.0
ÀTDS8 [kJmolÀ1
]
[a] XX=Pro–Pro, ProM-1, or ProM-2.
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1
Figure 8. Ligand-concentration-dependent H–15N HSQC chemical shifts for
W23 e-NH (of EnaH-EVH1) for the three ligands investigated. A strong slow-
exchange regime for the “wild-type” ActA-derived ligand and the ProM-2-
containing ligand was observed, whereas for the ProM-1-containing ligand
a moderate fast-exchange regime was observed. The blue spot corresponds
to the protein in the absence of any ligand, whereas the red spots corre-
spond to protein–ligand concentrations of 98 (model ligand) and 95%
(ProM-1 and ProM-2 ligands). The spots in between correspond to protein–
ligand concentrations of 25, 50, and 75%, as computed with the dissociation
constant determined by tryptophan-based FT (Table 2).
Figure 9. Chemical shifts of all trackable backbone NH protons plotted on:
A) the crystal structure of 1evh,[17] B) the model of EnaH-EVH1 with ProM-1,
or C) the ProM-2-containing ligand. Only the FPPPP core motif is displayed.
The shifts show that the ProM-1- and ProM-2-derived ligands occupy the
same binding groove as the model ligand. More shifts are visible for the
ProM-1 ligand, which indicates more complex binding behavior than those
of the model ligand and ProM-2 ligand.
ligands addressed the same binding groove as that of the
ActA-derived model peptide (Figure 8).
Moreover, we could detect a difference in the binding
1
modes for the ProM-1- and ProM-2-derived ligands in the H–
The scaffold ProM-2 described herein is an important addi-
tion to our set of proline-derived polycyclic dipeptide mimetics
(ProMs) recently developed in our laboratories.[12] We are opti-
mistic that the modular combination of different ProMs will
enable us to identify PPII secondary-structure mimetics that
are able to effectively and selectively bind to PRDs, that is, pro-
tein domains specialized in recognizing proline-rich motifs in
a PPII conformation. Moreover, the difunctional compounds
(including the epimeric scaffold epi-18, to which stereoselec-
tive access was discovered by accident) may also find future
applications as stereodefined scaffolds for the diversity-orient-
ed synthesis of meaningful compound libraries.
15N HSQC spectra. All ligands connect to the indol moiety of
Trp23 of EnaH-EVH1 by forming a hydrogen bond between
the e-NH of the tryptophan side chain and the “central carbon-
yl oxygen” of the ligand, that is, the central amide group that
connects the two pairs of prolines within the PP-PP motif of
the wild-type ligand (or the central amide bond of the ProM-
PP moiety in the modified ligands; cf. Figure 2). Although the
ProM-2-derived ligand showed the same chemical shift and
slow-exchange regime as the model peptide, the ProM-
1 ligand shifted differently and in a moderate fast-exchange
regime (Figure 9). This indicates a change in the important
contact to Trp23 for the different ligands, as suggested by mo-
lecular modeling (Figure 2).
Acknowledgements
Conclusion
This work was supported by the Deutsche Forschungsgemein-
schaft (DFG FOR 806). Valuable gifts of chemicals from Rock-
wood-Lithium GmbH and Evonik are gratefully acknowledged.
We also would like to thank Prof. G. Schwarz for access to an
Oxford Diffraction Supernova diffractometer and Kai Wirz for
the chromatographic purification of 1 on a Grace Reveleris
Flash system.
We developed a reliable synthesis of the new spirocyclic Pro–
Pro dipeptide mimetic ProM-2 in its Fmoc-protected form (1),
which was designed as a diproline unit locked in a PPII helix
conformation. By using vinylproline derivatives as key building
blocks, the target structure was assembled through HATU-
mediated peptide coupling and subsequent ruthenium-cata-
lyzed RCM. The developed synthesis allowed us to prepare
target compound 1 on a gram scale with about 14% overall
yield over 10 steps (longest linear sequence), starting from pro-
line. After incorporation of ProM-2 into a model peptide
known to bind to the EVH1 domain of Ena/VASP proteins, FT
and ITC measurements revealed that the new scaffold was
able to replace the highly conserved first two prolines of the
model ligand without loss in affinity. Furthermore, 1H–15N
HSQC spectra indicated that ProM-2 better mimicked the Pro–
Pro unit in this case, relative to the isomeric scaffold ProM-1.
Keywords: amino acids
· conformation analysis · helical
structures · protein–protein interactions · peptidomimetics
[1] a) B. N. Bullock, A. L. Jochim, P. S. Arora, J. Am. Chem. Soc. 2011, 133,
14220–14223; for selected reviews, see: b) O. Keskin, A. Gursoy, B. Ma,
R. Nussinov, Chem. Rev. 2008, 108, 1225–1244; c) D. Gonzµlez-Ruiz, H.
Gohlke, Curr. Med. Chem. 2006, 13, 2607–2625; d) Y. Pommier, J. Cher-
fils, Trends Pharmacol. Sci. 2005, 26, 138–145; e) A. Reayi, P. Arya, Curr.
Opin. Chem. Biol. 2005, 9, 240–247; f) T. Berg, Angew. Chem. Int. Ed.
2003, 42, 2462–2481; Angew. Chem. 2003, 115, 2566–2586.
Chem. Eur. J. 2015, 21, 8464 – 8470
www.chemeurj.org
8469
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[2] For selected recent reviews, see: a) M. Jesus PØrez de Vega, M. Martín-
Martínez, R. Gonzµlez-MuÇiz, Curr. Top. Med. Chem. 2007, 7, 33–62; b) J.
Vagner, H. Qu, V. J. Hruby, Curr. Opin. Chem. Biol. 2008, 12, 292–296;
c) A. Grauer, B. Kçnig, Eur. J. Org. Chem. 2009, 5099–5111; d) J. M.
Mason, Future Med. Chem. 2010, 2, 1813–1822.
[3] For selected references, see: a) R. E. Moellering, M. Cornejo, T. N. Davis,
C. Del Bianco, J. C. Aster, S. C. Blacklow, A. L. Kung, D. G. Gilliland, G. L.
Verdine, J. E. Bradner, Nature 2009, 462, 182–190; b) E. Ko, J. Liu, L. M.
Perez, G. Lu, A. Schaefer, K. Burgess, J. Am. Chem. Soc. 2011, 133, 462–
477; c) J. H. Lee, Q. Zhang, S. Jo, S. C. Chai, M. Oh, W. Im, H. Lu, H.-S.
Lim, J. Am. Chem. Soc. 2011, 133, 676–679.
[4] a) L. Ball, R. Kühne, J. Schneider-Mergener, H. Oschkinat, Angew. Chem.
Int. Ed. 2005, 44, 2852–2869; Angew. Chem. 2005, 117, 2912–2930;
b) Protein–Protein Interactions as New Drug Targets (Eds.: E. Klussmann,
J. Scott), Handbook of Experimental Pharmacology, Vol. 186, Springer,
Heidelberg, 2008.
[19] For related transformations with 1-propenyllithium-based cuprates, see:
a) K. F. McClure, P. Renold, D. S. Kemp, J. Org. Chem. 1995, 60, 454–457;
b) E. Grovenstein Jr., K. W. Black, S. C. Goel, R. L. Hughes, J. H. Northrop,
D. L. Streeter, D. VanDerveer, J. Org. Chem. 1989, 54, 1671–1679; c) F.
Nagaike, Y. Onuma, C. Kanazawa, H. Hojo, A. Ueki, Y. Nakahara, Y. Naka-
hara, Org. Lett. 2006, 8, 4465–4468.
[20] D. Rodríguez, M. Mulero, J. A. Prieto, J. Org. Chem. 2006, 71, 5826–
5829.
[21] a) M. E. Jung, M. A. Lyster, J. Chem. Soc. Chem. Commun. 1978, 315–
316; b) R. S. Lott, V. S. Chauhan, C. H. Stammer, J. Chem. Soc. Chem.
Commun. 1979, 495–496; c) F. ManfrØ, J. M. Kern, J. F. Biellmann, J. Org.
Chem. 1992, 57, 2060–2065.
[22] a) H. Bittermann, P. Gmeiner, J. Org. Chem. 2006, 71, 97–102; b) H. Bit-
termann, F. Bçckler, J. Einsiedel, P. Gmeiner, Chem. Eur. J. 2006, 12,
6315–6322.
[23] H. Wang, J. P. Germanas, Synlett 1999, 1, 33–36.
[5] a) L. Ball, R. Kühne, B. Hoffmann, A. Häfner, P. Schmieder, R. Volkmer-
Engert, M. Hof, M. Wahl, J. Schneider-Mergener, U. Walter, H. Oschkinat,
T. Jarchau, EMBO J. 2000, 19, 4903–4914; b) R. Aasland, C. Abrams, C.
Ampe, L. Ball, M. Bedford, G. Cesareni, M. Gimona, J. Hurley, T. Jarchau,
V. Lehto, M. Lemmon, R. Linding, B. Mayer, M. Nagai, M. Sudol, U.
Walter, S. Winder, FEBS Lett. 2002, 513, 141–144; c) L. Ball, T. Jarchau, H.
Oschkinat, U. Walter, FEBS Lett. 2002, 513, 45–52.
[6] I. Geisler, J. Chmielewski, Bioorg. Med. Chem. Lett. 2007, 17, 2765–2768.
[7] a) M. Dustin, M. Olszowy, A. Holdorf, J. Bromley, N. Desai, P. Widder, F.
Rosenberger, P. van der Merwe, P. Allen, A. Shaw, Cell 1998, 94, 667–
677; b) V. Laurent, T. Loisel, B. Harbeck, A. Wehman, L. Grçbe, B. Jock-
usch, J. Wehland, F. Gertler, M. Carlier, J. Cell. Biol. 1999, 144, 1245–
1258.
[24] a) D. Seebach, M. Boes, R. Naef, W. B. Schweizer, J. Am. Chem. Soc. 1983,
105, 5390–5398; for a review, see: b) D. Seebach, A. R. Sting, M. Hoff-
mann, Angew. Chem. Int. Ed. Engl. 1996, 35, 2708–2748; Angew. Chem.
1996, 108, 2881–2921.
[25] a) L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397–4398; b) L. A. Carpi-
no, A. El-Faham, F. Albericio, Tetrahedron Lett. 1994, 35, 2279–2282; for
reviews on peptide coupling, see: c) A. El-Faham, F. Albericio, Chem.
Rev. 2011, 111, 6557–6602; d) C. A. G. N. Montalbetti, V. Falque, Tetrahe-
dron 2005, 61, 10827–10852.
[26] G. Lesma, A. Colombo, A. Sacchetti, A. Silvani, Tetrahedron Lett. 2008,
49, 7423–7425.
[27] For reviews on microwave-assisted synthesis, see: a) C. O. Kappe,
Angew. Chem. Int. Ed. 2004, 43, 6250–6284; Angew. Chem. 2004, 116,
6408–6443; b) A. de La Hoz, A. Diaz-Ortiz, A. Moreno, Chem. Soc. Rev.
2005, 34, 164–178; c) Y. Coquerel, J. Rodriguez, Eur. J. Org. Chem. 2008,
1125–1132.
[28] a) P. Huy, H.-G. Schmalz, Synthesis 2011, 954–960; b) P. G. M. Wuts, T. W.
Greene, Greene’s Protective Groups in Organic Synthesis, 4th ed., Wiley-
Interscience 2007.
[29] G. Sunilkumar, D. Nagamani, N. P. Argade, K. N. Ganesh, Synthesis 2003,
15, 2304–2306.
[30] K. Voigtritter, S. Ghorai, B. H. Lipshutz, J. Org. Chem. 2011, 76, 4697–
4702.
[31] a) J. Bernstein, Polymorphism in Molecular Crystals, Clarendon, Oxford,
2002; b) R. Purohit, P. Venugopalan, Resonance 2009, 14, 882–893.
[32] A. L. Spek, Acta Crystallogr. Sect. D 2009, 65, 148–155.
[33] CCDC-1042396 (Z-9), 1042403 (10), 1042402 (12), 1042397 (14, poly-
morph 1), 1042400 (14, polymorph 2), 1042401 (14, polymorph 3),
1042399 (18), 1042395 (19), and 1042398 (epi-19) contain the supple-
mentary crystallographic data for this paper. These data can be ob-
tained free of charge from the Cambridge Crystallographic Data Center
via www.ccdc.cam.ac.uk/data_request/cif.
[34] a) E. T. Roussos, M. Balsamo, S. K. Alford, J. B. Wyckoff, B. Gligorijevic, Y.
Wang, M. Pozzuto, R. Stobezki, S. Goswami, J. E. Segall, D. A. Lauffenbur-
ger, A. R. Bresnick, F. B. Gertler, J. S. Condeelis, J. Cell Sci. 2011, 124,
2120–2131; b) F. Di Modugno, M. Mottolese, L. DeMonte, P. Trono, M.
Balsamo, A. Conidi, E. Melucci, I. Terrenato, F. Belleudi, M. R. Torrisi, M.
Alessio, A. Santoni, P. Nisticò, PLoS ONE 2010, 5, e15852; c) A. Toyoda,
H. Kawana, K. Azuhata, J. Yu, A. Omata, H. Kishi, M. Higashi, K. Harigaya,
Int. J. Oncol. 2009, 34, 53–60; d) S. Goswami, U. Philippar, D. Sun, A.
Patsialou, J. Avraham, W. Wang, F. Di Modugno, P. Nisticò, F. B. Gertler,
J. S. Condeelis, Clin. Exp. Metastasis 2009, 26, 153–159; e) U. Philippar,
E. T. Roussos, M. Oser, H. Yamaguchi, H.-D. Kim, S. Giampieri, Y. Wang, S.
Goswami, J. B. Wyckoff, D. A. Lauffenburger, E. Sahai, J. S. Condeelis,
F. B. Gertler, Development 2008, 15, 813–828.
[8] A. Goldstrohm, T. Albrecht, C. SuÇØ, M. Bedford, M. Garcia-Blanco, Mol.
Cell. Biol. 2001, 21, 7617–7628.
[9] a) B. Laggerbauer, S. Liu, E. Makarov, H.-P. Vornlocher, O. Makarova, D.
Ingelfinger, T. Achsel, R. Lührmann, RNA 2005, 11, 598–608; b) M. Kofler,
M. Schuemann, C. Merz, D. Kosslick, A. Schlundt, A. Tannert, M. Schaefer,
R. Luhrmann, E. Krause, C. Freund, Mol. Cell. Proteomics 2009, 8, 2461–
2473.
[10] a) C. M. Deber, F. A. Bovey, J. P. Carver, E. R. Blout, J. Am. Chem. Soc.
1970, 92, 6191–6198; b) N. Helbecque, M. H. Loucheux-Lefebvre, Int. J.
Peptide Protein Res. 2009, 19, 94–101; c) H. Okabayashi, T. Isemura, S.
Sakakibara, Biopolymers 1968, 6, 323–330; d) R. K. Dukor, T. A. Kiederl-
ing, Biopolymers 1991, 31, 1747–1761; e) R. K. Dukor, T. A. Kiederling, V.
Gut, Int. J. Peptide Protein Res. 2009, 38, 198–203; f) P. M. Cowan, S.
McGavin, Nature 1955, 176, 501–503.
[11] R. Kühne, H. Oschkinat, C. Brockmann, H.-G. Schmalz, 2008, GER, WO
2008/040332A1.
[12] a) J. Zaminer, C. Brockmann, P. Huy, R. Opitz, C. Reuter, M. Beyermann,
C. Freund, M. Müller, H. Oschkinat, R. Kühne, H.-G. Schmalz, Angew.
Chem. Int. Ed. 2010, 49, 7111 –7115; Angew. Chem. 2010, 122, 7265–
7269; b) C. Reuter, P. Huy, J.-M. Neudçrfl, R. Kühne, H.-G. Schmalz, Chem.
Eur. J. 2011, 17, 12037–12044; c) C. Reuter, M. Kleczka, S. de Mazan-
court, J.-M. Neudçrfl, R. Kühne, H.-. Schmalz, Eur. J. Org. Chem. 2014,
2664–2667; d) A. Soicke, C. Reuter, M. Winter, J.-M. Neudçrfl, N. Schlçr-
er, R. Kühne, H.-G. Schmalz, Eur. J. Org. Chem. 2014, 6467–6480.
[13] a) M. Krause, E. W. Dent, J. E. Bear, J. J. Loureiro, F. B. Gertler, Annu. Rev.
Cell Dev. Biol. 2003, 19, 541–564; b) A. S. Sechi, J. Wehland, Front. Biosci.
2004, 9, 1294–1310; c) F. Drees, F. B. Gertler, Curr. Opin. Neurobiol.
2008, 18, 53–59.
[14] a) M. Hamon, H. Bierne, P. Cossart, Nat. Rev. Microbiol. 2006, 4, 423–
434; b) J. A. Vµzquez-Boland, M. Kuhn, P. Berche, T. Chakraborty, G.
Domínguez-Bernal, W. Goebel, B. Gonzµlez-Zorn, J. Wehland, J. Kreft,
Clin. Microbiol. Rev. 2001, 14, 584–640; c) M. B. Goldberg, Microbiol. Mol.
Biol. Rev. 2001, 65, 595–626.
[35] J. H. Holtzman, K. Woronowicz, D. Golemi-Kotra, A. Schepartz, Biochem-
istry 2007, 46, 13541–13553.
[36] P. Schanda, E. Kupce, B. Brutscher, J. Biomol. NMR 2005, 33, 199–211.
[15] R. Opitz, Inhibition von Protein–Protein-Wechselwirkungen, Disserta-
tion, FU-Berlin, 2013.
[16] Sybyl-X Molecular Modelling suite, Certara, L. P.
[17] K. E. Prehoda, D. J. Lee, W. A. Lim, Cell 1999, 97, 471–480.
[18] a) T. Shono, H. Hamaguchi, Y. Matsumura, J. Am. Chem. Soc. 1975, 97,
4264–4268; b) T. Shono, Tetrahedron 1984, 40, 811 –850; c) M. Skrinjar,
L.-G. Wistrand, Tetrahedron 1991, 47, 573–582.
Received: December 15, 2014
Revised: March 11, 2015
Published online on April 23, 2015
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