Chiral organocatalysts based on lipopeptide micelles for aldol reactions in water

Article abstract of DOI:10.1039/c6cp08135e

A comprehensive study of the self-assembly in water of a lipopeptide consisting of a sequence of l-proline, l-arginine and l-tryptophan with a hydrocarbon chain has been performed. Fluorescence assays were used to determine the critical aggregation concentration. In situ small-angle X-ray scattering (SAXS) and molecular dynamics simulations showed the presence of spherical micelles with diameters around 6 nm. In agreement with these results, cryo-TEM images showed globular aggregates with diameters ranging from ≈4 nm up to ≈9 nm. Furthermore, the lipopeptide catalytic activity has been tested for the direct aldol reaction between cyclohexanone and p-nitrobenzaldehyde, and we have observed that the self-association of the organocatalyst played a critical role in the enhanced activity. Water affects the selectivity, and poor results are obtained under neat reaction conditions. The location of the catalytic groups at the lipopetide/water solvent interface also endowed unusual selectivity in the catalyzed aldol reactions. Under optimized reaction conditions, high yields (up to >99%), good enantioselectivity (ee up to 85%) and high diastereoselectivity (ds up to 92:8) were obtained.

Full text of DOI:10.1039/c6cp08135e

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Physical Chemistry Chemical Physics
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Chiral Organocatalysts Based on Lipopeptide Micelles for Aldol
Reactions in Water
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
c
a
d
e
B. M. Soares, A. M. Aguilar, E. R. Silva, M. D. Coutinho-Neto, I. W. Hamley, M. Reza, J.
e
a
Ruokolainen and W. A. Alves †
DOI: 10.1039/x0xx00000x
A comprehensive study of the self-assembly in water of a lipopeptide consisting of a sequence of L-proline, L-arginine and
L-tryptophan with a hydrocarbon chain has been performed. Fluorescence assays were used to determine critical
aggregation concentration. In situ small-angle X-ray scattering (SAXS) and molecular dynamics simulations showed the
presence of spherical micelles with diameters around 6 nm. In agreement with these results, cryo-TEM images showed
globular aggregates with diameters ranging from ≈ 4 nm up to ≈ 9 nm. Furthermore, the lipopeptide catalytic activity has
been tested for the direct aldol reaction between cyclohexanone and p-nitrobenzaldehyde, and we have observed that the
self-association of the organocatalyst played critical role in the enhanced activity. Water affects the selectivity, and poor
results are obtained under neat reaction conditions. The location of the catalytic groups at the lipopetide/water solvent
interface also endowed unusual selectivity in the catalyzed aldol reactions. Under optimized reaction conditions, high
yields (up to >99%), good enantioselectivity (ee up to 85%) and high diastereoselectivity (ds up to 92:8) were obtained.
www.rsc.org/
out in polar organic solvents such as DMSO, DMF or
chloroform. Therefore, one of the current challenges regarding
aldol reactions is replacing organic solvents (often volatile,
Introduction
Aldol reactions are one of the most important and investigated
transformations in organic synthesis because a new C-C bond
toxic and flammable) by environmentally friendly compounds
that retain the effectiveness of product conversion and
1
-3
is formed from two carbonyl compounds. This reaction
generates products that contain -hydroxyl carbonyl
1
8-21
selectivity.
Water is therefore a very attractive solvent for
a
β
promoting such reactions because, in addition to its obvious
environmental benefit, the presence of hydrophobic
interactions and mediation of hydrogen bonds could easily
skeleton, which is largely found in natural products with
outstanding pharmacological activities. Furthermore, in the
asymmetric version of the reaction, new stereogenic carbons
2
2-24
4
-8
promote self-assembly of catalyst superstructures,
can be generated with high levels of selectivity. In biological
systems, aldol reactions are promoted reversibly and
stereoselectively by a group of enzymes known as fructose 1,6-
whereas the high surface tension and high dielectric constant
of water potentially make reactions faster and more
2
1, 25-28
1
, 4, 9-11
selective.
bisphosphate aldolases, or simply aldolases.
1
first use in 1971,
Since its
2-14
The use of L-proline-derived organocatalysts containing long
hydrophobic chains has been reported as a strategy for
performing aldol reactions in solutions mixing water and
the amino acid L-proline has showed itself
as an efficient and versatile organocatalyst for aldol reactions.
Thus, the design and synthesis of highly efficient L-proline
1
2
9, 30
5- organic solvents.
A proposition emerging from these
studies is that amphiphilic species appear uniformly
distributed at the water-oil interfaces of H O droplets
derivatives as organocatalysts is still of considerable interest.
7
1
2
Many L-proline-catalyzed aldol reactions are usually carried
3
0
dispersed in the organic phase increasing the catalytic
surface area. Furthermore, organization into two-dimensional
chiral surfaces likely induces spatial ordering of reactants and
enhances stereoselectivity. Interestingly, when the reaction
does not favor emulsion production, satisfactory results are
a.
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo
André 09210-580, Brazil.
Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal
de São Paulo, Diadema 09972270, Brazil.
b.
3
0
c.
Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo 04023-
0
not observed due to the random distribution of the catalyst.
62, Brazil.
Compared to enzymes, supramolecular catalysts based on self-
assembled peptides appear as a promising strategy since they
are much more stable, cheaper and yet still allow catalyst sites
to be mimicked. In addition, they exhibit polymorphic richness
d.
Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD,
United Kingdom.
Department of Applied Physics, Aalto University School of Science, P. O. Box
e.
15100, FI- 00076, Finland.
Corresponding author: wendel.alves@ufabc.edu.br
†
Electronic Supplementary Information (ESI) available: spectroscopic, scattering and at the nanoscale and enable reactions in confined
3
1-34
molecular dynamics simulations data, and details on the aldol product
characterization. See DOI: 10.1039/x0xx00000x
environments with diverse geometrical constraints.
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Recently, a few research groups have proposed the use of Purity was determined at 95.43% and the molar mass was
peptide-based supramolecular assemblies for mimicking measured at 681.60 Da (expected: 681.98). Aqueous solutions
3
5, 36
24, 37
catalytic activity of aldolases.
Escuder et al.
and were prepared by dissolving PRW-C16 into ultrapure water
3
Miravet et al. have reported examples of aldol reactions (resistivity > 18 MΩ·cm at 25°C), obtained from a Mili-Q
8
-3
catalyzed by L-proline-derived amphiphilic peptides, where the system. Concentrations varied from 4.5
× 10 to 15 mM and
catalytic performance is activated by self-aggregation of the measurements performed with indicator paper revealed pH
catalyst within hydrogelated matrices. In this same way, Zhao values between ~ 6 (higher lipopeptide concentrations) and ~
3
9
et al. have shown that assembled prolinamide surfactants 7.5 throughout the concentration range. After mixing, samples
can enhance activity and stereoselectivity of aldol reactions in were submitted to ultrasonication (50°C, 15 minutes) to aid
nonpolar solvents, like benzene. These results provided new solubilization. In the following, solutions were kept in the dark,
insights into the role of the hydrophobic part of the at room temperature, for about 24 hours prior to
proline-functionalized organocatalysts on the observed high spectroscopic assays. Samples used for scattering and
reactivity and selectivity in water.
microscopy assays were kept refrigerated for a period of 4
Inspired by these previous investigations, we describe here a days before analysis.
study where the self-assembly of an L-proline-containing
lipopeptide (Figure 1) was explored for promoting aldol Critical aggregation concentration
reactions in excess water. The functional group of the
The critical aggregation concentration (CAC) of the lipopeptide
lipopeptide was made up from a tripeptide PRW sequence (P:
L-proline, R: L-arginine, W: L-tryptophan), which acts as a
catalyst site for reactions. This tripeptide was lipidated to a
C16:0 palmitoyl hydrocarbon chain at the carboxyl group of
the tryptophan amino acid, leading to a surfactant-like design.
Detailed investigations on the self-assembly of the PRW-C16
lipopeptide showed spontaneous ordering into spherical
micelles with diameters around 6 nm at concentrations as low
in water was determined through fluorescence assays. Two
methods were used for obtaining independent estimations on
the CAC. The first strategy was based on the intrinsic
40
fluorescence of the tryptophan side-chain. Aqueous PRW-C16
solutions were irradiated with excitation wavelength λexc = 280
nm and emission spectra were probed in the range 300 nm ≤
λem 450 nm. In the second strategy, pyrene was used as an
≤
extrinsic fluorophore for assessing the formation of peptide
-1
as 0.19 mmol L . In the following, these structures were
successfully applied as a supramolecular catalytic system for
direct aldol reactions between cyclohexanone and p-
nitrobenzaldehyde in excess water. Our results show, for the
first time, the viability of L-proline amphiphilic derivative
aggregates. Peptide mixtures were prepared across the same
concentration range described above into solutions containing
-4
10 wt%. Samples were then irradiated with λ
pyrene at 1 ×
338 nm and pyrene emission spectra were registered in the
exc
=
range 350 nm ≤ λem 550 nm. The pyrene fluorescence is
≤
micelles as
a
catalyst with excellent conversion and
41
significantly enhanced in nonpolar environments; thus, a
sudden growth on emission intensity is observed upon self-
assembly because chromophores remain trapped in the
hydrophobic core of peptide nanostructures. Fluorescence
data were recorded using a Carian Varian Ellipse spectrometer.
stereoselectivity. In contrast, poor results were obtained in the
absence of the lipopeptide assemblies. The excellent results
provided by the self-assembled structures demonstrate the
potential of lipopeptide assemblies to catalyze aldol reactions
in an environmentally-friendly manner.
Cryo-TEM imaging
Cryogenic transmission electron microscopy was performed as
2-44
Experimental
4
reported in our previous work.
Briefly, imaging was
Peptide samples
conducted on a JEOL JEM-3200FSC microscope, using
The lipopeptide was synthesized by CS Bio (Merlo Park, acceleration voltage of 300 kV. Slit width was kept at 20 eV
California) using TFA as a counter ion (catalog number and micrographs were recorded by a Gatan Ultrascan 4000
CS12677, batch O051). The sequence had a tripeptide as a CCD camera. Droplets containing about 3 µl solutions were
functional group with sequence PRW (P: L-proline, R: L- cast onto copper grids and then vitrified at – 180 °C in a 1:1
arginine and W: L-tryptophan) and it was lipidated by bonding ethane:propane mixture.
a C16:0 fatty acid chain to the carboxylic acid portion of the
tryptophan amino acid (henceforth, the lipopeptide will be Small-angle scattering assays
referred to as PRW-C16).
High-resolution small-angle X-rays scattering (SAXS) was
performed on the SWING beamline of synchrotron SOLEIL
(Saint Aubin, France). A HPLC-based bioSAXS setup was used to
flow about 50 ꢀl of sample across a 1 mm quartz capillary
during data acquisition. 60 frames, 1 s each, were grabbed
during the flow. Frames were compared and, if no radiation
damage was detected, they were averaged and background
subtracted. The beam energy was set at E = 12 KeV, the
sample-to-detector distance was SD = 1526.4 mm and data
Figure 1. Structure of the lipopeptide PRW-C16
.
2
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were recorded with an Aviex 1024
× 1024 detector (pixel size: simulation keeping the alkyl chains restrained followed by
-1
4
1.7
ꢀ
m). The q-range was scanned in the interval 0.1 nm
≤
5 nm . Complementary small-angle neutron scattering 5ns of equilibration time. All molecular dynamics simulations
q
unrestrained dynamics. All production data was collected after
-1
≤
5
7-59
(
SANS) data were obtained on the time-of-flight LOQ beamline were performed using the AMBER14 package.
6
Figures were
0
of ISIS spallation source (Didcot, UK). A 1 wt% solution rendered using VMD.
2
containing PRW-C16 was prepared into D O and loaded into a
Hellma banjo cell with pathlength l = 1 mm. A “white” neutron Reagents and aldol reactions
beam, with cross-section of 12 mm, irradiated the sample with
All reagents had analytical grade and, unless indicated to the
contrary, were used without further treatment.
wavelengths in the range 0.22 nm ≤ λ ≤ 1 nm. The sample-to-
detector distance was SD ~ 4 m and data was registered in the
Cyclohexanone was distillated under inert nitrogen
atmosphere prior to experiments. Aldol reactions were
monitored through thin layer chromatography (TLC), using 200
µm silica gel plates (Supelco) inspected under UV light or
revealed trough vanillin method. High resolution mass
spectroscopy (HRMS) was performed on a MicrOTOF-QII
-
1
-1
interval 0.1 nm
≤ q ≤ 2.5 nm . All scattering experiments
were conducted at 25 °C and scattering data treatment was
4
5
carried out by model fitting with SASFit program.
Molecular dynamics simulation setup
The PRW-C16 lipopeptide was modeled using the AMBER family (Bruker)
4
spectrometer.
High
performance
liquid
6
of force fields. The amber LIPID14 has been used for the chromatography (HPLC) was carried using a Shimadzu LC-10AD
palmitoyl chain while the peptide part was described using the apparatus equipped with an UV/Vis detector Shimadzu SPD-
4
7, 48
ff99SB-ILDN
updated parameter set. We used the standard 10AV and an integrator Shimadzu C-R8A, using a Chiralpak AD-
4
9
RESP charge scheme to derive charges for the alkyl section H column (hexane/2-propanol = 90/10), 30 °C, 254 nm, 1.0
using an acetyl cap. The ester bond connecting the peptide mL/min. NMR spectra were obtained with a Bruker DPX-300
and alkyl chain employed the same parameters used in instrument (Bruker BioSpin GmbH), operating at 300 MHz.
5
0
LIPID11 and LIPID14. The cyclohexanone molecule was Chemical shifts (δ) are shown in parts per million (ppm),
5
modeled using the GAFF parameter set and RESP charges. whereas coupling constants (J) are exhibited in Hz. Data were
1
We employed the Hartree-Fock method and a 6-31G* basis set normalized by the peak of the corresponding solvent used
5
in all RESP charge derivations. Tip3P water and Cl- counter during analysis.
2
ions were added to the system afterwards. Note that Cl- was
used instead of TFA as the counter ions in our simulations for General procedure for aldol reactions in water.
the sake of simplicity.
Cyclohexanone (1.7 mmol, 178 µL) was added to the catalyst
A typical spherical micelle initial condition for the simulation (7.3 µmol, 5.0 mg) in water (178 µL). Then, p-
5
3
was constructed using packmol from minimized PRW-C16 nitrobenzaldehyde (147.0 µmol, 22.0 mg) was added to the
conformations forming a circa 60 Å object from 160 individual mixture. After being stirred at room temperature for 2 days,
molecules. The reasoning for such aggregation number is given the mixture was extracted with ethyl acetate. The organic
in the results section. Ions were added to the ensure charge layer was concentrated to give crude aldol product.
1
neutrality using the xleap program from the AmberTools suite
3
H NMR (300 MHz, CDCl ): δ 8.22-8.18 (m, 2H, ArH); 7.51-7.47
of codes. Three configurations were considered: configuration (m, 2H, ArH); 5.47 (br s, 1H, CHOH-syn); 4.89 (dd, J = 7.5 Hz, 3.0
A had a PRW-C16 micelle in water while configurations B and C Hz, 1H, CHOH-anti); 4.08 (d, J = 3.0 Hz, 1H, CHOH-anti); 3.21 (d,
had a micelle in water/cyclohexanone mixtures using 575 J = 3.0 Hz, 1H, CHOH-syn); 2.66-2.30 (m, 1H, CHCHOH); 2.66-
cyclohexanone molecules. This number is close to the 2.30 (m, 2H, CH
2
C(O)); 2.16-1.24 (m, 6H, chex-H).
C NMR (50 MHz, CDCl ): δ 214.9 (C=O); 148.5 (ArC); 147.7
being 0.55 M and 0.66 M respectively. The resulting (ArC); 128.0 (2 x ArCH); 123.7 (2 x ArCH); 74.1 (CHOH); 57.3
1
3
cyclohexanone saturation concentration in water of 0.86 M,
3
2 2 2 2
concentration of PRW-C16 in these runs is roughly ten times (CHCHOH); 42.8 (CH ); 30.9 (CH ); 27.8 (CH ); 24.8 (CH ).
the CAC value measured in our experiments (see results).
Results and discussion
Simulation protocol
Structural determinations
Two thousand steps of steepest descent minimization was
followed by a short heating step of 140 ps to 300K using the
We first analyzed the self-assembly behavior of the PRW-C16
lipopeptide by determining its critical aggregation
concentration (CAC) in water through fluorescence assays. Our
first attempt to estimate the CAC was made by assessing the
intrinsic fluorescence of tryptophan available at the peptide
head of our lipopeptide. The presence of an indole
-1 chromophore in the tryptophan side-chain enabled direct
measurements of fluorescence and, since this amino acid is
54
Berendsen thermostat on a NVT ensemble. In this initial run
we used a 0.01 fs timestep and restrained all alkyl chains with
-2
a force constant of 10 kcal·Å . In the remainder of the
simulations we employed an NPT ensemble using the
Berendsen barostat with a 1.0 ps relaxation time and pressure
5
5
equals to 1 ATM, the Langevin thermostat with a 2 ps
5
6
collision frequency and a timestep of 0.02 fs. Shake was used
to constrain the hydrogen atoms to their initial positions. After
the initial heating step, we performed another 300 ps of
4
0, 61
widely-known to be sensitive to local polarity,
it was here
considered as a natural candidate for use as a probe in
aggregation studies. In Figure 2A, we show fluorescence data
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from PRW-C16 solutions illuminated with wavelength λexc = 280 To provide a more reliable estimation on CAC, we performed
nm, corresponding to the absorbance maximum of fluorescence experiments using solutions containing pyrene, a
6
tryptophan. The emission range exhibits a broad peak hydrophobic fluorophore whose quantum yield is strongly
2
4
1, 42
centered at
strongly correlated to peptide concentration (see inset in Fig. when aggregates appear in solution, the hydrophobic cores of
A). By plotting emission intensity as a function of log [peptide these particles provide nonpolar clusters where the
concentration], one clearly observes that fluorescence is fluorophore is trapped and its emission is raised. In Figure 2B
characterized by two distinct regimes. In the first domain, the intensity of pyrene emission at em = 394 nm is shown as a
λ
em
≈
357 nm, whose intensity appears to be dependent on polarity of local environment;
in this case,
2
λ
associated to dilute solutions, intensity increases regularly function of log [peptide concentration]. Again, two regimes are
upon peptide concentration in the mixture. On the other hand, clearly observed: in the dilute one, nearly-constant
the concentrated regime is typified by a systematic decay of fluorescence is found, indicating that lipopeptides remain in
fluorescence, which can be assigned to the inner-filter effect the monomeric form and no hydrophobic clusters are created
due to the tryptophan residue absorption or peptide in solution. On the other hand, in the concentrated range, one
6
3
aggregation in solution (or both). Unfortunately, the number observes sharp fluorescence growth and a linear relationship
of underlying parameters contributing for the emission between emission intensity and log [peptide concentration].
behavior is large and correction of filter effect across the Extrapolations from linear fits performed across the two
-1
concentration range is very complex; however, by investigating regimes reveal a crossover at 0.17 mmol L , which is
absorbance through UV-Vis assays (SI file, Fig. S2), one associated to the CAC. Also, this value is close to the the
observes saturation for peptide concentrations above ~ 0.1 separation between the emission domains found in Fig. 2A,
mM, close to the separation between the emission domains. suggesting that saturation in tryptophan absorbance is also
This finding suggests that, above this concentration, saturation related to appearance of aggregates in solution.
of detector leads to the strong decrease observed in emission Once the CAC was estimated, a sophisticated combination of
intensities.
in-situ techniques was used to characterize the morphology
and structure of self-assemblies in detail using experimental
and theoretical techniques. These assays included cryo-TEM
imaging from vitrified specimens and scattering experiments
performed in solutions similar to those where aldol reactions
were further investigated. In Figure 3, we show cryo-TEM
micrographs from mixtures containing the lipopeptide
-1
dissolved in water at a concentration of 15 mmol L , two
orders of magnitude above the CAC and representative of
catalyst concentrations used in the reactions below. One
observes that samples are mostly populated by nanostructures
with globular morphology and diameters between ≈
9 nm. A few structures with bigger volumes (sizes
4 nm and
15 nm)
≈
≈
and indefinite shapes – see detail in Figure 3C – are also found
in the medium. The hydrodynamic radius of particles in 15 mM
PRW-C16 solutions was estimated at 3.4 nm, with
polydispersity of about 12%, from dynamic light scattering
assays (SI file).
Information on the inner structure of the self-assemblies was
obtained from high-resolution SAXS. In Figure 4A, SAXS curves
from solutions prepared at different PRW-C16 concentrations
show scattering profiles characterized by oscillations across
the intermediate-to-high q-range. At moderate q-values, the
-
1
outline is dominated by a broad band centered at q ~ 1.7 nm ,
-1
-1
flanked by pronounced minima at q ~ 1 nm and q ~ 2.5 nm .
This shape of the data is coherent with globular structures and
the presence of sharp minima along with diffuse maxima, is
Figure 2. Fluorescence data used for estimating the CAC of the lipopeptide. (A) also consistent with high-symmetry particles suggesting high
Intensity of tryptophan fluorescence as a function of log [peptide concentration]. The
red line is a Lorentzian fit whose turning point has been ascribed to appearance of
aggregates in solution (see text for details). Inset: full-range fluorescence spectra from
PRW-C16 solutions. (B) Pyrene emission as function of log [peptide concentration]. The
64, 65
homogeneity of sizes and shapes in the solutions.
Despite
increasing concentration leads to better signal-to-noise ratios,
general features of data at higher q-values remain unchanged
intersection between straight lines fitted to data from different fluorescence regimes and support globular structures as dominant polymorphs in all
gives estimation of the CAC. Inset: fluorescence curves showing characteristic peaks solutions investigated.
associated to pyrene fluorescence. Intensities have been normalized to pyrene
emission in a peptide-free solution.
4
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Figure 3. Cryo-TEM micrographs from PRW-C16 solutions prepared at a concentration of
5 mM into water. (A) Samples appear populated by extensive numbers of globular
1
structures, most of them with diameters between ≈ 4 nm and ≈ 9 nm. (B) Magnified
image showing round micelles in detail. (C) Aggregates of micelles are highlighted by
arrows.
Conversely, some important differences are observed in the
low q-range and are evidence for different interacting regimes
as a function of peptide concentration. In the sample
containing the lowest amount of lipopeptide, 1.5 mM, a low q
upturn is found which has been ascribed to coexistence of
6
4
bigger structures in solution. At higher concentrations, 7.3
mM and 15 mM, a downturn of scattered intensities is
observed and appearance of a Bragg peak at small angle
6
4, 66
accounts for repulsive interactions between micelles.
To quantitatively describe these features and extract accurate
structural parameters, we performed model fitting of the data
4
5
using the SASFit program.
SAXS intensities in the
Figure 4. (A) SAXS data from lipopetide solutions at concentrations indicated. The
intermediate-to-high q-range has been fitted according to a spherical-shell model
intermediate-to-high q-range have been properly fitted
according to a spherical-shell form factor, convoluted with a leading to structural parameters described in Table 1. (B) 3D model of the PRW-C16
Gaussian radii distribution, in accordance with the presence of monomer, after energy minization (see text for details). Estimations of the geometric
characteristics of the molecule revealed a packing parameter ≈ 0.2. (C) Micelle model
micelles in the mixtures (see SI file for equations and further
obtained from geometrical constraints and association numbers revealed by SAXS data.
details). Values arising from this procedure are listed in Table 1
The model was built up using Packmol package and representations were rendered
and they reveal that the inner part of micelles have radii
using Pymol.
between 1.66 and 1.81 nm, with polydispersity of about 8%.
These values are in good agreement with the length estimated Still looking at Table 1, we see that thicknesses of spherical
for C16 alkyl chains, l
c
≈ 1.7 nm, and comply with previous work shells disclosed by SAXS are between 1.30 and 1.38 nm, with
on micelles formed from self-assembly of palmitoyl positive contrasts agreeing with their polar nature. These
6
7, 68
lipopeptides.
Furthermore, relative negative electronic values are consistent with the size of a tripeptide group (3 ×
contrasts of the cores are consistent with that expected for 0.34 = 1.02 nm) plus a TFA counterion shell. Putting these
7
0
6
8, 69
hydrophobic media, being also found in lipid chains.
findings together, we found that micelles have diameters of
about 6 nm, in close agreement with DLS data and consistent
with cryo-TEM imaging (Figure 3).
Table 1. Structural parameters obtained from model fitting of SAXS data (R: inner radius, σR: standard deviation of R; ꢁS: shell thickness, η
R
: core electronic contrast, η
S
: shell
electronic contrast; R : gyration radius; D: fractal dimension; RHS: hard sphere repulsion radius; F : volume fraction; V: micelle volume; n: association number).
g p
Mass fractal form
factor
Hard sphere structure
factor
Spherical shell form factor
Calculated parameters
Conc. (mM)
R (nm)
1.66
σ
R
(nm)
ꢁS (nm)
1.30
η
R
η
S
R
g
(nm)
D
2.54
---
RHS (nm)
---
F
p
V (nm³)
109
n
1
7
.5
.3
0.12
-1.67
-3.47
-4.95
1.61
3.63
4.95
13.6
---
---
145
164
181
1.76
0.14
0.12
1.33
11.8
0.18
0.21
123
1
5
1.81
1.38
---
---
9.5
136
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Journal Name
Self-assembly into spherical micelles is consistent with the constraints and association numbers were inputted in the
3
5
geometry of the PRW-C16 monomer, which presents a shape program Packmol for obtaining an approximate configuration
similar to an inverted cone (Figure 4B). for PRW-C16 micelles that was used as initial condition in our
In line with previous description of SAXS data, fitting at very simulations as described in the simulation setup section.
low q values required different strategies according to peptide The micelle simulation in water only (simulation A) over 50 ns
concentration used in the formulation. To fit this range for revealed a considerable variability in its shape. Cluster analysis
7
6
samples containing the lowest peptide concentration, 1.5 of the structures using a hierarchical clustering method
-1
mmol L , we introduced a mass fractal form factor with a having the C1-C15 distance as a metric (excluding intra-
radius of gyration of R = 13.6 nm and fractal dimension D = molecular pairs) showed that only two important topologies
.56 (see SI file for details). These measurements of size and with approximately equal weights dominate 68% of all
g
2
dimension are consistent with a few aggregates found in cryo- conformations. The C1-C15 (carbon one to carbon 15 from the
TEM images (Figure 3C), which were around 30 nm in extent alkyl chain) distance was chosen as it represents the average
and have an irregular shape. In samples prepared at higher radius of the inner hydrophobic part of the micelle when intra-
-1
-1
peptide concentrations, 7.3 mmol L and 15 mmol L , the molecular pairs are excluded. Figure 5 shows the average
small-angle data exhibits a Bragg peak arising from inter- cluster configurations that resulted from this analysis: panel A
particle repulsion. To fit this region, a hard sphere structure shows a single more cohesive but irregular structure, while
6
4, 66, 71
factor
was included in the model revealing repulsion panel B shows two micellar bodies with slightly different sizes.
radii of 11.8 and 9.5 nm, respectively. The volume fractions The second configuration is representative of the later stages
associated to the effective radii of the particles were found of the dynamics and formed spontaneously. The larger single
around 0.2. Complementary neutron scattering data structure shown in panel A is flatter in one dimension while
performed on samples containing 15 mM PRW-C16 dissolved in the smaller structures are more spherical in shape as seen on
2
D O – and thus exhibiting scattering contrasts very different cryo-TEM images.
from those observed in X-ray measurements – also indicated Interdigitation of the alkyl chains associated with the
core-shell structures with dimensions compatible with those monomer PRW-C16 charge character resulted in a rather
exhibited in Table 1 and the presence of inter-particle irregular micelle surface with increased surface area, which
repulsion (see SI file). We ascribe the emergence of this can be important to catalysis. Figure 6 shows the micelle using
repulsive regime at higher concentrations to increasing charge the surface representation for the amino acid moieties. As
density at the micelle surface. Indeed, arginine groups in expected, there is a large concentration of counter ions at the
functional group of lipopeptides remain protonated in solution micelle surface, with ions frequently being present in pockets
and the appearance of inter-micelle repulsion agrees with the closer to arginine residues.
growth of association number. Of course, such an increase in Radial distribution functions (RDFs or g(r)) using the C15 alkyl
the number of monomers per micelle also means that the carbon atom (C15 meaning carbon 15 from the alkyl residue)
density of proline catalyst sites also grows with concentration, as a reference revealed several interesting micellar structural
with strong repercussions on aldol reaction pathway as features (Figure S6). A value of g(r) greater than one means a
discussed below.
probability of finding a pair higher than in bulk (statistically
speaking) while a value less than one means a lower than in
bulk probability. The RDF between the C15 alkyl carbon and
water oxygen atoms revealed a drop in water density starting
at about 40 Å from the hydrophobic core.
Molecular Dynamics Simulations
To investigate the micelle structure in more detail we
performed molecular dynamics simulations. An aggregation
number range was estimated from molecular dynamics
simulations of a single PRW-C16 molecule in water and SAXS
data. The molecule surface area computed using the
7
2
2
Connolly approach gave an average value of 0.8 nm for the
2
whole system and 0.470 nm for the hydrophilic head. The
solvent exclusion volume computed from the solvent radial
3
distribution function gave values in the 0.8 to 0.9 nm range
3
for the whole system and 0.4 nm for the hydrophobic tail.
Simple geometrical considerations using an alkyl chain length
of 1.9 nm (l
calculate the packing parameter of the molecule at p = v
0.18, much lower than 0.33 which is the critical p
expected for self-assembly of surfactant-like moieties into
c
) and these estimated values allowed us to
/
(l
c
×
0
a ) ≈
7
3-75
spherical micelles.
From the solvent exclusion volume it is
A
B
possible estimate that micelles have association numbers
between n
≈
investigated. Information derived above for both geometric
121 and n
≈
170 across the concentration range Figure 5. Average representative cluster configurations from 50 ns simulations. The
amino acids are represented in gray while the alkyl chains are represented in red.
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simulation B having all cyclohexanone molecules on the
outside of the initial micelle while simulation while C had 50
cyclohexanone molecules at the center. Despite the
differences in initial conditions, both the final structures and
RDFs were nearly identical for the two simulations. Most
relevant to the results at hand, the RDFs indicate a similar
ordering in solution as in the water only case, with one
important difference: there is a strong preference for
cyclohexanone to be near or at the hydrophobic core of the
micellar structure as shown by Figure S7 and by the g(r)
presented in Figure S8. This generates a higher concentration
of cyclohexanone close to catalytic L-proline residues when
compared to bulk values, possibly allowing to a more efficient
catalysis. In fact, the RDFs presented in Figure S7 for proline
and ciclohexanone that have the same carbon 15 from the
alkyl chain as a reference show considerable overlap,
suggesting a kinetic enhancement allowed by the micellar
structure. In summary, our simulations suggest that the
Figure 6. Micelle structures after 15 ns (panel A) and 45 ns (panel B) of simulation time
for the water only simulation (simulation A). The amino acids are rendered using a
surface representation, the inner alkyl core using a bond representation in red and
chlorine ions are rendered in yellow.
Meanwhile the C1-C15 g(r) peaks at 1.4 nm, defining a micellar structure provides a unique arrangement for the aldol
hydrophobic core domain with C15-Wat(O) g(r) having only reaction, with higher local concentration of cyclohexanone
1
/3 of its bulk value at that distance. The RDFs also revealed close to the catalytic proline in a less hydrophilic environment.
5
7
that the arginine and proline residues remain at about the All presented analyses were made using the AmberTools 15
same distance from the core, with g(r) for the C15-Arg(NE) and suite of codes.
C15-Pro(N) peaking at about 2.4 nm. The g(r) for C15-Trp(NE)
distribution peaks at 1.8 nm, corroborating a picture that the Aldol Reactions
tryptophan ring interacts with the hydrophobic core of the
The catalytic activity of the PRW-C16 lipopeptide for the direct
asymmetric aldol reaction was investigated by performing a
model reaction using p-nitrobenzaldehyde and cyclohexanone
at room temperature, in the presence and absence of water.
The results are summarized in Table 2.
micelle. These RDFs are consistent with an inverted cone
configuration adopted by the PRW-C16 lipopeptides during the
dynamics. They are also consistent with the presence of a well-
defined hydrophobic core composed of alkyl chains and
tryptophan with a dimension in the 1.4-1.8 nm range. These
results are consistent with SAXS results for the inner radius
and with an ordered core shell structure present in the micelle.
Results for the water/cyclohexanone mixture simulations,
namely simulations B and C, indicate a similar atomistic
arrangement in solution as revealed by very similar RDFs to
the water only case (Figure S6). The overall structure of the
simulated object, however, changed considerably. The
addition of cyclohexanone lead to much more irregular shaped
micelles, with smaller aggregates separating from the larger
bodies. Simulations B and C differ in their initial condition, with
The reactions performed in aqueous media provided products
in excellent yields with good to excellent diastereoselectivities
and enantioselectivities for the anti aldol product. The best
observed catalytic efficiency is shown in entry 1 with full
conversion to anti-diastereomeric product, anti/syn
diastereomeric ratio of 92:8 and enantiomeric excess (ee) of
8
5%. By decreasing the catalyst loading from 5 to 1 mol%, the
reaction exhibited lower diastereoselectivity (entry 4, 87:13
ds).
a
Table 2. Direct asymmetric aldol reaction of p-nitrobenzaldehyde and cyclohexanone catalyzed by PRW-C16 lipopeptide.
b
c
c
d
Entry
Loading (mol%) Cyclohexanone (eq.)
H
2
O (eq.)
Conv. (%)
anti/syn
ee (%)
1
2
3
4
5
6
5
5
5
1
1
5
11.7
11.7
11.7
11.7
15.0
15.0
0.5
>99
>99
>99
>99
36
92:8
85
71
84
85
63
73
1.0
91:9
2.0
91:9
1.0
87:13
81:19
86:14
e
-
e
-
94
a
b
c
The reactions were performed at room temperature, for 2 days, with vigorous stirring; Water excess towards cyclohexanone (v/v); The diastereomeric ratios and
1
d
e
conversion were determined by H NMR analysis of the crude mixture; Determined by chiral-phase HPLC analysis of the anti isomer; Neat (no water addition).
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Different results were obtained when water was removed data, cryo-TEM imaging, and molecular dynamics simulations.
from the system and the cyclohexanone volume was Based on its amphiphilic nature, the peptide was found to be
increased. With 1 mol% loading of catalyst, both the an excellent catalyst for aldol reactions performed in water
conversion and stereoselectivity were reduced (entry 5, 36% under the optimal reaction conditions, where excellent
conversion, 81:19 ds, 63% ee). Using 5 mol% loading of diastereoselectivities (92:8) and enantioselectivities (85%) with
catalyst, there was an improvement in conversion but the very good conversions (>99%) were obtained by using as little
stereoselectivity remained lower in comparison with the tests as 5 mol% of catalyst. It is noteworthy that we did not use any
promoted in water (entry 6, 94% conversion, 86:14 ds, 73% additives in the experiments. A suggestive agreement between
ee).
reaction results and concentration effects computed by
The amphiphilic peptide showed better catalytic efficiency in molecular dynamics simulations of the cyclohexanone residues
aqueous media, by comparison with its performance in organic is noted.
media (neat cyclohexanone, entries 5 and 6), since the micelle
environment seems to provide an environment that enhances
Acknowledgements
catalysis. Therefore, water is responsible for optimizing the
catalyst and substrates approaching by forming a self-
This work was supported by FAPESP (grant no. 2015/24018-1)
assembling structure.
and CNPq (grant no. 302923/2015-2, 400239/2014-0). INCT in
The observed differences in results point to the existence of a
Bioanalytics (FAPESP grant no. 08/57805-2 and CNPq grant no.
structural feature directly involved in the reaction mechanism.
5
73672/2008-3) is kindly acknowledged for the grants. B.M.S.
It seems that the micelles revealed by cryo-TEM and SAXS data
and molecular dynamics simulations are responsible for the
catalysis promotion by the amphiphilic peptide. The effect of
phase transfer may contribute to enhancement of
stereoselectivity in aldol coupling reactions. Thus, the higher
activity of the lipopeptide in water is mainly due to the unique
environment around the proline residue that is similar to the
one present in a natural enzyme.
acknowledges CAPES for a doctoral fellowship and E.R.S. is
grateful to FAPESP for post-doctoral grants (Proc. 2013/12674-
6
). I.W.H. is recipient of a Royal Society-Wolfson Research
Merit Award and this work was supported by EPSRC Platform
Grant “Nanostructured Polymeric Materials for Healthcare”
(
EP/L020599/1). Synchrotron SOLEIL is kindly acknowledged
for awarding beamtime for SAXS experiments (proposal
0140919). E.R.S. and I.W.H. thank Steven Kirkham and Dr.
2
Corroborating the structural data previously presented for the
lipopeptide with the model aldol reaction results, we propose
based on molecular dynamics results that the micellar
aggregate formed in water concentrates the substrates in its
inner region, keeping the transition states mostly isolated from
water, affording, thereby, aldol products in excellent yields
with high diastereo- and enantioselectivities. In fact,
macroscopically, the reaction presents itself as an emulsion.
When it is performed in the absence of water (i.e., neat
cyclohexanone), the aldol reaction shows reduced yields and
stereoselectivity due to a lack of an aggregate that provides
Javier Perez for assistance during SAXS experiments at SWING
beamline. SANS measurements were performed at ISIS under
proposal RB1510029 and Dr. Stephen King is kindly
acknowledged for help during this beamtime. Prof. Alessandro
Rodrigues is kindly acknowledged for his help and support in
HPLC experiments. The anonymous Referee is also
acknowledged for leading us to consider the presence of inner-
filter effect and correcting discussion on tryptophan
fluorescence assays.
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