Uncovering Key Structural Features of an Enantioselective Peptide-Catalyzed Acylation Utilizing Advanced NMR Techniques

Article abstract of DOI:10.1002/anie.201608559

We report on a detailed NMR spectroscopic study of the catalyst-substrate interaction of a highly enantioselective oligopeptide catalyst that is used for the kinetic resolution of trans-cycloalkane-1,2-diols via monoacylation. The extraordinary selectivity has been rationalized by molecular dynamics as well as density functional theory (DFT) computations. Herein we describe the conformational analysis of the organocatalyst studied by a combination of nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC)-based methods that resulted in an ensemble of four final conformers. To corroborate the proposed mechanism, we also investigated the catalyst in mixtures with both trans-cyclohexane-1,2-diol enantiomers separately, using advanced NMR methods such as T₁relaxation time and diffusion-ordered spectroscopy (DOSY) measurements to probe molecular aggregation. We determined intramolecular distance changes within the catalyst after diol addition from quantitative NOE data. Finally, we developed a pure shift EASY ROESY experiment using PSYCHE homodecoupling to directly observe intermolecular NOE contacts between the trans-1,2-diol and the cyclohexyl moiety of the catalyst hidden by spectral overlap in conventional spectra. All experimental NMR data support the results proposed by earlier computations including the proposed key role of dispersion interaction.

Full text of DOI:10.1002/anie.201608559

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201608559
German Edition: DOI: 10.1002/ange.201608559
NMR Spectroscopy
Uncovering Key Structural Features of an Enantioselective Peptide-
Catalyzed Acylation Utilizing Advanced NMR Techniques
ˇ
Eliska Prochꢀzkovꢀ, Andreas Kolmer, Julian Ilgen, Mira Schwab, Lukas Kaltschnee,
Maic Fredersdorf, Volker Schmidts, Raffael C. Wende, Peter R. Schreiner,* and
Christina M. Thiele*
Abstract: We report on a detailed NMR spectroscopic study of
the catalyst-substrate interaction of a highly enantioselective
oligopeptide catalyst that is used for the kinetic resolution of
trans-cycloalkane-1,2-diols via monoacylation. The extraordi-
nary selectivity has been rationalized by molecular dynamics as
well as density functional theory (DFT) computations. Herein
we describe the conformational analysis of the organocatalyst
studied by a combination of nuclear Overhauser effect (NOE)
and residual dipolar coupling (RDC)-based methods that
resulted in an ensemble of four final conformers. To corrob-
orate the proposed mechanism, we also investigated the catalyst
in mixtures with both trans-cyclohexane-1,2-diol enantiomers
separately, using advanced NMR methods such as T1 relaxa-
tion time and diffusion-ordered spectroscopy (DOSY) meas-
urements to probe molecular aggregation. We determined
intramolecular distance changes within the catalyst after diol
addition from quantitative NOE data. Finally, we developed
a pure shift EASY ROESY experiment using PSYCHE
homodecoupling to directly observe intermolecular NOE
contacts between the trans-1,2-diol and the cyclohexyl moiety
of the catalyst hidden by spectral overlap in conventional
spectra. All experimental NMR data support the results
proposed by earlier computations including the proposed key
role of dispersion interaction.
acylation is a powerful tool for preparing optically pure
compounds for chemical syntheses.[2] For a long time, enzymes
played a key role in organic synthesis, for example, lipases or
acylases have been employed for acylation of structurally
different classes of secondary alcohols.[3] Within the last two
decades peptide-based catalysts have been discovered and
established as versatile low-molecular-weight alternatives to
enzymes.[4] They can form enzyme-like active sites and can
interact specifically with substrates.[4b,5] Such catalysts are
considered very efficient owing to their high structural
diversity and their ability to form multiple non-covalent
interactions simultaneously, for example, hydrogen bonds,
van der Waals as well as electrostatic interactions. Although
this biomimetic approach works well for a variety of racemic
alcohols, the effectivity of the kinetic resolution of trans-
cycloalkane-1,2-diols had been astonishingly low.
This situation changed with the introduction of the novel
lipophilic peptide-based catalyst Boc-l-(p-Me)-His-AGly-l-
Cha-l-Phe-OMe[6] (1), where AGly represents g-aminoada-
mantane carboxylic acid (Scheme 1). The designed oligopep-
tide contains a non-natural adamantyl amino acid to increase
rigidity and solubility in organic solvents. The nucleophilic N-
p-methylhistidine moiety is deemed catalytically active
during the enantioselective acetyl transfer.[4a] Finally, the
unnatural amino acid (S)-cyclohexylalanine is proposed to
enable dispersion interactions with the substrate that build
the basis for high selectivities.[7] To rationalize the origin of
the extraordinary high stereoselectivity of peptide catalyst 1,
molecular mechanics (MMFF) was employed and a dynamic
binding-pocket within the acylated tetrapeptide intermediate
was proposed.[7] In subsequent work,[7,8] the transition struc-
tures of the tetrapeptide–diol complex were optimized by
DFT [M06-2X/6-31 + G(d,p)] computations taking into
account medium-range correlation effects.[9] However, there
was no experimental evidence confirming the proposed
mechanism and explaining the extraordinary enantiodiscri-
mination up to now.
A
cyl transfer belongs to one of the most significant chemical
reactions occurring in biological systems as well as in organic
synthesis, where it is widely used in enantioselective catal-
ysis.[1] Kinetic resolution of chiral secondary alcohols through
[*] Dr. E. Prochꢀzkovꢀ, Dr. A. Kolmer, J. Ilgen, M. Schwab,
Dr. L. Kaltschnee, Dr. M. Fredersdorf, Dr. V. Schmidts,
Prof. Dr. C. M. Thiele
Clemens-Schçpf-Institut fꢁr Organische Chemie und Biochemie
Technische Universitꢂt Darmstadt
Alarich-Weiss-Strasse 16, 64287 Darmstadt (Germany)
E-mail: cthiele@thielelab.de
Herein, we investigate the conformational ensemble of
peptide catalyst 1 by a combination of NOE[10] and RDC[11]
data to extract NMR parameters directly connected to
molecular structure (distances, angles). Furthermore, the
differential interactions of 1 with the two enantiomers of
trans-cyclohexane-1,2-diol (2) are explored to gain insights
into the enantioselective recognition process.
To investigate 1 under conditions as close to the reaction
conditions as possible, as little as 2 mg of 1 were dissolved in
deuterated toluene (3.2 mmolLÀ1). As the signals were
severely broadened (see Supporting Information) at the
Dr. R. C. Wende, Prof. Dr. P. R. Schreiner
Institut fꢁr Organische Chemie
Justus-Liebig-Universitꢂt
Heinrich-Buff-Ring 17, 35392 Giessen (Germany)
E-mail: prs@uni-giessen.de
Dr. E. Prochꢀzkovꢀ
On leave from Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic
Flemingovo nꢀm. 2, 166 10, Prague (Czech Republic)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201608559.
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ensemble with the best agree-
ment between experimental
and calculated distances/struc-
tures was different for ROEs
and RDCs. Further attempts to
solve this problem by optimiz-
ing the MMFF structures at the
B3LYP/6-31 + G(d,p) level or
generating additional structures
using the OPLS2.1[20] force field
(as implemented in MacroMo-
del)[21] led to the same results. In
total we examined 660 struc-
tures, only 184 of which were
redundant (present in different
sets of structures obtained by
Scheme 1. Kinetic resolution using the tetrapeptide catalyst 1, (R,R)-trans-cyclohexane-1,2-diol is acety-
lated preferentially (left). The dynamic binding pocket in the acylium ion/catalyst adduct with (R,R)-2
proposed by computation (right);[7] dotted lines denote proposed H-bonds. Note the close proximity of
the cyclohexyl moiety of 1 to 2 (dispersion interactions).
different
methods).
Thus,
a change in strategy was neces-
sary.
temperature associated with the highest selectivity (S > 50 at
À208C), we conducted the experiment at 278C, the temper-
ature at which lines are sharp and selectivity is still high (S =
23 at room temperature). Quantitative distance information
was obtained from 1D selective NOE experiments[12] as well
as 2D EASY ROESYs (efficient adiabatic symmetrized
rotating frame Overhauser effect spectroscopy)[13] recorded
as a series of measurements differing in mixing-time values.
These served for the construction of PANIC[14] (peak
amplitude normalization for improved cross-relaxation)
plots, which permit the use of data points acquired with
much longer mixing times and improves the accuracy of
distances obtained. RDC data involve the coupling measure-
ments of isotropic as well as anisotropic samples. To obtain
the anisotropic samples the compound in question needs to be
oriented with respect to the magnetic field by using so called
alignment media. Few alignment media are compatible with
both toluene and 1. We succeeded in using an anisotropically
swollen gel of thermally cross-linked (via divinylbenzene)
The structures were analyzed in terms of dihedral angle
ranges present and clustered accordingly (see Supporting
Information). The fact that all six possible dihedral angle
ranges are populated in the computed structures for most of
the dihedral angles points toward a full coverage of the
conformational space by the computations. We decided to
employ a stepwise construction procedure and divided 1 into
two parts with rigid AGly as the obvious incision. For the “left
hand” side (Phe-Cha part) nine and for the “right hand” side
(His part) five dihedral angles were identified as relevant
parts of the “backbone”, respectively. Out of each dihedral-
angle range for any relevant dihedral angle, one structure was
chosen and the resulting ensemble was successively probed
using the ROE data for these junctions (for details see
Supporting Information). This led to four populated con-
formers for the Phe-Cha part and three populated conformers
for the His part. The ROE-derived ensemble of the Phe-Cha
part was then subjected to a multi-conformer single tensor
(MCST) evaluation using the RDC data of the respective
side.[19a] Although it is necessary to adjust the population of
the conformers to obtain a good fit of the RDC data, it is
nevertheless very reassuring that the conformers describing
the RDC data sufficiently well are among those obtained
from ROE data. The same cross check is impossible for the
His part, owing to a too small number of RDCs available. In
the next step, we joined the two parts: for which the global
information content of RDCs is most helpful. Additionally,
a number of inter-domain ROEs were also obtained, albeit
with larger errors (see Supporting Information). The four
conformers of the Phe-Cha part and the three conformers of
the His part were then probed with all RDC data in a MCST
analysis as well as with ROE data. According to the RDC
analysis, four conformers are populated (Figure 1); among
those, three are also populated in the ROE analysis (although
populations differ). Thus, we concluded that we have
obtained an ensemble of conformers that is in accordance
with ROE and RDC data. This ensemble confirms the pocket-
like structure of 1 proposed computationally (for comparison
see Supporting Information).
polystyrene[15] (27.4 mmolLÀ1 of 1). One-bond C H coupling
À
data (1JC-H/1DC-H) obtained from clean in-phase HSQC (CLIP-
HSQC)[16] spectra, as well as long-range coupling C H data
À
(nJC-H/nDC-H) obtained from HETLOC (determination of
heteronuclear long range couplings)[17] were used in the
structure elucidation process (see Supporting Information for
representative PANIC plots, ROE derived distances, and
RDCs). Having obtained these complementary structural
parameters, the next step in the structure elucidation process
was to find conformers or conformer ensembles that are in
agreement with the experimental data. For RDCs in flexible
organic compounds this is usually achieved by fitting the
experimental data via singular value decomposition (SVD)[18]
to ensembles of computed structures (e.g., DFT).[19] The
ensemble with the best fit is considered to be the most
probable (minimal) ensemble.
When considering the constitution of 1 as well as the
number of low-energy structures in the computations, appre-
ciable flexibility is expected. Therefore, we searched for an
ensemble of conformers that was in accordance with ROE
and RDC data simultaneously. All our attempts failed as the
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(X300KÀX253K) for (S,S)-2 is significantly higher (0.70) than
that for (R,R)-2 (0.35). These results indicate that after
a temperature change, the complex of 1 with R,R-(2) still
behaves similarly, on the other hand, the complex with (S,S)-2
displays larger changes. This could point towards a tighter
complex in the case of (R,R)-2.
To investigate whether structural changes occur in 1 after
diol addition, we measured quantitative selective 1D NOE
spectra (analyzed by PANIC plots). We extracted intra-
molecular distances and found significant changes between
1 alone and after the addition of 2. Again, as in the case for the
chemical shifts, we observed a dependence of intramolecular
distances on concentration (see Supporting Information),
thus, it is indispensable to compare samples with the same
concentration only. The most significant changes are observed
in the case of (R,R)-2 at the His part of 1 (H-26, H-27, and H-
28, see Supporting Information for corresponding PANIC
plots). Interestingly, this is also the moiety that is responsible
for the acetyl transfer. In Figure 2, these data are displayed as
Figure 1. Four final conformers of peptide 1 obtained by a combination
of ROE and RDC data.
After having verified the pocket-like shape of 1, we
attempted to acquire structural evidence for the enantiose-
lective recognition of diol 2 by 1. Mixtures of 1 with 2 in
[D8]toluene (both enantiomers separately) were prepared at
two different concentrations of 1 (19.3 and 38.6 mmolLÀ1): A
significant dependence of the chemical shifts on concentra-
tion was observed (see Supporting Information), thus, it is
essential to compare the samples at the same concentration.
To study the degree of aggregation between 1 and the
enantiomers of diol 2, we measured the T1 relaxation times
and conducted DOSY experiments.
In general, bigger molecules or molecular complexes tend
to display higher values of correlation time tC and this
quantity is directly connected to relaxation time constants.
Therefore, significant differences in T1 could be helpful in
deciding whether there is dynamic catalyst–substrate complex
formation. For this purpose, we selected the well-separated
NMR signal H-1 of 2 (CH proton neighboring the OH group;
chemical shift ca 3.3 ppm, see Supporting Information). The
T1 of 2 in a mixture with 1 is significantly lower (ca 2.5 s) than
for 2 alone (10.5 s). No significant difference in T1 between
the samples containing (R,R)-2 and (S,S)-2 was found though
(2.5 and 2.6 s, respectively). On the other hand, the T1 of
1 without and with 2 did not change significantly (1.58 and
1.60 s, respectively, see Supporting Information). T1 relaxa-
tion time data suggest that there is an interaction between
1 and 2, but no evidence was found for preferential binding of
one of the enantiomers.
Figure 2. Intramolecular distances of 1 without and with addition of 2
as extracted from quantitative 1D NOE measurements. (R,R)-2 addi-
tion (triangles) causes significantly larger changes in the structure of
1 than the addition of (S,S)-2 (circles). The error of measurement was
estimated to be not higher than 0.2 ꢃ.
To support this interaction hypothesis, we measured
DOSY spectra, where the chemical shift is correlated with
the diffusion coefficient. We hoped to see different behavior
of the enantiomeric diols 2 in mixtures with 1 in terms of
different complex formation. At room temperature, we
observed the same trend in both diol enantiomer mixtures.
The diffusion coefficient of 2 is approximately three times
higher than that of 1 (Ddiol = XDcat, with X ꢀ 3). We also
measured D at the same temperature at which the catalytic
reaction works best (À208C) and observed (Table 1) that DX
a correlation between intramolecular distances of 1 alone and
in mixtures with 2. After (R,R)-2 addition (Figure 2, trian-
gles), the H26–H28 distance is up to 0.6 ꢀ larger, on the other
hand, the H26–H27 distance is smaller. This means that an
addition of (R,R)-2 causes more significant structural changes
of 1 than (S,S)-2 (Figure 2, circles), in accordance with the
preferential acetylation of (R,R)-2.[6,7]
In 1D NOE spectra, it was also possible to identify
intermolecular contacts between 2 and the catalytically active
His part of 1 (see Supporting Information). This observation
led us to the idea to examine whether the observed
intermolecular interactions are in accordance with the
dispersion interactions[22] proposed by M06-2X/6-31 + G(d,p)
computations.[7] However, this necessitated the development
of a novel NMR spectroscopic method.
Table 1: The diffusivity ratios (X=Ddiol/Dcat.) of the catalyst 1 and diols 2
at two different temperatures (room temperature and the reaction
temperature).
Enantiomer
X300K
X253K
DX
As the aliphatic region in which we expected intermolec-
ular interactions (0.9–2.1 ppm) is overcrowded, we simplified
the spectra using homonuclear decoupling (pure shift
(R,R)-2
(S,S)-2
2.96
3.09
2.61
2.39
0.35
0.70
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Figure 3. Pulse sequence of PUSH-EASY-ROESY with decoupling in the F2 (A) and F1 (B) dimension. Narrow and wide rectangles represent 908
and 1808 pulses, respectively. Trapezoids with diagonal arrows correspond to low-power chirp pulses of small flip angle, which sweep frequency in
opposite directions simultaneously. Broad rectangles in gray are low- and high-field spinlocks; half-Gaussian shaped pulses were used as adiabatic
ramps. For more details see Supporting Information.
approach) to collapse broad multiplets into singlets.[23] We
used the PSYCHE[24] experiment (pure shift yielded by chirp
excitation) from which we expected the best compromise
between decoupling quality and sufficient sensitivity. As
homodecoupling simplified NMR signals assignment signifi-
cantly, the PSYCHE element was implemented into the
EASY ROESY pulse sequence. We thus developed two new
pure shift EASY ROESY NMR experiments (PUSH-EASY-
ROESY, Figure 3), which provide negligible offset depend-
ence combined with an efficient suppression of possible
TOCSY transfer artefacts and the improved signal resolution
from PSYCHE homodecoupling. In general, the experiment
was set up with one dimension decoupled (F1 or F2) and then
covariance processing[25] (along F2 or F1, respectively) was
employed to produce a doubly pure shift 2D spectrum.
Almost the same results were obtained for the decoupling of
either F1 or F2, albeit the decoupling in the indirect dimension
is less time consuming: the F1-decoupled PUSH-EASY-
ROESY was measured in 26 h whereas the F2-decoupled
experiment took significantly longer. Note that the improved
resolution asks for a compromise in sensitivity (typical
reduction to 5–20% of the intensity in conventional spectra).
The covariance-processed data provide significantly
better resolution (ultra-high-resolved spectra) than Fourier-
transform processed spectra (see Supporting Information)
and enabled us to detect the intermolecular interaction
between the two cyclohexyl moieties of 1 and 2 (Figure 4).
For illustration of the intermolecular interactions found we
used the computed structure of 1, which agrees well with
RDCs and ROE-based data (one of the four final structures
from Figure 1) and added (R,R)-2. The blue arrow in Figure 4
corresponds to the interactions found in PUSH-EASY-
ROESY as cross peaks (in the blue frames).
published computational studies. After the conformational
analysis of peptide catalyst 1 based on ROE and RDCs, we
Figure 4. Part of the F1-PSYCHE-decoupled EASY ROESY spectrum of
a mixture of 1 with (R,R)-2. For simplicity, 1D TSE-PSYCHE spectra are
shown along both dimensions.
In conclusion, we obtained detailed experimental NMR
spectroscopic data that support the results from previously
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of 2 (utilizing T1 measurements), we showed that catalyst–
substrate complex formation occurs; we found a four-times
lower T1 of 2 in a mixture with 1. These results were supported
by DOSY measurements, where we observed that (R,R)-2
forms a tighter interaction with 1 than (S,S)-2. From the
intramolecular distances extracted from NOE-based data, we
assigned significant structural changes in the His part of
1 after the addition of (R,R)-2. Finally, we developed two
novel NMR methods, namely EASY ROESY experiments
with PSYCHE homodecoupling implemented in the F1 or F2
dimension. These techniques enabled the detection of inter-
molecular dispersion interactions between 2 and the cyclo-
hexyl moiety of 1 despite the overcrowded NMR spectra. This
work supports the predicted structural properties, such as
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enantiodiscrimination of racemic diol 2, and crucial intermo-
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open a new field for study of catalyst–substrate complex
formation.
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Acknowledgements
We thank A.-C. Pçppler for a gift of the thermally cross-
linked PS gel and C. E. Mꢁller for performing some of the
initial computations. This work was supported by the DFG,
Priority Program “Dispersion” (SPP 1807, Schr 597/27-1) and
by ERC grant No. 257041 (to C.M.T.).
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Keywords: conformational analysis ·
enantioselective acylations · NMR spectroscopy ·
pure shift NMR · RDCs
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Received: September 1, 2016
Published online: && &&, &&&&
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Communications
NMR Spectroscopy
Mission possible: Advanced NMR spec-
troscopic data support the mechanism
proposed for a highly enantioselective
acylation reaction. The pocket-like struc-
ture of the catalyst is structurally affected
upon the addition of the preferentially
acetylated diol substrate and key disper-
sion interactions between catalyst and
substrate were uncovered by a newly
developed pure shift EASY ROESY
experiment.
E. Prochꢁzkovꢁ, A. Kolmer, J. Ilgen,
M. Schwab, L. Kaltschnee,
M. Fredersdorf, V. Schmidts, R. C. Wende,
P. R. Schreiner,*
C. M. Thiele*
&&&& — &&&&
Uncovering Key Structural Features of an
Enantioselective Peptide-Catalyzed
Acylation Utilizing Advanced NMR
Techniques
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!