Helical Polyisocyanopeptides as Lyotropic Liquid Crystals for Measuring Residual Dipolar Couplings

Article abstract of DOI:10.1002/chem.201700539

Residual dipolar couplings (RDC) emerged to be an important structural parameter for organic and biomolecules. Herein, a new helical polyisocyanopeptide (l,l-PIAF-OBn) that forms lyotropic liquid crystals (LLC) in CDCl₃ is proposed as a novel weakly orienting medium for acquiring residual dipolar couplings (RDCs) of organic molecules. We demonstrate its application for the structural elucidation of strychnine and triptolide.

Full text of DOI:10.1002/chem.201700539

A Journal of
Accepted Article
Title: Helical Polyisocyanopeptides as Lyotropic Liquid Crystals for
Measuring Residual Dipolar Couplings
Authors: Xinxiang Lei, Gao-Wei Li, Jiang-Ming Cao, Wen Zong, Li Hu,
Mao-Lin Hu, Ren Xiang Tan, and Han Sun
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To be cited as: Chem. Eur. J. 10.1002/chem.201700539
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Helical Polyisocyanopeptides as Lyotropic Liquid Crystals for
Measuring Residual Dipolar Couplings
Gao-Wei Li,[a] Jiang-Ming Cao,[a] Wen Zong,[c] Li Hu,[c] Mao-Lin Hu,[c] Xinxiang Lei,*[b] [c] Han Sun,*[d] and
Ren Xiang Tan*[a]
stable LLCs and simultaneously exhibit low critical concentration
are rather rare. Recently, the peptide-functionalized
polyisocyanides have been extensively explored, showing their
comprehensive applications to chirality sensing, drug-delivery
and LC formation.[9] Polyisocyanopeptides with well-defined
helical β-sheet architecture are thus promising candidates for
polymeric LC design. Based on the study mentioned above and
encouraged by our previous work on the development of
graphene oxide (GO) based LC phases for aligning small
molecules in polar solvents,[10] we designed and synthesized a
new dipeptide-based polyisocyanide, which has been proven to
be an effective and tuneable alignment medium for acquiring
RDCs in apolar solvents such as CDCl3.
Abstract: Residual dipolar coupling (RDC) emerged to be an
important structural parameter for organic molecules, as well as
biomolecules. Here, a new helical polyisocyanopeptide (L,L-PIAF-
OBn) that forms lyotropic liquid crystals (LLC) in CDCl3 is proposed
as a novel weakly orienting medium for acquiring residual dipolar
couplings (RDCs) of organic molecules. We demonstrate its
application to the structural elucidation of strychnine and triptolide.
As an information-rich NMR structural parameter, residual
dipolar coupling (RDC) has been widely employed in the
elucidation of constitution, relative configuration, and
conformation of organic molecules.[1] A combined approach
using RDC analysis and chiroptical methods was proposed and
demonstrated on the determination of the absolute configuration
of a number of small molecules.[2] In general, RDCs can be only
measured, if the molecule is partially aligned in an anisotropic
environment, such as lyotropic liquid crystal (LLC) phases and
stretched or compressed gels.[3] The method of reversible
compression/relaxation of flexible cross-linked gels introduced
by Gil et. al facilitates the measurement of residual dipolar
coupling and residual chemical shift anisotropy with high
efficiency and precision.[3f-h] As an alternative approach, LLCs
are good options for RDC measurements, as they can be freely
scaled by the variation of the concentrations and their ability to
quickly align the sample. The most employed LLC-based
alignment media for organic molecules are the α-helical
homopolypeptides PBLG, PELG, PCBLL,[4] ACHC-rich β-
peptides,[5] as well as recently introduced polymeric LLCs such
as polyguanidines,[6] polyacetylenes[7] and polyisocyanides.[8] An
effective and commonly used strategy in designing polymeric
LLCs as new alignment media is to create a confined system
which is composed of a stable polymer backbone. These self-
assembled helical polymers usually display high persistence
lengths that are capable of forming LLC media at relatively low
concentrations, and therefore can be translated into a moderate
degree of order for organic compounds.
To our knowledge, organic solution-based polymers that form
[a]
[b]
G. W. Li, J. M. Cao, Prof. R. X. Tan
Institute of Functional Biomolecules, State Key Laboratory of
Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210093,
P.R. China
E-mail: rxtan@nju.edu.cn
Prof. X. Lei
School of Pharmaceutical Sciences, South Central University for
Nationalities, Wuhan, 430074, P. R. China
E-mail: xinxianglei@aliyun.com
W. Zong, L. Hu, Prof. X. Lei, Prof. M. L. Hu
College of Chemistry & Materials Engineering, Wenzhou University,
Wenzhou, 325035, P. R. China
Figure 1. A) Basic principles employed in designing peptide-functionalized
polyisocyanides, which form stable LLC phases in CDCl3. B) Polymerization of
the monomer L,L-IAF-OBn, with the proposed hydrogen bonding network
between side chains for stabilizing the helical structure of the polymer.
[c]
[d]
Dr. H. Sun
In the first step, we designed four different polyisocyano-
dipeptides (PIPs) sharing the same chemical backbone. Figure 1
shows the polymerization procedure of the representative
monomer L-isocyanoalanyl-L-phenylalanine benzyl ester (L,L-
IAF-OBn) using an achiral nickel catalyst. Other PIPs could be
obtained using the same procedure. Polymerization details and
Leibniz-Institutfꢀr Molekulare Pharmakologie (FMP), Robert-
Roessle-Strasse 10, 13125 Berlin, Germany
E-mail: hsun@fmp-berlin.de
Supporting information for this article is given via a link at the end of
the document.
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the monomer synthesis are described in the Supporting
Information. Interestingly, we found that although all the
polymers presented in Figure 1A could be dispersed in CDCl3,
only poly(L-isocyanoalanyl-L-phenylalanine benzyl ester) (L,L-
PIAF-OBn) was able to quickly form liquid crystals. When
replacing one of the functional groups (R1 and R2, shown in
Figure 1A) in L,L-PIAF-OBn by other alkyl groups (e.g., L,L-
PIAF-OMe, L,L-PIAA-OBn and L,L-PIAF-ODc), the obtained
polymers did not form the LC phases. In addition, the UV-visible
In order to test the compatibility of L,L-PIAF-OBn with organic
molecules, we acquired RDCs of the highly rigid strychnine.
One-bond proton-carbon J-couplings (1JCH) in the isotropic
sample and the sum of the one-bond dipolar and J-couplings
(1TCH) in the presence of the LLC phase were collected using the
F2-coupled CLIP-HSQC experiment.[12] RDC values (1DCH) were
extracted from the obtained spectra using the relation
1DCH=1TCH-1JCH. The full 2D [1H-13C]-CLIP-HSQC spectra and
representative aromatic region of strychnine (Figure S2.2-3)
demonstrated the high quality of the spectrum with narrow lines,
which were recorded on a sample with 6.3 wt % L,L-PIAF-OBn.
The obtained RDC data range from -5.9 to +6.8 Hz (Table S2.2-
absorption spectrum of L,L-PIAF-OBn revealed
a distinct
shoulder peak at 370 nm (Figure S1-1), which was attributed to
the ordered packing of the helical main chain structure and
aromatic π-stacking interactions. Therefore, we conclude that
the π-π interactions formed by benzyl pendants in the side
chains play a key role in the formation of the meso-phase in
chloroform solution.
1
1). As an alternative approach, we measured the DCH couplings
of the analyte from the F1 dimension by using the J-scaled BIRD
HSQC (JSB-HSQC) experiment.[13] The RDC values extracted
from the JSB-HSQC experiment vary between -6.0 and +6.1 Hz,
which are very close to the data obtained from the CLIP-HSQC
experiment (Table S2.2-1 and S2.2-2). To verify the structure of
solute using the acquired RDCs, we calculated the alignment
We next investigated the possibility of employing L,L-PIAF-
OBn as a weak alignment medium for the RDC measurements.
At a concentration of 11.5 wt %, we observed a maximal
quadrupolar splitting of the deuterated CDCl3 solvent signal of
108 Hz. The formation of homogenous LC phase could be
additionally confirmed by the direct observation of strong
birefringence with crossed polarizers using the naked eye
(Figure 2B). Preparation of the anisotropic sample of L,L-PIAF-
OBn LCs was fast and straightforward, only requiring addition of
the polymers into the NMR tubes with the compound of interest
dissolved in the solvent. Notably, the equilibrium could be
reached in just a few minutes, and remained stable for several
months (Figure 2A). The excellent solubility and intrinsic low
viscosity enabled us to acquire high quality NMR spectra with
narrow lines. Additionally, we verified the spatial homogeneity of
the alignment in L,L-PIAF-OBn LCs by performing the deuterium
NMR 1D imaging experiment,[11] showing that the anisotropic
sample was well-equilibrated at different concentrations of the
polymer (Figure S2.1-1).
[14]
tensor using the singular value decomposition (SVD) method
with the program package MSpin.[15] Theoretically predicted
RDCs were calculated from the computed alignment tensor by
[16]
using the DFT-optimized structure
as the input and further
compared with the experimental determined ones. The quality
factors (Q factors) between experimental and predicted RDCs
were 0.15 (CLIP-HSQC data) and 0.16 (JSB-HSQC data),
respectively (Figure S2.2-4 and S2.2-9). In a different batch of
L,L-PIAF-OBn synthesis (L,L-PIAF-OBn*,Table S1-1; Figure
S2.2-10), the corresponding medium shows significantly higher
alignment strength with a quadrupolar coupling of ∆νQ = 151 Hz.
22 1DCH couplings of strychnine cover a favorable range of -15.2
to +21.2 Hz (Table S2.2-3). Furthermore, the full 2D spectra of
strychnine demonstrated the high quality of the spectrum with
narrow lines (Figure 3 and S2.2-11). The quality factors between
experimental and predicted RDCs were 0.09 (CLIP-HSQC data)
and 0.07 (JSB-HSQC data), respectively (Table 1). Currently,
factors that could influence the alignment strength are still
unknown and are under investigations in our lab.
Figure 2. A) Preparation of the alignment media for the RDC measurements in
5 mm NMR tubes. From left to right: Dried L,L-PIAF-OBn polymer powder;
Mixing of the LCs with solvent by shaking after 2 minutes; Addition of the
compound of interest dissolved in a small amount of solvent. B) Optical
micrograph of L,L-PIAF-OBn solution in
a NMR tube observed through
crossed polarizers, displaying the characteristic schlieren texture of LLC
phase. C) 1D 2H spectrum (25 oC, eight scans) of L,L-PIAF-OBn in CDCl3 at
11.5 wt %, with a deuterium splitting of 108 Hz (line width: 10.2 Hz).
One of the remarkable property of L,L-PIAF-OBn is its ability to
form LCs at very low concentrations. Comparable low critical
concentration of LCs as alignment medium has only been found
for a short water-based helical β-peptide.[5] However, until now
reasonable size of the residual dipolar coupling could be only
measured at relatively high concentration, as the ratio between
the quadrupolar coupling and residual dipolar coupling
measured in L,L-PIAF-OBn LCs is particularly high. For L,L-
PIAF-OBn we found that the ∆νQ observed in the 2H NMR
spectrum scaled up with the concentration of the dispersed
polymer, as shown in Figure S2.1-2.
Figure 3. Expanded representative aromatic region of strychnine in the
isotropic CDCl3 phase (blue contours, down-shifted 0.96 ppm in the 13C
dimension) and in anisotropic 6.0 wt % PIP LCs (red contours) acquired on a
600 MHz spectrometer (2H quadrupolar splitting of 151 Hz). The inserted trace
from the anisotropic 2D spectra (C11/H32, C12/H33, C13/H34 and C14/H35)
illustrates the favorable line widths achieved with L,L-PIAF-OBn LCs.
To further verify the scope of L,L-PIAF-OBn LC as a suitable
alignment medium for other natural compounds, a biologically
active triptolide was chosen as the analyte. RDCs for triptolide
were in the range of -4.9 to +2.8 Hz. We employed the same
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fitting procedure as described for strychnine, using the X-ray
Experimental details, data for strychnine, triptolide, and preparation of the
monomer are provided. (See Supporting Information)
[17]
structures of triptolide and 14-epi-triptolide
as the input. A
considerably lower quality factor (Q= 0.19 with CLIP-HSQC
data, 0.18 with JSB-HSQC data) was obtained for triptolide
(Figure S2.3-4 and S2.3-9), whereas the 14-epi-triptolide has a
significantly higher Q factor (Q= 0.68 with CLIP-HSQC data,
0.55 with JSB-HSQC data, Figure S2.3-5 and S2.3-10). This
result clearly demonstrates the value of the RDC data acquired
in the L,L-PIAF-OBn for the stereochemical elucidation of
triptolide.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21572164, 81421091, 81330079 and
91313303); HS and XXL thank the Sino-German Center for
Research Promotion (GZ1289). The authors thank Prof. Roberto
R. Gil sincerly for providing the pulse sequence of J-scaled
BIRD HSQC experiment. We think the Analytical & Measuring
Center, School of Pharmaceutical Sciences, SCUN for NMR test.
Table 1. Experimental RDCs (Hz) of strychnine in anisotropic 6.0 wt % PIP
LCs measured by using CLIP-HSQC and JSB-HSQC experiments,
respectively [a]
Keywords: polyisocyanodipeptides • liquid-crystalline medium •
residual dipolar couplings • NMR spectroscopy • small organic
molecules
Atom number
F2-coupled RDCs
F1-coupled RDCs
C12,H33
C21,H42
C13,H34
C14,H35
C11,H32
C23,H45
C22,H43
C22,H44
C5,H26
5.6
2.5
4.1
1.8
[1]
[2]
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-3.3
19.6
21.2
-10.0
-15.2
-5.7
-1.9
-11.0
-4.6
-1.4
5.6
-0.9
-5.7
11.6
-12.7
-0.5
-0.5
-11.5
9.9
-5.6
19.3
18.2
-10.2
-9.1
-9.1
-3.6
-9.8
-2.0
-2.0
3.4
C9,H31
C19,H40
C19,H41
C18,H38
C18,H39
C8,H30
C24,H46
C24,H47
C17,H36
C17,H37
C7,H29
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3.4
-3.8
0.8
0.8
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0.07
C6,H27
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1.1
0.08
[a] CLIP-HSQC and JSB-HSQC experiments were recorded with the same
anisotropic on a 600 MHz spectrometer.
In summary, we have presented L,L-PIAF-OBn LCs as a novel
and effective alignment medium, which enables to introduce a
low degree of alignment for RDC measurements of organic
molecules. Comparing dipeptide-based polyisocyanides with
different side chains suggested the high importance of the
aromatic side chains in LLC formation. Additional stabilization of
the helical structure was attained by the formation of hydrogen
bonds between amide and carbonyl groups in the introduced
peptide side chains. This work represents the first example of a
polymeric LLC which is stable at extremely low critical
concentration. Importantly, the excellent solubility and intrinsic
low viscosity allowed us to acquire good quality NMR spectra
with narrow lines. Taking different advantages of helical
polyisocyanopeptide as a tuneable mesogen, our future plan is
to increase the absolute RDC values for organic molecules by
increasing its persistence length of L,L-PIAF-OBn via different
catalysts and optimize the side chain of polyisocyanopeptides
for other organic solvents.
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Entry for the Table of Contents (Please choose one layout)
FULL PAPER
A
helical polyisocyanopeptide (L,L-
Gao-Wei Li, Jiang-Ming Cao, Wen Zong,
Li Hu, Mao-Lin Hu, Xinxiang Lei,* Han
Sun,* and Ren Xiang Tan*
PIAF-OBn) can fleetly form lyotropic
liquid crystal and adopt a striking low
critical concentration in chloroform.
The adjustable weakly alignment
medium is easily handle and allows for
acquiring residual dipolar couplings
(RDCs) of organic molecules.
Page No. – Page No.
Helical
Polyisocyanopeptides
Liquid Crystals
Residual
as
for
Dipolar
Lyotropic
Measuring
Couplings
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