Practical aspects of oligopeptide AAKLVFF as an alignment medium for the measurements of residual dipolar coupling of organic molecules

Article abstract of DOI:10.1002/mrc.4825

Practical aspects of the oligopeptide AAKLVFF as an alignment medium are discussed, including large-scale synthesis of the oligopeptide, detailed description of preparation of the alignment medium, and acquisition of the RDCs. The resulting orienting medium is stable and highly homogeneous with tunable alignment strength in methanol. We demonstrated its applicability on the stereochemical elucidation of two natural products, oridonin and α-santonin.

Full text of DOI:10.1002/mrc.4825

Received: 29 October 2018
DOI: 10.1002/mrc.4825
Revised: 3 December 2018
Accepted: 26 December 2018
S P E C I A L I S S U E T U T O R I A L
Practical aspects of oligopeptide AAKLVFF as an alignment
medium for the measurements of residual dipolar coupling
of organic molecules
Han Liu1 | Pian Chen1 | Xiao‐Lu Li2
| Han Sun2 | Xinxiang Lei1
1 School of Pharmaceutical Sciences, South
Abstract
Central University for Nationalities,
Practical aspects of the oligopeptide AAKLVFF as an alignment medium are
discussed, including large‐scale synthesis of the oligopeptide, detailed descrip-
tion of preparation of the alignment medium, and acquisition of the RDCs.
The resulting orienting medium is stable and highly homogeneous with
tunable alignment strength in methanol. We demonstrated its applicability
on the stereochemical elucidation of two natural products, oridonin and
α‐santonin.
Wuhan, China
2 Department of Structural Biology,
Leibniz‐Institut für Molekulare
Pharmakologie (FMP), Berlin, Germany
Correspondence
Xinxiang Lei, School of Pharmaceutical
Sciences, South Central University for
Nationalities, Wuhan 430074, China.
Email: xxlei@mail.scuec.edu.cn
KEYWORDS
Funding information
alignment media, oligopeptide, organic molecule, residual dipolar coupling, structural determination
National Natural Science Foundation of
China, Grant/Award Numbers: 21572164
and 21874158; the Sino‐German Center
for Research Promotion, Grant/Award
Number: GZ1289; Key Projects of Tech-
nological Innovation of Hubei Province,
Grant/Award Number: 2016ACA138
1 | INTRODUCTION
(PEO) are the gel‐based alignment media that have been
developed in recent years.[7] The liquid crystals include
RDC is a powerful structural parameter complementary
to the conventional NMR parameters such as scalar cou-
plings (J) and nuclear Overhauser effects (NOEs). RDCs
started to being applied for structure elucidation of bio-
molecules,[1,2] and were later adopted in organic structure
determination.[3,4] For their measurement, an anisotropic
environment preventing the analyte from tumbling
isotropically, is necessary.
Currently, two types of partially orienting media have
been frequently applied in the measurement of RDCs:
stretched/compressed polymer based gels and the lyotro-
pic liquid crystalline (LLC) phases.[5,6] Cross‐linked poly-
styrene (PS), poly (vinylacetate) (PVAc), cross‐linked poly
(dimethylsiloxane) (PDMS), poly (methyl methacrylate)
(PMMA), and the cross‐linked poly (ethylene oxide)
the high‐molecular weight homopolypeptides represented
by poly‐γ‐benzyl‐L/D‐glutamate (PBLG/PBDG),[3] poly‐γ‐
ethyl‐L/D‐glutamate (PELG/PEDG),[8] poly‐ε‐carboxy-
benzoyl‐L/D‐lysine (PCBLL/PCBDL),[9] and helically
chiral polymers such as polyguanidines exemplified by
poly‐(N‐methyl‐N′‐((R)‐1‐phenylethyl)guanidine((R)‐PP-
EMG),[10] polyarylacetylenes,[11–13] polyarylisocyani-
des,[14] polyisocyanopeptides,[15] as well as carbon‐based
graphene oxide (GO) sheets.[16,17]
In addition to the above‐mentioned liquid crystals, we
recently proposed an oligopeptide AAKLVFF as a novel
alignment medium in methanol, which enables to
acquire RDCs for intermediate and polar organic mole-
cules with high accuracy.[18] In the current study, we
present a detailed protocol for the large‐scale synthesis
Magn Reson Chem. 2019;1–7.
wileyonlinelibrary.com/journal/mrc
© 2019 John Wiley & Sons, Ltd.
1

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SPECIAL ISSUE TUTORIAL
of oligopeptide AAKLVFF, preparation of the anisotropic
phases and its application in the structural elucidation of
organic molecules.
texture under crossed polarizer (Figure 2a) as well as
characteristic 2H quadrupolar splitting of the solvent (Fig-
ures S2 and S3). The liquid crystalline phase of the
medium was formed at the critical concentration of
5 mg/mL and it was stable between[19] 0°C and 60°C.
Furthermore, the quadrupolar splitting of OD signal
ranging from 7.0 and 23.0 Hz scaled with the concentra-
tion of the peptide (12.0~34.5 mg/mL; Figure 2b), sug-
gesting that the alignment strength was tunable by
adjusting its concentration. Largest quadrupolar splitting
of OD signal (22.7 Hz) was obtained at a concentration
of 34.5 mg/mL. Higher concentration of the oligopeptide
led to an anisotropic phase with deteriorated spectro-
scopic properties for RDC measurements. Furthermore,
it is important to note that AAKLVFF oligopeptideis not
a suitable medium for aligning basic compounds, as the
liquid crystals were transformed into the gel state when
basic compounds were added. Although by neutralization
with trifluoroacetic acid (TFA) the deuterium splitting of
the solvent signal can be recovered (Figure 3), RDCs of
the molecules remains to be undetected (Figure S4).
2 | RESULTS AND DISCUSSION
2.1 | Synthesis of heptapeptide AAKLVFF
Synthesis of linear heptapeptide AAKLVFF was accom-
plished by the well‐established solid‐phase peptide syn-
thesis (SPPS) method using an automated peptide
synthesizer, with the Merrifield resin as solid support
and Fmoc‐protected amino acids as building blocks. The
scheme of synthetic route is shown in Figure 1. It is
important to note that Steps 1–3 were limited to the
attachment of the first amino acid, whereas the other
six amino acids were linked by Steps 4 and 5 in an itera-
tive way. After all seven amino acids were incorporated,
the resin was then liberated by cleavage reagents with
the crude peptide yielded. A further step of purification
was then performed to yield highly purified oligopeptide.
A molar mass of 795.35 (M + 1) consistent with the for-
mula (C41H62N8O8) was confirmed by mass spectrometry
(Figure S1).
2.3 | Preparation of the alignment
medium
The liquid crystalline phases of AAKLVFF were prepared
directly in the NMR tubes (5 mm). Three hundred micro-
liter MeOD‐d4 were added into a 5‐mm NMR tube,
followed by 17.25 mg oligopeptide, and another 200‐μL
MeOD‐d4 resulting in a concentration of 34.5 mg/mL.
2.2 | Characterization and orientational
properties of AAKLVFF liquid crystals
Oligopeptide AAKLVFF formed stable liquid crystalline
phase in methanol monitored by strong birefringence
FIGURE 1 Scheme of AAKLVFF synthesis: AA stands for an amino acid, R represents corresponding substituent groups of the seven
amino acids

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3
FIGURE 2 Characterization of
oligopeptide nanotube liquid crystals: (a)
photograph of birefringence exhibited
2
between crossed polarizers. (b) H NMR
spectrum of the anisotropic samples
(oligopeptide AAKLVFF dissolved in
MeOH) at different concentrations (the
quadrupolar splitting correspond to the
OD signal)
is too strong, the degree of alignment can be reduced by
diluting the anisotropic phase with MeOD‐d4.
2.4 | Structure elucidation
Oridonin is an ent‐kaurane diterpenoid with potential
applications for treating several diseases.[19,20] Here, 10.0‐
mg oridonin was aligned in the AAKLVFF medium
(12.0 mg/mL), which exhibited a moderate alignment
strength with a quadrupolar splitting of 7.0 Hz for the
OD signal. One bond proton carbon (1DCH) couplings were
extracted by subtracting the couplings measured under iso-
FIGURE 3 The quadrupolar splittings of OD signals of pure
oligopeptide (green, 27.8 Hz), aligned Aconitine (blue) and aligned
Aconitine which neutralized by 1% TFA (red, 14.8 Hz)
1
tropic and oriented environments, respectively. DCH
couplings of solute molecules were acquired through the
J‐scaled BIRD‐HSQC (JSB–HSQC) experiments.[21,22]
A
After the NMR tube was closed, the tube was turned up
and down several times to ensure that the oligopeptide
was well dissolved in MeOD‐d4. Shear action could accel-
erate the formation of the liquid crystalline phases. Com-
plete formation of the stable liquid crystalline phase took
around 2–3 days, which was stored at 4°C during the
equilibration process. Both color and flowability of the
medium are good indicators for judging its stability: the
color turned light blue and the flowability increased
when the alignment medium was stable (Figure S5).
Once the alignment medium was stable, analyte dissolved
in 100‐μL MeOD‐d4 was added into the NMR tube. Before
NMR measurements, the sample was turned up and
down again for several minutes. If the alignment strength
section of the overlaid two dimensional (2D) NMR spectra
of oridonin recorded under isotropic and anisotropic con-
ditions is shown in Figure 4. Although background signals
of the medium is visible in the anisotropic NMR spectrum,
they are well separated from the signals of the analyte mol-
ecule in the 2D spectrum.
1
Twenty‐four sets of DCH of oridonin ranging from
−6.5 to 25.4 Hz (Table S1) were used in the fitting against
the DFT‐optimized structure. Alignment tensor of the
molecule was calculated by singular value decomposition
(SVD) method as implemented in the RDC module of the
MSpin program.[23] Theoretically predicted RDCs were
calculated from the computed alignment tensor and com-
pared with the experimental ones. A reasonable good

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FIGURE 4 Expanded region of the
overlaid JSB–HSQC spectra of oridonin in
the isotropic CD3OD phase (blue
contours) and in anisotropic oligopeptide
(12.0 mg/mL) LCs (red contours)
1
FIGURE 5 Correlation between the experimental DCH and the
back‐calculated ones for oridonin in 12.0 mg/mL oligopeptide LCs
(quadrupolar splitting was 7.0 Hz for the OD signal). The RDC
fitting was performed with SVD method using MSpin software
package
FIGURE 6 Chemical structure and numbering of α‐santonin
18.0 mg/mL, Table 1). Experimental RDC datasets (Fig-
ures S6 and S7 and Tables S2 and S3) were used in the
fitting against the DFT‐refined structures of eight possible
relative configurations of α‐santonin that have been
reported by Hallwass et al. Lowest Q factors were obtained
for the correct stereoisomer SSSS (Figure 7), which are
0.053 (ΔQυ = 16 Hz) and 0.048 (ΔQυ = 11 Hz), respectively.
Other configurations have significantly larger Q factors,
which are all above 0.1. It is interesting to note that in
the previous study of α‐santonin aligned in a compressed
polymethylmethacrylate gel, correct assignment of the
quality factor of 0.137 was obtained, suggesting that the
experimentally measured RDCs were precise (Figure 5).
α‐Santonin (Figure 6), as a historically important
eudesmanolide, was previously employed by Hallwass et.
al as a test molecule for the RDC and residual chemical
shift anisotropy (RCSA)–based structure elucidation.[24]
In this study, we measured RDCs of α‐santonin at two dif-
ferent concentrations of the AAKLVFF phase (22.5 and

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TABLE 1 RDCs for α‐santonin at different alignment strengths
3 | EXPERIMENTAL PROCEDURES
RDCs (ΔQυ = 16 Hz)
RDCs (ΔQυ = 11 Hz)
Atom
number
3.1 | Equipment and materials for
synthesis
22.5 mg/mL
18.0 mg/mL
C2
20.1
−0.7
−9.1
−7.1
−7.2
3.9
15.7
−0.6
−7.3
−5.7
−5.5
3.1
NMR spectra were recorded on a Bruker DXR‐600 instru-
C3
ment (600.11 MHz for 1H, 150.91 MHz for 13C, and
C7
2
92.12 MHz for H NMR, respectively) equipped with a
C8
5‐mm BBO Prodigy cryo‐probe, BB‐(H‐F)‐D‐05‐Z (Bruker
Instruments Inc., Germany). Deuterated solvent (CD3OD)
were used to solubilize the samples. ESI‐MS was recorded
on a LC–MS system (Thermo Fisher, Santa Clara, Califor-
nia). Semipreparative reverse‐phase high‐performance
liquid chromatography (RP‐HPLC) was performed on an
Agilent Technologies 1260 Infinity II system equipped
C9
C10
C11
C13
C14
C15
−4.8
7.9
−3.7
5.9
4.2
2.4
−6.5
−4.9
with
a
diode array detector and C18 column
(250 × 9.4 mm, 5 μm, Agilent Technologies, Santa Clara,
California). Oligopeptide was automatically synthesized
by the Automated CS Bio peptide synthesizer (CSBio
(Shanghai) Ltd, CS136XT).
1‐Hydroxybenzotriazole
monohydrate
(HOBT,
purity > 97.0%), Trifluoroacetic acid (purity > 99.5%), N,
N‐diisopropylethylamine (DIEA, purity > 99.0%), and
triisopropylsilane (purity > 98.0%) were all purchased
from Aladdin (Shanghai) industrial corporation. The
amino acids were bought from CS Bio (Shanghai) Ltd.
N,N,N′,N′‐tetramethyl‐O‐(1H‐benzotriazol‐1‐yl)
uroniumhexafluorophosphate (HBTU, purity>99.0%) was
purchased from Adamas Reagent Co., Ltd. 2‐Chlorotrityl
chloride resin was bought from GL Biochem (Shanghai)
Ltd. Piperidine and the commonly used solvents were
bought from Sinopharm chemical reagent Co., Ltd with
the chromatographically pure acetonitrile bought from
Sigma Aldrich. The ultra‐pure water was prepared by
ourself.
FIGURE 7 Q factors of the RDCs for the eight possible
diastereomeric configurations of α‐santonin in 22.5‐ and 18.0‐mg/
mL oligopeptide LCs with the respective quadrupolar splittings of
16.0 and 11.0 Hz for the OD signal
relative configuration was only achieved by a combination
of RDC and RCSA, where RDC alone was not enough to
distinguish the correct configuration.
3.2 | The detailed synthetic route for
AAKLVFF
2.5 | Conclusion
For the synthesis of the AAKLVFF peptide, 0.948 mmol/g
2‐Chlorotrityl Chloride resin was used, together with
three equivalents of the first amino acid, two equivalents
of the other six amino acids, and 2.4 equivalents of both
HOBT and HBTU.
We discussed in this protocol the practical aspects of
AAKLVFF oligopeptide as a suitable alignment medium
for the acquisition of high quality RDC data and further
demonstrated its usage on the structure elucidation of
oridonin and α‐santonin. We noted little influence of
the background signals on the measurement of the RDCs
of different organic molecules. The alignment strength
can be easily scaled by varying the concentration of the
medium. In addition to the synthetic and purification
procedures of the AAKLVFF oligopeptide, we described
a detailed procedure for the preparation of the anisotropic
sample and discussed its drawbacks in acquisition of the
RDCs for basic compounds.
3.2.1 | Resin swelling
0.5 g resin was placed into the peptide synthesis vessel,
and then put into a wrist‐action shaker. The procedure
of automatic synthesis was set and started according to
the standard protocol. Fifteen milliliter N,N‐dimethyl
formamide (DMF) prepared in advance was added into

6
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the synthesis vessel, which was then shaken up and down
for 30 min to swell the resin. Afterwards, the solvent in
the synthesis vessel was drained out and the resin was
washed twice with freshly added DMF.
terminal of the former amino acid to form a peptide link-
age and the deprotection process of Step 3 was repeated
once an amino acid was coupled.
3.2.5 | Desiccation
3.2.2 | Substitution reaction
Upon successful coupling and deprotection, the resin
with the oligopeptide AAKLVFF attached was washed
with DMF for three times, followed by methanol and
DCM in the same way. Next, the resin was transferred
to a manually operated solid phase synthesis tube to be
vacuum desiccated (Shanghai senxin laboratory instru-
ment co. LTD, DZG‐6050) for 12 hr.
1.1018 g Fmoc‐Phe‐OH, 0.5902 g Fmoc‐Ala‐OH, 0.3218 g
Fmoc‐Val‐OH,0.3350 g Fmoc‐Leu‐OH, 0.4442 g Fmoc‐Lys
(Boc)‐OH, 1.5370 g HOBT, and 4.3140 g HBTU were
respectively dissolved in DMF, the amount of which
needed were 10 mL for the former two, 5 mL for the middle
three, and 50 mL for the last two. Next, 10 mL DIEA as well
as 30 mL piperidine were separately mixed with 40 and
120 mL DMF. With the manually prepared solutions ready,
the reagents were transferred to the corresponding storage
bottles, and procedures were set to continue next steps in
an automatic way according to the standard protocol.
Five milliliter Fmoc‐Phe‐OH/DMF solution, 5‐mL
DIEA/DMF, and 10 mL‐DMF were sequentially trans-
ferred into the reactor, together with that the contents in
the reaction vessel were mixed for 2 hr. When completed,
DMF was added, and the reaction system was washed for
four times in order to remove the residual substances. In
this step, the C‐terminus of Fmoc‐Phe‐OH was attached
to the resin through substitution reaction.
3.2.6 | Cleavage
Twenty milliliter cleavage solution (TFA/triisopropylsilane/
water = 95/2.5/2.5) was prepared and poured into the syn-
thesis tube and then kept for 2 hr. Subsequently, the filtra-
tion was carried out. The obtained filtrate was transferred
into a round‐bottom flask and evaporated at 40° C until
the volume was no longer reduced. The remained liquid
was transferred into a 50‐mL centrifuge tube with 20 mL
diethyl ether at 4°C, in order to precipitate the oligopeptide.
To isolate the product, centrifugation was made on a high‐
speed freezing centrifuge for 10 min at 7,500 rpm/min
under 4°C, which was repeated for three times. The precip-
itate was further dried in a vacuum dryer, and the crude
product was obtained.
3.2.3 | Deprotection
The solution inside the synthesis vessel was removed, and
10‐mL piperidine (20%)/DMF was then added, intensively
blended for 15 min, and drained eventually. Note that the
same procedure should be repeated once again before
next step. Afterwards, the resin was washed with DMF
and dichloromethane (DCM) for two times and dried nat-
urally. The product was washed again with DMF for
three times and dried as before. Above all, the Fmoc
group was deprotected by piperidine, and thus the amine
terminus was freed.
3.2.7 | Purification
Crude oligopeptide was dissolved in 50% acetonitrile
(MeCN)/water and purified by semipreparative RP‐HPLC
using the weak‐acid mobile phase MeCN‐H2O (27:73–
52:48, 25 min, v/v, 0.1% (v/v) TFA contained in both) for gra-
dient elution at a flow rate of 3.0 mL/min. Sample elution
was monitored via a UV/vis detector operating at 220 nm.
The purified product dissolved in HPLC solvents was prefro-
zen in a −80°C refrigerator for 2 hr and then freeze‐dried at
−60°C for 3–4 days. Ultimately, the white solid, which was
the purified oligopeptide, was obtained. Molecular weight
was determined by electro‐spray mass spectrometry.
3.2.4 | Continuous coupling reaction
Starting with the second amino acid, the building blocks
were sequentially added through an iterative process of
amino acid coupling and the removal of the protecting
group. Taking the coupling of the second one as an exam-
ple, 5‐mL solution of Fmoc‐Phe‐OH/DMF, HOBT/DMF,
HBTU/DMF, and DIEA/DMF each was transferred into
the reactor one after the other. The vessel was then shaken
up and down for 2 hr to make the reaction adequate. The
activated carboxyl group reacted with the exposed amino
3.3 | NMR experiments
2
The 1D H NMR spectra were collected at 293 K with
eight scans using the lock channel. For the J‐scaled BIRD
HSQC (JSB–HSQC) experiments, the conditions were as
follows: AQ 0.189 s, SW 10.0 ppm (1H) and 140 ppm
(13C). A total of 4 k data points were sampled in the direct

SPECIAL ISSUE TUTORIAL
7
[11] A. Krupp, M. Reggelin, Magn. Reson.Chem. 2013, 50, S45.
dimension over a spectral width of 10 ppm. A one‐bond
coupling constant was 145.0 Hz. LB 1 Hz for F2 and
0.3 Hz for F1 were applied before Fourier transformation.
All data were analyzed and processed using Bruker Top-
spin 3.5 pl 6 software.
[12] N. C. Meyer, A. Krupp, V. Schmidts, C. M. Thiele, M. Reggelin,
Angew. Chem. Int. Ed. 2012, 51, 8334.
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Tan, H. Sun, Chem. A Eur. J. 2017, 23, 7653.
3.4 | Structure optimization
[16] W. Zong, G. W. Li, J. M. Cao, X. X. Lei, M. L. Hu, H. Sun, C.
Griesinger, R. X. Tan, Angew. Chem. Int. Ed. 2016, 128, 3754.
The initial structure of oridonin was obtained from the
Cambridge Crystallographic Data Centre (CCDC), and
the optimization and frequency calculations were per-
formed by Gaussian 09 software package[25] using
B3PW91/6‐311 g(d) level of theory under the IEFPCM
solvation model with methanol parameters.
[17] X. X. Lei, Z. Xu, H. Sun, S. Wang, C. Griesinger, L. Peng, C.
Gao, R. X. Tan, J. Am. Chem. Soc. 2014, 136, 11280.
[18] X. X. Lei, F. Qiu, H. Sun, L. Bai, W. X. Wang, W. Xiang, H.
Xiao, Angew. Chem. Int. Ed. 2017, 56, 12857.
[19] D. Ye, C. Ding, N. Ye, Z. Liu, E. A. Wold, H. Chen, C. Wild, Q.
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ACKNOWLEDGEMENT
Q. Shen, J. Zhou, J. Med. Chem. 2013, 56, 5048.
[21] C. M. Thiele, W. Bermel, J. Magn. Reson. 2012, 216, 134.
This research work was financially supported by the
National Natural Science Foundation of China
(21874158, 21572164), Key Projects of Technological
Innovation of Hubei Province (No. 2016ACA138). X. L.
and H. S. thank the Sino‐German Center for Research
Promotion (GZ1289). The authors thank the Analytical
& Measuring Center, School of Pharmaceutical Sciences,
SCUN for their help with NMR measurements.
[22] M. Garcìa, E. Hellemann, P. Nolis, R. R. Gil, T. Parella, J. Org.
Chem. 2016, 81, 11126.
[23] A. Navarro‐Vázquez, Magn. Reson. Chem. 2012, 50, S73.
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[25] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A.
Petersson, H. Nakatsuji, X. Li, M. Caricato, A. Marenich, J.
Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P.
Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. Wil-
liams‐Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng,
A. Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J.
Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y.
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Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C.
Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M.
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09, Revision E.01, Gaussian, Inc., Wallingford CT 2016.
CONFLICTS OF INTEREST
There are no conflicts to declare.
ORCID
Xiao‐Lu Li https://orcid.org/0000-0002-0902-1823
Xinxiang Lei https://orcid.org/0000-0002-5635-5375
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How to cite this article: Liu H, Chen P, Li X‐L,
Sun H, Lei X. Practical aspects of oligopeptide
AAKLVFF as an alignment medium for the
measurements of residual dipolar coupling of
organic molecules. Magn Reson Chem. 2019;1–7.
https://doi.org/10.1002/mrc.4825
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