Application of the highly sensitive labeling reagent to the structural confirmation of readily isomerizable peptides

Article abstract of DOI:10.1007/s11418-020-01472-z

Thioamycolamide A (1) is a biosynthetically unique cytotoxic cyclic microbial lipopeptide that bears a d-configured thiazoline, a thioether bridge, a fatty acid side chain, and a reduced C-terminus. It has gained attention for its unique structure, and very recently we reported the total synthesis of 1 via a biomimetic route. The NMR spectra of synthetic 1 agreed with those of natural 1. However, structural identity between peptidic natural and synthetic compounds is often difficult to confirm by comparison of NMR spectra because their NMR spectra vary depending on the conditions in the NMR tube, which often result in the structural misassignment of peptidic compounds. Especially, our total synthesis based on the putative biomimetic route potentially gives 1 as a diastereomixture at the final step. The problem is that the diastereomers of peptidic mid-sized molecules often exhibit similar properties (such as NMR spectra and bioactivities), and their separation procedures are often laborious. Herein we report the structural confirmation of synthetic 1 by the LC–MS-based chromatographic comparison with the use of our highly sensitive labeling reagent l-FDVDA; the highly sensitive-advanced Marfey’s method (HS-advanced Marfey’s method). This work demonstrated the utility of our highly sensitive labeling reagent for the structural determination of not only scarce natural products but also readily isomerizable synthetic compounds.

Full text of DOI:10.1007/s11418-020-01472-z

Journal of Natural Medicines (2021) 75:339–343
https://doi.org/10.1007/s11418-020-01472-z
ORIGINAL PAPER
Application of the highly sensitive labeling reagent to the structural
confrmation of readily isomerizable peptides
Chengqian Pan1 · Takefumi Kuranaga1 · Hideaki Kakeya1
Received: 7 November 2020 / Accepted: 20 November 2020 / Published online: 2 January 2021
© The Japanese Society of Pharmacognosy 2021
Abstract
Thioamycolamide A (1) is a biosynthetically unique cytotoxic cyclic microbial lipopeptide that bears a d-confgured thiazo-
line, a thioether bridge, a fatty acid side chain, and a reduced C-terminus. It has gained attention for its unique structure, and
very recently we reported the total synthesis of 1 via a biomimetic route. The NMR spectra of synthetic 1 agreed with those
of natural 1. However, structural identity between peptidic natural and synthetic compounds is often difcult to confrm by
comparison of NMR spectra because their NMR spectra vary depending on the conditions in the NMR tube, which often
result in the structural misassignment of peptidic compounds. Especially, our total synthesis based on the putative biomimetic
route potentially gives 1 as a diastereomixture at the fnal step. The problem is that the diastereomers of peptidic mid-sized
molecules often exhibit similar properties (such as NMR spectra and bioactivities), and their separation procedures are often
laborious. Herein we report the structural confrmation of synthetic 1 by the LC–MS-based chromatographic comparison with
the use of our highly sensitive labeling reagent l-FDVDA; the highly sensitive-advanced Marfey’s method (HS-advanced
Marfey’s method). This work demonstrated the utility of our highly sensitive labeling reagent for the structural determination
of not only scarce natural products but also readily isomerizable synthetic compounds.
Keyword Natural products · Structural determination · Peptides · d-amino acid · Highly sensitive advanced marfey’s
method
Introduction
of the absolute confgurations are inevitable in the structural
determination of newly identifed peptide natural products.
Thioamycolamide A (1) is a biosynthetically unique cyto-
toxic cyclic microbial lipopeptide that bears a d-confgured
thiazoline, a thioether bridge, a fatty acid side chain, and a
reduced C-terminus (Fig. 1) [3]. It has gained attention for
its unique structure, and very recently we reported a concise
total synthesis of 1 based on the putative biosynthetic path-
way as summarized in Fig. 2 [4]. In general, peptides are
chemically constructed from its C-terminus to N-terminus.
Although the Fmoc-based solid-phase total synthesis of pep-
tide that bears reduced C-terminus has been reported [5],
peptide 1 possesses a thiazoline moiety, which was read-
ily isomerized through the cleavage of Fmoc group under
basic conditions [6–9]. Therefore, to avoid the isomeriza-
tion of thiazoline, the linear peptide 7 was synthesized from
Boc-l-cystine (6) under isomerization-suppressed manner in
solution-phase, and then cyclized by one-pot reduction/thio-
Michael addition under the neutral conditions to aford 1.
Although the NMR spectra of synthetic 1 agreed with
those of natural 1, the spectroscopic data of peptides may
Peptide mid-size molecules, which have intermediate prop-
erties between those of antibodies and small molecule drugs,
have gained much attention as ideal drug leads [1].The spe-
cifc bioactivities of peptide drug leads are exerted by their
unique structure, that often featured by the unusual amino
acids including d-amino acids [2]. Therefore, the analyses
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s1141
8-020-01472-z.
* Takefumi Kuranaga
tkuranaga@pharm.kyoto-u.ac.jp
* Hideaki Kakeya
scseigyo-hisyo@pharm.kyoto-u.ac.jp
1
Department of System Chemotherapy and Molecular
Sciences, Division of Bioinformatics and Chemical
Genomics, Graduate School of Pharmaceutical Sciences,
Kyoto University, Sakyo-ku, YoshidaKyoto 606-8501, Japan
Vol.:(0123456789)
1 3

340
Journal of Natural Medicines (2021) 75:339–343
separation procedures are often laborious. While the devel-
oped biomimetic route seemed to give 1 as a stereochemi-
cally pure form as with its biosynthesis, standard samples of
the diastereomers for chromatographic comparison to con-
frm its purity could not be prepared by this synthetic route.
Results and discussions
To establish the efciency of our bioinspired total synthesis,
we had to determine the purity of synthetic 1, which may be
readily isomerized similar to natural 1. Consequently, hav-
ing developed a rapid synthetic entry to 1, we then turned
our attention to the structural validation of the chemically
constructed 1. Herein we report the structural confrmation
of synthetic 1 by the combination of the chemical synthesis
and LC–MS-based chromatographic comparison.
Fig. 1 The structures and bioactivities of thioamycolamides A–E
(1–5) [3]
Among the strategies for the structural elucidation of
peptides, Marfey’s method [17] has been a powerful tool
not only for the structural study on the new natural prod-
uct but also the confrmation of the total synthesis [18].
Recently, we developed new modifed Marfey’s reagents
such as 1-fuoro-2,4-dinitrophenyl-5-l-valine-N,N-dimeth-
ylethylenediamine-amide (l-FDVDA) for “scarce natural
products” inspired by a scarce natural product yaku’amide B
[19]. The highly sensitive-advanced Marfey’s method (HS-
advanced Marfey’s method) by using new improved reagents
enabled the detection of component amino acids resulting
from mild chemical degradation of smaller amounts of pep-
tide, which would be useful for the structural analysis of
peptide 1 (Fig. 3).
In our preceding study (Fig. 4a) [4], frst, natural 1 was
carefully hydrolyzed (2 M HCl, 90 °C, 4 h) to determine
the stereochemistry of C5 and C8 by Marfey’s method [17].
Fig. 2 The outline of the total synthesis of 1 [4]
O
a)
S
N
H
vary depending on the solution conditions, and often result
in structural misassignment of peptidic natural products.
Structural identity between natural and synthetic compounds
is often difcult to confrm only by comparison of NMR
spectra because the NMR spectra of peptidic products varies
depending on the conditions in the NMR tube (e.g., concen-
tration, pH, and temperature) [10–15]. The essential task at
this stage was the structural confrmation of the chemically
constructed 1 because the thio-Michael addition used in the
synthesis may give 1 as a diastereomeric mixture due to
the absence of the chiral catalyst for thio-Michael addition.
Additionally, the thiazoline moiety may be isomerized not
only during the chemical conversions, but also during the
purifcation steps [6–9] Especially, diastereomers of peptidic
mid-sized molecules often exhibit similar properties (such
as NMR spectra [10–15] and bioactivities [16]), and their
N
H
+
OH
OH
H2N
H2N
NH
O
O
1
·strong acid
·high temperature
·long reaction time
high yield
high yield
L
-configuration
·weak acid
D
-configuration
low yield
low yield
·low temperature
·short reaction time
b)
inspired by scarce natural products
O
H
N
highly sensitive labeling reagents
· reduce sample loss
N
N
H
N
H
O
· prevent structural misassignment
application to isomerizable compounds
C-terminus of yaku’amide B
(this work)
Fig. 3 a The problem in the amino acid analyses of 1. b The outline
of this study
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Journal of Natural Medicines (2021) 75:339–343
341
a) previous study
6
R
O
6/9
S
1. 2 M HCl
11
14
5
O
90 °C
L
-DLA
OH
N
HN 4
OH
N
10
5/8
9
8
2. L-FDLA
H
9
2
NaHCO3
acetone
H2O
NH
16
3
R = Ph or S-
L
-DLA
Br
1
O
S
18
natural 1
1. 6 M HCl, 105 °C
21
Br
2. SOCl2, MeOH
O
O
O
H3N
OH
2
CD & NMR
2
HN
S
11
O
DMAP
O
S
1
Cl
O
O
pyridine
18
12
10
O
O
Br
NH2
NH2
Cl
F
NH
NH
11
O
O2N
NO2
O2N
NO2
L
-FDLA
L-DLA
b) this work
1. 6 M HCl, 105 °C
R
O
1. 2 M HCl
L
-DVDA
6/9
2. SOCl2, MeOH
90 °C
2
HN
L
-DVDA
1
OH
OH
5/8
N
3. L-FDVDA
2.
L-FDVDA
1
H
13
O
S
NaHCO3
acetone
H2O
NaHCO3
acetone
H2O
R = Ph or S-
L
-DVDA
O
18
14
LC-MS
Fig. 5 Synthesis of four possible isomers of thioether 14
O
O
N
N
N
H
N
H
F
NH
NH
NO2
nucleophilic aromatic substitution (SNAr) reaction leading
to 14a/14b. In the frst step of the synthesis of 14c/14d, the
hydroxy function of 15 was protected with a TBS group,
which can be cleaved simultaneously with a Boc group by
HCl. Second, reduction of the methyl ester of 19, followed
by the tosylation of the generated primary alcohol furnished
20. Then, 20 was converted to 14c/14d in the same fashion
as 14a/14b.
O2N
NO2
O2N
L
-DVDA
L
-FDVDA
Fig. 4 a Structural studies on 1 from a previous work; b Structural
studies on 1 in this work
Then the absolute confguration of C2 and C18 were evalu-
ated by a combination of the spectroscopic analyses (CD
[20] and NMR) and chemical synthesis. Although a single
plausible structure of 1 could be determined by these experi-
ments, a multi-milligram scale of 1, which was totally con-
sumed by the chemical degradation, was required for CD
and NMR analyses. Conversely, in this study (Fig. 4b), acid
hydrolysates of natural/synthetic 1 were derivatized with
the new labeling reagent l-FDVDA and the corresponding
derivatives were chromatographically compared with the
chemically synthesized standard samples. A minute amount
(<0.1 mg) of 1 was enough for the LC–MS-based chromato-
graphic comparison of the 2,4-dinitrophenyl-5-l-valine-N,N-
dimethylethylenediamine-amide (DVDA) derivatives (See
Fig. S1 in electronic supplementary materials for details
including structural confrmation of C5/C8 and C6/C9).
The synthesis of all four possible isomers of 14 is sum-
marized in Fig. 5. Treatment of the previously synthe-
sized thioethers 18a/18b [4] with HCl liberated the corre-
sponding amines, which were labeled with l-FDVDA by a
Finally, the key labeled thioether 14 derived from natu-
ral/synthetic 1 was chromatographically compared with
the standard thioethers 14a–d by LC–MS experiments.
The retention time of authentic 14 was matched with that
of 14a, confrming unambiguously the structure of natural/
synthetic thioamycolamide A was that shown for 1 (Fig. 6).
Moreover, no detectable peaks for 14b–14d were observed
14a
14b
14c
14d
14 from natural 1
14 from synthetic 1
Fig. 6 The LC–MS charts of thioether 14. See Supplementary Mate-
rials in the detail
1 3

342
Journal of Natural Medicines (2021) 75:339–343
in the LC–MS experiments of authentic samples even with
the use of the highly sensitive labeling reagent, confrming
the stereochemical purity of 1. The thioether bridge was ste-
reoselectively constructed in previous study, which strongly
supports our proposed thio-Michael addition pathway for the
thioether biosynthesis.
MeCN (100 μL), fltered through a membrane flter (SHI-
MADZU, TORAST™ DISC, PTFE 0.22 μm), and then ana-
lyzed by LC–MS [column: Cadenza CD-C18, 3.0×150 mm;
eluent: MeCN/H2O/formic acid = 50/50/0.1 to 100/0/0.1
(0–30 min), 0.2 mL/min; detection: ESI-positive].
In summary, the structural identity of natural and syn-
thetic 1 was confrmed by LC–MS experiments using our
improved new labeling reagent l-FDVDA. In the LC–MS
experiments, the stereochemical purity of chemically con-
structed 1 was verifed, which corroborates the efciency
of our bioinspired synthesis and provides insight into the
proposal that the thioether bridge can be stereoselectively
formed by thio-Michael addition. The utility of the newly
developed highly sensitive labeling reagent in the structural
determination of unstable natural and synthetic products was
also demonstrated. Further studies including the identifca-
tion of the thiomycolomide A (1) biosynthetic gene cluster
and detailed structure–activity relationship (SAR) studies
are currently underway and will be reported in due course.
Ester 19
To a solution of Boc-l-Ser-OMe (15) (500 mg, 2.28 mmol)
in DMF (10 mL) were added imidazole (466 mg, 6.84 mmol)
and TBSCl (516 mg, 3.42 mmol) at 0 °C. After being stirred
at room temperature for 0.5 h, saturated aqueous NH4Cl
(20 mL) was added to the reaction mixture. The resulting
solution was extracted with EtOAc (30 mL×2). The com-
bined organic layer was washed with brine (50 mL), dried
over MgSO4, fltered, and concentrated under reduced pres-
sure. The residue was purifed by silica gel column chro-
matography (EtOAc/hexane= 1:20) to aford 19 (719 mg,
1
95%) as a colorless oil: [α]20D = 7.0 (c 2.22, MeOH); H
NMR (500 MHz, CDCl3) δ 5.33 (d, J = 8.5 Hz, 1H), 4.34
(dt, J=8.8, 2.7 Hz, 1H), 4.03 (dd, J=10.0, 2.6, 1H), 3.81
(dd, J = 10.0, 3.0, 1H), 3.73 (s, 3H), 1.45 (s, 9H), 0.85
(s, 9H), 0.02 (s, 3H), 0.01 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 171.4, 155.6, 80.2, 63.9, 55.7, 52.4, 28.5, 25.8,
18.3, −5.4, −5.5; HRMS (ESI) calcd for C15H31NO5SiNa+
[M+Na]+ 356.1864, found 356.1845.
Materials and methods
1
General remarks H and 13C NMR spectra were recorded
on a JEOL ECA500 (500 MHz for 1H NMR and 125 MHz
for 13C NMR) spectrometer. Chemical shifts are denoted in
δ (ppm) relative to residual solvent peaks as internal stand-
Tosylate 20
1
ard (CDCl3, H δ 7.25, 13C δ 77.2). ESI–MS and LC–MS
experiments were recorded on a Shimadzu LCMS-IT-TOF.
Optical rotations were recorded on a JASCO P-2200 pola-
rimeter. High performance liquid chromatography (HPLC)
experiments were performed with a SHIMADZU HPLC sys-
tem equipped with an LC-20AD intelligent pump. All reac-
tions sensitive to air and/or moisture were conducted under
nitrogen atmosphere using dry, freshly distilled solvents,
unless otherwise noted. All reagents were used as supplied
unless otherwise stated. Analytical thin-layer chromatogra-
phy (TLC) was performed using E. Merck Silica gel 60 F254
pre-coated plates. Silica gel column chromatography was
performed using 40–50 μm Silica Gel 60 N (Kanto Chemi-
cal Co., Inc.).
To a solution of 19 (700 mg, 2.10 mmol) in THF (2 mL)
were added NaBH4 (238 mg, 6.30 mmol), LiCl (267 mg,
6.30 mmol), and MeOH (6 mL) at 0 °C. After being stirred
at 0 °C for 0.5 h and at room temperature for 1 h, aque-
ous citric acid solution (10%, w/w, 20 mL) was added to
the reaction mixture. The resulting solution was extracted
with CH2Cl2 (20 mL×2). The combined organic layer was
washed with saturated aqueous NaHCO3 (40 mL) and brine
(40 mL), dried over MgSO4, fltered, and concentrated under
reduced pressure to aford practically pure alcohol S1 in sup-
plementary material (664 mg, Figs. S5, S6, and S10) as a
colorless oil, which was used in the next reaction without
further purifcation.
To a stirred solution of the above alcohol S1 (500 mg, ca
1.64 mmol) in pyridine (10 mL) was added TsCl (650 mg,
3.41 mmol) at 0 °C. After being stirred at room temper-
ature overnight, saturated aqueous NH4Cl (40 mL) was
added to the reaction mixture. The resulting solution was
extracted with EtOAc (40 mL × 2). The combined organic
layer was washed with brine (80 mL), dried over MgSO4,
fltered, and concentrated under reduced pressure. The
residue was purifed by silica gel column chromatography
(EtOAc/hexane=1:10) to aford 20 (581 mg) as a colorless
Structural confrmation of 1
Natural 1 (0.1 mg) and synthetic 1 (0.1 mg) were hydro-
lyzed with 2 M HCl (100 μL) for 4 h at 90 °C under N2 and
then dried in vacuo, respectively. Each hydrolysate was dis-
solved in H2O (100 μL), and then 1 M NaHCO3 (20 μL) was
added to the solution. To the resulting solution was added
l-FDVDA (0.5% w/v in acetone, 20 μL), and then stirred at
50 °C for 2 h. The solution was cooled to room temperature,
neutralized with 2 M HCl (20 μL), evaporated, dissolved in
1
oil: [α]20D = − 2.2 (c 0.05, MeOH); H NMR (400 MHz,
1 3

Journal of Natural Medicines (2021) 75:339–343
343
CDCl3) δ 7.73 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz,
2H), 4.75 (d, J = 8.3, 1H), 4.03 (m, 2H), 3.80 (s, 1H), 3.62
(dd, J = 10.0, 3.7 Hz, 1H), 3.48 (dd, J = 10.0, 6.2 Hz, 1H),
2.39 (s, 3H), 1.36 (s, 9H), 0.77 (s, 9H), − 0.04 (s, 6H);
13C NMR (100 MHz, CDCl3) δ 155.1, 145.0, 132.6, 130.0,
128.0, 79.8, 67.9, 60.9, 50.4, 28.3, 25.8, 21.7, 18.1, 14.2,
5.6; HRMS (ESI) calcd for C21H37NO6SiSNa+ [M + Na]+
482.2003, found 482.1961.
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Acknowledgments This work was fnancially supported in part by a
Grant-in-Aid for Scientifc Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan [17H06401 (H.K.),
18K14396 (T.K.), and 19H02840 (H.K.)], and the Platform Project
for Supporting Drug Discovery and Life Science Research from the
Japan Agency for Medical Research and Development (AMED), Japan.
Contributions from the SUNBOR GRANT, the Daiichi-Sankyo Award
in The Society of Synthetic Organic Chemistry Japan, the Takeda Sci-
ence Foundation, and The Tokyo Biochemical Research Foundation to
T.K. are also greatly appreciated.
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(2020) Highly sensitive labeling reagents for scarce natural products.
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Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
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