Diastereoselective total synthesis and structural confirmation of surugamide F

Article abstract of DOI:10.1248/cpb.c18-00072

Surugamide F is a linear decapeptide (1) isolated along with the cyclic octapeptides surugamides A–E (2–6), from a marine-derived Streptomyces species. The linear peptide 1 is produced by two nonribosomal peptide synthetases (NRPSs) encoded in adjacent open reading frames, which are further flanked by an additional pair of NRPS genes responsible for the biosyntheses of the cyclic peptides 2–6. While the cyclic peptides 2–6 were identified to be cathepsin B inhibitors, the biological activity of the new metabolite 1 still remained unclear. In order to elucidate its unique biosynthetic pathway and biological activity in detail, we planned to develop an efficient synthetic route toward 1. Here we report the diastereoselective total synthesis of 1, utilizing 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. During this study, we found that the structural correction of 1 was required, due to the mislabeling of the commercially obtained 3-amino-2-methylpropionic acid, and the true structure of 1 was corroborated by the chemical synthesis and chromatographic comparison.

Full text of DOI:10.1248/cpb.c18-00072

Vol. 66, No. 6
Chem. Pharm. Bull. 66, 637–641 (2018)
637
Regular Article
Diastereoselective Total Synthesis and Structural Confirmation of
Surugamide F
,
a
a
b
b
Takefumi Kuranaga,* Atsuki Fukuba, Akihiro Ninomiya, Kentaro Takada,
b
,a
Shigeki Matsunaga, and Toshiyuki Wakimoto*
a
Faculty of Pharmaceutical Sciences, Hokkaido University; Kita-12, Nishi-6, Kita-ku, Sapporo 060–0812, Japan: and
Graduate School of Agricultural and Life Sciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–
b
8
657, Japan.
Received January 31, 2018; accepted March 7, 2018
Surugamide F is a linear decapeptide (1) isolated along with the cyclic octapeptides surugamides A–E
2–6), from a marine-derived Streptomyces species. The linear peptide 1 is produced by two nonribosomal
(
peptide synthetases (NRPSs) encoded in adjacent open reading frames, which are further flanked by an
additional pair of NRPS genes responsible for the biosyntheses of the cyclic peptides 2–6. While the cyclic
peptides 2–6 were identified to be cathepsin B inhibitors, the biological activity of the new metabolite 1 still
remained unclear. In order to elucidate its unique biosynthetic pathway and biological activity in detail, we
planned to develop an efficient synthetic route toward 1. Here we report the diastereoselective total synthesis
of 1, utilizing 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. During this study,
we found that the structural correction of 1 was required, due to the mislabeling of the commercially ob-
tained 3-amino-2-methylpropionic acid, and the true structure of 1 was corroborated by the chemical synthe-
sis and chromatographic comparison.
Key words marine natural product; structural elucidation; total synthesis; peptide
The marine-derived actinomycete Streptomyces sp. to total hydrolysis (6M HCl, 110°C, 4h), and then the cor-
α
JAMM992 produces surugamides A–E (2–6, Fig. 1b), along responding hydrolysates were treated with N -(2,4-dinitro-5-
with surugamide F (1, Fig. 1a). The cyclic octapeptides fluorophenyl)-L-valinamide (FDNP-Val). The FDNP-Val de-
2
–6 were initially isolated and identified to be cathepsin B rivative of AMPA (C, Chart 1c) was chromatographically
inhibitors. Subsequently, 1 was identified as a new linear compared to the authentic samples, which were obtained from
1)
2,3)
decapeptide from the same Streptomyces strain, although commercially available, optically active AMPAs. As a result,
its biological activity has not been evaluated yet. The draft the stereochemistry of AMPA in natural 1 was reported to be
genome sequence encodes four successive genes surA, surB, the (S) form (Chart 1d).
surC and surD, which are clustered and annotated as non-
We then turned our attention to the development of an
ribosomal peptide synthetases (NRPSs) with 18 A domains in efficient synthetic strategy for 1a because the previous syn-
total. The mutation experiment revealed that surA and surD thetic scheme generates an equal amount of the diastereomer
at both ends are responsible for the biosyntheses of 2–6. The 1b, which is difficult to separate from 1a. Concurrently, the
other genes, surB and surC, which are flanked by surA and reagent supplier notified us about the mislabeling of the op-
8)
surD are responsible for the biosynthesis of the structurally tically pure 3-amino-2-methylpropionic acids, which were
unrelated peptide 1. Since its intercalated NRPS gene archi- utilized as the standards for the stereochemical assignment
tecture is unprecedented, the new marine natural product 1 at- in the previous study (Chart 1). Thus, the stereochemistry of
tracted interest in terms of not only its unidentified biological AMPA required a structural correction. Herein, we report the
activities but also the biosynthetic mechanisms of 1 in relation first diastereoselective total synthesis of surugamide F (1), as
to the cyclic peptides 2–6.
well as the confirmation of the true structure of 1 by chemical
In the preceding study, we unveiled the structure of the synthesis. The details of the structural correction of 1 are also
linear peptide 1 by the combination of NMR, LC-MS/MS, described.
4
,5)
and Marfey’s analyses.
determined to possess four D-amino acids and a 3-amino- the requisite building blocks 9a and 9b were prepared (Chart
-methylpropionic acid (AMPA). Furthermore, the planar 2). The chiral 14a was synthesized from 11a, by using Evans’
The structure of peptide 1 was
At the outset of the diastereoselective total synthesis of 1,
2
9)
structure and stereochemistries of all amino acids except for asymmetric alkylation and azide reduction as key reactions.
AMPA, were validated by chemical synthesis. Specifically, Protecting group manipulation of 14a with trifluoroacetic acid
peptide 1 was chemically constructed by 9-fluorenylmethyl- (TFA), followed by Fmoc-Cl, led to 9a.
6
,7)
oxycarbonyl (Fmoc)-based solid-phase peptide synthesis using
racemic Fmoc-AMPA (9) as a building block, to yield the 11b, in the same manner. At this stage, the absolute configura-
The enantiomeric counterpart 9b was also synthesized from
10,11)
diastereomers (Chart 1a), which were separated by reversed- tion of 14b was confirmed by the Kusumi method
(Chart
phase HPLC. The stereochemically pure peptides were 3).
chromatographically and spectroscopically compared with
With the building blocks in hand, we commenced the solid-
natural 1 by LC-MS (Chart 1b) and NMR, respectively. For phase peptide syntheses of 1a and 1b from Fmoc-D-alanine-
the structural elucidation of AMPA, natural 1 was subjected loaded Wang-resin 7 (Chart 4a). The Fmoc group of 7 was de-
*
To whom correspondence should be addressed. e-mail: kuranaga@pharm.hokudai.ac.jp; wakimoto@pharm.hokudai.ac.jp
2018 The Pharmaceutical Society of Japan
©

6
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Chem. Pharm. Bull.
Vol. 66, No. 6 (2018)
Fig. 1. (a) Structure of Surugamide F (1); (b) Structures of Surugamides A–E (2–6)
(a) Previous structural confirmation of 1 by chemical synthesis. (b) LC-MS charts of synthetic and natural 1. (c) Marfey’s analysis of AMPA. (d) LC-MS charts of the
Marfey’s analysis of AMPA.
Chart 1.
tached by a treatment with piperidine, and then four rounds of
12)
N,N′-diisopropylcarbodiimide (DIC)/Oxyma -mediated amide
13)
α
coupling and piperidine-promoted N -deprotection were ap-
plied to 18, leading to the resin-bound pentapeptide 8. The
amine 8 was separately coupled with 9a and 9b to afford 10a
and 10b, and then Fmoc-based solid phase peptide synthesis
was re-applied to 10a and 10b for the syntheses of the resin-
bound decapeptides 20a and 20b. Finally, the treatment of 20a
and 20b with TFA–i-Pr SiH–H O (=90:5:5) simultaneously
3
2
achieved the global deprotection of the protecting groups and
the cleavage from the Wang resin, thus releasing crude 1a and
1b into the solution. After octadecyl silica (ODS)-HPLC puri-
fication, 1a and 1b were obtained in 36% yield and 38% yields
in 20 steps, respectively. The synthesized 1a and 1b were then
chromatographically compared with the natural 1 (Chart 4b),
confirming the true structure of the natural surugamide F (1),
Chart 2. Synthesis of Chiral AMPA

Vol. 66, No. 6 (2018)
Chem. Pharm. Bull.
639
as depicted in 1b.
In summary, the diastereoselective total synthesis of suru-
Experimental
General Methods H- and C-NMR spectra were re-
gamide F (1) was achieved, utilizing Fmoc-based solid-phase corded on a JEOL ECA 500 spectrometer (500MHz for
1
13
1
peptide synthesis was achieved. During this study, we found
that the structural correction of 1 was required, and we also residual solvent peaks as internal standards (CDCl , H δ 7.26,
corroborated the true structure of 1 by the chemical synthesis.
H-NMR). Chemical shifts are denoted in δ (ppm) relative to
1
3
13
C δ 77.0). Electrospray ionization (ESI)-MS spectra were
Based on the established synthetic pathway to 1, detailed bio- recorded on a Thermo Scientific Exactive mass spectrometer
logical and biosynthetic studies of 1 are currently underway or a SHIMADZU LCMS-2020 spectrometer. Specific rota-
and will be reported in due course.
tions were recorded on a JASCO P-1030 polarimeter. HPLC
experiments were performed with a SHIMADZU HPLC sys-
tem equipped with an LC-20AD intelligent pump. All reagents
were used as supplied unless otherwise stated. Analytical
TLC was performed using E. Merck Silica gel 60F254 pre-
coated plates. Column chromatography was performed using
4
0–50µm Silica Gel 60N (Kanto Chemical Co., Inc., Japan).
Procedure for Solid-Phase Peptide Synthesis (SPPS)
Step 1: To the solution of carboxylic acid (4eq) were added
DIC (4eq, 0.50M in NMP) and Oxyma (4eq, 0.50M in N,N-
dimethylformamide (DMF)). After 2min of pre-activation, the
mixture was injected into the reaction vessel. The resulting
mixture was stirred for 30min at 37°C.
Step 2: The resin in the reaction vessel was washed with
DMF (×3) and CH Cl (×3).
2
2
Step 3: The Fmoc group of the solid supported peptide was
removed with a 20% piperidine/DMF solution (10min, room
temperature).
Step 4: The resin in the reaction vessel was washed with
DMF (×3) and CH Cl (×3).
2
2
Amino acids were condensed onto the solid support by re-
peating Steps 1–4.
Carbamate 13a
Chart 3. Structural Confirmation of Chiral AMPA
To a solution of 11a [CAS 203454-44-8] (162mg,
(a) Diastereoselective total synthesis of 1. (b) LC-MS charts of synthetic and natural 1.
Chart 4.

6
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Chem. Pharm. Bull.
Vol. 66, No. 6 (2018)
0
.457mmol) in tetrahydrofuran (THF) (10mL) was added ous Na CO (9mL) at 0°C. After stirring overnight at room
2 3
Pd/C (10% on carbon, 80.2mg). The mixture was exposed to temperature, the mixture was evaporated to remove the THF.
a hydrogen atmosphere at room temperature. After stirring The aqueous solution was washed with Et O (twice), acidi-
2
overnight, the resulting mixture was filtered and concentrated fied with 1M HCl (to pH 2), and then extracted with EtOAc (3
to give 12a, which was used in the next reaction without fur- times). The combined organic layer was washed with brine,
ther purification.
dried over MgSO , filtered, and concentrated to give 9a [CAS
4
To a solution of 12a in CH Cl (4mL) at room tempera- 211682-15-4] (140mg, 83%).
2
2
ture were added Et N (0.13mL, 0.93mmol), N,N-dimethyl-
Peptide 20a
3
4
-aminopyridine (DMAP) (13.9mg, 0.114mmol), and p-TsCl
The Fmoc-D-Ala-Wang-resin 7 (82.4mg, 0.0503mmol) in a
(109mg, 0.569mmol). After stirring overnight, saturated aque- Libra tube (HiPep Laboratories) was suspended in a 20% pi-
ous NH Cl was added to the reaction mixture. The resulting peridine/DMF solution. After stirring at room temperature for
4
solution was extracted with EtOAc (3 times). The combined 10min, the reaction mixture was washed with DMF (×3) and
organic layer was washed with brine, dried over MgSO , fil- CH Cl (×3) to afford the amine 18, as the resin-bound form.
4
2
2
tered, and concentrated to give the crude tosylate, which was This resin was swelled in DMF for 1h, and then subjected to
used in the next reaction without further purification. 9 cycles [Fmoc-L-Val-OH, Fmoc-D-Ala-OH, Fmoc-L-Val-OH,
To a solution of the above tosylate in DMF (4mL) at room Fmoc-D-Leu-OH, 9a, Fmoc-L-Thr(t-Bu)-OH, Fmoc-L-Val-OH,
temperature was added NaN (132mg, 2.03mmol). After stir- Fmoc-D-Leu-OH, and Fmoc-L-Trp(Boc)-OH] of the SPPS pro-
3
ring at 40°C for 2d, saturated aqueous NaHCO was added tocol, to afford the peptide 20a as the resin-bound form.
3
to the reaction mixture. The resulting solution was extracted
with EtOAc (3 times). The combined organic layer was
Peptide 1a
To peptide 20a was added a mixture of TFA–H O–
2
washed with brine, dried over MgSO , filtered, and concen- i-Pr SiH=90:5:5 (1.0mL). After stirring for 30min, the reac-
4
3
trated to give the crude azide, which was used in the next tion mixture was filtered, and then washed with a mixture of
reaction without further purification.
TFA–H O–i-Pr SiH=90:5:5 (1.0mL). The filtrate was diluted
2
3
To a solution of the above azide in THF (8mL) were with Et O (20mL) and centrifuged (4°C, 3500G, 5min), and
2
added Boc O (0.19mL, 0.83mmol) and Pd/C (10% on carbon, then the Et O was removed by decantation. The obtained
2
2
8
0.7mg). The mixture was exposed to a hydrogen atmosphere crude peptide 1a was purified by reversed phase HPLC
at room temperature. After stirring overnight, the resulting (COSMOSIL 5C -MS-II 10×250mm) with MeOH–H O
1
8
2
1)
mixture was filtered and concentrated. The residue was pu- (=65:35) containing 0.05% TFA to afford 1a (18.9mg, 36%
rified by column chromatography (EtOAc–hexane=30:70) from 7). [α]D −6.6 (c=0.05, MeOH).
1
6
to afford 13a (80.4mg, 49% for 4 steps) as a colorless solid:
Peptide 20b
2
6
1
[
α] −51.7 (c=2.00, MeOH); H-NMR (500MHz, CDCl ) δ:
The Fmoc-D-Ala-Wang-resin 7 (86.9mg, 0.0530mmol) in a
.30 (dd, 2H, J=7.5, 7.5Hz), 7.23 (t, 1H, J=7.5Hz), 7.16 (d, Libra tube (HiPep Laboratories) was suspended in a 20% pi-
H, J=7.5Hz), 4.94 (br, 1H), 4.63 (m, 1H), 4.14 (m, 2H), 3.86 peridine/DMF solution. After stirring at room temperature for
D
3
7
2
(
m, 1H), 3.43 (m, 1H), 3.34 (m, 1H), 3.25 (d, 1H, J=13.0Hz), 10min, the reaction mixture was washed with DMF (×3) and
2
.75 (dd, 1H, J=10.0, 13.3Hz), 1.39 (s, 9H), 1.16 (d, 3H, CH Cl (×3) to afford the amine 18, as the resin-bound form.
2
2
13
J=7.5Hz); C-NMR (500MHz, CDCl ) δ: 175.6, 155.9, 153.2, This resin was swelled in DMF for 1h, and then subjected to
3
1
35.5, 129.6, 129.1, 128.4, 127.4, 79.3, 66.3, 55.5, 43.2, 38.9, 9 cycles [Fmoc-L-Val-OH, Fmoc-D-Ala-OH, Fmoc-L-Val-OH,
3
8.0, 28.5, 14.8; high resolution (HR)-MS (ESI) Calcd for Fmoc-D-Leu-OH, 9b, Fmoc-L-Thr(t-Bu)-OH, Fmoc-L-Val-OH,
+
+
C H N O Na [M+Na] 385.1734. Found 385.1734.
Fmoc-D-Leu-OH, and Fmoc-L-Trp(Boc)-OH] of the SPPS pro-
tocol, to afford the peptide 20b as the resin-bound form.
Peptide 1b
1
9
26
2
5
2
5
1
3b: [α] +57.0 (c=2.00, MeOH).
D
Acid 14a
To a solution of 13a (201mg, 0.555mmol) in THF (10mL)
Peptide 20b was combined with a mixture of TFA–H O–
2
at 0°C were added 30% aqueous H O2 (0.18mL) and i-Pr SiH=90:5:5 (1.0mL). After stirring for 30min, the reac-
2
3
LiOH·H O (36.8mg, 1.61mmol) in H O (3mL). After stir- tion mixture was filtered, and then washed with a mixture of
2
2
ring at 0°C for 2.5h, saturated aqueous Na SO and saturated TFA–H O–i-Pr SiH=90:5:5 (1.0mL). The filtrate was diluted
2
3
2
3
aqueous NaHCO were added to the reaction mixture. After with Et O (20mL) and centrifuged (4°C, 3,500G, 5min), and
3
2
stirring at room temperature for 2h, the mixture was evapo- then the Et O was removed by decantation. The obtained
2
rated to remove the THF. The aqueous solution was washed crude peptide 1a was purified by reversed phase HPLC
with CH Cl (twice), acidified with 1M HCl (to pH 3), and (COSMOSIL 5C -AR-II 10×250mm), with MeOH–H O
2
2
18
2
then extracted with EtOAc (3 times). The combined organic (=60:40 to 100:0 for 40min) containing 0.05% TFA, to af-
1
16
layer was washed with brine, dried over MgSO , filtered, and ford 1b (20.3mg, 38% from 7). [α]D −30.8 (c=0.05, MeOH).
4
concentrated to give 14a [CAS 190897-47-3] (105mg, 93%).
Acid 9a
Acknowledgments This work was partly supported by
TFA (3mL) was added to a solution of 14a (105mg, the Takeda Science Foundation, the Astellas Foundation for
0
.519mmol) in CH Cl (7mL) at room temperature. After stir- Research on Metabolic Disorders, the Naito Foundation, the
2
2
ring at room temperature for 2h, the resulting mixture was SUNBOR GRANT, the NOASTEC Foundation, the Aki-
concentrated and azeotroped with toluene (3 times) to afford yama Life Science Foundation, and Grants-in-Aid from the
the crude amine, which was used in the next reaction without Ministry of Education, Culture, Sports, Science and Tech-
further purification.
nology (MEXT) of Japan (JSPS KAKENHI Grant Numbers
Fmoc-Cl (167mg, 0.646mmol) in THF (2mL) was added to JP16703511, JP15547389, JP15597834, and JP14506930).
a solution of the above amine, in THF (6mL) and 10% aque-

Vol. 66, No. 6 (2018)
Chem. Pharm. Bull.
641
6
)
Structural revision of scarce peptide by chemical synthesis, see:
Kuranaga T., Sesoko Y., Sakata K., Maeda N., Hayata A., Inoue M.,
J. Am. Chem. Soc., 135, 5467–5474 (2013).
Conflict of Interest The authors declare no conflict of
interest.
7
)
Structural revision of scarce peptide by chemical synthesis, see:
Kuranaga T., Mutoh H., Sesoko Y., Goto T., Matsunaga S., Inoue
M., J. Am. Chem. Soc., 137, 9443–9451 (2015).
Supplementary Materials The online version of this ar-
ticle contains supplementary materials.
8
)
Ninomiya A., Katsuyama Y., Kuranaga T., Miyazaki M., Nogi Y.,
Okada S., Wakimoto T., Ohnishi Y., Matsunaga S., Takada K.,
ChemBioChem, 18, 1770 (2017).
Evans D. A., Connell B. D., J. Am. Chem. Soc., 125, 10899–10905
(
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