Total syntheses of nobilamides B and D: Application of traceless Staudinger ligation

Article abstract of DOI:10.1016/j.tet.2014.05.091

Nobilamide B is a long-acting antagonist of transient receptor potential vanilloid-1 (TRPV1), and is expected to show therapeutic potential for treatment of pain. This linear heptapeptide possesses a Z-didehydroaminobutanoic acid moiety at the C-terminus. Stereoselective construction of the didehydroamino acid moiety was successfully achieved by application of the traceless Staudinger ligation. The combination of solid-phase peptide synthesis and the Staudinger ligation allowed rapid access to not only nobilamide B, but also its macrocyclic analogue nobilamide D.

Full text of DOI:10.1016/j.tet.2014.05.091

Accepted Manuscript
Total syntheses of nobilamides B and D: application of traceless Staudinger ligation
Tomoya Yamashita , Hiroaki Matoba , Takefumi Kuranaga , Masayuki Inoue
PII:
S0040-4020(14)00797-2
10.1016/j.tet.2014.05.091
TET 25640
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 April 2014
Revised Date: 21 May 2014
Accepted Date: 23 May 2014
Please cite this article as: Yamashita T, Matoba H, Kuranaga T, Inoue M, Total syntheses of
nobilamides B and D: application of traceless Staudinger ligation, Tetrahedron (2014), doi: 10.1016/
j.tet.2014.05.091.
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Total syntheses of nobilamides B and D:
application of traceless Staudinger ligation
Leave this area blank for abstract info.
Tomoya Yamashita, Hiroaki Matoba, Takefumi Kuranaga and Masayuki Inoue*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan

1
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Tetrahedron
journal homepage: www.elsevier.com
Total syntheses of nobilamides B and D: application of traceless Staudinger ligation
Tomoya Yamashita, Hiroaki Matoba, Takefumi Kuranaga and Masayuki Inoue
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Nobilamide B is a long-acting antagonist of transient receptor potential vanilloid-1 (TRPV1),
and is expected to show therapeutic potential for treatment of pain. This linear heptapeptide
possesses a Z-didehydroaminobutanoic acid moiety at the C-terminus. Stereoselective
construction of the didehydroamino acid moiety was successfully achieved by application of
the traceless Staudinger ligation. The combination of solid-phase peptide synthesis and the
Staudinger ligation allowed rapid access to not only nobilamide B, but also its macrocyclic
analogue nobilamide D.
Available online
Keywords:
Peptides
2009 Elsevier Ltd. All rights reserved
.
Solid-phase synthesis
Staudinger ligation
Total synthesis
TRPV1 antagonist
1. Introduction
Development of a flexible synthetic route to the nobilamides
will make it possible to prepare a wider variation of nobilamide
analogues, thereby enabling a comprehensive structure-activity
relationship (SAR) study in search of optimized TRPV1
antagonists. To initiate such an investigation, the representative
nobilamide structures, linear nobilamide B (2) and macrocyclic
nobilamide D (4), were selected as the target molecules for
chemical construction. Here we report the efficient total
syntheses of 2 and 4 by a combination of solution-phase and
solid-phase strategies. In both syntheses, a traceless Staudinger
ligation was employed as the key reaction for stereoselective
construction of the ∆Abu moiety.
The vanilloid receptor TRPV1 belongs to the large family of
transient receptor potential (TRP) channels that comprise a
diverse group of non-voltage-gated cation channels.1 TRPV1
responds to multiple painful stimuli, including noxious heat
(>42 °C), extracellular pH below 5.5 and vanilloid toxins such as
capsaicin. A number of pharmacological experiments suggest
that selective TRPV1 antagonists hold great promise in
alleviating multiple modalities of pain.2
In 2011, a series of peptide natural products, designated as
nobilamides, were isolated and structurally determined by
Schmidt and co-workers. 3 Among them, nobilamide B (2,
Scheme 1), the linear heptapeptide, was shown to produce long-
term (> 1 h) inhibition of the TRPV1 channel. Schmidt proposed
that 2 acted as a long-lasting antagonist by forming a covalent
bond through nucleophilic 1,4-addition of the Cys residue of
TRPV1 to the Z-didehydroaminobutanoic acid (∆Abu) terminus.
In fact, nobilamide C (3) that lacks the ∆Abu moiety exhibited no
detectable inhibition. Interestingly, a structural change from the
propionamide at the N-terminus of 2 to the acetamide of
nobilamide A (1) significantly decreased the inhibitive potency,
while the macrocyclic hexaamide nobilamide D (4) was found to
be inactive.3 Therefore, small structural differences of the amino
acid residues and the C- and N-termini largely affected the
antagonistic activity.
2. Results and discussion
The retrosynthesis of nobilamides B and D is illustrated in
Scheme 1. Heptapeptide 2 was retrosynthetically disassembled
into unsaturated dipeptide 5 and pentapeptide 6 (Scheme 1).
Compound 6 would be synthesized from Fmoc-L-Val-Wang resin
74 by Fmoc-based solid-phase peptide synthesis (SPPS).5 The
macrocyclic structure of nobilamide D (4) was planned to be
constructed via lactamization rather than lactonization, because
the higher nucleophilicity of the nitrogen atom in comparison to
the oxygen atom was considered to be beneficial for facilitating
the cyclization.
retrosynthetically simplified into the branched peptide structure 8
by cleavage of the amide linkage between -Val and -Ala.
Compound 8 would be synthesized from the same 7 by SPPS.
Accordingly, cyclic hexapeptide 4 was
L
L
———
Corresponding author. Tel.: +81-3-5841-1354; fax: +81-3-5841-0568; e-mail: inoue@mol.f.u-tokyo.ac.jp

2
Tetrahedron
ACCEPTED MANUSCRIPT
Scheme 1. Structures and retrosyntheses of nobilamides.a
L-Phe
L-Val
D-Phe
Abu
O
BocHN
OAllyl
N
H
O
O
O
O
O
H
H
H
N
5
N
N
OH
R
N
N
N
N
+
H
H
H
H
O
O
O
O
OH
D-allo-Thr
D-Leu
L-Ala
O
O
O
H
N
H
nobilamide A (1): R = Me (IC50 = 1665 M)
nobilamide B (2): R = Et (IC50 = 275 M)
N
OH
N
H
N
N
H
H
O
O
O
OH
6
O
O
O
O
H
H
N
H
N
N
N
N
N
OH
O
H
H
H
O
O
O
OH
O
FmocHN
3
nobilamide C ( ) (inactive)
O
7
: Fmoc-L-Val-Wang resin
L-Phe
L-Val
O
H
O
O
O
O
H
N
H
H
N
O
N
N
OH
N
H
N
N
N
H
H
H
O
O
HN
O
O
O
O
O
H
H
N
N
L-Ala
O
O
NH2
O
D-allo-Thr
D-Phe
8
Abu
4
nobilamide D ( ) (inactive)
aBoc = t-butoxycarbonyl; Fmoc = 9-fluorenylmethyloxycarbonyl.
Scheme 2. Attempted Cu-mediated enamide formation.
Scheme 3. Application of traceless Staudinger ligation for
construction of the didehydroalanine structure.a
Stereoselective synthesis of the ∆Abu moieties of 5 and 8 is
the most important task in the development of the route to the
nobilamides.6 ,7 We recently demonstrated that the Cu-mediated
cross-coupling reaction was effective in E/Z-selective syntheses
of the didehydroisoleucine structures in the total synthesis of
highly unsaturated natural peptide yaku'amide A.8,9 For instance,
the coupling reaction between amide 9 and E-alkenyl iodide 10
was realized using the Buchwald reagent system [CuI, N,N'-
dimethylethylenediamine (11), Cs2CO3], resulting in formation of
the sterically congested tetrasubstituted olefin 12 of
didehydroisoleucine in the E-selective fashion (Scheme 2).
However, when iodide 13 was treated with 9 under the same
reaction conditions, alaninamide 9 was cleanly recovered, and
trisubstituted olefin 14 was not obtained. Judging from the
consumption of 13 during the reaction, iodide 13 appeared to be
involved in unproductive pathways, presumably due to its
chemical instability towards basic media. Thus, an alternative
coupling strategy was necessitated for construction of the Z-
didehydroaminobutanoic acid.
aBn = benzyl; Ph = phenyl; THF = tetrahydrofuran.

3
We planned to directly construct the requisite enamide by
Nobilamide B (2) was synthesized from 5 and 6 in 3 steps
ACCEPTED MANUSCRIPT
employing the traceless Staudinger ligation because of its
powerful and mild nature.10 Raines reported the high-yielding
generation of N-acetyl glycine derivative 17 from
phosphinothioester 15 and alkyl azides 16 under neutral aqueous
conditions (Scheme 3a).11 The model studies for assembly of the
unsaturated peptide using this strategy are depicted in Scheme 3b.
Use of alkenyl azide 18 in the presence of thioester 15 in aqueous
THF led to no formation of didehydroalanine 19, and the cysteine
derivative 20 was quantitatively produced. In this reaction,
intermediate 22, generated from 21, did not undergo hydrolysis
to give the desired 19. Instead, the strongly nucleophilic thiolate
of 22 intramolecularly added to the α,β-unsaturated ester, leading
to 23, hydrolysis of which provided the undesired 20. To
suppress the intramolecular Michael addition of the intermediate,
Bertozzi’s phenol derivative 2412 , 13 was next utilized for the
model reaction. By simply mixing 24 and alkenyl azide 18 in
aqueous THF, enamide 19 was produced in 88% yield, and no
Michael adduct 25 was observed, reflecting the low
nucleophilicity of the phenolate of the intermediate. These
studies clarified the importance of the structure of the activated
ester for effective enamide formation.
(Scheme 5). Removal of the Boc group of enamide 5 liberated
amine 34, which was then coupled with acid 6 using EDCI in the
presence of HOAt 17 and NaHCO3 to provide 35. Finally,
cleavage of the C-terminal allyl group of 35 with Pd(PPh3)4 and
PhSiH318 gave rise to nobilamide B (2) in 31% yield in 3 steps
after reversed-phase HPLC purification.
Scheme 5. Total synthesis of nobilamide B (2).a
1. 20% piperidine/DMF
2. Fmoc-D-allo-Thr-OH, HBTU, HOBt
O
R1
i-Pr2NEt, DMF, NMP
3. 20% piperidine/DMF
4. Fmoc-L-Phe-OH, HBTU, HOBt
i-Pr2NEt, DMF, NMP
FmocHN
O
7: R1 = Wang resin
MW-assisted SPPS
5. 20% piperidine/DMF
6. Fmoc-D-Leu-OH, HBTU, HOBt
i-Pr2NEt, DMF, NMP
7. 20% piperidine/DMF
8. Fmoc-D-Phe-OH, HBTU, HOBt
i-Pr2NEt, DMF, NMP
9. 20% piperidine/DMF
Before synthesis of the unsaturated dipeptide 5, its
component monomers 28 and 31 were prepared from the known
O
O
H
H
14
N
N
O
R1
compounds 26 and 29,
Phosphinophenol 26 was condensed with Boc-
respectively (Scheme 4).
-Ala-OH 27 by
R2HN
N
H
N
L
H
O
O
O
the action of DCC and DMAP, leading to 28. Alternatively, Z-
alkenyl azide 29 was hydrolyzed to afford carboxylic acid 30,
which was transformed to the corresponding allyl ester 31 using
allylbromide and K2CO3. Most importantly, the Staudinger
ligation reaction in dioxane/H2O at 100 °C successfully
transformed 28 and 31 into the Z-alkenyl enamide 5 in
stereoselective fashion (74% yield).
OH
: R1 = Wang resin, R2 = H
32
(EtCO)2O, CH2Cl2
33: R1 = Wang resin, R2 = EtCO
TFA, H2O, 49% (11 steps)
: R = H, R2 = EtCO
1
6
TFA, CH2Cl2
O
O
BocHN
OAllyl
H2N
OAllyl
N
N
H
H
O
O
5
34
Scheme 4. Synthesis of dipeptide 5. a
6 (1.0 eq), EDCI, HOAt, NaHCO3, CH2Cl2, DMF
O
O
O
O
H
H
H
N
N
N
OR3
N
N
N
N
H
H
H
H
O
O
O
O
OH
: R3 = Allyl
35
Pd(PPh3)4, PhSiH3, CH2Cl2, 31% (3 steps)
2: R3 = H
N
N
PF6
N
N
N
N
N
N
N
N
O
N
OH
HOBt
OH
HOAt
N
HBTU
aDMF
=
N,N-dimethylformamide; EDCI
=
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HBTU = O-(benzotriazol-1-
yl)-N,N,N,N-tetramethyluronium hexafluorophosphate; HOAt =
1-hydroxy-7-azabenzotriazole; HOBt = 1-hydroxybenzotriazole;
MW = microwave; NMP = N-methylpyrrolidone; TFA =
trifluoroacetic acid.
aDCC = N,N'-dicyclohexylcarbodiimide; DMAP =N,N-dimethyl-
4-aminopyridine.
The coupling partner 6 of thus obtained dipeptide 5 was
synthesized by means of microwave (MW)-assisted SPPS
(Scheme 5).15 Cycles of piperidine-promoted Nα-deprotection
and HOBt/HBTU-mediated amide coupling16 were employed on
Efficiency of the traceless Staudinger ligation strategy was
further demonstrated by the total synthesis of macrocyclic
nobilamide D (4, Scheme 6). Similar to the synthesis of 33, the
Ac-capped tetrapeptide 36 was elongated under MW-irradiation
Fmoc-
L-Val-Wang resin 7 using Fmoc-D-allo-Thr-OH, Fmoc-L-
Phe-OH, Fmoc-
D
-Leu-OH and Fmoc- -Phe-OH. The Nα-Fmoc
D
conditions on Fmoc-
L-Val-Wang resin 7 by using Fmoc-
D-allo-
of the elongated peptide was removed to afford 32. The free
amine of the N-terminus of 32 was in turn capped as the
propionamide, and the resin at the C-terminus was detouched
upon treatment with 95% aqueous TFA, furnishing pentapeptide
6 in 49% yield over 11 steps.
Thr-OH, Fmoc- -Phe-OH, Fmoc-
L
D-Phe-OH and Ac2O. The
secondary hydroxy group of 36 was then condensed with Z-
alkenyl azide 30 by the action of DCC and DMAP to yield
branched peptide 38 as the resin-bound form. Next, the Z-

4
Tetrahedron
ACCEPTED MANUSCRIPT
3. Conclusion
enamide moiety was constructed by the on-resin Staudinger
ligation. When 38 and 28 were heated to 60 °C under microwave
irradiation in NMP and H2O, didehydropeptide 39 was smoothly
formed. Treatment of 39 with 95% aqueous TFA realized the
removal of the Boc group and cleavage from the Wang resin,
leading to 8. Finally, the branched peptide 8 was cyclized using
PyBOP
macrolactamized product 4.
purification, nobilamide D (4) was obtained in 20% yield over 12
steps.
The total syntheses of nobilamides B and D (2 and 4) were
achieved for the first time (2: 15% overall yield in 13 steps from
7, 4: 20% overall yield in 12 steps from 7). The present route
features a new application of the traceless Staudinger ligation for
stereoselective construction of the Z-enamide structures (∆Abu).
The Staudinger ligation was successfully utilized in the solution-
phase for construction of 2 and on the solid-phase for synthesis
of 4, demonstrating its high versatility. Future research will
include incorporation of the present ligation strategy for synthesis
of other unsaturated peptide natural products, systematic SAR
studies of synthetic nobilamides and their artificial analogues,
and detailed investigations of the molecular mode of action of
synthetic TRPV1 antagonists.
19
and HOAt as a condensation reagent to deliver
After reversed-phase HPLC
The H- and 13C-NMR spectra of synthetic nobilamides 2 and
1
4 were in agreement with those of their natural counterparts, and
their absolute structures were further determined by MS/MS
experiments and Marfey’s analyses.20,21
Scheme 6. Total synthesis of nobilamide D (4).a
4. Experimental section
7 1. 20% piperidine/DMF
2. Fmoc-D-allo-Thr-OH, HBTU, HOBt
i-Pr2NEt, DMF, NMP
4.1. General information
3. 20% piperidine/DMF
4. Fmoc-L-Phe-OH, HBTU, HOBt
i-Pr2NEt, DMF, NMP
5. 20% piperidine/DMF
6. Fmoc-D-Phe-OH, HBTU, HOBt
i-Pr2NEt, DMF, NMP
7. 20% piperidine/DMF
8. Ac2O, CH2Cl2
4.1.1. General procedures
1H and 13C NMR spectra were recorded on a JEOL ECX 500
1
(500 MHz for H NMR, 125 MHz for 13C NMR) spectrometer, a
JEOL ECA 500 (500 MHz for 1H NMR, 125 MHz for 13C NMR)
1
spectrometer or a JEOL ECS 400 (400 MHz for H NMR, 100
O
O
H
H
MHz for 13C NMR). Chemical shifts are denoted in δ (ppm)
N
N
O
R1
relative to residual solvent peaks as internal standard (CDCl3, 1H
N
N
MW-assisted SPPS
H
H
O
O
O
δ 7.26, 13C δ 77.0; (CD3)2SO, H δ 2.50; 13C δ 39.5). IR spectra
1
OH
were recorded on a JASCO FT/IR-4100 spectrometer. MALDI-
TOF MS analyses were performed with a Shimadzu Biotec
36
Axima-TOF2
mass
spectrometer
using
α-cyano-4-
OH
N3
O
O
O
hydroxycinnamic acid as a matrix. ESI-TOF MS spectra were
recorded on a JEOL T100LP mass spectrometer. Optical
rotations were recorded on a JASCO P-2200 polarimeter. HPLC
was performed on a Jasco HPLC system equipped with a PU-
2089 Plus intelligent pump, a UV-2070 Plus UV detector. All
reactions were monitored by TLC on MERCK TLC plates silica
gel 60 F254 under UV light (254 nm), and/or developed by 10%
ethanolic phosphomolybdic acid or anisaldehyde solution (p-
anisaldehyde (50 mL), AcOH (10 mL), EtOH (900 mL) and
concentrated H2SO4 (50 mL)). Flash column chromatography
was performed using KANTO Silica gel 60 N (40-50 µm).
H
H
30 (3.0 eq)
O
N
N
O
R1
N
N
DCC, DMAP
DMF, THF
MW, 40 °C
H
H
O
O
O
N3
O
38
28 (3.0 eq), NMP
H2O, MW, 60 °C
O
O
H
H
N
N
O
N
R1
N
H
H
O
O
O
O
O
BocHN
H
O
N
O
NHR2
28
PPh2
O
4.1.2. Procedures for solid-phase synthesis
39: R1 = Wang resin, R2 = Boc
8: R1 = R2 = H
TFA, H2O
O
Solid-phase peptide synthesis was performed on a microwave
assisted peptide synthesizer EYELA MWS-1000. Standard
operation was shown as follows:
Step 1: Fmoc group of the solid supported peptide was removed
by 20% piperidine/DMF solution (10 min, room temperature).
Step 2: The reaction vessel containing the resin was washed by
DMF 5 times.
Step 3: To the solution of i-Pr2NEt (6 eq) in NMP was added a
solution of Fmoc-amino acid (3 eq), HBTU (3 eq), and HOBt (3
eq) in DMF. The mixture was injected to the reaction vessel, and
stirred for 20 min at 40 °C assisted with microwave energy 100
W.
PyBOP, HOAt
2,4,6-collidine
CH2Cl2, DMF
O
H
N
H
N
O
N
H
N
H
O
O
HN
O
7
20% (12 steps from
PyBOP
)
H
N
O
O
nobilamide D (4)
N
N
N
N
O
P
N
N
PF6
aPyBOP
hexafluorophosphate.
=
benzotriazol-1-yl-oxytripyrrolidinophosphonium
Step 4: The reaction vessel containing the resin was washed by
DMF 5 times.
Step 1～4 were repeated, and amino acids were condensed onto
the solid support.
4.2. Experimental procedures for Scheme 2–4.

5
4.2.1. Enamide 12. To a solution of amide 9 (199 mg, 1.06
mmol) and E-vinyl iodide 10 (340 mg, 1.27 mmol) in dioxane
(1.0 mL) were added Cs2CO3 (414 mg, 1.27 mmol), copper
(1H, m), 4.91 (1H, d, J = 6.8 Hz), 6.80 (1H, m), 7.10-7.20 (2H,
ACCEPTED MANUSCRIPT
m), 7.25-7.40 (11H, m); 13C NMR (100 MHz, CDCl3, 31P-
coupled, 1H-decoupled, observed signals) δ 18.0, 18.1, 28.3,
49.4, 79.8, 122.2, 126.3, 128.5, 128.6, 129.01, 129.05, 129.8,
129.99, 130.02, 133.7, 133.8, 133.9, 134.0, 135.2, 135.3, 152.4,
152.6, 154.9, 171.1; HRMS (ESI) calcd for C26H28NNaO4P
[M+Na]+ 472.1648, found 472.1634.
iodide
(60.8
mg,
0.319
mmol)
and
N,N’-
dimethylethylenediamine (11) (0.23 mL, 2.14 mmol) at room
temperature. After being stirred at 70 °C for 20.5 h, the reaction
mixture was filtered through a pad of silica gel. The solution was
concentrated, and the residue was purified with flash column
chromatography (EtOAc/hexane = 1/9 to 1/1) to afford enamide
12 (239 mg, 0.728 mmol, 69%) as a white solid: [α]D23 = -39.3 (c
0.58, CHCl3); IR (film) 3307, 2979, 1719, 1670, 1516, 1366,
4.2.5. Acid 30.
1
1170 cm-1; H NMR (500 MHz, CDCl3) δ 1.09 (3H, t, J = 8.0
To a stirred solution of 29 (2.70 g, 17.4 mmol) in EtOH (4.4 mL)
was added LiOH·H2O (2.92 g, 69.6 mmol) in H2O (13.1 mL) at
0 °C. The resulting mixture was warmed to room temperature
and stirred for 3 h. The reaction was quenched with 2 M aqueous
HCl (acidified to pH ~1), extracted with EtOAc (50 mL x 3) and
washed with brine (150 mL). Combined organic layer was dried
over Na2SO4 and filtered. Concentration afforded crude
carboxylic acid 30 (1.97 g, 15.5 mmol, 89%) as a white solid; IR
(film) 2847, 2606, 2511, 2129, 1669, 1628, 1426, 1262, 1140 cm-
1; 1H NMR (400 MHz, CDCl3) δ 1.85 (3H, d, J = 7.3 Hz), 6.46
(1H, q, J = 7.3 Hz), 11.6 (1H, brs); 13C NMR (100 MHz, CDCl3)
δ 13.4, 128.0, 130.0, 168.2; HRMS (ESI) calcd for C4H5N3NaO2
[M+Na]+ 150.0274, found 150.0268.
Hz), 1.27 (3H, t, J = 7.3 Hz), 1.39 (3H, d, J = 7.4 Hz), 1.44 (9H,
s), 1.80 (3H, s), 2.49 (2H, q, J = 7.3 Hz), 4.19 (2H, q, J = 7.4
Hz), 4.25 (1H, m), 5.04 (1H, m), 7.48 (1H, s); 13C NMR (125
MHz, CDCl3) δ 12.7, 14.1, 17.8, 19.4, 27.5, 28.3, 49.9, 60.7,
80.2, 120.9, 148.9, 155.6, 164.4, 171.0; HRMS (ESI) calcd for
C16H28N2NaO5 [M+Na]+ 351.1890, found 351.1881.
4.2.2. Amide 20.
To a solution of phosphinothioester 15 (34.0
mg, 0.124 mmol) in THF (0.46 mL) at room temperature was
added azide 18 (19.3 mg, 0.137 mmol) in THF (1.4 mL) by
cannula under Ar. After 10 min, H2O (0.62 mL) was added to
the mixture, which was stirred overnight at room temperature.
The solvent was then removed under reduced pressure, and the
residue was purified by flash column chromatography
(hexane/EtOAc = 10/1 to 0/1) to afford 20 (49.3 mg, 0.122
mmol, 98%) as a white foam: IR (film) 3252, 3056, 1739, 1666,
1547, 1438, 1372, 1187, 1121 cm-1; 1H NMR (400 MHz, CDCl3)
δ 1.24 (3H, t, J = 7.3 Hz), 2.07 (3H, s), 3.11 (1H, dd, J = 14.4,
4.0 Hz), 3.22 (1H, dd, J = 15.6, 7.8 Hz), 3.23 (1H, dd, J = 14.4,
7.0 Hz), 3.41 (1H, dd, J = 15.6, 5.0 Hz), 4.17 (2H, q, J = 7.3 Hz),
4.76 (1H, ddd, J = 7.3, 7.0, 4.0 Hz), 7.47-7.59 (6H, m), 7.70-7.78
(4H, m), 8.20 (1H, d, J = 7.3 Hz); 13C NMR (100 MHz, CDCl3,
4.2.6. Azide 31.
To a stirred solution of 30 (194 mg, 1.53 mmol) in DMF (3.1
mL) were added K2CO3 (232 mg, 1.68 mmol) and allyl bromide
(0.260 mL, 3.05 mmol) successively at 0 °C. This solution was
warmed to room temperature and stirred overnight. The resulting
mixture was quenched with saturated aqueous NH4Cl, extracted
with Et2O (20 mL x 3) and washed with brine (50 mL).
Combined organic layer was dried over Na2SO4 and filtered.
Concentration and flash column chromatography (hexane/Et2O =
10/1 to 5/1) afforded 31 (238 mg, 93%) as a pale yellow oil: IR
(film) 2126, 1720, 1640, 1387, 1347, 1272, 1253, 1132, 1054 cm-
1
31P-coupled; H-decoupled, observed signals) δ 14.1, 22.9, 30.6,
31.3, 36.1, 52.9, 61.6, 128.71, 128.77, 128.84, 128.89, 130.7,
130.88, 130.96, 130.98, 131.1, 131.7, 131.9, 132.33, 132.36,
132.38, 170.5, 170.6; HRMS (ESI) calcd for C20H24NNaO4PS
[M+Na]+ 428.1056, found 428.1037.
1; H NMR (400 MHz, CDCl3) δ 1.80 (3H, d, J = 7.3 Hz), 4.71
1
(2H, ddd, J = 6.0, 1.4, 1.4 Hz), 5.28 (1 H, ddd, J = 10.5, 2.7, 1.4
Hz), 5.36 (1H, ddd, J = 17.4, 2.7, 1.4 Hz), 5.95 (1H, dddd, J =
17.4, 10.5, 6.0, 6.0 Hz), 6.29 (1H, q, J = 7.3 Hz); 13C NMR (100
MHz, CDCl3) δ 13.0, 66.2, 119.0, 126.8, 128.5, 131.5, 162.3;
HRMS (ESI) calcd for C7H9N3NaO2 [M+Na]+ 190.0587, found
190.0583.
4.2.3. Enamide 19. To a solution of phosphinophenol 24 (24.0
mg, 0.0749 mmol) in THF (0.50 mL) at room temperature was
added vinyl azide 18 (15.1 mg, 0.107 mmol) in THF (0.62 mL)
by cannula under Ar. After 10 min, H2O (0.38 mL) was added to
the mixture, which was stirred overnight at room temperature.
The solvent was then removed under reduced pressure, and the
residue was purified by flash column chromatography
(hexane/EtOAc = 10/1 to 2/1) to afford 19 (10.4 mg, 88%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) δ 1.35 (3H, t, J =7.3 Hz),
2.13 (3H, s), 4.29 (2H, q, J = 7.3 Hz), 5.88 (1H, d, J = 1.4 Hz),
6.58 (1H, s), 7.74 (1H, brs); [CAS 23115-42-6].
4.2.7. Enamide 5. Azide 31 (11.0 mg, 0.0658 mmol) and
phosphinophenol 28 (22.5 mg, 0.0500 mmol) were placed in 2-
necked round bottom flask equipped with reflux condenser.
Dioxane (4.95 mL) and H2O (0.05 mL) were successively added
and inner air was purged with Ar. This solution was refluxed at
100 °C for 3 h. After cooled to room temperature, the solvent
was removed under reduced pressure. The residue was dissolved
in Et2O (15 mL), washed with 1 M aqueous NaOH (10 mL),
saturated aqueous NH4Cl (10 mL x 2) and brine (10 mL) (in
order to remove diphenylphosphinyl phenol). The organic layer
was dried over Na2SO4 and filtered. Concentration and flash
column chromatography (hexane/EtOAc = 10/1 to 1/2) afforded
5 (11.6 mg, 74%) as a white solid: [α]D25 -42 (c 0.60, CHCl3); 1H
NMR (400 MHz, CDCl3) δ 1.41 (3H, d, J = 6.9 Hz), 1.43 (9H, s),
1.75 (3H, d, J = 7.3 Hz), 4.28-4.33 (1H, m), 4.63 (2H, dt, J = 6.0,
1.4 Hz), 5.16 (1H, d, J = 7.3 Hz), 5.22 (1 H, ddt, J = 10.5, 1.4,
1.4 Hz), 5.33 (1H, ddt, J = 17.4, 1.4, 1.4 Hz), 5.90 (1H, ddt, J =
17.4, 10.5, 6.0 Hz), 6.83 (1H, q, J = 7.3 Hz), 7.67 (1H, brs);
[CAS 929112-05-0].
4.2.4. Phosphinophenol 28.
To a solution of 26 (373 mg, 1.34mmol) in CH2Cl2 (6.7 mL) was
added N-Boc-L-alanine (27) (421 mg, 2.23 mmol), DCC (462
mg, 2.24 mmol), and DMAP (1 portion) at room temperature.
After being stirred for 2.5 h, the reaction mixture was filtered
through a pad of celite. The solution was concentrated, and the
residue was purified with flash column chromatography (CH2Cl2)
to afford phosphinophenol 28 (568 mg, 1.26 mmol, 94%) as a
white foam: [α]D20 = -32.9 (c 0.15, CHCl3); IR (film) 2978, 1770,
1
1715, 1507, 1435, 1365, 1249, 1157, 1065, 746 cm-1; H NMR
(400 MHz, CDCl3) δ 1.26 (3H, d, J = 7.3 Hz), 1.45 (9H, s), 4.37

6
Tetrahedron
(Inertsil ODS-4 10×250 mm) with 37% MeCN containing 0.1%
ACCEPTED MANUSCRIPT
TFA to afford 2 (4.0 mg, 4.78 µmol, 31% from 5) as a white
4.3. Experimental procedures for Scheme 5.
23
solid: [α]D -15.6 (c 0.02, MeOH); IR (film) 3284, 2935, 1677,
1631, 1526, 1206, 1138 cm-1; H NMR (500 MHz, DMSO-d6)
1
4.3.1. Peptide 6. Preloaded resin Fmoc-L-Val-Wang-resin (7)
was purchased from Watanabe. Loading rate of Fmoc-
determined as follows:
and 13C NMR (125 MHz, DMSO-d6) data, see Table S1 in
Supplementary Material; HRMS (ESI) calcd for C43H61N7NaO10
[M+Na]+ 858.4372, found 858.4377.
L-Val was
To the dried resin (5.4 mg) was added 20% piperidine in DMF
(0.5 mL), and stirred for 30 min. The supernatant was diluted
with DMF (15 mL), and subjected to UV measurement at 301 nm.
The loading rate was determined to be 0.71 mmol/g from the
observed absorbance (1.999).
Fmoc-
cycles (Fmoc-
OH, and Fmoc-
protocol described above.
pentapeptide was deprotected via Step 1 ~ 2, and then treated
with 25% (EtCO)2O/CH2Cl2 solution (2.0 mL). After being
stirred for 5 min, the reaction vessel containing the resin was
washed with CH2Cl2 (× 5), DMF (× 5), and CH2Cl2 (× 5) to
afford resin-bound peptide 33.
4.4. Experimental procedures for Scheme 6.
4.4.1. Peptide 36. Preloaded resin Fmoc-L-Val-Wang-resin (7)
was purchased from Watanabe. Loading rate of Fmoc-
determined as follows:
L
-Val-Wang-resin (7, 0.200 mmol) was subjected to 4
-allo-Thr-OH, Fmoc- -Phe-OH, Fmoc- -Leu-
-Phe-OH) of the microwave assisted SPPS
Obtained Fmoc-protected
L-Val was
D
L
D
D
To the dried resin (4.7 mg) was added 20% piperidine in DMF
(0.5 mL), and stirred for 30 min. The supernatant was diluted
with DMF (15 mL), and subjected to UV measurement at 301 nm.
The loading rate was determined to be 0.66 mmol/g from the
observed absorbance (1.616).
Fmoc-
cycles (Fmoc-
L-Val-Wang-resin (7, 0.100 mmol) was subjected to 3
D
-allo-Thr-OH, Fmoc- -Phe-OH, and Fmoc-D-
L
To peptide 33 in Libra Tube was added 95% aqueous TFA
solution. After being stirred for 40 min, the reaction mixture was
filtered and washed with cleavage solution 3 times. The filtrate
was concentrated to afford crude peptide 6 and the residue was
dissolved in DMSO, and purified by reversed-phase HPLC
(Inertsil ODS-4 10×250 mm) with 45% 1-PrOH containing 1%
AcOH to afford peptide 6 (66.8 mg, 0.0980 mmol, 49% from 7)
as a white solid: [α]D21 -10 (c 0.87, MeOH); IR (film) 3290, 3061,
Phe-OH) of microwave assisted SPPS protocol described above.
Obtained Fmoc-protected tetrapeptide was deprotected via Step 1
~ 2 and added 25% Ac2O/CH2Cl2 solution (1.5 mL). After being
stirred for 5 min, the reaction vessel containing the resin was
washed with CH2Cl2 (× 5), DMF (× 5), and CH2Cl2 (× 5) to
afford resin-bound peptide 36.
2963, 2875, 1721, 1647, 1535, 1458, 1387 cm-1; H NMR (500
1
4.4.2. Peptide 38. To peptide 36 were added DCC (0.300 mmol),
DMAP (0.0100 mmol) and acid 30 (0.300 mmol) in THF/DMF =
2/1 (1.5 mL). After being stirred at 40 °C assisted with
microwave energy 100 W for 40 min, the reaction vessel
containing the resin was washed with CH2Cl2 (× 5), DMF (× 5),
and CH2Cl2 (× 5) to afford resin-bound peptide 38.
MHz, (CD3)2SO) δ 0.71 (3H, d, J = 6.0 Hz), 0.73 (3H, J = 6.4
Hz), 0.82-0.88 (9H, m), 1.34 (3H, d, J = 6.4 Hz), 1.15-1.19 (3H,
m), 1.96-2.08 (3H, m), 2.66-2.76 (2H, m), 2.95 (1H, dd, J = 13.7,
3.7 Hz), 3.06-3.09 (1H, m), 3.80-3.84 (1H, m), 4.19-4.23 (2H, m),
4.41 (1H, dd, J = 7.8, 7.8 Hz), 4.47-4.52 (1H, m), 4.61-4.65 (1H,
m), 4.75 (1H, brs), 7.14-7.30 (10H, m), 7.90-7.94 (3H, m), 8.13
(1H, d, J = 8.7 Hz), 8.31 (1H, d, J = 8.7 Hz); 13C NMR (125 MHz,
(CD3)2SO) δ 9.9, 18.0, 19.2, 19.9, 22.0, 22.9, 23.9, 28.4, 30.3,
37.3, 37.9, 41.2, 51.3, 53.7, 54.2, 57.2, 58.3, 67.3, 126.2, 128.0,
129.3, 129.4, 138.0, 138.2, 170.1, 171.3, 171.4, 171.8, 173.05,
173.08; HRMS (ESI) calcd for C36H51N5NaO8 704.3630 [M+Na]+,
found 704.3635.
This coupling reaction was repeated twice to complete the
reaction.
4.4.3. Peptide 39. To peptide 38 was added 39 (0.300 mmol) in
NMP/H2O = 99/1 (2.0 mL). After being stirred at 60 °C assisted
with microwave energy 100 W for 3 h, the reaction vessel
containing the resin was washed with DMF (× 5) and CH2Cl2 (×
5) to afford resin-bound peptide 39.
4.3.2. Nobilamide B (2). To a solution of enamide 5 (4.8 mg, 15.4
µmol) in CH2Cl2 (0.48 mL) was added TFA (0.12 mL) at 0 °C,
and the resultant solution was stirred at room temperature for 1.5
h. The reaction mixture was concentrated to afford crude amine
34, which was used in the next reaction without further
purification.
To a solution of 34 in CH2Cl2/DMF = 2/1 (0.30 mL) were added
peptide 6 (10.5 mg, 15.4 µmol), NaHCO3 (2.6 mg, 31.0 µmol),
HOAt (4.4 mg, 32.3 µmol), and EDCI (6.0 mg, 31.3 µmol) at
0 °C. The reaction mixture was stirred for 6 h. The reaction
mixture was diluted with EtOAc, washed with 1 M aqueous HCl,
saturated aqueous NaHCO3, H2O, and brine, and dried over
Na2SO4. The solution was concentrated to afford crude peptide
35, which was used in the next reaction without further
purification.
To a solution of 35 in CH2Cl2 (1.5 mL) wer added PhSiH3 (19.1
µL, 155 µmol), and Pd(PPh3)4 (18.0 mg, 15.6 µmol) at room
temperature. After being stirred for 6 h, the reaction mixture was
quenched with saturated aqueous NH4Cl, and separated. The
aqueous layer was acidified with 1 M aqueous HCl, extracted
with EtOAc/t-BuOH = 9/1, washed with brine, and dried over
Na2SO4. The solution was concentrated, the residue was
dissolved in DMSO, and purified by reversed-phase HPLC
4.4.4. Nobilamide D (4). To peptide 39 in Libra Tube was added
95% aqueous TFA solution. After being stirred for 40 min, the
reaction mixture was filtered and washed with cleavage solution
3 times. The filtrate was concentrated to afford crude peptide 8,
which was used in the next reaction without further purification.
To a solution of 8 in CH2Cl2/DMF = 9/1 (50 mL) was added
HOAt (27.1 mg 0.199 mmol), PyBOP (104 mg, 0.200 mmol),
and 2,4,6-collidine (53 µL, 0.402 mmol) at 0 °C. The reaction
mixture was allowed to warm to room temperature and stirred for
12 h. The solution was concentrated, and the residue was diluted
with EtOAc/t-BuOH = 9/1 and brine, washed with saturated
aqueous NaHCO3, 1 M HCl, brine, and dried over Na2SO4. The
solution was concentrated, the residue was dissolved in DMSO,
and purified by reversed-phase HPLC (Inertsil ODS-4 10×250
mm) with 37% MeCN containing 0.1% TFA to afford
nobilamide D (4) (14.1 mg, 0.0204 mmol, 20% from 7) as a
22
white solid: [α]D -66.3 (c 0.03, MeOH); IR (film) 3272, 2969,
1
1683, 1663, 1640, 1546, 1509, 1270 cm-1; H NMR (500 MHz,
DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table

7
S2 in Supplementary Material; HRMS (ESI) calcd for
ACCEPTED MANUSCRIPT
C36H46N6NaO8 [M+Na]+ 713.3269, found 713.3281.
Supplementary Material
Supplementary material data associated with this article can
be found, in the online version, at xxxx.
Acknowledgments
This work was supported financially by Funding Program for
Next Generation World-Leading Researchers [Japan Society for
the Promotion of Science (JSPS)] to M.I. and a Grant-in-Aid for
Young Scientists (B) (JSPS) to T.K.
References and notes
1. For reviews on TRPV1, see: (a) Premkumar, L. S.; Sikand, P. Curr.
Neuropharmacol. 2008, 6, 151; (b) Wu, L. J.; Sweet, T. B.; Clapham, D.
E. Pharmacol. Rev. 2010, 62, 381; (c) Iadarola, M. J.; Mannes, A. J.
Curr. Top. Med. Chem. 2011, 11, 2171.
2. (a) Szallasi, A.; Appendino, G. J. Med. Chem. 2004, 47, 2717; (b) Wong,
G. Y.; Gavva, N. R. Brain Res. Rev. 2009, 60, 267; (c) Kissin, I.;
Szallasi, A. Curr. Top. Med. Chem. 2011, 11, 2159.
3. Lin, Z.; Reilly, C. A.; Antemano, R.; Hughen, R. W.; Marett, L.;
Concepcion, G. P.; Haygood, M. G.; Olivera, B. M.; Light, A.; Schmidt,
E. W. J. Med. Chem. 2011, 54, 3746.
4. Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328.
5. Fmoc Solid Phase Peptide Synthesis - A Practical Approach; Chan, W.
C.; White, P. D. Eds.; Oxford University Press: Oxford, 2000.
6. For reviews on synthesis of didehydroamino acids, see: (a) Hamphrey, J.
M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243; (b) Bonauer, C.;
Walenzyk, T.; König, B. Synthesis 2006, 1.
7. β-Elimination has been frequently used for synthesis of didehydroamino
acid synthesis. For examples, see: (a) Stohlmeyer, M. M.; Tanaka, H.;
Wandless, T. J. J. Am. Chem. Soc., 1999, 121, 6100; (b) Sai, H.; Ogiku,
T.; Ohmizu, H. Synthesis, 2003, 201.
8. Kuranaga, T.; Sesoko, Y.; Sakata, K.; Maeda, N.; Hayata, A.; Inoue, M.
J. Am. Chem. Soc. 2013, 135, 5467.
9. For reviews on Cu-mediated enamide formation, see: (a) Evano, G.;
Blanchard, N.; Toumi, M. Chem. Rev. 2008, 108, 3054; (b) Ma, D.; Cai,
Q. Acc. Chem. Res. 2008, 41, 1450; (c) Surry, D. S.; Buchwald, S. L.
Chem. Sci. 2010, 1, 13; (d) Evano, G.; Theunissen, C.; Pradal, A. Nat.
Prod. Rep., 2013, 30, 1467. (e) Kuranaga, T.; Sesoko, Y.; Inoue, M. Nat.
Prod. Rep., 2014, 31, 514.
10. For an account, see: Mcgrath, N. A.; Raines, R. T. Acc. Chem. Res. 2011,
44, 752.
11. (a) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2,
1939; (b) Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2001,
3, 9.
12. Saxon, E.; Armstrong, J. I.; Bertozzi, C. R. Org. Lett. 2000, 2, 2141.
13. Representative works by Bertozzi and Ashfeld on the Staudinger ligation,
see: (a) Kiick, K. L.; Saxon, E.; Tirrell, D. A,; Bertozzi, C. R. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 19; (b) Lemieux, G. A.; de Graffenried, C. L.;
Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 4708; (c) Hang, H. C.; Yu,
C.; Pratt, M. R.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126, 6; (d)
Fernández-Suárez, M.; Baruah, H.; Martinez-Hernández, L.; Xie, K. T.;
Baskin, J. M.; Bertozzi, C. R.; Ting, A. Y. Nat. Biotechnol. 2007, 25,
1483; (e) Kosal, A. D.; Wilson, E. E.; Ashfeld, B. L. Chem. Eur. J. 2012,
18, 14444; (f) Kosal, A. D.; Wilson, E. E.; Ashfeld, B. L. Angew. Chem.,
Int. Ed. 2012, 51, 12036.
14. Kakimoto, M.; Kai, M.; Kondo, K. Chem. Lett. 1982, 525.
15. (a) Bacsa, B.; Horváti, K.; Bõsze, S.; Andreae, F.; Kappe, C. O. J. Org.
Chem. 2008, 73, 7532; (b) Yu, H.-M.; Chen, S.-T.; Wang, K.-T. J. Org.
Chem. 1992, 57, 4781.
16. Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Ziodrou, C. Synthesis
1984, 572.
17. Carpino, L. A. J. Am. Chem. Soc.1993, 115, 4397.
18. Dessolin, M.; Guillerez, M.-G.; Thieriet, N.; Guibé, F.; Loffet, A.
Tetrahedron Lett. 1995, 36, 5741.
19. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205.
20. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591.
21. See Supplementary Material for details.

ACCEPTED MANUSCRIPT
Supporting Information
Total syntheses of nobilamide B and D:
application of traceless Staudinger ligation
Tomoya Yamashita, Hiroaki Matoba, Takefumi Kuranaga, Masayuki Inoue*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
E-mail: inoue@mol.f.u-tokyo.ac.jp
18 pages
1H and 13C NMR chemical shifts of natural and synthetic nobilamide B
1H and 13C NMR chemical shifts of natural and synthetic nobilamide D
MS/MS spectrum of synthetic nobilamide B
S2
S3
S4
Marfey’s analysis of synthetic nobilamide B and D
1H and 13C NMR spectra of synthesized compounds
S5–S6
S7–S18
S1

ACCEPTED MANUSCRIPT
Table S1. 1H (500 MHz) and 13C (125 MHz) NMR chemical shift (ppm) for nobilamide B (2) in
DMSO-d6.
natural 2
synthetic 2
a
b
pos
δH
δC
δH
δC
∆Abu
NH
1
9.04
9.04
166.0
128.5
132.6
14.2
166.0
128.0
132.4
13.6
2
3
6.58
1.64
8.19
6.55
1.62
8.18
4
L
L
-Ala
-Val
NH
1
171.4
48.7
18.3
171.0
48.0
18.4
2
4.43
1.31
7.75
4.40
1.28
7.74
3
NH
1
171.0
57.7
31.1
19.5
18.3
170.8
57.3
32.0
19.4
18.1
2
4.30
2.03
0.88
0.86
8.14
4.28
2.00
0.87
0.83
8.13
3
4
5
D
-allo-Thr
NH
1
170.2
59.1
67.7
20.4
169.9
59.1
67.4
20.1
2
4.36
3.83
1.07
8.28
4.35
3.80
1.04
8.32
3
4
L
-Phe
NH
1
171.8
54.0
171.0
54.3
2
4.59
4.61
3
3.06/2.71
38.2
3.08/2.72
38.0
4
138.6
137.8
Ph
NH
1
7.02-7.30
7.92
126.7, 128.4, 129.7 7.14-7.31 126.0, 128.6,129.4
7.96
D
-Leu
-Phe
172.2
51.9
41.3
26.3
22.8
22.6
171.6
51.3
41.1
24.1
22.8
22.1
2
4.20
1.15
1.16
0.73
0.70
7.91
4.21
1.16
1.16
0.75
0.72
7.95
3
4
5
6
D
NH
1
171.6
54.5
172.0
53.7
2
4.48
4.50
3
2.93/2.69
37.7
2.94/2.69
37.4
4
138.5
137.8
Ph
1
7.00-7.30
128.0, 129.0, 126.7 7.14-7.31 128.6,129.4, 126.0
N-terminal
173.4
28.6
10.3
173.1
27.8
9.9
2
1.98
0.82
2.00
0.85
3
aChemical shifts were calibrated with the 9.04 ppm of NH in ∆Abu; bChemical shifts were calibrated
with the 166.0 ppm of C1 in ∆Abu.
S2

ACCEPTED MANUSCRIPT
Table S2. 1H (500 MHz) and 13C (125 MHz) NMR chemical shift (ppm) for nobilamide D (4) in
DMSO-d6.
natural 4
synthetic 4
b
a
pos
δH
δC
δH
δC
∆Abu
NH
1
8.29
8.29
163.2
126.0
133.8
15.3
163.2
126.5
134.4
15.5
2
3
6.71
1.64
7.94
6.71
1.64
7.96
4
L
L
-Ala
-Val
NH
1
170.1
50.0
17.9
170.1
50.4
18.2
2
4.28
1.30
7.51
4.27
1.29
7.53
3
NH
1
171.3
61.4
28.6
19.5
19.4
171.3
61.7
29.5
20.2
19.9
2
3.89
1.94
0.85
0.75
8.47
3.89
1.92
0.83
0.74
8.50
3
4
5
D
-allo-Thr
NH
1
168.1
57.6
73.9
17.9
167.8
58.0
74.0
18.2
2
4.15
4.72
1.36
8.42
4.15
4.72
1.35
8.45
3
4
L
-Phe
NH
1
171.9
55.0
171.7
54.6
2
4.47
4.46
3
3.05/2.84
38.2
3.04/2.83
38.1
4
137.7
137.9
Ph
NH
1
7.07-7.29
7.92
126.5, 128.0, 129.1 7.01-7.29 126.5, 128.1, 129.6
7.93
D
-Phe
172.0
54.6
170.8
53.7
2
4.48
4.48
3
2.74/2.52
37.6
2.72/2.51
38.5
4
137.4
137.9
Ph
1
7.07-7.29
1.70
126.2, 128.0, 129.0 7.01-7.29 126.5, 128.1, 129.6
N-terminal
169.9
22.9
169.7
23.2
2
1.70
aChemical shifts were calibrated with the 8.29 ppm of NH in ∆Abu; bChemical shifts were calibrated
with the 163.2 ppm of C1 in ∆Abu.
S3

ACCEPTED MANUSCRIPT
LC-MS/MS(ESI) spectrum of synthetic nobilamide B
Phe
Leu
317
Phe
464
Thr
Val
Ala
∆Abu
565
664
735
O
O
O
O
H
H
H
N
N
N
OH
N
N
N
N
H
H
H
H
O
O
O
O
OH
633
(+2H)
520
(+2H)
373
(+2H)
836
[M+H]+
S4

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Marfey’s analysis of synthetic nobilamides B (2) and D (4)
One portion of compound 2 or 4 in 6 M HCl (200 µL) was heated at 110 °C overnight. The
solution was concentrated and added H2O (100 µL), saturated aqueous NaHCO3 (120 µL), and 1%
L-FDAA/acetone solution (100 µL). After being stirred at 45 °C for 1 h, the reaction mixture was
added 6 M HCl (20 µL) and concentrated, the residue was dissolved in DMSO (150 µL), and
analyzed with reversed-phase UHPLC (Jasco X-LC system; column: kinetex 2.6µ C18 2.1×150 mm)
MeCN 10-50% with 0.05% TFA (1-17 min).
Standard sample
Compound 2
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Compound 4
D-Phe
L-Phe
L-Ala
L-Val
D-allo-Thr
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O
BocHN
OEt
N
H
O
14
O
BocHN
OEt
N
H
O
14
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O
Ph2P
S
O
OEt
N
H
O
20
O
Ph2P
S
O
OEt
N
H
O
20
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O
BocHN
O
PPh2
28
O
BocHN
O
PPh2
28
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COSY spectrum of synthetic 2
HSQC spectrum of synthetic 2
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HMBC spectrum of synthetic 2
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COSY spectrum of synthetic 4
HSQC spectrum of synthetic 4
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HMBC spectrum of synthetic 4
S18