Investigation for the cyclization efficiency of linear tetrapeptides: Synthesis of tentoxin B and dihydrotentoxin

Article abstract of DOI:10.1016/j.tet.2018.09.011

Investigation of the cyclization efficiency of N-methyl linear tetrapeptides using a molecular modeling study and chemical synthesis is described. The linear peptide with two N-methyl groups, MeAla-Leu-MePhe-Gly, forms γ-turn like conformation with the am

Full text of DOI:10.1016/j.tet.2018.09.011

Accepted Manuscript
Investigation for the cyclization efficiency of linear tetrapeptides: Synthesis of tentoxin
B and dihydrotentoxin
Ryota Sato, Kie Oyama, Hiroyuki Konno
PII:
S0040-4020(18)31066-4
10.1016/j.tet.2018.09.011
TET 29783
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 June 2018
Revised Date: 28 August 2018
Accepted Date: 4 September 2018
Please cite this article as: Sato R, Oyama K, Konno H, Investigation for the cyclization efficiency of
linear tetrapeptides: Synthesis of tentoxin B and dihydrotentoxin, Tetrahedron (2018), doi: 10.1016/
j.tet.2018.09.011.
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Investigation for the cyclization efficiency of
Linear tetrapeptides: Synthesis of tentoxin B
and dihydrotentoxin
Ryota Sato, Kie Oyama, Hiroyuki Konno*
Graduate School of Science and Engineering, Yamagata University

ACCEPTED MATNeUtraSheCdrRonIPT
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Investigation for the cyclization efficiency of linear tetrapeptides: Synthesis of
tentoxin B and dihydrotentoxin
Ryota Sato, Kie Oyama and Hiroyuki Konno*
Department of Biochemical Engineering, Graduate School of Science and Engineering,
Yamagata University, Yonezawa, Yamagata 992-8510, Japan
TEL&FAX: +81-238-26-3131
*Corresponding author
E-mail address: konno@yz.yamagata-u.ac.jp
Keywords: cyclic tetrapeptide, unusual amino acid, Fmoc-SPPS
ABSTRACT: Investigation of the cyclization efficiency of N-methyl linear
tetrapeptides using a molecular modeling study and chemical synthesis is described.
The linear peptide with two N-methyl groups, MeAla-Leu-MePhe-Gly, forms γ-turn like
conformation with the amine at N-terminus and the carbonyl at C-terminus in closer
proximity to give the desired cyclic tetrapeptide, dihydrotentoxin. In addition, synthesis
of tentoxin B by the combination of Fmoc solid-phase peptide synthesis and cyclization
in solution phase has been reported. An unusual amino acid, an
L-N-methyl-β-hydroxyphenylalanine derivative, which was assembled on solid support,
was prepared from ethyl cinnamate. Cyclic tetrapeptide formation and cleavage of
benzyl ether were optimized with DIPCI/HOBt/DIPEA and Et3SiH/Pd(OH)2,
respectively.
Introduction
Cyclic tetrapeptides are 12-membered cyclic molecules, which are expected
to have a variety of biological activities and functionalities, since cyclic tetrapeptides
have a rigid framework and fixed trajectory of the side chains of amino acid residues.
For instance, apicidin A and apicidin are members of a rare class of cyclic tetrapeptide
fungal metabolites of Fusarium pallidoroseum that have been shown to exhibit broad
spectrum cidal activity.1,2,3 Trapoxins A and B isolated from the culture broth of
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Helicoma ambiens RF-1023 are also epoxide-containing cyclic tetrapeptides. These
compounds can be attributed to histone hyperacetylation resulting from the inhibition of
histone deacetylase.4,5 However, it is difficult to obtain the desired cyclic tetrapeptides
without restriction, because treatment of linear tetrapeptide precursors with coupling
reagents proceeds to give the desired cyclic tetrapeptides generally in competition with
those leading to the formation of the corresponding cyclic octapeptides.6 The effective
cyclization of the corresponding linear peptides depends on the sequence of amino acids.
Therefore, natural products are role models to understand the cyclization potential.
Tentoxin B (1) was isolated from the fungus Phoma sp. derived from the giant
jellyfish Nemopilema nomurai by Jung et al. in 2012.7 Tentoxin B (1) is a cyclic
tetrapeptide
consisting
-N-methyl-β-hydroxyphenylalanine (MePhe(βOH)),
-leucine (Leu) and glycine (Gly). The chemical structure of tentoxin B (1) was
of
a
non-proteiogenic
amino
acid
L
L
L-N-methylalanine (MeAla),
determined by a combination of 2D-NMR experiments, Marfey’s method and the
modified Mosher rule. Tentoxin B (1) displays a weak suppressive effect on the
production of nitric oxide (NO) in murine macrophage cells without notable
cytotoxicity.7 As a pioneer work, Rich reported synthesis of tentoxin, dehydrated
product of tentoxin B (1), and conformational study by NMR in 1978.8,9 In this paper,
conformational analyses of various dehydro- and normal-cyclic tetrapeptides were
described in detail. On the other hand, dihydrotentoxin (2) was isolated from Alternaria
alternate by the Tresselt group.10 This was the first time that this cyclic peptide was
isolated as a natural substance in 1988 though its chemical synthesis has already
performed to show the crystal structure.11 The sequence of dihydrotentoxin (2),
cyclo[MeAla-Leu-MePhe-Gly], was confirmed by X-ray crystallographic analysis to
possess a cis-trans-cis-trans conformation with N-methyl peptide units in the trans
locations and two secondary amide groups in the cis conformation. This result identified
with Loiseau’s work,12 which is the prediction of conformational states of cyclic
tetrapeptides. As we were interested in tentoxin B (1) and related cyclic tetrapeptides,
synthetic study of tentoxin B (1) is unknown in the literature. A cis-trans-cis-trans
conformation of dihydrotentoxin (2) suggests a rigid framework and fixed trajectory and
therefore it is likely to cyclize the corresponding linear tetrapeptide with N-methyl
groups. In the present study, we report an investigation of the cyclization efficiency of
linear tetrapeptides, included dihydrotentoxin (2), with several N-methyl groups using a
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molecular modeling study and chemical synthesis and subsequently the first synthesis
of tentoxin B (1) (Fig. 1).
Fig. 1. Chemical structure of tentoxin B (1) and dihydrotentoxin (2)
Results and Discussion
Molecular modeling and conformational analysis can assist in identifying
linear precursor peptides that may adopt conformations that can proceed cyclization.13
Although conformational analyses of the cyclic tetrapeptides including tentoxin and
related compounds were investigated in detail, those of the linear peptide precursors had
rarely been attempted to the best of my knowledge. Therefore, we employed molecular
modeling to predict the linear sequences for cyclization to increase the probability of
cyclic tetrapeptide formation and attempted a molecular modeling study with the seven
linear precursors with N-methylamino acid residues by SPARTAN’14. The linear
peptides were designed as the sequence of Ala-Leu-Phe-Gly and using N-methylamino
acid residues in place of amino acid residues. Because these combination with linear
peptides 3-9 to analyze the conformation had never been reported. We adopted the Gly
residue as a C-terminus to avoid the epimerization of C-terminal residues during the
cyclization of chemical synthesis. The molecular mechanics calculation of the most
stable conformations of 3-9 were carried out using the SPARTAN’16 (conformation
distribution and MMFF). The chemical structures of 3-9 were drawn by Chem 3D Ultra
and located on the SPARTAN’16. The results shown in Table1 are derived from the
most stable conformations of 3-9. The second or third stable conformations of 3-9 were
similar results and therefore cyclization efficiency of the linear peptides was employed
by the conformational analyses. The results of the molecular modeling suggested that
the linear peptides were likely to be compatible with cyclization. The conformation of
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the linear tetrapeptides plays an important role in cyclization and accordingly
intramolecular hydrogen bonds can stabilize a folded conformation become favorable
for cyclization. Analysis of the linear peptide (3) with two N-methyl groups showed a
folded γ-turn like conformation with the amine at the N-terminus and carbonyl at the
C-terminus in closer proximity with a distance of about 3.8 Å (entry 1). In addition, the
linear peptides (4) and (5) with one N-methyl group formed a folded conformation by
three hydrogen bond networks (entries 2 and 3). A similar analysis of the linear peptides
(6) and (7) containing three N-methyl groups was also performed (entries 4 and 5). In
contrast, analysis of the linear peptides Ala-Leu-Phe-Gly (8) and
MeAla-MeLeu-MePhe-MeGly (9) suggested that it preferentially existed in an extended
conformation with a distance of 8.8 and 9.6 Å between the amino and carbonyl terminus
(entries 6 and 7). Based on these analyses, MeAla-Leu-MePhe-Gly (3) was expected to
efficiently give the designed cyclic tetrapeptide. These results indicated one of the
criteria for cyclization efficiency even if it is a semiempirical method in Spartan and
moreover those support conformational study of cyclic tetrapeptides (Table 1).
Table 1. Analysis of the conformations of the linear tetrapeptides using a molecular
modeling study
a
b
entry
Peptide sequence
compound hydrogen
bond
conformation
distance
R1(G) R2(F) R3(L)
R4(A)
Me
Me
H
(Å)
1
2
3
4
5
6
7
H
H
Me
H
H
H
4
3
3
2
3
2
0
3.8
4.8
5.2
5.6
6.9
8.8
9.6
3
4
5
6
7
8
9
γ-turn
γ-turn
H
Me
Me
Me
H
H
β-turn
H
Me
H
Me
Me
H
β-turn
Me
H
extended
extended
extended
H
Me
Me
Me
Me
a Preliminary assignment based on hydrogen bonding patterns.
b
The distance was measured from the carbonyl carbon of carbonyl terminal to the
amino terminal nitrogen.
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Conformational
analysis
of
selected
linear
tetrapeptides,
MeAla-Leu-MePhe-Gly (3) and MeAla-MeLeu-MePhe-MeGly (9), are shown in Fig. 2
(Fig. 2).
Fig. 2. Conformational analysis of the linear tetrapeptides MeAla-Leu-MePhe-Gly (3)
and MeAla-MeLeu-MePhe-MeGly (9) by SPARTAN’14; Conformer distribution,
molecular mechanics, MMFF were employed. Hydrogen bond: sky blue, the distance
between the amine and carbonyl in the terminus: yellow.
The linear peptides were synthesized on 2-chlorotrityl chloride (2-CTC)
resin (Table 2). As mentioned above, we selected the Gly residue as the C-terminus and
therefore Fmoc-Gly-OH or Fmoc-Sar-OH were anchored to the resin. Each
Fmoc-N-methylamino acid was prepared by the Aurelio’s protocol.14,15,16 After loading
of the first amino acid to the resin, synthesis of the linear peptides was performed using
standard Fmoc-SPPS. The crude linear peptides were cleaved from the resin with 20%
HFIP/CH2Cl2 followed by purification on RP-HPLC to give the designed linear
tetrapeptides 3-9 in 9-43% yields. Next, cyclization of the linear tetrapeptides was
performed in solution. The linear tetrapeptides 3-9 with DIPCI/HOBt/DIPEA in DMF
(10 mM) for 24 h were treated and then crude products were purified with preparative
HPLC to give the corresponding cyclic tetrapeptides (monomer), cyclic octapeptides
(dimer) and dodecapeptides (trimer). Predictably, the above-mentioned conditions were
used to cyclize MeAla-Leu-MePhe-Gly (3) to give cyclo[MeAla-Leu-MePhe-Gly] (2) in
40% yield. In this case, the cyclic octapeptide (dimer) (10) afforded in 15% yield (entry
1). In addition, treatment of the linear tetrapeptides 4-7 gave mixtures of monomer and
dimer products (entries 2-5). On the other hand, the cyclic tetrapeptide (19) was rarely
detected on HPLC as using the tetrapeptide Ala-Leu-Phe-Gly (8) (entry 6). Interestingly,
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cyclization of the four N-methyl tetrapeptide MeAla-MeLeu-MePhe-MeGly (9)
afforded the dimer (22) and the trimer (23) in 23% and 22% yields, respectively (entry
7). It is noted that a folded conformation with the N-methyl peptides and intramolecular
hydrogen bonds with the amide groups are essential elements for the cyclization process.
As expected from molecular modeling and conformational analysis, the two
N-methylated linear tetrapeptide would be converted to the designed cyclic tetrapeptide
(Table 2).
Table 2. Synthesis of the cyclic tetrapeptides
Entry
Linear
peptide (%)
3 (41)
Cyclic peptide (%)b
monomer
dimer
10 (15)
12 (10)
14 (17)
16 (23)
18 (33)
trimer
2 (40)a
11 (37)
13 (25)
15 (32)
17 (41)
-
-
-
-
-
-
1
2
3
4
5
6
7
4 (34)
5 (21)
6 (21)
7 (43)
8 (9)
19 (trace) 20 (20)
9 (23)
21 (trace) 22 (23) 23 (21)
a dihydrotentoxin (2)
bisolation yields
For an application of synthesis of a cyclic tetrapeptide natural product,
synthesis of tentoxin B (1) was attempted as depicted in Scheme 1. The linear
precursors 24-25 were prepared by Fmoc-SPPS starting from the Gly residue using
2-CTC resin followed by cleavage from the resin with 20% HFIP/CH2Cl2. It is possible
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to obtain the concern synthesized peptides by mild acidic condition from the 2-CT resin.
Macrolactamization of the linear peptides 24-25 with coupling reagents was attempted
to give tentoxin B (1) and its benzyl-protected derivative. Fmoc-N-MePhe(β-OH)-OH
(26) and its benzyl-protected compound (27) were synthesized from ethyl cinnamate
(28) (Scheme 1).
Scheme 1. Synthetic plan for tentoxin B (1)
Synthesis of Fmoc-N-MePhe(β-OH)-OH (26) is shown in Scheme 2.
Sharpless asymmetric dihydroxylation17 of ethyl cinnamate (28) using an AD mix-α
afforded the optical active diol in 86% yield and 94%ee and subsequently
chemoselective nosylation of the hydroxyl group at the α position gave sulfonate in
88% yield. After the exchange sulfonate to azide with stereo-inversion, Pd/C-catalyzed
hydrogenolysis in hydrogen atmosphere in the presence of (Boc)2O gave the ester (29)
in quantitative yield.18,19 Treatment of the ester (29) with LiAlH4 in refluxed THF
followed by Fmoc protection afforded the N-methylamino diol (30) in 85% over 2 steps.
Finally, primary alcohol selective oxidation of the N-methylamino diol (30) in the
presence of catalytic amount of DMN-AZADO20 successfully proceeded to give
Fmoc-N-MePhe(β-OH)-OH (26) in 62% yield (Scheme 2).
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Scheme 2. Synthesis of Fmoc-N-MePhe(β-OH)-OH (26).
The synthesis of tentoxin B (1) is shown in Scheme 3. Fmoc-Gly-OH was
linked onto 2-chlorotrityl resin (2-CT resin) by esterification with DIPEA and
subsequently removal of the Fmoc group by the treatment with 20% piperidine/DMF
afforded Gly-bounded resin. Coupling of Fmoc-N-MePhe(β-OH)-OH (26) with
HATU/HOAt followed by Fmoc deprotection afforded the dipeptide. The coupling
conditions of Fmoc-Leu-OH and Fmoc-MeAla-OH were needed for the optimization to
provide the linear tetrapeptide (24) after cleavage from the resin. In attempts with
several conditions, HATU/HOAt/DIPEA and subsequently PyBOP/HOBt/DIPEA
combinations as the coupling reagents were successful. The chemical yield of the linear
tetrapeptide (31) was 20% from the resin loading. It is noted that 24 was given in lower
yield when HATU/HOAt/DIPEA was selected instead of PyBOP/HOBt. HPLC profile
of the reaction mixture cleaved from resin showed 24 and several by-products. The
linear tetrapeptide (24) was cyclized under the condition of DIPCI/HOBt/DIPEA in
1
DMF for 20 h to give the cyclic tetrapeptide, tentoxin B (1) in 33% yield. The H and
13C NMR spectrum of synthetic 1 was identical to that of the natural product from the
Jung group.6 However, these results with low chemical yields were not satisfactory, so
we used the benzyl-protected derivative Fmoc-N-MePhe(β-OBn)-OH (27) for
Fmoc-SPPS and peptide cyclization shown in Scheme 4 and 5 (Scheme 3).
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Scheme 3. Synthesis of tentoxin B (1).
For the synthetic plan, we preferably selected the β-hydroxy ester (29) to
prepare the benzyl-protected derivative (27). After treatment of the ester (29) with
LiAlH4 in refluxed THF, Boc protection afforded the N-methyl diol (31) in 85% over 2
steps. To distinguish the two hydroxy groups of the N-methyl diol (31), primary and
secondary alcohols were protected as TBS and benzyl ethers, respectively. Subsequently,
TBS-deprotection with TBAF in THF afforded the alcohol (32) in 89% yield. After
successive deprotection of the Boc group with 4N HCl/AcOEt, the Fmoc protection
with Fmoc-OSu/Et3N in CH2Cl2 led to the Fmoc derivative in 81 % yield. Finally,
oxidation
of
free
primary
alcohol
with
Jones’
condition
gave
Fmoc-N-MePhe(β-OBn)-OH (27) in 87% yield (Scheme 4).
Scheme 4. Synthesis of Fmoc-N-MePhe(β-OBn)-OH (27).
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As depicted in Scheme 5, synthesis of the cyclic tetrapeptide (33) using
Fmoc-N-MePhe(β-OBn)-OH (27) was performed. The designed linear tetrapeptide (32)
containing N-MePhe(β-OBn) residue was synthesized by Fmoc-SPPS. Each coupling
condition for the peptide elongation was similar to those of synthesis of 24.
Consequently, chemical yield of the linear tetrapeptide (25) was 65% from resin loading.
In addition, cyclization of the linear tetrapeptide (25) with DIPCI/HOBt/DIPEA
smoothly proceeded to give the desired cyclic tetrapeptide (33) in 61% yield. As a result,
both chemical yields of the SPPS and the cyclization reaction were increased by using
the benzyl-protected derivative (27). Interestingly, the cyclic octapeptide was not
detected by HPLC or mass spectrometry (Scheme 5).
Scheme 5. Synthesis of cyclic tetrapeptide (33).
In a comparison of the HPLC profiles of reaction mixtures of cyclization
reactions at 20 h, the cyclization reaction of the linear tetrapeptide (24) afforded
tentoxin B (1) and several by-products. Although there were hardly shown dimeric and
trimeric cyclic peptides, many peaks were detected on HPLC (Fig. 3a). On the other
hand, treatment of the linear tetrapeptide (25) with DIPCI/HOBt/DIPEA clearly
afforded the corresponding cyclic tetrapeptide (33) (Fig. 3b). The benzyl protective
group of N-MePhe(β-OBn) residue was effective to give the desired cyclic tetrapeptide
(33) (Fig. 3).
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Fig. 3. Comparison of HPLC profiles of cyclization reactions. a) reaction mixtures of
the cyclization reaction from the linear peptide (24) to tentoxicin B (1) at 20 h. b)
reaction mixtures of the cyclization reaction from the linear peptide (25) to the cyclic
tetrapeptide (33) at 20 h. HPLC profiles with conditions of flow 1ml/min, 0-30 min
gradient from 10-90% MeCN/H2O with 1% TFA.
Finally, deprotection of the benzyl group of the cyclic tetrapeptide (33) was
optimized as depicted in Table 3. Treatment of the cyclic tetrapeptide (33) in the
presence of Pd/C or Pd(OH)2/C at hydrogen atmosphere has hardly reacted to yield the
desired tentoxin B (1) (entries 1 and 2). Using Pt/C as a catalyst, reduction of benzyl
group of the cyclic tetrapeptide (33) proceeded to give the cyclohexylmethyl derivative
(34) in 49% yield (entry 3). In addition, PtO2-catalyzed hydrogenation of the cyclic
tetrapeptide (33) afforded the cyclohexylmethyl derivative (34) and the
dicyclohexylmethyl derivative (35) in 57% and 20% yields, respectively (entry 4). Next,
we attempted Et3SiH21 as a hydrogen source. Treatment of the cyclic tetrapeptide (33)
with catalytic Pd(OH)2/C and Et3SiH (10 equiv.) in MeOH gave tentoxin B (1) in 24%
yield with the recovered starting material (33) in 70% yield (entry 5). By using Et3SiH,
deprotection of benzyl group of 33 preferentially proceeded and 34 and/or 35 were
rarely observed in this reaction condition. By screening of the solvent and temperature,
we confirmed MeOH/CH2Cl2 (1:1) and room temperature as the reasonable conditions
for hydrogenolysis of 33 (entry 6). The excess reagents, especially 100 equivalents of
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Et3SiH, may have accelerated this process (entry 7). As a result, tentoxin B (1) was
obtained by an Et3SiH-mediated reaction in catalytic Pd(OH)2/C and subsequent
purification of preparative RP-HPLC in 60% yield. Chemical structures of reductants 34
and 35 shown in Table 3 were anticipated by ESI-MS spectra (see supporting
information) (Table 3).
Table 3. Deprotection of benzyl group of cyclic tetrapeptide (35)
entry
catalyst
Pd/C
hydrogen
solvent
MeOH
products (%)a
1 (2) : 34 (1)
1
2
3
4
5
6
7
H2
Pd(OH)2/C
Pt/C
H2
H2
MeOH
1 (6) : 34 (1)
MeOH
1 (5) : 34 (49)
34 (57) : 35 (20)
1 (24)
PtO2
H2
MeOH
Pd(OH)2/C
Pd(OH)2/C
Pd(OH)2/C
Et3SiH (10 eq.)
Et3SiH (10 eq.)
Et3SiH (100 eq.)
MeOH
MeOH/CH2Cl2
MeOH/CH2Cl2
1 (27) : 35 (1)
1 (60) : 34 (5) : 35 (10)
aisolation yields
HPLC profile of the reaction mixture (Table 3, entry 7) and ESI-MS spectra
of the purified tentoxin B (1) are shown in Fig. 4. The characteristic ion peaks, m/z =
415.18 [M-OH]+, 433.18 [M+H]+, and 456.17 [M+Na]+ were detected by the
ESI-TOFMS spectra (positive mode) (Fig. 4).
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Fig. 4. (a) HPLC profile of deprotection of benzyl group (Table 3, entry 7). Reaction
mixture of the deprotection of benzyl group of the linear peptide (33). HPLC profiles
with conditions of flow 1 ml/min, 0-30 min gradient from 10-90% MeCN/H2O with 1%
TFA. (b) ESI-MS spectra of tentoxin B (1), [M-OH]+ m/z found 415.18, calcd. for
415.25, [M+H]+ m/z found 433.18, calcd. for 433.25, [M+Na]+ m/z found 456.17, calcd.
for 456.25.
The cytotoxicity of synthetic peptides 1-23 was performed against Hela cells by a
standard MTT assay using WST-8 protocols.22 Although all compounds at 200 µM
concentrations were evaluated against Hela cells RCM0007 (5000 cells), the cell death
ratio caused by these compounds was less than 10% and therefore showed practically no
cytotoxicity.
Conclusion
MeAla-Leu-MePhe-Gly (3) formed a γ-turn like conformation with the
amine at the N-terminus and carbonyl at the C-terminus in closer proximity using a
molecular modeling study and cyclization of 3, which efficiently gave
cyclo[MeAla-Leu-MePhe-Gly], dihydrotentoxin (2). We accomplished the first
synthesis of tentoxin B (1) by combination of Fmoc solid-phase peptide synthesis and
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cyclization in the solution phase. In this case, no dimer was detected. The two
N-methylated linear tetrapeptide is expected to lead to the designed cyclic tetrapeptide.
Experimental
General. All solvents were reagent grade. Melting points were determined on a AS
ONE ATM-02. Optical rotations were determined with a JASCO DIP-371 polarimeter at
the sodium D line. IR spectra of sample were obtained as films (NaCl) with a HORIBA
1
FT-720 spectrometer. H (400, 500 and 600 MHz) and 13C NMR (100, 125 and 150
MHz) were determined on a JNM-ECX400, JNM-ECX500 and ECZ600R/M1.
Chemical shifts are reported in ppm with reference to tetramethylsilane [1H NMR: TMS
(0.00)], or solvent signals [1H NMR: CDCl3 (7.26), MeOH-d4 (3.30); 13C NMR: CDCl3
(77.16), MeOH-d4 (50.05)]. Mass spectra were obtained using JEOL AccuTOF
JMS-T100LC (ESI-MS). Analytical TLC was performed on Merck Silica gel 60F254.
Crude products were purified by column chromatography on Silica Gel 60N [Kanto,
particle size, (spherical, neutral) 63-210 µm or 100-200 µm]. Analytical HPLC was
carried out with a COSMOSIL 5C18-AR-II column (4.6ID×150 mm) with a linear
gradient of MeCN (0.1% TFA) in H2O (0.1% TFA) at a run time of 30 min (flow rate of
1 ml/min), on a SHIMADZU SPD-10A as a UV-Vis detector, HITACHI L-6000 Pump
and HITACHI L-6200 Intelligent Pump. Preparative HPLC was performed with a
COSMOSIL 5C18-AR-II column (10ID x 250 mm) with a linear gradient of MeCN
(0.1% TFA) in H2O (0.1% TFA) at a run time of 30 min (flow rate of 2 mL/min), on a
SHIMADZU SPD-10Ai as a UV-Vis detector and HITACHI L-6200 Intelligent Pump.
UV measurements were recorded at a wavelength of 220 nm for the peptide analyses.
Absorbance for cytotoxicity assay was measured with the CORONA MTP-310Lab.
H-MeAla-Leu-MePhe-Gly-OH (3): To 2-chlorotrityl chloride resin (50.3 mg, 80 µmol)
pre-swollen in DMF for 30 min, was added a solution of Fmoc-Gly-OH (48.0 mg, 161
µmol) and iPr2EtN (42 µL, 241 µmol) in DMF (2 mL). The reaction mixture was gently
agitated at room temperature for 2 h. The resin was filtered and washed with DMF. The
peptidyl resin was treated with a solution of 20% piperidine in DMF and the mixture
agitated at room temperature for 30 min. The resin was filtered and washed with DMF.
To the peptidyl resin was added a mixture of Fmoc-N-MePhe-OH (96.9 mg, 241 µmol),
PyBOP (126 mg, 242 µmol), HOBt·H2O (37.0 mg, 242 µmol) and DIPEA (56 µL, 321
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µmol) in DMF (2 mL). The reaction mixture was agitated at room temperature for 2 h,
after which the resin was filtered and washed with DMF. The peptidyl resin was treated
with a solution of 20% piperidine in DMF and the mixture agitated at room temperature
for 30 min. The resin was filtered and washed with DMF. To the peptidyl resin was
added a mixture of Fmoc-Leu-OH (85.5 mg, 242 µmol), HATU (91.8 mg, 241 µmol),
HOAt (33.2 mg, 244 µmol) and DIPEA (56 µL, 321 µmol) in DMF (2 mL). The
reaction mixture was agitated at room temperature for 2 h, after which the resin was
filtered and washed with DMF. The peptidyl resin was treated with a solution of 20%
piperidine in DMF and the mixture agitated at room temperature for 30 min. The resin
was filtered and washed with DMF. To the peptidyl resin was added a mixture of
Fmoc-N-MeAla-OH (79.1 mg, 243 µmol), HATU (93.0 mg, 245 µmol), HOAt (33.4 mg,
245 µmol) and DIPEA (56 µL, 321 µmol) in DMF (2 mL). The reaction mixture was
agitated at room temperature for 2 h, after which the resin was filtered and washed with
DMF. The peptidyl resin was treated with a solution of 20% piperidine in DMF and the
mixture agitated at room temperature for 30 min. The resin was filtered and washed
with DMF. To the peptidyl resin was added 20% HFIP in anhydrous CH2Cl2 and
agitated at room temperature for 2 h. The mixture was filtered and purified by
preparative RP-HPLC to afford the linear tetrapeptide 3 (14.4 mg, 33.2 µmol, 41%) as
1
white fluffy flakes; H NMR (500 MHz, D2O): δ 7.12 (5H, m), 5.00 (1H, dd, J = 11.0,
5.0 Hz), 4.69 (1H, m), 4.60 (1H, m), 3.70 (3H, m), 3.17 (1H, m), 2.98 (1H, m), 2.87 (3H,
s), 2.50 (3H, s), 1.37 (3H, m), 1.25 (3H, d, J = 6.5 Hz), 0.74 (3H, d, J = 6.5 Hz), 0.72
13
(3H, d, J = 6.0 Hz). C NMR (125 MHz, D2O): δ 173.8, 173.1, 172.3, 168.9, 136.7,
129.1, 128.7, 127.0, 60.0, 57.0, 48.8, 41.2, 39.1, 33.2, 31.1, 30.5, 24.5, 22.4, 20.3, 15.6.
ESIHRMS (m/z): Calcd for C22H34N4NaO5 [M+Na]+ 457.2427; found 457.2425.
Dihydrotentoxin (2): To a solution of linear tetrapeptide 3 (6.2 mg, 14.3 µmol) in DMF
(1.4 ml) were added DIPCI (7 µl, 45.2 µmol), HOBt·H2O (11.3 mg, 42.4 µmol) and
DIPEA (10 µL, 57.4 µmol) and the mixture was stirred for 24h. The mixture was
purified by preparative RP-HPLC to afford the dihydrotentoxin 2 (2.4 mg, 5.6 µmol,
1
40%) as white fluffy flakes; H NMR (400 MHz, CDCl3): δ 7.69 (1H, br s), 7.31 (2H,
m), 7.29 (1H, m), 7.23 (2H, m), 7.16 (1H, brs), 4.92 (1H, dd, J = 13.6, 10.0 Hz), 4.28
(1H, d, J = 6.4 Hz), 3.68 (1H, d, J = 13.6 Hz), 3.52 (1H, d, J = 15.6 Hz), 2.96 (1H, m),
2.85 (3H, s), 2.80 (3H, s), 1.69 (1H, m), 1.56 (3H, d, J = 6.8 Hz), 1.40 (1H, m), 1.22
15

ACCEPTED MANUSCRIPT
(1H, m), 0.83 (3H, d, J = 6.0Hz), 0.79 (3H, d, J = 6.0Hz). 13C NMR (100 MHz, CDCl3):
δ 170.7, 172.2, 171.8, 170.2, 136.9, 129.0, 128.2, 127.2, 62.9, 57.2, 48.5, 44.5, 41.0,
34.4, 30.7, 30.1, 24.5, 22.6, 22.3, 15.6. IR (NaCl) νmaxcm-1: 3326, 2956, 1654, 1558,
1541, 1508, 1457, 1398, 1219. ESIHRMS (m/z): Calcd for C22H33N4O4 [M+H]+
417.2502; found 417.2502. The above-mentioned spectral data was identical to those of
the natural product from Tresselt group.7
H-MeAla-Leu-Phe-Gly-OH (4): white fluffy flakes; ESIHRMS (m/z): Calcd. for
C21H33N4O5 [M+H]+ 421.2451; found 421.2470.
H-Ala-Leu-MePhe-Gly-OH (5): white fluffy flakes; ESIHRMS (m/z): Calcd. for
C21H33N4O5 [M+H]+ 421.2451; found 421.2471.
H-MeAla-MeLeu-MePhe-Gly-OH (6): colorless oil; ESIHRMS (m/z): Calcd. for
C23H37N4O5 [M+H]+ 449.2764; found 449.2792.
H-MeAla-Leu-MePhe-MeGly-OH (7): colorless oil; ESIHRMS (m/z): Calcd. for
C23H36N4NaO5 [M+Na]+ 471.2583; found 471.2597.
H-Ala-Leu-Phe-Gly-OH (8): white fluffy flakes; ESIHRMS (m/z): Calcd for
C20H31N4O5 [M+H]+ 407.2294; found 407.2321.
H-MeAla-MeLeu-MePhe-MeGly-OH (9): colorless oil; ESIHRMS (m/z): Calcd. for
C24H39N4O5 [M+H]+ 463.2920; found 463.2902.
cyclo[MeAla-Leu-MePhe-Gly]2 (10): white fluffy flakes; ESIMS (m/z): Calcd for
C44H65N8O8 [M+H]+ 833.4925; found 833.4914.
cyclo[MeAla-Leu-Phe-Gly] (11): white fluffy flakes; ESIHRMS (m/z): Calcd. for
C21H31N4O4 [M+H]+ 403.2345; found 403.2352.
cyclo[MeAla-Leu-Phe-Gly]2 (12): as white fluffy flakes; ESIHRMS (m/z): Calcd. for
C42H61N8O8 [M+H]+ 805.4612; found 805.4593.
cyclo[Ala-Leu-MePhe-Gly] (13): white fluffy flakes; ESIHRMS (m/z): Calcd. for
C21H31N4O4 [M+H]+ 403.2345; found 403.2336.
cyclo[Ala-Leu-MePhe-Gly]2 (14): as white fluffy flakes; ESIHRMS (m/z): Calcd. for
C42H61N8O8 [M+H]+ 805.4612; found 805.4634.
cyclo[MeAla-MeLeu-MePhe-Gly] (15): white fluffy flakes; ESIHRMS (m/z): Calcd.
for C46H69N8O8 [M+H]+ 861.5238; found 861.5237.
cyclo[MeAla-Leu-MePhe-MeGly] (17): white fluffy flakes; ESIHRMS (m/z): Calcd.
for C23H35N4O4 [M+H]+ 431.2658; found 431.2643.
16

ACCEPTED MANUSCRIPT
cyclo[MeAla-Leu-MePhe-MeGly]2 (18): as white fluffy flakes; ESIHRMS (m/z): Calcd.
for C46H69N8O8 [M+H]+ 861.5238; found 861.5223.
cyclo[Ala-Leu-Phe-Gly] (19): white fluffy flakes; ESIHRMS (m/z): Calcd. for
C20H29N4O4 [M+H]+ 389.2189; found 389.2186.
cyclo[Ala-Leu-Phe-Gly]2 (20): as white fluffy flakes; ESIHRMS (m/z): Calcd. for
C40H56N8NaO8 [M+Na]+ 799.4119; found 799.4108.
cyclo[MeAla-MeLeu-MePhe-MeGly] (21): white fluffy flakes; ESIHRMS (m/z): Calcd.
for C24H36N4NaO4 [M+Na]+ 467.2634; found 467.2634.
cyclo[MeAla-MeLeu-MePhe-MeGly]2 (22): as white fluffy flakes; ESIHRMS (m/z):
Calcd. for C48H73N8O8 [M+H]+ 889.5551; found 888.5552.
cyclo[MeAla-MeLeu-MePhe-MeGly]3 (23): as white fluffy flakes; ESIHRMS (m/z):
Calcd. for C72H109N12O12 [M+H]+ 1333.8288; found 1333.8259.
(2S,3S)-2-(9-Fluorenylmethyloxycarbonyl(methyl)amino)-3-hydroxy-3-phenylpropan
-1-ol (30): To a solution of 29 (155 mg, 501 ꢀmol) in anhydrous THF (5 ml) were added
LiAlH4 (76.2 mg, 2.01 mmol) at 0°C and the mixture was refluxed under nitrogen
atmosphere for 3 h. The mixture was filtered on Celite-pad and filtrate was evaporated
in vacuo. After the residue was dissolved in CH2Cl2 (5 ml), Fmoc-OSu (178 mg, 528
µmol) and Et3N (140 µl, 1.00 mmol) were added to the solution and the mixture was
stirred for 17 h. AcOEt and H2O were added to the solution and organic layer was
washed with brine, dried over MgSO4 and evaporated in vacuo. The residue was
purified with column chromatography (CHCl3:MeOH = 95:5 v/v) to give 30 (171 mg,
1
424 ꢀmol, 85%) as a white solid; mp: 48-51 °C. H NMR (600 MHz, CDCl3): δ 7.75
(2H, d, J = 7.8 Hz), 7.55-7.19 (10H, m), 7.00 (1H, d, J = 3.6 Hz), 5.00 (1H, dd, J = 8.4,
3.6 Hz), 4.51-4.21 (3H, m), 4.12 (1H, m), 4.01 (2H, m), 3.85-3.67 (1H, m), 3.56-3.36
(1H, m), 2.57 (3H, s). 13C NMR (150 MHz, CDCl3): δ 157.0, 143.7, 141.5, 141.1, 128.2,
127.8, 127.5, 126.8, 126.3, 124.9, 119.8, 72.8, 67.3, 65.8, 61.6, 46.9, 34.0. IR (NaCl)
νmaxcm-1: 3403, 3064, 3013, 2952, 1678, 1478, 1450, 1404, 1329, 1218, 1152, 1035.
25
[α]D -16.7 (c 1.0, CHCl3). ESIHRMS (m/z): Calcd for C25H25NNaO4 [M+Na]+
426.1681; Found 426.1704.
(2S,3S)-2-(9-Fluorenylmethyloxycarbonyl(methyl)amino)-3-hydroxy-3-phenylpropan
oic acid (26): To a solution of 30 (46.7 mg, 116 ꢀmol) in toluene (2 ml) were added
17

ACCEPTED MANUSCRIPT
phosphate buffer solution (1.0 M, pH 6.8, 3 ml) and DMN-AZADO (2.3 mg, 10.8
µmol), NaClO2 (80.1 mg, 0.709 mmol) and NaOCl (100 µl, 161 µmol) and the mixture
was stirred for 3 h. Phosphate buffer solution (1.0 M, pH 2.3) and AcOEt were added to
the solution and organic layer was washed with brine, dried over MgSO4 and
evaporated in vacuo. The residue was purified with column chromatography
(CHCl3:MeOH = 80:20 v/v) to give 26 (29.9 mg, 71.6 ꢀmol, 62%) as a yellow solid;
mp: 105-108 °C. 1H NMR (600 MHz, CD3OD): δ 7.70 (2H, d, J = 6.6 Hz), 7.55 (1H, d,
J = 7.8 Hz), 7.37-7.28 (5H, m), 7.25-7.04 (5H, m), 5.17 (1H, d, J = 9.0 Hz), 4.44-4.28
(2H, m), 4.19-4.08 (2H, m), 4.00 (1H, t, J = 6.6 Hz), 2.57 (3H, s). 13C NMR (150 MHz,
CDCl3): δ 159.6, 156.1, 143.7, 141.1, 139.9, 128.6, 128.2, 127.5, 126.9, 125.8, 125.0,
119.7, 71.9, 67.7, 62.8, 46.7, 29.5. IR (NaCl) νmaxcm-1: 3370, 2926, 2360, 1683, 1601,
1489, 1450, 1402, 1310, 1219, 1167. [α]D29 -41.8 (c 1.4, CHCl3). ESIHRMS (m/z):
Calcd for C25H23NNaO5 [M+Na]+ 440.1474; Found 440.1452.
(2S,3S)-2-(t-Butyloxycarbonyl(methyl)amino)-3-hydroxy-3-phenylpropan-1-ol (31):
To a solution of 29 (2.93 g, 9.47 mmol) in anhydrous THF (95 ml) were added LiAlH4
(1.26 g, 33.2 mmol) at 0°C and the mixture was refluxed under nitrogen atmosphere for
6 h. The mixture was filtered on Celite-pad and filtrate was evaporated in vacuo. After
the residue was dissolved in H2O/CH3CN (1:1 v/v 95 ml), (Boc)2O (2.08 g, 9.53 mmol)
was added to the solution and the mixture was stirred for 34 h. AcOEt and H2O were
added to the solution and organic layer was washed with brine, dried over MgSO4 and
evaporated in vacuo. The residue was purified with column chromatography
1
(Hexane:AcOEt = 1:2 v/v) to give 31 (2.27 g, 8.07 mmol, 85%) as a colorless oil; H
NMR (500 MHz, CDCl3): δ 7.25-7.16 (5H, m), 4.83 (1H, dd, J = 7.5, 3.0 Hz), 4.53 (1H,
13
d, J = 3.5 Hz), 4.34 (1H, brs), 3.87-3.71 (3H, m), 2.37 (3H, s), 1.24 (9H, s). C NMR
(125 MHz, CDCl3): δ 156.4, 141.5, 127.8, 127.4, 126.4, 79.7, 72.7, 64.7, 61.6, 33.8,
28.0. IR (NaCl) νmaxcm-1: 3395, 2977, 1668, 1480, 1454, 1395, 1366, 1250, 1151, 1032.
24
[α]D -27.4 (c 1.0, CHCl3). ESIHRMS (m/z): Calcd for C15H23NNaO4 [M+Na]+
304.1525; Found 304.1546.
(2S,3S)-3-Benzyloxy-2-(t-butyloxycarbonyl(methyl)amino)-3-phenylpropan-1-ol (32):
To a solution of 31 (2.27 g, 8.07 mmol) in DMF (27 ml) were added imidazole (1.42 g,
20.9 mmol) and TBSCl (1.58 g, 10.5 mmol) at 0°C and the mixture was stirred for 18 h.
18

ACCEPTED MANUSCRIPT
Et2O and H2O were added to the solution and organic layer was washed with brine,
dried over MgSO4 and evaporated in vacuo. The residue was purified with column
chromatography (Hexane:AcOEt = 4:1 v/v) to give TBS ether (2.38 g, 6.02 mmol, 75%)
1
as a colorless oil; H NMR (500 MHz, CDCl3): δ 7.32 (1H, d, J = 7.5 Hz), 7.26-7.18
(4H, m), 4.94 (1H, d, J = 5.5 Hz), 4.33 (1H, brs), 4.00-3.85 (2H, m), 3.63 (1H, brs),
13
2.64 (3H, s), 1.33 (9H, s), 0.84 (9H, s), 0.04 (6H, s). C NMR (125 MHz, CDCl3): δ
156.1, 141.9, 127.9, 127.36, 126.3, 79.5, 74.8, 65.1, 61.3, 34.8, 28.2, 25.7, 17.9, -5.7. IR
(NaCl) νmaxcm-1: 3421, 2955, 2929, 1670, 1473, 1392, 1366, 1254, 1152. [α]D27 +7.6 (c
1.0, CHCl3). Calcd for C21H37NNaO4Si [M+Na]+ 418.2390; Found 418.2411. To a
solution of TBS ether (1.01 g, 2.55 mmol) in DMF (8.5 ml) were added NaH (184 mg,
7.67 mmol) and BnBr (760 µl, 6.40 mmol) at 0°C and the mixture was stirred under
nitrogen atmosphere for 15 h. AcOEt and H2O were added to the solution and organic
layer was washed with brine, dried over MgSO4 and evaporated in vacuo. The residue
was purified with column chromatography (Hexane:AcOEt = 6:1 v/v) to give benzyl
1
ether (1.00 g, 2.06 mmol, 81%) as a colorless oil; H NMR (500 MHz, CDCl3): δ
7.43-7.23 (10H, m), 4.74-4.24 (4H, m), 4.02 (2H, brs), 2.65 (3H, s), 1.34 (9H, s), 0.91
(9H, s), 0.07 (6H, s). 13C NMR (125 MHz, CDCl3): δ 155.1, 139.1, 137.8, 128.1, 128.0,
127.8, 127.4, 127.3, 127.3, 80.0, 78.5, 70.5, 65.3, 60.9, 28.8, 28.1, 25.6, 18.7, -5.6. IR
(NaCl) νmaxcm-1: 2955, 2929, 1693, 1472, 1455, 1390, 1365, 1351, 1323, 1254, 1152,
1110, 1067. [α]D24 +27.7 (c 1.0, CHCl3). ESIHRMS (m/z): Calcd for C28H43NNaO4Si
[M+Na]+ 508.2859; Found 508.2882. To a solution of benzyl ether (362 mg, 745 µmol)
in THF (4.0 ml) were added 1.0 M TBAF/THF (2.2 ml, 2.20 mmol) and the mixture
was stirred for 4 h. Saturated NH4Cl and AcOEt were added to the solution and organic
layer was washed with brine, dried over MgSO4 and evaporated in vacuo. The residue
was purified with column chromatography (Hexane:AcOEt = 2:1 v/v) to give alcohol
1
(32) (246 mg, 662 µmol, 89%) as a colorless oil; H NMR (500 MHz, CDCl3): δ
7.36-7.20 (10H, m), 4.76 (1H, d, J = 8.5 Hz), 4.38 (1H, dd, J = 19.5, 11.5 Hz), 4.19 (1H,
d, J = 11.5 Hz), 4.01-3.15 (3H, m), 2.48 (3H, s), 1.29 (9H, s). 13C NMR (125 MHz,
CDCl3): δ 155.9, 138.9, 137.7, 128.1, 128.0, 127.9, 127.6, 127.4, 127.1, 79.9, 79.2, 70.5,
64.5, 60.0, 34.0, 27.9. IR (NaCl) νmaxcm-1: 3445, 2975, 1685, 1480, 1454, 1393, 1365,
25
1249, 1151, 1065. [α]D +36.4 (c 1.0, CHCl3). ESIHRMS (m/z): Calcd for
C22H29NNaO4 [M+Na]+ 394.1994; Found 394.2022.
19

ACCEPTED MANUSCRIPT
(2S,3S)-3-Benzyloxy-2-(9-Fluorenylmethyloxycarbonyl(methyl)amino)-3-phenylprop
anoic acid (27): To a solution of 32 (236 mg, 635 µmol) in AcOEt (4.0 ml) were added
4 M HCl/AcOEt (4.0 ml) at 0°C and the mixture was stirred for 3 h. After the mixture
was evaporated in vacuo, the residue was dissolved in anhydrous CH2Cl2 (6.0 ml). Et3N
(180 µl, 1.29 mmol) and Fmoc-OSu (225 mg, 667 µmol) were added to the solution and
the mixture was stirred for 10 h. Saturated NH4Cl and AcOEt were added to the solution
and organic layer was washed with brine, dried over MgSO4 and evaporated in vacuo.
The residue was purified with column chromatography (Hexane:AcOEt = 1:1 v/v) to
1
give methylamine (253 mg, 513 µmol, 81%) as a colorless oil; H NMR (500 MHz,
CDCl3): δ 7.75 (2H, d, J = 7.5 Hz), 7.59 (1H, d, J = 6.5 Hz), 7.45-7.38 (6H, m),
7.36-7.32 (4H, m), 7.30-7.29 (4H, m), 6.87 (1H, brs), 4.85 (1H, d, J = 8.5 Hz), 4.44 (1H,
13
d, J = 12.0 Hz), 4.30 (2H, m), 4.16-4.01 (3H, m), 4.07-3.94 (2H, m), 3.55 (3H, s). C
NMR (125 MHz, CDCl3): δ 156.6, 143.8, 141.1, 138.9, 137.7, 128.4, 128.3, 128.2,
127.7, 127.5, 127.2, 126.9, 124.9, 124.5, 119.8, 80.3, 70.9, 67.2, 65.5, 60.3, 47.1, 34.2.
IR (NaCl) νmaxcm-1: 3445, 3030, 2949, 1685, 1478, 1451, 1403, 1320, 1243, 1216, 1154,
26
1065, 1028. [α]D -5.1 (c 1.0, CHCl3). ESIHRMS (m/z): Calcd for C32H31NNaO4
[M+Na]+ 516.2151; Found 516.1992. To a solution of methylamine (252.7 mg, 512
µmol) in acetone were added Jonesʼ reagent (excess) at 0°C and the mixture was stirred
for 30 min. The reaction was quenched by adding a 2-hydroxypropanol and filtered on
Celite-pad. AcOEt and H2O were added to the solution and organic layer was washed
with brine, dried over MgSO4 and evaporated in vacuo. The residue was purified with
column chromatography (CHCl3:MeOH = 95:5 v/v) to give 27 (227 mg, 447 µmol,
1
87%) as a white solid; mp: 69-72 °C. H NMR (500 MHz, CDCl3): δ 10.7 (1H, brs),
7.68-7.64 (2H, m), 7.56 (1H, m), 7.40-7.39 (1H, m), 7.32-7.12 (13H, m), 6.94 (1H, brs),
5.04 (1H, d, J = 8.5 Hz), 4.73-4.66 (1H, m), 4.40 (2H, brs), 4.21-4.17 (2H, m), 4.06 (1H,
dd, J = 14.0, 6.0 Hz), 2.67 (3H, s). 13C NMR (125 MHz, CDCl3): δ172.6, 156.1, 143.6,
143.4, 141.0, 137.1, 128.6, 128.4, 128.2, 127.8, 127.6, 127.5, 126.8, 124.9, 124.8, 119.7,
79.3, 71.0, 67.7, 65.7, 46.7, 33.7. IR (NaCl) νmaxcm-1: 3031, 1704, 1478, 1452, 1400,
1302, 1216, 1152, 1091, 1068. [α]D25 -34.9 (c 1.0, CHCl3). ESIHRMS (m/z): Calcd for
C32H30NNaO5 [M+Na]+ 508.2124; Found 508.2111.
H-MeAla-Leu-MePhe(βOH)-Gly-OH (31): To 2-chlorotrityl chloride resin (17.6 mg,
28.2 µmol) pre-swollen in DMF for 30 min was added a solution of Fmoc-Gly-OH
20

ACCEPTED MANUSCRIPT
(16.7 mg, 56.2 µmol) and DIPEA (15 µL, 86.1 µmol) in DMF (1 mL). The reaction
mixture was gently agitated at room temperature for 2 h. The resin was filtered and
washed with DMF. The peptidyl resin was treated with a solution of 20% piperidine in
DMF and the mixture agitated at room temperature for 30 min. The resin was filtered
and washed with DMF. To the peptidyl resin was added a mixture of
Fmoc-MePhe(βOH)-OH (26) (32.8 mg, 78.6 µmol), HATU (32.1 mg, 84.4 µmol),
HOAt (11.5 mg, 84.5 µmol) and DIPEA (20 µL, 115 µmol) in DMF (1 mL). The
reaction mixture was agitated at room temperature for 13 h, after which the resin was
filtered and washed with DMF. The peptidyl resin was treated with a solution of 20%
piperidine in DMF and the mixture agitated at room temperature for 30 min. The resin
was filtered and washed with DMF. To the peptidyl resin was added a mixture of
Fmoc-Leu-OH (29.9 mg, 84.6 µmol), HATU (32.1 mg, 84.4 µmol), HOAt (11.5 mg,
84.5 µmol) and DIPEA (20 µL, 115 µmol) in DMF (1 mL). The reaction mixture was
agitated at room temperature for 4 h, after which the resin was filtered and washed with
DMF. The peptidyl resin was treated with a solution of 20% piperidine in DMF and the
mixture agitated at room temperature for 30 min. The resin was filtered and washed
with DMF. To the peptidyl resin was added a mixture of Fmoc-MeAla-OH (27.5 mg,
84.5 µmol), PyBOP (49.0 mg, 94.2 µmol), HOBt·H2O (12.9 mg, 84.2 µmol) and
DIPEA (20 µL, 115 µmol) in DMF (1 mL). The reaction mixture was agitated at room
temperature for 3 h, after which the resin was filtered and washed with DMF. The
peptidyl resin was treated with a solution of 20% piperidine in DMF and the mixture
agitated at room temperature for 30 min. The resin was filtered and washed with DMF.
To the peptidyl resin was added 20% HFIP in anhydrous CH2Cl2 and agitated at room
temperature for 2 h. The mixture was filtered and purified by preparative RP-HPLC to
afford the linear tetrapeptide 31 (2.49 mg, 5.53 µmol, 20%) as white fluffy flakes;
ESIMS (m/z): Calcd. for C21H31N4NaO6 [M+Na]+ 473.24; found 473.16.
H-MeAla-Leu-MePhe(βOBn)-Gly-OH (25): To 2-chlorotrityl chloride resin (100 mg,
160 µmol) pre-swollen in DMF for 30 min, was added a solution of Fmoc-Gly-OH
(95.1 mg, 320 µmol) and DIPEA (84 µL, 482 µmol) in DMF (2 mL). The reaction
mixture was gently agitated at room temperature for 2 h. The resin was filtered and
washed with DMF. The peptidyl resin was treated with a solution of 20% piperidine in
DMF and the mixture agitated at room temperature for 30 min. The resin was filtered
21

ACCEPTED MANUSCRIPT
and washed with DMF. To the peptidyl resin was added a mixture of
Fmoc-N-MePhe(βOBn)-OH (27) (162 mg, 319 µmol), DIPCI (74 µl, 478 µmol),
HOBt·H2O (73.5 mg, 480 µmol) and DIPEA (84 µL, 482 µmol) in DMF (2 mL). The
reaction mixture was agitated at room temperature for 16 h, after which the resin was
filtered and washed with DMF. The peptidyl resin was treated with a solution of 20%
piperidine in DMF and the mixture agitated at room temperature for 30 min. The resin
was filtered and washed with DMF. To the peptidyl resin was added a mixture of
Fmoc-Leu-OH (170 mg, 481 µmol), HATU (183 mg, 481 µmol), HOAt (65.3 mg, 480
µmol) and DIPEA (84 µL, 482 µmol) in DMF (2 mL). The reaction mixture was
agitated at room temperature for 16 h, after which the resin was filtered and washed
with DMF. The peptidyl resin was treated with a solution of 20% piperidine in DMF
and the mixture agitated at room temperature for 30 min. The resin was filtered and
washed with DMF. To the peptidyl resin was added a mixture of Fmoc-N-MeAla-OH
(156 mg, 479 µmol), PyBOP (250 mg, 480 µmol), HOBt·H2O (73.6 mg, 481 µmol) and
DIPEA (84 µL, 482 µmol) in DMF (2 mL). The reaction mixture was agitated at room
temperature for 5 h, after which the resin was filtered and washed with DMF. The
peptidyl resin was treated with a solution of 20% piperidine in DMF and the mixture
agitated at room temperature for 30 min. The resin was filtered and washed with DMF.
To the peptidyl resin was added 20% HFIP in anhydrous CH2Cl2 and agitated at room
temperature for 2 h. The mixture was filtered and purified by preparative RP-HPLC to
1
afford the linear tetrapeptide 25 (54.8 mg, 101 µmol, 65%) as white fluffy flakes; H
NMR (500 MHz, D2O): δ 7.07-6.82 (10H, m), 4.82 (1H, t, J = 9.0 Hz), 4.71-4.42 (1H,
m), 4.30 (1H, dd, J = 11.0, 3.0 Hz), 4.05-3.88 (2H, m), 3.79-3.60 (3H, m), 2.64 (3H, s),
2.41 (3H, m), 1.26 (1H, m), 1.18 (3H, d, J = 7.0 Hz), 1.07 (1H, m), 0.70 (1H, m), 0.58
(3H, d, J = 7.0 Hz), 0.54 (3H, d, J = 6.0 Hz). 13C NMR (125 MHz, D2O): δ 172.7, 170.1,
163.0, 162.7, 136.9, 136.5, 129.1, 128.6, 128.5, 128.1, 117.5, 115.2, 79.0, 70.8, 61.7,
56.9, 48.3, 41.2, 39.0, 31.1, 31.0, 24.3, 22.4, 20.3, 15.6. ESIHRMS (m/z): Calcd for
C29H41N4O6 [M+H]+ 541.3026; found 541.3043.
cyclo[MeAla-Leu-MePhe(βOBn)-Gly] (33): To a solution of linear tetrapeptide 25
(34.5 mg, 63.8 µmol) in DMF (6.4 ml) were added DIPCI (30 µl, 194 µmol),
HOBt·H2O (29.4 mg, 192 µmol) and DIPEA (49 µL, 281 µmol) and the mixture was
stirred for 24 h. The mixture was purified by preparative RP-HPLC to afford the
22

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cyclo[MeAla-Leu-MePhe(βOBn)-Gly] 33 (20.2 mg, 38.6 µmol, 61%) as white fluffy
flakes; 1H NMR (600 MHz, CDCl3): δ 7.40-7.35 (5H, m), 7.32-7.27 (3H, m), 7.12 (2H,
d, J = 7.8 Hz), 6.87 (1H, brs), 6.40 (1H, brs), 4.70 (1H, brs), 4.66 (1H, d, J = 9.6 Hz),
4.43 (1H, d, J = 9.6 Hz), 4.37 (1H, d, J = 12 Hz), 4.34 (1H, m), 4.17 (2H, d, J = 12.0
Hz), 3.85 (1H, d, J = 9.6 Hz), 2.88 (3H, s), 2.80 (3H, s), 1.77 (1H, m), 1.51 (3H, d, J =
7.2 Hz), 1.42 (1H, m), 1.23 (1H, m), 0.85 (3H, d, J = 6.6 Hz), 0.84 (3H, d, J = 6.0 Hz).
13C NMR (150 MHz, CDCl3): δ 172.3, 171.4, 170.6, 170.1, 136.9, 136.5, 129.5, 129.1,
128.7, 128.3, 128.1, 127.5, 78.9, 70.7, 63.7, 57.8, 48.5, 45.9, 41.1, 32.4, 30.1, 24.5, 22.5,
22.4, 16.7. ESIHRMS (m/z): Calcd for C29H39N4O5 [M+H]+ 523.2920; found 523.2864.
Tentoxin B (1): To a solution of cyclic tetrapeptide 33 (15.9 mg, 30.4 µmol) in
MeOH/CH2Cl2 (1:1 v/v 2 ml) were added Et3SiH (485 µl, 3.04 mmol) and Pd(OH)2/C
(cat.) and the mixture was stirred for 24 h. The mixture was purified by preparative
RP-HPLC to afford the Tentoxin B (1) (7.9 mg, 18.3 µmol, 60%) as white fluffy flakes;
1
mp: 172-175 °C. H NMR (600 MHz, CD3OD): δ 7.88 (1H, brs), 7.86 (1H, brs), 7.46
(1H, d, J = 7.2 Hz), 7.36 (2H, t, J = 7.2 Hz), 7.31 (2H, t, J = 6.6 Hz), 4.98 (1H, d, J =
9.6 Hz), 4.64 (1H, m), 4.54 (1H, d, J = 7.8 Hz), 4.33 (1H, d, J = 9.0 Hz), 4.15 (1H, d, J
= 16.2 Hz), 4.01 (1H, dd, J = 15.6, 3.0 Hz), 2.87 (3H, s), 2.86 (3H, s), 1.72 (1H, m),
1.52 (3H, d, J = 7.8 Hz), 1.44 (1H, m), 1.28 (1H, m), 0.88 (3H, d, J = 6.0 Hz), 0.86 (3H,
d, J = 6.6 Hz). 13C NMR (150 MHz, CD3OD): δ 174.1, 173.6, 173.2, 172.6, 143.0,
129.8, 129.6, 128.0, 73.1, 66.3, 58.8, 49.8, 46.4, 42.1, 33.1, 30.7, 25.8, 23.0, 22.9, 16.8.
IR (NaCl) νmaxcm-1: 3328, 2957, 1638, 1523, 1402, 1218. [α]D26 -16.0 (c 0.15, MeOH).
ESIHRMS (m/z): Calcd for C22H32N4NaO5 [M+Na]+ 455.2270; found 455.2293. The
above-mentioned spectral data was identical to those of the natural product from Jung
group.6
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