Solid-Phase Synthesis of Triostin A Using a Symmetrical Bis(diphenylmethyl) Linker System

Article abstract of DOI:10.1021/acs.joc.5b01055

Triostin A is a symmetric bicyclic depsipeptide with very potent antitumoral activity because of its bisintercalation into DNA. In this study, we report a new synthetic strategy that exploits a structural symmetry of triostin A. First, we prepared a novel symmetric linker molecule that is labile under mildly acidic conditions and suitable for a solid-phase synthesis procedure. Two Cys units were attached to a linker-resin conjugate via their free thiol groups, and double deprotection and double coupling reactions were then applied to synthesize linear tetradepsipeptides. Subsequently, the key biscyclization of the tetradepsipeptides was performed on the resin, and the resulting cyclic octapeptide was detached from the linker-resin conjugate to give a peptide with two free thiols. Finally, triostin A was obtained by oxidizing the free thiols in solution to produce a disulfide. The yield was improved through exploration of two different solid-phase synthetic approaches under similar strategy. Mainly, this strategy was developed to enable the ease and rapid preparation of libraries of symmetric bicyclic depsipeptides. It also addresses several synthetic problems with our synthesis, including diketopiperazine (DKP) formation, poor cyclization yields and preparation of noncommercial N-methyl amino acids in good yields.

Full text of DOI:10.1021/acs.joc.5b01055

Article
pubs.acs.org/joc
Solid-Phase Synthesis of Triostin A Using a Symmetrical
Bis(diphenylmethyl) Linker System
Ganesh A. Sable and Dongyeol Lim*
Department of Chemistry, Sejong University, 209 Neungdong-ro, Gwangjin-gu, Seoul, Republic of Korea
S
* Supporting Information
ABSTRACT: Triostin A is a symmetric bicyclic depsipeptide
with very potent antitumoral activity because of its
bisintercalation into DNA. In this study, we report a new
synthetic strategy that exploits a structural symmetry of triostin
A. First, we prepared a novel symmetric linker molecule that is
labile under mildly acidic conditions and suitable for a solid-
phase synthesis procedure. Two Cys units were attached to a
linker-resin conjugate via their free thiol groups, and double
deprotection and double coupling reactions were then applied
to synthesize linear tetradepsipeptides. Subsequently, the key
biscyclization of the tetradepsipeptides was performed on the
resin, and the resulting cyclic octapeptide was detached from
the linker-resin conjugate to give a peptide with two free thiols. Finally, triostin A was obtained by oxidizing the free thiols in
solution to produce a disulfide. The yield was improved through exploration of two different solid-phase synthetic approaches
under similar strategy. Mainly, this strategy was developed to enable the ease and rapid preparation of libraries of symmetric
bicyclic depsipeptides. It also addresses several synthetic problems with our synthesis, including diketopiperazine (DKP)
formation, poor cyclization yields and preparation of noncommercial N-methyl amino acids in good yields.
because it allows various peptide analogues to be rapidly
synthesized by changing the amino acid at each position.
Because of the therapeutic significance of quinoxaline
antibiotics, we utilized our experience in peptide synthesis27−31
to develop an efficient solid-phase synthetic strategy for this
class of symmetric antibiotics, using triostin A synthesis as a
model. In developing our synthetic strategy, we considered the
following important points: (i) utilization of the structural C2
symmetry of this family of molecules; (ii) minimization of the
required number of synthetic steps while maximizing the use of
a solid support; and (iii) straightforward synthesis of libraries of
various bisintercalators by varying the amino acids and
chromophores.
Structurally, triostin A is a symmetric bicyclic octadepsipep-
tide composed of four N-Me amino acids, two ester linkages,
two chromophores and a disulfide backbone. These structural
elements make it sterically hindered or conformationally rigid
and therefore the use of a suitable linker system was necessary
to minimize the difficulties encountered in the solid-phase
synthesis of triostin A. Overall, the optimum synthesis
procedure includes preparing the appropriate linker, synthesiz-
ing commercially unavailable amino acids with suitable
protecting groups, choosing the first amino acid to couple to
the linker and utilizing the structural symmetry of triostin A to
reduce the number of synthetic steps. However, in the presence
INTRODUCTION
■
Triostin A (1),1,2 echinomycin (3),3 BE-22179 (4)4 and
thiocoraline (5)5,6 (Figure 1) are quinoxaline antitumor
antibiotics produced by marine bacteria. In 1961, triostin A
was isolated from a strain of Streptomyces aureus.1,7,8 Initial
studies showed that triostin A and echinomycin exhibit
significant antibacterial and antitumor activities because of
their bifunctional intercalation of two aromatic rings into
DNA.9−13 Triostin A is well-known to exhibit CpG selectivity
for bisintercalation into DNA, whereas it’s N-demethylated
derivative TANDEM (2) (Triostin A N-DEMethylated)14,15 is
known to be selective for TpA sequences.16−19 Initially, this
change in selectivity was attributed to differences in the
hydrogen-bonding abilities of these compounds; however, their
binding to DNA base pairs was later demonstrated to be driven
by both hydrogen-bonding and stacking interactions.10,20
In recent reports, triostin A activity against three human
cancer cell lines (breast MDA-MB-231, lung NSCLC A549,
colon HT-29) was tested. It exhibited cell-growth inhibitory
activities (GI50) as high as 10−7 M.21−23 Because of this activity
profile, many researchers have focused on developing various
synthesis methods for these types of peptides and on
understanding their mode of action and potential applications
in cancer therapy. To date, triostin A has been synthesized via
two solution-phase methods24−26 and one solid-phase
method.22 Generally, the solid-phase synthesis of these cyclic
depsipeptides is preferred over tedious solution-phase methods
Received: May 12, 2015
© XXXX American Chemical Society
A
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Figure 1. Structures of bicyclic bisintercalators 1−5.
Scheme 1. Synthesis of the Linker (9)
of two ester linkages and adjacent N-Me amino acids, peptide
syntheses are known to be challenging for several reasons,
including DKP formation,32,33 slow coupling reactions,23,34
difficult cyclization,35−37 and the risk of elimination reactions at
the Cys residue.32,38,39 Thus, in the present work, two different
approaches were explored to overcome or minimize these
problems, and triostin A was synthesized in a yield comparable
to those obtained by solid-phase synthesis reported to date.22
formation through a decomposition of the desired product
(byproducts include traces of the hydrolyzed product of 7 in
the presence of moisture trapped by reagents). Initially, this
reaction was performed in DMF under reflux; however, it was
too slow to achieve complete conversion of the starting
material. Next, the compound 8 was obtained by simple
mesylation41 of 7, followed by nucleophilic substitution42 with
4-hydroxybenzophenone under MW irradiation. Finally, the
desired linker 9 was obtained from 8 by selectively reducing
42
RESULTS AND DISCUSSION
two keto groups in the presence of NaBH4 and then
performing methyl ester hydrolysis.
■
Linker Synthesis. To develop a suitable linker system, the
following solid-phase synthesis requirements were considered:
(i) appropriate length of the spacer between the peptide and
polymer support, (ii) structural symmetry, (iii) lability under
mildly acidic conditions and stability under Fmoc deprotection
conditions, and (iv) ease of loading of the first amino acid to
the linker. On the basis of these requirements, a bisdiphe-
nylmethyl linker system was synthesized from the compound 6
(Scheme 1), which has C2 symmetry and two reactive
functional groups. The neat reaction of the ethylene
carbonatespacer with the phenolic hydroxyl of 6 under
microwave irradiation gave the symmetric disubstituted product
7 in 73% yield.40 The reaction was monitored at short time
intervals because longer treatments initiated byproducts
SPPS Synthesis. Because the N-Me-Cys residue was chosen
to be coupled to the linker-resin conjugate, Fmoc-N-Me-Cys-
OAllyl was synthesized from Alloc-N-Me-Cys(Trt)-OH43 by
sequentially performing Alloc deprotection, Fmoc protection,
allyl esterification and trityl deprotection reactions (Exper-
imental Section). The Trityl deprotected Fmoc-N-Me-Cys-
OAllyl was unstable for a longer storage as it undergoes S−S
dimerization; therefore, it was immediately utilized for the
SPPS synthesis without purification. The synthesis of Fmoc-
protected N-Me-Cys-OH was previously reported on the solid
support for rapid and diverse synthesis;43 however, we could
prepare it on a multigram scale via a solution-phase reactions
with high yield and purity.
B
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After the desired linker and Fmoc-N-Me-Cys-OAllyl were
prepared, triostin A was synthesized on the amino methyl resin
(Scheme 2). The symmetric linker 9 was first coupled to the
resin after the coupling of Fmoc-N-Me-Cys-OAllyl. The resin
was subsequently treated with acetic anhydride/DIPEA to cap
the unreacted sites.47,48
In the first synthesis attempt (Scheme 2), the Fmoc group of
11 was removed using 20% piperidine/DMF31 and Fmoc-Ala-
OH was coupled to the Cys residue using 7-azabenzotriazol-1-
yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/
1-hydroxy-7-azabenzotriazole (HOAt). However, during the
next Fmoc deprotection reaction, the resulting dipeptide, as
expected, underwent complete DKP formation in the presence
of the allyl ester of N-Me-Cys.
Scheme 2. Preparation of Resin-Bound Intermediate 12
To prevent DKP formation, a dipeptide was prepared
separately in solution and then used in the solid-phase
synthesis. Specifically, the dipeptide Fmoc-D-Ser-Ala-OH was
prepared in two steps (Experimental Section).49,50 The Fmoc
group of 11 was then removed, and Fmoc-D-Ser-Ala-OH was
coupled to the Cys residue using HATU/HOAt to give resin-
bound peptide 12. To prevent the free hydroxyl group of the
Ser residue from reacting to form byproducts, the reaction was
stopped immediately after a negative chloranil test result. After
the resin-bound peptide 12 was obtained, two different
approaches were tested to complete the synthesis of triostin A.
In Approach A (Scheme 2), the Fmoc group of tripeptide 12
was removed and 2-quinoxaline carboxylic (Qxc) acid was
coupled to the peptide by activation with HATU/HOAt.
However, 13a was obtained as a major byproduct. We
attempted to control this reaction stoichiometrically; however,
the second coupling reaction to the Ser free hydroxyl group
occurred too quickly because the conformation of the desired
product 13 imposed by the attached Qxc group might be
favorable for this side reaction. The unwanted product 13a was
then converted back to the desired product 13 by alcoholysis
on the resin in the presence of K2CO3 and allyl alcohol.51 Allyl
alcohol was used as a solvent to prevent the hydrolysis of the
allyl ester of the N-Me-Cys residue. This reaction was
effectively completed, but washing the resin with water to
remove K2CO3 led to the loss of some of the peptide from the
resin, as indicated by HPLC analysis. Next, Alloc-N-Me-Val-
OH was coupled to the growing peptide to obtain the resin-
bound peptide 14, and the Alloc and allyl protecting groups
were removed using [Pd(PPh3)4] and PhSiH3 in CH2Cl2. For
the biscyclization step, several reagents, including (benzotria-
zol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophos-
phate (BOP), HATU, 1-Ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide (EDC), diphenylphosphoryl azide (DPPA), N,N′-
bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl) and
Tetramethylfluoroformamidinium Hexafluorophosphate
(TFFH) were tested to obtain the resin-bound cyclic
octapeptide 15; however, in all cases, the monocyclic
tetradepsipeptide 15a and/or unidentified byproducts were
formed. Comparatively, the best biscyclization results were
obtained using N,N′-diisopropylcarbodiimide (DIC)/HOAt
with a DMSO/DMF (3:2) solvent mixture.22,38,52,53 The
peptide was cleaved from the resin by treatment with TFA/
TIS/CH2Cl2 (10:5:85), and the crude product was purified by
semipreparative HPLC to give the desired product 15 (7.1 mg,
4.0% overall yield from the Cys-loaded linker resin).
amino methyl resin in the presence of (Benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP)/N,N-Diisopropylethylamine (DIPEA) to obtain the
linker resin 10. Complete conversion was confirmed by a
negative Kaiser test,44 and the loading was determined to be
90% on a w/w basis.
For the peptide synthesis, the orthogonally protected Fmoc-
N-Me-Cys-OAllyl was immobilized on the linker resin 10 in
two steps; the activation of hydroxyl group of 10 to its
trichloroacetimidate using Cl3CCN/1,8-Diazabicyclo[5.4.0]-
undec-7-ene (DBU) followed by the substitution with the
free thiol of Fmoc-N-Me-Cys-OAllyl to obtain 11.45,46 The
immobilization of Fmoc-N-Me-Cys-OAllyl was confirmed by
FTIR spectroscopy, where the CN stretching band (1663
cm−1) of an intermediate trichloroacetimidate of 10 was
replaced by CO stretching band (1721 cm−1) of Fmoc-N-
Me-Cys-OAllyl. The loading level was determined to be
approximately 82% based on the weight increase of the dried
During the cyclization step, in addition to the desired
product 15, the cyclic tetradepsipeptide 15a was obtained as a
major byproduct. The structure of 15a was analyzed by NMR
and subsequently confirmed by mass analysis of its N-ethyl
maleimide (NEM) adduct (Supporting Information).54 With
this approach, the desired product was obtained in low yield;
C
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Scheme 3. Solid-Phase Synthesis of 15 (Approach A)
therefore, the synthesis approach was revised. The major
drawbacks of Approach A were as follows: (i) side-product
formation during the Qxc acid coupling; (ii) peptide loss from
the resin when it was washed with water to remove K2CO3 after
the alcoholysis reaction; and (iii) a relatively low biscyclization
yield along with the formation of the undesired monocyclic
tetradepsipeptide as a major byproduct. To address these
problems, the sequence of reactions in Approach A was
changed.
In Approach B (Scheme 4), the free hydroxyl group of the
Ser residue in 12 was blocked from reacting to form side
product 13a by coupling Alloc-N-Me-Val-OH to the peptide
before the Fmoc deprotection and Qxc acid coupling reactions.
The cyclization yield was assumed to be affected by the
presence of the Fmoc group and by the absence of Qxc acid
because the steric environment would otherwise be different.
Therefore, Alloc-N-Me-Val-OH was coupled to the free
hydroxyl group of 12 to obtain Fmoc-protected peptide 16.
Then, as in Approach A, the Alloc and allyl groups were
removed and biscyclization was performed to obtain the resin-
bound cyclic peptide 17. The HPLC yield of this cyclization
step was nearly 2.5 times larger than that of the cyclization step
in Approach A (Supporting Information).
Finally, the pure product 15 was oxidized to form triostin A
(1). The aerial oxidation55 of 15 in acetonitrile (ACN) or in
DMSO was slow. Therefore, the dithiol 15 was oxidized using
I2 in ACN/DMSO (2:1).56 The crude reaction mixture was
purified by semipreparative HPLC to obtain triostin A (1) in
80% yield. Similarly, the byproduct 15a was also oxidized to
obtain the dimer 15b, and its NMR spectrum was compared to
that of triostin A (Supporting Information).
During the synthesis, only one triostin A conformer of the
two possible conformers reported by Tulla-Puche, Albericio
and co-workers22 was obtained. Based on the characteristic
1
peaks of Cys-α (δ 5.79) and Cys-β (δ 3.28), the H NMR
spectrum of our product was consistent with that of the least-
polar conformer that they reported.22 The Ala N-H signal of
our synthetic sample (δ 6.74) is significantly different from the
previously reported (δ 6.45) data of triostin A.22 We observed
that the chemical shift of Ala N-H is dependent on the sample
concentration which indicates the Ala N-H is solvent exposed
(Supporting Information). It was known that the Ala N-H
group forms hydrogen bonds to the nucleic acid in the crystal
structure of a DNA-triostin A complex.12
CONCLUSIONS
■
Triostin A was synthesized via two different approaches using a
novel solid-phase synthesis strategy. The yields obtained were
comparable to those obtained via other solid-phase synthesis
procedures reported in the literature.57 To demonstrate the
synthesis of C2-symmetric antibiotics, a novel C2-symmetric
Next, the Fmoc group was removed and Qxc acid was
coupled to the peptide, which was then cleaved from the resin.
The crude product was purified by semipreparative HPLC to
give the desired product 15 (6.5 mg) in 9.1% overall yield.
D
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Scheme 4. Solid-Phase Synthesis of 15 (Approach B)
linker was prepared and employed in the synthesis of triostin A.
In this synthesis, each coupling, deprotection and cyclization
reaction on the resin was performed successfully in a
symmetrical manner, which considerably reduced the number
of synthetic steps. The biscyclization yields were improved
through the exploration of two different approaches. The solid-
phase strategy of using a C2-symmetric linker can be exploited
to prepare symmetric bicyclic depsipeptides and their libraries.
The syntheses of new analogues are underway in our laboratory
and will be presented in future reports.
EXPERIMENTAL SECTION
■
General Experimental. All reagents were obtained from
commercial suppliers and used without further purification, unless
specified. The aminomethyl resin (100−200 mesh, 1% DVB) was
purchased from Advanced ChemTech. Tetrahydrofuran (THF) and
dichloromethane (CH2Cl2) solvents were distilled over sodium and
1
benzophenone. H and 13C NMR spectra were collected at resonance
frequencies of 500.1 and 125.7 MHz, respectively. The solvents used
for NMR were DMSO-d6 or CDCl3 as indicated. The chemical shifts
for H NMR are reported in ppm from tetramethylsilane (0 ppm) or
referenced to the solvent (chloroform 7.26 ppm; DMSO-d6 2.49 ppm)
on the δ scale. Chemical shifts (δ) for 13C NMR spectra are referenced
to signals for residual deuterated solvents (chloroform-d 77.16 ppm,
1
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DMSO-d6 39.5 ppm). Multiplicities are reported by the following
abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (double doublet), brs (broad singlet), J (coupling
constants in hertz). Analytical reverse-phase high performance liquid
chromatography (RP-HPLC) was carried out using a C18 (4.6 × 150
mm) reverse-phase column at a flow rate of 1 mL/min with UV
detection at 214 and 254 nm. Linear gradients of CH3CN/H2O
solvents, each containing 0.1% TFA were used as follows: condition A
(50 to 100% CH3CN gradient over 20 min), condition B (30 to 80%
CH3CN gradient over 20 min), condition C (36 to 62% CH3CN
gradient over 25 min) and condition D (40 to 64% CH3CN gradient
over 25 min). For preparative HPLC, a C18 column (5 μm, 10 × 150
mm) was employed at a flow rate of 4 mL/min using the gradient
condition C and D. High resolution mass spectra (HRMS) were
recorded using two different instruments: (i) fast atom bombardment
ionization using a double-focusing magnetic sector mass analyzer (for
compounds 1, 15, 15a, NEM adduct of 15a and 15b), (ii) electrospray
ionization using an ion trap analyzer (for all other compounds). The
microwave heated reactions were performed in a CEM Discover
reactor. Only the strongest and/or structurally important absorptions
of IR spectra were reported in wavenumbers (cm−1). Reactions in
solution-phase were monitored by thin-layer chromatography (TLC)
performed on glass packed silica gel plates (60F-254) with UV light
and visualized with ninhydrin, p-anisaldehyde, phosphomolybdic acid
or KMnO4 solution stains. Flash column chromatography was
performed with silica gel (100−200 mesh) with the indicated solvent
system.
residue was purified by flash column chromatography on silica gel
using ethyl acetate/hexane as the eluent to afford the desired product
8 (6.1 g, 78% yield). 1H NMR (500 MHz, CDCl3) δ 7.84 (dd, J = 1.9,
7.1 Hz, 4H), 7.76 (m, 4H), 7.57 (m, 2H), 7.48 (t, J = 7.8 Hz, 4H),
7.28 (d, J = 2.3 Hz, 2H), 7.02 (m, 4H), 6.78 (t, J = 2.4 Hz, 1H), 4.42
(m, 4H), 4.40 (m, 4H), 3.92 (s, 3H). 13C NMR (125.7 MHz, CDCl3)
δ 195.6, 166.7, 162.3, 159.7, 138.4, 132.7, 132.4, 132.1, 130.8, 129.9,
128.4, 114.3, 108.5, 107.4, 66.9, 66.7, 52.5. HRMS m/z calcd for
C38H32O8Na [M + Na]+ 639.1995, found [M + Na]+ 639.1990.
3,5-Bis(2-(4-(hydroxy(phenyl)methyl)phenoxy)ethoxy)benzoic
acid (9). The compound 8 (6.0 g, 9.74 mmol) was dissolved in
methanol/toluene (2:1) and cooled to 0 °C. Into the reaction mixture
NaBH4 (3.11 g, 77.92 mmol) was added in portions and the mixture
was left stirred for 3 h at room temperature. The reaction mixture was
quenched by aqueous NH4Cl and organic solvents were removed on
rotary evaporator. The crude aqueous mixture was extracted with
EtOAc and the combined organic layers were washed with brine, dried
over anhydrous MgSO4 and the solvents were removed under a
vacuum. The crude product was precipitated using ether/hexane (1:3)
to obtain the desired methyl 3,5-bis(2-(4-(hydroxy(phenyl)methyl)-
1
phenoxy)ethoxy)benzoate as a white solid (5.95 g, 99% yield). H
NMR (500 MHz, CDCl3) δ 7.38−7.24 (m, 16H, overlapping with
solvent residual peak), 6.91 (d, J = 8.6 Hz, 4H), 6.74 (s, 1H), 5.82 (s,
2H), 4.32 (d, J = 4.1 Hz, 8H), 3.90 (s, 3H), 2.16 (bs, 2H). HRMS m/z
calcd for C38H36O8Na [M + Na]+ 643.2308, found [M + Na]+
643.2302. The obtained methyl 3,5-bis(2-(4-(hydroxy(phenyl)-
methyl)phenoxy)ethoxy)benzoate (5.9 g, 9.52 mmol) was dissolved
in MeOH/THF (3:1) and 2 N aqueous KOH solution was slowly
added to it. After 6 h stirring at room temperature, the reaction
mixture was acidified with 2 N HCl solution and volatiles were
removed on a rotary evaporator. The crude aqueous mixture was
extracted with EtOAc. The combined organic layers were washed with
brine, dried over anhydrous MgSO4 and the solvents were removed
under a vacuum to obtain the desired product 9 as a white solid (5.7 g,
99% yield). 1H NMR (500 MHz, CDCl3) δ 7.38−7.27 (m, 16H,
overlapping with solvent residual peak), 6.91 (m, 4H), 6.79 (t, J = 2.3
Hz 1H), 5.82 (s, 2H), 4.34−4.31 (m, 8H). 13C NMR (125.7 MHz,
CDCl3) δ 170.8, 159.9, 158.1, 144.1, 136.9, 131.3, 128.6, 128.1, 127.6,
126.6, 114.9, 114.1, 108.9, 108.2, 76.0, 67.1, 67.0. IR (neat) 3388,
2981, 1690, 1595, 1509, 1445, 1244, 1164, 1069, 1008, 698 cm−1;
HRMS m/z calcd. for C37H34O8Na [M + Na]+ 629.2151, found [M +
Na]+ 629.2147.
Fmoc-N-Me-Cys(Trt)-OH. The Alloc protecting group of Alloc-N-
Me-Cys(Trt)-OH (3.6 g, 7.81 mmol) was cleaved by treatment of
Pd(PPh3)4 (0.9 g, 0.781 mmol) and PhSiH3 (5.7 mL, 46.85 mmol) in
CH2Cl2 (180 mL) for 30 min at room temperature. The reaction
mixture was concentrated on a rotary evaporator and the crude
product was used for the next step without further purification. N-Me-
Cys(Trt)-OH (2.94 g, 7.79 mmol) was dissolved in dioxane/H2O
(1:1) (50 mL) and 2% aqueous Na2CO3 (50 mL) and the solution was
cooled to 4 °C. The solution of Fmoc-OSu (2.9 g, 8.57 mmol) in
dioxane (25 mL) was added slowly to the amino acid solution. The
mixture was stirred for 2 h at 4 °C and for further 16 h at 25 °C, with
the pH maintained between 9 and 10. The dioxane was removed and
the aqueous solution was acidified with 1 N HCl to pH 3. The crude
product was extracted with EtOAc (3 × 80 mL) and the combined
organic layers were washed with saturated aqueous NaCl (100 mL),
dried over (MgSO4), filtered, and concentrated under a vacuum.
Column chromatography (eluent, 40% ethyl acetate/hexane +1%
acetic acid) yielded Fmoc-N-Me-Cys(Trt)-OH (4.15 g, 89%) as a
white solid. 1H NMR data were identical to the data reported
previously.43
Linker Synthesis. Methyl 3,5-bis(2-hydroxyethoxy)benzoate (7).
In a round-bottom flask, a neat mixture of 6 (3.0 g, 17.85 mmol),
ethylene carbonate (5.72 g, 178.5 mmol) and K2CO3 (6.16 g, 44.64
mmol) were set to treat on Microwave reactor using open vessel
method. The mixture was stirred at 80 °C under a microwave (25 W)
irradiation for 30 to 40 min (20 min × 1, 10 min × 1 or 2 times) until
the disappearance of the starting material was confirmed by TLC. The
reaction mixture was diluted with water and extracted with ethyl
acetate (3 × 100 mL). The organic layers were combined and washed
with brine and then dried over anhydrous MgSO4. The solvent was
removed under a vacuum, and the residue was purified by flash column
chromatography on silica gel using ethyl acetate/hexanes as the eluent
1
to afford the desired product 7 (3.3 g, 73% yield). H NMR (500
MHz, CDCl3) δ 7.21 (d, J = 2.3 Hz, 2H), 6.69 (t, J = 2.3 Hz, 1H), 4.10
(t, J = 4.2 Hz, 4H), 3.97 (dd, J = 5.1 Hz, 9.2 Hz, 4H), 3.90 (s, 3H),
2.12 (t, J = 6.0 Hz, 2H). 13C NMR (125.7 MHz, CDCl3) δ 166.8,
159.8, 132.3, 108.3, 107.1, 69.8, 61.4, 52.4. HRMS m/z calcd for
C12H16O6Na [M + Na]+ 279.0845, found [M + Na]+ 279.0839.
Methyl 3,5-bis[2-(4-benzoylphenoxy)ethoxy]benzoate (8). To a
solution of 7 (3.3 g, 12.89 mmol) in anhydrous CH2Cl2 (90 mL) at 0
°C, was added triethylamine (8.77 mL, 64.45 mmol) followed by
mesyl chloride (2.99 mL, 38.67 mmol). The reaction temperature was
maintained at 0 °C during the first hour, followed by warming to room
temperature for another 2 h under N2 atmosphere with vigorous
stirring. At the end of reaction, the mixture was diluted with water and
extracted with dichloromethane. The combined organic layers were
washed with a 1 N aqueous hydrochloric acid solution, brine, dried
over MgSO4 and evaporated to afford the intermediate methyl 3,5-
bis(2-((methylsulfonyl)oxy)ethoxy)benzoate as a faint brown solid
1
(5.25 g, 99% yield). H NMR (500 MHz, CDCl3) δ 7.21 (d, J = 2.4
Hz, 2H), 6.68 (t, J = 2.3 Hz, 1H), 4.58 (m, 4H), 4.28 (m, 4H), 3.90 (s,
3H), 3.10 (s, 6H); HRMS m/z calcd for C14H20O10S2Na [M + Na]+
435.0396, found [M + Na]+ 435.0391. Next, to a solution of methyl
3,5-bis(2-((methylsulfonyl)oxy)ethoxy)benzoate (5.2 g, 12.62 mmol)
in DMF were added K2CO3 (6.09 g, 44.17 mmol) followed by 4-
hydroxybenzophenone (5.37 g, 27.13 mmol) and stirred at room
temperature for 5 min under N2 atmosphere. Then the reaction
mixture was stirred at 80 °C under a microwave (25 W) irradiation for
60 min (30 min × 2 times) until the disappearance of the starting
material was confirmed by TLC. The reaction mixture was diluted with
water and extracted with ethyl acetate (3 × 100 mL). The organic
layers were combined and washed with brine and then dried over
anhydrous MgSO4. The solvent was removed under a vacuum, and the
Fmoc-N-Me-Cys(Trt)-OAllyl. To a solution of Fmoc-N-Me-Cys-
(Trt)-OH (4.1 g, 6.84 mmol) in ACN (30 mL) at 0 °C was added
slowly N,N-diisopropylethylamine (1.78 mL, 10.25 mmol) followed by
allyl bromide (11.83 mL, 136.74 mmol). The mixture was stirred at
ambient temperature for 16 h until complete conversion was observed
on TLC. The reaction mixture was concentrated to remove ACN and
diluted with EtOAc (100 mL). The solution was subsequently washed
with water, 2.5% aqueous NaHCO3 and brine. The combined organic
F
DOI: 10.1021/acs.joc.5b01055
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
layer was dried over Na2SO4 and concentrated under reduced
pressure. The residue was purified by column chromatography (eluent
20% EtOAc/hexane) to afford Fmoc-N-Me-Cys(Trt)-OAllyl (3.45 g,
80%) as a hygroscopic pale yellow solid. 1H NMR (500 MHz, CDCl3)
δ 7.77(m, 2H)*, 7.58 (d, J = 7.2 Hz, 2H), 7.45−7.36 (m, 9H), 7.30−
7.21 (m, 10H, overlapping with solvent residual peak), 5.79−5.76 (m,
1H), 5.21−5.15 (m, 2H), 4.53−4.35 (m, 4H)*, 4.28 (t, J = 7.2 Hz
0.6H), 4.21−4.17 (m, 1H), 4.02 (bs, 0.4H), 2.86 (dd, J = 5.0, 13.2, Hz,
0.6H), 2.77 and 2.74* (s, 3H), 2.72−2.66 (m, 1H)*, 2.48* (m, 0.4H).
13C NMR (125.7 MHz, CDCl3) δ 169.7, 169.4*, 156.5, 156.0*, 144.6,
144.1, 141.5, 131.7, 131.6*, 129.8, 129.7*, 128.1, 127.8*, 127.2*,
126.9, 125.3, 125.2*, 120.2*, 120.1, 118.5*, 118.4, 67.9, 67.3, 65.9,
59.8, 59.3*, 47.3, 32.7, 31.3*, 31.2 (*minor rotamer). HRMS m/z
calcd. for C41H37NO4SNa [M + Na]+ 662.2341, found [M + Na]+
378.2336.
or chloranil tests and analyzed by RP-HPLC after cleavage of small
sample of the peptidyl resin (∼1 mg) using TFA/TIS/CH2Cl2
(10:4:86) cocktail. The peptide synthesis transformations and the
resin washings were performed at 25 °C.
Preparation of the Linker Resin (10). The amino methyl resin (1.0
g, 0.9 mmol/g) was swelled in DMF (10 mL) for 15 min. The linker
(9) (654 mg, 1.08 mmol) was activated using PyBOP (702 mg, 1.35
mmol) and DIPEA (469 μL, 2.70 mmol) in DMF (10 mL) and added
to the swelled resin. The progress of the reaction was monitored by
Kaiser test. After 5 h, the solution was removed from the reaction
vessel by filtration and the resin was washed with DMF (5 × 1 min)
and CH2Cl2 (5 × 1 min). The linker attachment was confirmed by IR
analysis and the loading was determined to be 90% (0.81 mmol/g) on
a w/w basis. IR (neat) 3657, 3339, 3025, 2980, 2924, 1663, 1593,
1509, 1492, 1451, 1380, 1246, 1165, 1071, 951, 757 cm−1.
Loading of Fmoc-N-Me-Cys-OAllyl on the Linker Resin to
Produce 11. To the preswelled linker resin 10 (1.0 g, 0.81 mmol/g)
in CH2Cl2 (10 mL) was added trichloroacetonitrile (2.45 mL, 24.3
mmol) and solution of DBU (72.4 μL, 0.49 mmol) in CH2Cl2 (5 mL).
The reaction mixture was shaken for 4 h at room temperature, filtered
and washed with CH2Cl2 (2 × 1 min), DMF (2 × 0.5 min) and
CH2Cl2 (3 × 1 min) sequentially. To the pre swelled trichlor-
oacetimidate resin, Fmoc-N-Me-Cys-OAllyl (1.29 g, 3.24 mmol) in dry
CH2Cl2 (8 mL) was added, followed by addition of TMSOTf (29.3
μL, 0.16 mmol) in dry CH2Cl2 (2 mL). The mixture was shaken for 12
h, filtered and the resin was washed with DMF (5 × 1 min) and
CH2Cl2 (5 × 1 min). The attachment of Fmoc-N-Me-Cys-OAllyl was
confirmed by IR analysis and loading was determined to be 82% on w/
w basis (1.33 mmol/g based on the loaded Cys). IR (neat) 3402, 3059,
3025, 2923, 1721, 1599, 1507, 1493, 1450, 1247, 1168, 1067, 1029,
758, 735 cm−1.
Solid-Phase Synthesis of Dithiol Triostin A 15 (Approach-A).
The Fmoc group of the resin 11 (0.33 mmol based on the loaded Cys)
was removed by 20% piperidine/DMF and the resin was washed with
DMF (4 × 1 min), CH2Cl2 (4 × 1 min). Fmoc-D-Ser-Ala (263 mg,
0.66 mmol) was coupled using HATU (251 mg, 0.66 mmol), HOAt
(89.8 mg, 0.66 mmol) and DIPEA (229 μL, 1.32 mmol) in DMF (3
mL) and the mixture was stirred for 1 h until negative Kaiser test was
observed. The mixture was filtered and the resin was washed with
DMF (5 × 1 min), CH2Cl2 (5 × 1 min) to obtain resin-bound peptide
12. A small portion of the resin was cleaved and the crude product was
analyzed by RP-HPLC (tR = 8.1 min, condition A). Next, the Fmoc
group was removed and the resin was washed with DMF (4 × 1 min)
and CH2Cl2 (4 × 1 min). The solution of 2-Quinoxaline carboxylic
acid (115 mg, 0.66 mmol), HATU (251 mg, 0.66 mmol), HOAt (89.8
mg, 0.66 mmol) and DIPEA (229 μL, 1.32 mmol) in DMF (3 mL)
was added to the resin and shaken for 50 min. Then the mixture was
filtered and the resin was washed with DMF (5 × 1 min), CH2Cl2 (5 ×
1 min) to obtain resin-bound peptide 13 (tR = 3.0 min, condition A)
along with 13a (tR = 6.5 min, condition A). Undesired resin-bound
peptide 13a was converted back to 13 by an alcoholysis reaction using
K2CO3 (45.5 mg, 0.33 mmol) and allyl alcohol (2.5 mL) with 16 h
shaking. Introduction of Alloc-N-Me-Val to the side chain of the Ser
residue was achieved by mixing the resin with the anhydride solution,
which was prepared in a separate flask by combining Alloc-N-Me-Val
(426 mg, 1.98 mmol), DIC (137 μL, 0.99 mmol), DMAP (3.66 mg,
0.03 mmol) and DIPEA (115 μL, 0.66 mmol) in THF (3 mL) at 0 °C
for 20 min. The mixture was shaken for 12 h, filtered and the resin was
washed with DMF (5 × 1 min) and CH2Cl2 (5 × 1 min) to obtain
resin-bound peptide 14 (tR = 18.8 min, condition B).
Fmoc-N-Me-Cys-OAllyl. To a round-bottom flask containing Fmoc-
N-Me-Cys(Trt)-OH (1.0 g, 1.56 mmol) in CH2Cl2 (10 mL) at 0 °C, a
solution of TFA and TIS in CH2Cl2 (20:5:75, 20 mL) was added
slowly. The solution was stirred for 1 h at room temperature. On
complete conversion of the starting material, the solvent was
evaporated under a reduced pressure. The residue was diluted with
water and extracted with EtOAc (3 × 60 mL). The combined organic
layers were washed with saturated aqueous NaCl (100 mL), dried over
(MgSO4), filtered, and concentrated under a vacuum. The crude
product was immediately used for the SPPS without further
1
purification. H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz,
2H), 7.62−7.58 (m, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz,
2H), 5.90−5.86 (m, 1H), 5.33−5.23 (m, 2H), 4.73 (q, J = 5.4 Hz, 9.8
Hz, 0.6H), 4.65−4.57* (m, 1.4H), 4.52−4.43 (m, 3H), 4.30 (t, J = 6.8
Hz, 0.6H), 4.24* (t, J = 5.6 Hz, 0.4H), 3.14 (m, 0.6H), 2.94−2.86 (m,
4H, overlapping)*, 2.53 (m, 1H)*, 1.48 and 1.34* (t, J = 10.0 Hz,
1H). 13C NMR (125.7 MHz, CDCl3) δ 169.5, 169.2*, 156.9, 156.1*,
144.0, 141.5, 131.6, 131.5*, 127.9, 127.2, 125.2, 124.9, 120.1, 119.1,
119.1*, 68.0, 67.7*, 66.2, 62.4, 62.3*, 47.4, 32.2, 23.9 (*minor
rotamer). HRMS m/z calcd. for C22H23NO4SNa [M + Na]+ 420.1245,
found [M + Na]+ 378.1239.
Fmoc-D-Ser-Ala-OH. Dicyclohexylcarbodiimide (0.76 g, 3.66 mmol)
was added to a solution of Fmoc-D-Ser-OH (1 g, 3.05 mmol) and N-
hydroxysuccinimide (0.37 g, 3.21 mmol) in dry THF (20 mL) at 0 °C
under N2 atmosphere. The mixture was stirred at room temperature
for 18 h and then cooled to 0 °C. The reaction mixture was
concentrated under a vacuum to obtain the crude product which was
used for next step without further purification. To a solution of H-Ala-
OH (0.3 g, 3.36 mmol) and triethylamine (0.51 mL, 3.67 mmol) in
ACN/H2O (1:1, 16 mL) was added a solution of Fmoc-D-Ser-OSu
(1.29 g, 3.05 mmol) in dry THF (8 mL) at 0 °C. The resulting mixture
was stirred for 6 h at room temperature. On complete conversion, the
solvent was removed under a vacuum and 10% citric acid solution was
added to the mixture. The aqueous solution was extracted with EtOAc
(3 × 60 mL). The combined organic layers were washed with brine
(40 mL), dried over Mg2SO4, filtered and concentrated to obtain the
crude product which was further purified by precipitation using a
mixture of CHCl3/hexane (1:9) to afford the desired product as a
1
white solid (1.2 g, quantitative). H NMR (500 MHz, DMSO-d6) δ
8.14 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74 (t, J = 6.4 Hz,
2H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.27 (d, J = 8.5
Hz, 1H), 4.88 (bs, 1H), 4.30−4.11 (m, 5H), 3.58−3.54 (m, 2H,
overlapping with residual water), 1.26 (d, J = 7.3 Hz, 3H). 13C NMR
(125.7 MHz, CDCl3) δ 174.0, 169.9, 155.9, 143.9, 140.8, 127.7, 127.1,
125.4, 125.3, 120.1, 65.8, 62.0, 56.8, 47.6, 46.7, 17.5 (minor aliphatic
impurities are observed). HRMS m/z calcd for C21H22N2O6Na [M +
Na]+ 421.1376, found [M + Na]+ 421.1370.
Solid-Phase Peptide Synthesis. Solid-phase peptide synthesis
was conducted in sintered glass vials using EYELA personal solid-
phase synthesizer (CCS-600). Solvents and soluble reagents were
removed by suction. Removal of the Fmoc group was carried out with
20% piperidine/DMF (10 mL/g, 2 × 8 min and 2 × 5 min). Washings
between deprotection, couplings steps were carried out with DMF (5
× 1 min) and CH2Cl2 (5 × 1 min) using 8 mL solvent/g of resin for
each wash. Reaction completion was determined by qualitative Kaiser
The Alloc and allyl protecting groups were removed by shaking the
resin with Pd(PPh3)4 (57.2 mg, 0.05 mmol) and PhSiH3 (406 μL, 3.30
mmol) in dry CH2Cl2 (3 mL) under nitrogen atmosphere for 30 min.
The process was repeated twice. For the final cyclization, DIC (229
μL, 1.65 mmol) was added to the resin solution in DMF/DMSO (3:2,
1.5 mL), followed by a solution of HOAt (135 mg, 0.99 mmol) and
DIPEA (57.4 μL, 0.33 mmol) in DMF/DMSO (3:2, 1 mL). The
mixture was shaken for 12 h, filtered and the resin was washed with
DMF (5 × 1 min) and CH2Cl2 (5 × 1 min) to obtain resin-bound
peptide 15 and 15a. The peptides were cleaved from the resin by
G
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The Journal of Organic Chemistry
Article
treatment of TFA/TIS/CH2Cl2 (10:4:86) (8 mL, 3 × 20 min) and the
combined solution was evaporated under a vacuum. The crude sample
was purified by semi preparative HPLC (condition C) to provide the
desired dithiol triostin A (15) (7.1 mg; 4.0% yield from the Cys loaded
resin) and the monocyclic tetradepsipeptide (15a) (21 mg; 12% yield
from the Cys loaded resin).
solution was evaporated under a vacuum. The crude sample was
purified by semi preparative HPLC (condition C) to provide the
desired dithiol triostin A (15) (6.5 mg; 9.1% yield from the Cys loaded
resin) and the monocyclic tetradepsipeptide (15a) (6.9 mg, 9.6% yield
from the Cys loaded resin).
Synthesis of Triostin A (1). To a solution of the dithiol triostin A
(15) (8.0 mg, 7.35 μmol) in dry ACN/DMSO (9:1, 1.6 mL) at 0 °C; a
solution of I2 (1.87 mg, 14.70 μmol) in dry ACN (0.8 mL) was added
dropwise over 30 min. The reaction mixture was stirred for 30 min at
ambient temperature to achieve complete conversion of the starting
material into the desired disulfide product, monitored by RP-HPLC.
The reaction mixture was lyophilized to remove organic solvents and
the crude residue was purified by semi prep HPLC (condition D) to
provide the desired triostin A (1) (6.3 mg, 80% yield). (RP-HPLC tR=
1
15: (tR= 12.5 min, condition B); H NMR (500 MHz, CDCl3) δ
9.75 (s, 1H, CH-Ar), 8.64 (d, 1H, J = 8.1 Hz, NH-Ser), 8.22 (d, 1H, J
= 7.9 Hz, CH-Ar), 8.16 (d, 1H, J = 7.7 Hz, CH-Ar), 7.89 (m, 2H, CH-
Ar), 6.84 (d, 1H, J = 9.6 Hz, NH-Ala), 5.53 (t, 1H, J = 7.2 Hz, CHα-
Cys), 5.31 (m, 1H, CHα-Ala), 5.08 (dd, 1H, J = 10.2 Hz, 16.8 Hz,
CHα-Ser), 4.60 (dd, 1H, 1H, J = 6.7 Hz, 9.7 Hz, CHβ-Ser), 4.23 (t, 1H,
J = 10.7 Hz, CHβ-Ser), 4.12 (d, 1H, J = 9.8 Hz, CHα-Val), 3.19 (m,
1H, CHβ-Cys), 3.04 (s, 3H, N-Me-Cys), 2.94 (s, 3H, N-Me-Val),
2.63−2.58 (m, 1H, CHβ-Cys), 2.28−2.07 (m, 1H, CHβ-Val), 1.56−
1.53 (m, 1H, SH-Cys), 1.43 (d, 3H, J = 6.5 Hz, CH3-Ala), 1.02 (d, 3H,
J = 6.3 Hz, CH3-Val), 0.97 (d, 3H, J = 6.5 Hz, CH3-Val). 13C NMR
(150 MHz, CDCl3) δ 172.5, 169.0, 168.9, 168.8, 163.4, 144.2, 143.9,
142.2, 140.4, 132.2, 131.2, 129.9, 129.5, 64.2, 63.7, 55.8, 50.9, 50.8,
50.8, 43.9, 30.8, 30.2, 28.5, 23.7, 19.7, 19.1 17.7. HRMS m/z calcd. for
C50H64N12O12S2Na, 1111.4106 [M + Na]+, found 1111.4104 [M +
Na]+. The compound 15 was also analyzed by 2D NMR spectroscopy
(COSY, HSQC, HMBC) for more structural confirmations (Support-
ing Information).
1
17.2 min, condition B). H NMR (CDCl3, 500 MHz) δ 9.72 (s, 1H,
CH-Ar), 8.63 (d, 1H, J = 8.4 Hz, NH-Ser), 8.21 (m, 1H, CH-Ar), 8.16
(m, 1H, CH-Ar), 7.92−7.86 (m, 2H, CH-Ar), 6.74 (bs, 1H, NH-Ala),
5.79 (t, 1H, J = 7.3 Hz, CHα-Cys), 5.29 (m, 1H, CHα-Ala), 5.06 (m,
1H, CHα-Ser), 4.59 (m, 1H, CHβ-Ser), 4.23 (t, 1H, J = 10.6 Hz, CHβ-
Ser), 4.18 (d, 1H, J = 9.8 Hz, CHα-Val), 3.28 (dd, 1H, J = 7.4 Hz, J =
13.8 Hz, CHβ-Cys), 3.05 (s, 3H, N-Me-Cys), 2.94 (s, 3H, N-Me-Val),
2.97−2.90 (m, 1H, CHβ-Cys, overlap with N-Me-Val), 2.30−2.26 (m,
1H, CHβ-Val), 1.41 (d, 3H, J = 6.6 Hz, CH3-Ala), 1.03 (d, 3H, J = 6.3
Hz, CH3-Val), 0.96 (d, 3H, J = 6.6 Hz, CH3-Val). HRMS m/z for
C50H62N12O12S2Na [M + Na]+ calcd 1109.3949, found 1109.3947.
Synthesis of Monocyclic Tetradepsipepide Dimer (15b). Pure
monocyclic tetradepsipeptide free thiol (15a) (16 mg, 0.029 mmol)
was oxidized by following the same procedure explained above to
obtain monocyclic tetradepsipeptide dimer (15b). The reaction
mixture was lyophilized to remove organic solvent and obtained
crude residue was redissolved in minimum amount of ACN/DMSO
(3:1) to purify by semi prep HPLC (condition D). Purification
provided monocyclic tetradepsipeptide dimer (15b) (12.1 mg) in 76%
1
15a: (tR= 11.6 min, condition B); H NMR (500 MHz, CDCl3) δ
9.64 (s, 1H, CH-Ar), 8.71 (d, 1H, J = 6.6 Hz, NH-Ser), 8.25 (d, 1H, J
= 8.1 Hz, CH-Ar), 8.05 (d, 1H, J = 8.1 Hz, CH-Ar), 7.96−7.89 (m,
2H, CH-Ar), 6.80 (d, 1H, J = 9.8 Hz, NH-Ala), 5.70 (dd, 1H, J = 6.0
Hz, 8.8 Hz, CHα-Cys), 5.30 (q, 1H, J = 6.7 Hz, 9.5 Hz, CHα-Ala), 5.06
(d, 1H, J = 10.5 Hz CHα-Val), 4.96 (m, 1H, CHβ-Ser), 4.89 (m, 1H,
CHα-Ser), 4.62 (dd, 1H, J = 1.9 Hz, 11.4 Hz, CHβ-Ser), 3.20−3.17 (m,
1H, CHβ-Cys), 3.05 (s, 3H, N-Me-Cys), 2.99 (s, 3H, N-Me-Val), 2.61
(m, 1H, CHβ-Cys), 2.37−2.35 (m, 1H, CHβ-Val), 1.52 (t, 1H, J = 9.3
Hz, SH-Cys), 1.34 (d, 3H, J = 6.6 Hz, CH3-Ala), 1.14 (d, 3H, J = 6.2
Hz, CH3-Val), 0.95 (d, 3H, J = 6.7 Hz, CH3-Val). 13C NMR (150
MHz, CDCl3) δ 172.9, 171.3, 169.3, 167.1, 164.4, 144.4, 143.6, 142.1,
140.3, 132.5, 131.5, 129.8, 65.7, 62.3, 55.3, 54.7, 45.3, 31.3, 30.9, 26.7,
22.5, 20.2, 18.7, 16.4. HRMS m/z calcd. for C25H33N6O6S, 545.2182
[M +H]+, found 545.2182 [M + H]+.
Solid-Phase Synthesis of Dithiol Triostin A 15 (Approach-B).
Introduction of Alloc-N-Me-Val to the side chain of the Ser residue
was achieved by mixing the peptidyl resin 12 (0.13 mmol based on the
loaded Cys) with the anhydride solution, which was prepared in a
separate flask by combining Alloc-N-Me-Val (168 mg, 0.78 mmol),
DIC (54.1 μL, 0.39 mmol), DMAP (1.58 mg, 0.013 mmol) and
DIPEA (22.6 μL, 0.13 mmol) in THF (1.5 mL) at 0 °C for 20 min.
The mixture was shaken for 12 h, filtered and the resin was washed
with DMF (5 × 1 min) and CH2Cl2 (5 × 1 min) to obtain resin-
bound peptide 16 (tR = 16.2 min, condition A). The Alloc and allyl
protecting groups were removed by shaking the resin with Pd(PPh3)4
(22.5 mg, 0.020 mmol) and PhSiH3 (160 μL, 1.30 mmol) in dry
CH2Cl2 (1.5 mL) under nitrogen atmosphere for 30 min. The process
was repeated twice and the resin was washed with DMF (5 × 1 min),
CH2Cl2 (5 × 1 min). For the cyclization step, DIC (90.1 μL, 0.65
mmol) was added to the resin solution in DMF/DMSO (3:2, 0.6 mL),
followed by a solution of HOAt (53.0 mg, 0.39 mmol) and DIPEA
(230 μL, 0.13 mmol) in DMF/DMSO (3:2, 0.4 mL). The mixture was
shaken for 12 h, filtered and the resin was washed with DMF (5 × 1
min) and CH2Cl2 (5 × 1 min) to obtain the resin-bound cyclic peptide
17 (tR = 17.9 min, condition A) and 17a (tR = 18.6 min, condition A).
For the final Qxc acid coupling, the Fmoc group of cyclized peptide
was removed and the resin was washed with DMF (4 × 1 min) and
CH2Cl2 (4 × 1 min). The solution of 2-Quinoxaline carboxylic acid
(45.3 mg, 0.26 mmol), HATU (98.8 mg, 0.26 mmol), HOAt (35.4 mg,
0.26 mmol) and DIPEA (67.8 μL, 0.39 mmol) in DMF (1.5 mL) was
added to the resin and shaken for 1 h. Then the mixture was filtered
and the resin was washed with DMF (5 × 1 min), CH2Cl2 (5 × 1 min)
to obtain resin-bound peptide 15 and 15a.
1
yield (RP-HPLC tR= 16.3 min, condition B). H NMR (CDCl3, 500
MHz) δ 9.65 (s, 1H, CH-Ar), 8.66 (d, 1H, J = 7.0 Hz, NH-Ser), 8.25
(m, 1H, CH-Ar), 8.04 (m, CH-Ar), 7.97−7.92 (m, 2H, CH-Ar), 6.38
(d, 1H, 10 Hz, NH-Ala), 6.07 (dd, 1H, J = 6 Hz, 9 Hz CHα-Cys), 5.22
(m, 1H, CHα-Ala), 5.04 (d, 1H, 10.5 Hz, CHα-Ser), 4.94−4.89 (m,
β
2H, CH2 -Ser, overlap), 4.60 (dd, 1H, J = 2.3, 11.2 Hz, CHα-Val), 3.27
(dd, 1H, J = 9 Hz, 13.3 Hz, CHβ-Cys), 3.06 (s, 3H, N-Me-Cys), 2.03
(s, 3H, N-Me-Val), 2.95 (dd, 1H, J = 6 Hz, 13.3 Hz, CHβ-Cys), 2.34−
2.32 (m, 1H, CHβ-Val), 1.32 (d, 3H, J = 6.7 Hz, CH3-Ala), 1.11 (d,
3H, J = 6.4 Hz, CH3-Val), 0.93 (d, 3H, J = 6.7 Hz, CH3-Val). 13C
NMR (150 MHz, CDCl3) δ 173.5, 171.2, 169.1, 167.4, 164.4, 144.0,
143.3, 142.4, 140.5, 132.7, 131.7, 129.8, 129.4, 65.74, 62.42, 54.84,
52.35, 45.23, 36.09, 31.46, 31.20, 26.74, 20.17, 18.72, 16.24. HRMS m/
z for C50H62N12O12S2Na [M + Na]+ calcd 1109.3949, found
1109.3949.
Preparation of NEM Adduct of 15a. To a pure sample of
monocyclic tetradepsipeptide (15a) (0.4 mg, 0.74 μmol) in ACN (100
μL) was added N-ethylmaleimide (10 mM in DMF, 100 μL) and the
mixture was stirred for 4 h at room temperature. The reaction was
monitored by RP-HPLC. Starting was completely disappeared and
new peaks were purified by analytical RP-HPLC. Desired NEM adduct
of 15a (tR= 12.1 min, condition B) was confirmed by mass analysis
(Supporting Information, Page no. S7). HRMS m/z for C31H40N7O8S
[M + H]+ calcd 670.2659, found 672.2659.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supporting Figures S1−S32 including copies of 1H, 13C and 2D
NMR, HPLC and, HRMS spectra for all required compounds.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b01055.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: dylim@sejong.ac.kr.
Finally, the peptides were cleaved from the resin by treatment of
TFA/TIS/CH2Cl2 (10:4:86) (4 mL, 3 × 20 min) and the combined
H
DOI: 10.1021/acs.joc.5b01055
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The Journal of Organic Chemistry
Article
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (2009-0077608 and 2012R1A1A2008046). HRMS
(ESI) data were obtained using the facilities of the Korea Basic
Science Institute.
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DOI: 10.1021/acs.joc.5b01055
J. Org. Chem. XXXX, XXX, XXX−XXX