Total synthesis and structural validation of cyclodepsipeptides solonamide A and B

Article abstract of DOI:10.1016/j.tet.2014.05.107

Microorganisms are an attractive source of new natural products with antimicrobial properties, and the marine environment constitutes a prolific resource of bioactive microorganisms. During a global research expedition (Galathea III), two depsipeptides, solonamide A and solonamide B, were isolated from the marine bacterium Photobacterium halotolerance and were found to inhibit virulence gene expression in the serious human pathogen, Staphylococcus aureus. They act by interfering with the agr quorum sensing system and show resemblance to the endogenous S. aureus quorum sensing peptide, autoinducing peptide I (AIP-I). To enable more comprehensive studies, we embarked on the chemical synthesis of solonamides A and B. The key synthetic steps were formation of the (R)-β-hydroxy-fatty-acids by stereo-selective aldol reactions and a cyclative macrolactamization, which proceeded under highly dilute conditions. Thus, the first total syntheses of the solonamides corroborated the originally assigned structures, and by changing the stereochemistry of the auxiliary in the aldol steps we gained access to the natural products as well as their β³-epimers.

Full text of DOI:10.1016/j.tet.2014.05.107

Tetrahedron xxx (2014) 1e12
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis and structural validation of cyclodepsipeptides
solonamide A and B
a
,
, ,
y
Betul Kitir y, Mara Baldry b, Hanne Ingmer b, Christian A. Olsen a
*
€
a Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kongens Lyngby, Denmark
b Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Microorganisms are an attractive source of new natural products with antimicrobial properties, and the
marine environment constitutes a prolific resource of bioactive microorganisms. During a global re-
search expedition (Galathea III), two depsipeptides, solonamide A and solonamide B, were isolated from
the marine bacterium Photobacterium halotolerance and were found to inhibit virulence gene expression
in the serious human pathogen, Staphylococcus aureus. They act by interfering with the agr quorum
sensing system and show resemblance to the endogenous S. aureus quorum sensing peptide, auto-
inducing peptide I (AIP-I). To enable more comprehensive studies, we embarked on the chemical syn-
Received 29 April 2014
Received in revised form 25 May 2014
Accepted 27 May 2014
Available online xxx
Keywords:
Natural products
Depsipeptides
Macrocycles
Quorum sensing
Virulence
thesis of solonamides A and B. The key synthetic steps were formation of the (R)-b-hydroxy-fatty-acids
by stereo-selective aldol reactions and a cyclative macrolactamization, which proceeded under highly
dilute conditions. Thus, the first total syntheses of the solonamides corroborated the originally assigned
structures, and by changing the stereochemistry of the auxiliary in the aldol steps we gained access to
the natural products as well as their b
3-epimers.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
depsipeptides, solonamides A and B, were discovered (Fig. 1).18 Both
of these macrocycles were shown to inhibit virulence of S. aureus by
interfering with the agr quorum sensing system,19 and access to
these compounds through total synthesis was therefore desirable. In
this present study, we report the first total syntheses of the solo-
namides, which confirm the initial structural assignment of both
natural product cyclodepsipeptides.
The Gram-positive bacterium Staphylococcus aureus is a major
human pathogen worldwide, being the leading cause of a variety of
infections, such as pneumonia, bacteremia, and endocarditis.1e5 The
pathogenicity of S. aureus is largely attributed to its ability to produce
a variety of toxins and virulence factors, including those believed to
induce immune responses and spread within the body.6e8 In many
cases, infections can be treated successfully using conventional an-
tibiotics, however, strains with resistance to known antibiotics have
emerged.9 Therefore, development of new therapeutic approaches
that suppress S. aureus pathogenicity through mechanisms that are
non-bactericidal, like anti-virulence approaches, could provide new
options for treating infections caused by resistant strains.10,11
A
promising example of such an approach is targeting of quorum
sensing (QS) pathways.12e14 Quorum sensing inhibitors (QSI) are not
intended to kill or inhibit the growth of a pathogen, but rather target
the expression or the activity of virulence factors,10,15e17 which have
been speculated to circumvent resistance development if non-
essential to the microbe. In a recent search for compounds that re-
duce virulence gene expression in S. aureus, two promising
Fig. 1. Structures of target compounds.
* Corresponding author. Tel.: þ45 4525 2105; e-mail addresses: cao@sund.ku.dk,
2. Results and discussion
cao@kemi.dtu.dk (C.A. Olsen).
y
Present address: Center for Biopharmaceuticals and Department of Drug Design
The solonamides were proposed to be macrocyclic depsipep-
tides containing four D- or L-amino acids and a b-hydroxy acid, the
and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Co-
penhagen, Denmark.
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.05.107
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2
B. Kitir et al. / Tetrahedron xxx (2014) 1e12
only difference between the two natural products being the length
of the hydrocarbon side chain in the -hydroxy acid moiety (Fig. 1).
We decided to first prepare a pair of diastereomeric macrocycles
using commercially available -amino acids and a racemic -hy-
droxy acid with the hydrocarbon length of solonamide B. This
would allow us to address the stereochemical assignment of the
and its epimer in 10%, which was found to be a satisfactory yield for
coupling and macrolactamization. The complete overlap observed
between the natural product (blue spectrum) and one of the syn-
thetic isomers (black spectrum) show that the original stereo-
b
a
b
chemical assignment of all
validation of the stereochemistry of the
chemical identity of the -hydroxy acid also needed validation
through chemical synthesis.
a-amino acids were correct. Thus, after
a
-
a-amino acids, the stereo-
amino acids as well as the disconnection/cyclization point in the
macrocycle.
b
Scheme 1. Solid-phase synthesis of peptide 3. Reagents and conditions: (a) piperidineeDMF (1:4); (b) DBUepiperidineeDMF (2:2:96); (c) Fmoc-D-Leu-OH (3 equiv), HATU
(2.95 equiv), 2,6-lutidine (6 equiv), DMF; (d) Fmoc-L-Phe-OH (3 equiv), HATU (2.95 equiv), 2,6-lutidine (6 equiv), DMF.
Scheme 2. Synthesis of diastereomeric mixture of solonamide B and b
3-epi-solonamide B. Reagents and conditions: (a) SnCl2 (0.1 equiv), ethyl diazoacetate (1 equiv), CH2Cl2; (b)
Bu4NBH4 (8 equiv), AcOH, MeOH; (c) LiOH (1.5 equiv, 1 M in H2O), THF; (d) BnBr (1.1 equiv), CsCO3 (0.6 equiv), DMF; (e) Boc-L-Leu-OH (1.2 equiv), DIC (2 equiv), DMAP (0.1 equiv),
CH2Cl2; (f) Pd/C (0.1%/w/w, 0.1 equiv), H2 (1 atm), EtOH; (g) C-terminally 2-chlorotrityl-resin-bound H-L-Phe-D-Leu-D-Ala (3), HATU (2 equiv), iPr2EtN (4 equiv), DMF; (h)
i
TFAeCH2Cl2 (1:1); (i) HATU (2 equiv), Pr2EtN (4 equiv), DMF (0.5 mM), room temperature, 16 h.
Our strategy for this initial synthesis was thus based on assembly
of two building blocks; resin-bound tripeptide H- -Phe- -Leu- -Ala-
OH (3; Scheme 1) prepared on a 2-chlorotrityl linker by standard
Fmoc chemistry and a dimer (5) containing the ester (‘depsi’) bond,
To achieve stereoselective total syntheses, we therefore pre-
pared the hydroxy acids by aldol reactions using chiral auxiliaries
(Scheme 3). We decided to obtain both enantiomers of both hy-
droxy acids, and thus first prepared the tert-butylglycine-derived
Evans’ auxiliary, (S)-4-(tert-butyl)oxazolidin-2-one.28 However,
this did not give rise to satisfactory diastereoselectivity in our
hands. Next, (R)- and (S)-4-benzylthiazolidine-2-thione were used
as auxiliaries in aldol reactions with butanal or hexanal (Scheme 3A
and B). Commercially available (S)-4-benzylthiazolidine-2-thione
(6) was acetylated to give 7, which furnished the aldol adducts 8
and 9 in diastereoselectivities of 6:1e3:1 (Scheme 3A). Removal of
L
D
D
which was obtained by coupling of Boc-
L
-leucine and (ꢀ)-
b-
hydroxyoctanoic acid (Hoa; 4) (Scheme 2). Incorporation of the ester
bond in the building block rather than having to rely on a macro-
lactonization in the final cyclization step was chosen as this has been
shown to be difficult and low yielding in similar syntheses.20 An
efficient three-step route afforded the racemic
b-hydroxyoctanoic
acid from hexanal and ethyl diazoacetate, first giving ethyl
b-
ketooctanoate under conditions reported by Holmquist and Ros-
kamp.21 An initial yield of 47% in this step was improved to 90% by
using excess of hexanal. Next, the ketone was reduced using excess
the auxiliary by hydrolysis afforded b-hydroxy acids (S)-10 and (S)-
11, for which the stereochemistry was confirmed by comparison of
specific optical rotations to literature values. The (R)-4-
Bu4NBH4 to give the ethyl
b-hydroxy-octanoate in 80% yield,
benzylthiazolidine-2-thione auxiliary was prepared from
D-phe-
whereas alternative reducing agents NaBH4, LiBH4 or NaBH(OAc)3
proved inefficient for this transformation. Finally, hydrolysis of the
ethylester gave the desired acid 4 in good overall yield. The car-
nylalanine by reduction to 12 ( -phenylalaninol), which was then
D
refluxed in the presence of CS2 under basic aqueous conditions to
give (R)-4-benzylthiazolidine-2-thione in good yield. Acetylation to
give compound 13 was followed by aldol reactions with butanal or
hexanal and cleavage of the auxiliary by hydrolysis as described
boxylate was then benzyl protected selectively and Boc-L-leucine
was coupled to the hydroxy group followed by catalytic hydroge-
nation to give building block 5 in 36% overall yield for three steps.
Coupling of 5 to resin 3 afforded a resin-bound pentamer, which
upon cleavage and direct macrocyclization under dilute conditions
using HATU as the coupling reagent22e26 gave a mixture of 2 and
vide supra (Scheme 3B). The stereochemistry of b-hydroxy acids
(R)-10 and (R)-11 were also validated by comparison of optical
rotation to literature values, showing that the acids had the ex-
pected configurations.
During purification of the aldol adducts by column chroma-
tography, partial hydrolysis was observed presumably due to the
slight acidity of the silica. This resulted in somewhat lowered yields
and purification difficulties due to co-elution of free auxiliary;
however, subsequent hydrolysis and purification afforded the pure
diastereoisomer
b
3-epi-2 (Scheme 2). The crude mixture of com-
pounds was subjected to preparative reversed-phase HPLC applying
multiple injections due to the poor solubility of these ‘lipo’-dep-
sipeptides.27 We succeeded in isolating two macrocyclic depsipep-
tides, which upon characterization by NMR and HRMS as well as
comparison with a 1H NMR spectrum of authentic solonamide B
b
-hydroxy acids.
With the -hydroxy acids in hand, we prepared dimeric building
blocks 18 and 19 for synthesis of the natural products and building
proved to be compound 2 and the diastereoisomer
b
3-epi-2 (Fig. 2).
b
Synthetic solonamide B (2) was obtained in 13% yield (from resin 3)
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B. Kitir et al. / Tetrahedron xxx (2014) 1e12
3
Fig. 2. Overlays of NMR spectra of synthetic materials with that of the natural solonamide B isolated from cell culture. Synthetic solonamide B from this experiment contains w4% of
3-epi-solonamide B. Solonmaide B and all additional compounds obtained from the stereoselective syntheses below were purified to >98% HPLC and NMR purity.
b
Scheme 3. Stereoselective synthesis of hydroxy acids Hha (10) and Hoa (11). Reagents and conditions: (a) AcCl (1.5 equiv), DMAP (0.1 equiv), NEt3 (1.5 equiv), CH2Cl2; (b) butanal or
i
hexanal (1 equiv), TiCl4 (1.8 equiv), PrEtN (1.8 equiv), CH2Cl2; (c) LiOH (4 equiv, 1 M, H2O), THF; (d) NaBH4 (2.3 equiv), I2 (1 equiv), CH2Cl2; (e) CS2 (5 equiv), KOH (5 M).
blocks 22 and 23 for synthesis of the corresponding
b
3-epimers.
Coupling of each to the resin-bound tripeptide (3) followed by
Three-step sequences afforded the dimer building blocks contain-
ing Boc-protected amines to enable concomitant N-terminal
deprotection during cleavage of linear pentamers from the resin in
the later stage of syntheses. Thus, benzylester protection, coupling
of Boc-L-Leu-OH, and hydrogenation furnished the desired four
dimers in good yields (Scheme 4).
cleavage/deprotection and macrolactamization of the crude linear
precursors afforded 1 and 2 (Scheme 4A) as well as b -
3-epi-1 and b3
epi-2 (Scheme 4B). Spectral data of 1 and 2 were in complete
agreement with those originally reported for the solonamides
isolated from natural sources, thus unequivocally validating the
original assignment of the structures of solonamides A and B.
Scheme 4. Synthesis of solonamides A and B as well as their
b b-hydroxy acids. Reagents and conditions: (a) BnBr (1.1 equiv), CsCO3
3-epimers using enantiomerically pure
(0.6 equiv), DMF; (b) Boc-L-Leu-OH (1.2 equiv), DIC (2 equiv), DMAP (0.1 equiv), CH2Cl2; (c) Pd/C (10% w/w), H2 (1 atm), EtOH; (d) resin-bound H-L-Phe-D-Leu-D-Ala-OH (3), HATU
i
i
(2 equiv), PrEtN (4 equiv), DMF; (e) TFAeCH2Cl2, 1:1; (f) HATU (2 equiv), PrEtN (4 equiv), DMF (0.1 mM).
Please cite this article in press as: Kitir, B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.107

4
B. Kitir et al. / Tetrahedron xxx (2014) 1e12
Furthermore, synthetic material for further biological testing was
obtained. However, although the cyclizations proceeded with full
conversion of linear precursors according to LCeMS analyses, the
isolated yields were relatively low. Since the identity of linear resin-
bound trimer (3), which was a common intermediate, had been
checked, we saw only three possible explanations, or a combination
thereof, for the low yields achieved. Although the linear peptides
were fully converted during macrocyclization, unindentified
byproducts were present in the crude pointing to difficulties during
the preceding dimer coupling or side reactions during cleavage. The
final reason for the low isolated yields could be incompatibility of
epi-2). This was surprising as the carboxylic acid and not the hy-
droxy group was activated in the coupling to give the dimer
building block. Thus, to test if the epimerization had occurred
during protecting group manipulation in the dimer (25) synthesis,
we coupled the
b-hydroxy acid directly on to the resin (3) followed
by coupling of Boc-
L-leucine. Unfortunately, a test cleavage of this
linear pentamer also revealed 4 compounds with the same mass,
which was puzzling since cleavage of the tripeptide precursor only
showed one peak in the LC-MS. Applying the follow-up solution-
phase synthesis approach, we were unfortunately not able to im-
prove the overall yields of macrocyclic peptides.
Scheme 5. Alternative solution-phase synthesis of solonamide B (2). Reagents and conditions: (a) TFAeCH2Cl2 (1:1), Et3SiH (10 equiv); (b) PyBOP (1.2 equiv), iPrEtN (1.2 equiv), DMF;
(c) Pd/C (0.1% w/w, 0.1 equiv), H2 (1 atm), EtOH; (d) HATU (2 equiv), iPrEtN (4 equiv), DMF.
highly lipophilic compounds with aqueous reversed-phase purifi-
cation, and the macrocycles in this study were poorly soluble in
both water and acetonitrile. In general, we were able to recover
more material when attempting purification by column chroma-
tography, however, this did not provide the final compounds in
satisfying purity for biological testing, and thus HPLC purification
was applied despite potential loss of material. In the future, we
therefore envision that purification by normal-phase preparative
HPLC may enable improved isolated yields of these compounds.
Nevertheless, we also decided to address the possible in-
efficiencies in the synthetic approach discussed above, and hence
devised an alternative solution-phase strategy. This would cir-
cumvent cleavage issues and reveal putative problems with the
peptide coupling applied to assemble the linear pentamer. Thus,
a tripeptide (26) was prepared in solution and dimer (25) was
synthesized from (R)-11 applying a different protecting group
strategy than described above. The hydroxy acid was first PMB-
Finally, the biological activity of our synthetic solonamide A and
B was verified and compared to the samples obtained by isolation
from culture. An agar diffusion assay was conducted18 that moni-
tored the expression of the S. aureus virulence genes hla (encoding
a-hemolysin) and spa (encoding Protein A) for both synthetic and
natural solonamides along with linear solonamide B as negative
control. Correlating with agr inhibition, we observed an increased
expression of spa and decreased expression of hla with the same
potency for the synthetic and natural solonamides, thus verifying
that the synthetic solonamides retain biological activity (Fig. 3).
protected, then coupled to Cbz-L-Leu-OH, finally deprotected un-
der acidic conditions to afford dimer 23 in 52% yield. A selection of
coupling reagents (i.e., DIC, EDC, HATU, and PyBOP) was tested for
condensation of 25 and 26, and PyBOP gave the best purity (Scheme
5). Unfortunately, purification at this stage proved to be difficult as
well, since column chromatography provided the linear pentamer
in impure form after attempts with several eluent systems.
Therefore we chose to continue with the crude pentamer and hy-
drogenation proceeded smoothly to give the linear precursor ready
for macrocyclization. It was decided to perform this important step
using two different strategies; (A) by slow addition of the linear
Fig. 3. Biological validation of the synthetic material compared with the natural iso-
lates. Agar plates containing the hla-lacZ (PC322) or spa-lacZ (PC203) reporter strains
from S. aureus were subjected to DMSO (20
mL) containing the noted compound
€
peptide to a solution of Hunig’s base and HATU using a syringe
(0.5 mg/mL). Vehicle (DMSO), linear solonamide B, and autoinducing peptide I (AIP-I)
were applied as controls.
pump as described by Ganesan and co-workers for similar macro-
cyles;29 and (B) by using dilute conditions (0.5e1 mM) as described
above. LCeMS analyses of the two reaction mixtures did not show
significant difference between the cyclizations and no oligomeri-
zation/dimerization were observed. However, upon purification
using preparative reversed-phase HPLC, four peaks were isolated
3. Conclusion
and analyzed, proving to be compounds 2 and b
3-epi-2 along with
In summary, we report the first total syntheses of macrocyclic
depsipeptide marine natural products solonamides A and B, which
validate the original structural assignments unequivocally. Our
synthetic efforts furthermore enabled preparation of unnatural
two additional isomers with the same mass as solonamide B and
similar proton NMR spectra. This indicated that the compound had
epimerized in two positions leading to four diastereoisomers, two
of which could be identified as 2 and its homologue epimerized at
synthetic analogues, which were epimerized at the
-hydroxy acid residue, by simply changing the stereochemistry of
b
3-position of the
b -
-hydroxy acid residue (b3
b
the eCHOCOe chiral center in the (R)-
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B. Kitir et al. / Tetrahedron xxx (2014) 1e12
5
the chiral auxiliaries used to prepare the
b
-hydroxy acids. A scale-up
4.2. Solid-phase peptide synthesis of 2-chlorotrityl-resin-
bound tripeptide (H- -Phe- -Leu- -Ala-O-Trt(Cl)-resin) (3)
strategy based entirely on solution-phase chemistry proved to be
more challenging than expected due to epimerization problems at
several stereocenters, but the most important challenge related to
these targets remain to be the final purification step.
L
D
D
Polystyrene 2-chlorotrityl chloride resin was added to a fritted
syringe and swelled in dry CH2Cl2. A solution of Fmoc- -Ala-OH
D
(1.2 g, 4 mmol, 2.5 equiv) and iPrEtN (1.4 mL, 8 mmol, 5 equiv) in dry
CH2Cl2 were added to the resin and the loading step was allowed to
proceed on a rocking table for 1 h. After washing with CH2Cl2 (ꢁ3),
the resin was capped with CH2Cl2eMeOHeiPrEtN (7:2:1) for 30 min
and washed with DMF (ꢁ3), MeOH (ꢁ3), and CH2Cl2 (ꢁ3). The Fmoc
group was removed with piperidineeDMF (1:4, 4 mL, 2ꢁ30 min)
and DBUepiperidineeDMF (2:2:96, 4 mL, 30 min), and the resin was
4. Experimental section
4.1. General
All chemicals and solvents were analytical grade and used
without further purification. Vacuum liquid chromatography (VLC)
was performed on silica gel 60 (particle size 0.015e0.040 mm).
Flash chromatography was performed on silica gel 60 (particle size
0.035e0.070 mm). UPLC-MS analyses were performed on a Phe-
washed as described above. Fmoc-
in DMF (6 mL) was preincubated for 10 min with 2,6-lutidine
(1.1 mL, 9.6 mmol, equiv) and HATU (1.8 g, 4.7 mmol,
D-Leu-OH (1.7 g, 4.8 mol, 3 equiv)
6
2.95 equiv) before addition to the resin and the reaction was allowed
to proceed on a rocking table for 17 h. After applying the standard
washing procedure outlined above the Fmoc group was removed
nomenex Kinetex column (1.7
m
m, 50ꢁ2.10 mm) using a Waters
Acquity ultra high-performance liquid chromatography (UPLC)
system. Gradient A with eluent I (0.1% HCOOH in water) and eluent
II (0.1% HCOOH in acetonitrile) rising linearly from 0% to 95% of II
during t¼0.00e5.20 min was applied at a flow rate of 0.6 mL/min.
with piperidineeDMF (1:4,
4
mL, 2ꢁ30 min) and
DBUepiperidineeDMF (2:2:96, 4 mL, 30 min) and then the resin
was again washed with DMF (ꢁ3), MeOH (ꢁ3), and CH2Cl2 (ꢁ3).
Preparative reversed-phase HPLC purification was performed on
Fmoc-L-Phe-OH (1.9 g, 4.8 mmol, 3 equiv), 2,6-lutidine (1.1 mL,
ꢀ
a C18 Phenomenex Luna column (5
m
m, 100 A, 250 mmꢁ20 mm)
9.6 mmol, 6 equiv), and HATU (1.8 g, 4.7 mmol, 2.95 equiv) in DMF
(6 mL) were preincubated for 10 min before addition to the resin and
the coupling reaction was allowed to proceed for 24 h before
washing with DMF (ꢁ3), MeOH (ꢁ3), and CH2Cl2 (ꢁ3). To check the
identity of the resin-bound peptide, a small sample of the resin
(100 mg, w0.86 mmol/g) was cleaved with TFAeCH2Cl2 (1:1, 2 mL,
2ꢁ30 min) and purification of the residue by preparative reversed-
phase HPLC afforded Fmoc-protected tripeptide (23 mg, 46%) as
using an Agilent 1260 LC system equipped with a diode array UV
detector and an evaporative light scattering detector (ELSD). Gra-
dient B with eluent III (watereMeCNeTFA, 95:5:0.1) and eluent IV
(0.1% TFA in acetonitrile) rising linearly from 45% to 95% of IV during
t¼5e35 min, then isocratically at 95% from t¼35e55 min was ap-
plied at a flow rate of 20 mL/min. Analytical HPLC was performed
ꢀ
on
a
C18 phenomenex Luna column (3
m
m, 100 A,
150 mmꢁ4.60 mm) using an Agilent 1100 series system equipped
with a diode array UV detector. Gradient C using eluent III and el-
uent IV rising linearly from 0% to 95% of IV during t¼2e20 min was
applied at a flow rate of 1 mL/min. High-resolution LCeDADeMS
was performed on an Agilent 1100 system equipped with a photo-
diode array detector (DAD) and coupled to an LCT orthogonal time-
of-flight mass spectrometer (Waters-Micromass, Manchester, UK)
with Z-spray electrospray ionization (ESI). Optical rotation was
measured on a Perkin Elmer Polarimeter with DMSO, CHCl3 or
CH2Cl2 as the solvents. Nuclear magnetic resonance (NMR) spectra
were recorded on a Varian Mercury 300 instrument (1H NMR and
13C NMR were recorded at 300 and 75 MHz, respectively). 1D and
2D NMR spectra were recorded on a varian INOVA 500 MHz in-
strument at 500 MHz for 1H and 125 MHz for 13C or on a Bruker
Ascend 400 MHz at 400 MHz for 1H and 100 MHz for 13C. All spectra
were recorded at 298 K. Correlation spectroscopy (COSY) spectra
was recorded with a relaxation delay of 1.5 s before each scan,
a spectral width of 6kꢁ6k, collecting 8 FIDs and 1kꢁ512 datapoints.
Heteronuclear single quantum coherence (HSQC) spectra were
recorded with a relaxation delay of 1.5 s before each scan, a spectral
width of 6kꢁ25k, collecting 16 FIDs and 1kꢁ128 datapoints. Het-
eronuclear 2-bond correlation (H2BC) spectra were recorded with
a relaxation delay of 1.5 s before each scan, a spectral width of
4kꢁ35k, collecting 16 FIDs at 295 K and 1kꢁ256 datapoints. Het-
eronuclear multiple-bond correlation (HMBC) spectra were recor-
ded with a relaxation delay of 1.5 s before each scan, a spectral
width of 6kꢁ35k, collecting 32 FIDs and 1kꢁ256 datapoints. Ro-
tating frame Overhauser effect (ROESY) spectra were recorded with
a relaxation delay of 2 s before each scan, a spectral width of 4kꢁ4k,
collecting 8 FIDs at 295 K, 1kꢁ256 datapoints, and a mixing time of
100 ms or 200 ms. Chemical shifts are reported in parts per million
relative to deuterated solvent peaks as internal standards (dH
DMSO-d6 2.50 ppm; dC DMSO-d6 39.52 ppm; dH CDCl3 7.26 ppm; dC
CDCl3 77.16 ppm). Coupling constants (J) are given in hertz (Hz).
Multiplicities of 1H NMR signals are reported as follows: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet.
a fluffy white material; 1H NMR (400 MHz, DMSO-d6)
d 8.21 (dd,
J¼14.9, 7.9 Hz, 2H), 7.88 (d, J¼7.5 Hz, 2H), 7.67 (dd, J¼16.3, 7.8 Hz,
3H), 7.41 (t, J¼7.5 Hz, 2H), 7.35e7.15 (m, 7H), 4.40e4.26 (m, 2H),
4.24e4.08 (m, 4H), 2.94 (dd, J¼13.4, 5.6 Hz, 1H), 2.80 (dd, J¼13.4,
9.6 Hz,1H),1.46e1.33 (m, 3H),1.29 (d, J¼7.32, 1H), 0.80 (d, J¼6.1, 3H),
0.76 (d, J¼6.1, 3H); 13C NMR (100 MHz, DMSO-d6)
d 174.4, 172.2,
171.6, 156.3, 144.3, 144.2, 141.1, 141.1, 138.2, 129.8, 128.5, 128.1, 127.5,
126.7, 125.8, 120.6, 66.2, 56.7, 50.8, 47.9, 47.0, 41.3, 38.1, 24.3, 23.7,
21.9, 17.4. The N-terminal Fmoc group was removed with piper-
idineeDMF (1:4, 4 mL, 2ꢁ30 min) and DBUepiperidineeDMF
(2:2:96, 4 mL, 30 min) and the resin was washed with DMF (ꢁ3),
MeOH (ꢁ3), and CH2Cl2 (ꢁ3) to give resin 3.
4.3.
-b-Hydroxyoctanoic acid ( -Hoa) (4)
To a solution of ethyl diazoacetate (3.3 mL, 31.6 mmol, 1 equiv)
in dry CH2Cl2 (55 mL) was added SnCl2 (600 mg, 3.2 mmol,
0.1 equiv) under nitrogen atmosphere. A solution of hexanal
(4.7 mL, 37.9 mmol, 1.2 equiv) in dry CH2Cl2 (65 mL) was added
dropwise to the suspension at room temperature. After evolution of
nitrogen had stopped (z2 h), the reaction was transferred to
a separatory funnel and quenched with brine (50 mL). The aqueous
phase was extracted with CH2Cl2 (3ꢁ50 mL), the organic layers
were collected, dried with MgSO4, filtered, and the solvent was
removed under reduced pressure. Purification by flash chroma-
tography (heptaneeEtOAc, 9:1) afforded the
90%) as pale yellow oil; Rf 0.45 (heptaneeEtOAc, 9:1); 1H NMR
(300 MHz, CDCl3)
4.11 (q, 2H), 3.35 (s, 2H), 2.46 (t, J¼7.4 Hz, 3H),
1.51 (t, 2H), 1.28e1.14 (m, 6H), 0.81 (t, 3H); 13C NMR (75 MHz,
CDCl3) 203.2, 167.5, 61.5, 49.5, 43.2, 31.3, 23.3, 22.6, 14.3, 14.1; in
agreement with previously reported data.27 To a solution of the
ketoester (1 g, 5.3 mmol, 1 equiv) in MeOHeAcOH (95$5, 35 mL) at
b-ketoester (5.88 g,
d
d
b
-
0
ꢂC was slowly added Bu4NBH4 (2.75 g, 10.7 mmol, 2 equiv). The
mixture was stirred at 0 ꢂC for 2 h and then another equivalent of
Bu4NBH4 was added, since TLC showed the reaction to be in-
complete. The mixture was then stirred at room temperature
Please cite this article in press as: Kitir, B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.107

6
B. Kitir et al. / Tetrahedron xxx (2014) 1e12
overnight. Another 5 equiv of Bu4NBH4 were added (1 equiv every
hour). The solvent was removed under reduced pressure and the
residue was dissolved in CH2Cl2 (50 mL), washed with 1 M aqueous
HCl (50 mL), which was back extracted with CH2Cl2 (50 mL). The
combined organic layers were dried with Na2SO4, filtered, and
concentrated under reduced pressure. Flash chromatography
155.4, 78.0, 70.6, 52.4, 44.0, 41.0, 33.4, 30.9, 28.1, 24.2, 23.9, 22.7,
13.8; MS calcd for C19H35NO6Naþ [MþNa]þ 396.2, found 396.2.
4.5. Diastereomeric mixture of solonamide B (2) and b
3-epi-
solonamide B (
b
3-epi-2)
(heptaneeEtOAc, 1:10/1:1) afforded the ethyl
yoctanoate (817 mg, 80%) as a colorless oil; Rf 0.36 (heptaneeEtOAc,
1:1); 1H NMR (300 MHz, CDCl3)
b
-hydrox-
Polystyrene
2-chlorotrityl-bound
L
-Phe-
D
-Leu-
D
-Ala
(3)
(0.13 mmol, 1 equiv) was added to a fritted syringe and swelled
with CH2Cl2. Dimer 5 (60 mg, 0.16 mmol, 1.2 equiv) was pre-
incubated for 10 min with PrEtN (85
d
4.15 (q, J¼7.1 Hz, 2H), 4.06e3.88
i
(m, 1H), 2.48 (dd, J¼16.4, 3.3 Hz, 1H), 2.37 (dd, J¼16.4, 8.8 Hz, 1H),
m
l, 0.53 mmol, 4 equiv) and
1.64e1.21 (m, 8H), 0.87 (t, 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3)
HATU (100 mg, 0.27 mmol, 2 equiv) before addition to the resin,
and the reaction was allowed to proceed for 7 h on a rocking table.
The resin was then washed with DMF (ꢁ3), MeOH (ꢁ3), and CH2Cl2
(ꢁ3) and treated with TFAeCH2Cl2 (1:1, 2 mL, 2ꢁ30 min) followed
by washing with CH2Cl2 (3ꢁ2 mL). All the fractions were pooled in
a round-bottomed flask, concentrated under reduced pressure, and
co-evaporated with toluene (ꢁ2), tolueneeCH2Cl2 (1:1, ꢁ2), and
hexaneeCH2Cl2 (1:1, ꢁ2) to furnish a crude linear precursor (40 mg,
51%), which was cyclized without purification. To a stirred solution
of crude linear peptide (40 mg) in DMF (140 mL) was added iPrEtN
d
173.3, 68.2, 60.9, 41.5, 36.7, 31.9, 25.4, 22.8, 14.4, 14.2; in agree-
ment with previous data.30 To the ethyl
b
-hydroxyoctanoate
(817 mg, 4.3 mmol, 1.0 equiv) in THF (22 mL) was added aqueous
LiOH (6.5 mL, 1.0 M, 1.5 equiv) and the mixture was stirred at room
temperature for 1 h. Then the THF was removed in vacuo and the
remaining solution was acidified with concentrated HCl, extracted
with EtOAc (3ꢁ30 mL), and the organic layers were dried with
Na2SO4, filtered, and concentrated to afford the b-hydroxy acid 4
(486 mg, 70%) as a colorless oil, which was used without further
purification; 1H NMR (300 MHz, CDCl3)
d
4.09e3.96 (m, 1H),
(46 ml, 0.26 mmol, 4 equiv) and HATU (50 mg, 0.13 mmol, 2 equiv)
2.55e2.38 (m, 2H), 1.62e1.15 (m, 8H), 0.89 (t, J¼6.3 Hz, 3H); 13C
and the reaction was stirred at room temperature for 16 h. The DMF
was evaporated and the residue was purified by preparative
reversed-phase HPLC to give the two diastereoisomers (5 mg and
4 mg, respectively, total yield of 23%) as white fluffy solids. For full
characterization, see Sections 4.34 and 4.36, respectively.
NMR (75 MHz, CDCl3)
d 178.1, 68.3, 41.3, 36.7, 31.9, 25.3, 22.8, 14.2;
in agreement with previously reported data.31
4.4. 3-(((S)-2-((tert-Butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)octanoic acid (5)
4.6. (S)-1-(4-Benzyl-2-thioxothiazolidin-3-yl)ethanone (7)
To an ice-cooled solution of 4 (284 mg, 1.8 mmol, 1 equiv) in
MeOH (2 mL) was added a solution of CsCO3 (347 mg, 1.1 mmol,
0.6 equiv) in water (1 mL). After 30 min, the solvents were removed
under reduced pressure and the CsCO3 salt was dissolved in DMF
(S)-4-Benzylthiazolidine-2-thione 6 (18.4 g, 88 mmol, 1 equiv),
DMAP (1.1 g, 8.8 mmol, 0.1 equiv) and NEt3 (18.4 mL, 132 mmol,
1.5 equiv) were dissolved in dry CH2Cl2 (240 mL) and cooled to 0 ꢂC.
AcCl (9.4 mL, 132 mmol, 1.5 equiv) was added dropwise and the
reaction was allowed to reach room temperature and stirred
overnight. The mixture was then quenched with satd NH4Cl
(200 mL), diluted with Et2O (300 mL), and the organic phase was
washed with satd CuSO4 (3ꢁ100 mL), water (100 mL), and brine
(100 mL), dried (MgSO4), filtered, and concentrated. Re-
crystallization of the crude material from EtOH afforded acetylated
(10 mL, 0.2 M). Benzyl bromide (232 ml, 2 mmol, 1.1 equiv) was
added and the resulting suspension was stirred for 16 h. The mix-
ture was then quenched with water (30 mL), the aqueous phase
extracted with EtOAc (3ꢁ30 mL), and the combined organic phases
were washed with water (5ꢁ20 mL) and brine (10 mL), dried over
Na2SO4, filtered, and concentrated. Without further purification,
benzylester (267 mg, 1.1 mmol, 1 equiv) and Boc-
1.3 mmol, 1.2 equiv) were dissolved in CH2Cl2 (15 mL, 0.2 M) at 0 ꢂC
and treated with DIC (344 l, 2.1 mmol, 2 equiv) and DMAP (13 mg,
L-Leu-OH (319 mg,
auxiliary 7 (18 g, 81%) as yellow needles; [
a
]
20 þ335ꢂ (c 13, CH2Cl2)
D
20
m
previously reported [
a]
þ211ꢂ (c 10, CHCl3);32 Mp 111e115 ꢂC
D
0.1 mmol, 0.1 equiv) allowing the mixture to reach room temper-
ature while stirring was continued for 16 h. The precipitate was
removed by filtration and washed with CH2Cl2 (10 mL). The com-
bined organic layers were washed with 1 M HCl (10 mL) and brine
(10 mL), dried (Na2SO4), filtered, and concentrated. The crude res-
idue was purified by flash chromatography (2e4% MeOH in CH2Cl2)
to give fully protected dimer (200 mg, 40%) as a colorless oil; Rf 0.48
previously reported 88e90 ꢂC;33 1H NMR (400 MHz, CDCl3)
d
7.37e7.12 (m, 5H), 5.31 (m, 2H), 3.31 (dd, J¼11.5, 7.2 Hz, 1H), 3.15
(dd, J¼13.2, 3.8 Hz, 1H), 2.97 (dd, J¼13.2, 10.6 Hz, 1H), 2.82 (d,
J¼11.5 Hz, 1H), 2.72 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 201.8,
170.8, 136.5, 129.5, 128.9, 127.2, 68.3, 58.3, 36.7, 27.1.
4.7. General procedure for the aldol reactions
(CH2Cl2eMeOH 96:4); 1H NMR (300 MHz, DMSO-d6)
d 7.53e7.27
(m, 5H), 7.19 (d, J¼7.7 Hz, 1H), 5.74 (s, 2H), 5.21e4.93 (m, 1H), 3.88
(ddd, J¼12.7, 11.2, 7.2 Hz, 2H), 2.76e2.52 (m, 2H), 1.66e1.40 (m, 7H),
1.35 (s, 9H), 1.28e1.09 (m, 2H), 0.80 (ddd, J¼13.6, 7.9, 4.7 Hz, 9H);
Acetylated auxiliary (1.7 equiv) was dissolved in CH2Cl2 (0.2 M).
TiCl4 (1.8 equiv) and iPrEtN (1.8 equiv) were added dropwise and
the resulting suspension was cooled to ꢃ78 ꢂC and stirred for 2 h.
The appropriate aldehyde (1 equiv) in CH2Cl2 (0.2 M) was then
added dropwise and the mixture was stirred at ꢃ78 ꢂC for 4e24 h.
The reaction was then quenched with satd NH4Cl (50 mL) and
water (50 mL) before Et2O (200 mL) was added. The organic phase
was washed with water (100 mL) and brine (100 mL), dried
(MgSO4), filtered, and concentrated to give a yellow crude material
(general selectivities obtained: (S,S)-(S,R) 6:1/3:1), which was
purified by column chromatography.
13C NMR (100 MHz, DMSO-d6)
d 170.2, 170.2, 155.4, 135.8, 128.7,
128.5, 128.5, 71.6, 66.7, 52.4, 41.8, 39.3, 33.9, 31.5, 28.4, 24.8, 23.0,
22.5, 22.0, 14.1; MS calcd for C26H41NO6Naþ [MþNa]þ 486.3, found
486.2. This dimer (200 mg, 0.43 mmol, 1 equiv) was dissolved in
EtOH (10 mL, 0.2 M) and degassed with N2. Palladium on activated
carbon (39 mg, 0.1 equiv, 0.1% w/w) was added and the mixture was
degassed with N2 again, before stirring under H2 atmosphere for
16 h. The catalyst was removed by filtration through by a pad of
Celite, which was washed with CH2Cl2 (50 mL). The solvents were
removed under reduced pressure affording title compound 5
(121 mg, 76%) as a colorless oil; 1H NMR (300 MHz, DMSO-d6)
4.8. (S)-1-((S)-4-Benzyl-2-thioxothiazolidin-3-yl)-3-
hydroxyhexan-1-one (8)
d
5.13e4.99 (m, 1H), 3.87 (m, 1H), 2.58e2.29 (m, 2H), 1.46 (m,
J¼23.1 Hz, 11H), 1.34 (m, J¼4.8 Hz, 9H), 1.22 (m, 2H), 0.82 (ddd,
Compound 7 was reacted with butanal according to General
J¼9.8, 6.5, 2.8 Hz, 9H); 13C NMR (75 MHz, DMSO-d6)
d
172.6, 171.4,
procedure
4.7
and
subsequent
flash
chromatography
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B. Kitir et al. / Tetrahedron xxx (2014) 1e12
7
(heptaneeEtOAc, 9:1/5:1) afforded the title compound 8 (2.2 g,
58%)asayellowoil; Rf 0.08(heptaneeEtOAc, 4:1); 1H NMR (400 MHz,
CDCl3) d 138.7, 129.2, 128.6, 126.5, 66.2, 54.2, 40.8; in agreement
with previously reported data.37
CDCl3)
d 7.36e7.07 (m, 5H), 5.33 (m, 1H), 4.19e4.02 (m, 1H),
3.67e2.77 (m, 6H),1.56e1.25 (m, 4H), 0.88 (t, J¼7.0 Hz, 3H); 13C NMR
4.14. (R)-1-(4-Benzyl-2-thioxothiazolidin-3-yl)ethanone (13)
(100 MHz, CDCl3)
d 201.6, 174.0, 136.5, 129.6, 129.1, 127.4, 68.4, 68.3,
45.6, 38.9, 36.9, 32.2, 18.8, 14.1; in agreement with previously re-
D
-Phenylalaninol 12 (13.2 g, 91 mmol, 1 equiv) was dissolved in
ported data.32
aqueous KOH (3 M, 200 mL). CS2 (26 mL, 436 mmol, 5 equiv) was
added and the solution was heated at reflux overnight. The solution
was extracted with CH2Cl2 (3ꢁ200 mL), dried (Na2SO4), filtered,
4.9. (S)-1-((S)-4-Benzyl-2-thioxothiazolidin-3-yl)-3-
hydroxyoctan-1-one (9)
and concentrated to afford (R)-4-benzylthiazolidine-2-thione
20
(15.6 g, 86%), which was used without further purification. [a]
D
Compound
7
was reacted with hexanal according to
ꢃ97ꢂ (c 13, CH2Cl2); Mp 86e87 ꢂC; 1H NMR (400 MHz, CDCl3)
d 8.08
General procedure 4.7 and subsequent flash chromatography
(br s, 1H), 7.46e7.16 (m, 5H), 4.54e4.43 (m, 1H), 3.57 (dd, J¼11.2,
7.7 Hz, 1H), 3.31 (dd, J¼11.2, 6.7 Hz, 1H), 3.03 (ddd, J¼34.4, 13.6,
(heptaneeEtOAc, 9:1/5:1) afforded the title compound 9 (2.2 g,
20
54%) as a yellow oil; [
CDCl3)
a]
ꢃ11ꢂ (c 14, CH2Cl2); 1H NMR (400 MHz,
7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3)
d 200.9, 135.9, 129.2, 129.1,
D
d
7.80 (s, 1H), 7.42e7.05 (m, 5H), 5.32 (m, 1H), 4.14e3.97 (m,
127.5, 65.2, 40.0, 38.1; in agreement with previously reported
data.38 The auxiliary (15.6 g, 75 mmol, 1 equiv), DMAP (912 mg,
7.5 mmol, 0.1 equiv), and NEt3 (16 mL, 112 mmol, 1.5 equiv) were
dissolved in dry CH2Cl2 (200 mL) and cooled to 0 ꢂC. AcCl (8 mL,
112 mmol, 1.5 equiv) was added dropwise and the reaction was
allowed to reach room temperature and stirred overnight. Then, the
reaction was quenched with satd NH4Cl (200 mL), diluted with Et2O
(200 mL), and the organic phase was washed with satd CuSO4
(3ꢁ100 mL), water (100 mL), and brine (100 mL), dried (MgSO4),
filtered, and concentrated to give the crude compound as a yellow
solid. Recrystallization from EtOH afforded the title compound 13
1H), 3.64e2.70 (m, 6H), 1.61e1.13 (m, 8H), 0.82 (m, 3H); 13C NMR
(100 MHz, CDCl3) 201.1, 173.4, 135.9, 129.2, 129.0, 127.5, 67.6, 65.1,
d
45.9, 40.2, 38.3, 36.9, 32.1,18.8,14.0; UPLC-MS gradient A tR 2.18 min
(>95%); MS calcd for C18H26NO2S2 [MþH]þ 352.1, found 352.1.
4.10. General procedure for the hydrolysis of aldol adducts
Aldol adduct (1 equiv) was dissolved in THF (0.2 M), aqueous
LiOH (1 M, 4 equiv) was added, and the mixture was stirred at room
temperature for 3 h. The THF was removed and the aqueous phase
was washed with EtOAc (3ꢁ100 mL), then acidified with 2 M HCl
and extracted with EtOAc (3ꢁ100 mL), dried (MgSO4), filtered, and
concentrated to afford the corresponding acid.
(11.7 g, 62%) as yellow needles; [a]
reported [
(400 MHz, CDCl3)
20 e48ꢂ (c 25, CH2Cl2), previously
D
20
a
]
e210ꢂ (c 10, CHCl3);38 Mp 87e89 ꢂC; 1H NMR
7.34e7.12 (m, 5H), 5.42e5.19 (m, 1H), 3.31
D
d
(dd, J¼11.5, 7.2, 0.9 Hz, 1H), 3.15 (dd, J¼13.2, 3.8 Hz, 1H), 2.97 (dd,
4.11. (S)-3-Hydroxyhexanoic acid (S-10)
J¼13.2, 10.6 Hz, 1H), 2.82 (d, J¼11.5 Hz, 1H), 2.73 (s, 3H); 13C NMR
(100 MHz, CDCl3)
31.8, 23.1.
d 201.6, 170.8, 136.5, 129.5, 128.9, 127.3, 68.2, 36.7,
Procedure 4.10 afforded the desired product (1.12 g, 79%) as
20
20
a colorless oil; [
a
]
þ21ꢂ (c 5, CH2Cl2), previously reported [
a]
D
D
þ29ꢂ (c 5, CHCl3);34 1H NMR (400 MHz, CDCl3)
d
3.99 (m, 1H), 2.45
4.15. (R)-1-((R)-4-Benzyl-2-thioxothiazolidin-3-yl)-3-
hydroxyhexan-1-one (14)
(ddd, J¼25.4, 16.5, 6.1 Hz, 2H), 1.53e1.25 (m, 4H), 0.87 (t, J¼8.7, 3H);
13C NMR (100 MHz, CDCl3)
d 177.8, 67.8, 41.1, 38.5, 18.7, 13.9; in
agreement with previously reported data.35
Compound 13 was reacted with butanal according to General
procedure
4.7
and
subsequent
flash
chromatography
4.12. (S)-3-Hydroxyoctanoic acid (S-11)
(heptaneeEtOAc, 9:1/5:1) furnished title compound 14 (2 g, 53%)
20
as a yellow oil; Rf 0.16 (heptaneeEtOAc, 4:1); [
a
]
ꢃ117ꢂ (c 6,
D
Procedure 4.10 afforded the desired product (951 mg, 95%)
CH2Cl2); 1H NMR (400 MHz, CDCl3)
d 7.51e7.19 (m, 5H), 5.42 (m,
20
20
colorless oil; [
a]
þ24.6ꢂ (c 5, CH2Cl2), previously reported [
a
]
1H), 4.26e4.12 (m, 1H), 3.73e2.84 (m, 6H), 1.68e1.36 (m, 4H), 0.97
D
D
þ14.2ꢂ (c 5, CHCl3);35 1H NMR (400 MHz, CDCl3)
d
4.02 (m, 1H),
(t, J¼7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 201.5, 173.9, 136.4,
129.5, 129.0, 127.3, 68.3, 68.2, 45.5, 38.8, 36.8, 32.0, 18.7, 14.0; UPLC-
2.57e2.37 (m, 2H), 1.60e1.18 (m, 8H), 0.86 (t, J¼6.7 Hz, 3H); 13C
NMR (100 MHz, CDCl3)
d
177.0, 68.3, 41.2, 36.4, 31.6, 25.1, 22.5, 14.0.
MS gradient
C
A
tR 2.39 min (>95%); MS calcd for
16H21NO2S2Naþ[MþNa]þ 346.1, found 346.2.
4.13.
D-Phenylalaninol (12)
4.16. (R)-1-((R)-4-Benzyl-2-thioxothiazolidin-3-yl)-3-
NaBH4 (6.7 g, 209 mmol, 2.3 equiv) and
D-phenylalanine (15 g,
hydroxyoctan-1-one (15)
91 mmol,1 equiv) were dissolved in dry THF (250 mL) and cooled to
0 ꢂC on an ice-water bath. Iodine (22 g, 91 mmol,1 equiv) in dry THF
(75 mL) was added dropwise and after gas evolution stopped, the
mixture was heated to reflux overnight. Then the mixture was
cooled to room temperature and MeOH was slowly added until
a clear solution emerged (z200 mL). Solvents were removed, the
resulting white paste was dissolved in aqueous NaOH (2 M,
300 mL), and the mixture was stirred at room temperature for 8 h.
The aqueous phase was extracted with CH2Cl2 (3ꢁ200 mL) and the
combined organic layers were dried (Na2SO4), filtered, and con-
Compound 13 was reacted with hexanal according to General
procedure
4.7
and
subsequent
flash
chromatography
(heptaneeEtOAc, 15:1/8:1) afforded title compound 15 (3.6 g,
20
44%) as a yellow oil; Rf 0.11 (heptaneeEtOAc 4:1); [
a
]
D
ꢃ87ꢂ (c 9,
20
CH2Cl2), previously reported [a]
ꢃ72.6ꢂ (c 9, CHCl3);39 1H NMR
D
(400 MHz, CDCl3)
d 7.37e7.03 (m, 5H), 5.33 (m, 1H), 4.12e4.03 (m,
1H), 3.65e2.75 (m, 6H), 1.64e1.15 (m, 8H), 0.83 (t, J¼8.1 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) d 201.5, 173.4, 136.4, 129.5, 129.0, 127.3, 68.4,
67.9, 45.9, 36.9, 36.4, 32.1, 31.8, 25.3, 22.6, 14.1.
centrated to afford -phenylalaninol 12 (13.2 g, 99%) as a white
D
viscous oil, which was used without further purification; [
a
]
20 þ20ꢂ
4.17. (R)-3-Hydroxyhexanoic acid (R-10)
D
(c 13, CH2Cl2), previously reported [
(400 MHz, CDCl3)
(dd, J¼10.7, 7.2 Hz, 1H), 3.10e2.96 (m, 1H), 2.72 (dd, J¼13.5, 5.2 Hz,
a
]
20 þ23ꢂ (c 10, H2O);36 1H NMR
D
d
7.19 (m, 5H), 3.56 (dd, J¼10.7, 3.8 Hz, 1H), 3.32
Hydrolysis of 14 following General procedure 4.10 furnished the
desired hydroxy acid (445 mg, 54%) as a colorless oil; [
a]
20 ꢃ18ꢂ (c
1H), 2.45 (dd, J¼13.5, 8.7 Hz,1H), 2.11 (br s, 3H); 13C NMR (100 MHz,
87, CH2Cl2), previously reported [
a]
20 ꢃ20ꢂ (c 2, CHCl3);2D8 1H NMR
D
Please cite this article in press as: Kitir, B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.107

8
B. Kitir et al. / Tetrahedron xxx (2014) 1e12
(400 MHz, CDCl3)
d
4.12e3.97 (m, 1H), 2.61e2.40 (m, 2H), 2.10 (s,
4.23. Benzyl (3S)-3-(((S)-2-((tert-butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)hexanoate (20)
1H), 1.61e1.31 (m, 4H), 0.94 (t, J¼7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3)
d 177.7, 67.9, 41.1, 38.5, 18.6, 13.9.
Subjecting (S)-10 to General procedures 4.19 and 4.20 followed
by purification by flash chromatography afforded the title com-
4.18. (R)-3-Hydroxyoctanoic acid (R-11)
pound (469 mg, 62%) as a colorless oil; Rf 0.59 (10% MeOH in
20
Hydrolysis of 15 following General procedure 4.10 furnished the
CH2Cl2); [
d
a]
þ72ꢂ (c 6, DMSO); 1H NMR (400 MHz, DMSO-d6)
D
desired hydroxy acid (1.7 g, 69%) as a colorless oil; [
a
]
20 ꢃ12ꢂ (c 5,
7.33e7.19 (m, 5H), 5.27e5.14 (s, 1H), 5.02 (s, 2H), 4.83 (d, J¼8.5 Hz,
D
20
CH2Cl2), previously reported [
a
]
D
ꢃ23ꢂ (c 2, CHCl3);40 1H NMR
1H), 4.16 (m, 1H), 2.67e2.41 (m, 2H), 1.66e1.17 (m, 16H), 0.87e0.73
(400 MHz, CDCl3)
d
4.02 (m, 1H), 2.57e2.37 (m, 2H), 1.60e1.18 (m,
(m, 9H); 13C NMR (100 MHz, DMSO-d6)
d
172.0, 170.3, 156.2, 136.3,
8H), 0.86 (t, J¼6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d
177.0, 68.3,
128.8, 128.5, 128.5, 78.6, 70.8, 65.9, 55.3, 40.0, 39.0, 36.0, 28.6, 21.1,
18.1, 14.1; HRMS calcd for C24H37NO6Naþ [MþNa]þ 458.2513, found
458.2528.
41.2, 36.4, 31.6, 25.1, 22.5, 14.0.
4.19. General procedure for benzylester protection
4.24. Benzyl (3S)-3-(((S)-2-((tert-butoxycarbonyl)amino)-4-
To an ice-cooled solution of hydroxy acid (1 equiv) in MeOH
(2 mL) was added a solution of CsCO3 (0.6 equiv) in water (1 mL).
After 30 min, the solvents were removed under reduced pressure
and the CsCO3 salt was dissolved in DMF (0.2 M). Benzyl bromide
(1.1 equiv) was added and the suspension was stirred overnight.
The mixture was then quenched with water (30 mL) and the
aqueous phase was extracted with EtOAc (3ꢁ30 mL). The combined
organic layers were washed with water (5ꢁ20 mL) and brine
(10 mL), dried over Na2SO4, filtered, and concentrated to give crude
benzylester, which was used without further purification.
methylpentanoyl)oxy)octanoate (21)
Subjecting (S)-11 to General procedures 4.19 and 4.20 followed
by purification by flash chromatography afforded the title com-
pound (394 mg, 76%) as a colorless oil; Rf 0.8 (CH2Cl2eMeOH, 9:1);
[
a
]
20 þ48ꢂ (c 10, DMSO); 1H NMR (400 MHz, DMSO-d6)
d 7.45e7.30
D
(m, 5H), 7.21 (d, J¼7.8 Hz, 1H), 5.26e5.01 (m, 3H), 3.90 (m, 1H),
2.77e2.54 (m, 2H), 1.75e1.16 (m, 20H), 0.84 (m, 1H); 13C NMR
(100 MHz, DMSO-d6) d 170.7, 170.3, 156.0, 136.6, 128.9, 128.4, 128.3,
78.5, 70.9, 40.0, 39.0, 33.9, 31.4, 28.5, 24.6, 24.4, 21.6, 21.1, 14.2;
HRMS calcd for
486.2845.
C
26H41NO6Naþ [MþNa]þ 486.2829, found
4.20. General procedure for dimer coupling
A mixture of Boc-
L-Leu-OH (1.2 equiv) and benzylester (1 equiv)
4.25. General procedure for Bn deprotection
was dissolved in CH2Cl2 (0.2 M) and treated with DIC (2 equiv) and
DMAP (0.1 equiv) at 0 ꢂC. The resulting mixture was allowed to
reach room temperature and stirred overnight. The forming pre-
cipitate was removed by filtration and washed with CH2Cl2 (10 mL).
The combined organic phase was washed with 1 M HCl (10 mL) and
brine (10 mL), dried with Na2SO4, filtered, and concentrated. The
crude oil was purified by flash chromatography (2e4% MeOH in
CH2Cl2) to afford the desired dimers.
The dimer (1 equiv) was dissolved in EtOH (0.2 M) and degassed
with N2. Palladium on activated carbon (0.1% wt, 0.1 equiv) was
added and the mixture was degassed with N2. The mixture was
stirred with H2 atm overnight. The catalyst was filtered by a pad of
Celite and washed with CH2Cl2 (50 mL). The solvents were removed
under reduced pressure affording the compounds.
4.26. (3R)-3-(((S)-2-((tert-Butoxycarbonyl)amino)-4-
4.21. Benzyl (3R)-3-(((S)-2-((tert-butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)hexanoic acid (18)
methylpentanoyl)oxy)hexanoate (16)
Deprotection of 16 by General procedure 4.25 afforded building
block 18 (220 mg, 42%) as a colorless oil; Rf 0.11 (heptaneeEtOAc,
Subjecting (R)-10 to General procedures 4.19 and 4.20 followed
by purification by flash chromatography (heptaneeEtOAc,
4:1/3:1) gave the title compound (609 mg, 61%) as an oil; Rf 0.8
1:1); [
a
]
D
20 ꢃ1.3ꢂ (c 3, CH2Cl2); 1H NMR (400 MHz, C6D6)
d 5.24e5.13
(m, 1H), 4.17 (m, 1H), 2.64e2.46 (m, 2H), 1.68e1.42 (m, 4H), 1.37 (s,
(heptaneeEtOAc,1:1); [
a
]
20 ꢃ50ꢂ (c 10, CH2Cl2); 1H NMR (400 MHz,
9H), 1.32e1.15 (m, 3H), 0.91e0.82 (m, 9H); 13C NMR (100 MHz,
D
CDCl3)
d
7.28 (m, 5H), 5.22 (m, 1H), 5.09e4.94 (m, 2H), 4.17 (m, 1H),
C6D6) d 177.4, 174.6, 157.6, 79.1, 71.1, 57.7, 42.0, 36.1, 28.1, 22.9, 22.7,
2.69e2.42 (m, 2H), 1.68e1.43 (m, 4H), 1.36 (s, 9H), 1.32e1.13 (m,
21.4, 18.1, 13.5; HRMS calcd for C17H31NO6Naþ [MþNa]þ 368.2043,
3H), 0.93e0.67 (m, 9H); 13C NMR (100 MHz, CDCl3)
d
172.7, 170.0,
found 368.2057.
155.3,135.7,128.6,128.4,128.4, 79.6, 71.2, 52.3, 39.2, 36.1, 28.3, 24.8,
22.9, 21.9, 18.3, 13.8; HRMS calcd for C24H37NO6Naþ [MþNa]þ
458.2513, found 458.2516.
4.27. 3(R)-3-(((S)-2-((tert-Butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)octanoic acid (19)
4.22. Benzyl (3R)-3-(((S)-2-((tert-butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)octanoate (17)
Deprotection of 17 by General procedure 4.25 afforded building
block 19 (275 mg, 87%) as a colorless oil; 1H NMR (400 MHz, CDCl3)
d
4.76 (m, 1H), 4.18 (m, 1H), 2.67e2.40 (m, 2H), 1.67e1.39 (m, 8H),
1.41e1.30 (m, 9H), 1.31e1.08 (m, 3H), 0.96e0.74 (m, 9H); 13C NMR
(100 MHz, CDCl3) 172.7, 170.0, 155.3, 79.7, 71.5, 52.3, 41.8, 41.7,
39.1, 33.8, 31.4, 28.3, 24.64, 22.9, 22.4, 22.4, 13.9; UPLC-MS gradient
A tR 2.51 min (>95%); MS calcd for C19H36NO6Naþ [MþNa]þ 396.2,
found 396.2.
Subjecting (R)-11 to General procedures 4.19 and 4.20 followed
by purification by flash chromatography afforded the title com-
pound (200 mg, 40%) as a colorless oil; Rf 0.84 (CH2Cl2eMeOH, 9:1);
d
[
a]
20 ꢃ46ꢂ (c 5, DMSO); 1H NMR (300 MHz, CDCl3)
d 7.53e7.27 (m,
D
5H), 7.19 (d, J¼7.7 Hz, 1H), 5.74 (s, 1H), 5.21e4.93 (m, 1H), 3.88 (ddd,
J¼12.7, 11.2, 7.2 Hz, 2H), 2.76e2.52 (m, 2H), 1.66e1.40 (m, 7H), 1.35
(s, 9H), 1.28e1.09 (m, 2H), 0.80 (ddd, J¼13.6, 7.9, 4.7 Hz, 9H); 13C
4.28. (3S)-3-(((S)-2-((tert-Butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)hexanoic acid (22)
NMR (100 MHz, CDCl3)
d
170.1, 170.0, 155.3, 135.7, 128.6, 128.4,
128.3, 71.5, 66.5, 52.3, 41.7, 39.1, 33.8, 31.4, 28.3, 24.6, 22.9, 22.4,
21.9,13.9; HRMS calcd for C26H41NO6Naþ [MþNa]þ 486.2826, found
486.2833.
Deprotection of 20 by General procedure 4.25 afforded building
block 22 (269 mg, 72%) as a colorless oil; [
a
]
20 þ5ꢂ (c 3, DMSO); 1H
D
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B. Kitir et al. / Tetrahedron xxx (2014) 1e12
9
NMR (400 MHz, DMSO-d6)
d
5.10 (m, 1H), 3.89 (m, 1H), 2.58e2.42
30, CH2Cl2); 1H NMR (400 MHz, CDCl3)
d
7.50e7.26 (m, 5H),
(m, 2H), 1.73e1.20 (m, 16H), 0.92e0.76 (m, 9H); 13C NMR (100 MHz,
5.38e5.04 (m, 3H), 4.44e4.20 (m, 1H), 2.73e2.47 (m, 2H), 1.79e1.20
DMSO-d6)
d
173.1, 172.0, 156.0, 78.6, 70.9, 52.5, 40.0, 39.0, 36.1, 28.6,
(m,11H), 0.92 (m, 9H); 13C NMR (100 MHz, CDCl3)
d
174.7,172.6,156.3,
24.7, 23.2, 21.9, 18.1, 14.2; HRMS calcd for C17H31NO6Naþ [MþNa]þ
136.1, 128.5, 128.2, 128.1, 71.6, 67.3, 52.7, 41.5, 41.2, 38.8, 33.8, 31.4,
31.4, 24.8, 24.7, 24.6, 22.9, 22.4, 21.7, 14.0; HRMS calcd for
368.2043, found 368.2060.
C
22H33NO6Naþ [MþNa]þ 430.2200, found 430.2212.
4.29. (3S)-3-(((S)-2-((tert-Butoxycarbonyl)amino)-4-
methylpentanoyl)oxy)octanoic acid (23)
4.32. H-L-Phe-D-Leu-D-Ala-OBn (26)
Deprotection of 21 by General procedure 4.25 afforded building
block 23 (244 mg, 79%) as a colorless oil; 1H NMR (400 MHz, DMSO-
Boc- -Ala-OBn (2.3 g, 8.2 mmol, 1 equiv) was stirred with
D
TFAeCH2Cl2 (1:1, 10 mL) and stirred for 2 h. The solvents were re-
moved and the mixture was co-evaporated with toluene (2ꢁ10 mL),
tolueneeCH2Cl2 (1:1, 2ꢁ10 mL), and hexaneeCH2Cl2 (1:1, 2ꢁ10 mL)
d6)
6.3 Hz, 2H), 1.61e1.09 (m, 20H), 0.96e0.68 (m, 9H); 13C NMR
(100 MHz, DMSO-d6) 173.1, 170.3, 156.0, 78.5, 70.9, 66.1, 52.5, 39.0,
d
5.18e4.98 (m, 1H), 3.93e3.80 (m, 1H), 2.66 (ddd, J¼24.1, 15.7,
d
and the mixture was used without further purification. Boc-D-Leu-
33.9, 31.4, 28.6, 24.6, 24.4, 23.1, 14.2; UPLC-MS gradient A tR
2.44 min (>95%); MS calcd for C19H36NO6 [MþH]þ 374.3, found
374.2.
OH (3.1 g, 13.5 mmol, 1.1 equiv) was dissolved in CH2Cl2 (70 mL).
HOBt (1.8 g, 13.5 mmol, 1.1 equiv) and DIC (2.1 mL, 13.5 mmol,
1.1 equiv) were added and the suspension was activated for 10 min.
The crude oil in CH2Cl2 (60 mL) was added to the mixture and was
stirred at room temperature 6 h and then quenched with brine
(100 mL). The aqueous phase was extracted with CH2Cl2 (3ꢁ50 mL).
The combined organic phases were washed with 2 M HCl (3ꢁ50 mL)
and satd aq NaHCO3 (50 mL) and then dried with MgSO4 and con-
centrated to a crude oil, which was purified byflash chromatography
4.30. (4R)-4-Methoxybenzyl 3-(((R)-2-(((benzyloxy)carbonyl)-
amino)-4-methylpentanoyl)oxy)octanoate (24)
(R)-11 (1 g, 6.2 mmol, 1.1 equiv) was dissolved in HPLC grade ac-
etone (40 mL). PMB-Cl (98 ml, 5.7 mmol, 1 equiv), TBAI (105 mg,
0.28 mmol, 0.05 equiv), and K2CO3 (1.2 g, 8.5 mmol, 1.5 equiv) were
added and the mixture was stirred at reflux for 16 h. Then, EtOAc
(50 mL) and water (50 mL) were added and the aqueous phase was
back extracted with EtOAc (2ꢁ50 mL). The combined organic phases
were dried over MgSO4, filtered, and concentrated to give a crude oil,
which was purified by flash chromatography (heptaneeEtOAc, 4:1)
to afford (R)-4-methoxybenzyl 3-hydroxyoctanoate (882 mg, 55%) as
(2e6% MeOH in CH2Cl2) affording a colorless oil (2.44 g, 76%). Boc-
D-
Leu- -Ala-OBn (2.4 g, 6.1 mmol, 1 equiv) was stirred with
D
TFAeCH2Cl2 (1:1, 10 mL) and stirred for 3 h. The solvents were re-
moved and the mixture was co-evaporated with toluene (2ꢁ10 mL),
tolueneeCH2Cl2 (1:1, 2ꢁ10 mL) and hexaneeCH2Cl2 (1:1, 2ꢁ10 mL)
and the mixture was used without further purification. Boc-L-Phe-
OH (2.5 g, 9.4 mmol, 1.1 equiv) and HOBt (1.3 g, 9.4 mmol, 1.1 equiv)
was dissolved in dry CH2Cl2 (50 mL). DIC (1.5 mL, 9.4 mmol,1.1 equiv)
was added and the suspension is stirred for 10 min. The crude oil in
CH2Cl2 (40 mL) was added to the solution and the mixture was
stirred at room temperature overnight and then quenched with
brine (100 mL). The aqueous phase was extracted with CH2Cl2
(3ꢁ50 mL). The combined organic phases werewashed with 2 M HCl
(3ꢁ50 mL), saturated aqueous NaHCO3 (50 mL) and then dried with
MgSO4 and concentrated. The crude oil was purified by flash chro-
a colorless oil; Rf 0.6 (heptaneeEtOAc,1:1); [
a
]
20 ꢃ5ꢂ (c 6, CH2Cl2); 1H
D
NMR (400 MHz, CDCl3)
d
7.31 (d, J¼8.7 Hz, 2H), 6.90 (d, J¼8.7 Hz, 2H),
5.10 (s, 2H), 4.02 (m, 1H), 3.82 (s, 3H), 2.99 (br s, 1H), 2.59e2.35 (m,
2H), 1.58e1.24 (m, 8H), 0.90 (t, J¼6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 172.9,159.7,130.2,127.8,114.0, 68.1, 66.3, 55.3, 41.5, 36.5, 31.7,
25.2, 22.6, 14.0; HRMS calcd for C30H41NO7Kþ [MþK]þ 319.1306,
found 319.1364. The PMB-ester (200 mg, 0.71 mmol, 1 equiv), DIC
(223
were dissolved in dry CH2Cl2 and stirred for 10 min at 0 ꢂC. Then Cbz-
-Leu-OH (208 mg, 0.79 mmol,1.2 equiv) was added and the mixture
ml, 1.43 mmol, 2 equiv), and DMAP (9 mg, 0.07 mmol, 0.1 equiv)
matography (2e4% MeOH in CH2Cl2) affording Boc-
Ala-OBn (2.5 g, 55%) as an oil; Rf 0.6 (CH2Cl2eMeOH, 95:5); 1H NMR
(400 MHz, DMSO-d6)
L-Phe-D-Leu-D-
L
was slowly heated to room temperature and allowed to proceed
under stirring for 16 h. The formed urea biproduct was removed by
filtration and washed with CH2Cl2 (20 mL). The combined organic
phase was washed with 1 M HCl (20 mL) and brine (20 mL), dried
over Na2SO4, filtered, and concentrated. The resulting residue was
purified by flash chromatography (2e4% MeOH in CH2Cl2) to afford
d
8.34 (d, J¼6.9 Hz, 1H), 8.07 (d, J¼8.6 Hz, 1H),
7.47e7.13 (m,10H), 6.98 (d, J¼7.8 Hz,1H), 5.17e4.97 (m, 2H), 4.31 (p,
J¼7.1 Hz, 2H), 4.16 (m, 1H), 2.81 (m, 2H), 1.42e1.14 (m, 15H),
0.90e0.64 (m, 6H); 13C NMR (100 MHz, DMSO-d6)
d 172.2, 172.0,
171.3, 155.3, 137.8, 136.0, 129.4, 129.2, 128.4, 128.0, 127.7, 126.2, 78.1,
65.9, 55.9, 50.2, 47.7, 40.9, 28.1, 23.8, 23.2, 21.4, 16.6. The fully pro-
tected tripeptide (900 mg, 1.7 mmol, 1 equiv) was then treated with
TFAeCH2Cl2 (1:1,10 mL) for 3 h. The solvents were removed and the
residue was co-evaporated withtoluene (2ꢁ10 mL), tolueneeCH2Cl2
(1:1, 2ꢁ10 mL), and hexaneeCH2Cl2 (1:1, 2ꢁ10 mL). The resulting
crude compound was purified by preparative RP-HPLC affording 26
the desired dimer (335 mg, 90%) as a colorless oil; Rf 0.76 (heptane-
20
eEtOAc, 1:1); [
d
a
]
ꢃ15ꢂ (c 27, CH2Cl2); 1H NMR (400 MHz, CDCl3)
D
7.35e7.10 (m, 8H), 6.81 (d, J¼6.7 Hz, 2H), 5.27e5.12 (m, 1H),
5.10e4.90 (m, 4H), 4.32e4.16 (m,1H), 3.72 (s, 3H), 2.65e2.38 (m, 2H),
1.70e1.30 (m, 5H), 1.19 (m, 6H), 0.81 (m, 9H); 13C NMR (100 MHz,
CDCl3)
d
172.4,170.1,159.7,155.9,136.3,130.3,128.7,128.2,128.1,128.1,
(580 mg, 79%) as a white solid; 1H NMR (400 MHz, DMSO-d6)
d 8.61
127.8,114.0, 71.7, 66.9, 66.4, 55.3, 52.7, 41.8, 39.1, 33.7, 31.4, 24.8, 24.6,
22.9, 22.4, 21.9, 13.9; HRMS calcd for C30H41NO7Naþ [MþNa]þ
550.2775, found 550.2789.
(dd, J¼13.3, 7.7 Hz, 2H), 8.13 (d, J¼4.9 Hz, 1H), 7.45e7.11 (m, 10H),
5.21e5.00 (m, 4H), 4.41e4.25 (m, 2H), 4.15e3.98 (m, 1H), 2.99 (m,
2H), 1.36e1.08 (m, 6H), 0.74 (dd, J¼10.9, 6.2 Hz, 6H); 13C NMR
(100 MHz, DMSO-d6) d 172.6, 171.9, 168.0, 136.4, 135.3, 129.9, 128.9,
4.31. (3R)-3-(((R)-2-(((Benzyloxy)carbonyl)amino)-4-
128.8, 128.5, 128.3, 127.5, 66.4, 53.7, 51.0, 48.1, 41.6, 37.7, 24.1, 23.5,
methylpentanoyl)oxy)octanoic acid (25)
21.9, 17.1; UPLC-MS gradient A tR 1.46 min (>95%); MS calcd for
C
C
25H33N3O4[MþH]þ 440.3, found 440.3; HRMS calcd for
Compound 24 (700 mg, 1.33 mmol, 1 equiv) was dissolved in dry
CH2Cl2 (10 mL) and cooled to 0 ꢂC. Et3SiH (2.2 mL, 13.8 mmol,
10 equiv) was added in one portion and TFA (10 mL) was added
slowly. The mixture was stirred at 0 ꢂC for 3 h whereafter the solvents
were removed and the residue was purified by flash chromatography
(heptaneeEtOAc, 3:1/2:1) to afforded the title compound 25
25H33N3O4[MþH]þ 440.2544, found 440.2552;
4.33. Solonamide A (1)
Polystyrene 2-chlorotrityl-bound
Fmoc-L-Phe-D-Leu-D-Ala
(600 mg, 0.51 mmol, 1 equiv) was added to a fritted syringe and
swelled with CH2Cl2 before the Fmoc group was removed with
(483mg, 89%) a colorless oil; Rf 0.2 (heptaneeEtOAc,1:1); [
a]
20 ꢃ8ꢂ (c
D
Please cite this article in press as: Kitir, B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.107

10
B. Kitir et al. / Tetrahedron xxx (2014) 1e12
piperidineeDMF (1:4, 4 mL, 2ꢁ30 min) and DBUepiperidineeDMF
(2:2:96, 4 mL, 30 min). Then the resin was washed with DMF (ꢁ3),
MeOH (ꢁ3), and CH2Cl2 (ꢁ3) and dimer 18 (200 mg, 0.56 mmol,
(233 mg, 0.39 mmol, 1 equiv) in DMFeCH2Cl2, 10:1 (25 mL) was
added by syringe pump over 4 h and stirred at room temperature
for additional 2 h. Then the solvents were removed and the residue
purified by preparative reversed-phase HPLC to afford solonamide
B 2 (3 mg, 1%). (B) To a stirred solution of crude linear precursor
(233 mg, 0.39 mmol, 1 equiv) in DMF (385 mL) was added iPrEtN
1.1 equiv) was preincubated for 10 min with iPrEtN (350
ml,
2.02 mmol, 4 equiv) and HATU (385 mg, 1.01 mmol, 2 equiv) before
addition to the resin and the reaction was allowed to proceed on
a rocking table for 24 h and the resin was washed with DMF (ꢁ3),
MeOH (ꢁ3) and CH2Cl2 (ꢁ3). The resin was treated TFAeCH2Cl2
(1:1, 2 mL, 2ꢁ30 min) followed by washing with CH2Cl2 (3ꢁ2 mL)
and all the fractions were pooled in a round-bottomed flask and
concentrated under reduced pressure. Co-evaporation with toluene
(ꢁ2), tolueneeCH2Cl2 (1:1, ꢁ2), and hexaneeCH2Cl2 (1:1, ꢁ2)
afforded the crude (201 mg, 64%) To a stirred solution the crude
linear precursor (108 mg, 0.19 mmol, 1 equiv) in DMF (190 mL) was
(269 ml,1.54 mmol, 4 equiv) and HATU (294 mg, 0.77 mmol, 2 equiv)
and the reaction was stirred at room temperature overnight. Con-
centration, purification of the crude residue by preparative
reversed-phase HPLC, and lyophilization gave cyclic depsipeptide
solonamide B (2 mg, 1%) as a white fluffy solid.
20
[
a
]
þ4ꢂ (c 2, DMSO); 1H NMR (400 MHz, DMSO-d6)
d 8.65 (d,
D
J¼5.6 Hz,1H), 8.61 (d, J¼3.2 Hz,1H), 7.49 (d, J¼8.9 Hz,1H), 7.30e7.16
(m, 5H), 7.06 (d, J¼9.7 Hz, 1H), 5.19e5.07 (m, 1H), 4.47 (td, J¼10.1,
4.3 Hz, 1H), 4.27 (m, 1H), 4.19 (m, 1H), 3.57 (m, 1H), 2.96 (dd, J¼13.4,
6.1 Hz, 1H), 2.78e2.64 (m, 2H), 2.12 (dd, J¼13.5, 10.2 Hz, 1H),
1.71e1.60 (m, 2H), 1.59e1.36 (m, 4H), 1.33 (d, J¼7.4 Hz, 3H), 1.25 (t,
J¼9.3 Hz, 7H), 1.06e0.95 (m, 1H), 0.90e0.81 (m, 9H), 0.71 (d,
J¼6.6 Hz, 3H), 0.54 (d, J¼6.4 Hz, 3H); 13C NMR (125 MHz, DMSO-d6)
added iPrEtN (130
ml, 0.74 mmol, 4 equiv). HATU (142 mg,
0.37 mmol, 2 equiv) was added and the reaction was stirred at room
temperature overnight, and then DMF is evaporated. The residue
was purified by preparative reversed-phase HPLC to give cyclic
depsipeptide (2.5 mg, 3%) as a white fluffy solid; 1H NMR (400 MHz,
DMSO-d6)
d
8.66 (d, J¼5.6 Hz, 1H), 8.62 (d, J¼3.1 Hz, 1H), 7.49 (d,
d 174.3, 171.6, 171.2, 170.2, 136.3, 129.2, 128.9, 126.4, 72.3, 55.8, 52.9,
48.8, 47.9, 39.2, 38.8, 36.1, 34.0, 30.9, 23.7, 22.9, 22.9, 21.8, 21.1, 20.4,
J¼8.9 Hz, 1H), 7.30e7.16 (m, 5H), 7.06 (d, J¼9.8 Hz, 1H), 5.23e5.11
(m, 1H), 4.48 (m, 1H), 4.27 (m, 1H), 4.19 (m, 1H), 3.57 (m, 1H), 2.96
(dd, J¼13.3, 6.1 Hz, 1H), 2.78e2.64 (m, 2H), 2.12 (dd, J¼13.5, 10.3 Hz,
1H), 1.71e1.59 (m, 2H), 1.58e1.40 (m, 4H), 1.33 (d, J¼7.4 Hz, 3H),
1.31e1.15 (m, 3H), 1.07e0.94 (m, 1H), 0.90e0.85 (m, 6H), 0.83 (d,
J¼6.4 Hz, 4H), 0.71 (d, J¼6.6 Hz, 4H), 0.54 (d, J¼6.4 Hz, 3H); UPLC-
MS gradient A tR 2.14 min (>95%). MS calcd for C30H46N4O6 [MþH]þ
559.3, found 559.3.
16.4, 13.5; UPLC-MS gradient A tR 2.35 min (>95%); MS calcd for
C
C
32H50N4O6 [MþH]þ 587.4, found 587.3; HRMS calcd for
32H50N4O6 [MþH]þ 587.3803, found 587.3813.
4.35.
b b
3-epi-Solonamide A ( 3-epi-1)
Polystyrene 2-chlorotrityl-bound H-
L
-Phe-
D
-Leu-
D-Ala
3
(0.38 mmol, 1 equiv) was added to a fritted syringe and swelled
4.34. Solonamide B (2)
with CH2Cl2. Dimer 22 (150 mg, 0.42 mmol, 1.1 equiv) was pre-
i
incubated for 10 min with PrEtN (243
ml, 1.5 mmol, 4 equiv) and
Procedure 1. Polystyrene 2-chlorotrityl-bound H-
-Ala-OH 3 (0.32 mmol, 1 equiv) was added to a fritted syringe and
swelled with CH2Cl2. Dimer 19 (130 mg, 0.35 mmol, 1.1 equiv) was
L
-Phe-
D
-Leu-
HATU (288 mg, 0.76 mmol, 2 equiv) before addition to the resin and
the reaction was allowed to proceed on a rocking table for 24 h and
the resin was washed with DMF (ꢁ3), MeOH (ꢁ3) and CH2Cl2 (ꢁ3).
The resin was treated TFAeCH2Cl2 (1:1, 2 mL, 2ꢁ30 min) followed
by washing with CH2Cl2 (3ꢁ2 mL) and all the fractions were pooled
in a round-bottomed flask and concentrated under reduced pres-
sure. Co-evaporation with toluene (ꢁ2), tolueneeCH2Cl2 (1:1, ꢁ2)
and hexaneeCH2Cl2 (1:1, ꢁ2) afforded the crude (108 mg, 50%) To
D
preincubated for 10 min with HATU (241 mg, 0.63 mmol, 2 equiv)
and iPrEtN (221
ml, 1.27 mmol, 4 equiv) before addition to the resin
and the reaction was allowed to proceed on a rocking table for
24 h and the resin was washed with DMF (ꢁ3), MeOH (ꢁ3) and
CH2Cl2 (ꢁ3). The resin was treated TFAeCH2Cl2 (1:1, 2 mL,
2ꢁ30 min) followed by washing with CH2Cl2 (3ꢁ2 mL) and all the
fractions were pooled in a round-bottomed flask and concen-
trated under reduced pressure. Co-evaporation with toluene (ꢁ2),
tolueneeCH2Cl2 (1:1, ꢁ2), and hexaneeCH2Cl2 (1:1, ꢁ2) afforded
a stirred solution of crude (108 mg, 0.19 mmol, 1 equiv) in DMF
i
(190 mL) was added PrEtN (130
ml, 0.74 mmol, 4 equiv). HATU
(142 mg, 0.37 mmol, 2 equiv) was added and the reaction was
stirred at room temperature overnight, and the DMF was evapo-
rated. The residue was purified by preparative reversed-phase
HPLC to give cyclic depsipeptide (4 mg, 4%) as a white fluffy
the crude linear precursor (127 mg, 66%). To a solution of the
i
crude in DMF (210 mL) were added PrEtN (146
m
l, 0.84 mmol,
4 equiv) and HATU (160 mg, 0.42 mmol, 2 equiv), and the dilute
reaction mixture was stirred at room temperature overnight. The
solvent was then removed under reduced pressure and the resi-
due was purified by preparative reversed-phase HPLC. Lyophili-
zation of the pure fractions gave cyclic depsipeptide 2 (1 mg, 2%)
as a white fluffy solid.
solid; 1H NMR (500 MHz, DMSO-d6)
d
8.34 (d, J¼7.3 Hz, 1H), 8.31 (d,
J¼5.4 Hz, 1H), 7.42 (d, J¼7.5 Hz, 1H), 7.32e7.18 (m, 6H), 4.85 (m, 1H),
4.37 (dd, J¼13.4, 7.6 Hz, 1H), 4.30 (m, 1H), 4.13 (p, J¼7.1 Hz, 1H), 3.87
(m, 1H), 2.91 (dd, J¼13.4, 7.2 Hz, 1H), 2.83 (dd, J¼13.4, 8.4 Hz, 1H),
2.54 (m, 2H), 2.39 (dd, J¼14.8, 4.0 Hz, 1H), 1.74 (m, 1H), 1.57 (m, 1H),
1.50 (m, 3H), 1.34 (m, 2H), 1.27 (m, 2H), 1.20 (d, J¼7.0 Hz, 3H), 1.00
(m, 1H), 0.87 (m, 6H), 0.83 (d, J¼5.9 Hz, 3H), 0.73 (d, J¼6.6 Hz, 3H),
Procedure 2. Dimer 25 (270 mg, 0.66 mmol, 1 equiv), PyBOP
i
(461 mg, 0.79 mmol, 1.2 equiv), and PrEtN (139
ml, 0.79 mmol,
0.64 (d, J¼6.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6)
d 172.0, 172.1,
1.2 equiv) were dissolved in DMF (5 mL) and stirred 10 min. Trimer
26 (320 mg, 0.73 mmol, 1.1 equiv) in DMF (5 mL) was added and
stirring was continued for 16 h at room temperature. Solvents were
removed and the crude pentamer (530 mg, 0.64 mmol,1 equiv) was
dissolved in EtOH (10 mL) and degassed with argon. Then, Pd/C
(106 mg, 0.1 equiv) was added and the mixture was again degassed
with argon. The mixture was stirred under an H2 atm overnight, the
catalyst was removed by filtration through a pad of Celite, and the
solvent was removed under reduced pressure to afford the linear
crude product as an oil. This was divided into two equal portions
and two cyclization procedures were performed. (A) HATU
171.0, 169.2, 169.2, 136.8, 128.1, 127.5, 126.0, 71.1, 55.0, 51.8, 51.5,
48.3, 39.3, 39.1, 38.2, 36.7, 34.5, 24.1, 23.3, 22.7, 22.0, 21.7, 20.6, 17.8,
1v7.5, 13.5. UPLC-MS gradient A tR 2.19 min (>95%); MS calcd for
C
C
30H46N4O6 [MþH]þ 559.3, found 559.3; HRMS calcd for
30H46N4O6 [MþH]þ 559.3490, found 559.3506.
4.36.
b
b
3-epi-Solonamide B ( 3-epi-2)
Polystyrene
2-chlorotrityl-bound
Fmoc-L-Phe-D-Leu-D-Ala
(190 mg, 0.08 mmol, 1 equiv) was added to a fritted syringe and
swelled with CH2Cl2 before the Fmoc group was removed with
piperidineeDMF (1:4, 4 mL, 2ꢁ30 min) and DBUepiperidineeDMF
(2:2:96, 4 mL, 30 min) and then the resin was washed with DMF
(294 mg, 0.77 mmol, 2 equiv) and iPrEtN (269
4 equiv) were dissolved in CH2Cl2 (20 mL), and linear peptide
ml, 1.54 mmol,
Please cite this article in press as: Kitir, B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.107

B. Kitir et al. / Tetrahedron xxx (2014) 1e12
11
(ꢁ3), MeOH (ꢁ3), and CH2Cl2 (ꢁ3). Dimer 23 (33 mg, 0.09 mmol,
1.1 equiv) was preincubated for 10 min with iPrEtN (51
l,
TSA was melted and 5-bromo-4-chloro-3-indolyl
b
-
D
-galactopyr-
m
anoside (X-gal) (150
m
gꢁmLꢃ1) and erythromycin (5
m
gꢁmLꢃ1
)
0.32 mmol, 4 equiv) and HATU (61 mg, 0.16 mmol, 2 equiv) before
addition to the resin and the reaction was allowed to proceed on
a rocking table for 24 h and the resin was washed with DMF (ꢁ3),
MeOH (ꢁ3), and CH2Cl2 (ꢁ3). The resin was treated TFAeCH2Cl2
(1:1, 2 mL, 2ꢁ30 min) followed by washing with CH2Cl2 (3ꢁ2 mL)
and all fractions were pooled in a round-bottomed flask and con-
centrated under reduced pressure. Co-evaporation with toluene
(ꢁ2), tolueneeCH2Cl2 (1:1, ꢁ2), and hexaneeCH2Cl2 (1:1, ꢁ2)
afforded the crude linear precursor (35 mg, 45%). To a stirred so-
lution of crude (35 mg, 0.058 mmol, 1 equiv) in DMF (60 mL) was
were added. For each small plate 800 mL of bacterial dilution were
mixed with 20 mL TEX (TSA, X-gal, and erythromycin), or 2 mL
bacterial dilution mixed with 50 mL TEX for large plates. Plates
were allowed to cool for 45 min. Once thoroughly dried, wells were
drilled into the agar. Test sample or control (20 mL) were added per
well and the plates were incubated at 37 ꢂC. Incubation was allowed
until the blue color appeared in the plates and this varied between
approximately 9e48 h.
Acknowledgements
added iPrEtN (40
ml, 0.23 mmol, 4 equiv) and HATU (44 mg,
0.12 mmol, 2 equiv) and the reaction was stirred at room temper-
ature overnight, after which DMF was evaporated. The residue was
purified by preparative reversed-phase HPLC to give cyclic dep-
sipeptide (2 mg, 7%) as a white fluffy solid; 1H NMR (500 MHz,
This work was supported by the Lundbeck Foundation (Young
Group Leader Fellowship, C.A.O.), the Danish Independent Research
Council-Natural Sciences (Steno Grant No. 10-080907, C.A.O.), the
Carlsberg Foundation and the European Community’s Seventh
Framework Programme (FP7/2007-2013) under grant agreement
Nꢂ289285 (M.B.). Novo Nordisk A/S is thanked for a generous do-
nation of peptide coupling reagents used in this work. We thank
Ms. Anne Hector and Dr. Charlotte H. Gotfredsen for assistance with
NMR spectroscopy and Ms. Tina Gustafsson for technical assistance
with UPLC-MS. Dr. Charlotte H. Gotfredsen and Louise Kjærulff are
gratefully acknowledged for providing NMR data of the natural
products. Professor Mads H. Clausen is gratefully acknowledged for
providing the (S)-4-benzylthiazolidine-2-thione auxilliary.
DMSO-d6)
d
8.34 (d, J¼7.3 Hz, 1H), 8.30 (d, J¼5.4 Hz, 1H), 7.42 (d,
J¼7.5 Hz, 1H), 7.31e7.16 (m, 6H), 4.85 (t, J¼12.0 Hz, 1H), 4.37 (dd,
J¼13.4, 7.7 Hz, 1H), 4.29 (m, 1H), 4.13 (m, 1H), 3.92e3.82 (m, 1H),
2.91 (dd, J¼13.4, 7.3 Hz, 1H), 2.83 (dd, J¼13.4, 8.4 Hz, 1H), 2.55 (dd,
J¼14.8, 8.0 Hz, 1H), 2.38 (dd, J¼14.8, 3.9 Hz, 1H), 1.74 (d, J¼7.4 Hz,
1H), 1.61 (d, J¼8.2 Hz, 1H), 1.50 (t, J¼8.0 Hz, 3H), 1.38e1.22 (m, 8H),
1.20 (d, J¼7.0 Hz, 3H), 1.05e0.93 (m, 1H), 0.89e0.79 (m, 9H), 0.73 (d,
J¼6.6 Hz, 3H), 0.64 (d, J¼6.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6)
d
172.2, 171.2, 170.1, 169.1, 136.7, 129.3, 128.6, 126.4, 71.4, 55.2, 52.1,
51.7, 48.5, 39.4, 39.3, 38.0, 36.8, 32.2, 31.1, 24.1, 24.0, 23.1, 23.0, 22.1,
22.0, 21.6, 20.6, 17.5, 13.6. UPLC-MS gradient A tR 2.29 min (>95%);
MS calcd for C32H50N4O6 [MþH]þ 587.4, found 587.3; HRMS calcd
for C32H50N4O6 [MþH]þ 587.3803, found 587.3822.
Supplementary data
Copies of NMR spectra are available. Supplementary data related
to this article can be found at http://dx.doi.org/10.1016/
j.tet.2014.05.107.
4.37. Linear solonamide B
References and notes
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DMSO-d6)
d
8.35e8.25 (m, 3H), 8.21 (d, J¼7.1 Hz, 1H), 7.27e7.14 (m,
5H), 5.08 (m, 1H), 4.65e4.54 (m, 1H), 4.31 (m, 1H), 4.15 (m, 1H), 3.92
(m, 1H), 2.94 (dd, J¼13.5, 5.8 Hz, 1H), 2.74 (dd, J¼13.4, 9.1 Hz, 1H),
2.47e2.33 (m, 2H), 1.78e1.47 (m, 3H), 1.44e1.32 (m, 5H), 1.29 (d,
J¼7.8 Hz, 3H), 1.26e1.12 (m, 6H), 0.89e0.85 (m, 9H), 0.82 (d,
J¼6.2 Hz, 3H), 0.77 (d, J¼6.1 Hz, 3H); 13C NMR (100 MHz, DMSO-d6)
d
174.4, 172.2, 171.1, 169.8, 168.8, 137.9, 129.7, 128.4, 126.7, 73.6, 54.3,
51.1, 50.8, 48.0, 41.3, 38.7, 33.3, 31.4, 24.4, 24.3, 24.2, 23.7, 22.5, 22.4,
22.4, 21.8, 17.3, 14.3; UPLC-MS gradient A tR 1.60 min (>95%); HPLC
gradient C tR 11.03 min (>95%); MS calcd for C32H52N4O7 [MþH]þ
605.4, found 605.3; HRMS calcd for C32H52N4O7 [MþH]þ 605.3909,
found 605.3910.
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B. Kitir et al. / Tetrahedron xxx (2014) 1e12
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Please cite this article in press as: Kitir, B.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.05.107