Solid-Phase Synthesis of Cyclic Depsipeptides Containing a Tyrosine Phenyl Ester Bond

Article abstract of DOI:10.1021/acs.orglett.6b02281

The first solid-phase strategy for the synthesis of cyclic depsipeptides containing a phenyl ester linkage in their structure is described. The key steps of the synthesis were the formation of the phenyl ester bond and the on-resin head-to-side-chain cyclization. The amino acid configuration significantly influenced the formation and the stability of the cyclic depsipeptides. The presence of a l-Tyr¹ and a d-Tyr⁷ led to the most stable sequences.

Full text of DOI:10.1021/acs.orglett.6b02281

Letter
pubs.acs.org/OrgLett
Solid-Phase Synthesis of Cyclic Depsipeptides Containing a Tyrosine
Phenyl Ester Bond
Cristina Roses, Cristina Camo, Kristy Vogels, Marta Planas,* and Lidia Feliu*
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LIPPSO, Department of Chemistry, University of Girona, Maria Aurelia Capmany 69, 17003 Girona, Spain
S
* Supporting Information
ABSTRACT: The first solid-phase strategy for the synthesis of cyclic depsipeptides containing a phenyl
ester linkage in their structure is described. The key steps of the synthesis were the formation of the
phenyl ester bond and the on-resin head-to-side-chain cyclization. The amino acid configuration
significantly influenced the formation and the stability of the cyclic depsipeptides. The presence of a L-
Tyr1 and a D-Tyr7 led to the most stable sequences.
engycins are cyclic lipodepsipeptides isolated from several
residue at positions 3 and 9. Initially, fengycins were reported to
F
Bacillus spp. that were first described in 1986.1 These cyclic
have a D-Tyr3/L-Tyr9 configuration,1,8 whereas a L-Tyr3/D-Tyr9
configuration was assigned to another family of related
lipopeptides named plipastatins.9 Nevertheless, a recent study
showed that fengycins and plipastatins have the same primary
structure, that is, a L-Tyr3/D-Tyr9 configuration.10 In this study,
we name the peptides as fengycins because this term is more
widely employed than plipastatins, and we use the correct
configuration (Figure 1).
compounds show interesting antifungal activity mainly against
filamentous fungi, and unlike other lipopeptides produced by
Bacillus spp., they are low hemolytic.1,2 Moreover, they have been
described as elicitors of defense responses in plants, making them
a promising alternative for the protection of crops against
phytopathogens.3
The fengycin family includes related compounds that share a
common cyclic structure with exceptional features.1,4−7 They are
decapeptides acylated at the N-terminus with a β-hydroxy fatty
acid tail (Figure 1). Eight of the amino acids form a macrolactone
in which the ester bond occurs between the phenol group of a
tyrosine (Tyr) and the carboxylic group of an isoleucine (Ile).
Until now, three isoforms of fengycins have been isolated, A, B,
and S, which differ on the amino acids at positions 4 and 6. There
has been some confusion over the configuration of the Tyr
Solid-phase synthesis has been established as a good strategy
for the obtention of cyclodepsipeptides.11−15 However, to the
best of our knowledge, the solid-phase synthesis of fengycins has
not yet been described. Moreover, cyclodepsipeptides that have
been synthesized on solid phase bear an ester bond involving the
β-hydroxyl group of a Thr or Ser residue. No examples have been
reported on the formation of ester bonds involving the phenol
group of a Tyr, which can be attributed to the high lability of
phenyl esters. In fact, until now, the preparation of fengycins was
only addressed by the group of Marahiel and Essen, and it
involves the solid-phase synthesis of the linear precursor
followed by chemoenzymatic cyclization in solution.16−18 Due
to the structural requirements of this last step, this approach is
restricted to the obtention of a limited number of analogues.
Based on these considerations, the synthesis of fengycins
constitutes an attractive synthetic challenge, not only for being
constrained cyclopeptides but also for containing a phenyl ester
in their structure.
Herein, we explore a suitable solid-phase approach for the
preparation of the macrolactone of eight amino acids present in
fengycins (Figure 1). In particular, we focused our attention on
the synthesis of compounds I and II (Figure 2). The structure of
Received: August 1, 2016
Figure 1. Structure of fengycins A, B, and S.
© XXXX American Chemical Society
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I contains amino acids with the configuration described for
fengycins, whereas II incorporates D-Tyr1 and L-Tyr7.
Val4, D-Tyr7). A three-dimensional orthogonal 9-fluorenylme-
thoxycarbonyl (Fmoc)/tert-butyl (tBu)/allyl (All) protecting
strategy was selected. According to our methodology, the linear
resin Boc-Tyr1-D-Thr2(tBu)-Glu(OtBu)-D-Val4-Pro-Gln(Tr)-D-
Tyr7(Wang)-OAll (1) was considered a precursor of BPC822.
Wang resin was used as solid support because it enables
incorporation of Tyr7 using a Mitsunobu reaction.19,20 Thus,
Fmoc-D-Tyr-OAll was anchored to the resin by treatment with
PPh3 and diisopropylazodicarboxylate (DIAD) in anhydrous
THF under stirring at room temperature for 24 h.19,20 As
determined with the Fmoc method, resin Fmoc-D-Tyr(Wang)-
OAll was obtained with a loading of 0.33 mmol/g. Moreover,
according to Marfey’s test, the Tyr residue did not racemize
under these conditions. Reaction time was considerably
shortened when the anchoring was carried out under microwave
irradiation. After only 30 min irradiation at 60 °C, the Fmoc-D-
Tyr(Wang)-OAll resin was obtained in a similar loading and no
racemization of the D-Tyr was observed. To the best of our
knowledge, this is the first example of a microwave-assisted
Mitsunobu reaction for the anchoring of Tyr onto a solid
support.
Elongation of the peptide sequence was performed by
sequential Fmoc removal and coupling steps. Fmoc group
removal was performed with piperidine/DMF (3:7). Amino acid
couplings were mediated by N,N′-diisopropylcarbodiimide
(DIPCDI) and ethyl 2-cyano-2-(hydroxyimino)acetate
(Oxyma) in DMF for 1 h. After completion of the sequence,
an aliquot of the resulting resin Boc-Tyr1-D-Thr2(tBu)-Glu-
(OtBu)-D-Val4-Pro-Gln(Tr)-D-Tyr7(Wang)-OAll (1) was
cleaved with TFA/H2O/triisopropylsilane (TIS) (95:2.5:2.5)
for 2 h, affording the expected linear peptide in 85% HPLC
purity, which was characterized by mass spectrometry.
Figure 2. General structure of fengycin analogues I and II.
The main concerns when planning a retrosynthetic analysis for
cyclopeptides I and II are the site of resin attachment and the
point of cyclization. The selection of these issues was determined
by the presence of the phenyl ester bond, whose formation is
especially challenging. Thus, the side chain of Tyr7 was selected
as the anchoring point to the resin, and the cyclization would
involve the formation of an amide bond between the carboxylic
acid of Tyr7 and the α-amino group of Ile8. Therefore, access to
these cyclic compounds would require (i) preparation of a linear
peptidyl resin with Tyr7 linked to the solid support, (ii) ester
bond formation, (iii) deprotection of the carboxylic and amino
groups involved in the cyclization step, and (iv) final on-resin
cyclization and cleavage.
Our investigations started with the solid-phase preparation of
cyclic depsipeptides I (Figure 2 and Scheme 1). In particular, we
assayed the synthesis of compound BPC822 (L-Tyr1, D-Thr2, D-
The unprotected phenol group of Tyr was esterified with
Alloc-Ile-OH. The allyloxycarbonyl (Alloc) group was chosen for
Scheme 1. Synthetic Strategy of Cyclodepsipeptide BPC822
B
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the protection of the α-amino group of Ile8 as an alternative to
the standard Fmoc or Boc protection because, on the one hand,
once the phenyl ester is formed the use of bases such as
piperidine is precluded due to the lability of the phenyl ester. On
the other hand, the Boc group cannot be removed without
releasing the peptide from the Wang resin. Moreover, the
conditions of the Alloc group removal using Pd(0) would allow
the simultaneous cleavage of the allyl ester group of Tyr7 before
the cyclization step. Esterification of resin 1 with Alloc-Ile-OH
was carried out, employing DIPCDI, DMAP, and N,N′-
diisopropylethylamine (DIEA) in DMF for 24 h. This reaction
was repeated twice to obtain the linear depsipeptidyl resin Boc-
Tyr1(O-Ile8-Alloc)-D-Thr2(tBu)-Glu(OtBu)-D-Val4-Pro-Gln-
(Tr)-D-Tyr7(Wang)-OAll (2). An aliquot of this resin was
treated under acidolytic conditions, yielding the expected linear
peptide in 84% HPLC purity. Next, the Alloc and allyl protecting
groups were removed using a catalytic amount of Pd(PPh3)4 in
the presence of PhSiH3 in CH2Cl2 for 4 h. After acidolytic
cleavage of an aliquot of Boc-Tyr1(O-Ile8-H)-D-Thr2(tBu)-
Glu(OtBu)-D-Val4-Pro-Gln(Tr)-D-Tyr7(Wang)-OH, the corre-
sponding peptide was obtained in 82% HPLC purity.
Scheme 2. Hydrolysis of the Ester Bond of BPC822
On-resin cyclization of this linear peptidyl resin was assayed
using [ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinyl-
phosphonium hexafluorophosphate (PyOxim), Oxyma, and
DIEA in DMF for 24 h. Acidolytic cleavage yielded the cyclic
depsipeptide BPC822 in 31% HPLC purity, as shown by ESI-
MS, where a peak at m/z 994.6 corresponding to [M + H]+ was
observed. The linear peptide precursor was not detected either
by HPLC or by mass spectrometry. To confirm the cyclic
structure of BPC822, the crude reaction mixture resulting from
acidolytic cleavage was treated with CH3OH/H2O/NH3 (4:1:1),
conditions that are known to hydrolyze ester bonds.21 HPLC and
mass spectrometry analysis of the resulting crude revealed only
the presence of the linear peptide resulting from the hydrolysis of
the ester bond in BPC822, H-Tyr1-D-Thr2-Glu-D-Val4-Pro-Gln-
D-Tyr7-Ile8-OH, and of the corresponding methyl ester H-Tyr1-
D-Thr2-Glu-D-Val4-Pro-Gln-D-Tyr7-Ile8-OMe (Scheme 2).
Therefore, this result demonstrated the formation of the cyclic
depsipeptide BPC822. Moreover, the structure of this
compound was verified by 1D and 2D NMR experiments.
This synthetic methodology was extended to the preparation
of the cyclic depsipeptides with general structure I: BPC824 (L-
Tyr1, D-Ser2, D-Val4 and D-Tyr7), BPC826 (L-Tyr1, D-Thr2, D-Ala4
and D-Tyr7), and BPC828 (L-Tyr1, D-Ser2, D-Ala4 and D-Tyr7)
(Figure 3). Mass spectrometry analysis showed that these cyclic
depsipeptides were the only products of the synthesis. Similarly
to BPC822, hydrolysis of the crude reaction mixtures confirmed
the cyclic structure of these compounds. Cyclic depsipeptides
BPC822, BPC824, BPC826, and BPC828 were purified by
column chromatography and obtained in purities ranging from
93 to >99%. These peptides were fully characterized by NMR.
Finally, the synthesis of cyclic depsipeptides type II bearing a
D-Tyr1 and a L-Tyr7 was also attempted following the above
methodology. This set included BPC830 (D-Tyr1, D-Thr2, D-Val4,
L-Tyr7), BPC832 (D-Tyr1, D-Ser2, D-Val4, L-Tyr7), BPC834 (D-
Tyr1, D-Thr2, D-Ala4, L-Tyr7), and BPC836 (D-Tyr1, D-Ser2, D-
Ala4, L-Tyr7) (Figure 3). Contrary to our expectations, the cyclic
depsipeptides were obtained together with a high amount of the
corresponding dimeric product and a linear peptide that did not
contain Ile8. The formation of the latter product revealed that the
cyclization was not complete and that the ester bond of the linear
precursor hydrolyzed, prompting the release of the Ile residue.
This result demonstrated that the cyclization of cyclic
Figure 3. Structure of cyclodepsipeptides.
depsipeptides with D-Tyr1 and L-Tyr7 is difficult and that they
are not stable, reinforcing the hypothesis by Honma and co-
workers that postulated a L and D configuration for these residues
in natural fengycins.10
Herein, we established a suitable strategy for the solid-phase
synthesis of head-to-side-chain cyclic depsipeptides containing a
phenyl ester linkage. Using a Fmoc/tBu/Alloc/allyl strategy, a set
of cyclic octadepsipeptides derived from fengycins was
successfully prepared. Our studies revealed the significance of
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DOI: 10.1021/acs.orglett.6b02281
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(20) Thutewohl, M.; Waldmann, H. Bioorg. Med. Chem. 2003, 11,
2591−2615.
(21) Cochrane, J. R.; Yoon, D. H.; McErlean, C. S. P.; Jolliffe, K. A.
Beilstein J. Org. Chem. 2012, 8, 1344−1351.
the amino acid configuration on the stability of the cyclic
depsipeptides, with those containing a L-Tyr1 and a D-Tyr7 being
the most stable. This study is the first example on the synthesis of
cyclic depsipeptides incorporating a phenyl ester bond and
represents a promising approach for the future total synthesis of
fengycins and of other natural compounds including this moiety.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.6b02281.
Experimental procedures and spectral data (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: marta.planas@udg.edu.
*E-mail: lidia.feliu@udg.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
C.R. was the recipient of a predoctoral fellowship from the
MINECO of Spain. This work was financed by Grant AGL2012-
■
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39880-C02-02. We acknowledge the Serveis Tecnics de Recerca
of the University of Girona for the ESI-MS, NMR, and HRMS
experiments.
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