A general solid phase method for the synthesis of depsipeptides

Article abstract of DOI:10.1039/c2ob26893k

Herein we describe the synthesis of depsipeptide sequences in which the backbone is composed of alternating esters and amides. Our methodology is based on the synthesis and protection of a depsidipeptide block, which is used as the growing unit for manual SPPS. We have explored Fmoc/OBzl and Fmoc/tBu SPPS strategies, and found the latter to be most compatible with our methodology.

Full text of DOI:10.1039/c2ob26893k

Organic &
Biomolecular Chemistry
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A general solid phase method for the synthesis of
depsipeptides†
Cite this: Org. Biomol. Chem., 2013, 11,
1167
Mary M. Nguyen, Nicole Ong and Laura Suggs*
Received 26th September 2012,
Accepted 21st December 2012
Herein we describe the synthesis of depsipeptide sequences in which the backbone is composed of alter-
nating esters and amides. Our methodology is based on the synthesis and protection of a depsidipeptide
block, which is used as the growing unit for manual SPPS. We have explored Fmoc/OBzl and Fmoc/tBu
SPPS strategies, and found the latter to be most compatible with our methodology.
DOI: 10.1039/c2ob26893k
www.rsc.org/obc
Lac-OEt repeats.6 Hydrogel formation from depsipeptides of
amyloid derivatives was dependent on the number and
Introduction
Depsipeptides are an interesting class of molecules that incor- location of the ester substitutions within the 10 residue
porate esters within a peptide backbone. Depsipeptides are sequence.4 Depsipeptides with three ester substitutions
readily found in nature, for example the marine depsipeptide yielded gel fibers that resembled large helical ribbons while
families of didemnins are isolated from Trididemnum solidum sequences with 1 ester substitution did not form a gel. A com-
and play an important role in the defense mechanisms of putational, molecular-mechanics model of a 12 residue, alter-
marine natural products.1 The synthesis of depsipeptides has nating sequence of lactic acid and lysine show the potential
previously been explored using solid phase peptide synthesis for regular folding patterns for depsipeptides with alternating
(SPPS). Kuilse et al. coupled α-hydroxy acid protected lactic ester and amide bonds.7 Limited work has been focused on
acid and mandelic acid to Boc-protected α-amino acids on a the synthesis of depsipeptides with regular repeats of esters.
Wang resin using DIC and DMAP.2 Spengler et al. devised a Here, we propose a synthetic methodology in which the depsi-
machine-assisted protocol for a family of depsipeptides based peptide sequence has regular, alternating esters between
on a sequence of 26 residues with up to 6 ester substitutions.3 charged peptide residues.
The highest yields (∼30%) of the final product were seen with
1 or 2 ester substitutions, while substitutions of 6 esters gave
relatively low yields (7%). Both bodies of work have shown that
single and multiple esters are successfully incorporated into a
Results and discussion
peptide backbone without modification of traditional SPPS
methods. Ester substitutions of peptide sequences are a
common strategy to analyze protein folding, function, and
self-assembly.4,5 Protein folding and self-assembly is governed
by a variety of supramolecular interactions, such as hydrogen-
bonding, pi–pi, electrostatic, or hydrophobic interactions
between peptides, peptide side chains, and/or protecting
groups. Despite the reduction of hydrogen bonding inter-
actions, depsipeptides are shown to have a stronger propensity
to form α-helices rather than β-sheets, as seen with Leu-Leu-
A general strategy towards the synthesis of depsipeptides
includes the synthesis of unique building blocks, and incor-
porating the building blocks into a peptide chain via tra-
ditional methods.8 Using the above strategy, we have
synthesized depsipeptides of varying lengths and sequences
(Fig. 1) using solid phase peptide synthesis (SPPS). We
designed our depsidipeptides with a Fmoc-protected N-termi-
nus, lactic acid (Lac) as the ester moiety to maintain hydropho-
bicity, and either lysine (Lys) or aspartic acid (Asp) as the
charged entities. Our system involves the synthesis of Fmoc-
depsidipeptides “building blocks” (Scheme 1). Synthesizing
the building blocks for SPPS requires protection at the N- and
C-terminus, as well as the peptide side chain. While a number
of protected peptides are commercially available, protecting a
depsipeptide building block is not as straightforward. Removal
of base-labile groups has been shown to affect the stereo-
chemistry of the ester bond2 while deprotection of acid-labile
groups may hydrolyze the ester bond. The synthesis of Fmoc-
dipeptides has been reported with O-pentafluorophenol (Pfp)-
The University of Texas at Austin, Department of Biomedical Engineering,
107 W Dean Keeton Street, Austin, TX 78712, USA. E-mail: m.nguyen@utexas.edu,
nicoleong@utexas.edu, Laura.Suggs@engr.utexas.edu; Fax: +1 512 471 0616;
Tel: +1 512 232 8593
†Electronic supplementary information (ESI) available: Experimental methods
for Fmoc-depsidipeptides 3a, 3b, 4c, and 4d and depsipeptides 5–9. 1H-,
13C-NMR, and MS/LCMS spectra for Fmoc-depsidipeptides 3a, 3b, 4c, and 4d.
HPLC spectra for depsipeptides 5–8. See DOI: 10.1039/c2ob26893k
This journal is © The Royal Society of Chemistry 2013
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Fig. 1 Depsipeptide library: we have synthesized depsipeptides 5–8 with alternating esters and charged peptides. All of the sequences are protected at the
N-terminal with an Fmoc group and are capped at the C-terminal with alanine (A). The sequences are named according to the residue (K = lysine, D = aspartic acid,
Lac = lactic acid) and total number of residues.
activated esters,9 which motivated us to take a similar strategy.
We synthesized the O-Pfp esters of Fmoc-Lys(Z)-OH and Fmoc-
Asp(OBzl)-OH then coupled them to lactic acid with DIPEA in
DCM. Our initial strategy was to use the Fmoc/OBzl strategy
for SPPS with these depsidipeptides, limiting the acidic
exposure to the last step of the reaction, i.e. cleaving from the
resin. The coupling reactions were monitored via TLC. The
samples were filtered and concentrated, and extracted with
ethyl acetate.
Upon purification via silica column chromatography in
either hexanes/ethyl acetate or DCM/methanol/acetic acid, we
found that Fmoc-peptide-OH remained in the sample mix-
tures. Purification by recrystallization was not successful.
We proceeded to protect the α-hydroxy acid with a benzyl
Scheme 1 Fmoc-depsidipeptide synthesis, whereby (a) Fmoc-peptide in DCM, group.10 Lac was dissolved in ethyl acetate with benzyl chloride
DIC/DMAP, 0 °C for 1 h, 16 hours at room temperature. Silica column in hexanes
and TEA. The mixture was refluxed for 5 hours, and the
and ethyl acetate (67–82%); (b) Pd/C in H2 at 5 or 15 psi for up to 16 h in dry
desired product was subsequently purified via distillation.
methanol, silica column in DCM and methanol (33–67%); (c) lactic acid in DCM
1H-NMR (not shown) matched that reported in the literature.10
with DIPEA 0 °C for 1 h, 16 hours at room temperature. Silica column in hexanes
and ethyl acetate. The synthesis of structures 110 and 39 have been described
Protections with the benzyl group required us to change the
original protection of the peptides used in the OPfp-activation
elsewhere.
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methods. With this strategy, we worked with OtBu- and Boc- block, to ensure the depsipeptide was stable under general
protected Fmoc-peptides, specifically coupling (1) with Fmoc- SPPS conditions. The resin was coupled initially with Lac and
Lys(Boc)-OH and Fmoc-Asp(OtBu)-OH in DCM with DIC and DIPEA in DCM for 2 hours and washed with DCM. Then
DMAP. The reactions were monitored by TLC. The samples Fmoc-Lys(Z)-OH was coupled with DIC and DMAP and washed
were filtered, reduced, and purified on silica in hexanes and with DMF and DCM. The depsidipeptide was coupled with
ethyl acetate. The benzyl-protecting group was removed in dry DIC and Oxymapure 3 times and cleaved in 1% TFA in DCM,
methanol with 10% palladium on activated carbon under with washings in between each coupling step. ESI/MS reveals
hydrogen and monitored with TLC. The deprotected sample that we were successful in synthesizing Fmoc-octodepsipep-
was purified on a silica column in DCM and methanol and tide, however a peak originating from the Fmoc-septadepsipep-
confirmed to be analytically pure with NMR and MS/LCMS. tide was observed. Further analysis with fragmented MS
The absence of broad peaks in the 1H-NMR spectrum suggests showed that Lac at the C-terminal was hydrolyzed. We also
the Fmoc-depsidipeptides are not racemized under the reac- observed that the internal esters were not disrupted during
tion conditions we used.
any of the SPPS methods nor were they ionized during the
Our depsipeptides are designed to have regularly alternat- analysis. To prevent hydrolysis of Lac at the C-terminal upon
ing esters and amides within their backbone. We have syn- cleaving, Fmoc-Ala-OH was used for the first coupling step. Of
thesized both charged and self-complementary sequences the four Fmoc-depsidipeptides synthesized via our methodo-
using SPPS with Lys, Asp, Ala, and Lac residues. SPPS pro- logy, (4a) and (4b) were successfully purified, and we pro-
ceeded with standard Fmoc strategies on a trityl chloride resin ceeded to use them with our optimized SPPS protocol using
(Scheme 2) and was monitored with ninhydrin. Preliminary Fmoc/OtBu strategies. This new strategy required us to opti-
tests were conducted with crude samples of 4d as the building mize the cleaving cocktail, as the mixture needed to simul-
taneously cleave the sequence off the resin and remove the Boc
or OtBu protecting groups without hydrolyzing the ester
bonds.
We tested four standard mixtures on Fmoc-K-Lac-8: A: TFA/
TIPS – 95/5; B: TFA/Water/TIPS – 95/2/3; C: TFA/DCM/TIPS –
95/2/3; D: TFA/DCM/TIPS – 50/48/2. Upon mixing for 3 hours,
the samples were precipitated in cold ether, cooled overnight,
and centrifuged. MALDI of the crude samples were very
similar among all of the cocktails and also suggest minimal
hydrolysis of the esters in all cocktails. We also synthesized a
16-mer with (4b), which was cleaved with mixture B. Again, no
internal esters were hydrolyzed, suggesting that longer
sequences can successfully be synthesized with our metho-
dology. We attempted to synthesize Fmoc-D-Lac-8 and
Fmoc-D-Lac-6. Both sequences did not precipitate well in ether
upon cleavage and were extracted in chloroform prior to purifi-
cation with HPLC. While ESI/MS (not shown) confirmed that
both were synthesized, neither sequence was successfully
purified.
SPPS on the trityl chloride resin gave very low yields (2–3%).
We proceeded to use a Fmoc-Ala-Wang resin and used the syn-
thesis methods as outlined in Scheme 2 with the exception of
eliminating the first coupling step with Lac (a). Under the
same Fmoc/OtBu strategies as described above, yields
increased to 30–40%. We proceeded to use Fmoc-Ala-Wang
resin for the synthesis of ionic, self-complementary depsipep-
tides Fmoc-K-Lac-D-A-8 and Fmoc-D-Lac-K-A-8, showing
additional evidence that our method can be used for a variety
of depsipeptide sequences.
Scheme 2 Synthesis of oligodepsipeptides via standard Fmoc-SPPS methods
with a trityl chloride resin, whereby (a) DIPEA (1 equiv.) for 1.5 hours with
mixing, followed by DMF and DCM washes; (b) 20% piperidine in DMF for
5 minutes (×3) followed by DMF and DCM washes; (c) DIC (4 equiv.) and DMAP
Conclusions
(0.01 equiv.) for 2 hours with mixing, followed by DMF and DCM washes; (d)
DIC (4 equiv.) and OxymaPure (0.1 equiv.) for 2 hours with mixing followed by
We have successfully synthesized depsipeptides with alternat-
DCM washes; (e) mixing with cleaving cocktail (A: TFA/TIPS – 95/5; B: TFA/
ing esters and amides of varying lengths and sequences. Our
SPPS method involves the synthesis of unique Fmoc-
Water/TIPS – 95/2/3; C: TFA/DCM/TIPS – 95/2/3; D: TFA/DCM/TIPS – 50/48/2)
for 3 hours followed by precipitation in cold ether or extraction in chloroform.
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depsidipeptides as the building block. Our strategy allows
for scale-up of the synthesis and can be used as a template
to incorporate α-hydroxy acids and peptide residues
not explored in this work. The sequences purified in
this article will be further explored for their tendency to
self-assemble and the results will be the subject of a future
publication.
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Acknowledgements
This work was supported by the Welch Foundation and
the Nation Institutes of Health (R21HL102806). We would
like to thank Cameron Peebles and Dr John Hardy for
productive discussions and for their time in revising
the manuscript. Also many thanks to the NMR Characteri-
zation Facility staff, Dr Ben Shoulders, Steve Sorey,
and Angela Spangelberg; the MS Facility and Karin Keller;
and the Protein and Metabolite Analysis Facility staff,
Michelle Gadush and Marvin Mercado. We would also like to
9 L. Kisfaludy and I. Schon, Synthesis-Stuttgart, 1983 (4), 325–327.
acknowledge Dr Brent Iverson and William Bell for their 10 W. Fan, X. Li, S. Qian, S. Wang and Y. Wu, Lett. Drug Des.
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