Total synthesis of daptomycin by cyclization via a chemoselective serine ligation

Article abstract of DOI:10.1021/ja4012468

A total synthesis of daptomycin, the first natural product antibiotic launched in a generation, was achieved. This convergent synthesis relies on an efficient macrocyclization via a serine ligation to assemble the 31-membered cyclic depsipeptide. The difficult esterification by the nonproteinogenic amino acid kynurenine was accomplished via the esterification of a threonine residue by a suitably protected Trp ester, followed by ozonolysis. This synthesis provides a foundation and framework to prepare varied analogues of daptomycin to establish its structure-activity profile.

Full text of DOI:10.1021/ja4012468

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Article
Total Synthesis of Daptomycin by Cyclization
via a Chemoselective Serine Ligation
Hiu Yung Lam, Yinfeng Zhang, Han Liu, Jianchao Xu, Clarence T. T. Wong, Ci Xu, and Xuechen Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja4012468 • Publication Date (Web): 05 Apr 2013
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Total Synthesis of Daptomycin by Cyclization via a Chemoselective
Serine Ligation
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Hiu Yung Lam, Yinfeng Zhang, Han Liu, Jianchao Xu, Clarence T. T. Wong, Ci Xu, Xuechen Li*
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China
KEYWORDS. Daptomycin, antibiotics, peptide ligation, lipodepsipeptide, cyclization.
ABSTRACT: A total synthesis of daptomycin, the first natural product antibiotic launched in a generation, was achieved.
This convergent synthesis relies on an efficient macrocyclization via a serine ligation to assemble the 31-membered cyclic
depsipeptide. The difficult esterification by the non-proteinogenic amino acid kynurenine was accomplished via the ester-
ification of a threonine residue by a suitably protected Trp-ester, followed by ozonolysis. This synthesis provides a founda-
tion and framework to prepare varied analogues of daptomycin to establish its structure-activity profile.
INTRODUCTION
produced from biosynthesis include mutations at D-Ala,
D-Ser, 3-mGlu and Kyn.6 Additional chemical modifica-
tions to improve the activity of daptomycin were confined
to the lipid chain and the δ-amino group of ornithine.1,8
An efficient route to chemically synthesize daptomycin,
that allows the assembly of the peptide sequence with
precision and flexibility, will make it possible to prepare a
wider variation of daptomycin analogues, thereby ena-
bling the establishment of a comprehensive SAR of dap-
tomycin to kickstart the search for optimized analogues.
However, a de novo chemical synthesis of daptomycin has
not yet been reported. The presence of two non-
proteinogenic amino acids, Kyn and 3-mGlu, embedded in
the cyclic peptide backbone, and the macrolactamization
of a 31-membered depsipeptidic ring render daptomycin a
challenging target for total synthesis. Herein, we describe
the first total synthesis of daptomycin, featuring a macro-
lactamization based on a serine-ligation developed in our
group.
The emergence of multidrug resistance against bacte-
rial pathogens has created an urgent need for the devel-
opment of effective antibiotics with new modes of action
against such resistant strains. Daptomycin is a lipodep-
sipeptide isolated from Streptomyces roseoporus that was
obtained from a soil sample from Mount Ararat (Turkey)
by scientists at Eli Lilly (Figure 1). Daptomycin has potent
bactericidal activity with a unique mode of action against
otherwise antibiotic-resistant Gram-positive pathogens,
including methicillin-resistant Staphylococcus aureus
(MRSA), vancomycin-resistant enterococci (VRE) and
vancomycin-resistant S. aureus.1 Daptomycin undergoes a
conformational change on binding to calcium ions, which
enables it to insert into bacterial membranes and induce
membrane leakage as its apparent mode of action.2 In
2003, daptomycin was officially approved by the FDA for
the treatment of skin infections caused by Gram-positive
pathogens. The development of drug-resistance to dap-
tomycin in pathogenic bacteria has so far not been ob-
served.3 The primary structure of daptomycin was estab-
lished by Debono et al. in 1968.4 It is a 13-amino acid cyclic
lipodepsipeptide that belongs to the nonribosomal pep-
tide family. It contains a 31-membered ring made up of 10
amino acids, and a linear 3-amino acid side chain modi-
fied with an n-decanoyl lipid at the N-terminus. Within
the sequence, there are two unnatural amino acids,
kynurenine (Kyn) and 3-methyl-glutamic acid (3-mGlu),
along with D-Asn, D-Ser and D-Ala (Figure 1).
H3N
D-Ala
O
O
H
N
NH
N
H
N
O
H
CONH2
O
NH
O
HO2C
O
O
HN
H
N
HO2C
O
N
H
N
H
Me
NH
O
O
O
HO
NH
O
CO2H
H
N
D-Asn
O
N
H
N
H
O
Daptomycin is the first natural product antibiotic
launched in a generation. Its distinct mechanism of action
renders daptomycin a new structural motif for the devel-
opment of new antibiotics. In this regard, the establish-
ment of the structure-activity-relationship (SAR) of dap-
tomycin will be invaluable in the search for daptomycin-
based next-generation antibiotics for additional clinical
applications and in preparation for future waves of the
development of bacterial resistance.5 However, only a lim-
ited number of daptomycin analogues with but a few vari-
ations have been produced via genetic engineering of the
non-ribosomal peptide synthetase (NRPS) in the dap-
tomycin biosynthetic pathway6, and by chemoenzymatic
synthesis7 and semi-synthesis.8 The daptomycin analogues
O
decanoyl side chain
O
D-Ser
CO2H
3-mGlu
Kyn
NH2
3-amino acid side chain
10-amino acid ring
Figure 1. The structure of daptomycin.
RESULTS AND DISCUSSION
Retrosynthesis. Our retrosynthetic analysis for dap-
tomycin synthesis is presented in Scheme 1. An anticipat-
ed challenge in the synthesis of the cyclic depsipeptide is
the macrolactamization.9,10 The development of new
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methods of peptide cyclization has attracted extensive
Page 2 of 9
Hackenberger to be applicable to the synthesis of an array
research efforts, because the conventional methods suffer
significant drawbacks.11 Prior to macrocyclization, the
limited solubility of the side-chain protected peptides in
most solvents makes the handling and purification of the
linear peptide precursor very difficult. Then the cycliza-
tion typically has to be performed under highly dilute
conditions (typically sub-millimolar concentrations) to
minimize dimerization and oligomerization, thereby lim-
iting the scale of each preparation.11 Furthermore, epimer-
ization at the C-terminus often accompanies slow peptide
cyclizations.
of cyclic heptapeptides.15 Yudin and co-workers have uti-
lized unprotected NH aziridine aldehyde, isocyanide and
linear peptides to achieve peptide cyclization efficiently.16
1
2
3
4
5
6
7
8
Recently, we have reported that an O-salicylaldehyde
ester could react with a 1,2-hydroxylamine moiety as
found in serine and threonine, to form an N,O-
benzylidene acetal-linked intermediate. The resultant
acetal group was readily removed by acidolysis to afford
an amide bond at the juncture to restore the natural pep-
tidic bond, resulting in an overall peptide ligation.17
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We further hypothesized that the C-terminal salic-
ylaldehyde ester of a side-chain unprotected peptide may
also be able to react with the N-terminal serine/threonine
of the same peptide intramolecularly, to realize a head-to-
tail peptide macrocyclization. Indeed, we were well aware
that in an intramolecular context, the presence of side-
chain unprotected functional groups (e.g. amines) could
complicate the chemoselectivity and efficiency of the liga-
tion. Thus, we sought to examine experimentally whether
the peptide cyclization of the side-chain unprotected lin-
ear daptomycin precursor could be achieved via the O-
salicylaldehyde ester-mediated intramolecular chemical
ligation at the serine/threonine site. Daptomycin serves as
an ideal target molecule to demonstrate the effectiveness
or constraints of this approach because of its representa-
tive amino acid composition, structural complexity, and
its therapeutic value.
H3N
O
O
H
N
NH
N
H
N
O
H
CONH2
O
NH
O
HO2C
O
O
HN
H
N
HO2C
O
N
N
H
H
Me
NH
O
O
O
HO
NH
O
CO2H
H
N
O
N
H
N
H
O
O
O
CO2H
NH2
1 (daptomycin)
serine ligation
-mediated cyclization
H3N
O
O
H
N
NH
N
H
N
H
O
CONH2
O
O
NH
O
HO2C
O
HN
H
N
HO2C
O
Upon the inspection of the daptomycin structure, we
decided to effect cyclization at Gly-DSer juncture using
the serine ligation (Scheme 1). We anticipated that the
requisite linear peptide precursor (cf. 2) could be obtained
via Fmoc-SPPS. Optimally, the depsipeptide ester bond
would be formed via on-resin esterification of Kyn (cf. 3).
N
H
N
H
Me
NH
O
O
O
HO
HN
NH2
O
CO2H
H
N
O
O
N
H
O
O
O
OHC
HO2C
NH2
2
The Synthesis of Kyn and (2S, 3R)-3-mGlu Build-
ing Blocks. Daptomycin contains two non-proteinogenic
amino acids: kynurenine (Kyn) and 3-methyl-glutamic
acid (3-mGlu) which are critical for bioactivity. Studies
have revealed that the Kyn residue is a key factor for en-
zymatic recognition during daptomycin biosynthesis, and
3-mGlu residue is essential for antibiotic activity.3,8,18
BocHN
O
O
H
N
NH
N
H
N
H
O
CONHTrt
O
NH
O
t-BuO2C
O
O
HN
H
N
t-BuO2C
O
N
H
N
H
Me
NH
O
O
O
t-BuO
HN
NHBoc
O
O
t-BuO2C
H
N
O
N
H
O
esterification
O
O
CO2t-Bu
NHBoc
3
Solid Phase Peptide Synthsis
H2
N
HN
O
O
O
H2N
CO2H
Kyn
H2
N
CO2
CO2
H
H
NHFmoc
CO2
H
H2
N
CO2H
H2
N
CO2H
3-mGlu
Scheme 1. The retrosynthetic strategy of daptomycin.
Chemical ligation-mediated cyclization presents an
alternative protocol which is more efficient than the tradi-
tional head-to-tail lactamization methods. Chemical liga-
tion-mediated peptide cyclization is less prone to epimer-
ization and is able to accommodate side-chain unprotect-
ed peptides. For example, native chemical ligation (NCL)
has already been applied to the synthesis of cysteine-
containing cyclic peptides.12,13 To extend the application
of this method to the preparation of non-cysteine con-
taining cyclic peptides, the use of thiol-containing unnat-
ural amino acids as cysteine surrogates, followed by effec-
tive desulfurization techniques14 has overcome this limit-
ing requirement of NCL.11 Alternatively, the Staudinger
ligation has been demonstrated by Kleineweischede and
Scheme 2. Synthesis of Fmoc-Kyn-OH and Fmoc-3-
mGlu-OH. Key: (a) (i) O3, -78 oC, CH2Cl2, 10 min; (ii) Me2S,
o
-78 C – rt, 2 h; (b) LiOH, H2O/THF, 18 h; (c) tBu-Br,
o
BTEAC, DMAC, K2CO3, 55 C, 24 h, 65% over two steps.
(d) TBAF, THF,
1
h,
84%; (e) NaIO4, RuCl3,
MeCN/CCl4/H2O, 1/1/2, 2 h, 82%; (f) 4 M HCl in dioxane, 0
oC – rt, 50 min; (g) Fmoc-OSu, Na2CO3, dioxane/H2O, 18
h, 54% over two steps.
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The preparations of Kyn and 3-mGlu are presented in
Having prepared the Kyn and 3-mGlu building
blocks, we proceeded to synthesize the daptomycin linear
precursor 3. According to the retrosynthetic plan (Scheme
1), 3 was expected to be readily constructed via standard
SPPS on a 2-chlorotrityl chloride resin. After the resin-
Gly-Asp(tBu)-DAla-Asp(tBu)-Orn(Boc)-Gly-Thr(OTBS)-
Asp(tBu)-DAsn(Trt)-Trp(Boc)-C9H19 11 was assembled via
standard Fmoc/tBu-SPPS, and the threonine residue was
desilylated to give 12 (Scheme 3), we encountered difficul-
ties in the coupling reaction of Fmoc-Kyn(Boc, CHO)-OH
4b with the resin-linked threonine-OH. A variety of cou-
pling reactions, including the use of common coupling
reagents, Mukaiyama esterification and Yamaguchi esteri-
fication, were tried in vain. It became apparent that the
on-resin esterification would be rather problematic. This
was probably due to the low reactivity of the Kyn moiety,
or the steric hindrance of the resin-linked peptide chain.
Scheme 2. Although Kyn derivatives have previously been
prepared from the corresponding Trp compounds by ozo-
nolysis in one step, this approach suffered from low yields
(20-33%) and the generation of a number of impurities.19
Due to these limitations, a multi-step synthetic route to
L-kynurenine via β-3-oxindolylalanines was recently de-
veloped.20
1
2
3
4
5
6
7
8
We suspected that the electron-rich indole moiety
suffered decomposition during ozonolysis of Trp and re-
sulted in poor yields. To verify this hypothesis, we sub-
jected both Fmoc-Trp-OH and Fmoc-Trp(Boc)-OH to
ozonolysis. Indeed, Fmoc-Kyn(CHO)-OH 4a was obtained
from Fmoc-Trp-OH in only 20% yield, a result similar to
that reported in the literature. However, Fmoc-Trp(Boc)-
OH was ozonolyzed to Fmoc-Trp(Boc, CHO)-OH 4b in
quantitative yield (99%). This approach is scalable and
provides ready access to the valuable Kyn moiety.
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The preparation of Fmoc-3-mGlu(tBu)-OH started
with compound 5.21 Ring opening of 5 with LiOH generat-
ed compound 6. The subsequent esterification of the γ-
carboxylic acid of 6 was at first problematic using tBuOH,
but compound 7 was finally obtained via the tert-butyl
bromide method22 in 65% yield over two steps. Subse-
quently, the TBS group of compound 7 was removed with
TBAF, followed by oxidation using sodium periodate and
ruthenium (III) chloride, to afford Boc-3-mGlu(tBu)-OH 9
in good yield. The Boc group of 9 was selectively removed
in the presence of the t-butyl ester by treatment with 4 M
HCl in dioxane. After re-protection with an Fmoc group,
the desired (2S, 3R)-methyl glutamic acid building block
10 suitable for Fmoc/tBu-SPPS was obtained with an
overall yield of 24% over six steps (Scheme 2).
Initial Synthetic Strategies toward Daptomycin.
Scheme 4. Second attempt toward daptomycin. Key:
(a) (i) Fmoc-AA-OH, HATU, DIEA, DMF, 80-95%; (ii)
DEA, CH2Cl2, 85-95%; (b) TBAF, THF, 90%.
To overcome the obstacles presented by the on-resin
esterification of Kyn, we decided to adopt a hybrid syn-
thesis strategy using both solid-phase and solution phase
synthesis. We envisaged that a fragment containing Kyn
(i.e. 16, Scheme 4) could be pre-assembled via solution
phase synthesis, and then joined with the resin-linked-
pentapeptide
(NH2-Orn(Boc)-Asp(tBu)-DAla-Asp(tBu)-
Gly) 17, to afford compound 13 (Scheme 4). Using this
strategy, we anticipated that the esterification reaction
conducted in the solution phase between compound 15
and Fmoc-Kyn(Boc, CHO)-OH 4b would be more favora-
ble. To construct compound 15, NH2-Gly-OAll was first
coupled with Fmoc-Thr(tBu)-OH with the aid of HATU.
The tBu group was removed under acidic conditions, fol-
lowed by re-protection with a TBS group. The Fmoc-
group of the resultant dipeptide was removed with di-
ethylamine (DEA). Sequentially, the peptide was homolo-
gated with Fmoc-Asp(tBu)-OH, Fmoc-DAsn(Trt)-OH,
Fmoc-Trp(Boc), and decanoic acid. After each coupling
and de-Fmoc step, the product was purified by flash
chromatography. After the desilylation of 14, we attempt-
ed the coupling of compound 15 with Fmoc-Kyn(Boc,
CHO)-OH 4b in solution. However, again, the results
were very disappointing.
Scheme 3. First attempt toward daptomycin. Key: (a)
(i) Fmoc-AA-OH, HATU, DIEA, DMF; (ii) piperidine,
DMF; (b) TBAF, THF, 1 h.
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Modified Route to Daptomycin. Having tried a Our synthesis of ester 26 started with glycine 18
range of coupling strategies and methods to engage 4b,
we conceded that the Kyn building block was not suitable
for direct esterification. Hence, an alternative strategy had
to be devised. At this point, we planned to take recourse
to using Trp for the esterification of the threonine resi-
due, after which Trp would be converted to Kyn at a later
stage. We were well aware that the structural complexity
presented by a large peptide fragment could complicate
the effort to achieve a clean transformation of Trp to Kyn
via ozonolysis. Therefore, a suitable Kyn-containing pep-
tide fragment of a minimal and robust structure was re-
quired.
(Scheme 5). Acidolysis of the Boc group of 18, followed by
coupling with Fmoc-Thr(tBu)-OH afforded the dipeptide
Fmoc-Thr(tBu)-Gly-OAll. Removal of the tBu group, fol-
lowed by silylation of the hydroxyl group gave rise to di-
peptide 19. This compound, after removal of the N-
terminal Fmoc group with DEA, was coupled with Fmoc-
Asp(tBu)-OH in 62% yield over two steps. The resultant
tripeptide 20 underwent Fmoc removal, diazo transfer24
and TBS deprotection to give tripeptide 23 in an overall
58% yield over three steps. Esterification of 23 with Fmoc-
Trp(Boc)-OH, with the aid of PyBOP, gave rise to ester 24
without epimerization, in an excellent yield (87%). After
palladium-catalyzed deallylation, ester 25 was subjected
to ozonolysis to convert Trp to Kyn. Gratifyingly, the reac-
tion proceeded without incident, providing ester 26 in an
overall 81% yield (Scheme 5). Overall, this late-stage ozo-
nolysis strategy could be a general route for the prepara-
tion of Kyn-containing peptides.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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42
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44
45
46
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48
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50
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53
54
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56
57
58
59
60
Firstly, we considered the simplest dipeptide Fmoc-
Thr[O-Trp(Boc)]-OH as a building block amenable to
ozonolysis. The Fmoc-Thr[O-Kyn(Boc, CHO)]-OH ob-
tained after ozonolysis, could be coupled with the resin-
linked growing peptide. In this plan, however, an O→N
acyl transfer23 may be a potential side reaction during the
subsequent SPPS from the threonine N-terminus. If the
tripeptide Fmoc-Asp(tBu)-Thr[O-Kyn(Boc, CHO)]-OH
were used as a preassembled building block, epimeriza-
tion at the threonine residue would be likely in the cou-
pling of this tripeptide to the resin-bound growing pep-
tide chain. At last, we settled on N3-Asp(tBu)-Thr-[O-
Kyn(Boc, CHO)-Fmoc]-Gly-OH 26 (Scheme 5) as the cou-
pling fragment via solution phase synthesis. The azide
group was selected to protect the N-terminus of Asp in
order to achieve orthogonality with Fmoc-SPPS.
BocHN
O
H
N
O
26
a
H2N
N
O
O
H
O
NHFmoc
t-BuO2C
t-BuO2C
HN
b
O
HN
O
O
17
BocHN
O
BocHN
O
O
H
N
O
H
N
N
H
N
O
O
H
O
NH
O
O
N
H
N
a
O
t-BuO2C
O
HN
H
O
NH
O
O
N3
t-BuO2C
O
HN
t-BuO2C
N
H
N3
t-BuO2C
O
O
HN
N
H
CO2t-Bu
O
t-BuO
HN
O
CO2t-Bu
O
H
N
NHFmoc
O
NHBoc
O
N
H
O
O
O
O
NBoc
CHO
CO2t-Bu
NBoc
CHO
27
28
BocHN
O
O
H
N
NH
N
H
N
H
O
O
CONHTrt
O
NH
O
O
c, a
t-BuO2C
O
O
HN
H
N
t-BuO2C
N
H
N
H
C9H19
NH
O
O
t-BuO
HN
NHBoc
O
CO2t-Bu
O
H
N
O
N
H
O
O
O
CO2t-Bu
NBoc
CHO
29
Scheme 6. Synthesis of the daptomycin linear pre-
cursor. Key: (a) Fmoc-SPPS; (b) HATU, DIEA, DMF, 45
min; repeat; (c) DTT, DIEA, DMF, 2 h, 100% conversion as
analyzed by LC-MS after the peptide was released from
the resin.
Scheme 5. Solution phase synthesis of a Kyn contain-
ing fragment. Key: (a) TFA, 30 min; (b) Fmoc-Thr(tBu)-
OH, EDCI, HOBt, DIEA, CH2Cl2, 8 h, 74% over two steps;
(c) 95% TFA, 30 min; (d) TBSCl, imidazole, DMF, 12 h,
70% over two steps; (e) DEA, CH2Cl2, 2 h, 80%; (f) Fmoc-
Asp(tBu)-OH, HATU, DIEA, DMF, 8 h, 77%; (g) DEA,
Next, the trityl-resin-linked pentapeptide (Fmoc-
Orn(Boc)-Asp(tBu)-DAla-Asp(tBu)-Gly) 17 was assembled
via standard Fmoc-SPPS, which was then successfully
coupled with N3-Asp(tBu)-Thr-[O-Kyn(Boc, CHO)-Fmoc]-
Gly-OH 26, using HATU as the coupling reagent. The re-
sultant resin-linked peptide was subsequently coupled
with Fmoc-3-mGlu(tBu)-OH 10 and Boc-DSer(tBu)-OH
under standard SPPS conditions to produce 28. After re-
CH2Cl2,
2 h, 100%; (h) Imidazole-1-sulfonyl azide,
CuSO4⋅5H2O, NaHCO3, MeOH, H2O, 2 h; (i) TBAF, AcOH,
THF, 4 h, 58% over two steps; (j) Fmoc-Trp(Boc)-OH,
PyBOP, DIEA, CH2Cl2, 12 h, 87%; (k) Pd(PPh3)4, N-
methylaniline, THF, 4 h, 85%; (l) (i) O3, -78oC, CH2Cl2; (ii)
Me2S, -78 oC to rt, 2 h, 95%.
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duction of the N-terminal azide group in 28 with dithio-
lecular weight and different functional groups compared
with the desired head-to-tail N,O-benzylidene acetal
product, and would be likely to be distinguishable and
separable by chromatography. Gratifyingly, we carefully
analyzed the crude cyclization mixture and did not ob-
serve any undesired head-to-side chain cyclic product.
Therefore, the second step in the ligation which restores
the natural peptidic bond at the coupling site by remov-
ing the acetal group by acidolysis yielded daptomycin as
the only product. Therefore, we have demonstrated that
the presence of unprotected side-chain functional groups
do not adversely impact the cyclization process. The suc-
cess of the daptomycin synthesis serves to showcase this
chemical ligation with N-terminal serine/threonine as an
alternative tool for synthesizing cyclic peptides. Indeed,
many naturally occurring cyclic peptides with promising
bioactivities, such as phakellistatin, lysobactin and kawa-
gucipeptin9-10, 29-30, contain serine or threonine, and these
targets could be disconnected for synthesis based on this
approach.
threitol (DTT), further peptide homologation via Fmoc-
SPPS afforded the whole linear sequence 29 successfully
(Scheme 6).
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6
7
8
Completing the Synthesis of Daptomycin via Ser-
ine Ligation. The side-chain protected linear peptide was
released from the trityl resin with AcOH/TFE/CH2Cl2 to
afford the peptide acid 30 with the side-chain protecting
groups intact. To set the stage for the final macrocycliza-
tion via serine ligation, a salicylaldehyde ester needed to
be installed at the C-terminal carboxyl group of com-
pound 30. The requisite peptide salicylaldehyde ester was
obtained through direct coupling between the peptide
acid and α,α-dimethoxy-salicylaldehyde mediated by Py-
BOP. Then, global deprotection of all side-chain protect-
ing groups with TFA/H2O/PhOH afforded peptide salic-
ylaldehyde ester 31 in 17% from the resin loading. Notably,
the formyl group on Kyn was also removed during the
acidolysis.
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With the linear side-chain unprotected peptide salic-
ylaldehyde ester 31 in hand, we were in a position to in-
vestigate the peptide cyclization via serine ligation
(Scheme 7). To this end, peptide 31 was dissolved in pyri-
dine acetate buffer (mol/mol, 1:1) at a concentration of 5
mM. The desired macrolactamization proceeded smooth-
ly, attaining a full conversion within 4 hours at room
temperature, and no side products were observed. With-
out isolation20, the N,O-benzylidene acetal linked cyclic
product was treated with TFA/H2O for 10 min, whereupon
daptomycin (1) was obtained in 67% yield after purifica-
tion by semi-preparative reverse-phase HPLC. It is
worthwhile to note that the cyclization was tried at a
range of concentrations from 5 mM to 50 mM; and at all
concentrations, the monomeric cyclic peptide was fur-
nished as the only product. This enables the effective scal-
ing up of the synthesis of cyclic peptides and makes this
strategy highly practical. To compare, we have also tried
the conventional lactamization approach to cyclize the
daptomycin linear precursor at the same Gly-DSer junc-
ture, using a side chain-protected linear peptide precur-
sor32. However, under all conditions attempted (HATU,
DIEA, DMF; DEPBT, DIEA, DMF) at a concentration of 5
mM, only a trace amount of cyclization, together with
dimerized and oligomerized products, was observed by
LC-MS analysis.
That the cyclization of 31 was successful indicated
that the functional groups of the unprotected side chain,
in particular, the amine group, did not interfere with the
serine ligation-mediated cyclization. Although the compe-
tition between the free amine of the side-chain and the N-
terminal serine for reacting with the aldehyde group of
the salicylaldehyde ester cannot be precluded, and in fact
could very well occur, the Schiff base of the free amine is
produced reversibly, and represents a transient and non-
productive intermediate species whose formation does
not consume the aldehyde and inhibit the desired macro-
lactamization.
The clean cyclization of 31 also indicated that the di-
rect aminolysis by the side-chain amine group in the Orn
residue of the peptide salicylaldehyde ester to afford a
head-to-side chain cyclized product, did not occur.31 The
serine/threonine mediated peptide cyclization reported
herein is a two-step process. The first step is hemiaminal
formation between the C-terminal salicylaldehyde ester
and the N-terminal serine/threonine to give an N,O-
benzylidene acetal cyclic peptide. If a head-to-side chain
cyclization via direct aminolysis were to occur under the-
se conditions, this product would have a different mo-
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Scheme 7. Completion of daptomycin via a serine li-
Page 6 of 9
from The University of Hong Kong (20101059015). We thank
gation-mediated cyclization. Key: (a) AcOH/TFE/DCM,
1.5 h; (b) (i) α,α-dimethoxy-salicylaldehyde, PyBOP, DIEA,
CH2Cl2, 2 h; (ii) TFA/H2O/PhOH, 1 h, 17% based on the
resin loading; (c) pyridine acetate, rt, 4 h; (d) TFA/H2O, 10
min, reverse-phase HPLC purification, 67%.
Prof. Chi-Ming Che for helpful discussions in the preparation
of the manuscript, Prof. Pauline Chiu for suggestions on edit-
ing, and Dr. Eva Fung of the Department of Chemistry at the
University of Hong Kong for ESI-HRMS work.
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8
ABBREVIATIONS
As daptomycin tends to aggregate, the 1H NMR of the
synthetic daptomycin (1) was recorded26 using the report-
ed optimal conditions27 (5% D2O in pH=7.8 PBS buffer).
The spectrum was in full accordance with those in the
literature.27 In addition, the identity and purity of the syn-
thetic material was in full agreement with authentic dap-
tomycin from a commercial source, when compared by
NMR spectroscopy and HPLC under the optimized HPLC
conditions.28
SPPS, solid phase peptide synthesis; HPLC, high performance
liquid chromatography.
9
REFERENCES
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(1) Baltz, R. H. Curr. Opin. Chem. Biol. 2009, 13, 144-151.
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In summary, we have described the first total synthe-
sis of cyclic depsipeptide daptomycin. The synthesis in-
volves a hybrid strategy using both solid phase and solu-
tion phase synthesis. The key features of the synthesis
include a practical synthesis of Fmoc-3-mGlu(tBu)-OH,
and a solution to the problem of Kyn preparation,19,20 in
which an N-Boc-protected Trp building block is ozo-
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gation of the structure-activity-relationship of daptomy-
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ASSOCIATED CONTENT
Supporting Information.
Experimental procedures, spectral and other characterization
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AUTHOR INFORMATION
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* xuechenl@hku.hk
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
This work was supported by the General Research Fund
(HKU703811P, 707412P) of the Research Grants Council of
Hong Kong, the National Basic Research Program of China
(973 Program, 2013CB836900), Peacock Program-Project
Development Fund (KQC201109050074A) and Seed Funding
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(26) A 600 MHz NMR spectrometer equpied with a cryoprobe
was used.
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(32) The synthesis of the side-chain protected linear peptide
precusor was similar to that described in the text, except
that Alloc-DSer(tBu)-OH was used instead of Boc-
DSer(tBu)-OH. The Alloc group was selectively removed
(Pd(PPh3)4, PhSiH3) with the side-chain protecting groups
intact, prior to the conventional lactamization.
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