Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position

Article abstract of DOI:10.1016/j.bmcl.2016.12.046

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu i

Full text of DOI:10.1016/j.bmcl.2016.12.046

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-activity relationship of daptomycin analogues with
substitution at (2S, 3R) 3-methyl glutamic acid position
a,
Du’an Lin a,1, Hiu Yung Lam a,1, Wenbo Han a, Nicole Cotroneo b, Bhaumik A. Pandya b, , Xuechen Li
⇑
⇑
a Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong Special Administrative Region
b Cubist Pharmaceutical, Inc., 65 Hayden Avenue, Lexington, MA 02421, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of
(2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial
activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of
three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln),
dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activ-
ities. The detailed synthesis of dimethyl glutamic acid was also reported.
Received 16 August 2016
Revised 23 November 2016
Accepted 15 December 2016
Available online xxxx
Keywords:
Daptomycin
Ó 2016 Elsevier Ltd. All rights reserved.
Structure-activity relationship
Solid phase peptide synthesis
Antibiotics
Methyl glutamic acid
Daptomycin, a first-in class antibiotic, was approved by the FDA
in 2003 for the treatment of skin and soft tissue infections (SSI)
caused by Gram-positive pathogens. It is active against highly
resistant bacterial strains including methicillin-resistant Staphylo-
coccus aureus (MRSA), vancomycin-resistant enterococci (VRE),
and vancomycin-resistant S. aureus.1 Until now, drug resistance
to daptomycin has been rarely found.2 Daptomycin has a unique
mode of action which is significantly different from that of other
currently used antibiotics. It may have multiple antibacterial
mechanisms. One of the most accepted ones is to disrupt mem-
brane upon binding to Ca2+ ions.3
The primary structure of daptomycin contains a 31-membered
ring composed of 10 amino acids. An exocyclic tail composed of
3 amino acids terminated by an N-terminal decanoyl lipid chain
is attached to the macrocycle via amide bond to threonine.4 The
presence of a non-proteinogenic amino acid in the sequence, (2S,
3R) 3-methyl glutamic acid (mGlu), has been found to be critical
to the bactericidal activity of daptomycin (grey box).5 Though the
exact function of (2S, 3R) mGlu has not yet been fully understood,
daptomycin analogue with (2S, 3R) mGlu substituted by Glu6 has
been found to lose the bactericidal activity by 16-fold, thus high-
lighting the importance of this particular amino acid. In this study,
we have further evaluated the biological significance of the (2S, 3R)
mGlu residue, by substituting it with other structurally relevant
moieties.
We have developed the first total chemical synthesis of dapto-
mycin in 2013, which allows for facile synthesis of daptomycin
analogues.7 We envisioned that modifications at the (2S, 3R) mGlu
position in the amino acid sequence of daptomycin would be valu-
able as it would allow us to study the role of (2S, 3R) mGlu via
chemical mapping as well as to search for potent compounds.
We began with the substitutions of (2S, 3R) mGlu with (2S, 3R)
methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R)
ethyl glutamic acid (eGlu) (Fig. 1) to examine the effect of substi-
tution at this position on the antibacterial activity. Prior to the total
synthesis, we had to first develop strategies towards the building
blocks suitable for solid phase peptide synthesis (SPPS), including
Fmoc-(2S, 3R) mGln-OH, Fmoc-(2S, 3R) eGlu(OtBu)-OH and Fmoc-
dimethyl Glu(OtBu)-OH. The syntheses of (2S, 3R) mGln and (2S,
3R) eGlu basically followed the literature reported method8 for
the synthesis of (2S, 3R) mGlu to afford Fmoc-(2S, 3R) mGln-OH
and Fmoc-(2S, 3R) eGlu(OtBu)-OH as two building blocks suitable
for Fmoc SPPS. However, synthesis of the dimethyl glutamic acid
building block was more challenging than originally anticipated.
Initially, our synthetic strategy towards dimethyl Glu started
with D-(À)-pantolactone (Scheme 1). The OH group of
D-(À)-pantolactone was successfully converted to the amino group
with an inversed stereochemistry to afford compound 4.9 Ring
opening of compound 4 was achieved by 33% HBr-AcOH to afford
⇑
Corresponding authors.
E-mail addresses: pandya.bhaumik@gmail.com (B.A. Pandya), xuechenl@hku.hk
(X. Li).
1
Equal contribution.
http://dx.doi.org/10.1016/j.bmcl.2016.12.046
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lin D., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.046

2
D. Lin et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Scheme 2. Base-catalyzed ring opening of compound 6.
O
O
O
TsO
NC
HO
TsCl
Pyridine
OBn
OBn
NHBoc
OBn
X
NHBoc
8
NHBoc
7
Pyr.SO3
TEA
RuCl3.xH2O
NaIO4
MeCN/CCl4/H2O (2:3:3)
O
DMSO
0oC
O
HO
O
OBn
NHBoc
OBn
NHBoc
O
9
12
EtOCOCl, TEA, THF
CH2N2, Et2O
Wittig reactions
X
X
O
O
O
Fig. 1. Structure of daptomycin and three analogues.
MeO
OBn
NHBoc
N2
O
OBn
NHBoc
MeO
OBn
NHBoc
O
11
10
13
O
O
O
Tf2O
Pyridine
CH2Cl2
HO
TfO
O
NaN3
DMF
N3
O
O
O
OH
O
O
OBn
2
3
O
NHBoc
O
H2
Pd/C
Scheme 3. Failed attempts towards synthesis of dimethyl glutamic acid.
H2N
HCl
33% HBr in AcOH
Br
O
OH
2N HCl in MeOH
NH2
HBr
4
5
tion was also attempted for installation of the extra carbon. The OH
of compound 7 was first oxidized to the corresponding aldehyde to
afford compound 12, followed by the Wittig reaction. However, a
variety of Wittig reagents have been used but again none of them
could give the desired olefin product 13.
X
Scheme 1. Synthesis of (S)-2-amino-4-bromo-3,3-dimethylbutanoic acid.
The failures of all these attempts forced us to re-design our syn-
thesis of the building block. A new strategy adopted an oxidative
oxazoline-oxazinone rearrangement followed by hydrogenation,
which was developed by Molinski (Scheme 4).12 Ring opening of
3,3-dimethylglutaric anhydride with (R)-(À)-2-phenylglycinol as
in toluene-dichloromethane solvent system provided amide 14 in
89% yield. The carboxylic acid of compound 14 was then protected
as the tert-butyl ester with the tert-butyl bromide method to afford
compound 15 in 73% yield. Conversion of compound 15 into oxazo-
line 16 using the reported diethylaminosulfur trifluoride (DAST)
condition12,13 proceeded in low yield (19%). Conversely, when
amide 15 was treated with 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ) and triphenylphosphine (PPh3), the desired oxazo-
line 16 could be obtained in a much higher yield (83%). Oxazoline
16 was then oxidized by selenium dioxide (SeO2) followed by a
concurrent rearrangement afforded oxazinone 17 in 67% yield.
Hydrogenation of compound 17 with high diastereoselectivity
(dr > 20:1) induced by the chiral phenyl auxiliary was achieved
by platinum dioxide (PtO2) in MeOH to afford compound 18 in
65% yield. Removal of chiral auxiliary was accomplished under
the hydrogenation condition to yield glutamic acid 19, which
was directly protected as the Fmoc-carbamate 20 (78% over 2
steps). The Fmoc-dimethyl Glu(OtBu)-OH (compound 20) served
as a suitable building block for solid phase peptide synthesis. The
new building block synthesis provided Fmoc-dimethyl Glu
(OtBu)-OH in 7 steps with a 18% overall yield. The sequence has
been shown to be robust and amenable to a large-scale synthesis.
(S)-2-amino-4-bromo-3,3-dimethylbutanoic acid 5. However, this
compound was highly unstable that it would easily undergo
cyclization and be converted back to compound 4. Thus compound
5 could not be used for further transformations. We thought that
the instability of compound 5 was due to the presence of the highly
reactive primary bromide.
To solve this problem, the strategy of the ring opening step was
revised (Scheme 2). The amino group of compound 4 was first pro-
tected with Boc to afford compound 6 in 81% yield. Ring opening
was then achieved by LiOH10 followed by Bn protection of the car-
boxylic acid to afford compound 7 in 70% yield over 2 steps.
In order to obtain the desired dimethyl Glu from compound 7,
an extra carbon had to be installed. Various strategies have been
adopted but none of them gave the desired product (Scheme 3).
The first attempt was to convert the OH group to CN which could
be further converted to a carboxylic acid. The OH of compound 7
was first converted to OTs 8 followed by the displacement by
NaCN. However, the displacement was not successful, probably
due to the steric hindrance of the two methyl groups. The next
attempt was to use Arndt-Eistert reaction.11 Compound 7 was first
oxidized to compound 9 with the aid of NaIO4 and RuCl3ÁxH2O.
Unfortunately, the key diazoketone intermediate 10 could not be
successfully obtained. Only the methyl ester of compound 11
was formed. As a result, this approach also failed. The Wittig reac-
Please cite this article in press as: Lin D., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.046

D. Lin et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
O
O
NHFmoc
1. Fmoc-SPPS
2. AcOH/TFE/CH2Cl2
BocHN
O
O
H
N
NBoc
O
N
H
N
H
HN
O
CONHTrt
O
NH
O
O
t-BuO2C
O
H
N
t-BuO2C
O
N
H
N
H
C9H19
NH
O
O
t-BuO
H
N
HN
NHBoc
O
CO2t-Bu
O
O
N
H
OH
O
O
R1
R2
R3
NBoc
CHO
O
21
16% based on the resin loading
H2N
1. PyBOP, DIEA, CH2Cl2
2. TFA/H2O/PhOH
O
O
HO
Scheme 4. Synthesis of Fmoc-dimethyl Glu (OtBu)-OH.
O
O
H
N
NH
N
H
N
H
HN
O
CONH2
O
O
NH
O
O
HO2C
H
N
With the building blocks Fmoc-(2S, 3R) mGln-OH and Fmoc-(2S,
3R) eGlu(OtBu)-OH and Fmoc-dimethyl Glu(OtBu)-OH in hands,
three daptomycin analogues with (2S, 3R) mGlu substituted by
(2S, 3S) mGlu, (2S, 3R) eGlu and dimethyl glutamic acid respec-
tively could be synthesized by the protocol we previously reported
(Scheme 5).7 The obtained analogues were purified and tested for
antibacterial activities. The new analogues generally displayed
diminished antibacterial activity (16–128 fold) than the parent
daptomycin (Table 1). The slight hindrance induced by replacing
the methyl group with an ethyl group caused 16-fold loss of activ-
ity, and conformational changes brought by the dimethyl group
due to angle compression (Thorpe-Ingold effect) led to 128 fold
loss of activity. Together with previous finding on Glu replace-
ment6 and a recent study by substitution of (2S, 3R) mGlu with
(2S, 3S) mGlu causing >40-fold loss,14,15 these results suggest that
(2S, 3R) mGlu in daptomycin functions in a compact and fixed con-
formation. Its functionality, steric size and absolute stereo-config-
uration are all critical for the daptomycin activity. Although the
precise role of (2S, 3R) mGlu in the mechanism of action is not
completely well understood, these findings via chemical mapping
provide insights at a molecular level. These analogues demonstrate
the utility of our fully synthetic approach to daptomycin. Future
daptomycin analogues will expand the SAR map with the intent
of elucidating the molecular mechanism of action.
HO2C
O
N
H
N
H
C9H19
NH
O
O
O
HO
HN
O
O
CONH2
H
O
N
NH2
N
H
O
O
O
R1
R2
OHC
2
3
(2S, 3R ) mGlu : R 1 = Me, R = H, R = OH
R3
NH2
2
3
(2S, 3R ) mGln : R 1 = Me, R = H, R = NH 2
O
(2S, 3R ) eGlu : R 1 = ethyl, R = H, R = OH
2
3
22
2
3
dime thyl Glu : R 1 = Me, R = Me, R = OH
1. pyridine acetate
rt, 4 h
66% (HPLC isolation)
H2N
2. TFA/H2O
O
N
O
H
N
NH
O
N
H
O
H
CONH2
O
NH
O
O
HN
HO2C
H
N
HO2C
O
N
H
N
H
C9H19
NH
O
O
O
HO
NH
O
CO2H
H
O
N
N
H
N
H
O
O
O
R1
R2
O
NH2
R3
Scheme 5. Synthesis of daptomycin analogues.
Table 1
MIC data of daptomycin analogues.
In summary, synthesis of daptomycin analogues with substitu-
tion of (2S, 3R) 3-methyl glutamic acid (mGlu) with other struc-
turally relevant amino acids would be important to study the
role of mGlu and to search for potent compounds. Herein we
reported a facile synthesis of dimethyl glutamic acid, which allows
for scalable synthesis of the building block of dimethyl glutamate
suitable for solid phase peptide synthesis (SPPS). Three dapto-
mycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) mGln,
(2S, 3R) eGlu and dimethyl Glu were synthesized and tested for
activity. Our studies have shown that even a subtle change to mGlu
caused dramatic loss of the activity. According to the proposed
mode of action of daptomycin in that it binds to Ca2+ ions prior
to aggregating on the membrane,3 the carboxylic acids in dapto-
mycin are responsible for sequestering calcium and thus orienta-
tion in space is important. Our findings suggest that it is not
sufficient to have acidic groups placed randomly in space rather
there is a network of interactions, yet to be fully understood, that
may impart activity and selectivity.
Daptomycin analogues
MIC against S. aureus 42, QC WT (lg/mL)
Daptomycin
0.5
32
8
(2S, 3R) mGlu ? (2S, 3R) mGln
(2S, 3R) mGlu ? (2S, 3R) eGlu
(2S, 3R) mGlu ? dimethyl Glu
64
Acknowledgments
We thank Dr. Jared Silverman for his continued support on
studies related to daptomycin. This work was supported by Cubist,
wholly owned subsidiary of Merck and Co.
A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12.
046.
Please cite this article in press as: Lin D., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.046

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D. Lin et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
8. Zhang S-Y, Li Q, He G, Nack WA, Chen G. J Am Chem Soc. 2013;135:12135.
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