Total Synthesis and Examination of Three Key Analogues of Ramoplanin: A Lipoglycodepsipeptide with Potent Antibiotic Activity

Article abstract of DOI:10.1021/ja039671y

The total synthesis and evaluation of three key ramoplanin aglycon analogues are detailed. The first (5a) represents replacement of the labile depsipeptide ester with a stable amide (HAsn² → Dap²) with removal of the HAsn pendant car

Full text of DOI:10.1021/ja039671y

Published on Web 01/08/2004
Total Synthesis and Examination of Three Key Analogues of Ramoplanin:
A Lipoglycodepsipeptide with Potent Antibiotic Activity
Yosup Rew, Dongwoo Shin, Inkyu Hwang, and Dale L. Boger*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received November 18, 2003; E-mail: boger@scripps.edu
Ramoplanin is a novel lipoglycodepsipeptide with potent anti-
bacterial activity that was isolated from the fermentation broth of
Actinoplanes sp. ATCC 33076 as a mixture of three closely related
compounds 1-3, of which 2 is the most abundant (Figure 1).1 The
structure was determined in 1989, and it was established that the
three compounds differ only in the acyl group attached to the Asn1
N-terminus.2 The ramoplanin complex is 2-10 times more active
than vancomycin against Gram-positive bacteria3 with a distinct
mode of action,4,5 and the ramoplanin A2 aglycon (4) is equally or
slightly more potent than the natural products in antimicrobial
assays.6 Ramoplanin disrupts bacterial cell wall biosynthesis where
it inhibits both the intracellular MurG-catalyzed (UDP-glcNAc
transferase) conversion of lipid intermediate I to II and the more
accessible extracellular transglycosylase-catalyzed polymerization
of lipid intermediate II to provide the cell wall peptidoglycan
backbone. It binds and sequesters the substrates, lipid intermediates
I and II (lipid II > lipid I),5 and the structural details of this
interaction are only now beginning to emerge.5,7,8 These two steps
immediately precede the transpeptidase-catalyzed cell wall cross-
linking reaction and a principal site of action of vancomycin. Thus,
cross-resistance between ramoplanin and other antibiotics including
vancomycin is not observed, and ramoplanin represents an excellent
candidate for more expansive clinical use (e.g., VREF and MRSA)
beyond its present introduction for topical infections. However, the
intrinsic instability of ramoplanin derived from a rapid hydrolysis
of the depsipeptide ester (IV administration) and its poor absorption
characteristics (oral administration) have limited its clinical poten-
tial.3 Nonetheless, ramoplanin is currently in Phase III clinical trials
for the oral treatment of intestinal vancomycin-resistant entero-
coccus faecium (VREF) and Phase II trials for nasal MRSA, neither
of which require absorption.
Figure 1. Structure of ramoplanin.
examination would facilitate subsequent studies. Herein, we report
the total synthesis and examination of these three analogues 5a-
5c.
Not only is the HAsn2 residue the most lengthy to secure,11 but
its incorporation into and maintenance throughout the late stages
of the synthesis constitute the most difficult challenge limiting the
ease of total synthesis.10 More significantly, it is the most fragile
linkage in the molecule and is central to the instability of the natural
products. Williams described the mild hydrolysis of ramoplanose
providing the inactive linear peptide.12 McCafferty and co-workers
reported the time-dependent decomposition of ramoplanin A2
aglycon in acidic solutions,7 and we have described depsipeptide
cleavage that occurs rapidly even under mild basic conditions (1%
Et3N-H2O, 25 °C, 40% hydrolysis in 2 min).10 Because it lies at
the end of the antiparallel â-sheet with the ester adopting a transoid
carbonyl eclipsed conformation analogous to that of an amide and
forms the link to the short flexible loop (residues 15-17),9 its
replacement with L-2,3-diaminopropionic acid (Dap) or L-2,4-
diaminobutyric acid (Dab) to provide 5a and 5b was expected to
have a minimal impact on the conformation but would preclude
hydrolysis (5a and 5b) and significantly reduce (5a) or preclude
(5b) â-elimination derived cleavage. Both would simplify the
synthetic challenges associated with assembling a library of
analogues and would be expected to improve in vivo stability.
The three-dimensional structure of ramoplanin A2 adopts a
U-shaped topology,9 forming two antiparallel â-strands (residues
2-7 and 10-14) connected by six transannular H-bonds and a
reverse â-turn (aThr8 and Phe9). The conformation is stabilized by
a hydrophobic cluster of aromatic side chains on residues 3, 9, and
11, providing a U-shape topology to the â-sheet with the â-turn at
one end (aThr8 and Phe9) and a flexible connecting loop at the
other (residues 15-17). We recently reported the first total synthesis
of the ramoplanin A2 aglycon designed to permit access to
analogues containing deep-seated changes in the natural product
structures.10 In addition to defining solutions to key challenges
presented by the preparation of synthetic 4, its deliberate late stage
convergent assemblage from three key subunits was anticipated to
facilitate the synthesis of an extensive series of analogues required
to probe the features that contribute to lipid intermediate I and II
binding and are critical for antimicrobial activity. Prior to embarking
on a comprehensive dissection of each structural feature where each
residue is examined, we identified three key analogues whose
9
10.1021/ja039671y CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 1041-1043
1041

C O M M U N I C A T I O N S
Scheme 1
The simplified pentapeptide subunits 12a and 12b, incorporating
the Dap2 and Dab2 amides in place of HAsn,2 were synthesized
from tripeptide 6 (Scheme 1),13 which in turn was obtained by
coupling Boc-Leu-D-Ala-OH10 with Chp-OBn10 (EDCI, HOAt, 20%
DMF-CH2Cl2, 0 °C, 12 h, 96%) followed by deprotection of the
benzyl ester (H2, 10% Pd/C, EtOH, 25 °C, 1 h). Fmoc-Dap-OBn
(9a)/Fmoc-Dab-OBn (9b), obtained from Fmoc-Dap(Boc)-OH/
Fmoc-Dab(Boc)-OH, were coupled with 7 using DEPBT (NaHCO3,
16% DMF-THF, 25 °C, 40 h, 67% for 10a/NaHCO3, THF, 25
°C, 36 h, 69% for 10b) to give only a single diastereomer of each
tetrapeptide 10a/10b, whereas pairs of diastereomers (>8:1),
resulting from competitive epimerization, were obtained with EDCI/
HOAt. Fmoc removal (Bu4NF, i-PrOH, DMF, 25 °C, 10 min,
sonication),10 coupling with Fmoc-Asn(Trt)-OH (EDCI, HOAt,
DMF, 0 °C, 48 h, 86% for 11a/20% DMF-THF, 64% for 11b),
and benzyl ester hydrogenolysis (H2, 10% Pd/C, EtOH, 25 °C, 1
h, 82-87%) provided 12.
The coupling of 12a/12b with heptapeptide 1410 provided 15a/
15b (DEPBT, NaHCO3, DMF, 0 °C, 3 days, 72-89% for 15a/
68-96% for 15b). The coupling reactions were carried out with
excess 12a/12b, leading to complete consumption of 14 because
residual 14 could not be removed from the product by either an
acid wash or chromatography. The intermediates 15a/15b were
isolated by successive washing with ice cold aqueous 0.5 N HCl,
water, and EtOAc, and were used for the next step without further
purification. Boc removal was accomplished under the mild
conditions (BCB, CH3CN, 0 °C, 3 h) that preserve the trityl
protecting group. In turn, the crude amines 16a/16b were coupled
with pentapeptide 17,10 to yield the macrocyclization substrates 18a/
18b (DEPBT, NaHCO3, DMF, 0 °C, 96 h, 75-87% for 18a/EDCI,
HOAt, NaHCO3, DMF, 0 °C, 72 h, 75-86% for 18b). The
conversion of 12 to 18 and the subsequent macrocyclization could
often be conducted without deliberate purification of the intermedi-
ates or even 18. Because conventional purification techniques (i.e.,
chromatography) typically resulted in substantial material losses
attributable to the poor physical properties of the 12-17 residue
peptides, the overall yields were much higher without the conven-
tional purifications. In instances when the macrocyclization precur-
sors 18a/18b, isolated in the same manner described for 15a/15b,
were not sufficiently pure for further reaction, we found that
membrane dialysis (MWCO ) 2000) in DMSO was the most
efficient and effective way to purify 18a/18b.
Successive Boc removal (BCB, CH3CN, 0 °C, 3 h), benzyl ester
hydrogenolysis (H2, 10% Pd/C, 10% DMF-EtOH, 25 °C, 10 h
for 18a/50% DMF-EtOH for 18b), and macrocyclization (EDCI,
HOAt, NaHCO3, 70% CH2Cl2-DMF, 0 °C, 3 days for 19a/EDCI,
HOAt, NaHCO3, DMF, 0 °C, 3 days for 19b) afforded the cyclic
peptide core 19a/19b. Fmoc removal under mild conditions (8 equiv
of Bu4NF, 10 equiv of i-PrOH, DMF, 25 °C, 1 h, sonication),10
Asn1 acyl side chain introduction (20,10 20% CH2Cl2-DMF, 90%
for two steps for 21a), and global deprotection of the trityl and
SES groups upon HF treatment (HF, anisole, 0 °C, 90 min, >75%)
provided 5a/5b. The amide analogues lacking the Asn1 side chain,
22a/22b, were also prepared by successive Fmoc removal (8 equiv
of Bu4NF, 10 equiv of i-PrOH, DMF, 25 °C, 1 h)10 and HF
deprotection of the trityl and SES groups (HF, anisole, 0 °C, 90
min).
A third key analogue 5c was prepared and assessed in which
the variable Asn1 lipid N-acyl group was replaced with a minimal
N-acetyl group. Semisynthetic modifications of the natural products
and their aglycons have established that removal of the side chain
unsaturation (hydrogenation) appears to have a minimal effect,6 but
the impact of the lipid side chain presence and its potential role
9
1042 J. AM. CHEM. SOC. VOL. 126, NO. 4, 2004

C O M M U N I C A T I O N S
Scheme 2
replaced with a more stable amide without impacting the in vitro
antimicrobial activity, the HAsn2 carboxamide found in 1-4 but
is absent in 5a does not appear to contribute directly to the
properties, and the lipid side chain of 1-4 contributes significantly
but is not essential, whereas even a simple methylene insertion into
the Dap2 residue of 5a abolishes activity.
Just as importantly, 5a proved to be completely stable to mildly
basic conditions (1% Et3N-H2O, 25 °C, 24 h, 0% disappearance)
that rapidly consume the ramoplanin aglycon (80% within 5 min
at 25 °C).10 The enhanced antimicrobial potency and physical
stability of 5a make it a promising lead compound in the search
for ramoplanin analogues with improved profiles and a more stable
and accessible macrocyclic template on which to conduct structure-
function studies.
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41101) and the
Skaggs Institute for Chemical Biology. We thank Drs. W. Jiang
and J. Wanner for early studies on 8-12.
Table 1. Antimicrobial Activity, Staphylococcus aureus
compound
MIC (µg/mL)
Supporting Information Available: Full experimental details and
compound characterizations (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
2, ramoplanin A2
4, ramoplanin A2 aglycon
1.56
0.78
0.39
6.25
5a
22a
5b
22b
5c
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was not known. This was addressed with the total synthesis of 5c
in Scheme 2. Removal of the Fmoc protecting group from 2310 (8
equiv of Bu4NF, 10 equiv of i-PrOH, DMF, 25 °C, 1 h, sonication),
the advanced synthetic intermediate we prepared enroute to the
ramoplanin aglycon 4,10 followed by treatment with Ac2O (DMF,
85% for two steps) provided the fully protected N-acetyl analogue
24. Global deprotection using HF (HF, anisole, 0 °C, 90 min)
furnished 5c (85%).
The antimicrobial activity of the key analogues 5a-5c and the
corresponding Asn1 free amines 22a/22b were compared with
ramoplanin A2 and its aglycon against Staphylococcus aureus
(ATCC 25923), which is among the least sensitive wild-type
bacteria, and the results are summarized in Table 1. The analogue
5a in which the backbone depsipeptide ester was replaced with an
amide retained or exhibited a slightly increased antimicrobial
potency (MIC ) 0.39 µg/mL) relative to the ramoplanin A2 aglycon
(MIC ) 0.78 µg/mL). In contrast, the depsipeptide amide analogue
5b containing the single additional methylene in the macrocycle
was inactive (MIC > 50 µg/mL), exhibiting a >100-fold loss in
activity relative to 4 and 5a. Interestingly, both 22a, the active
ramoplanin amide analogue lacking the Asn1 side chain, and the
ramoplanin aglycon N-acetyl analogue 5c were approximately 16-
fold less potent than the corresponding compounds containing the
natural side chain. Thus, the depsipeptide ester of 1-4 may be
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(13) EDCI ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,
HOAt ) 1-hydroxy-7-azabenzotriazole, DEPBT ) 3-(diethoxylphospho-
ryloxy)-1,2,3-benzotriazin-4(3H)-one, BCB ) B-bromocatecholborane.
JA039671Y
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