Alanine scan of [L-Dap2]ramoplanin A2 aglycon: Assessment of the importance of each residue

Article abstract of DOI:10.1021/ja068573k

In efforts that define the importance of each residue and that identify key regions of the molecule, an alanine scan of the ramoplanin A2 aglycon, a potent antibiotic that inhibits bacterial cell wall biosynthesis, is detailed. As a consequence of both it

Full text of DOI:10.1021/ja068573k

Published on Web 06/26/2007
Alanine Scan of [L-Dap2]Ramoplanin A2 Aglycon:
Assessment of the Importance of Each Residue
Joonwoo Nam, Dongwoo Shin, Yosup Rew, and Dale L. Boger*
Contribution from the 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 29, 2006; E-mail: boger@scripps.edu
Abstract: In efforts that define the importance of each residue and that identify key regions of the molecule,
an alanine scan of the ramoplanin A2 aglycon, a potent antibiotic that inhibits bacterial cell wall biosynthesis,
is detailed. As a consequence of both its increased stability (lactam vs lactone) and its “relative” ease of
synthesis, the alanine scan was conducted on [Dap2]ramoplanin A2 aglycon, which possesses antimicrobial
activity equal to or slightly more potent than that of ramoplanin A2 or its aglycon. Thus, 14 key analogues
of the ramoplanin A2 aglycon, representing a scan of residues 3-13, 15, and 17, were prepared enlisting
a convergent solution-phase total synthesis that consolidated the effort to a manageable level. The
antimicrobial activity of the resulting library of analogues provides insight into the importance and potential
role of each residue of this complex glycopeptide antibiotic.
Introduction
Because of the overuse of broad spectrum antibiotics in
medicine and agriculture, the increasing frequency of bacterial
resistance to existing drugs presents a serious threat to public
health.1 Such growing concerns have increased with the recent
reports of the emergence of multi-drug-resistant organisms
including methicillin-resistant Staphylococcus aureus (MRSA)
or methicillin-resistant enterococci that exhibit a reduced
vancomycin susceptibility.2 As a result, it has become increas-
ingly important to identify new antibiotics that can replace
vancomycin as the antibiotic of last resort for such resistant
bacterial infections.
Ramoplanin is a lipoglycodepsipeptide first isolated from the
fermentation broths of Actinoplanes sp. ATCC 33076 in 1984
(Figure 1).3 The ramoplanin complex, which consists of three
closely related compounds 1-3,4 was found to be 2-10 times
more active than vancomycin against Gram-positive bacteria
(500 strains) and maintains full activity against vancomycin-
resistant enterococci (VRE) (minimum inhibitory concentrations,
MIC ) 0.5 µg/mL) and all known strains of MRSA.5 Although
the mechanism of action of ramoplanin is not yet fully defined,
(1) Walsh, C. T. Nature 2000, 406, 775.
(2) (a) Pearson, H. Nature 2002, 418, 469. (b) Review: von Nussbaum, F.;
Brands, M.; Hinzen, B.; Weigand, S.; Ha¨bich, D. Angew. Chem., Int. Ed.
2006, 45, 5072.
(3) (a) Cavalleri, B.; Pagani, H.; Volpe, G.; Selva, E.; Parenti, F. J. Antibiot.
1984, 37, 309. (b) Pallanza, R.; Berti, M.; Scotti, R.; Randisi, E.; Arioli,
V. J. Antibiot. 1984, 37, 318.
Figure 1. Structure of the ramoplanins.
(4) (a) Ciabatti, R.; Kettenring, J. K.; Winters, G.; Tuan, G.; Zerilli, L.;
Cavalleri, B. J. Antibiot. 1989, 42, 254. (b) Kettenring, J. K.; Ciabatti, R.;
Winters, G.; Tamborini, G.; Cavalleri, B. J. Antibiot. 1989, 42, 268.
Reviews: (c) Walker, S.; Chen, L.; Hu, Y.; Rew, Y.; Shin, D.; Boger, D.
L. Chem. ReV. 2005, 105, 449. (d) Parenti, F.; Ciabatti, R.; Cavalleri, B.;
Kettenring, J. Drugs Exp. Clin. Res. 1990, 16, 451. (e) McCafferty, D. G.;
Cudic, P.; Frankel, B. A.; Barkallah, S.; Kruger, R. G.; Li, W. Biopolymers
2002, 66, 261.
it is a promising drug candidate for vancomycin-resistant
bacterial infections since it is known to function in a unique
manner.6 Both antibiotics inhibit bacterial cell wall biosynthesis,
but vancomycin prevents cell wall cross-linking by primarily
inhibiting the late stage transpeptidase-catalyzed step by binding
to its substrate D-Ala-D-Ala at the terminus of peptidoglycan
precursors,7 whereas ramoplanin inhibits the earlier stage
(5) (a) Review: Espersen, F. Curr. Opin. Anti-Infect. InVest. Drugs 1999, 1,
78. (b) Montecalvo, M. A. J. Antimicrob. Chemother. 2003, 51 (Suppl.
S3), iii31.
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J. AM. CHEM. SOC. 2007, 129, 8747-8755
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A R T I C L E S
Nam et al.
intracellular glycosyltransferase (MurG) and the more accessible
extracellular transglycosylase (PBP1b)-catalyzed steps by bind-
ing their substrates Lipids I and II, thereby precluding maturation
of the bacterial cell wall.6,8 Consequently, no cross-resistance
of ramoplanin with existing antibiotics, including vancomycin,
has yet been observed. Currently, ramoplanin is in phase III
clinical trials for topical, nasal, and GI infections.5,9 However,
the therapeutic applications of ramoplanin are limited because
of its poor pharmacokinetics: it is not orally absorbed and it is
not stable in plasma because of rapid hydrolysis of the labile
lactone.
Ramoplanin A1-A3 consists of a 49-membered ring com-
posed of 17 amino acids including 12 unnatural amino acids
and seven possessing the D-configuration. The initial structure
of ramoplanin was disclosed in 1989, and the three compounds
that make up the ramoplanin complex differ only in the lipid
side chains attached to the Asn1 N-terminus.4 Originally, the
two double bonds in the three different acyl groups were
assigned the cis-cis stereochemistry,4a and this has since been
corrected to be cis-trans.10 The C-terminal 3-chloro-4-hydrox-
yphenylglycine (Chp17) forms a lactone bond with the hydroxy
group of â-hydroxyasparagine (HAsn2). In 1991, the structure
of ramoplanose (4) was disclosed by Williams and co-workers,
and its composition was identical to ramoplanin A2 except for
the branched mannose trisaccharide (vs mannose disaccharide)
at Hpg11 and the stereochemistry of the acyl side chain (cis,-
trans- vs cis,cis-7-methyloctadi-2,4-enoic acid).11 Kurz and
Guba subsequently corrected the olefin stereochemistry of
ramoplanin A2 as cis-trans by 2D NMR in 1996.10 They also
established the Hpg6 and Hpg7 absolute stereochemistry and
found that the solution conformation consists of two antiparallel
â-strands (HAsn2-D-Hpg7 and D-Orn10-Gly14) stabilized by six
transannular H-bonds and a cluster of hydrophobic aromatic side
chains (D-Hpg3, Phe9, and Chp17) providing a U-shape topology
to the â-sheet with a reverse â-turn (aThr8-Phe9) at one end
and a more flexible connecting loop (Leu15-Chp17) at the other
end in the solution structure.
Figure 2. Structure of the enduracidins.
by the same mechanism as the ramoplanins12f (Figure 2). The
enduracidins and ramoplanins share a high degree of structural
similarity, including two antiparallel â-strands and a conserved
D-Hpg3-aThr8 region thought to be important for Lipid I and II
recognition and binding. Moreover, many of the remaining
residues in the enduracidins and ramoplanins are identical
(Hpg11, Gly14, D-Ala16) or represent conservative structural
departures (D-Ser12 vs D-aThr12, Dpg13 vs Hpg13, Hpg17 vs Chp17,
and Thr2 vs HAsn2). Even some of the significant departures
(Cit9 vs Phe9 and D-End10 vs D-Orn10) represent changes that
maintain the stereochemical and potential functional features
(D-End10 vs D-Orn10) of the ramoplanins. Recently, Marazzi and
co-workers disclosed a solution-phase conformation of the
enduracidins determined by NMR exhibiting only subtle struc-
tural differences between the enduracidins and ramoplanins.13
The enduracidins do not contain a di- or trisaccharide at Hpg11,
and the lipid side chains are longer than those found in the
ramoplanins. Enduracidin includes an additional basic residue
at End15 (vs Leu15) and an acidic residue at Asp1 (vs Asn1) that
are proximal and engaged in a transannular salt bridge, as well
as a flexible side chain at the Cit9, which is exposed to the
solvent (H2O/DMSO-d6, 4:1), whereas ramoplanin incorporates
a hydrophobic side chain at Phe9 forming a well-packed
hydrophobic core along with other aromatic side chains (D-Hpg3,
Chp17) and the lipid side chain.10 The net result is that the
characteristic ramoplanin hydrophobic core is disrupted within
the enduracidins. The significance of this difference is yet to
be defined, and it is not reflected in different transglycosylase
inhibition kinetics.12f Although less well characterized, jani-
emycin represents an additional member of the ramoplanin
family.14
Enduracidin A and B are additional members of the ramopla-
nin family that have been employed as feed additives and are
known to inhibit Gram-positive bacterial cell wall biosynthesis12
(6) (a) Somner, E. A.; Reynolds, P. E. Antimicrob. Agents Chemother. 1990,
34, 413. (b) Review: Reynolds, P. E.; Somner, E. A. Drugs Exp. Clin.
Res. 1990, 16, 385. (c) Bro¨tz, H.; Bierbaum, G.; Reynolds, P. E.; Sahl,
H.-G. Eur. J. Biochem. 1997, 246, 193. (d) Lo, M.-C.; Men, H.; Branstrom,
A.; Helm, J.; Yao, N.; Goldman, R.; Walker, S. J. Am. Chem. Soc. 2000,
122, 3540. (e) Lo, M.-C.; Helm, J. S.; Sarngadharan, G.; Pelczer, I.; Walker,
S. J. Am. Chem. Soc. 2001, 123, 8640. (f) Helm, J. S.; Chen, L.; Walker,
S. J. Am. Chem. Soc. 2002, 124, 13970. (g) Hu, Y.; Helm, J. S.; Chen, L.;
Ye, X.-Y.; Walker, S. J. Am. Chem. Soc. 2003, 125, 8736.
(7) Reviews: (a) Barna, J. C. J.; Williams, D. H. Annu. ReV. Microbiol. 1984,
38, 339. (b) Williams, D. H.; Bardsley, B. Angew. Chem., Int. Ed. 1999,
38, 1172.
(8) (a) Cudic, P.; Kranz, J. K.; Behenna, D. C.; Kruger, R. G.; Tadesse, H.;
Wand, A. J.; Veklich, Y. I.; Weisel, J. W.; McCafferty, D. G. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 7384. (b) Cudic, P.; Behenna, D. C.; Kranz, J.
K.; Kruger, R. G.; Wand, A. J.; Veklich, Y. I.; Weisel, J. W.; McCafferty,
D. G. Chem. Biol. 2002, 9, 897.
(9) Jones, R. N.; Barry, A. L. Diagn. Microbiol. Infect. Dis. 1989, 12, 279.
(10) Kurz, M.; Guba, W. Biochemistry 1996, 35, 12570.
(11) Skelton, N. J.; Harding, M. M.; Mortishire-Smith, R. J.; Rahman, S. K.;
Williams, D. H.; Rance, M. J.; Ruddock, J. C. J. Am. Chem. Soc. 1991,
113, 7522.
In 2002, we reported the first total synthesis of the ramoplanin
A2 and ramoplanose aglycon (5) confirming the assigned
(12) (a) Higashide, E.; Hatano, K.; Shibata, M.; Nakazawa, K. J. Antibiot. 1968,
21, 126. (b) Asai, M.; Muroi, M.; Sugita, N.; Kawashima, H.; Mizuno, K.;
Miyake, A. J. Antibiot. 1968, 21, 138. (c) Tsuchiya, K.; Takeuchi, Y. J.
Antibiot. 1968, 21, 426. (d) Hori, M.; Iwasaki, H.; Horii, S.; Yoshida, I.;
Hongo, T. Chem. Pharm. Bull. 1973, 21, 1175. (e) Iwasaki, H.; Horii, S.;
Asai, M.; Mizuno, K.; Ueyanagi, J.; Miyake, A. Chem. Pharm. Bull. 1973,
21, 1184. (f) Fang, X.; Tiyanont, K.; Zhang, Y.; Wanner, J.; Boger, D.;
Walker, S. Mol. BioSyst. 2006, 2, 69.
(13) Castiglione, F.; Marazzi, A.; Meli, M.; Colombo, G. Magn. Reson. Chem.
2005, 43, 603.
(14) (a) Meyers, E.; Weisenborn, F. L.; Pansy, F. E.; Slusarchyk, D. S.; von
Saltza, M. H.; Rathnum, M. L.; Parker, W. L. J. Antibiot. 1970, 23, 502.
(b) Linnett, P. E.; Strominger, J. L. Antimicrob. Agents Chemother. 1973,
4, 231.
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Alanine Scan of Ramoplanin Aglycon
A R T I C L E S
structure.15 Three key subunits composed of D-Hpg3-Phe9
(subunit A), Leu15-Asn1 (subunit B), and D-Orn10-Gly14 (subunit
C) were synthesized, sequentially coupled, and cyclized in a
solution-phase approach to the 49-membered macrocyclic core
of ramoplanin. Two macrocyclization sites, Phe9-D-Orn10 and
Gly14-Leu15, were examined that maximize the convergency of
the synthesis, minimize the use of protecting groups, prevent
late stage opportunities for racemization of carboxylate-activated
phenylglycine-derived residues, and benefit from a â-sheet
preorganization of an acyclic substrate for ring closure.16
Macrocyclization at the Phe9-D-Orn10 site additionally benefits
from closure at the corner of a â-turn incorporating a D-amine,17
while the alternative closure at the Gly14-Leu15 site represents
a nonhindered glycine site incapable of racemization. Additional
keys to the success of the approach were the choice of a SES
protecting group for Orn4 and Orn10 and Fmoc protection for
Asn1 furnishing orthogonal protecting groups to Boc, Cbz, and
benzyl ester deprotections yet capable of sequential and selective
removal in the presence of the unstable depsipeptide ester bond.
Deliberate final stage incorporation of the Asn1 lipid side chain
provided late stage access to all three ramoplanins as well as
side chain analogues of the aglycons. As such, the total synthesis
of the two minor components of the ramoplanin complex (A1
and A3) was achieved, confirming a reassigned cis-trans
stereochemistry for the lipid side chains.18
In 1998, Bro¨tz et al. reported the first direct evidence that
ramoplanin binds to a substrate involved in peptidoglycan
biosynthesis.21 In 2000, Walker and co-workers showed that
ramoplanin inhibits the transglycosylation step of peptidoglycan
biosynthesis by binding to Lipid II.6d Shortly following that,
insights into the interaction between ramoplanin A2 and a
peptidoglycan precursor (Park’s nucleotide) were disclosed by
McCafferty et al. using NMR studies suggesting that the
octapeptide sequence (D-Hpg3-D-Orn10),8a which is highly
conserved among ramoplanins and enduracidins, constitutes the
substrate recognition domain. Contemporary with these studies,
inhibition kinetic studies6g and NMR titration experiments6f
performed by Walker and co-workers suggest that the inhibitory
species binds with a stoichiometry of 2:1 ramoplanin/Lipid II,
and in a second NMR study, Lo et al. observed that ramoplanin
A2 exists as a mixture of monomer and dimer in methanol.6e
This latter series of studies led Walker to propose that Lipid II
may bind in a cleft formed by the dimerization of two
ramoplanin molecules and defined the dimer interface region
as D-Orn10-Hpg13.
Herein, we report an alanine scan of ramoplanin A2 aglycon
and the resulting antimicrobial properties of the derivatives in
efforts to define the importance of the individual residues and
to identify key regions of the molecule.22 As a consequence of
its “relative” ease of synthesis and its resulting stability (lactam
vs lactone), the alanine scan was conducted on the [Dap2]-
ramoplanin A2 aglycon (6) which possesses antimicrobial
activity equal to or slightly more potent than the authentic
ramoplanin A2 aglycon itself (Figure 3).19 Notably, the efforts
constitute a scan of residues 3-13, 15, and 17 conducted
implementing a convergent solution-phase total synthesis of the
14 key analogues.
This approach has since been extended to the synthesis of
two key analogues of ramoplanin containing an amide linkage
in place of the labile ester between HAsn2 and Chp17 in which
HAsn2 was replaced with L-2,3-diaminopropionic acid (Dap)
or L-2,4-diaminobutyric acid (Dab).19 The two derivatives are
both much more stable and synthetically more accessible than
the natural ramoplanin aglycon. In a subsequent mechanistic
analysis of these analogues, both amide linkage substitutions
as well as removal of the lipid chain did not affect Lipid II
binding, indicating that the residue 2 modifications and the acyl
side chain do not play an important role in substrate recognition
and binding.20 However, the antimicrobial activities of the Dab2
analogue and compounds containing truncated side chains were
not comparable to those of ramoplanin or the Dap2 analogue.
The ring-expanded 50-membered Dab2 analogue, but not the
49-membered Dap2 analogue, was found to aggregate exten-
sively in aqueous buffer where its increased conformational
flexibility perhaps permits the â-strands in the molecule to
associate in an intermolecular manner (aggregation),20 whereas
the lipid side chain presumably helps ramoplanin localize to
the bacterial cell membrane.20
Chemistry
Following the completion of the total synthesis of the natural
product aglycons15,18 and the identification of an active and
stable amide (vs depsipeptide ester) template, [Dap2]ramoplanin
A2 aglycon,19 we initiated and herein report an alanine scan of
the ramoplanin A2 aglycon enlisting [Dap2]ramoplanin A2 as
the template. These efforts include a full alanine scan of the
subunit A (D-Hpg3-Phe9) and C (D-Orn10-Gly14), which contain
the putative substrate recognition domain (D-Hpg3-D-Orn10)
proposed by McCafferty et al.,8a the dimer interface region (D-
Orn10-Hpg13) defined by Walker et al.,6e and D-Orn4 and D-Orn10,
which have been established to be important to the biological
properties of ramoplanin.6f,8a,15b,19 Selective semisynthetic modi-
fications of D-Orn4 or D-Orn10 are notorious for their low yields
or poor selectivity. Consequently, the identification of the
modified site is difficult and the results derived from their
examination have not been entirely conclusive.4c-e Therefore,
the site-specific incorporation of D-Ala4 and D-Ala10 by the
synthetic methods detailed herein unambiguously addresses
these issues. In addition and to more clearly elucidate the role
of the chlorine substitution at Chp17, a Hpg17 as well as an Ala17
analogue were prepared.
(15) (a) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud, P.-Y.; Boger, D. L. J. Am.
Chem. Soc. 2002, 124, 5288. (b) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud,
P.-Y.; Boger, D. L. J. Am. Chem. Soc. 2003, 125, 1877. (c) Review: Boger,
D. L. Med. Res. ReV. 2001, 21, 356-381.
(16) (a) Nonpolar solvents (CH2Cl2, CHCl3, EtOAc) should be used to take
advantage of the â-sheet preorganization. However, the linear substrates
typically proved insoluble in such solvents. Consequently, variable amounts
of DMF are added until the substrates were dissolved. (b) Maplestone, R.
A.; Cox, J. P. L.; Williams, D. H. FEBS Lett. 1993, 326, 95.
(17) (a) Rich, D. H.; Bhatnagar, P.; Mathiaparanam, P.; Grant, J. A.; Tam, J. P.
J. Org. Chem. 1978, 43, 296. (b) Brady, S. F.; Varga, S. L.; Freidinger, R.
M.; Schwenk, D. A.; Mendlowski, M.; Holly, F. W.; Veber, D. F. J. Org.
Chem. 1979, 44, 3101.
(18) Shin, D.; Rew, Y.; Boger, D. L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
11977.
(21) (a) Bro¨tz, H.; Bierbaum, G.; Leopold, K.; Reynolds, P. E.; Sahl, H.-G.
Antimicrob. Agents Chemother. 1998, 42, 154. (b) Bro¨tz, H.; Josten, M.;
Wiedemann, I.; Schneider, U.; Gotz, F.; Bierbaum, G.; Sahl, H.-G. Mol.
Microbiol. 1998, 30, 317.
(22) Chen, Y.; Bilban, M.; Foster, C. A.; Boger, D. L. J. Am. Chem. Soc. 2002,
124, 5431.
(19) Rew, Y.; Shin, D.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2004, 126,
1041.
(20) Chen, L.; Yuan, Y.; Helm, J. S.; Hu, Y.; Rew, Y.; Shin, D.; Boger, D. L.;
Walker, S. J. Am. Chem. Soc. 2004, 126, 7462.
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A R T I C L E S
Nam et al.
Scheme 1.23 Alanine-Substituted Subunit A Analogues and
Synthesis of [Ala5] Subunit A (10)
Figure 3. Structure of [Dap2]ramoplanin A2 aglycon (6) and the three
key subunits A-C.
The synthesis and assembly of three key subunits were
facilitated by the convergent nature of the solution-phase
approach (Figure 3). For example, each change within the A
subunit utilized the common [Dap2]B and C subunits for
completion of the synthesis, and this expedited the parallel
synthesis of the library. Similarly, each of the subunits A, B,
and C was also assembled in a convergent fashion (e.g., Ala3
through Ala5 were prepared by coupling the modified tripeptide
to a common Hpg6-Phe9 tetrapeptide), and this consolidated the
work to a manageable level, reducing the complexity of the
syntheses and number of new intermediates. Thus, each residue
analogue required at most five coupling reactions to assemble
the linear peptide, a macrocyclization, the side chain introduction
(two steps), and a final global deprotection (nine operations total)
enlisting unmodified subunits used in the synthesis of [Dap2]-
ramoplanin A2 aglycon.
subunit A (7), we assembled heptapeptides 8-14, which contain
all but Orn10 of the putative Hpg3-Orn10 recognition domain,
from the D-Hpg3-D-aThr5 tripeptide and Hpg6-Phe9 tetrapep-
tide.15 In turn, the tripeptide for 10 was obtained by the coupling
of Boc-D-Hpg3-D-Orn4(SES)-OH and H-D-Ala-OBn (72%).
Benzyl ester hydrogenolysis followed by coupling with H-Hpg6-
D-Hpg7-aThr8-Phe9-OBn gave the heptapeptide 10 (48%). In
similar fashion, the remainder of the alanine-substituted A
subunits was prepared (Supporting Information).
Synthesis of the C Subunit Ala Derivatives: Pentapeptide
D-Orn10-Gly14. The preparation of the pentapeptide 15,15 which
contains the entire dimer interface domain,6e and the synthetic
alanine-substituted C subunits (16-19) is summarized in
Scheme 2 along with details of the key [Ala10] derivative.
Coupling of HCl‚H-L-Hpg11-D-aThr12-OBn with Boc-D-Ala-OH
provided the tripeptide in 99% yield. Benzyl ester deprotection
and coupling with H-L-Hpg13-Gly14-OBn gave pentapeptide 16
(90% yield). The synthesis of remaining alanine-substituted C
subunits proceeded similarly (Supporting Information). Since
residue 14 is glycine, its replacement with an alanine was not
conducted.
Synthesis of the A Subunit Ala Derivatives: Heptapeptide
D-Hpg3-Phe9. The seven synthetic targets of the alanine-
substituted subunit A (8-14) and details of the preparation of
the [Ala5] analogue (10) are summarized in Scheme 1.23
Following the approach defined for synthesis of authentic
(23) EDCI ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,
HOAt ) 1-hydroxy-7-azabenzotriazole, DEPBT ) 3-(diethoxyphospho-
ryloxy)-1,2,3-benzotriazin-4(3H)-one, SES ) 2-trimethylsilylethanesulfonyl,
BCB ) B-bromocatecholborane.
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Alanine Scan of Ramoplanin Aglycon
A R T I C L E S
Scheme 2. 23 Alanine-Substituted Subunit C Analogues and
Synthesis of [Ala10] Subunit C (16)
Scheme 3. 23 Modified Subunit B and Its Analogues
potentially generating mixtures with a contaminate dechlorinated
side product if not closely monitored. In an effort to take
advantage of this observation and to reduce the number of
synthetic steps for the preparation of 23a, direct dechlorination
of the [Dap2]B subunit 20 or [Dap2]ramoplanin A2 aglycon itself
under various conditions was examined. However, the rate of
dechlorination was too slow, and the approach proved to be
impractical.
Synthesis of the B Subunit Derivatives: Pentapeptide
[L-Dap2]B. Within the B subunit, residue 2 was fixed as the
simplified and stable Dap2 where the side chain linkage is
attached to the coupled and similarly unmodified Asn1.19
Additionally, residue 16 already constitutes a D-Ala, and it was
not further modified. Rather, our interest focused on Leu15 as
well as Chp17, which was replaced with Hpg17 to directly assess
the role of the unusual chloride substituent as well as Ala17
(Scheme 3). The modified pentapeptide subunit 23, incorporating
the Hpg17 (or Ala17 for 22) residue and the Dap2 amide, was
obtained from an intermediate tripeptide, which in turn was
prepared by coupling Boc-Leu15-D-Ala16-OH with HCl‚H-Hpg17-
OBn (EDCI, HOAt, NaHCO3, 20% DMF-CH2Cl2, 0 °C, 20
h) followed by deprotection of the benzyl ester (H2, 10% Pd/C,
MeOH, 25 °C, 20 min, 100%). Fmoc-Dap2(NH2‚HCl)-OBn
obtained from Fmoc-Dap2(Boc)-OH (HCl-EtOAc) was coupled
with this tripeptide using 3-(diethoxyphosphoryloxy)-1,2,3-
benzotriazin-4(3H)-one (DEPBT)23 (NaHCO3, DMF, 0 °C, 1
h, then 25 °C, 52 h, 100%) to give a single diastereomer of the
corresponding tetrapeptide with no detectable racemization of
the sensitive Hpg17 residue. Fmoc removal (Bu4NF, i-PrOH,
DMF, 25 °C, 1 h, sonication), coupling the free amine with
Fmoc-Asn(Trt)-OH (EDCI, HOAt, DMF, 0 °C, 48 h, 61% for
two steps), and benzyl ester hydrogenolysis (H2, 10% Pd/C,
EtOH, 25 °C, 45 min, 100%) provided 23a.19 The original
pentapeptide subunit B and the simplified [Dap2]B subunit 20
were found to be sensitive to the benzyl ester deprotection step,
Synthesis of [Dap2,Alam]ramoplanin A2 Aglycon. The
convergent strategy developed for the total synthesis of the
ramoplanin and [Dap2]ramoplanin A2 aglycons was utilized for
the preparation of the alanine-substituted [Dap2]ramoplanin A2
aglycon analogues.19 For example, assemblage of the alanine-
substituted subunit A analogues (8-14) required coupling with
the common unmodified B and C subunits and maximized the
convergent nature of the synthesis. Representative of the final
stages of the analogue preparations, the synthesis of [Dap2,D-
Ala3]ramoplanin A2 aglycon begins with the treatment of 8 with
4 N HCl-dioxane followed by coupling of resulting amine with
the B subunit carboxylic acid 20a to provide 8b without
competitive â-elimination (Scheme 4). An excess of 20a was
employed to consume all 8a, which proved difficult to remove
from the product enlisting either an acid wash or chromatog-
raphy. Boc removal of 8b under mild conditions (B-bromocat-
echolborane t BCB, CH3CN, 0 °C, 3 h) that do not affect the
Asn1 trityl group gave the corresponding amine.19 The resulting
amine was then coupled with the unmodified subunit C (15a)
to yield 8c. Successive Boc removal, benzyl ester hydrogenoly-
sis, and macrocyclization provided the cyclic peptide core 8d
in superb yield attributable to the â-sheet preorganization of
the cyclization substrate as well as closure at a D-amine
terminus.17 Fmoc removal under specially developed conditions
(8 equiv of Bu4NF, 10 equiv of i-PrOH, DMF, 25 °C, 1 h,
sonication), acyl side chain introduction, and final global
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A R T I C L E S
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Scheme 4. 23 Synthesis of [Dap2,Ala3]ramoplanin A2 Aglycon (8g)
less crisp spectra that might be attributable to a conformational
heterogeneity (line broadening).
A 48-Membered Ramoplanin A2 Aglycon Analogue. One
of the more successful structural modifications of the ramoplanin
aglycon has been the replacement of L-HAsn2 with L-Dap2,
simplifying the synthesis and removing the hydrolysis labile
depsipeptide ester. Surprisingly, the analogous replacement with
L-Dab2, which results in a ring expansion to a 50-membered
ring, resulted in a >100-fold loss in antimicrobial activity and
provided an analogue with a propensity for aggregation in
aqueous buffer. As a consequence, we now examined the ring-
contracted 48-membered analogue 25g incorporating a D-Orn2
in place of L-HAsn2 where the side chain also incorporates an
amine for lipid side chain attachment, removing the need for
Asn1 (Scheme 5). The simplified B subunit 25a was prepared
and sequentially coupled with the authentic A (7a) and C (15a)
subunits (Scheme 5). To our disappointment, all efforts to
promote the macrocyclization of the linear peptide 25c following
C- and N-terminus deprotection failed to provide the macro-
cyclic peptide 25d. Presumably, the decreased flexibility of 25c
resulting from deleting the one methylene is sufficient to
preclude macrocyclization. These observations along with the
behavior of the 50-membered [Dap2]ramoplanin A2 aglycon
underscore the special role residue 2 plays in conveying
antimicrobial activity to the compounds and highlight how
remarkable the stable L-Dap2 for L-HAsn2 substitution is.
Antimicrobial Activity
The results of the antimicrobial assay of the analogues against
S. aureus alongside the ramoplanin complex (MIC ) 0.19 µg/
mL) and [Dap2]ramoplanin A2 aglycon (6, MIC ) 0.07 µg/
mL) are summarized in Figure 4. Analogous to observations
first reported for [Dap2]ramoplanin A2 aglycon (6) upon its
initial disclosure,19 it proved to be 2-3-fold more potent than
the natural ramoplanin complex and slightly more active than
the ramoplanin A2 aglycon.19,20 Perhaps the most important of
the comparisons to highlight first are the activities of the Ala4
and Ala10 analogues probing the importance of Orn4 and Orn.10
Semisynthetic modifications of these sites have indicated that
both contribute significantly to ramoplanin’s activity, but their
relative importance and potential role remained unclear.6f,8a,15b,19
Consistent with Walker’s observations with such semisynthetic
derivatives,6f the Ala10 analogue 16g (MIC ) 38 µg/mL) proved
to be 540-fold less active than 6, whereas the Ala4 analogue 9g
(MIC ) 3.1 µg/mL) was 45-fold less potent. This clearly
indicates that Orn10 is essential to the activity of ramoplanin
and plays a much more fundamental role than Orn4, although
both Ala10 and Ala4 are among the least effective of the Ala
analogues examined.
deprotection (HF, anisole, 0 °C, 90 min) yielded [Dap2,D-Ala3]-
ramoplanin A2 aglycon (8g).15 In a similar manner, each of the
ramoplanin A2 aglycon analogues was synthesized, and details
of their synthesis and their intermediates are provided in the
Supporting Information. The final products were purified by
reverse-phase HPLC to provide homogeneous materials that
were used for characterization and the biological evaluation of
the final products. However, it is notable that the final products
were obtained in purities ranging from 46-91% (av ) 73%,
see Supporting Information) even before this final purification.
The 1H NMR spectroscopic properties of each of the analogues
did not reveal a loss of conformational rigidity that might
contribute to a change or loss of antimicrobial activity, and only
Ala8, Ala10, Ala11, and most notably Ala17 exhibited slightly
Within the remainder of the putative Lipid II binding domain
proposed by McCafferty et al. (Hpg4-Orn10),8a the additional
largest impacts on activity were observed with D-Ala3 (vs
D-Hpg3, MIC ) 5.2 µg/mL), D-Ala7 (vs D-Hpg7, MIC ) 3.7
µg/mL), and Ala8 (vs aThr8, MIC ) 2.5 µg/mL), representing
75-fold, 50-fold, and 35-fold losses in antimicrobial activity,
respectively. Thus, both D-Hpg3 and D-Hpg7 proved more
significant than D-Orn4, and aThr8 approaches this level of
importance. Notably, the importance of aThr8 is unique among
the three aThr sites in ramoplanin, and each of the remaining
D-Ala/D-aThr substitutions (residues 5 and 12) resulted in e10-
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A R T I C L E S
Scheme 5.23 Attempted Synthesis of the 48-Membered
[D-Orn2]ramoplanin A2 Aglycon (25g)
Figure 4. Antimicrobial MIC values.
fold reductions in activity. Surprisingly, Ala6 (vs Hpg6, MIC )
0.9 µg/mL) as well as D-Ala5 (vs D-aThr5, MIC ) 0.16 µg/mL)
central to this region had the least impact on ramoplanin’s
properties, resulting in only 10-fold and 2-fold reductions in
antimicrobial activity. In fact, Hpg6 proved to be the least
important of the phenylglycines imbedded in the ramoplanin
structure and each of the Ala/Hpg substitutions elsewhere
resulted in >20-fold reductions in activity, and the Ala5 analogue
[Dap2, D-Ala5]ramoplanin A2 aglycon still proved to be slightly
more active than the natural ramoplanin complex despite its
2-fold reduction in activity versus 6.
The Ala9 analogue (MIC ) 0.6 µg/mL) with modification of
Phe9 resulted in a modest 8-9-fold reduction in antimicrobial
activity despite its role in capping the hydrophobic core and
important location at the corner of a â-turn in ramoplanins
structure adjacent to D-Orn10.10 Given that the Orn10 importance
almost certainly arises as a consequence of a key stabilizing
binding interaction with the diphosphate central to the structure
of Lipids I and II,6,8 it is surprising that the adjacent residue
Phe9, unlike Hpg11, would have such a modest effect. Nonethe-
less, it is consistent with the departure in structure observed
with the enduracidins which possess a solvent accessible Cit9
residue and suggests this may represent a useful modification
site for further functionalization or conjugation efforts.
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A R T I C L E S
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The Ala analogues spanning the dimerization domain defined
by Walker et al. and observed in MeOH (Hpg11-Gly14),6e which
resides adjacent to the key Orn10 and transannular to the putative
Lipid II recognition domain defined by McCafferty et al.,8a
proved especially interesting. Each of the Ala substitutions
resulted in a g10-fold reduction in antimicrobial activity, with
that of Ala11 (vs Hpg11, MIC ) 2.5 µg/mL) being the greatest
(35-fold), followed by Ala13 (vs Hpg13, MIC ) 1.4 µg/mL, 20-
fold), and D-Ala12 (vs D-aThr12, MIC ) 0.7 µg/mL, 10-fold).
In addition to illustrating a significant role that residues in this
potential dimerization domain may play in ramoplanin’s interac-
tion with its biological target(s), it also defines D-aThr12 as the
residue at which to probe such roles. In this regard, it is
interesting to note that the enduracidins incorporate a D-Ser12
in place of D-aThr12 and that the ramoplanin dimer structure
established by NMR places its residue 12 hydroxyls within 3.87
Å of one another on the same face of the dimerization interface.
Clearly, this represents a unique residue whose functionalization,
deliberate dimerization, or conjugation may be exploited to
probe mechanistic questions without perturbing the intrinsic
structure of the ramoplanin monomer or dimer.
interpreted this behavior to suggest that it is only the stereo-
chemistry at this center that might be important for its
antimicrobial activity and that residues 16-1, constituting a
more flexible loop including a D-Ala16 at one end of the
antiparallel â-sheet, do not directly interact with the biological
target Lipid I or II and do not play a direct functional role.
However, Phe9 and Chp17 do stabilize the intrinsic solution
structure of ramoplanin by forming a hydrophobic core buried
within the U-shaped conformation of the natural product and
may indirectly contribute to its properties. Significantly, the
results with Ala17 indicate that this residue, and perhaps the
adjoining residue Ala16, represent prime sites for modification
or conjugation in efforts to probe ramoplanin’s mechanism of
action. Finally, it is similarly interesting and surprising that the
conservative Ala15 replacement for Leu15 at the end of this
region of the molecule resulted in a 40-fold loss in antimicrobial
activity.
Discussion and Conclusions
A full alanine scan of [Dap2]ramoplanin aglycon (6), a
hydrolytically stable and slightly more potent analogue of the
ramoplanin aglycon (5), was conducted, providing insights into
the importance and potential role of each residue. By far the
most important residue in ramoplanin is D-Orn.10 Its replacement
with Ala10 resulted in a >500-fold reduction in antimicrobial
activity consistent with its proposed integral role in Lipid II
diphosphate binding. In contrast and more surprising to us, the
conserved Orn4 was found to be substantially less important
than Orn10, suggesting its role in binding Lipid I or II is not as
critical. Both these residues lie in the putative recognition and
binding domain proposed by McCafferty et al.8a Two of the
remaining residues in this region (residues 3-10) exhibit a larger
impact than Orn4 (D-Hpg3 and D-Hpg7), a third is comparable
(aThr8), and three appear much less important. Significantly,
two of these (D-aThr5, Hpg6) lie central to this putative Lipid
II recognition domain, and it is difficult to rationalize such a
marginal impact central to a contiguous binding interface. An
alternative explanation that might account for this behavior is
that Orn4 may not be involved in Lipid I or II substrate binding,
but rather that it may interact with the membrane phosphates
and collaborate with the lipid side chain to deliver and anchor
ramoplanin to the bacterial cell wall. In the monomer solution
structure of ramoplanin, Leu15, Ala16, the lipid side chain, and
perhaps Hpg13/Gly14 form a hydrophobic face on one side of
the molecule adjacent to Orn4 that could serve as the membrane
binding domain (Figure 5). As such, the enduracidin’s incor-
poration of End15 for ramoplanin’s Leu15 would represent
incorporation of an additional proximal membrane phosphate
binding residue spatially bracketing this hydrophobic face of
ramoplanin. The cluster of the remaining most prominent
residues including Orn10 (red, >100-fold reduction) and D-Hpg3,
D-Hpg7, aThr8, and Hpg11 (orange, 100-25-fold reduction)
traverses the opposite end of ramoplanin at one corner of its
reverse â-turn at aThr8-Phe9, potentially representing an alterna-
tive recognition domain for Lipid II. In the dimer structure, a
potential and similar discontiguous recognition motif is defined
by the red and orange residues, and Orn4 is found proximal to
the same hydrophobic face and lipid side chain. Although now
speculative, such roles will become clearer upon examination
of the derivatives herein for Lipid I or II binding in a
transglycosylation inhibition assay that can dissect such roles.20
Finally, the examination of several additional analogues
within the less well-defined Leu15-Asn1 segment, which adopts
a more flexible loop at one end of the ramoplanin structure and
contains depsipeptide ester as well as the lipid side chain, has
shed additional insights into its importance. Previously, we have
shown that the lipid side chain is essential for antimicrobial
activity (200-800-fold reductions),19,20 but it does not impact
Lipid II binding presumably serving to deliver or anchor
ramoplanin to the bacterial cell wall.20 Additionally, L-Dap2 (49-
membered ring), but not L-Dab2 (50-membered ring), effectively
replaces HAsn2 (49-membered ring) providing a stable amide
replacement for the labile depsipeptide ester.19,20 Herein, the
extensions of this work to an attempted replacement with D-Orn2
resulting in a ring-contracted 48-membered macrocyclic amide
revealed that the macrocyclization ring closure failed to provide
the core structure. The ease of 49-membered ring macrocy-
clization, the failure of the corresponding (more rigid) 48-
membered ring closure, and the aggregation of the 50-membered
ring system20 suggest a significant structural role for this corner
of ramoplanin structure and highlight how special the properties
of [Dap2]ramoplanin A2 aglycon are. Additionally, we probed
the importance of the adjacent Chp17 residue in this region. Its
replacement with Hpg17 representing the removal of the aromatic
chlorine substituent had virtually no impact on the antimicrobial
properties (MIC ) 0.09 µg/mL).24 Consistent with this lack of
functional role for the chlorine substituent, the enduracidins
incorporate Hpg17 in their structure at this site. More signifi-
cantly, the Ala17 replacement for Chp17 with 22g (MIC ) 0.3
µg/mL) had a similar lack of effect in the antimicrobial activity
resulting in only a modest 4-fold reduction in antimicrobial
potency. Despite the apparent significance of the structural
change, this Ala modification at residue 17 was among those
that had the least effect of any of the Ala substitutions although
it was among the residues including Phe9 that perturb the rigid
solution conformation characteristic of ramoplanin. We have
(24) In contrast, removal of the aromatic chlorines from vancomycin diminished
dimerization and altered its antimicrobial activity. See: (a) Harris, C. M.;
Kannan, R.; Kopecka, H.; Harris, T. M. J. Am. Chem. Soc. 1985, 107,
6652. (b) Gerhard, U.; Mackay, J. P.; Maplestone, R. A.; Williams, D. H.
J. Am. Chem. Soc. 1993, 115, 232.
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Alanine Scan of Ramoplanin Aglycon
A R T I C L E S
results in 200-800-fold reductions in antimicrobial activity but
has no effect on Lipid II binding or transglycosylase inhibition.20
This suggests that Leu15-HAsn2 resides adjacent to a membrane
anchoring center where this region constitutes a “relatively”
flexible loop at one end of the otherwise rigid U-shaped
antiparallel â-sheet. As mentioned above, it is also possible that
the proximal Orn4 collaborates with this lipid side chain by
capping this membrane binding domain with a positively
charged amine that binds membrane phosphates. We have also
shown and continue to highlight herein that HAsn2 may be
effectively replaced with Dap2, providing a hydrolytically stable
49-membered ring system in which an amide replaces the labile
depsipeptide ester. The HAsn2 â-carboxamide clearly does not
contribute to ramoplanin’s interaction with its biological target
(but may sterically hinder ester hydrolysis), and the Dap2 rigid
secondary trans amide bond assuredly mimics the analogous
trans ester conformation observed in the solution conformations
of ramoplanin and enduracidin. Although flexible, efforts to
expand19,20 or contract the macrocycle by a single carbon atom
at this site have failed or resulted in nonfunctional compounds,
suggesting this region plays important conformationally related
roles helping to confine ramoplanin to productive conformations.
With the possible exception of Leu15, the side chains of the
remainder of this “flexible” loop, Leu15-D-Ala16-Chp17, do not
appear to directly contribute to ramoplanin’s antimicrobial
activity. The surprisingly small impact of replacing Chp17 with
Ala17 (4-fold), the disparate Leu15 versus solubilizing End15
residues found in the ramoplanins versus enduracidins, and the
conserved minimal side chain at D-Ala16 suggest not only that
this “connecting” region of the molecule may represent a
membrane interacting region of the molecule, but also that it
may be a good site for modification or conjugation studies.
Finally, the impact of nearly each residue is so significant
that it is difficult to imagine deriving a simplified ramoplanin
by excision of a substantial portion of its structure (i.e.,
cyclization or alternative presentations of only the putative
recognition domain).8b The studies serve to define residues and
regions amenable to further funtionalizations for detailed
mechanistic studies in progress. Further insights into the
individual roles of each residue may be forthcoming from their
examination in assays establishing Lipid II binding or transg-
lycosylation inhibition.20
Figure 5. (A) Monomer and (B) dimer solution structures of ramoplanin
(red: >100, orange: 100-25, white: <25-fold reductions in antimicrobial
activity by alanine scan).
Within the residue 11-14 domain, all residues exhibit a
significant effect even though they lie outside McCafferty’s
putative Lipid II recognition domain. The magnitude of their
impact (10-40-fold) suggests a prominent role in establishing
ramoplanin’s activity. Although there may be many explanations
for this behavior, this would be consistent with their stabilization
of the ramoplanin dimerization interface observed by Walker
et al.6e It is noteworthy that the most significant of these residues
is Hpg11, which also lies adjacent to the critical Orn10 and is
the site of glycosylation. Given the impact of Hpg11, it is possible
that it plays a larger role in binding Lipid II than present models
suggest.
One of the most interesting regions of the molecule spans
residues 15-2. Attached to residue 2 is Asn1, which is external
to the cyclic ring system and which is acylated with the
unsaturated lipid side chain. We have suggested that this serves
to anchor the antibiotic in the bacterial cell wall, ensuring its
localization at its site of action.20 Consistent with this, removal
of the side chain or its replacement with a minimal acetyl group
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41101) and the
Skaggs Institute for Chemical Biology. We especially wish to
thank Dr. Asad Chavoshi in Professor Ghadiri’s Lab at the
Scripps Research Institute for conducting the HF deprotection
reactions.
Supporting Information Available: Full experimental details
and compound characterizations. This material is available free
of charge via the Internet at http://pubs.acs.org.
JA068573K
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