Total synthesis of the ramoplanin A2 and ramoplanose aglycon

Article abstract of DOI:10.1021/ja0212314

Full details of a convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon are disclosed. Three key subunits composed of residues 3-9 (heptapeptide 15), pentadepsipeptide 26 (residues 1,2 and 15-17), and pentapeptide 34 (residues 10-14) were prepared, sequentially coupled, and cyclized to provide the 49-membered depsipeptide core of the aglycon. Key to the preparation of the pentadepsipeptide 26 incorporating the backbone ester was the asymmetric synthesis of an orthogonally protected L-threo-β-hydroxyasparagine and the development of effective and near-racemization free conditions for esterification of its hindered alcohol (EDCI, DMAP, 0 °C). The coupling sites were chosen to maximize the convergency of the synthesis including that of the three subunits, to prevent late stage racemization of carboxylate-activated phenylglycine-derived residues, and to enlist β-sheet preorganization of an acyclic macrocyclization substrate for 49-membered ring closure. By altering the order of final couplings, two macrocyclization sites, Phe⁹-D-Orn¹⁰ and Gly¹⁴-Leu¹⁵, were examined. Macrocyclization at the highly successful Phe⁹-D-Orn¹⁰ site (89%) may benefit from both β-sheet preorganization as well as closure at a D-amine terminus within the confines of a β-turn at the end of the H-bonded antiparallel β-strands. A more modest, but acceptable macrocyclization reaction at the Gly¹⁴-Leu¹⁵ site (40-50%) found at the other end of the H-bonded antiparallel β-strands within a small flexible loop may also benefit from preorganization of the cyclization substrate, is conducted on a substrate incapable of competitive racemization, and accommodates the convergent preparation of analogues bearing depsipeptide modifications. Deliberate late-stage incorporation of the subunit bearing the labile depsipeptide ester and a final stage Asn¹ side-chain introduction provides future access to analogues of the aglycons which themselves are equally potent or more potent than the natural products in antimicrobial assays.

Full text of DOI:10.1021/ja0212314

Published on Web 01/23/2003
Total Synthesis of the Ramoplanin A2 and Ramoplanose
Aglycon
Wanlong Jiang, Jutta Wanner, Richard J. Lee, Pierre-Yves Bounaud, 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 October 1, 2002 ; E-mail: boger@scripps.edu
Abstract: Full details of a convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon are
disclosed. Three key subunits composed of residues 3-9 (heptapeptide 15), pentadepsipeptide 26 (residues
1, 2 and 15-17), and pentapeptide 34 (residues 10-14) were prepared, sequentially coupled, and cyclized
to provide the 49-membered depsipeptide core of the aglycon. Key to the preparation of the pentadep-
sipeptide 26 incorporating the backbone ester was the asymmetric synthesis of an orthogonally protected
L-threo-â-hydroxyasparagine and the development of effective and near-racemization free conditions for
esterification of its hindered alcohol (EDCI, DMAP, 0 °C). The coupling sites were chosen to maximize the
convergency of the synthesis including that of the three subunits, to prevent late stage racemization of
carboxylate-activated phenylglycine-derived residues, and to enlist â-sheet preorganization of an acyclic
macrocyclization substrate for 49-membered ring closure. By altering the order of final couplings, two
macrocyclization sites, Phe9-D-Orn10 and Gly14-Leu15, were examined. Macrocyclization at the highly
successful Phe9-D-Orn10 site (89%) may benefit from both â-sheet preorganization as well as closure at
a D-amine terminus within the confines of a â-turn at the end of the H-bonded antiparallel â-strands. A
more modest, but acceptable macrocyclization reaction at the Gly14-Leu15 site (40-50%) found at the
other end of the H-bonded antiparallel â-strands within a small flexible loop may also benefit from
preorganization of the cyclization substrate, is conducted on a substrate incapable of competitive
racemization, and accommodates the convergent preparation of analogues bearing depsipeptide modifica-
tions. Deliberate late-stage incorporation of the subunit bearing the labile depsipeptide ester and a final
stage Asn1 side-chain introduction provides future access to analogues of the aglycons which themselves
are equally potent or more potent than the natural products in antimicrobial assays.
Ramoplanin is a novel lipoglycodepsipeptide isolated from
the fermentation broths of Actinoplanes sp. ATCC 33076 (Figure
1).1 The structure was established in 1989 and found to be
composed of the three closely related compounds 1-3, of which
2 is the most abundant, that differ only in the structure of the
acyl group found on the Asn1 N-terminus.2 The ramoplanin
complex was found to be 2-10 times more active than
vancomycin against Gram-positive bacteria (500 strains), in-
cluding methicillin-resistant enterococci (MIC ) 0.5 µg/mL)
and all known strains of methicillin-resistant Staphylococcus
aureus (MRSA).3 Shortly after its disclosure, ramoplanin was
shown to disrupt bacterial cell wall biosynthesis where it inhibits
the action of intracellular UDP-glcNAc transferase (MurG) and
the conversion of lipid intermediate I to lipid intermediate II
(Figure 2).4 This inhibition was proposed to arise by ramoplanin
complexation of lipid intermediate I preventing its utilization
as a substrate.4,5 More recently, Walker has shown that
ramoplanin also inhibits the subsequent and more accessible
transglycosylase-catalyzed extracellular polymerization of lipid
intermediate II and forms self-associating 1:1 complexes with
close analogues of the substrate, lipid intermediate II, suggesting
this may represent the more relevant biological site of action.6
Further supporting this premise, Walker has shown that ramo-
planin binds a lipid intermediate II analogue better than the
corresponding lipid intermediate I analogue, that ramoplanin
also binds MurG (1:1, Kd ) 4 × 10-6 M), that the inhibition of
MurG cannot be overcome with additional substrate inconsistent
with a mechanism simply involving substrate depletion, and that
a ramoplanin analogue incapable of binding lipid intermediate
I still inhibits MurG but does not exhibit antimicrobial activity.
(4) Somner, E. A.; Reynolds, P. E. Antimicrob. Agents Chemother. 1990, 34,
413. Bro¨tz, H.; Bierbaum, G.; Reynolds, P. E.; Sahl, H.-G. Eur. J. Biochem.
1997, 246, 193. Review: Reynolds, P. E.; Somner, E. A. Drugs Exptl.
Clin. Res. 1990, 16, 385.
(5) Bro¨tz, H.; Josten, M.; Wiedemann, I.; Schneider, U.; Go¨tz, F.; Bierbaum,
G.; Sahl, H.-G. Mol. Microbiol. 1998, 30, 317.
(6) Lo, M.-C.; Men, H.; Branstrom, A.; Helm, J.; Yao, N.; Goldman, R.;
Walker, S. J. Am. Chem. Soc. 2000, 122, 3540. Helm, J. S.; Chen, L.;
Walker, S. J. Am. Chem. Soc. 2002, 124, 13970. For dimerization of
ramoplanin, see: Lo, M.-C.; Helm, J. S.; Sarngadharan, G.; Pelczer, I.;
Walker, S. J. Am. Chem. Soc. 2001, 123, 8640.
(1) Cavalleri, B.; Pagani, H.; Volpe, G.; Selva, E.; Parenti, F. J. Antibiot. 1984,
37, 309. Pallanza, R.; Berti, M.; Scotti, R.; Randisi, E.; Arioli V. J. Antibiot.
1984, 37, 318.
(2) Ciabatti, R.; Kettenring, J. K.; Winters, G.; Tuan, G.; Zerilli, L.; Cavalleri,
B. J. Antibiot. 1989, 42, 254. Kettenring, J. K.; Ciabatti, R.; Winters, G.;
Tamborini, G.; Cavalleri, B. J. Antibiot. 1989, 42, 268. Review: Parenti,
F.; Ciabatti, R.; Cavalleri, B.; Kettenring, J. Drugs Exptl. Clin. Res. 1990,
16, 451.
(3) Review: Espersen, F. Curr. Opin. Anti-Infect. InVest. Drugs 1999, 1, 78.
9
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J. AM. CHEM. SOC. 2003, 125, 1877-1887
1877

A R T I C L E S
Jiang et al.
Figure 2.
The structures were established by 2D NMR and found to
consist of a 49-membered ring composed of 17 amino acids in
which the C-terminal 3-chloro-4-hydroxyphenylglycine (Chp17)
forms a lactone bond with the hydroxy group of â-hydroxy-
asparagine (HAsn2).8 Twelve of the amino acids possess
nonstandard side chains and seven possess the D-configuration.
A related antibiotic ramoplanose, described by Williams in
1991,9 contains the identical depsipeptide core and the ramopla-
nin A2 Asn1 acyl side chain, but incorporates three versus two
D-mannose units on Hpg11. The high-resolution solution structure
of the well-defined conformation of ramoplanin A2, which
served to correct the olefin stereochemistry on the acyl side
chain and to allow for assignment of the Hpg6 and Hpg7 absolute
stereochemistry, is characterized by two antiparallel â-strands
(residues 2-7 and 10-14) stabilized by six transannular
H-bonds and a connecting reverse â-turn (aThr8-Phe9).8 The
conformation is further stabilized by a cluster of hydrophobic
aromatic side chains (residues 3, 9, and 11) providing a U-shape
topology to the â-sheet with the â-turn at one end and a flexible
connecting loop at the other (residues 15-17), Figure 3.
Although less well characterized, the enduracidins10 represent
closely related antibiotics acting by inhibition of bacterial cell
wall biosynthesis11 that had been disclosed much earlier than
the ramoplanins. Notably, they do not bear a Hpg11 di- or
trisaccharide and contain only subtle structural changes in the
stretch of residues 9-17 as well as at residues 1 (lipid side
chain) and 2 (Figure 1). In addition, the uncharacterized
antibiotic janiemycin has been reported to bear an amino acid
composition and biological properties suggesting it represents
an additional member of this class of natural products.12,13
Structural details of the interaction of ramoplanin with
peptidoglycan precursors are beginning to emerge from the
Figure 1.
These results suggest substrate recognition (lipid II > lipid I),
while unnecessary for MurG inhibition, is important for in vivo
antimicrobial activity. They also support the contention that
inhibition of extracellular transglycosidation (IC50 ) 0.25 µM,
MIC ) 0.1 µM) with lipid II as the substrate is the more relevant
site of action for observation of antimicrobial activity. These
two steps immediately precede the transpeptidase-catalyzed
cross-linking reaction and a principal site of action of vanco-
mycin. Thus, mechanism-based cross-resistance between ra-
moplanin and vancomycin is not observed, and ramoplanin
represents an excellent candidate for more expansive clinical
use beyond its introduction for topical infections.7 Ramoplanin
is presently in Phase III clinical trials for the oral treatment of
intestinal vancomycin-resisitant Enterococcus faecium (VREF)
and Phase II trials for nasal MRSA.3
(8) Kurz, M.; Guba, W. Biochemistry 1996, 35, 12570.
(9) 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.
(10) Hori, M.; Iwasaki, H.; Horii, S.; Yoshida, I.; Hongo, T. Chem. Pharm.
Bull. 1973, 21, 1175. Iwasaki, H.; Horii, S.; Asai, M.; Mizuno, K.;
Ueyanagi, J.; Miyake, A. Chem. Pharm. Bull. 1973, 21, 1184.
(11) Higashide, E.; Hatano, K.; Shibata, M.; Nakazawa, K. J. Antibiot. 1968,
21, 126. Asai, M.; Muroi, M.; Sugita, N.; Kawashima, H.; Mizuno, K. J.
Antibiot. 1968, 21, 138. Tsuchiya, K.; Takeuchi, Y. J. Antibiot. 1968, 21,
426.
(12) Meyers, E.; Weisenborn, F. L.; Pansy, F. E.; Slusarchyk, D. S.; von Saltza,
M. H.; Rathnum, M. L.; Packer, W. L. J. Antibiot. 1970, 23, 502.
(13) McCafferty, D. G.; Cudic, P.; Yu, M. K.; Behenna, D. C.; Kruger, R. G.
Curr. Opin. Chem. Biol. 1999, 3, 672.
(7) Jones, R. N.; Barry, A. L. Diagn. Microbiol. Infect. Dis. 1989, 12, 279.
9
1878 J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003

Ramoplanin A2 and Ramoplanose Aglycon
A R T I C L E S
pentadepsipeptide 26 (residues 1, 2 and 15-17), and pentapep-
tide 34 (residues 10-14) were sequentially coupled and cyclized
in a solution phase, convergent approach to the 49-membered
depsipeptide core of 1-3 (Figure 1). The indicated coupling
sites were chosen to maximize the convergency of the synthesis,
including that of the three subunits, to minimize the use of
protecting groups, to prevent late stage opportunities for
racemization of carboxylate-activated phenylglycine-derived
residues, and to enlist â-sheet preorganization of an acyclic
macrocyclization substrate18 for ring closure. Two macrocy-
clization sites, Phe9-D-Orn10 and Gly14-Leu15, were chosen
for examination. Macrocyclization at the Phe9-D-Orn10 site,
which was disclosed in our earlier communication,17 proved
unusually successful and represents a site found at the corner
of the â-turn at the end of the H-bonded antiparallel â-strands
(Figure 3). Consequently, closure at this site may benefit from
both â-sheet preorganization of the substrate as well as closure
at a D-amine terminus.19 The additional closure at the Gly14-
Leu15 site detailed herein lies within a small flexible loop at
the other end of the H-bonded antiparallel â-strands (Figure
3). In addition to also potentially benefiting from â-sheet
preorganization of the macrocyclization substrate, it represents
closure at a nonhindered site incapable of racemization. Pertinent
to projected future studies, this closure site may prove especially
useful for introducing alterations in the sensitive depsipeptide
ester found in the pentapeptide 26 which contributes to the
instability of the natural product. Deliberate late stage incorpora-
tion of this subunit bearing the labile depsipeptide ester and a
final stage Asn1 side chain introduction potentially provides
access to analogues of the aglycons which themselves have been
reported to be equally potent or more potent than the natural
products in antimicrobial assays. Also key to the implementation
of the approach was the judicious choice of the Orn4/Orn10 SES
protection and the Asn1 Fmoc protection providing orthogonal
protecting groups stable to Boc, Cbz, and benzyl ester depro-
tections, yet capable of sequential and selective removal in the
presence of the depsipeptide ester using conditions we intro-
duced in these or earlier unrelated studies.
Figure 3. Macrocyclization sites.
studies of McCafferty14 and these are defining the structural
features and sites within 2 (Figure 1) that contribute to
recognition, affinity, and selectivity for lipid intermediate I and
II binding.15 Most notably, the octapeptide sequence of Hpg3-
Orn10, which is highly conserved between the ramoplanins and
enduracidins, and the muramyl carbohydrate and adjacent
pyrophosphate of soluble lipid intermediate derivatives (PG
monomer) exhibit the greatest chemical shift changes and
majority of intramolecular nOe’s in a complex generated in vitro.
Insights derived from such studies14,15 and those of Walker6
combined with the work detailed herein provide the opportunity
to conduct detailed structure-function studies on this unique
class of natural products.16
Synthesis of the Hpg3-Phe9 Subunit (15). Heptapeptide 15,
the first of the three key subunits which contains all but Orn10
of the putative Hpg3-Orn10 recognition sequence,15 was as-
sembled as shown in Scheme 1 from the tripeptide 7 and
tetrapeptide 13 (EDCI, HOAt, DMF, 25 °C, 14 h, 76%)20
followed by Boc deprotection (HCl-dioxane). This coupling
was found to be sensitive to the reaction concentration (<40%
at <0.2 M vs >65% at g0.33 M) and cleanly provided 14
without deliberate protection of the free alcohols, and with no
evidence of competitive â-elimination or trace racemization.
Moreover, the heptapeptide 14 was sufficiently insoluble in
EtOH that a trituration recrystallization could be utilized for its
large scale purification albeit with a recovery somewhat lower
than a conventional chromatography. Tripeptide 7 was prepared
Herein, we provide full details of the first total synthesis of
the ramoplanin A2 and ramoplanose aglycon.17 Three key
subunits composed of residues 3-9 (heptapeptide 15), the
(18) Maplestone, R. A.; Cox, J. P. L.; Williams, D. H. FEBS Lett. 1993, 326,
95.
(14) 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.
(15) 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.
(16) Review: Chu, D. T. W.; Plattner. J. J.; Katz, L. J. Med. Chem. 1996, 39,
3853.
(17) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud, P.-Y.; Boger, D. L. J. Am.
Chem. Soc. 2002, 124, 5288. Review: Boger, D. L. Med. Res. ReV. 2001,
21, 356.
(19) 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. Rich, D. H.;
Bhatnagar, P.; Mathiaparanam, P.; Grant, J. A.; Tam, J. P. J. Org. Chem.
1978, 43, 296.
(20) EDCI ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,
HOAt ) 1-hydroxy-7-azabenzotriazole, HOBt ) 1-hydroxybenzotriazole,
PyBop ) benzotriazol-1-yl-oxytris-(pyrrolidino)phosphonium hexafluoro-
phosphate, HATU ) 2-(1H-7-azabenzotriazol)-1-yl-1,1,3,3-tetramethylu-
ronium hexafluorophosphate, DCC ) 1,3-dicyclohexylcarbodiimide, DPPA
) diphenylphosphoryl azide.
9
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A R T I C L E S
Jiang et al.
Scheme 1
Scheme 2. Improvements in the Preparation of
Fmoc-L-HAsn(Trt)-OBn (16)24
efficient synthesis of the Hpg3-Phe9 subunit which is ultimately
capped with Orn10 bearing a basic amine side chain lends itself
to systematic modification of the putative Hpg3-Orn10 recogni-
tion domain in efforts that could establish the importance and
potential role of each residue.
Synthesis of the Depsipeptide Subunit (26). Key to prepara-
tion of the pentadepsipeptide 26 incorporating the sensitive
backbone ester was the asymmetric synthesis of the orthogonally
protected L-threo-â-hydroxyasparagine 1624 and the subsequent
esterification of the hindered alcohol of 18 with 19.25 Since our
disclosure of the preparation of 16 which relied on a key
Sharpless asymmetric aminohydroxylation (AA) reaction,24 we
have improved its preparation in several subtle ways. These are
detailed in Scheme 2 highlighting the improvements which entail
conducting the Sharpless AA with 0.04 equiv of K2OsO2(OH)4/
0.05 equiv of (DHQD)2PHAL (lower amounts can result to
lower ee), increasing the reaction time of the amidation reaction,
and conducting the oxidative cleavage of the substituted anisole
in an EtOAc-CH3CN-H2O (1:1:8) solvent system with vigor-
ous mechanical stirring (vs magnetic stir bar) of the heteroge-
neous reaction mixture for larger scale reactions. The yield of
the amidation reaction utilized for the conversion of the methyl
ester to primary amide improved simply by increasing the
reaction time (sat. NH3-MeOH, 25 °C, 7-10 d, 62-77% vs
14 d, 99%). Attempts to conduct this transformation by
hydrolysis of the methyl ester (LiOH, THF/MeOH/H2O) fol-
lowed by amidation (PyBop, HOBt, i-Pr2NEt, 10 equiv NH4-
Cl, 58%)20,26 proceeded faster, but suffered competitive epimer-
ization and TBS deprotection especially when conducted on
large scales. Finally, a single step Boc and TBS group removal
was accomplished by treatment of Boc-L-HAsn(OTBS)-OBn
with 4 N HCl-EtOAc (30 equiv). This was followed by
treatment of the amine with FmocCl (1.5 equiv) and NaHCO3
(2 equiv) in 50% H2O-dioxane to provide Fmoc-L-HAsn-OBn
in an improved 92% yield and an intermediate that can be
purified by simple recrystallization on large scale.
by benzyl ester deprotection of 6 (H2, 10% Pd/C, MeOH, 25
°C, 2 h, 100%) obtained by coupling D-aThr-OBn21 with the
dipeptide 5 (EDCI, HOAt, 20% DMF-CH2Cl2, 0-10 °C, 3 h,
94%).20 In turn, 5 was obtained by benzyl ester deprotection of
4 (H2, 10% Pd/C, MeOH, 25 °C, 2 h, 98%) derived from
coupling Boc-D-Hpg-OH and D-Orn(SES)-OBn (EDCI, HOAt,
25% DMF-CH2Cl2, 0 °C, 6 h, 94%).20,22 Tetrapeptide 13 was
prepared from the dipeptides 8 and 10 secured by the coupling
of Boc-L-Hpg-OH with D-Hpg-OBn (EDCI, HOAt, 25% DMF-
CH2Cl2, 0-25 °C, 8 h, 93%)20 and Boc-L-aThr-OH21 with
L-Phe-OBn (EDCI, HOAt, 20% DMF-CH2Cl2, 0-25 °C, 18
h, 90%),20 respectively. Notably, benzyl ester hydrogenolysis
of 8 (H2, 10% Pd/C, MeOH, 25 °C, 2 h, 99%) followed by
coupling with 11 activated by DEPBT23 (NaHCO3, THF, 25
°C, 20 h, 83%) was accomplished with no detectable racem-
ization of the sensitive D-Hpg residue (>99% de) or â-elimina-
tion of the hindered L-aThr whereas alternative coupling reagents
gave lower conversions accompanied by substantial epimeriza-
tion even when the reaction was conducted at 0 versus 25 °C
[EDCI-HOAt (50%, 78% de); PyBop, NaHCO3 (57%, 75%
de); HATU, NaHCO3 (80%, 95% de)].20 This convergent and
(21) Beaulieu, P. L. Tetrahedron Lett. 1991, 32, 1031. Beaulieu, P. L.; Schiller,
P. W. Tetrahedron Lett. 1988, 29, 2019. Boc-L-aThr-OH was prepared from
L-aThr-OH by treatment with Boc2O, Na2CO3, THF/H2O (87%). HCI‚H-
D-aThr-OBn was prepared from Boc-D-aThr-OH by treatment with BnBr,
NaHCO3, DMF (89%); 4 N HCl-EtOAc (quant.).
(22) SES ) 2-trimethylsilylethanesulfonyl: Weinreb, S. M.; Demko, D. M.;
Lessen, T. A. Tetrahedron Lett. 1986, 27, 2099. Huang, J.; Widkanski, T.
S. Tetrahedron Lett. 1992, 33, 2657. Boc-D-Orn(SES)-OH, Boc-D-Orn-
(SES)-OBn, and D-Orn(SES)-OBn were prepared as described in the
Supporting Information.
(23) DEPBT ) 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one: Fan,
C.-X.; Hao, X.-L.; Ye, Y.-H. Synth. Commun. 1996, 26, 1455. Li. H.; Jiang,
X.; Ye, Y.-H.; Fan, C.; Romoff, T.; Goodman, M. Org. Lett. 1999, 1, 91.
(24) Boger, D. L.; Lee, R. J.; Bounaud, P.-Y.; Meier, P. J. Org. Chem. 2000,
65, 6770.
(25) Prepared from L-Chp-OH (Holdrege, C. T. U.S. Patent 3646024; Chem.
Abstr. 1972, 77, 34543) by treatment with Boc2O, Na2CO3, 25% H2O-
THF (98%); BnBr, NaHCO3, DMF (98%); CF3CONMeTBS, THF, 40-
45 °C (100%); H2, 10% Pd/C, EtOH (quant.).
(26) Wang, W.; McMurray, J. S. Tetrahedron Lett. 1999, 40, 2501.
9
1880 J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003

Ramoplanin A2 and Ramoplanose Aglycon
A R T I C L E S
Scheme 3
Scheme 4
Fmoc deprotection of 16 and coupling of 17 with Fmoc-L-
Asn(Trt)-OH (EDCI, HOAt, 17% DMF-CH2Cl2, 25 °C, 2.5 h,
81-90%)20 provided 18, and an intermediate that can purified
by simple recrystallization on large scale, Scheme 3. The key
esterification of 18 with 1925 required activation with EDCI20
conducted in the presence of DMAP27 catalyst (CH2Cl2, 0 °C,
1 h, 87%, >10:1 diastereoselection). A wide range of alternative
esterification protocols were examined including the use of
reactive, unhindered mixed anhydride activations, an acyl
fluoride activation, Mitsunobu esterification, Yamaguchi es-
terification, and the Corey-Nicolaou 2-pyridylthioester activa-
tion with metal catalysis. These and EDCI or DCC20 promoted
couplings in the absence of DMAP provided no product or
resulted in much lower conversions, lower diastereomeric ratios,
or suffered from competitive â-elimination. Even the EDCI-
DMAP, or a less satisfactory DCC-DMAP,20,27 reaction
required carefully controlled reaction conditions with higher
reaction temperatures (25 vs 0 °C), longer reaction times, or
more DMAP (0.5-2 equiv vs 0.15-0.2 equiv) offering less
satisfactory conversions. Boc removal (BCB,28 CH2Cl2, 0 °C,
2 h), coupling with 23 (EDCI, HOAt, 20% DMF-CH2Cl2, 0
°C, 1.5 h, 81%),20 buffered TBS deprotection (Bu4NF-HOAc,
THF, 0 °C, 30 min, 95%),29 and benzyl ester hydrogenolysis
(H2, 10% Pd/C, EtOH, 25 °C, 4.5 h, 94%) provided 26. Efforts
to shorten this sequence by first coupling 23 with L-Chp(OTBS)-
OBn followed by benzyl ester deprotection and esterification
with 18 have not been successful and an unbuffered Bu4NF
deprotection of 24 (no HOAc) led to competitive epimerization
of the Chp R-aminoester center. Given the effectiveness of this
preparation of 26, its simple adoption to incorprate amide or
even more stable carbon replacements of the sensitive dep-
sipeptide ester can be easily envisioned.
Synthesis of the Orn10-Gly14 Subunit (34). The preparation
of the final subunit 34 is detailed in Scheme 4. D-aThr-OBn21
was directly coupled with Boc-L-Hpg-OH to give dipeptide 27
(DEPBT,23 NaHCO3, THF, 0-25 °C, 18 h, 82%) devoid of any
diastereomeric contaminants. Boc deprotection (HCl-EtOAc)
and direct coupling of 28 with Boc-D-Orn(SES)-OH22 provided
tripeptide 29 (EDCI, HOAt, NaHCO3, 20% DMF-CH2Cl2,
0-15 °C, 4 h, 90%).20 Benzyl ester deprotection (H2, 10% Pd/
C, MeOH, 25 °C, 2 h, 98%) and coupling of 30 with 32 gave
pentapeptide 33 (EDCI, HOAt, NaHCO3, 50% DMF-CH2Cl2,
0-25 °C, 25 h, 90%).20 In turn, the dipeptide 32 was obtained
in two steps by coupling Boc-L-Hpg-OH and Gly-OBn hydro-
chloride to give dipeptide 31 (DEPBT,23 NaHCO3, THF, 0-25
°C, 19 h, 82%), followed by Boc deprotection (HCl-EtOAc).
Notably, C-terminus protection of D-aThr as its benzyl ester in
29 and elsewhere throughout the synthesis permitted hydro-
genolysis deprotection avoiding base-catalyzed â-elimination.
Similarly, coupling each sensitive Hpg-OH residue in 34, like
that throughout the synthesis, conducted upon activation with
DEPBT23 minimized epimerization especially when coupled
with a hindered and sensitive aThr amine terminus. For example,
DEPBT23 promoted coupling to provide 27 proceeded in good
yield with little or no epimerization (82%, 98% de) whereas an
EDCI-HOAt20 coupling was accompanied by significant ra-
cemization (80%, 80% de). Finally, the Orn10-Gly14 subunit
was initially prepared as the methyl ester30 but ultimately
implemented as the benzyl ester 33 permitting a cleaner ester
deprotection conducted on a substrate (i.e., 41) leading to Gly14-
Leu15 macrolactamization.
(27) Boger, D. L.; Chen, J.-H. J. Am. Chem. Soc. 1993, 115, 11 624. Boger, D.
L.; Chen, J.-H.; Saionz, K. W. J. Am. Chem. Soc. 1996, 118, 1629. DMAP
) 4-(dimethylamino)pyridine.
Assembly of Three Subunits and Macrocyclizations at the
Phe9-Orn10 and Gly14-Leu15 Sites. One of the strategic
advantages of a convergent synthesis employing three key
fragments was the opportunity to examine macrocyclization at
(28) BCB ) B-bromocatecholborane: Boeckman, R. K., Jr.; Potenza, J. C.
Tetrahedron Lett. 1985, 26, 1411.
(29) Boger, D. L.; Miyazaki, S.; Kim. S. H.; Wu, J. H.; Loiseleur, O.; Castle,
S. L. J. Am. Chem. Soc. 1999, 121, 3226. Boger, D. L.; Miyazaki, S.; Kim,
S. H.; Wu, J. H.; Castle, S. L.; Loiseleuer, O.; Jin, Q. J. Am. Chem. Soc.
1999, 121, 10004.
(30) Experimental details are provided in the Supporting Information.
9
J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003 1881

A R T I C L E S
Jiang et al.
Scheme 5
enhanced R-carbon acidity of the activated carboxylate derived
from 26 resulting from the use of a N-acyl versus carbamate
derivative. Only DEPBT23 promoted the coupling to provide
36 in superb yields (NaHCO3, DMF, 0 °C, 20 h, 50-68%) with
no competitive â-elimination, whereas all other alternative
coupling reagents and conditions surveyed over several years
provided predominantly â-elimination products. By way of
contrast, the best alternative to this coupling enlisted EDCI
(HOAt, 0-85% CH2Cl2-DMF, 0 °C, 18-40 h)20 and provided
36 in variable conversions of 0-19%. Little or no reaction was
observed when this DEPBT coupling was run in the absence
of added NaHCO3 (1.5 equiv with 1.5 equiv of DEPBT),
whereas the use of stronger tertiary amine bases led to
competitive and extensive â-elimination or hydrolysis of the
desired product. In addition, in early studies we found that
exposure of isolated 36 to MeOH to effect transfers, especially
on small scale, led to hydrolysis (methanolysis) of the labile
ester. As a consequence, the exposure of 36 and all subsequent
intermediates to MeOH or tertiary amine bases was avoided
although we do not know whether these precautions are
necessary. However, consistent with these observations and
expectations, we have found that treatment of either ramoplanin
(2) or the ramoplanin aglycon (48) with 1% Et3N-H2O results
in rapid hydrolysis of the depsipeptide ester to cleanly provide
the linear acyclic derivative18 (>90% isolated yield, 20 min;
80%, 5 min, 30%, 1 min)30 with little or no difference in the
rates of reaction. Boc removal was accomplished under mild
conditions (BCB,28 CH2Cl2, 0 °C, 30 min) that preserved the
trityl protecting groups. Although unnecessary, their mainte-
nance improved the detection, chromatographic, and solubility
properties of subsequent intermediates. Coupling of the crude
free amine with 34 (EDCI, HOAt, 25% DMF-CH2Cl2, 0 °C,
17 h, 60-82%)20 provided the key acyclic depsipeptide 37.
Successive Boc removal (BCB,28 CH3CN, 0 °C, 3 h), benzyl
ester hydrogenolysis (H2, 10% Pd/C, EtOH, 25 °C, 2 h), and
macrocyclization (EDCI, HOAt, 66-85% CH2Cl2-DMF, 1
mM, 0 °C, 18-20 h, 54-72% for 3 steps) afforded the cyclic
depsipeptide core 38. Presumably, â-sheet preorganization of
the cyclization substrate18 and closure at a D-amine terminus19
at the corner of a â-turn contributes to the superb conversions
for closure of the 49-membered ring (89% yield for macro-
cyclization using the purified amino acid). Given the unique
success of an initial ring closure attempt enlisting EDCI,20 the
subsequent optimization studies were conducted with the
examination of very few alternative reagents. Thus, although
this was not investigated in detail, small scale reactions with
either DEPBT23 (lower conversion) or DPPA20 (no reaction)
did not suggest they would be useful alternatives to EDCI.
Notably, the linear peptide 37 proved insoluble in most nonpolar,
aprotic solvents (CH2Cl2, CHCl3, EtOAc), slightly soluble in
CH3CN, and soluble in protic solvents (MeOH, EtOH). The
choices of solvents for the Boc deprotection (CH3CN) and
benzyl ester deprotection (EtOH) were dictated by this solubility
and the latter (EtOH vs MeOH) minimizes competitive ester
cleavage by the solvent under the reaction conditions.
either the Phe9-Orn10 or the Gly14-Leu15 site enlisting common
precursors altering only the order or site of fragment assembly.
At the onset of our work and despite the well-founded
macrocyclization site selections, the closure to provide the 49-
membered ring was unprecedented. Consequently, the conver-
gent fragment assembly provided maximum flexibility in
examining the macrocyclization in addition to providing the
strategic advantages commonly attributed to a convergent versus
linear (i.e., solid phase) synthesis. Assembly of three key
fragments and macrocyclization at the Phe9-Orn10 site, which
we judged most attractive to initially examine, are detailed in
Scheme 5. By far, the coupling of subunits 15 and 26 proved
to be the most challenging step of the synthesis. Carboxylate
activation of 26 typically resulted in preferential â-elimination
of the acyloxy substituent. This can be attributed to the
combination of a superb leaving group, the hindered nature of
the activated carboxylate resulting from its R,â,â-trisubstitution
and the large protecting groups (trityl and Fmoc), and the
In an effort to further streamline the synthesis, the N-terminus
Boc group of the Orn10-Gly14 subunit 33 was exchanged for a
Cbz protecting group such that the ester and amine deprotections
of 40 could be conducted in a single operation. Thus, Boc
deprotection of 34 (4 N HCl-EtOAc) followed by Cbz
9
1882 J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003

Ramoplanin A2 and Ramoplanose Aglycon
A R T I C L E S
Scheme 6
Scheme 7
precludes competitive racemization of the activated carboxylate.
As such, and based on these criteria alone, the two closures
might be expected to perform comparably. The distinction
between the two sites rests with Phe9-Orn10 lying at the corner
of a conformationally defined â-turn adjacent to the H-bonded
antiparallel â-strands whereas the Gly14-Leu15 site is embedded
in what appears to be a conformationally more flexible region
of the molecule. Benzyl ester deprotection of 36 (H2, 10% Pd/
C, EtOH, 25 °C, 4 h) followed by coupling with 35 provided
41 (EDCI, HOAt, NaHCO3, 2:1 CH2Cl2-DMF, 0 °C, 18-20
h, 76%).20 Boc deprotection (BCB,28 CH3CN, 0 °C, 3 h), benzyl
ester deprotection (H2, 10% Pd/C, EtOH, 25 °C, 2 h) followed
by macrocyclization (EDCI, HOAt, 75% CH2Cl2-DMF, 1 mM,
0 °C, 18-20 h) provided 38 in good conversions (40-50%).
This preliminary observation, which has not been subjected to
optimization efforts, suggests that closure at the Gly14-Leu15
site, while perfectly acceptable, may not be as facile as closure
at the Phe9-Orn10 site.
Acyl Side Chain Introduction and Completion of the Total
Synthesis. Having served their purpose admirably, the removal
of the orthogonal Fmoc protecting group with introduction of
the unsaturated acyl side chain followed by sequential or
simultaneous trityl and SES deprotections remained for comple-
tion of the total synthesis. Both were viewed as formidable
challenges to conduct in the presence of the sensitive depsipep-
tide ester. The latter SES deprotection in the presence of a
sensitive depsipeptide ester was a challenge we had addressed
earlier in the course of a total synthesis of the luzopeptins and
quinoxapeptins and solved by introducing a HF deprotection.31
Our selection of the orthogonal SES protecting group was based
on this experience and we assumed, but did not know, that the
Trt protecting groups would also be removed under these
conditions and tentatively hoped that the acyl side chain olefin
stereochemistry would be unaffected. As anticipated based on
a related experience in the total synthesis of thiocoraline and
protection (CbzCl, NaHCO3, 50% dioxane-H2O, 25 °C, 30 min,
78%) provided 39, Scheme 6. BCB28 Boc deprotection of 36
(CH2Cl2, 0 °C, 30 min) with isolation and purification of the
free amine followed by coupling with 39 provided 40 (EDCI,
HOAt, DMF, 0 °C, 26 h, 84%).20 Single step Cbz and benzyl
ester deprotection (H2, 10% Pd/C, 50% DMF-MeOH, 25 °C,
4 h) followed by EDCI promoted macrocyclization under the
conditions describe earlier provided 38 (66-71%) in competitive
conversions. However, the conversions were challenging to
reproducibly observe resulting in occasional competitive Fmoc
and Trt deprotections and ester cleavage under the hydrogenoly-
sis conditions especially if conducted for extended reaction
periods. Although these were perceived as solvable problems
complicated only by the insolubility properties of 40, this
coupled with the challenges of purifying 39 provided the
incentive for us to carry forward with material prepared as
detailed in Scheme 5.
Finally, the results of a brief study on macrocyclization at
the alternative Gly14-Leu15 site are shown in Scheme 7.
Although unnecessary for the completion of the total synthesis
of the ramoplanin aglycon, the examination of closure at this
site was of interest for several reasons. First, it opens the
opportunity to prepare analogues of the sensitive depsipeptide
region of 2 (Figure 1) enlisting an alternative coupling order
first assembling Hpg3-Gly14 as a common precursor, followed
by coupling with modified depsipentapeptide subunits and a final
key macrocyclization at the Gly14-Leu15 site. Just as interest-
ingly, it constitutes closure at the other end of the H-bonded
antiparallel â-strands at a site within a small flexible loop and
should benefit from â-sheet preorganization of the cyclization
substrate like closure at the Phe9-Orn10 site. Unlike the Phe9-
Orn10 site, it does not benefit from closure at a D-amine terminus
although the carboxylate terminus now lacks a L-side chain
substituent that may decelerate such ring closures. It also
(31) Boger, D. L.; Ledeboer, M. W.; Kume, M. J. Am. Chem. Soc. 1999, 121,
1098. Boger, D. L.; Ledeboer, M. W.; Kume, M.; Searcey, M.; Jin, Q. J.
Am. Chem. Soc. 1999, 121, 11 375.
9
J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003 1883

A R T I C L E S
Jiang et al.
Scheme 8
Scheme 9
subsequently examined the Fmoc deprotection utilizing Bu4NF
in the presence of EtSH.34 These conditions (2 equiv Bu4NF,
10 equiv EtSH, DMF, 25 °C, 3 min) did provide the desired
amine from 20 and 24, but the rapid reaction times made it
difficult to cleanly isolate the product free amine without
promoting cleavage of the depsipeptide and were not successful
when implemented with the macrocyclic precursor 38. Although
the inclusion of EtSH in this reaction mixture serves to trap the
released dibenzofulvene,35 we viewed its inclusion as one that
also serves to buffer the basicity of the reagent mixture. As
such, we examined several variations on such a buffered Bu4-
NF promoted Fmoc cleavage (e.g., PhSH, HOAc, CF3CH2OH)
and found that inclusion of the relatively hindered alcohol,
i-PrOH, in place of the thiol provided a superb new method of
Fmoc deprotection. Thus, treatment of 20 and 24 with Bu4NF
(2 equiv, 10 equiv i-PrOH, DMF, 25 °C, 15 min, sonication)
followed by acylation of the crude free amines with anhydride
43 (2 equiv, DMF, 25 °C, 16 h) provided 45 and 46 (90%) in
superb overall conversions. Through the course of these studies,
we established that this subsequent N-acylation was most
effective when conducted with the anhydride 43 permitting a
relatively easy purification of the products from the reaction
byproducts compared to acylation reactions conducted with the
corresponding carboxylic acid 42 (EDCI).20
Fmoc removal of 38 under these specially developed condi-
tions (8 equiv of Bu4NF, 10 equiv of i-PrOH, DMF, 25 °C, 1
h) that do not promote competitive Asn2 â-elimination followed
by side chain acylation of crude free amine with anhydride 43
(2 equiv, 20% CH2Cl2-DMF, 69% for 2 steps) provided 47 in
excellent overall conversions, Scheme 9. Notably, efforts to
purify and characterize the intermediate free amine were not
successful in early efforts on the Fmoc deprotection which we
tentatively ascribe to its instability. A single step HF deprotec-
tion31 of the trityl and SES groups (HF, anisole, 0 °C, 90 min,
82%) or sequential trityl (5% H2O-TFA, 25 °C, 5 h) and SES
deprotection (HF, 83%) provided the ramoplanin A2 and
ramoplanose aglycon 48 identical in all respects with an
authentic sample (1H NMR, HPLC, UV, MS).36,37
BE-22179 which bear a sensitive depsipeptide thioester,32
attempted Fmoc deprotection of 38 with piperidine (5% pip-
eridine-DMF, 0 °C, 20 min) simply led to multiple products
including cleavage of the depsipeptide ester by â-elimination
and/or ester aminolysis without generating perceptible amounts
of the desired macrocyclic free amine or the subsequent
acylation product 47 (5 equiv of 42, EDCI, HOAt, DMF-CH2-
Cl2, 0 °C, 18-24 h).20 Consequently, we examined a number
of alternatives to a conventional Fmoc deprotection and the
subsequent N-acylation reaction using advanced intermediates
bearing the sensitive depsipeptide ester (Scheme 8). On the basis
of the report33 that the Fmoc protecting group is sensitive to
certain hydrogenation conditions and our own observation that
24, 37, and 40 may have suffered slow competitive Fmoc
deprotection under extended reaction times for the benzyl ester
deprotection, the deprotection of 24 with H2 and Pd(OAc)2 was
examined and found satisfactory providing both benzyl ester
and Fmoc deprotection, albeit requiring a prolonged reaction
time (DMF, 16 h) notably without competitive Trt deprotection.
However, this Fmoc deprotection proved capricious to repro-
ducibly implement and provided only minor amounts of 45 or
the corresponding unstable amine when applied to 38 (H2, 2
equiv Pd(OAc)2, DMF, 25 °C, 14-46 h). In part, this may be
attributed to the unrecognized instability of the product free
amine and the use of subsequent nonoptimal acylation conditions
(5 equiv 42, EDCI, HOAt, DMF-CH2Cl2, 0 °C, 24 h).20 We
(32) Boger, D. L.; Ichikawa, S.; Tse, W. C.; Hedrick, M. P.; Jin, Q. J. Am.
Chem. Soc. 2001, 123, 561. Boger, D. L.; Ichikawa, S. J. Am. Chem. Soc.
2000, 122, 2956.
(33) Atherton, E.; Bury, C.; Sheppard, R. C.; Williams, B. J. Tetrahedron Lett.
1979, 3041.
(34) Ueki, M.; Amemiya, M. Tetrahedron Lett. 1987, 28, 6617. Ueki, M.;
Nishigaki, N.; Aoki, H.; Tsurusaki, T.; Katoh, T. Chem. Lett. 1993, 721.
(35) Carpino, L. A. Acc. Chem. Res. 1987, 20, 401.
9
1884 J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003

Ramoplanin A2 and Ramoplanose Aglycon
A R T I C L E S
In our hands, and unlike the conformational heterogeneity
alluded to by McCafferty,15 the ramoplanin aglycon 48 adopts
a single, rigid conformation in solution indistinguishable from
that of the natural product (1H NMR) although this was not
explored with 2D techniques. Similarly, we have not observed
stability differences between the natural product and the aglycon.
We do, like McCafferty, observe losses in material upon reverse
phase chromatography of 2 or 48. This poor recovery (ca. 50%),
in our assessment, is not derived from degradation (no other
products detected), but rather the poor physical properties of
the compounds including their complete and irretrievable
adherence to plastic and related materials. Consequently, we
do not view the role of the disaccharide subunit of ramoplanin
A2 as one that conveys stability or conformational rigidity to
the molecule,15 but rather one that, among other roles, may alter
and potentially improve solubility or physical properties. Thus,
the final, single step HF deprotection of 47 to provide the
aglycon 48 was most effectively conducted with a trituration
purification (EtOAc) of the final product avoiding a reverse
phase chromatography purification and the simple recovery
losses. Notably, cyclic depsipeptide intermediates bearing the
Fmoc, Trt, and SES protecting groups (e.g., 38) do not suffer
these same recovery properties and could be purified by standard
versus reverse phase chromatography, and 47 bearing the Asn1
acyl side chain and Trt/SES protecting groups could be purified
by reverse phase chromatography without analogous apparent
recovery losses.
Figure 4.
material loss that accompanies HPLC purification. Thus, the
use of anhydrous HF for deglycosidation of ramoplanin A1-
A3 is exceptionally effective providing the corresponding
aglycons in conversions as high as 92% and offers a superb
improvement over existing methods.
Reaction of 48 with SESCl (Et3N, DMF, -20 °C) provided
a mixture of 49 and 50 with the Orn4 amine reacting only
sluggishly under a wide range of reaction conditions, Scheme
10. Importantly, authentic 49 prepared in this manner proved
identical (1H NMR, HPLC, UV, MS) with the synthetic material
described above. Notably, 49 and 50 have proven to be key
derivatives of 2 useful in the definition of the site of action of
ramoplanin and the details of these and related studies will be
disclosed in due time.
Antimicrobial Activity of Key Derivatives. The results of
an antimicrobial evaluation of several of the key derivatives of
ramoplanin A2 that were prepared herein are summarized in
Figure 4 using Staphylococcus aureus (ATCC 25923) which is
among the least sensitive wild-type bacteria. Consistent with
the original36 and more recent subsequent observations,15 the
antimicrobial potency of ramoplanin A2 (2) and its aglycon 48
were not distinguishable with the latter typically displaying
slightly greater potency in our single assay. Consequently,
glycosidation does not contribute to intrinsic in vitro antimi-
crobial potency. Similarly, acycloramoplanin A2 and its aglycon,
in which the macrocyclic lactone was hydrolyzed, were inactive
(MIC > 128 µg/mL) and found to be >250- to 500-fold less
potent. Previous studies detailed an approximate 2000-fold loss
in antimicrobial activity upon hydrolysis of the macrocyclic
lactone of ramoplanin A215,18 and we see analogous drops in
the activity of not only this derivative, but its aglycon as well.
Similarly, 49, in which both the Orn4 and Orn10 δ-amino groups
are SES protected, was found to be inactive (MIC >128 µg/
mL, >500-fold loss in activity). Analogous observations were
recently disclosed by McCafferty15 with Orn4, Orn10-diacetyl
ramoplanin A2 which was found to be 500-fold less potent than
ramoplanin A2 highlighting the importance of these two basic
amines which are conserved among all members of this class
of antimicrobial natural products. Much more interestingly, the
mono SES derivative 50 (MIC ) 4 µg/mL), in which only the
Orn10 δ-amino group is protected and the Orn4 amine is not,
was 16-fold less potent than the free aglycon 48, but >32-fold
more potent than the diSES derivative 49. Clearly, both the Orn4
and Orn10 amines contribute to the antimicrobial activity and
the latter result suggests, but does not require, that the Orn4
free amine is more important than the Orn10 amine. Recent work
of Walker’s6 enlisting alanine derivatives of the Orn4 and Orn10
δ-amines, which maintain but move or extend the position of
the free amines, found that the Orn4-Ala was an active
antimicrobial agent (MIC ) 0.8 µg/mL) and bound peptidogly-
Deglycosidation of Ramoplanin and Functionalization of
the Ramoplanin A2 Aglycon. In an effort to secure comparison
samples of authentic 48 and preceding synthetic intermediates,
we examined methods for the deglycosidation of ramoplanin
and the subsequent functionalization of the ramoplanin A2
aglycon. Past protocols for the deglycosidation of ramoplanin
have relied on the treatment of the ramoplanin complex with
either: (1) trimethylsilyl iodide or trimethylsilyl chloride in the
presence of sodium iodide followed by hydrolysis, or (2) a
strong mineral acid (HCl) in the presence of a lower alcohol
(e.g., BuOH) under anhydrous conditions (entry 1).36 Typically,
pure ramoplanin A2 aglycon is obtained in 20-30% yield using
optimized conditions for these methods. In addition to low
conversions, the isolation of pure ramoplanin aglycon requires
a tedious HPLC purification that results in some loss of material
and contributes to the low conversions. We have found that the
use of anhydrous HF cleanly cleaves the dimannose sugar of
ramoplanin, without affecting the sensitive ester linkage or acyl
side chains.37 Thus, treatment of the ramoplanin complex with
anhydrous HF cleanly provided the A1-A3 aglycons contami-
nated only with mannose-derived reaction byproducts, Scheme
10. Reverse phase HPLC purification of the mixture which
serves to separate the A1-A3 aglycons (20-50% CH3CN-
HCOONH4 (aq, 0.05 M)) provided pure A1 (3%), A2 (46%),
A3 (3%), albeit with a recovery loss due to the physical
properties of the natural product aglycons. Enlisting pure
ramoplanin A2 (2), the HF deglycosidation followed by a simple
EtOAc trituration to remove the deglycosidation byproducts
provided the pure A2 aglycon (48) in 92% yield avoiding the
(36) Ciabatti, R.; Cavalleri, B. (BioSearch Italia S.p. A.) US patent 5491128;
Chem. Abstr. 1990, 112, 179 893.
(37) Wanner, J.; Tang, D.; McComas, C. C.; Crowley, B. M.; Jiang, W.; Moss,
J.; Boger, D. L. Bioorg. Med. Chem. Lett., in press. See also: Boger, D.
L.; Menezes, R. F.; Yang, W. Bioorg. Med. Chem. Lett. 1992, 2, 959.
9
J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003 1885

A R T I C L E S
Jiang et al.
Scheme 10
can lipid I analogues whereas the Orn10-Ala derivative was
nearly inactive (MIC ) 100 µg/mL) and did not bind to lipid
I peptidoglycan analogues implicating Orn10 versus Orn4 as the
more important of the two residues. Interestingly, the positively
charged amine of Orn4 flanks one side of the ramoplanin
solution structure whereas that of Orn10 flanks the other and
they lie at the opposite ends of the conserved putative pepti-
doglycan binding domain Hpg3-Orn10. Our observation of a
significant, but not complete loss of activity with 50 (16-fold
loss) suggests that the Orn10 free amine is important, but not
absolutely essential for activity. Further studies on these and
additional synthetic and semisynethtic derivatives should serve
to clarify the role of the Orn4 and Orn10 amines, the putative
Hpg3-Orn10 recognition domain, and offer insights into the site
and mechanism of action of ramoplanin.
enlisting some of the most effective coupling reagents and
unanticipated challenges associated with forming and maintain-
ing the depsipeptide ester were addressed as the key subunits
were assembled. In light of these findings, it is unlikely that a
linear solid phase synthesis of the ramoplanin aglycon could
be effectively implemented without the knowledge derived from
the work detailed herein. In anticipation of a continuation of
our efforts, the modular assemblage of 38 from three key
fragments makes the approach amenable to the systematic
preparation of analogues in which the role of each residue and
each side chain structural feature may be assessed without
conducting a complete linear solid-phase synthesis of each
individual compound. This superimposition of the parallel,
divergent synthesis of a library of ramoplanin analogues onto a
convergent total synthesis38 is in progress and will be disclosed
in due course.
Conclusions
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41101) and The
Skaggs Institute for Chemical Biology. We wish to thank Dr.
Inkyu Hwang for conducting the antimicrobial assays, Dr. S.
A solution phase convergent total synthesis of the ramoplanin
A2 and ramoplanose aglycon was developed defining two
strategically effective macrocyclization sites. In addition to the
design elements inherent in a convergent synthesis that were
highlighted in the Introduction, unexpected extensive epimer-
ization at many of the coupling sites were observed even when
(38) Chen, Y.; Bilban, M.; Foster, C. A.; Boger, D. L. J. Am. Chem. Soc. 2002,
124, 5431.
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1886 J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003

Ramoplanin A2 and Ramoplanose Aglycon
A R T I C L E S
Ichikawa for interim studies on the coupling of 15 and 26 and
macrocyclization studies of 38, Dr. P. Meier for contributing
to material supplies in interim studies, Dr. Mark O′Neil-Johnson
and Jason Moss for the training and assistance with each
anhydrous HF reaction.
1
Supporting Information Available: Full experimental details
and characterization (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
(Sequoia Sciences, San Diego, CA) for microscale H NMR
spectra, and Dr. Ciabatti (Biosearch Italia S.p.A.) for a supply
of the natural ramoplanin complex and a correlation sample of
the ramoplanin A2 aglycon. We wish to especially thank
Professor K. D. Janda for the use of the anhydrous HF apparatus
JA0212314
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J. AM. CHEM. SOC. VOL. 125, NO. 7, 2003 1887