Semisynthetic approaches to laspartomycin analogues

Article abstract of DOI:10.1021/np068062b

Laspartomycin C (1), a lipopeptide antibiotic related to amphomycin, consists of a cyclic peptide core and an aspartic acid unit external to the core and linking this to a Cis-2,3-unsaturated fatty acid. This was reported initially to be active against St

Full text of DOI:10.1021/np068062b

J. Nat. Prod. 2007, 70, 447-450
447
Semisynthetic Approaches to Laspartomycin Analogues
William V. Curran,*,† Richard A. Leese,† Howard Jarolmen,† Donald B. Borders,† Dominique Dugourd,‡ Yuchen Chen,‡ and
Dale R. Cameron‡
BioSource Pharm, Inc., Spring Valley, New York 10977, and MIGENIX Inc., VancouVer, BC, Canada V6S 212
ReceiVed October 27, 2006
Laspartomycin C (1), a lipopeptide antibiotic related to amphomycin, consists of a cyclic peptide core and an aspartic
acid unit external to the core and linking this to a C15-2,3-unsaturated fatty acid. This was reported initially to be active
against Staphylococcus aureus, and more recent studies have shown that it is active against VRE, VISA, and MRSA
isolates. The enzymatic cleavage of the fatty acid tail was accomplished with a deacylase produced by Actinoplanes
utahensis and resulted in two peptides, designated Peptide 1 and Peptide 2. Semisynthetic derivatives of both peptides
have been made, and the principal requirement for biological activity appears to be the presence of an acylaspartic acid.
Laspartomycin was originally reported by Umezawa et al. in 1968
as a lipopeptide antibiotic related to amphomycin with a 2,3-
unsaturated C15-fatty acid side chain.1 At that time, full chemical
and spectroscopic characterization was very difficult and the
structure has remained unknown until relatively recently.2 Subse-
quently glycinocin A was reported from another Streptomyces sp.
and is apparently identical with or a stereoisomer of laspartomycin
C (1).3 Laspartomycin C consists of a cyclic peptide core and an
aspartic acid external to the core linking the core to a C15-2,3-
unsaturated fatty acid. It was initially reported to be active against
Staphylococcus aureus, and current studies have shown that it is
active against vancomycin-resistant enterococci (VRE), methicillin-
resistant S. aureus (MRSA), and vancomycin-intermediate S. aureus
(VISA) isolates. The enzymatic cleavage of laspartomycin com-
ponents with a deacylase produced by Actinoplanes utahensis results
in two peptides4 shown below (Peptide 1 and Peptide 2), and both
of these peptides have been converted by synthetic modifications
to derivatives active against S. aureus. The purpose of this study
was to examine the influence of the side chain on the structure-
activity of this antibiotic. To this end, a variety of derivatives have
been prepared including the conversion of Peptide 1 to Peptide 2.
chain alkyl aromatic acyl side chains showed good activity (6 and
7), but when the aromatic portion was at the end of chain, the
compound was not active (8).
A number of new antibiotics were prepared by derivatizing the
laspartomycin cyclic peptide.5,6 The MIC values of these compounds
against S. aureus in Mueller Hinton broth were determined in the
presence and absence of 4 mM calcium chloride. Without the added
calcium the MIC values were significantly higher. In the case of
tsushimycin, which is closely related to amphomycin, this calcium
ion dependence of biological activity was explained by a confirma-
tion change stabilized by Ca2+ bound within the peptide ring and
the formation of a dimer that interacted with the bacterial cell
membrane. In this study the structure of tsushimycin was determined
in the presence of calcium ions by X-ray crystallography.8 The side
chains of the laspartomycin cyclic peptide contained acyl amino
acids, acyl dipeptides, and acyl tripeptides. The stereochemistry of
the amino acids and peptides was explored in the present investiga-
tion. The acyl groups were both aliphatic and aromatic. Some of
the structure-activity relationships observed are summarized in
Table 1. The length of the side chain was very important since the
octanoyl derivative was not active compared to the pentadecanoyl
compound (4 and 5). Derivatives containing p-substituted long-
Pentadecanoyl dipeptides showed that the L-aspartic was essential
for activity since the D-isomer was inactive (10). In addition,
aromatic amino acids had greater potencies (9, 11, and 12) and the
stereochemistry of this amino acid did not affect the results since
the D-phenylalanine was also active. The pentadecanoyl glycine
and alanine derivatives were not active (13 and 14), but the
corresponding L-lysine compound (15) did show moderate activity.
The importance of the L-aspartic side chain is also evident since
the pentadecanoyl derivative of Peptide 1 was inactive (16), and
surprisingly the corresponding L-glutamic acid (17) also had poor
activity. However one compound containing L-asparagine (18) did
show moderate activity. Appending the side chain of the lipodep-
sipeptide antibiotic daptomycin to Peptide 2 produced an inactive
derivative (27), whereas extending the lipid tail containing the
Dedicated to the late Dr. Kenneth L. Rinehart of the University of
Illinois at Urbana-Champaign for his pioneering work on bioactive natural
products.
* To whom correspondence should be addressed. Tel: 845-425-4778.
Fax: 845-425-3893. E-mail: Biosource@prodigy.net.
† BioSource Pharm, Inc.
‡ Migenix Inc.
10.1021/np068062b CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/27/2007

448 Journal of Natural Products, 2007, Vol. 70, No. 3
Notes
Table 1. Structure-Activity Relationships of Laspartomycin Analogues from Side-Chain Modifications
a MIC values are minimum inhibitor concentrations expressed as µg/mL. Assay organism was a methicillin-sensitive Staphylococcus aureus
assayed by the tube dilution method.
Experimental Section
pentadecanoyl rather than the decanoyl moiety (28) resulted in
moderate activity. Replacement of the aspartic acids in the core
peptide by asparagine or various amide functions resulted in
complete loss of activity.
In order to assess the effect of different types of linkers, the
corresponding long-chain ureas, sulfonamides, and a carbamate were
prepared (19, 20, 21, 22, 23, and 24) All of these compounds
showed reasonably potent biological activity (Table 1).
Catalytic hydrogenation of 1 afforded the dihydro-laspartomycin,
which had approximately the same in vitro potency as 1. Conversion
of laspartomycin C to the dihydroxy compound with dilute
potassium permanganate proceeded readily, but the product lacked
biological activity.
Table 2 shows representative laspartomycin analogues with
activity against staphylococci and entercococci that are resistant to
clinically used antibiotics. Thus it is clear from this work that
changing the side chain of laspartomycin to various sulfonyl or
carbamoyl derivatives produces compounds with interesting bio-
logical activity, but none were more potent than the parent antibiotic.
General Experimental Procedures. FABMS were obtained on a
VG model ZAB-2SE mass spectrometer. All semisynthetic compounds
were characterized also by HPLC/UV using a Hewlett-Packard Model
1100 instrument.
All the condensation reactions on Peptide 1 or Peptide 2 were carried
out in anhydrous dimethylformamide using either dicyclohexylcarbo-
diimide and 1-hydroxybenzotriazole or O-[(ethoxycarbonyl)cyano-
methyleneamino]-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TOTU)
as the coupling agents (Figure 1).
All of the intermediate acids either were available commercially or
were synthesized. The amino acid and peptide derivatives were prepared
mainly by the activated ester method using dicyclohexylcarbodiimide
and 1-hydroxybenzotriazole except for the tripeptide derivatives, which
were prepared using standard solid-phase techniques. The protecting
groups were removed by standard procedures using 95% trifluoroacetic
acid for the tert-butyl esters, N-tert-butyloxycarbonyl, and O-trityl
groups. Catalytic hydrogenation with 5% palladium on carbon was used
for removing benzyl and carbobenzoxy moieties. Examples are provided
in the Experimental Section.

Notes
Journal of Natural Products, 2007, Vol. 70, No. 3 449
MIC (µg/mL)a
Table 2. Activity of Laspartomycin Analogues against Resistant Strains of Bacteria
compound
side chain (R)
MSSA
MRSA
MRSE
VISA
VSE
VRE, MDR
5
CH3(CH2)13CO-L-Asp-
4
4
>64
4
4
4
8
4
8
4
4
4
16
8
>64
32
4
2-4
2
4
4
8
4
8
21
24
11
CH3(CH2)13NH-CO-L-Phe-L-Asp-
CH3(CH2)13SO2-L-Trp-L-Asp-
CH3(CH2)13CO-L-Phe-L-Asp-
a MIC values are minimum inhibitory concentrations for MSSA (methicillin-sensitive Staphylococcus aureus, Smith), MRSA (methicillin-resistant
S. aureus), MRSE (methicillin-resistant S. epidermidis), VISA (vancomycin-intermediate S. aureus), VRE (vancomycin-resistant enterococci), and
MDR (multidrug-resistant enterococci).
for compound 25 and freeze-dried to obtain 6.0 mg of white powder:
FABMS m/z 1339 (M + H)+ 1361 (M + Na)+, and 1377 (M + K)+
(calcd for C64H99N12O19, 1339.7).
Compound 5. A mixture of 3.0 mg of the benzyl derivative, 11 mg
of 5% palladium on carbon, and 1.0 mL of methanol was hydrogenated
at atmospheric pressure overnight (balloon technique). The mixture was
filtered through Celite, evaporated to dryness, slurried in water, and
lyophilized to give 2.0 mg of compound 5: FABMS m/z 1287 (M +
K)+ (calcd for C57H92N12O19K, 1287.6).
Dihydrolaspartomycin. A mixture of 21.3 mg of laspartomycin (1),
35 mg of 5% palladium on carbon, and 2.5 mL of methanol was
hydrogenated at atmospheric pressure overnight (balloon technique).
The mixture was filtered through Celite, evaporated to dryness, slurried
in water, and lyophilized to give 19.4 mg of the desired product:
FABMS m/z 1248 (M + H)+, 1270 (M + Na)+ (calcd for C57H93N12O19,
1249.7).
Dodecylcarbamoyl-Peptide 2 (19). A mixture of dodecylisocyanate
(0.0104 g, 0.043 mmol) and Peptide 2 (0.044 g, 0.043 mmol) was stirred
in DMF (0.60 mL). After 60 min at room temperature, a second 0.0104
mL aliquot of the isocyanate was added and stirred for 60 min. The
reaction was quenched, and the product was isolated on Sephadex LH-
20 as described for compound 25. Product-containing fractions were
pooled, methanol was removed under vacuum, and the product was
freeze-dried from aqueous solution. Yield: 32 mg of a white solid,
Figure 1. Peptide coupling with DCC and HOBT. The procedure
77% by HPLC (215 nm area %); FABMS m/z 1259 (M + Na)+ (calcd
was repeated with D-phenylalanine to produce compound 12.
for C55H89N13O19Na,1258.6).
Dodecyloxycarbonyl-Peptide 2 (22). A mixture of dodecylchlo-
The products from these reactions were purified by the procedures
outlined below, and identifications of the compounds were confirmed
by FABMS. Product purity from these procedures general ranged from
80 to 93% based on HPLC area percent when the chromatograms were
monitored at 215 nm.
Pentadecanoyl-L-aspartic Acid 4-O-Benzyl Ester. L-Aspartic acid
4-O-benzyl ester (0.2578 g. 1.156 mmol) was added to 2 mL of water
and 2 mL of tetrahydrofuran followed by 1 mL of saturated sodium
bicarbonate solution, and the mixture was stirred until solution was
complete. A slurry of pentadecanoyl hydroxybenzotriazole (0.2798 g,
0.758 mmol) in 5 mL of water and 5 mL of tetrahydrofuran was added,
and the reaction mixture was stirred at room temperature overnight.
The reaction mixture was poured into 20 mL of water and acidified to
pH 1.0 with 6 N hydrochloric acid. The resulting precipitate was chilled,
filtered, and dried to afford 0.2792 g of product, C22H41NO5: FABMS
m/z 448 (M + H)+, 470 (M + Na)+, 492 (M + 2Na - H)+ (calcd for
C22H41NO5, 448.3).
roformate (0.010 mL) and Peptide 2 (0.035 g, 0.034 mmol) in DMF
(0.80 mL) was stirred at room temperature. The reaction mixture was
diluted with 4 mL of methanol, and the product was isolated on a
Sephadex LH-20 column as in the preparation of compound 25.
Methanol was removed under vacuum from the product-containing
fractions; the residue was dissolved in 10 mL of 14% acetonitrile 0.10
M in ammonium phosphate (aqueous pH 7.2). This solution was
desalted by application to a styrene-divinylbenzene resin cartridge (0.5
g, Supelco EnviChrom-P, conditioned with 10 mL of acetonitrile and
6 mL of 14% acetonitrile. The sample-loaded cartridge was rinsed with
6 mL of salt-free 14% acetonitrile. The product was stripped off using
6 mL of salt-free 67% acetonitrile, which was removed under vacuum,
and the product was freeze-dried from aqueous solution. Yield: 11
mg of a white solid, 81% by HPLC (215 nm area %); FABMS m/z
1238 (M + H)+, 1260 (M + Na)+, 1276 (M + K)+ (calcd for
C55H89N12O20, 1237.6).
Hexadecylcarbamoyl-Peptide 2 (20). A mixture of Peptide 2
(0.0438 g) and hexadecylisocyanate (0.013 mL) in 0.50 mL of DMF
was stirred at room temperature. After 70 min, a second aliquot of the
isocyanate was added. The reaction mixture was quenched and the
product isolated on a Sephadex LH-20 column as described for
compound 25. Methanol was removed under vacuum from the product-
containing fractions, yielding 29 mg of a yellow solid. The product
was further purified by low-resolution reversed-phase chromatography.
The product was eluted with 40% acetonitrile 0.10 M in ammonium
phosphate (aqueous pH7.2). This fraction was diluted with an equal
volume of distilled water and desalted as described for compound 25.
Eighteen milliliters of 67% acetonitrile were necessary to elute the
product. Acetonitrile was removed under vacuum, and the product was
freeze-dried from aqueous solution. Yield: 24 mg of an off-white solid,
83% by HPLC (215 nm area %); FABMS m/z 1292 (M + H)+, 1314
(M + Na)+, 1330 (M + K)+ (calcd for C59H98N13O19, 1292.7).
Hexadecylsulfonyl-L-Phenylalanyl-Peptide 2 (24). Hexadecyl-
sulfonyl-L-phenyl alanine (60 mg., 0.132 mmol), hydroxybenzotriazole
(19 mg, 0.132 mmol), and dicyclohexylcarbodiimide (32 mg, 0.151
Pentadecanoyl-L-aspartic Acid 4-O-Benzylhydroxybenzotriazole
Ester. A mixture of pentadecanoyl-L-aspartic acid 4-O-benzyl ester
(0.2619 g 0.5851 mmol) 1-hydroxybenzotriazole (0.0895 g, 0.5851
mmol) and dicyclohexylcarbodiimide (0.1205 g, 0.5851 mmol) in 5.0
mL of tetrahydrofuran was stirred at room temperature overnight. The
reaction mixture was filtered and evaporated to dryness at reduced
pressure. The resulting oil was slurred in hexane to give a crystalline
product. Yield: 0.2933 g.
Pentadecanoyl-L-Aspartic Acid 4-O-Benzyl Derivative of Peptide
1. A mixture of Peptide 1 (14.8 mg, 0.0162 mmol) and diisopropyl-
ethylamine (0.023 mL, 0.1319 mmol) was added to 0.5 mL of
dimethylformamide and stirred at room temperature. To this was added
two 0.20 mL aliquots of a solution of the above hydroxybenzotriazole
ester (44.9 mg, 0.0794 mmol) at the beginning and after 2.5 h. After
5 h, water was added and the product adsorbed onto a 2.5 × 5.0 cm
styrene-divinylbenzene resin column (ENVI-Chrom P) and eluted with
0.05 M phosphate buffer (pH 7.2) in 10% and 25% acetonitrile.
Fractions containing the desired product were desalted as described

450 Journal of Natural Products, 2007, Vol. 70, No. 3
Notes
mmol) was stirred for 40 min in 0.67 mL of DMF. A 0.30 mL aliquot
of this solution was added to a solution of Peptide 2 (50 mg, 0.0488
mmol) in 0.40 mL of DMF. The progress of the reaction was monitored
by HPLC, and the product was isolated by chromatography in a similar
fashion to that described for compound 25. The product was a white
powder, 13 mg; FABMS m/z 1460.5 (M + H)+, 1482.4 (M + Na)+,
1498.4 (M + K)+ (calcd for C67H106N13O21S, 1460.7).
Hexadecylsulfonyl-L-Tryptophan Methyl Ester. A solution of
hexadecylsulfonyl chloride (260 mg, 0.80 mmol), tryptophan methyl
ester hydrochloride (254 mg, 1.0 mmol), and 0.34 mL of triethylamine
(2.45 mmol) in 2.0 mL of DMF was stirred at room temperature for
4.0 h. The mixture was diluted with 10 mL of 1.0 N HCl and extracted
with 20 mL of ethyl acetate. The ethyl acetate solution was washed
with water and saturated salt solution and dried over magnesium sulfate,
then evaporated to give beige crystals: yield 261 mg; FABMS m/z
507 (M + H)+ (calcd for C28H47N2O4S, 507.3).
Hexadecylsulfonyl-L-tryptophan. A mixture of hexadecylsulfonyl-
L-tryptophan methyl ester (260 mg, 0.514 mmol) and 0.50 mL of 1.0
N NaOH in 2.0 mL of methanol and 2.0 mL of tetrahydrofuran was
stirred at room temperature for several h. Thin-layer chromatography
indicated the reaction was incomplete. An additional 0.50 mL of 1.0
N NaOH was added and the mixture stirred until the reaction was
complete (18 h). The reaction was worked up as described in the above
example to afford 196 mg of product: FABMS m/z 493 (M + H)+
(calcd for C27H45N2O4S, 493.3).
MIC Determinations. MIC values against organisms were deter-
mined using the broth microdilution method in accordance with the
U.S. Clinical and Laboratory Standard Institute Guideline M7-A6 (7)
using cation-adjusted Mueller-Hinton broth with 50 µg/mL of calcium.
MSSA (methicillin-sensitive Staphylococcus aureus Smith, ATCC
19636), MRSA (methicillin-resistant S. aureus, ATCC 43300), MRSE
(methicillin-resistant S. epidermidis, ATCC 51625), VISA (vancomycin-
intermediate S. aureus, CDC HIP 5836), VRE/MDR (vancomycin-
resistant Enterococcus faecium, ATCC 51559), and VSE (vancomycin-
sensitive E. faecalis) ATCC 29212 were used. MIC values for
compounds in Table 1 were obtained by the tube dilution method against
a MSSA in Mueller-Hinton broth determined in the presence and
absence of 4 mM calcium chloride.
Acknowledgment. V. Boyd, L. Workman, D. Friedland, R. Siu,
D. Erfle, and E. Rubinchik are gratefully acknowledged for their
contributions to this work. R. Coulson and J. Clement are thanked for
their support and leadership. J. Sun and L. Wang are gratefully
acknowledged for their synthetic contributions. We are very grateful
to R. Simon for her synthetic assistance in providing the tripeptides
for two of the analogues. The additional research staff of both
MIGENIX, Inc. and BioSource Pharm, Inc. that supported this project
are also acknowledged.
Hexadecylsulfonyl-L-Tryptophanyl-Peptide 2 (25). Hexadecyl-
sulfonyl-L-tryptophan (90 mg, 0183 mmol), hydroxybenzotriazole (28
mg, 0.183 mmol), and dicyclohexylcarbodiimide (38 mg, 0.183 mmol)
was stirred for 40 min in 1.0 mL of DMF. A 0.30 mL aliquot of this
solution was added to a solution of Peptide 2 (94.5 mg, 0.0439 mmol)
in 0.20 mL of DMF. The progress of the reaction was monitored by
HPLC. At the completion of the reaction, the reaction mixture was
diluted with 5 mL of methanol and 1.5 M NH4OH was added to an
apparent pH of about 7. The filtered sample solution was applied to a
2.5 × 44 cm size exclusion column (Sephadex LH-20 fine, swelled in
methanol), which was eluted with methanol at about 0.8 mL/min. The
product eluted in about 25 mL of eluate starting at about 105 mL. The
methanol was removed from the product pool by evaporation under
vacuum at or below 30 °C. The solid residue was dissolved in about
12 mL of 10% acetonitrile buffered with 0.08 M ammonium phosphate
(aqueous pH 7.2). This solution was applied to a 2.5 × 5 cm styrene-
divinylbenzene resin column (Supelco ENVI-Chrom P resin) and eluted
with increasing concentrations of acetonitrile buffered with pH 7.2
ammonium phosphate. The product eluted in about 36 mL using 48%
acetonitrile as eluent. Acetonitrile was removed from this fraction by
evaporation under vacuum (prior to applying the sample to this resin,
the resin column was washed with 100 mL aliquots of 70% acetonitrile,
then 100% acetonitrile, and finally 20% acetonitrile.). The aqueous
solution of the sample was then applied to the column for desalting.
The column was rinsed with 28 mL of 21% acetonitrile (unbuffered),
and the desalted product was stripped from the column using 67%
acetonitrile. Acetonitrile was removed by evaporation under vacuum,
and the product was freeze-dried. Yield: 14 mg, white solid; FABMS
m/z 1499 (M + H)+, 1521 (M + Na)+, 1537 (M + K)+(calcd for
C69H107N14O21S, 1499.7).
References and Notes
(1) (a) Naganawa, H.; Hamada, M.; Maeda, K.; Okami, Y.; Takeuchi,
T.; Umezawa, H. J. Antibiot. 1968, 21, 55-62. (b) Naganawa, H.;
Takita, T.; Maeda, K.; Umezawa, H. J. Antibiot. 1970, 23, 423-
424.
(2) Borders, D. B.; Leese, R. A.; Jarolmen, H.; Francis, N. D.; Falla, T.
Abstracts of the 41st Interscience Conference on Antimicrobial
Agents and Chemotherapy, Chicago, IL, September 22-25, 2001, p
231, No. F-1154.
(3) Kong, F.; Carter, G. T. J. Antibiot. 2003, 56, 557-564.
(4) (a) Borders, D. B.; Francis, N. D.; Simon, R. J.; Fantini, A. A.
Abstracts of the 41st Interscience Conference on Antimicrobial
Agents and Chemotherapy, Chicago, IL, September 22-25, 2001, p
231, No. F-1156. (b) Borders, D. B.; Leese, R. A.; Jarolmen, H.;
Francis, N. D.; Fantini, A. A.; Falla, T.; Fiddes, J. C.; Aumelas A.
J. Nat. Prod. 2007, 70, XXXXX.
(5) Simon, R. J.; Curran, W. V.; Leese, R. A.; Borders, D. B.; Koontz
M. Z.; Lui, H. Abstracts of the 41st Interscience Conference on
Antimicrobial Agents and Chemotherapy, Chicago, IL, September
22-25, 2001, p 231, No. F-1155.
(6) Cameron, D. R.; Chen, Y.; Dugourd, D.; Sun, J.; Wang, L.; Borders,
D. B.; Curran, W. V.; Leese, R. A. Abstracts of Papers, 229th ACS
National Meeting, San Diego, CA, March 13-17, 2005; MEDI-358.
(7) National Committee for Clinical Laboratory Standards. Methods for
dilution antimicrobial susceptibility tests for bacteria that grow
aerobically; Document M7-A6; National Committee for Clinical
Laboratory Standards: Wayne, PA, 2003.
(8) Bunko´czl, G.; Ve´rtesy, L.; Sheldrick, G. M. Acta Crystallogr. 2005,
D61, 1160-1164.
NP068062B