Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A

Article abstract of DOI:10.1016/j.bmc.2013.06.064

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5- phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi.

Full text of DOI:10.1016/j.bmc.2013.06.064

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of potential threonine synthase
inhibitors: Rhizocticin A and Plumbemycin A
b
Mathias Gahungu a, Anthony Arguelles-Arias b, Patrick Fickers d, Astrid Zervosen a, , Bernard Joris ,
⇑
Christian Damblon c, André Luxen a
a Research Center of Cyclotron, University of Liège, Allée du 6 Août 8, Bât B30, B-4000 Sart-Tilman, Liège, Belgium
b Center of Protein Engineering, University of Liège, Bât B6, B-4000 Sart-Tilman, Liège, Belgium
c Department of Chemistry, University of Liège, Bât B6, B-4000 Sart-Tilman, Liège, Belgium
d Biotechnology and Bioprocess, Université Libre de Bruxelles, Av. Roosevelt 50, 1050 Bruxelles, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains
Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine
synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical
stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-
APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was syn-
thesized via the Still–Gennari olefination starting from Garner’s aldehyde. The Michaelis–Arbuzov reac-
tion was used to form the phosphorus–carbon bond. Oligopeptides were prepared using liquid phase
peptide synthesis (LPPS) and were tested against selected bacteria and fungi.
Received 14 May 2013
Revised 17 June 2013
Accepted 26 June 2013
Available online xxxx
Keywords:
Rhizocticin
Phosphonate antibiotic
Threonine synthase inhibitors
(S,Z)-APPA
Ó 2013 Elsevier Ltd. All rights reserved.
Plumbemycin
1. Introduction
a chemical synthesis of naturally occurring phosphonate antibiot-
ics; Rhizocticins (RZs, Fig. 1A) and Plumbemycins (PBs, Fig. 1B).
Today some strains of bacteria are resistant to all commercially
available antibiotics and the search for new drugs with alternative
cellular targets is highly needed. In this context, we have started
research on the phosphonate antibiotics. Phosphonates represent
an important class of organophosphorus compounds characterized
by the presence of one or more carbon-to-phosphorus (C–P) bonds.
Ciliatine (2-aminoethylphosphonic acid—AEP) was the first phos-
phonate discovered in 1959 by Kandatsu and Horigushi from
hydrolysates of ciliated protozoa.1 From this date, many other
phosphonates were gradually discovered or synthesized. Currently,
these compounds attracted the attention of researchers for their
use as reagents in organic synthesis, as herbicides or pesticides
in agriculture, as chemical warfare and most of all, for their
interesting medical properties. Indeed, we find among these
phosphonates; antibiotics,2–4 antivirals,5 antitumor compounds,6
antimalarial agents,7,8 antihypertensives,9 and many other. The
biological activity of some phosphonates stems from their
structural similarity to phosphate esters. Other members of this
class mimic carboxyl groups or the tetrahedral intermediates
formed during enzymatic metabolism.10 In this work, we develop
The structure of Rhizocticins (RZs) antibiotics was elucidated by
Rapp et al.11 and by Fredenhagen et al. using RZs isolated from cul-
ture media.12 Four different RZs have been isolated from a complex
culture media of Bacillus subtilis ATCC 6633: RZ-A = (S)-Arg-(S,Z)-
APPA, RZ-B = (S)-Val-(S)-Arg-(S,Z)-APPA, RZ-C = (S)-Ile-(S)-Arg-
(S,Z)-APPA and RZ-D = (S)-Leu-(S)-Arg-(S,Z)-APPA (Fig. 1A). It is
interesting to note that (S,Z)-APPA is the C-terminal component
of two other antibiotics: Plumbemycin A (PB-A) and Plumbemycin
B (PB-B), (Fig. 1B) isolated in 1976 from Streptomyces plumbeus by
Park, Hirota and Sakai.13 PB-A = (S)-Ala-(S)-Asp-(S,Z)-APPA and PB-
B = (S)-Ala-(S)-Asn-(S,Z)-APPA. The Z-APPA moiety of PBs was first
reported to be in a (R)-configuration based on the Clough–Lutz–Jir-
gensons (CLJ) rule.14 However, this observation was later contested
by Fredenhagen et al.12 who demonstrated by chiral HPLC analysis
and optical rotation measurement that (Z)-APPA has (S)-configura-
tion both in RZs and PBs. RZs are antifungal whereas PBs are anti-
bacterial. The selectivity of these compounds is due to the
recognition of amino acids attached to the (S,Z)-APPA moiety by
specific oligopeptide transport system.15 RZs and PBs are hydro-
lyzed by oligopeptidases into the active (S,Z)-APPA moiety and
inactive amino acids.
The target of the (S,Z)-APPA moiety of these antibiotics in
bacteria and fungi is threonine synthase (TS),16 the last enzyme
⇑
Corresponding author. Tel.: +32 4 366 3383; fax: +32 4 366 2946.
E-mail address: azervosen@ulg.ac.be (A. Zervosen).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.064
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2
M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
A
B
O
OH
O
P
OH
H
N
O
P
OH
H
N
O
OH
OH
H2N
N
H
OH
R
N
H
O
O
(S,Z)- APPA
R
O
(S,Z)- APPA
(S)-Ala (S)-Asp
(S)-Asn
PB-A : R= COOH
PB-B : R= CONH2
RZ-A : R = H
HN
RZ-B : R = (S)-Valyl
RZ-C : R = (S)-Isoleucyl
RZ-D : R = (S)-Leucyl
H2N
NH
O
O
(S)-Arg
O
C
Threonine HO
Synthase
P
4 steps
O
OH
O
O
OH
H3N
O
H3N
H3N
O
O
O
(S)-HSerP
(S)-Threonine
(S)-Aspartate
Figure 1. Chemical structures of (S,Z)-APPA and its containing oligopeptide phosphonate antibiotics (A) = Rhizocticins, (B) = Plumbemycins. (C) = threonine biosynthetic
pathway from (S)-aspartate.
of threonine biosynthetic pathway, which catalyses the formation
of (S)-threonine from the substrate (S)-homoserine- -phosphate,
(S)-HSerP (Fig. 1C). Since TS is absent in mammals, enzymes of this
pathway are potential targets for the development of new antibiot-
ics.17,18 In this work, we first present the stereoselective chemical
synthesis and characterization of the non-proteinogenic (S,Z)-APPA
(1H–1H COSY, 1H–13C HSQC). Importantly, the (Z)-geometry of
(S,Z)-2 was confirmed by the value of the coupling constant of
the doublet (d, JH–H = 11.4 Hz) corresponding to the olefinic pro-
c
3
ton CH@CH–COOMe at 5.78 ppm.
The (R,Z)-enantiomer of (S,Z)-2 was synthesized as above from
(S)-Garner’s aldehyde for comparative enantiomeric excess deter-
mination by chiral HPLC. In the HPLC chromatogram, the R-enan-
tiomer eluted before the S-enantiomer (tR = 7.8 min and 8.5 min,
respectively). The ee found for (S,Z)-2 was 94% and its optical rota-
8 as an analogue of (S)-homoserine-c-phosphate. Secondly, we de-
velop the synthesis of (S,Z)-APPA containing phosphonate antibiot-
ics (RZ-A and PB-A) in LPPS. Finally their biological evaluation is
done.
tion was measured as ½a D25
ꢁ
ꢀ27.3 (c = 0.91, CHCl3). Lit22, ½a D23
ꢁ
ꢀ32.6; (c = 1.4, CHCl3). For the (R,Z) enantiomer , ee = 96% and
the measured ½a 2D5
ꢁ
+28.5 (c = 1.03, CHCl3).
2. Chemical results and discussion
Having accomplish the stereo and enantioselective synthesis of
compound (S,Z)-2, the second step was the chemoselective reduc-
tion of its ester function to produce the corresponding (S,Z)-allyl
alcohol 3 using 3 equiv of DIBAL-H solution at ꢀ78 °C.22 Under
these conditions, 3 was obtained as a colorless oil with an excel-
2.1. Synthesis of (S,Z)-2-amino-5-phosphono-3-pentenoic acid:
(S,Z)-APPA 8
The natural (S,Z)-APPA 8 was isolated and characterized for the
first time by Park et al. as a constituent of PBs.13 In 1988, an at-
tempt for a chemical synthesis of this compound in order to assem-
ble the antibiotics PB-A and B was done by Natchev.19 However,
according to Fredenhagen’s observation,12 and based on the start-
ing material (E-4-bromocrotonaldehyde) used in this synthesis,
Natchev has probably synthesized the trans isomer (E)-APPA in
place of the required (Z)-APPA.
lent yield (90%) without the need of
purification.
a chromatographic
At the third step, (S,Z)-allyl alcohol 3 was converted to the cor-
responding (S,Z)-allyl bromide 4. Therefore, the alcohol 3 was con-
verted into the corresponding mesylate using MsCl and Et3N in
CH2Cl2 and in one flask manner, the mesylate was subjected in a
SN2 displacement using LiBr23 to furnish allyl bromide 4 as a color-
less oil. However, with our substrate, this reaction was not repro-
ducible and only moderated yields were obtained (60–70%). In
order to increase the yield of the reaction, we next tried the Appel
reaction by reacting allyl alcohol 3 with CBr4 (1.3 equiv) and PPh3
(1.5 equiv) in CH2Cl2 at 0 °C.24 Under these conditions, (S,Z)-allyl
bromide 4 was obtained as a colorless oil after a simple flash chro-
matography, with an excellent yield (93%).
The second key step to obtain (S,Z)-APPA 8 was the formation of
the phosphorous carbon bond using the classical Michaelis–Arbu-
zov reaction25 by heating (S,Z)-allyl bromide 4 in an excess of trim-
ethylphosphite P(OMe)3 at 80–100 °C. Under these conditions, the
reaction was complete after 6 h. The excess of P(OMe)3 was evap-
orated and the crude product purified by flash chromatography to
yield the oxazolidine phosphonate 5 in 82%. The expected mass of
phosphonate 5 was confirmed by HRMS. The 1D and 2D NMR spec-
tra confirmed also its structure (Supplementary data). It is impor-
tant to note that in the 1H decoupled 31P NMR spectrum, two peaks
Here, in order to overcome this stereoselectivity problem, we
planned to synthesize (S,Z)-APPA 8 in seven steps as shown in
(Scheme 1). The first key step was the stereo- and enantioselective
synthesis of the (S,Z)-2 ester starting from (R)-Garner’s aldehyde
120 and Still–Gennari reagent21 by following the protocol previ-
ously published by Shimamoto and Ohfune.22 Therefore, (CF3CH2-
O)2P(@O)CH2CO2Me was reacted with NaH at 0 °C in THF in
order to form the corresponding phosphonate anion in situ. To this
solution cooled at ꢀ78 °C was added 18-Crown-6 (5 equiv) and (R)-
aldehyde 1 (1 equiv) in THF at ꢀ78 °C.
After standard work up, the crude product was analyzed by 1H
NMR and according to the olefin proton integration, a mixture of
(S,Z)-2 ester and its (S,E) isomer was obtained with high Z-selectiv-
ity (Z/E >95/5). The two stereo isomers were separated by silica gel
column chromatography and pure (S,Z)-2 was obtained as a white
solid with good yield (82%). Finally, the structure of (S,Z)-2 and its
(E)-isomer was confirmed by 1D NMR (1H, 13C, DEPT) and 2D NMR
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M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
O
OH
COOMe
Boc
(b)
Boc
(a)
N
Boc
N
N
O
82 %
O
90%
O
( )-Garner's aldehyde
R
3
(S,Z)-2
1
(c)
93%
MeO
MeO
P
OMe
OMe
P
Br
Boc
O
O
N
Boc
(e)
Boc
(d)
N
N
H
O
79%
O
OH
82%
6
5
4
(f) 50%
HO
5 P
HO
OH
OH
OH
4
P
3
(h)
MeO
O
O
OMe
(g)
Fmoc
P
OH
1
N
H
H2N
2
69%
O
O
Boc
O
O
80%
N
H
(S,Z)-APPA
Fmoc-(S,Z)-APPA
OMe
8
9
7
Scheme 1. Seven steps synthesis of (S,Z)-APPA 8 starting with (R)-Garner’s aldehyde. Reagents and conditions: (a) (CF3CH2O)2P(@O)CH2CO2Me, 18-Crown-6, NaH, THF,
ꢀ78 °C; (b) DIBAL-H 1 M sol. in hexanes, toluene, ꢀ78 °C; (c) CBr4, PPh3, CH2Cl2, 0 °C; (d) P(OMe)3, 80–100 °C; (e) Dowex 50WX4 H+, MeOH/H2O:(9/1); (f) (i) H5IO6/CrO3,
CH3CN/H2O:(99/1), 0 °C; (ii) CH3I, K2CO3, CH3CN; (g) (i) 10% (CH3)3SiBr in CH2Cl2, and then MeOH/H2O:(9/1); (ii) 1 N LiOH, THF/H2O:(4/1); (h) Fmoc-OSu, NaHCO3, H2O/
acetone:(50/50).
with the same intensity were observed at 30.08 and 29.29 ppm be-
cause of the presence of two rotamers. After characterization, the
synthesis of 5 was scaled up to grams.
steps of its aqueous solution with CH2Cl2. We obtained the target
(S,Z)-APPA 8 as a white solid in good yield (80%) for the two steps
of deprotection followed by lyophilization.
As shown in Scheme 1, the fifth step was the opening of the oxa-
zolidine ring of phosphonate 5 to provide the alcohol 6 without re-
moval of the N-Boc-protective group. Thus, Dowex 50WX4 H+ resin
was used to achieve this selective ring opening in MeOH/H2O:(9/
1)26 and under these conditions Boc-phosphono amino alcohol 6
was obtained as a colorless oil after column chromatography
(79% yield). The structure of 6 was confirmed by 1D and 2D NMR
spectra. We observed that the 1H decoupled 31P NMR spectrum
of 6 gave a single peak at 30.14 ppm. This indicated that the phe-
nomenon of rotamers was suppressed with the opening of the oxa-
zolidine ring.
The alcohol 6 was then oxidized to the corresponding acid using
Zhao’s reagent.27,28 First, we tried a deprotection of the crude acid
using 10% (CH3)3SiBr in CH2Cl2 in order to form the target molecule
(S,Z)-APPA 8 directly. However, under these conditions, a red com-
plex mixture was obtained, probably because of the interaction be-
tween (CH3)3SiBr and the free carboxylic acid function. To
overcome this problem, we decided to convert the crude acid into
the corresponding methyl ester 7 using excess of CH3I in the pres-
ence of K2CO3; before deprotection. Pure methyl ester 7 was ob-
tained in yield of 50% as colorless oil after column
chromatography and was characterized by 1D and 2D NMR. The
1H decoupled 31P NMR spectrum of 7 gave a single peak at
28.99 ppm.
The expected mass of (S,Z)-APPA 8 was confirmed by LRMS and
HRMS. The 1H and 13C NMR spectra assignments were in agree-
ment with those of Z-APPA isolated by acidic hydrolysis of natural
PBs13 or RZs11,12 and were different from the 1H and 13C NMR spec-
tra of its trans isomer (S,E)-APPA published in the literature.30,31
The 1H NMR and COSY 1H–1H spectra of 8 in D2O showed the
olefinic protons at 6.04–5.93 ppm (m, 1H) for H-4 and at
3
5.64 ppm (td, JH–H = 10.3 Hz) for H-3, respectively. This value of
coupling constant between the olefinic protons of 8 indicated a
3
(Z) configuration. The H-2 signal appears at 4.75 ppm (d, JH–H
=
10.2 Hz) and the typical signal corresponding to CH2-P was a broad
multiplet that extends from 2.82 to 2.55 ppm. The 1H decoupled
31P NMR spectrum of 8 revealed a single signal at 19.20 ppm and
a transformation of this signal to a triplet of triplet in the
1H-coupled 31P NMR spectrum was observed. The 31P–1H HSQC
spectrum confirmed the simultaneous coupling of the phosphorus
and H-3, H-4 and H-5 protons. This explains why the signal was a
triplet of triplet. Importantly, the typical large coupling constant
1JC–P = 127.0 Hz at 28.38 ppm confirmed the CH2–P bond.11,13 It is
important to note that in the NMR data, published by Park et al.13
and by Rapp et al.11, for Z-APPA obtained by acidic hydrolysis of
PBs and RZs, both purified from bacterial cultures, the same value
(1JC–P = 127.0 Hz) was obtained in the 13C NMR spectrum. Further-
more, the 13C, the HSQC and the DEPT NMR spectra of 8 in D2O
support the structure shown in Scheme 1. Unfortunately, the
1JC–P coupling constant for E-APPA isomer is not published. Thus,
comparison of Z-APPA 8 with its E-APPA isomer based on the value
Finally, a deprotection of ester 7 was performed in two steps.
First, the simultaneous cleavage of the phosphonate methyl group
and the Boc protective group was achieved with Me3SiBr (10% in
CH2Cl2).29 Secondly, the methyl ester was saponified by LiOH.
The excess of LiOH was neutralized by Dowex 50WX4 H+ and after
filtration of the resin, the product was purified by several washing
1
of JC–P was not possible.
We have solved the problem of detecting the (S,Z)-APPA 8 in the
conventional UV coupled HPLC by protecting its amine function
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4
M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
with the Fmoc chromophore to produce Fmoc-(S,Z)-APPA 9 (69%
yield) for enantiomeric excess determination. For comparison,
the Fmoc-(R,Z)-APPA enantiomer of 9 was also synthesized from
(S)-Garner’s aldehyde following the same seven steps as in
Scheme 1. Thus, chiral HPLC of the two enantiomers was done
using Astec Chirobiotic T column. In the HPLC chromatogram, the
Fmoc-(S,Z)-APPA eluted before the Fmoc-(R,Z)-APPA enantiomer
and the enantiomeric excess of Fmoc-(S,Z)-APPA 9 was ee = 90%. Fi-
tetrafluoroborate (TBTU)34,35 or 3-(Diethoxyphos-phoryloxy)-1,2,3-
benzotriazin-4(3H) one (DEPBT), a coupling agent for peptidol
synthesis36 were used. Thus, the reaction was made in THF/DMF
between Boc-(S)-Arg(Pbf)-OH 10 and freshly deprotected 11 in
presence of TBTU or DEPBT as coupling agents and KHCO3 as base.
After preparative HPLC purification, the protected dipeptidol 13
was obtained with a yield of 65% for TBTU and 50% for DEPBT
coupling reagents.
Oxidation of 13 to the corresponding carboxylic acid using
H5IO6/CrO3 and subsequent esterification of the crude acid with
CH3I in presence of K2CO3 gave the fully protected ester 14 in mod-
erate yield (51%) after preparative HPLC purification. In order to
improve the yield, we have adopted a second approach: Boc-(S)-
Arg(Pbf)-OH 10 was coupled under the same conditions as de-
scribed above with fleshly prepared phosphono-aminoester TFA
salt 12 using TBTU coupling agent. However, a mild increase in
yield (3%) was observed.
Finally, deprotection of 14 to the target RZ-A 15 was performed
in three steps: First, the two phosphonate methyl groups and Boc
where removed using Me3SiBr, followed by aqueous MeOH.
According to MS (ESI+) analysis, the Pbf moiety resisted to Me3SiBr
and was cleaved in a second step using TFA/CH2Cl2/TIS/H2O:(19/
19/1/1).37 Finally, saponification of the methyl ester was per-
formed using LiOH. The crude product was purified by semi-pre-
parative C18-RP-HPLC and was lyophilized to give RZ-A 15 in
moderate yield (47%). The obtained product was characterized
with HRMS (ESI+), 1D NMR (1H, 31P and 13C, APT) and 2D NMR
(1H–1H COSY, 1H–13C HSQC). These NMR data confirmed that we
nally, the measured optical rotation of Fmoc-(S,Z)-APPA 9 was ½a D25
ꢁ
ꢀ15.9 (c = 0.46, EtOAc).
2.2. Synthesis of Rhizocticin A: RZ-A 15
Scheme 2 shows the peptide coupling reactions used to obtain
RZ-A 15 = (S)-Arg-(S,Z)-APPA. Arylsulfonyl protecting groups are
the best blocking groups for guanidine side chain of arginine. Their
relative acid lability increases in this order: Tos < Mts < Mtr < Pmc
< Pbf < MIS.32 Today, the Pbf protective group displays the best
deprotection kinetics among the Arg derivatives commercially
available. For this reason, commercially obtained Boc-(S)-
Arg(Pbf)-OH 10 was used to form phosphono-oligopeptides 13
and 14 in liquid phase. The advantage of this compound is that
the Boc and Pbf protective groups could be removed simulta-
neously by TFA solutions at the step of deprotection.33
To obtain fully protected dipeptide 14, two approaches were
followed. In the first approach, phosphono-aminoalcohol 11
obtained after TFA deprotection of compound 5 was used. The cou-
pling reagent N,N,N0,N0-tetramethyl-O-(benzotriazol-1-yl) uronium
5
quant.
O
(a)
O
P
OMe
OMe
OMe
O
P
OMe
BocHN
N
CF3COO
H3N
H
OH
OH
13
HN
11
Pbf
51%
(c)
N
NH
(b)
65%
H
O
H
N
O
OMe
OMe
P
Boc
HN
OH
O
H
N
Boc
N
COOMe
H
14
Pbf
N
H
NH
HN
68%
(b)
(d)
47 %
Pbf
10
N
NH
H
O
P
OMe
OMe
COOMe
O
P
OH
CF3COO
H3N
O
OH
H2N
OH
N
H
12
O
quant.
(a)
15
HN
H2N
7
NH
RHIZOCTICIN A
Scheme 2. Synthesis of dipeptide RZ-A = (S)-Arg-(S,Z)-APPA 15. Reagents and conditions: (a) TFA/CH2Cl2:(50/50); (b) TBTU or DEPBT, KHCO3, THF/DMF:(4/1); (c) (i) H5IO6/
CrO3, CH3CN/H2O:(99/1), 0 °C; (ii) CH3I, K2CO3, CH3CN; (d) (i) 10% (CH3)3SiBr in CH2Cl2 and then MeOH/H2O:(9/1); (ii) TFA/CH2Cl2/TIS/H2O:(19/19/1/1); (iii) 1 N LiOH; THF/
H2O:(4/1).
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M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
have successfully synthesized the desired RZ-A 15 (Supplementary
data). However, we have not found in the literature complete NMR
spectra of RZ-A for comparison.
hydrolysis of the formed TMS ester with aq MeOH. Secondly, the
benzyl and methyl esters functions of the crude corresponding
phosphonic acid were saponified with LiOH. After purification by
washing the aqueous solution three times with CH2Cl2, PB-A 21
was obtained as a white solid in good yield (80%) for the two steps
of deprotection. The 1H decoupled 31P NMR of PB-A 21 showed one
single peak at 22.39 ppm and the HRMS confirmed its expected
mass. The 1H NMR spectrum of PB-A 21 in D2O (400 MHz) con-
firmed that the obtained product was the tripeptide Ala-Asp-(Z)-
APPA. This spectrum was identical with that of PB-A purified from
bacterial cultures and published by Park et al. in 1977.13 Addition-
ally, we have completed the characterization of the obtained PB-A
21 by 13C and 2D NMR spectra (COSY 1H–1H and HSQC 1H–13C). The
assignments confirmed the structure of PB-A 21 shown in Scheme 3
(see experimental section and Supplementary data).
2.3. Synthesis of Plumbemycin A: PB-A 21
The same methodology used for the synthesis of RZ-A 15 was
applied to prepare another (S,Z)-APPA containing molecule; the
PB-A = (S)-Ala-(S)-Asp-(S,Z)-APPA 21. The synthetic route to PB-A
21 is shown in Scheme 3. The first step of peptide coupling reaction
was carried out using orthogonally protected Boc-(S)-Asp (Bn)-OH
16 and (S,Z)-APPA methyl ester 12 as starting materials. In this
case, T3P (propanephosphonic anhydride)38–4117 was used as cou-
pling reagent in place of TBTU. In accordance to the literature, the
use of T3P allowed an easier work-up and generally, higher yields
were observed.
Thus, dipeptide 18 was synthesized by treating an equimolar
mixture of the acid Boc-(S)-Asp(Bn)-OH 16 and 12 in DMF with
1.5 equiv of T3P 17 in presence of 2.5 equiv of N,N-diisopropyleth-
ylamine (DIEA) as base. After completion and standard work-up,
flash chromatography was used to isolate dipeptide 18 in good
yield (80%). The LRMS-(ESI+) spectrum of 18 confirmed the ex-
pected mass. The Boc protective group of Asp amino acid in dipep-
tide 18 was then removed by TFA. The obtained crude salt 19 was
used as obtained in a second peptide coupling reaction with Boc-
(S)-Ala-OH under the same conditions as described above. After
flash chromatography purification, the fully protected tripeptide
20 was obtained in 77% yield from 18.
3. Biological evaluation of synthetic RZ-A 15 and PB-A 21
RZs and PBs share a common mechanism of biological action.
They use the oligopeptide transport system to enter inside the cell,
where they are hydrolyzed by oligopeptidases into the active (S,Z)-
APPA 8 moiety and Arg for RZ-A or Ala-Asp for PB-A. (S,Z)-APPA 8 is
an inhibitor of threonine synthase42 and subsequently interferes
with threonine synthesis in sensitive yeasts and bacteria. In previ-
ous publications, antimicrobial activity was demonstrated for RZs
and PBs purified in small quantities from B. subtilis and S. plumbeus
culture supernatants, respectively.13,43,44 Indeed, antimicrobial
activities of PB-B with concentrations higher than 10 lg/mL
Finally, the target PB-A 21 = (S)-Ala-(S)-Asp-(S,Z)-APPA was
obtained after one pot deprotection in two steps. First, the
phosphonate methyl groups and the Boc were simultaneously
removed by treating compound 20 with CH3SiBr, followed by
against Escherichia coli, Bacillus subtilis and Pseudomonas ovalis
were described.44 According to Park, PB-A has almost the same
activity as PB-B.13 Crude RZ-A was active against various fungal
strains like Candida albicans and Saccharomyces cerevisiae (MIC:
O
OMe
O
OMe
P
H
N
O
P
OMe
COOH
H
Boc
OMe
CF3COO
N
(a)
Boc
N
H
COOMe
+
COOMe
H3N
COOBn
80 %
COOBn
16
12
18
quantitative
(b)
O
O
O
P
OMe
P
P
O
P
CF3COO
H3N
O
O
OMe
O
N
H
COOMe
O
17 = T3P
COOBn
19
(c)
77%
O
OMe
O
OH
P
P
O
OMe
O
H
N
OH
(d)
H
N
BocHN
N
H
COOMe
H2N
N
COOH
H
80 %
O
O
COOBn
COOH
20
21
PLUMBEMYCIN A
Scheme 3. Synthesis of tripeptide PB-A = (S)-Ala-(S)-Asp-(S,Z)-APPA 21 Reagents and conditions: (a) T3P 17 50% wt in DMF, DIEA, DMF; (b) TFA/CH2Cl2:(50/50); (c) Boc-(S)-
Ala-OH, T3P 17 50% wt in DMF, DIEA, DMF; (d) (i) 10% (CH3)3SiBr in CH2Cl2 and then MeOH/H2O:(9/1); (ii) 1 N LiOH, THF/H2O:(4/1).
Please cite this article in press as: Gahungu, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.064

6
M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
0.35
l
g/mL).43 In the present study, similar experiments were car-
Industries LTD). For polar compounds, Astec Chirobiotic T chiral
column (25 cm ꢂ 4.6 mm, 5 m, Sigma–Aldrich/Supelco) was used.
ried out with RZ-A 15 and PB-A 21 using experimental procedures
validated previously with antifungal and antibacterial compounds
(namely mycosubtilin and amylolysin).45,46 However, no signifi-
cant antimicrobial activities were observed against selected fungal
and bacterial strains even with concentrations up to 1000 times of
the MICs described in literature for PBs13,44 and for RZs,43
respectively.
Kugler et al. has shown that Thr like amino acids and some oli-
gopeptides could have an antagonistic effect on the antibiotic
activity of RZ-A.43 However, this was demonstrated with relatively
high amino acid concentration. Indeed, the biological effect of RZ-A
has been observed by Kluger et al. on Malt Extract Medium that
contains free Thr and Thr-containing peptides. Nevertheless, MICs
of synthesized RZ-A 15 and PB-A 21 were determined as described
in the literature (i.e., on Malt Extract Medium43 and Stephenson–
Wetham44 medium for RZ-A and PB-A, respectively). In addition,
MIC determinations were also performed in media that do not con-
tain any amino acid (YNB and Stephenson–Wetham for RZs and
PBs, respectively) to avoid neutralization of the biological effect
by Thr or Thr-containing peptides, but no antimicrobial activities
were observed.
l
The 1D (1H, 13C, 31P, DEPT or APT) and 2D (COSY, HSQC, HMBC)
NMR spectra were recorded on Brucker 250 or 400 MHz apparatus.
Chemical shifts are expressed in parts per million (ppm) and the
peak shapes are denoted as follow: s = singlet, d = doublet,
dd = doublet of doublet, t = triplet, q = quadruplet, m = multiplet,
br = broad; Spectra were recorded as solution in various solvants
(CDCl3, D2O, MeOD) and those were used as internal references.48
The NMR spectra were analyzed using MestReNova software. High
Resolution Mass Spectra (HRMS) were determined using an ESI-FT-
ICR mass spectrometer (SolariX, Brucker) utilizing H2O/CH3CN:(50/
50) as eluent. Low Resolution Mass Spectra (LRMS) were obtained
on a Thermoquest Finnigan TSQ 7000 spectrometer, utilizing elec-
tron spray ionization (ESI) and H2O/CH3CN:(50/50) eluent system
with 0.1% HCOOH added to this solvent. All starting materials
and reagents were generally purchased from Aldrich, Acros, Epsi-
lon, VWR or IRIS companies and used as such.
5.1.2. (S,Z)-tert-Butyl 4-(3-methoxy-3-oxoprop-1-enyl)-2,2-
dimethyloxazolidine-3-carboxylate (S,Z)-2
To an oily suspension of NaH 60% (2.1 g, 52.5 mmol) in THF
(90 mL), at 0 °C, under nitrogen was added, dropwise, a solution
of (CF3CH2O)2P(O)CH2CO2Me (10 mL, 52.3 mmol) in THF (60 mL).
The solution was stirred at 0 °C for 30 min and then cooled to
ꢀ78 °C. To this solution was added 18-Crown-6 (69 g, 261 mmol)
4. Conclusion
In this work, we present an efficient synthetic route for the
preparation of (S,Z)-APPA 8 and its derivatives phosphono-oligo-
peptides Rhizocticin A 15 and Plumbemycin A 21. Unfortunately,
the biological experiments done in this preliminary study show
that these chemically synthesized compounds had no antimicro-
bial activity against selected fungi and bacteria. Despite these dis-
appointing results, threonine synthase remains an interesting
target for the development of new antibiotics. However, until
now only racemic preparations of Z- and E-APPA were used in
enzymatic studies with TS.16,47 The synthetic scheme developed
in this work can be used for the preparation of different enantio-
mers of Z- and E-APPA. A detailed kinetic study of these molecules
with TS in vitro can help to clarify the absolute configuration of
APPA in biological active compounds in the future. The active
forms of APPA can then be introduced in various peptides using
automated solid-phase peptide synthesis (SPPS) and resulting pep-
tides can be used for biological experiments.
in THF (150 mL) and then (R)-Garner’s aldehyde
1 (9.2 g,
40.2 mmol) in THF (20 mL). The reaction mixture was stirred at
the same temperature for 2 h and the reaction quenched with
aqueous saturated NH4Cl solution. The reaction mixture was ex-
tracted with Et2O, dried over MgSO4, and concentrated under vac-
uum. The crude residue was purified by column chromatography
(silica gel, hexanes/Et2O:(1/4)) to give product (S,Z)-2 as white so-
lid. Yield (9.39 g, 82%). TLC (hexane/Et2O:1/1): Rf = 0.71. HPLC
(CH3CN/H2O:50/50, 0.7 mL/min): tR = 9.5 min. 1H NMR (250 MHz,
3
CDCl3) d (ppm) 6.22 (m, 1H, CH@CH–COOMe), 5.78 (d, JH–H
=
=
3
11.4 Hz, 1H, CH@CH–COOMe), 5.34 (s, 1H, CH-a), 4.23 (t, JH–H
3
4
7.8 Hz, 1 H, CH2aO), 3.71 (dd, JH–H = 9.2 and JH–H = 3.1 Hz, 1H,
CH2bO), 3.65 (s, 3H, COOCH3), 1.57 (s, 3H, C-CH3), 1.50–1.22 (m,
12H, C-CH3 and [C(CH3)3] Boc). 13C NMR (63 MHz, CDCl3) d
(ppm) 166.25 (COOMe), 152.26 (CO Boc), 151.86/151.24 (rotamers,
CH@CH–COOMe), 119.67/119.08 (rotamers, CH@CH–COOMe),
94.42/93.89 (rotamers, C-(CH3)2), 80.54/79.92 (rotamers,
([C(CH3)3]Boc), 69.03/68.80 (rotamers, CH2O), 56.61/55.58 (rota-
5. Experimental section
5.1. Chemistry
mers, CH-a), 51.38 (COOCH3), 28.34 ([C(CH3)3] Boc), 27.32/26.62
(rotamers, C-CH3), 24.92/23.75 (rotamers, C-CH3). The enantiomeric
excess of (S,Z)-2 was determined by HPLC (Chiral OD-H column,
i-PrOH/hexane:5/95), tR = 7.8 min for (R) enantiomer and tR =
8.5 min for (S) enantiomer in the HPLC chromatogram. The ee for
5.1.1. General information
All reactions were monitored by thin layer chromatography
(TLC) and/or High Performance Liquid Chromatography (HPLC).
The TLC were carried out on pre-coated TLC sheets POLYGRAM
SIL G/UV254 from MACHEREY-NAGEL GmbH. Spots were visualized
with UV light or by spraying a mixture of EtOH/H2SO4/p-anisalde-
(S,Z)-2 was 94% and its ½a D25
ꢀ27.3 (c = 0.91, CHCl3). For (R,Z)-2
ꢁ
enantiomer the ee = 96% and the measured ½a D25
ꢁ
+28.5 (c = 1.03,
CHCl3). LRMS-(ESI+), m/z: 286.2 [M+H]+, 308.1 [M+Na]+.
5.1.3. (S,Z)-tert-Butyl 4-(3-hydroxyprop-1-enyl)-2,2-
hyde:(18/1/1) with 20 lL of acetic acid. The HPLC chain from
dimethyloxazolidine-3-carboxylate 3
WATERS was equipated with an UV-Detector (PDA Waters 996).
HPLC analysis of synthesized compounds and their semi-prepara-
tive purifications were performed on XTerra RP 18 columns:
To a stirred solution of the ester (S,Z)-2 (6.01 g, 21.1 mmol) in
toluene (90 mL) at ꢀ78 °C under nitrogen was added a 1 M hex-
anes solution of DIBAL-H (63 mL, 63 mmol). The reaction mixture
was stirred at ꢀ78 °C for 2 h and then quenched by dropwise addi-
tion of MeOH (14 mL). The mixture was warmed to room temper-
ature; then ice (9 g) was added. The reaction mixture was stirred
for 30 min. To the resulting white suspension was added MgSO4
(23 g). After stirring for 30 min, the mixture was first filtered
through a pad of MgSO4 and then through silica gel to eliminate
trace of aluminates. The filtrate was concentrated under vacuum
(4.6 ꢂ 150 mm, 3.5
Waters), respectively. Preparative HPLC purifications was con-
ducted on column of type NOVASEP LC50.500.VE150 (Ø
lm, Waters) and (7.8 ꢂ 300 mm, 7.0
lm,
a
50 mm) with Merck silica gel 60 (0.015–0.040 mm) under a pres-
sure of 50 bars. Routine column chromatography was performed
on Acros silica gel (0.060–0.200 mm). The enantiomeric excesses
of non polar compounds were determined by HPLC on OD-H chiral
column (Chiralpack, 4.6 ꢂ 250 mm,
5 lm, Daicel Chemical
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M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
to give pure alcohol 7 as colorless oil. Yield (4.88 g, 90%). TLC
(hexane/AcOEt: 80/20): Rf = 0.20. HPLC (CH3CN/H2O:50/50,
0.7 mL/min): tR = 5.1 min. 1H NMR (250 MHz, CDCl3) d (ppm) 5.81
(m, 1H, CH@CH–CH2OH), 5.51 (t, 3JH–H = 10.6 Hz, 1H, CH@CH–CH2-
5.1.6. (S,Z)-tert-Butyl 5-(dimethoxyphosphoryl)-1-hydroxypent-
3-en-2-ylcarbamate 6
Oxazolidine phosphonate 5 (1.34 g, 3.84 mmol) was dissolved
in 90% MeOH/H2O (10 mL). To this solution was added Dowex
50XW H+ resin (1.34 g) previously washed with MeOH and H2O.
The suspension was stirred over night at room temperature. After
this time, TLC and HPLC analysis showed that the starting material
had completely reacted. Filtration of the resin and removal of the
solvent under vacuum provide a product which was purified using
column chromatography (silica gel, CHCl3/MeOH: 90/10) to afford
pure 6 as a colorless oil. Yield (0.94 g, 79%). TLC (CHCl3/MeOH: 9/1),
Rf = 0.23. HPLC (CH3CN/H2O: 50/50, 0.7 mL/min): tR = 3.6 min. 1H
3
4
OH), 4.89 (m, 1H, CH-a), 4.41 (dd, JH–H = 12.7 and JH–H = 8.7 Hz,
3
4
1H, CH2aOH), 4.03 (dd, JH–H = 9.0 and JH–H = 5.9 Hz, 1H, CH2aO),
3
4
3.87 (dd, JH–H = 12.9 and JH–H = 5.7 Hz, 1H, CH2bOH), 3.68 (dd,
3JH–H = 9.0 and JH–H = 1.8 Hz, 1H, CH2bO), 1.54 (s, 3H, C-CH3),
4
1.49–1.37 (m, 12H, C-CH3 and [C(CH3)3]Boc). 13C NMR (63 MHz,
CDCl3)
d (ppm) 152.36 (CO, Boc), 130.87 (CH@CH–CH2OH),
130.23 (CH@CH–CH2OH), 93.57 (C-(CH3)2), 81.12 ([C(CH3)3]Boc),
68.01 (CH2O), 57.69 (CH2OH), 53.64 (CH-a), 28.51 [C(CH3)3], Boc),
27.68 (C-CH3), 24.84 (C-CH3). LRMS-(ESI+), m/z: 258.2 [M+H]+,
NMR (400 MHz, CDCl3) d (ppm) 5.57–5.41 (m, 2H, CH@CH), 5.29
280.0 [M+Na]+.
(d, JN-H = 7.4 Hz, 1H, NH), 4.31 (s, 1H, CH-
a
), 3.81 (s, 1H, CH2OH),
3
3
3
3.66 (d, JP–H = 1.4 Hz, 3H, P-OCH3), 3.63 (d, JP–H = 1.4 Hz, 3H, P-
OCH3), 3.52 (m, 2H, CH2OH), 2.85 (m, 1H, CH2a-P), 2.58 (m, 1H,
CH2b-P), 1.32 (s, 9H, [C(CH3)3], Boc). 13C NMR (63 MHz, CDCl3) d
(ppm) 155.80 (CO, Boc), 133.03 (d, JC–P = 14.4 Hz, CH@CH–CH2P),
120.71 (d, JC–P = 10.7 Hz, CH@CH–CH2P), 79.71 ([C(CH3)3], Boc)
5.1.4. (S,Z)-tert-Butyl 4-(3-bromoprop-1-enyl)-2,2-
dimethyloxazolidine-3-carboxylate 4
2
The allyl alcohol 3 (4.4 g, 17 mmol) was dissolved in CH2Cl2
(30 mL) and then stirred under nitrogen at 0 °C. To this stirring
solution were added solid CBr4 (6.70 g, 21.3 mmol) followed by
PPh3 (7.10 g, 25.5 mmol). After stirring for 20 min, TLC analysis
showed that the starting allyl alcohol 3 had completely reacted.
The reaction mixture was added to a saturated solution of aq.
NaHCO3 and extracted with CH2Cl2. The combined organic layer
was washed with brine, dried over MgSO4, filtered and evaporated
under vacuum. The residue containing allyl bromide and Ph3P@O as
by-product was purified by flash chromatography (silica gel, hex-
ane/AcOEt: 70/30) to give pure bromide 4 as colorless oil. Yield
(5.09 g, 93%). TLC (hexane/EtOAc: 80/20): Rf = 0.53. HPLC (CH3CN/
H2O: 50/50, 0.7 mL/min): tR = 13.3 min 1H NMR (250 MHz, CDCl3)
3
3
3
65.00 (CH2OH), 53.09 (d, JC–P = 6.9 Hz, P-OCH3), 52.90 (d, JC–P
= 6.8 Hz, P-OCH3). 50.14 (s, CH- ), 28.46 ([C(CH3)3] Boc), 25.25 (d,
a
1JC–P = 139.3 Hz, CH2-P). 31P NMR (101 MHz, CDCl3)
d (ppm)
30.14. ½a 2D5
ꢁ
ꢀ91.7 (c = 0.88, CHCl3). LRMS-(ESI+), m/z calculated
for C12H24NO6P: 309.1; m/z: 309.9 [M+H]+, 209.7 [M-Boc+H]+.
5.1.7. (S,Z)-Methyl 2-(tert-butoxycarbonylamino)-5-
(dimethoxyphosphoryl) pent-3-enoate 7
The oxidant reagent was prepared by dissolving H5IO6 (1.14 g,
5 mmol) and CrO3 (2.3 mg, 0.12 mmol) in a volume of 99% aqueous
CH3CN (11.4 mL). Amino alcohol 6 (436 mg, 1.41 mmol) was dis-
solved in 99 % aq CH3CN (4 mL), cooled to 0 °C and treated with
the oxidant reagent (4 mL) in dropwise manner (30 min). After
an additional stirring of 3 h at 0 °C, the mixture was added to a
solution of saturated aq NaCl and extracted with ethyl acetate.
The combined organic layer was dried over MgSO4, filtered and
evaporated under vacuum to give crude acid. This crude acid was
dissolved in CH3CN (5 mL), and to this solution were added
K2CO3 (0.40 g, 2.89 mmol) and CH3I (0.5 mL, 8 mmol) at room tem-
perature. The mixture was left to react for 6 h and then solvents
were evaporated under vacuum. The residue was dissolved in
AcOEt and washed with an aqueous saturated solution of NaHCO3
and then with brine. The organic layer was dried over MgSO4, fil-
tered and then evaporated. Subsequent purification by column
chromatography (silica gel, CHCl3/MeOH: 90/10) provided pure
phosphonoester 7 as a colorless oil. Yield (228 mg, 50%). TLC
(CHCl3/MeOH: 9/1), Rf = 0.47. HPLC (CH3CN/H2O: 50/50, 0.7 mL/
3
d (ppm) 5.80 (s, 1H, CH@CH–CH2Br), 5.57 (t, JH–H = 10.2 Hz, 1H,
CH@CH–CH2Br), 4.69 (s, 1H-
a
), 4.38 (s, 1H, CH2aBr), 4.11 (dd,
4
3JH–H = 8.9 and JH–H = 6.1 Hz, 1H, CH2aO), 3.91 (s, 1H, CH2bBr),
3
4
3.70 (dd, JH–H = 8.9 and JH–H = 2.6 Hz, 1H, CH2bO), 1.60–1.39 (m,
15H, C-(CH3)2 and ([C(CH3)3], Boc).
5.1.5. (S,Z)-tert-Butyl 4-(3-(dimethoxyphosphoryl) prop-1-
enyl)-2,2-dimethyloxazolidine-3-carboxylate 5
The obtained (S,Z)-allyl bromide 4 (4.90 g, 15.32 mmol) was
mixed with an excess of P(OMe)3 (5 mL) in a double necked
50 mL round bottomed flask attached to a condenser, with dry-
ing tube. The flask was heated at 100 °C and allowed to stir
for 8 h. After this time, TLC analysis (hexane/EtOAc: 70/30)
showed that the starting material 4 (Rf = 0.64) had completely
reacted and a new compound formed (Rf = 0). A vacuum was
used to remove the excess of P(OMe)3 from the solution and
the crude product was purified by flash column chromatography
(silica gel, 30% EtOAc/Hexane and then 10% MeOH/EtOAc) to
yield pure phosphonate 5 as a colorless oil. Yield (4.39 g, 82%).
TLC (AcOEt/MeOH: 1/9), Rf = 0.54. HPLC (CH3CN/H2O:40/60,
0.7 mL/min): tR = 7.5 min. 1H NMR (400 MHz, CDCl3) d (ppm)
5.50 (m, 1H, CH@CH–CH2P), 5.37 (s, 1H, CH@CH–CH2P), 4.51 (s,
min): tR = 5.3 min. 1H NMR (400 MHz, CDCl3) d (ppm) 5.71 (m,
3
1H, CH@CH–CH2P), 5.50 (m, 1H, CH@CH–CH2P), 5.36 (d, JN–H
=
3
7.4 Hz, 1H, NH)), 4.95 (t, JH–H = 7.3 Hz, 1H, CH-a), 3.74 (m, 3H,
COOCH3), 3.71 (s, 6H, P-(OCH3)2), 2.92–2.60 (m, 2H, CH2-P),
1.40 (s, 9H, [C(CH3)3] Boc). 13C NMR (101 MHz, CDCl3) d (ppm)
2
171.53 (COOMe), 155.01 (CO, Boc), 128.38 (d, JC–P = 14.7 Hz,
3
CH@CH–CH2P), 124.47 (d, JC–P = 10.5 Hz, CH@CH–CH2P), 80.18
0.49H, CH-a), 4.41 (s, 0.47H, CH-a), 3.96 (m, 1H, CH2aO), 3.63-
3.55 (m, 7H, CH2bO and [P-(OCH3)2]), 3.02–2.23 (m, 2H, CH2-P),
([C(CH3)3] Boc), 52.89 (P-OCH3), 52.82 (P-OCH3), 52.74 (COOCH3),
1
51.43 (CH-
a
), 28.35 ([C(CH3)3], Boc), 25.45 (d, JC–P = 140.2 Hz,
CH2-P). ½a 2D5
ꢁ
ꢀ57.8 (c = 1.00, CHCl3). LRMS-(ESI+); m/z: 337.8
1.44 (m, 3H, C-CH3), 1.36 (s, C-CH3), 1.30 (m, 9H, [C(CH3)3]-
Boc).13
C NMR (63 MHz, CDCl3) d (ppm) 151.87 (CO Boc),
[M+H]+, 237.8 [M+H-Boc]+, 697.2 [2 M+Na]+. 31P NMR (101 MHz,
134.98/133.71 (rotamers, CH@CH–CH2P), 119.51/117.93 (rota-
mers, CH@CH–CH2P), 94.17/93.47 (rotamers, C-(CH3)2), 79.95
CDCl3) d = 28.99 ppm.
([C(CH3)3]Boc), 68.31 (rotamers, CH2O), 54.20 (CH-a), 52.90/
5.1.8. (S,Z)-2-amino-5-phosphonopent-3-enoic acid: (S,Z)-APPA
8
52.49 (2C, [P-(OCH3)2]), 28.45 (3C, [C(CH3)3], Boc), 27.42/26.49
(rotamers, C-CH3), 24.79/23.59 (rotamers, C-CH3), 24.71 (d,
1JC–P = 142 Hz, CH2P). 31P NMR (101 MHz, CDCl3) d (ppm) 30.08/
29.29 (rotamers). LRMS-(ESI+), m/z: 350.1 [M+H]+, 372.0
[M+Na]+. HRMS (ESI+), m/z calculated for C15H29NO6P [M+H]+
350.1727. Found 350.1725.
Fully protected phosphono ester 7 (102 mg, 0.3 mmol) was
dissolved in dried CH2Cl2 (1.8 mL) and TMSBr (0.2 mL) was added
at 0 °C under nitrogen. The solution was allowed to stir overnight.
The solvent and volatiles were evaporated under reduced pres-
sure. The residue was dissolved in 90% aq MeOH and the solution
Please cite this article in press as: Gahungu, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.064

8
M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
was allowed to stir for 1 h to ensure hydrolysis. The solvents
were concentrated under vacuum and the residue co-evaporated
with methanol and diethyl ether until a solid was obtained. This
solid was dissolved in H2O/THF:1/4 (2 mL) and then aq solution of
LiOH (1 M, 0.5 mL) was added and the reaction mixture stirred at
room temperature until the reaction was finished (HPLC monitor-
ing, 3 h).The mixture was neutralized with Dowex, 50W ꢂ 4 H+
resin, filtrated and evaporated to dryness. The residue was dis-
solved in water and pured in a funnel and washed with CH2Cl2
to yield the target compound (S,Z)-APPA 8 as a white solid after
lyophilisation. Yield (47 mg, 80%). TLC (n-Butanol/pyridine/
AcOH/H2O:15/12/3/10), Rf = 0.24. HPLC (gradient 100% CH3CN to
50 % H2O (0.1% TFA), 30 min, flow: 0.7 mL/min): tR = 12.4 min.
1H NMR (400 MHz, D2O): d(ppm) = 6.03 (m, 1H, CH@CH–CH2P),
AcOEt/MeOH: 90/10) to provide the dipeptidol 13 as a white solid.
Yield (1.87 g, 65%). TLC (AcOEt/MeOH: 1/9), Rf = 0.54. HPLC (CH3-
CN/H2O: 50/50, 0.7 mL/min): tR = 5.0 min. 1H NMR (400 MHz,
3
CDCl3) d (ppm) 7.43 (d, JN–H = 7.7 Hz, 1H, NH APPA ol), 6.37 (s,
3
3H, 3 ꢂ NH guanidine), 5.90 (d, JN–H = 6.4 Hz, 1H, NH Arg), 5.70–
5.60 (m, 1H, CH@CH–CH2P), 5.60–5.48 (m, 1H, CH@CH–CH2P),
4.75 (s, 1H, CH-
a APPA-ol), 4.22–4.03 (m, 2H, CH-a Arg and CH2-
3
3
OH), 3.74 (d, JH–P = 2.0 Hz, 3H, P-O-CH3), 3.71 (d, JH–P = 1.9 Hz,
3H), P-O-CH3), 3.66 (d, JH–H = 5.1 Hz, 2H, CH2OH), 3.23 (s, 2H,
3
CH2-NH, Arg), 2.95 (s, 2H, CH2, Pbf), 2.84–2.68 (m, 2H, CH2-P),
2.57 (s, 3H, CH3, Pbf)), 2.50 (s, 3H, CH3, Pbf), 2.08 (s, 3H, CH3,
Pbf)), 1.80 (s, 1H, CH2b Arg), 1.67–1.53 (m, 3H, CH2b and CH2
-
c
Arg), 1.45 (s, 6H, 2 ꢂ CH3, Pbf), 1.39 (s, 9H, [C(CH3)3], Boc). 13C
NMR (63 MHz, CDCl3) d 172.79 (CO Arg), 158.74 (Ar C–O Pbf),
156.65 (CO Boc), 156.01 (C guanidine), 138.27 (Ar C-SO2 Pbf),
3
5.56 (m, 1H, CH@CH–CH2P) 4.65 (d, JH–H = 9.5 Hz, CH–CH@CH),
2.8–2.5 (m, 2H, CH2P). 13C NMR (101 MHz, CDCl3) d (ppm)
132.52 (Ar–C Pbf), 132.17 (Ar–C Pbf), 131.72 (d, JC–P = 14.3 Hz,
2
5
2
3
172.35 (d, JC–P = 1.5 Hz, CO), 131.79 (d, JC–P = 10.6 Hz, CH@CH–
CH@CH–CH2P), 124.63 (Ar–C Pbf), 120.97 (d, JC–P = 10.6 Hz,
3
4
CH2P), 122.82 (d, JC–P = 12.7 Hz, CH@CH–CH2P), 50.71 (d, JC–P
=
CH@CH–CH2P), 117.52 (Ar–C Pbf), 86.41 (C-(CH3)2 Pbf), 79.74
2.0 Hz, CH–CH@CH), 28.36 (d, JC–P = 127.0 Hz, CH2P). 31P NMR
(101 MHz, D2O): d (ppm) = 19.20 (s). LRMS-(ESIꢀ), m/z: 194.1
[MꢀH]ꢀ.
([C(CH3)3], Boc), 63.99 (CH2OH), 54.37 (CH-
(OCH3)2], 49.16 (CH- APPA), 43.19 (CH2 Pbf), 40.10 (CH2-NH
Arg), 29.66 (CH2-b Arg), 28.58 (C-(CH3)2 Pbf), 28.28 ([C(CH3)3]
a Arg), 52.95 [(2C, P-
1
a
1
Boc), 25.68 (CH2-
c Arg), 24.75 (d, JC–P = 138.6 Hz, CH2-P), 19.37
5.1.9. Fmoc-(S,Z)-APPA 9
(CH3 Pbf)), 18.02 (CH3 Pbf), 12.48 (CH3 Pbf). 31P NMR (101 MHz,
(S,Z)-APPA
8
(23 mg, 0.12 mmol) and NaHCO3 (40 mg,
CDCl3) d
30.17 (s). LRMS-(ESI+), m/z: 718.3 [M+H]+, 740.0
0.49 mmol) were dissolved in 2 mL of water under stirring. A solu-
tion of Fmoc-OSu (41 mg, 0.12 mmol) dissolved in 2 mL of acetone
was then added. After being stirred at room temperature over-
night, the reaction mixture was concentrated. The crude product
was dissolved with 1 N HCl (20 mL) and then extracted with AcOEt
(3 ꢂ 20 mL). The organic layers were combined and evaporated un-
der vacuum to give a crude product, which was purified by HPLC
using semi-preparative XTerra column (eluent: CH3CN/H2O+0.1%
TFA: 50/50, flow = 3 mL/min) to afford Fmoc-(S,Z)-APPA 9 as a
white solid. Yield (24 mg, 69%). TLC (CHCl3/MeOH: 9/1), Rf = 0.59.
HPLC (CH3CN/H2O +0.1% TFA: 50/50, 0.7 mL/min): tR = 7.6 min. 1H
[M+Na]+. HRMS (ESI+), m/z calculated for C31H53N5O10PS [M+H]+,
718.3245. Found 718.3244.
5.1.11. Boc-(S)-Arg(Pbf)-(S,Z)-APPA-ester 14
5.1.11.1. Method A.
Boc-(S)-Arg(Pbf)-(S,Z)-APPA-ol 13
(360 mg, 0.50 mmol) was dissolved in CH3CN (3 mL) cooled to
0 °C and the H5IO6/CrO3 oxidant (3 mL) was added dropwise
(30 min). After an additional stirring of 3 h at 0 °C, the mixture
was added to a saturated solution aq NaCl and extracted with ethyl
acetate. The combined organic layers were evaporated under vac-
cum to give crude acid. This crude acid was dissolved in CH3CN
(5 mL) and to this solution were added K2CO3 (140 mg, 1.01 mmol)
and CH3I (0.5 mL) at room temperature. The mixture was left to re-
act for 6 h and then solvents were evaporated. The residue was dis-
solved in ethyl acetate and washed with saturated aqueous
NaHCO3. The two phases were separated and the aqueous phase
extracted with ethyl acetate two times. The combined organic lay-
ers were dried over MgSO4, filtered and evaporated under vaccum
to give a yellowish solid which was purified by preparative HPLC
(silica gel, AcOEt/MeOH: 90/10) to provide compound 14 as a yel-
lowish solid. Yield (192 mg, 51%).
3
NMR (250 MHz, MeOD) d (ppm) 7.74 (d, JH–H = 7.3 Hz, 2H Ar
Fmoc), 7.66–7.55 (m, 2H, Ar, Fmoc), 7.30 (m, 4H, Ar, Fmoc), 5.89–
5.69 (m, 1H, CH@CH–CH2P), 5.58 (m, 1H, CH@CH–CH2P), 5.03 (d,
3JH–H = 9.5 Hz, 1H, CH-
a), 4.31 (m, 2H, CH2O Fmoc), 4.19 (m, 1H,
CH–CH2O, Fmoc), 2.85–2.55 (br m, 2H, CH2-P). 31P NMR
(101 MHz, MeOD) d 24.35 (s). ½a D25
ꢀ15.9 (c = 0.46, AcOEt). LRMS-
ꢁ
(ESI+), m/z: 418.1 [M+H]+. HRMS (ESIꢀ), m/z calculated for C20H21-
NO7P [MꢀH]ꢀ, 416.0899. Found 416.0904. The enantiomeric ex-
cesses of Fmoc-(S,Z)-APPA were determined by HPLC (Chiral
Astec Chirobiotic T column, MeOH/acetate buffer pH 4.5:20/80,
flow = 1 mL/min). The ee of Fmoc-(S,Z)-APPA was 90% with
tR = 4.6 min for the major (S) enantiomer and tR = 7.2 min for the
(R) enantiomer.
5.1.11.2. Method B.
Phosphono ester 7 (68 mg, 0.20 mmol)
was deprotected with a 50% TFA solution in CH2Cl2. Boc-(S)-
Arg(Pbf)-OH (105 mg, 0.2 mmol) and the obtained TFA salt methyl
ester 12 were coupled by following the procedure 5.1.9. Yield
(102 mg, 68%). TLC (CH2Cl2/MeOH: 1/9), Rf = 0.40. HPLC (CH3CN/
H2O+0.1% TFA:40/60, 0.7 mL/min): tR = 16.4 min 1H NMR
(400 MHz, CDCl3) d (ppm) 7.74 (s, 1H, NH APPA), 6.32 (s, 3H, NH
5.1.10. Boc-(S)-Arg(Pbf)-(S,Z)-APPA-ol 13
Phosphonate oxazolidine 5 (1.75 g, 5 mmol) was deprotected
with a 50% TFA solution in CH2Cl2 at room temperature. After
30 min, TLC analysis (AcOEt/MeOH: 9/1) indicated that the reac-
tion was finished. The solvents were evaporated and toluene
(10 mL) was added to form a TFA azeotrope and was removed un-
der vacuum. The obtained crude TFA salt 11 was then dissolved in
DMF (6 mL) and was added to a stirring solution of Boc-(S)-
Arg(Pbf)-OH 10 (2.11 g, 4 mmol) in THF (24 mL). To this solution
was added successively TBTU (1.29 g, 4 mmol), solid KHCO3
(0.80 g, 8 mmol) and the reaction mixture was left to turn for
18 h at room temperature. After this time, the solvents were re-
moved under vacuum. The crude product was dissolved with ethyl
acetate (100 mL) and washed successively with saturated aqueous
NaHCO3, 0.5 N HCl and brine. The organic layer was dried over
MgSO4, filtered and evaporated under vacuum to give an off-white
foam, which was further purified by preparative HPLC (silica gel,
guanidine), 5.69 (s, 3H, CH@CH + NH Arg), 5.13 (s, 1H, CH-
a APPA),
4.19 (m, 1H, CH- Arg), 3.73 (s, 3H, COOCH3), 3.70 (m, 6H, P-
a
(OCH3)2), 3.21 (s, 2H, CH2-NH Arg), 2.93 (s, 2H, CH2 Pbf), 2.82 (m,
2H, CH2-P), 2.55 (s, 3H, CH3 Pbf), 2.48 (s, 3H, CH3 Pbf), 2.06 (s,
3H, CH3 Pbf), 1.79 (s, 1H, CH2-b Arg), 1.55 (m, 3H, CH2-b Arg and
CH2-c Arg), 1.43 (s, 6H, C(CH3)2], Pbf), 1.38 (s, 9H, [C(CH3)3], Boc).
13C NMR (101 MHz, CDCl3) d (ppm) 172.35 (CO), 170.84 (CO),
158.64 (Ar C–O Pbf)), 156.44 (CO Boc), 155.81 (C guanidine),
138.28 (Ar C-SO2 Pbf), 132.99 (Ar-C Pbf), 132.22 (Ar–C Pbf),
2
127.89 (d, JC–P = 14.2 Hz, CH@CH–CH2P), 124.53 (Ar–C Pbf),
3
124.28 (d, JC–P = 9.8 Hz, CH@CH–CH2P), 117.40 (Ar–C Pbf), 86.33
3
(C(CH3)2 Pbf), 79.87 ([C(CH3)3], Boc), 68.13 (CH), 53.07 (d, JH–P
=
3
1.4 Hz, P-OCH3), 53.00 (d, JH–P = 1.4 Hz, P-OCH3) 52.74 (OCH3),
Please cite this article in press as: Gahungu, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.064

M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
50.39 (CH-
a
APPA), 43.23 (CH2-NH Arg), 29.66 (CH2-b Arg), 28.58
Arg),
down to 0 °C and then T3P 50% wt in DMF, 0.37 mL, 0.62 mmol)
was added dropwise over 20 min. Then the reaction mixture was
stirred for 24 h at room temperature. The reaction was quenched
with water (20 mL) and then extracted with EtOAc (3 ꢂ 30 mL).
The organic layer was washed with saturated NaHCO3 (20 mL),
10% NaHSO4 (20 mL), saturated NaCl (30 mL), dried over MgSO4
and filtered before removing the solvent under vacuum. The crude
product was purified by flash chromatography (silica gel, 100%
EtOAc and then 10% MeOH/EtOAc) to yield the tripeptide 20 as a
white solid. Yield (202 mg, 81%). TLC (CHCl3/MeOH: 9/1),
Rf = 0.47. HPLC (CH3CN/H2O: 40/60, 0.7 mL/min): tR = 9.3 min. 1H
([C(CH3)3], Boc), 28.28 (C(CH3)2 Pbf), 25.81 (s), 25.24 (CH2-
c
1
25.11 (d, JC–P = 140.39 Hz, CH2P), 19.28 (CH3 Pbf), 18.83 (CH3),
17.92 (CH3 Pbf), 12.45 (CH3 Pbf). LRMS-(ESI+); m/z: 746.4 [M+H]+,
768.3 [M+Na]+. HRMS (ESI+), m/z calculated for C32H53N5O11PS
[M+H]+, 746.3194. Found 746.3194.
5.1.12. RZ-A = (S)-Arg-(S,Z)-APPA 15
RZ-A 15 was obtained in three steps as follow: (1°) Boc-(S)-
Arg(Pbf)-(S,Z)-APPA methyl ester dipeptide 14 (150 mg, 0.2 mmol)
was dissolved in dried CH2Cl2 (1.8 mL) and the solution was cold
down to 0 °C under nitrogen. Then, TMSBr (0.3 mL) was added
and the solution was allowed to stir 6 h at room temperature.
The volatiles were evaporated under reduced pressure and the res-
idue was dissolved in 90% aq MeOH (3 mL). The solution was al-
lowed to stir for 1 h to ensure hydrolysis and was concentrated.
(2°) The crude product was dissolved by TFA/CH2Cl2/TIS/H2O (9/
9/1/1, 4 mL) stirred 2 h at room temperatureto remove the Pbf
moiety and solvents were evaporated. (3°) The obtained crude
product was redissolved in H2O/THF:1/4 (2 mL) and then a solution
of LiOH (1 M, 0.5 mL) was added and the reaction mixture was stir-
red at room temperature until the reaction was finished (HPLC
monitoring). The mixture was neutralized with 0.2 N HCl, concen-
trated and purified on reverse phase semi preparative HPLC (C-18
Xterra column) using a gradient elution changed from 100% CH3CN
to 50% CH3CN/H2O +0.1% TFA: in 30 min, 3 mL/min yield 15 as a
white solid after lyophilisation . Yield (33 mg, 47%). HPLC (gradient
100% CH3CN to CH3CN/H2O +0.1% TFA: 50/50 for 30 min, 0.7 mL/
min):tR = 14.3 min. 1H NMR + COSY (400 MHz, D2O) d (ppm) 5.86
3
NMR (250 MHz, CDCl3) d 7.69 (d, JN–H = 7.6 Hz, 1H, NH APPA),
3
7.55 (s, 1H, NH Asp), 7.41 (d, JN–H = 3.4 Hz, 1H, NH Ala), 7.27 (s,
5H, Ar Bn), 5.66 (s, 2H, CH@CH–CH2P), 5.33 (s, 1H, NH), 5.05 (s,
3H, CH2O and CH-
a APPA), 4.84 (s, 1H, CH-a Asp), 4.10 (s, 1H,
CH- Ala), 3.68 (m, 9H, COOCH3 and P-(OCH3)2), 3.00 (m, 1H,
a
CH2-b Asp), 2.74 (m, 3H, CH2-b Asp and CH2P), 1.37 (s, 9H,
([C(CH3)3], Boc) 1.30 (m, 3H, CH3 Ala). 13C NMR (63 MHz, CDCl3)
d 172.91 (s), 171.30 (s), 170.57 (s), 169.99 (s), 155.54 (s), 135.44
(s), 128.50 (s), 128.25 (s), 128.21 (s), 128.10 (s), 127.87 (d,
J = 14.9 Hz), 124.96 (d, J = 12.1 Hz), 80.05 (s), 66.62 (s), 52.87 (t,
J = 6.7 Hz), 52.64 (s), 50.67 (s), 50.41 (d, J = 2.2 Hz), 49.10 (s),
1
35.74 (s), 28.27 (s), 25.48 (d, JC–P = 139.4 Hz), 18.24 (s), 18.24 (s),
15.22 (s). LRMS-(ESI+), m/z: 514 [M-Boc+H]+ 614.0 [M+H]+, 636.0
[M+Na]+ HRMS (ESI+), m/z calculated for C27H41N3O11P [M+H]+,
614.2473. Found 614.2468.
5.1.14. PB-A = (S)-Ala-(S)-Asp-(S,Z)-APPA 21
Boc-(S)-Ala-(S)-Asp(Bn)-(S,Z)-APPA methyl ester 20 (123 mg,
0.2 mmol) was dissolved in dried CH2Cl2 (1.8 mL) and the solution
was cold down to 0 °C under nitrogen. Then TMSBr (0.3 mL) was
added and the solution was allowed to stir 6 h at room tempera-
ture. The volatiles were evaporated under reduced pressure and
the residue was dissolved in 90% MeOH/H2O (3 mL). The solution
was allowed to stir for 1 h to ensure hydrolysis and 0.5 mL of a
1 N solution of LiOH in THF/H2O:4/1 (0.5 mmol) was added and
the reaction mixture stirred at room temperature until the reaction
was finished (6 h). The mixture was acidified with 0.2 N HCl
3
(m, 1H, CH@CH–CH2P), 5.64 (s, 1H, CH@CH–CH2P), 5.14 (t, JC–P
= 11.5 Hz, 1H, CH-
CH2-d Arg), 2.65 (dd, JH–H = 22.0 and JH–H = 8.0 Hz, 2H, CH2-P),
1.92–1.79 (m, 2H, CH2-b Arg), 1.75–1.49 (m, 2H, CH2-
Arg). 13C
NMR (63 MHz, D2O) d 173.67 (COOH APPA), 168.95 (CON Arg),
156.68 (C guanidine), 128.73 (d, JC–P = 10.7 Hz, CH@CH–CH2P),
a APPA), 3.98 (m, 1H, CH-a Arg), 3.16 (m, 2H,
3
4
c
2
3
124.41 (d, JC–P = 13.5 Hz, CH@CH–CH2P), 52.57 (CH-
a APPA),
1
50.89 CH-a Arg), 40.31 (CH2-d Arg), 28.04 (d, JC–P = 130.41 Hz,
CH2P) 27.01 (CH2-b Arg), 23.29 (CH2-
c
Arg). 31P NMR (101 MHz,
D2O) d (ppm) 21.98 (s). LRMS-(ESI+), m/z: 352.3 (M+H)+, HRMS
(ESI+), m/z calculated for C11H23N5O6P [M+H] +, 352.1380. Found,
352.1381
(20 mL), poured into a funnel and washed with CH2Cl2
(3 ꢂ 20 mL). The aqueous layer was concentrated under reduced
pressure and PB-A 21 was obtained as a white solid after lyophili-
sation. Yield (61 mg, 80%). HPLC (CH3CN/H2O +0.1% TFA): 50/50,
0.7 mL/min): tR = 2.3 min 1H NMR (400 MHz, D2O) d (ppm) 5.98
(m, 1H, CH@CH–CH2P), 5.76 (m, 1H, CH@CH–CH2P), 5.24 (d,
5.1.13. Tripeptide Boc-(S)-Ala-(S)-Asp (Bn)-(S,Z)-APPA methyl
ester 20
A solution of Boc-(S)-Asp (Bn)-OH 16 (165 mg, 0.51 mmol) and
the TFA salt of APPA methyl ester 12 (179 mg, 0.51 mmol) in DMF
(10 mL) was cooled in an ice bath before addition of DIEA
(0.175 mL, 2 mmol) under stirring. A solution of T3P 17 (50% wt
in DMF, 0.45 mL, 0.77 mmol) was then added slowly. The mixture
was removed from the cooling bath and allowed to stir overnight.
The reaction was quenched with water (30 mL), and then the aque-
ous phase was extracted three by EtOAc (3 ꢂ 30 mL) and water
(30 mL) and was stirred vigorously for 1 h. The two phases were
separated and the aqueous phase extract twice with EtOAc
(2 ꢂ 30 mL). The combined organic phases were washed with brine
(30 mL) and concentrated to afford the crude peptide, which was
purified by flash chromatography (silica gel, 100% EtOAc and then
10% MeOH in EtOAc) to yield dipeptide 18 as a yellowish solid.
Yield (221 mg, 80%). The expected mass for this compound was
3JH–H = 9.5 Hz, 1H, CH-
1H, CH-
a APPA), 4.91 (m, 1H, CH-a Asp), 4.26 (m,
a
Ala), 3.06–2.93 (m, 2H, CH2-b Asp), 2.86–2.66 (m, 2H,
CH2P), 1.63 (m, 3H, CH3 Ala). 13C NMR (63 MHz, CDCl3) d 174.15
(COOH APPA), 173.58 (COOH Asp), 171.46 (CON Asp), 170.60
2
(CON Ala), 128.04 (d, JC–P = 13.6 Hz, CH@CH–CH2P), 125.18 (d,
3JC–P = 10.2 Hz, CH@CH–CH2P), 50.99 (CH-
a
APPA), 49.60 (CH-
a
=
1
Asp), 49.23 (CH-
a
Ala), 36.42 (CH2-b Asp), 28.01 (d, JC–P
131.8 Hz, CH2P), 16.79 (CH3 Ala). 31P NMR (101 MHz, D2O) d
(ppm) 22.39 (s). LRMS-(ESI+), m/z: 382.1 [M+H]+. HRMS (ESIꢀ),
m/z calculated forC12H19N3O9P [MꢀH]ꢀ, 380.0864. Found
380.0867.
5.2. Biology
5.2.1. Antibacterial and antifungal tests in liquid media
confirmed by LRMS-(ESI+), m/z calculated for C24H35N2O10
P
Antifungal activity of RZ-A 15 was tested against Candida albi-
cans, Saccharomyces cerevisiae and Candida glabrata strains by a
microdilution method as previously described45,49 Briefly, indica-
tor strains were aerobically grown in 24-wells culture plates in
the presence of purified RZ-A 15 at concentrations ranging from
[M+H]+ 542.2; m/z: 543.1 [M+H]+, 565.0 [M+Na]+. The Boc
protective group of the obtained dipeptide 18 (221 mg, 0.41 mmol)
was deprotected with a 50% TFA solution in CH2Cl2 (6 mL) to
provide the TFA salt 19. This salt was dissolved in DMF (15 mL)
upon addition of solid Boc-(S)-Ala-OH (77 mg, 0.41 mmol) and
DIEA (0.14 mL, 0.82 mmol) under stirring. The solution was cooled
3.5
lg/L to 1050 lg/L in the appropriate culture medium. Each well
was seeded at an optical density at 600 nm (OD600) of 0.1. After
Please cite this article in press as: Gahungu, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.064

10
M. Gahungu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
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6. Clézardin, P. Bone 2011, 48, 71.
24 h of incubation under agitation at 28 °C, cell growth was
estimated by mean of OD600 measurements. Each experiment
was performed in triplicate in two complex medium namely:
YPD (10 g/L yeast extract, 20 g/L casein peptone, 20% dextrose)
and malt extract medium (20 g/L malt extract) as well as in two de-
fined medium: RPMI 1640 (Sigma–Aldrich) and YNB without ami-
no acids (Difco).
Similarly, antibacterial activity of PB-A 21 was tested against
Gram-positive (Bacillus subtilis, Listeria innocua) and Gram-nega-
tive bacteria (Escherichia coli, Pseudomonas aeruginosa). Cells were
grown in the presence of purified PB-A 21 at concentration ranging
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from 2 lg/L to 10 mg/L in the appropriate culture medium at 37 °C
for 24 h. Each experiment was performed in triplicate in two differ-
ent medium: LB (casein tryptone 10 g/L, yeast extract 5 g/L and
NaCl 10 g/L, agar 20 g/L) and Stephenson-Wetham medium (KH2-
PO4 1 g/L, MgSO4 7H2O 0.7 g/L, NaCl 1 g/L, (NH4)2HPO4 4 g/L, FeSO4
7H2O 0.03 g/L, glucose 5 g/L).
5.2.2. Antibacterial and antifungal tests on solid media
19. Natchev, A. I. Tetrahedron 1988, 44, 1511.
Antifungal and antimicrobial activity tests on plates were per-
formed as previously described by Halimi et al in 2010.46 Briefly,
sterile paper disks (6 mm diameter) containing different amounts
of RZ-A 15 or PB-A 21 were deposited on the surface of agar plates
containing culture medium previously seeded with the indicator
strains listed above. Antifungal and antibacterial activities were
estimated by measuring the diameter of the growth inhibition
zone obtained after 24 h of growth at 28 °C and 37 °C, respectively.
The same media (supplemented with 20 g/L agar), indicator strains
and concentrations of RZ-A 15 and PB-A 21 as those used for the
micro dilution method were used in agar diffusion tests.
20. Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361.
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The financial support from the Government of Burundi and
from the ULg’s PACODEL and ARD organizations is gratefully
acknowledged. Optical rotations were measured at the University
Henri Poincaré, Nancy-I and we thank Dr Françoise Chrétien for
this collaboration. The HRMS spectra and the elemental analyses
were carried out in the Mass Spectrometry Laboratory (GIGA Pro-
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Supplementary data
Supplementary data (1D NMR spectra: 1H, 31P, 13C, DEPT or APT
and 2D NMR spectra: 1H–1H COSY, 1H–13C HSQC of the key inter-
mediates and of all news compounds) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2013.06.064.
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Please cite this article in press as: Gahungu, M.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.06.064