Antifungal dipeptides incorporating an inhibitor of homoserine dehydrogenase

Article abstract of DOI:10.1002/psc.3060

The antifungal activity of 5-hydroxy-4-oxo-l-norvaline (HONV), exhibited under conditions mimicking human serum, may be improved upon incorporation of this amino acid into a dipeptide structure. Several HONV-containing dipeptides inhibited growth of human pathogenic yeasts of the Candida genus in the RPMI-1640 medium, with minimal inhibitory concentration values in the 32 to 64?μg?mL^?1 range. This activity was not affected by multidrug resistance that is caused by overexpression of genes encoding drug efflux proteins. The mechanism of antifungal action of HONV dipeptides involved uptake by the oligopeptide transport system, subsequent intracellular cleavage by cytosolic peptidases, and inhibition of homoserine dehydrogenase by the released HONV. The relative transport rates determined the anticandidal activity of HONV dipeptides.

Full text of DOI:10.1002/psc.3060

Received: 11 September 2017
DOI: 10.1002/psc.3060
Revised: 30 October 2017
Accepted: 13 November 2017
R E S E A R C H A R T I C L E
Antifungal dipeptides incorporating an inhibitor of
homoserine dehydrogenase
Andrzej S. Skwarecki1 Marta Schielmann2 Dorota Martynow2 Marcin Kawczyński1
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Aleksandra Wiśniewska1 Maria J. Milewska1 Sławomir Milewski2
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1 Department of Organic Chemistry, Gdańsk
University of Technology, 11/12 Narutowicza
The antifungal activity of 5‐hydroxy‐4‐oxo‐L‐norvaline (HONV), exhibited under conditions mim-
icking human serum, may be improved upon incorporation of this amino acid into a dipeptide
structure. Several HONV‐containing dipeptides inhibited growth of human pathogenic yeasts
of the Candida genus in the RPMI‐1640 medium, with minimal inhibitory concentration values
in the 32 to 64 μg mL−1 range. This activity was not affected by multidrug resistance that is
caused by overexpression of genes encoding drug efflux proteins. The mechanism of antifungal
action of HONV dipeptides involved uptake by the oligopeptide transport system, subsequent
intracellular cleavage by cytosolic peptidases, and inhibition of homoserine dehydrogenase by
the released HONV. The relative transport rates determined the anticandidal activity of HONV
dipeptides.
St, 80‐233 Gdańsk, Poland
2 Department of Pharmaceutical Technology
and Biochemistry, Gdańsk University of
Technology, 11/12 Narutowicza St, 80‐233
Gdańsk, Poland
Correspondence
Sławomir Milewski, Department of
Pharmaceutical Technology and Biochemistry,
Gdańsk University of Technology, 11/12
Narutowicza Str., 80‐233 Gdańsk, Poland.
Email: slamilew@pg.gda.pl
Funding information
National Science Centre, Grant/Award Num-
ber: UMO‐012/05/B/ST5/002910
KEYWORDS
antifungal agents, dipeptides, homoserine dehydrogenase, oligopeptide uptake
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1
INTRODUCTION
possible approaches to solve this problem could be the application of
the so‐called Trojan horse strategy, ie, conjugation of an enzyme inhib-
itor with a molecular nanocarrier.9 In the case of inhibitors of amino
acid structure, facilitated transport is possible after their incorporation
into oligopeptides, which are effectively taken up by oligopeptide per-
meases, demonstrating broad substrate specificity. Once internalized,
such oligopeptides are cleaved by intracellular peptidases to release
the inhibitor. Several examples of the successful application of this
strategy for the construction of antimicrobials have been reported in
the literature.10-13 Herein, we present results of our studies on synthe-
sis and biological properties of dipeptides incorporating HONV.
Several inhibitors of enzymes catalyzing particular steps of amino
acid biosynthesis pathways exhibit antifungal properties.1 One of
them is an antibiotic RI‐331 (5‐hydroxy‐4‐oxo‐L‐norvaline/2‐amino‐
5‐hydroxy‐4‐oxopentanoic acid, HONV), produced by Streptomyces
spp.2 This compound is active in vitro against several human
pathogenic yeasts and the plant pathogen Cladosporium fulvus but
has no effect against Aspergillus spp.3-5 Furthermore, RI‐331 was
shown to be effective in the treatment of systemic murine candidia-
sis, being well tolerated in mice.3,4 The molecular target of HONV is
homoserine dehydrogenase, the key enzyme in the fungi‐specific
biosynthetic pathway of L‐methionine, L‐isoleucine, and L‐threonine
biosynthesis.4-6 HONV acts as an enzyme‐assisted suicide inhibitor
of this enzyme.7 Interestingly, this compound also modulates a
glutathione‐related drug resistance of tumor cells.8
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2
RESULTS AND DISCUSSION
Rationale for the construction of
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2.1
Because homoserine dehydrogenase is a cytosolic enzyme, HONV
must be internalized, ie, has to cross the cytoplasmic membrane to
reach its target. The hydrophilic nature of its structure precludes fast
passive diffusion, so the only possibility is an active transport by one
of the amino acid permeases. These transporters, however, demon-
strate strict substrate specificity, usually limited to proteinogenic
amino acids. Therefore, the inefficient transport through the cytoplas-
mic membrane limits an antifungal activity of HONV. One of the
HONV‐containing dipeptides
The amino acid inhibitor of homoserine dehydrogenase (HONV;
Figure 1) was used for the construction of dipeptides as potential anti-
fungal agents. The rationale for the design of HONV‐containing
oligopeptides was based on the previous findings concerning the opti-
mization of structures of such compounds aimed at the maximization
of uptake by oligopeptide transport systems, namely, (1) a preference
J Pep Sci. 2018;24:e3060.
wileyonlinelibrary.com/journal/psc
Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.
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https://doi.org/10.1002/psc.3060

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SKWARECKI ET AL.
of powdered copper to give compound 4. Opening of the
1,3‐oxazolidin‐5‐one ring in 4 withTEA afforded N‐benzyloxycarbonyl
O‐acetyl HONV methyl ester 5, whereas treatment of 4 with HBr in
acetic acid gave HONV hydrobromide 1. Compound 5, after
deprotection of its amino functionality, was conjugated with one of
the N‐protected amino acid hydroxysuccinimide esters 6a‐h, to give
fully protected dipeptides 7a‐h. The final deprotection with 6M HCl
afforded hydrochlorides of dipeptides 8a‐h.
FIGURE 1 Structure of HONV
for dipeptides over the longer oligopeptides, (2) location of the
“warhead” as the C‐terminal amino acid, (3) aliphatic or aromatic amino
acids as N‐terminal components of a dipeptide.14-16 Following these
indications, 6 dipeptides with L‐alanine, L‐valine, L‐norvaline (Nva),
L‐leucine, L‐isoleucine, and L‐phenylalanine as the N‐terminal residues
were obtained. Additionally, Gly‐HONV and D‐Leu‐HONV were
synthesized and evaluated for comparative purposes.
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2.3
Antifungal in vitro activity of HONV dipeptides
Antifungal in vitro activity of HONV and 8 dipeptides containing this
compound as the C‐terminal amino acid was determined against
Candida albicans ATCC 10 231 cells in 3 growth media: the minimal
YNB medium containing ammonium sulfate as a nitrogen source, the
rich YPD medium, and the RPMI‐1640 medium recommended by Cen-
tral Laboratory Standards Institute (CLSI), the composition of which
mimics that of the low molecular weight compounds pool of human
serum. The determined minimal inhibitory concentration (MIC) values
are summarized in Table 1. Activity of HONV strongly depended on
growth medium composition. The lowest MIC value was noted in
YNB minimal medium, the 8‐fold higher one in rich YPD and the
highest one in RPMI‐1640. Dipeptides containing HONV were inactive
in YPD and demonstrated lower than RI‐331 activity in YNB, but 5 of
them (8b, 8d‐g) were more active than RI‐331 in RPMI‐1640. Dipep-
tide D‐Leu‐HONV (8h) was inactive in all 3 growth media.
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2.2
Chemical synthesis
The HONV‐containing dipeptides were synthesized by a multistep
procedure involving 2 main stages: synthesis of an appropriately
protected HONV and its conjugation with one of the N‐protected
amino acid hydroxysuccinimide esters, as shown in Scheme 1.
Synthesis of N‐benzyloxycarbonyl O‐acetyl HONV methyl ester 5
was performed from L‐aspartate as a starting substrate by the modified
procedure of Chang et al.17 Briefly, L‐aspartate after protection of its
α‐amino group with Z was converted into (S)‐3‐benzyloxycarbonyl‐4‐
methylcarboxy‐1,3‐oxazolidin‐5‐one 2 upon treatment with parafor-
maldehyde in the presence of p‐toluenesulfonic acid. Subsequent
treatment of 2 with oxalyl chloride, followed by the reaction with
diazomethane, afforded diazoketene 3. The diazo functionality was
exchanged for the O‐acetyl group derived from AcOH in the presence
The range of activity of HONV dipeptides in RPMI‐1640 medium
was appreciable (MICs in the 64 to >1024 μg mL−1 range), and the
SCHEME 1 Synthesis of HONV and
HONV‐containing dipeptides

SKWARECKI ET AL.
3 of 8
TABLE 1 Antifungal in vitro activity of HONV and its dipeptides
MIC, μg mL−1/mM
activity of Nva‐HONV and Leu‐HONV against Gu5 cells than against
Gu4 cells is additional evidence for the previously reported phenome-
non of enhanced susceptibility of yeasts overexpressing the ABC‐type
drug transporters to oligopeptidic antifungals.19 The C. albicans mutant
lacking oligopeptide permeases was resistant to HONV‐containing
dipeptides but not to HONV itself. The non‐albicans Candida strains
were generally more sensitive to dipeptides than to HONV, with
Candida parapsilosis being the least sensitive.
YNB‐AS
8/0.05
RPMI‐1640
YPD
64/0.44
HONV 1
256/1.74
>1024
Gly‐HONV 8a
Ala‐HONV 8b
Phe‐HONV 8c
Nva‐HONV 8d
Val‐HONV 8e
Ile‐HONV 8f
Leu‐HONV 8g
D‐Leu‐HONV 8h
256/1.25
64/0.29
>1024
>1024
128/0.58
512/1.74
64/0.26
64/0.26
128/0.5
64/0.25
>1024
>1024
>1024
64/0.26
32/0.13
128/0.5
32/0.12
1024/4.16
>1024
>1024
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>1024
2.4
Factors determining the activity of
>1024
>1024
HONV‐containing dipeptides
Minimal inhibitory concentration (MIC) values were determined against the
model Candida albicans ATCC 10 231 strain by a serial dilution method in 3
growth media. The values given correspond to 3 consistent results of
independent determinations.
The previous studies on mechanism of antifungal action of HONV
revealed that this amino acid is a strong inhibitor of homoserine
dehydrogenase, an enzyme catalyzing the key step in the fungi‐specific
biosynthetic pathway of amino acids of the aspartate family, namely,
L‐Met, L‐Ile, and L‐Thr.5 It is not surprising therefore that presence of
these amino acids in the growth medium prevents the growth
inhibitory effect of HONV, as shown in Table 3. The maximal
protection was provided by the mixture of 3 amino acids, but each of
them also exhibited some protective effect; the strongest one was
noted for L‐Met. The same phenomenon was observed for
HONV‐containing dipeptides, thus confirming that their antifungal
activity is a consequence of homoserine dehydrogenase inhibition by
the active component, ie, HONV. It is worth mentioning, therefore,
that the RPMI‐1640 medium contains the mixture of proteinogenic
amino acids, including L‐Met, L‐Ile, and L‐Thr at 0.1 to 0.33 mM
concentration level. This is probably too low to supplement deficiency
of these amino acids of biosynthetic origin in cells treated with HONV
or its dipeptides but enough to effectively compete with HONV for
respective amino acid permeases and thus prevent or strongly limit
its accumulation in these cells.
highest was found for 3 dipeptides containing Nva, Val, or Leu as the
N‐terminal amino acid residue (8d, 8e, and 8g; MIC = 64 μg mL−1).
A spectrum of anticandidal in vitro activity was determined for
dipeptides Nva‐HONV 8d and Leu‐HONV 8g and compared to that
of HONV 1. The set of yeasts of the Candida genus used in this study
comprised 6 C. albicans and 5 non‐albicans strains. The former included
C. albicans SC 5314, a mutant C. albicans PTR‐OPT strain lacking all
genes coding for di‐tripeptide and oligopeptide permeases of the
PTR and OPT families obtained from 5314 and 4 clinical strains: B3,
B4, Gu4, and Gu5. The Gu5 and B4 isolates are resistant to the
well‐known antifungal drug fluconazole (FLU), because of the
FLU‐induced overexpression of genes encoding multidrug efflux
pumps, CDR1 and/or CDR2 in the former and MDR1 in the latter.18
Their FLU‐sensitive counterparts, Gu4 and B3, respectively, exhibit a
basal expression of these resistance genes.
The MIC values determined in RPMI‐1640 medium are shown in
Table 2. Activity of HONV was generally low, with MICs = 256 or
512 μg mL−1. Presence of multidrug efflux pumps did not lower the
activity of either HONV or its dipeptides, thus suggesting that these
compounds are not substrates for the drug exporters. A slightly higher
The lack of activity of HONV‐containing dipeptides against the
C. albicans opt1‐opt5Δ ptr2Δ ptr22Δ mutant (Table 2) clearly indicates
that these compounds are transported to C. albicans cells by the
oligopeptide transport system, most probably by the di‐tripeptide
permeases Ptr2p and Ptr22p. Such an assumption is additionally
supported by the lack of activity of HONV‐containing dipeptides in
YPD medium. This medium contains an enzymatic digest of animal
protein (peptone), ie, a mixture of oligopeptides that effectively
compete with HONV dipeptides for the permease‐mediated transport,
thus preventing their intracellular accumulation, which is a prerequisite
for the possible interaction of HONV with its target.
TABLE 2 Anticandidal spectrum of HONV and its 2 dipeptides
MIC, μg mL−1/mM
HONV 1 Nva‐HONV 8d Leu‐HONV 8g
Candida albicans SC 5314 256/1.74
64/0.26
64/0.25
>1024
Candida albicans PTR‐OPT 256/1.74 >1024
Candida albicans Gu4
Candida albicans Gu5
Candida albicans B3
Candida albicans B4
Candida glabrata
256/1.74
256/1.74
256/1.74
256/1.74
256/1.74
256/1.74
512/3.48
512/3.48
256/1.74
64/0.26
32/0.13
64/0.26
64/0.26
64/0.26
32/0.13
128/0.52
32/0.13
64/0.26
64/0.25
32/0.12
64/0.25
64/0.25
32/0.12
64/0.25
128/0.5
64/0.25
64/0.25
The effectiveness of transport was assessed by determination of
the initial uptake rates of HONV or one of its dipeptides by C. albicans
ATCC 10 231 cells from buffered solutions. The pHs of buffers
(5.0 and 7.0) corresponded to those of the YNB‐AS and RPMI‐1640
medium, respectively. The uptake rates of HONV and the dipeptides
tested were constant for at least 15 to 20 minutes and then gradually
decreased. These properties allowed the determination of initial
uptake velocities. The rates were corrected for passive binding of the
compounds tested by measuring uptake in the presence of NaN3,
accounting for less than 3% of the initial rate of oligopeptide/amino
acid uptake.
Candida krusei
Candida parapsilosis
Candida tropicalis
Coriaria arborea
Minimal inhibitory concentration (MIC) values were determined by a serial
dilution method in RPMI‐1640 medium. The values given correspond to 3
consistent results of independent determinations.

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SKWARECKI ET AL.
TABLE 3 Influence of amino acids on anticandidal in vitro activity of HONV and its 2 representative dipeptides
MIC, μg mL−1
YNB‐AS
YNB‐AS + Met
YNB‐AS + Thr
YNB‐AS + Ile
YNB‐AS + (Met, Thr, Ile)
HONV 1
8
64
64
256
512
512
64
128
128
128
128
128
>1024
>1024
>1024
Ala‐HONV 8b
Nva‐HONV 8d
Minimal inhibitory concentration (MIC) values were determined against Candida albicans ATCC 10 231 by a serial dilution method in YNB‐AS medium. The
values given correspond to 3 consistent results of independent determinations.
TABLE 4 Uptake and cleavage rates of HONV dipeptides.
Uptake rate SD, nmol min−1 (mg dry weight)−1
Cleavage rate SD,
pH 5.0
pH 7.0
nmol min−1 (mg protein)−1
HONV 1
2.86 0.42
3.24 0.18
5.87 0.66
3.79 0.36
6.58 0.46
6.12 0.75
6.57 0.39
5.92 0.43
0.27 0.08
1.32 0.14
2.41 0.22
4.66 0.37
2.55 0.24
5.84 0.27
4.99 0.18
5.12 0.33
4.47 0.44
0.18 0.06
NA
Gly‐HONV 8a
Ala‐HONV 8b
Phe‐HONV 8c
Nva‐HONV 8d
Val‐HONV 8e
Ile‐HONV 8f
Leu‐HONV 8g
D‐Leu‐HONV 8h
14.5 0.56
12.3 0.82
13.3 0.87
16.4 1.44
18.9 1.83
15.1 0.44
17.6 1.33
0.5 0.04
Abbreviation: NA, not applicable.
Each value is the mean of 3 independent determinations standard deviation.
The results of the determination of the initial uptake rates are
presented inTable 4. The initial uptake velocities were generally higher
at pH 5.0 than at pH 7.0. This was expected, as amino acid and
oligopeptide permeases of C. albicans are ligand/H+ symporters,20,21
so their higher efficiency in acidic media is not surprising. All the dipep-
tides, except for D‐Leu‐HONV 8h, were taken up faster than HONV,
and generally, higher uptake rates were noted for dipeptides exhibiting
higher anticandidal in vitro activity (lower MIC values in Table 1).
The velocity of dipeptide cleavage by peptidases present in
cytosolic extracts prepared from C. albicans ATCC 10 231 cells was
also determined, and the data are presented in Table 4. Results of this
assay confirmed that all dipeptides but D‐Leu‐HONV 8h were rapidly
cleaved, with the rates in the 12.3 to 18.9 nmol min−1 (mg protein)−1
range. It is clear, therefore, that the cleavage rate is not a factor limiting
an anticandidal activity of HONV‐containing dipeptides. Conversely,
the uptake efficiency seems more important and confirms the previous
observations concerning other oligopeptide antifungals constructed
according to the “Trojan horse” strategy.16
targeting the homoserine dehydrogenase. Relative transport rates
determined the anticandidal activity of HONV dipeptides. It seems
that further optimization of the oligopeptide carrier for HONV might
be possible to get compounds exhibiting higher anticandidal activity
that the most active HONV dipeptides described in this work.
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4
EXPERIMENTAL
General
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4.1
All solvents and reagents were used as obtained from commercial
sources. 1H NMR and 13C NMR spectra were obtained at 500 and
125 MHz Varian Unity Plus spectrometers, and the deuterated
solvents were used as internal locks. Melting points were determined
on a melting point apparatus equipped with a thermometer and were
uncorrected. Column chromatography was performed with silica gel
(0.040‐0.063 mm) by using the indicated solvent systems. Optical
rotation was measured on an AUTOPOL V (Na D line) by using a
microcell of 10‐cm path length. High‐resolution mass spectra were
obtained using the Aqilent Technologies 6540 UHD Accurate – Mass
Q‐TOF LC/MS mass spectrometer.
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3
CONCLUSIONS
Incorporation of HONV into a dipeptide structure afforded com-
pounds demonstrating slightly higher than HONV itself anticandidal
in vitro activity in RPMI‐1640 medium, the composition of which
mimics that of the low molecular weight compounds pool of human
serum. The HONV‐containing dipeptides were transported to
C. albicans cells by oligopeptide permeases, possibly of the PTR sub-
family, and cleaved intracellularly by peptidases to release HONV,
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4.2
Synthesis
N‐Benzyloxycarbonyl‐L‐amino acids were prepared from L‐amino acids
according to the method of Bergmann and Zervas.22 N‐tert‐
Butoxycarbonyl‐L‐amino acids were prepared from L‐amino acids as
described.23,24 N‐Benzyloxycarbonyl‐ or N‐tert‐butoxycarbonyl‐L‐amino

SKWARECKI ET AL.
5 of 8
acid hydroxysuccinimide esters 6 were prepared from Z‐ or Boc‐L‐amino
acids following the methodology of Anderson et al.25
the resulting solution was washed with saturated NaHCO3 solution
and water. After being concentrated under vacuum, the yellow residue
was purified by flash chromatography, with hexane/ethyl acetate
7:2 v/v mixture as an eluent, to give 4 as a yellow oil (2.5 g, 50% yield).
1H NMR (CDCl3) δ:2.17 (s, 3H), 3.0 to 3.1 5(m, 1H), 3.30 to 3.40
(m, 1H), 4.35 to 4.38 (m, 1H), 4.60 to 4.65 (m, 2H), 5.10 to 5.25
(m, 2H), 5.4 (s, 1H), 5.45 to 5.60 (m, 1H), 7.35 to 7.40 (m, 5H).
MS‐ESI: m/z [M + H]+ found 335.1081. C16H17NO7 requires
335.1005.
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4.3
(S)‐3‐Benzyloxycarbonyl‐4‐methylcarboxy‐1,3‐
oxazolidin‐5‐one (2)
The mixture of N‐benzyloxycarbonyl‐L‐aspartic acid (0.1 mol, 26.7 g),
paraformaldehyde (0.2 mol; 6 g), and p‐toluenesulfonic acid (10 mmol,
1.9 g) in 200 mL of chloroform was heated at reflux for 2 hours, with
removal of water with a Dean‐Stark trap. After then, the solvent was
removed by evaporation, and the oily residue was dissolved in
100 mL of ethyl acetate, washed with saturated NaHCO3 solution
(2 × 10 mL) and water (2 × 10 mL), and dried over anhydrous MgSO4..
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4.6
Methyl (S)‐2‐benzyloxycarbonylamino‐5‐
acetoxy‐4‐oxopentanoate (5)
Oxazolidinone 4 (7.5 mmol, 2.5 g) was added to the solution of TEA
(25 mmol, 3.5 mL) in methanol (35 mL), at 0°C with stirring, under an
atmosphere of argon. After 1 hour, the mixture was warmed to room
temperature, and the stirring was continued for 12 hours. The solvent
and an excess of TEA were evaporated, to give the crude product 5.
The residue was dissolved in ethyl acetate (100 mL), and the resulting
solution was washed with 10% KHSO4 solution and water. After being
concentrated under vacuum, the yellow residue was purified by flash
chromatography, with hexane/ethyl acetate 2:3, v/v mixture as an
eluent, to give 5 as yellow crystals mp 65°C to 7°C; (1.5 g, 60% yield).
1H NMR (CDCl3) δ: 2.04 (s, 3H), 2.99 to 3.04 (dd, J = 4.4 and
18 Hz, 1H), 3.14 to 3.19 (dd, J = 4.4 and 18 Hz, 1H), 3.74 (s, 3H),
4.64 (s + m, 3H), 5.11 (s, 2H), 5.75 (d, 1H), 7.33 to 7.36 (m, 5H). MS‐ESI:
m/z [M + H]+ found 338.1197. C16H17NO7 requires 337.1162.
The solvent was evaporated to give
a colorless syrup, which
crystallized on standing overnight at 0°C. The final product was
crystallized from ethyl acetate to afford 24 g (90%) of 2; mp 82°C to
5°C, [a]20 + 120 (c3, MeOH). (lit. [a]20 + 125.7 (c3, MeOH); mp
D
D
82°C to 87°C.26
1H NMR (CDCl3, δ ppm): 3.05 to 3.10 (m, 1H), 3.30 to 3.40 (m,
1H), 4.36 to 4.40 (m, 1H), 5.10 to 5.25 (m, 2H), 5.30 to 5.35 (m, 1H),
5.40 to 5.45 (m, 1H), 7.35 to 7.45 (m, 5H); 13C NMR (CDCl3, δ ppm):
178.2, 175.1, 171.3, 152.8, 134.9, 128.6, 128.3, 78.2, 68.0, 51.4, 34.3.
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4.4
(S)‐3‐Benzyloxycarbonyl‐4‐(3‐diazo‐2‐
oxopropyl)‐1,3‐oxazolidin‐5‐one (3)
The mixture of (S)‐N‐benzyloxycarbonylo‐4‐carboxymethyl‐1,3‐
oxazolidin‐5‐one 2 (18 mmol, 5 g) and oxalyl chloride (10 mL) was
stirred for 15 hours at room temperature. An excess of chloride was
evaporated, hexane (10 mL) was added, and the mixture was evapo-
rated to dryness. This workup was repeated twice to remove the traces
of oxalyl chloride. The product, 5.4 g (100% yield), obtained as a color-
less oil, was dissolved in THF (20 mL, freshly distilled over LiAlH4) and
added dropwise to the ethereal solution of diazomethane, prepared
from N‐nitrozo‐N‐methylurea (0.15 mol, 16 g), maintaining the temper-
ature of the reaction mixture below −5°C. The mixture was then stirred
at 0°C for 10 hours under an atmosphere of argon. The resulting mix-
ture was evaporated under reduced pressure, and the yellow solid res-
idue obtained was purified by flash chromatography, with CHCl3 as an
eluent. After being concentrated under vacuum, the residue was crys-
tallized from ethyl acetate to give the diazoketene 3 as yellow crystals
(4.6 g, 85% yield); mp 94°C to 6°C; [α]D + 115.5 (c2, CHCl3).
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4.7
Methyl (S)‐2‐N‐[N‐alkoxycarbonyl‐2‐aminoacyl]
amino‐5‐acetoxy‐4‐oxopentanoate (7a‐h)
Compound 5 (500 mg, 1.5 mmol) was dissolved in 5 mL of HBr in
AcOH (45%), and the resulting solution was stirred for 1.5 hours at
room temperature. Removal of acetic acid by evaporation under
reduced pressure afforded a crude product as a bromide salt, which
was used in the next reaction without purification. The salt was
dissolved in 10 mL of THF, and 1 mL (7 mmol) of trimethylamine was
added. After 15 minutes of stirring at room temperature, an active
ester of one of the N‐protected amino acids 6a‐h (1.5 mmol) was
added as a solid, directly to the prepared solution. The mixture was
stirred at room temperature for 60 hours. After this time, the solvent
was removed under reduced pressure, and the residue was dissolved
in 10 mL of CHCl3 and washed twice with 10 mL of 10% aq. NaHSO4
and water. The organic layer was separated and dried under anhydrous
MgSO4. Subsequent filtration and evaporation of organic solvents
afforded the crude oily products, which were purified by column
chromatography (silica gel, hexane/ethyl acetate, 3:2 v/v).
1H NMR (CDCl3) δ:3.0 to 3.1 5(m, 1H), 3.30 to 3.40 (m, 1H), 4.33
to 4.35 (m, 1H), 5.05 to 5.20 (m, 3H), 5.25 to 5.35 (m, 1H), 5.40 to 5.55
(m, 2H), 7.35 to 7.40 (m, 5H).
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4.5
(S)‐3‐Benzyloxycarbonyl‐4‐(O‐acetyl‐3‐
hydroxy‐2‐oxopropyl)‐1,3‐oxazolidin‐5‐one (4)
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4.7.1
Methyl (S)‐2‐N‐[N‐tert‐butoxycarbonyl‐glycyl]
amino‐5‐acetoxy‐4‐oxopentanoate (7a)
The diazoketene 3 (15 mmol, 4.5 g) was dissolved in ethyl acetate
(175 mL), and acetic acid (17.5 mL) and powdered copper (14 mmol,
0.9 g) were added. The reaction mixture was stirred for 42 hours at
room temperature. The resulting suspension was filtered to remove
copper, and the remaining solution was evaporated to give the crude
product 4. The residue was dissolved in ethyl acetate (100 mL), and
Oil, 23% yield.
1H NMR (CDCl3) δ: 1.44 (s, 9H), 2.14 (s, 3H), 3.04 to 3.08 (dd, J = 4.4
and 18 Hz, 1H), 3.12 to 3.15 (dd, J = 4.4 and 18 Hz, 1H), 3.73 (s, 3H), 4.43
(m, 1H), 3.81 (s, 2H), 4.62 (s, 2H), 4.81 to 4.84 (m, 1H), 5.21 (br m, 1H),
7.03 (d, J = 7.8 Hz, 1H).

6 of 8
SKWARECKI ET AL.
|
18 Hz, 1H), 3.12 to 3.16 (dd, J = 4 and 18 Hz, 1H), 3.74 (s, 3H), 4.10
(m, 1H), 4.57 to 4.67 (AB, J = 17 Hz, 2H), 4.79 to 4.83 (m, 2H), 7.05
(d, J = 6.9 Hz, 1H).
4.7.2
Methyl (S)‐2‐N‐[N‐benzyloxycarbonyl‐(S)‐alanyl]
amino‐5‐acetoxy‐4‐oxopentanoate (7b)
White crystals mp 115°C to 8°C (CHCl3/hexane), 23% yield.
1H NMR (CDCl3) δ: 1.39 (d, J = 7.4 Hz, 3H), 2.14 (s, 3H), 3.04‐3.14
(AB, 2H), 3.73 (s, 3H), 4.24 (m, 1H), 4.62 (AB, 2H), 4.8 (br m, 1H), 5.11
(s, 2H), 5.4 (br m, 1H), 6.93 (d, J = 6.3 Hz, 1H), 7.29 to 7.36 (m, 5H).
|
4.8
(S)‐2‐N‐[2‐aminoacyl]amino‐5‐hydroxy‐4‐
oxopentanoic acid (8a‐h)
The protected dipeptide 7a‐h was dissolved in 10 mL of 6M HCl(aq)
,
|
4.7.3
Methyl (S)‐2‐N‐[N‐benzyloxycarbonyl‐(S)‐
and the solution was stirred at room temperature for 192 hours. After
this time, the mixture was concentrated under reduced pressure, and
the residue was purified by high‐performance liquid chromatography.
phenylalanyl]amino‐5‐acetoxy‐4‐oxopentanoate (7c)
White crystals mp 153°C to 5°C (CHCl3/hexane), 50% yield.
1H NMR (CDCl3) δ: 2.14 (s, 3H), 3.05 to 3.09 5 (m, 3H), 3.11 to 3.16
(dd, J = 6.3 and 16.2 Hz, 1H), 3.71 (s, 3H), 4.43 (m, 1H), 4.57 (s, 2H), 4.76
(m, 1H), 5.08 (s, 2H), 5.27 (d, J = 6.3 Hz, 1H), 6.77 (d, J = 6.8 Hz, 1H), 7.17
to 7.36 (m, 10H).
|
4.8.1
(S)‐2‐N‐glycylamino‐5‐hydroxy‐4‐oxopentanoic
acid (8a)
1H NMR (D2O) δ: 2.6 to 2.8 (m, 1H), 2.9 to 3 (m, 1H), 3.2 to 3.3 (m, 2H),
4.1 (t, J = 7 Hz, 1H), 4.25(m, 2H), 4.65 (m, 1H); 13C NMR (D2O) δ: 208,
175, 169, 69, 51, 42, 40. MS‐ESI: m/z [M + H]+ found 205.0657,
C7H12N2O5 requires 204.0746.
|
4.7.4
Methyl (S)‐2‐N‐[N‐tert‐butoxycarbonyl‐(S)‐norvalyl]
amino‐5‐acetoxy‐4‐oxopentanoate (7d)
Oil, 28% yield.
1H NMR (CDCl3) δ: 0.92 to 0.97 (dd, J = 7.3 and 14 Hz, 3H), 1.44
(s, 9H), 1.53 to 1.6 (m, 2H), 1.78 to 1.84 (m, 12), 2.16 (s, 3H), 3.04 to
3.08 (dd, J = 4.4 and 18 Hz, 1H), 3.12 to 3.16 (dd, J = 4.4 and 18 Hz,
1H), 3.74 (s, 3H), 4.07 (m, 1H), 4.64 (s, 2H), 4.80 to 4.83 (m, 1H),
5.06 (bs, 1H), 6.92 (d, J = 7.8 Hz, 1H).
|
4.8.2
(S)‐2‐N‐[(S)‐alanyl]amino‐5‐hydroxy‐4‐
oxopentanoic acid (8b)
1H NMR (D2O) δ: 1.4(d, J = 7 Hz, 3H), 2.6 to 2.8 (m, 1H), 2.9 to 3
(m, 1H), 3.9 (t, J = 6 Hz 1H), 4.18 (bs, 2H), 4.65 (m, 1H); 13C NMR
(D2O) δ: 207, 174, 173, 69, 52, 51.3, 43, 19.5. MS‐ESI: m/z [M + H]+
found 219.1012, C8H14N2O5 requires 218.0903.
|
4.7.5
Methyl (S)‐2‐N‐[N‐tert‐butoxycarbonyl‐(S)‐valyl]
amino‐5‐acetoxy‐4‐oxopentanoate (7e)
|
4.8.3
(S)‐2‐N‐[(S)‐phenylalanyl]amino‐5‐hydroxy‐4‐
Oil, 23% yield.
oxopentanoic acid (8c)
1H NMR (CDCl3) δ: 0.90 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz,
3H), 1.26 to 1.33 (m, 1H), 1.45 (s, 9H), 2.16 (s, 3H), 3.01 to 3.06
(dd, J = 4.4 and 18 Hz, 1H), 3.15 to 3.19 (dd, J = 4.4 and 18 Hz,
1H), 3.74 (s, 3H), 3.94 to 3.97 (m, 1H), 4.64 (s, 2H), 4.83 to 4.86
(m, 1H), 5.5 (d, J = 7.8 Hz, 1H), 6.81 (d, J = 6.8 Hz, 1H).
1H NMR (D2O) δ: 2.76 to 2.88 5 (m, 1H), 2.96 to 2.99 (m, 1H), 3.05
(AB, J = 7.3 Hz, 2H), 4.07 (t, J = 7.3 Hz, 1H), 4.14 (m, 2H), 4.54
(t, J = 5.6 Hz, 1H), 7.11 to 7.25 (m, 5H); 13C NMR (D2O) δ: 206, 175,
171, 137, 130, 128, 68, 55, 50.5, 44.5, 40. MS‐ESI: m/z [M + H]+ found
295.1195 C14H18N2O5 requires 294.1216.
|
4.7.6
Methyl (S)‐2‐N‐[N‐tert‐butoxycarbonyl‐(S)‐
|
4.8.4
(S)‐2‐N‐[(S)‐norvalyl]amino‐5‐hydroxy‐4‐
isoleucyl]amino‐5‐acetoxy‐4‐oxopentanoate (7f)
oxopentanoic acid (8d)
1H NMR (D2O) δ: 0.76 (m, 3H), 1.24 (m, 2H), 1.70 (m, 2H), 2.91
(bs, 2H), 3.89 (t, J = 6 Hz 1H), 4.18 (bs, 2H), 4.65 (m, 1H); 13C NMR
(D2O) δ: 208, 173.9, 170.1, 67.2, 53.0, 48.3, 38.9, 32.9, 17.6, 12.9.
White solid; mp 95°C to 7°C (CHCl3/hexane), 25% yield.
1H NMR (CDCl3) δ: 0.9 to 0.96 (m, 6H), 1.12 to 1.32 (m, 2H), 1.44
(s, 9H), 1.88 (m, 1H), 2.16 (s, 3H), 3.01 to 3.05 (dd, J = 3.9 and 18 Hz, 1H),
3.14 to 3.19 (dd, J = 3.9 and 18 Hz, 1H), 3.74 (s, 3H), 3.99 (m, 1H), 4.64
(s, 2H), 4.83 to 4.85 (m, 1H), 5.03 (br m, 1H), 6.8 (d, J = 6.5 Hz, 1H).
MS‐ESI: m/z [M
+
H]+ found 247.1245 C10H18N2O5 requires
246.1216.
|
4.7.7
Methyl (S)‐2‐N‐[N‐tert‐butoxycarbonyl‐(S)‐leucyl]
|
4.8.5
acid (8e)
(S)‐2‐N‐[(S)‐valyl]amino‐5‐hydroxy‐4‐oxopentanoic
amino‐5‐acetoxy‐4‐oxopentanoate (7g)
1H NMR (D2O) δ: 0.85 (d, J = 6.4 Hz, 3H), 0.86 (d, J = 6.4 Hz, 3H), 2.05
(octet, J = 6.4 Hz, 1H), 2.93 (bs, 2H), 3.63 (d, J = 5.4, 1H), 4.18 (bs, 2H),
4.68 (m, 1H); 13C NMR (D2O) δ: 208, 169.3, 67.2, 58.4, 48.3, 38.9,
30.1, 17.6, 16.9. MS‐ESI: m/z [M + H]+ found 247.2011; [M + Na]
269.2012. C10H18N2O5 requires 246.1216.
Oil, 25% yield.
1H NMR (CDCl3) δ: 0.93 to 0.96 (m, 6H), 1.42 to 1.49 (m, 1H),
1.44 (s, 9H), 1.62 to 1.69 (m, 2H), 2.15 (s, 3H), 3.04 to 3.14
(AB, 2H), 3.73 (s, 3H), 4.09 to 4.12 (m, 1H), 4.63 (s, 2H), 4.78 to
4.81 (m, 1H), 4.90 (d, J = 6.3 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H).
|
|
4.7.8
Methyl (S)‐2‐N‐[N‐tert‐butoxycarbonyl‐(R)‐leucyl]
4.8.6
(S)‐2‐N‐[(S)‐isoleucyl]amino‐5‐hydroxy‐4‐
amino‐5‐acetoxy‐4‐oxopentanoate (7h)
oxopentanoic acid (8f)
Oil, 23% yield.
1H NMR (D2O) δ: 0.8 (t, J = 7 Hz, 3H), 1.1 (d, J = 6.5 Hz, 3H), 1.25
(q, J = 7 Hz, 2H), 1.6 to 17 (m, 1H), 2.91 (bs, 2H), 3.65 (t, J = 6.7 Hz,
1H), 4.2 (bs, 2H), 4.8 (m, 1H); 13C NMR (D2O) δ: 206, 174, 170, 68,
1H NMR (CDCl3) δ: 0.93 to 0.95 (m, 6H), 1.44 to 1.49 (m, 1H), 1.44
(s, 9H), 1.65 to 1.67 (m, 2H), 2.15 (s, 3H), 3.01 to 3.05 (dd, J = 4.4 and

SKWARECKI ET AL.
7 of 8
58, 50, 36, 25, 15, 11.5. MS‐ESI: m/z [M + H]+ found 261.1448.
The 96‐well microtiter plates were also used for determination of
in vitro growth inhibitory activity in YPD and YNB‐based media.
Individual wells were inoculated with 5 × 103 cfu (colony forming
units) mL−1 of C. albicans cells from the overnight culture in YPD
medium. The inoculated plates were incubated 37°C for 24 hours,
and then turbidity was measured with a microplate reader at 531 nm,
as described above for the RPMI‐1640‐based assay.
C
11H20N2O5 requires 260.1372.
|
4.8.7
(S)‐2‐N‐[(S)‐leucyl]amino‐5‐hydroxy‐4‐
oxopentanoic acid (8g)
1H NMR (D2O) δ: 0.76 (d, J = 6.3 Hz, 3H), 0.77 (d, J = 6.3 Hz, 3H), 1.49
to 1.63 (m, 3H), 2.91 (bs, 2H), 3.82 (t, J = 6.7 Hz, 1H), 4.18 (bs, 2H), 4.62
(m, 1H); 13C NMR (D2O) δ: 206, 175, 174, 68, 53.5, 5044.542, 25, 23.
MS‐ESI: m/z [M
+
H]+ found 261.1291 C11H20N2O5 requires
|
260.1372.
5.3
Determination of peptide uptake rates
Candida albicans ATCC 10 231 cells grown exponentially in the
YNB‐AS medium were harvested by centrifugation (3000g, 5 min),
washed with 50mM potassium phosphate buffer (pH 5.0 or 6.5),
and suspended in the same buffer containing 1% glucose, to a final
cell density corresponding to A660 ≅ 1.0. The cell suspension was
incubated at 30°C. After 10 minutes, an HONV or dipeptide
solution was added, to give a final concentration of 100 μM. At
that moment and at 5‐minute intervals thereafter, 2‐mL samples
of the cell suspension were withdrawn, immediately filtered through
the Whatman GF/A filters, and the filtrates were used to determine
the residual amino acid/peptide concentration. Then, the 1‐mL
portions of the filtrates were taken and combined with 1.25‐mL
|
4.8.8
(S)‐2‐N‐[(R)‐leucyl]amino‐5‐hydroxy‐4‐
oxopentanoic acid (8h)
1H NMR (D2O) δ: 0.75 (d, J = 6.2 Hz, 3H), 0.79 (d, J = 6.2 Hz, 3H), 1.49
to 1.63 (m, 3H), 2.90 (bs, 2H), 3.84 (t, J = 6.7 Hz, 1H), 4.18 (bs, 2H), 4.62
(m, 1H). MS‐ESI: m/z [M + H]+ found 261.1611 C11H20N2O5 requires
260.1372.
|
4.9
(S)‐2‐amino‐5‐hydroxy‐4‐oxopentanoic acid (1)
Oxazolidinone 4 was dissolved in 10 mL of 6M HCl(aq), and the solution
was stirred at room temperature for 192 hours. After this time, the
mixture was concentrated under reduced pressure, and the residue
was purified by high‐performance liquid chromatography.
1H NMR (D2O) δ: 2.98 to 3.03 (dd, J = 6.5 and 18 Hz, 1H), 3.05 to
3.09 (dd, J = 4.4 and 18 Hz, 1H), 4.14 to 4.16 (dd, J = 4.4 and 6.3 Hz,
1H), 4.20 (s, 2H); 13C NMR (D2O) δ: 208.7, 169.8, 66.9, 53.9, 37.7.
MS‐ESI: m/z [M + H]+ found 148.0481 C5H9NO4 requires 147.0532.
aliquots of a solution containing 4% Na2B4O7
× 10 H2O and
0.8 mg mL−1 of 2,4,6‐trinitrobenzenesulfonate. The reaction was
performed at 37°C for 30 minutes. The A420 values were measured,
and the amino acid/peptide concentration was read from the
appropriate standard curve. Data were plotted as nanomoles of an
amino acid or oligopeptide, taken up by 1 mg (dry weight) of cells
versus time. The initial uptake velocities were determined from the
slopes of the linear part of the curves, in the 0‐ to 10‐minute
region.
|
5
BIOLOGICAL ASSAYS
|
5.1
Strains and culture conditions
|
5.4
Preparation of cell‐free extract
Reference and nonreference C. albicans strains used in this study are
listed in a table provided as the Table S1. Cells were grown at 30°C
in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) and
stored on YPD plates containing 2% agar. Susceptibility testing was
performed in YPD and other 2 media: (1) RPMI‐1640 without sodium
bicarbonate, with L‐glutamine + 2% glucose + 3.45% MOPS, pH
adjusted to 7.0; and (2) YNB‐AS (Yeast Nitrogen Base without amino
acids, contains ammonium sulfate 5 g L−1) + 2% glucose.
Candida albicans ATCC 10 231 cells from the overnight culture in YPD
were harvested by centrifugation and washed with cold 25mM potas-
sium phosphate buffer (pH 6.5). Cells were then suspended in a mini-
mal amount of the buffer and disrupted with the French press. The
resulting suspension was centrifuged (35 000g, 4°C, 45 min), and the
supernatant was used as a cell‐free extract for the determination of
peptide cleavage rates.
|
5.2
Susceptibility testing methods
|
5.5
Determination of peptide cleavage rates
The in vitro growth inhibitory activity of antifungals was quantified by
determination of MIC values by the serial 2‐fold dilution method, using
the 96‐well microtiter plates in 3 media: buffered RPMI‐1640, YNB‐
AS, and YPD. Conditions of the RPMI‐1640‐based assay were the
same as outlined in the CLSI recommendations,27 except for the end
point readout that was done by spectrophotometric determination of
cell density at 531 nm. Turbidity in individual wells was measured with
a microplate reader (Victor3; Perkin Elmer). MIC was defined as the
lowest drug concentration that gave at least an 80% decrease in
turbidity, relative to that of the drug‐free growth control.
The incubation mixtures, consisting of 10 mL of a 200‐μM peptide
solution in 50mM potassium phosphate buffer (pH 6.5) and 2 mL
of an appropriately diluted crude extract (final protein concentra-
tion, 0.1 to 0.5 mg mL−1), were incubated at 30°C. At 5‐minute
intervals, 2‐mL aliquots were withdrawn and heated at 100°C for
3 minutes. The resulting suspensions were centrifuged to remove
the protein precipitates, and the concentration of free amino acids
in the supernatant was determined by the Cd‐ninhydrin
procedure.28

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ACKNOWLEDGEMENTS
This work was supported by the National Science Centre, grant number
UMO‐012/05/B/ST5/002910. The helpful advice of Dr R. Mortimer is
gratefully acknowledged.
15. Payne JW. Drug delivery systems: optimizing the structure of peptide
carriers for synthetic antimicrobial drugs. Drugs Exp Clin Res.
1986;12:585‐594.
16. McCarthy PJ, Newman DJ, Nisbet LJ, Kingsbury WD. Relative rates of
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ORCID
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Med Chem. 1973;16:1277‐1280.
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