N5-Phosphonoacetyl-l-ornithine (PALO): A convenient synthesis and investigation of influence on regulation of amino acid biosynthetic genes in Saccharomyces cerevisiae

Article abstract of DOI:10.1016/j.bmcl.2011.02.081

A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N⁵-phosphonoacetyl-l-ornithine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model organism Saccharomyces cerevisiae found t

Full text of DOI:10.1016/j.bmcl.2011.02.081

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2351–2353
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N5-Phosphonoacetyl-L-ornithine (PALO): A convenient synthesis
and investigation of influence on regulation of amino acid biosynthetic genes
in Saccharomyces cerevisiae
Brad Johnson a, Rachel Steadman a, Krista D. Patefield b, Jeffrey J. Bunker b, Audrey L. Atkin b,
,
⇑
Patrick Dussault a,
⇑
a Departments of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA
b School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588-0666, USA
a r t i c l e i n f o
a b s t r a c t
A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N5-phosphonoacetyl-
L-orni-
Article history:
Received 16 November 2010
Revised 18 February 2011
Accepted 22 February 2011
Available online 26 February 2011
thine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model
organism Saccharomyces cerevisiae found that, in contrast to a previous report, PALO did not influence
growth rate or expression of genes involved in arginine metabolism.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
PALO
Ornithine transcarbamylase
Saccharomyces cerevisiae
Arginine biosynthesis
N5-Phosphonoacetyl-
L-ornithine (PALO, 1) is a bisubstrate
O
O
O
P
O-
O-
Phosophonoacetlyl-
transition-state analog which competitively inhibits ornithine
transcarbamylase (OTC) in vitro, blocking biosynthesis of citrulline
in both the urea and the arginine biosynthetic pathways (Fig. 1).1,2
Competitive inhibitors of amino acid metabolism have been key
components in studies ranging from arginine starvation in fungi
and bacteria to studies on OTC deficiency.3,4
PALO has been tested as a potential tool for in vivo metabolic
studies. It failed to inhibit citrulline biosynthesis in isolated rat
mitochondria or intact rat hapatocytes,5 and did not inhibit growth
of the bacterium Escherichia coli.6 These failures were attributed to
the inability of PALO to cross cell membranes. In contrast, Kinney
and Lusty showed that PALO induced arginine limitation in the
yeast Saccharomyces cerevisiae.4 The regulation of arginine metab-
olism in fungi is particularly important to understand because of
the role of this amino acid in protein synthesis, as a nitrogen source
and in signaling.2,7–10 We now report a short and scalable synthesis
of PALO, as well as results of investigations into the influence of
PALO on growth and arginine metabolism in S. cerevisiae.
-O
L-ornithine (PALO, 1)
N
H
+
+
NH3
-O2C
ornithine
O
O
NH2
OTC
NH3
-O
N
H
NH2
O
O
P
+
NH3
O-
O-
carbamoyl
phosphate
H2N
citrulline
O
Figure 1. PALO; OTC-catalyzed synthesis of citrulline.
ornithine,3,5 some of which are no longer commercially available.
As illustrated in Scheme 1, we found that temporary masking of
the amino acid of ornithine as the boroxazolidinone derivative en-
ables a rapid and scalable synthesis of PALO in highly pure form.
Reaction of L-ornithine with 9-borabicyclononane (9-BBN) fur-
nished the corresponding boroxazolidinone,12,13 which was di-
rectly condensed with dibenzylphosphonoacetic acid (2) to
furnish amide 3.14 We were unable to achieve deprotection of
the boroxazolidinone with aq HCl or methanolic ethylenedia-
mine,12 even though these conditions were successfully modeled
on the corresponding adduct of phenylalanine.15 Fortunately, dis-
solution of 3 in MeOH/CHCl3 resulted in slow but clean deprotec-
tion to amino acid 4.16 Hydrogenolysis of the benzyl esters
proceeded nearly quantitatively to furnish PALO (1) as a neutral
The first syntheses of PALO employed the direct functionaliza-
tion of copper-complexed ornithine. This strategy, while rapid, re-
quires a significant set of purification steps.1,2,5,11 Several more
traditional routes begin with partially protected derivatives of
⇑
Corresponding authors. Tel.: +1 402 472 6951; fax: +1 402 472 9402 (P.D.).
E-mail address: pdussault1@unl.edu (P. Dussault).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.081

2352
B. Johnson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2351–2353
O
O-
1) 9-BBN
O
O
P
OBn
OBn
L-ornithine
NH2
B
2) DCC, DMAP
N
H
O
O
P
OBn
OBn
3
HO
-O
(62%, 2 steps)
2
O
Pd/C,
HCO2H
(95%)
MeOH,
CHCl3
O
O
P
PALO (1)
OBn
OBn
NH3
(90%)
N
H
4
Scheme 1. Synthesis of PALO (1).
Figure 2. PALO has no effect on expression of the ARG5,6, CPA1, and TRP5 genes in
Saccharomyces cerevisiae. S. cerevisiae strains18,19 were inoculated from a saturated
culture into minimal medium supplemented with leucine, methionine, uracil and
molecule which was pure by 1H, 13C, and 31P NMR. The PALOÁNH4
salt could be prepared by elution from an ion-exchange column.
Kinney and Lusty reported that PALO increased expression of
CPA1, CPA2, HIS3, and TRP5 reporter constructs in the yeast
S. cerevisiae.4 The data were consistent with limitation of
arginine leading to specific up regulation of arginine biosynthetic
genes (CPA1 and CPA2), and a general amino acid starvation
response typified by up regulation of HIS3 and TRP5.
We tested whether treatment with PALO resulted in changes in
mRNA accumulation and/or translation of genes involved in amino
acid biosynthesis. We examined expression of two genes required
for arginine biosynthesis; ARG5,6, which encodes a protein in-
volved in the second and third steps of arginine biosynthesis from
glutamate, and CPA1, which encodes a subunit of arginine-specific
carbamyl-phosphate synthetase. Both of these genes are up regu-
lated specifically in response to arginine limitation. Expression of
TRP5, which encodes an enzyme that catalyzes the final step in
tryptophan biosynthesis, was also examined as an indicator of
the general amino acid starvation response. Quantitative northern
analysis was performed to measure mRNA abundance, a measure
of the combined rates of transcription and mRNA decay. Western
analysis was used to look at the level of protein synthesis for these
genes. This combined approach enabled direct evaluation of effects
on transcription, mRNA stability, and translation.
Although the yeast strains from the earlier studies were not
available,4 they were derived from the W303 genetic background,
which usually carry the can1-100 mutation.17 As CAN1 encodes
an arginine permease required for efficient uptake of arginine,
the can1-100 mutation is not well suited for the current experi-
ments. As a compromise, and to assess if PALO will be generally
useful as a metabolic tool in S. cerevisiae, the current data was ob-
tained using the BY4741 strain of S. cerevisiae.18 This strain is de-
rived from S288C, the strain used in the systematic sequencing
project, and it is the most commonly used genetic background
for genome-wide functional analyses.
histidine. Cultures were treated with 15
buffer, potassium phosphate buffer pH 6.8 alone (untreated), 1 mg/mL
both 15 M PALO and 1 mg/mL -arginine at the time of inoculation. Cells were
lM PALO in a potassium phosphate pH 6.8
L-arginine, or
l
L
incubated at 30 °C and then harvested at mid-log growth phase (OD600 0.4–0.6). (A)
Northern blots were prepared with total RNA extracted from BY474118,19 and
probed with oligolabeled DNA probes.20 DNA probes were generated using primer
sets for amplifying yeast open reading frames based on the sequences available
from the Saccharomyces Genome Database. Shown are representative phosphor-
images of a northern blot probed with radioactive ARG5,6, CPA1, TRP5, and SCR1
DNAs. SCR1 was used as a loading control. Quantitative results depicted are the
average of three replicate trials and are normalized to the SCR1 loading control. (B)
Western blots were prepared using protein extracts from S. cerevisiae YSC1178-
7500224, YSC1178-7502950 and YSC1178-7500415 strains19 expressing TAP-
fusion proteins Arg5,6-TAP, Cpa1-TAP, and Trp5-TAP, respectively, as well as
BY4741 (untagged control).21,22 The TAP-tagged proteins were detected using an
anti-TAP antibody. No proteins bound the anti-TAP antibody in the untagged
control. Duplicate polyacrylamide gels were stained with Coomassie blue for use as
loading controls. The corresponding loading controls are shown beneath the
western blots.
transcript did not show any variation in response to addition of
PALO, arginine or both. In summary treatment with PALO had
no effect on the abundance of ARG5,6, CPA1 or TRP5 mRNAs.
The possible effect of PALO on translation was investigated by
western analysis (Fig. 2B). If PALO affects translation, we expected
to see an increase in the production of Arg5,6p, Cpa1p, and Trp5p
in the presence of PALO. We also expected that addition of
L-arginine would reduce protein levels of Arg5,6p and Cpa1p, but
not Trp5p. We used cell extracts from strains (YSC1178-7500224,
YSC1178-7502950 and YSC1178-7500415, respectively) carrying
TAP-tagged alleles of ARG5,6, CPA1 and TRP5 for the western anal-
ysis. These strains produce a TAP-fusion protein for each gene
which can be detected on western blots with anti-TAP antibodies.
BY4741 was used as an untagged control because it is the parent
strain for the TAP-tagged strains. Cells were grown and treated in
the same manner as for the northern analysis. Measurement of
the signal intensity of the bands detected by western blotting
revealed no difference in the production of Arg5,6p, Cpa1p, or
If PALO affects transcription or mRNA stability, we expected to
observe an increase in expression of ARG5,6, CPA1, and TRP5
mRNA transcripts in the presence of PALO. We also expected that
addition of arginine would relieve the arginine limitation induced
Trp5p between strains treated with 15
lM PALO and the control.
As expected, addition of -arginine reduced production of Arg5,6p
L
and Cpa1p, but not of Trp5p regardless of whether PALO was added
or not. In summary, PALO had no effect on Arg5,6p, Cpa1p, and
Trp5p protein levels.
PALO did not affect the growth rate of BY4741 in minimal media
or nitrogen-limiting medium (data not shown), used in an attempt
to increase sensitivity to PALO by lowering intracellular arginine
concentrations. Growth in nitrogen-limiting medium induces cells
to use amino acids, including arginine, as a source of nitrogen.
These findings were consistent with the observations reported by
Kinney and Lusty.4
by PALO. Cells were grown and treated with either 15
synthesized PALO, 1 mg/mL -arginine, or 15 M of the synthe-
sized PALO and 1 mg/mL -arginine as described by Kinney and
lM of the
L
l
L
Lusty.4 An untreated culture was grown in the same media as
a control. The abundance of ARG5,6, CPA1, and TRP5 mRNAs was
measured by quantitative northern analysis (Fig. 2A). The abun-
dance of ARG5,6, CPA1, and TRP5 mRNAs in the PALO treated cells
was not significantly different from the untreated cells. The abun-
dance of ARG5,6 and CPA1, but not TRP5 mRNAs was lower in cells
treated with arginine regardless of whether the cells were treated
with PALO or not. Additionally, the abundance of the TRP5
In conclusion, we have developed an efficient synthesis of very
pure PALO by a route suitable for both analytical and preparative

B. Johnson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2351–2353
2353
8. Messenguy, F.; Dubois, C. Food Technol. Biotech. 2000, 38, 277.
9. Morris, S. M., Jr. J. Nutr. 2007, 137, 1602S.
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Petro, T. M.; Nickerson, K. W. Infect. Immun. 2009, 77, 1596.
11. Synthesis of PALO via a Cu(II)-ornithine complex: Slocum, R. D.; Richardson, D.
P. Plant Physiol. 1991, 96, 262.
12. Fields, S. C.; Dent, W. H., III; Erickson, R.; Parker, M. H.; Tromiczak, E. G. Org.
Lett. 2002, 4, 1249.
13. Details regarding the synthesis can be found in Supplementary data.
14. Koppel, G. A.; Kinnick, M. D. Tetrahedron Lett. 1974, 9, 711; Grison, C.; Coutrot,
P.; Comoy, C.; Balas, L.; Joliez, S.; Lavecchia, G.; Oliger, P.; Penverne, B.; Serre,
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applications. In contrast to a previous report describing PALO-
induced arginine starvation,4 we observed no response in
S. cerevisiae strain BY4741 to treatment with PALO (Fig. 2). The dis-
crepancy suggests either that not all yeast strains are sensitive to
PALO or that the earlier indications of arginine starvation resulted
from impurities in the samples of PALO employed. The current re-
sults are consistent with observations that PALO had no effect in
5,6
isolated rat mitochondria, intact rat hepatocytes or E. coli.
Acknowledgments
15. The boroxazolidinone from Phe is predicted to have a very similar log P as 3:
ChemDraw Ultra 11.01, CambridgeSoft, 2007.
16. Walker, W. H., IV; Rokita, S. E. J. Org. Chem. 2003, 68, 1563; Syed, B. M.;
Gustafsson, T.; Kihlberg, J. Tetrahedron 2004, 60, 5571. We are also unable to
explain the deprotection, but note that it is unaffected by oxygen, catalytic
acid, or catalytic base.
17. Thomas, B. J.; Rothstein, R. Cell 1989, 56, 619.
18. Brachmann, B. C.; Davies, A.; Cost, G. J.; Caputo, E.; Li, J.; Hieter, P.; Boeke, J. D.
Yeast 1998, 14, 115.
We thank Dr. Steven Fields (Dow Agro, Indianapolis, IN) for use-
ful discussions and a preprint describing investigations of PALO
synthesis. Support was provided by the University of Nebraska-
Lincoln Research Council and by NIH INBRE scholarships to R.S.
and J.B. This work was conducted in part, in facilities remodeled
with support from the NIH (RR0116544-01) and employing instru-
mentation purchased with NSF support (MRI 0079750 and CHE
0091975).
19. S. cerevisiae strains BY4741 (MATa his3-
D1 leu2-D0 met15-D0 ura3-D0,
American Type Culture Collection, Manassas, VA), YSC1178-7500224 (MATa
his3-
his3-
his3-
D
D
D
1 leu2-
1 leu2-
1 leu2-
D
D
D
0 met15-
0 met15-
0 met15-
D
D
D
0 ura3-
0 ura3-
0 ura3-
D
D
0 ARG5,6-TAP), YSC1178-7502950 (MATa
0, CPA1-TAP) and YSC1178-7500415 (MATa
D0, TRP5-TAP) were used. The YSC1178 strains
are from Open Biosystems Products (Huntsville, AL). BY4741 is the background
strain for the YSC1178 strains. S. cerevisiae strains were inoculated from a
saturated culture incubated at 30 °C in YPD in a tube roller in minimal medium
supplemented with leucine, methionine, uracil and histidine. Cultures were
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data, and NMR (1H, 13C, 31P) spectra for PALO and synthetic
precursors (PDF)) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.02.081.
treated with 15
potassium phosphate buffer pH 6.8 alone (untreated), 1 mg/mL
both 15 M PALO and 1 mg/mL -arginine at the time of inoculation. Cells were
l
M PALO in a 50 mM potassium phosphate pH 6.8 buffer,
L
-arginine, or
l
L
incubated at 30 °C and then harvested at mid-log growth phase (OD600 0.4–
0.6). Cells were observed for effects on growth via incubation at 30 °C in
nitrogen-limiting medium containing 2% dextrose, 1 mM ammonium sulfate,
References and notes
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