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An updated synthesis of Nd-phosphonoacetyl-L-ornit
hine Rachel Steadman1, Dr. Patrick
Dussault21Department of Biology, Nebraska
Wesleyan University, Lincoln Nebraska
68504 2Department of Chemistry, University of
Nebraska-Lincoln, Lincoln Nebraska 68588
Future Direction From the small amount of
material obtained thus far from STEP 3, attempts
to obtain PALO (STEP 4) have begun. Investigation
into literature suggests it may be possible to
employ an alternative procedure for STEP 3 or to
skip this step altogether, however time
constraints did not allow a thorough
investigation of these processes. Our future
work will focus on optimization of this route. We
believe the result will be a convenient and
efficient synthesis of PALO that will enhance the
accessibility of this important biochemical
reagent.
Results To date, we have been able to
successfully obtain compound 3, where the 9-BBN
protecting group has been cleaved from the main
structure. However, this product has proven to be
difficult to purify and poor yields create a lack
of availability of product to allow us to move on
to the final ethyl-cleavage.
The Goal The goal of the project was to find a
shorter synthetic route to PALO using an
inexpensive and commercially available starting
material.
Introduction Nd-phosphonoacetyl-L-ornithine,
better known as PALO is a transition state
analogue of ornithine transcarbamylase (OTC),
which is the second enzyme of the urea cycle.
When introduced into the urea cycle, PALO blocks
(L)-ornithine from reacting with
carbamoyl-phosphate to create citrulline, which
breaks down the urea cycle and allows isolation
of the OTC enzyme. Before PALO was
identified, the OTC enzyme was only able to be
isolated in very small yields and in poor purity,
and being able to study the OTC enzyme is a
crucial part of the study of OTC deficiency, a
rare disease that can cause hyperammonemia and
when found in infants usually cause death to half
of those diagnosed within a month, and another
25 dies before the age of five. Most survivors
suffer from brain damage that is a residue of a
comma that occurs within 72 hours of birth.
PALO has also been proven
to reduce biosynthesis in vivo, and plans have
been created to study arginine-starvation affects
on the expression of amino acid biosynthetic
pathways. Arginine synthesis in fungi is closely
related to the urea cycle, however arginine
synthesis begins with glutamate PALO was
originally synthesized in 1978 and in 1983 an
improved synthesis was published, however both
syntheses are long and their starting materials
are no longer available commercially.
Introduction Nd-phosphonoacetyl-L-ornithine,
better known as PALO, is a transition state
analogue and a potent inhibitor, of ornithine
transcarbamylase (OTC), which is the second
enzyme of the urea cycle.1 PALO blocks the
reaction of (L)-ornithine with carbamoyl-phosphate
to create citrulline, which breaks down the urea
cycle and allows isolation of OTC.2
Before PALO was identified, the OTC
enzyme was only isolable in very small amounts
and in poor purity. Being able to isolate the OTC
enzyme is essential in the study of OTC
deficiency, a rare disease that can cause
hyperammonemia. When found in infants, it usually
causes the death of 50 of those diagnosed within
a month, and another 25 die before the age of
five. Most survivors suffer from brain damage
that is an aftereffect of a coma that occurs
within 72 hours of birth.3 PALO
has also been proven to reduce arginine
biosynthesis in vivo, and research has begun to
study arginine-starvation effects through PALO on
the expression of amino acid biosynthetic
pathways. Arginine synthesis in fungi is closely
related to the urea cycle, however arginine
synthesis begins with glutamate4. PALO was
originally synthesized in 1978 and in 1983 an
improved synthesis was published, however both
syntheses are long and their starting materials
are no longer commercially available5.
Materials and Methods The challenge of preparing
PALO is functionalizing the sidechain amino group
in the presence of the amino acid. Using 9-BBN
and L-ornithine as starting materials, a
four-step synthesis was attempted. The key
feature of our approach is the protection of the
amino acid with a dialkylboron derivative using
methodology developed by Dent and coworkers6.
IR
PALO
Literature cited 1. Alewood, P.F., Hoogenraad,
N.J., Johns, R.B., and Sutherland, T. 1983. An
Improved Synthesis of Nd-(Phosphonoacetyl)-L-ornit
hine. Synthesis 5 403-404. 2. MetaCyc. 2008.
MetaCyc Pathway urea cycle. lt
http//biocyc.org/META/NEW-IMAGE?typePATHWAYobje
ctPWY-4984gt. Accessed 2008 July 15. 3. Walzer
M. Urea cycle disorders and other hereditary
hyperammonemic syndromes, in The Metabolic Basis
of Inherited Disease, 5th ed, edited by
Stanbury J.B., Wyngaarden J.B., Fredrickson D.S.,
Goldstein J.L., Brown M.S., New York,
McGraw-Hill, 1983, pp 402-438 4. Kinney, D.M.,
Lusty, C.J. 1989. Arginine Restriction Induced by
d-N-(Phosphonacetyl)-L-Ornithine Signals
Increased Expression of HIS3, TRP5, CPA1, and
CPA2 in Saccharomyces cerevisiae. Molecular and
Cellular Biology 94882-4887 5. Slocum, R., and
Richardson, D. 1991,Purification and
Characterization of Ornithine Transcarbamylase
from Pea (Pisum sativum L.) Plant Physiology 96
262-268 A two step synthesis involving complex
chemistry was published in 1991. 6. Dent III, W.,
Erickson, R., Fields, S., Parker, M., and
Tromiczak, E. 2002. 9-BBN An Amino Acid
Protecting Group for Functionalization of Amino
Acid Side Chains in Organic Solvents. Organic
Letters 4 1249-1251
1H NMR
Urea Cycle
Conclusions The described HCl-cleavage step does
create the desired product, compound 3, in
acceptable purity but in very poor yields. Both
Steps 1 and Step 2 have been optimized to give
moderate yield and purity. On the other hand,
while compound 3 has been successfully
synthesized, STEP 3 may not be the most efficient
path to produce the desired results. The
cleavage of the boron protecting group with
aqueous HCl does generate compound 3, the
phosphonate-protected form of PALO. However, to
date this reaction has proceeded in very poor
yield, and has proven unpredictable in terms of
our ability to isolate purified product.
The light red color indicates the path that is
blocked by PALO, while the dark red indicates
the enzyme itself that is isolated.
Acknowledgments University of Nebraska,
Lincoln Nebraska Wesleyan University Dr. Patrick
Dussault Members of the Dussault Group Dr.
Stephen Fields, Dow AgroSciences Dr. Matt
Sachs, Texas AM INBRE NIH Grant P20 RR016469