Synthesis of (R)- and (S)-2,3-diaminopropyl sulfate: Mechanism based inhibition of glutamate 1-semialdehyde aminomutase

Article abstract of DOI:10.1016/S0040-4039(00)00004-6

A short synthesis of each enantiomer of 2,3-diaminopropyl hydrogensulfate is described starting from (2R)- and (2S)-asparagine. The compounds serve as inhibitors for pyridoxal 5'-phosphate-dependent (2S)- glutamate 1-semialdehyde aminotransferase, a target for selective herbicides and antibacterial agents on the tetrapyrrole biosynthetic pathway. The (2R)- enantiomer, as predicted, serves as a potent irreversible inactivator. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00004-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1637–1641
Synthesis of (R)- and (S)-2,3-diaminopropyl sulfate: mechanism
based inhibition of glutamate 1-semialdehyde aminomutase
Khurshida Khayer,a Thierry Jenn,a Mahmoud Akhtar,a Robert John b and David Gani a,∗,†
aSchool of Chemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
bSchool of Biosciences, University of Wales, Cardiff, PO Box 911, Cardiff CF1 3US, UK
Received 15 October 1999; revised 17 November 1999; accepted 21 December 1999
Abstract
A short synthesis of each enantiomer of 2,3-diaminopropyl hydrogensulfate is described starting from (2R)-
and (2S)-asparagine. The compounds serve as inhibitors for pyridoxal 50-phosphate-dependent (2S)-glutamate
1-semialdehyde aminotransferase, a target for selective herbicides and antibacterial agents on the tetrapyrrole
biosynthetic pathway. The (2R)-enantiomer, as predicted, serves as a potent irreversible inactivator. © 2000 Elsevier
Science Ltd. All rights reserved.
In plants, 5-aminolevulinic acid (ALA), a precursor for tetrapyrroles such as heme and chlorophyll,
is synthesised from glutamate 1-semialdehyde through the action of pyridoxal 50-phosphate (PLP)
dependent glutamate semialdehyde aminomutase (EC 5.4.3.8).1 This enzyme is absent in mammals,
which instead synthesise ALA from glycine and succinyl CoA in a reaction catalysed by a different PLP-
dependent enzyme, δ-aminolevulinate synthase.2 Because no counterpart for the aminomutase exists in
animals, the enzyme is a potential target for selective herbicides and antibacterial agents. Glutamate
semialdehyde aminomutase (or aminotransferase, [GSA-AT]) is unusual in that it catalyses the exchange
of amino and oxo functionalities, a transamination reaction, within the same molecule. However, the
enzyme is structurally homologous to other aminotransferases3 and the catalytic mechanism is analogous
to classical transamination.4,5 Successful mechanism-based inhibitors of other aminotransferases have
been designed by introducing a good leaving group at a β-carbon in alkylamine and amino acid
substrate analogues. Such strategies lead to structures that resemble the true substrate sufficiently to
bind at the active site of the target enzymes and to undergo all of the catalytic steps of the natural
reaction up to, and including, proton abstraction from Cα.6 Elimination of the β-substituent at this
point in the reaction produces an ene imine, which is not a normal intermediate and which, eventually,
reacts irreversibly with the enzyme through covalent bond formation. Sulfate is both a good leaving
∗
†
Corresponding author.
Part of the work described here was performed at the University of St. Andrews.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00004-6
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group and carries a formal negative charge and is able to mimic a carboxy group in other PLP-
dependent enzymes. For example, the glutamate analogue, serine-O-sulfate, is an effective mechanism-
based inhibitor of glutamate oxaloacetate transaminase (aspartate aminotransferase)7,8 and glutamate
decarboxylase9,10 whereas, ethanolamine-O-sulfate, an analogue of the inhibitory neurotransmitter 4-
aminobutyrate (GABA), selectively inactivates 4-aminobutyrate aminotransferase.11 It occurred to us
that we could incorporate a similar strategy into the design of mechanism based inhibitors of GSA-AT, if
we could identify a potential activation mechanism.
Glutamate 1-semialdehyde aminomutase exists in two forms, the pyridoxamine-P form (EM) and the
pyridoxal-P form (EL). The catalytic sequence is initiated though the reaction of GSA and EM, resulting
in the formation of the C-1 aldimine of (2S)-GSA (Scheme 1, step 1).4,12,13 Abstraction of a C-40 pro-
S hydrogen from the coenzyme (step 2) gives the corresponding quinonoid intermediate, which upon
proton transfer to C-1 is converted to the 4,5-diaminovaleric acid (DAVA) 5-aldimine of EL (step 3). This
intermediate undergoes transaldimination by an as yet undetermined mechanism, possibly involving the
ε-amino group of Lys272, to give the DAVA, 4-Schiff’s base EL complex.
Scheme 1. Proposed mechanism for the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA).
R=CH2CH2CO2H
As DAVA reacts rapidly with free EL, it was expected that the structurally analogous 2,3-diaminopropyl
sulfate 2-Schiff’s base complex might undergo β-elimination (Scheme 2) and possibly inactivate the
enzyme.
Scheme 2. Expected initial reaction of 2,3-diaminopropyl sulfate with GSA-AT
2,3-Diaminopropyl sulfate was not known in the literature and, accordingly, a synthesis providing
access to each enantiomer of the potential suicide substrate was devised. Although the parent sulfate

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was unknown, platinum(II) complexes of 2,3-diamino-1-propanol14,15 have attracted attention as anti-
tumour agents. Okamoto et al.14 prepared racemic 2,3-diaminopropan-1-ol through the reaction of
the tetrahydropyran derivative of 2,3-dibromopropan-1-ol with sodium azide in DMSO, followed by
reduction with LiAlH4 in THF, or, H2S in methanol. Resolution of the racemic 2,3-diaminopropanol
was achieved through a modification of the method employed for 1,2-propane diamine.15 These methods
seemed unwieldy and so alternatives using chiral starting materials, which might additionally contain
selectively deuteriated sites, were examined.
N-Benzyloxycarbonyl (2S)-asparagine 2 was prepared using Schotten–Baumann conditions and was
converted to the β-amine 3 [mp 230–232°C {lit.,16 mp 228–230°C (decomp.)}] using the conditions
of Waki,16 phenyl iodosyl bis(trifluoroacetate) [PIFA] in a mixture of DMF and water, Scheme 3.
Subsequent protection of the free amino group using, again, benzylchloroformate gave the bis-Cbz
protected diamine 4 in 92% yield. Reduction of the carboxy group of 4 with borane in THF led not only
to low conversion to the required alcohol, but also to several by-products that hampered purification.
Alternative reductions under a range of conditions using LiAlH4 and either the free acid 4 or its methyl
ester (prepared through the action of diazomethane) were also unsatisfactory. The α-acid was, however,
successfully converted to the alcohol 5 through reduction of the preformed bis(N2,3-benzyloxycarbonyl)-
propanoic acid mixed isobutylcarbonic anhydride with NaBH4 at −25°C in moderate to good (68%)
overall yield. We originally preferred a strategy that would introduce sulfate before the removal of the
two Cbz protecting groups because we wished to use conventional chromatographic purification methods
for the product and, therefore, needed to keep the polarity of the synthetic intermediates to a minimum.
It was also anticipated that the target compounds themselves might be unstable such that samples for
enzyme assays would be generated immediately before use from the freshly purified protected precursor
through, for example, catalytic hydrogenolysis. However, all attempts to sulfate the protected diamino
alcohol 5 directly with chlorosulfonic acid in trifluoroacetic acid following the method of Previero17 or
several variations, were unsuccessful. Clearly the stability of the benzylurethane moieties under such
acidic conditions was suspect and, accordingly, it was decided to attempt the sulfation reaction on the
free diamino alcohol 6. Removal of the benzyloxycarbonyl protecting groups of compound 5 through
catalytic hydrogenation, Scheme 3, gave the required diamino alcohol 6 as its free base in 93% recovery
and this was converted into the dihydrochloride salt 7 for full characterisation purposes, mp 180–181°C
(lit.,14 172–174°C). The 13C NMR spectrum displayed only three signals (δ 61.98, 52.87 and 41.36)
consistent with the alcoholic CH2, α-CH and β-CH2 carbon atoms, respectively.
Scheme 3. Reagents and conditions. (i) PhCH2OCOCl, NaHCO3, H2O, 78%; (ii) (bis(trifluoroacetoxy)-iodo)benzene,
DMF/H2O, 64%; (iii) PhCH2OCOCl, NaHCO3, H2O, 92%; (iv) isobutylchloroformate, NMM, THF, −25°C; then NaBH4,
THF, 25°C, 68%; (v) H2, Pd/C, MeOH; 93%; (vi) ClSO3H, CH2Cl2, 45 h, 59%
Attempted sulfation of the diamino alcohol 6 using the method of Previero17 was again unsuccessful.
However, changing the solvent from trifluoroacetic acid to dichloromethane gave (2S)-2,3-diaminopropyl
hydrogen sulfate 7 as a hydrochloride salt in 59% yield. Recrystallisation from water/ethanol gave a white

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1
solid, mp 55–60°C, which was >90% pure as judged by H and 13C NMR spectroscopy.‡ The major
impurity was the non-sulfated starting material which was removed by ion exchange chromatography on
Dowex-1 (1×4–400) immediately before biological evaluation. The (R)-enantiomer 8,‡ was obtained in
an analogous manner starting from (2R)-asparagine, but in better overall yield, 32%.
Both enantiomers were tested for activity with the aminomutase in its aldehyde EL form18 and there
were significant differences which can only be due to the configuration at C-2. Nevertheless, both
caused the elimination of sulfate. The (2S)-enantiomer 7 underwent reversible transamination without
inactivating the enzyme. The coenzyme PLP was, of course converted to PMP such that further quantities
of PLP were required to sustain the transamination of 7. It was possible to characterise several of the
intermediate complexes of the substrate with the holoenzyme by UV-spectroscopy using rapid kinetic
techniques. For example, the transamination reaction was accompanied by a change in the initial 420 nm
absorbing EL form to one absorbing maximally at 340 nm. The rate change increased hyperbolically to a
limiting value with increasing concentrations of the sulfate ester 7 and the value of Kd was determined to
be 5±1 mM. The (2R)-enantiomer 8 caused rapid irreversible inactivation in a process that corresponded
with the release of sulfate ion. Intermediate complexes involving the enzyme-bound coenzyme could be
detected and these revealed a biphasic conversion of the EL form to one absorbing maximally at 520 nm.
KS was calculated to be 1.83±0.01 mM. The addition of succinic semialdehyde increased the amplitude
of the increase in A520 and the reaction became monophasic. Full details will be reported elsewhere.18
In conclusion, both enantiomers of 2,3-diaminopropyl hydrogen sulfate are processed by GSA-AT. The
(2R)-enantiomer is a novel suicide substrate while the (2S)-enantiomer is a highly useful mechanistic
probe. The synthesis described here will be useful for the preparation of chirally deuteriated isotopomers
starting from known deuteriated asparagines.
Acknowledgements
The authors thank the Universities of Birmingham and St. Andrews for financial support.
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(100, [M−2HCl−OSO3H]+) and 60 (11, [M+H−HCl−CH2OSO3H]+). Selected data for compound 8; (HRMS: found:
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1641
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