Chemoenzymatic synthesis of 1-deaza-pyridoxal 5′-phosphate

Article abstract of DOI:10.1016/j.bmcl.2010.01.002

The first synthesis of 1-deaza-pyridoxal 5′-phosphate (2-formyl-3-hydroxy-4-methylbenzyl phosphate) is described. The chemoenzymatic approach described here is a reliable route to this important isosteric pyridoxal phosphate analogue. This work enables elucidation of the role of the pyridine nitrogen in pyridoxal 5′-phosphate dependent enzymes.

Full text of DOI:10.1016/j.bmcl.2010.01.002

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1352–1354
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Bioorganic & Medicinal Chemistry Letters
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Chemoenzymatic synthesis of 1-deaza-pyridoxal 50-phosphate
*
Wait R. Griswold, Michael D. Toney
Department of Chemistry, University of California Davis, One Shields Avenue, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
The first synthesis of 1-deaza-pyridoxal 50-phosphate (2-formyl-3-hydroxy-4-methylbenzyl phosphate)
is described. The chemoenzymatic approach described here is a reliable route to this important isosteric
pyridoxal phosphate analogue. This work enables elucidation of the role of the pyridine nitrogen in pyr-
idoxal 50-phosphate dependent enzymes.
Article history:
Received 10 December 2009
Accepted 4 January 2010
Available online 7 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pyridoxal phosphate
Salicylaldehyde
Enzyme
Synthesis
Pyridoxal 50-phosphate (PLP) dependent enzymes comprise
more than 140 enzyme commission numbers.1 They catalyze a
wide variety of reactions including decarboxylation, racemization,
transamination, b elimination and retro aldol cleavages.1–3 Yet de-
spite this versatility, mechanistic similarities are found. All reac-
tions diverge from the common ‘external aldimine’ intermediate1
(Fig. 1) formed by the displacement of a lysine residue (which
forms the ‘internal aldimine’) by the amino group of the amino acid
substrate. Most reactions are thought to proceed through forma-
enzymatic) and the acidity of active site residue(s) interacting with
it (enzymatic).
Nonenzymatic transamination5 and racemization6 catalyzed by
the above mentioned analogues have been studied. However, the
constraints of active sites optimized for PLP preclude the use of
analogues structurally dissimilar from PLP in enzyme catalyzed
reactions. While N-methyl PLP, which contains a quaternized pyr-
idine nitrogen, has a long history of enzymatic study,1,7 the lack of
an isosteric salicylaldehyde analogue of PLP (‘deazaPLP’; 2-formyl-
3-hydroxy-4-methylbenzyl phosphate; 8, Scheme 1) has hampered
investigation of the role of the pyridine nitrogen in enzymatic reac-
tions. Surprisingly, the literature contains few attempts at obtain-
ing deazaPLP8 and, to the best knowledge of the authors, until now
there has been no successful synthesis reported.
tion of the ‘quinonoid’ intermediate,1–3 a C
by delocalization with the conjugated system of the cofactor
a carbanion stabilized
p
(Fig. 1). Indeed, the protonated pyridine nitrogen of the cofactor
acting as an electron sink in carbanion stabilization is considered
to be the hallmark of PLP catalyzed reactions.2
Recently, however, both experimental and computational stud-
ies call into question the crucial importance of the protonated pyr-
idine nitrogen.1,4a–c The structures of several enzyme active sites
preclude protonation of the pyridine nitrogen.4d,e Alternatively,
the protonated Schiff base, through electrostatic stabilization of
the carbanion, may play a central role (Fig. 1, structures A and B).1
Studies of catalysis by PLP analogues are a direct experimental
approach to this fundamental debate. At one extreme the pyridine
nitrogen of PLP and pyridine-based PLP analogues may be quatern-
ized, (1, Fig. 2), enforcing a positive charge. At the other extreme
are carbocyclic salicylaldehyde analogues (3, Fig. 2), which have
neither the potential for protonation nor the greater electronega-
tivity of nitrogen (vs carbon) in the aromatic ring. The charge of
the pyridine nitrogen on PLP itself (2, Fig. 2) depends on pH (non-
The general strategy employed here in the synthesis of dea-
zaPLP centers on oxidation and phosphorylation of triol 6 (Scheme
1) to the final product. Therefore a reliable route to 6 was sought
out. Reduction of lactone 5 would provide the desired compound.
A search of the literature revealed a previous published synthesis9
which proved to be a reliable route to lactone 5. Steps (i)–(iv) were
carried out as described in the original article except as noted.16 All
yields proved to be close to those published with the exception of
2, which provided only half the expected product.
Reduction of 5 with borane dimethyl sulfide provided triol 6
with yields of ꢀ50%. Attempts to obtain 6 by treatment of 5 with
LiBH4 proved unsuccessful. Phosphorylation of triol 6 by pyridoxal
kinase10,11 proved to be a facile and reliable route to the phosphor-
ylated triol with yields typically at 60–70%. Surprisingly, oxidation
of the phosphorylated triol by manganese dioxide to the final prod-
uct was not successful.
The presence of the negatively charged phosphate group
precluded the use of organic solvents for oxidation of the
* Corresponding author. Tel.: +1 530 754 5282; fax: +1 530 752 8995.
E-mail address: mdtoney@ucdavis.edu (M.D. Toney).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.002

W. R. Griswold, M. D. Toney / Bioorg. Med. Chem. Lett. 20 (2010) 1352–1354
1353
Figure 1. Carbanion formation and stabilization by PLP.
phosphorylated triol. On the other hand, water is a suboptimal sol-
vent choice for manganese dioxide oxidations due to competition
from solvent for adsorption to manganese dioxide12 and the pro-
pensity for over-oxidation to the acid. The obvious alternative
was to oxidize triol 6 to 7, followed by phosphorylation to 8.
Oxidation to the desired product occurred readily, but only with
freshly prepared manganese dioxide.13 Commercially available
manganese dioxide was ineffective and decreases in yield were
noted with the use of manganese dioxide over one week old. Yields
for 7 were 10–20%.
nating manganese with cation exchange resin was necessary. The
overall yield for 8 was 0.2%.
While the presence of two benzylic alcohols presented the com-
plication of a potential mixture of oxidation products13 from step
(vi), comparison of the UV–vis absorption spectra of salicylalde-
hyde and 3-hydroxybenzaldehyde with deazaPLP indicated that
the desired isomer was obtained. Successful enzymatic phosphor-
ylation of 7 further supports this conclusion. Final confirmation of
the structure of 8 was made by 1D nOe NMR experiments14 (see
Supplementary data for NMR and mass spectra).
Phosphorylation of 7 was carried out with pyridoxal kinase,
with yields similar to those with 6, although removal of contami-
To test the enzymatic binding properties of deazaPLP, apo
aspartate aminotransferase was prepared according to literature
methods15 and reconstituted with deazaPLP in a split-cell cuvette
to give difference absorption spectra due to deazaPLP binding.
The resulting spectra vs. time show a decrease in absorbance at
ꢀ350 nm and an increase in absorbance at ꢀ420 nm due to the
bathochromic shift on internal aldimine formation (Fig. 3). Fluores-
cence quenching (280 nm/340 nm) titrations of apo aspartate ami-
notransferase with deazaPLP indicate the dissociation constant to
be less than 1 lM (data not shown).
In summary, a reliable synthesis of the isosteric, carbocyclic
analogue of PLP (8) has been described. It binds tightly to aspartate
aminotransferase and forms the internal aldimine with the active
site lysine. The availability of this analogue will contribute to the
ongoing debate concerning the source of the catalytic prowess of
PLP enzymes.
Figure 2. Simplest pyridine and salicylaldehyde based analogues of PLP. H—A in
structure 2 denotes that the charge of the pyridine nitrogen may vary by pH
(nonenzymatic conditions) and the acidity of active site residue(s) interacting with
it (enzymatic conditions).
Scheme 1. Reagents and conditions: (i) HCL, formaldehyde, 80 °C; (ii) KOH, KMnO4, 5–10 °C; (iii) HCL, reflux; (iv) copper chromite, quinoline, 180 °C; (v) borane dimethyl
sulfide, THF, reflux; (vi) MnO2, ethyl acetate, rt; (vii) ATP, MgCl2, pyridoxal kinase.16

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W. R. Griswold, M. D. Toney / Bioorg. Med. Chem. Lett. 20 (2010) 1352–1354
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Virginia Tech University.
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12. Smith, M. B. Organic Synthesis; McGraw-Hill: New York, 2002.
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University Press: New York, 1998.
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16. Notes on synthesis. 1: (25 g) purchased from Alfa Aesar. 2: judged
sufficiently pure by 1H NMR to proceed without any purification. 3:
purified by anion exchange; acidification of fractions with concd HCL
precipitated out the desired compound. 4: judged sufficiently pure by 1H
NMR to proceed without any purification. Step (iv): after extraction with
ether, volume reduced by one half and extracted 3 times with 5 mL
portions of 1 M KOH. Combined extracts acidified with concd HCl to
precipitate crude product. 5: purified by silica gel: 100% ethyl acetate,
Rf = 0.8. Step (v): 2.2 equiv. of borane dimethyl sulfide added to solution of
5 (400 mM) in THF. Addition of borane dimethyl sulfide to THF solution
already at 60 °C doubled yields of 6. 6: purified by silica gel (1:1 hexane/
ethyl acetate to 100% ethyl acetate), Rf = 0.6 (in 100% ethyl acetate). Step
Figure 3. Difference UV–vis spectra vs. time (over ꢀ30 min) showing formation of
the internal aldimine of aspartate aminotransferase with deazaPLP.
Acknowledgment
This work was supported by Grant GM54779 from the National
Institutes of Health.
(vi): typically 50–100 mg MnO2 added to 1.5 mL 50–100 mM
6 in ethyl
acetate. Vigorously shaken for 20–25 min at room temperature. Extracted
3 times with 2–3 mL 100 mM KOH. Treated with cation exchange resin to
Supplementary data
remove manganese. Concentration of
6 estimated by using extinction
coefficient for phenol14 (1500 Mꢁ1 cmꢁ1). Step (vii): concentration of
7
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.002.
adjusted to 1 mM or less (estimated from 344 nm absorbance using
extinction coefficient of salicylaldehyde, 3300 Mꢁ1 cmꢁ1). Added fourfold
excess of ATP and MgCl2, adjusted solution to pH 8–9 and added
References and notes
pyridoxal kinase to 20 lM. Reaction wrapped in foil to exclude light
and stirred gently overnight at room temperature. Passed through a 10 kD
cut off filter to remove protein prior to purification. 8: purified by anion
exchange: 0–10 min, 100% water; 10–100 min, 0–20% 1 M NH4HCO3, pH
8.0; flow rate of 3.5 mL/min (column vol ꢀ25 mL). Final product
repeatedly lyophilized to remove NH4HCO3. Potassium salt prepared by
cation exchange followed by lyophilization. Final product stored at ꢁ80 °C.
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