Journal of the American Chemical Society
Article
features a four-enzyme combination to make the short-chain
fully saturated aliphatic aldehyde (3), a PLP-dependent
enzyme (Fub7) to connect 3 with an amino acid primary
metabolite O-acetyl-L-homoserine to generate the nitrogen-
containing heterocycle 4, and an oxidase to furnish the
aromatic pyridine ring at last. Many of these enzymes
characterized here can be useful reagents in biocatalysis and
synthetic biology.20 Among them, the C−C bond-forming
activity of Fub7 intrigued us to further explore its biocatalytic
potential.
We first tested Fub7 substrate specificity using different
linear aldehyde substrates with chain length varying from C3 to
C8. OAH was used as the limiting reagent in each reaction. To
facilitate product detection and quantification, we coupled this
assay to Fub9, enabling oxidation of any cyclic imine products
into chromophoric picolinic acids. As shown in Figure 7,
HPLC analysis indicates a series of 5-alkyl picolinic acids (9−
16) can be synthesized using this coupled enzymatic system
starting from OAH and aldehydes. Five compounds (10, 11,
and 14−16) were isolated from scaled-up, overnight reactions
(volume varies between 10 and 30 mL depending on the yield)
for structural characterization. Analysis of the reactions by LC-
MS did not reveal any accumulated tetrahydropicolinic acid
intermediates; therefore, the yield of each product is likely
limited by the substrate specificity of Fub7. Aldehydes bearing
bulky groups at the Cβ position are not favored by Fub7 as 2-
phenylacetaldehyde gave a low yield (product 15) and 2-
cyclohexylacetaldehyde was completely inactive (vide infra),
presumably due to increased steric hindrance impeding Fub7-
catalyzed enolization of aldehydes. Overall, Fub7 exhibits
somewhat relaxed substrate specificity toward linear aliphatic
aldehydes and prefers substrate chain length C4−C7.
This relaxed substrate specificity of Fub7 encouraged us to
repurpose its activity for the synthesis of 5-alkyl- and 5-dialkyl-
L-pipecolic acids by intercepting Fub7-synthesized cyclic imine
products through chemical reduction using NaBH3CN.
Compared to the reported method in which 5-alkyl-DL-pipcolic
acids were prepared through hydrogenation of 5-alkyl-picolinic
acids,6 our chemoenzymatic route is advantageous in that it
preserves the stereocenter at the C2 position (L-configuration)
originated from OAH. Furthermore, we reasoned that C−C
bond formation catalyzed by Fub7 should be stereoselective,
which can be leveraged to make L-pipecolic acids bearing a
quaternary stereocenter at the C5 position by starting from
asymmetric α-branched aldehydes.
As shown in Figure 8, a library of 5-substituted L-pipecolic
acids can be prepared chemoenzymatically. When the yield is
sufficiently high, the final products were purified and
structurally characterized by NMR as indicated in the figure.
Diastereomeric ratios were determined by either NMR or
LCMS when the compounds can be separated. As expected, for
linear aliphatic aldehydes substrates (entries 1−3), a pair of
2,5-diastereomers (cis and trans) was formed with the trans
isomers being the major products (Figure S13). For α-
branched aldehyde substrates (entries 5−14), Fub7 could
accept a series of α-methyl aldehydes with carbon chain length
C4−C7 preferred (products 20−24) but did not tolerate
aldehydes with ethyl or any other larger substitution groups at
Cα, which is also likely due to steric clash with enzyme active
site residues. It is noteworthy to mention that one of the
products, 5,5-dimethyl-L-pipecolic acid (20), is a natural
metabolite involved in flavunoidine biosynthesis.25 It was
proposed that 20 could be synthesized from OAH and α-keto-
isovalerate by the PLP-dependent enzyme FlvA and imine
reductase FlvB. Our result shown here shed more light on the
biosynthetic mechanism of 20 that the carbon nucleophile
substrate for FlvA might be isobutylaldehyde instead of α-keto-
isovalerate.
Moreover, reactions with asymmetric racemate α-methyl
aldehyde substrates (products 21−24) all convergently yielded
trans-5-alkyl-5-methyl-L-pipecolic acids. Because this C5
quaternary stereocenter can no longer epimerize, this result
strongly supports our hypothesis that Fub7 stereoselectively
catalyzes C−C bond formation between aldehyde-derived
enolate and PLP-bound vinylglycine ketimine. Notably,
product 24 is a 5,5,6-trialkyl-L-pipecolic acid with three
contiguous stereocenters, which are set through a cascade
reaction triggered by Fub7: stereoselective C−C bond
formation between 2,6-dimethyl-5-heptenal and OAH followed
by cyclization and dehydration; the newly formed imine
intermediate then undergoes a Pictet−Spengler-like reaction to
give the final bicyclic scaffold. Synthesis of 24 also
demonstrates that the reactive Fub7-synthesized imine
products can be leveraged to generate new scaffolds which
could open up new opportunities in constructing more
complex L-pipecolic acids.
Last but not least, Fub7 can also accept various α-branched
cyclic aldehydes (entries 15−20), leading to pipecolic acids
derived spirocycles (product 25−30). The low diastereomeric
ratios of product 29 and 30 indicate that significant substrate
desymmetrization (appending substitution groups) will be
required for Fub7 to achieve diastereoselectivity, as Fub7 does
not distinguish the re-face and si-face of the asymmetric enolate
substrates (entries 19 and 20) very well. However, additional
methyl substitution could not be accommodated (entries 21
and 22), which further suggests that the enzyme active site is
naturally tailored to best suit linear aliphatic aldehyde, such as
its natural substrate 3. Enlarging the active site by protein
engineering on Fub7 may be required to recognize bulkier
substrates.
Summary. In this work, we elucidated the biosynthetic
pathway for fusaric acid and characterized the enzymatic
activity of four key enzymes, Fub6−9. Among these, we
demonstrated the synthetic potential of Fub7 in synthesizing
either 5-alkyl-picolinic acids when coupled with Fub9 or
substituted pipecolic acids via a chemoenzymatic route. In
particular, the stereoselective C−C bond formation catalyzed
by Fub7 can be harnessed to stereoconvergently access 5-alkyl-
5-methyl-L-pipecolic acids with a high diastereomeric ratio.
The Fub7-enabled cascade reaction synthesizing 24 indicates
that the asymmetrically synthesized imine products from the
Fub7-catalyzed reaction can be potentially borrowed by many
other downstream pathways, leading to more complex
structural scaffolds. Our study also demonstrated how a
biosynthesis study could facilitate biocatalytic innovations.
Given the vast genetic potential of microbial genomes, more
PLP-dependent biosynthetic enzyme will be uncovered and
hold promise in biocatalysis and synthetic biology.
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* Supporting Information
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Experimental procedures and spectroscopic data (PDF)
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX