Breaking the Dogma of Aldolase Specificity: Simple Aliphatic Ketones and Aldehydes are Nucleophiles for Fructose-6-phosphate Aldolase

Article abstract of DOI:10.1002/chem.201701020

d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible.

Full text of DOI:10.1002/chem.201701020

A Journal of
Accepted Article
Title: Breaking the dogma of aldolase specificity: Simple aliphatic
ketones and aldehyde are nucleophiles for fructose-6-phosphate
aldolase
Authors: Raquel Roldán, Israel Sanchez-Moreno, Thomas Scheidt,
Virgil Hélaine, Marielle Lemaire, Teodor Parella, Pere Clapés,
Wolf-Dieter Fessner, and Christine Guérard-Hélaine
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To be cited as: Chem. Eur. J. 10.1002/chem.201701020
Link to VoR: http://dx.doi.org/10.1002/chem.201701020
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Breaking the dogma of aldolase specificity: Simple aliphatic
ketones and aldehyde are nucleophiles for fructose-6-phosphate
aldolase
Raquel Roldán§, Israel Sanchez-Moreno,§ Thomas Scheidt,§ Virgil Hélaine, Marielle Lemaire, Teodor
Parella, Pere Clapés*, Wolf-Dieter Fessner* and Christine Guérard-Hélaine*
Dedication ((optional))
Abstract: D-Fructose-6-phosphate aldolase (FSA) was probed for
extended nucleophile promiscuity by using a series of fluorogenic
substrates to reveal retro-aldol activity. Four nucleophiles ethanal,
propanone, butanone and cyclopentanone were subsequently
confirmed to be non-natural substrates in the synthesis direction
using the wild type enzyme and its D6H variant. This exceptional
widening of the nucleophile substrate scope offers a rapid entry, in
good yields and high stereoselectivity, to less oxygenated alkyl
ketones and aldehydes, which was hitherto impossible.
and electrophiles as substrates. In this sense, as repeatedly
reported in the literature, one of the major limitations of
aldolases is their strict specificity for their nucleophilic substrate,
where even small isosteric modifications usually result in a
dramatic decrease of activity up to several orders of
magnitude.[3] A further limitation is the fact that most of the
known aldolases prefer highly functionalized and polar
nucleophiles such as dihydroxypropanone phosphate (DHAP),
dihydroxypropanone (DHA), pyruvate, hydroxypyruvate[4] or
glycine.[1a] Clearly, nucleophilic components with lower functional
density are highly desirable in organic synthesis to broaden the
biocatalytic access towards a wider variety of more generic
chiral materials.
Aldolases are central biocatalytic tools for the bottom up
construction of functionalized carbon skeletons via asymmetric
aldol addition reactions. Their potential has been widely
demonstrated by a plethora of examples for the synthesis of
carbohydrates (including unusual deoxysugars, aminosugars,
iminocyclitols and phosphate sugars), amino acids and
ketoacids among many other classes of compounds.[1] Aldolases
catalyze the stereoselective addition of a nucleophile, via a
ketone enolate (i.e. class II enzymes) or an analogous enamine
(i.e. class I enzymes) onto an aldehyde electrophile.[2] This
carboligation process leads to the formation of a chiral aldol
product containing up to two new adjacent chiral centers of
known absolute configuration from unprotected substrates in
near neutral aqueous reaction medium and at mild ambient
conditions.
D-Fructose-6-phosphate aldolase from E. coli (FSA, EC 4.1.2.n)
or other microorganism sources[5] catalyzes the reversible
cleavage of D-fructose-6-phosphate (Fru6P) to DHA and D-
glyceraldehyde-3-phosphate (D-GA3P) (Scheme 1). Even
though D-GA3P is the electrophilic substrate with the highest
affinity known so far, FSA tolerates a wide variety of other
aldehydes. As the nucleophilic component, FSA also converts
DHA analogues such as hydroxypropanone (HA) and
6]
hydroxybutanone (HB) with comparable efficiencies.[4,
Remarkably, it accepts hydroxyethanal (HE) as an aldehyde
rather than
a
ketone nucleophile (Figure 1),[7] which is
unparalleled among the aldolases, with the exception of 2-
deoxy-D-ribose-5-phosphate aldolase (DERA), which uses
ethanal. More recently, the subtle nucleophile selectivity of wild-
type FSA was dramatically enhanced by minimalist protein
engineering.[8] Single or double active site mutations allowed an
efficient conversion of larger ketol components with up to seven
skeletal atoms. Consequently, FSA became one of the most
relevant aldolases for synthesis thus far because of its wide
substrate tolerance.
For highest synthetic utility aldolases not only must be
catalytically efficient and stereoselective but should also offer
broad substrate promiscuity by accepting diverse nucleophiles
[*]
Dr. I. S. Moreno, Dr. V. Hélaine, Pr. M. Lemaire, Dr. C. Guérard-
Hélaine
Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de
Chimie de Clermont-Ferrand, F-63000 Clermont–Ferrand, France.
E-mail : christine.helaine@univ-bpclermont.fr
Thomas Scheidt, Pr. W.-D. Fessner
Institut für Organische Chemie und Biochemie, Alarich-Weiss-Str. 4,
D-64287 Darmstadt, Germany.
E-mail : fessner@tu-darmstadt.de
R. Roldán, Pr. P. Clapés
Departamento de Química Biológica y Modelización Molecular,
Instituto de Química Avanzada de Cataluña IQAC-CSIC
Jordi Girona 18–26, 08034 Barcelona (Spain)
E-mail : pere.clapes@iqac.csic.es
Scheme 1. Reversible cleavage of D-fructose 6-phosphate (Fru6P) by FSA to
generate D-glyceraldehyde-3-phosphate (D-GA3P) and dihydroxypropanone
(DHA).
Dr. T. Parella
Servei de Ressonancia Magnetica Nuclear. Universitat Autonoma
de Barcelona, Bellaterra (Spain)
However, the invariable presence of the hydroxymethyl moiety in
[§]]
Those authors contributed equally to this work.
all these nucleophilic substrates is
a common structural
characteristic that FSA and transaldolase[6b] share with the
stereocomplementary set of the DHAP-dependent aldolases.[9]
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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On the hypothesis that for FSA the hydroxymethyl ketone motif
in the nucleophile substrates should not be an essential
functional requirement, in this work we explored the suitability of
simple unsubstituted aliphatic ketones as a replacement.
First, in a search for such a hidden ability of wild-type FSA (wt
FSA) to catalyze the cleavage of more generic aldol substrates
that lack the “essential” hydroxyl group within the ketol
nucleophile, we made recourse to a highly sensitive fluorogenic
assay, which is specific for the retro-aldol bond cleavage. This
assay technology is based on the non-fluorescent surrogate
substrate methodol (1a), which upon retro-aldol cleavage
liberates propanone (3a) plus the corresponding 6-
methoxynaphthaldehyde (2), which displays strong blue
fluorescence (Scheme 2).[10] This assay principle had been
developed earlier for the identification of catalytic antibodies[11]
and, more recently, for the evaluation of retro-aldolase catalysts
generated by de novo enzyme design.[12] When tested with 1a,
wt FSA was found to cause time-dependent and concentration-
dependent fluorescence development with typical Michaelis-
Menten kinetics, indicative that wt FSA at some degree will also
tolerate a non-hydroxylated moiety in the substrate. Interestingly,
basal activity was significantly lower than that of the commercial
38C2 antibody,[10, 12] which was used as a positive control, but
comparably higher than that of DERA from E. coli, which had
been demonstrated in synthesis direction to be active with
propanone as a replacement for its natural substrate ethanal.[3a]
It must be mentioned that fluorescence generation from 1a was
clearly originating from release of the aldehyde rather than
elimination of water to form the unsaturated ketone, because the
latter displays a green-shifted fluorescence maximum (also
verified by TLC control). Negative control experiments using
various proteins with bona fide inability to catalyze an aldol
reaction occasionally were found to demonstrate dehydratase
but no retro-aldolase activity (e.g., bovine serum albumine),[13]
while FSA, DERA and 38C2 showed retro-aldolase but no
dehydratase activity. Pure enantiomers of 1a, prepared by
kinetic resolution via lipase-catalyzed transacylation (see SI),[14]
clearly demonstrated that all three biocatalysts showed a
common preference for the (S)-configurated enantiomer, in line
Scheme 2. Fluorogenic retro-aldol assay tested with FSA for nucleophile
promiscuity.
Second, in the forward direction of aldol synthesis, unsubstituted
aliphatic carbonyl compounds (simple ketones and aldehydes)
were screened as potential nucleophiles. For this study, GA3P
was selected as the electrophilic substrate because of its
highest binding affinity for FSA (KM 0.8 mM),[17] which was
assumed to support the most efficient reaction kinetics.
Propanone, ethanal and butanone (3a, 4 and 5 respectively)
were selected for their structural analogy to the corresponding
active hydroxylated nucleophiles (HA, HE and HB). 2-Pentanone,
cyclobutanone, cyclopentanone and cyclohexanone (6, 7, 3g
and 3h respectively) were also included to verify the enzyme’s
maximum tolerance for the aliphatic chain length as well as its
compatibility for steric hindrance upon binding of their cyclic
homologues (Figure 1).
Figure 1. Known and new nucleophiles considered as substrates for FSA.
with the natural stereoselectivity of the enzymes at this
and the synthetic preference of the antibody
when using propanone.[16]
The selected seven nucleophile analogs 3a, 4-7, 3g-h were
incubated with wt FSA at 100 mM concentration using
commercial D,L-GA3P as the electrophile. The latter was applied
at 6 mM to avoid potential FSA inhibition by phosphorylated
substrate or products.[18] Based on relative intensities of product
spots from TLC reaction monitoring, wt FSA gave good
conversion rates with 3a, 4, 5, and 3g, while the other
nucleophiles 6, 7 and 3h, appeared to be unreactive or very
poor substrates, consistent with the above retro-aldol assays.
Several controls were performed to validate enzymatic active-
site catalysis and to exclude fortuitous unspecific catalytic
effects by surface protein residues. Thus, using propanone and
D,L-GA3P as the reference substrates (see SI), reactions were
incubated with enzyme samples of wt FSA that had been
inactivated by prior heat treatment or by pre-incubation with
acrolein or by using the catalytically inactive variant FSA K85M
instead, which lacks the essential catalytic Lys residue (class I
aldolase). No product formation was observed in these control
reactions. Moreover, when the generic reaction of 3a with D,L-
6, 15]
position[3a,
To further test the potential substrate scope, a panel of
representative methodol analogs containing various structural
modifications (1b–1h) was prepared by standard aldol
synthesis,[10a] including carbon backbone extension at both ends
of the ketone nucleophile, chain branching, unsaturation, and
cyclization (Scheme 2). The study indicated that wt FSA may
indeed have a low, but unambiguous activity with long chain
(beyond C5; 1d and e) and small-ring ketones (up to
cyclopentanone; 1g) as nucleophiles. Branching next to the
reactive carbonyl (1b, c and f) and larger ring structures (1h)
were not tolerated.
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GA3P was performed with wt FSA in the presence of 50 mM
Fru6P, TLC indicated a reduced conversion rate, which may be
interpreted as a consequence of both substrates (3a and Fru6P)
competing for the catalytic Lys residue. These experiments
confirmed that enzymatic catalysis of the reactions with the non-
natural nucleophiles proceeded in the FSA active site.
We next investigated the putative enantiopreference of FSA
towards D- and L-GA3P as a function of the nucleophile
component. Using 3a, 4, 5 and 3g the reaction progress was
monitored spectrophotometrically by assaying separately for
both D- and L-GA3P consumption using the auxiliary enzymes
glycerol phosphate dehydrogenase/triosephosphate isomerase
(GPDH/TPI) or L-GA3P reductase, respectively (see SI).[1e] To
correct for possible D-GA3P consumption during the syntheses
from its isomerization to DHAP by contaminant TPI remaining in
the FSA preparations, the DHAP concentration was also
analyzed independently. The L-GA3P conversions were always
above 80%, except for 5 that reached only 40%. On the other
hand, using D-GA3P, 3a and 5 gave 75% and 30% conversion
respectively, whereas no reaction was observed with the two
(R/S,S,S[a]
)
[a]
3g
11
82
-
[a] An isotopic exchange was observed in the C- to the carbonyl group (i.e.
the tertiary center) of compound 11. Hence, the configuration of this
asymmetric carbon is lost under the reaction conditions (pH 7.6-7.8).
NMR spectroscopic analysis confirmed the constitution of the
expected aldol products and revealed that the ketone products
in aqueous solution preferentially existed in their open-chain
forms whereas the aldehyde product 9 exists as the furanose
forms. NMR spectra of compound 10 clearly indicated that the
aldolisation of butanone exclusively occurred on C1. Absolute
configuration of the products is given by the chirality of L-GA3P
as an internal reference. The relative configuration of the newly
created asymmetric centers could be unequivocally assessed by
using various strategies depending on the compound considered.
In the case of aldehyde 9, expected to be L-2-deoxyxylose-5-
phosphate by the natural stereoselectivity of FSA, we compared
its 13C NMR spectrum with that of its commercial C3 epimer D-2-
deoxyribose 5-phosphate. Clearly two different spectra were
obtained excluding the presence of enantiomers, and thus
confirming the configuration for 9a (see SI Figure S2).
The relative stereochemistry of open chain aldol adducts 8, 10
and 11 was difficult to assess by NMR owing to the free rotation
of the bond between chiral carbons. Therefore, mild enzymatic
dephosphorylation was used to liberate the terminal hydroxyl
group. Compounds 12 and 13 were thus isolated (Scheme 3).
This allowed the formation of pyranose isomers in equilibrium
with the open chain species, and an unequivocal determination
of the relative stereochemical relationships among substituents
in the cyclic forms (Scheme 3). In this way compounds 8 and 10
were found to have the same relative configuration as in
compound 9. These structures also confirmed that the facial
attack at the aldehyde carbonyl group of the electrophilic
component during the FSA catalysis was unaffected by the
missing hydroxyl function in the nucleophile, fully in line with the
(S)-enantioselectivity observed for the retro-aldol cleavage of
FSA using the fluorogenic substrate analog 1a. In the case of 11,
the aldol addition creates two neighboring centers of chirality.
However, the tertiary stereocenter was found to epimerize under
the reaction conditions, which precluded an unequivocal
assessment of the stereopreference. However, considering a
similar mechanism to that observed for open-chain
hydroxyketols, which form a Z-configured hydroxy-enamine
nucleophile at the FSA K85, the rigid cyclopentanone ring would
impose an E-configured structure instead for geometrical
reasons (Figure 3). As a consequence, in both cases the -
proton of the nucleophile component would be abstracted from
the same enantiotopic face and as a result, the stereogenic
centers created from 8 and L-GA3P would be formed anti
selectively, whereas the contrary is true for the open chain
hydroxyketol nucleophiles. To unambiguously determine the
configuration of the second stereocenter, 14 was transformed
into a mixture of the methyl glycosides 15a-b by treatment with
methanol under acidic conditions to fix the pyranose ring
structure (Scheme 3).[19] The structural and conformational
other nucleophiles (see SI). This showed
a total FSA
enantiopreference towards L-GA3P with 3g and 4 nucleophiles.
To improve this situation, various FSA variants available in our
laboratory were evaluated. Among them, FSA D6H revealed to
be the best catalyst giving a complete conversion after 2 hours
with both GA3P enantiomers (see SI). In addition, this variant
displayed no electrophile enantiomer discrimination with 3a and
3g and very low with 4 and 5 as nucleophiles. As a general
tendency, the D6H variant gave higher reaction rates with any of
the electrophile enantiomers (see SI).
Owing to the complications from D-GA3P isomerisation occurring
from residual TPI contamination in the FSA samples (e.g., 15%
of DHAP was formed within 2 hours in the absence of
nucleophiles), for the characterization of products formed we
decided to rather carry out preparative syntheses using L-GA3P
and FSA D6H as catalyst. From reactions with nucleophiles 3a,
4, 5 and 3g performed on a 0.15-mmol scale the corresponding
four phosphorylated aldol products (8-11) were isolated in good
yields as their Na salts (Table 1).
Table 1. Small scale synthetic experiments conducted with L-GA3P as
electrophile
Nucleophile
(Nu-H)
Product
Nr. Yield [%] Configuration
d.e. [%]
>98
3a
8
85
(S,S)
4
5
9
83
85
(S,S)
(S,S)
>98
>98
10
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characterization of the two epimers 15a-b was performed by a
cyclopentanone to GA3P in good yields. It appears that the
thorough NMR study, including key NOE contacts through-space. presence of the phosphate ester moiety is necessary for the
Interestingly, 15a-b were recovered in only one configuration (R)
at the tertiary stereocenter, possibly directed by the
thermodynamic control of the acetalization. The stereochemistry
of compounds 15a-b, indicates that FSA D6H was fully (S)-
stereoselective for generation of the stereogenic center upon
addition to the electrophile.
reactivity since only the best known acceptor substrate for FSA,
namely GA3P, gave good results. The potential breadth of
further applications will be accessible, once more active enzyme
variants may be designed and become available. Work in this
direction is currently being carried out in our laboratories.
Acknowledgements
This project has received funding from the Auvergne council, the
Bundesministerium für Bildung und Forschung (BMBF grant
0315775B PT-J to W.-D.F.), Ministerio de Economía
y
Competitividad (MINECO) (grant CTQ2015-63563-R to P.C and
CTQ2015-64436-P to T.P.) and COST action CM1303 Systems
Biocatalysis. Anne Samland (Stuttgart University) is warmly
thanked for a gift of plasmid for expressing FSA K85M.
Keywords: fructose-6-phosphate aldolase • aldolisation •
enzyme • phosphorylated compounds • biocatalysis
Scheme 3. Cyclic isomers from phosphorylated and unphosphorylated
derivatives utilized for unambiguous stereochemical analysis.
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Figure 3. Schematic representation for FSA K85-enamine nucleophile
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D6H catalysis: A)
Z
configured FSA K85-hydroxylenamine and B)
E
[2]
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FSA D6H variant, by stereoselective addition of aliphatic
nucleophiles such as ethanal, propanone, butanone and
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10.1002/chem.201701020
Chemistry - A European Journal
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Fructose-6-phosphate aldolase (FSA)
Raquel Roldán, Israel Sanchez-Moreno,
Thomas Scheidt, Virgil Hélaine, Marielle
Lemaire, Teodor Parella, Pere Clapés*,
Wolf-Dieter Fessner* and Christine
Guérard-Hélaine*
Propan
one
Ethanal
is famous for being the only aldolase
that is able to convert different
nucleophiles bearing an alpha-
hydroxylated carbonyl group. Here we
uncover a hidden activity: FSA can
also catalyze aldol additions of
Hydroxy
ethanal
Hydroxy
butanone
Page No. – Page No.
Breaking the dogma of aldolase
specificity: Simple aliphatic ketones
and aldehyde are nucleophiles for
fructose-6-phosphate aldolase
aliphatic
propanone, ethanal, butanone and
even cyclopentanone, to
glyceraldehyde 3-phosphate thereby
providing a stereoselective access to
novel aldol adducts.
nucleophiles
such
as
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