Transketolase–Aldolase Symbiosis for the Stereoselective
Preparation of Aldoses and Ketoses of Biological Interest
Christine Gu8rard-H8laine,
aMaxime De Sousa Lopes Moreira,
aNadia Touisni,
aLaurence Hecquet,
aMarielle Lemaire,
aand Virgil H8laine
a,*
a Universit8 Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de Clermont-Ferrand, F-63000
Clermont-Ferrand BP 80026, F-63171 AubiHre, France Fax: (++33)-4-7340-7717; e-mail: virgil.helaine@uca.fr
Received: February 17, 2017; Revised: May 22, 2017; Published online: June 7, 2017
Supporting information for this article can be found under https://doi.org/10.1002/adsc.201700209. Abstract: Transketolase (TK) was used as
biocata-lyst to produce useful aldoses that serve as aldolase electrophiles to prepare monosaccharides of biolog-ical interest. Unconventionally, TK was implement-ed in a synthetic pathway, as in metabolism, i.e., without using a specially designed nucleophile such as hydroxypyruvate to shift the equilibrium. Unusu-ally, formaldehyde was taken as the general TK ac-ceptor substrate to generate aldehydes. Here it was demonstrated that this method can be generalized: the substrates and the aldolase were successfully varied, coupled with other enzymes in a one-pot, one-step green cascade reaction process involving four enzymes with atom economy. In addition, new assays were developed for both monitoring the progress of the reactions and assessing the purity of the synthesized products.
Keywords: aldolase; cascades; C–C coupling; phos-phorylated monosaccharides; transketolase
Stereoselective C–C bond formation is an essential step in organic chemical synthesis. For this purpose, enzymes play a key role, being able to catalyze reac-tions with atom economy in high yields under mild conditions and with good stereocontrol of the new
asymmetric centers created.[1]Of these enzymes,
aldo-lases, especially dihydroxyacetone phosphate
(DHAP) aldolases, have been the most widely used for ketose preparation in organic synthesis, by adding a three-carbon nucleophile to a wide range of alde-hyde electrophiles leading to a great number of
com-pounds of biological interest.[2] Since these enzymes
are strict with regard to their nucleophiles (DHAP or
borate analogues[3]), nowadays one main challenge is
to implement new and efficient accesses to
electro-philes.[4] The most recently discovered
dihydroxyace-tone (DHA) aldolase, fructose-6-phosphate aldolase
(FSA),[5]displays a comparable wide activity spectrum
toward the electrophile, but interestingly an unexpect-ed versatility as regards the nucleophile, broadening scope for the preparation of compounds inaccessible
up to now.[6] Another class of enzymes of particular
interest is ThDP-dependent transketolase (TK), which in vivo catalyzes the reversible transfer of a hy-droxyacetyl group between a ketose phosphate nucle-ophile and an aldose phosphate electrnucle-ophile. For syn-thetic applications, TK has been used with hydroxy-pyruvate (HPA) as nucleophile, rendering the
reac-tion irreversible by carbon dioxide release.[7] Many
useful ketoses have been prepared with this method.[8]
Both aldolases and TK are complementary C–C bond-forming or bond-breaking enzymes that can be coupled in sustainable stereoselective strategies to gain access to new phosphorylated monosaccharides. This concept was explored in the past in one example where an aldolase gave the TK aldehyde substrate, to
finally produce a phosphorylated ketose.[9] By
con-trast, the originality of the present work resides in the TK-biocatalyzed preparation of aldoses from a chiral pool of natural sugars as an electrophile source for al-dolases (Scheme 1). To the best of our knowledge, this approach has been explored only once with a
syn-thase to prepare a labeled dehydroquinic acid.[10]
Scheme 1. Strategy involving TK and aldolase as comple-mentary tools for stereoselective C–C bond formation.
Our strategy was first assessed by preparing d-er-ythrose-4-phosphate (E4P), a substrate of many en-zymes in central metabolism (transaldolase, transketo-lase, phosphoketotransketo-lase, fructose-1,6-diphosphate aldo-lase, etc.) and an important metabolite found particu-larly in the shikimate pathway. Since this latter route is found in plants and microorganisms but not in mammals, it has been extensively studied in order to design new antibiotics and herbicides, especially the enzyme involved in the first step: 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS) using
E4P as substrate.[11] The demand for synthetic routes
leading to E4P is thus strong and highly
challeng-ing.[12] In addition, E4P can serve as an electrophilic
substrate for aldolases, leading to complex higher sugar homologues of biological interest. We thus have designed a multi-enzymatic sequential process where (i) TK gives access to E4P starting from d-fructose-6-phosphate (F6P), and from the simplest aldehyde (formaldehyde), and then (ii) where E4P is involved as an electrophile in subsequent FSA-catalyzed reac-tions (Scheme 2). A one-pot, two-step process was necessary, since F6P is a substrate for both enzymes. Using three of the known FSA nucleophiles, namely DHA, hydroxyacetone (HA) and hydroxybutanone (HB), several products of particular interest, i.e.,
spe-cifically d-sedoheptulose-7-phosphate 2[13]and its
ana-logues (3[12b] and 4), could be efficiently synthesized.
Compound 2 is the precursor of many bioactive natu-ral compounds such as acarbose, an antidiabetic drug, validamycin, a crop protectant, various
mycosporine-like amino acid sunscreens[14] and the pyralomycins,
a group of antibiotics.[15]Finally, using glycolaldehyde
(GA) as FSA nucleophile, d-altrose-6-phosphate 5, an aldose obtained in the past as a by-product (14% yield),[16]can also be prepared.
Both for the synthesis of E4P and during the aldo-lase reactions, special attention was paid to total con-version of TK or FSA substrate (F6P and E4P, respec-tively) in order to avoid tedious purification of the final phosphorylated monosaccharide. Since TK was used here in a reversible reaction, formaldehyde was added in a sufficient amount to totally shift the equi-librium. Thus, the optimum quantity of formaldehyde was sought to complete the reaction in at most 8 h in order to prevent any degradation of E4P. The time course of the reaction was therefore followed by mon-itoring the appearance of DHA (Scheme 2) using glycerol dehydrogenase (GDH) in the presence of NADH (see the Supporting Information). The results indicated that the formaldehyde concentration must be below 400 mM due to TK inhibition. The use of 12 equivalents (100 mM) of formaldehyde gave a nearly total reaction (>95% of DHA formed) after 7 h reaction time, whereas use of 3 and 6 equivalents led to less than 90% of DHA formed, probably be-cause the thermodynamic equilibrium was reached (Supporting Information, Figures S1 and S2). Thus using the optimal conditions (12 equivalents), 200 mg of F6P were totally transformed into E4P 1. A simple precipitation was chosen for the purification of all the phosphorylated monosaccharides obtained, to avoid any product degradations. But purity had to be as-sessed because of the presence of NaCl, coming from pH adjustments. Thus, 170 mg of compound 1 were obtained as a white powder. To measure its purity, we
Scheme 2. TK-mediated synthesis of d-erythrose-4-phosphate and TK-FSA tandem reactions for the production of various hexose, heptuloses and octulose phosphates.
developed a new assay where prior conversion of E4P into F6P using TK in the presence of DHA was fol-lowed by the known F6P assay using glucose-6-phos-phate dehydrogenase (G6PDH)/phosphoglucose
iso-merase in the presence of NADP.[17] To the best of
our knowledge E4P has never before been assayed by this route (Scheme 3, part A). E4P purity was found to be 70%, corresponding to a 75% yield, showing this synthesis to be more powerful than those already
described.[18] 13C NMR spectra recorded at a low
con-centration (10 mg/mL) in D2O at acidic pH showed
the hydrated form as the main compound, as found in the literature.[19]
E4P was then used with the four FSA nucleophiles to prepare terminally phosphorylated sugars: d-sedo-heptulose-7-phosphate (S7P) 2 and analogues 3 and 4 as well as d-altrose-6-phosphate (A6P) 5. Quantities of DHA, HA, HB and GA were set as described by our group when G3P was the electrophile, i.e., 6, 6,
12 and 6 equivalents, respectively.[20] Reaction
prog-ress was followed by assaying E4P disappearance as established above. Interestingly, A6P was discovered to be a substrate of G6PDH, enabling its direct pro-duction to be monitored during its synthesis (Scheme 3, part B). After purification, the corre-sponding phosphorylated monosaccharides 2, 3, 4 and 5 were obtained as white powders. Their purities were determined using FSA-catalyzed retroaldolization coupled with the GDH assay of DHA, HA and HB produced in situ. As stated above, A6P was directly assayed with G6PDH. Thus, the purities of 2, 3, 4 and 5 were calculated to be 76, 53, 51 and 79%, respec-tively, corresponding to 70, 75, 65 and 61% respective yields.
To further demonstrate the versatility of our stereo-selective process, we explored an unprecedented
strat-egy where TK could produce both the electrophile and the nucleophile precursor for an aldolase. Thus, we decided to switch to a more general and sustaina-ble process (i) by designing a multienzymatic cascade, (ii) by diversifying both the substrate by choosing a TK nucleophile among the l-non-phosphorylated sugar series, and the aldolase by replacing FSA, and (iii) by using the second product released during the TK-catalyzed step (DHA), leading to a better atom economy. All these criteria could be met by imple-menting the process depicted in Scheme 4 as a proof of concept. Thus we started from l-sorbose, giving in situ another useful aldose: l-threose. DHA also formed was enzymatically phosphorylated to DHAP, involving an ATP recycling system catalyzed by
pyru-vate kinase (PK).[20]This parallel irreversible reaction
caused the equilibrium shift of the TK-catalyzed reac-tion, and thus enabled the use of only one equivalent of formaldehyde. DHAP in situ generation allowed us to introduce the most widely used DHAP-aldolase: RAbbit Muscle Aldolase (RAMA). This cascade led to compound 6, an l-heptulose-1-phosphate already known as an unusual product of fructokinase, but
never isolated and characterized.[21]The reaction was
carried out as a one-pot, one-step process, with all four enzymes and substrates mixed together at the 100 mg scale. A slight excess of l-sorbose over form-aldehyde was used, PEP being the default reagent. Monitoring the whole reaction progress was possible by assaying pyruvate appearance (see the Supporting Information). Although it has been demonstrated that phosphorylated compounds from the d-series are
better substrates for TK,[22] l-sorbose was,
remarka-bly, a TK substrate, and the reaction proceeded smoothly until complete disappearance of DHAP. The ketose phosphate 6 was then purified. The purity
Scheme 3. Spectrophotometric assays developed for monitoring the reactions and determining the purities of compounds by following the production of NADPH.
was found to be 50% corresponding to an 80% yield,
determined by 1H NMR using DMF as an internal
standard (retroaldolization with RAMA failed). We note that this methodology could be generalized to other TK substrates or other DHAP-aldolases.
We have successfully used TK in an unusual way with formaldehyde as new synthetic electrophile sub-strate to produce aldoses. In a sequential process (where the starting ketose is a substrate for both TK and FSA), or a domino process (when involving a DHAP aldolase), these latter compounds were then converted by aldolases into useful higher homologues. We have also shown that this new stereoselective method can be generalized to monosaccharides from the d- or l-series, phosphorylated or not, as starting compounds. In particular, in the one-pot multienzy-matic cascade process, TK efficiently provided both electrophile and nucleophile substrates for aldolase, in a synthesis with high atom economy. Interestingly, the set of compounds obtained in fair yields are re-ported to be involved in metabolism, or are analogues of natural compounds. Products 4 and 6 had never been prepared before, whereas compounds 1, 3 and 5 were obtained in a more straightforward way with better yields. In addition, new assays were developed enabling an easy quantification of most of the com-pounds prepared. Finally, an original, stereoselective, sustainable and straightforward approach was pro-posed that is generalizable for preparing new valuable compounds.
Experimental Section
Typical Procedure for Syntheses using FSA
To water (5 mL) containing d-E4P (0.12 mmol, 42 mg powder, 70% purity) and 6 equiv. (for DHA, HA or GoA)
or 12 equiv. (for HB) of aldolase nucleophile, adjusted to pH 7.3 with NaOH, were added 15 mg of FSA WT (1 U/mg) or FSAA129S (15 mg: 3.5 U/mg) when DHA was used. The reaction was stopped after approx. 5 h when E4P had totally disappeared, using the corresponding spectrophotometric assay (the sample being previously acidified to avoid retro-aldol reaction in the cuvet), except with GoA, where d-al-trose-6-phosphate was directly assayed (see protocol in the Supporting Information). The reaction was stopped by de-creasing the pH to 3, and the resulting precipitate was re-moved by centrifugation. The solution was then freeze-dried overnight. The solid obtained was re-suspended in 3 mL of water and centrifuged for 10 min at 10,000 rpm at 488C. The pellets were discarded and 18 mL of EtOH were added to the 3 mL of water (pH adjusted to 7). The solution was cen-trifuged and the pellets re-suspended in 3 mL of water. The same operation (water-ethanol) was repeated twice. The pel-lets were finally freeze-dried to yield each product (2, 3, 4 or 5) as a white powder. Purity was determined by the ap-propriate spectrophotometric assay (see the Supporting In-formation).
Typical Procedure for Syntheses using RAMA
To water (7 mL) containing l-sorbose (40 mg, 0.22 mmol), 25 mL of a solution of ThDP (10 mg/mL), 200 mL of a solu-tion of MgSO4 (10 mg/mL), 15 mL of CH2O (0.18 mmol),
320 mL of a PEP solution (0.47M, 0.15 mmol) and 80 mL of an ATP solution (50 mM), adjusted to pH 7.3 with NaOH, were successively added 15 mg of TK (14 U/mg), 1.25 mg of PK (200 U/mg), 2.5 mg of DHAK (6 U/mg) and 25 U of RAMA. The reaction was stopped after approx. 24 h when PEP was totally consumed and no DHAP could be detected. The solution was then freeze-dried. The solid was re-sus-pended in 1 mL of water. 6 mL of EtOH were added, and the mixture was centrifuged. The solid was washed twice again with the same composition (1/6 water/EtOH). Lastly, water was added, and after centrifugation the solid was dis-carded. The solution was freeze-dried to yield compound 6 as a white powder.
Scheme 4. Multi-enzymatic one-pot, one-step cascade reactions where TK provided both electrophile and nucleophile pre-cursors for aldolase.
Acknowledgements
This work was financially supported by the CNRS and the French Ministry of Research.
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