Chiral resolution through stereoselective transglycosylation by sucrose phosphorylase: Application to the synthesis of a new biomimetic compatible solute, (R)-2-O-α-d-glucopyranosyl glyceric acid amide

Article abstract of DOI:10.1039/c3cc47249c

Sucrose phosphorylase catalysed glycosylation of glyceric acid amide with complete regio- and diastereo-selectivity is studied. (R)-2-O-α-d- Glucopyranosyl glyceric acid amide was obtained in high yield from single-step transformation of racemic glyceric acid amide and sucrose. Non-productive binding of (S)-glyceric acid amide appeared to underlie strict enantiodiscrimination by the enzyme, thus supporting chiral resolutions based on stereoselective transglycosylation.

Full text of DOI:10.1039/c3cc47249c

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Chiral resolution through stereoselective
transglycosylation by sucrose phosphorylase:
application to the synthesis of a new biomimetic
compatible solute, (R)-2-O-a-D-glucopyranosyl
glyceric acid amide†
Cite this: Chem. Commun., 2014,
50, 436
Received 22nd September 2013,
Accepted 29th October 2013
DOI: 10.1039/c3cc47249c
Patricia Wildberger,a Lothar Breckerb and Bernd Nidetzky*a
www.rsc.org/chemcomm
Sucrose phosphorylase catalysed glycosylation of glyceric acid amide
with complete regio- and diastereo-selectivity is studied. (R)-2-O-a-D-
Glucopyranosyl glyceric acid amide was obtained in high yield from
single-step transformation of racemic glyceric acid amide and sucrose.
Non-productive binding of (S)-glyceric acid amide appeared to underlie
strict enantiodiscrimination by the enzyme, thus supporting chiral
resolutions based on stereoselective transglycosylation.
Scheme 1 Proposed mechanism of sucrose phosphorylase for kinetic
resolution of 2 and chiral synthesis of 3.
Biocatalytic transglycosylations constitute highly expedient trans-
formations in synthetic organic chemistry. Transglycosylations can through diastereoselective glycosylation has not so far been a useful
be applied broadly to the targeted modification of small molecules, option for biocatalytic synthesis.
polymers and even materials through covalent attachment of sugar
We herein provide a first-time example of enzymatic transglycosyla-
moieties.1–4 Chemically, transglycosylations proceed via transfer of tion with complete regio- and stereo-selectivity. When using sucrose (1)
a glycosyl residue from a donor glycoside onto a suitable functional as a glucosyl donor and racemic glyceric acid amide (2) as an acceptor
group, typically an alcohol, on an acceptor substrate (Scheme 1). (Scheme 1), sucrose phosphorylase was highly specific for formation of
Biocatalytic transglycosylations usually involve robust and (R)-2-O-a-D-glucopyranosyl glyceric acid amide (3) as a single diastereo-
co-substrate-independent enzymes that utilise inexpensive donor merically pure glucosyl transfer product. The enzymatic reaction thus
substrates. They are therefore regarded as practical methodology of supports different chiral resolution strategies for 2 in which synthesis of
glycoside synthesis that can be applied readily across scales. A number a structurally and stereochemically well-defined glycoside 3 or recovery
of glycosidic products prepared through biocatalytic transglycosyla- of an unreacted acceptor (S enantiomer of 2) is targeted. Based on the
tions have important industrial applications in the food, cosmetic and results of molecular docking analysis we explain how sucrose phos-
fine-chemical sectors.5,6 Despite these clear advantages, however, there phorylase achieves strict discrimination between R and S enantiomers
exists a significant gap in the scope of biocatalytic transglycosylations, of 2. We also provide evidence of the role of the acceptor’s 1,2-diol
in that known enzymes often lack acceptor substrate regioselectivity, group for substrate binding recognition and positioning for regio- and
stereoselectivity, or both.7 Site-heterogeneous glycosylation of acceptor stereo-selective catalytic glycosylation. Our findings suggest that sucrose
substrates offering more than a single reactive group severely restricts phosphorylase-catalysed chiral synthesis from racemic diols (for which
practicability of the method. Also, chiral resolution of racemic alcohols 2 is a representative example) could be an interesting novel strategy
for application of enzymatic transglycosylations in organic synthesis.
a Institute of Biotechnology and Biochemical Engineering,
The work of Franssen and co-workers has recently shown that the
Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
enzyme promotes 2-O-a-D-glucosylation of various diols with useful
E-mail: bernd.nidetzky@tugraz.at
stereoselectivity.8 Glycoside 3 was synthesised herein for the first time.
b
¨
Institute of Organic Chemistry, University of Vienna, Wahringerstraße 38,
A-1090 Vienna, Austria
It is a close structural analogue of (R)-2-O-a-D-mannopyranosyl glyceric
acid amide (4), also called Firoin-A, which was isolated from the
thermophilic bacterium Rhodothermus marinus. Firoin-A is an intra-
cellular compatible solute that the organism accumulates in response
to a high salt environment. Firoin-A is described as a useful stabiliser of
proteins in different applications, refolding processes in particular.9
† Electronic supplementary information (ESI) available: Experimental procedures
used; NMR spectra of 3; the CD spectrum of residual 2 isolated from the reaction
mixture; docking poses of R and S-forms of 1,2-diol acceptors glucosylated by
sucrose phosphorylase; time course of synthesis of 3; active-site superimposition
for sucrose phosphorylases from B. adolescentis and L. mesenteroides; table sum-
marising reaction conditions and product yields. See DOI: 10.1039/c3cc47249c
436 | Chem. Commun., 2014, 50, 436--438
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Sucrose phosphorylase promotes transglycosylation from sucrose intensive dipolar interaction, detected in the 2D NOESY spectrum.
in a two-step catalytic mechanism, depicted in Scheme 1, that The a-anomeric form of the glucopyranosyl moiety was determined
involves formation of a b-glucosyl enzyme intermediate.10 In the from the typical 13C and 1H chemical shifts as well as from the small
natural reaction of the enzyme, phosphate is the terminal glucosyl coupling constant (3.8 Hz) between the protons in positions one and
acceptor and a-D-glucose-1-phosphate is formed. In the absence of two of the glycoside (Fig. 1). Although the absolute configuration of
phosphate, when an alternative acceptor alcohol is present, a new C-20 could not be elucidated from the NMR data, the absence of
O-a-D-glycoside is synthesised, provided that the acceptor competes signal splitting of H-1 of the glycoside and H-20 of the aglycon
effectively with water for reaction with the enzyme intermediate. strongly hinted the presence of a diastereomerically pure glycoside
Acceptor 2 was synthesised chemically as described in the ESI† (d.e. Z99%). A racemic mixture would have led to a double set of
(Materials). Purified sucrose phosphorylase from Leuconostoc signals with slightly different chemical shifts (ESI,† Fig. S1).
mesenteroides, recombinantly produced in Escherichia coli as
In the next step, we applied chiroptical techniques for the deter-
described in the ESI† (Enzyme production), was used. We incubated mination of the transglycosylation product’s absolute configuration.
a mixture of 1 and 2 (each 500 mM) in the presence of an enzyme Due to unavailable reference data for the product, an indirect
(30 units of activity per mL; see the ESI† for methods and reaction approach was chosen. Residual 2 was recovered from the reaction
conditions used) and analysed samples from the reaction mixture mixture as described in the ESI† (conversion of 2 Z35%) and
using liquid chromatography. Release of fructose from conversion of the optical rotation [a]2D2 of the sample was determined to be +151.
1 exceeded by up to 2-fold the concurrent formation of glucose, The significant sign of the measurement can be compared with the
providing indirect evidence for the occurrence of transglycosylation literature where an optical rotation of [a]2D0 = ꢀ631 was reported for
next to a significant hydrolysis of the enzyme intermediate. The (R)-2.11 A circular dichroism spectrum of the sample (ESI,† Fig. S2)
product mixture in which about 45–50% of 2 has been consumed showed only one extremum at l = 230 nm with De = +15.7. These data
was analysed by NMR, and Fig. 1 summarises pertinent results. The can be compared to circular dichroism spectra of (R) and (S)-serine
glucosylation of 2 was confirmed and it was shown that only a single because serine shares some structural similarity with 2. The S
main transglycosylation product has been formed in the enzymatic enantiomer of serine shows a similar positive circular dichroism band
reaction. Identity of the fully stable product was established as 2-O-a- as the purified residual sample whereas the circular dichroism band
D-glucopyranosyl glyceric acid amide. The interglycosidic linkage was of (R)-serine is negative.12,13 In summary, therefore, strong evidence is
3
identified by the JH–C couplings determined from HMBC spectra presented supporting the suggestion that 2 recovered from the
showing interactions between the H-1 of the glycoside and C-20 of reaction with sucrose phosphorylase has S configuration. Stereo-
the aglycon, as well as between the C-1 of the glycoside and H-20 of chemical structure of the glucosylation product is assigned accord-
the aglycon. Furthermore, the two protons in these positions have an ingly as (R)-2-O-a-D-glucopyranosyl glyceric acid amide (3). Synthesis of
pure 3 necessitates that sucrose phosphorylase provides complete
regio- and diastereo-selectivity in the reaction with 2 which is a
combination of selectivity properties not previously observed in other
transglycosidase-catalysed conversions. Remaining 2 was recovered in
excellent yield from the reaction (>40%), suggesting the possibility of
kinetic resolution via stereoselective transglycosylation.
To gain a molecular interpretation of the phosphorylase’s remark-
able selectivity, we performed energy-minimised docking studies on the
high-resolution crystal structure of the enzyme (from Bifidobacterium
adolescentis)10 whereby the R and S enantiomers of 2 were placed
flexibly in the acceptor-binding pocket of the b-glucosyl enzyme inter-
mediate. Note: active-site residues of sucrose phosphorylase from
B. adolescentis are superimposable on the homology model of the
enzyme from L. mesenteroides (ESI,† Fig. S5). Fig. 2 illustrates the
obtained best-fit binding modes for (R)-2 and (S)-2. Reactive group
arrangement was highly plausible for the (R)-2 pose where the 2-OH
was well aligned for nucleophilic attack on the glucosyl anomeric
carbon, catalytically assisted by Glu-232 functioning as a general base,
consistent with the proposed catalytic mechanism of the enzyme.
The primary hydroxyl of (R)-2 was coordinated tightly by Asp-290 and
Fig. 1 Details of 2D NMR spectra recorded from the reaction mixture to
determine the product’s glycosidic linkage. TOCSY and COSY traces of the
Gln-345. The amide group of (R)-2 was bound weakly by comparison.
anomeric proton H-1 allow for determination of further protons in the glucosidic
moiety. The NOESY trace indicates spatial closeness of H-1 to H-20 and H-3ab0 For (S)-2 the predicted binding mode was characterised by a con-
of the glyceric acid amide aglycon. The concomitant trace of H-20 is shown.
spicuously large distance between the reactive carbon and oxygen,
clearly indicating that the (S)-2 pose was non-productive for reaction.
As in (R)-2, however, the primary hydroxyl of (S)-2 was accommodated
HSQC and HMBC traces indicate the 1JH–C and 3JH–C couplings of H-1 and
H-20, respectively. The long-range coupling over three bonds clearly
indicates the glycosidic bond to be formed with the secondary alcohol
through hydrogen bonding interactions with Asp-290 and Gln-345.
Other acceptors harbouring a terminal diol moiety (Fig. 3) were
of the glyceric acid amide. The signal group directly neighboured to those
of H-20 belongs to the H-2 of the not transformed substrate.
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of synthesis of 3 when 2 was present in equimolar or in 2.5-fold molar
excess over 1. Up to two-thirds of 1 were utilised for glucosylation of
(R)-2 while the remainder substrate was lost to hydrolysis. Product 3
was obtained in a maximum concentration of 240 mM. Quantitative
utilisation of the initial (R)-2 present was possible (ESI,† Fig. S4,
panel C). Glucosylation of glucose occurred to a minor extent under
these conditions (o5%). Production of 3 was lowered substantially
(r140 mM) upon decreasing the concentration of 2 to 400 mM, now
equaling the applied concentration of 1 (ESI,† Fig. S4, panel B), or
when using 1 (1000 mM) in excess over 2 (400 mM). Use of 2 in excess
over 1 is therefore supported. Procedures for efficient preparative
work-up of reaction mixtures from sucrose phosphorylase-catalysed
transglycosylations have previously been developed.5,14
Fig. 2 Best-fit docking poses for binding of (R)-2 (A) and (S)-2 (B) to the
covalent intermediate of sucrose phosphorylase having a b-glucosyl
residue linked to Asp-192 (PDB-entry 2gdv, molecule A).
Summarising, single-step diastereoselective glucosylation of 2
provides convenient access to the new biomimetic glycoside 3
through chiral synthesis. The same reaction is exploitable through
chiral resolution to obtain (S)-2 (ESI,† Fig. S4, panel C). Chiral
1,2-diols are important target molecules in organic chemistry, and
their preparation through chiral resolution is therefore of consider-
able interest.18 Relying on the selectivity of enzymes, biocatalytic
glycosylation might present an alternative to reported two-step
esterification processes catalysed by lipases/esterases.19
We acknowledge financial support from the Austrian Science
Fund FWF (project L586-B03); V. N. Belov (Max Planck Institute for
Biophysical Chemistry, Goettingen, Germany) for synthesis of 2;
S. Felsinger (Institute of Organic Chemistry, University of Vienna,
Fig. 3 Diastereoselectivity of sucrose phosphorylase in the glucosylation
of 1,2-diols correlated with the ratio of reactive atom distances (C1ꢁ ꢁ ꢁO2)
in (S)- and (R)-acceptor poses from molecular docking.
previously shown to be glucosylated by the enzyme with useful Austria) for recording several NMR spectra; and C. Araman and
stereoselectivity, resulting in 2-O-a-D-glucosidic products of high N. K. Chu (Institute of Biological Chemistry, University of Vienna,
diastereomeric purity.8,14 Molecular docking of (R)- and (S)-forms of Austria) for help in recording CD spectra.
glyceric acid and 3-methoxy-1,2-propanediol to sucrose phosphorylase
underpins the observed preference for reaction with one enantiomer of
the acceptor, suggesting this to be the (R)-form in each case (ESI,†
Fig. S3). In search of an interpretable structural parameter of enzyme
stereoselectivity in reactions with different acceptors, we compared
Notes and references
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