Expedient synthesis of C-aryl carbohydrates by consecutive biocatalytic benzoin and aldol reactions

Article abstract of DOI:10.1002/chem.201406156

The introduction of aromatic residues connected by a C-C bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., C-aril-sLex). In this work, an expedient asymmetric de novo synthetic route to new aryl carbohydrate derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented. First, the benzoin reaction of aromatic aldehydes to dimethoxyacetaldehyde is conducted, catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I. Then, the a-hydroxyketones formed are reduced by using NaBH₄ yielding the anti diol. After acetal hydrolysis, the aldol addition of dihydroxyacetone, hydroxyacetone, or glycolaldehyde catalyzed by the stereocomplementary d-fructose-6-phosphate aldolase and l-rhamnulose-1-phosphate aldolase is performed. Both aldolases accept unphosphorylated donor substrates, avoiding the need of handling the phosphate group that the dihydroxyacetone phosphatedependent aldolases require. In this way, 6-C-aryl-l -sorbose, 6-C-aryl-l-fructose, 6-C-aryl- l-tagatose, and 5-C-aryl-l-xylose derivatives are prepared by using this methodology.

Full text of DOI:10.1002/chem.201406156

DOI: 10.1002/chem.201406156
Full Paper
&
Biocatalytic Carbohydrate Analogues
Expedient Synthesis of C-Aryl Carbohydrates by Consecutive
Biocatalytic Benzoin and Aldol Reactions
Karel Hernꢀndez,[a] Teodor Parella,[b] Jesffls Joglar,[a] Jordi Bujons,[a] Martina Pohl,[c] and
Pere ClapØs*[a]
Abstract: The introduction of aromatic residues connected
by a CÀC bond into the non-reducing end of carbohydrates
is highly significant for the development of innovative struc-
using NaBH4 yielding the anti diol. After acetal hydrolysis,
the aldol addition of dihydroxyacetone, hydroxyacetone, or
glycolaldehyde catalyzed by the stereocomplementary d-
tures with improved binding affinity and selectivity (e.g., CÀ fructose-6-phosphate aldolase and l-rhamnulose-1-phos-
aril-sLex). In this work, an expedient asymmetric “de novo”
synthetic route to new aryl carbohydrate derivatives based
on two sequential stereoselectively biocatalytic carboligation
reactions is presented. First, the benzoin reaction of aromat-
ic aldehydes to dimethoxyacetaldehyde is conducted, cata-
lyzed by benzaldehyde lyase from Pseudomonas fluorescens
biovar I. Then, the a-hydroxyketones formed are reduced by
phate aldolase is performed. Both aldolases accept unphos-
phorylated donor substrates, avoiding the need of handling
the phosphate group that the dihydroxyacetone phosphate-
dependent aldolases require. In this way, 6-C-aryl-l-sorbose,
6-C-aryl–l-fructose, 6-C-aryl–l-tagatose, and 5-C-aryl-l-xylose
derivatives are prepared by using this methodology.
Introduction
rivatives have been found to be selective inhibitors of the
sodium-dependent glucose co-transporter 2 (SGLT2) with a po-
tential medicinal use for the treatment of type 2 diabetes.[4]
The synthesis of C-aryl carbohydrate derivatives is a complex
task that requires a number of cumbersome protection and
deprotection steps.[3a,5] The stereoselective aldol addition reac-
tion constitutes a straightforward and efficient bottom-up ap-
proach for the de novo construction of carbohydrate-like scaf-
folds.[1b,6] In that respect, biocatalytic carboligation reactions
have been recognized as a powerful methodology for such
transformations due to their high selectivity, their catalytic effi-
ciency, and the often uncompromised stereoselectivity.[7] New
and diverse structure types can be built up by a single or se-
quential combination of independent enzymatic carboligation
reactions in which the product of one reaction becomes the
substrate for the next one,[8] or even by convenient combina-
tion with chemical steps (e.g., Sharpless epoxidation).[9] Fur-
thermore, the existence of stereocomplementary enzymes with
similar substrate tolerance offers the potential of stereodiver-
gent product generation from a common pair of synthetic pre-
cursors.[8j,10]
Carbohydrates play essential roles in a broad range of signal-
ing and recognition processes; for example, bacterial and viral
infections, cancer metastasis, and inflammatory reactions.[1] The
introduction of aromatic residues at the non-reducing end of
carbohydrate structures, that is, C-aryl carbohydrates—different
from the C-aryl glycosides, in which the CÀC bond is connect-
ed to the reducing end—is highly significant for the develop-
ment of innovative oligosaccharide structures to improve the
binding affinity and the selectivity.[2] For instance, stabilization
of the core of sLex by an intramolecular hydrophobic contact
was found to be an important parameter in its affinity for E-se-
lectin.[3] Moreover, C-aryl carbohydrates themselves exhibit in-
teresting biological activities; for example, 5-C-aryl-l-xylose de-
[a] K. Hernꢀndez, Dr. J. Joglar, Dr. J. Bujons, Prof. Dr. P. Clapꢁs
Biotransformation and Bioactive Molecules Group
Instituto de Quꢂmica Avanzada de CataluÇa
IQAC-CSIC. Jordi Girona 18-26
08034 Barcelona (Spain)
Fax: (+34)932045904
E-mail: pere.clapes@iqac.csic.es
In this work, we endeavored to combine benzaldehyde lyase
from Pseudomonas fluorescens biovar I (BAL) with d-fructose-6-
phosphate aldolase (FSA) and l-rhamnulose-1-phosphate aldo-
lase (RhuA) for the asymmetric synthesis of 6-C -aryl-l-sorbose,
6-C-aryl-l-fructose, 6-C-aryl-l-tagatose, and 5-C-aryl-l-xylose, in-
cluding the deoxy derivatives.
[b] Dr. T. Parella
Servei de Ressonꢃncia Magnꢄtica Nuclear. Dept Quꢂmica
Universitat Autꢅnoma de Barcelona
08193 Bellaterra, Cerdanyola del Vallꢄs (Spain)
[c] Prof. Dr. M. Pohl
IBG-1: Biotechnologie
Forschungszentrum Jꢆlich GmbH
52425 Jꢆlich (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201406156.
Chem. Eur. J. 2015, 21, 1 – 13
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Results and Discussion
We first devised a strategy for the synthesis of the target com-
pounds consisting of a cross-benzoin reaction of aromatic al-
dehydes 1 to derivatives of acetaldehyde 2a–d, to furnish an
aryl-a,b-dihydroxyaldehyde after reduction of the carbonyl
group and hydrolysis of the acetal or oxidation of the primary
alcohol (Scheme 1). This aldehyde was the acceptor in a subse-
quent aldol addition of dihydroxyacetone (DHA; Scheme 1,
R2 =CH2OH), hydroxyacetone (HA; Scheme 1, R2 =CH3), or gly-
colaldehyde (Scheme 1, R2 =H) catalyzed by FSA or RhuA to
furnish the monosaccharide C-aryl derivatives (Scheme 1). FSA
uses unphosphorylated DHA and analogues as donor substra-
tes,[8h,l,11] which avoid the cumbersome and time-consuming
need of handling the phosphate group that the dihydroxyace-
tone phosphate-dependent aldolases require.[8a,b,I,j] Moreover,
the DHAP-dependent aldolase RhuA also accepts unphos-
phorylated DHA in the presence of borate buffer at practical
rates.[12]
For the benzoin reaction step, only dimethoxyacetaldehyde
(2a) and glycoladehyde (2b) were tolerated as acceptor sub-
strates (Scheme 1). Nevertheless, in preliminary experiments,
the oxidation of (1R,2R)-1-phenylpropane-1,2,3-triol (i.e., the
cross-benzoin product of benzaldehyde with 2b, after reduc-
tion with Zn(BH4)2) with 2-iodoxybenzoic acid (IBX), or (2,2,6,6-
tetramethylpiperidin-1-yl)oxidanyl (TEMPO)/ClOÀ, for example,
gave a complex mixture of compounds and this strategy was
therefore abandoned. Hence, dimethoxyacetaldehyde (2a) was
the selected acceptor for the rest of this study. Dimethoxyace-
taldehyde (2a) has been little exploited for synthetic purposes
in benzoin reactions and it has only been employed as an ac-
ceptor in combination with benzaldehyde,[13] 2,4-difluoroben-
zaldehyde, 4-methoxybenzaldehyde,[13] furan-2-carbaldehyde,[14]
and (benzyloxy)acetaldehyde donors[15] by using a variety of re-
actions conditions.
Scheme 2. BAL-catalyzed enantioselective cross CÀC bond formation be-
tween aromatic aldehydes and dimethoxyacetaldehyde (2a).
Thus, the reaction conditions for the benzoin reaction were
investigated for the BAL-catalyzed cross-coupling of com-
pounds 1a and 1t, respectively, to compound 2a (Scheme 2).
In an aqueous solution of Na2CO3 (0.1m, pH 8.0)/dimethyl sulf-
oxide (DMSO) (4:1 v/v), the cross-coupling products 4a (98%)
and 4t (95%), were formed after 24 and 72 h; respectively. In
both reactions the formation of the benzoins 3a and 3t were
observed immediately after the enzyme addition, which react
to form the cross-coupling products within the given reaction
times (Tables S1 and S2, as well as Figures S1 and S2, in the
Supporting Information). This finding was consistent with earli-
er studies.[13] For practical purposes, an aqueous–organic two-
phase system can be employed
for the in situ product recovery,
enabling the subsequent car-
bonyl reduction of compound 4
directly in the organic phase
without need for its isolation
and purification. In a 1:1 aque-
ous Na2CO3 (0.1m, pH 8.0)/tert-
butyl methyl ether (MTBE) two-
phase system,[15b] the cross-cou-
pling product was favored over
the homo-coupling one from
the beginning of the reaction in
comparison to the aqueous
Scheme 1. Multistep chemoenzymatic retrosynthesis of the monosaccharide aryl derivatives.
Na2CO3 (0.1 m, pH 8.0)/DMSO
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system (Tables S1–S4 and Figures S1–S4 in the Supporting In-
formation). This could be due to the distribution of the sub-
strates in the two phases: low concentration of the aromatic
donor aldehyde in the water phase (i.e., [1t]total =50 mm, dis-
tributed as [1t]aq =3 mm and [1t]orgMTBE =47 mm) and high
concentration of compound 2a in the water phase ([2a]aq =
200 mm) where BAL is located. However, the reaction rate was
slower than in the aqueous Na2CO3 (0.1m, pH 8.0)/DMSO mix-
ture (Tables S3 and S4 as well as Figure S3 and S4 in the Sup-
porting Information). An optimization study of the benzoin re-
action of compound 1t to furnish compound 2a with com-
plete conversion in 24 h was conducted (Tables S5–S7 and Fig-
ures S5–S7 in the Supporting Information). A substrate ratio of
[1t]/[2a]=1:10, [2a]=1m, 500 UBALmLÀ1, T=258C, and a 1:1
aqueous Na2CO3 (0.1m, pH 8.0)/MTBE (1:1) solvent mixture
gave the best results. These conditions were used for the rest
of the examples selected (Scheme 2).
stacking interaction) and against the protein surface. Replacing
the o-chlorophenyl moiety at the acceptor site by the bulkier
dimethoxymethyl substituent (i.e., compound 2a) would lead
to steric clashes with the protein, which could render difficult
or preclude the formation of the cross adduct. This steric effect
would be more important for larger ortho substituents, which
is consistent with the lack of reactivity observed for the cross-
benzoin reaction. However, the model for the meta-chloro-
phenyl cross-adduct shows that the Cl substituent can fit in
a subcavity located opposite to the acceptor side, which
would leave the path to the incoming acceptor aldehyde free
(Figure 1C). Alternatively, substituents at this position could be
located on the more open side of the cavity without compro-
mising the approach of the acceptor. Finally, the model for the
para-chlorophenyl cross-adduct does not show any clash with
the protein (Figure 1D), although steric restrictions can be ex-
pected for larger substituents. Concerning the rest of the
donor aldehydes, 1z (2-naphthaldehyde) was substrate, provid-
ing both homo- and cross-coupling products, whereas its
isomer 1aa (1-naphthaldehyde) and the more sterically de-
manding compound 1ab (phenanthrene-9-carbaldehyde) did
not react. Heterocycles such as compounds 1w (2-furalde-
hyde), 1x (thiophene-2-carbaldehyde), and 1ad (1H-indazole-
5-carbaldehyde) gave excellent results (Table 1). In contrast,
compounds 1y (1H-pyrrole-2-carbaldehyde) and 1ac (1H-inda-
zole-6-carbaldehyde) were ineffective as substrates for BAL.
These results were in agreement with the reported substrate
selectivity of BAL in homo-coupling reactions in aqueous
buffer/DMSO mixtures.[13,19] In this study, the target reaction
entails the aromatic aldehydes acting as donor substrates.
Their quality as acceptors was not investigated and can be op-
Our investigation into the substrate range of the BAL-cata-
lyzed cross-coupling reaction (Table 1) revealed that meta-sub-
stituted benzaldehydes furnished high conversions to the
Table 1. BAL-catalyzed cross-coupling of selected aromatic aldehydes
1 to products 2[a]
.
1
Cross[b] 3 Homo[c] 4 1 Cross[b] 3 Homo[c] 4 1 Cross[b] 3 Homo[c]
4
[d]
[d]
a
b
c
d
e
f
g
h
i
95
80
–
–
–
–
–
31
20
–
–
–
–
k
l
98
28
–
27
64
52
27
–
2
–
–
u
v
w
x
y
31
–
98
98
–
27
–
–
0
–
–
–
–
34
–
–
–
–
[d]
[d]
[d]
[d]
[d]
[d]
[d]
[d]
[d]
[d]
[d]
[d]
m
n
o
p
q
r
39
–
7
[d]
[d]
[d]
[d]
[d]
[d]
[d]
–
z
[d]
[d]
[d]
[d]
[d]
[d]
[d]
95
98
83
98
–
aa
ab
ac
[d]
[d]
53
–
–
[d]
[d]
11
–
s
t
–
98
–
[d]
j
ad 98
[a] Reaction conditions: ratio [1]/[2a]=1:10, [2a]=1m, 500 UBALmLÀ1
,
T=258C and sodium carbonate buffer (0.1m, pH 8.0, 2.5 mm MgSO4,
0.2 mm ThDP)/MTBE (1:1). [b] Cross-coupling. [c] Homo-coupling. [d] Not
detected by HPLC.
cross-coupling products (>80%), the para-substituted prod-
ucts provided moderate to low yields (27–64%), whereas, with
the ortho-substituted benzaldehydes, no cross-coupling prod-
uct was observed, with the exception of compound 1b (2-fluo-
robenzaldehyde), which is in agreement with the results ob-
tained for other thiamine diphosphate (ThDP)-dependent
lyases.[16] Aside from other electronic effects derived from the
differently substituted benzaldehydes, this could be related to
the steric limitations of the active-site cavity of BAL (Figure 1).
The crystal structure of BAL (Figure 1A) shows a funnel-shaped
entrance to its active site, which is narrower near the position
that would be occupied by the ortho substituents. An energy-
optimized molecular model of the 3c adduct bound to the thi-
amine cofactor (Figure 1B and Figure S36 IV in the Supporting
Information) shows that the adduct moiety could adopt a con-
formation with both o-chlorophenyl rings in an approximately
parallel disposition, tightly packed against each other (p–p
Figure 1. A) Crystal structure of the active site of BAL in complex with the in-
hibitor benzoylphosphonic acid methyl ester (PDB code 3D7K[17]). B–
D) Energy-optimized models of the covalently bound BAL–thiamin diphos-
phate intermediates of the 3c (B), 4k (C), and 4n (D) adducts. Residues con-
forming the active site of BAL (thin gray sticks), the thiamin diphosphate
moiety (thick green sticks), and the adduct moieties (thick orange sticks) as
well as the cavity accessible surface (gray, B–D) are shown. Models were
built from the crystal structure (A) and were optimized with Macromodel.[18]
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posite; for instance, compound 1v was a good acceptor but
not a donor substrate.[20]
porting Information). Reactions were performed directly in the
organic phase (i.e., MTBE) from the benzoin reaction and previ-
ous drying over anhydrous Na2SO4.
The examples 4a, 4b, 4g–k, 4t, 4w–x, and 4ad were scaled
up to 2 mmol. We assumed that the BAL-catalyzed reactions
were stereoselective, providing cross-coupling products with
absolute 2R stereochemistry, as reported for previous exam-
ples.[13] In the next step, different chemical reduction agents in
MTBE (Table 2) were tested with compound 4a (Scheme 3,
R1 =C6H5) as a model.
Attempts to hydrolyze acetal 6 in HCl (pH 1.0) or Dowex
8W50 H+ at 258C failed, leaving the starting material to be re-
covered intact. Increasing the temperature to 608C resulted in
product decomposition. Finally, the hydrolysis was accom-
plished by using aqueous 2m H2SO4/acetonitrile (5:1) at 258C
(Table 4 and Figure S11 in the Supporting Information). Neu-
Table 2. Reduction of the acyloin product 4a by using NaBH4, Zn(BH4)2,
and l-selectride in MTBE.
Table 4. Hydrolysis of the acetal group of compounds 5/6.
Reducing agent Reaction conversion [%] Ratio 5a/6a[a] (1R,2S)/(1S,2S)
NaBH4
Zn(BH4)2
l-selectride
>95
48
10:90
6:94
–
[b]
[c]
–
Product
Yield[a]
[a] Determined by HPLC (see Figure S8 in the Supporting Information).
The relative stereochemistry was assessed by NMR spectroscopy upon
formation of the corresponding 1,3-dioxolan-2-one derivatives (see Ta-
bles S8 and S9 as well as Figures S9 and S10 in the Supporting Informa-
tion for products S5a and S6a). [b] Prepared from ZnCl2 and NaBH4 in
THF.[21] [c] No reaction observed.
7a
7b
7g
7h
7i
7j
7k
7t
98
80
97
97
97
59
48
78
[b]
7w
7x
7ad
–
43
98
[a] Determined by HPLC. It was assumed that the syn (1R,2S)/anti (1S,2S)
ratio was identical to the reduced mixture 5/6. [b] The compound did not
survive the deprotection conditions.
Scheme 3. Chemical reduction of the acyloin products 4.
tralization to pH 7.5 with CaCO3 facilitated the removal of salt
from the medium as a precipitate of CaSO4. The product was
used directly in the next step.
NaBH4 in MTBE provided the best results, with remarkably
good stereoselectivity (from 26:84 to 3:97 (1R,2S)/(1S,2S)) and
high conversion (Table 3). The internal asymmetric induction of
the a-hydroxyketone 4 favored the anti-configured reduced
product. Notably, the reduction of the cross-benzoin addition
of benzaldehyde to glycoaldehyde with Zn(BH4)2 gave the syn-
configured (1R,2R)-1-phenylpropane-1,2,3-triol as major prod-
uct (10:90 anti/syn; see the Experimental Section and the Sup-
The last step was the enzymatic aldol addition reactions of
DHA, HA, and glycolaldehyde to the dihydroxyaldehydes 7
(Scheme 4) by using FSA wild-type, FSA A129S, and A129T var-
iants, and RhuA as catalysts.[8k,11a,22] As a result, twenty-four dif-
ferent carbohydrate derivatives (Table 5) were exemplarily ob-
tained by using this methodology.
Reaction conversions by using FSA wild-type and the A129S
variant for HA and DHA additions, respectively, were >95%.
This is consistent with the fact that FSA wild-type has the best
affinity for HA whereas FSA A129S has the best affinity for
DHA and that a-hydroxyaldehydes are well tolerated by these
biocatalysts.[8h,22] Moreover, as in N-Cbz-aminoaldehydes (Cbz=
carboxybenzyl) and (benzyloxy)acetaldehyde, which are ac-
ceptor substrates for FSA wild-type and variants, the arene
moiety could be oriented towards the entrance of the active-
site cavity.[11a,23] Overall yields, that is, four reaction steps and
the corresponding work-up steps and final purification, ranged
from 18 to 58% (Table 5). The previous optimized FSA biocata-
lyst for cross-aldol additions of glycolaldehyde, FSA A129X
(where X=T, V, and G variants),[11a] furnished overall yields of
11–50%. In this case, reaction conversions were not deter-
mined because of the presence of a- and b-anomers, which
Table 3. Reduction of the acyloin product 4 with NaBH4 in MTBE.
Starting material
Ratio 5/6[a] (1R,2S)/(1S,2S)
Reduction yield[b]
4a
4b
4g
4h
4i
4j
4k
4t
5:95
4:96
26:84
3:97
6:94
4:96
4:96
23:87
7:93
8:92
6:94
93
89
85
98
76
98
98
98
90
93
96
4w
4x
4ad
[a] Determined by HPLC. [b] The products were obtained as a mixture of
the diastereoisomers 5/6 with >90% purity by HPLC.
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the rate of the aldol formation,
compromising the stability of
the aldehyde acceptor and limit-
ing the final product yield.
RhuA
wild-type-catalyzed
aldol addition of DHA to com-
pounds 7a and 7k, was per-
formed in the presence of
borate buffer (200 mm). The
HPLC analysis of the reaction
mixture after 24 h revealed two
new peaks, which corresponded
to the aldol adduct epimers at
the C4 atom, that is, compounds
11 (i.e., l-fructose configured)
and 12 (i.e., l-tagatose config-
ured). Inversion of the stereo-
chemistry at the C4 atom has al-
ready been reported during
RhuA catalysis by using N-Cbz-
Scheme 4. Aldol addition of DHA, HA, and glycolaldehyde to compounds 7 catalyzed by FSA wild-type, FSA
A129S, and A129T variants, and RhuA. As examples, we performed the addition of DHA and glycolaldehyde to 7a, aminoaldehyde acceptors with
7b, 7g–k, 7t, 7x, and 7ad, of HA to 7a and 7h and of DHA to 7a and 7k.
identical stereochemistry to
compounds 7 at the C2 atom,
either by using DHA or the natu-
ral dihydroxyacetone phosphate donor.[12b,24] We ex-
Table 5. C-Aryl monosaccharide derivatives obtained.
pected the formation of the l-tagatose-configured
Product[a] Overall Product[c] Overall Product[d] Overall
Product[e]
Overall
yield[b]
aldol adduct 12 as a major adduct owing to the ther-
modynamically unfavored diaxial configuration of
compound 11 (1C4 conformation). However, the con-
trary was true, which was attributed to the equilibri-
um with the furanoid species that may favor the
furan 11 with trans vicinal diol configuration.
The structural characterization of the carbohydrate
derivatives 8, 9, and 10 (Figures S12–S35 in the Sup-
porting Information) indicate that the enzymatic car-
boligation steps were fully stereoselective within the
detection limits of NMR spectroscopy (i.e., >97%);
BAL rendered the 2R acyloins and FSA catalysts the
d-threo aldol adducts. This sequential two-step bio-
catalytic CÀC bond formation furnished the corre-
sponding (6S)-6-C-substituted-a-l-sorbose derivatives
8 and 9 and the (5S)-5-C-substituted-a/b-l-xylose de-
rivatives 10 (Table 5). In contrast, RhuA gave mixtures
of epimeric products at the C4 atom, furnishing the
(6S)-6-C-substituted-l-fructose derivatives 11 and the
(6S)-6-C-substituted-l-tagatose derivatives 12.
yield[b]
yield[b]
yield[b]
8a
49
9a
68
10a
25
11 a[h]/12a[i] 54
74:26
[f]
[f]
[f]
[f]
[f]
[f]
[f]
[f]
8b
8g
8h
8i
8j
8k
38
49
58
20
48
39
–
–
–
–
82
–
-
–
10b
10g
10h
10i
10j
10k
8 (11)[g]
16
–
–
–
–
–
–
–
–
–
–
[f]
[f]
[f]
[f]
[f]
[f]
9h
22 (50)[g]
23
[f]
[f]
–
–
–
[f]
f
23
24
[f]
[f]
11 k[h]/12k[i] 43
54:46
[f]
[f]
[f]
[f]
[f]
[f]
8t
8x
8ad
25
18
22
–
–
–
–
–
–
10t
10x
10ad
12
13
9 (32)[g]
–
–
–
–
–
–
[f]
[f]
[f]
[f]
[f]
[f]
[a] FSA A129S was used as catalyst. [b] Yields calculated from the starting aromatic al-
dehyde, four steps plus four work-up steps and final purification. All reactions were
performed under the same experimental conditions unless noted otherwise. Individual
optimization of the reaction conditions and purification steps was not performed.
[c] FSA wild-type was used as catalyst. [d] FSA A129T was used as catalyst. Addition of
glycolaldehyde by using
a ) unless noted otherwise.
syringe pump (0.25 mLhÀ1
[e] RhuA was used as catalyst in the presence of 200 mm borate buffer. [f] Not synthe-
sized. [g] Addition of glycolaldehyde in one portion. [h] Furanose-11a/11k/pyranose-
11a/11k (2.5:1). [i] Furanose-12a/12k/pyranose-12a/12k (1:1.5).
In summary, we have developed a new strategy for
the synthesis of uncovered C-aryl monosaccharide
overlapped with the acceptor substrate signal, making HPLC
peak integration difficult. Introduction of glycolaldehyde in
one portion gave in some instances better results than the
slow addition with a syringe pump.[11c] This could be related to
the instability of the dihydroxyaldehydes 7 under the reaction
conditions. A decrease in the acceptor amount was noticed
over time by HPLC, indicating a decomposition process proba-
bly due to water elimination of the benzylic alcohol. Conse-
quently, the slow incorporation of glycolaldehyde might limit
derivatives. Our study showed that complex carbohydrates can
be constructed in four steps, starting with simple and achiral
materials by using a combination of two enzymatic carboliga-
tion reactions and chemical reduction/deprotection steps.
Chem. Eur. J. 2015, 21, 1 – 13
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HPLC analyses: HPLC analyses were performed on a RP-HPLC
XBridge C18, 5 mm, 4.6 mm”250 mm column (Waters). The solvent
system used was: Solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) in
H2O; solvent B: 0.095% (v/v) TFA in CH3CN/H2O (4:1). The flow rate
was 1 mLminÀ1, detection was performed at l=215 nm and the
column temperature was 308C. The amounts of products and sub-
strates were quantified from the peak areas by using an external
standard methodology.
Experimental Section
Materials
Synthetic oligonucleotides were purchased from MWG-Biotech. Ar-
omatic aldehydes and dimethoxyacetaldehyde were purchased
from Sigma-Aldrich. MTBE, sodium borohydride, and active char-
coal were from Sigma-Aldrich. High-density IDA-Agarose 6BCL
nickel charged was from GE Healthcare Life Science. Water for ana-
lytical and preparative HPLC and for the preparation of buffers and
other assay solutions was obtained from an Arium pro ultrapure
water purification system (SartoriusStedim Biotech). All other sol-
vents used were of analytical grade. l-Rhamnulose-1-phosphate al-
dolase from E. coli (RhuA, 9.2 UmgÀ1; 1 unit catalyzes the cleavage
of 1 mmol of l-rhamnulose-1-phosphate per minute at 258C in
100 mm Tris-HCl buffer, pH 7.5, containing KCl (150 mm)), d-fruc-
tose-6-phosphate aldolase from E. coli (FSA) wild-type
(0.22 UmgÀ1), and the variants FSA A129S (9.4 UmgÀ1) and FSA
A129T (0.54 UmgÀ1; 1 U of enzyme catalyzes the synthesis 1 mmol
of d-fructose-6-phosphate per min from DHA and d,l-glyceralde-
hyde-3-phosphate at 258C in Gly–Gly buffer (50 mm, pH 8.0)) aldo-
lases were obtained in our laboratory by using previously de-
scribed procedures.[11a,12b,22]
NMR analysis: NMR spectra were recorded on Varian Mercury-400
spectrometer (400.13 MHz for 1H and 100.62 MHz for 13C) and
Bruker Avance-III-600 (600.13 MHz for 1H and 150.92 MHz for 13C)
1
spectrometers at 298 K. 1D (Conventional H, 13C, and selective 1D
NOESY) and 2D (COSY, multiplicity-edited HSQC, and 2D NOESY)
experiments were collected by using standard pulse programs
under routine conditions. The residual solvent signal was used as
the internal standard; chemical shifts (d) are expressed in parts per
million and coupling constants (J) in Hertz.
Activity assay of BAL: One unit of activity was defined as the
amount of BAL, which catalyzes the formation of 1 mmol of ben-
zoin per minute at 258C in 3-(N-morpholino)propanesulfonic acid
(MOPS) buffer, (50 mm; pH 8.0), containing ThDP (0.15 mm) and
MgSO4 (2.5 mm). The assay mixture consisted of benzaldehyde
(100 mm) dissolved in MOPS buffer (2.5 mL, 50 mm, pH 8.0) con-
taining ThDP (0.15 mm), MgSO4 (2.5 mm), and DMSO (25% v/v). Re-
actions were started by the addition of the enzyme (0.00875–
0.28 mg of lyophilized powder dissolved in MOPS buffer (0.5 mL,
50 mm, pH 8.0) containing ThDP (0.15 mm) and MgSO4 (2.5 mm)).
Reaction monitoring was as follows: At appropriate time intervals
(after 0, 2, 5, 8, 10, 15, and 20 min), samples (100 mL) were with-
drawn, diluted with methanol (900 mL), and directly analyzed and
quantified by HPLC; isocratic elution at 55% solvent B over 10 min.
Methods
Re-cloning of BAL: PCR amplification of the bal gene (1.7 kb) was
performed by using the plasmid pKK233-2/BAL[25] as template and
the primers bal (5–3) 5’-AGAGGAGAAATTAACCATGGCGATGATTA-
CAGGCGG-3’ and bal (3–5) 5’-CGGATCCTGCGAAGGGGTCCATGCC-
GAT-3’, which introduced the appropriated restriction sites (BseRI
and BamHI) for the subsequent cloning steps in a pQE-60.
Protein expression and purification: Expression and production
of BAL was performed as follows: The plasmid was transformed
into an E. coli strain M-15 [pREP-4] from QIAGEN and grown in LB
medium with ampicillin (100 mgmLÀ1) plus kanamycin (25 mgLÀ1) at
378C on a rotary shaker at 250 rpm. A final optical density at
600 nm (OD600) of 2–3 was usually achieved. An aliquot of the pre-
culture (12 mL) was transferred into a shake-flask (2 L) containing
Reactions catalyzed by BAL: General procedure
LB (0.4 L) with ampicillin (100 mgmLÀ1
)
plus kanamycin
Analytical scale: Reactions (4 mL total volume) were conducted at
25 8C and magnetically stirred at 250 rpm. The aromatic aldehyde
(0.2 mmol) was dissolved in MTBE (2 mL). Then, an aqueous
sodium carbonate buffer (1.66 mL, 50 mm, pH 8.0) containing BAL
(500 UmLÀ1), MgSO4 (2.5 mm), and ThDP (0.15 mm) was added. The
reaction was started by adding dimethoxyacetaldehyde (2 mmol,
340 mL of a commercial aqueous solution, 5.8m). Reaction monitor-
ing was as follows: at 24 h of reaction time samples of the organic
phase (10 mL) and the aqueous phase (10 mL) were withdrawn,
placed in separate vials, diluted with methanol (500 mL), and direct-
ly analyzed and quantified by HPLC by using a gradient elution
from 10 to 70% of solvent B over 30 min.
(25 mgmLÀ1) and incubated at 378C with shaking at 250 rpm.
During the middle exponential phase growth (DO600 ꢀ0.5), Isopro-
pyl b-d-1-thiogalactopyranoside (IPTG; 0.05 mm) was added and
the temperature was decreased to 258C while reducing the shak-
ing at 190 rpm to minimize potential inclusion bodies formation.
Cells from the induced-culture broths (4 L) were centrifuged at
12000 g for 10 min at 48C. The pellet was re-suspended with start-
ing sodium phosphate buffer (50 mm, pH 8.0), containing NaCl
(300 mm) and imidazole (10 mm) to a final cell density (OD600) of
ten. Cells were lysed by using a cell disrupter (Constant Systems).
Cellular debris was removed by centrifugation at 12000 g for
10 min. The clear supernatant was collected and purified by affinity
chromatography (IMAC) in an FPLC system (Amersham biosci-
ences). The crude supernatant was applied to a cooled HR 16/40
column (GE Healthcare) packed with HiTrap chelating support
(50 mL bed volume; Amersham Biosciences) and washed with the
start buffer (150 mL). The protein was eluted with sodium phos-
phate buffer (50 mm, pH 8.0) containing NaCl (300 mm) and imida-
zole (500 mm) at a flow rate of 3 mL minÀ1. Fractions containing
the recombinant protein were combined and dialyzed against
sodium phosphate buffer (10 mm, pH 7.0 containing ThDP
(0.15 mm) and MgSO4 (2.5 mm))[26] at 48C. The dialyzed solution
was frozen at À808C and lyophilized. The white solid obtained was
stored at À208C.
Scale-up: Reactions (40 mL total volume) were conducted at 258C
under magnetic stirring at 250 rpm. The aromatic aldehyde
(2 mmol) was dissolved in MTBE (20 mL). Then, sodium carbonate
buffer (16.6 mL, 50 mm, pH 8.0) containing BAL (500 UmLÀ1),
MgSO4 (2.5 mm), and ThDP (0.15 mm) was added. The reaction was
started by the addition of dimethoxyacetaldehyde (20 mmol,
3.4 mL of a commercial aqueous solution, 5.8m). Reaction monitor-
ing was carried out by HPLC as described for the analytical scale
reactions. After 24 h; NaCl was added to the reaction mixture up
to saturation, and the phases were separated. The aqueous layer
was extracted with MTBE (3”20 mL). The organic phases were
combined and dried over anhydrous Na2SO4. The product dissolved
in MTBE was used without further purification.
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(C5), 73.59 (C4), 70.47 (C3), 63.62 ppm (C1); ESI-TOF: m/z calcd for
C12H16O6Na: 279.0845 [M+Na+], C24H32O12Na: 535.1791 [2M+Na+];
found: 279.0840, 535.1793.
Synthesis of (1S,2S)-1-aryl-3,3-dimethoxypropane-1,2-diol
(6): General procedure
(R)-1-Aryl-2-hydroxy-3,3-dimethoxy-propan-1-one (4; 2 mmol) dis-
solved in MTBE (100 mL) was stirred at 250 rpm and 25 8C. To this
solution was added NaBH4 (4 mmol). After 4 h, the conversion of
the starting material was complete as determined by HPLC (as de-
scribed previously). Then, the MTBE was removed under reduced
pressure in a rotary evaporator. The residue containing the product
was dissolved in water (50 mL) and purified by chromatography.
The bulk stationary phase AMBERLITE XAD 1180 was packed into
a gravity glass column (50”2 cm) to give a final bed volume of
110 mL. The crude material (50 mL in water, divided into three ali-
quots) was loaded onto the column. Impurities were washed away
with H2O (500 mL), and the product was eluted with ethanol
(75 mL). Pure fractions were combined and the solvent was evapo-
rated. The product was used in the next reaction without further
purification.
(6S)-6-C-(2-Fluorophenyl)-a-l-sorbopyranose (8b): The title com-
pound was obtained according to the general procedure. Yield:
206.3 mg (white solid, 38%), >95% pure, as determined by HPLC;
retention factor k=3.4 (HPLC analyses were performed as de-
1
scribed previously); [a]D20 =À44.0 (c=5 in H2O); H NMR (400 MHz,
D2O): d=7.46–7.39 (m, 1H), 7.29 (dddd, J=8.3, 7.3, 5.4, 1.8 Hz, 1H),
7.13 (td, J=7.6, 1.2 Hz, 1H), 7.04 (ddd, J=10.7, 8.3, 1.2 Hz, 1H),
4.95 (d, J=10.1 Hz, 1H), 3.73 (d, J=9.6 Hz, 1H), 3.59 (d, J=11.8 Hz,
1H), 3.57 (d, J=9.6 Hz, 1H), 3.56 (dd, J=9.1 Hz, J=9.9 1H),
3.38 ppm (d, J=11.8 Hz, 1H); 13C NMR (101 MHz, D2O): d=162.11
(C8 arom), 159.67 (C7 arom), 130.68 (C10 arom), 129.12 (C12 arom),
124.57 (C11 arom), 115.60 (C9 arom), 98.04 (C2), 73.58 (C5, C4),
70.43 (C3), 67.74 (C6), 63.52 ppm (C1); ESI-TOF: m/z calcd for
C12H15FO6Na: 297.0750 [M+Na+], C24H30F2O12Na: 571.1603
[2M+Na+]; found: 297.0747, 571.1607.
(6S)-6-C-(3-Hydroxyphenyl)-a-l-sorbopyranose (8g): The title
compound was obtained according to the general procedure.
Yield: 264.7 mg (white solid, 49%), >95% pure, as determined by
HPLC; retention factor k=0.4 (HPLC analyses were performed as
described previously); [a]2D0 =À55.3 (c=5 in H2O); 1H NMR
(400 MHz, D2O): d=7.17 (t, J=7.9 Hz, 1H), 6.88 (dt, J=7.7, 1.2 Hz,
1H), 6.82 (dd, J=2.5, 1.6 Hz, 1H), 6.76 (ddd, J=8.2, 2.6, 1.0 Hz, 1H),
4.48 (d, J=9.9 Hz, 1H), 3.69 (t, J=9.4 Hz, 1H), 3.59 (d, J=11.8 Hz,
1H), 3.56 (d, J=9.7 Hz, 1H), 3.44 (dd, J=9.9, 9.2 Hz, 1H), 3.37 ppm
(d, J=11.8 Hz, 1H); 13C NMR (101 MHz, D2O): d=155.60 (C9 arom),
139.35 (C7 arom), 130.06 (C11 arom), 120.28 (C12 arom), 115.79
(C10 arom), 114.76 (C8 arom), 97.47 (C2), 74.45 (C6), 73.75 (C5),
73.54 (C4), 70.23 (C3), 63.56 ppm (C1); ESI-TOF: m/z calcd for
C12H16O7Na: 295.0794 [M+Na+], C24H32O14Na: 567.1690 [2M+Na+];
found: 295.0793, 567.1685.
Synthesis of (2S,3S)-3-aryl-2,3-dihydroxypropanal (7): Gener-
al procedure
(1S,2S)-1-Aryl-3,3-dimethoxypropane-1,2-diol (6; 2 mmol) was dis-
solved in acetonitrile (2 mL). To this solution, H2SO4 (10 mL of a 2m
aqueous solution) was added. The reaction was stirred at 250 rpm
and 258C. The formation of the aldehyde was monitored by HPLC,
as described previously. After 3 h the reaction was stopped by ad-
dition of CaCO3 powder to neutralize the acid. The solution was fil-
tered to remove precipitated CaSO4 and the aldehyde was used in
the next step without further purification dissolved in water
(50 mL).
Synthesis of (6S)-6-C-aryl-l-sorbose (8): General procedure
Dihydroxyacetone (4 mmol) was added to a solution of (2S,3S)-3-
aryl-2,3-dihydroxypropanal (7) (2 mmol) dissolved in H2O (50 mL)
adjusted to pH 7.5 with NaHCO3 (1m, pH 8.0). The reaction was
started by adding FSA A129S (47 UmLÀ1 reaction mixture). The re-
action was conducted at 258C with magnetic stirring at 250 rpm
and monitored by HPLC by using gradient elution from 0 to 60%
of solvent B over 30 min. When the aldehyde was consumed, the
reaction mixture was acidified to pH 3.0 with HCl (1m) and the FSA
started to precipitate. Then, K2SO4 was added up to saturation,
which helped to complete precipitation of the enzyme. The mix-
ture was filtered through Celite and the pellet was washed with
water (200 mL). Purification of the product was carried out as fol-
lows: The product was adsorbed onto activated charcoal (60 mL),
placed in a sintered glass funnel with a porosity of 3 mm (7”
4.5 cm), packed with water, and equipped with a vacuum system.
Then, the activated charcoal was washed with distilled H2O (1 L).
The product was eluted with a stepwise elution from 5:95 to 60:40
acetonitrile/H2O (i.e., twelve steps of 30 mL, increasing by 5% of
acetonitrile in each step). Fractions containing the product were
combined and concentrated to 40 mL under reduced pressure,
frozen at À808C, and lyophilized.
(6S)-6-C-(3-Methoxyphenyl)-a-l-sorbopyranose (8h): The title
compound was obtained according to the general procedure.
Yield: 330.9 mg (white solid, 58%), >95% pure, as determined by
HPLC; retention factor k=3.7 (HPLC analyses were performed as
described previously); [a]2D0 =À48.0 (c=5 in H2O); 1H NMR
(400 MHz, D2O): d=7.25 (dd, J=8.3, 7.5 Hz, 1H), 6.97 (t, J=1.2 Hz,
1H), 6.91–6.85 (m, 2H), 6.88 (ddd, J=8.3, 2.7, 1.0 Hz, 1H), 4.53 (d,
J=9.9 Hz, 1H), 3.70 (s, 3H), 3.70 (t, J=9.7 Hz, 1H), 3.59 (d, J=
11.9 Hz, 1H), 3.57 (d, J=9.5 Hz, 1H), 3.46 (dd, J=9.9, 9.2 Hz, 1H),
3.38 ppm (d, J=11.8 Hz, 1H); 13C NMR (101 MHz, D2O): d=158.94
(C9 arom), 139.24 (C7 arom), 129.93 (C11 arom), 120.95 (C12 arom),
114.50 (C10 arom), 113.51 (C8 arom), 97.91 (C2), 74.55 (C6), 74.11
(C5), 73.56 (C4), 70.44 (C3), 63.57 (C1), 55.30 ppm (C13); ESI-TOF:
m/z calcd for C13H18O7Na: 309.0950 [M+Na+], C26H36O14Na:
595.2003 [2M+Na+]; found: 309.0952, 595.2005.
(6S)-6-C-(3-Nitrophenyl)-a-l-sorbopyranose (8i): The title com-
pound was obtained according to the general procedure. Yield:
118.0 mg (white solid, 20%), >95% pure, as determined by HPLC;
retention factor k=3.8 (HPLC analyses were performed as de-
1
scribed previously); [a]D20 =À46.0 (c=5 in H2O); H NMR (400 MHz,
(6S)-6-C-Phenyl-a-l-sorbopyranose (8a): The title compound was
obtained according to the general procedure. Yield: 251.6 mg
(white solid, 49%), >95% pure, as determined by HPLC; retention
factor k=2.1 (HPLC analyses were performed as described previ-
ously); [a]2D0 =À43.3 (c=5 in H2O); 1H NMR (400 MHz, D2O): d=
7.37–7.25 (m, 5H), 4.55 (d, J=9.9 Hz, 1H), 3.72 (t, J=9.4 Hz, 1H),
3.60 (d, J=9.7 Hz, 1H), 3.57 (d, J=11.7 Hz, 1H), 3.49 (dd, J=9.9,
9.2 Hz, 1H), 3.38 ppm (d, J=11.8 Hz, 1H); 13C NMR (101 MHz, D2O):
d=137.43 (C7 arom), 128.57(C arom), 97.89 (C2), 74.69 (C6), 74.21
D2O): d=8.23–8.19 (m, 1H), 8.11 (ddd, J=8.3, 2.4, 1.1 Hz, 1H),
7.75–7.69 (m, 1H), 7.51 (d, J=8.0 Hz, 1H), 4.69 (d, J=9.9 Hz, 1H),
3.74 (t, J=9.5 Hz, 1H), 3.63 (d, J=11.9 Hz, 1H), 3.60 (d, J=9.9 Hz,
1H), 3.43 (dd, J=9.3, 9.2 Hz, 1H), 3.42 ppm (d, J=11.9 Hz, 1H);
13C NMR (101 MHz, D2O): d=147.90 (C9 arom), 139.75 (C7 arom),
134.73 (C12 arom), 129.68 (C11 arom), 123.61 (C10 arom), 122.74
(C8 arom), 98.08 (C2), 74.34 (C5), 73.77 (C6), 73.38 (C4), 70.39 (C3),
63.53 ppm (C1); ESI-TOF: m/z calcd for C12H15NO8Na: 324.0695
[M+Na+]; found: 324.0693.
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(6S)-6-C-(3-Cyanophenyl)-a-l-sorbopyranose (8j): The title com-
pound was obtained according to the general procedure. Yield:
271.1 mg (white solid, 48%), >95% pure, as determined by HPLC;
retention factor k=3.0 (HPLC analyses were performed as de-
J=9.4, 8.7, 4.8 Hz, 1H), 3.48–3.37 (m, 2H), 3.31–3.23 (m, 2H),
3.11 ppm (ddd, J=9.6, 8.7, 5.8 Hz, 1H); 13C NMR (101 MHz,
[D6]DMSO): d=139.92 (C13 arom), 133.82 (C10 arom), 133.07 (C7
arom), 127.05 (C11 arom), 122.92 (C9 arom), 119.88 (C8 arom),
109.42 (C12 arom), 98.19 (C2), 76.08 (C5), 74.74 (C4), 74.56 (C6),
71.23 (C3), 64.61 ppm (C1); ESI-TOF: m/z calcd for C13H17N2O6:
297.1087 [M+H+]; found: 297.1083.
1
scribed previously); [a]D20 =À52.7 (c=5 in H2O); H NMR (400 MHz,
D2O): d=7.73 (ddt, J=1.7, 1.0, 0.5 Hz, 1H), 7.64 (d, J=1.7 Hz, 1H),
7.62 (d, J=1.6 Hz, 1H), 7.44 (ddd, J=8.1, 7.4, 0.6 Hz, 1H), 4.59 (d,
J=9.8 Hz, 1H), 3.72 (t, J=9.4 Hz, 1H), 3.61 (d, J=12.0 Hz, 1H), 3.58
(d, J=9.6 Hz, 1H), 3.40 (d, J=11.9 Hz, 1H), 3.38 ppm (dd, J=9.2,
9.4 Hz, 1H); 13C NMR (101 MHz, D2O): d=139.10 (C7 arom), 133.01
(C12 arom), 132.58 (C10 arom), 131.77 (C8 arom), 129.43 (C11
arom), 119.33 (C13), 111.29 (C9 arom), 98.02 (C2), 74.30 (C5), 73.81
(C6), 73.36 (C4), 70.23 (C3), 63.53 ppm (C1); ESI-TOF: m/z calcd for
C13H15NO6Na: 304.0797 [M+Na+]; found: 304.0800.
Synthesis of (6S)-1-deoxy-6-C-aryl-l-sorbose (9): General pro-
cedure
Hydroxyacetone (4 mmol) was added to a solution of (2S,3S)-3-aryl-
2,3-dihydroxypropanal (7) (2 mmol) dissolved in H2O (50 mL) ad-
justed to pH 7.5 with NaHCO3 (1m, pH 8.0). The reaction was start-
ed by adding FSA wild-type (1.1 UmLÀ1 reaction mixture). The reac-
tion was conducted at 258C with magnetic stirring at 250 rpm and
monitored by HPLC by using gradient elution from 0 to 60% of
solvent B over 30 min. Reaction workup and purification was con-
ducted as described in the general procedure for the synthesis of
(6S)-6-C-aryl-l-sorbose.
(6S)-6-C-(3-Chlorophenyl)-a-l-sorbopyranose (8k): The title com-
pound was obtained according to the general procedure. Yield:
225.9 mg (white solid, 39%), >95% pure, as determined by HPLC;
retention factor k=4.9 (HPLC analyses were performed as de-
scribed previously); [a]2D0 =À3.3 (c=5 in H2O); 1H NMR (400 MHz,
D2O): d=7.42–7.21 (m, 4H), 4.53 (d, J=9.9 Hz, 1H), 3.71 (t, J=
9.4 Hz, 1H), 3.60 (d, J=11.9 Hz, 1H), 3.57 (d, J=10.0 Hz, 1H), 3.42
(t, J=9.4 Hz, 1H), 3.39 ppm (d, J=11.9 Hz, 1H); 13C NMR (101 MHz,
D2O): d=139.68 (C7 arom), 133.75 (C9 arom), 130.07 (C11 arom),
128.76 (C8 arom), 127.84 (C12 arom), 126.49 (C10 arom), 97.96 (C2),
74.21 (C6), 74.09 (C5), 73.44 (C4), 70.24 (C3), 63.56 ppm (C1); ESI-
TOF: m/z calcd for C12H15ClO6Na: 313.0455 [M+Na+],
C24H30Cl2O12Na: 603.1012 [2M+Na+]; found: 313.0452, 603.1015.
(6S)-1-Deoxy-6-C-phenyl-a-l-sorbopyranose (9a): The title com-
pound was obtained according to the general procedure. Yield:
328.5 mg (white solid, 68%), >95% pure, as determined by HPLC;
retention factor k=4.1 (HPLC analyses were performed as de-
1
scribed previously); [a]D20 =À49.3 (c=5 in H2O); H NMR (400 MHz,
D2O): d=7.35–7.25 (m, 5H), 4.53 (d, J=9.9 Hz, 1H), 3.64 (t, J=
9.3 Hz, 1H), 3.54 (dd, J=9.9, 9.2 Hz, 1H), 3.34 (d, J=9.5 Hz, 1H),
1.33 ppm (s, 3H); 13C NMR (101 MHz, D2O): d=137.16 (C7 arom),
128.96, 128.76, 128.25 8 (C arom), 98.00 (C2), 75.09 (C3), 74.75 (C6),
74.08 (C5), 73.42 (C4), 24.56 ppm (C1); ESI-TOF: m/z calcd for
C12H16O5Na: 263.0895 [M+Na+], C24H32O10Na: 503.1893 [2M+Na+];
found: 263.0897, 503.1895.
(6S)-6-C-(4-Hydroxy-3-methoxyphenyl)-a-l-sorbopyranose (8t):
The title compound was obtained according to the general proce-
dure. Yield: 135.1 mg (white solid, 22%), >95% pure, as deter-
mined by HPLC; retention factor k=0.1 (HPLC analyses were per-
formed as described previously); [a]2D0 =À43.3 (c=5 in H2O);
1H NMR (400 MHz, D2O): d=6.98 (d, J=1.9 Hz, 1H), 6.85–6.78 (m,
2H), 4.48 (d, J=9.9 Hz, 1H), 3.74 (s, 3H), 3.69 (t, J=9.5 Hz, 1H),
3.59 (d, J=11.3 Hz, 1H), 3.56 (d, J=9.4 Hz, 1H), 3.47 (dd, J=9.2,
9.3 Hz, 1H), 3.37 ppm (d, J=11.8 Hz, 1H); 13C NMR (101 MHz, D2O):
d=147.39 (C9 arom), 145.14 (C10 arom), 129.69 (C7 arom), 121.56
(C12 arom), 115.33 (C11 arom), 112.09 (C8 arom), 97.84 (C2), 74.54
(C6), 73.98 (C5), 73.53 (C4), 70.45 (C3), 63.59 (C1), 56.21 ppm (C13);
ESI-TOF: m/z calcd for C13H18O8Na: 325.0899 [M+Na+], C26H36O16Na:
627.1901 [2M+Na+]; found: 325.0895, 627.1904.
(6S)-1-Deoxy-6-C-(3-methoxyphenyl)-a-l-sorbopyranose
(9h):
The title compound was obtained according to the general proce-
dure. Yield: 442.6 mg (white solid, 82%), >95% pure, as deter-
mined by HPLC; retention factor k=5.0 (HPLC analyses were per-
formed as described previously); [a]2D0 =À46.0 (c=5 in H2O);
1H NMR (400 MHz, D2O): d=7.24 (ddd, J=8.2, 7.6, 0.5 Hz, 1H),
6.98–6.84 (m, 3H), 4.51 (d, J=9.9 Hz, 1H), 3.70 (s, 3H), 3.63 (t, J=
9.4 Hz, 1H), 3.51 (dd, J=9.9, 9.2 Hz, 1H), 3.33 (d, J=9.5 Hz, 1H),
1.33 ppm (s, 3H); 13C NMR (101 MHz, D2O): d=158.95 (C9 arom),
139.21 (C7 arom), 129.91 (C11 arom), 121.07 (C12 arom), 114.56 (C8
arom), 113.50 (C10 arom), 97.98 (C2), 75.06 (C3), 74.75 (C6), 74.11
(C5), 73.40 (C4), 55.27(C13), 24.55 ppm (C1); ESI-TOF: m/z calcd for
C13H18O6Na: 293.1001 [M+Na+], C26H36O12Na: 563.2104 [2M+Na+];
found: 293.1004, 563.2106.
(6S)-6-C-(2-Thiophene)-a-l-sorbopyranose (8x): The title com-
pound was obtained according to the general procedure. Yield:
95.5 mg (white solid, 18%), >95% pure, as determined by HPLC;
retention factor k=2.3 (HPLC analyses were performed as de-
1
scribed previously); [a]D20 =À41.3 (c=5 in H2O); H NMR (400 MHz,
D2O): d=7.37–7.32 (m, 1H), 7.11–7.06 (m, 1H), 6.97–6.91 (m, 1H),
4.86 (d, J=9.9 Hz, 1H), 3.69 (t, J=9.5 Hz,1H), 3.57 (d, J=11.9 Hz,
1H), 3.54 (d, J=9.5 Hz, 1H), 3.46 (dd, J=9.6, 9.2 Hz, 1H), 3.38 ppm
(d, J=11.8 Hz, 1H); 13C NMR (101 MHz, D2O): d=140.34 (C7 arom),
127.63 (C8 arom), 127.02 (C9 arom), 126.73 (C10 arom), 97.94 (C2),
74.87 (C5), 73.45 (C4), 70.33 (C6), 70.12 (C3), 63.45 ppm (C1); ESI-
TOF: m/z calcd for C10H14O6SNa: 285.0409 [M+Na+], C20H28O12S2Na:
547.0920 [2M+Na+]; found: 285.0404, 547.0915.
Synthesis of (5S)-5-C-aryl-l-xylose (10): General procedure
To a solution of (2S,3S)-3-aryl-2,3-dihydroxypropanal (7) (2 mmol)
dissolved in H2O (50 mL) adjusted to pH 7.5 with NaHCO3 (1m,
pH 8.0) and containing FSA A129T (2.7 UmLÀ1 reaction mixture),
glycolaldehyde (4 mmol, dissolved in 5 mL of H2O) was slowly
added with a syringe pump (0.254 mLhÀ1, 0.2 mmolhÀ1).[11c] The re-
action was conducted at 258C with magnetic stirring at 250 rpm
and monitored by HPLC by using gradient elution from 0 to 60%
of solvent B over 30 min. In some examples of this work, better re-
sults were obtained by adding the glycolaldehyde in one portion.
Reaction workup and purification was conducted as described in
the general procedure for the synthesis of (6S)-6-C-aryl-l-sorbose.
(6S)-6-C-[5-(1H-Indazole)]-a-l-sorbopyranose (8ad): The title
compound was obtained according to the general procedure.
Yield: 132.0 mg (white solid, 49%), >95% pure, as determined by
HPLC; retention factor k=1.3 (HPLC analyses were performed as
described previously); [a]2D0 =À35.3 (c=5 in DMSO); 1H NMR
(400 MHz, [D6]DMSO): d=12.91 (s, 1H), 7.99 (d, J=1.2 Hz, 1H), 7.68
(dt, J=1.4, 0.7 Hz, 1H), 7.41 (dt, J=8.6, 0.9 Hz, 1H), 7.35 (dd, J=
8.7, 1.5 Hz, 1H), 5.44 (s, 1H), 4.68 (d, J=4.8 Hz, 1H), 4.64 (d, J=
5.8 Hz, 1H), 4.59–4.51 (m, 2H), 4.44 (d, J=6.7 Hz, 1H), 3.55 (ddd,
(5S)-5-C-Phenyl-a/b-l-xylopyranose (10a): The title compounds
were obtained according to the general procedure. Yield: 95.5 mg
(white solid, 25%), >95% pure, as determined by HPLC; retention
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factor k1 =1.8, k2 =2.4 (HPLC analyses were performed as described
previously); [a]2D0 =À22.7 (c=5 in H2O, a/b 37:67); 1H NMR
(400 MHz, D2O): d=7.36–7.25 (m, 5H), 5.14 (d, J=3.6 Hz, 1H;
a anomer), 4.65 (d, J=8.0 Hz, 1H; b anomer), 4.59 (d, J=10.1 Hz,
1H; a anomer), 4.20 (d, J=9.3 Hz, 1H; b anomer), 3.69 (dd, J=9.8,
9.1 Hz, 1H; a anomer), 3.59 (dd, J=9.8, 3.8 Hz, 1H; a anomer),
3.51 (dd, J=9.8, 9.1 Hz, 1H; a anomer), 3.55–3.42 (m, 3H),
3.27 ppm (dd, J=9.1, 8.1 Hz, 1H; b anomer); 13C NMR (101 MHz,
D2O): d=137.22 (C6 arom, a anomer), 136.97 (C6 arom, b anomer),
128.44 (C arom), 96.21 (C1, b anomer), 92.41 (C1, a anomer), 78.21
(C5, b anomer), 75.43 (C4, b anomer), 74.27 (C2, b anomer), 74.07
(C4, a anomer), 73.90 (C3, b anomer), 73.81 (C5, a anomer), 72.61
(C3, a anomer), 71.59 ppm (C2, a anomer); ESI-TOF: m/z calcd for
C11H14O5Na: 249.0739 [M+Na+], C22H28O10Na: 475.1580 [2M+Na+];
found: 249.0737, 475.1582.
1H), 6.96–6.86 (m, 3H), 5.15 (d, J=4.1 Hz, 1H; a anomer), 4.63 (d,
J=8.7 Hz, 1H; b anomer), 4.57 (d, J=9.9 Hz, 1H; a anomer), 4.19
(d, J=9.2 Hz, 1H; b anomer), 3.70 (s, 1H), 3.68 (d, J=9.8 Hz, 1H;
a anomer), 3.59 (dd, J=9.8, 3.8 Hz, 1H; a anomer), 3.50–3.43 (m,
3H), 3.28 ppm (dd, J=9.8, 8.9 Hz, 1H;
b
anomer); 13C NMR
(101 MHz, D2O): d=158.96 (C8 arom), 139.08 (C6 arom, a anomer),
138.78 (C6 arom, b anomer), 129.98 (C10 arom), 120.88 (C11 arom,
a anomer), 120.62 (C11 arom, b anomer), 114.54 (C7 arom), 113.87
(C9 arom, a anomer), 113.24 (C9 arom, b anomer), 96.20 (C1,
b anomer), 92.41 (C1, a anomer), 78.05 (C5, b anomer), 75.41 (C4,
b anomer), 74.24 (C2, b anomer), 74.08 (C4, a anomer), 73.68 (C5,
a anomer), 72.59 (C3, a anomer), 71.56 (C2, a anomer), 55.29 ppm
(C12); ESI-TOF: m/z calcd for C12H16O6Na: 279.0844 [M+Na+],
C24H32O12Na: 535.1791 [2M+Na+]; found: 279.0840, 535.1790.
(5S)-5-C-(3-Nitrophenyl)-a/b-l-xylopyranose (10i): The title com-
pounds were obtained according to the general procedure. Yield:
119.2 mg (white solid, 23%), >95% pure, as determined by HPLC;
retention factor k1 =4.0, k2 =4.7 (HPLC analyses were performed as
described previously); [a]2D0 =À23.3 (c=5 in H2O, a/b 40:60);
1H NMR (400 MHz, D2O): d=8.18 (dddt, J=7.7, 2.2, 1.7, 0.5 Hz, 1H),
8.12 (dddd, J=8.3, 2.3, 1.7, 1.1 Hz, 1H), 7.74–7.67 (m, 1H), 7.52
(dddd, J=8.2, 7.7, 2.4, 0.4 Hz, 1H). 5.20 (d, J=4.2 Hz, 1H;
a anomer), 4.72 (d, J=10.0 Hz, 1H; a anomer), 4.69 (d, J=9.7 Hz,
1H; b anomer), 4.37 (d, J=9.32 Hz, 1H; b anomer), 3.72 (t, J=10.1,
1H; a anomer), 3.62 (dd, J=9.8, 4.2 Hz, 1H; a anomer), 3.53–3.43
(m, 3H), 3.30 ppm (t, J=9.5, 1H; b anomer); 13C NMR (101 MHz,
D2O): d=147.90 (C8 arom), 139.58 (C6 arom, a anomer), 139.17 (C6
arom, b anomer), 134.68 (C11 arom, a anomer), 134.38 (C11 arom,
b anomer), 129.82 (C10 arom), 123.76 (C9 arom), 122.79 (C7 arom,
a anomer), 122.57 (C7 arom, b anomer), 96.27 (C1, b anomer),
92.54 (C1, a anomer), 77.16 (C5, b anomer), 75.25 (C4, b anomer),
74.25 (C4, a anomer), 74.15 (C2, b anomer), 73.99 (C3, b anomer),
72.93 (C5, a anomer), 72.42 (C3, a anomer), 71.48 ppm (C2,
a anomer); ESI-TOF: m/z calcd for C11H13NO7Na: 294.0590 [M+Na+
]; found: 294.0592.
(5S)-5-C-(2-Fluorophenyl)-a/b-l-xylopyranose (10b): The title
compounds were obtained according to the general procedure.
Yield: 54.1 mg (white solid, 11%), >95% pure, as determined by
HPLC; retention factor k1 =3.1, k2 =3.7 (HPLC analyses were per-
formed as described previously); [a]2D0 =À32.0 (c=5 in H2O, a/
b 36:64); 1H NMR (400 MHz, D2O): d=7.41–7.34 (m, 1H), 7.29
(dddd, J=8.3, 7.3, 5.4, 1.8 Hz, 1H), 7.13 (tt, J=7.6, 1.5 Hz, 1H), 7.04
(ddd, J=10.7, 8.3, 1.2 Hz, 1H), 5.15 (d, J=3.9 Hz, 1H; a anomer),
4.97 (d, J=10.0 Hz, 1H; a anomer), 4.68 (d, J=8.0 Hz, 1H;
b anomer), 4.59 (d, J=9.8 Hz, 1H; b anomer), 3.71 (dd, J=9.8,
9.1 Hz, 1H; a anomer), 3.64–3.55 (m, 3H), 3.48 (t, J=9.2 Hz, 1H;
b anomer), 3.27 ppm (dd, J=9.4, 8.0 Hz, 1H; b anomer); 13C NMR
(101 MHz, D2O) d=162.18 (C7 arom, a anomer), 161.86 (C7 arom,
b
anomer), 159.74 (C6 arom, a anomer), 159.41 (C6 arom,
b anomer), 130.68 (C9 arom), 129.17 (C11 arom„ b anomer), 128.83
(C11 arom, a anomer), 124.71 (C10), 115.50 (C8), 96.31 (C1,
b anomer), 92.48 (C1, a anomer), 75.42 (C3, b anomer), 74.19 (C2,
b anomer), 73.31 (C4), 72.66 (C3, a anomer), 71.52 (C2, C5),
67.27 ppm (C5, a anomer); ESI-TOF: m/z calcd for C11H13FO5Na:
267.0645 [M+Na+], C22H26F2O10Na: 511.1392 [2M+Na+]; found:
267.0640, 511.1393.
(5S)-5-C-(3-Cyanophenyl)-a/b-l-xylopyranose (10j): The title com-
pounds were obtained according to the general procedure. Yield:
116.2 mg (white solid, 23%), >95% pure, as determined by HPLC;
retention factor k1 =3.7, k2 =4.3 (HPLC analyses were performed as
described previously); [a]2D0 =À18.7 (c=5 in H2O, a/b 32:68);
1H NMR (400 MHz, D2O): d=7.72–7.68 (m, 1H), 7.66–7.59 (m, 2H),
7.48–7.43 (m, 1H), 5.17 (d, J=4.2 Hz, 1H; a anomer), 4.67 (d, J=
8.0 Hz, 1H; b anomer), 4.65 (s, 1H; a anomer), 4.28 (d, J=9.1 Hz,
1H; b anomer), 3.70 (dd, J=9.8, 9.1 Hz, 1H; a anomer), 3.60 (dd,
J=9.8, 3.8 Hz, 1H; a anomer), 3.52–3.38 (m, 2H), 3.28 ppm (dd, J=
8.8, 8.3 Hz, 1H; b anomer); 13C NMR (101 MHz, D2O): d=138.92 (C6
arom, a anomer), 138.56 (C6, b anomer), 132.99 (C9, C11 arom,
a anomer), 132.71 (C9, C11 arom, b anomer), 131.80 (C7 arom,
a anomer), 131.52 (C7 arom, b anomer), 129.55 (C10 arom), 119.26
(5S)-5-C-(3-Hydroxyphenyl)-a/b-l-xylopyranose (10g): The title
compounds were obtained according to the general procedure.
Yield: 76.8 mg (white solid, 16%), >95% pure, as determined by
HPLC; retention factor k1 =1.9, k2 =2.4 (HPLC analyses were per-
formed as described previously); [a]2D0 =À26.0 (c=5 in H2O, a/
1
b 37:63); H NMR (400 MHz, D2O): d=7.18 (ddd, J=8.0, 7.6, 0.5 Hz,
1H), 6.89–6.83 (m, 1H), 6.82–6.74 (m, 2H), 5.14 (d, J=3.8 Hz, 1H;
a anomer), 4.64 (d, J=9.5 Hz, 1H; b anomer), 4.53 (d, J=9.9 Hz,
1H; a anomer), 4.15 (d, J=9.4 Hz, 1H; a anomer), 3.68 (dd, J=9.8,
9.1 Hz, 1H; a anomer), 3.58 (dd, J=9.8, 3.8 Hz, 1H; a anomer),
3.50–3.40 (m, 3H), 3.26 ppm (dd, J=9.4, 8.0 Hz, 1H; b anomer);
13C NMR (101 MHz, D2O): d=155.60 (C8 arom), 139.17 (C6 arom,
a anomer), 138.89 (C6 arom, b anomer), 130.10 (C10 arom), 120.22
(C11 arom, a anomer), 119.99 (C11 arom, b anomer), 115.84 (C7
arom), 114.76 (C9 arom, a anomer), 114.51 (C9 arom, b anomer),
96.18 (C1, b anomer), 92.39 (C1, a anomer), 77.97 (C5, b anomer),
75.39 (C4, b anomer), 74.24 (C2, b anomer), 74.04 (C4, a anomer),
73.88 (C3, b anomer), 73.60 (C5, a anomer), 72.58 (C3, a anomer),
71.55 ppm (C2, a anomer); ESI-TOF: m/z calcd for C11H14O6Na:
265.0688 [M+Na+], C22H28O12Na: 507.1478 [2M+Na+]; found:
265.0689, 507.1475.
(C12), 111.39 (C8 arom), 96.25 (C1,
b anomer), 92.50 (C1,
a anomer), 77.23 (C5, b anomer), 75.24 (C4), 74.15 (C2, b anomer),
73.96 (C3, b anomer), 72.98 (C5, a anomer), 72.42 (C3, a anomer),
71.47 ppm (C2, a anomer); ESI-TOF: m/z calcd for C24H26N2O10Na:
525.1485 [2M+Na+]; found: 525.1483.
(5S)-5-C-(3-Chlorophenyl)- a/b-l-xylopyranose (10k): The title
compounds were obtained according to the general procedure.
Yield: 126.9 mg (white solid, 24%), >95% pure, as determined by
HPLC; retention factor k1 =5.8, k2 =6.4 (HPLC analyses were per-
formed as described previously); [a]2D0 =À26.0 (c=5 in H2O, a/
b 39:61); 1H NMR (400 MHz, D2O): d=7.38–7.19 (m, 4H), 5.15 (d,
J=3.8 Hz, 1H; a anomer), 4.64 (d, J=9.0 Hz, 1H; b anomer), 4.57
(d, J=9.7 Hz, 1H; a anomer), 4.20 (d, J=8.9 Hz, 1H; b anomer),
3.68 (t, J=9.8, 1H; a anomer), 3.58 (dd, J=9.8, 4.5 Hz, 1H;
(5S)-5-C-(3-Methoxyphenyl)-a/b-l-xylopyranose (10h): The title
compounds were obtained according to the general procedure.
Yield: 256.4 mg (white solid, 50%), >95% pure, as determined by
HPLC; retention factor k1 =3.6, k2 =4.1 (HPLC analyses were per-
formed as described previously); [a]2D0 =À26.0 (c=5 in H2O, a/
1
b 37:63); H NMR (400 MHz, D2O): d=7.25 (ddt, J=8.1, 7.5, 0.8 Hz,
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a anomer), 3.50–3.39 (m, 3H), 3.27 ppm (dd, J=9.1, 8.1 Hz, 1H;
anomer); 13C NMR (101 MHz, D2O): d=139.53 (C6 arom,
3.51 (m, 1H; a anomer), 3.35–3.28 (m, 1H; a anomer), 3.27–3.18
(m, 2H; b anomer), 3.17–3.10 (m, 1H; a anomer), 3.06 ppm (ddd,
J=8.7, 7.6, 4.5 Hz, 1H; b anomer); 13C NMR (101 MHz, [D6]DMSO):
d=139.94 (C10 arom), 133.36 (C9 arom), 132.95 (C6, arom,
a anomer), 132.41 (C6 arom, b anomer), 126.73 (C12 arom,
a anomer), 126.51 (C12 arom, b anomer), 122.94 (C8 arom), 119.82
(C7 arom), 109.66 (C11 arom), 97.73 (C1, b anomer), 93.21 (C1,
a anomer), 78.38 (C5, b anomer), 77.25 (C4, b anomer), 76.12 (C4,
a anomer), 75.42 (C2, C3, b anomer), 73.77 (C3, C5, a anomer),
72.95 ppm (C2, a anomer); ESI-TOF: m/z calcd for C12H15N2O5:
267.0981 [M+H+]; found: 267.0982.
b
a anomer), 139.14 (C6 arom, b anomer), 133.71 (C8 arom), 130.13
(C10 arom), 128.87 (C11 arom), 127.89 (C7 arom, a anomer), 127.59
(C7 arom, b anomer), 126.45 (C9 arom, a anomer), 126.18 (C9
arom, b anomer), 96.21 (C1, b anomer), 92.45 (C1, a anomer), 77.54
(C5, b anomer), 75.30 (C4), 74.19 (C2, b anomer), 73.91 (C3,
b anomer), 73.24 (C5, a anomer), 72.49 (C3, a anomer), 71.51 ppm
(C2, a anomer); ESI-TOF: m/z calcd for C11H13ClO5Na: 283.0349
[M+Na+], C22H26Cl2O10Na: 543.0801 [2M+Na+]; found: 283.0347,
543.0805.
(5S)-5-C-(4-Hydroxy-3-methoxyphenyl)-a/b-l-xylopyranose (10t):
The title compounds were obtained according to the general pro-
cedure. Yield: 65.3 mg (white solid, 12%), >95% pure, as deter-
mined by HPLC; retention factor k1 =0.1, k2 =0.2 (HPLC analyses
were performed as described previously); [a]2D0 =À20.0 (c=5 in
H2O, a/b 35:65); 1H NMR (400 MHz, D2O): d=6.96–6.92 (m, 1H),
6.82–6.75 (m, 2H), 5.13 (d, J=4.0 Hz, 1H; a anomer), 4.63 (d, J=
8.9 Hz, 1H; b anomer), 4.52 (d, J=10.7 Hz, 1H; a anomer), 4.14 (d,
J=9.6 Hz, 1H; b anomer), 3.79 (s, 1H), 3.69 (t, J=10.7, 1H;
a anomer), 3.57 (dd, J=9.8, 4.0 Hz, 1H; a anomer), 3.52–3.40 (m,
3H), 3.25 ppm (dd, J=10.4, 8.9, 1H; b anomer); 13C NMR (101 MHz,
D2O): d=147.39 (C8 arom), 145.17 (C9 arom), 129.58 (C6 arom),
121.22 (C11 arom), 115.35 (C10 arom), 111.79 (C7 arom), 96.12 (C1,
b anomer), 92.23 (C1, a anomer), 78.03 (C5, b anomer), 75.40 (C2,
b anomer), 74.27 (C4, b anomer), 73.96 (C4, a anomer), 73.75 (C3,
b anomer), 73.65 (C5, a anomer), 72.60 (C3, a anomer), 71.59 (C2,
a anomer), 55.88 ppm (C12); ESI-TOF: m/z calcd for C12H16O7Na:
295.0794 [M+Na+], C24H32O14Na: 567.1690 [2M+Na+]; found:
295.0795, 567.1691.
Synthesis of (6S)-6-C-aryl-l-fructose (11) and (6S)-6-C-aryl-l-
tagatose (12): General procedure
Dihydroxyacetone (4 mmol) was added to a solution of (2S,3S)-3-
aryl-2,3-dihydroxypropanal (7) (2 mmol) dissolved in sodium borate
buffer (50 mL, 0.2m pH 7.5).[12b] Then, RhuA wild-type (46 UmLÀ1 re-
action mixture) was added and mixed again. The reaction was con-
ducted at 258C and stirred with a magnetic bar at 250 rpm. The re-
action was monitored by HPLC by using gradient elution from 0 to
60% of solvent B over 30 min as indicated above, until the alde-
hyde was nearly consumed. Reaction workup and purification was
conducted as described in the general procedure for the synthesis
of (6S)-6-C-aryl-l-sorbose.
(6S)-6-C-Phenyl-l-fructose (11a) and (6S)-6-C-phenyl-l-tagatose
(12a): The title compounds were obtained as a 76:26 mixture of
11 a/12a as a white solid according to the general procedure.
Yield: 277.3 mg (54%), >95% pure, as determined by HPLC; reten-
tion factor k1 =2.8, k2 =3.3; [a]2D0 =À10.0 (c=5 in H2O). Compound
11 a exists as an equilibrium mixture of 71% as fructofuranose and
29% as fructopyranose. Compound 12a exists of 40% as tagato-
furanose and 60% as tagatopyranose. ESI-TOF: m/z calcd for
C12H16O6Na: 279.0845 [M+Na+], C24H32O12Na: 535.1791 [2M+Na+];
found: 279.0846, 535.1795.
(6S)-6-C-Phenyl-a-l-fructopyranose (11a; pyran): 1H NMR
(400 MHz, D2O): d=4.81 (d, J=10.3 Hz, 1H), 4.06—4.00 (m, 1H),
3.97 (dd, J=3.3, 0.5 Hz, 1H), 3.96–3.87 (m, 1H), 3.66 (d, J=11.8 Hz,
1H), 3.28 ppm (d, J=11.6 Hz, 1H); 13C NMR (101 MHz, D2O): d=
104.85 (C2), 71.03 (C4), 70.98 (C6), 68.16 (C3), 67.78 (C5), 63.94 ppm
(C1).
(5S)-5-C-(2-Thiophene)-a/b-l-xylopyranose (10x): The title com-
pounds were obtained according to the general procedure. Yield:
58.9 mg (white solid, 13%), >95% pure, as determined by HPLC;
retention factor k1 =2.4, k2 =3.1 (HPLC analyses were performed as
described previously); [a]2D0 =À33.3 (c=5 in H2O, a/b 40:60);
1H NMR (400 MHz, D2O): d=7.38–7.33 (m, 1H), 7.08 (tdd, J=3.6,
1.3, 0.5 Hz, 1H), 6.97–6.92 (m, 1H), 5.12 (d, J=3.6 Hz, 1H;
a anomer), 4.91 (d, J=9.9 Hz, 1H; a anomer), 4.66 (d, J=8.0 Hz,
1H; b anomer), 4.55 (d, J=9.4 Hz, 1H; b anomer), 3.67 (dd, J=9.8,
9.1 Hz, 1H; a anomer), 3.55 (dd, J=9.8, 3.7 Hz, 1H; a anomer),
3.50–3.42 (m, 3H), 3.25 ppm (dd, J=9.3, 8.0 Hz, 1H; b anomer);
13C NMR (101 MHz, D2O): d=140.20 (C6 arom, a anomer), 139.55
(C6 arom, b anomer), 127.73 (C7 arom, a anomer), 127.53 (C7
arom, b anomer), 127.07 (C8 arom), 126.78 (C9, arom), 96.09 (C1,
b anomer), 92.37 (C1, a anomer), 75.28 (C4, b anomer), 74.90 (C4,
a anomer), 74.66 (C3, b anomer), 74.08 (C2, b anomer), 73.56 (C5,
b anomer), 72.49 (C3, a anomer), 71.43 (C2, a anomer), 69.28 ppm
(C5, a anomer); ESI-TOF: m/z calcd for C9H12O5SNa: 255.0303
[M+Na+], C18H24O10S2Na: 487.0709 [2M+Na+]; found: 255.0305,
487.0706.
(6S)-6-C-Phenyl-b-l-fructofuranose
(11a;
furan):
1H NMR
(400 MHz, D2O): d=4.68 (d, J=6.3 Hz, 1H), 4.16 (dd, J=7.8, 6.8 Hz,
1H), 3.93 (d, J=7.8 Hz, 1H), 3.87 (dd, J=6.8, 6.3 Hz, 1H), 3.37 ppm
(d, J=10.9 Hz, 2H); 13C NMR (101 MHz, D2O): d=101.55 (C2), 83.04
(C5), 75.82 (C3), 75.78 (C4), 74.46 (C6), 62.34 ppm (C1).
(6S)-6-C-Phenyl-a-l-tagatopyranose (12a; pyran): 1H NMR
(400 MHz, D2O): d=4.55 (d, J=9.9 Hz, 1H), 3.93–3.85 (m, 2H), 3.76
(t, J=9.7 Hz, 1H), 3.44 (d, J=18.6 Hz, 1H), 3.39 ppm (d, J=17.4 Hz,
1H); 13C NMR (101 MHz, D2O): d=98.31 (C2), 75.36 (C6), 70.98 (C5),
70.90 (C4), 69.83 (C3), 62.57 ppm (C1).
(5S)-5-C-[5-(1H-Indazole)]-a/b-l-xylopyranose (10ad): The title
compounds were obtained according to the general procedure.
Yield: 160.6 mg (white solid, 30%), >95% pure, as determined by
HPLC; retention factor k1 =0.8, k2 =1.4 (HPLC analyses were per-
formed as described previously); [a]2D0 =À46.7 (c=5 in DMSO, a/
(6S)-6-C-Phenyl-b-l-tagatofuranose
(12a;
furan):
1H NMR
(400 MHz, D2O): d=4.72 (d, J=5.5 Hz, 1H), 4.12 (t, J=5.5 Hz, 1H),
4.05–3.98 (m, 1H), 3.96–3.87 (m, 1H), 3.61 (d, J=11.6 Hz, 1H),
3.34 ppm (d, J=17.8 Hz, 1H); 13C NMR (101 MHz, D2O): d=98.55
(C2), 85.03 (C5), 81.35 (C3), 76.77 (C4), 73.12 (C6), 63.91 ppm (C1).
1
b 44:56); H NMR (400 MHz, [D6]DMSO): d=12.94 (s, 1H), 8.00 (d,
1.2 Hz, 1H), 7.64 (ddd, J=2.5, 1.6, 0.8 Hz, 1H), 7.43 (ddt, J=7.3, 6.4,
1.0 Hz, 1H), 7.30 (dd, J=8.7, 1.5 Hz, 1H), 6.63 (d, J=6.2 Hz, 1H;
b anomer), 6.34 (d, J=4.5, 1H; a anomer), 5.00 (t, J=4.0 Hz, 1H;
a anomer), 4.92 (dd, J=6.6, 4.5 Hz, 2H; b anomer), 4.74—4.69 (m,
2H), 4.66 (d, J=5.9 Hz, 1H; a anomer), 4.60 (d, J=9.7 Hz, 1H;
a anomer), 4.55 (d, J=6.6 Hz, 1H; a anomer), 4.46 (dd, J=7.7,
6.1 Hz, 1H; b anomer), 4.13 (d, J=8.8 Hz, 1H; b anomer), 3.59—
(6S)-6-C-(3-Chlorophenyl)-l-fructose (11k) and (6S)-6-C-(3-chloro-
phenyl)-l-tagatose (12k): The title compounds were obtained as
a 54:46 mixture of 11 k/12k as a white solid according to the gen-
eral procedure. Yield: 251.3 mg (43%), >95% pure, as determined
by HPLC; retention factor k1 =5.0, k2 =5.4; [a]2D0 =À10.0 (c=5 in
H2O). Compound 11 k exists as an equilibrium mixture of 71% as
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fructofuranose and 29% as fructopyranose. Compound 12k exists
of 17% as tagatofuranose and 83% as tagatopyranose. ESI-TOF: m/
ones reported for (1S,2R) and (1R,2R).[27] 1H NMR (400 MHz, D2O):
d=7.26-7.20 (m, 5H), 4.50 (d, J=6.5 Hz, 1H; (1S,2R)), 4.47 (d, J=
7.1 Hz, 1H; (1R,2R)), 3.74 (td, J=7.0, 3.2 Hz, 1H; (1R,2R)), 3.70 (d,
J=3.7 Hz, 1H; (1S,2R)), 3.62 (dd, J=11.9, 3.3 Hz, 1H; (1R,2R)), 3.43
(dd, J=11.8, 6.8 Hz, 1H; (1R,2R)), 3.35 (dd, J=11.8, 3.7 Hz, 1H;
(1S,2R)), 3.23 ppm (dd, J=11.8, 6.9 Hz, 1H; (1S,2R)); 13C NMR
(101 MHz, D2O): d=140.28 (C arom), 128.49 (C arom), 128.11 (C
arom), 127.07 (C arom), 75.36 (-CHOH-, (1S,2R)), 74.54 (-CHOH-,
(1R,2R)), 74.09 (Ph-CHOH-, (1S,2R)), 73.84 (Ph-CHOH-, (1R,2R)), 62.54
(-CH2OH, (1R,2R)), 62.39 ppm (-CH2OH, (1S,2R)).
z
calcd for C12H15ClO6Na: 313.0455 [M+Na+], C24H30Cl2O12Na:
603.1012 [2M+Na+]; found: 313.0457, 603.1010.
(6S)-6-C-(3-Chlorophenyl)-a-l-fructopyranose
(11k;
pyran):
1H NMR (400 MHz, D2O): d=4.79 (d, J=10.3 Hz, 1H), 4.05–3.98 (m,
1H), 3.93–3.85 (m, 1H), 3.89–3.81 (m, 1H), 3.63 (d, J=6.8 Hz, 1H),
3.29 ppm (d, J=11.6 Hz, 1H); 13C NMR (101 MHz, D2O) d=104.88
(C2), 72.99 (C4), 70.48 (C6), 68.14 (C3), 67.88 (C5), 63.87 ppm (C1).
(6S)-6-C-(3-Chlorophenyl)-b-l-fructofuranose
(11k;
furan):
1H NMR (400 MHz, D2O): d=4.68 (d, J=6.3 Hz, 1H), 4.14 (dd, J=
7.8, 6.8 Hz, 1H), 3.93 (d, J=7.8 Hz, 1H), 3.83 (dd, J=6.8, 6.2 Hz,
1H), 3.37 ppm (d, J=9.4 Hz, 2H); 13C NMR (101 MHz, D2O): d=
101.62 (C2), 82.89 (C5), 75.70 (C3), 75.66 (C4), 73.76 (C6), 62.30 ppm
(C1).
Acknowledgements
This work was supported by the Spanish MINECO (CTQ2012–
31605, CTQ2012–32436), the Generalitat de Catalunya (2009
SGR 00281), and ERA-IB MINECO (PIM2010EEI-00607,
EIB.10.012. MicroTechEnz-EIB., http://www.fkit.unizg.hr/miten)
and COST Action CM1303 Systems Biocatalysis. K. Hernꢀndez
acknowledges the CSIC for a JAE Predoctoral contract pro-
gram.
(6S)-6-C-(3-Chlorophenyl)-a-l-tagatopyranose
(12k;
pyran):
1H NMR (400 MHz, D2O): d=4.53 (d, J=9.8 Hz, 1H), 3.96–3.85 (m,
2H), 3.69 (t, J=9.4 Hz, 1H), 3.43 (d, J=9.1 Hz, 1H), 3.38 ppm (d, J=
9.0 Hz, 1H); 13C NMR (101 MHz, D2O): d=98.60 (C2), 74.78 (C6),
71.11 (C5), 70.77 (C4), 69.80 (C3), 63.87 ppm (C1).
(6S)-6-C-(3-Chlorophenyl)-b-l-tagatofuranose
(12k;
furan):
1H NMR (400 MHz, D2O): d=4.76–3.68 (m, 1H), 4.08 (t, J=5.5 Hz,
1H), 4.05–3.95 (m, 1H), 3.94–3.85 (m, 1H), 3.61 (d, J=11.7 Hz, 1H),
3.34 ppm (d, J=7.7 Hz, 1H); 13C NMR (101 MHz, D2O): d=98.33
(C2), 84.83 (C5), 81.36 (C3), 76.63 (C4), 72.48 (C6), 62.57 ppm (C1).
Keywords: aldolases · benzaldehyde lyase · carbohydrates ·
CÀC bond formation · enzyme catalysis
Synthesis of (1R,2R)-1-phenylpropane-1,2,3-triol (Benzoin
addition of benzaldehyde to glycolaldehyde; proof of con-
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magnetic stirring at 250 rpm. Benzaldehyde (10 mmol) was dis-
solved in MTBE (50 mL). Then, sodium carbonate buffer (50 mL,
50 mm, pH 8.0) containing BAL (500 UmLÀ1), MgSO4 (2.5 mm), and
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À808C was added. After 4 h the reaction mixture was allowed to
warm up to RT and a solution of HCl (1m 30 mL) was then added
dropwise. The solvent was removed under vacuum and the pH
value was adjusted to 7.0 with NaOH (1m). The solution was
frozen at À808C and lyophilized. Finally, the dry residue was ex-
tracted with AcOEt (3”30 mL), and the solvent was removed
under vacuum affording a white solid, which was identified as the
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Received: November 20, 2014
Published online on && &&, 0000
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Full Paper
FULL PAPER
Bottom-up sugars: C-Aryl carbohy-
drates on the non-reducing end exhibit
interesting biological activities. These
molecules have been prepared in four
synthetic steps combining cross-ben-
zoin and aldol addition reactions with
simple chemical transformation. By
using this methodology, C-aryl carbohy-
drate analogues of l-sorbose, l-xylose,
l-fructose, and l-tagatose are prepared
(see figure).
&
Biocatalytic Carbohydrate Ana-
logues
K. Hernꢀndez, T. Parella, J. Joglar,
J. Bujons, M. Pohl, P. Clapꢁs*
&& – &&
Expedient Synthesis of C-Aryl
Carbohydrates by Consecutive
Biocatalytic Benzoin and Aldol
Reactions
Chem. Eur. J. 2015, 21, 1 – 13
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ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ