FULL PAPERS

DOI: 10.1002/adsc.200900772

A Mutant d-Fructose-6-Phosphate Aldolase (Ala129Ser) with

Improved Affinity towards Dihydroxyacetone for the Synthesis
of Polyhydroxylated Compounds
Jos A. Castillo,a,b Christine Gurard-Hlaine,a,b Mariana Gutirrez,c
Xavier Garrabou,c Martine Sancelme,a,b Melanie Schrmann,e Tomoyuki Inoue,d
Virgil Hlaine,a,b Franck Charmantray,a,b Thierry Gefflaut,a,b Laurence Hecquet,a,b
Jesffls Joglar,c Pere Claps,c Georg A. Sprenger,d,e,* and Marielle Lemairea,b,*
a
Laboratoire SEESIB, Clermont Universit, Universit Blaise Pascal, 24 avenue des Landais, 63177 Aubire cedex, France
Fax: (+ 33)-(0)4-7340-7717; phone: (+ 33)-(0)4-7340-7584; e-mail: Marielle.Lemaire@univ-bpclermont.fr
b
CNRS, UMR 6504, 24 avenue des Landais, 63177 Aubire, France
c
Biotransformation and Bioactive Molecules Group, Instituto de Qumica Avanzada de CataluÇa – CSIC, Jordi Girona
18-26, 08034 Barcelona, Spain
d
Present address: Institute of Microbiology, Universitt Stuttgart, Allmandring 31, 70550 Stuttgart, Germany
Fax: (+ 49)-(0)711-685-65725; phone: (+ 49)-(0)711-685-65488; e-mail: g.sprenger@imb.uni-stuttgart.de
e
Institute for Biotechnology 1, Forschungszentrum Jlich, 52405 Jlich, Germany

Received: November 6, 2009; Revised: March 12, 2010; Published online: April 7, 2010

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/adsc.200900772.

Abstract: A mutant of d-fructose-6-phosphate aldo- makes them complementary biocatalysts allowing a

lase (FSA) of Escherichia coli, FSA A129S, with im-
proved catalytic efficiency towards dihydroxyacetone
(DHA), the donor substrate in aldol addition reac-
tions, was explored for synthetic applications. The
kcat/KM value for DHA was 17-fold higher with FSA
A129S than that with FSA wild type (FSA wt). On
the other hand, for hydroxyacetone as donor sub-
strate FSA A129S was found to be 3.5-fold less effi-
cient than FSA wt. Furthermore, FSA A129S also ac-
cepted glycolaldehyde (GA) as donor substrate with
3.3-fold lower affinity than FSA wt. This differential
selectivity of both FSA wt and FSA A129S for GA
control over donor and acceptor roles, which is par-
ticularly useful in carboligation multi-step cascade
synthesis of polyhydroxylated complex compounds.
Production of the mutant protein was also improved
for its convenient use in synthesis. Several carbohy-
drates and nitrocyclitols were efficiently prepared,
demonstrating the versatile potential of FSA A129S
as biocatalyst in organic synthesis.
Keywords: Ala129Ser mutant; carbohydrates; d-fruc-
tose-6-phosphate aldolase; dihydroxyacetone; nitro-
cyclitols

Introduction donor to furnish aldose-type products, opening new

Wild-type d-fructose-6-phosphate aldolase (FSA wt,
gene fsaA) from Escherichia coli, first described in
2001,[1,2] has attracted considerable attention due to
its remarkable catalytic potential in the asymmetric
synthesis of polyhydroxylated compounds.[3–7] This
enzyme was found to catalyze the 3S,4R (syn) stereo-
selective reversible aldol addition of dihydroxyace-
tone (DHA) on d-glyceraldehyde 3-phosphate (d-
G3P) to afford d-fructose 6-phosphate (d-F6P).[1] Fur-
thermore, it was later discovered by some of us that
FSA wt is able to accept glycolaldehyde (GA) as
strategies for biocatalytic aldol addition reactions.[7]
Therefore it can be considered both as a ketose- and
an aldose-aldolase. Mechanistically, it belongs to the
class I aldolase family (i.e., no metal cofactor re-
quired), in which an enamine (i.e., nucleophile) is
formed by reaction of the donor with a lysine residue
at the active site.[8] When compared with the well-
known DHAP-dependent aldolases, the hallmark of
FSA is its ability to catalyze direct aldol additions of
unphosphorylated DHA skipping a tedious, time-con-
suming and expensive dihydroxyacetone phosphate
(DHAP) synthesis. Moreover, it is the first uncovered

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 FULL PAPERS
Scheme 1. Examples of polyhydroxylated compounds
chemo-enzymatically prepared with FSA wt.[4–7]
aldolase that shows significant activity towards several
donor substrates, for example, hydroxyacetone (HA),
1-hydroxy-2-butanone (HB) and GA.[9] Recently,
some of us reported on a transaldolase B variant
(TalB F178Y)[10] that is also able to use DHA as
donor in aldol reactions and a double mutant (TalB
F178Y/R181E)[11] which exhibited improved affinity
towards non-phosphorylated acceptor aldehydes such
as d- and l-glyceraldehyde and GA. DHAP-depen-
dent rhamnulose aldolase (RhuA) has been described
to accept DHA in the presence of borate buffer.[12] In
addition, to alter RhuA donor substrate specificity
from DHAP to DHA, Sugiyama et al. reported the
development of an in vivo selection system for the di-
rected evolution of this aldolase.[13]
Due to its broad substrate tolerance, FSA wt has
been efficiently used to synthesize different poly-
hydroxylated compounds such as iminocyclitols and
carbohydrates (Scheme 1). Thus, the syntheses of d-
fagomine (1), deoxynojirimycin (DNJ, 2), 1,4,5-tri-
deoxy-1,4-imino-d-arabinitol (5-DDAB, 3) and ana-
logues were described by Claps and co-workers.[4,6]
Other illustrative iminocyclitols such as compound 4
were synthesized by Wong and co-workers.[5] In addi-
tion, the use of GA as donor provided the first biocat-
alytic strategy of self- and cross-aldol additions of GA
to prepare d-threose (5) and 5-deoxy-l-xylose (6).[7]
All along, these synthetic studies on FSA wt have
revealed different attractive features. Recombinant
FSA wt can be produced in high amount since it can
be efficiently expressed in E. coli. Moreover, the al-
dolase possesses an unusual thermostability, likely
due to its tight packing in a homodecamer form.[1–2,14]
Hence, FSA wt can be used with acceptable purity
after a simple heat treatment of a crude extract[1,4] or
even in whole cells.[5] Besides, the enzyme tolerates
organic co-solvents such as 10–20% dimethylforma-
mide and reactions can be performed at room temper-
ature or at lower temperature.[4]
As judged by the Vmax/KM values, FSA wt functions
~ 40-fold and ~ 20-fold better with HA and GA, re-
spectively, than with DHA.[7] This explains why HA is
1040 asc.wiley-vch.de  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Jos A. Castillo et al.
the donor substrate that provided the best reaction
rates and conversions to the aldol adducts.[3,5,6] There-
fore, aldehydes that are tolerated by FSA wt using
HA as donor may not react using DHA.[5–7] This low
affinity towards DHA is also the reason why the self-
aldol product of GA (i.e., d-threose, 5) was the only
product formed when DHA and GA were mixed in
the presence of FSA wt.[7] Since this peculiar reactivi-
ty map often limits some synthetic applicability, it was
regarded as interesting and significant to explore
novel FSA variants with distinct substrate specificity,
which complement FSA wt. In the course of investiga-
tions on the FSA function and mechanism, Schr-
mann and Sprenger envisaged a redesign of the FSA
active site with the aim to change the native FSA al-
dolase activity into transaldolase activity.[15,16] Accord-
ing to the crystallographic structure of FSA, the resi-
dues Leu107 and Ala129 were identified as putatively
involved in the binding site of the C-1 hydroxy group
of the donor substrate.[2] Hence, three mutants of
FSA wt were constructed by site-directed mutagene-
sis: two single A129S and L107N and a double
mutant L107N/A129S exchange.[15,16] Interestingly, al-
though no transaldolase activity was observed on any
of the mutants, the single A129S one gave a strikingly
improved kcat/KM towards DHA, d-G3P and d-F6P,
and potential complementary synthetic abilities to
those of FSA wt. It is likely that the serine residue
allows hydrogen bonding that stabilizes the DHA
donor substrate and also might be involved in the sta-
bilization of the Schiff-base intermediate.[2,15,16]
Regarding the interesting catalytic properties of
FSA A129S, a clear picture of its synthetic capabilities
has to emerge. To shed light on this point, in this
work we were prompted to study this FSA A129S
mutant for the synthesis of polyhydroxylated com-
pounds. Furthermore, kinetic parameters with differ-
ent substrates were measured to define precisely the
substrate spectrum of this biocatalyst.
Results and Discussion
Recently, we reported the kinetic parameters of FSA
wt for the retroaldol reaction of d-F6P and for aldol
reactions using DHA and HA as donors and GA as
donor and acceptor.[7] To exploit the catalytic proper-
ties of FSA A129S, the kinetic parameters for the
above-mentioned reactions, particularly for DHA and
HA as donors and GA as donor and acceptor, were
also determined. In addition, we have evaluated the
kinetic parameters for formaldehyde and 4-nitrobuta-
nal, as examples of acceptor substrates to evaluate
the potential effect of the mutation on the acceptor
affinity (Table 1).
As mentioned in the introduction for FSA wt, HA
and GA (Table 1, entries 2 and 3) were shown to be
Adv. Synth. Catal. 2010, 352, 1039 – 1046
A Mutant d-Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone

Table 1. Steady-state kinetic parameters of aldol and retroaldol reactions catalyzed by FSA wt and FSA A129S.

FSA wt FSA A129 S

Entry Substrate KM [mM] kcat[a,b] [s 1] kcat/KM [s 1 mM 1] KM [mM] kcat[a,c] [s 1] kcat/KM [s 1 mM 1]
1 DHA 32  2[d] 116[d] 4[d] 11  1 760 69
2 HA 17.4  0.5[d] 2527[d] 145[d] 22  3 899 41
3 GA donor 0.2  0.05[d] 16.5[d] 83[d] 0.18  0.02 4.4 25
4 GA acceptor 62.8  5.5[d] 16.5[d] 0.3[d] 12  5 4.4 0.4
5 4-Nitrobutanal 254  29 118 0.5 149  63 163 1
6 Formaldehyde ND[f] ND[f] ND[f] 77  6 27.7 0.4
7 G3P[e] 0.8 173 216 0.9 380 422
[a]
kcat = (Vmax/Vmax D-F6P) kcat D-F6P ; Vmax D-F6P is the value measured for cleavage of d-fructose-6-phosphate, see more details
in Supporting Information.
[b]
kcat D-F6P = 27 s 1.[16]
[c]
kcat D-F6P = 84 s 1.[16]
[d]
Values from Garrabou et al.[7]
[e]
Kinetic parameters for d,l-glyceraldehyde 3-phosphate were reported by Sprenger et al.[16]
[f]
ND: not done.

largely better donors than DHA (entry 1). FSA wt
has a KM value for DHA of 32 mM; the A129S var-
iant has an improved KM value of 11 mM. Therefore,
as judged by kcat/KM, the mutation induced a decrease
in the catalytic selectivity towards HA and GA
donors and an improvement towards DHA. In terms
of kcat/KM, it is noticeable that the activity towards
HA and GA was reduced by factors of 3.5 and 3.3, re-
spectively (entries 2 and 3). Interestingly, FSA A129S
functions 17-fold better than FSA wt with DHA. On
the other hand, the mutation induced no significant
effect on the acceptors since the catalytic activity was
found to be in the same range with 4-nitrobutanal as
well as with GA (entries 4 and 5). For both enzymes,
GA, 4-nitrobutanal and formaldehyde (entries 4, 5
and 6) were poor acceptors compared to d,l-G3P. Re-
markably, d,l-G3P is clearly the best acceptor for
both aldolases. In spite of the low kcat/KM good con-
versions and isolated yields were achieved with both
4-nitrobutanal and formaldehyde (vide infra).
These results point out that the mutant FSA A129S
is an improved biocatalyst for the aldol additions of
DHA to aldehydes. This differential reactivity of FSA
A129S as compared with FSA wt is of paramount im-
portance since it allows one to modulate donor and
acceptor roles in cascade self- and cross-aldol addition
reactions, particularly when using GA.
The production of enzymes for the development of
biosynthetic methodologies is of utmost importance.
In this connection, the production of FSA A129S was
improved using a classical plasmid vector carrying the
fsaA129S gene with a kanamycin resistance cassette
to have a continuous supply of the biocatalyst in large
quantities and at a reasonable cost (for details see
Supporting Information).
Synthetic Examples using FSA A129S as Biocatalyst:
Nitrocyclitols
4-Nitrobutanal has been widely used in our laboratory
to synthesize nitrocyclitols by a chemo-enzymatic
methodology using DHAP as donor substrate.[17–19]
The one-pot cascade process for nitrocyclitols prepa-
ration involved the formation of two carbon-carbon
bonds. First, the aldol reaction was catalyzed by d-
fructose 1,6-bisphosphate aldolase (FruA from rabbit
muscle, RAMA) in the presence of DHAP and this
was followed by an intramolecular Henry reaction.
This strategy furnished nitrocyclitols in a highly ste-
reoselective way. For example, nitrocyclitol (7) was
obtained (see Table 2, entry 1) as a major diastereo-
isomer in 60% isolated yield.[17]
For the sake of comparison, in the present work we
performed the two-step cascade carbon-carbon form-
ing process by the aldol addition of DHA to 4-nitro-
butanal catalyzed by FSA A129S. Interestingly, in
spite of the fact that 4-nitrobutanal was not a good
substrate for FSA A129S (Table 1, entry 5), the nitro-
cyclitol 7 was isolated in 73% isolated yield also as a
major diastereoisomer (Table 2, entry 1). 4-Nitrobuta-
nal was also submitted to aldol reaction with HA as
donor (Table 2, entry 2). In this case, the enzymatic
reaction was fully stereoselective, while the intramo-
lecular Henry reaction was not. This lower diastereo-
selectivity was probably due to the absence of the C-1
hydroxy group, which might direct the approach of
the nucleophilic nitro a-carbon to the carbonyl group.
The whole process gave a mixture of diastereoisomers
in 87% isolated yield, which were separated by classi-
cal chromatography furnishing nitrocyclitols 8, 9, 10
and 11 in 6%, 4%, 55% and 22% isolated yields, re-
spectively. The stereochemistries of these new nitrocy-
clitols were unequivocally assigned, based on the 3S-

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 FULL PAPERS Jos A. Castillo et al.

Table 2. Aldol additions catalyzed by FSA A129S.

Entry Acceptor Donor Product Isolated yield[a] and/or conversion[b]

1 DHA (7) 73%[a]

2 HA (8)–(11) (8) 6%;[a] (9) 4%;[a] (10) 55%;[a] (11) 22%[a]

3 DHA (12) 52%[a]

4 DHA (13) 68%[a]

5 DHA (14) 80%[b] ; 42%[a]

6 HA (15) 70%[b]

7 DHA (16) 81%[b] ; 67%[a]

8 HA (17) 86%[b] ; 68%[a]

9 GA (18) 38%[b,c]

[a]
Isolated yield.
[b]
Conversion of the aldehyde to product was determined by HPLC (for details see Supporting Information).
[c]
d-Threose formation (20% by HPLC) was observed arising from self-aldol addition of GA.

stereoselectivity of the aldolase and on high-field
NMR studies (coupling constants and NOESY experi-
ments).
An enantiomerically pure 2-hydroxy-4-nitrobuta-
nal[19] was submitted to aldolization with DHA
(Table 2, entry 3). In this case, the two-step cascade
chemo-enzymatic process was fully stereoselective
and nitrocyclitol 12 was isolated in 52% yield.
Remarkably, the aldol additions catalyzed by FSA
A129S gave generally higher isolated yields than
those with FruA and the synthetic process were con-
siderably simplified since a number of steps required
for DHAP preparation and phosphate group removal
of the resulting adduct were avoided.

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A Mutant d-Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone
Carbohydrates
As a first example of carbohydrates synthesis using
FSA A129S, l-erythrulose (13) was synthesized from
DHA and formaldehyde as starting materials. This
aldol reaction afforded l-erythrulose (13) in 68% iso-
lated yield as a unique enantiomer.
Interestingly, the aldol addition of DHA to GA
proceeded in 80% aldehyde conversion to d-xylulose
(14) (42% isolated yield not optimized, Table 2
entry 5). This was the most striking result since, using
FSA wt, d-threose was exclusively formed, arising
from the self-aldol addition reaction of GA.[6,7] This
differential reactivity was a consequence of the modi-
fied catalytic properties of FSA A129S (Table 1, en-
tries 1 and 3). Moreover, the aldol addition of DHA
to d-glyceraldehyde (d-GAL) furnished d-fructose
(16) in 67% isolated yield (Table 2, entry 7), while
FSA wt was nearly inactive to catalyze this aldol reac-
tion.[3] This might be due to better catalytic affinity of
FSA A129S for DHA which could afford a protection
of the donor active site against aldehyde inhibition.
This phenomenon has been previously demonstrated
for class I aldolases.[20]
Despite the lower catalytic activity of FSA A129S
towards HA as compared with FSA wt, the condensa-
tion of HA to GA and d-GAL proceeded with good
conversion to the corresponding 1-deoxy-d-xylulose
(15) and 1-deoxy-d-fructose (17) (Table 2, entries 6
and 8).[6, 7] In aldol additions involving GA (Table 2,
entries 5, 6 and 9), the side conversion to self-aldol
addition product, d-threose, was moderate (20%) as a
consequence of the reduced affinity of FSA A129S to
GA as donor and acceptor. The enzymatic condensa-
tion of DHA on d-threose was attempted, unfortu-
nately in a one-pot/two-step process where d-threose
was accumulated and then followed by addition of
DHA, the corresponding d-ido-hept-2-ulose was
never observed.
Finally, as ascertained by NMR spectroscopy and
within the limits of detection, FSA A129S was fully
stereoselective yielding the corresponding syn (3S,4R)
aldol adducts in all cases. As illustrated, the known l-
erythrulose, d-xylulose and d-fructose were exclusive-
ly formed.
Conclusions
From the kinetic data obtained with different sub-
strates, the mutant FSA A129S was found up to 17-
fold more efficient to condense DHA than FSA wt.
FSA A129S also accepted HA and GA as donor sub-
strates with 3.5- and 3.3-fold lower affinity, respective-
ly, than FSA wt. This differential selectivity of both
enzymes makes them complementary biocatalysts.
Hence, we have also demonstrated that the synthesis
Adv. Synth. Catal. 2010, 352, 1039 – 1046  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
of complex sugars and analogues such as d-xylulose
and d-fructose, not accessible via FSA wt catalysis,
was catalyzed by FSA A129S. Both aldolases will
expand the scope of carbohydrates and analogues
that can be prepared enzymatically and will open
novel synthetic opportunities for the carboligation
cascade synthesis of polyhydroxylated compounds.
Most importantly, the enzyme has shown a high dia-
stereoselectivity affording always the syn (3S,4R) con-
figuration. The use of an expression vector with an
added kanamycin resistance cassette led to a remark-
able improvement of the enzyme yields in comparison
to the system with ampicillin resistance alone. Thus,
FSA A129S production was improved for its use in
organic synthesis at multi-gram scale.
Experimental Section
For Kinetic Measurements
One unit of FSA cleaves 1 mmol of d-fructose 6-phosphate
per min to afford d-glyceraldehyde 3-phosphate and dihy-
droxyacetone at pH 7.5 (TEA 50 mM buffer) and 25 8C.
For 4-Nitrobutanal as Acceptor with FSA wt
Aldol addition reaction of DHA to 4-nitrobutanal: Measure-
ment of DHA was carried out with glycerol dehydrogenase
(GDH) from Cellulomonas sp. To a solution containing
DHA (100 mM) and FSA wt powder (1.5 mg to 3 mg, 1.2 U
to 2.4 U) in 100 mM tris-ethanolamine (TEA) buffer pH 7.5
at 25 8C, 4-nitrobutanal (in concentration varying from 40 to
200 mM) was added. The final volume was 1 mL. Then, ali-
quots (20 mL) were withdrawn at different times and diluted
in water (380 mL). Samples (20 mL) were then dissolved in a
solution (180 mL) composed of GDH (20 U) and NADH
(0.7 mM) in TEA buffer. The consumption of NADH was
monitored spectrophotometrically at 340 nm and 25 8C. One
mmol of NADH oxidized was equivalent to 1 mmol of
DHA.
Km and Vmax for 4-Nitrobutanal and Formaldehyde as
Acceptor with FSA A129S
Aldol addition reaction of DHA to 4-nitrobutanal or formal-
dehyde: Measurement of DHA was carried out with glycerol
dehydrogenase (GDH) from Cellulomonas sp. To a solution
containing DHA (30 mM) and FSA A129S powder (0.2 mg,
0.72 U for nitrobutanal and 0.4 mg to 1.3 mg, 1.4 U to 4.6 U
for formaldehyde) in 100 mM tris-ethanolamine (TEA)
buffer pH 7.5 at 25 8C, 4-nitrobutanal (in concentration vary-
ing from 50 to 120 mM) or formaldehyde (in concentration
varying from 20 to 200 mM) was added. The final volume
was 1 mL. Then, aliquots (20 mL) were withdrawn at differ-
ent times and diluted in water (80 mL). Samples (20 mL)
were then dissolved in a solution (980 mL) composed of
GDH (20 U) and NADH (0.2 mM) in TEA buffer. The con-
sumption of NADH was monitored spectrophotometrically
at 340 nm and 25 8C. One mmol of NADH oxidized was
equivalent to 1 mmol of DHA.
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Km and Vmax of donor substrates were measured as previ-
ously described.[7]
(1S,2S,3R,6R)-1-(Hydroxymethyl)-6-nitrocyclo-
hexane-1,2,3-triol (7)
4-Nitrobutanal (500 mg, 4.3 mmol) was dissolved in H2O
(50 mL). Then, dihydroxyacetone (320 mg, 3.6 mmol) and
FSA A129S aldolase powder (125 mg, 338 U) were added.
The reaction mixture was placed on a reciprocal shaker for
17 h at 25 8C (175 rpm). MeOH (50 mL) was added and the
mixture was centrifuged (20000 rpm, 40 min, 5 8C). The su-
pernatant was concentrated under reduced pressure, afford-
ing a yellow oil. The crude material was purified by silica
gel chromatography using CH2Cl2/MeOH (90:10) as eluent
to afford the desired nitrocyclitol 7; isolated yield: 538 mg
(73%). 1H NMR and 13C spectra and [a]20
D values were iden-
tical to those obtained previously in our group.[17] 1H NMR
(400 MHz, CD3OD): d = 4.79 (dd, 1 H, H-6, J6ax,5ax = 12.9,
J6ax,5eq = 4.0 Hz), 3.82 (d, Jgem = 11.0 Hz, 1 H, H-7), 3.73 (ddd,
J3,4ax = 11.6, J3,2 = 9.4, J3,4eq = 4.7 Hz, 1 H, H-3), 3.39 (d, J2,3 =
9.4 Hz, H-2), 3.35 (d, Jgem, = 11 Hz, 1 H, H-7’), 2.49–2.36 (m,
1 H, H-5eq) 2.03–1.99 (m, 1 H, H-5eq), 1.99–1.95 (m, 1 H, H-
4eq), 1.38–1.26 (m, 1 H, H-4ax); 13C NMR (100 MHz,
CD3OD): d = 86.4 (C-6), 77.1 (C-1), 75.2 (C-2), 71.0 (C-3),
61.9 (C-7), 29.6 (C-4), 24.7 (C-5); [a]20 D : + 22 (c 1.0,
MeOH).
1-Methyl-6-nitrocyclohexane-1,2,3-triols (8)–(11)
Hydroxyacetone (630 mg, 8.5 mmol) and FSA A129S aldo-
lase powder (250 mg, 675 U) were dissolved in water
(100 mL). 4-Nitrobutanal (1.1 g, 9.4 mmol) was added to the
reaction mixture. The reaction mixture was placed on a re-
ciprocal shaker for 18 h at 25 8C (175 rpm). The pH was
maintained between 7.5 and 8.5. MeOH (50 mL) was added
and the mixture was centrifuged (20000 rpm, 40 min, 5 8C).
The supernatant was concentrated under reduced pressure
at 25 8C. The crude material was purified by silica gel chro-
matography using CH2Cl2/acetone (6:4) as eluent to afford
the following nitrocyclitols 8–11; global yield: 87%.
(1S,2S,3R,6S)-1-Methyl-6-nitrocyclohexane-1,2,3-triol (8):
isolated yield: 100 mg (6%); Rf = 0.53 (CH2Cl2/acetone, 6:4);
mp 143–145 8C; [a]20 17.4 (c 1.0, MeOH); IR: n = 3435,
D:
3300, 2949, 2909, 2548, 2450, 1541, 1452, 1373, 1316, 1292,
1221, 1136, 1063, 1049, 1013, 970, 920, 862, 812, 750 cm 1;
1
H NMR (400 MHz, CD3OD): d = 4.71 (dd, J6,5ax = 10.4,
J6,5eq = 3.8 Hz, 1 H, H-6), 3.79 (dd, J3,4ax = 8.4, J3,2 = 4.5 Hz,
1 H, H-3), 3.69 (d, J2,3 = 4.5 Hz, 1 H, H-2), 2.52–2.41 (m, 1 H,
H-5ax), 1.98–1.89 (m, 1 H, H-4eq), 1.87–183 (m, 2 H, H-4ax
and H-5eq), 1.28 (s, 3 H, CH3); 13C NMR (100 MHz,
CD3OD): d = 90.2 (C-6), 75.9 (C-2), 75.0 (C-1), 71.6 (C-3),
27.3 (C-4), 23.7 (C-7), 22.3 (C-5); HR-MS (ES+): m/z =
214.0706, calcd. for [C7H13NO5 + Na+]: 214.0691.
(1R,2S,3R,6S)-1-Methyl-6-nitrocyclohexane-1,2,3-triol (9):
isolated yield: 62 mg (4%); Rf = 0.37 (CH2Cl2/acetone, 6:4);
[a]20 1
D : + 43 (c 1.0, MeOH); H NMR (400 MHz, CD3OD):
d = 4.68 (t, J6,5ax = 5.4, J6,5eq = 5.4, Hz, 1 H, H-6), 3.82 (ddd,
J3,4ax = 7.9, J3,2 = 7.0, J3,4eq = 4.4 Hz, 1 H, H-3), 3.66 (d, J2,3 =
7.0 Hz, 1 H, H-2), 2.14–2.09 (m, 2 H, H-5), 1.94–1.85 (m, 1 H,
H-4), 1.85–1.74 (m, 1 H, H-4’), 1.32 (s, 1 H, CH3); 13C NMR
(100 MHz, CD3OD): d = 92.0 (C-6), 77.6 (C-2), 73.7ACHTUNGRE(C-1),
1044 asc.wiley-vch.de  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Jos A. Castillo et al.
71.1ACHTUNGRE(C-3), 28.2 (C-4), 25.0 (C-5), 22.0 (C-7); HR-MS (ES ):
m/z = 190.0711, calcd. for [C7H12NO5]: 190.0715.
(1R,2S,3R,6R)-1-Methyl-6-nitrocyclohexane-1,2,3-triol
(10): isolated yield: 898 mg (55%); Rf = 0.34 (CH2Cl2/ace-
tone, 6:4); mp 162–164 8C; [a]20 D : + 21 (c 1.0, MeOH);
1
H NMR (400 MHz, CD3OD): d = 4.52 (dd, J6,5ax = 12.7,
J 6,5eq = 3.8 Hz, 1 H, H-6), 3.69 (ddd, J3,4ax = 11.6, J3,2 = 9.3,
J3,4eq = 5.2 Hz, 1 H, H-3), 3.02 (d, J2,3 = 9.3 Hz, 1 H, H-2), 2.41
(m, 1 H, H-5ax), 2.03–1.95 (m, 1 H, H-4eq), 1.92 (ddd, Jgem =
12.7, J3,4ax = 7.3, J5eq,6 3.8 Hz, 1 H, H-5eq), 1.36 (s, 3 H, CH3),
1.40–1.28 (m, 1 H, H-4ax); 13C NMR (100 MHz, CD3OD):
d = 91.6 (C-6), 80.0 (C-2), 74.7 (C-1), 70.9 (C-3), 29.6 (C-4),
25.0 (C-5), 23.0 (C-7); IR: n = 3505, 3428, 2982, 2914, 1539,
1452, 1369, 1330, 1298, 1260, 1220, 1190, 1128, 1109, 1074,
1038, 899, 876, 858, 779, 723, 694, 642 cm 1; elemental analy.
calcd. (%) for C7H13NO5 (Mw 191.18): C 43.98, H 6.85, N
7.33, O 41.84; found: C 44.00, H 6.94, N 7.46, O 41.60.
(1S,2S,3R,6R)-1-Methyl-6-nitrocyclohexane-1,2,3-triol
(11): isolated yield: 358 mg (22%); Rf = 0.24 (CH2Cl2/ace-
tone, 6:4); mp 125–127 8C; [a]20 D: 58 (c 1.0, MeOH);
1
H NMR (400 MHz, CD3OD): d = 4.55 (dd, J6,5ax = 9.7, J6,5eq
7.1 Hz, 1 H, H-6), 3.45 (ddd, J3,4ax = 11.3, J3,2 = 9.6, J3,4eq =
4.9 Hz, 1 H, H-3), 3.23 (d, J2,3 = 9.6 Hz, 1 H, H-2), 2.11–1.98
(m, 3 H, 2 H-5 and H-4eq), 1.47–1.34 (m, 1 H, H-4ax), 1.18 (s,
3 H, CH3); 13C NMR (100 MHz, CD3OD): d = 92.7 (C-6),
81.6 (C-2), 76.1 (C-1), 71.3 (C-3), 29.8 (C-4), 26.0 (C-5), 15.2
(C-7); IR: n = 3389, 2955, 2912, 1547, 1429, 1373, 1344, 1292,
1196, 1128, 1099, 1084, 1041, 1015, 984, 966, 947, 878, 781,
714 cm 1; elemental anal. calcd. (%) for C7H13NO5 (Mw
191.18): C 43.98, H 6.85, N 7.33, O 41.84; found: C 44.13, H
6.89, N 7.36, O 41.62.
(1S,2S,3R,4R,6R)-1-(Hydroxymethyl)-6-nitrocyclo-
hexane-1,2,3,4-tetraol (12)
To a solution of (R)-1,1-dimethoxy-4-nitrobutan-2-ol (30 mg,
0.17 mmol) in H2O (1 mL) was added a cation exchange
resin (Dowex 50 8, H+ form, 0.5 g). The suspension was
stirred at 45 8C for 1 h affording (R)-2-hydroxy-4-nitrobuta-
nal (quantitative by TLC). The resin was filtered off using
H2O (2 mL) as rinse. Then, the pH was adjusted to 7.5 with
1 M NaOH. To this solution were added DHA (15 mg,
0.17 mmol) followed by FSA A129S (40 mg lyophilized
powder, 98 U). The reaction mixture was placed on a recip-
rocal shaker for 17 h at 25 8C (175 rpm). MeOH (50 mL)
was added and the mixture was centrifuged (20000 rpm,
40 min, 5 8C). The supernatant was concentrated under re-
duced pressure. The residue was purified by silica gel chro-
matography using CH2Cl2/MeOH (8:2) as eluent, affording
the desired nitrocyclitol 12; yield: 19 mg (52%). The NMR
data were identical to those previously published.[19]
1
H NMR (400 MHz, CD3OD): d = 5.11 (dd, J6,5ax = 13.0,
J6,5eq = 4.0 Hz, 1 H, H-6), 4.08 (dd, J = 6.2, 3.3 Hz, 1 H, H-4),
3.85 (d, J2,3 = 9.8 Hz, 1 H, H-2), 3.82 (d, Jgem = 11.1 Hz, 1 H,
H-7), 3.70 (dd, J3,2 = 9.8, J3,4 = 3.3 Hz, 1 H, H-3), 3.36 (d, J =
11.1 Hz, 1 H, H-7’), 2.57 (dt, J5ax,5eq = 13.1, J5ax,6 = 13.1, J5ax,4 =
2.4 Hz, 1 H, H-5ax), 2.13 (td, J5eq,5ax = 13.2, J5eq,6 = 4.0, J5eq,4 =
3.9 Hz, 1 H, H-5eq); 13C NMR (100 MHz, CD3OD): d = 82.9
(C-6), 77.2 (C-1), 73.0 (C-2), 70.2 (C-3), 68.6 (C-4), 61.9 (C-
7), 32.1 (C-5); [a]20
D : + 9 (c 0.7, MeOH)
Adv. Synth. Catal. 2010, 352, 1039 – 1046
A Mutant d-Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone
l-Erythrulose (13)
Formaldehyde (676 mg of aqueous solution 37% w/w,
8.3 mmol) and dihydroxyacetone (750 mg, 8.3 mmol) were
dissolved in water (85 mL). Then FSA A129S powder
(300 mg, 810 U) was added to the solution. The pH was ad-
justed at 7.5. The mixture was shaked for 21 h at 150 rpm
and 25 8C. MeOH (85 mL) was added and the mixture was
centrifuged (20000 rpm, 10 8C, 40 min). The supernatant was
concentrated under reduced pressure at 30 8C. The crude
material was purified by silica gel chromatography using
CH2Cl2/MeOH (9:1) as eluent to afford l-erythrulose 14 as
colourless oil; yield: 676 mg (68%). The NMR spectra were
identical to those of a commercial l-erythrulose sample:
1
H NMR (400 MHz, D2O): d = 4.62 (d, J = 19.4 Hz, 1 H, H-
1), 4.54 (d, J = 19.4 Hz, 1 H, H-1’), 4.47 (dd, J = 4.3, 3.9 Hz,
1 H, H-3), 3.89 (dd, J = 12.2, 4.4 Hz, 1 H, H-4), 3.87 (dd, J =
12.2, 3.9 Hz, 1 H, H-4’); 13C NMR (100 MHz, D2O): d =
212.3 (C-2), 75.9 (C-3), 65.9 (C-1), 62.9 (C-4); [a]20
D : + 12 (c
1.0, H2O) [lit.21 [a]20
D : + 13 (c 2.44, H2O)].
d-Xylulose (14)
Dihydroxyacetone (111 mg, 1.2 mmol) and glycoladehyde
(59 mg, 1.0 mmol) were dissolved in NaHCO3 50 mM,
pH 7.5 buffer (12 mL). To this solution FSA A129S powder
(19 mg, 52 U) was added and the reaction mixture placed on
a reciprocal shaker (200 rpm) at 25 8C. When the limiting
substrate was consumed (~ 48 h) the reaction was filtered
through active charcoal to remove the enzyme. The filtrate
was adjusted to pH 5.0 with dilute HCl and freeze-dried.
The residue was purified by flash column chromatography
on silica gel with [ethyl acetate:CHCl3ACHTUNGRE(1:1):MeOH (9:1)] as
eluent. The oil obtained was then dissolved in water and
lyophilized to afford d-xylulose as a pale yellow oil; isolated
yield: 51 mg (42%); [a]20 D: 28 (c 1.1, H2O) [lit.22 [a]25D: 32
1
(c 2.7, H2O)]; H NMR (500 MHz, D2O) (mixture of cyclic
and acyclic forms): d = 4.54 (d, J = 19.4 Hz, 1 H), 4.43 (d, J =
19.4 Hz, 1 H), 4.35 (s, 1 H), 4.28 (m, 2 H), 4.17 (m, 1 H), 4.13
(dd, J = 9.5, 6.2 Hz, 1 H), 4.08 (dd, J = 9.5, 6.5 Hz, 2 H), 3.97
(m, 1 H), 3.94 (d, J = 5.6 Hz, 2 H), 3.79 (dd, J = 9.6, 4.2 Hz,
1 H), 3.60 (m, 1 H), 3.55 (m, 2 H), 3.51 (d, J = 11.1 Hz, 2 H),
3.46 (d, J = 12.1 Hz, 3 H); 13C NMR (101 MHz, D2O) (mix-
ture of cyclic and acyclic forms): d = 213.1, 105.9, 103.1, 80.7,
76.4, 76.0, 75.5, 75.0, 72.1, 72.1, 70.0, 66.2, 63.2, 62.6, 62.0.
d-Fructose (16)
Dihydroxyacetone (108 mg, 1.2 mmol) and d-glyceraldehyde
(86 mg, 0.96 mmol) were dissolved in NaHCO3 50 mM,
pH 7.5 buffer (12 mL). To this solution FSA A129S powder
(21 mg, 56 U, 68 mg protein) was added and the reaction
mixture placed on a reciprocal shaker (200 rpm) at 25 8C.
After 48 h the reaction was worked up and the crude isolat-
ed as described for compound 14. The flash column chroma-
tography over silica gel was eluted with (ethyl acet-
ACHTUNGREate:CHCl3 1:1):MeOH 9:1 to remove the non-converted
starting substrates. Then, the product was eluted with a mix-
ture of (ethyl acetate:CHCl3, 1:1):MeOH:H2O from 86:10:4
to 75:20:5. After concentration under vacuum, the residue
was lyophilized to afford d-fructose as a white solid; isolated
yield: 140 mg (81%); 1H NMR (500 MHz, D2O): d = 4.00 (d,
J = 3.5 Hz, 1 H), 3.93 (s, 1 H), 3.90 (s, 1 H), 3.88 (d, J =
Adv. Synth. Catal. 2010, 352, 1039 – 1046  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1.6 Hz, 2 H), 3.79 (dd, J = 10.1, 3.5 Hz, 1 H), 3.69 (m, 2 H),
3.58 (m, 4 H), 3.45 (m, 2 H); 13C NMR (101 MHz, D2O): d =
104.6, 101.6, 98.2, 82.1, 81.5, 80.8, 76.2, 75.5, 74.6, 69.8, 69.4,
67.7, 64.0, 63.5, 62.8, 62.5.
1-Deoxy-d-fructose (17)
Hydroxyacetone (79 mg, 1.1 mmol) and d-glyceraldehyde
(76 mg, 0.9 mmol) were dissolved in NaHCO3, 50 mM,
pH 7.5 buffer (11 mL). To this solution FSA A129S powder
(17 mg, 47 U) was added. Then the procedure and work up
were identical to that described for compound 14. The pu-
rification was similar to that described for d-fructose in the
following conditions: first, the column was eluted with
(ethyl acetate:CHCl3 1:1):MeOH 9:1 to remove the non-
converted starting substrates. Then, the product was eluted
with a mixture of (ethyl acetate:CHCl3 1:1):MeOH:H2O
from 80:16:4 to 70:20:10. After concentration under
vacuum, the residue was lyophilized to afford 1-deoxy-d-
fructose as a sticky white solid; isolated yield: 89 mg (68%);
[a]20
D: 87 (c 0.9, H2O) [lit.23 [a]25
D: 80 (c 2.7, H2O)]. 1H and
13
C NMR matched those reported by Jones et al.[23] 1H NMR
(500 MHz, D2O): d = 4.06 (m, 1 H), 4.01 (m, 1 H), 3.99 (m,
1 H), 3.80 (m, 2 H), 3.65 (dd, J = 18.2, 8.5 Hz, 2 H), 3.62 (m,
2 H), 3.59 (m, 2 H), 3.57 (m, 2 H), 2.28 (m, 1 H, keto form),
1.47 (m, 1 H, b-pyranose), 1.41 (m, 1 H, b-furanose), 1.36 (m,
1 H, a-pyranose); 13C NMR (101 MHz, D2O): d = 213.7,
105.3, 101.7, 98.3, 82.5, 81.4, 80.6, 80.2, 77.0, 76.5, 74.8, 73.0,
72.1, 71.1, 70.9, 70.7, 69.6, 69.4, 63.4, 63.0, 62.8, 61.3, 25.8,
24.7, 23.9, 21.9.
HPLC Measurements of Conversion of the Enzyme-
Catalyzed Reaction
For d-xylulose (14), 1-deoxy-d-xylulose (15), d-fructose
(16), 1-deoxy-d-fructose (17), d-arabinose (18), d-threose
(19), see Supporting Information.
Acknowledgements
JAC acknowledges the French foundation Vaincre Les Mal-
adies Lysosomales for a postdoctoral fellowship. MG and
XG acknowledge the I3P-CSIC predoctoral scholarship pro-
gram. This work was supported by the Spanish MCINN
CTQ2009-07359/BQU, La Marat de TV3 foundation (Ref:
050931), Generalitat de Catalunya DURSI 2005-SGR-00698
and ESF project COST CM0701. GS acknowledges the Deut-
sche Forschungsgemeinschaft which supported work of Mela-
nie S. and T.I. through SFB 380/B21. We thank Dr. Natalie
Trachtmann and Liliya Malikova for help with cloning of
vector pJFfsaA129S-kan.
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