Influence of N-amino protecting group on aldolase-catalyzed aldol additions of dihydroxyacetone phosphate to amino aldehydes

Article abstract of DOI:10.1016/j.tet.2005.12.031

This work examines the influence of N-protecting groups on the conversion and stereoselectivity of dihydroxyacetone phosphate (DHAP) dependent aldolase-catalyzed aldol additions of DHAP to N-protected-3-aminopropanal. Phenylacetyl-(PhAc-), tert-butyloxycarbonyl- (^tBoc-) and fluoren-9-ylmethoxycarbonyl- (Fmoc-)-3-aminopropanal were evaluated as substrates for d-fructose 1,6-bisphosphate aldolase from rabbit muscle (RAMA), and l-rhamnulose-1-phosphate aldolase (RhuA) and l-fuculose-1-phosphate aldolase (FucA), both from Escherichia coli. Using PhAc and ^tBoc ca. 70% conversions to the aldol adduct were achieved, whereas Fmoc gave maximum conversions of ca. 25%. The stereoselectivity of the DHAP-aldolases did not depend on the N-protected-3-aminopropanal derivative. Moreover, inversion of FucA stereoselectivity relative to that obtained with the natural l-lactaldehyde was observed. Both N-PhAc and ^tBoc adduct product derivatives were successfully deprotected by penicillin G acylase (PGA)-catalyzed hydrolysis at pH 7 and by treatment with aqueous TFA (6% v/v), respectively. However, the corresponding cyclic imine sugars could not be isolated, presumable due to the presence of a highly reactive primary amine and a keto group in the molecule, which lead to a number of unexpected reactions.

Full text of DOI:10.1016/j.tet.2005.12.031

Tetrahedron 62 (2006) 2648–2656
Influence of N-amino protecting group on aldolase-catalyzed aldol
additions of dihydroxyacetone phosphate to amino aldehydes
Jordi Calveras,a Jordi Bujons,a Teodor Parella,b Ramon Crehuet,a Laia Espelt,a
a
Jesus Joglar and Pere Clapes
a,
*
´
´
aInstitute for Chemical and Environmental Research-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
b
`
`
`
Servei de Ressonancia Magnetica Nuclear, Universitat Autonoma de Barcelona, Campus de Bellaterra, Barcelona, Spain
Received 18 November 2005; revised 14 December 2005; accepted 14 December 2005
Available online 9 January 2006
Dedicated to Professor Francisco Camps on the occasion of his 70th birthday.
Abstract—This work examines the influence of N-protecting groups on the conversion and stereoselectivity of dihydroxyacetone phosphate
(DHAP) dependent aldolase-catalyzed aldol additions of DHAP to N-protected-3-aminopropanal. Phenylacetyl-(PhAc-), tert-
butyloxycarbonyl- (tBoc-) and fluoren-9-ylmethoxycarbonyl- (Fmoc-)-3-aminopropanal were evaluated as substrates for D-fructose
1,6-bisphosphate aldolase from rabbit muscle (RAMA), and L-rhamnulose-1-phosphate aldolase (RhuA) and L-fuculose-1-phosphate
aldolase (FucA), both from Escherichia coli. Using PhAc and tBoc ca. 70% conversions to the aldol adduct were achieved, whereas Fmoc
gave maximum conversions of ca. 25%. The stereoselectivity of the DHAP-aldolases did not depend on the N-protected-3-aminopropanal
derivative. Moreover, inversion of FucA stereoselectivity relative to that obtained with the natural L-lactaldehyde was observed. Both
N-PhAc and tBoc adduct product derivatives were successfully deprotected by penicillin G acylase (PGA)-catalyzed hydrolysis at pH 7 and
by treatment with aqueous TFA (6% v/v), respectively. However, the corresponding cyclic imine sugars could not be isolated, presumable
due to the presence of a highly reactive primary amine and a keto group in the molecule, which lead to a number of unexpected reactions.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
In the course of our ongoing project on the chemo-
enzymatic synthesis of iminocyclitols we investigated
whether other N-blocking groups of the amino aldehyde
may also be suitable for the synthesis of these compounds.
Furthermore, alternative N-protecting groups to suit any
further synthetic strategies upon the N-protected amino-2-
keto-3,4-diols was also pursued. To this end, three
N-protecting groups for the model aldehyde 3-aminopro-
panal were selected: phenylacetyl (PhAc), tert-butyloxy-
carbonyl (tBoc) and fluoren-9-ylmetoxycarbonyl (Fmoc).
PhAc is structurally similar to Cbz, and it can be cleaved by
penicillin amidase (PGA)-catalyzed hydrolysis under mild
Aldolases are a class of lyases that catalyze stereoselective
aldol additions of aldehydes and ketones,1,2 constituting
powerful tools in the asymmetric synthesis of both
conventional and uncommon carbohydrates as well as
other complex hydroxylated products.1–4
We recently reported aldol additions of DHAP to
N-benzyloxycarbonyl (Cbz) amino aldehydes catalyzed by
D-fructose-1,6-diphosphate aldolase from rabbit muscle
(RAMA), and L-rhamnulose-1-phosphate aldolase (RhuA)
and L-fuculose-1-phosphate aldolase (FucA), both from
Escherichia coli.5,6 These enzymatic reactions afforded,
after cleavage of the phosphate group, the corresponding
2-keto-N-Cbz-amino-3,4-diols, which can be converted into
iminocyclitols, potent inhibitors of glycoprocessing
enzymes, by reductive amination.
t
and selective conditions.7 Removal of Boc requires acidic
conditions but less strenuous than simple amides like
acetyl.8 Fmoc group can be eliminated in the presence of
secondary amines, such as piperidine, by base induced
b-elimination.
Herein, we report on the reactivity and stereoselectivity of
RAMA, RhuA and FucA DHAP aldolases as catalysts
for aldol additions of DHAP to aldehydes: N-(PhAc)-(1),
N-(tBoc)- (2) and N-(Fmoc)-3-aminopropanal (3)
(Scheme 1). In this study, we focused on three aspects.
First, the influence of two reactions media, namely emulsion
Keywords: DHAP dependent aldolases; Aldol additions; Fmoc group; Boc
group; PhAc group; Penicillin amidase.
*
Corresponding author. Tel.: C34 93 4006112; fax: C34 93 2045904;
e-mail: pcsqbp@iiqab.csic.es
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.12.031

J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
2649
O
O
O
OH O
O
DHAP-aldolases
OPO3Na2
+ HO
H
OPO3Na2
R
N
R
N
H
H
OH
O
4–12
R:
O
1
2
3
Scheme 1. RAMA, RhuA and FucA DHAP aldolases catalyzed synthesis of products 4–12.
systems developed and assayed in previous works9 and
cosolvent DMF/H2O 1:4 mixtures, on the reaction conver-
sion to aldol adducts was investigated. Second, to assess the
stereoselectivity of the aldolases towards the three
N-protected-3-aminopropanal derivatives, the aldol adducts
were prepared under the best reaction conditions and their
structure and stereochemistry determined. Third, the
deprotection reactions of N-blocked-2-keto-aminodiol
adducts obtained in higher yield were studied.
Similarly to N-Cbz-3-aminopropanal,6 an inversion of
FucA stereoselectivity towards the N-protected amino
aldehydes 1–3 was observed.
The conversions and diastereomeric ratios shown in Table 1
remained constant up to 24 h of reaction, therefore it was
assumed that they reflect the final equilibrium compositions.
In a previous paper,6 we suggested that the stereochemical
outcome of the aldol addition of DHAP to Cbz-3-
aminopropanal catalyzed by FucA was thermodynamically
controlled. To assess if the diastereomeric ratios of the aldol
adducts generated from DHAP and PhAc-, tBoc- and Fmoc-
3-aminopropanal correlate with the thermodynamic stability
of the corresponding diastereoisomers, an extensive
exploration of the conformational space available to adducts
4–12 was carried out. Only the linear forms of these
products were considered, since the relative abundance by
NMR of the corresponding cyclic forms was always low.
The results of these calculations showed that the lowest
energy minima conformers of the (3S,4R), or (3R,4S),
diastereoisomers of adducts 4–12 were always more stable,
by approximately 1.5 kcal/mol, than the corresponding
(3S,4S), or (3R,4R), isomers independently of the protecting
group present. Assuming that the entropic contributions to
DG cancel out, this energetic difference suggests a predicted
(3S,4R):(3S,4S) or (3R,4S):(3R,4R) ratio close to 93:7 at
25 8C, in general good agreement with the ratios shown in
Table 1, particularly for the reactions catalyzed by RAMA.
The maximal deviation (67:33) was observed for the FucA
catalyzed reactions with substrates 1 and 2.
2. Results and discussion
2.1. Aldolase-catalyzed reactions
Aldol additions of DHAP to aldehydes 1–3 catalyzed by
RAMA, RhuA and FucA were investigated in two
reaction systems namely high water content emulsions
and DMF–H2O (1/4) cosolvent mixture (Table 1). Three
emulsion formulations were employed: H2O/C14E4/tetra-
decane, H2O/C14E4/hexadecane and H2O/C14E4/squalane
always in 90/4/6 wt%, where C14E4 is a technical
grade tetra(ethyleneglycol)tetradecyl ether surfactant
(C14H29(OCH2CH2)4OH), with an average of 4 mol of
ethylene oxide per surfactant molecule.5,9 Both PhAc and
t
the sterically more demanding Boc derivatives 1 and 2
were good substrates (Table 1, entries 1, 2, 4, 5, 7 and
8), the conversions to aldol adduct being similar to those
achieved with benzyloxycarbonyl (Cbz) N-protecting
group.5,6 The most hydrophobic and bulky Fmoc
derivative 3 was also tolerated as substrate, although it
gave the lowest conversions with the three aldolases
(Table 1, entries 3, 6 and 9).
Altogether, these results suggest that, under our reactions
conditions, the major products of the aldolic condensation
of aldehydes 1–3 with DHAP catalyzed by RAMA, RhuA
and FucA are those thermodynamically favoured, similarly
to what was previously observed for the condensation of
different N-Cbz-aminoaldehydes catalyzed by FucA.6 Thus,
while for RAMA and RhuA catalysts the stereofacial
selectivity observed with the natural substrates (glyceral-
dehyde-3-phosphate and L-lactaldehyde, respectively) was
conserved, the contrary was true for FucA and the main
adducts formed are those from the ‘wrong’ face attack (i.e.,
relative to that with the natural substrate, L-lactaldehyde).
Diastereomeric ratio of aldol adducts 4–12 were assessed by
NMR spectroscopy and are summarized in Table 1, last
column. The absolute configuration of the newly formed
stereogenic centers was assigned assuming that the
stereochemistry at the C-3 position depended exclusively
on the DHAP aldolase and was conserved upon reaction
with any electrophile.10–16 Hence, epimeric products at C-4
arose from attack on the inverted face of the N-protected-3-
aminopropanal carbonyl group relative to that on the natural
aldehyde. RAMA catalyst was the most stereoselective
towards the N-protected-3-aminopropanal derivatives, both
tBoc and Fmoc giving the highest diastereomeric excesses
(deO80%). The stereoselectivity of RhuA enzyme was
lower (de 40–60%) than that of RAMA, the de with both
PhAc and Fmoc being similar to that obtained with the
corresponding Cbz derivative.5 The NMR spectra of the
aldol adducts obtained with FucA catalyst were indis-
tinguishable from those observed with RhuA enzyme.
Docking simulations carried out with the different diastereo-
isomers of products 4–12 bound into the active centre of
RAMA and RhuA† suggest that in all cases the bulky
N-protecting group cannot get into the catalytic site of the
† We did not attempt to model the FucA complexes because of the
difficulty to predict the conformation of the flexible C-terminal tail of the
protein.17

2650
J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
Table 1. DHAP-dependent aldolase-catalyzed aldol addition of DHAP to N-protected-3-aminopropanal derivatives 1–3
Entry
Acceptor
aldehyde
Aldolase
(U mLK1
DHAP
concna (mM)
Conversion,b % (Time, h)
Bd
Product
Diastereomeric
ratio (C-4) R:S
)
Ac
OH
O
PhAc
OPO3Na2
N
1
2
3
4
5
6
7
1
2
3
1
2
3
1
RAMA 20
RAMA 20
RAMA 20
RhuA 0.4
RhuA 0.4
RhuA 0.4
FucA 8
86
97
66 (3)
66 (4)
25 (1)
47 (2)
63 (7)
15 (6)
72 (3)
65 (2)
70 (6)
19 (2)
28 (4)
45 (7)
15 (6)
71 (4)
89:11
93:7
H
OH
4
OH
O
Boc
OPO3Na2
OPO3Na2
OPO3Na2
OPO3Na2
OPO3Na2
OPO3Na2
N
H
OH
5
OH
O
Fmoc
PhAc
Boc
N
53
92:8
H
OH
6
OH
O
N
H
100
91
19:81
30:70
23:77
33:67
OH
7
OH
O
N
H
OH
8
OH
O
Fmoc
PhAc
N
53
H
OH
9
OH
O
N
H
45
OH
10
OH
O
Boc
OPO3Na2
OPO3Na2
N
8
9
2
3
FucA 8
FucA 8
45
53
70 (3)
20 (2)
70 (4)
18 (2)
33:67
21:79
H
OH
11
OH
O
Fmoc
N
H
OH
12
a Acceptor aldehyde (1.8 equiv molK1); reaction volume 5 mL. TZ25 8C.
b Molar percent conversion to the aldol adduct (4–12) with respect to the starting DHAP concentration, determined by HPLC from the crude reaction mixture
using purified standards.
c High water content emulsions. Reaction conversions to the corresponding aldol adduct in emulsions were similar regardless of the formulation used,
therefore, the mean values obtained in the three emulsion systems, H2O/C14E4/tetradecane, H2O/C14E4/hexadecane and H2O/C14E4/squalane 90/4/6 wt%, are
always given.
d DMF/H2O 1:4 v/v.
protein and probably remains at the entrance of the cavity,
partially exposed to the solvent. This is shown in Figure 1
for the major 3S,4R-isomers of adducts bearing N-PhAc,
tBoc and Fmoc bound in the active centre of RAMA and for
the corresponding 3R,4S- enantiomers in the active center of
RhuA. That would explain the little effect of the
N-protecting groups with different size and shape on the
stereochemical outcome of the reactions, since those groups
would remain far from the reactive atoms. In addition, it
also suggests that the lower yields observed for the Fmoc
containing adducts (6, 9, 12) could be due to the steric
hindrance arising from the bulkiest Fmoc moiety, which
could difficult the approach of the aldehyde group to the
reactive enzyme-bound DHAP.
2.2. Deprotection of the phenylacetyl and
tert-butyloxycarbonyl groups
t
The results obtained showed that both PhAc and Boc,
provided the highest reaction conversions with similar
stereoselectivities. At this point, it was also important to
establish proper reaction conditions for the N-protecting
group removal. To this end N-PhAc and N-tBoc amino-
polyols 4 and 5, and their corresponding unphosphated
derivatives, were treated with penicillin acylase at pH 7 and
aqueous trifluoroacetic acid, respectively.
t
Removal of PhAc and Boc was achieved quantitatively
under the aforementioned conditions (see Section 4). The
formation of the six-membered imine sugar, in equilibrium

J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
2651
Figure 1. Structures of aldol adducts bearing PhAc (brown), tBoc (green) and Fmoc (yellow) protecting groups docked into the active center of RAMA (left
panel) and RhuA (right panel). Structures on the left panel correspond to the (3S,4R) diastereoisomers, while those on the right panel correspond to the (3R,4S)
diastereoisomers.
with the corresponding unprotected aminoketopolyol, was
the product expected after the deprotection reaction.
However, the cyclic imine sugar could not be isolated in
any reaction condition. Hence, the NMR spectrum of the
residue obtained after PhAc removal of 4 and work up was
complex with signals that presumably belong to a number of
decomposition products since they can hardly be assigned to
any single structure. To avoid any possible influence of the
work up on the stability of the final product, the deprotection
reaction was performed into an NMR tube and monitored
continuously. This experiment confirmed the previous
observation: the signals corresponding to the product
disappeared while no major product was formed with
the exception of phenylacetic acid. On the other hand, the
deprotection of unphosphated derivative 13 gave the
hemiaminal mixture 15 as the major products. A possible
mechanism for the formation of 15 is outlined in Scheme 2.
The key step was the enolization of 14 and subsequent shift
of the ketone to position 3.
these compounds to acids, we decided to perform the
deprotection at the lowest possible TFA concentration. We
surveyed different TFA concentrations from 1 to 6% and we
found that 6% aqueous TFA lead to complete Boc cleavage
in 24 h. The reaction was monitored by NMR and, in
agreement with the expectations, the spectra revealed the
presence of the linear compound 16 in equilibrium with the
cyclic imine 17 (Scheme 3).
Nevertheless, the resulting imine could not be isolated and
after lyophilization, the NMR spectrum revealed a mixture
of decomposition products. Besides, when the reaction
mixture was left for more than 10 days, the solution turned
dark and no single compound could be assigned by NMR.
A similar result was also found for the tBoc deprotection of
unphosphated derivative of 5.
3. Conclusions
t
Deprotection of Boc blocking group was accomplished by
The results obtained demonstrated that DHAP-aldolases
tolerate a variety of N-protecting groups for the 3-amino-
propanal. The outcome of the reaction performance,
however, depended on the protecting group. Thus, ca.
70% conversion to aldol adduct were achieved with PhAc
aqueous TFA. It has been reported that treatment of
N-formyl-aminopolyol with aqueous acid at pH 1 efficiently
cleaves the amide, and that at pH 3 the same compound is
stable.8 On this basis and being aware of the sensitivity of
OH
O
O
OH
O
PGA
OH
OH
OH
H2N
H2N
N
H2O, pH=7
H
OH
14
OH
13
OH OH
OH
OH OH
O
OH
H
H2N
H2O
N
H
OH
OH
6
N
OH
6
N
5
2
5
2
7
+
OH
OH
7
4
3
OH
4
3
OH
OH
OH
OH
OH
15
Scheme 2. Deprotection of 13 by penicillin G acylase.

2652
J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
O
O
H
+
H3N
TFA 6% v/v
O
HO
HO
N
HN
O
OP
OH
OP
OH
OP
N
-H2O
OH
OH
H2O
+H2O
PO
OH
OH
OH
17
16
5
P=PO32-
In solution
H+
Lyophilization
decomposition products
Scheme 3. Deprotection of 5 by aqueous TFA (6%).
t
and Boc, similar to those obtained before with Cbz,
whereas Fmoc gave ca. 20% conversion. Modifications on
the protecting group structure, however, did not affect the
stereoselectivity of the aldolases to a significant extent, nor
the inversion of FucA stereoselectivity towards the
N-protected derivatives of 3-aminopropanal. N-PhAc and
tBoc adduct product derivatives were successfully depro-
tected by PGA-catalyzed hydrolysis at pH 7 and with
aqueous TFA (6% v/v) respectively. However, the
corresponding six-membered imine sugar could not be
isolated, even though under the mild reactions conditions
used with the PGA. When, N-PhAc was deprotected from
the unphosphated derivatives a five-membered iminocycly-
tol was identified. When both phosphated or Boc derivatives
were deprotected a complex NMR spectra were recorded
after lyophilization with signals that presumably belong to a
number of decomposition products. This behaviour may be
due to the presence of a highly reactive primary amine and a
keto group in the molecule, which lead to a number of
unexpected reactions. The situation was different with the
previously reported benzyloxycarbonyl group (Cbz).5,6 In
this case, the hydrogenolysis of the Cbz and the reductive
amination took place in one pot reaction, safely catching the
imine intermediate being less prone to side reactions.
Amidase, immobilized on Eupergitw C from E. coli (EC
3.5.1.11, 100 U gK1 immobilized preparation) was from
Fluka. Non-ionic polyoxyethylene ether surfactant with an
average of 4 mol of ethylene oxide per surfactant molecule
(C14E4) was from Albright and Wilson (Barcelona, Spain).
The precursor of dihydroxyacetone phosphate (DHAP),
dihydroxyacetone phosphate dimer bis (ethyl ketal), was
synthesized in our lab using a procedure described by Jung
et al.18 with slight modifications.
Molecular modelling. Molecular simulations were con-
ducted with the programs MOE (v. 2004.03, Chemical
Computing Group, Montreal). The conformational space of
all the possible diastereoisomers of adducts 4–6 was
exhaustively searched using the systematic conformational
search algorithm implemented in MOE, and the confor-
mations generated were minimized and ranked according to
their energy, as previously decribed.5 These energy
calculations were carried out using the implemented
MMFF94x force field with its standard atomic charges
and parameters,19 and the Born continuum solvation
model20–22 without cut-offs. Geometries were optimized
up to an RMS gradient !0.01.
Docked structures of the stereoisomers of products 4–6 in
the active centres of RAMA and RhuA were determined by
sampling the conformational space of the products in the
enzyme environment. The methodology used was similar to
that previously described.5 However, in this case we used
the above mentioned MMFF94x force field and the Born
continuum salvation model, with a smoothed cut-off
Protecting groups such as PhAc, tBoc and, Cbz provide also
a range of removal conditions to fulfill most of the required
orthogonalities for functional group manipulation on the
2-ketoaminodiols.
˚
between 14 and 15 A to model the nonbonded interactions.
4. Experimental
4.1. Materials
4.2. General methods
HPLC analyses. HPLC analyses were performed on a RP-
HPLC cartridge, 250!4 mm filled with Lichrospherew 100,
RP-18, 5 mm from Merck (Darmstadt, Germany). Samples
(50 mg) were withdrawn from the reaction medium,
dissolved with methanol to stop any enzymatic reaction,
and analyzed subsequently by HPLC. The solvent system
was the following: solvent A: 0.1% v/v trifluoroacetic acid
(TFA) in H2O, solvent B: 0.095% v/v TFA in H2O/CH3CN
1:4. Elution conditions for N-PhAc and N-tBoc derivatives:
isocratic 10% B during 2 min followed by a gradient from
10 to 33% B over 18 min; elution conditions for N-Fmoc
derivatives: gradient from 30 to 90% B over 30 min, always
Fructose-1,6-diphosphate aldolase from rabbit muscle
(RAMA; EC 4.1.2.13, crystallized, lyophilized powder,
19.5 U mgK1) was from Fluka (Buchs, Switzerland).
Rhamnulose 1-phosphate aldolase (RhuA; EC 4.1.2.19,
suspension 100 U mLK1) was kindly donated by Boehringer
Mannhein (Mannhein, Germany). L-Fuculose-1-phosphate
aldolase (FucA, EC 4.1.2.17, lyophilized 500–800 U gK1
´
)
was from Departament d’Enginyeria Quımica of the
`
Universitat Autonoma de Barcelona, produced from a
recombinant E. coli (ATCC no. 86984) and purified by
affinity chromatography. Acid phosphatase (PA, EC 3.1.3.2,
5.3 U mgK1) was from Sigma (St. Louis, USA). Penicillin

J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
2653
at a flow rate of 1 mL minK1 and detection at 215 nm.
Retention factors (k0) for the acceptor aldehydes and
condensation products are given below.
procedure described previously in our lab using Fmoc-
OSu as acylating agent.5 The compound was obtained as a
white solid (3.6 g, 90%, 99% pure by HPLC). The 1H NMR
spectrum matched that reported.25 13C NMR (75 MHz,
CDCl3, ppm): 157.3 (CONH), 143.7, 141.2, 127.6, 126.9,
124.9 (Fmoc), 66.5 (OCH2), 59.3 (CH2OH), 47.2 (NHCH2),
37.4 (CH), 32.5 (CH2CH2OH).
NMR analysis. High field 1H and 13C nuclear magnetic
resonance (NMR) analyses were carried out at the Servei de
`
`
`
Ressonancia Magnetica Nuclear, Universitat Autonoma de
Barcelona using an AVANCE 500 BRUKER spectrometer
for D2O solutions. Full characterization of the described
compounds was performed using typical gradient-enhanced
2D experiments: COSY, NOESY, HSQC and HMBC,
recorded under routine conditions. When possible, NOE
data was obtained from selective 1D NOESY versions using
a single pulsed-field-gradient echo as a selective excitation
method and a mixing time of 500 ms. When necessary,
proton and NOESY experiments were recorded at different
temperatures in order to study the different behaviour of the
exchange phenomena to avoid the presence of false NOE
cross-peaks that difficult both structural and dynamic
4.5. Synthesis of N-protected aminoaldehydes
The synthesis of N-protected amino aldehydes was achieved
by 2-iodoxobenzoic acid (IBX) oxidation method.26,27
Caution! IBX has been reported to detonate upon heavy
impact and/or heating over 200 8C. To a solution of the
N-protected amino aldehyde (10–19 mmol) in DMSO
(60–120 mL), IBX (24–48 mmol) was added. The reaction
was monitored by HPLC until no alcohol was detected. At
this point, the reaction mixture was diluted with water
(30–60 mL) and the mixture was extracted with ethyl
acetate (3!75–100 mL). The organic layers were pooled,
washed with NaHCO3 5% (w/w) (3!100 mL) and brine
(3!100 mL), dried over Na2SO4 and evaporated under
reduced pressure.
1
studies. H (300 MHz) and 13C NMR (75 MHz) spectra
´
were carried out at the Instituto de Investigaciones Quımicas
y Ambientales-CSIC.
Elemental analyses. Elemental analyses were performed by
`
the Servei de Microanalisi Elemental IIQAB-CSIC.
4.5.1. N-(3-Oxopropyl)-2-phenylacetamide (PhAc-amino-
propanal) (1). The title compound (1.04 g) was obtained as
a white solid in 52% yield by using the above general
4.3. Synthesis of N-protected amino aldehydes
1
procedure. HPLC k0Z7.05. H NMR (300 MHz, CDCl3,
The synthesis of N-protected amino aldehydes was carried
out in two steps. First, the N-protected 3-aminopropanol was
obtained and second the oxidation of the alcohol group to
aldehyde was performed.
ppm): dZ9.7 (1H, s, CHO), 3.5 (2H, s, CH2CO), 3.4 (2H, q,
NHCH2), 2.6 (2H, t, CH2CHO). 13C NMR (75 MHz, CDCl3,
ppm): 201.0 (CHO), 171.1 (CONH), 43.6 (NHCH2), 33.0
(CH2CHO).
4.5.2. tert-Butyl-3-oxoethylcarbamate (tBoc-aminopro-
panal) (2). The title compound (1.7 g) was obtained as a
pale yellow oil in 90% yield by using the above general
4.4. Synthesis of N-protected amino alcohols
4.4.1. N-(3-Hydroxypropyl)-2-phenylacetamide. To a
cooled (K20 8C) solution of 3-amino-1-propanol (9.2 mL,
122.5 mmol) in CH2Cl2 (40 mL) was added dropwise
phenylacetyl chloride (8 mL, 60.1 mmol) in CH2Cl2
(4 mL) and, simultaneously, an aqueous solution of NaOH
(2.4 g, 60 mmol) in water (8 mL) under vigorous stirring.
After the addition was complete, the reaction was kept at
K20 8C for 2 h and then allowed to warm to room
temperature under stirring overnight. Then, the crude
reaction mixture was evaporated under vacuum to dryness,
the residue was dissolved with ethyl acetate and washed
successively with citric acid 5% w/v (3!50 mL), NaHCO3
10% w/v (3!50 mL) and brine (3!50 mL). After being
dried over Na2SO4, the organic layer was evaporated under
reduced pressure to yield 3 as a white solid (8.1 g, 70%,
1
procedure. HPLC k0Z9.17. H NMR28 (300 MHz, CDCl3,
ppm): 9.7 (1H, s, CHO), 5.0 (1H, br, NH), 3.3 (2H, q,
NHCH2), 2.6 (2H, t, CH2CHO), 1.3 (9H, s, CH3). 13C
NMR (75 MHz, CDCl3, ppm): 201 (CHO), 155.7 (CONH),
79.3 (OC(CH3)3), 44.2 (NHCH2), 33.9 (CH2CHO), 28.3
(CH3).
4.5.3. Fluoren-9-yl-3-oxoethylcarbamate (Fmoc-amino-
propanal) (3). The title compound (1.7 g) was obtained as a
pale yellow solid in 98% yield by using the above general
procedure. HPLC k0Z9.42. The 1H NMR spectrum matched
that reported.29 13C NMR (75 MHz, CDCl3, ppm): 201.2
(CHO), 157.0 (CONH), 143.7, 141.2, 127.5, 126.9, 124.9
(Fmoc), 66.6 (OCH2), 47.1 (CH2CHO), 43.9 (CH), 34.3
(NHCH2).
1
99% pure by HPLC). The H and 13C NMR spectra were
consistent with those reported in the literature.23
4.6. Enzymatic aldol condensations
4.4.2. tert-Butyl-3-hydroxypropylcarbamate. To a solu-
tion of 3-amino-1-propanol (4.3 mL, 57.1 mmol) in CH2Cl2
(3 mL) at 25 8C was added dropwise a solution of (Boc)2O
(12.5 g, 57.1 mmol) in CH2Cl2 (7 mL). After 12 h, the
mixture was worked up as described above to yield 4 as a
Enzymatic aldol condensations in emulsions. Reactions
were carried out in 10 mL test tubes with screw caps.
The aldehyde (0.23–0.90 mmol), the oil (6% w/w) and the
surfactant (4% w/w) were mixed vigorously. Then, the
DHAP solution (0.13–0.50 mmol) at pH 6.9, freshly
prepared as described by Effenberger et al.,30 was added
dropwise while stirring at 25 8C with a vortex mixer. The
final reaction volume was 5 mL. Finally, RAMA (100 U),
RhuA (2 U) or FucA (40 U) was added and mixed again.
1
colourless oil (6.4 g, 64%, 99% pure by HPLC), whose H
and 13C NMR spectra matched those reported.24
4.4.3. Fluoren-9-yl-3-hydroxypropylcarbamate. The
synthesis of the title compound was performed by a

2654
J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
The test tubes were placed on a horizontal shaking bath
(100 rpm) at constant temperature (25 8C). The reactions
were followed by HPLC until the peak of the product
reached a maximum. The enzymatic reactions were
stopped by addition of MeOH. Then, the methanol was
evaporated and the aqueous solution washed with ethyl
acetate to remove the unreacted N-protected amino-
aldehyde. The aqueous layer was collected and lyophilized.
The residue was dissolved in water, adjusted to pH 3 with
trifluroacetic acid (TFA) and purified by reversed phase
HPLC on a Perkin-Elmer semipreparative 250!25 mm
column, filled with C18, 10 mm type stationary phase and
eluted using a CH3CN gradient (8–56% in 30 min; 24–72%
in 30 min for the Fmoc derivative) in 0.10% (v/v) aqueous
TFA. The best fractions were pooled, diluted, re-loaded
onto the column and eluted with a CH3CN gradient (0%
10 min and then 0–56% in 30 min) in plain water to
eliminate the TFA. The pure fractions were pooled and
lyophilized.
described above. 203 mg, 47%, 99.5% purity by HPLC
(k0Z3.81). [a]D20 C18.8 (c 1 in H2O/MeOH 5:95). 1H
NMR (500 MHz, D2O, ppm): d 4.56 (2H, dd, JZ5.9, 6.3,
18.9 Hz, CH2OP), 4.21 (1H, d, JZ1.4 Hz, CHOH), 4.03
(1H, br t, JZ7.7 Hz, CH(R)OH), 3.06 (2H, m, NHCH2),
1.64 (2H, br m, CH2), 1.30 (9H, s, CH3); minor signals
corresponding to the diastereoisomer 3S,4S: d 4.3 (1H, s,
CH(S)OH), 3.84 (1H, m, CHOH), 1.53 (2H, m, CH2). 13C
NMR (125 MHz, D2O, ppm): d 210.1 (CO), 158.0
(OCONH), 80.6 (C), 77.5 (CHOH), 68.9 (CHOH), 67.70
(CH2OP), 36.6 (CH2), 32.2 (CH2), 27.5 (CH3); minor
signals corresponding to the diastereoisomer 3S,4S: 77.6
(CHOH), 69.4 (CHOH), 68.4 (CH2OP). (Found: C, 31.83;
H, 5.62; N, 3.34. C11H20NNa2O9P$3/2H2O requires: C,
31.89; H, 5.60; N, 3.38%).
4.6.3. (3S,4R)-5,6-Dideoxy-{[(fluoren-9-ylmetoxy)carbo-
nyl]amino}-1-O-phosphonohex-2-ulose sodium salt and
(3S,4S)-5,6-dideoxy-{[(fluoren-9-ylmetoxy)carbonyl]-
amino}-1-O-phosphonohex-2-ulose sodium salt (6). The
title compounds were obtained as a mixture in a proportion
of 92:8, respectively, following the general methodology
described above. 31 mg, 23%, 99.5% purity by HPLC (k0Z
7.48 broad peak). Due to signal overlapping and for the
sake of simplicity, the NMR spectra were recorded for the
Enzymatic aldol condensations in mixtures water/dimethyl-
formamide 4:1. Reactions were carried out in 10 mL test
tubes with screw caps. The aldehyde (0.4–0.9 mmol) was
dissolved in DMF 20% (v/v). Then, the DHAP solution
(0.23–0.50 mmol), prepared as described above, was added
dropwise while mixing. The rest of the experimental
procedure was identical to that described for the reaction
in emulsions.
1
unphosphated derivatives of 6. H NMR (500 MHz, D2O,
ppm): d 7.84 (2H, d, JZ7.5 Hz, Ph), 7.66 (2H, d, JZ
7.4 Hz, Ph), 7.41 (2H, t, JZ7.4 Hz, Ph), 7.33 (2H, t, JZ
7.4 Hz, Ph), 4.50 (2H, dd, JZ19.3, 46.6 Hz, CH2OP), 4.37
(2H, m, CH2O), 4.22 (H, t, JZ6.8 Hz, CH–CH2O), 4.14
(H, d, JZ2.1 Hz, CH(R)OH), 4.01–3.98 (1H, m, CHOH),
3.29–3.22 (2H, m, NHCH2), 1.82–1.72 (2H, m, NHCH2-
CH2C(R)HOH). Signals corresponding to the diastereomer
(4S): 4.10 (H, d, JZ5.7 Hz, CH(R)OH), 3.87 (1H, br,
CHOH). 13C NMR (125 MHz, D2O, ppm): d 215.8 (CO),
161.4 (OCONH), 147.8 (C), 145.0 (C) 131.2, 130.6, 128.6,
123.4 (arom), 81.9 (CH(OH)CO), 73.8 (CH(OH)-
CH(OH)CO), 70.3 (CH2–O–CO), 70.1 (CH2OH), 48.22
(CH–CH2–O–), 41.0 (CH2), 36.8 (CH2). (Found: C, 22.36;
H, 1.15; N, 0.48. C21H22NNa2O9P$7NaCl$7CF3COONa
requires: C, 22.47; H, 1.19; N, 0.75%). Unphosphated
derivative: (Found: C, 62.28; H, 6.44; N, 3.64.
C21H23NO6$H2O requires: C, 62.52; H, 6.27; N, 3.45%).
The yields of the compounds 4–12 correspond to the
amounts from of the aldol enzymatic reactions at
semipreparative level. Most of them contained salts from
the purification process. The purification procedures were
not optimized.
4.6.1. (3S,4R)-5,6-Dideoxy-[(phenylacetyl)amino]-1-O-
phosphonohex-2-ulose sodium salt and (3S,4S)-5,6-
dideoxy-[(phenylacetyl)amino]-1-O-phosphonohex-2-
ulose sodium salt (4). The title compounds were obtained
as a mixture in a proportion of 89:11, respectively,
following the general methodology described above.
219 mg, 35%, 99.5% purity by HPLC (k0Z2.79). [a]D20
C6.7 (c 1 in H2O/MeOH 5:95) and [a]2D0 C12.9 (c 1 in
1
H2O/MeOH 1:1). H NMR (500 MHz, D2O, ppm): d 7.24
(5H, m, Ph), 4.55 (2H, dd, JZ6.3, 18.8 Hz, CH2OP), 4.27
(1H, d, JZ1.7 Hz, CHOH), 4.00 (1H, br t, JZ5.9 Hz,
CH(R)OH), 3.47 (2H, s, PhCH2), 3.18 (2H, t, JZ6.9 Hz,
NHCH2), 1.66 (2H, m, NHCH2CH2C(R)HOH); minor
signals corresponding to the diastereomer 3S,4S: d 3.79
(1H, m, CH(S)OH), 1.56 (2H, m, NHCH2CH2CH(S)OH).
13C NMR (125 MHz, D2O, ppm): d 211.1 (CO), 174.5
(OCONH), 134.9 (C ar), 128.9 (CH ar), 128.7 (CH ar),
127.1 (CH ar), 77.4 (CHOH), 69.0 (CHOH), 67.8 (CH2OP),
42.2 (CH2), 36.1 (CH2), 31.6 (CH2). (Found: C, 38.22; H,
4.62; N, 3.22. C14H18NO8Na2P$1/2H2O$1/2NaCl requires:
C, 37.92; H, 4.32; N, 3.16%).
4.6.4. (3R,4S)-5,6-Dideoxy-[(phenylacetyl)amino]-1-O-
phosphonohex-2-ulose sodium salt and (3R,4R)-5,6-
dideoxy-[(phenylacetyl)amino]-1-O-phosphonohex-2-
ulose sodium salt (7). The title compounds were obtained
as a mixture in a proportion of 81:19, respectively,
following the general methodology described above.
376 mg, 49%, 99.5% purity by HPLC (k0Z2.79). [a]D20
K12.0 (c 1 in H2O/MeOH 5:95). NMR spectra were
undistinguishable from those obtained for the corresponding
diastereoisomers 4. (Found: C, 34.31; H, 3.68; N, 2.47
C14H18NNa2O8P$CF3COONa NaCl requires: C, 34.36; H,
3.60; N, 2.50%).
4.6.2. (3S,4R)-5,6-Dideoxy-{[(tert-butyloxy)carbonyl]-
amino}-1-O-phosphonohex-2-ulose sodium salt and
(3S,4S)-5,6-dideoxy-{[(tert-butyloxy)carbonyl]amino}-1-
O-phosphonohex-2-ulose sodium salt (5). The title
compounds were obtained as a mixture in a proportion of
93:7, respectively, following the general methodology
4.6.5. (3R,4S)-5,6-Dideoxy-{[(tert-butyloxy)carbonyl]-
amino}-1-O-phosphonohex-2-ulose sodium salt and
(3R,4R)-5,6-dideoxy-{[(tert-butyloxy)carbonyl]amino}-
1-O-phosphonohex-2-ulose sodium salt (8). The title
compounds were obtained as a mixture in a proportion of
70:30, respectively, following the general methodology

J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
2655
described above. 115 mg, 37%, 99.5% purity by HPLC
(k0Z3.81). [a]D20 K11.8 (c 1 in H2O/MeOH 5:95). NMR
spectra were undistinguishable from those obtained for the
corresponding diastereoisomers 5. (Found: C, 30.53; H,
5.40; N, 3.14. C11H20NNa2O9P$1/2H2O$NaCl requires: C,
30.53; H, 5.12; N, 3.24%).
21.70; H, 1.72; N, 1.01%). Unphosphated derivative:
(Found: C, 61.12; H, 6.65; N, 3.13. C21H23NO6$3/2H2O
requires: C, 61.16; H, 6.35; N, 3.40%).
4.7. Removal of protecting groups
4.7.1. Removal of phosphate group. The phosphate group
of compounds 4–12 was removed by hydrolysis catalyzed
by acid phosphatase following the procedure described by
Bednarski et al.31 The reaction was followed by HPLC until
no starting material was detected. Then the crude was
desalted by HPLC and lyophilized.
4.6.6. (3R,4S)-5,6-Dideoxy-{[(fluoren-9-ylmetoxy)carbo-
nyl]amino}-1-O-phosphonohex-2-ulose sodium salt and
(3R,4R)-5,6-dideoxy-{[(fluoren-9-ylmetoxy)carbonyl]-
amino}-1-O-phosphonohex-2-ulose sodium salt (9). The
title compounds were obtained as a mixture in a proportion
of 77:23, respectively, following the general methodology
described above. 12 mg, 4%, 99.5% purity by HPLC (k0Z
7.48 broad peak). Due to signal overlapping and for the sake
of simplicity, the NMR spectra were recorded for the
unphosphated derivatives and were undistinguishable from
those obtained for the corresponding diastereoisomers 6.
(Found: C, 34.97; H, 4.23; N, 2.15. C21H22NNa2O9P$5H2-
O$2NaCl requires: C, 35.21; H, 4.50; N, 1.96%);
Unphosphated derivative: (Found: C, 59.21; H, 6.36; N,
3.07. C21H23NO6$9/4H2O requires: C, 59.22; H, 6.51; N,
3.29%).
4.7.2. Removal of N-phenylacetyl (PhAc) protecting
group by penicillin G acylase. Compound 4, or the
corresponding unphosphated analogue (0.320 mmol,
90 mg) was dissolved in plain water (5 mL). To this
solution was added penicillin amidase immobilized on
Eupergitw (100 mg). The pH was controlled by a pH meter
and maintained between 6.5 and 7 by additions of NaOH
0.1 M. Samples were withdrawn every 45 min and
analyzed by HPLC, a peak corresponding to the
phenylacetic acid appeared. When no signal of the starting
material was detected, the immobilized enzyme was
filtered off. Then, the phenylacetic acid was eliminated
by anion exchange chromatography on a Macroprep
High-Q support eluting with plain water. Finally, the
product, which was not retained under the elution
conditions, was lyophilized.
4.6.7. (3R,4R)-5,6-Dideoxy-[(phenylacetyl)amino]-1-O-
phosphonohex-2-ulose sodium salt and (3R,4S)-5,6-
dideoxy-[(phenylacetyl)amino]-1-O-phosphonohex-2-
ulose sodium salt (10). The title compounds were obtained
as a mixture in a proportion of 33:67, respectively,
following the general methodology described above.
255 mg, 35%, 99.5% purity by HPLC (k0Z2.79). [a]D20
K11.4 (c 1 in H2O/MeOH 5:95). NMR spectra were
undistinguishable from those obtained for the corresponding
diastereoisomers 4. (Found: C, 34.52; H, 4.32; N, 3.14.
C14H18NNa2O8P$3/2H2O NaCl requires: C, 34.27; H, 4.31;
N, 2.85%).
4.7.3. Removal of N-tert-butyloxycarbonyl (tBoc) pro-
tecting group by trifluoroacetic acid. Compound 5, or the
corresponding unphosphated analogue, (0.162 mmol) was
dissolved in plain water (9 mL). To this solution was added
an aqueous solution of TFA (1.2 mL, 1:1 TFA/H2O).
Samples were withdrawn every 45 min and analyzed by
HPLC. After 14 h, no signal of the starting material was
detected. Then the crude was lyophilized.
4.6.8. (3R,4R)-5,6-Dideoxy-{[(tert-butyloxy)carbonyl]-
amino}-1-O-phosphonohex-2-ulose sodium salt and
(3R,4S)-5,6-dideoxy-{[(tert-butyloxy)carbonyl]amino}-1-
O-phosphonohex-2-ulose sodium salt (11). The title
compounds were obtained as a mixture in a proportion of
33:67, respectively, following the general methodology
described above. 143 mg, 22%, 99.5% purity by HPLC
(k0Z3.81). [a]D20 K10.4 (c 1 in H2O/MeOH 5:95). NMR
spectra were undistinguishable from those obtained for the
corresponding diastereoisomers 5. (Found: C, 30.58; H,
5.63; N, 3.30. C11H20NNa2O9P$5/2H2O requires: C, 30.56;
H, 5.83; N, 3.24%).
Acknowledgements
Financial support from the Spanish C.I.C.Y.T. (PPQ2002-
04625-CO2-01 and BQU2003-01677) is acknowledged.
J.C. acknowledges the CSIC for the I3P predoctoral
´
scholarship. We are thankful to Prof. Josep Lopez-Santın
and the team of the Chemical Engineering Department at the
´
`
Universitat Autonoma de Barcelona for the supply of FucA
aldolase.
4.6.9. (3R,4R)-5,6-Dideoxy-{[(fluoren-9-ylmetoxy)carbo-
nyl]amino}-1-O-phosphonohex-2-ulose sodium salt and
(3R,4S)-5,6-dideoxy-{[(fluoren-9-ylmetoxy)carbonyl]-
amino}-1-O-phosphonohex-2-ulose sodium salt (12). The
title compounds were obtained as a mixture in a proportion
of 21:79, respectively, following the general methodology
described above. 41 mg, 22% (contained some salts), 99.5%
purity by HPLC (k0Z7.48 broad peak). Due to signal
overlapping and for the sake of simplicity, the NMR spectra
were recorded for the unphosphated derivatives and were
undistinguishable from those obtained for the corresponding
diastereoisomers 6. (Found: C, 21.70; H, 1.75; N, 0.82.
C21H22NNa2O9P$H2O$10NaCl$2CF3COONa requires: C,
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2005.12.
1
1
031. H NMR and 13C spectra of 4–12, H NMR spectrum
of the residue after work up and lyophilization of the
penicillin acylase-catalyzed removal of phenylacetyl
amino protecting group of 5; 1H NMR reaction monitoring
of the deprotection reaction using penicillin acylase and 1H
NMR spectrum of 15–17 are supplied.

2656
J. Calveras et al. / Tetrahedron 62 (2006) 2648–2656
16. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T.
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