Fructose-6-phosphate aldolase in organic synthesis: Preparation of D-fagomine, N-alkylated derivatives, and preliminary biological assays

Article abstract of DOI:10.1021/ol0625482

(Chemical Equation Presented) D-Fructose-6-phosphate aldolase (FSA) mediates a novel straightforward two-step chemo-enzymatic synthesis of D-fagomine and some of its N-alkylated derivatives in 51% isolated yield and 99% de. The key step is the FSA-catalyzed aldol addition of simple dihydroxyacetone (DHA) to N-Cbz-3-aminopropanal. The use of FSA greatly simplifies the enzymatic procedures that used dihydroxyacetonephosphate or DHA/esters. Some N-alkyl derivatives synthesized elicited antifungal and antibacterial activity as well as enhanced inhibitory activity, and selectivity against β-galactosidase and α-glucosidase.

Full text of DOI:10.1021/ol0625482

ORGANIC
LETTERS
2006
Vol. 8, No. 26
6067-6070
Fructose-6-phosphate Aldolase in
Organic Synthesis: Preparation of
D-Fagomine, N-Alkylated Derivatives,
and Preliminary Biological Assays
Jose´ A. Castillo,† Jordi Calveras,† Josefina Casas,† Montserrat Mitjans,‡
|
|
M. Pilar Vinardell,‡ Teodor Parella,§ Tomoyuki Inoue, Georg A. Sprenger,
Jesu´s Joglar,*,† and Pere Clape´s*,†
Institute for Chemical and EnVironmental Research (IIQAB)-CSIC, Jordi Girona
18-26, 08034 Barcelona, Spain, Department of Physiology, Faculty of Pharmacy,
UniVersity of Barcelona, AVgda. Joan XXIII s/n, 08028 Barcelona, Spain, SerVei de
Ressona`ncia Magne`tica Nuclear, UniVersitat Auto`noma de Barcelona, 08193
Bellaterra, Barcelona, Spain, and Institute of Microbiology, UniVersity of Stuttgart,
Allmandring 31, 70569 Stuttgart, Germany
pcsqbp@iiqab.csic.es
Received October 17, 2006
ABSTRACT
D-Fructose-6-phosphate aldolase (FSA) mediates a novel straightforward two-step chemo-enzymatic synthesis of
D-fagomine and some of its
N-alkylated derivatives in 51% isolated yield and 99% de. The key step is the FSA-catalyzed aldol addition of simple dihydroxyacetone (DHA)
to N-Cbz-3-aminopropanal. The use of FSA greatly simplifies the enzymatic procedures that used dihydroxyacetonephosphate or DHA/esters.
Some N-alkyl derivatives synthesized elicited antifungal and antibacterial activity as well as enhanced inhibitory activity, and selectivity against
â
-galactosidase and r-glucosidase.
D-Fagomine, (2R,3R,4R)-2-hydroxymethylpiperidine-3,4-diol
is a naturally occurring iminosugar that was first isolated
from buckwheat seeds of Fagopyrum esculentum Moench1
with remarkable biological properties. This iminosugar has
inhibitory activity against mammalian intestinal R-, â-glu-
cosidase and R-, â-galactosidase.2,3 Moreover, it appears to
have a potent antihyperglycemic effect on streptozotocin-
induced diabetic mice and potentiates glucose-induced insulin
secretion.4 Segraves et al.5 have found that C-alkylated 3,4-
di-epi-fagomines from Batzella sp. sponge have a good
antimicrobial activity against Staphylococcus epidermidis.
(2) Scofield, A. M.; Fellows, L. E.; Nash, R. J.; Fleet, G. W. J. Life Sci.
1986, 39, 645-650.
(3) Kato, A.; Asano, N.; Kizu, H.; Matsui, K.; Watson, A. A.; Nash, R.
J. J. Nat. Prod. 1997, 60, 312-314.
(4) Nojima, H.; Kimura, I.; Chen, F.-J.; Sugihara, Y.; Haruno, M.; Kato,
A.; Asano, N. J. Nat. Prod. 1998, 61, 397-400. Taniguchi, S.; Asano, N.;
Tomino, F.; Miwa, I. Horm. Metab. Res. 1998, 30, 679-683.
(5) Segraves, N. L.; Crews, P. J. Nat. Prod. 2005, 68, 118-121.
† Institute for Chemical and Environmental Research.
‡ University of Barcelona.
§ Universitat Auto`noma de Barcelona.
| University of Stuttgart.
(1) Koyama, M.; Sakamura, S. Agr. Biol. Chem. 1974, 38, 1111-1112.
10.1021/ol0625482 CCC: $33.50
© 2006 American Chemical Society
Published on Web 11/29/2006

Chemical synthetic approaches of D-fagomine and its
derivatives involve cumbersome protection-deprotection
reactions and chiral starting materials, and therefore, moder-
ate global yields are achieved.6,7 Recent syntheses of D-
fagomine and other stereoisomers have been described
starting from chiral D-serine-derived Garner aldehyde in six
to seven steps with global yields around 12%.8
Scheme 1. Chemo-enzymatic Synthesis of 3 and N-Alkylated
Derivatives
Stereodivergent asymmetric chemo-enzymatic methodolo-
gies are mostly based on dihydroxyacetone phosphate
(DHAP)-dependent aldolases. The key step is the stereo-
selective aldol addition of DHAP or DHA/arsenate (500 mM)
to synthetic equivalents of aminoaldehydes.9,10 Given the
toxicity of arsenate, it would be more attractive to find a
system allowing the use of “naked” DHA.11 Chemical
synthesis of DHAP involves several steps in ca. 70% overall
yield.12 Alternatively, enzymatic methods to generate DHAP,
which can be coupled with the aldol reaction, have also been
described.13 However, some limitations arising from the lack
of compatibility of conditions between the coupled enzymatic
reactions and the generation of complex mixtures that make
the product separation and purification difficult have been
observed.14
We report herein a straightforward procedure for the
stereoselective synthesis of D-fagomine and N-alkylated
derivatives using fructose-6-phosphate aldolase (FSA) as
biocatalyst and achiral easily accessible starting materials.
The key step in this synthetic scheme was the stereoselective
aldol addition of simple dihydroxyacetone (DHA) to 1
catalyzed by FSA (Scheme 1). FSA is a novel class I aldolase
from E. coli related to a novel group of bacterial transaldo-
lases, which catalyzes the aldol addition of DHA to glyc-
eraldehyde-3-phosphate. The cloning and overexpression in
E. coli DH5R of the gene encoding FSA and the biochemical
characterization was carried out for the first time by
Schu¨rmann et al.15 These authors16 reported aldol additions
of either DHA or hydroxyacetone to some hydroxyaldehydes
for the synthesis of sugar derivatives. The most interesting
feature of FSA is that utilizes DHA instead of either DHAP
or DHA/esters which greatly simplifies the chemo-enzymatic
strategies to R,â-dihydroxyketones.
In this work, after growing and disrupting the E. coli cells
the enzyme was purified easily by a heat treatment at 75 °C
during 40 min, centrifugation, and lyophilization of the
supernatant to yield a pale brown powder with 1.7 U mg-1.17
Further purification steps are not needed since they were not
crucial for the activity and stereoselectivity of the enzymatic
aldol addition.
(6) Fleet, G. W. J.; Fellows, L. E.; Smith, P. W. Tetrahedron 1987, 43,
979-990. Pandey, G.; Kapur, M. Tetrahedron Lett. 2000, 41, 8821-8824.
(7) Goujon, J.-Y.; Gueyrard, D.; Compain, P.; Martin, O. R.; Ikeda, K.;
Kato, A.; Asano, N. Bioorg. Med. Chem. 2005, 13, 2313-2324.
(8) Banba, Y.; Abe, C.; Nemoto, H.; Kato, A.; Adachi, I.; Takahata, H.
Tetrahedron: Asymmetry 2001, 12, 817-819. Takahata, H.; Banba, Y.;
Ouchi, H.; Nemoto, H.; Kato, A.; Adachi, I. J. Org. Chem. 2003, 68, 3603-
3607. Takahata, H.; Banba, Y.; Sasatani, M.; Nemoto, H.; Kato, A.; Adachi,
I. Tetrahedron 2004, 60, 8199-8205.
(9) Ziegler, T.; Straub, A.; Effenberger, F. Angew. Chem., Int. Ed. 1988,
27, 716-717. Von der Osten, C. H.; Sinskey, A. J.; Barbas, C. F., III;
Pederson, R. L.; Wang, Y. F.; Wong, C. H. J. Am. Chem. Soc. 1989, 111,
3924-3927. Pederson, R. L.; Wong, C. H. Heterocycles 1989, 28, 477-
480.
Preparation of 1 was carried out by previously described
procedures from 3-aminopropanol.10,18 The FSA-catalyzed
aldol addition of DHA to 1 was conducted at 4 °C in boric-
borate 50 mM pH 7 buffer containing 20% v/v DMF,
furnishing 79% reaction conversion by HPLC after 1 h (69%
isolated yield of 2). Glycylglycine 50 mM pH 7.0 buffer
can also be used yielding 82% reaction conversion after 1
h.11 Hence, FSA readily accepts DHA, and there is no need
to in situ generate DHA esters. Furthermore, FSA tolerates
organic solvents as other DHAP-dependent aldolases do.
D-Fagomine (3) was then obtained by selective catalytic
reductive amination10 of 2 (Pd/C, H2 50 psi) in 89% isolated
(10) Espelt, L.; Parella, T.; Bujons, J.; Solans, C.; Joglar, J.; Delgado,
A.; Clapes, P. Chem.sEur. J. 2003, 9, 4887-4899.
(11) Wong et al. have recently discussed the possibility of using DHA
as a substrate in the presence of borate buffer, presumably by in situ
formation of borate ester to permit the direct use of DHA in DHAP-aldolase-
mediated chemistry. See: Sujiyama, M.; Whalen, L. J.; Hong, Z.-Y.;
Greenberg, W.A.; Wong, C.-H. Book of Abstracts. 232nd ACS National
Meeting, San Francisco, Sep 10-14, 2006; American Chemical Society:
Washington, DC, 2006; ORGN-496. In our case, FSA uses DHA, and there
is no need for any ester, as demonstrated with reactions conducted in
glycylglycine buffer.
yield without further purification and 93:7 diastereomeric
20
ratio by NMR: [R]20 ) +20.4 (c 1.0, H2O) (lit.3 [R]D
)
D
+19.5 (c 1.0, H2O)). Further purification by cation-exchange
+
chromatography on CM-sepharose in NH4 form, eluted
isocratically with 0.01 M NH4OH, gave an excellent separa-
tion of 3 (83% recovery and de g99%) and a minor
diastereoisomer identified as D-2,4-di-epi-fagomine. This
(12) Jung, S.-H.; Jeong, J.-H.; Miller, P.; Wong, C.-H. J. Org. Chem.
1994, 59, 7182-7184. Gefflaut, T.; Lemaire, M.; Valentin, M.-L.; Bolte,
J. J. Org. Chem. 1997, 62, 5920-5922. Ferroni, E. L.; Ditella, V.;
Ghanayem, N.; Jeske, R.; Jodlowski, C.; Oconnell, M.; Styrsky, J.; Svoboda,
R.; Venkataraman, A.; Winkler, B. M. J. Org. Chem. 1999, 64, 4943-
4945. Charmantray, F.; El Blidi, L.; Gefflaut, T.; Hecquet, L.; Bolte, J.;
Lemaire, M. J. Org. Chem. 2004, 69, 9310-9312. Meyer, O.; Ponaire, S.;
Rohmer, M.; Grosdemange-Billiard, C. Org. Lett. 2006, 8, 4347-4350.
(13) Fessner, W.-D.; Sinerius, G. Angew. Chem., Int. Ed. 1994, 33, 209-
212. Sanchez-Moreno, I.; Francisco Garcia-Garcia, J.; Bastida, A.; Garcia-
Junceda, E. Chem. Commun. 2004, 1634-1635. van Herk, T.; Hartog, A.
F.; Schoemaker, H. E.; Wever, R. J. Org. Chem. 2006, 71, 6244-6247.
(14) Fessner, W.-D.; Walter, C. Top. Curr. Chem. 1996, 184, 97-194.
(15) Schu¨rmann, M.; Sprenger, G. A. J. Biol. Chem. 2001, 276, 11055-
11061.
(16) Schu¨rmann, M.; Sprenger, G. A. J. Mol. Catal. B: Enzym. 2002,
19, 247-252.
(17) One unit of U will synthesize 1 µmol of fructose-6-phosphate from
D-glyceraldehyde-3-phosphate and DHA per minute at pH 8.5 (glycylglycine
50 mM buffer) and 30 °C. The maximum specific activity observed for the
purified enzyme was 7.13 U mg-1
.
(18) Ocejo, M.; Vicario, J. L.; Badia, D.; Carrillo, L.; Reyes, E. Synlett
2005, 2110-2112.
6068
Org. Lett., Vol. 8, No. 26, 2006

compound arose from the re-face attack of the DHA-FSA
complex on the aldehyde, similar to that found with
D-fructose-1,6-bisphosphate aldolase.10 N-Alkylated deriva-
tives (4a-f) were easily obtained by catalytic reductive
amination (Pd/C, H2 50 psi) of a mixture of 3 and the
corresponding aldehyde. Most interestingly, we carried out
the preparation of 4a-f by reductive amination of a mixture
of 2 and the aldehyde in a one-pot reaction (Scheme 1). The
isolated yields obtained by this procedure were similar to
those achieved starting from 3.
Investigations of the synthetic abilities of FSA in organic
synthesis are currently in progress. We have found that this
enzyme accepts, among others, N-Cbz-glycinal and N-Cbz-
3-amino-2-hydroxypropanal, but R-alkyl-branched N-Cbz-
aminoaldehydes were not tolerated as substrates.
Table 1. Inhibition of Glycosidases by Compounds 3 and
4a-f
R-D-glucosidasea
â-D-galactosidaseb
compd
Kic (µM)
IC50 (µM)
Kid (µM)
IC50 (µM)
30.4
3
9.3 ( 0.8
126 ( 3.6
73.3 ( 2.0
27.3 ( 2.8
14.9 ( 1.2
16.4 ( 2.1
143 ( 5.4
13.2
151
35.9 ( 3.9
NIe
NIe
242 ( 14
140 ( 18
9.3 ( 0.7
691 ( 18
4a
4b
4c
4d
4e
4f
61.4
35.3
18.1
20.7
159
203
108
6.0
416
a From rice. An IC50 value of 350 µM has been reported for compound
3.3 The reason may lie in the different substrate used to evaluate the
glycosidase activity in the literature report (i.e., a disaccharide) and the
present work (i.e., p-nitrophenyl glucopyranoside). b From bovine liver.
c Competitive inhibition. d Noncompetitive. e No inhibition.
Antimicrobial activity of 3 and 4a-f was tested against
15 bacteria and 7 fungi (see the Supporting Information).
Compounds 3 and 4a-d,f did not show activity at concen-
trations below 256 µg mL-1, thus indicating much lower
potency than that found for antiseptics such as cationic
surfactants and biguanides.19 Interestingly, 4e gave minimum
inhibitory concentrations (MIC)s ranging from 32 to 64 µg
mL-1 against most of the Gram-positive bacteria tested,
whereas MICs of 128-256 µg mL-1 were observed for the
Gram-negative bacteria. Compound 4e was also rather potent
against fungi such as Aspergillus repens (32 µg mL-1) and
Cladosporium cladosporoides (64 µg mL-1) but less potent
(128-256 µg mL-1) against the rest of the fungi tested.
Since D-fagomine was reported to have inhibitory activity
on R-glucosidase,2,3 different chemical modifications have
been introduced to improve its activity and selectivity.
Goujon et al. synthesized R-1-C-substituted derivatives with
or without an N-butyl group, which exhibited higher inhibi-
tory activity than the parent compound against R-glucosidase
from rice.7 On the other hand, the substitution of the
piperidine ring for an azepane ring decreased the inhibitory
activity against both enzymes.20
Compounds 3 and 4a-f were inhibitors of R-D-glucosidase
from rice and â-D-galactosidase from bovine liver (Table 1),
whereas no inhibition was observed against R-D-glucosidase
from baker’s yeast, â-D-glucosidase from almond, R-D-
mannosidase from jack beans, and R-L-rhamnosidase from
Penicillium decumbens. Interestingly, among the N-alkylated
derivatives, 4d and 4e were the best inhibitors against R-D-
glucosidase, whereas 4e was a good inhibitor for the
â-galactosidase. As shown in Table 1, the activity of 4a-f
against R-glucosidase increased with the aliphatic chain
length up to nine carbon atoms (4d). Increasing the number
of carbon atoms to 12 (4e) or including an aromatic moiety
(4f) did not improve or caused a drastic decrease on the
inhibitory activity, respectively. Kinetic studies indicated that
active compounds behaved as competitive inhibitors of R-D-
glucosidase from rice. These results suggested that 3 may
interact with the subsite -1 of the two catalytic sites reported
for R-glucosidase family II21 and the N-alkyl substituent with
a convenient length may fit into subsite +1, but not with
the phenylethyl substitution.
Inhibition of â-D-galactosidase from bovine liver by 3 and
4a-f is also depicted in Table 1. For 4a-e, the higher the
hydrophobicity the lower the IC50 values, 4e being the most
active one. Inhibitory activity of 4e was similar to that
reported for the D-galacto isomer of 322 and higher than that
found for 3 or its R-1-C-ethyl derivative.23 Active compounds
inhibited this enzyme in a noncompetitive manner, with a
Ki value for 4e 4-fold lower than that for 3.
Finally, cytotoxicity of 3 and 4a-f was estimated by
determination of their hemolytic and protein denaturation
effects on human erythrocytes.24 The results showed that they
did not have activity, with the exception of 4e (see the
Supporting Information). The HC50 (i.e., concentration that
induces the hemolysis of 50% of the cells) for compound
4e was 138 ( 8 mg mL-1, whereas the denaturation index
(DI, i.e., hemoglobin denaturation induced by the compound)
was 5 ( 1. These values compare with commercial decyl-
glucoside (HC50 252 ( 6 mg mL-1 and DI 14.2).24
In summary, D-fagomine can be obtained stereoselectively
in two chemo-enzymatic asymmetric steps using inexpensive
achiral DHA and 1 as the starting materials and FSA from
E. coli as biocatalyst in 51% isolated yield and g99% de.
The N-alkylated derivatives of 3 were obtained for the first
time in one pot reaction from a mixture containing 2 and
the corresponding aldehyde. Preliminary results indicated that
4e elicited antifungal and antibacterial activity with slight
selectivity against Gram-positive bacteria. Overall, alkylation
of the nitrogen did not strongly improve the activity but
increased the selectivity toward glucosidase and galactosidase
(21) Kimura, A.; Lee, J.-H.; Lee, I.-S.; Lee, H.-S.; Park, K.-H.; Chiba,
S.; Kim, D. Carbohydr. Res. 2004, 339, 1035-1040.
(22) Asano, N.; Oseki, K.; Kizu, H.; Matsui, K. J. Med. Chem. 1994,
37, 3701-3706.
(23) Asano, N.; Nishida, M.; Miyauchi, M.; Ikeda, K.; Yamamoto, M.;
Kizu, H.; Kameda, Y.; Watson, A. A.; Nash, R. J.; Fleet, G. W. J.
Phytochemistry 2000, 53, 379-382.
(24) Mitjans, M.; Martinez, V.; Clapes, P.; Perez, L.; Infante, M. R.;
Vinardell, M. P. Pharm. Res. 2003, 20, 1697-1701.
(19) Castillo, J. A.; Pinazo, A.; Carilla, J.; Infante, M. R.; Alsina, M.
A.; Haro, I.; Clapes, P. Langmuir 2004, 20, 3379-3387.
(20) Li, H.; Bleriot, Y.; Chantereau, C.; Mallet, J.-M.; Sollogoub, M.;
Zhang, Y.; Rodriguez-Garcia, E.; Vogel, P.; Jimenez-Barbero, J.; Sinay, P.
Organ. Biomol. Chem. 2004, 2, 1492-1499.
Org. Lett., Vol. 8, No. 26, 2006
6069

inhibition. Compound 4d, which was not cytotoxic in the
red blood cell test, exhibited good inhibitory activity and
higher specificity against family II R-glucosidase than that
of the parent 3.
edge the CSIC I3P and UAs predoctoral scholarship pro-
grams, respectively. J.A.C. also acknowledges the COST D25
STSM for financial support.
Supporting Information Available: Experimental prepa-
rations, analytical data, biological data, and 1H and 13C NMR
spectra for all compounds; COSY, HSQC, and NOE spectra
for compound 4a. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. We thank Prof. Angels Manresa at
the Microbiology Department of the University of Barcelona
for her assistance and fruitful discussions on antimicrobial
activity. Financial support from the Spanish MEC (CTQ-
2005-00063 and CTQ2005-25182-E), La Marato´ de TV3
foundation (20060293), and Generalitat de Catalunya DURSI
2005-SGR-00698 is acknowledged. J.A.C. and J.C. acknowl-
OL0625482
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Org. Lett., Vol. 8, No. 26, 2006