D-fructose-6-phosphate aldolase-catalyzed one-pot synthesis of iminocyclitols

Article abstract of DOI:10.1021/ja073911i

A one-pot chemoenzymatic method for the synthesis of a variety of new iminocyclitols from readily available, non-phosphorylated donor substrates has been developed. The method utilizes the recently discovered fructose-6-phosphate aldolase (FSA), which is functionally distinct from known aldolases in its tolerance of different donor substrates as well as acceptor substrates. Kinetic studies were performed with dihydroxyacetone (DHA), the presumed endogenous substrate for FSA, as well as hydroxy acetone (HA) and 1-hydroxy-2-butanone (HB) as donor substrates, in each case using glyceraldehyde-3-phosphate as acceptor substrate. Remarkably, FSA used the three donor substrates with equal efficiency, with k_cat/K_M-values of 33, 75, and 20 M ^-1 s^-1, respectively. This level of donor substrate tolerance is unprecedented for an aldolase. Furthermore, DHA, HA, and HB were accepted as donors in FSA-catalyzed aldol reactions with a variety of azido- and Cbz-amino aldehyde acceptors. The broad substrate tolerance of FSA and the ability to circumvent the need for phosphorylated substrates allowed for one-pot synthesis of a number of known and novel iminocyclitols in good yields, and in a very concise fashion. New iminocyclitols were assayed as inhibitors against a panel of glycosidases. Compounds 15 and 16 were specific α-mannosidase inhibitors, and 24 and 26 were potent and selective inhibitors of β-N-acetylglucosaminidases in the submicromolar range. Facile access to these compounds makes them attractive core structures for further inhibitor optimization.

Full text of DOI:10.1021/ja073911i

Published on Web 11/07/2007
D-Fructose-6-Phosphate Aldolase-Catalyzed One-Pot
Synthesis of Iminocyclitols
Masakazu Sugiyama, Zhangyong Hong, Pi-Hui Liang, Stephen M. Dean,
Lisa J. Whalen, William A. Greenberg,* and Chi-Huey Wong*
Contribution from the Department of Chemistry and the Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received May 30, 2007; E-mail: wong@scripps.edu
Abstract: A one-pot chemoenzymatic method for the synthesis of a variety of new iminocyclitols from readily
available, non-phosphorylated donor substrates has been developed. The method utilizes the recently
discovered fructose-6-phosphate aldolase (FSA), which is functionally distinct from known aldolases in its
tolerance of different donor substrates as well as acceptor substrates. Kinetic studies were performed with
dihydroxyacetone (DHA), the presumed endogenous substrate for FSA, as well as hydroxy acetone (HA)
and 1-hydroxy-2-butanone (HB) as donor substrates, in each case using glyceraldehyde-3-phosphate as
acceptor substrate. Remarkably, FSA used the three donor substrates with equal efficiency, with kcat/KM-
values of 33, 75, and 20 M-1 s-1, respectively. This level of donor substrate tolerance is unprecedented for
an aldolase. Furthermore, DHA, HA, and HB were accepted as donors in FSA-catalyzed aldol reactions
with a variety of azido- and Cbz-amino aldehyde acceptors. The broad substrate tolerance of FSA and the
ability to circumvent the need for phosphorylated substrates allowed for one-pot synthesis of a number of
known and novel iminocyclitols in good yields, and in a very concise fashion. New iminocyclitols were
assayed as inhibitors against a panel of glycosidases. Compounds 15 and 16 were specific R-mannosidase
inhibitors, and 24 and 26 were potent and selective inhibitors of â-N-acetylglucosaminidases in the
submicromolar range. Facile access to these compounds makes them attractive core structures for further
inhibitor optimization.
Introduction
The polyhydroxylated pyrrolidines and piperidines, commonly
called iminocyclitols, are potent glycosidase and glycosyltrans-
ferase inhibitors, due to their mimicry of the transition state of
the enzymatic reactions.4 Glycosidases and glycosyltransferases
play key roles in metabolism, lysosomal catabolism, and
glycoprotein processing. Glycoproteins and glycoconjugates on
the cell surface regulate many significant biological events such
as viral infection, cell-cell recognition, and inflammation. As
a result, iminocyclitols have been attractive as drug candidates
for a number of diseases such as cancer, viral infection,
lysosomal storage disorders, and diabetes.5 Several examples
of important iminocyclitols are illustrated in Figure 1. The
elegant work of Schramm and co-workers has led to discovery
of iminocyclitol nucleoside derivatives that are exceptionally
potent inhibitors of purine nucleotide phosphorylase and MTAN
nucleosidase, illustrating the value of iminocyclitols in the design
of inhibitors of glycoprocessing enzymes.6 However, structure-
The field of biocatalysis involves the application of enzymes
as tools for organic synthesis.1 The catalytic power and
specificity of enzymes allows them to effect difficult transfor-
mations under mild conditions and with exceptional stereose-
lectivities. Many classes of enzymes have been exploited as
synthetic tools, and one of the most prominent is the aldolases,
which catalyze reversible formation of C-C bonds with high
enantioselectivities.2 A common limitation of enzymes as
synthetic catalysts, and of aldolases in particular, is due to their
substrate specificity. Substrates frequently need to be phospho-
rylated, and small changes in substrate structure typically lead
to significant reduction in activity. Lack of generality and
inability to use readily available substrates has limited the
practicality of aldolases as catalysts for organic synthesis.
Approaches to broadening the synthetic utility of aldolases
include directed evolution, substrate engineering, and discovery
of novel enzymes in nature.3
(4) (a) Winchester, B.; Fleet, G. W. J. Glycobiology 1992, 2, 199-210. (b)
Butters, T. D.; Dwek, R. A.; Platt, F. M. Chem. Rev. 2000, 100, 4683-
4696. (c) Wang, R.-W.; Qing, F.-L. Org. Lett. 2005, 7, 2189-2192. (d)
Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed. 1999, 38, 2300-2324. (e)
Ouchi, H.; Mihara, Y.; Takahata, H. J. Org. Chem. 2005, 70, 5207-5214.
(5) (a) Asano, N. Glycobiology 2003, 13, 93R-140R. (b) Dwek, R. A.; Butters,
T. D.; Platt, F. M.; Zitzmann, N. Nat. ReV. Drug DiscoVery 2002, 1, 65-
75. (c) Jacob, G. S. Curr. Opin. Struct. Biol. 1995, 5, 605 -611.
(6) (a) Schramm, V. L. Acc. Chem. Res. 2003, 36, 588-596. (b) Singh, V.;
Evans, G. B.; Lenz, D. H. Mason, J. M.; Clinch, K.; Mee, S.; Painter, G.
F.; Tyler, P. C.; Furneaux, R. H.; Lee, J. E.; Howell, P. L.; Schramm, V.
L. J. Biol. Chem. 2005, 280, 18265-18273.
(1) (a) Powell, K. A.; Ramer, S. W.; del Cardayre´, S. B.; Stemmer, W. P. C.;
Tobin, M. B.; Longchamp, P. F.; Huisman, G. W. Angew. Chem., Int. Ed.
2001, 40, 3948-3959. (b) Panke, S.; Held, M.; Wubbolts, M. Curr. Opin.
Biotechnol. 2004, 15, 272-279. (c) Tao, J.; Zhao, L.; Ran, N. Org. Process
Res. DeV. 2007, 11, 259-267.
(2) (a) Machajewski, T. D.; Wong, C.-H. Angew. Chem., Int. Ed. 2000, 39,
1352-1375. (b) Franke, D.; Hsu, C.-C.; Wong, C.-H. Methods Enzymol.
2004, 224-238.
(3) Dean, S. M.; Greenberg, W. A.; Wong, C.-H. AdV. Synth. Catal. 2007,
349, 1308-1320.
9
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J. AM. CHEM. SOC. 2007, 129, 14811-14817
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A R T I C L E S
Sugiyama et al.
Figure 1. Iminocyclitols. 1, DMDP; 2, â-hexosaminidase inhibitor; 3, fagomine; 4, DNJ.
Figure 2. Aldolase-catalyzed synthesis of iminocyclitols. (A) Original synthesis with D-fructose-1,6-diphosphate(FDP) aldolase; (B) synthesis with D-fructose-
6-phosphate aldolase (FSA).
activity relationships for iminocyclitol glycosidase inhibitors can
be difficult to elucidate, making rational inhibitor design a
frustrating exercise.7 This is well illustrated by the recent
observation by Fleet and co-workers that the L-enantiomers of
rationally designed D-iminocyclitols can be much more potent
inhibitors of the D-sugar processing enzymes.8 Considering this
situation, there is a need for facile methods of accessing a broad
range of iminocyclitol structures in sufficient quantities to allow
for synthesis of analogues, so that structurally diverse libraries
can be screened for inhibitory activity.
Among the reported methods of preparing iminocyclitols,9
chemoenzymatic synthesis using aldolases provides rapid access
to multiple scaffolds in a highly selective manner.10a-c The
dihydroxyacetone phosphate (DHAP)-dependent aldolases cata-
lyze the addition of DHAP to acceptor aldehydes, and are
capable of accepting aldehydes containing azide or N-Cbz-amine
substitutions, thus providing the corresponding azido- or N-Cbz-
amino polyhydroxy ketones. After removal of the phosphate
group with phosphatase, reduction of the azide or deprotection
of N-Cbz group followed by intramolecular reductive amination
affords a series of iminocyclitols (Figure 2A).10 However,
DHAP-dependent aldolases possess the drawback of strict donor
substrate specificity toward DHAP, and nonphosphorylated
dihydroxyacetone (DHA) cannot be used. The high cost and
instability of DHAP, as well as the requirement of a phosphatase
to remove the phosphate ester, reduce the practicality of the
process. Significant effort has been expended toward a more
practical access to DHAP,11 but it remains difficult. A more
desirable solution would be the elimination of the requirement
for DHAP, and facilitation of the use of readily available,
inexpensive DHA in its place. One approach to achieving this
end is directed evolution of the enzyme. Several examples of
the application of directed evolution to change the substrate
specificity or synthetic utility of an aldolase have been published
recently.12 Another approach to improving synthetic utility of
an aldolase is substrate or reaction engineering, as in our recent
application of borate to allow RhaD aldolase to accept DHA.13
A third route is the discovery of novel enzymes from nature
that catalyze the desired reaction.14 In parallel with these
biocatalytic efforts, many organocatalytic asymmetric aldol15
methods have been reported, including several that utilize DHA
or hydroxy acetone as donors.15b-f Not limited by the strict
substrate dependence of enzymes, a great deal of research in
organocatalytic aldol chemistry is focused on improving catalytic
efficiency and stereoselectivity to enzymelike levels.
(11) For chemical synthesis of DHAP, see: (a) Gefflaut, T.; Lemaire, M.;
Valentin, M.-L.; Bolte, J. J. Org. Chem. 1997, 62, 5920-5922. (b) Jung
S.-H.; Jeong, J.-H.; Miller, P.; Wong, C.-H. J. Org. Chem. 1994, 59, 7182-
7184. For recent enzymatic syntheses of DHAP, see: (c) Sa´nchez-Moreno,
I.; Garc´ıa-Garc´ıa, J. F.; Bastida, A.; Garc´ıa-Junceda, E. Chem. Commun.
2004, 1634-1635. (d) Schoevaart, R.; Van Rantwigk, F.; Sheldon, R. A.
Chem. Commun. 1999, 2465-2466. (e) Fessner, W.-D.; Sinerius, G. Angew.
Chem., Int. Ed. Engl. 1994, 33, 209-212.
(12) (a) Fong, S.; Machajewski, T. D.; Mak, C. C.; Wong, C.-H. Chem. Biol.
2000, 7, 873-883. (b) Hsu, C.-C.; Hong, Z.; Wada, M.; Franke, D.; Wong,
C.-H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9122-9126. (c) Woodhall,
T.; Williams, G.; Berry, A.; Nelson, A. Angew. Chem., Int. Ed. 2005, 44,
2109-2112. (d) Williams, G. J.; Woodhall, T.; Farnsworth, L. M.; Nelson,
A.; Berry, A. J. Am. Chem. Soc. 2006, 128, 16238-16247.
(7) Gloster, T. M.; Meloncelli, P.; Stick, R. V.; Zechel, D.; Vasella, A.; Davies,
G. J. J. Am. Chem. Soc. 2007, 129, 2345-2354.
(8) (a) Yu, C.-Y.; Asano, N.; Ikeda, K.; Wang, M.-X.; Butters, T. D.; Wormald,
M. R.; Dwek, R. A.; Winters, A. L.; Nash, R. J.; Fleet, G. W. J. Chem.
Commun. 2004, 1936-1937. (b) Asano, N.; Ikeda K.; Yu, L.; Kato, A.;
Takebayashi, K.; Adachi, I.; Kato, I.; Ouchi, H.; Takahata, H.; Fleet, G.
W. J. Tetrahedron: Asymmetry 2005, 16, 223-229.
(13) Sugiyama, M.; Hong, Z.; Whalen, L. J.; Greenberg, W. A.; Wong, C.-H.
AdV. Synth. Catal. 2006, 348, 2555-2559.
(9) (a) Ayad, T.; Genisson, Y.; Baltas, M. Curr. Org. Chem. 2004, 8, 1211-
1233. (b) Moriarty, R. M.; Mitan, C. I.; Branza-Nichita, N.; Phares, K. R.;
Parrish, D. Org. Lett. 2006, 8, 3465-3467.
(14) (a) Samland, A.; Sprenger, G. A. Appl. Microbiol. Biotechnol. 2006, 71,
253-264. (b) Schurmann, M.; Sprenger, G. A. J. Biol. Chem. 2001, 276,
11055-11061. (c) Schurmann, M.; Schurmann, M.; Sprenger, G. A. J. Mol.
Catal. B: Enzym. 2002, 19, 247-252.
(15) (a) Dalko, P. I.; Moisan, L. Angew. Chem., Int. Ed. 2004, 43, 5138-5175.
(b) Notz, W.; List, B. J. Am. Chem. Soc. 2000, 122, 7386-7387. (c)
Sakthivel, K.; Notz, W.; Bui, T.; Barbas, C. F. J. Am. Chem. Soc. 2001,
123, 5260-5267. (d) Cordova, A.; Notz, W.; Barbas, C. F. Chem. Commun.
2002, 3024-3025. (e) Fernandez-Lopez, R.; Kofoed, J.; Machuqueiro, M.;
Darbre, T. Eur. J. Org. Chem. 2005, 5268-5276. (f) Ramasastry, S. S. V.;
Zhang, H.; Tanaka, F.; Barbas, C. F. J. Am. Chem. Soc. 2007, 129, 288-
289.
(10) (a) Samland, A. K.; Sprenger, G. A. Appl. Microbiol. Biotechnol. 2006,
71, 253-264. (b) Whalen, L. J.; Wong, C.-H. Aldrichimica Acta 2006, 39,
63-71. (c) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong C.-H. Chem. ReV.
1996, 96, 443-474. (d) Kajimoto, T.; Liu, K. K.-C.; Pederson, R. L.; Zhong,
Z.; Ichikawa, Y.; Porco, J. A.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113,
6187-6196. (e) 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. (f) Liu, K. K.-C.; Kajimoto, T.; Chen, L.; Zhong, Z.; Ichikawa,
Y.; Wong, C.-H. J. Org. Chem. 1991, 56, 6280-6289.
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One-Pot Chemoenzymatic Synthesis of Iminocyclitols
A R T I C L E S
Table 1. Kinetic Valuesa for FSA with Three Donor Substrates,
Using Glyceraldehyde-3-phosphate as Acceptor Substrate
is unprecedented for an aldolase and suggests that FSA could
be a biocatalyst with an exceptionally broad substrate scope
and utility. This possibility was explored by preparing a range
of iminocyclitol products using FSA.
1
1
donor
K
M (mM)
k
cat (s-
)
k
cat/ KM (M-1 s-
)
dihydroxyacetone
hydroxy acetone
1-hydroxybutan-2-one
40 ( 18
11 ( 6
32 ( 17
1.31 ( 0.20
0.82 ( 0.15
0.63 ( 0.10
33
75
20
Initial preparative experiments were aimed at the synthesis
of 1,4-dideoxy-1,4-imino-D-arabitol, 8, a naturally occurring
five-membered iminocyclitol which inhibits R-glucosidases.17
FSA accepted azidoacetaldehyde and DHA to give a single
diastereomer of the aldol product 12 (Figure 2B). The aldol
product was subjected to reductive amination to afford 8. The
NMR spectra of the product matched that of authentic 8, and
no peaks from other diastereomers were observed, confirming
that FSA possesses the same (3S,4R) stereoselectivity as that
of D-fructose-diphosphate (FDP) aldolase.
The broad donor substrate specificity of FSA was exploited
to synthesize a large series of iminocyclitols (Table 2, 3),
including many that were not previously accessible by aldolase-
catalyzed methods, due to substrate tolerance restrictions. When
using azidoacetaldehyde as acceptor, FSA was able to efficiently
use HA and HB as donor substrates, to give methyl derivatives
13 or ethyl-substituted polyhydroxy ketones 14, respectively
(Figure 2). The reductive amination of azidoketoses was
diastereoselective (consistent with our previous reports10) with
hydrogen attacking the imine intermediate from the face opposite
to the axial OH-group being favorable.
A series of 2-azido aldehydes were used as acceptor aldehydes
to prepare five-membered iminocyclitols (Table 2), and N-Cbz
3-amino aldehydes were used to prepare six-membered imino-
cyclitols, as first described by Clape´s and co-workers16 (Table
3). Using HA and HB as donor substrates, azidoaldehydes 5,
17, and 22 were all accepted by FSA. Compounds 19 and 21
are deoxy- or ethyl derivatives of 2,5-dideoxy-2,5-imino-D-
mannitol (DMDP), 1,18 a naturally occurring â-glycosidase
inhibitor, and compounds 24 and 26 are derivatives of compound
2, a potent inhibitor of N-acetyl-â-hexosaminidase19 that may
be useful in the treatment of osteoarthritis. Interestingly, FSA
was highly enantioselective for 2-azido-3-substituted aldehydes
17 and 22, with (R)-aldehydes serving as better substrates than
(S)-enantiomers. Reactions with the N-Cbz amino aldehydes
were performed in DMF/water (1:4) cosolvent system because
of the low aqueous solubility of the substrate. The cosolvent
did not adversely affect enzyme activity, suggesting that organic
cosolvents are well-tolerated by FSA and may be used for other
substrates that have poor aqueous solubility. DHA and 27 were
condensed by FSA to yield one diastereomer 28, followed by
reductive amination to give the naturally occurring glucosidase
inhibitor fagomine 3. The specific rotation obtained for this
sample of 3 was +18.1°, whereas the reported value for 3
isolated from natural sources is +19.5o,20 suggesting the
enzymatically produced iminocyclitol has an enantiomeric
excess of 93%. A detailed analysis of the enantioselectivity and
a Values were obtained as described in the Experimental Section.
Here we report the application of fructose-6-phosphate
aldolase (FSA) from Escherichia coli14b for practical one-pot
synthesis of a broad range of iminocyclitols, in a procedure that
avoids the need for a phosphorylated substrate. Previous reports
from Sprenger and co-workers showed that FSA catalyzes the
reversible aldol reaction from D-fructose-6-phosphate to D-glyc-
eraldehyde-3-phosphate and the non-phosphorylated DHA and
also that it uses hydroxy acetone in the place of DHA.14c The
lack of strict donor substrate preference exhibited by FSA is
unusual for an aldolase. The sequence of FSA is also unusual
and is, in fact, more homologous to transaldolases, although
transaldolase activity was not observed for this enzyme.14b On
the basis of these findings, we hypothesized that this unique
aldolase could be used to synthesize iminocyclitols and related
compounds by using DHA (or other donors) instead of DHAP
as the donor substrate. During the course of our efforts, Clape´s
and co-workers reported the first application of FSA for the
synthesis of iminocyclitols, using a strategy similar to the one
outlined here to synthesize 3 and a series of N-alkyl deriva-
tives.16 In the present work, FSA was used to synthesize
iminocyclitols from DHA, HA, or HB, and a range of acceptor
substrates (Figure 2B). Finally, eliminating the need for DHAP
has enabled the development of a practical one-pot synthesis
of iminocyclitols by performing the aldol and reductive ami-
nation reactions in a single vessel. The new methods allowed
for extremely facile access to a broad range of iminocyclitols,
which were assayed against a panel of glycosidases.
Results and Discussion
To determine the catalytic efficiency of FSA with a variety
of donor substrates, kinetics experiments were performed with
dihydroxyacetone, hydroxy acetone, or 1-hydroxy-2-butanone
as donor, and D-glyceraldehyde-3-phosphate as acceptor. We
observed that DHAP was not a viable donor substrate for FSA,
which concurs with the original observations of Sprenger and
co-workers.14b Kinetic values were determined in the synthetic
direction by following the consumption of the acceptor D-glyc-
eraldehyde-3-phosphate (G-3-P) over the course of the reaction
by withdrawing aliquots at various time points and measuring
remaining G-3-P concentration by an enzyme-coupled assay
utilizing triosephosphate isomerase and R-glycerophosphate
dehydrogenase. Consumption of NADH, as measured by
absorbance at 340 nm, was equated with remaining G-3-P
concentration. Remarkably, the unnatural substrates HA and HB
were not merely tolerated, but were equally efficient substrates
when compared to the presumed endogenous substrate DHA,
as shown in Table 1. In fact, kcat/KM for HA was slightly higher
than for DHA, although all three substrates had catalytic
efficiencies within the same order of magnitude of each other.
This level of plasticity with respect to donor substrate structure
(17) (a) Fleet, G. W. J.; Nicholas, S. J.; Smith, P. W.; Evans, S. V.; Fellows, L.
E.; Nash, R. J. Tetrahedron Lett. 1985, 26, 3127-3130. (b) Fleet, G. W.
J.; Smith, P. W. Tetrahedron 1986, 42, 5685-5692.
(18) (a) Pederson, R. L.; Wong, C.-H. Heterocycles 1989, 28, 477-480. (b)
Ziegler, T.; Straub, A.; Effenberger, F. Angew. Chem., Int. Ed. Engl. 1988,
27, 716-717.
(19) (a) Liu, J.; Numa, M. M. D.; Liu, H.; Huang, S.-J.; Sears, P.; Shikhman,
A. R.; Wong, C.-H. J. Org. Chem. 2004, 69, 6273-6283. (b) Liu, J.;
Shikhman, A. R.; Lotz, M. K.; Wong, C.-H. Chem. Biol. 2001, 8, 701-
711. (c) Liang, P.-H.; Cheng, W.-C.; Lee, Y.-L.; Yu, H.-P.; Wu, Y.-T.;
Lin, Y.-L.; Wong, C.-H. ChemBioChem 2006, 7, 165-173.
(20) Kato, A.; Asano, N.; Kizu, H.; Matsui, K.; Watson, A. A.; Nash, R. J. J.
Nat. Prod. 1997, 60, 312-314.
(16) Castillo, J. A.; Calveras, J.; Casas, J.; Mitjans, M.; Vinaedell, M. P.; Parella,
T.; Inoue, T.; Sprenger, G. A.; Joglar, J.; Clapes, P. Org. Lett. 2006, 8,
6067-6070.
9
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A R T I C L E S
Sugiyama et al.
Table 2. Iminocyclitol Synthesis from Azidoaldehydes
diastereoselectivity of FSA-catalyzed reactions is in progress,
although it requires samples of the enantiomers, which are not
readily accessible except by multistep synthesis.
donor substrates for FSA than DHA from a synthetic standpoint,
because they reacted efficiently with all the azidoaldehydes or
N-Cbz amino aldehydes tested in this study. In contrast, DHA
was compatible only with 2-azidoaldehyde 5 and 3-[(benzy-
loxycarbonyl)amino]propionaldehyde 27, not with azido- or
Cbz-amino aldehydes containing branching at the 2-position.
The observation that the acceptor substrate tolerance of the
aldolase is dependent on the nature of the donor substrate is
intriguing. FSA has previously been noted to have significant
sequence homology (∼50%) to transaldolase B,14b which
catalyzes the reversible transfer of a DHA moiety from fructose-
6-phosphate to erythrose-4-phosphate, to form sedoheptulose-
7-phosphate and release glyceraldehyde-3-phosphate. The un-
usual substrate tolerance of FSA may be related to an evolutionary
origin as a transaldolase, an enzyme class that must accom-
modate four separate substrates in its active site over the course
of its catalytic cycle.
Deoxyfagomine 30 and the ethyl fagomine analogue 32 were
also synthesized. In order to synthesize 1-deoxynojirimycin
(DNJ) derivatives or 1-deoxymannojirimycin (mannoDNJ)
derivatives, both pure enantiomers of 3-[(benzyloxycarbonyl)-
amino]-2-hydroxypropionaldehyde 33 and 38 were prepared (see
Supporting Information). DNJ and its derivatives constitute an
important class of iminocyclitols, including N-butyl-DNJ (Mi-
glustat), which is a potent glucosylceramide synthase inhibitor.
It is used clinically for the treatment of the glycolipid storage
disorder, type I Gaucher disease.
As noted, FSA possesses unusually wide donor substrate
specificity, which is useful because strict donor substrate
specificities of aldolases have often limited their applications.
While the physiological role of FSA is still not entirely clear,
HA and HB are equally good substrates from a kinetic
standpoint compared to DHA and appear to be more versatile
Further optimization of FSA-catalyzed synthesis of iminocy-
clitols was achieved by developing methods for performing all
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One-Pot Chemoenzymatic Synthesis of Iminocyclitols
A R T I C L E S
Table 3. Iminocyclitol Synthesis from Cbz-Protected Aminoaldehydes
operations in a single pot (Figure 3). FSA-catalyzed aldolase
reactions were performed in sodium borate buffer (pH 7.6),
followed by the reductive amination in the same vessel, in the
presence of excess equivalents of diethylamine. The addition
of diethylamine protected the secondary amine of the resulting
iminocyclitols from further intermolecular reductive amination
by any residual aldehyde or ketone. Thus, the iminocyclitol
synthesis could now be completed in a one-pot fashion, without
isolation of intermediates. All iminocyclitols listed in Tables 2
and 3 were synthesized by the one-pot procedure, as well as
the two-step procedure. Analysis of crude product mixtures by
NMR showed small quantities of diastereomeric products,
typically in the range of 3-5%. This observation is in good
agreement with the 93:7 diastereoselectivity observed by Clape´s
and co-workers for preparation of compound 3.16 The major
source of yield loss was the difficulty in purification of these
polar molecules. Compounds 15, 21, 24, 26, 32, 37, and 42 are
previously unreported iminocyclitols, and preliminary inhibitory
activities toward a series of glycosidases were determined (Table
4).
Compounds 15 and 16 were found to be slightly more potent
inhibitors of R-mannosidase from jack beans than the known
inhibitor 8, with 26% and 14% inhibition at 1 µM, respectively.
While not exceptionally potent, 16 is quite specific for R-man-
nosidase, whereas 8 inhibits both R- (Ki 860 nM) and â-glu-
cosidases in addition to R-mannosidase. Further structural
elaboration of 16, which can be readily accessed by this synthetic
method, should lead to more potent and specific mannosidase
inhibitors. Such compounds may be useful for preventing
aberrant glycosylation in cancer. Compounds 24 and 26 are
potent and specific inhibitors of â-N-acetylglucosaminidases
from jack beans and human placenta. Individual Ki values were
determined to be in the submicromolar range in each case (Table
4). Compound 24 is a 320 nM inhibitor of the plant enzyme,
9
J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007 14815

A R T I C L E S
Sugiyama et al.
Figure 3. One -pot synthesis of iminocyclitols.
Table 4. Glycosidase Inhibition Activitiesa of Synthesized Iminicyclitols
enzymeb
3
8
15
16
19
21
24c
26c
35
37
R-Glu
3
0
14
25
34
6
100(58)
84(4)
81(9)
38
47
0
54 (4)
55 (4)
95 (26)
10
0
0
4
69 (0)
95 (14)
20
4
0
39
50
25
47
13
29
3
0
0
11
0
0
100 (61)
0.32
100 (59)
0.51
0
6
8
0
0
6
11
85 (2)
6
4
9
0
0
42
0
6
21
â-Glu
40
11
49
19
3
R-Man
â-Man
â-Gal
â-N-GlcNAc1
100 (37)
0.76
100 (68)
0.29
â-N-GlcNAc2
0
0
0
0
0
0
11
22
a Values represent percent inhibition at 200 µM compound. For the most potent inhibitors, percent inhibition was also determined at 1 µM, shown in
parentheses. b R-Glu, R-glucosidase from jack beans; â-Glu, â-glucosidase from almonds; R-Man; R-mannosidase from jack beans; â-Man, â-mannosidase
from snail; â-Gal, â-galactosidase from Aspergillus oryzae; â-N-GlcNAc,1 â-N-acetylglucosaminidase from jack beans; â-N-GlcNAc2, â-N-acetylglucosaminidase
from human placenta. c For compounds 24 and 26, Ki values against the two â-N-acetylglucosaminidases are shown in bold (µM).
whereas 26 is a 290 nM inhibitor of the human enzyme. These
structures may serve as readily accessible cores for optimization,
leading to discovery of new treatments for osteoarthritis.19c
Inhibition activities of new compounds toward R-galactosidase
from green coffee beans and R-fucosidase from bovine kidney
were also determined, but no significant inhibition was observed,
except compound 35 showed 48% inhibition against R-fucosi-
dase at 200 µM (data not shown).
allowed for the synthesis of a number of known and novel
iminocyclitols in relatively high yields, and in an extremely
concise fashion. Glycosidase inhibition studies of the new
compounds revealed that 24 and 26 are potent and specific
inhibitors of â-N-acetylglucosaminidases. This new synthetic
method renders these compounds accessible and will enable
optimization studies to develop more active inhibitors as
potential treatments for osteoarthritis and other disorders related
to glycoprocessing enzymes. Efforts are underway on the
expansion of the substrate tolerance of FSA, with the goal of
developing practical biocatalysts with very broad utility.
Conclusions
In conclusion, a newly discovered aldolase, FSA, has been
investigated as a catalyst for organic synthesis. Kinetic measure-
ments showed that FSA did not merely tolerate structural
variation in the donor substrate but also accepted hydroxy
acetone and 1-hydroxy-2-butanone equally well compared to
dihydroxyacetone, the presumed endogenous substrate. This
level of donor substrate tolerance is unprecedented for an
aldolase and makes FSA a practical catalyst for synthesis of a
wider range of compounds than is typically possible with an
aldolase. To illustrate the power of FSA as a biocatalyst, a one-
pot chemoenzymatic method for the synthesis of a variety of
iminocyclitols from readily available, non-phosphorylated donor
substrates has been developed. In addition to dihydroxyacetone,
the unnatural donors hydroxy acetone and 1-hydroxybutan-2-
one were also accepted in the FSA-catalyzed aldol reactions
with both azido- and Cbz-amino aldehyde acceptors. The
tolerance observed for both donor and acceptor suggests that
FSA has a significant amount of plasticity in its binding site,
which may be related to its high homology to transaldolases.
Directed evolution may allow for even greater substrate toler-
ance and synthetic utility. The broad substrate tolerance of FSA
Experimental Section
General. Solvents, starting materials, and reagents were used as
purchased without further purification. UV kinetic assays were per-
formed on a Beckman DU-650 spectrophotometer. Proton NMR spectra
(1H NMR) were recorded at 500 MHz using a Bruker 5 mm DCH
CryoProbe. Chemical shifts are expressed in parts per million (δ) and
are referenced to residual protium in the NMR solvent: CD2HOD, δ
3.31; DOH, δ 4.80. Carbon NMR (13C NMR) spectra were recorded at
125 MHz using the same probe. Chemical shifts (δ ppm) are referenced
to the carbon signal for the solvent: CD3OD, δ 49.05. Optical rotation
measurements were taken on a Perkin-Elmer 241 polarimeter with an
aperture of 2 mm using a microcell with 1 cm path length. Aldehyde
dehydrogenase, glycerophosphate dehydrogenase, and triosephosphate
isomerase were purchased from Sigma. For glycosidase inhibitor assays,
samples in 96-well plates were analyzed on a Packard Fusion Universal
Microplate Analyzer spectrophotometer.
Overexpression of His-Tagged FSA in E. coli. The mipB14b gene
encoding FSA was amplified from chromosomal DNA of E. coli W3110
by PCR with following two primers; MipB5Nde, 5′-gatgcatatggaact-
gtatctggatac-3′, and MipB3Xho, 5′-ggcctcgagaatcgacgttctgccaaacg-3′.
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14816 J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007

One-Pot Chemoenzymatic Synthesis of Iminocyclitols
A R T I C L E S
The amplified 660 bp fragment was cloned into the NdeI and XhoI site
of pET20b(+) vector (Novagene) to give pETFSA. E. coli BL21 (DE3)
was transformed with pETFSA, and transformants were cultivated in
LB medium containing 50 µg/mL carbenicillin, 10 µM IPTG at 37 °C
for 16 h. The recombinant FSA was expressed in the soluble fraction
and appeared as a major band on SDS-PAGE. The cells were harvested
by centrifuge, washed with saline, and used as whole cell catalysts.
His-tagged FSA was also purified by Ni2+ affinity column chroma-
tography. Cells were suspended in binding buffer (50 mM glycylglycine
buffer (pH 8.5), 300 mM NaCl, and 10 mM imidazole) and disrupted
by sonication. After removing debris by centrifuge, the soluble fraction
was applied onto a Ni2+ affinity column; the column was washed with
the binding buffer, and His-tagged FSA was eluted with elution buffer
(binding buffer containing 250 mM imidazole). The eluant was collected
and dialyzed against 20 mM HEPES/NaOH buffer (pH 7.5).
Kinetic Assays. A solution of D/L-glyceryaldehyde-3-phosphate was
obtained by deprotection of the commercially available D/L-glyceral-
dehyde-3-phosphate diethylacetal following the manufacturer’s protocol.
Donor substrate (dihydroxyacetone, hydroxy acetone, or 1-hydrox-
ybutan-2-one) was added in amounts varying from 5 to 120 mM to a
solution containing 3 mM D-glyceraldehyde-3-phosphate and 0.03 mg/
mL purified FSA in 50 mM glycylglycine buffer (pH 8.5) plus 1 mM
2-mercaptoethanol at 25 °C. Aliquots of 25 µL of these reactions were
removed every 2 min and quenched by addition to a solution containing
0.23 mM NADH and 8 units/mL R-glycerophosphate dehydrogenase-
triosephosphate isomerase in 50 mM Tris buffer (pH 7.5). Previous
work has shown that FSA is inhibited by Tris buffer, alleviating the
need for a separate quenching step.14b Absorbance at 340 nm was
measured, and consumption of NADH was equated to D-glyceraldehyde-
3-phosphate consumed. By measuring the consumption of d-glyceral-
dehyde-3-phosphate over the course of the reaction, the velocity of
the reaction at varying concentrations of donor was determined. The
kinetic parameters (KM, kcat) were obtained by nonlinear least-squares
curve-fitting using the GRAFIT program (Erithacus Software).
General Procedure for FSA-Catalyzed One-Pot Synthesis of
Iminocyclitols. To a solution of acceptor aldehyde (0.5 mmol) and
donor (DHA 9, hydroxy acetone 10, or 1-hydroxybutan-2-one 11, 1.0
mmol) in 50 mM sodium borate buffer (pH 7.6, 10 mL) were added
toluene (100 µL) and E. coli BL21 (DE3) cells harboring pETFSA
(0.3 g, wet weight). For the racemic acceptor aldehydes (entries 4-7),
acceptor aldehyde (1.0 mmol) and donor (10 or 11, 0.5 mmol) were
used. For the Cbz-protected amino aldehydes 27, 33, and 38 (entries
8-14), aldol condensations were performed in water/DMF (4:1)
cosolvent system instead of water. The reaction mixture was shaken at
room temperature for 6 h, and cells were removed by centrifuge. To
this mixture was added MeOH (10 mL), diethylamine (0.73 g, 10.0
mmol), and Pd(OH)2/C (10%, 50 mg), and the mixture was subjected
to hydrogenation (400 psi H2) overnight at room temperature. After
filtration over Celite and concentration, iminocyclitols were purified
by flash chromatography (CH2Cl2/MeOH/water/NH4OH).
General Procedure for Two-Step Synthesis of Iminocyclitols. The
FSA-catalyzed aldol reactions were carried out in HEPES/NaOH buffer
(100 mM, pH 7.5, 10 mL). After the aldol condensation and removal
of the cells, the enzymatic reaction solution was lyophilized, and the
residue was purified by flash chromatography (silica, CH2Cl2/MeOH)
to afford the azidoketones. The purified azidoketone was then dissolved
in 1.5 mL of MeOH and water (1:1), and Pd(OH)2/C (10%, 50 mg/
mmol substrate) was added. The reaction was hydrogenated (50 psi
H2) at room temperature for 12 h. Filtration over Celite and flash
chromatography (silica, CH2Cl2/MeOH/water/NH4OH) provided imi-
nocyclitols.
Enzymatic Inhibition Assays. Inhibitory activities of synthesized
iminocyclitols were evaluated toward R-glucosidase (from jack beans),
â-glucosidase (from almonds), R-mannosidase (from jack beans),
â-mannosidase (from snail), R-galactosidase (from green coffee beans),
â-galactosidase (from Aspergillus oryzae), R-fucosidase (from bovine
kidney), â-N-acetylglucosaminidase (from jack beans and human
placenta). All enzymes and corresponding p-nitrophenyl glycosides were
purchased from Sigma. Reactions were performed in 100 µL of reaction
mixture containing 50 mM HEPES/NaOH buffer (pH 6.8), 1 mM
p-nitrophenyl glycoside substrates, appropriate amounts (typically 0.5
to 1.0 U/mL) of glycosidases, and 200 µM (or 1 µM) iminocyclitols at
final concentrations, respectively. Increase of absorbance at 405 nm
was determined by a plate reader at 25 °C for 10 min. All assays were
performed in duplicate, and percent inhibition was calculated based
on the activities in the presence or absence of iminocyclitols. For the
compounds showing significant inhibitory activities at 200 µM, assays
were also performed at 1 µM. The Ki values of compounds 8, 24, and
26 were determined from a double reciprocal plot (1/V vs 1/[S]) to
give apparent Km values in the presence of inhibitors. The secondary
plot was generated by plotting the apparent Km values as a function of
inhibitor concentrations. Ki was calculated from the negative value of
the x-intercept of this plot.
Acknowledgment. We are grateful for support from the NIH
(GM044154). L.J.W. thanks the Skaggs Institute for Chemical
Biology for a postdoctoral fellowship. M.S. was supported by
Ajinomoto Co. Inc.
Supporting Information Available: Synthesis of aldehyde
acceptors, characterizations of all new compounds, and scanned
NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA073911I
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J. AM. CHEM. SOC. VOL. 129, NO. 47, 2007 14817