Looking-glass synergistic pharmacological chaperones: DGJ and L-DGJ from the enantiomers of tagatose

Article abstract of DOI:10.1021/ol201552q

The enantiomers of tagatose are converted to l-DGJ [a noncompetitive inhibitor of human lysosome α-galactosidase A (α-Gal A), K _i 38.5 μM] and DGJ [a competitive inhibitor of α-Gal A, K_i 15.1 nM] in 66% yield. l-DGJ and DGJ provide the first examples of pharmacological chaperones that (a) are enantiomeric iminosugars and (b) have synergistic activity with implications for the treatment of lysosomal storage disorders and other protein deficiencies.

Full text of DOI:10.1021/ol201552q

ORGANIC
LETTERS
2011
Vol. 13, No. 15
4064–4067
Looking-Glass Synergistic
Pharmacological Chaperones: DGJ and
L-DGJ from the Enantiomers of Tagatose
Sarah F. Jenkinson,†,‡ George W. J. Fleet,*,†,‡ Robert J. Nash,§ Yuriko Koike,
Isao Adachi, Akihide Yoshihara,^ Kenji Morimoto,^ Ken Izumori,^ and Atsushi Kato*,
Chemistry Research Laboratory, Department of Chemistry, University of Oxford,
Mansfield Road, OX1 3TA, United Kingdom, Oxford Glycobiology Institute, University
of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom, Phytoquest
Limited, Plas Gogerddan Aberystwyth, Ceredigion, SY23 3EB, United Kingdom,
Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan,
and Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki-cho,
Kita-gun, Kagawa 761-0795, Japan
george.fleet@chem.ox.ac.uk; kato@med.u-toyama.ac.jp
Received June 11, 2011
ABSTRACT
The enantiomers of tagatose are converted to L-DGJ [a noncompetitive inhibitor of human lysosome R-galactosidase A (R-Gal A), Ki 38.5 μM] and
DGJ [a competitive inhibitor of R-Gal A, Ki 15.1 nM] in 66% yield. L-DGJ and DGJ provide the first examples of pharmacological chaperones that
(a) are enantiomeric iminosugars and (b) have synergistic activity with implications for the treatment of lysosomal storage disorders and other
protein deficiencies.
This paper reports concise syntheses of L-DGJ 1L and
DGJ 1D from the enantiomers of tagatose in an overall
yield of 66%. L-DGJ 1L and DGJ 1D provide the first ex-
ample of both enantiomers of an iminosugar DGJ 1 acting
as pharmacological chaperones; enantiomeric glycosidase
inhibitors are well-established.
DGJ 1D is a competitive nanomolar- and L-DGJ 1L a
noncompetitive micromolar-inhibitor of a number of R-D-
galactosidases;1 similarly, DFJ 2L (the 6-deoxy analogue
of L-DGJ) is a competitive nanomolar, and its enantiomer 2D
a noncompetitive micromolar, inhibitor of R-L-fucosidases.2
(1) Kato, A.; Kato, N.; Kano, E.; Adachi, I.; Ikeda, K.; Yu, L.;
Okamoto, T.; Banba, Y.; Ouchi, H.; Takahata, H.; Asano, N. J. Med.
Chem. 2005, 48, 2036–2044.
† Department of Chemistry, University of Oxford.
‡ Oxford Glycobiology Institute, University of Oxford.
§ Phytoquest Limited.
ꢀ
€
(2) Bleriot, Y.; Gretzke, D.; Krulle, T. M.; Butters, T. D.; Dwek,
R. A.; Nash, R. J.; Asano, N.; Fleet, G. W. J. Carbohydr. Res. 2005, 340,
2713–2718.
University of Toyama.
^ Kagawa University.
r
10.1021/ol201552q
Published on Web 07/11/2011
2011 American Chemical Society

Figure 1. Enantiomeric iminosugar inhibitors of the same enzymes.
Many enantiomeric pairs of iminosugars inhibit the same
glycosidases; the natural products DMDP 3D and DAB
4D, and 4-C-methyl DAB 5D are competitive inhibitors of
D-glucosidases, whereas their L-enantiomers L-DMDP 3L,3
LAB 4L4 and 4-C-methyl-LAB 5L5 are more potent
noncompetitive inhibitors of the same enzymes (Figure 1).
molecule pharmacological chaperones to assist protein
folding and stabilization of their native conformation.8
Several iminosugars, including Miglustat 6 and isofago-
mine 7,9 are being tested as chaperones for Gaucher’s
disease (Figure 2);10,11 6 and LABNAc 8 have potential
as chaperones for late-onset Tay-Sachs disease.12 Also, 6
and isoLAB 913 partially rescue the defective F508del-
CFTR function in CF-KM4 cells14 and may provide a
strategy for the chemotherapy of cystic fibrosis.15
In 1999, Fan put forward the concept of active site-
specific chaperone-mediatedtherapy for lysosomal storage
diseases, using iminosugars to prevent misfolding and pre-
mature degradation of mutant enzymes in the endoplasmic
reticulum.16 DGJ [Amigal] 1D is a chaperone for the treat-
ment of Fabry’s disease, a lysosomal storage disorder
caused by an X-linked inherited deficiency of R-galactosi-
dase A (R-Gal A; EC 3.2.1.22). Deficiency of R-Gal A
activity results in the progressive accumulation of globo-
triaosylceramide (Gb3) in the lysosomes of vascular en-
dothelial cells.17 The severity of the disease is well correlated
Figure 2. Examples of iminosugars as pharmacological chaperones.
Iminosugars6 have a wide range of chemotherapeutic
applications,7 including their ability to act as small
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Org. Lett., Vol. 13, No. 15, 2011
4065

with the residual enzyme activity. Residual R-Gal A ac-
tivity in lymphoblasts derived from Fabry patients and in
tissues of R301Q or Q279E R-Gal A transgenic mice was
enhanced by treatment with DGJ [Amigal] 1D, a compe-
titive inhibitor of R-Gal A.18 DGJ acts as a pharmacolo-
gical chaperone thatstabilizesthenativefolding state inthe
endoplasmic reticulum (ER) by occupying the active site of
the mutant enzyme, thus allowing its maturation and traf-
ficking to the lysosome.19 Clinical trials of Amigal in patients
with Fabry disease are encouraging; other piperidine20 and
pyrrolidine inhibitors of R-Gal A, such as 10,21 are also
being evaluated. This paper reports that L-DGJ 1L shows
activity as a pharmacological chaperone and provides the
first example of both enantiomers of an iminosugar acting
as chaperones, and that its mode of action is synergistic
with that of DGJ. Concise syntheses of 1L and 1D from the
enantiomers of tagatose are also described
of more complex targets.25 L-DGJ 1L has previously been
prepared by multistep syntheses from Garner’s aldehyde.1,26
Reaction of D-tagatose 11D with acetone, copper(II)
sulfate and catalytic sulfuric acid gave the diacetonide 12D
22
[mp 55ꢀ58 ꢀC; [R]D þ66.3 (c, 1.04)]27 in 82% yield.
Esterification of the primary hydroxyl group in 12D with
triflic anhydride in dichloromethane in the presence of
pyridine gave the crystalline triflate 13D [mp 44ꢀ46 ꢀC;
22
[R]D þ43.6 (c, 0.89)] which, on treatment with sodium
22
azide in DMF, afforded the azide 14D [oil, [R]D þ44.5
(c, 1.01)] in 96% yield for the two steps. Removal of the
acetonide protecting groups in 14D with Dowex resin (50W
X8, Hþ) in water proceeded slowly to give 6-azidotagatose
21
15D [oil [R]D þ15.6 (c, 1.0 in MeOH)] as a 3:1 mixture of
anomers after 3 days in 86% yield; heating the reaction
mixture led to extensive decomposition. Hydrogenation of
the azide gave the corresponding amine which underwent a
highly stereoselective intramolecular reductive amination
25
to afford L-DGJ 1L [R]D ꢀ9.2 (c, 0.43 in H2O) in 97%
25
yield; for 1L as the HCl salt [R]D ꢀ54.8 (c, 0.16 in H2O).28
25
Scheme 1. Enantiomers of DGJ 1 from Tagatose 11
The enantiomer, DGJ 1D, {[R]D þ9.0 (c, 0.16 in H2O);
1D as HCl salt29 [R]D þ53.7 (c, 0.1 in H2O)} was synthe-
sized by an identical route from L-tagatose 11L.30 L-Taga-
tose, a less available hexose than its enantiomer 11D, is ac-
cessible by green biotechnology from L-psicose,31 L-sorbose,32
or galactitol.33
The biological activity of L-DGJ 1L was compared with
DGJ 1D. The inhibition constant (Ki) and the mode of
inhibition of DGJ and L-DGJ were determined by Line-
weaverꢀBurk plots. Whereas DGJ was a competitive
inhibitor of R-Gal A (Ki 15.1 nM), L-DGJ showed noncom-
petitive inhibition about ∼1000 times weaker (Ki 38.5 μM).
L-DGJ showed enhancement of activity in Fabry R301Q
fibroblasts. Treatment with L-DGJ for 3 days dose-
dependently increased intracellular R-Gal A activity, with
maximal increase of 10.8-fold at 10 mM (Figure 3B).
(25) Jones, N. A.; Rao, D.; Yoshihara, A.; Gullapalli, P.; Morimoto,
K.; Takata, G.; Hunter, S. J.; Wormald, M. R.; Dwek, R. A.; Izumori,
K.; Fleet, G. W. J. Tetrahedron: Asymmetry 2008, 19, 1904–1918.
(26) Karjalainen, O. K.; Passiniemi, M.; Koskinen, A. M. P. Org.
Lett. 2010, 12, 1145–1147.
The synthesis of L-DGJ 1L from D-tagatose 11D in 66%
overall yield requires introduction of an azide at C-6 fol-
lowed by an intramolecular reductive amination with the
carbonyl at C-2 (Scheme 1). D-Tagatose, until recently a
rare and expensive sugar, is now readily available from
galactose22 as a food substitute in soft drinks and ready to
eat cereals;23 it has been used as a chiron24 for the synthesis
(27) All specific rotations were determined using CHCl3 as solvent
unless otherwise indicated.
(28) Selected data for L-DGJ 1L: HRMS (ESIþve) found: 164.0917
(M þ Hþ); C6H14NO4 requires: 164.0917; [R]D ꢀ9.2 (c, 0.43 in H2O);
25
ν
max (thin film): 3384 (s, br, OH/NH); δC (D2O, 100 MHz): 49.6 (CH2),
59.4 (CH), 61.9 (CH2), 68.7 (CH), 69.8 (CH), 75.7 (CH); m/z (ESI þ ve):
164 (M þ Hþ, 100%). For L-DGJ 1L as HCl salt: [R]D ꢀ54.8 (c, 0.16 in
25
(18) Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.;
Martin, O. R.; Fan, J. Q. Eur. J. Biochem. 2000, 267, 4179–4186.
(19) Hamanaka, R.; Shinohara, T.; Yano, S.; Nakamura, M.; Yasuda,
A.; Yokoyama, S.; Fan, J. Q.; Kawasaki, K.; Watanabe, M.; Ishii, S.
Biochim. Biophys. Acta 2008, 1782, 408–413.
(20) Benjamin, E. E.; Flanagan, J. J.; Schilling, A.; Chang, H. H.;
Agarwal, L.; Katz, E.; Wu, X.; Pine, C.; Wustman, B.; Desnick, R. J.;
Lockhart, D. J.; Valenzano, K. J. J. Inherit. Metabol. Dis. 2009, 32, 424.
(21) Kato, A.; Yamashita, Y.; Nakagawa, S.; Koike, Y.; Adachi, I.;
Hollinshead, J.; Nash, R. J.; Ikeda, K.; Asano, N. Bioorg. Med. Chem.
2010, 18, 3790–3794.
(22) (a) Beadle, J. R.; Saunders, J. P.; Wajda, T. J. Process for
Manufacturing tagatose, US Patent 5,078796, January 7, 1992. (b) Rhimi,
M.; Chouayekh, H.; Gouillouard, I.; Maguin, E.; Bejar, S. Bioresour.
Technol. 2011, 102, 3309–3315.
(23) Skytte, U. P. Cereal Foods World 2002, 47, 224–227.
(24) Soengas, R.; Izumori, K.; Simone, M. I.; Watkin, D. J.; Skytte,
U. P.; Soetart, W.; Fleet, G. W. J. Tetrahedron Lett. 2005, 46, 5755–5759.
H2O); δH (D2O, 400 MHz): 2.88 (1H, a-t, H1, J 12.0), 3.40ꢀ3.45 (1H,
ddd, H5, J 0.8, 4.9, 8.8), 3.48ꢀ3.54 (1H, dd, H10, J 5.4, 12.5), 3.62ꢀ3.68
(1H, dd, H3, J 3.0, 9.7), 3.78ꢀ3.84 (1H, dd, H6, J 8.8, 12.2), 3.85ꢀ3.92
(1H, dd, H60, J 4.9, 12.2), 4.03ꢀ4.12 (1H, ddd, H2, J 5.4, 9.7, 11.4),
4.15ꢀ4.19 (1H, dd, H4, J 1.2, 2.9); δC (D2O, 100 MHz): 46.6 (C1), 59.6
(C6), 60.6 (C5), 65.2 (C2), 67.4 (C4), 72.4 (C3).
(29) Johnson, C. R.; Golebiowski, A.; Sundram, H.; Miller, M. W.;
Dwaihy, R. Tetrahedron Lett. 1995, 36, 653–654.
(30) Full details of the synthesis are given in the Supporting
Information.
(31) Rao, D.; Gullapalli, P.; Yoshihara, A.; Morimoto, K.; Takata,
G.; Jenkinson, S. F.; Fleet, G. W. J.; Izumori, K. J. Biosci. Bioeng. 2008,
106, 473–480.
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Granstrom, T. B.; Takada, G.; Izumori, K. J. Biosci. Bioeng. 2004, 97,
383–388. (b) Itoh, H.; Izumori, K. J. Ferment. Bioeng. 1996, 81, 351–353.
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1995, 79, 620–622.
4066
Org. Lett., Vol. 13, No. 15, 2011

Figure 3. Influence of DGJ 1D and L-DGJ 1L on R-Gal A activity in Fabry R301Q fibroblasts. (A) DGJ 1D was added to the culture
medium of R301Q cells at a concentration of 0.1ꢀ100 μM. (B) L-DGJ was added to the culture medium of R301Q cells at a
concentration of 10ꢀ10000 μM. (C) Combination of DGJ (0.1ꢀ10 μM) and L-DGJ (10ꢀ1000 μM). Cells were subsequently incubated
for 3 days; cell growth was not affected by the inclusion of DGJ and L-DGJ.
This enhancement of activity was similar to that observed
with 10 μM DGJ (Figure 3A). The in vitro inhibitory effect
of L-DGJ on human R-Gal A was ∼1000 times less than
DGJ. Wefurtherinvestigatedwhethera combination effect
was manifested upon Fabry R301Q fibroblasts when DGJ
(a competitive inhibitor) and L-DGJ (a noncompetitive
inhibitor) were administrated simultaneously. Very inter-
estingly, each enantiomer did not counteract the chaperone
effects of the other enantiomer but clearly showed doseꢀ
response synergistic effects (Figure 3C). These results indicate
that L-DGJ could be of therapeutic benefit to Fabry disease,
and that the combination of the competitive inhibitors and
noncompetitive inhibitors may be broadly applicable to
lysosomal storage disorders and other protein deficiencies.
In summary, this paper describes the concise synthesis of
DGJ 1D and L-DGJ1Lfrom L- and D-tagatose respectively
injustfour steps, and showsthe valueofthe enantiomersof
tagatose as chirons. As with other R-galactosidases L-DGJ
was found to be an inhibitor of R-Gal A, about 1000-fold
weaker than its enantiomer DGJ. L-DGJ was also found to
be a chaperone for R-Gal A, again about 1000 times less active
than DGJ. Noncompetitive inhibition by 1L is consistent
with binding at a different site than 1D which shows com-
petitive inhibition and binds at the active site. While the
inhibition of the same glycosidase by both enantiomers of
an iminosugar is well-known, this is the first example of
enantiomeric iminosugars acting as synergistic pharmaco-
logical chaperones for misfolded proteins; this result may
indicate that binding at the active site of the enzyme is not
necessary.
Acknowledgment. This work was supported in part by
the Program for Promotion of Basic Research Activities
for Innovative Biosciences (PROBRAIN) and Grant-in-
Aid for Scientific Research (C) from the Japanese Society
for the Promotion of Science (JSPS). We acknowledge
Ousty members for their help with microbial transforma-
tions.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
Org. Lett., Vol. 13, No. 15, 2011
4067