1-Deoxygalactonojirimycin-lysine hybrids as potent d-galactosidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.10.054

Cyclization by double reductive amination of l-arabino-hexos-5-ulose with suitably protected d- as well as l-lysine derivatives provided 1-deoxygalactonojirimycin lysine hybrids without any observable epimer formation at C-5. Modifications on the lysine m

Full text of DOI:10.1016/j.bmc.2008.10.054

Bioorganic & Medicinal Chemistry 16 (2008) 10216–10220
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-Deoxygalactonojirimycin-lysine hybrids as potent D-galactosidase inhibitors
a
b
Andreas J. Steiner a, Georg Schitter a, Arnold E. Stütz a, , Tanja M. Wrodnigg , Chris A. Tarling ,
*
Stephen G. Withers b, Katrin Fantur c, Don Mahuran d, Eduard Paschke c, Michael Tropak d
a Glycogroup, Institut für Organische Chemie, Technische Universität Graz, Stremayrgasse 16, A-8010 Graz, Austria
b Chemistry Department, University of British Columbia, 300-6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3
c Department of Pediatrics, Medical University of Graz, Auenbruggerplatz 30, A-8010 Graz, Austria
d Department of Laboratory Medicine and Pathobiology, Sick Kids Hospital, 555 University Avenue, University of Toronto, Ont., Canada M5G 1X8
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclization by double reductive amination of L-arabino-hexos-5-ulose with suitably protected D- as well
Received 24 September 2008
Revised 23 October 2008
Accepted 24 October 2008
Available online 26 October 2008
as
formation at C-5. Modifications on the lysine moiety by acylation gave access to lipophilic derivatives
which exhibited excellent -galactosidase inhibitory activities.
L-lysine derivatives provided 1-deoxygalactonojirimycin lysine hybrids without any observable epimer
D
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Iminoalditol
N-alkylation
Glycosidase inhibitor
Galactosidase inhibitor
Iminoalditol-amino acid hybrid
1. Introduction
preparation of glycosidase-recognizing arrays by immobilization
on surfaces we envisaged more convenient properties with com-
pounds providing a suitably positioned amine for tagging as well
as an additional ‘handle’ for chain-extensions.7
Relying on recent literature that lipophilic derivatives of various
iminoalditols such as N-butyl- (1a) and N-nonyl-1-deoxynojirimy-
cin (1b), as well as other examples such as adamantyl substituted
compounds8 have shown promise as molecular chaperones9 for the
experimental treatment of various lysosomal storage disease re-
lated cell lines, we have now extended our range of compounds
to meet structural requirements for these interesting applications.
Iminoalditols are well-known, (usually) competitive, glycosi-
dase inhibitors.1 Representatives of this class of compounds, for
example, glucosidase inhibitor 1, mannosidase inhibitor 2 as well
as galactosidase inhibitor 3 (Fig. 1) have found important roles as
biological probes such as in the investigation of glycoprotein trim-
ming glycosidases.2
Various N-alkylated derivatives including the N-butyl (1a),
N-nonyl (1b) and N-hydroxyethyl (1c) derivatives of compound 1
and the N-nonyl analogue (3a) of inhibitor 3 (Fig. 1) have become
pharmaceutical agents in the treatment of diabetes type II symp-
toms and other metabolic disorders including Gaucher’s disease.3
Earlier, it had been demonstrated that immobilized N-alkylated
iminoalditols can be employed as affinity ligands in glycosidase
isolation and purification protocols.4
2. Results and discussion
Initially pyranoid ulososide 4, prepared by Barili’s method10
was employed as the sugar component but isolated yields of this
compound ranging around 30% did not match our expectations.
Searching for a better yielding approach, acid treatment of com-
pound 4 in benzyl alcohol gave 5-ulofuranoside 5 (Scheme 1) in
essentially quantitative yield.
This turned out to be a very efficient reaction partner for the
amines employed in the catalytic hydrogenation/reductive amina-
tion step (Scheme 2).
Following up on our observation that some fluorescently la-
beled derivatives of the
D-glucosidase inhibitor 2,5-dideoxy-2,5-
imino-
D
-mannitol (DMDP) are powerful inhibitors exceeding the
parent compound’s activity by two orders of magnitude5 we have
reported the syntheses and glycosidase inhibitory activities of var-
ious pyranoid N-alkylated iminoalditols featuring fluorescent tags
such as dansyl moieties attached to simple N-substituents.6 Based
on their encouraging inhibitory activities and with a view to the
As amine components for the double reductive amination step,
partially protected D- as well as L-lysine derivatives featuring one
free amino group proved to be useful reaction partners in reactions
* Corresponding author. Tel.: +43 316 873 8244; fax: +43 316 873 8740.
E-mail address: stuetz@tugraz.at (A.E. Stütz).
with 5-ulohexoses11 to provide lysine-iminoalditol hybrids12
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.054

A. J. Steiner et al. / Bioorg. Med. Chem. 16 (2008) 10216–10220
10217
NMR. Likewise, reaction with tripeptide C gave exclusively
to derivative 8, albeit in 67% isolated yield.
D
-galac-
HO
HO
HO
HO
HO
HO
NR
NH
NR
Compounds 6, 7, as well as 8 were smoothly converted into the
corresponding N-dansyl derivatives by standard BOC removal
employing methanolic HCl and subsequent reaction of the free
amino group with dansyl chloride to give, after preparative TLC,
fluorescent inhibitors 6a, 7a, and 8a, respectively (Scheme 2).
For comparison in the glycosidase inhibition assays, D-lysine
derivative 9, was also prepared (89%) and converted into N-dansyl
derivative 9a (69%).
HO
OH
HO
OH
HO
OH
1
R = H
R = n-Bu
R = n-Non
R = (CH2)2OH
2
3
3a
R = H
R = n-Non
1a
1b
1c
Reaction of compound 5 with the a-amine of suitably protected
Figure 1. Structures of compounds 1–3.
lysine E gave (Scheme 3) an inseparable mixture of the iminosugar
substituted lysine methyl ester 10 (42%) and the lactone 11 (20%).
Conventional BOC removal and N-dansylation gave the corre-
sponding mixture of fluorescent derivatives 10a and 11a which
for analytical purposes was converted into stable amide 12 by
treatment with aqueous ammonia.
Inhibitory activities of the new compounds against a standard
b-galactosidase that also has b-glucosidase activity (Agrobacterium
sp. b-glycosidase) are summarized in Table 1. As previously6
observed, the presence of a dansyl residue was found beneficial
for inhibitory potencies when compared to the activity of unsubsti-
tuted parent compound 3. Conveniently, the second functional
‘handle’, the terminal carboxylic moiety of the amino acid, allows
for attachment, via linkers, to suitable surfaces.
OB
n
O
BnO
HO
O
O
OBn
OBn
BnO
OMe
HO
HO
OBn
4
5
Scheme 1. Rearrangement of compounds 4–5.
(Scheme 2) ready for a wide range of interesting follow-up
modifications.13
In preliminary experiments, compound 7a which also shows
inhibitory activity against human lysosomal b-galactosidase (IC50
,
Despite the complexity of this hydrogenation/reductive amina-
tion step which includes deprotection of both, the sugar unit as
well as the terminal lysine amino group followed by reductive ami-
nation at C-1 and subsequent intramolecular cyclization with for-
mation of a chiral center at C-5, isolated yields were fair and the
7.8 M) has exhibited significant improvements of the b-galactosi-
l
dase activity in lysosomal b-galactosidase deficient cell lines.
Detailed results will be published in due course.
stereoselectivities for the desired
D-galacto configuration were
3. Experimental
excellent in all cases probed due to the favorable influence of the
C-4 substituent.13
3.1. General methods
Thus, compound 5, with 6-N-benzyloxycarbonyl-2-N-BOC
methyl lysinate A as well as with the chain extended amide B gave
iminogalactitol derivatives 6 (70%) and 7 (73%), respectively. In
both cases, epimer formation at C-5 could not be detected by
Optical rotations were measured on a Perkin Elmer 341 polarim-
eter at the wavelength of 589 nm and a path length of 10 cm at
20 °C. NMR spectra were recorded on a Varian INOVA 500 operating
R
R
HN
O
HN
O
HO
HO
HO
HO
N
OM
e
N
NH
(CH2)5COOMe
6
6a
R = BOC
R = Dansyl
HO
OH
HO
OH
7
7a
R = BOC
R = Dansyl
+ BOC-L-Lys(Z)-NH(CH2)5COOMe
+ BOC
-
L
-Lys(Z)-OMe
O
B
O
A
OBn
OBn
BnO
HO
+ BOC
-
D
-Lys(Z)-OMe
+ BOC-
L-Pro-
L-Lys(Z)-L-Ala-OMe
5
C
D
R
N
R
HN
O
O
HO
HO
HO
HN
O O
HN
R = BO
N
OMe
HO
HO
HO
OMe
N
OH
8
8a
C
9
9a
R = BOC
R = Dansyl
OH
R = Dansyl
Scheme 2. Synthesis of iminoalditol-lysine hybrids 6–9.

10218
A. J. Steiner et al. / Bioorg. Med. Chem. 16 (2008) 10216–10220
HO
OH
OH
OM
O
HO
NH
N
e
R
10 R = BOC
HO
HO
OH
10a
O
+ H-
L
-Lys(BOC)-OMe
R = Dansyl
O
OH
NH2
O
OBn
OBn
E
BnO
+
N
HO
HO
OH
Dansyl NH
5
HO
12
N
O
O
R NH
11 R = BOC
11a
R = Dansyl
Scheme 3. Synthesis of iminoalditol-lysine hybrid 12.
Table 1
to 0.6 mM. Data were analyzed by direct fit to the Michaelis–Men-
ten equation describing reaction in the presence of inhibitors using
the program GraFit.
Inhibitory activities of compounds with Agrobacterium sp. b-glycosidase.
Compound
Ki [lm]
Compound
Ki [l
m]
Compound
Ki [lm]
The inhibitory activity of compound 7a with human lysosomal
b-galactosidase was evaluated using 4-nitrophenyl b-D-galactopy-
ranoside (5 mM) as the substrate and a lysosomal enzyme enriched
fraction derived from human placenta15 as the enzyme source.
Reactions were performed at 37 °C in pH 4.5 citrate phosphate buf-
fer (100 mM).
3
8
6a
100
1900
3.1
7a
8a
10
140
9a
12
21
170
at 500.619 MHz (1H), and at 125.894 MHz (13C). CDCl3 was em-
ployed for protected compounds and methanol-d4 for unprotected
inhibitors. Chemical shifts are listed in delta employing residual,
non-deuterated solvent as the internal standard. The signals of
the protecting groups were found in the expected regions and are
not listed explicitly. Electrospray mass spectra were recorded on
an HP 1100 series MSD, Hewlett Packard. Samples were dissolved
in acetonitrile or acetonitrile/water mixtures. The scan mode for
positive ions (mass range 100–1000 D) was employed varying the
fragmentation voltage from 50 to 250 V with best molecular peaks
observed at 150 V. MALDI mass spectra were recorded on a MALDI
Micro MX (Waters) time-of-flight instrument used in reflectron
mode with 2.3 m effective flight path. Analytical TLC was per-
formed on precoated aluminum plates silica gel 60 F254 (E. Merck
5554), detected with UV light (254 nm), 10% vanillin/sulfuric acid
as well as ceric ammonium molybdate (100 g ammonium molyb-
date/8 g ceric sulfate in 1 L 10% H2SO4) and heated on a hotplate.
Preparative TLC was performed on precoated glass plates silica gel
60 F254, 0.5 mm (E. Merck 5744). For column chromatography sil-
ica gel 60 (230–400 mesh, E. Merck 9385) was used.
3.3. General intramolecular reductive amination procedure
To a 0.03 m solution of 5 in MeOH/H2O (15:1, v/v), Pd(OH)2/C
(20%, 0.1 equiv) was added and the heterogeneous reaction mix-
ture was stirred under an atmosphere of hydrogen at ambient
pressure and room temperature for 1 h. One equivalent of the
respective peptide component was added and the reaction was
continued for 24 h. After filtration and removal of the solvent un-
der reduced pressure, the residue was purified by column chroma-
tography on silica gel (CHCl3/MeOH/concd NH4OH, 500:100:6, v/v/
v), yielding the target compound as a colorless syrup. The forma-
tion of L-altro-configured products was not observed in any of
the reported cases.
3.4. General procedure for BOC removal and N-dansylation
A 0.01 m solution of the respective BOC-protected iminosugar-
amino acid hybrid in MeOH/AcCl (30:1, v/v, prepared at 0 °C
10 min before use) was stirred for 20 h at ambient temperature.
The solvents were removed under reduced pressure and the resi-
due was dissolved in dry DMF, to give a 0.01 m solution. Triethyl-
amine (5 equiv) and dansyl chloride (1.1 equiv) were added, and
the resulting reaction mixture was stirred in a brown flask for
4 h at ambient temperature. Removal of the solvent under reduced
pressure and purification of the residue by preparative TLC (silica
gel, CHCl3/MeOH/concd NH4OH, 300:100:4, v/v/v, extraction with
MeOH) gave the desired target compound.
3.2. Kinetic studies
Agrobacterium sp. b-galactosidase/-glucosidase was purified and
assayed as described.14 Kinetic studies were performed at 37 °C in
pH 7.0 sodium phosphate buffer (50 mM) containing 0.1% bovine
serum albumin, using 7.2 ꢀ 10ꢁ5 mg/mL enzyme. Approximate val-
ues of Ki were determined using a fixed concentration of substrate,
4-nitrophenyl b-
D
-glucopyranoside (0.11 mM = 1.5 ꢀ Km) and
inhibitor concentrations ranging from 0.2 times to 5 times the Ki va-
lue ultimately determined. A horizontal line drawn through 1/Vmax
in a Dixon plot of this data (1/V vs [I]) intersects the experimental
line at an inhibitor concentration equal to ꢁKi. Full Ki determina-
tions where required, were performed using the same range of
inhibitor concentrations while also varying substrate (4-nitro-
phenyl glucoside) concentrations from approximately 0.015 mM
3.5. Benzyl 2,6-di-O-benzyl-b-L-arabino-hexofuranoside-5-
ulose (5)
A mixture of methyl (5R)-2,6-di-O-benzyl-5-C-benzyloxy-
a-L-
arabino-hexopyranoside (4) (2.86 g, 5.95 mmol), benzyl alcohol

A. J. Steiner et al. / Bioorg. Med. Chem. 16 (2008) 10216–10220
10219
(10 mL, 96 mmol) and p-toluenesulfonic acid monohydrate (1.0 g,
5.3 mmol) in CH2Cl2 (10 mL) was stirred at room temperature for
24 h. CH2Cl2 was added and the organic phase was washed with
satd aqueous NaHCO3, dried over Na2SO4 and filtered. Removal of
the solvents under reduced pressure and purification of the residue
by column chromatography on silica gel (cyclohexane/ethyl ace-
tate, 4:1, v/v) gave pure 5 (2.63 g, 5.86 mmol, 99%) as colorless
[C26H46N4O10]: m/z 574.677; ESIMS found: [M+H]+ 575.70,
[M+Na]+ 597.69.
3.9. Methyl N2-tert-butyloxycarbonyl-N6-(1,5-dideoxy-1,5-imino-
D-galactitol-1,5-diyl)-D-lysinate (9)
Following Section 3.3, compound 5 (210 mg, 0.468 mmol) was
crystals; mp 92–93 °C; ½a D20
ꢂ
+87.2 (c = 1.3, CH2Cl2); 1H NMR
reacted with BOC-
D-Lys(Z)-OMe (D, 1.25 equiv) to give iminoaldi-
(500 MHz, CDCl3): d = 4.99 (d, J1,2 = 4.4 Hz, 1H; H-1), 4.56 (dd,
J2,3 = 7.3 Hz, J3,4 = 6.3 Hz, 1H; H-3), 4.43 (d, J6a,6b = 18.1 Hz, 1H; H-
6a), 4.39 (d, 1H; H-4), 4.35 (d, 1H; H-6b), 3.91 (dd, 1H; H-2). 13C
NMR (125 MHz, CDCl3): d = 206.2 (C-5), 100.0 (C-1), 86.0, 83.1 (C-
2, C-3), 75.8 (C-4), 69.9 (C-6). Anal. Calcd for C27H28O6: C, 72.30;
H, 6.29. Found: C, 72.23; H, 6.35. MS: Calcd for [C27H28O6]: m/z
448.521; ESIMS found: [M+H]+ 449.57, [M+Na]+ 471.52.
tol (145 mg, 76%):
9
½
a 2D0
ꢂ
ꢁ5.8 (c = 2.4, MeOH); 1H NMR
(500 MHz, CD3OD): d = 4.09 (m, 1H), 3.98 (dd, J3,4 = 2.9 Hz,
J4,5 = 1.5 Hz, 1H; H-4), 3.80 (m, 3H; H-2, H-6a, H-6b), 3.23 (dd,
J2,3 = 9.3 Hz, 1H; H-3), 2.99 (dd, J1eq,1ax = 11.2 Hz, J1eq,2 = 4.9 Hz,
1H; H-1eq), 2.72 (m, 1H), 2.51 (m, 1H), 2.40 (m, 1H; H-5), 2.14
(dd, J1ax,2 = 10.3 Hz, 1H, H-1ax), 1.78 (m, 1H), 1.65 (m, 1H), 1.56–
1.28 (m, 4H); 13C NMR (125 MHz, CD3OD): d = 173.9 (C@O), 76.0
(C-3), 71.0 (C-4), 67.7 (C-2), 64.1 (C-5), 61.2 (C-6), 56.7 (C-1),
53.8, 52.6, 31.4, 23.7, 23.5. MS: Calcd for [C18H34N2O8]: m/z
406.480; ESIMS found: [M+H]+ 407.50, [M+Na]+ 429.58.
3.6. Methyl N2-tert-butyloxycarbonyl-N6-(1,5-dideoxy-1,5-imino-
D-galactitol-1,5-diyl)-L-lysinate (6)
3.10. Methyl N6-tert-butyloxycarbonyl-N2-(1,5-dideoxy-
titol-1,5-diyl)- -lysinate (10) and lactone 11
D-galac-
Following general procedure 3.3., compound
5
(135 mg,
L
0.301 mmol) was reacted with BOC-
L-Lys(Z)-OMe (A, 1.3 equiv)
ꢂ
to give iminoalditol 6 (86 mg, 70%): ½a D20
ꢁ16.2 (c = 0.9, MeOH);
1H NMR (500 MHz, CD3OD): d = 4.09 (m, 1H), 3.98 (dd,
J3,4 = 3.4 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.80 (m, 3H; H-2, H-6a, H-6b),
3.22 (dd, J2,3 = 9.3 Hz, 1H; H-3), 2.98 (dd, J1eq,1ax = 11.2 Hz,
J1eq,2 = 4.9 Hz, 1H; H-1eq), 2.73 (m, 1H), 2.50 (m, 1H), 2.40 (m,
1H; H-5), 2.14 (dd, J1ax,2 = 10.7 Hz, 1H, H-1ax), 1.77 (m, 1H), 1.66
(m, 1H), 1.56–1.30 (m, 4H); 13C NMR (125 MHz, CD3OD):
d = 173.9 (C@O), 76.0 (C-3), 71.1 (C-4), 67.8 (C-2), 64.1 (C-5), 61.3
(C-6), 57.1 (C-1), 53.8, 52.5, 31.3, 23.6, 23.5. MS: Calcd for
[C18H34N2O8]: m/z 406.480; ESIMS found: [M+H]+ 407.51,
[M+Na]+ 429.50.
Following Section 3.3, compound 5 was reacted with H-L-Lys
(BOC)-OMe (E) to give an inseparable mixture of iminoalditol 10
and the corresponding lactone 11 in a ratio of 2:1. Compound
10: 1H NMR (500 MHz, CD3OD): d = 3.95 (m, 1H; H-4), 3.90–3.78
(m, 3H; H-2, H-6a, H-6b), 3.70 (m, 1H), 3.20 (m, 1H; H-3), 3.04
(m, 3H), 2.68 (m, J4,5 = 1.5 Hz, 1H; H-5), 2.21 (dd, J1eq,1ax = 11.2 Hz,
J1ax,2 = 10.7 Hz, 1H; H-1ax), 1.85–1.32 (m, 6H); 13C NMR (125 MHz,
CD3OD): d = 173.5 (C@O), 75.3 (C-3), 71.7 (C-4), 68.5 (C-2), 65.3 (C-
5), 62.0, 61.5 (C-1, C-6), 52.9, 40.1, 29.7, 29.5, 22.7. MS: Calcd for
[C18H34N2O8]: m/z 406.480; ESIMS found: [M+H]+ 407.55,
[M+Na]+ 429.60. Lactone 11: 1H NMR (500 MHz, CD3OD): Charac-
teristic signals: d = 4.60 (dd, J5,6a = 4.0 Hz, J6a,6b = 11.7 Hz, 1H; H-
6a), 4.53 (dd, J5,6b = 4.9 Hz, 1H; H-6b); 13C NMR (125 MHz, CD3OD):
d = 172.8 (C@O), 76.5 (C-3), 72.0 (C-4), 69.2, 69.1, 63.0, 59.7 (C-1, C-
2, C-5, C-6), 53.2, 39.9, 29.7, 29.5, 23.4.
3.7. Methyl 6-[N2-(tert-butyloxycarbonyl)-N6-(1,5-dideoxy-
titol-1,5-diyl)- -lysinyl] aminohexanoate (7)
D-galac-
L
Following Section 3.3, compound 5 (175 mg, 0.39 mmol) was
reacted with BOC-L-Lys(Z)-NH-(CH2)5CO2Me (B, 1.2 equiv) to give
3.11. Methyl N2-dansyl-N6-(1,5-dideoxy-
-lysinate (6a)
D-galactitol-1,5-diyl)-
iminoalditol 7 (140 mg, 69%): ½a D20
ꢂ
ꢁ15.9 (c = 1.1, MeOH); 1H
L
NMR (500 MHz, CD3OD): d = 3.97 (dd, J3,4 = 3.4 Hz, J4,5 = 1.5 Hz,
1H; H-4), 3.95 (m, 1H), 3.80 (m, 3H; H-2, H-6a, H-6b), 3.21 (dd,
J2,3 = 9.3 Hz, 1H; H-3), 3.25–3.12 (m, 2H), 2.98 (dd,
J1eq,1ax = 11.2 Hz, J1eq,2 = 4.9 Hz, 1H; H-1eq), 2.72 (m, 1H), 2.50 (m,
1H), 2.38 (m, 1H; H-5), 2.32 (t, 2H), 2.11 (dd, J1ax,2 = 10.7 Hz, 1H,
H-1ax), 1.75–1.26 (m, 12H); 13C NMR (125 MHz, CD3OD):
d = 174.6, 174.0 (2 C@O), 76.1 (C-3), 71.1 (C-4), 67.8 (C-2), 64.2
(C-5), 61.3 (C-6), 56.8 (C-1), 55.0, 52.6, 38.9, 33.5, 32.2, 28.9,
26.2, 24.5, 23.7, 23.6. MS: Calcd for [C24H45N3O9]: m/z 519.641;
ESIMS found: [M+H]+ 520.66, [M+Na]+ 542.70.
Following Section 3.4, compound 6 (47 mg, 0.12 mmol) gave
fluorescent iminoalditol-lysine hybrid 6a (24 mg, 38%): ½a D20
ꢁ3.8
ꢂ
(c = 1.0, MeOH); 1H NMR (500 MHz, CD3OD): d = 3.95 (dd,
J3,4 = 3.4 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.76 (ddd, J1eq,2 = 4.9 Hz,
J1ax,2 = 10.3 Hz, J2,3 = 9.3 Hz, 1H; H-2), 3.73 (m, 1H), 3.70 (m, 2H;
H-6a, H-6b), 3.18 (dd, 1H; H-3), 2.82 (dd, J1eq,1ax = 11.2 Hz, 1H;
H-1eq), 2.40 (m, 1H), 2.30 (m, 1H), 2.27 (m, 1H; H-5), 1.99 (dd,
1H; H-1ax), 1.58–1.52 (m, 2H), 1.24–0.96 (m, 4H); 13C NMR
(125 MHz, CD3OD): d = 172.9 (C@O), 76.1 (C-3), 71.0 (C-4), 67.8
(C-2), 63.7 (C-5), 61.1 (C-6), 56.8 (C-1), 56.1, 52.2, 32.2, 23.1,
22.7. MS: Calcd for [C25H37N3O8S]: m/z 539.65; ESIMS found:
[M+H]+ 540.0, [M+Na]+ 562.2.
3.8. Methyl N-(tert-butyloxycarbonyl)-
-galactitol-1,5-diyl)- -lysinyl- -alaninate (8)
L
-prolinyl-N6-(1,5-dideoxy-
D
L
L
3.12. Methyl 6-[N2-dansyl-N6-(1,5-dideoxy-
-lysinyl]amino hexanoate (7a)
D
-galactitol-1,5-diyl)-
Following Section 3.3, compound 5 (139 mg, 0.310 mmol) was
reacted with BOC-L-Pro-L-Lys(Z)-L-Ala-OMe (C, 1.2 equiv) to give
L
iminoalditol 8 (87 mg, 48.8%): ½a D20
ꢂ
ꢁ55.7 (c = 0.6, MeOH); 1H
NMR (500 MHz, CD3OD): d = 4.42–4.20 (m, 3H), 3.98 (dd,
J3,4 = 3.4 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.81 (m, 3H; H-2, H-6a, H-6b),
3.50 (m, 1H), 3.40 (m, 1H), 3.22 (dd, J2,3 = 9.3 Hz, 1H; H-3), 2.99
(dd, J1eq,1ax = 11.2 Hz, J1eq,2 = 4.9 Hz, 1H; H-1eq), 2.75 (m, 1H),
2.55 (m, 1H), 2.42 (m, 1H; H-5), 2.16 (dd, J1ax,2 = 10.3 Hz, 1H,
H-1ax), 2.28–1.46 (m, 10H), 1.39 (d, 3H); 13C NMR (125 MHz,
CD3OD): d = 174.3, 173.3, 172.8 (3 C@O), 76.0 (C-3), 71.0 (C-4),
67.7 (C-2), 64.2 (C-5), 61.2 (C-6), 60.2, 56.7 (C-1), 53.2, 52.6,
49.1, 46.8, 32.2, 31.3, 23.7, 23.4, 23.4, 16.1. MS: Calcd for
Following Section 3.4, compound 7 (97 mg, 0.19 mmol) gave
fluorescent iminoalditol-lysine hybrid 7a (48 mg, 39%): ½a D20
ꢁ6.4
ꢂ
(c = 0.9, MeOH); 1H NMR (500 MHz, CD3OD): d = 3.95 (dd,
J3,4 = 3.4 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.76 (ddd, J1eq,2 = 4.9 Hz,
J1ax,2 = 10.7 Hz, J2,3 = 9.3 Hz, 1H; H-2), 3.70 (m, 2H; H-6a, H-6b),
3.56 (m, 1H), 3.17 (dd, 1H; H-3), 2.82 (dd, J1eq,1ax = 11.2 Hz, 1H;
H-1eq), 2.81 (m, 1H), 2.77 (m, 1H), 2.37 (m, 1H), 2.28 (m, 1H; H-
5), 2.27 (m, 2H), 2.23 (m, 1H), 2.00 (dd, 1H, H-1ax), 1.58–0.88
(m, 12H), 13C NMR (125 MHz, CD3OD): d = 174.6, 173.4 (2 C@O),

10220
A. J. Steiner et al. / Bioorg. Med. Chem. 16 (2008) 10216–10220
76.1 (C-3), 70.9 (C-4), 67.8 (C-2), 63.8 (C-5), 61.0 (C-6), 57.3 (C-1),
56.9, 52.3, 38.7, 33.5, 32.9, 28.5, 26.1, 24.4, 23.3, 22.9. MS: Calcd for
[C31H48N4O9S]: m/z 652.81; ESIMS found: [M+H]+ 653.79, [M+Na]+
675.75.
3.16. N6-Dansyl-N2-(1,5-dideoxy-
D-galactitol-1,5-diyl)-L-lysinea-
mide (12)
A mixture of 10a and 11a (10 mg, 0.019 mmol, 10a/11a = 7:3) in
aqueous ammonia (1 mL, 25%) was stirred for 2 h at 20 °C. Removal
3.13. Methyl (N-dansyl)-
L
-prolinyl-N6-(1,5-dideoxy-
D-galactitol-
of the solvent under reduced pressure yielded pure 12 (9.5 mg,
0.018 mmol, 96%) as green syrup; ½a D20
ꢁ13.8 (c = 0.5, MeOH);
ꢂ
1
1,5-diyl)- -lysinyl- -alaninate (8a)
L
L
NMR (500 MHz, CD3OD): d = 3.87 (dd, J5,6a = 5.9 Hz, J6a,6b = 11.7 Hz,
1H; H-6a), 3.85 (dd, J3,4 = 3.4 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.72 (dd,
J5,6b = 3.4 Hz, 1H; H-6b), 3.62 (ddd, J1eq,2 = 4.9 Hz, J1ax,2 = 10.2 Hz,
J2,3 = 8.8 Hz, 1H; H-2), 3.23 (m, 1H), 3.19 (dd, 1H; H-3), 3.01 (dd,
J1eq,1ax = 11.7 Hz, 1H; H-1eq), 2.84 (t, 2H), 2.74 (m, 1H; H-5), 2.05
(dd, 1H; H-1ax), 1.64–1.18 (m, 6H); 13C NMR (125 MHz, CD3OD):
d = 177.2 (C@O), 76.2 (C-3), 72.4 (C-4), 68.8 (C-2), 64.9 (C-5),
63.3, 62.6 (C-1, C-6), 51.3, 42.7, 29.6, 29.5, 23.7. MS: Calcd for
[C24H36N4O7S]: m/z 524.641; ESIMS found: [M+H]+ 525,70,
[M+Na]+ 547.69.
Following Section 3.4, compound 8 (53 mg, 0.092 mmol) gave
fluorescent iminoalditol-lysine hybrid 8a (20 mg, 31%):
½ ꢂ
a 2D0
ꢁ54.9 (c = 1.0, MeOH); 1H NMR (500 MHz, CD3OD): d = 4.44–4.28
(m, 3H), 3.97 (dd, J3,4 = 3.4 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.80 (m, 3H;
H-2, H-6a, H-6b), 3.58 (m, 1H), 3.40 (m, 1H), 3.21 (dd,
J2,3 = 9.3 Hz, 1H; H-3), 2.97 (dd, J1eq,1ax = 11.2 Hz, J1eq,2 = 4.9 Hz,
1H; H-1eq), 2.70 (m, 1H), 2.50 (m, 1H), 2.36 (m, 1H; H-5), 2.10
(dd, J1ax,2 = 10.7 Hz, 1H; H-1ax), 2.00–1.30 (m, 10H), 1.41 (d, 3H);
13C NMR (125 MHz, CD3OD): d = 173.3, 173.3, 172.6 (3 C@O),
76.1 (C-3), 71.1 (C-4), 67.9 (C-2), 64.1 (C-5), 61.7 (C-6), 61.2, 56.9
(C-1), 53.1, 52.5, 49.4, 48.3, 31.6, 31.2, 24.6, 23.6, 23.5, 16.2. MS:
Calcd for [C33H49N5O10S]: m/z 707.850; ESIMS found: [M+H]+
708.90, [M+Na]+ 729.95.
Acknowledgments
Financial support by the Austrian Fonds zur Förderung der Wis-
senschaftlichen Forschung (FWF), Vienna, (Project P18998-N17) as
well as by the Protein Engineering Network of Centres of Excel-
lence of Canada (PENCE) is gratefully acknowledged.
3.14. Methyl N2-dansyl-N6-(1,5-dideoxy-
-lysinate (9a)
D-galactitol-1,5-diyl)-
D
Following Section 3.4, compound 9 (95 mg, 0.24 mmol) gave
References and notes
fluorescent iminoalditol-lysine hybrid 9a (49 mg, 39%):
½ ꢂ
a 2D0
ꢁ27.9 (c = 1.8, MeOH); 1H NMR (500 MHz, CD3OD): d = 3.96 (dd,
J3,4 = 2.9 Hz, J4,5 = 1.5 Hz, 1H; H-4), 3.77 (ddd, J1eq,2 = 4.4 Hz,
J1ax,2 = 10.7 Hz, J2,3 = 9.3 Hz, 1H; H-2), 3.73 (m, 1H), 3.70 (m, 2H;
1. For example: Iminosugars
– From Synthesis to Therapeutic Applications;
Compain, P.; Martin, O. R., Eds.; Wiley: Chichester, 2007.; Martin, O. R.;
Compain, P., Eds. Curr. Top. Med. Chem. 2003, 3, 471.; Wrodnigg, T. M. From
lianas to glycobiology tools: Twenty-five years of 2,5-dideoxy-2,5-imino-D-
mannitol. In Timely Research Perspectives in Carbohydrate Chemistry; Schmid,
W., Stütz, A. E., Eds.; Springer: Vienna, New York, 2002; pp 43–76; Heightman,
T. D.; Vasella, A. T. Angew. Chem. Int. Ed. 1999, 38, 750; Iminosugars as
Glycosidase Inhibitors; Stütz, A. E., Ed.; Wiley-VCH: Weinheim, 1999.
2. Spiro, R. G. In Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G. W.,
Sinay, P., Eds.; Part II: Biology of Saccharides; Wiley-VCH: Weinheim, 2000;
Vol. 3, pp 65–79; Elbein, A. D.; Molyneux, R. J. In Iminosugars as Glycosidase
Inhibitors; Stütz, A. E., Ed.; Wiley-VCH: Weinheim, 1999; pp 216–251.
3. Diabetes: MiglitolÒ by BAYER, Gaucher: ZavescaÒ by Actelion.
4. De Raadt, A.; Ekhart, C.; Legler, G.; Stütz, A. E. In Iminosugars as Glycosidase
Inhibitors; Stütz, A. E., Ed.; Wiley-VCH: Weinheim, 1999; pp 207–215.
5. Hermetter, A.; Scholze, H.; Stütz, A. E.; Withers, S. G.; Wrodnigg, T. M. Bioorg.
Med. Chem. Lett. 2001, 11, 1339.
6. (a) Lundt, I.; Steiner, A. J.; Stütz, A. E.; Tarling, C. A.; Ully, S.; Withers, S. G.;
Wrodnigg, T. M. Bioorg. Med. Chem. 2006, 14, 1737; (b) Greimel, P.; Häusler, H.;
Lundt, I.; Rupitz, K.; Stütz, A. E.; Tarling, C. A.; Withers, S. G.; Wrodnigg, T. M.
Bioorg. Med. Chem. Lett. 2006, 16, 2067.
7. Steiner, A. J.; Stütz, A. E.; Wrodnigg, T. M.; Tarling, C. A.; Withers, S. G.;
Hermetter, A.; Schmidinger, H. Bioorg. Med. Chem. Lett. 2008, 18, 1922.
8. Yu, Z.; Sawkar, A. R.; Whalen, L. J.; Wong, C.-H.; Kelly, J. W. J. Med. Chem. 2007,
50, 94.
H-6a, H-6b), 3.20 (dd, 1H; H-3), 2.85 (dd, J1eq 1ax = 10.7 Hz, 1H;
,
H-1eq), 2.48 (m, 1H), 2.30 (m, 1H; H-5), 2.22 (m, 1H), 2.01 (dd,
1H; H-1ax), 1.62–1.50 (m, 2H), 1.30–0.98 (m, 4H); 13C NMR
(125 MHz, CD3OD): d = 172.5 (C@O), 76.0 (C-3), 70.9 (C-4), 67.7
(C-2), 64.0 (C-5), 61.0 (C-6), 56.7 (C-1), 55.8, 52.4, 32.0, 23.0,
23.0. MS: Calcd for [C25H37N3O8S]: m/z 539.65; ESIMS found:
[M+H]+ 541.00, [M+Na]+ 562.9.
3.15. Methyl N6-dansyl-N2-(1,5-dideoxy-
-lysinate (10a) and lactone 11a
D-galactitol-1,5-diyl)-
L
Following Section 3.4, a mixture of compounds 10 and 11 gave
a mixture of fluorescent iminoalditol-lysine hybrid 10a and the
corresponding lactone 11a in a ratio of 7:3. Compound 10a: 1H
NMR (500 MHz, CD3OD): d = 3.93 (dd, J3,4 = 3.4 Hz, J4,5 = 1.5 Hz,
1H; H-4), 3.90–3.70 (m, 3H; H-2, H-6a, H-6b), 3.62 (m, 1H),
3.17 (dd, J2,3 = 9.8 Hz, 1H; H-3), 2.96 (dd, J1eq,1ax = 11.2 Hz,
J1eq,2 = 4.9 Hz, 1H; H-1eq), 2.84 (t, 2H), 2.65 (m, 1H; H-5), 2.14
(dd, J1ax,2 = 10.7 Hz, 1H; H-1ax), 1.64–1.00 (m, 6H); 13C NMR
(125 MHz, CD3OD): d = 173.3 (C@O), 76.4 (C-3), 71.9 (C-4),
68.5 (C-2), 65.3 (C-5), 62.9, 61.4 (C-1, C-6), 52.8, 42.5, 29.2,
29.0, 23.0.
9. (a) Fan, J.-Q. In Iminosugars
– From Synthesis to Therapeutic Applications;
Compain, P., Martin, O. R., Eds.; Wiley: Chichester, 2007; pp 225–247; (b)
Compain, P.; Desvergnes, V.; Liautard, V.; Pillard, C.; Toumieux, S. In
Iminosugars – From Synthesis to Therapeutic Applications; Compain, P., Martin,
O. R., Eds.; Wiley: Chichester, 2007; pp 416–430.
10. Barili, P. L.; Berti, G.; Catalani, G.; D’Andrea, F. Gazz. Chim. Ital. 1992, 122, 135;
CAN 117:171848; AN 1992:571848.
11. For a comprehensive review of the method see: Baxter, E. W.; Reitz, A. B. Org.
React. 2002, 59, 1; for a typical example see: Zou, W.; Szarek, W. A. Carbohydr.
Res. 1993, 242, 311.
12. Stütz, A. E.; Steiner, A. J.; Wrodnigg, T. M. Eur. Pat. Appl. EP 2006-19545, 2008;
CAN 148:356085, AN 2008:369886.
13. Steiner, A. J.; Stütz, A. E.; Tarling, C. A.; Withers, S. G.; Wrodnigg, T. M.
Carbohydr. Res. 2007, 342, 1850.
MS: Calcd for [C25H37N3O8S]: m/z 539.65; ESIMS found: [M+H]+
540.0, [M+Na]+ 562.2.
Lactone 11a: 1H NMR (500 MHz, CD3OD): d = 4.54 (dd,
J5,6a = 4.0 Hz, J6a,6b = 11.7 Hz, 1H; H-6a), 4.38 (dd, J5,6b = 4.9 Hz,
1H; H-6b); 13C NMR (125 MHz, CD3OD): d = 172.7 (C@O), 75.2
(C-3), 71.5 (C-4), 69.0, 68.9, 65.1, 59.4 (C-1, C-2, C-5, C-6), 53.0,
42.7, 29.6, 29.1, 23.8.
14. (a) Prade, H.; Mackenzie, L. F.; Withers, S. G. Carbohydr. Res. 1998, 305, 371; (b)
Kempton, J. B.; Withers, S. G. Biochemistry 1992, 31, 9961.
15. Mahuran, D. J.; Lowden, J. A. Can. J. Biochem. 1980, 58, 287.