Iminoalditol-amino acid hybrids: synthesis and evaluation as glycosidase inhibitors

Article abstract of DOI:10.1016/j.carres.2007.03.024

Cyclization by double reductive amination of d-xylo-hexos-5-ulose with the terminal amino group of α-N-Boc-lysine methyl ester gave a 4:1-mixture of (1′R)-N-methoxycarbonyl-(1-N-Boc-amino)pentyl-1-deoxynojirimycin and the corresponding l-ido epimer wherea

Full text of DOI:10.1016/j.carres.2007.03.024

Carbohydrate Research 342 (2007) 1850–1858
Iminoalditol-amino acid hybrids: synthesis and evaluation
as glycosidase inhibitors
Andreas J. Steiner,a Arnold E. Stutz,a,* Chris A. Tarling,b Stephen G. Withersb and
¨
Tanja M. Wrodnigga
aGlycogroup, Institut fu¨r Organische Chemie, Technische Universita¨t Graz, Stremayrgasse 16, A-8010 Graz, Austria
bDepartment of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
Received 16 February 2007; received in revised form 19 March 2007; accepted 22 March 2007
Available online 28 March 2007
Abstract—Cyclization by double reductive amination of D-xylo-hexos-5-ulose with the terminal amino group of a-N-Boc-lysine
methyl ester gave a 4:1-mixture of (10R)-N-methoxycarbonyl-(1-N-Boc-amino)pentyl-1-deoxynojirimycin and the corresponding
L-ido epimer whereas D-lyxo-hexos-5-ulose furnished the desired N-alkylated 1-deoxymannojirimycin derivative without any obser-
vable epimer formation at C-5. By subsequent modification of the lysine moiety, additional chain-extended derivatives as well as
fluorescent compounds were obtained. All fluorescent iminoalditol-amino acid hybrids prepared in this study exhibited glycosidase
inhibitory activities better than or comparable to the parent compounds’.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Iminoalditol; Amino acid conjugate; Glycosidase inhibitor; Glucosidase; Mannosidase
HO
HO
HO
HO
1. Introduction
H
H
N
N
Iminoalditols are well-known, (usually) competitive,
glycosidase inhibitors.1 Representatives of this class of
compounds, for example, glucosidase inhibitor 1 and
mannosidase inhibitor 2 have found important roles as
biological probes such as in the investigation of glyco-
protein trimming glycosidases2 or as pharmaceutical
substances such as in the treatment of diabetes type II
symptoms (Miglitol by Bayer) and other metabolic dis-
orders including Gaucher disease (Zavesca by Actelion)
(Fig. 1). It was demonstrated that immobilized N-alkyl-
ated imino- alditols can be employed as affinity ligands
in glycosidase isolation and purification protocols.3
We have been interested in derivatives of such com-
pounds featuring ‘added value’ properties suitable for
analytical purposes. Recently, we have found that some
fluorescently labelled derivatives of the glucosidase
HO
OH
HO
OH
1
2
Figure 1.
inhibitor 2,5-dideoxy-2,5-imino-D-mannitol (or DMDP)
are powerful inhibitors exceeding the activity of the par-
ent compound by two orders of magnitude.4
In this context, we have reported syntheses and glyco-
sidase inhibitory activities of various N-alkylated imino-
alditols featuring fluorescent tags such as dansyl
moieties attached to simple N-substituents.5 Based on
the encouraging inhibitory activities of these com-
pounds, we envisaged more convenient properties with
compounds providing a suitably positioned amine for
tagging as well as an additional ‘handle’ for chain-exten-
sions and with a view to the preparation of glycosidase-
recognizing arrays by immobilization on surfaces.
*
Corresponding author. Tel.: +43 316 873 8744; fax: +43 316 873
8740; e-mail: stuetz@tugraz.at
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.03.024

A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
1851
2. Results and discussion
available from 4 by reaction with 12, which was pre-
pared from commercially available a-N-Boc-e-N-Cbz-
L-lysine by standard coupling with methyl 6-aminohex-
anoate. Removal of the Boc group followed by N-
dansylation gave compounds 13 and 14 (Schemes 2–6).
Compound 15, featuring two fluorescent moieties of
significantly different emission spectra in the same mole-
cule was prepared by saponification of methyl ester 13 to
the free acid followed by conventional coupling to the
NBD fluorophor in the presence of O-(benzotriazol-1-
Partially protected L-lysine derivatives featuring a free
terminal amino group were found to be useful reaction
partners in ring-closing reactions with 5-ulohexoses by
reductive amination6 to provide lysine-iminoalditol
hybrids (Scheme 1) ready for a wide range of useful
follow-up modifications.
Again, the presence of a dansyl residue attached to the
a-nitrogen in the L-lysine component was found to
improve inhibitory potencies whereas the second
functional ‘handle’, the terminal carboxylic moiety,
can clearly be employed for the attachment, via linkers,
to suitable surfaces.
yl)-N,N,N0,N0-tetramethyluronium
tetrafluoroborate
(TBTU) (Scheme 7).
In the D-manno series, (5R)-oxirane 167 (prepared in
44% overall yield from methyl a-D-mannopyranoside)
(5R)-Spiro-oxirane 3,7 readily available from methyl
a-D-glucopyranoside via Garegg reaction, per-O-acetyl-
ation, AgF-mediated8 5,6-ene formation (78%), and
m-CPBA oxidation (80%), gave by conventional Zem-
BOC
BOC
NH
NH
O
a
H
OH
N
plen deprotection and acidic hydrolysis known9
OMe
HN
Cbz
HN
Cbz
5
´
O
O
D-xylo-hexos-5-ulose (4). The latter was reacted with
a-N-Boc-e-N-benzyloxycarbonyl-L-lysine methyl ester
(5) at ambient pressure under an atmosphere of hydro-
gen in the presence of Pd(OH)2 (20%) on carbon.
Hydrogenolytic deprotection at the terminal amine fol-
lowed by double reductive amination was expected to
proceed with high diastereoselectivity at the newly
formed chiral centre at C-5 of desired inhibitor 6. Con-
trary to previous results with methyl 6-aminohexanoate,
which exclusively provided iminoglucitol 7 under the
same conditions, with the amino acid, a 4:1 mixture of
N-alkylated 1,5-dideoxy-1,5-imino-D-glucitol 6 and -L-
iditol 8 was obtained. Separation was found to be diffi-
cult on a preparative scale. Deprotection of the second-
ary amine in compound 6 followed by reaction with
dansyl chloride provided fluorescent iminoglucitol 9.
Chain-extended derivatives 10 and 11, were readily
12
Scheme 3. Reagents and conditions: (a) methyl 6-aminohexanoate
hydrochloride, NEt3, DMF, TBTU.
OMe
O
OH
HO
HO
a
HO
HO
O
N
OH
HO
OH
HO
OH
4
7
Scheme 4. Reagents and conditions: (a) methyl 6-aminohexanoate
hydrochloride, aq MeOH, NEt3, Pd(OH)2/C (20%), H2.
1
HNR
R2
O
HO
HO
HNR1
OH
N
R2
HO
HO
O
H2N
OH
+
O
HO
OH
HO
OH
Scheme 1.
OH
HO
HO
O
a
b, c
O
O
O
HO
HO
O
OMe
R1
AcO
AcO
OMe
R1
AcO
OMe
R1
OH
R1
R2
R2
R2
R2
HO
AcO
HO
3
R1=H, R2=OAc
16 R1=OAc, R2=H
4
R1=H, R2=OH
17 R1=OH, R2=H
Scheme 2. Reagents and conditions: (a) m-CPBA, CH2Cl2, aq NaHCO3; (b) NaOMe, MeOH, ꢀ30ꢁC; (c) H2O, Amberlite IR-120.

1852
A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
R5 NH
OMe
O
R3
BOC
OH
R4
NH
a
HO
HO
O
N
OMe
OH
HO
HN
+
R1
R1
O
Cbz
R2
R2
HO
HO
4
R1=H, R2=OH
17 R1=OH, R2=H
5
6
8
R1=H, R2=OH, R3=CH2OH, R4=H, R5=BOC
R1=H, R2=OH, R3=H, R4=CH2OH, R5=BOC
18 R1=OH, R2=H, R3=CH2OH, R4=H, R5=BOC
b, c
9
R1=H, R2=OH, R3=CH2OH, R4=H, R5=Dansyl
20 R1=OH, R2=H, R3=CH2OH, R4=H, R5=Dansyl
Scheme 5. Reagents and conditions: (a) MeOH, Pd(OH)2/C (20%), H2; (b) MeOH, AcCl; (c) NEt3, DMF, dansyl chloride.
R5 NH
O
1'
O
1''
HN
6'
OMe
BOC
5
OH
R3
R4
NH
O
a
H
6'
HO
HO
O
N
1''
N
1'
OMe
OH
HN
HO
+
5
1
R
R1
O
Cbz
R2
R2
HO
HO
4
R1=H, R2=OH
17 R1=OH, R2=H
12
10 R1=H, R2=OH, R3=CH2OH, R4=H, R5=BOC
11 R1=H, R2=OH, R3=H, R4=CH2OH, R5=BOC
19 R1=OH, R2=H, R3=CH2OH, R4=H, R5=BOC
b, c
13 R1=H, R2=OH, R3=CH2OH, R4=H, R5=Dansyl
14 R1=H, R2=OH, R3=H, R4=CH2OH, R5=Dansyl
21 R1=OH, R2=H, R3=CH2OH, R4=H, R5=Dansyl
Scheme 6. Reagents and conditions: (a) MeOH, Pd(OH)2/C (20%), H2; (b) MeOH, AcCl; (c) NEt3, DMF, dansyl chloride.
Dansyl NH
O
1'
Dansyl NH
O
O
O
1'
1'''
6'''
1''
1''
HN
HN
OMe
N
NO2
6'
N
6'
5
5
4
HO
HO
HO
HO
H
H
a, b
N
N
N
N
O
HO
OH
HO
OH
13
15
Scheme 7. Reagents and conditions: (a) NaOH, H2O/dioxane (1:1 v/v); (b) DMF, NEt3, 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexan-1-
aminium trifluoroacetate, TBTU.
was efficiently hydrolyzed to give known9 D-lyxo-hexos-
5-ulose 17. The latter furnished exclusively the D-manno
configured iminoalditols 18 and 19 when reacted with
L-lysine derivatives 5 and 12, respectively. Fluorescent
1-deoxymanojirimycin derivatives 20 and 21 were
prepared following the same protocols as for 9 and 13,
respectively.
Activities of compounds 9 and 13–15 in the D-gluco/L-
ido series as well as of compounds 20 and 21 in the D-
manno series and the respective parent compounds’ 1
and 2 are shown in Table 1. In the D-gluco series, the
new compounds prepared all exhibit better activities
than the parent compound against the enzyme probed.
Interestingly, despite the ‘wrong’ configuration at C-5,
the Ki value of 9.3 lM for L-ido configured iminoalditol
14 is essentially the same as for unsubstituted 1-deoxy-
nojirimycin (1). Similar cases of such insensitivity to this
particular configurational change at C-5 have previously
been observed with some 5-fluoro glycosyl fluorides as
diagnostic substrates for glycosidases.10 As previously
observed for related structures,5 the products in the
manno series, 20 and 21, turned out slightly less active
than parent compound 2. Nonetheless, the modifica-
tions under consideration here should be helpful tools
en route to applications of iminosugars in biochemical
investigations.

A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
Table 1. Ki values of compounds, b-glucosidase, Agrobacterium sp.
1853
3.2. Kinetic studies
Compd
Ki (lM)
Agrobacterium sp. b-glucosidase was purified and as-
sayed as described.11 Kinetic studies were performed at
37 ꢁC in pH 7.0 sodium phosphate buffer (50 mM) con-
taining 0.1% bovine serum albumin, using 7.2 · 10ꢀ5
mg/mL enzyme. Approximate values of Ki were deter-
mined using a fixed concentration of substrate, 4-nitro-
phenyl b-D-glucopyranoside (0.11 mM = 1.5 · Km) and
inhibitor concentrations ranging from 0.2 times to 5
times the Ki value 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 inhibi-
tor concentration equal to ꢀKi. Full Ki determinations
where required, were performed using the same range
of inhibitor concentrations while also varying substrate
(4-nitrophenyl glucoside) concentrations from approxi-
mately 0.015 to 0.6 mM. Data were analyzed by direct
fit to the Michaelis Menten equation describing reaction
in the presence of inhibitors using the program GraFit.12
a-Mannosidase from jack bean was purchased from
Sigma. Kinetic studies were performed at 25 ꢁC in pH
6.8 sodium phosphate buffer (50 mM) containing 0.1%
bovine serum albumin, using 3.7 · 10ꢀ3 mg/mL enzyme.
Approximate values of Ki were determined using a fixed
concentration of substrate, 4-nitrophenyl a-D-manno-
pyranoside (1.3 mM = 1.5 · Km) and inhibitor concen-
trations ranging from 0.2 times to 5 times the Ki value
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.
1
2
12
18a
2.6
6.2
9.3
4
9
13
14
15
20
21
60a
79a
a a-Mannosidase, jack beans.
In conclusion, we could demonstrate that cyclization
of hex-5-ulososes with the terminal amine of lysine is a
suitable method to provide iminoalditol-lysine hybrids.
Dansylation of the secondary amine in the lysine sub-unit
provided efficient inhibitors for the glycosidases probed
in this study. Based on previous work, it is expected that
immobilization via the carboxylate will not significantly
alter these activities thus providing new and selective
tools for the construction of micro-arrays with glycosi-
dase-scavenging and glycosidase-profiling properties.
3. Experimental
3.1. General methods
Melting points were recorded on a Tottoli apparatus
and are uncorrected. Optical rotations were measured
on a Perkin Elmer 341 polarimeter 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 operat-
ing at 500.619 MHz (1H), and at 125.894 MHz (13C).
CDCl3 was employed for protected compounds and
MeOH-d4 for unprotected inhibitors. Chemical shifts
are listed in delta employing residual, non-deuterated
solvent as the internal standard. The signals of the pro-
tecting 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 Da) was employed varying the fragmen-
tation voltage from 50 to 250 V with best molecular
peaks observed at 150 V. MALDI mass spectra were re-
corded on a MALDI Micro MX (Waters) time-of-flight
instrument used in reflectron mode with 2.3 m effective
flight path. Analytical TLC was performed on precoated
aluminium plates silica gel 60 F254 (E. Merck 5554), de-
tected with UV light (254 nm), 10% vanillin/sulfuric acid
as well as ceric ammonium molybdate (100 g ammo-
nium molybdate/8 g ceric sulfate in 1 L 10% H2SO4)
and heated on a hotplate. Preparative TLC was per-
formed on precoated glass plates silica gel 60 F254,
0.5 mm (E. Merck 5744). For column chromatography
silica gel 60 (230–400 mesh, E. Merck 9385) was used.
3.3. Methyl (5R)-2,3,4-tri-O-acetyl-5,6-anhydro-5-C-
hydroxy-a-D-xylo-hexopyranoside (3)
Methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-xylo-hex-5-eno-
pyranoside8 (2.00 g, 6.62 mmol) was dissolved in
CH2Cl2 (100 mL). Satd aq NaHCO3 (0.6 M, 40 mL)
and 3-chloroperbenzoic acid (70%, 2.00 g, 8.11 mmol,
1.2 equiv) were added and the mixture was stirred at
ambient temperature for 20 h. The organic layer was
washed with satd aq NaHCO3, dried (Na2SO4), filtered
and concentrated to about 10 mL under reduced pres-
sure at ambient temperature. Quick filtration over a
short plug of silica gel (cyclohexane/ethyl acetate, 2:1
v/v) yielded product 4 as one single diastereomer
20
(1.69 g, 5.31 mmol, 80%) as colourless syrup: ½aꢁD
1
+91.0 (c 0.5, CHCl3); H NMR (CDCl3): d 5.76 (dd,
1H, J2,3 = 9.8 Hz, J3,4 = 9.8 Hz, H-3), 5.54 (d, 1H, H-
4), 5.03 (d, 1H, J1,2 = 3.4 Hz, H-1), 5.01 (dd, 1H, H-
2), 3.44 (s, 3H, OCH3), 2.85 (d, 1H, J6a,6b = 4.2 Hz,
H-6a), 2.54 (d, 1H, H-6b); 13C NMR: d 98.5 (C-1),
79.1 (C-5), 71.0, 68.3, 66.8 (C-2, C-3, C-4), 56.8
(OCH3), 45.8 (C-6); Anal. Calcd for C13H18O9: C,
49.06; H, 5.70. Found: C, 49.00; H, 5.76.

1854
A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
3.4. D-xylo-Hexos-5-ulose (4)
(40 mL), Pd(OH)2/C (20%) (20 mg) was added and the
heterogeneous reaction mixture was stirred under an
atmosphere of hydrogen at ambient pressure for 44 h.
After filtration and removal of the solvent under
reduced pressure, the residue was purified by chromato-
graphy on silica gel (CHCl3/MeOH/concd NH4OH,
500:100:6 v/v/v), yielding pure product 6 (185 mg,
To a 2% methanolic solution of methyl (5R)-2,3,4-tri-
O-acetyl-5,6-anhydro-5-C-hydroxy-a-D-xylo-hexopyran-
oside (3) (400 mg, 1.26 mmol), 1.0 M NaOMe solution
(500 lL) was added dropwise at ꢀ30 ꢁC. After 3 h reac-
tion time at ꢀ30 ꢁC, water (30 mL) was added and the
resulting reaction mixture was concentrated to about
20 mL under reduced pressure. After addition of aceto-
nitrile (5 mL), ion exchange resin Amberlite IR 120 [H+]
was added to the reaction mixture, which was then stir-
red at 45 ꢁC for 20 h. Filtration and removal of the sol-
vents under reduced pressure gave compound 4 (220 mg)
as a slightly yellow foam. As previously observed, NMR
revealed a complex mixture of several tautomeric
forms.9 The compound was used for the next step with-
out further purification.
20
0.46 mmol, 41%) as colourless syrup: ½aꢁD ꢀ18.2 (c 3.4,
1
MeOH); H NMR (MeOH-d4): d 4.09 (m, 1H, H-20),
3.85 (m, 2H, J6a,6b = 11.7 Hz, H-6a, H-6b), 3.71 (s,
3H, OCH3), 3.47 (ddd, 1H, J1eq,2 = 4.9 Hz, J1ax,2
10.7 Hz, J2,3 = 8.8 Hz, H-2), 3.35 (dd, 1H, J3,4
=
=
9.3 Hz, J4,5 = 9.3 Hz, H-4), 3.14 (dd, 1H, H-3), 2.99
(dd, 1H, J1eq,1ax = 11.2 Hz, H-1eq), 2.79 (m, 1H, H-
60a), 2.58 (m, 1H, H-60b), 2.17 (dd, 1H, H-1ax), 2.12
(m, 1H, H-5), 1.82–1.30 (m, 6H, H-30a, H-30b, H-40a,
13
H-40b, H-50a, H-50b); C NMR: d 173.9 (C-10), 79.3
(C-3), 70.8 (C-4), 69.5 (C-2), 66.2 (C-5), 58.2, 56.4,
53.8, 52.2 (C-1, C-6, C-20, C-60), 51.5 (OCH3), 31.3
(C-30), 23.7, 23.6 (C-40, C-50); Anal. Calcd for
C18H34N2O8: C, 53.19; H, 8.43. Found: C, 53.22; H,
8.50.
3.5. N-[5-(Methoxycarbonyl)pentyl]-1-deoxynojirimycin
(7)
To a 1.5% solution of D-xylo-hexos-5-ulose (4) (510 mg,
2.60 mmol), methyl 6-aminohexanoate hydrochloride
(550 mg, 3.03 mmol, 1.2 equiv) and Et3N (200 lL,
145 mg, 1.4 mmol) in MeOH/H2O (2:1 v/v), Pd(OH)2/
C (20%, 50 mg) was added and the reaction mixture
was stirred under an atmosphere of hydrogen at ambient
pressure for 72 h. After filtration and removal of the sol-
vent under reduced pressure, the residue was purified by
chromatography on silica gel (CHCl3/MeOH/concd
NH4OH, 300:100:4 v/v/v), yielding product 7 (437 mg,
1.50 mmol, 58%) as colourless syrup. The formation of
the corresponding L-ido-configured product was not ob-
Side product 8 (46 mg, 0.11 mmol, 10%) was isolated
20
1
as a colourless syrup: ½aꢁD ꢀ12.8 (c 1.0, MeOH); H
NMR (MeOH-d4): d 4.09 (m, 1H, H-20), 3.85 (m, 2H,
H-6a, H-6b), 3.71 (s, 3H, OCH3), 3.57 (nr, 1H, H-2),
3.44 (nr, 1H), 3.31 (nr, 1H), 3.10 (nr, 1H, H-1eq),
2.90–2.64 (nr, 4H, H-1ax, H-5, H-60a, H-60b), 1.82–
1.30 (m, 6H, H-30a, H-30b, H-40a, H-40b, H-50a, H-
13
50b); C NMR: d 173.9 (C-10), 74.8 (C-3), 71.6 (C-4),
69.8 (C-2), 63.2 (C-5), 57.0, 54.0, 53.9, 51.9 (C-1, C-6,
C-20, C-60), 51.5 (OCH3), 31.3 (C-30), 26.2, 23.4 (C-40,
C-50); Anal. Calcd for C18H34N2O8: C, 53.19; H, 8.43.
Found: C, 53.12; H, 8.55.
20
served. ½aꢁD ꢀ7.0 (c 0.7, MeOH); 1H NMR (MeOH-d4):
d 3.96 (dd, 1H, J5,6a = 2.0 Hz, J6a,6b = 12.2 Hz, H-6a),
3.89 (dd, 1H, J5,6b = 2.9 Hz, H-6b), 3.66 (s, 3H,
OCH3), 3.58 (ddd, 1H, J1eq,2 = 4.9 Hz, J1ax,2 = 10.3 Hz,
3.7. Methyl N2-(dansyl)-N6-(1,5-dideoxy-D-glucitol-1,5-
diyl)-L-lysinate (9)
J2,3 = 8.8 Hz, H-2), 3.47 (dd, 1H, J3,4 = 9.3 Hz, J4,5
=
9.3 Hz, H-4), 3.25 (dd, 1H, H-3), 3.21 (dd, 1H,
J1eq,1ax = 11.7 Hz, H-1eq), 3.07 (m, 1H, H-60a), 2.88
(m, 1H, H-60b), 2.57 (m, 2H, H-1ax, H-5), 2.36 (t, 2H,
H-20a, H-20b), 1.70–1.60 (m, 4H, H-30a, H-30b, H-50a,
H-50b), 1.38 (m, 2H, H-40a, H-40b); 13C NMR: d 174.6
(C-10), 78.2 (C-3), 69.3, 68.1 (C-2, C-4), 66.2 (C-5),
56.1, 55.1, 52.5 (C-1, C-6, C-60), 50.9 (OCH3), 33.4 (C-
20), 26.4 (C-50), 24.5 (C-40), 23.3 (C-30); Anal. Calcd
for C3H25NO6: C, 53.59; H, 8.65. Found: C, 53.52; H,
8.70.
Acetyl chloride (0.5 mL, 0.55 g, 7.0 mmol) was slowly
added to dry MeOH (15 mL) at 0 ꢁC. After 10 min,
compound 6 (37 mg, 0.091 mmol) was added to this
mixture, which was then stirred for 20 h at ambient tem-
perature when TLC indicated complete conversion of
the starting material. The solvents were removed under
reduced pressure and the residue was dissolved in dry
DMF (15 mL). Et3N (60 lL, 44 mg, 0.43 mmol, 5 equiv)
and dansyl chloride (28 mg, 0.10 mmol, 1.1 equiv) were
added, and the reaction mixture was stirred in a brown
flask at ambient temperature for 4 h. Removal of the
solvent under reduced pressure and purification of the
residue by preparative TLC (CHCl3/MeOH/concd
NH4OH, 300:100:4 v/v/v, extraction with MeOH) gave
pure product 9 (32 mg, 0.059 mmol, 65%) as a bright
3.6. Methyl N2-(tert-butoxycarbonyl)-N6-(1,5-dideoxy-D-
glucitol-1,5-diyl)-L-lysinate (6) and methyl N2-(tert-
butoxycarbonyl)-N6-(1,5-dideoxy-L-iditol-1,5-diyl)-L-
lysinate (8)
20
green syrup: ½aꢁD +1.4 (c 0.3, MeOH); 1H NMR
To a mixture of D-xylo-hexos-5-ulose (4) (220 mg, 1.12
mmol) and 5 (590 mg, 1.50 mmol, 1.3 equiv) in MeOH
(MeOH-d4): d 3.67 (m, 2H, H-6a, H-6b), 3.64 (m, 1H,
H-20), 3.33 (ddd, 1H, J1eq,2 = 4.9 Hz, J1ax,2 = 10.2 Hz,

A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
1855
J2,3 = 8.8 Hz, H-2), 3.22 (dd, 1H, J3,4 = 9.3 Hz,
J4,5 = 9.3 Hz, H-4), 3.10 (s, 3H, OCH3), 3.01 (dd, 1H,
H-3), 2.73 (dd, 1H, J1eq,1ax = 11.2 Hz, H-1eq), 2.40 (m,
1H, H-60a), 2.26 (m, 1H, H-60b), 1.96 (dd, 1H, H-1ax),
1.93 (ddd, 1H, J5,6a = 2.0 Hz, J5,6b = 2.9 Hz, H-5),
1.52–1.43 (m, 2H, H-30a, H-30b), 1.24–0.80 (m, 4H, H-
NH4OH, 500:100:6 v/v/v), yielding pure compound 10
(0.30 g, 0.58 mmol, 9%) and an inseparable mixture of
10 and 11 (1.32 g, 2.54 mmol, 39%, 10/11 = 7:2 by inte-
1
gration over H NMR spectra), as colourless syrups.
20
1
Compound 10: ½aꢁD ꢀ10.8 (c 1.8, MeOH); H NMR
(MeOH-d4): d 3.96 (m, 1H, H-20), 3.87 (dd, 1H,
J5,6a = 2.9 Hz, J6a,6b = 12.2 Hz, H-6a), 3.84 (dd, 1H,
J5,6b = 2.9 Hz, H-6b), 3.65 (s, 3H, OCH3), 3.47 (ddd,
1H, J1eq,2 = 4.9 Hz, J1ax,2 = 10.7 Hz, J2,3 = 8.8 Hz, H-
2), 3.36 (dd, 1H, J3,4 = 9.3 Hz, J4,5 = 9.3 Hz, H-4),
3.25–3.12 (nr, 3H, H-3, H-600a, H-600b), 3.00 (dd, 1H,
J1eq,1ax = 11.2 Hz, H-1eq), 2.81 (m, 1H, H-60a), 2.59
(m, 1H, H-60b), 2.33 (t, 2H, H-200a, H-200b), 2.18 (dd,
1H, H-1ax), 2.13 (ddd, 1H, H-5), 1.80–1.30 (m, 12H,
H-30a, H-30b, H-40a, H-40b, H-50a, H-50b, H-300a, H-
300b, H-400a, H-400b, H-500a, H-500b); 13C NMR: d 174.6,
174.0 (C-10, C-100), 79.4 (C-3), 70.8 (C-4), 69.5 (C-2),
66.3 (C-5), 58.2, 56.5, 55.0, 52.3 (C-1, C-6, C-20, C-60),
50.9 (OCH3), 38.9 (C-600), 33.5 (C-200), 32.1 (C-30), 28.9
(C-500), 26.2, 24.5, 24.5, 23.7 (C-40, C-50, C-300, C-400);
Anal. Calcd for C27H28O5: C, 74.98; H, 6.53. Found:
C, 74.92; H, 6.60.
13
40a, H-40b, H-50a, H-50b); C NMR: d 172.5 (C-10),
79.4, (C-3), 70.9 (C-4), 69.6 (C-2), 66.0 (C-5), 58.3,
56.5, 55.9, 52.0 (C-1, C-6, C-20, C-60), 51.1 (OCH3),
31.9 (C-30), 23.0, 22.9 (C-40, C-50); ESI-MS Calcd for
[C25H37N3O8S]: m/z 539.65. Found: [M+H]+ 540.3,
[M+Na]+ 562.3.
3.8. Methyl 6-{[N6-(benzyloxycarbonyl)-N2-(tert-butoxy-
carbonyl)-L-lysyl]amino}hexanoate (12)
To a solution of commercially available (Fluka) N6-
(benzyloxycarbonyl)-N2-(tert-butoxycarbonyl)-L-lysine
(3.00 g, 7.89 mmol) and Et3N (3.0 mL, 2.2 g, 22 mmol,
2.7 equiv) in dry DMF (30 mL), O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium
tetrafluoroborate
(TBTU) (2.66 g, 8.28 mmol, 1.05 equiv) and methyl 6-
aminohexanoate hydrochloride (1.57 g, 8.64 mmol,
1.1 equiv) were added simultaneously and the reaction
mixture was stirred at ambient temperature for 20 min.
After removal of the solvent under reduced pressure,
the residue was dissolved in CH2Cl2, consecutively
washed with 6% aqueous HCl and satd aq NaHCO3,
dried (Na2SO4) and filtered. Removal of the solvent un-
der reduced pressure and chromatography (cyclohex-
ane/ethyl acetate, 2:1 v/v) gave 12 (3.99 g, 7.86 mmol,
1
Compound 11: H NMR (MeOH-d4): d 3.96 (m, 1H,
H-20), 3.81 (nr, 2H, H-6a, H-6b), 3.53 (nr, 1H, H-2),
3.02 (nr, 1H, H-1eq), 2.73 (m, 1H, H-60a), 2.65 (m,
13
1H, H-60b); C NMR: d 174.7, 174.0 (C-10, C-100),
74.7, 71.6, 70.1, 63.2 (C-2, C-3, C-4, C-5), 57.2, 54.5,
54.1, 51.6 (C-1, C-6, C-20, C-60), 50.8 (OCH3), 38.9 (C-
600), 33.5 (C-200), 32.1 (C-30), 28.9 (C-500), 26.2, 24.5,
24.5, 23.7 (C-40, C-50, C-300, C-400).
20
1
100%) as a colourless oil: ½aꢁD ꢀ9.6 (c 1.4, CHCl3); H
NMR (CDCl3): d 4.01 (m, 1H, H-2), 3.64 (s, 3H,
OCH3), 3.20 (m, 4H, H-6a, H-6b, H-60a, H-60b), 2.29
(t, 2H, H-20a, H-20b), 1.80 (m, 1H, H-3a), 1.66–1.28
(m, 11H, H-3b, H-4a, H-4b, H-5a, H-5b, H-30a, H-30b,
3.10. Methyl 6-{[N2-(dansyl)-N6-(1,5-dideoxy-D-glucitol-
1,5-diyl)-L-lysyl]amino}hexanoate (13)
Acetyl chloride (0.5 mL, 0.55 g, 7.0 mmol) was slowly
added to dry MeOH (25 mL) at 0 ꢁC. After 10 min, 10
(260 mg, 0.50 mmol) was added to the reaction mixture,
which was then stirred for at ambient temperature 40 h.
The solvents were removed under reduced pressure and
the residue was dissolved in dry MeOH (10 mL). Et3N
(200 lL, 145 mg, 1.4 mmol, 3 equiv) and dansyl chloride
(150 mg, 0.56 mmol, 1.1 equiv) were added, and the
resulting reaction mixture was stirred in a brown flask
at ambient temperature for 4 h. Removal of the solvent
under reduced pressure and chromatography on silica
gel (CHCl3/MeOH/concd NH4OH, 700:100:8 v/v/v)
gave pure product 13 (173 mg, 0.265 mmol, 53%) as
13
H-40a, H-40b, H-50a, H-50b); C NMR: d 174.3 (C-10),
172.3 (C-1), 54.6 (C-2), 51.7 (OCH3), 40.6 (C-6), 39.4
(C-60), 34.0 (C-20), 32.2 (C-3), 29.7 (C-5), 29.3 (C-50),
26.5 (C-40), 24.6 (C-30), 22.8 (C-4); Anal. Calcd for
C26H41N3O7: C, 61.52; H, 8.14. Found: C, 61.42; H,
8.21.
3.9. Methyl 6-{[N2-(tert-butoxycarbonyl)-N6-(1,5-dideoxy-
D-glucitol-1,5-diyl)-L-lysyl]amino}hexanoate (10) and
methyl 6-{[N2-(tert-butoxycarbonyl)-N6-(1,5-dideoxy-L-
iditol-1,5-diyl)-L-lysyl]amino}hexanoate (11)
20
bright green syrup: ½aꢁD +0.8 (c 0.7, MeOH); 1H
To a solution of D-xylo-hexos-5-ulose (4) (1.30 g,
6.63 mmol, 1.03 equiv) and 12 (3.28 g, 6.46 mmol) in
MeOH/H2O (40 mL, 3:1 v/v), Pd(OH)2/C (20%)
(120 mg) was added and the heterogeneous reaction
mixture was stirred under hydrogen at ambient pressure
for 96 h. After filtration and removal of the solvent un-
der reduced pressure, the residue was purified by chro-
matography on silica gel (CHCl3/MeOH/concd
NMR (MeOH-d4): d 3.77 (m, 2H, H-6a, H-6b), 3.65
(s, 3H, OCH3), 3.55 (m, 1H, H-20), 3.43 (ddd, 1H,
J1eq,2 = 4.9 Hz, J1ax,2 = 10.3 Hz, J2,3 = 8.8 Hz, H-2),
3.31 (dd, 1H, J3,4 = 9.3 Hz, J4,5 = 9.3 Hz, H-4), 3.09
(dd, 1H, H-3), 2.82 (dd, 1H, J1eq,1ax = 11.2 Hz, H-
1eq), 2.82–2.72 (m, 2H, H-600a, H-600b), 2.46 (m, 1H,
H-60a), 2.33 (m, 1H, H-60b), 2.27 (t, 2H, H-200a, H-
200b), 2.05 (dd, 1H, H-1ax), 2.01 (ddd, 1H,

1856
A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
J5,6a = 2.9 Hz, J5,6b = 2.4 Hz, H-5), 1.60–0.90 (m, 12H,
H-30a, H-30b, H-40a, H-40b, H-50a, H-50b, H-300a, H-
300b, H-400a, H-400b, H-500a, H-500b); 13C NMR: d 174.6,
173.6 (C-10, C-100), 79.4 (C-3), 70.8 (C-4), 69.6 (C-2),
65.9 (C-5), 58.2, 57.5, 56.5, 52.1 (C-1, C-6, C-20, C-60),
50.9 (OCH3), 38.7 (C-600), 33.5 (C-200), 33.1 (C-30), 28.5
(C-500), 26.1, 24.4, 23.3, 23.0 (C-40, C-50, C-300, C-400).
ESIMS Calcd for [C31H48N4O9S]: m/z 652.81. Found:
[M+H]+ 653.7, [M+Na]+ 675.7.
IR-120 [H+] and filtration, the solvents were removed
under reduced pressure. The residue was dissolved in
dry DMF (12 mL), Et3N (110 lL, 80 mg, 0.79 mmol,
4 equiv), O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyl-
uronium tetrafluoroborate (TBTU) (65 mg, 0.20 mmol,
1 equiv) and 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-
amino]hexan-1-aminium trifluoroacetate (80 mg, 0.20
mmol, 1 equiv) were added and the mixture was stirred
at room temperature for 40 min. Removal of the solvent
under reduced pressure and chromatography on silica
gel (CHCl3/MeOH/concd NH4OH, 700:100:16 v/v/v)
3.11. Methyl 6-{[N2-(dansyl)-N6-(1,5-dideoxy-L-iditol-
1,5-diyl)-L-lysyl]amino}hexanoate (14)
yielded product 15 (75 mg, 0.083 mmol, 42%) as a red
1
syrup: H NMR (MeOH-d4): d 3.76 (dd, 1H, J5,6a
2.9 Hz, J6a,6b = 12.2 Hz, H-6a), 3.72 (dd, 1H, J5,6b
=
=
Acetyl chloride (0.5 mL, 0.55 g, 7.0 mmol) was slowly
added to dry MeOH (10 mL) at 0 ꢁC. After 10 min, a
mixture of 10 and 11 (70 mg, 0.13 mmol, 10/11 = 7:2)
was added and the mixture was stirred at ambient tem-
perature for 16 h. The solvents were removed under
reduced pressure and the residue was dissolved in dry
MeOH (20 mL). Et3N (100 lL, 73 mg, 0.72 mmol,
5 equiv) and dansyl chloride (50 mg, 0.19 mmol,
1.4 equiv) were added, and the resulting mixture was
stirred in a brown flask at ambient temperature for
4 h. Removal of the solvent under reduced pressure
and purification of the residue by preparative TLC
(CHCl3/MeOH/concd NH4OH, 500:100:6 v/v/v, extrac-
tion with MeOH) gave pure 14 (10 mg, 0.015 mmol,
2.9 Hz, H-6b), 3.56 (m, 1H, H-20), 3.42 (m, 3H, H-2,
H-6000a, H-6000b), 3.30 (dd, 1H, J3,4 = 9.3 Hz, J4,5
9.3 Hz, H-4), 3.16 (t, 2H, H-1000a, H-1000b), 3.10 (dd,
1H, J2,3 = 9.3 Hz, H-3), 2.84 (m, 2H, H-600a, H-600b),
2.79 (d, 1H, J1eq,1ax = 11.2 Hz, J1eq,2 = 4.9 Hz, H-1eq),
2.40 (m, 1H, H-60a), 2.26 (m, 1H, H-60b), 2.13 (t, 2H,
H-200a, H-200b), 2.03 (dd, 1H, J1ax,2 = 10.7 Hz, H-1ax),
2.00 (ddd, 1H, H-5), 1.80–0.80 (m, 20H, H-30a, H-30b,
H-40a, H-40b, H-50a, H-50b, H-300a, H-300b, H-400a, H-
400b, H-500a, H-500b, H-2000a, H-2000b, H-3000a, H-3000b, H-
4000a, H-4000b, H-5000a, H-5000b); 13C NMR: d 174.8, 172.6
(C-10, C-100), 79.4 (C-3), 70.8 (C-4), 69.6 (C-2), 65.9 (C-
5), 58.3, 57.0, 56.5, 52.0 (C-1, C-6, C-20, C-60), 44.7
(C-6000), 39.1 (C-1000), 38.9 (C-600), 35.8, 32.5, 31.3, 29.1,
28.5, 26.5, 26.5, 26.2, 25.4, 23.1, 23.0 (C-30, C-40, C-50,
C-200, C-300, C-400, C-500, C-2000, C-3000, C-4000, C-5000); MAL-
DI-MS Calcd for [C42H61N9O11S]: m/z 900.07. Found:
[M]+ 900.3.
20
1
11%), as a green syrup: ½aꢁD ꢀ5.1 (c 0.4, MeOH); H
NMR (MeOH-d4): d 3.78 (dd, 1H, J5,6a = 4.9 Hz,
J6a,6b = 11.2 Hz, H-6a), 3.73 (dd, 1H, J5,6b = 6.8 Hz,
H-6b), 3.66 (s, 3H, OCH3), 3.63 (dd, 1H, J3,4
9.3 Hz, J4,5 = 5.4 Hz, H-4), 3.55 (m, 1H, H-20), 3.46
(ddd, 1H, J1eq,2 = 4.9 Hz, J1ax,2 = 9.3 Hz, J2,3
=
=
8.3 Hz, H-2), 3.35 (dd, 1H, H-3), 2.88 (m, 1H, H-5),
2.84–2.72 (m, 2H, H-600a, H-600b), 2.64 (dd, 1H,
J1eq,1ax = 12.2 Hz, H-1eq), 2.48 (dd, 1H, H-1ax), 2.41
(m, 1H, H-60a), 2.30 (m, 1H, H-60b), 2.27 (t, 2H, H-
200a, H-200b), 1.60–0.90 (m, 12H, H-30a, H-30b, H-40a,
H-40b, H-50a, H-50b, H-300a, H-300b, H-400a, H-400b, H-
3.13. Methyl (5R)-2,3,4-tri-O-acetyl-5,6-anhydro-5-C-
hydroxy-a-D-lyxo-hexopyranoside (16)
Methyl 2,3,4-tri-O-acetyl-6-deoxy-a-D-lyxo-hex-5-eno-
pyranoside8 (2.40 g, 7.94 mmol) was dissolved in
CH2Cl2 (60 mL). Satd aq NaHCO3 (0.6 M, 30 mL)
and 3-chloroperbenzoic acid (55%, 2.76 g, 8.80 mmol,
1.1 equiv) were added and the reaction mixture was stir-
red at ambient temperature for 3 h. The organic phase
was washed with satd aq NaHCO3, dried (Na2SO4), fil-
tered and concentrated to about 10 mL under reduced
pressure at ambient temperature. Quick filtration over
a short plug of silica gel (cyclohexane/ethyl acetate,
2:1 v/v) yielded 16 as the only diastereomer (1.92 g,
13
500a, H-500b); C NMR: d 174.7, 173.8 (C-10, C-100),
74.7 (C-3), 71.5 (C-4), 70.0 (C-2), 62.8 (C-5), 57.6,
56.4, 54.0, 51.6 (C-1, C-6, C-20, C-60), 50.8 (OCH3),
38.7 (C-600), 33.5 (C-200), 33.2 (C-30), 28.5 (C-500), 26.7,
26.1, 24.4, 23.1 (C-40, C-50, C-300, C-400); ESIMS Calcd
for [C31H48N4O9S]: m/z 652.81. Found: [M+H]+
653.3, [M+Na]+ 675.3.
20
3.12. N2-(Dansyl)-N6-(1,5-dideoxy-D-glucitol-1,5-diyl)-
N1-[6-({6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-
hexyl}amino)-6-oxohexyl]-L-lysinamide (15)
6.03 mmol, 76%) as colourless syrup: ½aꢁD +22.3 (c 0.9,
CHCl3); 1H NMR (CDCl3): d 5.80 (d, 1H, J3,4
=
10.7 Hz, H-4), 5.58 (dd, 1H, J2,3 = 3.4 Hz, H-3), 5.40
(dd, 1H, J1,2 = 2.0 Hz, H-2), 4.77 (d, 1H, H-1), 3.41 (s,
3H, OCH3), 2.85 (d, 1H, J6a,6b = 4.4 Hz, H-6a), 2.56
(d, 1H, H-6b); 13C NMR: d 100.3 (C-1), 79.9 (C-5),
70.1, 67.3, 64.3 (C-2, C-3, C-4), 56.4 (OCH3), 46.4 (C-
6); Anal. Calcd for C3H18O9: C, 49.06; H, 5.70. Found:
C, 48.93; H, 5.78.
To a solution of compound 13 (131 mg, 0.201 mmol) in
1,4-dioxane/H2O (10 mL, 1:1 v/v), 0.5 M aqueous
NaOH (1.0 mL) was added at 0 ꢁC and the resulting
mixture was stirred at ambient temperature for 16 h.
After neutralization with ion exchange resin Amberlite

A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
1857
3.14. D-lyxo-Hexos-5-ulose (17)
filtration and removal of the solvent under reduced pres-
sure, the residue was purified by chromatography on sil-
ica gel (CHCl3/MeOH/concd NH4OH, 400:100:5 v/v/v),
yielding compound 19 (1.73 g, 3.33 mmol, 56%) as col-
To a 1.5% methanolic solution of methyl (5R)-2,3,4-
tri-O-acetyl-5,6-anhydro-5-C-hydroxy-a-D-lyxo-hexopyr-
anoside (16) (610 mg, 1.92 mmol), 1.0 M NaOMe solu-
tion (500 lL) was added dropwise at ꢀ30 ꢁC. After 7 h
reaction time at ꢀ30 ꢁC, water (30 mL) was added and
the resulting reaction mixture was concentrated to about
20 mL under reduced pressure. After addition of aceto-
nitrile (8 mL), ion exchange resin Amberlite IR 120 [H+]
was added to the reaction mixture, which was then stir-
red at 45 ꢁC for 16 h. Filtration and removal of the sol-
vents under reduced pressure gave 17 (330 mg) as a
slightly yellow syrup. As previously reported, NMR
showed a complex mixture of several tautomeric forms
including hydrates.9 This was used for the next step
without further purification.
20
ourless syrup: ½aꢁD ꢀ20.3 (c 1.9, MeOH); 1H NMR
(MeOH-d4): d 3.96 (m, 1H, H-20), 3.92 (dd, 1H,
J5,6a = 2.4 Hz, J6a,6b = 12.2 Hz, H-6a), 3.89 (dd, 1H,
J5,6b = 2.9 Hz, H-6b), 3.87 (ddd, 1H, J1eq,2 = 3.9 Hz,
J1ax,2 = 2.0 Hz, J2,3 = 3.4 Hz, H-2), 3.71 (dd, 1H,
J3,4 = 9.3 Hz, J4,5 = 9.3 Hz, H-4), 3.65 (s, 3H, OCH3),
3.36 (dd, 1H, H-3), 3.25–3.12 (m, 2H, H-600a, H-600b),
3.06 (dd, 1H, J1eq,1ax = 12.2 Hz, H-1eq), 2.85 (m, 1H,
H-60a), 2.70 (m, 1H, H-60b), 2.62 (dd, 1H, H-1ax),
2.32 (t, 2H, H-200a, H-200b), 2.28 (ddd, 1H, H-5), 1.77–
1.25 (m, 12H, H-30a, H-30b, H-40a, H-40b, H-50a, H-
50b, H-300a, H-300b, H-400a, H-400b, H-500a, H-500b); 13C
NMR: d 174.7, 174.0 (C-10, C-100), 75.0, 68.0, 68.0 (C-
2, C-3, C-4), 66.0 (C-5), 57.5, 55.1, 55.0, 52.4 (C-1, C-
6, C-20, C-60), 50.9 (OCH3), 38.9 (C-600), 33.5 (C-200),
32.1 (C-30), 28.9 (C-500), 26.2, 24.5, 24.5, 23.5 (C-40, C-
50, C-300, C-400); Anal. Calcd for C24H45N3O9: C, 55.47;
H, 8.73. Found: C, 55.42; H, 8.80. The formation of
the corresponding L-gulo-configured product was not
observed.
3.15. Methyl N2-(tert-butoxycarbonyl)-N6-(1,5-dideoxy-
D-mannitol-1,5-diyl)-L-lysinate (18)
To a solution of D-lyxo-hexos-5-ulose (17) (200 mg,
1.02 mmol) and L-lysine derivative 5 (507 mg, 1.29
mmol, 1.3 equiv) in MeOH (40 mL), Pd(OH)2/C (20%)
(20 mg) was added and the heterogeneous mixture was
stirred under hydrogen at ambient pressure for 18 h.
After filtration and removal of the solvent under
reduced pressure, the residue was purified by chromato-
graphy on silica gel (CHCl3/MeOH/concd NH4OH,
500:100:6 v/v/v), yielding pure compound 18 (210 mg,
3.17. Methyl N2-(dansyl)-N6-(1,5-dideoxy-D-mannitol-
1,5-diyl)-L-lysinate (20)
Acetyl chloride (0.5 mL, 0.55 g, 7.0 mmol) was slowly
added to dry MeOH (15 mL) at 0 ꢁC. After 10 min, 18
(69 mg, 0.17 mmol) was added and the mixture was stir-
red at ambient temperature for 18 h. The solvents were
removed under reduced pressure and the residue was
dissolved in dry DMF (15 mL). Et3N (150 lL, 110 mg,
1.1 mmol, 6 equiv) and dansyl chloride (51 mg,
0.19 mmol, 1.1 equiv) were added, and the mixture was
stirred in a brown flask at ambient temperature for
5 h. Removal of the solvent under reduced pressure
and chromatography on silica gel (CHCl3/MeOH/concd
NH4OH, 500:100:6 v/v/v) gave product 20 (68 mg,
20
0.52 mmol, 51%) as colourless syrup. ½aꢁD ꢀ38.9 (c 1.4,
1
MeOH); H NMR (MeOH-d4): d 4.09 (m, 1H, H-20),
3.91 (dd, 1H, J5,6a = 2.9 Hz, J6a,6b = 12.2 Hz, H-6a),
3.87 (dd, 1H, J5,6b = 2.4 Hz, H-6b), 3.85 (ddd, 1H,
J1eq,2 = 3.9 Hz, J1ax,2 = 2.0 Hz, J2,3 = 2.9 Hz, H-2),
3.71 (s, 3H, OCH3), 3.68 (dd, 1H, J3,4 = 8.8 Hz, J4,5
=
9.3 Hz, H-4), 3.33 (dd, 1H, H-3), 3.01 (dd, 1H,
J1eq,1ax = 12.2 Hz, H-1eq), 2.81 (m, 1H, H-60a), 2.61
(m, 1H, H-60b), 2.52 (dd, 1H, H-1ax), 2.18 (ddd, 1H,
H-5), 1.82–1.30 (m, 6H, H-30a, H-30b, H-40a, H-40b,
20
13
H-50a, H-50b); C NMR: d 173.9 (C-10), 75.2, 68.3,
0.13 mmol, 74%) as a bright green syrup: ½aꢁD ꢀ13.6 (c
68.2 (C-2, C-3, C-4), 66.0 (C-5), 57.8, 55.1, 53.8, 52.3
(C-1, C-6, C-20, C-60), 51.4 (OCH3), 31.2 (C-30), 23.7,
23.5 (C-40, C-50); Anal. Calcd for C18H34N2O8: C,
53.19; H, 8.43. Found: C, 74.92; H, 6.60. The formation
of the corresponding L-gulo-configured product was not
observed.
0.8, MeOH); 1H NMR (MeOH-d4): d 3.84 (dd, 1H,
J5,6a = 2.4 Hz, J6a,6b = 11.8 Hz, H-6a), 3.81 (ddd, 1H,
J1eq,2 = 3.9 Hz, J1ax,2 = 2.0 Hz, J2,3 = 3.4 Hz, H-2),
3.76 (dd, 1H, J5,6b = 2.9 Hz, H-6b), 3.73 (m, 1H, H-
20), 3.64 (dd, 1H, J3,4 = 9.3 Hz, J4,5 = 9.3 Hz, H-4),
3.28 (dd, 1H, H-3), 3.21 (s, 3H, OCH3), 2.81 (dd, 1H,
J1eq,1ax = 12.2 Hz, H-1eq), 2.52–2.42 (m, 2H, H-60a, H-
60b), 2.40 (dd, 1H, H-1ax), 2.10 (ddd, 1H, H-5), 1.60–
1.54 (m, 2H, H-30a, H-30b), 1.30–1.02 (m, 4H, H-40a,
3.16. Methyl 6-{[N2-(tert-butoxycarbonyl)-N6-(1,5-di-
deoxy-D-mannitol-1,5-diyl)-L-lysyl]amino}hexanoate (19)
13
H-40b, H-50a, H-50b); C NMR: d 172.5 (C-10), 75.2,
To a solution of D-lyxo-hexos-5-ulose (17) (1.17 g,
5.96 mmol) and 12 (3.05 g, 6.01 mmol, 1.0 equiv) in
MeOH/H2O (40 mL, 3:1 v/v), Pd(OH)2/C (20%) (120
mg) was added and the heterogeneous mixture was stir-
red under hydrogen at ambient pressure for 120 h. After
68.2, 68.2 (C-2, C-3, C-4), 66.0 (C-5), 57.7, 55.8, 55.1,
52.1 (C-1, C-6, C-20, C-60), 51.1 (OCH3), 31.9 (C-30),
22.9, 22.7 (C-40, C-50); ESIMS Calcd for [C25H37-
N3O8S]: m/z 539.65. Found: [M+H]+ 540.6, [M+Na]+
562.6.

1858
A. J. Steiner et al. / Carbohydrate Research 342 (2007) 1850–1858
3.18. Methyl 6-{[N2-(dansyl)-N6-(1,5-dideoxy-D-manni-
tol-1,5-diyl)-L-lysyl]amino}hexanoate (21)
References
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Acetyl chloride (0.5 mL, 0.55 g, 7.0 mmol) was slowly
added to dry MeOH (15 mL) at 0 ꢁC. After 10 min, 19
(35 mg, 0.067 mmol) was added and the mixture was
stirred ambient temperature for 6 h. The solvents were
removed under reduced pressure and the residue was
dissolved in dry DMF (15 mL). Et3N (100 lL, 73 mg,
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d4): d 3.84 (dd, 1H, J5,6a = 2.4 Hz, J6a,6b = 11.7 Hz, H-
6a), 3.80 (ddd, 1H, J1eq,2 = 3.9 Hz, J1ax,2 = 2.0 Hz,
J2,3 = 3.4 Hz, H-2), 3.76 (dd, 1H, J5,6b = 2.9 Hz, H-
6b), 3.65 (s, 3H, OCH3), 3.64 (dd, 1H, J3,4 = 9.3 Hz,
J4,5 = 9.3 Hz, H-4), 3.54 (m, 1H, H-20), 3.27 (dd, 1H,
H-3), 2.80 (dd, 1H, J1eq,1ax = 12.2 Hz, H-1eq), 2.79 (m,
1H, H-60a), 2.70 (m, 1H, H-60b), 2.40 (m, 2H, H-600a,
H-600b), 2.34 (dd, 1H, H-1ax), 2.26 (t, 2H, H-200a, H-
200b), 2.02 (ddd, 1H, H-5), 1.60–0.95 (m, 12H, H-30a,
H-30b, H-40a, H-40b, H-50a, H-50b, H-300a, H-300b, H-
400a, H-400b, H-500a, H-500b); 13C NMR: d 174.6, 174.3
(C-10, C-100), 75.4, 68.4, 68.3 (C-2, C-3, C-4), 65.4 (C-
5), 57.8, 57.6, 55.2, 52.2 (C-1, C-6, C-20, C-60), 50.9
(OCH3), 38.6 (C-600), 33.5 (C-200), 33.5 (C-30), 28.4 (C-
500), 26.1, 24.4, 23.4, 22.9 (C-40, C-50, C-300, C-400);
ESIMS: Calcd for [C31H48N4O9S]: m/z 652.81. Found:
[M+H]+ 653.3, [M+Na]+ 675.3.
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Financial support by the Austrian Fonds zur Fo¨rderung
der Wissenschaftlichen Forschung (FWF), Vienna,
(Project P18998-N17) as well as by the Protein Engineer-
ing Network of Centres of Excellence of Canada
(PENCE) is gratefully acknowledged.