CuAAC-mediated diversification of aminoglycoside-arginine conjugate mimics by non-reducing di- and trisaccharides

Article abstract of DOI:10.1016/j.carres.2013.02.003

Di- and triguanidinylation of trehalose, sucrose, and melizitose has been achieved via a Huisgen-cycloaddition approach. They can serve as aminoglycoside-arginine conjugate mimics, which has been demonstrated by their biological profiles in assays against

Full text of DOI:10.1016/j.carres.2013.02.003

Carbohydrate Research 371 (2013) 61–67
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
CuAAC-mediated diversification of aminoglycoside–arginine
conjugate mimics by non-reducing di- and trisaccharides
a
a
a
a
Bernhard Westermann a,b, , Simon Dörner , Sebastian Brauch , Angela Schaks , Ramona Heinke ,
⇑
Sebastian Stark a, Floris L. van Delft c, Sander S. van Berkel a
a Leibniz-Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, 06120 Halle, Germany
b Martin-Luther University of Halle-Wittenberg, Institute of Chemistry, Kurt-Mothes-Str. 2, 06120 Halle, Germany
c Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Di- and triguanidinylation of trehalose, sucrose, and melizitose has been achieved via a Huisgen-cycload-
dition approach. They can serve as aminoglycoside–arginine conjugate mimics, which has been demon-
strated by their biological profiles in assays against Bacillus subtilis. For comparative studies,
tetraguanidinylated neamine and kanamycin derivatives have also been synthesized and evaluated.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 10 December 2012
Received in revised form 12 February 2013
Accepted 13 February 2013
Available online 24 February 2013
Keywords:
Aminoglycoside–arginine conjugate mimics
Click-reaction
Guanidinylation
1. Introduction
can discriminate between different pro- and eukaryotic RNAs mak-
ing them suitable for inhibiting the prokaryotic counterpart exclu-
The search for new strategies to face bacterial infections is an
everlasting battle due to the ability of these organisms to effi-
ciently adjust to current antibiotic treatments. Among the thera-
peutics which inhibit the prokaryotic protein biosynthesis,
polycationic aminoglycoside antibiotics (e.g., neamine 1, kanamy-
cin 2, streptomycin 4, neomycin 5, and paromomycin 8, see
Fig. 1) are among the most well established ones.1 Computational
and crystallographic studies suggest that the aminoglycosides bind
to the highly negatively charge RNA regions and prevent the pro-
ductive binding of divalent metal ions (Fig. 2A).2 Other aminogly-
coside targets are RNA/RNA binding protein (RBP) complexes,
which play a vital role in regulative processes such as nuclear
RNA-export, RNA-localization, translation, and degradation.3 The
binding of the RBP to its holoenzyme, which differs in large for
pro- and eukaryotic systems, occurs at arginine-rich sequences.
This recognition can thus be inhibited very efficiently by the poly-
cationic aminoglycosides (see, Fig. 2B). In this context it could be
shown, that arginine–aminoglycoside conjugates (AACs) can en-
hance the inhibition of prokaryotic transfer RNA maturation by
large extent, preventing the recognition of the peptidic and RNA-
subdomain.4,5
sively.6 Among these, the arginine-modified congeners 3 and 7
displayed better inhibition rates compared to 6, which has been as-
cribed to the higher flexibilities of the guanidinylated side arms.
In order to diversify these AACs, we set out to explore a two-
directional strategy for the production of AAC mimics. To this
end, we explored the possibility of employing disaccharide and tri-
saccharide scaffolds as core units for the attachment of guanidiny-
lated side chains. The attachment of the cationic side chains was
envisioned to be accomplished by a copper-catalyzed azide-alkyne
cycloaddition (CuAAC) approach starting from suitable azide-
containing aminoglycosides and alkyne-modified guanidine
(Fig. 3). To the best of our knowledge, this approach has not yet
been applied in the diversification of AACs. Our strategy entailed
the use of two non-reducing disaccharides (i.e., sucrose and treha-
lose), a non-reducing trisaccharide (melizitose), and aminoglyco-
sides neamine (1) and kanamycin 2. To validate the activity of
these modified AACs, a preliminary evaluation against Gram-
positive and Gram-negative bacteria was performed.
2. Results and discussion
In a seminal paper series by Lapidot and Litovchik it was
demonstrated that guanidinylated aminoglycosides 3, 6, and 7
As shown by various research groups, the scaffold of the natural
occurring aminoglycosides (e.g., neamine 1 resp. neomycin 5, and
kanamycin 2) can be significantly changed while retaining their
biological activities.7 In 2000, Lapidot and Litovchik were able to
demonstrate that arginine-modified aminoglycosides can increase
⇑
Corresponding author. Tel./fax: +49 (0) 345 5582 1340/1309.
E-mail address: bwesterm@ipb-halle.de (B. Westermann).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.02.003

62
B. Westermann et al. / Carbohydrate Research 371 (2013) 61–67
NHR
NH2
OH
O
HO
OH
NHR
O
HO
O
HO
HO
RHN
HO
H2N
HO
OH
O
H2N
O
O
NHR
RHN
NH2
1 Neamine
2 Kanamycin B (R = H)
KanaR5 (R = Arg)
3
NH
H2N
NHR
O
NH2
NH
NH
O
H2N
HO
HN
HO
HO
HO
OH
OH
HO
RHN
H2N
H2N
O
RHN
O
O
O
O
O
CHO
NHR
HO
NH2
O
O
HO
HO
Me
HO
HO
RHN
H2N
OH
O
HO
HO
O
OH
O
OH
O
OH
O
OH
NHMe
RHN
H2N
Neomycin B (R = H)
NeoG6 (R = CNHNH2)
NeoR6 (R = Arg)
8 Paromomycin
4 Streptomycin
5
6
7
Figure 1. Structures of commonly used aminoglycoside antibiotics.
Figure 2. (A) Left: complex between kanamycin A (green) and the 16S-rRna A Site (gray). PDB-ID: 2ESI5 (B) right: Crystal structure of aminoglycoside phosphotransferase IIIa
(gray) with ADP and neomycin B (green) complexed. PDB-ID: 2B0Q5. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article).
NH2
OH
NH2
O
H
N
HO
OH
NH2
HO
O
AACs
NH2
H2N
HO
+
HO2C
HO
O
O
H2N
NH2
NH
N3
HO
H
O
NH2
NH
N
O
+
AAC mimics
di/tri-
HO
saccharide
OH
Figure 3. Strategy for the synthesis of AAC mimics.
the antibiotic activity up to 500 fold.6b Utilizing peptide coupling
conditions up to six arginine residues could be attached to the tet-
racyclic framework of neomycin (5) yielding hexa-argininylated
neomycin 7 (NeoR6). Prompted by these results, we envisioned
the application of non-reducing di- and trisaccharide scaffolds for
the attachment of multiple guanidine residues. The CuAAC reaction
was envisioned as preferred ligation method for the coupling of the
different building blocks, as a result of the generally obtained high
yields, as well as the formation of a triazole ring which is consid-
ered an isostere for an amide group.8 To establish a suitable

B. Westermann et al. / Carbohydrate Research 371 (2013) 61–67
63
-
HSO4
OH
Cu(OAc)2
Na-ascorbate
OH
NH2
NH2
NH2
N
HN
N
N
O
HO
HO
N3
O
HO
HO
N
H
NH2
+
OH
tBuOH
82%
-
OH
HSO4
109
11
9
Scheme 1. Synthesis of guanidinylated glucose 11.
protocol with a minimum amount of protection/deprotection steps
we commenced with the formation of triazole 11 starting from 1-
azido glucose 9 and alkyne 10 (Scheme 1).9
24 (melizitose-based) all showed significantly lower activity com-
pared to kanamycin. Surprisingly, at a substrate concentration of
1 lM, both the kanamycin-based AAC mimic 30 and the sucrose-
The reaction went smoothly under classical CuAAC conditions,
to afford the coupled product 11 in 82% yield. This encouraging re-
sult forecasts that this protocol can most likely be used for the
straightforward synthesis of ACC mimics circumventing laborious
protection and deprotection steps of both the carbohydrate and
the guanidine moiety.
Continuing our studies, we used non-reducing trehalose 12, su-
crose 17, and melizitose 21 as carbohydrate moieties, which re-
quired no further treatment of the anomeric center. The primary
hydroxyl groups of these carbohydrates can be readily transformed
into the corresponding di- and triazides 13, 18, and 22, respec-
tively, by direct azidation of the unprotected carbohydrates with
PPh3/CCl4/NaN3 according to the published protocols (Scheme 2).10
Surprisingly, no substitution of the primary (neopentyl) hydroxyl
groups to the corresponding azides at C-1 of the fructose moieties
of sucrose and melizitose could be detected.
based AAC mimic 20, showed slightly increased activities in compar-
ison with kanamycin. This might suggest a different mode of binding
for the AAC mimics compared to the aminoglycosides. In addition,
these results indicate that different carbohydrates can be utilized
as suitable backbones for the orientation of the guanidine groups,
however, the biological activity is not dependent on the number of
guanidine groups attached. Further synthetic efforts to provide
new AAC derivatives with increased activities are currently under-
way. Moreover, modeling studies to gain more insights into the ori-
entation of the guanidine groups on the AACs are currently being
conducted in our laboratories.
3. Experimental section
3.1. General experimental information
An initial attempt to react alkyne 10 with trehalose diazide 13
turned out to be sluggish, leading to the formation of multiple
products according to TLC-analysis. To our satisfaction, changing
to the Boc-protected guanidine derivative 14,11 resulted in the de-
sired triazole 15 in a good yield (91%). Subsequent deprotection of
compound 15 with trifluoroacetic acid afforded the bis-guanidine
compound 16 which could be easily isolated as its trifluoroacetate
salt in 79% yield.
This protocol could also be applied for the synthesis of the pro-
tected di- and tri-guanidinylated sucrose and melizitose deriva-
tives 19 (quant.) and 23 (73%), respectively. Therefore, this
protocol can be considered robust for a variety of carbohydrate
scaffolds. The deprotection of the Boc groups on compounds 19
and 23 was carried out under similar conditions as described
above. The overall yields for these two-step processes were 61%
and 63% for 20 and 24, respectively.
In order to compare the biological data of known AACs with the
here reported AAC mimics we additionally prepared aminoglyco-
side derivatives 27 and 30 (Scheme 3) which allows for the direct
comparison with aminoglycosides 1 and 3. To this end, we used
tetraazides 26 and 29, which can be efficiently generated from neo-
mycin 5 and kanamycin 2.12,13 Following the two step CuAAC/
deprotection protocol, as described above, the corresponding
tetraguanidinylated derivates 27 and 30 could be obtained in rea-
sonable to good overall yields.
Biological evaluation of the guanidinylated compounds re-
vealed no activity against Gram-negative bacteria (Vibrio fischeri
assay14). However, pronounced activities could be observed
against Gram-positive bacteria (Bacillus subtilis assay15), as de-
picted in Table 1. The activity against the Gram-positive bacterial
strain Bacillus subtilis was measured by the percentage of growth
inhibition. All AAC mimics were examined at concentrations of 1
Unless otherwise stated all chemicals and solvents were ob-
tained from commercial sources and were used without further
purification. All 1H and 13C NMR spectra were recorded in either
CDCl3, methanol-d4, or D2O on either a 300 MHz Varian MER-
CURY-VX 300 apparatus (300 MHz for 1H NMR and 75 MHz for
13C NMR, respectively) or on a 400 MHz Varian MERCURY-VX
400 apparatus (400 MHz for 1H NMR and 100 MHz for 13C NMR,
respectively). Chemical shifts are reported in d values (ppm) with
tetramethylsilane (TMS) as internal standard. ESI-MS spectra were
obtained from an API-150EX spectrometer. HRMS spectra were re-
corded on an FT-ICR Bruker Apex III 70e mass spectrometer. Puri-
fication of the crude products by column chromatography was
performed on silica gel 60 (230–400 mesh, 0.040–0.063 mm),
Merck, Germany. TLC identification of products and reactants
was performed on silica gel coated aluminum foil (silica gel 60
F254 with fluorescence indicator), Merck, Germany. The antibacte-
rial activity against Bacillus subtilis was determined using a fluores-
cence-based antibacterial growth inhibition assay. The
fluorescence was measured on a GENios Pro plate reader (Fa. Te-
can, excitation, 510 nm; emission, 535 nm). The Bacillus subtilis
strain 168 (PAbrB-IYFP) [9] was maintained on TY medium (tryp-
tone-yeast extract) supplemented with 1% Bacto-tryptone, 0.5%
Bacto-yeast extract, 1% NaCl, and chloramphenicol (5 l
g mlꢀ1).
3.1.1. tert-Butyl {(1E)-{[(1-{[(2R,3S,4S,5R,6R)-6-({(2R,3R,4S,5S,
6R)-6-[(4-{[N0,N00-bis(tert-butoxycarbonyl)carbamimidamido]
methyl}-1H-1,2,3-triazol-1-yl)methyl]-3,4,5-trihydroxy-
tetrahydro-2H-pyran-2-yl}oxy)-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl]methyl}-1H-1,2,3-triazol-4-yl)methyl]amino}[(tert-
butoxycarbonyl)amino] methylidene}carbamate (15) General
Procedure A (GP-A)
Diazide 13 (98 mg, 0.20 mmol), N,N0-di-(tert-butyloxycar-
and 10
As can be deduced from the obtained data, at a substrate concen-
tration of 10 M all AAC mimics show a lower activity as compared
to unsubstituted kanamycin (5). Moreover, at a concentration of
M, AAC mimics 27 (neamine-based), 16(trehalose-based), and
lM using kanamycin as the standard.
bonyl)guanidine 14 (148 mg, 0.50 mmol),
a freshly prepared
0.08 M aq sodium ascorbate solution (2.00 mL, 0.16 mmol), and a
0.04 M aq copper(II) acetate solution (2.00 mL, 0.08 mmol) in
tert-butanol (10 mL) were combined and stirred vigorously at
room temperature. After completion of the reaction indicated by
l
1
l

64
B. Westermann et al. / Carbohydrate Research 371 (2013) 61–67
RHN
HN
R
RHN
OH
NR
N
NH
NBoc
NHBoc
N
N
HO
HO
OH
HO
O
RN
O
N
H
OH
O
R
HO
HO
OH
HO
O
O
14
O
N
OH
N
N
b)
12 R = OH (trehalose)
13 R = N310a
a)
OH
c)
15 R = Boc (91%)
16 R = H (79%)
RHN
NR
RHN
RN
HN
NBoc
R
N
NH
N
N
N
H
NHBoc
14
OH
R
N
HO
HO
O
O
N
OH
HO
N
O
O
O
b)
OH
HO
O
HO OH
OH
HO OH
17 R = OH (sucrose)
18 R = N310b
a)
19 R = Boc (quant.)
20 R = H (61%)
c)
RN
N
NHR
NR
R
NBoc
NHBoc
14
N
NH
N
N
H
NHR
OH
N
H
O
N
R
HO
O
O
OH
OH
N
N
HO
OH
O
HO
O
O
OH
O
OH
OH
b)
O
NR
NHR
HO
OH
R
O
HO
O
OH
HN
HO
OH
N
N
21 R = OH (melizitose)
22 R = N310c
a)
N
23 R = Boc (73%)
24 R = H (63%)
c)
Scheme 2. Synthesis of AAC mimics (16, 20, and 24 isolated as trifluoroacetate salts). Reagents: (a) NaN3, PPh3, CCl4; (b) Na-ascorbate, Cu(OAc)2, tBuOH; (c) TFA, DCM.
TLC analysis all volatiles were removed in vacuo. Purification by
column chromatography using a gradient of ethyl acetate/etha-
nol/water 70:30:0 > 70:15:8 afforded 15 as colorless solid
(180 mg, 91%). Mp >248 °C (decomposition); RF = 0.40 (ethyl ace-
nol-d4) d = 3.09 (t, J = 9.3 Hz, 2H), 3.31 (dt, J = 3.3, 1.6 Hz, 4H),
3.73 (t, J = 9.3 Hz, 2H), 4.13–4.22 (m, 2H), 4.49 (s, 4H), 4.55 (dd,
J = 14.5, 7.5 Hz, 2H), 4.69 (d, J = 3.7 Hz, 2H), 4.71–4.79 (m, 2H),
7.97 (s, 2H) ppm; 13C NMR (75 MHz, methanol-d4) d = 37.5, 52.3,
71.9, 72.8, 74.4, 95.9, 111.4, 125.5, 144.1, 158.8 ppm; ESI-MS m/
tate/ethanol/water = 70:15:8); ½a D23
ꢁ
+42.6 (c = 0.15, MeOH); 1H
NMR (300 MHz, methanol-d4) d = 1.41–1.54 (m, 36H), 3.08 (t,
J = 9.3 Hz, 2H), 3.31 (dt, J = 3.3, 1.6 Hz, 4H), 3.35 (dd, J = 9.5,
3.7 Hz, 2H), 3.71 (t, J = 9.3 Hz, 2H), 4.22 (t, J = 7.7 Hz, 2H), 4.46–
4.57 (m, 2H), 4.64 (m, 4H), 4.69–4.79 (m, 4H), 7.99 (s, 2H) ppm;
13C NMR (75 MHz, methanol-d4) d = 28.3, 28.6, 37.0, 52.4, 71.8,
72.8, 74.5, 80.5, 84.7, 96.1, 154.0, 157.4, 164.4 ppm; ESI-MS m/z:
987.6 [M+H]+, 1009.8 [M+Na]+; HRMS (ESI) calcd for C40H66N12O17-
Na 1009.4561 [M+Na]+, found 1009.4569.
z: 294.0 [M+2H]2+
,
587.3 [M+H]+; HRMS (ESI) calcd for
C
20H35N12O9 587.2644 [M+H]+, found 587.2652.
3.1.3. tert-Butyl {(1E)-{[(1-{[(2R,3S,4S,5R,6R)-6-{[(2S,3S,4S,5R)-
5-[(4-{[N0,N00-bis(tert-butoxycarbonyl)carbamimidamido]
methyl}-1H-1,2,3-triazol-1-yl)methyl]-3,4-dihydroxy-2-
(hydroxymethyl)tetrahydrofuran-2-yl]oxy}-3,4,5-
trihydroxytetrahydro-2H-pyran-2-yl]methyl}-1H-1,2,3-triazol-
4-yl)methyl]amino}[(tert-butoxycarbonyl)amino]
3.1.2. 6-[4-({[Amino(iminio)methyl] amino}methyl)-1H-1,2,3-
methylidene}carbamate (19)
triazol-1-yl]-6-deoxy-
({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
6-deoxy- -glucopyranoside bis(trifluoroacetate) (16) General
a
-
D
-glucopyranosyl 6-[4-
Diazide 18 (98 mg, 0.25 mmol), alkyne 14 (0.186 mg,
0.63 mmol), 0.08 M aq sodium ascorbate solution (2.50 mL,
0.20 mmol), and 0.04 M aq copper(II) acetate solution (2.50 mL,
0.10 mmol) in tert-butanol (10 mL) were reacted according to GP-
A. Purification by column chromatography using a gradient of ethyl
acetate/ethanol/water 70:30:0 > 70:20:10 afforded compound 19
as colorless oil (252 mg, quant.). RF = 0.37 (ethyl acetate/ethanol/
a
-D
Procedure B (GP-B)
Compound 15 (170 mg, 0.17 mmol) was treated with dichloro-
methane/trifluoracetic acid (5:1 v/v, 6 mL) and was stirred at room
temperature until TLC analysis indicated completion of the reac-
tion. All volatiles were removed under reduced pressure. Diguani-
dinylated 16 was obtained as white solid (110 mg, 79%) after
purification by column chromatography (RP18, H2O). M.p. 132-
water = 70:20:10);
½
a 2D7
ꢁ
+18.6 (c = 0.13, MeOH); 1H NMR
(400 MHz, methanol-d4) d = 1.44–1.54 (m, 36H), 3.16 (t,
J = 9.5 Hz, 1H), 3.31 (dt, J = 3.3, 1.6 Hz, 2H), 3.41–3.47 (m, 2H),
3.50–3.56 (m, 1H), 3.68–3.76 (m, 2H), 3.80 (t, J = 7.9 Hz, 1H),
134 °C; ½a 2D7
ꢁ
+70.8 (c = 0.12, MeOH); 1H NMR (300 MHz, metha-

B. Westermann et al. / Carbohydrate Research 371 (2013) 61–67
65
NR
RHN
HN
NR
N
N
N
H
N
N
N
RN
N
H
N3
O
N3
NHR
NR
N
O
N
N
N3
N
O
OH
O
OH
14
OH
OH
HO
N3
a)
HO
b)
N
N
NR
OH
OH
HN
N
2512
NHR
26 R = Boc (24%)
27 R = H (71%)
NHR
HN
NR
RN
HN
RHN
RN
NHR
NH
N3
N
N
N
N3
O
O
OH
OH
N
N3
N
N
N
14
N
O
HO
N
OH
N3
O
HO
O
a)
HO
O
O
HO
OH
OH
OH
OH
OH
NR
O
2813
HN
N
N
NHR
29 R = Boc (60%)
30 R = H (94%)
b)
OH OH
N
Scheme 3. Synthesis of guanidyl–aminoglycoside conjugates (27 and 30 isolated as trifluoroacetate salts). Reagents: (a) Na-ascorbate, Cu(OAc)2, tBuOH; (b) TFA, DCM.
Table 1
71.7, 72.2, 72.4, 73.0, 76.5, 77.0, 79.7, 93.2, 105.2, 115.4, 119.3,
Activity of AAC-mimics against Bacillus subtilis
125.5, 126.1, 144.4, 158.0, ppm; ESI-MS m/z: 294.4 [M+2H]2+
;
Inhibition (%) (c = 1
lM)
Inhibition (%) (c = 10 lM)
HRMS (ESI) calcd for
587.2642.
C
20H35N12O9 587.2644 [M+H]+, found
Kanamycin
40.9 ( 18.1)
16.4 ( 7.7)
48.1 ( 14.8)
28.1 ( 8.6)
24.9 ( 5.4)
50.2 ( 10.3)
99.3 ( 9.4)
30.9 ( 9.6)
54.1 ( 11.7)
49.4 ( 12.6)
36.7 ( 10.3)
57.9 ( 7.9)
16
20
24
27
30
3.1.5. tert-Butyl {(1Z)-{[(1-{[(2R,3S,4S,5R,6R)-6-{[(2S,4R,5R)-5-
[(4-{[N0,N00-bis(tert-butoxy
carbonyl)carbamimidamido]methyl}-1H-1,2,3-triazol-1-
yl)methyl]-3-({(2R,4S,5S,6R)-6-[(4-{[N0,N00-bis(tert-
butoxycarbonyl)carbamimidamido]methyl}-1H-1,2,3-triazol-1-
yl)methyl]-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl}oxy)-4-
hydroxy-2-(hydroxymethyl)tetrahydrofuran-2-yl]oxy}-3,4,5-
trihydroxytetrahydro-2H-pyran-2-yl]methyl}-1H-1,2,3-triazol-
4-yl)methyl]amino}[(tert-butoxycarbonyl)amino]
3.90–3.98 (m, 1H), 4.05–4.08 (m, 1H), 4.31 (dd, J = 14.1, 2.3 Hz, 1H),
4.35–4.42 (m, 1H), 4.45–4.57 (m, 3H), 4.67 (s, 2H), 4.84 (dd,
J = 14.4, 2.1 Hz, 2H), 5.32 (d, J = 3.5 Hz, 1H), 7.86 (s, 1H), 7.93 (s,
1H) ppm; 13C NMR (75 MHz, methanol-d4) d = 28.3, 28.6, 36.7,
37.1, 53.2, 54.2, 63.6, 72.8, 73.0, 73.3, 74.1, 77.5, 78.7, 80.5, 81.3,
84.6, 93.7, 105.9, 125.3, 126.0, 145.2, 145.4, 154.0, 157.4; 164.4,
164.5 ppm; ESI-MS m/z: 985.9 [MꢀH]ꢀ, 987.7 [M+H]+, 1009.9
[M+Na]+; HRMS (ESI) calcd for C40H66N12O17Na 1009.4561
[M+2H]2+, found 1009.4555.
methylidene}carbamate (23)
Triazide 22 (115 mg, 0.20 mmol), alkyne 14 (208 mg,
0.70 mmol), 0.08 M aq sodium ascorbate solution (3.00 mL,
0.24 mmol), and 0.04 M aq copper(II) acetate solution (3.00 mL,
0.12 mmol) in tert-butanol (20 mL) were reacted according to
GP-A. Purification by column chromatography using a gradient of
ethyl acetate/ethanol/water 70:30:0 > 70:15:8 afforded 23 as
white solid (215 mg, 73%). Mp >275 °C (decomposition); RF = 0.52
3.1.4. 6-[4-({[Amino(iminio)methyl]amino}methyl)-1H-1,2,3-
triazol-1-yl]-6-deoxy-
({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
6-deoxy- -glucopyranoside bis(trifluoroacetate) (20)
Compound 19 (50 mg, 50.7 mol) was treated with dichloro-
a-D-fructofuranosyl 6-[4-
(ethyl acetate/ethanol/water = 70:15:8);
½
a 2D7
+23.8 (c = 0.13,
ꢁ
MeOH); 1H NMR (300 MHz, methanol-d4) d = 1.35–1.63 (m, 54H),
3.06 (t, J = 9.5 Hz, 1H), 3.18 (t, J = 9.3 Hz, 1H), 3.27–3.33 (m, 6H),
3.34–3.52 (m, 3H), 3.71 (t, J = 9.3 Hz, 1H), 3.77–4.04 (m, 3H), 4.12
(d, J = 6.2 Hz, 1H), 4.24 (d, J = 9.5 Hz, 1H), 4.31–4.53 (m, 4H),
4.53–4.70 (m, 4H), 4.75 (br. s., 2H), 4.82 (br. s., 1H), 5.09 (d,
J = 3.7 Hz, 1H), 5.34 (d, J = 3.7 Hz, 1H), 7.81–8.08 (m, 3H) ppm;
13C NMR (75 MHz, methanol-d4) d = 28.2, 28.3, 28.6, 36.8, 37.0,
37.1, 52.1, 53.1, 53.9, 64.0, 72.0, 72.4, 72.8, 73.3, 74.5, 74.6, 76.8,
80.5, 82.4, 84.3, 84.5, 84.6, 93.7, 101.1, 106.8, 154.0, 157.4,
164.4 ppm; ESI-MS m/z: 1494.3 [M+Na]+; HRMS (ESI) calcd for
a
-D
l
methane/trifluoracetic acid (5:1 v/v, 6 mL) as described in GP-B.
After purification by column chromatography (RP18, H2O), Depro-
tected compound 20 was obtained as a colorless oil (21 mg, 61%).
½
a 2D8 +14.1 (c = 0. 30, MeOH); 1H NMR (300 MHz, D2O) d = 3.29 (t,
ꢁ
J = 9.4 Hz, 1H), 3.45–3.63 (m, 3H), 3.81 (t, J = 8.8 Hz, 2H), 4.01–
4.12 (m, 1H), 4.16 (d, J = 8.5 Hz, 1H), 4.27–4.39 (m, 2H), 4.47 (s,
2H), 4.53–4.58 (m, 3H), 4.62 (dd, J = 11.4, 8.2 Hz, 1H), 4.69 (d,
J = 7.9 Hz, 1H), 5.37 (d, J = 3.5 Hz, 1H), 7.96 (s, 1H), 8.08 (s, 1H)
ppm; 13C NMR (75 MHz, D2O) d = 37.1, 37.2, 52.7, 53.6, 62.2,
C
60H98N18O25Na 1493.6842 [M+Na]+, found 1493.6806.

66
B. Westermann et al. / Carbohydrate Research 371 (2013) 61–67
3.1.6. (2n)-6-[4-({[Amino(iminio)methyl]amino}methyl)-1H-
1,2,3-triazol-1-yl]-6-deoxy- -arabino-hexopyranosyl-(1?3)-
(3n)-6-[4-({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-
triazol-1-yl]-6-deoxy- -erythro-hex-2-ulofuranosyl 6-[4-
({[amino(iminio) methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
6-deoxy- -glucopyranoside tris(trifluoroacetate) (24)
60.5, 61.9, 65.6, 70.9, 71.0, 72.0, 76.2, 78.4, 97.6, 158.9 ppm; ESI-
a-
D
MS m/z: 408.2 [M+2H]2+
; HRMS (ESI) C28H48O6N24 calcd
408.2089, found 408.2086.
a-D
3.1.9. tert-Butyl {(1E)-[({1-[(2R,3R,4S,5S,6R)-2-
a
-D
{[(1S,2R,3R,4S,6R)-4,6-bis(4-{[N0,N00-bis(tert-
Compound 23 (180 mg, 0.18 mmol) was treated with dichloro-
methane/trifluoracetic acid (5:1 v/v, 12 mL) as described in GP-B.
After purification by column chromatography (RP18, H2O), depro-
tected compound 24 was obtained as white foam (139 mg, 63%).
butoxycarbonyl)carbamimidamido]methyl}-1H-1,2,3-triazol-1-
yl)-3-({(2S,3R,4S,5S,6R)-6-[(4-{[N0,N00-bis(tert-
butoxycarbonyl)carbamimidamido]methyl}-1H-1,2,3-triazol-1-
yl)methyl]-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl}oxy)-2-
hydroxycyclohexyl]oxy}-3,5-dihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-4-yl]-1H-1,2,3-triazol-4-
yl}methyl)amino][(tert-butoxycarbonyl)amino]methylidene}
carbamate (29)
Mp 133–135 °C; ½a D27
ꢁ
+31.3 (c = 0.14, MeOH); 1H NMR (300 MHz,
D2O) d = 3.11–3.22 (m, 3H), 3.30 (t, J = 9.5 Hz, 1H), 3.52
(dd, J = 10.2, 3.7 Hz, 1H), 3.56–3.62 (m, 1H), 3.72 (t, J = 9.3 Hz,
1H), 3.80 (t, J = 9.5 Hz, 1H), 3.87–3.97 (m, 1H), 3.97–4.18 (m, 4H),
4.27–4.44 (m, 2H), 4.47–4.57 (m, 6H), 4.62–4.72 (m, 1H), 4.73–
4.77 (m, 1H), 4.85–4.92 (m, 2H), 5.16 (d, J = 3.7 Hz, 1H), 5.33 (d,
J = 3.7 Hz, 1H), 7.98 (s, 1H), 8.03 (s, 1H), 8.09 (s, 1H) ppm; 13C
NMR (75 MHz, D2O) d = 37.1, 37.2, 49.9, 51.9, 52.6, 53.4, 62.5,
71.5, 71.7, 72.2, 73.5, 75.8, 79.9, 83.6, 93.1, 101.0, 105.2, 111.6,
115.4, 119.3, 123.2, 125.5, 126.2, 126.5, 144.1, 144.3, 144.4,
Tetraazido kanamycin 28 (88 mg, 0.15 mmol), alkyne 14
(199 mg, 0.67 mmol), 0.08 M aq sodium ascorbate solution
(3.00 mL, 0.24 mmol), and 0.04 M aq copper(II) acetate solution
(3.00 mL, 0.12 mmol) in tert-butanol (10 mL) were reacted accord-
ing to GP-A. Purification by column chromatography using a gradi-
ent of ethyl acetate/ethanol/water 70:30:0 > 70:15:8 afforded
compound 29 as a white solid (160 mg, 60%). Mp >295 °C (decom-
157.9, 158.0 ppm; ESI-MS m/z: 291.2 [M+3H]3+, 436.7 [M+2H]2+
,
871.5 [M+H]+; HRMS (ESI) calcd for C30H52N18O13 436.1975
[M+2H]2+, found 436.1971.
position); RF = 0.65 (ethyl acetate/ethanol/water = 70:15:8); ½a D27
ꢁ
+28.4 (c = 0.13, MeOH); 1H NMR (300 MHz, methanol-d4)
d = 1.37–1.56 (m, 72H), 2.48 (d, J = 3.3 Hz, 1H), 2.69 (t, J = 9.5 Hz,
1H), 2.82–3.04 (m, 1H), 3.13–3.22 (m, 1H), 3.26–3.33 (m, 8H),
3.44–3.56 (m, 1H), 3.63–3.70 (m, 1H), 3.70–3.81 (m, 1H), 3.81–
3.91 (m, 1H), 3.91–4.16 (m, 4H), 4.17–4.36 (m, 2H), 4.37–4.54
(m, 2H), 4.56–4.75 (m, 8H), 5.07–5.13 (m, 1H), 7.74–8.09 (m, 4H)
ppm; 13C NMR (75 MHz, methanol-d4) d = 28.2, 28.3, 28.6, 28.7,
31.1, 37.0, 58.3, 60.8, 61.3, 61.5, 61.7, 68.7, 69.4, 70.8, 70.9, 71.5,
73.4, 74.0, 74.4, 80.4, 80.5, 84.5, 84.6, 84.9, 154.0, 157.3, 157.4,
157.5, 164.4 ppm; ESI-MS m/z: 889.8 [M+2H]2+, 1779.0 [M+H]+;
HRMS (ESI) calcd for C74H120N24O27Na2 911.4270 [M+2Na]2+, found
911.4256.
3.1.7. tert-Butyl {(1E)-{[(1-{(2R,3R,4R,5S,6R)-2-
{[(1R,2R,3S,4R,6S)-4,6-bis(4-{[N0,N00-bis(tert-
butoxycarbonyl)carbamimidamido]methyl}-1H-1,2,3-triazol-1-
yl)-2,3-dihydroxy-cyclohexyl]oxy}-6-[(4-{[N0,N00-bis(tert-
butoxycarbonyl)carbamimidamido] methyl}-1H-1,2,3-triazol-
1-yl)methyl]-4,5-dihydroxytetrahydro-2H-pyran-3-yl}-1H-
1,2,3-triazol-4-yl)methyl]amino}[(tert-
butoxycarbonyl)amino]methylidene}carbamate (26)
Tetraazido neamine 25 (188 mg, 0.44 mmol), alkyne 14
(594 mg, 2.00 mmol), 0.08 M aq sodium ascorbate solution
(9.2 mL, 0.72 mmol), and 0.04 M aq copper(II) acetate solution
(9.2 mL, 0.36 mmol) in tert-butanol (20 mL) were reacted accord-
ing to GP-A. Purification by column chromatography using a gradi-
ent of dichloromethane/methanol 100:0 > 95:5) afforded
compound 26 as white solid (170 mg, 24%). Mp >315 °C (decompo-
3.1.10. (1S,2R,3R,4S,6R)-4,6-Bis[4-
({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
3-({6-[4-({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-
triazol-1-yl]-6-deoxy-b-
hydroxycyclohexyl 3-[4-
({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
3-deoxy-b- -glucopyranoside tetrakis(trifluoroacetate) (30)
Compound 29 (120 mg, 67.5 mol) was treated with dichloro-
D-glucopyranosyl}oxy)-2-
sition); RF = 0.18 (dichloromethane/methanol = 95:5); ½a D25
ꢁ
+21.7
(c = 0.13, CHCl3); 1H NMR (300 MHz, CDCl3) d = 1.34–1.56 (m,
73H), 2.12–2.34 (m, 1H), 2.51–2.62 (m, 1H), 2.80–3.04 (m, 1H),
3.19–3.32 (m, 1H), 3.88–4.00 (m, 1H), 4.10–4.43 (m, 5H), 4.44–
4.87 (m, 10H), 5.82–5.94 (m, 1H), 7.60–7.95 (m, 4H), 8.68–8.93
(m, 4H), 11.34–11.50 (m, 4H) ppm; 13C NMR (75 MHz, CDCl3)
d = 28.0, 28.3, 79.4, 79.5, 79.6, 80.2, 83.3, 83.5, 83.6, 152.9, 153.0,
155.9, 156.0, 156.1, 156.2, 163.1, 163.3 ppm; ESI-MS m/z: 1617.0
[M+H]+, 1638.0 [M+Na]+; HRMS (ESI) calcd for C68H110N24O22Na
1637.8119 [M+Na]+, found 1637.8105.
D
l
methane/trifluoracetic acid (5:1 v/v, 6 mL) as described in GP-B.
After purification by column chromatography (RP18, H2O), depro-
tected compound 30 was obtained as a white solid (91 mg, 94%).
Mp 140–142 °C; ½a D26
ꢁ
+33.0 (c = 0.12, MeOH); 1H NMR (300 MHz,
methanol-d4) d = 2.40–2.51 (m, 1H), 2.54–2.65 (m, 1H), 2.76 (t,
J = 9.3 Hz, 1H), 3.09–3.21 (m, 1H), 3.31 (dt, J = 3.3, 1.6 Hz, 8H),
3.41–3.55 (m, 1H), 3.64–3.71 (m, 1H), 3.71–3.81 (m, 2H), 3.86
(dd, J = 11.0, 3.7 Hz, 1H), 3.92–4.07 (m, 2H), 4.13–4.23 (m, 2H),
4.24–4.30 (m, 1H), 4.30–4.35 (m, 1H), 4.36–4.62 (m, 11H), 5.31
(d, J = 3.7 Hz, 1H), 7.88 (s, 1H), 7.99 (s, 1H), 8.04–8.08 (m, 1H),
8.14–8.20 (m, 1H) ppm; 13C NMR (75 MHz, methanol-d4)
d = 34.6, 37.2, 37.3, 49.9, 51.2, 60.1, 60.8, 61.0, 66.7, 68.2, 69.9,
70.3, 70.5, 70.8, 72.0, 72.9, 73.2, 73.4, 76.4, 81.1, 81.2, 99.1, 99.2,
116.2, 118.5, 125.2, 125.8, 126.3, 144.3, 144.4, 157.9, 158.0,
3.1.8. (1R,2R,3S,4R,6S)-4,6-Bis[4-
({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
2,3-dihydroxycyclohexyl 2,6-bis[4-
({[amino(iminio)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]-
2,6-dideoxy-a-D-glucopyranoside tetrakis(trifluoroacetate) (27)
Compound 26 (160 mg, 0.10 mmol) was treated with dichloro-
methane/trifluoracetic acid (5:1 v/v, 6 mL) as described in GP-B.
After purification by column chromatography (RP18, MeOH/
H2O = 30:70), deprotected compound 27 was obtained as a white
163.6, 163.9, 164.2, 164.5 ppm; ESI-MS m/z: 326.9 [M+3H]3+
,
489.7 [M+2H]2+; HRMS (ESI) calcd for C34H58N24O11 489.2353
[M+2H]2+, found 489.2357.
solid (90 mg, 71%). Mp 135–136 °C; ½a D27
ꢁ
+20.6 (c = 0.12, MeOH);
1H NMR (300 MHz, methanol-d4) d = 1.95–2.09 (m, 1H), 2.47 (d,
J = 13.2 Hz, 1H), 3.01 (t, J = 9.5 Hz, 2H), 3.27–3.33 (m, 8H), 3.82 (t,
J = 9.7 Hz, 1H), 3.92–4.04 (m, 1H), 4.11 (t, J = 9.7 Hz, 1H), 4.33–
4.69 (m, 12H), 5.86–6.01 (m, 1H), 8.04 (br s, 2H), 8.27 (br s, 2H)
ppm; 13C NMR (75 MHz, methanol-d4) d = 34.7, 37.5, 37.7, 37.8,
Acknowledgement
S.D. and R.H. are thankful to the Studienstiftung des deutschen
Volkes for PhD Grants.

B. Westermann et al. / Carbohydrate Research 371 (2013) 61–67
67
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