Synthesis of glycosidic (β-1′′→6, 3′ and 4′) site isomers of neomycin B and their effect on RNA and DNA triplex stability

Article abstract of DOI:10.3390/molecules24030580

Glycosidic (β-1′′→6, 3′ and 4′) site isomers of neomycin B (i.e., neobiosamine (β-1′′→6, 3′ and 4′) neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3′,4′-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4′-, 5,3′,4′- and 5,6,3′-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.

Full text of DOI:10.3390/molecules24030580

molecules
Article
Synthesis of Glycosidic (β-100→6, 30 and 40) Site
Isomers of Neomycin B and Their Effect on RNA
and DNA Triplex Stability
Lotta Granqvist, Ville Tähtinen and Pasi Virta *
Department of Chemistry, University of Turku, 20014 Turku, Finland;
ltgran@hotmail.com (L.G.); vpotah@utu.fi (V.T.)
* Correspondence: pamavi@utu.fi; Tel.: +358-503285719
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Roger Strömberg
Received: 4 January 2019; Accepted: 4 February 2019; Published: 6 February 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Glycosidic (
β
-100
→
6, 30 and 40) site isomers of neomycin B (i.e., neobiosamine (
β
-100
→
6, 30
and 40) neamines) have been synthesized in a straightforward manner. Peracetylated neomycin
azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6,
30,40-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted
to three different glycosyl acceptors (i.e., 5,6,40-, 5,30,40- and 5,6,30-tri-O-acetyl neamine azide).
Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave
the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation
and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these
aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.
Keywords: aminoglycosides; DNA- and RNA-triple helices; groove binders
1. Introduction
Among the small molecular ligands that target nucleic acids, aminoglycosides (AGs) deserve
special attention [1]. Their binding to a variety of nucleic acid targets has been extensively studied.
Binding to the ribosomal decoding site is the basis of AGs’ bactericidal effect [
2–5
]. The continuous
increase of antibiotic resistant infectious diseases maintains the interest around this RNA target,
and a significant effort has been paid to optimization and modification of existing AG-based lead
compounds to provide new potential antibacterial drugs [
also been reported for other structurally resembling binding sites in ribozymes [
regions of HIV RNAs (Trans Activation Response element (TAR), Reverse Response Element (RRE)
and Dimerization Initiation Site (DIS)) [ 12]). In these RNA targets, the binding sites are bulges and
6,
7]. Relatively high binding affinities have
7,8
] and important
9–
internal loops, which, in contrast to canonical double helices, are able to form appropriate hydrogen
bonds and electrostatic interactions with AGs. AGs can also act as groove binders that stabilize
DNA- and RNA-triple helices and their hybrids [13–17]. Among the AGs studied for the triplex
recognition, neomycin B has shown to be the most effective groove binder. The binding occurs to the
Watson–Hoogsteen groove, in which favorable contacts with rings II and IV (cf. Scheme 1), together
with the appropriate shape complementarity, may take place [14,17]. In each case, however, electrostatic
interactions dominate AGs’ affinity to nucleic acids, which makes the binding promiscuous and may
disturb detailed characterization of the binding motifs. Conformational adaption of the nucleic acid
targets (in particular RNA) disturbs modelling and discovery of specific ligands even further [1].
Chemically synthesized structural analogs of the known AG-ligands are hence important tools that
may be used for the exclusion analysis of the binding requirements.
Molecules 2019, 24, 580; doi:10.3390/molecules24030580
www.mdpi.com/journal/molecules

Molecules 2019, 24, 580
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In the present study, a straightforward synthesis of glycosidic (
β
-100
→
6, 30 and 40) site isomers of
neomycin B is described (Note: C6–, C30– and C40–substituted neamines are not neomycin class
AGs, i.e., 4,5-disubstituted deoxystreptamines, and the correct names for these compounds are
neobiosamine(
β
-C100
→
C6, 30 and 40)neamines). The key steps of the synthesis (Scheme 1) were
(1) acid-catalyzed thiolysis of neomycin azide [18], which gave useful intermediates for both glycosyl
donor and the acceptors, and (2) selective diacetylation of neamine azide, which after simple protecting
group manipulation afforded the glycosyl acceptors. Glycosylation between the neobiosamine donor
and triacetylated neamine acceptors resulted in the azide masked pseudo-tetrasaccharides, which, via
global deprotection, were converted to the desired site isomers of neomycin B (Scheme 2). In order
to evaluate how the changed glycosidic connection (
neobiosamine cores affects the groove binding, the stability of RNA and DNA triple helix models in
the presence of these structural analogs (and neomycin B) was studied by UV-melting profile analysis.
β
-100
→
30,40 or 6) between the neamine and
2. Results and Discussion
2.1. Synthesis of Glycosyl Donor 6 and Acceptors 14–16
Acid-catalyzed degradation of neomycin B azide (
Various conditions by using different Lewis acids (SnCl4, TMSOTf and BF3
(propylenedithiol, EtSH and TolSH) have been evaluated for the selective cleavage. According to
this data, was cleaved with thiophenol in the presence of BF3 OEt2, to yield an anomeric
) and 6,30,40-tri-O-acetyl
3
) has been studied in detail by Wu et al.
[18].
·
OEt2) and thiols
3
·
mixture of phenylthio 2,5,3040-tetra-O-acetyl 20,60-diazidoneobiosamine (
4
1,3,20,60-tetraazido neamine (5) in high yields (67% and 70%, respectively, iii in Scheme 1).
The anomeric mixture of phenylthio 2,5,3040-tetra-O-acetyl 20,60-diazidoneobiosamine (
4) may be
used as such as a glycosyl donor, but the mixture of the donor makes the control and monitoring of
the reaction (cf. Scheme 2) complex. Therefore, the phenylthioglycoside ( ) was hydrolyzed and the
resulted hemiacetal converted to pure -anomer of tricloroacetimidate . Overall, this leaving group
conversion (from 4 to 6) could be carried out in 86% yield (iv and v/Scheme 1).
The 5-OH group of neamine (
) is less nucleophilic than the 6-, 30- and 40-OH groups. For this
reason, selective triacetylation of N-protected neamines (e.g., from ) may be readily carried out
as described previously in several studies [19 20]. On using a smaller excess of acetic anhydride,
diacetylated neamines ( 10) may also be obtained, albeit the yield and ratio of the products ( 10
4
α
6
7
5
7
,
8–
8
,
9,
, 5
and monoacetylated neamines) are more sensitive to reaction conditions (temperature, reaction time,
and the addition rate and excess of acetic anhydride). Overnight reaction in pyridine in the presence of a
catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) and 2.5 equiv. of acetic anhydride (slowly
added to the reaction mixture) resulted in the desired diacetylated neamines
and 15% yield, respectively (
and 10 isolated as a mixture (4:6, n/n)) (iib/Scheme 1). The triacetylated
neamine was obtained as the major product (39% yield), but it could be readily converted back to
the starting material ( ) and the reaction repeated. It may be worth mentioning that a smaller excess
of acetic anhydride did not markedly improve the yields of 10. Moreover, the increased ratio of
8, 9 and 10 in 18%, 10%
9
5
7
8–
monoacetylated neamines disturbed the chromatographic isolation of the diacetylated products. Thus,
the slight ‘overacetylation’ was beneficial. The modest reactivity of the 5-OH group of the diacetylated
neamines (
groups to obtain 11
1.5 equiv. of levulinic anhydride in the presence of a catalytic amount of DMAP. The remained free
5-OH group of 11
13 was finally acetylated using an excess of acetic anhydride (5 equiv. iic/Scheme 1)
8, 9
and 10) was next utilized for the selective levulinoylation of the 30-, 6- and 40-OH
–13 (70% – quant., vii/Scheme 1). The reaction was carried out in pyridine using
–
and subsequent removal of the levulinoyl group with hydrazine hydrate (viii/Scheme 1) gave the
desired glycosyl acceptors 14–16 in 73–87% yield.

Molecules 2019, 24, 580
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Scheme 1. Reagents and conditions: i) CuSO4, TfN3, dichloromethane, H2O, iia-c) Ac2O (a: 10 equiv.,
b: 2.5 equiv., c: 5 equiv.), DMAP, pyridine, overnight at r.t. iii) PhSH, BF3
·
Et2O, DCM, iv)
◦
N-bromosuccinimide, trifluoroacetic acid, H2O, MeCN, dichloromethane, for 1h at 0 C–r.t., v) CCl3CN,
◦
−1
1.8-diazabicyclo(5.4.0)undec-7-ene, DCM, for 1h at 0 C, vi) 0.1 mol L NaOMe/MeOH, for 1h at r.t.,
vii) Lev2O, 4-(N,N-dimethylamino)pyridine, pyridine, overnight at r.t., viii) NH2NH2 AcOH, MeOH,
·
dichloromethane, for 3 h at r.t.
2.2. Glycosylation and Global Deprotection
A standard procedure using TMSOTf as a catalyst was applied for the glycosylation between
◦
acceptors 14
–
16 and donor
6
(1.5 equiv.). Each reaction was performed at
−
20 C (for 2 h) in dry
dichloromethane under nitrogen. For the purification reasons (the fully protected pseudo-tetrasaccharides
17 19 remained contaminated by traces of donor , despite a laborious chromatographic purification),
the glycosylation was followed by subsequent deacetylation (0.1 mol L−1 NaOMe in MeOH)
and the neomycin azides 20 22 could be isolated in acceptable yields (49–69%). The purity of
the products (20 22) was confirmed by reversed phase-high performance liquid chromatography
–
6
–
–

Molecules 2019, 24, 580
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(RP HPLC) analysis (Figure 1). Finally, the azide masks were removed by Staudinger reaction using
trimethylphosphine and aqueous ammonia, followed by elution through an ion exchange resin, to
give the glycosidic (β-C100→C6, C30 and C40) site isomers of neomycin B (23–25) in 58–83% yields.
Figure 1. RP HPLC profiles of the azide-masked neobiosamine neamines (20
An analytical RP HPLC (C18, 250 4.6 mm, 5 m) column, detection at = 220 nm, gradient elutions
from 10% to 80% (a–c) and from 10% to 60% (d) acetonitrile in H2O over 30 min (a–c) and 35 min (d).
–22). Conditions:
×
µ
λ
Scheme 2. Reagents and conditions: i) TMSOTf, DCM, 2 h at
1 h at r.t.; iii) 1: Me3P, toluene, water, dioxane, 4 h at r.t. under N2, 2: conc. ammonia, overnight at r.t.
−
20 ◦C; ii) 0.1 mol L−1 NaOMe/MeOH,

Molecules 2019, 24, 580
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2.3. The Effect of 23–25 on DNA and RNA Triplex Stability
The effect of the glycosidic site isomers of neomycin B (23
–25) and neomycin (
1) on the stability
of simple DNA and RNA triple helices was studied by UV-melting profile experiments (Table 1).
The DNA triple helix is consisted of a purine rich region of c-Myc promoter 1 [21], used as a model in
several previous studies [22
–
27]. The formation of this triple helix, consisting of CH+
G-C-triplets,
•
requires slightly acidic pH. The intramolecular RNA triplex is also a known model (in fact, this model
may exist as a mixture with its dimer [28]). The measurements were carried out using 2 µ
mol L−1 of
the oligonucleotides in a mixture of 10 mmol L−1 sodium cacodylate and 0.1 mol L−1 NaCl at pH 6.0
(both RNA and DNA model) and at pH 7.0 (RNA model only) in the presence of 5 and 10 eq. of the
AGs (
(triplex and duplex melting points) were extracted from the first and second inflection points of the
biphasic melting curves at = 260 nm, respectively (Figure 2). Consistent with the previous findings
1 and 23–
25). The temperature was changed at a rate of 0.2 ◦C min−1. The Tm3- and Tm2-values
λ
(affinity of neomycin follows the trend: DNA duplex < DNA triplex < RNA duplex ~ RNA triplex [17]),
AGs stabilized both the triplex and the duplex on the RNA model at pH 6.0, whereas basically only the
◦
◦
2.3 C). A slight selectivity of the
triplex on the DNA model (
stabilization on the RNA triplex vs. duplex may be observed at pH 7.0 (
vs. +1.1
1.9 ◦C). Most importantly, AGs’ effect on the triplex (both on RNA and DNA model) did
not show a marked difference when AGs are compared to each other. Only a slight variation was
observed. For example, on the DNA model, the stability of the triplex increased by
m = +18.6 ◦C,
), the C100 C40 (25),
∆
Tm3 vs.
∆
Tm2 = +11.1
−
18.6 C vs. +0.7
−
3
2
◦
∆
Tm vs.
∆
Tm = +6.9 7.8 C
−
−
∆
T
◦
◦
◦
+17.4 C, +15.8 C and +13.9 C in the presence of 10 equiv. of neomycin B (
1
→
C100
C100
→
C30 (23) and C100
C30 (23) and C100
) (
→
C6 (24) site isomers, respectively. On the RNA triplex, the effect with the
C6 (24) site isomers was, in fact, slightly higher compared to that with
Tm3 = +11.3 and +10.6 ◦C vs. +9.3 ◦C). Probably, the carbohydrate core and spatial
→
→
neomycin B (
1
∆
orientation of the amino groups on it do not play a marked enough role to result in discrimination
between the affinities of the glycosidic site isomers ( 23 25). This may be an unexpected observation
as, e.g., previous studies with kanamycin class aminoglycosides (kanamycin = a 4,6-disubstituted
deoxystreptamine consisted of neamine and -D-glucos-3-amine at C6) show a modest or no effect
1
,
–
α
on the poly U-A-U DNA triplex [14]. The effect of 24 (a 4,6-disubstituted deoxystreptamine) on
3
the DNA triplex was the smallest (
neomycin B (
∆
Tm = +13.9 ◦C) of the isomers (23
–
25), but still comparable to
1
,
∆Tm3 = +18.6 ◦C), the most effective aminoglycosidic DNA triplex groove binder to
date. The biosamine moiety, or the L-neosamine (Ring IV in Scheme 1), hence plays a marked role in
the groove binding despite the glycosidic site on the neamine core.
1,0
0,8
0,6
0,4
0,2
0,0
1,0
0,8
0,6
0,4
0,2
0,0
10
20
30
40
50
60
70
80
10
20
30
40
50
60
70
80
Figure 2. Representative melting profiles of the DNA and RNA triplex models in the presence of
aminoglycosides (AGs) (2
mol oligonucleotides in the presence of 5 eq. 1, 23–25, 10 mmol L−1 sodium
cacodylate, 0.1 mol L–1 NaCl, pH 6.0.
µ

Molecules 2019, 24, 580
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◦
Table 1. UV-melting experiments (Tm/ C).
RNA Triplex Model
AG
Tm3(◦C) pH 6.0
Tm2(◦C) pH 6.0
none
41.9
60.1
1 (5 eq)
23 (5 eq)
24 (5 eq)
25 (5 eq)
51.2 (+9.3)
53.2 (+11.3)
52.5 (+10.6)
51.4 (+9.5)
68.0 (+7.9)
66.7 (+6.6)
67.7 (+7.6)
68.0 (+7.9)
AG
Tm3(◦C) pH 7.0
Tm2(◦C) pH 7.0
none
22.6
60.1
1 (5 eq)
23 (5 eq)
24 (5 eq)
25 (5 eq)
29.4 (+6.9)
30.4 (+7.8)
30.4 (+7.8)
29.4 (+6.9)
62.0 (+1.9)
61.2 (+1.1)
61.6 (+1.5)
61.9 (+1.8)
DNA Triplex Model
AG
Tm3(◦C) pH 6.0
Tm2(◦C) pH 6.0
none
25.5
54.6
1 (5 eq)
40.1 (+14.6)
38.1 (+12.6)
36.6 (+11.1)
38.6 (+13.1)
44.1 (+18.6)
41.3 (+15.8)
39.4 (+13.9)
42.9 (+17.4)
56.3 (+1.7)
55.5 (+0.9)
56.3 (+1.7)
55.3 (+0.7)
56.3 (+1.7)
55.9 (+1.3)
56.9 (+2.3)
56.5 (+1.9)
23 (5 eq)
24 (5 eq)
25 (5 eq)
1 (10 eq)
23 (10 eq)
24 (10 eq)
25 (10 eq)
∆Tm-values in parentheses. Error limits for each Tm-value (an average of three temperature ramps) were less than
3
2
1 ◦C. Tm and Tm correspond to melting values of the triplex and duplex, respectively.
3. Materials and Methods
General Remarks
Pyridine, methanol and dichloromethane were dried over 3Å molecular sieves. Nuclear magnetic
resonance (NMR) spectra were recorded using a 500 MHz instrument. The chemical shifts for 1H and
13C-NMR resonances are given in parts of million from the residual signal of the deuterated solvents
(CD3OD and CD3Cl3). Mass spectra were recorded using electrospray ionization (ESI-TOF). The NMR
spectral data for all new compounds are showed in the Supplementary Materials.
2,5,30,40-Tetra-O-acetyl-20,60-diazido-1-phenylthio-
5.8 g, 5.6 mmol, synthesized according to the literature [18
α/β-neobiosamine (4). Peracetylated neomycin azide (3,
,
20]) and PhSH (0.64 mL, 6.2 mmol) were
◦
dissolved in dry dichloromethane (50 mL). The mixture was cooled down to 0 C, and BF3
·
Et2O
(2.1 mL, 17 mmol) was slowly added under nitrogen. The mixture was stirred first at 0 ◦C for 20 min,
and allowed then to warm up to room temperature. After overnight reaction, triethylamine (1.5 mL)
was added, the mixture was diluted with dichloromethane (150 mL), and washed with water (20 mL).

Molecules 2019, 24, 580
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The organic phase was dried over Na2SO4, filtered and evaporated to dryness. The residue was
purified by silica gel chromatography (30-40% EtOAc in petroleum ether) to give an anomeric mixture
of
4
(2.3 g, 67%,
α
/
β
= 7:3, v/v, pure fractions used for the NMR characterization) and 1,3,20,60-tetra
azido-6,30,40-tri-O-acetyl neamine (
5, 2.2 g, 70%).
4:
β
-anomer: H (500 MHz, CDCl3):
δ
7.52–7.50 (m,
1
2H), 7.34–7.28 (m, 3H), 5.74 (d, 1H, J = 4.7 Hz), 5.48 (t, 1H, J = 4.2 Hz), 5.06 (t, 1H, J = 2.7 Hz), 5.03
(d, 1H, J = 1.5 Hz), 4.73 (b, 1H), 4.50–4.47 (m, 3H), 4.26 (m, 1H), 4.14 (m, 1H), 3.64 (dd, 1H, J = 13.1
and 8.6 Hz), 3.42 (b, 1H), 3.22 (dd, 1H, J = 13.1 and 3.7 Hz), 2.20, 2.20, 2.18 and 2.09 (4
×
s, 3H each);
13C (125 MHz, CDCl3):
δ
170.7, 170.2, 169.9, 168.5, 134.5, 131.5, 129.1, 127.5, 98.5, 89.1, 78.4, 75.8, 73.9,
72.6, 68.8, 65.8, 63.0, 56.4, 50.7, 20.8, 20.8, 20.7, 20.6; HRMS (ESI-TOF) calculated for C25H30N6KO11S
1
[M + K]+: 661.1330, observed: 661.1332.
α
-anomer: H (500 MHz, CDCl3):
δ
7.54–7.52 (m, 2H), 7.38–7.33
(m, 3H), 5.43 (d, 1H, J = 2.9 Hz), 5.26 (dd, 1H, J = 5.1 and 3.0 Hz), 5.05 (dd, 1H, J = 2.9 and 2.8 Hz), 4.92
(d, 1H, J = 1.8 Hz), 4.73 (t, 1H, J = 2.0 Hz), 4.51 (dd, 1H, J = 12.1 and 2.6 Hz), 4.45 (dd, 1H, J = 7.3 and
5.2 Hz), 4.31 (m, 1H), 4.23 (dd, 1H, J = 12.1 and 5.1 Hz), 4.11 (ddd, 1H, J = 8.1, 4.4 and 1.8 Hz), 3.61 (dd,
1H, J = 13.0 and 8.2 Hz), 3.34 (dd, 1H, J = 2.1 and 2.0 Hz), 3.29 (dd, 1H, J = 13.0 and 4.4 Hz), 2.19, 2.18,
2.13 and 2.10 (4
×
s, each 3H); 13C (125 MHz, CDCl3):
δ 170.7, 170.3, 169.8, 168.6, 132.9, 132.2, 129.1,
128.3, 98.9, 88.7, 80.1, 76.5, 75.0, 73.5, 68.7, 65.7, 63.2, 56.4, 50.6, 20.9, 20.8, 20.7, 20.7; HRMS (ESI-TOF)
calculated for C25H30N6NaO11S [M + Na]+: 645.1591, observed: 645.1563.
2,5,30,40-Tetra-O-acetyl-20,60-diazido-neobiosamine trichloroacetimidate (
(0.77 g, 4.3 mmol) and trifluoroacetic acid (0.30 mL) were added to a two-phase mixture of
6
). N-bromosuccinimide (NBS)
4
(1.4 g,
2.2 mmol, as an anomeric mixture) in dichloromethane (15 mL), acetonitrile (7.5 mL) and H2O (15 mL) at
◦
0 C. The mixture was allowed to warm up to ambient temperature, stirred for 1 h, poured to saturated
NaHCO3 and extracted with dichloromethane. The organic layers were combined, dried over Na2SO4,
filtered and evaporated to dryness. The residue was purified by silica column chromatography (5%
MeOH in DCM) to give an anomeric mixture of the neobiosamine hemiacetal (1.1 g, 98%) as a white
1
foam. H-NMR (500 MHz, CDCl3)
δ
: 5.52 (dd, 0.25H, J = 10.3 and 4.1 Hz), 5.41 (d, 1H, J = 2.5 Hz), 5.23
(d, 1H, J = 4.5 Hz), 5.08 (dd, 0.25 H, J = 3.2 and 3.1 Hz), 5.05–5.02 (m, 1.25H), 5,00 (d, 0.25H, J = 2.7 Hz),
4.99 (d, 1H, J = 1.8 Hz), 4.75 (t, 0.25H, J = 2,3 Hz), 4.72 (dd, 1H, J = 1.9 and 1.8 Hz), 4.67 (dd, 1H, J = 7.5
and 4.5 Hz), 4.53 (m, 0.25H), 4.50 (dd, 1H, J = 11.6 and 2.3 Hz), 4.46 (t, 0.25H, J = 5.2 Hz), 4.38 (dd,
0.25H, J = 12.2 and 2.8 Hz), 4.31 (m, 1H), 4.28–4.21 (m, 1.25H), 4.15 (ddd, 1H, J = 8.3, 4.1 and 1.8 Hz),
4.11 (ddd, 0.25H, J = 8.5, 4.0 and 2.0 Hz), 3.66–3.60 (m, 1.25H), 3.46 (t, 0.25H, J = 2.4 Hz), 3.42 (d, 0.25H,
J = 4.1 Hz), 3.39 (d, 1H, J = 3.0 Hz), 3.36 (t, 1H, J = 1.9 Hz), 3.29 (dd, 0.25H, J = 13.1 and 4.1 Hz), 3.24
(dd, 1H, J = 13.1 and 4.1 Hz), 2.20, 2.18, 2.17, 2.13, 2.11 and 2.11 (each s, 15H); 13C-NMR (125 MHz,
CDCl3) δ: 171.0, 170.3, 169.9, 168.6, 100.2, 99.1, 98.6, 95.8, 79.3, 79.1, 76.4, 76.2, 74.4, 73.8, 73.5, 71.9,
68.8, 68.7, 65.8, 65.8, 64.5, 63.4, 50.8, 20.9, 20.8, 20.7, 20.7; MS (ESI-tOF): calculated for C19H26N6NaO12
[M + Na]+: 553.1506, observed 553.1488. The hemiacetal (1.0 g, 1.9 mmol) was dissolved in a mixture of
dich◦loromethane (1.0 mL) and trichloroacetonitrile (1.9 mL, 19 mmol). The mixture was cooled down
to 0 C and 1.8-diazabicyclo(5.4.0)undec-7-ene (DBU) (56 µL, 0.38 mmol) was added. The mixture was
stirred for 1 h at 0 ◦C and eluted as such (without a work up) through a silica gel column (1% Et3N,
50% EtOAc in petroleum ether) to give 1.2 g (91%, 84% overall yield from ) of the trichloroacetimidate
product ( 8.36 (s, 1H), 6.36 (s, 1H), 5.47 (d, 1H,
) as a white foam. 1H-NMR (500 MHz, CDCl3):
4
6
δ
J = 4.6 Hz), 5.06–5.04 (m, 2H) 4.73 (dd, 1H, J = 1.9 and 1.8 Hz), 4.68 (dd, 1H, J = 8.1 and 4.5 Hz), 4.53 (dd,
1H, J = 12.1 and 2.8 Hz), 4.45 (m, 1H), 4.22 (dd, 1H, J = 12.2 and 6.4 Hz), 4.19 (ddd, 1H, J = 8.6, 3.7 and
1,8 Hz), 3.63 (dd, 1H, J = 13.1 and 8,6 Hz), 3.35 (dd, 1H, J = 2.1 and 1.9 Hz), 3.21 (dd, 1H, J = 13.1 and
3.7 Hz), 2.20, 2.19, 2.18, 2.11 (each s, each 3H); 13C-NMR (125 MHz, CDCl3):
δ 170.8, 170.1, 169.8, 168.6,
160.3, 102.7, 98.4, 90.6, 80.2, 75.3, 73.9, 73.0, 68.7, 65.8, 64.0, 56.3, 50.7, 20.8, 20.8, 20.7, 20.6; MS (ESI-TOF):
calculated for C21H26Cl3N7NaO12 [M + Na]+: 696.0603, observed 696.0631.
Synthesis of diacetylated neamine azides (
added to a mixture of neamine azide (
stirred overnight at ambient temperature, concentrated, poured to saturated NaHCO3 and extracted
8
,
9
and 10). Acetic anhydride (2.3 mL, 24 mmol) was slowly
7, 4.0 g, 9.3 mmol) in pyridine (20 mL). The mixture was

Molecules 2019, 24, 580
8 of 13
with ethyl acetate. The organic layers were combined, washed with brine, dried over Na2SO3,
filtered and evaporated to dryness. The residue was purified by silica gel chromatography (40%
EtOAc in petroleum ether) to give three fractions of products. The first eluting compound was
6,30,40-tri-O-acetyl-1,03,20,60-tetra-azido-neamine (
n/n, 1.3 g, 25%) and the third fraction was pure 6,40-di-O-acetyl-1,3,20,60-tetra-azidoneamine (
5
, 2.0 g, 39%, the m0ain product). The second fraction
0
0
0
was a mixture of 3 ,4 -di-O-acetyl- (
9
) and 6, 3 -di-O-acetyl- (10) 1,3,2 ,6 -tetra-azidoneamines (
9:
10, 4:6,
8
δ
, 0.87 g,
5.32 (d,
1
18%). Each product was obtained as a white foam. Compound
8
: H (500 MHz, CDCl3):
1H, J = 3.4 Hz), 4.96 (t, 1H, J = 10.0 Hz), 4.91 (t, 1H, J = 9.7 Hz), 4.24 (ddd, 1H, J = 10.0, 5.0 and 2.8Hz),
4.12 (dd, 1H, J = 9.0 and 8.6 Hz), 3.86 (d, 1H, J = 3.3 Hz), 3.67 (ddd, 1H, J = 12.2, 9.5 and 2.9 Hz), 3.60
(dd, 1H, J = 10.1 and 3.7 Hz), 3.54 (ddd, 1H, J = 12.5, 10.0 and 4.6 Hz), 3.46–3.41 (m, 2H), 3.38–3.33
(m, 2H), 3.03 (b, 1H), 2.40 (ddd, 1H, J = 13.4, 4.6 and 4.5 Hz), 2.20 (s, 3H), 2.18 (s, 3H), 1.63 (ddd, 1H,
J = 12.6 Hz, each); 13C (125 MHz, CDCl3):
58.0, 51.0, 32.0, 20.9. A mixture of 9 + 10: H (500 MHz, CDCl3): δ 5.51 (dd, 0.4H, J = 10.3 and 9.4 Hz),
δ 171.4, 170.5, 98.9, 83.7, 74.9, 74.4, 72.3, 71.7, 69.5, 64.2, 58.6,
1
5.36 (d, 0.4H, J = 5.0 Hz), 5.34 (d, 0.6H, J = 3.7 Hz), 5.30 (dd, 0.6H, J = 10.1 and 9.7 Hz), 5.07 (dd, 0.4H,
J = 9.9 and 9.6 Hz), 4.94 (dd, 0.6H, J = 9.8 and 9.7 Hz), 4.37 (ddd, 0.4H, J = 10.2, 4.9 and 2.7 Hz), 4.17
(ddd, 0.6H, J = 10.1, 4.3 and 2.8 Hz), 3.77 (d, 0.4H, J = 2.7 Hz), 3.72–3.62 (m, 3.4H), 3.59–3.52 (m, 1.6H),
3.47–3.31 (m, 3.2H), 2.98 (b, 0.4H), 2.78 (d, 0.6H, J = 5.6 Hz), 2.43–2.36 (m, 1H), 2.22 (s, 1.8H), 2.20 (s, 1.8
H), 2.12 (s, 1.2 H), 2.08 (s, 1.2 H), 1.66–1.55 (m, 1H); 13C (125 MHz, CDCl3):
δ 172.1, 170.5, 170.2, 169.8,
99.0, 98.6, 83.7, 83.2, 75.9, 75.4, 74.9, 74.8, 74.4, 71.9, 71.3, 69.9, 69.5, 69.3, 61.72, 61.71, 59.7, 58.6, 58.4,
57.9, 53.5, 50.9, 50.8, 32.0, 31.9, 20.9, 20.8, 20.7, 20.6.
6,40-Di-O-acetyl-30-O-levulinyl-1,3,20,60-tetra-azidoneamine (11). Freshly prepared levulinic anhydride
(63 mg, 0.29 mmol) was added to a mixture of
8 (0.10 g, 0.20 mmol) in pyridine (2.0 mL) and a catalytic
amount of 4-(N,N-dimethylamino)pyridine was added. The mixture was stirred overnight at ambient
temperature, poured to saturate NaHCO3, and the product was extracted with dichloromethane.
The organic layer was separated, dried over Na2SO4 and evaporated to dryness. The residue was
purified by silica gel chromatography (5% MeOH in DCM) to give 120 mg (quant.) of the product
1
(
11) as a white foam. H-NMR (500 MHz, CDCl3):
δ
5.52 (dd, 1H, J = 10.1 and 9.9 Hz), 5.43 (d, 1H,
J = 3.6 Hz), 5.07 (t, 1H, J = 9.8 Hz), 4.94 (dd, 1H, J = 10.0 & 9.9 Hz), 4.34 (ddd, 1H, J = 7.9, 5.0 and
2.7 Hz), 3.88 (b, 1H), 3.69 (t, 1H, J = 9.5 Hz), 3.61 (dd, 1H, J = 10.6 and 3.6 Hz), 3.54 (ddd, 1H, J = 12.4,
10.2 and 4.4 Hz), 3.47 (dd, 1H, J = 9.8 and 9.2 Hz), 3.43–3.35 (m, 2H), 3.32 (dd, 1H, J = 13.5 and 5.2 Hz),
2.82 (m, 1H), 2.71 (m, 1H), 2.62 (m, 1H), 2.54 (m, 1H), 2.41 (ddd, 1H, J = 13.4, 4.5 and 4.5 Hz), 2.19 (s,
3H), 2.17 (s, 3H), 2.13 (s, 3H), 1.63 (ddd, 1H, J = 12.6 Hz. each); 13C (125 MHz, CDCl3):
δ 206.0, 171.9,
170.5, 170.0, 98.8, 83.2, 75.1, 74.4, 71.0, 69.6, 68.9, 61.8, 58.3, 57.9, 50.9, 37.6, 31.9, 29.7, 27.9, 20.8, 20.7;
HRMS (ESI-TOF) calculated for C21H28N12NaO10 [M + Na]: 631.1949, observed: 631.1924.
30,40-Di-O-acetyl-6-O-levulinyl-1,3,20,60-tetra-azidoneamine (12) and 6,30-Di-O-acetyl-40-levulinyl-1,3,20,60-
tetra-azidoneamine (13). The mixture of
11 (from
(500 MHz, CDCl3):
9
and 10 (1:2, n/n, 0.38 g, 0.74 mmol) was treated as described for
1
8
) above. 0.11 g (70%) of 12 and 0.27g (88%) of 13 were obtained as white foams. 12: H-NMR
δ
5.57 (d, 1H, J = 3.7 Hz), 5.48 (dd, 1H, J = 11.1 and 9.3 Hz), 5.04 (dd, 1H, J = 10.1
and 9.4 Hz), 4.94 (t, 1H, J = 9.9 Hz), 4.39 (ddd, 1H, J = 10.1, 5.0 and 2.8 Hz), 3.87 (d, 1H, J = 3.1 Hz),
3.76 (ddd, 1H, J = 9.4, 9.4 and 3.0 Hz), 3.53–3.42 (m, 2H), (t, 1H, J = 7.0 Hz), 3.50 (dd, 1H, J = 10.7 and
3.7 Hz), 3.39 (dd, 1H, J = 13.5 and 2.9 Hz), 3.31 (dd, 1H, J = 13.4 and 5.1 Hz), 2.93 (m, 1H), 2.81 (m, 1H),
2.66 (m, 1H), 2.57 (m, 1H), 2.40 (ddd, 1H, J = 13.3, 4.5 and 4.5 Hz), 2.21 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H),
1.64 (ddd, 1H, J = 12.5 Hz, each); 13C (125 MHz, CDCl3):
δ 207.8, 172.3, 170.2, 169.7, 98.4, 81.4, 75.8,
74.7, 70.6, 69.4, 69.3, 61.2, 58.5, 57.7, 50.8, 38.4, 31.9, 29.8, 28.1, 20.7, 20.6; HRMS (ESI-TOF) calculated
1
for C21H28N12NaO10 [M + Na]: 631.1949, observed: 631.1970. 13: H-NMR (500 MHz, CDCl3):
δ 5.52
(dd, 1H, J = 10.3 and 9.6 Hz), 5.38 (d, 1H, J = 3.6 Hz), 5.07 (t, 1H, J = 9.8 Hz), 4.94 (t, 1H, J = 9.8 Hz),
4.32 (ddd, 1H, J = 10.2, 4.9 and 2.8 Hz), 3.69 (m, 1H), 3.64 (dd, 1H, J = 10.6 and 3.7 Hz), 3.55 (ddd, 1H,
J = 12.6, 10.1 and 4.6 Hz), 3.48–3.36 (m, 3H), 2.76 (m, 2H), 2.51 (m, 2H), 2.41 (ddd, 1H, J = 13.5, 4.6 and
4.5 Hz), 2.18 (s, 6H), 2.14 (s, 3H), 1.63 (ddd, 1H, J = 12.5 Hz, each); 13C (125 MHz, CDCl3):
δ 206.2, 171.6,

Molecules 2019, 24, 580
9 of 13
170.3, 98.8, 83.5, 75.0, 74.4, 70.7, 69.6, 69.2, 61.8, 58.3, 57.9, 50.7, 37.7, 31.9, 29.6, 27.8, 20.7, 20.7; HRMS
(ESI-TOF) calculated for C21H28N12NaO10 [M + Na]: 631.1949, observed: 631.1961.
0
0
0
5,6,4 -Tri-O-acetyl-1,3,2 ,6 -tetra-azidoneamine (14). Acetic anhydride (85 µL, 0.90 mmol) was added to a
mixture of 11 (0.11 g, 0.18 mmol) and a catalytic amount of 4-(N,N-dimethylamino)pyridine in pyridine
(2.0 mL). The mixture was stirred at ambient temperature for 3 h, poured to saturated NaHCO3, and
the product was extracted with dichloromethane. The organic layers were combined, dried over
Na2SO4, and evaporated to dryness. The residue was dissolved in a mixture dichloromethane (6.0 mL)
and methanol (1.0 mL) and hydrazine acetate (34 mg, 0.36 mmol) was added. The mixture was stirred
at ambient temperature for 3 h, poured to 10% aqueous KH2PO4, and the product was extracted with
dichloromethane. The organic layers were combined, washed with brine, dried over Na2SO4, and
evaporated to dryness. The residue was purified by silica gel chromatography (2% MeOH in DCM) to
1
give 73 mg (73%) of the product 14 as a white foam. H-NMR (500 MHz, CDCl3):
δ 5.15 (dd, 1H,m
J = 9.8 Hz, both), 5.14 (d, 1H, J = 3.3 Hz), 4.95 (t, 1H, J = 10.0 Hz), 4.90 (dd, 1H, J = 9.7 and 9.6 Hz), 4.32
(m, 1H), 4.07 (t, 1H, J = 9.7 Hz), 3.69–3.64 (m, 2H), 3.51 (m, 1H), 3.39–3.24 (m, 4H), 2.44 (ddd, 1H, J = 13.3,
4.4 and 4.4 Hz), 2.15 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 1.64 (ddd, 1H, J = 12.8 Hz, each); 13C (125 MHz,
CDCl3):
δ 171.0, 169.9, 169.8, 98.9, 78.6, 74.1, 73.4, 72.1, 69.7, 69.6, 62.9, 58.7, 57.7, 51.0, 31.8, 20.9, 20.7,
20.5; HRMS (ESI-TOF) calculated for C18H24N12NaO9 [M + Na]: 575.1687, observed: 575.1680.
0
0
0
0
5,3 ,4 -Tri-O-acetyl-1,3,2 ,6 -tetra-azidoneamine (15). Compound 15 was synthesized from 12 as described
1
for 14 above. 12 (0.13 g, 0.22 mmol) gave 98 mg (87%) of the product (15) as a white foam. H-NMR
(500 MHz, CDCl3):
δ 5.46 (dd, 1H, J = 10.7 and 9.3 Hz), 5.26 (d, 1H, J = 3.8 Hz), 5.09–5.04 (m, 2H),
4.48 (ddd, 1H, J = 10.2, 4.8 and 2.8 Hz), 3.63–3.53 (m, 3H), 3.48 (m, 1H), 3.42–3.30 (m, 3H), 2.94 (d, 1H,
J = 6.3 Hz), 2.41 (ddd, 1H, J = 13.3, 4.4 and 4.4 Hz), 2.20 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 1.55 (ddd,
1H, J = 12.3 Hz, each); 13C (125 MHz, CDCl3):
δ 171.2, 170.0, 169.8, 98.7, 78.7, 76.2, 75.6, 69.8, 69.5, 69.3,
60.7, 60.3, 58.6, 50.7, 31.6, 20.9, 20.6, 20.6; HRMS (ESI-TOF) calculated for C18H24N12NaO9 [M + Na]:
575.1687, observed: 575.1668.
0
0
0
5,6,3 -Tri-O-acetyl-1,3,2 ,6 -tetra-azidoneamine (16). Compound 16 was synthesized from 13 as described
1
for 14 above. 13 (0.28 g, 0.47 mmol) gave 98 mg (75%) of the product (16) as a white foam. H-NMR
(500 MHz, CDCl3):
δ 5.28 (dd, 1H, J = 10.6 and 9.3 Hz), 5.18 (d, 1H, J = 3.8 Hz), 5.15 (t, 1H, J = 9.8 Hz),
4.95 (t, 1H, J = 10.1 Hz), 4.29 (m, 1H), 3.74 (ddd, 1H, J = 14.6, 10.1 and 4.6 Hz), 3.67 (t, 1H, J = 9.8 Hz),
3.67–3.54 (m, 4H), 3.34 (dd, 1H, J = 10.7 and 3.8 Hz), 3.09 (d, 1H, J = 6.8 Hz), 2.47 (ddd, 1H, J = 13.4, 4.6
and 4.6 Hz), 2.19 (s, 3H), 2.10 (s, 6H), 1.64 (ddd, 1H, J = 12.6 Hz, each); 13C (125 MHz, CDCl3):
δ 172.0,
169.9, 169.6, 98.9, 78.5, 74.1, 73.6, 73.1, 72.0, 69.9, 60.7, 58.5, 57.6, 50.8, 49.2, 31.9, 21.0, 20.6, 20.5; HRMS
(ESI-TOF) calculated for C18H24N12NaO9 [M + Na]: 575.1687, observed: 575.1676.
1,3,20,60,2000,6000 Hexa-azido-neobiosamine-(
β
-100
→
30) neamine (20). To a mixture of 14 (70 mg, 0.13 mmol)
and
6
(0.13 g, 0.19 mmol) in dry ◦DCM (2.0 mL), trimethylsilyl trifluoromethanesulfonate (14
µL,
0.075 mmol) was added at
−
20 C under N2. The resulting mixture was stirred for 2 h, then
quenched with Et3N (100
µL) and purified by silica gel chromatography (10% EtOAc/DCM) to
obtain peracetylated product (17, contaminated by traces from
6
). The residue 17 was then dissolved
in dry MeOH (2 mL) and sodium methoxide solution (2 mL of 0.2 mol L−1 solution in MeOH) was
added. After 1 h, the reaction solution was neutralized with a strong cation-exchange resin (Dowex
H+ 50W), filtered and evaporated to dryness. The residue was purified by silica gel chromatography
1
(10% MeOH/DCM) to obtain 56 mg (57%) of the product 20 as a white foam. H-NMR (500 MHz,
CD3OH): δ 5.64 (d, 1H, J = 3.7 Hz), 5.13 (s, 1H), 5.21 (d, 1H, J = 1.8 Hz), 4.64 (dd, 1H, J = 7.7 and 4.3 Hz),
4.27 (d, 1H, J = 4.3 Hz), 4.23-4.17 (m, 2H), 4.02 (ddd, 1H J = 8.3, 4.7 and 1.9 Hz), 3.94 (t, 1H, J = 3.4 Hz),
3.85-3.78 (m, 2H), 3.74 (dd, 1H, J = 12.1 and 3.7 Hz), 3.68-3.64 (m, 2H), 3.54-3.34 (m, 10H), 3.26 (t, 1H,
J = 9.4 Hz), 2.26 (ddd, 1H, J = 12.9, 4.4 and 4.4 Hz), 1.42 (ddd, 1H, J = 12.3 Hz, each); 13C (125 MHz,
CD3OH): δ 110.0, 99.7, 99.3, 83.3, 81.3, 81.2, 78.0, 77.9, 76.3, 75.8, 74.0, 73.1, 71.2, 71.1, 69.7, 64.8, 61.9,

Molecules 2019, 24, 580
10 of 13
61.9, 61.9, 60.8, 52.8, 52.5, 33.2; HRMS (ESI-TOF) calculated for C23H34N18NaO13 [M + Na]: 793.2450,
observed: 793.2427.
1,3,20,60,2000,6000 Hexa-azido-neobiosamine-(
β
-100
→
6) neamine (21). Compound 21 was synthesized from
15 (94 mg, 0.17 mmol) as described for 20 (from 14) above. 64 mg (49%) of 21 was obtained as a white
1
foam. H-NMR (500 MHz, CD3OH):
δ 5.56 (d, 1H, J = 3.8 Hz), 5.46 (d, 1H, J = 1.0 Hz), 5.10 (d, 1H,
J = 1.8 Hz), 4.46 (dd, 1H, J = 7.3 and 4.5 Hz), 4.24 (dd, 1H, J = 4.5 Hz and 1.0 Hz), 4.20–4.13 (m, 2H),
4.00 (ddd, 1H, J = 8.3, 4.7 and 1.9 Hz), 3.94 (t, 1H, J = 3.4 Hz), 3.87-3.83 (m, 2H), 3.78 (dd, 1H, J = 11.9
and 6.4 Hz), 3.68-3.61 (m, 3H), 3.55–3.61 (m, 7H), 3.40–3.35 (m, 2H), 3.18 (dd, 1H, J = 10.5 and 3.8 Hz),
2.32 (m, 1H), 1.51 (m, 1H); 13C (125 MHz, CD3OH):
δ 109.2, 100.2, 99.8, 83.6, 81.8, 81.7, 78.2, 77.6, 75.7,
74.6, 73.4, 72.7, 72.6, 71.3, 69.7, 65.0, 64.4, 61.9, 60.7, 60.3, 52.7, 52.5, 33.2; HRMS (ESI-TOF) calculated
for C23H34N18NaO13 [M + Na]: 793.2450, observed: 793.2478.
1,3,20,60,2000,6000 Hexa-azido-neobiosamine-(
β
-100
→
40) neamine (22). Compound 22 was synthesized from
16 (70 mg, 0.13 mmol) as described for 20 (from 14) above. 67 mg (69%) of 22 was obtained as a white
1
foam. H-NMR (500 MHz, CD3OH):
δ 5.63 (d, 1H, J = 3.8 Hz), 5.13 (d, 1H, J = 1.8 Hz), 4.99 (d, 1H,
J = 1.1 Hz), 4.57-4.55 (m, 1H), 4.19-4.16 (m, 3H), 4.03 (ddd, 1H, J = 8.5, 4.4 and 1.9 Hz), 3.96-3.91 (m,
2H), 3.82 (dd, 1H, J = 12.1 and 2.5 Hz), 3.72 (dd, 1H, J = 12.1 and 4.1 Hz), 3.69-3.63 (m, 2H), 3.57 (dd,
1H, J = 13.4 and 2.4 Hz), 3.52-3.38 (m, 7H), 3.36 (dd, 1H, J = 13.0 and 4.5 Hz), 3.26 (t, 1H, J = 9.4 Hz),
3.19 (dd, 1H, J = 10.5 and 3.8 Hz), 2.24 (ddd, 1H, J = 12.8, 4.3 and 4.3 Hz), 1.41 (ddd, 1H, J = 12.3 Hz,
each); 13C (125 MHz, CD3OH):
δ 110.0, 99.7, 99.5, 83.7, 81.3, 81.2, 78.0, 78.0, 76.4, 75.8, 74.6, 71.9, 71.3,
70.9, 69.7, 64.3, 62.1, 61.9, 61.9, 61.0, 52.5, 52.1, 33.3; HRMS (ESI-TOF) calculated for C23H34N18NaO13
[M + Na]: 793.2450, observed: 793.2467.
RP HPLC analysis of 20
an RP HPLC analysis. An aliquot of each compound was dissolved in a mixture of acetonitrile and
water (1:9, v/v) and injected to an analytical RP (C18, 250 4.6 mm, 5 m) column. Gradient elutions
from 10% to 80% acetonitrile in H2O over 30 min. (chromatograms a–c/Figure 1: purified products
20 23) and from 10% to 60% acetonitrile over 35 min. (chromatogram d/Figure 1: a mixture of 20 23
flow rate of 1.0 mL min−1, and detection at λ = 220 nm were used.
–23. The purity of the isolated products (20–23) was further confirmed by
×
µ
-
– ,
Neobiosamine (
-100 30) neamine (23). Trimethylphosphine (1 mol L−1 Me3P in toluene, 0.99 mL,
β
→
1.0 mmol) was added to a mixture of 20 (51 mg, 0.066 mmol) in water-dioxane (1:4, v/v, 2.0 mL).
The mixture was stirred at ambient temperature for 4 h under nitrogen and then concentrated ammonia
(1.0 mL) was added. After overnight reaction the mixture was evaporated to dryness. The crude
product was purifie−d1by an ion exchange chromatography (Dowex 1
×
2 200 anion exchanger resin,
activated by 2 mol L NaOH, eluent carbon dioxide free H2O) to give 30 mg (73%) of the product (23
)
1
as a yellowish powder. H-NMR (500 MHz, D2O):
δ
5.36 (d, 1H, J = 3.9 Hz), 5.24 (d, 1H, J = 0.8 Hz), 4.99
(d, 1H, J = 1.8 Hz), 4.62 (dd, 1H, J = 7.1 and 4.7 Hz), 4.40 (m, 1H), 4.23 (m, 1H), 4.05 (dd, 1H, J = 3.2 Hz,
both), 3.97 (m, 1H), 3.90 (dd, 1H, J = 12.5 and 2.8 Hz), 3.86–3.78 (m, 2H), 3.71–3.66 (m, 2H), 3.54 (t, 1H,
J = 9.2 Hz), 3.42 (t, 1H, J = 9.5 Hz), 3.32 (t, 1H, J = 9.3 Hz), 3.17 (t, 1H, J = 9.6 Hz), 3.08–3.01 (m, 3H),
2.94–2.84 (m, 4H), 2.75 (m, 1H), 2.00 (ddd, 1H, J = 12.9, 4.1 and 4.1 Hz), 1.23 (ddd, 1H, ddd, J = 12.1 Hz,
each); 13C (125 MHz, D2O):
δ 108.4, 100.3, 99.1, 86.2, 83.2, 81.5, 77.4, 76.0, 75.6, 75.2, 73.1, 72.2, 70.6, 69.8,
68.5, 60.3, 54.6, 52.6, 50.3, 49.3, 41.5, 41.1, 35.0; HRMS (ESI-TOF) calculated for C23H47N6O13 [M + H]:
615.3196, observed: 615.3221.
00
Neobiosamine (
β
-1
→
6) neamine (24). 24 was synthesized as described for 23 above. 61 mg (0.079 mmol)
1
of 21 gave 29 mg (58%) of 24 as a yellowish powder. H-NMR (500 MHz, D2O):
δ
5.34 (d, 1H, J = 3.9 Hz),
5.25 (s, 1H), 4.98 (d, 1H, J = 1.8 Hz), 4.62 (dd, 1H, J = 7.4 and 4.7 Hz), 4.38 (m, 1H), 4.20 (m, 1H), 4.04 (t,
1H, J = 3.2 Hz), 3.99 (m, 1H), 3.90 (dd, 1H, J = 12.6 and 2.6 Hz), 3.82–3.74 (m, 2H), 3.68–3.57 (m, 3H),
3.35–3.29 (m, 3H), 3.10–3.03 (m, 3H), 2.94 (dd, 1H, J = 13.5 and 4.1 Hz), 2.88–2.75 (m, 4H), 2.03 (ddd,
1H, J = 12.9, 4.1 and 4.1 Hz), 1.26 (ddd, 1H, J = 12.4 Hz, each); 13C (125 MHz, D2O):
δ 108.8, 100.4, 98.9,

Molecules 2019, 24, 580
11 of 13
85.9, 85.8, 81.3, 75.9, 75.4, 75.0, 73.4, 73.0, 72.4, 71.4, 70.5, 68.5, 60.2, 55.2, 52.6, 49.1, 48.9, 41.4, 41.0, 36.1;
HRMS (ESI-TOF) calculated for C23H47N6O13 [M + H]: 615.3196, observed: 615.3192.
00
0
Neobiosamine (
β
-1
→
4 ) neamine (25). 25 was synthesized as described for 23 above. 51 mg (0.066 mmol)
1
of 22 gave 32 mg (83%) of 25 as a yellowish powder. H-NMR (500 MHz, D2O):
δ
5.31 (d, 1H, J = 3.9 Hz),
5.09 (s, 1H), 4.97 (d, 1H, J = 1.6 Hz), 4.60 (dd, 1H, J = 7.0 and 4.6 Hz), 4.31 (m, 1H), 4.23 (m, 1H), 4.04 (m,
1H, 3.96-3.88 (m, 2H), 3.85–3.79 (m, 2H), 3.68–3.64 (m, 2H), 3.52 (dd, 1H, J = 9.2 Hz, both), 3.42 (t, 1H,
J = 9.4 Hz), 3.30 (t, 1H, J = 9.2 Hz), 3.16 (t, 1H, J = 9.6 Hz), 3.04 -2.98 (m, 3H), 2.90–2.81 (m, 4H), 2.72 (m,
1H), 1.99 (m, 1H), 1.22 (ddd, 1H, J = 12.4 Hz, each); 13C (125 MHz, D2O):
δ 108.1, 100.3, 99.2, 86.9, 81.5,
80.5, 77.4, 75.9, 75.7, 75.5, 73.0, 72.1, 71.9, 70.6, 68.5, 60.2, 54.9, 52.7, 50.3, 49.3, 41.2, 41.1, 35.5; HRMS
(ESI-TOF) calculated for C23H47N6O13 [M + H]: 615.3196, observed: 615.3202.
UV measurements. Melting curves (absorbance vs. temperature) were measured at 260 nm on
a Perkin-Elmer Lambda 35 UV/Vis spectrophotometer equipped with a multiple cell holder and a
Peltier temperature controller. An internal thermometer was additionally◦used to verify the validity
◦
of the target temperature. The temperature was changed from 10 to 80 C at a rate of 0.2 C min.
T
m values were determined as the maximum of the first derivate of the melting curve.
4. Conclusions
A straightforward synthesis of glycosidic (100
B has been described. The synthesis includes (1) acid-catalyzed thiolysis of neomycin azide (
obtain useful intermediates ( and ) for both the glycosyl donor ( ) and the acceptors (14 16), and
(2) selective diacetylation of neamine azide ( ), which after simple protecting group manipulation
(i.e., levulinoylation, acetylation and delevulinoylation) afforded the glycosyl acceptors (14 16).
Glycosylation between the neobiosamine donor ( ) and triacetylated neamine azide acceptors (14
was carried out in standard condition to give the azide masked pseudo tetrasaccharides (21
→
6, 30 and 40) site isomers (23
–
25) of neomycin
) to
3
4
5
6
–
7
–
6
–
16
)
)
–
22
in acceptable yields. The azide masks were removed by Staudinger reaction to obtain the desired
glycosidic site isomers of neomycin B (23 25). The potential of these aminoglycosides (23 25) to
stabilize DNA and RNA triple helix models was studied by UV-melting profile analysis. In each case a
–
–
0
0
marked stabilization was observed. The variable neobiosamine site (6-O, 3 -O or 4 -O) on the neamine
core did not show marked difference in the stability compared to neomycin B. It may be concluded
that the carbohydrate core and spatial orientation of the amino groups (i.e., spatial orientation of L and
D-neosamines provided by the variable glycoside site between neobioasamine and neamine) on it do
not play a marked enough role to result in discrimination between the affinities of the site isomers.
Supplementary Materials: The NMR spectral data for all new compounds are available online.
Author Contributions: L.G. performed the part of the aminoglycoside synthesis and UV-melting profile analysis,
V.T. performed part of the UV-melting profile analysis, P.V. (the corresponding author) supervised the project and
performed part of the aminoglycoside synthesis.
Funding: The financial support from the Academy of Finland (No. 251539, 256214 and 308931) is gratefully
acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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