Regioselective diazo-transfer reaction at the C3-position of the 2-desoxystreptamine ring of neamine antibiotics

Article abstract of DOI:10.1002/chem.201300912

Site-selective diazotation: A facile one-step modification at the 2-desoxystreptamine ring, is reported in diverse neamine antibiotics reaching high regioselectivities (see scheme; r.s.=regioselectivities). The diazo-transfer reaction takes place in aqueous solution, under mild conditions, and without any protective groups. Copyright

Full text of DOI:10.1002/chem.201300912

COMMUNICATION
DOI: 10.1002/chem.201300912
Regioselective Diazo-Transfer Reaction at the C3-Position of the
2-Desoxystreptamine Ring of Neamine Antibiotics
Andreas A. Bastian,[a] Eliza M. Warszawik,[a] Praveen Panduru,[a] Christoph Arenz,[b] and
Andreas Herrmann*[a]
Aminoglycosides represent one of the largest classes of
antibacterials with activity against Gram-positive and -nega-
tive bacteria. These compounds exert their antibacterial ac-
tivity by binding the decoding region (A-site) of the 16S
rRNA.[1] However, the increased bacterial resistance against
aminoglycoside antibiotics[2] fueled activities to modify these
scaffolds to obtain new active compounds.[3] In particular,
the neamine moiety (1, Figure 1) of these antibiotics attract-
ed major attention, since resistance is mainly based on the
introduction of phosphate- and acetyl groups at ring I and II
by the enzymes phosphotransferases (APHs) and acetyl-
transferases (ACCs).[4] Thus, chemical derivatizations of
these compounds were carried out to overcome bacterial re-
sistance or reduce toxicity.[5] However, these modifications
require multistep synthesis[3,5a,6] due to the presence of sev-
eral hydroxy and amino groups with similar reactivity.
Therefore, methods were established that enabled regiose-
lective introduction of functionalities in these antibiotics.
For example, selective modifications were enabled by the
application of noncovalent protective groups based on
metal–chelate complexes[5e,f,g] and RNA aptamers,[5h] or em-
ploying a chemoselective Staudinger reaction.[7] However,
Figure 1. Chemical structures of aminoglycoside antibiotics.
modification at the C3-position of the 2-DOS ring remains a
challenge and is so far limited to the utilization of enzymes[8]
or multistep synthesis. For example, Mobashery and co-
workers succeeded in the modification of this position in an-
tibiotic 1 to reduce the electrostatic interaction of the ami-
noglycoside with resistance enzymes.[6b] Unfortunately, this
modification required multistep synthesis and thus resulted
in low overall yield. To minimize the synthetic effort and
enable a facile access to new antibiotic derivatives, herein
we describe a one-step modification at the C3-position of
ring I applicable to different structurally complex neamine
antibiotics (Figure 1).
This synthetic shortcut is based on the application of the
diazo-transfer reagent, imidazole-1-sulfonyl azide
7
(Scheme 1), allowing regioselective azide introduction at the
least basic amino group at the 2-DOS ring in a diverse
range of neamine antibiotics. This cost-efficient and scalable
method does not require any protection or deprotection
steps and is performed in aqueous solution by using mild
conditions. The diazo-transfer reagent 7 that was applied in
this work has been recently introduced as a shelf-stable,
nonexplosive and water-soluble azide compound.[9] More-
over, it has proven to be a straightforward tool to convert
[a] Dr. A. A. Bastian, E. M. Warszawik, P. Panduru, Prof. A. Herrmann
Department of Polymer Chemistry
Zernike Institute for Advanced Materials
University of Groningen, Nijenborgh 4
9747 AG Groningen (The Netherlands)
Fax : (+31)50-363-440
E-mail: a.herrmann@rug.nl
[b] Prof. C. Arenz
Department of Chemistry, University of Berlin
Brook-Taylor-Str.2, 12489 Berlin (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300912.
Scheme 1. Diazo-transfer reaction by using imidazole-1-sulfonyl azide
7·HCl.
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free amines into azides through an aqueous diazo-transfer
reaction.
The crystalline and more stable hydrochloride salt, 7·HCl,
was applied to a wide range of molecules resulting in rea-
sonable reaction times and excellent yields.[9a] A further ad-
vantage of this reagent is that it has good functional-group
tolerance and is even reactive without any catalyst, such as
copper(II), nickel(II), or zinc(II).[9b] In 2011 van Hest et al.
applied imidazole-1-sulfonyl azide 7·HCl to introduce a
single azido group in proteins.[9b] It turned out that the N-
terminus could be selectively transformed, whereas more
basic amines of lysine residues were not converted at lower
pH and in absence of a metal catalyst.
Inspired by this work, we tested the applicability of 7·HCl
to the regioselective azide introduction to neomycin B (3),
which exhibits several amino groups with pKa values ranging
from 5.7 to 8.8 (Scheme 2).[10] In particular, the least basic
Figure 2. HPLC analysis of neomycin B 3 (NeoB) after transformation by
using eight equivalents of diazo-transfer reagent 7·HCl. (i–vi=number of
introduced azido groups).
by HPLC. As shown in Figure 2a, the transformation of neo-
mycin B (3) at pH 8 in the presence of copper(II) resulted
in a mixture of derivatives exhibiting three to six azido
groups. In contrast, in the absence of copper(II) and at neu-
tral pH values one azido group was introduced predomi-
nately reaching high conversion of neomycin B (82%) (Fig-
ure 2b and Table 1, entry 1).
Scheme 2. Regioselective azide introduction at the C3-position of neomy-
cin B 3 by using diazo-transfer reagent 7·HCl. The pKa values of the cor-
responding amino group are given in parentheses.[10]
amino group at the C3-position of the 2-DOS ring
(Scheme 2) seemed to be a valuable target. Thus, we ex-
plored the dependence of the reactivity of amino groups
within neomycin B (3) on the pH value and the presence of
copper(II) sulfate as a catalyst. All reactions were per-
formed in 10 mm sodium phosphate buffer by using eight
equivalents of diazo-transfer reagent 7·HCl for 18 h at room
temperature. The number of active amino groups was deter-
mined by mass spectrometry (see Figure S19 in the Support-
ing Information). Performing the transformation of 3 at
pH 8 in the presence of copper(II) resulted in the reaction
of all amino groups, whereas in the absence of the catalyst
only four amines seem to be reactive. Similar results were
obtained in previous studies when lysozyme was employed
as a substrate for 7·HCl.[9b] To investigate the influence of
the pH on the reactivity of 7·HCl with antibiotic 3 we car-
ried out the same set of reactions at pH 7. As demonstrated
by mass spectrometry up to five amino groups of 3 are still
reactive in the presence of copper(II), whereas in the ab-
sence of the catalyst only one amine was converted and
traces of a second amine reacted (see Figure S19 in the Sup-
porting Information). Despite the utilization of eight equiva-
lents of diazo-transfer reagent 7·HCl five out of six amino
groups seem not to be reactive under these conditions.
In the next step, the fraction of neomycin B derivatives
exhibiting one azido group was separated from the starting
Table 1. Transformation of antibiotics by using diazo-transfer reagent
7·HCl on a mmol scale.[a]
Entry
Antibiotic
Product
Reaction
t [h]
Conv.[e]
[%]
r.s.[f]
[%]
1[b]
2[c]
3[d]
4
5
6
3
3
3
1
2
4
5
6
8
8
8
18
20
40
40
40
40
40
40
82
63
81
94
46
60
96
97
90
88
90
96
96
90
97
98
9
10
11
12
13
7
8
[a] All reactions were performed on a 22 mmol scale of antibiotics in
water adjusted to pH 6.6 with 2m NaOH solution and 16 equivalents of
7·HCl at room temperature. [b] Performed in 10 mm sodium phosphate
buffer (pH 7) by using 8 equiv of 7·HCl. [c] Performed by using 16 equiv
of 7·HCl. [d] Performed by using 8 equiv of 7·HCl. [e] Conversions of an-
tibiotics to a mixture of corresponding antibiotic derivatives exhibiting
one azido group are given and were determined by HPLC analysis.
[f] Regioselectivities (r.s.) were determined by NMR spectroscopy from
the mixture of mono-azido antibiotic derivatives, which were separated
from the unconverted antibiotic by HPLC analysis.
Furthermore, we compared the conversion of neomycin B
(3) with 7·HCl under the most and least reactive conditions
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Neamine Antibiotics
COMMUNICATION
resulted in a high conversion of neomycin B to monoazido
derivatives with a regioselectivity of 90% for the modifica-
tion at the C3-position (Table 1, entry 3). In contrast, em-
ploying 16 equivalents of 7·HCl gave lower regioselectivity
(entry 2) and, additionally, formation of derivatives exhibit-
ing more than one azido group were observed (see HPLC
data, Figure S18 in the Supporting Information). Thus, the
formation of side products gave rise to the reduced forma-
tion of monoazido derivatives (entry 2).
To test whether different antibiotics exhibiting the nea-
mine moiety can be transformed selectively at the same po-
sition, we investigated azide introduction via 7·HCl with ne-
amine 1, amikacin 2, paromomycin 4, ribostamycin 5, and
apramycin 6 (Figure 1). Mass spectrometric analysis of the
transformation of all substrates confirmed the result previ-
ously obtained for neomycin B 3 showing high reactivity of
only a single amino group (see the Supporting Information,
Figure S21–S25). As proven by NMR spectroscopy, all ap-
plied aminoglycosides were transformed at the C3-position
of the 2-DOS ring, even when applying an excess of 16
equivalents of 7·HCl (see the Supporting Information, Figur-
es S8–S17). All substrates were successfully transformed
reaching a high regioselectivity of up to 98% for the modifi-
cation of the 2-DOS ring and conversions of the antibiotics
to the corresponding derivatives exhibiting one azido group
between 46 and 97% were obtained (Table 1, entries 4–8).
Thus, in all reactions still 3 to 54% unconverted neamine
antibiotics were found.
Figure 3. Sector of HSQC of neomycin B derivative 8 (a) and neomycin B
3 (b).
So far, all transformations were performed on a 22 mmol
scale of the antibiotics. To test the scalability of the regiose-
lective azide introduction, we performed the diazo-transfer
reaction on a mmol scale by using 16 equivalents of diazo-
transfer reagent 7·HCl. Moreover, the reaction was per-
formed at a ninefold higher concentration to decrease the
reaction volume. As shown in Table 2, the transformation of
antibiotics 1, 3, 4, 5, and 6 resulted in very high regioselec-
tivities and good conversions (entries 1–2 and 4–6). In con-
trast, the reaction of amikacin 2 resulted in low conversion,
even when performed at pH 7 (Table 2, entry 3). We assume
that the amino group at the C3-position of the DOS ring of
aminoglycoside 2 is more basic relative to the same position
of the other neamine antibiotics and, therefore, shows lower
reactivity. The reason could be that the amino group at the
C1-position of amikacin is acylated. This gives rise to easier
protonation of the N3-position due to less repulsive forces
since one positive charge in the N1-position is missing. In
previous studies, it was described for neomycin B that the
low pKa value of the amino group at the C3-position is due
to electrostatic repulsion caused by the positive charges of
the protonated amino groups at the C1 and C2’-positions.[10]
The regioselective introduction of the azido group at the
2-DOS ring is highly appealing, since this functionality can
be applied for further chemical diversifications through
Huisgen and Staudinger reactions.[11] Thus, they can be re-
acted with acetylenes in a click reaction resulting in 1,2,3-tri-
azoles and they allow the introduction of carbamates, pri-
mary amines, and amides.[11c] Since both transformations,
material by HPLC and the position of modification in anti-
biotic 3 was determined by 2D NMR spectra. As shown in
Figure 3a, the amino group at the C3-position at the 2-DOS
ring was transformed regioselectively resulting in derivative
8 (Scheme 2). In comparison to the HSQC spectrum of neo-
mycin B (Figure 3b) the corresponding JACTHNUGRTENUNG(C3-H) coupling
shows a remarkable shift to lower field proving the regiose-
lective azide introduction at this position. As shown in
Table 1, entry 1, a regioselectivity of approximately 90%
was determined by employing 1H NMR spectroscopy (see
the Supporting Information, Figure S5).
The selective transformation of the amino group at the
C3-position can be explained by differences of the basicity
of the amino groups. While five of six amines have a pKa
ranging from 7.55 to 8.8, the amino group at the C3-position
of the 2-DOS ring is the least basic one with a pKa of 5.74
(Scheme 2).[10] Therefore, only this amino group is accessible
for the reagent 7·HCl under our identified conditions.
Moreover, to avoid the use of aqueous buffer solutions,
the reaction was performed in water in the presence of
sodium hydroxide to adjust an initial pH of 6.6 in the reac-
tion mixture. Here, eight and 16 equivalents of imidazole-1-
sulfonyl azide 7·HCl were applied for the transformation of
antibiotic 3. It has to be noted that the pH is not changing
significantly during the applied reaction times of 20 and
40 h. As shown in Table 1, using eight equivalents of 7·HCl
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A. Herrmann et al.
Table 2. Transformation of antibiotics by using diazo-transfer reagent
25% ammonia (from 2:3:1 to 2:3:2 v/v/v) mixture. After evaporation of
the solvent the residue was resolved in water (3 mL) and traces of silica
were removed by filtration through 0.45 mm syringe filters. Lyophilization
yielded the 3C-azido aminoglycoside antibiotic.
7·HCl on a mmol scale.[a]
Entry
Antibiotic
Product
Conv.[d]
[%]
Yield[e]
[%]
r.s.[f]
[%]
1[b]
2
3
1
2
4
5
6
8
9
10
11
12
13
86
82
64
29
50
77
71
97
95
90
89
95
93
87 (63)
37 (36)
62 (55)
89 (78)
79 (68)
3[c]
4
Acknowledgements
5
6
This research was supported by the EU (ERC starting grant NUCLEOP-
OLY), the Netherlands Organization for Scientific Research (NWO-Vici,
NWO-Echo), and the Zernike Institute for Advanced Materials.
[a] All reactions were performed on a 0.22 mmol scale of antibiotics in
water adjusted to pH 6.6 with 2m NaOH solution and 16 equiv of 7·HCl
at room temperature. [b] Performed on a 5.5 mmol scale in water by
using 8 equiv of 7·HCl. [c] Performed at pH 7.0. [d] Conversions of anti-
biotics to mixture of corresponding antibiotic derivatives exhibiting one
azido group are given and were determined by HPLC analysis. In paren-
theses conversions are given by using 8 equiv of 7·HCl. [e] Isolated
yields. [f] Regioselectivities (r.s.) were determined by NMR spectroscopy
from the mixture of mono-azido antibiotic derivatives, which were sepa-
rated from the unconverted antibiotic by column chromatography.
Keywords: antibiotics
· diazo-transfer · drug design ·
modification · regioselectivity
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In conclusion, imidazole-1-sulfonyl azide 7 was successful-
ly applied as a diazo-transfer reagent for regioselective
azide introduction in neamine antibiotics avoiding any pro-
tective-group chemistry. We demonstrated that the reactivity
of amino groups within this important class of antibiotics
can be controlled by pH and addition of catalyst. Key to the
single specific transformation of the amino group at the C3-
position of the 2-desoxystreptamine ring is its low pKa
value. The resulting regioselectivity for six different neamine
antibiotics reached up to 98%. Moreover, we demonstrated
the scalability of the regioselective azide introduction per-
forming the reaction by using grams of neomycin B. Since
the amino group at the C3-position of the 2-DOS ring is a
target of the resistance-causing enzyme acetyltransferases
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Experimental Section
General procedure for the regioselective azide introduction in neamine
antibiotics: A solution of antibiotic sulfate salt (0.22 mmol) in water
(10 mL) was combined with a solution of imidazole-1-sulfonyl azide hy-
drochloride (737 mg, 3.52 mmol, 16 equiv) in water (10 mL). A pH value
of 6.6 of the reaction mixture was adjusted by addition of aq. 2m NaOH
solution and stirred at room temperature for 40 h before being washed
twice with dichloromethane (20 mL). The aqueous solution was concen-
trated under reduced pressure to remove dichloromethane residues and
freeze dried. The obtained crude mixture was purified by column chro-
Received: March 9, 2013
matography by using
a
homogeneous dichloromethane/methanol/aq.
Published online: && &&, 0000
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Neamine Antibiotics
COMMUNICATION
Drug Design
A. A. Bastian, E. M. Warszawik,
P. Panduru, C. Arenz,
A. Herrmann* ................ &&&& — &&&&
Site-selective diazotation: We report a
facile one-step modification at the 2-
desoxystreptamine ring in diverse nea-
mine antibiotics reaching high regiose-
lectivities (see scheme; r.s.=regiose-
lectivities). The diazo-transfer reaction
takes place in aqueous solution, under
mild conditions, and without any pro-
tective groups.
Regioselective Diazo-Transfer Reac-
tion at the C3-Position of the2-
Desoxystreptamine Ring of Neamine
Antibiotics
Chem. Eur. J. 2013, 00, 0 – 0
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www.chemeurj.org
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