Selective transformations of complex molecules are enabled by aptameric protective groups

Article abstract of DOI:10.1038/nchem.1402

Emerging trends in drug discovery are prompting a renewed interest in natural products as a source of chemical diversity and lead structures. However, owing to the structural complexity of many natural compounds, the synthesis of derivatives is not easily realized. Here, we demonstrate a conceptually new approach using oligonucleotides as aptameric protective groups. These block several functionalities by non-covalent interactions in a complex molecule and enable the highly chemo- and regioselective derivatization (>99%) of natural antibiotics in a single synthetic step with excellent conversions of up to 83%. This technique reveals an important structure-activity relationship in neamine-based antibiotics and should help both to accelerate the discovery of new biologically active structures and to avoid potentially costly and cumbersome synthetic routes.

Full text of DOI:10.1038/nchem.1402

ARTICLES
PUBLISHED ONLINE: 22 JULY 2012 | DOI: 10.1038/NCHEM.1402
Selective transformations of complex molecules
are enabled by aptameric protective groups
Andreas A. Bastian, Alessio Marcozzi and Andreas Herrmann
*
Emerging trends in drug discovery are prompting a renewed interest in natural products as a source of chemical diversity
and lead structures. However, owing to the structural complexity of many natural compounds, the synthesis of derivatives
is not easily realized. Here, we demonstrate a conceptually new approach using oligonucleotides as aptameric protective
groups. These block several functionalities by non-covalent interactions in a complex molecule and enable the highly
chemo- and regioselective derivatization (>99%) of natural antibiotics in a single synthetic step with excellent conversions
of up to 83%. This technique reveals an important structure–activity relationship in neamine-based antibiotics and should
help both to accelerate the discovery of new biologically active structures and to avoid potentially costly and cumbersome
synthetic routes.
ery recently, natural products have experienced a renaissance
in drug discovery. This trend is rationalized by a growing Regioselective acylation of neomycin B. For a straightforward
number of new natural sources, along with improved separ- proof-of-concept that RNA can act as an APG, well-
Results and discussion
V
a
ation methods and characterization techniques1,2. Another reason characterized aptamer–aminoglycoside complex was chosen:
for the high interest in natural products is the better chance of 23-mer RNA aptame′r (sequence, 5′-GGA-CUG-GGC-GAG-
finding a desirable bioactivity as a result of their natural scaffolds1,2. AAG-UUU-AGU-CC-3 , apt1) and neomycin B (Fig. 1b)23. X-ray
However, these frameworks are very often structurally complex and crystallography and nuclear magnetic resonance (NMR)
exhibit several chemically equivalent functionalities. Thus, regiose- measurements revealed that ring IV of the guest molecule 1 is not
lective modification of these molecular architectures is restricted involved in complex formation and extends into the solvent
to the application of genetically modified organisms1,2 and extensive (Fig. 1b)16. As shown in the left panel of Fig. 2a, reaction of acetic
semi- or total synthesis3–5. The latter route is very often character- acid N-hydroxysuccinimide (NHS) ester (3a, 10 equiv.) with the
ized by several protection and deprotection steps to modify specific apt1-protected neomycin B resulted mainly in the formation of
functional groups or sites of the target molecule. A much simpler the monoacetylated antibiotic, according to mass spectrometric
process to enable the selective derivatization of complex
natural products would be the introduction of macromolecular pro-
tective groups directly into these compounds. Owing to their
extended size they can, in parallel, protect several functionalities
of the natural products by non-covalent interactions and simul-
taneously allow other groups not involved in binding to be
reacted specifically.
a
b
R
II
O
O
HO
HO
HO
I
NH2
NH2
H2N
O
O
OH
III
IV
II
To test the feasibility of this approach, in the present study ribo-
nucleic acid (RNA) aptamers were introduced as non-covalent apta-
meric protective groups (APGs). As a promising class of compounds
with high affinity, specificity and stability, (deoxy)ribonucleic acids
are particularly well suited as APGs, as they can act as ligands for a
wide variety of low molecular weight compounds and macromol-
ecular architectures6,7, can be evolved through the well-established
in vitro systematic evolution of ligands by an exponential
enrichment (SELEX)8,9 process and can be produced by automated
NH2
OH
III
O
O
OH
NH2
I
1 Neomycin B, R = NH2
2 Paramomycin, R = OH
IV
OH
c
5
10
GC 10
CA
G10
A
A
A
5
5
G
G
G
A
G
5'-CUG GUCC
A
5'-UGUAGGGC
5'-GGACU
A
G
3'-GAC CGGG
3'-ACAUUUUG
A
3'-CCUGA
A
15 A
U
A
15
U
20
A
synthesis. Aptamers have been generated against amino acids10,11
,
20
U
G
20
15
nucleotides12, organic dyes8, porphyrin derivatives13, proteins14,
hydrophobic lipids15 and aminoglycoside antibiotics16–18, and of
these we chose aptamers for the last class of compounds.
Aminoglycosides offer a particularly attractive test for regioselective
derivatization using APGs, because the modification of aminoglyco-
side antibiotics that target prokaryotic 16S ribosomal RNA
(rRNA)18–20 has become a powerful tool to overcome bacterial
resistance21,22. Important examples based on the neamine scaffold
are the antibiotics neomycin B (1) and paromomycin (2) (Fig. 1a).
apt1
apt2
apt3
Figure 1 | Structures of aminoglycosides, protection mode and sequences
of APGs. a, Structure of representative aminoglycoside antibiotics. b, apt1
selectively protecting neomycin B (ref. 16) (green, carbon; red, oxygen; blue,
nitrogen; white, hydrogen). c, Sequences of applied APGs apt1, apt2 and
apt3. The arrows indicate accessible amino groups of neomycin B ring IV for
derivatization by applying acylation (blue arrows) and urea bond formation
(red arrows).
Zernike Institute for Advanced Materials, Department of Polymer Chemistry, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
e-mail: a.herrmann@rug.nl
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1402
ARTICLES
a
100
100
90
80
70
60
50
40
30
20
10
0
v
[M + Na]+
iv
90
80
70
60
50
40
30
20
10
0
NeoB
[M + Na]+
[M + H]+
i
[M + H]+
iii
[M + Na]+
iii
iv
i
[M + H]+
[M + H]+
[M + Na]+
NeoB
[M + Na]+
v
ii
[M + Na]+
[M + H]+
ii
ii
[M + H]+
vi
[M + H]+
[M + Na]+
600 640 680 720 760 800 840 880
600 640 680 720 760 800 840 880
m/z
m/z
NH2
b
O
O
HO
HO
NH2
NH2
HO
H2N
O
O
OH
1.5 eq. apt1
O
O
O
NH2
OH
NH
O
O
OH
NH
2 II
N
4a
O
O
HO
HO
I
NH2
NH2
OH
O
HO
H2N
O
O
O
O
3a (30 eq.)
O
OH
NH2
III
HN
NH2
OH
HO
HO
O
O
OH
NH2
NH2
NH2
O
O
HO
HO
IV
HO
H2N
not protected by APG
O
NH2
NH2
O
O
HO
OH
H2N
O
O
OH
O
O
O
1
OH
NH2
OH
N
O
O
OH
NH
NH2
OH
O
O
OH
4a
O
4b
OH
NH2
3a (1 eq.)
O
OH
c
CH3
4'''–H
1'''–H
1''–H
1'–H
3''–Hll
4a
(95%)
2a–H
2e–H
4a + 4b
1´–Hll
1´´´–H
3''–H
4.5
4'''–H
5'''–H
4.0
1'–H
6.0
5.5
5.0
3.5
3.0
2.5
2.0
ppm
Figure 2 | Comparison of acetylation of neomycin B (1) in presence and absence of APG apt1. a, Electrospray ionization mass spectra of the reaction
mixture of neomycin B (NeoB) with activated ester 3a (10 equiv.) in the presence (left) and absence (right) of apt1 (i–vi ¼ number of reacted amino groups
of 1; [M þ H]þ ¼ aminoglycoside ionized by protons; [M þ Na]þ ¼ aminoglycoside ionized by sodium cations). b, In the presence of apt1, neomycin B (1) is
monoacylated selectively to produce only 4a even when treated with a large excess of acylating agent. In the absence of the APG, a mixture of
monoacetylated products 4a and 4b is observed. c, 1H-NMR spectra of regioselectively monoacetylated neomycin B (4a) (upper) and a mixture of
regioisomers 4a and 4b (lower).
analysis and high-performance liquid chromatography (HPLC) (see groups within antibiotic 1. To obtain further insight into the
Supplementary Fig. S8). In stark contrast, the unprotected regioselectivity of transformations on 1, the reaction conditions
transformation of 1 under the same conditions, in which all the were optimized to yield the monoacetylated antibiotic for the
amino groups are reactive, yielded di-, tri-, tetra-, penta- and hexa- protected and non-protected transformations (Fig. 2b). The RNA–
acetylated products of 1 (Fig. 2a, right). These experiments indicate neomycin B complex was reacted with 30 equiv. of the NHS ester
clearly the ability of apt1 to inhibit the reactivity of multiple amino 3a and the unprotected antibiotic 1 was incubated with only 1
2
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1402
ARTICLES
R1
a
O
O
HO
HO
HO
1. APG
NH2
NH2
O
2.
O
O
3a R2 = Me
O
H2N
R2
O
3b R2 = iPr
1. APG
3
OH
3c R2 = 3-butynyl
7a R3 = OMe
7b R3 = H
7c R3 = NMe2
N
2.
R
N
C
NH2
OH
1 R1 = NH2
2 R1 = OH
R1
O
O
OH
O
O
NH2
O
O
HO
HO
R1
OH
NH2
NH2
HO
H2N
O
O
O
HO
HO
OH
R3
NH2
NH2
HO
H2N
O
O
O
NH2
OH
O
OH
NH
OH
NH
O
8a R1 = NH2 R3 = OMe
8b R1 = NH2 R3 = H
8c R1 = NH2 R3 = NMe2
R1 = OH R3 = OMe
O
NH
OH
R2
O
O
OH
OH
O
NH2
9
4a R1 = NH2 R2 = Me
OH
5
6
R
R
1 = NH2 R2 = iPr
1 = NH2 R2 = 3-butynyl
c
b
30
NH2
20
40
O
O
HO
30
HO
NH2
NH2
OH
O
50
HO
H2N
O
O
40
NH2
60
NH
50
O
HO
O
NH
OH
70
HO
O
O
OH
NH2
NH2
HO
60
H2N
O
O
80
2'''
3'''
O
NH2
OH
70
OH
90
1'''
NH2
OH
O
O
OH
NH
80
100
110
120
130
8a
90
OH
1'''
O
100
110
5'''
5
6'''
7.0 6.6 6.2 5.8 5.4 5.0 4.6 4.2 3.8 3.4 3.0 2.6 2.2 1.8
ppm
5.8 5.4 5.0 4.6 4.2 3.8 3.4 3.0 2.6 2.2 1.8 1.4 1.0
ppm
Figure 3 | Chemo- and regioselective transformation of amino groups of aminoglycosides 1 and 2, and structural confirmation of their derivatives 5 and 8a.
a, Selective transformations of the amino group in the C6 position of ring IV (left) using succinimide esters 3a–3c (30 equiv.) and of the amino group in the
C2 position of ring IV (right) employing isocyanates 7a–7c (15 equiv.). Modifications of antibiotics 1 and 2 were performed over 24 hours in 10 mM sodium
phosphate buffer at pH 6.9 containing 6.7 vol% DMF in the presence of APG (1.5 equiv.). b, HSQC spectrum (500 MHz, D2O) of 6′′′-N-2-methylpropanoyl
neomycin B (5). Arrows indicate the shift of specific signals caused by the regioselective transformation of the amino group in the C6 position of ring IV:
J(C6′′′–Ha) and J(C6′′′–Hb) coupling (red), J(C5′′′–H) coupling (blue) and J(C1′′′–H) coupling (black). c, HSQC (500 MHz, D2O) spectrum of 2′′′-N-((4-
methoxyphenyl)amino)carbonyl neomycin B (8a). Arrows indicate the shift of specific signals caused by the regioselective transformation of the amino group
in the C2 position of ring IV: J(C2′′′–H) coupling (red), J(C3′′′–H) coupling (blue) and J(C1′′′–H) coupling (black).
equiv. of 3a. After purification by HPLC, the monoacetylated allowed easy solubilization of the hydrophobic activated esters 3b
neomycin B products were characterized and regioselectivities were and 3c. In addition to these excellent regioselectivities, the APG-
determined by 1H-NMR spectroscopy (Fig. 2c). The RNA- mediated reactions exhibited high conversions of 76%, 71% and
protected aminoglycoside 1 was proved to be acetylated at the 83% for 4a, 5 and 6, respectively (Table 1).
amino group in the C6 position of ring IV with an extremely high
Two even shorter RNA sequences (apt2, 5′-CUG-CAG-UCC-
regioselectivity of 95% to form 4a, but in the absence of APG the GAA-AAG-GGC-CAG-3′, and apt3, 5′-UGU-AGG-GCG-AAA-
amino groups in the C6 positions of both ring II and ring IV were AGU-UUU-ACA-3′) (Fig. 1c) were also employed as APGs for
highly reactive. Thus, the unprotected neomycin B was converted antibiotic 1, and showed that the concept is not limited to a
mainly into two inseparable derivatives, 6′′′-N-acetylneomycin B single host–guest complex. These aptamers were chosen from a
(4a) and 6′-N-acetylneomycin B (4b), in a ratio of 9:11. These pool of 21 sequences generated by Wallis et al.9 in the same
results were confirmed by heteronuclear single quantum coherence SELEX experiment as that for apt1. Again, high regioselectivities
(HSQC) spectra (see Supplementary Fig. S6) and attached proton were obtained for the formation of 4a, reaching 94% for apt2 and
test measurements (see Supplementary Fig. S7). To test whether 95% for apt3 (Table 1). The conversions for the reactions were
apt1 can tolerate larger residues than the acetyl group, antibiotic 1 slightly lower than those for apt1, reaching 63% for apt2 and 62%
was functionalized using the NHS esters of isobutyric acid (3b) and using apt3. As shown in Table 1, apt2 was also successful in the
4-pentynoic acid (3c) (Fig. 3a). Again, remarkable regioselectivities synthesis of 5 and 6 with high regioselectivities (89% and
of 97% and 98% were achieved for 5 and 6, respectively (Table 1, 96%, respectively).
Fig. 3b). It is important that during the preparation of these
derivatives, apt1 even tolerated the addition of 6.7 volume per cent Urea bond formation at an alternative site. The APG strategy for
(vol%) of the organic solvent dimethylformamide (DMF), which the regioselective derivatization of natural products described here is
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Table 1 | Selective modification of aminoglycoside antibiotics.*
Compound
APG
R1
R2
R3
Conversion† (%)
Regioselectivity‡ (%)
4a
4a
4a
5
5
6
apt1
apt2
apt3
apt1
apt2
apt1
apt2
apt1
apt2
apt1
apt2
apt1
apt2
apt1
apt2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
OH
Me
Me
Me
i-Pr
i-Pr
3-butynyl
3-butynyl
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
76
63
62
71
59
83
67
44
45
52
37
55
61
95
94
95
97
89
98
96
.99
.99
.99
.99
92
93
97
92
6
8a
8a
8b
8b
8c
8c
9
OMe
OMe
H
H
N(Me)2
N(Me)2
OMe
OMe
54
55
9
OH
*All reactions were carried out using the general procedure (see Supplementary Information). †Maximal conversion observed for the antibiotic derivatives. Conversions were determined by HPLC.
‡Regioselectivity of antibiotic derivatives determined by 1H-NMR.
also amenable to different types of chemical transformation and
allows the modification of different sites. This is demonstrated
with urea bond formation at the amine in the C2 position of ring
Table 2 | Antimicrobial activity of modified aminoglycosides.
Compound*
Diameter† (mm)
MIC (mM)
IV of neomycin B by applying aromatic isocyanates 7a–7c
(Fig. 3a). In contrast, the applied NHS esters reacted exclusively at
the amino group in the C6 position. Phenyl isocyanates 7a–7c
were reacted with antibiotic 1 in the presence and in the absence
of APGs. Once again, the unprotected reaction of 1 yielded a
complex, inseparable mixture of derivatives (see Supplementary
Figs S9 and S10). However, when apt1 and apt2 were employed
under the same conditions, the transformation was accomplished
with high chemo- and regioselectivity (Fig. 3a,c and
Supplementary Figs S9 and S10). Indeed, the reaction of the
protected antibiotic 1 with isocyanates 7a and 7b resulted
exclusively in the single regioisomers 8a and 8b, respectively,
when apt1 and apt2 were employed (Table 1). The protected
1
16.8
16.1
13.2
13.9
11.5
12.7
12.9
15.1
3.1
3.1
1‡
2‡
4a
5
12.5
6.3
12.5
6.3
6.3
6.3
8a
8b
8c
*All compounds were used as heptafluorobutyric acid (HFBA) salts, except for neomycin B sulfate 1
(Sigma Aldrich). †The zones of inhibition of E. coli ATCC 25922 as determined by the Kirby–Bauer
disc method are given. For all compounds, the molar amount was kept constant at 17.5 nmol.
‡Antibiotic HFBA salt, purified by HPLC using the same conditions as those for neomycin B
derivatives 4a, 5 and 8a–8c.
transformation of the amine in the C2 position of ring IV with previously reported non-covalent protective group approaches24–
26. Neomycin B derivatives altered at ring IV have been described
isocyanate 7c yielded a slightly lower regioselectivity for 8c
(Table 1). Again, the urea derivatives 8a–8c were obtained with previously and were only obtained after extensive synthesis that
included more than 20 steps (see Supplementary Figs S11–S15)3.
good conversions that ranged from 37% to 61% (Table 1).
Our newly developed technology allows the production of such anti-
biotic derivatives modified at the same positions in a single reaction.
The preferential modification of two different reaction sites in
urea bond formation and acylation can be explained as a combi-
nation of two factors. The planar structure of the phenyl isocyanates
7a–7c, in contrast to the sterically more demanding structure of Investigation of antimicrobial activity. After the successful
NHS esters 3a–3c, allows relatively easy access to functionalities synthesis of novel neomycin B derivatives, compounds 4a, 5 and
that are partially protected, such as the amino group in the C2 8a–8c were investigated for their antimicrobial activities against
position of ring IV (Fig. 1b). In addition, in aqueous medium Escherichia coli ATCC 25922, which is a standard strain used to
isocyanates exhibit a higher reactivity towards amines attached to evaluate the efficiency of antibiotics3. Two methods, the Kirby–
secondary carbons than to primary ones. Thus, a detailed under- Bauer disc test and the determination of the minimal inhibitory
standing of the interaction between APG and the guest molecule concentration (MIC) (see Supplementary Information) were
and careful choice of the reaction conditions can allow the use of employed for this purpose. As shown in Table 2, all of the
the same APG for a range of modifications.
derivatives tested remained highly active antibiotics. Therefore, we
can exploit the APG technology directly to gain new structure–
Regioselective modification of paromomycin. Also, the APGs property relationships, as we have demonstrated that larger
presented here can be used to generate other drug derivatives with residues at the amino groups of ring IV are well tolerated and do
not significantly decrease antimicrobial activity.
a similar pharmacophore. For instance, the related antibiotic
paromomycin 2 could be converted into the corresponding urea
derivative 9 employing apt1 and apt2 (Fig. 3a). It was found that Conclusions
2 was also preferably transformed at the amino group in the C2 In conclusion, we demonstrate here the effective use of RNA
position of ring IV in good conversions of 54% and 55% and with sequences as non-covalent APGs for the highly chemo- and regio-
remarkable regioselectivities of 97% and 92%, applying apt1 and selective derivatization of complex molecules that bear several func-
apt2, respectively (Table 1, see Supplementary Fig. S5).
tional groups of similar reactivity. This was demonstrated
In light of the synthesized neomycin B and paromomycin deriva- successfully for the structurally complex aminoglycoside antibiotics
tives presented above, it should be apparent that the APG paradigm neomycin B and paromomycin with three chosen aptamer
decreases the number of synthetic steps necessary for the modifi- sequences. These natural antibiotics were modified in a single syn-
cation of complex molecules to the extreme and is superior to thetic step with substituents of variable size and hydrophobicities by
4
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1402
ARTICLES
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employing two different reactions, namely acylation and urea bond
formation, which proceeded with excellent regioselectivities (89% to
.99%) and very good conversions of up to 83%. However, it has to
be stressed that prior to the facile derivatization, the generation of
RNA aptamers by a SELEX experiment is necessary to obtain
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Received 17 February 2012; accepted 6 June 2012;
published online 22 July 2012
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Acknowledgements
This research was supported by the European Union (European Research Council
Starting Grant and Electronic Chemical Cell), the Netherlands Organization for Scientific
Research (NWO-Vici, NWO-Echo) and the Zernike Institute for Advanced Materials.
A.B. thanks P.v.d. Meulen for help in recording the NMR spectra, as well as M. Pudelko and
M. Bastian for useful discussions and suggestions.
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Author contributions
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A.B. performed the synthesis and characterization of all the compounds within this project
and participated in the design of the study. A.M. tested the antimicrobial activity of the
antibiotics and their derivatives. A.H. conceived and designed the study and co-wrote the
manuscript with A.B.
Additional information
The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints. Correspondence and requests for materials should be addressed to A.H.
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