Intramolecular proton transfer impact on antibacterial properties of ansamycin antibiotic rifampicin and its new amino analogues

Article abstract of DOI:10.1039/c2ob00008c

Intramolecular proton transfer in rifampicin (1) and its analogues 2-9 with the formation of zwitterions has been indicated by multinuclear NMR and crystallographic studies. Biological tests of 1-9 in combination with the analysis of ligand-protein intera

Full text of DOI:10.1039/c2ob00008c

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COMMUNICATION
Intramolecular proton transfer impact on antibacterial properties of
ansamycin antibiotic rifampicin and its new amino analogues†
a
a
a
a
b
Krystian Pyta, Piotr Przybylski,* Barbara Wicher, Maria Gdaniec and Joanna Stefańska
Received 2nd January 2012, Accepted 27th January 2012
DOI: 10.1039/c2ob00008c
Intramolecular proton transfer in rifampicin (1) and its ana-
logues 2–9 with the formation of zwitterions has been indi-
cated by multinuclear NMR and crystallographic studies.
Biological tests of 1–9 in combination with the analysis of
ligand–protein interactions have revealed the relationship
between the protonation site and extremely high antibacterial
activity.
Rifampicin (Scheme 1) belongs to a wide group of antibiotics
called rifamycins that have been at the top of clinically used
1,2
pharmaceuticals against tuberculosis over the last 35 years.
Antibacterially active rifamycins inhibit DNA-dependent RNA
polymerase (RNAP) by binding to the RNAP β subunit via
hydrogen bonds and hydrophobic interactions at a site about
Scheme 1 Rifampicin (1) and its amino analogues 2–9.
2+
3
1
2 Å away from Mg ions. The presence of a naphthalene ring
with quinone or hydroquinone oxygen atoms at C-1 and C-8 and
two hydroxyl groups at C-21 and C-23 of the ansa chain, which
have to be in a particular spatial arrangement, is essential for
4
antibacterial activity. The rifampicin molecule is relatively rigid
with conformational flexibility largely restricted by a system of
intramolecular hydrogen bonds. Moreover, owing to the presence
of both acidic (phenols) and basic (amine, hydrazone) groups
within the molecule, an intramolecular proton transfer can occur
resulting in neutral (phenolic) and zwitterionic (phenolate)
forms of this antibiotic (Scheme 2). This aspect of the rifampicin
5
structure, postulated already in 1975 by Ferrari and Gallo, has
been little studied and not taken into account in some later spec-
6,7
troscopic reports. Also the crystal structures of rifampicin sol-
vates, due to their low accuracy, were not reliable with respect of
neutral versus zwitterionic form of rifampicin present in the
8,9
crystal. Thus, it is not surprising that only the neutral form of
rifampicin, or its analogues, has been considered when
Scheme 2 Possible uncharged (phenolic) and zwitterionic (phenolate)
forms of 1 and 2–8.
constructing the ligand–protein interaction model on the basis of
the crystal structures of rifampicin–protein complexes. All
3
a
Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780
Poznan, Poland. E-mail: piotrp@amu.edu.pl; Fax: +48 618291505;
Tel: +48 61 8291252
Medical University of Warsaw, Department of Pharmaceutical
above considerations and earlier findings have prompted us to
explain the problem of localisation of protons from phenolic
groups within 1 and its derivatives and encouraged us to clarify
the biological role of the substituent at C-3 atom.
b
Microbiology, Oczki 3, 02-007 Warsaw, Poland
†
Electronic supplementary information (ESI) available: Experimental
details, FT-IR, 1D and 2D NMR data for 1–9, crystallographic data for
-CH CCl , 1-CH OH–H O and 2-CH OH–CH Cl , extended biologi-
The crystal structures of solvated forms of 1, 1-CH CCl and
3
3
1
3
3
3
2
3
2
2
1
-CH OH–H O, clearly showed that from aprotic CH CCl
3 2 3 3
cal data for compounds 1–9. CCDC 859792–859794. For crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/
c2ob00008c
solvent, 1 crystallized in the phenolic form with a proton
attached to O(8) (Fig. 1a) whereas when crystallized from
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Fig. 1 Crystal structures of: (a) 1-CH
3
3
CCl , (b) 1-CH
3
OH–H
2
O, (c) 2-CH
3
OH–CH
2
Cl
2
, indicating different localisation of the acidic proton from O
(8)–H phenol group within molecules in solid.
1
3
Fig. 2
2
C NMR spectra of: (a) 1 and (b) 2 and 9, recorded in protic and aprotic solvents with or without addition of H O.
methanol the phenolate form was obtained with protonated
N(40) atom of the piperazine group (Fig. 1b). The proton transfer
has a strong impact on a system of intramolecular interactions
within 1, as the collective system of four intramolecular hydro-
gen bonds can be formed solely in the phenolic form
= 174.5, δin py-d5 = 173.1, δ
ances indicate the localisation of the proton at the O(8) oxygen
= 172.8 ppm) reson-
in CD CN
CDCl3
3
and the presence of the phenolic form of 1. This conclusion is
1
1
13
also supported by the H NMR spectra and H– C HMBC cor-
relations (Fig. 8S†) which reveal δO(8)H resonances at 11.98
(CDCl ), 12.58 (py-d ) and 12.30 (CD CN) ppm and (H )spin–
(Table S1†). In turn, in the phenolate form, breaking of the intra-
3
5
3
8
molecular O(1)–H⋯O(15) bond involving the amide carbonyl
group increases the conformational freedom about the bond of
the amide group with the naphthalene fragment, strongly influen-
cing the conformation of the ansa chain and its orientation rela-
tive to the aromatic fragment. Thus, conformations of 1 in the
phenolic and phenolate forms should substantially differ, affect-
ing the docking process of this molecule at its RNAP binding
site. The crystal structure of the rifampicin derivative 2 (2-
CH OH–CH Cl , Fig. 1c) also reveals the phenolate form with a
proton attached to the N(38) atom of the amine group. This
group is involved in a strong intramolecular hydrogen bond with
the carbonyl oxygen atom of the amide fragment that is nearly
perpendicularly oriented with respect to the aromatic system
(C)spin couplings with neighbouring C-7, C-8 and C-9 carbons,
respectively. Furthermore, the δO(8)H value clearly demonstrates
the engagement of O(8)H group in the intramolecular hydrogen
bond with O(1) oxygen in all aprotic solvents, similar to the
crystal (Fig. 1a). However, in protic systems such as CD OD and
3
DMSO-d + H O, the C-8 resonances of 1 are shifted signifi-
6
2
cantly toward higher ppm values up to about 185 ppm (Fig. 2a).
This shift is a consequence of a proton transfer from O(8)H
group and the appearance of negative charge at O(8) delocalised
as a result of a strong resonance with the ketone group in para
position. Additional evidence of this proton transfer is provided
by the fact that the C-11 resonance of ketone moiety is strongly
shielded if compared with that of δC-11 region characteristic of 1
phenolic form in CDCl , py-d and CD CN. Now the question
3
2
2
(Fig. 1c). In effect, the rigidity of the ansa chain in 2 is increased
3
5
3
relative to that in 1.
of where the proton transferred from O(8)H phenolic group of 1
1
15
To study the proton transfer process in 1 and 2–9 the multi-
nuclear 1D and 2D NMR experiments were performed in sol-
vents of different dielectric constants with and without addition
of water (Fig. 2 and Fig. 1S–11S, Tables 3S and 4S†). Analysis
of the 165–200 ppm range for 1 (Fig. 2a) revealed that the pos-
ition of C-6, C-8 and C-11 signals in the spectra is strongly
dependent on the type of solvent used. In the aprotic solvents
in protic solvents is localised arises. As follows from H– N
HSQC and HMBC spectra of 1 in DMSO-d + H O (Fig. 1S
6
2
and 2S†) the nitrogen atom signals at: −30, −252, −255 and
−340 ppm are assigned to N(38), N(2) –H, N(39) and the posi-
+
tively charged N (40) atom, respectively. Thus, in protic sol-
vents, the proton of O(8)H is transferred to nitrogen N(40) and 1
exists in the phenolate form. As shown in Fig. 2a the addition of
such as CDCl , pyridine-d and CD CN C-11 (δ
= 195.3,
H O to 1 dissolved in aprotic solvents resulted in deshielding of
2
3
5
3
in CDCl3
δin py-d5 = 191.6 and δin
= 190.7 ppm) and C-8 (δin
δC-8 resonances, which is evidence for the proton transfer within
CD CN
3
2386 | Org. Biomol. Chem., 2012, 10, 2385–2388
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Table 1 Antibacterial activity MIC(μg mL− ) of 1, CIP and 2–9
1
Bacteria strain
1
2–8
9
CIP
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
0.008
0.016
0.008
0.008
0.008
0.5–2
1–2
0.5–2
0.5–2
0.008
0.016
0.008
0.008
0.008
0.5
0.5
0.5
0.5
0.5
0.125–0.25
1
and formation of the zwitterionic form and the key role of H O
2
molecules in this process. The uncharged and zwitterionic forms
of 1 (Scheme 2) in protic and aprotic solvents are reflected in the
respective crystal structures (Fig. 1a and b). A comparison of the
1
3
signals in the range 165–200 ppm between the C NMR spectra
of 1 (Fig. 2a) and 9 (Fig. 2b) indicated that 9 exists in the
phenolate form in protic solvents and in the phenolic one in
1
aprotic solvents, analogously to 1. In contrast to 1 and 9, the H,
1
3
15
C and N NMR data of derivatives 2–8 (Fig. 2b, 3S and 4S
and Tables 3S and 4S†) revealed in their structures the exclusive
presence of the phenolate form (Scheme 2), irrespective of
whether they are dissolved in protic or aprotic systems. The
proton transferred from the O(8)H group in structures 2–8 in
solution is localised, however at the N(38) atom of the highest
1
15
basicity as indicated by the H– N HSQC correlation
Fig. 4S†). Thus, the presence of the zwitterionic form and the
localisation of the transferred proton both in the crystal of 2
Fig. 1c) and in structures 2–8 in solution are consistent. Large
(
(
and pronounced differences in δC-11 and δC-8 shifts for phenolate
and phenolic forms exclude their simultaneous formation in
solution.
Comparison of antibacterial activity tests against Gram-(+)
strains of 1 and derivatives 2–9 (Tables 1 and 5S†) showed extre-
mely high activities of 1 and 9. Antibacterial activity of com-
pounds 2–8 is comparable to or even higher than that of the well
known antibacterial agent ciprofloxacin (CIP – Table 1).
The question of why compounds 2–8 reveal lower activity
than 1 and 9 arises. In attempt to answer this question the analy-
sis of possible interactions at the binding site to RNAP (Fig. 3)
was carried out. In the crystal structure reported by Campbell
3a
et al. the docking of 1 (yellow) at the binding site to RNAP is
mainly achieved via hydrogen bonds (yellow colour) between
the ansa-bridge groups and the amino acid residues as well as
via some hydrophobic interactions. As we have shown, in the
Fig. 3 Binding site to RNAP (green): (a) superposition of 1 phenolic
form (yellow), docked according to Campbell et al., with 1 zwitterio-
3a
nic form (grey), docked via and additional strong interaction with E445
,
(b) docked X-ray structure of 2. Other possible ligand–protein inter-
presence of H O, 1 exists as a zwitterion with a proton attached
actions are marked by yellow.
2
to N(40) atom and not in the uncharged phenolic form as
3
suggested earlier. Docking of the phenolate form of 1 into the
3
a
between the ansa-bridge groups and the amino acid residues of
crystal structure of RNAP (grey – Fig. 3a) and a simple
rotation around the C(38)–N(38) bond of the protonated pipera-
zine group revealed the possibility of formation of the earlier
3
a
RNAP, as reported by Campbell et al., are also possible.
Thus, as we have demonstrated, the presence of phenolate
form together with the localisation of the transferred proton at N
(40) atom of piperazine moiety is crucial for extremely high bio-
logical activity of 1 and 9 in comparison to that of the deriva-
tives 2–8.
overlooked relatively strong hydrogen bond (length 2.6 Å, angle
+
1
70°) between N(40) –H and the carboxylate group of E445.
This intermolecular hydrogen bond with the carboxylate of E445
should contribute to the extremely high antibacterial activity of 2
and 9, if one considers the lower activity of compounds 2–8, for
whose structures no similar interaction can be realised at the
RNAP binding site (Fig. 3b). It should be emphasized that when
Acknowledgements
1
and 2 were docked as zwitterions, in the form observed in their
K.P. wishes to thank the Foundation for Polish Science and
Adam Mickiewicz University Foundation for a fellowship. M.G.
crystal structures (Fig. 3a and b), the other important interactions
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Org. Biomol. Chem., 2012, 10, 2385–2388 | 2387

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and B.W. acknowledge support from the Polish Ministry of
Science and Higher Education (grant No. N N204 212940).
(c) I. Artsimovitch, M. N. Vassylyeva, D. Svetlov, V. Svetlov,
A. Perederina, N. Igarashi, N. Matsugaki, S. Wakatsuki, T. H. Tahirov and
D. G. Vassylyev, Cell, 2005, 122, 351–363.
4
5
A. Bacchi, G. Pelizzi, M. Nebuloni and P. Ferrari, J. Med. Chem., 1998,
41, 2319–2332.
Notes and references
P. Ferrari and G. G. Gallo, Farmaco, 1975, 30, 676–696.
1
E. Ngaimisi, S. Mugusi, O. Minzi, P. Sasi, K.-D. Riedel, A. Suda,
N. Ueda, M. Janabi, F. Mugusi, W. E. Haefeli, L. Bertilsson, J. Burhenne
and E. Aklillu, Clin. Pharmacol. Ther., 2011, 90, 406–413.
6 G. Tsukamoto, M. Taguchi and N. Aikawa, Chem. Pharm. Bull., 1980, 28,
2309–2317.
7 K. Bujnowski, L. Synoradzki, E. Dinjus, T. Zevaco, E. Augustynowicz-
Kopec and Z. Zwolska, Tetrahedron, 2003, 59, 1885–1893.
8 M. M. de Villiers, M. R. Caira, J. Li, S. J. Strydom, S. A. Bourne and
W. Liebenberg, Mol. Pharmaceutics, 2011, 8, 877–888.
2
3
H. G. Floss and T.-W. Yu, Chem. Rev., 2005, 105, 621–632.
(a) E. A. Campbell, N. Korzheva, A. Mustaev, K. Murakami, S. Nair,
A. Goldfarb and S. A. Darst, Cell, 2001, 104, 901–912; (b) E.
A. Campbell, O. Pavlova, N. Zenkin, F. Leon, H. Irschik, R. Jansen,
K. Severinov and S. A. Darst, EMBO J., 2005, 24, 674–682;
9 M. Gadret, M. Goursolle, J. M. Leger and J. C. Colleter, Acta Crystallogr.,
Sect. B: Struct. Crystallogr. Cryst. Chem., 1975, 31, 1454–1462.
2388 | Org. Biomol. Chem., 2012, 10, 2385–2388
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