Solid-state NMR analysis of calcium and D-mannose binding of BMY-28864, a water-soluble analogue of pradimicin A

Article abstract of DOI:10.1016/j.bmcl.2011.11.106

Pradimicin A (PRM-A) is a unique antibiotic with a lectin-like ability to recognize d-mannopyranosides (Man) in the presence of Ca²⁺ ion. BMY-28864 (1) is a water-soluble analogue of PRM-A, which has been extensively used for studies on the mode of Man recognition and antifungal action of pradimicins. Although it has been assumed that PRM-A and 1 bind Man in a similar fashion, direct experimental evidence has yet to be provided. In this report, we compared Ca²⁺ and Man binding of 1 with that of PRM-A through two solid-state NMR experiments. The solid-state ¹¹³Cd NMR analysis using ¹¹³Cd²⁺ ion as a surrogate for Ca²⁺ ion suggested the similarity in Ca²⁺ coordination of PRM-A and 1. The dipolar assisted rotational resonance (DARR) analysis using ¹³C- labeled 1 clearly showed that 1 as well as PRM-A binds Man near its carboxyl group. These results collectively indicate that the mode of binding of Ca ²⁺ ion and Man is nearly identical between PRM-A and 1.

Full text of DOI:10.1016/j.bmcl.2011.11.106

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1040–1043
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Bioorganic & Medicinal Chemistry Letters
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Solid-state NMR analysis of calcium and D-mannose binding of BMY-28864,
a water-soluble analogue of pradimicin A
Yu Nakagawa a, , Takashi Doi b, K. Takegoshi b, Yasuhiro Igarashi c, Yukishige Ito a,d,
⇑
⇑
a Synthetic Cellular Chemistry Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
c Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
d Japan Science and Technology Agency, ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Pradimicin A (PRM-A) is a unique antibiotic with a lectin-like ability to recognize D-mannopyranosides
(Man) in the presence of Ca2+ ion. BMY-28864 (1) is a water-soluble analogue of PRM-A, which has been
extensively used for studies on the mode of Man recognition and antifungal action of pradimicins.
Although it has been assumed that PRM-A and 1 bind Man in a similar fashion, direct experimental evi-
dence has yet to be provided. In this report, we compared Ca2+ and Man binding of 1 with that of PRM-A
through two solid-state NMR experiments. The solid-state 113Cd NMR analysis using 113Cd2+ ion as a sur-
rogate for Ca2+ ion suggested the similarity in Ca2+ coordination of PRM-A and 1. The dipolar assisted
rotational resonance (DARR) analysis using 13C-labeled 1 clearly showed that 1 as well as PRM-A binds
Man near its carboxyl group. These results collectively indicate that the mode of binding of Ca2+ ion
and Man is nearly identical between PRM-A and 1.
Received 14 November 2011
Revised 24 November 2011
Accepted 28 November 2011
Available online 6 December 2011
Keywords:
Antibiotic
Carbohydrate recognition
Natural product
Pradimicin
Ó 2011 Elsevier Ltd. All rights reserved.
Solid-state NMR
Pradimicin A (PRM-A, Fig. 1) and its congeners are the only
family of natural antibiotics with a lectin-like ability to recognize
Ca2+-mediated manner through its carboxyl group, and the cavity
consisting of -alanine moiety and ABC rings constitutes a Man
D
D
-mannopyranosides (Man) in the presence of Ca2+ ion.1,2 In recent
binding site (Fig. 2). These results suggest that D-alanine moiety
years, they have been attracting much attention as conceptually
novel drug candidates for human immunodeficiency virus
(HIV).3,4 PRM-A has been shown to block the virus entry to the host
cells and force the virus to progressively delete the envelope gly-
cans which shield against the host immune system. This dual mode
of antiviral action is ascribed to its specific binding to the high-
mannose-type glycans on the viral envelope.
Despite significance in terms of scientific interest as well as
therapeutic potential, the molecular basis of Man recognition by
pradimicins remains to be clarified. The essence of the problem lies
in their aggregate-forming propensity and complicated three-com-
ponent equilibrium in solution, which have hampered conven-
tional X-ray crystallographic and solution NMR analyses. Under
these circumstances, we have recently developed a new strategy
to analyze the ternary complex of PRM-A with Ca2+ ion and Man
in the solid state.5,6 The key of our analytical strategy is the use
of the aggregate of the ternary complex of PRM-A with Ca2+ ion
plays pivotal roles in both Ca2+ and Man binding of PRM-A.
In fact, these results are in accordance with the previous
structure–activity relationship studies of PRM-A. Replacement of
the D-alanine moiety with L-alanine or most of other D-amino acids
significantly diminished antifungal activity, which is correlated
with Man binding ability of pradimicins.7 The notable exceptions
were D-serine analogues (1, 2), which show significant antifungal
activity comparable to PRM-A (Fig. 1).8 Although additional
N-methyl group of BMY-28864 (1) does not impact the antifungal
activity, water solubility of 1 is significantly higher than those of
PRM-A and
2 (0.02 mg/mL for PRM-A, >20.0 mg/mL for 1,
0.26 mg/mL for 2 in phosphate buffered saline, pH 7.2).8,9 These
features have led 1 to be extensively used for studies on the mode
of Man recognition and antifungal action of pradimicins.2,10
Although it has been assumed a priori that PRM-A and 1 bind
Man in a similar fashion, the emerging importance of D-alanine
moiety of PRM-A requires direct experimental validation of this
hypothesis. To clarify this issue, we compared Ca2+ and Man bind-
ing of 1 with that of PRM-A through two solid-state NMR
experiments.
and methyl
a-D-mannopyranoside (Man-OMe), which enabled us
to analyze Ca2+ and Man binding of PRM-A by solid-state NMR
spectroscopy. Our studies revealed that PRM-A binds Man in a
In the previous study, we investigated the Ca2+ binding property
of PRM-A by employing solid-state 113Cd NMR spectroscopy.5
113Cd NMR spectroscopy has proven to be an excellent technique
for analysis of the Ca2+ environments present in biological systems,
⇑
Corresponding authors. Tel.: +81 48 467 9434; fax: +81 48 462 4680 (Y.N.);
tel.: +81 48 467 9430; fax: +81 48 462 4680 (Y.I.).
E-mail addresses: yu@riken.jp (Y. Nakagawa), yukito@riken.jp (Y. Ito).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.106

Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1040–1043
1041
OH
Me
CO2H
D-Ala
CO2H
D-Ser
O
NH
Me
O
NH
Me
HO
OH
14
HO
OH
O
O
O
O
A
MeO
MeO
C
B
Me
4'
Me
4'
O
2'
O
O
O
HO
HO
OH
OH
HO
OH
OH
O NHMe
O NMe
R
O
O
HO
HO
HO
OH
OH
Pradimicin A (PRM-A)
BMY-28864 (1): R = Me
2 : R = H
Figure 1. Structures of pradimicin A (PRM-A) and its serine analogues (1, 2).
because 113Cd2+ ion has an ionic radius similar to that of Ca2+ ion
and favorable characteristics for NMR analysis such as a spin of
1/2, reasonable sensitivity, and a broad chemical shift window
(>800 ppm).11,12 113Cd2+ ion was confirmed to serve as a good sur-
rogate for Ca2+ ion in Man binding of PRM-A, and successfully form
a ternary complex with PRM-A and Man-OMe.5 The solid aggregate
of the complex exhibited a sharp signal at d = –135 ppm in the
cross-polarization/magic angle spinning (CP/MAS) 113Cd NMR
spectrum, reflecting coordination of more than six oxygen ligands
to 113Cd2+ ion. Using this characteristic 113Cd signal as a guide, we
initially investigated Ca2+ binding of 1 by solid-state 113Cd NMR
spectroscopy.
BMY-28864 (1) was prepared according to the procedure re-
ported by Oki et al. using Actinomodura sp. TP-A0019.8,13 Although
1 formed little aggregates as a binary complex with 113Cd2+ ion due
to its high water solubility, the solid aggregate of the ternary com-
plex consisting with 113Cd2+ ion and Man-OMe was effectively ob-
tained.14 Comparison of the CP/MAS 113Cd NMR spectra of the
ternary complexes of PRM-A5 and 1 is shown in Figure 3. The latter
complex exhibited a sharper signal compared to the former. The
narrower line width of the signal probably reflects the higher
structural homogeneity of the ternary complex of 1. The 113Cd sig-
nal upfiled of d = ꢀ100 ppm suggests that 113Cd2+ ion in the ternary
complex of 1 as well as PRM-A is coordinated with more than six
oxygen ligands.11,12 Since 113Cd nucleus has a broad range of
chemical shifts (>800 ppm) that is sensitive to the nature, number,
and geometry of ligands coordinated to 113Cd2+ ion, it is reasonable
to assume that the slightly downfield signal of 1 (d = ꢀ129 ppm)
compared to that of PRM-A (d = ꢀ135 ppm) is ascribed to a subtle
difference in the 113Cd2+ environment in the ternary complexes.
The realistic implication of this result is that the Ca2+ coordination
in the ternary complex of 1 is almost similar to that of PRM-A.
Investigation of Man binding of PRM-A has been performed by
two-dimensional dipolar assisted rotational resonance (2D-
DARR)15,16 using 13C-enriched PRM-As.5,6 DARR, also known as RF
assisted diffusion (RAD),17 is a technique of solid-state NMR spec-
troscopy to detect weak 13C–13C couplings in the presence of
strong couplings derived from directly bound carbons. In 2D-DARR
spectra, dipolar interactions between 13C nuclei that are located
within 6 Å can be detected as cross peaks. In the DARR experiments
using the solid aggregates of the ternary complexes of 13C-labeled
PRM-As with Ca2+ ion and [13C6]Man-OMe, strong intermolecular
cross peaks were observed between the 13C signals for the
D-ala-
nine moiety of PRM-A and those for Man-OMe whereas the 13C sig-
nal for the N-methyl group at position 40 of PRM-A gave no
intermolecular cross peak.5,6 These observations clearly indicated
that PRM-A binds Man in its proximity to the D-alanine moiety.
In order to substantiate the specific close contact of Man with D-
(A)
*
*
Man-OMe
PRM-A
Ca2+
(B)
OH
HO
O
O
HO
HO
O
D-Ala
OMe
Me
MeO
HO
HN
NHMe
*
*
O
D-Xyl
Me
O
OH
O
14
HO
HO
Me
300
200
100
0
-100
-200
-300
2'
O
A
C
113Cd Chemical shift (ppm)
B
O
HO
O
Figure 3. Solid-state CP/MAS 113Cd NMR spectra of the ternary complexes of (A)
PRM-A5 and (B) 1 with 113Cd2+ ion and Man-OMe. One equivalent of 113CdCl2 and
250 equiv of Man-OMe were used for complete formation of the ternary complexes
with minimizing non-specific binding of 113Cd2+ ion to the aggregates.5 Chemical
shifts were calibrated in ppm relative to external solid Cd(ClO4)2ꢁ6H2O. The signals
with an asterisk are the spinning side bands of the 113Cd signals.
Ca2+-coordination
H-bond
CH/π interaction
Figure 2. Putative binding model of PRM-A with Man-OMe.6

1042
Y. Nakagawa et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1040–1043
13C-labeled position
=
OH
OH
CO2H
NH
Me
CO2H
NH
Me
O
O
HO
OH
HO
OH
O
O
O
O
20% H13CHO
NaBH3CN
Actinomadura sp.
TP-A0019
MeO
MeO
DL-[1-13C]Ser
Me
Me
CH3CN, H2O
O
O
O
O
HO
HO
OH
OH
HO
OH
OH
HO
O
O
O
O
NHMe
NMe
Me
HO
HO
OH
D-Ser analogue of PRM-A (2)
Scheme 1. Preparation of 13C-labeled 1.
OH
13C-labeled BMY-28864 (1)
serine moiety of 1, we planned to conduct DARR experiments using
13C-labeled 1.
C2 – C6 of
Man-OMe
C1 of
Man-OMe
(A)
To facilitate the DARR analysis, 13C-labeling of the
D
-serine car-
boxyl group and N-methyl group of 1 was carried out (Scheme 1).
Since a negligible amount of the -serine analogue of PRM-A (2) is
naturally produced by Actinomodura sp. TP-A0019, 13C-labeling of
the -serine carboxyl group was easily accomplished by feeding
D
D
of DL-[1-13C]serine. The 13CH3 group was then successfully intro-
duced to the amine group at position 40 by reductive methylation
using H13CHO (62%).5 The solution 13C NMR spectrum of resulting
13C-labeled 1 confirmed a high level of 13C-enrichment of the
targeted two carbons (Supplementary data). Figure 4 shows the
2D-DARR spectra of the ternary complex of 13C-labeled 1 with
Ca2+ ion and [13C6]Man-OMe at mixing times of 20 and 500 ms.
Whereas only intramolecular cross peaks were observed at the
mixing time of 20 ms (Fig. 4A), significant intermolecular cross
40
80
N-Me
of 1
120
160
peaks between the 13C signal for the
D-serine carboxyl group
(d = 176.3 ppm) and those for Man-OMe (d = 63.5, 68.4, 71–75,
101.2 ppm) were detected at the mixing time of 500 ms (Fig. 4B).
On the other hand, no intermolecular cross peak was detectable
for the 13CH3 group (d = 43.2, 48.6 ppm), which appeared as two
signals probably because of slow inversion at the asymmetric
nitrogen center. These observations indicate that 1 as well as
PRM-A binds Man near the carboxyl group.
CO2H
of 1
160
120
80
40
13C Chemical shift (ppm)
C2 – C6 of
Man-OMe
C1 of
(B)
In summary, we compared Ca2+ and Man binding of BMY-28864
(1) with that of PRM-A through solid-state 113Cd NMR and DARR
experiments. The results suggest that PRM-A and 1 are almost
the same in terms of Ca2+ coordination and Man binding site. This
is the first direct experimental evidence that PRM-A and 1 bind
Man in a similar fashion. Further investigations to understand
Man-OMe
why only pradimicins with D-alanine and D-serine moieties can
bind Man are currently underway.
N-Me
of 1
40
80
Acknowledgments
We thank Professor Toshikazu Oki for his generous support, and
Ms. Akemi Takahashi and Ms. Satoko Shirahata for technical assis-
tance. This research was partly supported by MEXT Grant-in-Aid
for Young Scientists (B) (22780109).
120
160
CO2H
of 1
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.106.
160
120
80
40
Figure 4. 2D-DARR spectra of the ternary complex of 13C-labeled 1 with Ca2+ ion
and
13C6]Man-OMe at the mixing times of (A) 20 ms and (B) 500 ms. Ten
equivalent of CaCl2 and 25 equiv of
13C6]Man-OMe were used for complete
formation of the ternary complex with minimizing non-specific binding of
[
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