Solid-state NMR spectroscopic analysis of the Ca2+-dependent mannose binding of pradimicinA

Article abstract of DOI:10.1002/anie.201007775

Aggregation facilitates analysis: The Ca²⁺-dependent mannose (Man) binding of the nonpeptidic carbohydrate binder pradimicinA (PRM-A) was investigated in the solid state. The use of PRM-A aggregates eliminated problems associated with the three-component equilibrium. A combination of ¹¹³CdNMR spectroscopy and 2D dipolar-assisted rotational resonance revealed the mannose-binding site of PRM-A and the crucial role of the Ca ²⁺ ion (see binding model). Copyright

Full text of DOI:10.1002/anie.201007775

Communications
DOI: 10.1002/anie.201007775
Carbohydrate Binding
2+
Solid-State NMR Spectroscopic Analysis of the Ca -Dependent
Mannose Binding of Pradimicin A**
Yu Nakagawa,* Yuichi Masuda, Keita Yamada, Takashi Doi, K. Takegoshi, Yasuhiro Igarashi,
and Yukishige Ito*
Pradimicins and benanomicins are closely related antibiotics
system by exposing cryptic immunogenic epitopes on the
virus surface. Both of these effects result from the specific
binding of PRM-A to Man residues of glycans on the viral
envelope.
[1]
isolated from actinomycetes. These compounds are unique
as nonpeptidic natural products with the lectin-like property
of being able to recognize d-mannopyranoside (Man) in the
2
+
[2]
presence of Ca ions. Recently, pradimicin A (PRM-A,
Scheme 1), the most commonmember of this family, has been
attracting much attention as a conceptually novel drug
There has been rapidly growing interest in small-size
“synthetic lectins” since Davis and co-workers demonstrated
that synthetic receptors can be used for the selective
[3]
[4]
candidate for human immunodeficiency virus (HIV). The
anti-HIV effect is ascribed to dual modes of action: PRM-A
blocks virus entry and triggers the action of the immune
recognition of carbohydrates in aqueous solution. The
biomimetic compounds that they have developed recognize
carbohydrates with all-equatorial substitution, including b-d-
glucopyranosides and N-acetyl-b-d-glucosamines, in a similar
manner to lectins and therefore have significant potential as
chemical tools in the field of glycomics. Although synthetic
receptors for Man are of particular benefit because of the
emerging biological significance of high-mannose-type oligo-
[
5]
saccharides, especially in protein-quality control, the devel-
opment of such compounds is challenging and still in its early
[
6]
stages. Under these circumstances, the action of PRM-A is a
groundbreaking concept for the design of synthetic receptors
for Man.
Given its scientific and therapeutic potential, it is highly
desirable to establish the molecular basis of Man recognition
by PRM-A. Until now, understanding about how PRM-A
2
+
recognizes Man in the presence of Ca ions has been limited.
The essence of the problem lies in the aggregation of the
1
3
Scheme 1. Structures of pradimicin A (PRM-A) and N- CH -PRM-A.
3
2
+
ternary PRM-A/Ca /Man complex and the complicated
three-component equilibrium, which have frustrated conven-
tional X-ray crystallographic and solution NMR spectroscop-
ic analyses. This situation led us to explore a conceptually
novel strategy for the solid-state analysis of the ternary
complex. Our strategy benefits from the aggregate-forming
propensity of PRM-A and eliminates the equilibrium prob-
lem. Herein, we report the interaction analysis of PRM-A
[
*] Dr. Y. Nakagawa, Dr. Y. Ito
Synthetic Cellular Chemistry Laboratory
RIKEN Advanced Science Institute
2-1 Hirosawa, Wako, Saitama, 351-0198 (Japan)
Fax: (+81)48-467-4680
E-mail: yu@riken.jp
yukito@riken.jp
2
+
with methyl a-d-mannopyranoside (Man-OMe) and Ca
ions through bipartite solid-state NMR spectroscopic experi-
Dr. Y. Masuda, K. Yamada, T. Doi, Prof. K. Takegoshi
Department of Chemistry, Graduate School of Science
Kyoto University (Japan)
2
+
ments. The results led us to propose an unprecedented Ca -
mediated binding model of PRM-A with Man.
Prof. Y. Igarashi
Biotechnology Research Center
Toyama Prefectural University (Japan)
Although several lines of evidence have indicated that two
2
+
molecules of PRM-A bind a single Ca ion, and the carboxy
group of PRM-Awas proposed as the putative binding site for
Dr. Y. Ito
2
+
[7]
Japan Science and Technology Agency
ERATO, Ito Glycotrilogy Project (Japan)
the Ca ion, clear experimental support for this theory has
yet to be provided. Furthermore, it remains unclear whether
[
**] We thank Prof. Toshikazu Oki for his generous support, and Akemi
Takahashi and Satoko Shirahata for technical assistance. This
research was partly supported by the Fund for Seeds of Collabo-
rative Research of RIKEN and a MEXT Grant-in-Aid for Young
Scientists (B) (22780109). Y.M. thanks JSPS for a postdoctoral
fellowship.
2+
the role of the Ca ion is solely to bridge two PRM-A
molecules, or whether it also participates in Man binding. To
1
13
address these issues, we planned a solid-state Cd NMR
1
13
2+
spectroscopic investigation with Cd ions with a spin of 1/2
2
+
as a surrogate probe for Ca ions with a spin of 7/2.
1
13
Cd NMR spectroscopy has proven to be an excellent
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201007775.
2
+
technique for the examination of the Ca environments
6
084
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Angew. Chem. Int. Ed. 2011, 50, 6084 –6088

1
13
2+
2+
2+
2+
present in biological systems because Cd and Ca ions
below). The structural similarity of Ca - and Cd -containing
[
8]
13
have the same formal charge and similar ionic radii. The
ternary complexes was confirmed by CP/MAS C NMR
experiments; solid samples of these complexes gave similar
spectra (see Supporting Information). Having confirmed that
1
13
attractive feature of the Cd nucleus is its very broad range
of chemical shifts (> 800 ppm), which results in high sensi-
tivity of the chemical shifts to the nature, number, and
2
+
2+
the Cd ion serves as a surrogate for the Ca ion in the Man
1
13
2+
113
geometry of ligands coordinated to the Cd ion. Thus, our
solid-state analysis started with the investigation of the role of
binding of PRM-A, we conducted CP/MAS
spectroscopic experiments with solid samples of the binary
Cd NMR
2
+
113
2+
113
2+
the Ca ion on the Man binding of PRM-A by cross-
polarization/magic angle spinning (CP/MAS)
spectroscopy.
PRM-A/ Cd and ternary PRM-A/ Cd /Man-OMe com-
plexes.
1
13
Cd NMR
1
13
113
2+
The Cd NMR spectrum of the binary PRM-A/ Cd
As a prerequisite, we confirmed the specific binding of
complex exhibited a broad signal around d = À50 ppm
2
+
PRM-A to Man-OMe in the presence of Ca ions. After
(Figure 1a). This chemical shift is similar to those
2
+
preparing the binary PRM-A/Ca
complex by adding
aqueous CaCl (1 equiv) to an equimolar mixture of PRM-
2
A and NaOH in water, we examined its coprecipitation with
Man-OMe. In the presence of Man-OMe (25 equiv), forma-
tion of the ternary complex was observed. We quantified the
incorporation of Man-OMe (PRM-A/Man-OMe 1:1.08) by
1
solution H NMR spectroscopic analysis of the mixture after
dissociation of the precipitated complex by acid treatment
(
see the Supporting Information). Methyl a-d-glucopyrano-
2
+
side (Glc-OMe) coprecipitated with the binary PRM-A/Ca
complex to a negligible extent (PRM-A/Glc-OMe 1:0.08),
2
+
which indicates that the binary PRM-A/Ca complex specif-
ically binds Man-OMe.
Under the complex-forming conditions, the PRM-A/Man-
2
+
OMe ratio in the ternary PRM-A/Ca /Man-OMe complex
was estimated to be 1:1 even in the presence of an excess
amount of Man-OMe (250 equiv); this result is inconsistent
[
7a]
113
113
2+
with the 1:2 ratio reported by Ueki et al.
A possible
Figure 1. Solid-state CP/MAS Cd NMR spectra of PRM-A/ Cd
complexes prepared a) without Man-OMe, b) with Man-OMe
25 equiv), and c) with Man-OMe (250 equiv). The signals with an
asterisk are the spinning side bands of the Cd signal at
explanation is that PRM-A might possess two Man-binding
sites with different affinities. Whereas Ueki et al. determined
the PRM-A/Man ratio by the phenol–sulfuric acid method,
(
1
13
d=À135 ppm.
2
+
without washing the aggregate of the ternary PRM-A/Ca /
Man-OMe complex, our complex-forming procedure
included an extensive washing process to eliminate non-
specific binding of Man-OMe; during this washing process,
Man-OMe might have been released from the weaker binding
site. The existence of two Man-binding sites in PRM-A is
supported by a previous spectroscopic study of Fujikawa
reported for solid cadmium compounds with two carboxy
groups, such as Cd(OAc) ·2H O
(d = À46 ppm),
Cd(O CCH CH CO )·2H O (d = À52 ppm), and [{Cd(o-
2
2
2
2
2
2
2
HOC H CO ) ·2H O} ] (d = À31 ppm), but quite different
6
4
2
2
2
2
from those reported for Cd(OH)2 (d = 158 ppm) with
hydroxy groups, [Cd(en) Cl ·H O] (d = 380 ppm; en =
[
9]
et al., who showed that one molecule of PRM-A binds two
molecules of Man in two separate steps. They proposed that
3
2
2
ethylenediamine) with amino groups, [Na Cd(edta)]
(d=102 ppm; EDTA = ethylenediaminetetraacetate) and
Cd(NH CH CO ) ·H O (d = 112 ppm) with amino/carboxy
groups, and [Cd(glycylglycine) ·2H O] (d = 169 ppm) with
carboxy/amino/amide groups.
the Cd ion binds to the carboxy group of PRM-A. This
observation supports the putative role of the Ca ion to
bridge the carboxy groups of two PRM-A molecules.
Considering that chemical exchanges are prohibited in the
solid state, and anisotropy effects are minimized by MAS, the
broadness of the signal may well be attributed to slightly
different chemical environments around the Cd ion owing
to structural heterogeneity of the binary PRM-A/ Cd
complex. On the other hand, the PRM-A/ Cd sample
prepared in the presence of Man-OMe (25 equiv) exhibited a
markedly sharper signal at d = À135 ppm along with a broad
signal almost identical to that observed in the absence of
Man-OMe (Figure 1b). The broad signal around d =
2
2
+
the binary PRM-A/Ca complex initially binds two mole-
2
+
cules of Man to form the ternary PRM-A/Ca /Man complex
with a ratio of 2:1:2, and that another two molecules of Man
are then incorporated to form the ultimate ternary complex
with a ratio of 2:1:4.
2
2
2
2
2
2
2
[
8a,10]
It therefore suggests that
1
13
2+
2
+
2+
Coprecipitation in the presence of Cd ions (1 equiv) and
Man-OMe or Glc-OMe (25 equiv) indicated that the binary
[
7b]
2
+
PRM-A/Cd complex also specifically binds Man-OMe
PRM-A/Man-OMe 1:0.46 versus PRM-A/Glc-OMe 1:0.02).
The PRM-A/Man-OMe ratio changed to 1:0.88 when
0 equivalents of Man-OMe were used, which indicates that
(
1
13
2+
5
2
+
113
2+
the precipitate formed in the presence of Cd ions was a
mixture of the ternary PRM-A/Cd /Man-OMe and binary
PRM-A/Cd complexes. This assumption was reinforced by
the presence of two signals with almost same area in the CP/
MAS Cd NMR spectrum of the PRM-A/ Cd complex
prepared with 25 equivalents of Man-OMe (Figure 1b; see
2
+
113
2+
2
+
1
13
113
2+
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Communications
À50 ppm almost disappeared when an excess amount of Man-
Although semisynthesis through detachment of the d-
alanine moiety followed by the introduction of C-enriched
1
3
OMe (250 equiv) was used (Figure 1c), which suggests that
the sharp signal at d = À135 ppm is derived from the ternary
d-alanine would be a possible approach to preparation of the
target C-enriched PRM-A, both cleavage and reconstruc-
1
13
2+
[11]
13
PRM-A/ Cd /Man-OMe complex. The clearly narrower
line width of the signal at d = À135 ppm probably reflects the
higher structural homogeneity of the ternary complex. More
notable is the marked upfield shift (> 80 ppm) of the signal
for the ternary complex with Man-OMe in comparison with
that of the binary complex, which does not contain Man-
OMe. This observation strongly indicates the occurrence of a
tion of the amide bond of PRM-A were found to be
problematic as a result of steric hindrance around the carboxy
[
15]
group.
Thus, we took advantage of the biosynthesis of
[16]
PRM-A
and used Actinomadura sp. TP-A0019 for the
1
3
preparation of PRM-A with a C-enriched d-alanine moiety.
Exogenous d-[ C ]alanine was successfully incorporated into
1
3
3
1
13
2+
change in Cd coordination upon Man-OMe binding. Since
PRM-A by inhibiting the supply of endogenous d-alanine
1
13
[17]
Cd signals upfield of d = À100 ppm are observed only for
through the addition of d-cycloserine,
an inhibitor of
1
13
2+
[8]
Cd coordinated with more than six oxygen ligands, it is
alanine racemase (see the Supporting Information).
1
3
reasonable to assume that Man-OMe coordinates as an
We obtained 2D DARR spectra of C-enriched PRM-A/
1
13
2+
2+ 13
additional ligand to the Cd ion in the binary PRM-A/
Ca /[ C ]Man-OMe at mixing times of 20 and 500 ms
6
1
13
2+
Cd complex. The realistic implication of these results is
(Figure 2). Whereas only intramolecular cross-peaks were
observed at the mixing time of 20 ms (Figure 2a), the
spectrum recorded at the mixing time of 500 ms (Figure 2b)
clearly showed intermolecular cross-peaks between carbon
signals for the d-alanine moiety of PRM-A (d = 20.0, 50.8,
179.8 ppm) and those for Man-OMe (d = 63.0, 68.2, 71–76,
2
+
that the role of the Ca ion in the Man-binding process of
PRM-A is twofold: it acts as a core to bridge two PRM-A
molecules and directly participates in Man binding.
The results of solid-state Cd NMR spectroscopic anal-
ysis suggested the possibility that Man is located in the
1
13
2
+
proximity of the Ca -bound carboxy group of PRM-A in the
ternary PRM-A/Ca /Man complex (Scheme 2). To obtain
2
+
2
+
Scheme 2. Model for the Ca -mediated binding of PRM-A with Man-
2+
OMe. We propose that in the ternary complex (PRM-A/Ca /Man-OMe
:1:2), a bridge structure consisting of the carboxy groups of two
2
2
+
PRM-A molecules and the Ca ion binds two molecules of Man-OMe
in a Ca -mediated manner.
2
+
more concrete experimental support for this assumption, we
performed two-dimensional dipolar-assisted rotational reso-
[
12]
nance (2D DARR) experiments for detection of the close
interaction of Man-OMe with the d-alanine moiety of PRM-
2
+
A, which contains the Ca -bound carboxy group. The DARR
method, also known as radiofrequency-assisted diffusion
[
13]
13
13
(
RAD), has been shown to detect weak C– C coupling
in the presence of strong coupling due to directly bound
[
14]
13
carbon atoms, and dipolar interactions between C nuclei
that are located within 6 ꢀ of one another can be detected as
cross-peaks in the 2D DARR spectrum. Therefore, we began
the 2D DARR investigation with the preparation of PRM-A
13
Figure 2. 2D DARR spectra of the ternary C-enriched PRM-A/
2+ 13
Ca /[ C ]Man-OMe complex at mixing times of a) 20 ms and
6
1
3
b) 500 ms. Red and blue circles indicate C signals derived from
1
3
13
13
with a C-enriched d-alanine moiety.
C-enriched PRM-A and [ C ]Man-OMe, respectively.
6
6
086 www.angewandte.org
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1
01.4 ppm). To eliminate the possibility that these cross-peaks
which PRM-A binds Man-OMe through coordination with
+
2
were simply derived from the accidental proximity of PRM-A
to Man-OMe in the solid sample, we carried out a control
experiment with N- CH -PRM-A (Scheme 1), which was
the Ca ion (Scheme 2).
2
+
In conclusion, we investigated the Ca -dependent man-
nose binding of PRM-A in the solid state. The analysis
strategy based on solid-state NMR spectroscopy enabled us to
avoid the problems associated with aggregation and the
complicated three-component equilibrium, which have ham-
pered conventional interaction analysis. On the basis of two
solid-state NMR spectroscopic experiments, we propose an
1
3
3
prepared by the reductive methylation of PRM-A with
1
3
[18]
[
C]formaldehyde. The 2D DARR spectra of the ternary
1
3
2+ 13
N- CH -PRM-A/Ca /[ C ]Man-OMe complex (Figure 3)
showed only intramolecular cross-peaks for [ C ]Man-OMe
3
6
1
3
6
1
3
(d = 64.3, 68.8, 71–76, 101.3 ppm) and for the CH group of
3
1
3
2+
N- CH -PRM-A (d = 42.8, 48.6 ppm), which was detected as
unprecedented Ca -mediated binding model of PRM-A with
3
two signals, probably because of slow inversion at the
asymmetric nitrogen center. No intermolecular cross-peak
was detectable even at the mixing time of 500 ms. This result
indicates that nonspecific binding of PRM-A with Man-OMe
is negligible, and the cross-peaks between carbon signals for
the d-alanine moiety of PRM-A and Man-OMe truly arises
from specific close interactions. Taken together, our results
indicate that Man-OMe is located within 6 ꢀ of the d-alanine
Man (Scheme 2). It is particularly significant that intermo-
lecular interactions between the d-alanine moiety of PRM-A
and Man-OMe were detected by 2D DARR. This result is a
rare example of the identification of the ligand-binding region
of a receptor by solid-state NMR spectroscopy, and more
importantly, the first solid evidence that the d-alanine moiety
of PRM-A is the Man-binding region. The present study
provides a clue toward the full elucidation of the molecular
basis of Man recognition by PRM-A. Further investigations
along this line are currently in progress.
2
+
moiety of PRM-A, which contains the Ca -bound carboxy
group. They strongly support our proposed binding model in
Received: December 10, 2010
Revised: March 18, 2011
Published online: May 19, 2011
Keywords: antibiotics · carbohydrates · natural products ·
.
receptors · solid-state NMR spectroscopy
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Supporting Information.
6
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