Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR

Article abstract of DOI:10.3762/bjoc.8.51

A novel reporter system, which is applicable to the ¹⁹F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein-ligand or protein-protein interactions of proteins fused or tagged to the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both ¹⁹F NMR signals of gluco/ manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between ¹⁹F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein-ligand interaction interfaces.

Full text of DOI:10.3762/bjoc.8.51

Synthesis of fluorinated maltose derivatives for
monitoring protein interaction by 19F NMR
Michaela Braitsch1, Hanspeter Kählig1, Georg Kontaxis2, Michael Fischer1,
Toshinari Kawada3, Robert Konrat*2 and Walther Schmid*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 448–455.
1Department of Organic Chemistry, University of Vienna, Währinger
Strasse 38, A-1090 Vienna, Austria, 2Department of Structural and
Computational Biology, Max F. Perutz Laboratories, University of
Vienna, Campus Vienna Biocenter 5, A-1030 Vienna, Austria and
3Graduate School of Life and Environmental Sciences, Kyoto
Prefectural University, Sakyo-Ku, Kyoto 606-8522, Japan
doi:10.3762/bjoc.8.51
Received: 16 January 2012
Accepted: 08 March 2012
Published: 27 March 2012
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
Email:
Robert Konrat* - robert.konrat@univie.ac.at;
Walther Schmid* - walther.schmid@univie.ac.at
Guest Editor: T. K. Lindhorst
* Corresponding author
© 2012 Braitsch et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
fluorination; 19F NMR; maltose-binding protein (MBP); maltose
derivatives; protein interaction
Abstract
A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses
2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to
the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/
manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding
isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative
affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect
agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation
between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand inter-
action interfaces.
Introduction
In recent years, we have witnessed significant improvements in and cryogenic-probe technology, unprecedented structural and
NMR spectroscopy, especially as a powerful tool for studying functional information can be obtained on biologically impor-
protein–ligand and protein–protein interactions [1,2]. Based on tant protein–ligand systems and protein complexes [2]. To over-
tremendous gains in sensitivity due to high-field spectrometers come the well-known and inherent problem of molecular weight
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Beilstein J. Org. Chem. 2012, 8, 448–455.
limitation of current NMR spectroscopy, which renders direct by using 2-F labeled maltose as a reporter system. The ratio-
protein observation of the interaction partners infeasible, an nale for choosing maltose lies in the fact that maltodextrin/
indirect observation technique for the detection of protein inter- maltose-binding protein (MBP) is a generally applicable protein
actions was recently established [3]. It utilizes the relaxation fusion tag with beneficial solution properties and therefore
properties of a small-molecular-weight reporter ligand that widely used in molecular biology [12,13].
reversibly binds to a ligand binding domain, which in turn
is fused to the interacting protein of interest. Subsequent MBP belongs to the family of periplasmic binding proteins,
protein–protein interaction leads to an additional increase of the which are involved in active transport processes of small mole-
molecular weight of the complex and can efficiently be probed cules into gram-negative bacteria through their function as an
by following the NMR relaxation changes of the ligand (e.g., initial high-affinity binding component; furthermore, these
selective T1 or T2, which reflect the effective molecular proteins participate as sensors for signaling during chemotaxis
weight). Due to this indirect detection scheme no isotope [14]. MBP binds maltodextrin and linear oligosaccharides of up
labeling of the protein interaction partners is required and to eight α(1→4)-linked glucose (Glc) units with micromolar
consumption of protein material is reduced.
affinities [15,16]. X-ray structural data (PDB ID codes
1-DMB and 1ANF) demonstrated that the MBP (370 residues,
The concept presented here relies on the development of an Mr = 41 kDa) consists of two globular domains joined by a
indirect 19F-detected NMR reporter system with possibilities for hinge-bending region, in which the ligand binding site is located
internal control for the study of protein-binding events. The in a cleft between the two domains. MBP exists in two different
benefits of fluorine (19F) NMR detection for ligand-based NMR conformations: The ligand-free “open” form, exposing the
screening applications as well as for 19F magnetic resonance binding site, and in the presence of a ligand, the “closed” form,
imaging (MRI) have been convincingly demonstrated in the trapping the ligand to provide contacts from both domains [17-
past [4-11]. The usage of the fluorine NMR alleviates most of 19]. The number of protein–sugar hydrogen bonds associated
the problems encountered with 1H observation, such as signal with maltose and MBP is 12, excluding those with water and
overlap and problems with the dynamic range. Additionally, the between glucose units. The reducing glucose unit (g1) makes
19F nucleus with 100% natural abundance and a magnetogyric about twice as many direct hydrogen bonds with MBP as the
ratio comparable to 1H is highly sensitive and, due to its large nonreducing glucose unit (g2) does (Figure 1). But there is
chemical shift anisotropy (CSA), very responsive to changes of some evidence for the importance of hydrogen bonds and
molecular weight that accompany the binding events.
van der Waals interactions for the oligosaccharide binding as
well [20-22].
We thus anticipate 19F detection to be a general and versatile
probe for indirect NMR studies of protein-binding and inter-
action events. Biological systems often require sophisticated
buffer systems for stabilization and solubility, thus leading
to severe spectral overlap and problems with the dynamic
range (e.g., intense buffer and solvent peaks). These drawbacks
are particularly present in the case of membrane-bound (or
attached) proteins, in which additional peaks originate from
membrane lipids and raise severe technical problems. However,
indirect detection techniques should always be cross-checked
with reference experiments and suitable controls, to demon-
strate selectivity of binding and to exclude systematic errors
(e.g., nonspecific binding or aggregation, and/or viscosity
changes due to increased protein concentration). Ideally, the
system of choice would thus be a mixture of reporter ligands
consisting of one 19F-labeled reporter ligand and another
chemically similar (also 19F-labeled) reference compound
lacking the affinity to the ligand binding domain.
Here we describe the possibility of monitoring protein interac-
tions by 19F NMR, known as fluorine chemical-shift anisotropy
and exchange for screening (FAXS) [5-7], with internal control
Figure 1: Schematic diagram of the network of hydrogen bonds in the
binding pocket of the complex between MBP and maltose (PDB ID
code 1ANF); hydrogen bonds are shown as dashed lines.
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Beilstein J. Org. Chem. 2012, 8, 448–455.
Specifically, the 2-OH and the 2′-OH moieties are involved in
an intricate hydrogen bonding network including the carboxy
group of Glu111 and Asp65 and the amino group of Lys15 and
Trp62, respectively. We thus decided to synthesize 2-19F-
labeled maltose. By replacing the OH group by fluorine and
modifying the stereochemistry at position 2, different binding
affinities of the anomeric mixture of the two resulting diastereo-
mers were expected (Figure 2). The gluco-type 2-F-maltose, in
which the fluorine atom occupies the equatorial position of g1
of maltose, should display comparable binding affinities to
maltose itself, whereas the manno-type 2-F-maltose was
Figure 2: 2-19F-Maltose reporter system: Nonstereoselective fluorine
labeling at the 2-position of maltose leads to a 2/1 mixture of two
epimeric forms [left: gluco-type; right: manno-type]. Only the gluco-type
isomer of 2-deoxy-2-fluoro-maltose retains the affinity to the maltose-
binding protein (MBP).
Results and Discussion
expected to lose its affinity due to the axial orientation of the Syntheses
fluorine atom. The synthesis of the 2-F-maltose reporter system was performed
following a modified protocol developed by Dax et al. [23,24].
This 19F-labeled reporter experiment (FAXS) [5-7] was addi- Starting from maltose (1), disaccharide α-bromide 3 was
tionally used to measure the relative binding affinities of obtained in excellent yield by a standard acetylation procedure
various fluorinated and nonfluorinated maltose derivatives to and subsequent treatment with hydrobromic acid in glacial
MBP in competitive titration experiments. The incorporation of acetic acid (Scheme 1) [25]. Treatment of bromide 3 with Zn
fluorine in different positions into maltose allows fine tuning of and N-methylimidazole [26] afforded the protected maltal
the carbohydrate affinities to the maltose-binding protein.
derivative 4, which was transformed to the target compounds by
Scheme 1: Syntheses of maltose derivatives; reagents and conditions: (a) Ac2O, Pyr, 97%; (b) HBr, AcOH, 99%; (c) Zn, N-methylimidazole, ethyl
acetate, 74%; (d) Selectfluor®, CH3NO2, 40%; (e) NaOMe, MeOH, 99%; (f) NH2NH2·HOAc, DMF, 94%; (g) DAST, CH2Cl2, 89%; (h) NaOMe, MeOH,
99%; (i) α,α-dimethoxytoluene, p-TosOH, DMF, 79%; (j) Ac2O, Pyr, 93%; (k) BH3·THF, Bu2BOTf, THF 56%; (l) microwave reaction, DAST, collidine,
CH2Cl2, 79%; (m) Pd/C, H2, ethylacetate, 64%; (n) NaOMe, MeOH, 75%.
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Beilstein J. Org. Chem. 2012, 8, 448–455.
utilizing Selectfluor® as a fluorinating agent [23,27,28] in a The synthesis of the galacto-type derivative 23 started from
nitromethane solution. The mixture of anomeric 2-fluoro peracetylated benzylidene maltose 11 [37]. Deprotection [37]
derivatives 5 with gluco- and manno-type stereochemistry was with acetic acid followed by microwave fluorination with
analyzed by 19F NMR, thus showing a gluco (α/β = 1/1) to DAST [34,35] yielded a mixture of fluorinated disaccharides:
manno (α/β = 2/1) ratio of 2/1. Final deprotection with sodium The desired product 22 [39] was isolated by column chromato-
methoxide yielded the deprotected fluoro-derivatives 6.
graphy and Zemplén deprotection yielded derivative 23.
Binding studies using the 2-F-maltose
Maltosyl fluoride 9 was obtained by deprotection of the
anomeric acetyl group of compound 2 with hydrazine acetate reporter system
[29] yielding derivative 7, followed by nucleophilic fluorin- The binding properties of the two stereoisomers of 2-19F-
ation with DAST [30,31] generating the diasteriomeric mixture labeled maltose (gluco- and manno-type) to the maltose-binding
8. The α-anomer was isolated by HPLC and subsequent protein and a MBP-V53 fusion protein comprising five V3
Zemplén saponification of the remaining acetate protecting modules of the LDL receptor in a linear tandem arrangement
groups yielded the α-maltosyl fluoride 9. However, the (V33333) were analyzed. As can be seen in Figure 3 and
β-maltosyl fluoride turned out to be rather unstable. Decompo- Figure 4, the stereoisomers of 2-F labeled maltose clearly ex-
sition of the unprotected fluorinated sugar to maltose and hibit different changes in the transverse relaxation rates upon
hydrofluoric acid started immediately in D2O-solution. There- addition of approx. 0.1 equiv of MBP. The significant change in
fore only the α-maltosyl fluoride was used for the binding line width was only observed for the interacting α-2-F-maltose.
studies. The regioselective reductive ring opening of benzyl- In contrast, the transverse relaxation remained nearly
idene acetals in the maltose derivative 11 was performed with a unchanged for the manno-type epimers and the β-gluco-type
complex of BH3/Bu2BOTf at −70 °C [32,33]. Fluorination with isomer. This observation corresponds to the anomeric prefer-
DAST [34,35] was performed in a sealed tube for 1 h at 80 °C ence described by Gehring et al. [40]. The numeric specifity of
under microwave conditions. The deprotection of the benzyl MBP with a 2.7-fold higher affinity for α- versus β-maltose was
group was achieved with Pd/C [36], followed by a Zemplén demonstrated by tritium NMR spectroscopy [40-42]. In addi-
saponification to obtain product 15. Starting from 4′,6′-O- tion, the β-anomer can be bound in two different modes, prob-
benzylidene maltose 10 [37], the primary alcohol was protected ably corresponding to the closed- and open-domain conforma-
as tert-butyldimethylsilyl ether followed by standard peracetyla- tions of MBP; but only the α-anomer complex has been
tion (Scheme 2). Treatment of the silyl protecting group with an observed in X-ray structures of MBP with maltose [21].
excess of Deoxofluor [38] yielded the 6-F-maltose derivatives
18. Final deprotection with acetic acid [37,39] and sodium Furthermore we used this technique for probing the interactions
methoxide yielded compound 20.
between 2-F-maltose and the MBP-V53 [43,44] fusion protein,
Scheme 2: Synthesis of the maltose- and galacto-type derivatives; reagents and conditions: (a) TBDMS-Cl, imidazole, DMF, 43%; (b) Ac2O, Pyr,
quant.; (c) Deoxofluor, CH2Cl2, 17%; (d) conc. AcOH, 73%; (e) NaOMe, MeOH, 40%, (f) Ac2O, Pyr, 93%; (g) conc. AcOH, 76%; (h) DAST, collidine,
CH2Cl2, 30%; (i) NaOMe; MeOH, quant.
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Beilstein J. Org. Chem. 2012, 8, 448–455.
Figure 3: 1-D 19F NMR: Experimental demonstration of differential
binding of gluco- and manno-type 2-F-labeled maltose (2 mM) in the
free form (A); bound to maltose-binding protein (200 µM) (B); and
bound to MBP-V53 fusion protein (200 µM) (C). Highlighted area
shows the gluco-type region. Spectra were recorded on a Bruker
Avance DRX 600 MHz spectrometer by using a conventional 1-D pulse
sequence. Up to 512 scans were acquired without signal suppression
via T2 relaxation filter.
Figure 4: 19F NMR expansion (of Figure 3) of the gluco-type region of
the 2-F-maltose reporter system.
performed with maltose, maltotriose, maltohexose and
cellobiose, as well as the artificial α-methyl glucoside. The
well-known ability of MBP to bind exclusively to linear
which has almost twice the molecular weight of MBP alone. maltooligosaccharides or maltodextrins of up to eight α(1→4)-
Upon the addition of 0.1 equiv again, the expected increase of linked glucose units was confirmed by competitive titration and
the transverse relaxation rate was observed through the specific 19F NMR experiments. The displacement of α-gluco-2-F-
and significant binding of the α-gluco-type isomer to the MBP- maltose was already observed by the addition of 0.04 equiv of
V53 fusion protein. The larger resulting molecular weight is maltose. Similar results were obtained for the malto-oligo-
reflected in a further (proportional) increase of the line broad- saccharides, maltotriose and maltohexose as well. In contrast,
ening (Figure 4). In a similar way, noncovalent protein–protein α-methyl glucoside and cellobiose showed no binding. To
interactions would increase the effective molecular weight by specify the precise hydroxy groups that are directly involved in
transient binding and result in a consequently increased line hydrogen bonding to MBP, further competition experiments
width, which can be quantified to derive affinities. This clearly were performed with different fluorinated maltose derivatives.
demonstrates both the binding selectivity of the α-gluco-type Change, i.e., reduction in the line width of the α-2-F-maltose
and the feasibility of the β-gluco-type and manno-type isomers, signal, could be observed if the competitor had a higher affinity
serving as internal reference compounds to rule out nonspecific than the α-2-F-maltose itself; caused by the release of α-2-F-
binding and interactions (e.g., changes in viscosity). It should maltose from the binding pocket of the maltose-binding protein.
be noted that the detection limit of protein binding improves An overview of the results of the titration experiments is shown
with decreasing ligand concentration, and thus even smaller in Figure 5. The stepwise addition of equivalent amounts of
protein and ligand concentrations can be used in the experiment single fluorinated maltose derivatives to the 2-F-maltose
[45]. Full exploitation of this effect, however, requires high reporter system allows a direct comparison of the relative affini-
performance 19F NMR probes (e.g., cryoprobes).
ties of the competitors to MBP. The 6-F-maltose is the most
efficient competitor with an affinity equal to maltose,
α-maltosyl fluoride and 6′-F-maltose. The 6′-F-“galacto”-
maltose derivative does not bind to MBP at all.
Relative affinity studies using the 2-F-maltose
reporter system
The 2-F-maltose FAXS reporter system [5-7] was further used
for studying the relative binding affinities of natural and artifi- Note that the competitive binding experiments shown in
cial maltose derivatives to MBP. The initial experiments were Figure 5 allow for the direct extraction of dissociation
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Beilstein J. Org. Chem. 2012, 8, 448–455.
fluorine–protein interaction patterns [48,49]. Shielded fluorine
atoms, due to their increased electron density, are preferentially
involved in direct hydrogen-bonding interactions with donor
groups of the protein. Although the 2-F fluorine is significantly
shielded (about −200 ppm), and thus an ideal hydrogen-bond
acceptor binding of 2-F-maltose is impaired due to the
hydrogen-bond donor activity of the 2-OH group (to the
carboxylate of Glu111). In that respect, introducing the fluorine
into the 6-position results in a smaller energetic penalty
(compared to the 2-F-maltose), because no direct H-bonds
between the ligand and MBP are involved, and only indirect
water-mediated interactions are concerned (data not shown).
Therefore the affinity is higher in that case. Similar arguments
apply in the other cases. It is, however, possible to “fine tune”
the affinity between the ligand binding domain and the reporter
ligand by using differently fluorinated maltose derivatives in
which different hydroxy groups are substituted by fluorine.
Thus the affinity of the reporter ligand can be “customized”
for ligand competition assays or for specific studies of
protein–ligand and protein–protein interactions to match the
affinities between the interaction partners. For example, small
affinities or proteins with a relatively low molecular weight are
more easily detected with high-affinity ligands, whereas
strongly interacting proteins or high-molecular-weight protein
ligands can be better studied with low-affinity ligands.
Conclusion
We have demonstrated that 2-deoxy-2-F-maltose can be effec-
tively used as a reporter system to study protein-binding inter-
actions by 19F NMR. The particular benefit of this novel
reporter system is the simultaneous accessibility of reference
molecules (nonbinding manno-type and β-gluco-type 2-F-
maltose isomers), which serve as internal standards, to rule out
nonspecific binding and interactions, and thus increasing the
reliability of this method. The 2-F-maltose reporter system was
used to study the ligand binding affinity to MBP. “Fine tuning”
Figure 5: Competitive titration with the 2-F-maltose reporter system
and 19F NMR: only the important section of the gluco-type isomers is
shown. (A) 2-F-maltose, (B) 2-F-maltose bound to MBP, (C–G) addi-
tion of 0.125 equiv of the following maltose derivatives: (C) 6-F-
maltose, (D) maltose, (E) α–maltosyl fluoride, (F) 6′-F-maltose, (G)
6′-F-“galacto”-maltose. Spectra were recorded on a Bruker Avance
DRX 600 MHz spectrometer by using a conventional 1-D pulse
sequence. Up to 128 scans were acquired without signal suppression
via T2 relaxation filter.
constants, as was shown by Dalvit and co-workers [46,47]. This by the regioselective fluorination of single hydroxy groups of
would offer additional valuable experimental possibilities for a maltose was used to define the important hydroxy groups that
quantitative analysis of protein–ligand interactions but is are responsible for the hydrogen bonding network and there-
beyond the scope of the present paper. Fluorinated substrate fore for binding to the protein. The results of the competitive
analogues perturb the hydrogen bonding network in the sub- titration are in perfect agreement with the X-ray data published
strate binding pocket to a certain extent. Therefore it is not [21] previously. Additionally, the different binding affinities of
always possible for the ligand to be bound with an optimal selectively 19F-labeled maltose derivatives to MBP illustrate
hydrogen-bonding geometry. These results are fully consistent how the recently established correlation between 19F chemical
with published X-ray data. For instance, in the case of 2-F- shift data and molecular interaction patterns [48,49] can be used
maltose, the 2-OH acts simultaneously as a hydrogen-bond to delineate details of protein–ligand interaction interfaces.
acceptor for the Nε of Lys15 and as a bond donor to the Together with efficient synthetic approaches to fluorinated
carboxylate of Glu111, and the 2-F fluorine can only be a derivatives, this offers exciting perspectives for rational
(limited) acceptor, thus leaving some of the H-bonds “frus- programs for drug design. Experiments to explore these possi-
trated”. It is worth comparing these findings with recently bilities are currently underway in our laboratories. Applications
reported correlations between 19F chemical shifts and of the reporter system to biological material inherently giving
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Beilstein J. Org. Chem. 2012, 8, 448–455.
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