Research Projects
Cyclodextrin Inclusion Complexes
Synthesis of functional cyclodextrin derivatives
Structure of
macromolecules by X-ray
crystallography
A. Cyclodextrin Inclusion
Complexes:
crystalline state and solution structures and properties
Cyclodextrins are natural, water soluble macrocyclic carbohydrate
molecules, oligomers of
α-D-glucose,
that possess a cavity where a plethora of hydrophobic molecules can be
encapsulated. The resulting host-guest inclusion complexes exhibit many
characteristics of receptor-substrate systems. The laboratory has a long
experience in inclusion complexes of cyclodextrins with various guest
systems:
Figure A1. The common cyclodextrins: α-cyclodextrin (αCD), β-cyclodextrin (βCD) and γ-cyclodextrin (γCD).
Packing Research on X-ray structures of β-cyclodextrin inclusion complexes with model compounds has revealed that the majority of them forms dimers, which pack in 4 major packing arrangements [1-11]. The hydrophobicity / hydrophilicity of the end groups of the guest that emerge from the primary sides of the host dimer determine the crystal packing. A characteristic example is the difference in packing between the complexes of long aliphatic mono-carboxylic acids and di-carboxylic acids (Figure A2).
Figure A2. Inclusion of long mono-carboxylic acids (left, CH packing)
and di-carboxylic acids (right, Intermediate packing) into
βCD.
It is suggested
that it is the hydrophobic end-methyl group of the former guests at one
of the primary faces of the βCD
dimer dictates the channel formation in order to shield that part of the
guest from the surrounding water molecules. The carboxylic groups found
entrapped in the channel, self-associate to carboxylic dimers, thus
stabilizing the whole system. In the dicarboxylic acids the
end-carboxylic groups interact with water molecules.
Of course, all rules have exceptions. Thus
flexibility on the part of the guest [1,2-bis(4-aminophenyl)ethane]
that acquires two different conformations in the cavity of the
βCD
dimer (Figure A3), results in a packing arrangement different that the
four defined above, which however, can be transformed approximately to
one of them, the
screw channel mode of packing [12 and references
therein].
Another guest, 4-pyridinealdazine, that has the tendency to form π-π dimers, on the one hand and H-bonds with water molecules on the other, is encapsulated into βCD with the accompanying water molecules and forms βCD trimers (Figure A4), detected for the first time [13], with host /guest ratio 3:2, a highly unusual stoichiometry.
Fig. A4. Two βCD trimers forming channels encompassing 4-pyridinealdazine dimers (grey) and the water clusters (pink) H-bonded to the guests.
II. Self-assembly in solution and solid state
Non-bonding interactions, H-bonding and organization of CD
supramolecular systems in the crystalline state and in aqueous solution
(Figures A5 and A6) have been extensively studied [14-18].
Fig. A5. Organization of cyclodextrins in dimers with guests 1 - 4 has
been detected in aqueous solutions [14].
Fig A6. Exothermic rotaxanation of Congo red in
γCD
and endothermic dimerization of the complex [16].
III. Structural and recognition studies on inclusion complexes of cyclodextrins
Complexes with pheromones [19-26] and several plant growth factors [27,
28] have resulted in applications on the
controlled release and
protection
the olive tree pheromones
Bactrocera
oleae and Prays
oleae [29]. The
induced
fit
exhibited by the cavities of permethylated
α-
and
β-cyclodextrins
that resulted in the resolution of the racemic mixture of
Bactrocera oleae (Scheme a1 and Figure A7)
is very remarkable. [30-31].
Fig. A7.
(Left): Structure in the crystal of the complex permethylated
α-CD/(R)-spiroacetal
selectively precipitated from the racemic mixture of the guest. (Right):
Structure of the complex permethylated
β-CD/(S)-spiroacetal.
selectively crystallised from the racemic mixture of the guest. Top:
Models of hosts and guests. Bottom: Differential electron density map of
the guests; van der Waals map of the hosts.
IV. Inclusion complexes with drug
Fig A9. The inclusion complex of the
antibacterial agent
triclosan in
βCD
forms dimers packing in channels.
The
dichlorophenyl ring of the guest enter the
cavities of
βCD
dimers from their primary sides,
whereas the hydrophilic chlorophenol ring
extends in the space between dimers.
Fig A10. From Left to right the structures of the inclusion complexes of
the
hypoglycemic drugs
TBM, TLZ and GLP in
βCD.
The mode of inclusion of the drugs is the same:
the hydrophobic groups enter
the cavities of two hosts, belonging to adjacent
βCD
dimers from
the primary sides, whereas the hydrophilic sulfonylurea moiety is
located between dimers, forming H-bonds with the hosts. The packing of
the
TBM, TLZ complexes is in channel mode, whereas in that of the GLP the
βCD
dimers
are placed further apart (Intermediate mode) due to the increased length
of the guest.
References
B. Synthesis of
functional cyclodextrin derivatives
I.
Modified CDs display novel properties
Synthetic modifications of cyclodextrins result in introduction
of functional groups that endow the host molecules with specific
properties [1-3] ( Figure B1), allow elongation of the host cavity to
capture long guest molecules (Figure A8) or the combination of the above
that permits direct inclusion in specific direction [4,5] (Figure B2).
Fig.
B1. Synthesis of mono-aminobenzoic acid-substituted β-cyclodextrins and
supramolecular self inclusion in the crystalline state [1].
Fig.
B2. Per(carboxyethylthio)-β-cyclodextrin encapsulates the elongated dye
methyl orange (left) in a sense opposite to that of β-cyclodextrin
(right) [5].
Per-6-modified CDs with guanidino or lysine-/arginine-like groups
(Figure B3) comprises a very interesting family of hosts that have the
ability to penetrate cell membranes, to transport molecules, but also
compact DNA and therefore perform transfection (Figure B4) [6,7]. These
hosts also act as encapsulators of hydrophilic phosphorylated
substrates, such as nucleotides (Figure B5 [8].
Fig B4. Guanidino and amino cyclodextrins penetrate cell membranes
(left) and perform DNA transfection that expresses GFP protein (right).
III.
Negatively charded CDs with
a range of applications
EDTA-type CDs, i.e. CDs modified with aminodiacetyl groups have been
synthesized, and characterized. Their main property is the ability to
coordinate with metal ions, especially lanthanides and particularly Gd(III).
The EDTA-CDs (Figure
B6)
coordinate with Gd(III) and fast exchanging water molecules to form
metal clusters that display high relaxivity values, especially at high
(100 MHz) magnetic fields.
Fig. B6. AEDTA
(left) and its Gd(III) complex [9]
as calculated by PM3 semiempirical methods.
IV.
Glycoclusters
Fig. B7. A representative cyclodextrin-based
glycocluster hosting a typical guest, t-butyl benzoic acid in the
cavity.
References
C.
Structure of macromolecules by X-ray crystallography
X-ray crystallography allows to “look” the detailed structure of biological macromolecules in three dimensions at the atomic level. This is essential in order to understand the macromolecules’ function (or malfunction in case of disease), propose reaction mechanisms of design drugs. Current and past projects include:
I. Binding of γ-cyclodextrin with Glucogen Phosphorylase b
Selective binding of
γ-cyclodextrin
with Glucogen Phosphorylase b
(GPb) at
the glucogen storage site of the enzyme (Figure C1) [1]. In order to investigate
whether the storage site can be exploited as target for modulating
hepatic glucose production,
α,
β
and
γ-cyclodextrins
(CDs) were identified as moderate mixed-type inhibitors of
GPb ( Ki
values 47.1, 14.1 and 7.4 mM for
α,
β
and
γ-CD,
respectively), the
γ-CD
being better than the others.
The structures of GPb with
β-
and
γ-CDs
provides an understanding of the binding, which is analogous to linear
maltopentaose (G5) and maltoheptaose (G7) oligomers namely, anchoring of
two glucose residues to GPb. However, the inhibition constant of
γ-CD
is almost 7-times higher than that of G7 and of
β
–CD even higher. The lower potency of
γ-CD
compared to G7 is explained by lack of H-bonds between the residues
γ-CD
that are next to the anchoring residues and the protein, due to its
round shape.
Figure C1. γCD (in red) binds to the storage site of the Glucose Phosphorylase b dimer.
II. Muscle proteins
Collaboration with the M. Wilmanns, EMBL-Hamburg and N. Pinotsis, Inst. Cancer Res., UK, on muscle proteins.
1. The structure of the amino terminus titin/telethonin complex [2]: Titin (the largest protein made by human cells) along with myosin and actin, plays a crucial role in the muscle function. The structure of the 2:1 titin:telethonin complex gives the details of the interaction of two titin molecules with telethonin, a small component of the the Z-disc of muscles (Figure C2). That leads to a model of titin interaction in the Z-disc: two titin molecules from different sarcomeric filaments cross-linked at their amino terminus via telethonin. The study may lead to new insight for some muscle and cardiac diseases; moreover, it may provide a molecular paradigm about the crosslinking of major sarcomeric filaments.
Fig. C2. A: ribbon representation of the two antiparallel titin immunoglobulin-like domains Z1 (blue, residues 1-98) and Z2 (cyan, residues 99-196 including the Z1-Z2 linker) cross-linked by telethonin (red) via extended antiparallel β-sheets between the three molecules (in two orientations). B: surface representation of the titin-telethonin-titin complex, in two orientations (in green, the telethonin domain 60-90 not participating in β-sheets).
2. The structure of the myomesin domains my9-my11 [44]. Each domain comprises a immunoglobulin-like and helix pattern. The above structure combined with the already available structure of the final my12-my13 domains of the carboxy terminus allows to make a model of how the myomesin filament attaches in the M-band of the muscle sarcomer (unpublished results).
III. Ferredoxins
Structure determination of the new family of 2[4Fe4S]
ferredoxins (Fds) from selected pathogenic bacteria as
Escherichia
coli (EcFd), Pseudomonas aeruginosa (PaFd), allochromatium
vinosum (alvinFd) in order to correlate their structure to the low and
widely different reduction potentials (–460 and –675 mV) of their two
metal clusters [4,5]. The structures of the above proteins along with
two new structures of mutated Alvin Fd, C57A and V13G (1.05 and 1.48 Å
resolution, respectively),
provide insight into the
significant effects of subtle structural differences of the protein and
solvent environment around the clusters on their electrochemical
properties. Namely, polar interactions of side chains and water
molecules with cluster II
sulfur atoms
(Figure 18),
which are absent in the environment of cluster
I (the cluster with the
lowest reduction potential) are correlated to the ca. 180-250 mV
difference between the reduction potentials of clusters
I and
II. The degree of
exposure of cluster I
leads towards less negative reduction potentials (E°). This is better
illustrated by V13G AlvinFd (high exposure, E° =
-594
mV) and EcFd (low exposure, E° =
-675
mV). In C57A AlvinFd
(Figure
C3),
the movement of the protein backbone, as a result of replacing the
non-coordinating Cys57 by
Fig. C3. The structure of the Alvin Fd variant, C57A (1.05 Å resolution, a). The environment of cluster II is comparable to that of the widely studied clostridia ferredoxin, (b), that possess two isopotential clusters (ca. –400 mV) although the former is buried inside the molecule. This is due to a water molecule (W6) entrapped in the interior of the protein that attracts side chains of polar residues close to the cluster II S-atoms. In contrast, cluster I is less polar than cluster II of AlvinFd and of clostridia ferredoxins.
Fig.
C4.
Solvent-accessibility surfaces color-mapped
according to the chemical nature of the surface atoms of a C57A, b V13G,
c PaFd, d EcFd: Nitrogen, oxygen, and carbon atoms are shown in blue,
red, and green, respectively. The Cys11SG atoms of cluster I are shown
in yellow at the central region of the surfaces. The exposed S1 atom of
cluster I of V13G is shown in magenta (b). The bulky sulfur atom of
Met13 of EcFd (d), in orange, limits the exposure of the Cys11SG atom
(yellow).
IV. RNA Complexes
Structure determination of complexes of RNA with antibiotics synthesized
in the
V. Proteins from marine demosponge Suberites domuncula
Molecular structure of natural
and appropriate variants of silicatein
and silicase from marine
demosponge Suberites domuncula for nanobiotechnology
applications,
as partner of the Marie Curie
Initial Training Network BIOMINTEC.
Silicateins are members of the cathepsin family of proteases with Cys
substituted by Ser residues. Silicase
belongs to the family of Carbonic anhydrase.
New crystallisation methods for biological macromolecules [47,
48].
As coordinator of the
Industry-Academia Partnerships and Pathways, Marie Curie project,
TOPCRYST, Dual Polarization Interferometry, pioneered by Farfield
Scientific Ltd., will be used to probe crystallisation at its most
crucial stages. This will allow to predict the outcome of
crystallisation trials when they are still at their earliest stages and
thus to rationally design and direct such experiments in order to lead
them to well-diffracting crystals.