Ditryptophan conjugation triggers conversion of biotin fibers into soft spherical structures

Article abstract of DOI:10.1002/anie.200705012

(Figure Presented) Trigger happy: Biotin and its methyl ester form long fibers in solution, which are transformed into soft spherical structures upon simple conjugation with ditryptophan dipeptide (see picture). Such morphogenesis is not achieved in a controlled fashion by other aromatic amino acids.

Full text of DOI:10.1002/anie.200705012

Communications
DOI: 10.1002/anie.200705012
Biotin Structures
Ditryptophan Conjugation Triggers Conversion of Biotin Fibers into
Soft Spherical Structures**
K. B. Joshi and Sandeep Verma*
Molecular self-assembly is a ubiquitous theme in natural
systems where constituent building blocks are recruited
following precise pairing patterns, to reveal an array of
hierarchical architectures that are primarily supported by
noncovalent interactions.[1,2] The functional role of such
structures in nature can be judged from the action of self-
assembled actin filaments, which are crucial for morpho-
genesis and cellular movement, self-organization of mitotic
spindles during cell division, and the construction of complex
viral capsids, to name but a few.[3–5] Inspiration derived from
natural systems and natural components is increasingly being
realized as a rich source of materials which imitate biological
structures and function.[6,7] Despite significant advances in
supramolecular chemistry and noncovalent synthesis, direct
morphological control of self-assembled structures remains
an enigma. Herein, we show that long biotin fibers can be
morphed into soft spherical structures by simple attachment
Information). Esterification of the carboxylic acid was
expected to modulate the effect of hydrogen bonding with
the ureido group.
Scanning electron microscopy (SEM) and atomic force
microscopy (AFM) of a freshly prepared solution of d-biotin
(1 mm, 50% aqueous methanol) revealed the formation of 1–
5 mm-long self-assembled fibrous structures (Figure 1a inset
and Figure 1b), which were further elongated to 10–100 mm
upon prolonged incubation for 10–12 days (Figure 1c). Such a
morphology was also supported by AFM analysis (Figure 1d).
Biotin methyl ester afforded long tubular structures, as
observed by AFM (Figure 1e), which was further confirmed
by SEM (Figure 1 f). The formation of these self-assembled
structures can be attributed to the propensity for hydrogen
bonding of the ureido and carboxylic groups, and the
interaction of the valeryl side chain with the ureido group
of biotin, while interaction at the ureido group will predom-
of the Trp–Trp dipeptide. The inspiration of using tryptophan inate for the biotin methyl ester. Intramolecular hydrogen
as a modifier of the biotin ultrastructure emanates from the
documented tryptophan interaction in the high-affinity
bonding of biotin in solution, with the participation of the
ureido carbonyl oxygen atom as a strong hydrogen-bond
biotin–avidin system, in which Trp indolic rings interact with acceptor, has been described.[15,16]
biotin, presumably through hydrophobic contacts or a charge-
The high-affinity biotin–avidin interaction has been
transfer mechanism.[8–10]
extensively investigated and mutagenesis studies implicated
contributions of three Trp residues, which control the excep-
tionally high binding constants observed in this system.[8–10] It
has been postulated that Trp residues control the equilibrium
enthalpy and entropic costs of the biotin–avidin interaction. A
crystal structure study of a model compound, in which biotin
is connected to an indole moiety through a trimethylene
linker, has suggested that a linked molecule occupies a cavity
formed by two indole rings, thus suggesting the importance of
hydrophobic interaction in the biotin–avidin complex.[17] In
view of the crucial roles described for Trp residues, we
decided to attach them to biotin to see the effect on the
morphology of biotin fibers.
A remarkable change from a fibrous structure to a
spherical morphology was observed in freshly prepared
samples of biotin–ditryptophan methyl ester (2; 1 mm, 50%
aqueous methanol) when analyzed by SEM on a copper
surface (Figure 2a). The spherical morphology was preserved
irrespective of surface variation to highly oriented pyrolytic
graphite (HOPG) or under wet conditions (Figure 2b, inset)
when observed by focused-ion-beam (FIB) high-resolution
SEM. Transmission electron microscopy (TEM) and AFM
further confirmed the transformation of biotin fibers to
spherical structures by simple attachment of ditryptophan
methyl ester (Figure 2c,d).
Biotin, a water-soluble vitamin, acts as a cofactor in a
number of important biochemical metabolic reactions and
pathways related to cell signaling, gene expression, and
chromatin structure.[11,12] The characterization of biotin has
revealed its extended structure in the crystalline state,
whereas multiple conformational ensembles ranging from
extended to folded states are preferred in solution.[13,14] The
latter structures are stabilized by hydrogen bonding between
the ureido group and the valeryl carboxylic acid side chain.
With this background, we became interested in probing the
ultrastructure of the biotin supramolecular assembly in
solution, by using free biotin and its methyl ester as candidate
molecules (Figure 1a; Scheme SS1 in the Supporting
[*] K. B. Joshi, Prof. Dr. S. Verma
Department of Chemistry, Indian Institute of Technology-Kanpur
Kanpur 208016 (UP) (India)
Fax: (+91)512-259-7436
E-mail: sverma@iitk.ac.in
Homepage: http://home.iitk.ac.in/~sverma
[**] K.B.J. thanks IIT-Kanpur for a predoctoral research fellowship. This
work was supported by a Swarnajayanti Fellowship in Chemical
Sciences to S.V. from the Department of Science and Technology,
India. We thank Prof. V. N. Kulkarni for use of the focused-ion-beam
facility, Prof. A. Sharma for the AFM facility, and ACMS, IIT-Kanpur,
for access to SEM.
Interestingly, the appearance of spherical structures was
almost instantaneous and without any indication of other
common self-assembled structures, such as fibrils, filaments,
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Figure 1. Self-assembly of d-biotin and d-biotin methyl ester (1 mm, 50% aqueous methanol). a) Molecular structure of biotin and its methyl
ester; inset: SEM image of d-biotin (fresh sample). b) AFM image of biotin fibers (fresh sample). c) SEM and d) AFM images of biotin fibers after
12 days incubation. e) AFM and f) SEM images of biotin methyl ester (fresh sample).
to provide a discernible change in the morphol-
ogy (Supporting Information), thus confirming a
crucial role for Trp–Trp dipeptide in controlling
the morphology of biotin fibers. As dipeptide
conjugation afforded interesting morphological
changes, we did not pursue the conjugation of
biotin with tripeptide or other higher homo-
logues.
It was possible to stain these self-assembled
structures with fluorescein dye to permit fluo-
rescence detection (Figure 2e). Dye-stained
microspheres released fluorescein and localized
the dye at the periphery of circular structures
when acidified to pH 4.5 (Figure 2 f, arrows). We
decided to investigate this property by SEM and
TEM analysis. Interestingly, SEM snapshots at
various pH values confirmed an abrasive effect
Figure 2. Microscopy images of biotinylated ditryptophan methyl ester (2). a) SEM
image of a fresh sample (1 mm, 50% aqueous methanol) displaying spherical ball-like
structures on a gold-coated copper stub. b) FIB-SEM image of structures on a HOPG
surface under wet (near-native) conditions. Inset: magnified view of a single spherical
structure. c) TEM image of a fresh sample. d) AFM image displaying a spherical
morphology on a mica surface. Fluorescence micrographs of e) spherical balls stained
with fluorescein, which reveals uniform, bright green structures, and f) release of dye
upon mild acidification of the solution (pH 4.5, 24 h incubation).
of the medium on the spherical structures
(Figure 3a–c). These observations revealed the
instability of the soft spherical structures in
acidic medium, possibly because of protonation
of the indole nitrogen atom(s).
The effect of urea on the aggregation process
of 2 was also investigated, as it may interact with
the ureido portion of the biotin structure. SEM
images displayed a catastrophic disintegrating
and tapes. Prolonged incubation refines the structure but the
gross morphology remains similar to that seen for the fresh
sample, which indicates the formation of shape-persistent
spherical balls under the influence of an aromatic dipeptide
(Supporting Information). Biotin–Trp methyl ester (1) failed
effect of urea (1 mm; 1 equiv) on the spherical balls, thus
suggesting a crucial role of the ureido group in the self-
assembly that is hindered by the addition of urea, possibly by
impairing the critical hydrogen-bonding contacts required for
the stability of spherical structures (Figure 3d).
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bisection, which suggests reasonable structural integrity that
allows them to withstand ion currents for short time periods
(Figure 4c). Interestingly, it was also possible to mill these
microspheres to create different shapes, thus revealing salient
features of the organization and structural integrity of the
core (Figure 4d–f).
The high-affinity biotin–avidin interaction in nature is
supported by the hydrogen bonding of serine and aspartic
acid side chains with the NH groups of the ureido ring, while
the tyrosine, asparagine, and serine side chains are within
hydrogen-bonding distance of the ureido carbonyl oxygen
atom.[8–10,18] Moreover, four Trp residues hydrophobically
interact with the tetrahydrothiophene ring and the valeryl
side chain of biotin. Individual interactions have also been
confirmed by mutagenesis experiments.[9,10] In view of the
various stabilizing noncovalent interactions, it can be envi-
sioned that the self-assembly observed in this study requires
an intricate interplay of hydrogen-bonding, van der Waals,
and p-stacking interactions in the biotinylated ester 2.
The participation of ureido, amido, and indolic NH
protons in the self-assembly process was qualitatively esti-
mated from the 1H NMR spectra of 2 taken in various ratios
of [D6]DMSO and H2O (v/v). The upfield chemical shifts of
the amido[19] and indolic protons was evident through the
incremental addition of H2O to [D6]DMSO (0–40% H2O;
Figure 5 and Table 1). A marked movement of key proton
Figure 3. Effect of pH and addition of urea on self-assembled spherical
structures from 2. a) TEM and b) SEM images of spherical balls at
pH 2, clearly revealing rough edges and abrasions (arrows). c) SEM
image of ruptured structure at pH 1. d) SEM image showing the
disintegrating effect of urea (1 mm) on the spherical balls.
Acid-induced abrasion of the spherical capsules provided
an impetus for us to probe the interior core of these soft
structures by FIB milling experiments. Although the imaging
aspects of FIB-SEM instrumentation are similar to those of
SEM, the technique provides superior control in producing a
finely focused ion beam of low current, which enables well-
defined milling and high-resolution imaging, or higher beam
currents for coarser and faster milling. We decided to mill
peptide microspheres to probe the properties of their
spherical core. A 500 ” 500-nm section was milled off the
surface to reach the interior of the structure (Figure 4a,b).
These microspheres were also amenable to simple FIB-aided
Figure 5. Solvent-dependent 1H NMR studies of 2. Changes in the
chemical shifts of amido, indolic, and aromatic protons (7.5 mgmLÀ1
in different solvent ratios (v/v) of [D6]DMSO and H2O (cf. Table 1).
)
resonances is suggestive of the crucial role of tryptophan
interaction in the supramolecular ensemble. Such upfield
shifts of aromatic protons may occur because of the partial
face-to-face arrangement of the aromatic side chain in
relation to the biotin moiety, thus causing shielding effects
that result from the spatially proximal aromatic ring.[20,21]
Furthermore, a remarkable upfield shift in indolic proton
resonances upon the addition of water also suggests an
inextricable role of hydrogen bonding in the self-assembled
Figure 4. Observation of the exterior and interior structure of spherical
balls by FIB milling. a) Intact isolated spherical structure. b) FIB
milling of a 500”500-nm cross-section, which suggests a stable
spherical core. c) Spherical ball bisected with a FIB of optimal
intensity. d–f) Various shapes patterned on the spheres by FIB milling,
which suggest stability of these self-assembled structures.
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Angewandte
Chemie
Dye-stained structures were examined under a fluorescence
Table 1: Comparison of chemical shifts of biotin–Trp–Trp methyl ester
(2) and biotin–Phe–Phe methyl ester (3).
microscope (Leica DM2500M) with a fluorescence illuminator and a
fluorescein filter (494/521 nm). This filter optimized visualization of
fluorescein-treated (positive resolution) compared with untreated
(negative resolution) spherical structures.
FIB-SEM: A HOPG surface was incubated in a solution of 2
(1 mm) for 12 h at 48C and the sample was directly mounted on the
stage under wet (native) conditions. An image was obtained with a
FIB scanning electron microscope dual-beam system (Nova 600
NanoLab, D97, FEI).
Peptide
0% water in [D6]DMSO
Indolic-NH
Amide-NH
Ureido-NH
N1H, N3H
6.35
NaH
NbH
8.36
8.39
NaH
10.90
–
NbH
10.80
–
2
3
7.90
7.93
6.36
40% water in [D6]DMSO
Indolic-NH
Amide-NH
Ureido-NH
N1H, N3H
6.30
NaH
7.72
7.99
NbH
8.00
8.36
NaH
10.52
–
NbH
10.54
–
2
3
Received: October 30, 2007
Revised: December 27, 2007
Published online: February 27, 2008
6.34
Keywords: biotin · conjugation · morphology · peptides ·
self-assembly
.
spherical structures. In contrast, biotinylated Phe–Phe dipep-
tide (3) and biotinylated Tyr–Tyr dipeptide exhibit either no
discernible change in morphology or irregular oblong mor-
phologies (Figures 9–11 in the Supporting Information), thus
indicating the crucial role of Trp–Trp dipeptide conjugation as
a morphological trigger that results in uniformly sized
spherical structures of biotin.
Thus, the conjugation of tryptophan dipeptide dramati-
cally influences and alters the morphology of biotin fibers into
spherical ball-like structures. Curiously, such morphogenesis
is not achieved in a controlled fashion by other aromatic
amino acids. This finding suggests that tryptophan elicits a
specific morphological response not only to aromatic hydro-
phobic interactions, but also perhaps because of its inherent
affinity for biotin, as demonstrated by key molecular contacts
operating in the biotin–avidin system. Interestingly, a capping
interaction of tryptophan with biotin has been proposed as
the molecular basis of slow streptavidin–biotin dissociation
kinetics.[22]
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Experimental Section
Fresh and aged peptide, biotin, biotin methyl ester, and N-biotiny-
lated peptide samples were imaged with an atomic force microscope
(Molecular Imaging, USA) operating in the acoustic ac mode, with
the aid of a cantilever (NSC 12(c), MikroMasch). Sample-coated mica
was dried for 30 min at room temperature before AFM imaging. SEM
images were acquired on an FEI Quanta 200 microscope equipped
with a tungsten-filament gun and operated at a working distance of
10.6 mm and 20 kV. For TEM, a fresh sample or a specimen incubated
for 12 days was placed on a 400-mesh copper grid. After 1 min, excess
fluid was removed and the grid was negatively stained with uranyl
acetate solution (2%). Excess stain was removed from the grid and
the samples were viewed with a JEOL 1200EX electron microscope
operating at 80 kV.
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