Monovinyl sulfone B-cyclodextrin. a flexible drug carrier system

Article abstract of DOI:10.1002/cmdc.201300385

Cyclodextrins have been conjugated to target various receptors and have also been functionalized with carbohydrates for targeting specific organs. However, this approach is based on a rigid design that implies the ad hoc synthesis of each cyclodextrin- targeting agent conjugate. We hypothesized that: 1) a modular design that decouples the carrier function from the targeting function leads to a flexible system, 2) combining the reactivity of the vinyl sulfone group toward biomolecules that act as targeting agents with the ability of cyclodextrin to form complexes with a wide range of drugs may yield a versatile system that allows the targeting of different organs with different drugs, and 3) the higher reactivity of histidine residues toward the vinyl sulfone group can be exploited to couple the cyclodextrin to the targeting system with a degree of regioselectivity. As a proof of concept, we synthesized a monovinyl sulfone b-cyclodextrin (module responsible for the payload), which, after coupling to recombinant antibody fragments raised against Trypanosoma brucei (module responsible for targeting) and loading with nitrofurazone (module responsible for therapeutic action) resulted in an effective delivery system that targets the surface of the parasites and shows trypanocidal activity.

Full text of DOI:10.1002/cmdc.201300385

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DOI: 10.1002/cmdc.201300385
Monovinyl Sulfone b-Cyclodextrin. A Flexible Drug Carrier
System
Teresa del Castillo,[c] Julia Marales-Sanfrutos,[a, b] Francisco Santoyo-Gonzꢀlez,[a]
Stefan Magez,[d] F. Javier Lopez-Jaramillo,*[a] and Jose A. Garcia-Salcedo[c]
Cyclodextrins have been conjugated to target various recep-
tors and have also been functionalized with carbohydrates for
targeting specific organs. However, this approach is based on
a rigid design that implies the ad hoc synthesis of each cyclo-
dextrin-targeting agent conjugate. We hypothesized that: 1) a
modular design that decouples the carrier function from the
targeting function leads to a flexible system, 2) combining the
reactivity of the vinyl sulfone group toward biomolecules that
act as targeting agents with the ability of cyclodextrin to form
complexes with a wide range of drugs may yield a versatile
system that allows the targeting of different organs with differ-
ent drugs, and 3) the higher reactivity of histidine residues
toward the vinyl sulfone group can be exploited to couple the
cyclodextrin to the targeting system with a degree of regiose-
lectivity. As a proof of concept, we synthesized a monovinyl
sulfone b-cyclodextrin (module responsible for the payload),
which, after coupling to recombinant antibody fragments
raised against Trypanosoma brucei (module responsible for tar-
geting) and loading with nitrofurazone (module responsible
for therapeutic action) resulted in an effective delivery system
that targets the surface of the parasites and shows trypanoci-
dal activity.
Introduction
Ever since Paul Ehrlich coined the magic bullet concept (i.e.,
drugs that go straight to their intended cell structure target
while leaving healthy tissues unaffected), much effort has been
devoted to the delivery of drugs to specific physiological sites
where pharmacological activity is required, particularly in the
field of cancer therapy.[1] The strategies for drug targeting are
frequently divided into passive targeting and active target-
ing.[2] Passive targeting is based on the physical and chemical
properties of the carrier and the differences in the biochemical
and physiological characteristics of the target tissue (i.e., en-
hanced permeability and retention (EPR) effect)[3] to achieve
a high target/non-target ratio at the site of action. However,
the random nature of this approach makes it difficult to con-
trol the process, and active targeting is an appealing alterna-
tive. Active targeting relies on the use of specific interactions
to promote not only the specificity, but also to increase the
local concentration of the therapeutic agent at the site of
action and, in some cases, to provoke its internalization medi-
ated by receptors.[4]
Given the inherent nanometer-scale functions of the biologi-
cal components of living cells, nanoscale materials are promis-
ing in medicine. Size is relevant in the context of drug delivery,
and carriers larger than 400 nm in diameter are captured by
the reticuloendothelial system.[5] Various nanoscale materials
have been described as carriers, and can be grouped into five
general categories: 1) nanospheres, 2) liposomes, 3) emulsions,
4) polymeric micelles, and 5) water-soluble polymeric carriers
including both naturally occurring polymers (i.e., proteins and
carbohydrates) and synthetic polymers.[2,4a,6]
[a] Dr. J. Marales-Sanfrutos,+ Prof. F. Santoyo-Gonzꢀlez, Dr. F. J. Lopez-Jaramillo
Departamento de Quꢁmica Orgꢀnica, Instituto de Biotecnologꢁa
Universidad de Granada, 18071 Granada (Spain)
E-mail: fjljara@ugr.es
[b] Dr. J. Marales-Sanfrutos+
Current address: Department of Chemistry
Chemistry Research Laboratory
University of Oxford, OX1 3TA Oxford (UK)
[c] Dr. T. del Castillo,+ Dr. J. A. Garcia-Salcedo
Hospital Universitario San Cecilio
Instituto de Investigaciones Biosanitarias de Granada
FIBAO, Granada (Spain)
In active targeting, interactions with the specific receptor
are mediated by biomolecules that act as targeting agents.
The use of antibodies in their native state as targeting agents,
either as fragments or as antibody mimics, is well established.[7]
In oncology the fact that cancer cells overexpress receptors to
maintain their high metabolic rates has also been exploited in
folate targeting[8] and in transferrin-mediated targeting.[9] How-
ever, regardless the targeting agent and the carrier, the cou-
pling of the elements comprising the drug delivery system is
an important issue. Methods of protein chemistry have been
successfully implemented for the direct conjugation of drugs
to antibodies.[10] For the particular case of immunotoxins, the
and
Instituto de Parasitologꢁa y Biomedicina Lꢂpez Neyra
CSIC, Granada (Spain)
[d] Dr. S. Magez
Laboratory for Cellular and Molecular Immunology
Vrije Universiteit Brussel, Brussels (Belgium)
and
Department of Molecular and Cellular Interactions
VIB, Brussels (Belgium)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300385.
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use of recombinant DNA techniques is a feasible approach to
obtain toxin–antibody systems as fusion proteins.[7b,10] In gen-
eral, nanoscale drug delivery systems have been conjugated to
targeting agents by classical chemical functionalization meth-
ods,[7a,9a,11,12] and by avidin–biotin affinity.[13] Another important
issue is the stability of the linkage, which may explain the fail-
ure of first-generation conjugates that led to premature release
of the drugs in the blood, or to no release at the site of action.
Second-generation linkers were designed to be cleaved within
intracellular organelles through decreased pH (acid-labile hy-
drazone)[7d] or a reductive environment.[14]
However, for drug delivery purposes the functionalization of
CDs with VS is an advantageous strategy because it renders
CDs into reactive species that yield a covalent bond (i.e., en-
hanced coupling stability) with any targeting element (en-
hanced target flexibility). In addition to the good reactivity
toward biomolecules, the reaction is straightforward (it in-
volves combining VSCD with the biomolecule in aqueous
media), takes place under mild conditions compatible with the
biological function of the targeting agent (aqueous buffer
even at 48C), and it does not yield by-products.[18b,19]
The synthesis of 6-deoxy-6-(2-hydroxyethyl) (vinylsulfonyl)-
methyl)amino-b-cyclodextrin (VSCD) was carried out in a two-
step procedure consisting of: a) microwave-assisted reaction of
6-O-monotosyl-6-b-cyclodextrin[20] with ethanolamine (1:20 sto-
ichiometry) in DMF to yield mono-6-(2-hydroxyethyl)amino-b-
cyclodextrin, and then b) introduction of VS by reaction with
divinyl sulfone (DVS) (Scheme 1). The overall yield of the syn-
thesis was 80%. This strategy to obtain mono-6-(alkylamino)-b-
cyclodextrin by displacement of the toluenesulfonyl group was
Cyclodextrins (CDs) have been identified as promising nano-
scale carriers.[15] They are cyclic oligosaccharides composed of
six, seven, or eight d-glucopyranose units referred to as a-, b-,
and g-cyclodextrin, respectively, linked by a(1!4) bonds en-
closing a lipophilic central cavity. They are well known for their
capacity to form inclusion complexes with a wide variety of
drugs.[16] In the context of active drug delivery, CDs have been
conveniently conjugated to target estrogen receptors, folate
receptors, transferrin receptors, and integrins, among others,
as well as functionalized with carbohydrates for targeting dif-
ferent organs.[15] This approach is based on the ad hoc synthe-
sis of each cyclodextrin–targeting agent conjugate and leads
to a rigid design that constrains the use of cyclodextrin in
active drug delivery.
We hypothesized that a modular design that decouples the
carrier function from the targeting function may lead to a flexi-
ble system. As a proof of concept, herein we report the synthe-
sis of a monovinyl sulfone functionalized b-cyclodextrin (VSCD)
that combines the capacity of CD to carry different drugs with
the good reactivity of the vinyl sulfone group toward the bio-
molecule, acting as targeting agent. Thus, the reaction of
VSCD with a His-tagged nanobody (i.e., a recombinant anti-
body fragment) raised against T. brucei and its loading with the
drug nitrofurazone yields a delivery system that targets the
surface of the parasite and that shows trypanocidal activity.
Scheme 1. Synthesis of 6-deoxy-6-(2-hydroxyethyl) (vinylsulfonyl)methyl)ami-
no-b-cyclodextrin (VSCD).
recently reported by Puglisi et al.,[21] who used similar micro-
wave conditions and a large molar excess of liquid amines
(1:150), the amine also acting as solvent. Two side reactions
may compromise the functionalization of mono-6-aminocyclo-
dextrin with DVS: 1) dimerization by intermolecular reaction of
both VS with two molecules of monoamino-b-cyclodextrin,
and 2) intramolecular reaction of both VS with the amino
group to yield a 1,1-dioxothiomorpholine derivative. The
former was prevented by using DVS in 2:1 molar excess, and
the latter by functionalizing the b-CD as a secondary amine.
Side reactions were minimized, as demonstrated by the fact
that the yield of the reaction of DVS with mono-6-(2-hydroxye-
thyl)amino-b-cyclodextrin was 85.8%.
Results and Discussion
Design and synthesis of a flexible carrier system
Flexibility, defined as the capacity to: 1) transport a wide range
of drugs (payload flexibility) and 2) form a stable link with any
targeting element (target flexibility), was envisioned as a modu-
lar design that decouples therapeutic function (drug), carrier
function (carrier element), and targeting function (biomole-
cule). The synthesis of such a flexible drug delivery system was
articulated on a b-cyclodextrin (b-CD) functionalized with
a vinyl sulfone group (VS) to exploit the well-known capacity
of CDs to form inclusion complexes with a wide variety of
compounds, and the good reactivity, under mild conditions, of
VS toward both amino and thiol groups naturally present in
biomolecules.[17] Recently, the functionalization of CDs with
a zinc porphyrin has been reported as a general strategy to
yield a multimodal system for combined therapy that takes ad-
vantage of the affinity of His and Trp residues for the metallo-
porphyrin center to couple CDs with a therapeutic protein.[18]
Selection of the in vitro model system to validate the proof
of concept
Trypanosomiasis was chosen as the model system to evaluate
the potential of VSCD as a flexible carrier. There are three fea-
tures that make trypanosomiasis attractive in this regard: 1) it
is a relevant disease with more than 10 million people infect-
ed, 2) the current therapeutic arsenal has serious limitations
and varied efficiency, as it relies on drugs that present an ap-
preciable risk of toxicity, and drug delivery is a clear approach
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for optimization,[22] and 3) trypanosomes are masters at eluding
the host immune response.[23] Our efforts were focused on
Human African trypanosomiasis (HAT, or sleeping sickness)
caused by T. brucei, because the mechanism of immune eva-
sion relies on the antigenic variation of the variant surface gly-
coprotein coating (VSG) of the parasite, making targeting a par-
ticular challenge.[23]
serving its capacity to interact with the conserved cryptic epi-
topes of T. brucei.
Construction of the drug delivery system
The modular design implies the assembly to the carrier ele-
ment of the modules responsible for therapeutic function and
for the targeting function in order to yield the drug delivery
system. In principle, the order of integration is flexible and is
determined by the features of both drug and targeting ele-
ment. The presence of groups reactive toward VS in the drug
means that the first reaction must involve coupling of VSCD to
the targeting element. Conversely, when the conditions used
to form the drug–CD inclusion complex may distort the func-
tionality of the targeting element (i.e., solvents, temperature,
pH, etc.), inclusion should be carried out as the initial step.
Nitrofurazone (5-nitro-2-furfurylidenesemicarbazone; NF)
was selected as payload. Although it is reported as an anti-
T. cruzi drug,[24] several features attracted our attention: 1) it is
active against T. brucei,[25] 2) it shows higher selectivity and
lower toxicity than Nifurtimox,[26] a classical anti-Chagas drug
currently used in combination therapy against sleeping sick-
ness,[27] and 3) it is expected to be encapsulated by VSCD, as
both hydroxymethylnitrofurazone and nitrofurazone have
been included in b-CD with similar Ka values, and the incorpo-
ration of hydroxymethylnitrofurazone in different substituted
CDs has been reported.[28]
Selection of the targeting element was constrained by the
fact that the antigenic variation of VSG makes T. brucei a diffi-
cult target for antibody-guided carriers, because conserved
epitopes are located at the inner part of the coating, inaccessi-
Coupling of VSCD to cAb-An33 nanobody
The coupling of VSCD to proteins consists of a Michael addi-
tion that involves a nucleophilic attack at the side chains of
Lys, Cys, and His residues by the VS, and the relative abun-
dance of these residues can compromise the regioselectivity of
the process. This issue may not be important for many of the
’-omic’ applications of vinyl sulfone chemistry. However, for
coupling the VSCD to a guidance system, regioselectivity is
crucial because if the linkage takes place at an unsuitable posi-
tion, it may prevent interaction with the target. Analysis of the
nanobody (Nb) structure (PDB code 1YC7) revealed that the re-
active points are three Lys residues, as the four Cys residues
are involved in two disulfide bonds (Figure 1).[31] A closer study
showed that only Lys75 is close enough to the antigen binding
loops to disrupt the interaction with the target, whereas Lys64
and Lys43 are at opposite sides of the molecule. The reactivity
of the Lys residues may be evaluated on the basis of the rela-
tionship between nucleophilic character and degree of proto-
nation (pKa) because amine groups with lower pKa values are
expected to be less protonated and behave as better nucleo-
philes, thus being more reactive.[32] The prediction of pKa gave
a value of ~10 for the three Lys residues, supporting a poor re-
gioselectivity of the reaction between Nb and VSCD. To over-
come such a limitation, the cAb-An33 nanobody was overex-
pressed as a poly-His tag fused protein. The poly-His tag is ex-
pected to act as a strong nucleophilic center, as the theoretical
pKa for His residues is 6.5.[33]
Figure 1. Antibodies and nanbodies. Conventional IgG antibodies consist of
two heavy chains (H) and two light chains (L). The Camelidae family produ-
ces heavy-chain-only antibodies, the variable antigen binding domains
(VHH) of which are termed nanobodies. Vinyl sulfone reactive residues of
the poly-His-tagged cAb-An33 nanobody (at right) are labeled, and areas re-
sponsible for interaction with the antigen are shown in light grey, toward
the top of the structure.
ble for standard antibodies (Figure 1). However, antibody frag-
ments consisting of the variable antigen binding domain of
the Camelid heavy-chain-only antibodies (i.e., nanobodies)
have been demonstrated to target conserved cryptic epi-
topes.[29] Additionally, nanobodies are well overexpressed in
E. coli, are highly soluble in aqueous solution, and show such
robust thermal and conformational stability that they have
been reported to retain significant antigen binding activity
even after incubation at 908C for 2 h.[30] In particular, the cAb-
An33 nanobody was chosen as a targeting element. It recog-
nizes the carbohydrate moiety of VSG, and its structure has
been solved at 1.6 ꢂ resolution, revealing a molecule 2.5 nm in
diameter and 4.4 nm in length.[29,31] Functionalization of the
monoaminocyclodextrin was carried out with DVS, the shortest
bis-vinyl sulfone, in order to minimize the size increase result-
ing from the coupling of VSCD to the nanobody, thereby pre-
To find the best reaction conditions, the poly-His-tagged
cAb-An33 Nb was reacted with VS-functionalized rhodamine B
(VSR).[17h] The reaction was carried out at two pH values and
two Nb/VSR stoichiometries, and the functionality of the cou-
pling was tested by incubation with parasites in vitro. Regard-
less of the stoichiometry, both pH 6 and 7.5 yielded the label-
ing of Nb (Figure 2a). However, a compromise between the
extent of labeling and functionality was found, as strong fluo-
rescence also correlates with multivalent labeling that may
yield nonfunctional Nb. It was found that Nb labeled with VSR
at pH 7.5 and a stoichiometry of 1:25 was fully functional and
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Figure 2. Reaction of the His-tagged cAb-An33 nanobody with vinyl sulfone
rhodamine and interaction with T. brucei. a) SDS PAGE of the His-tagged
nanobody having reacted with vinyl sulfone rhodamine B at two pH values
and two stoichiometries. b) Fluorescence resulting from the interaction of
T. brucei with the His-tagged nanobody having reacted with vinyl sulfone
rhodamine B at pH 7.5 and at a stoichiometry of 1:25 (nucleus and kineto-
plast are stained with DAPI).
targeted the entire surface of the parasite, validating the ex-
perimental approach (Figure 2b).
A finer-tuned approach was taken with VSCD. Different com-
binations of temperature, time, and reaction volume (i.e., final
concentrations of the reacting species) were assayed, and the
resulting products were analyzed by mass spectrometry (Sup-
porting Information S1). The best conditions were found to be
1 nmol Nb and 25 nmol VSCD in 60 mL, at 428C in reaction for
24 h to yield Nb_VSCD with an overall labeling of 45% (29%
single, 13% double, and 2% triple labeling). Harsher conditions
caused the sample to precipitate.
Figure 3. Detail of MALDI-ToF spectra of the two peptides bearing the poly-
His tag derivatized with VSCD resulting from tryptic digestion of Nb_VSCD
and Nb (control). The arrow indicates the expected m/z value.
corporated mainly via reaction with the poly-His tail. This
result is especially relevant, as His tags are widely used for the
purification of recombinant proteins, and in the context of
coupling via vinyl sulfone, it confers two additional advantag-
es: 1) a degree of regioselectivity that restricts the coupling to
other residues present in the biomolecules, and 2) reversibility
if the construction of the fussed protein contains a protease
cleavage site (typically for thrombin X) to remove the His tag.
To identify the residues that reacted with VS, two samples
consisting of Nb_VSCD and Nb control were submitted to the
Proteomic Facility (Universidad Autꢃnoma de Barcelona, Spain)
for digestion with trypsin and MALDI-ToF MS analysis. The
spectra were searched for the signals of the peptides bearing
Lys and His residues predicted by the in silico digestion of
both Nb and Nb_VSCD samples (Supporting Information S2).
As summarized in Table 1 and shown in Figure 3, the tryptic
digest of the sample Nb_VSCD displays a clear signal that coin-
cides with the m/z value expected for the peptide resulting
from the reaction between CDVS and the poly-His tail. Addi-
tionally, the MS data do not show the expected signal of the
peptides containing CD linked to Lys, except some traces for
Lys64 (Supporting Information S3), confirming that VSCD is in-
Inclusion of nitrofurazone
The fact that the formation of inclusion complexes between
hydroxymethylnitrofurazone and dimethyl-b-cyclodextrin have
been reported,[28] and that docking experiments have shown
the furan ring near the primary hydroxy group from CD[28a] led
us to hypothesize that NF could form an inclusion complex
with b-CD. Initial experiments aimed at forming inclusion com-
plexes revealed the low solubility of NF in aqueous solutions
as a significant obstacle that was overcome by including 1.5%
(v/v) DMSO in the medium. Although 1.5% DMSO doubles the
aqueous solubility of NF, T. brucei tolerance to DMSO is limit-
ed,[34] and the high solubility of NF in DMSO may prevent the
formation of an inclusion complex with CD. It has been report-
ed that the formation of the inclusion complex can be moni-
tored by the absorbance of NF at 260 nm.[28b] This is not feasi-
ble for VSCD when coupled to proteins, as aromatic residues,
particularly Phe, absorb in the 240–300 nm region, and disul-
fide bonds have an absorbance band close to 260 nm. Howev-
Table 1. Overview of the signals searched in the MALDI-ToF spectra and
summary of detection.
Peptide bearing the residue target of VSCD
m/z
Detected
(R)QAPGK43CDE(R)
2081.83
3655.56
3191.34
3916.34
3560.41
No
Trace
No
Yes
Yes
(R)EWVSSISSPGTIYYQDSVK64CDGR(F)
(R)DNAK75CDNTVYLQMNSLQR(E)
(R)SIREYWGQGTQVTVSSHHHHHHCD(–)
(R)EYWGQGTQVTVSSHHHHHHCD(–)
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er, the UV/Vis spectrum of NF shows an additional strong ab-
sorbance band at 371 nm that is not affected by the presence
of macromolecules (Supporting Information S4). The ratio A371
/
A280 was used as an indicator of the formation of the inclusion
complex. Thus, incubation of 5.4 nmol Nb_VSCD with
56.7 nmol NF in 300 mL phosphate-buffered saline (PBS) sup-
plemented with 1.5% DMSO and exhaustive dialysis against
PBS yielded a 1.4-fold increase in the A371/A280 ratio for the Nb_
VSCD+NF inclusion complex relative to that of Nb_VSCD, sup-
porting the loading of Nb_VSCD with NF to yield Nb_VSCD+
NF.
Biological assays
The therapeutic potential was evaluated by incubation of
20 mL of either Nb_VSCD+NF (i.e., 15 mm Nb_VSCD bearing
5 mm NF) or control experiments with 80 mL of a T. brucei cul-
ture in HMI-9 medium containing 2ꢄ105 parasitesmLꢀ1 for 5 h
at 378C and 5% CO2. The final concentration of DMSO in the
biological assay was 0.3% (v/v), which is well below the 0.42%
threshold reported as safe for T. brucei, and the growth of the
parasites was detected by Alamar Blue assay.[34] This assay
relies on the increase in fluorescence of a redox indicator in re-
sponse to chemical reduction of the growth medium resulting
from cell growth. Control experiments (PBS+NF and Nb+NF)
yielded basically the same results as the positive control of
parasite growth (PBS); only the culture in which the inclusion
complex Nb_VSCD+NF was added yielded a decrease in fluo-
rescence, revealing growth inhibition (Figure 4). The effective-
ness of Nb_VSCD+NF was further confirmed by studying its
effect on the growth kinetics of T. brucei. As shown in Figure 4,
treatment for 4 h was sufficient to kill a culture of 2ꢄ
105 parasitesmLꢀ1. These results demonstrate that both drug
and carrier are necessary to inhibit growth, and that NF is not
transported by the Nb unless it forms an inclusion complex
with CD.
Figure 4. Trypanocidal activity of the transporter loaded with nitrofurazone.
a) Alamar Blue assay of T. brucei after 7 h incubation with the inclusion com-
plex (Nb_VSCD+NF), with negative controls in which Nb_CD was replaced
by either PBS (PBS+NF) or Nb without CDVS (Nb+NF) or a positive control
(PBS); values <1 denote an oxidized environment resulting from the ab-
sence of growth. b) Effect of Nb_VSCD+NF on the growth kinetics of
T. brucei.
Experimental Section
General procedures: TLC was performed on Merck silica gel
60 F254 aluminum sheets. Flash column chromatography was per-
1
formed on silica gel (Merck, 230–400 mesh, ASTM). H and 13C NMR
spectra were recorded at room temperature on a Varian Direct
Drive (400, 500, and 600 MHz) spectrometer. Chemical shifts (d) are
given in ppm; J values are given in Hz. MALDI-ToF mass spectra
were recorded on an Autoflex Bruker spectrometer using HCCA
and NaI as matrices. Optical rotations were recorded on a Perki-
nElmer 341 polarimeter at room temperature. Vinyl sulfone derivat-
ized rhodamine (VSR) was obtained as described elsewhere.[17h] Ni-
trofurazone (5-nitro-2-furfurylidenesemicarbazone) was purchased
from Alfa Aesar.
Conclusions
The functionalization of b-CD with VS yields a drug carrier that
combines the reactivity of VS toward biomolecules with the
ability of CD to form complexes with a wide range of drugs.
This design decouples the carrier function from the targeting
function to yield a versatile drug delivery system that may
target various organs with different drugs upon coupling to
the suitable targeting biomolecule and formation of the inclu-
sion complex with the selected active principle. The reaction
of VSCD with the biomolecule is straightforward and can be
carried out without any special techniques. On the other hand
the high reactivity of the His residue toward VS can be exploit-
ed to couple His-tagged guidance systems with a degree of re-
gioselectivity that minimizes the disruptions of the interaction
with the target molecule. As a proof of concept, the reaction
of VSCD with Nb raised against T. brucei resulted in an effective
drug delivery system that targets the surface of the parasite
and shows trypanocidal activity when loaded with NF.
Synthesis of 6-deoxy-6-(2-hydroxyethyl) (vinylsulfonyl)methyl)a-
mino-b-cyclodextrin (VSCD): Ethanolamine (470 mL, 7.75 mmol)
was added to
a solution of 6-toluenesulfonyl-b-cyclodextrin
(500 mg, 0.38 mmol) in DMF (10 mL). The reaction mixture was irra-
diated at 500 W and 808C in a Milestone Star Microwave Labsta-
tion for 1 h. Acetone was added (100 mL), and the precipitate was
filtered in vacuo and washed with acetone (20 mL) and Et2O
(20 mL) to yield 6-deoxy-6-hydroxylethylamino-b-cyclodextrin as
a solid that was dried in vacuo at 508C for one day (410 mg,
90%).[20] Divinylsulfone (26 mL, 0.25 mmol) was added to a solution
of
6-deoxy-6-hydroxylethylamino-b-cyclodextrin
(150 mg,
0.13 mmol) in H2O (15 mL). The reaction mixture was kept at room
temperature for 1 h. The solvent was evaporated in vacuo (to
~8 mL). Acetone was added (50 mL), and the precipitate was fil-
tered in vacuo and washed with acetone (10 mL) and Et2O (10 mL).
The solid was dried in vacuo at 508C for one day (140 mg, 85%):
1
mp>2708C (dec); [a]D = +102 (c=1, H2O); H NMR (selected sig-
nals, [D6]DMSO, 300 MHz): d=6.90 (dd, J=16.6, 9.9 Hz, 1H), 6.20
(d, J=9.7 Hz, 1H, HC=cis), 6.17 ppm (d, J=16.6 Hz, 1H, HC=
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trans); 13C NMR ([D6]DMSO, 75 MHz): d=138.0, 129.8, 102.7, 82.2,
73.8, 73.1, 72.7, 60.6, 59.8, 57.0, 55.5, 51.0, 48.6 ppm; HRMS
(MALDI-ToF): [M+H]+ calcd for C48H83O37NS: 1296.428, found:
1296.452.
excitation wavelength of 535 nm and an emission wavelength of
590 nm.
Trypanolysis assays: A culture (400 mL) of 2ꢄ105 parasitesmLꢀ1 in
HMI-9 medium supplemented with 20% tetracycline-free FBS was
incubated at 378C and 5% CO2 with 100 mL test samples. After 4,
24, and 48 h incubation, the parasites were counted with a Neuba-
uer chamber.
Overexpression and purification of nanobody cAb-An33: Produc-
tion of the poly-His-tagged cAb-An33 nanobody (Nb) was per-
formed as described elsewhere.[31]
Coupling of Nb to VSCD to yield Nb_VSCD: A final reaction
volume of 60 mL containing 1 nmol Nb and 25 nmol VSCD in
100 mm HEPES pH 7.5 was incubated at 428C for 24 h. The sample
was thoroughly dialyzed against PBS to remove excess vinyl sul-
fone derivatized reagent and was used without further purification.
Abbreviations
CD, cyclodextrin; Nb, poly-His-tagged cAb-An33 nanobody; NF, ni-
trofurazone; DVS, divinylsulfone; VS, vinyl sulfone group; VSCD,
vinyl sulfone functionalized b-cyclodextrin; VSR, vinyl sulfone func-
tionalized rhodamine B; Nb_VSCD, Nb coupled to VSCD; Nb_VSR,
Nb coupled to VSCR.
Mass spectrometry analysis of Nb_VSCD: MS was carried out at
the Proteomic Facility at Universidad Autꢃnoma de Barcelona
(Spain). For localization of the VSCD in Nb, an aliquot of a solution
of either Nb or Nb_VSCC was reduced with DTT, derivatized with
iodoacetamide, and digested with trypsin (378C, 16 h). The result-
ing peptides were analyzed by MALDI-ToF MS (ABI-Sciex 4800) in
linear and reflection mode to cover a range between 700 and
6000 Da. The mass spectra were interpreted manually on the basis
of the in silico digestion of the sequence. The molecular masses of
the VSCD-derivatized peptides were calculated by adding the mo-
lecular mass of VSCD to the peptide with His or Lys.
Acknowledgements
The authors and indebted to Mr. Josꢃ Maceira for skillful techni-
cal assistance. This work was funded by grants CTQ2011-29299-
C02-01.A to F.S.-G. and SAF-2011-30528 to J.A.G.S. from the Minis-
terio de Economꢁa y Competitividad (Spain) and by the European
Union (NANOTRYP, FP7-HEALTH-2007-B-2.3.4-1:223048) to J.A.G.S.
and S.M.
Inclusion of nitrofurazone into Nb_VSCD: Nb_VS (5.4 nmol) in
300 mL PBS was incubated with a solution of nitrofurazone (4.5 mL,
12.6 mm in DMSO) at room temperature overnight. Excess nitrofur-
azone was removed by dialysis against PBS (3ꢄ100 mL). Inclusion
Keywords: bioconjugation · cyclodextrins · drug delivery ·
vinyl sulfones
was monitored by UV/Vis spectroscopy as the ratio A371/A280
.
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addition of a 3% SDS solution (50 mL). The plates were read on
a Tecan Infinite F200 reader (Tecan Austria GmbH, Austria) using an
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 383 – 389 388

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Received: September 26, 2013
Revised: November 20, 2013
Published online on December 13, 2013
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ChemMedChem 2014, 9, 383 – 389 389