Access to Versatile β-Cyclodextrin Scaffolds through Guest-Mediated Monoacylation

Article abstract of DOI:10.1002/chem.201503131

Herein, we report the selective mono-derivatization of heptakis[6-deoxy-6-(2-aminoethylsulfanyl)]-β-CD (1) through a guest-mediated covalent capture strategy. The use of guests functionalized with cleavable linkers enables the installation of an amine-orthogonal thiol group on the primary rim of 1 as a handle for further transformations to the β-CD scaffold. Applying this methodology, two novel monoderivatized β-CDs were obtained in good yield and high purity. Both of these monoacylated CDs were amenable to facile linker cleavage and further modification at the resulting thiol group. This methodology can be applied towards the synthesis heterofunctionalized β-CD constructs for analyte sensing, drug delivery, and other applications.

Full text of DOI:10.1002/chem.201503131

DOI: 10.1002/chem.201503131
Full Paper
&
Host–Guest Systems
Access to Versatile b-Cyclodextrin Scaffolds through
Guest-Mediated Monoacylation
Nesrin Vurgun, Rodolfo F. Gómez-Biagi, and Mark Nitz*[a]
Abstract: Herein, we report the selective mono-derivatiza-
tion of heptakis[6-deoxy-6-(2-aminoethylsulfanyl)]-b-CD (1)
through a guest-mediated covalent capture strategy. The
use of guests functionalized with cleavable linkers enables
the installation of an amine-orthogonal thiol group on the
primary rim of 1 as a handle for further transformations to
the b-CD scaffold. Applying this methodology, two novel
monoderivatized b-CDs were obtained in good yield and
high purity. Both of these monoacylated CDs were amenable
to facile linker cleavage and further modification at the re-
sulting thiol group. This methodology can be applied to-
wards the synthesis heterofunctionalized b-CD constructs for
analyte sensing, drug delivery, and other applications.
Introduction
zation of the b-CD primary rim with a desired functional group
often necessitates multiple synthetic steps. Usually, this is ach-
ieved by the monotosylation of a primary alcohol followed by
nucleophilic displacement. Optimizing the monotosylation of
CDs has been the subject of many studies.[14,31–33], However, se-
lectivity remains a challenge and difficult chromatographic
separations, and crystallizations, are required to remove side
products. This monoderivatization methodology enables the
introduction of a variety of substituents, such as a monoazide,
amine, hydroxylamine, or thiol, which can then be further
modified.[15,34,35] Amine-functionalized b-CD is a particularly de-
sirable scaffold, which has been conjugated to a variety of bio-
logically relevant molecules including glycodendrimers,[36,37]
peptides,[38] proteins,[39] and drugs[2].
b-CD is a naturally occurring cyclic oligomer comprised of
seven 1,4-a-linked d-glucopyranosyl units.[1] The formation of
b-CD host–guest complexes have been applied in a variety of
areas including drug delivery, as components in commercial
products and for enantiomeric separations.[1–7] While native b-
CDs possess this innate property, their applications are limited
due to their lack of functionality. In contrast, derivatized CDs
feature a broader range of applications including catalysis,[1,8]
polymer assembly,[9] molecular recognition of organic and bio-
macromolecules,[10–12] and targeted drug/DNA delivery.[2] While
a multitude of applications exist for functionalized b-CDs,
access to complex heterofunctionalized derivatives is currently
hindered by the lack of efficient synthetic methods.
To complement existing methodologies and to address
some of their inherent problems, we have developed a selec-
tive monoderivatization strategy based on the covalent cap-
ture of an activated guest by a nucleophilic b-CD. Covalent
capture involves a supramolecular recognition event that ac-
celerates the formation of a covalent bond between two reac-
tive components in comparison to the bimolecular reaction.[40]
If after reaction complex formation is prevented, significant se-
lectivity for monofunctionalization can be achieved. Covalent
capture with native CDs was initially demonstrated by Bender
et al., who observed a significant rate acceleration for the acy-
lation reaction of b-CD with m-nitrophenylacetate compared
to the rate of background ester hydrolysis, and has since been
successfully applied for CD-based catalysis.[41,42] This approach
has also been used to monofunctionalize a calix[6]arene core,
with various guests through a selective Huisgen cycloaddition
reaction.[43,44]
Derivatization of CDs may involve synthetic transformations
of the primary or secondary glucoside hydroxyls.[1,13–16] CDs can
be mono-, per-, or partially functionalized on the primary rim
using appropriate reagents and protecting groups.[13,14] An im-
pressive recent example has shown it is possible to orthogo-
nally functionalize all six positions of a-CD.[17] Synthetic strat-
egies that allow the selective modification of b-CD at the 2-,
3-, and 6- positions have also been reported.[18–23] Selective di-
or trisubstitution requires capping strategies, such as the use
of biphenyl-based disulfonates.[14,24] Other innovative methods
to regioselectively functionalize CDs include selective
diisobutylaluminium hydride (DIBAL-H)-promoted benzyl de-
protection,[25–27] tandem azide reduction/deprotection,[28] trity-
lations,[29] and introduction of biselectrophiles.[30] Monoderivati-
[a] N. Vurgun, R. F. Gómez-Biagi, Prof. Dr. M. Nitz
Department of Chemistry, University of Toronto
80 St. George Street, Toronto, Ontario
M5S 3H6 (Canada)
Using covalent capture, we reported the selective monoderi-
vatization of heptakis[6-deoxy-6-(2-aminoethylsulfanyl)]-b-CD
(1).[45] Host–guest complexation between 1 and a thioester
E-mail: mnitz@chem.utoronto.ca
functionalized
7-diethylaminocoumarin-3-carboxylic
acid
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201503131.
(DEAC) guest (first-generation guest, FFG) facilitated S!N acyl
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Scheme 1. Structure of DEAC-based guests, 2 and 3, featuring cleavable linkers and the general scheme proposed to access desired heterofunctionalized CDs
through the proposed methodology.
transfer with rate acceleration and high monoderivatization se-
lectivity (Scheme 1).[45] The high selectivity of this reaction is
Results and Discussion
the result of self-inclusion complex formation of the monoder-
Design and synthesis of DEAC-based guests with cleavable
ivatized product, which prevents association of additional acti-
linkers
vated guests and limits further acylations. While this provided
access to a high yield of monoderivatized 1, the scope of the
reaction was limited to direct covalent functionalization with
a DEAC guest. We, therefore, sought to amend this strategy to
increase the range of functional groups that could be incorpo-
rated.
CD 1 forms a stable, high affinity host–guest complex with 7-
diethylaminocoumarin-3-carboxylic acid (DEAC) (Ka =2.7”
106 mÀ1, NaHPO4 0.05m, pH 7.5).[54] To introduce a thiol on the
primary rim of 1, through a covalent capture strategy, two acti-
vated DEAC-based guests containing disulfide linkers, 2 and 3,
were synthesized (Scheme 1). Introduction of a thiol group to
CD 1 was desired due to the compatibility of thiols with aque-
ous ligation chemistry and applications in bioconjugation.[55–57]
Guests 2 and 3 contain alkyl linkers of differing length which
will affect the rate of reaction with CD 1 and the propensity to
form an intramolecular self-inclusion complex following mono-
derivatization.[45] The guests were activated as esters of salicylic
acid to increase the guests’ aqueous solubility and to provide
favorable electrostatic interactions between the activated ester
and the primary amines of 1.
Amine-functionalized CD 1 is a versatile scaffold, which has
been used for the design of fluorescent sensors,[10,45] synthetic
receptors,[46] investigated as a precursor to forming glycoclus-
ters,[47] controlling protein self-assembly,[48] DNA transfection,[49]
and as an inhibitor of anthrax lethal toxin.[50–53] In these exam-
ples, the amine groups of 1 were either per-functionalized, or
monofunctionalized nonselectively with desired reagents. The
installation of an amine-orthogonal functional group would
augment the utility of 1, by allowing for the synthesis of more
complex heterofunctionalized derivatives. Furthermore, the
higher affinity interactions of CD 1 with hydrophobic guests,
compared to native b-CD, could be exploited to design more
stable complexes.[54]
Synthesis of guests 2 and 3
In this work, we describe a strategy to monofunctionalize
1 that enables access to heterofunctionalized CD scaffolds.
Two activated disulfide-based linkers are investigated for mon-
oderivatization of 1, which after reduction and removal of the
guest molecule reveal a thiol for further elaboration of the b-
CD scaffold (Scheme 1).
DEAC was synthesized as previously described and activated
as a succinimide ester 4 (Scheme 2; see also the Supporting In-
formation).[45] Linker 5, was synthesized in 58% yield, in two
steps. Cystamine hydrochloride was oxidized with mCPBA and
the resulting sulfoxide was subsequently displaced with 3-mer-
captopropionic acid. To prepare the longer linker, disulfide ex-
change between 5-mercaptopentanoic acid and 2,2’-dithiodi-
pyridine was carried out, giving the activated disulfide 6 (74%
yield). The resulting activated disulfide (6), was then ex-
changed with cysteamine hydrochloride, to afford linker 7 in
73% yield. Succinimide activated DEAC 4, and linkers 5, or 7
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Scheme 2. Synthesis of guests 2 and 3: a) DIPEA, DMF, RT, o/n. 75% (8), 81%
(9). b) DCC, DMAP, DCM, 08C!RT, 4 h, then TFA/DCM 4:1, RT, 6 h, 74% (2).
c) DCC, DMAP, DCM, 08C!RT, o/n. 48%, then TFA/DCM 4:1, RT, 4 h, 41% (3).
DCC=N,N-dicyclohexylcarbodiimide; DIPEA=N,N-diisopropylethylamine;
DMAP=4-dimethylaminopyridine.
Scheme 3. Synthesis of b-CD derivatives through guest-mediated monoacy-
lation: a) 1 (1.0 equiv, 0.5 mm), 2 (1.0 equiv), borate buffer (0.1m, pH 8.5), RT,
30 min 68%; b) 1 (1.0 equiv, 0.5 mm), 3 (1.0 equiv), borate buffer (0.1m,
pH 8.5), RT, 3 h, 63%.
were condensed overnight in the presence of DIPEA to yield
compounds 8 and 9 in 75 and 81% yields, respectively. Com-
pound 8 was activated as a salicylate ester by coupling with
10 and, following careful trifluoroacetic acid (TFA) deprotec-
tion, gave guest, 2 in 74% yield over two steps. Guest 3 was
synthesized in an analogous manner. Protected intermediate
11 was synthesized in 48% yield from 9. Intermediate 11
proved to be more stable than guest 3 for long-term storage.
During TFA deprotection of 11, a side product resulting from
disulfide exchange was observed, accounting for the lower
yield in this step, 41%, for guest 3.
Monoderivatization with disulfide guest 3
Reaction of 1 (0.5 mm) with guest 3 (1.0 equiv) in borate buffer
(0.1m, pH 8.5) was complete in 3 h (Scheme 3, see also the
MALDI-TOF MS spectrum in Supporting Information Figure S2).
The reaction proceeded with high selectivity and only the
monoderivatized product, 13, was observed by MALDI-TOF MS.
Purification by (RP)-HPLC afforded the desired monoderivatized
product, 13, with a yield of 63%. We attribute the yield of this
reaction to the reduced stability of the disulfide of guest 3
which may have decomposed during purification. It should
also be noted that when reactant concentrations exceeded
2.0 mm, or equivalents of guest use were greater than
1.5 equivalents, the reaction selectivity decreased and diacylat-
ed product was observed. Below these thresholds, monoderiv-
atized CD 13 could be obtained with exceptional selectivity.
Monoderivatization reactions
Reactions were optimized by controlling the equivalents of
guest added, concentration of reagents in solution, and time
of reaction. These factors were varied to assess the selectivity
of reactions and to reduce reaction times. The reactions were
monitored by MALDI-TOF MS to qualitatively monitor the reac-
tion selectivity.
Kinetic analysis of monoderivatization reactions
Monoderivatization with disulfide guest 2
To understand differences in the selectivity between the two
monoderivatization reactions, and to determine the role of co-
valent capture in the reactions, the rates of both reactions
were followed by UV/Vis spectroscopy. Ethylenediamine (EDA)
was chosen to give a representative rate of aminolysis of
guests in the absence of complex formation as the second pKa
of EDA (7.63) is comparable to the least basic amine of 1 (pKa
7.37).[58] During these control experiments, EDA was used at an
equivalent concentration to the amines of 1. The release of the
salicylate leaving group was monitored at 300 nm (Supporting
Information, Figures S13 and S14). Upon acylation, the absorb-
ance spectra of the coumarin did not change significantly.
Reaction of 1 (0.5 mm) and guest 2 (1.0 equiv) in borate buffer
(0.1m, pH 8.5) gave the monoacylation product, 12, in 30 min
with a detectable amount of diacylated product (Scheme 3;
see also the MALDI-TOF MS spectrum in the Supporting Infor-
mation Figure S1). Guest 2 was readily solubilized upon addi-
tion to the reaction buffer in the presence of 1. At higher con-
centrations, or if more than one equivalent of 2 was used in
the reaction, the amount of over-acylated product formation
increased. Purification by reverse-phase (RP)-HPLC was success-
ful in removing over acylated products affording the desired
monoderivatized product, 12, in an isolated yield of 68%.
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In the presence of 1, the rate of salicylate release is in-
creased seven-fold for guest 2, and eight-fold for guest 3 com-
pared to the EDA control (Figure 1, see also the Supporting In-
formation, Figures S15 and S16). Taking into account the EDA
concentration, an effective concentration of 250 or 280 mm
Figure 2. Salicylate release over time measured by UV/Vis (300 nm) for the
aminolysis of guest 2 (2.0”10À5 m) or 3 (2.0”10À5 m) by CD 1 (2.0”10À4 m)
at 298 K in bicine buffer (0.1m, pH 8.5). Reactions were performed in the
presence or absence of competitive guest, adamantanecarboxylic acid
(2.0”10À4 m), or coumarin 8 or 9 (2.0”10À4 m) binding.
Figure 1. Salicylate release over time measured by UV/Vis (300 nm) for the
aminolysis of guest 2 (2.4”10À4 m) or 3 (2.4”10À4 m) by CD 1 (5.0”10À3 m)
or EDA (3.75”10À2 m) at 298 K, in borate buffer (0.1m, pH 8.5).
decreases in the observed rates are consistent with the extent
of competitive complex formation expected under the experi-
mental conditions and are consistent with a covalent capture
mechanism.
can be calculated for the amines on CD 1, for the reaction
with guest 2 or 3, respectively (see the Supporting Information
p. 26). These values of effective concentration are also compa-
rable to that observed in the reaction of CD 1 with guest FGG
(230 mm). This suggests that guests 2 and 3 are as effective as
FGG at facilitating the intramolecular amide bond formation
reaction with 1.[8,45,59]
ROESY- and DOSY-NMR studies of monoderivatized prod-
ucts
ROESY-NMR spectra of 12 and 13 were collected to detect the
presence of self-inclusion complexes. ROE cross-peaks between
1
In order to confirm that the rate accelerations observed are
due to host–guest complexation, kinetic experiments were re-
peated in the presence of a competitive guest. These experi-
ments were complicated by challenges of guest solubility. The
rate of guest 2 aminolysis by CD 1 was 2.5-fold slower in the
presence of 25-fold excess adamantane carboxylate (Support-
ing Information, Figure S17). Unfortunately, under the same
conditions, the rate of reaction could not be measured for
guest 3, due to unexpected changes in the absorbance spec-
trum likely due to aggregate formation (Supporting Informa-
tion, Figure S18). Additional experiments under more dilute
conditions with bicine buffer (0.1m, pH 8.5) showed the rate of
aminolysis of guest 2 by CD 1 was approximately two times
slower in the presence of a 10-fold excess of adamantane car-
boxylic acid or coumarin 8 (Figure 2; see also the Supporting
Information Figures S19 and S20). Similarly, for guest 3, the
rate of aminolysis was reduced by two- or five-fold in the pres-
ence of adamantane carboxylate or coumarin 9, respectively
(Figure 2; see also the Supporting Information Figure S22). The
the H resonances of the DEAC guest and the H-3s and H-5s of
the b-CD are diagnostic of inclusion complex formation. These
characteristic ROE cross-peaks were not observed for com-
pound 12 (Figure 3; see also the Supporting Information Figur-
es S23 and S24 and Table S1). This suggests that self-inclusion
of the covalently bound guest is not favorable. This observa-
tion may explain the multiply acylated products observed in
the monoderivatization reactions with guest 2; after monoacy-
lation, the covalently bound guest does not reside in the
cavity, allowing the binding of other activated guests and sub-
sequent acylations.
In contrast, ROE-cross-peaks between DEAC aromatic pro-
tons H-C and H-D and DEAC methyl protons, H-F, with the H-3
and H-5 protons of the CD are observed for compound 13
(Figure 3; see also the Supporting Information Figures S25 and
S26 and Table S2). Previously, similar ROE cross-peaks for the
reaction product of our first-generation guest FGG and 1 were
observed.[45] To rule out the possibility of intermolecular daisy-
chain formation, DOSY-NMR[60–61] was also carried out for CDs
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Figure 3. ROESY cross-peaks observed between the CD H-3 and H-5 protons and coumarin guest protons for CDs 12 (top) and 13 (bottom).
Linker cleavage and further derivatization
12 and 13 (10 mm, D2O, Supporting Information Figures S27–
S31). As the monomeric controls, inclusion complexes of cou-
marin 8 or 9 with CD 1 were prepared. The diffusion coeffi-
cients measured for the four coumarin ring protons in the
monoderivatized species were identical to those of the mono-
meric controls, which suggests that CDs 12 and 13 exist as
monomeric species in solution. These data support CD 13
adopting a self-inclusion complex and which disfavors multiple
acylation reactions during the functionalization reaction.
To assess the feasibility of further functionalization of 12 and
13, maleimide chemistry and disulfide exchange with dansyl-
modified reagents were investigated after reduction with
excess
aqueous
tris(2-carboxyethyl)phosphine
(TCEP)
(100 equiv) (Scheme 4). The desired thiols 14 and 16 were puri-
fied by cation-exchange chromatography (CM Sephadex C-25).
To facilitate the removal of the reduced coumarin guest, the
resin was washed with additional aqueous TCEP solution and
MeOH. The reduced CD was then eluted with 5% NH4OH. CDs
14 and 16 were stored under vacuum at RT to prevent oxida-
tion.
The suboptimal linker length of 2 or the geometry of the di-
sulfide bond may be factors which destabilize the self-included
complex in 12. While this does limit the inherent selectivity of
the reaction between 2 and 1 at high reagent concentrations,
the formation of multiply acylated products is successfully
curbed by using stoichiometric amounts of activated guest
under high dilution conditions. This issue is mitigated with the
longer linker design in guest 3 which allows covalently bound
guest 13 to adopt a favorable self-included conformation, ena-
bling monoacylation of 1 with exquisite selectivity.
Dansyl-functionalized maleimide 19 in DMSO was added to
a solution of CD 14 in water/acetic acid (pH 4.5), and incubat-
ed for 4 h. The desired CD construct 15 was purified by HPLC
1
and characterized by ESI-MS, and H NMR (Supporting Informa-
tion Figures S9 and S10). It should be noted that side products
were formed through the reaction of CD 1 amines with malei-
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Scheme 4. Linker cleavage and functionalization at the resulting thiol for CDs 12 and 13: a) Buffered TCEP (100 equiv) solution, RT, 20 min, 95% (14), 93%
(13); b) 19 (1.0 equiv), AcOH, DMSO/H2O, RT, 4 h, 29%; c) 22 (1.0 equiv), AcOH, DMSO/H2O, RT, 4 h, 47%.
mide 19, and these could be successfully removed during pu-
rification. The unexpected CD 1 amine reactivity may be attrib-
uted to their depressed pKa. Incubation with sub-stoichiometric
19 at 08C was found to reduce side-product formation.
Functionalization of the CD thiol through disulfide exchange
was investigated to increase the selectivity of the derivatiza-
tion. Dansyl 22, containing a pyridine-2-thiol leaving group, in
DMSO, was added to a solution of CD 16 in water/acetic acid
(pH 5), and incubated for 4 h. The desired CD construct 17 was
purified by HPLC and characterized by ESI-MS and 1H NMR
(Supporting Information Figures S11 and S12). As expected, no
side reactions were observed and the desired construct 17
could be isolated in good yields.
remaining primary amines. CDs 12 and 13 can, therefore, serve
as versatile scaffolds for the synthesis of heterofunctionalized
b-CDs to combine molecular sensing, targeted drug delivery or
catalysis capabilities with multivalent presentation of recogni-
tion sites for biomolecules. The general synthetic strategy de-
scribed here could, furthermore, be adapted to facilitate the
derivatization of other CDs by modifying the size of the includ-
ed guest, or enable generation of other scaffolds through the
use of different cleavable linker designs.
Experimental Section
General information
These reactions demonstrate that the envisioned linker
cleavage strategy is a feasible method for further functionaliz-
ing the CD 1 scaffold, and may enable its conjugation to
a range of desired groups such as biomolecules.
All chemicals or reagents were purchased from Sigma Aldrich or
Alfa Aesar. Dry solvents were purchased from Acros chemicals. All
reactions were performed under a nitrogen-atmosphere and at
1
room temperature, unless stated otherwise. H- and 13C NMR spec-
tra were recorded on a Varian 400 MHz, Agilent DD500 MHz, or
Bruker 400 MHz spectrometers at T=297 K. 1D 1H NMR and 2D
ROESY studies of monoderivatized CDs were recorded on an Agi-
lent DD700 MHz spectrometer. DOSY studies of CD inclusion com-
plexes and monoderivatized CDs were recorded on a Varian
500 MHz spectrometer. Absorption spectra and kinetics experi-
ments were measured on a Shimadzu UV-2401PC spectrophotome-
ter at T=298 K. Semi-preparative HPLC separations were per-
formed on a Waters 1525 Binary HPLC pump and Waters 2487 dual
l absorption detector using a Waters XBridgeTM Prep BEH130 C18
5 mm (10”250 mm) reverse-phase column. Analytical HPLC separa-
tions were performed on a Perkin–Elmer Series 200 pump and
Waters 2487 dual l absorption detector using an Agilent Zorbax
RX-C18 5 mm (4.6”250 mm) reverse-phase column. High resolution
mass spectra were obtained from an ABI/Sciex QStar mass spec-
Conclusion
In summary, guest-mediated monoacylation is an effective
strategy to install an orthogonal functional group on the pri-
mary rim of 1, with high selectivity, good yields, and short re-
action times. Aminolysis of guests 2 and 3 by 1 proceeds with
rate acceleration due to host–guest complexation, and demon-
strates a key principle of covalent capture. The high guest af-
finity of CD 1 allows for these conjugation reactions to pro-
ceed rapidly even under dilute conditions (low mm). Upon
linker cleavage, CDs 12 and 13 can be functionalized with a va-
riety of compatible compounds at the resulting thiol. In addi-
tion, the complex structures may be readily modified on the
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sorption/ionization time-of-flight mass spectrometer [MALDI-ToF
MS]) using a-cyano-4-hydroxycinnamic acid matrix. For the synthe-
sis of guests 2 and 3, and other CD derivatives, as well as analytical
data please refer to the Supporting Information.
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Heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-b-CD (1)
CD 1 was prepared according to a literature procedure.[54] 1H NMR
(400 MHz, D2O): d=5.16 (d, J=3.70 Hz, 7H), 4.06 (ddd, J=9.5, 6.5,
2.6 Hz, 6H), 3.96 (m, 7H), 3.68 (m, 14H), 3.30 (t, J=6.7 Hz, 14H),
3.18 (dd, J=14.6, 2.6 Hz, 6H), 3.05 (m, 7H), 3.02 ppm (t, J=6.7 Hz,
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Monoderivatization with activated guest 2
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CD
1 (32.3 mg, 0.018 mmol was dissolved in borate buffer
(42.0 mL, 0.1m, pH 8.5) to give a final concentration of 0.5 mm. Di-
sulfide guest 2 (8.9 mg, 0.018 mmol) was added, and the resulting
solution was vigorously stirred at RT for 30 min. The solution was
then lyophilized and the resulting solid was purified by reverse-
phase HPLC, to yield CD 12 a fluffy yellow solid. Isolated yield:
ˇ
ˇ
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1
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The authors kindly acknowledge the Natural Science and Engi-
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Keywords: covalent capture
systems · selective functionalization
· cyclodextrin · host–guest
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Received: August 8, 2015
Published online on December 4, 2015
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim