A convenient route to diversely substituted icosahedral closomer nanoscaffolds

Article abstract of DOI:10.1021/ja204488p

The design and synthesis of icosahedral polyhedral borane closomer motifs based upon carbonate and carbamate anchoring groups for biomedical applications are described. Dodecacarbamate closomers containing easily accessible groups of interest at their linker termini were synthesized via activation of the B-OH vertices as aryl carbonates and their subsequent reaction with primary amines. Novel dodecacarbonate closomers were successfully synthesized for the first time by reacting [closo-B₁₂(OH)₁₂]^2- with an excess of respective aryl chloroformates, utilizing relatively short reaction times, mild conditions and simple purification strategies, all of which had previously presented difficulties in closomer chemistry. This methodology for the 12-fold degenerate synthesis of carbonate and carbamate closomers will greatly facilitate further exploration of closomers as monodisperse nanomolecular delivery platforms.

Full text of DOI:10.1021/ja204488p

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pubs.acs.org/JACS
A Convenient Route to Diversely Substituted Icosahedral Closomer
Nanoscaffolds
Satish S. Jalisatgi, Vikas S. Kulkarni, Betty Tang, Zachary H. Houston, Mark W. Lee, Jr., and
M. Frederick Hawthorne*
International Institute of Nano and Molecular Medicine, School of Medicine, University of Missouri, 1514 Research Park Drive,
Columbia, Missouri 65211
S Supporting Information
b
ABSTRACT: The design and synthesis of icosahedral
polyhedral borane closomer motifs based upon carbonate
and carbamate anchoring groups for biomedical applications
are described. Dodecacarbamate closomers containing easily
accessible groups of interest at their linker termini were
synthesized via activation of the BÀOH vertices as aryl
carbonates and their subsequent reaction with primary
amines. Novel dodecacarbonate closomers were successfully
synthesized for thefirst time byreacting [closo-B12(OH)12]2À
with an excess of respective aryl chloroformates, utilizing
relatively short reaction times, mild conditions and simple
purification strategies, all of which had previously presented
Figure 1. Monodisperse nanomolecular closomer platform based
on B12 core: (a) schematic representation; (b) space filling model
of 12-fold degenerate carbamate closomer 3i.
2À
difficulties in closomer chemistry. This methodology for
the 12-fold degenerate synthesis of carbonate and carbamate
closomers will greatly facilitate further exploration of closomers
as monodisperse nanomolecular delivery platforms.
ester6,7 and ether8,9 derivatives of [closo-B12(OH)12]2À including
a recently communicated synthesis of a 12-fold degenerate azido
carboxylate ester closomer for ‘click chemistry’ approaches.10
Though ester and ether derivatives have demonstrated the utility
of the B122À core for use as a scaffold, simple and mild reaction
conditions are necessary for the successful utilization of clo-
somers as multifunctional carriers. Consequently, we report here
12-fold degenerate carbonate and carbamate closomers as useful
platforms for conjugation to bioreactive or other molecules of
interest.
Carbamates are widely used as protective groups for amines in
organic chemistry.11 They are easily synthesized by reacting
chloroformates with amines or more elegantly by nucleophilic
attack of amines on the carbonyl group of a carbonate ester.
In our earlier reports7 we have shown that closomer carboxylate
esters are relatively stable in aqueous media at moderately acidic
and alkaline pH. We envisioned that carbamates, when also
O-bonded to a B122À cage would be stable at physiological pH.
This prompted us to examine 12-fold degenerate carbonate
closomers as intermediates in carbamate closomer syntheses.
For the initial exploration we chose the p-nitrophenyl carbonate
since substituted aryl carbonates having strongly electron-with-
drawing substituents are preferred reactants for carbamate for-
mation from primary amines. Thus, closomer 2j was prepared by
reacting p-nitrophenyl chloroformate with 1 in the presence of
anhydrous pyridine and acetonitrile at room temperature for 72 h
to give the corresponding 12-fold degenerate carbonate closomer
ignificant advances have been made over the past decade in
S
the development of nanoscale pharmaceutical carriers to
enhance the in vivo efficacy of many pharmaceuticals currently
in clinical use.1,2 Attachment of many copies of pharmaceuticals
to nanocarriers such as liposomes, polymeric and metallic nano-
particles, micelles, dendrimers, etc., can be used to enhance the
effectiveness of a drug’s therapeutic or diagnostic functions.3
2À
Herein we present a novel nanocarrier based upon the B12
icosahedral borane scaffold. The chemical basis of these unique
nanocarriers is the discovery of the very stable and icosahedral
[closo-B12(OH)12]2À, 1, and its successful 12-fold derivatization
to produce general structures now known as closomers.4À6 The
syntheses of fully substituted borane closomers, in which linker
2À
groups emanate from a rigid B12 core, represent a novel
architecture at the interface of boron and carbon chemistries.
The 12-fold degenerate functionality of 1 is reflected in the
closomer species derived from it by simple organic reactions
characteristic of the hydroxyl group: carboxylate ester, ether, as
well as carbonate and carbamate ester formation reported here.
The resulting monodisperse closomers can accommodate closely
spaced radial substituents with variable size, hydrophilicity, ionic
charges, and solvation properties.6 A schematic representation of
such a system is shown in Figure 1.
One of the main objectives of this study was to develop a
universal toolbox that may be used to conjugate many copies of
substituents for therapeutic or diagnostic purposes as well as
others. Earlier, we reported the synthesis of 12-fold degenerate
Received: May 16, 2011
Published: July 18, 2011
r
2011 American Chemical Society
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|

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Table 1. Synthesis of 12-Fold Degenerate Carbonate
Closomers
precipitated from dichloromethane with diethyl ether to remove
the large excess of chloroformate used in the reaction. Benchtop
flash column chromatography using a gradient of dichloro-
methane and acetonitrile over neutral alumina gave the desired
12-fold degenerate carbonate in good purity although in reduced
yield along with some less than 12-substituted carbonate. The
yields of 12-fold carbonate closomers presented in Table 1 are
purified yields of 12-fold substituted carbonates only. Prelimin-
ary hydrolytic stability studies carried out in a DMSO/H2O
system12À14 indicated these carbonate closomers to be stable
between pH 3 and pH 11.
Once the carbonate closomers were obtained, their reactivities
toward n-butyl amine were examined. The reaction of carbo-
nates 2aÀ2e with n-butyl amine (60 mol equiv in acetonitrile)
required elevated temperature to obtain the corresponding
12-fold degenerate carbamates. At the same reagent concentra-
tions, carbonates 2fÀ2j were found to react with n-butyl amine at
room temperature to give the corresponding 12-fold degenerate
carbamate 3a. This led us to conclude that carbonate closomers
with substituents having Hammett σ constants of 0.37 and higher
were suitable for forming carbamates at room temperature. Due
to its great reactivity, described above, carbonate 2j could not be
routinely used. Alternatively, we chose carbonate 2f as a reagent
for its ease of synthesis, high reactivity toward amines, and long-
term stability.
The utility of the carbonate closomer, a precursor for 12-fold
degenerate carbamate closomers attached by suitable linkers,
depends upon the steric environment near the site of the reac-
tion. It was prudent to employ a flexible linker next to the ter-
minal amine group for reaction with the carbonate closomer 2f.
Typical reaction conditions involved reaction of carbonate
closomer 2f with an excess of the primary amine (60 mol equiv
corresponding to 5 mol equiv per vertex) in an appropriate
solvent (acetonitrile or DMF) at room temperature for 24À
120 h (Table 2). The progress of the reaction was monitored by
MS analysis and shifting of the 11B NMR singlet from À17.4
to À18.3 ppm. To explore the versatility of carbonate closomers
as intermediates, we prepared a variety of 12-fold degenerate
carbamate closomers shown in Table 2. These include simple
alkyl and aralkyl carbamates 3aÀ3c as well as peripherally
functionalized carbamates 3dÀ3k. Carbamates 3d and 3e have
alkyne and azido groups, respectively, on their periphery for click
chemistry applications. Carbamates 3f with a benzyl-protected
carboxylic acid function and 3g with a Boc protected primary
amino group are available for further conjugation to peptides or
bioactive molecules of interest through amide linkages. Carba-
mate closomer 3f and carbamate 3h having 12 sulfanilamide
groups on the periphery were synthesized using carbonate 2j
instead of carbonate 2f.
To further explore carbamate closomer chemistry, we synthe-
sized a series of 12-fold degenerate carbamates 3iÀ3k having a
fluorescent dansyl group on the closomer as a surrogate for a
bioactive molecule. The dansyl group (5-(dimethylamino)-
1-naphthalenesulfamido) absorbs strongly in the near UV region
and fluoresces in the visible region. The dansyl group is robust,
easily derivatized, and commonly used as a fluorescent marker
and sensor.15À18 In this series of 12-fold degenerate carba-
mate closomers, the linkers with variable length and functions
attached to dansyl were preassembled and then conjugated
with the B122À core in the final step. For example, the reac-
tive amine for carbamate 3i was synthesized in five steps
starting from commercially available tetraethylene glycol,
a Purified yield.
in 40% purified yield. This carbonate was highly reactive toward
amines but was not suitable for use in carbamate synthesis due to
its unstable nature and resulting low yield. This led us to other
12-fold degenerate carbonates for carbamate synthesis. Table 1
shows various carbonate closomers synthesized with substitu-
tents having Hammet Sigma constants ranging from À0.27
(p-OCH3) to 0.78 (p-NO2).
The 12-fold degenerate hydroxyl groups on the B12 icosahe-
dral surface pose unique challenges in synthesis. To achieve a
high yield of 12-fold degenerate carbonate closomer, the typical
reaction conditions involved reacting 1 with respective chloro-
formate (60 mol equiv) and pyridine (60 mol equiv) in acetoni-
trile (excess) at the refluxing temperature for 24 h (carbonates
2aÀ2f, method 1) or stirring at room temperature for 96 h
(carbonates 2gÀ2j, method 2). The reaction was monitored by
MS analysis and 11B NMR spectra of the reaction mixture. The
completion of the reaction was confirmed by the appearance of a
single peak around À17.4 ppm in the 11B NMR. Solvent was
removed under vacuum following filtration, and the residue was
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Table 2. Synthesis of 12-Fold Carbamate Closomers
Absorbance spectrum of the 12-fold degenerate dansyl clo-
somer 3i in acetonitrile exhibits an intense absorbance band in
the near-UV region (λmax = 221, 250, and 335 nm, ∈max = 5.24 Â
105, 1.93 Â 105, and 5.07 Â 104 MÀ1 cmÀ1, respectively) and a
strong fluorescence band in the visible region (λmax = 516 nm,
excitation at 335 nm). When compared to dansylamide, a mono-
dansyl model compound (absorbance λmax = 220, 250, and
335 nm, ∈max = 4.84 Â 104, 1.82 Â 104, and 4.07 Â 103
MÀ1 cmÀ1, respectively), closomer 3i shows approximately
12-fold increase in the absorbance. Similarly, on excitation at
335 nm in acetonitrile, the intensity of a fluorescence band at
516 nm was 12 times that of dansylamide, corresponding to 12
dansyl units on the closomer (please see Supporting Information
for details). Protonation of dansyl groups with triflic acid causes
the disappearance of this fluorescence band and the appearance
of a new fluorescence band with λmax= 338 nm for the protonated
dansyl group.21
In addition to closomer 3i, other 12-fold carbamate clo-
somers 3j and 3k having cleavable linkers were synthesized.
Carbamate 3j has a tetrapeptide Gly-Phe-Leu-Gly sequence
as a part of the linker. This tetrapeptide is very specific to
the cathepsin-B enzyme, which is overexpressed in breast
and other cancers.22À25 The carbamate 3k has a tetraethy-
lene glycol linker with an acid-labile carbamate function
attached to the terminal dansyl ethylene diamine moiety.
These carbamate closomers were synthesized analogously
to closomer 3i, made by building the respective linker with
a peripherally attached dansyl group and reacting it with
carbonate closomer 2f to give the corresponding 12-fold
carbamates in good yields.
In summary, we have shown the versatility of 12-fold degen-
erate carbonate and carbamate closomers for the attachment of a
wide variety of linkers. Such an approach can be utilized to
construct a delivery system carrying 12 copies of an active
pharmaceutical.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental proce-
b
dures and characterization data. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
hawthornem@missouri.edu
a Purified yield. b Carbonate 2j was used for the synthesis.
which was converted to a dimesylate derivative.19 Both of the
mesylate groups were then converted to azide using sodium
azide.20 This diazide was selectively reduced to give corre-
sponding monoamine azide in 65% yield. This mono amine
was reacted with dansyl chloride to give the corresponding
dansyl conjugated azide. The terminal azide was reduced to
the corresponding primary amine, and this amine was reacted
(12-fold excess per vertex) with carbonate 2f in dimethylfor-
mamide (DMF) at room temperature for 72 h. The crude
product was purified by benchtop size exclusion column
chromatography over lypophilic resin LH20 in methanol
and subsequent dialysis through 1000 MW membrane in
DMSO/water mixture to give the carbamate closomer 3i in
84% yield.
’ ACKNOWLEDGMENT
We gratefully acknowledge the support of this project by the
National Cancer Institute (Grant R21-CA114090). We thank
Brett Meers for MS analyses.
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