Journal of the American Chemical Society
Page 2 of 8
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mine (EDA), or diethylenetriamine (DETA) to yield secondary
diazeniumdiolate formation (Figures S6‒S9), possibly atꢀ
tributable to the absence of hydrogen bonding. Additional
evidence for the formation of CDꢀNONOates was provided by
the strong absorption peak at ~252 nm in the UVꢀVis spectra
of CDꢀHEDA7/NO (Figure 1c). A similar absorption peak
(around ~255 nm) was observed for each of the other CDꢀ
NONOates (Figure S10 and S11). Of note, an absorption peak
around 330‒360 nm, characteristic peaks of carcinogenic Nꢀ
nitrosamine species, was not observed, suggesting that these
CD derivatives did not form Nꢀnitrosamines during NO donor
synthesis. During the Nꢀdiazeniumdiolation step, NO first
reacts with a secondary amine to yield a nitrosamine radical
anion intermediate; subsequently, this intermediate reacts with
39
amineꢀmodified monoꢀsubstituted βꢀCD derivatives. These
CD derivatives were given the following nomenclatures: CDꢀ
HEDA, CDꢀPA, CDꢀMA, CDꢀEDA, or CDꢀDETA, based on
the primary amines employed in the reaction. To potentially
improve NO loading, the secondary hydroxyl groups of the βꢀ
CD were converted into bromo groups to yield heptakisꢀ6ꢀ
40
bromoꢀ6ꢀdeoxylꢀβꢀcyclodextrin (CDꢀBr7). Secondary amineꢀ
modified heptaꢀsubstituted βꢀCD derivatives were then syntheꢀ
sized by displacing bromide with primary amines to form CDꢀ
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HEDA7, CDꢀPA7, CDꢀMA7, CDꢀEDA7, and CDꢀDETA7.
Synthetic details and analytical characterization are provided
in Supporting Information (SI).
4
2,43
another molecule of NO to form the Nꢀdiazeniumdiolate.
High pressures (i.e., 10 bar) of NO are known to drive the
Scheme 1. Synthesis of secondary amine- and N-
diazeniumdiolate-functionalized CD derivatives. (a) Prep-
aration of secondary amine-modified CDs; reagents and
conditions: (i) TsOCl, NaOH, H O/CH CN, room temp.;
2
3
o
(
ii) Primary amine (RNH ), 75 C; (iii) Bromine, P(Ph) ,
2
3
o
DMF, 80 C; (iv) Primary amine (RNH ), DMF, room
2
temp. (b) Subsequent N-diazeniumdiolate formation.
R
a
NH
HO
6
OH
i
HO
O
O
O
i
i
OH HO
OH
O
OH
HO
O
HO
HO
R
=
CH3
NH
OCH
7
OH
O
O
2
OH
O
3
HO
NHCH
NHCH
2
2
CH
CH
2
2
OH
NH
OH
=
HO
2
O
OH
i
1
ii
Figure 1. (a) Synthetic route for CDꢀHEDA7/NO. (b) H
OH
R
OH
O
OH
OOH
OHO
O
i
v
NMR spectra for CDꢀHEDA7 (top line) and CDꢀHEDA7/NO
(bottom line). (c) UVꢀVis spectra for CDꢀHEDA7 (solid line)
and CDꢀHEDA7/NO (dash line).
NH
7
HO
O
OH
As shown in Figure 2a, the degradation of the N-
diazeniumdiolate upon protonation yields two moles of NO
and the parent secondary amine. This degradation is pHꢀ
dependent, and results in more rapid release at lower pH. Realꢀ
time NO release was measured using a chemiluminescenceꢀ
based nitric oxide analyzer (NOA). NO payloads and release
kinetics of the CDꢀNONOates were measured under physioꢀ
MeONa, 3d
R
R
N
N
N
CD
b
CD
H
1
0 bar NO
O
N
O Na
The resulting secondary amineꢀmodified CD derivatives
monoꢀ and heptaꢀsubstituted) were reacted with NO gas at
(
high pressures (10 bar) under strong alkaline conditions to
o
logical condition (pH 7.40, 37 C). The resulting NOꢀrelease
yield Nꢀdiazeniumdiolateꢀmodified CD derivatives (Scheme
1
parameters (i.e., NO payload, halfꢀlife of NO release, and conꢀ
version efficiency) are provided in Table 1. Representative
cumulative NOꢀrelease profiles of the N-diazeniumdiolateꢀ
modified CD derivatives are shown in Figures 2b and S12. In
general, the CDꢀNONOates exhibited tunable NO payload
capabilities (e.g., NO payloads from ~0.6 to ~2.4 ꢁmol/mg)
and variable NOꢀrelease kinetics (e.g., halfꢀlives spanning 0.7
to 4.2 h), depending on the type and amount of secondary
amines, and exterior chemical modification. The achievable
NO payloads with these NO donorꢀmodified CD derivatives
are significantly greater than previously reported NOꢀreleasing
1
b). The representative synthesis and sequent H NMR and
UVꢀVis characterization of CDꢀHEDA7/NO are provided in
Figure 1. Of note, only one –NHꢀ group is sufficiently facile to
react with NO, resulting from steric hindrance (Figure 1a).
Proton NMR indicated evidence for Nꢀdiazeniumdiolate NO
donorꢀmodification on the CDꢀHEDA7 backbone (Figure 1b).
Specifically, proton signals in the range of 2.72‒3.05 ppm
corresponding to methylene groups bound to secondary
amines were shifted downfield (2.90‒3.11 ppm), owing to
formation of hydrogen bonds between the terminal hydroxyl
groups and N-diazeniumdiolate functional groups. Similar
19
1
biopolymers (e.g., ~0.9 ꢁmol/mg for chitosan). Using the NO
payload data, we calculated secondary amine (NO precursor)
to N-diazeniumdiolate NO donor conversion efficiencies to
span 12‒41%. The less than expected conversion efficiency is
attributed to the proximity between the NO donor precursors
downfield shifts were also observed in the H NMR spectra of
other hydroxylꢀ or primary amineꢀterminated CDꢀNONOates
(Figures S1‒S5 for CDꢀHEDA/NO, CDꢀEDA/NO, CDꢀ
DETA/NO, CDꢀEDA7/NO and CDꢀDETA7/NO, respectively).
1
Of note, the H NMR spectra of methylꢀ and methoxylꢀ
(i.e., secondary amines) and oligosaccharide ring, sterically
terminated CDꢀNONOates (i.e., CDꢀMA/NO, CDꢀMA7/NP,
CDꢀPA/NO and CDꢀPA7/NO) revealed upfield shifts for their
methylene groups around the Nꢀdiazeniumdiolates after Nꢀ
hindering complete formation of the N-diazeniumdiolate NO
donor on each secondary amine.
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