2-Hydroxy-5-nitrobenzyl as a diazeniumdiolate protecting group: Application in no-releasing polymers with enhanced biocompatibility

Article abstract of DOI:10.1021/ol801883f

(Chemical Equation Presented) The 2-hydroxy-5-nitrobenzyl group is shown to be an effective protecting group for diazeniumdiolates. O²-(2- Hydroxy-5-nitrobenzyl)-substituted diazeniumdiolates display enhanced thermal stability, but efficiently release nitric oxide (NO) in pH 7.4 aqueous solutions. A lipophilic analogue incorporated into hydrophobic polymers shows NO surface flux rates comparable to that of the natural endothelium. Importantly, these polymer formulations also show significantly enhanced biocompatibility in vivo with use of a porcine implant model.

Full text of DOI:10.1021/ol801883f

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4593-4596
2-Hydroxy-5-nitrobenzyl as a
Diazeniumdiolate Protecting Group:
Application in NO-Releasing Polymers
with Enhanced Biocompatibility
Hua Xu,† Melissa M. Reynolds,‡ Keith E. Cook,§ Anthony S. Evans,† and John
P. Toscano*,†
Department of Chemistry, Johns Hopkins UniVersity, Baltimore, Maryland 21218,
Michigan Critical Care Consultants, Inc., Ann Arbor, Michigan 48103, and
Departments of Surgery and Biomedical Engineering, UniVersity of Michigan, Ann
Arbor, Michigan 48109
jtoscano@jhu.edu
Received August 12, 2008
ABSTRACT
The 2-hydroxy-5-nitrobenzyl group is shown to be an effective protecting group for diazeniumdiolates. O2-(2-Hydroxy-5-nitrobenzyl)-substituted
diazeniumdiolates display enhanced thermal stability, but efficiently release nitric oxide (NO) in pH 7.4 aqueous solutions. A lipophilic analogue
incorporated into hydrophobic polymers shows NO surface flux rates comparable to that of the natural endothelium. Importantly, these polymer
formulations also show significantly enhanced biocompatibility in vivo with use of a porcine implant model.
One of the most significant problems concerning the use of
synthetic biomaterials in blood-contacting applications is the
rapid onset of thrombosis (i.e., the formation of a blood clot).
Recent research in the field has focused on the development
of more thromboresistive surfaces.1 Strategies range from
design of more biocompatible hydrophobic surfaces to
grafting of drugs and biomolecules to polymer surfaces. A
completely thromboresistive surface must exhibit a range of
biological activity including inhibition of both fibrinogen
adsorption and its subsequent conversion to fibrin and a
reduction of adherence, aggregation, and release reactions
of platelets at the surface. The most thromboresistive material
known is the natural endothelium, which regulates the above
responses by a variety of mechanisms, including prostacyclin
release and nitric oxide (NO) generation. Many new bio-
materials are designed to mimic the properties of the
endothelium.
† Johns Hopkins University.
‡ Michigan Critical Care Consultants, Inc.
NO is continually released by the endothelium at a flux
of approximately 1 × 10-10 mol cm-2 min-1.2 This naturally
controlled-release has been shown to regulate vascular tone
§ University of Michigan.
(1) For a recent review, see: Wu, Y.; Meyerhoff, M. E. Talanta 2008,
75, 642–650.
10.1021/ol801883f CCC: $40.75
Published on Web 09/24/2008
2008 American Chemical Society

and also to inhibit platelet adhesion and aggregation.3-6
Much research has focused on the development of bioma-
terials that mimic the NO-releasing properties of the endot-
helium. Indeed, a variety of synthetic hydrophobic surfaces
that release NO at approximately the desired endothelium
flux all show reduced platelet adhesion and thus enhanced
thromboresistivity compared to NO-free analogues.1
Many NO-releasing biomaterials incorporate an NO donor
based on the diazeniumdiolate [N(O)dNO]- functional
group. Compounds containing this functional group have
proven useful as research tools in a variety of applications
requiring spontaneous release of NO.7 Anions such as
1-(N,N-dialkylamino)diazen-1-ium-1,2-diolates 1 are stable
as solid salts, but release up to 2 molar equiv of NO when
dissolved in aqueous solution at physiologically relevant
conditions in an acid-catalyzed dissociation reaction.8
Smith, Keefer, and co-workers first reported the prepara-
tion and study of several NO-releasing polymers containing
diazeniumdiolates in 1996.9 In one case, diazeniumdiolates
were noncovalently dispersed through water-soluble poly-
ethylene glycol (PEG) or biodegradable polycaprolactone
(PCL) matrices by simple mechanical mixing of the melted
polymer and a variety of different diazeniumdiolates. Al-
though diazeniumdiolates survived the blending process with
PEG (at 46 °C) with no appreciable decomposition, those
blended into PCL (at 60 °C) suffered 50% decomposition.
This observation highlights one of the major problems
associated with the development of NO-releasing biomate-
rials of this kind: free diazeniumdiolate anions decompose
readily even at only slightly elevated temperatures making
them impractical for use in standard polymer manufacturing
techniques such as extrusion and injection molding.
Several chemical strategies have been developed for the
protection of dialkylamino-substituted diazeniumdiolates 1.10
These approaches convert 1 to the stable prodrug (O2-
substituted diazeniumdiolate 2) by reaction with a variety
of alkylating or arylating agents that affix electrophilic groups
(R′ in Scheme 1) to the terminal oxygen. These “protected”
diazeniumdiolates can then be converted back to the NO-
releaser 1.
Scheme 1
.
The Dissociation of Diazeniumdiolate 1 and Its
Protection As Prodrug 2
into protein and peptide structures. Since the acid form (λmax
) ca. 320 nm) can easily be distinguished from the basic
form (λmax ) ca. 410 nm) by absorption spectroscopy,
this11,12 and related13,14 reagents have served as environ-
mentally sensitive probes. Reaction with nucleophiles is
initiated by deprotonation of the 2-hydroxy group (pKa )
ca. 7.4) to form a quinone methide intermediate that is
subsequently trapped by the nucleophile.
On the basis of this work, we reasoned that a modified
Koshland’s reagent could serve as a diazeniumdiolate
protecting group that could be removed at pH 7.4 in aqueous
solution. Thus, we synthesized precursor 3 (see the Sup-
porting Information) and examined its aqueous chemistry.
The pH dependence of the absorption spectra of NO
precursor 3, with λmax ) 310 nm for the protonated form
and 405 nm for the deprotonated form, is analogous to that
previously reported for 2-hydroxy-5-nitrobenzyl alcohol (see
the Supporting Information).11 As expected, at pH 7.4 3
decomposes cleanly to benzyl alcohol 4, amine, and NO as
shown in Scheme 2. The half-life (determined by UV-vis
Scheme 2
.
Decomposition of NO Precursor 3 and Its Lipophilic
Analogue 5
2-Hydroxy-5-nitrobenzyl bromide was first introduced by
Koshland et al. as a protein-modification reagent with
selectivity for tryptophan residues in 1964.11 Such modifica-
tion introduces the pH sensitive 4-nitrophenolic functionality
(2) Vaughn, M. W.; Kuo, L.; Liao, J. C. Am. J. Physiol. 1998, 274,
H2163–H2176
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(3) Gottenbos, B.; van der Mei, H. C.; Busscher, H. J. J. Biomed. Mater.
Res. 2000, 50, 208–214
(4) Razatos, A.; Ong, Y.-L.; Boulay, F.; Elbert, D. L.; Hubbell, J. A.;
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Sharma, M. M.; Georgiou, G. Langmuir 2000, 16, 9155–9158
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(5) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Meluleni, G.; Kim, E.;
Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001, 17,
absorption spectroscopy) of 3 in pH 7.4 solutions is 25 min
at room temperature and 5 min at 37 °C. For comparison,
1225–1233
.
(6) Arciola, C. R.; Montanaro, L.; Caramazza, R.; Sassoli, V.; Caveda-
gna, D. J. Biomed. Mater. Res. 1998, 42, 1–5
.
(7) For a recent review, see: Hrabie, J. A.; Keefer, L. K. Chem. ReV.
(11) Koshland, D. E., Jr.; Karkhanis, Y. D.; Latham, H. G. J. Am. Chem.
2002, 102, 1135–1154.
Soc. 1964, 86, 1448–1450
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(8) Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K. J. Am.
Chem. Soc. 2001, 123, 5473–5481.
(12) Horton, H. R.; Koshland, D. E., Jr J. Am. Chem. Soc. 1965, 87,
1126–1132
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(9) Smith, D. J.; Chakravarthy, D.; Pulfer, S.; Simmons, M. L.; Hrabie,
J. A.; Citro, M. L.; Saavedra, J. E.; Davies, K. M.; Hutsell, T. C.; Mooradian,
D. L.; Hanson, S. R.; Keefer, L. K. J. Med. Chem. 1996, 39, 1148–1156.
(10) Keefer, L. K. Curr. Top. Med. Chem. 2005, 5, 625–636.
(13) Horton, H. R.; Tucker, W. P. J. Biol. Chem. 1970, 245, 3397–
3401
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(14) Hojo, T.; Nakamura, H.; Tamura, Z.; Nakajima, T. Chem. Pharm.
Bull. 1983, 31, 3350–3352
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Org. Lett., Vol. 10, No. 20, 2008

the half-life of 2-hydroxy-5-nitrobenzyl bromide in pH 7.4
solutions is 5.5 min at room temperature and 1.5 min at 37
°C, consistent with the expectation that the diazeniumdiolate
is a poorer leaving group than bromide.15 At pH 11.6
decomposition is much more rapid; however, NO is not
observed (diazeniumdiolate 1 is stable under these basic
conditions) until the solution is acidified. Encouraged by
these promising results, we also synthesized (see the Sup-
porting Information) the lipophilic analogue 5 that could be
incorporated into hydrophobic polymers.
Because the protection of diazeniumdiolates is meant to
impart thermal stability to the NO-releasing functionality,
we performed qualitative thermal decomposition experiments
under conditions intended to mimic a typical polymer
extrusion process. The thermal stability of 5 was examined
by thermogravimetric analysis (TGA). Precursor 5 is stable
up to 90 °C, but lost ca. 9% of its weight over 2 h at 100
°C; heating from 100 to 170 °C over 2 min resulted in
another 3% weight loss. (For comparison, diazeniumdiolate
1 (R ) C8H17) is not stable above 60 °C.) These results
suggest that this diazeniumdiolate protection strategy should
hold up to extrusion conditions where high temperatures are
maintained for only a few minutes.
To demonstrate the feasibility of 2-hydroxy-5-nitrobenzyl
as a diazeniumdiolate protecting group that can be used in a
commercially viable NO-releasing polymer, we have exam-
ined formulations of the medical-grade polyurethane (PU)
Tecoflex blended with precursor 5. (Tecoflex is currently
used in the manufacturing of many blood and tissue contact
catheters and sensors.) Precursor 5 (2 wt %) was blended
into Tecoflex EG-80A by dissolving in THF. The resulting
polymer cocktail solution was cast inside 2.5 cm i.d. Teflon
rings and cured overnight under ambient conditions; the
resulting film thicknesses ranged from 150 to 200 µm.
Films were placed in pH 7.4 PBS solution at 37 °C and
NO surface flux was analyzed over the course of ap-
proximately 20 h, using a chemiluminescence analyzer.
Results are shown in Figure 1. Disappointingly, following a
bolus of NO production over the first 30 min, very little NO
was observed, i.e., only ca. 1% of the maximum possible
(NO release profile I, Figure 1a).16 However, when analogous
films were prepared that also incorporated 8 wt % of the
lipophilic salt, potassium tetrakis-4-chlorophenyl borate
(KTpClPB),17 the extremely promising NO release profile
II (Figure 1b) was observed. The amount of NO observed
is now approximately 60% of the maximum possible,
demonstrating the paramount importance of the added
lipophilic salt as has been reported previously.17 In addition,
an NO flux rate above 1 × 10-10 mol cm-2 min-1 is
maintained for over 20 h thus mimicking the NO-releasing
properties of the natural endothelium.
Figure 1. Nitric oxide surface flux observed at pH 7.4 and 37 °C
for a polymer containing (a) 2 wt % NO precursor 5 blended with
Tecoflex EG-80A and (b) 2 wt % NO precursor 5 and 8 wt %
KTpClPB blended with Tecoflex EG-80A.
The origin of the effect of added lipophilic salt has been
proposed to be related to a buffering effect.17 Proton-induced
decomposition of diazeniumdiolates within a polymer matrix
occurs as water diffuses in, leading to the formation of
secondary amines, which effectively raise the pH of the
polymer environment due to the formation of secondary
ammonium hydroxide moieties. In the case of lipophilic
diazeniumdiolates such as 1 (R ) C8H17), the secondary
ammonium hydroxide will be unable to diffuse out of the
polymer bulk due to hydrophobic interactions. This ultimately
leads to a retardation of the decomposition of diazeniumdi-
olates and an inability to recover all of the NO from
deprotected diazeniumdiolates in a reasonable amount of
time. By incorporating lipophilic anions into the polymer,
the alkali cations are able to pair with the hydroxide (and
likewise the lipophilic borate anions with the lipophilic
ammonium cations), and now the hydroxide is able to diffuse
out of the polymeric matrix and into the surrounding aqueous
buffer.
We also examined NO release profiles from polyvinyl
chloride (PVC) rods coated with PU containing our newly
developed NO donor 5. PVC tubing (0.027′′ i.d., 0.045′′ o.d.)
was capped with PU and smoothed before coating. Several
2-cm ends of 10-cm lengths of these rods were dip-coated
six times with a homogeneous polymer cocktail solution
containing Tecoflex SG-80A, NO-releasing agent 5, and
KTpClPB in THF. An additional thin layer of Tecoflex SG-
80A was applied as an overcoat. Coating integrity studies
were performed to ensure good adherence of the coating to
the substrate by soaking the rods in PBS buffer for 24 h
with a high velocity stir rate. The coatings were visually
examined pre- and postbuffer exposure. No difference in the
coating appearance was observed following this treatment.
Three different polymer cocktail solutions were used for
the dip-coating procedure to determine the effect of the
relative amounts of NO donor and lipophilic salt on NO
(15) Saavedra, J. E.; Srinivasan, A.; Bonifant, C. L.; Chu, J.; Shanklin,
A. P.; Flippen-Anderson, J. L.; Rice, W. G.; Turpin, J. A.; Davies, K. M.;
Keefer, L. K. J. Org. Chem. 2001, 66, 3090–3098.
(16) The lack of NO production over the course of 15 h (Figure 1a)
strongly suggests that NO precursor 5 does not leach from the polymer
into the aqueous solution. Further leaching studies are ongoing.
(17) Batchelor, M. M.; Reoma, S. L.; Fleser, P. S.; Nuthakki, V. K.;
Callahan, R. E.; Shanley, C. J.; Politis, J. K.; Elmore, J.; Merz, S. I.;
Meyerhoff, M. E. J. Med. Chem. 2003, 46, 5153–5161.
Org. Lett., Vol. 10, No. 20, 2008
4595

release profiles. These profiles, measured in pH 7.4 PBS
solutions at 37 °C, are shown in Figure 2. Comparison of
to implantation to ensure that the NO surface flux was in an
optimal range during the in vivo experiment. The rods
remained implanted for 8 h. Following removal, each rod
was rinsed gently with pH 7.4 PBS and examined visually;
digital images are shown in Figure 3 where a significant
Figure 2. Nitric oxide surface flux observed at pH 7.4 and 37 °C
for PVC rods coated with (a) 5 wt % NO precursor 5 and 5 wt %
KTpClPB blended with Tecoflex EG-80A, (b) 5 wt % NO precursor
5 and 20 wt % KTpClPB blended with Tecoflex EG-80A, and (c)
10 wt % NO precursor 5 and 20 wt % KTpClPB blended with
Tecoflex EG-80A.
Figure 3. Digital images of coated PVC rods after implantation
for 8 h. The portion of the rods to the left of the dotted lines was
exposed to flowing blood. The rods were prepared by dip-coating
in Tecoflex EG-80A cocktail solutions containing (a) 20 wt %
KTpClPB without any NO precursor 5 and (b) 20 wt % KTpClPB
with 5 wt % NO precursor 5.
profiles III and IV (Figure 2, panels a and b), where the
concentration of NO precursor 5 was kept constant and that
of KTpClPB was increased 4-fold, demonstrates that an
excess of KTpClPB is required to maximize NO release.
Comparison of profiles IV and V (Figure 2, panels b and c),
where the concentration of KTpClPB was kept constant and
that of NO precursor 5 was doubled, suggests that a polymer
matrix formulation with 20 wt % KTpClPB may be optimal
for NO release (i.e., doubling the amount of 5 doubles the
amount of NO produced).
To test the biocompatibility of materials incorporating NO
precursor 5, we examined polymer formulations using a
porcine implant model to evaluate thrombus formation.18
Four Tecoflex SG-80A-coated rods were prepared (two sets
of two, with each set containing a control rod without any
NO donor and another rod incorporating NO precursor 5).
The rods were prepared as described above by dip-coating
in polymer cocktail solutions containing (1) 20 wt %
KTpClPB without any NO precursor 5 and (2) 20 wt %
KTpClPB with 5 wt % NO precursor 5. One set was then
implanted in the femoral arteries and the other in the carotid
arteries of a 25 kg farm swine (see the Supporting
Information).
difference in thrombus formation can be seen. (Note that
only the portion of the rods to the left of the dotted lines in
Figure 3 was exposed to flowing blood.) The rods that
released NO (Figure 3b) have no observable thrombus
formation on their surfaces, whereas the control rods that
contained no NO precursor (Figure 3a) possess significant
areas of clots on their surfaces. These results, taken together
with the enhanced thermal stability imparted to the diazeni-
umdiolate functionality by 2-hydroxy-5-nitrobenzyl substitu-
tion, suggest that lipophilic NO precursor 5 may be useful
as a general additive to hydrophobic polymers to enhance
their thromboresistivity in blood-contacting biomedical ap-
plications.
Acknowledgment. We gratefully acknowledge the Na-
tional Science Foundation (CHE-0518406) for generous
support of this research.
Supporting Information Available: General experimental
and synthetic procedures, spectra, and details concerning the
porcine implantation procedure. This material is available
free of charge via the Internet at http://pubs.acs.org.
Given the NO release profiles shown in Figure 2, the rods
were soaked for 7 h in pH 7.4 PBS solution at 37 °C prior
(18) Frost, M. C.; Rudich, S. M.; Zhang, H.; Maraschio, M. A.;
Meyerhoff, M. E. Anal. Chem. 2002, 74, 5942–5947.
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