Hexamethylenetetramine carboxyborane: synthesis, structural characterization and CO releasing properties

Article abstract of DOI:10.1039/c6dt03856e

Carbon monoxide, although widely known as a toxic gas, has received great attention in the past few decades due to its promising role as a medical gas. Several classes of carbon monoxide releasing molecules (CORMs) have been synthesised with many of them having pharmacological activities under physiological conditions. Herein, we report the synthesis and structural characterization of the first example of amine carboxyborane that releases CO under physiological conditions without the aid of inducers. A representative compound hexamethylenetetramine carboxyborane (HMTA-CB) described here has a half-life of 2.7 days and gradually releases CO with the rate constant of 3.0 × 10^?6 s^?1. Its ability to promote cell growth shows the beneficial effect of slow CO release to supplement CO in small amounts over time.

Full text of DOI:10.1039/c6dt03856e

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DOI: 10.1039/C6DT03856E
DALTON TRANSACTIONS
ARTICLE
Hexamethylenetetramine carboxyborane: synthesis, structural
characterization and CO releasing property
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a,†
T. I. Ayudhya , C. C. Raymond , N. N. Dingra
DOI: 10.1039/x0xx00000x
Carbon monoxide, although widely known as a toxic gas, has received a wide interest in the past few decades due to its
promising role as a medical gas. Several classes of carbon monoxide releasing molecules (CORMs) have been synthesised
with many of them having pharmacological activities under physiological conditions. Here, we report synthesis and
structural characterization of the first example of amine carboxyborane that releases CO under physiological condition
without the aid of inducers. A representative compound hexamethylenetetramine carboxyborane (HMTA-CB) described
www.rsc.org/
-
6
-1
here has a half life of 2.7 days and gradually releases CO with the rate constant of 3.0x10 s . Its ability to promote cell
growth shows the beneficial effect of slow CO release to supplement CO in small amounts over time.
Another widely investigated group of CORMs are
boranocarbonates along with their ester and amide derivatives.
These compounds do not contain transition metals and are the first
Introduction
Carbon monoxide (CO) production from hemoglobin in vivo has
been known for many decades. Since the discovery of heme-
oxygenases that produce endogenous CO, it was postulated that CO
17
1
molecules reported to release CO under physiological conditions.
Many modifications on this group of compounds have been made
to fine-tune CO release as well as to lower toxicity towards
2-4
has cellular and physiological roles.
The effect of CO as a
1
8
biological systems. Recent advances on CORMs include improved
signalling molecule has also been investigated extensively. Despite
its reputation as a toxic gas, studies done in the animal models
show important physiological effects of heme oxygenases and CO in
vasodilation, longer cardiac and renal graft survival, preservation of
transplanted organs, anti-inflammatory, and anti-apoptotic
14,19,20
solubility and photo-inducible CO releasing properties.
While
not a significant number, various organic compounds have also
21,22
been identified to release CO.
Lewis base-carboxyboranes [R NBH COOH], also called amine
3
2
5
-9
carboxyboranes, are a class of molecules that have been known for
many decades. They show pharmacological effects such as anti-
properties.
Gaseous CO, however, is not a desired long-term therapeutic
agent due to poisoning by inhalation and the difficulty in
administration to desired sites without bypassing the circulatory
system. As a consequence, a group of molecules that are capable of
releasing CO, referred to as carbon monoxide releasing molecules
2
3-26
cancer, anti-inflammatory, and anti-arthritic activities.
Their
structural similarity to amino acids has given them the name boron
analogues of amino acids and studies have been done in synthesis
of peptides containing α-amino boronic acids mimicking the
transition states of enzymes
or as pharmaceutical
(
CORMs), have been developed. The major group of CORMs
25,27,28
compounds.
Despite these known pharmacological effects,
includes transition metal carbonyls such as ruthenium,
molybdenum, manganese, and iron-based complexes.
being the major group of CORM, metal carbonyl complexes have
low solubility in aqueous solutions which is a serious drawback as
therapeutic agents in the living systems. Various attempts such as
incorporation into ferritin protein cages or polymers have been
10-13
assumptions have been made that amine carboxyboranes do not
Despite
29
release CO. In this report, we introduce hexamethylenetetramine
carboxyborane (HMTA-CB), novel compound in the amine
a
carboxyborane class that releases CO slowly under physiological
conditions.
10,14,15
made to make insoluble metal carbonyls more soluble.
The
idea of attaching metal carbonyl complexes into proteins to make
artificial metalloproteins has also been introduced for controlled
delivery of CO.
Experimental
Materials and Instrumentation: All reagents and solvent used were
purchased from Acros Organics unless noted otherwise. All NMR
solvents were purchased from Cambridge Isotope Laboratories. Cell
cultures and media were obtained from ATCC. H, B, and C NMR
spectra were recorded on Bruker 300 MHz Avance III spectrometer.
IR and UV-Vis spectra were recorded on Thermo Scientific Nicolet
iS50 FT-IR and Evolution 220, respectively. Elemental analysis was
performed on PerkinElmer SeriesII CHNS/O Analyzer. CO
16
a.
1
11
13
School of Pure and Applied Sciences, Florida SouthWestern State College, Fort
Myers FL 33919, USA.
Chemistry Department, State University of New York at Oswego, Oswego NY
b.
1
3126, USA.
†Email: ndingra@fsw.edu
Electronic Supplementary Information (ESI) available: Figures, cif file, detailed
experimental procedures, IR, NMR, Mass sepctra. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
measurement in ppm was recorded using a Honeywell ToxiPro
Single Gas Detector. Fluorescence measurements for toxicity test Cell viability assay: Murine macrophage cell line, RAW264.7 (ATCC),
and ELISA were done using SpectraMax M2 plate reader.
Journal Name
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DOI: 10.1039/C6DT03856E
4
was seeded at 2x10 cells per well into a 96-well plate. They were
treated with varying concentrations of HMTA-CB in DMEM with
Synthesis: Trimethylamine carboxyborane was prepared using 10% FBS (ATCC), then incubated at 37 °C in 5% CO2 under a
27
published procedures. Hexamethylenetetramine carboxyborane humidified atmosphere for 24 hours. CellTiter-Blue reagent
HMTA-CB) was prepared as follows. Trimethylamine (Promega) was added to the culture and incubated 4 more hours
carboxyborane (117 mg, 1.00 mmol) and hexamethylenetetramine under the same conditions. Fluorescence signals (560Ex/590Em) were
280 mg, 2.00 mmol) were dissolved in THF (8.0 mL), protected measured using the plate reader.
(
(
o
from the atmosphere, and maintained at 67 C for 24 hours. The
solution was purged with N gas for 5 minutes and concentrated by TNF-α assay: RAW264.7 (ATCC) was used to test for LPS-induced
2
4
vacuum distillation leaving a white solid crude product (208 mg). TNF-α production and inhibition of that by HMTA-CB. 2x10 cells
The residue was purified by column chromatography on alumina (8 per well were seeded into a 96-well plate in DMEM with 10% FBS.
gram Redisep Rf neutral alumina by Teledyne). The column was Cells were treated with varying concentrations of HMTA-CB and
eluted with an ethyl acetate/acetone gradient elution. HMTA-CB incubated at 37 °C under humidified condition with 5% CO for 24
2
was collected from the second fraction as a white solid (58 mg, hours. LPS (ThermoScientific) was then added to
a
final
+
2
9%): HRMS (pos. ion ESI) m/z: [M] Calcd for C H BN O 198.1403; concentration of 100 ng/mL and incubated for 4 additional hours.
7
15
4 2
Found 198.1395; Anal. Calcd for C H BN O : C, 42.46; H, 7.63; N, The media containing secreted inflammatory cytokines were
7
15
4 2
1
2
8.29. Found: C, 42.63; H, 7.65; N, 28.32; H NMR in DMSO-d6 collected and TNF-α concentration in each sample was measured
shows δ 1.71 (m, 2H, BH ), δ 4.42 (d, 3H, axial CH , J = 12.2 Hz), δ using the ELISA assay (ThermoScientific).
2
2
4
.52 (d, 3H, equatorial CH , J = 12.6 Hz), δ 4.68 (s, 6H, CH ), δ 10.24
2 2
13
(
s, 1H, CO H); C NMR (DMSO-d6) δ 71.05 (C-3,6,7), δ 75.80 (C-
2
11
Results and discussion
Synthesis and characterization of HMTA-CB
2
1
,4,5); B NMR (DMSO-d6) δ -15.00 (br. s, 1B); FT-IR (solid, ATR)
-1
-1
-1
674 cm (C=O), 2379 cm (BH ), 2850-3300 cm (O-H).
2
HMTA-CB was prepared as mentioned in the procedure section
with a yield of 29% and >99% purity. Previous studies have shown
that trimethylamine of betaine-B was replaced by ammonia
through an amine exchange process at room temperature in a
X-ray Crystallography: Crystals were grown by recrystallization in
chloroform/hexane. Crystal data. C H BN O , 197.03,
orthorhombic, a = 10.4040(4), b = 11.3367(4), c = 16.7032(6) Å, U =
M
=
7
15
4 2
3
1
970.09(12) Å , T = 296 K, space group Pbca (no.61), Z = 8, 21034
2
7,31
pressurized cylinder.
This strategy was applied in the synthesis
reflections measured, 1727 unique (Rint = 0.0595), which were used
in all calculations. The final wR(F2) was 0.0966 (all data).
of HMTA-CB with a few modifications. Trimethylamine group from
trimethylamine carboxyborane was exchanged with HMTA as
shown in Scheme 1. The resulting white solid product is stable in air
Auto-decomposition measurement by NMR: HMTA-CB in D O at
2
‡
1
1
and highly soluble in water. The H NMR spectrum in DMSO-d6
exhibited a 6H singlet at δ 4.68 for the protons on C-2, C-4 and C-5
which are deshielded by the boron and carboxylic acid group. Two
protons on C-3, C-6, and C-7 are non-equivalent due to their
corresponding equatorial and axial positions of the rigid chair
conformation resulting in a pair of 3H doublets at δ 4.52 (J = 12.6
Hz) and δ 4.42 (J = 12.2 Hz), respectively. The carboxylic acid proton
in DMSO-d6 solvent is observed at δ 10.24 although this proton
1
2.0 mg/mL was incubated at 37 C in the standard NMR tube. H,
B, and C NMR spectra were recorded at the times indicated.
1
1
13
CO measurement by FT-IR: HMTA-CB (65.0 µmol) at the
concentration of 12.0 mg/mL in water was incubated at 37 C in a
sealed vessel for 1 hour. The gas phase in the head space of
decomposing HMTA-CB solution was transferred into the 10 cm IR
gas cell and the absorbance measured.
cannot be seen in D O solvent due to the acidic nature of this
2
3
0
group. Two protons on the boron atom show a very broad signal at
δ 1.71 which is likely due to rapid nuclear quadrupolar relaxation at
boron and a non-spherical symmetry. A broad triplet peak at -15.0
Mb assay: This was performed using the published procedure.
Under anaerobic condition, 1 mM horse Mb (Sigma-Aldrich) in 50
mM sodium phosphate buffer (pH 7.4) was deoxygenated using 3x
equivalent of sodium dithionite. Excess dithionite was removed
using a desalting column and Mb concentration measured. Deoxy-
Mb at the concentration of 72.9 µM was mixed with 55x HMTA-CB
in a cuvette, sealed, and incubated in 37 C. Visible spectra in the
range of 500-600 nm were recorded every 10 minutes to detect the
changes in absorption.
11
ppm on B NMR spectrum suggests the presence of a boron atom
Scheme 1. Synthesis of HMTA-CB by amine exchange process
CO measurement by Meter: Various amounts of HMTA-CB at the
concentration of 60 mM were incubated at 37 C in a sealed vessel
with 50 mL gas space. At specified times, liberated CO gas in ppm
was measured using a CO gas detector and the moles calculated
from the ppm number based on 50 mL volume.
2
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1
3
27
with two hydrogens. The C NMR spectrum in DMSO-d6 was also extremly acidic conditions and the later report describing that this
View Article Online
DOI: 10.1039/C6DT03856E
29
consistent with the assigned structure and showed two singlets at δ class of compounds does not show release of CO . Our results
7
7
5.80 due to C-2, C-4 and C-5 and at δ 71.05 due to C-3, C-6 and C- show HMTA-CB as the first amine carboxyborane to release CO
. The carbonyl carbon that is attached to boron atom is not under biocompatible conditions without induction by light, pH
13
observed on C NMR which is consistent with the previous changes, or chemical inducers such as dithionite.
3
2
reports. The compound was fully characterized by FT-IR that
shows the presence of carboxylic acid, B-H and C=O absorptions,
HRMS showing m/z 198.1395, and the elemental analysis reported
in the supporting information that was also in agreement with the
calculated values.
X-ray crystal structure
ORTEP (50% probability) represents the X-ray structure of
HMTA-CB on Figure 1. The packing diagram (Figure 1a) for this
molecular unit reveals no hydrogen bonding and the units are
discrete. Figure 1b shows single molecule with a slight distortion in
the hexamethylenetetramine portion of the molecule. C–N(1) bond
lengths are elongated at 1.515(1)–1.521(1)
Å compared to
1
.444(2)–1.484(2) Å for the other C–N bonds. The C–N–C angles are
also slightly smaller around N(1) compared to the other nitrogens.
This was anticipated since N(1) is joined to the B(1) atom through a
coordinate covalent bond and carries a fractional charge while the
other nitrogen atoms have standard covalent bonds. The metric
parameters about B(1) and C(1) are as expected for a carboxyborate
fragment.
(
a)
(b)
Figure 2. (a) CO production of HMTA-CB monitored by FT-IR. The gas phase
in the head space of decomposing 12 mg/mL HMTA-CB solution was
injected into the IR gas cell and the absorbance measured. The CO ro-
-
1
vibrational band from 2040 – 2250 cm confirmed the production of CO by
HMTA-CB. (b) CO-releasing property of HMTA-CB tested with traditional
Mb assay. Deoxygenated Mb was incubated with 55x equivalent of HMTA-
CB at 37 °C and conversion of deoxy-Mb to CO-Mb was monitored using
UV-Vis spectrometer every 10 minutes.
Figure 1. The X-ray structure of HMTA-CB: packing structure (a) and a single
molecule (b). Selected bond lengths (Å) and angles (º): B(1)–C(1) 1.611(2),
C(1)–O(1) 1.224(2), C(1)–O(2) 1.354(3), B(1)–N(1) 1.592(2), O(1)–C(1)–O(2)
In addition to measurement by IR spectroscopy, a myoglobin
assay was performed to measure the amount of CO produced from
HMTA-CB semi-quantitatively. Myoglobin assay has been the
standard method used in detecting and quantifying CO from various
CORMs. In this assay, myoglobin (Mb) was first deoxygenated by
treating with dithionite, and deoxy-Mb was mixed with HMTA-CB.
1
16.6(1), C(1)–B(1)–N(1) 112.6(1).
35
CO-releasing property of HMTA-CB
The CO releasing property of HMTA-CB was tested using several Excess HMTA-CB was used to observe CO saturation within a short
methods. A few reports in literature have suggested the use of IR period of time due to its slow releasing property. The mixture was
11,33,34
o
spectroscopy for CO measurement.
Analysis of the head space sealed in a cuvette, incubated at 37 C and the increase in two
air above the solution of HMTA-CB, after one hour incubation in absorption peaks at 540 and 580 nm were monitored. As seen in
o
water at 37 C, identified the presence of CO by the ro-vibrational Figure 2b, visible spectra taken at 0, 10, 20, 30, and 40 minutes
-
1
band at 2040 – 2250 cm (Figure 2a). This is in contrast to the early show
a
gradual transformation of deoxy-Mb to §carboxy-Mb
reports of amine carboxyboranes stating where CO is found only in resulting all the Mb saturated with CO within one hour.
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1
Figure 3. (a) Decomposition scheme of HMTA-CB in D
2
O at 37 C (b) H NMR of the HMTA-CB decomposition at 0 hour (with peak assignments), 1 hour and
2
4 hours. Disappearance of HMTR-CB peaks at 4.97, 4.73, and 4.69 ppm is compared with the appearance of HMTA peak at 4.84 ppm. Note: HDO solvent
13
peak overlaps with 3, 6, 7 equatorial protons. (c) C NMR spectra of HMTA-CB showing the two peaks from HMTA-CB at 70.26 and 75.24 ppm at 0 h and a
single peak of HMTA at 72.00 ppm. (d) B NMR spectra of the HMTA-CB decomposition at 0 hour, 1 hour and 24 hours that show –15.47 ppm peak
corresponding to BH and a peak at 19.54 ppm to B(OH)
11
2
3
.
While confirming that HMTA-CB releases CO by the above is the only new organic product detected showing all equivalent
methods, we tracked the products of decomposition in order to find carbons in a new singlet peak at δ 72.00 ppm (Figure 3c). In
possible metabolites. HMTA-CB dissolved in D O was incubated at addition to CO and HMTA as the products of interest, we conclude
2
o
3
(
(
7 C and auto-decomposition monitored with NMR spectroscopy that the boron species produced is boric acid which is identified by
Figure 3a). Our observation shows that hexamethylenetetramine
HMTA) is liberated from HMTA-CB. Since all protons in HMTA are
11
B NMR at δ 19.54 ppm (Figure 3d).
equivalent, the appearance of a singlet peak at δ 4.84 ppm signifies
HMTA released by decomposition of HMTA-CB molecule (Figure
CO-release rate
Although IR spectroscopy and Mb assay confirm the property of
HMTA-CB releasing CO, quantifying the amount of CO released
proved to be challenging because of the need for adequate
experimental setup for IR spectroscopy. Deoxy-myoglobin assay is
not suitable for long incubation times due to protein degradation
and the possibility of air contamination. Some reports have also
shown that dithionite used in the assay for deoxygenating
3
b). A new peak that corresponds to free HMTA emerges within
one hour and continues to grow while the peaks corresponding to
HMTA-CB protons disappear. Although the HDO solvent peak
overlaps with the equatorial protons from the amine part of HMTA-
CB in H NMR, it does not interfere with integration calculations.
The ratio of integrated peaks from
determining the percent of each species present and the average
amount of HMTA-CB decomposed in the first 24 hour was found to
be 17.5%. Calculations for initial hours prove to be difficult in terms
of integrating smaller differences between the peaks on H NMR.
Yet, NMR allows us to track the decomposition of HMTA-CB for
longer periods, i.e. days to weeks. The CO-releasing property of
1
1
H NMR was used for
§
36
myoglobin interferes with CO releasing process of CORMs. NMR
spectroscopy, on the other hand, provides us with quantitative
1
13
1
numbers but only indirectly since CO is not detected by H or
C
NMR. In order to better quantify the amount of CO freed from the
compound and to avoid complications from interferences, we
utilized
a commercially available CO meter to measure the
HMTA-CB observed over
a long period of time indicates a
concentration of liberated CO gas from the solution. Measurement
using a CO meter in ppm, after converting to the micromole
amount, was used for calculating the mole% of CO released based
mechanism similar to burst kinetics. Decomposition of HMTA-CB
happens at a higher rate and is linear during the initial stage up to 1
day, then the rate gradually gets lower showing 80% decomposition
by 12 days (ESI Fig. S18). After two weeks, the rate becomes
significantly slower and complete decomposition was recorded in
§
on the original amount of compound. The amount of CO detected
1
by the CO meter and percent decomposition measured by H NMR
13
are compared as seen in Figure 4a. The amount of CO-released is
2
1 days. Examining C NMR data indicates that the HMTA molecule
4
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relatively linear in this 24-hour comparison with approximately 1 CO-releasing property of HMTA-CB in different pH
ARTICLE
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DOI: 10.1039/C6DT03856E
mole% of HMTA-CB decomposing per hour in H O at the
physiological temperature of 37 C. Both methods display similar
2
The ability to release CO by CORMs depends on many external
36
factors. Some are more active in the presence of dithionite , or
mole% CO produced for up to 14 hours but the amount detected by
38
some have variable activities in different pH. We tested whether
1
H NMR diverges from the numbers measured by CO meter,
HMTA-CB will be affected by the pH of the environment and found
that CO-releasing property of HMTA-CB is not limited to the neutral
environment but generates CO in any pH ranging from 2 to 12
showing lower percent decomposition at longer hours. This
reduction could be attributed to the compressed gas in a small
volume inside an NMR tube disturbing the equilibrium, and
therefore causing HMTA-CB decomposition to slow down. As
manifested in Figure 4b, CO production by HMTA-CB is the first
order reaction and the rate constant based on the first 12 hours is
(
Figure 5). In fact, CO-release rate is the lowest in pH 7 but displays
higher rate as the pH shifts to extreme acidic or basic conditions. CO
is released at the highest rate in pH 2, as in stomach pH, with more
than double the amount in neutral condition while the alkaline
condition only increases slightly. Our result is consistent with the
earlier report about detecting CO signal from amine
carboxyboranes through the involvement of carbonyl intermediate
-6
-1
determined to be 2.97±0.04 x10 s . The observed half-life of
HMTA-CB measured by CO meter is 2.7 days which shows much
slower rate compared to the common metal carbonyl CORMs which
12
have half-lives of several minutes and a well-studied CORM-A1
27
that undergo hydrolysis in the solution with 1 M HCl. Changes in
17
which has a half-life of 21 minutes under similar conditions.
Slower CO release rate, with the half-life of 150 minutes, was later
the CO-release rate based on pH has also been described with a
similar boron family member, CORM-A1, where lowering pH
37
reported by applying a few modifications on CORM-A1. In contrast
to all earlier reports of CORMs, HMTA-CB is the first CORM that
shows slow CO release rate with the half-life extending to days. This
suggests that amine carboxyboranes such as HMTA-CB may be ideal
for conditions that require gradual release of CO and longer-lasting
CORMs in the system.
17
decreases the half-life of the compound. To find that HMTA-CB
molecule performs as CO-releasing molecule in different
environment of varying pH without the help of an inducer is
significant in the field of amine carboxyboranes where their CO-
releasing property was believed to be limited to extremely acidic
pH.
Figure 5. Percent CO generated by HMTA-CB in different pH monitored by
CO meter. Four micromole of HMTA-CB was dissolved in H O with adjusted
2
pH before incubation in 37C. After 12 hours, amount of CO gas in the head
space was analysed by CO meter. The ppm readings were then converted
to µmole.
Toxicity and CO-releasing property of HMTA-CB in cell culture
Based on the above findings, HMTA-CB undoubtedly releases CO
at an appreciable rate. Nonetheless, in order to assess the
compatibility with cellular environments, cytotoxicity of HMTA-CB
was tested using murine macrophage cells (RAW264.7) and the
CellTiter-Blue viability assay. Using the concentration range of 0.5 –
1
0 mM of HMTA-CB, none of them shows adverse effects on cell
Figure 4. Determination of the rate and rate constant (a) Comparison of
percent CO released measured by CO meter (red) and H NMR (blue) for 24
1
viability as seen on Figure 6. In fact, all cells treated with HMTA-CB
show slightly higher growth than untreated cells. The cells treated
with 1 mM H O , included as a control, display 44% viability after 24
hours. During the CO release process, the amine group,
hexamethylenetetramine (HMTA) in this case, is released from
HMTA-CB molecule. In order to see if there is any toxicity from
HMTA itself, a few controls with varying concentration of HMTA
were included in the test. These controls show a certain extent of
hours. Four micromole of HMTA-CB in 12 mg/mL concentration was
incubated in 37 C and amount of CO released was measured at specified
times. CO in ppm was recorded for measurement by meter while the
2
2
1
amount of HMTA formed was monitored by H NMR, and calculated to
%
CO released. (b) Rate vs amount of HMTA-CB plotted to determine the
rate order and constant. Various amount of HMTA-CB were incubated at 37
C for 12 hours and amount of CO produced was detected by CO meter

which is then converted to rate in µmole of CO produced per second.
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toxicity by HMTA with most cells losing viability at 2 mM ability of HMTA-CB to prevent cytokines producVtiioewnArbticyle Odnolisnee
concentration. Although HMTA-CB produces HMTA in the process dependent manner. All cells were still healtDhOyI:w10h.i1c0h39c/oCn6fDirTm0s38t5h6aEt
of releasing CO, harmful effect of HMTA is not seen in any of the reduction of TNF-α concentration is resulted exclusively from
samples suggesting that the cells are protected from the cytotoxic HMTA-CB and not from lowered number of cells due to toxicity.
effect of HMTA. Many CORMs have been reported to be non-toxic
3
9
to eukaryotic cells up to 500 µM concentration. We find that
HMTA-CB, instead of showing toxicity, promotes cell growth in any
concentration tested up to 5 mM. Lowering of viability below 100%
in the sample with 10 mM HMTA-CB could be due to alteration of
pH in the media as high concentration of carboxyboranes affects
the buffering capacity of the media.
Figure 7. Anti-inflammatory and cell toxicity effect of HMTA-CB tested on
murine macrophage cells (RAW264.7). LPS induced TNF-α production was
tested first and cell viability assay was performed on the same cells. [TNF-
α] concentration was normalized to LPS treated cells as 100% for ELISA
assay and untreated cells was considered 100% for viability test.
Conclusions
Figure 6. Cytotoxicity of HMTA-CB tested on murine macrophage cells,
The products determined from the culminated results of NMR,
RAW264.7. Various amount of HMTA-CB was added to the cells and
incubated in 5% CO at 37 °C for 24 hours before adding CellTiter-Blue
2
reagent. Cells were incubated with the reagent for 4 additional hours and
fluorescence readings (560Ex/590Em) were recorded to assess the viability.
Data was normalized using untreated cells as 100% viable.
CO meter, IR and myoglobin assay are shown in Scheme 2. This
suggests
a
general decomposition route for the amine
carboxyboranes producing CO, boric acid, and an amine group
which in this case is HMTA. Since HMTA is stable in water at neutral
§§
pH, subsequent decomposition of this molecule was not observed.
The exact mechanism of HMTA-CB molecule decomposition
remains to be elucidated, but we postulate that water initiates the
breakdown process since this compound in solid form is stable in air
In order to observe the CO-releasing property of HMTA-CB, we
tested for the CO-mediated inhibition of TNF-α production in
murine macrophages. It has been reported that lipopolysaccharide
and at 37 C for weeks. We also observed H in the decomposition
2
(
LPS), a cytotoxin produced from E. coli, induces production of
products as expected for borane compounds. According to Scheme
inflammatory factors in mammalian cells. Furthermore, this effect
can be counteracted by administration of CO or overexpression of
2
, carboxyboranes are suitable for use as a prodrug to protect drug
molecules such as HMTA in this case and release the drug slowly in
the body as it breaks down into CO, the drug, and boric acid which
has very low toxicity towards living systems. Since the
carboxyborane moiety has high solubility in water, it can also
improve the solubility of drugs which is an important
pharmacological parameter.
40-42
HO-1.
Thus, we used murine macrophage cells (RAW264.7) to
test the anti-inflammatory effect of CO produced by HMTA-CB.
Varying concentrations of HMTA-CB were directly added to the cell
culture to determine whether it inhibits production of LPS-induced
TNF-α. After 24 hour incubation with HMTA-CB, LPS was added to
stimulate the cells. Culture media were collected after 4-hour
incubation with LPS. Immediately after collecting the media
containing secreted inflammatory cytokines, these cells were
subjected to a viability test. The ELISA assay performed on the
culture media to determine secreted TNF-α concentration reveals
the property of HMTA-CB to inhibit TNF-α production. Calculated
TNF-α concentration in pg/mL was normalized taking 100% for LPS
induced cells without the CO source. For cell viability test,
untreated cells are regarded as 100%. While the cells do not secrete
TNF-α without LPS, significant amount of TNF-α was detected in
LPS-induced cells. Decrease in TNF-α concentration was observed
for increasing amount of HMTA-CB used (Figure 6) proving the
Scheme 2. Proposed scheme of CO-releasing process by HMTA-CB auto-
decomposition.
Amine carboxyboranes have long been recognized as
antineoplastic agents as well as the suppressors of cholesterol
and triglyceride syntheses. However, these effects were
6
| J. Name., 2012, 00, 1-3
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Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Journal Name
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DOI: 10.1039/C6DT03856E
rather than the effect of CO.
This new finding of their
1
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property to deliver CO in the biocompatible conditions may
shine a new light on carboxyboranes since the previously
reported cytoprotective effects could also have been resulted
from CO mediated signaling. Our newly discovered CORM,
HMTA-CB, is stable in air and has high solubility in aqueous
solutions making it easy to manage and avoids the needs for
organic solvents during biological testing. In addition, its slow
CO release rate makes it suitable for pharmacological studies
11.
12.
13.
that require gradual release of CO. Moreover, it does not 14.
require any form of activation to release CO but auto-
decomposes slowly at physiological pH and temperature. Lack
1
5.
of toxicity towards living cells may provide its greatest
advantage as a potential pharmacological compound as a CO-
releasing molecule. Promotion of cell survival by the slow
acting HMTA-CB demonstrates the beneficial effect of the
presence of low CO level on cell health.
16.
1
1
1
2
7.
8.
9.
0.
Acknowledgements
The authors thank M. Zeller for the X-ray data collection and
the NSF for funding the diffractometer (DMR-1337296) at
Youngstown State University. We would also like to show our
gratitude to Michael Witty from Florida SouthWestern State
College and Perry Pellechia from University of South Carolina
for helpful discussions.
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‡
Tested stability of solid HMTA-CB is at room temperature for
0
three months and at 37 C for one week. HMTA-CB solubility in
o
§
23.
water is 81 mg/mL at 24 C. Calculations can be found in the
Electronic Supplemental Information. Our experiment showed
that HMTA was stable in D O at 37 C for at least 6 days. NMR
spectra of this study can be found in Electronic Supplemental
Information.
§
§
o
2
24.
25.
2
2
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DOI: 10.1039/C6DT03856E
TOC Figure and Text
HMTA-CB is the first amine carboxyborane that yields CO under physiological conditions suitable for
utilization as a slow CO-releaser.