Green-Light-Induced PhotoCORM: Lysozyme Binding Affinity towards MnI and ReI Carbonyl Complexes and Biological Activity Evaluation

Article abstract of DOI:10.1002/ejic.201801055

Reaction of N-(2-pyridylmethylene)benzene-1,4-diamine (L) with [MBr(CO)₅] (M = Mn^I and Re^I) affords complexes of the type [MBr(CO)₃L] [M = Mn^I (1) and Re^I (2)]. Complex 1 releases CO upon i

Full text of DOI:10.1002/ejic.201801055

DOI: 10.1002/ejic.201801055
Full Paper
CO-Releasing Molecules
Green-Light-Induced PhotoCORM: Lysozyme Binding Affinity
towards MnI and ReI Carbonyl Complexes and Biological
Activity Evaluation
Ahmed M. Mansour*[a]
Abstract: Reaction of N-(2-pyridylmethylene)benzene-1,4-di- (CO)3L]) via the free catalyst [3+2] cycloaddition coupling. Com-
amine (L) with [MBr(CO)5] (M = MnI and ReI) affords complexes
of the type [MBr(CO)3L] [M = MnI (1) and ReI (2)]. Complex 1
releases CO upon illumination with light in the range of 525–
468 nm. No photo induced CO release is detected from 2. Reac-
tive azide complex 3, obtained by bromide's ligand exchange,
reacts with the electron-poor alkyne dimethyl acetylene dicarb-
oxylate giving triazolato complex 4 ([Mn(triazolateCOOMe,COOMe)-
plex 1 exhibits interesting antifungal activity (MIC = 30 nM)
against Candida albicans and Cryptococcus neoformans as well
as cytotoxic activity of 12.56 μg/mL against noncancerous
human embryonic kidney cells. Reactivity of the complexes to-
wards hen egg white lysozyme is studied by electrospray ioni-
zation mass spectrometry.
Introduction
induction are the biomedical prerequisites of photoCORMs for
the phototherapeutic CO applications. To decrease the detri-
mental problems of UV light and to increase the penetration
depth of the light into tissues, synthesis of visible-light trig-
gered CORMs is one of the major challenges in this research
area. For the class of metal carbonyl complexes, change of the
metal ion (rhenium, manganese, iron, ruthenium, molybdenum,
tungsten),[7–12] and the axial ligand as well as extension of the
ancillary ligand conjugation system led to modulation of the
M–CO that influence in turn the electron density and conse-
quently the energy of the incident light. Manganese(I) and ruth-
enium(II) carbonyl complexes have been widely studied in the
context of CORMs because of their rich photochemical proper-
ties.[7–12,14,15] By extending the conjugation system of the ancil-
lary ligand, Mascharak and his co-workers succeed in shift the
MLCT band of MnI and RuII photoCORMs into the visible region
and thus visible-light LEDs were used as excitation source.[16]
Carbon monoxide has a high affinity towards low oxidation-
state metal ions due to back donation from the metal to the π*
orbitals of CO. Mascharak's and Schatzschneider's groups sup-
posed that the MLCT transitions, close to the excitation wave-
length, reduce the electron density in the excited state and thus
decrease the affinity of CO to the metal center that results in
CO photo release.[17,18] However, the shift of MLCT band deep
into the visible-region may be paralleled with their instabilities.
For example, MnI CORMs bearing α,α′-diamine ligands with ex-
tended conjugated framework exhibited high sensitivity toward
low power visible-light (0.3–10 mW; λ ≥ 520 nm) even in the
solid-state.[19] The CO releasing properties of 2-phenyl azo-
pyridine MnI photoCORMs were investigated by Zobi et al.[20]
upon the illumination with red-light (λ ≥ 625 nm). While
low energy source-light was used, these photoCORMs release
0.1–0.7 mol of CO in the dark. In the contrast, dark stable
green-light (λ = 525 nm) induced MnI photoCORMs bearing ei-
Carbon monoxide, the silent killer, exhibits anti-inflammatory,
anti-proliferative and signaling properties[1] in the concentra-
tion range of 10–250 ppm.[2] Initially, an inhalation system (e.g.
Covox DS) was used to deliver quantitative amounts of CO. The
lack of target specificity of CO, administrated by the inhalation
system, requires the patient to be exposed to elevated levels of
CO to build up a realistic concentration in the target issue to
attain a desired biological effect. Carbon monoxide releasing
molecules (CORMs) were introduced as an alternative way to the
inhalation system to carry and liberate controlled quantities of
CO within the cellular systems. Organic (e.g. α,α-dialkylalde-
hydes,[3] and oxalates[4]) and organometallic (borano carboxyl-
ates,[5] sila-carboxylates,[6] and metal carbonyl complexes[7–12]
)
compounds have been investigated as CORMs. Different trig-
gering methods have been used to initiate the CO release. This
includes change of pH value,[7] and redox state,[8] thermal,[9]
enzymatic,[10] and photochemical ways.[11] At first, the ability
of [Mn2(CO)10] (CORM-1) and Fe(CO)5 to release CO upon the
exposure to a cold light was investigated by Motterlini et al.[12]
In 2008, a group of fac-Mn(CO)3 complexes bearing tris(pyraz-
olyl)methane derivatives was examined by Schatzschneider
group as photoinduced CO molecules.[11] Two years later, Ford
and co-workers gave the name “PhotoCORM” to the class of
compounds that is capable of release CO upon illumination.[13]
Solubility in water (or DMSO/water mixture), long-stability in
the dark, generation of nontoxic metabolites and visible-light
[a] Chemistry Department, Cairo University,
Faculty of Science, Gamma Street, Giza, Cairo 12613, Egypt.
E-mail: mansour@sci.cu.edu.eg
http://scholar.cu.edu.eg/?q=amansour/
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejic.201801055.
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ther N-(2-thiazolyl)-1H-benzotriazole-1-carbothioamide (CORM- nity of iClick (Inorganic Click) reaction for bioconjugation pur-
NS1)[14] or the anti-anxiety drug bromazepam[21] have been re- poses, reactive azide complex 3 is synthesized. Coupling of 3
cently published. The photoinduced process of CORM-NS1 was with a model alkyne gives triazolato complex 4.[30] DFT and
studied by commercial gas sensor at the excitation wavelength TDDFT calculations are carried out to get some information
of 525 nm. About one CO equivalent was released within about the origins of the observed electronic transitions. The
120 min. Therefore, finding of PhotoCORMs capable of release compounds are screened for their antimicrobial and cytotoxic-
CO by energy in the green region is recommended from the ity activity against the noncancerous human embryonic kidney
point of view.
cells (HEK293). As the drug delivery and pharmacological profile
of a biologically active compound are changed by some interac-
tions with the surface-accessible histidyl of proteins, it is essen-
tial to investigate the interactions between the complexes stud-
ied here, and the model enzyme hen egg white lysozyme
(HEWL). Finally, the question that should be considered and
answered in the field of CORMs, are PhotoCORMs stable in pres-
ence of biomolecules or not?
Few examples of ReI carbonyl complexes were found to be
CO releasers though they displayed exciting photophysical
properties and diverse applications such as luminescent biolog-
ical molecular probes and organic light-emitting diodes.[22] By
replacement the halide (X) from the coordination sphere of fac-
[Re(N-N)X(CO)3] with tris(hydroxymethyl)phosphine, a nontoxic
water-soluble complex capable of release CO upon illumination
at 405 nm was obtained.[23] The π-acid ligand labilizes the axial
CO ligand.[24] Some [ReBr(CO)3L] of 1,10-phenanthroline, capa-
ble of release CO upon the exposure to UV light, have been
published with Ford[25] and Mascharak.[26]
Results and Discussion
Synthesis and Characterization
Kinetics studies concerning the nature of the CO release
process and the type of the intermediates that might be formed
during the illumination of MnI tricarbonyl complexes have been
studied.[27–29] Solution IR and EPR spectroscopy as well as quan-
tum chemical calculations revealed the formation of Mn(CO)2
species upon illumination, followed by facile oxidation to
higher oxidation states in further dark processes.[27,28] Berends
and Kurz[28] as well as Schatzschneider[27] found that only one
CO molecule is photolytically released from MnI tricarbonyl
complexes, while the rest needs an additional dark process.
Femtosecond transient absorption UV pump/mid-IR probe
spectroscopic studies showed that one CO molecule was photo-
chemically liberated on very short timescales, but a fraction of
the molecules excited was shown to undergo geminate recom-
bination.[29]
The bidentate Schiff-base ligand (L) was synthesized by reaction
of 2-pyridine carboxaldehyde with one equivalent of 1,4-di-
amino-benzene in ethanol (Scheme 1).[31] NMR spectra and IR
chart of L are given in the supporting information (Figures S1
and S2). In the dark, reaction of L with [MBr(CO)5] (M = MnI
and ReI) gave fac-[MBr(CO)3L] [M = MnI (1) and ReI (2)]. The
characterization of the complexes was carried out by elemental
analysis, IR, ESI-MS and NMR tools (Figures S2–4). The ESI-MS
spectra of 1 and 2, in the positive mode, show unique peak at
m/z = 336.0165 and 468.0342 assigned to [M – Br]+, respec-
tively. The AT IR spectrum (Figure S2) of 1 shows symmetrical
and anti-symmetrical stretching modes of CO ligands at 2021,
1926 and 1901 cm–1. These vibrations are observed at 2016,
1902 and 1869 cm–1 in the spectrum of 2. Three 13CO signals
Here, the photoinduced CO releasing properties of com- are detected at δ = (222.9, 221.8, 220.2 ppm) and (197.2, 196.7,
plexes 1 and 2 (Scheme 1) are reported. To inspect the opportu- 187.4 ppm) in the NMR spectra of 1 and 2, respectively. The 1H
Scheme 1. Synthesis of N-(2-pyridylmethylene)benzene-1,4-diamine (L) and complexes 1–4.
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NMR spectrum of the free ligand shows two characteristic sig-
nals at δ = 8.64 (doublet) and 8.58 ppm (singlet) assigned to
pyridine-H6 and -CH=N- protons. These signals move downfield
to δ = (9.17, 8.74 ppm) and (9.02, 9.12 ppm) upon the complex
formation of 1 and 2, in that order. Therefore, the Schiff-base L
behaves as N,N-bidentate ligand towards MnI and ReI ions.
Abtraction of Br– from the coordination sphere of 1 and
treatment of the filtrate with excess sodium azide led to synthe-
sis of 3 (Scheme 1). The AT IR spectrum of 3 shows the charac-
teristic ν(N3) band at 2048 cm–1 (Figure S2). Three ν(CO) bands
are observed at 2012, 1930 and 1908 cm–1 in the IR spectrum
of 3. In comparison with 1, the symmetric stretching CO mode
moves to lower wavenumber upon introduction of azido ligand.
The positive mode mass spectrum of 3 displays two typical
peaks at m/z = 336.0163 {[M] – N3}+ and 714.0441 {2[M] – N3}+.
1
Like 1, the H NMR signals of pyridine-H6 and azomethine in 3
move downfield to δ = 9.10 and 8.85 ppm (Figure S5) compared
with the free ligand, which suggests the persistence of the
N,N-bidentate mode upon the complexation of azido ligand.
Stirring compound 3 with the electron-poor alkyne dimethyl
acetylene dicarboxylate for few days under the exclusion of
light, affords triazolato complex 4 via the free catalyzed [3+2]
cycloaddition reaction (Scheme 1). It was difficult to assign the
NMR signals and the type of the isomer formed [N(I)- or N(2)-
triazolate bonded][30] because of quadrupolar nuclear and long
relaxation time of 4. Suggestion of N(2) bound mode is based
on the previously reported data for the structurally related
mononuclear MnI complexes.[30] However, AT IR is a perfect
spectral tool to follow the cycloaddition coupling via the van-
ishing of ν(N3) mode and grown of ν(C=O) vibration. The AT IR
spectrum of 4 shows a strong ester ν(C=O) band at 1729 cm–1.
The νs(C≡ O) mode moves further to higher wavenumber
2035 cm–1 (4). In the positive mode, the ESI-MS spectrum of 4
exhibits unique peak at m/z = 521.0610 assigned to {[M]+H}+.
These spectral evidences reveal possibility of bioconjugation of
1 via iClick reaction.
Figure 1. UV/Vis spectral changes of 1 (in CH3SOCH3) upon photolysis at a)
525 nm for 0–30 and b) at 468 nm for 0–120 s after pre-incubation in the
dark for 16 h.
Photoactivatable CO-Release Properties
The electronic absorption spectrum (Figure 1) of 1, in DMSO,
displays a broad band at 437 nm with a tail extended to
570 nm. The assignment of the latter band has been done with
the aid of time-dependent density functional theory calcula-
tions using CAM-B3LYP/LANL2DZ method.[32] Based on the op-
timized geometries (Figure S6 & Table S1–3), the first 30 singlet
excited-states have been calculated (Table S4). The solvent ef-
fect (DMSO) has been introduced using the default continuum
model implemented in Gaussian03.[33] The calculated spectrum
of 1 is characterized by a lowest energy transition at 408 nm
corresponding to HOMO-1→LUMO/LUMO+2 transitions. As
shown in Figure 2, HOMO-1, which is 0.03 eV lower than HOMO,
results from d(Mn)/π(Br)/π(py) orbitals.
Figure 2. Selected frontier molecular orbitals and MLCT values of the ground-
state optimized complexes calculated by PCM(DMSO)/CAM-B3LYP/LANL2DZ.
1 was incubated for 16 h in the dark. A slightly change (4 %)
was monitored. This might be attributed to partial exchange of
the axial Br– with the coordinating solvent molecules. The pre-
The two LUMOs (LUMO and LUMO+2) are lying close to incubated solution was directly illuminated by 525 nm LED
HOMO with an energy gap of 2.67 eV. LUMO is mainly con- (λpeak = 515 nm, Δλ = 30 nm, 6.69 × 10–11 Einstein s–1).[34] An
tained upon π* of the phenyl ring, while LUMO+2 is a mixture isosbestic point is observed at 418 nm (Figure 1a) during the
of d(Mn)/π*(Br)/π*(CO). Hence, the lowest energy transition is a photolysis of 1 for 30 min. Switching from 525 nm excitation
combination of MLCT/d-d/π–π*. The aerated DMSO solution of wavelength to 468 nm (Figure 1b) gave rises to faster photo-
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process and similar photolysis side-view. The plateau is reached that is quantitatively estimated. About one and three CO
within two minutes. While myoglobin assay[32,35] suffers from equivalents are released from 1 upon the illumination at 525
some weakness points such as instability of the reduced species and 468 nm in that order. The values of the rate constant
for a long-time, interference from the highly colored metal and t1/2 are [(0.59 0.03) × 10–2 s–1, 2.01 0.21 min] and
carbonyls as well as CO release dependent on the quantity of [(0.40 0.03) × 10–3 s–1, 27.85 1.60 min] for 468 and 525 nm
the dithionite used as a reducing agent,[36] it is still a simple excitation wavelengths, respectively.
spectrophotometrically assay used to explore the photo-proc-
ess happened during illumination of metal carbonyls. To a buff-
ered standard reduced solution of myoglobin solution, 10 μL of
The calculated spectrum (Figure S7) of 2 shows three
transitions at 420, 289 and 242 nm corresponding to HOMO/
HOMO-1→, HOMO-4→ and HOMO-8→LUMO transitions, re-
spectively. The lowest energy transition at 420 nm (Figure 2)
has a ground state composed of d(Mn)/π(Br)/π(py), whereas the
excited state is of phenyl π* orbitals forming MLCT/d-d/π–π*.
No spectral change is observed in the electronic spectrum of 2
upon the exposure to light source in the range of 525–468 nm.
In addition, no photo induced CO release was detected under
the reduced conditions of the myoglobin assay and therefore,
complex 2 was not further examined for CO phototherapeutic
applications.
1 (10 μ
M
) was added. The myoglobin solution of 1 was illumi-
nated at excitation wavelengths, 525 and 468 nm (λpeak
=
455 nm, Δλ = 25 nm, 1.25 × 10–9 Einstein s–1). When kept in
the dark under the reduced condition of myoglobin assay, com-
plex 1 induced no spectral changes in the Q-band region (Fig-
ure S8). In both experiments (Figure 3), while two new bands
are grown at 540 and 577 nm during the photolysis, the inten-
sity of the band at 557 nm is decreased.
As shown in Figure 2, functionalization of 1 via the iClick
reaction results in blue-shift of the lowest energy transition
from 408 to 378 nm (complex 3). The HOMO→LUMO transition,
characterizes the lowest energy transition at 378 nm, is MLCT
towards the pyridine ring. Compared with the σ-donating Br–,
the HOMO–LUMO gap is increased by 0.2 eV upon iClick forma-
tion and consequently the MLCT band is blue-shifted.
Interaction with Protein
Interaction of some drugs with the cell components may give
the gate to cross the cell membrane. However, lack of knowl-
edge on how the drugs species communicate with the cell
components is the main challenge. Furthermore, it is well-
known that subsequent the administration of the biologically
active compounds, there is a highly prospect for interaction
with surface proteins containing histidyl side-chain. Such inter-
actions influence the drug delivery and pharmacological profile
of the biologically active compound. It is renowned that HEWL
could be used as biocompatible carrier of CORMs to deliver CO
into the living cells.[37] Metal carbonyl complexes was accom-
modated by HEWL at the selectively active surface His15 coordi-
nation site.[38] These arguments inspire the author to investi-
gate the reactivity of the studied complexes (1, 2) with a model
protein, HEWL. Affinity of HEWL (14303.8500 Da) towards 1 and
2 was studied at room temperature by positive mode electro-
spray ionization mass spectrometry. The photo triggered com-
plex 1 was investigated in the dark and upon the exposure to
the blue and green lights. In the dark, complex 1 interacts with
HEWL giving a weak peak at m/z = 1596.4309 Da (z = 9, Mnn+).
This fragment arises from the metalation of HEWL by manga-
nese ions. The intensity of the latter fragment increases upon
illumination at 468 nm for two minutes (Figure S9). Another
adduct peak [MnBrn+ and/or Mn(CH3SOCH3)n+] is also observed
at m/z = 1611.9668. Similar spectrum was recorded upon expo-
Figure 3. Relation between the concentration of MbCO [μ
upon the exposure of the myoglobin solution at a) 468 nm and b) 525 nm.
UV/Vis spectral changes in the Q-band region of myoglobin (60 μ in 0.1
PBS at pH 7.4) with sodium dithionite (10 m ) and complex 1 (10 μ ) under
a dinitrogen atmosphere upon photolysis at a) 468 nm and b) 525 nm.
M] and time (min.)
M
M
M
Pronounced changes are observed in the Q-band upon sure of HEWL/complex 1 to 525 nm LED. The intensities of the
the exposure to high energetic blue-light 468 nm LED (Fig- adduct peaks are lower than those recorded at the high energy
ure 3a) compared with green-light induced experiment excitation wavelength 468 nm. The obtained data compares
(Figure 3b). This is reflected in the number of CO equivalents with that obtained with CORM-NS1.[14] The powerful coordinat-
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ing ability of the selectively active His15 coordination site pre- releaser, two and 30 minutes were required for manganese(I)
vents HEWL to act as a biocompatible carrier for this class of complex to attain the plateau of the photoprocess upon the
photoCORMs. In other words, decomposition of MnI CORMs oc- illumination at 468 and 525 nm, respectively. Assignment of the
curs at the surface of HEWL as evidenced from the MnI adduct electronic transitions was done with the aid of time-dependent
peak. Two adducts peaks containing one (m/z = 1651.4297) and density functional theory calculations. To explore the possibility
two molecules of 2 (m/z = 1711.8691) are observed (Figure S9) of bioconjugation of MnI PhotoCORM, the well-known “iClick”
from reaction of HEWL with complex 2. This reveals non- reaction was applied via the synthesis of a reactive azido com-
covalent binding of 2 to protein via hydrogen-bonds and/or plex and then coupling with a model electron-poor alkyne. In
coulombic interactions.[39] However, in the absence of the crys- comparison, iClick reaction resulted in 30 nm blue-shift of the
tal structures, it is difficult to assign precisely the location of lowest energy transition. Subsequent the administration of
complex-HEWL binding.
CORM, there is a highly probability to bind to surface-accessible
histidyl of proteins. The question of stability of this class of
PhotoCORMs in presence of biomolecules was touched by ESI-
MS measurements. For this purpose, the model protein, hen
egg white lysozyme (HEWL) was used. Because of the powerful
coordinating ability of the surface selectively active His15 side-
chain, decomposition of MnI PhotoCORM occurs at the surface
of HEWL. In the contrast, the inactive CO releaser rhenium(I)
complex binds noncovalently to HEWL as previously reported
by other coordination compounds. MnI PhotoCORM exhibited
interesting antifungal activity with MIC value of 16 μg/mL
(equivalent to 30 nM) against C. albicans and C. neoformans as
well as cytotoxicity (MIC = 12.56 μg/mL) against noncancerous
human embryonic kidney cells (HEK293). Opposite action is no-
ticed upon complexation of the ligand with ReI and exchange
of the axial bromide in the coordination sphere with the tri-
azolate ring. Therefore, greatest potential, for further investiga-
tions is recommended to discuss the results in terms of stability
of the compounds in presence of biomolecules.
Antimicrobial Activity
The antimicrobial activity of Schiff-base ligand and complexes
(1, 2, 4) was evaluated in the dark using two fungi (C. albicans
and C. neoformans), gram-positive bacterium (S. aureus) as well
as four gram-negative bacteria (P. aeruginosa, K. pneumoniae,
E. coli, and A. baumannii). Standard broth microdilution as-
says[40] were used as endorsed by NCCLS for bacteria and yeasts
(M07-A8 and M27-A2) and standards of European Committee
on Antimicrobial Susceptibility Testing (EDef7.1). Initial screen-
ing was carried out at 32 μg/mL. The MIC value was determined
as the lowest concentration at which the growth was fully in-
hibited, defined by an inhibition = 80 % for C. albicans and
70 % for C. neoformans. The ligand and complexes exhibit no
or partially toxicity against the tested bacteria and fungi, except
complex 1, which shows interesting antifungal activity against
both fungi with MIC value of 16 μg/mL (equivalent to 30 nM).
Opposite action is noticed upon complexation of the ligand
with REI ion and exchange of the axial bromide with the tri-
azolate ring. Thus, the toxicity cannot be simply correlated to
the type of the axial ligand (Br or triazolate) and metal ion,
other variables such as size of the receptor sites, diffusion, and
combined effect of metal and ligands should be considered.
Experimental Section
Materials and Instruments: All manipulations were carried out in
argon atmosphere using standard Schlenk glassware's. The solvents
were degassed and purified using standard methods. The chemicals
were obtained from commercial sources and used as received.
Bromo penta-carbonyl manganese(I) and bromo pentacarbonyl
rhenium(I) were purchased from Sterm. Schiff-base ligand was syn-
thesized following the published procedure.[31] 1H and 13C NMR
spectra were recorded with Bruker-Avance 500 [1H, 500.13 MHz and
13C(1H), 125.77 MHz] and Bruker-Avance 400 [1H, 400.40 MHz;
13C(1H), 100.70 MHz] spectrometers. Assignments were done with
Cell Viability Assay
The cytotoxic activity of 1 was screened against noncancerous
human embryonic kidney cells (HEK293). Complex 1 exhibits cy-
totoxicity with MIC value of 12.56 μg/mL. Interaction of the
toxic compound with the blood components, RBCs, is vital to
examine the blood compatibility of the green-light triggered
PhotoCORM 1. The concentration at 10 % and 50 % haemolysis
(HC10 and HC50, respectively) was calculated by curve fitting
the inhibition values vs. log (concentration). The HC10 and HC50
values of 1 are found to be more than 32 μg/mL. Complex 1
shows good blood compatibility. Thus, greatest potential, for
further investigations by the biology groups, interested in
CORMs, is recommended to discuss the results in terms of sta-
bility of the compounds in presence of biomolecules.
1
the aid of {1H, H} COS90 and {1H, 13C} HSQC. UV/Vis. spectra were
recorded on an Agilent 8453 diode array spectrophotometer. IR
spectra were recorded in the solid state on a Nicolet 380 FT-IR spec-
trometer equipped with a smart iFTR accessory. Electrospray mass
spectra were run with a Thermo-Fisher Exactive Plus instrument
with an Orbitrap mass analyzer at a resolution of R = 70.000 and a
solvent flow rate of 5 μL min. Elemental micro-analysis was per-
formed with a Vario Micro Cube analyzer of Elementar Analysen-
systeme or an EA 3000 elemental analyzer from HEKtech.
Synthesis
Synthesis of Schiff-Base Ligand (L): The ligand (L) was synthesized
according to the literature method.[31] 2-pyridine carboxaldehyde
(3.21 g, 30 mmol) was added to the ethanolic solution of 1,4-
diaminobenzene (3.24 g, 30 mmol) and the reaction mixture was
stirred at room temperature for 90 min, where a yellow precipitate
was formed. The product was collected by filtration, washed several
Conclusions
The green-light triggered CO releasing properties of fac-MnI tri-
carbonyl complex functionalized with N,N-bidentate Schiff-base
ligand was reported. While the ReI analogue was inactive CO times with ethanol and then diethyl ether. Recrystallization from
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benzene gave golden-yellow crystals. IR (ATR, diamond): ν = 3387
small amount of silver azide was carefully filtered off. Solvent was
reduced under vacuum and the collected red precipitate was
washed several times with water and then hexane and was left
˜
(m, NH2), 3311 (m, NH2), 3201 (m, CH), 1625 (m, CC/CN), 1570, 1504,
1
1467, 1297, 1166, 823, 769 cm–1. H NMR ([D6]DMSO, 400.40 MHz):
δ = 8.64 [d, 3J(H,H) = 4.9 Hz, 1 H, py-H6], 8.58 (s, 1 H, CH=N), 8.08 (d, for drying under vacuum. Yield: 67 % (93 mg, 0.24 mmol). IR (ATR,
3
4
3JH,H = 7.9 Hz, 1 H, py-H3), 7.87 (td, JH,H = 7.5, JH,H = 1.2 Hz, 1 H,
diamond): ν = 3446 (m, NH2), 2048 (vs, N3), 2012 (vs, C≡ O), 1930
˜
3
3
py-H4), 7.41 (t, JH,H = 6.1 Hz, 1 H, py-H5), 7.22 (d, JH,H = 8.63 Hz,
(vs, C≡ O), 1908 (vs, C≡ O), 1624 (m, CC/CN), 1599, 1508, 1480, 1258,
3
2 H, ph-H2/H6), 6.61 (d, JH,H = 8.5 Hz, 2 H, ph-H3/H5), 5.38 (s, 2 H,
1169, 1030, 833 cm–1 1H NMR ([D6]DMSO, 400.40 MHz): δ = 9.10
.
NH2) ppm. 13C NMR ([D6]DMSO, 100.10 MHz): δ = 155.0 (CH=N),
(m, 1 H, py-H6), 8.85 (s, 1 H, CH=N), 8.23 (m, 2 H, py-H3/H4), 7.80
153.7 (py-C2), 149.4 (py-C6), 148.8 (H2N-C), 138.2 (py-C4), 136.7 (m, 1 H, py-H5), 7.29 (m, 2 H, ph-H2/H6), 6.71 (m, 2 H, ph-H3/H5),
(ph-C1), 124.5 (py-C5), 123.0 (ph-C2/C6), 120.3 (py-C3), 114.0
(ph-C3/C5) ppm.
5.67 (s, 2 H, NH2) ppm. 13C NMR ([D6]DMSO, 100.10 MHz): δ = 163.0
(CH=N), 155.2 (py-C2), 153.0 (py-C6), 149.9 (H2N-C), 140.6 (ph-C1),
139.4 (py-C3), 128.2 (py-C4), 127.8 (py-C5), 123.0 (ph-C2/C6), 113.3
(ph-C3/C5) ppm. ESI-MS (positive, acetone): m/z = 336.0163 {[M] –
N3+}, 714.0441 {2[M] – N3}+. C15H11MnN6O3·2H2O (414.03): C 43.49,
H 3.65, N 20.29; found C 43.80, H 3.74, N 18.63.
Synthesis of Complexes
1: In the dark, the ligand L (1.25 mmol, 246 mg) and bromo penta-
carbonyl manganese(I) (1.35 mmol, 371 mg) were dissolved under
argon in degassed anhydrous acetone (40 mL). The reaction mixture
was heated to reflux for 6 h. The volume of red-colored solution
was reduced under vacuum to 5 mL. Red-colored precipitate was
obtained upon addition of 50 mL of diethyl ether. The precipitate
was collected, washed with diethyl ether, and was dried in vacuo.
4: Dimethyl acetylenedicarboxylate (0.40 mmol, 57 mg) was added
to the acetone solution of 3 (0.13 mmol, 50 mg) and the reaction
mixture was stirred at room temperature for 3 d under exclusion of
light. The volume of the solvent was reduced under pressure and
then diethyl ether was added, whereupon red precipitate was
slowly formed. Filtration and washing with diethyl ether were done.
It was difficult to assign the NMR signals because of quadrupolar
nuclear and long relaxation time even with high-field instruments.
Yield: 87 % (457 mg, 1.09 mmol). IR (ATR, diamond): ν = 3334 (w,
˜
NH2), 3211 (w, NH2), 3073 (w, CH), 2021 (vs, C≡ O), 1926 (vs, C≡ O),
1901 (vs, C≡ O), 1623 (w, CN/CC), 1598 (m, CC/NH2), 1296, 1170, 832,
767 cm–1 1H NMR ([D6]DMSO, 500.13 MHz): δ = 9.17 (m, 1 H,
.
Yield: 68 % (47 mg, 0.09 mmol). IR (ATR, diamond): ν = 2955 (w,
˜
py-H6), 8.74 (s, 1 H, CH=N), 8.18 (m, 2 H, py-H3/H4), 7.72 (m, 1 H,
py-H5), 7.34 (m, 2 H, ph-H2/H6), 6.67 (m, 2 H, ph-H3/H5), 5.62 (s, 2
H, NH2) ppm. 13C NMR ([D6]DMSO, 125.75 MHz): δ = 222.9 (C≡ O),
221.8 (C≡ O), 220.2 (C≡ O), 164.1 (CH=N), 156.0 (py-C2), 153.9 (py-C6),
150.4 (H2N-C), 141.7 (ph-C1), 139.6 (py-C3), 128.9 (py-C4), 127.9 (py-
C5), 123.7 (ph-C2/C6), 113.8 (ph-C3/C5) ppm. ESI-MS (positive, acet-
one): m/z = 336.0165 {[M] – Br}+, 284.0219 {[M]-2CO – Br}+.
CH), 2035 (vs, C≡ O), 1931 (vs, C≡ O), 1729 (s, C=O), 1601 (m, CN/CC),
1508, 1439, 1225, 1162, 1091, 1030 cm–1. MS (positive, acetone):
m/z = 521.0610 {[M]+H}+. C21H17MnN6O7·2H2O (556.04): C 45.33, H
3.80, N 15.11; found C 45.28, H 3.75, N 14.43.
Density Functional Theory Calculations: Geometry optimization
of the complexes was performed by Becke 3-parameter (exchange)
Lee–Yang–Parr functional[41] and the effective core potential (ECP)
of the Hady and Wadt, LANL2DZ basis set. The absence of the imag-
inary vibrational modes characterized the obtained geometry as a
local minimum. Time-dependent DFT calculations were carried out
in the singlet state using hybrid exchange-correlation functional
CAM-B3LYP, with a long-range correction term, and LANL2DZ basis
set as well as the default continuum model (PCM) to introduce the
solvent effect. All the calculations were carried out using Gaus-
sian03 program package.[33]
C15H11BrMnN3O3 (416.11): calcd. C 43.30, H 2.66, N 10.10; found C
42.85, H 3.19, N 9.76.
2: A mixture of L (0.50 mmol, 100 mg) and [ReBr(CO)5] (0.55 mmol,
223 mg) was dissolved under argon in Schlenk flask containing
20 mL anhydrous degassed methanol. The mixture was heated to
reflux overnight, where a red-blood precipitate was formed. The
precipitate was collected, washed with methanol, diethyl ether, and
dried in vacuo. Yield: 65 % (183 mg, 0.33 mmol). IR (ATR, diamond):
ν = 3456 (m, NH2), 3352 (m, NH2), 2016 (vs, C≡ O), 1902 (vs, C≡ O),
˜
Myoglobin Assay: The number of CO equivalents released upon
illumination were determined using the standard myoglobin as-
say.[35] Illumination process was performed with custom-built LED
light sources, 468 nm (King-bright Elec. Co., 5000 mcd, part. no.
BL0106–15–299), and 525 nm (King-bright Elec. Co., 6500 mcd, part.
no. 34ZGC). Actinometry assay[34] was carried out to determine the
photo flow of the light source used. The cuvette was also positioned
perpendicular to the LED source light at a distance of 3 cm, where
the illumination was interrupted in regular intervals to take UV/Vis
spectra on an Agilent 8453 diode array spectrophotometer until no
more spectral changes were observed.
1869 (vs, C≡ O), 1622 (m, CC/CN), 1596, 1537, 1472, 1294, 1244, 1168,
770 cm–1 1H NMR ([D6]DMSO, 400.40 MHz): δ = 9.12 (s, 1 H, CH=
.
N), 9.02 (d, 3JH,H = 5.4 Hz, 1 H, py-H6), 8.27 (m, 2 H, py-H3/H4), 7.74
3
(m, 1 H, py-H5), 7.36 (d, JH,H = 8.7 Hz, 2 H, ph-H2/H6), 6.66 (d,
3JH,H = 8.7 Hz, 2 H, ph-H3/H5), 5.74 (s, 2 H, NH2) ppm. 13C NMR
([D6]DMSO, 100.10 MHz): δ = 197.2 (C≡ O), 196.7 (C≡ O), 187.4 (C≡ O),
163.9 (CH=N), 155.7 (py-C2), 152.8 (py-C6), 150.5 (H2N-C), 140.1 (ph-
C1), 139.5 (py-C3), 129.2 (py-C4), 128.5 (py-C5), 123.9 (ph-C2/C6),
113.3 (ph-C3/C5) ppm. ESI-MS (positive, acetone): m/z = 526.0762
{[M] – Br – CH3COCH3}+, 468.0342 {[M] – Br}+. C15H11BrN3O3Re·H2O
(565.04): C 31.86, H 2.32, N 7.43; found C 32.26, H 2.42, N 7.48.
Interaction with HEWL: The hen egg white lysozyme binding affin-
ity towards complexes (1,2) was investigated by orbitrap high reso-
lution mass spectro-meter (ThermoFisher Exactive plus orbitrap)
equipped with conventional ESI-source. The reaction mixture was
incubated for few minutes or 24 h at room temperature prior to
the measurements. ESI-MS spectra were measured by direct intro-
duction of the sample at a flow rate of 10 μL min–1. The working
conditions were as follows: spray voltage 3.80 KV, capillary voltage
45 V, and capillary temperature 320 °C. For acquisition, Thermo Xa-
clibur qual was used.
Caution: Organometallic compounds bearing azide group may be
exposed to unexpected violent decomposition. Scratching of com-
plexes contaminating with traces of sodium and/or silver azide
leads to explosive reactions. Therefore, handling and purification
with great care is so critical.
3: Silver trifluormethane sulfonate (0.50 mmol, 128 mg), dissolved
in a few drops of water, was added to acetone solution (20 mL) of
1 (0.36 mmol, 150 mg). The reaction mixture was stirred at room
temperature for 3 h, while the flask was protected from the light.
Silver bromide was filtered off. Sodium azide (1.00 mmol, 65 mg)
was added to the filtrate and stirring was continued for 15 h. A
Biological Activity Testing: The specifications of the biological ac-
tivity assays [evaluation of antimicrobial properties, cytotoxicity
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Acknowledgments
Ahmed Mansour thanks the Alexander von Humboldt Founda-
tion for Georg Forster postdoctoral fellowship. Antimicrobial
screening was performed by CO-ADD (The Community for Anti-
microbial Drug Discovery), funded by the Wellcome Trust (UK)
and The University of Queensland (Australia). Prof. Dr. Ulrich
Schatzschneider, Julius-Maximilians-Universität Würzburg, Ger-
many is acknowledged for giving the author some of the ana-
lytical facilities of the institution. Thanks also Dr. Ola Shehab,
Cairo University Faculty of Science, Chemistry Department for
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Received: September 4, 2018
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Full Paper
CO-Releasing Molecules
A. M. Mansour* ................................... 1–8
Green-Light-Induced PhotoCORM:
Lysozyme Binding Affinity towards
MnI and ReI Carbonyl Complexes
and Biological Activity Evaluation
Are manganese(I) photoCORMs stable studied green-light induced Photo-
in presence of biomolecules? Hen CORM were evaluated and compared
white egg lysozyme has been used as with the inactive CO-releasing ReI ana-
a model in this study. The antimicro- logue.
bial activity and cytotoxicity of the
DOI: 10.1002/ejic.201801055
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