Spectroscopic characterization of dipicolinic acid and its photoproducts as thymine photosensitizers

Article abstract of DOI:10.1016/j.saa.2020.118898

Dipicolinic acid (DPA), present in large amount in bacterial spores, has been proposed to act as an endogenous photosensitizer in spore photoproduct formation. The proposed mechanism involves a triplet-triplet energy transfer from DPA to thymine. However, up to now, no spectroscopic studies have been performed to determine the interaction between the endogenous compound and the nucleobase, probably due to its photolability in aqueous solutions. Here, triplet excited state properties of DPA are reported together with its bimolecular quenching rate constant by thymidine, k_q of ca. 5.3 × 10⁹ M^?1 s^?1. To run more reliable studies, a stable methyl ester derivative of DPA, which exhibits the same spectroscopic properties as the parent compound, is also described. Finally, DPA photoproducts are characterized. Studies of their triplet excited state properties have demonstrated that, interestingly, one of them is able to photosensitize thymidine triplet excited state formation.

Full text of DOI:10.1016/j.saa.2020.118898

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118898
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectroscopic characterization of dipicolinic acid and its photoproducts
as thymine photosensitizers
⁎
⁎
Giacomo Nardi, Mauricio Lineros-Rosa, Fabrizio Palumbo, Miguel A. Miranda , Virginie Lhiaubet-Vallet
Instituto Universitario Mixto de Tecnología Química, Universitat Politècnica de València, Consejo Superior de Investigaciones Científicas, Avenida de los Naranjos s/n, 46022, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2020
Received in revised form 21 August 2020
Accepted 26 August 2020
Available online 2 September 2020
Dipicolinic acid (DPA), present in large amount in bacterial spores, has been proposed to act as an endogenous
photosensitizer in spore photoproduct formation. The proposed mechanism involves a triplet-triplet energy
transfer from DPA to thymine. However, up to now, no spectroscopic studies have been performed to determine
the interaction between the endogenous compound and the nucleobase, probably due to its photolability in
aqueous solutions. Here, triplet excited state properties of DPA are reported together with its bimolecular
quenching rate constant by thymidine, kq of ca. 5.3 × 109 M−1 s−1. To run more reliable studies, a stable methyl
ester derivative of DPA, which exhibits the same spectroscopic properties as the parent compound, is also de-
scribed. Finally, DPA photoproducts are characterized. Studies of their triplet excited state properties have dem-
onstrated that, interestingly, one of them is able to photosensitize thymidine triplet excited state formation.
© 2020 Elsevier B.V. All rights reserved.
Keywords:
DNA
Laser flash photolysis
Photochemistry
Spores
Triplet excited state
1. Introduction
Spore photoproduct not only differs from the other dimers in its for-
mation but also in its repair process, because it cannot be reversed
DNA damage has attracted a huge interest over the years in connec-
tion with its harmful deleterious consequences linked to mutation and
cancer [1–3]. It is now well-established that in eukaryotic and vegeta-
tive prokaryotic cells, the main damages observed under direct UV radi-
ation are the bipyrimidinic lesions (Chart 1), i.e. cyclobutane pyrimidine
dimers (CPD) and (6–4) photoproducts (6-4PP). However, bacterial
spores have a singular response to UV with the sole formation of a pho-
toproduct, evidenced more than 50 years ago and tagged spore photo-
product (SP) [4]. This lesion contains a covalent methylene link
between two adjacent thymines and has been fully characterized by
NMR as 5-(R-thyminyl)-5,6-dihydrothymine (Chart 1) [5].
Despite the remarkable results reported over half a century, the
molecular mechanisms underlying the formation of SP are still not
fully understood. Dehydration, complexation of DNA by α/β-type
small, acid-soluble spore proteins (SASP), and the large amount
(~10–15% dry weight) of dipicolinic acid (DPA, Chart 2) have been
shown to be key factors in the photochemistry of bacterial spore DNA
[6,7]. From a chemical point of view, a mechanism of SP formation has
been proposed by Li using a deuterium labeling strategy, which points
toward an intramolecular H-atom transfer from the methyl group of
one thymine to the C6 carbon of the other one [8].
through the action of photolyases as CPD and 6-4PP do. Indeed, this
photoproduct is eliminated from DNA by a dark-repair mechanism
that involves the specific SP lyase enzyme, which belongs to the radical
S-adenosylmethionine (SAM) enzyme family [9,10,11]. Thus, bacterial
spore resistance to UVC radiation has been associated with the unique
photochemical behavior of its nucleic acid, which leads to exclusive for-
mation of SP, and with the ability of SP to undergo subsequently an ef-
ficient repair.
The possible role of DPA as an endogenous photosensitizer has been
addressed over the years, and its influence on SP or CPD formation has
been reported in the literature [6,7,12,13]. Addition of DPA to dry
films of isolated DNA or frozen solutions of thymidine increases the
yield of SP and the ratio of CPD to 6-4PP. In liquid solution, the presence
of DPA also improves significantly the yield of CPD in thymidine ex-
posed to UVC [6,7,12]. Altogether, the results available until now
strongly point toward a triplet-triplet energy transfer (TTET) between
DPA and thymine residues [12,14]. The involvement of the thymine
triplet excited state in SP formation has been further supported by the
experiments run on dry film photoreaction of thymidine in the presence
of pyridopsoralen derivatives or benzophenone under UVA irradiation
[15,16]. However, full characterization of the DPA photophysical prop-
erties has not been achieved yet, probably due to its photolability in so-
lution [14,17]. Specifically, a time-resolved study on the kinetics of the
interaction between DPA triplet excited state and thymidine would pro-
vide highly valuable information on the occurrence of TTET. Moreover,
in spite of its efficient photodegradation in solution, the structures and
⁎
Corresponding authors.
E-mail addresses: mmiranda@qim.upv.es (M.A. Miranda), lvirgini@itq.upv.es
(V. Lhiaubet-Vallet).
https://doi.org/10.1016/j.saa.2020.118898
1386-1425/© 2020 Elsevier B.V. All rights reserved.

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G. Nardi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118898
Chart 1. Structures of spore photoproduct (SP), cyclobutane thymine dimers and (6–4) photoproduct (6-4PP).
photosensitizing potential of DPA photoproducts have not been eluci-
dated. All these aspects are investigated in the present article, paying a
particular attention to the DPA triplet excited state behavior, in order
to shed some light on the peculiar photoreactivity of bacterial
spore DNA.
2.2.2. UPLC-MS/MS
Chromatography was performed on an ACQUITY UPLC system (Wa-
ters Corp.) with a conditioned autosampler at 4 °C. The separation was
carried out on an ACQUITY UPLC HSS T3 C18 column (150 mm × 2.1 mm
i.d., 1.8 μm). The column temperature was maintained at 40 °C. The
analysis was achieved using methanol:water (containing 0.01% formic
acid) 20:80 v:v as the mobile phase with a flow rate of 0.2 mL/min.
The injection volume was 1 μL. The Waters ACQUITY™ XevoQToF Spec-
trometer (Waters Corp.) was connected to the UPLC system via an
electrospray ionization (ESI) interface. The ESI source was operated in
positive ionization mode with the capillary voltage at 3.0 kV. The tem-
perature of the source and desolvation was set at 100 °C and 400 °C, re-
spectively. The cone and desolvation gas flows were 100 L h−1 and
800 L h−1, respectively. All data collected in Centroid mode were ac-
quired using Masslynx™ software (Waters Corp.). Leucine-enkephalin
was used as the lock mass generating an [M + H]+ ion (m/z
556.2771) at a concentration of 250 pg/mL and flow rate of 50 μL/min
to ensure accuracy during the MS analysis.
2. Material and methods
2.1. Chemicals
2,6-Pyridinedicarboxylic acid (dipicolinic acid, DPA), 5- and 6-
bromo-2-picoline, n-butyllithium, tributyltin chloride, β-carotene, xan-
thone, thymidine (Thd), sulfuric acid (98%) and chromium (VI) oxide
(CrO3) were from Sigma Aldrich; 6,6′-dimethyl-2,2′-bipyridine (1a)
was from TCI Europe. All compounds were used without further purifi-
cation. The reagent grade solvents were obtained from Scharlau and
used without further purification. Phosphate buffered saline (PBS) solu-
tions of 0.01 M (pH = 7.4) were prepared by dissolving tablets (Sigma-
Aldrich) in Milli-Q water. The model compounds Thy-Thy and
Thy<>Thy were synthesized as previously described in the literature
[18].
2.2.3. Microwaves reactor
Reactions promoted by microwaves were performed in a MARS (mi-
crowave accelerated reaction system) model unit (CEM Corporation,
2450 MHz) with Microwave Teflon liner (EasyPrep Temperature Con-
trol Vessel Assembly, CEM Corporation).
2.2. Instruments
2.2.4. Photoreactors
Irradiation in the UVC was performed by means of a multilamp
photoreactor equipped with fluorescent tubes of maximal output at
ca. 254 nm (Philips - TUV15W G15T8). UVB irradiation was performed
in the same photoreactor using lamps with a maximum output at
300 nm (from Luzchem).
2.2.1. NMR spectroscopy
1H NMR and 13C NMR were recorded on a 300 MHz spectrometer
(AV300, Bruker) operating in the Fourier transform mode. The chemical
shifts are reported in ppm (parts per million).
Chart 2. Chemical structures of dipicolinic acid (DPA), its methyl ester derivative (DMDP), photoproducts 1 and 2.

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3
2.2.5. UV–Vis absorption spectroscopy
CDCl3): δ = 165.8, 155.6, 147.7, 138.3, 125.6, 125, 53.1. HRMS (ESI+): Cal-
Absorption spectra of the samples were measured with a single
beam Varian UV–Vis model Cary 50 Scan spectrophotometer, using
1 cm pathway quartz cuvettes.
culated for C14H13N2O4 (M + H)+: 273.0875; Found: 273.0873.
2.3.3. Synthesis of 6,6′-dimethyl-2,3-bipyridine (2a)
6,6′-Dimethyl-2,3′-bipyridine was synthesized from the Stille
coupling reaction between 5-bromo-2-methylpyridine and 6-
tributylstannyl-2-methylpyridine, which was obtained from 6-bromo-2-
picoline following the procedure described in the literature [20]. 5-
Bromo-2-methylpyridine (170 mg, 1 mmol) and Pd(PPh3)4 (41 mg,
3.5% mol) were dissolved in dry toluene (4 mL) and 6-tributylstannyl-2-
methylpyridine (420 mg, 1.1 mmol) was added. The mixture was stirred
for 24 h under reflux. After evaporation of toluene under vacuum, the res-
idue was dissolved in dichloromethane and the palladium black was re-
moved by filtration through Celite. The filtrate was washed with
aqueous HCl (1 M) and transferred dropwise to an aqueous saturated so-
lution of Na2CO3. The resulting oil was extracted with CH2Cl2. The organic
phase was washed with brine, and the solvent was removed. The residue
was purified by silica gel chromatography using hexane:ethyl acetate
(1:1, v:v) as eluent to afford 2a (110 mg, 0.6 mmol, 60%). 1H NMR
(300 MHz, CDCl3): δ = 9.04 (d, J = 2 Hz, 1H), 8.23 (dd, J1 = 7.7 Hz,
J2 = 2 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.26 (d,
J = 7.7, 1H), 7.1 (d, J = 7.7 Hz, 1H), 2.62 (s, 3H), 2.61 (s, 3H). 13C NMR
(75 MHz, CDCl3): δ = 158.8, 158.6, 154.4, 147.5, 137.1, 134.8, 132.4,
123.1, 122.0, 117.3, 24.7, 24.2. HRMS (ESI+): Calculated for C12H13N2
(M + H)+: 185.1079; Found: 185.1084.
2.2.6. Laser flash photolysis (LFP)
Two laser flash photolysis systems were employed for the studies. For
266 and 355 nm excitation, experiments were carried out using the fourth
and third harmonic, respectively, of a pulsed Nd:YAG L52137 V LOTIS. The
laser flash photolysis system consisted of the pulsed laser, a 77250 Oriel
monochromator, and a photomultiplier (PMT) system made up of side-
on PMT, PMT housing, and a PMT power supply. The output signal from
the Tektronix oscilloscope was transferred to a personal computer for
study. The single pulses were ca. 10 ns duration, and the energy was ca.
15 mJ/pulse. All transient spectra were recorded using 1 × 1 cm2 quartz
cells with 4 mL capacity, and solutions were bubbled for 10 min with
N2, air, or O2, before acquisition. The absorbance of the samples was
kept in the range 0.30–0.40 at the laser excitation wavelength.
For quenching experiments, stock solutions of quenchers were pre-
pared so that it was only necessary to add microliter volumes to the
sample cell to obtain appropriate concentrations. A linear quenching
plot was obtained, and the resulting rate constant (kq) was calculated
from the slope of the Stern-Volmer plot: τ0/τ = 1 + kqτ0[Q] where τ0
(in s) is the triplet lifetime in the absence of quencher, τ is the triplet
lifetime (in s) in the presence of the quencher, and [Q] is the quencher
concentration in mol·L−1
.
2.2.7. Phosphorescence emission
(A)
Phosphorescence spectrum was obtained from a Photon Technology
International (PTI, TimeMaster TM-2/2003) spectrofluorometer
equipped with a pulsed Xe lamp. The apparatus was operated in time-
resolved mode, with a delay time of 0.5 μs. Compounds were dissolved
in ethanol, put in a quartz tube (5 mm of diameter) and cooled at 77 K.
These experiments were run under air atmosphere.
2.3. Synthesis
2.3.1. Dimethyl dipicolinate ester (DMDP)
Dipicolinic acid (250 mg, 1.50 mmol) was dissolved in MeOH
(250 mL), and concentrated H2SO4 (125 μL) was added dropwise to
the solution. After stirring under reflux for 24 h at 70 °C, the reaction
mixture was basified to a pH 7.8 using Na2CO3 and 250 mL of brine
was added. Methanol was evaporated under vacuum, and the obtained
aqueous solution was extracted with ethyl acetate (3×). The combined
organic layers were dried over anhydrous MgSO4, filtered and concen-
trated to afford DMDP as a white crystalline solid (280 mg, 1.43 mmol,
95%). No further purification was needed. 1H NMR (300 MHz, CDCl3):
δ = 8.32 (d, J = 7.8, 2H), 8.02 (t, J = 7.8 Hz, 1H), 4.02 (s, 6H). NMR
data coincide with those previously described in literature [19].
(B)
2.3.2. Synthesis of 6,6′-dimethyl-2,2′-bipyridinedicarboxylate (1)
6,6′-Dimethyl-2,2′-bipyridine (1a, 50 mg, 0.3 mmol) was placed into a
25 mL round-bottom flask cooled with an ice bath. Concentrated sulfuric
acid (3 mL) was added dropwise under stirring, followed by small por-
tions of the chromium (VI) oxide (170 mg, 1.7 mmol). The mixture was
stirred overnight at 70 °C. Afterwards, the solution was cooled to room
temperature and was slowly poured to 100 mL of methanol. After stirring
under reflux for 24 h at 70 °C, the reaction mixture was basified to a
pH 7.8 using Na2CO3 and 100 mL of brine was added. Methanol was evap-
orated under vacuum, and the obtained aqueous solution was extracted
with ethyl acetate (3×). The combined organic layers were dried over an-
hydrous MgSO4, filtered and concentrated to afford 1 (61 mg, 0.23 mmol,
75%). No further purification was needed. 1H NMR (300 MHz, CDCl3):
δ = 8.73 (dd, J1 = 7.9 Hz, J2 = 1.1 Hz, 2H), 8.15 (dd, J1 = 7.9 Hz, J2 =
1.1 Hz, 2H), 7.99 (t, J = 7.9 Hz, 2H), 4.0 (s, 6H). 13C NMR (75 MHz,
Fig. 1. (A) Normalized UV absorption spectra of DPA (black line) in PBS and DMDP (red
line) in H2O:MeCN (9:1, v:v), (B) Phosphorescence spectrum (λexc = 266 nm) of DPA
(black line) and DMDP (red line) in EtOH at 77 K.

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2.3.4. Synthesis of 6,6′-dimethyl-2,3′-bipyridinedicarboxylate (2)
2.4. Photolysis of DPA
6,6′-dimethyl-2,3′-bipyridine (2a) (50 mg, 0.3 mmol) was placed
into a round-bottom flask cooled with an ice bath. Concentrated sulfuric
acid (3 mL) was added dropwise under stirring, followed by small por-
tions of the chromium (VI) oxide (170 mg, 1.7 mmol). The mixture was
stirred overnight at 70 °C. Afterwards, the solution was cooled to room
temperature and was slowly poured to 100 mL of methanol in a teflon
vessel for microwave apparatus, which was then fixed to the rotor
mechanism of the microwave cavity. The most appropriate reaction
conditions were found for a temperature of heating of 80 °C (5 min)
and, then the sample was held for 10 min applying a radiation of 175/
125 W. The sample was cooled to room temperature and the vessel
was removed from the microwave cavity. The reaction mixture was ba-
sified to a pH 7.8 using Na2CO3 and 250 mL brine was added. Methanol
was evaporated under vacuum, and the obtained aqueous solution was
extracted with ethyl acetate 3 times. The combined organic layers were
dried over anhydrous MgSO4, filtered and concentrated under vacuum.
The resulting residue was subjected to flash chromatography using
ethyl acetate:hexane:dichloromethane (1:1:4, v:v:v) as eluent, com-
pound 2 was then crystallized from ethanol solution (Yield:70%).
1H NMR (300 MHz, CDCl3): δ = 9.30 (dd, J1 = 2.2 Hz, J2 = 0.8 Hz,
1H), 8.61 (dd, J1 = 8.2 Hz, J2 = 2.2 Hz, 1H), 8.28 (dd, J1 = 8.2 Hz,
J2 = 0.8 Hz, 1H), 8.19–8.14 (m, 1H), 8.02–7.96 (m, 2H), 4.04 (s, 3H),
4.03 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 165.6, 154.1, 148.9, 148.3,
138.4, 137.1, 136.1, 125.4, 124.8, 124.3, 53.2. HRMS (ESI+): Calculated
for C14H13N2O4 (M + H)+: 273.0875; Found: 273.0873.
Irradiation of DPA (3 mg/mL) was performed in PBS solution, under
N2 atmosphere, by means of a multilamp photoreactor equipped with 8
UVC tubes for 5 min. After removal of water under vacuum, the residue
was dissolved with 20 mL of methanol in a teflon vessel for microwave
apparatus. The esterification was performed as abovementioned (see
synthesis of 2). The photoreaction was monitored by high-
performance liquid chromatography using a reverse phase column
(C18 Mediterranea sea, 25 × 1.0 cm, acetonitrile:water, 60:40, v:v,
flow 0.7 mL/min) coupled with UV detection (λ = 254 nm).
2.5. Thymine cyclobutane dimers photosensitization by DMDP and
photoproduct 1
Nitrogen-purged solutions of Thy-Thy (0.5 mM) in acetonitrile:
water mixture (1:1, v:v) were irradiated with UVB light up to 60 min
in the presence or absence of DMDP or compound 1 (1.5 mM). Aliquots
were collected at different irradiation times and were analyzed by HPLC
(Agilent) equipped with a diode array detector (DAD), for all chromato-
grams the detection wavelength was 240 nm. Samples were analyzed
using a reverse phase Mediterranea Sea C18 (25 cm × 0.46 cm, 5 μm)
column. Chromatographic conditions for the analysis of the irradiations
of Thy-Thy alone and in the presence of DMDP were an isocratic mixture
of H2O (at pH 3, adjusted with trifluoroacetic acid): acetonitrile (80:20,
v:v) at a flow rate of 1 mL/min. The injection volume was of 10 μL. Irra-
diation of Thy-Thy with photoproduct 1 was analyzed using a gradient
elution. The column was equilibrated in H2O (at pH 3, adjusted with
trifluoroacetic acid): acetonitrile (80:20, v:v) at a flow rate of 1 mL/
min. The amount of acetonitrile was maintained for 20 min, then in-
creased from 20% to 100% in 5 min, kept at 100% for 5 min, restored to
the initial composition in 5 min, and finally maintained at this composi-
tion for 20 min. The injection volume was of 10 μL.
(A)
3. Results
3.1. Photophysics of DPA
The absorption spectrum of dipicolinic acid (DPA) in phosphate
buffer saline solution (PBS, pH 7.4) was centered at 270 nm (Fig. 1A,
black line) and extended up to ca. 295–300 nm. This low capability to
absorb in the UVB region would make DPA a poor sensitizer under the
conditions present at earth surface. Concerning fluorescence emission,
DPA only showed a very weak signal (data not shown), which could
not be accurately characterized due to the changes occurring during
measurements, associated with DPA photolability [14,17].
(B)
By contrast, phosphorescence emission upon excitation of DPA at
266 nm in EtOH glass at 77 K consisted of a band with maximum cen-
tered at ca. 410 nm (Fig. 1B). As this emission did not exhibit fine struc-
ture, the triplet excited state energy, ET, was estimated from the
wavelength corresponding to 20% of the highest intensity, obtaining a
value of ca. 328 kJ mol−1 [18,21]. This ET is clearly higher than that of
the triplet excited state of thymine in DNA with a value of ca.
267 kJ mol−1 [21,22], thus confirming the ability of DPA to photosensi-
tize the formation of cyclobutane thymine dimers by means of a triplet-
triplet energy transfer (TTET) mechanism [12,23].
Next, laser flash photolysis experiments were performed to obtain
3
⁎
key information on the DPA triplet excited state ( DPA ) at room tem-
perature in aqueous media. As mentioned above for the fluorescence
experiments, photodegradation was an important drawback to record
the transient absorption spectrum. Therefore, a point by point experi-
ment was run, using a fresh sample for each laser pulse (266 nm) i.e.
each recorded wavelength. Fig. 2A shows the spectra obtained in this
way for DPA in deaerated PBS, using fresh (black squares), one pulse
(red circles) or two pulses pre-irradiated solutions (blue triangles).
Fig. 2. (A) Transient absorption spectra of N2 degassed PBS solution of DPA obtained
changing the sample after each pulse/wavelength record, using fresh (■), one pulse (●)
or two pulses (▲) pre-irradiated solution. Inset: corresponding decay of DPA monitored
at 300 nm. (B) Decays monitored at 300 nm for deaerated PBS solution of DPA in the
presence of increasing amounts of Thd (0, 6.2 and 20 μM).