Blocking cyclobutane pyrimidine dimer formation by steric hindrance

Article abstract of DOI:10.1039/c6ob00382f

The efficiency of thymine (Thy) and uracil (Ura) to form cyclobutane pyrimidine dimers (CPDs) in solution, upon UV irradiation differs by one order of magnitude. This could to be partially related to the steric hindrance induced by the methyl at C5 in thymine. The aim of the present work is to establish the influence of a bulky moiety at this position on the photoreactivity of pyrimidines. With this purpose, photosensitization with benzophenone and acetone of a 5-tert-butyl uracil derivative (1) and the equivalent Thy (2) has been compared. Introduction of the tert-butyl group completely blocks CPD formation. Moreover, the mechanistic insight obtained by laser flash photolysis is in accordance with the observed photoreactivity.

Full text of DOI:10.1039/c6ob00382f

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Blocking cyclobutane pyrimidine dimer formation
by steric hindrance†
Cite this: Org. Biomol. Chem., 2016,
14, 4110
Victoria Vendrell-Criado,a Virginie Lhiaubet-Vallet,a Minoru Yamaji,b
M. Consuelo Cuquerella*a and Miguel A. Miranda*a
The efficiency of thymine (Thy) and uracil (Ura) to form cyclobutane pyrimidine dimers (CPDs) in solution,
upon UV irradiation differs by one order of magnitude. This could to be partially related to the steric
hindrance induced by the methyl at C5 in thymine. The aim of the present work is to establish the
influence of a bulky moiety at this position on the photoreactivity of pyrimidines. With this purpose,
photosensitization with benzophenone and acetone of a 5-tert-butyl uracil derivative (1) and the
equivalent Thy (2) has been compared. Introduction of the tert-butyl group completely blocks CPD for-
mation. Moreover, the mechanistic insight obtained by laser flash photolysis is in accordance with the
observed photoreactivity.
Received 17th February 2016,
Accepted 1st April 2016
DOI: 10.1039/c6ob00382f
www.rsc.org/obc
Introduction
Ultraviolet irradiation of DNA leads to the formation of lesions
such as cyclobutane pyrimidine dimers (CPDs). This hinders
replication and transcription of the biomacromolecule, which
may give rise to cytotoxicity and mutagenicity.1 Mechanisti-
cally, CPDs arise from a [2 + 2] photocycloaddition between
two adjacent pyrimidines (Pyr) in the same DNA strand.2,3
In solution, with monomeric derivatives, a fraction of Pyr is
promoted to the triplet excited state (3Pyr*) upon light absorp-
Scheme 1 Dimerization of pyrimidines to form cyclobutane pyrimidine
dimers.
3
tion and intersystem crossing (ISC). In turn, Pyr* reacts with
another Pyr in the ground state to give the final product
(Scheme 1, pathway a + b).4 In this context, the dimerization
efficiency (ϕDIM) of thymine (Thy) compared to uracil (Ura)
differs by one order of magnitude (ϕDIM = 0.0025 and 0.019,
respectively).4 This variance has been rationalized in terms of
a higher intersystem crossing quantum yield (ϕISC) and self-
quenching rate constant (kSQ) for Ura with respect to Thy
(0.40 vs. 0.18 and 2 × 109 M−1 s−1 vs. 6 × 108 M−1 s−1, respect-
ively). The disparity observed in the kSQ in spite of the struc-
tural similarity between these two Pyr could rely on the steric
hindrance associated with methyl substitution at C5; however
this has not been systematically investigated.
Scheme 2 Major photoproducts obtained upon photosensitization of
thymine derivatives by acetone and benzophenone.
Pyrimidine dimerization has also been studied after
irradiation with UVA light in the presence of typical triplet sen-
sitizers (Scheme 1, pathway a′ + b).2,5–7
aInstituto Universitario Mixto de Tecnología Química (UPV-CSIC), Universitat
Politècnica de València, Av. Los Naranjos s/n 46022, Valencia, Spain.
E-mail: mmiranda@qim.upv.es, mcuquere@itq.upv.es
Acetone photosensitization leads mainly to CPDs through
triplet–triplet energy transfer (TTET, Scheme 2); however,
benzophenone (BP) gives rise to mainly oxetane formation as a
result of a Paternò–Büchi reaction between the carbonyl
moiety of BP and the C5–C6 double bond of the Pyr
(Scheme 2).2,7–10 In this context, the aim of the present work is
bDivision of Molecular Science, Faculty of Science and Technology,
Gunma University, Kiryu Gunma 376-8515, Japan
†Electronic supplementary information (ESI) available: NMR spectra of syn-
thesized and isolated compounds. See DOI: 10.1039/c6ob00382f
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to determine the influence of steric hindrance on the photo-
sensitized dimerization that leads to CPDs. With this purpose,
the tert-butyl substituted uracil derivative 1 and its thymine
analog 2, have been selected to investigate their photo-
sensitization with BP (ET = 290 kJ mol−1) and acetone (ET
=
332 kJ mol−1) looking at the kinetic aspects and also at the
photoproduct distribution.
Scheme 3 Photoproducts isolated upon irradiation of 1 or 2 with
benzophenone.
Results and discussion
Direct irradiation of 1
Compound 1 displayed an ultraviolet spectrum in acetonitrile
similar to that of the parent pyrimidine, with a maximum at
260 nm. However, its direct irradiation differed from that of
Thy. Absorption of light by 1 provoked a progressive decrease
of the 260 nm peak, together with the appearance of a new
one at 314 nm. The presence of an isosbestic point at 280 nm
indicated the formation of one main photoproduct. This be-
havior was similar to that observed for the related compound
5-tert-butyl-2′-deoxyuridine that gives rise to the corresponding
Fig. 2 CPDs isolated after photosensitization of 2 by acetone.
The oxetane regiochemistry was determined on the basis of
the chemical shift of protons and carbons at C5 and C6. Thus,
Δδ (C5 and C6) was 35 and 11 ppm for 2b-1 and 2b-2, respec-
1,2-dihydrocyclobuta[d]pyrimidin-2-one through
a Norrish–
Yang photoreaction.11 Isolation and characterization of 1′
(Fig. 1) confirmed that under direct irradiation, 1 undergoes a tively, indicating that 2b-1 corresponds to the oxetane in which
oxygen is bonded to C6. The same trend, although less pro-
nounced due to the contribution of the tert-butyl moiety, was
observed for 1b-1 and 1b-2. The chemical shift observed for
H6 was consistent with this assignment; this proton was less
shielded in 1b-1 and 2b-1 (δ = 6.05 and 5.37 ppm, respectively),
than in 1b-2 and 2b-2, (δ = 5.18 and 4.87 ppm, respectively).
According to the product distribution, the bulky substituent at
C5 does not have a strong influence on the regiochemistry of
the reaction as it might be expected. Moreover, it is worth
noting that no CPDs were found among the photoproducts,
which means that the Paternò–Büchi reaction between BP and
the Pyr predominates over [2 + 2] photocycloaddition. To gain
further insight into this behavior, similar experiments were
performed increasing the proportion of Pyr in the medium to
favor TTET.8 Thus, solutions containing BP:1 (or 2) at
1 : 3 molar ratio were irradiated and the crude mixtures were
subjected to NMR. In the case of 1, the spectrum of the reac-
tion crude, showed a similar photoproduct distribution and
again evidenced the absence of CPDs even under these con-
ditions. On the contrary, the irradiation performed with the
reference compound 2 gave rise to additional photoproducts.
Their isolation and characterization allowed identifying the
cis–syn and trans–syn CPDs 2c-1 and 2c-2 (Fig. 2). The 2c-2
stereochemistry was established on the basis of the NOE effect
observed between the methyl (δ = 1.65 ppm) and the methyl-
ene protons (δ = 4.99 and 3.63 ppm). The CPD 2c-1 did not
show such interaction (see NMR spectra of 2c-1 in the ESI†).
Norrish–Yang process.
Steady state photolysis in the presence of benzophenone
An acetonitrile : water (11 : 3 v/v) solution of BP and 1 (or 2) at
a 2 : 1 molar ratio was irradiated with lamps emitting mainly
in the 310–390 nm range, the solvent was removed under
reduced pressure and the resulting mixture was analyzed by
NMR.
The 1H-NMR spectra of the reaction crudes clearly indicated
that, under these conditions, the tert-butyl uracil 1 is by far
less reactive than the reference thymine 2. In both cases,
the presence of two singlets located in the 5 to 6 ppm
range pointed to the formation of two oxetanes during the
photoreaction. The proportion of each oxetane, determined
from the integrals of the two singlets, was 50 : 50 for 1, and
35 : 65 for 2. Purification over silica column led to isolation of
the regioisomeric oxetanes 1b-1 and 1b-2 or 2b-1 and 2b-2
(Scheme 3).
Steady state photolysis in the presence of acetone
Photolysis of 1 at 310–390 nm in an acetone : water solution
Fig. 1 Spectral changes observed upon direct irradiation of 1 to give 1’.
(37 : 63 v/v) was performed for 18 h (72% conversion).
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Scheme 4 Photoproducts obtained after photosensitization of 1 with
acetone.
Then, the solvent was removed under vacuum yielding a
yellow oil. According to the analysis of the reaction crude
made by 1H-NMR, the progress of the reaction was very
limited, but new photoproducts were formed. The main one
Fig. 3 (A) Transient absorption spectrum at 1.3 μs obtained upon
was identified as the acetonyl derivative 1c-3 (Scheme 4), equi- 308 nm laser pulsing in acetone (0.6 M)/1 (1.6 mM) in ACN at 295 K.
Inset: time profile at 350 nm. (B) Rise rates for 31* plotted as a function
of the concentration of 1.
valent to the product previously found in thymidine photo-
sensitization by acetone.2 In addition, two uracil/5-tert-butyl
uracil heterodimers 1c-1 and 1c-2, together with photoproduct
1c-4, were isolated (Scheme 4). Clearly, 1c-1 and 1c-2 must
the energy transfer rate constant (kET) to the triplet energy gap
arise from the Norrish type II photofragmentation reported for
between the donor and acceptor:
similar 5-tert-butyl uracils, followed by [2 + 2] photocyclo-
1
addition of the resulting (less hindered) dealkylated product
with 1.
kET ¼ kD
ð1Þ
eꢀΔE=RT þ 1
Remarkably, no homodimers were obtained with this
photosensitization strategy in spite of the higher triplet energy
of acetone. Conversely, irradiation of 2 under similar experi-
mental conditions gave rise to homodimers 2c-1 and 2c-2
already obtained upon BP photosensitization. The results
observed with 2 are consistent with those previously described
for Thy and thymidine.2,5,7
where kD is the diffusion rate constant in liquid solutions.13
The interaction of 1 with BP was also investigated by determin-
ing the quenching rate constant of 3BP*. With this purpose,
BP solutions were prepared and increasing amounts of 1 and 2
for comparison were added. As shown in Fig. 4A, 1 barely
Laser flash photolysis
To gain mechanistic insight into the process, laser flash
photolysis experiments were performed. No transient signal
was observed upon direct excitation of 1, more likely due to its
very low triplet quantum yield and molar absorption coeffi-
3
cient. To circumvent this problem, 1* was photosensitized by
acetone upon excitation at 308 nm. The transient absorption
spectrum displayed a maximum at 350 nm, very similar to that
previously found for thymidine12 where the absorbance
change, ΔA350 was as small as 0.03 (Fig. 3A). Inset of Fig. 3A
depicts the triplet formation profile at 350 nm. The rise rate
varied with the concentration of 1. From the slope of the
linear plot kobsd vs. [1] (Fig. 3B), the quenching rate constant
of triplet acetone by 1 was determined to be kq = 5.5 × 108
M−1 s−1. This value indicates that the triplet energy of 1 is very
close to that of acetone (332 kJ mol−1), presumably within
Fig. 4 Decay profiles of the signal at 530 nm obtained after laser exci-
tation of BP in the presence of increasing amounts of (A) 1 (from 0 to
4–8 kJ mol−1, according to Sandros’ equation (1), which relates 2.6 mM) and (B) 2 (from 0 to 2.3 mM).
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quenches 3BP*, which is consistent with the low chemical reac- Isolation and identification of 1′
tivity exhibited by this compound upon photosensitization
Compound 1 (0.2 g, 0.9 mmol) was dissolved in acetonitrile
and irradiated at 254 nm through quartz. The progress of the
reaction was monitored by using a UV-Vis spectrophotometer.
When the UV band with λmax 314 nm, associated with 1′
reached its maximum, (4 h irradiation) the solvent was evapor-
ated under vacuum.
with BP. On the contrary, 2 displayed a significant quenching
of 3BP* in accordance with its reactivity. A rate constant of
3
1.5 × 108 M−1 s−1 was obtained for the BP*/2 interaction, which
is in the same range as the one observed for thymidine.8,9
1H NMR (300 MHz, DMSO-d6), δH: 7.70 (s, 1H), 4.58 (s, 2H),
3.70 (s, 3H), 3.12 (s, 2H), 1.40 (s, 6H). 13C NMR (75 MHz,
DMSO-d6): 181.7, 168.5, 157.2, 139.7, 128.4, 54.5, 52.2, 51.9,
50.0, 26.7. HRMS (ESI): m/z calcd for C11H15N2O3 [M + H+]
223.1075, found [M + H+] 223.1083.
Conclusions
Replacement of the C5 thymine methyl group with a bulky
tert-butyl substituent is associated with a marked decrease in
the photoreactivity under triplet photosensitization conditions
and completely blocks the formation of CPDs.
Isolation and identification of photoproducts obtained upon
irradiation of 1 and 2 with BP
An acetonitrile : water (11 : 3 v/v) solution of BP (28 mM) and 1
(or 2) at a 2 : 1 molar ratio was irradiated. Purification of photo-
products from 1 and 2 was achieved over silica gel column
using hexane : ethyl acetate 60 : 40 as the eluent. Two fractions
containing the two regioisomers of oxetanes were collected
and the solvent was evaporated.
Experimental
Synthesis of 1 and 2
Firstly, to a suspension of 13 mL of H2O containing 5-tert-butyl
uracil14 (1.0 g, 6.0 mmol) an aqueous 2 N KOH (13 mL) solu-
tion was added. The mixture was stirred at room temperature
until a clear solution was obtained. Ethyl bromoacetate (2 eq.)
was added and then heated to reflux overnight. The cold reac-
tion mixture was acidified with conc. HCl until pH 1 and the
crystallized product was filtered, washed with cold 1 N HCl
and water and dried in vacuo to give tert-butyl uracil-1-acetic
Oxetane 1b-1. 1H NMR (300 MHz, CD3OD), δH: 7.82 (d, J =
9 Hz, 2H), 7.62 (d, J = 9 Hz, 2H), 7.43–7.13 (m, 6H), 6.05 (s,
1H), 4.64 (d, J = 18 Hz, 1H), 4.29 (d, J = 18 Hz, 1H), 3.77 (s,
3H), 1.03 (s, 9H). 13C NMR (75 MHz, CD3OD): 171.5, 171.0,
153.5, 144.3, 143.0, 129.3, 128.9, 128.7, 128.4, 128.2, 127.5,
90.6, 86.1, 67.7, 52.8, 47.4, 36.6, 27.8. HRMS (ESI): m/z calcd
for C24H26N2O5Na [M + Na+] 445.1763, found [M + Na+]
445.1735.
1
acid (0.8 g, 59%). H NMR (300 MHz, DMSO-d6), δH: 11.18 (s,
1H), 7.36 (s, 1H), 4.39 (s, 2H), 1.19 (s, 9H). 13C NMR (75 MHz,
DMSO-d6): 169.8, 163.1, 150.8, 140.6, 119.8, 49.0, 32.5, 28.6.
HRMS (ESI): m/z calcd for C10H15N2O4 [M + H+] 227.1032,
found [M + H+] 227.1039.
Oxetane 1b-2. 1H NMR (300 MHz, CD3OD) δH: 7.43–7.20 (m,
10H), 5.18 (s, 1H), 4.64 (d, J = 18 Hz, 1H), 4.06 (d, J = 18 Hz,
1H), 3.81 (s, 3H), 1.13 (s, 9H). 13C NMR (75 MHz, CD3OD):
172.2, 170.7, 153.5, 145.4, 141.2, 129.7, 129.5, 128.9, 128.8,
126.7, 125.8, 92.4, 85.1, 62.1, 53.0, 49.9, 36.9, 24.9. HRMS
(ESI): m/z calcd for C24H26N2O5Na [M + Na+] 445.1739, found
[M + Na+] 445.1709.
Then, to a stirred solution of this compound (0.2 g,
1.1 mmol) or thymine-1-acetic acid (0.2 g, 0.9 mmol) in MeOH
(5 mL) H2SO4 was added (0.5 mL). The reaction mixtures were
heated to reflux overnight. The solvent was evaporated under
pressure. The solids were dissolved in AcOEt, washed with
brine (3×) and dried over MgSO4. The compounds were
obtained as white solids 1 (0.2 g, 90%) or 215 (0.2 g, 92%).
Compound 1. 1H NMR (300 MHz, DMSO-d6), δH: 11.26
(s, 1H), 7.40 (s, 1H), 4.54 (s, 2H), 3.69 (s, 3H), 1.21 (s, 9H).
13C NMR (75 MHz, DMSO-d6): 168.8, 163.0, 150.6, 140.4, 120.2,
52.2, 48.6, 32.5, 28.6. HRMS (ESI): m/z calcd for C11H17N2O4
[M + H+] 241.1181, found [M + H+] 241.1188.
Oxetane 2b-1. 1H NMR (300 MHz, CD3CN), δH: 8.39 (s, 1H),
7.61–7.54 (m, 4H), 7.42–7.37 (m, 2H), 7.35–7.18 (m, 4H), 5.37
(s, 1H), 4.32 (d, J = 18 Hz, 1H), 4.13 (d, J = 18 Hz, 1H), 3.71 (s,
3H), 1.39 (s, 3H). 13C NMR (75 MHz, CD3CN): 171.0, 170.3,
152.2, 143.5, 142.6, 129.6, 129.1, 128.9, 128.8, 126.7, 90.3, 89.7,
54.7, 53.4, 47.3, 20.3. HRMS (ESI): m/z calcd for C21H21N2O5
[M + H+] 381.1450, found [M + H+] 381.1441.
Oxetane 2b-2. 1H NMR (400 MHz, CD3CN), δH: 8.14 (s, 1H),
7.39–7.28 (m, 10H), 4.87 (s, 1H), 4.49 (d, J = 16 Hz, 1H), 3.77
(d, J = 16 Hz, 1H), 3.72 (s, 3H), 1.67 (s, 3H). 13C NMR (75 MHz,
CD3CN): 170.5, 169.6, 151.6, 144.8, 140.1, 129.1, 128.9, 128.5,
128.5, 126.3, 91.8, 77.3, 66.2, 52.7, 48.3, 23.3. HRMS (ESI): m/z
calcd for C21H20N2O5Na [M + Na+] 403.1270, found [M + Na+]
403.1255.
Compound 2. 1H NMR (300 MHz, DMSO-d6), δH: 11.42 (s,
1H), 7.51 (s, 1H), 4.49 (s, 2H), 3.70 (s, 3H), 1.77 (s, 3H). HRMS
(ESI): m/z calcd for C8H11N2O4 [M + H+] 199.0717, found
[M + H+] 199.0719.
Irradiation procedures
Irradiation was performed using a multilamp photoreactor
with lamps emitting at 254 nm through quartz or in the
310–390 nm range (with a maximum at 350 nm) through
Pyrex. All the solutions were bubbled with N2 prior to light
exposure.
Isolation and identification of the products obtained upon
photosensitization of 1b and 2b in the presence of acetone
Photoproducts obtained from 1. A solution of 1 or 2
(4.5 mM) in an acetone : water (37 : 63 v/v) solution was
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irradiated through Pyrex with lamps emitting in the The separation was carried out on an ACQUITY UPLC BEH C18
310–390 nm range (with a maximum at 350 nm). The crude column (50 mm × 2.1 mm i.d., 1.7 μm). A Waters ACQUITY™
reaction was subjected to reverse phase HPLC, and the photo- XevoQToF spectrometer (Waters Corp.) was connected to the
products were isolated using a 60 : 40 mixture of a 25 mM UPLC system via an electrospray ionization (ESI) interface. The
ammonium formiate aqueous solution and methanol as the ESI source was operated in positive ionization mode with the
eluent. The proportion of methanol was linearly increased capillary voltage at 1.5 kV.
from 40 to 70% within 35 min. Elution of the products was
monitored at 220 nm. According to the obtained mass 70% of
Laser flash photolysis
1 was consumed during the irradiation.
A pulsed Nd:YAG laser system, using 355 nm as the excitation
Photoproduct 1c-1. 1H NMR (400 MHz, CDCl3), δH: 4.97 (d,
wavelength or a Xe/HCl excimer laser (λexc = 308 nm) with a
pulse duration of 10 ns was used. The apparatus consisted of
J = 0.8 Hz, 1H), 4.93 (d, J = 0.8 Hz, 1H), 4.34 (d, J = 8 Hz, 1H),
4.09 (d, J = 8, 1H), 3.82–3.65 (s + m, 6 + 3H), 1.16 (s, 9H).
the pulsed laser, the Xe lamp, a monochromator and a photo-
13C NMR (75 MHz, CDCl3): 169.6, 169.5, 168.3, 166.3, 151.3,
multiplier. The output signal from the oscilloscope was trans-
ferred to a personal computer. Quartz cells of 1 cm optical
150.9, 62.7, 52.5, 50.7, 50.4, 46.9, 46.3, 41.1, 35.2, 29.7. HRMS
(ESI): m/z calcd for C18H25N4O8 [M + H+] 425.1659, found
path length were employed for the measurements.
[M + H+] 425.1672.
The triplet excited state absorption spectrum of 1 (1.6 mM)
Photoproduct 1c-2. 1H NMR (400 MHz, CD3CN), δH: 4.45 (d,
in acetonitrile was registered using acetone (0.6 M) as a photo-
J = 16 Hz, 1H), 4.11 (m, 3H), 3.84 (d, J = 20 Hz, 1H), 3.72 (s,
sensitizer upon excitation at 308 nm. The absorbance of the
3H), 3.67 (s, 3H), 3.57 (d, J = 16 Hz, 1H), 3.29 (d, J = 8, 1H),
sample at this wavelength was 1.6.
1.11 (s, 9H). 13C NMR (75 MHz, CD3CN): 170.2, 169.5, 166.9,
For quenching experiments 1 (from 0 to 2.6 mM) or 2 (from
153.5, 152.3, 61.7, 55.6, 55.4, 52.4, 48.0, 47.6, 37.3, 35.4, 25.9.
0 to 2.3 mM) were added to a BP solution in acetonitrile with
HRMS (ESI): m/z calcd for C18H25N4O8 [M + H+] 425.1658,
an absorption of 0.3 at 308 nm, and monitored at 530 nm.
found [M + H+] 425.1672.
Photoproduct 1c-3. 1H NMR (300 MHz, CD3OD), δH: 4.26 (d,
J = 15 Hz, 1H), 4.02 (d, J = 18 Hz, 1H), 3.74 (s, 3H), 3.71 (d, J =
6 Hz, 1H), 3.40 (d, J = 12.0 Hz, 1H), 3.26 (s, 1H), 2.61 (d, J =
Acknowledgements
18 Hz, 1H), 2.12 (s, 3H), 1.05 (s, 9H). 13C NMR (75 MHz,
Financial support by the Spanish Government (CTQ2012-
32621, CTQ2015-70164-P and contract JAE-Predoc 2011-00740
to V. V.-C.) and the Generalitat Valenciana (PROMETEOII/2013/
005) is gratefully acknowledged.
CD3OD): 208.8, 175.0, 170.8, 155.1, 52.6, 50.4, 49.1, 46.1, 37.4,
30.3, 26.9. HRMS (ESI): m/z calcd for C14H23N2O5 [M + H+]
299.1607, found [M + H+] 299.1601.
Photoproduct 1c-4. 1H NMR (400 MHz, CDCl3), δH: 4.28 (d,
J = 16 Hz, 1H), 4.05 (d, J = 20 Hz, 1H), 3.77 (s, 3H), 3.56 (dd, J =
16 Hz, 2H), 2.47 (t, J = 8 Hz, 1H), 1.12 (s, 9H). 13C NMR
(75 MHz, CDCl3): 169.6, 168.1, 151.7, 51.4, 48.7, 47.5, 44.7,
References
31.9, 27.2. HRMS (ESI): m/z calcd for C11H18N2O2 [M + H+]
243.1345, found [M + H+] 243.1361.
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9 V. Lhiaubet-Vallet, S. Encinas and M. A. Miranda, J. Am.
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Photoproduct 2c-2. 1H NMR (300 MHz, CDCl3), δH: 4.99 (d,
J = 18 Hz, 2H), 3.77 (s, 6H), 3.63 (d, J = 18 Hz, 2H), 3.52 (s, 2H),
1.65 (s, 6H). 13C NMR (75 MHz, CDCl3): 170.2, 169.5, 151.1,
63.4, 52.6, 48.8, 47.1, 24.7. HRMS (ESI): m/z calcd for
C16H21N4O8 [M + H+] 397.1359, found [M + H+] 397.1345.
UPLC-MS MS analysis
Liquid chromatography was performed on an ACQUITY UPLC 10 Q. H. Song, W. Z. Lin, S. D. Yao and N. Y. Lin, J. Photochem.
system (Waters Corp.) with a conditioned autosampler at 4 °C.
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