A bisubstrate reagent orchestrating adenosine triphosphate and L-tyrosine and making tyrosyl adenylate: Partial mimicking of tyrosyl-tRNA synthetase

Article abstract of DOI:10.1039/c8ob02866d

We report the development of a bisubstrate reagent that, similar to tyrosyl t-RNA synthetase (TyrTS), provides a surface for ATP and l-Tyr to render a pseudo-intramolecular reaction forming 5′-tyrosyl adenylate (tyrAd). The presence of the reagent in solu

Full text of DOI:10.1039/c8ob02866d

Organic &
Biomolecular Chemistry
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A bisubstrate reagent orchestrating adenosine
triphosphate and L-tyrosine and making tyrosyl
adenylate: partial mimicking of tyrosyl-tRNA
synthetase†
Cite this: Org. Biomol. Chem., 2018,
16, 9446
Harpreet Singh, Baljit Kaur, Harpreet Kaur and Palwinder Singh
*
We report the development of a bisubstrate reagent that, similar to tyrosyl t-RNA synthetase (TyrTS), pro-
vides a surface for ATP and L-Tyr to render a pseudo-intramolecular reaction forming 5’-tyrosyl adenylate
(tyrAd). The presence of the reagent in solution with TyrTS marred the enzymatic reaction and, noticeably,
tyrAd formed under the catalytic mode of the biomodel reagent was not picked up by TyrTS and hence
was not transferred to tRNA. A potential application of this reagent, which doesn’t allow the formation of
tyrosyl tRNA, may lie in an emerging therapeutic targeting the translation machinery of cells without inhi-
biting the normal workings of enzymes.
Received 16th November 2018,
Accepted 27th November 2018
DOI: 10.1039/c8ob02866d
rsc.li/obc
the denial of tRNA to free tyrAd (not bound to TyrTS)13,14 may
lead to the use of the reagent in mechanism-based drug
Introduction
Further to the skillful and intuitive work involving enzyme design, without inhibiting the enzyme.
modelled monosubstrate reagents,1–4 developing bisubstrate
Hypothesizing logically, a reagent capable of bringing ATP
reagents for bringing together two molecules and creating a and an amino acid into proximity and performing sequential
microenvironment for processing intramolecular reactions is transformations on the specific precursors may lead to the for-
highly enticing5–11 but challenging, as electronic and stereo- mation of aminoacyl adenylate. The analysis of crystal coordi-
chemical tuning of three participating molecules is needed. nates of TyrTS21 in association with L-Tyr and ATP showed the
While a number of bisubstrate enzymatic reactions, such as placement of adenine and phenolic units in the hydrophobic
those controlling protein synthesis,12 have been documented, pocket, with the tails projected into the polar region (Fig. 1A
the construction of similar artificial systems accomplishing a and S1†). The carboxylate group approaches the phosphorus
reaction between two reactants has been attempted mainly centre at a distance of 1.32 Å. In order to create a system
through supramolecular approaches.5,6,11 Culminating in the similar to the active site pocket of TyrTS, we chose an acridone
entry of Tyr into a growing protein chain12–14 and, sub- template as the hydrophobic surface, due to its capability for
sequently, utilizing the regulating role of specific phosphory- holding substituents without undergoing conformational
lated Tyr residues in cellular signals for cell proliferation, changes and the workings of previously functionalized
morphogenesis, migration and apoptosis,15 the bisubstrate acridones,22–25 for the synthesis of compounds 1, and 6–10
enzyme TyrTS has categorically been the target of several (Fig. 1B, S2–S59 and Chart S1†). The choice of Tyr and Phe was
chemotherapeutic agents.16–20 Alternatively but parallel to the to provide hydrophobicity at the other end of the molecule
enzymatic reaction, herein, it was planned to develop a system also, so that the adenine and aromatic rings of ATP and Tyr
wherein both ATP and L-Tyr bound simultaneously via a may interact at either of the termini.
designed receptor and where the temporary intramolecularity
may help in the formation of 5′-tyrosyl adenylate (tyrAd). The
competition of the reagent with TyrTS in making tyrAd and
Results and discussion
Chemistry
Department of Chemistry and Centre for Advanced Studies, University with Potential
The synthesis of compound 1 is depicted in Scheme 1.
Compounds 3 and 4 were prepared via the stepwise propargyla-
tion of compound 2. The reactions of compounds 3 and 4 with
for Excellence – Guru Nanak Dev University, Amritsar-143005, India.
E-mail: palwinder_singh_2000@yahoo.com; Fax: +91-183-2258820;
Tel: +91-183-2258802-09 x 3278
L-tyrosyl azide provided the respective compounds 5 and 1.
†Electronic supplementary information (ESI) available: NMR, mass, IR, and fluo-
rescence spectral data is supplied. See DOI: 10.1039/c8ob02866d
The syntheses of the other compounds shown in Fig. 1B were
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Fig. 1 (A) TyrTS, showing the active site pocket and binding with ATP and L-tyr. (B) Molecules for the present study.
also achieved by following a similar experimental protocol as
that used for the synthesis of compound 1 but using D-tyrosine
and D-phenylalanine for compounds 7, 10 and 9. Compound 8
was also prepared through the reaction protocol shown in
Scheme 1 but using L-Phe in place of L-Tyr. Compound 6 was
obtained via the hydrolysis of the ester moiety of 1 with LiOH
in acetone–water. All compounds were characterized with
NMR, mass, IR and spectral techniques (ESI†).
Fluorescence studies of the interactions of compounds with
nucleotides and amino acids
The high sensitivity of compound 1 to ATP and L-Tyr (Fig. 2
and S60†) was apparent from the relative change in the fluo-
rescence of the compounds in the presence of nucleotides and
amino acids, which made it an unambiguous choice for pursu-
ing further studies. Compound 1 was also responsive to GTP
(Fig. S61†) but not to UTP and CTP, whereas a partial decrease
in its fluorescence intensity occurred in the presence of ADP
and AMP (Fig. S62 and S63†). Phe and Trp also decreased the
fluorescence intensity of 1, whereas other amino acids showed
no effect (Fig. S64–S75†). It seems that the nucleobases of ATP,
GTP and the aromatic parts of Tyr, Trp and Phe interact with
the acridone moiety of 1 to change the fluorescence. The role
of the L-Tyr residue and the two arms of 1 in interacting with
ATP and L-Tyr were apparent from a comparison of the fluo-
rescence quenching results with those of compounds 7–10.
The mass peaks of 1. ATP and 1. L-Tyr adducts in ESI-HRMS
studies also support these interactions (Fig. S78–S81†).
Scheme 1 The synthesis of compound 1. Reaction conditions: (i) (a)
K2CO3, CuO, isoamyl alcohol, reflux, 42 h; (b) Conc. H2SO4, heat (ii)
K2CO3, propargyl bromide, KI, DMF, 60 °C, overnight. (iii) CuSO4·5H2O,
sodium ascorbate, t-butanol : H2O (9 : 1 v/v), 24 h, 60 °C.
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Fig. 3 (A) LC-MS data for tyrAd (pink trace), compared with an auth-
entic sample (blue trace),27 and (B) HRMS data for the pink trace in A,
corresponding to the mass of tyrAd (calcd m/z: 511.1336 [M + H]+).
Fig. 2 The fluorescence intensities of compounds (5 µM in DMSO–
H2O; 1 : 9; pH: 7.2) at 452 nm (λex: 260 nm): (X) ATP (25 equiv.) and L-Tyr
(4 equiv.) were added to 1, and 6–10; (Y) nucleotides and amino acids
(100 equiv. except L-Tyr (4 equiv.) and ATP (25 equiv.)) were added to 1
and (Z) ATP and L-Tyr (1 equiv. each of 1) were added to 1. The quantum
yield (Φ) of 1 for ATP was 0.44.
Kcat/Km = 11.0
Therefore, seamlessly, the biomodel reagent
appreciable catalytic efficiency for the formation of tyrAd,
when ATP and L-Tyr alone do not react without the use of a
coupling reagent.
1.7 × 103 M−1 s−1 (Fig. S98 and S99†).
exhibited
1
Therefore, compound 1 recognizes two critical components
of a biological system – ATP and L-Tyr. None of the D-amino
acids interacted with compound 1, thereby highlighting the
stereochemical selectivity of the compound. Moreover, a
stereochemical analogue of 1, compound 7, exhibited inter-
action with D-Tyr only (Fig. S82†), but it does interact with ATP
(Fig. S83†). The stability of the C11–C12 ester bond (see
Fig. 4B for numbering) in the presence of esterase26 ensured
Plausible mechanism for the compound 1 mediated formation
of tyrAd
The processivity of acridone and the two arms during reagent
operation was adjudged from the stepwise shifts in H1, H27,
1
H8, H14, H33–37, H20–24 and H28 signals in H NMR spectra
recorded at 25–40 °C (Fig. 4A and S100–S114†) upon the
addition of ATP, L-Tyr or L-Tyr and ATP. The OH signals of com-
pound 1 disappeared upon the addition of ATP (Fig. S109†)
but not when L-Tyr was added first (Fig. S114†), indicating that
Tyr stays away from the phenolic units of 1. The terminal part
of the phosphate unit of ATP seems responsible for extracting
phenolic OH. H20, 21, 23 and 24 and H33, 34, 36, and 37
signals were not affected when L-Tyr was added first to the
compound solution (Fig. S114†), supporting the placement of
Tyr towards the acridone moiety. H protons of the adenine
unit of ATP at δ = 8.50 and 8.15 ppm were shifted upfield when
L-Tyr was added to a solution of 1 and ATP. Apparently, similar
to their binding in the hydrophobic pocket of the active site of
TyrTS, the adenine and phenolic units of ATP and L-Tyr are
hooked to the acridone moiety through π–π interactions, and
their tails, placed in one direction over the two arms of 1,
come into proximity when the carboxyl group of Tyr is desir-
ably oriented to react over the phosphorus centre of ATP
the safety of compound
1 under biological conditions
(Fig. S84†), though the terminal ester moieties underwent
hydrolysis to give 6. Although compound 6 exhibited inter-
actions with ATP and L-Tyr (Fig. 2 and S85†), the better
response of 1 meant that it was preferred for use in the
present study. With a stoichiometry of 1 : 1 (Fig. S86–S89†) and
respective Ka values of 9.5 0.4 × 103 M−1 and 9.1 0.4 × 104
M
−1, the strong affinity of compound 1 for ATP and L-Tyr was
established. Intriguingly, the synergistic effects of ATP and
L-Tyr on the fluorescence quenching of 1 were noticed when a
mixture of ATP and L-Tyr (each at 1 equiv. of 1) led to a similar
decrease in the fluorescence intensity as was seen individually
for 25 equiv. of ATP and 4 equiv. of L-Tyr (Fig. 2).
The compound 1 mediated formation of tyrosyl adenylate
Since compound 1 tolerated both ATP and L-Tyr, expectedly, (Fig. 4B and S115†). The proposed interactions of ATP and
the LC-MS data of an incubated solution of 1, ATP, L-Tyr and L-Tyr with 1 justify the selectivity of the reagent for ATP/GTP
Mg(OAc)2 showed the formation of tyrAd (Fig. 3). and aromatic amino acids. Remarkably, the 1H NMR spectrum
Chromatographic purification of the reaction mixture provided of 1 + ATP + L-Tyr did not exhibit a change in the chemical
tryAd (65%) (Fig. S90†) along with an almost quantitative shifts of aromatic H protons of 1 (H1, H8 and phenolic Hs) at
recovery of compound 1 (90%). The kinetic parameters Km and 50 °C (Fig. S116†), indicating a disturbance to π–π interactions
1
Kcat of the reagent 1 catalyzed formation of tyrAd, calculated at higher temperatures. We did not observe changes in the H
via the change in tyrAd intensity in the LC-MS data from the NMR spectra of compounds 8 and 10 when ATP and/or L-Tyr
reaction mixture, were 7.4 1.0 × 10−4 M and 0.53 s−1, respect- were added to their respective solutions, signifying the role of
ively, and Kcat/Km was 7.1 1.0 × 102 M−1 s−1 (Fig. S91–97 and phenolic OH and the synergism in the two arms of compound
S99†). Noticeably, Kcat in the absence of Mg(OAc)2 was too low 1 for interactions with ATP and L-Tyr. L-Tyr did not alter the
to calculate. The respective constants for the TyrTS catalyzed chemical shifts of ATP H protons in the absence of compound
formation of tyrAd were 8.1 1.4 × 10−4 M and 9.3 s−1 with 1 (Fig. S110†).
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Fig. 5 HRMS signatures of: (A) a reaction mixture containing L-Tyr
(400 µM), ATP (490 µM) and 20 µL of diluted TyrTS in 2 ml of buffer
(3 µM HEPES, 50 mM MgCl2 and 80 mM KCl in ultrapure water); (B) solu-
tion A + 20 µL of tRNA; and (C) a reaction mixture containing ATP
(490 µM), L-Tyr (400 µM), TyrTS (20 µL) and compound 1 in 2 ml of
buffer showing the formation of tyrAd.
1093 [2M + Na + K–H]+), indicating the transfer of tyrAd from
TyrTS to tRNA. However, a mass spectrum of a reaction
mixture of ATP, L-Tyr, MgCl2, KCl, TyrTS and 1 in HEPES
buffer after incubation for 10 min at 37 °C exhibited a peak at
m/z = 593.0914, corresponding to the mass of tyrAd (calcd m/z:
593.0534 [M + 2Na + K–H]+) (Fig. 5C and S119†). In contrast to
the previous experiment (performed in the absence of 1), the
peak at m/z = 53 958 was absent in this spectrum, but peaks
for TyrTS at m/z = 52 879 and 74 320 were seen (Fig. S119A–C†).
Clearly, the presence of 10 µM compound 1 did not allow 50
nM TyrTS (enzyme conc. for the experiments) to make tyrAd.
Moreover, the addition of tRNA to this reaction mixture did
not affect the peak due to tyrAd at m/z = 593.0914 (Fig. S120†).
The tolerability of 1 for TyrTS was checked by varying the con-
centration of TyrTS and it was found that with 10 µM 1 the
enzymatic reaction was accomplished with >100 nM TyrTS
(Table 1). We inferred that compound 1 catalyzed the for-
mation of tyrAd that remains in the free state (not bound to
TyrTS) and the addition of tRNA to the reaction mixture did
not pick up free tyrAd, as the peak at m/z = 593.0940 is
unaffected (Fig. S119†). Additional observations (mass spectral
analysis) that a mixture of tyrAd (prepared in the lab),27 ATP,
MgCl2, KCl, TyrTS and tRNA in HEPES buffer did not result in
the transfer of tyrAd to either TyrTS or tRNA (Fig. S121†) also
Fig. 4 (A) Changes in the 1H chemical shifts of 1 (blue trace) upon the
addition of ATP (red trace) and L-Tyr (black trace). Solvent: DMSO–H2O– supported the preceding results that there was no interaction
DMSO-d6 (8 : 1 : 1 v/v); 25–40 °C. For all spectra, the reference peak of
DMSO is at 2.5461 ppm. (B) The proposed mechanism for the reaction
between ATP and L-Tyr.
of TyrTS/tRNA with free tyrAd. Apparently, reagent 1 interfered
Table 1 The interference of 1 in the TyrTS catalyzed formation of tyrAd
Study of the interference of 1 in the TyrTS catalyzed formation
of tyrAd
Formation of tyrAda
S. no.
Compound 1 (μM)
TyrTS (nM)
Freeb
Bound to TyrTSc
In order to assess the interference of compound 1 in the enzy-
matic reaction for tyrAd formation, we firstly ascertained the
TyrTS catalyzed formation of tyrAd and its subsequent transfer
to tRNA. Mass spectra of a solution of L-Tyr, MgCl2, KCl, ATP
and TyrTS showed a peak at m/z = 53 958 (Fig. 5A and S117†)
that was degraded to m/z = 52 879 (Fig. 5B, S118 and S118A†)
upon the addition of tRNA. Apparently, the m/z difference of
1079 between the mass peaks at m/z = 53 958 and 52 879
corresponds to the mass of two tyrAd molecules (calcd m/z:
1
2
3
4
5
10
10
10
10
5
50
75
100
125
50
✓
✓
✓
✗
✗
✗
✗
✓
✓
✗
a Mass spectral analysis: the enzyme catalyzed formation of tyrAd was
detected from the mass peak for a TyrTS-tyrAd adduct and the sub-
sequent transfer of tyrAd to tRNA. b Compound 1 mediated. c TyrTS
catalyzed.
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1
in the TyrTS mediated formation of tyrAd but it performed 1690 (CO); H NMR (400 MHz, DMSO-d6): δ 5.84–7.16 (br, 1H,
half the job of TyrTS.
exchangeable with D2O), 7.29–7.35 (m, 2H), 7.76 (d, J = 3.1 Hz,
2H), 8.21 (d, J = 7.9 Hz, 1H), 8.41–8.43 (m, 1H), 8.51 (d, J =
7.9 Hz, 1H), 11.93 (s, 1H, exchangeable with D2O); 13C NMR
(101 MHz, DMSO-d6): δ 114.97 (C), 118.63 (+ve, CH), 119.97
(+ve, CH), 120.23 (C), 120.60 (C), 122.01 (+ve, CH), 125.62 (+ve,
CH), 132.41 (+ve, CH), 134.10 (+ve, CH), 136.89 (+ve, CH),
139.91 (C), 141.20 (C), 169.14 (C), 176.52 (C); HRMS (micro
TOF-QII, MS, ESI): calcd for C14H9NO3 [M + H]+: 240.0655;
found: 240.0529.
Conclusions
Conclusively, compound 1 allowed the biogenic route to
5′-tyrosyl adenylate to be emulated, and it will incite the
design of other such reagents. The strategic plan involving the
partial mimicking of TyrTS may make the biomodel reagent
capable of breaking up the continuity of the protein synthesis
process at the step of tyrosyl tRNA formation, making it an
alternative therapeutic approach in place of the usual enzyme
inhibition, because enzyme inhibitory drugs, due to promis-
cuity or strong inhibition, are prone to side effects. Studies
into these applications of this reagent are underway.
Synthesis of prop-2-yn-1-yl 9-oxo-9,10-dihydroacridine-4-
carboxylate (3)
9-Oxo-9,10-dihydroacridine-4-carboxylic acid (1 g, 4.18 mmol)
was dissolved in 30 ml of DMF in a round bottom flask
(100 mL) fixed with a guard tube. K2CO3 (577 mg, 4.18 mmol),
propargyl bromide (0.37 ml, 4.18 mmol) and a catalytic
amount of KI were added in sequence. The reaction was
stirred overnight at 60 °C. After the completion of the reaction
(TLC), ethyl acetate was added to the reaction mixture, and it
was washed with distilled water. The organic layer was then
washed with a saturated solution of NaHCO3, dried over
Na2SO4 and concentrated under vacuum. The residue was puri-
fied by washing with diethyl ether to yield a yellow solid: yield
Experimental
General notes
Melting points were determined using capillaries and are
uncorrected. 1H and 13C NMR spectra were recorded using
Bruker 500 and 125 MHz NMR spectrometers, respectively,
using CDCl3 and/or DMSO-d6 as solvents. Chemical shifts are
given in ppm with TMS as an internal reference. J values are
given in hertz. Signals are abbreviated as: singlet: s; doublet: d;
double–doublet: dd; triplet: t; and multiplet: m. Mass spectra
were recorded using a Bruker micrOTOF Q II Mass spectro-
meter. In 13C/DEPT-135 data, +ve signs correspond to signals
due to CH3 or CH groups, while −ve signs symbolize signals
from CH2 groups, and signals from quaternary carbon are
absent (ab) in DEPT-135 spectra. Column chromatography was
performed with silica 60–120 mesh, and reactions were moni-
tored via thin layer chromatography (TLC) with silica plates
precoated with silica gel GF-254 (Qualigens, India). Some of
the reactions with the biomodel reagent and enzymatic reac-
tions were monitored with HRMS. IR, UV-vis, and fluorescence
spectral data were recorded using FTIR Agilent CARY 630 and
BIOTEK Synergy H1 Hybrid Reader instruments.
36%; ν (cm−1): 3257 (NH), 3201 (CH), 1694 (CO); 1H NMR
¯
(500 MHz, CDCl3): δ 2.60 (t, J = 2.4 Hz, 1H), 5.02 (d, J = 2.1 Hz,
2H), 7.32–7.26 (m, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.67–7.71 (m,
1H), 8.43–8.48 (m, 2H), 8.74–8.75 (m, 1H), 11.59 (s, 1H); 13C
NMR (126 MHz, CDCl3): δ 52.93 (−ve, CH2), 75.70 (+ve, CH),
112.85 (C), 117.60 (+ve, CH), 119.95 (+ve, CH), 121.60 (C),
122.47 (C), 122.57 (+ve, CH), 127.08 (+ve, CH), 134.08 (+ve,
CH), 134.57 (+ve, CH), 136.86 (+ve, CH), 140.04 (C), 141.91 (C),
167.50 (C), 177.70 (C); HRMS (micro TOF-QII, MS, ESI): calcd
for C17H11NO3: 278.0811 [M + H]+; found: 278.0844.
Synthesis of prop-2-yn-1-yl 9-oxo-10-(prop-2-yn-1-yl)-9,10-
dihydroacridine-4-carboxylate (4)
In a round bottom flask fixed with a guard tube, compound 3
(100 mg, 0.36 mmol) was dissolved in 10 ml of DMF. K2CO3
(75 mg, 0.54 mmol), propargyl bromide (0.05 ml, 0.54 mmol)
and a catalytic amount of KI were added in sequence. The reac-
tion was stirred overnight at 60 °C. After the completion of the
Synthesis of 9-oxo-9,10-dihydroacridine-4-carboxylic acid (2)
A mixture of 2-chlorobenzoic acid (10 g, 58.62 mmol), anthra- reaction (TLC), the reaction mixture was diluted with ethyl
nilic acid (8.04 g, 58.62 mmol), K2CO3 (12.14 g, 87.94 mmol) acetate and washed with distilled water. The organic layer was
and CuO (0.69 g, 8.79 mmol) in isoamyl alcohol was refluxed separated and passed through Na2SO4 and distilled off under
for 36 h. Isoamyl alcohol was distilled off on a rotary evapor- reduced pressure to yield a solid that was used without further
ator and hot water was added to the resulting mixture, fol- purification.
lowed by filtration. To the filtrate, conc. HCl was added to
cause precipitation. The solid product obtained after filtration
Synthesis of methyl 2-azido-3-(4-hydroxyphenyl)propanoate
was dried. The solid product was taken in a beaker and con- L-Tyrosine methyl ester hydrochloride (500 mg, 2.56 mmol)
centrated H2SO4 (100 mL) was added, and the mixture was was dissolved in MeOH (13 mL). K2CO3 (846.56 mg,
heated in a water bath for 4 h. The reaction mixture was then 3.07 mmol), CuSO4 (6.36 mg, 1 mol%) and imidazole sulpho-
cooled to room temperature and poured into distilled water, nyl azide hydrochloride (ISA.HCl) (643.89 mg, 3.07 mmol)
resulting in the formation of precipitates that were filtered to were added to the reaction mixture and it was stirred for 6 h at
obtain 9-oxo-9,10-dihydroacridine-4-carboxylic acid: green room temperature. After the completion of the reaction, it was
solid; yield 87%; mp >300 °C; ν (cm−1): 3226 (NH), 2881 (OH), quenched with water and extracted with ethyl acetate. The
¯
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organic layer was separated and dried over Na2SO4 and concen- using ethyl acetate–hexane (7 : 3) as eluent to isolate the
trated under vacuum. The residue was purified via column product: yield 10%; mp: 110–112 °C; [α]2D5 = −30° (0.5, CHCl3);
chromatography as a pale yellow liquid: yield 90%; 1H NMR 1H NMR (400 MHz, CDCl3): δ 3.14 (d, J = 6.7 Hz, 2H), 3.34–3.50
(400 MHz, CDCl3): δ 2.92–3.13 (m, 2H), 3.78 (s, 3H), 4.03 (dd, (m, 2H), 3.69 (s, 3H), 3.76 (s, 3H), 5.36 (m, 1H), 5.42–5.46 (m,
J = 8.7, 5.5 Hz, 1H), 5.03 (s, 1H), 6.79 (dd, J = 6.4, 2.3 Hz, 2H), 4H), 5.54–5.61 (dd, J = 9.90, 5.41 Hz, 1H), 6.25 (d, J = 8.3 Hz,
7.09 (dd, J = 6.4, 1.8 Hz, 2H), 7.26 (s, 1H); 13C NMR (101 MHz, 2H), 6.45 (d, J = 8.8 Hz, 2H), 6.56 (d, J = 8.3 Hz, 2H), 6.73 (s,
CDCl3): δ 36.84 (−ve, CH2), 52.65 (+ve, OCH3), 63.44 (+ve, CH), 1H), 6.77 (d, J = 8.45 Hz, 2H), 7.21 (d, J = 10.9 Hz, 1H), 7.24 (d,
115.54 (+ve, CH), 127.99 (C), 130.42 (+ve, CH), 154.91 (C); J = 7.75 Hz, 2H), 7.43–7.45 (m, 1H), 7.51 (t, J = 7.62 Hz, 1H),
HRMS (micro TOF-QII, MS, ESI): calcd for C10H11N3O3: 7.77 (s, 1H), 8.03 (d, J = 6.87 Hz, 1H), 8.28 (d, J = 7.55 Hz, 1H),
239.1139 [M + NH4]+; found: 239.0970.
8.51 (d, J = 7.90 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 37.62
(−ve, CH2), 37.76 (−ve, CH2), 49.54 (−ve, CH2), 53.18 (+ve,
OCH3), 53.25 (+ve, OCH3), 58.55 (−ve, CH2), 64.22 (+ve, CH),
64.35 (+ve, CH), 115.79 (+ve, CH), 116.01 (+ve, CH), 118.62
(+ve, CH), 121.27 (C), 121.78 (+ve, CH), 122.87 (+ve, CH),
Synthesis of (1-(3-(4-hydroxyphenyl)-1-methoxy-1-oxopropan-2-
yl)-1H-1,2,3-triazol-4-yl)methyl 9-oxo-9,10-dihydroacridine-4-
carboxylate (5)
Compound 3 (100 mg, 0.36 mmol) was dissolved in 18 mL of 123.21 (+ve, CH), 123.91 (C), 124.92, 125.12 (C), 125.41 (C),
t-butanol. To this solution, sodium ascorbate (3.57 mg, 125.66 (C), 127.08 (CH), 129.61 (CH), 129.94 (CH), 131.54 (CH),
5 mol%) in 1 mL of water, CuSO4·5H2O (1.79 mg, 2 mol%) in 134.06 (CH), 137.23 (CH), 141.85 (C), 142.47 (C), 142.82 (C),
1 mL of water and methyl 2-azido-3-(4-hydroxyphenyl)propano- 143.20 (C), 155.74 (C), 155.77 (C), 166.77 (C), 168.33 (C), 168.59
ate (79.78 mg, 0.36 mmol) were added in sequence and the (C), 178.68 (C); HRMS (micro TOF-QII, MS, ESI): calcd for
mixture was stirred for 14 h at 60 °C. The reaction was moni- C40H35N7O9: 758.2569 [M + H]+; found: 758.2670.
tored with TLC. After the completion of the reaction, the solu-
(S)-2-(4-((4-(((1-((S)-1-Carboxy-2-(4-hydroxyphenyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)carbonyl)-9-oxoacridin-10(9H)-yl)
methyl)-1H-1,2,3-triazol-1-yl)-3-(4-hydroxyphenyl)propanoic
acid (6)
tion was diluted with water and extracted with ethyl acetate.
The organic layer was dried over Na2SO4 and distilled under
reduced pressure to yield a solid compound that was purified
by washing with diethyl ether to yield a yellowish solid: yield
61%; mp: 197–200 °C; [α]2D5 = −65° (0.5, DMSO); ν (cm−1): 3293 Compound 1 (30 mg, 0.039 mmol) was dissolved in a
¯
(OH), 1742 (CO); 1H NMR (500 MHz, DMSO-d6): δ 3.40–3.48 2 : 1 mixture of acetone and water (5 mL of acetone, 2.5 mL of
(m, 2H), 3.71 (s, 3H), 5.53 (d, J = 4.64 Hz, 2H), 5.81 (dd, J = water) followed by the addition of LiOH (1.66 mg,
10.41, 5.3 Hz, 1H), 6.53 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 0.079 mmol). The reaction mixture was stirred at 35 °C and
2H), 7.35–7.41 (m, 2H), 7.79–7.87 (m, 2H), 8.25 (d, J = 7.9 Hz, monitored with TLC. After the completion of the reaction,
1H), 8.37 (d, J = 7.6 Hz, 1H), 8.41 (s, 1H), 8.58 (d, J = 7.9 Hz, acetone was removed using a rotary evaporator and the reac-
1H), 9.23 (s, 1H, exchangeable with D2O), 11.60 (s, 1H, tion mixture was cooled in an ice bath. 1 N HCl was added to
exchangeable with D2O); 13C NMR (125 MHz, DMSO-d6): δ 3.46 cause precipitation and the precipitate was collected via
1
(−ve, CH2), 53.35 (+ve, OCH3), 59.11 (−ve, CH2), 63.88 (+ve, vacuum filtration to procure the pure product: 35%; H NMR
CH), 114.63 (C), 115.53 (+ve, CH), 119.09 (+ve, CH), 120.84 (500 MHz, DMSO-d6): δ 3.05–3.04 (m, 4H, 2 × CH2), 5.30–5.45
(+ve, CH), 122.24 (C), 123.00 (+ve, CH), 125.70 (+ve, CH), (m, 5H, 2 × CH2), 5.63 (br, 1H), 6.38–6.47 (m, 6H, ArH), 6.86
126.06 (C), 126.43 (+ve, CH), 130.34 (+ve, CH), 133.54 (+ve, (d, 2H, ArH, J = 5.94 Hz), 7.30 (t, J = 6.38 Hz, 1H, ArH), 7.43 (t,
CH), 134.71 (+ve, CH), 137.10 (+ve, CH), 140.52 (C), 141.28 (C), J = 7.57 Hz, 1H, ArH,), 7.68 (s, 2H, ArH), 7.98 (d, J = 6.87 Hz,
141.87 (C), 156.60 (C), 166.80 (C), 169.40 (C), 176.90 (C); HRMS 1H, ArH), 8.04 (d, J = 6.0 Hz, 1H, ArH), 8.17 (d, J = 7.34 Hz, 1H,
(micro TOF-QII, MS, ESI): calcd for C27H22N4O6: 499.1612 ArH), 8.35 (s, 1H), 8.45 (d, J = 6.59 Hz 1H, ArH), 9.21 (s, 2 ×
[M + H]+; found: 499.1453.
OH), 13.22–13.89 (br, OH); 13C NMR (126 MHz, DMSO-d6): δ
35.90 (CH2), 36.10 (CH2), 48.58 (CH2), 58.54 (CH2), 63.71 (CH),
63.86 (CH), 114.95 (CH), 119.11 (CH), 121.49 (CH), 121.84 (C),
122.36 (C), 123.68 (CH), 124.92 (CH), 125.21 (CH), 125.81 (C),
125.96 (CH), 126.03(C), 129.39 (CH), 129.68 (CH), 130.31 (CH),
133.60 (CH), 136.24 (CH), 141.04 (C), 141.64 (C), 142.50 (C),
Synthesis of (1-(3-(4-hydroxyphenyl)-1-methoxy-1-oxopropan-2-
yl)-1H-1,2,3-triazol-4-yl)methyl 10-((1-(3-(4-hydroxyphenyl)-1-
methoxy-1-oxopropan-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-9-oxo-
9,10-dihydroacridine-4-carboxylate (1)
Compound 4 (50 mg, 0.16 mmol) was dissolved in 18 mL of 143.03 (C), 155.83 (C), 155.97 (C), 166.51 (CvO), 169.47
t-butanol. To this solution, sodium ascorbate (12.57 mg, (CvO), 169.73 (CvO), 176.84 (CvO); HRMS (micro TOF-QII,
40 mol%) in 1 mL of distilled water, CuSO4·5H2O (7.89 mg, MS, ESI): calcd for C38H31N7O9: 730.2256 [M + H]; found:
20 mol%) in 1 mL of water and methyl 2-azido-3-(4-hydroxy- 730.2250.
phenyl)propanoate (70.15 mg, 0.31 mmol) were added in
sequence and the reaction was stirred at 60 °C for 20 h. After
the completion of the reaction (TLC), the solution was diluted
with distilled water and extracted with ethyl acetate. Ethyl
acetate was distilled off under reduced pressure to yield a solid
compound that was purified with column chromatography 17%; mp: 105–107 °C; [α]2D5 = +40° (0.5, CHCl3).
(1-((R)-3-(4-Hydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1H-
1,2,3-triazol-4-yl)methyl 10-((1-((R)-3-(4-hydroxyphenyl)-1-
methoxy-1-oxopropan-2-yl)-1H-1,2,3-triazol-4-yl)methyl)-9-oxo-
9,10-dihydroacridine-4-carboxylate (7)
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(1-((S)-1-Methoxy-1-oxo-3-phenylpropan-2-yl)-1H-1,2,3-triazol-4- TyrAd was obtained as a thick oil: [α]2D5 = −18° (0.5, DMSO);
yl)methyl 10-((1-((S)-1-methoxy-1-oxo-3-phenylpropan-2-yl)-1H-
1,2,3-triazol-4-yl)methyl)-9-oxo-9,10-dihydroacridine-4-
carboxylate (8)
1HNMR (400 MHz, DMSO-d6 + D2O, 9 : 1): δ 8.43 (s, 2H), 8.09
(d, J = 4.6 Hz, 3H), 7.00 (d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.7 Hz,
2H), 5.86 (d, J = 6.4 Hz, 1H), 4.60–4.49 (m, 2H), 4.15 (t, J = 3.9
Hz, 1H), 3.99–4.04 (m, 1H), 3.92 (d, J = 8.4 Hz, 1H), 3.76–3.78
(m, 1H), 3.38 (q, J = 4.3 Hz, 1H), 2.98 (dd, J = 14.4, 4.3 Hz, 1H),
2.72 (dd, J = 14.4, 8.5 Hz, 1H); 13C DEPT NMR (101 MHz): δ
36.03 (−ve, CH2), 49.18 (+ve), 56.32 (+ve), 64.04 (−ve, CH2),
71.03 (+ve), 74.91 (+ve), 79.39 (+ve), 84.99 (+ve), 87.68 (+ve),
115.58 (+ve), 130.92 (+ve), 144.27 (+ve), 153.18 (+ve), 171.48
(CvO); HRMS (micro TOF-QII, MS, ESI): calcd for
C19H23N6O9P: 511.1336 [M + H]+; found: 511.1330.
Compound 4 (150 mg, 0.476 mmol) was dissolved in 54 ml of
t-butanol. To this solution, sodium ascorbate (37.72 mg,
40 mol%) in 3 mL of distilled water, CuSO4·5H2O (23.67 mg,
20 mol%) in 3 mL of distilled water and methyl (S)-2-azido-3-
phenylpropanoate (195.23 mg, 0.952 mmol) were added in
sequence and the reaction was stirred at 60 °C for 20 h; the
reaction was monitored with TLC. After the completion of the
reaction, the solution was diluted with distilled water and
extracted with ethyl acetate. Ethyl acetate was distilled off
under reduced pressure to yield a solid that was purified with
Conflicts of interest
column chromatography to isolate the product: 17%; mp:
1
120–123 °C; [α]2D5 = −50° (0.5, CHCl ); ν (cm−1): 1746 (CO); H
¯
3
There are no conflicts to declare.
NMR (400 MHz, CDCl3): δ 3.35–3.16 (m, 2H, CH2), 3.56–3.43
(m, 2H, CH2), 3.64 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 5.48–5.28
(m, 5H, 2CH2, CH), 5.60 (dd, J = 8.7, 6.4 Hz, 1H), 6.69 (d, 2ArH,
J = 6.9 Hz), 6.93 (s, 1H), 7.02 (d, 2ArH, J = 3.2 Hz), 7.17–7.06
Acknowledgements
(m, 6ArH), 7.34–7.28 (m, 2ArH), 7.41 (d, 1ArH, J = 8.2 Hz), We gratefully acknowledge SERB-DST, New Delhi. H. S. acknowl-
7.59–7.55 (m, 1ArH), 7.78 (s, 1H), 8.04 (dd, J = 7.5, 1ArH, 1.6 edges CSIR, New Delhi, and B. K. and H. K. acknowledge UGC
Hz), 8.43–8.40 (m, 1ArH), 8.66 (dd, 1ArH, J = 7.8, 1.4 Hz); 13C New Delhi, for their fellowships. UGC, New Delhi, is acknowl-
NMR (101 MHz, CDCl3): δ 38.36 (CH2), 38.77 (CH2), 49.98 edged for a grant under the UPE programme.
(CH2), 53.09 (CH), 53.14 (CH), 58.68 (CH2), 64.01 (CH3), 64.09
(CH3), 118.55 (CH), 121.42 (CH), 121.49 (CH), 122.36 (CH),
122.59 (C), 124.11 (C), 124.43(CH), 125.61 (C), 127.21 (CH), References
127.48 (CH), 127.57 (CH), 128.57 (CH), 128.74 (CH), 128.78
(CH), 128.85 (CH), 131.57 (CH), 133.68 (CH), 134.34 (C), 134.54
(C), 136.68 (CH), 142.00 (C), 143.00 (C), 143.27 (C), 143.38 (C),
1 D. J. Cram, Angew. Chem., Int. Ed. Engl., 1988, 27, 1009–
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