Phosphate linkers with traceable cyclic intermediates for self-immolation detection and monitoring

Article abstract of DOI:10.1039/d0cc06928k

Self-immolation (SI) is the key principle of ProTide nucleotide prodrugs such as remdesivir, which is currently used to treat COVID-19 patients. Developing novel tailor-made SI systems requires new analytical methods for the detection and monitoring of SI. We developed a robust method for SI analysis using novel phosphate-based SI linkers with NMR traceable cyclic intermediates to distinguish SI from alternative fragmentation pathways and to monitor cargo release in real time.

Full text of DOI:10.1039/d0cc06928k

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DOI: 10.1039/D0CC06928K
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Phosphate Linkers with Traceable Cyclic Intermediates for Self-
Immolation Detection and Monitoring
Eliška Procházková,a Petr Šimon,b Michal Straka,a Juraj Filo,c Michal Májek,c Marek Cigáňc and
Ondřej Baszczyňskib,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Self-immolation (SI) is the key principle of ProTide nucleotide for reaction monitoring by 31P NMR spectroscopy. Our approach
prodrugs such as remdesivir, which is currently used to treat COVID- provides both kinetic and structural data and can be used as a
19 patients. Developing of novel tailor-made SI systems requires diagnostic tool for screening newly developed SI linkers.10,11
new analytical methods for detection and monitoring the SI. We
Here, we present another advancement involving
developed a robust method for SI analysis using novel phosphate- phosphate-based SI linkers, more specifically, the development
based SI linkers with NMR traceable cyclic intermediates to of a new method for detecting and monitoring SI processes
distinguish SI from alternative fragmentation pathways and to based on cyclic phosphate intermediates, which are stable
monitor cargo release in real time.
enough for direct NMR detection. This method allows us to
clearly differentiate self-immolation from other processes, such
as hydrolysis or alternative decomposition with undesired side-
products, and thereby overcoming the limitations of optical
spectroscopy monitoring.
We propose the following SI mechanism for the newly
designed linkers 1–6: a) photoactivation, cleavage of the photo-
removable dimethoxy nitrobenzyl (DMNB) moiety and fast
release of CO2, yielding the activated intermediate I, b) self-
immolation: cyclisation and cargo release, affording the cyclic
intermediate cyc-I, and c) spontaneous cyc-I ring-opening,
providing the final product P (Fig. 1). To enhance the
functionality of our method, we used the most promising
ethylene glycol linker in compound 2 (X, Y = O) to study cargo
release in derivatives 7–10.
Self-immolative (SI) linkers are subjected to irreversible
fragmentation initiated by an external trigger and can be used
to develop smart materials (e.g., degradable polymers) or drug
delivery systems.1,2 In principle, the activated linker forms a
cyclic intermediate, which tends to release the leaving group
(cargo).3,4 Accordingly, phosphate-based SI linkers are more
versatile than their carbon analogues because phosphorus has
a higher valency (V) than carbon (IV), thus enabling an
additional substituent.5–7
SI processes are typically monitored by optical spectroscopy
using chromogenic/fluorogenic substrates.8 This approach is
versatile and easy to use, e.g., in high-throughput screenings of
structurally similar derivatives. However, optical spectroscopy
provides only limited data on complex reaction sequences. This
limited knowledge may lead to misinterpretation of spectral
data. In addition, SI itself has other drawbacks: 1) Enzymatic
activation of SI may alter the SI process as a result of substrate
specificity,9 and chemical activation requires carefully handling
chemical precursors and controlling both temperature and
activation time. 2) For each chromogenic/fluorogenic moiety,
the assay must be re-optimised. 3) Colourless leaving groups
cannot be monitored directly. To overcome these obstacles, we
recently proposed photo-activated, phosphate-based SI linkers
O
Spacer optimization
365 nm
O
O
P
O

HX
X
P
O
Y
O
Y
O
a)
-CO2
O
O
O
I
NO2
Intermediate
R
R
Cpd
1
2
3
4
5
6
7
8
X
O
O
NH
S
NH
S
O
O
O
Y
NH
O
O
O
NH
NH
O
O
O
R
H
H
H
H
H
H
OCH3
CH3
F
Cargo release
b)
Cargo optimization
O
O
X
P
O
Y
HX
P
O-
Y
O
a. Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences,
Prague 166 10, Czech Republic
c)
9
P
cyc-I
b. Faculty of Science, Charles University, Prague 128 43, Czech Republic
c. Department of Organic Chemistry, Faculty of Natural Sciences, Comenius
University, Bratislava 842 15, Slovakia.
Final product
Cyclic intermediate
10
O
O
NO2
Fig. 1 Chemical structure of phosphate-based linkers 1–10 studied in this work and the
proposed mechanism of self-immolation; a) photoactivation, and CO2 release; b)
cyclisation, and cargo release; c) cyclic intermediate cyc-I ring opening.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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The SI reactions of 1–6 were monitored by 31P NMR
Journal Name
Compounds 5 and 6 formed intermediates 5-IVaienwdAr6tic-IleuOpnloinne
spectroscopy, upon continuous irradiation, which revealed photoactivation, with no further spectralDcOhIa: n10g.e10(3F9i/gD.0SC1C40i6n9t2h8eK
alterations. Based on the results, these reactions were divided SI).
into three main classes: a) Successful SI process with phenol
release (1, 2), b) spacer self-cleavage without phenol release (3, we selected the ethylene glycol linker used in 2 as the best
4) and, c) photoactivation with no further reaction (5, 6). candidate for a more detailed mechanistic study of cargo
To demonstrate the practical use of the proposed method,
Self-immolation of 1 and 2 (Figs. 2A and 2B, respectively) release. We prepared derivatives 7–10 differing in para-
followed the proposed reaction pathway (Fig. 1). substituents on the phenyl ring (Fig. 1). In all cases, the para-
Photoactivation of 1 (δP +5.8 ppm) and 2 (δP –6.5 ppm) provided substituted phenol was released by SI upon irradiation, as
the corresponding intermediates 1-I and 2-I (δP +6.3 and –6.1 indicated by the formation of 2-cyc-I (18.1 ppm), (Fig. S15 in the
ppm, respectively), which consequently formed the cyclic ESI). As expected, para-substitution had a strong effect on the
intermediate cyc-I, as detected in 31P spectra14 (26.9 and 18.1 cyclisation rate, as shown by the formation of the
ppm, respectively). Interestingly, 2-cyc-I was formed ca. four corresponding intermediates I (Table 1). Compounds 7 and 8,
times faster than 1-cyc-I, thus yielding the final product 2-P in with the electron-donating OCH3 and CH3, respectively, had
90 minutes, while 1-P was formed overnight (Fig. S7 in the ESI). slower cyclisation rates than 9 and 10 and provided the highest
The detection of the cyclic intermediate clearly confirms that concentrations of I (ca. 35 % in 30 min. of irradiation). The
both compounds 1 and 2 release phenol by self-immolation results have also highlighted a strong correlation between
(Figs. S7–S11 in the ESI). More importantly, the presence of Hammett constants, the pKa of the corresponding phenols
cyclic intermediates could not be detected by optical (leaving groups), and the general ability of I to cyclise. The
spectroscopy, the conventionally used method.
better the leaving group was (in 10 with the electron-
Surprisingly, compounds 3 and 4, as single-atom alterations withdrawing NO2 group with p=0.81 and pKa=7.15), the lower
of 2, where X=O is substituted by NH and S, respectively, the concentration of the intermediate [I]rel and, accordingly, the
provided no cyclic intermediate or the desired final product P faster the cyclisation and cargo release were (Table 1).
(Figs. 2C and 2D, respectively). Moreover, an identical side Importantly, slow spontaneous hydrolysis of 10 could be easily
product, sideP (δP –5.4 ppm), was detected in both cases (Figs. misinterpreted for self-immolation, especially when using
S12–S13 in the ESI), indicating that the spacer was released optical spectroscopy, because both reaction mixtures become
instead of the phenol. For 3, this result can be explained by the yellow (Fig. 3).
inability of the spacer to cyclise due to protonation of the
To test whether photo-activation would account for any
terminal NH2. Instead, the system undergoes simple chemical discrepancies in our study, we checked the photochemical
hydrolysis of the spacer. For 4, we assume self-cleavage of the activation rates of 1–6, which were comparable within the
thiol spacer through a known thiirane mechanism.15,16 Spacer series (same order of magnitude), while the photolysis quantum
self-cleavage may find its application in the design of yields (QYs) of derivatives 3 and 5 differed slightly (Table S1 in
phosphorylating agents (e.g., amino acid or protein the ESI). Therefore, we performed a computational study to
phosphorylation) and antiviral prodrugs (e.g., ProTides assess this effect (Fig. 4 and ESI Section 2). Based on the
concept).17–21 Note, however, that no spacer self-cleavage was literature, we expected
a
correlation between the
observed in either 1 or 2.
experimentally obtained QYs and the calculated activation
energy ΔG(TS-S1) of excited-state hydrogen transfer (ESHT) in the
first singlet excited state S1.22
Fig. 2 A series of 31P NMR spectra of compounds 1–4 and their derivatives in 50% CACO*/DMSO (0.5 mM) recorded upon continuous irradiation by UV light (365 nm) at room
temperature. The patterns for compounds 5 and 6, where intermediates 5-I and 6-I do not provide any further reaction, are shown in ESI (Fig. S14). * CACO = cacodylate buffer (0.1
M, pH 7.4).
2 | J. Name., 2012, 00, 1-3
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Table 1 Effect of para-substituents on the rate of cargo release
DOI: 10.1039/D0CC06928K
NO2
O
O
O
O
OH
N
H
O
N
O

31P[b]
O
X
ESHT
G(TS-S )

1
26
[a]
30min.
Cpd
R
p
pKa
[I]rel
O
X
O
X
(ppm)
O
O
O
X
O
S
NH
Cpd
O
S1
2
7
H
OCH3
CH3
F
0
9.9827
10.5528
10.1427
9.9529
–6.52
0.31
1
2
6
O
O
,
4
,
TS
3
5
,
–0.28
–0.14
0.15
0.81
–6.11
–6.37
–6.43
–7.51
0.34
0.36
0.16
0[c]
Fig. 4 The transition state structures proposed for the photoactivation step. For
simplicity, the “ethylene glycol” spacers were substituted by a methyl group.
8
9
For further insight into the self-immolation of 1 and 2,
DFT calculations were performed. The reaction pathway was
pre-constructed based on previous studies.10,24 The
corresponding reactants, products, and intermediates were
minimised and interconnected by optimised transition
states, as described in ESI in detail. The following mechanism
was proposed.
10
NO2
7.1527
[a]
[b]
pKa values of para-substituted phenol which is cleaved off by SI process.
[c]
Measured as 0.5 mM solution in 50% CACO/DMSO at room temperature. The
formed intermediate 10-I is immediately and quantitatively converted into the cyclic
intermediate 2-cyc-I.
After the initiation, the photosensitive DMNB moiety is
cleaved off, and fast CO2 release provides intermediate I.13
The proposed structure of intermediate I is supported by the
calculated 31P NMR chemical shifts (Table S2 in ESI), which
differ from those with a carbonate/carbamate system. I then
deprotonates to (I-H)-, which is able to attack the central P
atom whilst forming a cyclic intermediate cyc-I(OPh) via TS1,
as shown in Fig. 5. The predicted barrier for this process is
32.5 (23.1) kcal/mol for 1 (2), respectively. This barrier
includes an energy penalty for I-OH deprotonation of 28 and
21 kcal/mol for 1 (2) estimated from the calculated pKa of 1
(2) (ESI). The barrier is lower for 1 than for 2 (Fig. 5), which
qualitatively explains why the SI process is much faster for 1
(see above). The intermediate cyc-I(OPh) lies approximately
7 kcal/mol under the pre-reaction complex and rapidly
cleaves the phenolate group – this process has a barrier of
only 1.9 (2.0) kcal/mol for 1 (2) (see TS2 in Fig. 5). For this
reason, the cyc-I(OPh) intermediate (Fig. 5) could be
observed in gas-phase IRPD studies10 but not in the solution-
state NMR experiments performed here. The resulting stable
cyc-I intermediate was detected in NMR experiments (Figs.
2 and 3 and Table S1 in ESI). Overall, the SI process is
exothermic (Fig. 5). 25
For compounds 1, 2 and 4, 6 (X = O and S, respectively), the
height of the barrier in S1 was effectively the same (ΔG(TS-S1)
= 8.4 and 8.3 kcal/mol, respectively), which indicates similar
reactivity and corresponds to the determined QYs. The QYs
of these four compounds ranged from 0.003 to 0.009, in line
with previously reported values for a similar compound with
a slightly lower barrier of 7.8 kcal/mol translated to a QY of
0.008.22,23 In turn, the predicted activation energy ΔG(TS-S1) of
compounds 3 and 5 (X = N) was 7.3 kcal/mol, which explains
the slightly higher QYs (0.019 and 0.016, respectively).
Intersystem crossing (ISC) into the triplet state T2 was
also considered. We calculated a spin orbit coupling of 38
cm-1, the same value for all three model groups (X = O, S and
NH; Fig. 4). This is approximately half of the value reported
for the simple o-nitrotoluene derivatives.22 Therefore, we
hypothesize that ISC is less important in our compounds 1–6
than in the previously reported o-nitrobenzenes (details in
ESI) and that the difference in reactivit y between the O/S
and N-bearing compounds primarily results from the
difference in activation energy at the S1 singlet excited state
(ΔG(TS-S1)).
In conclusion, we introduced a new practical and
straightforward method for SI detection. For this purpose,
we designed novel SI linkers, which form cyclic intermediates
detectable by NMR spectroscopy. We have shown that self-
immolation on phosphate-based linkers is highly sensitive to
the phosphorus environment, particularly to both ends of
the activated SI linker. As such, the attacking end should
have a high affinity to phosphorus. In addition, the cargo
release rate is significantly affected by the pKa of the cargo
itself. The cyclic intermediates are easy to monitor by NMR
spectroscopy, thus enabling us to differentiate self–
immolation from hydrolysis and/or detect the formation of
side products, which could possibly alter the SI reaction.
Ultimately, this approach and our linkers will pave the way
to design novel SI constructs useful in Medicine (e.g.,
Fig.
3
Self-immolation versus hydrolysis clearly distinguished by 31P NMR
spectroscopy. Detection of the cyclic products cyc-I and cyc-P provides direct
evidence of self-immolation, which could not be obtained when using optical
methods.
antiviral prodrugs
– new phosphoramidate ProTide
analogues) and Materials Science (e.g., self-degradable
smart polymers).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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DOI: 10.1039/D0CC06928K
Fig. 5 Mechanism of the self-immolation of 2 proposed by DFT calculations using B3LYP/def2TZVP level in implicit solvent water, see ESI. As an example, the optimised structures of
compound 2 corresponding to energy profile in black are shown. An energy profile of compound 1 is shown in grey.
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This work was supported by the Experientia Foundation
(O.B., Start-Up grant SG-2018-1), the Czech Science
Foundation (O.B., grant No. 20-25137Y), and the Slovak
Research and Development Agency (M.C., grant APVV-15-
0495). We thank Dr. Ondřej Gutten for calculation the pKa
constants. We would also like to acknowledge Květoslava
Kertisová and Kateřina Nováková, from the Mass
Spectroscopy Department at IOCB, for the HR-MS spectra,
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