Phosphate-Based Self-Immolative Linkers for Tuneable Double Cargo Release

Article abstract of DOI:10.1002/chem.202101805

Phosphorus-based self-immolative (SI) linkers offer a wide range of applications, such as smart materials and drug-delivery systems. Phosphorus SI linkers are ideal candidates for double-cargo delivery platforms because they have a higher valency than carbon. A series of substituted phosphate linkers was designed for releasing two phenolic cargos through SI followed by chemical hydrolysis. Suitable modifications of the lactate spacer increased the cargo release rate significantly, from 1 day to 2 hours or 5 minutes, as shown for linkers containing p-fluoro phenol. In turn, double cargo linkers bearing p-methyl phenol released their cargo more slowly (4 days, 4 hours, and 15 minutes) than their p-fluoro analogues. The α-hydroxyisobutyrate linker released both cargos in 25 minutes. Our study expands the current portfolio of SI constructs by providing a double cargo delivery option, which is crucial to develop universal SI platforms.

Full text of DOI:10.1002/chem.202101805

Accepted Article
Accepted Article
Title: Phosphate-Based Self-Immolative Linkers for Tunable Double
Cargo Release
Authors: Petr Šimon, Markéta Tichotová, María García Gallardo, Eliška
Procházková, and Ondrej Baszczynski
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202101805
Link to VoR: https://doi.org/10.1002/chem.202101805
01/2020

10.1002/chem.202101805
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Phosphate-Based Self-Immolative Linkers for Tunable Double
Cargo Release
Petr Šimon,ǂ, [a] Markéta Tichotová,ǂ,[a,b] María García Gallardo,[a] Eliška Procházková,*, [b] and Ondřej
Baszczyňski*, [a,b]
We dedicate this paper to the memory of Dr. John C. Martin (1951–2021), head of Gilead Sciences, to commemorate his
achievements in the development of anti-HIV drugs (acyclic nucleoside phosphonates, “Holy Trinity”: A. Holý, E. De Clercq, J. C.
Martin).
[a]
Dr. P. Šimon, M. Tichotová, M.G. Gallardo, Dr. O. Baszczyňski
Faculty of Science, Charles University
Prague 128 43, Czech Republic
E-mail: baszczyo@natur.cuni.cz
ǂ These authors contributed equally.
[b]
M. Tichotová, Dr. E. Procházková, Dr. O. Baszczyňski
Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences
Prague 166 10, Czech Republic
E-mail: prochazkova@uochb.cas.cz
Supporting information for this article is given via a link at the end of the document.
Abstract: Phosphorus-based self-immolative (SI) linkers offer a wide
range of applications, such as smart materials and drug delivery
systems. Phosphorus SI linkers are ideal candidates for double cargo
delivery platforms because they have a higher valency than carbon.
Accordingly, we designed a series of substituted phosphate linkers for
releasing two phenolic cargos through SI followed by chemical
hydrolysis. Suitable modifications of the lactate spacer increased the
cargo release rate significantly, from 1 day to 2 hours or 5 minutes, as
shown for linkers containing p-fluoro phenol. In turn, double cargo
linkers bearing p-methyl phenol released their cargo more slowly (4
days, 4 hours, and 15 minutes, respectively) than their p-fluoro
analogues. The α-hydroxyisobutyrate linker released both cargos in
25 minutes. Our study expands the current portfolio of SI constructs
by providing a double cargo delivery option, which is crucial to develop
universal SI platforms.
release have been studied[13] to pursue effective cancer
chemotherapy utilising complementary drug combination,[14]
antibiotic drug resistance[15] or novel fluorescent reporters/signal
amplification chemosensors.[5a,16] In addition, phosphorus linkers
enable us to monitor the reaction pathway by 31P NMR
spectroscopy in real-time,[17] providing detailed structural and
kinetic information, and to even detect the cyclic intermediates in
some cases,[18] confirming cargo release via Self-immolation,
more specifically intramolecular cyclisation, and not via chemical
hydrolysis.
Self-immolation, and consequently the rate of the cargo
release reaction, can be tuned up by varying substituents on the
phosphorus atom and is mainly directed by two effects: 1) the
Thorpe-Ingold[19] effect and 2) the pKa effect of the leaving groups.
The Thorpe-Ingold effect refers to the spacer – sterically
demanding substituents in the α-position accelerate
intramolecular cyclisation.[20] Conversely, the pKa effect is related
to the cargo – the more acidic the cargo is (lower pKa), the faster
the cargo will be released upon external activation.[18] Fast cargo
release systems are usually designed for biological applications.
However, slow SI may also be advantageous when constructing
slowly–reacting (decomposing) polymers[8,21] or developing
prodrugs with delayed cargo release (e.g., antibiotics).[22]
Notwithstanding these advances and the potential of SI linkers,
no phosphate-based, double cargo release system has been
reported yet.
Considering the above, we designed a novel set of
phosphate linkers bearing a lactate spacer (Figure 1) to attach
two cargos (double cargo option). We also fine-tuned the release
of both cargos by introducing a suitable modification in the lactate
spacer and by adjusting the pKa of the leaving groups. SI was
triggered by UV light (365 nm) and monitored by 31P NMR
spectroscopy. The structures of intermediates and products were
determined in situ by combining 13C and 31P NMR spectra. Our
findings indicate that SI linkers with controlled double cargo
release may be applied to develop novel combination therapies
and smart materials.
Introduction
Self-immolative (SI) linkers are chemical constructs subjected to
irreversible fragmentation triggered by external stimuli.[1] After
external activation, SI linkers disassemble (e.g., via cyclisation or
electronic cascade), thereby releasing a leaving group (cargo).
Triggered cargo release can be used in drug delivery[2]
(prodrugs,[3] antibody-drug conjugates,[4] and chemosensors[5]),
smart materials[6] (stimuli-responsive SI dendrimers[7] and
polymers[8]), or in vivo cell labelling,[9] thus highlighting the wide
range of applications of SI linkers. In turn, different structural
motifs, including carbamate[3,6,10] or phosphate,[11] can be used to
attach chemically variable cargos to the SI linker.
Phosphorus-based SI linkers can offer higher versatility
than their carbamate analogues because they allow us to attach
an additional substituent (a second cargo), which significantly
broadens their potential applications. For example, a double
cargo linker can be used to simultaneously tether a drug (warhead,
first cargo) and a reporter molecule (chromogenic molecule,
second cargo).[12] Systems combining double (multi) cargo
1
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Figure 1. Chemical structure of phosphate-based linkers 1–16 and the proposed mechanism of self-immolation; a) photoactivation; b) intramolecular cyclisation
leading to 1st cargo release; c) 2nd cargo release (via chemical hydrolysis).
also tested during reaction optimisation, albeit without
success.[26] The reaction progress was monitored by 31P NMR
spectroscopy (for NMR chemical shifts in CDCl3, see Table S9
in ESI).
Results and Discussion
We performed irradiation NMR experiments on 1 and 2,
in a previously optimised mixture of cacodylate buffer/DMSO
(1/1, v/v). Indeed, both pilot compounds 1 and 2 cyclised upon
UV light irradiation, releasing the cargos overnight (Figure 2).
Compound 1, bearing the electron-withdrawing (EWG) p-F
substituent, provided two 31P NMR signals at P = –13.02 and
–13.10 ppm, corresponding to two diastereoisomers resulting
from a stereogenic centre on the phosphorus atom, and carbon
in the lactate spacer. Upon UV light irradiation, DMNB was
cleaved off, and intermediate 1-I (P = –12.55 and –12.64 ppm)
was formed in 5 minutes (full conversion of 1 to 1-I in 15
minutes). The activated linker 1-I slowly cyclised and afforded
mono-phenyl products 1-P1 (P = –6.17 ppm) and 1-P2 (P = –
5.98 ppm) overnight. The more acidic p-F-phenyl substituent
(pKa 9.95[27]) was released by SI first (1-P1), while the phenyl
Double cargo system: proof of concept
Our initial strategy was to prepare the two model double cargo
linkers with a lactate spacer as proof of concept. Compounds
1 and 2 were prepared by phosphorylation[23] of 22 using the
corresponding in situ-generated diphenyl phosphoro-
chloridates 18 and 19 (Scheme 1) in dichloromethane (DCM)
at room temperature. Photocleavable dimethoxy nitrobenzyl
(DMNB)[24] ester 22 was prepared from DMNB-alcohol 20 and
the lactic acid 21 via acid-catalysed esterification in refluxing
toluene. Phosphorochloridates 18 and 19 were synthesized
from phenyl dichloridate 17 and the corresponding phenol in
toluene at room temperature (Scheme 1), and 1 and 2 were
synthesized using N-methylimidazole (NMI)[25] as
base/catalyst (Scheme 1c). Other bases, such as triethylamine
(TEA), pyridine, and 4-(dimethylamino)pyridine (DMAP), were
a
Scheme 1. Synthesis of the two model double cargo linkers 1 and 2 prepared for a proof of concept study. Reaction conditions: a) “the corresponding phenol”,
TEA, toluene, 25 °C, 12 hours; b) p-toluenesulfonic acid, toluene, reflux, 16 hours; c) diphenyl chlorophosphate 18 (for 1) or 19 (for 2) NMI, DCM, 25 °C, 12 hours.
2
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substituent (with higher pKa 9.98[28]
)
remained mostly
(Scheme 2). The lactate derivative 22 was treated with highly
reactive ethyl dichloridate 23 and with one equivalent of TEA
in toluene at room temperature to give the corresponding
phosphorochloridate 24 (not isolated). Toluene was removed
at low pressure, and crude 24 was subsequently used in
reactions with the corresponding phenols in DCM, in the
presence of TEA, to give 3–7. The reaction progress was
monitored by 31P NMR spectroscopy (for NMR chemical shifts
in CDCl3, see Table S10 in ESI).
unscathed (1-P2). A trace amount of the fully hydrolysed
product P (P = 0.06 ppm) was observed after 4 days.
Compound 2, with the electron-donating (EDG) p-Me group (P
= –12.92 and –13.09 ppm), also afforded the activated
intermediate 2-I (P = –12.57 ppm), but its cyclisation
proceeded significantly slower than that of 1-I. After 24 hours,
2-I was still the major component (Figure 2). The desired
products 2-P1 (P = –6.19 ppm) and 2-P2 (P = –6.02 ppm)
were identified, but the more acidic phenol (2-P2) was
preferentially released.
Both linkers (1 and 2 differing in R4, Figure 1) provided
the same mono-phenyl product P1 (1-P1 and 2-P1), albeit in
significantly different timescales (in 15 minutes and 24 hours,
respectively). Phenol release can be controlled by a nature of
the second cargo. The ability to attach cargos with different
release rates may be hence useful for further applications,
such as generating phosphorylated metabolites in biology
experiments and drug delivery, among others.
The structures of the two intermediates (1-I and 2-I) and
of products P1 and P2 were determined in situ by combining
13C and 31P NMR spectra. The key connectivity information
was derived from the 13C signal splitting caused by 13C-31P
spin-spin interaction (JCP), typically through two or three
chemical bonds, with JCP = 3–8 Hz (Table S3–8 in ESI). The
13C-19F spin-spin interactions (JCF = 4–244 Hz) enabled us to
easily identify the phenyl that was preferentially released from
1.
Figure 2. Figure Series of 31P NMR spectra of 5 mM 1 (left) and 2 (right) in
50% CACO/DMSO-d6 recorded before and after UV light irradiation (365
nm) at room temperature. In both cases, the more acidic phenol (p-F-
phenol in 1 and the unsubstituted phenol in 2) was released preferentially.
The high signal-to-noise ratios of the first spectra (the prior irrad row) are
caused by the lower solubility of the starting compounds in 50%
CACO/DMSO-d6.
pKa effect on single-cargo release
To better understand the pKa effect on cargo release, we
prepared a series of compounds 3–7 bearing only one
releasable phenolic cargo differing in para substitution
Scheme 2. Synthesis of lactate linkers 3–7. Reaction conditions: a) TEA, toluene, 25 °C, 12 hours, and then: b) “corresponding phenol”, TEA, DCM, 25 °C, 12
hours.
Compounds 3–7 released the phenolic cargo
successfully at various rates, and the same final product P2 (P
= –1.14 ppm) was detected in all cases (Figure 3). In 3 (EWG
NO2 group), the SI was so fast that 3-I was not detected, and
p-NO2-phenol was released in an hour. Moreover, we
observed spontaneous partial hydrolysis of 3 overnight, which
released p-NO2-phenol without photoactivation; therefore, no
SI could proceed (hydrolytic product hP, Figure 3, left).
Such a hydrolytic decomposition of 3 may be easily
misinterpreted for SI when using common optical methods.[18]
SI was slower In 4 than in 3, the intermediate 4-I was detected,
and the corresponding phenol was released in 24 hours. The
relative concentration of products 3-P2 and 4-P2 was
significantly higher than that of their unsubstituted phenol
counterpart 5. The SI reaction in 3, 4 and 5 matches the
corresponding pKa values of p-NO2, p-F, and p-H phenols
(7.15[28], 9.95, and 9.98, respectively). The lower the pKa of
phenol is, the faster SI will be. Conversely linkers bearing EDG
phenols with p-Me (6) and p-OMe (7) released the
corresponding phenol in 96 and 24 hours, respectively. The
higher pKa values of EDG-phenols in 6 and 7 (10.14[28] and
10.55[29], respectively) resulted in slower cargo release in 6; as
such, only traces of 6-P2 were detected overnight. Yet, in
contrast to 6, linker 7 did not fully match its pKa value and led
to a faster cargo release than 5. The 15-minute trace in Figure
3
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Figure 3. Series of 31P NMR spectra of compounds 3–7 (5 mM, 50% CACO/DMSO-d6) recorded upon UV light irradiation (365 nm) at room temperature.
3 shows the relative concentration of the final product P2 of 3–
7 as follows: 65%, 5%, 2%, 2% and 24%.
is similar among 3–7, which bear the same lactate spacer.
Thus, alterations in intermediate concentrations represent
differences in cyclisation rates. For example, in 30 minutes, we
obtain 0% of 3-I, meaning that cyclisation is fast and that
photoactivation is the rate-limiting step. Conversely, linkers 4–
7 provided 32% 4-I, 48% 5-I and 55% 6-I, and 18% 7- I in 30
refluxing toluene using a condenser trap to remove the reaction
water (Scheme 3). However, the synthetic approach used for
compounds 3–7 failed in the derivatives of α-
hydroxyisovalerate 8 and α-hydroxyisobutyrate 9, most likely
due to the degradation of the starting materials 26 and 27
(dehydration of branched acid esters 26 and 27). Therefore,
we came up with an alternative synthetic approach, similar to
1 and 2, generating the phosphorylating agent 25 in situ to
phosphorylate compounds 28 and 29 and thus yielding 8 and
9 (Scheme 3).
We monitored the cyclisation of 3–7 by 31P NMR spectra with
in situ irradiation (details in ESI) and extracted the
concentration profiles of intermediates within 60 minutes of UV
irradiation. Based on our previous study,[20] the photoactivation
minutes (kinetic curves in Figure S1 in ESI). The SI rates in 3–
7 did not fully match the pKa values of the leaving groups,
showing the following trend: 6 (p-Me) < 5 (p-H) < 4 (p-F) < 7
(p-OMe) < 3 (p-NO2). Most likely, the pKa value is not the only
parameter that affects SI. We may, nevertheless, speculate
that a resonance-based change of electron density on
phosphorus is mediated by p-OMe (free electron pair of
oxygen), which supports SI.
Spacer optimisation
To accelerate cargo release, we used the Thorpe-Ingold
effect[19] and modified the lactate spacer responsible for
cyclisation in 5 by introducing either a sterically demanding or
an additional -substituent. Thus, we prepared model α-
hydroxyisovalerate and α-hydroxyisobutyrate linkers (8 and 9,
respectively). For this purpose, photocleavable (DMNB)-esters
28 and 29 were synthesized from DMNB-alcohol 20 and the
corresponding carboxylic acids, α-hydroxyisovaleric 26 and α-
hydroxyisobutyric 27, via an acid-catalysed esterification in
Monochloridate 25 was prepared in toluene using TEA.
However, the phosphorylation of the sterically hindered
alcohols 28 and 29 was challenging. Several conditions
(solvents, bases and temperatures) were tested, but only
limited combinations gave the desired products, as monitored
by 31P NMR (see Table S11 in ESI). The secondary alcohol 28
yielded the desired product 8 only when using the DMAP base,
and the tertiary alcohol 29 gave product 9 when using NMI,
both of which in DCM.
Scheme 3. Synthesis of linkers 8 and 9 with branched substituents in  position. Reaction conditions: a) phenol, TEA, toluene, 25° C, 12 hours; b) p-
toluenesulfonic acid, toluene, reflux, 16 hours; c) for 8: compound 25, DMAP, DCM, 25 °C, 12 hours; for 9: compound 25, NMI, DCM, 25 °C, 12 hours.
4
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Figure 4. Series of 31P NMR spectra of linkers 5, and 8–9, (5 mM, 50% CACO/DMSO-d6) recorded upon UV light irradiation (365 nm) at room temperature.
Surprisingly, despite the increase of steric demands in the α
position, SI was not significantly better in 8 than in 5 (Figure 4).
Although traces of 8-P2 (P –0.60 ppm) were detected after 15
minutes, a comparable amount of P2 from both analogues (5
and 8) was detected within 24 hours. This finding contrasts with
our previous observation in the phosphoramidate linker
series,[20] wherein the increase in the α-substitution effect (Me
versus i-Pr) markedly increased the cargo release rate.
Conversely, linker 9 (P –10.99 ppm) increased the cargo
release considerably, with traces of 9-P2 (P –4.22 ppm) being
detected within 5 minutes. After 15 minutes, 9-P2 was the main
component in the reaction mixture. This trend is also clearly
visible in the concentration profiles of intermediates 5-I, 8-I and
9-I (Figure S1 in ESI), where 9-I remains at approximately 20%
(in 20–60 minutes), which means that 9-I cyclises more quickly
than 5-I and 8-I. In contrast, the concentration of 5-I and 8-I
increases from 40 to 70% (in 20–60 minutes), indicating
significantly slower cyclisation and, thus, slower cargo release.
and its limitations, encouraging us to design other double-
cargo systems with a wider range of release rates as the basis
for linkers with timed, double-cargo sequential release.
The third class of linkers, 10–13, containing diphenyl
substituted phosphorus, was prepared similarly to 1 and 2. In
situ-generated phosphorochloridates 18 and 19 (Scheme 1)
were directly used to phosphorylate 28 and 29 (Scheme 4), but
sterically hindered alcohols 28 and 29 showed low reactivity to
18 and 19. Thus, the reaction conditions required optimisation.
For α-hydroxyisovalerate 28, using DMAP in DCM was the
most effective approach. In contrast, for α-hydroxyisobutyrate
29, using one equivalent of TEA in DCM with DMAP catalysis
led to the highest yields (Scheme 4). Tertiary alcohol 29 did not
react under NMI conditions (as in 9), nor did DMAP (without
TEA) or TEA (without DMAP). This difference in reactivity is
likely caused by the low reactivity of diphenyl monochloridates
18 and 19 combined with the bulky alcohols 28 and 29. Hence,
synthetic access to asymmetric diphenol-phosphates with a
bulky alcohol spacer should be optimised for particular
substrates. In this study, we monitored the reaction progress
by 31P NMR spectroscopy (for NMR chemical shifts in CDCl3,
see Table S12 in ESI; for the synthetic note page S23).
Double cargo linkers with a tunable release rate
Our structure-activity relationship study, including spacer and
cargo modifications, provided us with deeper insights into SI
Scheme 4. Synthesis of branched double-cargo linkers 10–13. Reaction conditions: a) for 10 and 11: “diaryl-chlorophosphate 18 or 19”, DMAP, DCM, 25 °C, 12
hours; for 12 and 13: “diaryl-chlorophosphate 18 and 19”, respectively, TEA, DMAP (cat.), DCM, 25 °C, 12 hours.
5
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of 13 (EDG cargos, p-Me phenyl moiety) is similar to that of 2.
In general, linkers 1, 10, and 12 with EWG cargo yielded a
significantly higher amount of P1/P2 than their EDG cargo
counterparts 2, 11, and 13, respectively, as shown in Figures
All double-cargo linkers 10–13 displayed the desired properties,
covering a wide range of cargo release rates – from minutes to
days – and enabling sequential cargo release. Modifying the
lactate spacer increased SI significantly, from 24 hours in 1 and
2 to 2 hours in α-hydroxyisovalerate analogues 10 and 11 or to
15 minutes in their α-hydroxybutyrate counterparts 12 and 13,
as shown in Figures 2 and 5. The SI of 10–13 matches the
trend found in single-cargo linkers 5, 8 and 9 (Figure 4) where
butyrate spacer showed faster cyclisation (also supported by
the concentration profiles of the intermediates, Figure S1 in
ESI). Moreover, the relative concentrations of 12-I and 13-I are
approximately 15% and 35%, which proves the faster SI and
cargo release of p-F linker 12. We also noticed that the cargo
release rates of 10 and 12 (EWG cargos, p-F phenyl group)
are similar to those of 1 and 11 and that the cargo release rate
2
and 5 (see the 15-minute row). Moreover, the α-
hydroxyisovalerate analogues 10 and 11 were measured in
25% CACO/DMSO because of their low solubility in the 50%
solvent system (the solvent effect is shown in Figure S2, in ESI,
and is in line with our previous study[31]).
Interestingly, in 12, both phenolic cargos were released within
25 minutes, exclusively yielding the final product 12-P, while
compound 13 gave a mixture of 13-P2 and 13-P (=12-P) in 3
hours, showing similar characteristics to 11 but a faster release
rate. Importantly, 12 and 13 are stable in 50% CACO/DMSO
mixture at room temperature for seven days (Figure S3 in ESI),
which is enough time for most biological applications.
Figure 5. Series of 31P NMR spectra of compounds 10–13 (5 mM, 25% (10, 11) or 50% (12, 13) CACO/DMSO-d6) recorded upon UV light irradiation (365 nm) at
room temperature. The more acidic phenolic cargo is preferentially released, and the α-hydroxybutyrate linkers 12 and 13 released the cargo within minutes, while
the α-hydroxyisovalerate analogues 10 and 11 released the cargo within hours.
Negative controls
As a second negative control, we included three derivatives,
We examined three linkers, 14–16, differing in the spacer
32–34, bearing no SI spacer. All negative controls, 14–16 and
(lactate,
α-hydroxyisovalerate
and
α-hydroxybutyrate,
32–34, confirmed the importance of a suitable cargo
respectively) and bearing ethyl moieties instead of phenyl
groups serving as negative controls. We expected that the
photoactivation step would result in the formation of
intermediate I without any further change (no cargo release).
(appropriate leaving group) and a spacer, which responsible
for SI. Without them, no SI cascade is possible, as shown in
Figure S5, and Section 2 in ESI.
Scheme 5. Negative controls: linkers 14–16 bearing no releasable ethanol cargo (left), and linkers 32–34 with no spacer responsible for SI (right).
6
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Conclusion
(4), 56.26 (5’’-O-CH3), 56.12 (4’’-O-CH3), 20.32 ppm (2). HRMS (ESI+)
We developed a novel class of phosphate-based SI linkers with
a tuneable double-cargo release option. The first cargo is
released through SI, while the second cargo is released
through chemical hydrolysis. Suitable modifications of the
lactate spacer increased the cargo release rate significantly,
from 1 day to 2 hours or to 5 minutes, as shown for the linkers
containing p-F phenol 1, 10, or 12, respectively. In turn, the
double cargo linkers 2, 11, and 13, bearing p-Me phenyl,
released their cargo more slowly (4 days, 4 hours, and 15
minutes, respectively) than their p-F analogues. Our linkers
provide the: a) programmable release of the first cargo in 3
hours (10-P1, 11-P2); b) simultaneous release of both cargos
within 25 minutes (12-P); or c) sequential release of the first
cargo within 15 minutes (13-P2) followed by the second cargo
release within 3 hours (13-P). Linkers 12 and 13, with the
fastest cargo release rates, have shown satisfying stability in a
50% buffer/DMSO mixture for seven days at room temperature.
Overall, we are now able to drive the sequential release of two
cargos from minutes to days by controlling SI. Our systems
also provide phosphorylated products P1, P2, and P in a wide
range of time scales. These systems may thus find further
applications, such as the development of new smart materials
and multiple drug delivery or the generation of phosphorylated
species. Ultimately, our study pioneers a novel route for the
design of phosphorus-based, self-immolative systems for
double-cargo release.
calculated for C12H15O7NNa 308.07407, found [M+Na]+ 308.07381.
4,5-dimethoxy-2-nitrobenzyl (S)-2-hydroxy-3-methylbutanoate (28).
4,5-Dimethoxy-2-nitrobenzyl alcohol 20 (2.55 g, 12.0 mmol, 1.20 eq.)
and p-toluenesulfonic acid (172 mg, 1.00 mmol, 0.10 eq.) were
suspended in toluene (100 mL), subsequently adding (S)-2-hydroxy-3-
methylbutanoic acid (1.18 g, 10.0 mmol, 1.00 eq.) after 45 minutes of
azeotropic distillation. The mixture was refluxed overnight and washed
with saturated NaHCO3 (60 mL), water (60 mL), and saturated NaCl
(60 mL). The organic phase was dried with Na2SO4 and evaporated to
dryness in vacuo with silica-gel. The title compound was isolated by
normal-phase flash chromatography (DCM – methanol gradient on
silica-gel) followed by reverse-phase flash chromatography (water –
methanol gradient on C18 silica-gel). Yield 28 (1.68 g, 54%) of a red-
brown semisolid. 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.75 (s, 1H, 3’’),
7.01 (s, 1H, 6’’), 5.67 (d, JGEM = 14.3, 3a), 5.59 (d, JGEM = 14.3, 3b), 4.16
(d, 1H, J1-CH(iPr) = 3.5, 1), 4.00 (s, 3H, 5’’-O-CH3), 3.99 (s, 3H, 4’’-O-CH3),
2.14 (m, CHiPr), 1.07 (d, 3H, JCH3(iPr)-CH(iPr) = 7.0, CH3iPr), 0.92 ppm (d,
3H, J CH3(iPr)-CH(iPr) = 7.0, CH3iPr).13C NMR (100 MHz, CDCl3, 25 °C) δ
174.38 (2), 153.45 (5‘‘), 148.57 (4‘‘), 140.20 (2‘‘), 125.90 (1‘‘), 110.89
(6‘‘), 108.33 (3‘‘), 75.16 (1), 64.19 (3), 56.42−56.46 (m, 4’’-O-CH3 and
5’’-O-CH3), 32.26 (CHiPr), 18.78 and 16.03 ppm (CH3iPr). HRMS (ESI+)
calculated for C14H19O7NNa 336.10537, found [M+Na]+ 336.10506.
4,5-Dimethoxy-2-nitrobenzyl 2-hydroxy-2-methylpropanoate (29). 4,5-
Dimethoxy-2-nitrobenzyl alcohol 20 (2.13 g, 10.0 mmol, 1.00 eq.) and
2-hydroxy-2-methylpropanoic acid (1.04 g, 10.0 mmol, 1.00 eq.) were
suspended in toluene (100 mL), subsequently adding p-toluenesulfonic
acid (200 mg, 1.04 mmol, 0.10 eq.) at 25 °C. The mixture was refluxed
for 12 hours and evaporated to dryness in vacuo with silica-gel,
isolating the title compound by normal-phase flash chromatography
(DCM – methanol gradient on silica-gel) followed by reverse-phase
flash chromatography (water – acetonitrile gradient on C18 silica-gel).
Yield 29 (1.97 mg, 66%) of a greyish semisolid. 1H NMR (400 MHz,
CDCl3, 25 °C) δ 7.75 (s, 1H, 3’’), 6.98 (s, 1H, 6’’), 5.61 (s, 2H, 3), 3.99
and 3.98 (s, 6H, 5’’-O-CH3 and 4’’-O-CH3), 1.52 ppm (s, 6H, 1-(CH3)2).
13C NMR (100 MHz, CDCl3, 25 °C) δ 176.69 (2), 153.49 and 148.42 (5’’
and 4’’), 139.96 (2’’), 126.32 (1’’), 110.16 (6’’), 108.34 (3’’), 72.19 (1),
64.34 (3), 56.42 and 56.38 (5’’-O-CH3 and 4’’-O-CH3), 27.21 ppm (1-
(CH3)2). HRMS (ESI+) calculated for C13H17O7NNa 322.08972, found
[M+Na]+ 322.08944.
Experimental Section
General. All reagents were purchased from commercial suppliers and
used as received. 4,5-Dimethoxy-2-nitrobenzyl alcohol was purchased
from Fluorochem Ltd. (United Kingdom). All reactions were performed
under an inert argon atmosphere. Thin layer chromatography (TLC)
was performed on TLC aluminium sheets (silica-gel 60 F254; Merck).
Reaction progress was monitored using TLC and/or 31P NMR
spectroscopy in CDCl3. Flash-column chromatography was performed
on a Compact (ECOM s.r.o.) chromatography system using silica-gel
or C18 silica-gel 230–400 mesh, 60 Å (Merck KGaA, Germany). The
final products were recovered by solvent evaporation. All products were
viscous oils, semi-solids or non-crystalline solids. The reaction yields
were not optimised.
4,5-Dimethoxy-2-nitrobenzyl
fluorophenoxy)(phenoxy)phosphoryl)oxy)propanoate
(2S)-2-(((4-
(1). Phenyl
dichlorophosphate 17 (60 µL, 0.40 mmol, 1.00 eq.) was dissolved in
toluene (3 mL), subsequently adding TEA (100 µL, 0.72 mmol, 1.80
eq.) and 4-fluorophenol (45 mg, 40 mmol, 1.00 eq.) at 25 °C. The
mixture was stirred at 25 °C for 12 hours and evaporated to dryness in
vacuo. The solid residue was dissolved in DCM (3 mL) and 4,5-
dimethoxy-2-nitrobenzyl L-lactate 22 (110 mg, 0.39 mmol, 0.98 eq.),
subsequently adding NMI (35 µL, 0.44 mmol, 1.10 eq.) at 25 °C. The
mixture was stirred at 25 °C for 12 hours and evaporated to dryness in
vacuo. The title compound was isolated by normal-phase flash
chromatography (n-hexane – ethyl acetate gradient on silica-gel)
followed by reverse-phase flash chromatography (water – acetonitrile
gradient on C18 silica-gel). Yield 1 (76 mg, 36%) of a yellow oil. NOTE
– approximately 1:1 mixture of diastereomers. 1H NMR (400 MHz,
CDCl3, 25 °C) δ 7.71 (s, 1H, 3’’), 7.71 (s, 1H, 3’’), 7.35 (m, 2H, 3’), 7.29
(m, 2H, 3’), 7.14−7.24 (m, 10H, 4’, 2’’’, 2’), 7.02 (m, 2H, 3’’’), 6.97 (m,
2H, 3’’’), 7.00−7.01 (m, 2H, 6’’), 5.62 (dd, 1H, JGEM = 14.9, J4a-6’’ = 0.6,
4a), 5.61 (dd, 1H, JGEM= 14.7, J4a-6’’ = 0.6, 4a), 5.56 (dd, 1H, JGEM = 14.7,
J4b-6’’ = 0.6, 4b), 5.53 (dd, 1H, JGEM = 14.9, J4b-6’’ = 0.6, 4b), 5.15−5.24
(m, 2H, 1), 3.96 (s, 3H, 4’’-O-CH3), 3.95 (s, 3H, 4’’-O-CH3), 3.94 (s, 3H,
5’’-O-CH3), 3.92 (s, 3H, 5’’-O-CH3), 1.58−1.64 ppm (m, 6H, 2). 13C NMR
Synthesis
4,5-Dimethoxy-2-nitrobenzyl
L-lactate
(22).
4,5-Dimethoxy-2-
nitrobenzyl alcohol 20 (213 mg, 1.00 mmol, 1.00 eq.) and L-lactic acid
(90 mg, 1.00 mmol, 1.00 eq.) were suspended in toluene (50 mL),
subsequently adding p-toluenesulfonic acid (50 mg, 0.26 mmol, 0.26
eq.) at 25 °C. The mixture was refluxed for 12 hours, evaporated to
dryness in vacuo with silica-gel, and the title compound was isolated by
normal-phase flash chromatography (DCM – methanol gradient on
silica-gel) and followed by reverse-phase flash chromatography (water
– acetonitrile gradient on C18 silica-gel). Yield 22 (90 mg, 32%) of an
off-white semisolid. 1H NMR (400 MHz, DMSO-d6, 25 °C) δ 7.71 (s, 1H,
3’’), 7.21 (s, 1H, 6’’), 5.55 (d, 1H, JOH-3 = 5.9, -OH), 5.46 (d, 1H, JGEM
14.1, 4a), 5.39 (d, 1H, JGEM = 14.1, 4b), 4.25 (m, 1H, 1), 3.90 (s, 3H,
5’’-O-CH3), 3.87 (s, 3H, 4’’-O-CH3), 1.29 ppm (d, 3H, J2-1 = 6.8, 2). 13
NMR (100 MHz, DMSO-d6, 25 °C) δ 174.07 (3), 153.18 (5’’), 147.98
(4’’), 139.69 (2’’), 126.16 (1’’), 111.35 (6’’), 108.28 (3’’), 65.99 (1), 62.62
=
C
7
This article is protected by copyright. All rights reserved.

10.1002/chem.202101805
Chemistry - A European Journal
FULL PAPER
(100 MHz, CDCl3, 25 °C) δ 169.47 (d, J3-P = 5.0, 3), 169.38 (d, J3-P
4.7, 3), 160.13 (dd, J4’’’-F = 244.8, J4’’’-P = 1.1, 4’’’), 160.08 (dd, J4’’’-F
=
=
1.45 (dd, 3H, J2-1 = 7.0, J2-P = 0.6, 2), 1.25–1.28 ppm (m, 6H, -O-CH2-
CH3). 13C NMR (125 MHz, DMSO-d6, 25 °C) δ 169.35 (d, J3-P = 4.5, 3),
169.19 (d, J3-P = 4.9, 3), 154.83 (d, J1’ -P = 6.4, 1’), 153.20 and 153.15
(5’’), 148.16 and 148.11 (4’’), 144.43 and 144.28 (4’), 139.74 and
139.67 (2’’), 125.91 and 125.74 (3’), 125.25 and 125.21 (1’’), 121.01 (d,
J2’-P = 5.5, 2’), 120.93 (d, J2’-P = 5.5, 2’), 111.67 and 111.57 (6’’), 108.27
and 108.18 (3’’), 72.66 (d, J1-P = 5.6, 1), 65.49 (d, JCH2-P = 6.0, -O-CH2-
CH3), 65.38 (d, JCH2-P = 6.3, -O-CH2-CH3), 63.93 and 63.88 (4), 56.31
and 53.25 and 56.13 and 56.08 (4’’-O-CH3 and5’’-O-CH3), 18.80 (d, J2-
P = 5.6, 2), 18.71 (d, J2-P = 6.3, 2), 15.70−15.83 ppm (m, -O-CH2-CH3).
31P NMR (202 MHz, DMSO-d6, 25 °C) δ −8.15 and −8.18 ppm. HRMS
(ESI+) calculated for C20H23O12N2NaP 537.08808, found [M+Na]+
537.08765.
244.2, J4’’’-P = 1.1, 4’’’), 153.89 and 153.88 (5’’), 150.25 (d, J1’-P = 7.3,
1’), 150.16 (d, J1’-P = 7.3, 1’), 148.54 and 148.50 (4’’), 146.15−146.45
(m, 1’’’), 139.76−139.94 (m, 2’’), 130.04 and 129.91 (3’), 126.33 and
126.24 (1’’), 125.87 (d, J4’-P = 1.4, 4’), 125.78 (d, J4’-P = 1.5, 4’), 121.86
(d, J2’’’-P = 4.6, 2’’’), 121.70 (d, J2’’’-P = 4.9, 2’’’), 120.24 (d, J2’-P = 4.7, 2’),
120.20 (d, J2’-P = 5.4, 2’), 116.59 (d, J3’’’-F = 23.6, 3’’’), 116.43 (d, J3’’’-F
=
23.6, 3’’’), 110.43 and 110.37 (6’’), 108.31 and 108.30 (3’’), 73.65 (d,
J1-P = 6.1, 1), 73.64 (d, J1-P = 5.9, 1), 64.53 (4), 56.75 (5’’-O-CH3), 56.55
(4’’-O-CH3), 19.16 (d, J2-P = 5.9, 2), 19.13 ppm (d, J2-P = 6.2, 2). 31P
NMR (161 MHz, CDCl3, 25 °C) δ −12.33 ppm. HRMS (ESI+) calculated
for C24H23O10NFNaP 558.09358, found [M+Na]+ 558.09332.
4,5-Dimethoxy-2-nitrobenzyl
(2S)-2-(((4-
4,5-Dimethoxy-2-nitrobenzyl
(2S)-2-((ethoxy(4-
methylphenoxy)(phenoxy)phosphoryl)oxy)propanoate (2). Phenyl
dichlorophosphate 17 (60 µL, 0.40 mmol, 1.00 eq.) was dissolved in
toluene (3 mL), subsequently adding TEA (100 µL, 0.72 mmol, 1.80
eq.) and 4-methylphenol (45 mg, 0.40 mmol, 1.00 eq.) at 25 °C. The
mixture was stirred at 25 °C for 12 hours and evaporated to dryness in
vacuo. The solid residue was dissolved in DCM (3 mL) and 4,5-
dimethoxy-2-nitrobenzyl L-lactate 22 (114 mg, 0.40 mmol, 1.02 eq.),
adding NMI (40 µL, 0.50 mmol, 1.26 eq.) at 25 °C. The mixture was
stirred at 25 °C for 12 hours and evaporated to dryness in vacuo. The
title compound was isolated by normal-phase flash chromatography (n-
hexane – ethyl acetate gradient on silica-gel) followed by reverse-
phase flash chromatography (water – methanol gradient on C18 silica-
gel). Yield 2 (20 mg, 9%) of a yellowish oil. NOTE – approximately 1:1
mixture of diastereomers. 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.72 (s,
1H, 3’’), 7.71 (s, 1H, 3’’), 7.31–7.37 (m, 2H, 3’), 7.26–7.31 (m, 2H, 3’),
7.19–7.24 (m, 4H, 2’’’or 2’), 7.12–7.19 (m, 2H, 4’), 7.09–7.12 (m, 2H,
3’’’), 7.06–7.09 (m, 6H, 3’’’, 2’ or 2’’’), 7.02−7.04 (m, 2H, 6’’), 5.51–5.64
(m, 4H, 4), 5.16−5.25 (m, 2H, 1), 3.95−3.96 (m, 6H, 4’’-O-CH3), 3.92 (s,
3H, 5’’-O-CH3), 3.91 (s, 3H, 5’’-O-CH3), 2.32 (s, 3H, 4’’’-CH3), 2.28 (s,
3H, 4’’’-CH3), 1.61 (dd, 3H, J2-1 = 6.9, , J2-P = 0.9, 2), 1.61 ppm (dd, 3H,
J2-1 = 6.9, , J2-P = 0.8, 2). 13C NMR (100 MHz, CDCl3, 25 °C) δ 169.37
(d, J3-P = 4.9, 3), 169.34 (d, J3-P = 5.2, 3), 153.83 (5’’), 150.23−150.43
(m, 1’), 148.26 and 148.25 (4’’), 148.16 (d, J1’’’-P = 7.2, 1’’’), 148.07 (d,
J1’’’-P = 7.6, 1’’’), 139.54 and 139.51 (2’’), 135.29 (d, J4’’’-P = 1.5, 4’’’),
135.29 (d, J4’’’-P = 1.5, 4’’’), 130.26 (3’’’), 130.15 (3’), 126.47 and 126.45
(1’’), 129.83 (d, J4’-P = 1.6, 4’), 125.71 (d, J4’-P = 1.6, 4’), 120.15 (d, J2’-P
= 4.6, 2’), 120.10 (d, J2’-P = 4.5, 2’), 119.84 (d, J2’’’-P = 5.2, 2’’’), 119.78
(d, J2’’’-P = 5.1, 2’’’), 110.02 and 109.99 (6’’), 108.10 and 108.08 (3’’),
73.38 (d, J1-P = 5.8, 1), 64.28 (4), 56.64 (5’’-O-CH3), 56.39 (4’’-O-CH3),
fluorophenoxy)phosphoryl)oxy)propanoate
(4).
4,5-Dimethoxy-2-
nitrobenzyl L-lactate 22 (110 mg, 0.39 mmol, 1.00 eq.) was dissolved
in toluene (4 mL), subsequently adding ethyl dichlorophosphate 23 (50
µL, 0.40 mmol, 1.03 eq.) and TEA (100 µL, 0.72 mmol, 1.85 eq.) at
25 °C. The mixture was stirred at 25 °C for 12 h and evaporated to
dryness in vacuo. The solid residue was dissolved in DCM (4 mL),
subsequently adding 4-fluorophenol (45 mg, 0.40 mmol, 1.03 eq.) and
then TEA (100 µL, 0.72 mmol, 1.85 eq.). The reaction mixture was
stirred at 25 °C for 12 hours. The title compound was isolated after
evaporating volatiles by normal-phase flash chromatography (n-
hexane – ethyl acetate gradient on silica-gel) followed by reverse-
phase flash chromatography (water – acetonitrile gradient on C18
silica-gel). Yield 4 (30 mg, 15%) of slightly yellow oils. NOTE –
approximately 1:1 mixture of diastereomers. 1H NMR (500 MHz,
DMSO-d6, 25 °C) δ 7.71 (s, 1H, 3’’), 7.70 (s, 1H, 3’’), 7.23−7.24 (m, 2H,
6’’), 7.15−7.23 (m, 8H, 2’, 3’), 5.51 (d, 1H, JGEM = 13.9, 4a), 5.49 (d, 1H,
JGEM = 13.9, 4a), 5.48 (d, 1H, JGEM = 13.9, 4b), 5.46 (d, 1H, JGEM = 13.9,
4b), 5.09–5.19 (m, 2H, 1), 4.12–4.21 (m, 4H, -O-CH2-CH3), 3.89 (s, 3H,
5’’-O-CH3 or 4’’-O-CH3), 3.87 (s, 3H, 5’’-O-CH3 or 4’’-O-CH3), 3.86−3.87
(s, 6H, 4’’-O-CH3 and/or 5’’-O-CH3), 1.52 (d, 3H, J2-1 = 6.9, 2), 1.45 (dd,
3H, J2-1 = 6.9, J2-P = 0.6, 2), 1.20–1.25 ppm (m, 6H, -O-CH2-CH3). 13
C
NMR (125 MHz, DMSO-d6, 25 °C) δ 169.53 (d, J3-P = 4.6, 3), 169.36 (d,
J3-P = 5.4, 3), 159.12 (d, J4’-F = 241.5, 4’), 159.08 (d, J4’-F = 241.5, 4’),
153.26 and 153.24 (5’’), 148.17 and 148.15 (4’’), 146.14−146.31 (m, 1’),
139.74 and 139.71 (2’’), 125.40 and 125.38 (1’’), 121.68−121.94 (m, 2’),
116.53 (d, J3’-F = 23.7, 3’), 116.39 (d, J3’-F = 23.7, 3’), 111.62 and 111.57
(6’’), 108.32 and 108.29 (3’’), 72.28 (d, J1-P = 5.5, 1), 72.27 (d, J1-P
=
5.5, 1), 65.00 (d, JCH2-P = 6.2, -O-CH2-CH3), 64.91 (d, JCH2-P = 6.3, -O-
CH2-CH3), 63.86 and 63.83 (4), 56.34 and 53.29 and 56.17 and 56.15
(4’’-O-CH3 and 5’’-O-CH3), 18.89 (d, J2-P = 5.7, 2), 18.74 (d, J2-P = 6.2,
2), 15.74−15.85 ppm (m, -O-CH2-CH3). 31P NMR (202 MHz, DMSO-d6,
25 °C) δ −7.03 and −7.31 ppm. HRMS (ESI+) calculated for
C20H23O10NFNaP 510.09358, found [M+Na]+ 510.09302.
20.73 and 20.68 (4’’’-CH3), 19.01 (d, J2-P = 5.7, 2), 18.98 ppm (d, J2-P
=
5.9, 2). 31P NMR (161 MHz, CDCl3, 25 °C) δ −12.27 and −12.29 ppm.
HRMS (ESI+) calculated for C25H27O10NP 532.13671, found [M+H]+
532.13678.
4,5-Dimethoxy-2-nitrobenzyl
nitrophenoxy)phosphoryl)oxy)propanoate
(2S)-2-((ethoxy(4-
4,5-Dimethoxy-2-
4,5-Dimethoxy-2-nitrobenzyl
methylphenoxy)phosphoryl)oxy)propanoate
(2S)-2-((ethoxy(4-
(5). Ethyl
(3).
nitrobenzyl L-lactate 22 (110 mg, 0.39 mmol, 1.00 eq.) was dissolved
in toluene (4 mL), subsequently adding ethyl dichlorophosphate 23 (50
µL, 0.40 mmol, 1.03 eq.) and TEA (100 µL, 0.72 mmol, 1.85 eq.) at
25 °C. The mixture was stirred at 25 °C for 12 hours and evaporated to
dryness in vacuo. The solid residue was dissolved in DCM (4 mL),
subsequently adding 4-nitrophenol (56 mg, 0.40 mmol, 1.03 eq.) and
then TEA (100 µL, 0.72 mmol, 1.85 eq.). The reaction mixture was
stirred at 25 °C for 12 hours. The title compound was isolated after
evaporating volatiles by normal-phase flash chromatography (n-
hexane – ethyl acetate gradient on silica-gel) followed by reverse-
phase flash chromatography (water – acetonitrile gradient on C18
dichlorophosphate 23 (60 µL, 0.47 mmol, 1.00 eq.) was dissolved in
toluene (3 mL), subsequently adding TEA (100 µL, 0.72 mmol, 1.53
eq.) and phenol (44 mg, 0.47 mmol, 1.0 eq.) at 25 °C. The mixture was
stirred at 25 °C for 12 hours and evaporated to dryness in vacuo. Solid
residue was dissolved in DCM (3 mL), adding 4,5-dimethoxy-2-
nitrobenzyl L-lactate 22 (110 mg, 0.39 mmol, 0.83 eq.) and NMI (35 µL;
0.43 mmol, 0.91 eq.) at 25 °C. The mixture was stirred at 25 °C for 12
hours. The title compound was isolated (direct injection) by normal-
phase flash chromatography (n-hexane – ethyl acetate gradient on
silica-gel) followed by reverse-phase flash chromatography (water –
methanol gradient on C18 silica-gel). NOTE – approximately 1:1
1
silica-gel). Yield
3
(34 mg, 18%) of yellowish oils. NOTE
–
mixture of diastereomers. Yield 5 (20 mg, 11%) of a yellowish oil. H
approximately 1:1 mixture of diastereomers. 1H NMR (500 MHz,
DMSO-d6, 25 °C) δ 8.27 (m, 2H, 3’), 8.20 (m, 2H, 3’), 7.70 (s, 1H, 3’’),
7.67 (s, 1H, 3’’), 7.46 (m, 2H, 2’), 7.43 (m, 2H, 2’), 7.21 (s, 1H, 6’’), 7.15
(s, 1H, 6’’), 5.42–5.55 (m, 4H, 4), 5.18–5.27 (m, 2H, 1), 4.18–4.27 (m,
4H, -O-CH2-CH3), 3.89 (s, 3H, 5’’-O-CH3), 3.87 (s, 3H, 4’’-O-CH3), 3.86
(s, 6H, 4’’-O-CH3 and 5’’-O-CH3), 1.54 (dd, 3H, J2-1 = 7.0, J2-P = 0.5, 2),
NMR (500 MHz, DMSO-d6, 25 °C) δ 7.72 (s, 1H, 3’’), 7.71 (s, 1H, 3’’),
7.40 (m, 2H, 3’), 7.35 (m, 2H, 3’), 7.23 (s, 1H, 6’’), 7.17–7.22 (m, 7H, 2’,
4’, 6’’), 5.43–5.54 (m, 4H, 4), 5.08–5.18 (m, 2H, 1), 4.12–4.20 (m, 4H, -
O-CH2-CH3), 3.89 (s, 3H, 4’’-O-CH3 or 5’’-O-CH3), 3.88 (s, 3H, 4’’-O-
CH3 or 5’’-O-CH3), 3.87 (s, 3H, 4’’-O-CH3 or 5’’-O-CH3), 3.87 (s, 3H, 4’’-
O-CH3 or 5’’-O-CH3), 1.52 (dd, 3H, J2-1 = 6.8, J2-P = 0.4, 2), 1.45 (dd,
8
This article is protected by copyright. All rights reserved.

10.1002/chem.202101805
Chemistry - A European Journal
FULL PAPER
3H, J2-1 = 6.9, J2-P = 0.6, 2), 1.23 (td, 3H, JCH3-CH2 = 7.1, JCH3-P = 1.0, -
O-CH2-CH3), 1.22 ppm (td, 3H, JCH3-CH2 = 7.2, JCH3-P = 1.0, -O-CH2-CH3).
13C NMR (125 MHz, DMSO-d6, 25 °C) δ 169.53 (d, J3-P = 4.6, 3), 169.35
(d, J3-P = 5.3, 3), 153.25 and 153.23 (5’’), 150.12 (d, J1’-P = 6.4, 1’),
148.13 and 148.11 (4’’), 139.70 and 139.68 (2’’), 129.96 and 129.84
(3’), 125.42 (1’’), 125.35 and 125.22 (4’), 119.99 (d, J2’-P = 5.1, 2’),
119.89 (d, J2’-P = 4.5, 2’), 111.56 and 111.51 (6’’), 108.30 and 108.28
(d, 1H, JGEM = 14.9, 4a), 5.55 (d, 1H, JGEM = 14.9, 4b), 5.03−5.16 (m,
2H, 1), 4.19−4.31 (m, 4H, -O-CH2-CH3), 4.00 and 3.97 and 3.97 and
3.95 (s, 12H, 5’’-O-CH3 and 4’’-O-CH3), 3.79 (s, 3H, 4’-O-CH3), 3.75 (s,
3H, 4’-O-CH3), 1.67 (dd, 3H, J2-1 = 6.9, J2-P = 0.6, 2), 1.56 (dd, 3H, J2-1
= 7.0, J2-P = 0.9, 2), 1.35 (td, 3H, JCH3-CH2 = 6.3, JCH3-P = 1.2, -O-CH2-
CH3), 1.33 ppm (td, 3H, JCH3-CH2 = 6.3, JCH3-P = 1.2, -O-CH2-CH3). 13
C
NMR (100 MHz, CDCl3, 25 °C) δ 169.90 (d, J3-P = 4.2, 3), 169.53 (d, J3-
P = 5.4, 3), 156.77 and 156.75 (4’), 153.79 and 153.76 (5’’ or 4’’), 148.29
and 148.22 (4’’ or 5’’), 143.97 (d, J1’-P = 7.6, 1’), 143.92 (d, J1’-P = 7.2,
1’), 139.61 and 139.51 (2’’), 126.44 (1’’), 120.96 (d, J2’-P = 4.6, 2’),
120.88 (d, J2’-P = 4.6, 2’), 114.58 and 114.47 (3’), 110.11 and 110.03
(3’’), 72.17 (d, J1-P = 5.5, 1), 72.14 (d, J1-P = 5.5, 1), 64.83 (d, JCH2-P
=
6.4, -O-CH2-CH3), 64.76 (d, JCH2-P = 6.1, -O-CH2-CH3), 63.80 and 63.76
(4), 56.31 (4’’-O-CH3 or 5’’-O-CH3), 56.26 (4’’-O-CH3 or 5’’-O-CH3),
56.11−56.16 (m, 4’’-O-CH3 or/and 5’’-O-CH3), 18.87 (d, J2-P = 5.5, 2),
18.71 (d, J2-P = 6.3, 2), 15.70−15.84 ppm (m, -O-CH2-CH3). 31P NMR
(202 MHz, DMSO-d6, 25 °C) δ −7.14 and −7.47 ppm. HRMS (ESI+)
calculated for C20H24O10NNaP 492.10300, found [M+Na]+ 492.10263.
(6’’), 108.14 and 108.07 (3’’), 72.50 (d, J1-P = 5.3, 1), 72.48 (d, J1-P
=
5.1, 1), 65.18 (d, JCH2-P = 6.2, -O-CH2-CH3), 64.94 (d, JCH2-P = 6.1, -O-
CH2-CH3), 64.18 and 64.14 (4), 56.67 and 56.60 and 56.38 and 56.35
(5’’-O-CH3 and 4’’-O-CH3), 55.56 and 55.50 (4’-O-CH3), 19.16 (d, J2-P
=
5.4, 2), 18.99 (d, J2-P = 6.2, 2), 16.00 (d, JCH3-P = 6.4, -O-CH2-CH3),
15.95 ppm (d, JCH3-P = 6.6, -O-CH2-CH3). 31P NMR (161 MHz, CDCl3,
25 °C) δ –6.48, −6.67 ppm. HRMS (ESI+) calculated for C21H27O11NP
500.13162, found [M+H]+ 500.13106.
4,5-Dimethoxy-2-nitrobenzyl
(2S)-2-((ethoxy(4-
methylphenoxy)phosphoryl)oxy)propanoate (6). 4,5-Dimethoxy-2-
nitrobenzyl L-lactate 22 (110 mg, 0.39 mmol, 1.00 eq.) was dissolved
in toluene (3 mL), subsequently adding ethyl dichlorophosphate 23 (50
µL, 0.40 mmol, 1.03 eq.) and TEA (100 µL, 0.72 mmol, 1.85 eq.) at
25 °C. The mixture was stirred at 25 °C for 12 hours and evaporated to
dryness in vacuo. The solid residue was dissolved in DCM (3 mL),
adding 4-methylphenol (45 mg, 0.42 mmol, 1.08 eq.) and then TEA
(100 µL, 0.72 mmol, 1.85 eq.). The reaction mixture was stirred at 25 °C
for 12 hours. The title compound was isolated after evaporating
volatiles by normal-phase flash chromatography (n-hexane – ethyl
acetate gradient on silica-gel) followed by reverse-phase flash
chromatography (water – acetonitrile gradient on C18 silica-gel). Yield
6 (21 mg, 11%) of slightly yellow oil. NOTE – approximately 1:1 mixture
of diastereomers. 1H NMR (500 MHz, DMSO-d6, 25 °C) δ 7.72 (s, 1H,
4,5-dimethoxy-2-nitrobenzyl
(2S)-2-
((ethoxy(phenoxy)phosphoryl)oxy)-3-methylbutanoate
(8).
Ethyl
dichlorophosphate 23 (50 µL, 0.40 mmol, 1.00 eq.) was dissolved in
toluene (3 mL), subsequently adding TEA (100 µL, 0.72 mmol, 1.80
eq.) and phenol (40 mg, 0.43 mmol, 1.08 eq.) at 25 °C. The mixture
was stirred at 25 °C for 12 hours and evaporated to dryness in vacuo.
The solid residue was dissolved in DCM (2 mL), adding 4,5-dimethoxy-
2-nitrobenzyl (S)-2-hydroxy-3-methylbutanoate 28 (120 mg, 0.38 mmol,
0.95 eq.) and DMAP (100 mg, 0.82 mmol, 2.05 eq.) at 25 °C. The
mixture was stirred at 25 °C for 12 hours. The title compound was
isolated (direct injection) by normal-phase flash chromatography (n-
hexane – ethyl acetate gradient on silica-gel) followed by reverse-
phase flash chromatography (water – methanol gradient on C18 silica-
gel). Yield 8 (20 mg, 11%) of a yellowish oil. NOTE – approximately 1:1
mixture of diastereomers. 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.74 (s,
1H, 3’’), 7.72 (s, 1H, 3’’), 7.25–7.31 (m, 4H, 3’), 7.17–7.25 (m, 4H, 2’),
3’’), 7.71 (s, 1H, 3’’), 7.04–7.23 (m, 10H, 2’, 3’, 6’’), 5.52 (d, 1H, JGEM
=
13.8, 4a), 5.47 (d, 1H, JGEM = 13.8, 4b), 5.50 (d, 1H, JGEM = 14.0, 4a),
5.45 (d, 1H, JGEM = 14.0, 4b), 5.06–5.16 (m, 2H, 1), 4.11–4.19 (m, 4H,
-O-CH2-CH3), 3.89 (s, 3H, 5’’-O-CH3), 3.87 (s, 3H, 4’’-O-CH3), 3.87 (s,
3H, 4’’-O-CH3), 3.86 (s, 3H, 5’’-O-CH3), 2.27 (s, 3H, 4’-CH3), 2.24 (s,
3H, 4’-CH3), 1.51 (d, 3H, J2-1 = 6.9, 2), 1.45 (dd, 3H, J2-1 = 6.8, J2-P
=
0.5, 2), 1.22 (td, 3H, JCH3-CH2 = 7.0, JCH3-P = 1.0, -O-CH2-CH3), 1.21 ppm
(td, 3H, JCH3-CH2 = 7.1, JCH3-P = 1.0, -O-CH2-CH3). 13C NMR (125 MHz,
DMSO-d6, 25 °C) δ 169.56 (d, J3-P = 4.8, 3), 169.36 (d, J3-P = 5.3, 3),
153.25 (5’’), 148.13 and 148.10 (4’’), 148.00–147.86 (m, 1’), 139.69 and
139.65 (2’’), 134.51 (d, J4’-P = 1.5, 4’), 134.37 (4’), 130.22 and 130.09
(3’), 125.45 and 125.43 (1’’), 119.73 (d, J2’-P = 4.6, 2’), 119.64 (d, J2’-P
= 4.6, 2’), 111.55 and 111.47 (6’’), 108.29 and 108.26 (3’’), 72.12 (d, J1-
P = 5.5, 1), 72.10 (d, J1-P = 5.5, 1), 64.75 (d, JCH2-P = 6.2, -O-CH2-CH3),
64.68 (d, JCH2-P = 6.2, -O-CH2-CH3), 63.79 and 63.75 (4), 56.31 and
53.26 (4’’-O-CH3 or 5’’-O-CH3), 56.12–56.16 (m, 4’’-O-CH3 or/and 5’’-O-
7.13–7.17 (m, 2H, 4’), 7.12 (s, 1H, 6’’), 7.07 (s, 1H, 6’’), 5.66 (d, JGEM =
14.8, 3a), 5.60 (d, JGEM = 14.8, 3b), 5.60 (d, JGEM = 15.0, 3a), 5.54 (d,
JGEM = 15.0, 3b), 4.81–4.88 (m, 2H, 1), 4.21–4.33 (m, 4H, -O-CH2-CH3),
4.02 (s, 3H, 5‘‘-OCH3), 3.95 (s, 3H, 5‘‘-OCH3), 3.98 (s, 3H, 4‘‘-OCH3),
3.97 (s, 3H, 4‘‘-OCH3), 2.34 (m, 1H, CHiPr), 2.27 (m, 1H, CHiPr), 1.35 (td,
3H, JCH2-CH3 = 7.1, JCH3-P = 1.1, -O-CH2-CH3), 1.34 (td, 3H, JCH2-CH3
=
7.1, JCH3-P = 1.1, -O-CH2-CH3), 1.09 (d, 3H, JCH3(iPr)-CH(iPr) = 6.9, CH3iPr),
1.03 (d, 3H, JCH3(iPr)-CH(iPr) = 6.9, CH3iPr), 0.97 ppm (d, 6H, JCH3(iPr)-CH(iPr)
= 6.8, CH3iPr). 13C NMR (100 MHz, CDCl3, 25 °C) δ 169.23 (d, J2-P
=
1.6, 2), 168.86 (d, J2-P = 2.1, 2), 153.83 and 153.78 (5‘‘), 148.31 and
148.23 (4‘‘), 150.60 (1‘), 139.67 and 139.60 (2‘‘), 129.68 and 129.56
(3‘), 126.57 and 126.53 (1‘‘), 125.17 (d, J4’-P = 1.6, 4’), 125.11 (d, J4’-P
= 1.5, 4’), 120.02 (d, J2’-P = 4.6, 2’), 119.97 (d, J2’-P = 4.7, 2’), 110.37
and 110.27 (6‘‘), 108.12 and 108.04 (3‘‘), 80.78 (d, J1-P = 6.5, 1), 65.24
(d, JCH2-P = 6.3, -O-CH2-CH3), 64.99 (d, JCH2-P = 6.3, -O-CH2-CH3),
64.01 and 63.93 (3), 56.74 and 56.66 (5’’-O-CH3), 56.39 and 56.38 (4’’-
O-CH3), 31.78 (d, JCH(iPr)-P = 6.9, CHiPr), 31.69 (d, JCH(iPr)-P = 7.1, CHiPr),
18.47 and 18.37 and 16.76 and 16.61 (CH3iPr), 16.03 (d, JCH3-P = 7.4, -
O-CH2-CH3), 15.94 ppm (d, JCH3-P = 6.9, -O-CH2-CH3). 31P NMR (161
MHz, CDCl3, 25 °C) δ –6.67 ppm. HRMS (ESI+) calculated for
C22H28O10NNaP 520.13430, found [M+Na]+ 520.13434.
CH3), 20.26 and 20.21 (4’-CH3), 18.88 (d, J2-P = 5.5, 2), 18.74 (d, J2-P
=
6.3, 2), 15.81 (d, JCH3-P = 6.3, -O-CH2-CH3), 15.76 ppm (d, JCH3-P = 6.3,
-O-CH2-CH3). 31P NMR (202 MHz, DMSO-d6, 25 °C) δ −6.97 and −7.26
ppm. HRMS (ESI+) calculated for C21H26O10NNaP 506.11865, found
[M+Na]+ 506.11847.
4,5-Dimethoxy-2-nitrobenzyl
(2S)-2-((ethoxy(4-
methoxyphenoxy)phosphoryl)oxy)propanoate (7). 4,5-Dimethoxy-2-
nitrobenzyl L-lactate 22 (500 mg, 1.75 mmol, 1.00 eq.) was dissolved
in toluene (10 mL), subsequently adding ethyl dichlorophosphate 23
(250 µL, 1.98 mmol, 1.13 eq.) and TEA (300 µL, 2.16 mmol, 1.23 eq.)
at 25 °C. The mixture was stirred at 25 °C for 12 hours and evaporated
to dryness in vacuo. The solid residue was dissolved in DCM (10 mL),
adding 4-methoxyphenol (90 mg, 0.73 mmol, 0.42 eq.) and then TEA
(100 µL, 0.72 mmol, 0.41 eq.). The reaction mixture was stirred at 25 °C
for 12 hours. The title compound was isolated after evaporating
volatiles by normal-phase flash chromatography (DCM – methanol
gradient on silica-gel) followed by reverse-phase flash chromatography
(water – acetonitrile gradient on C18 silica-gel). Yield 7 (56 mg, 16%)
of a yellow oil. NOTE – approximately 1:0.7 mixture of diastereomers.
1H NMR (400 MHz, CDCl3, 25 °C) δ 7.74 and 7.73 (s, 2H, 3’’), 7.10−7.15
(m, 4H, 2’), 7.08 (s, 1H, 6’’), 7.05 (s, 1H, 6’’), 6.84 (m, 2H, 3’), 6.77 (m,
2H, 3’), 5.65 (d, 1H, JGEM = 15.1, 4a), 5.60 (d, 1H, JGEM = 15.1, 4b), 5.63
4,5-Dimethoxy-2-nitrobenzyl 2-((ethoxy(phenoxy)phosphoryl)oxy)-2-
methylpropanoate (9). Ethyl dichlorophosphate 23 (60 µL, 0.47 mmol,
1.00 eq.) was dissolved in toluene (3 mL), subsequently adding TEA
(100 µL, 0.72 mmol, 1.80 eq.) and phenol (45 mg, 0.48 mmol, 1.02 eq.)
at 25 °C. The mixture was stirred at 25 °C for 12 hours and evaporated
to dryness in vacuo. The solid residue was dissolved in DCM (3 mL),
adding 4,5-dimethoxy-2-nitrobenzyl (2-methyl)propanoate 29 (140 mg,
0.47 mmol, 1.00 eq.) and NMI (50 µL, 0.61 mmol, 1.30 eq.) at 25 °C.
The mixture was stirred at 25 °C for 12 hours and evaporated to
dryness in vacuo. The title compound was isolated by normal-phase
flash chromatography (n-hexane – ethyl acetate gradient on silica-gel)
9
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10.1002/chem.202101805
Chemistry - A European Journal
FULL PAPER
followed by reverse-phase flash chromatography (water – methanol
gradient on C18 silica-gel). Yield 9 (14 mg, 6%) of a yellowish oil. 1H
NMR (400 MHz, CDCl3, 25 °C) δ 7.72 (s, 1H, 3’’), 7.30 (m, 2H, 3’), 7.20
(m, 2H, 2’), 7.15 (m, 1H, 4’), 7.14 (s, 1H, 6’’), 5.63 (dd, JGEM = 14.9, J3a-
O-CH3), 3.92 (s, 3H, 5’’-O-CH3), 2.25−2.36 (m, 8H, CHiPr, 4’’’-CH3)
0.97−1.01 ppm (m, 12H, CH3iPr). 13C NMR (100 MHz, CDCl3, 25 °C) δ
168.64 (d, J2-P = 1.9, 2), 153.72 (5’’), 150.40 (d, J1’-P = 7.6, 1’), 150.36
(d, J1’-P = 7.0, 1’), 148.09−148.23 (m, 1’’’, 4’’), 139.49 and 139.45 (2’’),
135.06 (4’’’), 135.00 (d, J4’’’-P = 1.2, 4’’’), 130.12 and 129.97 (3’’’), 129.70
and 129.53 (3’), 126.46 and 126.43 (1’’), 125.37 and 125.30 (4’), 120.08
(d, J2’-P = 4.7, 2’), 119.92 (d, J2’-P = 4.9, 2’), 119.78 (d, J2’’’-P = 4.7, 2’’’),
119.59 (d, J2’’’-P = 4.7, 2’’’), 110.17 and 110.13 (6’’), 107.93 and
107.92(3’’), 81.53 (d, J1-P = 6.9, 1), 63.97 (3), 56.59 (5’’-O-CH3), 56.28
(4’’-O-CH3), 31.70 (d, JCH(iPr)-P = 7.1, CHiPr), 31.69 (d, JCH(iPr)-P = 7.1,
CHiPr), 20.62 and 20.58 (4’’’-CH3), 18.28 and 18.26 and 16.61 and
16.59 ppm (CH3iPr). 31P NMR (161 MHz, CDCl3, 25 °C) δ –11.91 and –
11.96 ppm. HRMS (ESI+) calculated for C27H31O10NP 560.16801,
found [M+H]+ 560.16748.
= 0.6, 3a), 5.57 (dd, JGEM = 14.9, J3b-6’’ = 0.6, 3b), 4.23 (m, 2H, -O-
6’’
CH2-CH3), 3.97 (s, 3H, 5’’-O-CH3), 3.97 (s, 3H, 4’’-O-CH3), 1.76 (s, 3H,
1-CH3), and 1.75 (s, 3H, 1-CH3), 1.32 ppm (td, JCH3-CH2 = 7.1, JCH3-P
=
1.2, -O-CH2-CH3). 13C NMR (100 MHz, CDCl3, 25 °C) δ 171.64 (d, J2-P
= 3.9, 2), 153.75 (5’’), 150.61 (d, J1’-P = 6.9, 1’), 148.20 (4’’), 139.62 (2’’),
129.54 (3’), 126.69 (1’’), 124.99 (4’), 120.10 (d, J2’-P = 4.7, 2’), 110.40
(6’’), 108.04 (3’’), 82.05 (d, J1-P = 6.2, 1), 64.71 (d, JCH2-P = 6.2, -O-CH2-
CH3), 64.50 (3), 56.69 and 56.36 (5’’-O-CH3 and 4’’-O-CH3), 26.56 (d,
JCH3-P = 5.5, 1-CH3), 26.08 (d, JCH3-P = 4.1, 1-CH3), 15.92 ppm (d, JCH3-
P = 7.0, -O-CH2-CH3). 31P NMR (161 MHz, CDCl3, 25 °C) δ –10.38 ppm.
HRMS (ESI+) calculated for C21H27O10NP 484.13671, found [M+H]+
484.13656.
4,5-Dimethoxy-2-nitrobenzyl
2-(((4-
fluorophenoxy)(phenoxy)phosphoryl)oxy)-2-methylpropanoate
(12).
4,5-dimethoxy-2-nitrobenzyl
fluorophenoxy)(phenoxy)phosphoryl)oxy)-3-methylbutanoate
(2S)-2-(((4-
(10).
Phenyl dichlorophosphate 17 (60 µL, 0.40 mmol, 1.00 eq.) was
dissolved in toluene (3 mL), subsequently adding TEA (100 µL, 0.72
mmol, 1.80 eq.) and 4-fluorophenol (45 mg, 40 mmol, 1.00 eq.) at 25 °C.
The mixture was stirred at 25 °C for 12 hours and evaporated to
dryness in vacuo. The solid residue was dissolved in DCM (3 mL),
adding 4,5-dimethoxy-2-nitrobenzyl (2-methyl)propanoate 29 (120 mg,
0.40 mmol, 1.00 eq.), TEA (100 µL, 0.72 mmol, 1.80 eq.) and a catalytic
amount of DMAP at 25 °C. The mixture was stirred at 25 °C for 1 hour
and evaporated to dryness in vacuo. The title compound was isolated
by normal-phase flash chromatography (n-hexane – ethyl acetate
gradient on silica-gel) followed by reverse-phase flash chromatography
(water – methanol gradient on C18 silica-gel). Yield 12 (21 mg, 10%) of
a yellowish oil. 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.70 (s, 1H, 3’’),
7.31 (m, 2H, 3’), 7.14−7.21 (m, 5H, 2’, 4’, 2’’’), 7.07 (s, 1H, 6’’), 6.98 (m,
2H, 3’’’), 5.58 (dd, JGEM = 14.9, J3a-6’’ = 0.6, 3a), 5.54 (dd, JGEM = 14.9,
J3b-6’’ = 0.6, 3b), 3.96 (s, 3H, 4’’-O-CH3), 3.94 (s, 3H, 5’’-O-CH3), 1.76
(s, 3H, 1-CH3), 1.75 ppm (s, 3H, 1-CH3). 13C NMR (100 MHz, CDCl3,
25 °C) δ 171.23 (d, J2-P = 3.9, 2), 159.77 (dd, J4’’’-F = 244.0, J4’’’-P = 1.5,
4’’’), 153.62 (5’’), 150.36 (d, J1’-P = 7.4, 1’), 148.25 (4’’), 146.30 (dd, J1’’’-
P = 7.7, J1’’’-F = 3.1, 1’’’), 139.71 (2’’), 129.64 (3’), 126.26 (1’’), 125.38 (d,
Phenyl dichlorophosphate 17 (60 µL, 0.40 mmol, 1.00 eq.) was
dissolved in toluene (3 mL), subsequently adding TEA (100 µL, 0.72
mmol, 1.80 eq.) and 4-fluorophenol (45 mg, 0.40 mmol, 1.00 eq.) at
25 °C. The mixture was stirred at 25 °C for 12 hours and evaporated to
dryness in vacuo. The solid residue was dissolved in DCM (2 mL),
adding 4,5-dimethoxy-2-nitrobenzyl (S)-2-hydroxy-3-methylbutanoate
28 (120 mg, 0.38 mmol, 0.95 eq.) and DMAP (100 mg, 0.82 mmol, 2.05
eq.) at 25 °C. The mixture was stirred at 25 °C for 12 hours. The title
compound was isolated (direct injection) by normal-phase flash
chromatography (hexanes – ethyl acetate gradient on silica-gel)
followed by reverse-phase flash chromatography (water – methanol
gradient on C18 silica-gel). Yield 10 (149 mg, 70%) of a yellowish oil.
NOTE – approximately 1:1 mixture of diastereomers. 1H NMR (400
MHz, CDCl3, 25 °C) δ 7.68 (s, 1H, 3’’), 7.68 (s, 1H, 3’’), 7.32 (m, 2H, 3’),
7.26 (m, 2H, 3’), 7.10–7.22 (m, 10H, 2’, 4’, 2’’’), 6.90–7.03 (m, 6H, 6’’,
3’’’), 5.51–5.61 (m, 4H, 3), 4.92 (dd, J1-CH(iPr) = 4.2, J1-P = 7.6, 1), 4.91
(dd, J1-CH(iPr) = 4.2, J1-P = 7.4, 1), 3.92−3.94 (m, 9H, 4’’-O-CH3), 3.91 (s,
3H, 5’’-O-CH3), 2.30 (m, 2H, CHiPr), 0.94–1.00 ppm (m, 12H, CH3iPr).
13C NMR (100 MHz, CDCl3, 25 °C) δ 168.56 (d, J2-P = 1.9, 2), 168.46
(d, J2-P = 1.9, 2), 159.70 (dd, J4’’’-F = 245.0, J4’’’-P = 1.2, 4’’’), 159.66 (dd,
J4’’’-F = 244.3, J4’’’-P = 1.5, 4’’’), 153.58 (5’’), 150.22 (d, J1’-P = 6.7, 1’),
150.15 (d, J1’-P = 6.6, 1’), 148.22 and 148.18 (4’’), 146.04−146.23 (m,
1’’’), 139.62 and 139.58 (2’’), 129.71 and 129.54 (3’), 126.09 and
126.00 (1’’), 125.47 and 125.39 (4’), 121.56 (dd, J2’’’-F = 8.6, J2’’’-P = 4.8,
2’’’), 121.35 (dd, J2’’’-F = 8.4, J2’’’-P = 5.4, 2’’’), 119.97 (d, J2’-P = 4.7, 2’),
J4’-P = 1.4, 4’), 121.61 (dd, J2’’’-F = 8.5, J2’’’-P = 4.7, 2’’’), 120.09 (d, J2’-P
=
4.7, 2’), 116.18 (d, J3’’’-F = 23.9, 3’’’), 110.54 (6’’), 108.02 (3’’), 83.28 (d,
J1-P = 6.6, 1), 64.67 (3), 56.59 (5’’-O-CH3), 56.32 (4’’-O-CH3), 26.39 (d,
JCH3-P = 5.0, 1-CH3), 26.28 ppm (d, JCH3-P = 4.9, 1-CH3). 31P NMR (161
MHz, CDCl3, 25 °C) δ –15.92 and –15.93 ppm. HRMS (ESI+)
calculated for C25H26O10NFP 550.12729, found [M+H]+ 550.12715.
119.82 (d, J2’-P = 4.6, 2’), 116.25 (d, J3’’’-F = 23.7, 3’’’), 116.03 (d, J3’’’-F
=
4,5-Dimethoxy-2-nitrobenzyl 2-(((phenoxy)(p-tolyloxy)phosphoryl)oxy)-
2-methylpropanoate (13). Phenyl dichlorophosphate 17 (66 µL, 0.44
mmol, 1.00 eq.) was dissolved in toluene (3 mL), subsequently adding
TEA (66 µL, 0.47 mmol, 1.07 eq.) and 4-methylphenol (45 mg, 0.42
mmol, 0.95 eq.) at 25 °C. The mixture was stirred at 25 °C for 12 hours
and evaporated to dryness in vacuo. The solid residue was dissolved
in DCM (2 mL), adding 4,5-dimethoxy-2-nitrobenzyl (2-
methyl)propanoate 29 (100 mg, 0.33 mmol, 0.75 eq.) and DMAP (60
mg, 0.49 mmol, 1.11 eq.) at 25 °C. The mixture was stirred at 25 °C for
12 hours and evaporated to dryness in vacuo. The title compound was
isolated by normal-phase flash chromatography (n-hexane – ethyl
acetate gradient on silica-gel) followed by reverse-phase flash
chromatography (water – methanol gradient on C18 silica-gel). Yield 13
(97 mg, 54%) of a yellowish oil. 1H NMR (400 MHz, CDCl3, 25 °C) δ
7.70 (s, 1H, 3’’), 7.30 (m, 2H, 3’), 7.20 (m, 2H, 2’), 7.16 (m, 4’), 7.11 (s,
1H, 6’’), 7.07−7.08 (m, 4H, 2’’’, 3’’’), 5.56 (s, 2H, 3), 3.96 (s, 3H, 4’’-O-
CH3), 3.92 (s, 3H, 5’’-O-CH3), 2.30 (d, 3H, JCH3-P = 0.8, 4’’’-CH3), 1.76
ppm (s, 6H, 1-(CH3)2). 13C NMR (100 MHz, CDCl3, 25 °C) δ 171.34 (d,
23.8, 3’’’), 110.35 and 110.30 (6’’), 107.94 (3’’), 81.59 (d, J1-P = 6.6, 1),
64.01 (3), 56.49 (5’’-O-CH3), 56.22 (4’’-O-CH3), 31.59 (d, JCH(iPr)-P = 7.2,
CHiPr), 18.23 and 18.20 and 16.48 and 16.44 ppm (CH3iPr). 31P NMR
(161 MHz, CDCl3, 25 °C) δ −11.96 and −11.98 ppm. HRMS (ESI+)
calculated for C26H28O10NFP 564.14294, found [M+H]+ 564.14368.
4,5-dimethoxyn-2-nitrobenzyl
(2S)-3-methyl-2-((phenoxy(p-
tolyloxy)phosphoryl)oxy)butanoate (11). Phenyl dichlorophosphate 17
(66 µL, 0.44 mmol, 1.00 eq.) was dissolved in toluene (3 mL),
subsequently adding TEA (66 µL, 0.47 mmol, 1.07 eq.) and 4-
methylphenol (45 mg, 0.42 mmol, 0.95 eq.) at 25 °C. The mixture was
stirred at 25 °C for 12 hours and evaporated to dryness in vacuo. The
solid residue was dissolved in DCM (2 mL), adding 4,5-dimethoxy-2-
nitrobenzyl (S)-2-hydroxy-3-methylbutanoate 28 (100 mg, 0.32 mmol,
0.73 eq.) and DMAP (60 mg, 0.49 mmol, 1.11 eq.) at 25 °C. The mixture
was stirred at 25 °C for 12 hours. The title compound was isolated
(direct injection) by normal-phase flash chromatography (hexanes –
ethyl acetate gradient on silica-gel) followed by reverse-phase flash
chromatography (water – methanol gradient on C18 silica-gel). Yield 11
(103 mg, 58%) of yellowish oil. NOTE – approximately 1:1 mixture of
diastereomers. 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.70 (s, 1H, 3’’),
7.70 (s, 1H, 3’’), 7.25−7.35 (m, 4H, 3’), 7.18−7.24 (m, 4H, 2’),
7.11−7.16(m, 2H, 4’), 7.02−7.11 (m, 10H, 2’’’, 3’’’, 6’’), 5.55−5.57 (m,
4H, 3), 4.92−4.95 (m, 2H, 1), 3.95 (s, 6H, 4’’-O-CH3), 3.92 (s, 3H, 5’’-
J2-P = 4.2, 2), 153.71 (5’’), 150.48 (d, J1’-P = 7.7, 1’), 148.25 (d, J1’’’-P
=
7.7, 1’’’), 148.12 (4’’), 139.51 (2’’), 134.89 (4’’’), 130.00 (3’’’), 129.56 (3’),
126.61 (1’’), 125.22 (d, J4’-P = 1.0, 4’), 120.14 (d, J2’-P = 4.7, 2’), 119.81
(d, J2’’’-P = 4.7, 2’’’), 110.31 (6’’), 107.93 (3’’), 83.06 (d, J1-P = 6.6, 1),
64.57 (3), 56.62 (5’’-O-CH3), 56.30 (4’’-O-CH3), 26.33 (d, JCH3-P = 5.2,
1-(CH3)2), 20.63 ppm (4’’’-CH3). 31P NMR (161 MHz, CDCl3, 25 °C) δ –
10
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15.90 ppm. HRMS (ESI+) calculated for C26H29O10NP 546.15236,
found [M+H]+ 546.15200.
(5’’-O-CH3), 56.38 (4’’-O-CH3), 26.30 (d, JCH3-P = 5.1, 1-(CH3)2), 16.00
ppm (d, JCH3-P = 7.1, O-CH2-CH3). 31P NMR (161 MHz, CDCl3, 25 °C) δ
–4.95 ppm. HRMS (ESI+) calculated for C17H27O10NP 436.13671,
found [M+H]+ 436.13666.
4,5-dimethoxy-2-nitrobenzyl
(S)-2-
((diethoxyphosphoryl)oxy)propanoate
(14).
4,5-Dimethoxy-2-
nitrobenzyl L-lactate 22 (114 mg, 0.40 mmol, 1.00 eq.) was dissolved
in dry DCM (3 mL), subsequently adding diethyl chlorophosphate 30
(60 µL, 0.41 mmol, 1.04 eq.) and TEA (100 µL, 0.72 mmol, 1.80 eq.) at
25 °C. The mixture was stirred at 25 °C for 36 hours and directly
injected to normal-phase flash chromatography system (hexanes –
ethyl acetate gradient on silica-gel), followed by reverse-phase flash
chromatography (water – methanol gradient on C18 silica-gel). Yield 14
Characterization of intermediates (I) and products identified after
irradiation with UV light
1-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 174.92 (d, J3-
= 5.1, 3), 160.88 (d, J4’’’-F = 242.6, 4’’’), 151.02−151.23 (m, 1’),
P
147.11−147.31 (m, 1’’’), 131.61 (3’), 127.40 (4’), 123.11−123.33 (m,
2’’’), 121.37 (d, J2’-P = 4.6, 2’), 121.35 (d, J2’-P = 4.7, 2’), 118.03 (d, J3’’’-
F = 23.7, 3’’’), 118.00 (d, J3’’’-F = 23.6, 3’’’), 78.25 (d, J1-P = 7.1, 1), 20.79
ppm (d, J2-P = 5.0, 2). 31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C)
δ −12.55 and −12.64 ppm. HRMS (ESI-) calculated for C15H13O6FP
339.04393, found [M-H]- 339.04370.
1
(18 mg, 11%) of a yellowish oil. H NMR (400 MHz, CDCl3, 25 °C) δ
7.75 (s, 1H, 3’’), 7.11 (s, 1H, 6’’), 5.66 (d, JGEM = 14.8, 3a), 5.62 (d, JGEM
= 14.8, 3b), 5.03 (m, 1), 4.10−4.22 (m, 4H, O-CH2-CH3), 4.03 (s, 3H,
5’’-O-CH3), 3.98 (s, 3H, 4’’-O-CH3), 1.64 (dd, 3H, J2-1 = 6.9, J2-P = 0.6,
1-Me), 1.36 (td, 3H, J2’-1’ = 7.1, JCH3-P = 1.0, O-CH2-CH3), 1.33 ppm (td,
3H, JCH3-1’ = 7.1, J2’-P = 1.0, O-CH2-CH3). 13C NMR (100 MHz, CDCl3,
25 °C) δ 170.13 (d, J2-P = 4.8, 2), 153.81 (5’’), 148.32 (4’’), 139.6 (2’’),
126.53 (1’’), 110.16 (6’’), 108.19 (3’’), 71.72 (d, J1-P = 5.4, 1), 64.32 (d,
JCH2-P = 5.9, O-CH2-CH3), 64.08 (d, JCH2-P = 6.2, O-CH2-CH3), 64.12 (3),
56.70 (5’’-O-CH3), 56.41 (4’’-O-CH3), 19.20 (d, J2-P = 5.5, 1-Me), 16.04
(d, JCH3-P = 6.9, O-CH2-CH3), 16.00 ppm (d, JCH3-P = 7.0, O-CH2-CH3).
31P NMR (161 MHz, CDCl3, 25 °C) δ –1.87 ppm. HRMS (ESI+)
calculated for C16H25O10NP 422.12106, found [M+H]+ 422.12143.
1-P1, 2-P1. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) 130.76
(3’), 124.66 (4’), 121.56 (d, J2’-P = 4.7, 2’), 74.19 (d, J1-P = 6.4, 1), 21.32
ppm (d, J2-P = 2.7, 2). 31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C)
δ −6.17 ppm. HRMS (ESI-) calculated for C9H10O6P 245.02205, found
[M-H]- 245.02221.
1-P, 2-P. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 180.53
(d, J3-P = 5.5, 3), 73.24 (d, J1-P = 5.5, 1), 21.39 ppm (d, J2-P = 4.3, 2).
31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −0.22 ppm. HRMS
(ESI-) calculated for C3H6O6P 168.99075, found [M-H]- 168.99092.
4,5-dimethoxy-2-nitrobenzyl
(S)-2-((diethoxyphosphoryl)oxy)-3-
methylbutanoate (15). 4,5-Dimethoxy-2-nitrobenzyl (S)-2-hydroxy-3-
methylbutanoate 28 (120 mg, 0.38 mmol, 1.00 eq.) was added to a
premixed solution of dry DCM (3 mL), diethyl chlorophosphate 30 (60
µL, 0.41 mmol, 1.08 eq.) and NMI (50 µL, 0.63 mmol, 1.66 eq.) at 25 °C.
The mixture was stirred at 25 °C for 12 hours. The title compound was
isolated (direct injection) by normal-phase flash chromatography
(hexanes – ethyl acetate gradient on silica-gel) followed by reverse-
phase flash chromatography (water – methanol gradient on C18 silica-
gel). Yield 15 (29 mg, 17%) of a yellowish oil. 1H NMR (400 MHz, CDCl3,
25 °C) δ 7.74 (s, 1H, 3’’), 7.13 (s, 1H, 6’’), 5.66 (dd, JGEM = 14.9, J3a-6’’
= 0.6, 3a), 5.61 (d, JGEM = 14.9, J3b-6’’ = 0.6, 3b), 4.74 (dd, J1-CH(iPr) = 4.4,
J1-P = 7.9, 1), 4.09−4.20 (m, 4H, O-CH2-CH3), 4.03 (s, 3H, 5’’-O-CH3),
3.97 (s, 3H, 4’’-O-CH3), 2.28 (m, CHiPr), 1.34 (td, 3H, JCH3-CH2 = 7.1,
JCH3-P = 1.1, O-CH2-CH3), 1.31 (td, 3H, JCH3-CH2 = 7.1, JCH3-P = 1.1, O-
CH2-CH3), 1.08 (d, JCH3(iPr)-CH(iPr) = 6.9, CH3iPr), 1.01 ppm (d, JCH3(iPr)-
CH(iPr) = 6.8, CH3iPr). 13C NMR (100 MHz, CDCl3, 25 °C) δ 169.45 (d, J2-
2-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 175.93 (d, J3-
=
5.4, 3), 151.10−151.27 (m, 1’), 148.83−149.08 (m, 1’’’),
P
136.88−136.99 (m, 4’’’), 131.80−131.90 (m, 3’’’), 131.52−131.60 (m, 3’),
127.31 and 127.29 (4’), 121.29−121.42 (m, 2’), 121.04−121.18 (m, 2’’’),
78.06 (d, J1-P = 6.6, 1), 21.39−21.46 (m, 4’’’-CH3), 20.83 ppm (d, J2-P
=
5.3, 2). 31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −12.57
ppm. HRMS (ESI-) calculated for C16H16O6P 335.06900, found [M-H]-
335.06906.
2-P2 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 179.17 (d,
J3-P = 7.3, 3), 151.50 (d, J1’’’-P = 7.0, 1’’’), 133.81 (4’’’), 131.10 (3’’’),
121.30−121.44 (m, 2’’’), 74.13 (d, J1-P = 6.0, 1), 21.41 (d, JCH3-P = 1.8,
4’’’-CH3), 21.33 ppm (d, J2-P = 3.1, 2). 31P NMR (202 MHz, 50%
CACO/DMSO-d6, 25 °C) δ −6.01 ppm. HRMS (ESI-) calculated for
C10H12O6P 259.03770, found [M-H]- 259.03787.
= 1.5, 2), 153.81 (5’’), 148.26 (4’’), 139.63 (2’’), 126.63 (1’’), 110.32
P
(6’’), 108.09 (3’’), 79.89 (d, J1-P = 6.2, 1), 64.22 (d, JCH2-P = 6.1, O-CH2-
CH3), 64.02 (d, JCH2-P = 6.2, O-CH2-CH3), 63.84 (3), 56.73 (5’’-O-CH3),
56.37 (4’’-O-CH3), 31.69 (d, JCH(iPr)-P = 7.0, CHiPr), 18.52 and 16.71
(CH3iPr), 16.05 (d, JCH3-P = 7.2, O-CH2-CH3), 15.97 ppm (d, JCH3-P = 7.0,
O-CH2-CH3). 31P NMR (161 MHz, CDCl3, 25 °C) δ –1.36 ppm. HRMS
(ESI+) calculated for C18H28O10NNaP 472.13430, found [M+Na]+
472.13438.
4-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C)
δ
123.11−123.33 (m, 2’), 117.90 (d, J3’-F = 23.7, 3’), 77.18 (d, J1-P= 6.4,
1), 66.90−67.06 (m, -O-CH2-CH3), 20.73−20.92 (m, 2), 16.84−16.99
ppm (m, -O-CH2-CH3). 31P NMR (202 MHz, 50% CACO/DMSO-d6,
25 °C) δ −7.05 and −7.38 ppm. HRMS (ESI-) calculated for C11H13O6FP
291.04393, found [M-H]- 291.04359.
4,5-dimethoxy-2-nitrobenzyl
methylpropanoate (16).
2-((diethoxyphosphoryl)oxy)-2-
4,5-Dimethoxy-2-nitrobenzyl (2-
5-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 176.47 (d, J3-
P = 5.6, 3), 175.40 (d, J3-P = 5.8, 3), 151.29 (d, J1’-P = 7.0, 1’), 151.25
(d, J1’-P = 6.7, 1’), 131.48 and 131.48 (3’), 127.04 and 127.01 (4’),
121.42 (d, J2’-P = 4.5, 2’), 121.37 (d, J2’-P = 4.6, 2’), 77.08 (d, J1-P = 6.6,
1), 77.06 (d, J1-P = 6.3, 1), 66.85 (d, JCH2-P = 6.5, -O-CH2-CH3), 66.81
methyl)propanoate 29 (120 mg, 0.40 mmol, 1.00 eq.) was dissolved in
dry DCM (3 mL), subsequently adding diethyl chlorophosphate 30 (60
µL, 0.41 mmol, 1.04 eq.) and NMI (50 µL, 0.63 mmol, 1.57 eq.) at 25 °C.
The mixture was stirred at 25 °C for 36 hours and directly injected into
a normal-phase flash chromatography system (hexanes – ethyl acetate
gradient on silica-gel), followed by reverse-phase flash
chromatography (water – methanol gradient on C18 silica-gel). Yield 16
(23 mg, 13%) of a yellowish oil. 1H NMR (400 MHz, CDCl3, 25 °C) δ
7.74 (s, 1H, 3’’), 7.19 (s, 1H, 6’’), 5.63 (s, 2H, 3), 4.12 (m, 4H, O-CH2-
CH3), 4.03 (s, 3H, 5’’-O-CH3), 3.97 (s, 3H, 4’’-O-CH3), 1.75 (s, 6H, 1-
(CH3)2), 1.32 ppm (td, 6H, JCH3-1’ = 7.1, JCH3-P = 1.0, O-CH2-CH3). 13C
NMR (100 MHz, CDCl3, 25 °C) δ 171.96 (d, J2-P = 3.9, 2), 153.78 (5’’),
148.23 (4’’), 139.67 (2’’), 126.81 (1’’), 110.48 (6’’), 108.09 (3’’), 80.92
(d, J1-P = 6.2, 1), 64.41 (3), 63.76 (d, JCH2-P = 6.1, O-CH2-CH3), 56.76
(d, JCH2-P = 6.3, -O-CH2-CH3), 20.86 (d, J2-P = 4.8, 2), 20.79 (d, J2-P
=
5.1, 2), 16.94 (d, JCH3-P = 6.7, -O-CH2-CH3), 16.91 ppm (d, JCH3-P = 6.7,
-O-CH2-CH3). 31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ
−7.14 and −7.53 ppm. HRMS (ESI-) calculated for C11H14O6P
273.05335, found [M-H]- 273.05358.
6-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 176.52 (d, J3-
P = 5.4, 3), 176.45 (d, J3-P = 5.8, 3), 149.09 (d, J1’-P = 7.1, 1’), 149.06 (d,
J1’-P = 7.3, 1’), 136.60 and 136.56 (4’), 131.78 and 131.77 (3’), 121.18
(d, J2’-P = 4.5, 2’), 121.13 (d, J2’-P = 4.6, 2’), 77.02 (d, J1-P = 6.5, 1), 77.00
(d, J1-P = 6.3, 1), 66.77 (d, JCH2-P = 6.4, -O-CH2-CH3), 66.74 (d, JCH2-P
=
11
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10.1002/chem.202101805
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6.3, -O-CH2-CH3), 21.43 and 21.42 (4’-CH3), 20.87 (d, J2-P = 4.9, 2),
20.80 (d, J2-P = 5.3, 2), 16.94 (d, JCH3-P = 6.7, -O-CH2-CH3), 16.91 ppm
(d, JCH3-P = 6.4, -O-CH2-CH3). 31P NMR (202 MHz, 50% CACO/DMSO-
d6, 25 °C) δ −6.96 and −7.37 ppm. HRMS (ESI-) calculated for
C12H16O6P 287.06900, found [M-H]- 287.06893.
11-P2. 13C NMR (125 MHz, 25% CACO/DMSO-d6, 25 °C) δ 176.85 (d,
J2-P = 3.1, 2), 151.92 (d, J1’’’-P = 6.4, 1’’), 131.79 (4’’’), 130.16 (3’’’),
120.63 (d, J2’’’-P = 4.9, 2’’’), 82.11 (d, J1-P = 6.9, 1), 32.05 (d, JCH(iPr)-P
=
5.2, CHiPr), 21.04 (4’’’-CH3), 19.94 and 18.48 ppm (CH3iPr). 31P NMR
(202 MHz, 25% CACO/DMSO-d6, 25 °C) δ –5.76 ppm. HRMS (ESI-)
calculated for C12H16O6P 287.06900, found [M-H]- 287.06927.
7-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 176.37–
176.47 (m, 3), 157.66 and 157.61 (4’), 144.89−145.04 (m, 1’), 122.57
(d, J2’-P = 4.4, 2’), 122.52 (d, J2’-P = 4.5, 2’), 116.33 and 116.24 (3’),
77.01 (d, J1-P = 6.8, 1), 77.00 (d, J1-P = 6.4, 1), 66.75 (d, JCH2-P = 6.4, -
O-CH2-CH3), 66.73 (d, JCH2-P = 6.4, -O-CH2-CH3), 56.91 and 56.84 (4’-
O-CH3), 20.87 (d, J2-P = 5.1, 2), 20.80 (d, J2-P = 5.3, 2), 16.94 (d, JCH3-P
= 6.4, -O-CH2-CH3), 16.92 ppm (d, JCH3-P = 6.3, -O-CH2-CH3). 31P NMR
(202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −6.68, −7.06 ppm. HRMS
(ESI-) calculated for C12H16O7P 303.06391, found [M-H]- 303.06411.
12-P and 13-P. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ
182.51–182.64 (m, 2), 81.41 (d, J1-P = 7.5, 1), 28.05 ppm (d, JCH3-P
=
3.7, 1-(CH3)2). 31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ –
2.68 ppm. HRMS (ESI-) calculated for C4H8O6P 183.00640, found [M-
H]- 183.00654.
13-P2. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 133.48
(4’’’), 131.04 (3’’’), 121.16 (d, J2’’’-P = 4.7, 2’’’), 81.97 (d, J1-P = 7.9, 1),
27.32 (d, JCH3-P = 2.2, 1-(CH3)2), 21.40 ppm (4’’’-CH3). 31P NMR
(202 MHz, 50% CACO/DMSO-d6, 25 °C) δ –9.30 ppm. HRMS (ESI+)
calculated for C11H14O6P 273.05335, found [M-H]- 273.05389.
3-P2, 4-P2, 5-P2, 6-P2 and 7-P2. 13C NMR (125 MHz, 50%
CACO/DMSO-d6, 25 °C) δ 179.87 (d, J3-P = 6.5, 3), 73.52 (d, J1-P = 5.4,
1), 62.42 (d, JCH2-P = 5.9, -O-CH2-CH3), 21.42 (d, J2-P = 3.2, 2), 17.33
=
7.5, -O-CH2-CH3). 31P NMR (202MHz, 50%
14-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 176.89 (J3-P
= 5.5, 3), 76.89 (d, J1-P = 6.0, 1), 65.76 (d, J1’-P = 5.6, 1’), 65.71 (d, J1’-P
= 5.4, 1’), 20.90 (d, J2-P = 5.0, 2), 16.97 (d, J2’-P = 6.6, 2’), 16.96 ppm (d,
J2’-P = 6.4, 2’). 31P NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ –
2.01 ppm. HRMS (ESI-) calculated for C7H14O6P 225.05335, found [M-
H]- 225.05355.
ppm (d, JCH3-P
CACO/DMSO-d6, 25 °C) δ −1.04 ppm. HRMS (ESI-) calculated for
C5H10O6P 197.02205, found [M-H]- 197.02187.
8-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 175.36 (d, J2-
P = 2.8, 2), 175.29 (d, J2-P = 2.8, 4), 151.31–151.53 (m, 1‘), 131.40 (3‘),
126.93 and 126.87 (4’), 121.45 (d, J2’-P = 4.4, 2‘), 121.38 (d, J2’-P = 4.4,
2‘), 85.66 (d, J1-P = 7.0, 1), 85.59 (d, J1-P = 6.2, 1), 66.66–66.84 (m, -O-
CH2-CH3), 32.29–32.46 (m, CHiPr), 20.11 and 20.08 and 18.04 and
17.86 (CH3iPr), 16.95 ppm (d, JCH3-P = 6.8, -O-CH2-CH3). 31P NMR (202
MHz, 50% CACO/DMSO-d6, 25 °C) δ −6.64 and −7.09 ppm. HRMS
(ESI-) calculated for C13H18O6P 301.08465, found [M-H]- 301.08489.
15-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 175.78 (d, J2-
P = 1.8, 2), 84.72 (d, J1-P = 6.8, 1), 65.65−65.75 (m, 1’), 32.36 (d, JCH(iPr)-
= 6.8, CHiPr), 20.14 (CH3iPr), 18.08 (CH3iPr), 17.01 (d, J2’-P = 6.3, 2’),
P
16.99 ppm (d, J2’-P = 6.4, 2’). 31P NMR (202 MHz, 50% CACO/DMSO-
d6, 25 °C) δ −1.52 ppm. HRMS (ESI-) calculated for C9H18O6P
253.08465, found [M-H]- 253.08479.
8-P2. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 178.68 (d,
J2-P = 3.1, 2), 82.29 (d, J1-P = 5.9, 1), 62.38 (d, JCH2-P = 5.8, -O-CH2-
CH3), 32.73 (d, JCH(iPr)-P = 5.2, CHiPr), 19.97 and 18.93 (CH3iPr), 17.37
ppm (d, JCH3-P = 8.0, -O-CH2-CH3). 31P NMR (202 MHz, 50%
CACO/DMSO-d6, 25 °C) δ −0.60 ppm. HRMS (ESI-) calculated for
C7H14O6P 225.05335, found [M-H]- 225.05350.
16-I. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 178.58 (d, J2-
= 6.6, 2), 86.18 (d, J1-P = 7.4, 1), 65.31 (d, J1’-P = 6.1, 1’), 27.53 (d,
P
JCH3-P = 3.6, 1-(CH3)2), 16.96 ppm (d, J2’-P = 6.9, 2’). 31P NMR (202 MHz,
50% CACO/DMSO-d6, 25 °C) δ −5.44 ppm. HRMS (ESI-) calculated for
C8H16O6P 239.06900, found [M-H]- 239.06903.
9-P2. 13C NMR (125 MHz, 50% CACO/DMSO-d6, 25 °C) δ 181.68 (2),
81.23 (d, J1-P = 7.9, 1), 62.21 (d, JCH2-P = 5.5, -O-CH2-CH3), 27.32 (d,
JCH3-P = 1.8, 1-(CH3)2), 17.33 ppm (d, JCH3-P = 7.7, -O-CH2-CH3). 31P
NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ –4.22 ppm. HRMS
(ESI-) calculated for C6H12O6P 211.03770, found [M-H]- 211.03777.
NMR Spectroscopy. For compound characterization, NMR spectra
were recorded on a Bruker Avance III spectrometer operating at 400
MHz for 1H, 100 MHz for 13C and 161 MHz for 31P. Irradiation NMR
experiments were performed on a Bruker Avance III spectrometer with
a broad-band cryo probe with an ATM module (5 mm CPBBO BB-
1
1H/19F/15N/D Z-GRD) operating at 500 MHz for H, 125.7 MHz for 13C
10-P1. 13C NMR (125 MHz, 25% CACO/DMSO-d6, 25 °C) δ 129.82 (3’),
123.01 (4’), 120.84 (d, J2’-P = 4.9, 2’), 82.20 (d, J1-P = 6.5, 1), 32.05 (d,
JCH(iPr)-P = 4.6, CHiPr), 19.92 and 18.47 ppm (CH3iPr). C2 and C1’ were
not detected. 31P NMR (202 MHz, 25% CACO/DMSO-d6, 25 °C) δ
−5.91 ppm. HRMS (ESI-) calculated for C11H14O6P 273.05335, found
[M-H]- 273.05330.
and 202 MHz for 31P. For NMR signal assignment, standard Bruker
pulse sequences were employed for both 1D (1H, 13C-APT, 31P) and 2D
(COSY, ROESY, HSQC, HMBC) NMR experiments at a corrected
temperature of 25 °C. The structure of each intermediate was
determined in situ, as recently reported.[17] All NMR data was
interpreted using Topspin 3.5. For reference, the following solvent
signals were used: DMSO-d6: 2.50 (1H) and 39.5 (13C) ppm; CDCl3:
7.27 (1H) and 77.0 (13C). 31P NMR spectra were referenced to H3PO4
with 0 ppm.
10-P and 11-P. 13C NMR (125 MHz, 25% CACO/DMSO-d6, 25 °C) δ
178.38 (2), 81.02 (d, J1-P = 6.5, 1), 31.65 (d, JCH(iPr)-P = 7.6, CHiPr), 20.39
and 17.74 ppm (CH3iPr). 31P NMR (202 MHz, 50% CACO/DMSO-d6,
25 °C) δ −0.99 ppm. HRMS (ESI-) calculated for C5H10O6P 197.02205,
found [M-H]- 197.02181.
For NMR experiments with in situ irradiation, a light emitting diode
(LED; Thorlabs, Germany) was used at 365 nm. The light was guided
into the spectrometer, directly into the NMR tube, via a multimode silica
optical fiber with 1-mm diameter, 0.39 NA, and a high amount of OH
(Thorlabs, Germany). The irradiation setup was described in detail in
our previous work.[30] For in situ irradiation NMR experiments, we used
0.5 mM solutions of the corresponding linkers 1−13 in a mixture of
cacodylate buffer (CACO) with DMSO-d6 (1:1, v/v or, in some case, 1:3,
v/v). All irradiation experiments were performed at room temperature.
11-I. 13C NMR (125 MHz, 25% CACO/DMSO-d6, 25 °C) δ 172.80 (m,
2), 148.82−149.01 (m, 1’’’), 135.57 (4’’’), 131.12 and 131.03 and 130.84
and 130.75 (3’’’, 3’), 126.25 (4’), 121.13 (d, J2’/2’’’-P = 4.8, 2’ or 2’’’),
120.90 (d, J2’/2’’’-P = 4.5, 2’ or 2’’’), 120.83 (d, J2’/2’’’-P = 4.6, 2’ or 2’’’),
120.55−120.67 (m, 2’ or 2’’’), 86.13−86.25 (m, 1), 31.74 (d, JCH(iPr)-P
=
7.2, CHiPr), 21.06 and 21.04 (4’’’-CH3), 19.99 and 19.97 and 17.54 and
17.53 ppm (CH3iPr). 31P NMR (202 MHz, 25% CACO/DMSO-d6, 25 °C)
δ –12.16 and –12.19 ppm. HRMS (ESI-) calculated for C18H20O6P
363.10030, found [M-H]- 363.10056.
Mass spectrometry. Mass spectra were measured on an LTQ Orbitrap
XL (Thermo Fisher Scientific) by electrospray ionisation (ESI).
12
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Acknowledgements
This work was supported by the Experientia Foundation (O.B.,
Start-Up grant SG-2018-1) and by the Czech Science
Foundation (O.B., grant No. 20-25137Y, and E.P. grant No. 21-
23014S). We would also like to acknowledge Kvetoslava
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Entry for the Table of Contents
Our phosphate-based self-immolative linkers suitable for
tunable double cargo release allow: 1) programmable slow-
release of the one cargo in 3 hours, 2) simultaneous release
of both cargos within 25 minutes or 3) sequential release of
the first cargo within 15 minutes followed by the second cargo
release within 3 hours. Optimized linkers are stable and may
find further applications in a design of innovative materials
and multiple drug delivery systems.
14
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