Photocaged and Mixed Photocaged Bioreversible-Protected ATP Derivatives as Tools for the Controlled Release of ATP

Article abstract of DOI:10.1002/ejoc.202001229

Adenosine triphosphate (ATP) is known as the universal energy source for cellular processes, in addition, ATP also plays an important role in inflammation and cell signaling. Extracellular ATP binds to purinergic receptors and initiates further immune responses. To investigate these processes in-depth and to understand the complex mechanism of purinergic signaling, chemical tools are necessary. Here we present the synthesis of different photocaged ATP derivatives and the investigation of the light-induced release of ATP depending on the different synthesized photocleavable protecting groups based on the 2-nitrobenzyl moiety. Furthermore, we also present the synthesis of a mixed protected ATP-derivative as an example for a novel class of lipophilically caged nucleoside triphosphates. This new type of compounds is protected with a highly lipophilic non-toxic bio-removable acyloxybenzyl group and a photocleavable group. This combination may allow both passive cell uptake and controlled release of ATP by irradiation with non-harmful UV light.

Full text of DOI:10.1002/ejoc.202001229

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Photocaged ATP
Photocaged and Mixed Photocaged Bioreversible-Protected ATP
Derivatives as Tools for the Controlled Release of ATP
Nils Jeschik,[a] Tobias Schneider,[a] and Chris Meier*[a]
Dedicated to Prof. Dr. Jürgen Heck on the occasion of his 70th birthday
Abstract: Adenosine triphosphate (ATP) is known as the uni- different synthesized photocleavable protecting groups based
versal energy source for cellular processes, in addition, ATP also on the 2-nitrobenzyl moiety. Furthermore, we also present the
plays an important role in inflammation and cell signaling. Ex- synthesis of a mixed protected ATP-derivative as an example
tracellular ATP binds to purinergic receptors and initiates fur- for a novel class of lipophilically caged nucleoside triphos-
ther immune responses. To investigate these processes in-depth phates. This new type of compounds is protected with a highly
and to understand the complex mechanism of purinergic lipophilic non-toxic bio-removable acyloxybenzyl group and a
signaling, chemical tools are necessary. Here we present the photocleavable group. This combination may allow both pas-
synthesis of different photocaged ATP derivatives and the inves- sive cell uptake and controlled release of ATP by irradiation with
tigation of the light-induced release of ATP depending on the non-harmful UV light.
Introduction
kinds of immune cells, including macrophages, T cells, or neu-
trophils. P2X receptors are activated solely by ATP and mediate
the influx or efflux of sodium, potassium, or calcium ions, while
ATP activated P2Y receptors modulate the activation of various
cellular enzymes.[12] The ATP-dependent activation of those P2
receptors promotes an inflammatory environment to fight path-
ogens or trigger tissue healing, for instance. To counteract the
pro-inflammatory signaling and to cancel the immune re-
sponse, the ectoenzymes CD39 and CD79 degrade ATP to
adenosine, which is the natural extracellular counterpart of
ATP.[13] An imbalance of this natural equilibrium between ATP
and adenosine can lead to a chronic pro-inflammatory environ-
ment, which is considered to cause chronic inflammatory dis-
eases.[13] For a better understanding of this complex purinergic
system, chemical tools are crucial to investigate distinct proc-
esses.
Adenosine triphosphate (ATP) is the universal energy source for
all kinds of cellular processes and provides a defined amount
of energy, that is stored in its phosphoanhydride bonds and
released upon their cleavage. This energy is used for specific
cellular processes like muscle contractions or biosynthesis of
cellular components. Furthermore, ATP also plays an important
role in inflammation and cell signaling.[1–5] In resting cells, the
extracellular ATP concentration is rather low (0.4–1 m
pared to the intracellular level (up to 10 m
M), com-
M
).[6] It was observed
that tissue damage, pathogens, or other cell stress led to a rapid
increase in the level of extracellular ATP and that this extracellu-
lar ATP triggers a proinflammatory environment.[7–9] This led to
further immune responses through signaling cascades, includ-
ing the activation of immune T-cells or inflammation. ATP is
released into the extracellular space by exocytosis of vesicles
from the cell interior or by active transport via membrane-
bound proteins. In the extracellular space, ATP binds to a wide
range of P2 purinergic receptors, which are divided into two
subsets: P2X and P2Y.[10,11] Both subsets are present on various
Photoremovable protection groups are promising candidates
for this kind of chemical tools.[14–16] By protecting appropriate
compounds with those photoremovable groups the ability to
release the protected substrate with exact control over time
and space is gained. The application of this concept to ATP may
enable the investigation of the effects triggered by extracellular
ATP directly and further on it may be possible to observe initi-
ated signaling events and immune responses.[17,18] Caged-ATP
was first synthesized and described by Kaplan and co-workers,
who protected the γ-phosphate with one photocleavable
2-nitrobenzyl group (NB-group). After photolytic cleavage of
the NB-group Kaplan and co-workers could observe the activa-
tion of a Na:K ion pump of human red blood cell ghosts.[19] In
contrast to the work of Kaplan and co-workers, we studied dif-
ferent kinds of electron donor substituted 2-nitrobenzyl moie-
ties to shift the absorption maximum towards lower energy
[a] N. Jeschik, T. Schneider, Prof. Dr. C. Meier
Organic Chemistry, Department of Chemistry, University of Hamburg,
Martin-Luther-Platz 6, 20146 Hamburg, Germany
E-mail: chris.meier@chemie.uni-hamburg.de
https://www.chemie.uni-hamburg.de/institute/oc/arbeitsgruppen/meier.html
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.202001229.
© 2020 The Authors. European Journal of Organic Chemistry published by
Wiley-VCH GmbH · This is an open access article under the terms of the
Creative Commons Attribution-NonCommercial-NoDerivs License, which
permits use, distribution and reproduction in any medium, provided the
original work is properly cited, the use is non-commercial and no modifica-
tions or adaptations are made.
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Scheme 1. Application of different photocaged ATP derivatives as potential extra- and intracellular precursors for ATP release.
(bathochromic shift) to avoid potential cell damage due to ity. With this new type of compounds, it should be possible to
usage of energy-intense UV-B or UV-C light.[20,21] With these generate a lock-in effect inside the cell, since the AB-group
photocleavable groups we wanted to study in the future which should be cleaved rapidly by esterase's after cell uptake and
protecting group is the most suitable for the application in the formed monoprotected MeNV-ATP does not efflux from the
extra- and intracellular context by irradiation with 365 nm UV- cells again. Furthermore, the formed MeNV-ATP should be bio-
light. Furthermore, we also planned to synthesize derivatives logically inactive until the remaining photocaging group is
of such donor substituted NB-masks which are derived from cleaved by light and ATP gets released.
acetophenone starting materials. With these photomasks, the
formation of a reactive and most likely toxic 2-nitrosobenzalde-
hyde by-product should be avoided due to the formation of a
Results and Discussion
less reactive ketone.[20] Moreover, we modified the photolabile
masks with lipophilic alkyl ether groups to potentially enable
passive diffusion through cell membranes (Scheme 1). With
Chemical Synthesis of Novel Caged ATP Compounds
these novel compounds not only extracellular but also intracel-
lular processes may be investigated in the future after light-
induced cleavage. To improve the lipophilicity and in contrast
to the work of Kaplan and co-workers our novel compounds
carry two photocleavable masks at ATPs γ-phosphate group in-
stead of only one, relating to our extensive experience and reli-
able protocols for the synthesis of γ-bis-protected nucleoside
triphosphates.[22,23]
As we have demonstrated earlier, γ-modified nucleoside tri-
phosphates can be synthesized in a three-step protocol, start-
ing from a nucleoside monophosphate and an appropriately
masked H-phosphonate.[24]
Following this protocol, the H-phosphonate was first acti-
vated by oxidative chlorination and subsequently reacted with
inorganic phosphate to yield a non-symmetric pyrophosphate.
Next, after stepwise activation this pyro-phosphate derivative
was coupled with a nucleoside monophosphate, yielding the
γ-masked nucleoside triphosphate. In a different approach to
obtain ꢀ- or γ-masked nucleoside di- or triphosphates, we also
For this reason and to preserve the comparability between
the individual prepared derivatives, all synthesized compounds
comprised two masks.
Finally, we demonstrated the versatility of our synthetic pro- reported on a highly reliable protocol using phosphoramidite
tocol in preparing mixed-type lipophilic photocaged-ATP by chemistry.[25,26] In the present work, we transferred both strate-
combining a simple photolabile group with an acyloxybenzyl gies to the synthesis of γ-bis-caged nucleoside triphosphates
(AB) mask known from our prodrug technology that, independ- by coupling of adenosine diphosphate with an appropriately
ently from the photocage, enhances the compound's lipophilic- masked phosphoramidite or H-phosphonate (Scheme 2).
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Scheme 2. Synthetic pathways for the preparation of γ-bis-photocaged ATP via phosphoramidite or H-phosphonate chemistry.
First, the different 2-nitrobenzyl derivatives 1–5 serving as of transformations and in a final decaging step the desired sub-
synthetic precursors for the photocleavable units were synthe- strate is released. Simultaneously the final step also provides a
sized starting from commercially available benzaldehyde and reactive nitroso aldehyde or less reactive nitroso ketone, de-
acetophenone derivatives. Table 1 summarizes the syntheses of pending on the used photocleavable group (Scheme 3).
these different photocleavable groups based on the 2-nitro-
benzyl moiety.
Table 1. Syntheses of different substituted photocleavable masks containing
the ortho-nitro moiety (NB: nitrobenzyl, NV: nitroveratryl, lip: lipophilic).
Scheme 3. Photoinduced cleavage of NB-protected ATP. Depending on the
used NB-derivative a reactive aldehyde or a less reactive ketone is formed.
The reactive and presumably toxic 2-nitrosobenzaldehyde
by-product from photo-decaging of groups 1–3 (Table 1, Entry
1–3) can be avoided by introduction of a methyl group at the
benzyl position (Table 1, Entry 4–5). Thus, a less reactive ketone
is formed after photolysis. Furthermore, the cleavage rate of
those secondary photocleavable groups is known to be faster
than the cleavage of primary 2-nitrobenzyl derivatives.[20] A ma-
jor disadvantage of these methyl groups is that they introduce
[a] 1.2 equiv. alkyl halide, 3.0 equiv. K2CO3, DMF, 50 °C, overnight. [b1] HNO3
a new stereogenic center into the target molecule. This led to
a complex mixture of diastereomers. While this complex mix-
ture can be avoided by an enantioselective reduction, the con-
(65 %), r.t., 48 h. [b2] HNO3 (65 %), Ac2O, 0 °C, 2 h. [c] 1–5–3.0 equiv. NaBH4,
MeOH/THF (1:1), 0 °C, 0.5–2 h.
The light-induced cleavage mechanism of these photore- figuration of this stereogenic center has no known influence
movable groups based on the 2-nitrobenzyl moiety is well regarding the mechanism or rate of the photocleavage reaction.
known and has been thoroughly investigated in the past.[27] By For that reason, so far, we did not perform a stereoselective
this mechanism, the 2-nitrobenzyl moiety undergoes a variety synthesis or separation of the diastereomers but did our synthe-
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Table 2. Syntheses of symmetric phosphoramidites 6–10 starting from the 2-nitrobenzyl derivatives 1–5.
Entry
2-Nitro-benzyl- derivative
R1
R2
Amidite
Yield [%]
1
2
3
4
5
1
2
3
4
5
H
H
H
CH3
CH3
–
6
7
8
9
83
92
92
–
3,4-OMe
3-OC8H17, 4-OMe
3,4-OMe
3-OMe, 4-OC8H17
10
–
[a] 1.0 equiv. dichloro-N,N-diisopropylaminphosphoramidite, 2.2 equiv. 2-nitrobenzyl alcohol 1–6, 2.5 equiv. Et3N, THF, 0 °C, 18 h.
ses with the stereomeric mixture, respectively. Additionally, to a steric interaction of the substituents is avoided. The corre-
increase the lipophilicity of the photocaged-ATP derivatives, sponding H-phosphonates were obtained in yields of 33 % (11)
two lipophilic masks were synthesized (3, 5), both containing and 72 % (12) by the reaction of diphenylphosphite with the
an octyl ether instead of the methoxy group (compare com- secondary alcohols in small excess, as shown in Table 3. Starting
pounds 2 and 4, Table 1). This was achieved by etherification of from diphenylphosphite (DPP), phenolate is replaced by the
vanillin (Table 1, Entry 3) or 3-hydroxy-4-methoxyacetophenone photo-removable group 4 or 5, most probably following an ad-
(Table 1, Entry 5), respectively, with 1-bromooctane under dition-elimination type mechanism. The moderate yields may
alkaline conditions followed by nitration and finally, reduction be explained by the potential similar leaving group tendencies
of the carbonyl group. In each case, the product was obtained of the secondary alcohol and the phenol, hence resulting in a
in nearly quantitative yields for both the etherification and re- possible reverse reaction. With phosphoramidites 6–8 or the
duction. The limiting step in both reaction procedures was the H-phosphonate 11–12 in hand, photocleavable ATP derivatives
nitration, which was achieved by stirring the vanillin derivative 13–17 were prepared by activation with dicyanoimidazole or
for two days in concentrated nitric acid and the ketone deriva- N-chlorosuccinimide of those precursors followed by coupling
tive for two hours in a mixture of concentrated nitric acid and with ADP in moderate to good yields as shown in Table 4.
acetic anhydride, respectively. In both nitration reactions, an
Table 3. Synthesis of H-phosphonates 11–12 starting from the related deriva-
tives 4–5.
ipso substitution of the carbonyl group with the nitronium ion
was observed, leading to the double nitrated side-product. The
desired nitrated regioisomers 26 and 29 were obtained in ap-
proximately 60 % yield in both cases after purification by flash
chromatography. Other nitration methods such as the standard
method using a mixture of nitric and sulfuric acid led to com-
plete decomposition of the starting materials, or in the case of
dilute nitric acid, to no observable conversion at all. Impor-
tantly, depending on the commercial availability of the starting
materials, not all these synthetic steps were necessary (Table 1).
Next, we prepared phosphoramidites 6–10 as precursors for
coupling reactions towards the photocaged-ATPs. As summa-
rized in Table 2, phosphoramidites 6–8 were synthesized in ex-
cellent yields. However, the synthesis of phosphoramidites con-
taining the methyl-substituted photocleavable groups 4 and 5
following the same protocol failed (Table 2, Entries 4 and 5).
In comparison with starting materials 1–3 the more sterically
demanding secondary benzyl alcohol in compounds 4 and 5 in
combination with the rather bulky N,N-diisopropylamino moi-
ety may have prevented the formation of phosphoramidites 9
and 10.
Entry
Secondary alcohol
R
Product
Yield [%]
1
2
4
5
3,4-OMe
3-OMe, 4-OC8H17 12
11
33
72
[a] 1.0 equiv. diphenylphosphite, 2.2 equiv. secondary alcohol derivative 11–
12, pyridine, r.t., 3 h.
Depending on the precursor, derivatives 13–17 were either
synthesized by a 3,4-dicyanoimidazol-mediated coupling of
phosphoramidites 6–8 with ADP and subsequent oxidation
with tert-butylhydroperoxide (Table 4, Entry 1–3) or by oxidative
chlorination of H-phosphonates 11–12 with N-chlorosuccinim-
ide and subsequent coupling with ADP (Table 4, Entry 4–5). All
photocaged-ATP derivatives were obtained in yields between
23–72 %. Comparing the different coupling strategies, it seems
that the phosphoramidite approach led to the photocaged ATP
in higher yields than the H-phosphonate approach. However,
the difference in R1 (H vs. CH3) may also have played a role in
To avoid the potential sterically hindrance during the phos-
phoramidite synthesis, we prepared symmetric H-phosphonates
from these methyl-substituted compounds as potential precur-
sors for the later coupling to photocleavable ATP.
In contrast to the phosphoramidites, H-phosphonates have this context. In terms of R2, compounds 15 and 17 comprising
no further bulky substituent at the phosphorus atom, and thus the lipophilic octyl ether (Table 4, Entry 3, 5) were obtained in
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Table 4. Syntheses of photocleavable ATP derivatives 13–17.
Entry
Starting material
Route
R1
R2
Product
Yield [%]
1
2
3
4
5
6
7
8
11
12
a
a
a
b
b
H
H
H
CH3
CH3
–
13
14
15
16
17
72
60
38
49
23
3,4-OMe
3-OC8H17, 4-OMe
3,4-OMe
3-OMe, 4-OC8H17
[a] 1.0 equiv. bis(tetrabutylammonium)-ADP, 1.5 equiv. amidite 6–8, 1.5 eq DCI sol. (0.25
M in CH3CN), 1 h, r.t., then 1.5 equiv. tBuOOH sol. (5.5 M in n-decane),
CH3CN, 20 min, r.t. [b] 1.0 equiv. H-phosphonate 11–12, 2.0 equiv. NCS, 1 h r.t. then 0.8 equiv. bis(tetrabutylammonium)-ADP, CH3CN, 1 h, r.t.
lower yields compared to the less lipophilic derivatives 13, 14
and 16. This observation can be explained by the lipophilic
octyl moiety that caused a negative impact on the required
solubility during the synthesis and also complicated the purifi-
cation by automated reverse phase chromatography. We as-
sumed that the increased amphiphilic properties of compounds
15 and 17 led to the formation of more complex structures
in aqueous media (e.g. micelles) thus resulting in a non-trivial
chromatographic purification of the compounds. Nonetheless,
all caged ATP derivatives 13–17 were successfully prepared via
the phosphoramidite or H-phosphonate route, and they were
next evaluated regarding the release of ATP by photolysis.
Photolytic Release of ATP from Caged ATP Derivatives
For these experiments, solutions of 1 μmol/mL were prepared
and irradiated with three 1300 mW UV-LEDs (365 nm, for more
details, see experimental section). After defined intervals, ali-
quots were analyzed via HPLC to determine the progress of ATP
formation (Figure 1). Based on the integral of the peaks as-
signed to starting material or ATP, half-lives and doubling times
were calculated respectively for each caged-derivative and the
released ATP 13–17 and the released ATP (Figure 2).
The photolytic cleavage was found to proceed stepwise for
all caged-ATP derivatives. First, the formation of an intermediate
with just one photocleavable mask was observed. During fur-
ther irradiation, this intermediate subsequently was cleaved
and released ATP. By comparison of the determined peak areas
during the photolysis of compound 17, it has to be considered,
that the determined start value of the peak area for compound
17 is higher than the determined end value for the formed
ATP since two UV-active groups are cleaved, thus lowering the
cumulated UV absorption at 254 nm (Figure 2, lower part). Fur-
thermore, the cleavage rate of the starting material and the
intermediate decreased during the photolysis and simultane-
ously the formation of ATP became slower (Figure 1, lower part).
Figure 1. Upper part: representative HPLC chromatograms showing the time-
course of the photolysis of lipMeNV-ATP (17) in PBS/CH3CN, 1:1 (pH 7.4, r.t.,
365 nm, 3 × 1300 mW). Due to the four different diastereomers of 17 that
are not fully resolved by this HPLC-method, the starting material appears as
a set of two peaks of different intensity. The main signals are labeled with
the corresponding compounds. Lower part: determined peak areas plotted
against photolysis time.
This fact can also be observed by comparison of the calculated high than the calculated half-live. We assume that this observa-
half-life and doubling times since all donor substituted caged- tion can be explained by the fact that the released nitroso by-
ATP derivatives showed a doubling time k more than twice as product absorbed the irradiated photons and thus the quan-
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noteworthy by direct comparison of the lipophilic NV and
MeNV derivatives 13 and 15. The fact that the methyl substitu-
ent in the benzyl position led to a faster cleavage compared to
their non-methylated derivatives is known in the literature.[19]
Importantly, from the work presented here, it becomes appar-
ent that the lipophilic modification in compound 17 almost
triples the cleavage rate compared to the MeNV derivative 16.
This rapid cleavage of the lipophilic MeNV photo group is a
promising feature opening up a new class of photolabile and
lipophilic masked nucleoside triphosphates. However, a poten-
tial disadvantage for the intracellular application of these lipo-
philic compounds is still the cleaved by-product. During the
photolysis reactions of lipophilic photocaged ATP 13 and 15,
the released by-product precipitated from the aqueous buffer,
which may lead to cytotoxicity or interfere with the assay read-
out by potential intracellular applications.
In addition, to study whether the release of ATP also oc-
curred by irradiation with visible light, the photolysis of the NB
and NV derivative 13 and 14 was repeated at 405 nm, since it
was reported that the NB-group can also be cleaved at
400 nm.[29] By usage of a 405 nm UV-light, with the exact power
output like the previously used 365 nm UV-source, a markedly
slower stepwise release of ATP, with half-lives of 102 s (NB) and
106 s (NV) for the ATP-derivatives 13 and 14 was observed.
Thus, although it is possible to cleave the NB and NV groups
with visible light, we would recommend the usage of 365 nm
UV-light for a rapid release of ATP, since the release is three to
four times faster than by usage of 405 nm light source. How-
ever, to avoid any potential cellular damages, it might be suit-
able to use visible light with the price of a slower release of
ATP than with UV-light.
Figure 2. Calculated half-lives (t1/2) and doubling times (k) for photolysis of
caged-ATP derivatives and corresponding release of ATP for each derivative,
respectively.
tum yield for the desired reaction is reduced. Only the non-
substituted NB-ATP 13 did not show this trend (Figure 2).
The observation that NB-groups are cleaved almost as
quickly as NV-groups by irradiation with 365 nm, despite poor
absorption at this wavelength, was described in the litera-
ture.[28] A possible explanation could be the equimolar release
of the respective nitroso by-product. During the photolysis of
the lipophilic MeNV-ATP derivative 17, we were able to observe
the formation of the respective by-product via HPLC monitoring
(Figure 1). As shown in Figure 3 the nitroso by-product has a
strong absorption at 365 nm, thus reducing the available pho-
tons for the desired cleavage reaction.
Combining Two Different γ-Masking Groups to Decouple
Photocleavage and Lipophilicity
To overcome this problem, we synthesized a next-generation
photocleavable and lipophilic ATP derivative, which gained its
lipophilicity from a bio-reversible acyloxybenzyl group (AB-
group) known from our TriPPPro-compounds, while a simple
photocleavable group was present to exhibit all beneficial fea-
tures of spatio-temporally controlled ATP-release. Since in the
previous photolysis experiments the MeNV group had proven
to be favorable over the other examined photocleavable masks,
derivative 22 was chosen as prototypic target molecule. The
synthesis of the asymmetric phosphoramidite precursor started
from commercially available bis(N,N-diisopropylamino)-chloro-
phosphine which was first reacted with the MeNV mask 4. Un-
expectedly and in contrast to what we had observed for the
symmetrical MeNV phosphoramidite 9, we experienced no diffi-
culties here and obtained phosphordiamidite 20 in excellent
yields up to 90 %. As one possible explanation, the chloro sub-
stituent may be replaced more easily in this starting material.
While, in the case of amidite 9, the first MeNV substituent can
interfere with the replacement of the second chloro substituent,
Figure 3. Measured UV/Vis spectra from the different non-lipophilic caged-
ATP derivatives (nitroso by-product: 2-nitroso-4-methoxy-5-octanoxyaceto-
phenon).
Since NB-ATP 13 did not show any relevant absorption at
365 nm, we assume that the corresponding nitroso by-product
also showed only a low absorption at 365 nm, whereby the
quantum yield is not affected.
In line with exceptions a more rapid cleavage of the methyl- here only one MeNV group was introduced and due to its free
substituted MeNV ATP derivative 16 compared to the NV-pro- rotation around the P–O-C-bond, it may be possible that an
tected ATP 14 was observed. This rapid cleavage is even more already present MeNV group more strongly restricts the second
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substitution reaction. The mixed phosphoramidite 21 was ase (PLE) in phosphate buffer (12 mM phosphate concentration,
obtained by DCI-mediated coupling of diamidite 20 with pH 7.4) at 37 °C. Aliquots were sampled at different time-points
4-(hydroxymethyl)phenyl decanoate 19, in almost quantitative and analyzed by RP-HPLC equipped with a diode array detector.
yield. The lipophilicity providing AB-mask 19 was synthesized In an independent second experiment, AB-MeNV-ATP 22 was
prior to esterification of the commercially available 4-hydroxy- subjected to photolysis following the protocol of the previous
benzyl alcohol 18 with decanoyl chloride. The last step towards experiments (Figure 1). The first experiment clearly showed that
the desired mixed AB-MeVN-ATP 22 was the DCI-mediated cou- the bioreversible AB-group was cleaved rapidly, but the photo-
pling with ADP. Unfortunately, we were unable to isolate the cleavable MeNV group was stable under this simulated intracel-
ATP derivative 22 after repeated purification steps. For this lular, esterase-containing conditions. The second experiment
reason, we again applied the H-phosphonate approach demonstrated, as expected, a rapid cleavage of the MeNV
(Scheme 4), which had proven favorable for the synthesis of the group by irradiation with UV-light, while the AB-group was sta-
symmetric MeNV protected derivatives 16 and 17.
ble under these conditions (Figure 4) The product in each ex-
periment was identified based on the corresponding UV/Vis-
spectra and retention times. After incubation of AB-MeNV-ATP
22 with esterase (PLE), the HPLC trace indicated the formation
of a less lipophilic compound, while the observed peak area for
the starting material was decreasing. In addition to the shift in
Scheme 4. Synthetic procedure for the preparation of AB-MeNV-ATP 22 via
the H-phosphonate approach. [A]: 1.1 equiv. 4-hydroxybenzyl alcohol 18,
1.0 equiv. decanoyl chloride, 1.0 equiv. Et3N, THF, 20 min 0 °C, then 2 h r.t.,
50 %; [B]: 1.0 equiv. DPP, 1.0 equiv. compound 4, 1.0 equiv. compound 19,
pyridine, 45 min –15 °C then 2 h r.t., 38 %; [C]: 1.0 equiv. H-phosphonate 23,
2.0 equiv. NCS, 1 h r.t. then 0.8 equiv. bis(tetrabutylammonium)-ADP, CH3CN
1 h, r.t., 26 %.
The corresponding mixed protected H-phosphonate 23 was
obtained starting from DPP after stepwise substitution of the
phenolate groups by first the MeNV-group 4 and next the AB-
group 19 in moderate yield. The coupling to the desired ATP
derivative 22 was achieved with 26 % yield, which is in the
same range as the lipophilic target compounds 17 (Table 4).
After the successful synthesis of this mixed γ-phosphate pro-
tected ATP derivative 22, the cleavage of each masking group
was examined independently. To test the bioreversibility of the
AB-mask, AB-MeNV-ATP 22 was incubated with pig-liver-ester-
Figure 4. Panel A – Time-course of release of MeNV-ATP from AB-MeNV-ATP
22 via esterase-triggered hydrolysis (PLE). Conditions: PLE concentration:
100 U/mL, c(AB-MeNV-ATP) = 6 mM, 37 °C, pH 7.4; Panel B – Time-course of
release of AB-ATP from AB-MeNV-ATP 22 during photolysis. Conditions:
3 × 1.3 W UV-LED, 365 nm ( 10 nm), 1 μmol/mL, r.t.; Peak areas were deter-
mined for panel A and B from measured RP-HPLC chromatograms (diode
array detector, 254 nm). Inside each graph, the UV-spectrum of the corre-
sponding formed product is shown. The presence of an absorption band at
>300 nm indicates that the observed compound still includes the MeNV-
masking unit.
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the retention time of 2.1 minutes, the emerging signal still including short or long alkyl ether moieties, target compounds
showed the typical absorption pattern of the MeNV group, indi- exhibiting different lipophilicities were obtained. All photo-
cating that
Indeed MeNV-ATP was formed (Figure 4 panel A). In contrast,
cleavable groups were readily cleaved by irradiation of the tar-
get compounds with 365 nm UV-light and released ATP quanti-
tatively. Moreover, we prepared a mixed protected ATP-deriva-
tive as an example for a new class of lipophilic photocaged
nucleoside triphosphates. In this new class of compound, pho-
tocaging and lipophilicity are no longer coupled as it included
one photocleavable MeNV-group and one acyloxybenzyl group
that provided lipophilicity, as known from our TriPPPro-com-
pounds. Furthermore, we demonstrated that in this mixed-type
masked ATP, both groups can be cleaved orthogonally, releas-
ing the corresponding mono-masked ATP-derivatives. In con-
trast to the prior synthesized lipophilic caged-ATP derivatives
no water-insoluble by-products are formed, and the amount of
potential toxic by-products is reduced by half. These properties
make the synthesized mixed protected ATP derivative a promis-
ing tool to study cellular ATP-dependent processes like inflam-
mation. Due to the enhanced lipophilicity, the mixed protected
ATP should be able to be passively uptaken by the cell so that
the potential assays are not influenced by invasive procedures
such as micro-injection.
the photolysis of the starting material 22 resulted in a minor
shift in retention time (1.1 minutes), which indicated that only
the MeNV was cleaved and that the AB-group was still cova-
lently bound to ATP, providing higher lipophilicity and thus
higher retention time compared to MeNV-ATP. Furthermore, in
contrast to the first experiment, here the UV/Vis spectra of the
emerging signal lack the typical MeNV absorption band (300–
400 nm), thus indicating the cleavage of the MeNV-moiety (Fig-
ure 4 panel B). Formation of fully de-masked ATP was excluded
since, for both experiments, the retention time of the product
signal does not match with the retention time of ATP, which
was proved by co-injection. The half-lives for AB-MeNV-ATP 22
were determined assuming simple decay as a mathematical
model for the received HPLC signals. For the esterase-triggered
cleavage of the AB-group (PLE, Figure 4, panel A), a half-life of
1.1 minutes was calculated and for the photolytic de-caging
reaction (panel B) the calculated half-life was 2.2 seconds. The
latter indicated that the photolysis of the MeNV group in com-
pound 22 occurred even faster than in the bis-caged-MeNV-
ATP 14 (t1/2 = 6.0 s). This difference in cleavage speed can be
explained by multiple possible reasons. First, the AB mask
showed no absorption >300 nm, which means that all irradi-
ated photons can be used for the desired cleavage reaction. In
contrast, during the photolysis of symmetrically protected bis-
MeNV-ATP 16, a competition between the cleavage of the
di-MeNV and the formed intermediate took place, which may
Experimental Section
General: All reactions except nitrations and reductions, were per-
formed in oven-dried glassware, under nitrogen atmosphere, and
with anhydrous solvents. THF, CH2Cl2, CH3CN, and pyridine were
dried with a MBraun (MB SPS-800) Solvent System and stored over
nitrogen and molecular sieves (3 or 4 Å). DMF was purchased from
increase the observed half-life. Furthermore, only one equiva- Acros Organics anhydrous over molecular sieves. Triethylamine was
heated to reflux over calcium hydride for two days and afterward
stored over molecular sieves under nitrogen atmosphere and in the
dark. All starting materials and reagents were purchased from
Sigma Aldrich, TCI, Acros, ABCR or Carbosynth and were used with-
out further purification. Ultrapure Water was obtained from a Sarto-
rius arium pro apparatus (Sartopore 0.2 μm, UV) and HPLC-grade
CH3CN, used for automated reversed-phase chromatography and
HPLC analysis, was purchased from VWR or Honeywell. Column
chromatography was carried out with technical grade solvents,
which were distilled prior to use, and silica gel MN 60 M (0.04–
0.063 mm) from Macherey-Nagel. Precoated thin-layer-sheets (ALU-
GRAM® Xtra SIL G/UV254, Macherey-Nagel) were used for thin-layer-
chromatography. Spots were visualized by staining with a mixture
of vanillin in sulfuric acid, acetic acid, and methanol, where possible.
P+III-containing compounds were stained with an alkaline perman-
ganate solution. Automated reversed-phase flash chromatography
was performed using prepacked MN RS 40 C18 ec columns on an
Interchim Puriflash 430 or a Büchi Sepacore system. For all auto-
mated reversed-phase chromatography, a gradient from 100 % ul-
trapure water to 100 % CH3CN within 20 min was used (flowrate:
20 mL/min). All NMR solvents were purchased from Euriso-Top or
Deutero. NMR spectra were recorded on Bruker instruments Fourier
HD 300, Avance I 400, DRX 500, or Avance III 600 at room tempera-
ture. All proton and carbon NMR spectra were calibrated by the
respective solvent signal. High-resolution mass spectra were meas-
ured with an Agilent 6224 ESI-TOF instrument und IR spectra were
obtained on a Bruker Alpha IR spectrometer.
lent of reactive and potential toxic nitroso-by-product was
formed during the photolysis of the mixed protected ATP 22 in
comparison to bis-MeNV-ATP 16, which liberated two equiva-
lents of this unwanted UV-absorbing by-product. This means,
that in the photolysis of 22 a minor number of photons was
absorbed, thus increasing the cleavage rate.
After successful cleavage of each mask in the previously de-
scribed independent experiments, we also demonstrated in one
single experiment that a stepwise cleavage of both masks is
feasible, as this is needed for potential cellular assays. For that,
we incubated AB-MeNV-ATP 22 first with PLE as described be-
fore and after complete cleavage of the AB-group we carried
out the photolysis and observed the quantitative formation of
ATP. Consequently, these studies demonstrated that this new
type of lipophilic bio-reversible masked and photocaged nucle-
oside triphosphate has promising properties for intracellular ap-
plications. On the one hand, lipophilicity can be specifically
modified by the bio-reversible AB mask, and the photocleava-
ble MeNV group may allow a precise and rapid release of the
nucleoside triphosphate after potential cell uptake.
Conclusion
We successfully applied phosphoramidite as well as H-phos-
phonate chemistry for the synthesis of different γ-bis-caged ATP
HPLC: Chromatograms were recorded on an Agilent 1260 Infinity II
derivatives based on 2-nitrobenzyl photocleavable groups. By with the following components: G7129A Vialsampler, G7111A Quat
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Pump, G7116A MCT, G7117C DAD HS. All adenosine-containing sig-
nals were detected at 254 nm with the following method: 0–25 min
stirred again for 30 minutes. All volatile components were removed
in vacuo. The crude product was purified by automated RP flash
column chromatography. Fractions were pooled and the solvent
was removed by lyophilization. The residue was dissolved in a mini-
mum of water and cations were exchanged to ammonium using
DOWEX (50WX8, 50–100 mesh), followed by a second automated
RP flash column chromatography.
5 % CH3CN in 2 m
M aq. tetrabutylammonium acetate buffer (pH
6.0) 5 % to 100 % CH3CN, 1mL/min.
Photolysis Studies: A solution of the test compound at 1 mg/mL
was dissolved in a 1:1-mixture of PBS (pH 7.4) and CH3CN in a small
glass vial (diameter: 11 mm). The solution was stirred with a small
stirring rod on a magnetic stirrer and the LED-UV source was placed
directly in front of the vial. The LEDs were arranged in a triangle on
a passive cooling apparatus (3 × 1400mW, NVSU233A-U365, Sahl-
mann Photochemical Solutions or 3 × 1400mW, NVSU233A-405,
Sahlmann Photochemical Solutions). After fixed intervals, aliquots
of 20 μL were analyzed via HPLC (injection volume: 7 μL; method:
see HPLC-section).
General Procedure 5: Synthesis of Caged-ATP via H-Phosphon-
ates: The corresponding H-phosphonate (1.0 equiv.) was dried in
high vacuo for one hour and subsequently dissolved in anhydrous
CH3CN. To the resulting solution, N-chlorosuccinimide (2.0 equiv.)
was added as a solid and the solution was stirred for one hour at
room temperature. Then, ADP (2.0–2.5 × tetrabutylammonium salt,
0.8 equiv.) was added and the solution was stirred for one hour at
room temperature. All volatile components were removed in vacuo.
The crude product was purified by automated RP flash column
chromatography. Fractions were pooled and the solvent was re-
moved by lyophilization. The residue was dissolved in a minimum
of water and cations were exchanged to ammonium using DOWEX
(50WX8, 50–100 mesh), followed by a second automated RP flash
column chromatography.
PLE Hydrolysis: To a mixture of 60 μL DMSO and 400 μL PBS (pH
7.4) 40 μL of a 50 m
ative in DMSO was added. To this solution, 30 μL PLE solution (100
U/mL) was added. Yielding a 6 m solution of the caged-ATP-deriv-
M stock solution of the related caged-ATP deriv-
M
ative and a final volume of 530 μL. The mixture was incubated at
37 °C and at fixed intervals, aliquots of 35 μL were analyzed via
HPLC (injection volume 30 μL, method: see HPLC-section).
Adenosine Diphosphate Tetrabutylammonium Salt (24): ADP
(Na-salt, 0.82 g, 1.7 mmol, 1.0 equiv.) was dissolved in 5 mL water
and transferred by ion-exchange chromatography on DOWEX
(50WX8, 50–100 mesh) into the protonated species. 12.2 mL (10 %
in water, 4.3 mmol, 2.5 equiv.) tetrabutylammonium hydroxide solu-
tion was added and the resulting solution was stirred for 30 min-
utes. The solvent was removed by lyophilization and the remaining
residue was purified by automated RP flash column chromatogra-
phy. The solvent was again removed by lyophilization and the resi-
due was dried in high vacuo. Yield 1.66 g (1.7 mmol, quant.) as a
Synthesis
General Procedure 1. Reduction of Carbonyl Groups: The respec-
tive aldehyde or ketone was dissolved in a 1:1 mixture of methanol
and THF under ambient atmosphere and cooled to 0 °C (amount
of volume listed in the appropriate synthesis protocol). To the re-
sulting solution sodium borohydride (1.5–3.0 equiv.) was added in
portions and the suspension was stirred for 1–3 hours at room tem-
perature. All volatile substances were removed in vacuo and the
remaining residue was taken up in water and ethyl acetate. The
aqueous layer was extracted with ethyl acetate. The combined or-
ganic layers were washed with brine, dried with sodium sulfate, and
concentrated in vacuo. After removal of the solvent, the desired
alcohol was obtained in high purity without further purification.
1
pale yellow hygroscopic solid. H-NMR (500 MHz, D2O): δ = 8.53 (s,
1 H), 8.19 (s, 1 H), 6.09 (d, 3JH,H= 5.8 Hz, 1 H), 4.75–4.70 (m, 1 H),
4.58 (dd, 3JH,H = 5.2 Hz, 3JH,H = 3.8 Hz, 1 H), 4.37–4.33 (m, 1 H), 4.28–
4.20 (m, 1 H), 4.19–4.12 (m, 1 H), 3.22–3.04 (m, 16 H, CH2), 1.67–
1.52 (m, 16 H, CH2), 1.39–1.24 (m, 16 H, CH2), 0.90 (t, 3JH,H = 7.4 Hz,
24 H, CH3); 13C-NMR (125 MHz, D2O): δ = 155.5, 152.7, 148.9, 139.9,
118.4, 86.8, 84.0 (d, 3JC,P = 9.1 Hz), 74.4, 70.1, 64.6 (d, 2JC,P = 5.4 Hz),
General Procedure 2: Synthesis of Phosphoramidites: Under
nitrogen atmosphere (diisopropylamino)dichlorophosphine was
dissolved in 5 mL anhydrous THF and added dropwise to an ice-
cold solution of the corresponding alcohol (2.1 equiv.) and dry tri- 58.0 (CH2), 23.0 (CH2), 19.1 (CH2), 12.8 (CH3); 31P-NMR (200 MHz,
2
2
ethylamine (3.0 equiv.) in 15 mL THF. The solution was stirred at
room temperature for 18 h in the dark. The precipitated solid was
removed by filtration and the filtrate was concentrated to dryness
in vacuo. Finally, the crude product was purified by column chroma-
tography on silica gel (PE/EtOAc, 10:1 or 1:1 + 5 % TEA).
D2O): δ = –8.23 (d, JP, P = 22.1 Hz), –11.2 (d, JP, P = 22.1 Hz).
(Diisopropylamino)dichlorophosphine (25): The synthesis of
compound 25 was performed as described in the literature. The
analytical data is in accordance with the literature.[30]
Bis(2-nitrobenzyl)-N,N-diisopropylphosphoramidite (6): The syn-
thesis of compound 6 was performed as described in the literature.
Purification was carried out by column chromatography with
PE/EtOAc, 1:1 + 5 % TEA on silica gel. The analytical data is in ac-
cordance with the literature.[31]
General Procedure 3: Synthesis of H-phosphonates: Under nitro-
gen atmosphere diphenylphosphite was dissolved in anhydrous
pyridine. Next, the corresponding alcohol was added and the solu-
tion was stirred for 2–3 h at room temperature. All volatile com-
pounds were removed in vacuo and the residue was co-evaporated
several times with toluene and CH2Cl2. Finally, the crude product
was purified by column chromatography on silica gel (PE/EtOAc,
2:1 to pure EtOAc).
Bis(4,5-dimethoxy-2-nitrobenzyl)-N,N-diisopropylphosphor-
amidite (7): The synthesis of compound 7 was performed as de-
scribed in the literature. Purification was carried out by column
chromatography with PE/EtOAc, 1:1 + 5 % TEA on silica gel. The
analytical data is in accordance with the literature.[31]
General Procedure 4: Synthesis of Caged-ATP via Phosphor-
amidites: The corresponding phosphoramidite (1.5 equiv.) and ADP
(as 2.0–2.5 tetrabutylammonium salt) were combined as solids, 3-Methoxy-4-octanoxybenzaldehyde (26): Under anhydrous con-
dried in high-vacuo for three hours, and subsequently dissolved in
ditions 3.00 g (19.7 mmol, 1.0 equiv.) vanillin and 8.35 g (59.2 mmol,
3.0 equiv.) freshly dried potassium carbonate was suspended in
50 mL dry DMF. To the resulting suspension 5.2 mL (5.8 g, 30 mmol,
1.5 equiv.) n-bromooctane were added. The mixture was stirred at
anhydrous CH3CN. To the stirred solution, DCI-activator (0.25 in
M
CH3CN) was added portion-wise (1 × 0.5 equiv., 4 × 0.25 equiv.) at
room temperature. Between each portion, the solution was stirred
for five minutes. After complete addition, the reaction was stirred 50 °C for 18 h. After full conversion 50 mL 1
M NaOH-soln. was
for another 30 minutes at room temperature. Afterward, tBuOOH
(5.5 in n-decane, 1.5 equiv.) was added and the solution was
added and the mixture was extracted three times with diethyl ether.
The combined organic layers were washed with brine, dried with
M
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2
3
sodium sulfate, and concentrated in vacuo. The remaining residue
6.9 Hz, JH,H = 16.4 Hz, 2 H, Ph-CH2-b), 4.06 (t, JH,H = 6.8 Hz, 4 H,
3
was purified on silica gel (CH2Cl2/EtOAc, 1:1). Yield: 5.0 g (19 mmol, CH2), 3.94 (s, 6 H, OMe), 3.82–3.74 (m, 2 H, NCH), 1.86 (p, JH,H
=
96 %) as a colorless solid. Rf = 0.41 (DCM/EtOAc, 1:1); m.p. 35 °C;
7.2 Hz, 4 H, CH2), 1.50–1.44 (m, 4 H, CH2), 1.39–1.25 (m, 16 H, 8 ×
CH2), 1.27 (d, JH,H = 6.9 Hz, 12 H, CH3), 0.88 (t, JH,H = 6.9 Hz, 6 H,
3
3
3
1H-NMR (400 MHz, CDCl3): δ = 9.84 (s, 1 H, CHO), 7.43 (dd, JH,H
=
4
4
8.1 Hz, JH,H = 1.9 Hz, 1 H, H-Ar), 7.40 (d, JH,H = 1.9 Hz, 1 H, H-Ar), CH3); 13C-NMR (100 MHz, CDCl3): δ = 154.4 (C-Ar), 147.2 (C-Ar), 138.8
3
3
3
6.96 (d, JH,H = 8.2 Hz, 1 H, H-Ar), 4.10 (t, JH,H = 6.9 Hz, 2 H, CH2),
(C-Ar), 131.6 (d, JC,P = 8.1 Hz, C-Ar), 109.5 (C-Ar), 109.2 (C-Ar), 69.7
3
2
2
3.92 (s, 3 H, OMe), 1.88 (p, JH,H = 7.3 Hz, 2 H, CH2), 1.50–1.43 (m, 2 (CH2), 62.7 (d, JC,P = 19.5 Hz, Ph-CH2), 56.5 (OMe), 43.7 (d, JC,P
=
H, CH2), 1.40–1.28 (m, 8 H, 4 × CH2), 0.88 (t, 3JH,H = 6.8 Hz, 3 H, CH3);
13C-NMR (100 MHz, CDCl3): δ = 191.7 (CHO), 154.4 (C-Ar), 150.0 (C-
Ar), 130.0 (C-Ar), 127.0 (C-Ar), 111.5 (C-Ar), 109.4 (C-Ar), 69.3 (CH2),
12.2 Hz, N-C), 31.9, 29.4, 29.3, 29.0, 26.0, 22.8 (6 × CH2), 24.9 (d,
3JC,P = 7.2 Hz, CH3), 14.2 (CH3); 31P-NMR (162 MHz, CDCl3): δ = 147.3;
IR (ATR): ν = [cm–1] = 3106, 2961, 2925, 2855, 1576, 1516, 1462,
˜
56.2 (OMe), 31.9, 29.4, 29.3, 22.8, 29.0, 26.0 (6 × CH2), 14.2 (CH3); IR 1432, 1399, 1370, 1323, 1125, 1020, 996, 976, 870, 818, 750, 724,
(ATR): ν = [cm–1] = 3338, 3080, 3005, 2964, 2766, 1681, 1669, 1583,
668; HRMS (ESI+): m/z calcd. for C38H62N3O10P + H+: 752.4246, found
752.4240.
˜
1507, 1464, 1405, 1390, 1266, 1197, 1132, 1065, 1011, 958, 790, 657,
571; HRMS (ESI+): m/z calcd. for C16H24O3 + H+: 265.1798, found
265.1799.
4,5-Dimethoxy-2-nitroacetophenone (28): Under ambient condi-
tions 10.18 g (56.47 mmol, 1.0 equiv.) 3,4-dimethoxyacetophenone
were dissolved in 30 mL acetic anhydride. The solution was slowly
added to an ice-cold solution of 60 mL conc. nitric acid (65 %) and
1.5 mL acetic anhydride. After complete addition, the solution was
stirred for one hour at room temperature. The solution was poured
carefully into 300 mL ice-water. After filtration, the precipitate was
washed with ice-cold water until the water was nearly colorless. The
solid was recrystallized from a mixture of water and ethanol (1:1).
Yield: 8.19 g (36.4 mmol, 64 %) as yellow needles. Rf = 0.45 (CH2Cl2);
5-Methoxy-2-nitro-4-octanoxybenzaldehyde (27): Under ambi-
ent conditions 45 mL conc. nitric acid (65 %) was cooled to 0 °C
and 1.77 g (6.70 mmol, 1.0 equiv.) of aldehyde 26 were added in
small portions. The resulting solution was stirred for two days at
room temperature. The reaction mixture was slowly poured into
200 mL ice water. The mixture was extracted with ethyl acetate
twice. The combined organic layers were washed with sat. sodium
bicarbonate solution and brine. The organic layer was dried with
sodium sulfate and concentrated in vacuo. The remaining residue
was purified on silica gel using a gradient (PE/CH2Cl2, 1:1 to pure
CH2Cl2). Yield: 1.19 g (3.85 mmol, 58 %) as a yellow solid. Rf = 0.30
(CH2Cl2); m.p. 67 °C; 1H-NMR (500 MHz, CDCl3): δ = 10.43 (s, 1 H,
1
m.p. 134 °C; H-NMR (400 MHz, [D6]DMSO): δ = 7.64 (s, 1 H, H-Ar),
7.22 (s, 1 H, H-Ar), 3.92 (s, 3 H, OMe), 3.90 (s, 3 H, OMe), 2.52 (s, 3
H, CH3); 13C-NMR (100 MHz, [D6]DMSO): δ = 199.3 (C=O), 153.2 (C-
Ar), 149.4 (C-Ar), 138.4 (C-Ar), 131.1 (C-Ar), 109.7 (C-Ar), 107.1 (C-Ar),
3
CHO), 7.59 (s, 1 H, H-Ar), 7.41 (s, 1 H, H-Ar), 4.14 (t, JH,H = 6.8 Hz, 2
56.6 (OMe), 56.3 (OMe), 30.0 (CH3). IR (ATR): ν = [cm–1] = 2973, 2931,
˜
3
H, CH2), 4.00 (s, 3 H, OMe), 1.90 (p, JH,H = 7.1 Hz, 2 H, CH2), 1.51–
2842, 1699, 1619, 1574, 1508, 1462, 1421, 1394, 1345, 1322, 1181,
1.45 (m, 2 H, CH2), 1.40–1.25 (m, 8 H, 4 × CH2), 0.88 (t, 3JH,H = 6.9 Hz, 1113, 1043, 1020, 953, 881, 788, 697, 666, 599, 536; HRMS (ESI+):
3 H, CH3); 13C-NMR (125 MHz, CDCl3): δ = 187.9 (CHO), 153.6 (C-Ar),
152.2 (C-Ar), 144.0 (C-Ar), 125.3 (C-Ar), 110.0 (C-Ar), 108.1 (C-Ar), 70.1
(CH2), 56.8 (OMe), 31.9, 29.4, 29.3, 28.9, 26.0, 22.8 (6 × CH2), 14.2
m/z calcd. for C10H11NO5 + H+: 226.0715, found 226.0707.
rac-1-(4,5-Dimethoxy-2-nitrophenyl)ethane-1-ol (4): The synthe-
sis was done according to general procedure 1; 0.95 g (4.2 mmol,
1.0 equiv.) of ketone 28 was dissolved in 30 mL THF/MeOH, 1:1 and
0.61 g (13 mmol, 3.0 equiv.) sodium borohydride was added. Yield:
0.94 g (4.1 mmol, 99 %) as a yellow solid. Rf = 0.12 (CH2Cl2); m.p.
(CH3); IR (ATR): ν = [cm–1] = 3013, 2960, 2914, 2852, 1685, 1602,
˜
1570, 1513, 1481, 1408, 1378, 1330, 1222, 1170, 1084, 1058, 1036,
1027, 983, 810, 756, 719, 607; HRMS (ESI+): m/z calcd. for C16H23NO5
+ H+: 310.1649, found 310.1648.
1
123 °C; H-NMR (400 MHz, [D6]DMSO): 7.53 (s, 1 H, H-Ar), 7.36 (s, 1
3
3
5-Methoxy-2-nitro-4-octanoxybenzyl Alcohol (3): The synthesis
was done according to general procedure 1; 0.75 g (2.4 mmol,
1.0 equiv.) of aldehyde 27 was dissolved in 30 mL THF/MeOH, 1:1
and 0.14 g (3.6 mmol, 1.5 equiv.) sodium borohydride was added.
Yield: 0.72 g (2.3 mmol, 96 %) as a yellow solid. Rf = 0.43 (CH2Cl2/
MeOH, 19:1); m.p. 69 °C; 1H-NMR (500 MHz, CDCl3): δ = 7.69 (s, 1 H,
H-Ar), 7.14 (s, 1 H, H-Ar), 4.94 (s, 2 H, Ph-CH2), 4.07 (t, 3JH,H = 6.8 Hz,
2 H, CH2), 3.98 (s, 3 H, OMe), 2.65 (s, 1 H, OH), 1.87 (p, 3JH,H = 7.2 Hz,
H, H-Ar), 5.47 (d, JH,H = 4.7 Hz, 1 H, OH), 5.26 (dt, JH,H = 4.4 Hz,
4JH,H = 6.2 Hz, 1 H, Ar-CH-CH3), 3.90 (s, 3 H, OMe), 3.84 (s, 3 H, OMe),
1.37 (d, JH,H = 6.3 Hz, 3 H, CH3); 13C-NMR (100 MHz, [D6]DMSO):
3
153.2 (C-Ar), 147.1 (C-Ar), 138.9 (C-Ar), 137.9 (C-Ar), 108.9 (C-Ar),
107.3 (C-Ar), 63.9 (C-Ar-CH-CH3), 56.0 (2 × OMe), 25.1 (CH3); IR (ATR):
ν = [cm–1] = 3287, 3113, 2965, 2928, 2897, 1614, 1580, 1523, 1499,
˜
1466, 1439, 1362, 1220, 1190, 1164, 1037, 1019, 970, 867, 796, 758,
659, 592. HRMS (ESI+): m/z calcd. for C10H13NO5+- H2O: 210.0766,
2 H, CH2), 1.50–1.44 (m, 2 H, CH2), 1.39–1.25 (m, 8 H, 4 × CH2), 0.88 found 210.0755.
(t, JH,H = 6.9 Hz, 3 H, CH3); 13C-NMR (125 MHz, CDCl3): δ = 154.4
(C-Ar), 147.7 (C-Ar), 139.9 (C-Ar), 132.0 (C-Ar), 111.4 (C-Ar), 109.4 (C-
3
Bis(1-(4,5-dimethoxy-2-nitrophenyl)ethyl)phosphonate (11):
The synthesis was done according to general procedure 3; 0.41 g
(1.8 mmol, 2.1 equiv.) of alcohol 4 was dissolved in 10 mL pyridine
and 0.17 mL (0.21 g, 0.90 mmol, 1.0 equiv.) diphenylphosphite was
added. Purification was done by column chromatography: PE/
EtOAc, 2:1 to EtOAc. Yield: 0.14 g (0.32 mmol, 31 %) as a yellow
Ar), 69.7 (CH2), 63.1 (Ph-CH2), 56.6 (OMe), 31.9, 29.4, 29.3, 29.0, 26.0
22.8 (6 × CH2), 14.2 (CH3); IR (ATR): ν = [cm–1] = 3538, 3103, 3013,
˜
2951, 2923, 2853, 1577, 1513, 1503, 1468, 1440, 1400, 1347, 1324,
1209, 1155, 1069, 1045, 975, 878, 810, 736, 716, 683, 506; HRMS
(ESI+): m/z calcd. for C16H25NO5+- H2O: 294.1705, found 294.1378.
1
solid and mixture of four diastereomers. H and 13C-NMR data are
Bis(5-methoxy-2-nitro-4-octanoxybenzyl)-N,N-diisopropylphos-
phoramidite (8): The synthesis was done according to general pro-
cedure 2; 0.19 mL (0.92 mmol, 0.19 g 1.0 equiv.) of compound 25
was added to 0.60 g (1.9 mmol, 2.1 equiv.) of alcohol 3 and 0.38 mL
(0.28 mmol, 0.28 g) triethylamine. Column chromatography was
done with PE/EtOAc, 10:1 + 5 % TEA. Yield: 0.64 g (0.85 mmol, 92 %)
as a yellow oil. Rf = 0.70 (PE/EtOAc + 5 % TEA, 10:1); 1H-NMR
(400 MHz, CDCl3): δ = 7.72 (s, 2 H, H-Ar), 7.39 (s, 2 H, H-Ar), 5.24
provided for one pure isolated diastereomer. Rf = 0.55 (EtOAc);
1H-NMR (600 MHz, CDCl3): δ = 7.46 (s, 1 H, H-Ar), 7.42 (s, 1 H, H-Ar),
1
7.04 (s, 1 H, H-Ar), 6.96 (d, JH,P = 703 Hz, 1 H, P-H), 6.94 (s, 1 H,
H-Ar), 6.27 (dq, 3JH,P = 9.6 Hz, 3JH,H = 6.3 Hz, 1 H, Ph-CH-P), 6.13 (dq,
3
3JH,P = 10.3 Hz, JH,H = 6.3 Hz, 1 H, Ph-CH-P), 3.96–3.88 (m, 12 H,
3
3
OMe), 1.66 (d, JH,H = 6.3 Hz, 3 H, CH3), 1.62 (d, JH,H = 6.3 Hz, 3 H,
CH3); 13C-NMR (150 MHz, CDCl3): δ = 154.1 (C-Ar), 153.9 (C-Ar), 148.3
(C-Ar), 148.3 (C-Ar), 138.7 (C-Ar), 138.6 (C-Ar), 132.4 (3JC,P = 3.9 Hz,
C-Ar), 132.2 (3JC,P = 5.2 Hz, C-Ar), 107.5 (C-Ar), 107.5 (C-Ar), 108.1 (C-
3
2
3
(dd, JH,P = 6.8 Hz, JH,H = 16.3 Hz, 2 H, Ph-CH2-a), 5.15 (dd, JH,P
=
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2
Ar), 108.1 (C-Ar), 71.0 (d, 2J
= 4.0 Hz, Ar-C-P), 70.5 (d, JC,P
=
1.38 (m, 2 H, CH2), 1.36 (d, 3JH,H = 6.2 Hz, 3 H, CH3), 1.31–1.26 (m, 8
C,P
3
3
5.0 Hz, Ar-C-P), 56.7,56.6, 56.5, 56.4 (OMe), 25.0 (d, JC,P = 3.4 Hz,
H, 4 × CH2), 0.86 (t, JH,H = 6.8 Hz, 3 H, CH3); 13C-NMR (75 MHz,
CH3), 24.7 (d, 3JC,P = 4.8 Hz,CH3); 31P-NMR (242 MHz, CDCl3): δ = 5.1;
[D6]DMSO): δ = 152.7 (C-Ar), 147.2 (C-Ar), 138.7 (C-Ar), 137.9 (C-Ar),
109.7 (C-Ar), 107.4 (C-Ar), 68.6 (CH2), 63.9 (Ph-C-OH), 56.0 (OMe),
31.2, 28.7, 28.6, 28.4, 25.3, 22.0 (6 × CH2), 25.1 (CH3), 13.9 (CH3); IR
IR (ATR): ν = [cm–1] = 2976, 2938, 2849, 1615, 1581, 1519, 1461,
˜
1378, 1330, 1273, 1172, 1105, 1041, 1000, 951, 873, 647, 599; HRMS
(ESI+): m/z calcd. for C20H25N2O11P + Na+: 523.1094, found 523.1053.
(ATR): ν = [cm–1] = 3301, 2926, 2854, 1613, 1579, 1513, 1465, 1445,
˜
1388, 1366, 1329, 1264, 1214, 1166, 1094, 1031, 1012, 896, 797, 723,
4-Methoxy-3-octanoxyacetophenone (29): Under anhydrous con-
ditions 0.97 g (5.8 mmol, 1.0 equiv.) 3-hydroxy-4-methoxyaceto-
phenone and 2.7 g (20 mmol, 3.5 equiv.) freshly dried potassium
carbonate was suspended in 50 mL dry DMF. To the resulting sus-
658; HRMS (ESI+): m/z calcd. for C17H27NO5 - H2O: 308.1862, found
+
308.1858.
Bis(1-(4-methoxy-2-nitro-5-octanoxyphenyl)ethyl)phosphonate
pension 2.7 mL (1.7 g, 30 mmol, 1.5 equiv.) n-bromooctane were (12): The synthesis was done according to general procedure 3;
added. The mixture was heated to 50 °C and stirred for 18 h. After
0.43 g (1.3 mmol, 2.1 equiv.) of alcohol 4 was dissolved in 10 mL
pyridine and 0.12 mL (0.15 g, 0.65 mmol, 1.0 equiv.) diphenylphos-
phite was added. Purification was done by column chromatogra-
phy: PE/EtOAc, 3:1 to PE/EtOAc, 2:1. Yield: 0.33 g (0.47 mmol, 72 %)
as a yellow oil and a mixture of four diastereomers. No single isomer
full conversion 50 mL 1 NaOH-sol. were added and the mixture
M
was extracted three times with diethyl ether. The combined organic
layers were washed with brine, dried with sodium sulfate, and con-
centrated in vacuo. The remaining residue was purified on silica gel
(CH2Cl2/EtOAc, 1:1). Yield: 1.45 g (5.21 mmol, 90 %) as a colorless was obtained pure, provided NMR-data is given for the obtained
solid. Rf = 0.40 (CH2Cl2/EtOAc, 1:1); m.p. 46 °C; 1H-NMR (500 MHz,
CDCl3): δ = 7.55 (dd, JH,H = 8.4 Hz, JH,H = 2.0 Hz, 1 H, H-Ar), 7.51
diastereomeric mixture. Rf = 0.30 (PE/EtOAc, 1:1); 1H-NMR (400 MHz,
CDCl3): δ = 7.57 (s, 2 H, H-Ar, 4 × ds), 7.11 (s, 2 H, H-Ar, 4 × ds), 6.37
(d, JH,P = 703 Hz, 1 H, P-H, 2 × ds), 6.29–6.11 (m, 2 H, Ar-CH-P),
3
4
3
3
1
(d, JH,H = 2.0 Hz, 1 H, H-Ar), 6.88 (d, JH,H = 8.2 Hz, 1 H, H-Ar), 4.06
3
(t, JH,H = 6.9 Hz, 2 H, CH2), 3.93 (s, 3 H, OMe), 2.56 (s, 3 H, CH3), 4.06–3.96 (m, 8 H, CH2 ), 3.93 (s, 3 H, OMe, 4 × ds), 1.90–1.83 (m, 8
3
3
1.85 (p, JH,H = 7.2 Hz, 2 H, CH2), 1.49–1.43 (m, 2 H, CH2), 1.38–1.25 H, CH2), 1.66 (d, JH,H = 6.1 Hz, 3 H, CH3, 4 × ds), 1.52–1.44 (m, 8 H,
(m, 8 H, H-Alkyl), 0.88 (t, 3JH,H = 7.1 Hz, 3 H, CH3); 13C-NMR (125 MHz,
CH2), 1.41–1.26 (m, 32 H, 8 × CH2), 0.90–0.87 (m, 12 H, CH3);
CDCl3): δ = 197.0 (C=O), 153.8 (C-Ar), 148.7 (C-Ar), 130.6 (C-Ar), 123.2 13C-NMR (100 MHz, CDCl3): δ = 153.8, 153.6 (C-5, ds), 148.6, 148.5,
(C-Ar), 111.7 (C-Ar), 110.3 (C-Ar), 69.2 (CH2), 56.2 (OMe), 31.9, 29.5,
148.3, 148.2 (C-Ar, ds), 138.7, 138.5, 138.2, 138.1 (C-Ar, ds), 132.2,
132.1, 132.0, 132.0, (C-Ar, ds), 108.7, 108.7, (C-Ar, ds), 107.8, 107.8,
29.3, 29.2, 26.1, 22.8 (6 × CH2), 26.4 (CH3), 14.2 (CH3); IR (ATR): ν =
˜
[cm–1] = 2962, 2940, 2921, 2851, 1671, 1583, 1507, 1464, 1444, 1424,
(C-Ar, ds), 70.9 (CH2), 69.7, 69.6 (d, JC,P = 2.8 Hz, Ar-C-P, ds), 56.4,
2
2
1344, 1286, 1215, 1185, 1143, 1072, 1003, 977, 877, 723, 642, 591; 56.3, 56.3, 56.2, (OMe, ds),29.4–29.3, 28.9 (d, JC,P = 3 Hz, CH3, ds),
HRMS (ESI+): m/z calcd. for C17H26O3 + H+: 279.1950, found 29.2, 25.9, 24.9, 24.8, 24.6, 24.6, (CH2, ds), 14.2, 14.1, (CH3, ds); 31P-
279.1960.
NMR (162 MHz, CDCl3): δ = 5.27, 4.56, 4.27; IR (ATR): ν = [cm–1] =
˜
2926, 2854, 1579, 1515, 1465, 1442, 1376, 1268, 1216, 1173, 1104,
4-Methoxy-2-nitro-5-octanoxyacetophenone (30): Under ambi-
ent conditions, 1.26 g (4.53 mmol, 1.0 equiv.) of ketone 29 were
dissolved in 23 mL acetic anhydride. The solution was slowly added
to an ice-cold solution of 30 mL conc. nitric acid (65 %) and 1.5 mL
1036, 944, 797, 722, 624, 522; HRMS (ESI+): m/z calcd. for
C
34H53N2O11P + Na+: 719.3285, found 719.3377.
γ-Bis(2-nitrobenzyl)ATP (13): The synthesis was done according to
acetic anhydride. After complete addition, the solution was stirred general procedure 4; 0.24 g (0.23 mmol, 1.0 equiv.) ADP 24, 0.13 g
for one hour at room temperature. The solution was poured care- (0.30 mmol, 1.3 equiv.) of phosphoramidite 6 were dissolved in
fully into 200 mL ice-water. The suspension was extracted twice
with CH2Cl2 and the combined organic layers were washed with sat.
sodium bicarbonate solution and brine, dried with sodium sulfate,
and concentrated in vacuo. The remaining residue was purified by
column chromatography on silica gel (CH2Cl2). Yield: 830 mg
(2.57 mmol, 57 %) as a yellow solid. Rf = 0.47 (CH2Cl2); m.p. 42 °C;
1H-NMR (400 MHz, CDCl3): δ = 7.60 (s, 1 H, H-Ar), 6.73 (s, 1 H, H-Ar),
10 mL dry CH3CN and 0.08 g (0.35 mmol, 1.5 equiv.) pyridinium
trichloroacetate and 64 μL (0.35 mmol, 1.5 equiv.) tert-butylhydro-
peroxide (5.5 M, in n-decan) were added. Yield: 0.13 g (0.16 mmol,
72 %) as a yellow cotton. Yield was calculated with two ammonium
counterions. 1H-NMR (600 MHz, D2O): δ = 8.40 (s, 1 H, H-Ar), 8.11
(s, 1 H, H-Ar), 7.98–7.93 (m, 2 H, H-Ar), 7.56–7.53 (m, 2 H, H-Ar),
3
7.53–7.48 (m, 2 H, H-Ar), 7.44–7.41 (m, 2 H, H-Ar), 5.89 (d, JH,H
=
3
3
2
4.09 (t, JH,H = 6.8 Hz, 2 H, CH2), 3.96 (s, 3 H, OMe), 2.49 (s, 3 H,
5.5 Hz, 1 H, H-1′), 5.41 (dd, JH,P = 8.3 Hz, JHH = 2.2 Hz, 2 H, Ph-
3
3
CH3), 1.87 (p, JH,H = 7.1 Hz, 2 H, CH2),1.49–1.42 (m, 2 H,CH2), 1.39– CH2-a), 5.37 (d, JH,P = 8.3 Hz, 2 H, Ph-CH2-b), 4.60–4.57 (m, 1 H, H-
3
3
3
1.25 (m, 8 H, 4 × CH2), 0.88 (t, JH,H = 7.0 Hz, 3 H, CH3); 13C-NMR
2′), 4.46 (dd, JH,H = 5.2 Hz, JH,H = 3.7 Hz, 1 H, H-3′), 4.33–4.28 (m,
(100 MHz, CDCl3): δ = 200.3 (C=O), 153.9 (C-Ar), 150.0 (C-Ar), 138.3
2 H, H-4′, 5′a), 4.26–4.20 (m, 1 H, H-5′b); 13C-NMR (150 MHz, D2O):
(C-Ar), 133.0 (C-Ar), 109.6 (C-Ar), 107.1 (C-Ar), 70.0 (CH2), 56.7 (OMe), δ = 153.5 (C-Ar), 148.9 (C-Ar), 148.0 (C-Ar), 146.0 (C-Ar), 145.8 (C-Ar),
3
36.5 (CH3), 31.9, 29.4, 29.3, 28.9, 25.9, 22.8 (6 × CH2), 14.7 (C-17); IR 140.5 (C-Ar), 134.5, 134.4 (C-Ar, rotamer), 130.7, 130.6 (d, JC,P
=
(ATR): ν = [cm–1] = 3095, 3001, 2919, 2852, 1697, 1574, 1517, 1503,
1471, 1442, 1394, 1352, 1284, 1220, 1184, 1120, 1050, 1004, 981,
957, 881, 794, 760, 547; HRMS (ESI+): m/z calcd. for C17H25NO5 + H+:
324.1805, found 324.1810.
4.7 Hz, rot.), 129.3, 129.2 (C-Ar, rot), 128.9, 128.6 (C-Ar, rot.), 124.9,
˜
3
124.8 (C-Ar, rot.), 118.0 (C-Ar), 87.2 (C-1′), 83.6 (d, JC,P = 9.4 Hz, C-
4′), 74.6 (C-2′), 70.0 (C-3′), 66.9 (m, Ph-CH2), 65.9 (m, C-5′); 31P-NMR
2
2
(242 MHz, D2O): δ = –10.5 (d, JP, P = 20.5 Hz, P-α), –11.9 (d, JP, P
=
17.0 Hz, P-γ), –23.0 (dd, 2JP, P = 20.6 Hz, 2JP, P = 16.9 Hz, P-ꢀ); IR (ATR):
rac-1-(4-Methoxy-2-nitro-5-octanoxyphenyl)ethane-1-ol (5):
The synthesis was done according to general procedure 1; 0.79 g
(2.5 mmol, 1.0 equiv.) of ketone 29 was dissolved in 40 mL THF/
MeOH, 1:1 and 0.33 g (8.6 mmol, 3.5 equiv.) sodium borohydride
was added. Yield: 0.78 g (2.4 mmol, 98 %) as a yellow solid. Rf =
ν = [cm–1] = 3047, 1689, 1641, 1608, 1523, 1445, 1340, 1237, 1127,
˜
1080, 1020, 993, 921, 815, 726, 462; HRMS (ESI+): m/z calcd. for
C24H26N7O17P3 + H+: 778.0676, found 778.0714.
γ-Bis(4,5-dimethoxy-2-nitrobenzyl)ATP (14): The synthesis was
0.23 (CH2Cl2); m.p. 42 °C; 1H-NMR (300 MHz, [D6]DMSO): δ = 7.52 (s, done according to general procedure 4; 0.13 g (0.23 mmol,
1 H, H-Ar), 7.34 (s, 1 H, H-Ar), 5.44 (d,3JH,H = 4.5 Hz, 1 H, OH), 5.26
1.5 equiv.) of phosphoramidte 7 and 0.15 g (0.16 mmol, 1.0 equiv.)
ADP 24 were dissolved in 10 mL dry CH3CN and 0.94 mL
(0.23 mmol, 1.5 equiv.) DCI-activator solution (0.25 M in CH3CN) and
3
3
(dq, JH,H = 4.6 Hz, JH,H = 6.2 Hz, 1 H, Ph-CH-CH3), 4.12–4.06 (m,2
3
H, CH2), 3.84 (s, 3 H, OMe), 1.75 (p, JH,H = 7.0 Hz, 2 H, CH2), 1.45–
Eur. J. Org. Chem. 2020, 6776–6789
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0.48 μL (0.27 mmol, 1.7 equiv.) tert-butylhydroperoxide (5.5
M
, in n-
(C-3′,ds), 65.0–64.9 (m, C-5′, ds), 55.9–55.8, 55.7, 55.5, 55.5, 55.4–
55.3, 54.1–55.1 (OMe, ds), 23.4–22.9 (CH3, ds); 31P-NMR (242 MHz,
[D4]MeOD): δ = –11.6 (d, 2JP, P = 19.2 Hz, P-α), –11.7 (d, 2JP, P = 20.8 Hz,
decan) were added. Yield: 89 mg (96 μmol, 60 %) as a yellow cotton.
Yield was calculated with two ammonium counterions. 1H-NMR
(600 MHz, D2O): δ = 8.40 (s, 1 H, H-Ar), 8.14 (s, 1 H, H-Ar), 7.40 (s,1
H, H-Ar), 7.38 (s,1 H, H-Ar), 6.80 (s,1 H, H-Ar), 6.73 (s, 1 H, H-Ar), 5.86
2
2
P-α), –14.1 (d, JP, P = 17.9 Hz, P-γ), –15.5 (d, JP, P = 16.9 Hz, P-γ),
–23.3 to –23.8 (m, P-ꢀ); IR (ATR): ν = [cm–1] = 3175, 1642, 1580,
˜
3
(d, JH,H = 5.3 Hz, 1 H, H-1′), 5.27–5.18 (m, 4 H, Ph-CH2), 4.54 (pt,
1518, 1453, 1379, 1272, 1218, 1172, 1127, 1077, 922, 874, 816, 794,
1 H, H-2′), 4.46 (pt, 1 H, H-3′), 4.38–4.33 (m, 1 H, H-5′a), 4.33–4.30 711, 642; HRMS (ESI–): m/z calcd. for C30H38N7O21P3– + H+: 926.1412,
(m, 1 H, H-4), 4.28–4.22 (m, 1 H, H-5′), 3.82 (s, 6 H,2 × OMe), 3.77 (s,
6 H,2 × OMe); 13C-NMR (150 MHz, D2O): δ = 152.7 (C-Ar), 152.6,
(C-Ar), 152.1 (C-Ar), 148.4 (C-Ar), 147.4 (C-Ar), 140.5 (C-Ar), 138.5 (d,
3JC,P = 12 Hz, C-Ar), 119.1 (C-Ar), 117.8 (C-Ar), 110.5 (C-Ar), 110.4 (C-
Ar), 107.5 (C-Ar), 107.5 (C-Ar), 87.1 (C-1′), 83.6 (C-4′), 74.5 (C-2′),69.3
(C-3′), 66.8 (Ph-CH2), 64.8 (C-5′), 56.2, 56.2, 55.9 (OMe); 31P-NMR
found 926.1443.
γ-Bis-O-(1-(4-methoxy-2-nitro-5-octanoxyphenyl)ethyl)ATP
(17): The synthesis was done according to general procedure 4b;
0.16 g (0.23 mmol, 1.0 equiv.) of H-phosphonate 11 was activated
with 62 mg (0.46 mmol, 2.0 equiv.) N-chlorosuccinimide and 0.23 g
(0.20 mmol 0.8 equiv.) ADP 24 was added. Yield: 53 mg (46 μmol,
23 %) as a yellow cotton in a mixture of four diastereomers. The
yield was calculated with two ammonium counterions. 1H-NMR
(600 MHz, [D4]MeOD): δ = 8.63, 8.62, 8.62, 8.60 (s, 1 H, H-Ar, ds.),
8.19, 8.18 (s, 1 H, H-Ar, ds.), 7.40–7.36 (m, 2 H, H-Ar), 7.06–6.93 (m,
2 H, H-Ar), 6.16–6.06 (m, 2 H, H-1′, Ph-CH-P, ds.), 4.69–4.64 (m, 1 H,
H-2′), 4.55–4.51 (m, 1 H, H-4′), 4.35–4.23 (m, 3 H, H-3′, 5′), 4.00–3.91
(m, 4 H, 2 × CH2), 3.91, 3.89, 3.87 (s, 6 H, 2 × OMe, ds.), 1.85–1.77
(m, 4 H, 2 × CH2), 1.64–1.53 (m, 6 H, 2 × CH3), 1.52–1.44 (m, 4 H,
2 × CH2), 1.42–1.27 (m, 16 H, 8 × CH2), 0.94–0.88 (m, 6 H, 2 × CH3);
13C-NMR (150 MHz, [D4]MeOD): δ = 155.1, 155.1, 154.9 (C-Ar, ds),
153.4 (C-Ar, ds), 149.6, 149.2 (C-Ar, ds), 148.0 (C-Ar), 137.9 (C-Ar), 131.
9 (C-Ar), 110.5, 110.1, 110.1 (C-Ar, ds), 108.6, 108.3 (C-Ar, ds), 91.2 (C-
1′), 83.7 (C-4′), 76.5 (C-2′), 73.9 (Ph-CH-O), 71.0 (C-3′), 70.0–69.9 (m,
C-5′), 65.9 (CH2), 56.7, 56.6, 56.5 (OMe, ds); 33.0, 30.6–30.1, 27.3–
2
2
(242 MHz, D2O): δ = –11.9 (d, JP, P = 20.5 Hz, P-α), –13.3 (d, JP, P
=
16.9 Hz, P-γ), –24.0 to –24.3 (m, P-ꢀ); IR (ATR): ν = [cm–1] = 3141,
˜
1679, 1521, 1327, 1275, 1218, 1169, 1127, 1066, 1019, 924, 813, 753,
710, 604; HRMS (ESI–): m/z calcd. for C28H33N7O21P3–: 896.0948,
found 896.0648.
γ-Bis(5-methoxy-2-nitro-4-octanoxybenzyl)ATP (15): The synthe-
sis was done according to general procedure 4; 0.16 g (0.21 mmol,
1.5 equiv.) of phosphoramidite 8 and 0.13 g (0.14 mmol, 1.0 equiv.)
ADP 24 were dissolved in 10 mL dry CH3CN and 0.85 mL
(0.21 mmol, 1.5 equiv.) DCI-activator solution (0.25
M
in CH3CN) and
0.44 μL (0.24 mmol, 1.7 equiv.) tert-butylhydroperoxide (5.5
M, in n-
decan) were added. Yield: 50 mg (45 μmol, 38 %) as a yellow cotton.
The yield was calculated with two ammonium counterions. 1H-NMR
(600 MHz, [D4]MeOD): δ = 8.65 (s, 1 H, H-Ar), 8.20 (s, 1 H, H-Ar), 7.55
(s, 1 H, H-Ar), 7.54, (s, 1 H, H-Ar), 7.25 (s, 1 H, H-Ar), 7.24(s, 1 H, H-
3
3
27.0, 23.7 (CH2), 24.7 (d, JC,P = 4.5 Hz, CH3), 24.6 (d, JC,P = 4.4 Hz,
3
3
CH3), 23.8 (d, JC,P = 4.3 Hz, CH3), 14.5 (CH3); 31P-NMR (242 MHz,
Ar), 5.99 (d, JH,H = 4.2 Hz, 1 H, H-1′), 5.57–5.46 (m, Ph-CH2), 4.50–
3
4.45 (m, 2 H, H-2′, 3′), 4.40–4.36 (m, 1 H, H-5′a), 4.30 (ddd, JH,H
=
[D4]MeOD): δ = –11.5 to –11.9 (m, P-α), –13.5 to –13.6 (m, P-γ),
2
2
2
2
11.5, JH,P = 6.0, JH,H = 2.4 1 H, H-5′b), 4.24–4.21 (m, 1 H, H-4′),
–15.5 (d, JP, P = 15.4 Hz, P-γ), –17.2 (d, JP, P = 16.5 Hz, P-γ), –23.4 to
–23.9 (m, P-ꢀ); IR (ATR): ν = [cm–1] = 3191, 2926, 2854, 1580, 1519,
4.00–3.97 (m, 4 H, 2 × CH2), 3.91 (s, 3 H, OMe), 3.90 (s, 3 H, OMe),
˜
3
1.80 (p, 3JH,H = 7.6 Hz, 4 H, 2 × CH2), 1.49 (p, JH,H = 7.1 Hz, 4 H, 2 ×
1442, 1379, 1329, 1271, 1217, 1175, 1104, 1079, 994, 871, 818, 757,
3
720, 624, 492; HRMS (ESI–): m/z calcd. for C44H65N7O21P3
:
–
CH2), 1.42–1.27 (m, 16 H, 8 × CH2), 0.91 (t, JH,H = 6.9 Hz, 6 H, 2 ×
CH3); 13C-NMR (150 MHz, [D4]MeOD): δ = 155.5 (C-Ar), 153.6 (C-Ar),
149.2 (C-Ar), 147.6 (C-Ar), 140.7 (C-Ar), 138.7 (C-Ar), 126.8 (C-Ar),
111.8 (C-Ar), 111.7 (C-Ar), 110.8 (C-Ar), 110.0 (C-Ar), 90.0 (C-1′), 85.4
(d, 3JC,P = 9.6 Hz, C-4′), 76.6 (C-2′), 71.1 (C-3′), 70.5 (d, 2JC,P = 9.6 Hz,
1120.3452, found 1120.3397.
4-(Hydroxymethyl)-phenyldecanoate (19): The synthesis of com-
pound 7 was performed as described in the literature. The analytical
data is in accordance with the literature.[25]
2
Ph-CH2), 68.0 (CH2), 65.4 (d, JC,P = 4.1 Hz, C-5′), 57.2, 56.8 (2 ×
OMe), 33.0, 30.5, 30.4, 30.1, 27.1, 23.7 (6 × CH2), 14.5 (CH3); 31P-NMR
O-(1-(4,5-Dimethoxy-2-nitrophenyl)ethyl)-bis(N,N-diisopropyl-
amin)phosphoramidite (20): Under anhydrous conditions a solu-
tion containing 0.30 g (1.3 mmol, 1.2 equiv.) of alcohol 4 and
0.45 mL (0.33 g, 3.3 mmol, 3.0 equiv.) anhydrous triethylamine in
10 mL anhydrous THF were added dropwise to an ice-cold solution
of 0.29 g (1.1 mmol, 1.0 equiv.) bis(N,N-diisopropylamino)-chloro-
phosphine in 10 mL anhydrous THF over a period of 30 minutes.
The resulting mixture was stirred at room temperature for 18 h. The
precipitate was removed and the filtrate was concentrated to dry-
2
(262 MHz, [D4]MeOD): δ = –11.8 (d, JP, P = 20.4 Hz, P-α), –13.3 (d,
2
2
2JP, P = 16.8 Hz, P-γ), –20.4 (dd, JP, P = 20.4 Hz, JP, P = 16.5 Hz, P-ꢀ);
IR (ATR): ν = [cm–1] = 3191, 2924, 2854, 1578, 1520, 1463, 1326,
˜
1275, 1217, 1128, 1067, 991, 872, 811, 754, 639; HRMS (ESI–): m/z
calcd. for C42H61N7O21P3–: 1092.3139, found 1092.3075.
γ-Bis-O-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl)ATP (16): The
synthesis was done according to general procedure 5; 0.20 g
(0.40 mmol, 1.0 equiv.) of H-phosphonate 11 was activated with
0.11 g (0.80 mmol, 2.0 equiv.) N-chlorosuccinimide and 0.31 g ness in vacuo. The remaining residue was purified on silica gel (PE/
(0.30 mmol 0.8 equiv.) ADP 24 was added. Yield: 0.14 g (0.15 μmol, EtOAc, 5:1 + 5 % TEA). Yield: 0.44 g (0.97 mmol, 88 %) as a yellow
49 %) as a yellow cotton in a mixture of four diastereomers. The
yield was calculated with two ammonium counterions. 1H-NMR
(600 MHz, [D4]MeOD): δ = 8.65, 8.64, 8.63, 8.61, (s, 1 H, H-Ar, ds.),
solid. Rf = 0.50 (PE/EtOAc, 5:1 + 5 % TEA); 1H-NMR (300 MHz, CDCl3):
3
δ = 7.56 (s, 1 H, H-Ar), 7.39 (s, 1 H, H-Ar), 5.51 (dq, JH,P = 12.4 Hz,
3JH,H = 6.2 Hz, 1 H, Ph-CH-O), 3.96 (s, 3 H, OMe), 3.93 (s, 3 H, OMe),
3
8.20, 8.19, 8.19 (s, 1 H, H-Ar, ds.), 7.43–7.29 (m, 2 H, H-Ar), 7.16–7.04 3.63–3.39 (m, 4 H, PNC-H), 1.53 (d, JH,H = 6.2 Hz, 3 H, CH3), 1.20
(m, 2 H, H-Ar), 6.15–6.02 (m, 2 H, H-1′, Ph-CH-P), 4.65–4.59 (m, 1 H,
(4 × d, 3JH,H = 6.6 Hz, 6 H, CH3); 13C-NMR (75 MHz, CDCl3): δ = 153.7
H-2′), 4.53–4.49 (m, 1 H, H-4′), 4.36–4.30 (m, 2 H, H-3′, 5′a), 4.29– (C-Ar), 147.5 (C-Ar), 139.4 (C-Ar), 138.1 (d, 3JC,P = 2.1 Hz, C-Ar), 109.6
4.21 (m, 1 H, H-5′b), 3.90, 3.88, 3.87, 3.83 (s, 12 H, OMe, ds.), 1.66– (C-Ar), 107.4 (C-Ar), 66.9 (d, 2JC,P = 17.7 Hz, Ph-CH2-P), 56.4, 56.3 (2 ×
1.51 (m, 6 H, 2 × CH3); 13C-NMR (150 MHz, D2O): δ = 154.1, 153.9,
OMe), 44.9, 44.7 (d, JC,P = 12.6 Hz, PNC), 25.2 (d, JC,P = 3.6 Hz,
153.7 (C-Ar, ds), 148.2, 148.2, 148.0, 147.9 (C-Ar, ds), 138.3, 138.1, CH3), 24.5–24.3 (m, CH3); 31P-NMR (162 MHz, CDCl3): δ = 148.8; IR
137.9 (C-Ar, ds), 132.7–132.6, 132.5–132.4, 132.1–131.9 (3 × m, C-Ar,
(ATR): ν = [cm–1] = 2966, 2929, 2868, 1519, 1497, 1462, 1452, 1438,
ds), 108.7, 108.7, 108.3, 107.6, 107.2, 107.1, 107.0, 106.8 (C-Ar, ds), 1319, 1272, 1219, 1184, 1114, 1099, 1051, 951, 916, 873, 794, 759,
88.1, 88.0, 87.9 (C-1′, ds), 84.1, 84.1 (C-4′, ds), 75.2, 75.1, 75.1 (C-2′,
672, 525; HRMS (ESI–): m/z calcd. for C22H40N3O5P + H+: 474.2733,
ds), 73.6–73.4, 73.0–72.8, 72.8–72.7 (3 × m, Ph-CH-O), 70.3, 70.2, 70.1 found 474.2730.
2
3
˜
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© 2020 The Authors. European Journal of Organic Chemistry
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Full Paper
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(1-(4,5-Dimethoxy-2-nitrophenyl)ethyl)-(4-decanoyloxybenzyl)-
phosphonate (23): Under anhydrous conditions 0.34 mL (0.41 g,
1.8 mmol, 1.0 equiv.) diphenylphosphite were dissolved in 20 mL
dry pyridine, cooled to –15 °C and mixed with 0.40 g (1.8 mmol,
1.0 equiv.) of compound 4. The solution was stirred for 45 min at
–15 °C, then 0.73 g (2.6 mmol, 1.5 equiv.) of compound 19 was
added stepwise. After complete addition, the reaction mixture was
δ = 8.64, 8.64, (s, 1 H, H-Ar, dia.), 8.18 (s, 1 H, H-Ar), 7.55–7.45 (m, 1
H, H-Ar), 7.36–7.18 (m, 3 H, H-Ar), 7.03–6.89 (m, 2 H, H-Ar), 6.28–
6.16 (m, 1 H, Ph-CH), 6.07–6.02 (m, 1 H, H-1′), 5.20–5.07 (m, 2 H, H-
2′, 3′ H), 4.60–4.52 (m, 1 H, H-4′), 4.49–4.40 (m, 1 H, H-5′a), 4.35–
4.18 (m, 3 H, H-5′b, Ph-CH2), 3.93, 3.86, 3.81, (s, 6 H, OMe, ds.), 2.61–
2.51 (m, 2 H, CH2), 1.77–1.70 (m, 2 H, CH2), 1.70–1.57 (m, 3 H, CH3,
3
ds.), 1.46–1.25 (m, 12 H, 6 × CH2), 0.90 (t, JH,H = 7.2 Hz, 3 H, CH3);
stirred for two hours at room temperature before all volatile com- 13C-NMR (150 MHz, [D4]MeOD): δ = 128.1 (C-Ar, ds), 120.9 (C-Ar, ds),
pounds were removed in vacuo. The residue was coevaporated first
with toluene and afterward with CH2Cl2. The residue was purified
by column chromatography on silica gel (PE/EtOAc, 1:1). Yield:
0.38 g (0.69 mmol, 38 %) as a yellow oil and a mixture of four
108.0 (C-Ar, ds), 106.6 (C-Ar), 87.4 (C-1′), 83.6 (C-4′), 74.9 (C-2′), 72.7
(Ph-CH-O), 69.9 (C-3′), 64.7 (m, C-5′), 68.4 (CH2), 55.0 (OMe, ds), 33.5,
29.1, 29.1, 29.0, 24.5, 22.2 (7 × CH2) 24.6 (d, 3JC,P = 4.4 Hz, CH3), 22.2
(Ph-CH-CH3), 14.5 (CH3); 31P-NMR (162 MHz, [D4]MeOD): δ = -11.0
diastereomers. The provided NMR data is given for the dominant to –11.3 (m, P-α). –13.3 to –14.0 (m, P-γ). –22.7 to –23.1 (m, P-γ); IR
diastereomer. Rf = 0.20 (PE/EtOAc, 1:1); 1H-NMR (300 MHz, CDCl3):
δ = 7.59 (s, 1 H, H-Ar), 7.34–7.28 (m, 2 H, H-Ar), 7.09–7.00 (m, 2 H,
H-Ar), 7.14 (s, 1 H, H-Ar), 6.95 (d, 1JH,P = 705 Hz, 1 H, P-H), 6.35–6.20
(m, 1 H, Ph-CH), 5.08–4.98 (m, 2 H, Ph-CH2), 4.01–3.98 (m, 6 H,2 ×
(ATR): ν = [cm–1] = 3175, 2928, 2854, 2684, 1673, 1606, 1579, 1457,
˜
1377, 1326, 1234, 1169, 1129, 1070, 1020, 991, 902, 810, 718; HRMS
(ESI–): m/z calcd. for C37H50N6O9P–: 975.2349, found 975.2280.
3
3
OMe), 2.55 (t, JH,H = 7.5 Hz, 2 H, CH2), 1.74 (p, JH,H = 7.4 Hz, 2 H,
CH2), 1.65 (m, 3 H, CH3), 1.45–1.11 (m, 12 H, 6 × CH2), 0.88 (t, 3JH,H
=
Acknowledgments
6.7 Hz, 3 H, CH3); 13C-NMR (75 MHz, CDCl3): δ = 172.3 (C-Ar), 154.0
(C-Ar), 151.2 (C-Ar), 148.4 (C-Ar), 139.8 (C-Ar), 132.8 (C-Ar), 129.3 (C-
Ar), 122.1 (C-Ar), 108.7 (C-Ar), 107.8 (C-Ar), 71.2 (Ph-CH), 67.0 (d,
2JC,P = 8.7 Hz, Ph-CH2), 56.5 (2 × OMe), 34.5 (CH2), 32.0 (CH2), 25.0
We are grateful for financial support provided by SFB1328,
Deutsche Forschungsgemeinschaft (DFG) and Universität Ham-
burg. Open access funding enabled and organized by Projekt
DEAL.
3
(CH2), 24.7 (d, JC,P = 4.9 Hz, CH3), 14.2 (CH3); 31P-NMR (162 MHz,
CDCl3): δ = 8.9, 8.9; IR (ATR): ν = [cm–1] = 2925, 2853, 1756, 1580,
˜
1517, 1461, 1375, 1330, 1271, 1166, 1104, 1020, 947, 872, 815, 793,
758, 558; HRMS (ESI+): m/z calcd. for C27H38NO9P + Na+: 574.2182,
found 574.2186.
Keywords: Caged compounds · Photocages · Bioorthogonal
Photo protecting group
(1-(4,5-Dimethoxy-2-nitrophenyl)ethyl)-(4-decanoyloxybenzyl)-
diisopropylphosphoramidite (21): The synthesis is based on gen-
eral procedure 4; 0.16 g (0.21 mmol, 1.5 equiv.) of alcohol 19 and
0.33 g (0.72 mmol, 1.3 equiv.) of diamidite 20 was dissolved in
15 mL dry THF, cooled to 0 °C and 1.8 mL (0.48 mmol, 1.0 equiv.)
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DCI-activator solution (0.25
M in CH3CN) was added. The solution
was stirred for one hour at room temperature. All volatile com-
pounds were removed in vacuo and the remaining residue was
purified by column chromatography on silica gel (PE/EtOAc, 5:1 +
5 % TEA). Yield: 0.29 g (0.45 mmol, 93 %) as a yellow oil and a
mixture of four diastereomers. The provided NMR data is given for
the dominant diastereomer. Rf = 0.75 (PE/EtOAc, 5:1 + 5 % TEA); 1H-
NMR (300 MHz, CDCl3): δ = 7.56 (s, 1 H, H-Ar), 7.38–7.31 (m, 3 H, H-
Ar), 7.08–7.01 (m, 2 H, H-Ar), 5.80–5.67 (m, 1 H, Ph-CH), 4.80–4.47
(m, 2 H, PH-CH2), 3.95–3.90 (m, 6 H, 2 × OMe), 3.63–3.44 (m, 2 H,
PNCH), 2.59–2.48 (m, 2 H, CH2), 1.81–1.67 (m, 2 H, CH2), 1.67–1.49
(m, 5 H, CH2, CH3), 1.47–1.24 (m, 10 H, 5 × CH2), 1.22–0.96 (m, 12
H, CH3), 0.91–0.81 (m, 2 H, H-CH3); 13C-NMR (100 MHz, CDCl3): δ =
172.5 (C-Ar), 153.7 (C-Ar), 150.0 (C-Ar), 147.7 (C-Ar), 139.0 (C-Ar),
137.0 (C-Ar), 128.8 (C-Ar), 128.1 (C-Ar), 121.6 (C-Ar), 109.5 (C-Ar),
107.4 (C-Ar), 67.8 (PhCH), 65.2 (Ph-CH2), 56.5, 56.5 (OMe), 43.2 (d,
2JC,P = 12.5 Hz, PNC), 34.5 (CH2), 24.8 (CH2), 24.8 (CH3), 29.6, 29.4,
29.3 (CH2), 25.1 (C-CH3), 14.3 (CH3); 31P-NMR (162 MHz, CDCl3): δ =
145.6, 144.5; IR (ATR): ν = [cm–1] = 2964, 2927, 2854, 1758, 1580,
˜
1518, 1460, 1394, 1364, 1332, 1271, 1198, 1183, 1164, 1103, 1012,
971, 919, 874, 793, 756, 597, 564; HRMS (ESI–): Could not be de-
tected neither in positive or negative mode.
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γ-(O-1-(4,5-Dimethoxy-2-nitrophenyl)ethyl)-(4-decanoyloxy-
benzyl)-ATP (22): The synthesis was done according to general
procedure 5; 55 mg (0.10 mmol, 1.0 equiv.) of H-phosphonate 23
was activated with 27 mg (0.20 mmol, 2.0 equiv.) N-chlorosuccinim-
ide and 0.08 g (0.08 mmol, 0.8 equiv.) ADP 24 was added. Yield:
24 mg (20 μmol, 26 %) as a yellow cotton in a mixture of four
diastereomers. 13C-signals were obtained from HSQC experiment,
due to the low amount of substance. 1H-NMR (400 MHz, [D4]MeOD):
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© 2020 The Authors. European Journal of Organic Chemistry
published by Wiley-VCH GmbH

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doi.org/10.1002/ejoc.202001229
EurJOC
European Journal of Organic Chemistry
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Received: September 11, 2020
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© 2020 The Authors. European Journal of Organic Chemistry
published by Wiley-VCH GmbH