A Modular Synthesis of Modified Phosphoanhydrides

Article abstract of DOI:10.1002/chem.201500838

Phosphoanhydrides (P-anhydrides) are ubiquitously occurring modifications in nature. Nucleotides and their conjugates, for example, are among the most important building blocks and signaling molecules in cell biology. To study and manipulate their biological functions, a diverse range of analogues have been developed. Phosphate-modified analogues have been successfully applied to study proteins that depend on these abundant cellular building blocks, but very often both the preparation and purification of these molecules are challenging. This study discloses a general access to P-anhydrides, including different nucleotide probes, that greatly facilitates their preparation and isolation. The convenient and scalable synthesis of, for example, ¹⁸O labeled nucleoside triphosphates holds promise for future applications in phosphoproteomics. Building the building blocks: This study discloses a general method for the functionalization of unprotected nucleotides and sugar phosphates with P-amidites in a highly modular way. The strategy facilitates the preparation of thiophosphate-containing nucleotides, ¹⁸O-labeled nucleoside triphosphates, and farnesylated nucleotides, as well as a range of dinucleoside polyphosphates and nucleotide sugars.

Full text of DOI:10.1002/chem.201500838

DOI: 10.1002/chem.201500838
Full Paper
&
Nucleotides |Very Important Paper|
A Modular Synthesis of Modified Phosphoanhydrides
Alexandre Hofer,[a] Gregor S. Cremosnik,[b] AndrØ C. Müller,[c] Roberto Giambruno,[c]
Claudia Trefzer,[c] Giulio Superti-Furga,[c] Keiryn L. Bennett,[c] and Henning J. Jessen*[a]
Abstract: Phosphoanhydrides (P-anhydrides) are ubiquitous-
ly occurring modifications in nature. Nucleotides and their
conjugates, for example, are among the most important
building blocks and signaling molecules in cell biology. To
study and manipulate their biological functions, a diverse
range of analogues have been developed. Phosphate-modi-
fied analogues have been successfully applied to study pro-
teins that depend on these abundant cellular building
blocks, but very often both the preparation and purification
of these molecules are challenging. This study discloses
a general access to P-anhydrides, including different nucleo-
tide probes, that greatly facilitates their preparation and iso-
lation. The convenient and scalable synthesis of, for exam-
ple, 18O labeled nucleoside triphosphates holds promise for
future applications in phosphoproteomics.
plings to obtain nucleotide oligophosphates,[6] this study un-
veils the true power of the concept. The broad applicability in
the synthesis of different established and novel nucleotide
conjugates and probes is showcased.
Introduction
Numerous important roles of nucleotides in biology are well-
established and it is difficult to identify areas in which nucleo-
tides and their conjugates are not critically involved.[1] As a con-
sequence, a great deal of research has been dedicated to the
development of nucleoside-derived drugs and nucleotide
probes as tools to study biological processes.[2] In addition to
modifications of the nucleobase and the sugar moiety, varia-
tions in the phosphate esters and anhydrides have been intro-
duced by chemical synthesis.[1a,3] Although there have been
promising recent developments in the field of phosphoanhy-
dride (P-anhydride) synthesis,[4] a flexible, straightforward, and
rapid synthetic approach to target different modified nucleo-
tides remains desirable. This unmet need motivated the devel-
opment of a phosphoramidite (P-amidite)[5]-based coupling
strategy that facilitates the rapid synthesis of a range of differ-
ent P-anhydride conjugates. Based on the idea of iterative cou-
Nucleotide probes that have been widely applied in chemi-
cal biology studies comprise such diverse structures as, for ex-
ample, phosphorothioate analogues of ATP and GTP in differ-
ent positions (a-S, b-S, g-S),[3b] and isotopically labeled probes,
such as the important radiolabeled nucleotides. Recently, non-
radioactive heavy isotope labels such as 18O have been pro-
posed as alternative tools for mass spectrometric profiling of
kinases.[7] In the future, and once this promising technology
finds more widespread application, further developments in
this direction will most likely lead to increased demand for
stable heavy isotope labeled nucleotides. In addition to the
terminally monosubstituted oligophosphates that have been
described so far, conjugates also exist in which the oligophos-
phate bridges two terminal organic residues. These structures
can be found, for example, in naturally occurring nucleoside di-
phosphate sugars,[8] dinucleoside polyphosphates,[9] prenylated
diphosphate saccharides,[10] and adenylated thiamine phos-
phates.[11] These can also exist as artificial structures, such as
the recently developed dye conjugates for single molecule
DNA sequencing,[12] nucleoside diphosphate prodrugs[13] or
photocaged nucleotides.[14] During the introduction and modi-
fication of the oligophosphate moiety, problems often arise
from low coupling efficiencies and extended reaction times,
hydrolysis and the requirement for excess reagents that com-
plicate purification of the polar products. These syntheses
have therefore been referred to as “arduous”[3k] and “notorious-
ly difficult to reproduce”.[4n] Such difficulties have significantly
hampered progress in many different areas of research that re-
quire access to the abundant phosphoanhydride conjugates.
In many instances, the preparation of the compounds is simply
restricted to specialized laboratories.
[a] A. Hofer, Prof. Dr. H. J. Jessen
Department of Chemistry, University of Zürich
Winterthurerstrasse 190
8057 Zürich (Switzerland)
E-mail: henningjacob.jessen@chem.uzh.ch
[b] G. S. Cremosnik
Chemistry Research Laboratory
University of Oxford, 12 Mansfield Road
Oxford OX1 3TA (UK)
[c] A. C. Müller, Dr. R. Giambruno, Dr. C. Trefzer, Prof. Dr. G. Superti-Furga,
Dr. K. L. Bennett
CeMM Research Center for Molecular Medicine
of the Austrian Academy of Sciences
Lazarettgasse 14, AKH BT 25.3
1090 Vienna (Austria)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201500838.
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Scheme 1. Anhydride bond formation using P-amidites under ambient coupling conditions provides access to a variety of interesting metabolites and probes
in a rapid, modular, and reproducible way.
In a recent study, it was shown that P-amidites can be che-
moselectively coupled to phosphate groups; thereby releasing
a PV-PV anhydride after oxidation and cleavage of fluorenyl-
methyl (Fm) groups that block the terminal phosphate
(Scheme 1).[6] Repetition of this procedure enables an iterative
elongation. The method is easy to implement because the re-
actions occur under ambient conditions without any drying of
solvents and reagents. Moreover, no protecting groups on the
nucleotides are necessary, the yields are usually high, and the
coupling reactions occur in less than 10 min. Purification of the
final products is often possible by simple precipitation. Conse-
quently, the method is well-suited to scale-up. An extension of
this method would greatly facilitate flexible and chemoselec-
tive P-anhydride-forming reactions to obtain such diverse mol-
ecules as heavy isotope labeled nucleotides, nucleoside di-
phosphate sugars, dinucleoside polyphosphates, internal and
terminal thiophosphate analogues, caged and membrane-per-
meable nucleotides, farnesyl oligophosphate conjugates, and
many others (1–8; Scheme 1).
actions were performed under ambient conditions (open flask,
wet solvents); under these conditions the acceptor phosphate
was completely consumed followed by hydrolysis of excess
donor. The comparatively fast hydrolysis of excess donor thus
enabled the use of unprotected acceptors, thereby substantial-
ly streamlining the synthesis. Rather than covering only a set
of different acceptors in combination with a single P-amidite
donor, a variety of P-amidite donors were combined with dif-
ferent acceptor phosphates to showcase the scope of this
straightforward protocol. It is important to note that residues
R in P-amidites can be easily introduced by well-established
chemistry from commercially available building blocks. With
these protocols, symmetric and asymmetric modifications of
the P-amidite can be introduced in a reliable way. Complemen-
tarily, many different acceptors such as nucleotides and sugar-
phosphates are either commercially available or can be readily
accessed by established protocols.
A set of P-amidite donors was chosen (Figure 1, upper sec-
tion). Amidite 9 is commercially available, but alternatively can
be prepared on a large scale in one step.[15] Amidite 10 was
obtained in one step from 18O labeled benzyl alcohol, which is
accessible on gram scale by hydrolysis of benzyl bromide with
H218O (see the Supporting Information). Amidite 11 has been
reported previously.[16] The preparation of the asymmetric P-
amidites was possible in two steps (see the Supporting Infor-
mation). The phosphate acceptors (Figure 1, lower section) are
commercially available.
Results and Discussion
Synthesis
The strategy described in this publication relied on a P-anhy-
dride bond-forming reaction in which a P-amidite was coupled
as an electrophile (donor) to a nucleophilic phosphate (accept-
or). The terms are chosen in analogy to glycosylation chemis-
try. Subsequently, the intermediate mixed PIII-PV anhydride was
oxidized or sulfurized to a PV-PV anhydride. Finally, the protect-
ing groups were removed if present on the anhydride or
donor, whereas the acceptor did not require protection. All re-
Table 1 gives an overview of the products that were synthe-
sized. As summarized in the experimental section, these syn-
theses are easy to conduct. Briefly, a soluble salt of the phos-
phate (tetrabutylammonium) in wet N,N-dimethylformamide
(DMF) was coupled with 1–2 equivalents of the P-amidite
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Figure 1. Overview of the acceptors and donors used in this study.
donor in the presence of an activator (4,5-dicyanomidiazole,
DCI; or ethylthiotetrazole, ETT)[17] under ambient conditions.
Progress of the reaction was monitored by 31P NMR spectro-
scopic analysis. After 1–30 min, the intermediate PIII-PV anhy-
dride was oxidized (2 min) and, if present, the protecting
groups were removed under basic conditions (usually 5 min).
The products were precipitated by addition of diethyl ether. In
cases where significant impurities were present, for example,
H-phosphonates or phosphates as a result of hydrolysis (see
the Supporting Information), further purification was possible
by chromatography (strong anion exchange or reverse phase).
Different conjugates of groups 1–8 were accessible in highly
competitive yields, purity, and time (Table 1; see Figure 2 and
the spectra section in the Supporting Information for a compre-
hensive 31P NMR spectroscopic analysis of all reactions). Nu-
cleoside thiophosphate conjugates were prepared from ami-
dite 9 in combination with S8-mediated oxidation.[18] The prod-
ucts (Table 1, entries 1–5) did not require further purification,
as judged by 31P NMR spectroscopic analysis, and ADP-bS was
readily synthesized on a 0.8 g scale. To synthesize UDP-a-S
(Table 1, entry 6), UMP-a-S 28 was coupled with amidite 9. Se-
lective functionalization of the oxygen rather than sulfur was
observed and oxidation required the use of tBuOOH; otherwise
the sulfur atom was additionally oxidized leading to decompo-
sition. In contrast to the terminally modified nucleotides
(Table 1, entries 1–5), UDP-a-S (entry 6) required purification by
strong anion exchange chromatography.
was not possible with fluorenylmethyl (Fm) groups. After cou-
pling, deprotection by hydrogenation under aqueous condi-
tions was therefore necessary. These conditions led to partial
hydrolysis of the protected P-anhydride. Whereas the overall
deprotection was very clean, hydrolysis led to the presence of
10–20% of the corresponding nucleoside diphosphate; this
was not considered problematic in assays that eventually re-
lease the NDP. The method summarized in entries 7–9 has
been scaled to produce up to 200 mg of 18O labeled nucleo-
side triphosphates with a purity of 80%. If required, the nu-
cleoside diphosphate (20%) can be removed by strong anion
exchange chromatography, but at the cost of reduced yield.
Cytosine was converted quantitatively into 5,6-dihydro uracil
under the hydrogenation conditions (Table 1, entry 9).
Table 1, entries 10 and 11 summarize the facile preparation
of nucleoside diphosphate prodrugs (AB)2-GDP and (AB)2-CDP.
The acetoxybenzyl group (AB) is the only known group that
can be conjugated to nucleoside diphosphates to render the
prodrugs cell permeable.[13] Once cells have taken up the mole-
cule, enzymes cleave the AB groups and release the bioactive
nucleoside diphosphate. Both (AB)2 prodrugs described herein
have been obtained in essentially quantitative yield (>95%)
within 15 min (coupling and oxidation) and in high purity
upon simple precipitation (see the Supporting Information).
Table 1, entries 12–14 show an easy preparation of photocaged
nucleotides.
The
[7-(diethylamino)coumarin-4-yl]methyl
(DEACM) photocage has been used extensively to exert control
over the spatiotemporal release of biologically important phos-
phates (e.g., inositol polyphosphates, and nucleotides).[16,19] By
using a mixed P-amidite 12, this group can now be introduced
within a few minutes to biologically relevant nucleoside di-
Table 1, entries 7–9 summarize the preparation of heavy iso-
tope (18O)-labeled nucleoside triphosphates with P-amidite 10.
The benzyl (Bn) protecting groups were chosen because facile
incorporation of 18O atoms from heavy water is enabled; this
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Table 1. Scope of P-amidite-based P-anhydride forming reactions.
Entry
Acceptor
Donor
Oxidant
Product
Yield [%]
Purification
1
2
3
4
5
6
9
9
9
9
9
9
15
16
19
20
22
28
S8
S8
S8
S8
ADP-bS
ATP-gS
GTP-gS
UDP-bS
CDP-bS
UDP-aS
71
57
53
74
91
33
95
44
95
52
95
44
95
95
44
30
63
95
95
quant.
92
88
75
36
83
precipitation
precipitation
precipitation
precipitation
precipitation
SAX
S8
tBuOOH
precipitation[b]
SAX
7
8
9
10
10
10
16
21
23
mCPBA
mCPBA
mCPBA
ATP-g(18O)2
precipitation[b]
SAX
precipitation[b]
SAX
precipitation
precipitation
RP-18 FC
UTP-g(18O)2
[c]
5,6-dihydro-UTP-g(18O)2
10
11
12
13
14
15
16
17
18
19
20
21
22
11
11
12
12
12
13
13
14[a]
14[a]
14[a]
14[a]
14[a]
14[a]
18
22
15
16
18
22
23
24
25
26
27
17
20
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
mCPBA
(AB)2-GDP
(AB)2-CDP
ADP-b-DEACM
ATP-g-DEACM
GDP-b-DEACM
CDP-b-farnesyl
CTP-g-farnesyl
UDP-a-D-Glc
UDP-a-d-GlcNAc
UDP-a-D-Man
UDP-a-d-Gal
AppppU
RP-18 FC
RP-18 FC
precipitation
precipitation
precipitation
precipitation
precipitation
precipitation
SAX
UppU
precipitation
[a] Acetyl groups were removed by treatment with aqueous ammonia or aqueous methanol/NEt3 after cleavage of the Fm protecting groups (piperidine).
[b] Precipitated material contained approximately 10–20% hydrolyzed XDP. Removal of XDP by SAX yielded highly enriched material, although with dimin-
ished yield (second line). [c] Cytidine was reduced under these conditions to 5,6-dihydrouridine.
and triphosphates such as adenosine and guanosine. After re-
moval of the Fm group in a few minutes under basic condi-
tions, the products were purified by RP-18 chromatography
because precipitation was difficult to achieve and was incom-
plete.
tion of uridine is not strictly required; however, amidite 14
with acetylated uridine can be purified and this is not the case
for the unprotected derivative. After coupling, the Fm and
acetyl groups were removed by treatment with piperidine and
either ammonia or triethylamine.
Similar to the AB nucleotides, lipidated nucleoside di- and
triphosphates have been proposed as potentially cell-permea-
ble analogues.[2b] P-amidite coupling can be used to obtain
such conjugates (Table 1, entries 15 and 16). As judged by
31P NMR spectroscopic analysis, b-farnesylated CDP (entry 15)
was obtained in essentially quantitative yield and high purity.
g-Farnesylated CTP, however, contained more impurities (15%
according to 31P NMR analysis, see the Supporting Information)
arising from partial hydrolysis. Again, coupling and oxidation
occurred within a few minutes and verification by mass spec-
trometry revealed that epoxidation of the double bonds in the
farnesyl residue had not occurred during oxidation of the PIII-PV
intermediate with mCPBA.
The high selectivity of the coupling reaction is showcased in
Figure 2 for the coupling of P-amidite 14 with N-acetyl glucos-
amine 1-phosphate to obtain uridine diphosphate N-acetyl glu-
cosamine (Table 1, entry 18). The proton-decoupled 31P NMR
spectrum of the reaction mixture after completed PIII-PV anhy-
dride coupling (Figure 2, spectrum II) shows that the only sig-
nificant side reaction is the hydrolysis of P-amidite 14. Com-
plete consumption of the unprotected sugar phosphate in wet
solvent can thus be achieved by using the reported conditions.
Precipitation after oxidation of the PIII intermediate enables the
removal of the majority of excess hydrolyzed 14 and yields
almost pure Fm-protected diphosphate intermediate (29;
Figure 2, spectrum III, mixture of diastereomers). After Fm-de-
protection, precipitation gives the free diphosphate 30 in es-
sentially pure form (Figure 2, spectrum IV). Product 31 is finally
obtained in a similarly high purity after removal of the acetyl
groups on the uridyl moiety followed by lyophilization of the
reaction mixture (Figure 2, spectrum V). This procedure does
not require any chromatography.
Application of the P-amidite protocol to obtain non-natural
analogues (Table 1, entries 1–16) was further extended to natu-
rally occurring conjugates. Most importantly, nucleoside di-
phosphate sugars were readily prepared by using P-amidite 14
(Table 1, entries 17–20). Here, one residue is acetylated uridine,
which enables facile conjugation to sugar phosphates. Acetyla-
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Figure 2. Reaction monitoring by proton-decoupled 31P NMR spectroscopy (recorded in 20% [D7]DMF under ambient conditions) for the coupling of P-amidite
14 to N-acetyl glucosamine 1-phosphate (25) to obtain UDP-a-d-GlcNAc (Table 1, entry 18). I) N-acetyl glucosamine 1-phosphate (25); II) addition of P-amidite
14 and activator (7 min); III) oxidation with mCPBA (2 min); IV) Removal of fluorenylmethyl (Fm) protecting group and precipitation; V) acetyl group cleavage
and precipitation. Abbreviations: Pip+ =piperidinium; TBA=tetra-n-butyl ammonium; Ac=acetyl; Fm=fluorenylmethyl; mCPBA=meta-chloroperbenzoic
acid.
Four different UDP sugars (glucose, N-acetyl glucosamine,
mannose, and galactose) were obtained in high yields (75% to
quantitative) and purity (60 to 86%, by HPLC and 31P NMR
analyses) by simple precipitation from the reaction mixture. Al-
though further purification by chromatography can be ach-
ieved, Hindsgaul and co-workers[4n] showed that nucleotide
sugars can be used in enzymatic glycosylations even in the
presence of byproducts and thus do not strictly require high
purity.
phosphorylation in vitro.[7,20] By using a 1:1 mixture of ATP and
heavy ATP, combined with a kinase of interest and a cellular ex-
tract, phosphorylation sites can be identified by proteolytic di-
gestion of proteins, phosphopeptide enrichment, and subse-
quent analysis by liquid chromatography mass spectrometry.
This method relies on the generation of doublets showing
a separation in mass of 6 Da. The next step was to study
whether g-(18O)2-ATP could be used in such assays to generate
a
mass shift of 4 Da.
A
synthetic peptide (biotin-
Finally, C2-symmetric (UppU) and nonsymmetric (AppppU)
dinucleoside polyphosphates with different P-anhydride chain
lengths can be easily accessed (Table 1, entries 21 and 22).
Again, it is evident that longer anhydride chains were more
challenging to prepare and thus the product required purifica-
tion by strong anion exchange chromatography. It is conceiva-
ble that this method could also be used to introduce thiophos-
phates as non-natural analogues.
GGEAIYAAPFKK-NH2) with the preferred ABL1 consensus motif
YxxP was phosphorylated in vitro by the tyrosine kinase Abel-
son murine leukemia viral oncogene homolog 1 (ABL1) using
ATP and g-(18O)2-ATP. Briefly, a 1:1 mixture of ATP and (18O)2-
ATP was added at a final concentration of 1 mm to a vial con-
taining immunopurified, constitutively active ABL1-PP. The re-
action was initiated by adding 5 mm of the synthetic ABL1
peptide at room temperature on a rotary shaker (see the Sup-
porting Information, Figure S1). After one hour and centrifuga-
tion, the supernatant was enriched for phosphopeptides by
immobilized metal affinity chromatography.[21] Samples were
analyzed with a Q-Exactive mass spectrometer coupled to
a Dionex Ultimate 3000 HPLC nanoflow system via a nanoelec-
trospray ion source using a liquid junction. The LC-MSMS anal-
ysis clearly identified the expected mass shift of 4 Da
Application to Phosphoproteomics
To demonstrate the utility of the prepared analogues, g-(18O)2-
labeled ‘heavy’ ATP of high purity (>95%) was prepared on
a 30 mg scale. It has been previously shown that (18O)4-labeled
ATP can be used in quantitative phosphoproteomics to study
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(Figure 3). Moreover, the reporter immonium ion, indicative of
a phosphotyrosine-containing peptide, was observed, also
with the expected mass shift of 4 Da (see the Supporting Infor-
mation, Figures S2 and S3).
of 20 mg; see the Supporting Information) and no loss of effi-
ciency regarding yield or purity of ADP-bS were observed. The
protocols described herein will thus make available a variety of
nucleotide probes on large scale that have been difficult to
obtain by other methods.
Along these lines, it is shown that synthetic g-(18O)2-labeled
ATP can be used in mass spectrometry-based proteomics to
identify and assign the phosphorylation sites introduced by an
exogenous kinase. Given the ease of preparation and thus the
ability to obtain large amounts (200 mg have been prepared
so far), this “heavy” ATP will be developed as an alternative to
expensive g-(18O)4-labeled ATP.
Conclusions
The described protocol, in combination with iterative oligo-
phosphate synthesis, provides a powerful toolbox that is suita-
ble for the generation of a multitude of oligophosphate conju-
gates (e.g., 1–8) and their analogues in a straightforward
manner. It is important to note that these couplings typically
occur within a few minutes under ambient conditions with
very little byproduct formation. This is in contrast to the major-
ity of other protocols; for example, the reaction times for nu-
cleotide sugar synthesis usually require multiple days to
ensure significant product formation.[8] Recent notable excep-
tions to this, however, do exist.[4n,q,22] Nonetheless, the simple,
reproducible, and modular coupling under ambient conditions
within a few minutes in approximately equimolar amounts of
donor and acceptor as described in this publication has been
a long-standing synthetic challenge. The sensitivity of the P-an-
hydride forming reaction to a 30-fold scale-up has been ana-
lyzed in the case of ADP-bS (0.6 g of starting material 9 instead
Experimental Section
General P-Anhydride Synthesis Procedure
Monophosphate TBA salt was dissolved in DMF/[D7]DMF. P-Amidite
(1.3–2.7 equiv) and activator (ETT or DCI; 2 equiv) were added and
the progress of the coupling was monitored by 31P NMR spectro-
scopic analysis. Subsequently, mCPBA (77%, 1.5–2.8 equiv) was
added in small portions at 08C. The product was obtained by pre-
cipitation with Et2O/hexane (5:1 v/v), centrifugation, washing of
the pellets with Et2O, and drying under high vacuum. Protecting
groups were removed under basic conditions (e.g., piperidine) and
Figure 3. MS2 spectra of the respective isolated and fragmented precursor ions A) non-phosphorylated synthetic peptide (m/z 738.87); B) normal phosphory-
lated synthetic peptide (m/z 778.87), and C) (18O)2-‘heavy’ phosphorylated synthetic peptide (m/z 780.87). For amino acid sequence identification, b-fragment
and y-fragment ions are annotated in red and blue, respectively. Sequence coverage of the synthetic peptide is indicated by the lower blue or upper red
lines next to the letter of the respective amino acid at the top right. The distance between the y6 and y7 ions corresponds to the mass of the modified or un-
modified tyrosine residue showing the expected additional mass shift of 4 Da for the (18O)2-‘heavy’ phosphorylated synthetic peptide (C).
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Acknowledgements
The authors would like to thank Jay S. Siegel and John Robin-
son for their continuous support. The work was funded by the
University of Zürich (Forschungskredit to H. J. J.) and the Swiss
National Science Foundation (Professorship PP00P2_157607 to
H. J. J.). Research in the Bennett laboratory at CeMM is sup-
ported by the Austrian Academy of Sciences and the Austrian
Science Fund FWF.
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Keywords: chemoselectivity · isotopic labeling · nucleotides ·
phosphorylation · polyanions
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Received: March 2, 2015
Published online on June 1, 2015
Chem. Eur. J. 2015, 21, 10116 – 10122
www.chemeurj.org
10122
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