Synthesis of 2-diphospho-myo-inositol 1,3,4,5,6-pentakisphosphate and a photocaged analogue

Article abstract of DOI:10.1039/c6ob00094k

Diphosphoinositol polyphosphates (inositol pyrophosphates, X-InsP₇) are a family of second messengers with important roles in eukaryotic biology. Their chemical synthesis and modification remains a challenging task due to the high density of ph

Full text of DOI:10.1039/c6ob00094k

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Synthesis of 2-diphospho-myo-inositol 1,3,4,5,6-
pentakisphosphate and a photocaged analogue†
Cite this: DOI: 10.1039/c6ob00094k
I. Pavlovic,a D. T. Thakora and H. J. Jessen*b
Received 13th January 2016,
Accepted 24th February 2016
DOI: 10.1039/c6ob00094k
www.rsc.org/obc
Diphosphoinositol polyphosphates (inositol pyrophosphates,
X-InsP7) are a family of second messengers with important roles in
eukaryotic biology. Their chemical synthesis and modification
remains a challenging task due to the high density of phosphate
groups arranged around the myo-inositol core. Here, a novel
approach is presented that facilitates the incorporation of the
diphosphate in the 2-position (2-InsP7) and that enables the intro-
duction of a photocage subunit.
myo-Inositol pyrophosphates are a family of ubiquitous mes-
senger molecules with broad impact on different cellular func-
tions.1 Since their initial discovery in 19932 it has been shown,
for example, that they are involved in the regulation of telo-
mere length, tumor growth, insulin secretion, weight gain, and
monitoring of cellular energy homeostasis.3 They provide a
link between signalling and metabolism4 and are specifically
regulated by a diverse set of kinases and phosphatases.5 While
in principle six different diphosphoinositol pentakisphos-
phates (X-InsP7, X position of the diphosphate on the myo-
inositol ring, see Fig. 1) could exist, only three of them have
Fig. 1 All six possible X-InsP7 (1–6).
inositol pyrophosphates has exclusively relied on hydro-
genations in order to remove up to thirteen protecting groups
in the last step without cleaving the P-anhydride.7b,d,8 However,
such conditions are not compatible with a wide range of modi-
fications that would be useful for studies into the function of
the inositol pyrophosphates. Consequently, there is a dearth of
synthetic tools for chemical biology applications, with non-
hydrolysable analogues as the only exceptions that have been
reported and applied by the groups of Fiedler and Potter.9
Recently, a novel approach has been disclosed and enabled
the synthesis of 5-InsP7 5 and a photocaged analogue without
the need for a hydrogenation in the last step.10 Moreover,
initial studies have demonstrated how such molecules can be
transported into living cells to study their biological func-
tion.10,11 Here, it is shown that the novel synthetic approach
can be applied to the synthesis of 2-InsP7 2 with the diphos-
phate moiety in the axial position, a challenge that has pre-
viously only been mastered by Falck and coworkers in 1997.8d
Additionally, it is shown that also fluorescent photocaged12
2-InsP7 17 can be prepared due to the novel deprotection con-
been identified in nature so far, namely 1-InsP7 1, 5-InsP7
5
and 6-InsP7 6. Addition of yet another phosphate group gives
rise to InsP8 of which 1,5-InsP8 and 5,6-InsP8 as well as the tri-
phosphate containing 5-InsP8 are occurring in nature.6 Even
so, there remains uncertainty as to whether the family of natu-
rally occurring inositol pyrophosphates has been exhaustively
described, as their structural assignment is difficult and
material for analysis is scarce.
There are several examples in which synthetic material has
been used to assign the naturally occurring regioisomers.
Moreover, a more detailed understanding of the specificities of
the dedicated kinases and phosphatases has been achieved
with such material.5a,6c,7 Previous synthetic work to obtain the
aDepartment of Chemistry, University of Zürich, Winterthurerstr. 190, 8057 Zürich,
Switzerland
bDepartment of Chemistry and Pharmacy, Albert-Ludwigs University Freiburg,
Albertstr. 21, 79104 Freiburg, Germany. E-mail: Henning.Jessen@oc.uni-freiburg.de
†Electronic supplementary information (ESI) available: Synthetic procedures
and spectra. See DOI: 10.1039/c6ob00094k
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ditions applied in the last step. This opens up the possibility the significant molecular crowding it was possible to isolate
to introduce a range of different alternative residues for func- hexakisphosphate 14 in 73% yield, underlining the efficacy of
tional and control studies, which were previously inaccessible.
P-amidite chemistry.15 The phosphate triester in the axial
The synthesis commenced with a selective benzoylation of 2-position was then deprotected selectively using hydrazine
the 2-position of myo-inositol via the ortho-benzoate as pub- acetate in acetic acid. These conditions effected hydrazone for-
lished previously by Potter and coworkers (see Scheme 1).13 mation and thus initiated Lev cleavage followed by 1,6 elimi-
Bis-acetonide formation, para-methoxybenzyl (PMB) protection nation of the benzyl adapter as a quinone methide. The
and cleavage of the benzoate furnished alcohol 10,8e which deprotected phosphate 14a was obtained in 87% crude yield
was phosphitylated with the novel P-amidite 11. This after precipitation and it was not purified further due to its
P-amidite contains two LevB protecting groups that link a levu- amphiphilic character. Next, the phosphoric anhydride was
linate (Lev) protecting group via a benzyl adapter (B) unit to generated through the intermediacy of a mixed P(III)–P(V) an-
the P-atom. The adapter enables the use of acyl protecting hydride followed by oxidation.16 Two different P-amidites 13
groups, such as Lev, that could otherwise not be stably con- and 15 were applied in these couplings to generate both an
nected to the phosphate moiety in 12a that results after oxi- exhaustively Fm protected 2-InsP7 16a and an analogue in
dation of the phosphite intermediate. Both acetonides and the which one of the Fm groups was replaced by a fluorescent di-
PMB groups were then removed under acidic conditions, ethylamino coumarine photocage (DEACM) 16b.10,17 All 11 or
which did not affect the LevB protecting groups on the phos- 12 Fm protecting groups were then cleaved in 5% piperidine
phate. The ensuing pentaol was exhaustively phosphitylated in DMF. After complete deprotection, the piperidinium salts
with bis-Fm-P-amidite 13 containing the 9-fluorenylmethyl (11 or 12 piperidinium counterions) of 2-InsP7 2 or DEACM
(Fm)14 protecting group and the phosphite triesters were oxi- 2-InsP7 17 precipitated from DMF upon addition of diethyl-
dized with meta-chloroperoxybenzoic acid (mCPBA). Despite ether. The precipitate was dissolved in methanol and addition
Scheme 1 Synthesis of 2-InsP7 2 and photocaged DEACM 2-InsP7 17 based on fluorenylmethyl (Fm) protection in combination with a recently
developed phosphate protecting group (LevB). Abbreviations: TFA: trifluoroacetic acid. DCM: dichloromethane. DCI: 4,5-dicyanoimidazole. mCPBA:
meta-chloro perbenzoic acid. Fm: fluorenylmethyl. DEACM: [7-(diethylamino)-coumarin-4-yl]methyl. DMF: dimethylformamide. pTSA: para-toluene
sulfonic acid. DMSO: dimethyl sulfoxide. TBAI: tetrabuthylammonium iodide. PMB: para-methoxybenzyl.
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in the last step. Consequently, the approach is highly modular
and allows for the generation of previously inaccessible ana-
logues. The application of fluorenylmethyl protection of phos-
phates facilitates basic deprotection, which is compatible with
the presence of a wide range of chemical functionalities.
Specifically, it is shown that a DEACM photocaging subunit
can be installed on the β-phosphate of the axial diphosphate
of 2-InsP7 2. Such DEACM photocages are very useful tools as
they are intrinsically fluorescent, can thus be tracked in living
cells and cleaved by irradiation with UV light. Applications of
such analogues to biological questions will be described else-
where. The synthetic 2-InsP7 2 was used in a modified mala-
chite green assay to study the specificity of a dedicated inositol
pyrophosphate phosphatase from yeast (Ddp1). 2-InsP7 2 is no
substrate, underlining the exquisite specificity of these
enzymes. More generally, access to 2-InsP7 2 will be helpful to
assign substrates and products of the enzymes involved in ino-
sitol pyrophosphate turnover. In addition, the new synthetic
strategy in combination with stereoselective approaches21 has
the potential to also make accessible modified asymmetric
inositol pyrophosphates for chemical biology applications.
Fig. 2 Purity analysis of 2-InsP7 and DEACM-2-InsP7 by gel electro-
phoresis (PAGE) and DAPI staining. Gel (a) Lane I: PolyP marker. Lane II:
empty. Lane III: 5-InsP7 (control). Lane IV: empty. Lane V: 2-InsP7 2. Gel
(b) Lane I: PolyP marker. Lane II: empty. Lane III: orange G dye. Lane IV:
empty. Lane V: DEACM-2-InsP7 17. PolyP serves as a standard for the
quality of separation and Orange G dye to track migration before stain-
ing. Gels were converted to grey-scale.
of excess sodium iodide led to precipitation of the sodium
salts of the products in high quality.
Acknowledgements
The quality of the material was checked by polyacrylamide gel
electrophoresis (PAGE) on dense gels followed by staining with
4,6-diamidin-2-phenylindol (DAPI).18 The gels underline the
high quality of the material with only little contamination of
InsP6 as hydrolysis product (Fig. 2). This impurity is easier to
assign by PAGE due to overlapping signals in the 31P NMR. Since
photocaged 2-InsP7 17 contains a chromophore, the quality was
additionally checked by reversed phase HPLC and only one peak
was found in this analysis (see the ESI† for the HPLC trace).
The synthetic material was then used in an enzymatic assay
to further study the specificity of diadenosine and diphospho-
inositol polyphosphate phosphohydrolase 1 from yeast (Ddp1).
This phosphatase has been shown previously to have a high
preference for cleavage of the diphosphate in 1-InsP7 1 over all
other positions but it has not been studied concerning the
cleavage of 2-InsP7 2.19 Recombinant purified Ddp1 was used
in a modified malachite green assay that measures phosphate
release from the inositol pyrophosphate according to literature
precedence.8b No significant phosphate release from 2-InsP7 2
was measured within 10 minutes incubation in the presence
of 100 ng Ddp1 at 37 °C, whereas 1-InsP7 1 was cleaved
efficiently (data not shown). Thus, 2-InsP7 2 is no substrate of
Ddp1, underlining the specificity of the phosphatases involved
in inositol pyrophosphate turnover, which was also recently
demonstrated for the phosphatase domain of Asp1 from
Schizosaccharomyces pombe.20
We thank A. Saiardi for kindly providing the plasmid encoding
Ddp1 and the Swiss National Science Foundation (SNF,
PP00P2_157607) for financial support.
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This paper describes a twelve-step synthesis of 2-InsP7 2 and a
photocaged analogue 17 that does not rely on a hydrogenation
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