Synthesis of modular headgroup conjugates corresponding to all seven phosphatidylinositol polyphosphate isomers for convenient probe generation

Article abstract of DOI:10.1002/ejoc.200900476

Phosphatidylinositol polyphosphate lipids (PIP_ns) play key roles in important: biological pathways, and defects in the signaling activities of these molecules have been implicated in a number of disease states. As such, it is necessary to under

Full text of DOI:10.1002/ejoc.200900476

FULL PAPER
DOI: 10.1002/ejoc.200900476
Synthesis of Modular Headgroup Conjugates Corresponding to All Seven
Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation
Denghuang Gong,[a] Heidi E. Bostic,[a] Matthew D. Smith,[a] and Michael D. Best*[a]
Keywords: Phosphoinositides / Lipid signaling / Probes / Phospholipids / Membranes / Bioorganic chemistry
Phosphatidylinositol polyphosphate lipids (PIPns) play key
roles in important biological pathways, and defects in the sig-
naling activities of these molecules have been implicated in
a number of disease states. As such, it is necessary to under-
stand the complex roles of these lipids in biological path-
ways, which often involve their actions as site-specific li-
gands that recruit receptors to the surfaces of cellular mem-
branes. However, the complex structures of PIPn family mem-
bers, of which seven biologically active isomers exist, compli-
cate studies. Derivatized analogs of the PIPn structures are
beneficial as chemical tools for elucidating signaling and
binding activities. Herein, we present an efficient approach
to probe the generation in which amino conjugates of PIPn
headgroups can be conveniently functionalized in the final
step of the synthesis to obtain a number of derivatized ana-
logs of use for different studies. In addition to the application
of this strategy to generate PI-(4,5)-P2 headgroup probes, we
also report the synthesis of PIPn–amine conjugates corre-
sponding to all seven naturally existing isomers. This ap-
proach will be invaluable for generating the range of probe
structures that is required to elucidate the intricacies of PIPn
signaling.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
stood. For example, numerous protein-binding modules
that target PIPns have been identified, including the PH,
PX, FYVE, ENTH, ANTH, FERM, Tubby, and
PROPPIN domains. However, the diversity present within
these families provides an impediment to detailed under-
standing, particularly with the PH domains, for which se-
quence homology searches predict over 250 members.[7]
Here, variations in binding details complicate full elucida-
tion of the affinities and specificities that each particular
receptor exhibits for the seven PIPn isomers. Furthermore,
diversity in the structural features required for binding also
exist, as certain proteins require the presence of the mem-
brane, whereas others show high affinity for isolated PIPn
headgroups.[9,16,17] In particular, certain PH domains con-
tain minimal domains for membrane penetration, and thus
interactions are believed to be primarily driven by headg-
roup association.[7,18] In such cases, simplified headgroup
analogs can be effective for probing and perturbing bind-
ing.[19]
The structural complexity of the PIPns complicates probe
synthesis, as these phospholipids contain phosphodiester-
linked myo-inositol headgroups that are phosphorylated at
every combination of the 3-, 4-, and 5-positions. Neverthe-
less, a number of effective approaches for PIPn synthesis
have been reported,[20–33] including the recent development
of peptide catalysts effective for stereoselective phosphory-
lation of different myo-inositol-based substrates.[34–38]
Whereas these strategies have resulted in the generation of
probes that have proven effective for characterizing binding,
the complex synthesis that is required for each reporter-
Introduction
The phosphatidylinositol polyphosphates (PIPns) are vi-
tal signaling lipids that regulate a litany of key cellular pro-
cesses.[1–7] As is common with signaling lipids, the PIPns act
as ligands for the recruitment of peripheral protein recep-
tors to membrane surfaces, events that control both protein
function and subcellular localization.[8–10] Signaling lipids
are particularly prominent in the regulation of crucial cellu-
lar processes as their activities transmit information from
membranes into cellular pathways. As such, defects in lipid-
signaling events have been implicated in the onset of dis-
eases including cancer, diabetes and leukemia.[11] A particu-
larly well-understood example pertains to the conversion
of phosphatidylinositol-4,5-bisphosphate [PI-(4,5)-P2] to
phosphatidylinositol-3,4,5-trisphosphate [PI-(3,4,5)-P3], the
forward and reverse reactions of which are catalyzed by
phosphoinositide 3-kinase (PI3K)[12] and the phosphatase
PTEN,[13] respectively. The product, PI-(3,4,5)-P3, binds
and activates protein kinase B (Akt), which activates nu-
merous pathways that control cell proliferation. Both
PI3K[14] and the tumor suppressor PTEN[15] are among the
most frequently mutated enzymes in tumorigenesis.
Whereas recent advances in the study of protein–PIPn
association have yielded the elucidation of important de-
tails, many aspects of these events remain poorly under-
[a] Department of Chemistry, the University of Tennessee,
Knoxville, TN 37996, USA
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900476.
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Eur. J. Org. Chem. 2009, 4170–4179

Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation
derivatized probe generally limits the scope of probes that (3,4,5)-P3 (1d) isomers are detailed in Scheme 2. First, inter-
can be produced. Herein, we report the design and synthesis
of modular PIPn headgroup scaffolds that can be conve-
niently derivatized for efficient access to a range of func-
tionalized headgroup analogs for use as probes.
mediates 3a–c, containing benzoyl groups at the location of
the eventual phosphate moieties, were synthesized in four
steps from myo-inositol as described previously.[22] PI-
(3,4,5)-P3 precursor 3d was generated by appending a third
benzoyl group onto compound 3c. The remaining hydroxy
groups in compounds 3a–d were then protected with BOM
groups to generate 4a–d, followed by benzoyl deprotection
to access 5a–d. Next, phosphoramidite chemistry was used
to install the phosphotriester moieties of 6a–d, followed by
silyl deprotection to produce 7a–d.
For the synthesis of headgroup analogs of PI-3P (1e), PI-
3,4-P2 (1f), and PI-3,5-P2 (1g), the corresponding interme-
diates 7e–g (Scheme 1), containing Bn rather than BOM
protecting groups, were synthesized in 9–12 steps as de-
scribed previously.[21] The synthesis of compounds 7a–g, in
which all functional groups are protected except for the P-
1 hydroxy moiety, set the stage for phosphodiester coupling
to produce 1a–g (Scheme 1). Here, compounds 7a–g were
each coupled to phosphoramidite reagent 8, obtained from
Cbz-protected aminohexanol and benzyloxybis(diisopropyl-
amino)phosphane, to install the phosphodiester linkage of
2a–g. Finally, global deprotection by hydrogenolysis yielded
quantitative access to modular PIPn–amine conjugates 1a–
g.
Results and Discussion
A particularly advantageous approach to the production
of a range of derivatized probes corresponding to a biomo-
lecule target is to install a reactive functional group that
can be modified in the final step of the synthesis. To im-
plement this strategy for the production of PIPn headgroup
probes, we designed modular analogs of type 1a–g
(Scheme 1), which consist of each PIPn headgroup tethered
to an amino group that can be conveniently functionalized
by coupling chemistry. The amino group is linked through
a phosphodiester moiety at the 1-position, similar to the
connection of the headgroup to the glycerolipid backbone
in the natural lipid. A similar derivatization approach has
previously proven effective in the development of tetherable
analogs of inositol 1,4,5-trisphosphate (IP3).[39–42] Finally, a
hexyl linker was installed between the headgroup and
amino moieties of 1a–g to introduce hydrophobicity and in
part mimic the non-polar properties of the lipid backbone.
Upon obtaining our initial PIPn–amine conjugate, that
These enantiomerically pure modular headgroup analogs corresponding to PI-(4,5)-P2 (1c), we set out to showcase
were synthesized as described below. the efficacy of this modular scaffold for probe development
In the synthesis of 1a–g, it was desirable to obtain these (Scheme 3). Derivatized PI-(4,5)-P2 headgroup analogs 9–
final compounds by a global deprotection in the final step 11 were conveniently synthesized in one step by coupling
through a clean hydrogenolysis process, an approach that the amine of 1c to the corresponding succinimidyl esters
has previously been advantageous.[24–26] Thus, we sought to (12a–c). We first introduced a tetraethyleneglycol (TEG)–
prepare these from precursors 2a–g, in which each func- biotin group to produce probe 9, of use for immobilizing
tional group is protected by a group that can be removed the headgroup onto surfaces or for pull-down studies. Re-
by hydrogenolysis, including amino protection using a ben- cently, we implemented this compound in the development
zyloxycarbonyl (Cbz) group, hydroxy moieties protected as of a microplate-based assay for the detection of protein–
benzyl (Bn) or benzyloxymethyl (BOM) ethers, and the PIPn headgroup binding interactions.[43] These studies pro-
phosphates masked as benzyl phosphotriesters.[24,26] The vided further evidence that proteins could bind to PIPn
initial synthetic routes to headgroup analogs corresponding headgroups with high affinity outside of the membrane en-
to the PI-4P (1a), PI-5P (1b), PI-(4,5)-P2 (1c), and PI- vironment, and showcased the efficacy of the described de-
Scheme 1. Synthetic route to modular PIPn headgroup–amine conjugates of all seven naturally occurring isomers as modular scaffolds.
Eur. J. Org. Chem. 2009, 4170–4179
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D. Gong, H. E. Bostic, M. D. Smith, M. D. Best
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Scheme 2. Synthesis of fully protected myo-inositol headgroup intermediates.
Scheme 3. Convenient coupling of PIPn headgroup–amine conjugates for modular access to derivatized probe structures.
rivatized headgroup analogs for probing and perturbing studies.[44] In addition, bifunctional probes exemplified by
these binding events. In addition, this approach shows great 11 are targeted for use in purifying, identifying and charac-
prospects for high-throughput characterization of protein– terizing PIPn-binding receptors from a complex sample
PIPn binding interactions.
such as a cell extract. This strategy for probe development
We have since further derivatized 1c to introduce other is inspired by the concept of activity-based protein profiling
reporter groups of use for investigations, such as the instal- (ABPP), which employs bifunctional analogs as probes for
lation of a benzophenone tag (10) for photo-crosslinking the mechanism-based collective labeling of enzyme targets
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Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation
Scheme 4. Synthesis of bifunctional reporter tag 15 for coupling onto PIPn headgroups.
based on their activity.[45–49] In the current approach, probe
11 contains both a photo-crosslinking group (benzophen-
one) for attachment to cognate receptors, as well as a sec-
ondary tag (azide) for purification of successfully cross-
linked proteins from a complex sample. Similar bifunctional
designs have previously proven effective for this pur-
pose,[50,51] including a recent study that utilized a phosphat-
idylcholine (PC) probe to identify receptors that target this
lipid.[52] The bifunctional tag of 11 was synthesized begin-
from a Pure Solv solvent delivery system purchased from Innov-
ative Technology, Inc. Column chromatography was performed by
using 230–400 mesh silica gel purchased from Sorbent Technol-
ogies. NMR spectra were obtained by using a Bruker AC250 spec-
trometer updated with a TecMag data collection system, a Varian
Mercury 300 spectrometer, and a Bruker Avance 400 spectrometer.
Mass spectra were obtained with JEOL DART-AccuTOF and ABI
DE Pro MALDI spectrometers with high-resolution capabilities.
Optical rotation values were obtained by using a Perkin–Elmer 241
polarimeter. The synthesis of initial modular PI-(4,5)-P2 scaffold
ning with benzophenone–lysine conjugate 13,[53] as shown 1c, including the procedures for intermediates 2c–6c and 8c as well
as biotin conjugate 9, were previously described.[43]
in Scheme 4. This was performed by Boc-deprotection and
direct azido transfer to azide 14, followed by ester hydroly-
sis to 15. We are currently implementing probe 11 for iden-
4-O-Benzyl-2,3,5,6-O-tetra(benzyloxymethyl)-1-O-(tert-butyldi-
phenylsilyl)-D-myo-inositol (4a): Diisopropylethylamine (1.0 mL,
tifying PIPn-binding proteins, studies that exemplify the
powerful advantages of chemical approaches employing
probes as tools for efficiently elucidating the intricacies of
complex biological binding and signaling events. Although
these probes may be subject to enzymatic modification dur-
ing biological studies, such as by phosphodiesterases, an ef-
fective remedy for this problem is to introduce non-hydro-
lyzable phosphorothioate groups.[54]
5.76 mmol) and benzyl chloromethyl ether (0.8 mL, 5.76 mmol)
were added to a solution of 3a (0.251 g, 0.48 mmol) in 1,2-dichloro-
ethane (10 mL). The reaction mixture was stirred at reflux for 24 h.
Diisopropylethylamine (1.0 mL, 5.76 mmol) and benzyl chlo-
romethyl ether (0.8 mL, 5.76 mmol) were added to the solution,
and the mixture was stirred at reflux for another 24 h. After the
reaction was finished, water was added and the mixture extracted
with dichloromethane (2ϫ50 mL). The combined organic phases
were dried with magnesium sulfate, filtered and concentrated. The
crude product was purified by chromatography on silica gel (hex-
ane/acetone, 5:1) to give 4a (0.275 g, 57%) as a syrup. [α]2D96 K
=
Conclusion
1
+8.3 (c = 1.0, CHCl3). H NMR (300 MHz, CDCl3): δ = 8.00 (d,
J = 7.2 Hz, 2 H), 7.71–7.80 (m, 4 H), 7.13–7.44 (m, 25 H), 6.95–
6.98 (m, 2 H), 6.86–6.89 (m, 2 H), 5.80 (t, J = 9.6 Hz, 1 H), 5.21
(d, J = 6.3 Hz, 1 H), 5.03–5.05 (m, 2 H), 4.75–4.86 (m, 5 H), 4.64
(d, J = 11.7 Hz, 1 H), 4.48 (d, J = 12.3 Hz, 1 H), 4.28–4.42 (m, 3
H), 4.14–4.22 (m, 2 H), 4.07 (d, J = 6.9 Hz, 1 H), 3.95–4.01 (m, 2
H), 3.78 (t, J = 9.3 Hz, 1 H), 3.51 (d, J = 9.9 Hz, 1 H), 3.44 (s, 1
H), 1.12 (s, 9 H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 165.7,
138.2, 137.9, 137.7, 136.2, 136.0, 134.2, 133.0, 133.0, 130.3, 130.1,
129.8, 128.5, 128.5, 128.3, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7,
127.5, 127.4, 127.4, 127.3, 97.3, 96.5, 95.4, 91.8, 79.2, 78.8, 74.7,
74.5, 73.5, 73.3, 70.7, 70.5, 69.4, 68.9, 27.4, 19.4 ppm. MALDI-
HRMS: calcd. for C61H66O11SiNa [M + Na]+ 1025.4267; found
1025.4282.
Whereas the signaling lipids that compose the phosphat-
idylinositol family play crucial roles in numerous cellular
processes, these activities are difficult to study due to the
complex synthesis required to access derivatized probes of
these structures. As such, versatile strategies for probe pro-
duction are imperative for generating the diverse probes
that are necessary to elucidate the complex details of lipid
signaling processes. The modular approach to probe gener-
ation presented herein has proven successful for the conve-
nient production of biologically active analogs of use for
direct characterization of protein–PIPn binding interactions.
We are currently extending these studies to scrutinize all
seven naturally occurring headgroup isomers.
5-O-Benzyl-2,3,4,6-O-tetra-(benzyloxymethyl)-1-O-(tert-butyldiphen-
ylsilyl)-D-myo-inositol (4b): Diisopropylethylamine (1.6 mL,
Experimental Section
General Experimental: Generally, reagents were purchased from
9.25 mmol) and benzyl chloromethyl ether (1.3 mL, 9.38 mmol)
were added to a solution of 3b (0.40 g, 0.765 mmol) in 1,2-dichloro-
ethane (16 mL). The reaction mixture was stirred at reflux for 24 h.
Acros or Aldrich and used as received. Dry solvents were obtained
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D. Gong, H. E. Bostic, M. D. Smith, M. D. Best
FULL PAPER
Diisopropylethylamine (1.6 mL, 9.25 mmol) and benzyl chlo-
romethyl ether (1.3 mL, 9.38 mmol) were added to the solution,
and the mixture was stirred at reflux for another 24 h. After the
reaction was finished, water was added and the mixture extracted
with dichloromethane (2ϫ50 mL). The combined organic phases
were dried with magnesium sulfate, filtered and concentrated. The
crude product was purified by chromatography on silica gel (hex-
ane/acetone, 5:1) to provide 4b (0.526 g, 69%) as a syrup. [α]2D96 K
(0.405 g, 87%) as a syrup. [α]2D96 K = +36.2 (c = 1.5, CHCl3). 1H
NMR (300 MHz, CDCl3): δ = 7.69–7.72 (m, 4 H), 7.14–7.39 (m,
26 H), 4.29–5.03 (m, 17 H), 4.01 (t, J = 9.3 Hz, 1 H), 3.91 (t, J =
9.6 Hz, 1 H), 3.81–3.84 (m, 2 H), 3.47 (dd, J = 9.9, 1.8 Hz, 1 H),
3.35 (t, J = 8.4 Hz, 1 H), 1.10 (s, 9 H) ppm. 13C NMR (75.5 MHz,
CDCl3): δ = 138.1, 137.9, 137.7, 137.1, 135.8, 135.7, 133.7, 133.5,
129.9, 129.6, 128.4, 128.3, 128.3, 128.2, 128.0, 128.0, 127.8, 127.8,
127.7, 127.6, 127.5, 127.3, 96.5, 95.8, 95.3, 92.9, 84.1, 79.1, 76.0,
74.7, 74.0, 73.3, 70.0, 69.6, 69.1, 69.1, 27.0, 19.3 ppm. MALDI-
1
= +15.7 (c = 1.9, CHCl3). H NMR (300 MHz, CDCl3): δ = 8.08
(d, J = 10.2 Hz, 2 H), 7.70–7.74 (m, 4 H), 7.11–7.44 (m, 25 H), HRMS: calcd. for C54H62O10SiNa [M + Na]+ 921.4004; found
6.93–6.96 (m, 4 H), 5.36 (t, J = 9.6 Hz, 1 H), 5.05 (d, J = 6.6 Hz,
1 H), 4.90 (d, J = 6.6 Hz, 1 H), 4.69–4.84 (m, 5 H), 4.19–4.49 (m,
11 H), 4.04 (d, J = 9.6 Hz, 1 H), 3.50–3.54 (m, 2 H), 1.09 (s, 9 H)
ppm. 13C NMR (75.5 MHz, CDCl3): δ = 166.0, 138.1, 137.7, 137.7,
137.6, 135.9, 135.8, 133.8, 132.8, 132.8, 130.1, 129.9, 129.8, 128.3,
128.2, 128.1, 127.8, 127.7, 127.6, 127.5, 127.5, 127.3, 127.3, 127.2,
127.2, 127.1, 96.7, 95.7, 95.5, 92.4, 79.2, 78.4, 76.6, 75.3, 75.2, 74.5,
73.9, 69.9, 69.7, 69.3, 69.1, 27.2, 19.3 ppm. MALDI-HRMS: calcd.
for C61H66O11SiNa [M + Na]+ 1025.4267; found 1025.4296.
921.4033.
2,6-O-Bis(benzyloxymethyl)-1-O-(tert-butyldiphenylsilyl)-D-myo-
inositol (5d): A 1% sodium hydroxide solution (2 mL) and dichloro-
methane (2 mL) were added to a solution of 4d (0.13 g,
0.134 mmol) in methanol (5 mL). The mixture was stirred at room
temp. under nitrogen for 18 h. The solvent was removed and the
residue purified by chromatography on silica gel (hexane/acetone,
4:1) to give 5d (0.66 g, 75%) as a syrup. [α]2D96 K = +60.5 (c = 2.6.
1
CHCl3). H NMR (400 MHz, CDCl3): δ = 7.68 (t, J = 7.2 Hz, 4
3,4,5-O-Tribenzyl-2,6-O-bis(benzyloxymethyl)-1-O-(tert-butyldiphen- H), 7.45–7.25 (m, 16 H), 4.94 (d, J = 6.8 Hz, 1 H), 4.75–4.65 (m,
ylsilyl)- -myo-inositol (4d): Diisopropylethylamine (0.43 mL, 3 H), 4.54–4.51 (m, 3 H), 4.42 (d, J = 11.6 Hz, 1 H), 4.30 (br. s, 1
D
2.6 mmol) and benzyl chloromethyl ether (0.36 mL, 2.6 mmol) were
added to a solution of 3d (0.236 g, 0.323 mmol) in 1,2-dichloroe-
thane (3 mL). The reaction mixture was stirred at reflux for 24 h.
After the reaction was finished, water was added and the mixture
H), 3.81–3.72 (m, 2 H), 3.66–3.54 (m, 3 H), 3.20–3.13 (m, 2 H),
2.75 (br. s, 1 H), 1.04 (s, 9 H) ppm. 13C NMR (100.6 MHz, CDCl3):
δ = 136.3, 136.2, 135.4, 135.3, 133.1, 129.6, 129.3, 128.1, 127.6,
127.5, 127.3, 127.1, 96.5, 96.2, 84.7, 82.6, 73.3, 72.2, 70.4, 70.0,
extracted with dichloromethane (2 ϫ50 mL). The combined or- 69.9, 26.5, 18.9 ppm. DART-HRMS: calcd. for C38H45O8Si [M –
ganic phases were dried with magnesium sulfate, filtered and con-
centrated. The crude product was purified by chromatography on
silica gel (hexane/acetone, 10:1) to yield 4d (0.158 g, 52%) as a
syrup. [α]2D96 K = +68.9 (c = 4.8, CHCl3). Other characterizations
match previous reports.[26]
H]– 657.2884; found 657.2888.
2,3,5,6-O-Tetrakis(benzyloxymethyl)-1-O-(tert-butyldiphenyl-
silyl)-D-myo-inositol 4-(Dibenzyl phosphate) (6a): Dibenzyl N,N-di-
isopropylphosphoramidite (0.16 mL, 0.489 mmol) was added to a
solution of alcohol 5a (0.220 g, 0.245 mmol) and 1H-tetrazole
2,3,5,6-O-Tetrakis(benzyloxymethyl)-1-O-(tert-butyldiphenyl- (1.63 mL, 0.735 mmol, 0.45  in acetonitrile) in dichloromethane
silyl)- -myo-inositol (5a): DIBAL-H (2.0 mL, 2.0 mmol) was added (10 mL) under nitrogen, and the mixture was stirred at room temp.
D
to a solution of 4a (0.27 g, 0.27 mmol) in dichloromethane (15 mL)
at –78 °C. After 90 min, ethyl acetate (2 mL) was added to quench
the reaction. The solution was partitioned between ethyl acetate
(60 mL) and water (80 mL), and the aqueous phase was extracted
with ethyl acetate (60 mL). The combined organic phases were
washed with brine (50 mL), dried with magnesium sulfate, filtered
and concentrated. The crude product was purified by chromatog-
for 18 h. The reaction solution was cooled to –60 °C, meta-chloro-
perbenzoic acid (mCPBA, 0.25 g, 57–86%) was added, and the
mixture was stirred at this temperature for 60 min before slowly
warming to room temp. by removal of the cooling bath. The solu-
tion was diluted to 60 mL with dichloromethane, and washed with
saturated sodium bicarbonate (2ϫ30 mL). The aqueous phase was
extracted with dichloromethane (60 mL). The combined organic
raphy on silica gel (hexane/acetone, 10:1) to give 5a (0.225 g, 91%) phases were dried with magnesium sulfate, filtered, concentrated,
as a syrup. [α]2D96 K = +16.5 (c = 1.0, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 7.77–7.83 (m, 4 H), 7.25–7.47 (m, 26 H), 5.19 (d, J =
6.6 Hz, 1 H), 5.01 (d, J = 6.9 Hz, 1 H), 4.83–4.93 (m, 7 H), 4.53–
4.66 (m, 5 H), 4.24–4.45 (m, 4 H), 4.06 (t, J = 9.6 Hz, 1 H), 3.97
(d, J = 9.9 Hz, 1 H), 3.69 (s, 1 H), 3.32–3.40 (m, 2 H), 1.17 (s, 9
and the residue was purified by chromatography on silica gel (hex-
anes/acetone, 7:1) to yield product 6a (0.258 g, 91%) as a syrup.
1
[α]2D96 K = +10.1 (c = 1.0, CHCl3). H NMR (400 MHz, CDCl3): δ
= 7.76 (d, J = 6.0 Hz, 2 H), 7.67–7.69 (m, 2 H), 7.17–7.38 (m, 34
H), 7.05–7.08 (m, 2 H), 5.19 (d, J = 6.3 Hz, 1 H), 5.12 (d, J =
H) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 138.2, 138.1, 137.8, 6.6 Hz, 1 H), 4.98–5.06 (m, 6 H), 4.62–4.86 (m, 8 H), 4.18–4.43 (m,
136.8, 135.9, 135.8, 133.8, 132.9, 130.0, 129.8, 128.4, 128.3, 128.2,
128.1, 127.9, 127.9, 127.7, 127.7, 127.6, 127.5, 127.4, 127.4, 127.2,
96.7, 96.5, 95.1, 92.7, 78.0, 75.0, 74.7, 74.4, 71.9, 70.1, 69.8, 69.1,
5 H), 4.10 (d, J = 12.3 Hz, 1 H), 3.91 (d, J = 9.6 Hz, 1 H), 3.65 (t,
J = 9.1 Hz, 1 H), 3.45 (s, 1 H), 3.35 (d, J = 10.2 Hz, 1 H), 1.09 (s,
9 H) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 138.3, 138.1, 138.1,
69.0, 27.2, 19.2 ppm. MALDI-HRMS: calcd. for C54H62O10SiNa 137.9, 136.0, 136.0, 135.8, 133.9, 132.7, 130.1, 129.9, 128.4, 128.3,
[M + Na]+ 921.4004; found 921.4011.
128.2, 128.2, 128.2, 128.0, 127.8, 127.8, 127.7, 127.7, 127.7, 127.5,
127.5, 127.4, 127.3, 127.2, 126.8, 97.0, 96.4, 95.3, 92.7, 77.7, 77.3,
77.2, 75.1, 74.3, 73.7, 70.6, 69.2, 69.1, 68.3, 27.2, 19.2 ppm. 31P
NMR (121.5 MHz, CDCl3): δ = 0.05 (s, 1 P) ppm. MALDI-
HRMS: calcd. for C68H75O13PSiNa [M + Na]+ 1181.4607; found
1181.4605.
2,3,4,6-O-Tetrakis(benzyloxymethyl)-1-O-(tert-butyldiphenyl-
silyl)-D-myo-inositol (5b): DIBAL-H (4.4 mL, 4.4 mmol) was added
to a solution of 4b (0.52 g, 0.518 mmol) in dichloromethane
(30 mL) at –78 °C. After 90 min, ethyl acetate (5 mL) was added to
quench the reaction. The solution was partitioned between ethyl
acetate (100 mL) and water (100 mL), and the aqueous phase was
extracted with ethyl acetate (80 mL). The combined organic phases
were washed with brine (80 mL), dried with magnesium sulfate,
filtered and concentrated. The crude product was purified by
chromatography on silica gel (hexane/acetone, 10:1) to provide 5b
2,3,4,6-O-Tetrakis(benzyloxymethyl)-1-O-(tert-butyldiphenyl-
silyl)-D-myo-inositol 5-(Dibenzyl phosphate) (6b): Dibenzyl N,N-di-
isopropylphosphoramidite (0.30 mL, 0.89 mmol) was added to a
solution of alcohol 5b (0.40 g, 0.445 mmol) and 1H-tetrazole
(3.0 mL, 1.34 mmol, 0.45  in acetonitrile) in dichloromethane
4174
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 4170–4179

Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation
(20 mL) under nitrogen, and the mixture was stirred at room temp.
for 18 h. The reaction solution was cooled to –60 °C, and mCPBA
(0.45 g, 57–86%) was added. The mixture was then stirred at this
temperature for 60 min and then slowly warmed to room temp.
following removal of the cooling bath. The solution was diluted to
80 mL with dichloromethane and washed with saturated sodium
hydrogen carbonate (2ϫ50 mL). The aqueous phase was extracted
with dichloromethane (60 mL). The combined organic phases were
dried with magnesium sulfate, filtered, and concentrated, and the
residue was purified by chromatography on silica gel (hexanes/ace-
127.8, 127.7, 127.6, 127.6, 127.6, 127.5, 96.8, 96.2, 95.7, 94.4, 83.3,
79.5, 79.5, 78.6, 78.5, 76.2, 75.3, 71.1, 70.4, 70.1, 69.7, 69.6, 69.3,
69.3, 69.2 ppm. 31P NMR (121.5 MHz, CDCl3): δ = 0.23 (s, 1 P)
ppm. MALDI-HRMS: calcd. for C52H57O13PNa [M + Na]+
943.3429; found 943.3421.
2,3,4,6-O-Tetrakis(benzyloxymethyl)-D-myo-inositol
5-(Dibenzyl
phosphate) (7b): A solution of fully protected inositol 6b (0.475 g,
0.41 mmol) in dimethylformamide (20 mL) was treated with tetra-
butylammonium fluoride trihydrate (0.414 g, 1.31 mmol) at room
temp. for 12 h. After the reaction was finished, the solution was
partitioned between ethyl acetate (100 mL) and water (100 mL).
The aqueous layer was further extracted with ethyl acetate
(2ϫ60 mL), and the combined organic layers were washed with
brine (80 mL), dried with magnesium sulfate and filtered. Follow-
ing concentration, the residue was purified by chromatography on
silica gel (hexanes/acetone, 3:1) to give alcohol 7b (0.324 g, 86%)
tone, 7:1) to yield product 6b (0.481 g, 93%) as a syrup. [α]2D96 K
=
+16.3 (c = 1.5, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.77 (d,
J = 6.0 Hz, 2 H), 7.67–7.70 (m, 2 H), 7.12–7.37 (m, 36 H), 5.23 (d,
J = 6.3 Hz, 1 H), 4.86–5.09 (m, 8 H), 4.56–4.76 (m, 6 H), 4.14–
4.49 (m, 8 H), 3.90 (d, J = 8.1 Hz, 1 H), 3.41 (s, 1 H), 3.31 (d, J =
9.9 Hz, 1 H), 1.10 (s, 9 H) ppm. 13C NMR (100.6 MHz, CDCl3):
δ = 138.3, 138.2, 138.1, 137.8, 136.2, 136.0, 135.8, 133.9, 132.7,
130.1, 130.0, 128.4, 128.3, 128.3, 128.2, 128.2, 128.2, 128.0, 127.8,
127.8, 127.7, 127.7, 127.6, 127.5, 127.5, 127.4, 127.4, 127.3, 127.3,
96.6, 96.5, 95.4, 92.8, 76.4, 75.8, 75.3, 74.6, 74.2, 70.6, 70.1, 69.4,
69.3, 69.3, 69.0, 27.3, 19.3 ppm. 31P NMR (121.5 MHz, CDCl3): δ
= 0.25 (s, 1 P) ppm. MALDI-HRMS: calcd. for C68H75O13PSiNa
[M + Na]+ 1181.4607; found 1181.4595.
1
as a syrup. [α]2D96 K = –21.1 (c = 1.5, CHCl3). H NMR (300 MHz,
CDCl3): δ = 7.17–7.31 (m, 30 H), 4.36–4.85 (m, 21 H), 4.24 (t, J =
9.6 Hz, 2 H), 4.14 (d, J = 3.3 Hz, 1 H), 3.83 (t, J = 9.3 Hz, 1 H),
3.67 (dd, J = 9.9, 2.1 Hz, 1 H), 3.52 (dd, J = 7.5, 3.0 Hz, 1 H) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 138.1, 137.8, 137.4, 136.9,
135.9, 135.9, 128.5, 128.4, 128.3, 128.3, 128.2, 127.9, 127.8, 127.8,
127.7, 127.7, 127.6, 127.5, 127.4, 96.8, 96.1, 95.7, 94.2, 83.1, 80.8,
80.7, 76.1, 76.1, 76.0, 75.9, 70.7, 70.3, 70.1, 69.7, 69.5, 69.4, 69.3,
69.2, 69.2 ppm. 31P NMR (121.5 MHz, CDCl3): δ = 0.06 (s, 1 P)
ppm. MALDI-HRMS: calcd. for C52H57O13PNa [M + Na]+
943.3429; found 943.3408.
2,6-O-Bis(benzyloxymethyl)-1-O-(tert-butyldiphenylsilyl)-D-myo-
inositol 3,4,5-Tris(dibenzyl phosphate) (6d): Dibenzyl N,N-diisopro-
pylphosphoramidite (0.40 mL, 1.22 mmol) was added to a solution
of alcohol 5d (0.134 g, 0.203 mmol) and 1H-tetrazole (4.0 mL,
1.8 mmol, 0.45  in acetonitrile) in dichloromethane (4 mL) under
nitrogen, and the mixture was stirred at room temp. for 18 h. The
reaction solution was cooled to –20 °C, and mCPBA (0.315 g, 57–
86%) was added. The mixture was warmed to room temp. and
stirred for 3 h. The solvent was removed and the residue purified
by flash chromatography on silica gel (hexanes/acetone, 8:1) to ac-
cess 6d (0.289 g, 99%) as a syrup. [α]2D96 K = +15.5 (c = 2.6, CHCl3).
1H NMR (400 MHz, CDCl3): δ = 7.76–7.67 (m, 4 H), 7.38–7.05
(m, 46 H), 5.15–4.88 (m, 12 H), 4.83–4.64 (m, 7 H), 4.54–4.48 (m,
2 H), 4.37–4.26 (m, 2 H), 4.08 (t, J = 12 Hz, 1 H), 3.98–3.91 (m, 2
H), 1.07 ppm. 13C NMR (100.6 MHz, CDCl3): δ = 138.2, 137.8,
136.2, 136.2, 136.1, 136.1, 136.0, 135.9, 135.9, 135.8, 135.7, 133.3,
132.5, 130.1, 130.0, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3,
128.2, 128.2, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8,
127.7, 127.6, 127.4, 127.3, 96.6, 96.0, 75.1, 75.1, 75.1, 73.4, 70.7,
69.9, 69.6, 69.5, 69.5, 69.4, 69.3, 69.2, 69.1, 69.0, 27.2, 19.4 ppm.
31P NMR (121.5 MHz, CDCl3): δ = 0.07, –0.04, –0.28 ppm.
2,6-O-Bis(benzyloxymethyl)-D-myo-inositol
3,4,5-Tris(dibenzyl
phosphate) (7d): A solution of fully protected inositol 6d (0.027 g,
0.019 mmol) in tetrahydrofuran (1 mL) was treated with tetrabu-
tylammonium fluoride trihydrate (0.018 g, 0.056 mmol) and stirred
at room temp. for 18 h. Following concentration, the residue was
purified by chromatography on silica gel (hexanes/acetone, 3:1) to
give alcohol 7d (0.019 g, 84%) as a syrup. [α]2D96 K = –15.4 (c = 0.8,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.32–7.15 (m, 40 H),
5.14–4.84 (m, 16 H), 4.70–4.60 (m, 5 H), 4.48–4.40 (m, 3 H), 4.28–
4.19 (m, 2 H), 3.85 (t, J = 9.4 Hz, 1 H), 3.48–3.44 (m, 1 H) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 137.6, 136.9, 136.1, 136.0,
135.9, 135.9, 135.8, 135.7, 135.6, 135.6, 128.5, 128.4, 128.3, 128.1,
128.1, 128.0, 128.0, 127.9, 127.8, 127.6, 97.1, 96.2, 82.4, 78.7, 76.8,
76.3, 75.4, 70.4, 70.1, 69.7, 69.6, 69.6, 69.5, 69.4, 69.3, 69.3 ppm.
31P NMR (121.5 MHz, CDCl3): δ = –0.04, –0.48, –0.58 ppm.
MALDI-HRMS: calcd. for C64H67O17P3Na [M + Na]+ 1223.3489;
found 1223.3498.
MALDI-HRMS: calcd. for C80H85O17P3SiNa [M
1461.4666; found 1461.4603.
+
Na]+
Cbz-Aminohexanol Phosphoramidite (8): 1H-Tetrazole (4.6 mL of a
0.45  solution in acetonitrile, 2.07 mmol) and benzyloxybis(diiso-
propylamino)phosphane (1.392 g, 4.11 mmol) were added to a
solution of cbz-aminohexanol (0.694 g, 2.76 mmol) in dichloro-
methane (30 mL) under nitrogen. This mixture was stirred at room
temp. for 2 h, at which point the solution was extracted with dichlo-
romethane (3ϫ80 mL) from saturated sodium hydrogen carbonate
(80 mL). The combined organic phases were dried with magnesium
sulfate, filtered, and concentrated, and the residue was purified by
chromatography on silica gel (hexane/ethyl acetate/triethylamine,
100:10:1). The resulting colorless liquid (0.983 g, 2.0 mmol) was
passed on to the next step without further purification.
2,3,5,6-O-Tetrakis(benzyloxymethyl)-D-myo-inositol
4-(Dibenzyl
phosphate) (7a): A solution of fully protected inositol 6a (0.255 g,
0.22 mmol) in dimethylformamide (10 mL) was treated with tetra-
butylammonium fluoride trihydrate (0.222 g, 0.70 mmol), and
stirred at room temp. for 12 h. After the reaction was finished, the
solution was partitioned between ethyl acetate (80 mL) and water
(80 mL). The aqueous layer was further extracted with ethyl acetate
(60 mL), and the combined organic layers were washed with brine
(60 mL), dried with magnesium sulfate and filtered. Following con-
centration, the residue was purified by chromatography on silica
gel (hexanes/acetone, 3:1) to give alcohol 7a (0.175 g, 86%) as a
syrup. [α]2D96 K = –18.7 (c = 1.0, CHCl3). 1H NMR (300 MHz,
General Procedure for Phosphoramidite Chemistry to Convert 2a–g
CDCl3): δ = 7.17–7.33 (m, 30 H), 4.82–5.07 (m, 11 H), 4.65–4.77 to 7a–g: Cbz-aminohexanol phosphoramidite 8 was added to a
(m, 6 H), 4.45–4.57 (m, 4 H), 4.26 (s, 1 H), 4.09 (d, J = 3.9 Hz, 1
H), 3.84 (t, J = 9.3 Hz, 1 H), 3.64–3.71 (m, 2 H), 3.52–3.56 (m, 1
solution of an alcohol of type 7a–g and 1H-tetrazole in dichloro-
methane (15 mL) under nitrogen, and the mixture was stirred at
H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 138.0, 137.8, 137.5, room temp. for 18 h. The reaction solution was cooled to –60 °C,
136.9, 136.0, 128.5, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8,
and mCPBA was added. The mixture was stirred at this tempera-
Eur. J. Org. Chem. 2009, 4170–4179
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4175

D. Gong, H. E. Bostic, M. D. Smith, M. D. Best
FULL PAPER
ture for 60 min and slowly warmed to room temp., after removal
of the cooling bath. The solution was diluted to 60 mL with dichlo-
romethane, washed with saturated aqueous sodium hydrogen car-
bonate (2ϫ30 mL), and the aqueous phase was extracted with
dichloromethane (60 mL). The combined organic phases were dried
with magnesium sulfate, filtered, and concentrated, and the residue
was purified by chromatography on silica gel to yield the corre-
sponding product of type 2a–g.
29.5, 29.4, 29.1, 25.5, 24.4 ppm. 31P NMR (121.5 MHz, CDCl3): δ
= 0.13, –0.03, –0.21, –0.56 ppm. MALDI-HRMS: calcd. for
C85H93NO22P4Na [M + Na]+ 1626.5032; found 1626.5042.
Benzyl (6-Benzyloxycarbonylaminohexyl) 2,4,5,6-O-tetra-(benzyl-
oxymethyl)-D-myo-inosit-1-yl Phosphate 3-(Dibenzyl phosphate)
(2e): Alcohol 7e (0.354 g, 0.442 mmol), 1H-tetrazole (2.4 mL,
1.06 mmol, 0.45  in acetonitrile), Cbz-aminohexanol phos-
phoramidite 8 (0.432 g, 0.884 mmol), and mCPBA (0.36 g, 57–
86%) were used. Purification by chromatography on silica gel (hex-
anes/acetone, 2:1) was used to yield product 2e (0.47 g, 88%) as a
syrup. 1H NMR (400 MHz, CDCl3): δ = 7.18–7.33 (m, 40 H), 4.77–
5.08 (m, 17 H), 4.50 (d, J = 8.8 Hz, 1 H), 4.30 (d, J = 8.8 Hz, 2
H), 4.03 (d, J = 9.2 Hz, 2 H), 3.86–3.92 (m, 2 H), 3.47–3.48 (m, 1
H), 3.10 (m, 2 H), 1.38–1.51 (m, 4 H), 1.18 (m, 4 H) ppm. 13C
NMR (100.6 MHz, CDCl3): δ = 156.4, 138.6, 138.3, 138.2, 136.7,
135.9, 135.7, 128.4, 128.4, 128.4, 128.3 128.0, 127.9, 127.8, 127.7,
127.7, 127.5, 127.2, 82.7, 79.9, 78.3 77.9, 76.1, 75.7, 75.6, 69.4, 69.3,
68.0, 66.5, 40.9, 30.0, 29.7, 26.1, 25.0, 24.9 ppm. 31P NMR
(121.5 MHz, CDCl3): δ = –1.57–1.37 (m, 2 P) ppm. MALDI-
HRMS: calcd. for C69H75NO14P2Na [M + Na]+ 1226.4555; found
1226.4502.
Benzyl
(6-Benzyloxycarbonylaminohexyl)
2,3,5,6-O-Tetrakis-
(benzyloxymethyl)-D-myo-inosit-1-yl Phosphate 4-(Dibenzyl phos-
phate) (2a): Alcohol 7a (0.173 g, 0.188 mmol), 1H-tetrazole
(1.25 mL, 0.564 mmol, 0.45  in acetonitrile), Cbz-aminohexanol
phosphoramidite 8 (0.23 g, 0.47 mmol), and mCPBA (0.191 g, 57–
86%) were used. Purification by chromatography on silica gel (hex-
anes/acetone, 3:1) was used to afford product 2a (0.181 g, 73%) as
a syrup. [α]2D96 K = –6.9 (c = 1.0, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 7.16–7.33 (m, 40 H), 4.60–5.07 (m, 24 H), 4.48 (s, 3
H), 4.19–4.24 (m, 2 H), 3.90 (t, J = 6.3 Hz, 2 H), 3.67 (d, J =
9.0 Hz, 2 H), 3.06–3.08 (m, 2 H), 1.17–1.46 (m, 8 H) ppm. 13C
NMR (100.6 MHz, CDCl3): δ = 156.3, 138.0, 137.9, 137.5, 136.7,
136.0, 135.9, 128.6, 128.5, 128.5, 128.3, 128.3, 128.1, 127.9, 127.8,
127.8, 127.7, 127.6, 127.6, 127.5, 96.7, 96.4, 95.5, 94.1, 79.0, 79.0,
78.2, 76.1, 76.0, 74.6, 70.7, 70.4, 70.3, 69.6, 69.3, 68.0, 68.0, 67.8,
67.8, 66.5, 40.8, 30.0, 29.9, 29.7, 26.0, 24.9 ppm. 31P NMR
(121.5 MHz, CDCl3): δ = 0.06 (d, J = 10.2 Hz, 1 P), 0.15 (s, 1
P) ppm. MALDI-HRMS: calcd. for C73H83NO18P2Na [M + Na]+
1346.4978; found 1346.4988.
Benzyl
(6-Benzyloxycarbonylaminohexyl)
2,5,6-O-Tris(benzyl-
oxymethyl)-D-myo-inosit-1-yl Phosphate 3,4-Bis(dibenzyl phosphate)
(2f): Alcohol 7f (0.37 g, 0.381 mmol), 1H-tetrazole (2.2 mL,
0.953 mmol, 0.45  in acetonitrile), Cbz-aminohexanol phos-
phoramidite 8 (0.372 g, 0.762 mmol), and mCPBA (0.36 g, 57–
86%) were used. Purification by chromatography on silica gel (hex-
anes/acetone, 3:1) was used to give product 2f (0.42 g, 80%) as a
syrup. 1H NMR (300 MHz, CDCl3): δ = 7.04–7.33 (m, 45 H), 4.73–
5.08 (m, 21 H), 4.62 (s, 1 H), 4.29–4.34 (m, 2 H), 4.07 (t, J = 9.6 Hz,
1 H), 3.84–3.88 (m, 2 H), 3.44–3.51 (m, 1 H), 3.09–3.11 (m, 2 H),
1.17–1.51 (m, 8 H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 156.4,
138.3, 138.0, 136.7, 136.0, 135.8, 135.8, 135.6, 128.5, 128.5, 128.3,
128.2, 128.1, 128.0, 127.8, 127.6, 127.5, 127.4, 127.2, 80.7, 79.6,
78.0, 77.8, 76.0, 75.8, 75.5, 69.8, 69.8, 69.6, 69.3, 68.1, 67.9, 66.4,
40.1, 30.8, 29.9, 29.6, 26.0, 24.9 ppm. 31P NMR (121.5 MHz,
CDCl3): δ = –0.211 (s, 1 P), –0.31 (d, J = 22.8 Hz, 1 P), –0.87 (s, 1
P) ppm. MALDI-HRMS: calcd. for C76H82NO17P3Na [M + Na]+
1396.4688; found 1396.4740.
Benzyl
(6-Benzyloxycarbonylaminohexyl)
2,3,4,6-O-Tetrakis-
(benzyloxymethyl)-D-myo-inosit-1-yl Phosphate 5-(Dibenzyl phos-
phate) (2b): Alcohol 7b (0.320 g, 0.348 mmol), 1H-tetrazole
(2.3 mL, 1.05 mmol, 0.45  in acetonitrile), Cbz-aminohexanol
phosphoramidite 8 (0.424 g, 0.869 mmol), and mCPBA (0.27 g, 57–
86%) were used. Purification by chromatography on silica gel (hex-
anes/acetone, 3:1) was used to access product 2b (0.256 g, 56%) as
a syrup. [α]2D96 K = –2.3 (c = 1.5, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 7.20–7.31 (m, 40 H), 4.86–5.11 (m, 15 H), 4.72–4.81
(m, 5 H), 4.49–4.67 (m, 6 H), 4.18–4.35 (m, 4 H), 3.92 (t, J =
6.0 Hz, 2 H), 3.63–3.67 (m, 1 H), 3.08 (dd, J = 12.9, 6.3 Hz, 2 H),
1.34–1.47 (m, 4 H), 1.18 (m, 4 H) ppm. 13C NMR (100.6 MHz,
CDCl3): δ = 156.4, 138.1, 138.1, 137.9, 137.4, 136.7, 135.9, 135.8,
128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 127.9, 127.8, 127.7, 127.7,
127.5, 127.4, 127.4, 96.4, 96.3, 95.6, 93.9, 80.6, 80.5, 75.9, 75.2,
75.1, 74.7, 70.3, 70.3, 70.2, 69.6, 69.5, 69.5, 69.3, 69.3, 68.0, 67.9,
67.8, 66.4, 40.8, 29.9, 29.9, 29.6, 26.0, 24.9 ppm. 31P NMR
(121.5 MHz, CDCl3): δ = –0.05 (d, J = 3.4 Hz, 1 P), 0.19 (d, J =
4.5 Hz, 1 P) ppm. MALDI-HRMS: calcd. for C73H83NO18P2Na
[M + Na]+ 1346.4978; found 1346.4985.
Benzyl (6-Benzyloxycarbonylaminohexyl) 2,4,6-O-Tris(benzyloxy-
methyl)-D-myo-inosit-1-yl Phosphate 3,5-Bis(dibenzyl phosphate)
(2g): Alcohol 7g (0.22 g, 0.227 mmol), 1H-tetrazole (1.3 mL,
0.568 mmol, 0.45  in acetonitrile), Cbz-aminohexanol phos-
phoramidite 8 (0.221 g, 0.453 mmol), and mCPBA (0.22 g, 57–
86%) were used. Purification by chromatography on silica gel (hex-
anes/acetone, 3:1) was used to provide product 2g (0.271 g, 92%)
as a syrup. 1H NMR (300 MHz, CDCl3): δ = 6.97–7.41 (m, 45 H),
4.48–5.17 (m, 21 H), 4.32–4.35 (m, 2 H), 4.09 (t, J = 9.0 Hz, 2 H),
3.85–3.86 (m, 2 H), 3.08–3.10 (m, 2 H), 1.15–1.38 (m, 8 H) ppm.
13C NMR (75.5 MHz, CDCl3): δ = 156.4, 138.4, 138.3, 138.0,
138.0, 137.2, 137.1, 137.1, 136.9, 136.9, 135.8, 135.8, 135.7, 135.7,
135.6, 135.5, 135.4, 128.5, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1,
128.1, 128.1, 128.0, 128.0, 127.8, 127.7, 127.7, 127.6, 127.5, 127.5,
127.5, 127.4, 127.3, 127.3, 78.1, 78.0, 78.0, 77.9, 77.9, 77.9, 77.9,
77.3, 75.7, 74.5, 69.6, 69.5, 69.3, 69.3, 69.2, 69.2, 66.4, 40.8, 29.6,
26.0, 24.8 ppm. 31P NMR (121.5 MHz, CDCl3): δ = –0.32 (d, J =
5.6 Hz, 1 P), –0.59 (d, J = 16.2 Hz, 1 P), –0.65 (s, 1 P) ppm.
Benzyl (6-Benzyloxycarbonylaminohexyl) 2,6-O-Bis(benzyloxymeth-
yl)-
Alcohol 7d (0.180 g, 0.15 mmol), 1H-tetrazole (1.0 mL, 0.45 mmol,
0.45  in acetonitrile), Cbz-aminohexanol phosphoramidite
D-myo-inosit-1-yl Phosphate 3,4,5-Tris(dibenzyl phosphate) (2d):
8
(0.183 g, 0.375 mmol), and mCPBA (0.78 g, 57–86%) were used.
Purification by chromatography on silica gel (hexanes/acetone, 3:1)
was used to afford product 2d (0.238 g, 99%) as a syrup. [α]2D96 K
=
–14.7 (c = 5.6, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.32–
7.17 (m, 50 H), 5.13–4.81 (m, 21 H), 4.76–4.52 (m, 5 H), 4.45–4.17
(m, 4 H), 3.94–3.82 (m, 2 H), 3.09–3.03 (m, 2 H), 1.46–1.18 (m, 8
H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 155.9, 137.6, 137.3,
136.3, 135.6, 135.6, 135.5, 135.3, 135.2, 135.2, 135.1, 128.0, 128.0,
127.9, 127.8, 127.8, 127.6, 127.2, 127.0, 126.9, 96.0, 95.7, 95.6, 81.9,
77.5, 75.1, 75.0, 74.8, 74.7, 74.3, 74.1, 74.0, 70.0, 69.9, 69.6, 69.3,
69.1, 69.0, 68.9, 68.9, 68.8, 67.7, 67.6, 67.5, 67.4, 66.0, 52.9, 40.4,
MALDI-HRMS: calcd. for C76H82NO17P3Na [M
1396.4688; found 1396.4722.
+
Na]+
General Procedure for the Global Deprotection of 2a–g to 1a–g by
Hydrogenolysis: Palladium hydroxide (20%) on charcoal was added
to a solution of a compound of type 2a–g in methanol (15–40 mL).
4176
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 4170–4179

Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation
The mixture was then stirred at room temp. with 1 atm of hydrogen
(balloon) for 3 d. The catalyst was removed by filtration and the
residue washed with methanol. The solvent was removed in vacuo,
and the crude product was dissolved in water and stirred with
Chelex 100 resin (Sigma, Na+ form) for 3 h. The resin was removed
by filtration and the filtrate lyophilized to give the corresponding
amino conjugate of type 1a–g.
P) ppm. MALDI-HRMS: calcd. for C12H29NO15P3 (free acid, [M
+ H]+) 520.0745; found 520.0730.
1-(6-Aminohexyl sodium phosphate)-D-myo-inositol 3,5-Bis(disodium
phosphate) (1g): Compound 2g (0.270 g, 0.197 mmol) and 20% pal-
ladium hydroxide on charcoal (0.30 g) gave amino conjugate 1g
(122 mg, 100%) as a white solid. [α]2D96 K = –4.8 (c = 0.65, H2O).
1H NMR (300 MHz, D2O): δ = 4.43 (s, 1 H), 3.89–4.03 (m, 7 H),
2.99 (t, J = 7.5 Hz, 2 H), 1.63–1.69 (m, 4 H), 1.41 (m, 4 H) ppm.
13C NMR (100.6 MHz, D2O): δ = 80.9, 78.1, 77.3, 73.9, 73.6, 72.9,
69.0, 42.1, 32.0, 29.2, 27.7, 27.0 ppm. 31P NMR (121.5 MHz, D2O):
δ = 4.05 (s, 1 P), 2.48 (s, 1 P), 0.91 (s, 1 P) ppm. MALDI-HRMS:
calcd. for C12H29NO15P3 (free acid, [M + H]+) 520.0745; found
520.0728.
1-(6-Aminohexyl sodium phosphate)-D-myo-inositol 4-(Disodium
phosphate) (1a): Compound 2a (0.18 g, 0.137 mmol) and 20% pal-
ladium hydroxide on charcoal (0.20 g) provided amino conjugate
1a (70 mg, 100%) as a white solid. [α]2D96 K = –7.0 (c = 0.64, H2O).
1H NMR (300 MHz, D2O): δ = 4.23 (s, 1 H), 4.12 (dd, J = 16.8,
9.0 Hz, 1 H), 3.88–3.98 (m, 3 H), 3.81 (t, J = 9.6 Hz, 1 H), 3.63
(dd, J = 9.9, 2.7 Hz, 1 H), 3.45 (t, J = 9.3 Hz, 1 H), 2.99 (t, J =
7.2 Hz, 2 H), 1.62–1.66 (m, 4 H), 1.40 (m, 4 H) ppm. 13C NMR
(100.6 MHz, D2O): δ = 78.8, 78.7, 78.7, 76.5, 74.1, 73.6, 69.0, 42.1,
32.2, 29.4, 27.8, 27.1 ppm. 31P NMR (121.5 MHz, D2O): δ = 5.67
1-[6-(14-Biotinamido-3,6,9,12-tetraoxatetradecanamido)hexyl
so-
dium phosphate]- -myo-inositol 4,5-Bis(disodium phosphate) (9):
D
Succinimidyl ester 12a was dissolved in dimethylformamide (1 mL),
and compound 1c (7 mg, 0.011 mmol), dissolved in triethylammo-
nium hydrogen carbonate (TEAB, 0.5 , 1 mL), was added. The
mixture was stirred at room temperature for 24 h. The solvent was
removed under reduced pressure, and the residue was washed with
acetone (4ϫ2 mL) and dried in vacuo. The resulting white solid
was dissolved in water, and the solution was stirred with Chelex
100 resin (Sigma, Na+ form) for 3 h. The resin was removed by
filtration, and the filtrate was lyophilized to give the product
(13 mg, 100%) as white solid. 1H NMR (300 MHz, D2O): δ = 4.56–
4.58 (m, 1 H), 4.38–4.41 (m, 1 H), 4.04–4.25 (m, 3 H), 3.82–3.95
(m, 4 H), 3.58–3.74 (m, 14 H), 2.93–3.40 (m, 14 H), 2.72–2.85 (m,
2 H), 2.24 (d, J = 6.9 Hz, 2 H), 1.33–1.79 (m, 16 H) ppm. 31P
NMR (121.5 MHz, D2O): δ = 5.79 (s, 1 P), 5.70 (s, 1 P), 0.87 (s, 1
P) ppm. MALDI-HRMS: calcd. for C32H61N4O22P3SH (free acid,
[M + H]+) 979.2784; found 979.2775.
(s,
1 P), 1.13 (s, 1 P) ppm. MALDI-HRMS: calcd. for
C12H28NO12P2 (free acid, [M + H]+) 440.1081; found 440.1088.
1-(6-Aminohexyl sodium phosphate)- -myo-inositol 5-(Disodium
D
phosphate) (1b): Compound 2b (0.25 g, 0.189 mmol) and 20% pal-
ladium hydroxide on charcoal (0.27 g) afforded amino conjugate
1b (96 mg, 100%) as a white solid. [α]2D96 K = –17.4 (c = 0.69, H2O).
1H NMR (300 MHz, D2O): δ = 4.23 (s, 1 H), 3.75–3.97 (m, 6 H),
3.57–3.61 (m, 1 H), 2.98 (t, J = 7.5 Hz, 2 H), 1.62–1.68 (m, 4 H),
1.40 (m, 4 H) ppm. 13C NMR (100.6 MHz, D2O): δ = 80.8, 78.6,
74.6, 73.8, 73.6, 73.3, 68.9, 42.1, 32.0, 29.2, 27.7, 27.0 ppm. 31P
NMR (121.5 MHz, D2O): δ = 4.62 (s, 1 P), 0.92 (s, 1 P) ppm.
MALDI-HRMS: calcd. for C12H28NO12P2 (free acid, [M + H]+)
440.1081; found 440.1063.
1-(6-Aminohexyl sodium phosphate)-D-myo-inositol 3,4,5-Tris(diso-
dium phosphate) (1d): Compound 2d (0.23 g, 0.143 mmol) and 20%
palladium hydroxide on charcoal (0.30 g) yielded amino conjugate
1d (88 mg, 100%) as a white solid. [α]2D96 K = –7.2 (c = 0.68, H2O).
1H NMR (400 MHz, D2O): δ = 4.04 (br. s, 2 H), 4.22–3.95 (m, 6
H), 3.02 (br. s, 2 H), 1.68 (br. s, 4 H), 1.44 (br. s, 4 H) ppm. 13C
NMR (100.6 MHz, D2O): δ = 78.2, 76.1, 75.3, 74.5, 70.9, 70.7,
66.3, 39.5, 29.4, 26.6, 25.1, 24.4 ppm. 31P NMR (121.5 MHz, D2O):
1-[6-(4-Benzoylbenzamido)hexyl sodium phosphate]-D-myo-inositol
4,5-Bis(disodium phosphate) (10): Compound 1c (8 mg,
0.0127 mmol) in triethylammonium hydrogen carbonate (TEAB,
0.5 , 0.85 mL) was added to a solution of succinimidyl ester 12b
(8 mg, 0.0254 mmol) in dimethylformamide (0.85 mL). Tetra-
hydrofuran (0.45 mL) was then added, and the mixture was stirred
at room temp. for 24 h. The solvent was removed under reduced
pressure, and the residue was washed with acetone (4ϫ2 mL) and
dried in vacuo. The white solid was dissolved in water and filtered,
and the filtrate was lyophilized to afford product 10 (12 mg, 100%)
as a white solid, containing a small amount of triethylammonium
buffer salt. 1H NMR (300 MHz, D2O): δ = 7.70–7.85 (m, 7 H),
7.57 (t, J = 7.8 Hz, 2 H), 4.25–4.28 (m, 2 H), 3.87–4.00 (m, 5 H),
3.69 (d, J = 9.0 Hz, 1 H), 3.40 (t, J = 6.6 Hz, 2 H), 1.64 (m, 4 H),
1.41 (m, 4 H) ppm. 31P NMR (121.5 MHz, D2O): δ = 3.47 (s, 1
P), 2.46 (s, 1 P), 0.91 (s, 1 P) ppm. MALDI-HRMS: calcd. for
C26H37NO17P3 [M + H]+ 728.1269; found 728.1245.
δ
= 2.75, 2.54, 1.78, 0.89 ppm. MALDI-HRMS: calcd. for
C12H31NO18P4Na [M + Na]+ 624.0389; found 624.0301.
1-(6-Aminohexyl sodium phosphate)- -myo-inositol 3-(Disodium
D
phosphate) (1e): Compound 2e (0.457 g, 0.34 mmol) and 20% palla-
dium hydroxide on charcoal (0.45 g) afforded amino conjugate 1e
(171 mg, 100%) as a white solid. [α]2D96 K = –2.3 (c = 0.72, H2O).
1H NMR (400 MHz, D2O): δ = 4.39 (s, 1 H), 3.93–4.03 (m, 4 H),
3.82 (dd, J = 21.2, 9.6 Hz, 2 H), 3.42 (t, J = 9.6 Hz, 1 H), 3.03 (t,
J = 7.2 Hz, 2 H), 1.67–1.73 (m, 4 H), 1.45–1.47 (m, 4 H) ppm. 13C
NMR (100.5 MHz, D2O): δ = 78.6, 76.7, 76.7, 74.8, 74.1, 73.7,
69.0, 42.1, 32.1, 29.3, 27.8, 27.1 ppm. 31P NMR (161.8 MHz, D2O):
δ = 6.86 (d, J = 7.9 Hz, 1 P), 2.69 (d, J = 3.2 Hz, 1 P) ppm.
MALDI-HRMS: calcd. for C12H28NO12P2 (free acid, [M + H]+)
440.1081; found 440.1078.
Bifunctional PI-(4,5)-P2–Benzophenone/Azide Conjugate 11: N-Hy-
droxysuccinimide (3 mg, 0.0254 mmol) and dicyclohexylcarbodiim-
ide (5.5 mg, 0.0254 mmol) were added to a solution of carboxylic
acid 15 (9.6 mg, 0.0254 mmol) in tetrahydrofuran (1 mL), and the
mixture was stirred at room temp. for 24 h. The reaction mixture
was filtered, and the filtrate was concentrated under reduced pres-
sure. The residue was washed with hexane/diethyl ether (1:1) and
dried in vacuo. The resulting white solid was dissolved in dimethyl-
formamide (1 mL), and compound 1c (8 mg, 0.0127 mmol) in tri-
ethylammonium hydrogen carbonate (TEAB, 0.5 , 1.3 mL) was
added. Tetrahydrofuran (0.5 mL) was added, and the mixture was
stirred at room temp. for 24 h. The solvent was removed under
reduced pressure, and the residue was washed with acetone
(5ϫ2 mL) and dried in vacuo. The resulting white solid was dis-
1-(6-Aminohexyl sodium phosphate)-D-myo-inositol 3,4-Bis(disodium
phosphate) (1f): Compound 2f (0.42 g, 0.306 mmol) and 20% palla-
dium hydroxide on charcoal (0.50 g) provided amino conjugate 1f
(191 mg, 100%) as white solid. [α]2D96 K = +3.4 (c = 0.60, H2O). H
1
NMR (400 MHz, D2O): δ = 4.47 (s, 1 H), 4.27 (dd, J = 18.0,
4.8 Hz, 1 H), 3.95–4.06 (m, 4 H), 3.87 (t, J = 9.6 Hz, 1 H), 3.57 (t,
J = 9.2 Hz, 1 H), 3.04 (t, J = 7.6 Hz, 2 H), 1.70–1.72 (m, 4 H),
1.46 (m, 4 H) ppm. 13C NMR (100.6 MHz, D2O): δ = 78.7, 78.3,
76.7, 76.5, 73.9, 73.4, 69.0, 42.1, 32.1, 29.3, 27.8, 27.0 ppm. 31P
NMR (121.5 MHz, D2O): δ = 5.89 (s, 1 P), 4.48 (s, 1 P), 1.05 (s, 1
Eur. J. Org. Chem. 2009, 4170–4179
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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D. Gong, H. E. Bostic, M. D. Smith, M. D. Best
FULL PAPER
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(Sigma, Na+ form) for 3 h. The resin was removed by filtration,
and the filtrate was lyophilized to give the product 11 (12 mg, 95%)
as a white solid. 1H NMR (300 MHz; D2O/CD3OD, 1:1): δ = 7.74–
8.00 (m, 7 H), 7.62 (t, J = 7.8 Hz, 2 H), 4.52 (t, J = 7.5 Hz, 1 H),
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1 P), 0.87 (s, 1 P) ppm. MALDI-HRMS: calcd. for C32H47N5O18P3
[M + H]+ 882.2124; found 882.2179.
Benzophenone-Lys(N3)-OMe (14): Benzophenone-Lys(Boc)-OMe
(13)[53] (112 mg, 0.238 mmol) was dissolved in dichloromethane
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stirring was continued for 2 h. The solvent was removed under re-
duced pressure, and the residue was dried under high vacuum for
2 h to remove excess trifluoroacetic acid. The residue was dissolved
in methanol (2.5 mL), and potassium carbonate [1.27 mL, 0.804 
(aqueous), 1.03 mmol] and copper sulfate·5H2O [238 µL, 0.01 
(aqueous), 10 mol-%] were added. Imidazole-1-sulfonyl azide hy-
drochloride[55] (60 mg, 0.286 mmol) was added, and stirring was
continued at room temp. for 3.5 h. The reaction mixture was ex-
tracted with ethyl acetate (2ϫ100 mL), which was subsequently
washed with 2  hydrochloric acid (50 mL). The organic phase was
dried with magnesium sulfate, filtered, and concentrated under re-
duced pressure. Column chromatography (silica gel; ethyl acetate/
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hexanes, 1:1) afforded 14 as a clear glass (91 mg, 97%). [α]2D96 K
=
+22.95(c = 2.296, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.94–
7.79 (m, 6 H), 7.63 (t, J = 7.4 Hz, 1 H), 7.53–7.48 (m, 2 H), 6.85
(d, J = 7.5 Hz, 1 H), 4.91–4.84 (m, 1 H), 3.82 (s, 3 H), 3.03 (t, J =
6.6 Hz, 2 H), 2.08–1.46 (m, 6 H) ppm. 13C NMR (75.5 MHz,
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was dissolved in tetrahydrofuran (2 mL). With stirring, 2  sodium
hydroxide (2 mL) was added, and stirring was continued for
70 min. At this point, the solution was neutralized to pH ≈ 4 with
Dowex® 50WX8-200H+ ion resin. The solution was then filtered,
and the filtrate was concentrated under reduced pressure to afford
15 as a clear solid (43 mg, quant.). [α]2D96 K = +17.97(c = 2.02,
CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.90–7.87 (m, 2 H),
7.78–7.73 (m, 4 H), 7.60 (t, J = 7.5 Hz, 1 H), 7.49–7.44 (m, 2 H),
4.79 (br. s, 1 H), 3.28 (t, J = 6.3 Hz, 2 H), 2.03–1.87 (m, 2 H),
1.63–1.53 (m, 4 H) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 196.1,
167.2, 140.4, 136.73, 136.69, 133.1, 130.13, 130.10, 128.5, 127.2,
51.0, 51.0, 31.5, 28.4, 22.7 ppm. DART-HRMS: calcd. for
C20H21N4O4 [M +H]+ 381.15628; found 381.15494.
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Received: May 4, 2009
Published Online: July 9, 2009
Eur. J. Org. Chem. 2009, 4170–4179
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4179