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Chemistry of Inositol Lipid Mediated Cellular Signaling.

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Chemistry of Inositol Lipid Mediated Cellular Signaling**
Barry V. L. Potter* and Dethard Lampe
It is now slightly more than a decade lular Ca” mobilization. This event,
since Michael Berridge and his collabo- which has spawned what is now one of
rators reported in Nuturr *‘. . . micromo- the most active fields of current biology,
lar concentrations of Ins(1,4.5)P3 ( 1 ~ - also stimulated a renaissance in inositol
uiJwinositol I ,4,5-trisphosphate) re- and inositol phosphate chemistry. The
lease Ca2 from a non-mitochondria1 synthesis of inositol polyphosphates
intraceIIuIar Ca’+ store in pancreatic presents a number of problems: the reacinar cells. Our results strongly suggest giospecific protection of inositol and the
t h a t this IS the same Ca” store that is optical resolution of the resulting prereleased by acetylcholine”. This obser- cursors, the phosphorylation of the
vation ushered in a new era in the field of polyol, removal of all phosphate prosignal transduction with the discovery of tecting groups without phosphate mia small-molecule second messenger link- gration, and finally the purification of
ing the spatially separated events of cell the water-soluble target polyanion.
surface receptor activation and intracel- With the solution of these problems over
I . Transmembrane Signaling
The cells in multicellular organisms must communicate with
each other in order to work together. They convey signals by
using substances such as hormones and neurotransmitters
(Fig. I ) . Lipophilic hormones such as steroids, for example. can
pass through the lipid bilayer of cell membranes and bind to
their target receptors within the cell. Many chemical messengers, however. are too hydrophilic to cross membranes. In order
to deliver their message they have to bind to specific receptors
on the outside of the cell membrane and activate mechanisms
that transmit the signal into the cell, a process known as
the last few years it is now possible to
look beyond the synthesis of naturally
occurring inositol polyphosphates,
whose number has been steadily increasing. to the design of chemically modified
inositol phosphate analogues with the
prospect of developing enzyme inhibitors, rationally modified receptor
ligands and antagonists. and perhaps,
through pharmacological intervention
in signal transduction pathways, even
the therapeutical agents of the future.
Keywords: inositol phosphates . second
messengers . signal transduction
transmembrane signaling or signal transduction. Several different classes of receptors are involved in signal transduction. If the
receptor is linked to an ion channel, the opening of this channel
can trigger the influx or efflux of ions into or out of the cell. A
change of ion concentration in the cytosol will then activate
cellular enzymes, evoking an ultimate response to the stimulatory cell surface ligand. Another class of receptor is the receptor
tyrosine kinase. which is intrinsically an enzyme. It is embedded
in the cell membrane and has binding sites able to recognize
extend s i w e
[*] Pi-or. Dr B. V L. Potter. Dr. D. Lampc
School 01‘ Pharmacy and Pharinacology. University of Bath
B:iih BH?i.4\i ( U K )
Telefax. Int. code + (1225)X26-114
e-mail. B V, I,. Potter;u
Ahhrcviauon\ used in thia rebiew: All = ally]. Bn = benzyl. Bz = benroyl.
C h = IR)-I-phcn)leihylcarhamoql. CbL = benzqloxycarbonyl. Gro = glyCL‘I’O.
M E M = mcthoxyethoxymethyl. MOM = methoxymethyl. M s =
nictl~!lwllon~l. Pi\ = pivaloyl. PMB = p-methoxyhen7yl. Prop = prop-lcii\l. 1’111 = phosphatidyl. Tf = tritluoromethanesulfonyl. TBDMS = terfh ~ r ~ g ’ l d i i i i c t l i ~ l ~All
t l ~references
to inositol refer to the mi’o isomer unless
oihevwiw >pccified. lnositol phosphates are abbreviated Ins. InsP. InsP,.
In\/’,. InaP,. and InsP, (inositol, inositol mono-. his-. Iris-. t e t r a k k
ind hexakisphosphate. respectively), with positioning of phosphate
groups. it\ indic;ttcd i n parentheses. Dcoxyfluoro aiialogues are identified by a
p i c h coii\istirig of a number indicating the position at which the fluorine
w h i u t i c i i i 11.1s taken place and theletter F. e.g. ?-F-lns(l,4.5)P,
= 7-deoxy-2Phosphorothioak analogues itre ,tmilnrilj
letter S, cg. Ins(1.4.5)P3-SS = inosttol 1.3-
Fig. 1. Getting the message across: signal transduction mechanisms
B. V. L. Potter and D. Lampe
agonists on the outer surface, and an active site on the inner
surface of the membrane. Binding of agonists activates the enzyme and leads to the phosphorylation of tyrosine residues on
target proteins within the cell. This signal transduction mechanism is used by many growth factors and hormones including
insulin.['] Recently, the role of specific protein-protein interactions in mediating the flow of information from receptor tyrosine kinases to ras proteins was elucidated.[']
Many water-soluble hormones make use of a signal transduction system whereby the receptor is coupled to the production of
an internal signaling molecule via an intracellular effector. After
the hormone binds to its receptor protein on the cell surface, a
membrane-bound GTP-binding protein (G-protein) . [ 3 - 5 1
which is associated with the receptor, is activated. The family of
G-proteins includes several members which regulate different
intracellular pathways. G-proteins are composed of three subunits designated x, p, and ? in order of decreasing mass. On
activation, guanosine diphosphate (GDP) bound to the a subunit is replaced by guanosine triphosphate (GTP), causing the
By subunit to dissociate from the activated x-GTP subunit. The
free subunits can now stimulate o r inhibit other membranebound enzymes acting as amplifiers (e.g. K + channels, C a 2 +
channels, adenylate cyclase (AC), guanylate cyclase (GC),
PtdIns(4,S)P2-specific phospholipase C (PtdIns-PLC)), which
in turn generate so-called second messengers on the cytosolic
side of the cellular membrane. The intrinsic GTPase activity of
the a subunit then hydrolyzes G T P to GDP, the x-GDP subunit
recombines with the B y subunit, and the G-protein returns to its
basal state.
1.1. Second Messengers
Second messengers identified so far include adenosine 3',5'cyclic monophosphate (CAMP), guanosine 3',5'-cyclic
monophosphate (cGMP), diacylglycerol (DAG), Ins( 1,4,5)P3,
and of course C a 2 + ions. Considerable current interest is also
focused upon phosphatidylinositol 3,4,S-trisphosphate as a potential second messenger (see Section 2). Recent studies suggest
that cyclic adenosine diphosphate ribose (cADP-ribose), a
metabolite of NAD', may also have Ca2+-mobilizingsecond
messenger properties,"
and the sphingolipid metabolite sphingosine 1-phosphate has been implicated as a potential second
messenger in signal transduction pathways activated by certain
1.2. The Role of Caz+ in Signal Transduction
The first indication that calcium ions play a role in the regulation of cellular events was an observation by Ringer in
1883.['01 On examining muscle tissue, he found that he could not
induce contraction when he replaced the tap water in his medium with distilled water. The missing component was found to be
Ca2 .
Today, it is known that a large number of different cellular
processes are controlled by protein phosphorylation induced by
changes in intracellular C a 2 + concentration, and the mobilization of C a 2 + has been found to be the primary function of many
Barry Potter was born in Brighton ( U K ) in 1953. He studiedchemistry
at the University of Oxford as an Exhibitioner of Worcester College,
graduating with,fi'rst-class honors and winning the Part II Thesis Prize
in Organic Chemistry. He completed his doctorate in bioorganic chemistry in 1981 in Oxford under Professor Gordon Lowe with research
on enzyme-catalyzed phosphoryl transjkr reactions using chiral
['60,'70,'80]phosphate esters. During that time he was a Graduate
Scholar and subsequentlji Junior Research Fellow of Wolfson College.
After postdoctoral research in nucleic acid chemistry and molecular
biology as a European Exchange Fellow of the Rojiul Society with
Professor Fritz Eckstein at the Max-Plunck-Institut ,fur E-uperimentelle Medizin in Glittingen, he accepted the post of Lecturer in
Biological Chemistry at the University of Leicester in 1984 and w ~ s
D. Lampe
B. v. L. Potter
awarded a Lister Institute Research Fellowship in 1987. In 1990 hc>
moved to the Chair ofMedicinu1 Chemistry in the School of Pharmacy and Pharmacology, University of Bath, where he is Lister
Institute Research Professor. He received his DSc.from Oxford University in 1993. HisJie1d.s ofresearch activity lie in biological
and medicinal chemistry, in particular enzyme mechunisms and nucleic acid and phosphorus chemistry. Special interests are the
chemistry of second messengers and the synthesis of receptor ligands and enzyme inhibitors.
Dethard Lampe was born in Delmenhorst (Germany) in 1963. He studied chemistry at the Universities of Hamburg and
Goftingen, where he obtained his diploma in clinical hiochemistr!: in 1989 under Projessor H.-D. Siiling. In the same year he
joined the research group of Dr. B. K L . Potter in Leicester on a Wellcome Trust Prize Studentship. H e moved to the School
of Pharmacy and Pharmacology at the University of Bath in 1990 and completed his PhD on the synthesis of novel itiositol
phosphate analogues in 1993. He continued research in Buth in the area of'inositol polyphosphates as a Wellcome Trust Prize
Fellow. A t the beginning of' I995 he moved to the Instituto de Quimica Orgunica General, CSIC (Prof: M . Martin-Lomasj in
Madrid, &$,here
he is involved in the synthesis of hiologically active oligosaccharides.
A n , ~ r i l -Chri?~.
l n t . E d E q I . 1995. 34. 1933-1972
Inositol Lipids
agonists. The cytosolic C a 2 + concentration can be regulated by
two different mechanisms:
1 . Many agonists operate by inducing a change in the potential difference across the membrane. This causes voltage-sensitive CnZ . channels in the cell membrane to open. As most Ca2+
within the cell is bound to membranes or proteins, the intracellular level of free Ca2+is low. Therefore there is a large gradient
in favor of influx of ions into the cell and the cytosolic C a 2 +
concentration is increased.
2. Other agonists mobilize sequestered Ca2 from intracellular stores so that the Ca" concentration in the cytosol is increased and Ca' +-dependent enzymes are activated. The cellular response effected depends on the type of cell targeted as well
as the nature of the agonist.
2. lnositol 1,4,5-Trisphosphate: A Second Messenger
I n 1850 Scherer isolated an optically inactive isomer of cyclohexanehexol from muscle. which he designated "Inosit"." ' I Just
over Icentury after this discovery and a few years prior to the
discovery ol' the now well-established second messenger 3',5'cyclic AMP by Rall and Sutherland,['2s'31 Lowell and Mabel
Hokin demonstrated that acetylcholine stimulates the turnover
of inositol-containing phospholipids in pancreas and brain cortex slices."" Folch had discovered the inositol-containing phospholipids in brain some ten years earlier.[1s1Later work showed
that the phospholipid effect described by the Hokins was observable in ;I number of other systems exposed to agonist stimulation, and this suggested a role for these lipids in stimulus-response coupling. In the late 1950s new analytical techniques
facilitated the separation of the inositol-containing phospholipids into phosphatidylinositol (PtdIns), phosphatidylinositol4-phosphate [PtdIns(4)P], and phosphatidylinositol-4,5-bisphosphote [ P t d I n ~ ( 4 , 5 ) P ~ ] [(Fig.
' ~ ~ 2). and the chemical
structures of these species were soon established by Ballou et
al." 'I Despite this progress, little headway was made in understanding the physiological significance of the stimulated
turnover of inositol-containing phospholipids in subsequent
years, primarily since too much attention was given to the major
phospholipid, PtdIns.
A key breakthrough, however, came in 1975 when Michell['81
realized the connection between agonist-stimulated phospholipid turnover and the increased intracellular CaZ' levels. which
had already been in focus with regard to regulation of a variety
of cellular functions. Despite this, there was no obvious contender for a chemical link between these events. Finally, at the
end of 2983, Berridge and his co-workers in Cambridge. UK,
and Frankfurt am Main, Germany,'19] identified the missing
second messenger in the form of a mjw-inositol polyphosphate
generated from the minor phospholipid PtdIns(4.5)P2 with the
statement: "micromolar concentrations of Ins( 1 .4,5)P3 (IDrnp-inositol 1.4.5-trisphosphate) release Ca2 ' from a non-mitochondrial intracellular C a 2 + store in pancreatic acinar cells.
Our results strongly suggest that this is the same C a 2 +store that
is released by acetylcholine".
These discoveries opened up a whole new chapter in signal
transduction and since then, progress in this field has been dramatic, as evidenced by the comment of Hokin:'*'] "the phosphoinositide field is currently the number one field in biochemistry in the number of citations (excluding molecular biology)".
Activity was still further stimulated by the mapping out of a
highly complex metabolic pathway for inositol phosphates,12'. 221 including the involvement of higher inositol phosphates such as InsP, and even the pyrophosphate-containing
InsP, and InsP, .[231Still more intriguing is the characterization
of the new inositol-containing phospholipids PtdIns(3)P,
Ptdlns(3,4)P2. and PtdIns(3,4,5)P3 (Fig. 2) formed after the
stimulation of a 3'-kinase in cells by growth factors.[*"] The
latter phospholipid. in particular, may be a second messenger in
its own right. This whole area is under considerable scrutiny and
appears to be a completely new branch of polyphosphoinositide-mediated signal transduction. Additionally. it was discovered that the cell nucleus appears to have its own autonomous
phosphoinositide signaling machinery.['" 2h1 Some of these
newer aspects of the polyphosphoinositide signaling pathway
have been reviewed.[27]Although a discrete molecule has yet to
be structurally identified, much excitement has been generated
recently by the discovery of a C a 2 + influx factor (CIF).[28,291
CIF appears to be a diffusable messenger that is released from
activated intracellular conipartments in stimulated cells and
stimulates C a 2 + influx across
2-02b*~po:the plasma membrane.[30~31] 2-02P0
Also, attention is drawn to reFig. 3. Thc second messenger ucent reports showing that the 1.4.5-trisphosphate.
affinity of the pPnicilliun,brevicompactiini metabolites adenophostin A and B for the Ins(1.4,5)P3 receptor is higher than that
- 341 (Fig. 3).
of Ins( 1 .4.5)p3
While most of the rapid progress of the last ten years has been
focused on biological aspects, it is clear that current and future
efforts are, and will be directed towards pharmacological intervention in the phosphoinositide signaling system. First syntheses of Ins(l,4.5)P3 were reported in 1986-1987, and these have
been followed by significant synthetic progress to the extent that
essentially all of the problems inherent to inositol phosphate
R1 = R2= R3= H;
R1= P O P . R2 = R3 = H;
PMl~(4,5)P2: R1 = R2 = PO:-,
R3 = H;
R 1 = R2 = H, R3 = PO:-
Ptdlns(3.4)P2: R' = R3 = PO$-, R2 = H;
Ptdlns(3p.5)P3:R1 = R2 = R3 = PO:-
Fig. 1. Locali<ui ol' phosphohpase C attack on inositol phospholipids
B. V. L. Potter and D. Lampe
synthesis have now been overcome. The complex nature of inositol phosphate metabolism, however. which is still being uncovered, is providing new targets for synthesis with regularity.
Moreover, receptors that bind second messengers, enzymes that
metabolize them, and the generative pathways responsible for
their formation are naturally seen as potential targets for rational drug d e ~ i g n . ‘ ~ ’ - ~Thus,
’ ~ the focus of synthetic activity is
now moving towards structurally modified inositol phosphates
with novel biological properties.
This review focuses primarily on recent chemical progress
in this area. Although attempts will be made to place the essential biological background in context, a completely comprehensive coverage of the biology of the past ten years is
well beyond the scope of this article and the reader is referred
to the many existing reviews[*” 3 8 - 4 7 1 and books[4n- 5 2 1 currently available on this topic. Several reviews and books on
more recent aspects of the synthesis of inositol phosphates have
Fo r information on synthetic approaches
prior to this, the reader is referred to two other major
of mJto-inositol in the following section. Proposals were made
that the system of stereospecific numbering should be adopted
for inositol
but these have not yet found widespread acceptance. A note on recent attempts to simplify
nomenclature and abbreviations has appeared.[621For a simplified discussion of this topic, the reader is referred to an excellent
3.2. myo-Inositol
myo-Inositol is a meso-cyclohexane hexol. or cyclitol. and
consequently possesses a plane of symmetry with five equatorial
hydroxyl groups and one axial hydroxyl group. The carbon
bearing the axial hydroxyl is designated C-2 and the other ring
carbons can be numbered from C-I to C-6 starting from a C-I
atom on either side of C-2 and proceeding around the ring in a
clockwise or anticlockwise fashion. According to convention,
an anticlockwise numbering in an asymmetrically substituted
inositol leads to a configurational D prefix, and a clockwise
numbering gives a substituted inositol with an L prefix. The
choice of prefix is normally determined by giving preference
to that which results in the lowest numbering of substituents.
Thus, in nzyo-inositol the symmetry plane is through C-2 and
3. Stereochemistry and Nomenclature of Inositol
3.1. Stereochemistry and Nomenclature
The substitution of one of the symmetry-related pairs of carbon atoms C-1, C-3 and C-4, C-6 gives rise to D and L enantiomers. Substitution of C-2 and/or C-5 clearly gives a meso
product and a prefix is inappropriate. In Figure 5 the enan-
After Scherer’s discovery of “Inosit”,[’ I ] the suffix -01 was
added, and when other isomers were discovered or synthesized
they were also named “inositols”. There are nine possible isomeric inositols, including one enantiomeric pair.[”g’ with the
prefixes scyllo-, n i p - , neo-, epi-, cis-, muco-, allo-, D-C/lirO-( )-,
and L-chiro-( -)- (Fig. 4). The large number of inositols and
their derivatives has caused difficult problems of nomenclature,
not all of which have been fully resolved. The publication of the
“1967 IUPAC Tentative Rules for Cyclitols”[601has gone a considerable way towards standardization, but difficulties still exist.
Some aspects of these rules are outlined in the treatment
Fig. 5. The enantiomers of n?jo-ino?itol1.4-hisphosphate.
tiomers of Ins( 1 ,4)P2 are shown. D - h ( 1 ,4)P2 is an h s ( l ,4,5)P3
metabolite formed by phosphatase action, and L-Ins(1 ,4)P2 is its
synthetic enantiomer. The numberings shown were chosen since
the alternatives, namely ~-Ins(3,6)P, and ~-Ins(3,6)P,. have
higher position numbers for the substituents. However, problems can arise when the IUPAC rules are adhered to strictly; in
particular, when a metabolic pathway is traversed, confusing
changes in nomenclature can occur. For example, o-n?yo-inositol-3,4-bisphosphate (name according to the IUPAC convention : L-myo-inositol-I ,6-bisphosphate) is metabolized by phosphatases to the monophosphate L-myo-inositol-1-phosphate
although the nonconventional name ~-nzyo-inositol-3-phosphate allows more immediate appreciation of this fact. An IUPAC recommendation allowing all biologically relevant compounds to be denoted as D derivatives has now been
proposed.[641This convention. where possible, is adopted in this
Fig. 4. The nine inosilol isomers.
Inositol Lipids
4. Biosynthesis and Metabolism of myo-Inositol
4.1. Biosynthesis of myo-Inositol
In humans the greater part of myo-inositol intake is
from plants and only a small fraction is biosynthesized. De
novo synthesis is. however, possible in both plants and
animals by the isomerization of D-glucose 6-phosphate
catalyzed by L-myo-inositol 1-phosphate synthase ( D - ~ J Y I inositol 3-phosphate synthase). This enzyme was purified to
homogeneity from yeast,[651 mammalian testis,166.6 7 1 and
The biosynthetic pathway has been reviewed[69,
and stereochemical aspects have been
Surprisingly. the i~-glucose-6-pIiosphateanalogue ~-glucose-6-phosphorothioate is not a substrate for L-myo-inositol-1 -phosphate synthetase.[”’
nijwlnositol is then generated by the action of inositol
L-rnyo-inositol-I-phosphate. The
monophosphatase is a key enzyme in the phosphatidylinositol
cycle since it hydrolyzes both isomers of Ins( 1 ) P ; the L isomer
arises by the above pathway and the D isomer by the hydrolysis
of phosphatidylinositol phospholipids. It was shown that this
single enzyme also dephosphorylates D- and ~ - I n s ( 4 ) Pand in
vitro I n ~ ( 5 ) P . [ ~mjw-lnositol
monophosphatase was purified
7 5 1 and an X-ray crystal structure of this enzyme
has now been
This enzyme is also of great current interest on account of
its sensitivity to inhibition by lithium ions. I t was observed in
197 1 that levels of free myo-inositol decreased significantly
in the brains of Li+-treated rats.[77]Currently, links between
the known therapeutic effects of lithium in the treatment of manic depression and its inhibition of myo-inositol
1-phosphatase are being actively sought. Since the brain does
not have iiccess to dietary nqv-inositol, which cannot cross
the blood brain barrier. the de novo biosynthetic pathway
via 1,-glucose 6-phosphate must be in effect. If the final
stage in this pathway, the dephosphorylation of L-Ins( 1)P,
is blocked by lithium ions, the brain is unable to replenish
its stores of phosphatidylinositol lipids, and the messenger system based on these lipids (see Section 4.2), which is used by
neurotransmitters in cell signaling to alter electrical activity.
is unable to operate. This is thought to be particularly important in hyperactive cells. as may also be the case in some
forms of
7 9 1 These important aspects have been reviewed,180 x21
The inhibition of inositol-1-phosphatase by Li’ ions[”] is
unusual in that it is uncompetitive. Li+ is thought to inhibit the
second step of the dephosphorylation reaction, namely the
breakdown of a phosphorylenzyme intermediate, possibly by
coordination to an attacking water molecule at the active site,
thus reducing its n ~ c l e o p h i l i c i t y . [ ~It~ .is~ clear
~ ] that a better
understanding of the molecular mechanism of t i inhibition
together w i t h the crystal structure of the enzyme and the extensive medicinal chemistry which has now been carried out will go
a long way towards assisting the rational design of mimics with
potential antimanic properties. Several potent, albeit competitive. enzyme inhibitors have now been synthesized (see Section 9 ) .
4.2. The Phosphatidylinositol Cycle
Inositol-containing phosphatides (Fig. 2) comprise less than
10 of the total phospholipid in animal cells. PtdIns is the most
abundant (over 90%) and is primarily located in the endoplasmic reticulum (ER). The polyphosphoinositides PtdIns(4)P and
PtdIns(4,S)P2 are. together with a small proportion of PtdIns,
located in the inner leaflet of the piasma membrane.
PtdIns isolated from plants has a different structure than that
from animal sources. (The development of plant phosphoinositide signaling is beyond the scope of this article, but has been
reviewed recently.[851)In plants the sn-2 glycerol carbon is
linked to the C,, fatty acid linoleic acid (18:2) and the sn-I
position is linked to fatty acids of the (16:O). (18:O). or (18:2)
form. In contrast, in animals arachidonic acid (20:4) at C-2 and
stearic acid (18:O) at C-1 predominate. PtdIns is phosphorylated
by a specific kinase (PtdIns 4-kinase) to give the 4-phosphate
PtdIns(4)P. which in turn is phosphorylated by another kinase
(PtdInsP 5-kinase) to generate the key lipid PtdIns(4,S)P2. On
the other hand, specific phosphomonoesterases can dephosphorylate PtdIns(4)P and PtdIns(4.5)P2 .1 x 6 1 Upon receptor activation the associated G-protein activates a membrane-bound
(or phosphoinositidase), phospholipase C,[”] which cleaves PtdIns(4,S)P2 into two second messengers, D-my-inositol 1.4,5 trisphosphate (Fig. 3) and 1.2-di-Oacylglycerol (DAG), which together form a bifurcating
signaling pathway. The hydrophilic Ins( 1,4,5)P3 diffuses into
the cytosol and activates the receptor of a Ca’ ’ channel on the
ER, resulting in the release of C a 2 + from an internal store (see
Section 5 ) .
The other product of this signaling pathway, DAG, was
demonstrated to activate protein kinase C (PKC) by Nishizuka
et al.[88.891 PKC was identified as the long-sought “receptor”
for the tumor-promoting phorbol esters.[901PKC was originally
thought to be a single entity, but it is now clear that a large
family of such protein kinase C isozymes exists.‘”’ - 9 3 1 DAG is
hydrophobic, remains in the plane of the membrane, and can be
metabolized by two major pathways involving a kinase or a
lipase. In the first case the fatty acid moieties are conserved and
DAG is phosphorylated to phosphatidic acid. which then combines with cytidine 5‘-monophosphate (CMP) to form CMPphosphatidate. which in turn can be coupled to a molecule of
inositol to reform PtdIns. This reaction is catalyzed by the enzyme PtdIns synthetase. In the second case the DAG is hydrolyzed by a lipase to give a monoacylglycerol with the release
of the eicosanoid precursor arachidonic :t~id,’’~]used in
prostaglandin synthesis. Structural aspects of the DAG limb of
the signaling pathway have been studied by using DAG anal o g u e ~ , [961
~ ~and
. the structural requirements for PKC activation by lipids have been reviewed.[971
Aside from the well-established phosphatidylinositol lipids
discussed above, whose signaling involvement is now clear, a
major new area has emerged recently due to the discovery of
three phospholipids bearing a phosphate group at the D-3 position of the inositol ring. These lipids are PtdIns(3)P, PtdIns(3,4)P2. and PtdIns(3,4,S)P3 (Fig. 2). While their source has
been the subject of some debate.[”’] it is now accepted that direct
phosphorylation of PtdIns(4,S)P2 by a 3-kinase (PtdIns-3K)[”J
is the main route of formation of PtdIns(3.4.5)P3 in stimulated
B. V. L. Potter and D. Lampe
cells. PtdIns(3)P and PtdIns(3,4)P2 are derived from the parent
PtdIns(3.4,5)P3 by phosphatase action.[24%
The 3-phosphorylated lipids, unlike PtdIns(4,5)P2, are not substrates for phospholipase C and thus cannot give rise to inositol phosphates as
signaling molecules.['011 PtdIns-3K activation is intrinsic to mitogenic signaling by growth Factor receptor tyrosine kinases[1°21
and has been implicated in signaling by many receptor and
non-receptor protein tyrosine k i n a ~ e s . ~ " ~ . A distinctive second messenger role for PtdIns(3.4,5)P3 is not yet apparent and
the hunt is on for an intracellular effector. It may be possible
that, like PtdIn~(4,5)P,,["'~~
PtdIns(3.4,5)P3 interacts with the
phorus, to yield inositol I ,2-cyclic-4,5-trisphosphate [Ins( I :2cyclic 4,5)P3], for example in thrombin-stimulated platelets.['07]
Initial reports led to the suggestion that Ins(1 :Zcyclic 4,5)P3 is
a second messenger in its own right,['0'] but it proved impossible to confirm some of the earlier r e ~ u l t s . [ ' ' The
~ ~ long half-life
of Ins(1 :2-cyclic.4.5)P3 in parotid cells is also not compatible
with a physiological function in C a 2 + release,["'] and its low
concentration in some stimulated cells argues against an important messenger role for this compound.["'] There is no reason
at present to assume that the production of Ins( 1 :2-cyclic 4,5)P3
is anything other than an unavoidable and nonfunctional consequence of the mechanism of action of phospholipase C.[''21
The role of Ins(l,4.5)P3 as a second messenger is now well
established. Two major routes of metabolism have been discovered. It was known for some time that Ins(l,4,5)P3 is metabo4.3. Metabolism of myo-Inositol 1,4,5-Trisphosphate
lized by the action of a 5-phosphatase; this was first demonstrated in erythrocyte^'"^] and subsequently in many other cells.[211
Once released inside the cell, a second messenger must be
There appear to be multiple types of this enzyme and both
efficiently deactivated in order to terminate its action and return
soluble. cytosolic forms and particulate. membrane-bound
the cell to a basal state in preparation for a new stimulus. Addiforms exist. Human platelet cytosol. for example, contains two
tionally, metabolites of the second messenger may well have
5-phosphatases, designated type I and type 11, and the particuphysiological activities within the cell. These aspects have come
late type I 5-phosphatase from human placental membranes has
under close scrutiny for Ins(l,4,5)P3 in the past few years, and
recently been purified and characterized.[' 14] Since the product
it has become clear that its metabolism to free inositol, which is
of the action of 5-phosphatase, Ins(1 ,4)P2. has no known physthen recycled into lipid synthesis. is quite complex, generating
iological role in this signaling pathway (there have, however,
many inositol phosphates which are often difficult to separate.
been reports that Ins( 1,4)P2acts as an activator of D N A polyOnly very basic features of this aspect of the PtdIns cycle will be
merasecr[' ''I and 6-pho~phofructo-l-kinase~"~~),
it seems clear
discussed here. For more details the reader should consult the
that this enzyme acts merely to terminate the C a 2 +mobilization
extensive reviews available."'. 22, l o b ] The important features of
signal. Ins(1 ,4)P2 is metabolized to Ins(4)P and subsequently
Ins(1 ,4.5)P3 metabolism are detailed in Figure 6.
converted to inositol, which reenters the cycle.
In 1985 the higher polyphosphate Ins(1,3,4,5)P4 was discovered" ' 71 closely followed by the 3-kinase enzyme," ''I which has
12'] sequenced, and cloned,['2'. '**I adding a
further complication to the metabolic pathway and initiating
speculations about the possible function of this molecule. Det I
spite fierce debate, no unambiguous role for this molecule has
yet been elucidated. Readers are referred to recent reports on the
- L 2 h 1 Considerable controversy has been raised by the
suggestion that Ins(1,3,4,5)P4 acts as a second messenger in its
~ h d i ) ~own right and may be responsible for mediating the entry of
extracellular C a 2 + through plasma membrane ion channels."271
No end to this debate is yet in sight.
t i
Whatever the function of Ins(1 ,3,4,5)P4, it appears to be metabolized by the same 5-phosphatase as Ins( 1,4,5)P3. However,
the situation is complicated by the existence of both soluble and
particulate forms of the enzyme and more than one subtype of
the former.['281 The particulate and type I soluble enzymes
cleave both substrates and Ins( 1,3,4,5)P4 is a better substrate,
but the type11 enzyme has a very poor affinity for the tetrakisphosphate.[". 12'. l2'I Th e product, Ins(1,3,4)P3, has also
been proposed to have a physiological function on the basis of
a report that it is a moderately potent mobilizer of intracellular
Fig. 6. Metabolism of inositol phosphates.
Ca2+.1'301However, this was not ~ o n f i r m e d ~ 'and
~ ' ] there is no
further evidence for a physiological role for this compound. The
The PLC-mediated cleavage of PtdIns(4,5)P2 is complicated
metabolism of Ins(1 ,3,4)P3 is tissue-dependent. The products,
by the fact that Ins(1,4,5)P3 is not the sole hydrophilic product.
Ins(1 ,3)P, and/or Ins(3,4)P2, are subsequently converted into
Attack by H,O on the phosphodiester linkage of PtdIns(4,5)P2
the monophosphates Ins( l)P and Ins(3)P, respectively, and
gives Ins(1 ,4,5)P3 and DAG. but cleavage may also be
these are ultimately dephosphorylated to inositol by inositol
accomplished by attack of the 2-hydroxyl group at phos-
Inositol Lipids
indicate that the conformation of this molecule varies with its
Studies on the action of Ins(1 ,3,4,5)P4 have been complicated
degree of protonation. At high pH the molecule assumes a conby the observation that in many tissues it can be converted to
Ins(1.4.5)P3 by a 3-pho~phatase.['~',
13" It is now thought that
formation having one axial and five equatorial phosphate
Insf, and InsP, are the physiological substrates of this enzyme
groups, whereas between pH 2 and 5 it adopts a conformation
with one equatorial and five axial phosphate groups. which is
since these compounds are attacked with much higher affinity
than Ins( 1 ,3.4,5)P4.['341Ins(1 ,3,4,5)P4 3-phosphatase was puristabilized by intramolecular hydrogen bonding.
fied from hepatocytes[' 341 where it is compartmentalized inside
the ER.[lJI
Other metabolic pathways have also emerged. It is now ap5. The Inositol 1,4,5-Trisphosphate Receptor
parent that Ins(1 ,3,4)P3 can be phosphorylated by a 5/6-kinase
to Ins(1 .3.4,6)P,,'I3'. 13'] which has a weak, but distinct, Ca"The inositol 1,4,5-trisphosphate receptor ( InsP3R) is now recognized to be a ligand-gated ion channel, activated by the bindmobilizing activity.[' 3 R . 391 The 5/6-kinase will also convert
ing of Ins(1,4,5)P3 and gating C a 2 + (Fig. 7). In the last five
Ins( 1 ,3,4)P3 to Ins(1 .3.4,5)P4. In animal cells Ins(l.3,4,5)P4 is
years much information has accrued concerning the molecular
phosphorylated by 5/6-kinase to Ins(1 ,3,4.5,6)P5, which is
structure of the InsP,R, its distribution in tissues and in the cell,
metabolized to three compounds, InsP, (phytic acid) and the
and its r e g ~ l a t i o n . [ "-'"]
In brain. Ins(1 .4.5)P3 receptors are
normally inseparable enantiomeric pair Ins(3,4,5,6)P4 and
highly expressed in cerebellar Purkinje cells, the hippocampus,
Ins( 1 .4,5.6)P4. Ins(1 ,4,5)P3 5/6-kinase was purified from liver
with an InsP,-linked affinity column.i14a1
striatum, and cerebral cortex. Peripherally. they were identified
Such higher inositol polyphosphates have long
been known to be present in plants and in avian
erythrocytes[57,I' where they function to provide
phosphate storage and to modulate the oxygen
affinity of hemoglobin. Their presence in mammalian cells, however, together with observations
indicating that these compounds may provide intracellular and extracellular signals in brain['41' 1421
indicate an interesting future for this area,[1431although it seems unlikely that these higher
polyphosphates are directly involved in signal
transduction. A physiological role for InsP, as an
inhibitor of iron-catalyzed hydroxyl radical formation was suggested.[1441InsP, is. surprisingly,
not the limit for phosphorylation. and evidence
has been put forward for the existence of the pyFig. 7. Schematic representation of the Ins(1.4.5)P, receptor (cross section).
rophosphate-containing higher polyphosphates
InsP, and I ~ S P , , [which
~ ~ ] as yet have not been
in the smooth muscle of arteries, uterus, and oviducts, as well as
securely structurally characterized. These compounds apparentin the contractile smooth muscle of the intestine and esophagus.
ly occur naturally in the slime mould Dicryisteliurn, but inositol
In Purkinje cells electron microscopy showed that the receptor
polyphosphate pyrophosphates can also be generated by
lyophilization of an inositol polyphosphate in the presence of a
is mainly associated with the smooth ER, but it is also present
in the rough ER and outer nuclear membrane.
phosphoryl donor.[231It seems certain that more surprises are in
InsP3R was purified in 1988 as two immunologically identical
To aid in the investigation of inositol polyphosphate metapolypeptides: an Ins( 1 ,4,5)P3-binding protein" i91 and the Pubolism in various tissues two major instrumental analytical
kinje cell enriched protein P400.I16o1 Since then, cloning of the
receptor cDNA from mouse,[161' 1 6 2 1 rat,['". '641 Duosophimethods were developed: one involves anion exchange
HPLC114'' with on-line enzymatic hydrolysis of the phosphates,
has shown that the general structure of
using a post-column reactor containing immobilized alkaline
the receptor, first deduced from mouse cDNA. appears to be
phosphatase. and molybdate detection of inorganic phoshighly conserved. The receptor protein binds Ins( 1 ,4,5)P3
phate.l14"l The other method relies on a metal-dye detection
activates Ca'+ channels, and reconstitution into lipid vesicles
allowed the demonstration of Ins( 1 ,4,5)P3-gated Ca'+ transsystem not requiring dephosphorylation, in which trivalent
port.['661The receptor is a homotetramer, as demonstrated by
transition metal ions are bound with high affinity to the dye
electron microscopy[167,1681 and cross-linking experiments.['h81
4-(2-pyridylam)resorcinol and inositol phosphate polyanThis method permits picomolar-range detection of
Each subunit binds one molecule of Ins( 1 ,4.5)P3. as revealed by
inositol phosphates.
examination of deletion
I' and InsP,R apparentSeveral NMR studies on inositol phosphates were also rely binds four molecules of Ins(1 ,4.5)P3 in a noncooperative fashported. Multinuclear N M R spectra were measured to confirm
ion;" 591 however, contradictory finding have also been reported,[1711
the structures and conformations of Ins( 1 ,4,5)P3
I ~ s ( I , ~ . ~ , ~ ) P15'1
, , I 'In~~"(.1 , 4 , 5 , 6 ) P , , [ ' ~InsP,,['S21
and a vaThere is no homology between the primary sequence of
riety of lower inositol phosphates.[1491The data on phytic acid
InsP,R and C a Z +channels on the plasma membrane."".
B. V. L. Potter and D. Lampe
but a significant partial homology does exist with the ryanodine
receptor of skeletal and cardiac muscle sarcoplasmic reticu1um.r'61.173,1741 InsP,R has a transmembrane domain close to
the C-terminus with long N-terminal and short C-terminal portions projecting into the cytoplasm. Examination of the hydropathy profile of the highly conserved amino acid sequence
suggests that the transmembrane domain has sixr1651or
eight['691membrane-spanning regions. The large N-terminal region incorporates some 650 amino acid residues, which are
highly conserved between species"".
and thought to
contain the critical sequence that forms the three-dimensional
Ins(1 ,4,5)P, binding site,[16y.I 7 O 1 since deletion of sequences
within this region abolished Ins(1 .4,S)P, binding activity. This
region is enriched in positively charged arginine and lysine
residues, which may have a role in binding the phosphate groups
of Ins(l.4,S)P3. A peptide labeled by an Ins(l,4.S)P3 photoaffinity ligand has been isolated whose sequence matches that of the
N-terminal 20 % of I ~ S P , R . [ ' ~Binding
of Ins( 1 ,4.S)P3 must
stimulate a large conformational change in the receptor, which
spans almost 1400 amino acid residues from the extreme N-term i n d Ins( 1 ,4.S)P3 binding site to the extreme C-terminal putative C a 2 + channel
The coupling region between the
Ins(l,4,5)P3 binding site and the C a Z + channel contains sites
that modulate the effect of Ins(1,4.S)P3 on channel opening.
Two serine residues present in this region can be phosphorylated
by CAMP-dependent protein kinase,[' 5 3 ,
' 7 6 1 which results
in uncoupling Ins(1 ,4,S)P3 binding from the opening of the
C a 2 + channel in the cerebellar receptor. The receptor also binds
equimolar amounts of ATP,[1681and ATP at low concentrations
has a potentiating effect upon Ins(1 ,4.S)P3-stirnulated C a 2 + release. Ca2' itself can also modulate channel opening, but by an
unknown mechanism.[' "I
InsP,R is known to exhibit significant heterogeneity as a result of alternative mRNA splicing, giving receptors that differ in
the coupling region and perhaps in the ligand binding domain.
The original cerebellar receptor is now termed Type 1. New
types of receptors encoded by separate genes were also reported.[164.' 781 While Type2 InsP,R shares a significant homology
with the Type 1 receptor, it has a higher affinity for Ins(1.4.S)P3
and is 48 amino acids shorter.r1641A Type3 InsP,R was characterized from rats[17y1and recently from humans" 1'' which has
63 and 64 YOidentity with the Type 1 and 2 receptors, respectively. It is expressed in kidney, in the gastrointestinal tract, and in
brain and binds Ins(1 ,3,4,S)P4 and InsP, as well as Ins(1 ,4,5)P3.
A partial sequence for the C-terminal domain of a Type4 receptor was reported.[156,1641 It is possible that the InsP,Rs identified so far may only be the first examples of a larger family of
receptors. If such receptors differ in their ligand binding affinities and specificity, they may offer an exciting prospect for the
selective targeting of drugs.
6. Synthesis and Activity of Inositol Analogues
lnositol is taken up by many ce11s[181-1831
and is incorporated into PtdIns by the enzyme PtdIns synthetase. The incorporation of synthetic analogues into phosphatidylinositol lipids offers
an attractive route for pharmacological intervention in the signaling pathway. Since a large number of growth stimuli are now
known to stimulate inositol lipid hydrolysis,['84] the presentation of fraudulent phospholipid species to the cell, especially
those which cannot be further phosphorylated. could inhibit
phospholipase C or reduce the availablity of PtdIns(4.S)P2, thus
affecting cell proliferation inter alia. A particular advantage of
this route is that problems intrinsic to the introduction of highly
charged phosphate-containing compounds into the cell are
avoided. Fluorinated inositol analogues have proved to be particularly attractive in this respect.
Kozikowski et al. prepared IDH&OH OH
Fig. 8. 1-Deoxy-l-fluoro-ni.i.o(Fig, 8) from 1~-3-O-methyl-clliinositol (1)
ro-inositol in five steps, including
fluorination with diethylaminosulfur trifluoride (DAST), via the 1.2 :5,6-di-O-isopropylidene
derivative. Direct regioselective fluorination of 1~-3-O-methylclziro-inositol with DAST followed by demethylation gave ID1,5-dideoxy-l ,S-difluoro-neo-inosito1."851 The same group had
earlier reported a ten-step synthesis of both enantiomers of 1-deoxy-l-fluoro-myo-inositolr1861
by DAST fluorination at the 2position of a suitably protected myo-inositol derivative and the
optical resolution of the resulting fluoro-scyllo-inositol intermediate. Recovery o f t h e myo-inositol configuration by Swern oxidation of the 1-hydroxyl group followed by stereospecific reduction of the keto function with L-selectride, gave, after
deprotection of the respective enantiomeric intermediates, IDand 1 L-1 -deoxy-l-fluoro-nqv-inositol.
The synthesis of ID- and 1L-I-deoxy-I-fluoro-myo-inositol
was also reported by Offer et a1.['87. '''1 Here, the problems of
recovering the m-yo-inositol configuration and optical resolution
were efficiently addressed in a single step by displacing a tosylate
group of a fluorinated scyh-inositol intermediate with (S)-(-)cesium camphanate. The resulting diastereoisomers were separated and deprotected to produce the D- and L-l-fluoro analogues of m>-o-inositol. The respective 2-,H-Iabeled analogues
were prepared by an exchange of the tritium radiolabel from
[2-3H]-ni.~o-inositolcatalyzed by inositol dehydrogenase.['891
1-Deoxy-1-fluoro-scyllo-inositol (2) (Fig. 9), the first fluorinated analogue of inositol,['yol was obtained from I-0-benzoyl3.4,~,6-tetra-~-benzyl-~.~o-inositol
by treatment with DAST at
elevated temperature and deblocking of the protected fluorinated scyllo-inositol intermediate. The preparation of 2 was subsequently described by another group.["'] Lowe and McPheer'y21
reported the synthesis of 2 and 2-deoxy-2-fluoro-myo-inositol
(3) together with their 2-3H-labeled derivatives 4 and 5.Fluorination of 1,3,4.S,6-penta-O-benzyl-myo-inosito~
with DAST
proceeded with inversion at the 2-position to give the fluorinat/lo-inositol, which was debenzylated with dry HBr to af-
2:X = H, Y= F:
3: X = F,Y= H:
4: X =3H, Y = F:
5: X = F, Y = 3H:
B : X= Y= F:
7: X = F, Y =CH20H
8: X = OH, Y = C Y F
9: X = SH,Y= H:
Fig. 9. C-2-rnoditied inositol analogues
I'hcm lrzl Ed t n p l 1995, 34. 1933 1972
Inositol Lipids
resulting in selective reaction at the I-position with inversion to
ford 2. In order to prepare 3, the configuration at C-2 was
give the myo-inositol configuration in 11. Demethylation gave
inverted by tritlation followed by displacement of the triflate
18.[1yh1D-3-Deoxy-nyo-inositol (19) was prepared from Lmoiety with sodium trifluoroacetate. Ester hydrolysis, reaction
3.4: 5,6-di-O-isopropylidene-2-O-methyl-chiro-inositol (12) by
of the product with DAST, and deprotection produced 3. Oxidatreatment with 1.1'-thiocarbonyldiimidazole. tributyltin hytion of 1.3,4,5.6-penta-O-benzyl-myo-inositol
with Jones'
Intermediate 12 was
dride. and finally boron tribromide."
reagent and reduction of the resulting inosose with [3H]NaBH,
also employed in the preparation of the ~-3-halogenoanalogues
gave predominantly the tritiated scyllo-inositol and some
20-22. The free hydroxyl group of this intermediate was dis1,3.4,5,6-penta-0-ben~yl-2-[~H]-m~~o-inositol.
Treatment with
placed with inversion of configuration by activation with
DAST and deprotection as before gave 4 and 5.['y21A synthesis
of 3 and 5 reported by another group[1891followed a route
triphenylphosphine and employing CCI,, Br?. or 1, as elecsimilar to the one outlined above.
trophilic sources of halogen. Deprotection of 13 (X = CI, Br, or
(6) was prepared by
I) with boron tribromide furnished the corresponding 11-3oxidizing 1-0-aIlyl-3,6-di-O-benzyl-4,5-0-isopropylidene-m,~o-h a l o g e n ~ i n o s i t o l s . ~ 'Compounds
23-25 were prepared by
employing mesylate 15, obtained from 12 via 14.[19'1 Introducinositol to the corresponding 2-inosose. which was fluorinated
with DAST to give the gem-difluoro derivative. Deprotection
tion of the azido group with inversion of configuration was
was carried out first by removal of the ally1 and isopropylidene
achieved by displacing the mesylate in 15 ( R = ethoxyethyl)
groups and subsequent h y d r o g e n ~ l y s i s . [ ' ~Yang
with sodium azide/hexamethylphosphoramide (HMPA). Acid
' ~ et al. used
hydrolysis furnished ~-3-azido-3-deoxy-n7~1~~-inositol
7-deoxy-2,,-fluoro-2,,-hydroxymethyl-~~~~o-inositol which could be converted into ~-3-amino-3-deoxy-nq~u-inositol
(7)[")" and 2-deoxy-2-C-fluoromethyl-r?~yo-inositoI (8) ,[1y41.
hydrochloride (24) by catalytic reduction in dilute HCI. The
The synthesis of the 2-mercapto [sulfanyl (IUPAC)] analogue
3-mercaptoinositol 25 was obtained by reaction of 15 (R = Ac)
9 of nzw-inositol was described:[Iy5]Selective iodination of the
with (CH,),NC(S)SNa/HMPA, followed by reduction with
2-position of racemic I .4,5,6-tetra-O-benzyl-~zyo-inositol
LiAIH,, and removal of the protecting groups. Oxidation of the
triphenylphosphineiimidazoleiiodineproceeded with inversion
hydroxyl group in 12 and treatment of the resulting ketone 16
of configuration. The free 3-hydroxyl group was silylated and
with MeMgBr resulted in an axial attack to give the protected
the equatorial iodine displaced with sodium benzylsulfide, thus
3-C-methyl derivative, which was deblocked a s described previreestablishing the myo-inositol configuration. Fluoride-induced
ously to yield 17.[1971
desilylation. debenzylation, acetylation of the crude product,
The growth inhibitory properties of these inositol analogues
purification. and methanolysis gave the thio analogue 9.
on wild-type NIH 3T3 cells and v-sis-transformed cells were
Kozikowski et al. reported the synthesis of a number of enanexamined. v-sis-Transformed cells exhibit an increased PtdIns
tiomerically pure inositol analogues modified at the 3-position
3'-kinase activity and can therefore be expected to be more
sensitive towards inhibitors of the PtdIns 3'-kinase signaling
(18- 25, Fig. 10),[1yh-2001
A two-step synthesis of 1D-3-deoxy3-fluoro-m,i~o-inositol(18)was possible by direct fluorination of
pathway than the nontransformed cells. Initial experiments['s5)
L-quebrachitol ( I ~-2-O-methyl-chiro-inositol, 10) with DAST,
showed that D-3-deoxy-3-fluoro-myo-inositol
(18) is a potent
and selective inhibitor of cell growth, although the potency was
decreased by about an order of magnitude in the presence of
nzyo-inositol in the growth medium. The fluoro analogue was
also found to act as a potent competitive inhibitor of [3H]-myoinositol uptake, suggesting that the compound is incorporated
into inositol phospholipids. In further
on a
number of C-3-modified D-my-inositol analogues, ~-3-azidomJwinositol (23) was found to be highly potent (IC50=
0.04 mM) and selective for v-sis-transformed cells (more than
1200-fold over the wild-type) in inositol-free medium. Addition
of nzyo-inositol. however, caused a considerable decrease in the
inhibitory properties of this compound, whereas the inhibition
of growth by ~-3-amino-3-deoxy-mpf~-Inositol
(24) was only
moderately affected. Also investigated were ~-3-deoxy-3methylene-. -3-(hydroxymethy1)-, -3-(iodomethy1)-. and -3RO OMSl5
phosphonomethyl-myo-inositol as well as the dimethyl ester of
the latter compound; none of these analogues displayed any
significant inhibitory activity.['"]
20: x = CI
Both D- and L-4-deoxy-4-fluoro-myo-inositol were obtained
21: X = Br
from DL-1.3,4,5-tetra-O-benzyl-myo-inositol
by selective tosylaH
DAST (giving
23: X = N3
cam24: X = NH; CIphanate (reestablishing the myo-inositol configuration by inver25:X-SH
sion at C-6 which becomes the new 2-position). The resulting
Fis 10. C'-3-~nod~fied
i)-iiil,o-inositols from ~-quebrachitol(10) (KoLikowski et
mixture of two diastereoisomers could be separated by HPLC o r
B. V. L. Potter and D. Lampe
partial crystallization. Deprotection afforded D- and L-4-deoxy4-fluoro-myo-inositol.[' "1 Whereas a 3H-radiolabel could be
introduced into the 2-position of the L isomer by employing
inositol dehydrogenase and [3H]-myo-inositol, the D isomer was
not a substrate for this enzyme. The radiolabeled D analogue
was prepared by oxidizing D-1 ,3,5,6-tetra-O-benzy1-4-deoxy-4fluoro-mp-inositol, reducing the resulting 2-inosose with
[3H]NaBH4, and removing the protecting groups. Syntheses of
racemic 4-deoxy-, 4-deoxy-4-fluoro-, and 4-deoxy-4-methyltnyo-inositol employing a precursor obtained from benzene by
microbial oxidation were also reported.[2011
6-O-Benzyl-l,2: 3.4-di-0-cyclohexylidene-myo-inositol was
employed in the preparation of 5-deoxy-5-fluoro-myo-inositol
(26) (Fig. 11) by two different groups. DAST fluorination gave
26: X = F,Y = H:
27: x = Y= I=:
28: X =GI. Y = H
29: X = Br, Y = H
30: X = H, Y = CI
31:X = H.Y = Br
32: X = H, YE I
33:X = Y= H
34: X = OCH3, Y = H:
Fig. 11. C-S-modified inositol analogues
the product resulting from inversion of configuration and as
well as minor amounts of that resulting from retention; the
latter was deprotected to 26.[2021
This low-yield method was
subsequently improved upon by inversion of configuration at
the 5-position prior to DAST fluorination.["7,
* O 3 ] Thus,
~ ~ 5-hydroxyl group folt ~ s y l a t i o n [ ' ~'"] ' ~ or t r i f l a t i o r ~ [ ~ofOthe
lowed by nucleophilic displacement with cesium propionate
afforded the neo-inositol derivative. After saponification, fluorination with DAST reestablished the nzyo-inositol configuration
and deprotection furnished 26.
Deuteriation studies revealed that the equatorial H-2 proton
of 26 is the one most rapidly exchanged.12031Incubation of 26
with inositol dehydrogenase and [2-3H]-myo-inositol led to an
exchange of the radiolabel, which was again incorporated at the
2-position of the fluorinated analogue.['sg1 The 5-deoxy-5,5difluoro analogue 27I2O3]was prepared from the 0x0 derivative
of 1,4,6-tri-0-benzyl-2,3-O-cyclohexylidene-neo-inositol,
an intermediate in the synthesis of 26.
Apart from the synthesis of 26 and 27 and myo-inositol analogues deoxygenated at positions l , 4, and 5, Baker et al. also
reported the preparation of further 5-deoxyhalogeno-myo- (28,
29) and epimeric neo-inositols (30-32),[2021as well as racemic
4-deoxy-4-fluoro-myo-inositol. DAST fluorinations generally
proceed with inversion of configuration,[2041but surprisingly,
fluorination of the intermediate 3-0-benzyl-l,2:5,6-di-O-cyclohexylidene-nzyo-inositol gave predominantly the product of
retention of configuration. This was rationalized as being due to
steric hindrance to the backside attack of fluoride ion on the
intermediate adduct with DAST.[2021
These inositol analogues were then evaluated as substrates
and inhibitors of PtdIns synthetase,[20s1as were a number of
C-3-modified D-nzyo-inositols.[2061 The enzyme showed very
stringent requirements for the cyclitol substrate. All compounds
examined were less effective substrates than myo-inositol. 5-Deoxy-5-fluoro-myo-inositol(26),
the best analogue found in these
studies, was incorporated into the cellular phospholipid at a rate
equal to 26 % of that of myo-inositol. Analogue 26 was further
phosphorylated to give the corresponding 5-fluoro-PtdIns(4)P
derivative but, not surprisingly, no lipid bisphosphate was detected. Other 5-deoxy-5-halogeno derivatives were very poor or
inactive substrates. The 5-deoxy analogue 33, however, was
found to be well recognized. In the series of C-3-modified
derivatives tested, D-3-amino- (24), D-3-fluoro- (18), and D-3deoxy-mjo-inositol(l9) as well as the 3-0x0 analogue were substrates for PtdIns synthetase,[20h]albeit > 90% less well tolerated than the natural substrate. The general conclusion derived
from these observations appears to be that substrate activity
diminishes with increased steric bulk of the substituent. In the
inhibition assay, the rank order of analogues was similar to their
effectiveness as substrates, with the exception of 5-deoxy-5,5difluoro-mvo-inositol (27) which was not a substrate for PtdIns
synthase but an effective inhibitor of the enzyme.
In another
the inhibition of incorporation of
[3H]Ins into PtdIns by inositol analogues modified at the I-, 2-.
3-, 4-. or 5-position was investigated. I-Deoxy-1-fluoro-scylloinositol (2) and 5-0-methyl-myo-inositol (34) were found to be
the most effective competitive inhibitors of both PtdIns synthase and a nucleotide-independent PtdIns/Ins exchange enzyme, whilst a number of other analogues also displayed significant inhibitory effects mainly on the exchange activity. Both
(4) and [2-3H]-2-deoxy2-fluoro-m.vo-inositol (5) are very poor substrates for the two
enzymes. However, once 5 was incorporated into the phospholipid, the corresponding analogues of PtdIns(4)P and
PtdIns(4,5)P2 could be detected.
Offer et a]. investigated the uptake of 3H-labeled monodeoxyf l u o r o - m ~ o - i n o s i t o l s [into
' ~ ~ ~thymocytes and their incorporation into the phospholipid.12081Only D-3-F-Ins 18, 5-F-Ins 26
and to a lesser extent D-6-F-Ins showed activity as substrates for
PtdIns synthase, and only 18 was both taken up actively into the
cells and incorporated into the phospholipid. However, it was
concluded that because of the overall selectivity for myo-inositol
both by the uptake mechanism and by PtdIns synthase,
monodeoxyfluoro analogues, unless made cell permeable, were
unlikely to be of any use as inhibitors of PtdIns(4,5)P2 formation or breakdown in intact cells.
Deoxy inositols were the subject of a report demonstrating
chemoenzymatic methods for the synthesis of selectively protected derivatives of 3-deoxy-epi-inositol and l-deoxy-scylloinositol, taking advantage of the preference of Cundidu cylinclrucea lipase for (R)-configurated esters.[20g1
7. Synthesis of Specific myo-lnositol Phosphates
Recognition of Ins(1 ,4,5)P3 as a second messenger has stimulated renewed interest in the chemistry of inositol phosphates.
Many inositol phosphates had already been synthesized before
this recent revival, and this earlier work has been dis~ u s s e d . [ ~ ' ~In spite of this, some of the older procedures used
were not entirely satisfactory. This was especially true for the
phosphorylation of polyols, where the presence of vicinal diols
A n j i m . C'hcm. lnr. Ed. EngI. 1995. 34. 1933- 1972
lnositol Lipids
makes simultaneous polyphosphorylation difficult and encourages the formation of cyclic five-membered phosphates (for exceptions see ref.[210]). Nevertheless, Ins(4,5)P2 was originally
synthesized in 1961.[2'11More recent work is covered in a new
derivatives into a pair of diastereoisomers, which is then separated by crystallization or chromatography. Until recently, the
procedures of Shvets,[21 9 , '201 employing orthoesters of D-mannose 35 (Fig. 13). were the only methods available. Recent
work, however, has highlighted the advantages of several other
resolving agents such as (S)-( )-0-acetylmandelic acid (36),
(R)-(+)-camphor dimethyl acetal (37) and its ( S ) isomer, Lmenthyl chloroformate (38), L-menthyloxyacetylchloride (39),
and (R)-(+)-I -phenylethyl isocyanate (40). Optical resolution
by formation of camphanates seems to be the most generally
useful method available at the present time. and both ( S ) - (-)camphanic acid chloride (41) and its ( R ) - (+) isomer have been
employed to obtain optically active inositol derivatives. Chiral
HPLC columns were used to resolve intermediates in syntheses
of Ins(1,3,4)P3[22'1and Ins(1,3,4.5)P,.f2221
7.1. General Synthetic Considerations
Five distinct problems are intrinsic to inositol polyphosphate
synthesis (Fig. 12): 1. Suitably protected derivatives must be
prepared to Facilitate later incorporation of phosphate groups at
selected positions. 2. Since the D enantiomer of Ins(l,4,5)P3 is
selecrve protecuon
( a d resoltAon)
ondabve W o r a k z a b o n
ard proechon(+ msoltAon)
Fig. 13. Agents employed in inositol phosphate synthesis t'oi- optical resolution
big 1 2 5ttpr in the sntheals ot inosltol phohphdtes
the natural messenger, such derivatives must be optically resolved. 3. An efficient phosphorylation strategy is required,
which overcomes the difficulties associated with cyclic phosphate foi-mation. 4. The phosphorylated intermediate must be
deprotected under conditions that preclude any migration of
phosphate groups onto neighboring hydroxyls. 5. The anionic
polyphosphate must be efficiently purified and separated from
minor potentially regioisomeric impurities, which might possess
act I vi t y .
7.1.I . Pvotrcting Cvoup Chemistry
Protection strategies for the synthesis of inositol polyphosphates already have a long historical development,[". "I but
several new approaches have been described.[21' -'"]
The subject has been discussed in two review articles.[53.541 The most
popular temporary and permanent hydroxyl protecting groups
are acetyl. benzoyl, benzyl, p-methoxybenzyl, allyl, isopropylidene, cyclohexylidene, and orthoformate. This will become apparent from examination of individual syntheses.
In a number of studies, enzymes such as esterases, lipases, and
proteases were examined for their ability to hydrolyze enantioand regioselectively racemic or meso carboxylic esters of n7yoi n ~ s i t o l , [ -' ~2261
~ 3-deoxy-epi-inosi to1 and 1 -deoxy-scj//o-inositol.[209]A new method is enzyme-catalyzed selective esterification, which was applied to resolve racemic 1.2: 5,6- and
1,2: 3,4-di-O-cyclohexylidene-n1~~o-inositol,[~~
', 2 2 8 1 and to obtain enantiomerically pure 1~-l-O-butyryl-4.6-di-O-benzoylmyo-ino~itoI.['~~]
The problem of optical resolution can be avoided altogether
by employing chiral starting materials, for example naturally
occurring compounds such as L-quebrachitol (lo), 1 ~ - 3 - 0 methyl-chiro-inositol (42), ( - )-quinic acid (43). D-glucurono6,3-lactone (44),and galactinol (45)(Fig. 14). The Ferrier reac-
7.1.2. Synthesis of Optically Active Compounds
If benzene or the meso compound myo-inositol are used as
starting material in the synthesis of inositol phosphates, optical
resolution is required to separate D and L isomers of the inositol
phosphate precursors. Most optical resolutions in the synthesis
of inositol phosphates rely on the conversion of racemic inositol
Fig. 14. C h i d starting materials for the synthesis of inoa~tolphosphates
B. V. L. Potter and D. Lampe
has been used to generate mpo-inositol derivatives from
~ - g a l a c t o s e , [ ~ ~- g' ~l u c o s e , [ 2341
~ ~ ~and
- in a biomimetic approach from methyl %-D-ghcopyranoside (46).[23s.
2361 A samarium diiodide promoted reductive carbocycliration of an intermediate derived from D-mannitol was employed to gain
access to enantiomerically pure mnyo-inositol derivative^.'^"]
7.1.3. Phosphovylating Agents
Phosphorylation of free hydroxyl groups in suitably protected inositol is performed primarily by two methods:
a) employing a Pv reagent in which the phosphorus atom is
already at the correct oxidation level. and b) phosphitylating
the hydroxyl group with a PUlreagent and then oxidizing the
resulting phosphite ester to give the protected phosphate ester
(Fig. 15).
53: R Et
R' 0
-- - --
55: R1 N C C Y C Y , FP iPr
56: ~1 En, FP pr
57: R* NCCH$Y, FP Et
58: Rl -En, FP Et
61: R CH.$YCN
62: R Me
60: R Me
Fig. 15 Pho3phorylating and phosphitylating reagent%
The advantages of Pv reagents for phosphorylation are the
relative stability of reagent and products. However, problems
have been encountered because of the low reactivity of the secondary hydroxyl groups of inositols; the phosphorylation of
vicinal diols has proved to be especially difficult. The intermediate phosphate triesters formed upon phosphorylation of the first
free hydroxyl group are prone to attack by the neighboring
second hydroxyl group. The bulkiness of most Pv reagents may
prevent a second reagent molecule from gaining access to the
phosphorylation site after monosubstitution of the diol has taken place. Cyclization reactions are therefore able to compete
successfully with substitution at the second hydroxyl group.
affording five-membered cyclic phosphates rather than the desired bisphosphates.
Phosphorus(v) oxychloride was first used as a phosphorylating reagent in 1857[L381and more recently in a stepwise phosphorylation of the vicinal 4,s-diol in mjo-inositol derivatives
with two P v reagents.[23y1Phosphorylation with bis(2,2,2trich1oroethyl)phosphorochloridate (47) only gave a mixture of
the 4- and 5-monophosphorylated inositols but not the bisphosphate presumably due to steric hindrance. When POCI, was
used, however, this mixture could be converted into the mixed
4,5-bisphosphate triesters. Phosphorochloridate 47 was also ap1944
plied in the preparation of Ins( 1,4,5)P3 and its 5-phosphorothioate analogue.[240- 2 4 2 1 Dianilidophosphorochloridate (48)
was used in the first reported synthesis of ~ - I n s ( l , 4 , 5 ) P , ; [ ~ ~ ~ ]
however, this phosphorylation method and the removal of the
protecting groups with isoamyl nitrite in pyridine/acetic acid/
acetic anhydride was unsatisfactory. Diphenyl phosphorochloridate (49) was employed in many phosphate ester preparations,
including an early synthesis of I ~ s ( ~ ) Pand
[ ~ the
~ ~synthesis
L-Ins( I)P.[2451
Phosphorodichloridates can react with two different partners,
thus forming a phosphate bridge between these moieties. As
phosphodiester links are common features of many natural
compounds (e.g. oligonucleotides and phospholipids), a number of phosphorodichloridates were developed for the synthesis
of these substances. N-Methylpyridinium phosphorodichloridate (50) was employed in the synthesis of Ins( 1 :2 - ~ y c l i c ) P . [ ~ ~ ~ ]
Polyphosphates and mixed anhydrides are widely used although they react nonspecifically and often lead to the formation of cyclic phosphates or other side products. Phosphoric
acid, potassium hydrogen phosphate, and pyrophosphoric acid
were all employed in syntheses of carbohydrate phosphates; the
latter was also used to phosphorylate epi- and mum-inositols.[24"
Tetrabenzyl pyrophosphate (TBPP, 51)[2481
is a commercially available crystalline solid formed by condensation of two
equivalents of dibenzyl phosphate with dicyclohexylcarbodiimide (DCC). It is the only Pv reagent that has been employed
successfully in inositol phosphate chemistry for the phosphorylation of vicinal dioIs.[24"-2s11For efficient phosphorylation
with TBPP, it is necessary to convert the free hydroxyl groups
into alkoxides. The strongly basic conditions can cause problems with some compounds and lead to decomposition.
PI" reagents are more reactive than most Pv reagents. Good
results were obtained by phosphitylation with Plli reagents and
subsequent oxidation in cases where Pv reagents failed to give
the desired products, for example phosphorylation of vicinal
diol precursors to Ins( 1 .4,5)P3. The formation of undesired
cyclic phosphodiesters is avoided. One further advantage of the
Pl" approach is that the phosphite triesters can be sulfoxidized
by elemental sulfur in pyridine'2s21 or by phenacetyl disulfide.[2s31thereby also making available phosphorothioates,
which were shown to be useful nonhydrolyzable analogues of
n u c l e o t i d e ~ [and
~ ~ ~inositol
phosphates (see Section 8.4).
Although phosphorochloridites are unstable towards
moisture, they have found widespread use in inositol phosphate synthesis. Dimethoxychlorophosphine (52) was employed in preparations of Ins(1 .4,5)P3. Ins(1 ,3,4.5)P4,
and Ins(1 .4,5.6)P,,[2ss1 diethoxychlorophosphine (53) for
Ins(1 ,2,4,5)P4 ,[2561 and di(2-cyanoethoxy)chlorophosphine (54)
for Ins( 1,3,4)P, .[2571
The more stable phosphoramidites are used under catalytic
activation by a weak acid ( 1 H-tetrazole). Compounds 55-57
are examples of these reagents. The use of di(benzy1oxy)(diethy1amino)phosphine (58) was considered to be less advantageous than 56, since the latter can be easily purified by column
3-DiethyIamino-2.4,3-benzodioxaphosphepane(59).[2s91 prepared by the reaction of hexaethylphosphorus triamide with
1,2-bis(hydroxymethyl)benzene,and the related dimethylamino
A I I ~ C I IC' .l i ~ wInr.
. Ed Eii,Fl. 1995. 34. 1933-1972
Inoaitol Lipids
compound 60 are increasingly applied in preparations of inosito1 phosphates. 2-Cyanoethoxy(diisopropylamino)chlorophosphane (61) and the corresponding methoxy derivative 62 have
also been extensively used, the former inter alia in syntheses of
Ins( 1.4,5)P., and 1ns(4,5)Pz . [ 2 h o - 2 h 2 1 The phosphoramidites initially obtained are usually converted into phosphite triesters
prior to oxidation. As bifunctional Pl" reagents, 61 and 62 are
also suitable for the preparation of phosphorothioate analogues
of inositol
- 2 6 5 1 The same is true of 63. which
was employed by van Boom et al. in syntheses of a PtdIns(4,5)P2
analogue12""l and an inositol-containing mycobacterial phospholipid.l'"'' Finally. the less conventional aminobicyclophosphane 64 was employed in the synthesis of inositol monophosphates.l'h81
The efficiency of different methods for the oxidation of phosphite triesters was studied;[""I in inositol phosphate chemistry
this transformation is usually effected with terf-butyl hydroperoxide (rBuOOH) or m-chloroperoxybenzoic acid (nz-CPBA).
7.1.4. Dep'protectionand PuriJication
In order t o itvoid migration of protected phosphate esters to
adjacent free hydroxyl groups it is essential to deblock the phosphate groups prior to the alcohols. When benzyl groups are used
for protection of both hydroxyls and phosphates. deprotection
in one step IS possible, since the benzylphosphate esters are
cleaved much more rapidly.[2701
Purification after the protecting
groups are removed with sodium/liquid ammonia usually involves anion-exchange chromatography. It may be possible,
however. to obtain the final compound without further purification by catalytic hydrogenolysis.
7.2. lnositol Monophosphates
Four possible uzyo-inositol monophosphates exist: the symmetrical 2- and 5-phosphates and the two enantiomeric pairs
inositol4(6)-phosphateand l(3)-phosphate. Many syntheses of
racemic and optically active mj'o- and other inositol monophosphates have been published prior to and after the demonstration
that the hydrolysis of brain phosphoinositides gives 1)Ins(l)P.['" '"I
Such earlier synthetic work has been revlcwcd,153.5 - 1
The D [ 2 - 3 . 2741 and L e n a n t i ~ m e r s [ of
~ ~Ins(1)P
were prepared from 1 L-quebrachitol (10) via 1 ~ - 3 , 45,6-di-O-cyclo:
hexylidene-2-O-methyl-c./2iro-inositol. For the synthesis of
ii-Ins(1) P , t h i h compound was oxidized to the inosose which
was stereoselectively reduced to give 11-1.2: 5,6-di-O-cyclohexylidcne-4-O-tnethyl-~~~~~~~-inositol.
In a six-step sequence, this
intermediatc was converted into the 1- d y l pentabenzoate. Desilylation. phosphorylation, and successive deblocking gave
u-lns(1)P. I n addition, L-Ins(1)P [ = ~ - I n s ( 3 ) P ] was obtained from 12 via the 1-tosylate. An elegant route to
u-Ins(1 ) P[ -'-'
involved resolution of inositol by formation
of the Miy0-iiiositol-D-camphor 2,3-acetal and selective phosphorylation of the 1-OH group with dibenzyl phosphorochloridate. [>-Ins(I )Pwas accessible by using the L-camphor dimethyl
acetal.["'hl Optically active Ins(1)P was also synthesized from
[resolved as the ( R ) (-)'
camphanate][2'01 and the corresponding pentaacetate (resolved
as the acid ~ x a l a t e ) , [which
~ ~ ~ ]was also used in the synthesis of
racemic I n ~ ( l ) P . [ DL-Ins(l)P
could be resolved on a chiral
GC column after persilyIati~n.[~'~l
Ins(2)P was first synthesized by taking advantage of the highly selective oxidation of my-inositol to scyllo-inosose by Acetobacrrr s u b o . ~ y d a n s . [A
~ ~more
~ ~ recent approach relied upon
selective dibenzylation of the 4- and 6-OH groups of rn,w-inositol orthoformate and phosphorylation of the free 2-OH groups
with TBPP.[2801
Synthesis of Ins(4)P was possible by selective benzoylation of
the 3-OH group of 1,2 :4,5-di-O-cyclohexylidene-nz~~1-inositoi,
phosphorylation of the remaining free hydroxyl group, and deprotection.[2'l.2201The corresponding 3-0-benzyl derivative
was also used to produce racemic and, after resolution via its
camphanate, optically active I n ~ ( 4 ) P . [ ' ~A~ short
synthesis of
DL-InS(4)P by chelation-controlled selective phosphorylation of
the monoanion of nijwinositol orthoformate'" ' I with TBPP
was reported.[280.2 8 2 1 Reaction of 1,2-O-cyclohexylidene-~z~~inositol with 1,3-dichloro-l, 1,3.3-tetraisopropyldisiloxanegave
selectively the 3,4-disiloxanylidene-inositol,which was benzoylated at the 6-position. Whereas reaction with TBPP resulted in
phosphorylation at the 6-position due to benzoate migration
and gave racemic Ins(4)P after deblocking. phosphitylation
with PCI, and benzyl alcohol. followed by oxidation and deprotection furnished Ins(5)P.[Zs31The latter was first prepared from
2-amino-2-deoxy-neo-inositol,[' which was obtained by hydrolysis of hygromycin A.
D-Ins( t .?-cyclic)P was synthesized from ~-3,4,5,6-tetra-Obenzyl-m?.o-inositol by reaction with 50.L2"h1
Direct phosphorylation of inositol with bicycloaminophosphane 64 followed by
oxidation of the resulting bicyclophosphorane. which has a fivecoordinate P atom. and acid hydrolysis gave mixtures of
Ins( 1)P, Ins(2)P, and Ins(l,2-cyclic)P separable by HPLC.[2681
7.3. Inositol Bisphosphates
The synthesis of racemic inositol 1.4-, 1.6-, and 4.5-bisphosphates by phosphorylation of di-0-cyclohexylidene-inositols
biscyclohexylidene acetals with phosphorochloridate was described as early as 1961
Enantiomerically pure bisphosphates were prepared by employing the optically active diacetals
obtained by resolution with orthoesters of ~ - m a n n o s e . [ ~ ~ ~ l
Ins( 1 ,3)P2 was synthesized by regioselective phosphorylation
of 2,4,6-tri-O-benzyl-rnyo-inositolusing chlorophosphate
2801 The 1.3-bisphosphorylated product was crystallized
from the mixture of 1.3- and 1,5-bisphosphorylated products.
Deprotection with lithium in liquid ammonia gave Ins( 1 ,3)P2.
Racemic Ins(l,4)P2 was prepared by phosphitylation of 1,2:4,5di-0-isopropylidene-nqwinositol with chlorophosphine 61,
conversion of the product into a bisphosphite ester, oxidation,
and d e p r o t e ~ t i o n . [ ~As
~ ~ Ian alternative to the resolution
of 1,2:4.5-di-O-cyclohexylidene-~1j~o-inositol
via orthoacetates,[2861formation of chromatographically separable dicamphanates gives access to the enantiomers of Ins(1 .4)P2.['"] A
short route to ins( 1,4)P2 from nip-inositol [,-camphor 2,3acetal was reported.[276.2871 D-Ins(1 ,4)P2 was also obtained
semisynthetically by periodate treatment of deacylated phos1945
B. V. L. Potter and D. Lampe
phoinositides.[2881The two enantiomers of Ins( 1 ,5)P,[2891were
synthesized from I>- and L-I ,2.4,6-tetra-U-benzyI-myo-inositoI
and 57. Resolution of the tetrabenzyl compounds obtained by
selective benzylation of 2,4,6-tri-U-benzyl-n~~~o-inositol
possible via the mono-( -)-camphanates. DL-InS(4,s)P2 was prepared from 1,2.3,4-tetra-O-benzyI-myo-inositol
by a PI1' approach.[26z.2901 Conduritol B derivatives obtained from benzene have also been used.12911
7.4. Inositol Trisphosphates
7.4.1. InositolI,4,5- Trisphosphate
Not surprisingly, most attention has been focused upon the
synthesis of Ins(1 ,4,5)P3, which is still by far the most important
inositol phosphate biologically. The first synthesis of DIns(1 ,4,5)P3 was reported by Ozaki et al. in 1986 (Fig. 16) .[2431
Fig. 17. Syntheses of Ins(1.4,5)P3 and Ins(1.4,5)PS3 by a P"' approach (Cooke et
al.) R=CH,CH2CN.
removed to give 73.Conversion to the fully protected derivative
74 required tin-mediated allylation of the 1-OH group, formation of the 4,5-acetal, and benzylation of the 2-OH group. Isomerization of the ally1 group in 74 and hydrolysis of acid-labile
protecting groups furnished triol 70,which was phosphitylated
with 61,converted into 75,and oxidized to 76.All protecting
groups were removed in one step by reaction with sodium in
liquid ammonia to give DL-InS(l,4.j)P3. When resolved D-[2'41
and L-1 ,2.4-tri-O-benzyl-myo-inositolwere employed, LIns(1 .4.5)P3[2611and D-Ins(l ,4,5)P3[2931
were obtained, respecti vely .
D- and ~-Ins(l,4,5)P,were also prepared from 79 (Fig. 18) by
phosphorylation with 48 to give 80,removal of the ketal, and
resolution of the resulting intermediate via 81.The I-hydroxyl
Fig. 16. The first synthesis of u-Ins(l,4,5)P3 (Oraki et al.)
4,5-Di-O-cyclohexylidene-m~o-inositol(65)was benzylated and the trans-4,5-acetal selectively removed to afford 66.
Allylation and acid hydrolysis of the remaining cis-acetal gave
the 1,2-dioI 67, which was resolved by conversion into the
diastereomeric esters 68.Regioselective allylation of D-67gave
69,which was benzykdted and deallylated to yield 70.Phosphorylation with 48 provided 71,and deblocking of phosphate and
hydroxyl groups with isoamyl nitrite and HJS % Pd-C, respectively, gave o-Ins(l,4,5)P3. The use of TBPP 51 or phosphoramidite 59 allowed significant improvements to this synthesis.[z92~
Racemic Ins( 1 ,4,5)P3 was subsequently synthesized[260,2 6 1 1
by modified routes to triol 701213.2141
and by employing a PlI'
approach (Fig. 17). Thus, 72 was dibenzylated and the acetals
s '
Fig. I X . Synthesis ofu-lns(l,4.5)P3utilizing mannose-orthoester resolution (Shvets
et a1.i
At1,q~w.Chrnr. I n t . Ed. Engl. 1995, 34. 1933 1972
Inositol Lipids
Phosphine 56 was proposed for the synthesis of inositol phosp h a t e ~ [ ~and
~ ' ] used to prepare Dr-Ins(l ,4,5)P3 from triol 70.
The intermediate phosphite esters were oxidized with ni-CPBA.
D-Ins( 1 .4,5)P3 was synthesized from 3,6-di-0-allyl-1J - 0 - c ~ clohexylidene-myo-inositol. The 4-OH group was benzylated
and the 5-OH group allylated to give 3.5,6-tri-0-allyl-4-0-benzyl-I ,2-O-cyclohexylidene-myo-inositol.
Hydrolysis of the I .2ketal, optical resolution via monomenthyloxyacetyl derivatives,
removal of the chiral auxiliary, dibenzylation, and deallylation
produced enantiomerically pure 70. Treatment with phosphine
58 and tBuOOH followed by hydrogenolysis gave DIns(I.4.5)P3 .[2y81
Ins(l.4,5)P3 was also synthesized by using a mixed Pll'/Pv
a p p r ~ a c h . [ ~ ~Phosphorylation
of 2,3.6-tri-0-benzyl-4,5B
31 with 47, removal of the keOBn
tal, and phosphorylation of the exposed vicinal diol gave a mixture of the 1,4- and 1,5-bisphosphates. The foriner crystallized
from the reaction mixture and was phosphitylated with
chlorophosphine 61, and the resulting phosphoramidite was
converted into the phosphate triester.[2601Deprotection afforded Iiis(1 ,4,5)P3.
3.6-Di- 0-benzyl-4,5 - bis(dibenzylphosphoryl)-myo-inositol
was employed in an approach to D-InS(1 ,4,5)Ps,
The enantiomers were resolved either as the 1 -0-monoinenthyloxyacetyl
derivatives or with a chiral HPLC column. Regioselective silylation of the equatorial hydroxyl group, benzoylation of the 2-hydroxyl group, and desilylation exposed the 1 -hydroxyl group
which was phosphitylated with PClJbenzyl alcohol. Oxidation,
hydrogenolysis, and alkaline treatment gave ij-Ins(1 .4,5)P3.
D-InS(1 ,4,5)P3 was also synthesized by using enantiomerically
pure 1,2 : 5,6-di-O-~yclohexylidene-~~~o-inositol.[~'~~
2 2 h 1 obtained either via the diastereomeric di(L-menthyloxyacetate) esters or by conversion into the diacetate or dibutyrate followed
by regio- and enantiospecific enzymatic deacylation.
The use of 2-(tev~-butyldimethyIsilyloxy)phenylacetylchloride for the selective protection of the I-OH group of 2,3-0-cyclohexylidene-myo-inositol
was the key step in another synthesis
of enantiomerically pure Ins(1 .4,5)P3 .[2yy1 Treatment of the reOBn
sulting mixture of diastereomeric esters with 1.3-dichloroFig. 1 Y . Synthesih of u-[ns(1,4,5)P, employing the xanthenyl protecting group
1.1,3,3-tetraisopropyldisiloxanegave the 4,5-O-silyl derivatives.
(Reese and Ward)
Methoxymethylation of the free 6-OH group, chromatographic
separation of the fully protected diastereoisomers. and removal
Phosphitylation with phosphine 57, oxidation, and deblocking
of acyl and silyl groups of the individual isomers followed by
gave m.-Ins( 1 .4,S)P3.[2y61 This route was also used to prepare
phosphorylation with 56/m-CPBA, and hydrogenolysis gave DD-Ins(1.4,5)P312961
by resolving diol 83 as its glucopyranosyl
and L-Ins(1 .4,5)P3, respectively.
derivative 88.l" bl
Regioselective 1-0-acylation of inositol was effected by perDiastereoisomeric camphanate derivatives of 2,3 :4,5-di-0borylation, transmetalation with bis(acety1acetonato)-di-ncyclohexylidene-6-0-benzyl-m~~o-inositol,
synthesized by selecbutyltin, and treatment of the dibutyltin intermediate with (-)tive benzylation of the corresponding racemic 1 , 6 - d i 0 1 , [ ~were
menthyl c h l ~ r o f o r m a t e .Separation
[ ~ ~ ~ ~ of the diastereoisomers
used to prepare D- and ~-6-0-benzyl-2,3-0-cyclohexylidene- by crystallization and treatment of the D isomer with 2,2/nyo-inositol. Phosphorylation of the tripotassium salts of these
dimethoxypropane gave D-1 -0-menthyloxycarbonyl-2.3: 4.5-difollowed by deprotecintermediates using TBPP 51[24y,270,2801
0-isopropylidene-rrzyo-inositol. Benzylation of the 6-OH group,
tion gave the enantiomers of Ins(1 .4,5)P3 .[25'.2 y 7 1
cleavage of the ester. and selective removal of the 4,5-acetal
A short synthesis of racemic Ins(1 ,4,5)P3 from 3.6-di-O-benafforded, after phosphorylation and hydrogenolysis, Dzoyl-inyo-inositol was reported.[2551Partial phosphitylation
Ins( 1 ,4,5)P3.
with chlorophosphite 52. acetylation of the 2-OH group, and
An elegant short route to D-Ins(1 .4,5)P3 was devised,[2871
emoxidation gave predominantly the 1,4,5-tris(dimethyl phosploying 2,3-0-(~-1,7,7-trimethyl[2.2.l]bicyclohept-2-ylidene)phate).
nzyo-inositol (89) (Fig. 20),[30'1 prepared from inositol and
Ins( 1,4,5)P,.
D-camphor dimethyl acetal by way of a precipitation-driven
groups of the cnantiomeric 1,2-diols obtained were then selectively phosphorylated with 48 to give 82. Successive removal of
N-phenyl- and benzyl protecting groups yielded the respective
enantiomers of Ins( 1,4,S)P3.[2y41Other phosphorylating methods are now preferred.['"]
Cyclopentylidene acetal 84 (from 83, Fig. 19) was employed
in a different approach in which silylation of the 1- and 4-hydroxyl groups afforded 85 and reaction with 2.7-dibromo-9phenyl-9-xanthenyl chloride gave triol 86 after desilylation.
B. V. L. Potter and D. Lampe
cyclic carbonate 94 derived from this diol was stereoselectively
oxidized to the a-epoxide 95. Regioselective ring opening of 95
with benzyl alcohol gave 96 and benzylation of the free hydroxyl
group afforded 97. Hydrolysis of the 1,2-carbonate, epoxidation
of the double bond, and reprotection of the cis-1.2-diol as an
isopropylidene acetal gave 98. The 8-epoxide was opened with
99 to give the protected inositol 100. Debenzylation and phosphorylation with TBPP afforded 101, which was deprotected to
give racemic Ins(1 ,4.5)P3. D- and ~ - I n s ( 1 , 4 , 5 ) P , [were
~ ~ ~ob~
tained by opening epoxide 95 with (R)-( )-src-phenethyl alcohol, giving diastereoisomers 102a and 102 b. which were separable by HPLC (Fig. 21 b).
Benzene was also used as starting material in a synthesis of
racemic Ins(1 ,4,5)P3 (Fig. 22) .["ll
trans-I ,2-Dihydroxycyclo-
O/N\ p ' 3 0
Fig. 30. Short route to u-lns(1,4.5)P3via camphor acetal 90 (Salamoncryk and
equilibrium reaction.[2751Reaction of 89 with five equivalents of
pivaloyl chloride afforded 90,which was benzylated to provide
91. After removal of the pivaloyl esters, phosphorylation with
phosphepane 60, and oxidation, 92 was obtained. which was
deblocked to give D-Ins( 1 ,4,5)P3.
A conceptually different approach was developed by Ley et
al.,i20'. 3023 who took advantage of the observation that microbiological oxidation of benzene by Pseudomoms putida affords
cis-I ,2-dihydroxycyclohexa-3,5-diene(93) (Fig. 21 a) .13031 The
p n
Fig. 22. Access to Ins(1.4,5)P3
from benzene via conduritol B derivative 107 and
with photooxidation of 103 as a key reaction (Carless and Busia).
Fig. 21. Approach to ri-Iiis(1.4.5)P., employing microbial oxidation of benzene
(Ley et al ): a ) synthesis of Ins(1.4.5)P3.
b) reaction of intermediate 95 giving
diastereomers separable by HPLC.
hexa-3J-diene (103) was prepared in 40% overall yield by Birch
reduction, bromination, frans-hydroxylation. acetylation, and
dehydrobromination. Protection of the hydroxyl groups as
methoxyethoxymethyl (MEM) ethers and [4 + 21 addition of
singlet oxygen furnished endoperoxide 104, which was reduced
stereospecifically to 105.[3041
Inversion at C-I by an oxidationreduction sequence gave the conduritol B derivative 107. Benzylation of 107 and cis-hydroxylation of the double bond afforded
108. which was regioselectively methoxyethoxymethylated to
give 109. Benzylation and removal of the M E M groups furnished 70, and, after phosphorylation with TBPP and hydrogenolysis. Ins( 1 ,4.5)P3.
Falck and Yadagiri used (-)-quinic acid (43) to prepare DIns( 1 .4,5)P3(Fig. 23) . [ 3 0 5 1 A four-step conversion gave ester 10.
Sequential protection of the I-hydroxyl group as a (1(trimethylsilyl)ethoxymethyl(SEM) ether, reduction of the ester, and selenylation of the resulting primary alcohol furnished
1 I I . Stereoselective [2.3] sigmatropic rearrangement of the
allylic selenoxide derived from 1 I 1 and benzylation generated
112 as a single stereoisomer. Transformation to silyl enol ether
Angcn.. C/7cm 1 n f . Ed. G i g / . 1995. 34. 1933-1972
lnositol Lipids
Other preparations of optically active13"'. 3')81 In s( 1,4,5)P3
were also published. including the synthesis of 'H-labeled Dand ins( 1 ,4,5)P3 by reduction of a protected inosose with
7.4.2. Other Inositol Tvisphosphates
Fig. 73 tnmtiospecilic synthesis of wlns(i,4,5)P, from (-)-quinic
(Falck and Yadagiri)
acid (43)
113 by ozonolysis and treatment with TBDMS triflate proceeded with nearly complete regiospecificity. Hydroboration of the
enol ether followed by oxidation furnished alcohol 114. which
on desilylation gave 115. TBPP phosphorylation and hydrogenolysis afforded D-InS(l ,4.5)P3.
Tegge and Ballou employed D- and L-chiro-inositol, derived
by demethylation of 1D-3-O-methyl-chiro-inositol (42) and L quebrachitol (lo), respectively, to obtain both enantiomers of
Ins( 1 ,4,5)P3 (Fig. 24).[3061 Formation of the cis,cis-dicyclohexylidene acetal 116 (from D-chiro-inositol) . benzylation, hydrolysis. and benzoylation gave mainly 117. Inversion of the
configuration at C-I via the triflate afforded 118, which was
debenzylated to give 119 and then converted into D-Ins(1 ,4,5)P3
by employing phosphine 56. The L isomer was synthesized similarly from i.-c,/ziro-inositol.
Fig. 2 1 Sgnthcqs of 1,-lns(1.4.5)P, from lo-3-0-methyl-~/~ir~~-inositol
(42) (Tegge
and Biillou)
i>-Ins(l:2-cyclic 4,5)P3 was synthesized by treatment of
50,["'] and (in low yield) from D-InS(l ,4,5)P3 by using l-ethyl3-(3-dimethylarninopropyl)~arbodiimide.[~'~~
In the latter reaction the product was isolated by HPLC and was shown to be
converted to Ins(1 ,4,5)P3 by treatment with acid.
Racemic Ins(2.4.5)P3 was prepared from 4.5-di-O-allyl-3.6di-0-benzyl-nzyo-inositol,which was selectively benzylated
at the I-position, deallylated, and phosphorylated with
TBPP.[250.l 5 DL- Ins(2.4,5)P3 was also obtained from benzene
via conduritol B
The synthesis of a fully protected. phosphorylated, and enantiomerically pure precursor of
~-Ins(2,4,5)P,was reported, but the final deprotection step was
not achieved.I3l 'I The same authors did. however, prepare
i>-Ins(2,4,5)P3 by phosphorylation of 3.6-di-0-benzyl- I .2-0cyclohexylidene-m>v-inositol with TBPP. removal of the acetals, and resolution via the diastereomeric 1 -menthyloxyacetates. The equatorial hydroxyl group was regiospecifically silylated and the 2-hydroxyl group was phosphorylated by treatment with PClJbenzyl alcohol followed by / BuOOH oxidati~n.['~
~ ] D- and ~-Ins(2,4,5)P,were prepared from L- and
D-1,3,4-trI-O-benzoyl-n?yo-inositol,respectively.13 21 which
were generated in turn from D- and ~ - ( . h i r ( ~ - i n o s i t o l . ' ~ ~ ~ '
Ins( I ,3.4)P3 was synthesized from 2,4.5-tri-O-benzyI-rnyoinositol, prepared from the corresponding 1.2 :4,S-di-O-cyclohexylidene acetal. Phosphitylation with chlorophosphine 54,
followed by oxidation and deprotection gave racemic
Ins( 1,3,4)P3,['571which was also prepared from the same precursor by using TBPP 5 1 ~ 2 s 0 ~ 2and
s 1 1 phosphine 56.[2s81
D-lns( 1 ,3,4)P3 was prepared by resolution of 5.6-di-0-benzyl3.4-di-0-p-methoxybenzyl-myu-inositol
using a chiral HPLC
column o r via the diastereomeric l-menthyloxyacetates.12211
Optically active 1,2:5,6-di-O-cyclohexylidene-m~o-inositol,
obtained either by an enzymatic process or by conversion into the
was used in another synthesis of
D-InS( 1 .3.4)P3 via ~-2,5,6-tri-O-benzyl-~?~~-in~~sitol.""~
Finally, Boehm and Prestwich synthesized both enantiomers of
Tns(1,3,4)P3 as well as radiolabeled [1-3H]-lns(l.3,4)P3
2.4.5-Tri-0-benzyl-inositolwas converted to the isopropylidene
ketal and resolved by means of diastereoisomeric (-)-camphanates. Saponification and oxidation of the alcohol gave the
I-inosose, which was reduced with [3H]NaBH, to give the alcohol with the equatorial OH group as the major product. Phosphorylation using TBPP followed by deprotection furnished D[1-3H]-Ins(l,3.4)P3. Despite the availability of Ins( 1 ,3,4)P3,
there has been some doubt about its Ca*+-niobilizing activity.
An unambiguous synthesis of the enantiomers of this compound has now been published.[3141
A synthesis of Ins( 1,4,6)P3[31s1relied upon diallylation of
1,2: 4,5-di-O-isopropylidene-m~winositoI.removal of the transketal, and tin-mediated p-methoxybenzylation of the 4,5-diol,
which gave mainly the desired 6-alkylated derivative. Benzyla1949
B. V. L. Potter and D. Lampe
tion of the 5-OH group, acid hydrolysis of the cis-ketdl, benzylation of the 1- and 2-OH groups, isomerization of the allyl
ethers, and removal of the resulting prop-I-enyl as well as the
p-methoxybenzyl groups gave the 1,4,6-triol, which was phosphorylated with 56/tBuOOH. Ins(1 ,4,6)P3 was also prepared by
another research group both as a racemate and in optically
I \
7.5. Inositol Tetrakisphosphates
Ins(1,3,4.5)P4 was first synthesized in racemic form from the
symmetrical inositol orthoformate 120 (Fig. 25). Chelationcontrolled monoallylation of 120 gave 121, which was benzylated to give 122. The allyl and orthoformate groups were removed
to furnish tetrol 123, which was phosphorylated with TBPP to
give 124 and Ins(1 ,3,4,5)P4 after catalytic hydrogenation.r2821A
similar route was adopted by another group[250'except that the
benzyloxymethyl group was used instead of the allyl group for
initial hydroxyl protection and a combined report has appeared.[280]2,6-Di-O-benzyl-myo-inositol
(123) was also used
for two P"' approaches to Ins(1 ,3,4,5)P4, employing either
60[3171or 56[2581as phosphitylating agents.
Fig. 25. Synthesis of Ins( chelation-controlled monoallylation of m w
inositol orthoformate (Billington et al.).
Orthoformate ester protection of myo-inositol was also a key
step in a synthesis by Vasella et al., which led to both enantiomers of Ins(1,3,4,5)P4 (Fig. 26).[2231 Benzylation of the
silylated orthoester 125[2811
gave the racemic monobenzyl compound 126, which was reacted with (R)-(
+)-I-phenylethyl isocyanate to give diastereoisomeric carbamate derivatives, which
could be separated as 127a and b after removal of the silyl
group. Subsequent benzylation of each diastereoisomer followed by removal of the carbamate and orthoformate groups
gave the enantiomers of 123, which were phosphorylated
with 56/m-CPBA and hydrogenolyzed to give D- and LIns( 1,3,4,5)P4. The enantiomeric dibenzylinositols 123 could also be derived by enantioselective monodeacylation of the symmetrical dibutyrate 128 formed by acylation and desilylation of
125. Thus, treatment of 128 with porcine liver esterase (PLE)
gave monobutyrate 129 in > 95 Yo enantiomeric excess. Dibenzylation, deacylation, and removal of the orthoester gave the
precursor (L-123)for L-Ins( 1 .3,4,5)P4. The D isomer was eventually obtained from 129 by a protection-deprotection sequence
7.5. I . Inositol1,3,4,5Tetvakisphosphate
; &H
Fig. 26. Chemoenzymatic route to the enantiomers ofIns(1.3,4,5)P4(Vasella et al.)
via 130 and 131. Surprisingly, ~-Ins(1,3,4,5)P, is a higher affinity ligand than D-InS(f ,3,4,5)P4 at Insf1,3,4,5)P4 binding sites in
pig cerebellum, and this difference in affinity is greater than that
between L-Ins(l ,4,5)P3 and D-Ins(l,4,5)P3 at the Ins(l,4,5)P3
Meek et al.12551employed a base-catalyzed isomerization of
the I-benzoyl group in the readily available myo-inositol 1,4dibenzoate to give inter aha the 2,4-dibenzoate. Phosphitylation
with chlorophosphane 52, oxidation, demethylation, and alkaline hydrolysis gave racemic Ins( 1,3,4,5)P4.
D-InS(1,3,4,5)P4 was synthesized by resolution of 3,4,5-tri-Obenzoyl-6-O-benzyl-m~~o-inositol,
prepared from the 1.2 :4,5-di0-cyclohexylidene ketal by selective benzoylation of the 3-OH
group, benzylation of the 6-OH group, removal of the trans-ketal, benzoylation of the 4- and 5-OH groups, and finally hydrolysis of the cis-1,2-ketal. Resolution by means of the monomenthyloxyacetyl derivatives and benzylation of the desired
diastereoisomer followed by deacylation afforded D-123,which
was phosphorylated with TBPP and deblocked to give DI n ~ ( 1 , 3 , 4 , 5 ) P , . ~ D-Ins(1
~ ~ ~ ] ,3,4,5)P4 was also prepared from
which was resolved
via its monomenthyloxyacetyl derivative. Chemoselective tinmediated allylation of the equatorial hydroxyl group, followed
by benzylation of the 2-hydroxyl group and deallylation gave
tetrol 123, which was phosphitylated with phosphine 58.[2981
D-1,2 :5.6-Di-O-cyclohexylidene-myo-inositol,obtained by
chemoenzymatic means, served a starting material for another
~'synthesis of D-InS(l .3,4,5)P4 via ~ - 1 2 3 . ' ~2261
A short route devised by Ozaki et a1.r2221
involved selective
benzoylation of inositol. The 1,3,4,5-tetrdbenzoate was isolated
as major product in 34% yield. Benzylation with benzyl trichloracetimidate and debenzoylation afforded the 2.6-protected
inositol, which was phosphitylated with phosphoramidite 59.
Resolution of the tetrabenzoate by means of chiral column
A n g m . Chwn. Int. Ed. Engl. 1995, 34. 1933-1972
Inositol Lipids
chromatography gave access to enantiomerically pure
Ins( 1 ,3.4,5)P4. The same group described the enantioselective
acylation o f inositol derivatives using tartaric acid monoester.’”’’ Thus. ‘I .3.5-tri-O-benzoyl-nz~~o-inositol,
from direct benzoylation of inositol, was transformed enantioselectively into the 4-tartrate and the 2- and 6-OH groups
were protected as silyl ethers. Separation of the fully protected
diastereoisomers followed by deacylation with ethylmagnesium
bromide. phosphorylation, and hydrogenolysis afforded 11Ins(i .3.4.5)~,.‘”*1
7.5.2. Other. Inositol Tetvakisphosphates
Racemic Ins( 1,4,5,6)P, was synthesized from the 1,2-O-isopropylidene ketal of rnyo-inositol by phosphitylation with 52
followed by hydrogen peroxide oxidation. Demethylation was
accomplished with bromotrimethylsilane, since HBr in acetic
acid led to premature hydrolysis of the ketal and phosphate
migration.[2s5’ DL-InS( I,4,S,6)P4 was also prepared by phosphitylation of the corresponding 1,2-ketal with chlorophosphine 61
i>-Ins(l,4,5.6)P4 was obtained by resolution via
the 0-camphanylidene-myo-inositol ~ i s - m o n o a c e t a l s . [ ~ ~ ~ ~
Ins( I.3,4.6)P4 was prepared by treating inositol with 1,3dichloro-l ,I .3.3-tetraisopropyldisiloxane,which gave the symmetrical 1.6 : 3.4-O-bis(disiloxanylidene) regioselectively. Benzoylation and removal of the siloxanes with H F produced the
2,5-dibenmate. which was phosphitylated with phosphoramidite 59.‘2x31
w-lns( 1 ,2.4.5)P4 was previously synthesized by benzoylation
of 1.2: 4.S-di-O-cyclohexylidene-m~~o-inositol,
hydrolysis of the
ketal, phosphorylation with 52/hydrogen peroxide. and stepwise deblocking.[2ss51
A similar strategy in a more recent synthesis employed the isopropylidene ketal and 53 as phosphitylating
agent.‘”h1 Conduritol B derivatives obtained from benzene
were also used to prepare Ins( 1 .2,4,5)P, ,[2y’1 and L-quebrachitol
served 21s starting material in a synthesis of ~-chiroIns(I . 2 , 3 . 5 ) ~ , . 1 ~ ~ ~ 1
age of the T H P ether. phosphorylation, and hydrogenolysis furnished ~-2-deoxy-Ins(l)P.[2761
By using 4,5-di-O-benzoyl-3,6-di-O-benzyl-I.2-O-cyclohexylidene-myo-inositol. ~~-2-deoxy-Ins(
1.4,5)P3 was preHydrolysis of the ketal was followed by tin-mediated
monomethoxymethylation of the 1-hydroxyl group. Deoxygenation at C-2 was accomplished by first oxidizing the hydroxyl group to give the inosose, which was then converted into the
tosyl hydrazone and reduced. The resulting hydrazine was removed with sodium acetate. Cleavage of MOM and benzoyl
protecting groups gave the 1.4.5-triol. which was phosphorylated with TBPP and deblocked.
u-3-deoxy-Ins(1 ,5,6)P3[325.3261 were prepared from ~-3-deoxy-r)z~~o-inosito~
(viburnitol). which was obtained from 0-diisopropylidene-Lquebrachitol, by Barton deoxygenation of the free hydroxyl
group, and removal of all protecting groups uith boron tribromide. On treatment with 2-methoxypropene;acid, viburnitol
gave a mixture of the 1,2:4.5- and the 1,2:5.6-di-O-isopropylidene derivatives. After benzylation, removal of the trms-ketals.
and benzoylation, the two regioisomers could be separated by
chromatography. Cleavage of the ketal of 4.5-di-O-benzoyl-6O-benzyl-3-deoxy-1,2-O-isopropylidene-~~~~~-inositol
by benzoylation ofthe I-hydroxyl group, protection of the axial
hydroxyl group as its ethoxyethyl ether. and removal of the
benzoate esters furnished the 1,4,5-triol, which was treated with
TBPP and deblocked to give ~-3-deoxy-Ins(1 .4,5)P3. ~ - 3 - D e oxy-Ins( 1 .S,6)P3 was prepared analogously from the other regioisomer.
.4,S)P3 133 (Fig. 27) was synthesized by utilizing precursors derived from benzene by microbial oxidation.[201.3271
The key step was the selective ring opening of the
8. lnositol Phosphate Analogues
Now that the second messenger properties of Ins( 1 ,4,5)P3 are
well established, attention has turned to the synthesis of inositol
phosphate analogues, which are hoped to have novel biological
properties. There is currently considerable interest in the concept of pharmacological intervention in cellular signaling pathways. If suitable cell-permeable receptor antagonists and enzyme inhibitors can be synthesized, some of these compounds
may have potential therapeutic value.
8.1. Deoxy Analogues
The racemic 2- and 6-deoxy analogues of Ins(1)P were synthesized in the search for inhibitors of inositol monophosphatase[32.51 (see Section 9). ~-2-Deoxy-Ins(l)Pwas prepared
by employing n-3,4,5.6-tetra-O-benzyl-myo-inositol,
which was
transformed into the 1-tetrahydropyranyl (THP) ether and
deoxygenated at C-2 by the Barton-McCombie method. Cleav-
132: X H
136: X -Me
138: X OMe
135:X F
137:X Me
Fig. 27. Synthesis of C-6-modified Ins(1,4.5)P, analogue5 via epowide 99
(Ley et al.).
intermediate epoxide 98 with LiAIH,. giving predominantly the
6-deoxy-myo-inositol derivative 132. ~-6-Deoxy-Ins(l.4,5)P~
was prepared from D-galactose by the Ferrier reaction. Interactions of this inositol phosphate with the Ins( I,4,5)P3 receptor
and metabolic enzymes were
although details of the
synthesis remain to be published.
Syntheses of 1~-2,3-dideoxy- and 1~-2,3,6-trideoxy-Ins(1 ,4,S)P3 from 1D-1,4,5-tri-O-benzoyl-6-O-benzyl-3-deoxymyo-inositol, an intermediate in the preparation of 11-3-deoxyIns(1 ,4.S)P3, were described.[326’ Earlier. racemic 2,3,6trideoxy-Ins(1 .4,S)P3 ( = DL-cyclohexane 1.2.4-trisphosphate)
had been synthesed by another
B. V. L. Potter and D. Lampe
8.2. Fluorinated Analogues of Inositol Phosphates
2-F-scyl/o-Ins( 1,4,S)P3
and 2,2-F2-1ns(1,4,S)P3
were both synthesized from 1-0-allyl-3,6-di-0-benzyl-4,S-O-isopropylidenemyo-inositol. Treatment with DAST resulted in monofluorination at the 2-position with inversion of configuration. and the
gem-difluoro derivative was obtained after oxidation to the 2inosose. Deblocking of the I-. 4-, and 5-OH groups, phosphorylation with TBPP, and hydrogenolysis gave 2-F-scylloIns( I ,4,5)P3 and 2,2-F2-Ins( 1.4,5)P3,
of these compounds by a similar approach have also been published.[’91~
3301 The enantiomers of 3,6-di-O-benzyI-2-deoxy2,2-difluoro-4,5-isopropylidene-myo-inositoI
were resolved by
separation of the camphanates. thus giving access to D- and
-. .
The protected scyllo-inositol 140 was used to prepare 2-FIns(l,4,5)P3 146 (Fig. 28).[3331Isomerization of the ally1 group
Fig. ?Y. Synthesis of optically active .i-F-lns(l.4,5)P,
152 (Kodkowski et al.)
Fig 28. Synthesis of 2-F-Ins(1,4.5)P,146 (Lampe and Potter)
of 140 gave the prop-I-enyl derivative 141. whose free 2-hydroxyl group was triflated giving 142. Displacement of the sulfonate with tetrabutylammonium fluoride furnished the fluoroinositol 143. Removal of acid-labile protecting groups followed
by treatment of the resulting trio1 144 with 56/tBuOOH gave 145
and, after deprotection, 146. An earlier attempt to synthesize
2-F-Ins(l .4,5)P3
from 140 using DAST failed because the reaction unexpectedly proceeded with retention of configurati~n.[~’~]
Treatment of D-3-deoxy-3-fluoro-myo-inositol
(18) (obtained
from L-quebrachitol as above) with 2-methoxypropene/camphorsulfonic acid (cat.) produced a mixture of the 1,2: 5.6- and
the desired 1,2:4,5-di-O-isopropylidene
acetal 147 (Fig. 29).
The latter was benzylated to 148, and cleavage of the frnizs-aceta1 followed by benzoylation gave 149. Hydrolysis of the remaining acetal. selective benzoylation of the equatorial hydroxyl group, and protection of the free axial hydroxyl group as its
ethoxyethyl derivative afforded 150. Removal of the benzoate
groups and phosphorylation with TBPP gave 151, which was
deblocked stepwise to yield ~-3-F-Ins(l.4,5)P3 152.[3341The
synthesis of ~-3-C-(trifluoromethyI)-Ins(1.4,S)P3 was recently
described by the same group.[’Z21
Racemic 6-F-Ins(l .4,S)P3 135 was accessible from benzene by
microbial oxidation via epoxide 98 (Fig. 27).120‘.3271R’1%
opening with LiAIH, gave mainly 134. Debenzylation, phosphorylation with TBPP, and deprotection with trimethylsilyl
bromide gave 135.
was employed
in syntheses of ?-F-scyllo-lns( 1,3,4)P3 and 2,2-F2-Ins(1 ,3,4)P3
Reaction with DAST proceeded with inversion of
configuration introducing an equatorial fluoro substituent.
Deallylation, TBPP phosphorylation, and hydrogenolysis afforded 2-F-scl,~No-Ins(l,3,4)P3.
The difluoro derivative was obtained similarly by fluorination of the 2-inosose. Synthesis of the
myo-inositol derivative was also attempted by inverting the configuration at C-2 prior to fluorination; however, the fluorinated
myo-inositol intermediate could not be selectively deallylated.13 131
8.3. Ring-Modified lnositol Phosphate Analogues
The versatile benzene-derived epoxide 98 (Fig. 27) was employed in syntheses of racemic 6-deoxy-6-methyl-Ins(I ,4,S)P3
137 and 6-0-methyl-Ins( 1 .4,S)P3139.1201.
3 0 2 , 3 2 7 1 Whereas ring
opening with methanolic sodium methoxide afforded mainly
136, the precursor for 137, reaction of 98 with lithium dimethyl(cyano)copper(i) gave 138, which can be used to prepare 139.
1.4,S)P3was also prepared and evaluated by
an 0th er group .I3
An Ins(1 ,4,S)P3
analogue with an equatorial 2-hydroxyl
group, DL-.wy/h-Ins( 1,2,4)P,, was prepared from 1-0-allyl-3,6di-O-ben~yl-4,S-O-isopropylidene-myo-inositol~~~~~
by trifla-
lnositol Lipids
tion of the 2-hydroxyl group followed by displacement of the
triflate moiety with cesium acetate.
Syntheses of chiro-1ns(2.3,5)P3 159, an Ins(1,4,5)P3 analogue
with inverted configuration at C-3. were reported by two
groups. Raceniic 159 (Fig. 30) was prepared from the conduri-
105: R H
- d:
dB I X ~Mb 8 n E
( W( Z R
o ) ~ O ~ ( O R ) ,
163: X = S
164: X = S
Fig. 31. Synthesis of L-~/iiru-Ins(l.4.6)P,167. L-[,/liro-Ins(7.3.j)f, 15Y. and their
trisphosphorothioate analogues from L-quehrachitol (Liu a n d Potter).
Fig. 30. Photoowid:ition approach to 1~~-~hiro-inosilol-2.3.5-trisphosphate
(Carless a n d Busia).
Other C-3-modified analogues reported include ~-3-chloro-.
~ - 3 - b r o m o - and
~-3-O-methyl-Ins(l,4.5)P3 .I3 Recently, the
first synthesis of an amino analogue of Ins(1 ,4.5)P3,D-3-amino3-deoxy-Ins( 1 ,4,5)P3, was published.r3391
to1 F derivative 105 (obtained from benzene, see Fig. 22), which
was benzylated to 153 and cis-hydroxylated to give the chiro8.4. Phosphorothioates
inositol derivative 154. Selective protection of the equatorial
hydroxyl group as its methoxymethyl ether (155) followed by
Phosphorothioate analogues of nucleotides have proved to be
benzylation gave the fully protected 156. Acidic hydrolysis furinvaluable in studies in mechanistic enzymology and molecular
nished 157, which was phosphorylated with TBPP to
biology[3401and in examining the stereochemistry of enzymatic
phosphoryl transfer
give 158, and hydrogenolysis gave DL-159.[3041 L-chiro3411 and phosphorothioate
Ins(2.3,5)~,["-1'.3 3 6 1 was synthesized from L-chiro-inositol
analogues of the established second messengers CAMP and
cGMP have been available for some time. Indeed, it should be
(Fig. 31 ). A selective tin-mediated benzylation yielded the key
noted that phosphorothioate substitution has generated the
intermediate 160 as the major product. Perbenzoylation and
cleavage of the benzyl ethers furnished 161. which was phosonly known competitive CAMP antagonists from the manhundreds of CAMP analogues, (&)-adenosine cyclic-3'.5'-monophosphitylated with either chlorophosphine 53 or phosphine 55.
phorothioate and the corresponding phosphorodithioate. The
Oxidation gave 162, which was deblocked to yield 159. The
corresponding thiophosphate 164 was prepared by sulfoxidaapplicability of these analogues to the inositol phosphate field
tion of the trisphosphite ester of I61 to give 163 followed
sterns from their resistance to phosphatase-catalyzed degradaby deprotection. Phosphitylation of intermediate 160 and
t i ~ n . ~ Already
~ ' ~ ] inositol phosphorothioates are emerging as
(su1f)oxidation of the resulting trisphosphite ester gave 165 and
the first partial agonists at the Ins(1 .4.5)P3 receptor (see Section
166. respectively, which were deblocked to afford ~-chiuo11. I ) . Synthesis of phosphorothioate analogues also provides
Ins( 1,4,6)P, 167 and its trisphosphorothioate analogue L-chiroan excellent method to introduce " S radiolabels. Since sulfoxiIns( 1.4,6)PS3 168.[3371
dation is usually the final synthetic step before deprotection
Carless and B u ~ i a [ ~reported
the synthesis of ~ ~ - 6 - F - c h i r o - and can be effected with elementary sulfur, the handling of
Ins(1.3.5)P1employing the cis-1.4-diol 105 (see Fig. 21). Benzylradioactive material can be kept to a minimum. Thus, Dation and cis-hydroxylation gave 1,4-di-O-benzyl-2,3-di-O- [3sS]Ins(1.4.5)PS3 was synthesized and used for enzyme[342]and
methoxyinethyl-c,hiro-inositol. Selective methoxymethylation of
receptor binding
in which it was found to label two
the equatorial hydroxyl group and DAST fluorination (which
different sites, the Ins(1 ,4,5)PJ receptor and possibly a different
proceeded with retention of configuration) furnished the fully
conformation of the receptor. Furthermore, since sulfur is a
protected fluorinated chiro-inositol. Hydrolysis of MOM
better nucleophile than oxygen, the selective attachment of reethers, TRPP phosphorylation. and hydrogenolysis gave DL-6porter groups such as fluorescent labels to phosphorothioates in
the presence of phosphates is possible.[344]
B. V. L. Potter and D. Lampe
8.4.1. Phosphovothioate Analogues of Inositol Mono- and
reaction with phenacetyl disulfide featured in another synthesis
of Ins( 1 ,4,5)P3-5S.[2531
Ins(1 .4,5)P3-1S was synthesized from 1-0-alIyl-2,3,6-tri-ODL-myo-Inositol 1-phosphorothioate [Ins(l)PS] was initially
by isomerization of the allyl group to give
obtained by treatment of inositol 1,2,4,5,6-pentaacetate with
-enyl derivative, which was then 4 3 thiophosphoryl chloride followed by ester hydrolysis.[2781A
P'l' chemistry. Removal of the propsynthesis of racemic as well as enantiomerically pure
I-enyl ether and phosphitylation of the I-hydroxyl group
Tns(l)PS[". 34s1 relied upon phosphitylation of the correspondfollowed by sulfoxidation and deprotections gave Ins(1 ,4,5)P3ing penta-0-benzylinositol (which could be resolved via camIns(1,4,5)P3-1S, which was used in the preparation of an
phanates) with phosphoramidite 61 and sulfoxidation. LIns(l
analogue carrying a fluorescent reporter group, was
Ins( 1)PS was also prepared by another
optically active form by employing ID-l-O-allyIDirect thiophosphorylation of, for example, DL-1,4,5,6-tetra2,3,6-tri-0-benzyl-rn~~o-inositol,[~~~~
DL-Ins(1 ,4,5)P3-lS was
0-acetyl-myo-inositol with thiophosphoryl chloride afforded a
reagent and
1 : 1 mixture of the endo and ex0 diastereoisomers of the protectphenacetyl
ed 3,2-cyclic-phosphorothioates,which was separated by reOther
D-6-deoxyverse-phase flash chromatography. Deacylation and precipitaIns(1 ,4.5)PS3 and ~-cl?iro-Ins(2,3,5)PS,[~~~.
3 5 2 1 as well as
tion with potassium chloride produced endo- and exo-rx-myoIns(l,3,5)PS3,
inositol 1 :2-~ycli~-phosphorothioate.[~~~~
A different approach
A different approach to the preparation of phosphorothioate
relied upon the monophosphitylation of 1,4,5,6-tetra-O-benzyIanalogues of Ins( 1 ,4,5)P3 was adopted by thiophosphorylating
myo-inositol at either the 1- or 2-position using 61, followed by
PtdIns and PtdIns(4)P using kinases in human erythrocyte
cyclization of the phosphoramidites to the e m - and mdoghosts and A T P Y S . [ This
~ ~ ~method
can naturally be adapted to
methoxyphosphites using tetrazole. Sulfoxidation gave a mixture
compounds. Thus,
of the respective protected diastereoisomeric phosphorothioates
produced 35Swhich were separated by chromatography and d e b l o ~ k e d . ~ ~ ' ~ ]
in both the
I n ~ ( l , 4 ) P S , [ ~and
~ ~ l n ~ ( 4 . 5 ) P S , [were
~ ~ ~ obtained
from DL4and
of phos1,2:4,5-di-O-isopropylidene-and 1,2,3,4-tetra-O-benzyI-myopholipase
give a
inositol, respectively.
mixture of inositol 1,4-bi~phosphate-5-[~~S]phosphorothioate
and inositol 1-phosphate 4.5-[35S]bisphosphorothioate,
8.4.2. Phosphovothioate Analogues of tnositol Trisphosphates
was shown to be resistant to 5-phosphatase.
nzyo-Inositol 1,4,5-trisphosphorothioate[Insfl .4,5)PS3, 781
(Fig. 17)[3481was the first Ins(l,4,5)P3 analogue reported and
8.4.3. Phosphovothioate Analogues of tnositol
has proved to be a valuable tool in the elucidation of the bioTetvakisphosphates
chemical role of Ins(1,4.5)P3 (see Section 12). Ins(l,4,5)PS3 was
prepared from triol 70, which was converted into the trisphosIns(1 ,3,4,5)P4-5S was obtained by phosphitylation of 2,6-diphoramidite by reaction with chlorophosphine 55 and then into
0-benzyl-I ,3,4-tris[(dibenzoxy)phospho]-rnyo-inositol with 56
trisphosphite 75 on reaction with tetrazole and 2-cyanoethanol.
and in situ sulfurization to produce the fully protected
Sulfoxidation to 77 and deprotection produced the trisphostns( 1,3,4,5)P4-5S analogue. Debenzylation yielded the 5-phosphorothioate 78. A second synthesis[2s31also employing triol 70
phorothioate derivative.[2s31
Ins(1,3,4,5)P4-3S 176 was prepared from 169 (Fig. 32). Hyinvolved phosphitylation with 56 to give the intermediate
drolysis of the ketals and tin-mediated para-methoxybenzylatrisphosphite triester. Sulfurization with phenacetyl disulfide
tion of the I-OH group gave 170, which was converted into 171
produced the phosphorothioate which was deblocked to
by simultaneous protection of the 4- and 5-OH groups as the
provide Ins(l,4,5)PS3.
isopropylidene ketal and benzylation of the 2-OH groups. Re2,3,6-Tri-0-benzyl-4,5-O-isopropylidene-~yo-inositol was
moval of acid-labile protecting groups and isomerization of the
used in syntheses of both the 5-phosphorothioate and the 4.5allyl ether gave 172, and phosphorylation gave the 1,4.5-trisphobisphosphorothioate analogues of Ins( I ,4,5)P3, Ins(1 ,4,5)P3sphate triester 173. Hydrolysis of the prop-1-enyl ether fur5S,[2403
2421 and Ins(l,4.5)P3-4,5S,.
Treatment of this intermedinished 174. Thiophosphorylation of the free 3-hydroxyl group
ate with phosphorochloridate 47 resulted in phosphorylation of
gave 175 and deblocking in sodium/liquid ammonia afforded
the 1-OH group. After removal of the ketal, phosphorylation
Ins(l ,3,4,5)P4-3s 176,[3551a potent inhibitor of Ins(1,3,4,5)P4
with 47 afforded a mixture of the 1.4- and 1,5-bisphosphate
3 - p h o ~ p h a t a s e . [ The
~ ~ ~ ]synthesis of enantiomerically pure
triesters, from which the 1,4-bisphosphate triester crystallized.
D-Ins( 1,3,4,5)P4-3s from L-quebrachitol was also reported.[3571
Phosphitylation of the free 5-hydroxyl group using chlorophosphine 61, sulfoxidation, and deblocking gave Ins(1 ,4,5)P3-5S.
Alternatively, the 4,5-diol obtained after acetal hydrolysis could
8.5. Phosphonate Analogues
be phosphitylated with 55 or 56, sulfoxidized, and deblocked to
give Ins( 1.4,5)P3-4,5Sz. The 4,5-bisphosphorothioate was used
The monoammonium salt of benzylphosphonic acid was
in the synthesis of the 4,5-cyclic pyrophosphate, obtained by
employed in the synthesis of inositol I ,4,5-tris(hydrogenphosdesulfurization with N-bromosuccinimide (NBS).[3491Phosphonate). Thus, phosphonylation of 70 with the salt of the actiphitylation of a fully benzyl-protected inositol 1,4-bisphosphate
vated acid. followed by debenzylation of the product with
with a free 5-hydroxyl group with phosphine 56 followed by
Angrw. Clietn. / ! i f , Ed. EngI. 1995. 34. 1933 1972
Inositol Lipids
Fig. 34. Preparation of the 5-methylphosphonate analogue of Ins( 1 .4.5)P3 employing phosphonylating agent 185 (vanBoom et al.)
Fig. 3 2 . Syiithesis of Ins(1,3.4.5)P,-3S 176 (Liu and Potter).
base provided the racemic 1,4,5-tris(hydrogenphosphonate) .[3sMl
The 5-methylenephosphonate analogue of Ins(1,4,5)P3, 180,
was also prepared (Fig. 33).[359JCompound 112, an intermediate in the synthesis of Ins(l,4,5)P3 from (-)-quinic acid (see
Fig. 23). was desilylated, and kinetically controlled addition of
(Fig. 34) and Ins(1 ,3,4,5)P4, by employing the new bifunctional
phosphonylating agents bis[6-(trifluoromethyl)benzotriazol-lyl]methylphosphonate (185) and (difluoromethy1)phosphonic
di(l,2,4-trIazolide), respectively.[360*3611 Thus, regioselective
benzylation of 181 gave the 6-benzyl ether as the main product.
Protection of the free 5-hydroxyl group by p-inethoxybenzylation and removal of the cis-ketal furnished 182, which was benzylated and deallylated to give 183. Phosphorylation of the 1and 4-OH groups with 56 and acidic removal of the p-methoxybenzyl group gave 184, which was phosphonylated with i85/
benzyl alcohol. Hydrogenolysis of 186 gave the 5-methylphosphonate analogue 187. Syntheses of racemic 3-methylphosphonate analogues of Ins(3,4)P2 and Ins(1 ,3,4)P3 were also
described.[3621Additionally, phosphonylating agent 185 was
used in syntheses of the methylphosphonate analogue of DIns(l)P13631and DL-myo-inositol 1-O-methylphosphonate 4 5
Syntheses of racemic m.yo-inosito1 5-methylphosphonate,
-4,5-bis(methylphosphonate), and -1,4,5-tris( methylphosphonate) were accomplished via the corresponding phosphinates,[365. 3661
Fig. 3 3 . Synthcm of the 5-methylenephosphonate analogue of Ins(l,4.5)P3 from
quinic acid (Falck et d.).
phenylselenyl bromide to the exocyclic alkene gave predominantly the anti-Markovnikov adduct. Oxidative elimination furnished the allylic bromide 177, which was converted into 178 by
Michaelis- Becker phosphorylation, hydroboration, and oxidative workup. Phosphorylation of the 1- and 4-hydroxyl groups
gave 179, which was deprotected to 180.
Also synthesized were the 5-methylphosphonate analogues and
the 5-(difluoromethyI)phosphonate analogues of Ins( 1 ,4,5)P3
8.6. “Caged” Analogues
The photochemically induced release of an active molecule of
physiological interest such as ATP, GTP, second messengers, or
even ions from an inactive precursor has considerable potential
for studies in cellular physiology, especially kinetic aspects,
since diffusional delay of agents into tissues is often a limitation.
Inactive precursors which on irradiation yield, for example,
a free second messenger are known as “caged” comp o u n d ~ [ ~3~6 8’1. and can be used for time-resolved measurements. Esterification of the three phosphate monoester groups
B. V. L. Potter and D. Lampe
of Ins( 1,4,5)P3 with 1(2-nitrophenyl)dia~oethane~”~~
caged inositol 1.4,5-tri~phosphate[~’~~
as a mixture of singly and
multiply caged compounds. The singly caged material was resolved into P-1. P-4, and P-5 isomers 188, 189, and 190, respectively, by HPLC (Fig. 35). All three compounds released
Fig. 36. Biotinylated C-2-modified Ins(1.4,5)P3 (Ozaki et al.).
Fig. 35. Regloisomers of caged lns(1,4.5)P3 (Trentham et
Ins(1.4,5)P3 on irradiation. 188 is a potent releaser of C a 2 + and
a substrate for 5-phosphatase. By contrast, 189 and 190 are
inactive in C a 2 + release and resistant to 5-phosphatase, and 190
is a 3-kinase inhibitor. Analogues 189 and 190 were used to
investigate the role of Ins(1 ,4,5)P3 in excitation-contraction
coupling in muscle.[37”3 7 2 1 The results indicate an essential role
for this compound in pharmacomechanical coupling in smooth
but not striated muscle. Studies in stomata1 guard cells have
shown that the reversible inactivation of K channels follows
the photolysis of caged Ins(1 ,4,5)P3 190.[3731
D-lnS(1,4,5)P3 analogues were successfully applied in the production and purification of antibodies against Ins(1 ,4,5)P3. by
allowing the preparation of Ins(l,4.5)P3-based immunogens and
an affinity
derivatives of
Ins( 1 ,4,5)P3,[3821 Ins(1 ,3,4,5)P4.[383]and InsP6[3841with an w-aminoalkyl group at
P-I were employed in the preparation of affinity matrices for the
purification of binding protein^[^^^.^^'] and [‘251]-labeled photoaffinity analogues.[l”. 386, 3871 Thus, [L251](azidosalicyl)aminopropyl-Ins(l,4,5)P3 194 (Fig. 37) was used to identify the major
8.7. Photoaffinity Analogues and Affinity Matrices
To aid studies on localization and purification of Ins( 1 .4,5)P3
receptors, it is desirable to synthesize photoaffinity analogues of
Ins( 1,4.5)P3. Initially, the preparation of an arylazide derivative
of Ins(1 ,4,5)P3 was
Thus, Ins( 1,4,5)P3 was coupled to p-azidobenzoic acid by using N,N’-carbonyldiimidazole.
Unfortunately, the site of location of the p-azidobenzoic acid
moiety was not determined, although hydroxyl group substitution was presumed, and a mixture was used. However. an irreversible inhibition of Ins(l,4,5)P3-induced CaZ release in
saponin-permeabilized photoirradiated macrophages was observed, which could be prevented by the presence of a large
excess of Ins(l,4,5)P3. This photoaffinity label was shown to
label three proteins in macro phage^.[^^^]
Acylation of the 2-OH group of Ins(l.4,5)P3 yielded anal o g u e ~ [ ~that
’ ~ ] were immobilized on Sepharose for the preparation of affinity matrices.’37712-0-p-Aminobenzoyl-Ins(l .4.5)P3
was utilized in the synthesis of the photoaffinity probe 191
(Fig. 36) carrying a biotin moiety as a nonradioactive marker.
This compound was successfully used to label the Ins(1 ,4,5)P3
5-phosphatase of erythrocyte ghosts.[3781The same research
group recently reported the preparation of affinity columns employing 2-substituted Ins(1 ,3,4,5)P4 and Ins(1,3,4,5,6)PS analogues and their application in the isolation of Ins(1 ,3,4,5)P4binding
(k)-(I R,3R,4R)-truns-N-(2-Aminoethyl)-3.4bis(phosphony1oxy)cyclohexane-I -carboxamide was used for the preparation of
an affinity matrix and a p h o t ~ l a b e I . [ but
~ ~ ~the
] utility of these
derivatives remains to be established. However, 6-0-substituted
194: X = ’261
196: Y = 3H
Fig. 37. Photoaffinity labeling by modification of the I-position of Ins(l.4,5)P3
(Prestwich et al.)
Ins(1 ,4.5)P3-binding protein in olfactory
With the help
of the new photoaffinity label [3H](benzoyldihydrocinnamyl)aminopropyl-Ins(l,4.5)P3 196, which was incorporated into the
rat cerebellar receptor at a substantially higher level than 194, it
was possible to purifiy and sequence a peptide matching specific
sequences in the N-terminal portion of the Ins( 1.4,5)P3 receptor.[’7s1 an area suggested to contain the Ins(1 ,4,5)P3 recognition site. An InsP, receptor isolated by these means was subsequently identified as the clathrin assembly protein AP-2.[3881
Syntheses of tetherable Ins(1,3,4)P3 and Tns(2,4,5)P3 derivatives
and their application in the preparation of bioaffinity matrices
were also described.[3891
Tethered ~ - I n s (.3,4,5)P4[23s3
2361 was prepared in a biomimetic synthesis involving Ferrier rearrangement of a suitably
protected D-glucose derivative (Fig. 38). Thus, the primary hydroxyl group in 46 was tritylated, the secondary alcohols were
Inositol Lipids
toxymethyl esters are rapidly cleaved intracellularly. releasing
the second messenger. A preliminary report on the synthesis of
membrane-permeable Ins(1 ,4,5)P3 and Ins( 1,3.4,5)P4
has been published.13921
8.9. Miscellaneous
A number of C-2-modified Ins(l.4,5)P3 analogues were prepared by chemical modification of the deacylated lipid 1-(sng l y c e r o - 3 - p h o s p h o ) - ~ - ~ ~ ~ - i n o s i t o l - 4 . 5 - b i s p h o s p hSynate.~~~~~
theses of racemic sulfonamide, sulfate, and carboxymethyl
analogues.[366.3y41 an enantiomerically pure hexadeoxy-I ,4,5tris(methylenesu1fonic acid) analogue of Ins( 1 ,4,5)P3.[3y51 and
the 3-methylenecarboxylate analogue of Ins( 1 ,3,4,5)P413y61
also described.
9. Inhibitors of Inositol Monophosphatase
Efforts towards the synthesis of inositol monophosphatase
inhibitors by the Merck, Sharpe and Dohme
have shown encouraging results. A number of active compounds have been prepared (Fig. 39), although they are not
202: R IPMB
203:R = P(O)(OBn),
Fig. 3X Biominietic synthesis of a precursor to a n Ins(1,3,4.5)P4affinity label
(Estuvci and Preslwich).
p-met hoxybenzylated, and the trityl group removed to produce
197. Swern oxidation and treatment of the resulting aldehyde
with acetic anhydride and base gave the (Z)-enol acetate 198,
which after Ferrier rearrangement furnished inosose 199.
Stereoselective reduction of the keto function with sodium
triacetoxyborohydride gave the inositol derivative 200. The
6- and 2-hydroxyl groups were then protected stepwise as their
benzyloxymethyl ethers to minimize acetyl migration. Basic
methanolysis gave 201, and condensation with benzyloxy{[3-(N-carbobenzyloxyam~no)propyl]oxy}(d~~sopropyl~mino)phosphaneketrazole followed by oxidation provided 202.
Cleavage of the PMB ethers and phosphorylation gave
203. hydrogenolysis of which afforded the P-I-tethered DIns(1 ,3.4,S)P4 204, used to synthesize an 1251-labeledaffinity
probe analogous to 192.
Cm = 50pM
cso =
Cm = 0.07pM
Ki = 6.3pM
= 0.61 pM
8.8. Lipophilic Analogues
Cell-permeable inositol phosphate analogues would obviously find considerable applications. To this end the synthesis of
racemic 2,3.6-tributyryI-Ins(1.4,5)P3 was addressed[3901 by
phosphorylation of 1.2,4-tri-O-butyryl-myo-inositol
with dianilidophosphorochloridate followed by removal of the phosphate protecting groups. The utility of this molecule, however,
remains to be established.
Masking the negative charges of anionic biomolecules is another strategy that has already been successfully employed in the
synthesis of a lipophilic CAMP analogue.[3911Here, the phosphate groups were esterified with acetoxymethyl bromide. Ace-
211: R = H
212: R = CHzOCOC(CH&
Fig. 39. lnostlol monophosphatase inhibitors
uncompetitive inhibitors like lithium. It was found that the hydroxyl groups in the 2- and 6-positions play very different roles
in the hydrolysis of Ins( l ) P by inositol monophosphatase. The
2-hydroxyl group is important for substrate recognition by the
enzyme (2-deoxy-Ins(l)P is only a weak substrate). whereas the
6-hydroxyl group is involved in the mechanism of phosphate
hydrolysis (6-deoxy derivatives like 205 have inhibitory proper-
B. V. L. Potter and D. Lampe
ties). The first approach to inhibitors was based on this observation, and by a "hydroxyl group deletion" strategy, ~-3,5,6-trideoxy-Ins(1)P 206 was synthesized and found to be a potent
inhibitor,[3971indicating that the 3- and 5-hydroxyl groups are
not necessary for enzyme recognition.
Inositol monophosphatase is known to be very unspecific,
and the enzyme is capable of hydrolyzing 2'-nucleotides including adenosine-2'-monophosphate (2'-AMP)[741as well as inosito1 monophosphates. When the molecular structures of 2'-AMP
and ~-3,5,6-tri-deoxy-Ins(l)P206 were superimposed, it was
concluded that substituents in the 6-position (the purine heterocycle in 2'-AMP) would be tolerated by the enzyme. This led to
the synthesis of 6-substituted 3,5-dideoxy-Ins(l ) P derivatives,
amongst them 207. the most potent monophosphatase inhibitor
reported to date.[3981
However, phosphate esters are biologically labile and tend to
have short half-lives due to hydrolysis by nonspecific phosphatases. Therefore, attempts to improve the stability of the
inhibitor by replacement of the phosphate ester with an isosteric, but stable, monophosphonate group have been made. Since
niyo-inositol 1-methylenephosphonate did not show any inhibitory properties, the search for inhibitors was based on hydroxymethylene phosphonate, which had been identified as a
weak inhibitor. In a series of analogues, the adamantyl ester 208
was the most potent inhibitor with a Ki value of 6.3 p ~Further
studies on hydroxymethylene bisphosphonic acid derivatiVeS1400. 403.4041 showed that compounds unrelated to the enzyme substrate like the tetralin derivative 209 can also be very
potent monophosphatase inhibitors. The 3-(3,4-dichlorobenzamido)benzyl derivative 210 is the most potent, nonhydrolyzable
inhibitor of inositol monophosphatase reported to date.[4021
The observation that the potent bisphosphonate 211[4031was
only poorly taken up into cells led to the development of prodrug 212 with reduced polarity.[4041Synthetic efforts of the
research group at Merck, Sharpe and Dohme have now been
extended towards derivatives and analogues of adenosine2 ' - m o n o p h o ~ p h a t e , [ and
~ ~ ~ molecular
modeling, kinetic. and
mutagenesis studies[4061are being employed to further elucidate
the enzyme mechanism. Attention should be drawn to a
report on the noncompetitive inhibition of inositol monophosphatase by the fungal metabolite K-76 monocarboxyhc acid
10. Structure- Activity Studies on Ins(1,4,5)P3
This section will address the consequences of structural modifications to Ins(1 ,4,5)P3 on its interactions with the three binding proteins-the Ins(1 .4,5)P3 receptor and the two metabolic
enzymes 5-phosphatase and 3-kinase.
10.1. Structural Requirements for CaZ+Release
The Ins(1 ,4,5)P3 receptor shows considerable stereo- and regiospecifity in its structural requirements for Ins( 1,4,5)P3 analogues in order to effect Ca2 release. However, of the three key
binding proteins Ins(1 ,4,5)P3 3-kinase appears to be the
most specific in its recognition of Ins(1,4,S)P3; Ins(1,4,5)P3 5phosphatase is much less specific. The specificity of the
Ins( 1,4,5)P3 receptor lies apparently between these two extremes.
The stereospecifity of the receptor is apparent since LIns(1,4,5)P3, [ = D - I ~ S ( ~ , S . ~ ) Pis, ] unable to mobilize
Ca2+.[13'.4081 The binding of this synthetic enantiomer to the
receptor is roughly 2000 times weaker than that of natural DIns(1 ,4,5)P3. [ 3 2 8 , 4 0 9 1 Likewise, the enantiomers of 2-deoxy-2,2difluoro-Ins( I ,4,5)P3 display very different agonist properties,[3331. 3321 ~-2,2-F,-Ins(l,4,5)P3was found to be a full agonist,
slightly less potent than Ins( 1,4,5)P3, whereas the L isomer is
only a very poor agonist.
Ins( 1 :2,4,5)P3, Ins(4,5)P2, and GroPtdIns(4,5)P2 were
shown to be 13-15 times,[t0g~4101
650 times,[4111and 3-10
times["'. 3 y 3 1 more weakly bound than Ins(1 ,4,5)P3. respectively; however, most other natural inositol phosphates including
Ins(1 )P, Ins(1: 2)P, Ins( 1 ,4)P2,["I
Ins(1 ,3,4)P3 , [ I 3 ' . 3281 In S(1 ,3,4.5,6)P5,"38.4121
and InsP, (phytic acid)[1311are ineffective
as agonists at the Ins(1 ,4,5)P3 receptor. Although the precise
role of Ins(1,3,4,5)P4 is still controversial, it was shown to mobilize Ca2+,[1381
probably however via the Ins(1 ,4,5)P3 recepIns(l.4,6)P3 was surprisingly found to be a full agonist
at the Tns(l.4,S)P3 r e ~ e p t o r . ' ~ ~
[ = LIns(1 ,3.4)P3]. obtained by an unambiguous route, was shown to
have Ca'+-mobilizing activity in Limulus photo receptor^,[^'^^
and this finding along with other data[4141helped to resolve a
long-standing confusion.
The Ca2+-releasingability of a number of semisynthetic 1substituted Ins( 1 ,4,5)P3 analogues[3931 showed that large
groups can be introduced at this position without major loss of
activity. Similar results were obtained when these compounds
were tested as inhibitors of aldolase A, an enzyme found to be
a potent isomer-selective binder of inositol polyph~sphates.[~'
Similarly, the 1-0-(3-aminopropyl) ester of Ins(1,4,5)P3 and a
derived photoaffinity analoguer3821were able to effect CaZt
release; the binding of both compounds was roughly eight times
weaker than that of Ins(1 ,4,5)P3. The binding of these derivatives by the receptor was equally good, and the photoaffinity
derivatives were successfully used to label Ins(1 ,4,5)P3 binding
sites."751 The 1 -phosphorothioate analogue of Ins(1 ,4,5)P3 as
well as fluorescently labeled Ins(1 ,4,5)P3-1S were also found to
be potent Ca2+-mobilizing a g ~ n i s t s . [ ~ ~ ~ ]
The introduction of large groups is also tolerated at the axial
2-position and does not affect the ability of the analogue to act
as a full agonist at the Ins(1 ,4,5)P3 receptor.[4101Deoxygenation
resulted in only a slight loss of activity,[4101and inversion of the
configuration of the hydroxyl-bearing carbon [scylloIns(1 ,2,4)P3[333]]gave a highly potent full a g o n i ~ t . [ ~D' ~L]- ~ - F Ins(1 ,4.5)P3[3331had the same potency as Ins(1,4,5)P3 in mobilizing sequestered Ca2 ions. Comparison with 2-F-scylloIns(l,4.5)P3 and 2,2-F2-Ins(1,4,5)P3(which are also full agonists
shows that the greater
at the Ins(1 .4,5)P3
structural similarity of 2-F-Ins(l .4,5)P3 to Ins(1 ,4,5)P3is reflected in its biological activity. The assumption that the 2-hydroxyl
group of Ins(1,4,5)P3 may interact with the receptor by acting as
a hydrogen bond acceptor rather than a donor[330,4171
appears to be strengthened : the fluoro substituent of 2-FIns(1 .4.5)P3 can no longer donate a hydrogen bond, but the ana+
.4ngc?l. Chcm. I n f . Ed En$
1995. 34, 1933-1972
Inositol Lipids
logue is still as potent as Ins(1 ,4,5)P3. ix-Ins(1.2,4.5)P4 was
pharmacologically evaluated and found to be the most potent
inositol tetrakisphosphate described to date.[2561
and Dn-3-Deoxy-lns( 1 .4,5)P3>13251 n-3-F-lns( 1 .4.5)P3
3-C-(trifluoromethyI)-lns(l ,4,5)P3[3221all mobilized C a 2 + as
full agonists. The potency of these compounds is similar to that
of Ins( 1,4,5)P3.Analogues with substituents at C-3 of increasing
steric bulk (3-chloro. 3-bromo. and 3-0-methyl) were found to
display decreasing activity with respect to receptor binding
and Ca' ' release.[200,3381 ~-chiro-Ins(2,3,5)P,, [ 3 3 5 , 3 3 6 1 an
Ins( 1 ,4,5)P3 analogue with an axial rather than equatorial 3-hydroxyl group. is a potent agonist for the mobilization of sequestered Ca' ' with an EC,, only 5 to 10 times higher than that
of Ins(l .4,5)P3.1335.4171The pharmacological properties of DL6-F-(.hilo-Iii~(2,3,5)P~[~"]
are not yet available. Ins(1 .3,4,5)P4,
the product of 3-kinase action on Ins(1.4,S)P3. was shown to
inhibit the binding of [3H]Ins(l .4,5)P3 to cerebellar membranes.
The receptor affinity of this tetrakisphosphate is. however,
about 60-fold lower than that of I n ~ ( 1 . 4 , 5 ) P , , [and
~ ~ ~a ~similar
decrease in potency was observed in Ca" release.[3z81By analogy. an axial phosphate group at the 3-position is likewise not
These results show that the 3-hydroxyl group
surprisingly plays a relatively insignificant role in receptor binding and Ca" release. Substituents at this position with increased steric demand (like an additional phosphate group),
however, reduce receptor binding properties considerably. It
has therefore been proposed that phosphorylation to
Ins( I .3,4.5)P4 may be the major regulatory mechanism for
Ins( 1.4,5)P3 receptor binding.["' Interestingly. a recently published study on ~-3-amino-3-deoxy-~ns(l
,4,5)P3 appears to
indicate that this compound is a pH-dependent partial agois[. I3 3 qi
Since thc early work of Berridge and I r ~ i n e [ ~the
' ~ 4,s-bis]
phosphate motif has been thought to be crucial for Ca2+-mobilizing activity. The phosphate group at C-4 appears to be essential for recognition by the Ins(1 ,4,5)P, receptor: all inositol
phobphates lacking this moiety are inactive (for exceptions see
ref. [420]), Ins(l .4.5)P3-4,5S2 is a potent Ca2+-mobilizingagonist. suggesting that at least phosphorothioate substitution at
C-4 is well tolerated.[3491However, no Ins( 1 ,4.5)P3 analogues
selectively modified at the 4-position have yet been studied, and
this is a clear area for future synthetic activity.
Ins( 1 .4,5)P3-5S[24'.2421
was found to bind with high affinity
to the Ins( 1 .4,5)P3 receptor and as a full agonist for mobilization
of sequestered Ca'+ is only about seven times less potent than
Ins( 1,4.5)P3. Similar to the trisphosphorothioate Ins(l,4,5)PS3,
Ins( 1,4.5)P,3-5S is not dephosphorylated by the 5-phosphatase
and is therefore able to produce a sustained release of Ca'+.
Only preliminary evaluations were carried out on the 5methylenephosphonate analogue 180, but these initial studies
showed that this compound releases C a 2 + from bovine adrenal
gland microsomes in a sustained fashion similar to other nonhydrolyzable analogues of Ins(l ,4,5)P3.[3591 5-Methylphosphonate 187 wah reported to antagonize Ins(l .4,5)P3-stimulated
Ca2 * release in a pH-dependent manner, and to act as a competitive inhibitor of ['H]Ins(l ,4,5)P3 binding to bovine adrenocortical microsomes.1360,3611 Full biological data for this compound and the 5-difluoromethylphosphonate analogue, which
is also supposed to act as a weak antagonist,[360,3 6 1 1are awaited
with interest, as these would be the first small-molecule antagonists of Ins(l ,4,5)P3 action. If these results can be confirmed,
then clearly the phosphate group at C-5 is crucial for the activity
of Ins(l,4.5)P3.
~-6-Deoxy-Ins(l,4,5)P, was found to be ii full agonist for
Ca2 release in permeabilized SH-SY5Y human neuroblastoma
cells and is roughly 70 times less potent than In~(l,4,5)P,.['~']
Other C-6-modified Ins( 1.4,5)P3 analogues were synthesized,
including the fluoro, methyl. and methoxy derivatives,[201. 302, 3 2 7 , 4 2 1 1 The biological properties of these compounds have not been reported to date except those of DL-6-0methyl-Ins(l ,4,5)P3. This derivative was found to mobilize
Ca" with an EC,, value over 200 times higher than that of
I n ~ ( l , 4 , 5 ) P , , [ ~a~ *result
similar to those obtained for other
6-0-substituted analogues.[3811DL-InS(1,4.5,6)P4 exhibited no
Ca"-releasing activity and did not appear to influence the
Ins( 1.4,5)P3-mediated C a Z + release at concentrations of up
to 10 pM.[I3*] Clearly, the 6-OH group plays a significant
role in receptor binding or fixing the conformation of the
5-phosphate group by hydrogen bonding. Substitution of
this hydroxyl group by both uncharged and charged groups
effectively abolishes activity. There is a clear need to examine
the potency of 6-F-Ins(1,4,5)P3, which remains as yet unevaluated.
The unnatural inositol phosphates Ins(2)P.11'21 Ins(2,4)P2,
and 3-deoxy-Ins(l.5,6)P3[3251
are all, like inositol. unable to release Ca". Ins(l:2cyclic 4,5)P3 was initially reported as being equipotent to
Ins( 1,4,5)P3 .['30.4241 and there were speculations that this cyclic
inositol phosphate may be a second messenger in its own
Further studies,['"] however, showed that Ins( 1 :2cyclic 4,5)P3 is only a weak agonist more than an order of magnitude less potent than Ins(1,4,5)P3. Ins(2,4,5)P3 was found to
effect Ca2+ release and is 12-68 times less potent than
Ins(l.4,5)p,,[109. 328.410,411.4251 Tegge et a1.[-112.4z51
the same EC,, value [roughly 30 times higher than that of
Ins(l.4,S)P3] for ~-Ins(2,4.5)P,and for D-chito-lns( I ,3,4)P3,an
analogue of Ins(2.4,5)P3 with an axial I-hydroxyl group. It thus
appears that the difference between the two compounds, an
equatorial rather than an axial hydroxyl group at C-I. is insignificant with respect to Ca2+-mobilizing activity. The L enantiomers of both compounds were much less active than the D
isomers and roughly 800 to 960 times less active than
Ins( 1.4.5)P3 .I3' ', 4 2 5 1 ~-3-Azido-3-deoxy-Ins(3,4.5)P,was considerably less potent than Ins(l.4,5)P3 in Ca2+ release studi e ~ ; [ ~ "its
] affinity for binding to the receptor is reduced by a
factor of 2000. The compound was found to mobilize 21 % of
sequestered Ca2+ at a concentration of 100 CLM, whereas
Ins(l,4,5)P3 released 53 % at 10 p ~The
. binding affinity of D2,3-dideoxy-Ins(l , 4 3 9 , to bovine adrenal cortices was only six
times lower than that of Ins( 1.4,5)P3, and its potency in effecting
C a 2 + release was reduced by a factor of 3.5. In sharp contrast,
additional deletion of the 6-hydroxyl group rebuked in a further
100-fold reduction in binding affinity, and ~-2,3,6-trideoxyIns(l.4.5)P3 was also a very poor receptor agonist with an EC,,
value more than 200 times higher than Ins(1,4,5)P3.[3261
This confirmed results of an earlier study in which the
trideoxy analogue, albeit in racemic form, had been shown to
effect Ca2 release from permeabilized smooth muscle cells
B. V. L. Potter and D. Lampe
with an EC,, value some 130 times higher than that of
Ins( 1,4,5)P3. [ 3 2 8 1
Benzene 1.2,4-trisphosphate was found to block the binding
of Ins( 1 ,4,5)P3 to adrenal cortex microsomes competitively with
an IC,, value of 34 VM. The affinity for the receptor is about
10000 less than that of Ins(1,4,5)P3, and no C a 2 + release could
be induced.[427 1 Benzene 1,2,4-tris(methylenephosphonate)was
likewise inactive in binding and C a 2 + release assays, as were
(1 R.2R,4R)-cyclohexane 1,2,4-trismethylenephosphate, 6 s (methylenephosphonate), -tris(methylenesulfonate). and the
(4s) isomer of the latter.[4281Racemic trissulfate, trissulfonamide, triscarboxymethyl, and trismethylphosphonate analogues of Ins(1,4,5)P3 did not show any biological activity;[365,3661 neither did the 4,5-dimethylenephosphonate
analogue of ~ ~ - I n s ( 4 , 5 ) .[4291
Inositol-1-phosphate-4.5-pyr o p h o ~ p h a t e [ ~and
~ ~ ]a n enantiomerically pure hexadeoxy1,4,5-tris(methylenesulfonic acid) analogue of Ins(l ,4,5)P3["']
were also found to be inactive.
A report by Schultz et al.[4201that cis,&-cyclohexane 1,3,5trisphosphate and related analogues with a 15bisphosphate
arrangement are able to effect C a 2 + release from isolated VdCuoles of Nrurospora crassa should be treated with caution, because Ins(1,3,5)P3 was shown not to act as an agonist in bovine
aortic smooth muscle
If these results were to be confirmed, it would imply that the vacuolar receptor of Nrurospora
crassa is different from mammalian receptors, which seems remarkable since Ins( 1,4.5)P3 receptors in different species have to
date been found to be highly similar. It seems possible that C a 2 +
contamination of the synthetic compound may be responsible
for this effect.
These results suggest that the vicinal4,5-bisphosphate moiety
is essential for receptor recognition and that the presence of an
additional phosphate group at C-1 greatly enhances Ca2+-mobilizing properties. The fact that Ins(2,4,5)P3 is also a full and
relatively potent agonist may possibly be explained by an interaction of the axial 2-phosphate with the receptor site normally
occupied by the equatorial 1-phosphate of Ins(1 ,4.5)P3. Clearly,
however, analogues can be synthesized with bulky substituents
at the 2-hydroxyl and the 1-phosphate groups. suggesting that
this area is probably relatively open to solvent. It remains to be
seen which substituents can be tolerated at the 3-position, but
the low activity of Ins(1,3,4,5)P4 makes it unlikely that this is an
attractive site for chemical modification (Fig. 40).
Relativelyuninpoiiarllor receptor recognition:
3deoxy-hs(l .4.5)P3 and BC-hs(1 ,4.5)P3as
well as ~ C hf mhs( 23.5 ) P~
are hry agonists.
Esserlialfor recognitlo%
however. mnserativs rmdi-
tications am tolerated:
hs(1,4,5)P34.5S and
hs(l.4.5)PS3 are active.
Esentlal for recognition by the receptor:
10.2. Structure-Recognition Studies on lns(1,4,5)P3
The specifity of 3-kinase is in many respects greater than
that of the Ins(1.4,5)P3 receptor itself;[461 lns(1,4,5)P3 and
Ins( 1 ,3,4,5)P4 are the only natural inositol polyphosphates
known to be recognized with high affinity.["']
Removal of the 1-phosphate group greatly decreases affinity
for the 3-kinase: Ins(4,5)P2 and Ins(2,4,5)P3 are both very poor
substrates for this e n ~ y m e , [ ~ ~ and
" ~ ~Ins(1
" ] : 2 cyclic 4,5)P3 is
not p h o ~ p h o r y l a t e d . [ ~The
~ " 1-phosphate group therefore appears to be essential for substrate recognition, which is in agreement with the observation that whilst both Ins(1,4,5)P35S[24L.4321
and lns(l.4,5)P3-4,5S,[4331 are substrates for
3-kinase. an additional phosphorothioate substitution at C-I is
not tolerated: Ins(1 ,4,5)PS3 is not a substrate.[434243s1
~ ~ - 2 - D e o x y - I n s,4,5)P3
effectively inhibits phosphorylation
of [3H]Ins(l,4,5)P, by 3-kina~e;[~l''the apparent K, value is
close to that of Ins(1 ,4,5)P3. Other analogues carrying bulky
substituents at C-2 were also recognized well by the enzyme.
However. no data indicating whether these analogues act as
substrates or inhibitors were given. Both ~ ~ - 2 - F - s c y l l o Ins( 1,4,5)P3 and ~ ~ - 2 , 2 - F , - I n s,4,5)P3
appeared to be substrates for 3 - k i n a ~ e . ' ~After
~ ~ ' resolution of the racemic gem-difluoride, however, the substrate properties of D- and ~-2,2-F,Ins(l,4.5)P3 were found to be quite different. Whereas the D
isomer is a substrate for 3-kinase. the L isomer is a potent inhibitor of this enzyme.[3321The reason for this inhibitory activity is not yet known; however. ~-2,2-F,-Ins(1,4,5)P3 represents
a novel lead in the design of further effective inhibitors for the
pharmacological intervention in the polyphosphoinositide
pathway and has the advantage that it does not mobilize intracellular Ca2 . 2-F-Ins(l ,4,5)P3 is also a good substrate for 3-kinase,I4I6] acting as a potent competitive inhibitor of the phosphorylation of [3H]Ins(1,4,5)P3 by this enzyme (apparent
Ki = 3.0 p ~ for
; comparison: 2-F-sc.yllo-lns(l,4,5)P3
and 2,2F,-Ins(l ,4,5)P3 have Ki values of 8.8 and 11.0 p ~ respectively).
As can be expected with C-3-modified Ins(l.4,5)P3 analogues, both ~ - 3 - F - I n s ( 1 . 4 , 5 ) P , [3341
~ ~ ~ . and L-chiroIns(2.3,5)P3 . [ 3 3 5 . 3361 a D-Tns(l ,4,5)P3 analogue with inverted
configuration at C-3, are resistant to phosphorylation by the
3-kinase, and they are potent inhibitors of this enzyme.[4361
Ins(1,3,4,5)P4, the product of Ins(1 .4.5)P3 phosphorylation by
3-kinase, is a weak inhibitor (IC,, = 90 p ~ ) .However.
based upon the Ic,, data it seems unlikely that sufficient Ins(1 ,3,4.5)P4 can accumulate to affect the rate of
Not very ilrportartfor reoeptor reccgnltion:
Ins(1 .4>5)P3phosphorylation significantly, suggesting
deoxy and fluom anakques are adive, as
are mnpoundscarrylrg larpe s&sUtue@
that it plays no role in any "feedback" mechanism.[32s1
In this position.
Phosphorylation of the 3-OH group results in effective
exclusion from the Ins(1,4.5)P3 receptor, and this could
be a regulatory mechanism to modulate Ins( 1,4,5)P3
Buky UllbtltuenSr am tolerated
binding.14' 71 Similarly, Ins( 1.3,4,5)P4-5s
was reported
position wiltmu mapr
"\ Inlosstheof1activily;
to be a competitive inhibitor of 3 - k i n a ~ e . ' ~ ' ~ ~
however, deletion
No compounds with selective modifications at the
4-position have been pharmacologically evaluated to
date. However, the affinity of Ins(l,4,5)P,-4,5S2 for 3kinase was ten times less than that of Ins(1 ,4,5)P35S,[433]indicating that the 4-phosphate group is important for substrate recognition.
.An,qrii'. Chenr. / / I / .
Ed, €ng/. 1995. 34, 1933-1972
lnositol Lipids
with erythrocyte ghost and brain cytosol 5-p1io~phatase.l~’~’
Unlike Ins( 1,4,5)PS,, DL-Ins(l ,4,S)P3-5S is a substrate for
was found that all compounds are competitive inhibitors of the
3-kinnse.1253.5321 but phosphorylation of this compound was
enzyme and substrates for 5-phosphatase. although the extent of
much slower than that of the natural substrate. The S hydrolysis varied. Surprisingly, ’-deoxy-Tns( 1 .4,5)P3 and most
methylenephosphonate analogue of Ins(1 .4,5)P3, reported to
of the other C-2-modified analogues showed an even greater
elicit a sustained release of Ca2+,[360.
3611 is presumably thereaffinity for 5-phosphatase than Ins(1 ,4.S)P3. 2-F-sc.jdlofore not a substrate for 3-kinase, although full biological details
Ins(l,4,S)P3 was found to be recognized but is a weaker subon this compound remain to be published. It appears that only
strate than Ins( 1 ,4,S)P3.13301
The 5-phosphatase substrate propconservative modifications are tolerated in this position.
erties of v- and ~-2,2-F,-Ins(1,4,5)P3 are quite different.
6-Deoxy-lns(l,4,5)P3 is one of the few compounds that is
Whereas D - ~ , ~ - F ~ - I ~ S ( is~ a, ~good
, S ) substrate.
the L enanrecognized by the highly selective 3-kina~e.I’~ The kinetics of
tiomer is a potent inhibitor of the enzyme.[”’] Since 5-phosits metabolism indicate that it is a substrate for this enzyme, and
phatase is known to be specific for the D isomer of Ins(1 ,4,5)P3,
the phosphorylation of Ins(1.4,5)P3 is inhibited competitively by
and analogues of the L enantiomer are usually not recognized
6-decixy-Ins( 1.4,S)P3. I t appears that hydroxyl group deletion
very well by this enzyme [cf. ~-Ins(l,4,S)P,“”~],this result is
remote from the site ofaction of the 3-kinase has no major effect
surprising. However, there were reports of ?-substituted anaon the binding properties of the substrate. In contrast to the
logues that also display Sphosphatase
and sur6-deoxy analogue, ~~-6-methoxy-Ins(
1,4,5)P3 showed a marked
drop in affinity (by roughly a factor of 120) for 3 - k i n a ~ e . [ ~ ~ * ]prisingly, the L isomer of 2-aminobenzoyl-Ins( 1 ,4,S)P3 was
found to be a potent inhibitor. Interactions of D L - ~ - F Since the hydrogen bonding potential at the 6-position is reIns(1
.4,5)P3 with 5-phosphatase were also studied. and this
moved in both analogues. it seems possible that the increased
derivative was found to be a moderately potent competitive
steric bulk a[ the 6-position of ~ ~ - h - m e t h o x y - I n s,4,S)P3
is the
inhibitor of this enzyme. It may be tentatively assumed that by
cause for the low affinity of this analogue. Once again it would
analogy with the difluoro analogue 2,2-FZ-Ins(1 .4,5)P3. the inbe useful to evaluate 6-fluoro-Ins( 1.4,S)P3 in this context.
hibitory effect of ~~-2-F-Ins(1,4,5)P,
is due to the presence of
uL-Cyclohexane i,2,4-trisphosphate is only a weak inhibitor
L-z-F-Ins(1 ,4,S)P3 in the racemic mixture. whereas the D isomer,
of [‘H]Ins(l ,4.5)P3 phosphorylation (ICso = 327 ~ L M ) . [ ~ ’ Sur*]
like D-2,2-F2-Ins(l ,4,5)P3, is probably a substrate for this enprisingly. benzene 1.2.4-trisphosphate was relatively well recogzyme. Thus, it may be concluded that the 2-hydroxyl group is
nized by 3-kinase (ICso = 6.1 ~ L Mwith
an affinity about ten
involved in substrate recognition by Ins( i .4S)P3 5-phosphatase,
times less than that of Ins( 1 .4,S)P3 .[4271o-2.3-Dideoxy- and Dalthough it does not appear to be an essential feature.
2,3,6-trideoxy-lns(l .4.5)P3 displayed K , values of 19 and 36 PM.
3-Deoxy-Ins(l ,4.S)P3 is a good substrate. binding with a
respectively. whereas various other multiply deoxygenated
slightly higher affinity than Ins(1,4.5)P3 itself.1J331and 3-Fanalogs did not show any interaction14281
(Fig. 41). InterestingIns(1,4,5)P3 is also a substrate for erythrocyte 5-phosly. 3-kinase is also inhibited by a d r i a m y ~ i n . [ ~ ” ~
p h a t a ~ e . In
~ ~contrast,
L - c I I ~ Y o - I ~ s ( ~ , ~which
. ~ ) P , can
be visualized as Ins( 1 ,4.5)P3 with inverted configuration at
Not irrportantforrecognition by
Site of enzyme action: 3deoxy-hs(l.4.5)P3,
C-3, was found to be a potent inhibitor.[3”, 3 3 6 . 4 3 6 1 This is
3-kinase: Pdeoxy-hs(l.4.5)F3 and
3-F-hs(t .4.5)P3 and ~-chlro-hs(23,5)P~
surprising, since the hydroxyl group at C-3 in this molecule
are all potent Inhlbitors.
some analogues cafr,4rg bukygroups
is remote from the site of attack of the enzyme. Two posshow anewngreateraffinltyhn
sible explanations for this phenomenon were given: The
conformation of ~-c~ziro-Ins(2,3,5)P~
in solution and/or
different from
The ,phosphalegmup appears
tobe anirrportardbutnotessenthat of Ins(1 ,4,5)P3 that although the analogue binds to the
Comenative modificationsare lolerated:
enzyme in a similar mode as 1ns(1.4,5)P3. the catalytic
hs(1,4.5)P3-5S Is phosphoiykted.
mechanism of the enzyme is interrupted. Alternatively. the
Delelionas in GdeOx~-hs(l,4,5)P3
inhibition may be the result of nonproductive binding of
tolerated; buky substituents howewr
the inverted substrate ~-chiro-Ins(2,3.5)P, in a rotated
appear to reduce the affinity.
In this arrangement the analogue would mimic
Fig. 41. Siru c i~ ire activity relationships Tor Ins(l.4.5)P3
three elements of Ins(l,4,5)P., correctly. namely the ring
pucker, the crucial vicinal 4.5-bisphosphate moiety (as the
2.3-bisphosphate pair), and the 3-hydroxyl group (as the
10.3. Structure-Recognition Studies on
4-hydroxyl group). The 5-phosphate group of L-chiroIns( 1,4,5)P.,-5-Phosphatase
Ins(2,3,5)P3 in this arrangement mimics an equatorial 2-phosphate group of an Ins( 1.4,5)P3 analogue, and such a phosphate
In contrast to the Ins(i,4.5)P3 receptor and the 3-kinase,
could presumably still bind reasonably well to the hydrophilic
Ins( 1,4,5)P, 5-phosphatase seems to be relatively nonspecific.
However. while many analogues bind to the phosphatase, only
pocket of the enzyme, which usually interacts with the equatorial i-phosphate group of 1ns(1,4,5)P3.In this inverted binding
a few appear to be substrates.
mode the axial 1 -hydroxyl group of ~-c/ziro-1tis(2,3,5)p3now
The I-phosphate group is an important feature in substrate
recognition by 5-phosphatase, since Ins(4,S)P2 is a very poor
mimics the axial 6-hydroxyl group of an Ins( 1 .4.5)P3 analogue.
As discussed below, the equatorial 6-hydroxyl group of
A number of racemic Ins( 1 .4,5)P3 analogues modified at the
Ins(l,4,5)P3 may play an important role in the mechanism of
2-hydroxyl group were examined for their ability to interact
5-phosphatase-catalyzed hydrolysis, and the inhibitory proper1961
B. V. L. Potter and D. Lampe
ties of ~-chir(~-Ins(2,3,5)P,
may possibly be ascribed to the pseudo-axial 6-hydroxyl group in the inverted binding conformation.
Phosphorothioate substitution at the 5-position creates a
potent inhibitor of Ins(1 ,4.5)P3-5-phosphatase, Ins( 1 .4,5)P35S.[241.2421 The 5-methylenephosphonate analogue of DIns( 1 .4,5)P3 is another long-lived agonist of C a 2 + mobilizat i ~ n . ~ ~The
~ ' ]sustained release of C a 2 + by this compound
indicates that it is not dephosphorylated and thus deactivated
by the Sphosphatase. Full data have yet to be published, and
details on interactions of the 5-methylphosphonate and 5-difluoromethylphosphonate analogues of Tns(1 ,4,5)P3r360.3611 are
also awaited with interest.
~-6-Deoxy-Ins(l,4.5)P3 r 2 3 inhibited the dephosphorylation
of [32P]Ins(1,4,5)P3by erythrocyte 5-phosphatase and is a moderately potent inhibitor of this enzyme. The nonselectivity of
this enzyme is underlined by the moderate reduction in affinity
of ~-6-deoxy-Ins(1 ,4,5)P3 (ca. twofold) and DL-6-methoxyIns(1 .4,5)P3 (ca. fourfold) for the aortic smooth muscle 5-phosp h a t a ~ e r relative
~ ~ ~ ' to the natural substrate. The resistance of
these two analogues to dephosphorylation confirms that the
minimal structural requirements for substrate hydrolysis by the
5-phosphatase include the vicinal 4.5-bisphosphate moiety and
a free 6-hydroxyl group. It is possible that the function of the
6-hydroxyl group of Ins(1,4,5)P3 is similar to the role that the
6-hydroxyl group of Ins(1)P plays in the dephosphorylation of
this compound by inositol monophosphatase; in other words,
the absence of this group has no significant effect on binding, but
its presence is essential for the enzyme to act on the substrate.
Whereas l n ~ ( 2 , 4 , 5 ) P , [ ~and
' ~ ~ Ins(1 :2-cyclic 4,5)P3[3101are
substrates for the 5-phosphatase, DL-Ins(l.4,5)PS3 is resistant to
degradation by this enzyme.[4401DL-Cyclohexane 1,2,4-trisphosphate is a weak competitive inhibitor (Ki = 124 mM), and no
inorganic phosphate release due to hydrolysis by the enand
zyme was detected.[3281Benzene 1,2,4-trispho~phate~~~'~
(1 R,2R,4S)-cyclohexane 1,2,4-tris(methylenesulfonate)[4281 were
found to inhibit the degradation of [3H]Ins(f ,4,5)P3 by 5-phosphatase surprisingly well, indicating that even radical structural
alterations can be tolerated by the enzyme. The trisphosphorothioates
ins( 1,4,5)PS3, Ins(l,3,5)PS3, and L-chiroIns(1,4,6)PS3 were found to be 5-phosphatase inhibitors of the
highest potency with submicromolar Ki values of 0.50,0.43, and
0.3 p ~ respectively.r3531
The latter two compounds d o not interact with 3-kinase, nor d o they mobilize Ca2
4421 These
analogues are therefore ideal tools for the selective inhibition of
5-phosphatase (Fig. 42).
11. Ins(1,4,5)P3 Partial Agonists and Antagonists
Only a small number of chemical agents are available for
intervention in the phosphatidylinositol signaling pathway,
which is underlined by the lack of a simple small-molecule
Ins(l,4,5)P3 antagonist. To aid the design of such an antagonist,
extensive structure-activity studies are required. In the rational
search for an agent having high affinity for the receptor yet not
releasing C a 2 + ,it is necessary to dissect the structural features
of Ins( 1 ,4,5)P3 that determine binding affinity from those that
trigger opening of the C a 2 + ion channel. Many naturally occurring and synthetic inositol phosphates display lower binding
affinity than Ins(1 ,4,5)P3 for the receptor, which can be correlated with correspondingly lower Ca2'-releasing ability. However,
no compound lacking a D-vicinal 4,5-bisphosphate grouping
has yet been found to release C a z + .Modifications at this locus
may be the way to design a small molecule that can directly
affect channel opening, providing that high binding affinity can
be maintained in such an analogue.
11.1. Partial Agonists at the Ins(1,4,5)P3Receptor
A number of partial agonists at the Ins(1 ,4,5)P3 receptor have
been identified: Ins(1 ,3,4,6)P4 was found to have moderate
affinity for the receptor and to give a maximal Ca2+ release only
some 80% of that of mobilized by Ins(1 ,4,5)P3. If the maximally
effective Ca2+-releasingconcentration of Ins(1,3,4.6)P4 is administered together with lns(1,4.5)P3, the EC,, value of the
latter is increased.rt381
This has led to the conclusion that the
C a 2 + released is not only from the same intracellular store, but
it also strongly indicates that Ins( 1,3.4,6)P4 and Ins( 1,4,5)P3
compete for the same receptor ~ i t e . [A~s Ins(
~ ~I .3,4,6)P4
, ~ ~ ~ ~
does not nominally possess the vicinal 4,5-bisphosphate moiety
normally required for agonist activity, it is not obvious
why this tetrakisphosphate should release Ca2+ . However, two
alternative binding conformations of Ins(l,3,4,6)P4 to the
Ins(l,4,5)P3 receptor can be visualized. in which a number
of important recognition features are mimicked, including
the 4,5-bisphosphate unit and the 1-phosphate group. It may
be that one or both of these binding conformations are the cause
of the partial agonist behavior exhibited by Ins(1 ,3,4,6)P4.
Chemical modification of Ins(1 ,3,4,6)P4 may provide a solution to this.
Weaker partial agonist behavior was found for 6-deoxyIns(l.4,5)PS3 and ~-~.Iiir0-Ins(2,3,5)PS,
. r 3 5 1 , 3521 Th eY
were able to mobilize only 42 and 34% of the
3aeoxy-hs(1,4,5)P3, 3-F-hs(l .4.5)f3 and hs(i 3 . 4 3 ) ~ ~ Not important for recognitionbv
5-phosphatase: 2deoxy-hs(l,4,5)P3
are good shsbates; however, inversion at
Ins(1 .4,5)P,-sensitive Ca2 pool, respectively. As weak
and other 8-modified analogues are
this positioncreates a potent inhibitor,
agonists they can be used to antagonize
Ins(1,4,5)P3-induced Ca2+ release, albeit at relatively
high concentrations. It should be noted that both compounds are C-3- or C-6-modified analogues of
Ins(1 ,4,5)P3, respectively, in addition to carrying phosphorothioate groups rather than phosphates in the I-, 4-,
Analogues Mdifiedat the
and 5-positions. As the weakest partial agonists, these
5-pC6itiOnlike hs(l,4.5)P3-5S
Deletion of the hydroxyl group
5-meU$snepbsphonate-hs(l .4.5)P3
compounds and ~-chivo-Ins(2.3,5)PS,in particular repreas in Gdeoky- and substihmonas in
am p0rem i ~ b i t o tof5-phosphatase.
sent key leads in the hunt for a small-molecule
,4,5)P3 gives
inhbitotsof 5-phosphatase.
Ins( 1 .4,5)P3 receptor antagonist. Partial agonist properties were also demonstrated for scylk,-Ins(l ,2,4,5)PS4.r4161
Fig. 42. Structure-activity relationships for Ins(1 .4.5)P3 S-phosphatase.
Clrivn. I n t . Ed. Enyl. 1995, 34. 1933-~1972
Inositol Lipids
11.2. Ins(l,4,5)P3 Receptor Antagonists
11.2.2. Decavanadate
Different vanadates were examined for their ability to inhibit
At present. none of the Ins(1 ,4,5)P3 analogues synthesized has
Ins(1 ,4,5)P3-receptor binding and Ca" release. It was found
shown any antagonistic properties apart from the 5-methylthat decavanadate (V,,O& at pH 7)['"] inhibits Ins(l ,4,5)P3phosphonate analogue of Ins( 1 ,4,5)P3[360,3611 and L-chiroinduced C a 2 + mobilization in permeabilized rat insulinoma
Ins(2,3,5)PS3.[4331 The latter, however, has significant intrinsic
and PC12 cells (1C5, 5 pM)'3101and SH-SY5Y cells (Ki
activity. The only molecules that have been clearly demonstrat1.2 p ~ ) , [ ' ~ and
' ] inhibits the binding of [3H]Ins(1.4.5)P, to its
ed to potently inhibit 1ns(1,4,5)P3-receptor binding are heparreceptor in cerebellar and adrenal cortex membranes.[4s' I Orin 1443 14I' and d e ~ a v a n a d a t e ; [ ' " ' ~ ~neither
- ~ ~ ~ ~of them,
thovanadate and oligovanadate on the other hand do not inhibit
however. shows specifity for the Ins(1 ,4,5)P3 r e c e p t ~ r . [ ~ ~ ~ . ' ~ ~ ]
Ins( 1,4,5)P3-receptor binding,[4501possibly because they are
and they are therefore of only limited use as pharmacological
unable to bridge the multiple Tns(1.4,S)P3 binding sites suggesttools .
ed by Meyer et a1.['"'
Decavanadate also suppresses
Ins( 1,3,4,5)P,-induced Ca2+ release from permeabilized SHSY5Y cells and inhibits Ins(1 .4.5)P3 5-phosphatase, 3-kiiiase,
11.2. I . Heparin
and Ins(1 .3,4,5)P4 5 - p h o s p h a t a ~ e .' II ~ ~
Heparin is a polysulfated polysaccharide with a molecular
Although decavanadate is a potent and competitive antagoweight between 6000 and 20000, depending on origin and
nist at the Ins(1.4,5)P3 receptor, the fact that its specifity is low
preparation. Initial indications that heparin could interact with
will most likely prevent it from becoming a useful tool to invesIns(1 ,4,5)P3 binding sites came from the observation that the
tigate the second messenger role of Ins(1 .4.S)P3. However.
cerebellar site first described by Worley et
is sensitive to
molecular modeling studies based upon decavanadate. and inheparin.["4.31Indeed, subsequently the protein representing this
deed heparin, may provide important leads.
site was significantly purified using heparin-agarose affinity
chromatography.[' 591 Heparin was also found to inhibit
Ins( I ,4,5)P3-induced C a 2 + release in hepatocyte~.[~~'1
pancreat12. Applications of Inositol Phosphate Analogues
ic cell^,[^'^^ and rat liver m i c ~ - o s o m e s .The
[ ~ ~potent
antagonism of Ins( 1 .4.5)P3-activated Ca2 release by heparin was
Apart from caged and photoaffinity analogues of Ins(1 ,4.5)P3
demonstrated to be both Competitive and reversible, with an
with obvious applications, 1ns(1,4.5)PS3 has proved to be the
affinity of heparin for the binding site of roughly 3
most versatile Ins( 1 ,4,5)P3 analogue synthesized to date.
It was also found that the heparin fragment TV84558-51 (averIns(1 ,4,5)P3 receptors recognize Ins( 1.4,S)PS3 with high affinity
in brain[409.4401
and l i ~ e r . [The
~ ~analogue,
a full intracellular
age M, = 5100) is as potent as heparin itself in inhibiting
["H]lns(l ,4.S)P3 binding, whereas smaller heparin fragments
Ca'+-releasing agonist, is only three to four times less potent
than Ins(1,4.5)P3 in a number of cells, including Xenopus
and less sulfated or unsulfated glycosaminoglycans (chondroitin
sulfate and hyaluronic acid) did not affect binding.[4431This is
o o ~ y t e s , permeabilized
~ ~ ~ ~ ~ ~ Swiss
~ ~ ]3T3 cells.1'31~4081
also the case for 0- and N-desulfated, N-reacetylated heparin:
h e p a t o ~ y t e s , [ ~pancreatic[461
- 4 6 3 1 and parotid[4641
acinar cells, skeletal muscle triads,[4651mouse lacrimal
N-desulfated heparin showed a decreased inhibitory activity at
and SH-SY5Y neuroblastonia cells.'4h1Ins(l.4,5)PS3 is resistant
the Ins( l,4.S)P3 receptor but not at the lns(1,3,4,5)P4 binding
to dephosphorylation by 5 - p h o ~ p h a t a s e [ ' ~4401
~ . and can prosite. whilst pentosan polyphosphate is another potent but
nonelective inhibitor at both Ins(1,4,5)P3 and Ins(1 ,3,4,5)P4
duce a sustained cellular Ca2 transient.[". 13s1 In S(l,4,5)PS,
binding sites."lR1 In contrast to the inhibitory effects of heparin
was the first competitive inhibitor of 5-phosphatase reportand related substances observed in many animal and plant tised,[432.438jbut is not bound by 3-kinase and does not compete
with Ins(1.4,5)P3 for this e n ~ y r n e . [ ' ~ ~
. ~1.4,5)PS3.
sues, Ins( 1.4,S)P3-induced Ca" mobilization in fungi is heparis resistant to all known routes of Ins(l.4,S)P3 metabolism, is
in-inseiisitive.llssl Heparin also inhibits Ins(1 .4,5)P3 3-kinase
activity (but not that of Ins(l,4,5)P3 5-pho~phatase).[~'~l
now commercially available, and is finding an increasing numspecific binding of Ins(1 ,3,4.5)P4 to cerebellar
ber of biological applications. as illustrated here.
and the ability of Ins(1,3.4,5)P4 to release C a 2 + from cerebellar
The possibility of synergistic interactions between Insmicro some^.[^' '1
(1,4.5)P3 and its metabolite, Ins(1,3,4,S)P4, in promoting C a 2 +
entry at the plasma membrane has been the subject of considerIt seems likely that the negatively charged sulfate groups of
heparin interact with the hydrophilic pockets in the receptor
able recent debate.['23'
' 2 6 1 Wh en a Ca2+-activatedK + current in singly internally perfused mouse lacrimal acinar cells was
binding site that usually accommodate the phosphate groups of
monitored by using a patch-clamp technique for whole-cell curlns(1.4.5)P.,. However, it is interesting to note here that myoinositol-l,4.S-trissulfate,in spite of carrying sulfate groups in
rent recording, addition of Ins(l,4,5)PS3 alone gave rise to a
the positions that are occupied by the phosphate groups in
single transient response, independent of external Ca2 and
typical of Ins(l,4,5)P3. In the presence of Ins(1,3,4,5)P4, howIns(l.4.5)P,. does not seem to have any antagonistic properAlthough fragments of lower molecular weight have
ever, this analogue gave a sustained response dependent upon
been investigated with some success.[4561their effectiveness is
external C a z + . The transient response of Ins(1.4,5)P3 is therestill an order of magnitude away from that of a true Ins(1 ,4,5)P3
fore presumed not to be aconsequence of rapid metabolism, and
analogue. Clearly, a closer study of the heparin-receptor interIns( 1 .3,4,5)P4 does not act by protecting Ins( 1 .4,S)P3 against
action is essential in the search for structural features that can
dephosphorylation by 5-phosphatase, for which it is also a subst rate ']
be incorporated into a small-molecule antagonist.
B. V. L. Potter and D. Lampe
When receptor activation in Xenopus oocytes is followed,
Intracellular Ca” oscillations are often observed in cells
complex spatial and temporal patterns of C a 2 + release in the
stimulated by extracellular agonists coupled to polyphosphoinositide hydrolysis, and the role and generative mechanisms of
form of regenerative circular and spiral waves can be observed.
Such spiral waves could be generated by injection of the nonmesuch oscillations have been the subject of significant recent intabolizable Ins(l,4,5)PS3 and were indistinguishable from reterest and ~ o n t r o v e r s y .Models
~ ~ ~ . of
~ ~the~ processes
underlyceptor-induced Ca2+ patterns.[4721Stimulation of Ca2+ release
ing these oscillations have been developed.[467,4681 Singly perby such a compound, however, did not result in gross alteration
fused mouse pancreatic acinar cells treated with 1ns(l,4,5)PS3
of the C a 2 + release pathway. This and other similar behavior
[or Ins(1,4,5)P3], applied through a patch pipette, were found to
suggested that the metabolism of Ins(1,4,5)P3 is not responsible
evoke repetitive pulses of Ca2 +-activated CI- current, similar in
for the cessation of C a 2 + activity. Clearly the Ins(l,4,5)P3 conamplitude and frequency to the response to acetylcholine, acting
centration remains relatively constant during the period of
through muscarinic receptors. Pulsatile C a 2 + release is possible,
regenerative wave stimulation. Regenerative activity is probatherefore. even at a constant level of the analogue, and prebly due to the cyclical nature of stimulation and inhibition
sumably therefore also of Ins(1,4,5)P3, which is an argument
of the Ins(1,4.5)P3 receptor channel as governed by cytoagainst a receptor-controlled oscillator in the generation of local
plasmic C a 2 + concentrations. Ins(l,4,S)PS3 has also been emmaxima in C a 2 + concentration, and a role for the periodic phosployed to study the influence of Ca” influx on Ca2+ waves
phorylation o r degradation of Ins( 1,4,5)P, ,[4621 Different types
induced by microinjection of the analogue. Receptor-activated
of cytosolic Ca” fluctuation patterns, mimicking those proC a 2 + influx was found to modulate the frequency and
duced by the natural agonist. can be generated by Ins(1,4,5)PS3
velocity of Ins(1 ,4,5)P3-dependent CaZ waves in Xenopus
depending on its concentration.[4631Low Ins( 1,4,5)PS, concenoocyte~.[~~~]
trations evoke repetitive local maxima in the Ca2 concentraMicroinjection of c G M P into unfertilized sea urchin eggs eliction, whereas at relatively high concentrations repetitive Ca’+
its activation by stimulating a rise in the intracellular free C a z +
waves are produced.
concentration by means of an unknown mechanism. This C a 2 +
When invertebrate microvillar photoreceptors are exposed to
release and activation are prevented in eggs in which the
light, C a 2 + release from the ER is effected by the intermediacy
Ins(l,4.5)P3-sensitive C a 2 + stores were emptied by prior injecof Ins(l.4,5)P3 acting through an Ins(1 ,4,5)P3 receptor. In vention of Insfl ,4.5)P3. Indeed, injection of Ins(l,4.5)PS3 into sea
tral photoreceptors of the horseshoe crab Limulus Ins(l,4,5)PS3
urchin eggs caused a Ca2+ transient of similar magnitude and
was used to investigate mechanisms that terminate Ins( 1,4,5)P,duration to that elicited by Ins(1 .4,5)P3. However, subsequent
induced Ca” mobilization. The action of Ins(1.4,5)P3 in these
injections of Ins( 1,4,5)PS, resulted in only small subsequent
cells is stereospecific for the D enantiomer and the second mesrepetitive Ca2+ transients in contrast to the much larger ones
senger is normally rapidly deactivated by metabolism. In the
elicited by Ins(1 ,4,5)P,. This is because, as a result of the nonhyLimztlus photoreceptor, Ins(l,4.5)PS3 generates sustained repetdrolyzable nature of Ins(l,4.5)PS3, the resting C a 2 + level is
itive oscillation of CaZ+-dependent membrane potential which
greater in Ins(1 .4,5)PS,-injected eggs. Thus, the Ca2
persists for tens of minutes.[4691Oscillations of membrane postore can be progressively depleted by successive injections of
tential[408]and Ca2+-dependent CIcan be generIns(1,4,5)PS,
ated by microinjection of Ins(l,4,5)PS3 into Xenopus oocytes.
Ins( 1,4,5)PS, was exploited to help distinguish functionally
Such oscillations are not sustained here, however, indicating
between Ins(1 ,4.5)P3-sensitive and -insensitive nonmitochondrithat factors other than metabolism are important in terminating
al MgATP-dependent C a 2 + pools in rat pancreatic acinar
the response. Ins(1,4,5)PS3-evoked membrane potential oscillaThe Ins(l,4,5)P3-sensitive C a 2 + pool was kept empty
tions differ from those generated by Ins( 1.4,5)P3, and resemble
by Ins(l,4,5)PS3 and Ca” reuptake occurred into the Insmore those of Ins(2,4,5)P3. The reason for this is not clear, but
(1 .4,5)P3-insensitive pool.
different mechanisms for setting up Ca’+ oscillations may be
Ins( 1,4,5)PS, and the related monophosphorothioate anapossible.[’261Oscillations in Ca2+-dependent CI- current inlogue Ins(1,4,5)P3-5S are potent 5-phosphatase inhibiduced by Ins(1,4,5)PS3 appear to resemble those induced by
tors[432. 4381 and have been employed to inhibit Ins(1 .4,S)P3
Ins(1,3,4,5)P4 rather than Ins(1 .4,S)P3
breakdown in electrically permeabilized SH-SY 5Y human
The free intracellular Ca2 concentration increased in a localneuroblastoma
Inhibition of 5-phosphatase-mediated
ized cellular area through the action of stimulus-induced elevametabolism of exogenously added S[32P]Ins(l,4,5)P, was about
tion of Ins(l,4,5)P3 can give rise to actively propagated C a 2 +
ten times greater than that of cell-membrane-derived
waves, which extend this signal to other parts of this cell.
[3H]Ins(l,4,5)P,, indicating the possibility of Ins(1 .4,5)P3 comIns( 1,4,5)PS, was employed to investigate the propagation of
in other words that homogenous mixing of
C a 2 + waves through the cytoplasm of Xenopus o o c y t e ~ . [ ~ ~ partmentation,
exogenously added and endogenously generated Ins( 1,4,5)P3
Ins(l.4,5)PS,-stimulated Ca2+ waves were found to be very
does not occur. Ins(l,4,5)P3-5S is a substrate for Ins(1,4,5)P3
similar to those induced by stimulation of acetylcholine recep3-kinase and inhibits the phosphorylation of [3H]Ins(1,4,5)P3by
tors and probably therefore result from the same intracellular
mechanisms. However, since Ins( l,4,S)PS3 cannot be hydrolyzed and is not readily metabolized by Xenopus o o ~ y t e s . [ ~ ~ ~ ]Intracellular C a 2 + release can be “quantized”, where the size
of the Ins( 1,4,S)P3-sensitive C a z + pool is apparently dependent
it can be inferred that such C a 2 + waves do not require fluctuaupon the concentration of Ins( 1,4.S)P3 .11241 Ins(1,4,5)PS3 has
tions in Ins(1 ,4,S)P, levels and may be derived by a mechanism
been employed in the investigation of quanta1 Ca” release in
dependent upon C a 2 + stimulation of Ins( 1,4,S)P,-induced Ca2’
permeabilized I i e p a t ~ c y t e s . [The
~ ~ ~failure
of submaximal conrelease.
A n p i , . Chcm. Inr.
Ed. En,$ 1995. 34, 1933-1972
lnositol Lipids
The first synthesis of a fluoro analogue of an inositol lipid
was described as early as 1982.I'
2-Deoxy-2-fluoro-lphosphatidyl-scj.Uo-inosito1 was obtained by condensation
of ~-fluoro-2-deoxy-3.4,5,6-tetra-0-benzyl-s~ll~~-inositol
the sodium salt of dipalmitoyl-L-r-phosphatidic acid, followed by hydrogenolysis. More recently, preparations of
D-3-deoxy-3-fluoro-I-phosphatidyl-nqwinositol and the respective 3-chloro derivative were reported.i488-4901 Th e former
PtdIns analogue displayed cytostatic properties, inhibiting
cell growth of NIH 3T3 cells at concentrations on the order
Of 100 pM.
The synthesis of 2-deoxy-Ptdlns was described.'""'] Racemic
was selectively acetylated at
the 1 -hydroxyl group. Deoxygenation was effected employing
the Robins' variation of the Barton- McCombie sequence:
thionocarbonate formation and radical reduction. Saponification and optical resolution of the resulting alcohol via its camphanates afforded ~-3,4,5,6-tetra-0-benzy~-2-deoxy-~n~~u-~nos~
tol, which was phosphitylated with the novel coupling agent
bis(diisopropylamino)(2-trimethylsilylethoxy)phosphine. Reaction with dipalmitoylglycerol and oxidation of the phosphite
triester gave the protected deoxy-Ptdlns analogue as a mixture
of diastereoisomers at phosphorus. Stepwise deblocking then
gave 2-deoxy-Ptdlns. which was used to study the specificity of
phospholipase C (PLC).
Syntheses of the PtdIns analogue!~o-inositol (DPPsI) were reported by two
In the first synthesis.[263.2641 111-2,3:5,6-di-0cyclohexylidene-4-0-methoxymethyl-myo-inositolwas phosphitylated with methoxy(diisopropy1amino)chlorophosphine
(61) and then converted directly to the protected 1.2-dipalmitoyl-srz-glycero-3-thiophospho-nzyo-inositol
by reaction with
1,2-dipalmitoyl-sr7-glycerol and sulfoxidation. Deprotection
with acid and demethylation with trimethylamine afforded
(RP+ S,)-DPPsI as a mixture of diastereoisomers. which was
used to examine the stereochemical mechanism by which
PtdIns-specific PLCs cleave their substrates. I n the second approach,[26s11,2-dipalmitoyl-sn-glycerol was reacted with 61 to
produce the phosphoramidite. which was then condensed with
Sulfoxidation afforded a mixture of the phosphorothioate diastereoisomers separable by chromatography. Deprotection produced the
individual diastereoisomers of 1,2-dipalmitoyl-sn-glycero-313. Syntheses of Inositol Lipids and Analogues
thiophospho-I -nzjwinositol. The synthesis of a number of
Ptdlns analogues with thiophosphoester bonds designed to inMuch of the earlier work on the synthesis of phosphatidylhibit PtdIns-PLC was also reported.[4921
inositol lipids and their analogues has been covered in a rePhosphonylating agent 185 was employed in syntheses
vie^,.["*"^ The synthesis of four stereoisomers of dipalinitoyl
of optically active phosphonate analogues of PtdIns and
phosphatidqlinositol to probe the substrate stereospecificity of
P t d I n ~ ( 4 ) P ~ " " ~as well as in the preparation of the
PtdIns 4-kinase was r e p ~ r t e d . ~ " ~ ~The
. " ' ~PtdIns(4.5)P2
phosdiastereomeric uncharged methylphosphonate analogues of
pholipid analogue 1 -0-(1,2-di-O-palmitoyl-sr~-glycero-3-phos-P t d I n ~ . [The
~ ~ phosphonate
analogue of Ptdlns has proved to
pho)-i>-Ins(4,5)P2was synthesized using a PI'' approach.['661
be a potent antiinflammatory and analgesic agent.[""] Phos(The preparation of the di-0-stearoyl phospholipid had been
phonate derivatives of PtdIns with a single alkyl chain in place
described previously by a Russian group,[Z'Y~48"4841
and the
of the diacylglycerol moiety were also described.[49s1 as were
isosteric methylenephosphonate and isopolar difluoro)A
work of this group in this area has been reviewed.["80~"Ss1
synthesis of optically active 1-0-(1.2-di-O-stearoyl-sn-glycero- methylenephosphonate analogues of PtdIns containing short3-phospho)-i>-lns(3,4.5)P, from ( -)-quinic
has been
chain ethers rather than lengthy acyl chains on the sn-1 and sn-2
complemented by a short synthesis of this PtdIns(3,4,5)P3 anaoxygens of the glycerol unit to improve the water solubility of
logue i n racemic
these compounds.[""61
centrations of Ins(1 ,4.5)P3 o r Ins( t ,4,5)PS, to empty the f a 2+
store completely was not due to stimulus deactivation or
to receptor desensitization. Use of the metabolically stable
Ins( 1,4.S)PS3 allowed C a 2 + efflux experiments to be performed a t il high cell density, at which metabolic degradation of Ins( 1,4,5)P, would normally have posed significant
Ins( 1 ,4.5)P3 activates a novel voltage-dependent K t conductance in rat C'A I hippocampal pyramidal
When injected into these cells, Ins(1,4,5)PS3 inhibited action potential firing. The metabolic stability of Ins(1,4,5)PS3 was crucial for
these observations. Ins( 1 ,4.5)P3 itself did not elicit this conductance, presumably because of its rapid enzymatic metabolism.
Thus. importantly. the use of Ins( 1,4.5)PS3may uncover activities 01' Ins( 1 .4,5)P3 that may not be observable experimentally
with the natural messenger because of rapid metabolism or the
slow diffusion of exogenously added agonist.
I n rabbit skeletal muscle triads. Ins(l,4,5)PS3 released up to
2 0 ' h of an actively loaded Ca'+ pool, although activation of
ryanodinc receptor CaZ channels was zero or mininial,[4hs1
raising the possibility that the Ca"-mobilizing activity may be
mediated by other channels o r mechanisms. Evidence was provided for two types of intracellular Ca2+-sensitive channels displaying fast activation kinetics, namely Ins( 1,4,5)P,-sensitive
and Ca' -sensitive channels. The kinetics of Ca2+ release by
Ins( 1.4,5)PS3in rabbit skeletal muscle SR were surprisingly fast
(20- 150 ms, depending upon agonist concentration), indicating
that Ins( 1 .4.5)P3 receptors of skeletal muscle may be kinetically
competent to bignal the raid elevation of cytosolic Ca2+ that
preceeds muscle
A role for Ins( 1 .4,5)P3 in red blood cells has yet to be established. although the turnover of polyphosphoinositides is well
known. In permeabilized human red blood cells Ins( 1,4,5)P3
evokes transient release of Ca2 ', disorganization of the spectrin
network, and reversible shape changes. as measured by immunofl~iorescence.["'~~
However, Ins( 1,4,5)PS, evokes sustained release of Ca" and irreversible shape changes and disorganization of the spectrin network. The polyphosphoinositide
signaling pathway evidently plays an important role in the shape
maintenance of' red blood cells.
B. V. L. Potter and D. Lampe
14. Glycosyl-Phosphatidylinositol Anchors
seems to be parasite-specific. Thus the design of drugs aiming at
this side chain may prove successful.
Some metabolic fragments of the protein-GPI complex
Many proteins are bound to biological membranes. Whereas
formed by the combined actions of specific endogeneous
integral membrane proteins have one or more hydrophobic doproteases and a glycosyl-phosphatidylinositol-specificphosphomains and are embedded to a great extent in the lipid bilayer,
other proteins are covalently attached to lipids which serve as
lipase C (GPI-PLC) were found to display certain biochemical
anchors to the cell membrane. It has been shown that many of
properties previously associated with crude preparations of puthese latter proteins are linked to the membrane by phostative insulin mediators (PIMs). (This GPI-PLC is distinct from
phatidylinositol anchors attached to the C-terminal amino acid
the PtdIns-PLC. which hydrolyzes PtdIns, PtdIns(4)P and Ptof the protein through an intervening glycan structure [glycosyldIns(4,5)P2 and is located on the inner membrane rather than
the cell surface.) PIMs are phosphorylated inositol-glycans
phosphatidylinositol (GPI) anchor]
5001More than 30 difgenerated after insulin stimulation of the cell surface receptor
ferent cell surface proteins with a GPI anchor have been identiand can mimic some biochemical properties of insulin in
fied (for the biosynthesis of GPI anchors see ref. [501]),
v i t r ~ . [ ~5 0"7 ~, , However, it is as yet still premature to regard
including hydrolytic enzymes (e.g. alkaline phosphatase and
mammalian antigens (e.g. Thy-I [5031),
inositol phosphate glycans as second messengers of insulin
and cell adhesion proteins. The generic structure of GPI anchors
action. Several groups directed their attention towards the
and related GPIs. Syntheses
is PtdIns-glucosamine-mannose,-phosphoethanolamine-pro- preparation of P I M s " ~ ~509-5'21
of fragments of mycobacterial inositol phospholipids were also
tein; however, modifications of the core mannosyl residues by
additional side chains are common and other variations (e.g. in
the fatty acid composition of PtdIns) have also been observed.
The discovery of GPI anchors has attracted the interest of
bioorganic chemists, and efforts to synthesize the GPI anchor of
15. Summary and Outlook
the variant surface glycoprotein (VSG) of the parasitic protoThe first evidence of a second messenger role for
' ~ ] 43) have been successzoan Trypanosoma b r ~ c e i [ ~ (Fig.
Ins(1 .4,5)P3[I9.4'91 catapulted the study of polyphosphoinositide metabolism and its effects from the position of a relatively
modest backwater to one of the most active fields at the forefront of modern biology. The resulting upsurge in interest in
cyclitol and inositol phosphate chemistry shows no current signs
of subsiding. Indeed, it is likely that a significant proportion of
future progress in inositol phosphate biology will be underpinned by the current interest in inositol phosphate chemistry.
Continuing development of synthetic inositol phosphate analogues with novel biological properties can be envisaged, including receptor antagonists, caged compounds, cell-penetrating compounds, affinity, photoaffinity, and spin-labeled inosito1 phosphate analogues, fluorescent probes, and inositol phosphates linked to affinity columns for the purification of
receptors and enzymes. The prospect of a whole new signaling
pathway based upon PtdIns(3,4,5)P3 opening up, the anticipation of the structural characterization of the as yet elusive
factor", and the advent of novel potent
ligands unrelated to Ins(1 ,4,5)P3 such as
Fig. 43. The GPI anchor of the variant surface glycoprotein of Trjpunosomo hruwi.
adenophostins (Fig. 44) together with the real possibility of deful.[50s.50h1 The survival of African trypanosomes depends on
the integrity of their cell-surface coat, which is formed by VSGs
arranging into tightly packed monolayers and which protects
the parasite from lytic factors of the host serum. By expressing
different VSGs at different times the parasite is able to evade the
host's immune response. This antigenic variation makes it difficult for the immune system to supply the appropriate antibodies
at the right time, and it also renders drugs against specific VSGs
ineffectual. Inhibition of the biosynthesis of the VSG GPI anchor, however, or drugs targeting specific features of this GPI
anchor, could prove successful. This explains the interest this
particular GPI anchor has attracted. Whereas the backbone
structure of the VSG GPI anchor is similar to that of other GPI
anchors, the a-galactose side chain of the VSG GPI anchor
Fig. 44. Adenophostin A
( R = H) and B ( R =
vising drugs of the future by pharmacological intervention in
signaling transduction pathways means that interdisciplinary
collaborations by chemists with cell biologists, biochemists, and
pharmacologists will be crucial to meet these new challenges and
apply the results.
A n f i ~ w .Cheni. Int. Ed. Enfit. 1995. 34. 1933-1972
Inositol Lipids
W1, tlltrnk thc,fbra Prize Fellowsliip ( t o D . L . )
m t l S. .I
Mills for CI critical reading qfthe manuscripi. B. K L. P.
is ( I Lisfcr In.siitute Rpserirch Professor.
German version:
Received: June 10. 1994 [A69IE]
C h i , f i i . 1995. 107. 20x5 -2125
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