Chemical Approach to Signal Transduction by Inositol Triphosphate

Berridge discovered that inositol 1,4,5-trisphophate (IP3) was generated at the cell surface in response to cell stimulation and functioned as a second messenger to release Ca2+ from internal stores. Ozaki et al. succeeded in the first total synthesis of optically active IP3 by 13 steps. He supported the signal transduction studies by supplying necessary reagents such as IP3, other IPx, phosphatidyl inositol, new synthetic methods and reagents. He discovered the regulators of Ca2+ release and consequent cellular processes. Chemical Approach to Signal Transduction by Inositol Triphosphate


Introduction
The fact that diacyl glycerol is second messenger was found by late Professor Yasutomi Nishizuka [1] and the fact that Inositol triphosphate (IP 3 ) is a second messenger was discovered by Michael Berridge who showed that it functioned to release Ca 2+ from internal stores. This bifurcating signaling system is of fundamental importance in regulating a wide range of cellular process.
Signals (first messenger) like light, noise, taste, odor, hormone, neurotransmitter, drug attach to the plasma membrane where they are recognized by cell surface receptors. Upon binding of the ligand to the appropriate receptor, activation of G protein activates in turn phospholipase C. Active phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) giving rise to two products: 1,2-diacylglycerol and inositol 1,4,5-triphosphate (IP 3 ). IP 3 stimulates the release of Ca 2+ from the intracellular stores in the endoplasmic reticulum through IP 3 receptor while regulating a wide range of cellular processes.

Why Plant Biosynthesize Inositol
The rice bran, wheat, corn contain much phytic acid (inositol hexaphosphate) as Ca salt. Plant make glucose by photo synthesis from carbon dioxide and water. Some of glucose is converted to inositol. Inositol is converted to phospholipids (PIP 2 ) and phytic acid. PIP 2 is converted to IP 3 and diacylglycerol. These two compounds are essential for signal transduction of plant. Plant makes phytic acid as storage of phosphorous. Phosphorous is an essential atom as fertilizer because ii is an essential atom to make nucleic acid, DNA. The seed store phosphorous atom as a store so that even when seed germinate at no phosphorous land [1].

Discovery of IP 3
Phospholipid was discovered by Bollow in 1961 [2] from bovine brain. The hypothesis of Michell [3] that the receptor controlled hydrolysis of phosphoinositides could be directly linked to cellular calcium mobilization. The observation by Berridge D-myo-inositol 1,4,5-trisphosphate (IP 3 ) act as a second messenger, a fundamental cell-signal transduction mechanism has been elucidated. IP 3 stimulates the release of Ca 2+ from the intracellular stores in the endoplasmic reticulum through IP 3 receptor while regulating a wide range of cellular processes .

Synthetic Competition of Inositol P
The discovery of inositol phosphate in particular IP 3 led to the dramatic stimulation for the synthesis of inositol phosphates. Many persons challenged the synthesis of inositol phosphate, starting from inositol, glucurolactone, phytic acid, arenas, quinic acid and L-quebrachitol.
A symposium; Inositol phosphates and Derivatives. Synthesis, biochemistry, and therapeutic potential was held by the division of carbohydrate Chemistry at the 200 th National meeting of the American Chemical Society, Washington DC, August 26-31.1990. ACS Symposium Series. 463 Edited by Allen B.Reitz was published.
The key problems in the synthesis of inositol phosphates are (1) synthesis and optical resolution of suitably protected inositol derivatives, (2) efficient phosphorylation of vicinal hydroxy groups.
In 1986, Ozaki et al succeeded in the first total synthesis of optically active myo-inositol tris (1,4,5) phosphate from myo-inositol by 13 steps [26]. At this report, phosphorylation yield of 2,3,6-tribenzyl myoinositol by dianilidephophotyl chloride isolation yield was only 10%. Then we have studied phosphorylation reagents and discovered new phsphorylation method Then we could get IP 3

Methods to Get Optically Pure Compound by 5 Different Methods › Separation of diastereomers
• L-mentoxy acetyl chloride gave best result, because desired product was crystal [26] › Starting from optically pure natural product
. This reagent was used for the synthesis of phosphofloridate analogues. Obtained phosphofloridates showed very interesting biological activity [56].

Discovery of Phosphonium Salt Methodology
This phosphonium salt methodology [59,60] provide a regioselective phospholylation. 1,2-Diol were phosphorylated regioselectively at C-1 with tribebzyl phosphite to give 1-dibenzyl phosphate 2-hyroxy free compound as shown in Figure 2. Other phosphory lating reagents do not have such selectivity. By using this free hydroxy group, we could get 2-acyl analog and IPx and PIPx. Three kind of combined reagents are possible.

P + TeCl 4
The reaction of an alcohol with a trialkylphophite in the presence of pyridinuum bromide per bromide proceed via the phosphonium salt (RO) 3 P + Br to afford the triester R1-OP(O)(OR) 2 , which can be converted to the phosphoric monoester by deprotection.
On the other hand, starting from dialkyl phosphoamidite (R0) 2 PNR,'the corresponding triester product ( The reactivity of phosphonium salt toward an alcohol seems to be between P III and P V , therefore we expected that the phosphonium salt methodology would provide a regioselective phosphorylation method. 1,2-Diol was phosphorylated regioselectively. Applying the phosphonium salt approach to the synthesis of phosphoinositides, the use of glyceryl phosphite, which was derived by the reaction of the glycerol derivatives with dimethylphosphoramidite in the presence of tetrazol, gave the protected PI(4,5)P2.

Finding of novel deacylation Methods
A Grignard reagent was used for deacylation without affecting the neighboring base-sensitive functional groups [76].

Use of Inositol Derivatives as Chiral Auxialiaries
• Diastereoselective addition of organometallics to keto esters [74].

2-substituted IP 3 analogs
• These were synthesized as shown in Figure 3. These analogs were used for the preparation of affinity columns [96].
The three phosphofluoridates thus prepared had potencies for inhibiting ( 3 H) InsP 3 binding to purified InsP 3 receptor that were less than for InsP 3 . Two analogues 44 and 40 were found to inhibit the dephosphorylation of ( 1 H) Ins P 3 by the 5-phsphatase with potencies similar to that for InsP 3 . Surprisingly, the inhibitory potency of 5-phosphofluoridate 44 toward 5-phosphosphatase was higher (about 20 fold) than those of InsP 3 and the another fluoridates 40 and 45.

Isolation and Characterization of Many IP 3 -Binding Proteins
The following many proteins were isolated by affinity column and characterizations were carried out [103,111].

Observation of Behaviors of IPx, PIPx and Ca 2+ Flux in the cells
IP 3 and PIPx are charged compounds. Therefore they do not readily penetrate through cell membranes. But it becomes permeable following salt formation with amine and this is a new method to put IP n or PIP n into the cell [127][128][129][130].
Ozaki synthesized about 20 fluorescent IP 3 , IP 4 , PIP, PIP 2 containing fluorescent amines with green and red colors (Figure 6), and introduced these tagged molecules into cells. He then used fluorescent microscopy.
He observed how and how fast the IP n or PIP n entered into the cell and how moved and how changed, metabolized and also observed a calcium flux (time, location, concentration) in NIH 3T3 Fibloblasts, when complexes of carrier and Ptd Ins (4,5) or Inos (1,4,5) P 3 were added extra cellular. He took more than a thousand pictures and movies.

Detection of Ca 2+ Flux
Fluoro-3 was used to measure intracellular calcium concentration. In case of IP 3 complex, Ca 2+ maximum peak (2.5×10 -7 M) was observed after 3.5 min. after addition of IP 3 . In case of PIPx complex, Ca 2+ maximum peak (4.2×10 -7 M) was observed after 6.5 min.

Discovery of DAB; Regulators of Ca 2+ Release and Cellular Response
In 1997, we identified 2-aminoethyl diphenylborinate (2-APB) as being an IP 3 receptor inhibiter and regulate IP 3 induced calcium release [131,132]. This discovery rose a substantial interest and had a great impact as it gained more than 600 citations and more than 1000 studies on 2-APB have been published so far. This was supported by increasing sales of 2-APB by Sigma-Aldrich as membrane-permeable modulator of calcium release. We aimed at generate better modulator of calcium release than 2-APB.
We synthesizes several 2APB analogues and measured their inhibitory activities on Store Operated Calcium Entry (SOCE) and IP 3 Induced Calcium Release (IICR).
2APB analogues presented in this study could be proven to be excellent lead compounds for many human diseases including heart disease [143,144], Alzheimer`s [145][146] and Huntington disease [148,149].
We found that boron compounds also can inhibit transglutaminase (Ca 2+ -dependent enzyme) [130]. There are many neurodegenerative disease, including Alzheimer`s disease, Huntington`s disease [136,149]. The boron compounds were found to be effective as inhibitor of acyl protein thioesterase [150].