2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34⁺ haematopoietic stem cells.

In this study we showed that 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)-TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte-specific cell surface markers showed a dramatic increase in expression in response to (R)-TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34(+) haematopoietic stem cells. Activation of the PI3K-AKT pathway and, to a lesser extent, the MEK-ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)-TEMOSPho-treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P-selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)-TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia.

Low pressure ethenolysis of renewable methyl oleate in a microchemical system.

A microchemical system for ethenolysis of renewable methyl oleate was developed, in which the dual-phase, microfluidic design enabled efficient diffusion of ethylene gas into liquid methyl oleate through an increased contact area. The increased mass transfer of ethylene favored the formation of desired commodity chemicals with significantly suppressed homometathesis when compared to the bulk system. In addition to higher selectivity and conversion, this system also provides the typical advantages of a microchemical system, including the possibility of convenient scale-up.

Effects of macrocycle size and rigidity on melanocortin receptor-1 and -5 selectivity in cyclic lactam alpha-melanocyte-stimulating hormone analogs.

The effects of the linker arm rigidity and size on melanocortin receptor selectivity were explored in a series of compounds using cyclic lactam alpha-melanocyte-stimulating hormone template. A variety of dicarboxylic acid linkers introduced between the alpha-amino group of His(6) and the epsilon-amino group of Lys(10) lead to high-affinity, selective human melanocortin receptor-1 and -5 (hMC1R and hMC5R) antagonists. The incorporation of hydrophilic functions into the linker arm was found to be unfavorable for both binding potency and receptor selectivity. Analogs 8 and 9 containing highly conformationally constrained hydrophobic linkers (m- and p-phthalic acids) were found to be selective nanomolar range hMC1R antagonists (IC(50) = 7 and 4 nm, respectively), whereas the employment of a small conformationally constrained linker (maleic acid) resulted in a high-affinity (IC(50) = 19 nm) and selective hMC5R antagonist (analog 12). These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-1 and -5 receptors.

Purification and structural determination of hematopoietic stem cell-stimulating monoacetyldiglycerides from Cervus nippon (deer antler).

A mixture of monoacetyldiglycerides was newly isolated from the chloroform extract of antlers of Cervus nippon, guided by the hematopoietic stimulation of stem cells. The structures of monoacetyldiglycerides were determined by various spectroscopic methods: FAB MS, CID tandem MS, and 1D and 2D NMR. A mixture of at least nine inseparable sn-3-monoacetyldiglycerides was identified: 1 [C(39)H(72)O(6) (C16 : 0/C18 : 1)], 2 [C(39)H(72)O(6) (C18 : 1/C16 : 0)], 3 [C(39)H(70)O(6) (C16 : 0/C18 : 2)], 4 [C(39)H(70)O(6) (C18 : 2/C16 : 0)], 5 [C(41)H(74)O(6) (C18 : 0/C18 : 2), 6 [C(41)H(74)O(6) (C18 : 2/C18 : 0)], 7 [C(41)H(74)O(6) (C18 : 1/C18 : 1)], 8 [C(43)H(74)O(6) (C18 : 0/C20 : 4)], and 9 [C(43)H(74)O(6) (C20 : 4/C18 : 0)]. Among these nine monoacetyldiglycerides in deer antlers, compound 3 was one of the major compounds and was efficiently synthesized from glycerol. Spectral data of synthetic monoacetyldiglyceride 3 were compared with the corresponding data for the mixture of natural monoacetyldiglycerides. The mixture of natural monoacetyldiglycerides from deer antlers showed potent activity on the hematopoiesis (stimulation index=1.40+/-0.05, p<0.02 at 1 microg/ml), and synthetic monoacetyldiglyceride 3 showed even better activity (stimulation index=1.54+/-0.12, p<0.001, at 1 microg/ml).

Stimulatory effects of monoacetyldiglycerides on hematopoiesis.

Monoacetyldiglycerides purified from deer antler, and identical synthetic compounds, have been shown to stimulate hematopoiesis. In the present study, we synthesized eight monoacetyldiglycerides, one of which, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (3), was structurally identical to one of the naturally occurring monoacetyldiglycerides and showed the most potent stimulation of hematopoiesis by colony forming unit in culture (CFUc) assay, having a stimulation index (SI) of 1.54+/-0.23 at a concentration of 1.0 microg/ml. Moreover, 1.0 microg/ml 3 showed potent growth-stimulation activities on megakaryocyte culture, long term culture of Lin(-)Sca-1(+) cells with irradiated MS-5 stromal cells (SI, 1.69+/-0.16), and on the number of cobblestone colonies (SI, 10.4+/-0.25). In a murine model, 3, at concentrations of 0.5, 5 and 50 mg/kg/d, i.p. and p.o., effectively stimulated hematopoiesis in vivo 7 d after syngenic bone marrow transplantation of irradiated C57BL/6 mice, when assayed by the colony forming units in spleen (CFUs) assay. These data suggest that monoacetyldiglycerides may have significant clinical potential for the acceleration of hematopoiesis.

Modification of concanavalin A-dependent proliferation by phosphatidylcholines isolated from deer antler, Cervus elaphus.

OBJECTIVE: The immunomodulatory effect of deer antler, which is used as traditional medicine, has been known, but the active component of antlers from Cervus elaphus has not been identified. In this study, we identified the immunomodulator from C. elaphus and examined its biological activities on the immune system. METHODS: To identify an immunomodulator, we used bioassay-guided fractionation after silica gel column chromatography. Structural analysis was performed with one- and two-dimensional nuclear magnetic resonance techniques and tandem mass spectrometry coupled with fast atom bombardment. RESULTS: The subfraction, phosphatidylcholines, isolated 70% ethanol extract of C. elaphus induced the proliferation of spleen cells in synergy with concanavalin A. According to the structural analysis, phosphatidylcholines were classified as a family (1,2-alkyl-sn-glycerol-3-phosphocholines) containing arachidonyl (C20:4), stearoyl (C18:0), oleoyl (C18:1), linoleoyl (C18:2), palmitoyl (C16:0), and myristoyl (C14:0) chains in their fatty acyl chains. Because the unsaturated fatty acids showed an inhibitory effect on the immune system, dialkyl phosphatidylcholines with different chain lengths from C10:0 to C20:0 that stimulate the proliferation of spleen cells were examined extensively. Among other saturated phosphatidylcholines used, dimyristoyl phosphatidylcholine (C14:0) induced the proliferation of spleen cells more efficiently, whereas dimyristoleoyl phosphatidylcholine (C14:1) effected little change in the proliferation of spleen cells. CONCLUSIONS: These data collectively suggest that phosphatidylcholines with saturated fatty acyl chains are immunostimulating factors. They may modify the proliferation of known mitogens. Further, chain length and saturation of the fatty acids may play important roles in the proliferation of spleen cells.

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH.

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.

Synthesis of novel lysophosphatidylcholine analogues using serine as chiral template.

Four novel lysophosphatidylcholine (lysoPC) analogues, (S)-N-stearoyl-O-phosphocholineserine methyl ester [(S)-1a], (R)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine [(R)-2a], (R)-N-stearoyl-O-phosphocholineserine methyl ester [(R)-1b], and (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine [(S)-2b], were synthesized starting from serine as a chiral template. These synthetic compounds exhibited greatly enhanced hyphal transition inhibitory activity in Candida as compared to the natural lysoPC.

Indium-mediated reductive elimination of halohydrins.

Olefin formation has been successfully carried out by reductive elimination reactions of halohydrins with Pd(PPh(3))(4)/In/InCl(3) in aqueous media.

An efficient synthesis of 2,5-diketopiperazine derivatives by the Ugi four-center three-component reaction.

A facile synthetic approach to 2,5-diketopiperazines 4 by the Ugi four-center three-component reaction using commercially available dipeptides as a bifunctional component, aldehydes, and isocyanides was described.

Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry.

The structural determination of sn-1 and sn-2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn-1 and sn-2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H](+) ions was shown to be greatly different. Moreover, the CID-MS/MS spectra of sodium-adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na - 103](+), [M + Na - 85](+), and [M + Na - 59](+), by which their regio-specificity can be differentiated.