Liquid chromatography/mass spectrometry based detection and semi‐quantitative analysis of INSL5 in human and murine tissues

Rationale

Insulin‐like peptide 5 (INSL5) is a hormone produced by enteroendocrine L‐cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field.

Methods

Peptides from purified murine L‐cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid‐phase extraction.

Results

Mass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L‐cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L‐cell stimulus.

Conclusions

The first LC/MS/MS‐based method capable of the detection and semi‐quantitative analysis of endogenous INSL5 using MS‐based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.     

Regioselective 1,3-Dipolar Cycloaddition Reactions of Unsymmetrical Münchnones (1,3-Oxazolium-5-olates) with 2- and 3-Nitroindoles. A New Synthesis of Pyrrolo[3,4-b]indoles

The unsymmetrical mesoionic münchnones 13 (3-benzyl-2-methyl-4-phenyl-1,3-oxazolium-5-olate) and 14 (3-benzyl-4-methyl-2-phenyl-1,3-oxazolium-5-olate) react with the N-protected 2- and 3-nitroindoles 1 (ethyl 2-nitroindole-1-carboxylate), 6 (3-nitro-1-(phenylsulfonyl)indole), and 17 (ethyl 3-nitroindole-1-carboxylate) in refluxing THF to afford in good to excellent yields the pyrrolo[3,4-b]indoles 15 (2-benzyl-1-methyl-3-phenyl-4-carboethoxy-2,4-dihydropyrrolo[3,4-b]indole), 16 (2-benzyl-3-methyl-1-phenyl-4-carboethoxy-2,4-dihydropyrrolo[3,4-b]indole), 18 (2-benzyl-1-methyl-3-phenyl-4-(phenylsulfonyl)-2,4-dihydropyrrolo[3,4-b]indole), and 19 (2-benzyl-3-methyl-1-phenyl-4-(phenylsulfonyl)-2,4-dihydropyrrolo[3,4-b]indole). In several cases the regiochemistry, which is opposite to that predicted by FMO theory, is very high and leads essentially to a single pyrrolo[3,4-b]indole; e.g., 6+13→19 in 74% yield.     

Synthesis of a novel dicyano abietane analogue: a potential antiinflammatory agent

From a structure-activity relationship perspective, the new abietane 5 having cyano groups at C-2 and C-13 and a phenolic ring C has been synthesized and evaluated biologically because the related compound 4 has high potency in inflammation models in vitro and in vivo. Compound 5 was synthesized from 8, which was obtained in five steps from the known compound 9, via an unexpected aromatization caused by the addition of PhSeCl and subsequent oxidation/elimination of the selenated intermediate 14 with H2O2.     

Studies on the Preparation of 2-Indolyl Triflates and Related Compounds

Upon treatment with trifluoromethanesulfonic acid anhydride (triflic anhydride), oxindole (4) is converted into 1-(trifluoromethanesulfonyl)indol-2-y1 trifluoromethanesulfonate (10) in 70% yield. Some attempted reactions of 1-(trifluoromethanesulfonyl)indole (13), conveniently prepared by treating indole (12) with n-butyllithium and triflic anhydride, are described.     

Mn(III)-based radical addition reactions of 2-nitroindole with activated CH compounds

2-Nitroindole undergoes addition reactions with the radicals generated from active CH compounds upon treatment with Mn(OAc)₃·2H₂O to afford the corresponding 3-substituted-2-nitroindoles in 27-66% yields. Products of the methylene addition reactions undergo a subsequent in situ Nef reaction to afford 2-oxoindolin-3-ylidenes.     

Structures of Three New (Phenylsulfonyl)Indole Derivatives

Abstract  

Three new 1-(phenylsulfonyl)indole derivatives, 3-acetyl-2-ethyl-1-(phenylsulfonyl)indole, (I), 2-benzyl-1-(phenylsulfonyl)indole, (II), and 3-trimethylsilylethynyl-1-(phenylsulfonyl)indole, (III), have been synthesized and their crystal structures determined by single crystal X-ray crystallography. (I), C₁₈H₁₇NO₃S, is triclinic with space group P-1 and cell constants: a = 8.6129(2) Å, b = 8.8366(2) Å, c = 11.0108(2) Å, α = 72.7920(1)°, β = 75.0120(1)°, γ = 77.2550(1)°, V = 763.79(3) ų and Z = 2. (II), C₂₁H₁₇NO₂S, is monoclinic with space group P21/n and cell constants: a = 10.8355(18) Å, b = 10.6987(19) Å, c = 14.472(3) Å, β = 75.0120(1), V = 1675.2(5) ų and Z = 4. (III), C₁₉H₁₉NO₂SSi, is monoclinic with, space group P21/c and cell constants: a = 12.9596(1) Å, b = 10.543(3) Å, c = 13.543(2) Å, β = 93.58°, V = 1846.8(6)ų and Z = 4. All three have the same (phenylsulfonyl)indole skeleton with different ligands attached to the 2- and 3-sites of the indole ring. The angle between the mean planes of the indole and benzyl groups is 76.9(6)° (I), 82.9(3)° (II), 89.6(2)° (III) and between the sulfonyl group with the indole and benzyl rings is 61.2(9)° (I), 59.0((5)° (II), 51.5(6)°, 42.5(8)° (III) and 51.5(6)°(I), 44.3(8)° (II), 47.2(8)° (III), respectively. In addition to the ligand substitutions, weak intermolecular C–H···O hydrogen bond interactions and π stacking interactions between the π orbitals of nearby indole and phenyl rings influence these twist angles. Comparison to a MOPAC AM1 computational calculation supports these observations.