Direct Quantitative Analysis of a 20 kDa PEGylated Human Calcitonin Gene Peptide Antagonist in Cynomolgus Monkey Serum Using In-Source CID and UPLC-MS/MS

PEGylation is a successful strategy to improve the pharmacokinetic and pharmaceutical properties of therapeutic peptides. However, quantitative analysis of PEGylated peptides in biomatrix by LC-MS/MS poses significant analytical challenge due to the polydispersity of the polyethylene glycol (PEG), and the multiple charge states observed for both the peptide and PEG moieties. In this report, a novel LC-MS/MS method for direct quantitative analysis of 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkey serum is presented. CGRP[Cit, Cit] is an investigational human calcitonin gene peptide receptor antagonist with amino acid sequence Ac -WVTH[Cit]LAGLLS[Cit]SGGVVRKNFVPT DVGPFAF-NH ₂ . In-source collision-induced dissociation (in-source CID) of 20 kDa PEGylated peptide was used to generate CGRP[Cit, Cit] fragment ions, among which the most abundant b ₈⁺ ion was selected and measured as a surrogate for the 20 kDa PEGylated peptide. A solid phase extraction (SPE) method was used to extract the PEGylated peptides from the biomatrix prior to the UPLC-MS/MS analysis. This method achieved a lower limit of quantitation (LLOQ) of 5.00 ng/mL with a serum sample volume of 100 μL, and was linear over the calibration range of 5.00 to 500 ng/mL in cynomolgus monkey serum. Intraday and interday accuracy and precision from QC samples were within ±15%. This method was successfully applied to a pharmacokinetic study of the 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkeys.     

Acylated Triterpene Saponins from Atroxima liberica Stapf

The four new acylated triterpene saponins 1 – 4 , isolated as two pairs of isomers and named libericosides A₁/A₂ and B₁/B₂, one pair of isomers 5 / 6 , the (Z)‐isomer libericoside C₂ ( 5 ) being new, one new sucrose ester, atroximoside ( 7 ), and eight known compounds were isolated from the roots of Atroxima liberica by repeated MPLC and VLC on normal and reversed‐phase silica gel. Their structures were elucidated on the basis of extensive 1D‐ and 2D‐NMR studies (¹H‐ and ¹³C‐NMR, DEPT, COSY, TOCSY, NOESY, HSQC, and HMBC) and mass spectrometry as 3‐O‐β‐D ‐glucopyranosylpresenegenin 28‐{O‐α‐L ‐arabinopyranosyl‐(1→3)‐O‐β‐D ‐xylopyranosyl‐(1→4)‐O‐α‐L ‐rhamnopyranosyl‐(1→2)‐4‐O‐[(E)‐3,4‐dimethoxycinnamoyl]‐β‐D ‐fucopyranosyl} ester ( 1 ) and its (Z)‐isomer 2 , 3‐O‐β‐D ‐glucopyranosylpresenegenin 28‐{O‐α‐L ‐arabinopyranosyl‐(1→4)‐O‐β‐D ‐xylopyranosyl‐(1→4)‐O‐α‐L ‐rhamnopyranosyl‐(1→2)‐O‐[O‐β‐D ‐xylopyranosyl‐(1→3)‐β‐D ‐glucopyranosyl‐(1→3)]‐4‐O‐[(E)‐3,4‐dimethoxycinnamoyl]‐β‐D ‐fucopyranosyl} ester ( 3 ) and its (Z)‐isomer 4 , 3‐O‐β‐D ‐glucopyranosylpresenegenin 28‐{O‐β‐D ‐xylopyranosyl‐(1→4)‐O‐α‐L ‐rhamnopyranosyl‐(1→2)‐O‐[6‐O‐acetyl‐β‐D ‐glucopyranosyl‐(1→3)]‐4‐O‐[(Z)‐3,4‐dimethoxycinnamoyl]‐β‐D ‐fucopyranosyl} ester ( 5 ), and 3‐O‐[(Z)‐feruloyl]‐β‐D ‐fructofuranosyl α‐D ‐glucopyranoside ( 7 ). Compounds 1 – 6 and the known saponins 8 / 9 were evaluated against the human colon cancer cells HCT 116 and HT‐29 and showed moderate to weak cytotoxicity.     

Solvation Properties of Aliphatic Alcohol–Water and Fluorinated Alcohol–Water Solutions for Amide Molecules Studied by IR and NMR Techniques

Solvation properties of aliphatic alcohol–water and fluorinated alcohol–water solutions were probed by amide molecules as solutes using infrared (IR) and ¹H and ¹³C NMR techniques. These include four alcohols: ethanol (EtOH), 2-propanol (2-PrOH), 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and three amides: N-methylformamide (NMF), N-methylacetamide (NMA) and N-methylpropionamide (NMP). The hydrogen bonds of the amide carbonyl oxygen with water are gradually weakened as the alcohol content increases. This decreases in the order of HFIP > TFE ≈ 2-PrOH > EtOH. In TFE– and HFIP–water solutions, the hydrogen bond between the amide amino hydrogen and water is also gradually broken with increasing x _A. This trend is more notable in the order of NMP > NMA > NMF. The hydrophobic moieties of the amide methyl and ethyl groups are solvated by the fluoroalkyl groups of fluorinated alcohols due to the hydrophobic interaction among them. Thus, the steric hindrance generated by the solvated alkyl group of amides promotes the breaking of the hydrogen bonds between amide and water.     

Harmonic vibrational frequencies: scale factors for pure, hybrid, hybrid meta, and double-hybrid functionals in conjunction with correlation consistent basis sets

Scale factors for (a) low (<1000 cm(-1)) and high harmonic vibrational frequencies, (b) thermal contributions to enthalpy and entropy, and (c) zero-point vibrational energies have been determined for five hybrid functionals (B3P86, B3PW91, PBE1PBE, BH&HLYP, MPW1K), five pure functionals (BLYP, BPW91, PBEPBE, HCTH93, and BP86), four hybrid meta functionals (M05, M05-2X, M06, and M06-2X) and one double-hybrid functional (B2GP-PLYP) in combination with the correlation consistent basis sets [cc-pVnZ and aug-cc-pVnZ, n = D(2),T(3),Q(4)]. Calculations for vibrational frequencies were carried out on 41 organic molecules and an additional set of 22 small molecules was used for the zero-point vibrational energy scale factors. Before scaling, approximately 25% of the calculated frequencies were within 3% of experimental frequencies. Upon application of the derived scale factors, nearly 90% of the calculated frequencies deviated less than 3% from the experimental frequencies for all of the functionals when the augmented correlation consistent basis sets were used.     

Emission-photoactivity cross-processing of mesoporous interfacial charge transfer in Eu3+ doped titania

Periodic mesoporous Eu(3+) doped titania materials were obtained through the EISA (Evaporation Induced Self Assembly) process. Eu(3+) ions, entrapped within the semi-crystalline walls of the highly porous framework, appear to be advantageous during the probing of surface photochemical reactions. Its emission intensity is very sensitive to the presence of physisorbed molecules, in gas or liquid phase, that reside within the pores. In particular, strong fluctuations in intensity of the (5)D(0)→(7)F(2) transition were observed under UV light exposure on the time scale of tens of seconds. The emission modulation dynamics show a strong correlation with the crystallinity of the titania matrix. Correlation of the emission with the photocatalytic activity of the semiconductor for photodegradation of an organic molecule is observed. A model is proposed to describe the involved mechanisms.     

Proton transfer events in GFP

Proton transfer is one of the most important elementary processes in biology. Green fluorescent protein (GFP) serves as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. Illumination initiates proton transfer through a 'proton-wire', formed by the chromophore (the proton donor), water molecule W22, Ser205 and Glu222 (the acceptor), on a picosecond time scale. To obtain a more refined view of this process, we have used a combined approach of time resolved mid-infrared spectroscopy and visible pump-dump-probe spectroscopy to resolve with atomic resolution how and how fast protons move through this wire. Our results indicate that absorption of light by GFP induces in 3 ps (10 ps in D(2)O) a shift of the equilibrium positions of all protons in the H-bonded network, leading to a partial protonation of Glu222 and to a so-called low barrier hydrogen bond (LBHB) for the chromophore's proton, giving rise to dual emission at 475 and 508 nm. This state is followed by a repositioning of the protons on the wire in 10 ps (80 ps in D(2)O), ultimately forming the fully deprotonated chromophore and protonated Glu222.     

The perfluorinated alcohols (F5C6)(F3C)2COH and (F5C6)(F10C5)COH: synthesis, theoretical and acidity studies, spectroscopy and structures in the solid state and the gas phase

The syntheses of the perfluorinated alcohols (F(5)C(6))(F(3)C)(2)COH (1) and (F(5)C(6))(C(5)F(10))COH (2) are described. Both compounds were prepared in reasonable yields (1: 65%, 2: 85%) by reacting the corresponding ketone with C(6)F(5)MgBr, followed by acidic work-up. The alcohols were characterized by NMR, vibrational spectroscopy, single-crystal X-ray diffraction, acidity measurements and gas-phase electron diffraction. A combination of appropriate 2D NMR experiments allowed the unambiguous assignment of all signals in the (19)F spin systems, of which that of 2 was especially complex. High acidity of the alcohols is indicated by acidity measurements as well as the calculated gas phase acidities. It is also supported by the crystal structure of 2, which exhibits only a single weak intermolecular hydrogen bridge with an O...O distance of 301 pm. This shows the low donor strength of the oxygen atom in the compound, which is partly compensated through formation of two intramolecular CF...H contacts of 220 and 232 pm length to the proton not involved in the hydrogen bridge. The pK(a) values in acetonitrile are 22.2 for 1 and 22.0 for 2; their calculated gas phase acidities are 1367 and 1343 kJ mol(-1) (MP2/TZVPP level).