Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

Structural studies on aryl-substituted enaminoketones and their thio analogues. Part II. Experimental and DFT calculated carbon-carbon coupling constants, (n)J(CC)'s (n = 1-3).

Spin-spin carbon-carbon coupling constants across one, two and three bonds, J(CC), have been measured for a series of aryl-substituted Z-s-Z-s-E enaminoketones and their thio analogues. As a result, a large set, altogether 178, of J(CC)s has been obtained. It consists of 82 couplings across one bond, 31 couplings across two bonds and 65 couplings across three bonds. Independently, the DFT calculations at the B3PW91/6-311++G(d,p)//B3PW91/6-311++G(d,p) level yielded a set of theoretical J(CC) values. A comparison of these two sets of data gave an excellent linear correlation with parameters a and b close to ideal; a = 0.9978 which is not far from unity and b = 0.22 Hz which is close to zero. The (1)J(CC) couplings determined for the crucial fragment of the molecules, i.e. -C=C-C=O (or -C=C-C=S), are: (1)J(C=C) approximately 68 Hz (67 Hz) and (1)J(C-C) = 60.5 Hz (60.0 Hz). The corresponding couplings found for the Z-s-Z-s-E isomer of the parent enaminoketone, 4-methylamino-but-3-en-2-one are 64.1 and 59.3 Hz, respectively. The most sensitive towards substitution of the oxygen atom by sulfur are two-bond couplings between the alpha-vinylic and aromatic C(ipso) carbon atoms, which attain 12 Hz in the enaminoketone derivatives and decrease to 5 Hz in their thio analogues.

Structural studies on aryl-substituted enaminoketones and their thio analogues. Part I. Analysis of high-resolution (1)H, (13)C NMR and (13)C CP MAS spectra combined with GIAO-DFT calculations.

A series of aryl-substituted enaminoketones and their thio analogues in CDCl(3) solution and in the solid state were studied by the use of high-resolution (1)H and (13)C as well as (13)C cross polarization magic angle spinning (CP MAS) NMR spectra in combination with gauge including atomic orbitals-density functional theory (GIAO-DFT) calculations performed at the B3PW91/6-311 + + G(d,p) level of theory using the B3PW91/6-311 + + G(d,p)-optimized geometries. The analysis of the (13)C NMR spectra in solution was done by using the Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) technique, whereas trends observed in the (13)C shielding constants, calculated for the compounds studied, were a great help in assigning most of the signals in the (13)C CP MAS NMR spectra. It was established on the basis of the experimental and theoretical NMR data that both groups of compounds exist in the form of Z-s-Z-s-E isomers in CDCl(3) solution as well as in the solid state, with the NH hydrogen atom involved in intramolecular hydrogen bonding. This conclusion is in agreement with the fact that some of the compounds studied reveal liquid-crystalline properties. Three-bond H, H and C, H coupling constants measured in solution played a crucial role in the structure elucidation.