Leif Grehn

New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.

Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

Synthesis, properties, and use of Nin-Boc-tryptophan derivatives

N in -Boc-protected tryptophan detivatines can be prepared with Boc2O–4-dimethylaminopyridine in MeCN; their properties and utility are demonstrated in the synthesis of a tryptophan-containing tetrapeptide related to substance P.

Synthesis of amino acid derivatives substituted in the backbone with stable isotopes for application in peptide synthesis

Starting from the corresponding precursors, 1 and 2, the two well known procedures for asymmetric synthesis of amino acids, those of Schollkopf and Oppolzer, were compared for the preparation of [1,2-13C2,15N]-labelled (substituted) Boc-leucine enantiomers. Although both gave the final products in comparable overall yields, analysis of optical purity by two independent chromatographic methods revealed that the two procedures differed considerably in this respect. The enantiomeric excess found was 97.2–97.4 and 99.7%, respectively. As a consequence, the latter method was preferred for the synthesis of a number of additional backbone-labelled Boc-derivatives of the three proteinogenic amino acids alanine, phenylalanine and tyrosine, including such deuteriated in the amino acid side-chain. These derivatives exemplify precursors suitable for chemical synthesis of specifically backbone-labelled peptides and should allow greater exploitation of the properties of the 13C and, especially, the 15N nuclei in structural studies.

Reductive cleavage of N-substituted aromatic amides as tert-butyl acylcarbamates

Synthetic and spectroscopic details relating to a set of heteroaromatic N-benzyl carboxamides and in particular the corresponding tert-butyl acylcarbamates are reported. These compounds were required to study the postulated effect of various heterocycles (pyridine and pyrazine with and without condensed benzene rings) on the cleavage of acyl–N bonds by reduction. All compounds were initially characterized by cyclic voltammetry (CV) which indicated various degrees of facilitated reduction, reflecting a direct influence of the heterocyclic component. Selected acylcarbamates were studied with respect to acyl–N bond cleavage by mild reducing agents, and selectively deacylated by activated aluminium and sodium borohydride. Conversion to acylcarbamates followed by reduction might therefore be a mild, efficient two-step procedure to effect cleavage of amides, allowing isolation of carbamates and with sodium borohydride also the corresponding alcohols.

Glutathione transferase activity with a novel substrate mimics the activation of the prodrug azathioprine.

Azathioprine is a prodrug that is widely used clinically as an immunosuppressive agent. The pharmacological action of azathioprine is associated with the release of 6-mercaptopurine by a reaction involving glutathione. This biotransformation of azathioprine is catalyzed by glutathione transferases (GSTs). The nonenzymatic reaction with glutathione is minimal in comparison with the GST-catalyzed process, but azathioprine is still a slow substrate in comparison with the most effective GST substrates. Novel GSTs with higher catalytic efficiency toward azathioprine could be useful in novel therapeutic applications; therefore, directed evolution of GSTs for enhanced activities is desirable. However, screening for variants having higher catalytic activity with azathioprine is a time-consuming process due to the low activity with this substrate. A new chromogenic and faster substrate, 1-methyl-4-nitro-5-(4-nitrophenylthio)-1H-imidazole (NPTI), has been synthesized and characterized by assays with several GSTs. The novel substrate mimicked azathioprine in the reaction with glutathione catalyzed by alpha class GSTs and, therefore, is a valuable surrogate in the screening of large mutant libraries. NPTI may also find use in the elucidation of the exact mechanism of immunosuppression effected by azathioprine where there is evidence that the imidazole moiety of azathioprine, rather than 6-mercaptopurine, is involved.

Mild, efficient cleavage of arenesulfonamides by magnesium reduction

The cleavage of arenesulfonamides via N-arenesulfonylcarbamates is achieved within a few minutes under ultrasonic conditions by reaction with magnesium in anhydrous methanol.

New reagents for exhaustive alkoxycarbonylation of amides and urethans. Di-1-adamantyl di- and tricarbornate

Di-1-adamantyl, di- and tricarbonate, and di-t-butyl tricarbonate were found to be useful reagents for the DMAP-catalyzed alkoxycarbonylation of amides and urethans.

1-TERT-BUTOXYCARBONYL-1-TOSYL-HYDRAZINE AS REAGENT FOR THE SYNTHESIS OF SUBSTITUTED HYDRAZINES WITH A SECONDARY ALKYL GROUP

Abstract Catalytic hydrogenolysis of 1-Boc-1-Ts-2-Z-hydrazine furnished 1-Boc-1-Ts-hydrazine as a stable crystalline solid. Although its nucleophilicity is considerably suppressed in comparison with that of non- or monoacylated hydrazines, it reacted under various conditions with a representative set of ketones to give the corresponding hydrazones in high yields. The aliphatic hydrazones were readily reduced to hydrazines with NaBH4, whereas the aromatic analogues for smooth reduction required the more powerful NaBH3CN. With one exception all the new compounds were crystalline and stable under normal conditions. The new reagent and an alkylated derivative retained satisfactory reactivity towards activated isocyanates to provide acyl ureas. Some characteristic features of the NMR- and IR-spectra of these novel compounds are briefly discussed.

Basicity of 3-aminopropionamidine derivatives in water and dimethyl sulphoxide. Implication for a pivotal step in the synthesis of distamycin A analogues

The acid-base properties of eight 3-aminopropionamidine derivatives R1R2N(CH2)2C((DOUBLE BOND)NH)NR3R4 (1, R1 = R2 = R3 = R4 = H; 2, R1 = R3 = R4 = H, R2 = Me; 3, R1 = R2 = R4 = H, R3 = Me; 4, R1 = R2 = H, R3 = R4 = Me; 5, R1 = Tos, R2 = R3 = R4 = H; 6, R1 = Tos, R2 = Me R3 = R4 = H; 7, R1 = Tos, R2 = R4 = H, R3 = Me; 8, R1 = Tos, R2 = H, R3 = R4 = Me; Tos = 4-toluenesulphonyl) related to the antiviral natural product distamycin A were investigated in water and dimethyl sulphoxide (DMSO). The measured pKa values for the ammonium function in 1–4 in water ranged between 7·48 and 7·73, whereas the corresponding values in DMSO were 9·4 ± 0·3. The amidinium moiety of these compounds displayed pKa values in the range 11·4–12·0 and 13·4–13·6 in water and DMSO, respectively. The tosylamide group in compounds 5–8 was deprotonated in the expected pH region and exhibited pKa values between 9·49 and 10·02 in water, but was considerably less acidic in DMSO (14·5 ≤ pKa ≤ 15·7). The behaviour of the amidinium cation of 5–8 in water and DMSO resembled that of 1–4. The measured pKa values are discussed and the solvent-induced pKa shifts are explained in terms of solvent and substituent effects. The observed pKa differences between the ammonium and the amidinium functions in 1–4 render these compounds suitable intermediates in an alternative synthesis of distamycin A.

Synthesis of 15N-labelled chiral Boc-amino acids from triflates: enantiomers of leucine and phenylalanine

An efficient synthesis of 15N-labelled chiral Boc-amino acids by triflate alkylation of di-tert-butyl [15N]imidodicarbonate is reported. Both enantiomers of Boc-leucine and -phenylalanine were synthesized from commercial α-amino acids of opposite configuration viaα-hydroxy carboxylic acids provided by diazotization, thus extending the scope of an earlier exploratory study. The high chiral purity of the final products was confirmed by HPLC. These labelled amino acid derivatives are suitable for direct application to the synthesis of labelled peptides.