Synthesis and properties of novel lipopeptides and lipid mimetics

Lipid mimetics, synthetic molecules that resemble natural lipids either structurally or functionally, have been developed as potential medicinal substances. They have been successfully applied in the development of drug and peptide delivery systems and for the development of inhibitors or lipid metabolizing enzymes. Phospholipase A2 is considered to be involved as the rate‒limiting step in the production of lipid mediators of inflammatory responses and, as such, it has been a target for drug design. A series of lipid mimetics including lipopeptides, amides and alcohols of lipidic α‒amino acids, have been tested by bulk and monolayer assay techniques. The findings suggested the direct interaction of the tested compounds with porcine pancreatic phospholipase A2. The inactivation of the enzyme occurred in a competitive manner. The most active compound 1 (2-amino-N-hexadecyl-L -hexanamide) showed an apparent IC₅₀ of 12 μ M and inhibitory power Z=13 in the monolayer assay. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Study of aminoglycoside-nucleic acid interactions by an HPLC method

The interactions of a number of aminoglycoside antibiotics with tRNA and DNA were studied by an HPLC method. based on tRNA and DNA peak size exclusion. Among the compounds studied (deoxystreptamine, neamine, neomycin B, kanamycin A, gentamicin A, netilmicin, streptomycin, and the synthetic neamine analogue BKN3), neomycin B and the synthetic analogue of neamine were proved to be the most potent binders.     

A simple method to separate base population and segregation effects in genomic relationship matrices

Background

Genomic selection and estimation of genomic breeding values (GBV) are widely used in cattle and plant breeding. Several studies have attempted to detect population subdivision by investigating the structure of the genomic relationship matrix G. However, the question of how these effects influence GBV estimation using genomic best linear unbiased prediction (GBLUP) has received little attention.

Methods

We propose a simple method to decompose G into two independent covariance matrices, one describing the covariance that results from systematic differences in allele frequencies between groups at the pedigree base (G_A^*) and the other describing genomic relationships (G_S) corrected for these differences. Using this decomposition and F_st statistics, we examined whether observed genetic distances between genotyped subgroups within populations resulted from the heterogeneous genetic structure present at the base of the pedigree and/or from breed divergence. Using this decomposition, we tested three models in a forward prediction validation scenario on six traits using Brown Swiss and dual-purpose Fleckvieh cattle data. Model 0 (M0) used both components and is equivalent to the model using the standard G-matrix. Model 1 (M1) used G_S only and model 2 (M2), an extension of M1, included a fixed genetic group effect. Moreover, we analyzed the matrix of contributions of each base group (Q) and estimated the effects and prediction errors of each base group using M0 and M1.

Results

The proposed decomposition of G helped to examine the relative importance of the effects of base groups and segregation in a given population. We found significant differences between the effects of base groups for each breed. In forward prediction, differences between models in terms of validation reliability of estimated direct genomic values were small but predictive power was consistently lowest for M1. The relative advantage of M0 or M2 in prediction depended on breed, trait and genetic composition of the validation group. Our approach presents a general analogy with the use of genetic groups in conventional animal models and provides proof that standard GBLUP using G yields solutions equivalent to M0, where base groups are considered as correlated random effects within the additive genetic variance assigned to the genetic base.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Synthesis, in vitro cytotoxicity and in vivo anti-inflammatory activity of long chain 3-amino-1,2-diols

The synthesis of long chain 3-amino-1,2-diols was carried out based on Sharpless asymmetric epoxidation of long chain allylic alcohols and regioselective nucleophilic ring opening by azido group. The in vitro cytotoxicity of the compounds prepared was evaluated against six solid tumor cell lines (A2780, H322, LL, WiDr, C26-10, UMSCC-22B). Free 3-amino-1,2-diols exhibited IC50 values between 1.45 microM and 32 microM. These compounds also presented interesting inhibition of carrageenin-induced paw edema in rats (85.3% - 79.6% at a concentration of 0.15 mmol/kg).     

Synthesis of 2‐oxoamides based on sulfonamide analogs of γ‐amino acids and their activity on phospholipase A2

A variety of lipophilic 2‐oxoamides containing sulfonamide analogs of γ‐amino acids as well as acyl sulfonamides of γ‐aminobutyric acid were synthesized. Their ability to inhibit intracellular GIVA cPLA₂ and GVIA iPLA₂ as well as secreted GV sPLA₂ was evaluated. The sulfonamide group seems a bioisosteric group suitable to replace the carboxyl group in 2‐oxoamide inhibitors of GVIA cPLA₂. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of enantiopure non-natural alpha-amino acids using tert-butyl (2S)-2-[bis-(tert-butoxycarbonyl)amino]-5-oxopentanoate as key-intermediate:the first synthesis of(S)-2-amino-oleic acid.

A general method for the synthesis of enantiopure non-natural alpha-amino acids is described. The key intermediate tert-butyl (2S)-2-[bis(tert-butoxycarbonyl)amino]-5-oxopentanoate was obtained from l-glutamic acid after suitable protection and selective reduction of the gamma-methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of delta,epsilon-unsaturated alpha-amino acids. This methodology was applied to the synthesis of (S)-2-amino-oleic acid.     

Synthesis of medicinally useful lipidicα-amino acids, 2-amino alcohols and diamines

Summary The lipidic-amino acids (LAAs) are non-natural-amino acids with saturated or unsaturated long aliphatic side chains. LAAs and their derivatives (lipid mimetics) together with the lipidic peptides represent a class of compounds which combine structural features of lipids with those of amino acids and peptides. Racemic LAAs may be prepared by classical methods and resolved by chemical or enzymatic methods. LAA amides and esters with saturated or unsaturated long chain amines and alcohols respectively, as well as lipidic dipeptide derivatives inhibit both pancreatic and human platelet phospholipase A₂. Lipophilic peptide derivatives are inhibitors of human neutrophil elastase. LAAs and their oligomers have been used as drug delivery system. A Lipid-Core-Peptide system has been designed and used as a combined adjuvant-carrier-vaccine system. A variety of lipid mimetics such as lipidic 2-amino alcohols, lipidic 1,2- and 1,3-diamines have been prepared based upon LAAs. Some of them are potent inhibitors of phospholipase A₂. A general approach to enantioselective synthesis of LAAs and lipid mimetics is based on the oxidative cleavage of 3-amino-1,2-diols obtained by the regioselective opening of enantiomerically enriched long chain 2,3-epoxy alcohols.Abbreviations Boc tert-butoxycarbonyl - BSA bovine serum albumin - CD circular dichroism - DET diethyl tartrate - DIBAL diisobutyl aluminum hydride - DMF N,N-dimethylformammide - HMPA hexamethylphosphoramide - HNE human neutophil elastase - LAA lipidic amino acid - LAAL lipidic amino alcohol - LH-RH luteinizing hormone-releasing hormone - LCP lipid-core-peptide - LDA lipidic diamine - LP lipidic peptide - MAP multiple antigenic peptide - PLA₂ phospholipase A₂ - TBHP tert-butyl hydroperoxide - THF tetrahydrofuran - TRH thyrotropin-releasing hormone - Z benzyloxycarbonyl