D-alanyl Residues Research Articles

Peptide-Cleavable Self-immolative Maytansinoid Antibody-Drug Conjugates Designed To Provide Improved Bystander Killing.

A new type of antibody–drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC’s linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate’s cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC’s cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.

Biogenesis of D-amino acid containing peptides/proteins: where, when and how?

Peptides and proteins are chiral molecules with their structure determined by the composition and configuration of the amino acids constituting them. Natural amino acids (except glycine) display two chiral types (l- and d-enantiomers). For example, the presence of octopine, a derivative of l-arginine and d-alanine in octopus, or peptidyl poly-d-glutamic acid in a bacterial cell wall was demonstrated in the 1920s and 1930s, respectively. Nevertheless, an old dogma in biology was that proteins (in a strict sense) are composed of amino acids in the l-configuration exclusively, until a d-alanyl residue was reported in a frog skin opioid peptide in the early 1980s, and since, numerous d-amino acid containing peptides (DAACPs) have been discovered in multicellular organisms. Several hypotheses may be formulated to explain the origin of a d-residue in the peptide/protein chain. It may result from different mechanisms such as incorporation of a d-amino acid, non-enzymatic racemisation associated with ageing or diseases and enzymatic posttranslational modification. In the last case, the DAACPs are synthesised via a ribosome-dependent manner, and a normal codon for l-amino acid is present in the mRNA at the position where the d-residue is processed in the mature peptide by peptidyl aminoacyl l-d isomerisation, a peculiar and subtle posttranslational modification. In this review, the different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l-d isomerisation.

Synthesis of the lipoteichoic acid of the Streptococcus species DSM 8747

The lipoteichoic acid (LTA) of the Streptococcus species DSM 8747 consists of a β- d-galactofuranosyl diacylglycerol moiety (with different acyl groups) that is linked via 6- O to a poly(glycerophosphate) backbone; about 30% of the glycerophosphate moieties carry at 2- O hydrolytically labile d-alanyl residues. As typical LTA for this array of compounds LTA 1a was synthesized. To this end, from d-galactose the required galactofuranosyl building block 5 was obtained. The anomeric stereocontrol in the glycosylation step with 1,2- O-cyclohexylidene- sn-glycerol ( 4) was based on anchimeric assistance, thus finally leading to the unprotected core glycolipid 16. Regioselective protection and deprotection procedures permitted the defined attachment of the pentameric glycerophosphate 3 to the 6-hydroxy group of the galactose residue. Introduction of four d-alanyl residues led after global deprotection and purification to target molecule 1a possessing on average about two d-alanyl residues at 2- O of the pentameric glycerophosphate backbone, thus being in close accordance with the structure of the natural material.

Synthesis of Staphylococcus aureus lipoteichoic acid derivatives for determining the minimal structural requirements for cytokine induction

For the investigation of the minimal structural requirements for cytokine induction, Staphylococcus aureus lipoteichoic acid derivatives with two, three, four, and five glycerophosphate backbone moieties, carrying each a d-alanyl residue, were needed. Based on two different glycerophosphate building blocks and 6b- O-phosphitylated gentiobiosyl diacylglycerol the desired target molecules (compounds 1– 4) could be readily obtained and provided for biological studies.

A Staphylococcus aureus lipoteichoic acid (LTA) derived structural variant with two diacylglycerol residues

Based on 1,2- O-isopropylidene- sn-glycerol five chiral building blocks containing differently modified glycerol residues were required for the synthesis of the target molecule 2. One of these building blocks is diacylglyceryl β-gentiobioside carrying a phosphite residue at 6b- O position. Ligation of these five building blocks led to the desired glycerol phosphate backbone to which d-alanyl residues were attached, thus generating after O-deprotection the target molecule 2, a bisamphiphilic structural variant of Staphylococcus aureus LTA. This compound displayed higher potency in terms of cytokine release by human blood leukocytes than the monoamphiphilic variant LTA.

Tripodal peptide hydroxamates as siderophore models. Iron(III) binding with ligands containing H-(alanyl)n-beta-(N-hydroxy)alanyl strands (n = 1-3) anchored by nitrilotriacetic acid.

Combining three units of one of H-(alanyl)n-beta-(HO)alanyl peptides (n = 1-3) with nitrilotriacetic acid affords tripodal peptide hydroxamate ligands (1L, 1D, 2LL, 2DL, and 3LLL, where each L or D denotes the L- or D-alanyl residue). These ligands form six-coordinate octahedral complexes (Fe-1L, Fe-1D, Fe-2LL, Fe-2DL, and Fe-3LLL) with iron(III) in aqueous near neutral pH solution, and the stability and the chirality of the complexes formed depend on the alanyl residues incorporated. Thus Fe-2LL is the most stable against attack of H+ and OH- ions and the least labile in the iron(III) removal by EDTA. The CD spectra show a predominance of the A configuration for Fe-1D, Fe-2LL, Fe-2DL, and Fe-3LLL, but the opposite delta configuration for Fe-1L. These ligands and their gallium(III) complexes are studied by 1H NMR spectroscopy in DMSO-d6 solution. CD and NMR spectral analysis, aided by molecular model examinations, indicates that critical factors in controlling the configuration and the stability of the complexes are (1) the hydroxamate-carrying alanyl residue, (2) the expanse of an interior space in the ligand, and (3) an interstrand amide NH hydrogen bond; the latter bonding is possible with ligands 2LL and 2DL. A microbial growth promotion activity test shows that ligands 1L, 2LL, and 3LLL all act as iron-transporting agents.

Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum.

Most bacteria synthesize muramyl-pentapeptide peptidoglycan precursors ending with a D-alanyl residue (e.g., UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala). However, it was recently demonstrated that other types of precursors, notably D-lactate-ending molecules, could be synthesized by several lactic acid bacteria. This particular feature leads to vancomycin resistance. Vancomycin is a glycopeptide antibiotic that blocks cell wall synthesis by the formation of a complex with the extremity of peptidoglycan precursors. Substitution of the terminal D-alanine by D-lactate reduces the affinity of the antibiotic for its target. Lactobacillus plantarum is a lactic acid bacterium naturally resistant to vancomycin. It converts most of the glycolytic pyruvate to L- and D-lactate by using stereospecific enzymes designated L- and D-lactate dehydrogenases, respectively. In the present study, we show that L. plantarum actually synthesizes D-lactate-ending peptidoglycan precursors. We also report the construction of a strain which is deficient for both D- and L-lactate dehydrogenase activities and which produces only trace amounts of D- and L-lactate. As a consequence, the peptidoglycan synthesis pathway is drastically affected. The wild-type precursor is still present, but a new type of D-alanine-ending precursor is also synthesized in large quantities, which results in a highly enhanced sensitivity to vancomycin.

The dacA gene of Bacillus stearothermophilus coding for D-alanine carboxypeptidase: cloning, structure and expression in Escherichia coli and Pichia pastoris.

The bacterial D-alanine carboxypeptidases (CPases) remove C-terminal D-alanyl residues from sugar-peptide cell wall precursors. The CPases have many characteristics in common with the high-M(r) penicillin-binding proteins (PBPs) whose inhibition by beta-lactam antibiotics is lethal. The CPases are attractive as model PBPs, because of their relatively lower M(r) and higher activity in vitro. We have cloned and sequenced the Bacillus stearothermophilus gene (dacA) coding for a membrane-bound CPase. The nucleotide (nt) sequence of the gene is homologous to that of the Escherichia coli and Bacillus subtilis dacA loci, which also code for membrane-bound CPases. E. coli host cells lysed when expression of B. stearothermophilus dacA was induced. The same coding sequence was expressed in the methylotrophic yeast, Pichia pastoris, using the alcohol oxidase-1 (AOX1) promoter. Over 100 micrograms/ml of CPase was efficiently secreted into the medium after induction by methanol, without adversely affecting this host. The yeast product is indistinguishable from the native enzyme in structure and activity. The ability to secrete large amounts of heterologous protein and the lack of endogenous peptidoglycan metabolism makes P. pastoris an attractive candidate for the production of PBPs.

Preparation and properties of the complex of Fe(III) with peptidoglycan monomer

The complex of Fe(III) with the peptidoglycan monomer (PGM) was prepared and characterised by chemical and physicochemical methods. The analyses showed that it contains two PGM molecules bonded with five Fe(III) ions. Mössbauer spectroscopy indicates that the iron oxy/hydroxy core has a dimeric or polymeric structure. Differential IR spectroscopy suggests complexation via carbonyl groups and NMR spectroscopy contribution of the carbonyl groups of D-isoglutaminyl, meso-diaminopimeloyl, D-alanyl residues and the carboxyl group of the C-terminal D-alanyl moiety of the peptidoglycan monomer. On the basis of the data obtained the possible structure of the Fe(III) PGM complex was proposed.

Biosynthesis of D-alanyl-lipoteichoic acid by Lactobacillus casei: interchain transacylation of D-alanyl ester residues.

Lipoteichoic acid (LTA) from Lactobacillus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D-[14C]alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. We propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.

STEREOCHEMICAL STUDIES ON CYCLIC PEPTIDES

Conformational analysis of cyclic pentapeptides having two intra-ring 3 leads to 1 hydrogen bonds has been carried out. It is found that the structure can easily be formed with trans planar peptide units without causing significant angular strain at the alpha-carbon atoms. Four different types of conformations designated Types I--IV are possible for the backbone structure. Details of these four types of conformations and also the accommodating possibility of these types for allglycyl and all-alanyl residues are presented. Three of the four types have relatively low energies for glycyl residues whereas the other one has a slightly higher energy. When alanyl residues are introduced at the five alpha-carbon atoms, the types that are energetically favourable depend upon the sequence of isomers. Energy calculations have also been carried out for the combinations of glycyl, L- and D-alanyl residues. The theoretical results are compared with available experimental observations both from solution and solid state studies.

Nonapeptide ethylamide inhibitors of the luteinizing hormone-releasing hormone (LH-RH) having a D-alanyl residue in position 6 and variations at positions 2 and 3

A series of ten analogues, of structure des-Gly10-[amino acid2, amino acid3,D-Ala6]-LH-RH ethylamide, was synthesized by solid-phase methods. L-Aromatic and alkylamino acids were substituted into position 2 and alkylamino acids into position 3. Highest in vitro inhibition of LH-RH action was obtained with analogues having aromatic residues in position 2. Des-Gly10-[Trp2,Leu3,D-Ala6]-LH-RH ethylamide inhibited the action of 0.6 ng/mL of LH-RH, in an isolated pituitary assay, at a dosage as low as 1 microgram/mL, and the corresponding Phe2 analogue inhibited the effect of 0.3 ng of LH-RH at 0.1 microgram/mL. The Trp2 analogue inhibited ovulation in rats at the dosage of 1.5 mg per rat.

Stereochemical studies on cyclic peptides: III. Conformational analysis of cyclotetrapeptides

A conformational study of cyclotetraalanyl has been made on the basis of contact criteria as well as from potential energy considerations. Using planar peptide units, it is possible to have ring closure only when the value of τ at the linking C a atoms is less than 100°, which is too small. However, when non-planarity of the peptide unit is introduced, a stable conformation is possible with l-alanyl residues for a four-fold symmetric structure, with conformational parameters (ω, ϕ, ψ) equal to (−12°, 90°, 125°). These are fairly close to the corresponding values for the α-helical conformation of poly- l-alanine. This supports the observation of Balasubramanian and Wetlaufer that this cyclic peptide bears close resemblance to the α-helix in its ORD characteristics. It is possible to have a similar structure with d-alanyl residues also for the inverse conformation (−ω, −ϕ, −ψ). However, structures with mixed l and d-residues are unlikely to occur, since they are not energetically favourable. When the four-fold symmetry is reduced to two-fold symmetry, the energy of the system increased appreciably, so that deviations from the highly symmetric structure with a four-fold axis are unlikely to occur. Similar studies made for cyclotetraglycyl indicate that this structure also has stable conformation with a four-fold symmetry axis. The conformational parameters (ω, ϕ, ψ) for this conformation are (−9°, 83°, 129°).