Synthesis of different types of dipeptide building units containing N- or C-terminal arginine for the assembly of backbone cyclic peptides.

Different types of dipeptide building units containing N- or C-terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). For cyclization in the N-terminal sequence N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N-terminal arginine to the N-alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z-Arg(Z)2-OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C-terminal arginine was prepared to synthesize BK-analogues with backbone cyclization in the C-terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM technology was applied using the Boc-Arg(Alloc)2-OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc-protected amino aldehyde, derived from Boc-Phe-OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid-phase synthesis in the same manner as amino acid derivatives.     

Synthesis of glucose-templated lysine analogs and incorporation into the antimicrobial dipeptide sequence kW-OBn

The synthesis of two glucose-templated (GlcT) lysine analogs GlcTK and GlcTk in which the side chain of D- and L-lysine (k and K) is conformationally constrained via incorporation into a D-glucose scaffold is described. A key-step in the synthesis is a high yielding, reductive ring opening of an exocyclic glucose-derived epoxide to form a alpha-hydroxy ester that can be converted into GlcTK and GlcTk. To demonstrate the use of these building blocks in peptide synthesis, we replaced D-lysine in the antimicrobial dipeptide sequence kW-OBn (W=L-tryptophan) and determined the antibacterial activity against various gram-positive and gram-negative organisms. Our results show that the replacement of D-lysine by unprotected GlcTk in dipeptide kW-OBn results in reduced antibacterial activity.     

The intercellular signaling activity of the Mycobacterium tuberculosis chaperonin 60.1 protein resides in the equatorial domain

The major heat shock protein, chaperonin 60, has been established to have intercellular signaling activity in addition to its established protein-folding function. Mycobacterium tuberculosis is one of a small proportion of bacteria to encode two chaperonin 60 proteins. We have demonstrated that chaperonin 60.1 from this bacterium is a very active stimulator of human monocytes. To determine structure/function relationships of chaperonin 60.1 we have cloned and expressed the apical, equatorial, and intermediate domains of this protein. We have found that the signaling activity of M. tuberculosis chaperonin 60.1 resides in the equatorial domain. This activity of the recombinant equatorial domain was completely blocked by treating the protein with proteinase K, ruling out lipopolysaccharide contamination as the cause of the cell activation. Blockade of the activity of the equatorial domain by anti-CD14 monoclonal antibodies reveals that this domain activates monocytes by binding to CD14. Looking at the oligomeric state of the active proteins, using native gel electrophoresis and protein cross-linking we found that recombinant M. tuberculosis chaperonin 60.1 fails to form the prototypic tetradecameric structure of chaperonin 60 proteins under the conditions tested and only forms dimers. It is therefore concluded that the monocyte-stimulating activity of M. tuberculosis Cpn60.1 resides in the monomeric subunit and within this subunit the biological activity is due to the equatorial domain.     

Combining a polar resin and a pseudo-proline to optimize the solid-phase synthesis of a 'difficult sequence'.

This paper describes the optimization of a synthesis of a difficult sequence related to a 12-mer sequence of a Pan DR epitope (PADRE). Elongation was followed by on-line monitoring of the N(alpha)-Fmoc removal adapted for the batch methodology. Studying the intrinsic factors related to the peptide-resin, such as substitution level, resin nature and backbone protecting group, has led to an increase in the elongation yield and purity of the crude peptide. Optimal elongation was obtained by combining a polar resin such as PEGA and a pseudo-proline as the backbone protecting group.     

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.     

Delayed fibril formation of amylin(20-29) by incorporation of alkene dipeptidosulfonamide isosteres obtained by solid phase olefin cross metathesis

The synthesis of a new peptidomimetic structure, the alkene dipeptidosulfonamide isostere, is described. The synthesis is based on a cross metathesis reaction between two allylic building blocks, both in solution and on the solid phase. This method was also applicable to the solid phase synthesis of alkene dipeptide isosteres. Derivatives of amylin(20-29) containing the alkene dipeptidosulfonamide isostere as well as the alkene dipeptide isostere were successfully synthesized using the solid phase cross metathesis method. Investigation of relations between structure and fibril formation of these amylin(20-29) derivatives showed retardation of fibril formation and altered secondary structures, compared to native amylin(20-29).     

Chemical synthesis and biological evaluation of an antimicrobial peptide gonococcal growth inhibitor

Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted “difficult sequences” within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H₅₀ (concentration of peptide that causes 50?% haemolysis) of 20 and 125?μM for two blood samples from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.     

Design, synthesis, and conformational analysis of azacycloalkane amino acids as conformationally constrained probes for mimicry of peptide secondary structures

Conformationally constrained amino acid and dipeptide units can serve in mimics of specific secondary structures for studying relationships between peptide conformation and biological activity. A variety of mimics are required to study systematically the structure-activity relationships in biologically relevant peptides. We present our efforts on the design, synthesis, and conformational analysis of a series of rigid surrogates of amino acid and dipeptide units for application within constrained peptide analogues, and for employment as inputs for combinatorial science. Conceived to be general and versatile, our methodology has delivered a variety of azacycloalkane and azabicycloalkane amino acids in enantiomerically pure form, via practical methods, from readily available and inexpensive starting materials.     

Conantokin-T. A gamma-carboxyglutamate containing peptide with N-methyl-d-aspartate antagonist activity

Conantokin-T, a 21-amino acid peptide which induces sleep-like symptoms in young mice was purified from the venom of the fish-hunting cone snail, Conus tulipa. The amino acid sequence of the peptide was determined and verified by chemical synthesis. The peptide has 4 residues of the modified amino acid, gamma-carboxyglutamate (Gla). The sequence of the peptide is: Gly-Glu-Gla-Gla-Tyr-Gln-Lys-Met-Leu-Gla-Asn-Leu-Arg-Gla-Ala-Glu-Val-Lys- Lys-Asn-Ala-NH2. Conantokin-T inhibits N-methyl-D-aspartate (NMDA) receptor-mediated calcium influx in central nervous system neurons. This observation suggests that like conantokin-G (a homologous Conus peptide with recently identified NMDA antagonist activity) conantokin-T has NMDA antagonist activity. A sequence comparison of conantokins-T and -G identifies the 4 Gla residues and the N-terminal dipeptide sequence as potential key elements for the biological activity of this peptide.     

Protein synthesis in rabbit reticulocytes: Evidence for the synthesis of initial dipeptides in the presence of pactamycin

The effects of pactamycin on peptide chain initiation were studied using a reconstituted system comprised of reticulocyte ribosomes, poly r(A-U-G) messenger and peptide chain initiation factors, and the transfer of methionine from precharged Met-tRNA_f^Met into di-, oligo- and polymethionine products was measured. In the presence of low concentrations of pactamycin (10^?6M), polymethionine synthesis in the above system was markedly reduced, and a concomitant increase in the synthesis of met-met and met-val dipeptides was observed. The latter dipeptide was presumably synthesized in response to endongenous hemoglobin messenger.Aurintricarboxylic acid and sparsomycin also inhibited polymethionine synthesis in response to poly r(A-U-G) messenger. Whereas aurintricarboxylic acid inhibited pactamycin induced dipeptide synthesis, sparsomycin had no significant effect on dipeptide synthesis in the presence of pactamycin.     

Design and synthesis of substrate-based inhibitors of botulinum neurotoxin type B metalloprotease

Botulinum toxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes, we synthesized a series of transition state analog inhibitors (TSI) in which the dipeptide cleavage site has been replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. Reduced amide, alpha-hydroxyamide, alpha-thio-amide, and hydroxyethylamine analogs of -Gln-Phe- were incorporated via solid phase peptide synthesis into 35-mer analogs of the minimal peptide substrate sequence. The synthesis, characterization, and inhibition kinetics for four series of compounds against holotoxin BoNT/B is described. The alpha-thiol amide derivatives of the 35-mer substrate were found to inhibit BONT/B in the low micromolar range.     

The chaperonin assisted and unassisted refolding of rhodanese can be modulated by its N-terminal peptide

Thein vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by theE. coli chaperonins, is modulated by the 23 amino acid peptide (VHQVLYRALVSTKWLAESVRAGK) corresponding to the amino terminal sequence (1–23) of rhodanese. In the absence of the peptide, a maximum recovery of active enzyme of about 65% is achieved after 90 min of initiation of the chaperonin assisted folding reaction. In contrast, this process is substantially inhibited in the presence of the peptide. The maximum recovery of active enzyme is peptide concentration-dependent. The peptide, however, does not prevent the interaction of rhodanese with the chaperonin 60 (cpn60), which leads to the formation of the cpn60-rhodanese complex. In addition, the peptide does not affect the rate of recovery of active enzyme, although it does affect the extent of recovery. Further, the unassisted refolding of rhodanese is also inhibited by the peptide. Thus, the peptide interferes with the folding of rhodanese in either the chaperonin assisted or the unassisted refolding of the enzyme. A 13 amino acid peptide (STKWLAESVRAGK) corresponding to the amino terminal sequence (11–23) of rhodanese does not show any significant effect on the chaperonin assisted or unassisted refolding of the enzyme. The results suggest that other sequences of rhodanese, in addition to the N-terminus, may be required for the binding of cpn60, in accord with a model in which cpn60 interacts with polypeptides through multiple binding sites.     

Expression and subcellular localization of cpn60 protein family members in Leishmania donovani

We have identified two diverged members of the cpn60 gene family in Leishmania donovani, causative agent of Indian Kala Azar. One of the genes, cpn60.1, although actively transcribed, is not expressed to detectable levels of protein in cultured L. donovani. The other gene, cpn60.2, which, compared with cpn60.1, shows a higher sequence conservation with the hsp60 genes from Trypanosoma brucei and Trypanosoma cruzi is expressed constitutively in cultured promastigotes. The abundance of the gene product, Cpn60.2, increases by 2.5-fold under heat stress and in axenic amastigotes of L. donovani. Cpn60.2 is also found enriched in mitochondrial cell fractions and localizes to the mitochondrial matrix. We conclude that Cpn60.2 is the major mitochondrial chaperonin in Leishmania.     

Sugar amino acid based peptide epoxyketones as potential proteasome inhibitors

This paper describes the synthesis and biological evaluation of nine epoxomicin-derived sugar amino acid containing peptide epoxyketones. The title compounds are assembled from six sugar amino acid dipeptide isosteres and are synthesized using solution-phase peptide synthesis protocols. Although neither of the compounds displays inhibitory activity towards any of the proteasome active sites, our approach holds promise towards the development of structurally new proteasome inhibitors. It is likely that the central sugar amino acid dipeptide isoster needs to be designed such that it closely resemble dipeptides at position P2 and P3 in proteasome substrates inhibitors, such as the Thr-Ile dipeptide present in the lead compound, epoxomicin.     

Synthesis and characterization of two potential impurities (des-ethyl-Ganirelix) generated in the Ganirelix manufacturing process

Controlling certain diseases using peptide drugs has remarkably increased in the past two decades. In this regard, a generic formulation is an upfront solution to fulfill market demands. Ganirelix, a leading peptide active pharmaceutical ingredient (API) primarily used as a gonadotropin-releasing hormone antagonist (GnRH), has established a potential market value worldwide. But its generic formulation mandates detailed impurity profiles from a synthetic source and contemplates the sameness of a reference-listed drug (RLD). Post-chemical synthesis and processing of Ganirelix, some commercial sources have revealed two new potential impurities among many known, which show the deletion of an ethyl group from the hArg(Et)₂ residue at the sixth and eighth positions, named des-ethyl-Ganirelix. These impurities are unprecedented in traditional peptide chemistry, and such monoethylated-hArg building blocks are not easily accessible commercially to synthesize these two impurities. Here, we have outlined the synthesis, purification, and enantiomeric purity characterization of the amino acids and their incorporation in the Ganirelix peptide sequence to synthesize these potential peptide impurities. This methodology will enable the convenient synthesis of side-chain substituted Arg and hArg derivatives in peptide drug discovery platforms.     

An imidazoline pseudodipeptide suitable for solid phase peptide synthesis.

This paper describes the synthesis of two diastereoisomers of an imidazoline dipeptide mimetic (a 4,5 dihydroimidazole-4-carboxylic acid), suitably protected for incorporation into solid phase peptide synthesis (SPPS) using the Fmoc protocol, from a phenylalanine-derived thioimidate and an alpha,beta-diaminopropanoic acid ester, followed by protecting group manipulation.     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides.

To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH4. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

An improved procedure for the synthesis of branched polyethylene glycols (PEGs) with the reporter dipeptide Met-betaAla for protein conjugation.

A new and more efficient route to the synthesis of branched PEG for protein conjugation, bearing a reporter dipeptide Met-betaAla, is described, which allows better purification of the final product by ion exchange chromatography. The product has the combined advantages of an 'umbrella-like' branched structure, which allows a better coverage of the protein surface, and the presence of the dipeptide Met-betaAla which has been used to detect the position of PEGylation within the peptide sequence.