Linkage of Ribonucleic Acid and Peptide

Nucleotide-peptide linkages were detected in a ribonucleic acid preparation obtained from yeast. Adenosihe-3′-phosphate combined with dinitrophenylated peptide was isolated after digesting the dinitrophenylated ribonucleic acid with enzymes. The peptide consisted of nine kinds of amino acid. In this preparation one peptide may be linked to the phosphate of terminal adenylic acid through its amino terminal, serine, as phosphoamide.     

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Synthesis and nucleophilic reactivity of a series of glutathione analogues, modified at the gamma-glutamyl moiety.

A series of GSH analogues with modifications at the gamma-glutamyl moiety was synthesized and purified by following peptide chemistry methodology. Benzyl, benzyloxycarbonyl and t-butyloxycarbonyl protective groups were used to protect individual amino acid functional groups. The formation of peptide bonds was accomplished through coupling of free amino groups with active esters, generated by reaction of the carboxylate functions with dicyclohexylcarbodi-imide and 1-hydroxybenzotriazole. The protecting groups in the tripeptides were removed in a single step by using Na in liquid NH3. Precautions were taken in order to prevent oxidation of the thiol function in the cysteine residue. Thus GSH analogues containing both L- and D-glutamic acid and L- and D-aspartic acid, coupled to cysteinylglycine through both the alpha- and the omega-carboxylate group, were synthesized. Also, decarboxy-GSH and deamino-GSH, lacking one functional group in the glutamate moiety, were prepared. The spontaneous non-enzyme-catalysed nucleophilic reaction of these GSH analogues with the electrophilic model substrate 1-chloro-2,4-dinitrobenzene showed appreciable rate differences, indicating the importance of intramolecular interactions in determining the nucleophilic reactivity of the thiol function in the cysteine residue. In particular, the free amino group in the gamma-L-glutamic acid residue appears to play a crucial role in activating the thiol group in GSH. In an adjacent paper [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] these results are compared with those obtained in a study on the ability of these GSH analogues to act as a co-substrate in the glutathione S-transferase-catalysed conjugation reaction with 1-chloro-2,4-dinitrobenzene.     

Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1′-Carbonyldiimidazole

A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1′-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.     

Yeast protein farnesyltransferase. pKas of peptide substrates bound as zinc thiolates.

Protein farnesyltransferase (PFTase) is a zinc metalloenzyme that catalyzes the posttranslational alkylation of the cysteine in C-terminal -Ca(1)a(2)X sequences by a 15-carbon farnesyl residue, where C is cysteine, a(1) and a(2) are normally aliphatic amino acids, and X is an amino acid that specifies selectivity for the farnesyl moiety. Formation of a Zn(2+) thiolate in the PFTase. peptide complex was detected by the appearance of an absorbance at 236 nm (epsilon = 15 000 M(-1) cm(-1)), which was dependent on the concentration of peptide, in a UV difference spectrum in a solution of PFTase and the peptide substrate RTRCVIA. We developed a fluorescence anisotropy binding assay to measure the dissociation constants as a function of pH for peptide analogues by appending a 2',7'-difluorofluorescein to their N-terminus. The electron-withdrawing fluorine atoms allowed us to measure peptide binding down to pH 5.5 without having to correct for the changes in fluorescence intensity that accompany protonation of the fluorophore. Measurements of the pK(a)s for thiol groups in free and bound peptide indicate that peptide binding is accompanied by formation of a zinc thiolate and that binding to PFTase lowers the pK of the peptide thiol by 3 units. In similar studies with the betaY310F mutant, the pK(a) of the thiol moiety was lowered by 2 units upon binding, indicating that the hydroxyl group in the conserved tyrosine helps stabilize the bound thiolate.     

Synthesis and Nucleic Acids Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the “cis-” homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

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 efficient and highly selective deprotection of N-Fmoc-alpha-amino acid and lipophilic N-Fmoc-dipeptide methyl esters with aluminium trichloride and N,N-dimethylaniline.

A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N-dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N-Fmoc-protected amino acid methyl esters and N-Fmoc-protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

Polymer-bound alkyltriazenes for mild racemization-free esterification of amino acid and peptide derivatives.

A novel tool for polymer-assisted solution phase (PASP) esterification of amino acid and peptide derivatives has been developed. When treated with carboxylic acids, polymer-bound alkyltriazenes react with a loss of nitrogen and transfer of the alkyl moiety to the carboxylate anion to form the corresponding alkyl esters. There are no limitations with regard to either the protecting groups or the nature of the amino acid. Furthermore no racemization occurs at the chiral centers of the amino acids as demonstrated by chiral GC-MS analyses. Alkyltriazene-resins were also applied successfully to the esterification of peptide acids and other peptidic structures, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). The triazene-mediated esterification reaction is exceptionally mild, and there is no need for prior activation of the carboxy groups. This method is therefore particularly suitable for the alkylation of complex peptidomimetic structures prone to racemization and for acid-sensitive structures.     

Conformational relationships between amino acids and their anticodons in the primitive decoding system

Detailed calculations of the conformational characteristics of a primitive decoding system are presented. A penta-nucleotide serves as the primitive tRNA (PIT) with a triplet of primitive anticodon (PAC) in a helical conformation. This molecular moiety has a cleft in the middle. An amino acid can comfortably nestle into the cleft. The conformation of this molecular association is stabilised by a few hydrogen bonds. The stereochemistry of the moiety restricts the conformational possibilities and the sidechain of the amino acid gets oriented at a proper position and in the correct direction to interact intimately with the PAC in the middle of the PIT. The model favours L-amino acids for beta-D-ribonucleotides. The location of the sidechain of the amino acid in the PIT gives a raison d'être for the important features of the organisation of nucleotide triplets for amino acids in the Genetic Code. The interaction of a few key amino acids with the different combinations of bases as PAC sequences has been studied and the stereochemical basis for the selection of the anticodons for amino acids is elucidated.     

Synthesis of differentially protected N-acylated reduced pseudodipeptides as building units for backbone cyclic peptides.

Backbone cyclization has become an important method for generating or stabilizing the bioactive conformation of peptides without affecting the amino acid side-chains. Up to now, backbone cyclic peptides were mostly synthesized with bridges between N-amino- and N-carboxy-functionalized peptide bonds. To study the influence of a more flexible backbone on the biological activity, we have developed a new type of backbone cyclization which is achieved via the N-functionalized moieties of acylated reduced peptide bonds. As described in our previous publications, the formation of N-functionalized dipeptide units facilitates the peptide assembly compared with the incorporation of N-alkyl amino acids. Besides the racemization-free synthesis of Fmoc-protected pseudodipeptide esters with reduced peptide bonds, the new type of backbone modification allows the use of a great variety of omega-amino- and alpha,omega-dicarboxylic acids differing in chain length and chemical properties. Best results for the coupling of the omega-amino- and alpha,omega-dicarboxylic acids to the reduced peptide bond were obtained by the formation of mixed anhydrides with alkyl chloroformates. Whereas the protecting group combination of Z/OBzl in the dipeptide unit and Boc/OtBu for the N-functionalized moiety leads to the formation of 2-ketopiperazine during hydrogenation, the combination of Fmoc/OtBu and Alloc/OAll is very suitable for the synthesis of backbone cyclic peptides on solid support.     

Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

Increased nucleophile reactivity of amino acid beta-naphthylamides in alpha-chymotrypsin-catalyzed peptide synthesis

Alpha-chymotrypsin-catalyzed acyl transfer from Boc-L-MetONp, Ac-L-TyrOEt, Bz-L-TyrOMe, Mal-L-PheOMe to the C-protected amino acids (L-AlaNH2, L-LeuNH2, L-ArgOMe and beta-naphthylamides of L-Arg, L-Leu, L-Ala and L-Glu) has been studied. Modification of the carboxylic groups with beta-naphthylamide was shown to increase the reactivity of nucleophiles in these reactions by a factor of more than 100 in comparison with amides and esters of the same amino acids. This effect can be accounted for by the effective formation of the nucleophile-acylenzyme complex due to hydrophobic interactions of the beta-naphthylamide moiety with the corresponding subsite of alpha-chymotrypsin. The reaction kinetics follows the scheme involving hydrolysis of the nucleophile-acylenzyme intermediate. The contribution of this pathway depends on the structures of both the acyl-group donor and the added nucleophile. The competitive inhibition by amino acid beta-naphthylamides is also observed. The results obtained show that modification of the COOH-group of added nucleophiles by beta-naphthylamide strongly affects the reactivity of these compounds in the alpha-chymotrypsin-catalyzed peptide synthesis.     

The origin of the protein synthesis mechanism

The origin and development of the protein synthesis mechanism is considered in four successive steps. The genetic code is supposed to be controlled by the relative amount (availability) of various amino acids and nucleotides on the one hand, and utility on each amino acid in the polypeptide. on the other hand. Thus, more simple (inutile) and abundant amino acids tended to correspond to codons which were rich in the less frequent base species, G and C. Features of primitive tRNA in the discrimination of amino acid are discussed. Primitive tRNA is proposed to have a discriminator site for amino acid and, separated from it, an anticodon site for interaction with nucleotides. A hypothetical course of subdivision of various nucleic acid species is proposed. In the scheme, mRNA and ribosomal RNA (rRNA) were derived from more primitive insoluble RNA. DNA appeared in the late, not first, step of the development. Several other aspects of evolutionary development of the whole protein synthesis mechanism, e.g., role of the discriminator site on primitive tRNA, modification and subdivision of code catalogue into a more precise specification of amino acids, and possible primordial interactions between tRNA and tRNA-binding sites on insoluble rRNA, are discussed.     

Racemization in the synthesis of polytripeptide models of a collagen.

Racemization in the synthesis of tripeptide intermediates and their polymers was investigated, using L -amino acid oxidase. Stepwise investigation of peptide intermediates showed no racemization during peptide coupling steps or deprotection of benzyl esters by hydrogenolysis. Saponification of one of the methyl esters produced some racemization. Preparation of active esters from N-protected tripeptide acids containing optically active C-terminal amino acid, with one exception, produced racemization. The fractionated polymers were found to contain less racemized amino acids than the crude products or starting monomeric tripeptides, indicating that the racemized sequences gave rise to lower molecular-weight oligomers. The sequences investigated were -Pro-Pro-Ala-, -Ala-Pro-Pro-, -Val-Pro-Pro-, -Pro-Pro-Leu-, -Pro-Gly-Leu-, -Pro-Gly-Phe-, -Pro-Gly-Val-, -Gly-Val-Pro-, -Phe-Pro-Gly-, -Leu-Pro-Gly-, and Ile-Pro-Gly-.     

Synthesis of Monomers Bearing at the 2′‐Position Cyanomethoxycarbonyl Group for Phosphoramidite Oligonucleotide Synthesis

A novel series of phosphoroamidites for the synthesis of 2′‐modified oligonucleotides was designed and synthesized on the base of 2′‐amino uridine and 2′‐amino arabinoadenosine. The amino groups in these compounds were acidified by bis‐cyanomethyl esters of different dicarbonic acids. Generated reactive linker groups containing cyanomethoxycarbonyl groups are stable under conditions of oligonucleotide synthesis but could be easily functionalised in post‐synthetic stage by treatment with compounds bearing primary amino groups.     

Synthesis and nucleic acids binding properties of diastereomeric aminoethylprolyl peptide nucleic acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the "cis-" homothymine aepPNA decamers with (2'R,4'R) and (2'S,4'S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2'R,4'S) and (2'S,4'R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2'R,4'R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Synthesis and Use of New Semispecific Substrates for Trypsin-Catalyzed Peptide Bond Formation

Two series of semispecific acyl donors, hydroxyalkyl esters of Z-Ala-OH and TV-modified carboxamidomethyl (Cam) esters of Z-Xaa-OH (Xaa = Ala, Leu, Phe) were synthesized as substrates for trypsin-catalyzed peptide synthesis. It follows from the specificity constants of these compounds, that the carboxamidomethyl derivatives are well accepted by trypsin due to favourable S₂′ – P₂′ interactions. These new substrates can be successfully used for the trypsin-mediated formation of dipeptide amides. The synthesis outcome depends on the amino acid in the P₁ position, the ability of the leaving group to provide efficient interactions with the enzyme subsite and the hydrophobicity of the nucleophilic amino acid amide. The modified Cam esters give better peptide yields in comparison to the unmodified ones.     

Ser-His catalyses the formation of peptides and PNAs

The dipeptide seryl-histidine (Ser-His) catalyses the condensation of esters of amino acids, peptide fragments, and peptide nucleic acid (PNA) building blocks, bringing to the formation of peptide bonds. Di-, tri- or tetra-peptides can be formed with yields that vary from 0.5% to 60% depending on the nature of the substrate and on the conditions. Other simpler peptides as Gly-Gly, or Gly-Gly-Gly are also effective, although less efficiently. We discuss the results from the viewpoint of primitive chemistry and the origin of long macromolecules by stepwise fragment condensations.     

A chemoenzymatic synthesis of deoxy sugar esters involving stereoselective acetylation of hemiacetals catalyzed by CALB

An extension of the scope of the chemoenzymatic strategy for the synthesis of stereochemically pure pyranose deoxy sugar esters of different carboxylic acids has been achieved. The objective of the work was to extend the strategy to the synthesis of furanose deoxy sugar derivatives and additionally, to N-Boc-protected amino acid esters. With all used carboxylic acids (deoxycholic acid, α-methoxyphenylacetic acid, N-Boc-l-phenylalanine and N-Boc-l-tyrosine) the lipase-catalyzed stereoselective acetylation of furanose or pyranose hemiacetal moiety as a key step afforded one desired stereochemically pure acetylated hemiacetal deoxy sugar ester in high de.