Enrique Pedroso

Diketopiperazine formation in solid phase peptide synthesis using p-alkoxybenzyl ester resins and Fmoc-amino acids

Summary Diketopiperazine formation rates under the usual conditions of a solid phase peptide synthesis cycle with Fmoc-amino acids have been studied on a p -alkoxybenzyl ester resin. Piperidine has been found to be an extremely efficient catalyst for the intramolecular aminolysis reaction.

Formation of aspartimide peptides in Asp-Gly sequences

Abstract The behaviour of protected H-Val-Lys-Asp-Gly-Tyr-Ile-OH towards imide formation was evaluated. The use of a carboxylic acid temporary protecting group is not a safe strategy for avoiding aspartimide formation during HF treatment. Piperidine showed a marked tendency to form aspartimides during Fmoc deprotection.

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3′-5′-cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di-GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEF-domain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.

Application of gel-phase 13C-NMR to monitor solid phase peptide synthesis

Abstract The results presented in this article show the general applicability of gel-phase 13C-NMR to monitor solid phase peptide synthesis on the most commonly used polystyrene-based resins. The optimal conditions to acquire gel-phase 13C-NMR spectra of copoly(styrene-l%-divinylbenzene) has been determined. The technique proved to be applicable to characterize polystyrene-based starting supports as well as to the determination of their degree of functionality and purity. The stage-by-stage 13C-NMR characterization of the growing peptide chain during the synthesis of tripeptide H-Asn-(N-Me)Ala-Thr-NH2 on a benzhydrylamine resin is described. The structural information, derived from this technique is relevant for the synthetic process as the spectra can be acquired under similar conditions. The characteristics of the spectra facilitate monitoring of coupling and deprotection reactions.

Convergent solid phase peptide synthesis. II. Synthesis of the 1–6 apamin protected segment on a NBB-resin. Synthesis of apamin

Abstract The article deals with the use of the NBB-resin for synthesis of protected segments followed by solid phase segment condenstaion. Solid phse synthesis on a NBB-resin of the segment 1–6 of apamin yielded either the (1–6) apamin-OH segment after photolysis or (1–6) apamin-NH-NH 2 after hydrazinolysis. The two protected segments were purified on Sephaex LH-20 followed by Bio-Beads S-X1 chromatography and respectively coupled onto a resin on which the 7–18 sequence of apamin was assembled stepwise with the standard solid phase procedure. On a portion of the resin, stepwise synthesis was continued to complete apamin. After HF treatment, deprotection of the cysteines, formation of the disulfide bonds and purification, biologically active apamin was obtained in the three cases.

Diketopiperazine formation in acetamido-and nitrobenzamido-bridgedpolymeric supports.

Abstract Diketopiperazine formation rates in deprotected Boc-D-Val-L-Pro-OCH 2 -resin( 1 ), Boc-D-Val-L-Pro-OCH 2 -Pab-resin( 2 ) and Boc-D-Val-L-Pro-OCH 2 -Nbb-resin( 3 ) have been studied under a variety of experimental conditions including those used on a solid phase peptide synthesis cycle.

A synthetic procedure for the preparation of oligonucleotides without using ammonia and its application for the synthesis of oligonucleotides containing 0-4-alkyl thymidines.

Abstract The preparation of 5′-O-dimethoxytrityl (DMT) and p -nitrophenylethyl (NPEOC, NPE) protected nucleosides linked to 4-(2-hydroxyethyl)-3-nitrobenzoic acid derivatives is described. These products attached to controlled-pore glass supports and together with DMT and NPE-protected nucleoside cyanoethyl phosphoramidites allows for the first time the preparation of short oligonucleotides containing the ammonia sensitive mutagenic bases 0-4-propyl and 0-4-butyl thymidines.

Convergent solid phase peptide synthesis. I. Synthesis of protected segments on a hydroxymethylphenyloxymethyl resin using the base labile FMOC α-amine protection. Model synthesis of LHRH.

Abstract Convergent solid phase peptide synthesis has been applied to yield LHRH. The segments 1–6 and 7–10 of LHRH were synthesized on a hydroxymethylphenyloxymethyl resin using the base labile Fmoc protecting group on the α-amines. The side chains were protected by HF labile groups. Purification of the segments was performed on Sephadex LH-20 columns and by HPLC on Silica Gel 60 columns. The two segments were then assembled on an α-aminobenzyl resin to yield entire sequence of LHRH. After HF treatment and standard purification on Sephadex G-15 and carboxymethylcellulose CM-52 the desired LHRH was obtained. Synthesis of the segments by the same strategy on carbazoyloxymethylphenyloxymethyl resin showed up unexpected difficulties.

A Simple Cytosine to G-Clamp Nucleobase Substitution Enables Chiral γ-PNAs to Invade Mixed-Sequence Double-Helical B-form DNA

Nature uses Watson–Crick base pairings as a means to store and transmit genetic information because of their high fidelity. These specific A–T (or A–U) and G–C nucleobase interactions, in turn, provide chemists and biologists with a general paradigm for designing molecules to bind to DNA and RNA. With knowledge of the sequence information, one can design oligonucleotides to bind to just about any part of these biopolymeric targets simply by choosing the corresponding nucleobase sequence according to these digital base-pairing rules. Although conceptually simple, such principles in general can only be applied to the recognition of single-stranded DNA or RNA, but not the double-stranded form. The reason is that in double-helical DNA (or RNA) not only are the Watson–Crick faces of the nucleobases already occupied, they are buried within the double helix. [1] Such molecular encapsulation imposes a steep energetic barrier on the designer molecules. To establish binding, not only would they need to be able to gain access to the designated nucleobase targets, which are blocked by the existing base pairs, they would also need to be able to compete with the complementary DNA strand to prevent it from re-annealing with its partner—a task that has rarely been accomplished by any class of molecules. To circumvent this challenge, most of the research effort to date has been focused on establishing principles for recognizing chemical groups in the minor and major groove instead because they are more readily accessible and energetically less demanding. [2] While impressive progress has been made on this front, especially in the development of triplex-forming oligonucleotides, [3–5] polyamides, [6–8] and zinc-finger-binding pep

Towards nucleopeptides containing any trifunctional amino acid (II)

Abstract Nucleopeptides with no restriction in the amino acid composition can be synthesized using stepwise solid-phase methodology. Best conditions for the protection of arginine and cysteine, as well as for the final deprotection treatment are established, and our conclusions can be extended to the preparation of any peptide–oligonucleotide hybrid. The association between certain side reactions and nucleopeptide sequences is also discussed.