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Biomedical subjects

F Albericio

Publications and source records attributed to F Albericio.

At least 19 recordsLinked to original sources
PubMed1998

"High-load" polyethylene glycol-polystyrene (PEG-PS) graft supports for solid-phase synthesis.

The choice of a polymeric support is a key factor for the success of solid-phase methods for syntheses of organic compounds and biomolecules such as peptides and oligonucleotides. Classical Merrifield solid-phase peptide synthesis (SPPS), performed on low cross-linked hydrophobic polystyrene (PS) beads, sometimes suffers from sequence-dependent coupling difficulties. The concept of incorporating polyethylene glycol (PEG) into supports for solid-phase synthesis represents a successful approach to alleviating such problems. Previous reports from our laboratories have shown the advantages of "low-load" PEG-PS (0.15-0.25 mmol/g) for SPPS. Herein, we demonstrate that the beneficial aspects of the PEG-PS concept can be extended with resins that have higher loadings (0.3-0.5 mmol/g).

Amino Acid Sequence↗
PubMed1998

Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.

Amino Acid Sequence↗
PubMed1998

Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles.

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka < 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.

Amino Acid Sequence↗
PubMed1997

Comparative evaluation of the synthesis and purification of transmembrane peptide fragments. Rat bradykinin receptor fragment 64-97 as model.

The 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin, receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of alpha-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor.

Amino Acid Sequence↗
PubMed1997

Biological activity and therapeutic potential of homologs of an Ii peptide which regulates antigenic peptide binding to cell surface MHC class II molecules.

An invariant chain peptide (murine Ii76-92; Ii-Key) is known to produce a 10 to 50 times baseline enhancement of the presentation of specific antigenic peptides to murine T cell hybridomas by cell surface MHC class II molecules. In order to define structure-activity relationships in Ii-key, homologs were synthesized with the following systematic variations: 1) N- and C-terminal truncations, 2) N-terminal acetylation and C-terminal amidation, 3) substitutions with 13 natural amino acids in each position of the shortest, fully active peptide, and then with an additional 12 nonnatural amino acids at certain 'pharmacophore' positions, 4) substitutions with D-amino acids and N-methyl-leucine, and 5) cyclical forms. More than 160 homologs were tested for effects on antigen presentation by the murine MHC class II alleles: A(d), Ak, E(d), or Ek. For some compounds, allele specificity between E(d) and Ek exceeded 1:20. D-Amino acid and/or N-methyl-leucine substitutions were accepted at some residue positions, leading to peptides with relatively long half-lives in mouse serum and low toxicities in mice. An Ii-Key homolog inhibited in vitro presentation of an internally processed hen egg lysozyme determinant to a specific T hybridoma. The best compounds can be tested in vivo for therapeutic applications: 1) immunosuppression upon release of antigenic peptide, and 2) vaccination or immunomodulation upon co-administration of a second antigenic peptide.

Amino Acid Sequence↗
PubMed1996

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides.

A novel approach for the preparation of oligonucleotides carrying amino groups at the 3'-end is described. Several CPG supports having aminoalkyl groups and 3'-amino-2',3'-dideoxynucleosides linked through base-labile carbamate linkages such as 2-(2-nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl were prepared using two different strategies. These supports are compatible to the standard solid phase phosphite-triester methodology and yield oligonucleotides containing amino groups at the 3'-end. Several properties of the 3'-amino oligonucleotides, such as nuclease resistance, hybridization, and preparation of oligonucleotide conjugates are discussed.

Carbamates↗
PubMed1995

Convergent solid-phase peptide synthesis. 12. Chromatographic techniques for the purification of protected peptide segments.

The purification of a range of protected peptide segments has been carried out using modified reversed-phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue.

Acetonitriles↗
PubMed1994

Chemical synthesis of a fully active transcriptional repressor protein.

Plasmid pLS1-encoded 45-amino acid transcriptional repressor CopG (formerly RepA) has been chemically synthesized. A one-step purification of the synthetic protein has been developed, which yields high levels of pure protein with low or no contamination of truncated products. We have compared some properties of the chemical CopG protein with those of the biologically purified CopG. The two proteins were indistinguishable in (i) their ability to generate specific protein-DNA complexes, (ii) their capacity to protect a restriction site included within the CopG DNA target, and (iii) in their in vitro capacity to specifically repress synthesis of copG mRNA.

Amino Acid Sequence↗
PubMed1993

Automated allyl cleavage for continuous-flow synthesis of cyclic and branched peptides.

An automated allyl cleavage scheme on a continuous-flow peptide synthesizer was used for the preparation of "head-to-tail" cyclic peptides, branched peptides, and multiple antigenic peptides. Standard allyl removal uses a suspended palladium catalyst. This approach is not feasible on a batch and continuous-flow peptide synthesizer due to problematic delivery of the insoluble palladium catalyst. Solvent conditions were examined and optimized to solubilize the catalyst, prevent undesired Fmoc deblocking and be compatible with sensitive amino acids (Trp and Met) and with glyco- and sulfopeptides. Protocols for a continuous-flow peptide synthesizer were modified using new conditions to carry out the allyl cleavage scheme for the facile preparation of complex peptides.

Allyl Compounds↗
PubMed1993

Antibodies cross-reactive with the scorpion-toxin II from Androctonus australis Hector elicited in mice by a synthetic peptide.

With the aim of developing active protection against the noxious effects provoked in humans by scorpion stings, the possibility of eliciting toxin reactive antibodies by immunization with a short peptide was assessed in mice. The amino acid sequence of residues 50 to 59 of Androctonus australis Hector toxin II was chosen, on the basis of previous results indicating that rabbit anti-(50-59) antibodies neutralize the biological effects of the parent toxin. The peptide was prepared by solid-phase synthesis procedures and used in different forms (free, linearly polymerized, coupled to KLH, coupled to a low-molecular weight B-lymphocyte activator) in order to immunize groups of non-congenic NMRI or congenic C57BL/6 mice. The reactivities of each serum with the peptide and with the toxin were assessed in ELISA. Strong reactivities with both the peptide (mean titer over 1:52,600) and the toxin (mean titer 1:800) were observed in all mice from the group that received the KLH-coupled peptide. However, mouse immune sera failed either to recognize the toxin in a liquid-phase radioimmunoassay or to neutralize the lethal effects of the toxin. The requirements, in terms of affinity and recognition of native conformation, for anti-peptide antibodies to display neutralizing properties are discussed.

Amino Acid Sequence↗
PubMed1993

Acidolytic cleavage of tris(alkoxy)benzylamide (PAL) "internal reference" amino acyl (IRAA) anchoring linkages: validation of accepted procedures in solid-phase peptide synthesis (SPPS).

Under the normal conditions of acidolytic cleavage/deprotection of tris(alkoxy)benzylamide (PAL) anchoring linkages in Fmoc solid-phase peptide synthesis (SPPS), product release occurs by a straightforward single-step pathway. A recently reported cleavage of the NH--alpha CH bond of an amino acyl residue adjacent to PAL [see Int. J. Peptide Protein Res. 38, 146-153 (1991)] could not be confirmed in novel experiments incorporating a double "internal reference" amino acid (IRAA) design. The results of the present work revalidate the widely accepted application of IRAAs to monitor yields in SPPS, and confirm the reliability of PAL methodology for the preparation of C-terminal peptide amides.

Amino Acids↗
PubMed1992

Racemization free coupling of peptide segments. Synthesis of an insect neuropeptide.

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.

Amino Acid Sequence↗
PubMed1992

RNA binding characteristics of a 16 kDa glycine-rich protein from maize.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.

Amino Acid Sequence↗
PubMed1991

Diurnal rhythm of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase.

Rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase exhibits a diurnal rhythm of enzyme activity which coincides with the diurnal rhythm of HMG-CoA synthase protein. The peaks of activity and protein (determined by SDS/PAGE and immunoblotting) both occur at D10 (the tenth hour of the daily 12 h dark cycle). The peak of mRNA levels (measured by slot-blot hybridization of liver RNA) is slightly advanced with respect to that of protein, by about 4 h, and shows a maximum at D6. Cytosolic HMG-CoA synthase activity and protein in rats fed on a normal diet were approx. 2-fold higher during the peak at D10 than in the nadir at D2. HMG-CoA synthase mRNA levels were approx. 4-fold higher during the peak at D6 than in the nadir at D2. These results point to a transcriptional and translational regulation of the cytosolic HMG-CoA synthase.

Amino Acid Sequence↗