Vicente Marchán

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.

AN IMPROVED SYNTHESIS OF N-[(9-HYDROXYMETHYL)-2-FLUORENYL]SUCCINAMIC ACID (HMFS), A VERSATILE HANDLE FOR THE SOLID-PHASE SYNTHESIS OF BIOMOLECULES

The optimization of a synthetic process for the preparation of HMFS handle, which is used for the solid-phase synthesis of peptides and oligonucleotides, is presented.

Conceptual “Heat‐Driven” Approach to the Synthesis of DNA Oligonucleotides on Microarrays

Abstract: The discovery of deoxyribonucleoside cyclic N‐acylphosphoramidites, a novel class of phosphoramidite monomers for solid‐phase oligonucleotide synthesis, has led to the development of a number of phosphate protecting groups that can be cleaved from DNA oligonucleotides under thermolytic neutral conditions. These include the 2‐(N‐formyl‐N‐methyl)aminoethyl, 4‐oxopentyl, 3‐(N‐tert‐butyl)carboxamido‐1‐propyl, 3‐(2‐pyridyl)‐1‐propyl, 2‐[N‐methyl‐N‐(2‐pyridyl)]aminoethyl, and 4‐methythiobutyl groups. When used for 5′‐hydroxyl protection of nucleosides, the analogous 1‐phenyl‐2‐[N‐methyl‐N‐(2‐pyridyl)]aminoethyloxycarbonyl group exhibited excellent thermolytic properties, which may permit an iterative “heat‐driven” synthesis of DNA oligonucleotides on microarrays. In this regard, progress has been made toward the use of deoxyribonucleoside cyclic N‐acylphosphoramidites in solid‐phase oligonucleotide syntheses without nucleobase protection. Given that deoxyribonucleoside cyclic N‐acylphosphoramidites produce oligonucleotides with heat‐sensitive phosphate protecting groups, blocking the 5′‐hydroxyl of these monomers with, for example, the thermolabile 1‐phenyl‐2‐[N‐methyl‐N‐(2‐pyridyl)]aminoethyloxycarbonyl group may provide a convenient thermo‐controlled method for the synthesis of oligonucleotides on microarrays.

Alternative Procedures for the Synthesis of Methionine-Containing Peptide−Oligonucleotide Hybrids

The synthesis of methionine-containing peptide−oligonucleotide hybrids has been found to be best accomplished by a stepwise solid-phase approach in which peptide assembly using the sulfoxide derivative of methionine is followed by elongation of the oligonucleotide chain using the phosphite triester methodology, ammonia deprotection, and reduction of the sulfoxide to thioether by reaction with N-methylmercaptoacetamide. Quantitative amino acid incorporation yields could not always be achieved when the order of assembly of the two moieties was reversed, i.e. by elongating the peptide chain on a resin-linked oligonucleotide in order to avoid exposure of the thioether function to oxidizing conditions.

Solution structure and stability of tryptophan-containing nucleopeptide duplexes.

Covalently linked peptide–oligonucleotide hybrids were used as models for studying tryptophan–DNA interactions. The structure and stability of several hybrids in which peptides and oligonucleotides are linked through a phosphodiester bond between the hydroxy group of a homoserine (Hse) side chain and the 3′‐end of the oligonucleotide, have been studied by both NMR and CD spectroscopy and by restrained molecular dynamics methods. The three‐dimensional solution structure of the complex between Ac‐Lys‐Trp‐Lys‐Hse(p3′dGCATCG)‐Ala‐OH (p=phosphate, Ac=acetyl) and its complementary strand 5′dCGTAGC has been determined from a set of 276 experimental NOE distances and 33 dihedral angle constraints. The oligonucleotide structure is a well‐defined duplex that belongs to the B‐form family of DNA structures. The covalently linked peptide adopts a folded structure in which the tryptophan side chain stacks against the 3′‐terminal guanine moiety, which forms a cap at the end of the duplex. This stacking interaction, which resembles other tryptophan–nucleobase interactions observed in some protein–DNA complexes, is not observed in the single‐stranded form of Ac‐Lys‐Trp‐Lys‐Hse(p3′dGCATCG)‐Ala‐OH, where the peptide chain is completely disordered. A comparison with the pure DNA duplex, d(5′GCTACG3′)–(5′CGTAGC3′), indicates that the interaction between the peptide and the DNA contributes to the stability of the nucleopeptide duplex. The different contributions that stabilize this complex have been evaluated by studying other nucleopeptide compounds with related sequences.

Incorporation of two modified nucleosides allows selective platination of an oligonucleotide making it suitable for duplex cross-linking.

Platinated oligonucleotides are promising tools for the control of gene expression, since they may target and cross-link nucleic acid chains. Here we describe a method for the preparation of platinated oligonucleotides that has proved able to selectively cross-link complementary sequences, making use of 5-methylcytidine analogs with thioether or imidazole groups attached to the 4-position. These nucleoside analogs were derivatized as phosphoramidites and introduced in oligonucleotide chains using standard phosphite triester chemistry. Different oligonucleotide sequences containing either one or two analogs appending from the 5′-end were synthesized and used in preliminary platination studies. The reaction of transplatin with oligonucleotides containing the thioether-modified nucleobase was fast, but generally afforded unstable adducts and complex reaction mixtures. The imidazole-containing oligonucleotides reacted with transplatin much more slowly, in particular at slightly basic pH, and it was found that the imidazole-modified cytosine was less reactive than the natural nucleobases. In contrast, transplatin selectively reacted with the thioether and imidazole groups of oligonucleotides containing the two cytosine analogs in neighboring positions, even in the presence of the four nucleobases and particularly three guanines, affording platinated oligonucleotides suitable for cross-linking.

Synthesis of Janus Compounds for the Recognition of G-U Mismatched Nucleobase Pairs

The design and synthesis of two Janus-type heterocycles with the capacity to simultaneously recognize guanine and uracyl in G-U mismatched pairs through complementary hydrogen bond pairing is described. Both compounds were conveniently functionalized with a carboxylic function and efficiently attached to a tripeptide sequence by using solid-phase methodologies. Ligands based on the derivatization of such Janus compounds with a small aminoglycoside, neamine, and its guanidinylated analogue have been synthesized, and their interaction with Tau RNA has been investigated by using several biophysical techniques, including UV-monitored melting curves, fluorescence titration experiments, and (1)H NMR. The overall results indicated that Janus-neamine/guanidinoneamine showed some preference for the +3 mutated RNA sequence associated with the development of some tauopathies, although preliminary NMR studies have not confirmed binding to G-U pairs. Moreover, a good correlation has been found between the RNA binding affinity of such Janus-containing ligands and their ability to stabilize this secondary structure upon complexation.

Stepwise Solid‐Phase Synthesis of Nucleopeptides

Phosphodiester‐linked peptide‐oligonucleotide conjugates (nucleopeptides) are obtained by stepwise solid‐phase procedures. The peptide is first assembled on a suitably derivatized solid matrix and the oligonucleotide is subsequently elongated at the free hydroxyl group of the linking amino acid. Temporary acid‐labile and permanent base‐labile protecting groups are combined. Careful choice of the protection scheme is required to prevent and minimize side reactions that may degrade the target molecule. Curr. Protoc. Nucleic Acid Chem. 31:4.22.1‐4.22.54.

Development of Green/Red-Absorbing Chromophores Based on a Coumarin Scaffold That Are Useful as Caging Groups

We report the design, synthesis, and spectroscopic characterization of a series of push-pull chromophores based on a novel coumarin scaffold in which the carbonyl of the lactone function of the original coumarin dyes has been replaced by the cyano(4-nitrophenyl)methylene moiety. The skeleton of the compounds was synthesized by condensation of a thiocoumarin precursor with the corresponding arylacetonitrile derivatives, and their photophysical properties were fine-tuned through the incorporation of electron-withdrawing groups (EWGs) like nitro and cyano at the phenyl ring, leading to absorption in the green to red region. Although fluorescence emission was weakened or even canceled upon introduction of two or three strong EWGs, the emission of the mononitro-containing coumarin derivatives in the red region upon excitation with green light is noticeable, as are their significantly large Stokes shifts. The new coumarin derivatives can be useful as photocleavable protecting groups, as demonstrated through the synthesis and characterization of a series of coumarin-based photocages of benzoic acid. Preliminary photolysis studies with green light have demonstrated that the structure of the coumarin chromophore influences the rate of the uncaging process, opening the way to exploiting these new coumarin scaffolds as caging groups that can be removed with visible light.

Sequential uncaging with green light can be achieved by fine-tuning the structure of a dicyanocoumarin chromophore

Abstract We report the synthesis and photochemical properties of a series of dicyanocoumarinylmethyl (DEAdcCM)‐ and dicyanocoumarinylethyl (DEAdcCE)‐based photocages of carboxylic acids and amines with absorption maximum around 500 nm. Photolysis studies with green light have demonstrated that the structure of the coumarin chromophore as well as the nature of the leaving group and the type of bond to be photocleaved (ester or carbamate) have a strong influence on the rate and efficiency of the uncaging process. These experimental observations were also supported by DFT calculations. Such differences in deprotection kinetics have been exploited to sequentially photolyze two dicyanocoumarin‐caged model compounds (e.g., benzoic acid and ethylamine), and open the way to increasing the number of functional levels that can be addressed with light in a single system, particularly when combining dicyanocoumarin caging groups with other photocleavable protecting groups, which remain intact under green light irradiation.