New phosphoramidite reagents for the synthesis of oligonucleotides containing a cysteine residue useful in peptide conjugation

The preparation is described of four 2-cyanoethyl-N,N-diisopropyl phosphoramidites of N-alpha-Fmoc-S-protected cysteine hydroxyalkyl amides. The phosphoramidites were used in solid-phase synthesis of 5'-cysteinyl oligonucleotides, useful intermediates in the preparation of peptide-oligonucleotide conjugates through reaction with a maleimide peptide or with a peptide thioester via "native ligation".     

Hydrophilic fluorescein derivatives: useful reagents for liposome immunolytic assays

Hydrophilic derivatives of fluorescein containing hydroxyalkyl substituents were synthesized and encapsulated within liposomes. The fluorophores showed significantly more retention with time than did fluorescein, carboxyfluorescein, or calcein. Unlike calcein, the fluorophores are minimally susceptible to fluorescence quenching by Co2+. The utility of these compounds as immunodiagnostic reagents was demonstrated by encapsulating 5(6)-carboxyfluorescein trismethylolamide in haptenized liposomes which were used in an immunoassay for digoxin.     

New useful reagents for peptide synthesis. Insoluble active esters of polystyrene-bound 1-hydroxybenzotriazole.

Insoluble 1-hydroxbenzotriazole-bound polystyrene was prepared through a series of chemical modifications of commercially available polystyrene. Reaction of 3-nitro-4-chlorobenzyl alcohol or of 3-nitro-4-chlorobenzyl bromide with polystyrene in the presence of aluminium trichloride yielded (3-nitro-4-chloro)benzylated polystyrene. Upon reaction with hydrazine it was converted to (3-nitro-4-hydrazine) benzylated polystyrene which was cyclized, by acidolysis, to yield 1-hydroxybenzotriazole-bound polystyrene. This was coupled, using N, N' -dicyclohexylcarbodiimide as the coupling agent, to many N-blocked amino acid derivatives, yielding polymeric polystyrene-bound active esters.Such derivatives are highly reactive and their efficacy in the synthesis of several peptides, including that of the tetrapeptide Boc-L-Leu-L-leu-L-Val-0bzl-L-Tyr-0Bzl and of thyrotropin-releasing hormone was demonstrated.     

Polymer-supported silyl cyanide and silyl azide: useful reagents for solid-phase applications

The support of a delicate reagent on a solid matrix allows for better and safer handling of the reagent itself. Because we had an interest in silicon-based supported reagents(1) we turned our attention to a polymer-supported trialkylsilyl cyanide and trialkylsilyl azide starting from a commercially available trialkylsilane resin. The supported cyanide was obtained with excellent yield and proved to be shelf-stable. This supported reagent was reacted with a series of aldehydes and ketones yielding the corresponding polymer-supported cyanohydrins in good-to-excellent yields. A stability study on a model cyanohydrin demonstrated that these supported intermediates also can be stored for a prolonged time. For the last step, a cleavage strategy that could release either cyanohydrins or alpha-hydroxy esters was adopted. Finally, we prepared a polymer-supported trialkylsilyl azide, which also proved to be shelf-stable.     

Polymer-supported reagents for multi-step organic synthesis: application to the synthesis of sildenafil

Sildenafil 1 (Viagra), a well known and commercially important pharmaceutical drug, has been prepared using polymer-supported reagents in a multi-step, convergent process resulting in a clean and efficient preparation without the need for conventional purification methods.     

Fluorescein isothiocyanates: improved synthesis and purity. Spectral studies

Modification of the methods of synthesis by direct reaction of the isomeric aminofluoresceins with a limited excess of thiophosgene in the presence of calcium carbonate has yielded the 5- and 6-isothiocyanatofluoresceins of high purity. The ir and nmr spectra of these isomers are described.     

BOP‐OXy,BOP‐OBt,and BOP‐OAt: novel organophosphinic coupling reagents useful for solution and solid‐phase peptide synthesis

Stand‐alone coupling reagents derived from bis(2‐oxo‐3‐oxazolidinyl)phosphorodiamidic chloride show efficient performance in solution and SPPS. In particular, the Oxyma Pure (Luxembourg Biotech., Tel Aviv, Israel) derivative shows the additional advantage of being highly soluble in DMF and even fairly soluble in CH₃CN, which can extend its use for the synthesis of complex peptides. These new stand‐alone coupling reagents have the advantage of not bearing any counteranion such as PF₆ or BH₄, whose presence can jeopardize the purification of final peptides prepared in solution. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Studies in C-terminal sequencing: New reagents for the synthesis of peptidylthiohydantoins

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Baileyet al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50°C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.     

New reagents and solid support for automated oligonucleotide synthesis

The optimal system for the rapid, efficient, convenient, and economical synthesis and purification of synthetic oligonucleotides has been advancing. By recognizing the very rapid reaction kinetics and taking advantage of an efficient, low volume delivery system, cycle times have decreased to about 5.5 minutes, without compromising synthesis performance. A new set of base protecting groups for cyanoethylphosphoramidite nucleoside monomers have been developed, which decreases the post-synthesis time requirements. A particular form of polystyrene has also been developed as a solid support for automated oligonucleotide synthesis. Typical sequencing or PCR primers (20mers) now require less than 2 hours for synthesis and 2 hours for cleavage and deprotection.     

Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.

Trinucleotide phosphoramidites representing codons for all 20 amino acids have been prepared and used in automated, solid-phase DNA synthesis. In contrast to an earlier report, we show that these substances can be used to introduce entire codons into oligonucleotides in excess of 98% yield, and are ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.     

Design,solid phase synthesis and evaluation of cationic ferrocenoyl peptide bioconjugates as potential antioxidant enzyme mimics

Synthetic C-terminal amidated cationic ferrocenoyl peptide bioconjugates Fc-Orn-Orn-Orn (1) and Fc-Tyr-Orn-Orn-Orn (2) were rationally designed as superoxide dismutase (SOD) mimics based on the structure of the iron SOD from Escherichia coli. Ferrocenoyl peptide bioconjugates 1, 2 and ferrrocenecarboxylic acid (4) were subsequently evaluated as SOD mimics and as inhibitors of peroxynitrite-mediated tyrosine nitration. Due to their cationic character, ferrocenoyl peptide bioconjugates 1 and 2 exerted an acceptable SOD activity (EC₅₀ = 575 μM and 310 μM, respectively) in comparison with 4 (EC₅₀ = 1.4 mM). The C-terminal amidated cationic peptide Ac-Tyr-Orn-Orn-Orn (3), designed as marker of peroxynitrite, was used to evaluate the inhibitory activity of 1 and 4 towards peroxynitrite-mediated tyrosine nitration. Both compounds proved to inhibit the nitration especially the cationic ferrocenoyl peptide bioconjugates 1. The ferrocene moiety of conjugate 2 displayed a strong inhibitory activity of peroxynitrite-mediated nitration of the neighboring tyrosine.     

New reagents and methods for the synthesis of internal and 3'-labeled DNA

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.     

Radio-LC-MS for the characterization of 99mTc-labeled bioconjugates

This report describes the first example of using radio-LC-MS for determining the composition of (99m)Tc radiopharmaceuticals at the tracer level. The in-line radiometric detector is a useful addition to a standard LC-MS and provides direct correlation between the MS data and the radioactive species in a radiopharmaceutical kit. Complexes [(99m)Tc(HYNICtide)(tricine)(L)] (RP444, L = TPPTS; RP445, L = TPPDS; and RP446, L = TPPMS) were prepared using a decayed generator eluant. All the ternary ligand (99m)Tc complexes show the expected monoprotonated molecular ions, (M + 1)(+), and diprotonated molecular ions, (M + 2)(2+). The LC-MS spectral data support the proposed structure and are consistent with those obtained for their corresponding (99)Tc analogues. Ternary ligand complexes [(99m)Tc(HYNICtide)(tricine)(L)] (L = ISONIC-HE and ISONIC-Sorb) are neutral, and the molecular weights are also lower than that of RP444. Using a fresh generator eluant (24 h prior elution), only 1-2 mCi of (99m)Tc [(7 x 10(-)(12))-(1.5 x 10(-)(11)) mol of technetium complex] are required to obtain a reasonably clean mass spectrum. Radio-LC-MS is a quick and accurate analytical tool for characterization of (99m)Tc radiopharmaceuticals at the tracer level.     

Design and synthesis of reagents for phage display screening of dehalogenases

Bifunctional molecules containing both a biotin and a substrate unit have been designed and synthesized for phage display screening of mutant libraries of haloalkane dehalogenase enzymes. The molecules were assembled using a convergent modular synthetic strategy. One molecule was synthesized to evaluate the concept of covalent capture and a second for screening of phage libraries for enantioselectivity.     

Arylsulfonyltetrazoles, new coupling reagents and further improvements in the triester method for the synthesis of deoxyribooligonucleotides.

The modified triester approach has been further improved and refined to the synthesis of defined sequences of deoxyribo-oligonucleotides. Improvements include arylsulfonyltetrazoles as faster and milder condensing agents, benzenesulfonic acid to avoid depurination during deblocking of trityl protecting groups and improved chromatographic procedures for purification of triester intermediates and purification of the final product containing 3'-5' phosphodiester linkages.