Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.     

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl amino protecting strategy.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.     

Synthesis of oligonucleotides containing 2''-deoxy-6-thioguanosine at a predetermined site.

A new approach has been devised for the synthesis of oligonucleotides containing 2'-deoxy-6-thioguanosine [d(s6G)]. The synthesis of oligonucleotides containing d(s6G) requires special protection and deprotection strategies to prevent the thione functionality from undergoing oxidation and hydrolysis. Previous attempted syntheses have neglected to address this problem. By using the cyanoethyl protecting group for the thione and phenoxyacetyl for the exocyclic amino group, it was possible to deprotect oligonucleotides with a mixture of sodium hydroxide and sodium hydrogen sulfide without any significant conversion of d(s6G) to deoxyguanosine. Application of this strategy will allow investigation of the structural as well as biological activity of d(s6G)-containing oligonucleotides.     

The aspartimide problem in Fmoc-based SPPS. Part III.

A newly developed Fmoc-Asp derivative, Fmoc-Asp beta-(2,3,4-trimethyl-pent-3-yl) ester, has been tried in the Fmoc-based SPPS of H-Val-Lys-Asp-Xaa-Tyr-Ile-OH, a well-established peptide model for studying base-catalysed aspartimide formation. When synthesizing the hexapeptide incorporating Gly, Arg(Pbf), Asn(Mtt), Asp(OtBu) or Cys(Acm) for Xaa, considerable amounts of aspartimide-related by-products were to be expected. The Asp(3) beta-carboxy protecting group and the duration of exposure to bases were varied. By-product formation could be reduced by incorporation of the new Asp derivative more efficiently than by introducing the less bulky Asp(OMpe). Significant improvements were observed in cases of prolonged contact with piperidine or DBU. Both beta-carboxy protecting groups were superior to the standard Asp(OtBu) which was also included in this study, but the additional stabilization gained by our new protecting group was valuable especially in syntheses of long peptides or difficult sequences.     

Nucleosides and nucleotides. 126. Incorporation of a mutagenic nucleoside, 5-formyl-2′-deoxyuridine, into an oligodeoxyribonucleotide

An oligodeoxyribonucleotide containing 5-formyl-2′-deoxyuridine (1) was synthesized using a new protecting group of the formyl group.     

Solid-phase synthesis of a nucleopeptide from the linking site of adenovirus-2 nucleoprotein, -Ser(p5'CATCAT)-Gly-Asp-. Convergent versus stepwise strategy.

The synthesis of a nucleopeptide with the sequence -Ser(p5'CATCAT)-Gly-Asp- has been undertaken by either convergent or stepwise solid-phase strategies, both of which use base-labile permanent protecting groups. The coupling of phosphitylated protected peptides onto oligonucleotide-resins did not afford the desired nucleopeptide, which was nevertheless obtained after oligonucleotide elongation at the hydroxyl group of the resin-bound peptide and deprotection under mild basic conditions. A preliminary study on the stability of different nucleopeptides to bases is also reported.     

2-C-Methyluridine modified hammerhead ribozyme against the estrogen receptor

A new synthesis of 2′-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2′-hydroxyl group. Comparison to previous protecting strategies and analysis of stability under 5′-DMTr removing conditions are discussed. The synthetic incorporation of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estrogen receptor α protein (ER-α), and transfection experiments in MCF-7 cell line are also presented.     

Properties and applications of the (2-nitrofluoren-9-yl)methoxycarbonyl group.

This paper presents a new protecting group, the (2-nitrofluoren-9-yl)methoxycarbonyl group. Investigations on the properties of this new modification of the Fmoc-system, such as the solvent-dependent photochemical cleavage, and enhanced lability towards bases, are described, as well as UV-kinetic measurements of the cleavage reaction. In addition, the incorporation of the (2-nitrofluoren-9-yl)methoxycarbonyl group into two peptides, and a sequence-dependent photochemical cleavage reaction are reported.     

4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new orthoester-type protecting group for the 2'-hydroxyl function in the chemical synthesis of RNA

We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2'-hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for oligonucleotide synthesis. This group was introduced to the 2'-hydroxyl group of appropriately protected nucleoside derivatives in good yields under mild acidic conditions. Post-synthetic conversion of the moiety of this protecting group with an amine resulted in formation of a new amide moiety that is more stable to acid deprotection in aqueous solution, but it can still be easily removed by treatment with acids in organic solvents. In this article, we also describe the stability of not only the original and modified protecting groups but also internucleotidic phosphate linkages of protected RNA intermediates under deprotection conditions.     

A new 2'-hydroxyl protecting group for the automated synthesis of oligoribonucleotides.

We have developed a new type of 2'-hydroxyl protecting group for the automated machine synthesis of RNA oligomers: a 2-hydroxyisophthalate formaldehyde acetal (HIFA). The unique feature of this protecting group is that, as the bis ester, it is relatively stable to the acidic conditions that are used for repeated removal of dimethoxytrityl groups during chain elongation, but the final deprotection step in alkali, which cleaves the chain from the support and removes the base and phosphate protecting groups, converts it to the bis carboxylate and this can be removed relatively rapidly by treatment with mild acid. Conversion of the bis ester to the bis carboxylic acid increases the rate of acid-catalyzed hydrolysis of the acetal by 42-fold at pH 1, and, possibly, by 1320-fold at pH 3. The bis ester is 112 times more stable than the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp) towards hydrolysis at pH 1, while the bis acid is only 2.35 times more stable than Fpmp at pH 3. In synthesis of the dimers UpU and UpG, with a coupling time of 5 min, the dimethoxytrityl cation assay indicated coupling yields of > 98%.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Prevention of chain cleavage in the chemical synthesis of 2''-silylated oligoribonucleotides.

Strong aqueous ammonium hydroxide used to remove N-acyl protecting groups from synthetic oligoribonucleotides causes removal of some alkylsilyl protecting groups from 2'-hydroxyls and leads to chain cleavage. This problem is most severe when 30% ammonium hydroxide is used and substantially reduced but still detectable when 3:1 ammonium hydroxide-ethanol is used. We have virtually eliminated this unwanted cleavage by incorporating the labile phenoxyacetyl amino protecting group on adenosine and guanosine. The N-benzoyl protecting group remains adequate for cytidine nucleosides. Synthetic oligoribonucleotides containing these N-acylated nucleosides and 2'-t-butyldimethylsilyl or 2'-triisopropylsilyl protecting groups can be deacylated by room temperature treatment in saturated anhydrous methanolic ammonia (8-12 h) without causing any detectable chain cleavage.     

A New Protecting Group Strategy for Amino Groups in Oligonucleotide Chemistry: CEOC Group

Abstract

A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.     

Ninhydrin as a reversible protecting group of amino-terminal cysteine.

The proximity of the alpha-amine and beta-thiol of alpha-amino terminal-cysteine (NT-Cys) residues in peptides imparts unique chemical properties that have been exploited for inter- and intra-molecular ligation of unprotected peptides obtained through both synthetic and biological means. A reversible protecting group orthogonal to other protection strategies and reversible under mild conditions would be useful in simplifying the synthesis, cleavage, purification and handling of such NT-Cys peptides. It could also be useful for the sequential ligation of peptides. To this end, we explored tri-one chemistry and found that ninhydrin (indane-1,2,3 trione) reacted readily with cysteine or an NT-Cys-containing peptide on- or off-resin at pH 2-5 to form Ninhydrin-protected Cys (Nin-Cys) as a thiazolidine (Thz). The Thz ring, protecting both the amino and thiol groups in Nin-Cys, completely avoids the formylation and Thz side reactions found during hydrofluoric acid (HF) cleavage when N-pi-benzyloxymethyl histidine groups are present. Nin-Cys is stable during coupling reactions and various cleavage conditions with trifluoroacetic acid or HF, but is deprotected under thiolytic or reducing conditions. These properties enable a facile one-step deprotection and end-to-end-cyclization reaction of Nin-Cys peptides containing C-terminal thioesters.     

Cell surface mucin-type glycoproteins and mucin-like domains.

Cell surface mucins and mucin-like domains comprise a diverse and heterogeneous group of cell surface glycoproteins. The heterogeneity results from both genetic variations in the polypeptides and carbohydrate differences. Mucins form extended rods from the cell surface. The mucin domains apparently serve a protective function, protecting the glycoproteins from cell surface proteolysis and protecting the cells from attack by other cells. Biosynthesis of mucin oligosaccharides is initiated near the transit of the proteins from the endoplasmic reticulum to the Golgi and proceeds rapidly during passage to the cell surface. In some carcinomas a second O-glycosylation pathway adds new oligosaccharides to the glycoproteins after they have reached the cell surface, presumably during recycling.     

4,5-BIS(ETHOXYCARBONYL)-[1,3]DIOXOLAN-2-YL AS A NEW ORTHOESTER-TYPE PROTECTING GROUP FOR THE 2′-HYDROXYL FUNCTION IN THE CHEMICAL SYNTHESIS OF RNA

We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2′-hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for oligonucleotide synthesis. This group was introduced to the 2′-hydroxyl group of appropriately protected nucleoside derivatives in good yields under mild acidic conditions. Post-synthetic conversion of the moiety of this protecting group with an amine resulted in formation of a new amide moiety that is more stable to acid deprotection in aqueous solution, but it can still be easily removed by treatment with acids in organic solvents. In this article, we also describe the stability of not only the original and modified protecting groups but also internucleotidic phosphate linkages of protected RNA intermediates under deprotection conditions.     

A new water-soluble N-protecting group, 2-(phenyl(methyl)sulfonio)ethyloxycarbonyl tetrafluoroborate, and its application to solid phase peptide synthesis in water.

A new water-soluble N-protecting group, 2-[phenyl(methyl)sulfoniolethyloxycarbonyl tetrafluoroborate, has been prepared and its application to solid phase peptide synthesis in water has been studied. Leu-enkephalin amide was successfully synthesized in water by the solid phase method using this protecting group.     

Synthesis of RNA oligomer using 9-fluorenylmethoxycarbonyl (Fmoc) group for 5'-hydroxyl protection

An approach using a new combination of protecting groups in RNA oligomer synthesis is proposed, in which 5'-hydroxyl group of ribose moiety is temporarily protected with the alkaline labile 9-fluorenylmethoxycarbonyl (Fmoc) group and the 2'-hydroxyl group is protected with the acid labile 1-ethoxyethyl (EE) group. The adoption of this method presented great selectivity in removing the 5'-hydroxyl protecting group and facilitated the RNA oligomer synthesis. A RNA pentamer was synthesized by the phosphotriester method in solution.     

A convenient solid-phase method for synthesis of 3'-conjugates of oligonucleotides.

We present a new procedure for the preparation of 3'-conjugates of oligonucleotides through solid-phase synthesis. A suitable universal solid support was readily prepared using a series of peptide-like coupling reactions to incorporate first a spacer and then an L-homoserine branching unit. The N-alpha-position of the homoserine carries an Fmoc protecting group that is removed by treatment with piperidine to liberate an amino group suitable for attachment of the conjugate (e.g., small organic molecule, fluorescent group, cholesterol, biotin, amino acid, etc.) or for assembly of a short peptide. The side-chain hydroxyl group of the homoserine carries a trityl protecting group. After TFA deprotection, the hydroxyl group acts as the site for oligonucleotide assembly. An additional spacer, such as aminohexanoyl, may be incorporated easily between the conjugate molecule and the oligonucleotide. A number of examples of synthesis of 3'-conjugates of oligonucleotides and their analogues are described that involve standard automated oligonucleotide assembly and use of commercially available materials. The linkage between oligonucleotide and 3'-conjugate is chirally pure and is stable to conventional ammonia treatment used for oligonucleotide deprotection and release from the solid support. The homoserine-functionalized solid support system represents a simple and universal route to 3'-conjugates of oligonucleotides and their derivatives.     

(2-Azidomethyl)phenylacetyl as a new, reductively cleavable protecting group for hydroxyl groups in carbohydrate synthesis

The (2-azidomethyl)phenylacetyl group (AMPA) is described as a new protecting group for carbohydrates. AMPA was introduced to carbohydrate hydroxyl groups in the presence of DCC, while its removal was conveniently achieved via Lindlar catalyst-catalyzed hydrogenation that had no influence on other protecting groups including benzyl, acyl, acetal and ketal.