Synthesis of peptide-siRNA conjugates via internal sulfonylphosphoramidate modifications and evaluation of their in vitro activity.

Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide-siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide-siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3'-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.

A Streptococcus pneumoniae Type 2 Oligosaccharide Glycoconjugate Elicits Opsonic Antibodies and Is Protective in an Animal Model of Invasive Pneumococcal Disease.

Invasive pneumococcal diseases (IPDs) remain the leading cause of vaccine-preventable childhood death, even though highly effective pneumococcal conjugate vaccines (PCVs) are used in national immunization programs in many developing countries. Licensed PCVs currently cover only 13 of the over 90 serotypes of Streptococcus pneumoniae (Sp), so nonvaccine serotypes are a major obstacle to the effective control of IPD. Sp serotype 2 (ST2) is such a nonvaccine serotype that is the main cause of IPD in many countries, including Nepal, Bangladesh, and Guatemala. Glycoconjugate vaccines based on synthetic oligosaccharides instead of isolated polysaccharides offer an attractive alternative to the traditional process for PCV development. To prevent the IPDs caused by ST2, we identified an effective ST2 neoglycoconjugate vaccine candidate that was identified using a medicinal chemistry approach. Glycan microarrays containing a series of synthetic glycans resembling portions of the ST2 capsular polysaccharide (CPS) repeating unit were used to screen human and rabbit sera and identify epitope hits. Synthetic hexasaccharide 2, resembling one repeating unit (RU) of ST2 CPS, emerged as a hit from the glycan array screens. Vaccination with neoglycoconjugates consisting of hexasaccharide 2 coupled to carrier protein CRM197 stimulates a T-cell-dependent B-cell response that induced CPS-specific opsonic antibodies in mice, resulting in killing of encapsulated bacteria by phagocytic activity. Subcutaneous immunization with neoglycoconjugate protected mice from transnasal challenge with the highly virulent ST2 strain NCTC 7466 by reducing the bacterial load in lung tissue and blood.

Catalytic asymmetric synthesis of 4-nitropyrazolidines: an access to optically active 1,2,3-triamines.

The first catalytic enantio- and diastereoselective synthesis of 4-nitropyrazolidines is presented. Asymmetric hydrogen-bonding activation of nitro-olefins facilitated the 1,3-dipolar cycloaddition with hydrazones, affording optically active 4-nitropyrazolidines containing three continuous stereogenic centers as a single diastereomer in up to 99% ee. Furthermore, it is demonstrated that the optically active 4-nitropyrazolidines can be applied as precursors for the synthesis of highly interesting 1,2,3-triamines.

Catalytic asymmetric diaziridination.

The first catalytic enantio- and diastereoselective synthesis of diaziridines is presented. Aziridination of N-tosyl aldimines applying modified hydroxylamine under asymmetric phase-transfer catalysis furnished optically active diaziridines. The diaziridines are formed as a single diastereomer in up to 96% ee, containing two orthogonal N-protecting groups, which can be deprotected selectively.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Catalytic enantioselective oxaziridination.

The first catalytic enantioselective synthesis of oxaziridines is presented. The oxidation of aryl and alkyl aldimines with m-CPBA under organocatalytic conditions using cinchona alkaloid-derived catalysts furnished optically active oxaziridines in good yields and high enantioselectivities (up to 94% ee). Mechanistic investigations indicate a stepwise enantioselective oxidation process.

Organocatalytic strategies for the construction of optically active imidazoles, oxazoles, and thiazoles.

This study demonstrates the first enantioselective synthesis of hydroxyalkyl- and aminoalkyl-substituted imidazoles, oxazoles, and thiazoles. The approach developed utilizes a highly effective one-pot reaction cascade that consists of either an organocatalytic epoxidation or aziridination of α,ß-unsaturated aldehydes coupled with a [3+2]-annulation, in which amidines, ureas, or thioureas act as effective 1,3-dinucleophilic species. The methodology described benefits from low catalyst loadings, commercially and readily available starting materials, and mild reaction conditions.

Asymmetric one-pot sequential organo- and gold catalysis for the enantioselective synthesis of dihydropyrrole derivatives.

A direct asymmetric one-pot synthesis of optically active 2,3-dihydropyrroles from propargylated malononitrile and N-Boc-protected (Boc = tert-butoxycarbonyl) imines is presented. The approach is based on a bifunctional organocatalytic Mannich-type reaction and a subsequent gold-catalyzed alkyne hydroamination and isomerization. The compatibility of both catalytic systems is presented and the overall transformation results in good yields (up to 70 %) with high selectivities (endo/exo > 10:1) and enantioselectivities (up to 88 % ee). The absolute configuration of the final products is unambiguously established by X-ray analysis. To highlight the synthetic potential of the accessed heterocyclic compounds, their transformation into 1-pyrrolines, which represent direct precursors of pyrrolidines, is presented.

A cleavable azide resin for direct click chemistry mediated enrichment of alkyne-labeled proteins.

The direct conjugation of a labeled proteome to a cleavable azide resin utilizing the copper-catalyzed azide alkyne cycloaddition is demonstrated. The procedure omits the classical streptavidin- and biotin-based affinity enrichment step and represents an operationally simpler, cheaper and less contaminated alternative for protein purification.

An organocatalytic one-pot cascade incorporating the Achmatowicz reaction affording 3-pyrone derivatives.

The development of an organocatalytic one-pot cascade for the annulation of simple starting materials: α,ß-unsaturated aldehydes, hydrogen peroxide, ß-carbonyl compounds and NBS to furnish optically active 3-pyrones in good yield and with excellent enantioselectivity is presented. Further diversification of the obtained products is demonstrated by selective reductive transformations.

A practical electromediated ipso-hydroxylation of aryl and alkyl boronic acids under an air atmosphere.

An efficient electromediated aerobic ipso-hydroxylation reaction of aryl and alkyl boronic acids has been developed. Furthermore, mechanistic insight into the role of superoxide anions in this reaction has also been provided based on electrochemical studies and experimental results.

An asymmetric organocatalytic approach towards allylic amines and beta-keto amino compounds.

The enantioselective organocatalysed addition of beta-keto benzothiazolesulfones to N-Boc-protected imines, leading to intermediates easily transformed into optically active allylic amines or beta-keto amino compounds, is presented.