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The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Flexible manufacturing systems are needed in the pharmaceutical industry due to the future challenges of volatility, uncertainty, complexity, and ambiguity [1]. Compared to traditional manufacturing systems that dominate the pharmaceutical industry today, processes that are better able to react to changes in the types of products being sold, the volume of sales, and the facilities needed for production will be an asset to an organization. Many emerging technologies are inherently adaptable, though the types of flexibility exhibited vary widely. The nature of these emerging technologies is examined here, and the case is made that flexibility should be valued as much as cost and time when selecting technologies, even though flexible manufacturing systems require extra time and money initially. An outlook on this type of manufacturing is shared with the assertion that flexible development and manufacturing would help reduce overall cost and better serve patient needs following the initial investment period. In the next decade, concentrated effort is needed from industry, academia, suppliers, and regulators to enable more agile and nimble pharmaceutical development and manufacturing.

Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.

Quantification of oligonucleotides is commonly performed by UV absorption or fluorescence based assays. However, chemical modifications may change the UV absorbance or fluorescence of oligonucleotides, making their accurate quantification difficult. Alternatively, atomic spectrometry can be used for direct quantification of phosphorus content in oligonucleotides, and their concentration can then be calculated using the phosphorus to DNA/RNA nucleotide mass ratio. Nevertheless, these methods require labor-intensive sample preparations, and thus have relatively low sample analysis throughput. In order to meet the need to quantify large libraries of synthetic structurally-modified oligonucleotides in drug discovery, a high-throughput inductively coupled plasma–mass spectrometry (ICP-MS) method has been developed in this study. Specifically, a high-performance liquid chromatography (HPLC) system is utilized in automated flow injection (FI) mode and coupled to an ICP-MS instrument to provide rapid and accurate quantification of phosphorus content in synthetic small interfering RNAs (siRNAs). Isobaric interferences at 31 P + are avoided by monitoring the 31 P 16 O + molecular ion under optimized ICP-MS conditions. The use of a well-plate autosampler reduces sample injection cycle time and adds flexibility to the system by allowing direct analysis of aqueous samples in 96/384 well-plates without sample preparation. A throughput of 39 s per sample has been realized. The method requires minimal sample consumption (1–100 μL), which overcomes a major drawback of the conventional methods and makes it possible for practical use of oligonucleotide screening. Other advantages of this approach include excellent sensitivity (LOD = 136 ng L − 1 as phosphorus), large linearity range and reduced solvent usage. The FI-ICP-MS method provides the capability of accurate quantitation of a large number of oligonucleotide samples with significantly improved sample analysis throughput over conventional methods.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

Process research and development of a synthetic route towards a novel renin inhibitor (1) is described. The highly convergent synthetic route provided 1 in 15% yield on multikilogram scale with a longest linear sequence of 11 steps. The use of catalytic hydrogenation features prominently in our design. The proper choice of N-methylpyridone surrogate was also important, and we describe a method for the easy conversion of 2-methoxypyridines to N-methylpyridones using cheap and readily available reagents.

The use of ambient pressure desorption ionization mass spectrometry for the rapid analytical support of process and pharmaceutical development is demonstrated. The ability of direct analysis in real time (DART) technology to analyze both active pharmaceutical ingredients (APIs) and intermediates without sample preparation or the development of LC-based separations provided critical experimental results with minimal time required for method development. The utility and versatility of DART is shown for applications such as degradation studies, analysis of high-throughput catalyst screens, preparative-scale chromatography fractions, and impurity determination.

The realization of polymer conjugate-based RNA delivery as a clinical modality requires the development and optimization of novel formulations. Although many literature examples of polymer conjugate-based SiRNA delivery systems exist, the protocols described herein represent a robust and facile way of screening any poly(amine)-based polymer system for SiRNA delivery. In this chapter, we describe the synthetic methods used to prepare poly(amide) polymers using a controlled polymerization method, as well as the preparation of the resulting targeted SiRNA polymer conjugates. In addition, detailed methods are provided for the characterization of the biodegradable poly(peptides) as well as the polymer conjugate that ensues.

The realization of polymer conjugate-based RNA delivery as a clinical modality requires the development and optimization of novel formulations. Although many literature examples of polymer conjugate-based SiRNA delivery systems exist, the protocols described herein represent a robust and facile way of screening any poly(amine)-based polymer system for SiRNA delivery. In this chapter, we describe the synthetic methods used to prepare poly(amide) polymers using a controlled polymerization method, as well as the preparation of the resulting targeted SiRNA polymer conjugates. In addition, detailed methods are provided for the characterization of the biodegradable poly(peptides) as well as the polymer conjugate that ensues.

The insertion of carbon dioxide into the Mn−O bond of fac-(CO)3(dppe)MnOCH3 (1) was observed to occur instantaneously at −78 °C by in situ infrared spectroscopy. The product of carboxylation of 1, fac-(CO)3(dppe)MnOC(O)OCH3 (2), underwent decarboxylation with a first-order rate constant of 1.49 × 10-4 s-1 at 23 °C. The kinetic parameters for this process were determined by trapping the intermediate produced upon CO2 extrusion, complex 1, with COS to provide the very stable fac-(CO)3(dppe)MnSC(O)OCH3 (3) derivative. The structure of 3 was determined by single-crystal X-ray diffraction analysis, establishing the presence of the Mn−S bond.

Topochemical reduction of CaEu2Ti2O8, a new n=1 mixed Ruddlesden–Popper/Dion–Jacobson phase prepared by divalent ion-exchange of NaEuTiO4, yields the non-defective n=2 Ruddlesden–Popper phase Eu2CaTi2O7.

We describe the synthesis of a polycatenated cyclic polymer, a structure that resembles a molecular charm bracelet. Ruthenium-catalyzed ring-opening metathesis polymerization of an amino-containing cyclic olefin monomer in the presence of a chain transfer agent generated an alpha,omega-diazide functionalized polyamine. Cyclization of the resulting linear polyamine using pseudo-high-dilution copper-catalyzed click cyclization produced a cyclic polymer in 19% yield. The click reaction was then further employed to remove linear contaminants from the cyclic polymer using azide- and alkyne-functionalized scavenging resins, and the purified cyclic polymer product was characterized by gel permeation chromatography, (1)H NMR spectroscopy, and IR spectroscopy. Polymer hydrogenation and conversion to the corresponding polyammonium species enabled coordination and interlocking of diolefin polyether fragments around the cyclic polymer backbone using ruthenium-catalyzed ring-closing olefin metathesis to afford a molecular charm bracelet structure. This charm bracelet complex was characterized by (1)H NMR spectroscopy, and the catenated nature of the small rings was confirmed using two-dimensional diffusion-ordered NMR spectroscopy.

Hydrazines and hydroxylamines have been found to be excellent nucleophiles for the palladium-catalyzed dynamic asymmetric allylic amination of vinyl epoxide, with good yields and enantioselectivities of up to 97% ee. This method is applicable to acyclic and heterocyclic amines and was applied toward a five-step synthesis of (R)-piperazic acid.

Hydrazines and hydroxylamines have been found to be excellent nucleophiles for the palladium-catalyzed dynamic asymmetric allylic amination of vinyl epoxide, with good yields and enantioselectivities of up to 97% ee. This method is applicable to acyclic and heterocyclic amines and was applied toward a five-step synthesis of (R)-piperazic acid.

[reaction: see text]. Olefin metathesis has been employed in the efficient syntheses of a [2]catenane with the templation being provided by the recognition between a secondary ammonium ion and a crown ether. In one approach, a crown ether precursor has been clipped around an NH2+ center situated in a macrocyclic ring, yielding the mechanically interlocked compound. In the other approach, the reversible nature of olefin metathesis allows for a magic ring synthesis to occur wherein two free macrocycles can be employed as the stationary materials, leading to the formation of the same [2]catenane.

A strategy for the formation of mechanically interlocked polymers is presented. Ring-closing olefin metathesis has been shown to provide a very high yielding route to [c2]daisy-chains suitably functionalized to allow their one-step conversion to bisolefins which can be used as monomers in ADMET polymerizations to afford mechanically interlocked polymers. Metathesis, in two different guises is making a hitherto unreachable goal in synthesis a reality.

Topochemical reduction of CaEu2Ti2O8, a new n=1 mixed Ruddlesden–Popper/Dion–Jacobson phase prepared by divalent ion-exchange of NaEuTiO4, yields the non-defective n=2 Ruddlesden–Popper phase Eu2CaTi2O7.