Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide.

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general. In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN. This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part I: Separation of small molecules.

Nowadays, environmental problems are drawing the attention of governments and international organisations, which are therefore encouraging the transition to green industrial processes and approaches. In this context, chemists can help indicate a suitable direction. Beside the efforts focused on greening synthetic approaches, currently also analytical techniques and separations are under observation, especially those employing large volumes of organic solvents, such as reversed-phase liquid chromatography (RPLC). Acetonitrile has always been considered the best performing organic modifier for RPLC applications, due to its chemical features (complete miscibility in water, UV transparency, low viscosity etc); nevertheless, it suffers of severe shortcomings, and most importantly, it does not fully comply with Environmental, Health and Safety (EHS) requirements. For these reasons, alternative greener solvents are being investigated, especially easily available alcohols. In this work, chromatographic performance of the most common solvents used in reversed-phase chromatography, i.e., acetonitrile, ethanol and isopropanol, have been compared to a scarcely used solvent, dimethyl carbonate (DMC). The analytes of interest were two small molecules, caffeine and paracetamol, whose kinetics and retention behaviour obtained with the four solvents have been compared, and all contributions to band broadening have been assessed. Results about kinetic performance are very promising, indicating that a small amount (7 % v/v) of DMC is able to produce the same efficiency as a 2.5-times larger ACN volume (18 % v/v), and larger efficiency than alcohols. This paper reports, for the first time, fundamental studies concerning the mass transfer phenomena when DMC is used as an organic solvent in RPLC, and, together with the companion paper, represents the results of a research whose final aim was to discover whether DMC is suitable for chromatographic applications both in linear and preparative conditions.

Microwave-assisted enzymatic synthesis of geraniol esters in solvent-free systems: optimization of the reaction parameters, purification and characterization of the products, and biocatalyst reuse.

Various geraniol esters act as insect pheromones and display pharmacological activities, especially as neuroprotective agents. Therefore, the search for synthetic strategies alternative to traditional chemical synthesis could help designing ecofriendly routes for the preparation of such bioactive compounds. Hence, this work aims at the microwave-assisted enzymatic synthesis of geranyl esters in solvent-free systems. The process variables were optimized for the synthesis of geranyl acetoacetate, achieving 85% conversion after 60 min using a 1:5 substrates molar ratio (ester to geraniol), 80 °C and 8.4% of Lipozyme 435 lipase without removal of the co-produced methanol. On the other hand, a 95% conversion was reached after 30 min using 1:6 substrates molar ratio, 70 °C and 7% lipase in the presence of 5Å molecular sieves for the methanol capture. In addition, the lipase showed good reusability, maintaining the same activity for five reaction cycles. Finally, under the above optimized conditions, other geraniol esters were successfully synthetized such as the geranyl butyrate (98%), geranyl hexanoate (99%), geranyl octanoate (98%), and geranyl (R)-3-hydroxybutyrate (56%). These results demonstrate the microwave-assisted lipase-catalyzed transesterification in a solvent-free system as an excellent and sustainable catalytic methodology to produce geraniol esters.

Photoredox Cross-Dehydrogenative Coupling of N-Aryl Glycines Mediated by Mesoporous Graphitic Carbon Nitride: An Environmentally Friendly Approach to the Synthesis of Non-Proteinogenic α-Amino Acids (NPAAs) Decorated with Indoles.

Indole-decorated glycine derivatives are prepared through an environmentally benign cross-dehydrogenative coupling between N-aryl glycine analogues and indoles (yield of ≤81%). Merging heterogeneous organocatalysis and photocatalysis, C-H functionalization has been achieved by selective C-2 oxidation of N-aryl glycines to afford the electrophilic imine followed by Friedel-Crafts alkylation with indole. The sustainability of the process has been taken into account in the reaction design through the implementation of a metal-free recyclable heterogeneous photocatalyst and a green reaction medium. Scale-up of the benchmark reaction (gram scale, yield of 69%) and recycling experiments (over seven runs without a loss of efficiency) have been performed to prove the robustness of the protocol. Finally, mechanistic studies were conducted employing electron paramagnetic resonance spectroscopy to unveil the roles of the photocatalyst and oxygen in the formation of odd-electron species.