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The following review of Green Chemistry principles along with its history, advantages and its relationship to Solid Phase Peptide Synthesis comes from  Fernando Albericio,, and Beatriz Garcia De La Torre,, at Peptide Sciences Laboratory South Africa. The solid phase has features that work with the Green concept. For example, it uses a single reactor, can synthesize any amount of peptide you want (low concentration μmols), has no mechanical loss, no intermediate purification, provides easy work-up, high yield, high purity, better purification, and allows parallel simultaneous synthesis in the very same conditions. In addition, Green Chemistry principles and the processes mentioned above save time, energy, cost, and solvents with less impact on the environment and human health.

Green Chemistry

Green chemistry has been previously practised in many chemical syntheses in the past, however, the concert and definition of Green Chemistry were first developed in the 1990s as the “design of chemical products and processes to reduce or eliminate the use and generation of hazardous substances”[1, 2]. The most crucial aspect of Green Chemistry is the word “design”, since it involves new ideas and careful planning. The twelve green chemistry principles were initiated in 1998 by Anastas and Warner, and they can be simplified as the following: Waste prevention, Atom Economy, Less hazardous chemical synthesis, Designing safer chemicals, Safer solvents and auxiliaries, Design for energy for energy efficient, Use of renewable feedstock, Reduce derivatives, Catalysis, design for degradation, Real-time analysis for pollution prevention, and Inherently safer chemistry for accident prevention, these are explained further in literature[2-5]. These principles or “design rules” should be complied to whenever a new product is being designed, produced, or used, to eliminate hazardous adverse consequences. The Green Chemistry ideology calls for a development of new strategies for chemical synthesis, processing, and use of chemicals that are less hazardous and would be safe for humans and environment[6]. The green chemistry approach has many benefits, it allows chemist to achieve sustainability, it protects the environment, good for human health, it cost effective/profitable. Most importantly, the green chemistry is well adopted by many chemistry sectors including scholars and researchers in the academia, research centres and industries worldwide[6-9].

Green Solid Phase Peptide Synthesis

The commonly used strategy for the synthesis of peptides in nowadays is solid phase peptide synthesis (SPPS), pioneered in 1963, by R. Bruce Merrifield[10]. Since its introduction, it has revolutionized the peptide chemistry, and Merrifield was honoured by an award of Nobel Prize in Chemistry in 1984, and this caused a high synthesis of peptide for pharmaceutical market[10, 11]. The solid phase has features that agrees with Green concept. For example, uses a single reactor, can synthesize any amount of peptide you want (low concentration μmols), no mechanical loss, no intermediate purification, easy work-up, high yield, purity is high and better purification, allow parallel simultaneous synthesis in the very same conditions, all these processes mentioned above save time, energy, cost effective, automated, saves solvents and not too bad for environment and human health[10, 12]. Also, the solid support used in SPPS, 2-chlorotrityl chloride (CTC) resin can be re-used few times with small sized peptides, although this is not a universal protocol, and the cleavage of tBu protected peptide from CTC resin is done under mild conditions (1−2% of TFA in DCM)[13, 14]. Although SPPS is a good strategy, however it also has a major drawback, which violate green chemistry concept, like the use of harsh acids/bases for deprotection of protecting groups, cleaving of peptide from resin, hazardous solvents for swelling of resin, coupling, excess washing, and the purification generate a lot of solvent waste[8].
The SPPS which involves no solvent or water as solvent will be the best strategy, however due to solubility issues of some chemicals or reagents used in SPPS this is not easily done but possible[12, 15, 16]. Also, other peptide sequences are difficult to synthesize under these conditions, so it is best idea to substitute hazardous solvents with greener solvents that would still work well with SPPS and produce excellent results. The commonly used solvents for SPPS are dichloromethane (DCM), N,N-dimethylformamide (DMF), and methylpyrrolidone NMP which in green chemistry guides are classified as extremely hazardous[17, 18]. Our group have reported various greener solvents such as 2-MeTHF (2-methyltetrahydrofuran) and CPME (cyclopentyl methyl ether), ethyl acetate, γ-valerolactone (GVL), -formylmorpholine (NFM) as alternatives[19-23]. Also, our group is not focusing only on greening the solvents, but they are extensively working on greening the whole SPPS chemicals used and reagents[15, 24, 25]. The atom economy of SPPS is highly negative, our group is also researching about this topic[26].

Green Ethers to Precipitate Peptides

The two famous strategies of SPPS are Boc/benzyl and Fmoc/t-butyl after peptide synthesis, are both depending on acidolysis cleavage for the simultaneous removal of protecting groups and release of peptide from resin[27-29]. This process is commonly known as “global deprotection” which is carried out in harsh acid (e.g., TFA) only when peptide has no protecting groups, or in presence of scavengers (TIS/H2O) to capture carbocation obtained from release of protecting groups.
Usually a cold (-20o C) diethyl ether (DEE) is used to precipitate the peptide out of the acidic solution while keeping non-volatile scavengers and any other non-polar protecting groups by-product in solution[30]. The precipitated peptide is washed with cold DEE, centrifuge and collect precipitate, this is repeated 3 times. This repeated washing helps to remove any residual scavengers[31]. Although DEE is the most widely used ether but has a low flash point and boiling point (45 C and 35 C, respectively) and a low temperature of auto ignition, and it is prone to forming peroxides and is not regarded as a green solvent[17, 32, 33]. There are many different ethers that have been tried in replacement of DEE. The peroxide free methyl tert-butyl ether (MTBE) was investigated as an alternative. However, MTBE posed serious drawbacks such as of low solubility, low flash point, unstable under acidic conditions, and sometimes causes the tert-butylation of Met or Trp of the released peptide, especially when severe HF or trifluoromethanesulfonic acid (TFMSA) is used for global deprotection (acidolytic cleavage)[30].
Since the DEE and MTBE are no longer suitable precipitating ether, 2-MeTHF and CPME are used as alternatives. 2-MeTHF introduced by Pawlas and co-workers, can be used alone or in mixtures in n-heptane, and shows good recovery of the peptide after final global deprotection[34, 35]. Unfortunately, 2-MeTHF is not best ether since it still has issues like low flash point, not stable under acidic conditions, forms peroxide easily, insufficient recovery from water[36]. CPME is also accepted as a green precipitating ether, and it free from the above-mentioned drawbacks about other ether solvents. CPME is characterized by favourable environmental, health and safety (EHS) properties, including a high flash point (10 C), the high boiling point (1060 C), stable in acidic/basic conditions, and hardly form peroxides[32, 37, 38]. Various peptide has been reported to be precipitated by CPME and they showed excellent results except for Leu-enkephalin which was dissolved in CPME and did not precipitate. Another important thing to mention about CPME is that there was no alkylation generated by the cyclopentyl carbocation as evidenced by LC-MS data[35, 38]. These results are consistent with the study of Watanabe et al., that shows CPME is stable in acidic conditions, also stable at room temperature for 8h in TFA[37]. Additionally, CPME was found to preserve the structural morphologies of CTC resin beads, allowing the TCT resin to be recycled[39]

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