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Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles
© Hojo et al 2011
- Received: 6 June 2011
- Accepted: 25 August 2011
- Published: 25 August 2011
Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids.
- Peptide Synthesis
- Protected Amino Acid
- Acid Nanoparticles
In current industrial processes, the synthesis of organic compounds usually by dissolving material compounds in organic solvents is widely used. More stringent rules regulating the use and disposal of organic solvents are increasingly restricting chemical synthesis alternatives, and there is an urgent need to develop non-hazardous alternatives in order to achieve green sustainable chemistry [1, 2]. Reduction of the use of harmful organic solvents is required to clean the chemical synthesis processes, reduce waste, and ensure safety.
As a result of major advances in molecular biology and medicine, the importance of the peptides in biological processes has been attracting attention, and the need to accelerate the supply of peptide by chemical synthesis is rapidly increasing. However, a great deal of organic solvent is consumed in chemical peptide syntheses because of the multiple condensation steps in organic solvents. With their E-factors , a useful measure of the potential environmental acceptability of chemical processes, that are estimated to be 100 times larger than that of normal synthetic compounds, chemical peptide syntheses are almost incomplete in terms of environmental friendliness. Therefore, development of a method for organic solvent-free peptide synthesis is strongly desired. If water is substituted for organic solvents, the use of organic solvents can be reduced radically. In conventional organic synthesis, it is common to dissolve reactant molecules in solvents to carry out the reaction efficiently. Peptide synthesis can be carried out in water via chemical conversion of protected amino acids to water-soluble forms [4–7]. In this case, however, additional conversion steps are required in total synthetic process, and it is not desirable in terms of preparation costs, resource saving, and energy conservation. Thus, development of simplified techniques other than chemical conversion is urgently needed for environmentally friendly peptide synthesis.
Chemical peptide synthesis is classified into two types: solid-phase synthesis and solution-phase synthesis. Choosing the best amino acid derivatives is one of the most important, and sometimes difficult, aspects of peptide synthesis. Currently, Fmoc-amino acids are routinely used building blocks for solid-phase peptide synthesis [8, 9]. In solution-phase synthesis, tert-butyloxycarbonyl (Boc)-protected amino acids are commonly used. Both Fmoc- and Boc-amino acids are highly soluble in ordinary organic solvents. These molecules are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. We have developed technology using these common water-insoluble protected amino acids processed into water-dispersible nanoparticles to allow synthesis in water, and have reported that the use of water-dispersible nanoparticles is effective in dramatically reducing reaction time in water [10–12]. This review describes a method for solid-phase and solution-phase peptide synthesis in-water using water-dispersible protected amino acid nanoparticles.
Development of solid-phase peptide synthesis in water using Fmoc-protected amino acids
In-water solid-phase coupling reaction using water-dispersible nanoparticles a
Yield [%] b
Protocol for in-water, solid-phase synthesis using nanoparticles
1 min × 5
Water-dispersible Fmoc-amino acid nanoparticle
WSCD, HONB, DIEA
1 min × 5
Aqueous 50% EtOH
1 min × 2
0.1 N NaOH aqueous/90% EtOH
5 min × 3
Aqueous 50% EtOH
1 min × 2
Grinding condition and particle size of water-dispersible Fmoc-Ala-OH nanoparticles a
Bead Size [mm]
Particle Size [nm]
0.4% Triton X-100 solution
292 ± 119
0.2% Triton X-100 solution
217 ± 89
0.2% Triton X-100 solution
362 ± 157
0.1% Triton X-100 solution
484 ± 225
0.01% Triton X-100 solution
2581 ± 954
Thus, to summarize our results, we specifically developed efficient in-water reaction using water sparingly soluble protected amino acids in which dispersed in water, and verified their utilities by successful solid-phase synthesis of Leu-enkephalinamide in water. General solid-phase syntheses are based on reactions of solids and liquids, while this method is based on reactions between a solid reactant and an aqueous nanocolloidal reactant. These results suggest that our method can be applied to every organic reaction. There are some advantages of using the water-dispersed nanoparticulate reactants over the water-soluble reactants. In the case of in-water synthesis using water-soluble protected amino acids, after the in-water reaction the excess reactants remain dissolved in water, causing possibilities of water contamination. In contrast, after in-water reaction using nanoparticles, the nanoparticles could be easily separated from the resin by filtering through a microfilter (Figure 1). The particles in the filtrate could also be easily separated from the water by centrifugation or by using salting-out technique. Thus, the nanoparticle-based technology described here leads a major step forward in the development of environmentally friendly solid-phase synthesis method.
Development of solution-phase peptide synthesis in water using water-dispersible submicron particles of Boc-protected amino acids
In-water coupling reaction using water-dispersible nanoparticles a
Yield [%] b
Yield and purity of in-water synthetic peptides a
Yield [%] b
Purity [%] b
This method is superior to conventional methods in terms of cost effectiveness, resource conservation, and environment friendliness. Because, after a reaction, synthetic protected peptides precipitate and can be isolated easily by filtration. That is, the use of this method will simplify manufacturing processes.
Consumption of organic solvents can be radically reduced by developing alternative methods that utilize water. There are obviously many potential advantages in replacing the traditional organic solvent-based methods with the water in synthetic chemistry. Recently, many new approaches are being developed, including a catalytic reaction process in water. Many of these new approaches, however, are limited to fields of research in laboratories, and there are a few applications to industrial processes, because only water-soluble materials can be used for them. In general, many organic compounds of synthetic intermediates for industrial use are poorly soluble in water, and therefore inadequate for reactions in water. Establishment of a technique that effectively accelerates reactions in water will make it possible to use water in industrial processes in many chemical synthesis plants, including those for compounds poorly soluble in water. This is the first technique reported that focuses on the conversion of insoluble reacting molecules themselves to water-dispersible nanoparticles. Control on the production of chemical substances, including the establishment of emission regulations, has been strengthened more than ever for environmental protection on a global scale. We hope that development of this technology will contribute to the construction of a sustainable industrial structure.
This work was supported in part by a Grant-in-Aid for Scientific Research and by an "Academic Frontier" Project for Private Universities: matching fund subsidy from the Japanese Ministry of Education, Culture, Sports, Science and Technology, 2006-2010.
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