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Microwave Assisted Green Solid-Phase Peptide Synthesis using #-Valerolactone (GVL) as Solvent Ashish Kumar, Yahya E Jad, Jonathan M Collins, Fernando Albericio, and Beatriz de la Torre ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01531 • Publication Date (Web): 29 Apr 2018 Downloaded from http://pubs.acs.org on April 30, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Sustainable Chemistry & Engineering

Microwave-Assisted Green Solid-Phase Peptide Synthesis using γValerolactone (GVL) as Solvent

Ashish Kumar1,2, Yahya E. Jad2, Jonathan M. Collins3, Fernando Albericio,1,4,5*, Beatriz G. de la Torre6* 1.

School of Chemistry and Physics, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South Africa

2.

Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZuluNatal, University Road, Westville, Durban 4001, South Africa

3.

CEM Corporation, 3100 Smith Farm Road, Matthews, North Carolina 28104, United States

4.

Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, Barcelona 08028, Spain

5.

CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, Barcelona 08028, Spain

6.

KRISP, College of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The increasing demand for synthetic peptides in various research fields raises a challenge, namely the development of greener methods for their preparation. Here we report on an ecofriendly SPPS methodology. Substitution of the hazardous solvent DMF by the biomassderived organic solvent GVL, together with the application of microwave-assisted automated SPPS, allowed the synthesis of peptides with a wide range of lengths and high purity using polystyrene- and polyethylene glycol-based resins. The yields achieved were comparable to those obtained with standard methodologies. To date, this is the greenest approach reported in terms of the solvent used, waste generated, and energy efficiency.

KEYWORDS: Sustainable Chemistry, Green Solvent, Peptide, Automatic synthesis


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INTRODUCTION Since the preparation of the first peptide (Gly-Gly)1 by Fischer in 1903, peptide chemistry2 has come a long way. Among the milestones achieved is the development of protecting groups3 and activating reagents4, and in particular the revolution brought about by Merrifield in 1963 when he reported Solid-Phase Peptide Synthesis (SPPS) 5. This approach consists of the sequential introduction of Nα-protected amino acids on an insoluble polymeric support, which acts as C-terminal protecting group, followed by the removal of the Nα-protection6. The significance of SPPS was that a peptide whose synthesis in solution previously took months could now be accomplished in only a few hours. First, the so-called Boc (tert-butoxycarbonyl)/benzyl chemistry was developed6. This strategy involves the use of TFA (trifluoroacetic acid) for the removal of the temporary amino protecting group (Boc) and strong acids such as anhydrous HF (hydrogen fluoride) for final deprotection and cleavage6. Although this method had a long record of successful use, its performance became an issue because Teflon labware was needed for handling anhydrous HF and increasingly demanding safety and environmental regulations were brought into force for the use of HF7. Then, in the 1970s, Carpino reported a new concept of amino protecting group fluorenylmethoxycarbonyl (Fmoc)8, which was labile under basic conditions, thereby facilitating the development of the Fmoc/tBu approach for SPPS, which is the main approach used nowadays9. In the standard Fmoc/tBu methodology, piperidine is used to remove the temporary amino protecting group and TFA for the final deprotection and cleavage. This development marked the start of the Peptide Boom: new resins and linkers, orthogonal protecting groups, coupling reagents, synthetic strategies, automatic synthesizers, and as result the democratization of the peptide world10, thus allowing researchers from diverse fields access to peptides.



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The inherent straightforward nature of the SPPS strategy has fueled the development of new scientific areas based on the use of peptides. This has brought about an increasing demand for these molecules. In this regard, chemists have channeled their efforts into fine-tuning synthetic processes; however, little attention has been devoted to the Green concept11. SPPS is based on the use of a large excess of reagents, which are later removed by simple filtration and exhaustive washing steps between each reaction. Therefore the consumption of solvents is a major bottleneck of SPSS. Indeed, this bottleneck is not exclusive of peptide chemistry, as reflected in a GSK survey carried out in 200712 that stated that solvents account for 8090% of the non-aqueous masses in the manufacturing of Active Pharmaceutical Ingredients (APIs). A more recent study by the same company indicated that organic solvents account for 56% of the material used in the manufacturing of drug substance13. The use of water as a solvent is highly attractive in the context of green chemistry and its application in an SPPS strategy has been addressed by several groups14-16. However, this approach has encountered various problems. One issue is that polystyrene (PS)-based resins do not swell in an aqueous medium, while another is that poor or null aqueous solubility of the standard Fmoc amino acid derivatives calls for the development of new protecting groups. With this in mind, we have been working for many years toward the substitution of DMF, the main solvent used in SPPS. DMF is a highly reprotoxic solvent, being classified as a Substance of Very High Concern (SVHC), but is considered difficult to replace.17 Our initial efforts focused on the use of less hazardous solvents such as acetonitrile (ACN) and tetrahydrofuran

(THF)18, and

later

on

solvents

considered

green,

including

2-

methyltetrahydrofuran (2-MeTHF)19, cyclopentylmethylether (CPME)19, γ-valerolactone (GVL)20, and N-formylmorpholine (NFM)20 (Figure 1). Unfortunately, CPME showed poor solubility properties of the protected amino acids and coupling reagents and was therefore not compatible with SPPS.


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Figure. 1. Chemical structures of solvents. Nevertheless, the other three solvents performed well, solubilizing almost all commercial Fmoc-amino acids, diisopropylcarbodiimide (DIC) and ethyl cyano(hydroxyimino)acetate (OxymaPure)19. Our first study demonstrated that 2-MeTHF used only for the coupling step performed better than DMF for the synthesis of short peptides containing hindered amino acids in both PS and the total polyethyleneglycol (PEG), ChemMatrix resin, with a similar performance in terms of racemization19. However, a full green strategy using 2-MeTHF for the removal of Fmoc and for coupling steps, together with ethyl acetate (EtOAc) for the washing, only performed well when ChemMatrix resin was used and the Fmoc removal step was carried out at 40°C21. This study exemplified that, in many cases, the bottleneck of SPPS lies in Fmoc removal rather than the coupling step. Given the difficulty to remove Fmoc using 2-MeTHF, a systematic study of this step using green solvents in both solution and solid-phase modes was carried out. GVL and NFM were found to be the most adequate solvents for the removal step in both PS and ChemMatrix resins22. Finally, the performance of GLV or NFM as the solvent in all synthetic and washing steps during the preparation of short peptides containing hindered amino acids was good when ChemMatrix resin was used. However, while the results were not totally acceptable for PS resin, they were better than when 2-MeTHF was used20. Parallel to our work, North and co-workers obtained similar results using propylene carbonate (PC) as a green solvent23. Thus, PC performs well in solution for the synthesis of



di- and tri-peptides and for the synthesis of small peptides in solid-phase using ChemMatrix resin. However, PC has poor swelling properties when used with PS resins. Microwave irradiation (MW) is now widely used for the synthesis of small organic molecules24 and also peptides25. In this regard, here we turned our attention to the use of MW to develop a completely green methodology for SPPS26. The benefits of MW include faster reaction rates (time and energy saving) and higher purity of the crude product. By obtaining a cleaner product, the corresponding purification steps are subsequently shortened (time- and solvent-saving). Furthermore, the amount of solvent used during the synthesis is considerably less than in conventional (manual or automatic) synthesis. These features make MW a valuable “green” tool in terms of saving energy, time and solvents27. In this context, a full green solid-phase peptide synthesis (GSPPS) strategy compatible with both ChemMatrix and PS resins is required for both small peptides and medium-large peptides. Here we report SPPS using only GVL as green solvent, combined with the use of a MW-assisted peptide synthesizer (Liberty Blue, CEM). We based our study on the HighEfficiency Solid-Phase Peptide Synthesis (HE-SPPS) MW-assisted protocol developed by one of our labs28. Briefly, the protocol involves 2 min of coupling (Fmoc-AA-OH, DICOxyma Pure, 5 equivalents) and 1 min of deprotection (20% piperidine), both at 90oC, and no washes between coupling and deprotection steps. This optimized protocol translated into a total consumption of 14 mL of solvent per cycle with a duration of 4 min. In comparison with manual synthesis, this protocol allows a considerable saving in terms of both solvent and time. For instance, in a standard manual synthesis recently published in the context of green chemistry, 42 mL of solvent was needed per cycle and the total duration was 170 min.23


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MATERIALS AND METHODS All reagents and solvents were obtained from commercial suppliers and used without further purification. Resins and amino acids were purchased from Iris. OxymaPure and COMU were a

gift

from

Luxembourg

Technologies.

γ-Valerolactone

(GVL)

and

N,N-

Diisopropylethylamine (DIEA) were purchased to Sigma-Aldrich. Organic solvents (DMF, CH2Cl2) and HPLC quality acetonitrile (CH3CN) were supplied by Merck. Milli-Q water was used for RP-HPLC analyses. Analytical HPLC was performed on an Agilent 1100 system using a PhenomexLunaC18 (3 µm,4.6 × 50 mm) column, with flow rate of 1.0 mL/min and UV detection at 220 nm. Buffer A: 0.1% trifluoroacetic acid in H2O; buffer B: 0.1% trifluoroacetic acid in CH3CN. LC-MS was performed on a Shimadzu 2020 UFLC-MS using a YMC-Triart C18 (5 µm, 4.6 × 150 mm) column. Buffer A: 0.1% formic acid in H2O; buffer B: 0.1% formic acid in CH3CN. Side reaction of amino-terminal group with γ-valerolactone (GVL). H-Gly-Phe-Leu-NH2 (H-GFL-NH2) and H-Leu-Phe-Gly-NH2 (H-LFG-NH2) syntheses were carried out manually in 0.1-mmol scale in a plastic syringe fitted with a polyethylene porous disk and using FmocRink Amide AM-PS resin (0.74 mmol/g). The coupling was performed using 3 equivalents of the corresponding Fmoc-amino acid, diisopropyl carbodiimide( DIC) and OxymaPure for 1 h. Fmoc was removed using a solution of 20% piperidine in DMF. After completion of the respective tripeptides, the resins were divided into three parts and subjected to the following conditions: i) treatment with GVL for 12 h at room temperature; ii) treatment with GVL for 10 min at 90oC (MW); and iii) treatment with 20% piperidine in GVL for 12 h at room temperature. The peptides were then cleaved from the resin using a mixture of TFA/TIS/H2O (95:2.5:2.5) for 1 h and precipitated using cold diethyl ether. Purity was determined by analytical HPLC.



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MW-assisted SPPS. All syntheses were carried out under MW conditions using a CEM Liberty Blue system on a 0.1 mmol scale and a 5-fold excess of reagents. Solutions of 0.2 M Fmoc-amino acid, 0.5 M DIC, 1.0 M Oxyma and 20% of piperidine were prepared in GVL. Fmoc-Gly-Wang PS resin (0.71 mmol/g) was used for the synthesis of

65-74

ACP peptide and

H-Rink Amide ChemMatrix resin (0.45 mmol/g) for the syntheses of JR peptide, ABC 20mer and Thymosin. The last two were also were synthesized on Fmoc-Rink Amide AM-PS resin (0.74 mmol/g). 65-74ACP peptide was synthesized using the standard cycle provided by the manufacturer, with a 125 s coupling and 65 s deprotection, both at 90oC, as well as the following cycles: i) extended deprotection (95 s); ii) extended coupling (165 s); iii) extended deprotection (95 s) and coupling (165 s). The rest of the peptides were synthesized using only the last cycle. The peptides were cleaved respectively from the resin by treatment with TFA-TIS-H2O (95:2.5:2.5) for 1 h at room temperature. They were then precipitated by addition of chilled diethyl ether, taken up in water or 10% acetic acid. Finally, the crude peptides were analyzed by HPLC and LS-MS (See SI for detailed gradient for each peptide).

RESULTS AND DISCUSSION The choice of solvent was the first challenge for the development of a MW-assisted automated methodology. Our previous experience indicated that 2-MeTHF presented problems during the deprotection step that could be solved by the application of MW22. Nevertheless, this solvent was discarded for this study because of its low boiling point (80oC) (the physical properties of the solvents are summarized in Table 1). In contrast, the use of GVL and NFM in the traditional SPPS (manual methodology) gave promising results for both PS- and PEG-based resins20. Taking into account that the results were slightly poorer for NFM and that this reagent has a melting point (20-23oC) near to room temperature, we


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discarded it on the basis of the potential risk of solidification inside the instrument, which would cause the blocking pipes and shutting down of valves. We then chose to focus on GVL because its physical-chemical properties are highly compatible with the use of MW. Its melting and boiling point are adequate (-31oC and 207-208oC respectively) and its flash point (96 oC)29 is higher than that of DMF (58oC). Additionally, GVL has the advantage that it is a biomass-derived organic solvent29. Table 1. Solvent properties.

Density (g/ml)

Melting point (oC)

Boiling point (oC)

Flash Point (oC)

Viscosity (cP)

DMF

0.95

−31

153

58

0.92

2-MeTHF

0.86

−136

78-80

−10

0.60

GVL

1.05

−31

207-208

96

1.86

NFM

1.15

20-23

236-237

125

7.83

First, we checked the ability of GVL to reach a fixed elevated temperature in comparison to DMF (the standard solvent used for SPPS and for MW-SPPS) by applying different amounts of MW power. In this regard, no significant difference was found between the two solvents, thereby indicating that GVL could be a promising substitute for DMF. From a strictly chemical point of view, we had to address an important issue regarding GLV, namely the opening of its cycle by amines30 (Figure 2. upper panel). For this purpose, we synthesized a model tripeptide Gly-Phe-Leu anchored to a Rink-amide PS-resin which was subjected to three stress conditions: (i) GVL for 12 h at 25°C; (ii) GVL for 10 min at 90°C (MW); and (iii) 20% piperidine-GVL for 12 h at 25°C. In the three cases, two extra peaks appeared in the HPLC analysis (Figure 2). The conversion of the tripeptide under conditions (i) was around 15% (Figure 2a); however, the process accelerated and increased when the temperature was raised (25% of conversion), as can be appreciated in HPLC trace c (Figure



2). In the case of piperidine treatment (iii), the amount of side products formed (70% conversion) was a serious concern (Figure 2D). The mass analysis of the two peaks revealed that the one at 5.7 min (peak (2) in Figure 2) corresponded to the acyl tripeptide, as we expected. The second one at 8.6 min (peak (3) in Figure 2), with a mass 196 units higher than the tripeptide, was identified as the trifluoroacetyl derivative of the previous side product (Figure S2). These observations imply that a subsequent reaction of the side product took place during the cleavage step.

Figure 2.

GVL ring opening by amines (upper panel). GVL secondary reaction under

different conditions: HPLC trace of: a. model tripeptide H-Gly-Phe-Leu-NH2. b. after 12 h in GVL at room temperature. c. after 10 min under MW at 90oC. d. after 12 h in 20% piperidine in GVL (middle panel). Chemical structure of the tripeptide and by-products found (lower panel).

It is important to bear in mind that the model tripeptide Gly-Phe-Leu-resin has a residue of Gly at the N-terminal that is not hindered and therefore prone to side reactions. When the same experiment was carried out with the retro peptide, Leu-PheGly-resin, no side reactions occurred (Figure 3A). Finally, we also evaluated the


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extension of the side reaction when the tripeptide H-Gly-Phe-Leu-NH2 was prepared with GVL in the MW-assisted peptide synthesizer (Liberty Blue, CEM) using the standard cycle, i.e. coupling by DIC-OxymaPure, at 90oC for 2 min and deprotection by 20% piperidine at 90oC for 1 min and using GVL in all steps, including washing. The analysis after the cleavage showed that, in the unfavourable case of Gly as the Nterminal amino acid, the purity of the tripeptide was 95% (Figure 3B).

Figure 3. HPLC trace of: A. model tripeptide H-Leu-Phe-Gly-NH2 after 12 h in 20% piperidine in GVL. B. model tripeptide H-Gly-Phe-Leu-NH2 prepared with GVL in the MWassisted peptide synthesizer.

We then proceeded our study taking as model peptide

65-74

ACP (H-VQAAIDYING-OH), a

difficult peptide sequence used for evaluating SPPS protocols. Four protocols based on HESPPS using GVL in all steps were tested: (a) standard (125 s coupling, 65 s deprotection); (b) extended deprotection (95 s); (c) extended coupling (165 s); and (d) extended deprotection (95 s) and coupling (165 s). A Gln deletion was observed as the main impurity for all syntheses using Gly-Wang-PS resin (Figure S3). In comparison, the two extended protocols (b, c) showed improved quality of the target compound, while the protocol with the double extension (d) outperformed the rest. The synthesis under the same conditions (d) using DMF



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showed a comparable profile (Figure 4, Panel A). It is important to highlight that no peaks corresponding to reaction with GVL were detected in any of the protocols. Second, we addressed the synthesis of the difficult Jung-Redemann (JR) decapeptide31 (HWFTTLISTIM-NH2). This sequence has proven much more demanding than the

65-74

ACP

peptide and is currently used as model for developing synthetic strategies32-34. Two syntheses were carried out on Rink-amide ChemMatrix resin (Figure S4) following the protocol d described above, one in DMF and the other in GVL. The HPLC analysis of the crude peptides obtained in each case showed the same profile but with a higher purity for GVL (68%) compared to DMF (57%) (Figure 4, Panel B). Again, no peaks, corresponding to side reaction with GVL were found.

Figure 4. HPLC profile of the crude peptides. A.

65-74

ACP synthesis in DMF (upper) and

GVL (lower). B. JR decapeptide synthesis in DMF (upper) and GVL (lower).

To further demonstrate the general application of this methodology, and taking into account that the examples of GSPPS described in the literature involved relatively small peptides (< 10 mer), the synthesis of two medium-large size peptides was undertaken in both PS- and PEG-based resins. Thus, syntheses of ABC Peptide (H-VYWTSPFMKLIHEQCNRADGNH2, 20 mer)35 and Thymosin (H-SDAAVDTSSEITTKDLKEKKEVVEEAEN-NH2, 28


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mer)36 were performed using the extended protocol. Although these two peptides are considered complex sequences, all the amino acids were added by single coupling and without special MW conditions to prevent secondary reactions such as aspartimide formation, δ-lactamization of Arg, and epimerization of Cys and His37. Similarly, a standard cleavage protocol in TFA/TIS/H2O (95:2.5:2.5) without extra scavengers was used. Following this synthesis protocol, the HPLC traces of the crude peptides obtained in each synthesis (Figure. 5) were of good quality. The analysis of the peaks in ABC 20 peptide revealed that the main impurity corresponded to the oxidized peptide (peptide mass +16), probably due to Met oxidation during cleavage. In the case of the synthesis on PS, one extra peak (* in Figure. 5) appeared in the HPLC profile, showing a mass equal to a His deletion (Figure S5). We believe that this is an occasional failure, not related to the methodology. Regarding the synthesis of Thymosin, the purity of the crude peptides obtained were clearly superior in the case of ChemMatrix resin, as expected36 (Figure S6).38.

Figure 5. HPLC profile of crude peptides: ABC 20 synthesis on polystyrene (upper) and ChemMatrix (lower) resin. Thymosin synthesis on polystyrene (upper) and ChemMatrix (lower) resin. In conclusion, here we developed a highly efficient green methodology for SPPS. The approach involves the total substitution of the hazardous DMF by GVL, an aprotic solvent



produced from carbohydrate-based biomass with low toxicity and good biodegradability. Our results demonstrate the full compatibility of the solvent with the automation of the process by means of a MW-assisted SPPS protocol. The MW conditions are substantially greener than other methodologies in terms of energy efficiency (approximately 5 min per cycle), and waste of solvents (15 ml per cycle). In addition, the new solvent in combination with MW protocols is applicable to both PS- and PEG-based solid supports. These results reveal the promise of GVL for SPPS ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: HPLS and MS of the synthesized peptides AUTHOR INFORMATION Corresponding authors *E-mail: [email protected]; [email protected] ORCID Beatriz G de la Torre: 0000-0001-8521-9172 Fernando Albericio: 0000-0002-8946-0462 Notes The authors declare no competing financial interest ACKNOWLEDGMENTS The work was funded in part by the following: National Research Foundation (NRF) (CSUR Grant No: 105892 and Blue Sky’s Research Programme # 110960) and the University of


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KwaZulu-Natal (South Africa); and the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) (CTQ2015-67870-P) and the Generalitat de Catalunya (2017 SGR 1439) (Spain). REFERENCES 1. Fischer, E.; Fourneau, E., Ueber einige Derivate des Glykocolls. Ber. Dtsch. Chem. Ges. 1901, 34 (2), 2868-2877, DOI 10.1002/cber.190103402249 2. Goodman, M.; Cai, W.; Smith, N. D., The bold legacy of Emil Fischer. J. Pept. Sci. 2003, 9 (9), 594-603 DOI 10.1002/psc.476. 3. Isidro-Llobet, A.; Alvarez, M.; Albericio, F., Amino Acid-Protecting Groups. Chem. Rev. 2009, 109 (6), 2455-2504, DOI 10.1021/cr800323s. 4. El-Faham, A.; Albericio, F., Peptide Coupling Reagents, More than a Letter Soup. Chem. Rev. 2011, 111 (11), 6557-6602, DOI 10.1021/cr100048w. 5. Merrifield, R. B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85 (14), 2149-2154, DOI 10.1021/ja00897a025. 6. Lloyd-Williams, P.; Albericio, F.; Giralt, E., Chemical Approaches to the Synthesis of Peptides and Proteins. CRC: 1997; p 297 pp. 7. Muttenthaler, M.; Albericio, F.; Dawson, P. E., Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis. Nat. Protoc. 2015, 10 (7), 1067-1083, DOI 10.1038/nprot.2015.061. 8. Carpino, L. A.; Han, G. Y., 9-Fluorenylmethoxycarbonyl function, a new base-sensitive aminoprotecting group. J. Amer. Chem. Soc. 1970, 92 (19), 5748-5749, DOI 10.1021/ja00722a043. 9. Behrendt, R.; White, P.; Offer, J., Advances in Fmoc solid-phase peptide synthesis. J. Pept. Sci. 2016, 22 (1), 4-27, DOI 10.1002/psc.2836. 10. Jad, Y. E.; El-Faham, A.; de la Torre, B. G.; Albericio, F., CHAPTER 18 Solid-Phase Peptide Synthesis, the State of the Art: Challenges and Opportunities. In Peptide-based Drug Discovery: Challenges and New Therapeutics, The Royal Society of Chemistry: 2017; pp 518-550, DOI 10.1039/9781788011532-00518. 11. Anastas, P.; Eghbali, N., Green Chemistry: Principles and Practice. Chem. Soc. Rev. 2010, 39 (1), 301312, DOI 10.1039/B918763B. 12. Constable, D. J. C.; Jimenez-Gonzalez, C.; Henderson, R. K., Perspective on Solvent Use in the Pharmaceutical Industry. Org. Process Res. Dev. 2007, 11 (1), 133-137, DOI 10.1021/op060170h. 13. Henderson, R. K.; Jimenez-Gonzalez, C.; Constable, D. J. C.; Alston, S. R.; Inglis, G. G. A.; Fisher, G.; Sherwood, J.; Binks, S. P.; Curzons, A. D., Expanding GSK's solvent selection guide - embedding sustainability into solvent selection starting at medicinal chemistry. Green Chem. 2011, 13 (4), 854-862, DOI 10.1039/C0GC00918K. 14. Galanis, A. S.; Albericio, F.; Grotli, M., Solid-phase peptide synthesis in water using microwaveassisted heating. Org. Lett. 2009, 11 (20), 4488-4491, DOI 10.1021/ol901893p. 15. Hojo, K.; Ichikawa, H.; Fukumori, Y.; Kawasaki, K., Development of a Method for the Solid-Phase Peptide Synthesis in Water. Int. J. Pept. Res. Ther. 2008, 14 (4), 373-380, DOI 10.1007/s10989-008-9145-0. 16. Hojo, K.; Shinozaki, N.; Hidaka, K.; Tsuda, Y.; Fukumori, Y.; Ichikawa, H.; Wade, J. D., Aqueous microwave-assisted solid-phase peptide synthesis using Fmoc strategy. III: Racemization studies and waterbased synthesis of histidine-containing peptides. Amino Acids 2014, 46 (10), 2347-2354, DOI 10.1007/s00726014-1779-y. 17. Dunn, P. J., The importance of green chemistry in process research and development. Chem. Soc. Rev. 2012, 41 (4), 1452–1461, DOI 10.1039/C1CS15041C 18. Jad, Y. E.; Acosta, G. A.; Khattab, S. N.; de la Torre, B. G.; Govender, T.; Kruger, H. G.; El-Faham, A.; Albericio, F., Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives. Org. Biomol. Chem. 2015, 13 (8), 2393-2398, DOI 10.1039/c4ob02046d. 19. Jad, Y. E.; Acosta, G. A.; Khattab, S. N.; de La Torre, B. G.; Govender, T.; Kruger, H. G.; El-Faham, A.; Albericio, F., 2-Methyltetrahydrofuran and cyclopentyl methyl ether for green solid-phase peptide synthesis. Amino Acids 2016, 48 (2), 419-426, DOI 10.1007/s00726-015-2095-x.



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GRAPHICAL ABSTRACT

A combination of Nature and Technology helping to make greener the synthesis of peptides



GRAPHICAL ABSTRACT

A combination of Nature and Technology helping to make greener the synthesis of peptides


Page 18 of 18

Microwave Assisted Green Solid-Phase Peptide ... - ACS Publications

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