Greening the Solid-Phase Peptide Synthesis Process. 2-MeTHF for

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Greening the Solid-Phase Peptide Synthesis Process. 2MeTHF for the Incorporation of the First Amino Acid and Precipitation of Peptides after Global Deprotection Othman Al Musaimi, Yahya E Jad, Ashish Kumar, Ayman El-Faham, Jonathan M Collins, Alessandra Basso, Beatriz G de la Torre, and Fernando Albericio Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00335 • Publication Date (Web): 21 Nov 2018 Downloaded from http://pubs.acs.org on November 21, 2018

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Organic Process Research & Development

Greening the Solid-Phase Peptide Synthesis Process. 2-MeTHF for the Incorporation of the First Amino Acid and Precipitation of Peptides after Global Deprotection

Othman Al Musaimi,1,2 Yahya E. Jad,1,2 Ashish Kumar,1,2 Ayman El-Faham,3,4 Jonathan M. Collins,5 Alessandra Basso,6 Beatriz G. de la Torre2* Fernando Albericio1,3,7,8* 1Peptide

Science Laboratory, School of Chemistry and Physics, University of

KwaZulu-Natal, Durban 4001, South Africa;

2KwaZulu-Natal

Research

Innovation and Sequencing Platform (KRISP), School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4041, South Africa; 3Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; 4Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Alexandria 21321, Egypt; 5CEM Corporation, 3100 Smith Farm Road, Matthews, North Carolina 28104, United States; 6Purolite, Llantrisant Business Park, Llantrisant, CF72

8LF,

United

Kingdom;

7CIBER-BBN,

Networking

Centre

on

Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, University of Barcelona, 08028 Barcelona, Spain; 8Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain

ACS Paragon Plus Environment

Organic Process Research & Development 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

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Table of Contents (TOC) figure

Current Work

Previous Work

Amide Bond Formation

1st AA Incorporation Current Work

Precipitation


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Abstract In solid-phase peptide synthesis, dichloromethane is the predominant solvent used to incorporate the first amino acid on 2-chlorotrityl chloride resin (via nucleophilic substitution) and

Wang

resin

(via

activation

with

carbodiimide

in

the

presence

of

4-

dimethylaminopyridine). However, legal authorities have restricted the use of this solvent as it is considered hazardous and a potential occupational carcinogen. Therefore, there is a need for an alternative that is easy to handle and poses less risk for the environment and more importantly for human health. Herein, we describe 2-methyltetrahydrofuran as a greener alternative for the incorporation of the first protected amino acids on both 2-chlorotrityl chloride and Wang resins. The amounts of several amino acids loaded on 2-chlorotrityl chloride and Wang resins using dry dichloromethane or 2-methyltetrahydrofuran were comparable. In addition, the use of 2-methyltetrahydrofuran rendered acceptable racemization and dipeptide formation in the case of Wang resin. This solvent can also be used to replace diethyl ether and tert-butyl methyl ether in the peptide precipitation step performed after final global deprotection. On the basis of our findings, 2-methyltetrahydrofuran emerges as a good alternative to the hazardous solvents currently used for amino acid incorporation and peptide precipitation. Furthermore, and taking into account that 2-methyltetrahydrofuran proved effective for the elongation of the peptide in a solid-phase mode, we propose 2-methyltetrahydrofuran as a universal solvent for all solid-phase peptide synthesis steps, namely incorporation of the first amino acid, assembly of the peptide chain, and precipitation of the final peptide.



Keywords:

2-MeTHF;

incorporation;

precipitation;

solid-phase

Page 4 of 25

peptide

synthesis;

racemization; dipeptide formation

Introduction Peptides are key molecules in the drug design sector, finding broad applications, such as in bioactive molecules and drug carriers.1-3 Synthetic peptides are usually prepared by the 9fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) approach,4-5 which starts with the immobilization of the first amino acid through the carboxylic group onto a polymeric support known as a resin.6 The removal of Fmoc, which is protecting the α-amino group, then takes place, facilitating the coupling of additional amino acids until the desired peptide is assembled.6-9 For the preparation of unprotected C-terminal acid peptides, the final step in the synthetic process involves cleaving the peptide from the resin with trifluoroacetic acid (TFA), with concomitant removal of the protecting groups, followed by a precipitation process using a non-polar solvent, which is usually an ether.7,10 Figure 1 shows a schematic representation of the SPPS process.


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Wang

HO

Cl

CTC Current work

Incorporation of first amino acid / 2-MeTHF

AA1 O

Fmoc

Linker

a: Fmoc removal / 2-MeTHF

AA1 O

H

Linker

b: Coupling of second amino acid / 2-MeTHF

Fmoc

AA2

AA1 O

Linker

AA1 O

Linker

Previous work

a and b for n times & a

H

AAn+2

AA2

Cleavage and global deprotection

H

AAn+2

AA2

AA1 OH Current work

Precipitation / 2-MeTHF

Purification and Characterization Figure 1. Schematic representation of the SPPS process

For the preparation of C-terminal acid peptides, the most common resins used are 2chlorotrityl chloride (CTC)11 and p-alkoxybenzyl alcohol (Wang).12 Although the incorporation mechanisms for the first Fmoc-amino acid differ between these resins, CH2Cl2 is the solvent of choice for both. CTC resin was designed for the preparation of protected peptides through the release of the peptide using a low content of TFA (1-3%). However, it can also be used to prepare unprotected peptides when cleavage is carried out with a high TFA content ( 90%), similarly to Wang resin. In this case, the peptide cleaved from these two resins is usually precipitated with diethyl ether (DEE) or tert-butyl methyl ether (MTBE). However, CH2Cl2, DEE, and MTBE are classified as hazardous chemicals, and their use has raised concerns regarding the risks to human health and the environment.13-15 In addition, the



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US Environmental Protection Agency (EPA) has restricted the use of CH2Cl2.16 In this context, the search for safer alternatives is timely.15,17-19 Furthermore, MTBE shows instability in acidic conditions and triggers undesired tert-butylation reactions of the cleaved peptide, thereby influencing the purity of the final product.20 For the precipitation step, cyclopentyl methyl ether (CPME) was successfully introduced by our group as a green alternative to the hazardous DEE and MTBE.21 One of the principles of green chemistry is the minimization of the number of solvents required during a synthetic process from a cost standpoint.22 In addition, limiting resin exposure to different solvents may help to preserve the morphological properties of the polymeric support by minimizing osmotic shock, thereby enhancing the efficiency of the synthesis.23 Due to the poor solubility of Fmoc-amino acids in CPME,24 it cannot be used for the entire SPPS processes, hence the need for a “universal” solvent. Herein, we report on 2-methyltetrahydrofuran (2-MeTHF) as a greener solvent for incorporating the first amino acid onto CTC and Wang resins, as well as for precipitating the peptide released after the cleavage step. 2-MeTHF is derived from biomass-renewable resources. It is a byproduct in the production process of furfuryl alcohol from furfural, which is

derived

from

corncobs

or

bagasse25-27,

or

it

can

be

produced

by

a

dehydration/hydrogenation pathway of levulinic acid, which is derived from the degradation of cellulose.ß26-27 2-MeTHF has a relatively high boiling point (82⁰C) and good thermal stability.28 Most importantly, it shows considerable stability under acidic conditions.26, 29 It is worth mentioning that 2-MeTHF is easily degraded upon exposure to sunlight and air (probably by oxidation and ring opening pathway), which are also considered advantages especially in the case of spillage.26,28 To the best of our knowledge, 2-MeTHF has not been classified under the International Conference

on

Harmonization of

Technical

Requirements


for

Registration

of

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Pharmaceuticals for Human Use (ICH) classes to date. However, Antonucci and et al.30 from Merck Research Laboratories have concluded that 2-MeTHF is not genotoxic or mutagenic. Furthermore, Sanofi’s solvent selection guide, which is based on safety, health, environmental, quality, and industrial constraints, has classified 2-MeTHF under the “recommended” class.13 Results and discussion a) Incorporation of the first amino acid onto CTC / Wang resins The evaluation of a solvent for use in solid-phase peptide chemistry should consider the following parameters: resin swelling; dielectric constant; ability to dissolve protected amino acids and coupling reagents; toxicity; and also price.a From a strictly chemical perspective, the conversion yield and the absence of side-reactions, such as racemization and overactivation resulting in the double incorporation, are crucial properties for new solvent candidates. 2-MeTHF has a dielectric constant (7.0)29,31 close to that of CH2Cl2 (8.9).32 In addition, all Fmoc-amino acids show good solubility in 2-MeTHF.24 These observations make 2-MeTHF a strong candidate to replace CH2Cl2 as solvent for the incorporation of protected amino acids onto CTC and Wang resins—the most common solid supports used for the preparation of Cterminal acid peptides. For the CTC resin (1.0 mmol/g, supplier’s specification), the following 10 amino acids were considered: Fmoc-Arg(Pbf)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Glu(OtBu)OH; Fmoc-Gly-OH; Fmoc-Leu-OH; Fmoc-Phe-OH; Fmoc-Pro-OH; Fmoc-Ser(tBu)-OH; and Fmoc-Tyr(tBu)-OH. Amino acids were selected considering several parameters: (i) no side chain protecting group, such as Gly, Leu, Phe, and Pro; (ii) poor solubility, such as Asn(Trt), Gln(Trt), and Phe; (iii) hindered side-chains, such as Arg(Pbf), Asn(Trt), Gln(Trt).

a

Prices can decrease dramatically depending on worldwide consumption.


In


addition, this selection comprised the most common protecting groups (tBu, Pbf, and Trt). Incorporation was carried out in parallel using 2-MeTHF and CH2Cl2 and using 2 equiv. of the protected amino acid, in the presence of N,N-diisopropyethylamine (DIEA) for 1 h at room temperature. It is important to highlight that conditions were not optimized, in order to better appreciate the differences between the two solvents. The loading values obtained for all the amino acids using 2-MeTHF were in good agreement with those achieved with CH2Cl2 (Figure 2). Furthermore, some amino acids showed higher values when the former was used. Of note, 2-MeTHF (water content

Greening the Solid-Phase Peptide Synthesis Process. 2-MeTHF for

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