Green Transformation of Solid-Phase Peptide Synthesis - ACS

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The Green Transformation of Solid-Phase Peptide Synthesis Yahya E Jad, Ashish Kumar, Ayman El-Faham, Beatriz G de la Torre, and Fernando Albericio ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06520 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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ACS Sustainable Chemistry & Engineering

The Green Transformation of Solid-Phase Peptide Synthesis Yahya E. Jad,a,b Ashish Kumar,a,b Ayman El-Fahamc,d Beatriz G. de la Torre,a,b* and Fernando Albericioa,c,e,f*

a

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

b

KRISP, College of Health Sciences, University of KwaZulu-Natal, 719 Umbilo Rd, Umbilo, Durban 4041, South Africa

c

Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

d

Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt

ACS Paragon Plus Environment

1

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

e

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Department of Organic Chemistry, University of Barcelona, Martí i Franqués 111, Barcelona 08028, Spain

f

CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and

Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, Barcelona 08028, Spain

Corresponding Author * Beatriz G. de la Torre, e-mail: [email protected] * Fernando Albericio, e-mail: [email protected] or [email protected]

ABSTRACT

Peptides play key roles in medicinal chemistry, being found in therapeutics and diagnostic agents, among others. Furthermore, their synthetic protocols have also been optimized for other fields such as the solid-phase organic synthesis of other


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organic compounds. However, the classical protocols for peptide synthesis fall short of Green Chemistry parameters. The excess of reagents and solvents used in classical protocols is costly. Moreover, the widely used hazardous chemicals pose a threat to the environment and to human health. In this review, we examine peptide synthesis in the context of green chemistry and address its pros and cons. Furthermore, we discuss several attempts to make peptide synthesis greener. These attempts have followed two major pathways, namely reducing the amount of material used or replacing hazardous materials for friendlier ones. Although this work focuses mostly on the solid-phase peptide synthesis approach, a brief description of some reagents, solvents, methods or strategies used in solution is given because these compounds could be easily adapted to the solidphase mode.

KEYWORDS Green chemistry, peptide synthesis, solid-phase synthesis, parallel synthesis, green solvents


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Introduction The development of chemical synthesis throughout the 20th century marked a significant turning point for civilization. Chemical industries play a critical role in a wide range of fields, ranging from agriculture to the production of paint, fuel, and pharmaceutics, among others. However, these revolutionary new products brought with them pollution issues, which threaten life on this planet. Thus, by the beginning of the 1990s, the field of Green Chemistry, or sustainable chemistry, had emerged as an approach to significantly reduce pollution.1 This movement attracted

the

attention

of

academic

scholars,

industrial

companies,

and

governmental and non-governmental organizations worldwide. Later, in 1998, the twelve principles of Green Chemistry2 were introduced, taking into account all aspects of the chemical process, the toxicity of the products, solvents, reagents and catalysts used, safety issues, and the energy required for the whole transformation.1,3 In this context and in their own mission to fight disease and improve quality of life, pharmaceutical industries were undoubtedly one of the sectors most involved


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in adopting Green Chemistry.4,

5

One of the main processes in this sector is the

production of “Active Pharmaceutical Ingredients” (APIs). These products can be divided into two main categories, namely chemicals and biologics, on the basis of the way they are produced. Peptides are an important class of APIs because they show high and specific biological activities.6 From the synthesis of the first peptide carried out by Fischer7 at the beginning of the last century until the 1950s and 1960s, when Nobel Prize laureate Du Vigneaud synthesized the first active peptide, oxytocin,8 peptides were produced by mixing solutions of the different components and reagents and then performing the corresponding workup—a process that greatly resembles that used in standard organic synthetic techniques. In 1963, R. Bruce Merrifield reported a completely new strategy, namely solid-phase peptide synthesis (SPPS). Although widely criticized initially,9 mainly by European chemists,10 his approach brought about a revolution in the field and it has since fueled the development of peptides for the pharmaceutical market.6 In acknowledgement of his work, Merrifield received the 1984 Nobel Prize in Chemistry. Merrifield’s idea was very simple, interpreting the synthesis


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of peptides with more than two amino acids by successive steps of peptide bond formation (coupling) and the removal of protecting groups. Merrifield envisaged a permanent protecting group for the C-terminal that is bound to a polymer, which was initially polystyrene, and then the successive peptide bonds are accomplished by removal of the temporary amino protecting group and coupling of the incoming amino

acid,

whose

amino

(temporary)

and

side-chains

(permanent)

are

conveniently protected. Once the elongation of the total sequence has been completed, the totally unprotected peptide is released from the polymer through a chemical treatment (Figure 1).11


6

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OH

AA

PG

R

X

1- Coupling reagent 2- Washing from the resin

R

AA

PG

1- Removal of protecting group 2- Washing from the resin H

H

P

E

P

R

AA

T

I

D

R

E

PG

PG

Cleavage and global deprotection H

P

E

P

T

I

D

E

X

Peptide ready for characterization and purification

Figure 1. Solid-phase peptide synthesis (SPPS)

Depending on the protecting groups used (Figure 2), the SPPS methodology is divided into two main strategies: (i) tert-butoxylcarbonyl (Boc)/benzyl (Bzl); and (ii) fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu). The former uses trifluoroactic acid (TFA) to remove the temporary Boc protecting group and strong acids such as anhydrous HF or trifluoromethanesulfonic acid (TFMSA) for global deprotection


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and cleavage.12 In contrast, the second strategy uses bases such as piperidine to remove the Fmoc group and TFA for global deprotection and cleavage.13 The (Fmoc)/tert-butyl strategy, which is more versatile and based on orthogonal protecting groups, has proved amenable to being extended through the introduction of additional orthogonal protecting groups for the synthesis of more complex peptides, such as cyclic or branched peptides.14

O

O

O

O Bzl

Boc

Fmoc

t

Bu

Figure 2. Common protecting groups for SPPS

SPPS techniques are normally used for the preparation of peptides in research and for the vast majority of

peptide-based APIs in multiKg scale,6 with the

exception of those whose drug master file was approved in the 1980s and 1990s and for which solution synthesis was indicated as the method of preparation.


8

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SPPS in the green chemistry court Although the importance of making SPPS greener is widely acknowledged, there are only a few reports in the literature about peptide synthesis under green conditions.15-17 While writing this review, we found that the number of publications addressing “peptide synthesis and green chemistry” has increased since 2011 (Figure 3).

Publication number per year 25 20 15 10 5 0 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

Figure 3. Number of publications per year using Scifinder (this survey was done on 10th December 2018 using “peptide synthesis” and “green chemistry” as keywords)


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Advantages of SPPS from the perspective of green chemistry The following features of the solid-phase mode are associated with the Green concept (Figure 4): (i) a single reactor is used for all reactions, thereby minimizing the cleaning processes and also saving time and energy; (ii) absence of matter transfer and therefore no mechanical losses; (iii) simple work-up processes, avoiding extraction techniques, which are both costly in terms of time and solvent use; (iv) no purification of intermediates, with the resulting savings of solvent, yield, and time; (v) possibility of miniaturization, which, in research programs, allows the synthesis of the required amount (very few µmols) for testing, without the need of “feeding refrigerators” with the “leftovers”; (vi) high yields because of the use of excess reagent to ensure quantitative conversions, thus optimizing the work scale and with the consequent saving of raw materials; (vii) high purity, which translates into straightforward purification (fewer solvents and high yields); (viii) possibility of parallelization, which allows the simultaneous preparation of a set of compounds using the same conditions; (ix) possibility of automatization– fully in research programs or partially in production–with time and energy savings;


10

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and (x) as a corollary of the previous point, optimization of man/womanpower and also time.

Figure 4. SPPS in the green chemistry court

An additional feature of the solid-phase mode is that the solid support can be recycled in the case of 2-chlorotrityl chloride (CTC) resin18.19 This resin allows the release of tBu protected peptides under very mild acid conditions (1–2% of TFA in DCM). Therefore, after treatment with 1–2% TFA to release the protected peptide, a further treatment of the resin with a higher amount of TFA (5–10%) will ensure the full release of all remaining peptide chains. The hydroxytrityl chloride resin can then be activated again with thionyl or acetyl chloride (Scheme 1).19 However, this is not an universal protocol and can be used only with smallmedium sized peptides and only a few times, because the resin can undergo mechanical degradation.19


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Cl Cl

SOCl2

SPPS

Cl HO

Cl Peptide

Peptide

Scheme 1. Recycling of CTC resin

Taking a decapeptide at research scale as a reference, the synthesis will possibly take between 5 and 10 days in solution mode (considering that the average time of a reaction in solution is one day, the maximum yield achievable will be two amino acids per day, which involves coupling and deprotection steps) and between 90 min and 5 h in automatic mode depending on the automatic synthesizer. The former will involve a full-time employee for the whole period, while the latter will call for only a few minutes (time devoted to programming the instrument), the weighing of reagents being common to both modes.


12

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Drawbacks of SPPS from the perspective of green chemistry SPPS has a low score on the following Green Chemistry metrics: Atom Economy; Environmental (E)-factor; and Process Mass Intensity (PMI). Unlike the traditional calculation of percentage yield, which deals only with the yield obtained against the expected one, Green Chemistry uses

20

metrics to reflect the amount

of materials involved in the whole chemical process. Atom Economy, defined as how much of the mass present in the reactants remains in the final product,21 is highly negative in the current context of SPPS. Thus, for the introduction of a residue of Arg using Fmoc-Arg(Pbf)-OH, Atom Economy is 0.24, and for Gly, which does not require a side-chain protecting group, it drops to 0.19 using Fmoc-Gly-OH.

This issue can only be properly

addressed through a revolutionary change in the current paradigm of SPPS—a change that implies minimizing the use of protecting groups. The E-factor, defined as the ratio of total mass of waste to the mass of the isolated product, and PMI, defined as the ratio of the total mass of materials to


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the mass of the isolated product, are strongly penalized by the excess of reagents and, more importantly, by the large amounts of solvents used. In this regard, at the research scale (low), the excess of reagents can be compensated by the pros of the method. In contrast, during the large-scale production of peptides, careful optimization of each coupling allows the use of 1.25–1.5 equiv. of reagent, which can be considered acceptable. However, the most important weakness of SPPS is related to the environmental impact of the excess and the characteristics of solvents used for coupling, deprotection, and washings before and after each step. The consumption of solvents can become a severe issue during the industrial production of peptides (Figure 4). The solvents most widely used solvents in peptide synthesis, namely DMF, NMP, and DCM, are classified as hazardous substances according to several guidelines, and calls have been made for their substitution.22 Furthermore, some of the reagents are also considered hazardous, as exemplified by the classical benzotriazole coupling reagents, which have been considered explosive since 11 September 2001. The deprotection solution frequently used for the removal of the


14

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Boc group in the Boc/Bzl strategy is also considered dangerous. TFA can cause severe burns and related issues upon waste disposal.23 Although TFA can be replaced by solutions of HCl in organic solvents, which could be greener alternatives for TFA in DCM solution,24 this alternative would require further optimization. Regarding the peptide cleavage and global deprotection, TFMSA also causes severe burns while HF is toxic, causes severe burns, and requires special vessels.23 Therefore, and in the context of the Green Chemistry, the Fmoc/tBu strategy could be considered greener than the Boc/Bzl approach as it uses smaller amounts of hazardous reagents (Figure 4).

Greening the SPPS Non-conventional SPPS strategies Parallel synthesis SPOT synthesis and tea bag synthesis, both SPPS strategies especially designed for parallelization, are of interest from the Green Chemistry viewpoint. The former is carried out on a membrane, which allows the preparation of very small amounts of peptides, with a low consumption of both reagents and washing


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solvents. In contrast, the tea bag strategy brings together two synthetic concepts: parallel

and

simultaneous.

Thus,

deprotection

and

washing

solutions

are

simultaneously carried out for all syntheses, and the coupling steps are grouped on the basis of the incoming protected amino acids.

Thus, all syntheses

incorporating the same amino acid are also performed simultaneously. This approach results in a reduction in the amount of solvent used.

SPOT (Franks’s strategy) First introduced by Frank and Döring in 1988,25 SPOT synthesis enables the parallel synthesis of peptides on a membrane as simple as a cellulose disk.26 It is a straightforward synthetic process in which the reagents and solvents can be easily added by “spotting” them via an ordinary micropipette onto the cellulose disk (Figure 5). This approach has also been adapted for automatic synthesis purposes.


16

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P E P T I D E Linker Cellulose disk

Figure 5. SPOT technique

SPOT can be considered a green process because it is rapid, efficient in terms of yields and purity, and highly economical with respect to cost and use of reagents and solvent alike. The cost of peptides produced by SPOT synthesis is approximately 1% of that of those prepared on conventional resin.27 Regarding the consumption of solvents, manual spotting is feasible with a volume of 0.1 µL. However, during automated spotting, the volume can decrease to 10 nL, which can be delivered with precision to any position of the membrane. It has been demonstrated that the manual synthesis of 96 15-mer peptides can be performed


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with 2.5-3 L of DMF, which can be translated to 1.74 to 2.00 mL of DMF for a cycle of 96 peptides.28 The smallest spot yielded peptide in nmol quantity, which was sufficient for isolation, characterization, and screening, all of which can often be done on the same membrane. Finally, the automatization of this technology has also been reported.29

Tea bag (Houghten’s strategy) The tea bag strategy was first introduced by Richard A. Houghten in 1985 as a method for the simultaneous synthesis of peptides.30 In this technique, synthesis is carried out in polypropylene mesh bags, filled with resin beads, which are sealed and labeled for identification. The mesh size of the tea bags must be smaller than the resin beads in order to retain them but must allow soluble regents and solvents to enter and leave readily, thus facilitating reactions and washings. During the synthetic process, bags are placed in the same reactors (a bottle) for common chemical steps, e.g. deprotection of α-amino function, washings, and coupling with the same amino acid. In the coupling step, the bags are arranged systematically on the basis of the amino acids to be coupled and


18

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treated simultaneously in separate reaction vessels with appropriate amino acids in the presence of coupling reagents (Figure 6). This technique allows the preparation of large numbers of peptides simultaneously, thus allowing a great saving of solvents since the same solvent is used in all steps to synthesize all the peptides.

Washing

Teabag Fmoc removal Resin

Washing

AA1

AA2

AA3

AA4

Coupling

Figure 6. Tea bag approach for SPPS

Liquid-phase peptide synthesis (LPPS) Although LPPS should not strictly be considered part of the SPPS concept, both approaches share common features and LPPS is also based on a polymer, which also precipitates at the end of the process.


Following work initiated by

19


Page 20 of 63

Merrifield with the SPPS approach, LPPS was first reported in the early 1970s by Bayer and Mutter. In LPPS, insoluble resins are replaced by a soluble polymeric support.31 This support is soluble in halogenated and ether solvents, and less soluble in polar solvents such as MeOH or EtOH. Therefore, the coupling/deprotection steps are carried out in solvents such as DCM, CHCl3 or THF, followed by either precipitation by MeOH or MeCN,32,33 or aqueous workup (Figure 7).34 LPPS combines the advantages of SPPS with those of classical solution chemistry. For instance, catalytic hydrogenation can be used for LPPS but it is not compatible with SPPS.35 Moreover, the use of a fluorous support allows monitoring of the coupling/deprotection steps by

19F

NMR.34 Recently, Takahashi

and co-workers developed a branched-chain anchor molecule that significantly enhances the solubility of long peptides and operational efficiency. This enhancement is reflected by the amount of solvent consumed. In this regard, only 174 mL of solvent was used per 1 g of peptide obtained vs. 658 mL in case of the precipitation approach and 2183 mL in case of SPPS.36 Furthermore,


20

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the green solvent cyclopentylmethyl ether (CPME) was reported as an alternative for the hazardous solvent CHCl3 in the most recent AJIPHASE® protocol.36 LPPS

SPPS

Homogeneous reaction between the reagents and the soluble resin

Heterogeneous reaction between the reagents and insoluble resin

The excess of reagent easily removed by aqueous workup

The excess of reagent easily removed by filteration and washing

Vacuum

Figure 7. LPPS vs. SPPS

Avoiding hazardous solvents Solvent role As the major components of any chemical reaction,37 solvents are perhaps the most active area to be tackled from a green perspective. A survey by GSK in 2007 stated that solvents account for 80–90% of the non-aqueous masses in API manufacturing.38 A more recent survey by the same company in 2011 concluded that organic solvents account for 56% of the material used to manufacture a pharmaceutical substance.39 The relevance of solvents is also addressed in the


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principles of “Green Chemistry”, a field that deals with the design of chemical products and processes that do not involve or that minimize the use of hazardous substances3. Green Chemistry has received much attention from the scientific community in recent years. Several solvent selection guides provided by various pharmaceutical companies and collaborative groups have produced rankings of solvents on the Green Chemistry scale.40-42 This classification was carried out by analyzing the EHS (Environmental, Health, and Safety) profile of each substance and then ranking the solvents into four categories, namely recommended, problematic, hazardous, and highly hazardous.22 Problematic solvents can be used in research and/or kilolabs but they require specific control of the waste. The use of hazardous solvents is tightly regulated during the scale-up, while the use of highly hazardous solvents is to be avoided even in the laboratories. Given this scenario, the identification of alternatives to hazardous and highly hazardous solvents is paramount.22 Thus the underlying concept of a green solvent is the substitution43 the hazardous substances by those with a better EHS profile or bio-solvents produced from renewable resources.37


22

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Solvent-free peptide synthesis Given that solvents are the major component of peptide syntheses and that they are hazardous substances, solvent-free synthesis offers the greenest option. In another words, the best solvent is no solvent at all.44,45 For peptide synthesis, Lamaty and co-workers reported a solvent-free synthesis using ball-milling. Urethane-protected α-amino acid N-carboxyanhydride (UNCA) derivatives were coupled to amino acid esters, achieving a good yield (Figure 8). This method was also used to produce high yields of the sweetener aspartame.46 Later, the same research group reported that Boc-AA-NCA and Boc-AA-OSu can be coupled to α-amino acid alkyl ester salts in the presence of sodium bicarbonate and a minimal amount of ethyl acetate (EtOAc) to produce di- to pentapeptides in high yields. This method is also suitable for gram-scale reactions.47

R O

PG N O O

Figure 8. Urethane-protected α-amino acid N-carboxyanhydride (UNCA)


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More recently, the same group expanded the scope of this methodology. The optimization of the coupling step concluded that EDC/Oxyma is the best condition. However, the conversion was low, and the standard deviation was very high. These observations indicated that the reaction was not homogenous. Therefore, a liquid additive was added to increase the homogeneity. After screening several solvents, EtOAc showed the best results, achieving 93% conversion and 3% standard deviation.48 To the best of our knowledge, EtOAc is a green solvent, and it has been used in trace amounts for this methodology. A further study has been reported comparing this approach with classical ones, namely Boc-solution and Fmoc-SPPS. The comparison can be summarized as follows:49 Yield: Ball-milling > SPPS > solution Purity: SPPS > Ball-milling > solution Environmentally: Ball-milling > solution >>> SPPS

Landeros and Juaristi reported the use of Mg-Al hydrotalcite for solvent-free ball-milling peptide synthesis. In addition to inexpensive materials, easy workup,


24

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and good to excellent yield, this approach has the further advantage that Mg-Al hydrotalcite can be recycled by heating at 100°C.50 Furthermore, Jain and co-workers reported microwave-assistant solvent-free peptide synthesis using diisopropylcarbodiimide (DIC)/ N-hydroxy-5-norborneneendo-2,3-dicarboxyimide (HONB) as coupling reagent and DIEA as a base.51 Several bioactive peptides were successfully synthesized in good to excellent yields (53–92%) using this methodology.51

Aqueous peptide synthesis Although the disposal or recycling of “chemically contaminated” water is not a straightforward process, water could be the paradigm of a green solvent. Indeed, water is the greenest solvent.22 However, due to the lack of solubility of Boc or Fmoc amino acid derivatives in water, several strategies to overcome this problem have been developed.


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Water-soluble protecting group The first attempt to replace Boc and Fmoc by other water-soluble protecting groups was first achieved by Tesser and Balvert-Geers in 1975. In this regard, these authors reported methylsulfonylethyloxycarbony (Msc) as a water-soluble Nprotecting group for peptide synthesis in solution chemistry.52 Later, other protecting

groups,

such

as

2-phosphonioethoxycarbonyl

(Peoc)53

and

2-

(triphenylphosphonio)isopropyloxycarbonyl (Ppoc), were also reported for solution chemistry.

For

SPPS

application,

Hojo

and

co-workers

described

2-

[phenyl(methyl)sulfonio]ethoxycarbonyl (Pms),54,55 2-ethanesulfonylethoxycarbonyl (Esc)56 and 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps)56 (Figure 9). These protecting groups can be removed under basic conditions like those used for the Fmoc group. For SPPP, Hojo reported the synthesis in combination with poly(ethyleneglycol)-polystyrene (PEG-PS) resin,57 which is more compatible with aqueous media.58 TritonX-100 was added to increase the swelling of the resin and to inhibit peptide aggreagation.54 During the development of these protecting groups, full PEG resin such as ChemMatrix resin was not commercially available.59 The water-soluble coupling reagent 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide


26

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hydrochloride (EDC.HCl) and HONB were used. The N-terminal protecting group was removed using an aqueous solution of NaHCO3, Na2CO3 or NaOH in 50% aqueous ethanol. However, some of these protecting groups have a number of drawbacks, such as instability in the case of the onium salt-based protecting group (Pms) or inefficient removal at aqueous solution in the case of Esc. Although these strategies showed great scientific merit, the yields and purities of the model peptide Leu-enkephalin synthesized using these protecting groups were much lower than those commonly achieved by the standard Fmoc SPPS protocol, which uses organic solvents.17 X O S O

X O

P

O

O

O Msc

O O Pms

O S O Esc

O O

Ppoc X = Cl or Br

Peoc X = Cl or Br

BF4 S

P

O O

O S Na O O


O S O

O O

Sps

27


Page 28 of 63

Figure 9. Water-soluble N-protecting groups Recently, Knauer and co-workers described a novel strategy to adapt SPPS to water medium.60 They introduced a water-solubility enhancing functional group, SO3- in particular, into the Fmoc group. Other variable functional groups, such PO32-, NMe3+, and CN, were also attempted. These amino acid derivatives can be prepared in a one-step reaction by stirring concentrated sulfuric acid with Fmoc amino acid derivatives for 24 h at room temperature to produce Smoc amino acid derivatives in high yields (Scheme 2).

This idea of sulfonation of

the Fmoc protecing group was first introduced by the Merrifield laboratory, when they proposed 9-(2-sulfo)Fmoc (Sulfmoc) as a strategy for the purification of synthetic peptides.61 O3S

O

H N

O

H2SO4, 24 h, rt OH

O

95% yield

H N

O O3S

Fmoc-Ala-OH

O OH

O Smoc-Ala-OH

Scheme 2. Synthesis of Smoc-AA-OH


28

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The aqueous solubility of other more widely used protecting groups, such as Trt, tBu, Boc, and Cbz, was also enhanced by attaching SO3- to them (Figure 10). A further advantage of the Smoc group is that changes in fluorescence intensity can be measured, thus allowing monitoring of the coupling efficiency. This new strategy was applied to the SPPS of H-Val-Gly-Gly-Val-Gly-OH, in which only water was used in the entire synthetic process. Coupling was performed by Smoc-AA-OHs (3 equiv.), EDC (2.8 equiv.), HOSu (3 equiv.) and NaHCO3 as a base in a minimum amount of water for 1 h at room temperature on a polyethylene (PEGA) resin. Capping with 2-sulfoacetic acid or 4-sulfobenzoic acid was performed after each coupling step and before removal of the Smoc group by 10% ammonia solution. After cleaving the peptide from the resin, it was passed through an ion exchange column to obtain the target peptide, while the side products were retained on the column, taking advantage that the terminated peptides were capped with a 2-sulfoacetyl moiety.


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SO3

SO3 O3S

O O O3S

O

O

O

O3S

Sboc

O

SO3

SulfoCbz SO3 Sulfu-Trt

Smoc O3S

O3S t

S

S O3S

t

BuS

S BuS

BzS

Figure 10. New protecting groups for Sulfo-tool SPPS

Aqueous nanoparticle peptide synthesis Hojo and co-workers also reported another innovative approach based on the conversion of the sparingly soluble Boc or Fmoc amino acids into nanoparticles to overcome the solubility problem. In theory, nanoparticles have a higher surface area, which leads to increased homogenous mixing of these particles with the resin in water, thus allowing a smooth progression of the reaction.15,17 The nanoparticles were prepared using a planetary ball mill in the presence of PEG as a dispersion agent. TFA62 and 0.1 N NaOH 90% aqueous EtOH63 were used as deprotection solution for the Boc and Fmoc strategy, respectively. Again, PEGgrafted resin, Triton X-100 solution for washing, and microwave heating were also


30

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used

in

the

synthesis

of

various

peptides,

including

Leu-enkephalin,

dermorphinamide, and ACP (65-74).15,62-64 This strategy shows that the use of the commercial Boc and Fmoc amino acids has the major advantage of achieving acceptable yields.

Microwave-aqueous peptide synthesis Microwave-assisted organic synthesis (MAOS) was also used to overcome the poor solubility of conventional protected amino acids in water. Heating is one of the common approaches used to increase the solubility and reaction rate. In this regard, the microwave can be considered a greener approach than convenient heating methods because the energy is transferred directly to the reaction mixture rather than to the reaction vessel and then to the reaction mixture.65,66 For peptide synthesis in solution, Jain and co-workers67 reported MAOS in neat water, i.e. no zwitterionic detergent was added. While for SPPS, we reported a microwaveassisted SPPS protocol using water for coupling and deprotection in the presence


31


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of Triton X-100 to improve the solubility of the amino acid derivatives in water, as well as the swelling properties of the resin.68 After evaluating a number of protecting groups and coupling reagents, the best protocol was found to involve Boc amino acids, a PEG-based resin, EDC.HCl/HONB as a coupling method (other additives such as HOBt and OxymaPure did not give satisfactory results in this case).68

Surfactants as solubilizing agents for peptide synthesis TritonX-100 has been used independently by Hojo54 and Galanis68 to enhance resin swelling and aqueous solubility in the previously mentioned protocols. However,

other

parameters,

such

as

water-soluble

protecting

groups,

nanoparticles, and microwave-heating, were also used in these protocols. Later, Lipshutz and co-workers69 reported peptide synthesis in aqueous media at room temperature. TPGS-750-M (Figure 11) was used as a surfactant70 in combination with COMU as coupling reagent and 2,6-lutidine as a base. Although it was not expected that COMU could be used with water because of its high tendency to hydrolyze,71,72 these conditions gave a very high coupling yield when several


32

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amino acid derivatives were used, as well as when alkyl acid/amine derivatives were employed. TPGS-750-M serves to dissolve water-insoluble substance, as well as acting as a nanoreactor in aqueous medium. Furthermore, the aqueous media can be recycled after extracting the peptide by isopropyl acetate. This is then reflected in a significant decrease in solvent consumption. O O O

O O

O

O 16

Figure 11. TPGS-750-M

Later, the same group reported a tandem deprotection/coupling sequence for aqueous peptide synthesis. This novel protocol started by dissolving Cbz amino acid in a 2 wt % solution of TPGS-750-M. Cbz was removed by Pd/H2 for 2 h, followed by the addition of the next amino acid with the previously optimized coupling condition. This protocol was used to prepare a 10-mer peptide [Cbz-DPhe-Pro-Val-Orn(Boc)-Leu-D-Phe-Pro-Val-Orn(Boc)-Leu-OMe].73 In summary, this method has several advantages in the context of Green Chemistry because: (i) it does not involve hazardous solvents; (ii) it minimizes solvent consumption by


33


Page 34 of 63

combining two steps into one; and (ii) it uses green conditions for the deprotection step because Pd/H2 can be considered green on the basis of the recyclability of the catalyst and the low cost.74 However, this approach has not been applied to SPPS to date.

Others Collins and co-workers75 reported an attempt to reduce the amount of organic toxic solvent by using water in washing steps (again it is important to highlight that 90% of the solvent consumed during SPPS occurs during washing steps). This approach showed the promise of saving great amounts of solvent. However, it was not successful due to the poor solubility of Fmoc amino acids and the formation of an adduct during its removal. Therefore, (1,1-dioxobenzo[b]thiophene2-yl)methyloxycarbonyl amino acids (Bsmoc)76 was proposed as substitute of the Fmoc protecting group (Figure 12). Comparison of the structures of Bsmoc with Fmoc indicates that the presence of the SO2 moiety might be responsible for aqueous solubility. Bsmoc was removed by 5% piperazine/0.1 M HOBt in DMF.


34

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All washing steps were performed using water and isopropanol—both green solvents.75

O

S O

O O

Figure 12. Structure of Bsmoc amino acid

Sarma and co-workers77 reported the synthesis of dipeptide in solution using water extracted from banana (WEB). First, dry banana peels were burned to ash, then water was added to the ash and mixed well, and the solution was then filtered to produce WEB. The optimized protocol involved using trace amounts of ethylene glycol (ca. 5%) and EDC.HCl as coupling reagent, but no addition of base.

Non-aqueous green peptide synthesis The previous section discussed the advantages of using aqueous solutions for peptide synthesis. However, this approach has some disadvantages that limit its use in peptide synthesis. In this regard, PS is the most widely used resin in


35


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SPPS, but it shows poor swelling properties in water.59 Furthermore, most protecting groups that are used in standard peptide synthesis protocols (e.g. Fmoc, Boc, Z, Trt…. etc) have highly hydrophobic structures and are therefore not prone to being soluble in water. These characteristics limit the use of these protecting groups in aqueous peptide synthesis in general, and in solid-phase synthesizers in particular, because they can precipitate on the system, which can lead to blockages in the tubing and filters of the reaction vessels.75 Moreover, it limits the efficiency of standard coupling reagents. For instance, the preparation of the model peptide Z-Phg-Pro-NH2 using EDC.HCl/OxymaPure as coupling reagent in water yielded only 25% conversion.78 Some peptide syntheses in aqueous media involve HONB as an additive to the EDC carbodiimide. However, the use of HONB is accompanied by a side reaction, similar to that which occurs when HOSu is used (Scheme 3).79


36

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O

O

N O O O

H2N

HN Prot

O

O

H N Prot

+1

Rn

N O

Rn+1

NH O Rn

Rn

N

O

Lossen

O

C

OH

NH

Rearrangment

O

O Rn R

R

R O NH NH O

H N Prot Rn+1

O N O

H N

O

+1 NH2 Rn

O

Rn

NH R

O

O

O

NH2 Prot

NH O

-CO2 O

Rn O

NH O

O

Rn R

Rn

OH

NH

R

R

Scheme 3. Mechanism of insertion of β-Ala-amino acid via the Lossen rearrangement

As mentioned earlier, the solvents of choice for SPSS in order of preference are DCM, followed by DMF and NMP. Therefore, the development of green solvents that are more environment-friendly and suitable for the SPPS protocol is still a high priority. The first attempt to replace DMF in SPPS was made by our group in 2009. In this regard, acetonitrile (MeCN) was used for coupling, deprotection and washing steps. Furthermore, MeCN achieved better coupling efficiency than DMF and a mixture of DMF/DCM.80 However, MeCN is not a


37


Page 38 of 63

green solvent.22 It can be considered friendlier than DMF because it is not included in the list substances of very high concern (SVHC)81, and it is classified on some recent selection guides as a mandatory solvent.22,82 In fact, it was initially reported that MeCN could be a greener alternative to DMF.83 Later in 2013, Watson and co-workers84 screened a range of green solvents to replace DMF and DCM in solution. In their study, a variety of coupling reactions between alkyl and aryl acids and amines were evaluated. It was concluded that dimethyl carbonate, 2-methyltetrahydrofuran (2-MeTHF) and EtOAc are suitable alternatives for DMF and DCM, especially when COMU is used as coupling reagent.84 See Figure 13 for the new set of solvents tested for peptide synthesis. Later, our group evaluated various solvents with the aim to make the peptide synthesis greener. We first examined 2-MeTHF and CPME for the amide bond formation reaction.85 Later, we reported a green synthetic protocol in which DMF and DCM were substituted by 2-MeTHF for coupling/deprotection steps and EtOAc for washing steps in combination with the ChemMatrix resin.86 For PS resin, we have recently evaluated other solvents such as γ-valerolactone (GVL), a biomass-


38

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derived organic solvent, and N-formylmorpholine (NMF).87 In conclusion, we optimized a green protocol compatible with PS and ChemMatrix resins and in which GVL was used as a solvent for all steps, in combination with microwave heating.88 The peptides obtained showed similar purities, comparable to those obtained with standard methodologies. Furthermore, this green approach showed superior performance in terms of solvent and energy consumption, and waste generated. The only drawback observed was a side reaction during Fmoc removal from the Gly residue. This side reaction was efficiently circumvented using FmocAA-Gly-OH (Scheme 4) (in preparation).

O

O

O H2N

OH Peptide

H N

Base

O Peptide

O

Scheme 4. Side reaction between GVL and the Gly residue during SPPS

North and co-workers89 reported propylene carbonate (PC) as a green solvent for peptide synthesis. It was successfully used for peptide synthesis in solution via the Boc/Bzl approach. Furthermore, it has also been utilized for the FmocSPPS of a 9-mer peptide on ChemMatrix resin. In both approaches, PC was


39


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used in all steps (coupling, deprotection and washing).89 PC can also be recycled because it is not contaminated by other solvents since the protocol involves only one solvent. Lopez and co-workers90 at Novartis studied N-butylpyrrolidinone (NBP) in depth as a greener alternative for DMF in SPPS. NBP is a promising candidate for green polar aprotic solvents because it has been classified as non-reproductively toxic, non-mutagenic and biodegradable.91 During its evaluation for peptide synthesis, it showed good swelling properties for PS and a similar performance to DMF with respect to dissolving Fmoc amino acids. In addition, NBP also showed a satisfactory performance during Fmoc removal steps. Thus, it is no surprise that the yield and purity of peptides obtained by Fmoc-SPPS performed in NBP are comparable with those achieved with DMF.90 Cyrene™ has also been reported for the amide bond formation reaction.92 It is a bio-based solvent that has been put forward as a substitute for toxic petrochemical-derived solvents.93 Cyrene™ has been successfully used as a


40

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solvent for the synthesis of dipeptides using HATU as coupling reagent, achieving good yields. However, its evaluation for SPPS has not been described to date.92 H O

O

N

-Valerolactone (GVL)

O N-Formylmorpholine (NFM)

O

2-Methyltetrahydrofuran (2-MeTHF)

O

O O

O

O

N

Propylene carbonate (PC)

O

N-Butylpyrrolidinone (NBP)

O

O

Cyrene

Figure 13. New set of green solvents for peptide synthesis

During the evaluation of green solvents for SPPS, we noted that the Fmoc removal steps were not as straightforward as originally expected. The wide use of the Fmoc group in peptide synthesis is due to its stability under basic conditions during coupling steps, while it is easily removed under mild basic conditions. We therefore screened a range of green solvents for Fmoc removal. In conclusion, GVL emerged as a promising candidate for Fmoc removal steps due to its high performance with both PS and ChemMatrix.94 Furthermore, Di Gioia and co-


41


Page 42 of 63

workers developed a green condition for Fmoc removal in solution by using triethylamine in an ionic liquid solvent [Bmim][BF4].95 Of note, piperidine has been classified as a controlled substance. However, this issue can be easily overcome by replacing piperidine by 4-methylpiperidine.96 TFA also requires a green substitute. This solvent is used mainly in the final step, namely cleavage and global deprotection in Fmoc-SPPS. However, TFA is corrosive and causes skin burns.23 Palladino and Stetsenko reported 0.1 N HCl in hexafluoroisopropanol (HFIP) or trifluoroethanol (TFE) as promising candidates for the cleavage and global deprotection step.24

However, these cleavage

approaches still require optimization and the particular problem of protecting the Arg residue should be addressed. Pbf, which is the protecting group of choice, shows excessive stability to some of these conditions. Diethyl ether (DEE) and to a lesser extent methyl tert-butyl ether (MTBE) are the most commonly used solvents for peptide precipitation after acidic cleavage of the peptide from the resin. However, both compounds have been classified as hazardous chemicals. Therefore, greener alternatives are called for. Our group


42

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has reported CPME (Figure 14) as an efficient greener alternative for DEE and MTBE.97 Our motivation was driven by the poor dissolving property of amino acids in CPME.85 CPME showed the same performance as DEE and MTBE in terms of recovery of the cleaved peptide and purity (no extra peaks were observed in the HPLC of the crude peptide).

O

Figure 14. Cyclopentyl methyl ether (CPME) Finally, our group has demonstrated that 2-MeTHF is also a highly efficient substitute for DEE and MTBE for peptide precipitation.98 It shows the same performance–recovery and purity–as DEE, MTBE, and CPME. Furthermore, 2MeTHF efficiently substitutes DCM during the incorporation of the first Fmoc protected amino acid onto Wang and CTC resins, which are the most common supports used for the preparation of C-terminal acid peptides.98 In both cases, the incorporation yield using 2-MeTHF was similar to that achieved with DCM. Furthermore, in the case of Wang resin where DIC and N,N-dimethylaminopyridine


43


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(DMAP) are used for first amino acid incorporation, the degree of racemization was similar to that obtained with DCM.98

Conclusions and Perspectives: Transforming chemical processes into greener procedures is a mandate from society. In this regard, while working hard to improve social wellbeing in terms of better medicines, more secure transport, more comfortable domestic utilities, etc., chemists should remain steadfast in their mission to protect the environment and human health.

In this context, peptide chemists also have a role to play,

namely by optimizing green synthetic protocols.

In addition to reducing the

amount of material require for a synthetic procedure—which will be reflected in a reduction in both cost and waste—and avoiding hazardous materials, peptide chemists should fully embrace the green concept. When Frank and Houghten independently developed the SPOT and tea bag techniques for the simultaneous synthesis of peptides, they were probably unaware of the green perspective of their research. Today the community acknowledges that both techniques fully meet the criteria of Green Chemistry.


44

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In the context of recycling, not only should this process be applied to solvents, thus implying the use of solvents with a moderate boiling point and which are therefore friendlier, but also to resins, such as CTC. A major reduction in Atom Economy will call for major efforts to be channeled into the development of a totally new concept of peptide synthesis. This concept is likely to consist of minimizing the presence of protecting groups following a similar approach to that used for native chemical ligation.99 “Greening” the coupling step will also require considerable efforts, because converting the carboxylic acid into a reactive species is a demanding reaction. In terms of waste, the use of (U)NCA emerges as highly convenient because the only side product is CO2. However, the preparation of this reagent requires the concourse of harmful reagents, such as COCl2, and their derivatives. However, the impact of the use of (U)NCA on multiKg synthesis has not yet been studied. The use of benzotriazole derivatives is restricted the basis of the potential explosivity of this family of reagents.100 To date, the literature describes EDC.HCl,


45


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OxymaPure, and COMU as the greenest compatible coupling reagents.74 However, as mentioned earlier, further efforts in this field are required.101 At the research scale, some green solvents (GVL, NBP, 2-MeTHF, CP) have shown excellent performance for coupling and deprotection steps, as well as for swelling the resin and dissolving amino acids. The synthesis of large peptides (up 28 amino acids) has been reported using GVL. When C-terminal acid peptides are required, 2-MeTHF can be used in all steps of the synthesis: incorporation of the first amino acid, removal of Fmoc, coupling of the incoming protected amino acid, and peptide precipitation after global deprotection and cleavage. For precipitation, the use of CPME is also an excellent choice. In most cases, ChemMatrix resin outperforms PS when used with green solvents. However, the large-scale synthesis of peptides is still carried out mostly with PS resins. In this regard, we believe that the production of peptide-based APIs using PS requires further optimization of all steps. It would also be convenient to address the use of mineral acids for the cleavage steps, because TFA, HF, and TFMSA are all hazardous chemicals to distinct


46

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degrees. Although HCl solution in organic solvents can replace TFA, more work is needed, and the removal of Pbf from Arg will probably be the main obstacle to be tackled. We envisage the development of a new protecting group for Arg in order to fulfill green requirements. Green Chemistry is defined as the ‘‘design of chemical products and processes to reduce or eliminate the use and generation of hazardous substances”.3 For most chemical processes, including peptide synthesis, it is unlikely that such substances can be completely eliminated. However, it is the duty of chemists to

reduce the use of these products In this regard, Green Chemistry should fulfill the ancestral Quechua concept of Sumak Kawsay (Good Living), which seeks to achieve a balance with nature, taking only what is necessary. In other words, this approach is based on the idea of sustainability and not only on economic growth.

AUTHOR INFORMATION Author Contributions


47


Page 48 of 63

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. (match statement to author names with a symbol)

Funding Sources Any funds used to support the research of the manuscript should be placed here (per journal style).

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

The work in the laboratories of the authors was funded in part by the following: National

Research

Foundation

(NRF),

Nanotechnology

for

Sustainable

Development Platform (University of KwaZulu-Natal), and the University of KwaZulu-Natal (South Africa); the International Scientific Partnership Program ISPP at King Saud University (ISPP# 0061) (Saudi Arabia); and the Spanish


48

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Ministry of Economy, Industry and Competitiveness (MINECO) (CTQ2015-67870P) and the Generalitat de Catalunya (2017 SGR 1439) (Spain).

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Graphical abstract

Solid-phase peptide synthesis, which is the most commonly used approach on peptide synthesis, from green chemistry point of view


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Author biographies

Yahya E. Jad received his BSc (2009) and MSc (2013) from Alexandria University, Egypt. Later, he received his PhD degree in pharmaceutical chemistry from University of KwaZulu-Natal, Durban, South Africa under supervision of Professor Fernando Albericio. Currently, he is a postdoctoral fellow at peptide sciences lab under the supervision of Professors Beatriz de la Torre and Fernando Albericio. His research interests focus on peptide synthesis methodologies such as peptide coupling reagents and greening of peptide synthesis.

Ashish Kumar obtained his M.S. Pharm degree in Natural product (Pharmacy) at National Institute of Pharmaceutical Education and Research (NIPER) Kolkata India and Ph.D in Pharmacy at Jadavpur University (India), under the supervision of Professor Sibabrata Mukhopadhyaya and Professor Tarun Jha. He is currently working as a postdoctoral fellow at the University of KwaZulu Natal (Durban, South Africa). He moved from isolation and characterization of natural product to solid phase peptide synthesis in search of green solvent substitute for current strategies.


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Ayman El-Faham has been Professor of Organic Chemistry at the College of Science, King Saud University, since 2011. He received his BSc and MSc degrees from Alexandria University and his PhD degree in organic chemistry as a joint project between the University of Massachusetts (USA) and the University of Alexandria. In 2010, he received his D.Sc. degree in peptide synthesis (Cambridge Recommendation). His major scientific efforts have been concentrated on the development of new coupling reagents, first in the laboratory of Professor Carpino, and since 1993 in collaboration with Professor Albericio. Lastly, he is involved in developing GSPPS strategies.He has heavily worked in drug discovery by using relevant scaffolds, such as the triazine.

Beatriz G. de la Torre obtained her PhD from the University of Barcelona (Spain). After a dilated career in Spain, she is presently Research Professor at KRISP, College of Health Sciences, University of KwaZulu-Natal (Durban, South Africa). She has been working extensively on glyco, nucleo-, and lipopeptides. Lastly, her scientific interests are focused more on the discovery of new antimicrobial


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peptides, including those for antituberculosis, peptidebased vaccines, and peptidebased drug-delivery systems. She is also involved in developing GSPPS strategies

Fernando Albericio has developed his academic and professional career in Europe, USA, Latin America, Asia, and presently in Africa (Research Professor at the University of KwaZulu-Natal (Durban, South Africa)). His major research interests cover practically all aspects of peptide synthesis, as well as the synthesis of peptides and small molecules with therapeutic activities, especially tuberculosis, infectious diseases, and cancer. His group is also involved in the development of new strategies for drug delivery. He is deeply involved in the development of the third mission of the University: the transference of knowledge and technology to society. He was cofounder and General Director of the Barcelona Science Park and is presently cofounder of BioDurban.


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Green Transformation of Solid-Phase Peptide Synthesis - ACS

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