N-Butylpyrrolidinone as Alternative Solvent for Solid-Phase Peptide

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N-BUTYLPYRROLIDINONE AS ALTERNATIVE SOLVENT FOR SOLID PHASE PEPTIDE SYNTHESIS JOHN LOPEZ, Stefan Pletscher, Andreas Aemissegger, Christoph Bucher, and Fabrice Gallou Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00389 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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Organic Process Research & Development

N-BUTYLPYRROLIDINONE AS ALTERNATIVE SOLVENT FOR SOLID PHASE PEPTIDE SYNTHESIS John Lopez,* Stefan Pletscher, Andreas Aemissegger,¥ Christoph Bucher, and Fabrice Gallou Novartis Pharma AG, Novartis campus, WSJ-145-5.51, CH-4056, Basel, Switzerland. ¥

Current address: Bachem AG, Hauptstrasse 144, CH-4416 Bubendorf, Switzerland.

ACS Paragon Plus Environment

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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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KEYWORDS:

DMF replacement for solid phase peptide synthesis, N-Butylpyrrolidinone,

Green solid phase peptide synthesis. ABSTRACT. By means of a systematic approach, several green solvent candidates were tested for their feasibility to replace the reprotoxic dimethylformamide (DMF) as a solvent used in solid phase peptide synthesis (SPPS). According to the results presented in this paper it is clear that Nbutylpyrrolidinone (NBP) is the best green solvent candidate to replace DMF in SPPS.


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INTRODUCTION There is an increasing interest in the pharmaceutical industry to use therapeutic peptides. This is due to several factors which include the rise of alternative routes of administration, new methods to favor peptide stability1, and the fact that peptides are used as lead compounds to create non-peptide structures called peptidomimetics, where these kinds of molecules are able to mimic protein-protein interactions2. The majority of the marketed peptide drugs, are produced by chemical methods3, and more often by solid phase peptide synthesis (SPPS)4, even though the practice of SPPS requires the use of significant amounts of toxic process solvents5 like; DMF, NMP, DMA and DCM. The high consumption of solvents is due to two main factors: Firstly, the large number of chemical steps necessary to produce a peptide and secondly, the repetitive washings. Since in SPPS there is no purification after each chemical step it is necessary to remove excess reagents from the media before proceeding to the next step, and this is achieved by extensive resin washes that generate a large amount of solvent waste. There are an increasing number of publications dealing with the reduction of toxic waste generated by SPPS process. For example, by using micro reactors in a type of continuous flow chemistry6, or by the use of optimized reaction conditions7 in special equipment.

Nevertheless, both these approaches

mentioned still use toxic solvents. Due to their reprotox characteristics, polar aprotic solvents like DMF, NMP and DMA, have been labelled by REACH-ECHA as a Substances of Very High Concern (SVHC). For that reason, the replacement of those solvents in any kind of manufacturing processes is an opportunity to mitigate potential supply risks resulting from the REACH-ECHA classification8. In the field of amide bond formation in solution, Watson9 proposed to use DMC, EtOAc and Me-THF as green alternatives for replacing DMF and DCM. More recently, in parallel to our investigational work, the group of Hunt10 demonstrated the successful use of non-reprotoxic NBP in several type of common organic reactions, for example,


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in the Menschutkin reaction, the Heck reaction, the benzylation of sodium acetate and the Biginelli reaction as an example of heterocycle synthesis chemistry performed in solution. In the field of SPPS, after Albericio11 synthesized a model peptide produced on ChemMatrix resin and using DICI/Oxyma as the coupling system, Albericio found that the best replacement for DMF as the SPPS solvent was Me-THF. For polystyrene based supports, a more recent publication by Albericio12 suggested that in spite of their lower performance as SPPS solvents; γValerolactone and N-formylmorpholine could both be used as green solvents for SPPS. Another green solvent that was proposed as replacement for DMF in both solution and solid phase peptide synthesis is propylene carbonate (PC). The potential use of PC was exemplified by the Fmoc based SPPS of bradykinin13 performed in a ChemMatrix type resin. The most attractive scenario to reduce the generation of any kind of waste in peptide synthesis would be to use solvent-free conditions, for example, by the application of mechanoenzymatic conditions14. The feasibility of the mechanoenzymatic concept was tested in the synthesis of dipeptides on small scale and further work on this approach to demonstrate its real potential in the large scale manufacture of more complex peptides is required. Another approach, addressed by numerous research groups around the world, has employed water as the solvent of choice in order reduce use of toxic solvents. Several complex solutions have been provided, for example, the use of specially designed protecting groups to facilitate the solubility of hydrophobic amino acid derivatives in water,15 or by the use of microwave assisted amino acid couplings with Boc amino acids16 or enzymatic mediated coupling reactions performed aqueous media17. Although very worthy, none of the aqueous options are compatible with polystyrene based resins which render them out of scope of the main focus of our investigational work.


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Octreotide (Sandostatin®) is a cyclic octapeptide that is manufactured on large scale and commercialized by Novartis. Octreotide is used in the treatment of acromegaly and gigantism.18 Similar to other large-scale SPPS processes, the manufacture of 1.0 kg of a peptide with the size of Octreotide generates between 2 to 3 tons of solvent waste that is mainly comprised of DMF. The goal of the current investigational work was to evaluate the feasibility of replacing DMF with a less toxic solvent in order to provide a regulatory and technical solution. The regulatory hurdle was that the new solvent should be in agreement with ICH guidelines, Novartis internal green chemistry process recommendations and should not be included in any of the lists of the REACH-ECHA regulation. The technical challenge was that the use of the new solvent should be able to produce peptides with similar quality as if performed using DMF. Although commercial availability and price of the solvent are critical in long-term implementation, we kept our investigations at the technical level and did not exclude potential candidates based on price criteria and/or availability. REDUCING COMPLEXITY The large scale manufacture of peptides by SPPS methodology is a multidimensional challenge where the peptide chemist has to take into account several parameters before proposing any manufacturing process for example: The length and difficulty of the target peptide, the potential need of non-standard reactions to be executed while the peptide is attached to the support, the kind of solid support to be used, the type of SPPS strategy to be used (i.e. Boc chemistry vs Fmoc chemistry), the initial degree of resin loading, the target scale to be produced, the compatibility of the available equipment with the target scale and manufacturing strategy, etc. Due to this complexity, the definition of what is a suitable solvent for large scale SPPS is not an easy task and does not have a “unique right answer”. A way of reducing the complexity is to give priorities to those parameters that fulfil the needs of the manufacturing industry, for example:


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RESIN POLYMER TYPE From the point of view of the polymeric support, SPPS can be executed on a variety of supports with structural properties that allow execution of SPPS in a wide range of solvents from organic to aqueous. For example, Tentagel®, ChemMatrix® and PS. Tentagel® based resins have a polymeric base structure that is a composite of polyethylene glycol (PEG) in a low crosslinked polystyrene (PS) network. The combination of PEG and PS gives Tentagel® resins a wider range of swelling in polar solvents, but at the same time it is hygroscopic and the backbone is more reactive, compared to PS, giving the risk of backbone damage and therefore leakage of PEG or PEG derivatives reducing the process yield and purity19. Another emerging polymeric support is ChemMatrix® which is made from 100% PEG and it is claimed that ChemMatrix® resins are more appropriate for the synthesis of large peptides and proteins20. However, to our knowledge there are no examples of large scale manufacturing of peptides using ChemMatrix® resin. The classical support used in large scale manufacture of peptides is polystyrene crosslinked with 1-2% of divinylbenzene (hereafter called just polystyrene or PS). The popularity of PS arises from a variety of factors. It has excellent mechanical and chemical stability, a broad flexibility in terms of availability of a wide range of loading levels. PS is also a commodity that is used as starting material in other areas of the manufacturing industry, giving PS an important advantage of low cost and high bulk availability. For those reasons, this investigation was conducted with the final target of finding a non-reprotoxic solvent that would be compatible with PS based resins. COUPLING STRATEGY Due to cost, efficiency and thermal safety in the large scale manufacture of peptides, we gave priority to finding a solvent that yields results in terms of coupling efficiency similar to those obtained with DMF while using the coupling system carbodiimide/oxyma.


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SWELLING OF THE RESIN The ability of a solvent to keep the resin swollen during the complete SPPS process is a key parameter for a successful process. In our experience, the measurement of the swelling properties can produce erratic results, especially if done with too small amounts of resin and/or requiring having a “dry” state of the resin21, therefore, we systematically use the same simplified method as described in the experimental section. In the field of understanding resin swelling there is a remarkable scientific work dealing with the creation of a computational model to predict swelling properties of green solvents with several types of resins22. The validity of the method was successfully demonstrated with an example of the Ugi reaction. Even if it is clear that a solvent must be able to swell the resin, it is also worth to remark that an excessive resin swelling is also a drawback for a solvent: 1) The higher the level of swelling, the smaller amount of peptide can be produced in the same volume of SPPS reactor. 2) The higher the level of swelling, the more solvent that is necessary to obtain a uniform suspension for SPPS, increasing the already high solvent consumption of SPPS.

For the purpose of this

investigation work, the solvents assessed had to produce a level of swelling of not more/not less of 30% to that of DMF. PROPOSED SOLVENTS TO BE TESTED The solvents included in Solve nt ID Solvent name

BP (C°)

Polarity*

Type of Solvent

Polarity Level

Protic/Aprotic

1

(S)-(-)-Limonene

177

0.046

Hydrocarbon

Low polarity

Aprotic

2

(R)-(+)-Limonene

177

0.046

Hydrocarbon

Low polarity

Aprotic

3

Toluene

111

0.099

Hydrocarbon

Low polarity

Aprotic

4

Diethylcarbonate (DEC)

127

0.185

Carbonate

Low polarity

Aprotic

5

Methyltetrahydofuran (Me-THF)

80

0.187

Ether

Low polarity

Aprotic

6

Anisole

153

0.198

Ether

Low polarity

Aprotic

7

Tetrahydrofuran (THF)

66

0.207

Ether

Medium polariy

Aprotic

8

Propyl acetate

102

0.210

Ester

Medium polariy

Aprotic

9

EtOAc

77

0.228

Ester

Medium polariy

Aprotic


8

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10

Butyl acetate (ButOAc)

126

0.241

Ester

Medium polariy

Aprotic

11

Tributyl phosphate

289

0.253

Organophosphate

Medium polariy

Aprotic

12

γ-Valerolactone (GVL)

207

0.301

Lactone

High Polarity

Aprotic

13

Propylene carbonate (PC)

246

0.301

Carbonate

High Polarity

Aprotic

14

Tetramethylurea (TMU)

176

0.315

Amide

High Polarity

Aprotic

15

N,N,N',N'-Tetraethyl sulfamide (TES)

250

0.318

Sulfamide

High Polarity

Aprotic

16

N-Butylpyrrolidone (NBP)

243

0.323

Amide

High Polarity

Aprotic

17

Triethyl phosphate

215

0.324

Organophosphate

High Polarity

Aprotic

18

1,3-Dimethyl-2-imidazolidinone (DMI)

225

0.325

Amide

High Polarity

Aprotic

19

Methyl ethyl ketone (MEK)

79

0.327

Ketone

High Polarity

Aprotic

20

222

0.336

Amide

High Polarity

Aprotic

21

N-Ethylpyrrolidone (NEP) 1,3-Dimethyl-3,4,5,6-tetrahydro-2pyrimidinone (DMPU)

247

0.352

Amide

High Polarity

Aprotic

22

N-Methylcaprolactam (MCL)

247

0.352

Amide

High Polarity

Aprotic

23

N-Methylpyrrolidone (NMP)

204

0.355

Amide

High Polarity

Aprotic

24

N,N-Dimethylacetamide (DMA)*

165

0.377

Amide

High Polarity

Aprotic

25

Dimethylformamide (DMF)

153

0.386

Amide

High Polarity

Aprotic

26

tert-Butanol

82

0.389

Alcohol

High Polarity

Protic

27

Sulfolane

285

0.410

Organosulfur

Very High polarity

Aprotic

28

3-Methyl-2-oxazolidinone (MOL)

266

0.438

Carbamate

Very High polarity

Aprotic

29

Dimethyl sulfoxide (DMSO)

189

0.444

Organosulfur

Very High polarity

Aprotic

30

Acetonitrile (ACN)

82

0.460

Nitrile

Very High polarity

Aprotic

31

Ethylene glycol

197

0.713

Alcohol (Diol)

Very High polarity

Protic

32

Isopropyl acetate

88

n.a

Ester

n.a

Aprotic

33

88

n.a

Ester

n.a

Aprotic

34

tert-Butyl acetate Dipropylene glycol (DMM)

175

n.a

Ether

n.a

Aprotic

35

p-Cymene

178

n.a

Hydrocarbon

n.a

Aprotic

36

N,N-Diethyl-meta-toluamide (DEET)

285

n.a

Amide

n.a

Aprotic

37

Isobutyl acetate

118

n.a

Ester

n.a

Aprotic

dimethylether

Table 1 were used for the stepwise evaluation of their suitability as replacements for DMF in SPPS. The selection of the candidates was performed following internal Novartis guidelines and published solvent selection guides23. In all tests DMF and NMP were included as reference solvents.

Solvent ID

Solvent name

BP (C°)

Polarity*

Type of Solvent

Polarity Level

Protic/Aprotic

1

(S)-(-)-Limonene

177

0.046

Hydrocarbon

Low polarity

Aprotic

2

(R)-(+)-Limonene

177

0.046

Hydrocarbon

Low polarity

Aprotic

3

Toluene

111

0.099

Hydrocarbon

Low polarity

Aprotic

4

Diethylcarbonate (DEC)

127

0.185

Carbonate

Low polarity

Aprotic

5

Methyltetrahydofuran (Me-THF)

80

0.187

Ether

Low polarity

Aprotic


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6

Anisole

153

0.198

Ether

Low polarity

Aprotic

7

Tetrahydrofuran (THF)

66

0.207

Ether

Medium polariy

Aprotic

8

Propyl acetate

102

0.210

Ester

Medium polariy

Aprotic

9

EtOAc

77

0.228

Ester

Medium polariy

Aprotic

10

Butyl acetate (ButOAc)

126

0.241

Ester

Medium polariy

Aprotic

11

Tributyl phosphate

289

0.253

Organophosphate

Medium polariy

Aprotic

12

γ-Valerolactone (GVL)

207

0.301

Lactone

High Polarity

Aprotic

13

Propylene carbonate (PC)

246

0.301

Carbonate

High Polarity

Aprotic

14

Tetramethylurea (TMU)

176

0.315

Amide

High Polarity

Aprotic

15

N,N,N',N'-Tetraethyl sulfamide (TES)

250

0.318

Sulfamide

High Polarity

Aprotic

16

N-Butylpyrrolidone (NBP)

243

0.323

Amide

High Polarity

Aprotic

17

Triethyl phosphate

215

0.324

Organophosphate

High Polarity

Aprotic

18

1,3-Dimethyl-2-imidazolidinone (DMI)

225

0.325

Amide

High Polarity

Aprotic

19

Methyl ethyl ketone (MEK)

79

0.327

Ketone

High Polarity

Aprotic

20

222

0.336

Amide

High Polarity

Aprotic

21

N-Ethylpyrrolidone (NEP) 1,3-Dimethyl-3,4,5,6-tetrahydro-2pyrimidinone (DMPU)

247

0.352

Amide

High Polarity

Aprotic

22

N-Methylcaprolactam (MCL)

247

0.352

Amide

High Polarity

Aprotic

23

N-Methylpyrrolidone (NMP)

204

0.355

Amide

High Polarity

Aprotic

24

N,N-Dimethylacetamide (DMA)*

165

0.377

Amide

High Polarity

Aprotic

25

Dimethylformamide (DMF)

153

0.386

Amide

High Polarity

Aprotic

26

tert-Butanol

82

0.389

Alcohol

High Polarity

Protic

27

Sulfolane

285

0.410

Organosulfur

Very High polarity

Aprotic

28

3-Methyl-2-oxazolidinone (MOL)

266

0.438

Carbamate

Very High polarity

Aprotic

29

Dimethyl sulfoxide (DMSO)

189

0.444

Organosulfur

Very High polarity

Aprotic

30

Acetonitrile (ACN)

82

0.460

Nitrile

Very High polarity

Aprotic

31

Ethylene glycol

197

0.713

Alcohol (Diol)

Very High polarity

Protic

32

Isopropyl acetate

88

n.a

Ester

n.a

Aprotic

33

88

n.a

Ester

n.a

Aprotic

34

tert-Butyl acetate Dipropylene glycol (DMM)

175

n.a

Ether

n.a

Aprotic

35

p-Cymene

178

n.a

Hydrocarbon

n.a

Aprotic

36

N,N-Diethyl-meta-toluamide (DEET)

285

n.a

Amide

n.a

Aprotic

37

Isobutyl acetate

118

n.a

Ester

n.a

Aprotic

dimethylether

Table 1: List of Solvents It is noteworthy that if the polarity of the solvent is plotted against its boiling point as described by Hunt10, the usual toxic solvents used in SPPS like DMF, NMP and DMA all are located in the same region of the plot. That region, dubbed the “Golden Zone” is defined by the polarity range of 0.3-0.4. Interestingly that golden zone is also populated by other greener


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solvents that are included in the current investigation work. Note: For solvents 31-37 there was no polarity data available and are therefore not included in Figure 1.

Figure 1: Plot Boiling point vs Polarity RESIN SWELLING TEST RESULTS The first solvent selection criterion applied was the ability of the solvent to swell a polystyrene based resin (in this case, Aminomethyl-PS (AMRES)). AMRES was selected as the support for the swelling test based on the fact that AMRES is used in several of our large scale manufacturing process. The results of the swelling test are shown in Table 2. Solvent

Solvent

ID 1

Anisole

Swelling

Solvent

(ml/g)

ID

9.1

19

Solvent

Swelling (ml/g)

DMM

4.6


11


Solvent

Solvent

ID

Swelling

Solvent

(ml/g)

ID

Solvent

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Swelling (ml/g)

2

DMI

8.5

20

p-Cymene

2.2

3

NEP

8.2

21

MOL

2.0

4

DMPU

8.0

22

CAN

2.0

5

THF

7.9

23

TES

2.0

6

NBP

7.4

24

MCL

1.9

7

Me-THF

7.2

25

DMSO

1.6

8

Propyl acetate

6.8

26

Ethylene glycol

1.6

9

TMU

6.8

27

γ-Valerolactone

1.6

10

DEC

6.5

28

DEET

1.6

11

Toluene

6.4

29

PC

1.6

12

DMF

6.3

30

t-Butanol

1.6

13

NMP

6.2

31

t-Butyl acetate

1.6

14

EtOAc

6.0

32

Tributylphosphate

1.6

15

MEK

5.6

33

Trietylphosphate

1.6

16

Butyl acetate

5.2

34

Sulfolane

1.6

17

Isobutyl acetate

4.9

35

(S)-(-)-Limonene

1.6

18

Isopropyl acetate

4.8

36

(R)-(+)-Limonene

1.6

Table 2: Resin swelling results The solvents in Table 2 are divided into 3 groups as follows: Group 1: Solvents 1-14 (Marked in green), solvents included in this group are considered as solvents with good resin swelling properties. All solvents in this group were subject to further testing.


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Group 2: Solvents 15-19 (Marked in Yellow), solvents included in this group are considered as solvents with borderline resin swelling properties. Solvents in this group were considered for potential future evaluation but were not tested further within the scope of this investigation work. Group 3: Solvents 20-36 (Marked in Red): These solvents have very poor resin swelling capacity, and were therefore not included in further testing. In this investigation work, MEK and t-Butanol were included as learning tools, since due to their reactivity they cannot be considered as potential solvents for SPPS. MEK was excluded from further testing based on its reactivity. SOLUBILITY OF SPPS REAGENTS A good SPPS solvent is able to dissolve all reagents that are involved in SPPS. For the purpose of this investigation, the following exemplary materials were used: Amino acids Fmoc-Gln(Trt)OH and Fmoc-Gly-OH; coupling reagents such as diisopropylcarbodiimide (DICI); additives, such as oxyma; and related by-products generated during the SPPS process. In the case of the coupling system DICI/oxyma, the by-product diiosopropylcarbodiimide (DIU) is generated, and in an ideal solvent, this by-product should remain soluble during the coupling reaction and should be removed to prevent resin clogging, which has a direct impact in the efficiency of the washings and then in the quality of the produced peptide. Table 3 summarizes the results of the solubility tests and it can be seen that solvents like anisole and toluene that have a good resin swelling ability, are not able to dissolve properly SPPS related reagents, such as DIU. Interestingly in this test, DMF, THF and Me-THF only showed partial solubility for DIU, but in typical practice of SPPS, those solvents require more extensive resin washings to remove undesired rest of reagents.


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Reagents

Solvent Solvent name ID Fmoc-Gln(Trt)OH

Fmoc-GlyOH

Oxyma

DICI

DIU

1

Anisole

I

I

S

S

I

2

DMI

S

S

S

S

S

3

NEP

S

S

S

S

S

4

DMPU

S

S

S

S

S

5

THF

S

S

S

S

Ps

6

NBP

S

S

S

S

S

7

Me-THF

S

S

S

S

Ps

8

Propyl acetate

S

I

S

S

I

9

TMU

S

S

S

S

S

10

DEC

I

I

S

S

I

11

Toluene

I

I

I

S

I

12

DMF

S

Ps

S

S

Ps

13

NMP

S

S

S

S

S

14

EtOAc

S

I

S

S

I

25

DMSO

S

S

S

S

S

Table 3: Solubility of SPPS reagents: S = Soluble; Ps = Partially soluble; I = Insoluble STUDY OF THE COUPLING REACTION The current manufacturing process for octreotide typically requires a complete coupling time of 2-3 h. This coupling time is very representative of other carbodiimide mediated couplings performed at large scale. For productivity and quality reasons longer coupling times are avoided. In order to evaluate if the candidate solvent could complete the coupling reaction in the desired time of 2h; Fmoc-Leu-OH (1.0 eq) was coupled to H-Phe-OMe (1.0 eq) using


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Diisopropylcarbodiimide (1.0 eq) and Oxyma (1.0 eq) as the coupling system. The progress of the reaction was followed by HPLC measuring the residual amount of Fmoc-Leu-OH at different times. Propyl acetate and diethyl carbonate (DEC) were not included in the coupling test due to their poor solubilizing ability of SPPS reagents.

Figure 2: Study of the coupling reaction As can be seen in Figure 2, when compared to DMF, the coupling reaction is very fast if performed in non-polar/borderline aprotic solvents like anisole, toluene, Me-THF, ethyl acetate and THF.

It was also found that in all those solvents the coupling was accompanied by


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precipitation of DIU and/or the dipeptide from the reaction mixture. This observation is in agreement with the results of the solubility test results. In polar aprotic solvents like: NMP, TMU, DMI, NEP and NBP the coupling reaction is slower compared to when performed in DMF, but this small difference can be overcome by reaction parameters optimization. As shown in Figure 1, DMSO gives the slowest coupling reaction and therefore it will not considered as a candidate for replacing DMF. The solvents tested for coupling efficiency were also tested for their efficiency in the Fmoc cleavage reaction. STUDY OF THE FMOC CLEAVAGE REACTION The next step in the solvent selection process was to monitor the performance in the Fmoc cleavage reaction. A polymer supported octapeptide bearing an Fmoc group was treated with a 20% v/v solution of 4-methylpiperidine prepared in the solvent candidate, and subsequently small portions of resin were taken at different times and washed immediately. The peptide was cleaved from the support using 3.0% v/v TFA in dichloromethane and the amount of unprotected peptide was evaluated by HPLC (See Figure 3).


16

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Figure 3: Study of the Fmoc cleavage reaction Figure 3 shows that for non-polar aprotic solvents like; EtOAc, Anisole, Me-THF, THF and Toluene, the Fmoc cleavage reaction was slow since it was not complete after 30 min. For those solvents, it could be convenient to consider the use of a stronger Fmoc cleavage reagent, for example, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)24 instead of 4-methyl piperidine. The poor performance of Me-THF as reagent for Fmoc cleavage was recently reported and it was proposed to use γ-Valerolactone25 (GVL) as green alternative for that reaction. As clearly shown in Figure 3, the Fmoc cleavage reaction done in THF is very slow, in comparison with DMF were the cleavage of the Fmoc is complete in less than five minutes, in THF at the same time just 36.6% of the peptide is deprotected, if one allows the reaction to procced for longer time, like for example the time limit of 30 minutes, even there the cleavage reaction was not complete (98.5%) this poor performance in the Fmoc cleavage reaction will be reflected in the generation of more


17


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deletion sequences specially in the manufacture of larger peptides . In the case of polar aprotic solvents like DMSO, DMPU, NEP, DMI and NBP the Fmoc cleavage reaction is complete in less than 5 min, this is very similar to when performed in DMF. At this point of our investigation, the list of 14 solvents that passed the swelling test was shorted again based on the following arguments: Anisole, Propyl acetate, DEC, Toluene and EtOAc, were excluded because of their poor solubility of SPPS reagents. In the case of Me-THF26 (previously reported as potential alternative for replacing DMF in SPPS), due to its borderline solubility of SPPS reagents, precipitation of DIU during coupling and its poor performance in the Fmoc cleavage reaction, it was also excluded. In the case of THF, this solvent showed good results in the swelling test, borderline results in the solubility test and good results in the coupling reaction test, but it showed poor performance in the Fmoc cleavage reaction. In summary in the case of THF due to its poor performance in the cleavage reaction, its borderline solubility of SPPS reagents and its suspected toxicity (H351), THF was also excluded for the final test even if THF is already recommended as DMF replacement27. The solvents listed in Table 4 were carried to the next selection test. DMSO was maintained in the list only as a negative control. Solvent

CAS

Structure

MW

BP

Viscosity

number

(g/mol)

(°C)

(cP, 25 °C)

DMF

68-12-2

73.09

153

0.8

TMU

632-22-4

116.16

177

1.5


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DMI

80-73-9

114.15

225

1.9

NBP

3470-98-2

141.21

243

4.0

DMPU

7226-23-5

128.17

246

2.9

DMSO

200-664-3

78.13

189

2.0

Table 4: List of selected solvents for SPPS use test SPPS USE TEST

Figure 4: Use test for the solid phase peptide synthesis of an Octreotide intermediate

In this test a linear octapeptide that is involved in the manufacturing process of Octreotide was first synthesized using DMF as solvent (See Figure 4). The synthesis was performed on a fully automatic peptide synthesizer. After the peptide synthesis was complete, the resin was washed, dried and then a sample of the peptide resin was treated with 3% TFA in DCM. The cleavage solution was analyzed by HPLC (Figure 5) and LC-MS (Table 5 ). The same procedure was repeated with each solvent candidate. The HPLC profile of each experiment is shown in Figure 5.


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Figure 5: HPLC profile of SPPS use test ANALYSIS OF THE RESULTS The LC-MS analysis of the crude peptides generated in each solvent system showed that: 1) All crudes have a similar impurity profile and 2) LC-MS data indicates that most of the impurities were shorter versions of the target peptide (TP), mainly deletions of one amino acid (See Table 5 ). By-product description Solvent/Ionic material TP minus Boc TP minus Cys(StBu)_1 TP minus Cys(StBu)_2 TP minus Lys(Boc) TP minus Phe TP Epimer TP minus Thr TP plus Fmoc Product purity (Area %)1 Sum of unknowns Product purity (Area %)2

DMF 8.9 4.2 1.3 2.2 3.3 1.0 0.0 3.5 0.0 73.1 2.5

NBP 11.7 1.9 6.8 0.0 3.3 2.6 1.5 3.7 2.3 66.1 0.0

TMU 3.2 1.2 1.3 5.5 1.6 0.9 0.8 6.1 5.4 73.1 0.9

DMI 3.6 3.2 10.2 0.0 3.2 1.4 2.1 4.4 0.0 70.7 1.3

DMSO 14.0 3.5 4.1 3.8 8.0 6.1 0.0 7.7 0.0 34.9 17.9

DMPU 6.2 1.7 7.3 4.1 7.0 4.3 0.0 11.0 0.0 43.1 15.3

86

80

78

78

52

51


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SPPS yield (%)3

100

93

103

99

87

85

Table 5: Analysis of LC-MS results from SPPS use test; 1) HPLC purity; 2) HPLC purity without solvent and Boc deprotected; 3) Yield based on resin weight gain, non-corrected for peptide content. Since the solvents tested at this level demonstrated to be efficient in the Fmoc cleavage reaction, the origin of deletion sequences could be assigned to incomplete coupling reactions. As demonstrated by the coupling experiments, TMU, DMI and NBP are solvents where the coupling reaction is slightly slower compared to DMF. In this situation it is expected to have more deletion sequences when compared to DMF. In the case of solvents where the reaction is very slow, like for example, DMSO and DMPU, it is expected to have more deletion sequences and therefore poor crude quality compared to the rest of the tested solvents. In order to have a clear idea of the quality of the crude generated by each solvent, the reported purity of each crude is done after the removal of peaks assigned to solvents/ionic material and addition of the peaks assigned to target peptide that have prematurely lost the Boc protecting group during the cleavage reaction. None of those peaks are related to the performance of the candidate solvent but due to sample preparation. The purity of the crudes are as follows: 1) DMF (86 %); 2) NBP (80%); 3) TMU and DMI (78%); 4) DMSO (52%) and 5) DMPU (51.0%). EXPERIMENTAL PART SWELLING TEST 1.0 g of amino methyl resin (AMRES) was placed in a 10 mL calibrated graduate cylinder (error limit ±0.1 mL), to which the candidate solvent was added giving a final volume of 10 mL. The suspension was gently stirred and allowed to settle for 3.0 h. The final volume reached by the resin was measured and reported as mL/g.


21


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SOLUBILITY TEST To 2 mmol of each reagent was added 10 mL of the candidate solvent. All mixtures were stirred at the same speed and temperature (22-23 °C) for 10 min. Subsequent visual observation of the state of the mixture qualitatively reported as Soluble (S), partially soluble (Ps, most of the solids are dissolved but still a cloudy suspension is observed) and Insoluble (I, no apparent dissolution). COUPLING TEST To a 25 mL round bottom flask were added sequentially Fmoc-Leu-OH (707 mg, 2 mmol), Oxyma (285 mg, 2.0 mmol) the mixture was dissolved in 9 mL of the candidate solvent, then DICI is added (310 µL, 2.0 mmol), the mixture was stirred at room temperature (22-25 °C) for 10 min then a solution of H-Phe-OMe (358 mg, 2.0 mmol) in 1 mL of the candidate solvent was added. The progress of the coupling reaction was followed by HPLC by taking samples at 15, 30, 45 and 120 min. In each chromatogram just the area of Fmoc-Leu-OH and the area of the dipeptide were integrated, and then the reported is the area % of residual Fmoc-Leu-OH. FMOC CLEAVAGE TEST To each of 3 micro reactors was added 10 mg of a polymer supported peptide bearing an Fmoc group, the 3 micro reactors were added to a round bottom flask with 20.0 mL of the candidate solvent and stirred for 30 min for resin swelling. After the swelling was time was completed then the 3 micro reactors were transferred to a round bottom flask with 20 mL a 20% v/v solution of 4-methyl-piperidine* prepared in the candidate solvent, after 5 min one micro reactor was taken out from the solution and immediately washed with pure solvent candidate and then washed with DCM, the same procedure was applied to the two remaining micro reactors at 10 and 30 min respectively.


22

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The washed resin samples were treated with 0.5 mL of a 3.0 % v/v solution of TFA in DCM, from the cleavage solution an aliquot of 100 µL were diluted with 1.0 mL of acetonitrile and then the amount of unprotected peptide was evaluated by HPLC and LC-MS.

In each

chromatogram just the area of the peptide without the Fmoc and the area of the peptide with the Fmoc protecting group were integrated, and then the reported is the area % of the residual peptide without the Fmoc protecting group. *The use of 4-methylpiperidine is preferred to conventional piperidine due the fact that piperidine is a controlled substance and 4-methylpiperidine is not. The controlled status of piperidine makes its use a complicated task for the development laboratories. SPPS USE TEST A linear octapeptide that is used in the manufacturing process of octreotide was prepared by SPPS. The synthesis was performed in an automatic peptide synthesizer (AAPPTEC® focus XC): The synthesis was started with 3.028 g of amino methyl resin (AMRES) with a loading of 1.3 mmol/g (3.9 mmols). Each Fmoc protected building block (1.5 eq) was coupled in the candidate solvent using the coupling system DICI/HOBt (1.5 eq), after 2.0 h of coupling time the reaction mixture was drained and the suspension was washed with the candidate solvent, the Fmoc protecting group was cleaved and then the resin was washed with the solvent candidate, the full cycle was done at 25 °C.

The same cycle was repeated with each Fmoc protected

building block. After the peptide elongation was complete the resin was washed sequentially with; candidate solvent (3x15mL), isopropanol (3x15mL) and isopropyl ether (3x15mL), then the peptide resin was dried under vacuum at 35°C and the weight of the peptide resin was recorded and used to calculate SPPS yield based on resin weight gain. In order to determine the purity on the crude peptide, a small portion of the dried resin was treated with 3% trifluoroacetic


23


Page 24 of 32

acid in dichloromethane. Those conditions were chosen to cleave the peptide from the solid support while still having the side chain protecting groups and also to avoid other side reactions that are due to the cleavage reaction and not related to the solvent used in the SPPS. Each crude peptide was analyzed by HPLC and LC-MS. CONCLUSIONS After executing a systematic evaluation of properties including: resin swelling, solubility of SPPS related reagents, performance on the Fmoc cleavage reaction, performance in the coupling reaction and preparing a linear octapeptide that is used as intermediate in the manufacture of Octreotide , it was found that: most of the 34 tested solvents were invalidated due to their poor ability to swell polystyrene based resin and/or their poor capacity to dissolve SPPS related reagents. According to the results of the the Fmoc experiments, it was found that the Fmoc cleavage reaction is effective in polar aprotic solvents like DMF, DMI, DMPU TMU, NMP DMSO and showed to be very slow in medium to apolar aprotic solvents like for example Anisole, THF, Me-THF, Toluene, and EtOAc whilst the coupling reaction is preformed faster in medium to apolar aprotic solvents like for example Anisole, THF, Me-THF, Toluene, and EtOAc. One could suggest that an ideal SPPS requires two kinds of solvents: a non-polar aprotic solvent for the coupling reaction like for example Me-THF, THF, EtOAc and a polar aprotic solvent like for example NBP for the Fmoc cleavage reaction. But from the regulatory perspective, a mixed scenario is more complicated to implement in a commercial manufacturing process compared with replacing one single solvent. Additionally the generation of mixed solvent waste makes recycling more complicated and expensive.


24

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According to our results NBP is a solvent that could be used as replacement of DMF as solvent in the large scale SPPS manufacture of Octreotide and other peptides29. NBP is compatible with the most popular polystyrene based resins. For the synthesis of a linear octapeptide it was demonstrated that use of NBP as a solvent produces peptide crudes with similar quality compared to using DMF. This situation facilitates the switch from DMF to NBP for most SPPS processes from the regulatory perspective, as it offers the benefits of direct 1:1 replacement. Most of the impurities found in the synthesis using NBP were deletion sequences. Those deletion sequences are likely due to slower reaction rates of the activated amino acids in NBP. Our SPPS team at Novartis is working on fine tuning reaction conditions to make NBP even more attractive as a replacement of reprotoxic DMF. In this endeavor we are using Design of Experiments (DoE) models to find optimized reaction parameters that could be applied to the large scale production of peptide drugs. The important difference in viscosity (NBP has a viscosity of 4.0 cP (25 °C) and DMF has a viscosity of 0.8 cP (25 °C)) is enhanced in small peptide synthesizers. Although chemically compatible, due to the high viscosity of NBP, the transfers of solution from one point to another in an automatic synthesizer is slower and therefore the definition of cycles has to be adapted to assure full transfers. In large scale equipment, given the larger dimensions, the high viscosity of NBP is not a technical problem. Additionally the high viscosity of NBP could be the part of the explanation for the observed slower coupling rates of NBP compared to DMF. A potential further development work to make NBP even more appropriated solvent for large scale SPPS, could be to

consider the use of heated SPPS, increasing process temperature could have

beneficial effects for SPPS as demonstrated be the academic scientific literature with the use of


25


microwave assisted SPPS30.

Page 26 of 32

Using microwave in large scale is still an important technical

challenge but performing conventionally heated SPPS is feasible. With the increased number of implementations of NBP, and the phasing out, or at least reduction of DMF, it is expected that the demand in NBP will grow, triggering volume effect and reduction of its price.

Although TMU and DMI also gave good results and neither are included in the REACH regulation, both solvents have hazard statements of being reprotoxic and therefore those solvents should not be considered as nontoxic alternatives to replace DMF. From our experimental results, it is clear that NBP is the best candidate to replace DMF as solvent for SPPS. The crude peptide generated by a SPPS performed in NBP has a slightly lower purity (80 %) when compared to a crude generated in DMF (86 %), but the impurity profile is similar. The main impurities found in the crudes of NBP are deletion sequences and the Novartis SPPS team is working on developing optimized SPPS conditions to make NBP the best solvent choice for greener SPPS. ABBREVIATIONS 2-CTCl, 2- chlorotrityl chloride; ACN, acetonitrile; AMRES, aminomethyl resin; Boc, tertbutyloxycarbonyl; CHAD, chemical and analytical development; DEC, diethylcarbonate; DEET, N,N-diethyl-meta-toluamide; DICI, diisopropylcarbodiimide; DIU, diisopropylcarbodiimide; DMA, dimethylacetamide; DMC, dimethylcarbonate; DCM, dichloromethane; DMF, dimethylformamide; DMI, 1,3-dimethyl-2-imidazolidinone; DMM, di(propyleneglycol)dimethylether; DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone; DMSO, dimethylsulfoxide; DoE, design of experiments; DVB, divinylbenzene; ECHA,


26

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european chemicals agency; EtOAc, ethyl acetate; Fmoc, fluorenemethyloxycarbonyl; ICH, international council for harmonisation; MCL, N-methylcaprolactam; MEK, methyl ethyl ketone; Me-THF, 2-methyltetrahydrofurane; MOL, 3-methyl-2-oxazolidinone; NBP, Nbutylpyrrolidinone; NEP, N-ethylpyrrolidinone; NMP, N-methylpyrrolidinone; NMP, Nmethylpyrrolidinone; Oxyma, cyano-hydroxyimino-acetic acid ethyl ester; PC, propylene carbonate; PEG, polyethyleneglycol; PS, polystyrene; REACH, registration evaluation authorization of chemicals; SPPS, solid phase peptide synthesis; TES, N,N,N',N'-tetraethyl sulfamide; THF, tetrahydrofuran; TMU, tetramethyl urea; CONFLICTS OF INTEREST “There are no conflicts to declare”. ACKNOWLEDGEMENTS Special thanks to Dr. Daniel Kaufmann for his support in this project, thanks to development chemist peers for the productive discussions and recommendations in the beginning of the project, to Timothy Woodcock for his patience correcting the manuscript and thank you to the reviewers of the manuscript.


27


Page 28 of 32

AUTHOR INFORMATION Corresponding Author John Lopez*.: [email protected] Phone: +41 79 536 87 09 Novartis Pharma AG, Novartis campus, WSJ-145-5.51, CH-4056, Basel, Switzerland.; REFERENCES

1 Albericio, F.; Kruger H. G. Therapeutic peptides. Future Med. Chem. 2012, 4, 1527-1531. 2 Feng, Y.; Z, Wang, S.; Burgess, K. SNAr Cyclizations to form cyclic peptidomimetics of βturns. J. Am. Chem. Soc. 1998, 120, 10768-769. 3 Zompra, A.; Galanis, A.; Werbitzky, O.; Albericio, F. Manufacturing peptides as active pharmaceutical ingredients. Future Med. Chem. 2009, 1, 361-377. 4 Merrifield, B. Solid phase peptide synthesis. I The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149–2154. 5 Synthesis of Peptides and Peptidomimetics:

Methods of Organic Synthesis:

ed. M.

Goodman, A. Felix, L. Moroder and C. Toniolo, Thieme, Stuttgart, 2004, vol. E22a. 6 Flögel, O.; Codée,J.; Seebach, D.; Seeberger,P. Microreactor synthesis of β-peptides. Angew. Chem., Int. Ed. 2006, 45, 7000-7003.


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Page 29 of 32 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


7 Simon, D.; Heider, P.; Adamo, A.; Vinogradov, A.; Mong, S.; Li, X.; Berger,T.; Policarpo, R.; Zhang, C.; Zou,Y.; Liao, X.; Spokoyny, A.; Jensen,K.; Pentelute, B. Rapid Flow-Based peptide sythesis. Chem. Bio. Chem. 2014, 15, 713-720. 8 Bergkamp, L.; Herbatschek, N. Regulating Chemical Substances under Reach: The choice between Authorization and Restriction and the case of Dipolar Aprotic Solvents. Rev. Euro. Comp. Int. Env. Law, 2014, 23, 221-245. 9 MacMillan, D.;

Murray, J.; Sneddon, H.;

Jamieson, C.; Watson, A. Evaluation of

alternative solvents in common amide coupling reactions: replacement of Dichloromethane and N,N-dimethylformamide. Green Chem. 2013, 15, 596-600. 10 Sherwood, J.; Parker, H.; Moonen, K.; Farmer, T.; Hunt, A. N-Butylpyrrolidinone as a dipolar aprotic solvent for organic synthesis. Green Chem. 2016, 18, 3990-3996.

11 Jad, Y.; Acosta, G.; Govender, T.; Kruger, H.; El-Faham, A.; de la Torre, B.; Albericio, F. Grenn Solid-Phase Peptide Synthesis 2. 2-Methyltetrahydrofuran and Ethyl acetate for solid phase under Green Conditions. ACS Sustainable. Chem. Eng. 2016, 4, 6809-6814. 12 Kumar, A.; Jad, Y.; El-Faham, A.; de la Torre, B.; Albericio, F. Green solid-phase peptide synthesis 4. γ-Valerolactone and N-formylmorpholine as green solvents for solid phase peptide synthesis. Tetrahedron Lett. 2017, 58, 2986-2988. 13 Lawrenson, S.; Arav, R.; North, M. The greening of peptide synthesis. Green Chem, 2017, 19, 1685-1691.


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14 a) Hernandez, J.; Ardila-Fierro, K.; Crawford, D.; James, S.; Bolm, S. Mechanoenzymatic peptide and amide bond formation. Green Chem, 2017, 19, 2620-2025; b) Bonnamour,J.; Metro,T.; Martinez, J.; Lamaty, F. Enviromentally benign peptide synthesis using liquid-assisted ball-milling: application to the synthesis of Leu-enkephalin. Green Chem, 2013, 15, 1116-1120.

15 a) Hojo, K.; Maeda, M.; Tanakamaru, N.; Mochida, K.; Kawasaki, K. Solid phase peptide synthesis in water. Protein Pept. Lett. 2006, 13, 189-192; b) Wang, Q.; Wang, Y.; Kurosu. M. A new Oxyma derivative for nonracemizable amide-forming reactions in water. Org Lett. 2012, 14, 3372-3375.

16 Galanis, A.; Albericio, F.; Grotli, M. Solid-Phase peptide synthesis using microwave assisted heating. Org Lett, 2009, 20, 4488-4491. 17 a) Schellenberger, V.; Jakubke. H. Protease-Catalized kinetically controlled peptide synthesis. Angew. Chem. Int. Ed. Engl. 1991, 30, 1437-1449; b) Cortes-Clerget, M.; Berthon,J.; Krolikiewicz-Renimel, I.; Chaisemartin, L.; Lipshutz, B. Green Chem, 2017,19, 4263-4267 18 Katznelson, L.; Atkinson, J.; Cook, D.; Ezzat, S.; Hamrahian, A.; Miller, K. Medical guidelines for clinical practice for the diagnosis and treatment of Acromegaly.

Endocr Pract,

2011; 17(Suppl 4). 19 Solid-Phase Synthesis and Combinatorial Technologies; P. Seneci; Wiley-Interscience , 2000, 4-38.


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20 García-Ramos, Y.;

Paradís-Bas, M.; Tulla-Puche, J.; Albericio, F. ChemMatrix for

complex peptides and combinatorial chemistry. J. Pept. Science, 2010, 16, 675-678. 21 Santini, R.; Griffith, M.; Qi, M. A measure of solvent effects on swelling of resins for solid phase peptide synthesis. Tetrahedron Lett. 1998, 39, 8951-8954. 22 Lawrenson, S.; North, M.; Peigneguy, F.; Routledge, A. Greener solvents for solid phase synthesis. Green Chem, 2017, 19, 952-962. 23 Prat, D.; Hayler, J.; Wells, A. A survey of solvent selection guides. Green Chemistry, 2014, 16, 4546-4551.

24 Ralhan, K.; KrishnaKumar, V.; Gupta, S. Piperazine and DBU: a safer alternative for rapid and efficient Fmoc deprotection in solid phase peptide synthesis. RSC Adv. 2015, 5, 104417-104425.

25 Jad, Y.; Govender, T.; Kruger, H.; El-Faham, A.; de la torre, B.; Albericio, F. Green Solid-Phase Peptide Synthesis (GSPPS) 3. Green Solvents for Fmoc Removal in Peptide Chemistry. Org. Process. Res. Dev. 2017, 21, 365-369.

26 Jad, Y.; Acosta, G.; Khattab, S.; de la Torre, B.; Govender, T.; Kruger, H.; El-Faham, A.; Albericio, F. 2-methyltetrahydrofuran and cyclopentyl methyl ether for green solid phase peptide synthesis. Amino acids, 2016, 48, 419-426.


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27 Jad, Y.; Acosta, G.; Khattab, S.; de la Torre, B.; Govender, T.; Kruger, H.; El-Faham, A.; Albericio, F. Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives. Org. Biomol. Chem, 2015, 13, 2393-2398. 28

The use test that is disclosed in this investigation work was done first at the 5.6 g (3.9 mmol) and then the best results were repeated successfully at preparative scale (data not disclosed) of 36 g (25 mmol) at that scale the setup of the synthesizer was easier since due to the larger tubbing diameter the liquid transfers were easier. 29 After the successful demonstration that NBP can be used as solvent in the SPPS manufacturing process of Octreotide, the solvent was tested with the synthesis of other peptides, (results not published), confirming the same good results as found for Octreotide, with one exception, during the coupling of Fmoc-Arg(Pbf)-OH, it was found that in order to achieve a complete coupling it was necessary double and in some cases triple couplings, it was rationalized the inefficient Arg coupling could be explained by Lactamization of Arg during its activation in NBP, according to NMR studies this side reaction also occurs in DMF but since the coupling is faster in DMF, thenit is possible to have complete couplings with singles or double couplings. The lactamization problem is well known in the scientific literature but is not fully solved. 30 Yu H-M.; Chen, S-T.; Wang K-T. Enhanced coupling efficiency in solid phase peptide synthesis by microwave irradiation. J. Org. Chem.1992; 57: 4781–4784. .


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N-Butylpyrrolidinone as Alternative Solvent for Solid-Phase Peptide

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