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Green Solid-Phase Peptide Synthesis (GSPPS)-III. Green solvents for Fmoc removal in peptide chemistry Yahya E Jad, Thavendran Govender, Hendrik G. Kruger, Ayman El-Faham, Beatriz G de la Torre, and Fernando Albericio Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00439 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on January 30, 2017

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Organic Process Research & Development

Green Solid-Phase Peptide Synthesis (GSPPS)III. Green solvents for Fmoc removal in peptide chemistry Yahya E. Jad,a Thavendran Govender,a Hendrik G. Kruger,a Ayman El-Faham

b,c

Beatriz G. de

la Torre,a,d* and Fernando Albericioa,b,e,f,g* a

Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South Africa. b

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

c

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

School of Laboratory of Medicine and Medical Sciences, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South Africa.

e

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

ACS Paragon Plus Environment

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f

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

g

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

KEYWORDS. Solid-phase peptide synthesis, Fmoc, protecting groups, green solvents, γ-valerolactone, peptide aggregation.

ABSTRACT.

DMF (N,N-dimethylformamide) is the most widely used solvent for Fmoc-SPPS, which is the conventional method of choice for peptide synthesis. It is usually employed for coupling, washing and Fmoc-removal steps. We have recently reported greener solvents such as 2-MeTHF (2-methyltetrahydrofuran) and CPME (cyclopentyl methyl ether) as environmentally friendlier alternatives to DMF. However, the Fmoc-removal step with these green solvents was not optimal. Thus, deletion peptides and Nα-Fmoc terminal peptides were observed upon TFA (trifluoroacetic acid) cleavage of the peptide from the resin during SPPS of a challenging aggregated peptide. Herein, Fmoc removal using a solution of 20% piperidine in a range of green solvents is reported. After evaluation of the resin’s swelling (PS and ChemMatrix resins), and Fmoc deprotection in solution and on solid-phase for these two resins, it was found that γvalerolactone can replace DMF for Fmoc removal steps during SPPS on both resins, PS or ChemMatrix. Furthermore, N-formylmorpholine showed an excellent performance with ChemMatrix resin.


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Introduction A successful peptide synthesis completely depends on a suitable protecting group scheme together with the most convenient coupling reagent. Peptides are synthesized by repetitive coupling and deprotection steps.1-2 Although it is common thought that the coupling step is crucial for successful peptide synthesis, incomplete deprotection step(s) will also jeopardize the synthesis.3 In solid-phase peptide synthesis (SPPS), peptides are prepared following either the N → C or C → N directions, the first approach is not commonly used because it is usually associated with side reactions such as the formation of diketopiperizines or oxazolones.4-6 Therefore, most peptides are synthesized in the C → N direction, where temporary Nα protecting groups are normally removed several times during the synthesis while the C-terminal is permanently “protected” via its attachment to the resin. Although many temporary protecting groups were reported and utilized for masking the α-amino functionality,2 the Fmoc group is the most widely employed in SPPS and also finds some applications in solution chemistry.7 It was firstly introduced by Carpino in the early 1970s8-9 and has been routinely applied for SPPS since 1978.10-11 It displays exquisite chemical properties in that it can be removed under mild basic conditions, while being stable against the primary amino component that is to be acylated, during the coupling step. Although it can also be removed by Lewis acid such as AlCl312-13 or catalytic hydrogenation,14 but the mostly used approach is solutions of secondary amines such as piperidine, piperazine, or morpholine in polar aprotic solvents as the removal cocktail. The most common method involves 20% piperidine in DMF for a few minutes.15 Moreover, replacement of piperidine with 4-methylpiperidine was also reported and revealed similar efficiencies than piperidine but it has found only a few applications,16 possibly due to financial considerations.


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Piperazine is another six membered cyclic secondary amine and it shows a maximum solubility of 6% in DMF or NMP (N-methyl-2-pyrrolidone). Therefore, 10% piperazine in a mixture of EtOH (ethanol) and NMP (10:90) was reported and utilized in SPPS.17 The kinetics of Fmoc removal in 5% piperazine + 2% DBU (1,8-diazabicycloundec-7-ene) in DMF were recently evaluated and it exhibited faster reaction than 10% piperazine in EtOH/NMP (10:90) and 20% piperidine in DMF, mainly due to the presence of DBU, which is more basic than piperidine (pKa of piperidine 11.12 vs pKa of DBU 13.5).18 Solvents are the major component of the deprotection cocktail, as they represent at least 80% of the deprotection solution. This estimation is in accordance with a survey accomplished by GSK (GlaxoSmithKline) in 2007 about the percentage of solvent used during manufacturing of API (Active Pharmaceutical Ingredients) wherein it represented 80-90% of the non-aqueous masses.19-20 In this regards, it is important to take into account that DMF and NMP are the most widely used solvents but are listed on the solvent substitution request list since they are hazardous chemicals.20-23 Most importantly, the use of these two solvents is likely to be restricted by REACH (Registration, Evaluation, Authorization and Restriction of Chemicals)24 since they are listed as SVHC (substances of very high concern).25 Our group have also used MeCN (acetonitrile), which is also not a green solvent22 but is classified as a “recommended alternative to DMF” in some solvent guides,20-21, 23 for Fmoc removal in SPPS with the ChemMatrix resin.26 Our research was expanded to include THF, 2-MeTHF and CPME, with the latter two classified as green solvents,27-28 for solution synthesis and SPPS.29-30 These solvents were only used during the coupling step while DMF was still employed during the deprotection and washings steps. Very recently while we were attempting the establishment of a global green solvent SPPS technology, the difficulties and


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limitations associated with 2-MeTHF as solvent for the piperidine cocktail were clearly evident.31 These previous results have confirmed the key role of Fmoc removal in assuring good yields and purities of the final peptides. With this in mind, an extensive study of 20% piperidine in various solvents for Fmoc removal in solution and in SPPS have been performed. Results and discussion The following green solvents20 were considered: N-formylmorpholine, 2-methyltetrahydrofuran, cyclopentyl methyl ether, isosorbide dimethyl ether, EtOAc (ethyl acetate), dimethyl carbonate, γ-valerolactone, IPA (isopropanol) and α,α,α-trifluorotoluene. The performance of these solvents during Fmoc deprotection was compared to DMF, which is mostly used for Fmoc removal. The swelling of the PS (polystyrene) and ChemMatrix resins, which are commonly used, were evaluated for these solvents since this plays a critical role in SPPS. Thus, a good swelling facilitates the accessibility of the reagents to the functional group in the growing chain.32 To best of our knowledge, DMF is the most commonly used solvent in SPPS because its good ability to swell resins very well.33 The results in Table 1 shows that all solvents, except CPME and IPA, gave acceptable swelling for the ChemMatrix resin (> 5 mL/g). Regarding the PS resin, isosorbide dimethyl ether and γ-valerolactone resulted in similar swelling than the conventional solvent DMF (> 5 mL/g). A second block of solvents, 2-MeTHF, CPME, EtOAc, dimethyl carbonate and α,α,α-trifluorotoluene resulted in less swelling of the PS resin (but > 3 mL/g). Finally, N-formylmorpholine, and IPA exhibited the worst resin swelling performance.


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Table 1. Swelling of PS and ChemMatrix resins in various solvents Swelling (mL/g)b Solvent a

PS

ChemMatrix

DMF

5.25

8.25

N-Formylmorpholine

2.75

8.25

2-MeTHF

3.75

5.25

CPME

3.75

3.75

Isosorbide dimethyl ether

5.25

7.75

EtOAc

3.25

5.50

Dimethyl carbonate

3.75

6.75

γ-Valerolactone

5

8.25

IPA

2.75

3.75

α,α,α-Trifluorotoluene

3.25

6.25

a

Green: green solvent; Red: non-green solvent: b Green: good swelling, yellow: moderate, and red: low.

Removal of the Fmoc group was investigated both in solution and on solid-phase (PS and ChemMatrix resins). In this work, the merit of solution mode is only to evaluate the effect of solvents on Fmoc removal by avoiding other factors such as the swelling of the resins and the peptide aggregation. Therefore, a simple amino acid Fmoc-Phe-OH (0.1 mmol) was dissolved in 800 µL of the corresponding solvent and 100 µL of toluene as an internal standard. Then, 200 µL


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of piperidine was added and the reaction was terminated at different times [0 min (before adding the piperidine), 2, 4, 6, 8, 10, 15, 20, 25 and 30 min]. To best mimic a “realistic” solid-phase synthesis case, a Fmoc-peptide bonded to a resin which is known to undergo aggregation was chosen to study the deprotection. Our group has previously identified that during the synthesis of the aggregation prone model peptide ACP (65-74),34 the intermediate Fmoc-Ile-Asp(tBu)-Tyr(tBu)-Ile-Asn(Trt)-Gly-NH-RinkAmide-PS-resin required longer reaction times (∼15 min, 20% piperidine in 2-MeTHF) for deprotection.27 The approach followed is depicted in Scheme 1. H-Asp(tBu)-Tyr(tBu)-Ile-Asn(Trt)-Gly-NHRinkAmide-resin was prepared using PS and ChemMatrix resins and standard Fmoc-SPPS method.30 DMF was used during the synthesis because it is the most convenient solvent so far. Fmoc-Ile-OH was attached and then treated with 20% piperidine solution in the corresponding solvent for 45 s. This reduced time was chosen to enable differentiation based on the kinetics of the deprotection reaction in each solvent whilst taking into account that in solid phase, due to the use of larger excess of the deprotection reagent, reactions are generally faster.31


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Scheme 1. Fmoc removal study on solid-phase synthesis.


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Then, Boc-Gly-OH was coupled to the deprotected amino group, followed by complete removal of intact Fmoc-groups using the most potent solution, i.e 20% piperidine in DMF for 10 min.35 Then, Boc-Leu-OH was coupled to those amino groups that were deprotected during the second piperidine treatment. Upon simultaneous Boc deprotection and cleavage of the two peptides from the resin, the crude mixture was analyzed with HPLC. The ratio H-Gly-Ile-Asp-Tyr-Ile-Asn-GlyNH2 vs H-Leu-Ile-Asp-Tyr-Ile-Asn-Gly-NH2 provides information about the performance of the solvent during the first deprotection step where more Gly peptide indicates faster and more effective Fmoc removal (Scheme 1). Table 2. Fmoc removal in solution and SPPS by using 20% piperidine solution in different solvents.a Fmoc removal Solid Phase (%)d

solution Solventb

t (min)c

PS

ChemMatrix

DMF

2

97.7

65.5

N-Formylmorpholine

4

29.3

66.9

2-MeTHF

10

13.2

15.9

CPME

10

4.9

26.3

Isosorbide dimethyl ether

15

2.3

47.3

EtOAc

15

33.9

9.2

Dimethyl carbonate

15

30.4

4.6

γ-Valerolactone

4

43.3

89.1

IPA

> 30 mine

1

3.2


6

12.1

31.1

a

Green: Good performance, yellow: moderate, and red: low, b Green: green solvent; Red: nongreen solvent. c Time required to complete the reaction in solution experiment, d Yield is


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calculated by the ration of H-Gly-Ile-Asp-Tyr-Ile-Asn-Gly-NH2 vs H-Leu-Ile-Asp-Tyr-Ile-AsnGly-NH2, e 16.6% of starting materials after 30 min stirring. After evaluation of the solvent impact on Fmoc removal in solution and solid-phase conditions, the following conclusions can be drawn (Table 2):

•

In solution where effect of resin’s swelling and peptide aggregation are avoided, excellent results were also obtained with N-formylmorpholine, γ-valerolactone, and α,α,α-trifluorotoluene in addition to the non-green solvent DMF. This is in agreement with the E1CB mechanism of the reaction. Thus, in IPA, which has higher dielectric constant than N-formylmorpholine (19.9 vs 15.1, respectively), and lower than DMF, and valerolactone (38.0 and 36.5, respectively)36-37,

38

the reaction was slow. This can be

interpreted because IPA is a polar protic solvent. The rest of the esters and ethers revealed slower removal kinetics.

•

In solid-phase, DMF was overall the best solvent, while γ-valerolactone also gave excellent results for both resins.

•

PS resin performed worse than ChemMatrix in all solvents which can be interpreted by the swelling nature of the ChemMatrix resin in comparison to the PS one, except for DMF, EtOAC and dimethylcarbonate.

With respect to solvent-type, the following observations can be drawn:

•

N-Formylmorpholine

demonstrated

excellent

performance

in

solution.

Poorer

performance of this solvent with PS can be attributed to its low resin swelling capacity. However, N-Formylmorpholine performed better with the ChemMatrix resin due to the good swelling features of this resin in this solvent.


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•

For the ether solvents, 2-MeTHF displayed superior performance in both solution and solid-phase with the PS resin. However, CPME and isosorbide dimethyl ether showed better performance for the ChemMatrix resin, especially isosorbide dimethyl ether also due to better swelling properties when compared to 2-MeTHF. Overall, 2-MeTHF showed a promising performance in peptide synthesis corroborating the results described by MacMillan et al.39 and our group.30

•

In case of ester solvents, γ-valerolactone showed superior results in all parameters: solution, and solid-phase for both resins. Although, PS swelled less in EtOAc and dimethylcarbonate than the ChemMatrix resin, faster reactions were observed for PS, which can be explained by the peptiyl-resin solvation effect.33 In fact, ester solvents showed better performance than the others green solvents with PS.

•

IPA showed the worst result both in solution and solid-phase. That agrees with other results obtained from our laboratory where we have replaced DMF with IPA.27

•

α,α,α-Trifluorotoluene displayed good performance for solution and SPPS for the ChemMatrix resin only.

N-Formylmorpholine, γ-valerolactone, and α,α,α-trifluorotoluene were evaluated for complete Fmoc removal to be sure that these green solvents can replace DMF in a general way. Therefore, a similar experiment as described in Scheme 1 was performed but with 7 min Fmoc removal time instead of 45 s. DMF and 2-MeTHF were also included for comparison purpose, because DMF is the mostly used solvent in this type of reactions while the green 2-MeTHF was recently employed for Fmoc removal reactions.28


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γ-Valerolactone exhibited an excellent performance in the same range of DMF with both resins. N-Formylmorpholine showed an excellent performance with ChemMatrix resin only. and 2MeTHF and α,α,α-trifluorotoluene showed moderate to low performance with both resins (Table 3). Table 3. Fmoc removal in SPPS by using 20% piperidine/solvent after 7 minutes. a Fmoc removal (%) Solvent b

PS

ChemMatrix

DMF

100

100

N-Formylmorpholine

47.9

96.3

2-MeTHF

27.5

29.5

γ-Valerolactone

95.5

100


47.7

58.6

a

Green: Good performance, yellow: moderate, and red: low, b Green: green solvent; Red: nongreen solvent.

Conclusions This work has exemplified that the removal of the Fmoc group from the Nα-amino function in both solution and solid-phase modes is more demanding than commonly thought and is very much solvent depending. In solution and only taking into account green solvents, the removal of the Fmoc group from a simple amino acid such as Phe can take place in 4 min when N-formyl morpholine and γvalerolactone are used. Another convenient medium for the removal of Fmoc in 6 min is α,α,αtrifluorotoluene.


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In solid-phase synthesis, overall ChemMatrix gave better results than PS. Using PS resin, γvalerolactone, N-formylmorpholine, and in this case EtOAc and dimethyl carbonate provided the best results, while the ether solvents showed to be totally incompatible with this resin. For the ChemMatrix resin, N-formylmorpholine and γ-valerolactone, followed by α,α,αtrifluorotoluene are the most efficient solvents. Although CPME did not give a good result for the coupling step,30 this solvent and isosorbide dimethyl ether rendered good results for the Fmoc removal. The linear ester based solvents ethyl acetate and dimethyl carbonate gave disappointing results. The overall conclusion with respect to SPPS is that γ-valerolactone could be an excellent green alternative to DMF for both resins while N-formylmorpholine is compatible with ChemMatrix resin. N-Formylmorpholine has the drawback of its price and that its stability should be demonstrated, because the presence of the formyl moiety always represents a potential drawback in peptide synthesis. Although, EtOAc and dimethylcarbonate were used by MacMillan et al.39 for amide bond formation reactions and no side-reactions were reported, the use of esters solvents (γ-valerolactone, ethyl acetate, and dimethyl carbonate) should be further investigated to assure that transesterication or methyl carbamate formation will not jeopardize its use in peptide chemistry especially when we take into our consideration the recent report by Wong et al.40 about the stability of γ-valerolactone. Although γ-valerolactone has been recently used as a green solvent for acidic hydrolysis of carbohydrates into 5-(hydroxymethyl)furfural, levulinic acid and formic acid. In overall, it has been demonstrated that green solvents can be an excellent substitution for conventional solvents in peptide synthesis.


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ASSOCIATED CONTENT Electronic Supplementary Information (ESI) available: [Experimental details and HPLC chromatograms] and these materials are available free of charge via the Internet at http://pubs.acs.org.”

AUTHOR INFORMATION Corresponding Author * B. G. de la Torre. E-mail: [email protected]. * F. Albericio. E-mail: [email protected] or [email protected].

Present Addresses †If an author’s address is different than the one given in the affiliation line, this information may be included here. Author Contributions 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) ACKNOWLEDGMENT


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This work was funded in part by the National Research Foundation (NRF), the University of KwaZulu-Natal (South Africa), and the Generalitat de Catalunya (2014 SGR 137) and MEC (CTQ2015-67870-P). Additionally, the authors thank the Deanship of Scientific Research at King Saud University for funding this work through the Prolific Research Group Program (PRG1437-33; Saudi Arabia). Finally, the authors thank Y. Luxembourg (Luxembourg Bio Technologies Ltd) for continuous support of this study. ABBREVIATIONS API,

Active

Pharmaceutical

Ingredients;

COMU,

1-[(1-(cyano-2-ethoxy-2-

oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexafluorophosphate; Fmoc, 9-fluorenylmethyloxycarbonyl; GSK, GlaxoSmithKline; REACH, Registration, Evaluation, Authorization and Restriction of Chemicals; SPPS, solid-phase peptide synthesis; SVHC, substances of very high concern. REFERENCES

1.

El-Faham, A.; Albericio, F., Peptide coupling reagents, more than a letter soup. Chem.

Rev. 2011, 111 (11), 6557-602. 2.

Isidro-Llobet, A.; Alvarez, M.; Albericio, F., Amino acid-protecting groups. Chem. Rev.

2009, 109 (6), 2455-504. 3.

Larsen, B. D.; Holm, A., Incomplete Fmoc deprotection in solid-phase synthesis of

peptides. Int. J. Pept. Protein Res. 1994, 43 (1), 1-9.


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Thieriet, N.; Guibé, F.; Albericio, F., Solid-Phase Peptide Synthesis in the Reverse (N →

C) Direction. Org. Lett. 2000, 2 (13), 1815-1817. 5.

Henkel, B.; Zhang, L.; Bayer, E., Investigations on Solid-Phase Peptide Synthesis in N-

to-C Direction (Inverse Synthesis). Liebigs Ann. 1997, 1997 (10), 2161-2168. 6.

De Marco, R.; Spinella, M.; De Lorenzo, A.; Leggio, A.; Liguori, A., C → N and N → C

solution phase peptide synthesis using the N-acyl 4-nitrobenzenesulfonamide as protection of the carboxylic function. Org. Biomol. Chem. 2013, 11 (23), 3786-3796. 7.

Behrendt, R.; White, P.; Offer, J., Advances in Fmoc solid-phase peptide synthesis. J.

Pept. Sci. 2016, 22 (1), 4-27. 8.

Carpino, L. A.; Han, G. Y., 9-Fluorenylmethoxycarbonyl function, a new base-sensitive

amino-protecting group. J. Am. Chem. Soc. 1970, 92 (19), 5748-5749. 9.

Carpino, L. A.; Han, G. Y., 9-Fluorenylmethoxycarbonyl amino-protecting group. J. Org.

Chem. 1972, 37 (22), 3404-3409. 10. Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J., A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonylamino-acids. J. Chem. Soc., Chem. Commun. 1978, (13), 537-539. 11. Chang, C.-D.; Meienhofer, J., Solid-phase peptide synthesis using mild base cleavage of Nαfluorenykmethyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int. J. Pept. Protein Res. 1978, 11 (3), 246-249.


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12. Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G., New Strategies for an Efficient Removal of the 9-Fluorenylmethoxycarbonyl (Fmoc) Protecting Group in the Peptide Synthesis. Eur. J. Org. Chem. 2000, 2000 (4), 573-575. 13. Di Gioia, Maria L.; Leggio, A.; Le Pera, A.; Liguori, A.; Perri, F.; Siciliano, C., Alternative and Chemoselective Deprotection of the α-Amino and Carboxy Functions of NFmoc-Amino Acid and N-Fmoc-Dipeptide Methyl Esters by Modulation of the Molar Ratio in the AlCl3/N,N-Dimethylaniline Reagent System. Eur. J. Org. Chem. 2004, 2004 (21), 44374441. 14. Maegawa, T.; Fujiwara, Y.; Ikawa, T.; Hisashi, H.; Monguchi, Y.; Sajiki, H., Novel deprotection method of Fmoc group under neutral hydrogenation conditions. Amino Acids 2009, 36 (3), 493-499. 15. Fields, G. B., Methods for Removing the Fmoc Group. In Peptide Synthesis Protocols, Pennington, M. W.; Dunn, B. M., Eds. Humana Press: Totowa, NJ, 1995; pp 17-27. 16. Hachmann, J.; Lebl, M., Alternative to Piperidine in Fmoc Solid-Phase Synthesis. J. Comb. Chem. 2006, 8 (2), 149-149. 17. Collins, J. M.; Porter, K. A.; Singh, S. K.; Vanier, G. S., High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS). Org. Lett. 2014, 16 (3), 940-943. 18. Ralhan, K.; KrishnaKumar, V. G.; Gupta, S., Piperazine and DBU: a safer alternative for rapid and efficient Fmoc deprotection in solid phase peptide synthesis. RSC Advances 2015, 5 (126), 104417-104425.


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Green Solid-Phase Peptide Synthesis (GSPPS) 3 ... - ACS Publications

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