Green solid-phase peptide synthesis 4. γ-Valerolactone and N-formylmorpholine as green solvents for solid phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2017.06.058

Herein, we report the use of γ-valerolactone (GVL) and N-formylmorpholine (NFM) as DMF substitutes in polystyrene based SPPS. The solubility of selected amino acids and coupling reagents were studied in GVL and NFM, followed by their use in the successful synthesis of Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH₂) and Aib-ACP decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂).

Full text of DOI:10.1016/j.tetlet.2017.06.058

Accepted Manuscript
Green Solid-Phase Peptide Synthesis 4. γ-Valerolactone and N-Formylmorpho-
line as Green Solvents for Solid Phase Peptide Synthesis
Ashish Kumar, Yahya E. Jad, Ayman El-Faham, Beatriz G. de la Torre,
Fernando Albericio
PII:
S0040-4039(17)30789-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.06.058
TETL 49050
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 May 2017
15 June 2017
19 June 2017
Please cite this article as: Kumar, A., Jad, Y.E., El-Faham, A., de la Torre, B.G., Albericio, F., Green Solid-Phase
Peptide Synthesis 4. γ-Valerolactone and N-Formylmorpholine as Green Solvents for Solid Phase Peptide Synthesis,
Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.06.058
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Green Solid-Phase Peptide Synthesis 4. γ-
Valerolactone and N-Formylmorpholine as
Green Solvents for Solid Phase Peptide
Synthesis
Leave this area blank for abstract info.
Ashish Kumar, Yahya E. Jad, Ayman El-Faham, Beatriz G. de la Torre, Fernando Albericio
Green Solid-Phase Peptide
Decapeptide
Synthesis of Aib-ACP
GVL
(Previous work)
Polystyrene
resin
2-MeTHF
Decapeptide
Decapeptide
NFM

1
Tetrahedron Letters
journal homepage: www.els evier.com
Green Solid-Phase Peptide Synthesis 4. γ-Valerolactone and N-Formylmorpholine as
Green Solvents for Solid Phase Peptide Synthesis
Ashish Kumara,b, Yahya E. Jadb, Ayman El-Fahamc,d, Beatriz G. de la Torre*b,e, Fernando Albericio*a,b,c,f,g
a School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa
b School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
c Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
d Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, 12321 Alexandria, Egypt
e School of Laboratory of Medicine and Medical Sciences, University of KwaZulu-Natal, Durban 4001, South Africa
f
Department of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain
g CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona 08028, Spain
*Corresponding Authors: Beatriz G. de la Torre : e-mail: Garciadelatorreb@ukzn.ac.za; Fernando Albericio: phone: 0312603097; e-mail:
Albericio@ukzn.ac.za
ARTICLE INFO
ABSTRACT
Article history:
Received
Herein, we report the use of γ-valerolactone (GVL) and N-formylmorpholine (NFM) as DMF
substitutes in polystyrene based SPPS. The solubility of selected amino acids and coupling
reagents were studied in GVL and NFM, followed by their use in the successful synthesis of
Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH2) and Aib-ACP decapeptide (H-Val-
Gln-Aib-Aib-Ile-Asp-Tyr-Ile-Asn-Gly-NH2).
Received in revised form
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Solid-phase peptide synthesis
Green solvents
Polystyrene resin
GVL
NFM
Over the past decade, there have been a variety of governmental
and industrial efforts to eliminate, replace, recycle or minimize
the use of solvents in pharmaceutical and chemical industries.
This effort has been driven by the desire to reduce human health
impacts, process safety risks, and multiple impacts to the
environment.1, 2 The idea of using ‘‘green’’ solvents is the goal to
minimize the environmental impact resulting from their use in
chemical production, together with the use of bio-solvents
derived from renewable resources. Therefore, substitution of
hazardous solvents with green solvents is better for the
environment as well as human, health and safety.3, 4
Since peptides play a crucial role in pharmaceutical and
medicinal research, their synthesis has been a major focus for
over a century.5 Peptides can be synthesized using two main
strategies: solution phase and solid-phase peptide synthesis
(SPPS).6-8 The protocol for SPPS, as initially developed by
Merrifield,9 is widely based on the use of a solid support, Nα
protected amino acids, and the activation of carboxyl groups
using coupling reagents.5 The SPPS approach currently
dominates peptide synthetic protocols because it allows the use
of excess reactants to secure quantitative yields in each step. The
excess reagents and/or by-products can easily be removed by
filtration and washing steps using an appropriate solvent.10
Therefore, the consumption of solvents is a key issue in SPPS.
The most commonly used solvents are N,N-dimethylformamide
(DMF), dichloromethane (DCM) and N-methylpyrrolidone
(NMP). Several selection guides for green chemistry classify
these as hazardous solvents.11-15 Furthermore, the use of DMF in
the pharmaceutical industry will probably be restricted by the
Regulation for Registration, Evaluation, Authorisation and
Restriction of Chemicals (REACH).16 Therefore, the search for
greener alternatives in SPPS is necessary.7
During the last few years, our group and others have
investigated substitutes for DCM and DMF in SPPS.7,
10, 17
However, swelling of the classical and most used, polystyrene
(PS) resin limits the use of solvents other than DMF, DCM and
NMP. On the other hand, the ChemMatrix resin, which was
reported in 2006 by our group,18 shows better swelling properties
than the PS resin in polar as well as non-polar solvents, and
therefore is compatible with a broader range of solvents. Thus in
2009, our group explored MeCN as an alternate for DMF using
DIPCDI/OxymaPure as coupling reagents during ChemMatrix
SPPS.19 Later, we investigated the use of THF, however, MeCN
and THF are not considered as green solvents.11, 13 In the next
screen of solvents, 2-methyl tetrahydrofuran (2-MeTHF),
cyclopenthyl methyl ether (CPME) and isopropyl alcohol (IPA)
were assayed. While CPME and IPA did not show compatibility
with either of the two resins,7 we proposed a successful green
solid phase peptide synthesis (GSPPS) by employing
DIPCDI/OxymaPure in 2-MeTHF as coupling conditions, 20%
piperidine in 2-MeTHF for Fmoc removal and ChemMatrix resin
as the solid support with additional washing steps using EtOAc.7
The most problematic step in this strategy was Fmoc removal due
to peptide aggregation. An optimized Fmoc removal was

2
Tetrahedron
achieved at a slightly higher temperature (40 °C). This strategy
failed when the PS based resin was used.7
This short sequence containing two Aib residues in a row is
known to increase the possibility of side-product (des-Aib-
pentapeptide)7 formation due to slow reaction as a result of steric
hindrance.30 Therefore, the peptide model Aib-enkephalin
pentapeptide was prepared on the Fmoc-RinkAmide-AM-PS
resin (0.1 g, 0.74 mmol/g) using the synthetic protocols described
in Table 1. Excellent results were obtained using protocol B
where GVL was used during the coupling steps, resulting in
99.2% purity of Aib-pentapeptide with 0.8% of des-Aib-
pentapeptide by HPLC. Although protocol C rendered the
product in lower purity than protocol A (93.1% vs 97.8%, Table
2, entries 1, and 3), it still can be considered as a promising result
when the synthetic difficulty of this peptide is taken into
consideration.
This prompted a parallel study of Fmoc removal using 20%
piperidine employing different green solvents including γ-
valerolactone (GVL), N-formylmorpholine (NFM), dimethyl
carbonate, dimethyl isosorbide ether, EtOAc, and α,α,α-
trifluorotoluene. As a result, it was found that GVL and NFM
showed good results for Fmoc removal with the ChemMatrix
resin and promising results with the PS resin.20
To expand the scope of the GSPPS strategy, herein we report
the use of GVL and NFM with the PS resin. To the best of our
knowledge, this represents the first report employing green
solvents on PS; previous reports from our group and others dealt
with ChemMatrix or PEG-PS resins.7, 10, 17, 21-23
GVL is a naturally available chemical24 and has recently been
described as an excellent green solvent candidate.25 As
demonstrated in detail by Horvath and co-workers,24 GVL is
renewable, has a low mp (-31°C), high bp (207°C), a definitive
but acceptable smell for easy recognition of leaks and spills, non-
toxicity and high solubility in water to assist biodegradation.
Further, it does not hydrolyze under neutral conditions or form
any measurable amount of peroxides in a glass flask kept under
air for weeks, making it safe for large scale use. Due to the high
solubility in water, GVL could be extracted from the reaction
mixture using water and then separated via distillation since it
does not form azeotrope with water.24 Most importantly, GVL
can be efficiently produced from biomass, preferentially from
lignocelluloses.26-28 On the other hand, NFM was reported as an
alternative to the toxic solvent NMP.29
Table 2. HPLC purities of Aib-enkephalin pentapeptide
assembled on the Fmoc-Rink Amide-AM-PS resin. a
Entry
Protocol
Pentapeptide (%)b
des-Aib (%)
1
2
A
B
C
97.8
99.2
93.1
2.1
0.8
3.6
3
a
Same conditions and solvents used for the synthesis as in
b
Table 1. Determined by HPLC using the following conditions:
linear gradient of 20 → 40% 0.1% TFA in a CH3CN/0.1% TFA
mixture in H2O over 15 min, with a flow rate of 1.0 mL/min−1
and detection at 220 nm using a Phenomex C18 (3 µm, 4.6 × 50
mm).
The solubility of reagents can limit the use of solvents in
automatic peptide synthesizers.7 Thus, we first investigated the
solubility of different Fmoc-AA-OH compounds and OxymaPure
in GVL or NFM and compared this with DMF. It was found that
the amino acids (Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aib-OH,
Fmoc-Tyr(tBu)-OH), showed good solubility (> 0.2 M) in GVL
and NFM. The only exception was Fmoc-Phe-OH which showed
Following the above results, the more demanding model
peptide Aib-ACP decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-Tyr-
Ile-Asn-Gly-NH2)7, 17, 31 was synthesized using the PS resin. This
modified version of model peptide ACP(65-74)30 is the most used
peptide for determining the efficiency of new SPPS protocols i.e.,
coupling reagents, resins; heating conditions or, as for this case,
solvent systems. The results in Table 3 show excellent purities
for the two greener syntheses, with protocol C proving the best
(94.4%).
a solubility of 0.16 M in GVL. It has been reported that peptide
synthesizers generally use 0.1 or 0.2 M7,
10
which is in fair
agreement with our results. Furthermore, OxymaPure showed
promising solubility in GVL and NFM (> 0.5 M).
Table 3. HPLC purities of Aib-ACP decapeptide assembled
on the Rink Amide-AM- PS resin. a
Next, we investigated the performance of GVL and NFM in
the key peptide coupling step Three different solvent system
protocols were used as illustrated in Table 1: the standard DMF
based protocol (A); GVL for coupling steps only (B) and NFM
for coupling steps only (C).
Entry
Protocol
Decapeptide (%)b
des-Aib (%)
1
2
A
B
C
93.8
89.7
94.4
6.2
1.4
5.6
Table 1. SPPS protocols used for this study.a
3
a
GVL (only
for coupling)
(B)
NFM (only
for coupling)
(C)
Same conditions and solvents used for the synthesis as in
b
Table 1. Determined by HPLC using the following conditions:
Standard
(A)
linear gradient of 10 → 50% 0.1% TFA in a CH3CN/0.1% TFA
mixture in H2O over 15 min, with a flow rate of 1.0 mL/min−1
and detection at 220 nm using a Phenomex C18 (3 µm, 4.6 × 50
mm).
2×DMF
2×DCM
2×DMF
2×DMF
2×DCM
2×DMF
2×DMF
2×DCM
2×DMF
Washing
Deprotection
Washing
20% piperidine/DMF 7 min
The results obtained by performing coupling reactions with
GVL or NFM encouraged us to completely avoid DMF and
replace it by either GVL or NFM during washing and Fmoc
removal steps. The peptide model Aib-ACP-decapeptide was
manually assembled on the Fmoc-RinkAmide-AM-PS resin to
evaluate these two protocols (Table 4). The HPLC purities of
Aib-ACP decapeptide with GVL (protocol D) and NFM
(protocol E) were almost identical (Table 5, entries 1 and 2,), but
lower than that of protocol B and C (Table 3, entries 2, 3).
However, the overall conclusion from this experiment is both
GVL and NFM rendered this difficult peptide in purities higher
than when 2-MeTHF and EtOAc (Table 5, entry 3) was
2×DMF
2×DCM
2×DMF
2×DMF
2×DCM
2×GVL
2×DMF
2×DCM
2×NFM
Fmoc-AA-OH/DIC/OxymaPure (3 equiv) for 1 h
Coupling
DMF
GVL
NFM
Washing
2×DMF
2×GVL
2×NFM
a
Red indicates hazardous solvents, while green indicates
green solvents.
The efficiency of GVL and NFM for SPPS was evaluated by
synthesizing the relatively difficult model peptide Aib-enkephlin
pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH2) on the PS resin.17

3
2 Pollet, P.; Davey, E. A.; Ureña-Benavides, E. E.; Eckert, C. A.;
Liotta, C. L. Green Chem. 2014, 16, 1034.
employed for the synthesis of the same peptide with the PS-
resin.7
3
Capello, C.; Fischer, U.; Hungerbühler, K. Green Chem. 2007, 9,
Byrne, F. P.; Jin, S.; Paggiola, G.; Petchey, T. H.; Clark, J. H.;
927.
4
Table 4. Green SPPS protocols used for this study.
GVL in all steps
(D)
NFM in all steps
(E)
Farmer, T. J.; Hunt, A. J.; McElroy, C. R.; Sherwood, J. Sustain. Chem.
Process. 2016, 4, 1.
5
6
7
Coin, I.; Beyermann, M.; Bienert, M. Nat. Protoc. 2007, 2, 3247.
Albericio, F.; Kruger, H. G. Future Med. Chem. 2012, 4, 1527.
Jad, Y. E.; Acosta, G. A.; Govender, T.; Kruger, H. G.; El-Faham,
Washing
2×GVL
2×NFM
20% piperidine/GVL
1×1 min + 1 ×7 min
20% piperidine/NFM
1×1 min + 1 ×7 min
Deprotection
A.; de la Torre, B. G.; Albericio, F. ACS Sustainable Chemistry &
Engineering 2016, 4, 6809.
Washing
Coupling
3×GVL
3×NFM
8
Albericio, F. Curr. Opin. Chem. Biol. 2004, 8, 211.
Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
Jad, Y. E.; Acosta, G. A.; Khattab, S. N.; Beatriz, G.; Govender,
Fmoc-AA-OH/DIC/OxymaPure (3 equiv) for 1 h
GVL NFM
9
10
T.; Kruger, H. G.; El-Faham, A.; Albericio, F. Amino Acids 2016, 48, 419.
11
12
Prat, D.; Hayler, J.; Wells, A. Green Chem. 2014, 16, 4546.
Alfonsi, K.; Colberg, J.; Dunn, P. J.; Fevig, T.; Jennings, S.;
Table 5. HPLC purities of Aib ACP- decapeptide in green
solvent protocols.a
Entry
Johnson, T. A.; Kleine, H. P.; Knight, C.; Nagy, M. A.; Perry, D. A. Green
Chem. 2008, 10, 31.
Protocol
Decapeptide (%)b
52.0
13
Henderson, R. K.; Jiménez-González, C.; Constable, D. J.; Alston,
1
2
GVL in all steps
S. R.; Inglis, G. G.; Fisher, G.; Sherwood, J.; Binks, S. P.; Curzons, A. D.
Green Chem. 2011, 13, 854.
NFM in all steps
54.0
25.0c
14
Prat, D.; Pardigon, O.; Flemming, H.-W.; Letestu, S.; Ducandas,
3
2-Me-THF in all steps
V. r.; Isnard, P.; Guntrum, E.; Senac, T.; Ruisseau, S. p.; Cruciani, P. Org.
Process Res. Dev. 2013, 17, 1517.
a
Same conditions and solvents used for the synthesis as in
b
Table 4. Determined by HPLC using the following conditions:
15
Shukla, L.; Shuster, L. E.; Sneddon, H. F. Green Chem. 2016, 18, 3879.
16 Regulation (EC) No 1907/2006 of the European Parliament and of
Alder, C. M.; Hayler, J. D.; Henderson, R. K.; Redman, A. M.;
linear gradient of 10 → 50% 0.1% TFA in a CH3CN/0.1% TFA
mixture in H2O over 15 min, with a flow rate of 1.0 mL/min−1
and detection at 220 nm using a Phenomex C18 (3 µm, 4.6 × 50
mm). cData extracted from ref 7.
the Council of 18 December 2006 concerning the Registration, E.,
Authorisation and Restriction of Chemicals (“REACH”)
17
Jad, Y. E.; Acosta, G. A.; Khattab, S. N.; Beatriz, G.; Govender,
T.; Kruger, H. G.; El-Faham, A.; Albericio, F. Org. Biomol. Chem. 2015, 13,
2393.
In summary, we evaluated GVL and NFM as greener solvent
alternatives in SPPS. GVL and NFM both show good solubility
when compared to DMF. They also showed excellent coupling
efficiencies when Aib-enkephalin pentapeptide and Aib-ACP
decapeptide were synthesized in combination with the PS resin,
DIC/OxymaPure as coupling reagent and DMF for washing and
deprotection steps. Both solvents were then used to completely
substitute DMF for successful synthesis of the same peptide
models using the PS resin, resulting in higher purity than when 2-
MeTHF was used. As reported in our previous publications,7,20
and which was also confrmed here, we believe that the
impurities come from the Fmoc-removal step, which is often
the key step in the SPPS strategy.20,32 Additionally, in the
case of the GVL syntheses, we did not detect any major
side-reaction associated with opening of the ring and further
acylation of the amino function.33 To the best of our
knowledge, this is the first report applying a green solvent
protocol with the PS resin. The overall conclusion drawn from
this study is that GVL and NFM may be promising green
solvents for SPPS in combination with the PS resin.
18
García-Martín, F.; Quintanar-Audelo, M.; García-Ramos, Y.;
Cruz, L. J.; Gravel, C.; Furic, R.; Côté, S.; Tulla-Puche, J.; Albericio, F. J.
Comb. Chem. 2006, 8, 213.
19
M.; Côté, S.; Royo, M.; Albericio, F. J. Pept. Sci. 2009, 15, 629.
20 Jad, Y. E.; Govender, T.; Kruger, H. G.; El-Faham, A.; de la
Torre, B. G.; Albericio, F. Org. Process Res. Dev. 2017, 21, 365.
21 Jad, Y. E.; Khattab, S. N.; de la Torre, B. G.; Govender, T.;
Kruger, H. G.; El-Faham, A.; Albericio, F. Molecules 2014, 19, 18953.
22 Hojo, K.; Hara, A.; Kitai, H.; Onishi, M.; Ichikawa, H.; Fukumori,
Y.; Kawasaki, K. Chem. Cent. J. 2011, 5, 49.
Acosta, G. A.; del Fresno, M.; Paradis‐Bas, M.; Rigau‐DeLlobet,
23
24
Galanis, A. S.; Albericio, F.; Grøtli, M. Org. Lett. 2009, 11, 4488.
Horváth, I. T.; Mehdi, H.; Fábos, V.; Boda, L.; Mika, L. T. Green
Chem. 2008, 10, 238.
25
26
Duan, Z.-Q.; Hu, F. Green Chem. 2012, 14, 1581.
Alonso, D. M.; Wettstein, S. G.; Dumesic, J. A. Green Chem.
2013, 15, 584.
27
2010, 66, 1078.
Fegyverneki, D.; Orha, L.; Láng, G.; Horváth, I. T. Tetrahedron
28
29
Yan, K.; Yang, Y.; Chai, J.; Lu, Y. Appl. Catal. B 2015, 179, 292.
Westbye, P. In Agricultural formulations with acyl morpholines
and polar aprotic co-solvents; Google Patents, 2014.
30 Paradís-Bas, M.; Tulla-Puche, J.; Albericio, F. Chem. Soc. Rev.
2016, 45, 631.
31 Subirós‐Funosas, R.; Prohens, R.; Barbas, R.; El‐Faham, A.;
Albericio, F. Chem. Eur. J. 2009, 15, 9394.
Acknowledgments
32.
Peptide Res., 19969, 106..
Kates, S.F.; Solé, N.S.; Beyermann, M.; Barany, B.; Albericio, F.
This work was funded in part by the following: National
Research Foundation (NRF) and the University of KwaZulu-
Natal (South Africa); and the International Scientific Partnership
Program ISPP at King Saud University (ISPP# 0061) (Saudi
33. Chalid, M.; Heeres, H.J.; Broekhuis, A.A. J. App. Polymer Sci.,
2012, 123, 3556.
Arabia). MEC (CTQ2015-67870-P), the Generalitat de
Catalunya (2014 SGR 137), We thank Luxembourg Biotech for
Supplementary Material
the gift of coupling reagents.
Supplementary data associated with this article can be found,
in the online version, at
References and notes
1 Curzons, A.; Constable, D.; Cunningham, V. Clean Products and
Processes 1999, 1, 82.

4
Tetrahedron
Highlights
•
•
•
•
Green SPPS strategy using polystyrene (PS)
resin
GVL and NFM as excellent DMF
substitutes in SPPS
Solubility of reagents studied in GVL and
NFM
SPPS of Aib-enkephalin pentapeptide and
Aib-ACP decapeptide using GVL and NFM