2D green SPPS: Green solvents for on-resin removal of acid sensitive protecting groups and lactamization

Article abstract of DOI:10.1039/c9gc00898e

Aiming at greener synthesis of complex peptides we report that conventional one-dimensional (1D) green solid-phase peptide synthesis (SPPS) is elevated to a two-dimensional (2D) concept in which side chains in peptide resins are functionalized by suitable green tactics. Specifically, we disclose on-resin deblocking using trifluoroacetic acid (TFA)/triisopropylsilane (TIS) in EtOAc/MeCN used in a synthesis of a melanocortin receptor agonist comprising (i) 1D green SPPS (ii) 2D green SPPS by an on-resin TFA/TIS in EtOAc/MeCN deprotection of Lys(Mtt) and Asp(O-2-PhiPr) followed by a lactamization using PyBOP/DIEA in NBP/EtOAc (iii) TFA cleavage followed by green precipitation using 4-methyltetrahydropyran (MTHP)/n-heptane. A further application for our green deprotection protocol was found in peptide fragment cleavages off CTC resins.

Full text of DOI:10.1039/c9gc00898e

Green Chemistry
View Article Online
View Journal
COMMUNICATION
2D green SPPS: green solvents for on-resin
removal of acid sensitive protecting groups and
Cite this: DOI: 10.1039/c9gc00898e
lactamization†
Received 15th March 2019,
Accepted 23rd April 2019
Jan Pawlas, * Biljana Antonic, Marika Lundqvist, Thomas Svensson, Jens Finnman
and Jon H. Rasmussen
DOI: 10.1039/c9gc00898e
rsc.li/greenchem
Aiming at greener synthesis of complex peptides we report that peptide API.11 The need for the greening of the peptide syn-
conventional one-dimensional (1D) green solid-phase peptide syn- thesis has been recognized12 as evidenced by notable
thesis (SPPS) is elevated to a two-dimensional (2D) concept in advances for example within solventless,13,14 catalytic15,16
which side chains in peptide resins are functionalized by suitable and biocatalytic17,18 peptide synthesis. As Merrifield’s SPPS19
green tactics. Specifically, we disclose on-resin deblocking using is the standard method to make peptides20 the most substan-
trifluoroacetic acid (TFA)/triisopropylsilane (TIS) in EtOAc/MeCN tial greening efforts have been devoted to this technique,21
used in a synthesis of a melanocortin receptor agonist comprising focusing primarily on substituting the standard SPPS
(i) 1D green SPPS (ii) 2D green SPPS by an on-resin TFA/TIS in solvent DMF22,23 by environmentally friendlier alternatives.
EtOAc/MeCN deprotection of Lys(Mtt) and Asp(O-2-PhiPr) fol- Nevertheless, green SPPS has thus far not encompassed the
lowed by a lactamization using PyBOP/DIEA in NBP/EtOAc (iii) TFA synthesis of peptides of length and complexity available to
cleavage followed by green precipitation using 4-methyl- the conventional synthetic methods24,25 and consequently,
tetrahydropyran (MTHP)/n-heptane. A further application for our the adoption to commercial manufacturing where the most
green deprotection protocol was found in peptide fragment clea- profound reduction of environmental burden could be
vages off CTC resins.
achieved, has been lacking. For example, despite the numer-
ous examples of cyclic26 and branched27,28 therapeutic pep-
tides to our knowledge there are no greener SPPS approaches
to these classes of compounds. Further, despite the fact that
saving costs constitutes a key driver towards adoption of GC
by the pharmaceutical supply chain,29 utilizing green SPPS
would be far more costly than using conventional methods in
the art. Specifically, most of the green SPPS methods either
rely on uncommon amino acid (AA) protecting group (PG)
schemes30,31 or require using a fully polyethylene glycol
(PEG) based solid-support such as ChemMatrix,32 either in
aqueous33,34 or organic,35–37 reaction media. Unfortunately,
the PEG resins are not only significantly more expensive than
the polystyrene/divinylbenzene (PS/DVB) based polymers38
ordinarily used in peptide manufacturing, but also exhibit a
much higher swelling in TFA than PS/DVB resins do, which is
a nuisance during cleavages of peptide resins as it drives up
the solvent need. On the other hand, the greener SPPS
methods which used PS/DVB resins have either resulted in
poorer peptide purities than syntheses on PEG resins,39 or
utilized γ-valerolactone (GVL),40,41 N-formylmorpholine
(NFM),40 N-butylpyrrolidinone (NBP)42 and 2-methyl-
tetrahydrofuran (2-MeTHF)43 as reaction media, all of which
are considered too expensive for use in peptide manufactur-
ing (see ESI†).
Over the past few decades significant progress in the advance-
ment of Green Chemistry (GC) has been made1 including
defining metrics for the assessment of greenness of chemical
processes.2,3 The pharmaceutical manufacturing sector has
been among the industries most prominently involved in
encouraging the implementation of GC, exemplified for
instance by published key GC areas worth pursuing from
pharmaceutical manufacturers perspective.4 A class of phar-
maceuticals currently experiencing a renewed interest are
therapeutic peptides, with >60 peptide medicines on the
market and additional >150 in development.5 In fact, in 2017
alone six peptide drugs received an FDA approval6 and a
further growth of this market is expected.7,8 Nevertheless,
although methods to manufacture peptide drugs on commer-
cial scales are readily available,9,10 Kopach recently noted that
from the standpoint of sustainability a significant unmet
challenge is the 3000–15 000 kg of waste containing hazar-
dous reagents and solvents required to produce a kg of a
PolyPeptide Group, Limhamnsvägen 108, PO BOX 30089, 20061 Limhamn, Sweden.
E-mail: jap@polypeptide.com
†Electronic supplementary information (ESI) available: Experimental details and
solvent pricing estimates. See DOI: 10.1039/c9gc00898e
This journal is © The Royal Society of Chemistry 2019
Green Chem.

View Article Online
Communication
Green Chemistry
We set out to develop cost-efficient, environmentally higher rate of Mtt deblocking, however the Orn loss was also
friendly methods for the synthesis of complex peptides by ele- slightly increased (entries 5 and 6). We next tried to increase
vating green SPPS from a one-dimensional (1D) to a two- the rate of Mtt removal by mild heating, evaluating a series of
dimensional (2D) concept in which peptide resins are deriva- green solvents at 45 °C/1 h (entries 7–11). While EtOAc,
tized by suitable green protocols. Targeting a greener entry to 2-MeTHF and dimethylcarbonate (DMC) gave poorer conver-
both branched and cyclic peptides we started our 2D green sions (entries 7, 9 and 10), a high rate of Mtt removal was
SPPS assessment by examining on-resin removal of PGs from attained (entries 8 and 11) in MeCN and anisole albeit the
basic side chains of diamino AAs such as Lys and Orn. Among latter solvent also suffered from an appreciable loss of Orn
the numerous PG schemes for these AAs44 we chose to test the (∼8%). The chemoselectivities observed in the on-resin Orn
common acid induced removal of the 4-methyltrityl (Mtt) (Mtt) removals may be affected by numerous reaction para-
group, typically carried out using TFA or AcOH/trifluoro- meters including resin swelling as well as polarity, dielectric
ethanol in dichloromethane (DCM).45 As DCM is a suspected constant and dipole moment of the solvent. The reasons for
carcinogen23 on-resin Mtt removal constitutes a suitable opportu- the different activities of the tested solvents in Mtt removal
nity for greening. As the substrate we used Fmoc-Orn(Mtt) and Fmoc-Orn-NH2 leaching are currently unknown and will
bound to an aminomethylstyrene (AMS) functionalized PS/ be examined in future investigations. Based on the initial
DVB resin via Ramage (RMG) linker,46 which we used instead solvent screening we examined MeCN and MeCN/EtOAc at pro-
of the more common Rink amide (RAM)47 counterpart as the longed reaction times (2 × 1 h) using 2% and 4% TIS all of
latter is known to be hampered by lower yield48 and formation which gave >99% Mtt removal and only a minimal loss of Orn
of impurities49 during peptide resin cleavages. Nevertheless, (entries 12–15). As adequate resin swelling is a prerequisite for
on-resin Mtt deblocking employing RMG linker is complicated suitable reaction kinetics,51,52 we examined swelling of an AMS
by the limited stability of peptide–RMG bonds in TFA/DCM50 resin in MeCN/EtOAc and MeCN. Thus, we found that from
meaning that the greener protocols ideally also had to exhibit the swelling standpoint, using MeCN/EtOAc (∼7 ml g−1) over
better chemoselectivity than the standard Mtt removal con- MeCN (∼2 ml g−1) could be beneficial. In fact, it is worth
ditions. We first tested a conventional deblocking in DCM, noting that swelling in EtOAc was ∼6 ml g−1 meaning that in
using 2% TFA and 2% TIS as the scavenger. After 1 h at rt, the MeCN/EtOAc the swelling was higher than in both neat
Mtt removal was essentially quantitative however in keeping solvents.
with the known lability of peptide–RMG bond,50 the deblock-
With suitable green conditions for on-resin Mtt removal in
ing was accompanied by ∼10% loss of Fmoc-Orn-NH2 (Table 1, hand we examined the scope of the PG deblocking using a
entry 1). In an attempt to replace the hazardous DCM we exam- series of Fmoc-AA(PG)-RMG AMS resins (Table 2). Deblocking
ined two inexpensive green solvents EtOAc and MeCN,23 which of Lys(Trt) using TFA in DCM, which gave >99% conversion,
resulted in a sluggish Mtt removal, requiring 76 h for ∼65% was accompanied by a significant loss of Fmoc-Lys-NH2 (entry
and ∼98% deblocking, respectively (entries 2 and 3). We next 1). On the other hand, with TFA in MeCN/EtOAc (entries 2 and
tested a 1 : 1 mixture of these solvents (entry 4) which gave a 3) the Trt deblocking was more sluggish than the PG removals
rate of Mtt removal comparable to MeCN (∼98%) and a loss of of the more acid-labile Mtt44 in Table 1. The removal of the
Orn similar to EtOAc (1%). An attempt to accelerate the rate of O-2-phenyl isopropyl (O-2-PhiPr) group53 from Asp proceeded
Mtt removal by raising TFA concentration did result in a to >99% using both DCM and MeCN/EtOAc protocols, with the
Table 1 Assessment of conditions for removal of Mtt group from Fmoc-Orn(Mtt) RMG resina
a Hazardous DCM in red, greener solvents in green. b After 16 h the resin was drained, analyzed and the Mtt removal was continued for 60 h
using a new batch of solvents and reagents.
Green Chem.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Green Chemistry
Communication
Table 2 Assessment of conditions for PG removal from Fmoc-AA(PG) RMG resins
loss of Fmoc-Asp-NH2 being higher in DCM than in MeCN/ kinetics and solubility of starting materials. Instead of piper-
EtOAc (entries 4 and 5). While with TFA/DCM Cys(Trt) and Cys idine, the standard base for Fmoc removals, we used the less
(Mmt) were fully deprotected after 1 h at rt (entries 6 and 8),54 hazardous 4-methylpiperidine (4-MP)42,65 and to offset the
both Cys PGs were fairly stable with TFA in MeCN/EtOAc higher price of 4-MP it was used at 5% (v/v) instead of 20%
(entries 7 and 9). The good stability towards TFA in MeCN/ which is customary for piperidine.66 Diisopropylcarbodiimide
EtOAc could be valuable as it enables deblocking of Orn/Lys (DIC)/Oxyma,67 deemed suitable for most SPPS couplings,68
(Mtt) and Asp(O-2-PhiPr) without affecting Cys(PG),55 which was used throughout except for His3 as a higher level of His
otherwise requires conditions using hazardous dichloroethane racemization using DIC/Oxyma was reported.69 For the His3
(DCE)23 as solvent.56 Finally, exposing His(Trt)57 to the coupling a DIC/HONB/HOBtxH2O protocol was used instead
deblocking protocols revealed a higher Trt loss with TFA in which we previously used for Fmoc-His(Trt)-OH couplings
DCM (entry 10), than upon using the MeCN/EtOAc conditions with minimal racemization.70 To keep the consumption of
(entry 11).
expensive NBP to a minimum lower NBP amounts (10% v/v)
To demonstrate the usefulness of our green PG deblocking were used in washes after Fmoc removals. Furthermore,
method a suitable model peptide was synthesized. We chose washes after couplings were omitted entirely71 and replaced
melanocortin receptor agonist Ac0-Nle1-c[Asp2-His3-Phe4-Arg5- by quenching the coupling mixtures with i-PrOH to prevent any
Trp6-Lys7]-NH2 (1)58,59 and devised an approach comprising interference with the subsequent Fmoc removal step. The syn-
(i) 1D green SPPS of the requisite peptide resin (ii) on-resin thesis commenced with 1.4 mmol of an Fmoc-RMG AMS resin
deblocking of Lys(Mtt) and Asp(O-2-PhiPr) (iii) on-resin lacta- carrying out all Fmoc removals for 2 × 15 min and couplings
mization involving the deprotected Lys and Asp (iv) cleavage for 30 min (Scheme 1). The progress of couplings was checked
of the peptide off the solid support followed by isolating 1 by by ninhydrin test72 revealing full conversion in all couplings
precipitation. Regarding 1D green SPPS we sought a protocol except for His3 which was recoupled. The synthesis gave the
that would not be more costly than a conventional SPPS in expected amount of resin 2 (102% of theory) as well as satisfac-
DMF. As 1D green SPPS methods rely on materials and/or pro- tory HPLC (purity 90%) and MS ([M + H]+ calcd 1042.559;
tocols that are more expensive than standard SPPS (vide found 1042.557) analyses of the crude product. D-His content
supra) we decided to utilize an approach in which the green of the crude material was 0.3% (chiral GC-MS) which is not
dipolar aprotic NBP60 is used with an inexpensive, less polar only appreciably lower than His racemization for green SPPS in
green cosolvent.61 Such green solvent was EtOAc which can MeCN73 and water,74 but is also below D-His contents attained
be sourced renewably,23 swells PS/DVB resins well,52 and with coupling agents specifically designed to suppress racemi-
facilitates good kinetics in solution phase couplings.62 In zation.69 The total volume of SPPS waste was 2.5 L 27% of
fact, using NBP with EtOAc not only lowers the cost of the which was NBP, rendering the solvent cost comparable to a syn-
synthesis but also reduces the high viscosity of NBP, which is thesis carried out in DMF. It is worth noting that the environ-
a drawback of using this DMF substitute in its neat form.42 mental profile as well as solvent economy of the SPPS could be
Regarding use of EtOAc in green SPPS it is worth noting that further improved by recycling EtOAc, a well established
Albericio et al. used EtOAc, together with 2-MeTHF, for green process75 aided here by the large bp difference between EtOAc
SPPS of Aib-enkephalin pentapeptide and Aib-ACP deca- (77 °C) and the two other major liquid constituents of the
peptide.36 Nevertheless, these EtOAc and 2-MeTHF based waste in NBP (243 °C) and 4-MP (124 °C).
SPPS protocols required ChemMatrix resin as the polymer
Next, for the second dimension, conventional (Table 1,
support as syntheses on a PS resin afforded crude peptides in entry 1) and green (entry 14) conditions for Lys(Mtt) and Asp
poor HPLC purities. Fmoc SPPS63 in NBP/EtOAc (1 : 1) was (O-2-PhiPr) deblocking on resin 2 were evaluated, proceeding
used for Fmoc removals and AA couplings which, together to >99% in both cases. The desired deprotections were
with mild conventional heating,64 secured suitable reaction accompanied by a partial deblocking of His(Trt) which none-
This journal is © The Royal Society of Chemistry 2019
Green Chem.

View Article Online
Communication
Green Chemistry
Scheme 1 1D green SPPS of peptide resin 2. Reagents and conditions: (i) a) Fmoc removal: 5% 4-MP in NBP/EtOAc, 1 : 1, 45 °C, 2 × 15 min, (b) resin
wash: 4 × NBP/EtOAc, 1 : 9, 45 °C, 5 min each; (ii) coupling: R-COOH/Oxyma/DIC (1 : 1 : 3) in NBP/EtOAc, 1 : 1, 45 °C, 30 min; (iii) (a) capping: 10
equiv. AcOH, (b) quench, i-PrOH/EtOAc (1 : 9); (iv) His coupling: Fmoc-His(Trt)-OH/HONB/HOBtxH2O/DIC (1 : 1 : 0.15 : 3) in NBP/EtOAc, 1 : 1, 45 °C, 2
× 30 min.
theless did not impair the subsequent on-resin cyclization
(vide infra). Next, TFA/TIS/H2O (90 : 5 : 5) cleavages were carried
out on the deblocked resins followed by precipitation of the
crude peptides. The HPLC profiles of these crudes were essen-
tially indistinguishable, albeit, in agreement with the Orn
leaching caused by Table 1, entry 1 deblocking, a ∼10% lower
amount of crude peptide was isolated from the cleavage of
resin exposed to TFA/DCM.
Table 3 Assessment of solvents for peptide precipitation after TFA
cleavage of resin 2
With the goal of using green solvents throughout the entire
synthesis of 1 we briefly assessed the step of crude peptide iso-
lation, normally carried out by a precipitation after TFA clea-
vage/global deprotection of peptide resin. Recently, it has been
To finalize the synthesis of the model peptide 1 we sub-
noted that the commonly used ethers such as diethyl ether jected resin 2 to TFA/TIS/EtOAc/MeCN, 2 : 2 : 48 : 48 at 45 °C
(DEE) and methyl tert-butyl ether (MTBE) can be replaced by (Scheme 2). Three 1 h deprotections were carried out as after
greener counterparts such as cyclopentyl methyl ether the second deblocking, an analysis showed a small amount of
(CPME)76 and 2-MeTHF.77 We evaluated these solvents Lys and Asp PGs still present on the resin. As various para-
together with MTHP, proposed as environmentally friendlier meters such as means of agitation80 may affect SPPS kinetics
alternative to hazardous ethers in various applications.78 Thus, we propose that during process development, the PG deblock-
resin 2 was cleaved using TFA/TIS/H2O (90 : 5 : 5) and the ing conversion is checked before proceeding to the next step of
HPLC profiles of peptides isolated in subsequent precipi- the synthesis. The deprotected resin was converted to the free
tations were all comparable, indicating that the solvents exam- base (DIEA) and cyclized using PyBOP81/DIEA (1 : 2) in NBP/
ined did not take part in side reactions involving the peptide. EtOAc (1 : 1). After confirming that the lactamization pro-
On the other hand, crude peptide amounts were all slightly ceeded to completion (ninhydrin test) the cyclized peptide was
lower with the greener solvents (Table 3, entries 2–4), than cleaved off the resin using TFA/TIS/H2O (90 : 5 : 5) and isolated
with DEE (entry 1). As green peptide precipitations using by precipitation in MTHP/n-heptane (1 : 4). The crude material
CPME, 2-MeTHF and MTHP are also hampered by higher cost was desalted and isolated by lyophilization yielding peptide 1
of these solvents (see ESI†) we examined the inexpensive in 62% overall yield, 95% HPLC purity and expected MS ([M +
n-heptane79 as a cosolvent which revealed that employing H]+ calcd 1024.548; found 1024.549) analysis.
MTHP/n-heptane (1 : 4) gives a comparable amount of peptide
as DEE (Table 3, entry 5).
Finally, in a search for further applications for our green de-
protection protocol we examined cleavages of peptide frag-
Green Chem.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Green Chemistry
Communication
Scheme 2 2D green SPPS of peptide resin 3 followed by TFA cleavage and isolation of peptide 1. Reagents and conditions: (i) TFA/TIS/EtOAc/
MeCN, 2 : 2 : 48 : 48, 45 °C, 3 × 1 h; (ii) 2 equiv. PyBOP/DIEA, 1 : 2 in EtOAc/NBP, 1 : 1, rt, 16 h; (iii) TFA/TIS/H2O, 90 : 5 : 5, rt, 2 h; (iv) precipitation in
MTHP/n-heptane, 1 : 4; (v) desalting and lyophilization.
ities, for example in on-resin deblocking of Lys/Orn(Mtt) and
Asp(O-2-PhiPr) which was done without cleaving the acid labile
peptide–RMG and Cys–Trt bonds appreciably, while a His
coupling using Fmoc-His(Trt)-OH was carried out with
minimal racemization. At last, a cleavage of a CTC peptide
resin in green MeCN/EtOAc was carried out, paving the way for
Scheme 3 Cleavages of Fmoc-Gly-Gly CTC resin.
the use of GC in the convergent synthesis of larger peptides.
ments off chlorotrityl chloride (CTC) resins. Such cleavages are
typically carried out using various acidic reagents in DCM, for
Conflicts of interest
example utilizing 1–2% TFA in DCM,82 AcOH/TFE/DCM
(1 : 1 : 8)83 or HFIP/DCM (1 : 4)84 and this chemistry has been
used in convergent synthesis of larger peptides,85 and also
implemented in manufacturing of peptide therapeutics.86
We used Fmoc-Gly-Gly CTC resin as a substrate and
exposed it to 1% TFA/TIS in DCM and EtOAC/MeCN (1 : 1) for
1 h at rt (Scheme 3). In both cases the dipeptide was released
in high yield indicating that DCM could be replaced with the
greener protocol. It is worth noting that 2-MeTHF, used as a
greener alternative to DCM in the attachment of AAs to CTC
resins,77 afforded a lower yield of the dipeptide than DCM and
EtOAc/MeCN, respectively.
There are no conflicts to declare.
References
1 H. C. Erythropel, J. B. Zimerman, T. M. de Winter,
L. Petitjean, F. Melnikov, C. H. Lam, A. W. Lounsbury,
K. E. Mellor, N. Z. Janković, Q. Tu, L. N. Pincus,
M. M. Falinski, W. Shi, P. Coish, D. L. Plata and
P. T. Anastas, Green Chem., 2018, 20, 1929.
2 R. A. Sheldon, Green Chem., 2017, 19, 18.
3 F. Roschangar, Y. Zhou, D. J. C. Constable, J. Colberg,
D. P. Dickson, P. J. Dunn, M. D. Eastgate, F. Gallou,
J. D. Hayler, S. G. Koenig, M. E. Kopach, D. K. Leahy,
I. Mergelsberg, U. Scholz, A. G. Smith, M. Henry, J. Mulder,
J. Brandenburg, J. R. Dehli, D. R. Fandrick, K. R. Fandrick,
F. Gnad-Badouin, G. Zerban, K. Groll, P. T. Anastas,
R. A. Sheldon and C. H. Senanayake, Green Chem., 2018, 20,
2206.
4 M. C. Bryan, P. J. Dunn, D. Entwistle, F. Gallou,
S. G. Koenig, J. D. Hayler, M. R. Hickey, S. Hughes,
M. E. Kopach, G. Moine, P. Richardson, F. Roschangar,
A. Steven and F. J. Weiberth, Green Chem., 2018, 20, 5082.
5 J. L. Lau and M. K. Dunn, Bioorg. Med. Chem., 2018, 26,
2700.
Conclusions
We reported a concept of 2D green SPPS, which elevates the
standard 1D green SPPS to now encompass the synthesis of
more complex peptides by means of on-resin derivatization.
2D green SPPS was illustrated by a synthesis of a melanocortin
receptor agonist consisting of (i) 1D green SPPS of the requisite
peptide resin (ii) green on-resin removal of acid labile PGs and
on-resin cyclization (2D green SPPS) (iii) TFA cleavage/green
precipitation of the target peptide. The key aspects of the
green peptide synthesis herein are that (i) conventional sol-
vents were replaced with greener counterparts without notably
increasing the costs of raw materials (ii) the use of greener sol-
vents was accompanied by attaining valuable chemoselectiv-
6 O. A. Musaimi, D. A. Shaer, B. G. de la Torre and
F. Albericio, Pharmaceuticals, 2018, 11, 42.
This journal is © The Royal Society of Chemistry 2019
Green Chem.

View Article Online
Communication
Green Chemistry
7 K. Fosgerau and T. Hoffmann, Drug Discovery Today, 2015, 35 G. A. Acosta, M. del Fresno, M. Paradis-Bas, M. Rigau-
20, 122.
DeLlobet, S. Cote, M. Royo and F. Albericio, J. Pept. Sci.,
2009, 15, 629.
36 Y. E. Jad, G. A. Acosta, T. Govender, H. G. Kruger, A. El-
Faham, B. G. de la Torre and F. Albericio, ACS Sustainable
Chem. Eng., 2016, 4, 6809.
8 A. Henninot, J. C. Collins and J. M. Nuss, J. Med. Chem.,
2018, 61, 1382.
9 T. Bruckdorfer, O. Marder and F. Albericio, Curr. Pharm.
Biotechnol., 2004, 5, 29.
10 R. Lax, Chim. Oggi, 2016, 34(November), 20.
11 M. E. Kopach, Curr. Opin. Green Sustainable Chem., 2018,
11, 54.
37 S. B. Lawrenson, R. Arav and M. North, Green Chem., 2017,
19, 1685.
38 B. Yan, Comb. Chem. High Throughput Screening, 1998, 1, 215.
12 S. Datta, A. Sood and M. Török, Curr. Org. Synth., 2011, 8, 39 Y. E. Jad, G. A. Acosta, T. Govender, H. G. Kruger, A. El-
262.
Faham, B. G. de la Torre and F. Albericio, ACS Sustainable
Chem. Eng., 2016, 4, 6809.
40 A. Kumar, Y. E. Jad, A. El-Faham, B. G. de la Torre and
F. Albericio, Tetrahedron Lett., 2017, 58, 2986.
13 O. Maurin, P. Verdié, G. Subra, F. Lamaty, J. Martinez and
T.-X. Métro, Beilstein J. Org. Chem., 2017, 13, 2087.
14 Y. Yeboue, B. Gallard, N. Le Moigne, M. Jean, F. Lamaty,
J. Martinez and T.-X. Métro, ACS Sustainable Chem. Eng., 41 A. Kumar, Y. E. Jad, J. M. Collins, F. Albericio and
2018, 6, 16001.
B. G. de la Torre, ACS Sustainable Chem. Eng., 2018, 6,
8034.
15 T. Krause, S. Baader, B. Erb and L. J. Gooßen, Nat.
Commun., 2016, 7, 11732.
16 Z. Liu, H. Noda, M. Shibasaki and N. Kumagai, Org. Lett.,
2018, 20, 612.
17 A. Goswami and S. G. Van Lanen, Mol. Biosyst., 2015, 11, 338.
18 M. Schmidt, A. Toplak, P. J. L. M. Quaedflieg and
T. Nuijens, Curr. Opin. Chem. Biol., 2017, 38, 1.
19 R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149.
20 D. M. M. Jaradat, Amino Acids, 2018, 50, 39.
42 J. Lopez, S. Pletscher, A. Aemissegger, C. Bucher and
F. Gallou, Org. Process Res. Dev., 2018, 22, 494.
43 The scope of this green SPPS method is limited by the use
of NaOH in MeOH/2-MeTHF in Fmoc removal steps see:
A. Přibylka, V. Krchňák and E. Schütznerová, Green Chem.,
2019, 21, 775.
44 A. Isidro-Llobet, M. Álvarez and F. Albericio, Chem. Rev.,
2009, 109, 2455.
21 (a) Y. E. Jad, A. Kumar, A. El-Faham, B. G. de la Torre and 45 A. Aletras, K. Barlos, D. Gatos, S. Koutsogianni and
F. Albericio, ACS Sustainable Chem. Eng., 2019, 7, 3671; P. Mamos, Int. J. Pept. Protein Res., 1995, 45, 488.
(b) K. G. Varnava and V. Sarojini, Chem. – Asian J., 2019, 14, 46 R. Ramage, S. L. Irving and C. McInnes, Tetrahedron Lett.,
1088. 1993, 34, 6599.
22 https://www.echa.europa.eu/documents/10162/5d7b909d- 47 M. S. Bernatowicz, S. B. Daniels and H. Köster, Tetrahedron
ef93-4fb3-8ca0-60e4e799e0e6. Lett., 1989, 30, 4645.
23 F. P. Byrne, S. Jin, G. Paggiola, T. H. M. Petchey, J. H. Clark, 48 M. Noda, M. Yamaguchi, E. Ando, K. Takeda and
T. J. Farmer, A. J. Hunt, C. R. McElroy and J. Sherwood,
Sustainable Chem. Processes, 2016, 4, 1.
24 J. M. Palomo, RSC Adv., 2014, 4, 32658.
K. Nokihara, J. Org. Chem., 1994, 59, 7968.
49 P. Stathopoulos, S. Papas and V. Tsikaris, J. Pept. Sci., 2006,
12, 227.
25 K. E. Schwieter and J. N. Johnston, J. Am. Chem. Soc., 2016, 50 It has been reported that in 30 min at rt TFA/DCM (3 : 97)
138, 14160.
cleaves peptides off RMG linker bound resins quantitat-
ively, see: F. Guillier, D. Orain and M. Bradley, Chem. Rev.,
2000, 100, 2091.
26 A. Zorzi, K. Deyle and C. Heinis, Curr. Opin. Chem. Biol.,
2017, 38, 24.
27 A. Pini, C. Falciani and L. Bracci, Curr. Protein Pept. Sci., 51 R. Santini, M. C. Griffith and M. Qi, Tetrahedron Lett., 1998,
2008, 9, 468. 39, 8951.
28 E. M. Bech, S. L. Pedersen and K. J. Jensen, ACS Med. 52 S. B. Lawrenson, M. North, F. Peigneguy and A. Routledge,
Chem. Lett., 2018, 9, 577. Green Chem., 2017, 19, 952.
29 V. R. Veleva, B. W. Cue and S. Todorova, ACS Sustainable 53 C. Yue, J. Thierry and P. Potier, Tetrahedron Lett., 1993, 34,
Chem. Eng., 2018, 6, 2. 323.
30 K. Hojo, M. Maeda and K. Kawasaki, Tetrahedron Lett., 54 Barlos et al. have shown that using 1.0% TFA in DCM/TES
2004, 45, 9293.
(95 : 5) 4–5% S(Trt) and 100% S(Mmt) is deprotected in
30 min at rt, see: K. Barlos, D. Gatos, S. Koutsogianni,
O. Hatzi and N. Koch, Int. J. Pept. Protein Res., 1996, 47, 148.
31 S. Knauer, T. M. L. Roese, O. Avrutina and H. Kolmar, WO,
2016/050764 A1, 2016.
32 F. Garcia-Martin, M. Quintanar-Audelo, Y. Garcia-Ramos, 55 Upon using suitable scavengers Cys(Trt) is stable at
L. J. Cruz, C. Gravel, R. Furic, S. Cote, J. Tulla-Puche and
F. Albericio, J. Comb. Chem., 2006, 8, 213.
33 A. S. Galanis, F. Albericio and M. Grotli, Org. Lett., 2009, 11,
4488.
34 R. De Marco, A. Tolomelli, A. Greco and L. Gentilucci, ACS
Sustainable Chem. Eng., 2013, 1, 566.
high TFA concentrations: P. Stathopoulos, S. Papas,
M. Sakka, A. G. Tzakos and V. Tsikar, Amino Acids, 2014,
46, 1367.
56 T. Wang, J. Fan, X. X. Chen, R. Zhao, Y. Xu, D. Bierer,
L. Liu, Y. M. Li, J. Shi and G. M. Fang, Org. Lett., 2018, 20,
6074.
Green Chem.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Green Chemistry
Communication
57 P. Sieber and B. Riniker, Tetrahedron Lett., 1993, 34, 323.
72 E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook,
58 P. Grieco, P. Balse-Srinivasan, G. Han, D. Weinberg,
Anal. Biochem., 1970, 34, 595.
T. MacNeil, L. H. T. Van der Ploeg and V. J. Hruby, J. Pept. 73 G. A. Acosta, M. del Fresno, M. Paradis-Bas, M. Rigau-
Res., 2003, 62, 199.
59 Bradley et al. recently prepared 1 using a method hinging
DeLlobet, S. Côté, M. Royoa and F. Albericio, J. Pept. Sci.,
2009, 15, 629.
on on-resin removal of (i) Asp(O-2-PhiPr) using TFA/DCM 74 K. Hojo, N. Shinozaki, K. Hidaka, Y. Tsuda, Y. Fukumori,
(ii) Lys(VeZ) using tetrazine/DMF, see: M. Staderini, H. Ichikawa and J. D. Wade, Amino Acids, 2014, 46, 2347.
A. Gambardella, A. Lilienkampf and M. Bradley, Org. Lett., 75 https://www.tradebesolventrecycling.com/ethyl-acetate-
2018, 20, 3170. suppliers.
60 J. Sherwood, H. L. Parker, K. Moonen, T. J. Farmer and 76 O. Al Musaimi, Y. E. Jad, A. Kumar, J. M. Collins, A. Basso,
A. J. Hunt, Green Chem., 2016, 18, 3990.
61 J. Pawlas, M. Nilsson, B. Antonic, B. T. Svensson,
B. G. de la Torre and F. Albericio, Curr. Opin. Green
Sustainable Chem., 2018, 11, 99.
J. Finnman and J. H. Rasmussen, Poster PM1 at TIDES, 77 O. Al Musaimi, Y. E. Jad, A. Kumar, A. El-Faham,
Boston, MA, U.S.A, May 2018.
62 D. S. MacMillan, J. Murray, H. F. Sneddon, C. Jamieson and
A. J. B. Watson, Green Chem., 2013, 15, 596.
63 R. Behrendt, P. White and J. Offer, J. Pept. Sci., 2016, 22, 4.
64 M. A. Fara, J. J. Díaz-Mochón and M. Bradley, Tetrahedron
Lett., 2006, 47, 1011.
65 J. Hachmann and M. Lebl, J. Comb. Chem., 2006, 8, 149.
66 N. Zinieris, L. Leondiadis and N. Ferderigos, J. Comb.
Chem., 2005, 7, 4.
67 R. Subirós-Funosas, R. Prohens, R. Barbas, A. El-Faham
and F. Albericio, Chem. – Eur. J., 2009, 15, 9394.
68 F. Albericio and A. El-Faham, Org. Process Res. Dev., 2018,
22, 760.
J. M. Collins, A. Basso, B. G. de la Torre and F. Albericio,
Org. Process Res. Dev., 2018, 22, 1809.
78 https://www.kuraray.eu/en/produkte/product-ranges/mthp/.
79 The environmental status of n-heptane is comparable to
2-MeTHF, see ref. 23.
80 I. Alshanski, M. Bentolila, A. Gitlin-Domagalska, D. Zamir,
S. Zorsky, S. Joubran, M. Hurevich and C. Gilon, Org.
Process Res. Dev., 2018, 22, 1318.
81 B. Castro, J. R. Dormoy, G. Evin and C. Selve, J. Chem. Res.,
Synop., 1977, 7, 182.
82 M. Góngora-Benítez, M. Cristau, M. Giraud, J. Tulla-Puche
and F. Albericio, Chem. Commun., 2012, 48, 2313.
83 K. Barlos, D. Gatos and W. Schäfer, Angew. Chem., Int. Ed.
Engl., 1991, 30, 590.
69 J. E. Jad, S. N. Khattab, B. G. de la Torre, T. Govender,
H. G. Kruger, A. El-Faham and F. Albericio, Org. Biomol. 84 R. Bollhagen, M. Schmiedberger, K. Barlos and E.J. Grell,
Chem., 2014, 12, 8379. Chem. Soc., Chem. Commun., 1994, 2559.
70 J. Pawlas, Proceedings of the 34th European Peptide 85 P. Athanassopoulos, K. Barlos, D. Gatos, O. Hatzi and
Symposium and the 8th International Peptide Symposium,
Leipzig, Germany, 2016, p. 60.
71 For an example of a SPPS protocol in which washes after
couplings are eliminated see: J. M. Collins, K. A. Porter,
S. K. Singh and G. S. Vanier, Org. Lett., 2014, 16, 940.
C. Tzavara, Tetrahedron Lett., 1995, 36, 5645.
86 H. Zhang, S. E. Schneider, B. L. Bray, P. E. Friedrich,
N. A. Tvermoes, C. J. Mader, S. R. Whight, T. E. Niemi,
P. Silinski, T. Picking, M. Warren and S. A. Wring, Org.
Process Res. Dev., 2008, 12, 101.
This journal is © The Royal Society of Chemistry 2019
Green Chem.