Environmentally Friendly SPPS II: Scope of Green Fmoc Removal Protocol Using NaOH and Its Application for Synthesis of Commercial Drug Triptorelin

Article abstract of DOI:10.1021/acs.joc.0c00599

With the growing necessity to consider environmental impacts when synthesizing peptide-based drugs and to expand upon the recently published short communication report, we herein present a thorough evaluation of a green Fmoc removal protocol. Our protocol avoids the use of hazardous components (using piperidine as a base and dichloromethane (DCM) and N,N-dimethylformamide (DMF) as solvents) and relies on the utilization of the green mineral base NaOH in combination with the greener solvent 2-methyltetrahydrofuran (2-MeTHF) mixed with MeOH. For the original Fmoc removal cocktail (solvents ratio of 1:1), we evaluated the impact of quality/purity of the used 2-MeTHF, scale-up, ratio of 2-MeTHF/MeOH, utilized hydroxide, temperature, and reaction time. An alternative 3:1 protocol was examined using various amino acids, and only Gly required the optimization of the Fmoc removal cocktail composition. The optimized protocol used to remove Fmoc from Gly residue was proved by the synthesis of Leu-enkephalin. We also investigated the stability of the conventional amino acid side-chain-protecting groups, t-Bu, Boc, Trt, and Pbf, and the formation of aspartimide as an undesirable side reaction that occurs during Fmoc solid-phase peptide synthesis (SPPS). The applicability of this synthesis strategy was documented by evaluating the SPPS of a commercial drug used for prostate and breast cancer treatments - decapeptide triptorelin.

Full text of DOI:10.1021/acs.joc.0c00599

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Article
Environmentally friendly SPPS II: Scope of green Fmoc removal protocol
using NaOH and its application for synthesis of commercial drug triptorelin
Adam Pribylka, Viktor Krchnak, and Eva Schütznerová
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00599 • Publication Date (Web): 10 Jun 2020
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The Journal of Organic Chemistry
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Environmentally friendly SPPS II: Scope of green Fmoc remo-
val protocol using NaOH and its application for synthesis of
commercial drug triptorelin
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Adam Přibylka, † Viktor Krchňák, †,‡ and Eva Schütznerová †,*
†Department of Organic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, 771 46 Olomouc, Czech
Republic. E-mail: eva.schutznerova@upol.cz.
‡Department of Chemistry and Biochemistry, 251 Nieuwland Science Center, University of Notre Dame, Notre
Dame, Indiana 46556, United States
KEYWORDS: green chemistry, solid-phase peptide synthesis, protecting groups, Fmoc, 2-methyltetrahydrofuran,
sodium hydroxide, triptorelin, aspartimide
ABSTRACT: With the growing necessity to consider environmental impacts when synthesizing peptide-based drugs and
to expand upon the recently published short communication report, we herein present a thorough evaluation of a green
Fmoc removal protocol. Our protocol avoids the use of hazardous components (using piperidine as a base and DCM and
DMF as solvents) and relies on the utilization of the green mineral base NaOH in combination with the greener solvent 2-
MeTHF mixed with MeOH. For the original Fmoc removal cocktail (solvents ratio of 1:1), we evaluated the impact of qual-
ity/purity of the used 2-MeTHF, scale-up, ratio of 2-MeTHF/MeOH, utilized hydroxide, temperature, and reaction time.
An alternative 3:1 protocol was examined using various amino acids, and only Gly required the optimization of the Fmoc
removal cocktail composition. The optimized protocol used to remove Fmoc from Gly-residue was proved by the synthe-
sis of Leu-enkephalin. We also investigated the stability of the conventional amino acid side-chain protecting groups, t-
Bu, Boc, Trt, and Pbf, and the formation of aspartimide as an undesirable side reaction that occurs during Fmoc solid-
phase peptide synthesis (SPPS). The applicability of this synthetic strategy was documented by evaluating the SPPS of a
commercial drug used for prostate and breast cancer treatments - decapeptide triptorelin.
mixture.10,11 Regarding peptide synthesis, among others,
there are two key chemical strategies - solution-phase and
INTRODUCTION
While “small-molecule” drugs may suffer from a reduced
target selectivity, peptide-based pharmaceuticals exhibit
high biological activity and high specificity.1 However, the
interest of the pharmaceutical industry in peptide chem-
istry has been reduced due to the low oral bioavailability
and low stability of these “Nature’s pharmaceuticals”
under physiological conditions. Recent advances in a field
of chemical modification and drug-delivery technology
led pharmaceutical companies to refocus their attention
to peptide-based drugs, which is documented by the in-
creasing number of therapeutic peptides on the market
and drug candidates in several clinical and preclinical
trials.2-6 The commercial production of peptides used as
active pharmaceutical ingredients (APIs) is scaling up to
multi-ton quantities.7-9 Moreover, peptides are expected
to play an important role as future therapeutics, and
therefore, their manufacturing processes represent an
environmental issue.
solid-phase peptide synthesis (SPPS) as well as their com-
binations, which are referred to as hybrid synthesis meth-
ods.12-14 These approaches include the consumption of
large amounts of solvents that are used during the prepa-
ration and purification steps, and in SPPS methods, sol-
vents are also used to wash the resin. The most often
employed solvents used in SPPS are undoubtedly DMF
and DCM. Both of these solvents are considered hazard-
ous according to several green selection guides,15 and
DMF is likely to be restricted by REACh.16 SPPS is used
not only in research laboratories but also in industry. For
this reason, environmentally friendly approaches are
highly desirable. Notable, greener SPPS belongs to key
green chemistry research areas identified by ACS Green
Chemistry
Institute
Pharmaceutical
Roundtable
(GCIPR).17
SPPS was introduced in 1963 by B. R. Merrifield,18 who
was awarded the Nobel Prize for this finding. The meth-
odology starts with the immobilization of the first pro-
tected amino acid (AA) via the carboxylic terminus onto
insoluble support (resin), which then plays the role of
The primary environmental concerns of the processes
used to manufacture every API involve solvents since
solvents are the major component of the reaction
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Scheme 1. Repetitive nature of SPPS – deprotection/coupling cycle
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the C-terminal protecting group (PG). The peptide chain
assembly is performed by a repetitive stepwise process
based on repeating the coupling and deprotecting cycles
of N-protected AAs, which is alternated with washing
steps (Scheme 1 was adapted from reference19). When the
peptide elongation process is complete, the whole proce-
dure is accomplished by the final cleavage of the synthe-
sized peptide from the resin concurrently with the global
deprotection of the side-chain PGs.13,20 In combination
with the appropriate PGs,21 SPPS became a widespread
practical tool used in peptide synthesis. Although both
coupling reagents22-24 and resins have evolved since then,
SPPS has not changed much until now. There is a collec-
tion of polyethylene glycol (PEG)-based solid supports,
with advantages in the swelling of solvents from medium
to high polarities, which range from toluene to water.25
Despite that, the most popular solid support is still a
polystyrene (PS) resin cross-linked with divinylbenzene
(1-2% DVB), and the main reason is the financial aspect.
There have been attempts to make SPPS more envi-
ronmentally friendly, which follows the current trend of
green chemistry principles.26 Greening SPPS is an exten-
sively studied process, and the term green SPPS (GSPPS)
should be used only after each aspect of the performance
is addressed under green conditions. The number of pub-
lications dedicated to this topic per year has increased
since 2011,27 and the relevance of this field is proven by the
fact that since 2018 till now, four reviews summarizing the
results of developing GSPPS were published.27-30 Specifi-
cally, performing SPPS in water, supercritical carbon
dioxide and alternative organic solvents was clearly sum-
marized in the review by Lawrenson.28 Varnava and Saro-
jini extended their summary of the literature by also con-
sidering solvent-free techniques as well as evolving the
use of greener and safer reagents.29 And perhaps because
Albericio’s group published most of the original papers,
they offered an insider perspective by comprehensively
examining all the aspects of SPPS in the context of green
chemistry studied so far, including steps such as the final
cleavage from the resin and precipitation of peptides.27
And very recently, they reviewed greening of each indi-
vidual step of the Fmoc/t-Bu-SPPS process.30
vironmental, health, and safety) profile15 enables the pro-
cess to keep the advantages of traditional solid-phase
synthesis, including the utilization of traditional com-
mercially available reagents.
Alternative solvents were most intensively studied by
Albericio and co-workers.31-40 They investigated less prob-
lematic alternatives, such as MeCN and THF, only for the
coupling step in combination with a PEG-based resin.31
However, also studied greener alternative 2-MeTHF ex-
hibited a good solubility for all the Fmoc-AAs and acyla-
tion agents as well as good swelling abilities for both the
PS- and PEG-based resins.32 2-MeTHF was used as a uni-
versal solvent for the SPPS steps, including the attach-
ment of the first AA onto both the Wang41 and chlo-
rotrityl chloride (CTC)42 resins (PS-based resins), the
assembly of the peptide chain,32 and the precipitation
step36. However, the Fmoc removal step requiring an
elevated temperature appeared to be critical to the final
crude purity of the synthesized peptide.35 To look for
suitable green solvents for Fmoc removal, Albericio’s
group evaluated γ-valerolactone (GVL) and N-
formylmorpholine (NFM).33 However, both solvents ex-
hibited some limitations. While NFM performed well only
with the PEG-based resin, GVL exhibited good results
when it was combined with both PS-34 and PEG-based
resins in amide bond formation,33 and was compatible
with microwaves.39 GVL was also a suitable solvent for the
immobilization of the first AA onto the Wang resin (but
not suitable for the CTC resin).37 However, the solvent
reacts with the less hindered Gly residues because the
ring opens under the Fmoc removal conditions, resulting
in acylation as a side reaction. The authors solved this
issue by the incorporation of the corresponding Fmoc-
AA-Gly-OH dipeptides.38 Another green solvent, cyclo-
pentyl methyl ether (CPME), was shown to be a good
precipitating ether for use in SPPS from both a chemical
point of view43 and also from a morphological perspec-
tive.40 However, CPME exhibits poor solubility of Fmoc-
AAs.32 In addition to Alberico’s group, Lawrenson at al.44
studied the utilization of propylene carbonate (PC) on a
PEG support in SPPS. More recently, Lopez et al.45 from
Novartis Pharma AG published a systematic evaluation of
more than thirty green solvents in combination with a PS-
based resin, and their best green candidate for SPPS was
N-butylpyrrolidinone (NBP). The concept of green solvent
mixtures for SPPS (GM-SPPS) introduced by Ferrazzano
and Cabri et al.46 relying on combinations of cyrene, sul-
The utilization of water as the greenest solvent requires
using unusual hydrophilic PGs, or the traditional Fmoc-
and Boc-protected AAs have to be transformed by na-
nosizing into water-dispersible nanoparticles, etc. Mean-
while, the simple substitution of the harmful solvents
DMF and DCM by a green solvent with a better EHS (en-
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folane, or anisol with dimethyl carbonate or diethyl car-
al., which explored water-dispersible Fmoc-AAs in com-
bination with PEG-based resins.75,76
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bonate enabled to tailor a media of optimal properties.
Since Fmoc removal is one of the two-repeating-steps
of SPPS (coupling/deprotection), the green performance
would have huge environmental impacts. Fmoc47-49 is an
α-amino PG utilized by the Fmoc/t-Bu SPPS strategy,50,51
which is the most commonly used SPPS approach, where
the α-amino group is protected with the base-labile Fmoc
and AA side chains with acid-labile groups.52 This meth-
odology superseded the original one introduced by Merri-
field, relying on Boc/Bzl protection.
The removal of Fmoc is typically carried out under
mildly basic conditions, and piperidine is almost exclu-
sively used as a reagent for Fmoc removal in SPPS. Piperi-
dine is toxic and scored 6.9 points (yellow/usable) on a
scale 0 – 10 in GSK’s acid and base selection guide.53
Moreover, piperidine is classified as a controlled sub-
stance serving as an intermediate for the synthesis of
illegal narcotic drugs.54-56 The main reported obstacle of
using piperidine is the mediation of aspartimide for-
mation, a notorious sequence, and conformation-sensitive
side reaction that occurs in SPPS.57,58 Last but not least,
piperidine is also an expensive reagent.
Attempts to make the Fmoc removal step greener were
dedicated to the utilization of lower concentrations of
piperidine59 or piperidine alternatives, such as methyl
piperidines,60-62 DBU,63,64 piperazine,58,62 a mixture of
piperazine and DBU,65 4-methylpiperazine,61 a series of
amines,66 or morpholine.67 All these piperidine alterna-
tives were studied only in DMF. Notable, Lopez et al.
were the first researchers to combine a green solvent and
an alternative reagent for Fmoc cleavage (4-
methylpiperidine in NBP).45 More recently, Pawlas et al.
also used 4-methylpiperidine in a mixture of green sol-
vents NBP/EtOAc68 and DMSO/EtOAc.69 On the other
hand, most of the papers dedicated to greening SPPS still
used 20% piperidine in the evaluated solvent.
By focusing on greening the SPPS process, step-by-step,
and considering that the Fmoc removal step is also crucial
for SPPS to be successful, we were interested in possible
means of improving this aspect. At the beginning of 2019,
we published a short communication describing a new
green protocol for removing the Fmoc group. The cleav-
age cocktail consisted of 0.2 M NaOH in a mixture of 2-
MeTHF and MeOH (1:1).19 The green inorganic base
NaOH scored an 8 according to GSK’s guide53 and was
also generally recognized as safe (GRAS) by the FDA.70
The utilization of NaOH in solid-phase synthesis is not
new; it has a long history since R. B. Merrifield used sa-
ponification by aq. NaOH in EtOH in his revolutionary
paper published in 1963.18 Our research group have used
NaOH in a mixture of solvents (THF/MeOH) for the ester
hydrolysis of heterocycles immobilized via amides71 on a
Rink resin,72 and as a cleavage cocktail of ester-linked
compounds on a Wang resin.73 However, the lability of
the Fmoc group toward NaOH has been reported already
in 1980,49,74 and to best of our knowledge, we have not
found any evidence of the utilization of this inorganic
base in SPPS. The only exception is the work by Hojo et
In our green Fmoc removal protocol, we substituted the
typically utilized THF71,73 for the greener alternative 2-
MeTHF.77 2-MeTHF is a bio-based solvent that can be
produced from renewable resources derived from ligno-
cellulosic biomass, such as furfural or levulinic acid, and
moreover, 2-MeTHF is biodegradable.77,78 Furthermore, 2-
MeTHF has an acceptable toxicological profile for phar-
maceutical chemical process development.79 However, the
assessment of 2-MeTHF by selection guides is not ambig-
uous (classified as recommended/green by Sanofi,80 usa-
ble/yellow by Pfizer,81 yellow by GCIPR and GSK, hazard-
ous/red by Astra Zeneca82). To conclude, it is a preferable
solvent substitute in comparison to DMF and DCM. 2-
MeTHF was shown to be superior to DMF in SPPS when it
was used only in the coupling step, but Fmoc removal was
still performed with piperidine/DMF.35 In the case of the
fully green solvent performance using only 2-MeTHF in
combination with the widely used PS resin, the Fmoc
removal step was not optimal.33
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In our previously published short communication,19 the
usability of the Fmoc removal protocol was documented
on selected single Fmoc-AAs attached to a Rink resin and
also on the synthesis of pentapeptide Leu-enkephalin
amide. Moreover, the protocol is compatible with PS-
based resin, the AA racemization was excluded, and also
the comparison of NaOH with other organic bases, such
as piperidine, morpholine, and DBU, was accomplished.
However, other features are required to be addressed in
order to make this protocol applicable for the sustainable
manufacturing of therapeutic peptides. Additionally, this
protocol has the potential to become commonly used not
only in GSPPS, but also in green solid-phase organic syn-
thesis (GSPOS) of peptidomimetics and nature-inspired
heterocycles utilizing Fmoc-AAs as building blocks. Here,
we report our green Fmoc removal protocol in terms of
the optimization of the cleavage conditions, the stability
of the acid-labile PGs, and the formation of aspartimide as
an undesired side-product. The applicability of this pro-
tocol was documented by synthesizing the commercial
peptide drug triptorelin. Triptorelin (pGlu-His-Trp-Ser-
Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2) is a synthetic decapep-
tide and belongs to a family of gonadotrophin-releasing
hormone (GnRH) analogs. Triptorelin is an agonist to the
luteinizing hormone-releasing hormone (LH-RH) recep-
tor, possesses greater potency than the native hormone,
and is resistant to proteolysis. Triptorelin is a commercial
peptide-based drug with the following clinical applica-
tions: prostate cancer, breast cancer, endometriosis, uter-
ine fibroids, assisted reproduction, endometrial thinning,
and central precocious puberty.83,84
RESULTS AND DISCUSSION
Optimization of the Fmoc removal conditions. To
investigate the scope and limitation of the presented
protocol, numerous Fmoc removal experiments (Tables 2
– 8) were tested on the model compound (resin 2) pre-
pared according to Scheme 2. Briefly, Fmoc-Ala-OH (or
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Table 1. Specification overview of the evaluated 2-MeTHF with different qualities
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2
3
4
5
6
7
8
Label
Vendor
Sigma-Aldrich
VWR
Grade
Declared purity
≥99.5%
Stabilizer BHTa
150 – 400 ppm
150 – 400 ppm
% Waterb % Water by K. F.c
A
B
ReagentPlus®
≤1.0
0.0124 ± 0.0005
0.0132 ± 0.0009
GPR RECTAPUR®
≥99.0%
≤0.03
for synthesis
C
Merck
EMPLURA®
~98%
>100 ppm
≤0.1
0.0059 ± 0.0009
b
aBHT is the abbreviation for butylhydroxytoluene (2,6-di-tert-butyl-4-methylphenol). Water content declared by the supplier.
cWater content determined by coulometric Karl Fisher titration from 5 measurements.
9
Scheme 2. Evaluation of the Fmoc removal condi-
tions using a Fmoc-Ala (or Fmoc-Gly) attached to a
Rink resina
not reveal any significant difference (the spectra are
shown in the Supporting Information).
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In the next step, the efficiencies of NaOH-based cleav-
age cocktail of various concentrations consisting of a
mixture of MeOH with 2-MeTHFs mentioned above of
different qualities were evaluated. Quantitative Fmoc
removal was achieved using 0.3 M NaOH (entries 1 – 3,
Table 2) as well as 0.2 M NaOH (entries 4 – 6), irrespec-
tively of the purity of 2-MeTHF. At a very low concentra-
tion of NaOH, 0.05 M (entries 10 – 12), all 2-MeTHFs pro-
vided incomplete Fmoc removal (81 – 91%). To conclude,
the differences between evaluated 2-MeTHFs were negli-
gible. From a practical point of view, all three 2-MeTHFs
performed similarly and are suitable for the presented
protocol.
aReagents and conditions: (i) 50% piperidine in DMF, rt, 15
min; (ii) Fmoc-AA-OH, DIC, HOBt, DCM/DMF (1:1), rt, 1 h;
(iii) NaOH, 2-MeTHF/MeOH, -2 – +40 °C, 1 – 15 min, or DBU
in MeTHF, rt, 15 min (for details, see Tables 2 – 8); (iv) 50%
TFA/DCM, rt, 30 min.
Table 2. Effect of the NaOH concentration and 2-
MeTHF source on the Fmoc removal from Fmoc-Ala-
Rink 2a
Entry
1
2
3
4
5
cNaOH
0.3 M
0.3 M
0.3 M
0.2 M
0.2 M
0.2 M
0.1 M
0.1 M
0.1 M
0.05 M
0.05 M
0.05 M
2-MeTHF
4:5
Fmoc-Gly-OH for Table 8) as the first AA was attached to
Rink amide resin (1) in the traditional manner (using
hazardous components DMF, DCM, piperidine). Then the
resin 2 was exposed to a NaOH solution under green
conditions and subsequently reacted with a second Fmoc-
AA to form Fmoc-dipeptide (3). After the TFA-mediated
cleavage of the product from the resin, the ratio of 4:5 was
estimated by HPLC at 300 nm. To obtain better chroma-
tographic separation of Fmoc-Ala-NH2 (4) and the dipep-
tide (5), Fmoc-Phe was used as the second AA. The HPLC
ratio of 4 and 5 after TFA cleavage revealed the amount of
Fmoc removed, i. e., a >99% presence of 5 indicated the
quantitative NaOH-mediated removal of the Fmoc group.
Typically, the experiments were performed on an analyti-
cal scale using the volume of the Fmoc removal cocktail 1
mL, the amount of the treated resin in each experiment
was 20 mg, and exposure to NaOH cocktail was 15 min if
not stated otherwise.
A
B
C
A
B
C
A
B
C
A
B
C
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
2:98
<1:>99
<1:>99
19:81
6
7
8
9
10
11
12
9:91
12:88
aAll the reactions were performed using 2-MeTHF/MeOH 1:1
at 25 – 26 °C.
To prove our protocol to be applicable also for the syn-
thesis of peptides at a larger (preparative) scale regardless
of the quality of used 2-MeTHF, the efficiency of Fmoc
removal was evaluated using the cocktail of 0.2 M NaOH
in a mixture of 2-MeTHF (A – C) and MeOH (1:1) on 250
mg and 1 g of Fmoc-Ala-Rink resin 2. All three studied 2-
MeTHFs achieved quantitative Fmoc removal for both
evaluated scales.
Other experiments were focused on changing the ratio
of 2-MeTHF and MeOH in the Fmoc removal cocktail.
The utilized solvents play different roles. MeOH is im-
portant for dissolving NaOH, but at the same time, it
causes the resin to shrink. On the other hand, 2-MeTHF
ensures the swelling of the resin. To achieve better resin
By exploring the possibilities of optimizing the Fmoc
removal cocktail, several aspects needed to be addressed.
In the beginning, we turned our attention to the composi-
tion of the cocktail. We were interested in the impact of
the quality of the used 2-MeTHF since vendors offer vari-
ous purities for different prices (Table 1). We purchased
high-quality 2-MeTHFs from Sigma-Aldrich (purity
≥99.5%, for a simplification we use label A) and VWR
(≥99.0%, label B). Additionally, we also evaluated low-
cost EMPLURA from Merck (reportedly, its purity exceeds
1
98%, label C).85 First, H NMR of each 2-MeTHF type did
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swelling, the solutions of 2-MeTHF with MeOH in the 0.2 M NaOH in 2-MeTHF/MeOH 1:1 (Table 5). While 1
1
ratio of 1:1, 3:1, and 4:1 and all without NaOH addition
were studied (an evaluation of the resin swelling86 is in-
cluded in the Supporting Information). Next, the efficien-
cy of Fmoc removal by using cocktails of NaOH with a 2-
MeTHF/MeOH ratio of 3:1, and 4:1, was studied on the
model Fmoc-Ala-Rink resin 2 (Table 3). 0.2 M NaOH in
both studied solvent ratios provided a quantitative Fmoc
cleavage (entries 1 and 2), but the solubility of NaOH in
the 4:1 solution was slightly reduced. Decreasing the con-
centration to 0.1 M NaOH showed incomplete Fmoc
cleavage (entry 6), indicating a worsening trend (compare
to entry 9 in Table 2). Therefore, to find out if more con-
centrated (0.2 M) NaOH cocktail with the ratio of sol-
vents 4:1 tolerates the presence of higher water content,
which could increase the solubility of NaOH in cleavage
cocktail, three additions of water up to 20% (v/v) were
evaluated (entries 3 – 5). In all three cases, Fmoc was
quantitatively removed; however the use of 20% addition
of water into cleavage cocktail led to the formation of an
emulsion (entry 5), which is not suitable for synthesis.
The Fmoc removal cocktail containing 5% addition of
water (entry 3) was also successfully applied for the Fmoc
removal step at a preparative scale (up to 1 g of resin).
Another notable point is the reaction temperature. To
easily observe a potential improvement or deterioration
of Fmoc removal efficiency, a low concentrated (0.05 M)
NaOH-based cleavage cocktail providing only 88% effi-
ciency at room temperature (entry 2 in Table 4) was se-
lected for this study. The lower temperature of -2 °C
slowed the reaction to achieve only 21% (entry 1), whereas
the elevated temperature of 40 °C (entry 3) removed
Fmoc almost quantitatively.
and 5 min reactions resulted in partial Fmoc cleavage,
only 32% and 73%, respectively (entries 1 and 2), 10 min
treatment already led to quantitative Fmoc removal (en-
try 3), and 15 min Fmoc removal is a guarantee (entry 4).
In the next step, the effect of the utilized hydroxide was
evaluated. Potassium hydroxide and sodium hydroxide
scored an 8 according to GSK’s guide53 and are both GRAS
by the FDA. On the other hand, lithium hydroxide re-
ceives only a 5.7 on the scale of greenness and is not
GRAS.70 For this study, 2-MeTHF B was mixed with
MeOH to achieve ratios of 1:1 and 3:1. Under these condi-
tions, NaOH provided quantitative Fmoc removal, and
KOH reached very similar results (entries 3 and 4 in Table
6). In contrast, LiOH was significantly less soluble than
the other studied hydroxides, and also, its efficiency of
Fmoc removal was only 53% and 65% (entries 5 and 6).
For all these reasons, LiOH is not a suitable alternative.
To make our protocol greener, the possibility of exchang-
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
ing MeOH for EtOH, alcohol with
a better EHS
profile,85,87 was tested. Probably due to the significantly
lower solubility of NaOH in EtOH than in MeOH, even
0.3 M (the highest concentration studied) or lower con-
centrations of NaOH provided, at most, only 41% Fmoc
removal (entry 1, Table 7). To increase the solubility of
NaOH, water was added into the studied mixture. The
Fmoc cleavage cocktail containing 3% or 10% addition of
water improved the outcome to 65% and 79%, respective-
ly (entries 2 and 3). However, the higher addition of water
(20%) into the mixture led to the formation of emulsion,
providing a low efficiency of Fmoc removal (entry 4).
Therefore, MeOH cannot be replaced by EtOH as greener
alcohol.
In an attempt to accelerate the Fmoc removal step, the
reaction time was evaluated as another parameter of the
protocol. The experiments were performed by applying
Table 5. Effect of the reaction time on the Fmoc re-
moval from Fmoc-Ala-Rink 2a
Entry
Time
1 min
5 min
10 min
15 min
4:5
68:32
27:73
<1:>99
<1:>99
Table 3. Effect of the ratio of 2-MeTHF/MeOH on the
Fmoc removal from Fmoc-Ala-Rink 2a
1
2
3
4
Entry cNaOH
2-MeTHF/MeOH
Water
addition
0%
0%
5%
10%
20%
0%
4:5
aAll the reactions were performed using 0.2 M NaOH in 2-
1
0.2 M
0.2 M
0.2 M
0.2 M
0.2 M
0.1 M
3:1
4:1
4:1
4:1
4:1
4:1
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
3:97
MeTHF B with MeOH 1:1 at 25 °C.
2
3
4
5
6
Table 6. Effect of the utilized hydroxide on the Fmoc
removal from Fmoc-Ala-Rink 2a
aAll the reactions were performed at 24 – 26 °C. For all these
Entry Hydroxide 2-MeTHF/MeOH
4:5
1
NaOH
NaOH
KOH
KOH
LiOH
LiOH
1:1
3:1
1:1
3:1
1:1
3:1
<1:>99
<1:>99
3:97
2:98
47:53
35:65
experiments, 2-MeTHF C was used.
2
3
4
5
6
Table 4. Effect of the temperature on the Fmoc re-
moval from Fmoc-Ala-Rink 2a
Entry
1
2
3
Temperature
-2 °C
4:5
79:21
12:88
2:98
aAll the reactions were performed using 0.2 M hydroxide
(prepared from a 4.7 M resp. 12.5% aq. stock solution; result-
ing water content in the Fmoc cleavage cocktail was 4%) in
2-MeTHF B with MeOH at 26 °C.
25 °C
40 °C
aAll the reactions were performed using 0.05 M NaOH in 2-
MeTHF C with MeOH 1:1.
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Table 7. Effect of exchanging MeOH for EtOH on the
Fmoc removal from Fmoc-Ala-Rink 2a
Table 8. The Fmoc removal from Fmoc-Gly-Rink 2a
using NaOH or DBU as basea
1
2
3
4
5
6
7
8
Entry 2-MeTHF/EtOH Water addition
4:5
Entry
Base
2-MeTHF/
MeOH
Water
addition
0%
0%
0%
0%
0%
5%
5%
4a:5a
1
2
3
4
1:1
1:1
1:1
1:1
0%
3%
10%
20%
59:41
35:65
21:79
53:47
1
0.3 M NaOH
0.2 M NaOH
0.2 M NaOH
0.1 M NaOH
0.2 M NaOH
0.2 M NaOH
0.2 M NaOH
1% DBU
1:1
1:1
1:1
1:1
3:1
3:1
4:1
100:0
100:0
17:83
19:81
2
3
4
5
6
7
8
9
5:95b
19:81
aAll reactions were performed at 23 – 24 °C using 0.3 M
NaOH in 2-MeTHF C with EtOH 1:1, and with or without an
appropriate amount of water addition.
14:86
<1:>99
3:97
<1:>99
<1:>99
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
To conclude this section, the optimal and also recom-
mended concentration of NaOH for achieving a quantita-
tive Fmoc removal is 0.2 M. The ratio of 2-MeTHF and
MeOH in cleavage cocktail may range from 1:1 to 4:1. The
recommended reaction time for quantitative Fmoc re-
moval is 15 min; however, 10 min should also be suitable.
NaOH can be eventually substituted by KOH, but not by
LiOH. The presence of MeOH in the Fmoc removal cock-
tail is necessary, mainly to serve as a solvent for dissolving
NaOH. However, MeOH cannot be replaced with greener
alcohol, such as EtOH.
0%
0%
0.5% DBU
aFor all these experiments, 2-MeTHF C was used, and the
reaction temperature was 25 °C except for entry 3. bThe reac-
tion was performed at 40 °C.
To determine if this unexpected behavior of Gly will al-
so manifest with bases other than NaOH, we investigated
DBU for removing Fmoc from the Fmoc-Gly residue. In
contrast to NaOH, 1%, and 0.5% DBU (entries 8 and 9)
were the concentrations evaluated to remove Fmoc com-
pletely.
According to green solvent selection guides, MeOH is a
less desirable solvent in the mixture of 2-MeTHF/MeOH,
but its role is inherent. Since MeOH could not be ex-
changed with EtOH, we tried to use the minimum MeOH
amount necessary. Therefore, we evaluated 3:1 (2-
MeTHF/MeOH) an optimized (0.2 M NaOH) protocol for
all the previously studied19 Fmoc-AAs (Gly, Phe, Lys(Boc),
Pro, Ser(Ot-Bu), Thr(Ot-Bu), Tyr(Ot-Bu), Asp(Ot-Bu),
Glu(Ot-Bu), Val, Leu, and Ile), and we newly extended the
protocol used for Trp(Boc) and His(Boc) and compared it
to the original 1:1 (2-MeTHF/MeOH) protocol. The com-
plete Fmoc removal was achieved using both protocols for
all the tested Fmoc-AAs with only one exception – Gly,
which was not in accordance with our previous results.19
Although Gly is, from a structural point of view, the sim-
plest AA, it cannot be considered a typical AA, and there-
fore, an in-depth study was required. The results are
summarized in Table 8. While 0.2 M NaOH ensured
quantitative Fmoc removal for other AAs, in the case of
Fmoc-Gly-Rink resin 2a (Scheme 2), the efficiency of
Fmoc removal was only 81% (entry 2). Increasing or de-
creasing the hydroxide concentration provided very simi-
lar results (entries 1 and 4). The expected trend was ob-
served at elevated temperature. The ratio of the cleaved
Fmoc group increased to 95% when the reaction was
performed at 40 °C (entry 3). Considering the solvent
content of the Fmoc cleavage cocktail, the utilization of
the 3:1 protocol to achieve better swelling than the 1:1
protocol did not have a significant influence on the out-
come (entry 5). Surprisingly, the 5% addition of water
into the 3:1 mixture (entry 6) resulted in quantitative
Fmoc removal. This composition of the Fmoc cleavage
To verify the optimized protocol for the Fmoc-Gly resi-
due, we synthesized the pentapeptide Leu-enkephalin
amide with a structure of H-Tyr-Gly-Gly-Phe-Leu-NH2,
which contains two troublesome Gly residues in the se-
quence. For the synthesis, a protocol used in a previously
published short communication report was adopted.19
Acylation was carried out in 2-MeTHF B, which was also
used for the Fmoc removal step. The ratio of the solvents
was 3:1 to achieve better swelling of the resin and also to
provide an alternative to the previous 1:1 protocol. When
the Fmoc group from Gly was being removed, the opti-
mized protocol with the 5% addition of water was ap-
plied. For the Fmoc removal of both Fmoc-Gly residues,
we evaluated at an analytical scale (with ca. 20 mg of
resin) all three protocols: 1:1, 3:1, and 3:1 + 5% addition of
water (see the chromatograms in the Supporting Infor-
mation). It should be noted that removing Fmoc from the
Fmoc-Gly residue with Gly in the position of the third AA
at solvent ratios of 1:1 and 3:1 + 5% addition of water was
quantitative, whereas, in the case of the 3:1 protocol, a
significant amount of the Fmoc-derivative was detected.
Removing Fmoc from the following Gly residue in the
position of the fourth AA was even more difficult. Both
protocols without water addition partly failed, and only
the 3:1 protocol with 5% addition of water removed Fmoc
quantitatively at an analytical scale (20 mg of resin).
However, the preparative performance of using 0.5 g of
Fmoc-Gly-Gly-Phe-Leu-NH-Rink resin was carried out,
and the Fmoc removal process required one repetition. It
is worth to mention that we have not noticed such diffi-
culties previously when synthesizing Leu-enkephalin
amide.19 The reason might be that the used 2-MeTHF
solvent with an EMPLURA grade obtained from Sigma-
Aldrich (not from Merck) contained a higher amount of
water. In any case, the optimized SPPS protocol for
cocktail was also successfully applied at
a semi-
preparative scale (up to 1 g of resin), and therefore, it was
afterward included in the utilized methods for the syn-
thesis of triptorelin. Further increasing the ratio of 2-
MeTHF up to 4:1 did not form a clear solution (entry 7).
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Scheme 3. Design of the first PG stability experimenta
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
aReagents and conditions: (i) Fmoc-Ala-OH, HOBt, DIC, DMAP, DCM/DMF (1:1), rt, on; (ii) 50% piperidine in DMF, rt, 15 min;
(iii) Fmoc-Rink linker, HOBt, DIC, DCM/DMF (1:1) rt, 1 h; (iv) Fmoc-AA(X-PG)-OH, HOBt, DIC, DCM/DMF (1:1), rt, 1 h; (v) 50%
TFA/DCM, rt, 30 min; (vi) 0.2 M NaOH, 2-MeTHF/MeOH (1:1), rt, 30 min.
removing Fmoc from Gly residues and final isolation pro-
cess, as described in the previous short communication
report,19 produced the target pentapeptide in a crude
purity of 99% and an overall yield of 72% (refer to the
group of Lys (10) or aromatic nitrogen of Trp (12). The
combination of the racemic Rink linker and two optically
pure AAs (20) resulted in a mixture of two diastereomers
(see the chromatograms in the Supporting Information).
In the second approach, the Fmoc-AAs (Figure 1) were
immobilized on the Wang linker (21, Scheme 4). Then,
the Fmoc group was removed using piperidine/DMF, and
the liberated amino group was reacted with 4-nitroben-
zensulfonyl chloride to ensure the good absorbance and
1
chromatogram and H NMR spectrum in the Supporting
Information). These values proved the good applicability
of this novel Fmoc removal protocol comparable to the
traditional protocol that uses piperidine and DMF with
DCM as solvents.
Stability of AA side-chain protecting groups. In
Fmoc/t-Bu SPPS, the AA side-chain functionalities are
protected with orthogonal acid-labile PGs.21 To evaluate
the stability of these PGs toward our Fmoc removal cock-
tail, we designed two sets of experiments involving side-
chain PGs t-Bu, Boc, Trt, and Pbf by utilizing Fmoc-
Ser(Ot-Bu)-OH (6, Figure 1), Fmoc-Tyr(Ot-Bu)-OH (7),
Fmoc-Asp(Ot-Bu)-OH (8), Fmoc-Glu(Ot-Bu)-OH (9),
Fmoc-Lys(Boc)-OH (10), Fmoc-His(Boc)-OH (11), Fmoc-
Trp(Boc)-OH (12), Fmoc-Asn(Trt)-OH (13), Fmoc-
Gln(Trt)-OH (14), Fmoc-His(Trt)-OH (15), and Fmoc-
Arg(Pbf)-OH (16).
In the first set of experiments, the Fmoc-AA(X-PG)-Rink-
linker-Ala-Wang resin models (18, Scheme 3) were syn-
thesized. The Rink linker ensured reliable chromato-
graphic separation, and in addition, it is also a good
chromophore as well as the Fmoc group. Treatment with
the TFA cleavage cocktail (conditions (v)) resulted in the
liberation of Fmoc-AA(XH)-NH2 (19) and PG removal
from the side chain. On the other hand, the NaOH cock-
tail (conditions (vi)) cleaved only the ester-based attach-
ment of Ala to the Wang resin and also removed the
Fmoc group, while the PGs of all the studied AAs stayed
intact (20) except for Boc on Fmoc-His (11). On the con-
trary, Trt, as another studied acid-labile PG of His (15)
remained intact. Interestingly, Boc was stable to NaOH
treatment when serving as protection of aliphatic amino
Figure 1. Structures of selected Fmoc-AAs with side-
chain protection
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Scheme 4. Design of the second PG stability experimenta
Page 8 of 16
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
aReagents and conditions: (i) Fmoc-AA(X-PG)-OH, HOBt, DMAP, DIC, DCM/DMF (1:1), rt, on; (ii) 50% piperidine in DMF, rt, 15
min; (iii) 4-Ns-Cl, lutidine, DCM, rt, 2.5 h; (iv) 50% TFA/DCM, rt, 30 min; (v) 0.2 M NaOH, 2-MeTHF/MeOH (1:1), rt, 30 min.
also better chromatographic retention of the cleaved
compounds (23, 24). An analytical amount of resin 22 was
treated using acidic (conditions (iv)) and basic (condi-
tions (v)) cleavage cocktails, and the presence/absence of
the PGs was evaluated using HPLC-MS. Obtained results
were in agreement with previous experiments. The TFA
cocktail caused the cleavage of material from the resin
and, also, the removal of the PGs (23). On the other hand,
NaOH cleaved the compounds from the resin, and the
PGs remained intact (24). Only in the case of Fmoc-
His(Boc) the material without PG was detected, which
can be avoided using Trt for the side chain protection.
Notably, the methanolic NaOH cleavage cocktail caused
re-esterification into methyl ester derivatives 24b, and
saponification led to the formation of carboxylic acids 24a
(see the chromatograms in the Supporting Information).
Compatibility with amide- and ester-based types of
attachments. The NaOH Fmoc removal cocktail is com-
patible only with the amide-based attachment, which is
achieved, for example, via the Rink amide resin, resulting
in peptide amides such as GnRH analogs. However, the
stability of tert-butyl esters (Asp(Ot-Bu) and Glu(Ot-Bu))
led us to assume that the ester linkage formed via the
trityl-based CTC resin might be more stable than the
Wang linkage due to steric hindrances, allowing for the
preparation of terminal carboxylic acid peptides. This
hindered resin has already been shown to be convenient
for avoiding diketopiperazine formation as a side reac-
tion.88 Therefore, we performed the following sequence of
experiments (Scheme 5). Fmoc-Ala-OH was attached to
the CTC resin (25), and the loading was determined by
HPLC to be 0.44 mmol/g. The exposure to the NaOH
cocktail was followed by the reaction with Fmoc-OSu,
and the loading decreased to only 0.002 mmol/g, indicat-
ing that almost no material remained attached to the
resin. To conclude, the ester-type of attachment formed
via the CTC resin is not compatible with the presented
green protocol.
Aspartimide formation. To evaluate the effect of the
NaOH treatment on this serious side reaction in SPPS, a
susceptible peptide sequence was synthesized in a tradi-
tional manner (Scheme 6) and subsequently exposed to
an extended treatment of the base. The evaluated hex-
apeptide was a fragment of the scorpion toxin II H-Val-
Lys-Asp-Gly-Tyr-Ile-NH2, which is particularly prone to
aspartimide formation.58,89 Our green Fmoc removal pro-
tocol was examined and compared to a reagent typically
used for Fmoc removal – piperidine, and green base DBU.
Both piperidine and DBU58,63,64 are known to induce as-
partimide formation. Piperidine, as a nucleophilic base,
also causes subsequent adduct formation. Scheme 6,
which shows the pathway of aspartimide (29) and the by-
products (30 – 33) formation, was adapted according to a
reference.58 First, the Fmoc-hexapeptide using standard
Fmoc-SPPS protocols was prepared, and then, the Fmoc
group was cleaved using 50% piperidine in DMF to obtain
peptide 28. This polymer-supported peptide was subse-
quently exposed to 50% piperidine in DMF, 3% DBU in
DMF or 2-MeTHF, and 0.2 M NaOH in 2-MeTHF/MeOH
1:1 and 3:1 at room temperature for 24 h. The products
were cleaved by 50% TFA/DCM for 1 h to secure the
cleavage of the Boc and t-Bu PGs. The cleaved compounds
were analyzed by HPLC-MS (see the chromatograms in
the Supporting Information). The results showed that
piperidine induced substantial degrees of both aspar-
timide and base adduct formation, and only 13% of the
target peptide remained intact. DBU caused 37%, resp.
16% aspartimide formation. The chromatogram obtained
for the extended treatment with NaOH, the 1:1 protocol,
was almost identical to this obtained for the untreated
one, indicating no aspartimide formation. In
Scheme 5. Evaluation of the stability of the trityl-
based CTC resin toward the NaOH cocktaila
aReagents and conditions: (i) Fmoc-Ala-OH, DIEA, dry DCM,
rt, on, first determination of loading; (ii) 0.2 M NaOH, 2-
MeTHF/MeOH (1:1), rt, 30 min; (iii) Fmoc-OSu, DCM, rt, 1 h,
second determination of loading.
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Table 9. Methods used for the preparation of the model decapeptide triptorelina
1
2
3
4
5
6
7
8
Method
Swelling
(A)
3x DCM
3x DMF
(B)
(C)
3x 2-MeTHF
(D)
3x 2-MeTHF
3x 2-MeTHF
Deprotection
50% PIP/DMF
rt, 15 min
0.2 M NaOH
2-MeTHF/MeOH (1:1)b
rt, 15 min
0.2 M NaOH
0.2 M NaOH
2-MeTHF/MeOH (1:1)b 2-MeTHF/MeOH (3:1)b
rt, 15 min
rt, 15 min
5x 2-MeTHF
Washing
Coupling
5x DMF
3x DCM
3x 2-MeTHF
3x DCM
5x 2-MeTHF
9
Fmoc-AA-OH, HOBt, DIC
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
DMF/DCM (1:1)
rt, 1 h
DMF/DCM (1:1)
rt, 1 h
2-MeTHF
rt, 1 h
2-MeTHF
rt, 1 h
Washing
3x DMF
3x DCM
3x DMF
3x DCM
3x 2-MeTHF/MeOH
(1:1)
3x 2-MeTHF/MeOH
(1:1)
3x 2-MeTHF
3x 2-MeTHF
aReaction steps performed using hazardous components are in red color, and environmentally friendly approaches are in
green. b0.2 M NaOH in 2-MeTHF/MeOH 3:1 + 5% addition of water was used to remove Fmoc from the Gly residue.
Scheme 6. Synthesis of model peptide 28 and base-
mediated formation of aspartimide and by-productsa
another difficult structural moiety in Arg with the guani-
dine unit. The synthesis was carried out using Fmoc-AAs
on a Rink amide PS/DVB-based resin.72 The swelling,
deprotecting, coupling, and washing steps are given in
Table 9. The traditional protocol (method A) relied on the
utilization of hazardous reagents DMF, DCM, and piperi-
dine and served as a standard for comparison. The com-
bination of traditional acylation and green deprotection
(method B) was included to simplify the evaluation of the
green Fmoc removal impact on the crude purity of the
final decapeptide. As well two fully green protocols
(methods C and D) using 2-MeTHF for coupling and
NaOH in 2-MeTHF/MeOH, 1:1, and 3:1, for deprotection
were evaluated.
The decapeptides synthesized according to methods A
– D were cleaved from the resin by a mixture of TFA/H2O
(95:5). The cleavage cocktail was then evaporated, and the
peptides were precipitated by diethyl ether. Isolated
1
decapeptides were characterized by HRMS and H NMR.
The crude purities were determined from the HPLC traces
and are summarized in Table 10. One example of chroma-
togram illustrating the presence of side-product is shown
in Figure 2. Yields were calculated from NMR spectra with
respect to the resin loading. The identity of prepared
decapeptides was proven using a commercially available
triptorelin standard purchased by AAPPTec (see the
aReagents and conditions: (i) 50% piperidine in DMF, rt, 15
min; (ii) Fmoc-AA-OH, DIC, HOBt, DCM/DMF (1:1), rt, 1 h;
repeating steps (i), (ii) until the completed elongation of the
desired hexapeptide; (iii) base; (iv) nucleophilic base.
1
chromatograms and H NMR spectra in the Supporting
Information). Note that the SPPS process proceeded well
for all four methods until the penultimate step – the
Fmoc removal from His residue – before the last acylation
step with pGlu. The treatment with NaOH resulted in the
formation of earlier eluted impurity/side-product with
the same mass spectrum. The ratios of side-product to
product are shown in Table 10. In the case of method D,
when the 3:1 protocol was used, 30% of the side-product
was formed. Therefore, this side-product was isolated
from this mixture and characterized. The HRMS spectra
the case of 3:1 protocol, an additional peak (12%, with a
higher retention time) with the same mass as that of line-
ar hexapeptide 28 (88%) without a PG, presumably the β-
peptide (33), was detected.
Synthesis of commercial peptide triptorelin. To
document the applicability of the presented green Fmoc
removal protocol, we used the SPPS method to synthesize
the commercial drug decapeptide triptorelin (pGlu-His-
Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2). This commer-
cial peptide has a challenging sequence since it includes
AAs with heterocyclic side-chain substitutions, namely,
two Trp (one as D-isomer) and His. Furthermore, there is
1
were comparable to those of triptorelin, but H NMR
showed slight differences in the aromatic part. The com-
parison suggested the correct peptide sequence with
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Page 10 of 16
Table 10. Purities and yields of the decapeptide trip-
torelin prepared using different methods
the crude peptide showed only one peak corresponding to
1
2
3
4
5
6
7
8
the main product with no side-product occurrence (see
the Supporting Information). These results indicated that
the side-product formation is caused by the presence of
His. Therefore His was incorporated into entirely unrelat-
ed peptide Leu-enkephalin to evaluate its impact on the
Method
Crude
puritya
96%
Purity including
side-product
SP:Pb
Yieldc
A
B
96%
77%
0:100
6:94
83%
71%
72%
side-product
formation.
Polymer-supported
Leu-
C
72%
57%
83%
87%
82%
83%
17:83
30:70
0:100
53%
48%
80%
enkephalin was acylated with Fmoc-His(Boc)-OH in a
traditional manner, and the Fmoc removal was carried
out using the green 3:1 protocol. Interestingly, no side-
product was detected (see Supporting Information).
Based on this experiment, it was confirmed that our green
Fmoc removal protocol can be applied for the preparation
of His-containing peptides. Furthermore, we have proved
that the formation of the side-product in the synthesis of
triptorelin is sequence-dependent. It has to be noted that
to entirely eliminate the formation of the undesired trip-
torelin side-product, 3% DBU in 2-MeTHF should be
applied as a green alternative for removing Fmoc from the
His residue (protocol E, Table 10) resulting in decapeptide
of 83% purity. Obtained yields ranged from 48 to 83%
depending on used protocol (see Table 10). The combina-
tion of green Fmoc removal and traditional acylation
(Method B) provided comparable yield to traditional
approach (Method A) indicating that green Fmoc removal
step did not cause substantial decrease of peptide yield.
Both protocols employing fully green solvent performance
(Methods C and D) led to lower yields (53% and 48%,
respectively) suggesting that green acylation in 2-MeTHF
might be responsible for this decrease. Using DBU in 2-
MeTHF only for Fmoc removal from His residue did not
significantly affect isolated yield (compare Methods A and
E).
D
Ed
9
aPurities were estimated from the HPLC traces obtained at
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b
220–400 nm. SP:P means the ratio of side-product to prod-
c
uct. Yields were calculated from the NMR spectra of the
decapeptides following cleavage with TFA/H2O (95:5) at rt
for 4 h, evaporation with N2, and precipitation with diethyl
d
ether. Modified Method A, where Fmoc removal from His
residue was performed using 3% DBU in 2-MeTHF at rt for 15
min.
1.5e+1
1.4e+1
triptorelin
(70%)
1.3e+1
side-product
1.2e+1
1.1e+1
1.0e+1
9.0
0.55
0.52
(30%)
8.0
2.19
0.48
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.18
0.94
-1.25e-7
-1.0
-2.0
-3.0
Time
-0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
4.40
4.60
4.80
5.00
5.20
5.40
Figure 2. HPLC chromatogram of triptorelin prepared
using Method D.
differences in chemical shifts of His and Trp signals (see
1H NMR spectra in the Supporting Information). To elim-
inate potential racemization of His, we prepared trip-
torelin by incorporating D-His instead of L-His using a
traditional protocol to obtain a peptide containing a D-
isomer. The chromatographic co-elution of the isolated
side-product with the triptorelin sequences having L- or
D-His residues clearly eliminated the racemization of His.
Also, the racemization studied on a simpler dipeptide
model (according to Scheme 2, when first AA = His in-
stead of Ala) did not indicate any epimerization. Keeping
on mind the lability of Boc PG on His under NaOH
treatment, triptorelin with incorporated Trt protected His
was prepared using traditional protocol up to the critical
step and Fmoc from His(Trt) residue was cleaved using 3:1
green protocol. Regardless of used His side chain PG, the
identical side-product was formed, indicating no effect of
the PG. To eliminate a potential cis/trans isomerism,
solutions of isolated triptorelin and side-product in
DMSO were exposed to 80 °C for 2.5 h. However, no
transformation occurred, and both compounds were sta-
ble under these conditions. To prove that the formation
of side-product is connected to the presence of His in
triptorelin sequence, modified decapeptide replacing His
for Phe was prepared in a traditional protocol up to the
step of Fmoc removal from Phe residue, and subsequently
exposed to green Fmoc removal cocktail, the 3:1 protocol,
which was more prone to the formation of the side-
product. The synthesis was accomplished by the coupling
with pGlu, and decapeptide was isolated. HPLC trace of
To conclude, the presented method for green Fmoc re-
moval was successfully applied in SPPS of commercial
peptide triptorelin and proved to be amenable for the
production of peptides.
CONCLUSIONS
Following the short communication published in January
2019, in this full paper, we address further aspects of a
new green Fmoc removal protocol employing mineral
base NaOH. All features of the presented protocol were
evaluated in detail on selected model compound contain-
ing Fmoc-Ala, and optimized conditions were subse-
quently screened also for other Fmoc-AAs. Notably, the
polymer-supported Fmoc-Gly residue required a specific
protocol utilizing a 5% addition of water in the Fmoc
removal cocktail. Further, the AA side-chain PGs, such as
t-Bu, Boc, Trt, and Pbf, proved their stability by remaining
intact after exposure to NaOH-based Fmoc removal cock-
tail with only one exception of Boc group protecting His.
Using a hexapeptide fragment of scorpion toxin II, it was
confirmed that our evaluated green protocol does not
induce aspartimide formation, which is another ad-
vantage over using hazardous piperidine. Furthermore,
the protocol is compatible with the most commonly used
PS-based resin when the amide-type of attachment uses
the Rink amide resin. Wang and CTC resins are not com-
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The Journal of Organic Chemistry
Purification was carried out using semipreparative HPLC
patible since their attachment via an ester is unstable
1
2
3
4
5
6
7
8
Agilent on YMC-Actus Pro 20 x 100 mm C18 reverse phase
column, 5 µm particles. Mobile phases: 0.1% TFA in HPLC
grade water (A) and gradient grade acetonitrile for HPLC (B).
A gradient was formed from 10% to 50% in 6 min, with a flow
rate of 15 mL/min.
under the NaOH treatment. Despite this limitation, there
are numerous peptide amides produced on a large scale,
such as analogs of oxytocin and LH-RH. To document the
applicability, the commercially produced decapeptide
triptorelin, a synthetic agonist analog of LH-RH, was
synthesized using several green protocols. The crude
purities of the prepared decapeptide ranged from 57 to
83% were acceptable regarding the one achieved with the
traditional protocol using hazardous piperidine in combi-
nation with hazardous solvents DMF and DCM.
1
All H NMR experiments were performed at magnetic field
strengths of 9.39 T (with operating frequencies 399.78 MHz)
at ambient temperature (20 °C). All the spectra were refer-
enced relative to the signal of DMSO (δ = 2.49 ppm).
9
HRMS analyses were performed using UPLC Dionex Ulti-
mate 3000 equipped with Orbitrap Elite high-resolution
mass spectrometer Thermo Exactive plus operating at full
scan mode (120,000 FWMH) in the range of 100 – 1800 m/z.
The settings for electrospray ionization were as follows: oven
temperature of 150 °C and the source voltage of 3.6 kV. The
acquired data were internally calibrated with diisooctyl
phthalate as a contaminant in MeOH (m/z 391.2843). Prior
UPLC separation (column Phenomenex Gemini C18, 2 x 50
mm, 3 μm particles), the samples diluted in MeOH were
injected by direct infusion into the MS using an autosampler.
Isocratic elution was performed using the mobile phase
formed of acetonitrile/isopropyl alcohol/10 mM ammonium
acetate (40:5:55), and a flow rate was 0.3 mL/min.
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EXPERIMENTAL SECTION
HRMS of triptorelin prepared using protocol A:
HRMS (ESI-TOF) m/z calculated for C64H82N18O13 [M+H]+
1311.6382, found 1311.6387; [M-H]- 1309.6225, found 1309.6221.
HRMS of triptorelin prepared using protocol B:
HRMS (ESI-TOF) m/z calculated for C64H82N18O13 [M+H]+
1311.6382, found 1311.6384; [M-H]- 1309.6225, found 1309.6226.
HRMS of triptorelin prepared using protocol C:
HRMS (ESI-TOF) m/z calculated for C64H82N18O13 [M+H]+
1311.6382, found 1311.6387; [M-H]- 1309.6225, found 1309.6225.
HRMS of triptorelin prepared using protocol D:
Determination of water content in evaluated 2-MeTHFs was
performed using coulometric Karl Fischer titration on 831 KF
Coulometer (Metrohm) with CombiCoulomat frit Karl Fisch-
er reagent (Merck).
HRMS (ESI-TOF) m/z calculated for C64H82N18O13 [M+H]+
1311.6382, found 1311.6387; [M-H]- 1309.6225, found 1309.6230.
HPLC and NMR spectra are included in the Supporting In-
formation.
Experimental Procedures
Material and Methods
Fmoc removal of the Fmoc Rink amide resin and subse-
quent acylation with Fmoc-AA-OH (Resin 2):
Solvents were used without further purification. The Rink
amide resin (100-200 mesh, 1% DVB, 0.57 mmol/g), Wang
linker (100-200 mesh, 1% DVB, 0.9 mmol/g) and 2-
chlorotrityl chloride resin (100-200 mesh, 1% DVB, 0.85
mmol/g) were used. The synthesis was carried out on Dom-
ino Blocks (www.torviq.com) in disposable polypropylene
reaction vessels.
The Fmoc Rink amide AM resin, resin 1 (1 g, 0.61 mmol/g, 100
– 150 mesh), was washed with DCM (3× 10 mL) and DMF (3×
10 mL) and treated with a solution of 50% piperidine in DMF
(10 mL) at rt for 15 min. The resin was thoroughly washed
with DMF (5× 10 mL) and DCM (3× 10 mL). Then, a solution
of Fmoc-AA (2 mmol), HOBt•H2O (2 mmol, 306 mg) and DIC
(2 mmol, 313 μL) in 10 mL of DCM/DMF (1:1) was added, and
the reaction slurry was shaken at rt for 1 h. The resin was
washed with DMF (3× 10 mL) and DCM (3× 10 mL).
All reactions were carried out at ambient temperature
(~24 °C) unless stated otherwise. The volume of wash solvent
was 10 mL per 1 g of resin unless stated otherwise. For wash-
ing, resin slurry was shaken with the fresh solvent for at least
1 min before changing the solvent. After adding a reagent
solution, the resin slurry was manually vigorously shaken to
break any potential resin clumps. Resin-bound intermediates
were dried by a stream of nitrogen for prolonged storage
and/or quantitative analysis.
Example of the treatment with NaOH in 2-
MeTHF/MeOH:
Resin 2 (ca. 20 mg) was washed 3× with 2-MeTHF and treat-
ed with 0.2 M NaOH (10.6 μL of 50% (w/v) NaOH ~ 18.94 M
NaOH aq. solution) in 2-MeTHF/MeOH (1:1, v/v; 1 mL) for 15
min at rt. The resin was then washed 5× with 2-MeTHF and
cleaved by 50% TFA in DCM for 30 min at rt, and the cleaved
compound was extracted into MeOH for the HPLC-MS
measurement.
For the LC-MS analysis, a sample of resin (~5 mg) was treat-
ed with 50% TFA in DCM for 30 min or with H2O/TFA (5:95)
for 1 h, the cleavage cocktail was evaporated by a stream of
nitrogen, and cleaved compounds extracted into 1 mL of
MeOH.
Evaluation of NaOH, KOH and LiOH as hydroxides:
A 4.735 M, resp. 12.5% (w/v) aqueous solution of NaOH was
prepared by dissolving the stock solution (50% (w/v) NaOH
~ 18.94 M NaOH aq. solution) in water. Appropriate amounts
of solids were dissolved in water to prepare the 4.735 M, resp.
12.5% (w/v) aqueous stock solutions of KOH and LiOH. For
each hydroxide, 42.2 µL of a 4.735 M (12.5%) aqueous solu-
tion was added to 960 µL of a solvent to achieve an appropri-
ate ratio of 2-MeTHF and MeOH. The percentage water
content in the Fmoc removal cocktail was approximately 4%
in all the cases.
The LC-MS analyses were carried out using UPLC Waters
Acquity equipped with PDA and QDa detectors. The system
comprised XSelect® HSS T3 (Waters) 3 x 50 mm C18 reverse
phase column XP, 2.5 µm particles. Mobile phases: 10 mM
ammonium acetate in HPLC grade water (A) and gradient
grade acetonitrile for HPLC (B). A gradient was mainly
formed from 20% to 80% of B in 4.5 min, kept for 1 min, with
a flow rate of 0.6 mL/min. The MS ESI operated at cone
voltage 25 V, and at probe temperature 600 °C and source
temperature 120 °C.
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Page 12 of 16
Example of the treatment with DBU in 2-MeTHF:
A sample of resin 26 was washed with DCM (5× 3 mL) and
1
2
3
4
5
6
7
8
MeOH (3× 3 mL) and dried with nitrogen. Then, 5 mg of the
resin was cleaved with 50% TFA in DCM for 30 min. The
cleavage cocktail was evaporated by a stream of nitrogen,
and the cleaved compound was extracted into 1 mL of
MeOH. This sample of the Fmoc derivative was analyzed by
HPLC, and the quantity was compared with the analysis of
the standard (Fmoc-Ala-OH; concentration of 0.25 mg/mL).
The loading of the resin was determined by an external
standard method by the integration of the UV response at
300 nm.
Resin 2 (ca. 20 mg) was washed 3× with 2-MeTHF and treat-
ed with 1% DBU (10 μL) in 2-MeTHF (990 μL) for 15 min at rt.
The resin was then washed 5× with 2-MeTHF and treated by
50% TFA in DCM for 30 min at rt. The cleaved compound
was extracted into MeOH for the HPLC-MS measurement.
Immobilization of Fmoc-AA-OH onto the Wang resin
(resin 21):
Wang resin 17 (250 mg, 0.9 mmol/g, 100 – 200 mesh) was
washed with DCM (3× 3 mL). A solution of Fmoc-AA (0.5
mmol), HOBt•H2O (0.5 mmol, 77 mg), DMAP (0.12 mmol, 15
mg) and DIC (0.5 mmol, 78 μL) in 2.5 mL of DCM/DMF (1:1)
was added and the reaction slurry was shaken overnight at rt.
The resin was then washed with DMF (3× 3 mL) and DCM
(3× 3 mL).
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Treatment with NaOH in 2-MeTHF/MeOH (resin 27):
Resin 21 (ca. 100 mg) was washed 3× with 2-MeTHF and
treated with 0.2 M NaOH (21.1 μL of 50% (w/v) NaOH ~ 18.94
M NaOH aq. solution) in 2-MeTHF/MeOH (1:1, v/v; 2 mL) for
30 min at rt. The resin was then washed 5× with 2-MeTHF.
Acylation of amino group with Fmoc-Rink amide linker
(after Fmoc removal and subsequent acylation with
Fmoc-AA = resin 18) or with Fmoc-AA (resin 3):
Reaction with Fmoc-OSu (resin 26):
Resin 22 (ca. 100 mg) was washed 3× with DCM, and a solu-
tion of Fmoc-OSu (0.5 mmol, 169 mg) in 1 mL of DCM was
added to the resin. The resulting slurry was shaken at rt for 1
h. The resin was subsequently washed 5× with DCM, and the
resin loading was quantified.
Resin (250 mg) acylated with Fmoc-AA-OH was after Fmoc
removal washed with DCM (3× 3 mL) and treated with a
solution of Fmoc-Rink amide linker or Fmoc-AA- OH (0.5
mmol) with HOBt•H2O (0.5 mmol, 77 mg) and DIC (0.5
mmol, 78 μL) in 2.5 mL of DCM/DCM (1:1) and the reaction
slurry was shaken at rt for 1 h. The resin was washed with
DMF (3× 3 mL) and DCM (3×3 mL).
SPPS of Leu-enkephalin amide by starting with the
Fmoc-Rink amide resin:
Green Fmoc removal by 0.2
MeTHF/MeOH:
M
NaOH in 2-
Cleavage of the product from the resin by 50% TFA/DCM
and preparation of the sample for HPLC-MS (com-
pounds 19 and 23):
The resin (0.5 g) was washed 3× with 2-MeTHF (≥99.0%
purity purchased from VWR) and treated with 0.2 M NaOH
(52.8 μL of 50% (w/v) NaOH ~ 18.94 M NaOH aq. solution)
in 2-MeTHF/MeOH (3:1, v/v; 5 mL) or (3:1, v/v; 4.75 mL) + 5%
H2O (250 μL) at rt for 15 min. The resin was then washed 5×
with 2-MeTHF.
An analytical amount of resin 18 or resin 22 (ca. 5 mg) was
transferred into an Eppendorf tube and treated with 1 mL of
a solution of 50% TFA in DCM for 30 min at rt. The TFA
solution was evaporated by a stream of nitrogen. The cleaved
compound was extracted into 1 mL of MeOH.
Acylation with Fmoc-AA:
Cleavage from the resin by 0.2 M NaOH and preparation
of the sample for HPLC-MS (compounds 20 and 24):
The resin (0.5 g) was washed 3× with 2-MeTHF. A solution of
Fmoc-AA (1 mmol), HOBt•H2O (1 mmol, 154 mg) and DIC (1
mmol, 156 μL) in 5 mL of 2-MeTHF was added to the resin,
and the reaction slurry was shaken at rt for 1 h. Then, the
resin was washed 3× with 2-MeTHF, 3× with 2-
MeTHF/MeOH (1:1), and 3× with 2-MeTHF.
An analytical amount of resin 18 or resin 22 (ca. 10 mg)
placed in a plastic syringe with a frit was treated with 1 mL of
a solution of 0.2 M NaOH (10.6 μL of 50% (w/v) NaOH ~
18.94 M NaOH aq. solution) in 2-MeTHF/MeOH (1:1, v/v; 1
mL) for 30 min at rt. The resin was filtered off, and the ob-
tained solution was neutralized with AcOH and diluted with
MeOH to achieve a ratio of 1:1.
SPPS of triptorelin by starting with the Fmoc-Rink amide
resin:
The synthesis method was carried out according to methods
A – D by starting with 0.5 g of the Fmoc-Rink amide resin.
For methods B – D, 2-MeTHF (GPR RECTAPUR® for synthe-
sis, ≥99.0%) purchased from VWR was used in both the
acylation and Fmoc removal steps.
Reaction with 4-Ns-Cl (resin 22):
After Fmoc removal, resin 21 (250 mg) was washed with DCM
(3× 3 mL), and a solution of 4-Ns-Cl (0.75 mmol, 165 mg) and
2,6-lutidine (0.83 mmol, 95 μL) in 2.5 mL of DCM was added
to the resin. The resulting slurry was shaken at rt for 2.5 h
and subsequently washed with DCM (3× 3 mL).
Acylation with Fmoc-Arg(Pbf)-OH in methods C and D:
Due to the low reactivity of Fmoc-Arg(Pbf)-OH with the H-
Pro-Gly-NHRink resin, the acylation in 2-MeTHF had to be
repeated 2× – 3× at rt for 1 h to obtain quantitative conver-
sion. In the traditional protocol using DMF/DCM (1:1) at rt
for 1 h, the acylation step did not require repetition.
Immobilization of Fmoc-Ala-OH onto the CTC resin
(resin 26):
CTC resin 25 (125 mg, 0.85 mmol/g, 100 – 200 mesh) was
washed with anhydrous DCM (3× 3 mL). A solution of Fmoc-
Ala-OH (0.2 mmol, 63 mg) and DIEA (0.5 mmol, 88 μL) in 3
mL of anhydrous DCM was added, and the reaction slurry
was shaken overnight at rt. The resin was washed with DMF
(3× 3 mL) and DCM (3× 3 mL), and the resin loading was
quantified.
Acylation with pyroglutamic acid in methods C and D:
Due to the low solubility of pyroglutamic acid in 2-MeTHF,
the acylation step was performed by adding 10% DMF into 2-
MeTHF at rt for 1 h.
Quantification of the resin loading:
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The Journal of Organic Chemistry
(8) Bray, B. Innovation: Large-Scale Manufacture of Peptide
Therapeutics by Chemical Synthesis. Nat. Rev. Drug Discov.
2003, 2 (7), 587-593.
Cleavage and isolation of triptorelin:
1
2
3
4
5
6
7
8
250 mg of resin-supported pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-
Arg-Pro-Gly-NH-Rink was washed 3× with MeOH and 3×
with DCM. The resin was treated with 3 mL of a solution of
TFA/H2O (95:5) at rt for 4 h. The TFA solution was collected,
and the resin was washed with DCM. The combined extracts
were concentrated under a stream of nitrogen, and the crude
peptide was precipitated with diethyl ether (3 mL), filtered
and subsequently lyophilized.
(9) Mulder, K. C. L.; Viana, A. A. B.; Xavier, M.; Parachin, N. S.
Critical Aspects to be Considered Prior to Large-Scale Produc-
tion of Peptides. Curr. Protein Pept. Sci. 2013, 14 (7), 556-567.
(10) Capello, C.; Fischer, U.; Hungerbuehler, K. What is a
green solvent? A comprehensive framework for the environmen-
tal assessment of solvents. Green Chem. 2007, 9 (9), 927-934.
(11) Constable, D. J. C.; Jimenez-Gonzalez, C.; Henderson, R. K.
Perspective on Solvent Use in the Pharmaceutical Industry. Org.
Process Res. Dev. 2007, 11 (1), 133-137.
9
ASSOCIATED CONTENT
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(12) Goodwin, D.; Simerska, P.; Toth, I. Peptides as therapeu-
tics with enhanced bioactivity. Curr. Med. Chem. 2012, 19 (26),
4451-4461.
(13) Tulla-Puche, J.; El-Faham, A.; Galanis, A. S.; de Oliveira,
E.; Zompra, A. A.; Albericio, F. Methods for the peptide synthesis
and analysis. John Wiley & Sons, Inc.: 2015; pp 11-73.
(14) Zompra, A. A.; Galanis, A. S.; Werbitzky, O.; Albericio, F.
Manufacturing peptides as active pharmaceutical ingredients.
Future Med. Chem. 2009, 1 (2), 361-377.
(15) Byrne, F. P.; Jin, S.; Paggiola, G.; Petchey, T. H. M.; Clark,
J. H.; Farmer, T. J.; Hunt, A. J.; McElroy, C. R.; Sherwood, J. Tools
and techniques for solvent selection: green solvent selection
guides. Sustainable Chem. Processes 2016, 4, 7-1-7/24.
(16) Regulation (EC) Number 1907/2006 of the European Par-
liament and of the Council of 18 December 2006.
https://echa.europa.eu/regulations/reach/understanding-reach.
(17) Bryan, M. C.; Dunn, P. J.; Entwistle, D.; Gallou, F.; Koenig,
S. G.; Hayler, J. D.; Hickey, M. R.; Hughes, S.; Kopach, M. E.;
Moine, G.; Richardson, P.; Roschangar, F.; Steven, A.; Weiberth,
F. J. Key Green Chemistry research areas from a pharmaceutical
manufacturers' perspective revisited. Green Chem. 2018, 20 (22),
5082-5103.
(18) Merrifield, R. B. Solid phase peptide synthesis. I. The syn-
thesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85 (14), 2149-
2154.
(19) Pribylka, A.; Krchnak, V.; Schutznerova, E. Environmen-
tally friendly SPPS I. Application of NaOH in 2-
MeTHF/methanol for Fmoc removal. Green Chem. 2019, 21 (4),
775-779.
(20) Jaradat, D. M. M. Thirteen decades of peptide synthesis:
key developments in solid phase peptide synthesis and amide
bond formation utilized in peptide ligation. Amino Acids 2018,
50 (1), 39-68.
(21) Isidro-Llobet, A.; Alvarez, M.; Albericio, F. Amino Acid-
Protecting Groups. Chem. Rev. 2009, 109 (6), 2455-2504.
(22) Albericio, F.; El-Faham, A. Choosing the Right Coupling
Reagent for Peptides: A Twenty-Five-Year Journey. Org. Process
Res. Dev. 2018, 22 (7), 760-772.
Supporting Information. This material is available free of
charge via the Internet at http://pubs.acs.org. H NMR spec-
1
tra of different used 2-MeTHFs and prepared peptides (Leu-
enkephalin amide and triptorelin), swelling properties of PS-
Rink resin in chosen solvents, HPLC chromatograms (select-
ed steps in SPPS of Leu-enkephalin amide, stability evalua-
tion of side-chain PGs, aspartimide formation, prepared
peptides), HRMS data of prepared peptides (PDF).
AUTHOR INFORMATION
Corresponding Author
ORCID
Adam Přibylka: 0000-0003-3701-5460
Viktor Krchňák: 0000-0001-5688-4069
Eva Schütznerová: 0000-0003-0186-5981
Author Contributions
The manuscript was written through contributions of all
authors. / All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by the Department of Chemis-
try and Biochemistry, University of Notre Dame, and re-
search grant 18-17978Y from the Czech Grant Agency
(GACR). We thank Jana Skopalová, Ph. D., for determination
of water content by coulometric Karl Fisher titration.
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