Environmentally friendly SPPS I. Application of NaOH in 2-MeTHF/methanol for Fmoc removal

Article abstract of DOI:10.1039/c8gc03778g

Focusing on the step-by-step transformation of the traditional solid-phase peptide synthesis (SPPS) into an environmentally friendly process, we herein report the elimination of environmentally hazardous components (piperidine, DMF and DCM) from this technique. We developed a synthetic protocol that employs sodium hydroxide in a 2-MeTHF/MeOH mixture for Fmoc group cleavage and uses 2-MeTHF alone as the coupling solvent. The protocol was developed with the most frequently used PS/DVB-based resin. This synthetic strategy was used to prepare Leu-enkephalin amide, and the results (crude purity 99%, yield 62%) were fully comparable to those achieved with the traditional protocol using piperidine, DMF and DCM.

Full text of DOI:10.1039/c8gc03778g

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Environmentally friendly SPPS I. Application of NaOH in 2-
MeTHF/methanol for Fmoc removal
Received 00th January 20xx,
Accepted 00th January 20xx
Adam Přibylka, Viktor Krchňák, and Eva Schütznerová*a
a
a,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
Focusing on step-by-step transformation of traditional solid-phase
peptide synthesis (SPPS) to an environmentally friendly process,
we herein report the elimination of environmentally hazardous
components (piperidine, DMF and DCM) from this technique. We
developed a synthetic protocol that employs sodium hydroxide in
a 2-MeTHF/MeOH mixture for Fmoc group cleavage and uses 2-
MeTHF alone as the coupling solvent. The protocol was developed
with the most frequently used PS/DVB-based resin. This synthetic
Fmoc removal
Rn
Rn
Rn
H
H
Fmoc
N
H
N
H
NH2
L
L
N
N
N
n
n
n
H
H
H
O
O
O
coupling with Fmoc-AA-OH
n = n + 1
L = Rink amide linker
R = side chain of AA
strategy was used to prepare Leu-enkephalin amide, and the Scheme 1 Repetitive nature of Fmoc-SPPS on Rink resin.
results (crude purity 99 %, yield 62 %) were fully comparable to
those achieved with a traditional protocol using piperidine, DMF
and DCM.
Only after each of these steps can be referred to as “green”
can we use the term GSPPS. Our long term aim is to focus on
each step of SPPS in order to make it „green“. This short
communication evaluates green alternatives for Fmoc removal
step, it is the first manuscript in a sequence and therefore we
included „I“ in the title.
Peptide-based drugs are experiencing a renaissance, and they
are expected to play an even more important role in the future
of the pharmaceutical industry. Therapeutic peptides are
1
-3
4
being produced on a multiton scale, and therefore the
environmental impacts of peptide manufacturing processes
are an important issue. Among others, there are two
predominant methodologies used for peptide chemical
The most commonly used solvents in SPPS are DMF and
6
DCM, which are both considered hazardous, and DMF is likely
7
to be restricted by REACh. The solvent is the main pollutant in
5
synthesis - solution-phase peptide synthesis and solid-phase
the process of preparing every API because the solvent is the
peptide synthesis (SPPS) as well as their combinations. Thus,
SPPS has been applied not only in research but also in the
pharmaceutical industry. For this reason, environmentally
friendly approaches are highly desirable.
The SPPS involves two repetitive steps: Fmoc removal and
coupling with Fmoc protected amino acid (Scheme 1).
However, the entire process includes the selection of the type
of resin, the linker for immobilization of the carboxy terminal
amino acid, and the amino acid protecting groups as well as
their syntheses, cleavage of the N-alpha protecting group, the
selection of reagents for the coupling procedures, the final
cleavage and the purification.
8
major component of a reaction mixture. Based on this, early
efforts to make SPPS more environmentally friendly were
9-14
focused on the use of green solvents.
Thus far, green
solvents have been shown to be compatible rather with more
10,12
expensive PEG-based resins (polyethylene glycol),
which is
not used in the manufacturing industry. The application of
green solvents in all steps of SPPS using PS/DVB-based resin,
which has been the most common polymer support since
15
11
times of Merrifield, lead at first only to moderate success.
Recent results published by Lopez at al. showed that N-
butylpyrrolidinone could be alternative for DMF in SPPS
14
compatible with PS-based resin.
The next environmentally critical step is Fmoc removal13
due to the quantity of reagent used in this repeated step.
Piperidine, almost exclusively used reagent, is a hazardous
chemical that scored 6.9 points on a scale of 0-10 in GSK´s acid
and base selection guide. Toxic piperidine is also a controlled
substance because it can be abused as an intermediate in the
a. 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.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
b.
1
6
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synthesis of narcotic drugs.17,18 There are greener alternatives
known to cleave Fmoc group, such as morpholine (scored 7.5)
R
O
R
H
i, ii
H
iii, ii
H
H
View Article Online
N
Fmoc
N
N
N
Fmoc
L
N
L
Fm
oc N L
DOI: 10.1039/C8GC03778G
H
H
O
O
1
9,20
and DBU (7.3).
Methyl derivatives of piperidine and
L = Rink amide linker
R = side chain of AA
1
2
2
1,22
22
piperazine,
piperazine itself
and mixtures of
piperazine/DBU23 have already been applied in SPPS as
alternatives to piperidine but always in solutions with DMF.
Recently, Lopez at al. evaluated several green solvents in SPPS
and in Fmoc removal step in combination with 4-methyl
piperidine. We have chosen to perform our evaluation in 2-
MeTHF,24 which was used in SPPS for the first time by
Albericio.9
Scheme 2 Optimization of Fmoc removal conditions on model
Fmoc-Ala attached to Rink resin. Reagents and conditions: i) 50%
piperidine in DMF, rt, 15 min; ii) 0.2 M Fmoc-Ala-OH, DIC, HOBt,
DCM/DMF (1:1), rt, on; iii) evaluated base (see Table 1).
1
4
also with hazardous piperidine, which is used almost
exclusively for Fmoc removal. The Fmoc cleavage experiments
were carried out on a model Rink amide PS/DVB resin acylated
with Fmoc-Ala-OH. First, we evaluated conventional DMF-
containing cocktails of each organic base, and then the DMF
was replaced with 2-MeTHF. Different concentrations were
tested to identify the mildest conditions that provided
complete Fmoc cleavage.
To address the rate of Fmoc removal, we subjected a
sample of Fmoc-Ala Rink amide resin to the Fmoc removal
cocktail, washed the resin and subsequently acylated with
Fmoc-Ala-OH (Scheme 2). After cleavage from the resin with
With exception of 4-methyl piperidine,14 greener
alternatives to traditionally used piperidine in Fmoc-SPPS have
never been studied in combination with green solvents.
Moreover, published works in the field of reportedly “green”
SPPS have suffered limited applicability and are only suitable
1
0,12
for PEG-based resins.
Publications focusing on PS-based
resins did not afford final compounds with sufficient purity
when green solvents were used for the Fmoc removal and
washing steps.11
Here, we report the successful use of the green inorganic
base sodium hydroxide (scored 8 in GSK´s guide), which has
not previously been used in SPPS despite the fact that the
5
0% TFA/DCM, we analyzed the sample by LC/MS. The
absence of Fmoc-Ala-NH indicated quantitative removal, and
its presence indicated incomplete removal. The ratio of Fmoc-
Ala-NH to Fmoc-Ala-Ala-NH indicated the extent of the
cleavage (Table 1).
2
lability of Fmoc to this inorganic base has been known since
0,25
1
980.2 In our previous work, we utilized NaOH solution in a
2
2
mixture of solvents (THF and MeOH) in the ester hydrolysis of
heterocycles attached to Rink resin, and the attachment via
A conventional Fmoc removal protocol utilizing 50%
piperidine in DMF (entry 1) was compared to a method
involving 2-MeTHF (entry 2). Both procedures gave complete
cleavage. Then, we examined greener alternatives to
piperidine. Even though they are structurally very similar,
morpholine in DMF enabled Fmoc removal (entry 3), but 50%
morpholine in 2-MeTHF resulted in only 53% Fmoc
2
6
the amide was stable since ester immobilization to Wang
resin leads to cleavage from polymer support under these
basic conditions.27 Alkaline hydrolysis represents an integral
2
8-30
part of SPPS cleavage protocols.
We compared the performance of NaOH solution with
selected green organic bases, such as morpholine and DBU and
Table 1 The rates of Fmoc cleavage from Fmoc-Ala attached to Rink resin using various bases.
Entry
Base
Solventa
DMF
2-MeTHF
DMF
2-MeTHF
2-MeTHF
2-MeTHF
DMF
2-MeTHF
Time
Temp.
rt
rt
rt
rt
rt
50 °C
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
1´:2´b
<1:>99
<1:>99
<1:>99
47:53
<1:>99
11:89
<1:>99
<1:>99
<1:>99
<1:>99
10:90
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
21:79
1
2
3
4
5
6
7
8
9
50% piperidine
50% piperidine
50% morpholine
50% morpholine
50% morpholine
50% morpholine
5% DBU
5% DBU
1% DBU
0.5% DBU
0.1% DBU
0.5 M NaOH
0.5 M NaOH
0.2 M NaOH
0.2 M NaOH
0.1 M NaOH
0.02 M NaOH
15 min
15 min
15 min
15 min
1 h
15 min
15 min
15 min
15 min
15 min
15 min
30 min
30 min
30 min
15 min
15 min
15 min
2-MeTHF
2-MeTHF
2-MeTHF
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
THF/MeOH (1:1)
2-MeTHF/MeOH (1:1)
2-MeTHF/MeOH (1:1)
2-MeTHF/MeOH (1:1)
2-MeTHF/MeOH (1:1)
2-MeTHF/MeOH (1:1)
rt
a
b
The volume of Fmoc removal cocktail was 1 mL, the amount of treated resin in each experiment was 20 mg. The ratio of 1´:2´ cleaved
from resins 1 and 2 was estimated from the LC traces at 300 nm.
2
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cleavage (entry 4). Extended the reaction time to 1 h
provided complete cleavage (entry 5), and increasing the
temperature to 50 °C for 15 min cleaved 89% of the Fmoc
group (entry 6). DBU (5%) in both DMF and 2-MeTHF
Since racemization represents the most serious side-
reaction in peptide bond formation, we evaluated the
integrity of chiral centre on model dipeptide amide H-Phe-
3
1
2
Phe-NH prepared using our protocol for Fmoc removal and
(
entries 7 and 8) was successful, so we investigated lower
2-MeTHF as solvent for coupling.
We synthesized
1
concentrations of DBU in 2-MeTHF, and we reached the limit
at a concentration of 0.1% DBU (entry 11). In addition to the
bases traditionally used for Fmoc removal, we introduce a
new method using sodium hydroxide as a suitable base for
Fmoc cleavage. The starting reaction conditions for the Fmoc
cleavage were 0.5 M NaOH in a mixture of THF and MeOH
diastereomers (LL and LD) and H NMR spectra showed
differences in amide shifts (area 7.5 – 8.9 ppm, spectra in
Supporting Information) and methylene shifts (area 2.6 - 3.2
ppm). The spectra showed no evidence of second
diastereomer. We evaluated the racemization also by more
sensitive reverse phase ion-pair HPLC separation. The
(
(
1:1) (entry 12). Next, we replaced the THF with 2-MeTHF
entry 13). We then tested both lower concentrations of
analysis of LL dipeptide (t
of the DL diastereomer only in traces (0.3%). As well as the
analysis of DL dipeptide (t = 11.7 min) showed insignificant
presence (0.05%) of LL diastereomer (see the Supporting
Information for details).
R
= 6.7 min) revealed the presence
base and shorter cleavage times. Lowering the NaOH
concentration to 0.02 M resulted in only 79% Fmoc cleavage
R
(entry 17). The Fmoc cleavage cocktail was always prepared
freshly from concentrated aqueous solution of NaOH
To document the utility of 0.1 M NaOH in 2-
MeTHF/MeOH for Fmoc cleavage, we synthesized Leu-
(experimental procedures are in Supporting Information).
The lowest concentration of sodium hydroxide suitable
2
enkephalin amide H-Tyr-Gly-Gly-Phe-Leu-NH and compared
for Fmoc removal from Fmoc-Ala attached to Rink PS resin
our new protocol with the conventional protocol using
piperidine in DMF. At the same time, we confirmed the
results of Albericio that 2-MeTHF alone is a suitable solvent
(
(
Table 1) was tested with various model Fmoc-amino acids
AAs) bearing diverse substituents (R) followed by the
9
,13
reaction with Fmoc-Ala-OH. The results are listed in Table 2.
Selected Fmoc-AAs behaved very similarly to Fmoc-Ala
attached to Rink, and a concentration of 0.1 M ensured
quantitative removal in all cases.
for acylation reactions.
However, after each acylation
step, the resin had to be washed with a mixture of MeOH/
Table 3 Protocols used in the preparation of pentapeptide
amide Leu-enkephalin.a
Table 2 Fmoc removal conditions for selected amino acid
models attached to Rink resin using 0.1 M NaOH.a
Step
Standard
A)
2-MeTHF
only for Fmoc
removal (B)
3x 2-MeTHF
2-MeTHF (C)
(
Entry
Fmoc-AA
Gly
Phe
Lys(Boc)
Pro
Ser(OtBu)
Thr(OtBu)
Tyr(OtBu)
Asp(OtBu)
Glu(OtBu)
Val
R
H
1´:2´b
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
<1:>99
Wash.
3x DCM
3x 2-MeTHF
1
2
3
4
5
6
7
8
9
3
x DMF
CH
(CH
2
4
-C
-NHBoc
6 5
H
Fmoc
removal
50%
piperidine
DMF
rt, 15 min
5x DMF
3x DCM
0.1 M NaOH
2-MeTHF/MeOH
(1:1)
rt, 15 min
5x 2-MeTHF
3x DCM
0.1 M NaOH
2-MeTHF/MeOH
(1:1)
rt, 15 min
5x 2-MeTHF
2
)
cyclic
CH -OtBu
CH(CH )OtBu
(CH )-C OtBu
CH -CO tBu
(CH -CO tBu
CH(CH
CH CH(CH
CH(CH )C
2
3
Wash.
Coupl.
2
6 4
H
2
2
0.2 M Fmoc-AA-OH, HOBt, DIC
2
)
2
2
DMF/DCM
DMF/DCM (1:1)
rt, 1 h
2-MeTHF
rt, 1 h
3 2
)
(
1:1)
1
1
1
0
1
2
rt, 1 h
3x DMF
Leu
Ile
2
3
)
2
Wash.
3x DMF
3x DCM
3x 2-MeTHF
3x 2-MeTHF/MeOH
3
2
H
5
3
x DCM
a
The volume of Fmoc removal cocktail was 1 mL; the amount
(
1:1)
of treated resin in each experiment was 20 mg; the reaction
3
x 2-MeTHF
b
was performed at rt for 15 min. The ratio of 1´:2´ cleaved
a
Reaction steps performed using hazardous solvents are in
red, and environmentally friendly approaches are in green.
from resins 1 and 2 was estimated from the LC traces at 300
nm.
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Table 4 Purities and yields of pentapeptide amide Leu-
enkephalin prepared using different protocols.
the probably most critical steps in the transformation of
standard Merrifield peptide synthesis to an environmentally
View Article Online
DOI: 10.1039/C8GC03778G
friendly process. This protocol is not compatible with Wang
linker, however, there are numerous peptide amides
prepared on large production scales (analogs of oxytocin,
vasopressin, LH-RH). There are several other issues in the
route to GSPPS that need to be addressed, including the
nature of the resin, the potential for recycling, and the
cleavage from the resin under green conditions, etc.
Protocol
Puritya
99%
99%
Yieldb
75%
69%
62%
A
B
C
99%
a
The crude purities were estimated from the LC traces from
diod area 220-400 nm. Yields were calculated from the
NMR spectra of the pentapeptides following cleavage with
b
5
0% TFA/DCM, 1.5 h, rt, evaporation with N
2
and
precipitation with diethyl ether.
Conflicts of interest
There are no conflicts to declare.
2
-MeTHF to remove the residual Fmoc-AA-OH used for the
acylation (Table 3, washing step after acylation). The Fmoc-
amino acid derivatives and the coupling reagents showed
good solubility in 2-MeTHF and it showed good swelling of
the PS resin. The synthesis was performed on Rink amide
PS/DVB-based resin,32 since PS-based polymer supports are
Acknowledgements
This research was supported by the Department of Chemistry and
Biochemistry, University of Notre Dame and by research grant 18-
1
7978Y from the Czech Grant Agency (GACR). We thank Martin
the most widely used. The protocols are described in Table
Grepl, Ph.D., for the HPLC separation.
3
. Standard protocol A using DCM and DMF as solvents and
piperidine as the Fmoc cleavage reagent was used for
comparison, protocol B is the new Fmoc removal method,
but for the coupling reactions, we used DCM and DMF as
solvents, and new protocol C avoids using hazardous
solvents and reagent.
Notes and references
1
2
3
4
5
6
D. J. Craik, D. P. Fairlie, S. Liras, D. Price, Chem. Biol. Drug
Des., 2013, 81, 136.
K. Fosgerau, T. Hoffmann, Drug Discovery Today, 2015, 20,
Pentapeptides synthesized according to procedures A, B
and C were cleaved from their resins by 50% TFA/DCM.
Then, the cleavage cocktail was evaporated, and the
peptides were precipitated by diethyl ether. Crude purities
were determined from the LC traces, and crude yields were
1
22.
A. A. Zompra, A. S. Galanis, O. Werbitzky, F. Albericio,
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calculated from the H NMR spectra. The NMR spectra
1
9, 4451.
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prepared peptides were optically pure. The results are
summarized in Table 4.
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Leu-enkephalin extended by Fmoc-Ala-OH using our NaOH
Fmoc cleavage protocol and 2-MeTHF for coupling in
excellent purity/yield (99%/72%).
1
Conclusions
We successfully eliminated the most environmentally
hazardous solvents (DMF and DCM) and reagent (piperidine)
used in traditional SPPS and replaced them with 2-MeTHF as
a sole coupling solvent and a solution of NaOH in 2-
MeTHF/MeOH for Fmoc cleavage. To document the
applicability of this new protocol, we synthesized a model
peptide, Leu-enkephalin amide, with high purity, yield and
full optical integrity. Our protocol is compatible with the
most commonly used PS-based resin. Comprehensive
experimental data including further optimization of the
cleavage cocktail, testing of all amino acids and synthesis of
difficult peptide sequences as well as commercially
manufactured peptides will be included in a full paper.
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