Green Solid-Phase Peptide Synthesis (GSPPS) 3. Green Solvents for Fmoc Removal in Peptide Chemistry

Article abstract of DOI:10.1021/acs.oprd.6b00439

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

Full text of DOI:10.1021/acs.oprd.6b00439

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Full Paper
Green Solid-Phase Peptide Synthesis (GSPPS)-III.
Green solvents for Fmoc removal in peptide chemistry
Yahya E Jad, Thavendran Govender, Hendrik G. Kruger,
Ayman El-Faham, Beatriz G de la Torre, and Fernando Albericio
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00439 • Publication Date (Web): 30 Jan 2017
Downloaded from http://pubs.acs.org on January 30, 2017
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Green Solid-Phase Peptide Synthesis (GSPPS)-
III. Green solvents for Fmoc removal in peptide
chemistry
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a
a
a
b,c
Yahya E. Jad, Thavendran Govender, Hendrik G. Kruger, Ayman ElꢀFaham Beatriz G. de
a,d*
a,b,e,f,g*
la Torre, and Fernando Albericio
a
Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZuluꢀNatal,
University Road, Westville, Durban 4001, South Africa.
b
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh
11451, Saudi Arabia.
c
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
Alexandria 21321, Egypt.
d
School of Laboratory of Medicine and Medical Sciences, University of KwaZuluꢀNatal,
University Road, Westville, Durban 4001, South Africa.
e
Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1ꢀ11, 08028ꢀ
Barcelona, Spain.
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CIBERꢀBBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, Baldiri Reixac 10, 08028ꢀBarcelona, Spain
g
School of Chemistry and Physics, University of KwaZuluꢀNatal, University Road, Westville,
Durban 4001, South Africa.
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KEYWORDS.
Solidꢀphase peptide synthesis, Fmoc, protecting groups, green solvents, γꢀvalerolactone, peptide
aggregation.
ABSTRACT.
DMF (N,Nꢀdimethylformamide) is the most widely used solvent for FmocꢀSPPS, which is the
conventional method of choice for peptide synthesis. It is usually employed for coupling,
washing and Fmocꢀremoval steps. We have recently reported greener solvents such as 2ꢀMeTHF
(
2ꢀmethyltetrahydrofuran) and CPME (cyclopentyl methyl ether) as environmentally friendlier
alternatives to DMF. However, the Fmocꢀremoval step with these green solvents was not
α
optimal. Thus, deletion peptides and N ꢀFmoc terminal peptides were observed upon TFA
(
trifluoroacetic acid) cleavage of the peptide from the resin during SPPS of a challenging
aggregated peptide. Herein, Fmoc removal using a solution of 20% piperidine in a range of green
solvents is reported. After evaluation of the resin’s swelling (PS and ChemMatrix resins), and
Fmoc deprotection in solution and on solidꢀphase for these two resins, it was found that γꢀ
valerolactone can replace DMF for Fmoc removal steps during SPPS on both resins, PS or
ChemMatrix. Furthermore, Nꢀformylmorpholine showed an excellent performance with
ChemMatrix resin.
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Introduction
A successful peptide synthesis completely depends on a suitable protecting group scheme
together with the most convenient coupling reagent. Peptides are synthesized by repetitive
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1ꢀ2
coupling and deprotection steps. Although it is common thought that the coupling step is
crucial for successful peptide synthesis, incomplete deprotection step(s) will also jeopardize the
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synthesis.
In solidꢀphase peptide synthesis (SPPS), peptides are prepared following either the N → C or C
→ N directions, the first approach is not commonly used because it is usually associated with
4ꢀ6
side reactions such as the formation of diketopiperizines or oxazolones. Therefore, most
α
peptides are synthesized in the C → N direction, where temporary N protecting groups are
normally removed several times during the synthesis while the Cꢀterminal is permanently
“
protected” via its attachment to the resin. Although many temporary protecting groups were
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reported and utilized for masking the αꢀamino functionality, the Fmoc group is the most widely
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employed in SPPS and also finds some applications in solution chemistry. It was firstly
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ꢀ9
introduced by Carpino in the early 1970s and has been routinely applied for SPPS since
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1978.
It displays exquisite chemical properties in that it can be removed under mild basic
conditions, while being stable against the primary amino component that is to be acylated, during
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the coupling step. Although it can also be removed by Lewis acid such as AlCl3
or catalytic
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hydrogenation, but the mostly used approach is solutions of secondary amines such as
piperidine, piperazine, or morpholine in polar aprotic solvents as the removal cocktail. The most
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common method involves 20% piperidine in DMF for a few minutes. Moreover, replacement
of piperidine with 4ꢀmethylpiperidine was also reported and revealed similar efficiencies than
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piperidine but it has found only a few applications, possibly due to financial considerations.
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Piperazine is another six membered cyclic secondary amine and it shows a maximum solubility
of 6% in DMF or NMP (Nꢀmethylꢀ2ꢀpyrrolidone). Therefore, 10% piperazine in a mixture of
1
7
EtOH (ethanol) and NMP (10:90) was reported and utilized in SPPS. The kinetics of Fmoc
removal in 5% piperazine + 2% DBU (1,8ꢀdiazabicycloundecꢀ7ꢀene) in DMF were recently
evaluated and it exhibited faster reaction than 10% piperazine in EtOH/NMP (10:90) and 20%
piperidine in DMF, mainly due to the presence of DBU, which is more basic than piperidine
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(
pKa of piperidine 11.12 vs pKa of DBU 13.5). Solvents are the major component of the
deprotection cocktail, as they represent at least 80% of the deprotection solution. This estimation
is in accordance with a survey accomplished by GSK (GlaxoSmithKline) in 2007 about the
percentage of solvent used during manufacturing of API (Active Pharmaceutical Ingredients)
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wherein it represented 80ꢀ90% of the nonꢀaqueous masses.
In this regards, it is important to
take into account that DMF and NMP are the most widely used solvents but are listed on the
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solvent substitution request list since they are hazardous chemicals.
Most importantly, the
use of these two solvents is likely to be restricted by REACH (Registration, Evaluation,
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Authorization and Restriction of Chemicals) since they are listed as SVHC (substances of very
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high concern). Our group have also used MeCN (acetonitrile), which is also not a green
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20ꢀ21, 23
solvent but is classified as a “recommended alternative to DMF” in some solvent guides,
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for Fmoc removal in SPPS with the ChemMatrix resin. Our research was expanded to include
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THF, 2ꢀMeTHF and CPME, with the latter two classified as green solvents,
for solution
29ꢀ30
synthesis and SPPS.
These solvents were only used during the coupling step while DMF was
still employed during the deprotection and washings steps. Very recently while we were
attempting the establishment of a global green solvent SPPS technology, the difficulties and
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limitations associated with 2ꢀMeTHF as solvent for the piperidine cocktail were clearly
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evident.
These previous results have confirmed the key role of Fmoc removal in assuring good yields and
purities of the final peptides. With this in mind, an extensive study of 20% piperidine in various
solvents for Fmoc removal in solution and in SPPS have been performed.
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Results and discussion
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The following green solvents were considered: Nꢀformylmorpholine, 2ꢀmethyltetrahydrofuran,
cyclopentyl methyl ether, isosorbide dimethyl ether, EtOAc (ethyl acetate), dimethyl carbonate,
γꢀvalerolactone, IPA (isopropanol) and α,α,αꢀtrifluorotoluene. The performance of these solvents
during Fmoc deprotection was compared to DMF, which is mostly used for Fmoc removal.
The swelling of the PS (polystyrene) and ChemMatrix resins, which are commonly used, were
evaluated for these solvents since this plays a critical role in SPPS. Thus, a good swelling
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facilitates the accessibility of the reagents to the functional group in the growing chain. To best
of our knowledge, DMF is the most commonly used solvent in SPPS because its good ability to
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swell resins very well. The results in Table 1 shows that all solvents, except CPME and IPA,
gave acceptable swelling for the ChemMatrix resin (> 5 mL/g). Regarding the PS resin,
isosorbide dimethyl ether and γꢀvalerolactone resulted in similar swelling than the conventional
solvent DMF (> 5 mL/g). A second block of solvents, 2ꢀMeTHF, CPME, EtOAc, dimethyl
carbonate and α,α,αꢀtrifluorotoluene resulted in less swelling of the PS resin (but > 3 mL/g).
Finally, Nꢀformylmorpholine, and IPA exhibited the worst resin swelling performance.
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Table 1. Swelling of PS and ChemMatrix resins in various solvents
b
Swelling (mL/g)
a
Solvent
PS
ChemMatrix
8.25
DMF
5.25
2.75
3.75
3.75
5.25
3.25
3.75
5
NꢀFormylmorpholine
8.25
2
ꢀMeTHF
5.25
CPME
3.75
Isosorbide dimethyl ether
EtOAc
7.75
5.50
Dimethyl carbonate
γꢀValerolactone
IPA
6.75
8.25
2.75
3.25
3.75
α,α,αꢀTrifluorotoluene
6.25
a
b
Green: green solvent; Red: nonꢀgreen solvent: Green: good swelling, yellow: moderate, and
red: low.
Removal of the Fmoc group was investigated both in solution and on solidꢀphase (PS and
ChemMatrix resins). In this work, the merit of solution mode is only to evaluate the effect of
solvents on Fmoc removal by avoiding other factors such as the swelling of the resins and the
peptide aggregation. Therefore, a simple amino acid FmocꢀPheꢀOH (0.1 mmol) was dissolved in
800 µL of the corresponding solvent and 100 µL of toluene as an internal standard. Then, 200 µL
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of piperidine was added and the reaction was terminated at different times [0 min (before adding
the piperidine), 2, 4, 6, 8, 10, 15, 20, 25 and 30 min].
To best mimic a “realistic” solidꢀphase synthesis case, a Fmocꢀpeptide bonded to a resin which is
known to undergo aggregation was chosen to study the deprotection. Our group has previously
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identified that during the synthesis of the aggregation prone model peptide ACP (65ꢀ74), the
intermediate FmocꢀIleꢀAsp(tBu)ꢀTyr(tBu)ꢀIleꢀAsn(Trt)ꢀGlyꢀNHꢀRinkAmideꢀPSꢀresin required
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longer reaction times (∼15 min, 20% piperidine in 2ꢀMeTHF) for deprotection.
The approach followed is depicted in Scheme 1. HꢀAsp(tBu)ꢀTyr(tBu)ꢀIleꢀAsn(Trt)ꢀGlyꢀNHꢀ
RinkAmideꢀresin was prepared using PS and ChemMatrix resins and standard FmocꢀSPPS
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0
method. DMF was used during the synthesis because it is the most convenient solvent so far.
FmocꢀIleꢀOH was attached and then treated with 20% piperidine solution in the corresponding
solvent for 45 s. This reduced time was chosen to enable differentiation based on the kinetics of
the deprotection reaction in each solvent whilst taking into account that in solid phase, due to the
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use of larger excess of the deprotection reagent, reactions are generally faster.
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Scheme 1. Fmoc removal study on solidꢀphase synthesis.
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Then, BocꢀGlyꢀOH was coupled to the deprotected amino group, followed by complete removal
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of intact Fmocꢀgroups using the most potent solution, i.e 20% piperidine in DMF for 10 min.
Then, BocꢀLeuꢀOH was coupled to those amino groups that were deprotected during the second
piperidine treatment. Upon simultaneous Boc deprotection and cleavage of the two peptides from
the resin, the crude mixture was analyzed with HPLC. The ratio HꢀGlyꢀIleꢀAspꢀTyrꢀIleꢀAsnꢀGlyꢀ
NH vs HꢀLeuꢀIleꢀAspꢀTyrꢀIleꢀAsnꢀGlyꢀNH provides information about the performance of the
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solvent during the first deprotection step where more Gly peptide indicates faster and more
effective Fmoc removal (Scheme 1).
Table 2. Fmoc removal in solution and SPPS by using 20% piperidine solution in different
a
solvents.
Fmoc removal
d
solution
Solid Phase (%)
b
c
Solvent
t (min)
PS
ChemMatrix
DMF
2
97.7
29.3
13.2
4.9
65.5
66.9
15.9
26.3
47.3
9.2
NꢀFormylmorpholine
ꢀMeTHF
4
2
10
CPME
10
Isosorbide dimethyl ether
EtOAc
15
2.3
15
33.9
30.4
43.3
1
Dimethyl carbonate
γꢀValerolactone
IPA
15
4.6
4
89.1
3.2
e
> 30 min
6
α,α,αꢀTrifluorotoluene
12.1
31.1
a
b
Green: Good performance, yellow: moderate, and red: low, Green: green solvent; Red: nonꢀ
c
d
green solvent. Time required to complete the reaction in solution experiment, Yield is
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calculated by the ration of HꢀGlyꢀIleꢀAspꢀTyrꢀIleꢀAsnꢀGlyꢀNH vs HꢀLeuꢀIleꢀAspꢀTyrꢀIleꢀAsnꢀ
2
e
GlyꢀNH , 16.6% of starting materials after 30 min stirring.
2
After evaluation of the solvent impact on Fmoc removal in solution and solidꢀphase conditions,
the following conclusions can be drawn (Table 2):
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•
In solution where effect of resin’s swelling and peptide aggregation are avoided,
excellent results were also obtained with Nꢀformylmorpholine, γꢀvalerolactone, and
α,α,αꢀtrifluorotoluene in addition to the nonꢀgreen solvent DMF. This is in agreement
with the E1CB mechanism of the reaction. Thus, in IPA, which has higher dielectric
constant than Nꢀformylmorpholine (19.9 vs 15.1, respectively), and lower than DMF, and
3
6ꢀ37 38
valerolactone (38.0 and 36.5, respectively)
,
the reaction was slow. This can be
interpreted because IPA is a polar protic solvent. The rest of the esters and ethers
revealed slower removal kinetics.
•
•
In solidꢀphase, DMF was overall the best solvent, while γꢀvalerolactone also gave
excellent results for both resins.
PS resin performed worse than ChemMatrix in all solvents which can be interpreted by
the swelling nature of the ChemMatrix resin in comparison to the PS one, except for
DMF, EtOAC and dimethylcarbonate.
With respect to solventꢀtype, the following observations can be drawn:
• NꢀFormylmorpholine demonstrated excellent performance in solution. Poorer
performance of this solvent with PS can be attributed to its low resin swelling capacity.
However, NꢀFormylmorpholine performed better with the ChemMatrix resin due to the
good swelling features of this resin in this solvent.
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•
For the ether solvents, 2ꢀMeTHF displayed superior performance in both solution and
solidꢀphase with the PS resin. However, CPME and isosorbide dimethyl ether showed
better performance for the ChemMatrix resin, especially isosorbide dimethyl ether also
due to better swelling properties when compared to 2ꢀMeTHF. Overall, 2ꢀMeTHF
showed a promising performance in peptide synthesis corroborating the results described
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by MacMillan et al. and our group.
•
In case of ester solvents, γꢀvalerolactone showed superior results in all parameters:
solution, and solidꢀphase for both resins. Although, PS swelled less in EtOAc and
dimethylcarbonate than the ChemMatrix resin, faster reactions were observed for PS,
3
3
which can be explained by the peptiylꢀresin solvation effect. In fact, ester solvents
showed better performance than the others green solvents with PS.
•
•
IPA showed the worst result both in solution and solidꢀphase. That agrees with other
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7
results obtained from our laboratory where we have replaced DMF with IPA.
α,α,αꢀTrifluorotoluene displayed good performance for solution and SPPS for the
ChemMatrix resin only.
NꢀFormylmorpholine, γꢀvalerolactone, and α,α,αꢀtrifluorotoluene were evaluated for complete
Fmoc removal to be sure that these green solvents can replace DMF in a general way. Therefore,
a similar experiment as described in Scheme 1 was performed but with 7 min Fmoc removal time
instead of 45 s. DMF and 2ꢀMeTHF were also included for comparison purpose, because DMF is
the mostly used solvent in this type of reactions while the green 2ꢀMeTHF was recently
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employed for Fmoc removal reactions.
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γꢀValerolactone exhibited an excellent performance in the same range of DMF with both resins.
NꢀFormylmorpholine showed an excellent performance with ChemMatrix resin only. and 2ꢀ
MeTHF and α,α,αꢀtrifluorotoluene showed moderate to low performance with both resins (Table
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3).
a
Table 3. Fmoc removal in SPPS by using 20% piperidine/solvent after 7 minutes.
Fmoc removal (%)
b
Solvent
PS
ChemMatrix
100
DMF
100
47.9
27.5
95.5
47.7
NꢀFormylmorpholine
96.3
2
ꢀMeTHF
29.5
γꢀValerolactone
100
α,α,αꢀTrifluorotoluene
58.6
a
b
Green: Good performance, yellow: moderate, and red: low, Green: green solvent; Red: nonꢀ
green solvent.
Conclusions
α
This work has exemplified that the removal of the Fmoc group from the N ꢀamino function in
both solution and solidꢀphase modes is more demanding than commonly thought and is very
much solvent depending.
In solution and only taking into account green solvents, the removal of the Fmoc group from a
simple amino acid such as Phe can take place in 4 min when Nꢀformyl morpholine and γꢀ
valerolactone are used. Another convenient medium for the removal of Fmoc in 6 min is α,α,αꢀ
trifluorotoluene.
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In solidꢀphase synthesis, overall ChemMatrix gave better results than PS. Using PS resin, γꢀ
valerolactone, Nꢀformylmorpholine, and in this case EtOAc and dimethyl carbonate provided the
best results, while the ether solvents showed to be totally incompatible with this resin.
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For the ChemMatrix resin, Nꢀformylmorpholine and γꢀvalerolactone, followed by α,α,αꢀ
trifluorotoluene are the most efficient solvents. Although CPME did not give a good result for
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0
the coupling step, this solvent and isosorbide dimethyl ether rendered good results for the Fmoc
removal. The linear ester based solvents ethyl acetate and dimethyl carbonate gave disappointing
results. The overall conclusion with respect to SPPS is that γꢀvalerolactone could be an excellent
green alternative to DMF for both resins while Nꢀformylmorpholine is compatible with
ChemMatrix resin.
NꢀFormylmorpholine has the drawback of its price and that its stability should be
demonstrated, because the presence of the formyl moiety always represents a potential drawback
39
in peptide synthesis. Although, EtOAc and dimethylcarbonate were used by MacMillan et al.
for amide bond formation reactions and no sideꢀreactions were reported, the use of esters
solvents (γꢀvalerolactone, ethyl acetate, and dimethyl carbonate) should be further investigated to
assure that transesterication or methyl carbamate formation will not jeopardize its use in peptide
4
0
chemistry especially when we take into our consideration the recent report by Wong et al.
about the stability of γꢀvalerolactone. Although γꢀvalerolactone has been recently used as a
green solvent for acidic hydrolysis of carbohydrates into 5ꢀ(hydroxymethyl)furfural, levulinic
acid and formic acid.
In overall, it has been demonstrated that green solvents can be an excellent substitution for
conventional solvents in peptide synthesis.
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ASSOCIATED CONTENT
Electronic Supplementary Information (ESI) available: [Experimental details and HPLC
chromatograms] and these materials are available free of charge via the Internet at
http://pubs.acs.org.”
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AUTHOR INFORMATION
Corresponding Author
*
B. G. de la Torre. Eꢀmail: garciadelatorreb@ukzn.ac.za.
* F. Albericio. Eꢀmail: albericio@ukzn.ac.za or albericio@ub.edu.
Present Addresses
†
If an author’s address is different than the one given in the affiliation line, this information may
be included here.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. ‡These authors contributed equally. (match statement to
author names with a symbol)
ACKNOWLEDGMENT
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This work was funded in part by the National Research Foundation (NRF), the University of
KwaZuluꢀNatal (South Africa), and the Generalitat de Catalunya (2014 SGR 137) and MEC
(
CTQ2015ꢀ67870ꢀP). Additionally, the authors thank the Deanship of Scientific Research at
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King Saud University for funding this work through the Prolific Research Group Program (PRGꢀ
1437ꢀ33; Saudi Arabia). Finally, the authors thank Y. Luxembourg (Luxembourg Bio
Technologies Ltd) for continuous support of this study.
ABBREVIATIONS
API,
Active
Pharmaceutical
Ingredients;
COMU,
1ꢀ[(1ꢀ(cyanoꢀ2ꢀethoxyꢀ2ꢀ
oxoethylideneaminooxy)ꢀdimethylaminoꢀmorpholinomethylene)]methanaminium
hexafluorophosphate; Fmoc, 9ꢀfluorenylmethyloxycarbonyl; GSK, GlaxoSmithKline; REACH,
Registration, Evaluation, Authorization and Restriction of Chemicals; SPPS, solidꢀphase peptide
synthesis; SVHC, substances of very high concern.
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procedures to the synthesis of difficult sequences. Nat. Protocols 2007, 2 (12), 3247ꢀ3256.
3
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8. Taken from Vogel’s Textbook of Practical Organic Chemistry, 5th ed.
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