Successful development of a method for the incorporation of Fmoc-Arg(Pbf)-OH in solid-phase peptide synthesis using: N -butylpyrrolidinone (NBP) as solvent

Article abstract of DOI:10.1039/c9gc03784e

NBP has proved an excellent alternative solvent to the hazardous DMF for SPPS. Here we studied the incorporation of Fmoc-Arg(Pbf)-OH, one of the most problematic amino acids, into a growing peptide chain. The poor performance of this amino acid is attributed to the formation of a fully inactive δ-lactam, which causes a reduction in yield and very often the concomitant formation of the corresponding des-Arg peptides. This problem is exacerbated when NBP is used as solvent, presumably because of its high viscosity, which impairs the penetration of the coupling cocktail into the resin. To tackle this issue, we propose the following strategy for the safe introduction of Fmoc-Arg(Pbf)-OH in SPPS at 45 °C, keeping excesses to a minimum: 1.75 equiv. of the protected amino acids, 1.8 equiv. of DIC, and 1.5 equiv. of OxymaPure. The cornerstone of the strategy is to carry out in situ activation. In this regard, Fmoc-Arg(Pbf)-OH and OxymaPure dissolved in NBP were added to peptidyl-resin, allowed to reach the 45 °C, then half the DIC was added and left for 30 min, followed by the other half and some extra Fmoc-Arg(Pbf)-OH. During the entire process, the temperature was kept at 45 °C, with the double purpose of reducing the viscosity of NBP, thus facilitating the penetration of the coupling cocktail into the resin, and speeding up the coupling itself. It is envisaged that this strategy could be widely used to improve the performance of SPPS, including the industrial preparation of peptides using this approach.

Full text of DOI:10.1039/c9gc03784e

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DOI: 10.1039/C9GC03784E
Green Chemistry
ARTICLE
Successful Development of a Method for the Incorporation of
Fmoc-Arg(Pbf)-OH in Solid-Phase Peptide Synthesis using N
Butylpyrrolidinone (NBP) as Solvent
Received 00th January 20xx,
Accepted 00th January 20xx
*
a
a,b
b
c
Beatriz G. de la Torre, Ashish Kumar, Mahama Alhassan, Christoph Bucher, Fernando
*b,d,e
*c,
DOI: 10.1039/x0xx00000x
Albericio,
John Lopez
NBP has proved an excellent alternative solvent to the hazardous DMF for SPPS. Here we studied the incorporation of
Fmoc-Arg(Pbf)-OH, one of the most problematic amino acids, into a growing peptide chain. The poor performance of this
amino acid is attributed to the formation of a fully inactive -lactam, which causes a reduction in yield and very often the
concomitant formation the corresponding des-Arg peptides. This problem is exacerbated when NBP is used as solvent,
presumably because of its high viscosity, which impairs the penetration of the coupling cocktail into the resin. To tackle
this issue, we propose the following strategy for the safe introduction of Fmoc-Arg(Pbf)-OH in SPPS at 45C, keeping
excesses to a minimum: 1.75 equiv. of the protected amino acids, 1.8 equiv. of DIC, and 1.5 equiv. of OxymaPure. The
cornerstone of the strategy is to carry out in situ activation. In this regard, Fmoc-Arg(Pbf)-OH and OxymaPure dissolved in
NBP were added to peptidyl-resin, allow to reach the 45C, then half the DIC was added and left for 30 min, followed by
the other half and some extra Fmoc-Arg(Pbf)-OH. During the entire process, the temperature was kept at 45C, with the
double purpose of reducing the viscosity of NBP, thus facilitating the penetration of the coupling cocktail into the resin,
and speeding up the coupling itself. It is envisaged that this strategy could be widely used to improve the performance of
SPPS,
including
the
industrial
preparation
of
peptides
using
this
approach.
has been key for peptides to be considered alternative drugs
6
Introduction
to the so-called small molecules. Although in its early days
7
SPPS was heavily criticized by Merrifield’s colleagues, the
power of this approach has been widely exploited, first as a
potent research tool and later as the strategy of choice for the
The number of peptide-based drugs approved by the two most
important regulatory agencies, namely the Food and Drug
Administration (FDA) and the European Medicines Agency
8
, 9
1
-4
production of most of the peptides on the market.
In this
(EMA), has risen notably in recent years. With respect to the
regard, the establishment of the multi kilogram-scale
production of enfuvirtide (T-20 or Fuzeon), a first-in-class
membrane fusion inhibitor for the treatment of HIV, is
considered the validation of SPPS for the production of
validation of the role of the peptides in the drug arena, more
important than the number of peptides on the market (more
1
than 60 ) is the number of peptides in clinical phases (more
1
than 170 ). From a structural point of view, peptides either on
1
0
peptides. Enfuvirtide is a 36-amino acid peptide that was
synthesized by a hybrid solid phase–solution strategy, in which
three protected peptides were prepared in solid phase and
then assembled in solution. Although this compound did not
become the expected commercial blockbuster, its
development marked a breakthrough. At that time, it became
clear that almost any peptide could be synthetically affordable,
mostly using SPPS. As a minor, but important, consequence of
the bulk synthesis of enfuvirtide was that the high
consumption of protected amino acids and chlorotrityl
chloride (CTC) resin—the solid support used for the
preparation of the protected fragments—leaded the cost of
these materials fell, thereby bringing about a certain
democratization of the peptide field.
the market or in active development are characterized by
increasing complexity. Thus, those containing more than 30
amino acids, several cycles, and/or pending fatty or
polyethylenglycol chains are common in the various drug
discovery phases. Indeed, this is exemplified by the peptides
approved by the FDA in 2017, an exceptional year for the
peptide harvest, with six peptide approvals.
There is no doubt that the solid-phase peptide synthesis (SPPS)
strategy developed by the Nobel Laurate R. Bruce Merrifield
5
a
KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), School of
Laboratory Medicine and Medical Sciences, College of Health Sciences, University
of KwaZulu-Natal, Durban 4041, South Africa.
b
Peptide Science Laboratory, School of Chemistry and Physics, University of
KwaZulu-Natal, Durban 4001, South Africa.
Novartis Pharma AG, Novartis Campu Campus, WSJ-145-5.51, CH-4056 Basel,
Interesting, the most expensive protected amino acid currently
on the market, Fmoc-Arg(Pbf)-OH, was one of the exceptions
in the enfuvirtide sequence. However, to be fully honest, it is
also important to clarify that the synthesis of Fmoc-Arg(Pbf)-
c
Switzerland.
d
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine, Barcelona Science Park, University of Barcelona, 08028 Barcelona,
Spain.
e
Department of Organic Chemistry, University of Barcelona, 08028 Barcelona,
Spain.
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OH is probably the most complex of all common protected such as Etelcalcetide, Abaloparatide, and Semaglutide, among
View Article Online
1
1
5
DOI: 10.1039/C9GC03784E
others approved by the FDA in recent years. Arg is also key
amino acids.
From a sustainability perspective, SPPS has a number of pros for the development of the so-called Cell-Penetrating Peptides
2
4
and cons. The pros include the following: (i) easy (CPPs). It is important to note that Fmoc-Arg(Pbf)-OH, which
manipulations, as all reactions take place in the same reactor, is the most used building block for the introduction of Arg into
and therefore no isolation of intermediates is needed and no SPPS, is the most expensive of all protected proteinogenic
major cleaning procedures are required; (ii) easy work-up, amino acids and, like all Arg derivatives, it undergoes an
because the excess of reagents and soluble side-products are important side-reaction upon activation. In this regard, once
removed by filtration and washings, thereby allowing the use the carboxylic group of the protected Arg is activated, -lactam
of an excess of reagents, thereby facilitating high conversion formation takes place, with the corresponding formation of a
yields; (iii) use of solvents of high boiling point; and (iv) six-member ring (Scheme 1), which blocks the incorporation of
possibility of partial and even full automatization. On the other Arg into the growing peptide sequence. Although this side-
hand, the cons encompass the following: (i) the same use of reaction does not modify the final peptide, it often leads to the
reagent excess, although this is mitigated in large scale formation of deletion peptides, in which Arg is missing. To
production, where only slight excesses of the reagents are mitigate this, repetitive couplings of protected Arg are carried
used; (ii) very poor atom economy [e.g., 0.24 for Fmoc- out, a procedure that increases the cost of the whole process.
Arg(Pbf)-OH and 0.19 for Fmoc-Gly-OH]; and (iii) the large This drawback is exacerbated during the industrial preparation
volume of solvents used for washing the resin after each of Arg-containing peptides, where minimum excesses are used
synthetic step.
and repetitive couplings have a penalty in time, solvent
In this last regard, the large consumption of solvents and the consumption, and cost.
fact that N,N-dimethylformamide (DMF) has been by far the
most widely used solvent in SPPS are the main drawbacks in
greening the strategy. DMF is reprotoxic and, like other polar
aprotic solvents, such as N-methylpyrrolidone (NMP), and
dimethylacetamide (DMA), has been labeled by REACH-ECHA
as a Substance of Very High Concern (SVHC) and is therefore a
focus for replacement. To tackle this issue, several research
1
2-15
groups
and pharmaceutical industries participating in the
ACS Green Chemistry Institute Pharmaceutical Roundtable
1
6
have shown interest in identifying green solvents for SPPS. In
recent years, our groups have been working independently to
identify an alternative solvent to DMF. In this regard, one
Scheme 1. Activation of Fmoc-Arg(Pbf)-OH with the concomitant
formation of δ-lactam.
group
methyltetrahydrofurane (Me-THF) and
have been shown to be suitable alternatives to DMF.
other group has proposed the use of N-butylpyrrolidone (NBP)
has
developed
strategies
in
which
2-
Results and discussion

-valerolactone (GVL)
1
7-21
The
The present study was carried using DIC and OxymaPure as coupling
2
5
2
2
cocktail in DMF and NBP as solvents. OxymaPure is the additive of
C). According to OECD test methods, choice for the most peptide syntheses and has been proposed by
NBP is non-mutagenic, non-reprotoxic and biodegradable and the ACS Green Chemistry Institute Pharmaceutical Roundtable as
for the same purpose. NBP is a polar aprotic solvent with a
high boiling point (243

12
is therefore friendlier than other amide-based solvents of the greenest aditive.
related structure, such as DMF and NMP.
2
2
In our previous work, and after a careful evaluation of 34 To study the activation kinetics of Fmoc-Arg(Pbf)-OH and the
non-hazardous solvents, NBP was shown to outperform the subsequent ẟ-lactam formation, an equimolar mixture of the three
other solvents tested in terms of resin swelling, Fmoc removal reagents, Fmoc-Arg(Pbf)-OH, DIC, and OxymaPure, was prepared in
efficiency, and coupling yield and thus emerges as a strong the two solvents, DMF and NBP, at room temperature (RT). In the
candidate to replace DMF. NBP was successfully applied for absence of any nucleophile, the behavior observed in both solvents
the semi-industrial synthesis of the linear precursor of the
octapeptide Octreotide. Although the purity of the crude
peptide synthesized with NBP was slightly inferior to that
prepared with DMF (80% vs. 86%), the impurity profile was
similar in both cases. This observation reinforces the notion of
the appropriateness of NBP to achieve greener SPPS.
was very similar (Figure 1A). The activation in DMF was slightly
faster than in NBP, as was ẟ-lactam formation. When the same
experiment was carried out at 60C (Figure 1B), the activation and
ẟ-lactam formation were much faster than at RT, as expected.
Furthermore, the previous tendency was inversed and although
both solvents gave similar results, the complete conversion of
Fmoc-Arg(Pbf)-OH into its δ-lactam was slightly faster in NBP.
Next, the previous experiment at RT was repeated in the presence
of a nucleophile, the peptide H-Gly-Phe-Leu anchored to a Rink
amide polystyrene resin. This reaction mixture allowed us to study
Here, in an attempt to generalize the use of NBP, we studied
the compatibility of this solvent with the coupling of Fmoc-
Arg(Pbf)-OH. Arg is an important residue and has been object
2
3
of several methodological studies.
The relevance of this
amino acid lies in its presence in many peptide-based APIs,
2
| J. Name., 2012, 00, 1-3
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the competition between coupling and ẟ-lactam formation by the HPLC analysis of the supernatant and the compoVsieitwioAnrticloefOntlhinee
DOI: 10.1039/C9GC03784E
o
A
RT
B
6
0 C
peptide after cleavage from the resin (Scheme 2).
Figure 1. HPLC analysis of the activation kinetics and ẟ -lactamization of Fmoc-Arg(Pbf)-OH with DIC and OxymaPure in DMF and NBP at A)
RT and B) 60C (see methods section for HPLC conditions). Note: The peak corresponding to Fmoc-Arg(Pbf)-OH could be due to unreacted
amino acid or mostly to instability of the activated species [Fmoc-Arg(Pbf)-Oxyma] under aqueous HPLC conditions.
Scheme 2. Reaction of the activated Fmoc-Arg(Pbf)-OH with a
tripeptidyl resin.
For these sets of experiments, Fmoc-Arg(Pbf)-OH and OxymaPure
were dissolved in the corresponding solvent, and DIC was added at
a ratio of 1:1:1 at a concentration 0.075 M. Next, 1.5 equiv. of the
mixture was added to the resin, which had been previously washed
with the solvent, which was removed by filtration. HPLC analysis of
the supernatant showed almost negligible formation of ẟ-lactam
when DMF was used, in comparison with NBP (Figure 2). In
contrast, analysis of the cleaved peptide showed more than 98%
incorporation of Arg(Pbf) when DMF was used, but less of 50% in
the case of NBP (Figure 2).
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that the reaction had reached a plateau, where no fuVrtiehweArrtcicoleuOpnlilninge
was possible. Thus, as this temperature (6D0OIC: 1)0o.1n03t9h/Ce9oGnCe03h7a8n4Ed
Figure 2. HPLC analysis of the reaction described in Scheme 2 using showed rapid consumption of reagent in both the coupling and side
DMF and NBP as solvents at RT (See note at Figure 1 legend and reaction, and on the other hand could be considered a limit for
Material and Methods section for HPLC conditions).
industrial purposes, the fine-tuning should be attempted using a
strategy other than merely an increase in temperature.
1
7
As pointed out in the previous study carried out with NBP the As mentioned earlier, the higher viscosity of NBP may explain its
higher viscosity of NBP vs. DMF (4.0 cP vs. 0.8 cP at 25C) could poorer performance in coupling because of difficulty reaching the
explain the different performance of the coupling reaction, since reactive points inside the resin. In this regard, it was envisaged that
the viscosity of the solvent may prevent the active species from -lactam formation was taking place and was accelerated during
reaching the functional groups inside the resin. Thus, with this idea the time taken to reach the temperature after the addition of the
in mind, we repeated the same experiment at 45C in order to solution mixture to the resin at RT. With this idea in mind, we
reduce the effect of viscosity during the coupling. Analysis of the attempted a new strategy. Using 25% of the total NBP needed for
supernatants revealed that both solvents rendered similar results in the coupling, the resin was swelled and allowed to equilibrate at
terms of ẟ-lactam formation (Figure 3). However, analysis of the 45C, followed by the addition of the mixture solution containing
cleaved peptide showed an almost quantitative incorporation of the Fmoc-Arg(Pbf)-OH, OxymaPure, and DIC (1:1:1, 1.5 equiv.) in the
protected amino acid in the case of DMF and a substantial remaining 75% volume of NBP (0.075M final concentration). This
improvement in case of NBP, from 44.9% to 85.5% of coupling procedure was the so-called pre-conditioning strategy.
(
Figure 3).
Figure 3. HPLC analysis of the reaction described in Scheme 2 using
DMF and NBP as solvents at 45C. (See note at Figure 1 legend and
Material and methods section for HPLC conditions)
Figure 4 HPLC analysis of the reaction described in Scheme 2 using
NBP as solvent. A) at 60C. B) pre-conditioning at 45C (See note at
Although these results represented a notable improvement, they Figure 1 legend and Material and methods section for HPLC
were far from optimal. In order to fine-tune the coupling reaction in conditions).
NBP to achieve quantitative incorporation of Fmoc-Arg(Pbf)-OH into
the peptidyl-resin, the next experiments were performed only in Under this new strategy, analysis of the reaction supernatant
NBP. Thus, in view of the favorable effect of heating in the previous showed that not all activated protected amino acid had been
o
experiment, the temperature was increased to 60 C. Interestingly, consumed after 2 h, either because of coupling or conversion to -
analysis of the supernatant showed that no active species remained lactam, as indicated by the presence of Fmoc-Arg(Pbf)-OH, which
after 30 min of reaction and therefore no further coupling was derived from the active species hydrolyzed in the reaction mixture
possible after that time. Nevertheless, analysis of the cleaved or during the HPLC analysis (Figure 4B).
peptide showed that the incorporation of the protected Arg Analysis of the peptide after cleavage from the resin showed a
reached 93% (Figure 4A).
Although these results showed a satisfactory increase in the
coupling reaction, the lack of active species in a short time meant
coupling of 92.3%. On the other hand, these pre-conditioning
conditions at 45C almost matched (92.3% vs. 93.0%) the best
coupling performance, which were achieved at 60C and clearly
4
| J. Name., 2012, 00, 1-3
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better than the previous experiment at 45C (85.5% vs 92.3%, place when the peptidyl-resin is already solvated inVNiewBPArtaictle4O5nlinCe,
Figure 3). The observation that no full conversion to ẟ-lactam had and therefore the peptide bond formationDOdIo: 1e0s.1n0o39t/Cco9mGCp0e3t7e84soE
been reached using the last conditions supported the idea that fine unfavorably with ẟ-lactam formation.
tuning them could render better results. In other words, the Encouraged by this result, the next experiment was designed under
presence of some Fmoc-Arg(Pbf)-OH, presumably from Fmoc- the same concentration, temperature and equivalent as before but
Arg(Pbf)-Oxyma, indicated that the coupling was able to proceed modifying the way in which DIC was added. The total amount DIC
further.
was divided into two equal portions, the first was added when the
peptidyl-resin and Fmoc-Arg(Pbf)-OH/OxymaPure mixture in the
NBP solution reached 45C and the second after 30 min of reaction.
In this case, the coupling achieved was slightly better (98.7%)
(
Figure 6B). Although slight, this increase was considered positive
because it showed that this strategy does not impair the process
and thus fine-tuning can be attempted.
Figure 5. HPLC analysis of the reaction described in Scheme 2 using
NBP as solvent by “in situ” activation. A) at concentration 0.075M.
B) at concentration 0.15M. (See note at Figure 1 legend and
Material and methods section for HPLC conditions).
In view of the improved results achieved by pre-conditioning the Figure 6. HPLC analysis of the reaction described in Scheme 2 using
temperature of the resin, the further experiments were carried out NBP as solvent by “in situ” activation using Fmoc-Arg(Pbf)-
doing this pre-conditioning, adding a solution with the total amount OH/DIC/Oxyma 1:1.2:1 ratio. A) DIC addition in one portion. B) DIC
(
1:1, 1.5 equiv) of Fmoc-Arg(Pbf)-OH and OxymaPure to the resin to addition in two portions. (See note at Figure 1 legend and Material
the resin. Then, once the mixture had reached 45C, the total and methods section for HPLC conditions).
amount of DIC (1.5 equiv.) was added. This procedure, which was
The rationale behind this experiment was to protect Fmoc-Arg(Pbf)-
called "in situ" activation, was run as described previously at
reagents concentration of 0.075M but also using the same
equivalents at a concentration of 0.15 M, a value very similar to
that used in production mode SPPS. These assays highlighted the
importance of concentration, since the one performed at 0.075 M
gave a coupling yield of 88.4% while that at 0.15 M was 95.1%. In
both cases, but particularly in the second, a large amount of
unreacted Fmoc-Arg(Pbf)-OH was found when the supernatants
were analyzed (Figure 5). Therefore, to force the coupling of the
remaining protected amino acid, the next experiment was
performed by increasing the amount of coupling reagent, i.e. DIC,
leading the ratio of reagents Fmoc-Arg(Pbf)-OH/DIC/Oxyma to
OH from conversion into ẟ-lactam. As shown in Scheme 3, when
half the DIC (0.90 equiv.) is added, a part of Fmoc-Arg(Pbf)-OH is
converted to its O-acyl isourea derivative [Fmoc-Arg(Pbf)-DIiU],
(
max 0.90 equiv.), which immediately reacts with the rest of the
Fmoc-Arg(Pbf)-OH (0.60 equiv.) to give the symmetrical anhydride
[Fmoc-Arg(Pbf)] O] and/or with OxymaPure to give the Oxyma
active ester [Fmoc-Arg(Pbf)-Oxyma]. Then, the [Fmoc-Arg(Pbf)]
[
2
2
O
can react either with the amino peptidyl-resin or with Oxyma pure.
In either case, one molecule of Fmoc-Arg(Pbf)-OH (0.60 equiv.) will
be released and this will be activated when the second portion of
DIC is added (Blue pathway in Scheme 3).
1
:1.2:1 (this means 1.8 equiv of DIC with respect to the growing
peptide)following the same procedure as before. This increment in
the DIC ratio yielded a satisfactory improvement of the result,
raising coupling to 98.2% (Figure 6A). This result could be because
using this in situ activation, the formation of the Oxyma ester take
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that obtained by standard protocols using DMF as sVoielvweAnrttic(leFOignulirnee
).
8
DOI: 10.1039/C9GC03784E
Figure 8. HPLC Analysis of the lineal precursor of the cyclic RGD
peptide. Green: total synthesis in NBP, Arg coupled under
Scheme 3. Synthetic pathway involved in the coupling of Fmoc- methodology developed. Red: standard synthesis in DMF. Grey:
Arg(Pbf)-OH.
Des-Arg peptide synthesized as standard.
Finally, the same strategy was attempted but adding some fresh
Fmoc-Arg(Pbf)-OH together with the second portion of DIC. Only
Experimental
0
.25 equiv. of the protected amino acid with respect to the growing
Materials
peptide was added, resulting in a total usage of 1.75 equiv. of the
protected Arg. In this case, the coupling reaction reached 100%.
All reagents and solvents were purchased from commercial
suppliers and used without further purification. Fmoc amino acids,
Fmoc-Rink Amide AM resin (loading 0.74 mmol/g) and 2-CTC resin
were purchased from Iris Biotech. NBP (TamiSolveNxG) was
purchased from Eastman, OxymaPure was a gift from Luxembourg
Biotech., and N,N-Diisopropylethylamine (DIEA) and 4-
methylpiperidine were supplied by Sigma-Aldrich. Organic solvents
Dimethylformamide (DMF), 2-Methyltetrahydrofuran (2-MeTHF),
cyclopenthylmethyl eter (CPME) and HPLC quality acetonitrile
(
3
CH CN) were purchased from Merck. Milli-Q water was used for
RP-HPLC analyses. Analytical HPLC was performed on an Agilent
TM
1
100 system using a Phenomenex Aeris C18 (3.6μm, 4.6 × 150
mm) column, with flow rate of 1.0 mL/min and UV detection at 220
nm. Chemstation software was used for data processing. Solvent A:
0
2 3
.1% TFA in H O; Solvent B: 0.1% TFA in CH CN. LC-MS was
performed on a Shimadzu 2020 UFLC-MS using an YMC-Triart C18
Figure 7. HPLC analysis of the reaction described in Scheme 2 using (5 μm, 4.6 × 150 mm) column. Solvent A: 0.1% formic acid in H O;
2
NBP as solvent by “in situ” activation using Fmoc-Arg(Pbf)-
solvent B: 0.1% formic acid in CH CN.
3
OH/DIC/Oxyma 1:1.2:1 ratio. DIC addition in two portions, 0.25
equiv of protected Arg in the second DIC addition. (See note at
Figure 1 legend and Material and methods section for HPLC
conditions).
δ-Lactam formation study
1
. 0.1 M solution of Fmoc-Arg(Pbf)-OH/DIC/OxymaPure (1:1:1) was
prepared in NBP or DMF, 10-μL sample was taking at 0, 30, 60, and
20 min. mixture and diluted to 1 mL with CH CN. From this
To validate this procedure, first the assay was performed studying
the coupling over a more hindered amino acid, since Gly lacks of
side chain. The choice was Val because it is a β-branched amino
acid, thus the coupling reaction is more difficult. The tripeptide H-
Val-Phe-Leu-NH-Rink-amide-resin was prepared and Fmoc-Arg(Pbf)-
OH was coupled following the last protocol described above.
Incorporation exceeded 99%.
1
3
solution 1 μL was injected and analyzed by analytical RP-HPLC.
General Method: 30–95% B into A in 15 min.
2
. From the different coupling conditions (Table 1) 10-μL sample of
the supernatant was taking at 0, 30, 60, and 120 min. mixture and
diluted to 1 mL with CH CN. From this solution 1 μL was injected
and analyzed by analytical RP-HPLC. General Method: 30–95% B
Next, the strategy was applied to the synthesis of the lineal into A in 15 min.
3
26
precursor of the Cilengitide, the cyclic RGD peptide. The sequence
H-DfKRG-OH was synthesized on chlorotrityl (CTC)-resin and the
peptide was obtained with excellent purity without the presence of
the des-Arg peptide. The quality of the crude was comparable to
SPPS
6
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All peptides were assembled manually in plastic syringes fitted with using a ratio Fmoc-AA-OH/DIC/Oxyma 1:1:1, 1.5 equViievw. AinrticNleBOPnlioner
a polyethylene porous disc, according to Fmoc/tBu methodology by DMF at 45 °C for 1h. The different conditionDsOfI:o1r0c.1o0u3p9l/iCn9gGoCf0F3m78o4cE-
mean of DIC/ Oxyma as coupling agents. Fmoc removal was carried Arg (Pbf)-OH are detailed in Table 1. Peptides were cleaved from
out by treatment with a solution of 20% of 4-methylpiperidine. The the resin, by treating with TFA/TIS/H2O (95:2.5:2.5) for 1 h at rt and
solvent was NBP and DMF for comparison.
The tripeptidylresin (H-GFL-resin) was prepared on Rink-amide-PS quantification was 10–25% B into A.
loading 0.74 mmol/g), coupling of each residue was performed
precipitated with chilled CPME. HPLC analysis method for coupling
(
Table 1: Coupling conditions for Fmoc-Arg(Pbf)-OH
Fmoc-Arg(Pbf)-OH/DIC/ Oxyma
[Fmoc-Arg(Pbf)-OH]
(M)
Entry
Ratio
Eq.
T (°C)
1
2
3
4
1:1:1
1:1:1
1:1:1
1.5
1.5
1.5
0.075
0.075
0.075
RT
45°C
60°C
1
1.5
1.5
1.5
0.075
0.075
0.15
0.15
0.15
0.15
45°C
45°C
45°C
45°C
45°C
45°C
1:1:1
1:1:1
1:1:1
2
2
5
6
7
8
9
2
3
4
1
.5:1.8:1.5
.5:1.8:1.5
1
1
1
:1.2:1
:1.2:1
:1.2:1
1
1
.75:1.8:1.5
1
Pre-conditioned resin in solvent at 45°C
2In situ activation:
Pre-conditioned resin in Fmoc-Arg (Pbf)-OH/Oxyma in solvent at 45°C, followed by DIC addition.
3
4
Pre-conditioned resin in Fmoc-Arg (Pbf)-OH/Oxyma in solvent at 45°C, followed by addition:
t=0 0.6 equiv of DIC
t=30 min 0.6 equiv of DIC
Pre-conditioned resin in Fmoc-Arg (Pbf)-OH/Oxymas in solvent at 45°C, followed by addition:
t=0 0.6 equiv of DIC
t=30 min 0.25 eq. Fmoc-Arg(Pbf)-OH + 0.6 equiv of DIC
RGD and desArgRGD peptides were synthesized on 2-CTC resin intramolecularly, leading to its -lactam derivative. The kinetic
0.05 mmol scale, loading 0.56 mmol/g). The incorporation of the comparison of -lactamization in DMF and NBP in the presence of a
(
first amino acid was done with Fmoc-Gly-OH (1.5 equiv) in 2-Me- stoichiometric ratio of DIC/oxymapure proved only slightly superior
THF in presence of base DIEA (10 equiv) for 2 hours, then capping in DMF than NBP at RT but was much faster in the green solvent
with MeOH (50µL) for 30 min at room temperature. The rest of when the temperature was raised to 60C. When the assay was
amino acids were coupled as described above and Arg was performed in the presence of the nucleophile, the peptidyl-resin, to
incorporated following the conditions Entry 9 described in Table 1. study the competition between δ-lactam formation and
Peptides were cleaved from the resin, by treating with TFA/TIS/H2O incorporation of the protected amino acid to the resin, the results
(
95:2.5:2.5) for 1 h at rt and precipitated with chilled CPME. The revealed the key role of solvent viscosity for the coupling reaction.
solid was dissolved in 10% aqueous AcOH and liophilized to render While at RT, the incorporation of the protected Arg was almost
2
7mg of a floppy solid (87% overall yield). HPLC analysis method
quantitative in DMF and less than 50% in NBP, at 45C the
was 5–20% B into A. The peptides were characterized by LCMS.
incorporation using NBP exceeded 85%.
Conclusions
After systematically modifying the different parameters that could
influence the coupling reaction, such as temperature, concentration
The potential suitability of NBP as a green solvent candidate to or mode of addition of the reagents, a successful protocol was
substitute the DMF in SPPS was reported in a previous study. In the established. Thus, the optimal temperature was fixed at 45C, the
present work, we focused on the coupling of Fmoc-Arg(Pbf)-OH, a concentration of the reagents at 0.15 M and the Fmoc-Arg(Pbf)-
highly problematic amino acid because of its ability to react OH/DIC/oxymapure ratio at 1:1.2:1. The procedure involved pre-
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conditioning of the NBP solution containing the protected Arg, 15.
oxymapure and the peptidyl-resin at the fixed temperature, then
addition of half the DIC and after 30 min the other half, together
Y. E. Jad, A. Kumar, A. El-Faham, B. G. de laVieTwoArrrteicleaOndnlinFe.
Albericio, ACS Sustainable ChemD. OEIn: 1g0..,1023091/C99, G7C,033768741E-
3683.
A. Isidro-Llobet, M. N. Kenworthy, S. Mukherjee, M. E.
Kopach, K. Wegner, F. Gallou, A. G. Smith and F.
Roschangar, J. Org. Chem., 2019, 84, 4615-4628.
Y. E. Jad, G. A. Acosta, S. N. Khattab, B. G. de la Torre, T.
Govender, H. G. Kruger, A. El-Faham and F. Albericio,
Amino Acids, 2016, 48, 419-426.
with 0.25 equiv. of Fmoc-Arg(Pbf)-OH.
16.
Thus, using NBP as a solvent, it was possible to safely introduce
Fmoc-Arg(Pbf)-OH in SPPS under conditions compatible with 17.
industrial requirements, namely moderate temperature and
keeping excesses to a minimum: 1.75 equiv. For the protected
amino acids, 1.8 equiv. of DIC, and 1.5 equiv. Of oxymapure. It is 18.
envisaged that this procedure will be further applied for the
incorporation of other protected amino acids in a SPPS approach.
Y. E. Jad, G. A. Acosta, S. N. Khattab, G. Beatriz, T.
Govender, H. G. Kruger, A. El-Faham and F. Albericio,
Amino acids, 2016, 48, 419-426.
1
9.
O. Al Musaimi, Y. E. Jad, A. Kumar, A. El-Faham, J. M.
Collins, A. Basso, B. G. de la Torre and F. Albericio, Org.
Process Res. Dev, 2018, 22, 1809-1816.
F. Albericio, A. Kumar, Y. E. Jad, J. Collins and B. G. de la
Torre, 2018.
A. Kumar, A. Thompson-Adewumi, K. Nandhini, J. M.
Collins, F. Albericio and B. G. de la Torre, Org. Process Res.
Dev, 2019, 23, 1096-1100.
J. Lopez, S. Pletscher, A. Aemissegger, C. Bucher and F.
Gallou, Org. Process Res. Dev, 2018, 22, 494-503.
T.-L. Hwang, K. Ranganathan, Y.-Q. Fang, R. D. Crockett, S.
Osgood and S. Cui, Org. Process Res. Dev, 2018, 22, 1007-
Finally, this work has evidenced that the study of the supernatant of
a SPPS reaction can bring key information to fine-tune the overall
process.
2
0.
1.
2
Conflicts of interest
There are no conflicts to declare
2
2.
3.
Acknowledgements
2
The work was funded in part by the following: the National
Research Foundation (NRF) (# 105892 and Blue Sky’s Research
Programme # 110960) and the University of KwaZulu-Natal
1014.
24.
C. Allolio, A. Magarkar, P. Jurkiewicz, K. Baxová, M.
Javanainen, P. E. Mason, R. Šachl, M. Cebecauer, M. Hof
and D. Horinek, Proc. Natl. Acad. Sci., 2018, 115, 11923-
(
South Africa); and the Spanish Ministry of Science, Innovation,
and Universities (RTI2018-093831-B-100) and the Generalitat
11928.
de Catalunya (2017 SGR 1439) (Spain).
2
5.
6.
R. Subirós‐Funosas, R. Prohens, R. Barbas, A. El‐Faham
and F. Albericio, Chem. Eur. J., 2009, 15, 9394-9403.
M.-M. Carlos, R. Florian and K. Horst, Anti-Cancer Agents
in Medicinal Chemistry, 2010, 10, 753-768.
2
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8
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