Microwave-assisted TFA cleavage of peptides from Merrifield resin

Article abstract of DOI:10.1002/psc.1191

Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5min microwave-assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI-MS conditions. Copyright

Full text of DOI:10.1002/psc.1191

Research Article
Received: 13 July 2009
Revised: 28 August 2009
Accepted: 7 September 2009
Published online in Wiley Interscience: 10 November 2009
(www.interscience.com) DOI 10.1002/psc.1191
Microwave-assisted TFA cleavage of peptides
from Merrifield resin
Alicja Kluczyk,∗ Magdalena Rudowska, Piotr Stefanowicz
and Zbigniew Szewczuk
Microwave-assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner
reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses
of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used
in product cleavage are not compatible with off-resin monitoring of the reaction progress. The application of MW irradiation
in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and
the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well
as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in
synthetic bioactive peptides was analyzed using LC-MS and MS/MS experiments. In a 5 min microwave-assisted TFA reaction
at low temperature, the majority of product is released from the resin, making the analytical scale MW-assisted procedure a
method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility
c
with HPLC and ESI-MS conditions. Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: microwave-assisted synthesis; SPPS; Merrifield resin; cleavage; protecting groups
Introduction
in use, especially in the synthesis of peptide conjugates when
nonstandard reaction conditions are often required [18–22]. The
standard acidolysis of the Merrifield linker requires strong acids
such as liquid HF, TFMSA or HBr/TFA [23]. As these acids have
to be removed before the analysis of the product, the procedure
is inconvenient for reaction monitoring or pilot syntheses on a
small scale (10–50 mg of the resin). The TFA : DCM (1 : 1) mixture
was used by Stadler and Kappe [24] as the cleavage reagent in
microwave irradiation (500 W, 30 min) to remove a number of
carboxylic acids from the Merrifield resin. No degradation of the
polymer support was detected, but the high reaction temperature
(120 ◦C) seems to be not compatible with SPPS because of the
possibility of peptide degradation [25].
In our research on peptide–heterocycle conjugates [26,27] we
usedmicrowave-assistedcleavagefromWang resintomonitorthe
heterocycle formation. The product cleavage was complete after
five irradiation steps (1 min) in TFA/H2O (95 : 5) mixture, which
was confirmed by incubation of the same resin sample in TFA for
further 2 h and HPLC analysis of the evaporated filtrate. Owing to
highabsorbanceofthequinoxaline–peptideconjugate,theHPLC-
UV is a very sensitive method of product detection (A. Kluczyk,
unpublished results). We have found that, after the microwave-
assisted cleavage, there is no product left on the resin; therefore,
thereleaseofproductfromWangresiniscompletein5 minat20 W
irradiation energy, which is in agreement with literature data [15].
However, there are classes of peptide conjugates, which require
a more stable linker to the solid support for a successful synthesis.
Since 1992, when Wang described the use of the kitchen
microwave oven in SPPS [1], a number of reports have been
published that show advantages of microwave irradiation in
organic chemistry, including peptide synthesis (loading of the
resin [2–6], coupling of amino acids [7,8] or in cleavage of peptides
from solid support [9]).
The application of microwaves in cleavage of peptides from
solid support attracts a lot of attention; however, the number
of available reports is limited, and in most cases the product is
removed from the resin in a classical way [8,10–14]. A typical
procedure was recently described by Harris et al. [15], who
synthesized peptides on Wang and Rink amide polystyrene (PS)
resin and released the products from the resin by treatment
with TFA/TIS/H2O/EDT (94 : 1 : 2.5 : 2.5, v/v/v/v) either at room
temperature (rt) for 2–3 h or under microwave irradiation (18 min,
10 W, maximum temperature 35 ◦C). Microwave irradiation was
also applied to the cleavage of highly lipidated peptide from
2-chlorotrityl support with TFA/H2O (1 : 1, v/v; 150 W, 20 min at
80 ◦C) [16]. It was found that the cleavage of the peptide from the
resinproceedswellusinglow-powermicrowaveirradiation.Brandt
et al. [17] compared three different resins: PEGA (poly(ethylene
glycol)polyacrylamide), PS, TG (tentagel) and three handles:
rink amide and two kinds of BAL (backbone amide linker) in
the microwave-assisted synthesis of the phosphorylated 15-mer
peptide. To release the product from the resin, a small sample
of resin was treated with TFA/H2O (19 : 1) or with TFA/TFMSA
(9 : 1) for 2 h at rt, and also under microwave irradiation for 5 min
at 60–80 ◦C. It was demonstrated that all resins performed well
when heated with strong acids and only the PEGA resin started to
degrade at 60 ◦C in TFA/TFMSA.
∗
Correspondence to: Alicja Kluczyk, Faculty of Chemistry, University of Wroclaw,
F. Joliot-Curie 14, 50-383 Wroclaw, Poland. E-mail: kluczyk@wchuwr.pl
Despite the advantages of Fmoc peptide synthesis strategy
on Wang resin, the Merrifield resin and Boc/Bzl method is still
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie14, 50-383 Wroclaw,
Poland
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J. Pept. Sci. 2010; 16: 31–39
Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.

KLUCZYK ET AL.
In our research on solid-phase imidazole-containing peptides, we
used the Merrifield resin, and, to monitor the reaction progress,
we attempted to use the microwave-assisted TFA cleavage from
this resin.
very low MW-assisted cleavage from peptidyl resin 2, we repeated
this synthesis using commercially available Boc-Ile-Merrifield resin
(peptide 2a).
We investigated the efficiency of the method using model
peptides with various C-terminal amino acids to determine
the influence of the steric hindrance on product release from
Merrifield resin (R):
Synthesis of peptides 5–8
The syntheses were published elsewhere [28–30].
Cleavage from Merrifield resin
Boc-Gly-Phe-Ala-Gly-R
Boc-Gly-Phe-Ala-Ile-R
Boc-Gly-Ile-Ala-Phe-R
Boc-Gly-Phe-Ala-Val-R
[peptide 1]
[peptide 2]
[peptide 3]
[peptide 4]
Standard procedure (room temperature)
A sample of resin (50 mg) was placed in polypropylene syringe
reactor and was treated with TFA/TFMSA/cresol (25 : 3 : 2, 1 ml) for
2.5 h at rt. The resin was removed by filtration and rinsed with TFA.
The combined filtrates were added to cold diethyl ether and the
precipitated peptide was collected by centrifugation.
All the peptides contain the Phe residue to facilitate the moni-
toring of the cleavage progress by HPLC with UV detection. The
sequences were also designed to avoid the possible incomplete
side-chain protection removal interference in product analysis.
To determine the effect of microwave-assisted TFA cleavage on
protectinggroups,weselectedfourpeptidylresinsusedpreviously
for the synthesis of IL-1 inhibitors (peptides 5 and 8) [28], TGFβ
fragments (peptide 6) [29] and tuftsin analogs (peptide 7) [30]:
Microwave-assisted procedure
A sample of resin (50 mg) in a 2-ml polypropylene syringe reactor
was treated with TFA/TIS/H2O (95 : 2.5 : 2.5, 1 ml) for 15 min at
rt. Then the reactor was transferred into microwave synthesizer
cavity and subjected to microwave irradiation with gas cooling
(pressure of 20 psi was maintained during irradiation) for a total
time of 15 min (15 × 1 min) at 30 W with magnetic stirring, and a
temperature limit of 50 ◦C. Between irradiations, the sample was
cooled in an ice-water bath for 2 min. Finally, the solution was
removed and the resin was washed with TFA. The combined
filtrates were evaporated in a stream of nitrogen. In all the
experiments, the 2-ml syringe reactors with perforated Parafilm
caps were used.
Boc-Pro-Gly-Gly-Gly-Val-Thr(Bzl)-Lys(Z)-Phe-Tyr(Bzl)-Phe-R
Boc-Arg(NO2)-Thr(Bzl)-Pro-Lys(Z)-Val-R
[peptide 5]
[peptide 6]
Ac-Lys(Z)-Arg(NO2)-Pro-R
[peptide 7]
[peptide 8]
Boc-Val-Thr(Bzl)-Lys(Z)-Phe-Tyr(Bzl)-Phe-Ile-Thr(Bzl)-Gly-Ser(Bzl)-Glu(OBzl)-R
In this work, we present the study on the microwave-assisted
method of peptide cleavage from Merrifield resin using TFA, which
could be used not only for the final release of the product but also
in a fast off-resin method of reaction monitoring compatible with
ESI-MS and HPLC.
ForUV-HPLCexperiments,thetotalreactiontimewasprolonged
to 30 min, with the cleavage mixture replaced every five steps (5
irradiations by 1 min).
When using different types of reaction vessels (standard CEM
glass microwave vials, 5-ml syringe reactors, etc.), it might be
necessary to modify the reaction conditions to keep the reaction
temperature below 50 ◦C (shorter reaction time, more efficient
cooling, etc.).
Materials and Methods
Reagents
All solvents and reagents were used as supplied, exceptfor TFMSA,
which was distilled at atmospheric pressure prior to use. Boc-
amino acid derivatives and Boc-amino acid–loaded resins were
synthesized in-house. The Merrifield support (2% cross-linked
chloromethylated copolymer of styrene – divinyl benzene) was
purchased from Reanal, Hungary and loaded using the cesium
carbonate procedure [31]; the loading was calculated according
Standard procedure (elevated temperature, 45 ◦C)
A sample of resin (50 mg) was treated with TFA/TIS/H2O
(95 : 2.5 : 2.5, 1 ml) for 15 min at rt, then for 30 min at 45 ◦C in
a water bath with occasional shaking. The solution was separated
and the resin was washed with TFA. The combined filtrates were
evaporated in a stream of nitrogen.
to the mass difference and was in the range of 0.6–0.7 mmol g−1
.
Boc-Ile attached to Merrifield resin (loading 0.52 mmol g−1), used
for synthesis of peptide 2a, was purchased from Amino Tech.
N,N,Nꢁ,Nꢁ-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluorob-
orate (TBTU) and TFA were purchased from Iris Biotech.
After peptide cleavage, the deprotected resins were immedi-
ately prepared for further analysis by consecutive washing with
DCM, TEA/DCM (1 : 9), DCM, DCM/MeOH (1 : 1) and MeOH and
drying in vacuo.
All the microwave-assisted reactions were carried out in a
monomode microwave Discover CEM apparatus, equipped with
IR temperature sensor and gas cooling system. The reactor was
used in Power-Time mode (a set power value was applied for a
specific time, with the temperature safety limit of 50◦).
Peptide Analysis
HPLC
Prior to HPLC analysis, all peptides were dissolved in MeOH : water
(1 : 1) solution. The analysis was performed using a Thermo
Separation HPLC system with UV detection (210 nm) on a Vydac
ProteinRP C18column(250×4.6 mm, 5 µm), withgradientelution
of 0–80% B in A (A = 0.1% TFA in water; B = 0.1% TFA in
acetonitrile/H2O, 4 : 1) over 45 min (flow rate 1 ml min−1, rt).
Peptide Synthesis
Synthesis of peptides 1–4
SPPS was performed manually in polypropylene syringe reactors
(Intavis AG) equipped with polyethylene filters, according to
standard Boc/Bzl procedure. As the preliminary results indicated a
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Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2010; 16: 31–39

MW-ASSISTED TFA CLEAVAGE FROM MERRIFIELD RESIN
Elemental analysis
The carbon, nitrogen and hydrogen content of the deprotected
resins was established using VarioEL III CHNS analyzer (Elementar
Analysensysteme, Germany) in the Laboratory of Elemental
Analysis and Environmental Studies at the Faculty of Chemistry,
University of Wroclaw.
Infrared spectroscopy
The samples of deprotected resins were ground and mixed with
KBr. The IR spectra for KBr tablets were recorded on an FTIR IFS
66/ S spectrometer (Bruker, Germany) in Laboratory of Infrared
Spectroscopy at the Faculty of Chemistry, University of Wroclaw.
Mass spectrometry
Figure 1. The HPLC chromatograms obtained after cleavage of peptide 3
(main peak) from Merrifield resin using presoaking in TFA and consecutive
5-min MW 30-W irradiations.
Mass spectra were recorded on a Bruker micrOTOF-Q mass
spectrometer and apex ultra 7T FT-ICR mass spectrometer with
electrospray ionization in the Laboratory of Mass Spectrometry at
the Faculty of Chemistry, University of Wroclaw. The samples were
dissolved in a 50 : 50 acetonitrile–water mixture containing 0.1%
HCOOH.
the resin was rinsed with TFA/H2O and again irradiated using a
fresh portion of TFA/H2O. The procedure was repeated six times.
The respective filtrates (from irradiation stage and washing step)
were evaporated and subjected to HPLC analysis. As shown in
Figure 1, the intensity of peptide signal decreased in samples from
consecutive experiments, but the product signal did not disappear
even after 30 min of irradiation. The same tendency was observed
for other model peptides.
The initial experiments showed the increase in peptide content
during the second irradiation, which indicated that a time period
is required for the proper swelling of the resin (a dry sample was
used in these experiments). To allow proper penetration of the
resin by TFA and to improve the yield in the next experiments,
we incubated the resin prior to the first irradiation step in the
cleavage mixture for 15 min at rt. As indicated by the ‘15-min
TFA’ chromatogram in Figure 1, during this time the product is
practically not released from the support. The soaking period also
allows for a safe release of gaseous products of TFA-induced Boc
group decomposition.
The temperature of the reaction has to be controlled, both by
reactor IR temperature probe and the external metering, because
TFA and its solutions strongly absorb the microwave energy. In
a separate experiment, after each irradiation step, a protected
digital temperature probe was inserted into the reaction mixture.
The conditions (microwave power, time) were adjusted to keep
the reaction temperature below 50 ◦C. Before the next irradiation
step, the syringe reactor was placed into ice-water bath for 2 min
to readjust the initial reaction conditions. A similar approach
(intermediate cooling step) was used previously by Bacsa et al.
[10] for synthesis of model peptides.
The LC–MS analysis was performed using Agilent 1200 HPLC
systemcoupledtomicrOTOF-Qmassspectrometer.Forseparation,
a RP C18 Zorbax (50 × 2.1 mm, 3.5 µm) column was used, with
gradient elution of 0–100% B in A (A = 0.1% HCOOH in water; B =
0.1% HCOOH in acetonitrile) over 30 min (flow rate 0.1 ml min−1
room temperature).
,
Results and Discussion
We investigated the efficiency of the product release from the
Merrifield resin by analyzing the changes in resin weight and
elemental composition, as well as the changes in IR spectra. To
concentrate on the peptide removal from Merrifield support using
the microwave-assisted TFA procedure, we used model peptides
1–4, which did not require protection of side chains. The resins
deprotected in TFA with microwave irradiation were compared
with other portions of the same batch, subjected to standard
cleavage (TFA/TFMSA/cresol). Both samples were neutralized,
washed and dried to reduce the interference from acids used
in deprotection.
The model tetrapeptides were synthesized on Merrifield resin
using a standard Boc procedure. The resins loaded with longer
fully protected peptides were selected from the research group
repository [28–30].
Optimization of the Protocols of MW-assisted Cleavage
In the earlier experiments, we investigated the influence of
microwave power on cleavage efficiency. Samples of peptide
3 were removed from the Merrifield support during 5-min
microwave irradiations of 20, 30, 35 and 40 W. The HPLC analysis
of cleaved peptide indicated that the increase in MW power
by over 30 W does not result in higher removal yield, but
causes overheating of the mixture. Therefore, we used the 30-
W microwave power setting to release peptides from the resin in
further experiments.
MW-assisted Cleavage Versus Standard TFMSA Reaction
To compare the yields of cleavage of the peptides from Merrifield
support with and without MW radiation, we evaluated the amount
of released peptides using the previously established UV-HPLC
procedure, and the level of resin deprotection, using elemental
analysis, weight comparison and IR spectroscopy. We selected
peptides of different lengths (from tripeptide to undecapeptide)
and sequences to investigate the influence of the C-terminal
residue on cleavage efficiency (peptides 5–8).
We also optimized the time of MW irradiation. A sample of
peptidyl resin 3 was suspended in TFA/H2O and irradiated with
30 W MW for 5 min. Then the cleavage solution was filtered,
The standard reaction used the TFA/TFMSA/cresol mixture, with
areactiontimeof2.5 h.Theproductwasprecipitatedincolddiethyl
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J. Pept. Sci. 2010; 16: 31–39
Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

KLUCZYK ET AL.
Figure 2. The comparison of HPLC peak areas of peptides 1–4 cleaved from the Merrifield resin using standard TFMSA procedure, TFA with microwave
irradiation (30 W, 15 × 1 min) and TFA at elevated temperature 45 ◦C (water bath). Fifty-milligram resin samples from the same synthetic batch were used
in each case.
ether and separated by centrifugation. In the microwave-assisted
procedure, the resins were treated with TFA/TIS/H2O solution
for 15 min at rt and then for 15 min (15 × 1 min, with cooling
between irradiations) at 30 W. Afterward, the cleavage mixture
was evaporated. All the peptides released from Merrifield resin
were analyzed by HPLC with UV detection and mass spectrometry
(MS).
In case of peptides 5–8, we observed that not all protecting
groups were removed at the same time and to the same extent,
which resulted in several signals in HPLC chromatograms and was
confirmed by MS analysis. Model peptides 1–4 were designed
specially for UV-based analytical procedure; hence, a direct
comparison of chromatographic peak intensity was possible.
As shown in Figure 2, the TFMSA treatment releases more
product from the Merrifield resin than in the MW-assisted TFA
reaction; however, in the case of peptide 1, with C-terminal
Gly residue, the difference is minimal. The branched C-terminal
residues (Ile in case of peptide 2 and Val in peptide 4) seem to be
more resistant to microwave-assisted TFA cleavage.
Figure 3. The HPLC profiles of peptide 4 removed from the Merrifield resin
using TFA in water bath at elevated temperature and in MW reactor.
Comparison of the TFA Cleavage at 45 ◦C and the MW
Procedure
We compared the results obtained for four model tetrapeptides
with different sequences, and in each case the microwave-assisted
cleavage resulted in more product release (Figure 2). The observed
differences in reaction yield under microwave irradiation as
compared to conventional heating (water bath) are in agreement
with several reports on possible nonthermal effects in microwave-
assisted reactions [24,32–35].
To investigate the influence of thermal effects on TFA cleavage of
the product from Merrifield resin, the reactions were run without
MWirradiation. Allsamplesweretreatedwiththecleavagemixture
at rt for 15 min to achieve proper swelling. The peptide resins were
subjected to the reaction conditions (TFA/H2O/TIS mixture) for
30 mininthermostatedwaterbathat45 ◦C, whereasanothersetof
resinsamplesunderwentthemicrowave-assistedprocedure(30 W
15×1 min, coolingbetweenirradiations). Forthethermalreaction,
the 30-min period was selected to compensate for the prolonged
exposition of the microwave sample to TFA during the cooling
steps. In the microwave-assisted procedure, the temperature did
not exceed 50 ◦C (measured after irradiation).
Figure 3 shows the HPLC profiles of the crude peptide 4 (H-
Gly-Phe-Ala-Val-OH) obtained from the resins incubated with
TFA in a water bath and with MW irradiation. It is evident
that the yield of product is much higher for the MW-assisted
reaction than in the case of conventional heating (water bath).
Elemental Composition and Weight Analysis of the Resin
Taking into account the partial deprotection of side-chain groups
(peptides 5–8) as well as the solubility of hydrophilic model
peptides in diethyl ether, we investigated the resin left after the
cleavage to determine the completeness of peptide removal.
The elemental composition of all resin samples was analyzed
and the difference in nitrogen content was determined, as in
the peptidyl Merrifield resin the synthesized peptide is the only
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Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2010; 16: 31–39

MW-ASSISTED TFA CLEAVAGE FROM MERRIFIELD RESIN
loss is significantly higher after TFMSA treatment, whereas in other
cases the MW-assisted procedure is more efficient. The theoretical
loss values were calculated according to the loading of the resins,
whereas the samples after cleavage taken from the same synthetic
batch and neutralized and dried by the same procedure after the
cleavage show the real difference between the methods. Actually,
in the case of longer peptides, both the weight difference and
the elemental analysis results may be affected by the fact that the
protective groups could be removed from the peptide when it is
still attached to the resin. This may be responsible for the results
for peptides 5 and 8, the longest in the series, where the nitrogen
loss is relatively small, but the weight difference does not differ
much from the other samples.
The results show that the efficiency of the cleavage reaction
depends on the C-terminal amino acid and the length of peptide.
The MW-assisted reaction is affected by the amino acid residues
withstericalhindrance.Itisvisiblethatthepeptideisremovedfrom
the resin by TFA with microwave assistance, and the prolongation
of the reaction time should increase the yield to the level of TFMSA
cleavage.
Table 1. Nitrogen loss and weight difference in peptidyl resins
deprotected using standard TFMSA and microwave-assisted (MW)
TFA (30 W, 15 × 1 min) procedures
Mass difference between
Nitrogen loss
(%)
peptidyl resin before and
after cleavage (mg)
C-terminal
residue
Peptide TFMSA
MW
TFMSA
MW
Theorya
Gly
Ile
1
2
93.3
87.7
94.3
93.9
71.4
100.0
81.3
18.4
35.0
65.5
10.9
8.8
12.8
18.6
19.4
18.3
14.2
18.4
17.8
5.9
15.9
12.7
13.5
15.4
18.8
13.5
14.4
11.5
26.3
10.3
12.8
10.1
14.1
26.1
12.5
18.7
14.8
25.4
2a
3
Phe
Val
43.7
24.2
12.8
26.0
19.3
21.1
5
4
6
Pro
Glu
7
8
21.1
Fifty-milligram resin samples from the same synthetic batch were used
in each case.
a Calculatedaccordingtotheloadingoftheresinandpeptidesequence.
Infrared Analysis of the Resin
component containing nitrogen. The weight difference between
the loaded and deprotected resins was measured (50 mg samples
were used). The theoretical weight loss was calculated according
to the resin loading and the peptide sequence (Table 1).
All the resins, after removing peptides with TFMSA method or
MW irradiation, were analyzed using IR spectroscopy. The spectra
of respective peptidyl resins were used as references. The IR
spectroscopy was frequently applied to the monitoring of solid-
phase reactions [3,36–38].
The composition analysis shows that the MW-assisted TFA
reaction leaves more peptide on the resin than the TFMSA
procedure; however, the difference depends strongly on the C-
terminal residue, for example, peptides with C-terminal Val and
Ile residues are more resistant to MW-assisted cleavage, whereas
peptide 7 (Ac-Lys-Arg-Pro-OH) in general shows poor cleavage
efficiency. The results obtained for peptide 2, significantly lower
than for other peptides, prompted us to repeat the synthesis using
The bands, both at 1450 and 1943 cm−1, were attributed to
aromatic bonds vibrations of Merrifield resin [36] and could be
treated as a baseline. The band at 1680 cm−1 corresponds to
absorption of amide carbonyl group and another characteristic
band at 1740 cm−1 is attributed to the ester carbonyl group
vibrations. The selection was confirmed after comparing the IR
spectraofcommerciallyavailablefreeMerrifieldresinandtheresin
loaded with Boc-Ile. Figure 4 shows that the carbonyl bands have
the largest intensity for peptide resin and practically disappear
in samples after cleavage both with the classical method and
MW irradiation. These bands are more intensive for resin after
microwave-assisted cleavage than after classical method, which
suggests that MW-assisted release of the peptides from Merrifield
resin is less efficient; however, both methods remove a significant
a commercial Boc-Ile-Merrifield resin (loading 0.52 mmol g−1
,
peptide 2a). As shown in Table 1, the results obtained for
theseindependentlysynthesizedpeptidesarepracticallyidentical,
confirming that the C-terminal Ile residue is responsible for the
poor cleavage efficiency in the MW-assisted procedure.
The weight difference between the peptidyl resin and the resin
after the cleavage is less pronounced in the analyzed samples. For
the pairs of samples that contain C-terminal Ile and Val, the weight
Figure 4. The comparison of IR spectra: resin loaded with the peptide 1 (thin line), the resin after TFMSA deprotection (dotted line) and TFA–MW
deprotection (thick line).
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KLUCZYK ET AL.
Figure 5. Top panel: the LC–MS chromatogram of crude peptide 5 obtained after 15-min MW-assisted TFA cleavage from Merrifield resin (total ion
current, positive ion mode). Chromatographic peaks A–E correspond to forms of peptide 5 with various protection level, peaks F–J represent methylated
forms of compounds A–E. Panels A–E: mass spectra of peaks of deprotected (A) and partially protected (B–E) forms of peptide 5.
amount of the product. A similar conclusion was obtained after
the analysis of IR spectra of other resin pairs, with samples 5, 7 and
8 showing the smallest changes, which is in agreement with the
results of weight analysis.
involving the transfer of the sample from microwave reactor to the
cooling bath is tedious, and the commercially available microwave
synthesizerswithinternalthermostating(liketheCoolMatesystem
introduced recently by CEM) might be of use in this application.
Although MW irradiation is not superior to the TFMSA cleavage
in terms of yield, the advantage of using microwave-assisted TFA
protocol for Merrifield resin lies in the possibility of working with a
minute amount of the resin (few milligrams), removing the solvent
by evaporation and analyzing the product, which allows for mon-
itoring of reaction progress. The standard precipitation of product
after TFMSA cleavage in diethyl ether is not suitable for very small
samples and hydrophobic products, frequently soluble in ether.
Theresultsshowthatthe5-minirradiationat30 Wremovesmostof
the product, and the sample could be used after dilution for direct
electrospray mass spectrometry analysis. Such a procedure could
accompany the classical Kaiser test or even replace it if reactions
do not involve the amino group. A similar effect could be obtained
through the HF procedure, which, however, requires complicated
equipment and special handling due to safety reasons.
MW-assisted Removal of the Protecting Groups
We also investigated the release of the amino acid protecting
groups used in Boc/Bzl peptide synthesis strategy during MW-
assistedcleavageofthepeptidefromMerrifieldresin.Thereactions
were performed using small samples of peptidyl resin 5 (Boc-
Pro-Gly-Gly-Gly-Val-Thr(Bzl)-Lys(Z)-Phe-Tyr(Bzl)-Phe-R), which was
selected because of the presence of Z (benzyloxycarbonyl) and
benzyl ether (both aromatic and aliphatic type) protecting groups.
The Boc (t-butoxycarbonyl) protecting group is removed by
TFA during the 15-min preincubation period [39]. The analysis
of the HPLC profiles of the products after TFMSA and MW-
assisted cleavage indicated that after the MW procedure the peaks
observed in the chromatogram appear at much longer retention
times than those from the TFMSA cleavage sample, suggesting a
higher hydrophobicity caused by the not-complete deprotection.
The LC–MS analysis performed after 15 min of irradiation
revealed a small amount of the expected product (peak A in
The increase in the amount of peptidyl resin is possible, but
requires a very strict temperature control with shorter irradiation
steps to avoid overheating of the reaction mixture. The procedure
c
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Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2010; 16: 31–39

MW-ASSISTED TFA CLEAVAGE FROM MERRIFIELD RESIN
Figure 6. The mass spectra of peptide 5 after MW-assisted TFA cleavage from Merrifield resin after 5, 10 and 15 min of irradiation (negative ion mode).
whereas the peptide with one benzyl group removed and the fully
protected form were found in peaks D and E, respectively.
InthemassspectrumextractedfrompeakIsignalscharacteristic
for peptide containing two benzyl groups could be observed;
Table 2. Calculated monoisotopic masses and m/z values for the
possible partially protected forms of peptide 5
Protecting
M
Protecting groups
removed
groups
left
however, additional signals corresponding to the methyl ester
of the peptide were also present. The m/z value of the fully
deprotected peptide with an additional methyl group was also
evident in peak F (m/z 543.8). Peaks G and H represent the two
possible combinations of benzyl ether and methyl ester. The
methylated products are probably artifacts, formed when the
sample was dissolved in methanol before LC–MS analysis.
To confirm the order of removal of the protecting groups, we
investigated the samples collected in separate experiments after
5, 10 and 15 min of the MW irradiation, and analyzed them in
negative ion mode to avoid the differences in ionization efficiency
of Nα and Nε amino groups, as well as to simplify the spectrum
due to the lack of multiple charge ions (Figure 6).
We found that during MW-assisted TFA cleavage of the peptide
from the Merrifield support the removal of the protecting groups
is incomplete. After 5 min of 30-W microwave irradiation, the
dominant signal in MS spectrum represents the peptide with all
side-chain protecting groups (Figure 6). During the next stage of
theremovalprocedure, theblocking groupsaregraduallyreleased
from the peptide, with the Z group being the most labile group,
which could be concluded from the relative intensity of the signals
corresponding to peptides without the Z or one Bzl group. The
fully deprotected peptide appeared in the sample after 10 min of
MW irradiation, but its abundance was low and did not increase
much in the sample irradiated for a further 5 min (Figure 6).
Signal (g mol−1
)
m/z
A
1071.5
358.2 [M+3H]3+
536.8 [M+2H]2+
1072.5 [M+H]+
–
Z and 2Bzl
Z and Bzl
B
C
1161.6
1251.7
581.8 [M+2H]2+
Bzl
1162.6 [M+H]+
418.3 [M+3H]3+
626.8 [M+2H]2+
1252.7 [M+H]+
Z
2Bzl
D
E
1295.7
1385.7
1296.7 [M+H]+
Bzl
–
Z and Bzl
1386.7 [M+H]+
Z and 2Bzl
The letters A–E indicate identified signals in LC–MS chromatogram
(Figure 5).
Figure 5) and most of the possible forms containing combinations
ofprotectinggroups(Table 2andFigure 5). Thepartiallyseparated
peak B represents two isomeric peptides with a single benzyl
protection left (on Thr or Tyr residues). The most abundant peak
C corresponded to the peptide containing two benzyl groups,
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KLUCZYK ET AL.
further reactions, or, in the case of partially protected product
mixture, subjected to hydrogenation, in order to complete the
protecting group removal with no risk of product deterioration.
The partial removal of protecting groups may be inconvenient
when the TFA cleavage is used to confirm the product formation,
but in our opinion the advantages of short reaction time and
the possibility of immediate analysis of the released product by
ESI-MS, compensate for the effort spent in calculating the formulas
of possible forms of product.
Conclusions
We have demonstrated that peptides could be successfully
removed from Merrifield resin using TFA in a microwave-assisted
procedure. Although the cleavage is not as efficient as the TFMSA
method, most of the product is released from the resin during
a 5-min reaction. The fact that the amount of peptide removed
from the resin in MW-assisted reaction is higher than that in
reaction run at 45 ◦C (conventional heating) suggests a possibility
of nonthermal effects. The quick removal of the product from
Merrifield support using TFA makes the procedure developed by
us a method of choice in monitoring the reactions carried out on
Merrifield resin due to the short reaction time and compatibility
with HPLC and ESI-MS conditions.
Figure 7. The MS/MS spectrum of the ion m/z = 1162.6, corresponding
to peptide 5 with one protective benzyl group preserved (FT-ICR, collision
energy of 30 eV). The asterisks indicate the daughter ion series produced
when the benzyl group is located on Tyr residue.
The intensity of the signal of peptide with only one Bzl group
left increases significantly with the irradiation time. There are
two possible locations of that benzyl group – in the side chain of
threonine or tyrosine residues. It seems that the remaining group
is quite stable, because of a gradual increase in M+Bzl signal, but
there are practically no changes in abundance of the completely
deprotected peptide (signal M) after prolonged MW irradiation. As
phenolic ethers are more sensitive to acidic environment [39], we
suppose that the Bzl group is removed from tyrosine.
In order to confirm this hypothesis, we investigated the
collision-induced dissociation (CID) of the ion m/z = 1162.6
using a high-resolution FT-ICR mass spectrometer. To facilitate the
fragment identification, the experiment was performed in positive
ion mode. Figure 7 shows the fragmentation spectra obtained by
CID-MS/MS of the singly charged precursor ion m/z = 1162.6,
corresponding to peptide 5 with one remaining benzyl group. The
relative abundance of b8 and b8-H2O daughter ions, containing
the Thr(Bzl) fragments, could be compared to that of respective
ions formed from peptide with Tyr(Bzl) (marked with asterisks).
The results indicate that the signal at m/z = 1162.6 comes from
the mixture of two isomeric ions:
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