Development of a new microwave-assisted cleavable backbone amide linker (BAL): A comparative study

Article abstract of DOI:10.1039/b919365k

A thorough comparative study to demonstrate the properties of a new microwave labile backbone amide linker is presented. A cyclic pentapeptide, cyclo(Trp-Gln-Gly-β-Ala-Phe), was used as a model and synthesized following 16 different conditions. The new backbone amide linker is stable towards acid and base at r.t., and can be cleaved at elevated temperature in trifluoroacetic acid under microwave irradiation, avoiding the use of aggressive reagents like HF. The new linker is compatible with Boc- as well as Fmoc-strategy and allows the cleavage of acid labile side chain protective groups at r.t., prior to cleavage of the cyclic pentapeptide from the resin. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b919365k

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COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Development of a new microwave-assisted cleavable backbone amide linker
(BAL): a comparative study†
Stijn Claerhout,a Thibault Duche`ne,b Dirk Tourwe´b and Erik V. Van der Eycken*a
Received 17th September 2009, Accepted 31st October 2009
First published as an Advance Article on the web 19th November 2009
DOI: 10.1039/b919365k
A thorough comparative study to demonstrate the properties
of a new microwave labile backbone amide linker is presented.
A cyclic pentapeptide, cyclo(Trp-Gln-Gly-b-Ala-Phe), was
used as a model and synthesized following 16 different condi-
tions. The new backbone amide linker is stable towards acid
and base at r.t., and can be cleaved at elevated temperature
in trifluoroacetic acid under microwave irradiation, avoiding
the use of aggressive reagents like HF. The new linker is
compatible with Boc- as well as Fmoc-strategy and allows
the cleavage of acid labile side chain protective groups at r.t.,
prior to cleavage of the cyclic pentapeptide from the resin.
of pharmacophoric groups which can interact with the corre-
sponding receptors.4 In this way, they form useful leads for the
development of new drugs as was successfully demonstrated by
the examples of Octreotide5 and Cyclosporin A.6
Linkers with an aldehyde functionality, to which the first amino
acid is anchored via reductive amination, have become a widely
used tool in SPPS, since their first description by Barany et al.7
in the mid 1990’s. This so-called Backbone Amide Linker (BAL)
approach, in which the growing peptide chain is anchored to a
solid support via a backbone amide nitrogen instead of via its
Ca-carboxy group, allows the limitations linked to C-terminal
anchoring or side chain anchoring to be overcome and gives access
to these C-terminal modified and cyclic peptides.8
Introduction
Since the description of this first BAL-approach, numerous
variations have been described, giving access to a variety of C-
terminal modified peptides and allowing the use of several cleavage
conditions.3
Since the introduction of the concept of Solid Phase Peptide
Synthesis (SPPS) by Merrifield in 1963,1 this technique has evolved
to a very efficient procedure for the synthesis of peptides and
even small proteins. The breakthrough of combinatorial chemistry
has increased the need for solid phase synthesis, even outside
the domain of peptides, e.g. for the synthesis of small organic
molecules.2
The acid sensitivity of the BAL is mainly determined by the
stability of the formed carbenium ion after treatment with acid.
The substitution pattern (e.g. the influence of methoxy groups on
the benzene ring), the planarity of the aromatic core and the peri-
effect define the ease in which the carbocation is formed.9 These
factors have led to the development of many different BAL’s with
various acid sensitivities.3 Less electron-rich analogues than the
original trisalkoxy BAL such as dialkoxy,10,11 alkoxy-hydroxy12
and monoalkoxy13–15 benzaldehyde, indole,16 naphthalene,9,17,18
thiophene19 and triazene20 based handles have also been used as
core structures for SPPS by the BAL-strategy.3,21 Also photolabile
BAL’s have been developed which are not acid sensitive.22
However, these described linkers still show some limitations.
Their synthesis is not always straightforward and the alkoxy
substituents on the aromatic core often lead to steric hindrance
which tends to make the acylation of the attached secondary amine
more difficult.3,7,23
A comparison is given between the monoalkoxy-BAL 5-(4-
formylphenoxy)pentanoic acid 114 and a new meta-dialkoxy-BAL
5-(4-formyl-2-methoxyphenoxy)pentanoic acid 2 which shows
interesting properties in combination with microwave irradiation
(Fig. 1). These properties were studied during the synthesis of
a model cyclic peptide cyclo(Trp-Gln-Gly-b-Ala-Phe), which was
previously prepared in a study using BAL linker 1.24
In the original design of Merrifield for SPPS, the growing
peptide chain is attached to the resin through its C-terminal
carboxyl group and the peptide chain is elongated towards its
N-terminus. After cleavage from the resin, C-terminal carboxylic
acids or amides are obtained. Nevertheless, within the context
of pharmaceutical development of peptides, the demand for
biologically relevant analogues of peptides where the C-terminus
is modified to other functionalities such as alcohols, ethers, esters,
N-alkylamides, hydrazides, trifluoromethyl ketones, aldehydes,
mercaptoalkylamides, thioesters, thioamides and cyclic peptides
is rising.3 Modifications of the C-terminus, as well as its use
to form cyclic peptides, give access to new classes of peptides
that represent potentially efficient therapeutic agents.3 These
modifications increase the bioavailability of peptides by protecting
them against enzymatic degradation and by increasing their ability
to cross biological barriers. By fixing some conformations of
peptides through cyclization, their receptor affinity and selectivity
in comparison with their linear analogues, can be increased. Small
cyclic peptides are also very good scaffolds for the introduction
The monoalkoxy-BAL 1 has the advantage of having a high
acid stability, so that Boc- as well as Fmoc- and allyl-protective
groups can be used. Due to this orthogonality, peptide cyclization
as well as the removal of acid sensitive side-chain protective groups
can be performed on-resin (Scheme 1). The disadvantage of this
monoalkoxy-BAL 1 is the need of very strong acidic cleavage
conditions as anhydrous HF or a mixture of HBr in trifluoroacetic
aLaboratory for Organic and Microwave-Assisted Chemistry (LOMAC),
Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan
200F, B-3001 Leuven, Belgium. E-mail: erik.vandereycken@chem.
kuleuven.be
bLaboratory for Organic Chemistry (ORGC), Vrije Universiteit Brussel
(VUB), Pleinlaan 2, B-1050 Brussel
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on enabling technologies for organic synthesis.
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Fig. 1 The monoalkoxy-BAL 1 and the meta-dialkoxy-BAL 2.
Scheme 1 Synthesis of cyclo(Trp-Gln-Gly-b-Ala-Phe) applying the BAL-strategy. PG, PG¢ = protective group; PG = Boc or Fmoc; PG¢= trityl when
Fmoc-strategy was used; no PG¢ when Boc-strategy was used.
acid (TFA),24 which is inappropriate for combinatorial issues.
The long reaction times are also a major drawback. The acid
sensitivity of BAL-linkers based on a benzaldehyde structure can
be increased by the use of electron donating substituents, e.g. one
or two methoxy groups.3 However, a general problem inherent to
the BAL-strategy is the difficult coupling of the second amino acid
to the secondary amine,7,23 leading to long reaction times. This
problem is more severe when the electron donating substituent
is in the ortho-position towards the aldehyde function, a position
which assures the best resonance stabilization of the carbenium ion
that is formed during cleavage. These problems can be addressed,
for instance by using an ortho-phenolic hydroxyl which allows an
intramolecular O–N acyl transfer reaction, but also the application
of microwave irradiation19,25 has been reported.
We now propose a new linker 2 which offers the same advantages
as linker 1, but whose acid sensitivity has been fine tuned by the
introduction of a methoxy substituent in the meta position towards
the aldehyde function, so that it is stable during TFA treatment
at r.t., but it becomes sufficiently labile upon TFA treatment
under microwave irradiation, and does not have an increased
steric hindrance for the coupling of the second amino acid to
the secondary amine.
pentapeptide, cyclo(Trp-Gln-Gly-b-Ala-Phe), was used as a model
and synthesized under 16 different conditions. This allowed a
profound comparison of the applied conditions. This reference
peptide has been prepared using BAL linker 1 and a Boc-strategy
in 14% crude yield and 97% purity after HF cleavage from the
solid support.24
In order to investigate the acid sensitivity of the new BAL 2 and
to compare it with that of the existing BAL 1, the cyclic peptide was
assembled on both BAL-linkers as shown in Scheme 1. Microwave-
assisted cleavage of the peptide from the resin applying linker 1
required a rather high temperature (Table 1, entry 6). Although
the cleavage was complete at 120 ◦C, LCMS-analysis revealed
that the peptide contains many impurities. Attempts to lower the
cleavage temperature by increasing the TFA concentration resulted
in incomplete or no cleavage of the peptide (Table 1 entries 1–5).
In contrast, cleavage of the peptide linked with BAL 2, required
only 80 ◦C to have complete cleavage (Table 1 entry 8), while linker
2 was stable towards TFA at r.t. (Table 1, entry 7)
The yields of the peptides, linked via linker 1 and cleaved upon
microwave irradiation, were very low as were the purities (Table 2,
entries 9, 11, 13, 15). Every synthesized peptide was also cleaved
using HF at 0 ◦C, in order to make a profound comparison
(Table 2, entries 2, 4, 6, 8, 10, 12, 14 and 16). These experiments
showed that when the cyclic peptides were cleaved from the resin
with HF, they were isolated with acceptable yields and purities
(Table 2, entries 10, 12, 14, 16). Therefore we can conclude that
the elevated temperature needed to cleave the cyclic peptide from
Results and discussion
To demonstrate the properties of the new microwave labile
BAL 2, a thorough comparative study was performed. A cyclic
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Table 1 Acid stability of linker 1 and 2 towards TFA at different
at room temperature was compared with coupling at elevated
temperature under microwave irradiation.26–28 Except for the
deprotection of the amino acids and the reductive amination, all
coupling steps were done applying microwave irradiation. The
completeness of the coupling reactions was followed with our
recently developed DESC test29 for the colorimetric detection of
resin bound primary and secondary amines.
temperatures under microwave irradiation
Entry Linker Conditionsa
Cleavageb?
1
2
1
1
rt, TFA–DCM (95 : 5), 1 h
MW, 80 ◦C, TFA–TIS (95 : 5),
50 W, 30 min
No
No
3
4
5
6
1
1
1
1
MW, 90 ◦C, TFA–TIS (95 : 5),
50 W, 30 min
No
The microwave-assisted reductive amination, necessary for the
coupling of the first amino acid to the resin-bound linker, was
already described in literature.19,25 However, when the reductive
amination was performed under microwave irradiation at a ceiling
temperature of 60 ◦C for 2 ¥ 40 min we detected at the end of the
peptide synthesis, next to the presence of the correct sequence, also
a slight amount of deletion peptide, missing the C-terminal Phe. A
possible explanation for this phenomenon is an over reduction of
the aldehyde to the alcohol by NaCNBH3. During the following
amino acid coupling step, b-Ala reacts with this alcohol and
further peptide synthesis leads to a deletion sequence lacking the
first amino acid Phe. Therefore we decided to run this reaction
under conventional conditions. Comparable yields and purities
were obtained for the peptides synthesized applying conventional
coupling conditions or coupling under microwave irradiation via
Boc- or Fmoc-strategy (Table 2, compare entry 1 with 5 and entry
3 with 7). This demonstrates that the application of microwave
irradiation can speed up the synthesis without loss of yield and
purity compared to conventional coupling conditions.26–28
Finally, the two mainly used N-protective groups for peptide
synthesis, Boc and Fmoc were evaluated. Comparing the results
of the peptides synthesized via Boc- and Fmoc-strategy, we notice
that both protective groups are compatible with the new linker
(Table 2, entries 1–8). Orthogonal deprotection of acid labile
protective groups, prior to release of the peptide, is possible, as
the new linker is stable towards TFA at r.t. (Table 1, entry 7).
When Fmoc N-protected amino acids were used, the amide of
the Gln side chain was trityl protected. The trityl group was
MW, 100 ◦C, TFA–DCM (6 : 4),
80 W, 30 min
Traces
MW, 100 ◦C, TFA–TIS (95 : 5),
50 W, 30 min
Yes, incomplete
Yes, complete
MW, 120 ◦C, TFA–DCM (1 : 4),
20 min
7
8
2
2
rt, TFA–DCM (95 : 5), 1 h
MW, 80 ◦C, TFA–TIS (95 : 5),
50 W, 40 minc
No
Yes, completec
a The peptide was synthesized following the Boc-strategy applying mi-
crowave irradiation for the coupling and cyclization reactions (see materials
and methods). b The cleavage efficiency was determined by repeating the
applied cleavage conditions twice and checking (HPLC) the filtrate for
the presence of cleaved amino acid. c When the temperature and TFA
concentration was lowered, incomplete cleavage was observed.
the resin under microwave irradiation in TFA is detrimental to the
peptide.
Table 2 in contrast shows that the new linker 2 does not need
HF for cleavage to obtain good yields and purities (Table 2, entries
1, 3, 5 and 7). Despite the fact that mostly (Table 2, entries
1-8) a slightly higher yield was observed using HF cleavage, it
is obvious that cleavage in TFA applying microwave irradiation
◦
at a temperature of 80 C represents a more convenient method
than the use of liquid HF. The ease of cleavage under microwave
irradiation conditions justifies the loss of yield compared to the
hazardous HF conditions.
Microwave irradiation also accelerates the amino acid coupling
reaction rates considerably.26–28 Therefore, conventional coupling
Table 2 Results of the investigated variations given in Scheme 2
Entry Linker Coupling conditions a(MW/Conv.) Boc/Fmocb Cleavage conditions cHF or MW Crude yieldd (%) Purity after cleavagee’c (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
MW
MW
MW
MW
Conventional
Conventional
Conventional
Conventional
MW
MW
MW
Fmoc
Fmoc
Boc
MW
HF
MW
HF
MW
HF
MW
HF
MW
HF
MW
HF
MW
HF
MW
HF
36
58
40
54
39
21
33
48
75
65
71
90
72
51
78
53
7
70
10
69
20
90
25
72
Boc
Fmoc
Fmoc
Boc
Boc
Fmoc
Fmoc
Boc
Very lowf
47
Very lowf
33
MW
Boc
Conventional
Conventional
Conventional
Conventional
Fmoc
Fmoc
Boc
Very lowf
33
Very lowf
23
Boc
a MW-coupling: 60 ◦C, 50 W, 20 min (see methods and materials for detailed conditions). Conventional coupling: r.t., 2 h (see methods and materials
for detailed conditions). b Fmoc-Gln-OH was side chain trityl protected. c Cleavage conditions for linker 2: MW, 80 ◦C, 45 min, 60 W, TFA–TIS (95 : 5).
Cleavage conditions for linker 1: MW, 120 ◦C, 15 min, 100 W, TFA–DCM–TIS (20 : 75 : 5). Cleavage conditions with HF for linker 2 and 1: HF(liq)–
p-cresol–p-thiocresol (9 : 0,5:0,5), 0 ◦C, 90 min. d Yields are based on the loading of the aminomethylated polystyrene resin (0.2 mmol g-1). e Purity was
determined by HPLC with UV detection at 215 nm. f According to LCMS 5–10% of the cyclic peptide was present in the mixture next to many unidentified
side compounds.
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Scheme 2 Investigated variations for the synthesis of the model peptide cyclo (Trp-Gln-Gly-b-Ala-Phe). Reagents and conditions: Conventional synthesis
(blue): (a) Linker 1 or 2 (3 equiv), HOBt (3 equiv), DIC (3 equiv), r.t., 24 h; (b) NaBH3CN (5 equiv), H-L-Phe-OAll.HCl (5 equiv), DMF–MeOH
(9 : 1), r.t., 18 h; (c) Fmoc- or Boc-b-Ala-OH (5 equiv), DIC (2,5 equiv), CH2Cl2–DMF (9 : 1), r.t., 2 ¥ 24 h; (d) Fmoc- or Boc AA-OH (3 equiv), HOBt
(3 equiv), DIC (3 equiv), r.t., 3 h; (e) Pd(PPh3)4 (5 mol%), PhSiH3 (24 equiv), dry CH2Cl2, 3 h; (f) PyBOP (3 equiv), DIEA (6 equiv), r.t., 2 ¥ 4 h; (g)
conditions are given in Table 1. Microwave-assisted synthesis (green): (a) HOBt (3 equiv), DIC (3 equiv), CH2Cl2–DMF (9 : 1), linker (3 equiv), 60 ◦C,
20 min; (b) NaBH3CN (5 equiv), H-L-Phe-OAll.HCl (5 equiv), DMF–MeOH (9 : 1), r.t., 2 ¥ 8 h; (c) Fmoc- or Boc-b-Ala-OH (10 equiv), DIC (5 equiv),
CH2Cl2–DMF (9 : 1), MW, 2 ¥ 40 min; (d) Fmoc- or Boc AA-OH (3 equiv), HOBt (3 equiv), DIC (3 equiv), MW, 60 ◦C, 20 min; (e) Pd(PPh3)4 (5 mol%),
PhSiH3 (24 equiv), dry CH2Cl2, 3 h; (f) PyBOP (3 equiv), DIEA (6 equiv), 2 ¥ 40 min; (g) conditions are given in Table 1.
removed before release of the cyclic peptide from the support
upon treatment with a TFA–TIS (95 : 5) mixture at r.t. This did
not lead to a decreased yield (Table 2, entries 1 and 5) indicating
that orthogonal deprotection prior to release of the peptide is
possible with the new BAL 2.
In summary, a new tailor made acid labile linker 2 was developed
for the BAL solid phase methodology. The new linker is compatible
with Boc- as well as Fmoc-strategy and allows the cleavage of acid
labile side chain protective groups at r.t., but is cleavable under
acidic conditions (TFA–TIS, 95 : 5) at elevated temperature (80 ◦C)
upon microwave irradiation. The new linker 2 was thoroughly
compared with the existing linker 1, the last requiring very strong
acidic conditions (HF) for the release of the peptide from the solid
support. Additionally, it was once more demonstrated that the use
of microwave irradiation for the coupling reactions reduces the
coupling time leading to a seriously decreased total reaction time
for the synthesis of cyclic pentapeptides.
Gln was only side-chain protected in the Fmoc method, as Fmoc-
Gln(Trt). The aminomethylated polystyrene resin (0.2 mmol g-1,
100–125 mm) was obtained from RAPP Polymere. Peptides
were synthesized using the conditions described in Scheme 2.
Microwave irradiation (2.45 GHz) was performed in a dedicated
CEM-Discover monomode microwave apparatus. All experiments
were carried out in sealed microwave process vials (10 mL).
A temperature control was performed using external infrared
sensor. After completion of the reaction, the vial was cooled to
25 ◦C via air jet cooling before opening. Conventional peptide
synthesis was carried out manually in syringes containing a PE
frit (MultiSyntech Gmbh, Germany) for filtration.
Synthesis of monoalkoxy-BAL 5-(4-formylphenoxy)pentanoic
acid 1
Step 1. To a solution of 4-hydroxybenzaldehyde (10 g,
82 mmol, 1 equiv) in DMF (100 mL), ethyl 5-bromovalerate
(15.57 mL, 98.4 mmol, 1,2 equiv) and K2CO3 (16.84 g, 123 mmol,
1.5 equiv) were added. The reaction mixture was stirred at room
temperature for 19 h under argon atmosphere. After reaction
diethyl ether, (300 mL) and water (200 mL) were added and the
layers were separated. The water phase was washed two times more
with diethyl ether. The combined organic layers were dried over
MgSO4, and the solvent was removed under reduced pressure.
A white product was obtained, which still contains the excess
Materials and methods
Amino acid derivatives, PyBOP (benzotriazol-1-yl-oxytripyrro-
lidinophosphonium hexafluorophosphate), DIC (diisopropylcar-
bodiimide), HOBt (1-hydroxy-benzotriazole) and DIEA (N,N-
diisopropylethylamine) were purchased from Iris Biotech or
NovaBiochem. Amino acids were Na-Fmoc or -Boc protected.
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ethyl 5-bromovalerate (invisible with TLC). The pure product
was obtained after crystallization from pentane–ethyl acetate
(300 mL : 80 mL). After keeping the solution in the freezer for
one night white to colorless crystals were obtained (17.42 g, 85%).
Boc deprotection: the resin was treated at r.t. with TFA–CH2Cl2–
TIPS (40 : 55 : 5, 5 mL) for 2 ¥ 15 min.
The solution was filtered, and the resin was washed with DMF
(3¥), CH2Cl2 (3¥) and MeOH (3¥). The DESC test was positive.
Fmoc-or Boc-AA-OH (3 equiv), HOBt (3 equiv) DIC (3 equiv)
were mixed for 2 min and added to the resin. The mixture was
irradiated for 20 min at a ceiling temperature of 60 ◦C and
50 W maximum power. The mixture was filtered, and the resin was
washed with DMF (3¥), CH2Cl2 (3¥) and MeOH (3¥). Completion
of the couplings was confirmed by a negative DESC test.29
Step 2. The product obtained after step one was poured in
MeOH (200 mL) and NaOH (1M aq. solution, 209 mL, 3 equiv)
was added. The reaction mixture was stirred for 12 h at room
temperature and was subsequently acidified with 1M HCl to pH =
2. The formed precipitate was filtered and recrystallized from
diethyl ether (12.37 g, 80%, overall yield = 68%).
1H NMR (300 MHz, CDCl3): d 9.88 (s, 1H), 7.82 (d, J = 8.70,
2H), 6.97 (d, J = 8.60, 2H), 4.07 (t, J = 4.35 Hz, 2H), 2.44 (t, J =
5.19, 2H), 1.86 (m, 4H).
5) C-terminal deprotection. See conventional synthesis
6) Cyclization. PyBop (93 mg, 3 equiv) and DIEA (59mL,
6 equiv) were suspended in DMF (3 mL) and stirred for 10–15 min.
The resin was added to the mixture and irradiated for 40 min at
13C NMR (75 MHz, CDCl3): 191.04, 179.16, 164.01, 132.07,
129.81, 114.73, 67.75, 33.52, 28.35, 21.29.
◦
a ceiling temperature of 60 C and 60 W maximum power. The
HRMS C12H14O4 calc. 222,0892 found 222.0902.
mixture was filtered, and the resin was washed with DMF (3¥),
CH2Cl2 (3¥) and MeOH (3¥). The cyclization and washing steps
were repeated once. Completion of the coupling was confirmed by
a negative DESC test.29
Synthesis of meta-dialkoxy-BAL
5-(4-formyl-2-methoxyphenoxy)pentanoic acid 2
The procedure followed for the synthesis of compound 2
was identical as for compound 1, except that 4-hydroxy-
3-methoxybenzaldehyde (vanillin) was used instead of 4-
hydroxybenzaldehyde. Overall yield = 64%.
Synthesis of cyclic peptide using conventional conditions
1) Loading of the resin. The synthesis was performed as
described above, with the exception that the resin was shaken for
24 h at r.t. instead of irradiated.
1H NMR (300 MHz, CDCl3): d 9.83 (s, 1H), 7.41 (m, 2H), 6.94
(d, 6 Hz, 1H), 4.12 (t, J = 4.65 Hz), 3.89 (s, 3H), 2.47 (t, J =
5.52 Hz, 2H), 1.88 (m, 4H).
2) Reductive amination. H-Phe-OAll.HCl (72,6 mg, 5 equiv)
and NaBH3CN (19 mg, 5 equiv) were suspended in DMF–MeOH
(8 : 2, 3 mL), and the suspension was added to the loaded resin
and shaken for 18 h. The solution was filtered, and the resin was
washed with DMF (3¥), CH2Cl2 (3¥) and MeOH (3¥).
13C NMR (75 MHz, CDCl3): 191.5, 179.7, 154.4, 150.2, 130.4,
127.24, 111.8, 109.7, 68.9, 56.3, 33.9, 28.6, 21.7.
HRMS C13H16O5 calc. 252,9977 found 252.1002.
Synthesis of cyclic peptide applying microwave irradiation
3) Acylation with symmetrical anhydride. The synthesis was
performed as described above, with the exception that the resin
was shaken for 2 ¥ 24 h at r.t. instead of irradiated.
1) Loading of the resin applying linker
1 or 2. The
aminomethylated polystyrene resin (0.20 mmol g-1, 100–125 mm,
300 mg) was washed with CH2Cl2 and gave a positive DESC test.27
5-(4-Formylphenoxy)pentanoic acid 1 (39.9 mg, 3 equiv) or 5-(4-
formyl-2-methoxyphenoxy)pentanoic acid 2 (45.3 mg, 3 equiv),
HOBt (24.3 mg, 3 equiv), DIC (30 mL, 3 equiv) were mixed in
CH2Cl2 (2.5 mL) for 2 min and added to the resin. The mixture
4) Peptide elongation. The synthesis was performed as de-
scribed above, with the exception that the resin was shaken for 3 h
at r.t. instead of irradiated.
5) C-terminal deprotection. Pd(PPh3)4 (5 mol%) and PhSiH3
(24 equiv) were suspended in dry CH2Cl2 and stirred for 10–15 min.
The resin was added to the mixture and shaken at r.t. for 3 h. The
solution was filtered, and the resin was washed with DMF (3¥),
CH2Cl2 (3¥) and MeOH (3¥).
◦
was irradiated for 20 min at a ceiling temperature of 60 C and
50 W maximum power. The solution was filtered, and the resin was
washed with DMF (3¥), CH2Cl2 (3¥) and MeOH (3¥). Completion
of the coupling was confirmed by a negative DESC test.29
6) Cyclization. The synthesis was performed as described
above, with the exception that the resin was shaken for 2 ¥ 4 h
at r.t. instead of irradiated.
2) Reductive amination. See conventional conditions.
3) Acylation with symmetrical anhydride. Fmoc- or Boc-
bAla-OH (Fmoc: 186 mg, Boc: 113 mg, 10 equiv) and DIC
(47.8 mL, 5 equiv) were suspended in CH2Cl2–DMF (9 : 1, 3 mL)
and preactivated for 10–15 min and added to the resin prepared
according to step 1. The mixture was irradiated for 40 min at
Release from the support
Applying microwave irradiation for linker 1. The resin was
treated with TFA–TIPS (95 : 5) for 1 h at r.t. to remove the side-
chain protective trityl group from Gln, in cases where the Fmoc
protective group was used. No side chain protective group was
used with the Boc-strategy.
The resin bound cyclic peptide was suspended in TFA–CH2Cl2–
TIPS (20 : 75 : 5) and irradiated for 15 min at a ceiling temperature
of 120 ◦C and 100 W maximum power. The resin was filtered and
washed with TFA and CH2Cl2 (3¥). The combined phases were
◦
a ceiling temperature of 60 C and 60 W maximum power. The
mixture was filtered, and the resin was washed with DMF (3¥),
CH2Cl2 (3¥) and MeOH (3¥). The coupling and washing steps
were repeated once. Completion of the coupling was confirmed by
a negative DESC test.29
4) Peptide elongation. a) Fmoc deprotection: the resin was
treated at r.t. with piperidine–DMF (1 : 4, 5 mL) for 2 ¥ 5 min; b)
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concentrated, and the crude peptide was precipitated with diethyl
ether, filtered, dissolved in acetic acid and lyophilised.
4 M. Royo, W. Van Den Nest, M. del Fresno, A. Frieden, D. Yahalom,
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Applying microwave irradiation for linker 2. The synthesis was
performed as for linker 1 with the exception that TFA◦–TIPS (95 : 5)
was used for 45 min at a ceiling temperature of 80 C and 60 W
maximum power.
Applying HF for linker 1 and 2. The resin was treated with
TFA–TIPS (95 : 5) to remove the side-chain protective trityl group
from Gln, in cases where the Fmoc protection group was used. No
side chain protective group was used with the Boc-strategy.
9 M. Pittelkow, U. Boas and J. B. Christensen, Org. Lett., 2006, 8(25),
5817–5820.
10 U. Boas, J. Brask, J. B. Christensen and K. J. Jensen, J. Comb. Chem.,
◦
The resin bound cyclic peptide was treated for 90 min at 0 C
2002, 4(3), 223–228.
11 C. T. Bui, A. M. Bray, F. Ercole, Y. Pham, F. A. Rasoul and N. J. Maeji,
Tetrahedron Lett., 1999, 40(17), 3471–3474.
12 T. Okayama, A. Burritt and V. J. Hruby, Org. Lett., 2000, 2(13), 1787–
1790.
13 W. Gu and R. B. Silverman, Org. Lett., 2003, 5(4), 415–418.
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15 G. T. Bourne, W. D. F. Meutermans, P. F. Alewood, R. P. McGeary, M.
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with liquid HF in the presence of p-cresol and p-thiocresol. After
removal of the HF under reduced pressure, the crude peptide was
precipitated in anhydrous ether, filtered, dissolved in acetic acid
and lyophilised.
Cyclo(Trp-Gln-Gly-b-Ala-Phe)
Analytical HPLC was performed using a GRACE C18 reversed-
phase column (0.21 ¥ 15 cm) on an Agilent 6110 instrument,
configured with a quaternary pump and a UV-DAD detector. UV
detection was at 215 nm and linear gradients of CH3CN and 0,1%
aqueous CHOOH were run at 0.2 mL min-1 flow rate from 1 : 9 to
1 : 0 over 20 min. Tr= 13.6 min., MS (ESI-MH+) = 590.2 g mol-1.
16 K. G. Estep, C. E. Neipp, L. M. S. Stramiello, M. D. Adam, M. P.
Allen, S. Robinson and E. J. Roskamp, J. Org. Chem., 1998, 63(16),
5300–5301.
17 U. Boas, J. B. Christensen and K. J. Jensen, J. Comb. Chem., 2004, 6(4),
497–503.
18 M. Pittelkow, U. Boas, M. Jessing, K. J. Jensen and J. B. Christensen,
Org. Biomol. Chem., 2005, 3(3), 508–514.
19 M. Jessing, M. Brandt, K. J. Jensen, J. B. Christensen and U. Boas,
J. Org. Chem., 2006, 71(18), 6734–6741.
20 S. Braese, S. Dahmen and M. Pfefferkorn, J. Comb. Chem., 2000, 2(6),
710–715.
Acknowledgements
The authors thank the I.W.T. (Institute for the Promotion of
Innovation by Science and Technology in Flanders) for financial
support (SBO-project IWT 040083). They are grateful to the
company Milestone (Via Fatebenefratelli, 1/524010 Sorisole (BG)
Italy) for giving the opportunity to evaluate their Multimode
microwave instrument in the course of this project.
21 P.-O. Norrby and K. J. Jensen, Int. J. Pept. Res. Ther., 2006, 12(4),
335–339.
22 R. Minkwitz and M. Meldal, QSAR Comb. Sci., 2005, 24(3), 343–353.
23 J. Alsina, K. J. Jensen, F. Albericio and G. Barany, Chem.–Eur. J., 1999,
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24 G. T. Bourne, S. W. Golding, W. D. F. Meutermans and M. L. Smythe,
Lett. Pept. Sci., 2001, 7(6), 311–316.
25 M. Brandt, S. Gammeltoft and K. J. Jensen, Int. J. Pept. Res. Ther.,
2006, 12(4), 349–357.
26 B. Bacsa, K. Horvati, S. Bosze, F. Andreae and C. O. Kappe, J. Org.
Chem., 2008, 73(19), 7532–7542.
27 M. Erdelyi and A. Gogoll, Synthesis, 2002, (11), 1592–1596.
28 F. Rizzolo, G. Sabatino, M. Chelli, P. Rovero and A. M. Papini,
Int. J. Pept. Res. Ther., 2007, 13(1-2), 203–208.
29 S. Claerhout, D. S. Ermolat’ev and E. V. Van der Eycken, J. Comb.
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