Orthogonal protection of peptides and peptoids for cyclization by the thiol-ene reaction and conjugation

Article abstract of DOI:10.1021/jo500427c

Cyclic peptides and peptoids were prepared using the thiol-ene Michael-type reaction. The linear precursors were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either cycloadditions (Diels-Alder, Cu(I)-catalyzed azide-alkyne) or the same Michael-type reaction as for cyclization.

Full text of DOI:10.1021/jo500427c

Article
pubs.acs.org/joc
Orthogonal Protection of Peptides and Peptoids for Cyclization by
the Thiol−Ene Reaction and Conjugation
Xavier Elduque, Enrique Pedroso, and Anna Grandas*
̀ ̀
Departament de Química Organica i IBUB, Facultat de Química, Universitat de Barcelona, Martí i Franques 1-11, 08028 Barcelona,
Spain
S
* Supporting Information
ABSTRACT: Cyclic peptides and peptoids were prepared using the thiol−ene Michael-type reaction. The linear precursors
were provided with additional functional groups allowing for subsequent conjugation: an orthogonally protected thiol, a
protected maleimide, or an alkyne. The functional group for conjugation was placed either within the cycle or in an external
position. The click reactions employed for conjugation with suitably derivatized nucleoside or oligonucleotides were either
cycloadditions (Diels−Alder, Cu(I)-catalyzed azide−alkyne) or the same Michael-type reaction as for cyclization.
common alternatives, but other “click” reactions, and
particularly the Cu(I)-catalyzed azide−alkyne cycloaddition,
are becoming increasingly frequent.
INTRODUCTION
■
With respect to the linear counterparts, conformationally
constrained peptides offer increased bioavailability and affinity
for the target.1 Cell uptake has been shown to increase if
cyclization is accompanied by the formation of internal H-
bonds, which reduces the cost of amide bond desolvation.2,3
Suitably derivatized cyclic peptides can be used as scaffolds
(cyclic decapeptide RAFT4 is an example) for the assembly of
different moieties (epitopes, drugs, ligands, labels, etc.) at
predetermined positions, affording biomolecules with a degree
of structural organization much higher than the one accessible
through derivatization of a linear peptide. Furthermore, cyclic
peptides hold promise to interfere with protein−protein or
protein−RNA interactions (refs 5−7 are some examples of
recent publications on these subjects).
A survey of the latest literature shows that the synthesis of
conjugates of cyclic peptides is an active subject of research.
In a large number of papers, the affinity of αVβ3 integrin8 for
RGD-containing cyclic peptides9 (RGD = ArgGlyAsp) is
exploited to develop new methodologies both for the selective
targeting and the imaging of tumor cells/tissues and vessels
expressing that cell adhesion protein. Additionally, conjugates
of cyclic peptides with biological interest (immunosuppressive,
epitope mimicking, antimicrobial, antifungal, or cell penetrat-
ing, among others) or nanotube precursors have been
prepared to improve pharmacological properties10−15 and to
investigate new materials.16,17
There are very few reports on the synthesis and application
of [cyclic peptide]−oligonucleotide conjugates. Hamilton and
co-workers18 linked cyclic tetrapeptides to guanine-rich
oligonucleotides that assembled onto a parallel tetraplex.
The distribution of the peptide ligands on the vertexes of the
top tetrad provided an α-chymotrypsin inhibitor that
effectively interacted with the protein surface. In a related
approach, the covalent attachment of peptides to the 5′ ends
of two guanine-rich oligonucleotides provided, in the presence
of potassium cations, a parallel G-quadruplex with two loop
mimics on its surface.19 Cyclic peptides have been covalently
linked to the sense strand of a siRNA duplex, which facilitated
transport of the oligonucleotide through the cell membrane
and inhibited gene expression.20 More recently, disulfide-
containing citrullinated peptides have been linked to various
oligonucleotides and immobilized on plates for diagnostic
ELISA assays.21
In this paper, we describe methodology allowing for the
straightforward synthesis of [cyclic peptide]−oligonucleotide
conjugates. Model peptides (and a peptoid) cyclized using the
Michael-type maleimide−thiol reaction22 and incorporating a
functional group allowing for subsequent conjugation were
prepared. Then, oligonucleotides were linked to different
positions (internal or external) of the cyclic polyamide using
various click reactions, namely the Diels−Alder and Cu(I)-
Amide-linked units form most of these peptides, while some
incorporate unnatural linkages. As to the conjugation
chemistry, formation of peptide bonds and the Michael-type
reaction between a maleimide and a thiol are the most
Received: November 5, 2013
Published: March 11, 2014
© 2014 American Chemical Society
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catalyzed Huisgen cycloadditions or, again, the Michael-type
thiol−ene. We believe that easy access to [cyclic peptide]−
oligonucleotide conjugates will pave the way for new studies
aiming at assessing whether cyclic peptides facilitate the cell
uptake of oligonucleotides and promote interactions with cell
machineries different from those driven by the linear
oligomers. To our knowledge, whether the gene silencing or
splice-switching properties of oligonucleotides linked to cyclic
peptides differ from those of [linear peptide]−oligonucleotide
conjugates has never been assessed.
Scheme 1. (A) General Procedure for the Synthesis of
Peptides Cyclized Using the Thiol−Maleimide Reaction.
(B) General Structure of the Three Types of Cyclic
Peptides (or Peptoid) Prepared in This Work, Derivatized
a
for Conjugation. (C) Schematic Structure of the Cyclic
Molecules Showing the Functional Group for Conjugation
and Combinations of Functional Groups (R1, R2, and R3,
Suitably Protected when Required) on the Resin-Linked
Peptide/Peptoid Precursor for Each of the Alternatives
b
Tested (a−d)
RESULTS
■
As recently described by the group,22 peptides can be cyclized
by reaction between a maleimide and a thiol. Cyclic peptides
can be obtained from linear precursors after the TFA
treatment that deprotects the peptide chain following stepwise
elongation on a solid matrix, using S-trityl-protected cysteine
and a maleimide moiety (Scheme 1A). Since the maleimide
moiety is labile to nucleophiles such as piperidine, which is
used at every synthesis cycle to remove the Fmoc temporary
protecting group, it can only be installed at the N-terminus.
For introduction at any other position of the chain the
maleimide has to be protected, for which purpose an Fmoc-
lysine derivative with 2,5-dimethylfuran-protected 3-maleimi-
dopropanoic acid linked to the ε-amine can be used.23 In this
case, the cyclic peptide is not obtained after the reaction with
TFA but after the thermal treatment that deprotects the
maleimide.
Cyclic peptides can be derivatized for conjugation in three
different ways (see Scheme 1B). In cyclic peptides types I and
II, the functional group for conjugation (depicted as a
triangle) is outside the cycle, and the cycle involves,
respectively, amino acids placed at either the N-terminus of
the peptide chain (the group for conjugation is then placed at
the C-terminus) or the C-terminus (and the group for
conjugation is at the N-terminus). In type III cyclic peptides
the group for conjugation is within the cycle.
Linear precursors incorporating a free and a protected
maleimide are needed to synthesize maleimido-derivatized
cyclic peptides. The free maleimide, which will form the cycle,
can only be placed at the N-terminal end, while the protected
moiety can occupy any other position. As stated above, if the
protected maleimide is closer to the C-terminus than the thiol
involved in cyclization, the result is a type I cyclic peptide
(Scheme 1C, alternative a). These peptides can be conjugated
with either thiol- or diene-derivatized molecules.
The precursors of cyclic peptides derivatized with thiols
must incorporate two orthogonally protected cysteine
residues. The cysteine residue involved in cyclization can be
protected with the trityl group (Scheme 1C, alternatives b and
c), but the best option to protect the cysteine residue
involved in conjugation depends on its position within the
chain (see below). Both thiol-derivatized cyclic peptides types
II and III have been prepared and conjugated with a
maleimido-containing compound.
The use of different reactions for cyclization and
conjugation is less complicated from a chemical point of
view and has been evaluated with a peptoid chain (peptoid
units are N-alkylglycines). Even though the thiol−ene reaction
is probably much quicker than the reduction of azides by
thiols,24,25 we preferred not to expose the azide to the
presence of a free thiol. Hence, an azide-containing molecule
was used for conjugation with the alkyne-derivatized, type III
a
Cyclization was accomplished by a Michael-type maleimide-thiol
b
reaction in all cases. Two of those groups had to react with each other
and generate the cycle, and the third provided the functionality for
conjugation with another molecule (in this work, nucleoside or
oligonucleotide). Their relative position within the chain determined
the type of cyclic molecule that was obtained (I, II or III).
Abbreviations: Fm = 9-fluorenylmethyl; Mal = maleimide; Prot mal
= (2,5-dimethylfuran)-protected maleimide; Tr = trityl.
cyclic peptide. Cyclization can be carried out with no risk in
the presence of an alkyne (Scheme 1C, alternative d), which
will not react with either the thiol or the maleimide.
Peptides were prepared by solid-phase synthesis using the
standard Fmoc/tBu methodology. All were assembled on the
Rink amide MBHA (p-methylbenzhydrylamine) resin and
were thus obtained as C-terminal carboxamides.
Acylation with 3-maleimidopropanoic acid after chain
elongation allowed a free maleimide to be attached to the
peptide N-terminus.
These compounds were conjugated with suitably derivatized
oligonucleotides (or nucleoside), as shown in Schemes 2−5 in
more detail. Diene- and maleimido-containing oligonucleo-
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a
Scheme 2. Synthesis of Type I Cyclic Peptide 2 and Conjugation with Oligonucleotide dT5
a
Abbreviations: Pbf =2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.
tides (dT5, dT = 2′-deoxythymidine) were synthesized
following the described procedures.26,27
Schemes 2−4 illustrate in more detail the different steps
required to obtain cyclic peptides types I to III and the
corresponding conjugation products.
Mass spectrometric analysis confirmed presence of the StBu
group in 6, its loss after reaction with tris(2-carboxyethyl)-
phosphine (TCEP) to yield 16 (see structure in Scheme 3c),
and loss of the three N-terminal amino acids when 6 was
digested with thrombin. Instead, the mass of 7 was lower than
that of 6, and the product remained undigested upon
treatment with thrombin. Final confirmation of the presence
of a free maleimide in 7 was obtained from reaction with
diene-dT5, which afforded the [disulfide cyclic peptide]−
oligonucleotide conjugate 8. As expected, the Diels−Alder
reaction is fully compatible with the presence of the disulfide.
Even though the thiol−maleimide reaction slightly predomi-
nated over disulfide formation, the relative amounts of the
products obtained after maleimide deprotection are too
similar to consider this alternative a good synthetic method.
In the second group of assays (Scheme 3b), the N-terminal
cysteine was protected with the acetamidomethyl (Acm)
group. Here the problem was that none of the conditions
tested for its deprotection (reactions with either Ag+, iodine,
or Tl3+) provided clean crudes with the target cyclic peptide
(16) as the main product (see the Experimental Section).
We next evaluated the combination of protecting groups
shown for peptide-resin 12 (Scheme 3c), using commercially
available N-Boc-Cys(Fm)−OH. In this case, treatment with
TFA deprotected the N-terminus and the other functional
groups, except those on the N-terminal cysteine and the
maleimide, to yield 13. Maleimide deprotection and the
thiol−maleimide reaction that furnished the cycle (14) took
place simultaneously, and as described in the literature,28
treatment with piperidine removed the Fm protecting group
and provided the disulfide (15). The cyclic peptide with the
free thiol (16) was obtained after reduction with TCEP, and
subsequent reaction with maleimido-dT5 afforded conjugate
17. Hence, the 9-fluorenylmethyl group is fully compatible
with maleimide deprotection and peptide cyclization by the
Michael-type thiol−ene reaction. Even though removal of the
Fm group is accompanied by thiol to disulfide oxidation,
which adds a reduction step, this group is suitable to mask the
cysteine side chain provided that the so-protected cysteine is
placed at the N-terminus. Peptide elongation using the
Fmoc/tBu approach rules out any other possibility.
Synthesis and Conjugation of a Type I Cyclic
Peptide. In the first alternative (aimed to provide type I
cyclic peptides, a in Scheme 1B), the fully protected peptide
precursor (1) incorporated two maleimides, one free (at the
N-terminus) and one protected (at the C-terminus), and a
trityl-protected cysteine residue (Scheme 2). Therefore, the
TFA treatment that was carried out after assembly of the
linear precursor removed all protecting groups except that on
the C-terminal maleimide and, as previously described,22
furnished the partially protected cyclic peptide 2. Maleimide
thermal deprotection (retro-Diels−Alder reaction) was
performed subsequently in the presence of 5′-diene-dT5,
which afforded the target conjugate 3.
Synthesis and Conjugation of a Type II Cyclic
Peptide. The goal of the second alternative (b in Scheme
1B) was to obtain a peptide with a free thiol group for
conjugation at the N-terminus of the chain (type II cyclic
peptide). This required a precursor with two differently
protected cysteines and a protected maleimide (Scheme 3).
The cysteine that had to participate in cyclization was
protected with the TFA-labile trityl group, and it was
necessary to assess which thiol protecting group was suitable
for the other cysteine. This group had to remain stable during
the treatments required to obtain the cycle, that is, TFA
treatment and maleimide deprotection.
We first evaluated the possibility of protecting the side
chain of the N-terminal cysteine with the StBu and
acetamidomethyl groups, whose Fmoc derivatives are
commercially available and which are TFA-stable (Scheme
3a,b). In preliminary experiments, both Fmoc-Cys(StBu)-OH
and Fmoc-Cys(Acm)-OH proved to be stable to maleimide
deprotection conditions (heating in a microwave oven a
solution of the compound in 1:1 MeOH/H2O for 90 min at
90 °C). Yet, when peptide 5 was submitted to these reaction
conditions, two products were obtained (Scheme 3a): the
target compound (6) with the thiosuccinimide-containing
cycle and a peptide (7) with a disulfide bridge and a free
maleimide (the 6:7 ratio was 1:1 when the peptide was heated
for 30 min, and 3:2 when the reaction was extended to the 90
min typically required for full maleimide deprotection27).
Synthesis and Conjugation of Type III Cyclic
Polyamides (Peptide and Peptoid). Two different type
III cyclic polyamides were synthesized (alternatives c and d in
Scheme 1B). Here the functional group for conjugation
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Scheme 3. Assays Carried out To Obtain Cyclic Peptide 16 (Type II, Alternative b in Scheme 1) Using Different Thiol
a
Protecting Groups for the N-Terminal Cysteine (the One To Be Involved in Conjugation)
a
(a) Deprotection of the maleimide of peptide 5 afforded two products, 6 and 7, in a similar ratio. The latter furnished conjugate 8 by reaction with
diene-dT5. (b) Removal of the Acm cysteine protecting group from 11 yielded complex mixtures of products in all the conditions tested. (c) Cyclic
peptide 16 was prepared by combining Tr and Fm as cysteine protecting groups and conjugated with maleimido-dT5. Abbreviations: Acm =
acetamidomethyl; Fm = 9-fluorenylmethyl.
appended from a residue placed within the cycle. In one case,
the polyamide was a peptide and the functional group for
conjugation a thiol (20), while in the other it was a peptoid
derivatized with an alkyne (24).
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Scheme 4 depicts the preparation and conjugation of cyclic
peptide 20. The C-terminal cysteine was protected with the
troduce maleimide moieties at other positions of the chain, 2-
[(2,5-dimethylfuran-protected) maleimide]-1-aminoethane23
should be employed. In the absence of two orthogonal
maleimide protecting groups, this precludes use of maleimides
for subsequent conjugation.
Scheme 4. Steps Involved in the Preparation of Cyclic
Peptide 20 and Conjugate 21
Chain assembly furnished the fully protected peptoid 22,
from which all protecting groups except that on the
maleimide were removed by treatment with TFA. Maleimide
deprotection and cyclization took place simultaneously to
afford 24. Finally, the Cu(I)-catalyzed Huisgen reaction
between cyclic peptoid 24 and AZT (2′,3′-dideoxy-3′-
azidothymidine) provided the target conjugate (25). The
azide-containing molecule was in this case a simple nucleoside
analogue, but the high success of the Cu(I)-catalyzed alkyne−
azide cycloaddition has prompted many groups to develop
methodology for the preparation of azide-derivatized
oligonucleotides (see, for instance, refs 32−34) circumventing
the incompatibility between azides and phosphite-based
reagents.35,36
DISCUSSION
■
We have shown that, by adequately combining differently
protected cysteines, free and protected maleimides, and an
alkyne and choosing their relative position in the chain, it is
possible to easily obtain cyclic peptides derivatized with a
functional group allowing for subsequent conjugation, for
instance, with oligonucleotides.
In all cases, cyclization was carried out using the thiol−ene
Michael-type reaction. For this purpose, in all peptide
precursors one of the cysteine residues was masked with
the TFA-labile trityl group, so that the acidic treatment that
follows chain elongation deprotected its thiol. When a free
maleimide had been appended to the N-terminus of the chain,
this unmasked thiol reacted with the maleimide to form the
cycle (peptides 2 and 19) during the TFA deprotection
step.22 Whenever a masked maleimide was incorporated into
the chain, an additional deprotection step was required. Then,
thermal removal of the maleimide protecting group (retro-
Diels−Alder reaction) and cyclization took place simulta-
neously (peptide 14 and peptoid 24).
trityl group, which reacted with the N-terminal, free
maleimide upon treatment with TFA. Under these conditions,
the free thiol gave the Michael-type reaction and left
untouched the internal, StBu-protected cysteine residue. In
other words, the free thiol reacted much faster with the
maleimide than with the StBu-protected cysteine. The side
chain of the internal cysteine of the cyclic peptide (19) was
deprotected by reaction with TCEP and, after purification,
reacted with 5′-maleimido-dT5 to yield conjugate 21.
As required for conjugation with a suitably derivatized
molecule, cyclic peptides had to incorporate an additional
functional group. In this work, this group was either a
maleimide, a thiol, or an alkyne.
With the cyclization strategy that we have used, the only
possibility to use a maleimide for conjugation is to assemble a
peptide precursor with a free maleimide at the N-terminus (to
form the cycle) and a protected maleimide at any other
position. Placing the lysine carrying the protected maleimide
at the C-terminus (as in 1) furnishes type I cyclic peptides,
with amino acids out of the cycle and the maleimide for
conjugation at the C-terminal end. It is reasonable to surmise,
on the basis of the results here described, that placing that
lysine within the cycle will successfully provide cyclic peptides
with an internal maleimide for conjugation. Maleimide-
derivatized cyclic peptides (and any maleimide-containing
compound) can be conjugated with diene-derivatized
molecules, as in the synthesis of 3, where maleimide
deprotection and conjugation took place simultaneously. It
is also worth mentioning that, even though the proof of
principle experiments here described were carried out with
simple oligonucleotide sequences, the compatibility of
oligonucleotides of different length and composition27 with
Scheme 5 shows the preparation of a cyclic peptoid and the
structure of the derived conjugate. On-resin assembly of the
peptoid was carried out using the submonomer procedure,29
also on the Rink amide MBHA resin. As shown in the upper
part of Scheme 5, chain elongation involves two subsequent
steps, namely acylation with bromoacetic acid and reaction
with an amine. Three of the amines required to synthesize
peptoid 23 were commercially available (see the list in the
Supporting Information), and S-Tr-protected cysteamine and
N-Boc-1,2-diaminoethane were prepared following described
procedures.30,31 Cyclization with an N-terminal maleimide
required incorporation of a maleimide-containing monomer,
and 3-maleimidopropanoic acid is the cheapest alternative.
However, since we had observed that the quality of cyclic
peptide crudes was a bit lower when cyclization took place
upon treatment with TFA,22 we incorporated the 2,5-
dimethylfuran-protected maleimidopropanoic acid.27 To in-
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Scheme 5. Synthesis of Cyclic Peptoid 24 and Conjugate 25
the reaction conditions in which maleimides are deprotected
suggests that any oligonucleotide can be linked to cyclic
peptides. Alternatively, maleimide-derivatized cyclic peptides
could be conjugated with thiols.
with the free thiol resulting from the deprotection of Cys(Tr)
(or if it does, the extent of the reaction is negligible). As
stated before, at room temperature and under these strongly
acidic conditions, formation of the cycle by reaction between
the free thiol and the free maleimide is much quicker. Yet
(see above and Scheme 3a), this is not the case when the
peptide has to be heated.
Thiol-derivatized cyclic peptides can be conjugated with
maleimido-oligonucleotides, as in the case of 17 and 21.
Type III cyclic peptoids derivatized for conjugation by the
Cu(I)-catalyzed Huisgen cycloaddition were also synthesized.
In this case, the key monomers were 3-(2,5-dimethylfuran-
protected)maleimidopropanoic acid,27 S-tritylcysteamine,30
and propargylamine. Use of S-Fm-cysteamine instead of
propargylamine would provide peptoids cyclized by the
thiol-maleimide reaction and derivatized with a thiol group
for conjugation.
As to the use of alkynes for conjugation, the triple bond
can be installed at any position of the polyamide chain. It is
neither affected by any of the peptide/peptoid synthesis
reagents nor by the thiol-maleimide cycle-forming reaction.
We have prepared a [cyclic peptoid]−nucleoside conjugate to
verify that synthesis proceeded as expected, and the triple
bond was introduced in an internal position by reacting
propargylamine with the haloacylated resin-linked, growing
peptoid chain. Commercially available alkyne-containing L-
amino acids or alkynoic acids allow triple bonds to be
attached to peptides in a straightforward manner, which is
also the case for derivatization with azides.
When the cyclic peptide is to be derivatized with a thiol for
conjugation, the choice of protecting group for the cysteine
providing this thiol depends on its position in the chain. If the
goal is a type II cyclic peptide, the best alternative among
those we have examined is to block the thiol with the Fm
group, which is compatible with maleimide deprotection and
cyclization and can be removed with no formation of
byproducts. The only drawback is that removal of the Fm
group by reaction with piperidine is accompanied by disulfide
formation, an additional reduction step being thus necessary
prior to conjugation. N-Terminal derivatization of a peptide
with a thiol was here accomplished by introducing a suitably
protected cysteine residue, but an S-blocked mercaptoacetic
acid should also work. Use of cysteine derivatives has the
advantage that the amino function can be further derivatized,
allowing for the attachment a fluorophore, for instance, or any
reporter group.
Conversely, the synthesis of cyclic peptides with the
cysteine that provides the thiol for conjugation at any other
position of the chain (not at the N-terminus) is much more
straightforward (this is the case of type III peptide 20, but the
same protection scheme could provide type I peptides). That
cysteine can indeed be protected with the StBu group, which
remains stable during the acidic treatment that removes most
of the protecting groups from the peptide and does not react
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dissolved in anhyd NMP (500 μL per 100 mg of resin) for 3 h. After
this reaction, the resin was washed again (DCM, DMF and MeOH,
3× each). Cleavage from the resin and removal of most permanent
protecting groups was accomplished by reaction with TFA at room
temperature (see details below, and Scheme 5), which was followed
by washings with the cleavage mixture and DCM. Solvents and
reagents were removed from the filtrate by means of a N2 stream
until the solution became an oil. Cold diethyl ether (10 volumes)
was added, and the mixture was centrifuged (10 min, 5 °C, 4800
rpm) and decanted. The ether addition plus centrifugation procedure
was repeated three times. The crude was analyzed and purified by
HPLC, quantified by UV spectroscopy (phenoxy group: ε270 (M−1
cm−1) = 1450), and characterized by mass spectrometry.
CONCLUSIONS
■
In summary, we have broadened the scope of applications of
peptides cyclized making use of the maleimide−thiol reaction
by introducing an additional group that allows for conjugation
with oligonucleotides (or other molecules) utilizing click
reactions and shown that this methodology can also be
employed with peptoids. The method here described is based
on the adequate choice of protection for maleimides and
thiols, as well as suitably placing maleimides, thiols, or alkynes
within the peptide/peptoid chain. These synthetic alternatives
could also provide other cyclic polyamide scaffolds derivatized
for conjugation, such as peptide nucleic acids.
HPLC. Unless otherwise stated, the following conditions were
used. Semipreparative HPLC, acidic conditions: Jupiter Proteo
column; 3 mL/min; solvent A: H2O + 0.1% TFA; solvent B:
ACN + 0.1% TFA. Analytical HPLC, acidic conditions: Phenomenex
C18, 4 μm, Jupiter Proteo column; 1 mL/min; solvent A: H2O +
0.045% TFA); solvent B: ACN + 0.036% TFA. Analytical HPLC
(also used for purification at a small scale), buffered medium:
Kromasil C18, 10 μm column; 1 mL/min; solvent A: 0.05 M TEAA
(triethylammonium acetate) in water; solvent B: 1:1 H2O/ACN.
MW-Promoted Polyamide Deprotection and Cyclization. The
[protected maleimido]-containing polyamide was dissolved in a 1:1
(v/v) MeOH/H2O mixture at a concentration between 25 and 100
μM in a microwave flask. The flask was sealed and heated in a
microwave oven (Biotage InitiatorTM Classic) at 90 °C for 90 min
(with stirring and internal probe for temperature monitoring).
Methanol was then removed under reduced pressure, and the
resulting aqueous solution was analyzed by HPLC. Alternatively,
methanol removal was followed by lyophilization, and the crude was
redissolved in water for HPLC analysis.
Assessment of Cyclization: Reaction with H2O2. Formation of a
cyclic product was confirmed by reacting the peptide, either linear or
cyclic, with a dilute solution of H2O2 (3.5%) for 1 h, and subsequent
MS analysis.23 When the peptide is linear, H2O2 oxidizes the free
thiol to sulfonic acid (and the mass increases 48 units), whereas
when the peptide is cyclic the thioether is oxidized to sulfoxide (and
the mass increases 16 units). This method cannot be used when the
peptide to be analyzed incorporates a Cys(StBu) residue because this
disulfide is oxidized to different products. ESI mass spectrometric
analysis showed formation of mixtures of products with and without
loss of the StBu group. When analysis was performed in the negative
mode, the main group of peaks corresponded to loss of the StBu
group and oxidation of the thiol to sulfonic acid (+ 3 oxygen atoms,
M + 48).
Assessment of Cyclization: Digestion with Thrombin. Reaction
with thrombin can also be used to assess circularity. Thrombin
cleaves the Arg-Gly bond in linear GRG peptide sequences, but not
when this sequence is within a cycle. Twenty micrograms of peptide
and 5 u of thrombin, dissolved in a 1:2 PBS/H2O solution (PBS
buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM
KH2PO4, pH = 7.23), were incubated at room temperature
overnight. The crude was readily analyzed by MALDI-TOF MS.
Deprotection of Cys(StBu) and Reduction of Disulfide Bridges.
TCEP (500 equiv) was added to a 0.3 mM solution of the
polyamide in H2O (1−3 mL). The pH was adjusted to 5 with an
aqueous dilute NaOH solution, and the mixture was stirred at room
temperature for 3 h. Water was removed by lyophilization and the
crude analyzed by HPLC.
It has been reported that subtle differences in the
conjugation chemistry may have considerable impact on the
biological outcome.37−39 Therefore, it is of interest to develop
chemical tools giving access to differently linked conjugates.
This manuscript describes methodology allowing oligonucleo-
tides to be linked to cyclic peptides, either to an internal
position, or to any of the two ends of a chain in which only
some of the residues of the peptide are part of a chemically
stable cycle. Both cyclic peptides and oligonucleotides (natural
or modified) are relevant chemical goals that hold promise
from the therapeutic and diagnostics point of view. So far
there is lack of information as to the differential effect of
linking oligonucleotides to cyclic peptides rather than the
linear counterparts. Work on this subject is now in progress in
the group, and results will be published in due time.
EXPERIMENTAL SECTION
■
General Methods. Peptide Synthesis. All peptides were
assembled on the Rink Amide resin, which was treated and washed
with DCM (3×), DMF (3×), 20% piperidine/DMF (2 × 15 min),
DCM (3×), DMF (3×), and MeOH (3×) prior to chain elongation.
Incorporation of the first amino acid on the solid matrix and peptide
elongation were carried out using 3 equiv of Fmoc-amino acid,
HOBt·H2O, and DIPC (DIPC = N,N′-diisopropylcarbodiimide)
dissolved in the minimum amount of DCM and a few drops of DMF
(90 min), which was followed by washing with DCM, DMF, and
MeOH (3×). In case the coupling was not complete, as assessed by
the Kaiser test, it was repeated using 2 equiv of the reagents. Fmoc
groups were removed by reaction with 20% piperidine/DMF (1 × 3
min + 1 × 10 min) followed by washing (DMF, DCM). Activation
of 3-maleimidopropanoic acid was carried out with DIPC (90 min
reaction time). Even though activation with DIPC and HOBt·H2O
seems not to cause any harm on the maleimide, use of only the
carbodiimide is safer to prevent addition of HOBt to the maleimide
of maleimidopropanoic acid.
Reaction with a TFA/H2O/TIS mixture (TIS = triisopropylsilane)
at room temperature cleaved the peptide from the resin and removed
most permanent protecting groups (see below and Schemes 2−4 for
details). Filtrate and washings (DCM) were collected and
concentrated under a N2 stream, and cold diethyl ether was added
to the resulting oil. The mixture was centrifuged (10 min, 5 °C, 4800
rpm) and decanted. This procedure was repeated three times.
Peptides were quantified by UV spectroscopy from the absorbance of
tyrosine residues at 280 nm (ε280 M−1 cm−1 = 1480).
Diels−Alder Cycloadditions. The maleimido- or protected
maleimido-containing peptide and diene-dT5 were dissolved in
water, and the amounts required to carry out the reaction (typical
reagent ratio was 1:1) were taken from each of these solutions and
mixed. This mixture was diluted with water (or water and methanol,
to end up with a 1:1 proportion, if MW-promoted maleimide
deprotection and conjugation were to take place simultaneously) to
reach a reagent concentration between 30 and 300 μM. The
maleimide + diene mixture was reacted overnight at 37 °C. The
[protected maleimide] + diene mixture was reacted in a MW oven
Peptoid synthesis. Peptoid 22 was also assembled on the Rink
Amide resin, which was treated and washed as described above for
peptide synthesis. For the acylation step, bromoacetic acid (25
equiv) was dissolved in anh. DMF in a round-bottom flask, the
solution was cooled to 0 °C, and DIPC (25 equiv) was added
dropwise. After 5 min, the solution was poured onto the resin in the
syringe, and the mixture was stirred (shaker) under an Ar
atmosphere for 45 min. The resin was filtered and washed (DCM,
DMF, iPrOH, and DCM, 3× each), and the amination step was
carried out by reaction with the corresponding amine (20 equiv)
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for 90 min at 90 °C. Solvents were removed by evaporation under
reduced pressure and/or lyophilization, and reaction crudes were
analyzed by HPLC. Conjugates were purified by HPLC and
characterized by MALDI-TOF MS.
Michael-Type Reactions. Aliquots of aqueous solutions containing
the required amounts of maleimido-containing compound and the
corresponding thiol derivative were mixed (final concentration of
polyamide: 50−150 μM). The mixture was stirred at room
temperature under an Ar atmosphere overnight. After lyophilization,
reaction crudes were analyzed by HPLC. Conjugates were purified
by HPLC and characterized by MALDI-TOF MS.
Deprotection of the Maleimide of 5. Compound 5 (100
nmol) was dissolved in H2O/MeOH (4 mL) to yield a 25 μM
solution, which was heated in a MW oven as described in the
General Methods. Two different products, namely 6 and 7, were
obtained (no trace of 5 was detected in any case) in a ratio that
varied depending on the reaction time: the 6:7 ratio was 49:51 after
30 min heating, 56:44 after 60 min, and 59:41 after 90 min.
Compunds 6 and 7 were separated using analytical acidic HPLC
conditions and characterized by MALDI-TOF MS before and after
reaction with thrombin (see below). For an additional proof of
structure, samples of 6 and 7 were subsequently reacted as described
below to yield compounds 16 and 8, respectively, which were also
characterized by MS.
Assays To Assess the Stability of Cysteine Derivatives to
Microwave-Promoted Maleimide-Deprotection Conditions. The
stabilities of Fmoc-Cys(StBu)-OH, Fmoc-Cys(Acm)-OH, and Boc-
Cys(Fm)-OH were assessed by dissolving some 5−10 mg of the
product in MeOH/H2O 1:1 (v/v) and heating the resulting solution
at 90 °C for 90 min in the microwave oven. Solvent was removed
Cyclic H-Cys(StBu)-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys*-NH2,
6. Analytical HPLC: acidic conditions, linear gradient from 5 to
50% of B in 30 min, tR = 20.1 min. MALDI-TOF MS (DHB,
positive mode): m/z 1258.9 [M + H]+, 1280.9 [M + Na]+, 1296.8
[M + K]+; M calcd for C54H79N15O14S3 1257.5. MALDI-TOF MS
after reaction with H2O2 (DHB, positive mode): m/z 1186.7 [M −
StBu + 16 + H]+, 1234.7 [M − StBu + 16 + 48 + H]+, 1290.7 [M +
32 + H]+; M + 32 calcd for C54H79N15O16S3 1289.5, M − StBu + 16
calcd for C50H71N15O15S2 1185.5. MALDI-TOF MS after reaction
with thrombin (DHB, positive mode): m/z 876.4 [M − [Cys(StBu)-
Gly-Arg] + Na]+, 892.4 [M − [Cys(StBu)-Gly-Arg] + K]+; M −
[Cys(StBu)-Gly-Arg] calcd for C39H51N9O11S 853.3.
Cyclic H-Cys-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys*-NH2, 16.
Compound 6 (67 nmol) was reacted with TCEP according to the
general procedures. Compound 16 was obtained in 76% yield (based
on the HPLC trace). Analytical HPLC: acidic conditions, linear
gradient from 5 to 50% of B in 30 min, tR = 16.1 min. MALDI-TOF
MS (DHB, positive mode): m/z 1170.7 [M + H]+, 1192.7 [M +
Na]+, 1208.7 [M + K]+; M calcd for C50H71N15O14S2 1169.5.
MALDI-TOF MS after reaction with H2O2 (DHB, negative mode):
m/z 1232.6 [M + 48 + 16 − H]−; M + 48 + 16 calcd for
C50H71N15O18S2 1233.5. MALDI-TOF MS after digestion with
thrombin (DHB, positive mode): m/z 876.5 [M − [Cys-Gly-Arg]
+ Na]+, 892.5 [M − [Cys-Gly-Arg] + K]+; M − [Cys-Gly-Arg] calcd
for C39H51N9O11S 853.4.
Cyclic H-Cys*-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys(Prot.Mal.)-
NH2, 7. Analytical HPLC: acidic conditions, linear gradient from 5
to 50% of B in 30 min, tR = 16.7 min. MALDI-TOF MS (DHB,
positive mode): m/z 1168.8 [M + H]+, 1190.7 [M + Na]+, 1206.7
[M + K]+; M calcd for C50H69N15O14S2 1167.5. MALDI-TOF MS
after reaction with H2O2 (DHB, negative mode): m/z 1264.7 [M +
48 + 48 − H]−; M + 48 + 48 calcd for C50H71N15O20S2 1265.4.
MALDI-TOF MS after digestion with thrombin (DHB, positive
mode): m/z 1168.8 [M + H]+, 1190.7 [M + Na]+, 1206.7 [M + K]+;
M calcd for C50H69N15O14S2 1167.5. No digestion was detected.
Conjugate 8, [Cyclic H-Cys*-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-
Lys(Mal.)-NH2] + [diene-dT5]. Compound 7 (15 nmol) and diene-
dT5 (25 nmol) were dissolved in water (65 μL) and allowed to react
overnight at room temperature, yielding the desired product 8 (8.8
nmol, 59% conjugation and purification yield). 8 Was purified using
acidic analytical HPLC conditions. Analytical HPLC: buffered
medium, linear gradient from 5 to 60% of B in 30 min, tR = 23.0
min. MALDI-TOF MS (THAP, negative mode): m/z 2785.0 [M −
H]−, 2822.9 [M − 2H + K]−; M calcd for C106H144N25O50P5S2
2785.8.
H-Cys(Acm)-Gly-Arg-Gly-Cys-Tyr-Ala-Tyr-Lys(Prot.Mal.)-NH2,
10. Fmoc-Cys(Acm)-OH was incorporated onto 34 μmol of peptide-
resin Fmoc-Gly-Arg(Pbf)-Gly-Cys-Tyr(tBu)-Ala-Tyr(tBu)-Lys-
(Prot.Mal.)-resin and the Fmoc group removed to yield 9. Treatment
with TFA/TIS/H2O 95:2.5:2.5, 2 × 1 h and purification at the
semipreparative scale (linear gradient from 25 to 70% of B in 30
min, tR = 6.2 min) afforded a white solid corresponding to the
desired peptide 10 (11.4 μmol, 34%). Analytical HPLC: acidic
conditions, linear gradient from 5 to 50% of B in 30 min, tR = 19.0
min. MALDI-TOF MS (DHB, positive mode): m/z 1170.7 [M −
furan − Acm + H]+ 1241.8 [M − furan + H]+; M calcd for
C59H84N16O16S2 1336.6, M − furan calcd for C53H76N16O15S2
1
under reduced pressure, and the crude was analyzed H NMR. In all
cases, a spectrum with the same set of signals as for the commercial
compound was obtained, with one exception: partial loss of the tBu
signal in the case of Boc-Cys(Fm)-OH, due to partial loss of the Boc
group upon heating.
Cyclic Maleimido*-Lys-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys-
(Prot.Mal.)-NH2, 2 (the Label * Refers to the Units Linked in
the Cycle). Peptide 2 was assembled on 110 mg of resin (f = 0.65
mmol/g). Treatment of peptide-resin 1 with TFA/TIS/H2O 90:5:5,
2 × 1 h, followed by purification at the semipreparative scale (linear
gradient from 15 to 35% of B in 30 min, tR = 15.1 min), afforded 2
as a white solid (12.1 μmol, 17%). Analytical HPLC: acidic
conditions, linear gradient from 10 to 50% of B in 30 min, tR
=
16.2 min. MALDI-TOF MS (DHB, positive mode, DHB = 2,5-
dihydroxybenzoic acid): m/z 1347.4 [M − furan + H]+, 1369.4 [M
− furan + Na]+; M calcd for C66H91N17O18S 1441.6, M − furan
calcd for C60H83N17O17S 1345.6. MALDI-TOF MS after reaction
with H2O2 (DHB, positive mode): m/z 1362.8 [M − furan + 16 +
H]+; M − furan + 16 calcd for C60H83N17O18S 1361.6. MALDI-TOF
MS after digestion with thrombin (DHB, positive mode): m/z
1347.3 [M + H]+, 1369.3 [M + Na]+, 1385.2 [M + K]+; M calcd for
C66H91N17O18S 1441.6, M − furan calcd for C60H83N17O17S 1345.6.
No digestion was observed.
Conjugate 3 [Cyclic Maleimido*-Lys-Gly-Arg-Gly-Cys*-Tyr-
Ala-Tyr-Lys(Mal.)-NH2] + [Diene-dT5]. Diene-dT5 and 2 (15 nmol
each) were reacted in a 1:1 MeOH/H2O solution (500 μL, 30 μM)
in a MW oven for 90 min at 90 °C. HPLC Analysis of the crude
showed that the extent of the cycloaddition was 90%. Analytical
HPLC: buffered medium, gradient from 5% to 60% of B in 30 min,
tR = 22.3 min. MALDI-TOF (THAP, negative mode, THAP = 2,4,6-
trihydroxyacetophenone): m/z 2962.7 [M − H]−; M calcd for
C116H158N27O53P5S 2963.9.
H-Cys(StBu)-Gly-Arg-Gly-Cys-Tyr-Ala-Tyr-Lys(Prot.Mal.)-NH2,
5. Fmoc-Cys(StBu)-OH was incorporated onto 121 μmol of Fmoc-
Gly-Arg(Pbf)-Gly-Cys(Tr)-Tyr(tBu)-Ala-Tyr(tBu)-Lys(Prot.Mal.)-
resin, and the Fmoc group removed to yield 4. 4 Was deprotected
and cleaved by reaction with TFA/TIS/H2O 95:2.5:2.5, 2 × 1 h, and
purified at the semipreparative scale (linear gradient from 25 to 50%
of B in 30 min, tR = 9.6 min). A white solid corresponding to the
desired peptide (5) was obtained (28 μmol, 23%). Analytical HPLC:
acidic conditions, linear gradient from 5 to 50% of B in 30 min, tR =
22.7 min. MALDI-TOF MS (DHB, positive mode): m/z 1258.9 [M
− furan + H]+; M calcd for C60H87N15O15S3 1353.6, M − furan calcd
for C54H79N15O14S3 1257.5. MALDI-TOF MS after reaction with
H2O2 (DHB, negative mode): m/z 1216.6 [M − furan − StBu + 48
− H]−, 1264.5 [M − furan − StBu + 48 + 48−H]−; M + 48 calcd
for C60H87N15O18S3 1401.6, M − furan − StBu + 48 calcd for
C50H71N15O17S2 1217.5. MALDI-TOF MS after digestion with
thrombin (DHB, positive mode): m/z 876.4 [M − furan −
[Cys(StBu)-Gly-Arg] + Na]+, 892.4 [M − furan − [Cys(StBu)-Gly-
Arg] + K]+; M calcd for C45H59N9O12S 949.4, M − furan −
[Cys(StBu-Gly-Arg] calcd for C39H51N9O11S 853.3.
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1240.5. MALDI-TOF MS after reaction with H2O2 (DHB, negative
mode): m/z 1169.0 [M − furan − Acm-H]−, 1217.0 [M − furan −
Acm + 48−H]−, 1233.0 [M − furan − Acm + 48 + 16−H]−; M +
48 calcd for C59H84N16O19S2 1384.5, M − furan − Acm + 48 calcd
for C50H71N15O17S2 1217.5.
Cyclic H-Cys(Acm)-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys*-NH2,
11. Compound 10 (100 nmol) was dissolved in the H2O/MeOH
mixture (1 mL, 100 μM) and heated in a MW oven according to the
general procedures, yielding the desired product 11 (30 nmol, 30%
cyclization and purification yield). Compound 11 was purified using
acidic analytical HPLC conditions. Analytical HPLC: acidic
with H2O2 (DHB, positive mode): m/z 1202.7 [M − Fm + 32 +
H]+, 1380.9 [M + 32 + H]+; M + 32 calcd for C64H81N15O16S2
1379.5.
Disulfide Dimer of Cyclic H-Cys-Gly-Arg-Gly-Cys*-Tyr-Ala-
Tyr-Lys*-NH2, 15. Compound 14 (92 nmol) was dissolved in 33%
piperidine in DCM (500 μL) and the mixture reacted for 45 min.
The solvent was then removed under reduced pressure and
coevaporated with acetonitrile. The crude was purified using
analytical HPLC acidic conditions (linear gradient from 5 to 50%
of B in 30 min, tR = 17.8 min), yielding 15 (41.6 nmol, 45% reaction
and purification yield). MALDI-TOF MS (DHB, positive mode): m/
z 1170.6 [monomer + H]+, 2338.3 [M + H]+; M calcd for
C100H114N30O28S4 2336.9.
Cyclic H-Cys-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys*-NH2, 16.
Compound 15 (27 nmol) was reacted with TCEP as described
above. Compound 16 was obtained in 89% yield (based on the
HPLC trace) and purified using analytical HPLC conditions.
Analytical HPLC: acidic conditions, linear gradient from 5 to 50%
of B in 30 min, tR = 15.8 min. MALDI-TOF MS (DHB, positive
mode): m/z 1171.1 [M + H]+, 1193.0 [M + Na]+; M calcd for
C50H71N15O14S2 1169.5.
Conjugate 17 [Cyclic H-Cys-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-
Lys*-NH2] + [Maleimido-dT5]. [Protected maleimido]-dT5 (25
nmol) was dissolved in the 1:1 MeOH/H2O solution (1 mL, 25
μM) and heated in the microwave oven for 60 min at 90 °C.
Methanol was removed under reduced pressure. HPLC analysis of
the crude (buffered medium) showed that maleimide deprotection
had taken place at an 85% extent. This crude was used immediately
for the conjugation reaction. Analytical HPLC: buffered medium,
linear gradient from 5 to 60% of B in 30 min, tR = 18.3 min.
MALDI-TOF MS (THAP, negative mode): m/z 1661.7 [M − H]−;
M calcd for C56H72N11O38P5 1661.3.
Mal-dT5 (5 nmol) was mixed with 16 (4 nmol) dissolved in water
(375 μL) and reacted overnight. The desired product (17) was
obtained in 73% yield (based on the HPLC trace). Analytical HPLC:
buffered medium, linear gradient from 5 to 60% of B in 30 min, tR =
22.0 min. MALDI-TOF MS (THAP, negative mode): m/z 2830.2
[M − H]−; M calcd for C106H143N26O52P5S2 2830.8.
conditions, linear gradient from 5 to 50% of B in 30 min, tR
=
16.0, 16.2 min (2 diastereomers). MALDI-TOF MS (DHB, positive
mode): m/z 1241.7 [M + H]+, 1263.7 [M + Na]+, 1279.7 [M + K]+;
M calcd for C53H76N16O15S2 1240.5. MALDI-TOF MS after reaction
with H2O2 (DHB, positive mode): m/z 1203.2 [M − Acm + 32 +
H]+; M + 32 calcd for C53H76N16O17S2 1272.5, M − Acm + 32 calcd
for C50H71N15O16S2 1201.6.
Assays To Remove the Acm Group from Peptide 11.
Reaction with AgOTf. Compound 11 (30 nmol) was dissolved in
TFA (1 mL), and AgOTf (1 mg, 3.9 μmol) and anisole (430 μL, 3.9
μmol) were added. The mixture was allowed to react for 1 h, after
which time solvent was removed under reduced pressure and the
crude redissolved with DTT (1 mg, 6.5 μmol) in a 1:1 mixture of
H2O/AcOH (1.5 mL). After 30 min, the solution was frozen and
lyophilized. The crude was then redissolved in water and analyzed,
yielding a very complex HPLC profile. Integration of the HPLC
trace and MALDI-TOF MS analysis showed that the desired product
was obtained in less than 5% yield.
Reaction with I2. Compound 11 (500 nmol) was dissolved in a
9:1 H2O/AcOH mixture (100 μL), and I2 (100 μL of a 50 mM
solution in MeOH) was added. The mixture was allowed to react for
4 h. The reaction was quenched with 0.2 M sodium ascorbate (20
μL), and methanol was removed under reduced pressure. Neither
HPLC analysis nor MALDI-TOF MS of the crude showed the
presence of the desired product, but instead one resulting from the
addition of one to four iodines to the peptide.
Reaction with Tl(OAc)3. Compound 11 (50 nmol) was dissolved
in TFA (500 μL), and a solution of Tl(OAc)3 (390 μg, 100 nmol)
and anisole (10.9 μL, 100 nmol) in TFA (10 μL) was added. The
mixture was reacted for 1 h at 0 °C, and the solvent was removed
with a N2 stream. 10% acetic acid in water (1 mL) and chloroform
(1 mL) were added, and the two fractions were collected separately.
HPLC and MALDI-TOF analysis of the two fractions showed the
presence of multiple products, none of which could be identified.
H-Cys(Fm)-Gly-Arg-Gly-Cys-Tyr-Ala-Tyr-Lys(Prot.Mal.)-NH2,
13. Boc-Cys(Fm)-OH was incorporated onto 31 μmol of Fmoc-Gly-
Arg(Pbf)-Gly-Cys(Tr)-Tyr(tBu)-Ala-Tyr(tBu)-Lys(Prot.Mal.)-resin.
Treatment with TFA/TIS/H2O 95:2.5:2.5, 2 × 1 h, and purification
at the semipreparative scale (linear gradient from 30 to 60% of B in
30 min, tR = 9.5 min) afforded a white solid corresponding to the
desired peptide 13 (9.6 μmol, 31%). Analytical HPLC: acidic
conditions, linear gradient from 30 to 60% of B in 30 min, tR = 7.0
min. MALDI-TOF (DHB, positive mode): m/z 1348.9 [M − furan
+ H]+, 1370.8 [M − furan + Na]+, 1386.8 [M − furan + K]+; M
Cyclic Lys*-Gly-Arg-Gly-Cys(StBu)-Tyr-Ala-Tyr-Cys*-NH2, 19.
The synthesis of 18 was carried out on 110 mg of resin (f = 0.65
mmol/g). After reaction with TFA/TIS/H2O 90:5:5, 2 × 1 h, and
purification at the semipreparative scale (linear gradient from 25 to
50% of B in 30 min, tR = 9.8 min), 19 was obtained as a white solid
(8.7 μmol, 11%). Analytical HPLC: acidic conditions, linear gradient
from 20 to 60% of B in 30 min, tR = 11.7 min. MALDI-TOF MS
(DHB, positive mode): m/z 1259.1 [M + H]+; M calcd for
C54H79N15O14S3 1257.5. MALDI-TOF MS after reaction with H2O2
(DHB, positive mode): m/z 1186.6 [M − StBu + 16 + H]+, 1306.7
[M + 32 + 16 + H]+, M − StBu + 16 calcd for C50H71N15O15S2
1185.5; M + 32 + 16 calcd for C54H79N15O17S3 1305.5. MALDI-
TOF MS after digestion with thrombin (DHB, positive mode): m/z
1259.2 [M + H]+, 1281.2 [M + Na]+, 1297.2 [M + K]+; M calcd for
C54H79N15O14S3 1257.5. No digestion was observed.
Cyclic Lys*-Gly-Arg-Gly-Cys-Tyr-Ala-Tyr-Cys*-NH2, 20. Com-
pound 19 (100 nmol) was reacted with TCEP as described in the
General Methods. Compound 20 was obtained in 80% yield (based
on the HPLC trace) and purified using acidic analytical HPLC
conditions. Analytical HPLC: acidic conditions, linear gradient from
10 to 50% of B in 30 min tR = 13.5 min. MALDI-TOF MS (DHB,
positive mode): m/z 1170.9 [M + H]+; M calcd for C50H71N15O14S2
1169.5.
calcd for C70H89N15O15S2 1443.6,
M − furan calcd for
C64H81N15O14S2 1347.6. MALDI-TOF MS after reaction with
H2O2 (DHB, negative mode): m/z 1214.4 [M − furan − Fm +
48 − H]−, 1232.6 [M − furan − Fm + 48 + 16 − H]−; M + 48 +
16 calcd for C70H89N15O19S2 1507.6, M − furan − Fm + 48 + 16
calcd for C50H71N15O18S2 1233.5.
Cyclic H-Cys(Fm)-Gly-Arg-Gly-Cys*-Tyr-Ala-Tyr-Lys*-NH2,
14. Compound 13 (400 nmol) was dissolved in the H2O/MeOH
mixture (4 mL, 100 μM), heated in a MW oven according to the
general procedures, and purified using analytical HPLC conditions to
yield 14 (184 nmol, 46% cyclization and purification yield).
Analytical HPLC: acidic conditions, linear gradient from 30 to
60% of B in 30 min, tR = 4.8 min. MALDI-TOF MS (DHB, positive
mode): m/z 1348.8 [M + H]+, 1370.8 [M + Na]+, 1386.7 [M + K]+;
M calcd for C64H81N15O14S2 1347.6. MALDI-TOF after reaction
Conjugate 21 [Cyclic Lys*-Gly-Arg-Gly-Cys-Tyr-Ala-Tyr-
Cys*-NH2] + [Maleimido-dT5]. Compound 20 (10 nmol) and
[protected maleimido]-dT5 (10 nmol) were dissolved in a 3:1
MeOH/H2O solution (500 μL, 20 μM) and heated in a MW oven
for 90 min at 90 °C. Methanol was removed under reduced pressure.
The desired product (21) was obtained in 77% yield (based on the
HPLC trace). Analytical HPLC: buffered medium, linear gradient
from 5 to 50% of B in 30 min, tR = 14.9 min. MALDI-TOF MS
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(THAP, negative mode): m/z 2829.5 [M − H]−; M calcd for
C106H143N26O52P5S2 2830.7.
CH2-NH2); 1.44 (s, 9H, CH3) ppm; 13C NMR CDCl3, 100 MHz) δ
156.2, 79.2, 43.2, 41.8, 28.4 ppm; ESI MS (positive mode) m/z
161.13 [M + H]+; M calcd for C7H16N2O2 160.12.
[Protected maleimido]-[N-(CH2 CH2 NH2 )Gly]-[N-
(CH2CH2Phenol)Gly]-[N-(CH2CH2OH)Gly]-[N-(Propargyl)Gly]-[N-
(CH2CH2Phenol)Gly]-[N-(CH2CH2NH2)Gly]-[N-(CH2CH2OH)Gly]-
[N-(CH2CH2SH)Gly]-Gly-NH2, 23. Peptoid 23 was assembled on
100 mg of resin (f = 0.72 mmol/g). Treatment of the peptoid-resin
22 with TFA/m-cresol/TIS/H2O 90:5:2.5:2.5, 2 × 1 h, followed by
purification at the semipreparative scale (linear gradient from 20 to
40% of B in 30 min, tR = 12.0 min), afforded 23 as a white solid (3.0
μmol, 4.1%). Analytical HPLC: acidic conditions, linear gradient
from 20 to 40% of B in 30 min, tR = 9.7 min. MALDI-TOF MS
(THAP/CA, positive mode, CA = citric acid): m/z 1194.7 [M −
furan + H]+, 1290.8 [M + H]+; M calcd for C60H83N13O17S 1289.6,
M − furan calcd for C54H75N13O16S 1193.5. MALDI-TOF MS after
reaction with H2O2 (THAP/CA, negative mode): m/z 1240.7 [M −
furan + 48 − H]−; M − furan + 48 calcd for C54H75N13O19S 1241.5.
Cyclic Maleimido*-[N-(CH2CH2NH2)Gly]-[N-(CH2CH2Phenol)-
Gly]-[N-(CH2CH2OH)Gly]-[N-(Propyne)Gly]-[N-(CH2CH2Phenol)-
Gly]-[N-(CH2CH2NH2)Gly]-[N-(CH2CH2OH)Gly]-[N-(CH2CH2SH)-
Gly]*-Gly-NH2 24. Compound 23 (100 nmol) was dissolved in the
H2O/MeOH solution (1 mL, 100 μM) and heated in a MW oven
according to the general procedures. MeOH was removed under
reduced pressure. The desired product (24) was obtained in 79%
yield (based on the HPLC trace). Analytical HPLC: acidic
ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC traces of the compounds prepared, commercially
available building blocks used for peptide and peptoid
assembly, and 1H and 13C NMR spectra of compounds
synthesized according to described procedures. This material
is available free of charge via the Internet at http://pubs.acs.
org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: anna.grandas@ub.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by funds from the Ministerio de
́
Economia y Competitividad (Grant No. CTQ2010-21567-
conditions, linear gradient from 20 to 40% of B in 30 min, tR
=
C02-01 and the project RNAREG, Grant No. CSD2009-
00080, funded under the programme CONSOLIDER
INGENIO 2010), and the Generalitat de Catalunya
(2009SGR-208). X.E. was a recipient fellow of the MINECO.
15.8 min. MALDI-TOF MS (THAP/CA, positive mode): m/z
1194.7 [M + H]+; M calcd for C54H75N13O16S 1193.5. MALDI-TOF
MS after reaction with H2O2 (THAP/CA, positive mode): m/z
1210.7 [M + 16 + H]+; M + 16 calcd for C54H75N13O17S 1209.5.
Conjugate 25 [Cyclic Maleimido*-[N-(CH2CH2NH2)Gly]-[N-
(CH2CH2Phenol)Gly]-[N-(CH2CH2OH)Gly]-[N-(Propyne)Gly]-[N-
(CH2CH2Phenol)Gly]-[N-(CH2CH2NH2)Gly]-[N-(CH2CH2OH)Gly]-
[N-(CH2CH2SH)Gly]*-Gly-NH2] + AZT. A 0.5 mM solution of
CuSO4 in water (50 μL, 25 nmol) and a 12 mM solution of sodium
ascorbate in water (50 μL, 600 nmol) were mixed in a microwave
flask under an Ar atmosphere and stirred during 5 min. Then TBTA
(tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) (50 μL of a 0.5
mM solution in MeOH:H2O 3:2, 24 nmol), AZT (3′-azido-3′-
deoxythymidine) (110 μL of a 2 mM aqueous solution, 200 nmol),
and compound 24 (100 μL of a 290 μM aqueous solution, 29 nmol)
were added. The mixture was heated in a microwave oven for 1 h at
90 °C. MeOH was then removed under reduced pressure. The
desired product (25) was obtained in 90% yield (based on the
HPLC trace, calculated from compound 24). Analytical HPLC:
acidic conditions, linear gradient from 5 to 50% of B in 30 min, tR =
17.1 min. MALDI-TOF MS (DHB, positive mode): m/z: 1462.4 [M
+ H]+, 1484.3 [M + Na]+; M calcd for C64H88N18O20S 1460.6.
S-Trityl Cysteamine Hydrochloride. Trityl chloride (279 mg,
1.6 mmol) and cysteamine hydrochloride (200 mg, 1.8 mmol) were
dissolved in DMF (1.5 mL) and stirred overnight at room
temperature. The reaction was then quenched with cold water (10
mL), and the precipitated formed was filtered and washed with cold
water (3 × 5 mL). A white solid (480 mg, 85% yield) was obtained:
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