On-Resin Conjugation of Diene-Polyamides and Maleimides via Diels-Alder Cycloaddition

Article abstract of DOI:10.1021/acs.joc.5b00592

The reaction between maleimides and resin-linked diene-polyamides allows the latter to be used in the preparation of conjugates. Conjugation takes place by reacting the insoluble, hydrophobic diene component either with water-soluble dienophiles or with dienophiles requiring mixtures of water and organic solvents. Experimental conditions can be adjusted to furnish the target conjugate in good yield with no need of adding large excesses of soluble reagent. In case protected maleimides are used, maleimide deprotection and Diels-Alder cycloaddition can be simultaneously carried out to render conjugates with different linking positions. On-resin conjugation is followed by an acidic treatment that removes the polyamide protecting groups with no harm to the cycloadduct, in contrast with the unreacted diene that is indeed degraded under these conditions. Cycloadducts incorporating suitable functional groups can undergo subsequent additional conjugation reactions in solution to furnish double conjugates.

Full text of DOI:10.1021/acs.joc.5b00592

Article
pubs.acs.org/joc
On-Resin Conjugation of Diene−Polyamides and Maleimides via
Diels−Alder Cycloaddition
Omar Brun, Xavier Elduque, Enrique Pedroso, and Anna Grandas*
̀ ̀
Departament de Química Organica, Facultat de Química and IBUB, Universitat de Barcelona, Martí i Franques 1-11, 08028
Barcelona, Spain
S
* Supporting Information
ABSTRACT: The reaction between maleimides and resin-
linked diene−polyamides allows the latter to be used in the
preparation of conjugates. Conjugation takes place by reacting
the insoluble, hydrophobic diene component either with
water-soluble dienophiles or with dienophiles requiring
mixtures of water and organic solvents. Experimental
conditions can be adjusted to furnish the target conjugate in
good yield with no need of adding large excesses of soluble
reagent. In case protected maleimides are used, maleimide
deprotection and Diels−Alder cycloaddition can be simultaneously carried out to render conjugates with different linking
positions. On-resin conjugation is followed by an acidic treatment that removes the polyamide protecting groups with no harm to
the cycloadduct, in contrast with the unreacted diene that is indeed degraded under these conditions. Cycloadducts incorporating
suitable functional groups can undergo subsequent additional conjugation reactions in solution to furnish double conjugates.
as cyclooctynes) react with azides in the absence of Cu(I),15,16
in other words in biocompatible conditions, but the reaction
gives a mixture of regioisomers.
INTRODUCTION
■
Chemical reactions allowing natural or synthetic biomolecules
to be appended with reporter groups (such as fluorescent tags,
spin labels, or biotin), radical-generating reagents, metal
complexes, cell-penetrating entities, or specific recognition
motifs have been the focus of extensive research over the past
few decades.1 In the resulting new entities, commonly referred
to as conjugates, biomolecules are expected to be endowed with
new or improved properties. In order to prevent the results of
conjugate-involving experiments from being misinterpreted, as
well as premature loss of conjugation synergies, conjugates
should have a defined structure and be stable.
One of the oldest and most extensively used conjugation
reactions is the Michael-type reaction between a thiol and a
maleimide.2 It takes place quickly and in high yield, is water
compatible, does not require any catalyst, and does not
generate byproducts. Therefore, it fulfills the basic requirements
of click reactions.3 Yet, the thiol−maleimide reaction generates
two diastereomers, and different recent publications have
revealed that the resulting succinimide may undergo thiol
exchange and hydrolysis.4−7 The Cu(I)-catalyzed Huisgen
cycloaddition between an azide and an alkyne has become the
par excellence click reaction after it was described that Cu(I)
accelerates the cycloaddition and prevents formation of the 1,5-
regioisomer.8,9 The addition of Cu(I)-stabilizing entities
accelerates the reaction and prevents formation of oxidizing
species10 and has been key in the preparation of all types of
conjugates linked through stable 1,2,3-triazoles. However,
complete elimination of the metal catalyst may be difficult to
achieve,11,12 and optimization of reaction conditions may be
highly demanding.13,14 Suitably modified strained alkynes (such
The Diels−Alder (DA) cycloaddition has been successfully
used to prepare bioconjugates. It offers the advantage of being
totally chemoselective and byproduct free and can be
conducted in (and be accelerated by) water, and hence, it
can also be classified as a click reaction. The DA cycloaddition
is known to furnish a mixture of isomers in which those with
the endo configuration usually predominate.17 With respect to
the Michael-type and Cu(I)-catalyzed Huisgen cycloaddition, it
offers the advantage of providing stable conjugates that do not
undergo exchange with reagents present in biological media
and that of not requiring metal catalysts if performed in
aqueous media, respectively.18−20 Moreover, cycloadducts
formed in DA reactions are smaller than those formed in
copper-free azide−cyclooctyne reactions, especially in the case
of cyclooctynes activated by mono- or dibenzo substitution.
Bioconjugates have been obtained in solution from DA
reactions, affording both carbon−carbon21−29 and carbon−
heteroatom bonds30−32 as well as using the inverse electron
demand version.33−36 The DA reaction is in principle
reversible,37 but most cycloadducts are sufficiently stable to
withstand chemical manipulation and use in physiological
media because the retro-DA reaction typically requires fairly
harsh conditions.37
In principle, any of the two reacting groups (diene or
dienophile) can be linked to either of the conjugate
Received: March 16, 2015
Published: May 18, 2015
© 2015 American Chemical Society
6093
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
components. In the case of molecules routinely prepared by
solid-phase synthesis, such as peptides and oligonucleotides, the
most straightforward alternative is to introduce the additional
group required for conjugation while they are still linked to the
solid matrix. Yet, the compatibility of dienes and dienophiles
with the reaction conditions that deprotect the oligomer and
cleave the oligomer−resin bond has to be considered. For
instance, maleimides (which are the dienophiles most
commonly used in bioconjugations) are stable to the acidic
treatment that deprotects peptides but not to the reaction with
ammonia that deprotects oligonucleotides. As a result,
maleimido−oligonucleotides can only be on-resin assembled
provided that the maleimide moiety is protected.38 Dienes are
not degraded by the ammonia treatment, and dienes
conjugated with electron-withdrawing groups remain stable to
polyamide acidic deprotection conditions.39,40 However, alkyl-
substituted 1,3-dienes, which react more quickly with
dienophiles in prototype DA cycloadditions, are in principle
degraded by strong acids (Scheme 1).
Therefore, the DA cycloaddition between dienophiles and
diene−polyamides is the only combination in which one of the
reagents is not easily available, since the instability of 1,3-
diene−polyamides to acids precludes their solid-phase synthesis
using standard procedures.
With the aim of extending the DA-based conjugation
toolbox, we decided to examine the possibility of attaching
alkyl-substituted 1,3-dienes to resin-linked peptides and PNAs
and carrying out the DA cycloaddition on the solid matrix. This
alternative is simple from the operational point of view, of
general application, and offers the advantage of preventing
dienes from being exposed to acid-containing deprotection
mixtures and diene−polyamides from having to be purified
before the conjugation step. The final deprotection treatment
takes place after formation of the cycloadduct, which remains
stable under these conditions. Moreover, cycloadducts derived
from the reaction between 1,3-dienes and maleimides are more
stable than those formed by reacting maleimides and
furans.26,42,43
However, the fact that the dienophile-containing moiety
might possibly be a water-soluble biomolecule raised some
doubts as to the feasibility of this synthetic alternative. To our
knowledge, on-resin DA reactions44 have always been carried
out in organic solvents, even on hydrophilic resins.45 The use of
water is indeed advantageous, because the reaction takes place
more quickly and in milder conditions than in organic
solvents,18−20 but the outcome of cycloadditions between
water-soluble maleimide-containing compounds and hydro-
phobic diene-[protected polyamide]-resins performed in water
(or solvents with a high water content) was not obvious.
Here we describe that such on-resin DA reactions in water
(Scheme 2a) do provide the target conjugates, allowing diene−
polyamides to be used in DA reactions. We also report that the
scope of conjugation alternatives can be broadened by using
protected maleimide derivatives and simultaneously carrying
out maleimide deprotection and conjugation (Scheme 2b).
This approach has been used to obtain conjugates with different
linking sites and in reactions involving maleimides derivatized
with tagging agents such as fluorophores and biotin, which are
sparingly soluble in water. In case conjugates assembled by on-
resin DA cycloaddition incorporate appropriate functional
groups, an additional chemoselective conjugation reaction can
be carried out to yield doubly derivatized molecules.
Scheme 1. Bioconjugation Reactions Involving Alkyl-
a
Substituted 1,3-Dienes and Maleimides
a
General structures of the reagents and cycloadduct, and summary of
the permitted derivatizations.
There are no precedents in the literature reporting the
synthesis and use of diene−PNAs (peptide nucleic acids) in DA
cycloadditions, and no general alternative for the solid-phase
synthesis of alkyl-substituted 1,3-diene−peptides (or any
diene−polyamide whose final deprotection reaction requires
strong acids) has so far been described. The few reports
involving diene−peptides have some limitations. The Madder
group could obtain furan-derivatized peptides by solid-phase
synthesis provided that an aromatic residue was placed next to
the furan-containing building block;41 otherwise, furan was
reduced by the triisopropylsilane (TIS) scavenger typically
present in the final peptide deprotection mixture. This group
also reacted maleimides with resin-linked furans, but the
resulting cycloadducts partially underwent reversion to the
starting materials.42 The Waldmann group prepared 1,3-diene−
peptides by solid-phase assembly on a sulfamylbutyryl linker
resin using Fmoc−amino acids (Fmoc = 9-fluorenylmethox-
ycarbonyl).26 However, their methodology is only compatible
with glycine at the C-terminus and Fmoc−amino acids with
side chain protecting groups removable under very mild acidic
conditions, which excludes the presence of some trifunctional
amino acids because the required derivatives are not
commercially available.
RESULTS AND DISCUSSION
■
Preliminary Experiments, Choice of the Solid Matrix,
and Analysis of the Results. It is well known that conjugated
polyenes react with acids and undergo addition reactions. As
stated above, dienes conjugated with electron-withdrawing
groups such as sorbic acid can be submitted to peptide
deprotection conditions with no harm to the conjugated
system,39,40 but alkyl-substituted 1,3-dienes cannot be
presumed to exhibit the same behavior. To definitely assess
whether alkyl-substituted 1,3-dienes would or not withstand
typical peptide deprotection conditions, H2CCH−CH
CH−CH2−CH2−CO-Ala-Tyr(tBu)-Lys(Boc)-Rink amide
resin (Boc = tert-butoxycarbonyl) was treated with a
95:2.5:2.5 TFA/TIS/water mixture for 2 h and the resulting
crude analyzed by HPLC-MS. The crude was very complex, and
the main peaks were peptides linked to products resulting from
hydration and reduction of the 1,3-diene, as well as peptides
containing only fragments of the diene linker chain (see
Supporting Information, section 3).
6094
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
Scheme 2. General Schemes for (a) Solid-Phase DA Cycloaddition between Maleimides and Diene−Polyamides and (b) the on-
a
Resin Conjugation between Protected Maleimides and Diene−Polyamides
a
In both cases, the table below each of the schemes shows the moieties combined in the different conjugates. *Linear or cyclic peptides, with the
protected maleimide appending from different positions (see structures in Table 2). Prot. Mal. = 2,5-dimethylfuran-protected N-substituted
maleimide; TFA = trifluoroacetic acid.
As we intended to conduct the cycloadditions in an aqueous
medium, solid supports made up of polystyrene and poly-
ethylene glycol chains seemed the most convenient for this
study since they swell in water, and nowadays are standard
resins for the solid-phase assembly of peptides and PNAs.
Therefore, NovaSyn TGR and TentaGel resins with the Rink
amide-type functionality were used (see Experimental Section).
All PNAs and peptides were assembled on these resins using
the standard Fmoc-based methodology (Fmoc/Bhoc mono-
mers in the case of PNAs46 and Fmoc/tBu in the case of
peptides;47 Bhoc = benzhydryloxycarbonyl). Then the resin-
linked polyamides were derivatized with either (E)-4,6-
heptadienoic acid or 3-maleimidopropanoic acid depending
on whether they were intended to remain resin attached or be
used as the soluble maleimide-containing component in the DA
reaction, respectively.
The outcome of conjugation reactions was examined by
HPLC analysis of the crude, and the major products were
collected and identified by MALDI-TOF MS. No attempt was
made to separate the isomers resulting from the cycloaddition,
which, as expected for the size of the molecules, in most cases
coelute (see HPLC traces at the Supporting Information). As
stated above, both the thiol−maleimide Michael-type addition
and the Cu-free Huisgen cycloaddition yield mixtures of
isomers that are used as such. Isolation and testing of one of the
isomers may become an important issue for biomedical
applications but not at this preliminary level. Yields of
conjugates (Tables 1 and 2) were calculated as the ratio of
the area of the conjugate peak to the sum of the areas of all
peaks. Since impurities generated during the solid-phase
polyamide assembly also contribute to the total area, reported
yields are likely lower than the actual conjugation yields. In
reactions with protected peptide−resins it is convenient to
determine conjugation yields from the areas of the peaks as
appearing on HPLC traces recorded at 220 nm, since only at
this wavelength the unreacted peptide can be observed and
quantified. On the contrary, for conjugations with PNA−resins
260 nm is the wavelength of choice. In case conjugates were
isolated, the isolation yield was determined on the basis of the
loading of Fmoc−polyamide−resin prior to incorporation of
the diene and accounts for the overall conjugation, depro-
tection, and purification yield.
On-Resin DA Reactions Involving Water-Soluble
Maleimides. The initial set of experiments had different
aims: first, to confirm that water could be a suitable solvent for
the cycloaddition; second, to identify the mildest possible
6095
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
Table 1. Results of the on-Resin Conjugations between Maleimides and Diene−Polyamides
entry
reagents
equiv of Mal.
solvent
[Mal.] (mM)
time (h)
temp. (°C)
yield (%)
1
2
3
4
5
6
7
1 + 5
1 + 5
1 + 5
1 + 5
2 + 5
3 + 6
4 + 7
1.5
1.5
1.5
3
H2O
20
20
20
40
40
40
40
70
70
24
24
24
24
24
40
40
65
65
65
65
65
74
48
62
79
69
92
72
1:1 H2O/DMSO
H2O
H2O
H2O
H2O
H2O
3
3
5
*
a,c,g,t = PNA residues with appending adenine, cytosine, guanine, and thymine, respectively; Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl.
Table 2. Results of the on-Resin Reactions Involving Protected Maleimides and Diene−Polyamides
b
a
entry
reagents
equiv of [Prot. Mal.]
solvent
[Prot. Mal.] (mM)
time (h)
temp. (°C)
yield (%)
isolated yield (%)
1
2
3
4
5
6
7
8
8 + 5
3
5
5
3
5
5
3
H2O
H2O
H2O
H2O
H2O
H2O
40
40
40
40
40
40
40
40
24
24
24
24
24
24
24
24
65
65
65
65
65
65
65
65
82
58
55
67
9 + 7a
10 + 7a
11 + 5
11 + 7
11 + 7a
12 + 5
12 + 7a
28
30
c
n.d.
60
88
72
65:35 H2O/DMSO
65:35 H2O/DMSO
48
19
5
a
b
c
Conjugation yields are HPLC based. Prot. Mal. = 2,5-dimethylfuran-protected maleimide-containing reagent (see also Table 1). Not determined:
complex HPLC trace (see Supporting Information, page S21).
conditions for the conjugation reaction allowing one to use a
minimum excess of the maleimido-derivatized compound and
not requiring extremely long reaction times. The conditions
reaching the best balance between these variables would be
chosen as our standard protocol for reactions in water (Scheme
2a). Table 1 assembles the results of these experiments, in
which water-soluble maleimido−PNA 1 and resin-linked, diene-
derivatized peptide 5 were chosen as model compounds.
The first two experiments (entries 1 and 2) showed that the
reaction between a water-soluble dienophile and a hydrophobic
immobilized diene takes place in a higher extent in water than
in a water−organic solvent (DMSO = dimethyl sulfoxide)
mixture and confirm the favorable effect of water even in these
heterogeneous reaction conditions.
The next step was finding suitable conditions to avoid the
long conjugation time used in the first experiments. Different
combinations of temperature, time, concentration, and
equivalents of the maleimide−PNA 1 were tested (Table 1,
entries 3 and 4), from which we concluded that the yield of the
reaction with 3 equiv of 1 (40 mM concentration) for 24 h at
65 °C was very satisfactory. Although the reaction time was
somewhat long, reaction conditions were fairly mild and it was
not necessary to use a great excess of maleimido−PNA.
With these results in hand, we decided to further explore the
possibilities of the methodology (entries 5−7). The same
6096
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
peptide−resin (5) was reacted with a longer PNA (2), and
soluble maleimido−peptides (3, 4) were reacted with diene-
derivatized, resin-linked PNAs (6, 748).
this conjugation methodology. When 11 was reacted with
immobilized diene-[protected-PNA]−resin 7 (Table 2, entry
5), a complex crude was obtained (see Supporting Information,
page S21) and the conjugation yield could not be determined.
We surmised that the yield was not good and decided to
evaluate whether the introduction of a short spacer (one
glycine) between the protected PNA chain and the diene
allowed the cycloaddition yield to increase (Table 2, entry 6).
The yield of the conjugation reaction reached 60%, and the
conjugation crude was cleaner (see Supporting Information,
page S22). Hence, moving away the diene reactive point and
the hydrophobic polyamide may be useful to increase
conjugation yields in difficult cases. Subsequent reactions
were carried out with the insoluble dansyl fluorophore 12,
which was reacted with both 5 and 7a (Table 2, entries 7 and
8). In this case a 65:35 (v/v) water/DMSO mixture had to be
used (in which 12 was still sparingly soluble). We were gladly
surprised to see that HPLC analysis of the reaction crudes
showed rather high conversions, in particular, taking into
account that cycloadditions were not being carried out in water
but in water/organic solvent mixtures. Both conjugates were
purified and isolated in 48% and 19% yield, respectively.
Thinking about possible reasons for such good conjugation
yield, the 12 + 5 reaction was repeated and the Eppendorf tube
containing the mixture of reagents was exposed to UV light
(254 nm). We could then see that the fluorophore seemed to
be adsorbed onto the resin beads surface (see Supporting
Information, page S24), presumably because of hydrophobic
interactions between the apolar molecule and the carbon-rich
chains of the polymer. This could increase the effective
concentration of 12 near the reactive points and likely
compensate for the decrease of reaction rate expected for a
DA reaction in an organic solvent/water mixture, thus
accounting for the good yields. These results demonstrate
that the on-resin DA reaction can also be carried out with
molecules sparingly soluble and even not soluble in water,
either by heating the aqueous suspensions to get saturated
solutions or by dissolving the dienophile in mixtures with an
organic cosolvent and high water content.
Polyamide Double Conjugates. PNAs interact with
complementary sequences in nucleic acids with higher affinity
than natural oligonucleotides but do not enter cells.54 The
covalent attachment of cell-penetrating peptides to PNAs
allows one to overcome this obstacle, but the fate of the
conjugate within cells cannot be monitored unless a suitable tag
is attached to the molecule. PNAs linked to both fluorophores
and carrier peptides are thus interesting molecules, and we
anticipated that the on-resin DA cycloaddition could be a
valuable tool for their straightforward synthesis. For this proof
of principle experiment, cysteine and the diene were
subsequently coupled to a protected peptide−resin (Scheme
3), and the double derivatization was achieved after a DA
reaction on the solid support and a Michael-type reaction in
solution.
A preliminary experiment was run to assess the stability of
the Trt group, typically used to protect the cysteine side chain,
under the conditions of the DA cycloaddition. 3-Maleimido-
propanoic acid (4 equiv) was incubated in water with Fmoc-
Cys(Trt)-Gly-Arg(Pbf)-Gly-Ser(tBu)-Tyr(tBu)-Glu(OtBu)-
Ala-Tyr(tBu)-Lys(Boc)-resin for 24 h at 65 °C, which was
followed by extensive washing, treatment with a 94:2.5:1:2.5
TFA/H2O/TIS/EDT mixture for 2 h at rt, and HPLC analysis
of the crude (see Supporting Information, page S27). MALDI-
It was observed that an increase in the length of the reagents
(either the soluble or the immobilized moiety) had a negative
impact on the extent of the conjugation reaction, which could
be offset by slightly increasing the excess of soluble maleimide.
Use of Protected Maleimides To Obtain Conjugates
with Different Linking Sites. It has been described that
differently linked conjugates may have different biological
effects.49−52 Standard solid-phase stepwise synthesis may
provide hybrid polyamides, either peptide−PNA or PNA−
peptide, where the C-terminus of one component is linked,
either directly or by means of an additional linker, to the N-
terminus of the other. The experiments described here so far
furnish conjugates in which the N-termini of the two
polyamides are linked through the cycloadduct resulting from
the DA reaction and thus give access to different hybrid
polyamides.
As stated above, maleimides can be protected with 2,5-
dimethylfuran,38 which allows maleimides to be placed at any
position of peptide chains assembled using Fmoc−amino
acids.43 We also described that maleimide deprotection and
maleimide-involving click reactions (Diels−Alder, Michael
type) can take place simultaneously.43 If the one-pot
deprotection + conjugation reaction could also be performed
on a solid support, the scope of the on-resin conjugation via DA
cycloaddition might be enlarged, making it possible to link the
diene not only to the end of another chain but also to internal
positions. This would generate “branched” or “T-shaped”
conjugates otherwise difficult to prepare.
To examine whether this alternative was feasible (Scheme
2b), (2,5-dimethylfuran)-protected 2-hydroxyethylmaleimide38
(8) was reacted with peptide−resin 5 (entry 1 of Table 2).
HPLC analysis of the crude showed that the target conjugate
was formed in 82% yield when using the same reaction
conditions as for entries 4−6 of Table 1.
This good result prompted us to tackle the synthesis of more
challenging conjugates, either involving more complex reagents
or with the aim of preparing conjugates with different linking
sites. Cyclic peptide 9,53 with a C-terminal protected-maleimide
(shown in Table 2 as R), was reacted with 7a (see below) to
yield a conjugate in which the C-terminal residue of a cyclic
peptide was linked to the N-terminus of a PNA. This reaction
proceeded in 58% yield, and the conjugate was isolated in 28%
yield (Table 2, entry 2). Then peptide 10, with a protected
maleimide in the middle of the peptide chain, was also reacted
with 7a to render the desired “branched/T-shaped” conjugate
also in good yield (Table 2, entry 3). On the basis of these
results, we can conclude that the use of [protected-
maleimido]−peptides allows synthesizing conjugates with
different linkage patterns.
On-Resin Conjugation Reactions Involving Reagents
Sparingly Soluble in Water. We also wondered whether
molecules sparingly soluble or even nonsoluble in water could
be employed in this methodology. To answer this question, two
reporter molecules incorporating protected maleimides, biotin
and the dansyl fluorophore, were chosen for the first assays.
[Protected-maleimide]−biotin 11 is not soluble in water at
room temperature but can be solubilized by heating at 150 °C
for a few seconds, with no precipitation after cooling. Reaction
of 11 with 5 proceeded in 67% yield (Table 2, entry 4), which
showed that sparingly soluble molecules could also be used in
6097
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
Scheme 3. Double Conjugation Affording Compound 17, in Which a Peptide and a Fluorophore Are Covalently Linked to a
a
PNA
a
The on-resin DA cycloaddition linked the fluorophore to the diene−peptide, and the solution conjugation reaction (thiol−ene Michael-type
reaction) provided the peptide−PNA linkage. Trt = trityl.
TOF mass spectrometric analysis of the major peak confirmed
that the Trt group remained stable under the conjugation
reaction conditions, since incorporation of 3-maleimidopropa-
noic acid had not taken place.
such as peptides and PNAs. The presence of water in the
reaction medium allows the reaction to proceed within a
reasonable time with no need of heating to very high
temperatures (24 h or less at 65 °C). Good to very good
conjugation yields can be attained using a small excess of
dienophile (1.5−5 equiv), which allows this precious reagent
not to be wasted. Yet, this must be balanced by prolonging the
reaction time.
Thereafter, the double derivatization was carried out
(Scheme 3). More in detail, diene−peptide−resin 13 was
prepared by solid-phase assembly of the decapeptide using the
standard Fmoc/tBu chemistry and incorporation of (E)-4,6-
heptadienoic acid. The 1,3-diene-derivatized peptide was then
reacted with the commercially available maleimido−fluoro-
phore 1-[2-(maleimido)ethyl]-4-[2-(3,4-dihydro-2H-benzopyr-
an-6-yl)-5-oxazolyl]-pyridinium triflate (14). As compound 14
was not very soluble in the 65:35 water/DMSO mixture, even
less than 12, a suspension of 14 (3 equiv) was reacted with
diene−peptide−resin 13. After 24 h at 65 °C, the resin-linked
conjugate was treated with 94% TFA (in the presence of
scavengers, see above) to yield 15, and the resulting crude was
analyzed by HPLC (page S29 of the Supporting Information).
The DA reaction yield was 77%. In parallel, two lysine residues
and 3-maleimidopropanoic acid were added to the PNA−resin
from which 7 had been obtained to yield maleimido−PNA 16
after the final deprotection and HPLC purification. Finally, 15
and 16 (1:1.3 relative molar ratio) were reacted in solution at
room temperature, and HPLC analysis of the crude (page S30
of the Supporting Information) showed that in 2.5 h the
Michael-type reaction had already gone to completion.
Conjugate 17 was purified (23% overall synthesis and isolation
yield) and its identity assessed by mass spectrometric analysis.
On-resin bioconjugations have been also performed using
azide−alkyne cycloadditions, most often catalyzed by Cu(I),
and experimental conditions vary a lot between the different
reports. On the basis of those of three different groups12,55,56 it
can be said that (i) reactions are always carried out in mixtures
of water and organic solvents and not pure water, (ii) Cu(I)-
catalyzed reactions can satisfactorily take place at 60 °C and in
1 h or less if performed in a MW oven (using 2.3 equiv of
soluble reagent)55 or overnight at room temperature in case
activated alkynes are used (4 equiv of soluble reagent was
employed in this case),12 and (iii) copper-free azide−alkyne
cycloadditions at room temperature can require from 30 min to
16 h (using 20 equiv of soluble reagent).56 Therefore, our
reaction conditions (excess of soluble reagent, reaction time,
temperature) do not substantially exceed those required by the
most typical cycloadditions and broaden the scope of
possibilities for solid-supported DA cycloadditions. Reaction
times could likely be shortened by using more reactive dienes,
either cyclic or with more alkyl substituents (or both).28
The DA reaction is chemoselective and compatible with
PNA and peptide functionalities and their protecting groups
and provides a mixture of cycloadducts that remains stable to
the acidic treatment that deprotects the polyamide. We never
observed any undesired reaction (such as hydrolysis of the
CONCLUSIONS
■
In summary, the on-resin DA cycloaddition in water (or high
water content mixtures) between a 1,3-diene and a maleimide is
a suitable alternative to prepare stable conjugates of polyamides
6098
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
DCM (3×). This resin is supplied without an Fmoc protecting group
on the amine, so no piperidine treatment is needed prior to chain
elongation. Incorporation of the first amino acid onto the resin and
peptide assembly were accomplished by using 3 equiv of both Fmoc−
amino acid, HOBt·H2O (1-hydroxybenzotriazole), and DIPC (N,N′-
diisopropylcarbodiimide), all dissolved in a minimal amount of DCM
and a few drops of NMP (N-methylpyrrolidone), for 90 min at room
temperature. Coupling was followed by washing with DCM, DMF,
and MeOH (3 × each). In case the coupling was not complete, as
assessed by the Kaiser test,59 it was repeated using 2 equiv of the
reagents. The resin was always allowed to swell in DCM for 2 min
before Fmoc removal. Removal of the Fmoc groups was effected by
reaction with 20% piperidine/DMF (1 × 3 min + 1 × 10 min),
followed by washing with DCM (3×), DMF (3×), and DCM (3×).
No capping steps were performed, except after incorporation of the
last amino acid and prior to incorporation of (E)-4,6-heptadienoic acid
or 3-maleimidopropanoic acid. The capping step was effected by
reaction with Ac2O/2,6-lutidine/DMF (v/v/v 5:6:89 mixture, 2 × 5
min) and followed by washing with DMF (3×) and DCM (3×). At
this level, the substitution degree was determined by taking a carefully
weighed aliquot, removing the Fmoc group with piperidine, and
quantitating the amount of 9-fluorenylmethylpiperidine formed (λmax
= 301 nm, ε = 7800).60 This value was taken as the substitution degree
of the resin-linked diene−polyamides in conjugation experiments (see
below), without any further correction for the addition of the diene,
and used to determine the overall synthesis and purification yield for
the isolated conjugates. Fmoc-Gly-Arg(Pbf)-Gly-Ser(tBu)-Tyr(tBu)-
Glu(OtBu)-Ala-Tyr(tBu)-Lys(Boc)-resin, substitution degree: 0.17
mmol/g.
Activation of 3-maleimidopropanoic acid was carried out with DIPC
only (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. (E)-4,6-Heptadienoic acid was coupled in the same
conditions as amino acid residues (DIPC + HOBt). Both the
diene−acid and 3-maleimidopropanoic acid were coupled to the
peptide by using 10 equiv of the appropriate reagents.
Deprotection of N-terminal maleimido−peptides and cleavage from
the resin was generally carried out by reaction with a v/v/v 95:2.5:2.5
TFA/H2O/TIS mixture (1−2 mL) for 2 h at room temperature. In
case of cysteine-containing peptides (such as conjugate 15), a v/v/v/v
94:2.5:1:2.5 TFA/H2O/TIS/EDT mixture (EDT = 1,2-ethanedithiol)
was used. Filtrate and washings (TFA, no scavengers) were collected
and concentrated by blowing N2 over the mixture, and diethyl ether
was added to the resulting oil or semisolid (3−5 mL). Water was
added to dissolve the peptide (2−3 mL), the biphasic mixture was
shaken, and the two phases were allowed to separate. The organic
phase was discarded, and new diethyl ether was added. This washing
procedure was repeated 3 times. The aqueous phase was then
lyophilized and the peptide analyzed and purified by HPLC.
succinimide ring) on the diene−maleimide cycloadducts, and to
our knowledge it has not been described.
The DA cycloaddition can be carried out both with
maleimides (many of which are commercially available) and
with protected maleimides using aqueous solutions of the
dienophile and even suspensions in water/DMSO mixtures.
Therefore, this methodology is not limited to hydrophilic
dienophiles. Use of protected maleimides broadens the scope of
possible targets and allows conjugates with different linking
sites to be obtained. In this case, deprotection and conjugation
take place simultaneously.
As to the resin-linked reagent, polyamides are assembled on a
hydrophilic solid matrix using standard Fmoc-protected
building blocks and synthesis procedures, with no sequence
restriction. The diene, which can be easily prepared in two
steps from cheap starting materials,57,58 is then incorporated at
the N-terminus. Since this is followed by conjugation, the diene
is never exposed to acids, and the diene−polyamide is not
purified. This is an advantage over solution convergent
syntheses, where additional time is required to purify the
diene-containing moiety prior to conjugation.
Double derivatization of polyamides can be easily carried out
by incorporating into the polyamide the diene and a building
block containing a functional group allowing for a second click
conjugation reaction, such as the Michael-type reaction. Since
the first conjugation takes place on the fully protected diene−
polyamide, a cysteine residue with a TFA-labile side chain
protecting group can be the thiol-providing unit. No thiol−
maleimide reaction will occur during the first conjugation
because the thiol remains masked, and the second conjugation
reaction can be performed after the TFA treatment. In a proof
of concept experiment, the on-resin DA cycloaddition was used
to attach a fluorophore to a peptide, and a Michael-type
reaction in solution linked the fluorophore−peptide conjugate
to a maleimido−PNA.
As a modular approach, the methodology here described can
easily provide libraries of polyamide conjugates, polyamides
derivatized with reporter groups, or both. It is finally worth
mentioning that since the DA reactions are conducted in
aqueous media, conjugation yields are good enough not to
require the addition of metal catalysts, thus precluding the need
of assessing their complete absence in the target conjugate.
EXPERIMENTAL SECTION
■
General Materials and Methods. Acid-free DCM (dichloro-
methane) was obtained by filtration through basic alumina. Ethyl (E)-
hepta-4,6-dienoate was synthesized as described in ref 57, (E)-hepta-
4,6-dienoic acid as described in ref 58, exo-(2,5-dimethylfuran)-
protected 3-maleimidoethylamine (TFA salt) as described in ref 43,
and exo-(2,5-dimethylfuran)-protected 3-maleimidoethanol and 3-
maleimidopropanoic acid as described in ref 38.
N-Terminal diene−peptide−resins were stored at 8 °C until
conjugation was effected.
PNA Synthesis. PNAs were assembled on the TentaGel R RAM
resin (derivatized with a Rink amide linker). Prior to chain elongation
the resin was washed with DCM (3×), DMF (3×), MeOH (3×), and
DCM (3×), treated with 20% piperidine/DMF (1 × 3 min + 1 × 15
min), and washed with DCM (3×), DMF (3×), and DCM (3×).
Incorporation of the PNA monomers was effected by reaction with 4
equiv of both the Fmoc monomer and COMU (1-[(1-(cyano-2-
ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethyl-
ene)] methanaminium hexafluorophosphate) and 8 equiv of DIPEA
(N,N-diisopropylethylamine), all dissolved in a minimal amount of
NMP. This mixture was preactivated for 1 min before being poured
onto the resin and allowed to react for 90 min at rt. Coupling was
followed by washing with DCM, DMF, and MeOH (3 × each). In case
the coupling was not complete, as assessed by the Kaiser test, it was
repeated as before. The resin was always allowed to swell in DCM for
2 min before Fmoc removal. Removal of the Fmoc groups was
performed by reaction with 20% piperidine/DMF (1 × 3 min + 1 × 10
min), followed by washing with DCM (3×), DMF (3×), and DCM
MALDI-TOF mass spectra were recorded using reflector, unless
otherwise indicated. The THAP (2,4,6-trihydroxyacetophenone)
matrix was prepared by dissolving 10 mg of THAP in 1 mL of a 1:1
(v/v) H2O/ACN mixture (ACN = acetonitrile). The DHB (2,5-
dihydroxybenzoic acid) matrix was prepared by dissolving 10 mg of
DHB in 1 mL of a 1:1 (v/v) H2O/ACN mixture containing 0.1% of
TFA.
Polyamide Synthesis. Polyamides were manually assembled in a
polyethylene syringe fitted with a polypropylene disk. The DCM used
in polyamide synthesis was acid free (passed through a basic alumina
column).
Peptide Synthesis. Peptides were assembled on the NovaSyn TGR
resin derivatized with a Rink amide linker, which was washed with
DCM (3×), DMF (N,N-dimethylformamide, 3×), MeOH (3×), and
6099
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
(3×). A capping step was carried out after incorporation of each
monomer, as described above, to ensure that no trace of free amines
was left. After the polyamide chain was elongated, the substitution
degree was determined by taking a carefully weighed aliquot, removing
the Fmoc group with piperidine and quantitating the amount of 9-
fluorenylmethylpiperidine formed (λmax = 301 nm, ε = 7800).60 This
value was taken as the substitution degree of the resin-linked diene−
polyamides in conjugation experiments (see below), without any
further correction for the addition of the diene (or the diene and one
glycine as in 7a), and used to determine the overall synthesis and
purification yield for the isolated conjugates. Fmoc-t-t-Lys(Boc)-resin,
substitution degree: 0.17 mmol/g. Fmoc-c(Bhoc)-a(Bhoc)-t-a(Bhoc)-
g(Bhoc)-c(Bhoc)-g(Bhoc)-t-t-t-c(Bhoc)-resin, substitution degree:
0.073 mmol/g.
Activation of 3-maleimidopropanoic acid and (E)-4,6-heptadienoic
acid was effected as for the synthesis of peptides. Storage of the diene−
PNA−resins and cleavage of the maleimido−PNAs were also the same
as for the peptide−resins.
exo-(2,5-Dimethylfuran)-Protected Biotin−Maleimide (11). Bio-
tin−NHS ester (NHS = N-hydroxysuccinimide) (154 mg, 0.49 mmol)
was dissolved in DMF (2 mL), and exo-(2,5-dimethylfuran)-protected
3-maleimidoethylamine·TFA (207 mg, 0.59 mmol, 1.2 equiv) was
added. After complete dissolution of all products triethylamine (83 μL,
0.59 mmol, 1.2 equiv) was added and the mixture stirred at 40 °C for
18 h. The solvents were evaporated in vacuo, and the crude product
was washed twice with DCM (HPLC quality) to obtain a white
powder solid (104 mg, 45%).
three or four times over the reaction course. The Eppendorf tube was
placed in a sand bath heated at the desired temperature, and the
reaction was allowed to proceed with no additional stirring.
After the appropriate reaction time (see Tables 1 and 2), the
Eppendorf content was filtered through a 2 mL syringe equipped with
a filter, and the resin was washed with H2O (3×), ACN (3×), DMF
(3×), H2O (3×), ACN (3×), and DCM (3×) and allowed to air dry.
In the case of sparingly soluble maleimide-containing reagents (biotin
and dansyl derivatives), 0.1% TFA was added to the H2O and ACN
washings to increase their solubilizing properties. After washing the
resin, the conjugate was cleaved as described in the polyamide
synthesis section, then analyzed by HPLC, and characterized by
MALDI-TOF mass spectrometry (see Supporting Information for
yields and HPLC profiles of the crudes, section 4). Yields are also
indicated in Tables 1 and 2. In some cases (see tables) conjugates were
purified (conditions and HPLC traces of purified products are shown
in the Supporting Information).
Additional Data for Conjugation Experiments Reported in
Tables 1 and 2. 1 + 5 (Table 1, entry 1): 8.5 mg of 5, 106 μL of
H2O. 1 + 5 (Table 1, entry 2): 8.1 mg of 5, 101 μL of 1:1 H2O/
DMSO. 1 + 5 (Table 1, entry 3): 6.7 mg of 5, 83 μL of H2O. 1 + 5
(Table 1, entry 4): 5.6 mg of 5, 70 μL of H2O. 2 + 5 (Table 1, entry
5): 7.7 mg of 5, 97 μL of H2O. 3 + 6 (Table 1, entry 6): 2.8 mg of 6,
36 μL of H2O. 4 + 7 (Table 1, entry 7): 5.1 mg of 7, 47 μL of H2O. 8
+ 5 (Table 2, entry 1): 6.1 mg of 5, 80 μL of H2O. 9 + 7a (Table 2,
entry 2): 16.5 mg of 7a, 151 μL of H2O. 10 + 7a (Table 2, entry 3):
2.7 mg of 7a, 25 μL of H2O. 11 + 5 (Table 2, entry 4): 5.3 mg of 5, 66
μL of H2O. 11 + 7 (Table 2, entry 5): 5.6 mg of 7, 51 μL of H2O. 11 +
7a (Table 2, entry 6): 7.4 mg of 7a, 67 μL of H2O. 12 + 5 (Table 2,
entry 7): 12.0 mg of 5, 150 μL of H2O. 12 + 7a (Table 2, entry 8):
16.5 mg of 7a, 150 μL of H2O.
Double-Conjugation Experiments (Scheme 3). 13 + 14:
Commercially available fluorophore 14 (3.7 mg, 6 μmol, taking into
account that it was 90% pure) was suspended in 150 μL of a 65:35 (v/
v) H2O/DMSO mixture, and this suspension was poured into an
Eppendorf tube containing diene−peptide−resin 13 (12.0 mg, 2 μmol
of diene−peptide). The cycloaddition reaction was carried out at 65
°C for 24 h, after which time the resin was filtered and washed as
indicated above. Deprotection and cleavage was performed by reaction
with a 94:2.5:1:2.5 mixture of TFA/H2O/TIS/EDT for 2 h at room
temperature, and filtrates and washings were collected and treated as
indicated above (peptide synthesis section) to yield crude 15. Crude
15 was purified by HPLC and characterized by MALDI-TOF MS.
15 + 16: Maleimido−PNA 16 (1 μmol, 1.3 equiv) and compound
15 (0.8 μmol) were reacted in H2O (200 μL; maleimido−PNA
concentration: 4 mM) for 2.5 h at 37 °C. The target conjugate (17)
was purified by HPLC (0.47 μmol of pure 17 was isolated) and
characterized by MALDI-TOF MS (see Supporting Information).
Overall yield (from Fmoc−peptide−resin, peptide derivatization with
the diene, two conjugation reactions, and two purification steps): 23%.
1H NMR (400 MHz, DMSO-d6) δ: 7.78 (t, J = 5.9 Hz, 1H), 6.41 (s,
1H), 6.36 (s, 2H), 6.35 (s, 1H), 4.30 (m, 1H), 4.13 (m, 1H), 3.40 (t, J
= 6.3 Hz, 2H), 3.16 (q, J = 6.1 Hz, 2H), 3.12−3.06 (m, 1H), 2.86 (s,
2H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 1.99 (t,
J = 7.4 Hz, 2H), 1.64−1.56 (m, 1H), 1.53 (s, 6H), 1.50−1.39 (m, 3H),
1.35−1.22 ppm (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 174.8,
172.2, 162.7, 140.6, 86.9, 61.0, 59.2, 55.4, 52.2, 39.8, 37.7, 35.9, 35.1,
28.2, 28.0, 25.0, 15.7 ppm. HRMS (ESI, positive mode): M calcd for
C22H30N4O5S 462.1937, m/z found 463.2000 [M + H]+, 485.1815 [M
+ Na]+.
exo-(2,5-Dimethylfuran)-Protected Dansyl−Maleimide (12). Dan-
syl chloride (31 mg, 0.115 mmol, 1.33 equiv) was dissolved in DMF
(6.5 mL), and DIPEA (38 μL, 0.215 mmol, 2.5 equiv) and exo-(2,5-
dimethylfuran)-protected 3-maleimidoethylamine·TFA (30 mg, 0.086
mmol) were added. The reaction mixture was stirred for 1 h at rt and
then taken to dryness in vacuo. DCM (20 mL) was added to dissolve
the resulting oil, and this organic phase was washed with 1% aq HCl (3
× 15 mL). The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated in vacuo (33 mg, 82%). The product was
used without further purification.
1H NMR (400 MHz, CDCl3) δ: 8.51 (d, J = 8.5 Hz, 1H), 8.21 (dd,
J = 12.9, 8.6 Hz, 2H), 7.52 (m, 2H), 7.16 (d, J = 7.5 Hz, 1H), 6.25 (s,
2H), 5.29−5.26 (m, 1H), 3.57−3.51 (m, 2H), 3.13 (q, J = 6.0 Hz,
2H), 2.86 (s, 6H), 2.55 (s, 2H), 1.62 ppm (s, 6H). 13C NMR (101
MHz, CDCl3) δ: 187.2, 175.0, 151.9, 140.7, 134.4, 130.4, 129.8, 129.4,
128.4, 123.3, 119.0, 115.2, 87.6, 52.4, 45.4, 41.1, 38.1, 15.8 ppm
(impurities detected at 1.26 ppm in the 1H NMR correlate with
impurities at 29.7 and 21.8 ppm as seen by gHSQC). HRMS (ESI,
positive mode): M calcd for C24H27N3O5S 469.1671, m/z found
470.1745 [M + H]+, 939.3406 [2M + H]+.
Conjugation. General Procedure. The appropriate amount (2−
17 mg, see below) of resin-linked, fully protected diene−polyamide
(PNA or peptide) was carefully weighed into a 500 μL Eppendorf tube
(the μmoles of diene were calculated on the basis of the weigh and
substitution degree of the polyamide−resin, see above). The
maleimide-containing reactant was dissolved (or suspended in the
solvent), poured into the Eppendorf, and allowed to react (solvent,
reaction time, temperature, molar excess, and concentration of the
reagent for each particular case are indicated in Tables 1 and 2 and
summarized also in the Supporting Information, Table S1, page S7).
The Eppendorf tube was sonicated immediately after the addition of
the dienophile to ensure homogeneity of the mixture (until it was
observed that all beads were covered by the maleimide solution) and
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC and mass spectrometric characterization data of the
different compounds (polyamides and conjugates), assessment
of the diene stability to polyamide deprotection conditions,
results of the conjugation assays, and 1H and 13C NMR spectra
of compounds 11 and 12. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.joc.5b00592.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: anna.grandas@ub.edu
Notes
The authors declare no competing financial interest.
6100
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101

The Journal of Organic Chemistry
Article
(33) Schooch, J.; Wiessler, M.; Jaschke, A. J. Am. Chem. Soc. 2010,
̈
ACKNOWLEDGMENTS
■
132, 8846−8847.
This work was supported by funds from the Ministerio de
(34) Lang, K. L.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.;
Chin, J. W. Nat. Chem. 2012, 4, 298−304.
́
Economia y Competitividad (grants CTQ2010-21567-C02-01
and CTQ2014-52658-R, and the project RNAREG, grant
CSD2009-00080, funded under the programme CONSOL-
IDER INGENIO 2010) and the Generalitat de Catalunya
(2009SGR-208). O.B. and X.E. were recipient fellows of the
MINECO.
(35) Lukinavicius, G.; et al. Nat. Chem. 2013, 5, 132−139.
(36) Wu, H.; Yang, J.; Seckute, J.; Devaraj, N. K. Angew. Chem., Int.
Ed. 2014, 53, 5805−5809.
(37) Kwart, H.; King, K. Chem. Rev. 1968, 68, 415−447.
́
(38) Sanchez, A.; Pedroso, E.; Grandas, A. Org. Lett. 13, 4364−4367.
(39) Blaskovich, M. A.; Kahn, M. J. Org. Chem. 1998, 63, 1119−1125.
(40) Ogbu, C. O.; et al. Bioorg. Med. Chem. Lett. 1998, 8, 2321−2326.
(41) Hoogewijs, K.; Deceuninck, A.; Madder, A. Org. Biomol. Chem.
2012, 10, 3999−4002.
REFERENCES
■
(1) Kalia, J.; Raines, R. T. Curr. Org. Chem. 2010, 14, 138−147.
(2) Weltman, J. K.; Szaro, R. P.; Frackelton, A. R., Jr.; Dowben, R.
M.; Bunting, J. R.; Cathou, R. E. J. Biol. Chem. 1973, 248, 3173−3177.
(3) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2005−2021.
(4) Alley, S. C.; Benjamin, D. R.; Jeffrey, S. C.; Okeley, N. M.; Meyer,
D. L.; Sanderson, R. J.; Senter, P. D. Bioconjugate Chem. 2008, 19,
759−765.
(5) Baldwin, A.; Kiick, K. L. Bioconjugate Chem. 2011, 22, 1946−
1953.
(42) Hoogewijs, K.; Buyst, D.; Winne, J. M.; Martins, J. C.; Madder,
A. Chem. Commun. 2013, 2927−2929.
́
(43) Elduque, X.; Sanchez, A.; Sharma, K.; Pedroso, E.; Grandas, A.
Bioconjugate Chem. 2013, 24, 832−839.
(44) Yli-Kauhaluoma, J. Tetrahedron 2001, 57, 7053−7071.
(45) Graven, A.; Meldal, M. J. Chem. Soc., Perkin Trans I 2001, 3198−
3203.
(46) Braasch, D. A.; Nulf, C. J.; Corey, D. A. Synthesis and
purification of peptide nucleic acids. Curr. Protoc. Nucleic Acid
Chemistry 2002, 9:4.11.1−4.11.18.
(6) Shen, B.-Q.; et al. Nat. Biotecnol. 2012, 30, 184−189.
(7) Strop, P.; et al. Chem. Biol. 2013, 20, 161−167.
(8) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057−3064.
(47) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35,
161−224.
(48) Good, L.; Awasthi, S. K.; Dryselius, R.; Larsson, O.; Nielsen, P.
E. Nat. Biotechnol. 2001, 19, 360−364.
(9) Rostovsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, B. K.
Angew. Chem., Int. Ed. 2002, 41, 2596−2599.
(49) Wang, Y.-S.; Youngster, S.; Grace, M.; Bausch, J.; Bordens, R.;
Wyss, D. F. Adv. Drug Delivery Rev. 2002, 54, 547−570.
(50) Dhalluin, C.; Ross, A.; Huber, W.; Gerber, P.; Brugger, D.; Gsell,
B.; Senn, H. Bioconjugate Chem. 2005, 16, 518−527.
(51) Putta, M. R.; Zhu, F.-G.; Wang, D.; Bhagat, L.; Dai, M.;
Kandimalla, E. R.; Agrawal, S. Bioconjugate Chem. 2010, 21, 39−45.
(10) Chan, T. R.; Hilgraf, R.; Sharpless, K: B.; Fokin, V. V. Org. Lett.
2004, 6, 2853−2855.
(11) Jayaprakash, K. N.; Peng, C. G.; Butler, D.; Varghese, J. P.;
Maier, M. A.; Rajeev, K. G.; Manoharan, M. Org. Lett. 2010, 12, 5410−
5413.
(12) Wenska, M.; Alvira, M.; Steunenberg, P.; Stenberg, Å.; Murtola,
́
(52) Monso, M.; de la Torre, B. G.; Blanco, E.; Moreno, N.; Andreu,
M.; Stromberg, R. Nucleic Acids Res. 2011, 39, 9047−9059.
̈
D. Bioconjugate Chem. 2013, 24, 578−585.
(13) Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108, 2952−3015.
(14) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int.
Ed. 2009, 48, 9879−9883.
(53) Elduque, X.; Pedroso, E.; Grandas, A. J. Org. Chem. 2014, 79,
2843−2853.
(54) Nielsen, P. E. Chem. Biodivers. 2010, 7, 786−804.
(55) Pourceau, G.; Meyer, A.; Vasseur, J.-J.; Morvan, F. J. Org. Chem.
2009, 74, 6837−6842.
(15) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046−15047.
(16) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.;
Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 16793−16797.
(17) Martin, J. G.; Hill, R. K. Chem. Rev. 1961, 61, 537−562.
(18) Rideout, D. C.; Breslow, R. J. Am. Chem. Soc. 1980, 102, 7817−
7818.
(56) Singh, I.; Freeman, C.; Heaney, F. Eur. J. Org. Chem. 2011,
6739−6746.
(57) Willems, L. I.; Verdoes, M.; Florea, B. I.; van der Marel, G. A.;
Overkleeft, H. S. ChemBioChem 2010, 11, 1769−1781.
(58) Baillie, L. C.; Batsanov, A.; Bearder, J. R.; Whiting, D. A. J.
Chem. Soc., Perkin Trans. 1 1998, 3471−3478.
(59) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595−598.
(19) Otto, S.; Engberts, J. B. F. N. Org. Biomol. Chem. 2003, 1, 2809−
2820.
(20) Pirrung, M. C. Chem.Eur. J. 2006, 12, 1312−1317.
(60) Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.;
Makofske, R. C.; Chang, C.-D. Int. J. Pept. Protein Res. 1979, 13, 35−
42.
(21) Seelig, B.; Jaschke, A. Tetrahedron Lett. 1997, 38, 7729−7732.
̈
(22) Hill, K. W.; et al. J. Org. Chem. 2001, 66, 5352−5358.
(23) Pozsgay, V.; Vieira, N. E.; Yergey, A. Org. Lett. 2002, 4, 3191−
3194.
(24) Fruk, L.; Grondin, A.; Smith, W. E.; Graham, D. Chem.
Commun. 2002, 2100−2101.
(25) Tona, R.; Haner, R. Chem. Commun. 2004, 1908−1909.
̈
(26) Dantas de Araujo, A.; Palomo, J. M.; Cramer, J.; Seitz, O.;
́
Alexandrov, K.; Waldmann, H. Chem.Eur. J. 2006, 12, 6095−6109.
́
(27) Marchan, V.; Ortega, S.; Pulido, D.; Pedroso, E.; Grandas, A.
Nucleic Acids Res. 2006, 34, e24.
(28) Steven, V.; Graham, D. Org. Biomol. Chem. 2008, 6, 3781−3787.
(29) Borsenberger, V.; Howorka, S. Nucleic Acids Res. 2009, 37,
1477−1485.
(30) Li, Q.; Dong, T.; Liu, X.; Lei, X. J. Am. Chem. Soc. 2013, 135,
4996−4999.
(31) Samoshin, A. V.; Hawker, C. J.; Read de Alaniz, J. ACS Macro
Lett. 2014, 3, 753−757.
(32) Jouanno, L.-A.; Chevalier, A.; Sekkat, M.; Perzo, N.; Castel, H.;
Romieu, A.; Lange, N.; Sabot, C.; Renard, P.-Y. J. Org. Chem. 2014, 79,
10353−10366.
6101
DOI: 10.1021/acs.joc.5b00592
J. Org. Chem. 2015, 80, 6093−6101