A "catch-and-release" protocol for alkyne-tagged molecules based on a resin-bound cobalt complex for peptide enrichment in aqueous media

Article abstract of DOI:10.1002/chem.201400056

The development of new and mild protocols for the specific enrichment of biomolecules is of significant interest from the perspective of chemical biology. A cobalt-phosphine complex immobilised on a solid-phase resin has been found to selectively bind to a propargyl carbamate tag, that is, "catch", under dilute aqueous conditions (pH 7) at 4-°C. Upon acidic treatment of the resulting resin-bound alkyne-cobalt complex, the Nicholas reaction was induced to "release" the alkyne-tagged molecule from the resin as a free amine. Model studies revealed that selective enrichment of the alkyne-tagged molecule could be achieved with high efficiency at 4-°C. The proof-of-concept was applied to an alkyne-tagged amino acid and dipeptide. Studies using an alkyne-tagged dipeptide proved that this protocol is compatible with various amino acids bearing a range of functionalities in the side-chain. In addition, selective enrichment and detection of an amine derived from the "catch and release" of an alkyne-tagged dipeptide in the presence of various peptides has been accomplished under highly dilute conditions, as determined by mass spectrometry. Catch and release: The specific enrichment of alkyne-tagged molecules has been achieved by using cobalt beads. A cobalt complex bound to a resin was found to selectively "catch" a propargyl carbamate tag under dilute aqueous conditions (see scheme). Upon acidic treatment of the alkyne-cobalt complex, the Nicholas reaction was induced to "release" an amine. Studies using an alkyne-tagged dipeptide proved its compatibility with various amino acids and peptides under highly dilute conditions.

Full text of DOI:10.1002/chem.201400056

DOI: 10.1002/chem.201400056
Full Paper
&
Peptides
A “Catch-and-Release” Protocol for Alkyne-Tagged Molecules
Based on a Resin-Bound Cobalt Complex for Peptide Enrichment
in Aqueous Media
Ayako Miyazaki,[a] Miwako Asanuma,[a] Kosuke Dodo,[a] Hiromichi Egami,[a] and
Mikiko Sodeoka*[a, b]
Abstract: The development of new and mild protocols for
the specific enrichment of biomolecules is of significant in-
terest from the perspective of chemical biology. A cobalt–
phosphine complex immobilised on a solid-phase resin has
been found to selectively bind to a propargyl carbamate
tag, that is, “catch”, under dilute aqueous conditions (pH 7)
at 48C. Upon acidic treatment of the resulting resin-bound
alkyne–cobalt complex, the Nicholas reaction was induced
to “release” the alkyne-tagged molecule from the resin as
a free amine. Model studies revealed that selective enrich-
ment of the alkyne-tagged molecule could be achieved with
high efficiency at 48C. The proof-of-concept was applied to
an alkyne-tagged amino acid and dipeptide. Studies using
an alkyne-tagged dipeptide proved that this protocol is
compatible with various amino acids bearing a range of
functionalities in the side-chain. In addition, selective enrich-
ment and detection of an amine derived from the “catch
and release” of an alkyne-tagged dipeptide in the presence
of various peptides has been accomplished under highly
dilute conditions, as determined by mass spectrometry.
Introduction
Considerable research has been carried out in this field to
overcome the harsh, denaturing and destructing conditions
that are usually required to disrupt the strong biotin–avidin in-
teraction and “release” the molecule from the beads.[4] For ex-
ample, various types of cleavable linkers between the biotin
tag and the site of attachment on the probe molecule, such as
disulfide,[5] diazobenzene,[6] hydrazone,[7] protease-labile,[8] acid/
base-labile[9] and photolabile[10] cleavable linker systems, have
been introduced (Scheme 1b). On the other hand, the reduc-
tion in bioactivity and membrane permeability of bioactive
molecules arising from the attachment of biotin to a larger
linker has been overcome by the development of click reac-
tions, such as the Sharpless–Meldal reaction (Scheme 1c).[11]
The incorporation of cleavable linkers circumvents the issue of
having to use harsh conditions to disrupt the biotin–avidin in-
teraction after the click reaction (Scheme 1d). Nevertheless, as
these procedures involve multiple steps, further development
of a direct, mild and selective method for the efficient catch
and release of biomolecules is of significant interest from
a chemical biology perspective.
The selective enrichment of biomolecules is important in
chemical proteomics and pharmaceutical drug research to elu-
cidate small molecule–protein interactions and to gain a better
understanding of biological processes.[1] In particular, the
“catch-and-release” approach for the specific enrichment of
a biomolecule from complex mixtures, for example, cell lysates,
is a widely used technique that has found many applications
in chemical biology.[2] Affinity purification, such as the use of
avidin or streptavidin beads to “catch” biotin-labelled mole-
cules has been used in many proteomic studies to identify the
binding site of bioactive molecules in proteins (Scheme 1a).[3]
[a] Dr. A. Miyazaki,+ Dr. M. Asanuma, Dr. K. Dodo, Dr. H. Egami,++
Prof. Dr. M. Sodeoka
Sodeoka Live Cell Chemistry Project
ERATO, Japan Science and Technology Agency (JST)
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
and Synthetic Organic Chemistry Laboratory, RIKEN
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
[b] Prof. Dr. M. Sodeoka
Recent advances in click chemistry have led to the recogni-
tion of the alkyne moiety as a useful bio-orthogonal tag due
to its selective reactivity towards specific functional groups,
along with minimal structural perturbation upon introduction
into a bioactive molecule. One of the most prominent exam-
ples is the copper-mediated Huisgen cycloaddition reaction
between an azide and an alkyne, which has led to the success-
ful installation of various functionalities, such as fluorophores
and biotin.[11] Alkynes are also known to selectively react with
octacarbonyldicobalt(0), [Co2(CO)8], to form stable alkyne(hexa-
RIKEN Center for Sustainable Resource Science
2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)
E-mail: sodeoka@riken.jp
[+] Current address: Institute of Transformative
Bio-Molecules (WPI-ITbM), Nagoya University
Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan)
++] Current address: School of Pharmaceutical Sciences
University of Shizuoka, 52-1 Yada, Suruga-ku
Shizuoka 422-8526 (Japan)
[
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201400056.
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ried out in organic solvents on
a millimolar scale and Fe(NO3)3
was used as an oxidant to re-
lease the alkyne-derivatised
phospholipid from the cobalt–
phosphine
resin.
Covering
a broad range of biomolecules,
we have been independently in-
vestigating the development of
a mild and non-oxidative catch-
and-release protocol based on
alkyne–cobalt chemistry and re-
ported the preliminary results of
model studies in 2011.[19] Herein
we report full details of the de-
velopment of an efficient and
mild catch-and-release method
for the enrichment and detec-
tion of alkyne-tagged molecules
by using a polymer-supported
cobalt complex, which allows re-
actions to be carried out in
Scheme 1. Common strategies for catch-and-release reactions using the biotin–avidin system.
dilute
aqueous
media
(Scheme 3).
carbonyl)dicobalt complexes upon loss of two carbonyl li-
Results and Discussion
gands, and this chemistry has been extensively studied since it
was first reported over 50 years ago. The broad scope of
alkyne–cobalt chemistry is demonstrated by the vast number
of reports on alkyne protection, the Nicholas reaction,[12,13]
which makes use of the stabilised propargylic cation, and the
Pauson–Khand reaction, which yields cyclopentenone
rings,[14,15] as well as the activity of the complex as an anti-
tumour agent.[16] Cobalt–alkyne complexes have been mainly
studied in the solution phase with organic solvents, with its re-
activity in the solid phase first being reported by Gibson and
co-workers in 1999.[17] A polymer-bound triphenylphosphine
moiety was used to immobilise an alkyne–cobalt complex in
THF at 508C. A hydroxy moiety
To establish a method for the selective enrichment of alkyne-
tagged biomolecules, for example, peptides and proteins,
catch-and-release reactions that are highly selective with re-
spect to the alkyne are required. The reactions should be car-
ried out under mild conditions in aqueous media at low molar
concentrations and low temperature, as well as be compatible
with a wide range of functional groups. Thus, we envisioned
that the use of an alkyne(carbonyl)cobalt complex as a key in-
termediate would meet the above criteria. It is well known
that [Co2(CO)8] and its phosphine complex react readily and se-
lectively with alkynes, and the resulting complexes are usually
on the alkyne was acylated on
the resin and then the alkyne
was released under oxidative
conditions (Scheme 2a). In addi-
tion, an enyne was subjected to
the Pauson–Khand reaction on
a phosphine-linked resin to gen-
erate a bicyclic cyclopentenone
(Scheme 2b).
In 2010, Brown and co-work-
ers reported the purification of
an alkyne-modified phospholipid
by using [Co2(CO)8] and phos-
phine-functionalised silica gel via
the formation of an alkyne–
cobalt complex in methanol/
chloroform at a high tempera-
Scheme 2. Organic transformations of a polymer-bound alkyne(carbonyl)cobalt(0)–phosphine complex in organic
ture.[18] The reactions were car- solvents.
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reaction of alkyne 1 with [Co2(CO)7(PPh3)] in THF at room tem-
perature afforded alkyne–cobalt complex 4.
The stability and reactivity of alkyne–cobalt complex 4 in
aqueous media were monitored by HPLC with UV detection at
280 nm. Upon treatment of cobalt complex 4 (50 mm) with 5%
trifluoroacetic acid (TFA) in 5% MeCN/H2O, the Nicholas reac-
tion proceeded smoothly to provide the corresponding amine
2 in 98% yield at 48C (Scheme 5). In the absence of TFA, com-
plex 4 appeared to be stable under neutral aqueous condi-
Scheme 3. Catch-and-release protocol in aqueous media at high dilution
based on alkyne–cobalt chemistry.[19]
bench-stable at ambient temper-
ature in the absence of strong
oxidising agents. So far, the ma-
jority of the studies conducted
on alkyne–cobalt complexes
have been in organic solvents,
and information on their proper-
ties in aqueous media has been
rather limited. After investigating
the reactivity and stability of
cobalt complexes in water, we
decided to study a polymer-sup-
Scheme 5. Reactivity of cobalt complex 4.
ported cobalt complex and
a propargyl carbamate tag, an-
ticipating that it would form the polymer-supported alkyne–
cobalt complex A linked by a phosphine ligand (Scheme 3). In
the presence of a Brønsted acid, the bound molecule is expect-
ed to be released as an amine derivative through the Nicholas
reaction.[20] By prior coordination of a phosphine resin to
cobalt, we envisaged that the stabilised cobalt–phosphine
resin would lead to efficient alkyne–cobalt complexation under
aqueous conditions. Release by a Brønsted acid overcomes the
issue of having to use oxidative conditions to decomplex the
cobalt moiety, which have a tendency to cause damage to cer-
tain amino acid residues, such as the sulfur-containing cysteine
moieties in peptides. Furthermore, many of the nucleophilic
functionalities that are present in amino acid side-chains are
known to be relatively stable under acidic conditions.
tions. Other additives, such as tetrabutylammonium fluoride,[21]
ethylenediamine,[22] 2-aminoethanol[22] and cyclohexylamine,[23]
which are known to decomplex alkyne–cobalt complexes in or-
ganic solvents, showed diminished decomplexation ability with
complex 4 in water. When free alkyne 1 was treated with TFA,
no reaction was observed, which indicates that amine 2 was
indeed obtained by the Nicholas reaction from complex 4. The
Nicholas reaction is expected to selectively cleave the CꢀO
bond at the a position of the alkyne(phosphine)cobalt com-
plex. The cobalt stabilises the positive charge at the a position,
which is subsequently captured by a water molecule. This is
supported by the observation that upon treatment of complex
4 with TFA, LC-MS revealed the formation and gradual decom-
plexation of cobalt complex 5, which is the other product aris-
ing from the Nicholas reaction.
Thus, we prepared dansyl derivative 1 bearing a triethylene
glycol linked propargyl carbamate tag as a model substrate
(Scheme 4). The propargyl carbamate tag was introduced into
amine 2 by using the activated carbonate 3, and subsequent
Encouraged by these results, we next attempted to prepare
the polymer-supported cobalt complex in the solution phase.
We employed the TentaGel PS-PEG (polystyrene-polyethylene
glycol) resin as it has been used as a polymer support for cata-
lysts in aqueous media and is also compatible with organic sol-
vents due to its good swelling ability. TentaGelꢁ-supported
phosphine 6 was synthesised according to a reported proce-
dure,[24] and subsequently the brown phosphine–cobalt com-
plex 7a was prepared by mixing 6 and [Co2(CO)8] in THF at
room temperature.[17c,19] The reaction of cobalt complex 7a
supported on TentaGel with alkyne 1 (2 mm) was carried out in
aqueous HEPES buffer (pH 7.0)[25] containing 0.5% Triton X-100.
As expected, the reaction proceeded to provide alkyne(phos-
phine)cobalt complex
8 after 6 h at room temperature
(Scheme 6). The cobalt–phosphine complex 7a and its alkyne
complex 8 were characterised by IR spectroscopy. The IR ab-
sorptions of the carbonyl groups of the polymer-supported
complexes 7a and 8 showed good correlation with those of
Scheme 4. Synthesis of the model dansyl substrate 1 and its cobalt com-
plex.
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good swelling properties, were also investigated. A phosphine
linker was attached to each resin and was subsequently treat-
ed with [Co2(CO)8] in accord with Scheme 6. Cobalt–phosphine
complexation was detected in most cases, as shown by similar
IR absorptions with respect to the TentaGelꢁ resin (see Figur-
es S4 and S5 in the Supporting Information). However, amine 2
was detected in low yields upon treatment of the alkyne–
cobalt resins 8 based on PEGA, Toyopearl and ChemMatrix
with 5% TFA (Table 1, entries 3–5). This may be attributed to
differences in the physical properties of the resins, such as
swelling ability, stability and compatibility with cobalt. In addi-
tion, according to HPLC, a significant amount of leaching of
resin material was observed after acidic treatment with other
resins, including CLEAR.[27] When glass beads (non-porous silica
microspheres) were used, cobalt–phosphine complexation re-
sulted in the beads having a purple appearance, which sug-
gests that oxidation of [Co2(CO)8] had occurred. Overall, Tenta-
Gel provided the most efficient and reproducible results for
catch-and-release and was used in further studies.[28]
Scheme 6. Synthesis of the alkyne–cobalt complex 8 supported on Tenta-
Gelꢁ PS-PEG in water.
[Co2(CO)7(PPh3)], the corresponding non-polymer-supported
complex 4 and similar known cobalt complexes (see Figure S4
in the Supporting Information).[17c,19,26]
The above results prompted us to investigate the catch-and-
release reaction of alkyne 1 on cobalt beads 7a at a low molar
concentration (50 nmol scale, 50 mm) and low temperature
(48C). The catch reaction was monitored by HPLC of the super-
natant after removal of the beads by centrifugation, and the
subsequent release reaction was separately monitored to
detect amine 2 (Table 1). Pleasingly, alkyne 1 reacted with Ten-
taGel-based cobalt beads 7a at low concentration and low
Further optimisation of the reaction conditions for catch-
and-release was carried out. When cobalt beads 7a were used
directly for alkyne complexation without any pre-washing of
the beads, varying amounts of amine (0–20%) were observed
after analysing the supernatant and washing solutions after
alkyne complexation. This is presumed to be due to the slight-
ly acidic environment arising from the presence of cobalt ions
on the resin, leading to release of the amine from the alkyne–
cobalt complex. Consequently, pre-washing of the beads after
the complexation of [Co2(CO)8] with the phosphine beads was
attempted, and it was found that the pre-washing of cobalt
beads 7a with HEPES buffer (pH 7.0) led to an improvement of
the alkyne catch to 66% (Table 1, entry 6). In addition, the
catch yield was improved to 73% when 1.25% EtOH/HEPES
buffer (pH 7.0) was used as solvent (Table 1, entry 7). Subse-
quent treatment of alkyne–cobalt beads 8 with 5% TFA led to
71% release of amine 2. In addition, to wash out non-specific
binding molecules more efficiently, the washing conditions fol-
lowing the formation of the alkyne–cobalt complex 8 were ex-
amined. Hardly any alkyne 1 was detected after the release re-
action with TFA when alkyne–cobalt beads 8 were washed
with 50% EtOH/HEPES buffer (pH 7.0) followed by 50% MeCN/
HEPES buffer and 5% MeCN/H2O after the catch reaction.
To examine the selectivity of the catch-and-release reactions,
a mixture of equimolar amounts of alkyne-tagged substrate
1 and a non-alkyne benzyl derivative 9 was treated with cobalt
resin 7a, pre-washed with HEPES buffer (Scheme 7).[29] Again,
77% of alkyne 1 was captured by the cobalt resin, with 96%
of the control compound 9 and 23% of alkyne 1 being recov-
ered in the supernatant and washing solution. Subsequent
treatment of the resin with 5% TFA generated the Nicholas
product 2 in a yield of 67%. Neither the benzyl derivative 9
nor alkyne 1 was detected after release. These results indicate
that the cobalt beads are highly selective towards the alkyne
molecule and that specific enrichment has been achieved.
With the success of the model substrate in hand, we decid-
ed to extend this methodology to biomolecules such as amino
acids and peptides. Alkyne-derivatised phenylalanine 10 and
Table 1. Results of the catch and release of alkyne 1 with various resins.[a]
Entry Resin
Catch of alkyne Release of
Recovery of
1 [%]
amine 2 [%]
alkyne 1 [%]
1
2
3
4
TentaGel 7a 57
CLEAR 7b 75
PEGA 7c 96
Toyopearl 79
7d
59
72
19
14
5
8
4
1
5
ChemMatrix 50
7e
6
0
6[b,c] TentaGel 7a 66
7[b,c,d] TentaGel 7a 73
70
71
0
0
[a] Standard procedure: cobalt beads 7 in alkyne 1 solution (50 mm) in
0.5% Triton X-100/HEPES buffer (pH 7.0) rotated at 48C for 12 h. After re-
moval of the supernatant and washing of the beads with 0.5% Triton X-
100/HEPES buffer (pH 7.0, 10 times), the beads were treated with 5% TFA
in 5% MeCN/H2O and rotated at 48C for 24 h. [b] Cobalt beads 7 pre-
washed with HEPES buffer (pH 7.0) before alkyne complexation (catch).
[c] Washing conditions after catch: 50% EtOH/HEPES buffer (three times),
50% MeCN/HEPES buffer (three times) and 5% MeCN/H2O (twice). [d]
Alkyne 1 solution (50 mm) in 1.25% EtOH/HEPES buffer (pH 7.0).
temperature, and 57% of the alkyne was complexed to the
cobalt resin after it had been washed 10 times with HEPES
buffer (pH 7.0) containing 0.5% Triton X-100. Finally, amine 2
was obtained in a yield of 59% after treatment of the resin
with 5% TFA for 24 h at 48C (Table 1, entry 1).
Other resins such as CLEAR (cross-linked ethoxylate acrylate
resin), PEGA (polyacrylamide-PEG resin), Toyopearl (methacrylic
polymer resin) and ChemMatrix (100% PEG-based resin), which
have been demonstrated to be hydrophilic and to exhibit
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Catch-and-release reactions
were performed with alkyne-
tagged amino acid 10 and di-
peptide 11 under the optimised
conditions. Amino acid 10 was
dissolved in HEPES buffer
(pH 7.0) containing 0.5% Triton
X-100. As a result of the hydro-
phobicity of dipeptide 11 and
thus its insolubility in HEPES
buffer containing 0.5–1% Triton
X-100, dipeptide 11 was dis-
solved in 30% EtOH/HEPES
buffer solution[30] and was used
as the catch solution. Catch was
performed by mixing each
alkyne solution (50 mm) with
cobalt beads 7a for 12 h at 48C.
After removing the supernatant
Scheme 7. Catch and release of an alkyne-tagged molecule 1 using TentaGelꢁ PS-PEG resin in the presence of
control compound 9.
lysine-phenylalanine dipeptide 11 were prepared according to
Scheme 8. To monitor the progress of the reactions by HPLC
with UV detection at 215 nm, substrates bearing CBz (Z)
groups were selected as substrates.
and washing the beads,[31] HPLC analysis of the supernatant
and washing solutions with UV detection at 215 nm indicated
that 79 and 81% of amino acid 10 and dipeptide 11, respec-
tively, had been bound to the cobalt beads (Scheme 9).[32]
Pleasingly, subsequent treat-
ment of alkyne–cobalt beads 17
and 18 with 5% TFA for 24 h at
48C followed by washing of the
beads resulted in release of
amines 13 and 16 in yields of
67 and 77%, respectively. Al-
kynes 10 and 11 were not ob-
served after acidic treatment of
the
corresponding
alkyne-
bound cobalt resins 17 and 18.
The catch-and-release reac-
tion of dipeptide 11 was also
monitored by mass spectrome-
try (Figure 1). Figure 1a shows
the mass spectrum of the initial
solution containing alkyne-
tagged dipeptide 11 ([M+H]+
at m/z=818.40 and [M+Na]+
at m/z=840.38) prior to cobalt
complexation. After complexa-
tion, alkyne–cobalt beads 18
were subsequently washed to
remove any non-complexed
molecules. Figure 1b shows the
mass spectrum of the sixth
washing of the cobalt beads
after the catch reaction, which
no longer contains any starting
alkyne 11. After treatment of
the alkyne–cobalt beads 18
with 5% TFA, the supernatant
was removed and the beads
were again washed with 50%
Scheme 8. Synthesis of alkyne-tagged amino acid 10 and dipeptide 11.
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plexation and almost quantita-
tive recovery of the following
derivatised amino acids (side-
chain functionality): N-Bz-alanine
19 (methyl), N-Z-phenylalanine
20 (phenyl), N-Ac-tryptophan 21
(indole), N-Bz-arginine 23 (guani-
dine),
N-Bz-asparagine
24
(amide), Na-Bz-lysine 25 (amine),
N-Bz-serine 26 (hydroxy), N-Z-ty-
rosine 27 (phenol) and N-Bz-me-
thionine 28 (thioether). Further-
more, the free carboxylic acid
group appeared to have no sig-
nificant effect on the reaction.
Approximately 10% loss of N-Bz-
histidine 22 (imidazole) was ob-
served, which suggests that
a small percentage of histidine
had coordinated to the cobalt
Scheme 9. Catch and release of amino acid 10 and dipeptide 11.
metal centre. Interestingly, N-Bz-
cysteine 29, which contains
a free thiol group, was not de-
tected in either the supernatant or the washing solution, even
after treatment of the resin with TFA. This suggests that the
free thiol had either been oxidised or had coordinated to the
cobalt centre.[33] Although N-Bz-cysteine 29 did not appear to
have a major effect on the outcome of the catch and release
of dipeptide 11 on a 50 mm scale, protection of the thiol
moiety prior to the reaction would be desirable to prevent any
transformation or uptake by cobalt. In biological experiments,
cysteine residues in peptides are usually alkylated with alkylat-
ing agents, such as iodoacetamide, to prevent oxidation of the
thiol and formation of disulfide bonds. Thus, the catch and re-
lease of dipeptide 11 was carried out in the presence of N-Bz-
S-carbamoylmethyl-cysteine 30. Good recovery of the thiol-
protected derivative 30 was observed after the catch reaction,
which indicates that capping of free thiols is effective in pre-
venting any potential side-reactions and thus shows compati-
bility with cobalt. Upon treatment of the alkyne–cobalt resin
18 with 5% TFA, amine 16 was successfully detected in good
yield in the presence of all the amino acids that were investi-
gated. Overall, these results suggest that this catch-and-release
protocol is compatible with a range of functionalities that are
present in peptides. Apart from cysteine residues containing
the thiol group, amino acid residues containing oxygen and ni-
trogen functionalities that can potentially coordinate to cobalt
appeared to have almost no or minimal effect on the cobalt
beads. Sulfur functionalities were found to be nearly unaffect-
ed under the catch-and-release conditions when they are pres-
ent as thioethers.
Figure 1. Mass spectra of solutions from the catch and release of dipeptide
11 (50 mm). a) Initial solution with alkyne-tagged dipeptide 11 (50 mm) prior
to cobalt complexation (catch). b) Sixth washing solution of cobalt beads 18
after catch. c) First washing solution after TFA treatment. The mass spectra
were recorded with a LTQ Orbitrap XL spectrometer.
MeCN/H2O to provide amine 16 ([M+H]+ at m/z=736.39) as
shown in Figure 1c.
The compatibility of this catch-and-release protocol with var-
ious amino acids containing a range of functional groups in
their side-chain was then investigated by using dipeptide 11
(Table 2). To easily detect the compounds by HPLC, N-deriva-
tised (benzoyl (Bz), CBz (Z) or acyl (Ac)) amino acids were used
to investigate their tolerability. Dipeptide 11 (50 mm) was
mixed with an equimolar amount of each derivatised amino
acid (50 mm) in 30% EtOH/HEPES buffer. Cobalt beads 7a were
added to the solution, which was rotated, so as not to damage
the beads, at 48C for 12 h. HPLC analysis of the supernatant
and washing solutions showed good degrees of alkyne com-
The scope and practicality of this reaction were then ex-
plored by carrying out the catch and release of dipeptide 11 in
the presence of various peptides at low molar concentrations.
A solution of alkyne-tagged dipeptide 11 (1.25 mm, 1.25 nmol)
and Waters MassPREP peptide mixture containing approxi-
mately 0.4 mm (ca. 400 pmol) of nine peptides (RASG-1 (A), an-
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Table 2. Compatibility of the catch and release of dipeptide 11 with vari-
ous amino acids.
Table 3. Peptide sequences for the peptides used to investigate the
scope of catch-and-release reactions with dipeptide 11.
Entry Amino acid
(functional group)
Catch of
alkyne 11
[%]
Recovery of
amino acid
[%]
Release of
amine 16
[%]
Peptide
Sequence
RASG-1[a]
A
B
C
D
E
F
G
H
I
RGDSPASSKP
DRVYIHP
angiotensin frag. 1–7[a,b]
bradykinin[a,b]
angiotensin II[a,b]
angiotensin I[a,b]
renin substrate[a]
enolase T35[a]
enolase T37[a]
melittin[a]
1
N-Bz-alanine
19 (Me)
78
100
100
100
88
87
95
100
94
87
83
98
82
86
96
76
61
RPPGFSPFR
DRVYIHPF
DRVYIHPFHL
2
N-Z-phenylalanine 20 82
(Ph)
DRVYIHPFHLLVYS
WLTGPQLADLYHSLMK
YPIVSIEDPFAEDDWEAWSHFFK
GIGAVLKVLTTGLPALISWIKRKRQQ
pyroELYENKPRRPYIL
3
N-Ac-tryptophan
21 (indole)
80
82
80
81
77
78
76
79
78
4
N-Bz-histidine
22 (imidazole)
N-Bz-arginine
23 (guanidine)
N-Bz-asparagine
24 (amide)
neurotensin[b]
J
5
100
100
100
100
100
100
0
[a] Peptides in Waters MassPREP peptide mixture (see Figure 3 in the Ex-
perimental Section). [b] Peptides used in the catch-and-release reaction
shown in Figure 2.
6
7
Na-Bz-lysine
25 (amine)
8
N-Bz-serine
alkyne-tagged peptides were thoroughly washed out from the
resin after catch according to the absence of any related peaks
in the mass spectra of the sixth washing and the washings
after release (see Figure 3b,c in the Experimental Section).[34]
These results correlate with the observation that this catch-
and-release protocol is compatible with a range of amino acids
containing various functionalities.
26 (hydroxy)
N-Z-tyrosine
27 (phenol)
9
10
11
12
N-Bz-methionine
28 (thioether)
N-Bz-cysteine
29 (thiol)
N-Bz-S-carbamoylmeth- 80
yl-cysteine 30 (thioeth-
er)
85
Following the success of the compatibility of the catch-and-
release protocol with various peptides, it was decided to lower
the concentration of the alkyne-tagged molecule even further.
A solution of dipeptide 11 (0.5 mm, 500 pmol) and a peptide
mixture containing angiotensin fragment 1–7 (B; 5 mm), brady-
kinin (C; 20 mm),[35] angiotensin II (D; 5 mm), angiotensin I (E;
5 mm) and neurotensin (J; 5 mm; Table 3) in 30% EtOH/HEPES
buffer was rotated with cobalt beads 7a at 48C for 12 h. The
resulting beads were washed to remove any unreacted alkyne
11 and other non-alkyne-containing peptides present in large
excess. The alkyne–cobalt–resin 18 was then treated with 5%
TFA followed by washing of the beads. Mass spectrometric
analysis of the initial solution before cobalt complexation, the
supernatant after catch and washings of the alkyne–cobalt
beads after both catch and release was carried out and the
spectra are shown in Figure 2. The eighth washing of cobalt
beads 18 after catch revealed the absence of alkyne 11 and
other peptides, which indicates that non-specific-binding mol-
ecules had been successfully washed out from the beads (Fig-
ure 2c). Amine 16 was detected in the washings of the beads
after release with 5% TFA. These results indicate that catch
and release of dipeptide 11 proceeded at a concentration of
0.5 mm in the presence of other peptides each existing in over
10-fold excess with respect to the target molecule to generate
amine 16 after release from the resin. Hence, specific enrich-
ment had occurred at high dilution with no significant interfer-
ence from the other peptides. Furthermore, when the concen-
tration of dipeptide 11 was lowered to 50 nm (50 pmol) in the
presence of 5 mm of each peptide B, D, E and J (20 mm for bra-
dykinin (C)) under the same conditions, successful release of
amine 16 was detected by mass spectrometry (see Figure S1 in
the Supporting Information). It is worth mentioning that mass
spectrometric analysis of the supernatant after alkyne com-
giotensin fragment 1–7 (B), bradykinin (C), angiotensin II (D),
angiotensin I (E), renin substrate (F), enolase T35 (G), enola-
se T37 (H) and melittin (I); peptide sequences shown in
Table 3) in 30% EtOH/HEPES buffer was rotated with cobalt
beads 7a at 48C for 12 h. The resulting beads were washed to
remove any unreacted alkyne 11 and other non-alkyne-con-
taining peptides. Alkyne–cobalt–resin 18 was then treated
with 5% TFA and washed afterwards. Mass spectrometric anal-
ysis of the washing solutions after TFA treatment revealed the
presence of amine 16, which demonstrates that catch and re-
lease had proceeded smoothly in the presence of other pep-
tides. Pleasingly, amino acid residues that contain metal-coor-
dinating functionalities such as arginine (R), serine (S), lysine
(K), tyrosine (Y), histidine (H) and tryptophan (W) in the pep-
tides did not appear to interfere with the reaction, and all non-
Chem. Eur. J. 2014, 20, 8116 – 8128
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such as carboxylic acid, hydroxy,
phenol, amine, amide, indole,
imidazole, guanidine and thio-
ether groups, were found to be
compatible under the estab-
lished reaction conditions and
the desired amine was released
in good yields. Finally, when the
catch-and-release protocol was
carried out with a peptide mix-
ture under highly diluted condi-
tions (0.5 mm or 50 nm), non-
alkyne-containing peptides were
successfully washed out from
the resin and selective enrich-
ment of the amine was clearly
detected by mass spectrometry.
Overall, these results demon-
strate that the propargyl carba-
mate tag selectively coordinates
to the cobalt resin under dilute
aqueous conditions at low tem-
perature. The alkyne tag is easily
prepared and relatively small in
size, which makes it a good can-
didate for incorporation into bio-
active molecules without drasti-
cally changing their activities.
This protocol is operationally
Figure 2. Mass spectra of solutions from the catch and release of dipeptide 11 (0.5 mm) in a peptide mixture con-
taining angiotensin frag. 1–7 (B), bradykinin (C), angiotensin II (D), angiotensin I (E) and neurotensin (J; Table 3).
a) Initial solution of alkyne-tagged dipeptide 11 (0.5 mm) and peptide mixture (5 mm each of peptides B, D, E and
J, and 20 mm of peptide C) prior to cobalt complexation (catch). b) Supernatant after catch showing unreacted
peptides and cobalt complexes of angiotensin analogues B, D and E. c) Eighth washing solution of cobalt beads
18 after catch. d) First washing solution after TFA treatment. The mass spectra were recorded with a LTQ Orbitrap
XL spectrometer.
plexation showed peaks for the other peptides that were con-
sidered to not form complexes with the cobalt resin. Interest-
ingly, cobalt adduct ions ([M+56]+) were observed in the su-
pernatant for angiotensin analogues (Figure 2b), which sug-
gests the formation of [MꢀH+Co]2+ and [M+Co]3+ ions[36] ac-
cording to the theoretical masses (see Figures S2 and S3 in the
Supporting Information). This phenomenon may arise from the
presence of oxidised cobalt ions coordinating to the specific
amino acid sequence of angiotensin analogues containing ar-
ginine and histidine residues.
simple and enables the release of the target molecule under
acidic conditions that are relatively mild compared with oxida-
tive conditions. It is anticipated that this strategy has the po-
tential to be bio-orthogonal and applicable for the specific en-
richment of alkyne-tagged biomolecules in a range of complex
peptide mixtures, including cell lysates, and thus may prove to
be useful in identifying the target proteins of bioactive mole-
cules.
Experimental Section
General
Conclusion
All reactions and manipulations involving organometallic com-
pounds were performed under an inert atmosphere of dry nitro-
gen. Dehydrated tetrahydrofuran (THF), methanol (MeOH) and di-
chloromethane (CH2Cl2) were purchased from Wako Pure Chemical
Industries, Ltd. TentaGel was purchased from HiPep Laboratories
Co., Inc. The phosphine ligand supported on TentaGel was synthes-
ised according to the literature.[24] [Co2(CO)8] was purchased from
Tokyo Chemical Industry Co., Ltd. (TCI) and Kanto Chemical Co.,
Inc. [Co2(CO)7(PPh3)] was synthesised according to the literature.[26g]
Peptides (angiotensin I, angiotensin II, angiotensin frag, 1–7, brady-
kinin and neurotensin) were purchased from Peptide Institute Inc.
All other reagents were used as purchased from commercial sour-
ces. Water was prepared with a Millipore Milli-Q Advantage A10 in-
strument. TLC analysis was performed on silica gel 60 F254-coated
glass plates (Merck). Visualisation was accomplished by means of
ultraviolet (UV) irradiation at 254 nm, iodine or by using 12-molyb-
do(VI)phosphoric acid/ethanol solution, potassium permanganate,
In conclusion, the selective catch and release of propargyl car-
bamate tagged molecules has been investigated by using
model compounds, including amino acid and peptide deriva-
tives. At high dilution and low temperature, alkyne-tagged
molecules were selectively coordinated to a resin-bound
cobalt complex, followed by release of the molecules as
amines by the Nicholas reaction using TFA in aqueous media.
A control experiment using non-alkyne-containing molecules
demonstrated that the cobalt resin has a high selectivity for
alkyne-tagged molecules. Furthermore, non-specific binding
molecules were thoroughly washed out from the resins after
the catch reaction. Catch-and-release reactions of an alkyne-
tagged peptide in the presence of amino acids bearing various
functionalities also proceeded smoothly. A range of oxygen, ni-
trogen and protected sulfur functionalities in amino acids,
Chem. Eur. J. 2014, 20, 8116 – 8128
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ninhydrin and heat as developing agents. Flash column chroma-
tography was performed with silica gel N-60 (40–100 mm) pur-
chased from Kanto Chemical Co., Inc. or Chromatorex (N-H-type
silica gel) purchased from Fuji Silysia Chemical, Ltd. 1H, 13C and
31P NMR spectra were recorded at room temperature on a JEOL
JNM-AL400 or JEOL JNM-ECP-500 spectrometer at 400 or 500, 100
or 125 and 160 MHz, respectively. J values are reported in Hz and
chemical shifts (d) in ppm relative to tetramethylsilane (TMS),
chloroform or H3PO4. IR spectra were recorded on a Thermo Nico-
let iS5 spectrometer. Only diagnostic absorptions are listed in the
characterisation data. HPLC was performed on a DIONEX UltiMate
3000 instrument with the following equipment: pump, DGP-3600;
auto-sampler, WPS-3000; flow manager, FLM-3x00; detector, VWD-
3x00 measured at 215 or 280 nm. Mass spectra were acquired on
a Bruker microTOF-QII-RSL or ThermoScientific LTQ Orbitrap XL spec-
trometer. Electrospray ionisation (ESI) Orbitrap mass spectra were
acquired in positive ion mode for a m/z range of 280–1600. The FT
resolution was set at 100000. Mass spectra were analysed with
a Qual Browser instrument (Thermo Fisher Scientific). Optical rota-
tions were recorded on a JASCO P-2200 Polarimeter using sodium
low-pressure light (sodium D line 589 nm) with a 50 mm path
length; concentrations are given as g per 100 mL.
as a colourless oil (77 mg, 81%). Rf =0.11 (N-H SiO2; CH2Cl2/MeOH,
99:1); [a]2D6 = +15.6 (c=0.5 in CHCl3); 1H NMR (400 MHz, CDCl3):
d=2.78 (brs, 2H; NH), 3.01–3.09 (m, 2H; CH2), 3.30–3.58 (m, 16H;
CH2), 4.47 (dd, 3J(H,H)=6.9, 14.3 Hz, 1H; CH), 5.01–5.07 (m, 2H;
CH2), 5.91 (d, 3J(H,H)=8 Hz, 1H; NH), 7.17–7.34 (m, 10H; CArH),
7.58 ppm (brs, 1H; NH); 13C NMR (100 MHz, CDCl3): d=39.3 (CH2),
41.4 (CH2), 56.1 (CH), 66.8 (CH2), 69.9 (CH2), 70.0 (CH2), 70.2 (CH2),
70.6 (CH2), 72.7 (CH2), 126.8 (CAr), 128.0 (CAr), 128.2 (CAr), 128.5 (CAr),
128.6 (CAr), 129.4 (CAr), 129.6 (CAr), 136.5 (CAr), 137.0 (CAr), 156.0 (C=
O), 171.1 ppm (C=O); IR (neat): n˜max =3303, 2868, 1712, 1659,
1528 cmꢀ1; HRMS (ESI+): m/z calcd for [C25H35N3O6 +H]+: 474.2599;
found:474.2657.
Benzyl prop-2-yn-1-yl (S)-13-oxo-15-phenyl-3,6,9-trioxa-12-
azapentadecane-1,14-dicarbamate (10)
To a solution of azide 12 (100 mg, 0.2 mmol) in MeOH (2 mL) was
added Pd/C–ethylene diamine complex (9 mg) at room tempera-
ture under nitrogen. After placing the reaction mixture under hy-
drogen, the mixture was stirred for 6 h. After filtration of the reac-
tion mixture through a pad of Celite, the filtrate was concentrated
under reduced pressure and used in the next step without further
purification. The residue was dissolved in CH2Cl2 (1.5 mL) and trie-
thylamine (82 mL, 0.59 mmol) and 3 (49 mg, 0.22 mmol) were
added at room temperature. The solution was stirred for 24 h, fol-
lowed by the addition of a saturated solution of aqueous NaHCO3.
After extraction with ethyl acetate (3ꢂ10 mL), the combined or-
ganic layers were washed with brine, dried over Na2SO4 and con-
centrated under reduced pressure. Flash column chromatography
(N-H SiO2; CH2Cl2/MeOH, 1:0 to 99:1) of the residue afforded the
title compound 10 as a colourless oil (79 mg, 71%). Rf =0.47 (N-H
SiO2; CH2Cl2/MeOH, 99:1); [a]2D6 = +5.9 (c=1 in CHCl3); 1H NMR
Cobalt beads were rotated by using a MACSmixTM Tube Rotator.
Compounds 1–9 have been synthesised and characterised previ-
ously.[19]
Synthesis
Benzyl (S)-1-azido-13-oxo-15-phenyl-3,6,9-trioxa-12-azapenta-
decane-14-carbamate (12)
N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide
(288 mg,
4
(400 MHz, CDCl3): d=2.44 (t, J(H,H)=2.5 Hz, 1H; CꢁCH), 3.06 (d,
1.5 mmol), DIPEA (0.52 mL, 3 mmol), 4-(dimethylamino)pyridine
(18 mg, 0.15 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine
(0.21 mL, 1.05 mmol) were added to a solution of N-Z-l-phenylala-
nine (299 mg, 1 mmol) in CH2Cl2 (10 mL) at room temperature.
After stirring the mixture for 24 h, the mixture was diluted with
ethyl acetate. The organic layers were washed with aqueous 1n
HCl, a saturated aqueous solution of NaHCO3, distilled water and
brine, dried over Na2SO4 and concentrated under reduced pressure.
Flash column chromatography (N-H SiO2; CH2Cl2) of the residue af-
forded the title compound 12 as a colourless oil (322 mg, 65%).
Rf =0.34 (N-H SiO2; CH2Cl2); [a]2D5 = +6.3 (c=1 in CHCl3); 1H NMR
(400 MHz, CDCl3): d=3.06–3.10 (m, 2H; CH2), 3.35–3.71 (m, 16H;
CH2), 4.41 (dd, 3J(H,H)=6.9, 14.3 Hz, 1H; CH), 5.09 (d, 3J(H,H)=
3J(H,H)=5.5 Hz, 2H; CH2), 3.34–3.58 (m, 16H; CH2), 4.40 (d,
3J(H,H)=7 Hz, 1H; CH), 4.65 (d, 4J(H,H)=2.5 Hz, 2H; CH2CꢁC),
3
5.06–5.10 (m, 2H; CH2), 5.52 (d, J(H,H)=6.5 Hz, 1H; NH), 5.61 (brs,
1H; NH), 6.44 (brs, 1H; NH), 7.18–7.34 ppm (m, 10H; CArH);
13C NMR (100 MHz, CDCl3): d=39.1 (CH2), 39.3 (CH2), 40.9 (CH2),
52.5 (CH2CꢁCH), 56.1 (CH), 67.0 (CH2), 69.7 (CH2), 70.0 (CH2), 70.2
(CH2), 70.4 (CH2), 70.5 (CH2), 74.7 (CꢁCH), 78.5 (CꢁCH), 127.0 (CAr),
128.1 (CAr), 128.3 (CAr), 128.6 (CAr), 128.7 (CAr), 129.5 (CAr), 136.3 (CAr),
136.7 (CAr), 155.7 (C=O), 155.9 (C=O), 170.9 ppm (C=O); IR (neat):
n˜max =3296, 2870, 1709, 1660, 1520 cmꢀ1; HRMS (ESI+): m/z calcd
for [C29H37N3O8 +Na]+: 578.2473; found:578.2484.
3
4.0 Hz, 2H; CH2), 5.51 (d, J(H,H)=7.0 Hz, 1H; NH), 6.32 (brs, 1H;
Methyl (S)-2-{(S)-2,6-bis[(benzyloxycarbonyl)amino]hexanami-
do}-3-phenylpropanoate (14)
NH), 7.20–7.39 ppm (m, 10H; CArH); 13C NMR (100 MHz, CDCl3): d=
39.1 (CH2), 39.3 (CH2), 50.7 (CH2), 56.4 (CH), 67.0 (CH2), 69.6 (CH2),
70.1 (CH2), 70.3 (CH2), 70.61 (CH2), 70.63 (CH2), 70.8 (CH2), 127.0
(CAr), 128.1 (CAr), 128.3 (CAr), 128.6 (CAr), 128.7 (CAr), 129.4 (CAr), 136.3
Na-Z-l-Lysine (1.68 g, 6 mmol) was dissolved in a solution of
NaHCO3 (24 mL, 1n) and NaOH (4.8 mL, 4n). The solution was
cooled to 08C and benzyl chloroformate (1.01 mL, 9 mmol) was
added dropwise and the reaction mixture stirred for 1 h at 08C.
The reaction mixture was allowed to warm to room temperature
and stirred for an additional 24 h. It was then acidified to pH 2–3
by the slow addition of aqueous 1n hydrochloric acid. After extrac-
tion with ethyl acetate (3ꢂ10 mL), the combined organic layers
were washed with brine, dried over Na2SO4 and concentrated
under reduced pressure. N-(3-Dimethylaminopropyl)-N’-ethylcarbo-
diimide (575 mg, 3 mmol), DIPEA (0.45 mL, 2.6 mmol) and 1-hydro-
benzotriazole (324 mg, 2.4 mmol) were added to a suspension of
Na-Z,Ne-Z-l-lysine (829 mg, 2 mmol) and l-phenylalanine methyl
ester hydrochloride (518 mg, 2.4 mmol) in CH2Cl2 (10 mL) at room
temperature. After stirring the mixture for 2 h, the mixture was di-
luted with ethyl acetate. The organic layers were washed with dis-
(CAr), 136.6 (CAr), 155.9 (C=O), 170.7 ppm (C=O); IR (neat): n˜max
=
3304, 2868, 2100. 1712, 1657, 1526 cmꢀ1; HRMS (ESI+): m/z calcd
for [C25H33N5O6 +Na]+: 522.2323; found:522.2327.
Benzyl (S)-1-amino-13-oxo-15-phenyl-3,6,9-trioxa-12-azapen-
tadecane-14-carbamate (13)
Pd/C–ethylenediamine complex (9 mg) was added to a solution of
azide 12 (100 mg, 0.2 mmol) in MeOH (2 mL) at room temperature
under nitrogen. After placing the reaction mixture under hydrogen,
the mixture was stirred for 2 h. After filtration of the reaction mix-
ture through a pad of Celite, the filtrate was concentrated under
reduced pressure. Flash column chromatography (N-H SiO2; CH2Cl2/
MeOH, 1:0 to 98:2) of the residue afforded the title compound 13
Chem. Eur. J. 2014, 20, 8116 – 8128
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tilled water, a saturated aqueous solution of NaHCO3, distilled
water, aqueous 0.1m citric acid, distilled water and brine, dried
over Na2SO4 and concentrated under reduced pressure. Flash
column chromatography (SiO2; hexane/ethyl acetate, 2:1 to 1:1) of
the residue afforded the title compound 14 as a colourless amor-
phous solid (599 mg, 58%). Rf =0.33 (SiO2; hexane/ethyl acetate,
1:1); [a]2D7 = +22.0 (c=1 in CHCl3); 1H NMR (400 MHz, CDCl3): d=
1.23–1.78 (m, 6H; CH2), 3.02–3.15 (m, 4H; CH2), 3.66 (s, 3H; CH3),
4.08–4.14 (m, 1H; CH), 4.83 (dd, 3J(H,H)=6.0, 13.8 Hz, 1H; CH),
SiO2; CH2Cl2/MeOH, 9:1); [a]2D7 =ꢀ4.8 (c=1 in CHCl3); 1H NMR
(500 MHz, CDCl3): d=1.21–1.67 (m, 6H; CH2), 2.81–3.56 (m, 22H;
CH2, NH2), 4.06–4.07 (m, 1H; CH), 4.66–4.68 (m, 1H; CH), 5.00–5.02
(m, 4H; CH2), 5.21 (m, 1H; NH), 5.59–5.60 (m, 1H; NH), 7.12–
7.28 ppm (m, 15H; CArH); 13C NMR (125 MHz, CDCl3): d=22.0 (CH2),
29.3 (CH2), 31.9 (CH2), 38.5 (CH2), 39.3 (CH2), 40.2 (CH2), 40.9 (CH2),
54.5 (CH), 54.9 (CH), 66.7 (CH2), 67.1 (CH2), 69.7 (CH2), 69.8 (CH2),
70.0 (CH2), 70.4 (CH2), 70.5 (CH2), 71.1 (CH2), 126.9 (CAr), 128.0 (CAr),
128.2 (CAr), 128.3 (CAr), 128.58 (CAr), 128.63 (CAr), 129.5 (CAr), 136.3
(CAr), 136.7 (CAr), 136.8 (CAr), 156.3 (C=O), 156.8 (C=O), 171.1 (C=O),
171.8 ppm (C=O); IR (neat): n˜max =3303, 2867, 1704, 1645,
1527 cmꢀ1; HRMS (ESI+): m/z calcd for [C39H53N5O9 +H]+: 736.3916;
found:736.3894.
3
4.92–5.07 (m, 4H; CH2), 5.43 (d, J(H,H)=7.5 Hz, 1H; NH), 6.44 (d,
3J(H,H)=7.5 Hz, 1H; NH), 7.07 (d, 3J(H,H)=7 Hz, 2H; CArH) 7.19–
7.32 ppm (m, 13H; CArH); 13C NMR (100 MHz, CDCl3): d=22.1 (CH2),
29.4 (CH2), 31.9 (CH2), 37.8 (CH2), 40.3 (CH2), 52.5 (CH3), 53.2 (CH),
54.7 (CH), 66.8 (CH2), 67.2 (CH2), 127.3 (CAr), 128.17 (CAr), 128.22 (CAr),
128.3 (CAr), 128.58 (CAr), 128.64 (CAr), 128.7 (CAr), 129.3 (CAr), 135.7
(CAr), 136.2 (CAr), 136.6 (CAr), 156.2 (C=O), 156.7 (C=O), 171.3 (C=O),
171.9 ppm (C=O); IR (neat): n˜max =3308, 2948, 1692, 1658,
Dibenzyl prop-2-yn-1-yl (14S,17S)-14-benzyl-13,16-dioxo-3,6,9-
trioxa-12,15-diazahenicosane-1,17,21-tricarbamate (11)
1526 cmꢀ1
;
HRMS (ESI+): m/z calcd for [C32H37N3O7 +Na]+:
Pd/C–ethylene diamine complex (7 mg) was added to a solution of
azide 15 (114 mg, 0.15 mmol) in MeOH (1.5 mL) at room tempera-
ture under nitrogen. After placing the reaction mixture under hy-
drogen, the mixture was stirred for 4 h. After filtration of the reac-
tion mixture through a pad of Celite, the filtrate was concentrated
under reduced pressure and used in the next step without further
purification. The residue was dissolved in THF (1.2 mL) and 4-(di-
methylamino)pyridine (27 mg, 0.225 mmol), DIPEA (78 mL,
0.45 mmol) and 3 (41 mg, 0.19 mmol) were added at room temper-
ature. The solution was stirred for 6.5 h, followed by the addition
of a saturated aqueous solution of NaHCO3. After extraction with
ethyl acetate (3ꢂ10 mL), the combined organic layers were
washed with brine, dried over Na2SO4 and concentrated under re-
duced pressure. Flash column chromatography (N-H SiO2; CH2Cl2/
MeOH, 1:0 to 99.5:0.5) of the residue afforded the title compound
11 as a colourless amorphous solid (103 mg, 84%). Rf =0.81 (N-H
SiO2; CH2Cl2/MeOH, 95:5); [a]2D7 =ꢀ8.9 (c=1 in CHCl3); 1H NMR
(400 MHz, CDCl3): d=1.22–1.83 (m, 6H; CH2), 2.45 (brs, 1H; Cꢁ
CH), 2.99–3.60 (m, 20H; CH2), 4.10–4.11 (m, 1H; CH), 4.60–4.66 (m,
3H; CH2, CH), 5.01–5.12 (m, 5H; CH2, NH), 5.58–5.59 (m, 1H; NH),
6.67–6.69 (m, 1H; NH), 7.17–7.34 ppm (m, 15H; CArH); 13C NMR
(100 MHz, CDCl3): d=22.0 (CH2), 29.6 (CH2), 38.6 (CH2), 39.5 (CH2),
41.1 (CH2), 52.6 (CH2), 54.6 (CH), 55.2 (CH), 66.9 (CH2), 67.4 (CH2),
69.7 (CH2), 70.1 (CH2), 70.4 (CH2), 70.6 (CH2), 70.7 (CH2), 74.8 (Cꢁ
CH), 78.7 (CꢁCH), 127.2 (CAr), 128.3 (CAr), 128.5 (CAr), 128.7 (CAr),
128.8 (CAr), 129.5 (CAr), 136.3 (CAr), 136.8 (CAr), 155.1 (C=O), 155.9 (C=
O), 170.7 (C=O), 171.6 ppm (C=O); IR (neat): n˜max =3290, 2943,
1686, 1640, 1531 cmꢀ1; HRMS (ESI+): m/z calcd for [C43H55N5O11 +
Na]+: 840.3790; found:840.3754.
598.2524; found:598.2519.
Dibenzyl (14S,17S)-1-azido-14-benzyl-13,16-dioxo-3,6,9-trioxa-
12,15-diazahenicosane-17,21-dicarbamate (15)
An aqueous 10% NaOH solution (0.68 mL) was added to a solution
of methyl ester 14 (100 mg, 0.17 mmol) in THF/MeOH (1.7 mL, 1:1)
at 08C. After stirring for 30 min at 08C, aqueous 1n HCl s was
added. After extraction with ethyl acetate, the combined organic
layers were washed with brine, dried over Na2SO4 and concentrat-
ed under reduced pressure. N-(3-Dimethylaminopropyl)-N’-ethylcar-
bodiimide (49 mg, 0.26 mmol), DIPEA (91 mL, 0.52 mmol), 4-(dime-
thylamino)pyridine (3 mg, 0.03 mmol) and 11-azido-3,6,9-trioxaun-
decan-1-amine (37 mL, 0.19 mmol) were added to a solution of the
residue in CH2Cl2 (1.7 mL) at room temperature. After stirring the
mixture for 45 h, the mixture was diluted with ethyl acetate. The
organic layers were washed with aqueous 1n HCl, a saturated
aqueous solution of NaHCO3, distilled water and brine, dried over
Na2SO4 and concentrated under reduced pressure. Flash column
chromatography (SiO2; ethyl acetate) of the residue afforded the
title compound 15 as a colourless amorphous solid (90 mg, 70%).
Rf =0.62 (SiO2; ethyl acetate); [a]2D7 =ꢀ6.6 (c=1 in CHCl3); H NMR
1
(400 MHz, CDCl3): d=1.19–1.74 (m, 6H; CH2), 2.99–3.16 (m, 4H;
CH2), 3.35–3.66 (m, 16H; CH2), 4.03–4.10 (m, 1H; CH), 4.57–4.66 (m,
1H; CH), 5.00–5.07 (m, 4H; CH2), 5.52–5.53 (m, 1H; NH), 6.62–6.67
(m, 1H; NH), 7.16–7.33 ppm (m, 15H; CArH); 13C NMR (100 MHz,
CDCl3): d=22.3 (CH2), 29.5 (CH2), 32.0 (CH2), 39.4 (CH2), 39.5 (CH2),
40.4 (CH2), 50.8 (CH2), 54.5 (CH), 55.1 (CH), 66.8 (CH2), 67.3 (CH2),
69.5 (CH2), 69.6 (CH2), 70.1 (CH2), 70.3 (CH2), 70.6 (CH2), 70.7 (CH2),
70.8 (CH2), 127.1 (CAr), 128.3 (CAr), 128.4 (CAr), 128.67 (CAr), 128.72
(CAr), 128.74 (CAr), 129.4 (CAr), 136.3 (CAr), 136.7 (CAr), 156.5 (C=O),
156.7 (C=O), 170.6 (C=O), 171.7 ppm (C=O); IR (neat): n˜max =3297,
Protocol for the catch-and-release reactions
Catch and release reaction of alkyne 1 at 48C at 50 mm
(Table 1, Entry 6)
2942, 2106, 1682, 1642 cmꢀ1
[C39H51N7O9 +Na]+: 784.3640; found:784.3604.
;
HRMS (ESI+): m/z calcd for
Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1 [17c,19]
pre-washed with
)
HEPES buffer (pH 7.0) was added to a solution of alkyne 1 (50 mm,
1 mL) in HEPES buffer (pH 7.0) containing 150 mm NaCl, 10 mm
HEPES-Na, and 0.5% Triton X-100 at 48C. The mixture was rotated
for 12 h at the same temperature, and then centrifuged. After re-
moval of the supernatant, the beads were washed with 50% EtOH/
HEPES buffer (3ꢂ1 mL), 50% MeCN/HEPES buffer (3ꢂ1 mL), 5%
MeCN/H2O (2ꢂ1 mL). The supernatant and the washing solutions
were subjected to HPLC to determine the conversion. To the beads
was added 5% TFA solution in 5% MeCN/H2O (1 mL) and the mix-
ture was rotated at 48C for 24 h. After removal of the supernatant,
the beads were washed with 50% MeCN/H2O (1 mL). The superna-
Dibenzyl (14S,17S)-1-amino-14-benzyl-13,16-dioxo-3,6,9-
trioxa-12,15-diazahenicosane-17,21-dicarbamate (16)
Pd/C–ethylene diamine complex (2.4 mg) was added to a solution
of azide 15 (40 mg, 0.053 mmol) in MeOH (0.53 mL) at room tem-
perature under nitrogen. After placing the reaction mixture under
hydrogen, the mixture was stirred for 6 h. After filtration of the re-
action mixture through a pad of Celite, the filtrate was concentrat-
ed under reduced pressure. Flash column chromatography (N-H
SiO2; CH2Cl2/MeOH, 1:0 to 95:5) of the residue afforded the title
compound 16 as a colourless oil (39 mg, quant.). Rf =0.34 (N-H
Chem. Eur. J. 2014, 20, 8116 – 8128
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8125
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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tant and washings were subjected to HPLC to determine the yields
of products. The conversions and yields of all materials were calcu-
lated with reference to standard curves. HPLC [GL Sciences Inertsil
ODS-3, H2O/MeCN (0.1% TFA)=86/14 (0–10 min)!77/23 (10–
25 min), 100 mLminꢀ1, 280 nm, t (compound 2)=10.1 min, t (com-
pound 1)=23.8 min].
beads was added 5% TFA solution in 5% MeCN/H2O (1 mL) and
the mixture was rotated at 48C for 24 h. After removal of the su-
pernatant, the beads were washed with 50% MeCN/H2O (1 mL).
The supernatant and washings were subjected to HPLC to deter-
mine the yields of products. The conversions and yields of all ma-
terials were calculated with reference to standard curves. HPLC [GL
Sciences Inertsil ODS-3, H2O/MeCN (0.1% TFA)=90/10 (0–8 min) !
0/95 (8–18 min) ! 90/10 (18–20 min), 100 mLminꢀ1, 215 nm, t
(compound 16)=16.9 min, t (compound 11)=18.9 min]. The initial
solution, supernatant and washings were desalted with ZipTip C18
(Merck Millipore) and loaded to a metal-coated glass capillary
nanoelectrospray tip (HUMANIX) prior to mass spectrometry. The
mass spectra were taken on LTQ Orbitrap XL.
Selective catch-and-release reaction of alkyne 1 in the pres-
ence of control material 9 (Scheme 7)
Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1) pre-washed with
HEPES buffer (pH 7.0) was added to a solution (1 mL) of alkyne
1 (50 mm) and control material 9 (50 mm) in HEPES buffer (pH 7.0)
containing 150 mm NaCl, 10 mm HEPES-Na, and 0.5% Triton X-100
at 48C. The mixture was rotated for 12 h at the same temperature,
and then centrifuged. After removal of supernatant, the beads
were washed with 50% EtOH/HEPES buffer (3ꢂ1 mL), 50% MeCN/
HEPES buffer (3ꢂ1 mL), 5% MeCN/H2O (2ꢂ1 mL). The supernatant
and the washing solutions were subjected to HPLC to determine
the conversion. To the beads was added 5% TFA in 5% MeCN/H2O
(1 mL) and the mixture was rotated at 48C for 24 h. After removal
of the supernatant, the beads were washed with 50% MeCN/H2O
(1 mL). The supernatant and washings were subjected to the HPLC
analysis and the yields of all materials were calculated. The conver-
sions and yields of all materials were calculated with reference to
standard curves. HPLC [GL Sciences Inertsil ODS-3, H2O/MeCN
(0.1% TFA)=86/14 (0–10 min) ! 77/23 (10–25 min) ! 61/39 (25–
35 min), 100 mLminꢀ1, 280 nm, t (compound 2)=10.1 min, t (com-
pound 1)=23.8 min, t (compound 9)=31.0 min].
Selective catch-and-release reaction of dipeptide 11 in the
presence of derivatised amino acids (Table 2)
For the synthesis of derivatised amino acids, see the Supporting In-
formation. Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1
) pre-
washed with HEPES buffer (pH 7.0) was added to a solution (1 mL)
of dipeptide 11 (50 mm) and derivatised amino acid (50 mm) in 30%
EtOH/HEPES buffer (pH 7.0) containing 150 mm NaCl and 10 mm
HEPES-Na at 48C. The mixture was rotated for 12 h at the same
temperature, and then centrifuged. After removal of the superna-
tant, the beads were washed with 50% EtOH/HEPES buffer (3ꢂ
1 mL), 50% MeCN/HEPES buffer (3ꢂ1 mL), 5% MeCN/H2O (2ꢂ
1 mL). The supernatant and the washing solutions were subjected
to HPLC to determine the conversion. To the beads was added 5%
TFA solution in 5% MeCN/H2O (1 mL) and the mixture was rotated
at 48C for 24 h. After removal of the supernatant, the beads were
washed with 50% MeCN/H2O (1 mL). The supernatant and wash-
ings were subjected to HPLC to determine the yields of products.
The conversions and yields of all materials were calculated with ref-
erence to standard curves. HPLC [GL Sciences Inertsil ODS-3, H2O/
MeCN (0.1% TFA)=90/10 (0–8 min) ! 0/95 (8–18 min) ! 90/10
(18–20 min), 100 mLminꢀ1, 215 nm, t (compound 16)=16.9 min, t
(compound 11)=18.9 min, t (compound 19)=14.4 min, t (com-
pound 20)=17.8 min, t (compound 21)=15.1 min, t (compound
22)=6.7 min, t (compound 23)=11.5 min, t (compound 24)=
7.3 min, t (compound 25)=7.3 min, t (compound 26)=9.1 min, t
(compound 27)=16.2 min, t (compound 28)=15.7 min, t (com-
pound 29)=15.4 min, t (compound 30)=13.6 min].
Catch and release reaction of amino acid 10 at 48C at 50 mm
(Scheme 9)
Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1) pre-washed with
HEPES buffer (pH 7.0) was added to a solution of amino acid 10
(50 mm, 1 mL) in HEPES buffer (pH 7.0) containing 150 mm NaCl,
10 mm HEPES-Na, and 0.5% Triton X-100 at 48C. The mixture was
rotated for 12 h at the same temperature, and then centrifuged.
After removal of the supernatant, the beads were washed with
50% EtOH/HEPES buffer (3ꢂ1 mL), 50% MeCN/HEPES buffer (3ꢂ
1 mL), 5% MeCN/H2O (2ꢂ1 mL). The supernatant and the washing
solutions were subjected to HPLC to determine the conversion. To
the beads was added 5% TFA solution in 5% MeCN/H2O (1 mL)
and the mixture was rotated at 48C for 24 h. After removal of the
supernatant, the beads were washed with 50% MeCN/H2O (1 mL).
The supernatant and washings were subjected to HPLC to deter-
mine the yields of products. The conversions and yields of all ma-
terials were calculated with reference to standard curves. HPLC [GL
Sciences Inertsil ODS-3, H2O/MeCN (0.1% TFA)=90/10 (0–8 min) !
0/95 (8–18 min) ! 90/10 (18–20 min), 100 mLminꢀ1, 215 nm, t
(compound 13)=15.7 min, t (compound 10)=17.9 min].
Selective catch-and-release reaction of dipeptide 11 in the
presence of Waters MassPREP peptides (Figure 3)
Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1) pre-washed with
HEPES buffer (pH 7.0) was added to a solution (1 mL) of dipeptide
11 (1.25 mm) and Waters MassPREP peptide mixture containing ap-
proximately 0.4 mm each of 9 different peptides (RASG-1, angioten-
sin fragment 1–7, bradykinin, angiotensin II, renin substrate, eno-
lase T35, enolase T37, and melittin) in 30% EtOH/HEPES buffer
(pH 7.0) containing 150 mm NaCl and 10 mm HEPES-Na at 48C. The
initial solution was subjected to mass spectrometry and the mix-
ture was rotated for 12 h at the same temperature, and then cen-
trifuged. After removal of the supernatant, the beads were washed
with 50% EtOH/HEPES buffer (3ꢂ1 mL), 50% MeCN/HEPES buffer
(3ꢂ1 mL), 5% MeCN/H2O (2ꢂ1 mL). The supernatant and the
washing solutions were subjected to mass spectrometry to deter-
mine the composition. To the beads was added 5% TFA solution in
5% MeCN/H2O (1 mL) and the mixture was rotated at 48C for 24 h.
After removal of the supernatant, the beads were washed with
50% MeCN/H2O (1 mL). The initial solution, supernatant and wash-
ings were desalted with ZipTip C18 (Merck Millipore) and loaded to
Catch and release reaction of dipeptide 11 at 48C at 50 mm
(Scheme 9)
Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1) pre-washed with
HEPES buffer (pH 7.0) was added to a solution of dipeptide 11
(50 mm, 1 mL) in 30% EtOH/HEPES buffer (pH 7.0) containing
150 mm NaCl and 10 mm HEPES-Na at 48C. The mixture was rotat-
ed for 12 h at the same temperature, and then centrifuged. After
removal of the supernatant, the beads were washed with 50%
EtOH/HEPES buffer (3ꢂ1 mL), 50% MeCN/HEPES buffer (3ꢂ1 mL),
5% MeCN/H2O (2ꢂ1 mL). The supernatant and the washing solu-
tions were subjected to HPLC to determine the conversion. To the
Chem. Eur. J. 2014, 20, 8116 – 8128
www.chemeurj.org
8126
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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Figure 3. Mass spectra of solutions from the catch and release of dipeptide
11 in Waters MassPREP peptide mixture (peptides A–I, Table 3). a) Initial solu-
tion of alkyne-tagged dipeptide 11 (1.25 mm) and Waters MassPREP peptide
mixture (approx. 0.4 mm each of RASG-1 (A), angiotensin fragments 1–7 (B),
bradykinin (C), angiotensin II (D), angiotensin I (E), renin substrate (F), enola-
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a LTQ Orbitrap XL spectrometer.
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a metal-coated glass capillary nanoelectrospray tip (HUMANIX)
prior to mass spectrometry. The mass spectra were taken on LTQ
Orbitrap XL.
Selective catch-and-release reaction of dipeptide 11 in the
presence of peptides (Figure 2)
Cobalt complex 7a (10 mg, ~0.23 mmolgꢀ1) pre-washed with
HEPES buffer (pH 7.0) was added to a solution (1 mL) of dipeptide
11 (0.5 mm) and a peptide mixture containing 5 mm each of angio-
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20 mm of bradykinin in 30% EtOH/HEPES buffer (pH 7.0) containing
150 mm NaCl and 10 mm HEPES-Na at 48C. The initial solution was
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12 h at the same temperature, and then centrifuged. After removal
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H2O (2ꢂ1 mL). The supernatant and the washing solutions were
subjected to mass spectrometry to determine the composition. To
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pillary nanoelectrospray tip (HUMANIX) prior to mass spectrometry.
The mass spectra were taken on LTQ Orbitrap XL.
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Keywords: alkynes · cobalt · peptides · solid-phase synthesis
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[29] The catch efficiency improved relative to previously reported results
upon pre-washing cobalt beads 7a with HEPES buffer prior to the
catch (see ref. [<19]).
[30] The composition of the catch solvent had a significant effect on the
catch yield. When 40% EtOH/HEPES buffer solution and 50% EtOH/
HEPES buffer solution were used to dissolve dipeptide 11, the catch
yield deteriorated to 57 and 5%, respectively.
[31] Beads were washed with 50% EtOH/HEPES buffer (pH 7.0) three times,
50% MeCN/HEPES buffer three times and 5% MeCN/H2O twice.
[32] A small amount of amine 13 (6%) was detected after alkyne complexa-
tion with amino acid 10 and a negligible amount of amine 16 (~1%)
was detected after the catch of dipeptide 11.
[23] T. Sugihara, M. Yamada, H. Ban, M. Yamaguchi, C. Kaneko, Angew. Chem.
1997, 109, 2884–2886; Angew. Chem. Int. Ed. Engl. 1997, 36, 2801–
2804.
[24] a) Y. Uozumi, Bull. Chem. Soc. Jpn. 2008, 81, 1183–1195; b) H. Danjo, D.
Tanaka, T. Hayashi, Y. Uozumi, Tetrahedron 1999, 55, 14341–14352.
[25] The buffer contains 150 mm NaCl and 10 mm HEPES–Na (pH 7.0) in
milli-Q water.
[26] The cobalt complexes were identified by comparison of their IR spectra
with the reported IR spectra of triphenylphosphine complexes and
other analogous complexes. See, a) P. Szabo, L. Fekete, G. Bor, J. Orga-
nomet. Chem. 1968, 12, 245–248; b) L. M. Bower, M. H. B. Stiddard, J. Or-
ganomet. Chem. 1968, 13, 235–239; c) G. O. Evans, C. U. Pittman, Jr., R.
McMillan, R. T. Beach, R. Jones, J. Organomet. Chem. 1974, 67, 295–314;
d) R. A. Dubois, P. E. Garrou, K. D. Lavin, H. R. Allcock, Organometallics
1986, 5, 460–466. The peak at 1886 cmꢀ1 indicates that the ionic
cobalt complex [Co(CO)3L2]+[Co(CO)4]ꢀ was located on the polymer,
see: S. E. Gibson, C. Johnstone, A. Stevenazzi, Tetrahedron 2002, 58,
4937–4942 and ref. [17c]. Attempts were made to quantify the loading
of cobalt–phosphine complexes on the resin by using 31P NMR spec-
troscopy. However, the presence of cobalt led to broadening of the
[33] Upon mixing N-Bz-histidine 22 and N-Bz-cysteine 29 with the phos-
phine–TentaGel resin bearing no cobalt, hardly any loss of material was
observed according to HPLC. This suggests that both histidine and cys-
teine have a tendency to be modified or coordinated to the metal
centre in the presence of cobalt.
[34] The low concentration of each peptide (approx. 0.4 mm each) in the
Waters MassPREP peptide mixture made it rather difficult to detect and
monitor the fate of each peptide in the initial catch and subsequent
washing solutions by mass spectrometry (see Figure 3 in the Experi-
mental Section).
[35] As a result of the low ionisation of bradykinin in the presence of other
peptides, the concentration was increased by a factor of four to im-
prove its detection by mass spectrometry.
[36] a) M. J. Pushie, A. R. S. Ross, H. J. Vogel, Anal. Chem. 2007, 79, 5659–
5667; b) E. Constantino, J. Tortajada, M. Sodupe, L. Rodrꢄguez-Santiago,
J. Phys. Chem. 2008, 112, 12385–12392.
Received: January 6, 2014
Published online on April 30, 2014
Chem. Eur. J. 2014, 20, 8116 – 8128
www.chemeurj.org
8128
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