Preparation of novel alkylated arginine derivatives suitable for click-cycloaddition chemistry and their incorporation into pseudosubstrate- and bisubstrate-based kinase inhibitors

Article abstract of DOI:10.1039/b922928k

Efficient strategies for the introduction of arginine residues featuring acetylene or azide moieties in their side chains are described. The substituents are introduced in a way that maintains the basicity of the guanidine moiety. The methodology can be used e.g. for non-invasive labeling of arginine-containing peptides. Its applicability is demonstrated by the introduction of 'click' handles into a Protein Kinase C (PKC) pseudosubstrate peptide, and the subsequent preparation and evaluation of a novel bisubstrate-based inhibitor based on such a peptide.

Full text of DOI:10.1039/b922928k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Preparation of novel alkylated arginine derivatives suitable for
click-cycloaddition chemistry and their incorporation into pseudosubstrate-
and bisubstrate-based kinase inhibitors†
Jeroen van Ameijde,a Alex J. Poot,a Loek T. M. van Wandelen,a Angelique E. M. Wammes,a Rob Ruijtenbeek,b
Dirk T. S. Rijkersa and Rob M. J. Liskamp*a
Received 2nd November 2009, Accepted 9th January 2010
First published as an Advance Article on the web 4th February 2010
DOI: 10.1039/b922928k
Efficient strategies for the introduction of arginine residues featuring acetylene or azide moieties in their
side chains are described. The substituents are introduced in a way that maintains the basicity of the
guanidine moiety. The methodology can be used e.g. for non-invasive labeling of arginine-containing
peptides. Its applicability is demonstrated by the introduction of ‘click’ handles into a Protein Kinase C
(PKC) pseudosubstrate peptide, and the subsequent preparation and evaluation of a novel
bisubstrate-based inhibitor based on such a peptide.
Recently, our group described a strategy for the preparation of
several different functionalised arginine building blocks suitable
Introduction
for incorporation into peptides.9 In that synthesis, an alternative
guanylation procedure was employed using amines. In the method
described here, alcohols are used for the required modified arginine
residues, thus widening the scope for preparation of such building
blocks.
Because of its permanent positive charge at physiological pH, the
amino acid arginine often plays a crucial role in the interactions
of peptides and proteins, e.g. in kinase substrates,1 carbohydrate
binding domains,2 nucleic acid binding domains3 and cationic
antimicrobial peptides.4
There is a growing need for methods to selectively and efficiently
attach auxiliary moieties to peptides, e.g. for the introduction of
probes used in proteomics protein ‘fishing’ experiments or for the
attachment of radioactive ligands.5 Obviously, it is particularly
advantageous if such moieties can be introduced into unprotected
peptides with minimal change to the peptide’s structure and
properties.
Here, we present a strategy to introduce substituted arginine
residues suitable for the well-known chemoselective acetylene–
azide ‘click’ reaction.6 This reaction can be carried out on
unprotected peptides virtually irrespective of the sequence.7 The
modified arginine residues described here bear azido or acetylene
groups on the guanidine side chain that are introduced by alkyla-
tion, and therefore the often crucial basicity of the guanidine side
chain is maintained. In addition, the azido-containing building
blocks have ethyleneglycol spacers of variable length, which allows
fine tuning of the structural requirements of a modified peptide
for biological activity. Finally, this methodology is compatible
with routine Fmoc–tBu solid phase peptide synthesis. In contrast,
most methods currently described for the preparation of alkylated
arginine residues,7 do not offer a general, convenient strategy
for monomers with suitable protection for solid phase peptide
synthesis.8
As an interesting application, the preparation of functionalised
kinase substrate peptides is described, which contain arginine
residues close to the phosphorylation site. These ‘clickable’
peptides can then be chemoselectively ligated to ATP binding site
inhibitors, yielding so-called bisubstrate-based inhibitors.10,11 Such
inhibitors feature both high affinity, due to the ATP-competitive
inihibitor part, as well as high selectivity as a result of the peptide
part, which can bind to the high selectivity but low affinity peptide
binding groove of kinases.
As an example of the applicability of the novel arginine
derivatives, a series of modified peptides will be described here
based on a sequence we have recently12 identified as a suitable
peptidic component for the preparation of isozyme-selective
Protein Kinase C (PKC) bisubstrate-based inhibitors. Then, one
of these modified peptides is attached to an ATP-competitive
inhibitor using ‘click’ chemistry to afford a novel bisubstrate-based
inhibitor. The PKC family is involved in diseases such as cancer
and diabetes, and have attracted attention as potential drug targets
for a long time.13 Homology between the twelve isoforms of the
PKC family is very high, in particular for their catalytic domains,
and selective inhibition is a difficult issue, making preparation of
selective inhibitors a challenging but rewarding prospect.
Results
aMedicinal Chemistry and Chemical Biology, Faculty of Science, Utrecht
University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. E-mail:
r.m.j.liskamp@uu.nl; Fax: 31 302536655; Tel: 31 302537396
Chemistry
bPamgene International BV, P.O. Box 1345, 5200 BJ ‘s-Hertogenbosch, The
Netherlands
The synthesis of several Fmoc-protected arginine derivatives is
outlined in Scheme 1 (routes A and B). As can be seen, we
decided on employing pyrazole guanylation14 to convert a suitably
protected ornithine derivative into the corresponding arginine
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of all new compounds, and HPLC chromatograms and mass
spectra of all peptides. See DOI: 10.1039/b922928k/
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building blocks. The pyrazole derivatives used in this methodology
usually react cleanly and can be conveniently furnished with a side-
chain using a Mitsunobu reaction.15
material only occurred after stirring for 24 h at room temperature.
During subsequent acidic work-up, the trimethylsilyl group was
cleaved, after which the alkylated arginine building blocks 5a–e
could be obtained in good yields (61–82%).
Next, we evaluated the applicability of these substituted arginine
building blocks in solid phase peptide synthesis. Initial attempts
to incorporate building block 5a–e into several peptide sequences
on the solid phase led to low coupling efficiency in many cases,
depending on coupling conditions (coupling reagents, added
equivalents and solvent) and on the peptide sequence. Since the
corresponding non-alkylated arginine building block has been
coupled successfully in the past,18 the difficulties observed here
were surprising.
When the coupling was carried out in DCM, the solvent could
be evaporated and the reaction mixture analysed. This led to the
isolation of side product 8 in near-quantitative yield (Scheme 2).
This product can be explained by nucleophilic attack of the
guanidine NH on the intermediate activated ester, leading to a
favourable six-membered ring, and can be considered as a 6-
membered ring derivative of the Freidinger lactam.19 Because this
cyclisation was clearly very efficient under the conditions where
side product analysis was possible, it can be assumed to be the
cause of low coupling yield, since the coupling reagent is present
in all cases. As stated above, the efficiency of the cyclisation in
this case was surprising, since the corresponding Boc-protected
non-alkylated arginine may be coupled without problems. In all
The required pyrazole derivatives 3a–e were prepared by treating
commercially available N,N-bis-Boc-guanylpyrazole 1 with either
propargyl alcohol 2a, or known azido oligoethyleneglycols 2b–
e,16 in the presence of DEAD and triphenylphosphine (Scheme 1,
route A). Although these Mitsunobu alkylations proceeded slowly,
clean product formation was observed in all cases, and reagents
3a–e were obtained in 70–86% yield after purification by column
chromatography.
For the preparation of building blocks suitable for Fmoc–
tBu solid phase peptide synthesis, arginine derivatives 5a–e were
envisaged. These could be conveniently prepared from commercial
Fmoc-Orn(Boc)-OH 4 (Scheme 1, route B). First, the Boc group
was removed using a saturated solution of HCl in diethylether.
TFA could also be used but was tedious to remove completely,
which is deleterious for the subsequent step, generally leading
to lower overall yields. After deprotection, the carboxylic acid
was protected in situ as a trimethylsilyl ester by treatment
with N,O-bis(trimethylsilyl)acetamide in the presence of DiPEA.
These conditions were inspired by a paper on guanylation with
trifluoromethyl sulfonamides.17 The resulting precipitates went
into solution virtually instantaneously, after which guanylating
agents 3a–e were added. Although considerable conversion was
observed on TLC after 3 h, complete consumption of starting
Scheme 1 Synthesis of the “Arg-Pro” building block.
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Table 1 Solid phase evaluation of building block 5a
Equiv.
5a
Yield
Resin/product
Cond.a (%)b
2
5
2
5
5
5
5
5
A
A
B
B
C
D
E
E
<10
15
<10
41
<10
28
>95
>95
5
E
>95
Scheme 2 Side product formation.
2
5
5
5
A
A
B
E
<10
<10
34
probability, the guanidine protecting group selected is important,
and we have indeed found that Pbf-protected alkylated arginine
residues may be coupled efficiently.9
>95
On evaluation of coupling conditions (Table 1), we found
that good efficiency (>90% as judged by subsequent Fmoc-
determination) could be achieved by treating the resin with a
solution of 5 equivalents of 5a–e in DCM followed by addition
of 5 equivalents of DIC. As expected, coupling conditions where
a base was present in the reaction mixture enhanced the efficiency
of the side reaction, leading to lower yields (conditions A and
B). Therefore, neutral coupling conditions using DIC are clearly
preferred. However, even without base, the presence of merely
the coupling reagent appeared sufficient to promote significant
side product formation (conditions C and D). Even using the
preferred procedure (condition E), there was competition between
coupling and cyclisation, and an excess of monomer was required.
However, since in this case there was no pre-activation time,
the probability of reaction by coupling was increased, leading
to an optimal coupling efficiency. This procedure worked well
for coupling the monomers to bare Rink-resin20 and to glycine
or leucine modified resin, and to the peptide sequence Arg-Trp-
Gln-Trp-Arg derived from anti-microbial peptide Lactoferricin
B21 leading to H-Arg(R)-Arg-Trp-Gln-Trp-Arg-NH2.
However, when the building blocks were coupled to a proline
residue, poor conversion was observed even with these optimised
conditions, presumably due to the increased steric demand of the
proline nucleophile, which allows more time for the side reaction
to occur.
In order to devise a generally applicable alternative method-
ology for the incorporation of these modified arginine building
blocks, two alternative strategies were envisaged based on ei-
ther incorporation of dipeptide building blocks or orthogonal
deprotection of a complete peptide followed by guanylation. Both
methods were evaluated as part of the preparation of a series of
modified peptides based on a sequence which we have recently
described as a suitable component of bisubstrate-based inhibitors
of individual PKC isozymes.12 The peptide sequence of this
inhibitor was identified from a microarray containing hundreds of
a Conditions: A 5a/BOP/DiPEA in NMP added as a solution. B
5a/HOAt/HATU/DiPEA in NMP added as a solution. C 5a/DIC in
NMP added as a solution. D 5a/DIC in DCM added as a solution. E 5a
in DCM added as a solution, followed by DIC. In all cases the amount of
coupling reagent added corresponded to the number of equivalents of 5a.
Where a base was used, its amount corresponded to two times the number
of equivalents of 5a. b Yields are determined through spectrophotometric
measurement of the amount of Fmoc cleavage product after treatment of
a known quantity of resin with 20% piperidine in NMP.
kinase (peptide) substrates by investigating the phosphorylating
activity of three highly homologous PKC isozymes. It was shown
that this peptide sequence was phosphorylated more strongly by
PKCz compared to PKCa and PKCq.
The first synthetic strategy used dipeptide building blocks
containing a modified arginine residue. These building blocks were
prepared as shown in Scheme 1 (route C). Dipeptides 7a–d were
prepared first by the coupling of unprotected proline, which is
present at the C-terminus of the modifed arginine in the inhibitor
peptide sequence, to the succinimidyl ester of commercially
available Fmoc-Orn(Boc)-OH 4. Then, after acidolysis of the Boc-
group in the resulting dipeptide 6, a guanylation was performed
using azido-reagents 3a–d in a similar procedure as the preparation
of arginine building blocks 5a–e following route B. The reaction
proceeded cleanly in all cases and after column chromatography,
dipeptides 7a–d were obtained in 78–81% yield.
In order to prepare the desired inhibitor peptides, sequence 9
was first prepared using solid phase peptide synthesis (Scheme 3,
route A). Then, dipeptide building blocks 7a–d were coupled under
normal coupling conditions with excellent efficiency within 45 min
as judged by the Kaiser test22 and Fmoc-determination. Synthesis
of the remainder of the peptide proceeded uneventfully and after
cleavage from the resin, a single main product was observed on
RP-HPLC which could be purified by preparative HPLC, after
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Scheme 3 Solid phase synthesis of pseudosubstrates 12a–d.
which the identity of peptides 12a–d was confirmed by mass
spectrometry.
In an alternative approach (Scheme 3, route B), peptide 13
was prepared containing an Aloc-protected ornithine residue.
This protecting group was selectively removed on the solid phase
using [Pd(PPh3)4] and a scavenger. After this deprotection, the
peptide was treated on the solid phase with a solution of pyrazole
derivative 3c and triethylamine in THF. Reaction overnight at
room temperature resulted in a negative Kaiser test, indicating
successful guanylation to the alkylated arginine derivative in 11b.
The peptide was then cleaved and purified using preparative
HPLC, affording peptide 12b.
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Scheme 4 Synthesis of bisubstrate based inhibitor 15.
Table 2 Inhibition data of compounds 12b, 14 and 15
IC50/mMa
Biological activity
We decided to evaluate the biological activity of the peptides
containing a modified arginine residue by converting one of
them into a potential bisubstrate-based inhibitor for PKC. Thus,
peptide 12b was treated with ATP-competitive inhibitor 14 in
the presence of copper sulfate and sodium ascorbate under
microwave irradiation. This led to clean conversion into the desired
bisubstrate-based inhibitor 15 in 62% yield after purification by
preparative HPLC (Scheme 4).
Enzyme
Substrateb
14
12b
15
PKCa
CREB1
KPCB
MARCS
CREB1
KPCB
MARCS
CREB1
KPCB
1.8 0.6
2.9 0.9
1.9 0.5
—
—
—
2.9 0.3
2.3 0.3
2.9 0.4
—
—
—
—
—
—
—
—
—
—
—
—
PKCz
PKCq
0.33 0.029
0.23 0.016
0.59 0.026
0.81 0.019
0.45 0.012
0.17 0.029
Inhibitor 15 was applied in a dynamic peptide microarray-
based kinase inhibition assay using three highly homologous PKC
isozymes (PKCa, z and q). PKCa is a subject of cancer research
because of its ability to regulate phospholipase D,23 PKCz is
involved in the immune system24 and long-term memory,25 and
PKCq has recently emerged as a target in T cell leukaemias.11 The
MARCS
a If no value is given, no inhibition was observed up to 10 mM. Errors are
given as standard error of the mean over six experiments. b CREB1 (cAMP
response element binding protein 126-138, EILSRRPSYRKIL), KPCB
(PKC splice isoform b-II 19-31, RFARKGSLRQKNV), MARCS (myris-
toylated alanine-rich C-kinase substrate 152-164, KKKKKRFSKKSF)
and NCF1 (neutrophil cytosol factor 1 296-308, RGAPPRRSSIRNA).
R
Pamchipꢀ microarray setup used employs chips made of a highly
porous material onto which several hundreds of peptides derived
from endogenous kinase substrates are immobilised in discrete
spots.12,26 The porosity of the surface allows mixtures containing a
kinase, co-factors and fluorescent antibodies against phosphory-
lated amino acid residues for detection, to be pumped through the
solid support of the microarray, so that fluorescence pictures may
be taken from the top-side of the chip while the analyte droplet
with bulk background fluorescence is underneath the surface.
By taking pictures at discrete time intervals, the development of
fluorescence, and therefore the progress of phosphorylation, can
be monitored in real-time, and kinetic data on the kinase reaction
may be obtained.
summarised in Table 2. As can be seen, attachment of the peptide
containing the modified arginine residue has a marked effect
on both the affinity and selectivity of the resulting bisubstrate-
based inhibitor. The IC50 values obtained for 15 were significantly
better than those of ATP-competitive inhibitor 14. It should be
noted that for the inhibition experiments, a high concentration
of ATP (100 mM) was used to obtain strong signals throughout
the experiments, and Ki values can be expected to be in the low
nanomolar range. We previously demonstrated that structures
such as bisubstrate-based inhibitor 15 are competitive inhibitors
both with respect to the ATP- and peptide-binding site.12 Therefore
the Ki may be estimated using the Cheng–Prusoff equation27
The inhibition data for 15 derived from initial velocities (vini)
measured in the presence of different inhibitor concentrations is
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(Ki = IC50/(1+[S]/Km). Employing a Km,ATP of 5 mM previously
determined for the assay described here,12 this leads to a Ki of 8–
39 nM. The peptide part (12b) itself was unable to significantly
inhibit any of the PKC isozymes at the concentrations tested
(10 nM–10 mM).
With respect to the selectivity, ATP-competitive inhibitor 14
only inhibits PKCa and PKCq, whereas bisubstrate-based ini-
hibitor 15 does not inhibit PKCa any more, and instead has affinity
for PKCz and PKCq. Gratifyingly, this matched the selectivity
profile originally observed for this particular peptide sequence
as noted above. This clearly demonstrates the influence of the
peptide part on the inhibitory properties of 15. It is interesting
to note that the previously studied12 bisubstrate-based inhibitors,
which contained a neutral amino acid residue instead of the
positively charged modified arginine monomer in 15, did not show
any inhibitory activity against PKCz, and instead were selective
towards PKCq.
reagent. Column chromatography was carried out using Silicycle
UltraPure silicagel (40–63 mm). For optical rotations [a]D values
are expressed as 10-1 deg cm2 g-1. FTIR spectra were recorded on
a PerkinElmer SpectrumTM 100 system using the ATR accessory.
1H NMR spectra were recorded on a Varian G-300 (300.1 MHz)
spectrometer and chemical shifts are given in ppm relative to TMS
(0.00 ppm). 13C NMR spectra were recorded using the attached
proton test (APT) sequence on a Varian G-300 (75.5 MHz) spec-
trometer and chemical shifts are given in ppm relative to the solvent
signal. Electrospray ionisation mass spectrometry was performed
on a Shimadzu LCMS-QP8000 single quadrupole bench-top mass
spectrometer in positive ionisation mode. MALDI-TOF analysis
was performed on a Kratos Axima CFR apparatus with human
ACTH(18 - 39) (monoisotopic M + H+ 2465.198), bovine insulin
oxidized B chain (monoisotopic M + H+ 3494.651), or bovine
insulin (monoisotopic M + H+ 5730.609) as external references
and a-cyano-4-hydroxycinnamic acid as the matrix. Preparative
HPLC was carried out on a Gilson preparative HPLC system
equipped with a reverse phase C8 column (Alltech Altima XL
Both the improved affinity and changed selectivity profile
demonstrate the power of the novel bisubstrate-based inhibitors
such as 15, and therefore the usefulness of the alkylated charged
arginine residues described in this paper.
˚
C8, 100 A, 10 mm, 250 ¥ 22 mm) using a linear gradient of 100%
buffer A (0.1% TFA in H2O–MeCN 95 : 5 v/v) to 100% buffer
B (0.1% TFA in H2O–MeCN 5 : 95 v/v) in 40 min at a flow
rate of 11.5 mL min-1. Analytical HPLC was carried out on a
Shimadzu HPLC workstation using a reverse phase C8 column
Conclusions
˚
(Alltech Altima XL C8, 90 A, 5 mm, 250 ¥ 4.6 mm) using a linear
We have successfully prepared a series of guanylating reagents
and building blocks for the introduction of arginine derivatives
functionalised with ‘click’ handles on variable spacers. The
monomeric building blocks may be used in certain sequences
under carefully controlled coupling conditions, however, with
demanding sequences, coupling efficiency may be low. Alterna-
tively, dipeptide building blocks are easily accessible and can be
incorporated without problems. Furthermore, it is also possible to
employ the guanylating reagents described in this paper to directly
derivatise a complete peptide by incorporating orthogonally
protected ornithine residues at the desired locations, followed by
treatment with one of the pyrazole derivatives described here.
The resulting modified peptides may be used to attach different
cargos to arginine-containing peptides in an orthogonal way using
click-cycloaddition chemistry. This was illustrated by the incorpo-
ration of an ATP-competitive inhibitor, leading to bisubstrate-
based kinase inhibitor 15, and can be easily extended to the
introduction of, for example, fluorescent labels and radionuclide
chelators.
gradient of the same buffers as above in 20 min at a flow rate
of 1.0 mL min-1. Microwave assisted reactions were conducted in
closed reaction vessels using a Biotage Initiator microwave reactor
equipped with a temperature and pressure sensor for monitoring
reaction conditions.
General procedure A
N,N-Bis-Boc-1-guanylpyrazole 1 (620 mg, 2.0 mmol), alcohol 2a–
e (2.0 mmol) and triphenylphosphine (786 mg, 3.0 mmol) were
dissolved in THF (15 mL). At 0 ◦C diethyl azodicarboxylate
(471 mL, 3.0 mmol) was added dropwise over a period of 30 min.
The reaction mixture was stirred at room temperature for 16 h.
EtOAc (30 mL) and water were added and the layers separated.
After washing with brine, the organic layer was dried with Na2SO4
and concentrated under reduced pressure. The resulting crude
product was purified by column chromatography.
The thus developed bisubstrate-based kinase inhibitor was
tested for affinity and selectivity towards three highly homologous
PKC isozymes. The resulting compound showed improved affinity
and a highly interesting shift in selectivity.
General procedure B
Fmoc-Orn(Boc)-OH 4 (454 mg, 1.0 mmol) was dissolved in a
mixture of DCM (4.0 mL) and a saturated solution of HCl in
diethylether (4.0 mL). After stirring at room temperature for
20 min, a white precipitate had formed and the reaction mixture
was evaporated to dryness. The resulting crude HCl salt was
suspended in DCM (5.0 mL) and DiPEA (348 mL, 2.0 mmol)
was added. After addition of N,O-bis(trimethylsilyl)acetamide
(489 mL, 2.0 mmol), all precipitates went into solution instantly.
After stirring at room temperature for 15 min, the guanylating
agent 3a–e (1.0 mmol) was added and the reaction mixture was
stirred for 24 h at room temperature. After addition of DCM
(20 mL), washing with 1 M KHSO4 (20 mL) and brine (20 mL),
and drying on Na2SO4, the solvents were evaporated. The resulting
crude product was purified by column chromatography.
Experimental
General remarks
All reactions were carried out at ambient temperature unless stated
otherwise. All reagents were used as supplied from commercial
sources unless stated otherwise. THF was distilled from LiAlH4
prior to use. DCM, NMP and DiPEA were stored on molecular
˚
sieves (4 A) prior to use. Rf values were determined by thin layer
chromatography (TLC) on Merck precoated silicagel 60F254 plates.
Spots were visualized by UV-quenching, ninhydrin or Hanessian’s
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General procedure C
N-(2-(2-Azidoethoxy)ethyl)-N,N¢-bis-Boc-1-guanylpyrazole 3b
Fmoc-Orn(Boc)-Pro-OH 6 (552 mg, 1.0 mmol) was dissolved in
a mixture of DCM (5.0 mL) and sat. HCl in Et2O (5.0 mL), and
stirred for 15 min, after which the solvents were evaporated. The re-
sulting white solid was suspended in dry THF (10 mL) after which
DiPEA (523 mL, 3.0 mmol) and N,O-bis(trimethylsilyl)acetamide
(734 mL, 3.0 mmol) were added. This mixture was stirred for
10 min at room temperature, at which point a clear solution
was obtained. Guanylating agent 3a–d (1.0 mmol) was added and
the reaction mixture stirred for 24 h at room temperature, after
which the solvent was evaporated. The residue was suspended
in EtOAc (10 mL), washed with 1 M HCl (10 mL) and dried
on Na2SO4. The resulting crude product was purified by column
chromatography.
This compound was prepared according to General procedure A.
The crude product was purified using column chromatography
(EtOAc–hexanes, 1 : 4 v/v) yielding 3c (706 mg, 84%) as a clear,
colorless oil. (Found C, 51.2; H, 6.7; N, 23.1. C18H29N7O5 requires
C, 51.05; H, 6.9; N, 23.15); Rf 0.74 (EtOAc–hexanes, 1 : 1 v/v);
n
max/cm-1 2101 (N3), 1726 (CO), 1664 (CN); dH (300 MHz; CDCl3;
Me4Si) 1.12 (9 H, s, C(CH3)3), 1.34 (9 H, s, C(CH3)3), 3.10 (2 H,
t, J 4.9, CH2), 3.42 (2 H, t, J 5.0, CH2), 3.62 (2 H, t, J 5.4, CH2),
3.77 (2 H, t, J 5.0, CH2), 6.26 (1 H, m, ArH), 7.52 (1 H, t, J 0.8,
ArH), 7.82 (1 H, s, ArH); dC (75.5 MHz; CDCl3; Me4Si) 27.4,
27.6 (C(CH3)3), 47.4, 50.4, 68.4, 69.0 (CH2), 81.9, 82.5 (C(CH3)3),
=
=
108.5, 129.7, 142.7 (ArC), 152.1 (C N), 157.2 (C O); m/z (ESI)
(M + H+. C18H30N7O5 requires 424.2), 424.5.
N-(2-(2-(2-Azidoethoxy)ethoxy)ethyl)-N,N¢-bis-Boc-1-
guanylpyrazole 3c
General procedure D
R
The peptide was prepared on Tentagelꢀ S RAM resin on a
This compound was prepared according to General procedure A.
The crude product was purified using column chromatography
(EtOAc–hexanes, 1 : 4 v/v) yielding 3d (660 mg, 70%) as a clear,
colorless oil. (Found C, 51.3; H, 7.3; N, 21.1. C20H33N7O6 requires
C, 51.4; H, 7.1; N, 21.0); Rf 0.66 (EtOAc–hexanes, 1 : 1 v/v);
0.080 mmol scale using Fmoc–tBu solid phase chemistry. Agita-
tion was achieved by bubbling with nitrogen. Fmoc-deprotection:
The resin was treated with 20% piperidine in DMF (2.0 mL, 3 ¥
8 min), followed by washing with DMF (2.0 mL, 3 ¥ 2 min) and
DCM (2.0 mL, 3 ¥ 2 min). A positive Kaiser test22 indicated
successful deprotection. Coupling: The resin was treated with a
solution of Fmoc-Xxx-OH (0.32 mmol), HBTU (0.32 mmol),
HOBt (0.32 mmol) and DiPEA (0.64 mmol) in DMF (2.0 mL)
for 30 min. The resin was washed with DMF (2.0 mL, 3 ¥
2 min) and DCM (2.0 mL, 3 ¥ 2 min). A negative Kaiser test
indicated successful coupling. Capping: The resin was treated with
a solution of Ac2O (0.5 M), DiPEA (0.125 M) and HOBt·H2O
(0.015 M) in DMF (2.0 mL, 2 ¥ 10 min). The resin was washed
with DMF (2.0 mL, 3 ¥ 2 min) and DCM (2.0 mL, 3 ¥ 2 min).
A negative Kaiser test indicated successful capping. Cleavage and
deprotection: The resin was stirred with a mixture of TFA (9.5 mL),
TIS (0.25 mL) and H2O (0.25 mL) for 4 h at room temperature,
after which it was removed by filtration and rinsed with TFA
(3.0 mL). The crude peptide was precipitated from the combined
filtrate by dropwise addition to a cold (-20 ◦C) mixture of MTBE
and hexanes (40 mL, 1 : 1 v/v). After centrifugation (3000 rpm,
5 min) and decantation, the pellet was washed with ether (3 ¥
40 mL). The resulting crude peptide was dissolved in water and
lyophilized.
n
max/cm-1 2102 (N3), 1726 (CO), 1665 (CN); dH (300 MHz; CDCl3
Me4Si) 1.25 (9 H, s, C(CH3)3), 1.49 (9 H, s, C(CH3)3), 3.32 (2 H, t,
J 5.1, CH2), 3.55 (6 H, m, 3 ¥ CH2), 3.75 (2 H, t, J 5.6, CH2), 3.90
(2 H, t, J 5.2, CH2), 6.38 (1 H, dd, J 1.1 and 1.6, ArH), 7.66 (1 H,
dd, J 0.5 and 1.1, ArH), 7.95 (1 H, s,ArH); dC (75.5 MHz; CDCl3;
Me4Si) 27.9, 28.2 (C(CH3)3), 48.1, 50.9 (NCH2), 60.0, 70.2, 70.4,
70.7 (OCH2), 82.4, 83.0 (C(CH3)3), 108.8, 130.4, 143.2 (ArC),
+
=
=
152.6 (C N), 157.7 (C O); m/z (ESI) (M + H . C20H34N7O6
requires 468.3), 468.4.
N-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-N,N¢-bis-Boc-1-
guanylpyrazole 3d
This compound was prepared according to General procedure
A. The crude product was purified using column chromatography
(MeOH–DCM, 0.5 : 99.5 v/v) yielding 3e (800 mg, 78%) as a clear,
colorless oil. (Found C, 51.6; H, 7.3; N, 19.3. C22H37N7O7 requires
C, 51.65; H, 7.3; N, 19.2); Rf 0.66 (EtOAc–hexanes, 1 : 1 v/v);
n
max/cm-1 2102 (N3), 1727 (CO), 1664 (CN); dH (300 MHz; CDCl3;
Me4Si) 1.27 (9 H, s, C(CH3)3), 1.50 (9 H, s, C(CH3)3), 3.37 (2 H, t,
J 5.1, CH2), 3.59 (10 H, m, 5 ¥ CH2), 3.75 (2 H, t, J 5.6, CH2), 3.91
(2 H, t, J 5.2, CH2), 6.40 (1H, dd, J 1.1 and 1.6, ArH), 7.68 (1 H,
d, J 1.1, ArH), 7.98 (1 H, s, ArH); dC (75.5 MHz; CDCl3; Me4Si)
27.4, 27.6 (C(CH3)3), 47.6, 60.4 (NCH2), 68.3, 69.7, 69.9, 70.3
N-(2-Azidoetyl)-N,N¢-bis-Boc-1-guanylpyrazole 3a
This compound was prepared according to General procedure A.
The crude product was purified using column chromatography
(EtOAc–hexanes, 1 : 9 v/v) yielding 3b (610 mg, 86%) as a clear,
colorless oil. (Found C, 50.9; H, 6.5; N, 25.6. C16H25N7O4 requires
C, 50.65; H, 6.6; N, 25.8); Rf 0.76 (EtOAc–hexanes, 1 : 2 v/v);
(OCH2), 81.9, 82.4 (C(CH3)3), 108.4, 130.0, 142.7 (ArC), 152.1
+
=
=
(C N), 157.2 (C O); m/z (ESI) (M + H . C22H38N7O7 requires
512.3), 512.1.
n
max/cm-1 2105 (N3), 1724 (CO), 1660 (CN); dH (300 MHz; CDCl3;
N,N¢-Bis-Boc-N-propargyl-1-guanylpyrazole 3e
Me4Si) 1.26 (9 H, s, C(CH3)3), 1.48 (9 H, s, C(CH3)3), 3.63 (2 H,
t, J 6.1, CH2N3), 3.84 (2 H, t, J 6.1, CH2NBoc), 6.41 (1 H, m,
ArH), 7.67 (1 H, t, J 0.7, ArH), 7.95 (1H, s, ArH); dC (75.5 MHz;
CDCl3; Me4Si) 27.6 and 27.8 (C(CH3)3), 47.4, 49.6 (CH2),
82.6, 83.2 (C(CH3)3), 109.0, 130.0, 143.2 (ArC), 152.1 (C N),
157.3 (C O); m/z (ESI) (M + H . C16H26N7O4 requires 380.2),
This compound was prepared according to General procedure A.
The crude product was purified using column chromatography
(EtOAc–hexanes, 1 : 4 v/v) yielding 3a (550 mg, 79%) as a white
solid. (Found C, 58.9; H, 6.8; N, 16.3. C17H24N4O4 requires C, 58.6;
H, 6.9; N, 16.1); Rf 0.80 (EtOAc–hexanes, 1 : 1 v/v); nmax/cm-1 1737
and 1709 (CO), 1653 (CN); dH (300 MHz; CDCl3; Me4Si) 1.30
=
+
=
379.8.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 1629–1639 | 1635
©

View Article Online
≡
(9 H, s, C(CH3)3), 1.50 (9 H, s, C(CH3)3), 2.27 (1 H, s, CH), 4.48
54.5 (Ca), 66.6, 69.1,69.8, 70.0, 70.3 (Fmoc-CH2 and OCH2), 82.9
≡
(2 H, s, CH2C ), 6.41 (1 H, s, ArH), 7.70 (1 H, s, ArH), 7.95 (1 H, s,
(C(CH3)3), 119.8, 125.0, 126.9, 127.5, 128.1, 141.1, 143.7 (ArC),
+
=
=
ArH); dC (75.5 MHz; CDCl3; Me4Si) 27.5, 27.7 (C(CH3)3), 37.9
(CH2), 72.8 ( CH), 77.4 (C CH), 82.3, 83.4 (C(CH3)3), 109.0,
129.8, 143.2 (ArC), 151.4 (C N), 156.9 (C O); m/z (ESI) (M +
143.9 (C N), 151.4, 155.9, 176.8 (C O); m/z (ESI) (M + H .
C37H52N7O10 requires 754.4), 753.9.
≡
≡
=
=
H+. C17H25N4O4 requires 349.2), 349.2.
Fmoc-Nx-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-ethyl)-Nx,Nx¢-
bis-Boc-L-arginine 5d
Fmoc-Nx-(2-azidoethyl)-Nx,Nx¢-bis-Boc-L-arginine 5a
This compound was prepared according to General procedure
B. After purification by column chromatography (MeOH–DCM,
4 : 96 v/v), 5e (503 mg, 63%) was obtained as a white foam. (Found
C, 58.8; H, 6.8; N, 12.55. C39H55N7O11 requires C, 58.7; H, 6.95;
N, 12.3); Rf 0.51 (MeOH–DCM, 1 : 9 v/v); [a]2D0 +6.0 (c 0.21 in
MeOH); dH (300 MHz; CDCl3; Me4Si) 1.46 (9 H, s, C(CH3)3), 1.48
(9 H, s, C(CH3)3), 1.72 (4 H, br m, CbH2 and Cg H2), 3.32 (4 H, m,
CdH2 and CH2), 3.57 (14H, m, 7 ¥ CH2), 4.19 (1 H, t, J 6.9, CaH),
4.36 (3 H, m, Fmoc-CH and Fmoc-CH2), 6.00 (1 H, br s, Fmoc-
NH), 7.29 (4 H, m, ArH), 7.61 (2 H, m, ArH), 7.34 (2 H, d, J 7.8,
ArH), 11.6 (2 H, br s, OH and NH); dC (75.5 MHz; CDCl3; Me4Si)
24.4 (Cb), 27.9 (C(CH3)3), 29.9 (Cg ), 47.1 (Fmoc-CH), 48.3 (Cd),
50.4 (CH2), 54.1 (Ca), 66.6, 68.9, 69.7, 69.9, 70.3, 70.4 (Fmoc-CH2
and CH2), 82.2 (C(CH3)3), 119.8, 125.0, 126.9, 127.5, 141.1, 143.7
This compound was prepared according to General procedure
B. After column chromatography (MeOH–DCM, 4 : 96 v/v), 5b
(411 mg, 62%) was obtained as a white foam. (Found C, 59.7;
H, 6.4; N, 15.0. C33H43N7O8 requires C, 59.5; H, 6.5; N, 14.7); Rf
0.35 (MeOH–DCM, 1 : 9 v/v); [a]2D0 +8.3 (c 0.28 in MeOH); dH
(300 MHz; CDCl3; Me4Si) 1.46 (9 H, s, C(CH3)3), 1.48 (9 H, s,
C(CH3)3), 1.77 (4 H, br m, CbH2 and Cg H2), 3.33 (2H, s, CdH2),
3.51 (2 H, s, CH2), 3.69 (2 H, s, CH2), 4.19 (1 H, m, CaH), 4.33
(3 H, m, Fmoc-CH and Fmoc-CH2), 5.96 (1 H, br s, Fmoc-NH),
7.29 (4 H, m, ArH), 7.60 (2 H, m, ArH), 7.73 (2 H, d, J 7.1, ArH),
11.7 (2 H, br s, NH and OH); dC (75.5 MHz; CDCl3; Me4Si)
24.5 (Cb), 27.8 (C(CH3)3), 29.9 (Cg ), 47.0 (Fmoc-CH), 49.9 (CH2),
53.3 (Cd), 55.9 (Ca), 66.7 (Fmoc-CH2), 83.6 (C(CH3)3), 119.7,
=
=
=
125.0, 126.8, 127.5, 141.0, 143.7 (ArC), 143.8 (C N), 141.8, 155.9,
(ArC), 143.9 (C N), 151.3, 155.8, 176.0 (C O); m/z (ESI) (M +
+
176.1 (C O); m/z (ESI) (M + H . C33H44N7O8 requires 666.3),
H+. C39H56N7O11 requires 798.4), 798.9.
=
666.2.
Fmoc-Nx,Nx¢-bis-Boc-Nx-propargyl-L-arginine 5e
Fmoc-Nx-(2-(2-azidoethoxy)-ethyl)-Nx,Nx¢-bis-Boc-L-arginine 5b
This compound was prepared according to General procedure
B. After column chromatography (MeOH–DCM, 5 : 95 v/v), 5a
(518 mg, 82%) was obtained as a white foam. (Found C, 64.5;
H, 6.6; N, 8.7. C34H42N4O8 requires C, 64.3; H, 6.7; N, 8.8); Rf
0.45 (MeOH–DCM, 1 : 9 v/v); [a]2D0 +4.5 (c 0.27 in MeOH); dH
(300 MHz; CDCl3; Me4Si) 1.34 (9 H, s, C(CH3)3), 1.35 (9 H, s,
This compound was prepared according to General procedure
B. After purification by column chromatography (MeOH–DCM,
4 : 96 v/v), 5c (451 mg, 64%) was obtained as a white foam. (Found
C, 59.2; H, 6.5; N, 13.5. C35H47N7O9 requires C, 59.2; H, 6.7;
N, 13.8); Rf 0.31 (MeOH–DCM, 1 : 9 v/v); [a]2D0 +7.2 (c 0.20 in
MeOH); dH (300 MHz; CDCl3; Me4Si) 1.47 (9 H, s, C(CH3)3), 1.48
(9 H, s, C(CH3)3), 1.81 (4 H, m, CbH2 and Cg H2), 3.30-3.71 (10 H,
br m, CdH2 and 4 ¥ CH2), 4.19 (1 H, t, J 7.0, CaH), 4.34 (3 H, m,
Fmoc-CH and Fmoc-CH2), 6.02 (1 H, br s, Fmoc-NH), 7.30 (4
H, m, ArH), 7.60 (2 H, m, ArH), 7.73 (2 H, d, J 7.3, ArH), 12.2
(2 H, br s, NH and OH); dC (75.5 MHz; CDCl3; Me4Si) 24.2 (Cb),
27.9 (C(CH3)3), 30.0 (Cg ), 47.0 (Fmoc-CH), 48.3 (Cd), 50.4 (CH2),
54.1 (Ca), 66.7, 69.0, 69.4 (Fmoc-CH2 and CH2), 83.8 (C(CH3)3),
b
g
≡
C(CH3)3), 1.64 (4 H, m, C H2 and C H2), 2.21 (1 H, br s, CH),
a
≡
3.21 (2 H, br s, CH2C ), 4.05 (1 H, m, C H), 4.21 (3 H, m, Fmoc-
CH and Fmoc-CH2), 5.78 (1 H br d, J 5.2, Fmoc-NH), 7.18 (4
H, m, ArH), 7.44 (2 H, m, ArH), 7.60 (2 H, d, J 7.4, ArH), 11.6
(2H, br s, NH and OH); dC (75.5 MHz; CDCl3; Me4Si) 25.3 (Cb),
27.9 (C(CH3)3), 30.9 (C ), 36.8 ( CH), 41.4 (C ), 45.0 (CH2C ),
47.0 (Fmoc-CH), 54.2 (C ), 66.5 (Fmoc-CH2), 67.7 (C CH), 82.8
g
d
≡
≡
a
≡
(C(CH3)3), 119.7, 124.9, 126.8, 127.4, 141.0, 143.6 (ArC), 143.8
(C N), 152.9, 155.8, 156.2, 175.8 (C O); m/z (ESI) (M + H .
+
=
=
=
119.8, 125.0, 126.9, 127.5, 141.1, 143.7 (ArC), 143.9 (C N), 151.9,
+
=
155.8, 176.2 (C O); m/z (ESI) (M + H . C35H48N7O9 requires
C34H43N4O8 requires 635.3), 635.5.
710.4), 710.8.
Fmoc-Orn(Boc)-Pro-OH 6
Fmoc-Nx-(2-(2-(2-azidoethoxy)ethoxy)-ethyl)-Nx,Nx¢-bis-Boc-L-
arginine 5c
Fmoc-Orn(Boc)-OH
4
(909 mg, 2.0 mmol) and N-
hydroxysuccinimide (230 mg, 2.0 mmol) were dissolved in
dry DCM (10 mL). After cooling to 0 ◦C, EDCI·HCl (383 mg,
2.0 mmol) was added and the resulting mixture stirred for 3 h
at room temperature. After washing with 1 M HCl (10 mL) and
brine (10 mL), and drying on Na2SO4, the solvent was evaporated
and the crude activated ester redissolved in DMF (10 mL).
H–Pro–OH (253 mg, 2.2 mmol) and DiPEA (383 mL, 2.2 mmol)
were added and the reaction mixture was stirred overnight at
room temperature. After evaporation to dryness, the residue was
redissolved in EtOAc (10 mL), washed with 1 M HCl (10 mL)
and dried on Na2SO4. Column chromatography (MeOH–DCM,
5 : 95 v/v) yielded 6 (625 mg, 57%) as a white foam. (Found C,
65.6; H, 6.8; N, 7.3. C30H37N3O7 requires C, 65.3; H, 6.8; N, 7.6);
This compound was prepared according to General procedure
B. After purification by column chromatography (MeOH–DCM,
8 : 92 v/v), 5d (460 mg, 61%) was obtained as a white foam. (Found
C, 58.8; H, 6.8; N, 13.1. C37H51N7O10 requires C, 58.95; H, 6.8;
N, 13.0); Rf 0.41 (MeOH–DCM, 1 : 9 v/v); [a]2D0 +7.3 (c 0.24 in
MeOH); dH (300 MHz; CDCl3; Me4Si) 1.45 (9 H, s, C(CH3)3), 1.47
(9 H, s, C(CH3)3), 1.79 (4 H, m, CbH2 and Cg H2), 3.29 (4 H, m,
CdH2 and CH2), 3.61 (10 H, m, 5 ¥ CH2), 4.19 (1 H, m, CaH), 4.32
(3 H, br m, Fmoc-CH and Fmoc-CH2), 6.07 (1 H, s, Fmoc-NH),
7.28 (4 H, m, ArH), 7.59 (2 H, m, ArH), 7.59 (2 H, m, ArH), 11.0
(2 H, br s, NH and OH); dC (75.5 MHz; CDCl3; Me4Si) 24.4 (Cb),
27.9 (C(CH3)3), 30.0 (Cg ), 47.0 (Fmoc-CH), 48.3 (Cd), 50.4 (CH2),
1636 | Org. Biomol. Chem., 2010, 8, 1629–1639
This journal is
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©

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Rf 0.23 (MeOH–DCM, 1 : 9 v/v); dH (300 MHz; CDCl3; Me4Si)
1.42 (9 H, s, C(CH3)3), 1.40-2.21 (8 H, br m, Pro-CbH2, Pro-Cg H2,
Orn-CbH2 and Orn-Cg H2), 3.07 (2 H, m, Orn-CdH2), 3.68 (2 H,
m, Pro-CdH2), 4.08 (3 H, m, Fmoc-CH and Fmoc-CH2), 4.55 (2
H, m, Pro-CaH and Orn-CaH), 5.06 (1 H, br s, Boc-NH), 6.79 (1
H, br s, Fmoc-NH), 7.26 (4 H, m, ArH), 7.58 (2 H, m, ArH), 7.73
(2 H, d, J 7.4, ArH), 7.80 (1 H, br s, OH); dC (75.5 MHz; CDCl3;
Me4Si) 24.9, 25.7 (Orn-Cb and Pro-Cb), 28.3 (C(CH3)3), 28.7, 29.0
(Pro-Cg and Orn-Cg ), 37.0 (Fmoc-CH), 40.1, 47.1 (Pro-Cd and
Orn-Cd), 52.3, 59.1 (Pro-Ca and Orn-Ca), 66.9 (Fmoc-CH2), 79.3
Fmoc-Nx-(2-(2-(2-azidoethoxy)ethoxy)-ethyl)-Nx,Nx¢-bis-Boc-
Arg-Pro-OH 7c
This compound was prepared according to General Procedure C.
Column chromatography (MeOH–DCM, 5 : 95 v/v) yielded 7c
(687 mg, 81%) as a white foam. (Found C, 59.15; H, 6.5; N, 13.2.
C42H58N8O11 requires C, 59.3; H, 6.9; N, 13.2); Rf 0.35 (MeOH–
DCM, 1 : 9 v/v); [a]2D0 -32.8 (c 0.10 in MeOH); dH (300 MHz;
CDCl3; Me4Si) 1.46 (9 H, s, C(CH3)3), 1.48 (9 H, s, C(CH3)3),
1.58-2.23 (8 H, br m, Pro-CbH2, Pro-Cg H2, Arg-CbH2 and Arg-
Cg H2), 3.20-3.80 (16 H, br m, 6 ¥ CH2, Pro-CdH2 and Arg-CdH2),
4.16 (1 H, m, Fmoc-CH), 4.28 (2 H, d, J 7.2, Fmoc-CH2), 4.55 (2
H, m, Pro-CaH and Arg-CaH), 6.72 (1 H, br s, Fmoc-NH), 7.32
(4 H, m, ArH), 7.61 (2 H, m, ArH), 7.74 (2 H, d, J 7.1, ArH),
9.60 (2 H, br s, OH and NH); dC (75.5 MHz; CDCl3; Me4Si)
28.0 (C(CH3)3), 24.5, 24.9, 28.7, 29.2 (Pro-Cb, Pro-Cg , Arg-Cb and
Arg-Cg ), 39.7 (Pro-Cd and Arg-Cd), 50.4 (CH2), 47.0, 52.3 (Pro-Ca
and Arg-Ca), 59.3 (Fmoc-CH), 66.8, 69.2, 69.7, 70.0 (Fmoc-CH2
and CH2), 82.5 (C(CH3)3), 119.7, 125.1, 127.0, 127.5, 141.1, 143.6
(C(CH3)3), 119.7, 125.2, 126.9, 127.5, 141.1, 143.7, 143.9 (ArC),
+
=
156.4, 171.6, 174.2 (C O); m/z (ESI) (M + H . C30H38N3O7
requires 552.3), 551.8.
Fmoc-Nx-(2-azidoethyl)-Nx,Nx¢-bis-Boc-Arg-Pro-OH 7a
This compound was prepared according to General procedure C.
Column chromatography (MeOH–DCM, 5 : 95 v/v) yielded 7a
(615 mg, 81%) as a white foam. (Found C, 60.0; H, 6.5; N, 14.8.
C38H50N8O9 requires C, 59.8; H, 6.6; N, 14.7); Rf 0.36 (MeOH–
DCM, 9 : 1 v/v); [a]2D0 -25.6 (c 0.19 in MeOH); dH (300 MHz;
CDCl3; Me4Si) 1.47 (9 H, s, C(CH3)3), 1.48 (9 H, s, C(CH3)3),
1.66-2.25 (8 H, m, Pro-CbH2, Pro-Cg H2, Arg-CbH2 and Arg-Cg H2),
3.20-3.80 (8 H, br m, 2 ¥ CH2, Pro-CdH2 and Arg-CdH2), 4.18 (1 H,
m, Fmoc-CH), 4.28 (2 H, d, J 6.1, Fmoc-CH2), 4.55 (2 H, m, Pro-
CaH, Arg-CaH), 6.61 (1 H, br s, Fmoc-NH), 7.32 (4 H, m, ArH),
7.60 (2 H, m, ArH), 7.73 (2 H, d, J 7.5, ArH), 9.58 (2 H, br s, OH
and NH); dC (75.5 MHz; CDCl3; Me4Si) 24.9 (Pro-Cb and Arg-
Cb), 28.0 (C(CH3)3), 26.2, 28.6 (Pro-Cg and Arg-Cg ), 43.5 (Pro-Cd
and Arg-Cd), 46.9 (Fmoc-CH), 46.7, 50.0 (CH2), 52.0, 59.2 (Pro-
Ca and Arg-Ca), 66.9 (Fmoc-CH2), 80.1, 83.2 (C(CH3)3), 119.7,
=
=
(ArC), 143.9 (C N), 151.9, 156.2, 170.9, 173.6 (C O); m/z (ESI)
(M + H+. C42H59N8O11 requires 851.4), 851.6.
Fmoc-Nx-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-ethyl)-Nx,Nx¢-
bis-Boc-Arg-Pro-OH 7d
This compound was prepared according to General Procedure C.
Column chromatography (MeOH–DCM, 5 : 95 v/v) yielded 7d
(701 mg, 78%) as a white foam. (Found C, 58.9; H, 7.15; N, 12.8.
C44H62N8O12 requires C, 59.05; H, 7.0; N, 12.5); Rf 0.28 (MeOH–
DCM, 1 : 9 v/v); [a]2D0 +4.6 (c 2.86 in MeOH); dH (300 MHz;
CDCl3; Me4Si) 1.46 (9 H, s, C(CH3)3), 1.48 (9 H, s, C(CH3)3),
1.57-2.22 (8 H, br m, Pro-CbH2, Pro-Cg H2, Arg-CbH2 and Arg-
Cg H2), 3.33 (4 H, m, 2 ¥ CH2), 3.61 (16 H, m, 6 ¥ CH2, Pro-CdH2
and Arg-CdH2), 4.17 (1 H, t, J 6.9, Fmoc-CH), 4.30 (2 H, d, J 6.9,
Fmoc-CH2), 4.56 (2 H, m, Pro-CaH and Arg-CaH), 6.43 (1 H, br s,
Fmoc-NH), 7.34 (4 H, m, ArH), 7.59 (2 H, m, ArH), 7.74 (2 H, d,
J 7.4, ArH), 8.13 (2 H, br s, NH and OH); dC (75.5 MHz; CDCl3;
Me4Si) 28.1 (C(CH3)3), 24.9, 28.6, 29.4, 29.5 (Pro-Cb, Arg-Cb, Pro-
Cg and Arg-Cg ), 47.0, 52.1 (Pro-Ca and Arg-Ca), 50.5 (Pro-Cd and
Arg-Cd), 59.2 (Fmoc-CH), 66.8, 69.1, 69.9, 70.2, 70.5 (Fmoc-CH2
and CH2), 79.9, 82.3 (C(CH3)3), 119.8, 125.1, 127.0, 127.6, 141,1,
=
125.1, 126.9, 127.5, 141.1, 143.6 (ArC), 143.9 (C N), 156.3, 170.9,
+
=
174.1 (C O); m/z (ESI) (M + H . C38H51N8O9 requires 763.4),
763.3.
Fmoc-Nx-(2-(2-azidoethoxy)-ethyl)-Nx,Nx¢-bis-Boc-Arg-Pro-OH
7b
This compound was prepared according to General procedure C.
Column chromatography (MeOH–DCM, 8 : 92 v/v) yielded 7b
(637 mg, 79%) as a white foam. (Found C, 59.7; H, 6.6; N, 14.1.
C40H54N8O10 requires C, 59.5; H, 6.75; N, 13.9); Rf 0.35 (MeOH–
DCM, 1 : 9 v/v); [a]2D0 -22.7 (c 0.10 in MeOH); dH (300 MHz;
CDCl3; Me4Si) 1.46 (9 H, s, C(CH3)3), 1.48 (9 H, s, C(CH3)3),
1.63-2.19 (8 H, br m, Pro-CbH2, Pro-Cg H2, Arg-CbH2 and Arg-
Cg H2), 3.23-3.80 (12 H, br m, 4 ¥ CH2, Pro-CdH2 and Arg-CdH2),
4.25 (3 H, m, Fmoc-CH and Fmoc-CH2), 4.56 (2 H, m, Pro-CaH
and Arg-CaH), 6.98 (0.5 H, d, J 7.9, Fmoc-NHa), 7.14 (0.5 H,
d, J 7.1, Fmoc-NHb), 7.23 (4 H, m, ArH), 7.59 (2 H, m, ArH),
7.72 (2 H, d, J 7.4, ArH), 10.5 (2 H, br s, OH and NH); dC
(75.5 MHz; CDCl3; Me4Si) 27.8 (C(CH3)3), 24.7, 24.9, 28.6, 29.0
(Pro-Cb, Pro-Cg , Arg-Cb and Arg-Cg ), 40.4 (Pro-Cd and Arg-Cd),
50.3 (CH2), 46.8, 52.1 (Pro-Ca and Arg-Ca), 59.1 (Fmoc-CH),
66.6, 68.9, 69.0 (Fmoc-CH2 and CH2), 79.2, 82.4, 83.0 (C(CH3)3),
119.5, 125.0, 125.1, 126.8, 127.3, 127.9, 128.7, 140.9, 143.5 (ArC),
=
=
143.7 (ArC), 143.9 (C N), 151.9, 156.0, 170.7, 173.3 (C O); m/z
(ESI) (M + H+. C44H63N8O12 requires 895.5), 895.3.
Monoethyleneglycol peptide 12a
This compound was prepared according to General procedure D.
Preparative RP-HPLC yielded 12a (27.6 mg, 20%, 94% per step) as
a white solid m/z (ESI) (M + 2H2+. C77H135N27O18 requires 863.0),
863.1; Rt = 17.6 min.
Diethyleneglycol peptide 12b
Method 1. This compound was prepared according to General
procedure D. Preparative RP-HPLC yielded 12b (39.6 mg, 28%,
95% per step) as a white solid.
Method 2. This compound was prepared according to General
procedure D, incorporating Fmoc-Orn(Aloc)-OH at the guanyla-
tion site. The Aloc-group was removed by treatment of the resin
with a solution of PhSiH3 (1.6 mmol) and [Pd(PPh3)4] (8 mmol) in
=
143.7 (C N), 151.8, 152.8, 156.0, 156.2, 162.7, 170.8, 170.9, 174.0,
+
=
174.2 (C O); m/z (ESI) (M + H . C40H55N8O10 requires 807.4),
807.1.
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NMP (2.0 mL) for 45 min while bubbling through Ar. The resin
was then washed with NMP (2.0 mL), diethyldithiocarbamate
trihydrate (2.0 mL), a 20% solution of DiPEA in NMP (2.0 mL)
and DCM (2.0 mL). The deprotection was repeated three times.
Guanylation was achieved by treatment with a solution of N,O-
(bistrimethylsilyl)acetamide (0.32 mmol), DiPEA (0.32 mmol)
and 3e (0.32 mmol) in DCM (2.0 mL) for 48 h. The resin was
then washed and the peptide cleaved according to the protocols
described in General procedure D.
analysed by BioNavigator software (Pamgene, ‘s-Hertogenbosch,
The Netherlands).
Notes and references
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m/z (ESI) (M + 2H2+. C79H139N27O19 requires 885.0), 885.4.
Triethyleneglycol peptide 12c
3 R. Y. Tan and A. D. Frankel, Proc. Natl. Acad. Sci. U. S. A., 1995,
92, 5282; J. Koeck, M. Nassal, K. Deres, H. E. Blum and F. von
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This compounds was prepared according to General procedure D.
Preparative RP-HPLC yielded 12c (29.6 mg, 20%, 94% per step) as
a white solid m/z (ESI) (M + 2H2+. C81H143N27O20 requires 907.0),
907.2; Rt = 17.9 min.
4 D. I. Chan, E. J. Prenner and H. J. Vogel, Biochim. Biophys. Acta,
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Tetraethyleneglycol peptide 12d
This compound was prepared according to General procedure D.
Preparative RP-HPLC yielded 12d (49.0 mg, 41%, 96% per step) as
a white solid m/z (ESI) (M + 2H2+. C83H147N27O21 requires 929.1),
929.3; Rt = 17.9 min.
5 C.-B. Yim, O. C. Boerman, M. de Visser, M. de Jong, A. C. Dechesne,
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Bisubstrate-based inhibitor 15
Peptide 12b (10.0 mg, 5.6 mmol), CuSO4·5H2O (1.4 mg, 5.6 mmol),
staurosporine mimic 1412 (2.5 mg, 5.6 mmol) and sodium ascorbate
(1.1 mg, 5.6 mmol) were dissolved in a 1 : 1 v/v tert-BuOH : H2O
(5.0 mL). The resulting reaction mixture was heated under
microwave irradiation to 80 ◦C for 20 min.28 After lyophilization,
the peptide was purified by preparative HPLC. Lyophilization of
pure fractions afforded 15 (7.7 mg, 62%) as a red powder m/z
(MALDI) (M + H+. C107H164N31O21 requires 2219.3), 2219.6; Rt =
18.6 min.
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One 96 well plate was used for each PKC isozyme, which was
fully blocked by treatment with 2% BSA (aq) for 15 min prior
to use. The inhibitor of interest was dissolved in DMSO, after
which serial dilutions were made in 20% aqueous DMSO at
10 times the final concentrations. Then, to all 96 wells were
added a mixture of water (6.25 mL), a 0.5 mg mL-1 solution of
phosphatidylserine (Fluka BioChimika) in water (10 mL), Abl
buffer (New England Biolabs) (2.5 mL), a 0.1 mg mL-1 solution
of BSA (New England Biolabs) in water (0.25 mL), a solution
of anti-PKA antibody #2261 (Cell Signalling Technologies) in
buffer (0.25 mL), a 4 mg mL-1 solution of FITC labelled Goat-anti-
Rabbit antibody (Santa Cruz Biotechnology) in buffer (0.25 mL),
and 20 ng kinase (Invitrogen) in 0.5 mL buffer. The inhibitor
solution, or 20% DMSO (aq) for the positive controls, (2.5 mL)
were added, as well as a 1 mM solution of ATP in water, or water
for the negative controls (2.5 mL). All inhibitor concentrations
were tested in triplicate for each isozyme. Samples were placed on
R
the PamChipꢀ 96 STK-array plate (Pamgene, ‘s-Hertogenbosch,
The Netherlands) and during 60 min incubation at 30 ◦C, real time
images were taken automatically every 2.5 min. All images were
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©