'Clickable' 2,5-diketopiperazines as scaffolds for ligation of biomolecules: Their use in Aβ inhibitor assembly

Article abstract of DOI:10.1039/c4ob00541d

The synthesis of 1,3,6-trisubstituted-2,5-diketopiperazine scaffolds bearing up to three 'clickable' sites for further oxime bond or alkyne-azide cycloaddition ligations is described. The orthogonally Boc/Alloc protected DKP precursors prepared from l-lysine residues and an aminohexyl arm are efficiently prepared on a gram scale by sequentially using Fukuyama-Mitsunobu alkylation, dipeptide coupling and diketopiperazine ring formation as key steps. These scaffolds, with their glyoxylyl, aminooxy, alkynyl or azido functions, are ready-to-use platforms for biomolecular assembly. Their potentiality in this field was proved through the chemoselective ligation of Aβ-binding motifs, the KLVFFA peptide and the curcumin molecule. The inhibitory effect of these conjugates on Aβ amyloid fibril formation is reported using thioflavin T fluorescence assays and AFM observation. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00541d

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
‘Clickable’ 2,5-diketopiperazines as scaffolds for
ligation of biomolecules: their use in Aβ inhibitor
assembly†
Cite this: Org. Biomol. Chem., 2014,
12, 4964
E. Dufour,a L. Moni,b L. Bonnat,a S. Chierici*a and J. Garciaa
The synthesis of 1,3,6-trisubstituted-2,5-diketopiperazine scaffolds bearing up to three ‘clickable’ sites for
further oxime bond or alkyne–azide cycloaddition ligations is described. The orthogonally Boc/Alloc pro-
tected DKP precursors prepared from L-lysine residues and an aminohexyl arm are efficiently prepared on
a gram scale by sequentially using Fukuyama–Mitsunobu alkylation, dipeptide coupling and diketopipera-
zine ring formation as key steps. These scaffolds, with their glyoxylyl, aminooxy, alkynyl or azido functions,
are “ready-to-use” platforms for biomolecular assembly. Their potentiality in this field was proved through
the chemoselective ligation of Aβ-binding motifs, the KLVFFA peptide and the curcumin molecule.
The inhibitory effect of these conjugates on Aβ amyloid fibril formation is reported using thioflavin T
fluorescence assays and AFM observation.
Received 11th March 2014,
Accepted 9th May 2014
DOI: 10.1039/c4ob00541d
www.rsc.org/obc
ligation of biomolecules has been reported.6 Our research group
being interested in this field,7 we focused on such DKPs
Introduction
2,5-Diketopiperazines (DKPs), derived from the condensation (Fig. 1). We prepared then a series of DKPs allowing oxime
of two α-amino acids with functionalized side chains, are bond or alkyne–azide cycloaddition ligations, which are com-
attractive scaffolds for molecular assembly design and have monly used for the conjugation of biomolecules such as
been extensively used for the rational design of drugs and pep- peptides.8
tidomimetics.1,2 DKPs in which additional functional groups
We chose a cyclo(Lys–Lys) as a scaffold to benefit from the
are introduced at the lactam nitrogen positions can display up chemically addressable amino side chains and then easily
to four functionalities in a well-defined spatial manner. In this introduced diverse ‘clickable’ functionalities. In addition, the
context, DKPs bearing functionalities such as an amine (e.g. use of natural lysines as starting materials is a way to control
derived from Lys or Orn) and/or a carboxylic acid (e.g. derived the spatial orientation of the conjugated molecules in a cis
from Asp or Glu) are of particular interest. Considering the
variety of orthogonal protecting groups for amines and the
chemical orthogonality between amine and carboxylic
acid functions, they can be regioselectively and diversely
addressed. G. Gellerman et al. have used this strategy to con-
struct a DKP library for combinatorial chemistry.3,4 Orthogonal
chemical reactions can also be used to modify a DKP core.
One example has been reported by M. Tullberg et al. with a
DKP scaffold bearing reaction sites for hydroxylation, Heck
reaction, and Huisgen cycloaddition.5 However, to the best of
our knowledge, no ‘ready-to-use’ DKP for chemoselective
aDépartement de Chimie Moléculaire, CNRS UMR 5250, ICMG FR 2607, Equipe
I2BM, Université Joseph Fourier, 570 rue de la Chimie, BP 53, 38041 Grenoble
cedex 09, France. E-mail: sabine.chierici@ujf-grenoble.fr
bUniversità degli Studi di Genova, Dipartimento di Chimica e Chimica Industriale,
Via Dodecaneso 31, 16146 Genova, Italy
†Electronic supplementary information (ESI) available: Kinetics of Aβ40 fibril
formation in the presence of DKPs 1b, 18, 20 and 21, copies of 1H and 13C NMR
spectra, and copies of MS and HRMS. See DOI: 10.1039/c4ob00541d
Fig. 1 Chemoselective addressable DKPs targeted in this study.
4964 | Org. Biomol. Chem., 2014, 12, 4964–4974
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
position. The ‘clickable’ DKPs such as DKPs A can be used to thesis of such head-to-tail DKPs.1,3,4,10,11 Very similar DKP
ligate two copies of the selected biomolecule possessing the scaffolds resulting from the condensation of two orthogonally
complementary ‘clickable’ functionality. The aminohexyl arm protected lysine residues with Alloc, CBz or Fmoc groups and
at the N1-position of DKPs A is used to improve the solubility bearing a carboxymethyl group in the N1-position have been
in the aqueous media in which are usually performed chemo- reported.3a In this case, as in most cases, reductive amination
selective ligation of biomolecules. This amino arm may also be using aldehydes (e.g. glyoxylic aldehyde) is employed for
used as an additional orthogonally ‘clickable’ site, as shown in Nα-alkylation of the first amino acid partner. In our case, a
DKPs B, to introduce a detecting agent or other functionalities Fukuyama–Mitsunobu reaction12,13 is applied for the
depending on the desired application.
Nα-monoalkylation of the first lysine partner using hydroxyl
In this paper, we report the synthesis of these new DKPs via derivatives instead of aldehydes as alkylating agents. The nitro-
an orthogonally protecting group strategy from DKPs 1 as benzenesulfonyl (Ns) group used as both a protecting and an
precursors (Fig. 1). We also investigate, through the ligation of activating group on the primary amines ensures the conversion
well-known Aβ-binding motifs (the KLVFFA peptide and into the secondary amines and prevents the formation of
the curcumin molecule),9 their potentiality as scaffolds in Aβ the tertiary amines often produced as by-products under
inhibitor assembly.
reductive amination conditions with no sterically hindered
agents.
Thus, the easily prepared Nε-Alloc protected lysine methyl
ester 2a was first protected as 2-nitrobenzenesulfonamide
with a slight excess of 2-nitrobenzenesulfonyl chloride in the
presence of Et3N. The sulphonamide intermediate 3a, which
was obtained in 91% yield, was then alkylated under the
Mitsunobu conditions (DIAD, PPh3) using the 6-aminohexanol
derivative, BocHN(CH2)6OH. After removal of the Ns group via
aromatic nucleophilic substitution by thiophenol in the pres-
ence of K2CO3, the Nα-alkylated lysine aminoester 5a was
obtained in 90% yield from 3a after chromatography purifi-
cation. The same sequence was applied to the commercially
available Nε-Boc protected lysine aminoester 2b using AllocHN-
(CH2)6OH as an alkylating agent to obtain 5b with a similar
yield (88% from 2b).
Results and discussion
Synthesis of the DKP precursors
We first prepared the orthogonally protected DKPs 1a and 1b
(P1, P2 = Boc or Alloc, Scheme 1). The strategy used is solution-
phase synthesis, i.e. Nα-alkylation of the first lysine partner
(2a or 2b), condensation in solution of this Nα-alkylated Nε-
protected aminoester (5a or 5b) with the second Nα,ε-protected
lysine partner, and then intramolecular cyclization of the
resulting dipeptide. This strategy has been proved for the syn-
In the literature, Nα-Boc protected amino acids are often
used as the second partners. The cyclization is then realized
under basic conditions after removal of the Nα-Boc group
under acidic conditions. We chose to use commercially avail-
able Nα-Fmoc protected lysine residues. The coupling of the
Fmoc-Lys(Alloc)-OH to the Nα-alkylated lysine aminoester 5a
was carried out using HATU as a coupling agent, which is
known to be efficient for coupling of secondary amines. The
dipeptide 6a was thus efficiently obtained in 83% yield.
Finally, removal of the Fmoc group and intramolecular cycliza-
tion of 6a were realized in one pot using a mixture of piperi-
dine in dichloromethane14 to afford the DKP precursor 1a in
87% yield. The DKP 1b was obtained in the same manner
from Fmoc-Lys(Boc)-OH and 5b in 78% yield (two steps).
The strategy developed yielded the orthogonally protected
DKPs 1a and 1b on a gram scale in more than 60% overall
yield. The protected amino alcohol arms 4a and 4b, used to
introduce the additional functionality into the N1-position of
the DKP core, were easily prepared from the commercially
available 6-aminohexanol and the corresponding chloro-
formates. Starting from commercially available or readily
prepared amino acids, this approach may be applied to
the synthesis of other orthogonally protected DKPs with
Dde or CBz groups. In addition, by using Fmoc-Lys(Dde)-OH
as the second lysine partner from 5a or 5b, DKPs with three
orthogonally protecting sites can be also obtained.
Scheme 1 Synthesis of the orthogonally protected DKP precursors 1.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4964–4974 | 4965

View Article Online
Paper
Organic & Biomolecular Chemistry
Synthesis of the ‘clickable’ DKPs
The orthogonally protected DKP 1a was used as the common
precursor of the ‘clickable’ DKPs 9, 11, 13, 15 bearing glyoxylyl,
aminooxy, alkynyl, or azido functions, respectively (Scheme 2).
Except for 15, these functions were easily introduced by N-acyl-
ation of the Alloc deprotected intermediate 7a15 using the acti-
vated carboxylic acids 8, 10 and 12. We preferred to use
N-hydroxysuccimide esters as ‘ready-to-use’ activated species
but the formation in situ of the activated species can also been
realized.
In 9, the glyoxylyl functions were introduced by coupling
the Boc-Ser(tBu)-OSu N-hydroxysuccinimide ester 8 on the free
lysine residues of 7a. The Boc-Ser(tBu)-OH residue is com-
monly used as a masked glyoxylic acid equivalent in peptide
chemistry to attach glyoxylyl groups to the N-terminus or to
Nε-lysine residues of peptides. Indeed, after removal of the Boc
and tBu groups, periodate oxidation of the serine residue is a
simple way to obtain the desired α-oxo aldehyde group.16 The
DKP 9 was then obtained in 26% overall yield (three steps).
Loss of product during the last RP-HPLC purification step
(only 42% yield) accounts for this modest yield, while the
Scheme 3 Synthesis of an orthogonally ‘clickable’ DKP from 1b.
HPLC analysis of the oxidation reaction mixture shows a total
conversion to 9.
For DKP 11, the aminooxy functions were incorporated as
Boc-protected aminooxyacetyl ester 1017 with 76% yield. We
preferred for storage keeping the protecting groups due to the
high reactivity of the free aminooxy functions towards electro-
philic agents. Oxime ligation being performed under acidic
conditions in the presence of aldehydes, removal of the Boc
acid sensitive protecting groups of 11 will be accompanied by
in situ ligation reactions. The DKP 13 was similarly obtained
from 7a and 4-pentynoic acid succinimidyl ester 12, after Boc
removal of its N-aminohexyl arm, in 78% yield after
purification.
Finally, DKP 15 bearing azido functions was efficiently
obtained (62% yield) by converting the free amino lysine
residues of 7a into azides using the hydrogen chloride salt of
imidazole-1-sulfonyl azide 14 as
a stable diazotransfer
reagent,18,19 followed by Boc removal.
The DKP 1b was employed to access an orthogonally ‘click-
able’ DKP that presents one azido group and two aminooxy
precursors (compound 16, Scheme 3).
Removal of the Alloc group of 1b was performed, as for 1a,
using the Pd0/PhSiH3
procedure, affording the DKP 7b
15
in more than satisfactory yield (91%). The free amino function
of 7b was then converted into azide as mentioned above for
15. Then, the aminooxy functions were introduced, as Boc-
aminooxyacetic acid succinimide ester (compound 10), on the
lysine side chains after removal of their Nε-Boc protecting
groups. The DKP 16 was obtained in three steps with 57%
yield. It is worth noting that the same strategy may be applied
for 7a to obtain the DKP bearing one aminooxy and two azido
groups tethered on the lysine side chains.
Aβ inhibitor assembly
Synthesis. The ‘clickable’ DKPs 11, 15 and 16 were applied
in biomolecular assembly design through the ligation of
Aβ-binding motifs, the KLVFFA peptide and the curcumin
molecule (Scheme 4). Our research group is interested in the
field of Alzheimer’s disease inhibitors. In a previous study, we
Scheme 2 Synthesis of the ‘clickable’ DKPs from 1a.
4966 | Org. Biomol. Chem., 2014, 12, 4964–4974
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
the Boc group at the N-1 position and by in situ ligation of the
KLVFFA peptide leading to 18 in 55% yield. The reaction was
monitored by RP-HPLC analysis and a total conversion of 11
was obtained after 1 h reaction at 37 °C.
In order to obtain 20, two copies of the curcumin alkynyl
derivative 1921 were attached on “azido” DKP 15 using the
Cu(I)-catalyzed alkyne–azide cycloaddition (CuAAC). We
employed the classical CuAAC combination of copper sulfate
and sodium ascorbate as a reductive agent. To avoid the pre-
cipitation of 19 due to its strong hydrophobicity, we performed
the reaction in the presence of DMF as an organic solvent. We
also added THPTA22 as a water-soluble Cu1-stabilizing ligand.
Under these conditions, however, the ligation product 20 was
obtained in only 9% yield after RP-HPLC purification. The
ligation followed by RP-HPLC showed a total conversion rate of
19 after only 1 h and the low yield is due to loss of product
during the purification step.
The orthogonally ‘clickable’ DKP 16 was employed to
prepare the mixed KLVFFA/curcumin DKP 21. Two copies of
the KLVFFA sequence 17 were first ligated via oxime bond fol-
lowed by CuAAC ligation of one copy of the curcumin deriva-
tive 19. If the first ligation step of peptides was realized with a
satisfactory yield of 47%, the second ligation step gave 21 in
only 23% yield after purification, justifying the 10% overall
yield obtained. As mentioned above for 20, the poor solubility
of the product after the ligation of the curcumin molecule in
the solvents used for RP-HPLC purification could be involved.
In vitro inhibition studies of Aβ40 fibril formation. The
inhibitory effect on Aβ40 fibril formation of DKPs 18, 20 and 21
was evaluated using thioflavin T (ThT) fluorescence assays.
Synthetic fibrils made from Aβ40 monomers and ThT, a
specific dye of the characteristic cross-β-sheet structure of
fibrils, are commonly used in vitro for screening inhibitors.23
The Aβ40 peptide (50 µM) and ThT (10 µM) were co-incubated
in phosphate buffer at 37 °C with a concentration range of
each DKP (1 to 50 μM) and the ThT fluorescence at 485 nm
was measured each day during a period of two weeks (see
ESI†). Results summarized in Fig. 2 show the relative change
of ThT fluorescence (as compared to Aβ with no inhibitor)
Scheme 4 Chemoselective ligation of Aβ inhibitors.
showed that ligation of two copies of KLVFFA or curcumin on
a cyclodecapeptide scaffold leads to more potent inhibitors
than the Aβ-binding motifs alone.20 Built to present at least
two ligation sites, we were interested in evaluating our DKPs in
this field.
To assemble the KLVFFA sequence, we chose the oxime
bond ligation method from the DKP 11 that presents two
masked aminooxy sites. The KLVFFA sequence bearing as a
complementary function a glyoxylyl group at its N-terminus
was first prepared (compound 17, Scheme 4). The SKLVFFA
intermediate was assembled on a solid support and its N-ter-
minus serine residue was oxidized in the solution phase using
sodium periodate. The ligation was performed in the presence
of 50% TFA in a mixture of water–acetonitrile to ensure the
solubility of both precursors and limit the aggregation of 17.
The removal of Boc protecting groups from the aminooxy
moieties of 11 was accompanied by concomitant removal of
Fig. 2 Aβ40 (50 μM) co-incubated with DKPs and controls at the indi-
cated molar concentration. Values are the maximal ThT fluorescence
intensity at 485 nm obtained after 7 days compared to that of the
control (Aβ40 with no inhibitor). Results are the mean standard devi-
ation of three experiments.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4964–4974 | 4967

View Article Online
Paper
Organic & Biomolecular Chemistry
Experimental
Materials and methods
Protected amino acids, especially H-Lys(Boc)-OMe 2b, PyBOP®
and HATU®, were purchased from Calbiochem-Novabiochem
and 2-chlorotritylchloride® resin from Advanced ChemTech
Europe. Other reagents were obtained from Sigma-Aldrich or
Acros Organics.
N-Hydroxysuccinimide esters 8, 10 and 12 were synthesized
as previously described.17,7a Curcumin derivative 19 was
prepared as reported in the literature from curcumin and
propargyl bromide.21 Imidazole-1-sulfonyl azide hydrochloride
14 was synthesized as reported.18
Silica plated aluminum sheets (Silica gel 60 F254) were
used for thin-layer chromatography (TLC) and spots were
detected by UV (254 nm) or by a solution of 1% ninhydrine in
EtOH. For flash chromatography, silica gel 60 (230–400 mesh)
was used. RP-HPLC analyses and purifications were performed
on Waters equipment consisting of a Waters 600E controller, a
Waters 2487 Dual Absorbance Detector, and a Waters In-Line
Degasser. UV absorbance was monitored at 214 nm and
250 nm simultaneously. The analytical column (Nucleosil C18,
particles size 3 μm, pore size 120 Å, 30 × 4 mm2) was operated
at 1 mL min−1 using linear A–B gradients in 20 min run time
(solvent A, H2O containing 0.1% trifluoroacetic acid (TFA);
solvent B, CH3CN containing 9.9% H2O and 0.1% TFA). The
preparative column (Delta-Pak™ 300 Å 15 μm C18 particles,
200 × 25 mm2) was operated at 22 mL min−1 using linear A–B
gradients in 30 min run time.
Mass spectra were obtained by electrospray ionization
(ESI-MS) on an Esquire 3000 (Bruker) spectrometer in positive
mode. The multiply charged data produced by the mass
spectrometer on the m/z scale were converted to the molecular
weight. High resolution mass spectra (HRMS) were recorded
by the ICOA (Orléans, France) on a Q-Tof MaXis in positive
mode.
Fig. 3 AFM images (height data) of protofilaments from Aβ40 (50 μM)
after 8 days of incubation at 37 °C with: (A) no inhibitor, (B) 10 μM of 18,
and (C) 10 μM of 20.
after 7 days of incubation, from which a maximum of fluo-
rescence was observed. DKP 1b with no ligand and the ligands
alone (KLVFFA peptide and curcumin) were also tested under
the same conditions.
At 10 µM concentration, scaffold 1b and the KLVFFA
peptide did not significantly reduce ThT fluorescence signal
whereas the DKP 18 bearing two copies of the KLVFFA peptide
showed a strong inhibitory activity for a 10-fold lower ratio.
Indeed, at 1 µM concentration, the fluorescence signal was
reduced by about 80% in the presence of 18.
This anti-amyloidogenic effect of 18 is consistent with the
atomic force microscopy (AFM) observation (Fig. 3). In the
control sample of Aβ40 with no inhibitor (Fig. 3A), numerous
and long fibrils were formed, whereas in the presence of 18 at
10 µM concentration, only a few and shorter fibers were
observed (Fig. 3B). The benefit of the dimeric presentation of
the KLVFFA motif in DKP 18 is in concordance with previous
studies that demonstrated the increased efficiency of KLVFFA
multimers against Aβ peptide aggregation.20,24,25 Especially,
the activity of conjugate 18 has a similar activity to that of a
cyclodecapeptide scaffold presenting two KLVFFA copies for
which we had obtained 90% of inhibition of Aβ40 fibril for-
mation for the same Aβ/compound molar ratio.20
For the curcumin conjugate 20, the inhibitory activity was
much lower compared to 18 since only 60% of inhibition was
observed for a 10-fold higher concentration (Fig. 2). This result
was confirmed by the AFM image (Fig. 3C). We had obtained a
similar inhibition when two curcumin ligands were ligated
onto a cyclodecapeptide scaffold.20 Moreover, compared to the
inhibitory effect of the curcumin ligand alone at the same con-
centration of 10 μM, 20 presents only a slight benefit. With the
mixed DKP 21 at 10 μM, surprisingly, no inhibitory effect on
Aβ40 fibril formation was shown by the ThT assay (Fig. 2). A
5-fold higher concentration was necessary to obtain 50% of
inhibition (see ESI†). In this case, we assume that the assay is
affected by the very low solubility of 21 in the aqueous phos-
phate buffer used.
NMR spectra were recorded on BRUKER Avance 400 and
Avance III 500 spectrometers. Chemical shifts are expressed in
ppm and calculated taking the solvent peak as an internal
reference. Coupling constants are in Hz and signals are
described using the usual abbreviations: br (broad), s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), etc. 1D and
2D NMR techniques such as COSY and 1H, 13C HSQC have
been used for spectral assignments.
In vitro inhibition studies of Aβ40 fibril formation were
performed using thioflavin T (ThT) assays and atomic force
microscopy (AFM) as described previously.20
Nα-Allyloxycarbonyl-L-lysine methyl ester or H-Lys(Alloc)-
OMe (2a). Boc-Lys-OH (3.0 g, 12.20 mmol) was dissolved in
120 mL of water–dioxane (1 : 1) and Et3N (3.40 mL,
24.50 mmol) was added. The solution is cooled in an ice bath
and allylchloroformate (1.95 mL, 18.20 mmol) was added drop-
wise. After 3 h of stirring at rt, 50 mL of water was added and
the pH was adjusted to 2 with 1 M HCl. The mixture was
extracted three times with EtOAc and the combined organic
phases were washed with water and with brine and dried over
Through these results, especially through conjugate 18, the
relevance of the DKP scaffold to build efficient in vitro Aβ fibril
inhibitors is highlighted. This scaffold is of particular interest
in this field considering its potentiality to cross the blood–
brain barrier.26 In the dimers of ligands such as 20, for which
the inhibition is quite similar to the ligand alone, the DKP
core may be of special interest as cargo to transport the
ligands into the brain.
4968 | Org. Biomol. Chem., 2014, 12, 4964–4974
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Na2SO4. After filtration and concentration of the solvent under precipitated in diethyl ether to afford 4a (7.91 g, 85% yield) as
1
vacuum, the residue Boc-Lys(Alloc)-OH was dissolved in 20 mL a white powder. H NMR (400 MHz, CDCl3): δ 4.55 (br s, 1H,
of MeOH and chlorotrimethylsilane (4 mL, 31.50 mmol) was NH), 3.71–3.59 (m, 2H, CH2OH), 3.16–3.09 (m, 2H, CH2NH),
added dropwise.27 The solution was stirred overnight at rt and 1.63–1.32 (m, 17H, 4 × CH2, tBu).
concentrated under vacuum. The residue was triturated with
6-(N-Allyloxycarbonyl)aminohexan-1-ol (4b). To a solution of
diethyl ether to afford the hydrochloride salt of 2a (3.10 g, 6-aminohexan-1-ol (3 g, 25.65 mmol) and Et3N (9 mL,
92%). ESI-MS calcd for C11H20N2O4 244.29; found 245.1. 64.05 mmol) in DCM (120 mL) was added slowly at 0 °C allyl-
3
3
1H NMR (400 MHz, CDCl3): δ 6.01–5.82 (ddt, J = 5.5 Hz, J = chloroformate (4.60 mL, 43.70 mmol). The mixture was then
3
2
3
10.5 Hz, J = 17 Hz, 1H, vCHallyl), 5.29 (dd, J = 1.5 Hz, J = stirred overnight at rt and washed with water, with an aqueous
2
3
17 Hz, 1H, vCH2allyl), 5.20 (dd, J = 1.5 Hz, J = 10.5 Hz, 1H, solution brought to pH 2 with 1 M HCl, and brine. The
vCH2allyl), 4.79 (br s, 1H, NHε), 4.55 (d, 3J = 5.5 Hz, 2H, organic phase was dried over Na2SO4, filtered and con-
3
OCH2allyl), 3.72 (s, 3H, Me), 3.46 (dd, J = 6.0 and 7.0 Hz, 1H, centrated under reduced pressure to give 4b (4.68 g, 91%).
CHα), 3.18 (dd, J = 7.0 and 13.0 Hz, 2H, CH2ε), 1.83–1.35 (m, 1H NMR (400 MHz, CDCl3): δ 5.91 (ddt, 3J = 5.5 Hz, 3J =
3
6H, CH2β, CH2γ, CH2δ). 13C NMR (125 MHz, CDCl3): δ 170.02, 10.5 Hz, J = 17 Hz, 1H, vCHallyl), 5.29 (dd, J = 1.5 Hz, J =
3
2
3
2
3
157.17, 132.88, 117.71, 65.86, 53.53, 53.27, 50.35, 40.40, 29.69, 17 Hz, 1H, vCH2allyl), 5.20 (dd, J = 1.5 Hz, J = 10.5 Hz, 1H,
3
28.84, 21.98.
vCH2allyl), 4.74 (s, 1H, NH), 4.55 (d, J = 5.5 Hz, 2H, OCH2-
Nα(Ns)Lys(Alloc)-OMe (3a). A solution of o-nitrobenzosulfo- allyl), 3.63 (t, 3J = 6.5 Hz, 2H, CH2OH), 3.18–3.12 (m, 2H,
nyl chloride (3.20 g, 14.46 mmol) in 4 mL of dioxane was CH2NH), 1.61–1.45 (m, 4H, 2 × CH2), 1.43–1.28 (m, 4H,
added dropwise at 0 °C to a solution of 2a (2.9 g, 10.33 mmol) 2 × CH2).
and Et3N (4.3 mL, 30.10 mmol) in 100 mL of water–dioxane
Nα(N-Boc(aminohexyl))-Lys(Alloc)-OMe (5a). To a solution of
(1 : 1) cooled in an ice bath. The solution was stirred for compound 3a (3.58 g, 8.33 mmol) in 50 mL anhydrous THF,
30 min at 0 °C and overnight at rt. Water was added and the PPh3 (3.50 g, 13.33 mmol), 4a (2.90 g, 13.33 mmol) and DIAD
pH was adjusted at 2 with 1 M HCl. The mixture was extracted (2.62 mL, 13.33 mmol) were respectively added. The reaction
three times with EtOAc and the combined organic phases were was stirred at rt under argon and was monitored by TLC
washed twice with a saturated aqueous solution of NaHCO3, (EtOAc–cyclohexane, 2 : 1). After completion of the reaction
once with brine, and dried over Na2SO4. After concentration (3 h), the solvent was removed under vacuum and the residue
under vacuum, 3a was obtained (4.03 g, 91%) as a yellow oil. was purified by column chromatography (30 to 40% EtOAc in
ESI-MS calcd for C17H23N3O8S 429.44; found 430.1. 1H NMR cyclohexane) to obtain the desired compound from the
(400 MHz, CDCl3): δ 8.11–8.01 (m, 1H, HAr), 7.97–7.88 (m, 1H, Mitsunobu reaction in mixture with 4a. This mixture was dis-
HAr), 7.80–7.65 (m, 2H, 2 × HAr), 6.13 (d, 3J = 9.0 Hz, 1H, solved in 80 mL anhydrous DMF and K2CO3 (6.72 g,
NHα), 5.99–5.83 (ddt, 3J = 5.5 Hz, 3J = 10.5 Hz, 3J = 17.0 Hz, 1H, 48.66 mmol) was added. The solution was degassed with
2
3
vCHallyl), 5.29 (dd, J = 1.5 Hz, J = 17.0 Hz, 1H, vCH2allyl), argon for 10 min and PhSH (2.49 mL, 24.33 mmol) was intro-
2
3
5.20 (dd, J = 1.5 Hz, J = 10.5 Hz, 1H, vCH2allyl), 4.79 (br s, duced dropwise. After 3 h of stirring under argon, water was
1H, NHε), 4.55 (d, 3J = 5.5 Hz, 2H, OCH2allyl), 4.16 (td, 3J = added and the reaction mixture was extracted three times with
3
5.0 Hz, J = 9.0 Hz, 1H, CHα), 3.47 (s, 3H, Me), 3.17–3.15 (m, diethyl ether. The combined organic phases were dried over
2H, CH2ε), 1.90–1.72 (m, 2H, CH2β), 1.57–1.37 (m, 4H, CH2γ, Na2SO4, filtered and concentrated under vacuum. The residue
CH2δ). 13C NMR (125 MHz, CDCl3): δ 171.54, 156.49, 147.84, was purified by column chromatography (50 to 100% EtOAc in
134.24, 133.79, 132.98, 130.61, 125.79, 117.80, 65.68, 60.54, cyclohexane) to provide 5a (3.32 g, 90%). ESI-MS calcd for
1
56.69, 52.50, 40.57, 32.73, 29.36, 22.25.
C22H41N3O6 443.58, found 444.3. H NMR (400 MHz, CDCl3):
Nα(Ns)-Lys(Alloc)-OMe (3b). The procedure described above δ 5.91 (ddt, 3J = 5.5 Hz, 3J = 10.5 Hz, 3J = 17 Hz, 1H, vCHallyl),
2
3
2
for 3a was applied from 2b (2.1 g, 7.08 mmol) to give 3b 5.29 (dd, J = 1.5 Hz, J = 17 Hz, 1H, vCH2allyl), 5.19 (dd, J =
(3.09 g, 98% yield) as an oil. ESI-MS calcd for C18H27N3O8S 1.5 Hz, 3J = 10.5 Hz, 1H, vCH2allyl), 4.85 (br s, 1H, NHε),
445.48, found 446.1. 1H NMR (400 MHz, CDCl3): δ 8.10–8.00 4.62–4.50 (m, 3H, OCH2allyl, NHBoc), 3.73 (s, 3H, Me),
(m, 1H, HAr), 7.95–7.85 (m, 1H, HAr), 7.77–7.66 (m, 2H, 2 × 3.26–3.14 (m, 3H, CHα, CH2ε), 3.13–3.06 (m, 2H, CH2NHBoc),
HAr), 6.11 (d, 3J = 7.0 Hz, 1H, NHα), 4.57 (br s, 1H, NHε), 2.63–2.53 (m, 1H, CH2NHα), 2.53–2.41 (m, 1H, CH2NHα),
4.20–4.08 (m, 1H, CHα), 3.46 (s, 3H, Me), 3.09–3.07 (m, 2H, 1.74–1.6 (m, 2H, CH2β), 1.60–1.24 (m, 21H, 6 × CH2, tBu).
CH2ε), 1.84–172 (m, 2H, CH2β), 1.53–1.35 (m, 13H, CH2γ, 13C NMR (100 MHz, CDCl3): δ 175.72, 156.42, 156.15, 133.17,
CH2δ, tBu).
117.69, 79.12, 68.11, 65.57, 61.44, 51.92, 48.23, 40.88, 32.96,
6-(N-tert-Butyloxycarbonyl)aminohexan-1-ol (4a). To a solu- 30.12, 29.95, 29.81, 28.58, 26.70, 23.06, 21.17.
tion of 6-aminohexan-1-ol (5 g, 42.70 mmol) in 200 mL of
Nα(N-Alloc(aminohexyl))-Lys(Boc)-OMe (5b). The procedure
MeOH, di-tert-butyl dicarbonate (10.83 g, 49.80 mmol) was described for 5a was applied for 3b (2.5 g, 5.60 mmol) and 4b
added. After 8 h of stirring at rt, the solution was concentrated (1.80 mg, 8.95 mmol) to afford after column chromatography
under vacuum. The residue was dissolved in DCM (250 mL), (40 to 50% EtOAc in cyclohexane) the compound from the
washed with an aqueous solution brought to pH 3 with 1 M Mitsunobu reaction in mixture with 4b. Nosyl removal of the
HCl and brine. The organic layer was dried over Na2SO4, fil- compound from the Mitsunobu reaction was realized on this
tered and concentrated under vacuum. The residue was then mixture as described below to give after column chromato-
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4964–4974 | 4969

View Article Online
Paper
Organic & Biomolecular Chemistry
graphy (50 to 100% EtOAc in cyclohexane) the compound 5b
c[Nε-Allyloxycarbonyl-L-lysinyl-N(,)α((N-tert-butyloxycarbonyl)-
1
(2.2 g, 90% yield). H NMR (400 MHz, CDCl3): δ 5.98–5.91 (m, aminohexyl)-Nε-allyloxycarbonyl-L-lysinyl] or DKP (1a). Com-
3
1H, vCHallyl), 5.29 (d, J = 17.0 Hz, 1H, vCH2allyl), 5.20 (d, pound 6a (4.0 g, 4.56 mmol) was dissolved in 25mL of a piper-
3J = 10.5 Hz, 1H, vCH2allyl), 4.73 (br s, 1H, NHε), 4.60–4.50 idine–DCM solution, 1 : 4. The mixture was stirred for 2 h at rt.
(m, 3H, OCH2allyl, NHBoc), 3.71 (s, 3H, Me), 3.27–3.13 (m, 3H, After addition of DCM, the organic layer was washed twice
CHα, CH2ε), 3.12–3.04 (m, 2H, CH2β), 2.62–2.48 (m, 1H, with 10% citric acid solution, once with water and brine. The
CH2NHα), 2.48–2.33 (m, 1H, CH2NHα), 1.65–1.27 (m, 23H, 7 × organic layer was dried over Na2SO4, filtered and concentrated
CH2, tBu). 13C NMR (125 MHz, CDCl3): δ 176.05, 156.29, under reduced pressure. The crude was purified on column
155.96, 133.05, 117.55, 79.02, 65.42, 61.45, 51.66, 48.11, 40.97, chromatography (2 to 10% MeOH in DCM) to afford com-
40.36, 33.18, 30.05, 29.91, 29.88, 28.44, 26.22, 26.52, 23.08.
pound 1a (2.47 g, 87%). RP-HPLC (5 to 100% solvent B) tR =
Nα(Fmoc)-Lys(Alloc)-N(,)α(N-Boc(aminohexyl))-Lys(Alloc)-OMe 13.6 min. ESI-MS calcd for C31H53N5O8 623.78, found 624.3.
(6a). To a mixture of Fmoc-Lys(Alloc)-OH (5.78 g, 12.78 mmol) 1H NMR (400 MHz, CDCl3): δ 6.54 (br s, 1H, NHlactam),
and HATU (4.86 g, 12.78 mmol) in anhydrous DCM (60 mL) 5.99–5.83 (m, 2H, 2 × vCHallyl), 5.29 (dd, 2J = 1.5 Hz, 3J =
2
3
was added DIEA (3.56 mL, 20.44 mmol). The solution was 17 Hz, 2H, 2 × vCH2allyl), 5.20 (dd, J = 1.5 Hz, J = 10.5 Hz,
stirred for 30 min under argon, and then a solution of 5a 2H, 2 × vCH2allyl), 5.00 (s, 2H, 2 × NHε), 4.60–4.50 (m, 5H,
(2.27 g, 5.11 mmol) in 20 mL anhydrous DCM was added. The 2 × OCH2allyl, NHBoc), 3.95–3.86 (m, 2H, 2 × CHα), 3.86–3.76
solution was stirred under argon and the completion of the (m, 1H, CH2N), 3.25–3.15 (m, 4H, 2 × CH2ε), 3.14–3.00 (m, 2H,
reaction was monitored by TLC (EtOAc–cyclohexane, 2 : 1). CH2NHBoc), 2.90–2.79 (m, 1H, CH2N), 2.03–1.90 (m, 2H,
The mixture was diluted with DCM and was washed with a CH2β), 1.68–1.87 (m, 2H, CH2β), 1.58–1.27 (m, 25H, 8 × CH2,
saturated aqueous solution of NaHCO3, water and brine. The tBu). 13C NMR (100 MHz, CDCl3): δ 168.49, 166.20, 156.43,
organic layer was dried over Na2SO4, filtered, concentrated 155.99, 133.01, 117.34, 78.84, 65.30, 59.34, 55.33, 53.43, 44.72,
under vacuum and the residue purified by column chromato- 40.58, 35.41, 32.65, 29.80, 29.59, 29.16, 28.35, 26.81, 26.41,
graphy (40 to 50% EtOAc in cyclohexane) to give 6a (3.73 g, 26.29, 22.70, 22.39.
83%). RP-HPLC (5 to 100% solvent B) tR = 18.4 min. ESI-MS
c[Nε-tert-Butyloxycarbonyl-L-lysinyl-N(,)α((N-allyloxycarbonyl)-
calcd for C47H67N5O11 878.06, found 878.4. 1H NMR (400 MHz, aminohexyl)-Nε-tert-butylcarbonyl-L-lysinyl] or DKP (1b). The
CDCl3): δ 7.76 (d, 3J = 7.5 Hz, 2H, 2 × HAr), 7.59 (d, 3J = 7.5 Hz, same procedure as described above for 1a was applied from 6b
2H, 2 × HAr), 7.40 (t, 3J = 7.5 Hz, 2H, 2 × HAr), 7.31 (t, 3J = (2.5 g, 2.80 mmol) to give 1b (1.49 g, 83%) after purification by
7.5 Hz, 2H, 2 × HAr), 5.98–5.81 (m, 2H, 2 × vCHallyl), 5.62 (d, column chromatography (2 to 5% MeOH in DCM). RP-HPLC
3
2
3
1H, J = 7.0 Hz, NHα), 5.27 (dd, 2H, J = 1.5 Hz, J = 17.0 Hz, (5 to 100% solvent B) tR = 14.5 min. ESI-MS calcd for
2 × vCH2allyl), 5.18 (dd, 2H, 2J = 1.5 Hz, 3J = 10.5 Hz, 2 × C32H57N5O8 639.82, found 640.5. H NMR (400 MHz, CDCl3):
1
vCH2allyl), 4.99 (br s, 2H, 2NHε), 4.69 (br s, 1H, NHBoc), δ 6.55 (s, 1H, NHlactam), 5.96–5.87 (m, 1H, vCHallyl), 5.30 (dd,
4.63–4.56 (m, 1H, CHα), 4.53 (d, 3J = 5.5 Hz, 4H, 2 × OCH2- 2J = 1.5 Hz, 3J = 17.2 Hz, 1H, vCH2allyl), 5.20 (dd, 3J = 10.5 Hz,
3
allyl), 4.47–4.30 (m, 3H, CHα, CH2Fmoc), 4.21 (t, J = 7.0 Hz, 1H, vCH2allyl), 4.81 (s, 1H, NH), 4.75–4.60 (br s, 2H, 2 × NH),
1H, CHFmoc), 3.69 (s, 3H, Me), 3.46–3.28 (m, 2H, CH2N), 4.55 (d, 3J = 4.9 Hz, 2H, OCH2allyl), 3.93–3.74 (m, 3H, 2 × CHα,
3.26–3.01 (m, 6H, 2 × CH2ε, CH2NHBoc), 2.14–2.02 (m, 1H, CH2N), 3.20–3.05 (m, 6H, 2 × CH2ε, CH2NHAlloc), 2.89–2.81
CH2N), 1.86–1.30 (m, 29H, 10 × CH2, tBu).
(m, 1H, CH2N), 2.04–1.26 (m, 38H, 10 × CH2, 2 × tBu).
Nα(Fmoc)-Lys(Boc)-N(,)α(N-Alloc(aminohexyl))-Lys(Boc)-OMe 13C NMR (125 MHz, CDCl3): δ 167.96, 166.10, 156.42, 156.20,
(6b). The procedure described for 6a was applied to Fmoc-Lys 133.17, 117.68, 79.35, 79.27, 65.54, 59.56, 55.87, 44.81, 40.95,
(Boc)-OH (4.86 g, 10.36 mmol) and 5b (1.84 g, 4.14 mmol) to 40.23, 35.75, 32.86, 29.91, 28.56, 26.95, 26.50, 26.37, 23.11,
give 6b (3.5 g, 95%) after column chromatography (50 to 60% 22.66.
EtOAc in cyclohexane). RP-HPLC (5 to 100% solvent B) tR
=
c[L-Lysinyl-N(,)α((N-tert-butyloxycarbonyl)aminohexyl)-L-lysinyl]
18.9 min. ESI-MS calcd for C48H71N5O11 894.11, found 894.6. or DKP (7a). To a solution of 1a (2.12 g, 3.40 mmol) in 120 mL
1H NMR (400 MHz, CDCl3): δ 7.78 (d, 3J = 7.5 Hz, 2H, 2 × HAr), of anhydrous DCM under argon, PhSiH3 (8.4 mL, 68 mmol)
3
3
7.62 (d, J = 7.5 Hz, 2H, 2 × HAr), 7.42 (t, J = 7.5 Hz, 2H, 2 × followed by Pd(PPh3)4 (79 mg, 68 μmol) were added. The
HAr), 7.33 (t, 3J = 7.5 Hz, 2H, 2 × HAr), 6.03–5.81 (m, 1H, mixture was stirred for 2 h under an inert atmosphere, and
vCHallyl), 5.66 (br s, 1H, NHα), 5.31–5.22 (m, 1H, vCH2allyl), then 10 mL of MeOH was added. The solvent was removed
3
5.18 (d, J = 10.5 Hz, 1H, vCH2allyl), 5.05 (br s, 1H, NHAlloc), under vacuum and the crude was purified by column chrom-
4.81 (s, 1H, NHε), 4.76 (s, 1H, NHε), 4.69–4.58 (m, 1H, CHα), atography (2 to 20% MeOH in DCM in the presence of 5%
4.57–4.50 (m, 2H, OCH2allyl), 4.49–4.31 (m, 3H, CHα, Et3N) to give 7a (1. 38 g, 89%) as a white powder. RP-HPLC
3
CH2Fmoc), 4.24 (t, J = 7 Hz, 1H, CHFmoc), 3.72 (s, 3H, Me), (5 to 100% solvent B) tR = 9.3 min. ESI-MS calcd for
3.49–3.32 (m, 1H, CH2N), 3.27–3.01 (m, 6H, 2 × CH2ε, C23H45N5O4 455.64, found 456.4. RMN 1H (400 MHz, DMSO-
CH2NHAlloc), 2.15–2.00 (m, 1H, CH2N), 1.91–1.31 (m, 38H, d6): δ 8.30 (s, 1H, NHlactam), 6.74 (s, 1H, NHBoc), 3.83–3.80 (m,
10 × CH2, 2 × tBu). 13C NMR (125 MHz, CDCl3): δ 171.36, 156.37, 1H, CHα), 3.76–3.72 (m, 1H, CHα), 3.65–3.57 (m, 1H, CH2N),
156.08, 143.20, 141.33, 133.10, 127.73, 127.07, 125.18, 120.00, 2.91–2.85 (m, 3H, CH2NHBoc, CH2N), 2.61–2.54 (m, 4H,
117.46, 79.09, 67.07, 65.37, 53.42, 52.29, 51.00, 47.18, 40.74, 2 × CH2ε), 1.88–1.47 (m, 4H, 4 × CH2β), 1.46–1.15 (m, 25H,
40.34, 33.33, 29.84, 28.69, 28.46, 26.32, 26.09, 23.84, 22.32.
8 × CH2, tBu). RMN 1H (125 MHz, DMSO-d6): δ 166.72, 165.95,
4970 | Org. Biomol. Chem., 2014, 12, 4964–4974
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
155.57, 77.29, 58.98, 54.46, 43.97, 35.11, 32.10, 31.17, 30.79, Compound 10 (95 mg, 0.33 mmol) was then added and the
29.38, 28.28, 26.52, 26.04, 25.98, 22.34, 22.15. solution was stirred at rt for 3 h (completion of the reaction
c[Nε-tert-Butyloxycarbonyl-L-lysinyl-N(,)α(N-aminohexyl)-Nε- was checked by analytical RP-HPLC). The solvent was removed
tert-butylcarbonyl-L-lysinyl] or DKP (7b). The same procedure under reduced pressure and the residue was taken up in DCM,
described above for 7a was applied from 1b (1.2 g, 1.84 mmol) washed with a 5% aqueous NaHCO3 solution, with water and
to give 7b (930 mg, 91%) after purification (5 to 10% MeOH in brine. The organic layer was dried on Na2SO4, filtered and con-
DCM in the presence of 1% of Et3N). RP-HPLC (5 to 100% centrated under vacuum. The crude was then purified by
solvent B) tR = 11.6 min. ESI-MS calcd for C28H53N5O6 555.75, column chromatography (2 to 6% MeOH in DCM) to afford 11
found 556.3. 1H NMR (400 MHz, CDCl3): δ 6.61 (s, 1H, (67 mg, 76%). RP-HPLC (5 to 100% solvent B) tR = 13.8 min.
NHlactam), 4.73 (s, 1H, NHBoc), 4.68 (s, 1H, NHBoc), 3.98–3.78 ESI-MS calcd for C37H67N7O12 801.97, found 802.5. 1H NMR
(m, 3H, 2 × CHα, CH2N), 3.21–3.02 (m, 4H, 2 × CH2ε), (500 MHz, CDCl3): δ 8.52 (br s, 1H, ONH), 8.33 (br s, 1H,
2.91–2.77 (m, 1H, CH2N), 2.76–2.59 (m, 2H, CH2NH2), ONH), 8.14 (br s, 1H, NHε), 7.99 (br s, 1H, NHε), 7.37 (br s, 1H,
1.97–1.22 (m, 38H, 10 × CH2, 2 × tBu).
NHlactam), 4.66 (br s, 1H, NHBoc), 4.31 and 4.29 (2 s, 2 × 2H,
DKP (9). Compound 7a (46 mg, 0.1 mmol) was dissolved in 2 × CH2ONH), 3.94–3.90 (m, 1H, CHα), 3.87–3.85 (m, 1H,
1 mL of DCM and the pH was adjusted to 9 with DIEA. Com- CHα), 3.81–3.75 (m, 1H, CH2N), 3.28–3.32 (m, 4H, 2 × CH2ε),
pound 8 (79 mg, 0.22 mmol) was added and the mixture was 3.08–3.05 (t, 3J = 7.0 Hz, 2H, CH2NHBoc), 2.88–2.82 (m, 1H,
stirred at rt. The reaction was monitored by analytical CH2N), 1.99–1.93 (m, 2H, 2 × CH2β), 1.81–1.68 (m, 2H, 2 ×
RP-HPLC. After 3 h of stirring, the mixture was diluted with CH2β), 1.68–1.27 (m, 43H, 8 × CH2, 3 × tBu). 13C NMR
AcOEt. The organic layer was washed once with 10% citric acid (125 MHz, CDCl3): δ 169.36, 169.30, 168.13, 166.21, 158.03,
solution, water and brine. The organic layer was dried on 156.21, 82.95, 82.82, 79.19, 76.08, 76.03, 59.47, 55.61, 44.86,
Na2SO4, filtered and concentrated under vacuum. The residue 40.52, 38.65, 38.52, 35.33, 32.45, 30.00, 28.74, 28.54, 28.26,
was purified by column chromatography (2 to 5% MeOH in 26.98, 26.59, 26.46, 22.82, 22.44.
DCM) to afford the intermediate DKP (79 mg, 84%) in which
DKP (13). Compound 7a (40 mg, 0.088 mmol) was dissolved
two Boc-Ser(tBu) are ligated on the lysine residues. RP-HPLC in 2 mL of DMF and the pH was adjusted to 9 with DIEA. Com-
(5 to 100% solvent B) tR = 16.6 min. ESI-MS calcd for pound 12 (51 mg, 0.264 mmol) was added and the solution
1
C47H87N7O12 942.24, found 942.7. H NMR (400 MHz, CDCl3): was stirred at rt. The solvent was removed after 3 h under
δ 6.82 (br s, 1H, NHlactam), 6.71–6.68 (m, 2H, 2 × NHε), 5.46 (br reduced pressure and the residue was purified by column
s, 2H, 2 × NHαserine), 4.62 (br s, 1H, NHBoc), 4.18–4.11 (br s, chromatography (0 to 6% MeOH in DCM) to afford the DKP
2H, 2 × CHαserine), 3.91–3.85 (m, 1H, CHαlysine), 3.85–3.79 (m, intermediate (46 mg, 85% yield). RP-HPLC (5 to 100% solvent
2H, CHαlysine, CH2N), 3.77–3.69 (m, 2H, 2 × CH2βserine), B) tR = 11.9 min. ESI-MS calcd for C33H53N5O6 615.81, found
1
3
3.43–3.33 (m, 2H, 2 × CH2βserine), 3.31–3.19 (m, 4H, 2 × CH2ε), 616.3. H NMR (400 MHz, CDCl3): δ 7.86 (d, 1H, J = 2.4 Hz,
3
3.07 (t, J = 6.9 Hz, 2H, CH2NHBoc), 2.85–2.75 (m, 1H, CH2N), NHlactam), 6.60 (br s, 1H, NHε), 6.45 (br s, 1H, NHε), 4.66 (br s,
2.04–1.88 (m, 2H, 2 × CH2βlysine), 1.84–1.62 (m, 2H, 2 × 1H, NHBoc), 5.00 (s, 2H, 2 × NHε), 3.92–3.88 (m, 1H, CHα),
CH2βlysine), 1.61–1.24 (m, 43H, 8 × CH2, 3 × Boc), 1.17 (s, 18H, 3.85–3.83 (m, 1H, CHα), 3.80–3.73 (m, 1H, CH2N), 3.28–3.24
2 × CH2OtBu). 13C NMR (100 MHz, CDCl3): δ 170.82, 170.72, (m, 4H, 2 × CH2ε), 3.10–3.02 (m, 2H, CH2NHBoc), 2.88–2.81
167.68, 165.97, 156.03, 155.55, 79.99, 79.07, 73.87, 73.84, (m, 1H, CH2N), 2.51–2.47 (m, 4H, 2 × CH2Csp), 2.40–2.36 (m,
61.95, 61.91, 59.39, 55.61, 54.38, 44.76, 40.57, 38.99, 35.52, 4H, 2 × CH2CO), 2.04–2.01 (m, 2H, 2 × CHalcyne), 1.98–1.90
32.68, 29.90, 29.19, 29.04, 28.43, 28.34, 27.46, 26.89, 26.48, (m, 2H, 2 × CH2β), 1.81–1.67 (m, 2H, 2 × CH2β), 1.65–1.25 (m,
26.37, 22.95, 22.60.
25H, 8 × CH2, tBu). 13C NMR (100 MHz, CDCl3): δ 171.57,
This intermediate DKP (79 mg, 0.084 mmol) was dissolved 171.52, 168.43, 166.19, 156.20, 83.19, 83.15, 79.21, 69.65,
in a mixture of TFA–DCM (1 : 1, 8 mL). The solution was 69.59, 59.49, 55.68, 44.81, 40.65, 39.28, 35.55, 35.38, 35.36,
stirred for 2 h at rt and the solvents were removed under 32.60, 29.99, 29.36, 29.12, 28.55, 26.98, 26.57, 26.46, 22.97,
vacuum. The residue was taken up in water and lyophilized to 22.55, 15.15, 15.13. Boc removal of this intermediate (36 mg,
give the deprotected DKP, in which serine residues are ligated 0.058 mmol) was then performed using a mixture of TFA–DCM
on the lysine side chains, as TFA salt (53 mg, 73%). RP-HPLC (1 : 1, 3 mL) for 2 h. After removal of solvents under vacuum
(5 to 40% solvent B) tR = 4.5 min. ESI-MS calcd for C24H47N7O6 and trituration in diethyl ether, 13 (34 mg, 92% yield) was
529.67, found 530.5. This DKP (27 mg, 0.031 mmol) was dis- obtained as TFA salt. RP-HPLC (5 to 100% solvent B) tR
=
1
solved in 4 mL of a CH3CN–H2O–TFA (3 : 1.9 : 0.1). NaIO4 8.6 min. ESI-MS calcd for C28H45N5O4 515.69, found 516.4. H
3
(200 mg, 0.93 mmol) was added and the mixture was stirred NMR (500 MHz, CD3OD): δ 3.94 (dd, J = 3.8 and 5.4 Hz, 1H,
for 45 min at rt. The solution was purified by preparative CHα), 3.89 (dd, 3J = 4.7 and 6.5 Hz, 1H, CHα), 3.75–3.69 (m,
RP-HPLC (5 to 40% solvent B) to give 9 (8 mg, 42%) after 1H, CH2N), 3.25–3.18 (m, 4H, 2 × CH2ε), 3.08–3.03 (m, 1H,
lyophilization, as TFA salt. RP-HPLC (5 to 40% solvent B) tR
5 min. ESI-MS calcd for C22H37N5O6, 2H2O 503.59, found CH2Csp), 2.40–2.36 (m, 4H, 2 × CH2CO), 2.99 (t, 3J = 2.4 Hz,
504.4.
2H, 2 × CHalcyne), 2.03–1.39 (m, 20H, 10 × CH2). 13C NMR
=
CH2N), 2.93 (t, 3J = 7.5 Hz, 2H, CH2NH2), 2.48–2.45 (m, 4H, 2 ×
DKP (11). Compound 7a (50 mg, 0.11 mmol) was dissolved (125 MHz, CD3OD): δ 173.97, 173.93, 169.62, 168.72, 83.57,
in 4 mL of DMF and the pH was adjusted to 9 with DIEA. 83.54, 70.48, 70.41, 61.11, 56.54, 46.05, 40.59, 40.04, 39.83,
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4964–4974 | 4971

View Article Online
Paper
Organic & Biomolecular Chemistry
36.63, 36.09, 36.08, 33.51, 29.99, 29.93, 28.37, 27.74, 27.24, yield. RP-HPLC (5 to 100% solvent B) tR = 14.2 min. ESI-MS
26.94, 24.07, 23.59, 15.82, 15.80.
calcd for C32H57N9O10 727.85, found 728.5. 1H NMR (400 MHz,
DKP (15). Compound 7a (73 mg, 0.16 mmol) was dissolved CDCl3): δ 8.38 (br s, 1H, NH), 8.19 (br s, 2H, 2 × NHε), 8.00 (br
in 4 mL of DCM, and K2CO3 (156 mg, 1.14 mmol) dissolved in s, 1H, NH), 7.29 (br s, 1H, NH), 4.32 and 4.31 (2d, 2 × 2H, 2 ×
2 mL of water, followed by ZnCl2 (4.4 mg, 0.032 mmol), was CH2ONH), 3.95–3.79 (m, 3H, 2 × CHα, CH2N), 3.88–3.75 (m,
added. Then compound 1418 (100 mg, 0.48 mmol) dissolved in 1H, CH2N), 3.33–3.30 (m, 4H, 2 × CH2ε), 3.25 (t, 2H, 3J = 6.8
4 mL of MeOH was added slowly. The mixture was stirred for Hz, CH2N3), 2.88–2.81 (m, 1H, CH2N), 2.05–1.93 (m, 2H, 2 ×
2 h at rt. The solvents were removed under vacuum and the CH2β), 1.85–1.68 (m, 2H, 2 × CH2β), 1.65–1.23 (m, 34H, 8 ×
residue was taken up in H2O. The pH was adjusted to 2 with CH2, 2 × tBu). 13C NMR (100 MHz, CDCl3): δ 169.46, 168.09,
1 M HCl and the aqueous phase was extracted three times with 166.16, 158.05, 83.10, 82.97, 76.15, 76.10, 59.48, 55.68, 51.45,
DCM. The organic layers were pooled, washed with water, 44.74, 38.69, 38.51, 35.30, 32.28, 28.84, 28.76, 28.28, 26.98,
brine, and dried over Na2SO4. After filtration and concen- 26.55, 26.51, 22.85, 22.29.
tration under vacuum, the crude material was purified by
Peptide (17). The protected S(tBu)K(Boc)LVFFA was auto-
column chromatography (0 to 5% MeOH in DCM) to afford the matically assembled on 2-chlorotritylchloride resin® (200 mg,
DKP intermediate (64 mg, 78% yield). RP-HPLC (5 to 100% 0.6 mmol g−1) using Fmoc/tBu strategy on a Syro II synthesizer
solvent B) tR = 14.6 min. ESI-MS calcd for C23H41N9O4 507.63, using PyBOP as a coupling agent. The peptide was deprotected
1
3
found 508.3. H NMR (400 MHz, CDCl3): δ 7.24 (d, J = 2.5 Hz, and released from the resin by treatment for 1 h with 5 mL of
1H, NHlactam), 4.56 (br s, 1H, NHBoc), 3.94–3.90 (m, 1H, CHα), a TFA–TIS–H2O (95/2.5/2.5) solution. The residue obtained
3.89–3.86 (m, 1H, CHα), 3.85–3.79 (m, 1H, CH2N), 3.31–3.27 after evaporation was precipitated in DCM–Et2O and the crude
(m, 4H, 2 × CH2ε), 3.09–3.06 (m, 2H, CH2NHBoc), 2.86–2.79 material was purified by preparative RP-HPLC (5 to 100% B) to
(m, 1H, CH2N), 2.03–1.93 (m, 2H, 2 × CH2β), 1.84–1.44 (m, afford the SKLVFFA peptide (50 mg, 0.048 mmol) in 40% yield
14H, 7 × CH2), 1.42 (s, 9H, tBu), 1.36–1.24 (m, 4H, 2 × CH2γ). (TFA salt). RP-HPLC (5 to 100% solvent B) tR = 10.3 min.
13C NMR (100 MHz, CDCl3): δ 168.06, 165.94, 156.17, 79.25, ESI-MS calcd for
C41H62N8O9 810.98, found 811.6. The
59.45, 55.66, 51.25, 51.22, 44.90, 40.66, 35.62, 32.64, 30.04, SKLVFFA peptide (45 mg, 43.3 µmol) was dissolved in 800 µL
28.72, 28.54, 28.46, 26.99, 26.61, 26.48, 23.11, 22.70. This inter- of CH3CN and water containing 0.1% TFA (1.2 mL), then
mediate (50 mg, 0.098 mmol) was dissolved in a TFA–DCM NaIO4 (98 mg, 45 µmol) was added. The solution was stirred
(1 : 1, 4 mL) mixture. After 2 h of stirring at rt, the solution was for 30 min at rt. The reaction mixture was directly purified by
concentrated under reduced pressure and the crude was puri- preparative RP-HPLC (20 to 70% solvent B) to give 17 (31 mg,
fied by preparative HLPC (5 to 80% solvent B in 20 min run 34.0 µmol) in 79% yield (TFA salt). RP-HPLC (5 to 60%
time) to afford 15 (41 mg, 0.078 mmol), as TFA salt, in 80% solvent B) tR = 14.2 min. HRMS (ESI) calcd for C40H57N7O9,
yield. RP-HPLC (5 to 100% solvent B) tR = 11.9 min. ESI-MS H2O [M + H]+ 798.4396, found 798.4392.
1
calcd for C18H33N9O2 407.51, found 408.4. H NMR (500 MHz,
DKP (18). To a solution of DKP 11 (5.4 mg, 7.3 µmol) in
3
CD3OD): δ 3.98 (dd, J = 3.8 and 7.1 Hz, 1H, CHα), 3.93 (dd, 1.3 mL of TFA–H2O–CH3CN (5 : 3 : 2), peptide 17 (14 mg,
3J = 4.9 and 7.9 Hz, 1H, CHα), 3.76–3.70 (m, 1H, CH2N), 15.6 µmol) was added and the mixture was heated for 1 h at
3.36–3.33 (m, 4H, 2 × CH2ε), 3.05–3.10 (m, 1H, CH2N), 2.93 37 °C. The mixture was injected in preparative RP-HLPC (20 to
(t, 3J = 7.6 Hz, 2H, CH2NH2), 2.06–1.99 (m, 1H, 1 × CH2β), 80% solvent B) to give 18 (9.0 mg, 4.0 µmol) in 55% yield, as
1.94–1.84 (m, 2H, 2 × CH2β), 1.82–1.75 (m, 1H, 1 × CH2β), TFA salt. RP-HPLC (20 to 80% B) tR = 12.1 min. HRMS (ESI)
1.73–1.35 (m, 16H, 8 × CH2). 13C NMR (125 MHz, CD3OD): calcd for
δ 169.56, 168.69, 61.09, 56.48, 52.23, 52.18, 46.07, 40.60, 36.35, 1013.0831.
C102H153N21O22 [M +
2H]2+ 1013.0822, found
33.25, 29.57, 29.47, 28.41, 27.76, 27.28, 26.97, 23.97, 23.69.
DKP (20). DKP 15 (3.4 mg, 6.5 µmol) and curcumin 19
DKP (16). The procedure described above for the synthesis (6.4 mg, 16 µmol) were dissolved in 400 μL of DMF and the
of 15 was applied from 7b (120 mg, 0.216 mmol) to afford the solution was degassed with argon (solution 1). To a degassed
intermediate bearing one azido function and two free lysine CuSO4·5H2O (3.2 mg, 13.0 µmol) solution in water (100 μL),
residues (112 mg, 0.184 mmol) in 85% (TFA salt, 2 steps). THPTA (12.9 mg, 32.5 mmol) was added followed by sodium
RP-HPLC (5 to 100% solvent B) tR = 8.16 min. ESI-MS calcd for ascorbate (13.0 mg, 66.0 µmol) previously dissolved in 300 μL
C18H35N7O2 381.52, found 382.4. This intermediate (110 mg, of water (solution 2). Solution 2 was added to solution 1 and
0.181 mmol) was dissolved in 4 mL of DMF and the pH was the mixed solution was degassed and stirred at rt under argon.
adjusted to 9 with DIEA. Compound 10 (121 mg, 0.42 mmol) The reaction was followed by analytical RP-HPLC and the reac-
was added and the solution was stirred at rt. The completion tion mixture was purified by preparative HLPC (20 to 100%
of the reaction was checked by analytical RP-HPLC and the solvent B) to give 20 as TFA salt (0.91 mg, 0.68 µmol) in 10%
solvent was removed under reduced pressure. The residue was yield. RP-HPLC (15 to 100% solvent B) tR = 13.4 min. HRMS
dissolved in EtOAc and washed once with a 10% citric acid (ESI) calcd for C66H77N9O14 [M + H]+ 1220.5663, found
aqueous solution and brine. The organic layer was dried over 1220.5662.
Na2SO4, filtered and concentrated under reduced pressure.
DKP (21). To a solution of DKP 16 (3.3 mg, 4.5 µmol) in
The residue was purified by column chromatography (0 to 5% 1 mL of TFA–H2O–CH3CN (5 : 3 : 2), the peptide 17 (10 mg,
MeOH in DCM) to afford 16 (88 mg, 0.121 mmol) in 57% 11 µmol) was added and the mixture was heated at 37 °C. The
4972 | Org. Biomol. Chem., 2014, 12, 4964–4974
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
reaction was monitored by analytical RP-HPLC and the reac-
tion mixture was purified by preparative HLPC (5 to 100%
solvent) to give, as TFA salt, the intermediate in which two
peptides are ligated (4.7 mg, 2.1 µmol) in 47% yield. RP-HPLC
(5 to 100% solvent B) tR = 13.6 min. ESI-MS calcd for
2011, 13, 4838. In this paper, the synthesis of a N,N′-dipro-
pargyl 2,5-diketopiperazine derived from glycine residues is
reported to prepare by ‘click chemistry’ rotaxanes but this
scaffold could be used to ligate biomolecules.
7 (a) G. M. Galibert, P. Dumy and D. Boturyn, Angew. Chem.,
Int. Ed., 2009, 48, 2576; (b) O. Renaudet, J. Garcia,
D. Boturyn, N. Spinelli, E. Defrancq, P. Labbé and P. Dumy,
Int. J. Nanotechnol., 2010, 7, 738.
8 (a) M. Sletten and C. R. Bertozzi, Angew. Chem., Int. Ed.,
2009, 48, 2; (b) J. Kalia and R. T. Raines, Curr. Org. Chem.,
2010, 14, 138.
9 (a) M. A. Findeis, G. M. Musso, C. C. Arici-Muendel,
H. W. Benjamin, A. M. Hundal, J. J. Lee, J. Chin, M. Kelley,
J. Wakefield, N. J. Hayward and S. M. Molineaux, Biochemi-
stry, 1999, 38, 6791; (b) Y. Porat, A. Abramowitz and
E. Gazit, Chem. Biol. Drug. Des., 2006, 67, 27.
10 A. S. M. Ressurreição, A. Bordessa, M. Civera, L. Belvisi,
C. Gennari and U. Piarulli, J. Org. Chem., 2008, 73,
652.
11 C. J. Dinsmore and D. C. Beshore, Tetrahedron, 2002, 58,
3297.
12 (a) T. Fukuyama, C.-K. Jow and M. Cheung, Tetrahedron
Lett., 1995, 36, 6373; (b) T. Kan and T. Fukuyama, Chem.
Commun., 2004, 353.
13 O. Demmer, I. Dijkgraaf, M. Schottelius, H.-J. Wester and
H. Kessler, Org. Lett., 2008, 10, 2015.
C102H151N23O22 2051.44, found 2051.4. This intermediate
(4.7 mg, 2.1 µmol) and curcumin 19 (1 mg, 2.52 µmol) were
‘clicked’ using the same procedure as described for the syn-
thesis of 20, and the DKP 21 (1.2 mg, 0.49 µmol) was obtained
after RP-HPLC purification (5 to 100% solvent B) in 23% yield.
RP-HPLC (15 to 100% solvent B) tR = 13.9 min. HRMS (ESI)
calcd for
1229.1504.
C126H173N23O28 [M
+
2H]2+ 1229.1483, found
Conclusions
We have developed new “ready-to-use” DKP scaffolds for
chemoselective ligations. They are prepared via an orthogonal
protecting group strategy from Boc/Alloc DKP precursors
which are efficiently prepared on a gram scale and which can
be easily functionalized with ‘clickable’ functions. In addition,
we have proved their relevance for assembling peptides and
organic molecules through the synthesis of potential inhibi-
tors of Aβ amyloid aggregation, the DKP core being of particu-
lar interest in the Aβ’s target domain due to its potentiality to
cross the blood–brain barrier. Our current efforts are devoted
to expanding the functional diversity of these scaffolds,
especially by exploring other chemoselective and orthogonal
ligation protocols.
14 P. Besada, L. Mamedova, C. J. Thomas, S. Costanzi and
K. A. Jacobson, Org. Biomol. Chem., 2005, 3, 2016.
15 M. Dessolin, M. G. Guillerez, N. Thieriet, F. Guibé and
A. Loffet, Tetrahedron Lett., 1995, 36, 5741.
16 O. El-Mahdi and O. Melnyk, Bioconjugate Chem., 2013, 24,
735.
17 S. Foillard, M. O. Rasmussen, J. Razkin, D. Boturyn and
P. Dumy, J. Org. Chem., 2008, 73, 983.
18 E. D. Goddard-Borger and R. V. Stick, Org. Lett., 2007, 9,
3797 (additions and corrections to this article in Org. Lett.,
2011, 13, 2514).
19 (a) For a more safe and at large-scale protocol than
Goddard-Borger’s protocol to prepare imidazole-1-sulfonyl
azide see: H. Ye, R. Liu, D. Li, Y. Liu, H. Yuan, W. Guo,
L. Zhou, X. Cao, H. Tian, J. Shen and P. G. Wang, Org. Lett.,
2013, 15, 18; (b) For more safe alternatives to the hydrogen
chloride salt as the hydrogen sulfate salt see: N. Fischer,
E. D. Goddard-Borger, R. Greiner, T. M. Klapötke,
B. W. Skelton and J. Stierstorfer, J. Org. Chem., 2012, 77,
1760.
Acknowledgements
This work was supported by the ‘Region Rhône-Alpes’ and has
been partially funded by the labex ARCANE (ANR-11-
LABX-0003-01) and the ‘Association France Alzheimer’. We are
grateful to NanoBio (Grenoble) for the facilities of the
Synthesis Platform, and to ICMG platforms (B. Gennaro for
NMR, L. Fort and R. Guéret for mass spectroscopy).
Notes and references
1 A. D. Borthwick, Chem. Rev., 2012, 112, 3641.
2 M. Marchini, M. Mingozzi, R. Colombo, I. Guzzetti,
L. Belvisi, F. Vasile, D. Potenza, U. Piarulli, D. Arosio and
C. Gennari, Chem. – Eur. J., 2012, 18, 6195.
20 M. Ouberai, P. Dumy, S. Chierici and J. Garcia, Bioconjugate
Chem., 2009, 20, 2123.
3 (a) G. Gellerman, E. Hazan, T. Brider, T. Traube, A. Albeck
and S. Shatzmiler, Int. J. Pept. Res. Ther., 2008, 14, 183; 21 W. Shi, S. Dolai, S. Rizk, A. Hussain, H. Tariq, S. Haverick,
(b) G. Gellerman, E. Hazan, M. Kovaliov, A. Albeck and
S. Shatzmiler, Tetrahedron, 2009, 65, 1389.
W. L’Amoreaux, A. El Idrissi, P. Banerjee and K. Raja, Org.
Lett., 2007, 9, 5461.
4 G. Gellerman, US Pat, 20100210474 A1, 2010.
5 M. Tullberg, M. Grotli and K. Luthman, J. Org. Chem., 2007,
72, 195.
6 E. V. Dzyuba, L. Kaufmann, N. L. Löw, A. K. Meyer,
H. D. F. Winkler, K. Rissanen and C. A. Schalley, Org. Lett.,
22 (a) V. Hong, S. I. Presolski, C. Ma and M. G. Finn, Angew.
Chem., Int. Ed., 2009, 121, 10063; (b) C. Besanceney-Webler,
H. Jiang, T. Zheng, L. Feng, D. Soriano del Amo, W. Wang,
L. M. Klivansky, F. L. Marlow, Y. Liu and P. Wu, Angew.
Chem., Int. Ed., 2011, 50, 8051.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 4964–4974 | 4973

View Article Online
Paper
Organic & Biomolecular Chemistry
23 (a) H. LeVine, Protein Sci., 1993, 2, 404; (b) H. LeVine, 26 M. Teixido, E. Zurita, M. Malakoutikhah, T. Tarrago
Methods Enzymol., 1999, 309, 274.
24 G. Zhang, M. J. Leibowitz, P. J. Sinko and S. Stein, Bioconju-
gate Chem., 2003, 14, 86.
25 S. M. Chafekar, H. Malda, M. Merkx, E. W. Meijer,
D. Viertl, H. A. Lashuel, F. Baas and W. Scheper, ChemBio-
Chem, 2007, 8, 1857.
and E. Giralt, J. Am. Chem. Soc., 2007, 129,
11802.
27 (a) B.-C. Chen, A. P. Skoumbourdis, P. Guo, M. S. Bednarz,
O. R. Kocy, J. E. Sundeen and G. D. Vite, J. Org. Chem.,
1999, 64, 9294; (b) J. Li and Y. Sha, Molecules, 2008, 13,
1111.
4974 | Org. Biomol. Chem., 2014, 12, 4964–4974
This journal is © The Royal Society of Chemistry 2014