Synthesis of a tripodal scaffold for solid phase synthesis of artificial receptors

Article abstract of DOI:10.1002/ejoc.200701104

A convergent synthesis for the preparation of a new orthogonally protected tripodal scaffold has been developed. The scaffold was successfully coupled to a Tentagel solid support and further derivatised at the three attachment points demonstrating its possible use for combinatorial chemistry. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200701104

FULL PAPER
DOI: 10.1002/ejoc.200701104
Synthesis of a Tripodal Scaffold for Solid Phase Synthesis of Artificial
Receptors
Steven E. Van der Plas,[a] An Gea,[a] Sara Figaroli,[a] Pierre J. De Clercq,[a] and
Annemieke Madder*[a]
Keywords: Receptor / Solid-phase synthesis / Combinatorial chemistry / Peptides / Protecting groups
A convergent synthesis for the preparation of a new orthogo-
nally protected tripodal scaffold has been developed. The
scaffold was successfully coupled to a Tentagel solid support
and further derivatised at the three attachment points dem-
onstrating its possible use for combinatorial chemistry.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
are organized in a parallel way.[5] Next to comparable inter-
actions between the aromatic moieties, extra stabilisation is
achieved by intramolecular H-bonding as shown in Fig-
ure 2.
Introduction
The design of new artificial receptors requires suitable
scaffolds that allow incorporation of various chemical func-
tionalities and at the same time preorganise the different
built-in residues. By equipping such a scaffold with an an-
chor point, allowing attachment to a solid support, combi-
natorial methods can be used to generate libraries of pos-
sible artificial receptor candidates. Screening for affinity
towards particular ligands can then enable identification of
a synthetic receptor. Earlier developed tripodal scaffolds are
mostly based on a rigid skeleton that imposes a specific
orientation on the attached functionalities.[1] Only a few
flexible tripods have been created to date, among which the
pentaerithryltetramine scaffold developed by Virta.[2]
Our group has also made a contribution in this area
using the flexibility of the pentaerythritol skeleton. In an
attempt to design a non-rigid, yet conformationally con-
strained template molecule, three aromatic residues were in-
troduced.[3] Molcular modelling studies suggested a pre-
ferred parallel orientation for the three benzylic chains in
1 (see Figure 1). However, when using scaffold 1 for the
construction of multipodal peptides, various problems
arose due to the presence of benzylic ether bonds. More-
over, hydrolysis of ester linkages between the scaffold and
peptide chains was observed.[4] With these problems in
mind we now wish to report on the convergent synthesis of
the racemic tripodal scaffold 2, omitting sensitive function-
alities and providing amino groups as anchoring points.
Molecular modelling showed that also in this case the arms
Figure 1. Structure of previously developed scaffold 1 and structure
of the new tripodal scaffold 2.
[a] Department of Organic Chemistry, Ghent University,
Krijgslaan 281, 9000 Ghent, Belgium
Fax: +32-9-264-4998
Figure 2. Results of molecular modelling studies on a simplified
version of 2.[5] Two energetically favoured conformations in H2O
are shown. The dotted lines represent intramolecular H-bonding.
E-mail: Annemieke.Madder@UGent.be
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Solid Phase Synthesis of Artificial Receptors
duction should be achieved using hydrazine in the presence
of preactivated Raney-Ni catalyst, TLC-monitoring showed
the reduction not to be fully complete, with some mono-
and di-nitriles still present. Changing the reaction variables
and the type of catalyst did not result in a significant im-
provement. However, the crude product could be used as
such and the impurities could be removed on a later stage
in the synthesis.
Results and Discussion
Scaffold 2 is characterized by a central core 5 (Scheme 1)
easily synthesized via a previously described method.[6] Al-
though according to this procedure, complete nitrile re-
The three different aromatic moieties were synthesized
starting from 4-aminobenzoic acid (Scheme 2). After pro-
tection of the amine,[7] the carboxylic acids were activated
as the pentafluorophenyl ester derivatives 6, 7 and 8 for
further coupling with 5.
Scheme 1. Synthesis of the core.
Scheme 2. Synthesis of the orthogonally protected legs.
Scheme 3. Assembly of scaffold 2.
Scheme 4. Solid-phase synthesis of a possible library member.
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To maximize the yield of monoderivatisation in the first Fmoc deprotection was observed. The use of alternative
step, three equiv. of triamine 5 were used and activated 6 coupling reagents such as DIC with HOAt, although avoid-
was added over a period of 8 h (Scheme 3). Separation of ing Fmoc deprotection, gave much lower loading values
the monopodal product from the excess starting material (0.059 mmolg–1). Finally, optimized conditions were found
and the side products was achieved via chromatography using the PyBOP/DIEA protocol at room temp. with only
using a CH2Cl2/MeOH/NH3 mixture. The two other aro- 1.5 equiv. of 2. A loading of 0.084 mmolg–1 was achieved.
matic residues, 7 and 8, were coupled in a similar fashion Under these conditions only 12% Fmoc deprotection was
with comparable yields.
observed. The remaining free aliphatic spacer amines were
Deprotection of the silyl ether was achieved with cata- selectively capped with AcOH/PyBOP/DIEA while less then
lytic amounts of (Ϯ)-CSA and only a very small amount of 2% of deprotected 2 was acylated.
Boc deprotection (Ͻ 1%) was observed. Oxidation to the
After attaching 2 to the solid support, deprotection of
carboxylic acid 2 was carried out using Jones reagent. the different carbamate groups gives rise to arylamines with
Gram quantities of 2 are easily available via this synthetic a low nucleophilicity. Activating 12 equiv. of the desired
route.
amino acid in the presence of 6 equiv. DIC for 30 min at
[11]
In order to facilitate monitoring of reactions on solid 0 °C in CH2Cl2
generates the symmetrical anhydrides
support, a photocleavable linker 10 was used which releases that gave a clean coupling.[12] Next to performing the NF31
the product upon irradiation at 365 nm.[8] Only small test,[13] completeness of the reaction was checked via ES-
amounts of resin are needed (typically 1 mg or less) and the MS (after photolytic cleavage) and the coupling was re-
solution obtained upon irradiation can be directly analyzed peated when necessary.
by LC-MS. Using 10, in some cases yellow coloration of
The Fmoc group was removed first where after Fmoc-
the solid support was observed, lowering the sensitivity of PheOH was coupled, Fmoc-deprotected and capped. A tan-
the TNBS-test.[9] This problem could be circumvented by dem-deprotection with the ternary system Pd0/PhSiH3/
using the NF31-test which has a higher sensitivity.[10] Boc- (FmocAla)2O gave incomplete deprotection and coupling.
γ-aminobutyric acid was used as a spacer to minimize steric However, after Alloc deprotection with Pd0 as a catalyst
hindrance when performing coupling reactions to the sec- and PhSiH3 as scavenger, FmocAlaOH was attached, de-
ondary amine of 10. It was noted that the use of Fmoc-γ- protected and capped[14] (see Scheme 4).
aminobutyric acid led to undesired alkylation of the spacer
Finally, the Boc group was deprotected with 50% TFA/
upon Fmoc deprotection with piperidine.
CH2Cl2. FmocGlyOH was coupled and deprotected to give
Following Boc deprotection, coupling to the solid sup- 12 in good purity as judged from LC (Figure 3) and ES-
port was first tested using a threefold excess of 2 in the MS (Figure 4) analysis. It has been further shown that the
presence of PyBOP (3 equiv.) and DIEA (9 equiv.) yielding order of aniline deprotection for the Boc and Alloc groups
a loading of 0.077 mmolg–1 (maximum theoretical loading can be switched. We realize that including the Boc group
is 0.164 mmolg–1). LC control of the reaction mixture excludes the use of functionalized Fmoc/tBu amino acids in
showed that a small amount of Fmoc-deprotected com- the previous strand(s). This can be circumvented by first
pound was present. In an attempt to improve coupling deprotecting the Boc group of 11 and then coupling a N-α-
yields, raising the reaction temperature to 50 °C or per- ivDde-protected amino acid, after which the current
forming a double coupling only resulted in a slightly higher Scheme can be applied in a Fmoc/tBu protocol with func-
loading of 0.084 mmolg–1 (51%). Moreover, increased tionalized amino acids.[15]
Figure 3. LC at 214 nm of the crude tripodal peptide obtained after photolysis of 12.
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Solid Phase Synthesis of Artificial Receptors
Figure 4. ES-MS+ of the crude tripodal peptide obtained after photolysis of 12.
following abbreviations were used to explain the multiplicities: s
Conclusions
singlet, d doublet, t triplet, q quadruplet, m multiplet, br. broad.
Infrared spectra (IR) were recorded on a Perkin–Elmer 1600 series
FTIR spectrometer and reported in wave numbers (cm–1). Samples
were prepared as a thin films (neat) on KBr plates. Elemental
analyses were performed by SIARE at the Université Pierre &
Marie Curie, Paris, France. High-Resolution Mass Spectra
(HRMS) were recorded by Amberlab at Ghent University on a
Thermo Finnigan MAT95XP-Trap tandem mass spectrometer or
by the Rega institute at the University of Leuven on a ThermoFin-
nigan LCQ MSn ion trap. Low Resolution Mass Spectra were re-
corded with an atmospheric pressure electrospray-ionisation (ESI)
Hewlett–Packard 5988 A mass spectrometer. HPLC analyses were
performed on an Agilent 1100 Series instrument with a Phe-
nomenex Luna C18(2) column (250ϫ4.6 mm, 5µ at 35 °C) using a
flow rate of 1 mL/min and with the following solvent systems: 0.1%
TFA in H2O (A) and MeCN (B). Unless otherwise stated the col-
umn was flushed for 3 min with 100% A, then a gradient from 0
to 100% B over 15 min was used, followed by 5 min of flushing
with 100% B. Photolyses were carried out with a 4 W Bioblock
Scientific compact UV lamp set at 365 nm. Melting point ranges
were determined with an Electrothermal 9100 melting point appa-
ratus.
In conclusion an efficient route towards orthogonally
protected tripodal scaffold 2 has been developed. A particu-
lar feature of the new template molecule is its highly flexi-
ble, though conformationally preorganisable nature. Immo-
bilisation onto a solid support and further derivatisation
of the different attachement points has been achieved. The
development of artificial receptors using 2 is currently un-
der investigation.
Experimental Section
General Methods: All solid-phase reactions on at most 20 mg of
resin were performed in polypropylene Chromabond columns of
1 mL with a polyethylene frit, closed at the bottom with a B7 sep-
tum from Aldrich. Solid-phase reactions on a larger scale were per-
formed in a peptide vessel protected against light with aluminium
foil and comprising a sintered glass funnel and a 3-way stopcock
for easy filtration and washing. All solution phase reactions were
conducted under an inert atmosphere of argon gas in oven dried
glassware. The reactions were monitored by thin layer chromatog-
raphy (TLC) using SIL G-25 UV254 pre-coated silica gel plates
(0.25 mm thickness). The TLC plates were visualized using an anis-
aldehyde (5% anisaldehyde in ethanol with 1% sulfuric acid) or a
PMA (5% phosphomolybdic acid in ethanol) solution. Flash col-
umn chromatography was performed using BIOSOLVE silica gel
(0.063–0.200 mm particle size). NMR spectra were recorded at
500 MHz or 300 MHz for proton and at 125 MHz or 75 MHz for
carbon nuclei in [D]chloroform, [D6]DMSO, [D4]MeOD or [D6]-
aceton. Chemical shifts are reported in units of parts per million
(ppm), referenced relative to the residual 1H or 13C peaks of the
Materials: All amino acids and the solid support Tentagel-S-NH2
were purchased from NovaBiochem. DMF extra dry was pur-
chased from Aldrich. DMF peptide grade was purchased from Bi-
osolve. All chemicals were purchased and used without any further
purification, except tetrahydrofuran (THF), which was distilled
from Na/benzophenone prior to use and dichloromethane, which
was distilled from CaH2.
Triamine 5: In a two-necked flask of 500 mL, 4 (22.0 g, 68.9 mmol)
was dissolved in 250 mL of pure ethanol and 13.2 mL of water. To
this mixture, NaOH (10.6 g, 26.0 mmol) was added, after which the
mixture was cooled down to 0 °C. After stirring for half an hour,
used solvent as internal standards ([D]chloroform: 1H 7.26 and 13
C
77.16; [D6]DMSO: 1H 2.50 and 13C 39.52; [D4]MeOD: 1H 3.31 and hydrazine monohydrate (26.4 mL, 544 mmol) was added. In the
13C 49.00; [D6]aceton: 1H 2.05 and 13C 29.84 and 206.26). The
meantime a Raney-Ni slurry (6.2 g, 53 mmol, Merck, 50% active
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S. E. Van der Plas, A. Gea, S. Figaroli, P. J. De Clercq, A. Madder
FULL PAPER
catalyst in water) was washed thoroughly with water and pure etha-
nol in a little flask. This suspension was added to the reaction
mixture over a period of 2 h, after which the solution was allowed
to warm up to room temp. After 1 h of stirring, the reaction mix-
ture was refluxed during 2 h. The reaction mixture was filtered off
over celite and the filtrate was concentrated under reduced pressure.
Toluene was added to the residue to precipitate the NaOH. The
base was filltered off and the filtrate was evaporated under reduced
pressure. This action was repeated until no more precipitation was
formed. A light yellow oil was obtained (19.6 g, 59.2 mmol, 86%).
1H NMR (300 MHz, CDCl3): δ = 3.12 (s, 2 H), 3.10 (br. s, 6 H),
2.64 (t, J = 6.8 Hz, 6 H), 1.34 (m, 6 H), 1.15 (m, 6 H), 0.84 (s, 9
H), 0.03 (s, 6 H) ppm. 13C + APT (75 MHz, CDCl3): δ = 66.4
(CH2), 43.0 (CH2), 39.3 (C), 31.1 (CH2), 27.4 (CH2), 25.8 (CH3),
reduced pressure. The resulting powder was chromatographed with
pentane/EtOAc (9:1) to give 7 (26.8 g, 69.4 mmol, 83%) as a white
1
powder; m.p. 137–139 °C. H NMR (COSY, 300 MHz, CDCl3): δ
= 8.17 (ddd, J = 9.4/2.4/2.0 Hz, 2 H), 7.75 (ddd, J = 9.4/2.4/2.0 Hz,
2 H), 5.97 (ddt, J = 17.3/10.5/5.7 Hz, 1 H), 5.40 (ddd, J = 17.1/2.9/
1.5, 1 H), 5.30 (ddd, J = 10.4/2.4/1.2 Hz, 1 H), 4.70 (ddd, J = 5.9/
1.3/1.3 Hz, 2 H). 13C NMR (APT, 75 MHz, CDCl3): δ = 162.1 (C),
152.6 (C), 143.9 (C), 132.3 (CH), 131.9 (CH), 121.3 (C), 118.3
(CH ), 117.9 (CH), 66.4 (CH ) ppm. IR (KBr): ν = 3355 (w), 1759
˜
2
2
(s) cm–1. LRMS (ESI–): m/z = 386.1 [M – H]–. C17H10F5NO4
(387.05): calcd. C 52.73, H 2.60, N 3.62; found C 52.65, H 2.78, N
3.45.
4-(Fluorenylmethoxycarbonylamino)benzoic Acid (8a): In a 5 L
flask, 4-aminobenzoic acid (40.7 g, 0.297 mol) was dissolved in
810 mL water. NaHCO3 (68.0 g, 0.809 mol) was then added, fol-
lowed by the addition of 810 mL of dioxane and FmocOSu (100 g,
0.297 mol). After stirring for 4 h, 400 mL water was added. The
solution was stirred for another 44 h and was then acidified with
1  HCl solution until pH 3 was reached. The white precipitate was
filtered off an dried with P2O5 under vacuum (102.9 g, 0.286 mol,
18.1 (C), –5.7 (CH ) ppm. IR (KBr): ν = 3358 (s), 2929 (s), 2857
˜
3
(m) 1568 (m), 1472 (m), 1386 (w), 1328 (m) cm–1. LRMS (ESI+):
332.3 [MH+]
Synthesis of the Orthogonally Protected Scaffold Legs
4-(tert-Butoxycarbonylamino)benzoic Acid (6a): See ref.[7]
Pentafluorophenyl 4-(tert-Butoxycarbonylamino)benzoate (6): To a
solution of 6a (10.0 g, 42.0 mmol) in THF (65 mL) was added
pentafluorophenol (9.37 g, 50.4 mmol), DMAP (260 mg,
2.11 mmol) and molecular sieves. After stirring the solution for
10 min at 0 °C the coupling reagent DCC (10.5 g, 50.4 mmol) was
added and the ice-bath was allowed to warm up to room temp.
After 18 h the reaction mixture was filtered off and the filtrate was
concentrated under reduced pressure. The resulting orange precipi-
tate was chromatographed with pentane/CH2Cl2 (1:1) to give 6
1
96%); m.p. 276–278 °C. H NMR (COSY, 500 MHz, [D6]DMSO):
δ = 10.0 (br. s, 1 H), 7.91 (d, J = 7.5 Hz, 2 H), 7.84 (d, J = 8.1 Hz,
2 H), 7.75 (d, J = 7.5 Hz, 2 H), 7.54 (br. s, 2 H), 7.43 (dd, J = 7.4/
7.4 Hz, 2 H), 7.35 (ddd, J = 7.4/7.4/0.9 Hz, 2 H), 4.53 (d, J =
6.4 Hz, 2 H), 4.33 (t, J = 6.4 Hz, 1 H). 13C NMR (APT, 75 MHz,
[D6]DMSO): δ = 166.9 (C), 153.2 (C), 143.7 (C), 143.2 (C), 140.8
(C), 130.4 (CH), 127.7 (CH), 127.2 (CH), 125.2 (CH), 125.1 (C),
120.2 (CH), 117.4 (CH), 65.8 (CH ), 46.5 (CH) ppm. IR (KBr): ν
˜
2
= 3342 (w), 1711 (s), 1678 (m), 1529 (s), 1238 (s), 739 (s) cm–1.
LRMS (ESI–): m/z = 358.5 [M – H]–. C22H17NO4 (359.12): calcd.
C 73.53, H 4.77, N 3.90; found C 73.40, H 4.93, N 3.93.
1
(13.9 g, 34.4 mmol, 82%) as a white powder; m.p. 124–126 °C. H
NMR (COSY, 300 MHz, CDCl3): δ = 8.12 (ddd, J = 8.4/2.4/1.8 Hz,
2 H), 7.54 (ddd, J = 9.3/2.3/1.9 Hz, 2 H), 6.77 (br. s, 1 H), 1.52 (s,
9 H). 13C NMR (APT, 75 MHz, CDCl3): δ = 162.1 (C), 151.9 (C),
144.4 (C), 132.2 (CH), 120.7 (C), 117.6 (CH), 81.7 (C), 28.2 (CH3)
Pentafluorophenyl 4-[(9H-Fluoren-9-yl)methoxycarbonylamino]ben-
zoate (8): To a solution of 8a (10.0 g, 27.8 mmol, 1 equiv.) in THF
(45 mL) was added pentafluorophenol (7.23 g, 38.9 mmol), DMAP
(173 mg, 1.40 mmol) and molecular sieves. After stirring the solu-
tion for 10 min at 0 °C, the coupling reagent DCC (8.11 g,
38.9 mmol) was added and the ice-bath was allowed warm until
room temp. After 18 h the reaction mixture was filtered off and the
filtrate was concentrated under reduced pressure. The resulting
light-yellow precipitate was recrystallized from EtOAc. Then the
precipitate was taken into THF and refluxed. When everything was
dissolved, the solution was slowly cooled in an ice-bath. The white
precipitate was filtered off and the filtrate was evaporated to give
8 (10.8 g, 20.6 mmol, 74%) as a white powder; m.p. 194–196 °C.
1H NMR (COSY, 300 MHz, CDCl3): δ = 8.14 (ddd, J = 8.8/2.4/
1.8 Hz, 2 H), 7.80 (d, J = 7.5 Hz, 2 H), 7.63 (d, J = 7.5 Hz, 2 H),
7.55 (d, J = 8.6 Hz, 2 H), 7.44 (dd, J = 7.2/7.2 Hz, 2 H), 7.35 (ddd,
J = 7.5/7.5/1.1 Hz, 2 H), 6.90 (s, 1 H), 4.62 (d, J = 6.4 Hz, 2 H),
4.28 (t, J = 6.4 Hz, 1 H) ppm. 13C NMR (APT, 75 MHz, CDCl3):
δ = 162.1 (C), 152.8 (C), 143.7 (C), 143.5 (C), 141.4 (C), 132.3
(CH), 128.0 (CH), 127.2 (CH), 124.8 (CH), 121.3 (C), 120.2 (CH),
ppm. IR (KBr): ν = 3355 (w), 1758 (s), 1736 (m) cm–1. LRMS
˜
(ESI–): m/z = 402.7 [M – H]–. C18H14F5NO4 (403.08): calcd. C
53.61, H 3.50, N 3.47; found C 53.71, H 3.67, N 3.30.
4-(Allyloxycarbonylamino)benzoic Acid (7a): In a 2 L flask, 4-ami-
nobenzoic acid (1 g, 7.29 mmol) was dissolved in 10 mL of dioxane
and 10 mL of water. DIEA (2.6 mL, 14.9 mmol) and NaHCO3
(1.8 g, 21.8 mmol) were added. Finally, allyl chloroformate
(0.78 mL, 7.33 mmol) was added and the reaction is stirred over-
night. A 1  HCl solution was added drop wise until there was no
more precipitate forming. The white powder was filtered off and
dried with P2O5 under reduced pressure to provide 7a (1.35 g,
6.10 mmol, 83%); m.p. 199–201 °C. 1H NMR (COSY, 500 MHz,
[D6]DMSO): δ = 10.07 (br. s, 1 H), 7.83 (ddd, J = 9.3/2.3/1.8 Hz,
2 H), 7.74 (ddd, J = 9.3/2.3/1.9 Hz, 2 H), 5.95 (ddt, J = 17.3/10.5/
5.5 Hz, 1 H), 5.35 (ddd, J = 17.3/3.3/1.6, 1 H), 5.21 (ddd, J = 10.4/
2.9/1.3 Hz, 1 H), 4.58 (ddd, J = 5.5/1.3/1.3 Hz, 2 H). 13C NMR
(APT, 75 MHz, [D6]acetone): δ = 167.3 (C), 153.9 (C), 144.5 (C),
133.9 (CH), 131.6 (CH), 125.3 (C), 118.3 (CH), 117.9 (CH2), 66.0
117.9 (CH), 67.2 (CH ), 47.0 (CH) ppm. IR (KBr): ν = 3343 (w),
˜
2
(CH ) ppm. IR (KBr): ν = 3334, 1702, 1685, 1600, 1532, 1315,
˜
2
1755 (s), 1708 (m) cm–1. LRMS (ESI+): m/z = 590.4 [M + MeCN
1230 cm–1. LRMS (ESI–): m/z = 220.2 [M – H]–. C11H11NO4
(221.07): calcd. C 59.73, H 5.01, N 6.33; found C 59.43, H 4.95, N
6.58.
+ H]+.
Synthesis of Silyl Ether 9: i) To a solution of 5 (11.182 g,
33.7 mmol) in CH2Cl2 (70 mL) was added Et3N (1.475 mL,
10.5 mmol) and molecular sieves. To the resulting mixture, a solu-
tion of the activated ester 6 (4.247 g, 10.5 mmol) in CH2Cl2
(60 mL) was slowly added via the syringe pump over a period of
8 h. The solution was then stirred overnight and afterwards concen-
trated under reduced pressure. The residual oil was chromato-
graphed with gradient elution where the amount of MeOH(NH3)
Pentafluorophenyl 4-(Allyloxycarbonylamino)benzoate (7): To
a
solution of 7a (19.2 g, 83.6 mmol) in THF (225 mL) was added
pentafluorophenol (19.2 g, 104.5 mmol), DMAP (480 mg,
3.93 mmol) and molecular sieves. After stirring the solution for
10 min at 0 °C the coupling reagent DCC (21.6 g, 104.5 mmol) was
added and the ice-bath is allowed to melt. After 18 h the reaction
mixture was filtered off and the filtrate was concentrated under
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Solid Phase Synthesis of Artificial Receptors
in CH2Cl2 was varied from 5–25%. The desired diamine was ob-
tained as a light-yellow foam (3.182 g, 55%); m.p. 66–68 °C. Finally
the column was washed with pure MeOH(NH3) to recover the ex-
cess starting material (6.512 g or 55%). 1H NMR (COSY,
500 MHz, CDCl3): δ = 8.26 (br. s, 1 H), 7.63 (d, J = 8.8 Hz, 2 H),
7.36 (d, J = 8.6 Hz, 2 H), 6.77 (t, J = 5.6 Hz, 1 H), 3.25 (m, 2 H),
3.16 (s, 2 H), 2.53 (t, J = 6.6 Hz, 4 H), 1.43–1.36 (m, 11 H), 1.28–
1.05 (m, 10 H), 0.77 (s, 9 H), –0.10 (s, 6 H). 13C NMR (APT,
75 MHz, CDCl3): δ = 167.1 (C), 152.9 (C), 142.0 (C), 128.5 (C),
128.0 (CH), 117.6 (CH), 80.3 (C), 66.4 (CH2), 42.9 (CH2), 40.7
(CH2), 39.2 (C), 31.1 (CH2), 28.3 (CH3), 27.1 (CH2), 25.8 (CH3),
(CH ) ppm. IR (KBr): ν = 3304 (w), 1755 (s), 1708 (m) cm–1.
˜
3
LRMS (ESI+): m/z = 1095.5 [M + H]+. HRMS (ESI+): calcd. for
[C62H78N6O10Si + H]+: 1095.56215, found 1095.56582.
Synthesis of Carboxylic Acid 2: i) The silyl ether 9 (2.95 g,
2.70 mmol) was dissolved in a 1:1 mixture of CH2Cl2 (45 mL) and
MeOH (45 mL). Then (Ϯ)-CSA (125 mg, 0.539 mmol, 20 mol-%)
was added and the reaction mixture is stirred overnight. The reac-
tion was monitored by reversed-phase HPLC at 262 nm and if there
was more than 6% starting material, then an extra portion (Ϯ)-
CSA (31 mg, 0.13 mmol, 5 mol-%) was added. After another 4 h
the mixture is concentrated under reduced pressure in the presence
of silica gel. The light-yellow powder is chromatographed with a
gradient elution where the amount of MeOH is varied from 5 to
23.4 (CH ), 18.1 (C), –5.6 (CH ) ppm. IR (KBr): ν = 3304 (w),
˜
2
3
1755 (s), 1708 (m) cm–1. LRMS (ESI+): m/z = 551.3 [M + H]+.
HRMS (ESI+): calcd. for [C29H54N4O4Si + H]+: 551.39924, found
551.39871.
1
6% to give the alcohol (2.33 g, 88%); m.p. 153–155 °C. H NMR
(COSY, 500 MHz, MeOD): δ = 7.81 (d, J = 7.5 Hz, 2 H), 7.75–
7.65 (m, 8 H), 7.56–7.42 (m, 6 H), 7.38 (dd, J = 7.4/7.4 Hz, 2 H),
7.29 (ddd, J = 7.4/7.4/0.9 Hz, 2 H), 5.96 (ddt, J = 17.2/10.6/5.5 Hz,
1 H), 5.34 (ddd, J = 17.2/3.1/1.6 Hz, 1 H), 5.20 (ddd, J = 10.5/2.6/
1.5 Hz, 1 H), 4.60 (ddd, J = 5.7/1.4/1.4 Hz, 2 H), 4.48 (d, J =
6.4 Hz, 2 H), 4.27 (t, J = 6.6 Hz, 1 H), 3.30 (m, 8 H), 1.59–1.50
(m, 6 H), 1.48 (s, 9 H), 1.33–1.22 (m, 6 H) ppm. 13C NMR (APT,
75 MHz, MeOD): δ = 169.8 (C), 155.5 (C), 155.3 (C), 154.8 (C),
145.2 (C), 143.9 (C), 143.5 (C), 143.4 (C), 142.7 (C), 134.1 (CH),
129.8 (C), 129.3 (C), 129.2 (CH), 129.1 (CH), 128.9 (CH), 128.2
(CH), 126.2 (CH), 121.0 (CH), 119.1 (CH), 119.0 (CH), 118.9
(CH), 118.0 (C), 81.7 (C), 67.9 (CH2), 66.6 (CH2), 48.4 (CH), 41.7
(CH2), 40.3 (C), 32.0 (CH2), 28.7 (CH3) 24.2 (CH2) ppm. IR (KBr):
ii) To a solution of the diamine (6.176 g, 11.2 mmol) in CH2Cl2
(60 mL) was added Et3N (0.72 mL, 5.1 mmol) and molecular si-
eves. To the resulting mixture, a solution of activated ester 7
(1.974 g, 5.1 mmol) in THF (14 mL) was slowly added via the sy-
ringe pump over a period of 8 h. The solution was then stirred
overnight and afterwards concentrated under reduced pressure. The
residual oil was chromatographed with gradient elution where the
amount of MeOH(NH3) in CH2Cl2 was varied from 3% to 10%.
The desired mono-amine was obtained as a light-yellow foam with
a
54% (2.091 g) yield; m.p. 113–115 °C. 1H NMR (COSY,
300 MHz, CDCl3): δ = 7.78 (br. s, 1 H) 7.73 (d, J = 8.6 Hz, 2 H),
7.70 (d, J = 8.6 Hz, 2 H), 7.42 (d, J = 8.3 Hz, 2 H), 7.38 (d, J =
8.6 Hz, 2 H), 715 (br. s, 1 H), 6.64 (br. s, 1 H), 6.57 (br. s, 1 H),
5.96 (ddt, J = 17.2/10.5/5.7 Hz, 1 H), 5.36 (ddd, J = 17.2/2.9/1.5 Hz,
1 H), 5.26 (ddd, J = 10.3/2.4/1.3 Hz, 1 H), 4.66 (ddd, J = 5.6/1.3/
1.3 Hz, 2 H), 3.38–3.31 (m, 4 H), 3.22 (s, 2 H), 2.62 (t, J = 6.8 Hz,
2 H), 1.53–1.43 (m, 13 H), 1.35–1.18 (m, 8 H), 0.84 (s, 9 H), –0.03
(s, 6 H). 13C NMR (APT, 75 MHz, CDCl3): δ = 167.2 (C), 167.1
(C), 153.2 (C), 152.6 (C), 141.5 (C), 141.0 (C), 132.3 (CH), 129.3
(C), 128.8 (C), 128.1 (CH), 128.0 (CH), 118.4 (CH2),118.2 (CH),
118.0 (CH), 80.9 (C), 66.6 (CH2), 66.0 (CH2), 42.9 (CH2), 40.8
(CH2), 39.4 (C), 31.5 (CH2), 30.8 (CH2 or C), 28.3 (CH3), 27.1
(CH2), 25.8 (CH3), 23.3 (CH2), 18.1 (C), –5.6 (CH3) ppm. IR
ν = 3304 (w), 1755 (s), 1708 (m) cm–1. LRMS (ESI+): m/z = 981.4
˜
[M + H]+. HRMS (ESI+): calcd. for [C56H64N6O10 + Na]+:
1003.4559, found 1003.4576.
ii) The alcohol (4.35 g, 4.43 mmol) was dissolved in acetone
(28 mL) and the solution was cooled in an ice bath. Jones reagent
was prepared by dissolving CrO3 (4.01 g, 40.1 mmol) in a mixture
of acetone (3.4 mL) and water (11.6 mL). The freshly prepared
Jones reagent (6.7 mL, 2.67 ) was added slowly to the reagent mix-
ture in two portions. After one hour another 1.7 mL Jones reagent
(1 equiv.) was added. The reaction was stirred for another 2 h and
then 2-propanol (10 mL) was added. The mixture was extracted
twice with diethyl ether and 6 times with EtOAc, where after the
organic phase was concentrated under reduced pressure in the pres-
ence of silica gel (20 g). The yellow powder was chromatographed
with gradient elution where the percentage of MeOH in CH2Cl2
was varied from 5 to 15%) to give carboxylic acid 2 (3.39 g, 77%);
m.p. 155–157 °C. 1H NMR (COSY, 300 MHz, [D6]DMSO): δ =
12.17 (br. s, 1 H), 9.96 (s, 1 H), 9.91 (s, 1 H), 9.56 (s, 1 H), 8.33
(br. s, 3 H), 7.96 (d, J = 7.4 Hz, 2 H), 7.80–7.68 (m, 8 H), 7.55–
7.45 (m, 6 H), 7.41 (dd, J = 7.4/7.4 Hz, 2 H), 7.33 (ddd, J = 7.4/
7.4/1.1 Hz, 2 H), 5.97 (ddt, J = 17.0/10.4/5.5 Hz, 1 H), 5.36 (ddd,
J = 17.2/3.3/1.7 Hz, 1 H), 5.23 (ddd, J = 10.4/2.8/1.4 Hz, 1 H), 4.61
(ddd, J = 5.4/1.3/1.3 Hz, 2 H), 4.49 (d, 6.9 Hz, 2 H), 4.31 (t, 6.9 Hz,
1 H), 3.15 (s, 6 H), 1.51–1.41 (m, 6 H), 1.40–1.28 (s, 15 H), 1.40–
1.28 (s, 6 H). 13C (APT, 75 MHz, MeOD): δ = 180.4 (C), 169.7 (C),
155.5 (C), 154.8 (C), 145.2 (C), 144.0 (C), 143.5 (C), 143.4 (C),
142.7 (C), 134.1 (CH), 129.7 (C), 129.2 (CH), 128.9 (CH), 128.2
(CH), 126.2 (CH), 121.0 (CH), 119.1 (CH), 119.0 (CH), 118.8
(CH), 118.0 (C), 81.2 (C), 67.9 (CH2), 66.6 (CH2), 48.4 (CH), 41.2
(CH2), 32.9 (CH2), 28.7 (CH3), 25.5 (CH2) ppm. IR (KBr): 3304
(w), 1755 (s), 1708 (m) cm–1. LRMS (ESI–): m/z = 992.9 [M –
H]–. HRMS (ESI+): calcd. for [C56H62N6O11 + H]+: 995.4537,
found 995.4549.
(KBr): ν = 3304 (w), 1755 (s), 1708 (m) cm–1. LRMS (ESI+): m/z
˜
= 755.0 [M + H]+. HRMS (ESI+): calcd. for [C29H54N4O4Si +
H]+: 754.45695, found 754.45493.
iii) To a solution of the mono-amine (2.09 g, 2.77 mmol) in THF
(34 mL) was added DIEA (0.48 mL, 2.76 mmol) and 8 (2.90 g,
5.52 mmol). The solution was then stirred overnight and afterwards
concentrated under reduced pressure. The residual yellow foam was
chromatographed with gradient elution where the amount of
MeOH in CH2Cl2 was varied from 2% to 4%. In this way the
product 9 was collected as a white powder (2.70 g, 2.47 mmol,
1
89%); m.p. 134–136 °C. H NMR (COSY, 300 MHz, CDCl3): δ =
7.81 (br. s, 1 H), 7.74 (d, J = 7.7 Hz, 2 H), 7.72–7.65 (m, 6 H), 7.63
(br. s, 1 H), 7.59 (d, J = 7.5 Hz, 2 H), 7.45–7.30 (m, 8 H), 7.26
(ddd, J = 7.4/7.4/0.9 Hz, 2 H), 7.15 (br. s, 1 H), 6.95 (br. s, 2 H),
6.86 (t, J = 5.5 Hz, 1 H), 5.86 (ddt, J = 17.2/10.6/5.7 Hz, 1 H), 5.28
(ddd, J = 17.2/3.0/1.5 Hz, 1 H), 5.18 (J = 10.4/2.5/1.2 Hz, 1 H),
4.58 (ddd, J = 5.7/1.4/1.4 Hz, 2 H), 4.46 (d, J = 6.6 Hz, 2 H), 4.21
(t, J = 6.9 Hz, 1 H), 3.26 (br. s, 6 H), 3.13 (s, 2 H), 1.46 (s, 9 H),
1.36 (br. s, 6 H), 1.09 (br. s, 6 H), 0.77 (s, 9 H), –0.08 (s, 6 H). 13C
(APT, 75 MHz, CDCl3): δ = 167.4 (C), 153.5 (C), 152.6 (C), 143.7
(C), 141.5 (C), 141.3 (C), 141.0 (C), 132.3 (CH), 129.1 (C), 128.6
(C), 128.1 (CH), 127.9 (CH), 127.2 (CH), 125.0 (CH), 120.1 (CH),
118.4 (C), 118.4 (CH), 118.3 (CH), 118.1 (CH), 81.0 (C), 67.1
(CH2), 66.6 (CH2), 65.6 (CH2), 47.0 (CH), 40.8 (CH2), 39.3 (C), Solid-Phase Syntheses: After each step the resin was washed with
30.8 (CH2), 28.3 (CH3), 25.8 (CH3), 23.1 (CH2), 18.1 (C), –5.63 three times DMF/MeOH/CH2Cl2 successively, unless differently
Eur. J. Org. Chem. 2008, 1582–1588
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1587

S. E. Van der Plas, A. Gea, S. Figaroli, P. J. De Clercq, A. Madder
FULL PAPER
described. For the coupling reactions, the amount of solvent added
was such that a concentration of ca. 0.1  of coupling species was
achieved. The loading was determined by treating an amount of
resin with a 20% piperidine/DMF solution for 20 min. Then the
absorbance of the piperidine-fulvene adduct was measured at
300 nm. From a correlation curve, the concentration of the adduct
was retrieved from which the proper loading could be calculated.
washed after which the capping was repeated for 20 min. The
ninhydrin test was negative. The loading was 0.074 mmol/g and the
amount of Fmoc deprotection is 12%. Thus the total loading was
0.084 mmol/g which correlated (maximal theoretical loading is
0.164 mmolg–1) to a yield of 51%. LRMS (ESI+, after photocleav-
age in CH3CN): m/z = 1023 [M – tBu + H]+, 1079 [M + H]+,
1101.5 [M + Na]+. HRMS (ESI+): calcd. for the Fmoc-deprotected
11: [C45H60N8O9 + H]+: 857.45556, found 857.45427
Fmoc Deprotection: To the resin was added a 20% piperidine/DMF
solution (10 mL/g resin) and the resin was shaken for 1 min. The
solution was drained and the resin was washed. This step was re-
peated for 5 and 8 min.
Supporting Information (see also the footnote on the first page of
1
this article): Copies of H and 13C spectra of compounds 2, 5, 6,
7, 8, 9 and intermediates in Scheme 3.
Boc Deprotection: To the resin was added a 50% TFA/CH2Cl2 solu-
tion (10 mL/g resin) and the suspension was shaken for 5 min. The
solution was drained and the resin was washed three times with
CH2Cl2. This step was repeated once for a period of 25 min and
afterwards the washing included CH2Cl2/10% Et3N in CH2Cl2/
MeOH/CH2Cl2.
Acknowledgments
Financial assistance of the Institute for the Promotion of Innova-
tion through Science and Technology in Flanders (IWT-Vlaand-
eren) and of the FWO-Vlaanderen (KAN 1.5.186.03 and
G.0.47.04) is gratefully acknowledged. This research has further
been supported by a Marie Curie Early Stage Research Training
Fellowship of the European Community’s Sixth Framework Pro-
gramme under contract number MEST-CT-2005-020643.
Alloc Deprotection: To the resin was added PhSiH3 (25 equiv.) in
CH2Cl2 after which the (PPh3)4Pd catalyst was added (10 mol-%).
The suspension was shaken for 10 min. After draining the resin is
washed with CH2Cl2/MeOH/CH2Cl2.
Phtotocleavage of the Resin: A small amount of resin (1 mg or less)
was suspended in 100 µL of CH3CN in a small glass tube. The tube
was placed under UV light of 365 nm and after 3 h the solution
was analysed via LC, ES-MS or LC-MS. When the amount of resin
was increased, the irradiation time could be decreased enabling a
fast analysis.
[1] a) V. del Amo, L. Siracusa, T. Markidis, B. Baragana, K. M.
Bhattarai, M. Galobardes, G. Naredo, M. N. Perez-Payan,
A. P. Davis, Org. Biomol. Chem. 2004, 2, 3320–3328; b) L. S.
Sonntag, S. Ivan, M. Langer, M. A. Conza, H. Wennemers,
Synlett 2004, 7, 1270–1272; c) T. Opatz, R. M. J. Liskamp, Org.
Lett. 2001, 3, 3499–3502; d) X. T. Zhou, A. U. Rehman, C. H.
Li, P. B. Savage, Org. Lett. 2000, 2, 3015–3018; e) P. Kocis, O.
Issakova, N. F. Sepetov, M. Lebl, Tetrahedron Lett. 1995, 36,
6623–6626.
Synthesis of the Photocleavable Linker 10: See ref.[8]
Coupling of the Photocleavable Linker 10 to Tentagel-NH2: Tent-
agel-NH2 (539 mg, 135 µmol) was swollen in DMF (5.3 mL) and
then the solvent was drained. The photocleavable linker 10
(210 mg, 404 µmol) was dissolved in 4 mL DMF and the solution
was added to the pre-swollen resin. The coupling reagent PyBOP
(520 mg, 404 µmol) and the base DIEA (145 µL, 808 µmol) were
added. The suspension was shaken for 3 h. After draining, the resin
was washed. The NF31 test was used to monitor the completeness
of the reaction. By measuring the UV absorbance of the piperi-
dine–fulvene adduct at 300 nm, the loading was determined to be
0.21 mmolg–1.
[2] P. Virta, M. Leppänen, H. Lönnberg, J. Org. Chem. 2003, 69,
2008–2016.
[3] N. Farcy, H. De Muynck, A. Madder, N. Hosten, P. J.
De Clercq, Org. Lett. 2001, 3, 4299–4301.
[4] A. Gea, N. Farcy, N. Hosten, J. Martins, P. De Clercq, A. Mad-
der, Eur. J. Org. Chem. 2006, 18, 4135–4146.
[5] A full conformational analysis was performed using Macro-
Model V 6.0, force field MM2. To reduce calculation times the
carboxylic acid linker was simplified as a methylgroup and the
amine protecting groups were simplified as acetyl protections.
[6] D. Imbert, F. Thomas, P. Baret, G. Serratrice, D. Gaude, J. L.
Pierre, J. P. Laulhère, New J. Chem. 2000, 24, 281–288.
[7] F. Mu, S. L. Coffing, D. J. Riese, J. David, J. Med. Chem. 2001,
44, 441–452.
Coupling of the Spacer Boc-GABA: First, the resin with the photo-
cleavable linker (3 g, 0.75 mmol) was Fmoc-deprotected. Then the
spacer Boc-GABA (475 mg, 2.25 mmol) and the coupling reagent
PyBOP (1.17 g, 2.25 µmol) were added to the pre-swollen resin.
DMF was added and the suspension was shaken until the reagents
were dissolved. The base DIEA (785 µL, 4.5 mmol) was added
where after the reaction vessel was shaken for 3 h. After draining
and washing the resin, approximately 1 mg of the beads were sub-
jected to a NF31 test which was negative.
[8] C. P. Holmes, J. Org. Chem. 1997, 62, 2370–2380.
[9] W. S. Hancock, J. E. Battersby, Anal. Biochem. 1976, 71, 260–
264.
[10] A. Madder, N. Farcy, Eur. J. Org. Chem. 1999, 11, 2787–2791.
[11] The anhydrides are insoluble in CH2Cl2 and after activation
DMF is added until a clear solution is obtained.
[12] R. Pascal, R. Sola, F. Labéguère, P. Jouin, Eur. J. Org. Chem.
2000, 3755–3761.
Coupling of 1 to the Solid Anchor to Form Construct 11: The resin
(14 mg, 3 µmol) was Boc-deprotected. A solution of 2 (4.5 mg,
4.5 µmol) and PyBOP (2.3 mg, 4.5 µmol) in 0.15 mL DMF was
prepared. When homogeneous, the solution was transferred to the
pre-swollen resin and after adding DIEA (2.4 µL, 13.5 µmol) the
suspension was shaken for 18 h. The resin was drained and washed
thoroughly. The TNBS test turned the beads dark-yellow and with
NF31 we got red beads. The remaining free spacer-amino groups
were capped for 25 min with a 0.1  solution of AcOH and PyBOP
in the presence of 2 equiv. of DIEA. The resin was drained and
[13] S. E. Van der Plas, A. Madder, Tetrahedron Lett. 2007, 48,
2587–2589.
[14] N. Thieriet, P. Gomez-Martinez, F. Guibe, Tetrahedron Lett.
1999, 40, 2505–2508.
[15] S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W.
Bycroft, W. C. Chan, Tetrahedron Lett. 1998, 39, 1603–1606.
Received: November 22, 2007
Published Online: February 5, 2008
1588
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 1582–1588