An efficient synthesis of an orthogonally protected aromatic diamine as scaffold for tweezer receptors with two different arms

Article abstract of DOI:10.1002/ejoc.200900536

The synthesis of a new versatile template, 3-(N-Boc-aminomethyl)-5-(N-Fmoc- aminomethyl)benzoic acid (1), was developed and is hereby reported. The key feature of the synthetic strategy is the selective mono reduction of a symmetric diazide using a biphas

Full text of DOI:10.1002/ejoc.200900536

FULL PAPER
DOI: 10.1002/ejoc.200900536
An Efficient Synthesis of an Orthogonally Protected Aromatic Diamine
as Scaffold for Tweezer Receptors with Two Different Arms
Hannes Y. Kuchelmeister[a] and Carsten Schmuck*[a]
Keywords: Protecting groups / Template synthesis / Peptides / Solid-phase synthesis / Receptors
The synthesis of a new versatile template, 3-(N-Boc-amino-
methyl)-5-(N-Fmoc-aminomethyl)benzoic acid (1), was de-
veloped and is hereby reported. The key feature of the syn-
thetic strategy is the selective mono reduction of a symmetric
diazide using a biphasic reaction mixture. The mono re-
duction allows the sequential introduction of two orthogonal
protecting groups – e.g., Fmoc and Boc. This building block
can be used as a scaffold for the solid-phase synthesis of di-
podal or cyclic peptidic receptors. It allows the preparation
of tweezer receptors with unsymmetrically substituted side
arms and therefore for the implementation of an enhanced
structural and functional diversity in comparison to symmet-
ric tweezers.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
dium but still on-bead revealed selective tripeptide binding
with moderate affinities (K ≈ 103 –1). For a more pro-
nounced use of unsymmetrical tweezers, additional scaf-
folds are needed which allow the introduction of two dif-
ferent side arms while keeping the synthetic effort as small
as possible. We decided to develop a novel scaffold for un-
symmetrical tweezer receptors. Therefore we designed and
prepared the orthogonally protected template 1 (Figure 1)
which allows the solid-phase peptide synthesis (SPPS) of
artificial hosts with two different side arms. Other examples
of orthogonally protected scaffolds (based on β-amino acids
or pentaerythritol, for instance) have recently been reported
e.g. by Heinonen or Madder, respectively.[7] A convenient
synthesis of the dipodal scaffold 1 is reported herewith as
well as the solid-phase synthesis of a first prototype of a
tweezer receptor 11 with two different side arms.
Introduction
The design of new sophisticated artificial receptors has
led to the need for templates that are preorganized and yet
flexible enough to allow for efficient binding of the sub-
strate. If such a scaffold is equipped with a suitable anchor
group it is possible to generate libraries of potential recep-
tors on solid support using combinatorial methods.[1] Con-
sequent screening then identifies the best receptors for a
given substrate.
Synthetic host molecules with two arms, so-called
tweezer receptors, have proven to feature selective substrate
binding.[2] Additional interactions between host and guest
often lead to superior results in comparison to their one
armed analogs.[3] Thereby the template plays a decisive role
in the binding behavior of the receptor: it defines its flexibil-
ity as well as the angle and distance between the two side
chains.[3b,4] Both Boc- and Fmoc-protected derivatives of
bis(aminomethyl)benzoic acid have shown to perform ex-
traordinarily well as building blocks for the solid-phase syn-
thesis of artificial peptidic and peptidomimetic hosts.[3b,5]
However, most of the reported host systems are symmetric
compounds with two identical side arms. In order to intro-
duce a greater extent of functional and structural diversity
the design of more elaborate receptors with two different
arms is desirable. One of the few examples of unsymmetric
tweezer receptors was recently presented by Kilburn et al.[6]
A combinatorial library of receptors for a biological impor-
tant tripeptide was synthesized using a guanidinium scaf-
fold and screened on bead. Binding studies in aqueous me-
Figure 1. Orthogonally protected template 1.
Results and Discussion
Besides an acid group that can be attached to a resin, the
newly developed template 1 incorporates each an Fmoc and
a Boc protected amine – two orthogonal protecting groups
that are well established in SPPS. The design therefore of-
fers a synthetic strategy on solid support that follows the
standard Fmoc procedure during the preparation of the
first arm (with the need, however, to avoid Boc or tBu
[a] Institut für Organische Chemie, Universität Duisburg-Essen,
Universitätsstr. 7, 45141 Essen, Germany
Fax: +49-201-183-4259
E-mail: carsten.schmuck@uni-due.de
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Scaffold Synthesis for Tweezer Receptors
groups in the side chains of the amino acids in the first
arm). After Boc deprotection the second side chain may
then be synthesized, again, by the standard Fmoc pro-
cedure.
Scheme 1 describes the preparation of 3-(N-Boc-amino-
methyl)-5-(N-Fmoc-aminomethyl)benzoic acid (1). The
preparation of diazide 5 starting from 2 is known in litera-
ture and has been achieved accordingly. The synthesis could
be carried out in batches of 5 grams with the commercially
available 3,5-dimethylbenzoic acid (2) as starting material.
Acid 2 was reacted to give the corresponding acyl chloride
with oxalyl chloride and catalytic amounts of dimethyl-
formamide (DMF).[8] In situ reaction with the solvent
methanol gave the corresponding methyl ester 3. The intro-
duction of one bromine substituent to each methyl group
(4) was carried out in a Wohl–Ziegler bromination with N-
bromosuccinimide (NBS) and azobis(isobutyronitrile)
(AIBN) in carbon tetrachloride.[9] The reported yield of
84% could not be reproduced. We obtained dibromide 4 in
67% yield after purification. Side products mainly consisted
of starting material and the mono-brominated derivative.
Increasing the amount of N-bromosuccinimide to more
than 2.2 equiv. led to the formation of higher brominated
products which complicated the purification of 4. Elon-
gation of the reaction time, exchange of the solvent to
methyl formate and/or UV irradiation did not improve the
yield. However, in the original literature procedure no data
on the purity of the dibromide are provided. A moderate
yield of a subsequent reaction using 4 as the starting mate-
rial suggests that 4 was not completely pure, questioning
the reported high yield.
Scheme 1. Synthesis of the orthogonally protected template 1.
The introduction of the amine functionalities was
achieved by a classical Staudinger reaction. The bromines
were quantitatively substituted by heating 4 with sodium ethyl ether and an aqueous phase containing 5% HCl. The
azide in DMF to afford the diazide 5, which can then be generation of the iminophosphorane intermediate 6 takes
reduced to the corresponding amine with triphenylphos- place in the organic phase while the hydrolysis takes place
phane.[10] The complete reduction of the diazide with tri- at the phase interface. The resulting amine 7 is immediately
phenylphosphane in methanol to the corresponding di- protonated by HCl and transferred as ammonium salt into
amine works without problems (93% yield). However, any the aqueous phase where it cannot be reduced further as
attempt to selectively functionalize one of the two amino the triphenylphosphane is only soluble in the organic phase
groups failed.
(Scheme 2).
Attempts to protect symmetric diamines with just one
We could successfully adopt this procedure for the syn-
equivalent of protecting group are inefficient leading to thesis of diazide 5 (yield 61%) and even further improve the
product mixtures of unprotected, mono- and bis-protected yield by addition of cyclohexane (CyHex) to the ether
product.[11] Sometimes the diprotected compound is even phase. This way the yield for the mono reduction increased
favoured due to solubility reasons. A simultaneous reaction to 86% (cf. 93% yield for the complete unselective re-
with two different protecting groups is also possible but at duction of both azide groups to the diamine). The addition
best gives rise to a statistical distribution of products which of cyclohexane decreases the polarity of the organic layer
often goes along with difficult purification steps. Both vari- favouring the phase transfer of the protonated amine and
ants were tested on the diamine obtained after the double thus decreasing the amount of double reduction. The amine
reduction of diazide 5 and did not lead to satisfying results. 8 was isolated as the trifluoracetic acid (TFA) salt by means
Thus, in order to create an asymmetry we chose to carry of reversed-phase MPLC. The only side products were the
out a selective mono reduction of the diazide 5 to the corre- diamine and compounds in which the methyl ester was hy-
sponding azidoamine 8. This kind of reaction has been re- drolyzed to the free carboxylic acid. Thus, the mono re-
ported to the best of our knowledge so far only for two duction of the diazide 5 in a biphasic reaction mixture pro-
examples, one aliphatic and one polyethylene glycol spaced vides an elegant and facile approach to a diamine scaffold
diazide.[12] These reductions were carried out with tri- in which the two amines can be individually functionalized
phenylphosphane in a two-phase system composed of di- further. The next step of the synthesis is the protection of
Eur. J. Org. Chem. 2009, 4480–4485
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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H. Y. Kuchelmeister, C. Schmuck
FULL PAPER
As depicted in Scheme 3 this building block was then used
for the solid-phase synthesis of the unsymmetric tweezer re-
ceptor 11. The synthesis was carried out according to stan-
dard Fmoc protocol.[13] Completion of coupling and depro-
tection steps were monitored by the Kaiser test.[14] After
swelling the MBHA resin (1.3 mmol/g) in dichloromethane
(DCM), the template was attached with PyBOP as coupling
reagent in 3% N-methylmorpholine (NMM)/DMF using
2.5 equiv. of each reactant. The coupling step was repeated
with another equivalent of the reactants to assure complete
conversion of all accessible amino groups on the resin. The
resin was then treated with 10 equiv. of acetic anhydride and
NMM in DMF to acetylate any remaining free amino
groups. After deprotection of the Fmoc group with 20% pi-
peridine/DMF the first arm – consisting of the three amino
acids glutamine, phenylglycine and alanine – was synthe-
sized. The deprotection time after the glutamine coupling
was kept short in order to prevent pyroglutamate forma-
tion.[15] The last amino acid of the first arm carries a Cbz
protecting group at the N-terminus which is stable under the
conditions needed for the following Boc deprotection (50%
TFA/DCM). The resin was then washed with DCM and 3%
NMM/DMF to assure that no amino group is present as
TFA salt which could lead to trifluoracetylation during the
next coupling step. Afterwards lysine was attached to the
template utilizing the more reactive HATU instead of
PyBOP.[16] The coupling step was carried out twice in order
to ensure quantitative conversion of the sterically demanding
amine functionality of the template.
Scheme 2. Selective mono reduction of diazide 5 in a two-phase
system consisting of an organic and an acidic aqueous phase.
the free amine in 8 with di-tert-butyl dicarbonate (Boc2O)
and sodium hydrogen carbonate under standard conditions
in methanol to afford the Boc-protected product 9. Re-
duction of the remaining azide function in 9 and saponifica-
tion of the methyl ester were carried out in a one-pot syn-
thesis. We abstained from a purification step after the re-
duction because the amine proved to be unstable during the
column chromatography on silica. The resulting compound
10 was isolated via reversed-phase MPLC with 0.1% NEt3
added to the solvent and obtained as zwitterion after lyo-
philisation. The last step is the protection of the amine with
Fmoc-Cl and sodium hydrogen carbonate in dioxane/water
to give the orthogonally protected template 1. Approxi-
mately two grams of the product could easily be obtained
from each batch after a final purification via flash column
chromatography on silica gel.
The next amino acid, again lysine, and the artificial argi-
nine analog 12 were coupled following standard procedure
again. Amino functions in the side chains were all protected
with Cbz groups. 12 was incorporated into the receptor as
an efficient binding motif for carboxylates. Its preparation
and application have been described elsewhere.[17] After the
Scheme 3. Solid-phase peptide synthesis of the unsymmetric tweezer receptor 11.
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Eur. J. Org. Chem. 2009, 4480–4485

Scaffold Synthesis for Tweezer Receptors
are reported in parts per million (ppm) relative to the deuterated
solvents CDCl3 or [D6]DMSO. The following abbreviations are
used for peak multiplicities: s, singlet; d, doublet; t, triplet; m, mul-
tiplet; br, broad. Coupling constants (J values) are expressed in
Hertz [Hz]. HR-ESI-mass spectra were received by using a Bruker
MicroTOF focus. EI-mass spectra were obtained by using a Finni-
gan MAT 90. Melting points were obtained in open glass capillary
tubes using an apparatus from Büchi and are quoted uncorrected.
resin was thoroughly washed and dried the tweezer was
cleaved from the solid support with 10% trifluormethane-
sulfonic acid (TFMSA)/TFA. After a first crude purifica-
tion via reversed-phase MPLC with 0.1% TFA the receptor
11 could be obtained as TFA salt in 45% yield and with
85% purity according to HPLC analysis [SupelcosilTM LC-
18 column (25 cmϫ4.6 mm, 5 µm), solvent: 5 min water
(0.1% TFA), then within 35 min to methanol (0.1% TFA),
flow rate 1 mL/ min, retention time TR = 26.4 min, peak
integration based on detection at 250 nm]. Hence, even
though our synthesis incorporates a nonpeptidic template,
two nonstandard amino acids (Phg and 12) and an amino
acid which often can cause problems in SPPS (glut-
amine)[18] the unsymmetrical tweezer 11 was obtained in
good yields and purity. Also with respect to the efficient
synthesis of 1, this opens the way for a further exploration
of this new tweezer class. Binding studies of the prototype
11 with anionic oligopeptides as guests are currently un-
derway in our laboratory and will be reported in due time.
Methyl 3,5-Dimethylbenzoate (3): To a solution of 3,5-dimethylben-
zoic acid (5.00 g, 0.33 mol, 1 equiv.) in dry methanol (100 mL) a
catalytic amount of dry dimethylformamide and oxalyl chloride
(9.50 mL, 1.00 mol, 3 equiv.) were slowly added dropwise under ar-
gon atmosphere at 0 °C. After stirring for 5 h at 50 °C the solvent
was evaporated under reduced pressure. The crude product was
purified by flash chromatography on silica gel (cyclohexane/ethyl
acetate, 1:1) to give the esterified product 3 as a white solid (4.92 g,
90%). Rf = 0.83 (SiO2, hexane/ethyl acetate, 1:1), m.p. 30 °C. FT-
IR (KBr disk): ν = 3344 (w), 2975 (w), 2093 (m), 1714 (s), 1668
˜
(m), 1604 (w), 1521 (m), 1436 (w), 1314 (s), 1220 (s), 1158 (m), 863
1
(w), 771 (w) cm–1. H NMR (400 MHz, CDCl3): δ = 2.36 (s, 6 H,
CH3), 3.90 (s, 3 H, OMe), 7.18 (dd, 4J1 = 4J2 = 1.7 Hz, 1 H, CHar),
7.66 (d, 4J = 1.7 Hz, 2 H, CHar) ppm. 13C NMR (100 MHz,
CDCl3): δ = 21.1 (CH3), 51.9 (OMe), 127.3 (CHar), 130.0 (Cq),
134.5 (CHar), 138.0 (Cq), 167.4 (CO2) ppm. MS (pos. EI): m/z cal-
Conclusions
+·
culated for C10H12O2 [M+·] 164.083; found 164.1.
The new dipodal scaffold 1 could successfully be synthe-
sized with an overall yield over seven steps of 34%. The key
step is the selective mono reduction of a symmetric diazide
taking advantage of the different solubility of starting mate-
rial, reducing agent and mono reduction product in a bi-
phasic reaction mixture. Our improved protocol might also
be useful for other diazides and their conversion into orthog-
Methyl 3,5-Bis(bromomethyl)benzoate (4): To a refluxing solution
of 3 (5.20 g, 0.31 mol, 1 equiv.) in carbon tetrachloride (100 mL)
N-bromosuccinimide (12.40 g, 0.70 mol, 2.2 equiv.) and a catalytic
amount of 2,2Ј-azobisisobutyronitrile were added and stirred for
48 h. After cooling to room temperature the resulting precipitate
was removed by filtration, washed with carbon tetrachloride and
rejected. The yellow-orange filtrate was washed with a saturated
onally protected diamines. Template 1 can thus be easily syn- aqueous solution of sodium hydrogen carbonate (50 mL) and brine
(50 mL). The colorless organic phase was dried with sodium sulfate
and the solvent was evaporated under reduced pressure. The raw
product was purified by flash chromatography on silica gel (cyclo-
hexane/ethyl acetate, 19:1) affording the dibrominated 4 as a white
solid (6.83 g, 67%). Rf = 0.67 (SiO2, hexane/ethyl acetate, 1:9), m.p.
thesized in gram quantities starting from commercially avail-
able compounds. We could also show that 1 can be used in a
modified SPPS Fmoc-protocol for the synthesis of unsym-
metrical tweezers such as 11 with two different side arms.
99 °C. FT-IR (KBr disk): ν = 2117 (w), 1723 (s), 1435 (m), 1315
˜
(m), 1212 (s), 994 (m), 898 (w), 771 (m), 697 (s) cm–1. 1H NMR
Experimental Section
(400 MHz, CDCl3): δ = 3.93 (s, 3 H, OMe), 4.50 (s, 4 H, CH2), 7.61
(dd, 4J1
=
4J2 = 1.7 Hz, 1 H, CHar), 7.99 (d, 4J = 1.7 Hz, 2 H,
General Remarks: The starting materials and reagents were used as
obtained from the commercial suppliers unless otherwise stated.
Solvents were dried and distilled before use. All reactions were car-
ried out in oven dried glassware. Lyophilization was carried out in
an Alpha 1–4 2D plus freeze drying apparatus from Christ. Analyt-
ical TLC was carried out on SiO2 aluminium foils 60 F254 from
Merck and C18 SiO2 aluminium foils ALUGRAM RP-18W/
UV254 from Macherey–Nagel. Column chromatography was done
on columns packed with ICN Silica with a spherical size of 32–
63 µm from Biomedicals GmbH. Reversed-phase column
chromatography was done with an Isco Inc. Combi Flash MPLC
apparatus with RediSep C-18 reversed-phase columns. Analytical
HPLC was done with a Dionex HPLC apparatus consisting of a
P680 HPLC pump, an ASI-100 automated sample injector and a
CHar) ppm. 13C NMR (100 MHz, CDCl3): δ = 31.8 (CH2), 52.4
(OMe), 130.0 (CHar), 131.4 (Cq), 133.8 (CHar), 138.9 (Cq), 165.9
(CO2) ppm. MS (pos. EI): m/z calculated for C10H10Br2O2 [M+·]
+·
319.904; found 319.9.
Methyl 3,5-Bis(azidomethyl)benzoate (5): A suspension of 4 (7.30 g,
22.65 mmol, 1 equiv.) and sodium azide (5.89 g, 90.76 mmol,
4 equiv.) in dimethylformamide (100 mL) was stirred at 65 °C for
3 h. The solution was diluted with water (300 mL) and extracted
with ethyl acetate (5ϫ200 mL). The combined organic phases were
dried (Na2SO4) and the solvent was removed under reduced pressure.
The brown, oily raw product was purified by flash column
chromatography on silica gel (cyclohexane/ethyl acetate, 5:1) to give
the yellowish, oily product 5 (5.52 g, 99%). Rf = 0.55 (SiO2, hexane/
UVD 340U UV-detector with
a
SupelcosilTM LC-18
ethyl acetate, 1:9), m.p. 99 °C. FT-IR (KBr disk): ν = 2952 (w), 2091
˜
(25 cmϫ4.6 mm, 5 µm) column. Commercially available HPLC (w), 1718 (s), 1607 (w), 1303 (m), 1216 (s), 1116 (m), 1007 (w), 860
grade solvents were used as eluents. The IR spectra were recorded
on a FT-IR 1600 spectrometer from Perkin–Elmer. Bands were
quoted in cm–1 and the following abbreviations are used: w weak,
m medium, s strong, br. broad. 1H and 13C NMR spectra were
recorded on an AVANCE 400 MHz and a DMX 600 MHz spec-
trometer from Bruker at ambient temperature. The chemical shifts
(m), 765 (s), 718 (w), 610 (w) cm–1. 1H NMR (400 MHz, CDCl3): δ
= 3.94 (s, 3 H, OMe), 4.43 (s, 4 H, CH2), 7.48 (dd, 4J1
= =
4J2
1.6 Hz, 1 H, CHar), 7.96 (d, 4J = 1.6 Hz, 2 H, CHar) ppm. 13C NMR
(100 MHz, CDCl3): δ = 52.4 (OMe), 54.1 (CH2), 128.9 (CHar), 131.4
(Cq), 131.8 (CHar), 136.7 (Cq), 166.1 (CO2) ppm. MS (pos. EI): m/z
+·
calculated for C10H10N6O2 [M+·] 246.087; found 246.1.
Eur. J. Org. Chem. 2009, 4480–4485
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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H. Y. Kuchelmeister, C. Schmuck
FULL PAPER
Methyl 3-(Aminomethyl)-5-(azidomethyl)benzoate, TFA Salt (8): To
a solution of 5 (4.00 g, 16.24 mmol, 1 equiv.) in diethyl ether
(120 mL) cyclohexane (40 mL) and 5% aqueous hydrochloric acid
(160 mL) were added. The mixture was cooled to 0 °C, tri-
phenylphosphane (4.26 g, 16.24 mmol, 1 equiv.) was added and the
solution was stirred for 8 h at 0 °C and for 40 h at ambient tem-
perature. The phases were separated and the organic layer was ex-
tracted once with 5% aqueous hydrochloric acid (80 mL). The
combined aqueous phases were freeze dried in vacuo. The raw
product was purified by MPLC on C18 reversed-phase silica gel
(20% water/methanol to 40% water/methanol in 40 min, 0.1%
TFA) to give 8 as white solid (4.43 g, 86%). Rf = 0.66 (C18 RP
SiO2, water/methanol, 1:1 + 0.1% TFA), m.p. 138 °C. FT-IR (KBr
(CHar), 126.8 (CHar), 126.9 (CHar), 138.7 (Cq), 139.4 (Cq), 155.7
(NHCO2) ppm; the COOH signal could not be detected. HR-MS
(pos. ESI): m/z calculated for C14H20N2O4Na+ [M
+
Na+]
303.1315; found 303.1315.
3-(N-Boc-aminomethyl)-5-(N-Fmoc-aminomethyl)benzoic Acid (1):
Fmoc-Cl was recrystalized from toluene/cyclohexane. To a solution
of 10 (1.38 g, 4.92 mmol, 1 equiv.) and sodium hydrogen carbonate
(1.12 g, 13.29 mmol, 2.7 equiv.) in dioxane/water (5:2, 70 mL)
Fmoc-Cl (1.91 g, 7.38 mmol, 1.5 equiv.) was added and the mixture
was stirred for 5 h. Water (80 mL) was added and the pH was ad-
justed to 6 with 5% aqueous hydrochloric acid. The resulting sus-
pension was extracted with ethyl acetate (5ϫ250 mL). The com-
bined organic phases were dried with sodium sulfate and the sol-
vent was removed under reduced pressure. The raw product was
purified by flash chromatography on silica gel (ethyl acetate, then
ethyl acetate/methanol, 9:1) to give 1 as white solid (1.91 g, 80%).
Rf = 0.58 (SiO2, ethyl acetate), m.p. 184–185 °C. FT-IR (KBr disk):
disk): ν = 2969 (br. w), 2098 (w), 1710 (m), 1671 (s), 1423 (w), 1313
˜
(w), 1237 (m), 1176 (s), 889 (w), 836 (w), 779 (m), 722 (m) cm–1.
1H NMR (400 MHz, [D6]DMSO): δ = 3.89 (s, 3 H, OMe), 4.15 (s,
4
2 H, CH2), 4.61 (s, 2 H, CH2), 7.73 (dd, J1 = 4J2 = 1.6 Hz, 1 H,
CHar), 7.97 (dd, 4J = 1.6 Hz, 1 H, CHar), 8.26 (dd, 4J = 1.6 Hz,
1 H, CHar), 8.39 (br. s, 3 H, NH3+) ppm. 13C NMR (100 MHz,
[D6]DMSO): δ = 41.6 (CH2), 52.2 (OMe), 52.6 (CH2), 128.7 (CHar),
129.3 (CHar), 130.2 (Cq), 133.5 (CHar), 135.2 (Cq), 137.0 (Cq), 165.5
ν = 3340 (w), 2366 (m), 1688 (s), 1540 (m), 1476 (w), 1250 (m), 1162
˜
1
(w), 1055 (w), 783 (w) cm–1. H NMR (400 MHz, [D6]DMSO): δ
3
= 1.39 (s, 9 H, tBu), 4.15 (d, J = 6.0 Hz, 2 H, CH2), 4.20–4.25 (m,
3 H, Fmoc-CH + CH2), 4.31 (d, 3J = 7.0 Hz, 2 H, Fmoc-CH2),
7.28–7.45 (m, 6 H, 4ϫFmoc-CHar + CHar + NH), 7.68–7.76 (m,
+
(CO2) ppm. HR-MS (pos. ESI): m/z calculated for C10H13N4O2
[M + H+ – TFA] 221.1033; found 221.1033.
4 H, Fmoc-CHar), 7.88 (d, J = 7.5 Hz, 2 H, CHar), 7.94 (dd, 3J1 =
3
Methyl 3-(N-Boc-aminomethyl)-5-(azidomethyl)benzoate (9): Sodium 3J2 = 6.1 Hz, 1 H, NH) ppm. 13C NMR (100 MHz, [D6]DMSO): δ
hydrogen carbonate (0.54 g, 6.43 mmol, 1.2 equiv.) and 8 (1.70 g, = 28.1 (Boc-CH3), 43.1 (CH2), 43.6 (CH2), 46.6 (Fmoc-CH), 65.5
5.36 mmol, 1 equiv.) were dissolved in water/methanol (5:1, 50 mL)
and Boc anhydride (1.40 g, 6.43 mmol, 1 equiv.) was added. After
stirring for 20 h water (100 mL) was added and the mixture was
extracted with ethyl acetate (3ϫ75 mL). The combined organic
phases were dried with sodium sulfate and the solvent was removed
under reduced pressure. The raw product was purified by flash col-
umn chromatography on silica gel (cyclohexane/ethyl acetate, 7:3) to
afford 9 as white solid (1.85 g, 90%). Rf = 0.84 (SiO2, hexane/ethyl
(Fmoc-CH2), 77.8 (Boc-Cq), 119.9 (CHar), 120.0 (CHar), 121.3 (Cq),
125.1 (CHar), 126.3 (CHar), 126.3 (CHar), 127.0 (CHar), 127.2
(CHar), 127.5 (CHar), 128.8 (CHar), 139.7 (Cq), 140.2 (Cq), 140.6
(Cq), 143.8 (Cq), 155.7 (NHCO2), 156.2 (NHCO2) ppm; the COOH
signal could not be detected. HR-MS (pos. ESI): m/z calculated for
C29H30N2O6Na+ [M + Na+] 525.1996; found 525.1997.
Solid-Phase Peptide Synthesis of the Receptor (11): The reaction
was carried out in a flask equipped with a frit on a Heidolph Rota-
max 120 shaker. MBHA resin (58 mg, 1.3 mmol/g) was swollen in
DCM (5 mL) for 1 h and washed with DMF (3ϫ5 mL). The tem-
plate 1 (95 mg, 0.19 mmol, 2.5 equiv.) was attached to the resin
by shaking for 5 h under argon atmosphere with PyBOP (98 mg,
0.19 mmol, 2.5 equiv.) in 3% NMM/DMF (5 mL) and consequent
washing with DMF (3ϫ5 mL). The coupling and washing steps
were repeated with 1 equiv. PyBOP (39 mg, 0.08 mmol) and 1
(38 mg, 0.08 mmol). Afterwards the resin was treated with acetic
anhydride (71 µL, 0.75 mmol, 10 equiv.) and NMM (83 µL,
0.75 mmol, 10 equiv.) in DMF (5 mL) for one hour. Fmoc depro-
tection was achieved by shaking the resin in 20% piperidine/DMF
(5 mL) for 15 min twice followed by washing with DMF
(6ϫ5 mL). Fmoc-Gln-OH (69 mg, 0.19 mmol, 2.5 equiv.), Fmoc-
Phg-OH (70 mg, 0.19 mmol, 2.5 equiv.) and Cbz-Ala-OH (42 mg,
0.19 mmol, 2.5 equiv.) were coupled in the same manner without
repetition of the coupling and washing steps. The duration of the
Fmoc deprotection step of Fmoc-Gln-OH was reduced to two
times 10 min. Boc deprotection of the template was achieved by
shaking the resin in 50% TFA/DCM (5 mL) for 15 min twice fol-
lowed by washing with DCM (3ϫ5 mL) and 3% NMM/DMF
(3ϫ5 mL). Then Fmoc-Lys(Cbz)-OH (95 mg, 0.19 mmol,
2.5 equiv.) was coupled with HATU (69 mg, 0.18 mmol, 2.4 equiv.)
in the same manner as described above. The coupling was carried
out twice. Then the second Fmoc-Lys(Cbz)-OH (95 mg,
0.19 mmol, 2.5 equiv.) and 12 (97 mg, 0.19 mmol, 2.5 equiv.) were
coupled with PyBOP again. The resin was washed with DCM
acetate, 1:1), m.p. 64–66 °C. FT-IR (KBr disk): ν = 3343 (w), 2984
˜
(w), 2092 (m), 1713 (s), 1675 (m), 1520 (m), 1435 (w), 1276 (m), 1222
(s), 1156 (s), 1122 (m), 929 (w), 862 (m), 773 (m), 706 (m), 617 (m)
1
cm–1. H NMR (400 MHz, CDCl3): δ = 1.47 (s, 9 H, tBu), 3.93 (s,
3 H, OMe), 4.38 (d, 3J = 5.6 Hz, 2 H, CH2), 4.40 (s, 2 H, CH2), 7.45
(s, 1 H, CHar), 7.89 (s, 1 H, CHar), 7.93 (s, 1 H, CHar) ppm. 13C
NMR (100 MHz, CDCl3): δ = 28.3 (Boc-CH3), 44.1 (CH2), 52.3
(OMe), 54.2 (CH2), 79.8 (Boc-Cq), 128.1 (CHar), 128.2 (CHar), 131.1
(Cq), 131.3 (CHar), 136.3 (Cq), 140.4 (Cq), 155.8 (NHCO2), 166.4
(CO2) ppm. HR-MS (pos. ESI): m/z calculated for C15H20N4O4Na+
[M + Na+] 343.1377; found 343.1383.
3-(N-Boc-aminomethyl)-5-(aminomethyl)benzoic Acid (10): To a
solution of 9 (1.72 g, 5.37 mmol, 1 equiv.) in methanol/water (10:1,
30 mL) triphenylphosphane (1.41 g, 5.37 mmol, 1 equiv.) was
added. After stirring for 20 h lithium hydroxide (1.29 g,
53.69 mmol, 10 equiv.) and water (27 mL) were added and the mix-
ture was stirred for additional 24 h. The solvent was concentrated
to a few mL under reduced pressure, water (50 mL) was added and
the resulting suspension was extracted with ethyl acetate (50 mL).
The aqueous phase was freeze dried in vacuo. The raw product was
purified by MPLC on C18 reversed-phase silica gel (water to 40%
water/methanol in 30 min, 0.1% NEt3) to give 10 as white solid
(1.40 g, 93%). Rf = 0.68 (C18 RP SiO2, water/methanol, 1:1 + 0.1%
TFA), m.p. Ͼ 250 °C. FT-IR (KBr disk): ν = 3344 (m), 2979 (w),
˜
2093 (m), 1713 (s), 1676 (s), 1520 (s), 1276 (m), 1222 (s), 1157 (s),
863 (m), 775 (m) cm–1. 1H NMR (400 MHz, [D6]DMSO): δ = 1.40
3
(s, 9 H, tBu), 3.69 (s, 2 H, CH2), 4.10 (d, J = 5.9 Hz, 2 H, CH2), (3ϫ5 mL), methanol (3ϫ5 mL) and DCM (3ϫ5 mL) and dried
7.12 (s, 1 H, CHar), 7.32 (t, 3J = 5.7 Hz, 1 H, NH), 7.61 (s, 1 H,
CHar), 7.68 (s, 1 H, CHar) ppm. 13C NMR (100 MHz, [D6]DMSO):
δ = 28.2 (Boc-CH3), 43.5 (CH2), 45.0 (CH2), 77.6 (Boc-Cq), 126.2
under reduced pressure for one hour. In order to cleave the product
the resin was shaken for 3 h in 10% TFMSA/TFA (5 mL) and
washed with TFA (5 mL). The red-brown filtrate was collected and
4484
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© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 4480–4485

Scaffold Synthesis for Tweezer Receptors
[4]
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concentrated in high vacuum at room temperature. Diethyl ether
(20 mL) was added and the resulting suspension was centrifuged.
The supernatant solvent was decanted and the solid was washed
with diethyl ether and centrifuged again. The raw product was dis-
solved in little methanol, water (30 mL) and TFA (1 mL) were
added and the mixture was freeze dried in vacuo. The resulting
solid was purified by MPLC on C18 reversed-phase silica gel (water
to methanol in 45 min, 0.1% TFA) to give 11 as voluminous white
solid (51 mg, 45%). HPLC: 5 min water (0.1% TFA), then in
35 min to methanol (0.1% TFA), 1 mL/ min, TR = 26.4 min
[6]
[7]
J. Shepherd, T. Gale, K. B. Jensen, J. D. Kilburn, Chem. Eur. J.
2006, 12, 713–720.
(250 nm), m.p. 245 °C. FT-IR (KBr disk): ν = 3291 (w), 1661 (s),
For additional examples see: a) H. De Muynck, A. Madder, N.
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˜
1536 (s), 1429 (m), 1282 (w), 1197 (m), 1130 (s), 986 (s), 721 (w),
610 (m) cm–1. 1H NMR (600 MHz, [D6]DMSO): δ = 1.25–1.41 (m,
8 H), 1.47–1.57 (m, 5 H), 1.58–1.64 (m, 1 H), 1.66–1.77 (m, 2 H),
3
3
1.79–1.93 (m, 2 H), 1.98 (t, J = 8.3 Hz, 1 H), 2.12 (t, J = 7.9 Hz,
1 H), 2.70–2.79 (m, 4 H, CH2), 3.95–4.09 (m, 2 H), 4.19–4.37 (m,
7 H), 5.58 (d, 3J = 7.9 Hz, 1 H, CH), 6.76–6.84 (m, 1 H), 6.86–6.91
(m, 1 H), 7.17–7.21 (m, 1 H), 7.23 (s, 1 H), 7.25–7.38 (m, 5 H), 7.42
3
(t, J = 7.4 Hz, 2 H), 7.63 (s, 2 H), 7.92–8.01 (m, 1 H), 8.13 (br. s,
3
3 H, NH3+), 8.32–8.36 (m, 1 H), 8.38 (br. s, 2 H, NH2), 8.42 (t, J
3
[8]
= 5.8 Hz, 1 H), 8.52–8.62 (m, 3 H), 8.63–8.67 (m, 1 H), 8.74 (d, J
= 7.2 Hz, 1 H, NH), 8.77 (d, 3J = 7.6 Hz, 1 H, NH), 8.85 (br. s,
3
2 H, NH2), 8.97 (d, J = 7.5 Hz, 1 H, NH), 9.14 (d, 3J = 8.1 Hz,
1 H, NH), 11.64 (br. s, 1 H, NH), 12.30 (s, 1 H, NH) ppm. 13C
NMR (150 MHz, [D6]DMSO): δ = 17.2 (CH3), 22.0 (CH2), 22.3
(CH2), 26.4 (CH2), 26.6 (CH2), 27.8 (CH2), 27.9 (CH2), 31.0 (CH2),
31.1 (CH2), 38.5 (CH2), 44.7 (CH2), 44.8 (CH2), 47.8 (CH), 51.8
(CH), 52.5 (CH), 52.7 (CH), 55.7 (CH), 56.0 (CH), 112.7 (CHar),
115.4 (CHar), 124.6 (CHar), 126.6 (CHar), 127.4 (CHar), 127.7
(CHar), 128.3 (CHar), 128.4 (CHar), 128.8 (CHar), 132.1 (CO), 134.3
(CO), 137.6 (CO), 138.4 (CO), 139.1 (CO), 139.4 (CO), 155.3 (CO),
158.3 (CO), 158.6 (CO), 159.9 (CO), 167.0 (CO), 167.8 (CO), 167.9
(CO), 169.3 (CO), 170.8 (CO), 171.5 (CO), 173.6 (CO) ppm. HR-
[9]
[10]
[11]
+
MS (MALDI-TOF): m/z calculated for C47H70N17O10 [M – 5
TFA + H+] 1032.548; found 1032.556.
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2711.
Acknowledgments
Financial support of our work from the Deutsche Forschungsge-
meinschaft (DFG) and the Fonds der Chemischen Industrie is
gratefully acknowledged. H. Y. K. would like to thank the Studien-
stiftung des Deutschen Volkes for a PhD scholarship.
[13] Due to the use of Boc as the second protection group on the
template, in the first arm of the receptor amino acid side chains
have to be Cbz and benzyl-protected instead of Boc or tBu.
However, these amino acids are commercially available. Alter-
natively, of course our synthetic method allows to also intro-
duce other combination of protection groups onto the template
which might be more useful for a specific application.
[14] E. Kaiser, R. L. Colescott, Anal. Biochem. 1970, 34, 595–598.
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A Practical Approach, 1st ed., Oxford University Press, New
York, 1999.
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Received: May 14, 2009
Published Online: July 27, 2009
Eur. J. Org. Chem. 2009, 4480–4485
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4485