A versatile, modular platform for multivalent peptide ligands based on a dendritic wedge

Article abstract of DOI:10.1002/ejoc.200901045

A general methodology for the synthesis of multifunctional AB₂, AB₃, AB₄, and AB₅ dendritic wedges is described based on an orthogonal protection strategy. Asymmetric polyamide dendrons that possess N-terminal cysteine residues at the periphery were quantitatively functionalized with C-terminal thioester peptides using native chemical ligation. Conjugation of biologically relevant groups at the focal point resulted in a series of related structures with a highly controlled valency (2-5) that can directly be used in a systematic study on the strength of multivalent interactions. Using our modular approach various ligands, functional groups and spacers can readily be combined in order to generate a toolbox for the development of smart biomaterials used in molecular medicine and imaging.

Full text of DOI:10.1002/ejoc.200901045

FULL PAPER
DOI: 10.1002/ejoc.200901045
A Versatile, Modular Platform for Multivalent Peptide Ligands Based on a
Dendritic Wedge
Edith H. M. Lempens,[a][‡] Brett A. Helms,[a][‡] Andrea R. Bayles,[a] Maarten Merkx,[a] and
E. W. Meijer*[a]
Keywords: Dendrimers / Peptides / Multivalency / Modular approach / Native chemical ligation / Orthogonal functionaliza-
tion
A general methodology for the synthesis of multifunctional
AB2, AB3, AB4, and AB5 dendritic wedges is described based
on an orthogonal protection strategy. Asymmetric polyamide
dendrons that possess N-terminal cysteine residues at the pe-
riphery were quantitatively functionalized with C-terminal
in a series of related structures with a highly controlled val-
ency (2–5) that can directly be used in a systematic study on
the strength of multivalent interactions. Using our modular
approach various ligands, functional groups and spacers can
readily be combined in order to generate a toolbox for the
thioester peptides using native chemical ligation. Conjuga- development of smart biomaterials used in molecular medi-
tion of biologically relevant groups at the focal point resulted
cine and imaging.
and divergent growth strategies.[5] While these desymme-
trized dendrimers possess a highly controlled surface func-
tionalization, they are limited by synthetic procedures based
on either 1 Ǟ 2 branching or 1 Ǟ 3 branching. As a result
the number of end groups increases exponentially with each
generation.[6] For many biomedical applications, however,
an arithmetic branching scheme is more attractive for eluci-
dating and quantifying the fundamental aspects of multi-
valent ligand interactions for targeting. We present here a
new type of bifunctional building block that enables the
modular synthesis of multivalent peptide dendritic wedges
with arithmetic control over the degree of branching (Fig-
ure 1).
The design of our biocompatible platform allows an opti-
mal presentation of multiple functional groups and is in-
spired by Newkome’s polyamide dendrimers.[7] In contrast
to previously reported strategies, the number of ligands in-
creases arithmetically, allowing a systematic study on the
nature and strength of multivalent interactions. Two, three,
four or five ligands can be presented at the periphery of a
polyamide dendritic wedge, while another functional group
is site-specifically introduced at the focal point. Poly(ethyl-
ene glycol) (PEG) units are used to append reactive cysteine
residues to the dendritic core. Besides the fact that the
length of this spacer can be easily tuned to inter-receptor
distances, PEG-dendrimer hybrids are clinically approved
and have shown to decrease toxicity and increase circu-
lation times in vivo.[8] N-Terminal cysteine residues at the
PEGylated wedge periphery were used for the introduction
of multiple peptide-based ligands via their C-terminus using
native chemical ligation, a highly efficient and specific cou-
pling reaction.[9] Minor modifications will even allow the
use of other ligation reactions such as oxime or “click”
Introduction
Over the past two decades, multiple synthetic strategies
have been developed to arrive at functional dendrimers. The
ability of this type of macromolecule to present multiple
groups at the surface, while maintaining a well-defined
structure with low polydispersity makes them ideal vehicles
for use in biomedical applications.[1] Inherent to their struc-
ture are opportunities to employ multivalent interactions to
yield ligands with high affinity and specificity for biological
targets. Beyond that, the preparation of well-defined multi-
functional dendrimers – for example, by conjugation of
therapeutic agents, targeting groups and radio- or fluores-
cent labels onto a single platform – is limited by the fact
that most dendritic molecules possess uniformly protected
functional groups in a spherical (or cylindrical) topology.
This symmetry-related synthesis constraint is inherently
contrary to generating molecular complexity within a sin-
gle, well-defined molecular species.[2] Methodologies for the
preparation of dendrimers with asymmetric functionaliza-
tion have appeared recently: for example, by conjugation of
functional units (e.g. biotin or chromophores) at the focal
point of dendritic wedges;[3] by merging two separate
wedges using click chemistry, native chemical ligation or
Diels–Alder reactions;[4] or by a combination of convergent
[a] Laboratory of Chemical Biology, Department of Biomedical
Engineering, Eindhoven University of Technology,
P. O. Box 513, 5600 MB Eindhoven, The Netherlands
Fax: +31-40-245-1036
E-mail: e.w.meijer@tue.nl
[‡] Edith H. M. Lempens and Dr. Brett A. Helms contributed
equally to this work.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901045.
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E. W. Meijer et al.
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Figure 1. A modular approach towards multifunctional peptide wedges. Besides the valency (R1) of the ligands conjugated to the periph-
ery, also the linker length (R2) and the functional group at the focal point (R3) are easily varied.
chemistry. The related synthesis of the labeled pentameric of tert-butyl acrylate to nitroethane in the presence of Tri-
peptide wedge (AB5) has been published elsewhere since it ton-B (Scheme 1). Catalytic reduction of the nitro group
uniquely mimics the multivalent architecture of peptides using Raney nickel and H2 in ethanol afforded 2. This AB2
fused to the five pIII domains at the head of M13 bacterio- synthon is a key building block and can be used as a start-
phage. As a result, the affinity of phage-display-derived ing point for the synthesis of AB4 dendron 5 and the AB5
peptides can be significantly enhanced by restoring the dendron.
multivalent binding character using the AB5 dendrimer dis-
Therefore 2 was treated with acryloyl chloride followed
play.[10] In this work, we elaborate the complete synthetic byanother Michael addition to nitroethane and a catalytic
route to biotin and fluorescein labeled multivalent peptide reduction step, producing the aminotetraester 5 in good
constructs (AB2, AB3, AB4, and AB5) for direct use in solid overall yield. Aminotriester 6 is commercially available and
phase binding assays and fluorescence microscopy, respec- was used directly for further synthesis. With these three ba-
tively. The scope of the approach is demonstrated by pres- sic core structures in hand, the following series of transfor-
enting examples with a variety of targeting peptides.
mations were performed on each molecule to arrive at the
labeled multivalent peptide dendrons. Protection of the free
amine at the focal point with an Fmoc-group was followed
by removal of the tert-butyl protecting groups at the periph-
Results and Discussion
An important requirement for the realization of our syn- ery using formic acid. The polyacid was activated to tetra-
thetic strategy is the use of robust and distinct orthogonal fluorophenyl esters, which have a unique 1H NMR chemical
protecting groups that allow functionalization in the pres- shift that confirms a complete reaction. Conjugation of the
ence of other reactive groups. Poly(ethylene glycol) (PEG) tetrafluorophenol activated wedge with cysteine-function-
units were used to link acid-labile trityl-protected cysteine alized PEG 12 resulted in an orthogonally protected den-
residues to the periphery, while an orthogonal base-labile dritic structure. After removal of the Fmoc protecting
fluorenylmethoxylcarbonyl (Fmoc) group at the focal point group, various labels can be coupled to the focal point using
allowed us to manipulate the material so as to incorporate uronium-based HOAt-mediated condensation. In this man-
a variety of anchoring groups. The branched core of the ner, both 6-(biotinylamino)caproic acid and 5-carboxyfluo-
dimeric structure (AB2) was built up via a Michael addition rescein were successfully coupled via HATU activation to
112
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Modular Platform for Multivalent Peptide Ligands
Scheme 1. Synthesis and structure of AB2 (2), AB3 (6) and AB4 (5) polyamide dendritic core molecules.
all wedges. After deprotection of the trityl groups present peptides (RQIKIWFQNRRMKWKK) originating from the
on the cysteines, reverse phase HPLC purification was used Drosophila transcription factor Antennapedia spontane-
to obtain the desired compounds in high purity. Figures ously internalize exogeneous macromolecules.[14] Conjuga-
S7–S12 (see Supporting Information) show the LC-MS tion of three of these 16-amino-acid peptides to the fluores-
characterization of Cys2-Biotin (16a), Cys3-Biotin (16b) and cein-labeled AB3 wedge makes it a valuable construct for the
Cys4-Biotin (16c) as well as Cys2-Fluorescein (16d), Cys3- development of new therapeutic approaches (Figure 2, b).
Fluorescein (16e) and Cys4-Fluorescein (16f). All com- A biotin-labeled dimeric wedge functionalized with the type
pounds show a single sharp peak in the chromatogram indi-
1
collagen-binding domain of bone sialoprotein
cating Ͼ95% purity and their masses correspond to the cal- (NGVFKYRPRYFLYKHAYFYPPLKRFPVQ)[15]
was
culated molecular weights. As an example, the complete also prepared and characterized (Figure 2, c). This result
synthesis route for the preparation of a biotin-labeled tetra- shows that even these long, 28-amino-acid peptides can be
meric wedge functionalized with peptides is outlined in efficiently coupled to these dendritic structures and might
Scheme 2.
be useful for the initiation of bone mineralization after joint
To assess the general applicability of our approach, dif- injury. Finally, using the methodologies above, we synthe-
ferent peptides with various lengths and sequences were cou- sized the AB5 dendron with collagen type I binding peptides
pled to the labeled AB2, AB3, AB4, and AB5 constructs. (HVWMQAPGGG) and a fluorescein label at the focal
Thereto, a variety of biomedically relevant targeting pep- point.[10] The examples illustrate that our approach is in-
tides were selected from the literature and prepared with a deed quite versatile allowing conjugation of a variety of
C-terminal thioester using Fmoc-mediated solid phase pep- peptides to a series of ABx dendritic wedges.
tide synthesis on a safety-catch resin.[11] Peptides containing
the amino acid sequence Gly-Arg-Gly-Asp-Ser (GRGDS),
which mimics the integrin binding motif of adhesive pro-
Conclusions
teins in the extracellular matrix (e.g. fibronectin) mediate ad-
hesion and spreading of cells.[12] Synthetic multivalent mole-
The importance of peptide-mediated targeting and/or
cules displaying these small peptide fragments are therefore trafficking of biomolecules in living systems is becoming
of high interest for tumor targeting, imaging angiogenesis increasingly apparent. Mechanistic investigations into these
or the development of new biomaterials.[13] Native chemical complex interactions in vitro and in vivo have been limited
ligation of an Ac-GRGDS-containing thioester peptide to by the lack of suitable multifunctional molecular probes, in
the biotin-labeled AB4 dendritic wedge showed full conver- particular when these interactions involve multivalent li-
sion after 16 h. LC-MS analysis was used to characterize gands. We have addressed this deficiency through the devel-
the crude ligation mixture (Figure 2, a). Cell-penetrating opment of a polyamide dendritic platform for the presenta-
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Scheme 2. Chemical synthesis of a biotin-labeled tetrameric dendritic wedge functionalized with peptide ligands to cellular targets. a) 9-
Fluorenylmethyl succinimidyl carbonate, DIPEA, DCM. b) HCO2H. c) TFP, PPTS, DCC, DCM. d) Trt-C(Trt)-OSu, Et3N, DCM. e)
PPh3,10% H2O in THF. f) Trt-C(Trt)-PEG-NH2, DIPEA, DCM. g) 20% Piperidine in DCM. h) 6-(Biotinylamino)caproic acid, HATU,
DIPEA, DMF. i) TFA, triisopropylsilane, H2O. j) Peptide thioester, thiophenol, benzyl mercaptan, 6  guanidine, 70 m Tris, pH 8.0.
tion of peptide (or protein) ligands with controlled valency targeted drug delivery, molecular imaging and material sci-
(ABx: x = 2 to 5), spacer length, primary sequence along ence.
with suitable labels for many biological investigations, in-
cluding binding assays via introduction of biotin moieties
or fluorescence microscopy via fluorescein dyes. The syn-
thesis presented here relies on an orthogonal protecting
group strategy that was inspired by what is used in routine
Experimental Section
General: Unless otherwise stated, all solvents used were reagent
1
grade and all chemicals were used as supplied. H and 13C NMR
solid phase synthesis of linear peptides. With it comes ac-
spectra were acquired on a Varian 400-MR spectrometer at 400
cess to a toolbox of AB2, AB3, AB4, and AB5 wedges that
and 100 MHz, respectively, in deuterated solvents containing 0.5%
enables a systematic study on the role of valency in de-
(v/v) TMS; chemical shifts are listed in ppm relative to TMS as an
internal standard. ESI-MS spectra were recorded on an Applied
termining the strength of multivalent interactions in chemi-
cal biology. As quantitative functionalization of the periph-
Biosystems Single Quadrupole Electrospray Ionization Mass Spec-
ery with peptides ranging from 5–28 amino acids was read-
trometer API-150EX in positive mode. Reversed-phase HPLC was
performed on a Shimadzu LC-8A HPLC system by using a VY-
ily achieved using native chemical ligation, this approach is
also expected to provide access to multivalent protein archi- DAC protein & peptide C18 column.
tectures via ligation of recombinant protein thioesters – al-
Di-tert-Butyl 4-Methyl-4-nitroheptanedioate (1): A mixture of nitro-
though, our approach readily lends itself to other efficient
bioorthogonal ligation chemistries. The molecular-level in-
formation gleaned by probes is considered a crucial first
ethane (1.46 g, 19.4 mmol) and tert-butyl acrylate (10.0 g,
78.0 mmol) in EtOH (30 mL) was cooled in an ice bath before the
dropwise addition of Triton-B (2 mL). The mixture was allowed to
step for the further development of systems used in peptide- gradually warm to room temp. over 16 h. An additional portion of
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Modular Platform for Multivalent Peptide Ligands
Figure 2. LC-MS characterization of crude labeled multivalent peptide constructs. a) Biotin-labeled tetrameric wedge conjugated with
cell adhesion peptides (calcd. mass: 5023 Da). b) Fluorescein-labeled trimeric wedge conjugated with cell-penetrating peptides (calcd.
mass: 9158 Da). c) Biotin-labeled dimeric wedge conjugated with peptides corresponding to the type 1 collagen-binding domain of bone
silaoprotein (calcd. mass: 8815 Da).
Triton-B (2 mL) was added to the reaction mixture, which was then
stirred at room temp. for a total of 48 h. After removing the vola-
tiles in vacuo, the product was recovered after recrystallization
from EtOH/H2O as a colorless solid (5.78 g, 90%). 1H NMR
(CDCl3, 400 MHz): δ = 2.35–2.07 (m, 8 H, CH2CH2), 1.53 (s, 3 H,
CH3), 1.44 [s, 18 H, C(CH3)3] ppm. 13C NMR (CDCl3, 100 MHz):
= 2977, 2932, 1724, 1607, 1456, 1421, 1392, 1366, 1306, 1255, 1147,
952, 848, 757 cm–1. ESI-MS: calcd. C16H32NO4+: m/z 302.23; found
m/z 302.18.
Di-tert-butyl 4-Acryloylamino-4-methylheptanedioate (3): A solu-
tion of 2 (5.00 g, 16.6 mmol) and Et3N (4.20 g, 41.5 mmol) in
DCM (50 mL) was cooled in an ice bath before adding a solution
of acryloyl chloride (1.50 g, 16.6 mmol) in DCM (20 mL) dropwise
over 30 min via an addition funnel. The reaction mixture was al-
lowed to gradually warm to room temp. overnight. After 16 h, the
volatiles were removed in vacuo and the mixture dissolved in
EtOAc (100 mL). The organic layer was washed with water
(3ϫ50 mL), satd. NaHCO3 (3ϫ50 mL) and brine (50 mL) before
drying over Na2SO4. After concentration, the crude mixture was
purified by flash chromatography using 2:1 hexane/EtOAc eluent.
The product was isolated as a colorless solid (4.43 g, 75%). 1H
NMR (CDCl3, 400 MHz): δ = 6.20 (d, J = 16.8 Hz, 1 H, HCC),
6.07 (s, 1 H, NH), 6.00 (dd, J1 = 16.8, J2 = 9.8 Hz, 1 H, CHCO),
5.56 (d, J = 9.9 Hz, 1 H, HCC), 2.29 (t, J = 8.0 Hz, 4 H, CH2CO),
2.09 (m, 2 H, NCCH2), 1.94 (m, 2 H, NCCH2), 1.43 [s, 18 H,
δ = 171.3, 89.9, 81.0, 34.3, 30.1, 28.0, 21.7 ppm. IR: ν = 2979,
˜
2934, 1728, 1539, 1454, 1367, 1308, 1152, 947, 847, 759 cm–1. EI-
MS: calcd. C16H29NO6+: m/z 331.20; found m/z 258, 220, 202, 173,
155, 127, 109, 57, 41.
Di-tert-butyl 4-Amino-4-methylheptanedioate (2): A mixture of 1
(5.50 g, 16.6 mmol) and Raney nickel (2 cm3) in EtOH (100 mL)
was purged with argon for 30 min before hydrogenation in a Parr
reactor (pH2 = 70 psi). Caution: Raney nickel catalyst is pyrophoric!
After 8 h, the reaction mixture was carefully filtered through Celite
under an argon atmosphere. After removal of the volatiles in vacuo,
the title compound was dried with P2O5 yielding a colorless low
melting solid (5.00 g, 100%) which was subsequently stored at
1
–20 °C until needed. H NMR (CDCl3, 400 MHz): δ = 2.27 (t, J =
8.2 Hz, 4 H, CH2CO), 1.65 (t, J = 8.2 Hz, 4 H, NCCH2), 1.44 [s, C(CH3)3], 1.35 (s, 3H. CH3) ppm. 13C NMR (CDCl3, 100 MHz): δ
18 H, C(CH3)3], 1.02 (s, 3 H, CH3) ppm. 13C NMR (CDCl3, = 173.3, 164.9, 131.9, 125.7, 80.7, 55.5, 33.5, 30.3, 28.1, 23.6 ppm.
100 MHz): δ = 173.3, 80.2, 50.7, 37.5, 30.5, 28.0, 27.2 ppm. IR: ν
IR: ν = 3311, 3063, 2975, 2936, 1721, 1658, 1620, 1539, 1459, 1367,
˜
˜
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E. W. Meijer et al.
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1304, 1249, 1149, 1115, 991, 955, 849, 810 cm–1. ESI-MS: calcd.
1229, 1210, 1144, 1092, 1060, 934, 922, 848, 760, 740 cm–1.
MALDI-TOF-MS: calcd. C31H41NO6Na+: m/z 546.29; found m/z
546.45.
C19H34NO5+: m/z 336.24; found m/z 336.20.
(tBuO)4-NO2 (4): A solution of nitroethane (362 mg, 4.82 mmol),
acrylamide 3 (3.60 g, 10.13 mmol) and N,N,NЈ,NЈ-tetramethylgua-
nidine (TMG, 500 µL) in THF (20 mL) was heated at 50 °C for
48 h, during which the reaction progress was monitored by THF-
SEC. After removal of the volatiles in vacuo, the residue was dis-
solved in ethyl acetate (150 mL) and the organic layer washed with
water (4ϫ50 mL), 1.0  HCl (4ϫ50 mL), satd. NaHCO3
(4ϫ50 mL) and brine (50 mL). The organic layer was dried with
Na2SO4 and passed through a short plug of silica before concen-
trating. The crude reaction mixture was purified by flash
chromatography using a gradient elution of 1:1 to 2:3 hexane/
EtOAc. The product was obtained as a colorless solid (3.37 g,
89%). 1H NMR (CDCl3, 400 MHz): δ = 5.92 (s, 2 H, NH), 2.3–
2.0 (m, 20 H, CH2), 1.88 (m, 4 H, NO2CCH2), 1.51 (s, 3 H,
NO2CCH3), 1.44 [s, 36 H, C(CH3)3], 1.28 (s, 6 H, NHCCH3) ppm.
13C NMR (CDCl3, 100 MHz): δ = 173.2, 170.3, 90.4, 80.7, 55.5,
(tBuO)3-NHFmoc (7b): Yield of 89% from 6 (2.00 g, 4.81 mmol).
1H NMR (CDCl3, 400 MHz): δ = 7.76 (d, J = 7.6 Hz, 2 H, CHar),
7.59 (d, J = 7.2 Hz, 2 H, CHar), 7.39 (t, J = 7.3 Hz, 2 H, CHar),
7.32 (t, J = 7.2 Hz, 2 H, CHar), 5.02 (s, 1 H, NH), 4.38 (d, J =
7.1 Hz, 2 H, CH2O), 4.18 (t, J = 7.2 Hz, 1 H, CHCH2O), 2.31–
1.18 (m, 6 H, CH2CO), 2.00–1.85 (m, 6 H, NCCH2), 1.43 [s, 27 H,
C(CH3)3] ppm. 13C NMR (CDCl3, 100 MHz): δ = 172.7, 154.2,
144.0, 141.3, 127.7, 127.1, 125.0, 120.0, 80.7, 65.9, 56.5, 47.3, 30.1,
29.7, 28.1 ppm. IR: ν = 3356, 2977, 2935, 1724, 1524, 1451, 1392,
˜
1367, 1314, 1244, 1148, 1082, 948, 847, 758, 739 cm–1. MALDI-
TOF-MS: calcd. C37H51NO8Na+: m/z 660.35; found m/z 660.23.
(tBuO)4-NHFmoc (7c): Yield of 100% from 5 (920 mg, 1.21 mmol).
1H NMR (CDCl3, 400 MHz): δ = 7.76 (d, J = 7.7 Hz, 2 H, CHar),
7.62 (d, J = 7.7 Hz, 2 H, CHar), 7.39 (t, J = 7.1 Hz, 2 H, CHar),
7.31 (t, J = 7.6 Hz, 2 H, CHar), 5.91 (s, 2 H, NH), 5.75 (s, 1 H,
NH), 4.37 (d, J = 6.6 Hz, 2 H. CH2O), 4.19 (t, J = 6.6 Hz, 1 H,
CHCH2O), 2.3–1.8 (m, 24 H, CH2), 1.42 [s, 36 H, C(CH3)3], 1.26
(overlapping s, 6H + 3 H, CH3) ppm. 13C NMR (CDCl3,
100 MHz): δ = 173.2, 172.6, 155.0, 144.1, 141.3, 127.6, 127.0, 125.2,
119.9, 80.6, 80.5, 65.9, 55.3, 54.6, 47.3, 34.6, 33.3, 32.0, 30.3, 28.0,
34.7, 33.3, 31.6, 30.3, 28.1, 23.6, 21.8 ppm. IR: ν = 3332, 2979,
˜
2937, 1729, 1655, 1539, 1453, 1392, 1368, 1305, 1256, 1153, 953,
847, 758 cm–1. ESI-MS: calcd. C40H72N3O12+: m/z 786.50; found
m/z 786.33.
(tBuO)4-NH2 (5): A mixture of (tBuO)4-NO2 4 (1 mmol) and
Raney nickel (0.25 cm3) in EtOH (50 mL) was purged with argon
for 30 min before hydrogenation in a Parr reactor (pH2 70 psi). Af-
ter 8–16 h, the reaction mixture was carefully filtered through Ce-
lite under an argon atmosphere. After removal of the volatiles in
vacuo, the title compounds were dried with P2O5 yielding a color-
less oil (0.74 g, 98%), which were subsequently stored –20 °C until
27.8, 24.2, 23.7 ppm. IR: ν = 3330, 3065, 2977, 2935, 1724, 1652,
˜
1531, 1451, 1423, 1392, 1367, 1304, 1249, 1147, 1113, 1085, 947,
846, 757, 740 cm–1. MALDI-TOF-MS: calcd. C55H83N3O12Na+:
m/z 999.60; found m/z 1000.45.
General Procedure for the Synthesis of (HO)n-NHFmoc Wedges:
The title compounds were prepared from (tBuO)n-NHFmoc
(1 mmol) after 8–16 h stirring at room temp. in formic acid
(10 mL). After removal of formic acid in vacuo, colorless solids
were obtained; yields 95–100%.
1
needed. H NMR (CDCl3, 400 MHz): δ = 6.61 (s, 2 H, NH2), 2.33
(t, J = 7.7 Hz, 4 H, CH2CON), 2.22 (t, J = 7.9 Hz, 8 H, CH2COO),
2.08 (m, 4 H, NH2CCH2), 1.85 (m, 8 H, NHCCH2), 1.42 [s, 36 H,
C(CH3)3], 1.24 (s, 6 H, NHCCH3), 1.23 (s, 3 H, NH2CCH3) ppm.
13C NMR (CDCl3, 100 MHz): δ = 173.4, 172.6, 80.6, 55.4, 36.1,
(HO)2-NHFmoc (8a): Yield of 100% from 7a (2.52 g, 4.81 mmol).
1H NMR [D6]DMSO, 400 MHz): δ = 7.86 (d, J = 7.7 Hz, 2 H,
CHar), 7.69 (d, J = 7.7 Hz, 2 H, CHar), 7.39 (t, J = 7.3 Hz, 2 H,
CHar), 7.30 (t, J = 7.0 Hz, 2 H, CHar), 7.00 (s, 1 H, NH), 4.20 (br.,
2 H, CH2O), 4.18 (overlapping t, 1 H, CHCH2O), 2.13 (br., 4 H,
CH2CO), 1.95 (br., 2 H, NCCH2), 1.68 (br., 2 H, NCCH2), 1.08 (s,
3 H, CH3) ppm. 13C NMR [D6]DMSO, 100 MHz): δ = 175.1,
154.8, 144.4, 141.2, 128.1, 127.5, 125.7, 120.5, 65.2, 53.9, 47.2, 33.5,
33.3, 33.2, 31.7, 30.4, 28.1, 25.3, 23.6 ppm. IR: ν = 3285, 2977,
˜
2933, 1726, 1648, 1541, 1453, 1391, 1366, 1304, 1254, 1146, 1113,
1037, 951, 921, 847, 758 cm–1. MALDI-TOF-MS: calcd.
C40H74N3O10+: m/z 756.53; found m/z 756.68.
(tBuO)3-NH2 (6): Commercially available.
General Procedure for the Preparation of (tBuO)n-NHFmoc Wedges:
A mixture of (tBuO)n-NH2 (1 mmol) and DIPEA (3.3 mmol) in
DCM (2 mL) was cooled in an ice-bath before the addition of 9-
fluorenylmethyl succinimidyl carbonate (1.1 mmol). After 30 min,
the reaction was removed from the ice bath and the reaction
warmed to room temp. After 4 h, the crude reaction mixture was
concentrated, redissolved in EtOAc (100 mL) and washed with
satd. NaHCO3 (3ϫ100 mL). After drying over MgSO4, filtration
and concentration, the crude product was purified by flash
chromatography using a gradient elution of chloroform to chloro-
form containing 2–5% MeOH. The title compounds were obtained
as colorless solids; yields 80–100%.
29.1, 23.7 ppm. IR: ν = 3356, 3200, 3063–2900, 1719, 1693, 1536,
˜
1450, 1413, 1343, 1292, 1264, 1233, 1212, 1183, 1171, 1129, 1093,
960, 947, 817, 756, 737 cm–1. MALDI-TOF-MS: calcd.
C23H25NO6Na+: m/z 434.17; found m/z 434.31.
(HO)3-NHFmoc (8b): Yield of 95% from 7b (2.34 g, 3.67 mmol).
1H NMR [D6]DMSO, 400 MHz): δ = 7.90 (d, J = 7.3 Hz, 2 H,
CHar), 7.72 (d, J = 7.7 Hz, 2 H, CHar), 7.41 (t, J = 7.3 Hz, 2 H,
CHar), 7.32 (t, J = 7.3 Hz, 2 H, CHar), 7.02 (s, 1 H, NH), 4.26–
4.16 (overlapping signals, 2 H, 1 H, CHCH2O), 2.18–2.08 (m, 6 H,
CH2CO), 1.85–1.76 (m, 6 H, NCCH2) ppm. 13C NMR [D6]DMSO,
100 MHz): δ = 174.5, 154.2, 144.0, 140.8, 127.7, 127.1, 125.4, 120.1,
(tBuO)2-NHFmoc (7a): Yield of 80% from 2 (2.00 g, 6.63 mmol).
This material could also be recrystallized from pentane/Et2O. 1H
NMR (CDCl3, 400 MHz): δ = 7.76 (d, J = 7.3 Hz, 2 H, CHar),
7.59 (d, J = 6.6 Hz, 2 H, CHar), 7.39 (t, J = 7.0 Hz, 2 H, CHar),
7.31 (t, J = 7.5 Hz, 2 H, CHar), 4.96 (s, 1 H, NH), 4.37 (br., 2 H,
CH2O), 4.19 (t, J = 6.6 Hz, 1 H, CHCH2O), 2.23 (br., 4 H,
64.9, 55.8, 46.829.2, 28.1 ppm. IR: ν = 3348, 2962, 1742, 1701,
˜
1542, 1451, 1406, 1292, 1246, 1178, 1092, 954, 826, 741 cm–1.
MALDI-TOF-MS: calcd. C25H27NO8Na+: m/z 492.16; found m/z
492.09.
(HO)4-NHFmoc (8c): Yield of 100% from 7c (1.18 g, 1.21 mmol).
CH2CO), 2.02 (br., 2 H, NCCH2), 1.87 (br., 2 H, NCCH2), 1.44 [s, 1H NMR [D6]DMSO, 400 MHz): δ = 12.00 (s, 4 H, OH), 7.86 (d,
18 H, C(CH3)3], 1.24 (s, 3 H, CH3) ppm. 13C NMR (CDCl3, J = 7.3 Hz, 2 H, CHar), 7.70 (d, J = 7.7 Hz, 2 H, CHar), 7.39 (t, J
100 MHz): δ = 172.9, 154.5, 144.0, 141.3, 127.6, 127.0, 125.0, 119.9,
= 7.3 Hz, 2 H, CHar), 7.31 (t, J = 7.3 Hz, 2 H, CHar), 7.23 (s, 2 H,
80.5, 65.9, 54.4, 47.4, 33.6, 30.2, 28.1, 23.8 ppm. IR: ν = 3330, NH), 6.99 (s, 1 H, NH), 4.17 (br., 2 H, 1 H, CHCH2O), 2.11 (m,
˜
3049, 2977, 2940, 1725, 1696, 1531, 1452, 1421, 1366, 1308, 1259,
8 H, CH2CO), 2.01 (m, 8 H, NCCH2), 1.82 (m, 2 H, CH2), 1.69
116
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 111–119

Modular Platform for Multivalent Peptide Ligands
(m, 4 H, 2 H, CH2), 1.24 (s, 3 H, CH3), 1.08 (s, 6 H, CH3) ppm. stirred at room temp. for 16 h and then loaded onto a column for
13C NMR [D6]DMSO, 100 MHz): δ = 175.1, 172.6, 154.9, 144.4,
141.1, 128.0, 127.5, 125.8, 120.5, 79.6, 54.4, 47.2, 34.6, 33.3, 31.2,
purification via flash chromatography using an eluent of 2%
MeOH in DCM. The product was recovered as a colorless glass
(1.10 g, 92%). 1H NMR (CDCl3, 400 MHz): δ = 7.37–7.34 (m, 6
H, CHar), 7.20–7.12 (m, 24 H, CHar), 6.40 (t, J = 3.7 Hz, 1 H,
NH), 3.71–2.90 (m, 29 H, CH2, CH), 2.79 (d, J = 5.5 Hz, 1 H,
NHTrt), 2.22 (m, 2 H, CH2S) ppm. 13C NMR (CDCl3, 100 MHz):
δ = 172.7, 145.5, 144.6, 129.7, 128.9, 127.9, 127.8, 126.7, 126.6,
71.5, 70.70, 70.67, 70.63, 70.55, 70.53, 70.46, 70.19, 70.02, 69.7,
29.1, 23.9, 23.8 ppm. IR: ν = 3331, 2964, 1703, 1633, 1535, 1450,
˜
1414, 1384, 1258, 1216, 1185, 796, 756, 740 cm–1. MALDI-TOF-
MS: calcd. C39H51N3O12Na+: m/z 776.35; found m/z 776.25.
General Procedure for the Synthesis of (TFP)n-NHFmoc Wedges: To
a mixture of (HO)n-NHFmoc (1 mmol), 2,3,5,6-tetrafluorophenol
(TFP, nϫ1.25 mmol) and PPTS (nϫ0.1 mmol) in DCM ([R-
CO2H] = 0.15–0.25 ) was added DCC (nϫ1.25 mmol) in small
portions over 30 min. After 16 h, the reaction mixture was filtered
through a short plug of Celite to remove the urea byproduct. The
crude reaction mixture was then purified by flash chromatography
using a gradient elution of chloroform with up to 15% Et2O. The
title compounds were obtained as colorless solids; yields 76–94%.
(TFP)2-NHFmoc (9a): Yield of 94% from 8a (412 mg, 1 mmol). 1H
NMR (CDCl3, 400 MHz): δ = 7.77 (d, J = 7.3 Hz, 2 H, CHar),
7.61 (d, J = 7.7 Hz, 2 H, CHar), 7.40 (t, J = 7.5 Hz, 2 H, CHar),
7.33 (t, J = 7.5 Hz, 2 H, CHar), 7.00 (m, 2 H, HCCF), 4.65 (s, 1
H, NH), 4.54 (br., 2 H, CH2O), 4.20 (t, J = 5.5 Hz, 1 H, CHCH2O),
2.62 (br., 4 H, CH2CO), 2.14 (br., 2 H, NCCH2), 2.02 (br., 2 H,
NCCH2), 1.24 (s, 3 H, CH3) ppm. 13C NMR (CDCl3, 100 MHz):
δ = 169.2, 154.4, 146.0 [ddt, JF,C = 249 Hz], 143.8, 141.4, 140.6
(ddd, JF,C = 248 Hz), 129.6 (dt), 127.7, 127.1, 124.8, 120.0, 103.3
(t, JF,C = 46 Hz), 65.6, 54.2, 47.4, 33.2, 28.2, 24.0 ppm. 19F NMR
66.7, 56.5, 38.8, 36.7 ppm. IR: ν = 3312, 3084, 3056, 3031, 2867,
˜
2100, 1669, 1594, 1520, 1489, 1445, 1347, 1284, 1246, 1103, 1032,
884, 851, 744, 701 cm–1. MALDI-TOF-MS: C55H64N5O7S+: m/z
938.44; found m/z 938.48.
Trt-C(Trt)-PEG-NH2 (12): Trt-C(Trt)-PEG-N3 11 (940 mg,
1.00 mmol) was dissolved in THF (18 mL) before the addition of
H2O (2 mL), followed by PPh3 (328 mg, 1.25 mmol) in small por-
tions over 30 min. The reaction mixture was stirred at room temp.
for 6 h, after which the IR band at 2100 cm–1 for the azide had
completely disappeared. The reaction mixture was concentrated in
vacuo before purification by flash chromatography using
a
multistage eluent of 2% MeOH in DCM to 10% MeOH in DCM
to 10% MeOH in DCM containing 5% isopropylamine. The de-
sired compound was obtained as a colorless glass (900 mg, 99%).
1H NMR (CDCl3, 400 MHz): δ = 7.37–7.34 (m, 6 H, CHar), 7.20–
7.12 (m, 24 H, CHar), 6.43 (t, J = 3.7 Hz, 1 H, NH), 3.71–2.80 (m,
29 H, CH2, CH), 2.78 (d, J = 5.5 Hz, 1 H, NHTrt), 2.25 (m, 2 H,
CH2S), 1.8 (br. s, 2 H, NH2) ppm. 13C NMR (CDCl3, 100 MHz):
δ = 172.7, 145.5, 144.6, 129.7, 128.9, 127.9, 127.8, 126.7, 126.6,
73.2, 71.5, 70.56, 70.53, 70.50, 70.46, 70.28, 70.18, 69.68, 66.7, 56.5,
(CDCl3, 376 MHz): δF = –142.0, –156.1 ppm. IR: ν = 3405, 3074,
˜
2936, 1785, 1709, 1645, 1522, 1487, 1451, 1416, 1379, 1301, 1271,
1230, 1178, 1086, 1065, 953, 839, 759, 740, 716 cm–1. MALDI-
TOF-MS: calcd. C35H25F8NO6Na+: m/z 730.15; found m/z 730.38.
41.8, 38.8, 36.7 ppm. IR: ν = 3306, 3084, 3056, 3030, 2867, 1667,
˜
(TFP)3-NHFmoc (9b): Yield of 80% from 8b (520 mg, 1.11 mmol).
1H NMR (CDCl3, 400 MHz): δ = 7.79 (d, J = 7.3 Hz, 2 H, CHar),
7.59 (d, J = 6.6 Hz, 2 H, CHar), 7.41 (t, J = 7.2 Hz, 2 H, CHar),
7.33 (t, J = 7.3 Hz, 2 H, CHar), 7.01 (m, 3 H, HCCF), 4.65–4.53
(m, 3 H, NH, CH2O), 4.20 (t, J = 6.6 Hz, 1 H, CHCH2O), 2.70–
2.55 (m, 6 H, CH2CO), 2.32–2.20 (m, 6 H, NCCH2) ppm. 13C
1595, 1520, 1489, 1445, 1347, 1291, 1245, 1100, 1033, 940, 902,
848, 744, 701 cm–1. MALDI-TOF-MS: calcd. C55H66N3O7S+: m/z
912.45; found m/z 912.46.
General Procedure for the Synthesis of [Trt-C(Trt)-PEG-NH]n-
NHFmoc Wedges: A solution of (TFP)n-NHFmoc (1 mmol), Trt-
C(Trt)-PEG-NH2 (nϫ1.05 mmol) and DIPEA (nϫ3.15 mmol) in
DCM ([TFP-activated ester] = 0.25 ) was stirred at room temp.
for up to 4 h and subsequently purified by flash chromatography
using a gradient elution of 5% to 10% MeOH in DCM. The prod-
ucts were obtained as a colorless glass; yields 73–98%.
NMR (CDCl3, 100 MHz): δ = 169.1, 154.2, 146.1 (ddt, JF,C
=
256 Hz), 143.8, 141.7, 140.7 (ddd, JF,C = 43 Hz), 129.6 (m), 127.9,
127.2, 124.7, 120.1, 103.8 (t, JF,C = 248 Hz), 65.9, 56.7, 54.9, 53.0,
47.5, 29.9, 27.5 ppm. 19F NMR (CDCl3, 376 MHz): δ = –142.0,
–156.1 ppm. IR: ν = 3395, 3081, 2949, 1783, 1708, 1645, 1521,
˜
1487, 1451, 1271, 1178, 1094, 1065, 954, 840, 760, 741, 716 cm–1. [Trt-C(Trt)-PEG-NH]2-NHFmoc (13a): Yield of 98% from 9a
MALDI-TOF-MS: calcd. C43H27F12NO8Na+: m/z 936.14; found
m/z 936.11.
(150 mg, 214 µmol). 1H NMR (CDCl3, 400 MHz): see Figure S1
ppm. 13C NMR (CDCl3, 100 MHz): δ = 173.1, 172.8, 154.5, 145.5,
144.6, 144.1, 141.3, 129.7, 128.9, 127.9, 127.8, 127.6, 127.0, 126.7,
126.6, 125.1, 119.9, 71.5, 70.5, 70.4, 70.2, 70.1, 69.8, 69.7, 66.7,
(TFP)4-NHFmoc (9c): Yield of 76% from 8c (400 mg, 0.66 mmol).
1H NMR (CDCl3, 400 MHz): δ = 7.73 (d, J = 7.2 Hz, 2 H, CHar),
7.58 (d, J = 7.2 Hz, 2 H, CHar), 7.37 (t, J = 7.4 Hz, 2 H, CHar),
7.28 (t, J = 7.4 Hz, 2 H, CHar), 6.98 (m, 4 H, HCCF), 5.59 (s, 2
H, NH), 5.44 (s, 1 H, NH), 4.36 (br., 2 H, CH2O), 4.18 (t, J =
6.8 Hz, 1 H, CHCH2O), 2.68 (t, J = 7.6 Hz, 8 H, CH2CO), 2.48
65.7, 56.5, 54.5, 47.4, 39.3, 38.8, 36.7, 34.4, 31.4 ppm. IR: ν = 3315,
˜
3056, 2869, 1722, 1652, 1595, 1526, 1489, 1446, 1348, 1246, 1085,
1033, 940, 903, 849, 742, 699 cm–1. MALDI-TOF-MS: calcd.
C
133H151N7O18S2Na+: m/z 2222.06; found m/z 2222.15.
(m, 4 H, CH2), 2.18 (br., 4 H, CH2), 2.08 (m, 4 H, CH2), 1.96 (br., [Trt-C(Trt)-PEG-NH]3-NHFmoc (13b): Yield of 73% from 9b
1
4 H, CH2), 1.30 (s, 6 H, CH3), 1.25 (s, 3 H, CH3) ppm. 13C NMR
(CDCl3, 100 MHz): δ = 172.9, 169.4, 155.1, 146.0 (ddt, JF,C
249 Hz), 143.8, 141.3, 140.4 (ddd, JF,C = 248 Hz), 129.5 (dt), 127.7,
127.0, 124.9, 120.0, 103.2 (t, JF,C = 46 Hz), 66.0, 55.2, 54.8, 47.3,
34.7, 33.0, 32.0, 28.3, 24.5, 23.8 ppm. 19F NMR (CDCl3,
(200 mg, 220 µmol). H NMR (CDCl3, 400 MHz): see Figure S2.
13C NMR (CDCl3, 100 MHz): δ = 173.0, 172.8, 145.8, 144.9, 144.2,
141.5, 129.8, 129.1, 128.0, 127.8, 127.7, 127.1, 126.9, 126.8, 125.4,
120.1, 71.6, 70.7, 70.3, 70.2, 69.8, 69.7, 66.8, 57.0, 56.5, 47.4, 39.4,
=
38.9, 36.9, 31.2, 30.7 ppm. IR: ν = 3309, 3054, 2869, 1721, 1652,
˜
376 MHz): δ = –142.0, –156.1 ppm. IR: ν = 3399, 3282, 3070, 2935,
1523, 1489, 1446, 1348, 1244, 1097, 1033, 941, 902, 851, 743, 702
cm–1. MALDI-TOF-MS: calcd. C190H216N10O26S3Na+: m/z
3175.05; found m/z 3173.83.
˜
1786, 1703, 1660, 1521, 1487, 1452, 1386, 1305, 1267, 1178, 1092,
1064, 954, 840, 760, 739, 716 cm–1. MALDI-TOF-MS: calcd.
C63H51F16N3O12Na+: m/z 1368.32; found m/z 1368.22.
[Trt-C(Trt)-PEG-NH]4-NHFmoc (13c): Yield of 75% from 9c
1
Trt-C(Trt)-PEG-N3 (11): A mixture of azido-PEG-amine 10 (Poly-
pure, Norway, 446 mg, 1.27 mmol), Trt-C(Trt)-OSu (938 mg,
1.33 mmol) and Et3N (386 mg, 3.81 mmol) in DCM (5 mL) was
(150 mg, 110 µmol). H NMR (CDCl3, 400 MHz): see Figure S3.
13C NMR (CDCl3, 100 MHz): δ = 173.4, 172.8, 145.5, 144.6, 144.1,
140.1, 129.6, 128.9, 127.9, 127.8, 127.1, 126.7, 126.6, 119.9, 71.5,
Eur. J. Org. Chem. 2010, 111–119
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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117

E. W. Meijer et al.
FULL PAPER
70.5, 70.1, 69.7, 66.7, 56.5, 55.3, 50.4, 39.3, 38.8, 37.6, 36.7, 34.3,
each containing 0.1% TFA, to give the biotinylated compounds
as colorless solids or the fluorescein labeled compounds as yellow
solids.
31.1, 23.7 ppm. IR: ν = 3311, 3058, 2873, 1656, 1595, 1541, 1490,
˜
1447, 1348, 1249, 1220, 1106, 1033, 905, 772, 744, 698 cm–1.
MALDI-TOF-MS: calcd. C259H303N15O36S4Na+: m/z 4353.60;
found m/z 4352.45.
[Cys-PEG-NH]2-Biotin (16a): Yield of 48% from 14a over two
steps. ESI-MS: calcd. C58H110N10O19S3: m/z 1347.77; found m/z
1347.0 (Figure S7).
General Procedure for the Synthesis of [Trt-C(Trt)-PEG-NH]n-NH2
Wedges: A solution of [Trt-C(Trt)-PEG-NH]n-NHFmoc (1 mmol)
in 20% (v/v) piperidine/DCM (10 mL) was stirred at room temp.
for 1 h. The reaction mixture was diluted with DCM (50 mL) be-
fore washing with a 1.0  phosphate buffer containing 1.0  NaCl
at pH 5.5 (5ϫ50 mL). After drying and concentration, the crude
product mixture was purified by flash chromatography using 3%
MeOH in DCM, followed by 10% MeOH in DCM containing up
to 5% isopropylamine to elute the title compounds; yields 95–
100%.
[Cys-PEG-NH]3-Biotin (16b): Yield of 26% from 14b over two
steps. ESI-MS: calcd. C77H147N13O27S4: m/z 1815.34; found m/z
1814.2 (Figure S8).
[Cys-PEG-NH]4-Biotin (16c): Yield of 21% from 14c over two
steps. ESI-MS: calcd. C108H206N18O37S5: m/z 2509.25; found m/z
2508.7 (Figure S9).
[Cys-PEG-NH]2-Fluorescein (16d): Yield of 52% from 14a over two
steps. ESI-MS: calcd. C64H97N7O21S2: m/z 1364.64; found m/z
1364.8 (Figure S10).
[Trt-C(Trt)-PEG-NH]2-NH2 (14a): Yield of 95% from 13a (440 mg,
200 µmol). 1H NMR (CDCl3, 400 MHz): see Figure S4. 13C NMR
(CDCl3, 100 MHz): δ = 173.2, 172.7, 145.5, 144.6, 129.7, 128.9,
127.9, 127.8, 126.7, 126.6, 71.5, 70.5, 70.4, 70.2, 70.1, 69.8, 69.7,
[Cys-PEG-NH]3-Fluorescein (16e): Yield of 38% from 14b over two
steps. ESI-MS: calcd. C83H134N10O29S3: m/z 1832.23; found m/z
1832.8 (Figure S11).
66.7, 56.5, 53.4, 51.5, 39.3, 38.8, 37.5, 36.7, 31.3, 27.2 ppm. IR: ν
˜
[Cys-PEG-NH]4- Fluorescein (16f): Yield of 42% from 14c over two
steps. ESI-MS: calcd. C114H193N15O39S4: m/z 2526.13; found m/z
2526.4 (Figure S12).
= 3312, 3057, 3031, 2869, 1652, 1595, 1532, 1489, 1446, 1348, 1287,
1246, 1184, 1098, 1032, 922, 904, 848, 743, 699 cm–1. MALDI-
TOF-MS: calcd. C118H141N7O16S2Na+: m/z 1999.99; found m/z
2000.03.
Solid-Phase Peptide Synthesis of Peptide Thioesters: For the prepa-
ration of three peptides with a C-terminal thioester using Fmoc
chemistry the procedure of Ingenito et al.[11] was followed. 4-Sulfa-
mylbutyryl AM resin (1 g, 1.1 mmol, Novabiochem) was suspended
in CH2Cl2 (10 mL) before the addition of 9.0 equiv. of DIPEA
(1.65 mL) and 3.0 equiv. of Fmoc-Gly-OH (0.98 g). The mixture
was stirred for 10 min at room temperature followed by cooling to
–20 °C. After 20 min, 3.0 equiv. of PyBOP (1.72 g) were added as
a solid and the mixture was stirred overnight. The resin was then
washed with CH2Cl2, DMF, MeOH and dried in vacuo. The extent
of incorporation was quantified by the Fmoc release UV assay and
showed a loading of 0.68 mmol/g. Peptide assembly on the Fmoc-
Gly-Sulfamylbutyryl resin was performed using an automated pep-
tide synthesizer (Prelude, Protein technologies) following the stan-
dard Fmoc peptide synthesis protocol (100 µmol scale). Each
amino acid was allowed to couple for two times 20 min and the N-
terminus was acetylated using a mixture of pyridine, acetic anhy-
dride and DMF (1:1:3). Alkylation of the peptide-acylsulfonamide
resin was performed by swelling the resin in THF (2 mL) before
the addition of 1  TMS-CH2N2 (50:50, v/v, hexane/THF). After
stirring for 2 h, the resin was washed with THF and DMF. Ethyl-
3-mercaptopropionate (40 equiv., 1  in DMF) and sodium thio-
phenate (0.4 equiv.) were added and stirred for 24 h. The resin was
filtered and washed with DMF. The filtrate containing the peptide
was collected and dried in vacuo. The cleaved, protected peptides
were subsequently treated with 96% TFA, 2% H2O and 2% triiso-
propylsilane and stirred for 2 h. Cold diethyl ether was added and
the mixture was stored for 2 h at –30 °C. The precipitate formed
after centrifugation was dissolved in water and lyophilized. Purifi-
cation was done by preparative RP-HPLC using a gradient of ace-
tonitrile in water (both containing 0.1% TFA).
[Trt-C(Trt)-PEG-NH]3-NH2 (14b): Yield of 100% from 13b
(150 mg, 48 µmol). 1H NMR (CDCl3, 400 MHz): see Figure S5.
13C NMR (CDCl3, 100 MHz): δ = 173.3, 172.8, 145.5, 144.6, 129.6,
129.0, 128.9, 128.2, 127.9, 127.8, 126.7, 126.6, 125.3, 71.5, 70.5,
70.4, 70.3, 70.1, 69.9, 69.6, 69.4, 66.7, 58.3, 56.5, 39.3, 38.8, 36.7,
32.0, 30.3, 29.8 ppm. IR: ν = 3412, 3055, 3021, 2926, 2052, 1650,
˜
1525, 1490, 1447, 1397, 1349, 1247, 1222, 1102, 1033, 940, 745, 703
cm–1. MALDI-TOF-MS: calcd. C175H207N10O24S3+: m/z 2930.75;
found m/z 2929.99.
[Trt-C(Trt)-PEG-NH]4-NH2 (14c): Yield of 100% from 13c
(200 mg, 46 µmol). 1H NMR (CDCl3, 400 MHz): see Figure S6.
13C NMR (CDCl3, 100 MHz): δ = 173.3, 172.7, 145.5, 144.6, 129.7,
128.8, 127.9, 127.8, 126.7, 126.6, 71.5, 70.5, 70.4, 70.3, 70.1, 69.8,
69.7, 69.5, 66.7, 56.5, 53.4, 39.3, 38.8, 36.7, 30.5, 27.9 ppm. IR: ν
˜
= 3306, 3055, 3025, 2887, 1651, 1530, 1489, 1446, 1363, 1303, 1246,
1225, 1099, 1032, 938, 743, 698 cm–1. MALDI-TOF-MS: calcd.
C
224H293N15O34S4Na+: m/z 4108.33; found m/z 4107.85.
General Procedure for the Synthesis of Biotin- and Fluorescein-Lab-
eled [Cys-PEG-NH]n-NHR Wedges: A solution of [Trt-C(Trt)-
PEG-NH]n-NH2 (10 µmol) in DMF (500 µL) was added to a solu-
tion of 6-(biotinylamino)caproic acid (30 µmol) or alternatively 5-
carboxyfluorescein (Novabiochem, 30 µmol) and DIPEA (90 µmol)
in DMF (500 µL). This mixture was cooled in an ice bath before
the addition of HATU (30 µmol) as a solid. The reaction mixture
warmed to room temp. gradually. After 12 h, the reaction mixture
was diluted with DCM (50 mL) and the organic layer washed with
a 1:4 dilution of satd. NaHCO3 (4ϫ50 mL) and then H2O
(50 mL). At this stage, MALDI-TOF MS was used to determine
extent of reaction. In all cases, greater than 75% conversion was
observed. After drying and concentration, the reaction mixture was
immediately subjected to trityl deprotection as follows. An ice bath
cooled solution (5 mL) of 95% TFA-containing 2.5% (v/v) of tri-
isopropylsilane and H2O was used to dissolve the crude [Trt-C(Trt)-
PEG-NH]n-NH-R labeled wedges (10 µmol). After 30 min of stir-
ring at 4 °C, the volatiles were removed in vacuo and the residue
taken up into H2O (20 mL). The aqueous layer was washed with
Et2O (4ϫ20 mL) before freeze drying. The labeled dendrons were
purified by RP-HPLC using gradient of 15% to 45% ACN in H2O,
Ac-GRGDSGG-SR: Gradient RP-HPLC: 10–20% in 20 min. ESI-
MS: Calcd. m/z 762.6; found m/z 762.3 (Figure S13).
Ac-RQIKIWFQNRRMKWKKGGG-SR: Gradient RP-HPLC: 15–
30% in 45 min. ESI-MS: Calcd. 2576.0 m/z; found m/z 2575.6 (Fig-
ure S14).
Ac-NGVFKYRPRYFLYKHAYFPPLKRFPVQGG-SR: Gradient
RP-HPLC: 15–35% in 45 min. ESI-MS: Calcd. m/z 3868.4; found
m/z 3867.7, and 3979.4 (TFA adduct) (Figure S15).
118
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Multivalent Peptides via Native Chemical Ligation: For these reac-
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dendritic wedge were used. Thiophenol was employed as the trans-
thioesterification catalyst with benzyl mercaptan as the in situ re-
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pH 8.0) before adding the peptide thioesters, also in the ligation
buffer. The addition of thiols brought the overall pH to 7.6–7.8.
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(Ac-GRGDSGGC)4-Biotin: ESI-MS: Calcd. m/z 5023; found m/z
5024.
(Ac-RQIKIWFQNRRMKWKKGGGC)3-Fluorescein:
ESI-MS:
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(Ac-NGVFKYRPRYFLYKHAYFYPPLKRFPVQGGC)2-Biotin:
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Supporting Information (see also the footnote on the first page of
this article): 1H NMR spectra of compounds 13 and 14 and ad-
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Received: September 11, 2009
Published Online: November 27, 2009
Eur. J. Org. Chem. 2010, 111–119
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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