A new dendrimer scaffold for preparing dimers or tetramers of biologically active molecules

Article abstract of DOI:10.1016/j.tetlet.2005.01.026

Synthesis of a new scaffold derived from iminodipropionic acid for the preparation of peptide dimers and tetramers is described.

Full text of DOI:10.1016/j.tetlet.2005.01.026

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1463–1465
A new dendrimer scaffold for preparing dimers or tetramers
of biologically active molecules
Natarajan Raju,* Rama S. Ranganathan, Mike F. Tweedle and Rolf E. Swenson
Bracco Research, USA, Inc., 305 College Road East, Princeton, NJ 08540, USA
Received 23November 2004; revised 3January 2005; accepted 7 January 2005
Available online 22 January 2005
Abstract—Synthesis of a new scaffold derived from iminodipropionic acid for the preparation of peptide dimers and tetramers is
described.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Synthesis of the scaffold (Scheme 1)
Enhanced potency can be achieved by multivalent dis-
play of ligands as antagonists or agonists of biological
processes.1 Constructs bearing multiple copies of a
ligand often display dramatically increased affinity and
specificity relative to the monomer due to avidity and
cooperativity effects.2,3 Recently, increased emphasis
has been placed on utilizing constructs capable of multi-
ple interactions with either different epitopes of a single
molecule of a biomolecular target or with the same epi-
tope on more than one molecule of the target. The strat-
egy has been applied to problems in cancer imaging,4
cancer therapy,5 cancer radiotherapy,6 and as immuno-
gens,7,8 biosensors,9 and artificial proteins.10 Biosyn-
thetic methods have been recently disclosed to prepare
the scaffolds that can carry several copies of the peptides
in immunotherapy and gene therapy for cancer.11
We chose iminodipropionic acid,15 as the central core
for the dendrimer. Bisaddition of benzylamine to t-
butylacrylate under standard conditions was unsuccess-
ful, but a neat Michael addition reaction of benzylamine
to t-butyl acrylate in the presence of 1 mol%
Yb(OTf)3,16 enabled this transformation in 90% yield,
to provide 1a. Debenzylation of 1a, to obtain 1b, fol-
lowed by protection with N-(9-fluorenylmethoxycarbon-
yloxy)succinimide yielded the protected amine 1c.
Alternatively, the amine 1b was coupled to Fmoc-Gly-
OH to furnish the amide 2a which in turn was selectively
deprotected to give the acid 3. The t-butyl groups were
removed from 1c to provide 1d. Crude acids 1d and 3
were purified by recrystallization from acetonitrile and
both were obtained as colorless solids. Bisacid 3 was
coupled with 2.2 equiv of 2b to yield the corresponding
tetraester 4a which was smoothly converted to tetraacid
4b. The intermediates 1d and 4b were set for coupling to
the protected TKPPR peptide to provide a dimer or
tetramer construct, respectively.
The delivery of ligands to the binding site as polyamino-
amide (PAMAM) dendrimers is well documented.12 We
describe here an efficient synthesis of a scaffold that
utilizes a polyamide approach for dimeric or tetrameric
presentation of ligands, with the added ability to append
a detectable tag, such as Oregon green (for binding stud-
ies). We applied this scaffold to the antagonist,
TKPPR,13 of the Tuftsin receptor.14
3. Preparation of protected Tuftsin receptor antagonist
H-Adoa-Thr(t-Bu)-Lys(Boc)Pro-Pro-Arg(Pmc)-Ot-Bu (7)
The fully protected core sequence Fmoc-Thr(t-Bu)-Lys-
(Boc)-Pro-Pro-Arg(Pmc)-Ot-Bu (5), was prepared as
follows: H-Arg(Pmc)-Ot-Bu was coupled to Z-Pro-OH
using HATU/(i-Pr)2NEt. Removal of the Z-group by
hydrogenation followed by another coupling with
Keywords: Iminodipropionic acid; Dimer; Tetramer; Dendrimer;
Oregon Green.
*
Z-Pro-OH
Z-Lys(Boc)-OH and Fmoc-Thr(t-Bu)-OH were then
provided
Z-Pro-Pro-Arg(Pmc)-Ot-Bu.
Corresponding author. Tel.: +1 609 514 2420; fax: +1 609 514
2446; e-mail: natarajan.raju@bru.bracco.com
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.026

1464
N. Raju et al. / Tetrahedron Letters 46 (2005) 1463–1465
NHR1
N
CO2P
CO2P
R
i
v
N
1b
POOC
COOP
CO2C4H9-t
O
R
P
R1
P
1a = Bn
-C4H9-t
-C4H9-t
ii
2a = Fmoc
-C4H9-t
vi
1b = H
iii
iv
2b
=
H
-C4H9-t
1c = Fmoc
1d = Fmoc
-C4H9-t
H
COOP
COOP
O
O
H
NHR1
COOH
NHR1
N
N
O
N
O
N
iv
vii
N
2a
COOP
COOP
O
O
H
N
COOH
3 R1 = Fmoc
R1
P
4a
=
Fmoc
-C4H9-t
H
iv
4b = Fmoc
Scheme 1. Reagents and conditions: i) BnNH2, 0.45 eq; Yb(OTf)3, 1.0 mol%, 100 °C, 2 h, 90%; ii) H2, Pd(OH)2, MeOH–water (95:5), 100%; iii)
Fmoc-OSu; THF/water, 65%; iv) TFA/CH2Cl2 (1:1); 2 h, 75%; v) Fmoc-Gly-OH, HATU, (iPr)2NEt, CH2Cl2; vi) 10% piperidine in acetonitrile,
30 min., 85%; vii) 2b, HATU, (iPr)2NEt, CH2Cl2, 65%.
sequentially coupled to the growing peptide chain. At
this stage the fully protected peptide 5 was purified by
silica gel column chromatography [50% overall yield;
DCM–MeOH (9:1)]. Treatment of 5 with piperidine in
acetonitrile afforded H-Thr(t-Bu)-Lys(Boc)-Pro-Pro-
Arg(Pmc)-Ot-Bu, which was acylated with 8-Fmoc-
Fmoc
N
R3
N
R2
R2
ii, iii, iv
R4
R4
i
1d
O
O
O
O
O
O
8
9
O
O
O
H
N
H
N
O
N
O
R2
N
O
R2/R4
FmocHN
R3HN
N
O
O
N
O
O
v
H
ii, iii, iv
H
N
4b
N
R2
R2/R4
N
O
O
N
O
O
R2
R2/R4
O
O
R2
R4/R2
10
11a, 11b
O
O
8, 10, 11a R = Adoa-Thr( Bu)-Lys(Boc)-Pro-
t
2
O
R3 =
Pro-Arg(Pmc)-O Bu
t
F
F
9, 11b R4= Adoa -Thr-Lys-Pro-Pro-Arg-OH
HO
O
OH
Scheme 2. Reagents and conditions: i) 7 (2.2 eq.), HATU, (iPr)2NEt, CH2Cl2; ii) 10% piperidine in acetonitrile, 30 min; iii) Oregon Green-OSu,
DMF; iv) TFA/anisole/water (95:5:1); v) 7 (4.4 eq.), HATU, (iPr)2NEt, CH2Cl2.

N. Raju et al. / Tetrahedron Letters 46 (2005) 1463–1465
1465
amino-3,6-dioxaoctanoic acid (Fmoc-Adoa) to give
Fmoc-Adoa-Thr(t-Bu)-Lys(Boc)Pro-Pro-Arg(Pmc)-Ot-
Bu (6). Removal of the Fmoc—protecting group from 6
resulted in H-Adoa-Thr(t-Bu)-Lys(Boc)Pro-Pro-Arg-
(Pmc)-Ot-Bu (7). Both 6 and 7 were purified by silica
gel column chromatography (9:1—DCM/MeOH for 6
and 8:2—DCM/MeOH for 7). This procedure provided
rapid access to the suitably protected peptide 7 for
coupling to a dimeric/tetrameric cores.
ligands. We are currently exploring other routes to in-
crease the number of displayed peptide units in a con-
trolled manner and applications to the Tuftsin
receptor antagonist and other bioactive molecules.
References and notes
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The linker acid 1d was coupled to amine 7 to give the
protected dimer 8, whose Fmoc group was removed
(piperidine/acetonitrile). The fluorescent tracer Oregon
GreenTM was appended to the secondary amine function
using Oregon Green-OSu ester. The protecting groups
were removed under standard conditions and the crude
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the required dimer 9 in 48% overall yield for the final
three steps. The structure of fluorescent dimer 9 was
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(Scheme 2).
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The tetra acid 4b when subjected to coupling with amine
7, following similar protocols as above, resulted in 10,
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poration of the fluorescent tag with Oregon Green-OSu
ester yielded compound 11a (R2 = Adoa-Thr(t-Bu)-Lys-
(Boc)-Pro-Pro-Arg(Pmc)-Ot-Bu, R3 = Oregon Green)
the penultimate protected peptide tetramer bearing the
reporter. Deprotection of 11a under standard conditions
resulted in the desired fluorphore-labeled peptide tetra-
mer 11b. Purification of crude 11b by preparative HPLC
yielded the pure compound, containing four units of the
biologically active peptide, in 24% overall yield. The
structure was confirmed by NMR, MS, and analytical
data.
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`
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Thus, we have increased the versatility of the imino-
dipropionic acid-based scaffold as a carrier for multiple