12 Multivalent Peptide Dendrimers

H. Malda, M.H.P. van Genderen, T.M. Hackeng, E.W. Meijer

Molecular Bioengineering & Molecular Imaging, Laboratory of Macromolecular and Organic Chemistry

Introduction Results of Ligation

Goal Ligation RGD-peptide to G1-Cys4 and G2-Cys8
The synthesis of a multivalent construct consisting of a The Arg-Gly-Asn (RGD) motif is known to bind strongly to integrins
dendrimer and multiple peptides and functional probes, either at the cell surface. Ligation of the thioester peptide to dendrimers
reversible or non-reversible, and testing their suitability for gives exclusively the fully modified dendrimer.
biomedical applications. (AcGRGDSGGC)4-G1 3243.2 (AcGRGDSGGC)8-G2 6627.1

5+

Multivalency and Dendrimers 6+ 8+

Multivalency is a phenomenon wherein the interactions of multiple

2000 4000 6000 8000 4000 6000 8000 10000
Mass Mass

4+ 9+
ligands to multiple receptors is stronger than the corresponding 3+
7+
5+
6+
monovalent interactions. Dendrimers are regularly branched (star 2+ 10+ 4+

like), monodisperse, molecules with a high number of endgroups 500 1000

m/z
1500 2000 500 1000
m/z
1500 2000

via which multivalency can be explored. They promise to be good

candidates for biomedical applications as they are uniform in size Apart from the RGD-motif, a lot of other peptides have been
and relatively small. In this research modified poly (propylene successfully ligated to dendrimers.
imine) dendrimers are used.
Introduction of probes via maleimide- thiol reaction
Native Chemical Ligation After ligation, the thiol of cysteine is free for a reaction with e.g.
Native Chemical Ligation is a commonly used synthetic method for maleimides. In this way, additional functional probes or peptides
the coupling of two peptide fragments. In short, this technique can be introduced at the dendrimer.
makes use of a thioester-terminated (oligo)peptide-fragment as Fluorescent probe: Oregon Green-maleimide
O

well as an (oligo)peptide-fragment with a terminal cysteine

O
NH
HN

SH
4+
HN O
O NH
O
O
NH
HN

O O

residue. Combined under mild conditions, this yields a native

O
HN NH
HN NH HN O

N NH2
O NH
O
OH
OH
O NH2

O N HO O
HN

HN
NH

O
O OH

O NH

R
O
O

peptide bond.
NH F O
O F HN

HN O COOH
HOOC O
O O NH
O S

HOOC S O O
O N
N NH
HN
O
O

F F HO F
N
N
F OH

Spontaneous
F

Chemical
OH
HO F
O
- HS
S
O

reaction Rearrangement + +
NH

5 3
HN N

O O O O
N
S O
O
HN
S
O O
O

O O OH O COOH
HOOC O NH

NH F O
O F HN

+ S O N
H3 N
O
O

SR H
HN O

Oregon Green
O NH

6+ 2+
O OH
HO O

O
NH

O NH2 HN
OH

S
OH O NH2
NH2 O
HN O
O NH HN NH

maleimide
HN NH

O HN
O
NH

Figure 1: Native Chemical Ligation

HN O

N O NH

O
NH
HN

R
O
O

Magnetic Resonance Imaging-probe: DTPA-maleimide

Material and methods (RGD)4(DTPA)4-G1 5897.8
O

Synthesis Cysteine Dendrimers

O

O HN
NH

HN O
O NH

N O
O

O NH

4+
HN

O HN NH
O NH
HN O
HN NH

O NH2

The synthesis of Cysteine dendrimer is accomplished according to

NH2 O OH
OH COOH
HOOC NH COOH
HOOC HN O OH
HO

HN
O N

5+
N HN O
O NH HOOC
COOH O N

3+
N O
NH N
N HN
O COOH
HOOC

scheme 1, yields are reasonable to good.

O NH
HN O O COOH
HOOC HN O NH
O

N
O S

N
4000 6000 8000 10000
N
S O
N
N

O O
NH
Mass HN
O

OH O
O

OH
6+
N
N

O OH O O
NH
O

OH HO N
HN
O S
N

O O O TFA HOOC HN
O O
S
NH
O
HN O O
NH COOH

O
O COOH
H Ph H Ph Et3SiH
HOOC O
N

Ph DSC
NH

N H Ph N Ph O N HN
O N

HO acetonitrile N N Ph NH2 N water

N O HOOC

DTPA-maleimide has
COOH HN O

NH2
O
2+
O NH N

H
N O OH

N HO O NH COOH

Ph Ph
HOOC HN COOH

O
HOOC OH

S Ph S H NH2 O
OH O
HN O
NH2

S
Ph Ph
been synthesized by
O NH HN NH

Ph triethylamine SH
HN NH

HN
NH

Ph Ph CH2Cl2 Ph Ph O
O

Ph Ph 500 1000 1500 2000 2500 HN O

Anouk Dirksen
O NH

NH
HN

H2N NH2
m/z O
O

NH2
O HS
HS O
H2N O HN O
SH HN HN

Near Future Work and Conclusions

SH NH2 HS
NH2
H2N HS O NH
O SH HN
HN H2N N O
O NH N
N
SH H HS
O
H SH
H2N N N O N N NH2
NH2 N N NH2 N N HN
H H2N N
O O H O
HS HS
N SH N H
SH HN N

Reversible binding of functional groups

N N
SH NH2
HS O O O
O H
H2N N N N N N
H N N NH2 H 2N H N N
H2N N N H
N N NH2 O SH HS
H HS
O HN
SH HN O N
NH O N HS
NH NH NH2
O
NH SH

In the near future, investigation will be done in the use of oxime

O SH NH2
H2N SH
NH2 SH O NH2
O O NH NH NH SH
O SH
NH2 SH O
H2 N
H2N NH2

G1-Cys4 G2-Cys8 G3-Cys16

728.4 1598.4 3337.1 chemistry to reversible link drugs to the dendrimer. While lowering
the pH (like in endosomes), the oxime-bond will be hydrolyzed and
Scheme 1: Synthesis of Dendrimers drugs will be released.
O OH O O
O OH N

Thioester Peptide Synthesis (tBoc-mediated SPPS)

OH
OH
OH
moderate pH OH
O
OCH3 O OH O H2 N
low pH OCH3 O OH O
CH3
O CH3
OH
1. Boc-Leu-OH, HBTU, DIEA, DMF 1. Trt-MPA-OH, HBTU, DIEA, DMF NH2 OH
2. TFA H2N 2. TFA, iPr3SiH, H2O NH2
N
H2N H

Scheme 3: Oxime Chemistry

Dendrimers can be modified with a number of peptides via Native
O R O
H 1. Boc-AA-OH, HBTU, DIEA, DMF H
HS N 2. TFA S N
N H2N N
H H
O O O
Chemical Ligation and subsequently with other functional groups
O R O
via a maleimide-thiol reaction. In the near future, the biomedical
Repetitive addition H
of amino acids
followed by
N
H
O
S

O
N
NH2
= [AA]n-MPAL applicability will be tested for several of these systems. Also, we
cleavage with HF n
will look into the possibility of attaching functionalities in a
Purification by reversed phase HPLC
reversible way.
Scheme 2: Synthesis of thioester on C-terminus of peptides