A Facile Synthesis of NODASA-Functionalized Peptide

Article abstract of DOI:10.1055/s-0035-1561970

Herein, we report a mild and efficient synthesis of a NODASA-functionalized peptide, which was initiated with a Michael addition reaction between monomethyl fumarate and 1,4,7-triazacyclononane.

Full text of DOI:10.1055/s-0035-1561970

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©
Georg Thieme Verlag Stuttgart · New York
2016, 27, A–D
A
letter
Synlett
J. Dutta et al.
Letter
A Facile Synthesis of NODASA-Functionalized Peptide
Jyotibon Duttaa
Praveen K. Chinthakindia
H
O
N
OH
Peptide
Per I. Arvidssona,c
Beatriz G. de la Torrea
Hendrik G. Krugera
O
N
N
H
Ga
NH
HN
N
N
O
Thavendran Govendera
HO
O
OH
Tricia Naicker*a
-
On- and off-resin synthetic approach
Fernando Albericio*a,b,d,e,f
- Potential PET imaging agent
-
-
Peptide-functionalized NODASA
Overall 84% yield
a
Catalysis and Peptide Research Unit, School of Health Sciences,
University of KwaZulu-Natal, Durban 4001, South Africa
School of Chemistry and Physics, University of KwaZulu-Natal,
b
Durban 4001, South Africa
naickert1@ukzn.ac.za
albericio@ukzn.ac.za
c
Science for Life Laboratory, Drug Discovery & Development
Platform & Division of Translational Medicine and Chemical
Biology, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden
d
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials
and Nanomedicine, Barcelona Science Park, 08028 Barcelona,
Spain
Department of Chemistry, College of Science, King Saud
e
University, P.O. Box 2455,Riyadh 11451, Saudi Arabia
Department of Organic Chemistry, University of Barcelona,
f
08028-Barcelona, Spain
Received: 28.01.2016
Accepted after revision: 07.03.2016
Published online: 30.03.2016
cost-effective alternative to the radionuclides produced by
4,7
a cyclotron.
Bifunctional chelators (BFC) are typically
DOI: 10.1055/s-0035-1561970; Art ID: st-2016-b0057-l
used for the development of radiolabeling; focusing on the
in vivo target specificity and specific metal-binding capa-
bilities. BFC contain two parts; one is a functional group
which can readily react with the targeting molecule (e.g.,
peptides, antibodies, and nanoparticles) as well as provide
a stable covalent bond to it and the other is to form a strong
complex with the metal ion ensuring its undesired release
within the recipient.8
Abstract Herein, we report a mild and efficient synthesis of a NODA-
SA-functionalized peptide, which was initiated with a Michael addition
reaction between monomethyl fumarate and 1,4,7-triazacyclononane.
Key words bifunctional chelators, NOTA, NODASA, PET, peptide
In radiopharmaceutics, chelators are small organic mol-
ecules that can form stable coordination complexes with
radiometals (radioactive isotopes of various metals) and
Significant development has been achieved in the field
of oncology in the context of radiolabeled peptides for tis-
1
9
can be delivered to the target of interest in vivo. Based on
sue localization and therapy. In comparison to small mole-
the type of radiometal-ion selectivity it can either be used
in disease diagnosis or therapeutics. In disease diagnosis;
metal ions comprising of positron- or γ-emitting radionu-
cules, antibodies, and proteins as a carrier, peptides show
distinctive advantages. They are reasonably lower molecu-
lar weight, easier to synthesize with the development of
solid-phase synthesis, compatible to couple with various
chelators, bind many significant biological targets, display
excellent tumor penetration, and shows rapid clearance
from the recipient. Moreover, as a therapeutic, it shows
minimal side effects compared to conventional drugs and it
is non immunogenic.10
2
clides and are used in positron emission tomography
3
,4
(
(
PET) and single-photon emission computed tomography
5
SPECT), respectively. The α- and β-ion generating radio-
isotopes are widely employed as therapeutics in cancer
6
treatment. Due to its noninvasive, quantitative, and highly
specific nature, PET imaging has become a powerful tool for
diagnostic or therapeutic purposes. Some of the common
A well-known BFC, 1,4,7-triazacyclononane-N′,N′,N′′-tri
acetic acid (NOTA); is a cyclic polyamino carboxylic ligand
68
radiometals which can be used in PET imaging are Ga,
6
4
86
89
44
1
64
Cu, Y, Zr, and Sc metal ions. Among various radiome-
which is one of the first and most efficient chelators of Cu
6
8
67/68
2,11
tals used for PET, Ga is of choice due to its availability
from enduring Ge/ Ga generator systems which offers a
and
Ga,
therefore much research and development
68
68
2
into its derivatives has been done. Contrary to its populari-
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D

B
Synlett
J. Dutta et al.
Letter
ty, a large excess of NOTA is required for the complexation
with a peptide (Figure 1) as well as the high cost associated
with its commercial form.12
reaction (Scheme 2). Subsequently, monomethyl fumarate
was attached to the peptide on resin, and the addition with
1,4,7-triazacyclononane was attempted, however, this reac-
tion did not yield the product.
COOH (NOTA)
R
N
N
H
HN
NHHN
COOH
(NODASA)
NH
N
OH
O
Br
O
O
N
N
COOH
COOH (NODAGA)
O
O
R
N
R
NH
H
O
O
O
OH
expected product
R = Bn
O
HOOC
O
O
Figure 1 NOTA, NODASA, and NODAGA
O
R
N
R
N
H
DIPEA
H
O
O
N
H
R = Me
NH
HN
NOTA derivatives namely NODASA and NODAGA (Figure
1
) contain an additional carboxylic acid moiety within the
macrocycle allowing the core to better saturate the hexa-
dentate coordination of the metal ion as well as provide a
Scheme 2
9
site for attachment of a peptide.
Next, a Michael addition reaction between monomethyl
fumarate and 1,4,7-triazacyclononane (Scheme 3) was car-
ried out in solution. To our delight, the reaction proceeded
to the desired product and proved to be regioselective for
the C3 position of the alkane and was confirmed using 2D
NMR spectroscopy. It was recrystallized in DMF with a yield
of 94%. This product (1) was also confirmed by LC–MS (pos-
itive mode, at m/z = 260). In order to prevent self-polymer-
ization of 1 during the amide coupling reaction, protection
of the free secondary amines with Fmoc-OSu was carried
out.
Compound 2 was then easily coupled to the model pep-
tide YGGF with standard coupling reagents, DIC/Oxyma-
Pure. The reaction was monitored by cleaving a small por-
tion of the resin and further analysis via HPLC. Complete
conversion into the desired diastereomeric product was ob-
served at m/z = 1127. It was then subject to a standard Fmoc
deprotection to yield compound 3 which was monitored by
HPLC and confirmed by LC–MS.
CO2CHPh2
Ph2HCO2C
CO2H
HO2C
Br
N
NH
HN
hydrolysis
N
H
NHHN
HO2C
HO2C
CO2H
CO2H
N
N
+
NH
N
O
CO2H
NH
HN
HO2C
O
Br CO2CH2Ph
hydrolysis
Scheme 1 NODASA13 and NODAGA14 synthesis
In literature noncommercially available bis(diphenyl
methyl) d,l-bromosuccinate and α-bromoglutaric acid 1-
tert-butyl ester 5-benzyl are typically described for the
synthesis of NODASA and NODAGA, respectively (Scheme
13
14
1
). Hence, there is a need to provide a more convenient syn-
The resulting free secondary amines were then success-
fully alkylated on resin with tert-butyl bromoacetate in the
presence of DIPEA. Complete conversion into the product
was observed using analytical HPLC and LC–MS (positive
mode) and showed its corresponding m/z = 799 for com-
pound 4. Thereafter, base hydrolysis of the ester was
achieved on resin with LiOH in THF–MeOH to furnish deriv-
ative 5 which was confirmed from analytical HPLC with
100% conversion of the ester. The tert-butyl groups were re-
moved simultaneously with cleavage of the peptide from
thetic approach for these functionalized chelator deriva-
tives and their coupling to peptides. Herein, we report a
new and facile synthetic route for the preparation of pep-
tide-functionalized NODASA on solid phase from readily
available and cheaper starting material.
The model peptide, YGGF from the parent peptide YGGFL,
is a part of the enkephalin sequence. Coupling of each ami-
no acid was carried out with HBTU/DIPEA in DMF and each
step was monitored by the ninhydrin test. Single coupling
for one hour with excess of Fmoc-protected amino acids
were sufficient for completion of the reaction. The pure
peptide product was observed in analytical HPLC when a
small portion of the peptide was cleaved from the resin.
Initially, the NODASA synthesis was carried out with the
coupling of 4-(benzyloxy)-3-bromo-4-oxobutanoic acid to
a peptide on resin followed by addition of 1,4,7-triazacyclo-
nonane; this was unsuccessful due to the HBr elimination
the resin using TFA/H O/TIS. The final product 6 was ana-
2
lyzed by analytical HPLC followed by LC–MS characteriza-
tion. Analytical HPLC showed 100% conversion into the
product with its corresponding m/z = 785 and an isolated
yield of 84%.15
An application of the conjugated functionalized peptide
product was metal chelation with cold gallium. A period of
30 minutes at room temperature was adequate for GaCl in
3
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Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D

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Synlett
J. Dutta et al.
Letter
H
O
N
OH
R
O
N
N
O
N
O
Peptide
1
. DIC/OxymaPure, DMF
Peptide
O
N
1
. CH2Cl2/DIPEA
H
O
O
O
+
NH
OH
HN
2
. Fmoc-OSu/NaHCO3
2. 20% piperdine/
0.5 M HOBt in DMF
(quantative yield)
O
N
NHHN
R
1
2
R = H (94% yield)
R = Fmoc (82% yield)
3
t-Bu-Br-acetate/DIPEA in NMP
quantative yield)
(
H
H
H
O
N
O
N
O
N
OH
OH
O
Peptide
Peptide
Peptide
LiOH/THF, MeOH
(quantative yield)
O
95% TFA/2.5% H O/2.5% TIS
O
O
2
N
N
N
84% yield
N
O
N
N
O
N
O
N
O
N
O
HO
O
OH
O
O
O
O
6
5
4
Scheme 3
the presence of NaOAc for the complete complexation of
NODASA–YGGF (6). The stability of gallium complexation
was further analyzed by 500-fold excess of EDTA. However,
EDTA was challenged for 0, 30, 60, 120, 180, and 240 min
showing no significant release of gallium from the complex
confirming the resistance towards trans chelation.
In this study we described a facile seven-step synthesis
and purification of NODASA with a model peptide. The
combination of on- and off-resin synthetic approach was
employed. We also successfully demonstrated the efficient
conjugation of cold gallium to this NODASA–YGGF chelator
as a potential PET imaging agent. The synthetic route pro-
vides a cheap and simple alternative to commercially avail-
able functionalized NODASA in the current market and
could be applied to various peptides of choice.
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(
(
(
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1
Acknowledgment
(
This work was funded in part by the following: National Research
Foundation (NRF) and the University of KwaZulu-Natal. Luxembourg
Biotech Ltd. is acknowledged for the generous gift of coupling re-
agents.
(10) (a) Liu, S. Chem. Soc. Rev. 2004, 33, 445. (b) Fani, M.; Maecke, H.
R.; Okarvi, S. M. Theranostics 2012, 2, 481.
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Supporting Information
(
(
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1561970.
S
u
p
p
ortioIgnfrm oaitn
S
u
p
p
ortioIgnfrm oaitn
14) Eisenwiener, K.-P.; Prata, M. I. M.; Buschmann, I.; Zhang, H.-W.;
Santos, A. C.; Wenger, S.; Reubi, J. C.; Mäcke, H. R. Bioconjug.
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References and Notes
(15) General Procedure for the Synthesis of 1-Amino-2-benzyl-
1
5-[4,7-bis(2-tert-butoxy-2-oxoethyl)-1,4,7-triazonan-1-yl]-
(
1) Price, E. W.; Orvig, C. Chem. Soc. Rev. 2014, 43, 260.
11-(4-hydroxybenzyl)-1,4,7,10,13-pentaoxo-3,6,9,12-tetra-
(2) Lattuada, L.; Barge, A.; Cravotto, G.; Giovenzana, G. B.; Tei, L.
azahexadecan-16-oic Acid (5)
The functionalized peptide on resin 4; 0.0125 mmol was
swelled in 1.0 ml CH Cl for 5 min followed by filtration, and 1.0
Chem. Soc. Rev. 2011, 40, 3019.
2
2
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D

D
Synlett
J. Dutta et al.
Letter
mL of 1 M LiOH (dissolved in MeOH and THF in 1:1 ratio) was
added. The reaction was carried out for a period of 30 min at
room temperature. The completion of the reaction was moni-
tored by cleaving an aliquot of the compound from the resin
and checked by LC–MS as well as analytical HPLC. The resin was
washed with about 5.0 mL of THF (2×), DMF (2×), and CH Cl
was evaporated with the aid of N2 gas bubbling through the
mixture. The peptide was then precipitated in 5.0 mL of ice-cold
Et O. The precipitated peptide was centrifuged and the Et O
2
2
solution was decanted. It was then dissolved in 1.0 mL of water
and freeze dried without further purification which gave a yield
of 84%. The purity of the synthesis was checked by analytical
RP-HPLC which showed 100% purity and characterized by LC–
2
2
(
2×) consecutively. Compound 5 (0.0125 mmol) was depro-
tected and cleaved from the resin using a cocktail of 1.0 mL
TFA/H O/thioanisole (95:2.5:2.5) over h. The resin was
removed by filtration and washed with 1 mL TFA. Further, TFA
+
MS. HRMS (ESI ): m/z calcd. for C36H49N O [M + H]: 785.3464;
8
12
2
found: 785.3434.
2
©
Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, A–D