Solid-phase synthesis and fluorine-18 radiolabeling of cycloRGDyK

Article abstract of DOI:10.1039/c6ob01636g

Solid-phase peptide synthesis, head-to-tail cyclization, and subsequent radiolabeling provided a reproducible, simple, rapid synthetic method to generate the cyclic peptide radiotracer cRGDyK([¹⁸F]FBA). Herein is reported the first on-resin cyclization and ¹⁸F-radiolabeling of a cyclic peptide (cRGDyK) in an overall peptide synthesis yield of 88% (cRGDyK(NH₂)) and subsequent radiolabeling yield of 14 ± 2% (decay corrected, n = 4). This approach is generally applicable to the development of an automated process for the synthesis of cyclic radiolabeled peptides for positron emission tomography (PET).

Full text of DOI:10.1039/c6ob01636g

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Solid-Phase Synthesis and Fluorine-18 Radiolabeling of CycloRGDyK.
Ryan A. Davis,a-c Kevin Lau,a,b Sven H. Hausner,a-c and Julie L. Sutcliffea-d*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
(109.77 min).[2] Thus, developing methods to rapidly synthesize
radiolabeled peptides is important to facilitate rapid radiotracer
development and ultimately translation to the clinic.[1-4]
The arginine-glycine-aspartic acid (RGD)-containing peptides,
known to target αv-integrins have emerged as promising peptide
radiotracers for imaging many diseases especially cancer where
over expression of αv-integrins is associated with more aggressive
phenotypes.[3-6] Unfortunately, linear RGD-peptides can be prone to
proteolytic degradation. Efforts have been made to cyclize them to
improve in vivo stability and binding selectivity. Kessler and co-
workers’ pioneered the study of cyclopentapeptide cRGDfV (1),
which has high affinity and selectivity for the αvβ3-integrin.[7] The N-
methylated valine version of this peptide is now marketed by Merck
under the tradename Cilengitide and has shown initial promise as
an αvβ3-targeted antiangiogenic therapy.[3] Various amino acid
replacements and modifications, illustrated in Figure 1, have been
Solid-phase peptide synthesis, head-to-tail cyclization, and
subsequent radiolabeling provided a reproducible, simple,
rapid synthetic method to generate the cyclic peptide
radiotracer cRGDyK([18F]FBA). Herein is reported the first on-
resin cyclization and 18F-radiolabeling of a cyclic peptide
(cRGDyK) in an overall peptide synthesis yield of 88%
(cRGDyK(NH2)) and subsequent radiolabeling yield of 14 ± 2%
(decay corrected, n = 4). This approach is generally applicable
to the development of an automated process for the synthesis
of cyclic radiolabeled peptides for positron emission
tomography (PET).
studied in attempts to further enhance this cyclopentapeptide’s
[3,8-11]
properties,
including an 18F-labeled dimeric derivative of 4.[12]
Several approaches to the radiolabeling of cRGDyK (4) have been
reported including: the use of activated ester N-succinimidyl-4-
PET is a minimally invasive imaging technique that has greatly
improved medical diagnosis through its ability to detect, diagnose,
and follow treatment of cancer and various other diseases by
providing three-dimensional (tomographic) whole-body imaging.[1]
To visualize the extent of disease in vivo with PET, a radiotracer is
injected, typically intravenously. Peptide-based radiotracers are
becoming a major focus because of the ready ability to be tuned for
specificity, affinity, and desirable pharmacokinetic properties,
allowing imaging as early as 1 hour post-injection.[2] The biological
half-life of peptides also matches the radioactive half-life of 18F
[
18F]-fluorobenzoate ([18F]SFB,[13] activated ester p-nitrophenyl-2-
fluoropropionate ([18F]NFP,[11] conjugation of 2-[18F]-fluorodeoxy-
glucose ([18F]FDG),[14] and reaction of aldehydes to form oximes.[15]
NH2
HN
NH2
NH
NH
HN
O
O
O
N
H
O
NH
HN
OH
N
O
O
H
HO
NH
HN
OH
O
HO
NH HN
O
N
H
O
NH HN
O
O
O
R'
O
HO
O
R
NH2
1
5
)
cRGDfV ( ), R = CH2Ph,
R' = CH(CH3
R' = (CH2)4NH2
)
cRGDfK(galacturonopyranoside)(
2
a.Radiochemistry Research and Training Facility
cRGDfK ( ), R = CH2Ph,
2
cRGDyV (3), R = CH2PhOH, R' = CH(CH3)2
b.Department of Biomedical Engineering
4
cRGDyK ( ), R = CH2PhOH, R' = (CH2)4NH2
c. Department of Internal Medicine, Division of Hematology and Oncology
d.Center for Molecular and Genomic Imaging
Figure 1. CycloRGD pentapeptides.
University of California, Davis, 2921 Stockton Blvd. Sacramento, CA 95817, USA
*Email: jlsutcliffe@ucdavis.edu, FAX: 1-916-734-7572, Phone: 1-916-734-5536.
Electronic Supplementary Information (ESI) available: [NMR, ESI-HRMS, and HPLC].
See DOI: 10.1039/x0xx00000x
The synthesis of cyclic peptides has required head-to-tail
cyclizations either done in solution phase or using solid phase
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peptide synthesis (SPPS).[16-20] These cyclizations often required very cleavage as used by the majority of researchers doing on-resin-
cyclizations with 4-methylbenzhydryl- amine resin (MBHA) and (2)
to help increase cleavage yield while maintaining short cleavage
times following the on-resin radiolabeling step.[13-16] The resin was
preloaded with the aspartic acid (D), connected via its side-chain
and protected as the allyl ester for orthogonal deprotection and
cyclization. The use of an orthogonally 1-(4,4-dimethyl-2,6-
dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)-sidechain protected
dilute conditions, large excess of carboxylic acid activating agents
(3-20 equivalents), and long reaction times varying from 6-18 hours
up to 5-10 days.[16-20] The reactions, were plagued with side
reactions, were poor yielding, lacked the ability to be readily
modified for radioisotope incorporation, and in most cases,
required multiple purifications. Most synthetic methods towards
cyclic peptides suffered from cyclodimerization, oligomerization, or
racemization of the C-terminal residue.[16-20] Furthermore,
aspartimide formation, N-acetylation, piperidine amide formation,
and tetramethylguanidinium formation were also observed as
byproducts during cyclic peptide synthesis.[16-20]
lysine provided
a handle for attachment of the radiolabel.
Protected resin-bound linear yKRGD pentapeptide 6 (Scheme 1)
was built by reiterative coupling and deFmocing using 3 equivalents
of N-terminally Fmoc-protected amino acids, activated with HATU
(2.8 equiv) and diisopropylethylamine (DIPEA, 6 equiv) for coupling,
and subsequent deprotection with 20% piperidine in N,N-
dimethylformamide (DMF). After completion of 6, the allyl ester of
aspartic acid (D) was removed by tetrakis (triphenylphosphine)
palladium(0) and triphenylsilane, followed by the removal of the N-
terminal Fmoc. A small-scale test-cleavage, was performed in order
to characterize the partially-deprotected linear-peptide of 6 (allyl
ester and Fmoc removed); it also, revealed that the ivDde-group
In order to streamline the synthesis, researchers have focused on
the solid phase cyclization step. Cyclopentapeptide, cRGDfK (2) was
cyclized on solid support using
2
equivalents of 1-
[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluoro-phosphate (HATU) and a reaction time of 16
hours to afford
a a fully
46% yield.[21] Since this synthesis,
migrated to the N-terminus during the acidic cleaveage.[41]
A
automated synthesis and cyclization of RGD-peptides has been
demonstrated, resulting in 40% yields. However, the automated
reanalysis by HPLC of the purified linear-NH2-yK(ivDde)-RGD
exhibited no further ivDde migration, providing further evidence
procedure required a total of 8 equivalents of activating agent and that the cleavage conditions were responsible for the ivDde-
migration event.[41] Fortunately, this was only relevant to the test
cleavage as the final radiolabeled cyclic peptide would be fully
constructed before encountering the acidic cleavage solution.
cyclization times between 2-6 hours.[20]
The use of SPPS in radiochemistry has been beneficial for site-
specific addition of a ‘prosthetic group’ a small synthon to which
the radioisotope is attached to in the first step, followed by the
coupling with a peptide in a subsequent step.[22-24] Among the wide
range of prosthetic groups available,[22,23] 18F]fluoropropionic acid
[
([18F]FPA) and [18F]fluorbenzoic acid ([18F]FBA), or their respective
activated esters [18F]NFP, and [18F]SFB are commonly used to
accomplish peptide radiolabeling.[22-27] Recent advances to the
introduction of fluorine-18 into peptides include click chemistry,
particularly the 1,3-dipolar cycloaddition reaction[28] and its copper-
free derivatives[29,30] utilizing ring-strain promoted cycloadditions
between cyclooctynes and azides or the use of the inverse-electron
demand Diels-Alder reactions between tetrazines and the strained
trans-cyclooctene.[31,32] Additionally, fluoride capturing reactions
based on peptide modifications allow for stable peptide-Si-18F,
peptide-B-18F, and peptide-chelator-Al-18F bonds to be generated
rapidly and efficiently.[23,33-38] Collectively, these studies indicate a
correlation between the prosthetic group and the biodistribution of
the radiotracer,[39,40] highlighting the importance of the ability to
test different prosthetic groups on a radiolabeled peptide.
Peptide radiolabeling on solid support has been carried out
successfully in the Sutcliffe laboratory with the goal to reduce the
overall synthetic complexity and number of purifications steps by
allowing excess reagents and byproducts to be rinsed away.[25-27]
Additionally, SPPS provides a pseudodilution effect which can aid
with cyclization, and permit radiolabeling of small amounts of
peptide, a current challenge of fluorine-18 radiochemistry. In
addition, the solid phase synthetic strategy is amenable to
automation. Thus, to further evaluate the capabilities of SPPS-
based chemistries for the entire synthetic preparation of peptide
radiotracers, this paper describes the use of SPPS-technology to
synthesize, cyclize, and radiolabel the clinically relevant cyclic
peptide cRGDyK (4).
Scheme 1. Synthesis of cRGDyK(4).
The on-resin cyclization of partially-deprotected linear-peptide of
6 was done via activation with a single equivalent of HATU and two
equivalents of DIPEA to minimize capping and possible
dimer/oligomerization byproducts (Scheme 1). The on-resin
cyclization was evaluated by removing the peptide from the resin
with a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIPS),
and water (95/2.5/2.5) to afford compound 7, which was then
converted to cRGDyK (4) by deprotection of lysine’s ivDde-group
with 2% hydrazine in solution in a nearly quantitative manner.
Alternatively, preparation of 4 was also confirmed by first removing
the ivDde-group with 2%-hydrazine in DMF, followed by cleavage
from the resin and concomitant removal of other side-chain
protecting groups using TFA-TIPS-water solution to attain 4 in the
same yield (98%) (Scheme 1).
A series of test reactions were performed to monitor the kinetics
of the solid-support cyclization and formation of ivDde-protected
cRGDyK (7) following removal from the resin and HPLC analysis.[41]
Contrary to literature data,[16-21] the on-resin-cyclization reaction
was very rapid with almost complete transformation of 6 to 7 after
5 min with no observable byproduct formation.[41] Cyclized product
(7) was obtained in this manner and purified with an overall yield of
90% as determined from the initial loading capacity of the resin.
Compound 7 was confirmed by mass spectroscopy and NMR-
The synthesis of 4 was carried out using fluorenylmethyloxy-
carbonyl (Fmoc)-chemistry to build the peptide with acid labile side-
chain protecting groups on acid sensitive resin (Novasyn®TGA). This
resin was selected to (1) avoid the harsh hydrofluoric acid (HF)
2 | J. Name., 2012, 00, 1-3
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analysis and conclusively deduced using heteronuclear multiple- cRGDyK([19F]FBA) 9 in a competition assay against iodine-125
bond correlation spectroscopy (HMBC) (Figure 2). The HMBC-NMR
labeled 125I-c(RGDyK) to primary human brain capillary endothelial
spectrum of 7 confirms the point of ring closure for the on-resin cells, which was also measured to be 17 nM.[43] Furthermore, for
cRGDyK (4), an IC50 of 4 nM is reported in the literature,[44] which is
cyclization by exhibiting heteronuclear coupling between the
α-
in close agreement to our obtained value of 1 nM for the same
compound.
proton of tyrosine and the carbonyl carbon of aspartic acid. The
HMBC-NMR spectrum further shows the 3-bonding coupling
For the synthesis of radiolabeled cRGDyK([18F]FBA) (10) (Scheme
3), [18F]FBA was synthesized from ethyl-4-(trimethylammonium
triflate) benzoate by reacting [18F]-fluoride in the presence of
through the heteroatom-nitrogen from the ε-protons of lysine’s
cryptand,
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-
hexacosane (KryptofixTM [K222]) and potassium carbonate at 100
̊C
in dimethylsulfoxide (DMSO) for 15 minutes (Scheme 3), followed
by hydrolysis of the ethyl ester with sodium hydroxide solution (0.5
M) to give [18F]FBA. The generated [18F]FBA was quenched with
hydrochloric acid, cartridge purified (C18-SepPak), concentrated,
Figure 2. HMBC-spectrum of compound
carbon coupling to -protons of lysine and the carbonyl carbon of aspartic acid
coupled to the -proton of tyrosine.
7
. The spectrum shows the ivDde sp2-
ε
α
Scheme 3. Synthesis of [18F]FBA and radiolabeled cRGDyK([18F]FBA)(10).
side-chain to the sp2-hybridzed carbon of ivDde group, indicating
that the cyclized product does contain the lysine ivDde protection.
For on-resin radiolabeling and the preparation of the non-
radioactive cold fluorine-19 standard, upon cyclization of 6 the
ivDde-group of lysine was removed with 2% hydrazine in DMF to
and added to the 5 mg of pre-swelled peptidyl resin (8) (0.49 mg,
0.79 µmol of peptide (8)).[27] Following our well established solid-
phase radiolabeling procedure for linear peptides,[27] a brief 30
minute coupling of [18F]FBA (96-150 mCi) to 8 via activation of
[
18F]FBA in situ by HATU (5 mg) and DIPEA (10 µL) and a short 30
obtain the cyclic peptide, with the free
ε-amine of lysine (8), still
minute cleavage/deprotection with TFA-TIPS mixture, generated
cRGDyK([18F]FBA) (10) in 14 ± 2% (n = 4) decay corrected yield.
Compound 10 was obtained in >99% radiochemical purity after
purification by reverse-phase HPLC in a synthesis time of 90
minutes from [18F]FBA. Notably, even before purification, crude
product 10 did not contain radioactive byproducts (Figure 3).
attached to the resin (Scheme 2). Compound 8 was first coupled to
Scheme 2. Synthesis of cold standard cRGDyK([19F]FBA)(
9).
non-radioactive 4-[19F]-fluoro-benzoic acid ([19F]FBA) to provide a
cold standard to confirm the identity of the
18F]-peptide
radiolabeled with [
18F]FBA. Upon coupling 19F]FBA, global radioactive (black) and UV (220 nm, blue) HPLC trace of 10, co-injected with cold
Figure 3. A. Semi-preparative radioactive HPLC trace of crude 10
.
B. Analytical
C. Analytical
[
radioactive (black) and UV (220 nm, blue) HPLC trace of purified 10
.
[
standard 9.
deprotection and simultaneous removal of the peptide from the
solid support with TFA-TIPS solution, the non-radioactive product 9
was obtained in 80% yield based on the original loading capacity of
the resin (Scheme 2). In addition to being used as a cold standard,
cRGDyK([19F]FBA) 9 was also evaluated by a competitive ELISA for
binding to integrin αvβ3 in the presences of the natural ligand
vitronectin.[42] As expected, 9 exhibited good affinity to integrin
αvβ3 with an IC50 = 17 nM. This value matches the one reported for
Taken together the overall synthetic approach, including rapid
on-resin cyclization and radiolabeling, followed by a single HPLC
purification
cRGDyK([18F]FBA) (10) in excellent purity and high specific activity of
240±14 GBq/ mol. The radiolabeling results obtained here,
provided
the
cyclic
radiolabeled
peptide
µ
compared well to other previously reported radiolabeling reactions
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of cyclic-RGD peptides. Radiolabeling of 5 with [18F]NFP, proceeded
in a 29±5% overall radiochemical yield after decay correction with a
200±18 minutes total reaction time including HPLC purification and
required a considerable amount of peptide (5 mgs, 5.7µmol).[11] The
solution-phase radiolabeling of cRGDyK(4) with [18F]FDG to make a
derivative similar to galactoRGD(5) was done by modifying the
lysine sidechain by attachment of a Boc-aminooxy acetic acid to the
ε-amine. Upon removal of the Boc-group, the oxyamine could react
with the sugar aldehyde of [18F]FDG.[14] However, this radiolabeling
required 1 hour at a low pH = 1.5-2.5 and a temperature of 100 C
to obtain an isomeric mixture of E/Z-oximes in 41% decay-corrected
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yield.[14] Similarly,
a
novel aldehyde,
[
18F]-fluoro-PEG-
nicotinicaldehyde prepared in four steps has been coupled to
various cyclic peptide oxyamines in TFA/water solution at 70 C for
30 min to afford the peptide oximes with radiochemical decay
corrected yields ranging from 21-35%.[15] These methods, however,
used between 2-5 mg of purified peptide. The most comparable
radiolabeling method to the one reported here was carried out by
Chen and co-workers where the ε-amine of the lysine in cRGDyK(4)
(0.1 mg, purified peptide) was labeled with [18F]SFB in less than 2
hours with 20-25% decay corrected yield and specific activity of 230
GBq/µmol.[13]
18. J. A. Camarero, J. J. Cair
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1
23. O. Jacobson, D. O. Kiesewetter, X. Chen, Bioconjugate
Chem., 2015, 26, 1-18.
In conclusion, the presented study has synthesized cRGDyK (4),
completely on solid support and demonstrated rapid, efficient and
convenient on-resin cyclization, in high purity. The presented SPPS
and cyclization has circumvented formation of byproducts that have
been observed in previous head-to-tail cyclizations: piperidine
24. S. M. Okarvi, Eur. J. Nucl. Med., 2001, 28, 929-938.
25. J. Marik, S. H. Hausner, L. A. Fix, M. J. Gagnon, and J. L.
Sutcliffe, Bioconjugate Chem., 2006, 17, 1017-1021.
26. J. L. Sutcliffe-Goulden, et al., Eur. J. Nucl. Med. Mol. Imag.,
2002, 29, 754-759.
amide formation, N-acetylation, aspartimide
formation,
27. S. H. Hausner, N. Bauer, L. Y. Hu, L. M. Knight, and J. L. Sutcliffe,
J. Nucl. Med., 2015, 56, 784-790.
28. J. Marik, and J. L. Sutcliffe, Tetrahedron Lett., 2006, 47, 6681-
6684.
29. R. D. Carpenter, S. H. Hausner, and J. L. Sutcliffe, ACS Med.
Chem. Lett., 2011, 2, 885-889.
30. J. Dommerholt, et al., Angew. Chem. Int. Ed., 2010, 49, 9422-
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31. R. Rossin, et al., Angew. Chem. Int. Ed., 2010, 49, 3375-3378.
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cyclodimerizations, and oligiomerizations as well as capping due to
large excess of uronium derived activating agents.[16-20] In addition
the new method improved on previous low yields, shortened the
cyclization time, and allows for the precursor peptide to remain
fully protected on-resin until after the last synthetic step of
radiolabeling, which does not require low pH or harsh conditions
and conserves precious and potentially costly peptide material
allowing simple purification by a single HPLC injection. More
importantly, this procedure is amendable to automation of cyclic-
radiolabeled peptides with the ability to readily change the
prosthetic group allowing for fine tuning of pharmacokinetic
properties. The ability of SPPS to be adapted for continuous-flow
systems and automated peptide synthesis allows further
exploitation of this technology to the synthesis of radiolabeled
peptides with increasing complexity, where rapid site-specific
incorporation of the radioisotope is required. To date, this study is
to our knowledge the first reported fully on-resin cyclization and
radiolabeling of a cyclic-peptide radiotracer. This solid-support
cyclization and radiolabeling can be automated for future
application of other cyclic-radiolabeled peptides for PET-imaging.
40. S. Richter, et. al., Bioconjugate Chem., 2015, 26, 201-212.
41. See Supporting Information.
Notes and references
This work was supported by the Office of Science, United States
Department of Energy Grant: DE-SC0008385. Grant for 800MHz
instruments from NIH: NIH PR1973.
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