The backbone N-(4-azidobutyl) linker for the preparation of peptide chimera

Article abstract of DOI:10.1021/ol402150m

A robust synthetic strategy for the introduction of the N-(4-azidobutyl) linker into peptides using standard SPPS techniques is described. Based on the example of Cilengitide it is shown that the N-(4-azidobutyl) group exerts similar conformational restra

Full text of DOI:10.1021/ol402150m

ORGANIC
LETTERS
2013
Vol. 15, No. 17
4572–4575
The Backbone N‑(4-Azidobutyl) Linker for
the Preparation of Peptide Chimera#
†,‡,§
†
†
ꢀ
ꢀ
´
Jesus Garcıa, Jaume Adan, David Meunier,
Ana I. Fernandez-Llamazares,
Francesc Mitjans, Jan Spengler,*,†,‡,§ and Fernando Albericio*,†,‡,§,^
Institute for Research in Biomedicine (IRB), PCB, 08028-Barcelona, Spain,
CIBER-BBN, PCB, 08028-Barcelona, Spain, Biomed Division, Leitat Technological
Center Institution, PCB, 08028-Barcelona, Spain, Department of Organic Chemistry,
University of Barcelona, 08028-Barcelona, Spain, and School of Chemistry,
University of KwaZulu Natal, 4001-Durban, South Africa
albericio@irbbarcelona.org; jan.spengler@irbbarcelona.org
Received July 29, 2013
ABSTRACT
A robust synthetic strategy for the introduction of the N-(4-azidobutyl) linker into peptides using standard SPPS techniques is described. Based
on the example of Cilengitide it is shown that the N-(4-azidobutyl) group exerts similar conformational restraints as a backbone N-Me group and
allows conjugation of a desired molecule either via click chemistry or;after azide reduction;via acylation or reductive alkylation.
The site-specific covalent attachment of “unnatural”
moieties, such as fluorophores, radiolabels, affinity labels,
or polymers, to peptides has proven useful for a wide
variety of applications.1 The conjugation of peptides with
a desired molecule is typically performed at the N-terminus
or at naturally occurring side-chain functional groups.2
Cyclic peptides or even some linear peptides without
derivatizable groups often require the introduction of
additional residues, such as Lys or Cys, to support side-
chain-selective conjugation. In the case of cyclic peptides,
additionalamino acidscannot be introduced, and findinga
suitable position for amino acid replacement is not straight-
forward. Along these lines, bio-orthogonal conjugation
methods that target unnatural amino acids are becoming
valuable alternatives to the more commonly used Lys- and
Cys-based strategies, as they do not involve cumbersome
protection and deprotection protocols.3 However, each of
the techniques currently available for peptide modification
have specific drawbacks, and generally there is a lack of
widely usable and flexible methods.
We envisaged that modification of the backbone amide
groups with a functionalized N-substituent may be a
valuable addition to the chemist’s toolbox to perform pep-
tide conjugation. Kessler’s group has shown that the
introduction of N-Me groups in peptide ligands can opti-
mize their activity and receptor selectivity as a result
of conformational modulation.4 Furthermore, backbone
N-Me groups are common structural motifs in many
bioactive peptides isolated from natural sources.4
# Dedicated to Professor Klaus Burger on the occasion of his 75th
birthday.
† Institute for Research in Biomedicine (IRB), PCB.
‡ CIBER-BBN, PCB.
Here we describe the N-(4-azidobutyl) group as linker
for the attachment of molecules. This N-substituent can be
Leitat Technological Center Institution, PCB.
§ University of Barcelona.
^ University of KwaZulu Natal.
(3) (a) Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10,
253–262. (b) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009,
48, 6974–6998.
(4) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem.
Res. 2008, 10, 1331–1342.
(1) (a) Lee, S.; Xie, J.; Chen, X. Biochemistry 2010, 49, 1364–1376.
(b) Shu, J. Y.; Panganiban, B.; Xu, T. Annu. Rev. Phys. Chem. 2013, 64,
631–657.
(2) Gauthier, M. A.; Klok, H.-A. Chem. Commun. 2008, 2591–2611.
r
10.1021/ol402150m
Published on Web 08/22/2013
2013 American Chemical Society

the five amino acids in its cyclic structure, three (RGD) are
essential for binding to the receptor, D-Phe is involved in
hydrophobic interactions, and NMeVal has no derivatiz-
able functional group.8 Substitution of NMeVal by Lys led
to cyclo[RGDfK], one of the most conjugated peptide
ligands which is used in a number of biomedical applica-
tions.9 However, a decrease in biological activity has to be
taken into account when replacing NMeVal by Lys, as the
N-Me group of Val promotes constraints that stabilize the
RGD motif in its preferred Rvβ3-binding conformation.10
We report on the synthesis of an analog of cyclo-
[RGDfNMeV] (1) in which the N-Me group of Val is
replaced by the N-(4-azidobutyl) group (2), with minimal
perturbation of the original conformation. By preparing
various PEG conjugates from 2, we show that our linker
allows conjugation onto cyclic peptides under full conser-
vation of their amino acid sequence.
Scheme 1. Peptide Modification through the N-(4-Azidobutyl)
Linker
To obtain the N-azidoalkylated cyclopeptide (2), its
linear pentapeptide precursor (3) was prepared by stepwise
solid-phase peptide synthesis (SPPS) on 2-chlorotrityl
chloride (CTC) resin and then cleaved for subsequent
cyclization and side-chain deprotection (Scheme 2). Posi-
tioning of the N-alkylated residue in the middle of the
sequenceof 3 minimizes sterichindranceduring cyclization
and is expected to facilitate this process as a result of
backbone preorganization.11
introduced into a resin-bound peptide by reductive alkylation
with 4-azidobutanal, providing an azide onto which alkyne-
functionalized molecules can be grafted by Cu(I)-catalyzed
1,3-dipolar cycloaddition (Scheme 1). Alternatively, the azide
group can be reduced to an amine, onto which molecules can
be conjugated via amide bond formation or via reductive
alkylation. The azide function is stable to common deprotec-
tion protocols used in peptide synthesis and chemically inert
to side-chain functional groups,5 thereby minimizing side
reactions and simplifying protection schemes.
The N-(4-azidobutyl) group was introduced into the
resin-bound peptide by reductive alkylation with 4-azido-
butanal in the presence of NaBH3CN. The reaction was
tested with various amounts of aldehyde; with 1.5 equiv,
most N-terminal Val was exclusively N-monoalkylated.
Taking advantage of the low reactivity of this secondary
amine, the small amount of unreacted resin-bound pep-
tide was capped with Ac2O in order to facilitate the final
purification. The foreseeable challenging step was the
coupling of Fmoc-D-Phe onto N-(4-azidobutylated) Val.
The acylation of this sterically demanding residue did not
take place under conditions reported to be efficient for
coupling D-Phe onto NMeVal. Stronger activation meth-
ods, such as PyBOP/HOAt and HATU/HOAt, also failed
to form the desired product. Finally, this coupling was
achieved by activating Fmoc-D-Phe with bis(trichloromethyl)-
carbonate (BTC) in the presence of 2,4,6-trimethylpyridine.12
After three prolonged couplings (15 h), acylation was al-
most complete and no epimerization was detected (HPLC).
Further peptide elongation and cleavage afforded penta-
peptide 3, which was easy to cyclize with EDC and catalytic
amounts of 4-DMAP. The Pbf- and tBu- groups were then
removed, and RP-HPLC purification rendered 2 in 17%
overall yield.
A few years back, Kirshenbaum et al. showed that
N-azidopropyl groups are straightforward to incorporate
in peptoid sequences using an azido amine as a submonomer
reagent, and that azide-functionalized peptoids can be used
as substrates for azideꢀalkyne cycloaddition reactions.6
However, the submonomer approach is only efficient for
the preparation of N-substituted Gly oligomers. Also worth
mentioning is that there is no reported example in which a
peptide with a backbone N-azidoalkyl substituent has been
obtained.
To demonstrate the applicability of our N-(4-azidobutyl)
linker strategy, Cilengitide was chosen as a model. This
Arg-Gly-Asp (RGD)-peptide is a good example of the
difficulty involved in preparing conjugates of small cyclic
peptides that do not offer attachment sites and/or that are
not amenable to structural modification while preserving
biological activity. The RGD-cyclopeptide sequence of
Cilengitide, cyclo[RGDfNMeV], is the result of systematic
research to constrain the RGD motif in its optimum
conformation for binding to the Rvβ3-integrin receptor,
which is overexpressed in various malignant cancers
and in tumor neovasculature.6 The functionalization of
RGD-cyclopeptide ligands that target this receptor is of
great interest, as it allows the conjugation of suitable
chemical entities for tumor imaging and therapeutics.7
However, Cilengitide cannot be conjugated as it is. Among
(8) (a) Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. J. Acc.
Chem. Res. 2009, 42, 969–980. (b) Chen, X. Theranostics 2011, 1, 28–29.
(9) Mas-Moruno, C.; Rechenmacher, F.; Kessler, H. Anti-cancer
Agents Med. 2010, 10, 753–768.
(5) Brase, S.; Gil, C.; Knepper, K.; Zimmermann, V. Angew. Chem.,
Int. Ed. 2005, 44, 5518–5240.
(6) (a) Jang, H.; Fafarman, A.; Holub, J. M.; Kirshenbaum, K. Org.
Lett. 2005, 7, 1951–1954. (b) Holub, J. M.; Jang, H.; Kirshenbaum, K.
Org. Biomol. Chem. 2006, 4, 1497–1502.
(10) Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. J. Acc. Chem.
Res. 2009, 42, 969–980.
€
(11) Dechantsreiter, M. A.; Planker, E.; Matha, B.; Lohof, E.;
€
Holzemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. J. Med.
Chem. 1999, 42, 3033–3040.
(7) Liu, Z.; Wang, F.; Chen, X. Drug Dev. Res. 2008, 69, 329–339.
(12) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509–524.
Org. Lett., Vol. 15, No. 17, 2013
4573

Scheme 2. Small-Scale Synthesis of
Cyclo[RGDf(N-CH2CH2CH2CH2N3)V] (2)
Scheme 3. Large-Scale Synthesis of
Cyclo[RGDf(N-CH2CH2CH2CH2N3)V] (2)
that the acylation of the N-alkylated residue was achieved
on a small scale using BTC, we consider that the detour
from solid-phase to solution chemistry in the synthesis of
2 was necessary because of the additional steric hindrance
exerted by the β-branched side chain of Val and that this
change may not be a general requirement for the synthesis
of other N-(4-azidobutylated) peptides.
It is reasonable to assume that the incorporation of an
N-(4-azidobutyl) group into a cyclic peptide will exert
the same conformational restrictions as a backbone N-Me
group. The conformation of small cyclic peptides is dictated
by their backbone stereochemistry and by the presence of
N-alkyl groups, rather than by the interactions with or
among the amino acid side chains.14 To test this notion, we
performed a detailed NMR study of cyclo[RGDfNMeV] (1)
and its N-(4-azidobutylated) analog (2). Both peptides had
very similar HN-, HR-, and CR-chemical shifts, and their
amide protons had almost identical temperature coefficients
(Δδ/ΔT) and very similar vicinal scalar coupling constants
[3J(HNꢀHR)] (see Supporting Information). The close re-
semblance of these NMR parameters, which are highly sensi-
tive to conformational changes, indicates that replacement
of the N-Me group of 1 by our linker provided a minimal
perturbation of its conformational state.
To demonstrate the applicability of the N-(4-azidobutyl)
linker, we prepared several conjugates of 2 with PEG.
Conjugate 11 was obtained from 2 by Cu(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition with a polydisperse
PEG-alkyne (2 KDa). To obtain conjugates 12 and 13,
the azido group of 2 was first reduced to an amine with
the mild Zn/NH4Cl reducing system. The resulting N-(4-
aminobutylated) cyclopeptide (10) was acylated with a
polydisperse PEG-COOSu derivative (2 KDa) to yield
conjugate 12, whereas reductive alkylation of 10 with a
polydisperse PEG-propionaldehyde (2 KDa) furnished con-
jugate 13. The optimized conditions for each transformation
are shown in Scheme 4. Due to the polydispersity of PEG,
However, when the SPPS of pentapeptide 3 was per-
formed in a larger amount of resin (>3 g) and/or with a
higherfunctionalization(>0.50mmolgꢀ1), the yieldswere
not as satisfactory. To obtain larger amounts of 3, an
efficient double SPPS scheme was developed using the
CTC resin for elongation, de- and reattachment of fully
protected peptide, and final elongation again (Scheme 3).
In this approach, the Val-Arg(Pbf)-Gly sequence was
assembled followed by reductive alkylation of its N-terminus
with 4-azidobutanal, as previously described. At this
stage, the peptide was cleaved with 2% TFA in CH2Cl2, and
its C-terminus was protected as a methyl ester. The coupling
between Fmoc-D-Phe and the N-azidoalkylated peptide seg-
ment (6) was performed in solution using the BTC method.
This procedure allowed us to obtain the desired peptide (7),
which was isolated in 48% yield. Then, the methyl ester
of 7 was hydrolyzed under basic conditions in the presence
of CaCl2, which is reported to suppress Fmoc- decomposi-
tion.13 Using this additive, the methyl ester was saponified
with no detectable Fmoc- decomposition (HPLC-MS). The
desired peptide (8) was isolated by simple aqueous extrac-
tion and loaded again onto the CTC resin. The incorpora-
tion of 8 took place with an acceptable yield (i.e., 80%
peptide incorporation for an expected functionalization
of 0.10 mmol gꢀ1). Further peptide chain elongation
and cleavage from the resin yielded pentapeptide 3, which
was cyclized and deprotected as described above. After
RP-HPLC purification, the N-azidoalkylated cyclopeptide
(2) was obtained in 18% overall yield.
With the synthesis of 2, we demonstrate that N-(4-
azidobutylated) peptides are accessible using standard
SPPS protocols that are compatible with common protect-
ing groups used in peptide synthesis. Taking into account
(13) (a) Thern, B.; Rudolph, J.; Jung, G. Angew. Chem., Int. Ed. 2002,
41, 2307–2309. (b) Thern, B.; Rudolph, J.; Jung, G. Tetrahedron Lett.
2002, 43, 5013–5016.
(14) Pascal, R.; Sola, R. Tetrahedron Lett. 1998, 39, 5031–5034.
Org. Lett., Vol. 15, No. 17, 2013
4574

Scheme 4. Synthesis of PEG-Conjugates 11ꢀ13
Figure 1. CD spectra of cyclo[RGDfNMeV] (1), cyclo[RGDf(N-
CH2CH2CH2CH2N3)V] (2), and compounds 11ꢀ13 in H2O at a
concentration of 0.5 mM.
Table 1. Adhesion Inhibition Assays of Cyclo[RGDfNMeV] (1)
and Compounds 11ꢀ13a
conditions had to be carefully optimized in order to facilitate
the RP-HPLC purification of the PEG-conjugates (11ꢀ13).
The PEG-conjugates (11ꢀ13), the N-azidoabutylated
cyclopeptide (2), and cyclo[RGDfNMeV] (1) were analyzed
by circular dichroism (CD), a sensitive technique to monitor
changes in a peptide secondary structure. All compounds
showed a positive band between 212 and 216 nm (λmax) anda
negative band between 230 and 238 nm (λmin) (Figure 1). CD
measurements on peptides 2 and 11ꢀ13 are consistent with
data on the peptide 1, which has a conformation featuring
two inverse γ (γi) turns and a γ turn.10
Rvβ3 þ Rvβ5
Rvβ3
HUVEC
DAOY
on VN
HUVEC
on FB
DAOY
on FB
compd
on VN
1
0.37
8.90
3.06
3.40
2.69
75.21
28.76
49.56
0.076
0.53
0.23
0.41
0.44
4.36
1.77
2.71
11
12
13
a IC50 values are given in μM.
The biological activity of the PEG-conjugates (11ꢀ13)
and cyclo[RGDfNMeV] (1) was evaluated in cell adhesion
inhibition assays. All compounds were tested for their
capacity to inhibit the integrin-mediated adhesion of
HUVEC endothelial and DAOY glioblastoma cells to
their immobilized ligands vitronectin (VN) and fibrinogen
(FB) (Table 1). For all the cell/ligand systems, all the
compounds inhibited cell adhesion in a concentration-
dependent manner, and the same pattern of inhibitory
activities was observed. The PEG-conjugates (11ꢀ13)
showed IC50 values in the low μM range, albeit inferior
to those of 1. The decreased inhibitory activity of 11ꢀ13
withrespect totheparentpeptide(1) may be attributableto
an interference of the PEG chain with the RGDꢀreceptor
interaction, resulting in lower binding affinities. Indeed,
the reduced biological activity of peptides upon attach-
ment of a bulky PEG chain is an issue of major concern,
especially in the case of small peptides.15
the azide, our linker is compatible with side-chain protec-
tion strategies, linkers, and resins commonly used in pep-
tide synthesis. Moreover, the chemical versatility of the
azide function, which can be reduced to an amine prior to
conjugation, allows for the flexible design of peptide
conjugates. Along these lines, the possibility of using click
chemistry in the conjugation step is an advantageous
feature, since it permits conjugation in the presence of
side-chain functional groups and thus implies a minimal
requirement for protection. On the basis of all these
considerations, we strongly believe that our N-(4-azidobutyl)
linker will have broad utility in peptide chemistry and will
widen the application of established conjugation methods.
Acknowledgment. This work has been partially sup-
ported by CICYT (CTQ2012-30930), the Generalitat de
Catalunya (2009SGR 1024), the and the IRB.
In conclusion, we have shown that a backbone N-(4-
azidobutyl) group can be incorporated into a peptide using
standard SPPS techniques and allows conjugation at a late
stage of the synthesis. Due to the orthogonal properties of
Supporting Information Available. Experimental de-
tails of the syntheses, cellular assays, characterization
data, and copies of the HPLC, HRMS, and NMR spectra
of selected compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(15) Chatterjee, J.; Mierke, D. F.; Kessler, H. Chem.;Eur. J. 2008,
14, 1508–1517.
The authors declare no competing financial interest.
(16) Veronese, F. M.; Mero, A. Biodrugs 2008, 22, 315–329.
Org. Lett., Vol. 15, No. 17, 2013
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