Tackling lipophilicity of peptide drugs: Replacement of the backbone n -methyl group of Cilengitide by N -oligoethylene glycol (N -OEG) chains

Article abstract of DOI:10.1021/bc4003844

Cilengitide is an RGD-peptide of sequence cyclo[RGDfNMeV] that was was developed as a highly active and selective ligand for the α _vβ₃ and α_vβ₅ integrin receptors. We describe the synthesis of three analogues of this peptide in which the N-Me group has been replaced by N-oligoethylene glycol (N-OEG) chains of increasing size: namely N-OEG₂, N-OEG₁₁, and N-OEG₂₃, which are respectively composed of 2, 11, and 23 ethylene oxide monomer units. The different N-OEG cyclopeptides and the original peptide were compared with respect to lipophilicity and biological activity. The N-OEG₂ analogue was straightforward to synthesize in solid phase using an Fmoc-N-OEG₂ building block. The syntheses of the N-OEG ₁₁ and N-OEG₂₃ cyclopeptides are hampered by the increased steric hindrance of the N-substituent, and could only be achieved by segment coupling, which takes place with epimerization and thus requires extensive product purification. All the N-OEG analogues were found to be more hydrophobic than the parent peptide, and their hydrophobicity was systematically enhanced upon increasing the length of the OEG chain. The N-OEG₂ cyclopeptide displayed the same capacity as Cilengitide to inhibit the integrin-mediated adhesion of HUVEC endothelial, DAOY gliobastoma, and HT-29 colon cancer cells to their ligands vitronectin and fibrinogen. The N-OEG₁₁ and N-OEG ₂₃ analogues also inhibited cell adhesion to these immobilized ligands, but their IC₅₀ values dropped by 1 order of magnitude with respect to the parent peptide. These results indicate that replacement of the backbone N-Me group of Cilengitide by a short N-OEG chain provides a more lipophilic analogue with a similar biological activity. Upon increasing the size of the N-OEG chain, liophilicity is enhanced, but synthetic yields drop and the longer polymer chains may impede targeted binding.

Full text of DOI:10.1021/bc4003844

Communication
pubs.acs.org/bc
Tackling Lipophilicity of Peptide Drugs: Replacement of the
Backbone N‑Methyl Group of Cilengitide by N‑Oligoethylene Glycol
(N‑OEG) Chains
Ana I. Fernandez-Llamazares,†,‡ Jaume Adan,∥ Francesc Mitjans,∥ Jan Spengler,*,†,‡
́
and Fernando Albericio*,†,‡,⊥,§
‡
†Institute for Research in Biomedicine (IRB) Barcelona, CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
∥
Nanomedicine, and Biomed Division, Leitat Technological Center Institution, Barcelona Science Park, Baldiri Reixac 10, 08028
Barcelona, Spain
⊥Department of Organic Chemistry, University of Barcelona, Martí i Franques
́
1-11, 08028 Barcelona, Spain
§School of Chemistry & Physics, University of KwaZulua-Natal, 4001 Durban, South Africa
S
* Supporting Information
ABSTRACT: Cilengitide is an RGD-peptide of sequence cyclo-
[RGDfNMeV] that was was developed as a highly active and selective
ligand for the αvβ3 and αvβ5 integrin receptors. We describe the
synthesis of three analogues of this peptide in which the N-Me group
has been replaced by N-oligoethylene glycol (N-OEG) chains of
increasing size: namely N-OEG2, N-OEG11, and N-OEG23, which are
respectively composed of 2, 11, and 23 ethylene oxide monomer units.
The different N-OEG cyclopeptides and the original peptide were
compared with respect to lipophilicity and biological activity. The N-
OEG2 analogue was straightforward to synthesize in solid phase using
an Fmoc-N-OEG2 building block. The syntheses of the N-OEG11 and
N-OEG23 cyclopeptides are hampered by the increased steric
hindrance of the N-substituent, and could only be achieved by
segment coupling, which takes place with epimerization and thus
requires extensive product purification. All the N-OEG analogues were found to be more hydrophobic than the parent peptide,
and their hydrophobicity was systematically enhanced upon increasing the length of the OEG chain. The N-OEG2 cyclopeptide
displayed the same capacity as Cilengitide to inhibit the integrin-mediated adhesion of HUVEC endothelial, DAOY gliobastoma,
and HT-29 colon cancer cells to their ligands vitronectin and fibrinogen. The N-OEG11 and N-OEG23 analogues also inhibited
cell adhesion to these immobilized ligands, but their IC50 values dropped by 1 order of magnitude with respect to the parent
peptide. These results indicate that replacement of the backbone N-Me group of Cilengitide by a short N-OEG chain provides a
more lipophilic analogue with a similar biological activity. Upon increasing the size of the N-OEG chain, liophilicity is enhanced,
but synthetic yields drop and the longer polymer chains may impede targeted binding.
ethyl,5,6 N-allyl,5,7,8 N-butyl,9 and N-guanidylbutyl10]. In the
n peptide-based medicinal chemistry, backbone N-methyl-
Iation is an established tool to improve pharmacological field of N-backbone cyclic peptides, which are usually prepared
using N-functionalized building blocks [e.g., N-aminoalkyl, N-
carboxyalkyl, and N-sulfanyl],11−15 the solution synthesis and
purification of N-alkylated dipeptide building units have been
described numerous times.11,12,16−21
Very recently, we showed that peptides bearing an N-
triethylene glycol (N-TEG) chain are straighforward to access
using standard solid-phase techniques.22 With our method-
ology, we synthesized several N-TEG analogues of Sansalva-
mide A peptide, all of which were found to be slightly more
lipophilic than their corresponding N-Me homologues. Since
properties of peptide drugs. The introduction of backbone N-
Me groups has allowed to optimize the activity and selectivity
of numerous peptide ligands as a result of conformational
modulation.1 Furthermore, the introduction of N-Me residues
into peptides increases their hydrophobicity, proteolytic
resistance, and membrane permeability, which can enhance
their bioavailability, thus amplifying their therapeutic poten-
tial.1,2 Surprisingly, little research has been conducted on
modifying the peptide backbone with other N-alkyl sub-
stituents. Besides the so-called peptoids (N-substituted glycine
oligomers)3 and N-backbone cyclic peptides introduced by
Gilon,4 only a few examples have been reported in which other
N-alkylated peptides have been accessed, and such examples are
limited to modification with small N-alkyl groups [i.e., N-
Received: August 20, 2013
Revised: October 20, 2013
Published: December 13, 2013
© 2013 American Chemical Society
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Figure 1. Structure of cyclo[RGDfNMeV] (1) and the N-OEG cyclopeptides (2−4) synthesized and tested.
lipophilicity is a key pharmacological parameter,23 we found it
Scheme 1. Synthesis of the Fmoc-N-OEG Val Derivatives
(6a−6c)
a
desirable to know if such an enhancement in lipophilicity would
also be observed upon incorporation of an N-oligoethylene
glycol (N-OEG) chain into other peptides. We also sought to
evaluate the effect of such structural modification on biological
activity in more detail. To investigate this, we chose Cilengitide
(1) as model peptide. This peptide is an RGD-peptide of
sequence cyclo[RGDfNMeV] that was developed as a highly
active and selective ligand for the αvβ3 and αvβ5 integrin
receptors.24
In this work, we have investigated the effect of replacing the
backbone N-Me group of Cilengitide by different N-OEG
chains on its lipophilicity and biological activity. Herein we
describe synthesis of three analogues of 1 in which the N-Me
group of Val has been replaced by N-OEG chains of increasing
length, namely, N-OEG2, N-OEG11, and N-OEG23, which are
respectively composed of 2, 11, and 23 repeating ethylene oxide
monomer units (Figure 1).
In a first attempt to synthesize cyclopeptides 2−4, we
adopted the strategy that we had employed for the synthesis of
the N-TEG Sansalvamide A peptide analogues. Such an
approach involved the use of Fmoc-protected N-TEG building
blocks in the solid-phase synthesis of the linear peptide
precursors, which were cyclized in solution. To prepare the
required Fmoc-N-OEG Val derivatives (6a−6c), valine tert-
butyl ester was subjected to reductive alkylation with a suitable
aldehyde.25 In all cases, the reductive alkylation mixture
consisted of unreacted starting material, N-monoalkylated
product, and N,N-dialkylated product. The latter two were
found to be inseparable, but after Fmoc-protection of the
amino group, the desired products (5a−5c) could be isolated
by flash chromatography. Remarkably, the increased length of
the N-OEG chain did not prevent the acylation of the
secondary amine with Fmoc-Cl and did not hamper the
purification of 5a−5c by flash chromatography. Finally, acidic
cleavage of the tert-butyl ester yielded 6a−6c in 45−54%
overall yield (Scheme 1).
The solid-phase peptide synthesis (SPPS) of the linear
pentapeptides 9a−9c was performed on the 2-chlorotrityl
chloride (CTC) resin by Fmoc-/tBu- chemistry (Scheme 2).
The use of a low functionalization (0.2−0.3 mmol/g) was
aimed to facilitate the sterically hindered coupling step.26 In all
the pentapeptides (9a−9c), the N-OEG Val residue was placed
in the middle of the sequence to render Gly at the C-terminus,
which minimizes steric hindrance during cyclization and rules
a
Reagents and conditions: a. R-CHO (1.1 equiv), NaBH3CN (1.34
equiv), MeOH/AcOH 99:1; b. Fmoc-Cl (1.2 equiv), DIEA (2.0
equiv), DCM; c. TFA/DCM 1:1.
out the possibility of epimerization in this step. The Fmoc-N-
OEG Val derivatives (6a−6c) were coupled onto the peptidyl-
resin in a 3-fold excess using DIPCDI/OxymaPure activation.
With these conditions, the coupling of 6a and 6b took place
efficiently and with no detectable epimerization, whereas the
coupling of 6c was found to be hampered by its longer N-
OEG23 chain and required PyBOP/HOAt as a stronger
activation method, although the coupling did not proceed to
completion. After having assembled the three N-OEG
tripeptidyl-resins, we investigated the coupling of Fmoc-D-Phe
using bis(trichloromethyl)carbonate (BTC) as activating
reagent.27,28 It is important to note that this coupling is
hampered not only by the N-substituent, but also by the β-
branched side-chain of Val. With this activation method, resin-
bound N-OEG2 Val was completely acylated after two
treatments (15 h) with the in situ generated acid chloride of
Fmoc-D-Phe (as checked by HPLC analysis of a cleaved resin
sample). The use of BTC also allowed for coupling Fmoc-D-
Phe onto resin-bound N-OEG11 Val, but the conversion was
low and did not improve after a third coupling cycle. However,
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Scheme 2. Synthesis of Cyclo[RGDfNMeV] (1) and the N-OEG Cyclopeptides 2−3
for the N-OEG23 peptidyl-resin, no coupling could be achieved.
We tested other activation methods, but all of them failed to
form the desired amide bond. Thus, we discontinued the
synthesis of pentapeptide 9c by this approach. For the resin-
bound N-OEG2 and N-OEG11 peptides, further peptide chain
elongation and cleavage afforded the desired pentapeptides (9a
and 9b), which were cyclized with EDC and catalytic amounts
of 4-DMAP. Cyclization proceeded smoothly and, after removal
of side-chain protecting groups with 95% TFA in the presence
of H2O and TIS, the N-OEG2 and N-OEG11 cyclopeptides (2
and 3) were isolated by semipreparative RP-HPLC.
Thus, the synthetic strategy shown in Scheme 2 allows the
efficient preparation of peptides bearing an N-OEG chain of 2
ethylene oxide units, and enables us to obtain peptides with an
11-monomer unit N-OEG chain, though in very low amount.
However, the synthesis of our target N-OEG23 pentapeptide
(9c) was not found feasible, since no coupling of Fmoc-D-Phe
onto resin-bound N-OEG23 Val could be achieved. To avoid
the difficult formation of the D-Phe-(N-OEG11)Val and D-Phe-
(N-OEG23)Val amide bonds in solid phase, we sought to
prepare suitable Fmoc-D-Phe-(N-OEG)Val dipeptides in
solution and use them as solid-phase building blocks. To
prepare the required dipeptides (8b and 8c), valine tert-butyl
ester was subjected to reductive alkylation with a suitable
aldehyde, and the resulting N-OEG11 or N-OEG23 Val
derivatives were reacted with the acid chloride of Fmoc-D-
Phe. This procedure enabled us to obtain dipeptides 7b and 7c
with no detectable epimerization, and acidic cleavage of the tert-
butyl ester yielded the desired building blocks, 8b and 8c
(Scheme 3).
In the SPPS of the N-OEG11 and N-OEG23 pentapeptides
(9b and 9c), dipeptides 8b and 8c were coupled onto the
peptidyl-resin using PyBOP/HOAt activation (Scheme 4).
Since these dipeptides are time-consuming to synthesize, only
1.0 equiv was coupled. After 3 h coupling, further PyBOP (1.0
equiv) and DIEA (1.0 equiv) were added and the reaction was
allowed to proceed overnight. These couplings proceeded with
acceptable efficiency. However, epimerization of C-activated
dipeptides can occur through various mechanisms and is known
to be a major disadvantage of segment peptide synthesis.29 In
our case, considerable epimerization took place at the
dipeptidic C-terminal Val residue. The occurrence of
epimerization was confirmed by the presence of two peaks
with the mass of the desired product in the HPLC spectra of a
cleaved peptidyl-resin sample. The high degree of epimerization
observed was expectable, as the epimerization of C-activated
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Scheme 3. Synthesis of the Fmoc-D-Phe-(N-OEG)Val
Dipeptides (8b−8c)
Scheme 4. Synthesis of the N-OEG Cyclopeptides 3−4 Using
a Dipeptide Building Block
a
a
Reagents and conditions: a. R-CHO (1.1 equiv), NaBH3CN (1.34
equiv), MeOH/AcOH 99:1; b. Fmoc-D-Phe-Cl (1.5 equiv), DIEA (2.5
equiv), DCM; c. TFA/DCM 1:1.
peptides is favored by itself (no carbamate as protecting group
of the C-terminal residue) and, in this case, by the presence of
D- and/or N-alkyl residues.30,31 Further peptide elongation and
cleavage from resin yielded pentapeptides 9b and 9c epimerized
at Val. After cyclization and deprotection, the desired N-OEG11
and N-OEG23 cyclopeptides (3 and 4) were separated from
their nondesired Val epimers by semipreparative RP-HPLC.
For the two isolated N-OEG11 cyclopeptides, the stereoisomer
having a lower retention time was found to coelute with
stereochemically pure cyclo[RGDf(N-OEG11)V] (3), which
had been previously prepared using Fmoc-N-OEG11 Val (6b)
as building block. Analogously, the isolated N-OEG23 cyclo-
peptide with a lower RP-HPLC retention time was assumed to
be the product with the desired stereochemistry, cyclo[RGDf-
(N-OEG23)V] (4) (see Supporting Information).
To study the effect of the N-OEG chain on lipophilicity, we
coinjected the N-OEG cyclopeptides (2−4) and cyclo-
[RGDfNMeV] (1) onto a C18 column and compared their
RP-HPLC retention times.32 This is reported as a reliable
method to estimate the relative hydrophobicities of a series of
modified analogues.33 The RP-HPLC behavior of a compound
depends on its hydrophobic interactions with the nonpolar
stationary phase: the more hydrophobic a compound is, the
stronger its retention on the column. All the N-OEG analogues
(2−4) were found to be more lipophilic than 1, and their
lipophilicity was systematically enhanced upon increasing the
size of the N-OEG chain (Figure 2). These results can be
explained by the amphiphilic nature of OEG.34,35 Although
OEG-modified peptides often show better aqueous solubility
than their corresponding nonmodified peptides, this is due to
the polymer chain preventing intermolecular aggregation, and
not due to an increase in hydrophilicity.36,37 Along these lines,
the attachment of an OEG chain to peptides can improve their
solubility in organic solvents.38,39
consistent with our previously reported findings for the N-
OEG analogues of the totally aliphatic Sansalvamide A peptide:
for both model peptides, replacement of a backbone N-Me
group by an N-OEG chain provides a higher lipophilicity.
Therefore, we can reasonably expect that N-Me-for-N-OEG
substitution will also increase lipophilicity for other N-
methylated peptide scaffolds. Such an enhancement in
lipophilicity can be conveniently exploited for improving the
absorption of peptide drug candidates that are too hydrophilic
to cross biological membranes via transcellullar passive
diffusion,40,41 which is the most common transport route for
peptides. Indeed, the covalent modification of peptides by
attaching lipophilic moieties has proven to be an effective
approach to improve their intestinal permeability42−46 and oral
bioavailability.47,48
The parent peptide, cyclo[RGDfNMeV] (1), and its N-OEG
analogues (2−4) showed no degradation when incubated in
human serum at 37 °C over a period of 48 h (see Supporting
Information). The high enzymatic stability of 1−4 was
expected, since cyclic peptides show increased resistance to
proteolytic cleavage, and the presence of D- and N-alkyl
residues confers them further stability.49
The biological activity and selectivity of cyclo[RGDfNMeV]
(1) and the N-OEG cyclopeptides (2−4) were evaluated in cell
adhesion inhibition assays (Table 1). Adhesion studies were
carried out with HUVEC endothelial, DAOY glioblastoma, and
Our findings upon incorporation of N-OEG into Cilengitide,
which has two ionizable side-chain functionalities, are
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Figure 2. HPLC chromatogram obtained after coinjection cyclo[RGDfNMeV] (1) and the N-OEG cyclopeptides (2−4) on a C18 column, linear
gradient from 10% to 50% ACN over 8 min.
Table 1. Adhesion Inhibition Assays of Cyclo[RGDfNMeV]
(1) and the N-OEG Cyclopeptides (2−4)
HPLC purification was required to separate the desired cyclic
products from their nondesired diastereoisomers.
a
All the N-OEG analogues (2−4) were found to be more
hydrophobic than the parent peptide (1), and their hydro-
phobicity was systematically enhanced upon increasing the
length of the OEG chain. The N-OEG2 cyclopeptide (2)
displayed the same capacity as Cilengitide (1) to inhibit
integrin-mediated cell adhesion, but the inhibitory activities of
the N-OEG11 and N-OEG23 cyclopeptides (3 and 4) were 1
order of magnitude lower. Taken together, the results show
that, in the case of the cyclic pentapeptide Cilengitide,
substitution of a backbone N-Me group by a short N-OEG
chain provides a more lipophilic analogue with a similar
biological activity. Upon increasing the length of the OEG
chain, lipophilicity is enhanced, but the synthesis is not efficient
and steric hindrance may impede targeted binding. Thus,
replacement of a backbone N-Me group by a short N-OEG
chain is a feasible way to enhance the lipophilicity of peptide
drug candidates, which can result in a better membrane
permeability via passive diffusion, and may not have a negative
impact on biological activity. Considering that backbone N-Me
groups are common structural motifs in many biologically
active peptides, we want to communicate that modification at
this position is a valuable alternative to introduce chemical
diversity or alter pharmacologically important parameters when
modification at any other position of the peptide is not wished
or possible.
vitronectin (VN)
fibrinogen (FB)
HUVEC
DAOY
on VN
αvβ3 +
αvβ5
HT-
on VN
αvβ3 +
αvβ5
29 on HUVEC DAOY
VN
αvβ5
on FB
αvβ3
on FB
αvβ3
compound
cyclo[RGDfNMeV]
(1)
0.37
2.69
3.11
2.17
n.d.
n.d.
0.076
0.036
1.38
0.44
0.14
4.55
2.31
cyclo[RGDf(N-
OEG2)V] (2)
0.42
3.62
cyclo[RGDf(N-
OEG11)V] (3)
12.42
22.30
171.5
143.1
cyclo[RGDf(N-
OEG23)V] (4)
0.28
a
IC50 values are given in μM.
HT-29 colon cancer cells using vitronectin (VN) or fibrinogen
(FB) as ligands. For HUVEC and DAOY cells, adhesion to
their ligand VN is mediated by integrins αvβ3 and αvβ5, whereas
their adhesion to FB is only mediated by integrin αvβ3. In
contrast, the adhesion of HT-29 cells to VN is only αvβ5-
dependent. For all the cell/ligand systems, the compounds
inhibited cell adhesion in a concentration-dependent manner,
resulting in the expected sigmoid curves. The inhibitory
activities of the N-OEG2 cyclopeptide (2) were very similar
to those of 1, which indicates that replacement of its N-Me
group by a short N-OEG2 chain does not affect binding affinity.
In contrast, the N-OEG11 and N-OEG23 analogues (3 and 4)
inhibited cell adhesion with less potency, which is probably due
to their longer OEG chains interfering with the RGD-receptor
interaction. Indeed, the reduced biological activity of peptides
upon attachment of a bulky polymer chain is an issue of major
concern, especially in the case of small peptides.50
In summary, the analogue of Cilengitide bearing an N-OEG2
chain instead of a backbone N-Me group (2) was
straightforward to synthesize in solid phase. The acylation of
resin-bound N-OEG2 amines can be achieved by activating the
following amino acid with BTC, and the protocol is compatible
with the acid-labile CTC resin. However, for resin-bound N-
OEG11 and N-OEG23 residues, this method does not work. The
increased steric hindrance of their N-substituents impedes the
acylation in solid phase (1 and 10 atom-chains for 1 and 2,
versus 38 and 74 atom-chains for 3 and 4). We were able to
obtain the N-OEG11 and N-OEG23 cyclopeptides (3 and 4) by
using a dipeptidic building block, but considerable epimeriza-
tion took place during dipeptide coupling, and extensive RP-
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details of the syntheses, cellular assays, serum
stability assays, characterization data, HPLC traces and HRMS
data of the pure cyclopeptides (1−4), and copies of the NMR
spectra of selected compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: jan.spengler@irbbarcelona.org. Phone: (+34) 93 403
71 27, Fax: (+34) 93 403 71 26.
*E-mail: albericio@irbbarcelona.org. Phone: (+34) 93 403 70
88, Fax: (+34) 93 403 71 26.
Notes
The authors declare no competing financial interest.
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Communication
aminoalkyl) amino acids and N-(ω-carboxyalkyl) amino acids. J. Org.
Chem. 62, 411−416.
(14) Bitan, G., Muller, D., and Gilon, C. (1997) Building units for N-
backbone cyclic peptides: 4. Synthesis of protected Nα-functionalized
alkyl amino acids by reductive alkylation of natural amino acids. J.
Chem. Soc., Perkin Trans. 1, 1501−1510.
ACKNOWLEDGMENTS
■
This work was partially supported by CICYT (CTQ2012-
30930) and the Generalitat de Catalunya (2009SGR 1024).
The Institute for Research in Biomedicine (IRB) Barcelona, the
Barcelona Science Park, and Leitat Technological Center are
also acknowledged for their support. Ana I. Fernan
Llamazares thanks Ministerio de Educacion y Ciencia for a
FPU fellowship. We thank Barcelona Science Park (Mass
Spectrometry Core Facility, Nuclear Magnetic Resonance Unit)
for the facilities. We thank to Dr. Thomas Bruckdorfer (IRIS
Biotech) for fruitful discussions.
́
dez-
(15) Falb, F., Yechezkel, T., and Gilon, C. (1999) In situ generation
of Fmoc-amino acid chlorides using bis-(trichloromethyl)carbonate
and its utilization for difficult couplings in solid-phase peptide
synthesis. J. Pept. Res. 53, 507−517.
́
(16) Muller, B., Besser, D., Kleinwachter, P., Arad, O., and
̈
̈
Reissmann, S. (1999) Synthesis of N-carboxyalkyl and N-aminoalkyl
functionalized dipeptide building units for the assembly of backbone
cyclic peptides. J. Pept. Res. 54, 383−393.
ABBREVIATIONS
(17) Schumann, C., Seyfarth, L., Greiner, G., and Reissmann, S.
(2000) Synthesis of different types of dipeptide building units
containing N- or C-terminal arginine for the assembly of backbone
cyclic peptides. J. Pept. Res. 55, 428−435.
■
4-DMAP, 4-dimethylaminopyridine; ACN, acetonitrile; BTC,
bis(trichloromethyl)carbonate; CTC, 2-chlorotrityl chloride;
DIEA, N,N′-diisopropylethylamine; DIPCDI, N,N′-diisopropyl-
carbodiimide; EDC·HCl, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride; FB, fibrinogen; MeOH, methanol;
OEG, oligoethylene glycol; OxymaPure, ethyl-2-cyano-2-
(hydroxyimino)acetate; TEG, triethylene glycol; VN, vitronec-
tin
(18) Besser, D., Muller, B., Agricola, I., and Reissmann, S. (2000)
̈
Synthesis of differentially protected N-acylated reduced pseudodipep-
tides as building units for backbone cyclic peptides. J. Pept. Sci. 6, 130−
138.
(19) Schumann, C., Seyfarth, L., Greiner, G., Paegelow, I., and
Reissmann, S. (2002) Synthesis and biological activities of new side
chain and backbone cyclic bradykinin analogues. J. Pept. Res. 60, 128−
140.
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