The synthesis of β-peptides containing guanidino groups

Article abstract of DOI:10.1016/S0960-894X(01)00027-0

The synthesis of the β-peptide 1 by the postsynthetic modification of the corresponding amino-containing peptide 3 is described. The potential of 1 to act as a template for the ligation of complementary negatively-charged peptides is discussed.

Full text of DOI:10.1016/S0960-894X(01)00027-0

Bioorganic& Medicinal Chemistry Letters 11 (2001) 689±691
The Synthesis of ꢀ-Peptides Containing Guanidino Groups
Ke Wen,a Hyunsoo Han,b,* Timothy Z. Ho€man,b Kim D. Jandab,*
and Leslie E. Orgela,*
aThe Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92186, USA
bDepartment of Chemistry, The Scripps Research Institute and The Skaggs Institute for Chemical Biology,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 9 August 2000; accepted 9 January 2001
AbstractÐThe synthesis of the b-peptide 1 by the postsynthetic modi®cation of the corresponding amino-containing peptide 3 is
described. The potential of 1 to act as a template for the ligation of complementary negatively-charged peptides is discussed. # 2001
Published by Elsevier Science Ltd.
Oligomers of b-amino acids (b-peptides) fold into well-
de®ned secondary structures including helices, sheets
and turns that are analogous to the well-known sec-
ondary structures of proteins.1À3 Some b-peptides com-
posed of as few as six amino acids adopt a stable helical
conformation designated as the L+2, 31 or 14 helix
which is characterized by a perfect three-residue geo-
metricrepeat with a 5 A pitch.4 This regular repeat
should make it possible to design peptides in which
selected side chains lie on a straight line parallel to the
helix axis. Such molecules might act as templates for the
ligation of `complementary' b-peptides as do some a-
peptides.5 Here, we describe the synthesis of a positively
charged peptide of this nature that could, in principle,
facilitate the ligation of complementary negatively
charged b-peptides (Fig. 1).
The guanidino group plays an important role in biolo-
gically active proteins, peptides and peptidomimetics,6
particularly in arginine-containing peptides and pro-
teins. Guanidine and its simple derivatives are strongly
basic(the p Ka of the guanidinium ion is approximately
13.5), and are fully protonated under physiological
conditions. Since positive charge of guanidino groups
and their potential to form hydrogen bonds serve as a
basis for speci®c interactions between biological mole-
cules we decided to synthesize peptide 1 as a potential
template.
Attempts to use the b-amino acid derivative 2 with a
protected guanidino group as a building block in
b-peptide chain elongation failed. We surmise that the
bulky Boc functionality used as a protecting group on
the guanidino group of 2 may have prevented access of
the required amino functionality to the activated car-
boxylate group. Peptide 1, therefore, could only be
obtained post-synthetically from peptide 3 by convert-
ing its amino moieties to guanidino groups. The synth-
eses of the b-peptide amides 3 and 4 were carried out
*Corresponding author. Fax: +1-619-552-8285; e-mail: orgel@salk.edu
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00027-0

690
K. Wen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 689±691
Figure 1. Schematic representation of proposed template-directed ligation. The positive charged b-peptide 1 serves as a template by aligning com-
plementary negative charges on substrate peptides. The arrow indicates the position of the new peptide bond.
using Fmocsolid-phase peptide synthesis on Applied
Biosystems' AMNH2 resin. b-Amino acid derivatives 5
and 6 were employed as building blocks. Compound 5
was synthesized by a published procedure7 in 29% yield.
Compound 6 was synthesized by an extension of a pre-
viously published synthesis8 from one of our
laboratories in an overall yield of 38% (Scheme 1).
NMR and mass spectroscopy were used to characterize
these compounds.9
simultaneously removed the Bocprotetcing groups.
Crude peptides were obtained after cleavage with puri-
ties of 33% for 3, and 27% for 4, determined by RP-
HPLC on a C18 column.11 The purities of the puri®ed
peptides were above 95% for 3 and 4. The presence of
several peaks close to each other in front of the major
peaks (product peaks) in the HPLC pro®les indicates
that the coupling reactions were incomplete. The pur-
i®ed peptides were water soluble. They were character-
ized by electron spray ionization MS (3 MH+ 1141, 3
MNa+ 1163; 4 MH+ 1681, and 4 MNa+ 1703), and (3
M+TFAÀ 1253; 4 M+TFAÀ 1793). All the measured
masses agreed with theoretical values.
In the course of the syntheses of peptides 3 and 4, the
®rst amino acid (5) was attached to the resin very e-
ciently. The next seven residues in the peptide sequences
were incorporated using the standard protocol.10 How-
ever, the coupling and deprotection eciency decreased
after the formation of the octapeptide, possibly due to
the folding of the peptide in the swelling solution. To
overcome this diculty, in subsequent elongation steps
we carried out two successive couplings using prolonged
coupling times and also a prolonged deprotection time
(standard coupling time 45±60 min and deprotection
time 3 min; the coupling time employed in these steps
was 120 min and the deprotection time was 6 min). The
N-terminal b-amino groups of these peptides were
acetylated after the completion of chain elongation. The
peptides were cleaved from the resin using TFA, which
The circular dichroism spectra of 3 in pure water, pH 7
bu€er, pH 12 bu€er and methanol solution (0.28 mM)
were recorded (Fig. 2) in an attempt to characterize its
conformation. The spectrum in water has a minimum at
203 nm and a maximum at 186 nm, indicating the pre-
sence of a well-de®ned secondary structure. The blue-
shift of the maximum and minimum relative to those
reported for other b-peptides,12 was possibly caused by
the amino groups in the backbone chain, which are
protonated under the conditions of the experiment.
When the pH was increased, slight red shifts of the
peaks were observed. These might arise from the
deprotonation of the amino groups. The peak intensity
decreased at high pH values, perhaps indicating a
decrease in the percentage of the peptide that has the
de®ned secondary structure. The same behavior was
observed in methanol solution. The shape of the curve
suggests that the peptide forms a 12/10/12 helix,12 or a
Scheme 1. (a) Ref 7; (b) Ph3P (1.1 equiv), H2O (5 equiv), THF; (c)
(Boc)2O (2 equiv); (d) 10% Pd/C, MeOH; (e) Fmoc-Cl (1.1 equiv),
DIEA (1.1 equiv), CH2Cl2; (f) TFA; (g) (Boc)2O (1.5 equiv), DIEA (3
equiv); (h) 8 (1.1 equiv), DIEA (3 equiv).
Figure 2. CD spectra of 0.28 mM b-peptide 3 in aqueous solution at
di€erent pHs or in methanol. The mean residue ellipticity is given in
deg.cm2/dmol.

K. Wen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 689±691
691
hair-pin structure.3 An NMR study designated to give
detailed structural information is in progress.
for performing the solid-state peptide syntheses, and Dr.
Barbara Chu for helpful discussion. K.W. is grateful to
the NASA Specialized Center of Research and Train-
ing (NSCORT/Exobiology), for a postdoctoral fellow-
ship. Financial support of this research from the
Skaggs Institute for Chemical Biology is gratefully
acknowledged.
In the guanidinylation reaction of resin-bound 3 (4-
methylbenzhydrylamine resin), a number of reagents
were explored. The most commonly used reagent 7, 1H-
pyrazole-carboxamidine, failed, but reagent 8, N,N0-di-
Boc-N00-tri¯ylguanidine, was used successfully.13,14 The
guanidinylation of the amino groups was carried out in
a 1 M solution of 8 in dichloromethane in the presence
of at least a 4-fold excess of triethylamine. After 6 days,
only two, on average, of the four amino groups in 3
were converted to guanidino groups. However, this
References and Notes
1. Krauthauser, S.; Christianson, L. A.; Powell, D. R.; Gell-
man, S. H. J. Am. Chem. Soc. 1997, 119, 11719.
2. Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinon,
B.; Oberer, L.; Hommel, V.; Widmer, H. Helv. Chim. Acta
1996, 79, 913.
3. Seebach, D.; Schreiber, J. V.; Abele, S.; Daura, X.; van
Gunsteren, W. F. Helv. Chim. Acta 2000, 83, 34.
4. Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015.
5. Lee, D. H.; Granja, J. R.; Martinez, J. A.; Severin, K.;
Ghadiri, M. R. Nature 1996, 382, 525.
intermediate product after cleavage from the resin with
15
tri¯uoromethanesulfonicacid,
could be further guani-
dinylated in solution as described below to give the
desired product.
A more ecient synthesis, entirely in homogeneous
solution, was then developed. The guanidinylating
reagent 8 is usually used in nonpolar solvents, such as
dichloromethane. However, since 3 is insoluble in di-
chloromethane, methanol was chosen as the solvent.
Peptide 3 (1 mg) was added to a mixture of 0.75 mL of
diisopropylethylamine and 150 mL of a 0.5 M solution
of 8 in methanol. The solution was allowed to stand for
6 days at 45 ꢀC. Dialysis of the reaction mixture followed
by RP-HPLC puri®cation yielded peptide 1 in 31% yield
(electron spray ionization MS, MH+ 1309, M+TFAÀ
1421; theoretical values MH+ 1309, M+TFAÀ 1421).
6. Berlinck, R. G. S. Nat. Prod. Rep. 1996, 377.
7. Davies, S. G.; Ichihara, O. Tetrahedron: Asymmetry 1991,
2, 183.
8. Han, H.; Yoon, J.; Janda, K. D. J. Org. Chem. 1998, 63,
2045.
9. Compound 6 [a]rDt +8.5 (methanol). H NMR (400 MHz,
ꢀ
1
CDCl3) d 1.23 (br. s, 3H), 1.46 (s, 9H), 4.21±4.47 (m, 5H), 5.67
(br. s, 1H), 7.28±7.32 (m, 2H), 7.39 (t, J=7.2 Hz, 2H), 7.58±
7.60 (m, 2H), 7.75 (d, J=7.2 Hz, 2H). FABMS: MNa+ 463.
Theoretical value 463.
10. Beck-Sickinger, A. G.; Durr, H.; Jung, G. Peptide Res.
1991, 4, 88.
The methods described above should make available a
variety of b-peptides containing 2-guanidino-3-amino-
butanoicaicd residues. They should prove useful in
template-directed synthesis and perhaps as high anity
aptomers for proteins and other biomolecules.
11. HPLC condition: 0.1% TFA H2O/acetonitrile gradient
(0±40% acetonitrile in 30 min) at a ¯ow rate of 1 mL/min.
12. Seebach, D.; Abele, S.; Gademan, K.; Guichard, G.; Hin-
termann, T.; Jaun, B.; Matthews, J. L.; Schreiber, J. V.;
Oberer, L.; Hommel, V.; Widmer, H. Helv. Chim. Acta 1998,
91, 932.
13. Feichtinger, K.; Zaft, C.; Sings, H. L.; Goodman, M. J.
Org. Chem. 1998, 63, 3804.
14. Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.;
Goodman, M. J. Org. Chem. 1998, 63, 8432.
Acknowledgements
The authors wish to thank Dr. Steven Koerber for
carrying out the CD measurements, Jill Meisenhelder
15. Thompson, P. E.; Keah, H. H.; Gomme, P. T.; Stanton,
P. G.; Hearn, M. T. W. Int. J. Pept. Prot. Res. 1995, 46, 174.