A New Class of Foldamers Based on cis-γ-Amino-L-proline

Article abstract of DOI:10.1021/ja0398621

A synthetic method for the preparation of conformationally constrained γ-peptides derived from γ-amino-L-proline is described. The methodology allows the independent buildup of the peptide backbone and the introduction of sequential variations by reactions with the γ-amino group of γ-aminoproline. Both alkyl- and acyl-substituted γ-peptides have been prepared and studied by CD and NMR. Conformational restrictions due to the cyclic structure of the monomer give rise to long-range interactions that are indicative of secondary structures even in aqueous solution. Interresidue NOEs suggest a concatenation of turns that, in a permissive solvent, could give rise to an isolated hydrogen bond ribbon, flanked and protected by proline rings.

Full text of DOI:10.1021/ja0398621

Published on Web 04/21/2004
A New Class of Foldamers Based on cis-γ-Amino-L-proline1,2
Josep Farrera-Sinfreu,†,‡ Laura Zaccaro,‡ David Vidal,§ Xavier Salvatella,†
Ernest Giralt,†,| Miquel Pons,*,§,| Fernando Albericio,*,†,| and Miriam Royo*,‡,|
Contribution from the Barcelona Biomedical Research Institute, Combinatorial Chemistry Unit,
and Laboratory of Biomolecular NMR, Barcelona Science Park, UniVersity of Barcelona,
Josep Samitier 1, 08028-Barcelona, Spain, and Department of Organic Chemistry, UniVersity of
Barcelona, Mart´ı i Franque´s 1, 08028-Barcelona, Spain
Received November 29, 2003; E-mail: mroyo@pcb.ub.es; albericio@pcb.ub.es; mpons@qo.ub.es
Abstract: A synthetic method for the preparation of conformationally constrained γ-peptides derived from
γ-amino-L-proline is described. The methodology allows the independent buildup of the peptide backbone
and the introduction of sequential variations by reactions with the R-amino group of γ-aminoproline. Both
alkyl- and acyl-substituted γ-peptides have been prepared and studied by CD and NMR. Conformational
restrictions due to the cyclic structure of the monomer give rise to long-range interactions that are indicative
of secondary structures even in aqueous solution. Interresidue NOEs suggest a concatenation of turns
that, in a permissive solvent, could give rise to an isolated hydrogen bond ribbon, flanked and protected by
proline rings.
Introduction
this term, adding the restriction that ordered structures formed
by these compounds should be stabilized by a collection of
Biopolymers encompass a broad range of structures and are
involved in key aspects of our understanding of life. In nature,
there are only three major biopolymer backbones, proteins,
nucleic acids, and polysaccharides. Therefore, the development
of new oligomers that mimic natural biopolymers should provide
a powerful tool to obtain compounds with potential applications
in life sciences. One particularly relevant aspect of natural
biopolymers is the variability introduced by combining a limited
set of monomers in a well-defined sequence. The foldamer field
is directly inspired by this philosophy. The term foldamer, which
was first coined by Gellman,3 is used to describe those unnatural
oligomers that in solution fold into a conformationally ordered
state. Some authors have suggested a more rigid definition for
noncovalent interactions between nonadjacent monomer units.4
Foldamers can be classified either by their secondary structure4
or by the backbone type. Various examples of foldamers of the
latter type have been described in the literature, including
peptoids,5 vinylogous polypeptides,6 peptide nucleic acids,7
oligoureas,8 oligopyrrolinones,9 oligo(phenylene ethylenes),10
aedamers,11 guanidines,12 and â-3,13 and γ-peptides.14
â-Peptides are probably the most extensively studied fol-
damers.15 The success of these compounds is a result of three
factors: their resemblance to R-peptides, the diverse range of
(4) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem.
ReV. 2001, 101, 3893-4011.
(5) (a) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell,
D. A.; Banville, S.; Ng, S.; Wang, S.; Rosenberg, S.; Marlow, C. K.;
Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen, F. E.;
Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367-9371. (b)
Armand, P.; Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.; Barron,
A. E.; Truong, K. T.; Dill, K. A.; Mierke, D. F.; Cohen, F. E.; Zuckermann,
R. N.; Bradley, E. K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4309. (c)
Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.; Bradley,
E. K.; Truong, K. T.; Dill, K. A.; Cohen, F. E.; Zuckermann, R. N. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 4303.
† Barcelona Biomedical Research Institute.
‡ Combinatorial Chemistry Unit.
§ Laboratory of Biomolecular NMR.
| Department of Organic Chemistry.
(1) A preliminary report describing part of this work was presented at the 17th
American Peptide Symposium: Giralt, E.; Royo, M.; Kogan, M.; Crespo,
L.; Sanclimens, G.; Farrera, J.; Pons, M.; Albericio, F. Proline: a key
building block in “de novo” designed peptide molecules. In Peptides
Proceedings of the 17th American Peptide Symposium; Houghten, R., Lebl,
M., Eds.; American Peptide Society: San Diego, CA, 2001; pp 432-434.
(2) Abbreviations: Ac, acetyl; MeCN, acetonitrile; Ac2O, acetic anhydride;
Amp, cis-4-amino-L-proline or (2S,4S)-4-amino-pyrrolidine-2-carboxylic
acid; Boc, tert-butoxycarbonyl; Dab, R,γ-diaminobutyric acid; DCM,
dichloromethane; DHB, 2,5-dihydroxybenzoic acid; DIEA, N,N-diisopro-
pylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMF, N,N-dimeth-
ylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; (2S,4S)-Boc-Amp(Fmoc)-
OH, (2S,4S)-Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic acid; HOAc,
acetic acid; HOBt, 1-hydroxy-1,2,3-benzotriazole; HR-ESI: high-resolution
electro spray; iV, isovaleryl; MALDI-TOF, matrix-assisted laser desorption
ionization, time-of-flight; MBHA, p-methylbenzhydrylamine; MeOH,
methanol; MPLC, medium-pressure liquid chromatography; PhAc, phen-
ylacetyl; RP-HPLC, reversed-phase high performance liquid chromatog-
raphy; SPOS, solid-phase organic synthesis; SPPS, solid-phase peptide
synthesis; TBME, tert-butylmethyl ether; TFA, trifluoroacetic acid; TFE,
2,2,2-trifluoroethanol; amino acid symbols denote the L-configuration.
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J. AM. CHEM. SOC. 2004, 126, 6048-6057
10.1021/ja0398621 CCC: $27.50 © 2004 American Chemical Society

A New Class of Foldamers Based on cis-γ-Amino-L-proline
A R T I C L E S
stabilized secondary structures that they form,3,16 and the
different applications found (e.g., antimicrobials,17 Trojan
carriers,18 or other biological activities19).
different types of secondary structures induced and, as a result,
in the possible applications of these materials.21,22 The increased
number of degrees of freedom in γ-peptides, as compared to
R- and â-peptides, can be reduced by the use of cyclic
monomers,7b,c,23 which increase the stability of regular secondary
structures. The cyclic monomers described to date show less
diversity than the linear ones, and there is not much distinction
between a conserved backbone and a diversity-carrying side
chain.13g,23g,h,24
Even though γ-peptides represent the natural next step for
the generation of a new family of foldamers based on the amide
backbone, only a few examples of γ-peptides have been reported
in the literature,20 and these systems are based on linear amino
acids with substituents at different backbone positions.14,21
Different substitution patterns introduce diversity into the
In the work described here, we developed a synthetic strategy
to obtain two new families of γ-peptides formed by the cyclic
monomer cis-γ-amino-L-proline. The backbone in these peptides
contains amide/peptide bonds between the carboxyl function
and the γ-amino function of successive residues. The R-amino
group is left free for the introduction of different substituents,
either during the synthesis of the oligomers (obtaining hetero-
oligomers) or as a final functionalization step (obtaining
homooligomers). The independent buildup of the backbone and
side-chain sequences leads to a very high level of synthetic
versatility. We explored both acylation and alkylation for the
introduction of the side chains to give NR-acyl-γ-peptides and
NR-alkyl-γ-peptides, respectively.
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CD and NMR studies were used to elucidate the structural
features of these new families of compounds in different
solvents, and we compared these features with those of acyclic
γ-peptides based on R,γ-diaminobutyric acid (Figure 1).
Results and Discussion
Synthesis and Characterization of γ-Hexapeptides. To
study the influence on the secondary structure of the different
types of linkage between the side chain and the γ-peptide
backbone, we prepared two families of γ-peptides: NR-acyl-
γ-peptides and NR-alkyl-γ-peptides. An Fmoc/Boc combined
solid-phase strategy was chosen, where Fmoc was the temporary
protecting group for the γ-amino group of each monomer and
Boc was the semipermanent protecting group for the R-amino
group through which the side chain was introduced.25 The same
(22) This effect is more extensively studied in the field of â-peptides; for
examples, see: (a) Krauthau¨ser, S.; Christianson, L. A.; Powell, D. R.;
Gellman, S. H. J. Am. Chem. Soc. 1997, 119, 11719-11720. (b) Abele,
S.; Seiler, P.; Seebach, D. HelV. Chim. Acta 1999, 82, 1559-1571. (c)
Seebach, D.; Abele, S.; Sifferlen, T.; Ha¨nggi, M.; Gruner, S.; Se´ller, P.
HelV. Chim. Acta 1998, 81, 2218-2243. (d) Abele, S.; Guichard, G.;
Seebach, D. HelV. Chim. Acta 1998, 81, 2141-2155. (e) Abele, S.; Vo¨gtli,
K.; Seebach, D. HelV. Chim. Acta 1999, 82, 1539-155. (f) Seebach, D.;
Schreiber, J. V.; Abele, S.; Daura, X.; van Gunsteren, W. F. HelV. Chim.
Acta 2000, 83, 34-57. (g) Schreiber, J. V.; Seebach, D. HelV. Chim. Acta
2000, 83, 3139-3151. (h) Seebach, D.; Schreiber, V.; Arvidsson, I.;
Frackenpohl, J. HelV. Chim. Acta 2001, 84, 271-279. (i) Gla¨ttli, A.; Daura,
X.; Seebach, D.; van Gunsteren, W. F. J. Am. Chem. Soc. 2002, 124,
12972-12978. (j) Langenhan, J. M.; Guzei, I. A.; Gellman, S. H. Angew.
Chem., Int. Ed. 2003, 42, 2402-2405.
(23) In â-peptides, S. H. Gellman has already proposed the use of cyclic
monomers to reduce the degree of freedom. In addition to ref 16f,k,i,m,
see: (a) Huck, B. R.; Langenhan, J. M.; Gellman, S. H. Org. Lett. 1999,
1, 1717-1720. (b) Barchi, J. J., Jr.; Huang, X.; Appella, D. H.; Christianson,
L. A.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 2000, 122, 2711-
2718. (c) Wang, X.; Espinosa, J. F.; Gellman, S. H. J. Am. Chem. Soc.
2000, 122, 4821-4822. (d) Huck, B. R.; Fisk, J. D.; Gellman, S. H. Org.
Lett. 2000, 2, 2607-2610. (e) Chung, Y. J.; Huck, B. R.; Christianson, L.
A.; Stanger, H. E.; Krautha¨user, S.; Powell, D. R.; Gellman, S. H. J. Am.
Chem. Soc. 2000, 122, 3995-4004. (f) Porter, E. A.; Wang, X.; Schmitt,
M. A.; Gellman, S. H. Org. Lett. 2002, 4, 3317-3319. (g) Le Plae, P. R.;
Fisk, J. D.; Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc.
2002, 124, 6820-6821. (h) Park, J.-S.; Lee, H.-S.; Lai, J. R.; Kim, B. M.;
Gellman, S. H. J. Am. Chem. Soc. 2003, 125, 8539-8545.
(20) (a) Hanessian, S.; Luo, X.; Schaum, R.; Michnick, S. J. Am. Chem. Soc.
1998, 120, 8569. (b) Hanessian, S.; Luo, X.; Schaum, R. Tetrahedron Lett.
1999, 40, 4925-4929. (c) Woll, M. G.; Lai, J. R.; Guzei, I. A.; Taylor, S.
J. C.; Smith, M. E. B.; Gellman, S. H. J. Am. Chem. Soc. 2001, 123, 11077-
11078.
(21) (a) Hintermann, T.; Gademann, K.; Bernhard, J.; Seebach, D. HelV. Chim.
Acta 1998, 81, 983-1002. (b) Brenner, M.; Seebach, D. HelV. Chim. Acta
2001, 84, 1181-1189. (c) Brenner, M.; Seebach, D. HelV. Chim. Acta 2001,
84, 2155-2166. (d) Seebach, D.; Brenner, M.; Rueping, M.; Schweizer,
B.; Jaun, B. Chem. Commun. 2001, 207-208. (e) Seebach, D.; Brenner,
M.; Rueping, M.; Jaun, B. Chem.-Eur. J. 2002, 8, 573-584.
(24) (a) Woll, M. G.; Fisk, J. D.; Le Plae, P. R.; Gellman, S. H. J. Am. Chem.
Soc. 2002, 124, 12447-12452. (b) Raguse, T. L.; Lai, J. R.; LePlae, P. R.;
Gellman, S. H. Org. Lett. 2001, 3, 3963-3966. (c) Huck, B. R.; Fisk, J.
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9037.
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A R T I C L E S
Farrera-Sinfreu et al.
Figure 1. Monomers used in these studies: (left) cis-γ-amino-L-proline and (right) L-R,γ-diaminobutyric acid. Peptide synthesis was carried out through the
γ-amino group, to give the γ-peptide backbone. The R-amino group allows diversity to be introduced and mimics the protein R-amino acid side chains.
Figure 2. Synthesis of the NR-alkyl-γ-hexapeptides and the NR-acyl-γ-hexapeptides (synthesis of homooligomeric systems and of oligomers with different
side chains for both cases). In the case of the homooligopeptides (strategy 1), we first synthesized the peptide backbone using the Fmoc strategy and
functionalized the R-amino groups at the end. In the case of the heterofunctionalized γ-hexapeptides (strategy 2), we introduced the corresponding side chain
after each monomer coupling. For the acylation of the R-amino groups to obtain NR-acyl-γ-hexapeptides, we used the corresponding acid, DIPCDI, and
HOBt in DMF (step c for strategy 1 and steps iv and ix for strategy 2). The NR-alkyl-γ-hexapeptides were obtained by alkylation of the R-amino group of
each monomer, through a reductive amination reaction (step c for strategy 1 and steps iv and ix for strategy 2) with the corresponding aldehyde and NaBH3-
CN in 1% HOAc/DMF.
strategy allows the synthesis of either homooligomeric γ-pep-
tides (identical side chains) or heterooligomeric γ-peptides (with
different side chains). In the case of γ-peptide homooligomers,
the backbone was synthesized first, and, after removal of all
Boc protecting groups, all of the side chains were introduced
at the same time. This strategy was carried out for NR-acyl-γ-
peptides and NR-alkyl-γ-peptides (Figure 2, strategy 1). In the
case of the sequentially functionalized γ-peptides, different side
chains were introduced after the coupling of each monomer
(Figure 2, strategy 2).
The γ-peptide backbone was prepared from the protected
amino acid (2S,4S)-Fmoc-4-amino-1-Boc-pyrrolidine-2-carbox-
ylic acid [(2S,4S)-Boc-Amp(Fmoc)-OH] using DIPCDI with
HOBt. The reaction was monitored by the ninhydrin test.26 The
(25) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the
Synthesis of Peptides and Proteins; CRC: Boca Raton, FL, 1997.
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A New Class of Foldamers Based on cis-γ-Amino-L-proline
A R T I C L E S
Figure 3. Chemical structures of the cis-γ-aminoproline and R,γ-diaminobutyric acid peptide oligomers synthesized in this work.
side chain of NR-acyl-γ-peptides was introduced using the
corresponding carboxylic acid and the coupling reagents de-
scribed above. In this case, the reaction was followed either by
the chloranil27 or by the De Clercq28,29 tests which detect
secondary amines. For NR-alkyl-γ-peptides, the alkyl group was
introduced by reductive amination using the corresponding
aldehyde and NaBH3CN. This reaction also was monitored by
the chloranil test.
chemical structures of the γ-hexapeptides prepared in this work
(1-7, 11, and 12) are shown in Figure 3.
Circular Dichroism. The CD spectra of the γ-peptides
reported here were obtained in different solvents: H2O, MeOH,
and TFE in the range 190-250 nm. Peptides were studied in
the concentration range 100-1000 µM. The CD spectra are
independent of concentration, indicating that aggregation does
not occur. The CD spectra of peptides 1, 2, 4, 5, 11, and 12 are
shown in Figure 4. The largest differences in CD spectra are
between γ-peptides derived from γ-aminoproline with acyl or
alkyl side chains. Compounds 2 and 4 belong to the NR-acyl-
γ-peptide family. The CD spectrum of compound 2 in H2O
shows two minima at 204 and 217 nm and a zero crossing at
ca. 200 nm. The CD spectrum of 2 in TFE also shows two
minima, but these are slightly shifted to 202 and 223 nm. In
MeOH, the CD spectrum shows only one negative maxima at
204 nm. The CD spectrum of peptide 4 in H2O shows only one
negative maxima at 207 nm and a zero crossing at ca. 197 nm;
this is similar to the spectrum obtained in MeOH. The CD
spectrum in TFE shows a broad negative band with a negative
maxima at 207 nm. By comparison with the CD spectrum of 2
in the same solvent, the wide band can be interpreted as resulting
from the overlap of the two minima observed for this peptide.30
Peptides 1 and 5 are representative of the NR-alkyl-γ-peptides
derived from γ-aminoproline and show CD patterns that are
markedly different from those of 2 and 4. The CD spectra of 1
and 5 each show a positive maxima at ca. 200 nm in the three
The γ-peptide family based on R,γ-diaminobutyric acid was
prepared following the same synthetic strategy using NR-Boc-
Nγ-Fmoc-L-diaminobutyric acid.
At the end of the synthesis, and after removal of the Fmoc
group, the N-terminal amino group was acetylated. However,
in a few cases, the terminal amino group was kept free (4, 7) to
increase solubility in H2O. Peptides were cleaved from the resin
by acidolytic treatment with anhydrous HF. The purity of the
crude γ-peptides ranged from 70% to 95% as determined by
HPLC. Compounds were purified to >95% homogeneity by
preparative reversed-phase HPLC prior to characterization by
electrospray and/or MALDI-TOF mass spectrometry. All com-
pounds gave results consistent with the desired products. The
(26) The γ-amino functions of these building blocks anchored on the resin give
a clear positive Kaiser ninhydrin test, so this test is useful in controlling
the synthesis of this kind of peptide. Kaiser, E.; Colescott, R L.; Bossinger,
C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 594-598.
(27) Christensen, T. Acta Chem. Scand. 1979, 33, 760-766.
(28) Madder, A.; Farcy, N.; Hosten, N. G. C.; De Muynck, H.; De Clercq, P.;
Barry, J.; Davis, A. P. Eur. J. Org. Chem. 1999, 11, 2787-2791.
(29) Although the De Clercq test seems more sensitive than the the chloranil
test (De Clercq gives slightly positive when chloranil gives negative), the
final product whose synthesis has been controlled by just the chloranil test
is good to excellent.
(30) This different intensities of the CD bands between 2 and 4 could be related
to the presence of an aromatic side chain in 4 or to the increased bulkiness
of the substituents.
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A R T I C L E S
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Figure 4. Circular dichroism spectra for peptide hexamers (1, 2, 4, 5, 11, and 12) in (A) H2O, (B) MeOH, and (C) TFE at 25 °C. Data were normalized
for γ-peptide concentration and number of monomer groups.
solvents studied. Compound 1 in H2O at pH ) 12.1 (results
not shown) and compound 5 in methanol both show a weak
negative maxima at ca. 220-230 nm. The CD spectrum of
peptide 5 in TFE shows a more intense minimum at ca. 220
nm, thus giving a CD pattern more similar to that observed for
the NR-acyl-γ-peptide family.
Peptide 12 (derived from R,γ-diaminobutyric acid, with a free
R-amino group) presents a CD pattern similar to that of the
NR-alkyl-γ-peptides derived from γ-aminoproline. The corre-
sponding acylated form (peptide 11) has completely different
CD spectra in H2O and TFE. In TFE, peptide 11 gives rise to
a CD spectrum resembling that of the acylated γ-peptides
derived from γ-aminoproline. In H2O, on the other hand, 11
shows a completely different spectrum that is characterized by
a maximum at ca. 210 nm.
The CD pattern in each family of γ-peptides was compared
Figure 5. Circular dichroism spectra for the different peptide oligomers
to those observed for monomeric model compounds that were
in H2O at 25 °C. The data have been normalized for γ-peptide concentration
representative of each class. The CD spectra in H2O of each
γ-peptide family resemble that of its corresponding model
compound when the data are normalized in terms of the number
of amide chromophores. The CD spectral features observed for
alkyl and acyl peptides should therefore be attributed mainly
to interactions involving the additional amide chromophore in
the second family (Figure 5). This situation is not unexpected
given that interactions between chromophores responsible for
the CD spectra are distance-dependent and the interactions
between the backbone and side-chain amide bonds in the NR-
acyl-γ-peptides are equivalent to the interaction between
adjacent backbone peptide bonds in R-peptides. It is these latter
interactions that give rise to the well-known CD spectra of
natural peptides.
and number of monomer groups.
γ-aminoproline peptides, the CD signal at around 220 nm
changes from positive (in H2O) to negative (in MeOH and TFE).
Interestingly, the absence of amide chromophores in the side
chains indicates that the observed changes in the CD spectra
reflect a different conformation of the peptide backbone in H2O
than in TFE or MeOH. Backbone cyclization has a major effect
on the structure of γ-peptides, as deduced from the CD spectra
of 11 and 2 in H2O and TFE. The CD spectrum of 11 changes
from having a positive maximum in H2O to one characterized
by two minima, while the spectra of 2 show two minima in all
solvents studied. This behavior suggests that the spectra with
two minima could correspond to a structural feature that is
preserved in all solvents in the peptide with a cyclized backbone,
but is only present in TFE when the backbone is not restrained.
Within each family, the CD spectra show significant solvent
dependency. In acylated peptide 2, derived from γ-aminoproline,
the long wavelength minimum is shifted from 217 to 225 nm
on changing solvent from H2O to TFE. The corresponding peak
appears as a shoulder in MeOH. More strikingly, in alkylated
The large differences in the CD spectra between alkylated
and acylated peptides 11 and 12 suggest some structural role
9
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A New Class of Foldamers Based on cis-γ-Amino-L-proline
A R T I C L E S
Figure 6. 1H NMR spectrum of γ-peptide 2 in H2O plus 10% D2O. The inset shows an expansion of the carboxamide region. Labeled peaks correspond
to different isomers. The relative intensities are (a)1:(b)2.8:(c)0.46:(d)3.4:(e)10.8.
for the amide group in the side chain, at least in the peptides
and the relative stabilities of the trans and cis isomers are the
same as in the monomer (3:1).
formed by noncyclic γ-amino acids.
Nuclear Magnetic Resonance. The H NMR spectrum of
1
The presence of strong hydrogen bonds involving the main-
chain amide groups can be ruled out on the basis of the
γ-peptide 2 in H2O/D2O (9:1) is shown in Figure 6.31 The
presence of different sets of signals is evident, and this
corresponds to different conformations in slow exchange. On
considering the carboxamide signals (inset in Figure 6), it can
be seen that at least five different conformations are present.
The different conformations can be traced to the presence of
possible side-chain cis-trans isomers for each of the side-chain
amide groups. This situation was confirmed by the observation
of two isomers in 3:1 relative populations in the model monomer
10, which contains an acetyl side chain, and a single population
for monomer 9 with a free R-amino group (spectra not shown).
observation of the same temperature coefficients (-6 ppb K-1
)
for all of the observed amide protons, a fact that suggests a
flexible structure.
However, the 3JNH-γ couplings of all internal amide protons
show values of around 7.5 Hz, which are slightly higher than
the expected 6.5 Hz for a freely rotating group, as observed for
the N-terminal amide proton. In addition, the presence of a
number of interresidue NOEs suggests a preferred average
conformation of proline γ-peptides.
NOE spectra were obtained in the rotating frame as the global
correlation tune of the peptides studied gave only very weak
NOEs in the laboratory frame. In addition, strong overlap due
to the highly repetitive sequence of the peptides studied
prevented complete sequential assignment of the backbone
protons. However, the observation of an NOE in the NR-acyl
family between protons located in opposite faces of the proline
ring was interpreted as evidence of interresidue interactions.
Indirect NOE (three-spin effects) can be ruled out in rotating
frame experiments as the resulting cross-peaks would have
The number of possible isomers in the hexamer is 64 (26).
However, the chemical shift of the C-terminal carboxamide
depends mainly on the conformation of the side chain of the
last residues. The relative populations of the four major isomers
observed in the carboxamide region are close to 9:3:3:1. These
are the expected populations if we consider that the conforma-
tions of neighboring side chains are independent of each other
(31) Peptides were practically not soluble in CDCl3, but were soluble in H2O,
and some of them were soluble in MeOH and TFE.
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A R T I C L E S
Farrera-Sinfreu et al.
Figure 7. Top: structural model for peptide 2 deduced from NMR data. Bottom: related structure for peptide 2 derived by reorientation of the N- and
C-terminal proline residues, with the observed NOE contacts indicated.
opposite signs. Interresidue NOEs are observed between protons
γ and â′, R-â′ and γ-δ′, where the “prime” symbol denotes
the proton trans to the R proton of the proline ring. γ-Peptides
containing a free or an alkylated R-amino group show a slightly
different pattern of interresidue NOEs. The R-â′ strong NOE
is still observed. However, the â′-γ NOE is not observed in
the free peptide 1 and is very weak in the methylated peptide
5. Additional strong NOEs are observed between protons â, R,
and δ′.
in the structures shown in Figure 7 by rotation of the C- and
N-terminal proline groups. Due to the evidence of flexibility,
no attempt to derive a single structure using restrained mini-
mization was attempted. The suggested structure for peptide 2
in H2O is an idealized model for the folding of proline-derived
γ-peptides. The structure can be described as a series of turns
in which the two amide bonds connected to the R and γ positions
of each proline are in the same plane, which is perpendicular
to the average plane of the proline rings. In aqueous solution,
however, hydrogen bonds are not formed, a situation indicated
by the temperature coefficients and the coupling constants
measured on NH residues. In the minimized structure, protons
The observed interresidue NOEs cannot be explained as
interactions between consecutive residues. However, the NOEs
are consistent with short distances between residues i and i+2
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6054 J. AM. CHEM. SOC. VOL. 126, NO. 19, 2004

A New Class of Foldamers Based on cis-γ-Amino-L-proline
A R T I C L E S
cm2 dmol-1), calculated per mol of total amide groups (chromophores)
present in the different molecules.
R and γ in prolines i and i+2 are at 5 Å. This structure also
accounts for the observed coupling constant of 7.5 Hz JNH-γ
3
NMR Spectroscopy. NMR spectra were acquired on a Bruker 500
spectrometer. γ-Hexapeptides were dissolved in H2O/D2O (9:1) at
concentrations of ∼3 mg/mL at 25 °C apart from the temperature
experiments, which were collected between 5 and 30 °C under the
control of a Eurotherm variable-temperature unit with an accuracy of
0.1 °C. Two-dimensional spectra (COSY, TOCSY, NOESY, and
ROESY) were recorded employing standard pulse sequences with the
number of acquisitions typically set to 64 for the COSY, NOESY, and
ROESY and 32 for the TOCSY experiments. Presaturation was used
to suppress the water resonance. TOCSY spectra were recorded with
an isotropic mixing time of 70 ms. NOESY spectra were acquired with
mixing times of 150 and 400 ms. ROESY spectra were collected with
mixing times of 150 and 200 ms. The temperature coefficients of the
amide protons for compound 2 were studied by acquiring monodimen-
sional spectra at six different temperatures between 278 and 303 K in
5° increments and are reported in -ppb/K. All spectra were processed
with VNMR on an PC computer.
as the dihedral angle between the two bonds is close to 0°.
Concluding Remarks
The γ-amino-L-proline is a very convenient building block
for the preparation of γ-peptides. The use of two orthogonal
protecting groups for both amino groups of the building block
leads to a flexible synthesis strategy for alkyl- and acyl-
substituted γ-peptides, allowing a convenient method for the
preparation of both homo- and heterooligomers.
The CD spectra of alkylated peptides suggest that proline-
containing γ-peptides show solvent-dependent secondary struc-
tures. In addition, comparison of the CD spectra of γ-peptides
prepared from γ-amino-L-proline and γ-aminobutyric acid in
different solvents indicates that the proline-derived γ-peptides
retain in aqueous solutions a conformation that is present only
in TFE solution in the linear analogues. NMR spectra in aqueous
solution show interresidue NOEs that are consistent with a
folded structure in which vectors perpendicular to the mean
plane of adjacent proline rings are alternatively pointing up and
down and, moreover, the peptide bonds are in a common plane
perpendicular to those of the proline ring.
The turn-like conformation induced by the γ-aminoproline
skeleton could stabilize one of the smallest conceivable â-me-
ander structures, which could prove useful as a model for an
isolated, â-sheet-like, hydrogen bond ribbon.
Work is in progress to characterize the biological properties
of proline γ-peptides which, as shown in this work, are readily
accessible from a synthetic point of view with a versatility that
opens the way to a variety of applications.
General Procedures. Solid-Phase Synthesis. Peptide syntheses were
performed manually in a polypropylene syringe fitted with a polyeth-
ylene porous disk. Solvents and soluble reagents were removed by
suction. Washings between deprotection, coupling, and subsequent
deprotection steps were carried out with DMF (5 × 1 min) and DCM
(5 × 1 min) using 10 mL of solvent/g of resin each time.
Fmoc Group Removal. (i) DMF (5 × 1 min); (ii) piperidine/DMF
(2:8) (1 × 1 min + 2 × 15 min); (iii) DMF (5 × 1 min).
Boc Group Removal. (i) DCM (5 × 1 min); (ii) TFA/DCM (4:6)
(1 × 1 min + 1 × 30 min); (iii) DCM (5 × 1 min); (iv) DIEA/DCM
(5:95) (3 × 3 min); (v) DCM (5 × 1 min).
Solid-Phase γ-Peptide Backbone Elongation. All syntheses were
carried out with MBHA resin (1 g) by an Fmoc/Boc combined solid-
phase strategy. Couplings of Boc-Amp(Fmoc)-OH (1.58 g, 5 equiv)
and Fmoc-Dab-OH (1.53 g, 5 equiv) were carried out with DIPCDI
(540 µL, 5 equiv) and HOBt (472 mg, 5 equiv) in DMF for 2 h at 25
°C. After the coupling, the resin was washed with DMF (5 × 1 min)
and DCM (5 × 1 min). Couplings were monitored by the Kaiser test.
Synthesis of Homo Nr-Acyl-γ-hexapeptides. After the NR-Boc
groups had been removed, acylation of the R-amino groups was carried
out using RCOOH (30 equiv, 5 equiv for each amine), DIPCDI (540
µL, 30 equiv), and HOBt (472 mg, 30 equiv) in DMF for 2 h at 25 °C.
The resin was washed with DMF (5 × 1 min) and DCM (5 × 1 min).
The acylation was monitored by the chloranil test.
Experimental Section
Materials and Equipment. (2S,4S)-Fmoc-4-amino-1-Boc-pyrroli-
dine-2-carboxylic acid and NR-Boc-Nγ-Fmoc-L-diaminobutyric acid
were obtained from Neosystem (Strasbourg, France), and MBHA resin
(0.7 mmol/g) was supplied by Calbiochem-Novabiochem AG. DIPCDI
was obtained from Fluka Chemika (Buchs, Switzerland), and HOBt
was from Albatross Chem, Inc. (Montreal, Canada). Solvents for peptide
synthesis and RP-HPLC equipment were obtained from Scharlau
(Barcelona, Spain). Trifluoroacetic acid was supplied by KaliChemie
(Bad Wimpfen, Germany). Other chemicals were obtained from Aldrich
(Milwaukee, WI) and were of the highest purity commercially available.
All commercial reagents and solvents were used as received. HF was
obtained from Air Products and Chemicals, Inc. (Allentown, Canada),
and the equipment was from Peptide Institute Inc., Minoh, Osaka, Japan.
Analytical RP-HPLC was performed using Shimadzu (Kyoto, Japan)
or Waters (Milford, MA) chromatography systems with reversed-phase
Kromasil C18 (250 × 4 mm) 10 µm and Symmetry C18 (150 × 4.6
mm) 5 µm columns with UV detection at 220 nm. Semipreparative
RP-HPLC was performed on a Waters (Milford, MA) chromatography
system using Vydac C8 (1 × 25 cm, 10 µm) and Symmetry C8 (3 ×
10 cm, 5 µm) columns. Compounds were detected by UV absorption
at 220 nm. Mass spectra were recorded on a MALDI Voyager DE RP
time-of-flight (TOF) spectrometer (Applied Biosystems, Framingham).
DHB was used as a matrix and was purchased from Aldrich.
Synthesis of Hetero Nr-Acyl-γ-hexapeptides. Once the monomer
had been introduced by coupling of the corresponding protected
monomer, the NR-Boc protecting group was removed and the acyl
function was introduced as above. After removal of the Nγ-Fmoc
protecting group, the reactive sequence was repeated.
Synthesis of Homo Nr-Alkyl-γ-hexapeptides. After the Boc groups
had been removed, alkylation of the R-amino group was performed by
on-resin reductive amination using RCHO (30 equiv, 5 equiv for each
amine) and NaBH3CN (232 mg, 30 equiv) in 1% HOAc in DMF for
2 h. After the reductive amination, the resin was washed with DMF
(5 × 1 min) and DCM (5 × 1 min). The alkylation was monitored by
the chloranil test.
Synthesis of Hetero Nr-Alkyl-γ-hexapeptides. This transformation
was carried out by the sequential acylation methodology, but in this
case the alkylation conditions outlined above were used.
Acetylation. The end terminal Nγ-amino Fmoc protecting group was
removed and was terminated with Ac2O (337 µL, 5 equiv) and DIEA
(607 µL, 5 equiv) in DMF for 2 h. The resin was washed with DMF
(5 × 1 min) and DCM (5 × 1 min).
Circular Dichroism. CD measurements were obtained using a Jasco
model 720 spectropolarimeter at 25 °C. Spectra were obtained in fused
quartz cells with path lengths in the range 0.1-1 mm. Peptide samples
as TFA salts were dissolved in an appropriate amount of the selected
solvent [(H2O (pH in all cases was in the 6.0-6.5 range), MeOH, and
TFE)]. A baseline correction was measured with only solvent in the
cell. Data are expressed in terms of mean residue ellipticity, [θ] (deg
Acidolytic Cleavage with HF. The peptide resin was washed with
MeOH (3 × 1 min), dried, and treated with HF in the presence of 10%
anisole for 1 h at 0 °C. Peptides were precipitated with cold anhydrous
TBME, dissolved in HOAc, and then lyophilized.
9
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A R T I C L E S
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Ac-(γAmp)6-NH2 (1): The crude peptide was purified by preparative
HPLC using a linear gradient of MeCN (containing 1% of TFA) and
H2O (containing 1% of TFA). The purity of each fraction was verified
by analytical HPLC and MALDI-TOF and showed that the peptides
were 95-99% pure. 1H NMR [H2O/D2O (9:1), 500 MHz]: δ ) 1.848-
1.945 (m, 5H; 5Hâ); 1.962 (s, 3H; CH3); 1.998-2.052 (m, 1H; Hâ);
2.587-2.647 (m, 5H; Hâ′); 2.673-2.719 (m, 1H; Hâ′); 3.017-3.057
(m, 4H; 4Hδ); 3.001 (dd, Jδ-δ′ ) 12.0 Hz and Jδ-γ ) 4.5 Hz; 1H,
Hδ); 3.200 (dd, Jδ-δ′ ) 12.0 Hz and Jδ-γ ) 4.5 Hz; 1H, Hδ); 3.334-
4.160 (t, JR-â,â′ ) 9 Hz; 1H, HR); 4.226 (t, JR-â,â′ ) 9 Hz; 1H, HR);
4.450-4.511 (m, 1H, Hγ); 4.570-4.645 (m, 5H, 5Hγ); 7.525 (s, 1H,
carboxamide); 8.054 (s, 1H, carboxamide); 8.300 (d, JNH-γ ) 5.5 Hz;
1H, amide Nt); 8.900 (bs.); MS calcd for C38H65N13O7, [M + H]+
816.5202; HR-ESI found, [M + H]+ 816.5212; MALDI-TOF found,
[M + 3H]+ 818.03, [M + Na]+ 840.08, and [M + K]+ 856.09.
Ac-[γAmp(Nr-CH2CH2Ph)]6-NH2 (6): The crude peptide was
purified by preparative HPLC using a linear gradient of MeCN
(containing 1% of TFA) and H2O (containing 1% of TFA). The purity
of each fraction was verified by analytical HPLC and MALDI-TOF
3.383 (m, 4H; Hδ′); 3.398-3.450 (m, 2H; Hδ′); 4.011 (t, JR-â
7.5 Hz; 4H, 4HR); 4.081 (t, JR-â ) 7 Hz, 1H; HR); 4.190 (dd, JR-â
)
)
1
and showed the peptides to be 95-99% pure. H NMR (H2O-D2O
7 Hz and JR-â′ ) 9 Hz, 1H; HR); 4.337-4.427 (m, 6H; 6Hγ); 7.274
(s, 1H; carboxamide); 7.895 (s, 1H; carboxamide); 8.138 (d, JNH-γ
(9:1), 500 MHz): 1.951-2.091 (m, 18H, 6Hâ′ and 6 × CH2); 2.800-
3.117 (m, 18H, 6Hâ and 6 × CH2); 3.268-3.780 (m, 12H, 6Hδ and
6Hδ′); 4.088-4.427 (m, 12H, 6HR and 6Hγ); 7.252-7.402 (m, 30H,
aromatics); 7.464 (s, 1H, carboxamide); 8.007 (s, 1H, carboxamide);
8.264-8.276 (d, JNH-γ ) 6 Hz; 1H, Nt amide); MALDI-TOF calcd
for C80H101N13O7, [M + H]+ 1355.79; found, [M + H]+ 1359.83,
[M + Na]+ 1382.07, and [M + K]+ 1397.05.
)
5.5 Hz, 1H; N-terminal amide); MS calcd for C32H53N13O7, [M + H]+
732.4263; HR-ESI found, [M + H]+ 732.4239; MALDI-TOF found,
732.97 [M + H]+, 754.99 [M + Na]+, and 771.97 [M + K]+.
Ac-[γAmp(Nr-Ac)]6-NH2 (2): The crude peptide was purified by
preparative HPLC using a linear gradient of MeCN (containing 1% of
TFA) and H2O (containing 1% of TFA). The purity of each fraction
was verified by analytical HPLC and MALDI-TOF and showed that
the peptides were 95-99% pure. 1H NMR [H2O/D2O (9:1), 500 MHz]:
1.900-1.983 (m, 6H; Hâ′); 1.973 (s, 3H; Nt methyl); 2.091-2.105
(m, 18H; side-chain methyls); 2.572-2.685 (m, 6H; Hâ); 3.490-3.570
(m, 6H; 6Hδ); 3.916-4.010 (m, 6H; 6Hδ′); 4.328-4.372 (m, 6H; 6HR);
4.442-4.499 (m, 6H; 6Hγ); 7.087 (s, 1H; carboxamide); 7.855 (s, 1H;
carboxamide); 8.126 (d, JNH-γ ) 6.5 Hz, 1H; N-terminal amide); 8.499
(m, JNH-γ around 7.5 Hz, 5H; central amides); these NMR data
correspond to the majority species, although there are other minority
species. MS calcd for C44H65N13O13, [M + Na]+ 1006.4717; HR-ESI
found, [M + Na]+ 1006.4765; MALDI-TOF found, [M + H]+ 984.57,
[M + Na]+ 1006.60, and [M + K]+ 1022.56.
H-{γAmp[Nr-CH2CH2CH(CH3)2]-γAmp(Nr-CH2CH2Ph)}3-NH2
(7): The crude peptide was purified by preparative HPLC using a linear
gradient of MeCN (containing 1% of TFA) and H2O (containing 1%
of TFA). The purity of each fraction was verified by analytical HPLC
1
and MALDI-TOF and showed the peptides to be 95-99% pure. H
NMR (H2O-D2O (9:1), 600 MHz): 0.883-0.919 (m, 18H, 6 × CH3
isopropylic); 1.494-1.598 (m, 6H, 3 × CH2 isopropyl); 1.613-1.686
(m, 3H, 3 × CH isopropyl); 2.007-2.157 (m, 6H, 6Hâ′); 2.200-2.267
(m, 6H, 3 × CH2 phenyl); 2.900-3.144 (m, 12H, 6Hâ and 3 × CH2
phenyl linkage to N); 3.172-3.315 (m, 6H, isopropylic 3 × CH2 linkage
to N); 3.404-3.659 (m, 6H, 6Hδ); 3.760-3.922 (m, 6H, 6Hδ′); 4.253-
4.410 (m, 6H, 6HR); 4.700-4.900 (m, 6H, 6Hγ); 7.291-7.429
(m, 15H, aromatics); 7.554 (s, 1H, carboxamide); 8.060 (s, 1H,
carboxamide); 8.963-8.973 (d, JNH-γ ) 6 Hz; 1H, amide); 9.016-
9.039 (t, JNH-γ ) 7.2 Hz; 2H, central amides); 9.061-9.072 (d,
JNH-γ ) 6.6 Hz; 1H, amide); 9.184 (bc., 1H, Nt amine); MALDI-TOF
calcd for C69H105N13O6, [M + H]+ 1211.83; found, [M + H]+ 1212.79,
[M + Na]+ 1234.76, and [M + K]+ 1250.37.
Ac-[γAmp(Nr-PhAc)]6-NH2 (3): The crude peptide was purified
by preparative HPLC using a linear gradient of MeCN (containing 1%
of TFA) and H2O (containing 1% of TFA). The purity of each fraction
was verified by analytical HPLC and MALDI-TOF and showed that
the peptides were 95-99% pure. MALDI-TOF calcd for C80H89N13O13,
[M + H]+ 1439.67; found, [M + H]+ 1440.28, [M + Na]+ 1464.61,
and [M + K]+ 1479.57. This peptide could be synthesized, but it proved
impossible to obtain the CD and NMR data for solubility reasons.
H-[γAmp(NriV)-γAmp(Nr-PhAc)]3-NH2 (4): The crude peptide
was purified by preparative HPLC using a linear gradient of MeCN
(containing 1% of TFA) and H2O (containing 1% of TFA). The purity
of each fraction was verified by analytical HPLC and MALDI-TOF
and showed the peptides to be 95-99% pure. 1H NMR [H2O/D2O (9:
1), 500 MHz]: 0.910-0.959 (m, 18H; isovaleric methyls); 1.270 (s,
1H; primary amine NH2); 1.800-1.950 (m, 6H; 6Hâ′); 2.037-2.087
(m, 3H; three isovaleric CH); 2.138-2.240 (m, 6H; three isovaleric
methylenes CH2); 2.452-2.567 (m, 6H; 6Hâ); 3.464-3.536 (m, 6H;
6Hδ′); 3.656-3.719 (m, 6H; three CH2 linkage to phenyl); 3.799-
3.896 (m, 6H; 6Hδ); 4.227-4.335 (m, 6H; 6HR); 4.363-4.478 (m,
6H; 6Hγ); 7.162 (s, 1H; carboxamide); 7.187-7.271 (m, 15H; aromatic
protons); 7.811 (s, 1H; carboxamide); 8.561 (d, JNH-γ ) 7.5 Hz, 1H;
amide); 8.616-8.679 (m, 4H; central backbone amides); 8.874 (d,
JNH-γ ) 8 Hz, 1H; amide); these NMR data correspond to the major
species, although there are other minor species. MALDI-TOF calcd
for C69H93N13O12, [M + H]+ 1295.71; found, [M + H]+ 1296.88,
[M + Na]+ 1318.88, and [M + K]+ 1334.85.
H-[γAmp(Nr-Ac)]-NH2 (8): This compound was synthesized in the
solid phase using a Boc/Fmoc strategy. The synthesis was carried out
on 350 µmol of MBHA resin at a substitution level of 0.7 mmol/g.
The Boc-Amp(Fmoc)-OH monomer was first coupled onto the resin
by using the same Fmoc/tBu strategy used above. When the monomer
was coupled to the resin, the NR-Boc protecting group was removed
and the R-amino group was acetylated using Ac2O (170 µL, 5 equiv)
and DIEA (305 µL, 5 equiv) in DMF for 2 h. The Fmoc group was
removed and the peptide cleaved from the resin. The crude peptide
was precipitated with anhydrous diethyl ether, dissolved in HOAc, and
lyophilized. The crude peptide was not purified by preparative HPLC
because the product was obtained in good yield and with excellent
purity. The purity of the sample was verified by analytical HPLC and
1
MALDI-TOF. H NMR [H2O/D2O (9:1), 500 MHz]: 2.010 (s, 3H,
CH3); 2.131 (s, 2H, NH2); 2.175 (ddd, Jâ-â′ ) 15 Hz, Jâ-R ) 4.2 Hz,
Jâ-γ ) 4.2 Hz; 1H, Hâ); 2.745 (ddd, Jâ′-â ) 15 Hz, Jâ′-R ) 9.6 Hz,
Jâ′-γ ) 6.6 Hz; 1H, Hâ′); 3.860 (dd, Jδ-δ′ ) 12 Hz, Jδ-γ ) 2.4 Hz;
1H, Hδ); 4.044 (dd, Jδ′-δ ) 12 Hz, Jδ′-γ ) 6 Hz; 1H, Hδ′); 4.116
(dddd, Jγ-â′ ) 6.6 Hz, Jγ-δ′ ) 6 Hz, Jγ-â ) 4.2 Hz, Jγ-δ ) 2.4 Hz;
1H, Hγ); 4.521 (dd, JR-â′ ) 9.6 Hz, JR-â ) 4.2 Hz; 1H, HR); 7.291
(s, 1H, carboxamide); 8.017 (s, 1H, carboxamide); MS calcd for C7-
H13N3O2, [M + H]+ 172.1080; HR-ESI found, [M + H]+ 172.1073;
MALDI-TOF found, [M + H]+ 171.96, [M + Na]+ 193.94, and
[M + K]+ 209.90.
CH3CH2CO-(γAmp)-NH2 (9): This compound was synthesized in
the solid phase using a Boc/Fmoc strategy with 350 µmol of MBHA
resin at a substitution level of 0.7 mmol/g. The Boc-Amp(Fmoc)-OH
monomer was first coupled onto the resin by a general Fmoc/tBu
strategy. The coupling of Boc-Amp(Fmoc)-OH (790 mg, 5 equiv) was
carried out with the same protocol used above. When the monomer
Ac-[γAmp(Nr-Me)]6-NH2 (5): The crude peptide was purified by
preparative HPLC using a linear gradient of MeCN (containing 1% of
TFA) and H2O (containing 1% of TFA). The purity of each fraction
was verified by analytical HPLC and MALDI-TOF and showed the
1
peptides to be 95-99% pure. H NMR [H2O/D2O (9:1), 500 MHz]:
1.986 (s, 3H, CH3 acetyl); 2.053-2.141 (m, 4H, Hâ′); 2.201-2.211
(m, 2H, Hâ′); 2.824 (s, 9H, 3 × CH3); 2.845 (s, 3H, CH3); 2.901 (s,
3H, CH3); 2.911-2.974 (m, 9H, 6 Hâ and one CH3); 3.402-3.569
(m, 6H, 6Hδ); 3.612-3.779 (m, 6H, 6Hδ′); 3.991-4.086 (m, 4H, 4HR);
9
6056 J. AM. CHEM. SOC. VOL. 126, NO. 19, 2004

A New Class of Foldamers Based on cis-γ-Amino-L-proline
A R T I C L E S
had been coupled to the resin, the Fmoc group was removed and the
acylation of the γ-amino group was carried out using CH3CH2COOH
(130 µL, 5 equiv), DIPCDI (270 µL, 5 equiv), and HOBt (236 mg, 5
equiv) in DMF for 2 h. After the coupling was complete, the NR-Boc
protecting group was removed. Peptides were cleaved from the resin
with anhydrous HF in the presence of 10% anisole for 1 h at 0 °C. The
crude peptide was precipitated with anhydrous diethyl ether, dissolved
in HOAc, and lyophilized. The crude peptide was not purified by
preparative HPLC because the target compound was obtained in good
yield and with excellent purity. The purity of the sample was verified
by analytical HPLC and MALDI-TOF. 1H NMR [H2O/D2O (9:1), 500
MHz]: 1.075 (t, JCH3-CH2 ) 7.8 Hz; 3H, CH3); 2.001 (s, 1H, NH);
2.161 (ddd, Jâ′,â ) 14.4 Hz, Jâ′,R ) 7.2 Hz, Jâ′,γ ) 7.2 Hz; 1H, Hâ′);
2.236 (q, JCH2-CH3 ) 7.8 Hz; 2H, CH2); 2.800 (ddd, Jâ,â′ ) 13.8 Hz,
Jâ,R ) 7.2 Hz, Jâ,γ ) 7.2 Hz; 1H, Hâ); 3.405 (dd, Jδ-δ′ ) 12 Hz,
Jδ-γ ) 5.4 Hz; 1H, Hδ); 3.657 (dd, Jδ′-δ ) 12 Hz, Jδ′-γ ) 7.2 Hz; 1H,
Hδ′); 4.480-4.518 (m, 2H, HR and Hγ); 7.407 (s, 1H, carboxamide);
7.952 (s, 1H, carboxamide); 8.110 (s, 1H, amide); MS calcd for
C8H15N3O2, [M + H]+ 186.1237; HR-ESI found, [M + H]+ 186.1232;
MALDI-TOF found, [M + H]+ 186.03, [M + Na]+ 208.00, and
[M + K]+ 223.97.
Jδ′-γ ) 6.6 Hz; 1H, Hδ′); 4.401-4.449 (m, 2H, HR and Hγ); 7.098 (s,
1H, carboxamide); 7.778 (s, 1H, carboxamide); 8.011 (d, JNH-γ
)
5 Hz; 1H, amide). These NMR data correspond to the majority
compound; in the NMR spectrum is observed another minority species
corresponding to the cis isomer in the side chain with relative population
3:1. MS calcd for C10H17N3O3, [M + Na]+ 250.1162; HR-ESI found,
[M + Na]+ 250.1150; MALDI-TOF found, [M + H]+ 227.92,
[M + Na]+ 249.94, and [M + K]+ 265.86.
Ac-[Dab(Nr-Ac)]6-NH2 (11): The crude peptide was purified by
preparative HPLC using a linear gradient of MeCN (containing 1% of
TFA) and H2O (containing 1% of TFA). The purity of each fraction
was verified by analytical HPLC and MALDI-TOF and showed the
1
peptides to be 95-99% pure. H NMR [H2O/D2O (9:1), 500 MHz]:
1.788-1.878 (m, 6H, Hâ); 1.971-2.060 (m, 27H; 6Hâ, 3H backbone
methyls and 18H side-chain methyls); 3.236-3.325 (m, 12H, Hγ);
4.138-4.180 (m, 4H, HR); 4.196-4.238 (m, 2H, HR); 7.093 (s, 1H,
carboxamide); 7.695 (s, 1H, carboxamide); 7.966 (t, JNH-γ ) 5.5 Hz;
1H, amide Nt); 8.128-8.162 (m, 5H, backbone amides); 8.326-8.374
(m, 6H, side-chain amides); MS calcd for C38H65N13O13, [M + Na]+
934.4717; HR-ESI found, [M + Na]+ 934.4741; MALDI-TOF found,
[M + Na]+ 936.34 and [M + K]+ 952.30.
CH3CH2CO-[γAmp(Nr-Ac)]-NH2 (10): This compound was syn-
thesized in the solid phase using a Boc/Fmoc strategy. The synthesis
was carried out on 350 µmol of MBHA resin at a substitution level of
0.7 mmol/g. The coupling of Boc-Amp(Fmoc)-OH (790 mg, 5 equiv)
was carried using the same protocol described above. The NR-Boc
protecting group was removed, and the acylation of the R-amino group
was carried out using Ac2O (84 µL, 5 equiv) and DIEA (155 µL, 5
equiv) in DMF for 2 h. The Fmoc group was removed, and the acylation
of the γ-amino group was carried out using CH3CH2COOH (130 µL,
5 equiv), DIPCDI (270 µL, 5 equiv), and HOBt (236 mg, 5 equiv) in
DMF for 2 h. The peptide was cleaved from the resin with anhydrous
HF in the presence of 10% anisole for 1 h at 0 °C. The crude peptide
was precipitated with anhydrous diethyl ether, dissolved in HOAc, and
lyophilized. The crude peptide was not purified by preparative HPLC
because the compound was obtained in good yield and with excellent
purity. The purity of the sample was verified by analytical HPLC
Ac-(Dab)6-NH2 (12): The crude peptide was purified by preparative
HPLC using a linear gradient of MeCN (containing 1% of TFA) and
H2O (containing 1% of TFA). The purity of each fraction was verified
by analytical HPLC and MALDI-TOF and showed the peptides to be
1
95-99% pure. H NMR [H2O/D2O (9:1), 600 MHz]: 2.002 (s, 3H,
CH3 acetyl); 2.031-2.172 (m, 12H, Hâ); 3.290-3.350 (m, 6H, Hγ);
3.399-3.485 (m, 6H, Hγ); 3.537-3.567 (m, 2H, amines); 3.631-3.658
(m, 2H, amines); 3.757-3.793 (m, 2H, amines); 3.959-4.056 (m, 6H,
HR); 7.396 (s, 1H, carboxamide); 7.935 (s, 1H, carboxamide); 8.110
(t, J ) 5 Hz; 1H, amide Nt); 8.440-8.700 (m, 5H, backbone amides);
MS calcd for C26H53N13O7, [M + H]+ 660.4263; HR-ESI found,
[M + H]+ 660.4269; MALDI-TOF found, [M + H]+ 661.87,
[M + Na]+ 683.88, and [M + K]+ 699.88.
Acknowledgment. The work was partially supported by funds
from CICYT (BQU2002-02047 and BQU2003-00089), Gener-
alitat de Catalunya (Grup Consolidat and Centre de Refere`ncia
en Biotecnolog´ıa), and Barcelona Science Park.
1
and MALDI-TOF. H NMR [H2O/D2O (9:1), 500 MHz]: 1.081 (t,
JCH3-CH2 ) 7.8 Hz; 3H, CH3); 1.974-2.019 (m, 1H, Hâ); 2.116 (s,
3H, methyl NR); 2.212-2.238 (q, JCH2-CH3 ) 7.8 Hz; 2H, CH2); 2.618
(ddd, Jâ,â ) 13.8 Hz, Jâ,R ) 9 Hz, Jâ,γ ) 6 Hz; 1H, Hâ); 3.527 (dd,
Jδ-δ′ ) 11.1 Hz, Jδ-γ ) 5.4 Hz; 1H, Hδ); 3.975 (dd, Jδ′-δ ) 11.1 Hz,
JA0398621
9
J. AM. CHEM. SOC. VOL. 126, NO. 19, 2004 6057