Self-assembling cyclic tetrapeptide from alternating C-linked carbo-β-amino acid [(S)-β-Caa] and α-aminoxy acid [(R)-ama]: A selective chloride ion receptor

Article abstract of DOI:10.1021/jo901923q

(Graph Presented) A cyclic tetrapeptide is prepared from alternating (S)-β-Caa (C-linked carbo-β-amino acid) and (R)-Ama (R-aminoxy acid). Extensive NMR (in CDCl₃ solution) and mass spectral (MS) studies show its halide binding capacity, with a special affinity to the chloride ion. At higher concentration it was found to form molecular aggregates as evidenced from transmission electron microscopic and atomic force microscopic analysis, confirming the formation of nanorods.

Full text of DOI:10.1021/jo901923q

pubs.acs.org/joc
Self-Assembling Cyclic Tetrapeptide from Alternating C-Linked
Carbo-β-amino Acid [(S)-β-Caa] and r-Aminoxy Acid [(R)-Ama]:
A Selective Chloride Ion Receptor
Gangavaram V. M. Sharma,*,† Vennampalli Manohar,† Samit Kumar Dutta,‡
Bojja Sridhar,§ Venna Ramesh, Ragampeta Srinivas, and Ajit C. Kunwar*,‡
†Organic Chemistry Division III, ‡Centre for Nuclear Magnetic Resonance, §National Centre for
Mass Spectrometry, and Inorganic and Physical Chemistry Division, Indian Institute
of Chemical Technology (CSIR), Hyderabad 500 607, India
esmvee@iict.res.in; kunwar@iict.res.in
Received September 11, 2009
A cyclic tetrapeptide is prepared from alternating (S)-β-Caa (C-linked carbo-β-amino acid) and
(R)-Ama (R-aminoxy acid). Extensive NMR (in CDCl3 solution) and mass spectral (MS) studies
show its halide binding capacity, with a special affinity to the chloride ion. At higher concentration it
was found to form molecular aggregates as evidenced from transmission electron microscopic and
atomic force microscopic analysis, confirming the formation of nanorods.
Introduction
for the surfeit of activity on cyclic β- and hybrid peptides.
Further, Yang et al.6 have reported a new class of cyclic
Design and synthesis of β- and hybrid peptides from
R- and homologous unnatural amino acids with interesting
secondary and higher order structures has been a fascinating
area of research in biopolymers.1 For the first time in 1972,
Hassal et al.2 predicted the cyclic tetrapeptide, derived from
1:1 R- and β-amino acids, to form nanotubular structures.
Since a vast number of naturally occurring cyclic peptides
were shown to display biological activities,3 research on the
cyclic peptides from unnatural amino acids has drawn con-
siderable attention. The observation from Seebach et al.4 on
stacking through intermolecular H-bonding in a cyclic
β-tetrapeptide and the findings of Ghadiri et al.5 on the
transmembrane channels in β-peptides has been responsible
(1) (a) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S.
Chem. Rev. 2001, 101, 3893–4011. (b) Seebach, D.; Matthews, J. L. Chem.
Commun. 1997, 2015–2022. (c) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173–
180. (d) Kirschenbaum, K.; Zuckerman, R. N.; Dill, D. A. Curr. Opin. Struct.
Biol. 1999, 9, 530–535. (e) Stigers, K. D.; Soth, M. J.; Nowick, J. S. Curr.
Opin. Chem. Biol. 1999, 3, 714–723. (f) Smith, M. D.; Fleet, G. W. J. J. Pept.
Sci. 1999, 5, 425–441. (g) Cheng, R. P.; Gellman, S. H.; De Grado, W. F.
Chem. Rev. 2001, 101, 3219–3232. (h) Venkatraman, J.; Shankaramma, S. C.;
Balaram, P. Chem. Rev. 2001, 101, 3131–3152. (i) Cubberley, M. S.; Iverson,
€ €
B. L. Curr. Opin. Chem. Biol. 2001, 5, 650–653. (j) Martinek, T. A.; Fulop, F.
Eur. J. Biochem. 2003, 270, 3657–3666. (k) Seebach, D.; Beck, A. K.;
Bierbaum, D. J. Chem. Biodivers. 2004, 1, 1111–1239. (l) Goodman, C. M.;
Choi, S.; Shandler, S.; DeGrado, W. F. Nature Chem. Biol. 2007, 3, 252–262.
(m) Hecht, S., Huc, I., Eds. Foldamers: Structure, Properties and Applications;
Wiley-VCH: Weinheim, Germany, 2007.
DOI: 10.1021/jo901923q
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Published on Web 01/22/2010
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2010 American Chemical Society

Sharma et al.
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peptides derived from R-aminoxy acids,6a R-aminoxy acids
with R-amino acids,6b and small organic molecules6c as selective
chloride ion receptors.7 The advantage of the design in these
peptides, unlike the R-andβ-cyclic peptides, is that the aminoxy
amide8 NH has higher acidity over the normal amide NH and,
hence, becomes a better binder of anions. Similarly, Le Grel
et al.9a reported aza β3-cyclopeptides, as a different class of
cyclic peptides, from aza-β3-amino acids.9b,c In continuation of
our studies10 on peptides from unnatural amino acids with
carbohydrate side chains, herein we report the synthesis and
structural investigations of linear peptide 4 and cyclic tetrapep-
tide 5 (Figure 1) from 1:1 alternating (S)-β-Caa 1 (C-linked
carbo β-amino acid) and R-aminoxy acid 2 (R-Ama) using
NMR, mass, MD, transmission electron microscopy (TEM),
and atomic force microscopy (AFM).
Unlike the oligomers of R-aminoxy acid (Ama), which
provided a novel class of very robust foldamers, stabilized
by N-O turns,8 the peptides reported11 by our group pre-
pared from (R)-Ama and (R)-β-Caa with 1:1 alternation,
demonstrated a “new motif” for a 12/10-mixed helix. In these
oligomers, the conformational preference of β-Caa governs
that of (R)-Ama, driving it to behave more like a (S)-β2-amino
acid, thus resulting in pseudo β2/β3-peptides. This investiga-
tion is also pertinent, as we were intrigued by the fact that side
chains play a special role in controlling the secondary struc-
tures.11 To further explore the competing conformational
preferences of these monomers, we present our studies
on cyclic peptide derived from alternating (S)-β-Caa and
(2) Hassal, C. H. In Chemistry and Biology of Peptides: Proceedings of the
Third American Peptide Symposium; Meinhoffer, J., Ed.; Ann Arbor Sci.:
Ann Arbor, MI, 1972; pp 153-157.
(3) Liskamp, R. M. J.; Rijkers, D. T. S.; Bakker, S. E. Bioactive Macro-
cyclic Peptides and Peptide Mimetics. In Modern Supramolecular Chemistry:
Strategies for Macrocycle Synthesis; Diederich, F., Stang, P. J., Tykwinski,
R. R., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2008.
(4) Seebach, D.; Matthews, J.; Meden, A.; Wessels, T.; Baerlocher, C.;
McCusker, L. B. Helv. Chim. Acta 1997, 80, 173–182.
(5) (a) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.;
Khazanovich, N. Nature 1993, 366, 324–327. (b) Clark, T. D.; Buehler, L. K.;
Ghadiri, M. R. J. Am. Che, Soc. 1998, 120, 651–656. (c) Bong, D. T.; Clark,
T. D.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 988–
1011.
(6) (a) Yang, D.; Qu, J.; Li, W.; Zhang, Y.-H.; Ren, Y.; Wang, D.-P.; Wu,
Y.-D. J. Am. Chem. Soc. 2002, 124, 12410–12411. (b) Yang, D.; Li, X.; Sha,
Y.; Wu, Y.-D. Chem.;Eur. J. 2005, 11, 3005–3009. (c) Li, X.; Shen, B.; Yao,
X.-Q.; Yang, D. J. Am. Chem. Soc. 2007, 129, 7264–7265.
(7) (a) Gerenscser, G. Chloride Transport Coupling in Biological Mem-
branes and Epithelia; Elsevier: Amsterdam, 1984. (b) Aschroft, F. M. Ion
Channels and Disease; Academic Press: New York, 2000.
FIGURE 1. Structures of hybrid peptides 3-5.
(R)-Ama. In contrast to the linear oligomers with alternating
(R)-β-Caa and an (R)-Ama, which could not be cyclized, the
cyclization of tetrapeptide with alternating (S)-β-Caa and an
(R)-Ama was rather facile. This observation is analogous to
the inference drawn by Le Grel et al.9 in aza-β3-peptides.12
Similar observations were made by Yang et al.13 in the
oligomers of R-aminoxy acids where the chirality switch from
homo- to heterochiral resulted in helical scaffolds and cyclized
systems, respectively. As the amide protons of the R-aminoxy
acid are known to be better H-bond donors while interacting
with anions,6b,8 in the present study, the anion-binding pro-
perties of cyclic-[(S)-β-Caa-(R)-Ama-(S)-β-Caa-(R)-Ama] 5
(Figure 1) have been explored in detail. Further, the cyclic
peptide 5 has favored the molecular assembly, and we have
shown the formation of nanorods.
(8) Li, X.; Yang, D. Chem. Commun. 2006, 3367–3379.
(9) (a) Salaun, A.; Mocquer, C.; Perochon, R.; Lecorgne, A.; Le Grel, B.;
Potel, M.; Le Grel, P. J. Org. Chem. 2008, 73, 8579–8582. (b) Cheguillaume,
A.; Salaun, A.; Sinbandhit, S.; Potel, M.; Gall, P.; Baudy-Floc’h, M.;
Le Grel, P. J. Org. Chem. 2001, 66, 4923–4929. (c) Cheguillaume, A.;
Doubli-Bounoua, I.; Baudy-Floc’h, M.; Le Grel, P. Synlett 2000, 331–334.
(d) Le Grel, P.; Salaun, A.; Potel, M.; Le Grel, B.; Lassagne, F. J. Org. Chem.
2006, 71, 5638–5645.
Results and Discussion
(10) (a) Sharma, G. V. M.; Ravinder Reddy, K.; Radha Krishna, P.;
Ravi Sankar, A.; Narsimulu, K.; Kiran Kumar, S.; Jayaprakash, P.;
Jagannadh, B.; Kunwar, A. C. J. Am. Chem. Soc. 2003, 125, 13670–13671.
(b) Sharma, G. V. M.; Ravinder Reddy, K.; Radha Krishna, P.; Ravi Sankar,
A.; Jayaprakash, P.; Jagannadh, B.; Kunwar, A. C. Angew. Chem., Int. Ed.
2004, 43, 3961–3965. (c) Sharma, G. V. M.; Nagender, P.; Radha Krishna, P.;
Jayaprakash, P.; Ramakrishna, K. V. S.; Kunwar, A. C. Angew. Chem., Int.
Ed. 2005, 44, 5878–5882. (d) Sharma, G. V. M.; Jadhav, V. B.; Ramakrishna,
K. V. S.; Jayaprakash, P.; Narsimulu, K.; Subash, V.; Kunwar, A. C. J. Am.
Chem. Soc. 2006, 128, 14657–14668. (e) Srinivasulu, G.; Kiran Kumar, S.;
Sharma, G. V. M.; Kunwar, A. C. J. Org. Chem. 2006, 71, 8395–8400.
(f) Sharma, G. V. M.; Babu, B. S.; Ramakrishna, K. V. S.; Nagendar, P.;
Kunwar, A. C.; Schramm, P.; Baldauf, C.; Hofmann, H.-J. Chem.;Eur. J.
2009, 15, 5552–5566.
Synthesis of Hybrid Peptides 3-5. The peptides 3-5
(Figure 1) were synthesized in solution phase from the
(12) (a) De Santis, P.; Morosetti, S.; Rizzo, R. Macromolecules 1974, 7,
52–58. (b) Khazanovich, N.; Granja, J. R.; McRee, D. E.; Milligan, R. A.;
Ghadiri, M. R. J. Am. Chem. Soc. 1994, 116, 6011–6012. (c) Hartgerink,
J. N.; Granja, J. R.; Milligan, R. A.; Ghadiri, M. R. J. Am. Chem. Soc. 1996,
118, 43–50. (d) Clark, T. D.; Buriak, J. M.; Kobayashi, K.; Isler, M. P.;
McRee, D. E.; Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 8949–8962.
(e) Bong, D. T.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 2163–2166.
ꢀ
(f) Rosenthal-Aizman, K.; Svensson, G.; Unden, A. J. Am. Chem. Soc. 2004,
126, 3372–3373. (g) Ashkenasy, N.; Horne, W. S.; Ghadiri, M. R. Small 2006,
1, 99–101.
(13) Li, X.; Wu, Y.-D.; Yang, D. Acc. Chem. Res. 2008, 41, 1428–1438.
(11) Sharma, G. V. M.; Manohar, V; Dutta, S. K.; Subash, V; Kunwar,
A. C. J. Org. Chem. 2008, 73, 3689–3698.
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SCHEME 1. Synthesis of Peptides 3-5a
aKey: (a) N2H4 .H2O, CH3OH, rt, 2 h; (b) aq 4 N NaOH, CH3OH, 0 °C to rt, 2 h; (c) HOBt, EDCI, DIPEA, dry CH2Cl2, 0 °C-rt, 5 h; (d) CF3COOH,
3
dry CH2Cl2, 0 °C-rt, 2 h; (e) EDCI, pentafluorophenol (PFP), dry CH2Cl2, 0 °C-rt, 2 h; (f) DIPEA, dry CH3CN, 70 °C, 3 h.
monomers 1 and 6. Accordingly, ester 1 (Scheme 1) was
subjected to hydrolysis with aq 4 N NaOH at room temp-
erature to afford the acid 7 in 92% yield. Coupling of the acid
7 with the ester 2 (prepared from 611 with hydrazine hydrate
in methanol) in the presence of EDCI and HOBt in CH2Cl2
at room temperature for 5 h furnished the dipeptide 3 in 67%
yield.
Base (aq 4 N NaOH) hydrolysis of dipeptide 3 gave the
acid 9, while the corresponding amine salt 10 was derived
from 3 on exposure to CF3COOH in CH2Cl2 for 2 h. The
thus-derived acid 9 was then coupled (EDCI, HOBt,
DIPEA) with amine 10 in CH2Cl2 for 5 h to furnish the
pseudo β2- and β3-tetrapeptide 4 in 52% yield. Reaction of 4
with NaOH in CH3OH gave the corresponding acid 11
(92%), which on reaction with pentafluorophenol14 in the
presence of EDCI in CH2Cl2 at room temperature for 2 h
furnished the ester 12. Further, ester 12 on exposure to
CF3COOH in CH2Cl2 for 2 h was converted into the
corresponding amine salt 13. Slow addition of a solution of
13 into acetonitrile containing DIPEA at 70 °C for 2 h
furnished the cyclic tetrapeptide 5 in 55% yield.
Conformational Analysis. The NMR studies on peptides 4
(3.5 mM) and 5 (1.3 mM) were undertaken in CDCl3 solution.
For the linear tetrapeptide 4, it was difficult to obtain detailed
structural information due to the presence of several isomers and
the resulting broadening of the spectral lines and exchange
peaks in the ROESY experiments. For the major isomer, lack
1
of dispersion in the H NMR and non distinctive values of
3JNH-CβH ≈ 8 Hz for the β-Caa residues suggest either an averag-
ing over several structures or absence of a well-defined structure.
This was further confirmed from the solvent titration studies
(adding 33% v/v of DMSO-d6 in CDCl3 solution), wherein
only one amide proton (NH(3)) appeared to be H-bonded as
is reflected in the small change in its chemical shift (Δδ) of
FIGURE 2. (A) Characteristic NOE correlations: β-CaaNH/
β-CaaCRH(pro-S), β-CaaNH/β-CaaC4H, β-CaaC4H/β-CaaCRH-
(pro-S), β-CaaNH/AmaCRH, β-CaaNH/Ama CβH, AmaNH/
β-CaaCRH(pro-R), AmaNH/β-CaaCβH and AmaNH/AmaCRH
are depicted as 1-8, respectively, in the partial structure of peptide
5. (B) One of the lowest energy structures derived from the res-
trained MD studies, where the backbone is highlighted with a band.
0.41 ppm.15 However, several medium range NOE correla-
tions such as NH(1)/C4H(1), CβH(1)/NH(2), CRH(1)/NH(2),
NH(3)/C4H(3), CβH(3)/NH(4), CRH(3)/NH(4), C4H(1)/
NH(2), C4H(3)/NH(4), and NH(2)/NH(3) were observed.
(14) Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.;
Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913–941.
(15) See the Supporting Information.
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FIGURE 3. Negative ion ESI mass spectrum of cyclic peptide 5.
FIGURE 5. (A) Concentration dependence of amide protons and
(B) CRHs of β-Caa of peptide 5 and (C) change of 3JCRH(pro-S)-CβH
with concentration in peptide 5.
C(O)-N-Cβ-CR (φ) ≈ 120° and N-Cβ-CR-C(O) (θ) ≈
-60°. Unlike for the linear analogue from (R)-Ama/(R)-
β-Caa, where the N-O turns are conspicuous by their absence,
these dihedral angles along with the NOE correlations,
β-CaaNH/Ama CRH, β-Caa NH/Ama CβH, Ama NH/
β-Caa CβH, Ama NH/β-Caa CRH(pro-R), and Ama NH/
Ama CRH (Figure 2A), support an 8-membered N-O turn
between carbonyls and amides of the two β-Caa residues
[β-Caa(CO) -β-Caa0(NH) and β-Caa0(CO)-β-Caa (NH)]. One
of the lowest energy structures derived from the restrained
molecular dynamics (MD) studies is shown in Figure 2, where
the backbone bracelet-like structure is highlighted with a band.
Very similar structures have been observed by Yang et al.13 for
16- and 24-membered macrocycles from cyclic-(Ama)4 and
cyclic-(Ama)6, respectively, and a 21-membered macrocycle
of cyclic-(Ama-R amino acid)3.
Anion-Binding Studies. Anion-binding properties were in-
vestigated with the help of NMR and mass spectrometry.
Negative-ion electrospray ionization of a methanolic solution
of the cyclic peptide 5 mixed with NH4F, NH4Cl, NH4Br,
NH4I, NaNO2, NaNO3, NaN3, Na2SO4, NaHSO4, Na3PO4,
Na2HPO4, NaH2PO4, KHCO3, and K2CO3 solutions gave
rise to the spectrum shown in Figure 3. The spectrum shows
FIGURE 4. (A) Change in chemical shift of amide protons and
(B) Job plot of peptide 5 upon addition of anions (Cl-, Br-, and I-).
As discussed above, a facile macrocyclization of 4 is
suggestive of the proximity of two termini. The presence of
only one set of peaks from (R)-Ama and (S)-β-Caa in 5 is
consistent with 2-fold molecular symmetry in the NMR time
scale. The amide protons for the (S)-β-Caa residue appeared
at 8.35 ppm, suggesting their participation in H-bonding.
This was further confirmed from the solvent titration studies,
wherein a very small change in its chemical shift (Δδ) of
0.09 ppm was observed.15 For (S)-β-Caa, the large 3JNH-CβH
(∼10 Hz) and NOE correlations β-Caa NH/β-CaaCRH(pro-S)
and β-CaaNH/β-CaaC4H suggest the dihedral angles around
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FIGURE 6. Dihedral angle N-Cβ-CR-C(O) (θ) of (S)-β-Caa (shown in red) in (A) dilute solution (1.3 mM), θ ≈ -60° and (B) concentrated
solutions (∼100 mM), θ ≈ 60° forming nanorod like structure in CDCl3 (the H-bonds arerepresented by blue dashed lines; the solid and dashed
arrows in magenta represent strong and weak intensity NOE correlations, respectively).
the peaks corresponding to [5 þ Cl]- (m/z 695), [5 þ Br]-
(m/z 739), [5 þ I]- (m/z 787), [5 þ NO3]- (m/z 722), and [5 þ
HSO4]- or [5 þ H2PO4]- (m/z 757).
Cl- > Br- > I- pattern. These values of the Ka are much
smaller than those found in cyclic hexapeptides by Yang
et al.6b presumably due to smaller ring size in 5. The linear
peptide 4 displayed very poor binding with these halide
anions.15
It is noteworthy that the abundance of [5 þ Cl]- is higher
as compared to other adduct ions, suggesting stronger
affinity of Cl- toward the cyclic peptide 5. To investigate
the nature of the adduct ion complex, the MS/MS spectrum
of [M þ Cl]- (m/z 695) was examined. The spectrum15 shows
mainly [M - H]- (m/z 659) at various collision energies.
This suggests that a loosely bound noncovalent ion-
molecule complex is formed between Cl- and the peptide
under the given experimental conditions. NMR titration
studies provide further evidence for the anion binding
(Figure 4), wherein sequential addition of the halogen ions
(Cl-, Br-, I-) reveals significant changes in the spectra with
the signals of the aminoxy amides shifting downfield, while
those of the β-Caa amide protons show upfield shift
(Figure 4).16 These results imply that the aminoxy amides in
the 5-anion complex are H-bonded, unlike in the free peptide
5. The NMR studies of the halogen binding15 are comparable
with the data reported by Yang et al.,6b implying identical
mode of binding. Thus, the 8-membered N-O turn gets
disrupted (β-CaaNH shifts upfield) and the acidic aminoxy
NH forms strong H-bond with the Cl- (large downfield shift
observed upon complexation). A Job plot of the amide proton
chemical shift against the mole fraction of the ions provided
emphatic support for a 1:1 complex of the Cl- ion with 5
(Figure 4).16b,17 In conformity with mass spectrometry data,
the complex with I- was rather weak, and no definitive
information on it could be obtained. However, the complex
with the Br- ion appears to be a 1:1 complex. The association
constant (Ka) was determined by a titration method reported
by Kelly et al.18 The complex is very selective for Cl-, with an
association constant (Ka) of about 513 ( 70 M-1. The
Studies on Molecular Association and Nanorod Assembly.
Changes in the chemical shifts as a function of the concentra-
tion provide a handy tool for studying molecular association.
We have observed very substantial changes in chemical shifts
of both the amide and CR protons of (S)-β-Caa (Figure 5),
supporting molecular association. Amide protons display large
downfield shifts (1.58 ppm and 0.88 ppm for (R)-Ama and (S)-
β-Caa, respectively) implying their involvement in H-bonding.
A close look at 1H NMR spectra with varying concentration
revealed that in addition to changes in the chemical shifts, the
3
coupling constant JCRH(pro-S)-CβH also changes as a function
of concentration (from a value of 12.4 to 4.6 Hz in 1.3 and
151.5 mM solution in CDCl3, respectively), indicating a definite
conformational change in backbone conformation.
3
Two small JCRH-CβH values (4.1 and 4.6 Hz in 151.5 mM
solution in CDCl3) and the weakening the NOEs of β-CaaCRH-
(pro-S) with both β-CaaNH and β-CaaC4H, compared to that
in dilute solutions, gives strong evidence of change in the dihedral
angle θ from ∼-60° (Figure 6A) to ∼60° (Figure 6B). This in
turn disrupts the 8-membered N-O turn and leaves the amide
and carbonyl groups in axial disposition, facilitating the inter-
molecular H-bonding (Figure 6). A plausible model has been
presented in Figure 6B. The side chains thus occupy equatorial
position on the exterior of the flat, disklike peptide ring, as
observed for other β-peptides.5
Additional evidence for the molecular association is garne-
red from the presence of m/z 1319 ([2M - H]-), a dimeric peak
of 5 in the MS/MS.15 Very large intensity of the [M - H]-
(m/z659) peak in the MS/MS suggests that the dimeric complex
of the peptide is loosely bound.15
Transmission electron microscopy (TEM) was used to
obtain further insight of the molecular association and self-
assembly of the peptide 5 into nanorods (Figure 7). Aliquots
of the sample were applied to previously cleaved mica and
left to evaporate. The samples were subsequently floated
on a water-methanol mixture and collected over 400 mesh
Ka value for the [5 þ Br]- complex is about 111 ( 11 M-1
.
These observations are in line with the mass spectrometric
data, where the halogen ion-5 complex intensities follow the
(16) (a) Macomber, R. S. J. Chem. Educ. 1992, 5, 375–378. (b) Blanda,
M. T.; horner, J. H.; Newcomb, M. J. Org. Chem. 1989, 54, 4626–4636.
(17) Gil, V. M. S.; Oliveira, N. C. J. Chem. Educ. 1990, 67, 473–478.
(18) Kelly, T. R.; Kim, M. H. J. Am. Chem. Soc. 1994, 116, 7072–7080.
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FIGURE 7. (A) TEM image of the nanorods formed by 5 from a 1 mM solution in H2O-MeOH. (B) Magnified image of the micrograph.
(C) TEM image of nanorods formed by 5 from diluted solution (1 μM). (D) Magnified portion from image C showing single nanorod of width
∼120 nm.
FIGURE 8. (A) AFM image of peptide 5 from 50 μM solution in MeOH. (B) A topographic profile of (A). (C) Section analysis of (B).
Cu/Rh grids, which were then negatively stained with 2%
uranyl acetate solution. Under these conditions, as can be
seen from Figure 7, both 2D and 3D structures are formed
probably due to piling of individual rods at 1 mM concentra-
tion. The nanorods are mainly observed as single entities or
grouped into rods. However, when dilute (1 μM) samples were
taken, the peptides self-assembled into rods of length in the
micrometers range with a width of about 150 nm (Figure 7C).
The self-assembly of molecules into rodlike structures was
also observed by atomic force microscopy (AFM) (Figure 8).
At 50 μM concentration and longer incubation time, rodlike
structures with a shape similar to those observed by TEM are
formed. The height of the nanorods was obtained by doing
section analysis of the observed image. The aggregates
heights for the peptide deposited onto a mica substrate from
a fresh 50 μM solution of 5 in MeOH (Figure 8) is found to be
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in the range of 10-20 nm. Different heights were observed
probably due to piling of individual rods as aggregates at
1 mM concentration.
stirred at 0 °C under N2 atmosphere for 15 min, treated with the
above crude amine 2 and DIPEA (0.50 mL, 2.90 mmol), and
stirred at room temperature for 5 h. The reaction mixture was
quenched with satd aq NH4Cl (10 mL) at 0 °C and diluted with
CHCl3 (10 mL). It was sequentially washed with 1 N HCl
(10 mL), water (10 mL), and aq NaCl solution (10 mL). The
organic layer was dried (Na2SO4) and evaporated to give the
residue, which was purified by column chromatography
(60-120 mesh silica gel, 50% ethyl acetate and petroleum ether)
Both TEM and AFM techniques, in spite of the use of two
different substrates (Cu/Rh grid and mica respectively),
provide equivalent nanorod structures. As depicted in
Figure 6, intermolecular association of macrocyles give rise
to nanostructured rods at 1 mM concentration. The surfaces
of these assemblies appear smooth and uniform throughout
the length, which suggests that the rods are made up of tens
of tightly packed peptide nanorods aligned parallel to each
other. Based on a “hierarchical” process proposed by Dory
and co-workers,20 it is believed that it involves an assembly of
individual peptide nanorods over several generations to end
up in the formation of nano- to micro- structured rods, which
most probably happens due to noncovalent interactions
among the nanorods.
to afford 3 (0.62 g, 67%) as a white solid: mp 95-96 °C; [R]D
=
þ86.6 (c 0.26, CHCl3); IR (KBr) 3267, 2988, 2930, 1751, 1736,
1
1670, 1660, 1550, 1168, 1055, 1021, 889, 855 cm-1 ; H NMR
(500 MHz, CDCl3) δ 9.34 (s, 1H, NH-2), 5.90 (d, J = 3.9 Hz, 1H,
C1H-1), 5.11 (br, 1H, NH-1), 4.58 (d, J = 3.9 Hz, 1H, C2H-1),
4.51 (q, J = 7.2 Hz, 1H, CRH-2), 4.31 (m, 1H, CβH-1), 4.23
(m, 2H, -CH2), 4.10 (br, 1H, C4H-1), 3.76 (br, 1H, C3H-1), 3.41
(s, 3H, OCH3-1), 2.54 (dd, J = 5.8, 15.0 Hz, 1H, CRH(pro-S)-1),
2.39 (dd, J = 5.0, 15.0 Hz, 1H, CRH(pro-R)-1), 1.66 (d, J =
7.2 Hz, 3H, CβH-2), 1.50, 1.48 (2s, 6H, Acetonide-CH3), 1.43
(s, 9H, BOC), 1.31 (t, J = 7.2 Hz, 3H, -CH3); 13C NMR
(150 MHz, CDCl3, 278 K): δ 172.0, 168.1, 156.0, 111.8, 104.6,
83.8, 81.2, 80.0, 79.6, 79.2, 61.3, 57.3, 47.6, 36.0, 28.2, 26.7, 26.2,
Conclusions
The present study demonstrates that, unlike the peptides
from (R)-β-Caa/(R)-Ama, (S)-β-Caa/(R)-Ama can easily
be cyclized. NMR and mass spectral studies reveal that the
symmetric cyclic peptide 5, with two N-O turns, shows
selective and appreciable binding affinity with the chloride
ion, while the linear tetrapeptide 4 does not display this
capability. NMR studies as a function of solute concentration
provide ample support of the intermolecular H-bonding,
which results in the nanomolecular assembly of these cyclic
peptides explored in detail by TEM and the AFM studies.
This new class of pseudo β2- and β3-peptides from (S)-β-Caa
and (R)-Ama mayaid in the designof diverse peptide scaffolds
with desired functional features.
16.2, 14.1; HRMS (ESIþ) m/z calcd for C21H36N2O10 (Mþ
þ
Na) 499.2267, found 499.2256.
Boc-(S)-β-Caa-(R)-Ama-(S)-β-Caa-(R)-Ama-OEt (4). A so-
lution of 3 (0.2 g, 0.42 mmol) as described for 7 gave Boc-(S)-
β-Caa-(R)-Ama-OH (9; 0.17 g, 90%) as a white solid, which was
used for further reaction without any purification.
A solution of 3 (0.18 g, 0.38 mmol) and CF3COOH (0.4 mL)
in CH2Cl2 (2 mL) was stirred at 0 °C to room temperature for
2 h. Solvent was evaporated under reduced pressure, and the
resulting salt 10 was dried under high vacuum and used as such
without any further purification.
A solution of 9 (0.17 g, 0.38 mmol), HOBt (0.06 g, 0.45 mmol),
and EDCI (0.08 g, 0.45 mmol) in dry CH2Cl2 (2 mL) was stirred
at 0 °C for 15 min and treated with the above-obtained amine
TFA salt 10 and DIPEA (0.10 mL, 0.57 mmol) under nitrogen
atmosphere for 5 h. Workup as described for 3 and purification
by column chromatography (60-120 mesh silica gel, 1.8%
CH3OH in CHCl3) afforded 4 (0.16 g, 52%) as a white solid:
Experimental Section
The peptides were synthesized following standard solution-
phase peptide coupling methods19 using 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxy-
benzotriazole (HOBt) as coupling agents and dry CH2Cl2 as
solvent. Saponification of tetrapeptide using NaOH in MeOH
gave free acid, and treatment with pentafluorophenol (PFP) in
CH2Cl2 using EDCI as esterification agent gave activated ester.
This activated ester was subjected to Boc deprotection using TFA
in CH2Cl2. The TFA salt of activated ester was taken in CH3CN
(0.01 M) and slow addition into N,N-diisopropylethylamine
(DIPEA; 1.5 equiv) in CH3CN (0.003 M) at 70 °C, after aqueous
workup and chromatographic purification furnished the product
5 in 55% yield.
mp 94-96 °C; [R]20 = þ137.4 (c 0.25 in CHCl3); IR (KBr)
D
3431, 2985, 2934, 1682, 1552, 1456, 1374, 1166, 1082, 1021, 888,
856 cm-1 ; 1H NMR (600 MHz, CDCl3, 278 K) δ 10.32 (s, 1H,
NH-2), 9.89 (s, 1H, NH-4), 7.81 (d, J = 8.2 Hz, 1H, NH-3), 5.91
(d, J = 3.9 Hz, 1H, C1H-3), 5.91 (d, J = 3.9 Hz, 1H, C1H-1),
5.38 (d, J = 7.7 Hz, 1H, NH-1), 4.59 (d, J = 3.9 Hz, 1H, C2H-3),
4.59 (d, J = 3.9 Hz, 1H, C2H-1), 4.54 (m, 1H, CRH-4), 4.48 (m,
1H, CβH-3), 4.30 (m, 1H, C4H-3), 4.28 (m, 1H, CRH-2), 4.26
(m, 1H, C4H-1), 4.21(m, 2H, ethyl ester (-CH2CH3) 4.17
(m, 1H, CβH-1), 3.74 (d, J = 3.3 Hz, 1H, C3H-1), 3.74 (d,
J = 3.3 Hz, 1H, C3H-3), 3.37 (s, 6H, OCH3-1, OCH3-3), 2.50
ꢁ
(m, 1H, CRH-1), 2.50 (m, 1H, CRH-1), 2.48 (m,1H, CRH-3),
Boc-(S)-β-Caa-(R)-Ama-OEt (3). Asolutionofester121 (0.80g,
2.13 mmol) in methanol (4 mL) was treated with aq 4 N NaOH
solution (4 mL) at 0 °C to room temperature. After 2 h, methanol
was removed and adjusted pH to 2-3 with aq 1 N HCl solution at
0 °C and extracted with EtOAc (3 ꢀ 10 mL). The organic layer was
dried (Na2SO4) and concentrated to give 7 (0.71 g, 92%) as a white
solid, which was used for further reaction without any purification.
A solution of acid 7 (0.70 g, 1.93 mmol), HOBt (0.31 g, 2.32
mmol), and EDCI (0.44 g, 2.32 mmol) in CH2Cl2 (5 mL) was
ꢁ
2.48 (m, 1H, CRH-3), 1.50 (s, 6H, acetonide-CH3), 1.48 (m, 3H,
CβH-4), 1.45 (m, 3H, CβH-2), 1.43 (s, 9H, BOC), 1.32 (s, 6H,
acetonide-CH3) 1.30(m, 3H, ethyl ester (-CH2CH3); 13C (150
MHz, CDCl3, 278 K): δ 171.1, 171.7, 169.1, 168.1, 156.5, 111.8,
111.7, 104.8, 104.7, 84.2, 83.9, 82.9, 81.3, 81.2, 80.6, 80.2, 80.0,
79.1, 61.3, 57.5, 57.4, 47.4, 45.9, 36.7, 36.4, 29.6, 29.3, 28.3, 26.7,
26.2, 16.8, 16.2, 14.0; HRMS (ESIþ) m/z calcd for C35H58N4O17
(Mþ þ Na) 829.3694, found 829.3669.
Cyclic-[(S)-β-Caa-(R)-Ama-(S)-β-Caa-(R)-Ama] (5). A solu-
tion of 4 (0.14 g, 0.17 mmol) as described for 7 gave Boc-(S)-
β-Caa-(R)-Ama-(S)-β-Caa-(R)-Ama-OH (11; 0.12 g, 92%) as a
white solid, mp 96-97 0 °C.
(19) (a) Bodanszky, M.; Bodanszky, A. The Practices of Peptide Synthesis;
Springer-Verlag: New York, 1984. (b) Grant, G. A. Synthetic Peptides: A User’s
Guide; W. H. Freeman: New York, 1992. (c) Bodanszky, M. Peptide Chemistry:
A Practical Textbook; Springer-Verlag: Berlin, 1993.
A solution of 11 (0.12 g, 0.16 mmol) was dissolved in CH2Cl2
(4 mL) at 0 °C and treated with pentafluorophenol (0.06 g,
0.32 mmol) and EDCI (0.06 g, 0.32 mmol) sequentially under
nitrogen atmosphere. The resulting solution was stirred at room
(20) Leclair, S.; Baillargeon, P.; Skouta, R.; Gauthier, D.; Zhao, Y.;
Dory, Y. L. Angew. Chem., Int. Ed. 2004, 43, 349–353.
(21) Sharma, G. V. M.; Reddy, V. G.; Reddy, K. R. Tetrahedron:
Asymmetry 2002, 13, 21–24.
J. Org. Chem. Vol. 75, No. 4, 2010 1093

Sharma et al.
JOCArticle
temperature for 2 h. The reaction mixture was diluted with
CHCl3 (20 mL) and washed with 1 N HCl (20 mL) and saturated
NaCl solution (20 mL). The solvent was removed under reduced
pressure and residue washed with hexane to give 12 (0.14 g) in
93% yield. Crude 12 (0.14 g, 0.15 mmol) was deprotected as
described for 10 to furnish amine salt 13, which was dissolved in
CH3CN (0.01 M, 14.5 mL) and added slowly to a solution of
DIPEA (0.04 mL, 0.22 mmol) in CH3CN (73 mL, 0.003 M) at
70 °C for 2 h. Workup as described for 3 and purification by
column chromatography (60-120 mesh silica gel, 2.0% CH3OH
in CHCl3) furnished 5 (0.05 g, 55%) as a white solid: mp
255-257 °C; [R]D = -50.4 (c 0.25, CHCl3); IR (KBr) 3416,
3281, 2988, 2928, 2854, 1704, 1659, 1567, 1380, 1260, 1232, 1164,
acetonide-CH3), 1.44 (d, J = 7.0 Hz, 3H, CβH-Ama), 1.31
(s, 6H, acetonide-CH3); 13C NMR (150 MHz, CDCl3, 278 K) δ
172.8, 167.5, 112.0, 104.8, 83.4, 82.5, 80.9, 80.6, 57.5, 29.3, 26.7,
17.4, 14.2; HRMS (ESIþ) m/z calcd for C28H44N4O14 (Mþ þ H)
661.2932, found 661.2920.
Acknowledgment. We thank CSIR, New Delhi, for the
research fellowships (V.M., S.K.D., and V.R.).
Supporting Information Available: General experimental
procedures, protocols for NMR, mass, TEM, AFM, and MD
1
studies, detailed mass spectral studies, copies of H NMR and
13C NMR data for peptides 3-5, copies of TOCSY and ROESY
spectra of peptides 4 and 5, graph showing solvent dependence
of NH chemical shifts at varying concentration of DMSO-d6 in
CDCl3, distance constraints used in MD calculations, copies of
mass spectra for peptide 5, structural studies of peptide 5, and
chemical shift of amide protons of 5 at different temperatures in
DMSO-d6 solvent. This material is available free of charge via
the Internet at http://pubs.acs.org.
1079, 1025, 956, 860 cm-1 1H NMR (600 MHz, 0.5 mg in
;
600 μL, concn 1.3 mM, CDCl3, 303 K) δ 9.62 (s, 1H, NH-
Ama), 8.35 (d, J = 10.2 Hz, 1H, NH-Caa), 5.88 (d, J = 3.9 Hz,
1H, C1H), 4.58 (d, J = 3.9 Hz, 1H, C2H), 4.57 (m, 1H, CβH-
Caa), 4.39 (q, J = 7.0 Hz, 1H, CRH-Ama), 4.08 (dd, J = 3.5, 8.4
Hz, 1H, C4H), 3.61 (d, J = 3.5 Hz, 1H, C3H), 3.39 (s, 6H,
OCH3), 2.58 (dd, J = 3.7, 16.7 Hz, 1H, CRH (pro-R)-Caa), 2.52
(dd, J = 12.3, 16.7 Hz, 1H, CRH (pro-S)-Caa), 1.52 (s, 3H,
1094 J. Org. Chem. Vol. 75, No. 4, 2010