Cyclotetrapeptides with alternating D-Ala residues: Synthesis and spectroscopic studies

Article abstract of DOI:10.1016/S0022-2860(03)00207-2

Three cyclotetrapeptides, c[Leu-D-Ala-Xaa-D-Ala], where Xaa is Leu (P1), Lys (P2) and Glu (P3) were synthesized and studied by ¹H and ¹³C NMR and CD spectroscopy. These cyclotetrapeptides exhibit similar coupling constants, ³J_HNHα, in the range of 8.56-9.93 Hz, commonly observed for β-turn structures. All amide proton chemical shifts for P1, P2 and P3 exhibited linear dependence on temperature with moderate temperature coefficients ranging from -3.1 to -9.8 ppb/K. Amide proton signal broadening was observed for all residues in P1, P2 and P3, indicating that they are solvent accessible. The number of resonance observed for P1 was half of the total counts, indicating a C2 symmetric conformation. P2 and P3 exhibit similar CD in solvents of varying dielectric constants and dilutions, with characteristic positive CD bands at ca. 210 and 222 nm, which correspond to a β-turn type structure. Small CD/temperature effect was also observed with isodichroic points, consistent with conformational stability and a well-populated cyclotetrapeptide energy state. These heterochiral cyclotetrapeptides consisting of alternating D-Ala residues adopt stabilized open β-turn conformations and may be useful as a ligand template for further functionalization.

Full text of DOI:10.1016/S0022-2860(03)00207-2

Journal of Molecular Structure 655 (2003) 59–68
www.elsevier.com/locate/molstruc
Cyclotetrapeptides with alternating D-Ala residues: synthesis
and spectroscopic studies
a,
Maria Ngu-Schwemlein , Zhe Zhou , Toni Bowie , Rebecca Eden
b
a
a
*
aDepartment of Chemistry, University of South Alabama, Mobile, AL 36688, USA
bDepartment of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
Received 2 January 2003; revised 10 March 2003; accepted 18 March 2003
Abstract
Three cyclotetrapeptides, c[Leu-D-Ala-Xaa-D-Ala], where Xaa is Leu (P1), Lys (P2) and Glu (P3) were synthesized and
studied by 1H and 13C NMR and CD spectroscopy. These cyclotetrapeptides exhibit similar coupling constants, 3JHNHa, in the
range of 8.56–9.93 Hz, commonly observed for b-turn structures. All amide proton chemical shifts for P1, P2 and P3 exhibited
linear dependence on temperature with moderate temperature coefficients ranging from 23.1 to 29.8 ppb/K. Amide proton
signal broadening was observed for all residues in P1, P2 and P3, indicating that they are solvent accessible. The number of
resonance observed for P1 was half of the total counts, indicating a C2 symmetric conformation. P2 and P3 exhibit similar CD
in solvents of varying dielectric constants and dilutions, with characteristic positive CD bands at ca. 210 and 222 nm, which
correspond to a b-turn type structure. Small CD/temperature effect was also observed with isodichroic points, consistent with
conformational stability and a well-populated cyclotetrapeptide energy state. These heterochiral cyclotetrapeptides consisting
of alternating D-Ala residues adopt stabilized open b-turn conformations and may be useful as a ligand template for further
functionalization.
q 2003 Elsevier Science B.V. All rights reserved.
Keywords: Cyclotetrapeptides; Circular dichroism; Nuclear magnetic resonance spectroscopy; Beta structure
1. Introduction
experienced by their relatively more flexible linear
congeners. Some naturally occurring cyclopeptides
that exhibit biological activities includes the cytotoxic
tetrapeptides, Tentoxin [1] and the AM and HC toxins
[2–4], the antibiotic dodecapeptide, Gramicidin S [5]
and the ionophoric depsipeptide, Valinomycin [6]. In
general, macrocyles are attractive template for
attaching donor atoms for selective ligand–substrate
interactions. Moreover, the size of the macrocycle can
be tailored to encapsulate substrates of varying sizes
as demonstrated by the studies conducted on the
crown ethers and aza crowns [7,8].
Small cyclopeptides have been reported to exhibit
interesting biological activities that are frequently
more specific and of higher efficacy than their linear
analogues. This is generally attributed to the inherent
conformational constraints experienced by the small
cyclic peptide, which enhances selective molecular
interactions and recognition as opposed to that
*
Corresponding author. Tel.: þ1-251-460-7424; fax: þ1-251-
460-7359.
E-mail address: mngu@usouthal.edu (M. Ngu-Schwemlein).
0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0022-2860(03)00207-2

60
M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
The cyclotetrapeptide scaffold, a conformationally
Dihedral angles (f) were calculated from the Karplus
3
rigid 12-membered macrocyclic structure with four
donor oxygen atoms and four nitrogen atoms, offers
structural and chemical features that are favorable for
metal ion chelation. In addition, the ease in chemical
modification of the peptide backbone offers additional
versatility in changing the donor atoms, allowing the
design of macrocyclic derivatives with selectivity for
different types of metal ions based on the ligand
preferences and rates of complex formation of
individual metal ions. Moreover, a cyclotetrapeptide
can be synthesized, incorporating the natural amino
acid, Trp. The indole heterocycle of Trp can function
as an intrinic fluorophore to signal metal ion binding.
Additionally, chromophoric groups can be covalently
and strategically attached to the macrocyclic template
to act as a signal for ligand–substract interactions.
Therefore, fluorophore tagged cyclopeptides and their
derivatives are attractive candidates as chemosensors.
We are interested in studying the secondary
structural preference and stability of small cyclopep-
tides containing D-Ala residues to explore the potential
of using the heterochiral cyclopeptide as a ligand
template for attaching fluorophores. The incorporation
of alternating D-residues should enhance the stability
of the peptide backbone towards hydrolysis by
proteases. Additionally, heterochiral peptide
sequences, compared to their homochiral analogues,
have been shown to have a greater tendency to form
b-turns, which are stable under a variety of environ-
ments [9,10]. In this paper, we describe the synthesis
and spectroscopic studies of three cyclotetrapeptides
containing D-Ala residues and functionalized side
chain groups for fluorophore attachment.
equation [11], JHNHa ðHzÞ ¼ A cos2 u þ B cos u þ
C; where A ¼ 6:4; B ¼ 21:4 and C ¼ 1:9; and u ¼
lf 2 608l [12]. Temperature dependence of the amide
hydrogen chemical shifts was studied for every 10 K
increment from 303 to 353 K.
MALDI-TOF mass spectra were recorded on a
Perseptive Biosystem MALDI-TOF mass spec-
trometer at Louisiana State University. The matrix
compound was 2,5-dihydroxybenzoic acid (DHB).
Crude cyclotetrapeptides were purified on a Vydac
˚
C-4 reversed-phase column (300 A). The mobile
phase was H2O/0.1% trifluoroacetic acid, TFA (A),
and CH3CN (50%)/isopropanol (50%)/0.1% TFA (B)
delivered by the Rainin Dynamax HPLC system with
UV monitoring at 220 nm. CD measurements were
carried out on an Aviv 62DS Spectrophotometer.
Spectra were recorded using 1 mg/ml solutions of P2
and 0.5 mg/ml solutions of P3 by sampling every
1 nm with an averaging time of 1 s, employing a
0.1 cm path length quartz cell. DPC in methanol was
prepared as 1 mM solutions. The spectra were
smoothed by the Savitzky–Golay algorithm.
2.2. General method employed for the preparation
of P1-3
Cyclotetrapeptides P1-3 were prepared by con-
vergent synthesis as shown in Fig. 1 for P1. The linear
tetrapeptides were synthesized using the N a-t-Boc/
Ca-O-phenacyl ester protecting group combination.
Amino acid and peptide couplings were mediated by
1-hydroxy-7-azabenzotriazole(HOAT)/1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide
(EDCI)/N-
methylmorpholine (NMM) in dichloromethane or
by N-{(dimethylamino)-H-1,2,3-triazolo[4,5-b]pyri-
din-1-ylmethylene}-N-methylmethanaminium hexa-
fluorophosphate N-oxide (HATU)/collidine in
dimethylformamide following the procedure reported
earlier [13]. Head-to-tail cyclization of the tetrapep-
tides was accomplished by cyclization of the penta-
fluorophenyl (Pfp) ester of the linear precursor,
Leu-D-Ala-Xaa-D-Ala-O-Pfp, using the Schmidt
method [14]. The crude peptides were purified by
semi-preparative reversed-phase HPLC and charac-
terized by MALDI-MS. The details are given below
and Tables 1–3 shows the 1H and 13C NMR
assignments for each cyclotetrapeptide.
2. Experimental
2.1. Materials and methods
All chemicals were obtained from commercial
suppliers and used without further purification. All
spectra were obtained on peptide samples prepared in
(CD3)2SO or CDCl3/CF3CD2OH (8:1), 5 mg/ml, on a
Bruker 300 spectrometer, with TMS as an internal
standard. Sequential assignments of all H and 13C
1
resonances were accomplished by through bond
connectivities from HSQC and HMBC spectra.

M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
61
Fig. 1. Synthesis of P1 by convergent approach.
2.3. Cyclo[Leu-D-Ala]2 P1
t-Boc-Leu-D-Ala-O-phenacyl ester (81%). Reaction
of half of the dipeptide with Zn in AcOH gave t-Boc-
Leu-D-Ala-OH, and deprotection of the second half
with TFA/CH2Cl2 (7:3) gave TFA.Leu-D-Ala-O-
phenacyl ester. Coupling of these dipeptides with
Convergent approach to tetrapeptide preparation:
coupling of the protected D-Ala and Leu, with
HOAT/EDCI/NMM in CH2Cl2 gave the dipeptide,

62
M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
Table 1
1H and 13C Chemical Shifts (ppm) of P1 in CDCl3/CF3CD2OH
Table 3
1H and 13C chemical shifts (ppm) of P3 in (CD3)2SO
Residue
HN
Ha
Hb
Hg
Hd
Residue
HN
Ha
Hb
Hg
Hd
Leu-1,3
Ala-2,4
6.65
6.75
4.36
4.43
1.56
1.29
1.56
0.89/0.92
Leu-1
Ala-2
Glu-3
Ala-4
7.23
8.16
7.27
8.05
4.2
4.3
4.2
4.3
1.44
1.12
1.75
1.12
1.44
2.16
0.87/0.84
Residue
Leu-1,3
Ala-2,4
CyO
173.7
174.1
Ca
Cb
Cg
Cd
50.9
48.4
37.0
13.7
24.7
22.5/22.1
Residue
Leu-1
Ala-2
Glu-3
Ala-4
CyO
Ca
Cb
Cg
Cd
172.94
173.02
172.45
172.69
50.98
38.2
24.2
22.6/22.1
47.0/47.1
51.72
14.3/14.2
24.6
29.7
173.76
HOAT/EDCI/NMM in CH2Cl2 gave the tetrapeptide
in 81% yield. C-terminal deprotection with Zn in
AcOH, followed by transesterification with penta-
fluorophenyl trifluoroacetate, and treatment with
TFA/CH2Cl2 (7:3) gave TFA.Leu-D-Ala-Leu-D-Ala-
O-Pfp (85%, over 3 steps).
47.0/47.1
14.3/14.2
2.4. Cyclo[Leu-D-Ala-Lys-D-Ala] P2
Cyclo[Leu-D-Ala-Lys-D-Ala] was purified on a
2
˚
Vydac C-4 column (10 £ 250 mm , 300 A, 5 mm), to
Cyclization of the tetrapeptide: a solution of
(0.13 g,
give a white fluffy peptide. Conditions were 5% B to
15% B over 35 min with a flow rate of 5 ml/min. RP-
HPLC tr ¼ 27:1 min: MALDI-MS [M þ H]þ/384
(calculated 384.6). [M þ Na]þ/405 (calculated
405.6).
TFA.Leu-D-Ala-Leu-D-Ala-O-Pfp
0.2 mmol), DMAP (0.16 mmol) in anhydrous dioxane
(200 ml) and trifluoroethanol (4.5 ml) was treated at
85 8C with a solution of pyridine (3 ml) in dioxane
(100 ml), over 17 h. After a further 4 h of stirring, the
solution was concentrated under reduced pressure.
The residue was dissolved in chloroform and
extracted with 10% HCl, sat. NaCl, sat. NaHCO3,
H2O, sat. NaCl, and dried over Na2SO4 and
evaporated. Recrystallization from chloroform and
methanol (1:1) gave fine white needles of cyclo[Leu-
D-Ala-Leu-D-Ala] (24 mg, 33%). The characteriz-
ation of this hydrophobic cyclotetrapeptide by
optimized MALDI-MS techniques has been described
[15]. MALDI-MS [M þ Na]þ/392 (calculated 392.6).
2.5. Cyclo[Leu-D-Ala-Glu-D-Ala] P3
Cyclo[Leu-D-Ala-Glu-D-Ala] was purified on a
2
˚
Vydac C-4 column (10 £ 250 mm , 300 A, 5 mm), to
give a white fluffy peptide. Conditions were 10% B to
25% B over 30 min with a flow rate of 5 ml/min. RP-
HPLC tr ¼ 18:7 min: MALDI-MS [M þ H]þ/385
(calculated 385.5). [M þ Na]þ/406 (calculated
406.5).
3. Results and discussion
Table 2
1H and 13C chemical shifts (ppm) of P2 in (CD3)2SO
3.1. Synthesis
Residue HN
Ha
Hb
Hg
Hd
He
The preparation of the pentafluorophenyl ester
derivatives of the tetrapeptides were readily con-
ducted using the general N a-t-Boc/Ca-O-phenacyl
ester protecting groups [13]. This convergent
approach allows for the mixing and matching of
dipeptides for [2 þ 2] fragment couplings to prepare
the tetrapeptide analogs. Head-to-tail cyclization of
the tetrapeptide to form the constrained 12-membered
ring was conducted under dilute conditions to avoid
Leu-1
Ala-2
Lys-3
Ala-4
7.14 4.36
8.30 4.39
7.21 4.24
8.25 4.40
1.43
1.12
1.45
0.88/0.84
1.61/1.49 1.26/1.17 1.52
1.12
2.76
Ce
Residue CyO Ca
Cb
Cg
Cd
Leu-1
Ala-2
Lys-3
Ala-4
173.0 50.9
38.2
24.1
22.5/22.1
172.8 46.9/46.8 14.1/14.0
172.7 52.3 28.9
172.6 46.9/46.8 14.1/14.0
22.1
26.7
38.5

M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
63
cyclodimerizarion [16]. Although cyclization of these
tetrapeptides is facilitated by the presence of D-Ala,
which stabilize turn structures, this reaction turned out
to be the yield-limiting step for the preparation of
these three cyclotetrapeptides. The hydrophobic side
chain protecting group (N e-2-Cl-CBZ or O g-Bzl) of
the cyclopeptide precursors was removed by hydro-
genation over Pd/C in trifluoroethanol. Although P1
was readily purified by crystallization, the pure
peptide was insoluble in most common solvents. P2
and P3 did not readily crystallize and were purified by
reversed-phase HPLC.
supports a well-populated low energy cyclotetra-
peptide backbone conformation since inter-convert-
ing multiple conformations would have yielded an
average coupling constant of lower value (,7 Hz)
3
[17]. Phi angle (f) constraints from JHNHa values,
as calculated from the Karplus equation, yielded
similar values for all three cyclopeptides. These
large phi angle values (Table 4) are typically
observed in peptides adopting b-turn structures.
However, the interpretation of the above computed
phi angles for structure analysis must be taken with
caution as low level conformational averaging is
3
not precluded from the observed JHNHa coupling
constants values.
3.2. NMR studies
To further investigate the secondary structure of
this cyclotetrapeptide scaffolding, the temperature
dependence of the amide protons were studied
by conducting variable temperature 1H NMR
experiments at 10 K intervals from 303 to 353 K. All
amide proton chemical shifts for P1, P2 and P3
exhibited linear dependence on temperature,
suggesting no significant conformational changes
occurred as the temperature is raised. The coefficient
for the change in chemical shift per degree K for each
amide proton is shown in Table 4. The chemical shifts
of the non-exchangeable protons did not exhibit any
significant temperature dependence. In general, coef-
ficients greater than 3 ppb/K indicate solvent exposed
amide protons [18]. The chemical shift of the amide
protons of Ala residues (range from 23.1 to
23.7 ppb/K) have a moderate, and slightly lower
temperature dependence than that of the other
residues (25.1 to 29.8 ppb/K) in the cyclotetrapep-
tides. The intermediate temperature coefficients
observed for Ala residue do not exclude the possibility
of their participation in weak or transient intramole-
cular or intermolecular hydrogen bonding. However,
amide proton signal broadening was observed for all
amide protons (Fig. 3) indicating that they are solvent
accessible.
1
Sequential assignments of all H and 13C reson-
ances for P1 (Table 1), P2 (Table 2) and P3 (Table 3)
were accomplished by through bond connectivities
from HSQC and HMBC spectra. The 1H and 13C
resonances for P1 were half of the total counts,
indicating that the peptide macrocylic ring adopts a
C2 symmetry. The chemical shifts of the amide proton
and the carbonyl carbon of Ala are more deshielded
than that of Leu. The low field resonance observed for
the amide proton of Ala in P2 and P3, (8.1–8.3 ppm)
parallels that reported for the NH of the i þ 3 residue
(,8 ppm) in type II b-turns [17]. In order to
investigate the dipolar couplings between protons,
˚
which are within 3 A of each other in the low energy
conformation of the peptide, NOESY as well as
ROESY spectra were acquired. Relatively strong
cross peaks between all four amide protons, NH(i),
and the alpha proton of the adjacent residue,
Ha(i þ 1), are observed in the NOESY and ROESY
spectra of P1, P2 and P3 (Fig. 2), which is indicative
of a cyclotetrapeptide backbone consisting of trans
peptide bonds. Unfortunately, no meaningful quanti-
tative distance geometrical values can be derived from
these spectra at this time since the distance between
any two protons in these peptides cannot be
unambiguously assigned. An analogue of these
peptides containing an indole ring is in preparation
and the results of the distance geometry analysis will
be reported then.
3.3. CD studies
P2 and P3 showed similar CD spectra with
characteristic positive CD bands at ca. 190, 210 and
222 nm and negative bands at ca. 200 and 238 nm
(Fig. 4a and b). The CD spectra of these heterochiral
peptides show a marked negative, red shifted, npp
3
The JHNHa coupling constants for P1-3 are as
shown in Table 4. All four residues in each
cyclotetrapeptide exhibit a relatively large coupling
constant value ranging from 8.56 to 9.93 Hz. This

64
M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
Fig. 2. (a) ROESY spectrum of P1, (i) NHAla $ Ha(Leu): (ii) NHLeu $ Ha(Ala), (b) NOESY spectrum of P2, (i) NHAla2 $ Ha(Leu1): (ii)
NHAla4 $ Ha(Lys3): (iii) NHLys3 $ Ha(Ala2): (iv) NHLeu1 $ Ha(Ala4), and (c) ROESY spectrum of P3, (i) NHAla2 $ Ha(Leu1): (ii) NHAla4
Ha(Glu3): (iii) NHGlu3 $ Ha(Ala2): (iv) NHLeu1 $ Ha(Ala4)
$
.
band at 238 nm which is attributable to the contri-
bution by the D-Ala residues. Two positive npp bands
at 210 and 222 nm resembles the mirror image of the
a-helix similar to that reported for peptides turns
containing the sequence L-Pro-D-Xaa [19]. The ppp
transitions occur as a moderately strong positive band
at ca. 190 nm and a negative, red shifted, band at
200 nm. The general CD profile of both peptides
corresponds to a b-turn type structure. Comparative
data for P1 was not obtained because it was insoluble
in common solvents. Relatively small CD/tempera-
ture effect is observed for P2 and P3, accompanied by

M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
65
Table 4
3JHNHa coupling constants and temperature coefficients of amide chemical shifts
Cyclopeptide
Residue
Leu-1
Ala-2
Xaa-3
Ala-4
9.52
P1
P2
P3
3JHNHa
9.93
21208
3.1
9.52
9.93
21208
3.1
f ð^208Þ
2110/ 2 1308
5.1
2110/ 2 1308
5.1
2Dd=DT (ppb/K)
3JHNHa
9.44
8.74
9.50
8.84
f ð^208Þ
2110/ 2 1308
3.2
2100/ 2 1408
9.4
2110/ 2 1308
3.1
2100/ 2 1408
9.8
2Dd=DT (ppb/K)
3JHNHa
9.45
8.56
9.47
8.82
f ð^208Þ
2110/ 2 1308
3.7
2100/ 2 1408
8.9
2110/ 2 1308
3.5
2100/ 2 1408
9.0
2Dd=DT (ppb/K)
moderate decrease in molar ellipticity at higher
temperatures, with common isodichroic points at
205, 226 and 246 nm, which is consistent with
conformational stability and a single well-populated
cyclotetrapeptide energy state. These peptides exhibit
similar circular dichroism spectra in solvents of
different polarity (Fig. 5a). Decrease in solvent
polarity is accompanied by a decrease in molar
ellipticity, with a relatively larger decrease of the
positive band at 210 nm. Conversely, a moderate
increase in molar ellipticity at 210 and 222 nm is
observed following peptide dilution in water (Fig. 5b).
The heterochiral cyclotetrapeptide CD is indepen-
dent of the change of one side chain of the L-amino
acid from Lys (P2) to Glu (P3). It is also almost
independent of solvent type and concentration, unlike
the solvent dependent cyclotetrapeptides reported by
Tamaki et al. [20]. This result is consistent with
previous reports that b-turn structures are stable in
various conditions, including strong solvents. Gier-
asch et al. has reported that intramolecular hydrogen
bond does not play a major role in stabilizing b-turn
structures, although it is necessary to maintain g-turn
structures [17]. The above CD data show that there is
1
Fig. 3. Amide proton region of the H NMR spectra of P2 and P3-temperature shifts and line broadening.

66
M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
Fig. 4. Variable Temperature CD spectra of (a) P2, and (b) P3 in trifluoroethanol/water (1:1), from 263 to 353 K at 10 K increments as indicated
by arrow.
no remarkable difference in the conformations of
these cyclotetrapeptides when the solvent, concen-
tration or one of the L-amino acid is varied.
by intramolecular hydrogen bondings) b-turn struc-
,
3
ture. The large coupling constant values, JHNHa
supports a preferred peptide backbone structure with
considerable rigidity. Consistent with the general
parallel observed for preferred conformations of
cyclized turn models, peptides containing defined
inserts of alternating D-Ala residues maybe useful in
initiating this variant b-turn structure. Additionally,
cyclotetrapeptides with such a motif maybe useful as a
molecular template for attaching chemical groups or
chromophores, with applications in molecular recog-
nition or chemosensing studies. We propose that this
stable heterochiral cyclotetrapeptide backbone struc-
ture could be particularly useful for further
4. Conclusion
We have reported the synthesis of three cyclote-
trapeptides consisting of alternating D-Ala residues.
Nuclear magnetic resonance and circular dichroism
spectroscopy showed that these cyclotetrapeptides
with alternating D-Ala residues are structurally similar,
with a well-populated cyclotetrapeptide backbone
energy-state, which resembles an open (not stabilized

M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
67
Fig. 5. CD spectra of (a) P2 in water, 1 mM DPC, methanol, TFE, TFE/THF (1:1), and; (b) P3 at 3.5, 0.7, 0.35, 0.17, 0.08 mM in water,
sequentially as indicated by arrow.
functionalization and might be useful for the develop-
ment of peptide-based ligands.
helpful discussions with Dr. Karl Markos of Tulane
University.
References
Acknowledgements
[1] M. Santolini, C. Minoletti, J.M. Gomis, C. Sigalat, F. Andre,
F. Haraux, Biochemistry 41 (2002) 6008–6018.
This work was supported by the National Science
Foundation, grant # CHE 0071632. We also wish to
express our appreciation to Kari GreenChurch and
Steven Macha of Louisiana State University for
the MALDI TOF mass spectra. We acknowledge
[2] H. Otani, K. Kohmoto, M. Kodama, Can. J. Bot. 73 (1995)
S453–S458. Suppl. 1 A–D.
[3] M. Miyashita, T. Nakamori, T. Murai, T. Yonemoto, H.
Miyagawa, M. Akamatsu, T. Ueno, C. Naturforsch,
Z. Naturforsch. C 56 (2001) 1029–1037.

68
M. Ngu-Schwemlein et al. / Journal of Molecular Structure 655 (2003) 59–68
[4] Z. Jiang, M.O. Barret, K.G. Boyd, D.R. Adams, A.S.F. Boyd,
J.G. Burges, Phytochemistry 60 (2002) 33–38.
[13] X. Wang, W. Daly, P. Russo, M. Ngu-Schwemlein, Bioma-
cromolecules 2 (4) (2001) 1214–1219.
[5] E.J. Prenner, R.N.A.H. Lewis, R.N. McElhaney, Biochim.
Biophys. Acta 1462 (1999) 201–221.
[14] U. Schmidt, A. Lieberknecht, H. Griesser, J. Tabiersky, J. Org.
Chem. 47 (1982) 3261–3264.
[6] W.L. Duax, J.F. Griffin, D.A. Langs, G.D. Smith, P.
Grochulski, V. Pletnev, V. Ivanov, Biopolymers 40 (1996)
141–155.
[15] K.B. Green-Church, P.A. Limbach, Anal. Chem. 70 (1998)
5322–5325.
[16] M. El Haddadi, F. Cavelier, E. Vives, A. Azmani, J. Verducci,
J. Martinez, J. Peptide Sci. 6 (2000) 560–570.
[17] G.D. Rose, L.M. Gierasch, J.A. Smith, Adv. Protein Chem. 37
(1985) 1–109.
[7] C.J. Pedersen, J. Inclus. Phenom. Mol. Recognit. Chem. 12
(1992) 7–10.
[8] J.S. Bradshaw, R.M. Izatt, Acc. Chem. Res. 30 (1997)
338–345.
[18] H. Kessler, Angew. Chem. Int. Ed. 21 (1982) 512–523.
[19] A. Perczel, M. Hollosi, in: G.D. Fasman (Ed.), Circular
Dichroism and Conformational Analysis of Biomolecules,
Plenum Press, New York, 1996, pp. 285–380.
[20] T. Makoto, S. Komiya, M. Yabu, E. Watanabe, S. Akabori, I.
Muramatsu, J. Chem. Soc., Perkins Trans. I 23 (1997)
3497–3500.
[9] C.M. Wilmot, J.M. Thorton, J. Mol. Biol. 203 (1988)
221–232.
[10] C.M. Venkatachalam, Biopolymers 6 (1968) 1425–1436.
[11] M. Karplus, J. Chem. Phys. 30 (1959) 11–15.
[12] A. Pardi, M. Billiter, K. Wu¨thrich, J. Mol. Biol. 180 (1984)
741–751.