Nucleation and stability of hydrogen-bond surrogate-based α-helices

Article abstract of DOI:10.1039/b612891b

We have reported a new class of artificial α-helices in which a pre-organized α-turn nucleates the helical conformation [R. N. Chapman, G. Dimartino, and P. S. Arora, J. Am. Chem. Soc., 2004, 126, 12252 and D. Wang, K. Chen, J. L. Kulp, III, and P. S. Arora, J. Am. Chem. Soc., 2006, 128, 9248]. This manuscript describes the effect of the core nucleation template on the overall helicity of the peptides and demonstrates that the macrocycle which most closely mimics the 13-membered hydrogen-bonded α-turn in canonical α-helices also affords the most stable artificial α-helix. We also investigate the stability of these synthetic helices through classical helix-coil parameters and find that the denaturation behavior of HBS α-helices agrees with the theoretical properties of a peptide with a well-defined and stable helix nucleus. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b612891b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Nucleation and stability of hydrogen-bond surrogate-based a-helices†
Deyun Wang, Kang Chen, Gianluca Dimartino and Paramjit S. Arora*
Received 6th September 2006, Accepted 2nd October 2006
First published as an Advance Article on the web 17th October 2006
DOI: 10.1039/b612891b
We have reported a new class of artificial a-helices in which a pre-organized a-turn nucleates the helical
conformation [R. N. Chapman, G. Dimartino, and P. S. Arora, J. Am. Chem. Soc., 2004, 126, 12252
and D. Wang, K. Chen, J. L. Kulp, III, and P. S. Arora, J. Am. Chem. Soc., 2006, 128, 9248]. This
manuscript describes the effect of the core nucleation template on the overall helicity of the peptides
and demonstrates that the macrocycle which most closely mimics the 13-membered hydrogen-bonded
a-turn in canonical a-helices also affords the most stable artificial a-helix. We also investigate the
stability of these synthetic helices through classical helix–coil parameters and find that the denaturation
behavior of HBS a-helices agrees with the theoretical properties of a peptide with a well-defined and
stable helix nucleus.
which sacrifice side-chain functionality to nucleate helical
Introduction
conformations.10–12 Modifying side-chains renders them unavail-
The a-helix is the most common protein secondary structure
able for molecular recognition; moreover, the resulting tether
and is intimately involved in biomolecular recognition. Artificial
blocks at least one face of the target helix. In previous reports,
mimics of a-helices have the potential to regulate these molecular
we demonstrated that the HBS a-turn can stabilize the a-
processes.1 We are developing a new approach for the preparation
of stable artificial a-helices from short peptide sequences.2,3 This
approach is centered on the helix–coil transition theory in peptides,
which suggests that the energetically demanding organization
of three consecutive amino acids into the helical orientation
inherently limits the stability of short a-helices.4,5 Our method
affords preorganized a-turns to overcome this intrinsic nucleation
barrier and initiate helix formation.6–8 We prepare preorganized
a-turns by replacing a main-chain hydrogen-bond between the
helical structure in short alanine-rich2 and biologically-relevant
sequences.3 We have also demonstrated that HBS a-helices can
target their expected protein receptors with high affinities.13
In the HBS approach, the macrocycle mimicking the N-terminal
a-turn nucleates the a-helix. In our preliminary studies we replaced
the 13-membered hydrogen-bonded ring that characterize a-
helices with a 13-membered macrocycle, based on the conjecture
that this ring size may be best at nucleating the helix. In this
paper we fully examine the effect of the macrocycle structure on
the nucleation and stability of the helix. These studies are critical
for the further development of these artificial a-helices as tools
for biomolecular recognition; the experiments described here also
test our basic design principle: is the N-terminal a-turn properly
designed for effective nucleation of the helix? If our initial design
is correct, we would predict that a slight change in the pattern of
hydrogen-bond acceptors in the macrocycle should significantly
disrupt the stability of the a-helix.
=
C O of the ith amino acid residue and the NH of the i + 4th
amino acid residue with a carbon–carbon bond derived from
a ring closing metathesis reaction (Fig. 1).9 The hydrogen-bond
surrogate (HBS) approach allows full access to the helix surface
due to the internal placement of the cross-link. Our method,
therefore, differs significantly from the commonly employed
side-chain cross-linking methods for helix stabilization,
In this manuscript, we also inspect the thermal denaturation
properties of the synthetic helices and derive a nucleation constant
based on the Zimm–Bragg helix–coil theory. We find that the HBS
method sets the nucleation constant to unity, in accordance with
the theory and our original hypothesis. These studies suggest that
HBS helices could become unique probes for the examination of
parameters that describe helix formation.
Results and discussion
Fig. 1 Nucleation of short a-helices by replacement of an N-terminal i
and i + 4 hydrogen bond with a carbon–carbon bond.
Effect of nucleation macrocycles on overall helicity
The nucleation studies presented here were performed on two short
peptides 1 and 2 (Fig. 2) derived from apoptotic protein BAK
BH3 and c-Jun coiled-coil domains, respectively.3,14,15 These two
biologically relevant sequences are unstructured at physiological
conditions, providing good models for studying the efficiency of
HBS macrocycles for helix nucleation. In a previously reported
Department of Chemistry, New York University, New York, NY 10003,
USA. E-mail: arora@nyu.edu
† Electronic supplementary information (ESI) available: Synthesis and
characterization of HBS helices, and estimation of the helix–coil transition
in HBS helices. See DOI: 10.1039/b612891b
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containing 13- and 14-membered rings 3b, 3d, 4b and 4d (Fig. 2).
(We were unable to isolate sufficient amounts of the cis-alkene
isomers for the present studies.) We reasoned that the hydrogenated
compounds should be less helical than the trans-alkene analogs
owing to the extra degrees of freedom in the macrocycle.
Characterization by circular dichroism spectroscopy
The HBS helices presented here were synthesized as reported;3,9
detailed synthetic procedures and characterization are included
in the Supplementary Information.† We began structural charac-
terization of 3b–d and 4b–d with circular dichroism spectroscopy.
The CD spectra were obtained in phosphate-buffered saline (PBS)
and in a solution of 10% trifluoroethanol (TFE) in PBS. Fig. 3
shows the CD spectra of HBS a-helices 3a–d and 4a–d along with
their unconstrained peptide counterparts 1 and 2, respectively, in
10% TFE in PBS. A summary of the circular dichroism results is
listed in Table 1. Unconstrained peptides 1 and 2 display spectra
typical of unstructured or slightly helical peptides. Importantly, all
HBS a-helices (3a–d and 4a–d) display double minima at 208 and
222 nm, and maxima near 190 nm consistent with those observed
for canonical a-helices. Percent helicity is calculated from the
ratio [h]222/[h]max, where [h]max = −23 400 for 10-residue a-helices.3
Table 1 also shows the ratio of the 222 nm and 208 nm bands,
which is occasionally used as an additional gauge of a-helicity.
However, the origin and effect of peptide sequence on this ratio
remains ill-defined.16 Nevertheless, we were pleased to observe that
the ratios of the 222 nm and 208 nm bands in the HBS a-helices are
close to the values (1.25–1.75) expected for canonical a-helices.17
The CD studies show that HBS analogs 3c and 4c containing
14-membered macrocycles are significantly less helical than their
Fig. 2 (a) Determination of the optimum nucleation macrocycle in HBS
helices. (b) Control peptides and HBS a-helices designed to determine the
effect of the nucleation macrocycle on the overall helicity.
study, we utilized extensive circular dichroism and NMR studies
to demonstrate that the 13-membered HBS analogues (3a and 4a)
of peptides 1 and 2 do indeed adopt stable a-helical structures.3 In
the current manuscript, we present characterization on analogues
that contain 14-membered rings (3c and 4c) in which the hydrogen
bond acceptor (carbonyl groups) functionality is offset by one
atom. (We intended to prepare both the 12-membered and the 14-
membered macrocycle analogs; however, the 12-membered ring
requires a 3-butenoic acid residue (a b,c-unsaturated acid) at the
N-terminus that undergoes alkene isomerization rather than ring-
closing metathesis.) We hypothesized that a minor change in the
macrocycle may have a big impact on the stability of the helix,
thus supporting our original design principles that a precise a-
turn mimic is critical for helix nucleation.
The metathesis-derived macrocycle affords high yields of the
trans-alkene product, and our previous studies have mainly utilized
this alkene isomer. To fully determine the effects of the macrocycle
on nucleation, we also prepared the hydrogenated HBS analogs
Fig. 3 Circular dichroism spectra of (a) 1 (black), 3a (red), 3b (green), 3c
(pink), 3d (blue) and (b) 2 (black), 4a (red), 4b (green), 4c (pink), 4d (blue).
The CD spectra were obtained in 10% TFE–PBS.
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Table 1 Summary of circular dichroism data for peptides 1 and 2 and
HBS a-helices 3a–d and 4a–d
experiments. The goal of these studies is to determine how a
small change in the nucleation macrocycle may affect the helicity
of the peptide several residues away from the macrocycle. NMR
spectroscopy allows several different means for gauging a-helical
structure in peptides including evaluation of key medium and long
range NOEs, coupling constants and φ angle values, temperature-
dependence of amide NH chemical shifts, and rates of amide
proton exchange.18 We used all these experiments to probe the
structure of 3b as compared to 3a.3 The NMR studies fully support
the CD data and unambiguously suggest that both 3a and 3b
adopt defined a-helical structures and that the alkene-based HBS
a-helix is only slightly superior to the hydrogenated analog in helix
stability.
A combination of 2D TOCSY, DQF-COSY and NOESY
spectroscopy was used to assign 1H NMR resonances for the
HBS helices.18 The NOESY spectra of 3a and 3b show remarkable
similarity; this similarity is also captured in the correlation charts
for 3a and 3b (Fig. 4). Sequential NN (i and i + 1) NOESY cross-
peaks, a signature of helical structure, were observed for both
sequences as shown in the NOE correlation charts (Fig. 4). The
NOESY spectrum further reveals several nonsequential medium
range NOEs, for example, daN(i, i + 3), daN(i, i + 4) and dab(i,
i + 3), that provide unequivocal evidence for the helical structure
in 3b.
Helix
1
Buffer
[h]222/[h]208
Helicity (%)a
PBS
10% TFE–PBS
PBS
10% TFE–PBS
PBS
10% TFE–PBS
PBS
10% TFE–PBS
PBS
10% TFE–PBS
PBS
10% TFE–PBS
PBS
10% TFE–PBS
PBS
0.41
0.65
1.17
1.40
1.79
1.71
0.87
1.19
0.94
1.55
0.08
0.25
0.82
1.29
0.87
1.25
0.81
1.17
0.94
1.17
14
20
46
68
46
66
24
36
23
36
6
13
32
48
31
43
26
34
28
34
3a
3b
3c
3d
2
4a
4b
4c
4d
10% TFE–PBS
PBS
10% TFE–PBS
PBS
10% TFE–PBS
a Percent helicity was measured from the ratio [h]222/[h]max, where [h]max
−23 400.3
=
13-membered ring analogues 3a and 4a. These results are consis-
tent with the hypothesis that the macrocycle which most closely
mimics the 13-membered hydrogen-bonded a-turn in canonical a-
helices should afford the most stable artificial a-helix. Interestingly,
the CD studies suggest that the hydrogenated and trans-alkene
HBS helices are similar in overall helicity (Fig. 3 and Table 1). The
two sets of CD spectra are superimposable except for the 208 nm
bands, which are less negative in the hydrogenated series. This
result is significant as it indicates that a saturated hydrocarbon
link may be sufficient to nucleate an a-helix.
Table 2 summarizes the NMR results obtained for 3b and
3
compares them to those previously reported for 3a. The JNHCHa
coupling constant provides a measure of the φ angle and affords
intimate details about the local conformation in peptides and
3
proteins.18 The JNHCHa values typically range between 4–6 Hz
(−70 < φ < −30) for a-helices, and a series of three or more
coupling constants in this range are indicative of the a-helical
structure. As observed with 3a, all coupling constants and the φ
angles for 3b fall in the range expected for a-helical peptides with
the exception of coupling constants for residues at the termini (Q1
and Y10).
Characterization by NMR spectroscopy
The amide proton chemical shift is temperature sensitive and
the magnitude of this shift is indicative of the extent to which the
particular amide proton is hydrogen-bonded.20 If an amide proton
exchanges slowly with a temperature coefficient more positive than
−4.5 ppb K−1, it is considered to be hydrogen-bonded. We find
that most amide temperature coefficients in helix 3b fall within the
range considered indicative of hydrogen-bonded amides and that
We were surprised to find that hydrogenation of the alkene did
not effect the stability of these artificial helices. These CD studies
provide important insights into the overall conformation of the
oligomer but do not offer detailed information about the peptide
structure. To learn about the behavior of individual residues in
3b in comparison to 3a, we performed comprehensive NMR
Table 2 Summary of NMR data for HBS helices 3a and 3b
Residues
Q1
V2
R4
Q5
L6
A7
E8
I9
Y10
3JNH–CaH/Hz
Calculated φ/◦
3a
3b
3a
8.4
9.1
−95
4.0
3.3
4.4
5.9
4.8
4.8
4.4
4.4
3.3
3.3
4.8
5.1
6.0
6.2
6.9
6.6
−81
−79
a
−58
−62
−65
−62
−52
−65
−74
3b −103
−52
−73
−65
−62
−52
−67
−76
Temperature coefficient/ppb DK−1
3a
3b
−4.45
−8.88
2.70
−2.64
−5.69
−4.50
−2.98
−3.26
−5.85
−4.73
−7.94
11.1
27.0
1.13
0.74
1.47
0.88
2.30
0.6
1.1
2.94
2.72
3.94
3.64
−4.42
−5.66
−4.60
−2.83
16.6
9.3
−3.20
−3.68
11.0
3.3
1.13
1.65
1.48
2.20
H/D exchange constant x 10−5/s−1 3a
8.0
2.2
1.6
3.7
3.2
3b
80.7
1.64
0.63
2.18
0.69
∼1.0
2.62
0.95
3.51
3.96
1.3
1.8
3.2
Protection factor (log kch/kex)b,c
Stabilization, −DG/kcal mol−1b
3a
3b
3a
3b
2.09
2.18
2.80
2.92
1.90
2.20
2.54
2.95
1.60
1.85
2.12
2.47
1.94
1.74
2.33
2.33
c
a Calculated according to the Karplus equation.18,21 b Calculated using the spreadsheet at http://hx2.med.upenn.edu/download.html. kex: measured
exchange rates, kch: intrinsic chemical exchange rate.
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Fig. 4 NOESY correlation charts and cross-sections of NOESY spectra for 3a (a, c) and 3b (b, d). The alanine-3 residues in both artificial helices
are N-alkylated. Filled rectangles indicate relative intensity of the NOE cross-peaks. Empty rectangles indicate NOE that could not be unambiguously
assigned because of overlapping signals.
the values are consistent with those observed with 3a (Fig. 5 and
Table 2).
exchange rate constants, kch, for an unstructured peptide of the
same sequence, to assess individual protection factors (log kch/kex)
and the corresponding free energies of protection (DG). The
predicted intrinsic chemical exchange rates, protection factors, and
the free energy of protection were calculated using the spreadsheet
at http://hx2.med.upenn.edu/download.html, and are shown in
Table 2. The data indicate that HBS a-helices 3a and 3b contain a
highly stable hydrogen-bonded network with significant protection
factors and associated free energies of protection (0.7–3.9 kcal
mol−1).3 Such a degree of stabilization is typically observed for
buried amide protons in proteins but not in short peptides.10,25
Overall, the NMR studies confirm that hydrogenated analog 3b
adopts a stable a-helical structure very similar to the one observed
for 3a.
Backbone amide hydrogen–deuterium exchange offers a sen-
sitive tool for examining protein structure and dynamics.22,23
The amide exchange rates for unstructured peptides in aqueous
solution are often too fast to measure; however if the amide
hydrogen is protected from exchange, i.e. through hydrogen
bonding, the exchange rates can slow down by several orders
of magnitude. Relative rate constants for the H–D exchange,
along with the temperature coefficients, provide important insights
regarding the involvement of individual amino acid residues
in intramolecular hydrogen bonds. Fig. 5b and 5d show H–D
exchange curves for helix 3a and 3b, respectively. The tabulated
exchange rates for 3a and 3b are shown in Table 2. The data in
Fig. 5 show that the individual hydrogen-exchange rates in these
helices can be determined precisely.24 The measured exchange rate
constants, kex, can be compared to the predicted intrinsic chemical
In summary, the circular dichroism and NMR results reported
here show that proper alignment of hydrogen bond donors and
acceptors is critical for the nucleation of stable a-helices, and
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Fig. 5 Temperature-dependent chemical shifts (a, c) and hydrogen–deuterium exchange plots (b, d) for backbone amide protons in 3a and 3b.19
that the 13-membered macrocycle affords the optimum nucleation
template. We also find that the double bond (which formally
replaces a carbonyl group in the peptide chain) in the nucleation
macrocycle can be hydrogenated while retaining helix stability.
This result will allow us to utilize the alkene group as a synthetic
handle for N-terminal functionalization of the HBS a-helices.
provide a rough estimation of the nucleation constant; detailed
studies aimed at deriving the nucleation constant in N- and
C-capped polyalanine helices will be described in due course.29
To estimate the nucleation constant, r, in HBS helices, the
thermal stabilities of 3a and 3b were investigated by monitoring
the temperature-dependent change in the intensity of the 222 nm
bands in the CD spectra (Fig. 6a). We observe a gradual increase
in the signal intensity at 222 nm with temperature, which indicates
helix unwinding at high temperatures. Nevertheless, we find that
HBS helices show a remarkable degree of thermal stability as the
two p◦eptides retain roughly 70% of their maximum CD signal
at 85 C. Although the HBS helices are conformationally stable
at high temperatures, they can be denatured with concentrated
guanidinium chloride.2 CD spectroscopy indicates that HBS helix
3a retains only 25% of its maximum helicity in 8 M GuHCl
(Supplementary Information, Figure S18†). We attribute the CD
signal at 8 M GuHCl ([h]222 = −4500) to the contribution of
the nucleation macrocycle to the overall CD spectra. All CD
studies were performed at 100 lM peptide concentrations. Fig. 6b
HBS a-helices and classical helix-coil parameters
Formation of stable helical structures from disordered polypep-
tides depends on two parameters: r which reflects helix nucleation
and s which describes propagation.4,5,26 The nucleation constant
which refers to the organization of three consecutive amino
acid residues in an a-turn is typically very low (10−3–10−4) in
unconstrained peptides, disfavoring helix formation.27,28 The intent
behind our hydrogen-bond surrogate approach was to afford
prenucleated helices such that r would be ∼1. Here we describe
preliminary studies to obtain a nucleation constant for HBS a-
helices based on the thermal stability of 3a and 3b. These studies
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Fig. 6 (a) Effect of temperature on the stability of HBS a-helices 3a and 3b. (b) Concentration dependence of mean residue ellipticity of HBS helices
at 25 ◦C. The CD spectra were obtained in 10% TFE–PBS. (c) Normalized thermal denaturation curves for HBS helices. (d) Comparison of theoretical
denaturation curves as a function of different nucleation constant, r, values with the normalized experimental denaturation curve. The theoretical curves
were obtained by simulating eqn (1) and (2).
shows the effects of peptide concentration on the mean residue
ellipticity and suggests that HBS helices are not aggregating in the
50–300 lM concentration range.
is the number of residues in the peptide beyond the macrocycle. The
simulations in Fig. 6d agree with the theory and experiments that
for a low r value, helicity is highly dependent on temperature but
that the helix is less perturbed as a function of temperature at high
r values. Comparison of the experimental thermal denaturation
curves with the simulation suggests that the nucleation constant,
r, is close to unity in HBS helices (Fig. 6c,d).
r|sm − 1|
f =
(1)
m
s − 1 + r|s − 1|
ꢀ
ꢁ
msm+1 − msm + s
DH
RT0
DH
s = s0 exp
−
RT
(2)
f =
(3)
m
m (s − 1) (s − 1)
To ascertain the effect of temperature on the fractional helicity
of the compounds, we normalized the temperature denaturation
curves in Fig. 6a such that the [h]222 value at 5 ◦C for each peptide
was assigned as the maximum fractional helicity value for that
peptide (Fig. 6c). These experimental denaturation curves can
be compared with the plots of fractional helicity as a function
of temperature at various theoretical r values (Fig. 6d). These
plots were obtained by simulating eqn (1) which represents a
simplified version of the Zimm–Bragg model for a helix that can
only denature in one direction (Supplementary Information†).30
Fractional helicity can then be correlated with the propagation
parameter, s, and temperature through eqn (2), as propagation
is largely an entropic phenomenon.31 The enthalpic contribution
(DH) to helix formation was set to a value of −1000 cal mol−1 in
eqn (2).32 In these calculations we utilized a value of s0 = 1 at T0 =
277 K to estimate a nucleation constant for these heteropolymers; v
The broad melting transition in HBS helices is consistent with
r = 1.33 This r value (and the broad transition) implies a
noncooperative case in which each unit behaves independently.
This situation suggests that the helix–coil transition does not
depend on the value of r. This reasoning led us to consider
transition in HBS helices with a modified Ising model in which
one end is always locked in the helix state. A complete discussion
on this subject is presented in the Supplementary Information†.
For this modified case, we can use eqn (3) to define fraction helicity
as a function of temperature. Fig. 7 compares the simulated curve
from eqn (3) to the experimental denaturation curve for helix 3a
and suggests that HBS helices are good models for an oligomer
that is locked in a helical conformation at one end.
This helix–coil analysis suggests that HBS helices would provide
a unique opportunity to obtain propagation constants for amino
acids in pre-nucleated helices. Determination of propagation
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Synthesis of HBS a-helices
Detailed synthetic procedures and characterization of HBS
helices and intermediates are reported in the Supplementary
Information.†
CD spectroscopy
CD spectra were recorded on an AVIV 202SF CD spectrometer
equipped with a temperature controller using 1 mm length cells
and a scan speed of 5 nm min−1. The spectra were averaged
over 10 scans with the baseline subtracted from analogous
conditions as those for the samples. The samples were prepared
in phosphate buffered saline (2 mM phosphate, 27 mM sodium
chloride and 0.5 mM potassium chloride, pH 7.4), containing
0%–10% trifluoroethanol, with a final peptide concentration of
100 lM. The concentrations of unfolded peptides were determined
by UV absorption of the tyrosine residue at 275 nm in 6.0 M
guanidinium hydrochloride aqueous solution. The helix content
of each peptide was determined from the mean residue CD at
222 nm, [h]222 (deg cm2 dmol−1) corrected for the number of amino
Fig. 7 Comparison of HBS helices to a theoretical oligomer with one
end always locked in the helix state (eq. 3). The experimental denaturation
curve of HBS helices agrees well with the simulated curve from eq. 3.
values in simple, nucleated and well-defined a-helices would signif-
icantly enhance our understanding of peptide and protein folding,
and has been a long-term goal of several research groups.16,34–36 We
are in the process of determining these propagation values in HBS
helices; the results of these studies will be reported in due course.
acids. Percent helicity was calculated from the ratio [h]222/[h]max
,
where [h]max = (−44 000 + 250T)(1 − k/n) = −23 400 for k = 4.0
and n = 10 (number of amino acid residues in the peptide).3,12,37
Conclusions
Temperature dependent amide proton chemical shift measurements
This manuscript outlines our continuing efforts to prepare well-
defined short a-helices as potential tools in disruption of biomolec-
ular interactions and as well-defined probes for examining the
biophysics of helix formation. Evaluation of several different
nucleation templates suggests that the hydrogen-bond surrogate-
based macrocycle that most closely resembles the 13-membered
hydrogen-bonded ring in canonical a-helices is optimum in these
artificial helices. This article also described our initial efforts to
characterize HBS helices in terms of classical helix–coil param-
eters. We find that these artificial a-helices mimic the theoretical
properties of a peptide with a well-defined and stable helix nucleus.
All experiments were carried out on a Bruker AVANCE 500 MHz
spectrometer equipped with an inverse TXI probe and 3D gradient
control. Samples of peptide 3a and 3b were prepared by dissolving
2 mg peptide in 450 lL PBS buffer, 30 lL D2O and 120 lL TFE-d3,
and adjusting the pH of the solution to 3.5 by adding 1 M HCl.
The 1D proton spectra or 2D TOCSY spectra (when overlapping
is severe) were employed to read the chemical shifts of the amide
protons. Solvent suppression was achieved with a 3919 Watergate
pulse sequence. The temperature ranged from 5 ◦C to 45 ◦C
at 5 ◦C intervals. At each temperature, the sample was allowed
to equilibrate for 15 minutes. The temperature was calibrated
precisely by measurement of peak separation in 100% methanol
(<30 ◦C) or 80% ethylene glycol in DMSO (>30 ◦C). The chemical
shifts were calibrated with internal standard tetramethylsilane
(TMS).
Experimental
General
Commercial-grade reagents and solvents were used without
further purification except as indicated. All Fmoc amino acids,
peptide synthesis reagents, and Rink Amide MBHA resin were
obtained from Novabiochem (San Diego, USA). All other reagents
were obtained from Sigma-Aldrich (St. Louis, USA). Reversed-
phase HPLC experiments were conducted with 4.6 × 150 mm
(analytical scale) or 21.4 × 150 mm (preparative scale) Waters
C18 Sunfire columns using a Beckman Coulter HPLC equipped
with a System Gold 168 Diode array detector. The typical flow
rates for analytical and preparative HPLC were 1 mL min−1 and
8 mL min−1, respectively. In all cases, 0.1% aqueous trifluoroacetic
acid and acetonitrile buffers were used. Proton and carbon NMR
spectra of monomers were obtained on a Bruker AVANCE
400 MHz spectrometer. Proton NMR spectra of HBS peptides
were recorded on a Bruker AVANCE 500 MHz spectrometer.
High-resolution mass spectra (HRMS) were obtained on a
LC/MSD TOF (Agilent Technologies). LCMS data was obtained
on an Agilent 1100 series LC/MSD (XCT) electrospray trap.
2D NMR spectroscopy
Spectra of peptide 3a and 3b (samples prepared as described
above) were recorded on a Bruker Avance 500 at 20 C. All 2D
◦
spectra were recorded by collecting 4092 complex data points in
the t2 domain by averaging 64 scans and 256 increments in the
t1 domain with the States-TPPI mode. All TOCSY experiments
are performed with a mixing time of 80 ms on 6000 Hz spin lock
frequency, and all NOESY with a mixing time of 200 ms. The
data were processed and analyzed using the Bruker TOPSPIN
program.38 The original free induction decays (FIDs) were zero-
filled to give a final matrix of 2048 by 2048 real data points. A 90◦
sine square window function was applied in both dimensions.
Amide H–D exchange study
Lyophilized samples of peptides 3a and 3b from the above
experiments were dissolved in 600 lL of D2O–TFE-d3 mixture
4080 | Org. Biomol. Chem., 2006, 4, 4074–4081
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(80 : 20) to initialize the H–D exchange. The pH of the solution
was confirmed. Spectra were recorded on a pre-shimmed Bruker
AVANCE 500 MHz spectrometer. The recorded temperature was
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◦
20 C both inside and outside the probe. The dead time was ca.
2 minutes. The intensity changes for each amide proton were
determined by monitoring either the HN peaks on 1D spectra
or the cross-peaks between HN and Ha on 2D TOCSY spectra
when overlapping was severe. The peak height data were fit into
one phase exponential equation to get the exchange rate constants
using GraphPad Prism 4.0 program.39
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19 HBS helices 3a and 3b contain arginine residues at the 4th position. The
amide protons of these residues display substantial upfield shifts. Please
see reference 3 for a discussion on the characteristics of R4 residues.
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Acknowledgements
We thank Professors Neville Kallenbach and Alexander Volo-
godskii for their invaluable advice. P.S.A is grateful for finan-
cial support from the NIH (GM073943), the donors of the
American Chemical Society Petroleum Research Fund, Research
Corporation (Cottrell Scholar Award), and NYU (Whitehead
Fellowship). We thank the NSF for equipment Grants MRI-
0116222 and CHE-0234863, and the NCRR/NIH for Research
Facilities Improvement Grant C06 RR-16572.
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