Reversible α-helix formation controlled by a hydrogen bond surrogate

Article abstract of DOI:10.1016/j.tet.2011.12.068

Strategically placed covalent linkages have been shown to stabilize helical conformations in short peptide sequences. Here we report the synthesis of a stabilized α-helix that utilizes an internal disulfide linkage. Structural analysis indicates that the

Full text of DOI:10.1016/j.tet.2011.12.068

Tetrahedron 68 (2012) 4434e4437
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Reversible
a
-helix formation controlled by a hydrogen bond surrogate
*
*
Stephen E. Miller, Neville R. Kallenbach , Paramjit S. Arora
Department of Chemistry, New York University, New York, NY 10003, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 October 2011
Received in revised form 24 November 2011
Accepted 21 December 2011
Available online 29 December 2011
Strategically placed covalent linkages have been shown to stabilize helical conformations in short pep-
tide sequences. Here we report the synthesis of a stabilized -helix that utilizes an internal disulfide
linkage. Structural analysis indicates that the dynamic nature of the disulfide bridge allows for the re-
versible formation of an -helix through oxidation and reduction reactions.
Ó 2011 Elsevier Ltd. All rights reserved.
a
a
Keywords:
Foldamer
Stabilized a-helix
Helix inducer
Hydrogen bond surrogate
Disulfide bridge
1. Introduction
Methods that provide reversible control over protein secondary
structure formation have proven to be useful for probing protein
structure and interactions.1e4 In this report, we describe the design of
a helix stabilized by a reversible covalent linkage. The strategy builds
on our reported efforts to prepare internally-constrained
a-helix
mimetics, termed hydrogen bond surrogate (HBS) helices.5,6 HBS
helices contain a covalent bond in place of an intrastrand hydrogen
bond between main chain atoms in canonical a-helices (Fig. 1a). We
have previously demonstrated that alkene, alkane, and thioether
linkages can serve to nucleate and stabilize the desired conformation
in short peptides.7e9 Herein we show that replacement of the main
chain hydrogen bond with a disulfide group provides facile access to
reversibly stabilized a
-helices (Fig. 1a).1,10,11 The disulfide linkage en-
forces slightly different dihedral requirements than the hydrocarbon
bridges;12 however, our biophysical studies suggest that conforma-
tional stability of dsHBS helix is similar to those of HBS helices. dsHBS
helices are readily formed by oxidizing bis-thiol peptides inwhich the
twosulfhydrylgroupsformallyoccupytheiandiþ4positions(Fig.1b).
2. Results and discussion
For these preliminary studies we designed a short helix based
on the p53 activation domain.13,14 The bis-thiol p53 peptide 1 was
synthesized on solid phase as shown in Scheme 1. Peptide 3 was
Fig. 1. (a) Hydrogen bond surrogate (HBS) a-helices feature a covalent bond in place of
* Corresponding authors. Tel.: þ1 212 998 8757 (N.R.K.); tel.: þ1 212 998 8470
(P.S.A.); e-mail addresses: nrk1@nyu.edu (N.R. Kallenbach), arora@nyu.edu (P.S.
Arora).
the intramolecular hydrogen bond between the i and iþ4 residues. (b) In dsHBS
a-
helices the main chain hydrogen bond is replaced with a disulfide linkage, and can be
reversibly formed from a bis-thiol peptide.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.12.068

S.E. Miller et al. / Tetrahedron 68 (2012) 4434e4437
4435
Scheme 1. Solid phase synthesis of bis-thiol 1 and dsHBS a-helix 2.
prepared using Fmoc chemistry on Rink amide resin.15 Coupling of
3 with bromoacetic acid followed by treatment with S-trityl-2-
mercaptoethylamine provided 4. Sequential coupling of Fmoc-
Glu(OtBu)eOH, Fmoc-Gln(Trt)eOH, and bromoacetic acid, treat-
ment with triphenylmethyl mercaptan followed by cleavage from
resin afforded bis-thiol peptide 1. We evaluated the potential of
iodine10 and DMSO16 to oxidize HPLC-purified 1 to dsHBS 2 and
found DMSO in 10% TFE/ammonium bicarbonate buffer (pH 6) to
provide efficient conversion to the desired product (Fig. 2).
peaks, a signature of helical structure, were observed for 2 as
depicted in Fig. 4. The NOESY spectrum further reveals medium to
weak (i, iþ3) and (i, iþ4) CH
aeNH cross peaks that support an a-
helical conformation in 2, although spectral overlap prevented as-
signment of some key cross-peaks. The NMR spectra, peak assign-
ments, and the NOESY correlation chart are included in the
Supplementary data.
3. Conclusion
The conformation of the peptides was examined by circular
dichroism and 2D NMR spectroscopies. CD studies were per-
formed in dilute phosphate buffered saline to obtain a measure of
their helical content (Fig. 3). CD spectroscopy suggests that, as
expected, bis-thiol 1 is weakly helical in aqueous solutions while
We have developed a new class of hydrogen bond surrogate
helices in which the conformation can be reversibly controlled
through oxidation of bis-thiols or reduction of the resulting disul-
fide bridge. We find that DMSO-mediated oxidation provides effi-
cient conversion to the desired compound. CD and NMR
spectroscopies provide strong support for our hypothesis that the
dsHBS 2 provides a CD signature typical of a canonical
a-helix,
with a double minima near 208 and 222 nm and a maximum at
190 nm.17e21 Upon treatment with 1.5 equiv of tris(2-
carboxyethyl)phosphine (TCEP), a reducing agent, dsHBS 2 loses
its conformational preference. The CD traces of the bis-thiol pep-
tides are similar to that of linear p53 peptide analog (6: Ac-
QEGFSDLWKLLS-NH2).
disulfide bridge provides conformationally-defined
a-helices. In
ongoing efforts we are utilizing dsHBS helices to probe biophysical
parameters related to the helix-coil transition; the results of these
studies will be reported in due course.
We performed 2D NMR experiments to further establish
the conformation of 2. NMR studies were performed in 20% TFE-
d3 in PBS (pH 3.5) on a Bruker 500 MHz spectrometer. The or-
ganic co-solvent was needed to solubilize the compound at the
low millimolar concentrations needed for NMR studies. Tri-
fluoroethanol, along with other organic co-solvents, is known to
enhance helicity of peptides.22e24 The %helicity of 2 increases by
1.5-fold in the presence of 20% TFE as compared to the pure
aqueous solution according to CD spectroscopy (Supplementary
data). The shapes of the curves, as well as the 208/222 nm ra-
4. Experimental section
4.1. General
Commercial-grade reagents and solvents were used without
further purification, except as indicated. Dry DMF was obtained
using an Innovative Technology PureSolv solvent drying system. All
reactions were either stirred or mechanically shaken at room
temperature, except as indicated. After each step, the resin was
sequentially washed with DMF (3ꢀ5 mL), MeOH (3ꢀ5 mL), and
DCM (3ꢀ5 mL). Microwave irradiation was performed in the CEM
Discover single-mode reactor with controlled power, temperature,
time, and stirring settings. NMR experiments were performed using
a Bruker AVANCE 500 MHz spectrometer. Reversed-phase HPLC
experiments were conducted with 4.6ꢀ150 mm (analytical scale)
or 21.4ꢀ150 mm (preparative scale) Waters C18 Sunfire columns
tio used to evaluate
a-helicity remain constant in the two sol-
vents.17,18 Thus, CD spectroscopy suggests that a different helical
conformation is not being stabilized by the addition of the co-
solvent.
A set of 2D TOCSYand NOESY spectra was used to assign 1H NMR
resonances for 2. Sequential NHeNH (i and i þ1) NOESY cross-

4436
S.E. Miller et al. / Tetrahedron 68 (2012) 4434e4437
Fig. 4. Short-range (dashed arrows) and medium-range (black arrows) NOEs observed
for dsHBS 2. 2D NMR spectra were acquired in 20% trifluoroethanol-d3/PBS at 20 ꢂC.
using a Beckman Coulter HPLC equipped with a System Gold 168
Diode array detector. HPLC buffers consisted of 0.1% aqueous tri-
fluoroacetic acid and 0.1% trifluoroacetic acid in acetonitrile. Mass
spectra were obtained by either an Agilent 1100 series LC/MSD
(XCT) electrospray trap or a Bruker UltraflexXtreme MALDI-TOF/
TOF. The peptide concentrations for the CD and NMR studies
were calculated using absorbance of the tryptophan residue
(5560 cmꢁ1
M
ꢁ1 at 280 nm).
4.2. Synthesis of bis-thiol peptide 1
Parent peptide 3 (0.25 mmol) was synthesized on a CEM Liberty
microwave peptide synthesizer using standard Fmoc solid phase
chemistry with Knorr Amide MBHA resin. The resin bearing 3 was
transferred to a fritted polypropylene SPE tube, washed, and dried
under vacuum. A solution of preactivated bromoacetic acid (0.17 g,
1.25 mmol, 5 equiv), HOBt (0.18 g, 1.25 mmol, 5 equiv), DIC
(0.20 mL, 1.25 mmol, 5 equiv) in 3 mL dry DMF was added to the
resin containing 3. After 3 h, the resin was washed and treated with
S-trityl-2-mercaptoethylamine (0.45 g, 1.25 mmol, 5 equiv) and
DIEA (0.66 mL, 3.37 mmol, 15 equiv) in 3 mL dry DMF. After 3 h, the
resin containing 4 was washed. The resin was dried under vacuum
for 1 h, transferred to a microwave tube, capped, and purged with
N2 for 1 h.
Fig. 2. DMSO-mediated conversion of bis-thiol 1 to dsHBS 2.
To the microwave tube containing dried 4 on resin, a solution of
preactivated Fmoc-Glu(tBu)eOH (2.13 g, 5.0 mmol, 20 equiv), HOAt
(0.35 g, 2.5 mmol, 10 equiv), DIC (0.66 mL, 5.0 mmol, 20 equiv) in
4 mL dry DMF was added. The reaction mixture was subjected to
microwave irradiation at 60 ꢂC for 45 min, after which the resin was
transferred to an SPE tube and washed. The resin was treated with
20% piperidine in NMP (2ꢀ20 min) for Fmoc deprotection, washed
and dried under vacuum.
A solution of preactivated Fmoc-
Gln(Trt)eOH (0.76 g, 1.25 mmol, 5 equiv), HBTU (0.42 g, 1.13 mmol,
4.5 equiv) in 4.1 mL 5% DIEA/NMP was added to the resin. After 3 h,
the resin containing 5 was washed and dried under vacuum.
Resin bound 5 was subjected to Fmoc deprotection and bro-
moacetylation as described above. A solution of triphenylmethyl
mercaptan (0.35 g, 1.25 mmol, 5 equiv) and DIEA (0.66 mL,
3.75 mmol, 15 equiv) in 3 mL dry DMF was added to the resin. After
3 h, the resin was washed and dried under vacuum. The dried resin
was treated with 81.5% TFA, 5% H2O, 5% thioanisole, 5% phenol, 2.5%
Fig. 3. Circular dichroism spectra of peptides 1, 2, and 6. The CD spectra were obtained
in phosphate buffered saline. TCEP¼tris(2-carboxyethyl)phosphine. Calculated % hel-
icity values: 1: 22%, 2: 55%, 2þTCEP: 22%, and 6: 14%.

S.E. Miller et al. / Tetrahedron 68 (2012) 4434e4437
4437
EDT, 1% TIPS. After 2 h, the mixture was filtered and concentrated in
vacuo. The crude solid was washed with cold ether and dried under
N2. HPLC purification (gradient of 5e75 acetonitrile/water in
45 min) and lyophilization yielded bis-thiol peptide 1 as a white
powder (10 mg). [MþH]þ calculated: 1555.73; [MþH]þ observed:
1555.71; analytical HPLC tR: 9.3 min (gradient of 5e95 acetonitrile/
water in 15 min).
domain by averaging 72 scans and 512 increments in the t1 domain
with the States-TPPI mode. TOCSY experiments were performed
with a mixing time of 80 ms on a 6000 Hz spin lock frequency. For
NOESY, mixing time of 200 ms was used. The data were processed
and analyzed using the Bruker TOPSPIN program. The original free
induction decays (FIDs) were zero-filled to give a final matrix of
1024 by 1024 real data points. A 90ꢂ sine-square window function
was applied in both dimensions.
4.3. Synthesis of dsHBS 2
Acknowledgements
Bis-thiol peptide 1 (5 mg) was dissolved in 6.3 mL of 0.1 M
aqueous ammonium bicarbonate solution (buffered to pH 6 with
TFA) containing 20% DMSO and 10% TFE. After 15 h, the mixture was
frozen and lyophilized to obtain a colorless oil. HPLC purification
(gradient of 5e65 acetonitrile/water in 45 min) and lyophilization
yielded dsHBS 2 (1 mg, 20%) as a white powder. [MþH]þ calculated:
1553.71; [MþH]þ observed: 1553.72; analytical HPLC tR: 9.5 min
(gradient of 5e95 acetonitrile/water in 15 min).
This work was financially supported by the National Science
Foundation (CHE1027009 to N.R.K.) and the National Institutes of
Health (GM073943 to P.S.A.). S.E.M. thanks the New York University
Department of Chemistry for a Margaret Strauss Kramer Pre-
doctoral Fellowship. Support from the National Science Foundation
(CHE-0958457) in the form of an instrumentation grant is grate-
fully acknowledged.
4.4. Synthesis of S-trityl-mercaptoethylamine
Supplementary data
hydrochloride25
2D NMR and circular dichroism spectra of 2. Supplementary
data related to this article can be found online at doi:10.1016/
j.tet.2011.12.068.
Cysteamine hydrochloride (1.00 g, 8.8 mmol, 1 equiv) was dis-
solved in DMF/DCM (1:1, 50 mL), followed by the addition of trityl
chloride (3.68 g, 13.2 mmol, 1.5 equiv). After 3 h, insoluble material
was filtered and washed with DCM (3ꢀ10 mL). The crude product
was concentrated in vacuo and subsequently re-constituted and re-
concentrated (3ꢀ50 mL DCM). Purification by flash chromatogra-
phy (10% MeOH/DCM) yielded 2.35 g of off-white solid (75% yield).
References and notes
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Rf: 0.60 (1:9 MeOH/DCM). 1H NMR (CDCl3):
d
¼2.36e2.60 (m, 4H),
5.25 (br s, 3H), 7.19e7.56 (m, 15H). 13C NMR (CDCl3):
67.05, 126.88, 128.08, 129.53, 144.44.
d
¼32.08, 39.57,
4.5. Circular dichroism spectroscopy
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Soc. 1991, 113, 9391e9392.
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equipped with a temperature controller using 1 mm length cells
and a scan speed of 5 nm/min. The spectra were averaged over 10
scans with the baseline subtracted from analogous conditions to
those of the samples. The samples were prepared in 0.1ꢀ phos-
phate buffered saline (13.7 mM NaCl, 1 mM phosphate, 0.27 mM
KCl, pH 7.4), with the final peptide concentration of 50
monitor the effects of a reducing agent on the helix conformation,
a sample of dsHBS 2 was prepared with 75 M TCEP in the buffer
mM. To
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conditions; reduction of the disulfide bond to bis-thiol was con-
firmed by mass spectroscopy. The helix content of each peptide was
determined from the mean residue CD at 222 nm,
[q]
222
(deg cm2 dmolꢁ1) corrected for the number of amino acids. Percent
helicity was calculated from the ratio
[
q
]
222/[
q
]
, where
max
[
q
]
¼(ꢁ44,000þ250T)(1ꢁk/n)¼ꢁ25,170 for T¼25 (ꢂC), k¼4.0,
max
and n¼12 (number of amino acid residues).7,20,23
4.6. 2D NMR spectroscopy
Spectra of dsHBS 2 were recorded on a Bruker Avance 500 at
20 ꢂC. The sample was prepared by dissolving 1 mg of 2 in 300
L of
m
20% TFE-d3 in PBS (pH 3.5) in a Shigemi NMR tube. All 2D spectra
were recorded by collecting 2048 complex data points in the t2