Peptide design. Helix-helix motifs in synthetic sequences

Article abstract of DOI:10.1039/a700624a

Two centrally positioned α-aminoisobutyryl (Aib) residues have been used to stabilize distinct heptapeptide helical segments in the 15-residue synthetic sequence Boc-Met-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe. The helices are connecte

Full text of DOI:10.1039/a700624a

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Peptide design. Helix–helix motifs in synthetic sequences
Arindam Banerjee,a S. Raghothama b and P. Balaram*,a
a
Molecular Biophysics Unit and b Sophisticated Instruments Facility, Indian Institute of
Science, Bangalore-560012, India
Two centrally positioned á-aminoisobutyryl (Aib) residues have been used to stabilize distinct
heptapeptide helical segments in the 15-residue synthetic sequence Boc-Met-Ala-Leu-Aib-Val-Ala-Leu-
Acp-Val-Ala-Leu-Aib-Val-Ala-Phe-OM e. The helices are connected by the flexible linker å-aminocaproic
acid (Acp). N M R studies in CD Cl3 establish helical conformations for both independent segments as
evidenced by N H –N H nuclear Overhauser effects (N OEs). The peptide strongly aggregates in CD Cl3 with
the N H groups of M et(1) and Ala(2) participating in intermolecular hydrogen bonds. In (CD 3)2SO two
solvated helical segments are supported by N M R results. Solvent dependent breakdown of aggregates on
addition of (CD 3)2SO to CD Cl3 solutions is suggested by analysis of chemical shifts and temperature
coefficients of N H protons. The observation of several interhelical N OEs in CD Cl3, relatively few N OEs
in 10% (CD 3)2SO–CD Cl3 and their absence in (CD 3)2SO provides a means of inferring helix orientations.
While an antiparallel arrangement resulting in closed aggregate formation is suggested in CD Cl3, a
parallel solvated arrangement is favoured in (CD 3)2SO.
Introduction
The success of de novo protein design rests on the ability to
construct peptide sequences with predictable folding patterns
which then assemble to form compact tertiary structures.1,2 In
aqueous solutions, the driving forces for maximizing tertiary
interactions are hydrophobic in nature. The construction of
amphipathic secondary structures then provides a convenient
means of achieving compact polypeptide conformations with a
largely hydrophobic core.3,4 The secondary structural elements
are themselves constructed using well-established principles of
helix stabilization in short peptides5–7 and the less widely
investigated strategies for β-hairpins.8,9 The difficulties of pre-
cisely designing well packed cores has led to many designed
proteins which have molten globule like characteristics.10–13 The
use of metal ion and organic templates to organize structured
peptide segments has proved promising.14–20
Fig. 1 Interconversions between antiparallel, close packed helical
cylinders and open extended forms are illustrated. Two molecules are
schematically shown to indicate the nature of intramolecular ‘helix like’
hydrogen bonds observed in crystals of the peptide Boc-Val-Ala-Leu-
Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe.25
CO groups of a neighboring molecule, which constitute the end
of the N-terminal segment and the beginning of the C-terminal
segment, respectively. Intramolecular folding would have been
expected to yield a U-shaped structure in which the two helices
are roughly antiparallel, with an interhelical angle close to 0Њ.
In order to address the question of conformational variability
about the linking Acp residue, we have carried out detailed
NMR investigations of the 15 residue peptide sequence Boc-
Met-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-Ala-
Phe-OMe (1) in organic solvents [CDCl3 and (CD3)2SO], which
establish solvent dependent changes in structures and aggrega-
tion states. The peptide sequence was chosen such that both
helical modules (1–7 and 9–15) have centrally positioned Aib
residues. The use of Met and Phe as the N- and C-terminal
residues respectively, facilitates NMR assignments without
changing the apolar character of the peptide and affords the
possibility of observing assignable NOEs, diagnostic of tertiary
interactions.
An alternative approach to synthetic polypeptide design
which is being investigated in this laboratory uses well-defined
secondary structural modules which are soluble in organic solv-
ents. The use of α-aminoisobutyric acid (Aib) and related α,α-
dialkylated amino acids to stabilize helices up to 16 residues has
been demonstrated by crystallographic and spectroscopic
studies.21–24 The subsequent assembly of apolar peptide helices
into helix–linker–helix motifs has also been reported.25 In this
approach peptide chain folding is directed by the use of a limit-
ed number of conformationally constrained amino acids. Sub-
sequent interactions of secondary structural elements are
determined by the stereochemical properties of the linking
segments, hydrogen bonding and side chain packing inter-
actions. The solubility of these systems in relatively inert solv-
ents like chloroform and methanol permits folding processes to
be studied in the absence of a strong hydrophobic driving force.
The crystal structure determination of the helix–linker–helix
peptide (Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-
Aib-Val-Ala-Leu-OMe) reveals that the two helices make an
angle of approximately 180Њ, with the two cylindrical modules
being staggered about the connecting ε-aminocaproic (Acp)
segment (Fig. 1).25 This arrangement in crystals is stabilized by
a continuous chain of intermolecular ‘helix-like’ hydrogen
bonds involving the NH groups of one molecule and the central
Experimental
Peptide synthesis
Peptide 1 was synthesized by conventional solution phase
methods by using a racemization free fragment condensation
strategy. The Boc group was used for N-terminal protection and
the C-terminus was protected as a methyl ester. Deprotections
were performed using 98% formic acid or saponification,
respectively. Couplings were mediated by dicyclohexylcarbo-
diimide–1-hydroxybenzotriazole (DCC–HOBT). All the inter-
1
mediates were characterized by H NMR (400 MHz) and thin
* E-Mail: pb@mbu.iisc.ernet.in
J. Chem. Soc., Perkin Trans. 2, 1997
2087

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layer chromatography (TLC) on silica gel and used without fur-
ther purification. The final peptide was purified by medium
pressure liquid chromatography (MPLC) and high performance
liquid chromatography (HPLC) on reverse phase C-18 columns
and fully characterized by 400 MHz 1H NMR.
CδH and CγH), 1.98 (Val CβH), 2.12 (Met CεH), 2.25 (Met
CβH), 2.30 (Acp CαH), 2.66 (Met CγH), 3.18 (Acp CεH), 3.28
(Acp CεH), 3.65 (OCH3), 3.80 (1H, Val CαH), 4.07–4.11 (3H,
Met CαH, Ala CαH, Leu CαH), 4.24 (1H, Ala CαH), 4.40 (1H,
Leu CαH), 6.32 (1H, Met NH), 7.13 (1H), 7.15 (1H), 7.31 (1H),
7.34 (1H), 7.58 (1H) (Ala, Leu, Val NH), 7.22 (1H, Acp NH),
7.37 (Aib NH).
Boc-Met-Ala-Leu-Aib-OMe 2. To 1.65 g (4.11 mmol) of Boc-
Ala-Leu-Aib-OMe,26 10 ml of 98% formic acid was added and
the removal of Boc group monitored by TLC. After 8 h, the
formic acid was removed in vacuo. The residue was taken in
water (20 ml) and washed with diethyl ether (2 × 20 ml). The
pH of the aqueous solution was then adjusted to 8 with sodium
hydrogen carbonate and extracted with ethyl acetate (3 × 30
ml). The extracts were pooled, washed with saturated brine,
dried over sodium sulfate, and concentrated to 5 ml of a highly
viscous liquid that gave a positive ninhydrin test. The tripeptide
free base was added to an ice-cooled solution of Boc-Met-OH
(0.93 g, 3.64 mmol) in 10 ml of dimethylformamide (DMF ),
followed by 0.74 g (3.7 mmol) of DCC and 0.49 g (3.65 mmol)
of HOBT. The reaction mixture was stirred for 3 d under nitro-
gen atmosphere. The residue was taken in ethyl acetate (60 ml)
and dicyclohexylurea (DCU) was filtered off. The organic layer
was washed with 2  HCl (3 × 50 ml), 1 sodium carbonate
(3 × 50 ml) and brine (2 × 50 ml), dried over sodium sulfate and
evaporated in vacuo to yield 1.36 g (70%) of a gummy material.
δH (400 MHz, CDCl3) 0.90–0.93 (Leu CδH), 1.43 (Ala CβH),
1.46 (Boc CH3), 1.50 and 1.53 (Aib CβH3), 1.59 (Leu CβH and
CγH), 1.93 (Met CβH), 2.12 (Met CεH), 2.60 (Met CγH), 3.71
(OCH3), 4.19 (Ala CαH), 4.38–4.42 (Met CαH and Leu CαH),
5.27 (Met NH), 6.67 (Ala NH and Leu NH), 6.77 (Aib NH).
Boc-Met-Ala-Leu-Aib-OH 3. To 1.26 g (2.37 mmol) of 2 in
15 ml of methanol, 3 ml of 2  NaOH were added and the
reaction was monitored by TLC. On completion of the reaction
after 10 h methanol was evaporated, the residue was taken in
water and washed with diethyl ether the aqueous layer was acid-
ified with cold 2  HCl to pH 2 and extracted with ethyl acetate
(3 × 30 ml). The organic extracts were pooled, dried over
anhydrous sodium sulfate and evaporated in vacuo to yield 1.2 g
(70%) of a white solid. δH (400 MHz, CDCl3) 0.88–0.92 (Leu
CδH), 1.34 (Ala CβH), 1.44 (Boc CH3), 1.50, 1.54 (Aib CβH3),
1.63 (Leu CβH, CγH), 1.93 (Met CβH), 2.09 (Met CεH), 2.54
(Met CγH), 4.34 (1H, Ala CαH), 4.52–4.56 (2H, Met CαH, Leu
CαH), 5.45 (1H, Met NH), 7.23 (1H, Aib NH), 7.52 (1H), 7.93
(2H) (Ala NH, Leu NH).
Boc-Met-Ala-Leu-Aib-Val-Ala-Leu-OMe 4. Boc-Val-Ala-
Leu-OMe26 (1.1 g, 2.65 mmol) was deprotected with 98% for-
mic acid and worked up as reported in the preparation of 2.
This was coupled with 1.2 g (2.25 mmol) of 3 in 15 ml of DMF
using 0.48 g (2.40 mmol) of DCC and 0.30 g (2.25 mmol) of
HOBT under nitrogen atmosphere. After 3 d the reaction was
worked up as described for 3 to yield the crude peptide as a
solid (1.17 g, 1.44 mmol). The peptide was purified on a reverse
phase C-18 MPLC column using methanol–water mixtures.
δH (400 MHz, CDCl3) 0.90 (Leu CδH), 0.95 (Leu CδH), 1.01 (Val
CγH), 1.45 (Ala CβH), 1.49 (Aib CβH), (Ala CβH), 1.58 (Boc
CH3), 1.72 (Leu CβH, CγH), 1.95 (Val CβH), 2.14 (Met CεH),
2.50 (Met CβH), 2.66 (Met CγH), 3.70 (OCH3), 4.10 (2H), 4.16
(1H), 4.23 (2H), 4.56 (1H) (Ala, Leu, Met, Val CαH), 5.67 (1H,
Met NH), 6.71 (1H), 6.76 (1H), 7.20 (1H), 7.22 (1H), 7.59 (1H)
(Ala, Leu, Val NH), 7.33 (1H, Aib NH).
Boc-Val-Ala-Phe-OMe 6. Boc-Val-Ala-OH 26 (2.82 g, 10
mmol) was taken in 15 ml of DMF and coupled to H-Phe-OMe
isolated from 5.30 g (24.5 mmol) of the corresponding methyl
ester hydrochloride using DCC (2.08 g, 10.4 mmol) and HOBT
(1.35 g, 10 mmol). The reaction was worked up after 3 d as
described for 2 to yield a white solid (2.08 g, 5.7 mmol, 57%).
δH (400 MHz CDCl3) 0.90 (Val CγH), 1.34 (Ala CβH), 1.44
(Boc CH3), 2.11 (Val CβH), 3.10 (Phe CβH), 3.70 (OCH3), 3.91
(Val CαH), 4.46 (Phe CαH), 4.82 (Ala CαH), 5.05 (Val NH),
6.56 (Ala NH), 6.56 (Phe NH), 7.09 (Phe ring H), 7.28 (Phe
ring H).
Boc-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe 7. The peptide 6
(1.08 g, 2.34 mmol) was deprotected with 98% formic acid and
worked up as reported in the preparation of 2. This was
coupled to 0.97 g (2 mmol) of Boc-Val-Ala-Leu-Aib-OH 26
using DCC (0.42 g, 2.1 mmol) and HOBT (0.27 g, 2 mmol). The
reaction was worked up after 3 d as described for 2 to yield the
crude peptide as a white solid (0.88 g, 1.06 mmol). The peptide
was purified on a reverse phase C-18 MPLC column using
methanol–water mixtures. δH (400 MHz, CDCl3) 0.89 (Leu
CδH), 0.95 (Leu CδH), 1.04 (Val CγH), 1.47 (Ala CβH), 1.49
(Aib CβH), (Ala CβH), 1.58 (Boc CH3), 1.64–1.70 (Leu CβH and
CγH, Acp CδHs and CγH), 1.98 (Val CβH), 2.12 (Met CεH), 2.25
(Met CβH), 2.30 (Acp CαH), 2.66 (Met CγH), 3.18 (Acp CεH),
3.28 (Acp CεH), 3.65 (OCH3), 3.80 (1H, Val CαH), 4.07–4.11
(3H, Met CαH, Ala CαH, Leu CαH), 4.24 (1H, Ala CαH), 4.40
(1H, Leu CαH), 6.32 (1H, Met NH), 7.13 (1H), 7.15 (1H), 7.31
(1H), 7.34 (1H), 7.58 (1H) (Ala, Leu, Val NH), 7.22 (1H), Acp
NH), 7.37 (Aib NH).
Boc-Met-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-
Val-Ala-Phe-OMe 1. The peptide 5 (0.87 g, 0.94 mmol) was
saponified using 15 ml of methanol and 2 ml of 2  NaOH
under nitrogen atmosphere and worked up as described for 3.
This was coupled to H-Val-Ala-Leu-Aib-Val-Ala-Phe-OMe
[isolated from 8 (0.80 g, 0.96 mmol) as described in the prepar-
ation of 2] using 0.11 g of DCC and 0.2 g of HOBT under
nitrogen atmosphere. After 3 d the reaction was worked up as
usual to yield 0.83 g of the crude peptide. The peptide was
purified on
a reverse phase C-18 MPLC column using
methanol–water mixtures. The peptide was further subjected to
HPLC purification on a Lichrosorb reverse phase C-18 HPLC
column (4 × 250 mm, particle size 10 µm, flow rate 1.5 ml minϪ1
)
and eluted on a linear gradient of methanol–water (70–95%)
with a retention time of 23 min. Mp 109–110 ЊC. The peptide
was homogeneous on a reverse phase C-18 (5 µm) column and
fully characterized by NMR (see results).
Spectroscopic studies
All NMR studies were carried out on a Bruker AMX-400 spec-
trometer at the Sophisticated Instruments Facility. All two-
dimensional NMR experiments were carried out using a pep-
tide concentration of 3.7 m. Concentration dependent 1D
NMR experiments were performed over the range 6–0.05 m
and the probe temperature was maintained at 298 K. Reson-
ance assignments were made using two-dimensional double
quantum filtered COSY spectra (1K data points, 512 experi-
ments, 48 transients, spectral width 4500 Hz). Two-dimensional
NOESY and ROESY spectra were acquired using 1024 points,
512 increments, 64 transients and mixing time 300 ms. All two-
dimensional data sets were zero filled to 1024 points with a 90Њ
phase shifted squared sinebell filter in both the dimensions.
Coupling constants were calculated from one-dimensional
experiments.
Boc-Met-Ala-Leu-Aib-Val-Ala-Leu-Acp-OMe 5. The peptide
4 (1.16 g) was saponified using 15 ml of methanol and 2 ml of 2
 NaOH under nitrogen atmosphere and worked up as
reported in the preparation of 3. This was coupled to H-Acp-
OMe that was isolated from 0.48 g (2.64 mmol) of the corres-
ponding hydrochloride using DCC (0.56 g, 2.80 mmol) and
HOBT (0.36 g, 2.67 mmol) under nitrogen atmosphere. The
reaction was worked up after 2 d as described for 2 to yield 0.88
g (75%) of a white solid. δH (400 MHz CDCl3) 0.89 (Leu CδH),
0.95 (Leu CδH), 1.04 (Val CγH), 1.47 (Ala CβH), 1.49 (Aib CβH,
Ala CβH), 1.58 (Boc CH3), 1.64–1.70 (Leu CβH and CγH, Acp
2088
J. Chem. Soc., Perkin Trans. 2, 1997

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CD spectra were recorded on a JASCO J-500 spectropolari-
meter equipped with a DP-501N data processor using 1 mm
path length cuvettes. Scan speeds were generally kept at 5 nm
minϪ1 and the time constant at 8 or 16 s. Spectra were averaged
over several scans (typically 4–8). The baseline was obtained
under similar conditions as the sample. All spectra are baseline
subtracted, converted to a uniform scale of molar ellipticity
and replotted. Temperature was kept at 20 ЊC. The sample
concentration used was 181 µ.
Results and discussion
NMR studies of peptide 1 were carried out in CDCl3, CDCl3–
(CD3)2SO (90:10) and (CD3)2SO. Complete assignment of the
1H NMR spectrum in CDCl3 and (CD3)2SO was performed
using a combination of COSY and NOESY/ROESY. The rep-
resentative NOESY spectra shown in Fig. 2 illustrate observed
inter-residue connectivities which permit sequence specific
assignments. Chemical shifts for the various resonances are
summarized in the Table 1. The temperature coefficients of the
chemical shifts of the various NH resonances (dδ/dT ) and the
3
α
JH N C H , vicinal coupling constants in the three solvent systems
are listed in Table 2. The observed NOEs in the three solvent
systems are summarized in Fig. 3.
Conformational analysis in chloroform
In CDCl3, a succession of strong dN N (i,i ϩ 1) (NiH
NOE connectivities were observed over the segments 1–7 and
9–15. The corresponding dαN (Cα H
Ni ϩ 1H) NOEs are
Ni ϩ 1H)
i
weak or not observed (Fig. 2 and 3). These observations sup-
port the formation of two helical segments that are separated
by the flanking Acp residue.
The effect of adding a strong hydrogen bond accepting solv-
ent like (CD3)2SO to CDCl3 solutions of the peptide 1 on the
amide NH chemical shifts is summarized in Fig. 4. Normally,
addition of small amounts of (CD3)2SO results in monotonic
downfield shifts of exposed NH groups in peptides, leaving
solvent shielded NH groups largely unaffected. This permits
delineation of intramolecularly hydrogen bonded NH groups in
peptides using solvent titration experiments which have found
Fig. 2 Partial 400 MHz 1H–1H NOESY spectrum of the peptide 1 in
CDCl3. CαH᎐NH NOEs (top panel) and amide NH᎐NH NOEs (bot-
tom) panel are indicated. Specific resonance assignments are indicated
on the 1D spectra using one letter code. Several dαN (top) and dN N
(botttom) are also labelled. Inter-helix NOEs are underlined (top
panel).
Met 1 Ala 2 Leu 3 Aib 4 Val 5 Ala 6 Leu 7 Acp 8 Val 9 Ala 10 Leu 11 Aib 12 Val 13 Ala 14 Phe 15
ND
dαN (i,i+3) CDCl3
NN (i,i+1) 90% CDCl3
dNN (i,i+1) (CD3)2SO
αN (i,i+1) CDCl3
d
ND
ND
ND
ND
ND
d
dαN (i,i+1) (CD3)2SO
dNN (i,i+2) CDCl3
dNN (i,i+2) CDCl3
dNN (i,i+2) CDCl3
dαN (i,i+2) CDCl3
dαN (i,i+3) CDCl3
dαN (i,i+3) CDCl3
dαN (i,i+3) 90% CDCl3
ND
strong
medium
weak
NO NOE
not determined due to resonance overlap
Fig. 3 A summary of inter-residue NOEs for the peptide 1 in three solvent systems
J. Chem. Soc., Perkin Trans. 2, 1997
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Table 1 Characteristic 1H NMR parameters for the peptide 1
δ valuesa
Residues
Met1
NH
CαH
CβH
CγH
CδH
Others
6.87
(7.09)
4.07
(3.98)
1.98
(2.46)
2.61
(1.81)
ε-CH3,
2.07
(2.01)
Ala2
Leu3
Aib4
Val5
Ala6
Leu7
Acp8
8.13
(8.04)
7.38
(7.99)
7.35
(8.14)
7.55
(7.29)
7.51
(7.80)
7.87
(7.47)
7.36
4.04
(4.26)
4.09
(4.16)
—
(—)
3.81
(3.88)
4.23
(4.20)
4.33
1.48
(1.49)
1.72
(1.61)
1.56
(1.49)
2.18
(1.93)
1.53
(1.46)
1.76
(1.58)
1.62, 1.76
(1.47)
1.68
(1.61)
0.90, 0.94
(0.84)
1.07, 1.08
(0.87)
1.70
(1.58)
1.62, 1.70
(1.24)
0.84, 0.89
(0.86)
1.62
(4.17)
2.24, 2.60
(2.15)
ε-CH2
2.96, 3.71
(3.02)
(7.43)
(1.40)
Val9
8.46
(7.87)
8.37
(8.07)
7.05
(7.82)
7.86
(8.02)
7.54
(7.06)
7.62
(7.77)
7.83
3.53
(4.07)
4.03
(4.22)
4.20
(4.12)
—
(—)
4.05
(4.06)
4.40
(4.24)
4.75
(4.44)
2.21
(2.15)
1.45
(1.44)
1.79
(1.60)
1.52
(1.48)
2.27
(2.02)
1.39
(1.46)
3.05, 3.25
(2.91, 3.01)
1.09, 0.99
(0.83)
Ala10
Leu11
Aib12
Val13
Ala14
Phe15
1.69
(1.60)
0.95, 0.98
(0.85)
1.02, 1.03
(0.78)
aromatic H
7.18–7.31
(7.19–7.30)
(8.13)
aChemical shifts of proton resonances in CDCl3 and, in parentheses, (CD3)2SO.
Table 2 1H HNMR parameters for NH groups in peptide 1
dδ/dT a (ppb KϪ1
)
3JH N C b/Hz
αH
CDCl3–(CD3)2SO
(9:1)
CDCl3–(CD3)2SO
(9:1)
Residue
CDCl3
(CD3)2SO
CDCl3
(CD3)2SO
Met1
Ala2
Leu3
Aib4
Val5
15.9
18.4
3.7
Ϫ0.2
9.2
1.0
6.4
2.4
8.4
6.7
Ϫ2.3
3.3
8.1
1.2
9.0
7.9
2.2
6.7
5.1
4.3
5.6
3.4
4.0
3.6
3.4
4.5
5.4
4.7
5.1
1.6
4.3
6.3
<2
<2
ND
<2
<2
ND
ND
5.0
6.2
—
Ϫ0.6
—
—
5.5
0.8
5.8
4.9
8.1
7.1
Ϫ1.1
3.9
5.8
ND
5.7
ND
4.0
<2
<2
7.6
ND
6.2
7.8
ND
3.0
2.9
7.6
—
<2
Ala6
4.0
9.0
4.5
8.1
6.9
6.5
—
<2
ND
<2
Leu7
Acp8
Val9
Ala10
Leu11
Aib12
Val13
Ala14
Phe15
—
ND
7.1
7.1
6.3
7.7
8.2
1.9
3.7
6.1
a
Temperature coefficients in CDCl3, 90% CDCl3–10% (CD3)2SO and (CD3)2SO. b Coupling constants in CDCl3, 90% CDCl3–10% (CD3)2SO and
(CD3)2SO. ND = not determined due to resonance overlap.
widespread use in the literature.27,28 The results in Fig. 4 are
striking, with several NH groups showing anomalous
behaviour. Met1 NH and Ala2 NH which are expected to be
solvent exposed in the N-terminal helix move upfield up to a
(CD3)2SO concentration of ca. 5%; at higher (CD3)2SO concen-
tration both these NH groups show monotonic downfield shifts
with saturation being observed at a (CD3)2SO concentration of
ca. 40%. In a 310-helical structure the NH groups of the first two
residues from the N-terminus are exposed to the solvent. In
most of the examples of helical peptides studied so far, addition
of (CD3)2SO to CDCl3 solutions of the peptide results in a
steady downfield shift of the NH resonances for the residues 1
and 2. The strikingly unusual behavior of the Met1 and Ala2
NH groups of peptide 1 is suggestive of peptide aggregation at
the concentrations (3.7 m) used in the NMR experiments.
Indeed, helical peptides can associate in apolar solvents by
hydrogen bond formation between exposed amino terminal NH
groups and carboxy terminal CO groups. This kind of aggreg-
ation results in infinite head-to-tail hydrogen bonded arrays in
crystals.29–31
The variation of NH chemical shifts as a function of peptide
concentration over the range 6–0.05 m in CDCl3 is summar-
2090
J. Chem. Soc., Perkin Trans. 2, 1997

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Fig. 6 Free radical (TEMPO) induced broadening of amide protons
of peptide 1 in CDCl3. Relative broadening of only four amide protons
are indicated
Fig. 4 Solvent dependence of chemical shifts of amide proton reson-
ances of peptide 1 at varying concentrations of (CD3)2SO in CDCl3
Table 3 Observed long range NOEs for the peptide 1 in two solvent
systemsa
CDCl3
Phe15 CαH
Ala14 CαH
Met1 CβH
Ala14 CβH
Ala14 CβH
Ala14 CαH
Leu7 CαH
Ala6 CαH
Met1 CβH (m)
Met1 NH (s)
Ala6 CαH
Leu7 CαH
Leu7 CαH
Leu7 CαH
Val9 CαH
Ala6 CαH
Leu7 CαH
Val9 NH (m)
Val9 NH (w)
Ala10 NH (w)
Ala10 CβH (w)
Ala6 CβH (s)
Val9 CβH (s)
Val9 CγH (s)
Phe15 ringH (m)
Met1 NH (m)
Ala2 CβH (m)
Ala2 NH (m)
Val9 CβH (m)
Ala10 NH (m)
90% CDCl3–10% (CD3)2SO
Ala6 CαH
Ala10 NH (w)
Val9 NH (w)
Ala6 CαH
Val9 NH (w)
Leu7 CαH
a
s = strong, m = medium, w = weak.
solvents, no such distinction is readily made in CDCl3). NH
groups of intermediate dδ/dT values may arise due to the for-
mation of weak inter- or intra-molecular hydrogen bonds.32–34
The NMR results suggest that the peptide is composed of
two independent helical segments as evident from NOE data.
The molecule aggregates strongly in CDCl3 via intermolecular
hydrogen bonds involving Met1 and Ala2 NH groups. The size
of the aggregate is likely to be small (possibly dimers), because
the NMR spectra reveal relatively sharp resonances for various
side chain and backbone (particularly CαH) resonances. The
nature of the aggregate will be considered subsequently.
Fig. 5 Concentration dependence of NH chemical shifts of peptide 1
in CDCl3
If peptide 1 contains two independent helical segments, NH
groups of residues 1,2 and 9,10 should not be involved in
intramolecular hydrogen bonds making them available for
intermolecular interactions as exemplified in the crystal struc-
tures of a related 15 residue peptide.25 The solvent and concen-
tration dependence of NH chemical shifts shown in Figs. 4 and
5 suggest that the behavior of Met1, Ala2 and Val9, Ala10 NH
resonances are distinctly different. The chemical shift changes
observed for Val9 and Ala10 NH groups are much smaller than
those observed for Metl and Ala2 NH protons. The relative
solvent accessibilities of these four NH groups were further
probed using the paramagnetic nitroxide 2,2,6,6-tetramethyl-
piperidin-1-oxyl (TEMPO) as a perturbant. Fig. 6 shows add-
ition of free radical results in much larger broadening for Met1
ized in Fig. 5. The most dramatic concentration dependencies
are observed for the Met1 and Ala2 NH groups. This clearly
supports their involvement in intermolecular hydrogen bond-
ing. The temperature coefficients (dδ/dT ) listed in Table 2 reveal
that the largest temperature dependencies are observed for
Met1 and Ala2 NH groups. In an apolar, nonhydrogen bonding
solvent like CDCl3 large dδ/dT values have generally been
attributed to NH groups involved in intermolecular hydrogen
bonds which are readily broken up upon heating. Low dδ/dT
values may arise from strongly intramolecularly hydrogen
bonded NH groups or from solvent exposed NH groups. (Note
that while distinction between intramolecular hydrogen bonded
and free NH groups are possible in strongly hydrogen bonding
J. Chem. Soc., Perkin Trans. 2, 1997
2091

View Article Online
cantly larger values in the latter, indicative of substantial
deviation of the dihedral angle φ from an ideal helical φ angle.
Conformations in CDCl3–(CD3)2SO (90:10)
While the NMR data in CDCl3 favors aggregated structures
involving antiparallel intramolecular helix orientation, the data
in (CD3)2SO favor a more extended arrangement of the two
highly solvated helical segments. An examination of the chem-
ical shift dependence of the NH resonance in CDCl3–(CD3)2SO
mixtures suggests that aggregates formed by intermolecular
hydrogen bonding involving Met1 and Ala2 NH groups are
broken at (CD3)2SO concentration > 5%. This is undoubtedly
due to the ability of (CD3)2SO to complete for hydrogen bond-
ing to NH groups. The anomalous behavior of Met1 and Ala2
NH groups in solvent titration experiments provides a conveni-
ent means of studying this disaggregation upon addition of
(CD3)sSO. The NH chemical shifts over the range 5–10%
(CD3)2SO lead to the expectation that while the hydrogen bond-
ed aggregate may be disrupted, intramolecular organization
might still be maintained. ROESY spectra in (CD3)2SO–CDCl3
(10:90) show several strong dN N connectivities in both the hel-
ical segments with relatively weak inter-residue dαN NOEs (Fig.
Fig. 7 Partial 400 MHz 1H–1H NOESY spectrum of the peptide 1 in
CDCl3. Some observed long range CαH ←→CβH/CαH ←→CγH NOE
connectivities are indicated.
3
α
3). The observed JH N C H values are also very close to those
determined in CDCl3 suggesting that helical conformations are
indeed maintained for residues 1–7 and 9–15. The temperature
coefficients (dδ/dT ), Table 2, in this solvent mixture are particu-
larly noteworthy. Four NH resonances, Met1, Ala2, Val9 and
Ala10 have dδ/dT > 7 ppb KϪ1 which is significantly greater
than the values observed for the remaining NH groups. This
observation argues for the high degree of exposure to the solvent
of the NH groups of the residues 1,2,9 and 10. Of the remain-
ing residues, Val5 NH, Leu7 NH and Val13 NH have dδ/dT
values of 5.5–5.8 ppb KϪ1 suggestive of a moderate degree of
solvent exposure. This may be indicative of preferential inter-
action of (CD3)2SO at helix sites susceptible to solvation. In the
mixed solvent system only three long range NOEs (Table 3)
and Ala2 NH resonances as compared to Val9 and Ala10 NH
resonances. The remaining NH resonances in peptide 1 are
largely unaffected over the range of radical concentrations
studied, confirming their shielded nature. This result strongly
suggests that the degree of solvent exposure for Met1 and Ala2
is much greater than the Val9 and Ala10 NH groups.
Inspection of the NOESY spectra of peptide 1 in CDCl3
revealed several long range NOEs, summarized in Table 3. The
observed NOEs between residues 7 and residues 9,10 and resi-
due 6 and residues 9,10 may be interpreted in terms of an
approximate antiparallel orientation of the two helical seg-
ments about the central Acp hinge. Interestingly, NOEs are
observed between protons of residue 1 and 14 and 15 and res-
idues 2 and 14 (see also Fig. 7). While it is tempting to assign
the long range NOEs between the C- and N-terminal end of
the molecule as arising through folded structures which brings
the two extremities of the molecule in close proximity, the pos-
sibility of these NOEs arising due to intermolecular inter-
actions cannot be ruled out. In order to probe solvent effect on
peptide aggregation and structure NMR studies were carried
out in (CD3)2SO and in a (CD3)2SO–CDCl3 (10:90) mixture.
could be detected. These are Ala6 CαH
Ala10 NH, Ala6
Val9 NH, suggesting
CαH Val9 NH and Leu7 CαH
that the two helical segments are orientated in approximately
antiparallel fashion, while the limited number of long range
NOEs precludes the very small values of interhelical angle. The
data are consistent with a folded geometry about the Acp hinge.
The disappearance of several long range NOEs in CDCl3 upon
the addition of 10% (CD3)2SO established that interaction
between protons 1/2 and 14/15 are probably a consequence of
aggregation.
The NMR results establish that broadly helical conform-
ations are maintained for the segments 1–7 and 9–15 in CDCl3,
(CD3)2SO–CDCl3 (10:90) and (CD3)2SO. The orientation of
the helices about the central flexible Acp hinge is however sensi-
tive to solvent composition as established by the observation
of NOEs between residues 6/7 and 9/10 in CDCl3 and
(CD3)2SO–CDCl3, (10:90) whereas no interhelical interactions
are detected in (CD3)2SO. The peptide is strongly aggregated in
CDCl3 with the addition of a small amount of (CD3)2SO result-
ing in dissociation, a feature inferred from the solvent depend-
ence of NH chemical shifts. This type of self-assembly form-
ation in relatively apolar non-hydrogen-bonding solvents has
been reported in the literature and the nature of the solvent
plays a crucial role for noncovalent association,36 which is
entropically disfavored but enthalpically favored due to the
formation of intermolecular hydrogen bonds. The fact that
sharp well-resolved resonances are observed at high concentra-
tions of CDCl3 favors the formation of very small aggregates
with dimers being an attractive possibility. A schematic model
to rationalize the observed NMR data is presented in Fig. 8.
The formation of closed dimeric aggregates formed by
hydrogen bonding between exposed NH groups of the N-
terminal helical segments and the exposed CO groups of the C-
terminal helical segments is postulated to account for the strong
Conformation in (CD3)2SO
Several strong dN N connectivities could be assigned in NOESY
spectra of the peptide in (CD3)2SO (Fig. 3). Some sequential
connectivities could not be detected due to resonance overlap.
The observed intraresidue dαN (NiH
Cα H) NOEs are sig-
i
nificantly more intense relative to dN N NOEs as compared to
CDCl3. Further all the inter-residue dαN NOEs are very weak or
unobserved. These results favor helical conformations for the
1–7 and 9–15 segments. Interestingly, no long range NOEs
could be detected in (CD3)2SO suggesting the absence of con-
formation which involves antiparallel orientation of the two
helices. The temperature coefficients (dδ/dT ) observed in
(CD3)2SO are all relatively high with the exception of Val13
NH. This suggests that most of the peptide NH groups of the
peptide 1 are reasonably accessible in (CD3)2SO, although the
overall helical folding pattern is supported by the NOE data.
Fraying of peptide helices and consequent solvent invasion of
the backbone are likely to be favored in a strong hydrogen bond
donating solvent like (CD3)2SO. Indeed, crystallographic and
NMR evidence for fragility of short peptide helices in
(CD3)2SO has been presented.35 A comparison of the JH N C H
3
α
values in CDCl3 and (CD3)2SO (Table 2) establishes signifi-
2092
J. Chem. Soc., Perkin Trans. 2, 1997

View Article Online
Fig. 8 Schematic representations of (a) solvent dependent structural
changes of the peptide 1; (b) possible aggregation patterns for peptide 1
in CDCl3
Fig. 9 A comparison of CD spectra of the peptide 1 in methanol,
TMP (trimethyl phosphate) and TFE (2,2,2-trifluoroethanol)
Conclusions
concentration dependence of NH chemical shifts of Met1 NH
and Ala2 NH resonances. In this model Val9 and Ala 10 NH
groups are solvent exposed, but do not participate appreciably
in intermolecular interactions. Indeed, concentration depend-
ence of NH chemical shifts for these residues are substantially
lower than that observed for Met1 and Ala2 NH protons. An
alternative aggregated species which may be considered is the
open structure, illustrated in Fig. 8. This may be ruled out since
such species would be expected to propagate to large sizes, a
requirement that is not supported by the observed narrow
NMR resonances. The effects of addition of a strong hydrogen
bonding solvent like (CD3)2SO are also rationalized in Fig. 8.
While small amounts of (CD3)2SO successfully disrupt inter-
molecular hydrogen bonding, the peptide retains an overall
folded super-secondary structure, consistent with the observ-
ation of a limited number of interhelical NOEs in (CD3)2SO–
CDCl3 (10:90). In pure (CD3)2SO solutions, there is no evi-
dence for compact arrangements of helices and it is likely that
conformations similar to that observed in the related peptide
Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-
Ala-Leu-OMe (UV15) are obtained. In that case, crystal struc-
ture determination reveals an approximate 180Њ interhelical
angle.25
The present study establishes that antiparallel conformations
may be populated for a designed helix–linker–helix motif Boc-
Met-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-
Ala-Phe-OMe in an apolar solvent like CDCl3. Peptide associ-
ation through head-to-tail hydrogen bonding may in fact stabil-
ize an antiparallel orientation. Strongly hydrogen bonding solv-
ents like (CD3)2SO not only disrupt peptide aggregation but
also favor formation of noncompact structures in which the
helical segments extend away from one another, probably in a
manner similar to that in crystals of a related peptide UV15. In
the solid state the formation of intermolecular hydrogen bonds
between all CO and NH groups which do not participate in
intramolecular hydrogen bond formation is likely to be the driv-
ing force for the extended (open) orientation. Such open struc-
tures may also be facilitated in (CD3)2SO, by the ability of the
solvent to interact strongly with the exposed NH groups. The
present study demonstrates that designed synthetic peptides can
mimic helical hairpin structures under defined conditions. The
use of a conformationally mobile linking segment results in the
complete absence of stereochemical control at the hinge region.
The use of conformationally constrained residues in segments
linking secondary structural elements is presently being
explored in order to permit greater orientational control in the
designed super secondary structural motifs. The present study
establishes that compact polypeptide structures possessing a
limited level of tertiary interactions can indeed be obtained in
designed synthetic peptides in nonaqueous media. Further
elaboration to larger systems may be of relevance in studies
aimed at mimicking helical hairpins in membrane proteins.
Circular dichroism
The CD spectra of the peptide 1 in methanol, TMP and TFE
are shown in Fig. 9. The peptide 1 displays CD spectra which
are identical in nature but of varying intensities in the three
solvents. The long wavelength component of the π–π* band
appears at 203 nm in TFE and at 202 nm in both methanol and
TMP. The n–π* transition appears as a shoulder at 220 nm in
all the three different solvents. The spectra resemble those
observed for well-characterized helical peptides of similar
length.25,37 In TFE, both π–π* and n–π* bands are more intense
indicating greater helicity in that solvent. The lack of any dra-
matic solvent dependence of the CD spectra suggests that the
overall backbone conformation of the two independent helical
modules is retained in solvent media of widely different polarity
and hydrogen bonding ability. Detailed conclusions regarding
the nature of the peptide helix (310 or α) and the extent of the
helicity are fraught with uncertainty, because of the problem of
subtle conformational heterogeneity in solution 38–40 and the
difficulty of providing definitive conformational interpretation
of relative intensities of the far UV CD bands in short peptide
helices.41–43
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Paper 7/00624A
Received 28th January 1997
Accepted 13th May 1997
2094
J. Chem. Soc., Perkin Trans. 2, 1997