Stereochemical analysis of higher α,α-dialkylglycine containing peptides. Characterization of local helical conformations at dipropylglycine residues and observation of a novel hydrated multiple β-turn structure in crystals of a glycine rich peptide

Article abstract of DOI:10.1021/ja970596z

The peptide Boc-Gly-Dpg-Gly-Gly-Dpg-Gly-NHMe (1) has been synthesized to examine the conformational preferences of Dpg residues in the context of a poor helix promoting sequence. Single crystals of 1 were obtained in the space group P2₁/c with

Full text of DOI:10.1021/ja970596z

12048
J. Am. Chem. Soc. 1997, 119, 12048-12054
Stereochemical Analysis of Higher R,R-Dialkylglycine
Containing Peptides. Characterization of Local Helical
Conformations at Dipropylglycine Residues and Observation of
a Novel Hydrated Multiple â-Turn Structure in Crystals of a
Glycine Rich Peptide
,‡
Isabella L. Karle,*,§ Ramesh Kaul, R. Balaji Rao, S. Raghothama,† and
P. Balaram*,‡
Contibution from the Laboratory for the Structure of Matter, NaVal Research Laboratory,
Washington, DC 20375-5341, Department of Chemistry, Banaras Hindu UniVersity, Varanasi
221005, India, Sophisticated Instruments Facility, Indian Institute of Science, Bangalore 560012,
India, and Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
ReceiVed February 24, 1997X
Abstract: The peptide Boc-Gly-Dpg-Gly-Gly-Dpg-Gly-NHMe (1) has been synthesized to examine the conformational
preferences of Dpg residues in the context of a poor helix promoting sequence. Single crystals of 1 were obtained
in the space group P21/c with a ) 13.716(2) Å, b ) 12.960(2) Å, c ) 22.266(4) Å, and â ) 98.05(1)°; R ) 6.3%
for 3660 data with |Fo| > 4σ. The molecular conformation in crystals revealed that the Gly(1)-Dpg(2) segment
adopts φ, ψ values distorted from those expected for an ideal type II′ â-turn (φGly(1) ) +72.0°, ψGly(1) ) -166.0°;
φDpg(2) ) -54.0°, ψDpg(2) ) -46.0°) with an inserted water molecule between Boc-CO and Gly(3)NH. The Gly-
(3)-Gly(4) segment adopts φ, ψ values which lie broadly in the right handed helical region (φGly(3) ) -78.0°, ψGly(3)
) -9.0°; φGly(4) ) -80.0°, ψGly(4) ) -18.0°). There is a chiral reversal at Dpg(5) which takes up φ, ψ values in
the left handed helical region. The Dpg(5)-Gly(6) segment closely resembles an ideal type I′ â-turn (φDpg(5)
)
+56.0°, ψDpg(5) ) +32.0°; φGly(6) ) +85.0°, ψGly(6) ) -3.0°). Molecules of both chiral senses are found in the
centrosymmetric crystal. The C-terminus forms a hydrated Schellman motif, with water insertion into the potential
6 f 1 hydrogen bond between Gly(1)CO and Gly(6)NH. NMR studies in CDCl3 suggest substantial retention of
the multiple turn conformation observed in crystals. In solution the observed NOEs support local helical conformation
at the two Dpg residues.
Introduction
extended (φ ∼ ψ ∼ 180°) regions of φ, ψ space.10 This situation
was in marked contrast to the prototype R,R-dialkyl residue Aib,
which appeared an almost obligatory helical residue.4,5 Sub-
sequent studies with heteromeric sequences of variable length
revealed several examples of Dpg and its homolog R,R-
dibutylglycine (Dbg) in helical conformations.11-15 A particular
interesting example is the coexistence of both conformations
in single crystals of the tripeptide Boc-Leu-Dpg-Val-OMe.16
Theoretical calculations suggest that for higher dialykylglycines
pronounced energy minima exist in both the helical and fully
extended regions of the conformational space. There appears
R,R-Dialkylated amino acids have been widely used to
introduce backbone conformational restraints in synthetic pep-
tides.1,2 The prototype residue R-aminoisobutyric acid (Aib)3
stabilizes helical conformations in diverse sequences.4,5 The
higher dialkyl glycines like diethylglycine (Deg), di-n-propy-
lglycine (Dpg), and di-n-butylglycine (Dbg) have been less
extensively investigated. Initial interest in the use of higher
R,R-dialkyl residues in peptide design stemmed from the fact
that fully extended (φ ∼ ψ ∼ 180°, C5) conformations were
observed in short homooligopeptides,6-9 an observation ratio-
nalized by theoretical calculations which suggested energy
minima in both helical (φ ) (60°, ψ ) (30°) and fully
(7) Bonora, G. M.; Toniolo, C.; DiBlasio, B.; Pavone, V.; Pedone, C.;
Benedetti, E.; Lingham, I.; Hardy, P. M. J. Am. Chem. Soc. 1984, 106,
8152-8156.
† Sophisticated Instruments Facility, Indian Institute of Science.
‡ Molecular Biophysics Unit, Indian Institute of Science.
§ Naval Research Laboratory.
(8) Benedetti, E.; Barone, V.; Bavoso, A.; DiBlasio, B.; Lelj, F.; Pavone,
V.; Pedone, C.; Bonora, G. M.; Toniolo, C.; Leplawy, M. T.; Kaczmarek,
K.; Redlinski, A. Biopolymers 1988, 27, 357-371.
Banaras Hindu University.
(9) Toniolo, C.; Benedetti. E. Macromolecules 1991, 24, 4004-4009.
(10) Barone, V.; Lelj, F.; Bavoso, A.; DiBlasio, B.; Grimaldi, P.; Pavone,
V.; Pedone, C. Biopolymers 1985, 24, 1759-1767.
X Abstract published in AdVance ACS Abstracts, November 15, 1997.
(1) Balaram, P. Curr. Opin. Struct. Biol. 1992, 2, 845-851.
(2) Benedetti, E.; Toniolo, C. Polymeric Materials Encyclopedia 1996,
8, 6472-6481.
(11) DiBlasio, B.; Pavone, V.; Isernia, C.; Pedone C.; Benedetti, E.;
Toniolo, C.; Hardy, P. M.; Lingham, I. J. Chem. Soc, Perkin Trans. 2 1992,
2, 523-526.
(3) (a) Abbreviations used: Aib, R-aminoisobutyric acid; Deg, R,R-
diethylglycine; Dpg, R,R-di-n-propylglycine; Dbg, R,R-di-n-butylglycine;
Boc, tert-butyloxycarbonyl; OMe, methyl ester; OBzl, benzyl ester; NHMe,
methylamide. (b) IUPAC-IUB Commission on Biochemical Nomenclature.
Biochemistry 1970, 9, 3471-3479.
(4) Prasad, B. V. V.; Balaram, P. CRC Crit. ReV. Biochem. 1984, 16,
307-347.
(5) Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747-6756.
(6) Benedetti, E.; Toniolo, C.; Hardy, P. M.; Barone, V.; Bavoso, A.;
Di Blasio, B.; Grimaldi, P.; Lelj, F.; Pavone, V.; Pedone, C.; Bonora, G.
M.; Lingham, I. J. Am. Chem. Soc. 1984, 106, 8146-8152.
(12) Karle, I. L.; Rao R. B.; Prasad, S.; Kaul, R.; Balaram, P. J. Am.
Chem. Soc. 1994, 116, 10355-10361.
(13) Karle, I. L.; Gurunath, R.; Prasad, S.; Kaul, R.; Rao R. B.; Balaram,
P. J. Am. Chem. Soc. 1995, 117, 9632-9637.
(14) Karle, I. L.; Rao R. B.; Kaul, R.; Prasad, S.; Balaram, P. Biopolymers
1996, 39, 75-84.
(15) Karle, I. L.; Gurunath, R.; Prasad, S.; Rao R. B.; Balaram, P. Int.
J. Peptide Protein Res. 1996, 47, 376-382.
(16) Prasad S.; Mitra S.; Subramanian, E.; Velmurugan, D.; Rao, R. B.;
Balaram, P. Biochem. Biophys. Res. Commun. 1994, 198, 424-430.
S0002-7863(97)00596-9 CCC: $14.00 © 1997 American Chemical Society

Multiple â-Turn Structure
J. Am. Chem. Soc., Vol. 119, No. 50, 1997 12049
to be a pronounced dependence on the bond angle N-CR-CO,
τ (with τ ∼ 110° in helices and τ ∼ 103° in fully extended
structures).2,8 Available data thus suggests that the conforma-
tions of the higher dialkylglycines may be modulated by subtle
environmental effects. In order to probe systematically the
relationship between backbone conformation and sequence
context, we have chosen to conformationally characterize a wide
range of Deg/Dpg/Dbg containing peptides. In this report, we
describe the conformation in crystals and in solution of the
glycine rich protected hexapeptide Boc-Gly-Dpg-Gly-Gly-Dpg-
Gly-NHMe (1).
Glycine rich sequences have been chosen since glycine is
the most conformationally flexible residue and has a relatively
low propensity for occurring in helical structures.17 The
juxtaposition of Gly and the R,R-dialkylated residue Aib in short
sequences has led to interesting stereochemical consequences
in both designed18 and natural sequences like trichogin A IV19
and synthetic fragments.20,21 X-ray diffraction studies establish
that both Dpg residues at position 2 and 5 favor local helical
conformations in crystals. The peptide adopts a novel, hydrated
multiple turn structure. In the apolar solvent CDCl3, NMR data
are consistent with a major population of conformers resembling
that observed in crystals, while in the strongly interacting solvent
(CD3)2SO, a mixed population of partially folded and extended
structures is supported by the experimental evidence.
The ethyl acetate extract was dried over anhydrous sodium sulfate and
evaporated in Vacuo to yield 5.68 g (18 mmol, 78%) of dipeptide acid
2.
Boc-Gly-Dpg-Gly-OBzl (3). 3.16 g (10 mmol) of peptide 2 was
dissolved in DMF (5 mL). Glycine benzyl ester-p-toluene sulfonate
(6.74 g, 20 mmol) was added to it followed by DCC (2.0 g,10 mmol)
and HOBt (1.35 g, 10 mmol). The reaction was stirred at room
temperature for 3 days. DCU was filtered, and the work up was similiar
to one described for 1. The peptide 3 was obtained as a yellowish
solid weighing 3.9 g (8.5 mmol, 85%): 400 MHz 1H NMR (CDCl3, δ
ppm) 0.8, 0.95 (6H, t, Dpg CδH3), 1.12, 1.25 (4H, m, Dpg CγH2), 1.45
(9H, s, Boc CH3), 1.6, 2.45 (4H, m, Dpg CâH2), 3.75 (2H, d, Gly CRH),
4.12 (2H, d, Gly CRH), 5.14 (1H, t, Gly NH), 5.20 (2H, s, -CH2), 6.5
(1H, t, Gly NH), 7.18 (1H, s, Dpg NH), 7.35 (5H, m, Phe).
Boc-Gly-Dpg-Gly-OH (4). Peptide 3 (1.85 g, 4 mmol) was
saponified using 15 mL of MeOH and 4 N NaOH (8 mL). The reaction
was monitored by TLC. After 3 days the reaction was worked up as
described for 2 to yield 1.3 g (3.5 mmol, 87.5%) of the tripeptide acid
4 as a white solid.
H2N-Gly-Dpg-Gly-OBzl (5). 3 (1.4 g, 3 mmol) was taken in 8 mL
of 98% formic acid, and the reaction mixture tightly stoppered. The
reaction was monitored by TLC. After 8 h, formic acid was evaporated
in vacuo, and the residue was dissolved in water. The solution was
washed with ether (2 × 30 mL). The aqueous layer was neutralized
with sodium carbonate solution and extracted with ethyl acetate. The
organic extract was dried over anhydrous sodium sulfate and evaporated
in Vacuo to yield 0.72 g (2 mmol, 66%) of free amine tripeptide 5 as
a gum.
Experimental Section
Boc-Gly-Dpg-Gly-Gly-Dpg-Gly-OBzl (6). 4 (0.75 g, 2 mmol) was
dissolved in DMF (4 mL) and to it was added 0.72 g (2 mmol) of 5,
followed by DCC (0.4 g, 2 mmol) and HOBt (0.27 g, 2 mmol). The
reaction was stirred at room temperature for 5 days. DCU was filtered
and the work up was similar to one described for 1 to yield 1.14 g (1.6
mmol, 80%) of the hexapeptide ester 6 as a white solid: mp 136-138
The peptide was assembled by conventional solution phase procedure
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 (OMe). Glycine was protected as a benzyl
ester (OBzl). Deprotections were performed using 98% formic acid,
while saponifications were carried out using 4 N sodium hydroxide
solution and methanol. Couplings were mediated by dicyclohexylcar-
bodiimide-1-hydroxybenzotriazole (DCC/HOBT). Dpg and Dpg‚-
OMe‚HCl were synthesized as described previously.22 All intermediate
1
°C; 400 Mhz H NMR (CDCl3, δ ppm) 0.88-0.9 (12H, t, Dpg 2,5
CδH3), 1.2-1.3 (8H, m, Dpg 2,5 CγH2), 1.49 (9H, s, Boc CH3), 1.85
(4H, m, Dpg 2 CâH2), 2.05 (4H, m, Dpg 5 CâH2), 3.65 (2H, d, Gly 6
CRH), 3.85-3.95 (2H, d, Gly 3/Gly 4 CRH), 4.10 (2H, d, Gly 1 CRH),
5.25 (2H, s, -CH2), 5.65 (1H, t, Gly 1 NH), 6.78 (1H, s, Dpg 2 NH),
6.89 (1H, s, Dpg 5 NH), 6.92 (1H, t, Gly 6 NH), 7.45 (5H, m, Phe),
7.41 (1H, t, Gly 3 NH), 7.82 (1H, t, Gly 4 NH).
1
peptides were characterized by H NMR (80 MHz, 400 MHz), thin
layer chromatography (TLC) and used without further purification.
Synthesis of Peptides. Boc-Gly-Dpg-OMe (1). Boc-Gly-OH (5.25
g, 30 mmol) was dissolved in dichloromethane (DCM) (25 mL). H2N-
Dpg-OMe (5.20 g, 30 mmol) obtained from its ester hydrochloride was
added, followed by DCC (6.0 g, 30 mmol). The reaction mixture was
stirred at room temperature for 2 days. DCM was removed in vacuo.
The residue was taken up in ethyl acetate (about 25 mL). The
precipitated dicyclohexylurea (DCU) was filtered. The organic layer
was washed with an excess of brine solution, 2 N HCl (2 × 50 mL),
1 M sodium carbonate solution (2 × 50 mL), and again with brine.
The solution was then dried over anhydrous sodium sulfate and
evaporated in vacuo. The dipeptide 1 was obtained as light yellow
Boc-Gly-Dpg-Gly-Gly-Dpg-Gly-NHMe (7). Peptide 6 (0.5 g, 7
mmol) was dissolved in dry MeOH (10 mL), and methyl amine (CH3-
NH2) gas was passed till saturation (about 2 h). MeOH was evaporated,
and the peptide 7 was obtained as a yellow solid: yield 0.3 g (0.5
mmol, 71%); mp ) 141-143 °C.
The crude peptide was purified on a reverse phase MPLC C18 column
(40-60 µ) using a gradient of MeOH/H2O. Homogeneity of the peptide
was susequently demonstrated by analytical HPLC on a reverse phase
(C18, 5 µ) column. The peptide was characterized by complete
assignment of 400 MHz 1H NMR spectra, by 125 MHz 13C NMR, and
by its FAB mass spectrum: 400 MHz 1H NMR (CDCl3, δ ppm) 0.89-
0.93 (12H, t, Dpg 2/5 CδH3), 1.21-1.3 (8H, m, Dpg 2/5CγH2), 1.45
(9H, s, Boc CH3), 1.85, 1.75 (4H, m, Dpg 2 CâH2), 2.10, 1.83 (4H, m,
Dpg 5 CâH2), 2.78 (3H, d, NH 7 Me), 3.88-3.86 (2H, d, Gly 3/Gly
4/Gly 6 CRHs), 3.66 (2H, d, Gly 1 CRH), 5.60 (1H, t, Gly 1 NH), 6.68
(1H, s, Dpg 2 NH), 6.96 (1H, s, Dpg 5 NH), 7.13 (1H, t, Gly 6 NH),
7.53 (1H, t, Gly 3 NH), 7.92 (1H, t, Gly 4 NH), 7.24 (NH 7 Me). 125
MHz 13C NMR (CDCl3, δ ppm): 14.06-14.09 (4C, Cδ,δ′ Dpg 2/5),
16.45-16.56 (4C, Cγ,γ Dpg 2/5), 26.06 (1C, NHCH3), 28.11 (3C, Boc
CH3s), 35.11, 35.71 (4C, Câ,â′ Dpg 2/5), 43.19, 43.84, 44.05, 45.30
(4C, CR Gly 1/3/4/6), 62.60, 63.06 (2C, CR Dpg 2/5), 81.15 [1C,
(CH3)3C], 157.3 (1C, Boc CdO), 170.22, 170.59, 170.63, 171.37,
173.97, 174.67 (6C, CdO); mass spectral data M + Na+ ) 665, Mcalcd
) 642.
1
gum weighing 7.6 g (23 mmol, 76%): 80 MHz H NMR (CDCl3, δ
ppm) 0.8, 0.93 (6H, t, Dpg CδH3), 1.26 (4H, m, Dpg CγH2), 1.5 (9H,
s, Boc CH3), 2.4, 2.3 (4H, m, Dpg CâH2), 3.70 (3H, s, OCH3), 3.75
(2H, d, Gly CRH), 5.50 (1H, t, Gly NH), 6.75 (1H, s, Dpg NH).
Boc-Gly-Dpg-OH (2). Peptide 1 (7.6 g, 23 mmol) was saponified
using MeOH (25 mL) and 4 N NaOH (10 mL). The reaction mixture
was stirred at room temperature, and its course followed by TLC. After
4 days, MeOH was evaporated, and the residue taken in water. The
aqueous solution was washed with ether (2 × 40 mL). The aqueous
layer was neutralized with 2 N HCl and extracted with ethyl acetate.
(17) Richardson, J. S.; Richardson, D. C. In Prediction of Protein
Structure and Principles of Protein Conformation. Fasman, G. D., Ed.;
Plenium Press: New York, pp 1-95.
(18) Karle, I. L.; Banerjee, A.; Bhattacharjya, S.; Balaram, P. Biopolymers
1996, 38, 515-526.
(19) Toniolo, C.; Peggion, C.; Crisma, M.; Formaggio, F.; Shui, X.;
Eggleston, D. S. Nature Struct. Biol. 1994, 1, 908-914.
(20) Gurunath, R.; Balaram, P. Biopolymers 1995, 35, 21-29.
(21) Monaco, V.; Formaggio, F.; Crisma, M.; Toniolo, C.; Shui, X.;
Eggleston, D. S. Biopolymers 1996, 39, 31-42.
Spectroscopic Studies. All NMR studies were carried out on a
Bruker AMX-400 spectrometer. Peptide concentrations were in the
range of 7-8 mM and the probe temperature was maintained at 298
K. Resonance assignments were done using two-dimensional ROESY
(22) Prasad, S.; Rao, R. B.; Balaram, P. Biopolymers 1995, 35, 11-20.

12050 J. Am. Chem. Soc., Vol. 119, No. 50, 1997
Table 1. Crystal and Diffraction Parameters
Karle et al.
Table 3. Hydrogen Bonds
empirical formula
color, habit
crystal size (nm)
crystal system
space group
C30H59N7O10
colorless, very fragile wafers
0.05 × 0.40 × 1.3
monoclinic
N‚‚‚O (Å)
O‚‚‚O (Å)
H‚‚‚O (Å)
H‚‚‚O (Å)
type
donor
acceptor
lateral, intermol
head-to-tail
N1
N2
N3
N4
N5
N6
N7
W1
W1
W2
W2
O4a
O5b
W1
O1
O2
W2
O4
3.147
2.893
2.854
3.091
3.345c
2.886
2.954
2.850
2.809
2.756
2.865
2.43
2.00
2.01
2.30
2.51c
2.10
2.08
2.01
1.96
1.67
2.16
P21/c
unit cell dimensions
a ) 13.716(2) Å
b ) 12.960(2) Å
c ) 22.266 Å
â ) 98.050(10)°
3918.2(11) Å3
4
4 f 1
4 f 1
4 f 1
volume
Z
formula weight
density (calc)
absorption coeff
F(000)
O0
O6d
O1
677.8
1.149 Mg/m3
0.713 mm-1
1472
O3a
a Symmetry equivalent at 2-x, 0.5+y, 1.5-z. b Symmetry equivalent
at x, 0.5-y, -0.5+z. c Long values for hydrogen bonds. d Symmetry
equivalent at 2-x, -0.5+y, 1.5-z.
Table 2. Torsional Anglesa (deg) for Peptide 1
residue
φ
ψ
ω
ø1
ø2
Boc
Gly(1)
Dpg(2) -54
Gly(3)
Gly(4)
Dpg(5) +56
Gly(6)
175
+72 -166 -171
-46 -173
-9 172
-18 -179
+32 179 179, -56
-3 -179
177, +63
178, -170
-179, 177
-78
-80
+85
a The torsional angles for rotation about bonds of the peptide
backbone (φ,ψ,ω) and about the bonds in the Dpg side chains (ø1, ø2)
are described in IUPAC-IUB commission on Biochemical Nomen-
clature.3b
spectra.23 2D data were collected in phase sensitive mode using the
time proportional phase incrementation method. Data points numbering
512 with 64 transients were collected. Spectral widths were in the
range of 4500 Hz with a spin lock time of 300 m s. Zero filling was
done to yield data sets of 1K × 1K using square sine bell window.
X-ray Studies. Single crystals of peptide 1 were grown from
methanol-water solution by slow evaporation. The crystal and
diffraction parameters are listed in Table 1. X-ray data were collected
on a Siemens automated diffractometer with Cu radiation in the θ/2θ
mode, constant scan speed of 10 deg/m, scan width of 1.3° + 2θ (R1-
R2), 2θmax ) 116° (resolution 0.9 Å) for a total of 5363 unique
reflections and 3360 reflections with intensities greater than 4σ(F). The
structure was solved by direct phase determination. Full-matrix
anisotropic least-squares refinement was performed on the parameters
for the C, N, and O atoms. The positions of the four H atoms on W1
and W2 were found in a difference map and refined isotropically. The
remaining 55 H atoms were placed in idealized positions and allowed
to ride with C or N atom to which each was bonded. The final R
values were R ) 6.33% for 3360 data and RW ) 6.40%.
Figure 1. Conformation of the hexapeptide 1 in the crystal. Dashed
lines indicate intramolecular hydrogen bonds. W1 and W2 are the water
molecules inserted into the molecule.
) -120°; φi+2 ) -80°, ψi+2 ) 0°). Gly(3)-Gly(4) adopts φ,
ψ values which lie broadly in the right handed helical region.
There is a chiral reversal at Dpg(5) which now adopts φ, ψ
values in the left handed helical region. The Dpg(5)-Gly(6)
segment closely resembles an ideal type I′ â-turn (φi+1 ) +60°,
ψi+1 ) +30°; φi+2 ) +90°, ψi+2 ) 0°). Inspection of the
hydrogen bonding pattern suggests that the Dpg(2)-Gly(3)-Gly-
(4) segment forms one turn of a 310 helix stabilized by two
intramolecular 4 f 1 hydrogen bonds[Gly(1)CO‚‚‚Gly(4)NH,
N‚‚‚O 3.09 Å; Dpg(2)CO‚‚‚Dpg(5)NH, N‚‚‚O 3.34 Å]. The
Dpg(5)-Gly(6) segment forms a type I′ â-turn stabilized by a 4
f 1 hydrogen bond between Gly(4)CO‚‚‚NHMe, N‚‚‚O 2.95
Å. At the N-terminus, a water molecule (W1) is inserted into
the Gly(1)-Dpg(2) type II â-turn resulting in the absence of a
4 f 1 intramolecular hydrogen bond [Boc CO‚‚‚Gly(3)NH,
N‚‚‚O 4.30 Å]. Water insertion into â-turn structures is
commonly observed in peptides.28 The observed distortion in
backbone dihedral angles from the idealized values expected
in type II/II′ â-turns is clearly a consequence of the water
insertion into the backbone. Interestingly, a potential 6 f 1
hydrogen bond between Gly(1)CO‚‚‚Gly(6)NH is disrupted by
insertion of a water molecule (W2) resulting in an N‚‚‚O
distance of 4.60 Å. The Dpg(2)-Gly(3)-Gly(4)-Dpg(5) segment
forms an RRRRRRRL motif (RR-residue in right handed helical
conformation and RL-residue in left handed helical conforma-
tion). Such conformational features have been termed as
Schellman motifs and are frequently found at the C-terminus
end of R-helical structures in proteins.29-32 In synthetic
Results and Discussion
Crystal Structure of 1. Figure 1 shows a view of the
molecular conformation determined in crystals. The backbone
conformational parameters are summarized in Table 2. While
the hydrogen bond parameters are listed in Table 3. Since
peptide 1 is achiral it crystallizes in a centrosymmetric space
group and contains molecules of both hands (chiral senses). Only
one sign of the dihedral angles is listed in Table 2, and only
one hand is shown in Figure 1.
Inspection of the torsional angles reveals that the Gly(1)-
Dpg(2) segment adopts φ, ψ values somewhat distorted from
those expected for an ideal type II′ â-turn (φi+1 ) +60°, ψi+1
(23) Wuthrich, K. NMR of Proteins and Nucleic Acids; John Wiley and
Sons: New York, 1986.
(24) Kopple, K. D.; Schamper, T. J. J. Am. Chem. Soc. 1972, 94, 3644-
3646.
(25) Kopple, K. D.; Go, A.; Pilipauskas, D. R. J. Am. Chem. Soc. 1975,
97, 6830-6838.
(26) Pitner, T. P.; Urry, D. W. J. Am. Chem. Soc. 1972, 94, 1399-1400.
(27) Iqbal, M.; Balaram, P. J. Am. Chem. Soc. 1981, 103, 5548-5552.
(28) Karle, I. L. Biopolymers 1996, 40, 157-180.
(29) Schellman, C. In Protein Folding; Janenicke, R., Ed.; Elsevier/North
Holland Biochemical Press: Amsterdam, 1980; pp 53-61.
(30) Milner-White, E. J. J. Mol. Biol. 1988, 199, 503-511.

Multiple â-Turn Structure
J. Am. Chem. Soc., Vol. 119, No. 50, 1997 12051
Table 4. Observed Dpg Backbone Conformations in Peptide Crystal Structures
torsional anglesa
peptides
φ
ψ
τ
ref
Tfa-Dpg-DBH
mol. A
mol. B
176.0
177.0
179.5
172.5
173.1
(54.8
(59.9
(48.3
176.0
62.8
175.9
174.8
169.4
-179.4
179.0
(39.4
(10.2
(32.9
-180.0
39.6
101.1
101.1
103.0
102.3
105.4
108.0
116.0
111.0
102.0
111.0
113.4
113.4
107.0
105.0
113.0
109.0
112.7
110.6
109.9
109.1
104.1
107.2
109.9
110.8
6
6
Tfa-(Dpg)2-DBH
For-Met-Dpg-Phe-OMe
Tfa-(Dpg)3-DBH
37
11
Boc Leu-Dpg-Val-OMe
mol. A
mol. B
16
Boc-Ala-Dpg-Ala-OMe
Boc-Ala-Dpg-Ala-NHMe
Boc-Leu-Dpg-Leu-Ala-Leu-Aib-OMe
66.2
19.3
40
40
12
-50.6
-55.0
-53.0
-43.0
-56.0
-46.0
-51.0
-52.0
-53.0
178.0
-52.0
-54.0
56.0
-14.2
-43.0
-43.0
-37.0
-47.0
-35.0
-47.0
-51.0
-50.0
171.0
-44.0
-46.0
32.0
mol. Ab
mol. Bb
mol. Ac
Boc-Aib-Ala-Leu-Ala-Leu-Dpg-Leu-Ala-Leu-Aib-OMe
Boc-Gly-Dpg-Ala-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe
Boc-Gly-Dpg-Leu-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe
Boc-Gly-Dpg-Pro-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe
Boc-Gly-Dpg-Gly-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe
Boc-Gly-Dpg-Gly-OH
12
13
13
13
14
39
39
Boc-Val-Val-Ala-Leu-Gly-Dpg-Gly-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe
Boc-Gly-Dpg-Gly-Gly-Dpg-Gly-NHMe
this study
a Nomenclature follows ref 3b. τ is the bond angle N-CR-C′. b Triclinic. c Orthorhombic.
peptides, the presence of an achiral residue at the C-terminus
of a helix frequently results in chiral reversal of φ, ψ values
resulting in the formation of RL terminated helices.33-35 In the
present structure, the use of achiral residues results in a reversal
of screw sense at the C-terminus. Formally this structure may
be viewed as a case where a right handed helical twist is
immediately followed by a left handed helical segment. Indeed,
such ambidextrous helical molecules have recently been char-
acterized in crystals of a 14 residue peptide helix containing
two contiguous helical segments of opposite chirality.36
Dpg Residue Conformations. Table 4 summarizes observed
Dpg backbone conformations in crystals and also lists the bond
angle at the CR atom (τ). Theoretical calculations suggest a
strong bond angle dependence of the backbone φ, ψ values with
τ < 105° resulting in extended conformations and τ > 105°
yielding helical conformations.2,10 While the majority of
examples listed in the table follow this correlation, τ values
∼105° appear to accomodate both types of local conformations.
The number of examples of Dpg in helical structures is clearly
growing in diverse sequence environments suggesting that Dpg
and related residues may be largely helix stabilizers, with fully
extended forms requiring specific “local factors” for stabiliza-
tion.
Figure 2. Packing of the distorted helices into sheets. Projection down
the a axis. Dashed lines show the head-to-tail N2-O5 hydrogen bonds
and the lateral N1-N4 hydrogen bonds between helices. The water
molecules W1 and W2 also participate in forming lateral bridges of
hydrogen bonds between helices (not shown).
Crystal Packing. The molecules of 1 contain multiple turns
of varying conformational types, resulting in a structure that is
more compact than a helix of comparable length. Nevertheless,
the peptide CO and NH groups which are internally hydrogen
bonded are the same as those that would have participated in
such interactions in a 310/R-helical structures. The molecules
assemble into columns by head-to-tail hydrogen bonding
between N2 and O5, a motif adopted by almost all 310- and
R-helices (Figure 2).5 The columns pack into layers with
antiparallel columns and lateral hydrogen bonds between N1
and O4. Water molecules W1 and W2, in addition to forming
bridges between O0 and N3 as well as O1 and N6 in individual
helices, also connect neighboring helices by lateral hydrogen
bonds with O6 and O3, respectively. There are no hydrogen
bonds between layers, that is, in the direction perpendicular to
the view in Figure 2.
(31) Nagarajaram, H. A.; Sowdhamini, R.; Ramakrishnan, C.; Balaram,
P. FEBS Lett. 1993, 321, 79-83.
(32) Aurora, R.; Srinivasan, R.; Rose, G. D. Science 1994, 264, 1126-
1130.
(33) Karle, I. L.; Flippen-Anderson, J. L.; Uma, K.; Balaram, P. Int. J.
Pept. Protein Res. 1993, 42, 401-410.
(34) Rajashankar, K. R.; Ramakumar, S.; Mal, T. K.; Jain, R. M.;
Chauhan, V. S. Angew. Chem., Int. Ed. Engl. 1996, 35, 765-768.
(35) Banerjee, A.; Datta, S.; Pramanik, A.; Shamala, N.; Balaram, P. J.
Am. Chem. Soc. 1996, 118, 9477-9483.
(36) Banerjee, A.; Raghothama, S.; Karle, I. L.; Balaram, P. Biopolymers
1996, 39, 279-285.
NMR Studies. Sequence specific assignments were easily
achieved using sequential interresidue nuclear Overhauser effects

12052 J. Am. Chem. Soc., Vol. 119, No. 50, 1997
Karle et al.
Figure 3. Partial 400 MHz 1H NMR spectra in CDCl3 showing the effect of TEMPO on the line width of NH resonance in peptide 1. Resonance
assignments are indicated. TEMPO concentrations (M) are marked on the spectra. Peptide concentration was 10.4 mM. [X ) Dpg].
Table 5. NMR Parameters for Peptide 1
NH
residue
CDCl3 (CD3)2SO ∆δa (ppm) -dδ/dtb (ppb/K)
Gly (1)
Dpg(2)
Gly(3)
Gly(4)
Dpg(5)
Gly(6)
5.60
6.68
7.53
7.92
6.96
7.13
7.24
7.15
7.73
8.22
8.03
7.73
8.03
7.49
0.89
1.15
0.51
0.20
0.30
0.31
0.12
8.2
4.9
6.1
4.4
3.8
6.1
3.7
NH(7)Me
a ∆δ is the chemical shift difference for NH protons in CDCl3 and
21.05% (CD3)2SO/CDCl3. b dδ/dt (ppb/K) values in (CD3)2SO.
measured using 2D ROESY methods.23 The Gly, Dpg, and
methylamide NH protons can be readily differentiated on the
basis of their multiplet patterns. In both solvents, the methy-
lamide NH proton was unambiguously assigned to the only
quartet resonance, while the Gly(1)NH was easily identified by
virtue of its high field position in both solvents. The chemical
shifts of the backbone NH protons are summarized in Table 5.
The number of intramolecularly hydrogen bonded NH groups
were delineated using radical induced line broadening24,25 and
solvent induced perturbation of chemical shifts26 in CDCl3-
(CD3)2SO mixtures.27 Figure 3 shows the effect of the free
radical TEMPO on the line widths of the various NH resonances.
It is clear that while Gly(1), Dpg(2), and Gly(3) NH groups are
exposed to the solvent, the remaining four NH groups are
inaccessible. All NH groups show downfield shifts on addition
of small amounts of the hydrogen bonding solvent (CD3)2SO
to CDCl3 solutions. The magnitude of chemical shift changes
(∆δ) on going from CDCl3 to 21.05% (CD3)2SO-CDCl3 are
again diagnostic of peptide NH accessibility. From the data
summarized in Table 5, it is evident that the first three NH
groups also have larger ∆δ values confirming their solvent
accessible nature. It is thus clear that the peptide favors a folded
conformation involving the NH groups of residues 4-6 and
the terminal NHMe in intramolecular hydrogen bonding. Figure
4 shows the NH-NH region of the ROESY spectrum of 1 in
CDCl3, while Figure 5 illustrates observed CRH T NH NOEs.
Sequential NiH T Ni+1H (dNN) NOEs are observed over the
entire length of the peptide strongly indicating a continuous
stretch of backbone φ, ψ values in the helical regions of
conformational space. It may be noted that in short peptides
NOEs are usually observed only up to distances of e3.5 Å.
Figure 4. Partial 400 MHz ROESY spectrum of peptide 1 in CDCl3
showing NiHTNi+1H NOEs. [X ) Dpg].
Observation of sequential dNN connectivities thus provides a
strong constraint on the structure. Together with the fact that
the four C-terminus NH groups are hydrogen bonded, the NOE
results could, in principle, be interpreted in terms of a continuous
R-helical conformation over the entire peptide segment as the
predominant structure in CDCl3 solution. This conclusion is
however at variance with the observed crystal structure. Close
examination of the solid state conformation suggests that
residues Dpg(2), Gly(3), Gly(4), Dpg(5), and Gly(6) do indeed
have φ, ψ values lying in the 310/R-helical regions of confor-
mational space (Table 2) with the two C-terminal residues
forming a chiral reversal resulting in a change of the helix
handedness. Thus sequential dNN connectivities over the 2-6
segment are in fact consistent with this conformation. Indeed
the only “nonhelical” residue in the crystal state structure is
Gly(1) with a Gly(NH)‚‚‚Dpg(2)NH distance of 4.6 Å. (All
other NiHTNi+1H distances lie between 2.4 and 2.9 Å.) The
observation of a dNN NOE of moderate intensity between Gly-
(1) and Dpg(2) could be indicative of conformational averaging
between Type II/II′ and Type III/III′ conformation at the Gly-
(1)-Dpg(2) segment. Such a process requires a flip of the

Multiple â-Turn Structure
J. Am. Chem. Soc., Vol. 119, No. 50, 1997 12053
Figure 5. Partial 400 MHz ROESY spectrum of peptide 1 in CDCl3 showing CRHTNi+1H NOEs. [X ) Dpg].
peptide bond between the residues 1 and 2, which can be
achieved by correlated rotation about ψ (Gly1) and φ (Dpg 2).
It should also be noted that if the water invasion of the peptide
backbone is ignored, then intramolecular hydrogen bonds of
the 4 f 1/6 f 1 type will result in solvent shielding of all but
the two N-terminal NH groups. Indeed in the crystal the
potential type II/II′ â-turn at the Gly(1)-Dpg(2) segment is
distorted by hydration, as is the Schellman motif29-32 at the
C-terminus.
The NMR data in CDCl3 solution is thus broadly consistent
with a significant population of the multiple turn conformation
seen in crystals. Further support for this conclusion is obtained
from the medium range NOEs Gly(4)NHTGly(6)NH (w)
(Figure 4), Gly(1)CRHTGly(3)NH (w), and Gly(1)CRHTGly-
(4)NH (w) (Figure 5). The corresponding distances in the
crystal structure are 4.21, 3.77, and 4.21 Å, respectively. An
unusual, strong NOE is also observed between the Boc CH3
resonance (1.45 δ) and the cluster of CRH resonance of Gly(3),
Gly(4), and Gly(6) centred at (3.86-3.88δ) (data not shown).
Figure 6. Partial 400 MHz ROESY spectrum of peptide 1 in (CD3)2-
SO showing NiHTNi+1H NOEs. [X ) Dpg].
Interestingly, the crystal structure places one of the Boc CH3
groups relatively close to the Gly(4) CRH2 group. (The shortest
Boc CH3‚‚‚Gly(4)CRH distance is 2.52 Å.)
backbone NH groups in peptide 1 (Figure 7). The NMR results
thus favour a largely multiple turn conformation in CDCl3 which
is unfolded and solvated in (CD3)2SO. In CDCl3 both Dpg
residues adopt conformations lying in the helical regions of φ,
ψ space.
The temperature coefficients of NH chemical shifts in (CD3)2-
SO solution are summarized in Table 5. While Gly(1), Dpg-
(2), and Gly(3) have high dδ/dt values (>4.5 ppb/K), a very
high value of 6.1 ppb/K is also observed for Gly(6)NH. this
clearly suggests that in a strongly solvating medium, solvent
invasion destabilizes intramolecularly hydrogen bonded back-
bone conformations. The dδ/dt values observed for Gly(4)-
NH, Dpg(5)NH, and NHMe groups lie between 3.7-4.4 ppb/
K, which are suggestive of only moderately solvent shielded
protons. Figure 6 shows the NH-NH region of the ROESY
spectrum of 1 in (CD3)2SO. Although all sequential dNN
connectivities can be identified, the NOE magnitudes appear
to be significantly smaller than in CDCl3. This is particularly
true for the ROESY cross peaks for the segment residues 2-6.
Together with the temperature coefficients, the NOE data
suggest a diminished population of folded compact conforma-
tions and support unfolding by solvation. These conclusions
are reinforced by the observation of very strong exchange cross
peaks between residual water protons in (CD3)2SO and all the
Conclusions
A novel multiple turn conformation is established in single
crystals for the peptide Boc-Gly-Dpg-Gly-gly-Dpg-Gly-NHMe.
The observed structure provides examples of hydrated TypeII/
II′ turns, Type III turns, and a chiral reversal to establish a 6 f
1 hydrogen bonded π-turn, in a hydrated Schellman motif. While
five out of six residues lie in right or left handed helical regions
of φ, ψ space, the molecule as a whole forms a relatively
compact folded structure. NMR studies support substantial
retention of the crystal state conformation in the apolar solvent
CDCl3 with key NOEs and solvent delineation of shielded NH
groups favoring the multiple turn structure. A particularly
interesting feature of the crystal structure is the adoption of

12054 J. Am. Chem. Soc., Vol. 119, No. 50, 1997
Karle et al.
Figure 7. Partial 400 MHz ROESY spectrum of peptide 1 showing CRHTNi+1H connectivities. Strong exchange cross peaks between residual
water protons in (CD3)2SO and all the backbone NH groups are indicated. [X ) Dpg].
helical φ, ψ values for the two Dpg residues even in the context
of a very poor helix promoting segments containing as many
as four Gly residues, including two central contiguous glycines.
Folded conformations at higher dialkylglycine residues must
be considered in interpreting structure-activity relationships for
biologically active peptides. Indeed, studies of chemotactic
tripeptide analogs have shown high biological activity when the
central Leu residue is replaced by dialkylglycine containing both
cyclic and acyclic side chains.37,38 In the case of 1-aminocy-
cloalkane-1-carboxylic acids, helical conformations have been
exclusively reported, whereas for linear dialkyl residues both
helical and extended conformations have been crystallographi-
cally characterized.21 The present results suggest that the local
helical conformations are likely to be significantly more
important even for the higher dialkylglycines. Indeed, the
segment Gly-Dpg-Gly has been crystallographically character-
ized in 3, 6, 10, and 14 residues peptides, with the sole example
of an extended conformation being observed for the Dpg residue
in the tripeptide Boc-Gly-Dpg-Gly-OH.39 The conditions under
which fully extended conformations can be preferentially
stabilized remain to be firmly established. A clear understanding
of the factors that determine conformational state of the higher
dialkylglycines will be of great value in the control of backbone
stereochemistry in peptide design.13,14
Acknowledgment. This research was supported in part by
National Institute of Health Grant GM-30902, the Office of
Naval Research, and the Department of Science and Technology,
India. R.K. acknowledges award of a Research Associateship
from the Department of Biotechnology, India.
Supporting Information Available: Tables of atomic
coordinates, bond lengths, bond angles, anisotropic temperature
factors, and H atom coordinates for 1 (7 pages). See any current
masthead page for ordering and Internet access information.
JA970596Z
(37) Dentino, A, R.; Raj, P. A.; Bhandary, K. K.; Wilson, M. E.; Levine,
M. J. J. Biol. Chem. 1991, 266, 18460-18468.
(38) Prasad, S.; Rao, R. B.; Bergstrand, H.; Lundquist, B.; Becker, E.
L.; Balaram, P. Int. J. Pept. Protein Res. 1996, 48, 312-318.
(39) Datta, S.; Kaul, R.; Rao, R. B.; Shamala, N.; Balaram, P. J. Chem.
Soc., Perkin Trans. 2 1997, in press.
(40) Crisma, M.; Valle, G.; Toniolo, C.; Rao, R. B.; Prasad, S.; Balaram,
P. Biopolymers 1995, 35, 1-9.