Analysis of designed β-hairpin peptides: Molecular conformation and packing in crystals

Article abstract of DOI:10.1039/c3ob25777k

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned ^D

Full text of DOI:10.1039/c3ob25777k

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Analysis of designed β-hairpin peptides: molecular
conformation and packing in crystals†
Cite this: DOI: 10.1039/c3ob25777k
Subrayashastry Aravinda,a Upadhyayula S. Raghavender,a Rajkishor Rai,b
Veldore V. Harini,a Narayanaswamy Shamala*a and Padmanabhan Balaram*b
The crystal structures of several designed peptide hairpins have been determined in order to establish
features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has
been induced using a centrally positioned DPro-Xxx segment (Xxx = LPro, Aib, Ac6c, Ala; Aib = α-aminoiso-
butyric acid; Ac6c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-
DPro-LPro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-DPro-LPro-Leu-Phe-Val-OMe (2, polymorphic forms
labeled as 2a and 2b), Boc-Leu-Val-Val-DPro-LPro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-DPro-Aib-Leu-
Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe
(5) and Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II’
β-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The
angle of twist between the two antiparallel strands lies in the range of −9.8° to −26.7°. A detailed analy-
sis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association:
parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation
modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in
the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of rele-
vance in modeling multiple modes of association, which may provide insights into the structure of insolu-
ble polypeptide aggregates.
Received 23rd April 2012,
Accepted 12th April 2013
DOI: 10.1039/c3ob25777k
www.rsc.org/obc
association. The crystallographic characterization of well-
Introduction
designed peptide β-hairpins,4 also provides an opportunity to
Considerable recent interest has focused on the modes of examine modes of aggregation of peptide molecules, which
association of extended polypeptide strands (β-strands) in crys- may be useful in considering models for fibrillar structures
tals.1 The observed modes of association provide insights into formed by insoluble segments that are of relevance in under-
possible structures for amyloid fibrils and polypeptide aggre- standing the structure of amyloid deposits.5 Constrained
gates associated with a range of disease pathologies.2 Cyclic dipeptide templates which preferentially adopt type I′/II′ β-turn
peptide mimics have been structurally characterized and the conformations have been successfully used in the design of
observed packing arrangements have been correlated with the β-hairpin structures in synthetic peptides. The type I′ and II′
association of extended polypeptide strands in amyloid oligo- β-turns are characterized by specific Ramachandran angles at
mers.3 The packing motifs in crystals of acyclic peptide hair- the dipeptide segment (ideal values for type I′ β-turns are ϕi+1
pins can also provide insights into modes of β-strand = 60°, ψi+1 = 30°, ϕi+2 = 90°, ψi+2 = 0° and for type II′ β-turns are
ϕi+1 = 60°, ψi+1 = −120°, ϕi+2 = 90°, ψi+2 = 0°).6 These confor-
mations may be readily imposed by the choice of residues in
aDepartment of Physics, Indian Institute of Science, Bangalore 560 012, India.
which the conformational options are limited. The D-Pro
(DPro) residue has emerged as a unit of choice, since the
E-mail: shamala@physics.iisc.ernet.in; Fax: +91-80-23602602; Tel: +91-80-22932856
bMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
covalent constraints of pyrrolidine ring formation restricts
E-mail: pb@mbu.iisc.ernet.in; Fax: +91-80-23600683; Tel: +91-80-22932337
ϕDPro to values ≈+60° 30°.7 The two favored regions of con-
†Electronic supplementary information (ESI) available: CIF files, main chain
and side chain torsional angles for peptides 1–6 (Tables S1–S4), hydrogen bond formational space correspond to, ψ = +30 10° and −120°
10°, both of which are compatible with the occurrence of DPro
parameters for 1–6 (Tables S5–S7), twist and aromatic interaction parameters for
at the i + 1 position of type I′/II′ β-turns.4a The DPro-Xxx
1–6 (Tables S8 and S9). The asymmetric and packing figures for peptides 1–6
(Fig. S1–S8). 1H NMR spectra of peptides 1–6 and expansion of NH region
segment, where any L-residue is placed at the i + 2 position,
(Fig. S9–S10). CCDC 821276, 821280, 821281, 821277, 821278, 821279, 821275
strongly favors type II′ β-turns (Fig. 1).4a Achiral residues like
and 821274. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c3ob25777k
Gly and α,α-dialkylated residues (Aib and Ac6c) facilitate the
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conformational preference may be exploited to construct
prime turns for β-hairpin nucleation in DPro-Aib (type II′
β-turn)4b and Aib-DXxx (type I′ β-turn)17 segments. The recent
crystal structure of an octapeptide, Boc-Leu-Val-Val-Aib-DPro-
Leu-Val-Val-OMe, illustrates an example of a type I′ β-turn
nucleated hairpin (Boc: tert-butoxycarbonyl, OMe: methyl
ester).18 As part of an ongoing project aimed at the synthetic
and structural characterization of peptide β-hairpins and
multi-stranded β-sheets, we describe in this report hairpin con-
formations in crystals of the following model peptide
sequences incorporating central DPro-LPro and DPro-Xxx seg-
ments (where Xxx is Ala, Aib and Ac6c). Boc-Leu-Phe-Val-DPro-
LPro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-DPro-LPro-Leu-Phe-
Val-OMe (2, polymorphic forms labeled as 2a and 2b),
Boc-Leu-Val-Val-DPro-LPro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-
Val-DPro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled
as 4a and 4b), Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe (5)
and Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (6). A detailed
consideration of the observed packing motifs is presented, pro-
viding insights into the modes of aggregation of antiparallel
β-strand structures.
Fig. 1 Ramachandran plot showing β-sheet region (gray), conformational
space accessible to Aib (green) and L-proline and D-proline (blue). The ideal con-
formations of residues i + 1 and i + 2 in a type II’ β-turn are indicated by a red
arrow.
Results and discussion
Structures of β-hairpins
formation of both type I′/II′ β-turns, when used at the i + 2
position in DPro-Xxx segments.4b,8a The type II′ β-turn in a Crystals of peptides 1–6 consist of two molecules in the asym-
DPro-LPro unit was initially characterized in the crystal struc- metric unit. All molecules adopt type II′ β-turn nucleated,
ture of a tripeptide.9 Robinson’s group has been instrumental β-hairpin conformations. The turn segment in peptides 1–3 is
in using the β-hairpin motif to design protein epitope DPro-Pro and in peptides 4 to 6, DPro-Xxx (where Xxx is Aib(4),
mimetics by transplanting hairpin loop sequences from folded Ac6c(5) and Ala(6)) (Fig. 2). The individual hairpins of the pep-
proteins onto hairpin-stabilizing DPro-LPro templates.10 In the tides 1–6 (16 independent octapeptide molecules) adopt very
process, they have been successful in designing and optimiz- similar backbone conformations, as indicated by the super-
ing β-hairpin mimetics, which are well suited for the design of position of the backbone atoms (N, Cα, C) of the residues 2–7
inhibitors of both protein–protein and protein–nucleic acid (r.m.s.d. of 0.132 Å, Fig. 3). Residues 1–3 and 6–8 adopt confor-
interactions.11 Subsequently, the DPro-LPro segment has been mations corresponding to β-strands, whereas residues 4–5
used in nucleating hairpin structures in synthetic antigenic occur in the conventional type II′ β-turn conformation, with
peptides.12 In particular, β-hairpin shaped peptidomimetics the exception of molecule A in peptide 6. Main chain and side
based on the membranolytic host-defense peptide protegrin I chain torsional angles are summarized in the ESI Tables S1–
containing loop sequences linked to a DPro-LPro template sta- S4.† A scatter plot of backbone torsional angles for all 8 resi-
bilizing the hairpin conformations have been shown to dues in the various β-hairpin structures is shown in Fig. 4.
possess potent antimicrobial activity in a mouse septicemia While most residues are fairly tightly clustered in ϕ, ψ space,
infection model.13 Peptide hairpins have also been developed significant variation is observed for the C-terminus Val
as novel biomaterials, especially in the rational design of residue. Fraying of the inter-strand hydrogen bonds at the
amphiphilic sequences which can form functionally useful hairpin terminus is a common feature. Three intramolecular
hydrogels.14 We have been systematically studying the confor- hydrogen bonds are observed to stabilize the β-hairpin confor-
mational properties of synthetic oligopeptides containing mation in all the individual molecules, with four hydrogen
centrally positioned DPro-LPro segments in order to bonds being observed only in the case of peptide 6 (molecule
unambiguously establish the conformational properties of A). The individual molecules are stabilized by three cross-
potential hairpin peptides. The NMR structural characteriz- strand hydrogen bonds (N13⋯O16, N16⋯O13, and N18⋯O11)
ation of a β-hairpin in the synthetic peptide, Boc-Leu-Phe- and (N23⋯O26, N26⋯O23, and N28⋯O21 (ESI Tables S5–S7†).
Val-DPro-LPro-Leu-Phe-Val-OMe, has been reported.15
Residue 5 in molecule A, peptide 6 (DPro(4)-Ala(5)) is a Rama-
The ability of α,α-dialkylated residues, of which the Aib chandran outlier, with backbone torsion angles (ϕ = −138.3°,
residue is the prototype, to be restricted to local helical regions ψ = −13.8°) lying clearly in the disallowed region (Fig. 4).
(right-handed α-helix, αR: (ϕ, ψ) = (−60°, −30°) and left-handed Inspection of the turn segment reveals this is the only case
α-helix, αL: (ϕ, ψ) = (60°, 30°) is well established.16 This where the DPro(4) residue adopts ϕ, ψ values (ϕ = 84.6°, ψ =
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Fig. 2 Individual molecular conformations of the peptides 1–6. Top panel shows molecule A and bottom panel shows molecule B. The turn region is shown in ball-
and-stick (green) and the cocrystallized water molecules as spheres (red).
Fig. 4 Distribution of backbone torsion angles (ϕ, ψ) in peptide hairpins rep-
resented on a Ramachandran map. Data from 18 peptide structures, corres-
ponding to 32 independent hairpin molecules. The superposition of the type II’
β-turn segment in 31 molecules (RMSD = 0.104 Å; Val(3)CO⋯NHLeu(6) = 3.01
0.07 Å) is shown. The distorted turn segment in peptide 6 molecule A is shown
Fig. 3 (a) Superposition of the backbone atoms (N, Cα, C) of the individual
β-hairpin molecules of the peptides 1–6 (RMSD = 0.132 Å). Carbon atoms are
colored differently for each molecule, whereas nitrogen and oxygen atoms are
colored blue and red, respectively. (b) Side view of the β-hairpin molecules.
separately. Backbone torsion angles at DPro(4) and Ala(5) in 6 deviate consider-
ably from the clusters observed in the other 31 examples. The hydrogen bond
distances are Val(3)CO⋯NHAla(5) = 2.770 Å and Val(3)CO⋯NHLeu(6)NH
3.143 Å. The Val(3)CO⋯NHXxx(5) distances in the other 31 examples are 3.22
0.09 Å.
=
−73.5°) corresponding to a classical γ-turn structure,19 stabi-
lized by a 3 → 1 hydrogen bond between the Val(3)CO and Ala-
(5)NH group (N⋯O = 2.77 Å, H⋯O = 2.08 Å and ∠N–H⋯O =
136.8°). In peptide 6 molecule A, the turn segment is signifi-
cantly distorted from an ideal type II′ structure, with Val(3) CO
potentially accepting two simultaneous hydrogen bonds (3 →
1,4 → 1). Interestingly, the two antiparallel strands are per-
fectly registered with all four inter-strand hydrogen bonds
being formed. Torsional strain at the Ala(5) residue in peptide
6 molecule A must undoubtedly be compensated by energeti-
cally favorable interactions, both intramolecular and inter-
molecular. The twist of the individual hairpin molecules is
evaluated as a virtual torsion angle between Cα atoms of resi-
dues 3–6 (ESI Table S8†). The twist of the individual hairpins
in all the hairpin structures is right-handed and is observed to
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be in the range −10° to −20°. Molecule B in peptide 6 is solvent in the asymmetric unit. The DPro-Pro segment in the
slightly more twisted than the other individual molecules in peptides 1, 2a, 2b and 3 is not solvated, presumably because of
the series (−26.7°).
the lack of free NH groups.
Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (1). The lone
water molecule, cocrystallized with the peptide molecule in the
Side chain conformation and interactions
By convention, the signs of χ1, χ2, χ3 and χ4 torsions decide the asymmetric unit, helps in bridging the molecules related by
Cγ-exo (UP) and Cγ-endo (DOWN) pucker of the pyrrolidine ring the translation symmetry (O1w (x, y, z) and N17 (x, y + 1, z) and
(−, +, −, +corresponding to Cγ-exo (UP) and +, −, +, − corres- O15 (x, y + 1, z)) by the formation of hydrogen bonds (ESI
ponding to Cγ-endo (DOWN)).20 Accordingly, the DPro(4) Table S5 and Fig. S2†).
residue is observed to adopt a Cγ-endo (DOWN) pucker in 1,
Boc-Leu-Tyr-Val-DPro-LPro-Leu-Phe-Val-OMe (2a and 2b). In
2a, 3 (molecule A) 4a, 4b and 5. Cγ-exo (UP) puckering is both the polymorphic crystal forms, three fully occupied, co-
observed for the DPro(4) in 3 (molecule B) and 6. The Pro(5) crystallized water molecules are observed in the asymmetric
residue adopts a Cγ-endo (DOWN) in 1, 2a, 2b, and 3 (molecule unit. The water molecules co-crystallized with the peptide in
A) and Cγ-exo (UP) in 3 (molecule B). Aromatic–aromatic inter- 2a, help in bridging symmetry related molecules by hydrogen
actions17,21 are observed between the Phe residues present on bonds (O2w(x + 1, y, z) and O15(x, y, z), O2w(x, y, z) and N27-
the antiparallel strands of all the peptides except peptide 3, (x, y, z)) and also interact with the hydroxyl group of the Tyr
where the strands did not have Phe residues. The interaction residue (O1w(x, y, z) and O1t(x, y, z), O3w(x, y, z) and O2t(x, y,
parameters for the π⋯π interactions are mostly T-shaped or z)). In contrast, in 2b the co-crystallized water molecules are
inclined (ESI Table S9†). Notably, in peptide 6 molecule A, the observed to form peptide backbone hydrogen bonds with the
aromatic sidechains Phe(2) and Phe(7) adopt different back- molecule-B (O2w(x, y, z)⋯O24(x, y, z) and O1w(x, y, z)⋯O25-
bone conformations as compared to all other peptides con- (x, y, z)) and with the Tyr(2) OH (O3w(x, y, z)⋯O1t(x, y, z)).
taining a facing pair of phenyl rings at this non-hydrogen O1w and O3w also form water–water hydrogen bonds. O1w is
bonding site in the β-hairpins. It is observed that the Phe resi- observed to hold the translationally related molecules by
dues, in the case of peptide 6, are involved in a network of hydrogen bonds (O1w(x, y, z)⋯O25(x, 1 + y, z)), whereas O2w
interactions across symmetry-related molecules, as discussed water molecule is observed to bridge 21 screw related peptide
subsequently.
molecules (O2w(x, y, z)⋯O1t(−x, y + 1/2, 1 − z)) in a direction
perpendicular to the propagation of β-sheet hydrogen bonds
(approximately along the b-axis) as opposed to peptide 1. The
Packing of the β-hairpins in crystals
In crystals the two molecules in the asymmetric unit, of all the turn segment is not hydrated in both the independent mole-
peptides 1–6, are inclined (approximately) orthogonally with cules in the case of 2a, whereas in 2b molecule-B shows a
respect to each other. The angle between the two molecules in strong peptide–water hydrogen bond with the backbone carbo-
the asymmetric unit is calculated by fitting a mean plane nyl oxygen of the turn segment (O2w(x, y, z)⋯O24(x, y, z)),
passing through the Cα atoms of the residues 2–7 of the indi- which in turn forms a hydrogen bond with peptide molecule
vidual molecules (ESI Table S8†). The two molecules in the (ESI Table S5 and Fig. S3†).
asymmetric unit are inclined to each other at an angle of ∼52°
Boc-Leu-Val-Val-DPro-LPro-Leu-Val-Val-OMe (3). The two
to ∼77°, with the exception of peptide 3, where an approximate molecules in the unit cell of peptide 3 pack as two indepen-
parallel orientation of the molecules is observed (∼37°). dent and separate β-sheets. The packing of the individual
Peptide 6 shows an almost perfect orthogonal arrangement molecules, along the crystallographic a-axis, is stabilized by
with the angle of inclination being ∼86° (ESI Fig. S1†). Two the formation of three intermolecular peptide–peptide hydro-
strong intermolecular hydrogen bonds (N12⋯O22 and gen bonds (ESI Table S6†). A bifurcated hydrogen bond is
N22⋯O12) are observed connecting the two molecules in the observed at the N-terminus of both the molecules (N11(x, y, z)
asymmetric unit in all the peptides (ESI Tables S5–S7†). Hydro- and O17(1 + x, y, z), N12(x, y, z) and O17(1 + x, y, z), N21-
gen bonds (intramolecular and intermolecular), involving (x, y, z) and O27(−1 + x, y, z), N22(x, y, z) and O27(−1 + x, y,
peptide backbone NH and CO groups, stabilize the packing of z)). This brings the N-terminus of one β-hairpin mole-
the molecules in the plane of the sheet. Packing of the mole- cule in register with the C-terminus of the next translated
cules perpendicular to the plane of the sheet is mediated by molecule. The overall packing in the crystals can be
peptide–solvent hydrogen bonds. Cocrystallized solvent mole- described as corresponding to that of the “parallel” β-sheet
cules help in stabilizing the molecular conformation by (ESI Fig. S4†).
forming hydrogen bonds with the peptide backbone. The
Boc-Leu-Phe-Val-DPro-Aib-Leu-Phe-Val-OMe (4a and 4b). The
cocrystallized solvents in 1, 2a, 2b, 4a, 4b, 5 and 6 are observed octapeptide 4a crystallized with two molecules in the
to be involved in bridging the symmetry related molecules asymmetric unit and a cocrystallized dioxane and a water
along the direction of the intramolecular and intermolecular molecule.2 In the crystals, the dioxane molecule helps in
hydrogen bonds. The structure of peptide 3 is devoid of bridging the peptide molecules related by symmetry with
solvent, leading to increased interpeptide hydrogen bonding hydrogen bonds (O2S(x, y, z) and N25(−1 + x, y, z)) (ESI
between the symmetry related molecules. This is the only Table S6†). The water molecule is involved in bridging the
example of a β-hairpin crystal structure with no cocrystallized translated molecules (O2w (x, y, z) and O15(1 + x, y, 1 + z)).
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In peptides 1–3 the design of the β-turn using the L-Pro −z + 1/2)). The terminal methoxy ester group of molecule-B is
residue at the i + 2 position precludes any solvation at the also oriented such that it is also involved in hydrogen bond
central peptide unit of the turn region. In contrast the β-turn with O2w (O2w⋯O28(−x + 2, y + 1/2, −z + 1/2)). The 1,1 disub-
in 4a reveals solvation of the central DPro-Aib peptide unit. stituted cyclohexane rings of the Ac6c residues, in both the
This is a common feature in β-turns observed in protein polymorphic forms of 5, adopt almost ideal chair confor-
structures.22 In contrast, 4b was observed to have a single mations, the amino group occupying an axial position8 (ESI
water molecule in the asymmetric unit. The cocrystallized Fig. S6†).
water in 4b is seen to be involved in bridging translated
Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (6). Peptide
6
molecules by hydrogen bonding with the peptide backbone crystallized in the orthorhombic space group P212121, with two
(O1w(x, y, z)⋯O15(x, y, z), and O1w(x, y, z)⋯N27(−1 + x, y, peptide molecules and two water molecules in the asymmetric
−1 + z)). One intermolecular hydrogen bond between the trans- unit, whereas the polymorphic form (also P212121) previously
lated molecules is also observed to be involved in stabilizing described crystallized with two peptide molecules, one isopro-
the packing of the molecules (O27(x, y, z)⋯N27(1 + x, y, 1 + panol and three water molecules in the asymmetric unit.4b The
z)) (ESI Table S6†). In 4b unlike 4a, there is no stabilizing crystals of the polymorphic form were obtained by slow evapo-
interaction mediated by solvent, in a direction perpendicular ration from a mixture of isopropanol–water, whereas the crys-
to the β-sheet hydrogen bonds. It can be deduced that the tals of 6 were grown from dimethylformamide (DMF) to which
packing of the molecules is very robust in both the crystal small amount of water was added. In both the cases thin
forms and the cocrystallized solvents occupy the void space needles were formed. The puckering of the DPro(4) residue is
in the crystals, effectively stabilizing the packing by getting different in both the forms. The DPro(4) residue is Cγ-exo (UP)
involved in favorable hydrogen bonding interactions with the puckered in 6 and Cγ-endo (DOWN) in the polymorphic form
peptide backbone. It is likely that the dioxane molecules (molecule-A). Peptide 6 has the two molecules oriented at
might have escaped from the crystals over the long period of approximately 86°. The molecule-A is observed to form all the
storage. The symmetry related layers of the peptide molecules four possible intramolecular cross-strand hydrogen bonds for
in 4b retain all the characteristics of molecular packing and an octapeptide hairpin (N11⋯O18, N13⋯O16, N16⋯O13 and
hydrogen bonds, despite the absence of the bridging solvent N18⋯O11), whereas molecule-B is observed to form only three
mediated hydrogen bond, connecting the screw-related layers cross-strand intramolecular hydrogen bonds (N23⋯O26,
as in 4a. This feature is concomitant with shrinkage in the N26⋯O23 and N28⋯O21) (ESI Table S7†). Cocrystallized water
unit cell axis corresponding to the dimension of the dioxane molecules are observed to form hydrogen bonds with the back-
molecule and a slight rearrangement of the symmetry-related bone atoms, DPro(4) CO in molecule-A (O1w⋯O14) and Ala(5)
molecules, with denser packing along a direction perpendicu- NH in molecule-B (O2w⋯N15), of the β-turn segment. Three
lar to the β-sheet (ESI Fig. S5†).
peptide–peptide backbone cross-strand intermolecular hydro-
Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe (5). The asym- gen bonds stabilize packing along the a-axis (N12⋯O22,
metric unit of 5 contains three cocrystallized water molecules, O12⋯N22, O17⋯N27). The adjacent hairpins are also con-
of which one is disordered (the water molecule O3w is dis- nected by solvent mediated peptide–water hydrogen bonds
ordered over two positions O3w and O3wa, with occupancies (O1w(x, y, z)⋯N17(1/2 + x, 3/2 − y, 2 − z), O1w(x, y, z)⋯O15(1/2
of 0.42 and 0.58). These three water molecules act as inter- + x, 3/2 − y, 2 − z), O2w(x, y, z)⋯O27(−x, 1/2 + y, 3/2 − z)). In
molecular solvent bridges in the orthorhombic form 5, as com- contrast, in the polymorphic form the two molecules in the
pared to the water and dioxane molecules in the monoclinic asymmetric unit are oriented at an angle of approximately 67°
form.8a Three water molecules bridge free CO and NH groups with respect to each other. Extended packing of this asym-
in the neighboring symmetry related molecules, forming a metric unit is achieved through a complex intermolecular
complex network of intermolecular hydrogen bonds, stabiliz- hydrogen bonding pattern, involving two bridging water mole-
ing the packing in crystals. O1w and O3w link adjacent mole- cules, O1w and O2w. O2w is also involved in association of
cules related by translational symmetry along the molecule-A and its symmetry-related neighbors along the
crystallographic a-axis direction via the strand segment. The b-axis. A third water molecule, O3w, forms hydrogen-bonded
asymmetric units are linked along [1 0 0] direction, by a direct bridges along the c-direction between Ala(5) NH and Val(8) CO
interpeptide hydrogen bond N27⋯O17(x + 1, y, z) and three of symmetry related molecule-B. The lone isopropanol mole-
water bridges, involving N17⋯O1w⋯O25(x
− 1, y, z), cule appears to fill cavities and is hydrogen bonded only to
O17⋯O3w*⋯O25(x − 1, y, z) and O15⋯O3wa*⋯O1w⋯O25(x − O3w4b (ESI Fig. S7†). It is observed that the π⋯π interactions
1, y, z), respectively (ESI Table S7†). The distance between extend beyond being restricted to the individual molecules in
O3w* and O1w is 2.02 Å, whereas it is 4.748 Å between O3w* the case of peptide 6. The Phe(2) residue of molecule-A in 6 is
and O3wa* (* indicates two disordered positions of the water involved in two additional T-shaped aromatic interactions with
molecule O3w). Along the crystallographic b-axis, the central one translated molecule (x, 1 + y, z)(Rcen = 6.617 Å, Rclo
=
peptide NH (Ac6c,NH) of molecule-B is hydrogen bonded to 4.637 Å, γ = 51.06°) and one symmetry related molecule (−x,
water molecule O2w, which is further anchored by two hydro- y + 1/2, −z) (Rcen = 6.437 Å, Rclo = 4.220 Å, γ = 52.82°). (Rcen: cen-
gen bonds to O18 and O27 of symmetry related molecules troid–centroid distance, Rclo: shortest distance between two
(O2w⋯O18(−x + 1, y + 1/2, −z + 1/2), O2w⋯O27(−x + 2, y + 1/2, carbon atoms of the interacting rings and γ: interplanar
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angle). In turn, the Phe(2) of molecule-B is involved in a
weaker π⋯π interaction with a 21-screw symmetry related mole-
cule-A (Rcen = 7.155 Å, Rclo = 5.083 Å, γ = 72.56°). All of the
above weakly polar, π⋯π interactions observed in the structure
of 6 are a result of the unusual orientation (completely ortho-
gonal) of the molecules in the asymmetric unit. As already
noted molecule-A has all its backbone cross-strand intramole-
cular hydrogen bonds satisfied. A ladder of aromatic inter-
actions appears to be involved in the further stabilization of
the packing of the molecules in the crystals of peptide 6 (ESI
Fig. S8†).
Indexing of crystal faces and insights into the growth of
crystalline plates of β-hairpins
Aggregation of molecules in solution to form nuclei that grow
along specific directions must precede the formation of single
crystals. Nucleation and crystal growth are poorly understood
phenomena, at a molecular level. In the case of peptides the
development of the principles for the design of sequences that
facilitate crystal formation is not readily achieved. In the case
of conformationally restricted peptides, considerable success
has been realized in the crystallization of hydrophobic helical
Fig. 5 Indexing of the crystal faces of peptides (a) 1, (b) 3 and (c) 4b. Top
panel shows the crystal with the macroscopic faces indexed and the planes indi-
cated as yellow lines with the direction indicated. Bottom panel shows the weak
interactions in the peptides for peptides 1 and 3. The direction of escape of
solvent dioxane molecules in the polymorphic forms of peptide 4 is coincident
with the length of the crystal.
sequences.4a,16f–h,23 Attempts to correlate observed crystal sym- that β-turn segments approach each other in the symmetry
metries with models for nucleation and growth have been related layers. The DPro-Pro segment is not hydrated in both
reported.24 Hydrophobic β-hairpins have been more recalci- the molecules. The only possible weak interaction is that of
trant to crystallization, often yielding thin, two-dimensional CδH⋯O type involving the Pro residues.25 A close-up view of
plates. This morphology suggests that while aggregate for- the weak interactions observed in the turn segment provides
mation in two dimensions is facile, presumably mediated by clues to the growth of the crystal faces (Fig. 5). It is clear that,
antiparallel β-strand hydrogen bonds, ordered packing along apart from hydrogen bonding, which is the stabilizing inter-
the third dimension is less favored. To test this hypothesis, action along the b-axis, weak interactions of the CH⋯O type
face indexing of selected peptide β-hairpin crystals has been may contribute to the packing along the c-axis. The preferen-
carried out.
tial growth of the crystal along the b and c direction may
Boc-Leu-Phe-Val-DPro-L-Pro-Leu-Phe-Val-OMe (1). The plate arise from these contributing factors. On the other hand, the
like single crystal on which the diffraction data was collected interactions along the perpendicular direction that is along
had the macroscopic dimensions of 0.80 × 0.12 × 0.01 mm. It the a-axis, are purely apolar (van der Waals), corresponding
is clear that two dimensions, length and breadth, correspond to the weak interactions of the projecting side chains of the
to the well developed faces of the crystal. The peptide crystal- strand residues; consequently growth along this face is sig-
lized in the monoclinic space group P21, with two molecules nificantly slower.
in the asymmetric unit. The cell parameters of the crystal are
Boc-Leu-Val-Val-DPro-LPro-Leu-Val-Val-OMe (3). The crystals
a = 14.403 Å, b = 18.932 Å, c = 25.490 Å and β = 105.67°. of 3 were assigned to the triclinic space group P1 with two
Indexing of the faces reveals that the (010) plane corresponds molecules in the asymmetric unit. All the crystals were found
to the b-axis (18.962 Å); it is collinear with the length of the to be needles. The single crystal on which the diffraction data
crystal (0.80 mm). All the intramolecular and intermolecular was collected had the macroscopic dimensions of 0.49 × 0.09
hydrogen bonds are formed along the b-axis. The cocrystal- × 0.05 mm. The cell parameters of the crystal are a = 9.922 Å,
lized water molecule also forms hydrogen bonds along the b = 11.229 Å, c = 26.423 Å and α = 87.15°, β = 89.44°, γ =
b-axis. The (100) plane could be indexed to the a-axis 73.28°. Two of the macroscopic faces could be indexed,
(14.403 Å), which is along the thickness of the crystal corresponding to the two crystallographic axes. The (100)
(0.01 mm). The (001) planes could be indexed to the c-axis plane corresponds to the breadth (0.09 mm) of the crystal
(25.490 Å); it is collinear with the breadth of the crystal and is aligned along the a-axis (9.922 Å). The (001) plane cor-
(0.12 mm). The two molecules in the asymmetric unit are responds to the crystallographic c-axis (26.423 Å) and is
inclined to each other at an angle of ≈60°. Molecule A is observed to be collinear with thickness of the crystal
oriented, with its extended strands approximately along the (0.05 mm). The length of the crystal (0.49 mm) could be
a-axis, while the strands in molecule B lie along the c-axis. indexed to correspond to the (121) and (121) planes. Mole-
Fig. 5 shows the projection of the molecules along the bc- cules are approximately oriented along the (121) planes and
plane, corresponding to the [100] direction. The two layers of the set of intra- and intermolecular hydrogen bonds propa-
molecules shown are related by a 21 screw. It can be seen gate along the planes (Fig. 5). This can be taken
Org. Biomol. Chem.
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approximately along the bc-plane. There are no free NH
groups in the crystal structure, as all the amide nitrogen
atoms are involved in hydrogen bonds. The two molecules
form separate β-sheets. There is no cocrystallized solvent in
the structure and the molecules are packed in parallel
fashion in the crystals. The orientation of the molecules in
the adjacent asymmetric units and the interactions between
them would decide the growth along (100) and (001) planes.
The molecules in the adjacent asymmetric units, along the
ab-plane or (001) plane are oriented with their β-turn seg-
ments, DPro-Pro, overlapping with each other. CδH⋯O hydro-
gen bonds, involving the Pro residues, appear to be
stabilizing interactions in this direction, perpendicular to the
hydrogen bond direction. The interactions in the asymmetric
unit are between the termini of the hairpins, tert-butoxycarbo-
nyl (Boc) and methyl ester groups (OMe), with the two mole-
cules oriented with their termini facing each other. The
interactions along the b-axis or (010) planes are essentially
apolar, involving the projecting side chains of the β-strand
residues.
Fig. 6 Schematic illustration of the packing of β-hairpin molecules in crystals.
Monomers can self-assemble into dimers in three different ways. Further assem-
bly into tetramers is a consequence of space group symmetry, non-crystallo-
graphic symmetry, hydrophobic interactions and weak interactions. Interactions
in the plane of the sheets are invariably hydrogen bonds, whereas the inter-
actions in perpendicular directions are weak and hydrophobic in nature.
Boc-Leu-Phe-Val-DPro-Aib-Leu-Phe-Val-OMe (4b). The habit
of the crystal of 4b can be classified as ‘platy’. The dimensions
of the crystal used for collecting X-ray diffraction data are 0.40
× 0.20 × 0.04 mm. The cell parameters obtained were a =
18.095 Å, b = 23.032 Å, c = 18.637 Å and β = 117.47°. The association of the molecules with the nearest neighbors which
peptide crystallized in the monoclinic space group P21, with forms the basis for the classification, as illustrated schemati-
two molecules in the asymmetric unit oriented orthogonally cally in (Fig. 6). Representative examples are chosen to highlight
(∼60°) to each other. The (100) plane, a-axis (14.318 Å), was the gross features of packing arrangements. Parallel packing
indexed along the length of the crystal (0.40 mm). This corres- corresponds to a peptide molecule being in register with a sym-
ponds to the direction of hydrogen bonding, intra- and inter- metry related, translated molecule. This is the simplest type of
molecular, in the crystals. The (010) plane, b-axis (18.992 Å), packing and is observed in all the sequences crystallizing in the
was indexed along the breadth of the crystal (0.20 mm). The triclinic space group P1. The other example is where the two
(101) plane was indexed along the thin dimension of the peptide molecules associate adjacently, with the edge of one
crystal (0.04 mm). The polymorphic form (4a) of the above hairpin in register with the β-turn segment of the next hairpin.
crystal has the following cell parameters a = 18.41 Å, b = This is observed in only one β-hairpin, the decapeptide Boc-
23.22 Å, c = 19.24 Å and β = 118.04°. Noticeable differences can Met-Leu-Phe-Val-DPro-Ala-Leu-Val-Val-Phe-OMe4b (space group
be seen in the cell dimensions of the a- and c-axes. The cocrys- P1). Both the parallel and antiparallel packing arrangements are
tallized dioxane molecule observed in 4a is not present in 4b. observed in the triclinic space group P1. The “orthogonal”
The crystal used in the structure solution and refinement of packing is a special case, wherein the two peptide molecules
the sequence 4a was observed to be no longer transparent. It associate through β-sheet like hydrogen bonds at an angle with
can be seen that the dioxane molecules in the structure of 4a respect to each other (∼60°). This arrangement is unusual, in
lie on the (200) plane. The (200) plane is along the length of the sense of packing of molecules, in the asymmetric unit. Mul-
the crystal of 4b, hence it is possible that dioxane molecules tiple molecules in the asymmetric unit are usually observed to
could have escaped from the crystal after a prolonged time, possess non-crystallographic symmetry. In all the cases, where
concomitant with a corresponding reduction in the a-axis by multiple molecules were observed in the asymmetric unit (Z′ >
0.315 Å (Fig. 5), corresponding to a decrease in the volume of 1) a pseudo 2-fold axis is invariably observed. Grossly, it lies in
∼369 Å3.
the middle of the interpeptide intermolecular cross-strand
hydrogen bonds and more or less collinear with a crystallo-
graphic two fold axis. β-hairpin peptide sequences containing
all α-amino residues and possessing Leu-Phe-Val as β-strand
Classification of peptide β-hairpins based on their
self-assembly in crystals
Broadly, peptide β-hairpins can be classified into three types segments have been, without exception, observed to be ortho-
based on the way in which the individual peptide molecules gonally packed, and usually crystallize in space groups which
associate with each other in crystals. The three classes corres- possess a 21 screw. Table 1 provides a summary of the β-hairpin
pond to (i) parallel packing, (ii) anti-parallel packing, and (iii) crystal structures available in the literature and those presented
orthogonal packing. Though the individual β-strands of the here and their classification based on the packing of the mole-
β-hairpins are antiparallel in all the three cases, it is the local cules observed in the solid state.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem.

Table 1 Classification of β-hairpin peptide crystal structures
No. Sequence
Space group Z/Z′a β-turn
Cocrystallized solvent
Type II′ 2 H2O
Type II′ 1 H2O
Type I′ 3 H2O
Type II′ 1 CH3OH
Packing in crystals Reference
1
2
3
4
Boc-Leu-Val-Val-DPro-Gly-Leu-Val-Val-OMe. a = 9.739 Å, b = 11.579 Å, c = 26.253 Å,
α = 98.39°, β = 120.44°, γ = 107.80°
P1
2/2
2/2
4/2
2/1
Parallel
29a
29b
29c
29d
Boc-Leu-Val-Val-DPro-Ala-Leu-Val-Val-OMe. a = 9.942 Å, b = 11.240 Å, c = 25.882 Å,
α = 86.14°, β = 95.62°, γ = 104.72°
P1
Parallel
Boc-Leu-Val-βPhe-Val-DPro-Gly-Leu-βPhe-Val-Val-OMe. a = 19.059 Å, b = 19.470 Å,
c = 21.077 Å
P212121
P21
Antiparallel
Parallel
Boc-βPhe-βPhe-DPro-Gly-βPhe-βPhe-OMe. a = 9.854 Å, b = 10.643 Å, c = 25.296 Å,
β = 100.39°
5
6
Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe. a = 10.004 Å, b = 13.724 Å, c = 51.214 Å
Boc-Leu-Val-βVal-DPro-Gly-βLeu-Val-Val-OMe. a = 34.184 Å, b = 10.673 Å, c = 18.965 Å,
β = 120.44°
P212121
C2
4/1
4/1
Type I′
Type I′
4 H2O
1 H2O
Parallel
Antiparallel
17
29e
7
8
9
Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe. a = 9.883 Å, b = 12.238 Å, c = 46.861 Å
Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe. a = 19.107 Å, b = 23.905 Å, c = 28.450 Å
Boc-Leu-Val-Val-DPro-Aib-Leu-Val-Val-OMe. a = 9.882 Å, b = 11.288 Å, c = 15.734 Å,
α = 107.545°, β = 90.017°, γ = 104.261°
P212121
P212121
P1
4/1
8/2
1/1
Type II′ 1 H2O
Type II′ 3 H2O + 1 isopropanol
Type II′ 1 H2O + 1 DMF
Parallel
Orthogonal
Parallel
4b
4b
4b
10
11
12
13
14
Boc-Met-Leu-Phe-Val-DPro-Ala-Leu-Val-Val-Phe-OMe. a = 12.153 Å, b = 24.100 Å,
c = 27.990 Å, α = 101.02°, β = 102.51°, γ = 104.62°
P1
4/4
4/2
4/2
4/2
2/2
Type II′ 4 H2O
Antiparallel
Parallel
4b
Boc-Leu-βPhe-Val-DPro-Gly-Leu-βPhe-Val-OMe. a = 19.555 Å, b = 11.352 Å,
c = 28.912 Å, β = 101.909°
P21
P21
P21
P1
Type II′ 2 Ethanol
Type II′ 1 Dioxane + 1 H2O
Type II′ 3 H2O
29f
8a
Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe. a = 18.143 Å, b = 24.858 Å,
c = 18.449 Å, β = 117.02°
Orthogonal
Orthogonal
Parallel
Boc-Leu-Phe-Val-DPro-Ac8c-Leu-Phe-Val-OMe. a = 15.039 Å, b = 25.900 Å,
c = 19.083 Å, β = 108.498°
8a
Boc-Leu-Val-γAbu-Val-DPro-Gly-Leu-γAbu-Val-Val-OMe. a = 9.742 Å,
b = 10.842 Å, c = 31.473 Å, α = 89.46°, β = 83.28°, γ = 78.85°
Boc-Leu-Val-Val-DPro-δAva-Leu-Val-Val-OMe. a = 9.678 Å, b = 11.967 Å, c = 52.228 Å
Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe. a = 9.558 Å, b = 26.278 Å, c = 27.434 Å
Boc-Leu-Phe-Val-DPro-ψPro-Leu-Phe-Val-OMe. a = 34.646 Å, b = 15.337 Å,
c = 25.553 Å, β = 103.387°
Type II′ 2 H2O
29g
15
16
17
P212121
P212121
C2
4/1
4/1
8/2
C13 ring 1 H2O + 1 CH3OH
C12 ring 1 Tetrahydrofuran (THF) Parallel
Type II′ 3 H2O Orthogonal
Parallel
29h
29i
29j
18
19
Boc-Leu-Val-Val-Aib-DPro-Leu-Val-Val-OMe. a = 11.0623 Å, b = 18.7635 Å,
c = 16.6426 Å; β = 102.37°
P21
P21
2/1
4/2
Type I′
2 DMF
Parallel
18
Boc-Leu-Phe-Val-DPro-Pro-Leu-Phe-Val-OMe (1) a = 14.4028 Å, b = 18.9623 Å,
c = 25.4903 Å, β = 105.674°
Type II′ 1 H2O
Orthogonal
Present study
20
21
Boc-Leu-Tyr-Val-DPro-Pro-Leu-Phe-Val-OMe (2a) a = 19.086 Å, b = 26.216 Å, c = 28.015 Å P212121
8/2
4/2
Type II′ 3 H2O
Type II′ 3 H2O
Orthogonal
Orthogonal
Present study
Present study
Boc-Leu-Tyr-Val-DPro-Pro-Leu-Phe-Val-OMe (2b) a = 14.318 Å, b = 18.992 Å,
c = 25.157 Å, β = 105.59°
P21
22
23
24
Boc-Leu-Val-Val-DPro-Pro-Leu-Val-Val-OMe (3) a = 9.922 Å, b = 11.229 Å,
c = 26.423 Å, α = 87.15°, β = 89.44°, γ = 73.28°
P1
2/2
4/2
4/2
Type II′ No solvent
Type II′ 1 H2O + 1 dioxane
Type II′ 1 H2O
Parallel
Present study
Present study
Present study
Boc-Leu-Phe-Val-DPro-Aib-Leu-Phe-Val-OMe (4a) a = 18.410 Å,
b = 23.219 Å, c = 19.242 Å, β = 118.036°
P21
P21
Orthogonal
Orthogonal
Boc-Leu-Phe-Val-DPro-Aib-Leu-Phe-Val-OMe (4b) a = 18.095 Å, b = 23.031 Å,
c = 18.637 Å, β = 117.471°
25
26
Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe (5) a = 18.880 Å, b = 24.579 Å, c = 28.780 Å P212121
8/2
8/2
Type II′ 3 H2O
Type II′ 4 H2O
Orthogonal
Orthogonal
Present study
Present study
Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (6) a = 19.042 Å, b = 23.627 Å, c = 28.591 Å
P212121
a Z: number of molecules in the unit cell; Z′: number of molecules in the asymmetric unit.

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Conclusions
Experimental section
Peptide synthesis
The successful crystallization and study of a large number of
model peptide hairpin sequences points towards a strategy for The peptides were synthesized by classical solution phase
the creation of multi-stranded β-sheet structures.26 So far, the methods by using a racemization-free fragment condensation
trials to crystallize three and four stranded β-hairpins contain- strategy. The tert-butyloxycarbonyl (Boc) group was used to
ing conformationally constrained DPro-Xxx segments have not protect the N-terminus. Deprotections were performed using
been successful. Designed β-hairpins constructed with predo- 98% formic acid and saponification for the N- and C-terminal
minantly apolar amino acids display high solubility in organic protection groups, respectively. Couplings were mediated by
solvents and do not show a pronounced tendency to aggregate. N,N′-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole
The crystallographic studies on model β-hairpin peptide (HOBt). All the intermediates were characterized by 1H NMR
sequences and understanding the factors responsible for the (80 MHz) and TLC (silica gel, chloroform–methanol 9 : 1), and
growth of crystals and deciphering the interactions responsible were used without further purification. The final peptides were
for certain types of packing arrangements provides a first step purified by reverse phase, medium-pressure liquid chromato-
in the design and construction of model polypeptide graphy (C18) and high performance liquid chromatography
sequences that can fold into completely β-sheet structures. In (HPLC) on a reverse phase C18 column (5–10 μ, 7.8 × 250 mm)
addition, these motifs can also serve as templates to generate using methanol–water gradients. The purified peptides were
1
novel folds with a mixture of α-helical and β-hairpin frag- characterized by electrospray or MALDI and 500 MHz H NMR
ments.27 The observed packing modes for the β-strand seg- spectra (ESI Fig. S9 and S10†) (peptide 1: MNaobs = 1067,
+
+
ments may be of relevance in modeling multiple modes of Mcalc = 1044; peptide 2: MNaobs = 1083, Mcalc = 1060; peptide
association that may provide insights into the structure of 3: MNaobs+ = 971; Mcalc = 948; peptide 4: MNaobs+ = 1055, Mcalc
+
insoluble polypeptide aggregates. A detailed understanding of = 1032; peptide 5: MNaobs = 1095, Mcalc = 1072; peptide 6:
the nucleation and growth of crystals of β-hairpin peptides will MNaobs+ = 1041.7, Mcalc = 1018.5).
undoubtedly benefit from an analysis of the modes of peptide
association in solution. While considerable efforts have been
X-ray diffraction
expended on conformational analysis in solution, peptide Single crystals suitable for X-ray diffraction were obtained by the
association in this class of molecules remains to be slow evaporation of solutions of the peptides in a range of
investigated.
solvent conditions. Colorless single crystals for the peptides 1–3
Table 2 Crystal and diffraction parameters for the peptides 1–3
1
2a
2b
3
Empirical formula
Crystal habit
Crystal size [mm]
Crystallizing solvent
Space group
C
Plates
0.80 × 0.12 × 0.01
Methanol–water
P21
56H84N8O11.5
C56H83N8O13.5
Plates
0.40 × 0.20 × 0.04
Methanol–water
P212121
C56H83N8O13.5
Plates
0.49 × 0.09 × 0.05
Methanol–water
P21
C48H84N8O11
Plates
0.49 × 0.09 × 0.05
Methanol–water
P1
a [Å]
b [Å]
c [Å]
14.403 (8)
18.962 (1)
25.490 (1)
105.67 (4)
6702.8 (7)
4/2
19.086 (4)
26.216 (5)
28.015 (6)
14.318 (8)
18.992 (9)
25.157 (1)
105.59 (4)
6589.5 (6)
4/2
9.922 (3)
11.229 (4)
26.423 (9)
87.15 (6), 89.44 (6), 73.28 (7)
2816.1 (1)
2/2
α, β, γ [°]
Volume [Å3]
14 018.0 (5)
8/2
Z/Z′
Co-crystallized solvent
Molecular weight
Density [g cm−3] [calc.]
F (000)
One water molecule
1053.31
1.044
2272
Cu Kα
Three water molecules
Three water molecules
1084.30
1.093
2332
Cu Kα
None
949.23
1.119
1032
Mo Kα
21
1084.81
1.028
4668
Mo Kα
21
Radiation
Temperature [°C]
21
21
θ Range [ο]
3.60–71.83
ω + ϕ
1.55–20.18
ω
1.82–71.74
ω + ϕ
1.54–23.26
ω
Scan type
Measured reflections
Unique reflections
Observed reflections [|F| > 4σ(F)]
Final R [%]
33 680
12 699
6142
9.59
22.51
0.925
0.28/−0.21
4/1349
14 663
7212
2093
34 052
12 393
4214
12.49
29.29
1.075
0.37/−0.27
4/1397
27 419
8021
2366
10.58
23.54
0.932
0.37/−0.27
9/1202
9.89
Final wR2 [%]
Goodness-of-fit (S)
20.64
1.032
0.32/−0.23
9/1351
Δρmax/Δρmin [e Å−3
]
Restraints/parameters
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Organic & Biomolecular Chemistry
Table 3 Crystal and diffraction parameters for the peptides 4–6
4a
4b
5
6
Empirical formula
Crystal habit
Crystal size [mm]
Crystallizing solvent
Space group
a [Å]
C
Plates
57H88N8O12.5
C55H84N8O11.5
Plates
0.40 × 0.20 × 0.04
Dioxane–water
P21
18.095 (4)
23.032 (5)
18.637 (5)
117.47 (2)
6891.2 (3)
4/2
C58H88N8O11
Plates
0.54 × 0.16 × 0.01
Methanol–water
P212121
18.880(3)
24.579(4)
C54H78N8O12
Needles
0.50 × 0.02 × 0.01
DMF–water
P212121
18.7511 (9)
23.3396 (11)
28.1926 (13)
0.80 × 0.48 × 0.10
Dioxane–water
P21
18.410 (2)
23.220 (3)
19.240 (3)
118.06 (1)
7260.0 (1)
4/2
b [Å]
c [Å]
28.780(5)
α, β, γ [°]
Volume [Å3]
13 355(4)
8/2
12 338.3 (10)
8/2
Z/Z′
Co-crystallized solvent
Molecular weight
Density [g cm−3] [calc.]
F (000)
One dioxane and one water molecule One water molecule Three water molecules Two water molecules
1085.35
0.993
2344
Mo Kα
21
1041.30
1.004
2248
Cu Kα
21
1099
1.092
4736
MoKα
21
1031.24
1.110
4432
Cu Kα
21
Radiation
Temperature [°C]
θ Range [ο]
1.20–27.35
ω
4.64–71.82
ω + ϕ
1.1–20.8
ω
2.46–72.27
ω + ϕ
Scan type
Measured reflections
Unique reflections
Observed reflections [|F| > 4σ(F)] 2772
69 713
13 869
40 004
12 050
4518
88 591
7648
5737
73 135
12 534
3798
Final R [%]
Final wR2 [%]
10.12
20.61
0.865
0.14/−0.14
11/1349
9.15
20.96
0.985
0.40/−0.15
7/1343
12.49
22.62
0.961
0.29/−0.25
0/1424
12.08
28.98
1.041
0.36/−0.33
0/1334
Goodness-of-fit (S)
Δρmax/Δρmin [e Å−3
]
Restraints/parameters
and 5 were grown from methanol–water, 4 from dioxane–water, summarized in Tables 2 and 3. The structures of all the octapep-
and 6 from DMF–water, respectively. Initially, all the X-ray dif- tide sequences 1–6 were solved by direct methods using
fraction data on the crystals of peptides 1–6 were collected on a SHELXD,28 which combines ‘peak list optimization’ with the
Bruker AXS SMART APEX CCD diffractometer (equipped with ‘minimal function’ involving dual space recycling. All the struc-
sealed tube Mo Kα source, λ = 0.71073 Å). All the crystals were tures were observed to have two molecules in the asymmetric
preserved. With the availability of a rotating-anode X-ray single unit. The structures were refined against F2, with full-matrix
crystal diffractometer equipped with intense CuKα (λ
=
least squares methods by using SHELXL-9728 for peptides 1, 2b,
1.54178 Å) radiation at the Indian Institute of Science, it was felt 4a, 4b, 5 and 6, and the two molecules were treated as two separ-
necessary to improve the data quality of all the crystals by a ate blocks in the case of peptides 2a and 3. The final values after
recollection of the diffraction data on a Bruker AXS ULTRA the refinement of crystal structures are shown in Tables 1 and 2.
APEXII CCD diffractometer (rotating anode X-ray generator). It CCDC 821276 (1), 821280 (2a), 821281 (2b), 821277 (3), 821278
was found that only a few of the crystals retained crystallinity, as (4a), 821279 (4b), 821275 (5) and 821274 (6) contain the sup-
the time gap between the data recollection was approximately 2 plementary crystallographic data for this paper.
years. The crystal of 2a was decaying and had started growing
whitish in color. The crystal did not diffract in the X-ray beam.
The preserved crystals were checked for any crystal showing
signs of crystallinity. It was observed that a few of the crystals
Acknowledgements
retained signs of crystallinity under the polarizing microscope; This research was supported by a grant from the Council of
the rest were no longer transparent. One of the crystals was Scientific and Industrial Research (CSIR) and a program grant
picked to recollect the X-ray diffraction data. Surprisingly, the from the Department of Biotechnology, India, in the area of
crystal could be unequivocally indexed in a cell corresponding to Molecular Diversity and Design, India. S.A. and V.V.H. thank
the monoclinic space group (P21), in contrast to the orthorhom- the CSIR for a Research Associateship and a Senior Research
bic cell parameters in 2a. It was clear that this was a new crystal Fellowship respectively. U.S.R. thanks the University Grants
form (2b) and the cell dimensions of 2b corresponded closely to Commission for a Senior Research Fellowship.
peptide 1. Diffraction data was also collected on a new crystal of
peptide 4a; the earlier crystal had lost crystallinity and did not
diffract in the X-ray beam. It was observed that the cell dimen-
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