Hydrophobic peptide channels and encapsulated water wires

Article abstract of DOI:10.1021/ja9083978

Peptide nanotubes with filled and empty pores and close-packed structures are formed in closely related pentapeptides. Enantiomorphic sequences, Boc- ^DPro-Aib-Xxx-Aib-Val-OMe (Xxx = Leu, 1; Val, 2; Ala, 3; Phe, 4) and Boc-Pro-Aib-^DXxx-Aib-^DVal-OMe (^DXxx = ^DLeu, 5; ^DVal, 6; ^DAla, 7; ^DPhe, 8), yield molecular structures with a very similar backbone conformation but varied packing patterns in crystals. Peptides 1, 2, 5, and 6 show tubular structures with the molecules self-assembling along the crystallographic six-fold axis (c-axis) and revealing a honeycomb arrangement laterally (ab plane). Two forms of entrapped water wires have been characterized in 2: 2a with d _O...O = 2.6 A and 2b with d_O...O = 3.5 A. The latter is observed in 6 (6a) also. A polymorphic form of 6 (6b), grown from a solution of methanol-water, was observed to crystallize in a monoclinic system as a close-packed structure. Single-file water wire arrangements encapsulated inside hydrophobic channels formed by peptide nanotubes could be established by modeling the published structures in the cases of a cyclic peptide and a dipeptide. In all the entrapped water wires, each water molecule is involved in a hydrogen bond with a previous and succeeding water molecule. The O-H group of the water not involved in any hydrogen bond does not seem to be involved in an energetically significant interaction with the nanotube interior, a general feature of the one-dimensional water wires encapsulated in hydrophobic environements. Water wires in hydrophobic channels are contrasted with the single-file arrangements in amphipathic channels formed by aquaporins.

Full text of DOI:10.1021/ja9083978

Published on Web 12/31/2009
Hydrophobic Peptide Channels and Encapsulated Water Wires
Upadhyayula S. Raghavender,† Kantharaju,‡ Subrayashastry Aravinda,†
Narayanaswamy Shamala,*,† and Padmanabhan Balaram*,‡
Department of Physics and Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560012, India
Received October 2, 2009; E-mail: shamala@physics.iisc.ernet.in; pb@mbu.iisc.ernet.in
Abstract: Peptide nanotubes with filled and empty pores and close-packed structures are formed in closely
related pentapeptides. Enantiomorphic sequences, Boc-DPro-Aib-Xxx-Aib-Val-OMe (Xxx ) Leu, 1; Val, 2;
Ala, 3; Phe, 4) and Boc-Pro-Aib-DXxx-Aib-DVal-OMe (DXxx ) DLeu, 5; DVal, 6; DAla, 7; DPhe, 8), yield
molecular structures with a very similar backbone conformation but varied packing patterns in crystals.
Peptides 1, 2, 5, and 6 show tubular structures with the molecules self-assembling along the crystallographic
six-fold axis (c-axis) and revealing a honeycomb arrangement laterally (ab plane). Two forms of entrapped
water wires have been characterized in 2: 2a with dO· · ·O ) 2.6 Å and 2b with dO· · ·O ) 3.5 Å. The latter is
observed in 6 (6a) also. A polymorphic form of 6 (6b), grown from a solution of methanol-water, was
observed to crystallize in a monoclinic system as a close-packed structure. Single-file water wire
arrangements encapsulated inside hydrophobic channels formed by peptide nanotubes could be established
by modeling the published structures in the cases of a cyclic peptide and a dipeptide. In all the entrapped
water wires, each water molecule is involved in a hydrogen bond with a previous and succeeding water
molecule. The O-H group of the water not involved in any hydrogen bond does not seem to be involved
in an energetically significant interaction with the nanotube interior, a general feature of the one-dimensional
water wires encapsulated in hydrophobic environements. Water wires in hydrophobic channels are contrasted
with the single-file arrangements in amphipathic channels formed by aquaporins.
tubular structures in the solid state.4 The recent interest in crystal
Introduction
structures that contain pores with molecular dimensions of 8-10
Tubular structures are formed from both cyclic and acyclic
peptides in the solid state. The original observation of a hybrid
cyclic peptide stacking into a cylindrical structure by Karle et
al.1 was followed nearly two decades later by the elegant work
of Ghadiri and co-workers on cyclic peptides, demonstrating
nanotube formation in a cyclic sequence, cyclo-[DAla-Glu-DAla-
Gln2-], containing amino acid residues with alternating chirality.2
Subsequent work established the ability of analogous sequences
to form tubular structures.3 More recently, Gorbitz has developed
a class of dipeptides with hydrophobic side chains which form
Å stems from their ability to serve as mimics for biological
structures that are involved in transmembrane transport of small
molecules and ions. Porous organic structures have also attracted
considerable attention because of their potential in the areas of
separation science, adsorption, and catalysis.5 The selectivity
of pores in organic solid-state structures is defined by the inner
dimensions of the tube and the chemical nature of the groups
that line the interior of the pore. As demonstrated by the work
of the groups of Ghadiri and Gorbitz, peptides provide an
excellent scaffold for the creation of tubular structures.
In the course of studies aimed at delineating the structure-
directing properties of DPro residues, we serendipitously en-
countered the formation of a tubular structure in the pentapeptide
Boc-DPro-Aib-Leu-Aib-Val-OMe (1).6 The peptide adopts a
folded conformation stabilized by three intramolecular hydrogen
bonds. The DPro(1)-Aib(2) segment forms a Type II′ ꢀ-turn,
while the Aib(2)-Aib(4) segment folds into a short stretch of a
† Department of Physics.
‡ Molecular Biophysics Unit.
(1) Karle, I. L.; Handa, B. K.; Hassall, C. H. Acta Crystallogr., Sect. B
1975, 31, 555–560.
(2) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.;
Khazanovich, N. Nature 1993, 366, 324–327.
(3) (a) Ghadiri, M. R.; Granja, J. R.; Buehler, L. K. Nature 1994, 369,
301–304. (b) Khazanovich, N.; Granja, J. R.; McRee, D. E.; Milligan,
R. A.; Ghadiri, M. R. J. Am. Chem. Soc. 1994, 116, 6011–6012. (c)
Ghadiri, M. R.; Kobayashi, K.; Granja, J. R.; Chadha, R. K.; McRee,
D. E. Angew. Chem., Int. Ed. Engl. 1995, 34, 93–95. (d) Engels, M.;
Bashford, D.; Ghadiri, M. R. J. Am. Chem. Soc. 1995, 117, 9151–
9158. (e) Clark, T. D.; Buehler, L. K.; Ghadiri, M. R. J. Am. Chem.
Soc. 1998, 120, 651–656. (f) Bong, D. T.; Clark, T. D.; Granja, J. R.;
Reza Ghadiri, M. Angew. Chem., Int. Ed. 2001, 40, 988–1011. (g)
Bong, D. T.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 2163–
2166. (h) Fernandez-Lopez, S.; Kim, H.-S.; Choi, E. C.; Delgado, M.;
Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger,
D. A.; Wilcoxen, K. M.; Ghadiri, M. R. Nature 2001, 412, 452–455.
(i) Sanchez-Quesada, J.; Isler, M. P.; Ghadiri, M. R. J. Am. Chem.
Soc. 2002, 124, 10004–10005. (j) Horne, W. S.; Stout, C. D.; Ghadiri,
M. R. J. Am. Chem. Soc. 2003, 125, 9372–9376.
(4) (a) Gorbitz, C. H.; Gundersen, E. Acta Crystallogr., Sect. C 1996, 52,
1764–1767. (b) Gorbitz, C. H. Chem.sEur. J. 2001, 7, 5153–5159.
(c) Gorbitz, C. H. New J. Chem. 2003, 27, 1789–1793. (d) Gorbitz,
C. H. Chem.sEur. J. 2007, 13, 1022–1031. (e) Gorbitz, C. H.; Rise,
F. J. Pept. Sci. 2008, 14, 210–216.
(5) (a) Kitagawa, S.; Kitaura, R.; Noro, S.-I. Angew. Chem., Int. Ed. 2004,
43, 2334–2375. (b) Scanlon, S.; Aggeli, A. Nano Today 2008, 3, 22–
30. (c) Bonifazi, D.; Mohnani, S.; Llanes-Pallas, A. Chem.sEur. J.
2009, 15, 7004–7025. (d) Yang, Z.; Lu, Y.; Yang, Z. Chem. Commun.
2009, 17, 2270–2277.
(6) Raghavender, U. S.; Aravinda, S.; Shamala, N.; Kantharaju; Rai, R. K.;
Balaram, P. J. Am. Chem. Soc. 2009, 131, 15130–15132.
9
10.1021/ja9083978  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 1075–1086 1075

A R T I C L E S
Raghavender et al.
Figure 1. Crystal lattice (honeycomb pattern) of linear, acyclic, hydrophobic pentapeptides 1 (left, Xxx ) Leu) and 2a (right, Xxx ) Val), revealing a
two-dimensional array of nanotubes (projection down c-axis). (a) Peptide 1 shows hollow tubes (diameter ∼5.2 Å). (b) Peptide 2a shows water-filled channels
(diameter ∼7.5 Å).
right-handed 310 helical structure. The molecule is then left with
only one free amide NH group of Aib(2). In crystals, this lone
NH group forms an intermolecular hydrogen bond to a peptide
carbonyl, CO of Leu(3). The formation of crystals in the space
group P65 reveals a helical array of peptides about the c-axis
linked by a single hydrogen bond to their neighbors. This
arrangement results in the formation of a hollow tube for the
column of molecules arranged about the crystallographic six-
fold axis (c-axis). Further assembly of these tubular structures
results in a crystal lattice that contains a two-dimensional array
of peptide nanotubes (Figure 1). The inner diameter of the pore,
which is unoccupied, is ∼5.2 Å. The inner lining is entirely
hydrophobic, a property of the sequence, with the isobutyl
groups of Leu(3) pointing into the pore. Modeling studies
suggest that even water molecules cannot be accommodated in
such a pore, as a result of severe short contacts with the atoms
lining the inner walls. We therefore turned to a peptide with a
shorter side chain at position 3, Boc-DPro-Aib-Val-Aib-Val-
OMe (2), which also crystallized in the space group P65.
Structure determination revealed a pore diameter of ∼7.5 Å,
with a one-dimensional wire of water molecules occupying the
hollow tube (Figure 1). The crystal structure of 2 was determined
for two crystal forms which differed only in the structures of
water wires. Peptide 2 provides a well-characterized example
of a one-dimensional array of water molecules connected by
hydrogen bonds, with no significant stabilizing contacts with
the surrounding walls of the encapsulating molecular tube.6 In
order to examine the role of residue 3 in generating peptide
nanotubes, we synthesized the analogues Boc-DPro-Aib-Xxx-
Aib-Val-OMe (Xxx ) Ala, 3; Phe, 4). As part of a program to
study the packing effects in peptide racemates, we examined
the enantiomeric sequences, Boc-Pro-Aib-DXxx-Aib-DVal-OMe
(DXxx ) DLeu, 5; DVal, 6; DAla, 7; DPhe, 8). Peptides 5-8
also provided an opportunity to examine the crystal structures
of the enantiomers of peptides 1-4. In a previous study of
diproline peptides, we have reported the utility of attempting
to crystallize peptides and their enantiomers under a variety of
conditions which facilitate the search for polymorphic forms.7
In this report, we describe the structures of peptides 1-8.
Peptide 6, Boc-Pro-Aib-DVal-Aib-DVal-OMe, provides an ex-
ample of a situation where two polymorphic forms are char-
acterized with one revealing a tubular structure while the other
established a close-packed arrangement. The characterization
of water wires in the crystal forms of 2 and 6 has also prompted
us to examine models for water molecules in hydrophobic tubes
formed by the cyclic peptides of Ghadiri and co-workers and
the dipeptides of Gorbitz.3d,4c The results establish that hydro-
phobic tubes constructed from peptide scaffolds can provide a
sequestering environment for one-dimensional water wires,
permitting detailed structural characterization.
Single-file water wires that traverse hydrophobic channels
are of special interest in building models for understanding
proton and water translocation across biological membranes.
The structural properties of ordered water arrays assume
increasing significance in view of the growing recognition of
the role of water in confined spaces which are routinely
encountered in cellular phenomena.8
Results and Discussion
Molecular Conformation in Crystals. Ten independent crystal
structures were determined for the peptide series Boc-DPro-Aib-
Xxx-Aib-Val-OMe (1-4) and the enantiomeric series Boc-Pro-
Aib-DXxx-Aib-DVal-OMe (5-8). For peptides 2 (Xxx ) LVal)
and 6 (Xxx ) DVal), two independent crystal forms were
characterized. Forms 2a and 2b were very similar, differing only
in the nature of the water incorporated into the pores formed in
the hexagonal space group P65. In contrast, for peptide 6, two
distinct polymorphic forms were obtained. In 6a, molecules pack
into a hexagonal space group (P61) with a central pore, while
6b is a close-packed form (space group C2). Relevant crystal
and diffraction parameters are listed in Table 1. Figure 2 shows
the molecular conformation for all the ten cases. Backbone
dihedral angles are summarized in Table 2. In all cases, the
peptide molecules adopt a folded conformation stabilized by
three intramolecular 4f1 hydrogen bonds: Xxx(3) NH · · ·OC,
Boc(0) and Aib(4) NH · · ·OC, DPro(1) and Val(5) NH · · ·OC
for 1-4; DXxx(3) NH · · ·OC, Boc(0) and Aib(4) NH · · ·OC, and
Pro(1) and DVal(5) NH · · ·OC DXxx(3) for 5-8 (see Table 2
footnotes and Supporting Information Tables S5 and S6 for
details of hydrogen bond parameters). Figure 3 illustrates the
superposition for the four structures in each enantiomeric set
and also provides the Ramachandran plot for both sets of
peptides. The backbone folding pattern for the pentapeptides
may be classified as a Type II′ ꢀ-turn followed by a single right-
handed 310 helical turn (1-4) and a Type II ꢀ-turn followed by
a left-handed 310 helical turn (5-8). These crystallographic
(7) (a) Chatterjee, B.; Saha, I.; Raghothama, S.; Aravinda, S.; Rai, R.;
Shamala, N.; Balaram, P. Chem.sEur. J. 2008, 14, 6192–6204. (b)
Saha, I.; Chatterjee, B.; Shamala, N.; Balaram, P. Biopolymers (Peptide
Sci.) 2008, 90, 537–543.
(8) Ball, P. Chem. ReV. 2008, 108, 74–108.
9
1076 J. AM. CHEM. SOC. VOL. 132, NO. 3, 2010

Water Wires in Hydrophobic Peptide Channels
A R T I C L E S
Table 1. Crystal Parameters, Unit Cell Dimensions, and Final R-Factors for Peptides 1-8
Boc-DPro-Aib-Xxx-Aib-Val-OMe
1
2a
2b
3
4
Xxx
Leu
Val
Val
Ala
Phe
empirical formula
crystal size (mm3)
crystallizing solvent
space group
a (Å)
C30H53N5O8
0.4 × 0.21 × 0.03
methanol/water
P65
24.3673(9)
24.3673(9)
10.6844(13)
5494.09(7)
6.37
C29H51N5O8
0.45 × 0.12 × 0.07
ethyl acetate/pet. ether
P65
24.2920(13)
24.2920(13)
10.4830(11)
5357.67(7)
5.5
C29H51N5O8
0.45 × 0.12 × 0.07
ethyl acetate/pet. ether
P65
24.3161(3)
24.3161(3)
10.4805(1)
5366.62(1)
6.2
C27H47N5O8
0.45 × 0.29 × 0.20
methanol/water
P212121
12.2403(8)
15.7531(11)
16.6894(11)
3218.09(4)
4.4
C33H48N5O8
0.60 × 0.38 × 0.39
acetonitrile/water
P212121
10.3268(8)
18.7549(15)
18.9682(16)
3673.73(5)
4.7
b (Å)
c (Å)
V (Å3)
final R (%)
final wR2 (%)
14.5
15.5
18.4
12.5
13.2
Boc-Pro-Aib-DXxx-Aib-DVal-OMe
5
6a
6b
7
8
DXxx
DLeu
DVal
DVal
DAla
DPhe
empirical formula
crystal size
crystallizing solvent
space group
a (Å)
C30H53N5O8
0.6 × 0.4 × 0.16
dioxane/water
P61
24.4102(8)
24.4102(8)
10.6627(7)
C29H51N5O8
C29H51N5O8
0.75 × 0.01 × 0.19
methanol/water
C2
20.7278(35)
9.1079(15)
19.5728(33)
94.207(3)
3685.1(11)
6.6
C27H47N5O8
0.34 × 0.18 × 0.14
methanol/water
P212121
12.2528(12)
15.7498(16)
16.6866(16)
C33H48N5O8
0.51 × 0.29 × 0.42
dioxane/water
P212121
10.3354(5)
18.7733(10)
18.9820(10)
0.56 × 0.10 × 0.04
ethyl acetate/pet. ether
P61
24.3645(14)
24.3645(14)
b (Å)
c (Å)
10.4875(14)
ꢀ (°)
V (Å3)
5502.3(4)
5.4
14.9
5391.60(8)
7.5
18.1
3220.2(5)
4.7
12.8
3683.1(3)
5.1
15.3
final R (%)
final wR2 (%)
17.6
observations establish the robustness of the backbone conforma-
tion adopted by the pentapeptide sequences, in which structural
rigidity is conferred by the N-terminus DPro/LPro-Aib segment
and Aib residue at position 4. The role of the residue at position
3 in determining packing in crystals can therefore be investigated.
Packing in Crystals. Peptide enantiomers 1 and 5 crystallize
in the space groups P65 and P61, respectively, as illustrated in
Figure 4. Neighboring molecules are linked by a single
intermolecular hydrogen bond (Aib(2)NH · · ·OC Val(3)), form-
ing a superhelix about the six-fold axis, resulting in a central
hollow tube, decorated with the methyl groups of the Boc
protecting group and the leucine side chain. The tubular peptide
structures assemble into a two-dimensional array. The central
pore has a diameter of ∼5.2 Å, estimated by the procedure
and there is no co-crystallized solvent. Symmetry-related peptide
molecules also show evidence of a significant aromatic-aromatic
interaction between projecting Phe residues (Figure 6).10
The 10 crystal structures determined for peptides 1-8
establish that peptide nanotube formation is critically determined
by the size of the side chain at position 3. Tubular structures
could be demonstrated when Xxx ) Leu/Val in both the
enantiomeric series. Packing into hexagonal space groups P61
and P65 permits the formation of a hydrophobic channel which
runs through the crystal along the six-fold axis, with the side-
chain atoms of residue 3 projecting inward. The solvent
occupancy of the channel depends on the steric bulk of the side
chains. In the case of Leu residues, the size of the isobutyl group
reduced the diameter of the channel to ∼5.2 Å, precluding the
entry of even water molecules. Despite the presence of empty
pores which run through the lattice, this packing arrangement
appears to be stable in crystals, being observed in both peptides
1 and 5. In contrast, peptides 2 and 6, which possess the smaller
valine side chain, show evidence for both solvent-filled hydro-
phobic channels and close-packed polymorphs, which lack the
pores running through the crystal. The observation suggests that
the presence of hydrophobic channels with diameter significantly
greater than 5.5 Å may be unstable unless occupied by solvent.
The formation of both porous and close-packed polymorphs
should then be determined by the kinetics of nucleation of the
crystal. If a very small side chain is placed at position 3, as in
9
D
proposed by Miyazawa. Peptide 6 (Xxx ) Val) crystallizes
in two distinct polymorphic forms. Form 6a, illustrated in Figure
5, displays a packing arrangement almost identical to that
observed in 1, 2, and 5. The shortening of the side chain at
position 3 results in expanding the diameter of the tubular
peptide structures to ∼7.6 Å. As in the case of the enantiomeric
peptide 2, water molecules are identifiable in the hydrophobic
channel forming a one-dimensional water wire, which will be
considered later. Interestingly, in the polymorphic form 6b,
peptide molecules crystallize in a close-packed arrangement in
the monoclinic space group C2, with two water molecules (full
occupancy), which form bridges between symmetry-related
molecules. A further reduction of the size of the residue at
position 3 in peptides 3 (Xxx ) Ala) and 7 (DXxx ) DAla)
results in close-packed structures in the space group P212121,
with no solvent molecules (Figure 5). The introduction of a
bulky aromatic side chain at position 3 in peptides 4 (Xxx )
D
the case of peptides 3 (Xxx ) Ala) and 7 (DXxx ) Ala), the
anticipated pore size should be ∼10 Å. Formation of such a
large diameter channel would tend to be energetically unfavor-
able when compared to the observed close-packed structure.
As anticipated, the bulky side chain at position 3 precludes the
D
Phe) and 8 (DXxx ) Phe) results in the formation of crystals
in the space group P212121. Here the molecules are close-packed
(9) Miyazawa, T. J. Polym. Sci. 1961, 55, 215–231.
(10) Aravinda, S.; Shamala, N.; Das, C.; Sriranjini, A.; Karle, I. L.; Balaram,
P. J. Am. Chem. Soc. 2003, 125, 5308–5315.
9
J. AM. CHEM. SOC. VOL. 132, NO. 3, 2010 1077

A R T I C L E S
Raghavender et al.
Figure 2. Molecular conformation and the asymmetric unit of the peptides (a) Boc-DPro-Aib-Xxx-Aib-Val-OMe (Xxx ) Leu, 1; Val, 2a/2b; Ala, 3; Phe,
4) and (b) Boc-Pro-Aib-DXxx-Aib-DVal-OMe (DXxx ) DLeu, 5; DVal, 6a/6b; DAla, 7; DPhe, 8). Xxx residues are shown as spheres (green). Intramolecular
4f1 hydrogen bonds are shown as dashed lines.
Table 2. Backbone Torsion Angles for Peptides 1-8a
Boc-DPro-Aib-Xxx-Aib-Val-OMe
1
2a
2b
Xxx ) Val
3
4
Xxx ) Leu
Xxx ) Val
Xxx ) Ala
Xxx ) Phe
φ
ψ
ω
φ
ψ
ω
φ
ψ
ω
φ
ψ
ω
φ
ψ
ω
DPro(1)
51.6 -134.1 -174.1
52.9 -136.6 -174.0
-24.2 -177.9 -59.3 -24.7 -177.8 -59.6
-1.1 165.7 -73.6 -1.2 162.6 -73.9
53.9 -137.0 -174.2
55.1 -146.7 -175.4
61.4 -139.8 -176.7
Aib(2) -57.9
Xxx(3) -72.7
Aib(4) -57.3
-24.2 -178.0 -61.8
-20.8 -178.6 -54.6 -35.5 -170.6
-1.2
162.4 -68.7
-8.8
167.2 -70.8
-17.9
-16.9
-42.4
178.4
178.4
179.6
-29.3 -177.3 -55.5
-32.0 -175.6 -55.4
-31.6 -175.7 -54.4
-31.0 -177.5 -71.2
Val(5)
-90.1
133.3
176.5 -85.3
130.2
175.7 -85.7
130.3
175.8 -93.0
135.0
178.9 -68.2
Boc-Pro-Aib-DXxx-Aib-DVal-OMe
5
6a
6b
DXxx ) DVal
7
8
DXxx ) DLeu
DXxx ) DVal
DXxx ) DAla
DXxx ) DPhe
φ
ψ
ω
φ
ψ
ω
φ
ψ
ω
φ
ψ
ω
φ
ψ
ω
Pro(1)
-50.5
58.6
73.2
133.5
22.9
0.4 -165.4
31.8 176.1
173.9 -52.5
136.5
24.9
1.2 -163.0
32.6 175.5
174.1 -63.3 151.8
170.5 -55.1
146.7
20.7
8.5 -167.3
30.7 177.6
175.2 -61.4 139.8
176.4
170.6
Aib(2)
DXxx(3)
Aib(4)
DVal(5)
179.2
58.9
73.5
55.2
177.5
47.6
73.7
63.3
91.0
46.5
168.9
61.8
68.9
54.7
178.8
54.8
71.0
71.6
68.7
35.3
7.2 -169.7
17.4 -178.2
56.7
18.9
178.6
16.5 -178.3
87.5 -132.9 -175.8
84.5 -130.5 -176.4
48.7 -167.6
93.1 -134.8 -179.0
43.3
177.7
a Mean values for hydrogen bond parameters for peptides 1-4: intramolecular hydrogen bonds, N · · ·O ) 3.111 Å, H · · ·O ) 2.314 Å, ∠N-H· · ·O )
157.35°; intermolecular hydrogen bonds, N · · ·O ) 2.942 Å, H · · ·O ) 2.094 Å, ∠N-H· · ·O ) 167.54°. Mean values for hydrogen bond parameters for
peptides 5-8: intramolecular hydrogen bonds, N · · ·O ) 3.079 Å, H · · ·O ) 2.283 Å, ∠N-H· · ·O ) 157.91°; intermolecular hydrogen bonds, N · · ·O )
2.976 Å, H · · ·O ) 2.134 Å, ∠O ) 166.53°.
9
1078 J. AM. CHEM. SOC. VOL. 132, NO. 3, 2010

Water Wires in Hydrophobic Peptide Channels
A R T I C L E S
Figure 3. (a) Superposition of the backbone atoms (N, CR, C, O) and ꢀ-carbon of Xxx residues in the pentapeptides 1-4 (rmsd ) 0.18 Å) and 5-8 (rmsd
) 0.25 Å). (b) Ramachandran plots indicating the backbone torsion angles adopted by the peptides 1-4 and 5-8.
formation of a tubular structure, as demonstrated for peptides
temperature for several weeks. The c-axis dimension (∼10.48
Å) in the hexagonal unit cell results in four water molecules in
the unit cell (six peptides) for 2a, while in 2b there are three
water molecules. In the derived water wire models each water
makes two hydrogen bond contacts, one donor and one acceptor,
to near neighbors (water molecules). The second O-H bond
of the water molecule points toward the hydrophobic wall of
the channel. The range of H · · ·H contacts between the water
molecule and the channel walls is 2.9-4.9 Å, suggesting that
the interactions between the embedded water wire and the
surrounding hydrophobic tube must be extremely weak. Water
molecules can execute a rotational motion about the six-fold
axis (hydrogen bond direction), contributing entropically to the
net free energy of stabilization of the observed structure.6 The
crystal structure of peptide 6a (DXxx ) DVal) and the geometry
of the water wire are identical to those observed in 2b (Figure
7a).
D
4 (Xxx ) Phe) and 8 (DXxx ) Phe).
Water Wires in Hydrophobic Channels. In the pentapeptide
series, Boc-DPro-Aib-Xxx-Aib-Val-OMe, and the enantiomeric
series, Boc-Pro-Aib-DXxx-Aib-DVal-OMe, the independent crys-
tal structure determinations of peptides 2a/2b (Xxx ) Val) and
6a (DXxx ) DVal) provide a direct observation of one-
dimensional arrays of water molecules. In all three cases, the
tubular channel running through the crystal is occupied by water
molecules. Refinements of the crystal structures lead to partial
occupancies of oxygen atoms at sites along the six-fold axis.
Inspection of O · · ·O distances permits the building of a water
wire model which is stereochemically acceptable, with no short
O· · ·O contacts. In 2a, water molecules arranged along the six-
fold axis are separated by an O · · ·O distance of 2.6 Å, while in
2b the O · · ·O separation is 3.5 Å. The crystal structure of 2b
was obtained after the crystal of 2a was stored at room
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A R T I C L E S
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adjacent water molecules is now ∼3.2 Å, and each water
molecule is involved in two hydrogen bond interactions (one
donor/one acceptor) with near neighbors. The resulting water
wire structure is almost identical to that described above for
peptides 2a, 2b, and 6a. Re-examination of the electron density
for the entrapped water molecules may provide some further
insights.
Dipeptides with free amino and carboxylic acid groups
have been shown by Gorbitz to crystallize in hexagonal space
groups, incorporating tubular channels which run through the
crystal.4 The dipeptide Val-Ile yielded crystals in the space
group P61 with one molecule in the asymmetric unit. Crystal
structure solution using low-temperature data (T ) 105 K)
revealed a co-crystallized water molecule, disordered over
two sites, with partial occupancies of 0.24 and 0.14.4c
Interestingly, the distance between the two disordered water
molecules is 0.9 Å, with the c-axis dimension of ∼10.314
Å, corresponding to the repeat spacing about the six-fold axis.
This length (c-axis) corresponds closely to the c-axis dimen-
sion of the pentapeptides 2a, 2b, and 6a.6 A water wire can
therefore be modeled along the c-axis, which satisfies the
steric criteria for adjacent molecules. The resultant model
yields four water molecules in the unit cell (Figure 9).
Inspection of the water environment establishes that the walls
of the nanotube formed by the dipeptides are composed
exclusively of the isopropyl side chains of Val residues. The
distance between the oxygen atoms of the water molecules
and the hydrogen atoms lining the tube interior lies between
2.97 and 3.71 Å. As in the case of 2a, 2b, and 6a, the water
molecules occupy unique environments. The symmetry of
the space group is not manifested in the water wires within
the hydrophobic nanotubes of the peptide crystals. The
resultant peptide stoichiometry would then be six peptide/
four water molecules.
One-dimensional arrays of water molecules (“wires”) are
also involved in the single-file transport of water through
the aquaporins.11 Elegant biochemical and crystallographic
work on aquaporins, which form water-permeable channels
across biological membranes, has revealed an architecture
which may be compared with the hydrophobic peptide
channels described above.11 Figure 10 illustrates the water
wire characterized in the crystal structure of the human
aquaporin 4 (AQP4).11e The O · · · O distances between
hydrogen-bonded pairs are 2.31-2.96 Å, very close to that
observed in peptide 2a. In the case of aquaporins, one
face of the tubular channel is lined by hydrophobic side
chains of Val, Leu, and Ile residues, while the other face is
composed of polar groups which can hydrogen bond to the
water molecules. The distances between the water oxygen
atoms and the apolar methyl groups which are closest to the
waters range 3.65-4.84 Å, very close to those observed in
the hydrophobic peptide nanotubes. The amphipathic nature
of the lining of the APQ4 channel may be of some relevance
in precluding proton transport while facilitating the perme-
Figure 4. Encapsulated water wire in the peptide 6a, Boc-Pro-Aib-DVal-
Aib-DVal-OMe. The peptide backbone is shown as sticks, and the water
molecules are shown as spheres. The lone intermolecular hydrogen bond
between the peptide molecules and the water-water hydrogen bonds are
shown as dashed lines.
The unexpected characterization of the water wires in the
hydrophobic channels formed by the linear, protected, apolar
pentapeptides prompted us to re-examine the nature of the
solvent structure in the previously reported peptide nanotubes.
The work of Ghadiri and co-workers established that cyclic
octapeptides containing alternating L,D residues formed disc-
like structures, which self-assemble to form tubular arrays.3 In
these molecules the peptide bonds are approximately parallel
to the long axis of the nanotube, with the amino acid side chains
projecting outward. Stacking of disc-like peptides is facilitated
by multiple hydrogen-bonding interactions between neighboring
molecules in the peptide cyclo-[(Phe-D,MeNAla)4-], with an
internal tube diameter of ∼10 Å. Ghadiri et al. noted, “The
tubular cavity is filled with partially disordered water mol-
ecules”. They added, “water molecules near the hydrophobic
domains are considerably more disordered, displaying only a
weak residual electron density. The observed electron density
for water is the time-average of water molecules binding at
several overlapping sites, which suggests a facile movement of
loosely held water molecules within the cavity.” 3c In the
reported structure, the peptide crystallized in the space group
I422 with one molecule in the asymmetric unit. The deposited
coordinates for five water molecules lead to the O · · ·O separa-
tions shown in Figure 8a. It may be noted that four water
molecules, Ow1 to Ow4, are arranged in a linear fashion with
O· · ·O separations of 1.58-1.67 Å. A significantly larger
separation of 5.12 Å is observed between Ow4 and Ow5. In
this model, the water molecule Ow5 would appear to be isolated,
lacking either donor or acceptor for hydrogen bond interactions.
The immediate environment of Ow5 reveals a cluster of phenyl
rings from symmetry-related molecules which surround the
water molecule. The observed distances between the oxygen
atom and the nearest atom of the phenyl rings lie between 4.6
and 5.0 Å, suggesting the absence of any appreciable non-
covalent interactions involving this lone water molecule (Figure
8b). This led to the consideration of an alternate model for the
single-file water molecules encapsulated within the hydrophobic
channels formed by the assembly of the cyclic peptide. Insertion
of two additional oxygen atoms between Ow4 and Ow5 leads
to the arrangement shown in Figure 8c. In this model, the O · · ·O
distances between the water molecules are completely disal-
lowed sterically. The stereochemically acceptable distances may
therefore be derived by considering alternate water molecules
along the axis of the hydrophobic channel. This arrangement is
illustrated in Figure 8d. The O · · ·O distances between the
(11) (a) Fujiyoshi, Y.; Mitsuoka, K.; De Groot, B. L.; Philippsen, A.;
Grubmuller, H.; Agre, P.; Engel, A. Curr. Opin. Struct. Biol. 2002,
12, 509–515. (b) Engel, A.; Nielsen, S. J. Physiol. 2002, 542, 3–16.
(c) Nielsen, S.; Frokiaer, J.; Marples, D.; Kwon, T.-H.; Agre, P.;
Knepper, M. A. Physiol. ReV. 2002, 82, 205–244. (d) Khalili-Araghi,
F.; Gumbart, J.; Wen, P. C.; Sotomayor, M.; Tajkhorshid, E.; Schulten,
K. Curr. Opin. Struct. Biol. 2009, 19, 128–137. (e) Ho, J. D.; Yeh,
R.; Sandstrom, A.; Chorny, I.; Harries, W. E. C.; Robbins, R. A.;
Miercke, L. J. W.; Stroud, R. M. Proc. Natl. Acad. Sci. U.S.A. 2009,
106, 7437–7442.
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1080 J. AM. CHEM. SOC. VOL. 132, NO. 3, 2010

Water Wires in Hydrophobic Peptide Channels
A R T I C L E S
Figure 5. Molecular packing of the peptides in crystals: (a) 5 and (b) 6a, viewed down the c-axis; (c) 6b, viewed down the b-axis; and (d) 7, viewed down
the a-axis.
ation of neutral water molecules, a key feature of the
biological function of the protein. It should be noted that
rapid transport of protons by the Grotthuss mechanism would
involve correlated rotation of the water molecule along the
wire, resulting in shifts of the hydrogen-bonded protons.
The area of water and proton transport across biological
membranes has been a subject of considerable interest in recent
years.12 The structure and dynamics of water molecules embed-
ded in hydrophobic channels and carbon nanotubes has been
extensively investigated by theoretical methods.13 The ability
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Raghavender et al.
Figure 6. (a) Molecular packing of the peptide 8, Boc-Pro-Aib-DPhe-Aib-DVal-OMe, viewed down the a-axis. (b) Aromatic-aromatic (DPhe-DPhe) interactions
(see ref 10 for definition of parameters) in the peptide 8: Rcen ) 6.05 Å, Rclo ) 4.15 Å, and γ ) 49.61°. Centroid-centroid distance is shown as dashed line.
Figure 7. Model of a water wire inside the hydrophobic channel of the peptide 6a, Boc-Pro-Aib-DVal-Aib-DVal-OMe. The residues lining the inner wall
of the pore are shown as sticks (DVal in green and Boc protecting group in gray). Crystallographically (space group P61) generated water molecules are
shown as spheres. The hydrogen bonds between the water molecules are shown as dashed lines (magenta). Non-covalent weak interactions of the water
oxygens with the peptide heavy atoms are also indicated as dots (orange). Distances marked are in angstroms.
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Water Wires in Hydrophobic Peptide Channels
A R T I C L E S
Figure 8. Cyclic peptide cyclo-[(Phe-D,MeNAla)4-](from ref 3c, redrawn using coordinates obtained from Cambridge Structure Database22). (a) Asymmetric
unit as in the crystal structure, with five disordered water positions shown. (b) Environment of the water oxygen Ow5, revealing the zero-coordination. (c)
Proposed structure with two additional oxygen atoms between Ow4 and Ow5. (d) Proposed water wire model inside the peptide. Distances between atoms
are shown as dashed lines (magenta, values given in angstroms).
of single-file water wires to transport protons across narrow
hydrophobic channels was first investigated by the multistate
empirical valence bond method by Voth and co-workers.14
These studies suggested that the rate of proton transport could
be dramatically enhanced upon constricting a hydrophobic
channel to dimensions where only a single-file water wire could
be sterically accommodated. Molecular dynamics simulations
have yielded valuable insights into the properties of water wires
in apolar environments. In cavities of large dimensions,
distinctive hydrogen-bonded clusters of water structures may
be stabilized.15 An intriguing recent report provides crystal-
lographic support for novel structures for a hydrated proton
inside a hydrophobic nanotube formed in crystals of a
carborane acid.16
Clusters of water molecules have been found embedded
within membrane-spanning segments of a number of channel-
forming proteins. A particularly interesting example of this is
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Figure 9. Linear dipeptide Val-Ile (from ref 4d, redrawn using coordinates obtained from Cambridge Structure Database22). (a) Environment of the four
water molecules in the unit cell within the tubular peptide. The peptide atoms are shown as sticks. The shortest distances of the water oxygen with the atoms
lining the pore interior are shown as dashed lines (magenta, values given in angstroms). (b) Modeled water wire inside the dipeptide Val-Ile.
the recent structure determination of the influenza virus proton
channel, which is formed by the M2 protein.17 The M2 channel
has a relatively poor conduction rate of less than 104 protons/s,
which is significantly lower than those of the other proton
channels. The structural work suggests the absence of a large
pore and does not provide evidence for a continuous water wire
(12) (a) Day, T. J. F.; Schmitt, U. W.; Voth, G. A. J. Am. Chem. Soc.
2000, 122, 12027–12028. (b) Ermler, U.; Fritzsche, G.; Buchanan,
S. K.; Michel, H. Structure 1994, 2, 925–936. (c) Seibold, S. A.; Mills,
D. A.; Ferguson-Miller, S.; Cukier, R. I. Biochemistry 2005, 44,
10475–10485. (d) Cui, Q.; Karplus, M. J. J. Phys. Chem. B 2003,
107, 1071–1078. (e) Lamoureux, G.; Klein, M. L.; Berne`che, S.
Biophys. J. 2007, 92, L82–L84. (f) Luecke, H.; Schobert, B.; Richter,
H. T.; Cartailler, J. P.; Lanyi, J. K. J. Mol. Biol. 1999, 291, 899–911.
(13) (a) Lu, D.; Voth, G. A. J. Am. Chem. Soc. 1998, 120, 4006–4014. (b)
Voth, G. A. Acc. Chem. Res. 2006, 39, 143–150. (c) Swanson, J. M. J.;
Maupin, C. M.; Chen, H.; Petersen, M. K.; Xu, J.; Wu, Y.; Voth,
G. A. J. Phys. Chem. B 2007, 111, 4300–4314.
(14) Brewer, M. L.; Schmitt, U. W.; Voth, G. A. Biophys. J. 2001, 80,
1691–1702.
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Water Wires in Hydrophobic Peptide Channels
A R T I C L E S
Figure 10. Experimentally observed single-file water inside the human aquaporin AQP4 (ref 11e, drawn using the cooordinate set PDB ID 3GD8 from the
Protein Databank23 using PyMol24). Shown in sticks are the residues forming the amphipathic faces of the water channel. The hydrogen-bonding distances
between the water oxygens in the water wire and with the residues of the hydrophilic face are shown as dashed lines (distances given in Å). The distances
of the water oxygens closest to the hydrophobic face of the channel are indicated as dotted lines.
spanning the membrane. Molecular dynamics studies of the M2
transmembrane domain provide evidence for water penetration
deep into the transmembrane helix regions.18 The infiltration
of water molecules into the membrane channel specific for alkali
cations has also been established in recent structural work on
the NaK channel from Bacillus cereus.19
molecules aligned along the long axis of the tube. The absence
of strong interactions with the walls of the tube in the case of
peptide nanotubes would permit rapid rotation of water mol-
ecules about the axis of the tube. Such a motion must contribute
to rotational entropy, which in turn may be a factor resulting in
a lower overall free energy for the encapsulated water wire.20
Rotational motion in a direction perpendicular to the tube axis
should permit interchange of donor/acceptor interactions in a
concerted fashion, providing a mechanism for Grotthuss-type
proton transport, which has been favored as a mechanism for
proton conduction in ordered water arrays.12,14,20,21 In contrast,
the architecture of the aquaporin water channels anchors the
second O-H bond of the water molecule in a strong hydrogen
bond interaction with the polar side chains which line the
channel.11e Water hops between hydrogen-bonding sites may
then be a mechanism for the single-file transport through the
protein pores. The two crystal forms of peptide 2 (2a and 2b)
characterized earlier provided an example of both short hydrogen
bond (dO· · ·O ) 2.6 Å) and long hydrogen bond (dO· · ·O ) 3.5
Å) arrangements of the water wires. The latter was obtained by
the transformation of the originally formed crystals. Peptide 2
and its enantiomorph 6 present examples of multiple crystal
forms, and different packing arrangements could be obtained.
The structure of peptide 6a reveals a porous lattice with water
Conclusions
The water wires considered above consist of a one-
dimensional assembly of water molecules which participate as
a donor/acceptor in hydrogen bond interactions with preceding
and succeeding water molecules. Two classes of O · · · O
distances have been observed: 2.6-2.9 Å in the case of peptide
2a, the dipeptide Val-Ile,4c and the human aquaporin AQP411e
and 3.2-3.5 Å in the case of peptides 2b, 6a, and cyclic peptide
nanotubes reported by Ghadiri and co-workers.3c In the case of
hydrophobic tubes, the second hydrogen atom points toward
the walls of the tube, but in all cases discussed above, except
the aquaporins, there does not appear to be any strong interaction
involving this O-H bond. Tubular peptide channels considered
above have diameters in the range 5.2-7.7 Å, with the water
(15) (a) Blanton, W. B. B.; Gordon-Wylie, S. W.; Clark, G. R.; Jordan,
K. D.; Wood, J. T.; Geiser, U.; Collins, T. J. J. Am. Chem. Soc. 1999,
121, 3551–3552. (b) Barbour, L. J.; Orr, G. W.; Atwood, J. L. Chem.
Commun. 2000, 859–860. (c) Custelcean, R.; Afloroaei, C.; Vlassa,
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Moorthy, J. N.; Natarajan, R.; Venugopalan, P. Angew. Chem., Int.
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M. I. Chem. Commun. 2002, 62–63. (f) Pal, S.; Sankaran, N. B.;
Samanta, A. Angew. Chem., Int. Ed. 2003, 42, 1741–1743. (g) Ghosh,
S. K.; Bharadwaj, P. K. Inorg. Chem. 2003, 42, 8250–8254. (h) Ma,
B.; Sun, H.; Gao, S. Angew. Chem., Int. Ed. 2004, 43, 1374–1376.
Byl, O.; Liu, J.-C.; Wang, Y.; Yim, W. L.; Johnson, J. K.; Yates,
J. T., Jr. J. Am. Chem. Soc. 2006, 128, 12090–12097. Mascal, M.;
Infantes, L.; Chisholm, J. Angew. Chem., Int. Ed. 2006, 45, 32–36.
(16) Stoyanov, E. S.; Stoyanova, I. V.; Tham, F. S.; Reed, C. A. J. Am.
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(20) (a) Pomes, R.; Roux, B. Biophys. J. 1996, 71, 19–39. (b) Hummer,
G.; Rasaiah, J. C.; Noworyta, J. P. Nature 2001, 414, 188–190. (c)
Berezhkovskii, A.; Hummer, G. Phys. ReV. Lett. 2002, 89064503.
(21) (a) Agmon, N. Chem. Phys. Lett. 1995, 244, 456–462. (b) Pomes, R.;
Roux, B. Biophys. J. 2002, 82, 2304–2316. (c) Tajkhorshid, E.; Nollert,
P.; Jensen, M. A.; Miercke, L. J. W.; O’Connell, J.; Stroud, R. M.;
Schulten, K. Science 2002, 296, 525–530. (d) Burykin, A.; Warshel,
A. Biophys. J. 2003, 85, 3696–3706. (e) Chakrabarti, N.; Tajkhorshid,
E.; Roux, B.; Pomes, R. Structure 2004, 12, 65–74. (f) Dellago, C.;
Hummer, G. Phys. ReV. Lett. 2006, 97, 245901. (g) Takhashi, R.;
Wang, H.; Lewis, J. P. J. Phys. Chem. B 2007, 111, 9093–9098. (h)
Alexiadis, A.; Kassinos, S. Chem. ReV. 2008, 108, 5014–5034. (i)
Kofinger, J.; Hummer, G.; Dellago, C. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 13218–13222. (j) Parthasarathi, R.; Elango, M.; Subrama-
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(22) Allen, F. H. Acta Crystallogr., Sect. C 2002, 58, 380–388.
(23) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J.; Meyer, E. E., Jr.;
Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi,
M. J. Mol. Biol. 1977, 112, 535.
(17) Stoufer, A. L.; Acharya, R.; Salom, D.; Levine, A. S.; Costanzo, D. L.;
Soto, C. S.; Tereshko, V.; Nanda, V.; Stayrook, S.; DeGrado, W. F.
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(18) (a) Yi, M.; Cross, T. A.; Zhou, H. X. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 13311–13316. (b) Khurana, E.; Peraro, M. D.; DeVane,
R.; Vemparala, S.; DeGrado, W. F.; Klein, M. L. Proc. Natl. Acad.
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[M + K]+; 2 and 6, 598.5 [M + H]+ (Mcal ) 597.4 Da), 620.5 [M
+ Na]+, 636.2 [M + K]+; 3 and 7, 570.4 [M + H]+ (Mcal ) 569.3
Da), 592.2 [M + Na]+; 4 and 8, 646.4 [M + H]+ (Mcal ) 645.4
Da); 668.4 [M + Na]+, 684.4 [M + K]+.
entrapped within hydrophobic channels, while 6b crystallizes
in a monoclinic form in a close-packed arrangement. In contrast,
we have thus far been able to obtain only one crystal form which
contains empty hydrophobic channels. In the case of peptide 1
and the enantiomorph 5, the side chains of isobutyl groups
project inside the channel interior, thereby limiting the diameter
of the pore. Close-packed structures are formed when the
projecting side chains are reduced in size, as in the case of
peptide 3 (Xxx ) Ala) and the enantiomorph 6 (DXxx ) DAla),
or increased, as in the case of peptide 4 (Xxx ) Phe) and the
Colorless crystals were grown by the slow evaporation method
for peptides 1-8. The crystals of 1, 3, 6b, and 7 were grown from
methanol solution, to which a small amount of water was added.
Peptide crystals for 2 and 6a were obtained from a mixture of ethyl
acetate/petroleum ether. Crystals for 4 were grown from a mixture
of acetonitrile/water, and those of 5 and 8 were obtained from a
solution in dioxane, to which a small amount of water was added.
The X-ray diffraction data collected for the crystals of 1-8 are
summarized in Table 1 (see Supporting Information for details).
The structures were solved by direct methods using SHELXS-97
and refined against F2, with full-matrix least-squares methods by
using SHELXL-97. The final values after the refinement of crystal
structures are shown in the Table 1 (see Supporting Information
Tables S1-S6 for details).
D
enantiomorph 8 (DXxx ) Phe). Water-filled pores have thus
far been characterized only in the case of projecting isopropyl
side chains for the Val residue in the peptides 2 and 6. Further
characterization of the water structures inside the hydrophobic
tubes may provide additional insights into the properties of water
wires. The incorporation of other guest molecules and further
tuning of the channel diameter and properties of the walls using
modified amino acids remains to be explored.
Acknowledgment. This work is supported by a grant from the
Council of Scientific and Industrial Research (CSIR), India and a
program grant from the Department of Biotechnology (DBT), India,
in the area of Molecular Diversity and Design. U.S.R. and S.A.
thank the University Grants Commission and CSIR, India, for a
Senior Research Fellowship and Research Associateship, respec-
tively. Kantharaju is supported by the award of a DBT Postdoctoral
Fellowship from the DBT, India. X-ray diffraction data were
collected at the CCD facility under IRHPA program of the
Department of Science and Technology, India and by the Indian
Institute of Science, Bangalore, India.
Experimental Section
The peptides for the studies were synthesized by solution-phase
procedures using a racemization-free fragment condensation strat-
egy. The tert-butoxycarbonyl (Boc) group was used for N-terminal
protection, and the C-terminal was protected as a methyl ester. The
Boc deprotection was performed using 98% formic acid and methyl
ester by saponification. The final step in the synthesis of peptides
was achieved by the fragment condensation of Boc-DPro-OH/Boc-
Pro-OH with H2N-Aib-Xxx-Aib-Val-OMe (Xxx ) Leu, Val, Ala,
Phe, and its D-isomers) using N,N′-dicyclohexylcarbodiimide
(DCC)/1-hydroxybenzotriazole (HOBt) as the coupling reagent. All
the intermediates were characterized by electrospray ionization mass
spectrometry (ESI-MS) on a Bruker Daltonics Esquire-3000 instru-
ment and thin-layer chromatography (TLC) on silica gel and used
as such for the final step. The final peptides were purified by
reverse-phase medium-pressure liquid chromatography (RP-MPLC,
C18, 40-60 µm) and high-performance liquid chromatography
(HPLC) on a reverse-phase C18 column (5-10 µm, 7.8 mm × 250
mm) using methanol/water gradients. The purified peptides were
characterized by ESI-MS. Mass spectral data (m/z): peptides 1 and
5, 612.2 [M + H]+ (Mcal ) 611.4 Da), 634.2 [M + Na]+, 650.5
Supporting Information Available: X-ray crystallographic
characterization details (Tables S1-S6) and CIF files for 1, 2a,
2b, 3, 4, 5, 6a, and 6b. This material is available free of charge
via the Internet at http://pubs.acs.org. CCDC deposition numbers
for peptides are 728333 (1), 728334 (2a), 728335 (2b), 749313
(3), 749314 (4), 749317 (5), 749318 (6a), 749319 (6b), 749315
(7), and 749316 (8).
JA9083978
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