R.S. Roy
P. Balaram
Conformational properties
of hybrid peptides containing
a- and x-amino acids*
Authors' affiliations:
Key words: ax sequences; b-peptides; c-peptides; hybrid peptides;
R.S. Roy and P. Balaram, Molecular
peptide conformations
Biophysics Unit, Indian Institute of Science,
Bangalore 560 012, India.
Abstract: This review briefly surveys the conformational properties
P. Balaram, Chemical Biology Unit, Jawaharlal
of guest x-amino acid residues when incorporated into host
Nehru Centre for Advanced Scientific Research,
a-peptide sequences. The results presented focus primarily on the
Jakkur Campus, Jakkur PO, Bangalore 560 004,
India.
use of b- and c-residues in ax sequences. The insertion of
additional methylene groups into peptide backbones enhances the
range of accessible conformations, introducing additional
Correspondence to:
P. Balaram
torsional variables. A nomenclature system, which permits ready
Molecular Biophysics Unit
comparisons between a-peptides and hybrid sequences, is defined.
Indian Institute of Science
Crystal structure determination of hybrid peptides, which adopt
Bangalore 560 012
India
Tel.: 91 80 22932337/91 80 23602741
helical and b-hairpin conformations permits the characterization
of backbone conformational parameters for b- and c-residues
Fax: 91 80 23600683/91 80 23600535
inserted into regular a-polypeptide structures. Substituted b- and
E-mail: pb@mbu.iisc.ernet.in
c-residues are more limited in the range of accessible
conformation than their unsubstituted counterparts. The achiral
b,b-disubstituted c-amino acid, gabapentin, is an example of a
stereochemically constrained residue in which the torsion angles
about the Cb–Cc (h1) and Ca–Cb (h2) bonds are restricted to the
gauche conformation. Hybrid sequences permit the design of
novel hydrogen bonded rings in peptide structures.
The conformational properties of peptides derived from
a-amino acid residues are well established. The Ramachandran (/,w) dihedral angles and intramolecular hydrogen
Dates:
Received 5 December 2003
Revised 22 December 2003
bonding patterns serve as descriptors of polypeptide backbone structures. These parameters provide a convenient
Accepted 18 January 2004
means of identifying and classifying secondary structural
*Dedicated to the memory of Arno. F. Spatola.
features in peptides and proteins (1,2). Limited attention
To cite this article:
has been focused on the stereochemistry of the polypeptides
Roy, R.S. & Balaram, P. Conformational properties of
containing higher x-amino acid (3), until the finding of the
hybrid peptides containing a- and x-amino acids*.
J. Peptide Res., 2004, 63, 279–289.
groups of Seebach and Gellman that the oligo-b-peptides
Copyright Blackwell Munksgaard, 2004
adopt novel folded structures, with hydrogen bonding
279
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
patterns distinct from those observed in poly-a-peptides
widely observed hydrogen bonded structures have N-termi-
(4–11). The term x-amino acid is used to refer to the entire
nus acceptor C¼O groups (C¼Oi) interacting with C-termi-
family of residues generated by homologation of the back-
nus donor NH-groups (Ni+n) H). For n ¼ 2 to 4 we get the well
bone. The growing body of work on peptides containing
known c-turn (C7), b-turn(C10) and a-turn (C13) structures.
b-amino acid residues points to a tendency of oligomeric
Repetition of these hydrogen bonded structures can lead to
sequences to adopt well defined structures, despite the
regular helices notably 2.27, 310 and a-helical structures.
possibility of enhanced conformational freedom, because of
Propogation of a helical structure is necessarily contingent
the presence of an additional torsional variable (h) about the
upon adoption of specific backbone (/,w) angles (1). The n ¼ 1
a
b
C –C bond. More recent studies have expanded the range
structure results in the formation of an eight-membered ring
of the b-amino acids used in the design of synthetic peptides
(hydrogen bond directionality reversed, N fi C) which is
and have extended the exploration of conformational space
feasible only if the central peptide unit adopts a cis-geometry
to studies of oligomers of c- and d-amino acid residues
(17). The n ¼ 5 structure, the (p-turn) which is stablized by
(12,13). The use of x-amino acid residues in conjunction
6 fi 1 hydrogen bonding is less common and is often ob-
with a-residues permits systematic exploration of the ef-
served at the C-terminus end of helices, resulting in the for-
fects of introducing additional backbone atoms into well
mation of Schellman motifs (18). The p-helix which results
characterized a-peptide structures. Modification of the
from repetition of C16 hydrogen bonded turns is intrinsically
backbone of a- peptides can result in proteolytically stable
unstable; its larger diameter resulting in poor packing in the
sequences; a property of some importance in the design of
helix interior, with attendant loss of favorable nonbonded
analogs of biologically active sequences. Arno Spatola re-
interactions. In all cases, the directionality of the hydrogen
viewed this area two decades ago; introducing the concept
bond with respect to the chain direction (C ‹ N) is the same.
of peptide bond surrogates and the psi-bracket nomencla-
The most striking finding of the early work on oligomeric
ture for backbone modified structures (14). We present here
b-peptides is the observation of regular structures with
a brief overview of ongoing studies on ax hybrid sequences
hydrogen bonding patterns, which run in both directions,
in our laboratory.
with respect to the polypeptide chain. Figure 1 illustrates
five regular structures which have been experimentally
realized or theoretically postulated for oligomeric b-pep-
Conformational Properties of Hybrid axSequences
tides. The 8- and 12-helices have a hydrogen bond direction
Torsion angle and hydrogen bond nomenclature
directions (N fi C) are reversed. Figure 2 provides a sum-
(C ‹ N), which is the same as that observed in a-peptides,
whereas in the C10 and C14 structures the hydrogen bond
mary of the potential hydrogen bonds in two residue ax
The nomenclature of the canonical hydrogen bonded struc-
sequences. In hybrid sequences, the insertion of additional
tures found in poly-a-peptides is summarized in Table 1. All
atoms may perturb the formation of intramolecularly
Table 1. Canonical a- and b-polypeptide structures
Backbone torsion
angles (helices)
Hydrogen-bonded ring size
and directionality
Turn/helixa
C7, C ‹ N
c-Turn, 2.27-helix
)78
59
(1)
C10, C ‹ N
b-Turn, 310-helix
)49
)26
(1,15)
C13, C ‹ N
a-Turn, a-helix
)57
)47
(1,15)
C16, C ‹ N
p-Turn, p-helix
)57
)70
(1,15)
C8, C ‹ N
8-Helix
120
0
(6)
/ (°)
h (°)
)72
w (°)
References
C10, N fi C
10-Helix
64
59
75
(16)
C12, C ‹ N
12-Helix
95
)94
103
(11)
C14, N fi C
14-Helix
)134
60
)140
(11)
a. Note that multiple turn types are possible which are characterized by different sets of /, w
angles. Only specific turn types give rise to the repetitive helical structures.
280
J. Peptide Res. 63, 2004 / 279–289
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
Figure 1. Novel helices formed by oligob-peptides. The structures have been
generated using insight II program using
theoretically or experimentally determined
backbone torsion angles. Views obtained
perpendicular to the helix axis and down the
helix axis are shown. The values used for
models are: 8-helix: / ¼ 120, h ¼ )72,
w ¼ 0; 10-helix: / ¼ 64, h ¼ 59, w ¼ 75,
12-helix: / ¼ 100.2, h ¼ )95.5, w ¼ 103.8;
14-helix: / ¼ )134.4, h ¼ 60, w ¼ )139.9.
For the 10/12-helix see ref. (25).
8-Helix
Figure 2. Potential hydrogen bonded rings in
ax-hybrid sequences. Dipeptide segments
which have been observed in crystal structures are circled. Segments characterized by
nuclear magnetic resonance are enclosed in
squares.
10-Helix
Ci = O H
12-Helix
N i+3
β
γ
δ
α
10
11
12
13
β
11
12
13
γ
12
13
δ
13
14
N
β
γ
δ
α
8
9
10
11
14
β
9
10
11
12
14
15
γ
10
11
12
13
15
16
δ
11
12
13
14
H
N
N
O
O
H
C ←N
αα,C 10
N
= C i+1
α
H
O
O
12/10-Helix
Ni H O
α
H
14-Helix
N H
O
N→ C
ββ, C10
hydrogen-bonded structures. It should be noted that if the
reveal that the 10- and 13-membered hydrogen bonded rings
number of backbone atoms are considered, correspondence
commonly observed in a-peptide structures may be mim-
may be obtained between ax sequences and a stretch of
icked by specific hybrid combinations. For example, the
a-residues. For example, ad or bc segments result in inser-
conventional a-peptide b-turn may be mimicked by a bb
tion of nine atoms into a peptide backbone and may be
dipeptide, with a reversal of the direction of the intramo-
formally viewed as similar to a stretch of three contiguous
lecular hydrogen bond. Figure 2 also highlights the hydro-
a-residues (Fig. 3).
gen bonded rings, which have thus far been definitively
Inspection of the sizes of the hydrogen bonded rings for
various combinations of residues in dipeptide segments
characterized by X-ray diffraction in crystals or inferred
from nuclear magnetic resonance (NMR) data in solution.
J. Peptide Res. 63, 2004 / 279–289
281
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
Figure 3. Definition of backbone torsion
angles. (A) Gly-Gly-Gly (a,a,a) segment,
(B) Gly-dAva (ad) segment, (C) b-HGly-cAbu
(bc) segment.
In describing conformations of x-amino acid residues and
acids, comfortable accommodation into extended strand
relating observed stereochemical characteristics to the vast
structures require the dihedral angles (hn) about the central
data available on a-peptides, it is convenient to follow a
C–C bonds to adopt a largely trans(180) conformation. This
backbone torsion angle nomenclature, which readily per-
requirement may be anticipated to be particularly stringent
mits direct comparison. In this description, / and w con-
for b-residues, while in the case of the higher x-amino acids
x
tinue to refer to the torsion angles about the N–C and
the possibility of compensating torsion angles about con-
Ca–CO bonds. Torsions about polymethylene chains are
tiguous C–C bonds may permit greater variation. Earlier
denoted by the angles h1,h2,…,hn, with increasing subscript
studies from this laboratory have explored the use of DPro–
numbers referring to the progression of the chain from the
Xxx sequences in nucleating b-hairpin structures (19). We
N-terminus end to the C-terminus. Residue numbers may
have systematically investigated the effect of inserting
be indicated as superscripts, e.g. hin for the ith residue. Note
x-residues at different positions of the strands. Table 2
that in the conventional nomenclature for b and higher x
illustrates observed torsion angles of five peptide hairpins
amino acids the Cb/Cx atom precedes the Ca atom, when
containing facing x-residues. In all cases, good interstrand
the chain is read from the N-terminus.
hydrogen bonds between facing antiparallel strands have
been observed. Figure 4 illustrates representative b-hairpin
structures in a hybrid a/b-sequences. Notably, in the
Extended structures: b-sheets and b-hairpins
structure of octapeptide Boc-Leu-Val-b3-HVal-DPro–Gly-b3HLeu–Val-Val-OMe, the b3-HVal3 residue adopts a gauche
a-Residues can be incorporated into extended strand struc-
conformation about the Ca–Cb bond (h ¼ +65), which is the
tures which assemble into sheets by backbone hydrogen
only example, thus far reported, where such a large devi-
bonding involving proximal strands. The backbone dihedral
ation from a generally observed trans value is established
angles for the b-sheet structure lie in the region
(22). In this case, an almost fully extended w-value
/ ¼ )120 ± 30 and w ¼ 120 ± 30. In the case of x-amino
(w ¼ )175) is observed for b3-HVal3, presumably to restore
282
J. Peptide Res. 63, 2004 / 279–289
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
Table 2. Torsion angles (/, h, w) of b,c-residues in crystals of hybrid peptide hairpins
Torsion angles (°)
Peptide sequences
a
b,c-Residues
Boc–L-V-bF–V-DP-G-L-bF-V-V-OMe
Boc -bF-bF-DP-G-bF-bF-OMe
Boc–L-V-bV- DP-G-bL-V-V-OMe
Boc-L-bF-V-DP-G-L-bF-V-OMe
h1
h2
References
w
bF3
)116
168
)
106
bF8
)109
172
)
128
bF1
)128
163
–
115
bF2
)113
153
–
110
bF5
)84
171
–
115
bF6
)99
166
)
147
bV3
)131
65
–
)175
bL6
)136
)178
)
115
bF2(A)
)141.1
150.2
–
158.8
bF2(B)
)145.4
160.5
–
151.1
bF7(A)
)101
166.7
–
118.5
)91.9
166.3
)
115.5
c Abu3(A)
)118.7
107.2
172.9
c Abu3(B)
)116.2
)172.8
176.3
bF7(B)
Boc L- V-cAbu- V-DPro - Gly - L -cAbu -V-V-OMe
/
)158
(20)
(21)
(22)
b
b
105.9
c Abu8(A)
94.1
)174.8
)65.1
143.4
c Abu8(B)
)178.8
)171.3
)135.3
131.5
a. For simplicity, b-HPhe, b-HVal and b-HLeu are abbreviated as bF, bV and bL respectively.
b. I.L. Karle personal communication.
A
C
the hydrogen bonding interaction. Insertion of b- residues
into b-sheet structures changes the nature of hydrogen
bonding faces of the strands. In the case of a-residues, CO
and NH groups alternately point inward and outward,
whereas in b-residues the CO groups are directed towards
one face and the NH groups face the opposite direction,
resulting in the formation of Ôpolar sheetsÕ (Fig. 4) (21,23).
Furthermore, the insertion of additional atoms into the
strand backbone place the sidechains of successive b-residues on the same face of the sheets as opposed to a-peptide
B
sheets in which they lie in opposite directions (Fig. 4).
Thus, b-amino acid residues may be used to generate peptide assemblies with a distinctly different hydrogen bonding
pattern when compared with a-peptides. Furthermore,
scaffolds with a unique disposition of sidechains may be
generated.
b-Residues may also be inserted into turn segments of
hairpins. In the peptide Boc-Leu-Val-Val-
D
Pro-b3-HPhe–
Leu-Val-Val-OMe, b -HPhe is placed at the i + 2 position of
3
the b-turn in order to assess the effect of expansion of the
nucleating turn to yield a 11-membered hydrogen bonded
ring (22). NMR studies reveal the observation of several
Figure 4. (A) A crystalline b-hairpin with b-amino acids in the strands,
Boc-b3-HPhe-b3-HPhe-DPro-Gly-b3-HPhe-b3-HPhe-OMe. (B) Infinite
b-sheets formed by the peptide. (C) A sideview illustrating the sidechain
orientation ref. (21).
critical nuclear Overhauser effects (NOEs), characteristic of
a b-hairpin with a nucleating hybrid ab turn. The higher
x-amino acids may also be inserted into the strand
J. Peptide Res. 63, 2004 / 279–289
283
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
segments of hairpins as examplified by the structure of the
b-hairpins containing x-amino acids in the strand segments
decapeptide sequence Boc-Leu-ValcAbu-Val-DPro-Gly-Leu-
have yielded crystals, only infrequently. We have thus far
cAbu-Val-Val-OMe, in which two facing cAbu residues are
being unable to crystallize hairpins with expanded turn
observed in antiparallel strands of a b-hairpin structure in
segments. Interpretation of NMR data is sometimes ham-
the crystal (I.L. Karle, personal communication). Attempts
pered by resonance overlap, which obscures critical inter-
have also been made to examine the role of x-amino acid
strand NOEs between facing x-amino acids in synthetic
insertion into the turn segment of peptide hairpins, specif-
model peptides.
D
Pro–Xxx sequences. NMR
The effect of insertion of x-amino acids into the strand
analysis provides evidence of a hairpin structure for
segments of b-hairpins on the thermodynamic stability of
the peptide Boc-Leu-Val-Val-DPro-dAva–Leu-Val-Val-OMe,
the fold have not been assessed thus far. Preliminary studies
with a nucleating ad turn (24). Further evidence is desirable
with the designed peptides suggest that the b3-residues
for the definitive delineation of the effect of inserting higher
strongly favor conformations accomodated into extended
x-amino acids into b-hairpin structures. The rate of accu-
strands, with several examples of stable hairpins having been
mulation of crystal structure data for b-hairpin peptides has
(2) demonstrated in solution. It is likely that the stability of
been slow, because of the low tendency of b-sheets to form
registered anti-parallel hairpins may be compromised by the
single crystals, suitable for X-ray diffraction. In our hands,
insertion of higher x-amino acids into strands, as conform-
ically at the i + 2 position of
Figure 5. Molecular conformation of hybrid helices in crystals. (a) Eight-residue peptide (Boc-Leu-Aib-Val-b-HGly-cAbu-Leu-Aib-Val-OMe).
(B) Eleven-residue peptide, (Boc-Leu-Aib-Val-b-HGly-cAbu-Leu-Aib-Val-Ala-Leu-Aib-OMe). (C) The hybrid dipeptide segments are shown (top)
Val-b-HGly, (middle) b-HGly- cAbu and (bottom) cAbu- Leu [ref. (26)].
284
J. Peptide Res. 63, 2004 / 279–289
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
Table 3. Torsion angles (/, h, w) of b- and c-residues in crystals of hybrid peptide helices
Torsion angles (°)
Peptide sequencesa
b,c-Residues
/
h1
Boc-L-U-V-bG-cAbu- L-U-V-OMe
bG4
)130
76
cAbu5
)108
58
bG4
)103
78
)
)107
cAbu5
)121
62
57
)121
bV5
)125.9
76.2
)
)124
bF6
)88.5
81
)
)118.6
Boc-L-U-V-bG-cAbu- L-U-V-A-L-U-OMe
Boc - V - A - F - U -bV-bF-U-V-A-F-U-OMe
w
References
–
)162
(26)
66
)169
h2
(26)
b
a. For simplicity, b-HPhe, b-HVal and b-HGly are abbreviated as bF, bV and bG respectively.
b. I.L. Karle personal communication.
ational entropy considerations may offset the enthalpic
tains 12 and 14 atoms, respectively. In these structures, the
advantages of cross strand hydrogen bond formation.
torsion angles lie in the following regions of conformational
space: 14-helix: / ¼ )134, h ¼ 60, w ¼ )140; 12-helix:
/ ¼ 95, h ¼ )94, w ¼ 103.
Notably, the h-values lie close to the gauche conformation
Helical structures
in
both
cases.
While
unambiguous
crystallographic
As depicted in Fig. 1, oligomeric sequences of b-amino acids
characterization has been achieved for 12 and 14 helical
have been shown to fold into the 12 and 14 helical struc-
structures of homooligomers of the constrained b-amino
tures characterized by hydrogen bonded rings which con-
acids of ACPC (trans 2-aminocyclopentanecarboxylic acid)
Figure 6. Interproton distances for a,b and c-residues present as guests into host a-peptide helical and b-sheet conformations. b-sheet (top) and
helical (bottom). (A) a-Residues, (B) b-residues and (C) c-residues. In (A) and (B), Phe and b3-HPhe residues have been chosen from crystal structures,
while in (C), the cAbu residue is represented. For the sake of clarity, the dcb and dca distances are not indicated by arrows, in the helical conformation
and dNc distance is not indicated by arrow in the sheet conformation.
J. Peptide Res. 63, 2004 / 279–289
285
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
Table 4. Conformation Sensitive Interproton Distances (A°)a
Distances (A°)
b-Sheet (X-ray)
Helix (X-ray)
torsion angles about the Ca–Cb and Cb–Cc lie close to an ideal
gauche conformation. Interestingly, the /, w values for the
b- and c-residues are both somewhat extended, but have the
a-Residues
same sign (negative for a right-handed helical twist).
daNN
4.2
2.8
daaN
2.2
3.4
daNa
2.7
b-Residues
2.6
Recent work in our laboratory suggests that the incorporation of contiguous b-residues into helical a-peptide
structures can be accomplished without any dramatic
b
dbNN
4.8
3.5
structural perturbation. The structure of the peptide Boc-
dbbN
4.1
3.9
Val-Ala-Phe-Aib-b3-HVal-b3-HPhe-Aib-Val-Ala-Phe-Aib–OMe
dbaN
2.2, 3.1
2.2, 3.2
determined by X-ray diffraction, reveals a continuous helix
dbNb
2.8
2.7
stabilized by intramolecular hydrogen bonds (I.L. Karle
dbNa
2.6, 3.0
2.5, 3.5
personal communication).
2.4, 2.8
2.5, 2.8,
dbba
The solution conformational analysis in solution of
b- and c-residues in peptides depend critically on the
c
c-Residues
dcNN
6.1
4.4
observation of specific NOEs. Figure 6 shows a perspective
dccN
4.1, 4.7
3.7, 4.9
overview of a, b and c-residues when incorporated into host
dcbN
3.7, 4.2
4.3, 4.4
a-peptide sequences that closely resembles a-helical and
dcaN
2.2, 3.0
2.2, 3.0
b-sheet structures. Relevant interproton distances are
dcN c
2.4, 2.7
2.3, 2.7
defined. Table 4 lists the interproton distances for b- and
dcNb
2.5, 3.0
2.7, 3.6
c-residues accommodated into extended strand and helical
dcNa
4.2, 4.5
3.0, 4.1
conformation in host regular structures.
dccb
2.1–2.8
2.3–2.8
dcba
2.2–2.8
2.3–2.8
dcca
2.5–3.3
3.0–4.1
a. Distances calculated for /, h, w values observed in
specific secondary structures using crystallographically
determined coordinates. Values are averaged over several residues. The superscripts indicate the residue type
while the subscripts indicate the atom type.
b. Distances crystallographically determined for b3-residues.
c. dccb , dcba and dcca distances are given as ranges which
represent the upper and lower limits in the c-Abu residue.
Constrained x-amino acids
The use of x-amino acids in which torsions about rotatable
bonds are restricted by freezing into cyclic structures are
useful in probing the promotion of specific conformations in
oligopeptides. Figure 7 shows some representative structures
of amino acids with restricted torsional freedom. The successful characterization of the 12 and 14 helical structures of
oligo-b-peptides by X-ray diffraction are striking examples of
and ACHC (trans 2-aminocyclohexanecarboxylic acid), the
the utility of introducing conformational constraints (8–11).
10 helix and the mixed 10/12 helix have thus far not been
Nipecotic acid, a b-amino acid which incorporates both / and
observed in crystals. The mixed 10/12 helix has been
h constraints, has been introduced into a reverse turn motif,
postulated in the structure of the synthetic peptide
where a heterochiral dinipecotic acid segment occupies the
(H2N-b3-HVal-b2-HAla-b3-HLeu-b2-HVal-b3-HAla-b2-HLeu-b3-
i + 1 and i + 2 positions of a C12 turn (27).
HVal-b2-HAla-b3-HLeu–OH) from NMR data (25). In hybrid
(S)-H-b3-HPro–OH, derived by backbone homologation of
sequences, an important question to be addressed is whether
L-proline, is a readily available amino acid in which the
the x-amino acid can be accommodated into the helical fold
torsion angle / ¼ )60 ± 20. Ordered structures of homoo-
generated in the host a-peptide sequences. Two early crystal
ligomeric sequences of b3-homoproline and b2-homoproline
structures of 8- and 11-residue peptide helices demonstrated
(nipecotic acid) have been inferred from circular dichorism
that the bGly-cAbu segment could be incorporated into a
(CD) data (28). In principle, (S)-H-b3-HPro–OH residue can
helical fold, without significant perturbation of the secon-
be readily accommodated in the i + 2 position in expanded
dary structure (26). Figure 5 shows a view of the helices
b-turns in ab sequences.
formed and also illustrates the expanded hydrogen bonded
Trans-3–ACPC
(trans–3–aminocyclopentanecarboxylic
rings of the ab,bc, and ca dipeptide segments. Table 3 lists
acid) is a constrained c-amino acid. Trans-3–ACPC has been
the conformational parameters for the b- and c-residues
incorporated into the strand segment of a parallel sheet
incorporated in host a-peptide structures. In all the cases, the
structure nucleated intramolecularly using a d-prolyl–(1,1-
286
J. Peptide Res. 63, 2004 / 279–289
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
Figure 7. The structures of constrained x-amino acid residues. Relevant torsion angles which are restricted are indicated.
Figure 8. The structures and nomenclature of acyclic, substituted x-amino acid residues. The prototype residue Aib is also shown.
dimethyl)-1,2-diaminoethyl unit as the turn segment (29).
dialkylated amino acid, a-aminoisobutyric acid (Aib) the
3-Aminobenzoic acid which is a c-amino acid with
prototype residue in this class, has been shown to adopt an
h1 ¼ h2 ¼ 180, is comfortably accomodated into the strand
extremely limited range of (/,w) values, resulting in the
segment of model hairpin structures as established by NMR
stabilization of ordered structures in short sequences
studies (30).
(19,31). By extension, substitution along the carbon back-
The addition of substituents at the Ca-carbon atom in a-
bone of acyclic x-amino acid residues should restrict tor-
amino acids restricts the range of sterically allowed con-
sional freedom. In the case of b and c-amino acids multiple
formations about the torsional angles / and w. The Ca,a
possibilities of substitution exist permitting selective con-
J. Peptide Res. 63, 2004 / 279–289
287
Roy and Balaram. Hybrid peptides containing a- and x-amino acids
formational restrictions to be imposed about the /, h, w
ture of the tripeptide Boc-b3-HPhe–Gpn-Phe-OMe reveals a
torsional angles (see Fig. 8 for the nomenclature for sub-
compact folded conformation stabilized by two intramo-
stituted acyclic b and c-residues).
lecular hydrogen bonds; C13 (C ‹ N), encompassing the bc
The widely used anti-epileptic drug gabapentin [1-(ami-
dipeptide segment and C10 (N fi C) encompassing the ba
nomethyl) cyclohexaneacetic acid, Gpn] is an achiral b,b-di-
dipeptide segment (Vasudev, P.G., Ananda, K., Shamala, N.
substituted c-amino acid (c3,3) Fig. 8. The pair of geminal
and Balaram, P., Unpubl. data).
b
substituents at the central C -carbon atom restrict the range
of conformations available about the torsion angles h1 and h2.
The determination of several X-ray structures of gabapentin
Conclusions
derivatives reveals the almost exclusive adoption of gauche–
gauche conformations about the Cc–Cb(h1) and Cb–Ca(h2)
Systematic exploration of the conformational characteris-
bonds (32). Preliminary studies in our laboratory suggest that
tics of hybrid dipeptide segments enumerated in Fig. 2 may
Gpn residues in hybrid sequences promote obligatory re-
prove valuable in formulating approaches for the insertion
versed of chain direction, even in the absence of stabilizing
of such segments into host a-peptide sequences in the de-
intramolecular hydrogen bonds as exemplified in the crystal
sign of analogs of biologically active peptides. The insertion
structure of Boc–Gpn-b3-HPhe–Leu-OMe (Ananda, K., Ara-
of b, c and d residues provides a convenient means for
vinda, S., Shamala, N. and Balaram, P., Unpubl. data).
replacing amide bonds by Ôsurrogate ethylene unitsÕ and for
perturbing the local structure in helices and b-sheets by
insertion of additional atoms into peptide backbones.
Analogs, which confer site-directed proteolytic stability
(35), without perturbation of the overall fold in biologically
active sequences, may be of special importance. The
growing body of literature on naturally occurring, biolo-
Crystal structure determination of N-protected dipeptide
gically active peptides containing diverse b-amino acid
acids and esters like Piv- Pro-Gpn–OH, Boc–Aib–Gpn–OH,
residues adds relevance to attempts to explore the con-
Boc–Gly–Gpn–OH, Boc–Aib–Gpn–OMe establishes the
formational characteristics of peptides containing the
folding of the peptide chain at the Gpn residue (33).
higher homologs of a-amino acids (36).
L
Novel helical incipient structures have been crystallographically demonstrated in short sequences containing
Acknowledgements: We thank Anindita Sengupta for help in
c-amino acids. Seebach et al. have established a 2.614-helical
generating Figs 1, 4–6. We are grateful to Dr Isabella Karle, Prof.
structure in a tetrapeptide containing c
N. Shamala, Prema G. Vasudev and Dr K. Ananda for providing
2,3,4
-amino acids (34).
unpublished results. We are grateful to Prof. Dieter Seebach and
Dr Magnus Rueping for kindly providing the coordinates of the
NMR structure of a 10/12-helix. Work in this area has been supported by grants from the Department of Biotechnology in the
area of Molecular Diversity and Design. RSR thanks the CSIR for
In short c-peptide sequences evidence of nine-membered
award of a Senior Research Fellowship.
hydrogen bonded rings have also been obtained (33,34).
The use of hybrid sequences permits characterization of
novel hydrogen bonded rings in short peptides. The struc-
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