Unusual folding propensity of an unsubstituted β,γ-hybrid model peptide: Importance of the C-H?O intramolecular hydrogen bond

Article abstract of DOI:10.1002/chem.201300630

The single-crystal X-ray diffraction analysis of a β,γ-hybrid model peptide Boc-β-Ala-γ-Abu-NH₂ revealed the existence of four crystallographically independent molecules (A, B, C and D conformers) in the asymmetric unit. The analysis revealed t

Full text of DOI:10.1002/chem.201300630

FULL PAPER
Everywhere U-turn: The characterisa-
tion of non-functionalised b-turn-like
structures across an unsubstituted b,g-
hybrid peptide unit is reported. X-ray
diffraction analysis revealed the exis-
tence of four crystallographically inde-
pendent molecules. Unusual b-turn-
like folded structures predominate and
the U-shaped conformers (see figure)
are seemingly stabilised by an effective
Peptidomimetics
P. Venugopalan,
R. Kishore* ................... &&&& — &&&&
Unusual Folding Propensity of an
Unsubstituted b,g-Hybrid Model
Peptide: Importance of the C H···O
Intramolecular Hydrogen Bond
ꢀ
ꢀ
unconventional C H···O intramolecu-
lar hydrogen bond.
Chem. Eur. J. 2013, 00, 0 – 0
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DOI: 10.1002/chem.201300630
Unusual Folding Propensity of an Unsubstituted b,g-Hybrid Model Peptide:
ꢀ
Importance of the C H···O Intramolecular Hydrogen Bond
Paloth Venugopalan[a] and Raghuvansh Kishore*[b]
ꢀ
Abstract: The single-crystal X-ray dif-
fraction analysis of a b,g-hybrid model
peptide Boc-b-Ala-g-Abu-NH2 re-
vealed the existence of four crystallo-
graphically independent molecules (A,
B, C and D conformers) in the asym-
metric unit. The analysis revealed that
unusual b-turn-like folded structures
predominate, wherein the conforma-
tional space of non-proteinogenic b-
Ala and g-Abu residues are restricted
to gauche-gauche-skew and skew-
gauche-trans-skew orientations, respec-
tively. Interestingly, the U-shaped con-
formers are seemingly stabilised by an
effective unconventional C H···O in-
tramolecular hydrogen bond, encom-
passing a non-covalent 14-membered
ring-motif. Taking into account the
signs of torsion angles, these conform-
ers could be grouped into two distinct
categories, A/B and C/D, establishing
the incidence of non-superimposable
non-chiral one-component peptide
model system, that is, “mirror-image-
like” relationships. The natural occur-
rence of b-Ala and g-Abu entities in
various pharmacologically important
molecules, coupled with their biocom-
patibilities, highlight how the non-func-
tionalised b,g-hybrid segment may
offer unique advantages for introducing
and/or manipulating a wide spectrum
of biologically relevant hydrogen
bonded secondary structural mimics in
short synthetic peptides.
stereogeometrical features across
a
Keywords: hydrogen bonds · pepti-
des · peptidomimetics · unsubstitut-
ed hybrid peptides · X-ray diffrac-
tion
Introduction
The design and construction of diversely folded peptide con-
formations through readily available non-proteinogenic un-
ꢀ
ꢀ
substituted b- and g-amino acid residues, that is, CONH
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ACHTUNGTRENNUNG(CH2)2 CONH (b-Ala) and CONH ACHTUNTGERN(NUGN CH2)3 CONH (g-
Abu) moieties, have attracted much attention.[1,2] As pre-
sented in Figure 1, incorporation of the higher homologues
of the proteinogenic non-chiral Gly residue introduces addi-
b
a
ꢀ
tional one and two torsion angles, q (or m), across the C C
b
g
b
a
ꢀ
ꢀ
bond in b-Ala, and q1 and q2 across the C C and C
C
bonds in g-Abu, in the peptide backbone. The additional
torsion angles introduced by b-Ala and g-Abu in peptides
can exert dramatic structural effects on the global molecular
conformations, the potentials of which are yet to be exploit-
ed for designing novel biomimetic three-dimensional struc-
tures.[1,2] Our systematic conformational analysis,[1i] together
with those reported by other researchers,[1,2] strongly suggest
that the insertion of methylene units through b-Ala or g-
Abu residues in synthetic a,b- and a,g-hybrid peptides can
Figure 1. The chemical structures of unsubstituted model peptides Boc-
Aaa-g-Abu-NH2 where Aaa=Gly (1), b-Ala (2) or g-Abu (3) residue.
The arrows indicate main-chain torsion angles across the Gly (f, y), b-
Ala (f, q, y) and g-Abu (f, q1, q2, y) residues.
encourage a range of unusual biologically relevant hydrogen
bonded secondary structural features. In addition, the natu-
ral occurrence of b-Ala and g-Abu moieties in the animal
and plant kingdoms,[1,2] coupled with their proteolytic resist-
ance,[3,4] not only signify their biocompatibility but also po-
tential convenience for developing more effective pharma-
ceutical therapeutics and specific peptidomimetics capable
of exhibiting substantially altered biological response(s).
Although, it is widely known that methylene units usually
adopt more favourable all-trans or all-anti conformations,[5a]
nevertheless, excessive repulsive or attractive interactions
between neighbouring amide groups may enforce more
[a] P. Venugopalan
Department of Chemistry, Panjab University
Sector 14, Chandigarh–160 014 (India)
[b] Dr. R. Kishore
Protein Science and Engineering Division
CSIR-Institute of Microbial Technology
Sector 39-A, Chandigarh–60 036 (India)
E-mail: kishore@imtech.res.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201300630.
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Folding Propensity of an Unsubstituted b,g-Hybrid Peptide
FULL PAPER
complimentary folded or unfolded structural features across
b,g-hybrid peptide Boc-b-Ala-g-Abu-NH2 (2) with the pre-
sumption that its “simplicity”, that is, lack of side-chains,
and “biocompatibility”, that is, folding and resistance to pro-
teolysis,[3] could be fundamental for obtaining a better un-
derstanding of sequence–conformation relationships. The
analysis establish coexistence of four crystallographically in-
dependent molecules, A, B, C and D, in the asymmetric
unit, and the unusual chain-reversal facilitates the fabrica-
[5b]
ꢀ
ꢀ
ꢀ
ꢀ
the CONH ACHTUNGTRENNUNG(CH2)n CONH moieties (n=2 or 3). Re-
sults of widespread structural investigations of b-Ala con-
taining hybrid peptides have demonstrated that the residue
preferably adopts three distinct conformations, that is, the
folded gauche (q~ ꢁ60ꢁ208), the semi-extended skew (q~
ꢁ120ꢁ208) and the fully-extended trans (qꢂ ꢁ180ꢁ208)
orientations.[1i,j] On the other hand, under these conditions,
g-Abu exhibits strong bias for folded gauche (q1 and/or q2 ~
ꢁ60ꢁ208) dispositions.[2] In fact, we recently demonstrated
that even in the absence of a C-terminal amide group, g-
ꢀ
tion of an unconventional C H···O intramolecular hydrogen
bond.[7] We highlight that the observed U-shaped topology
closely resembles a typical hydrogen bonded b-turn struc-
ture wherein the folded b-Ala and g-Abu residues occupy
analogous left- and right-corner positions, respectively.
ꢀ
ꢀ
ꢀ
Abu, that is, the CONH ACHTNUGTRENUNG(CH2)3 COOH moiety, adopted
a folded conformation.[2g] The existing literature inclined us
to suggest that experimental characterisation of a fully-ex-
tended g-Abu residue, that is, fꢂq1 ꢂq2 ꢂyꢂ ꢁ180ꢁ208
conformation, could be rather demanding.[2g] We believe
that subtle modulation of folded structural features by in-
serting limited methylene units through b-Ala and/or g-Abu
residue(s) in appositely designed peptides could be construc-
tive for expanding the range of accessible secondary struc-
tural components.[1,2]
Results and Discussion
The single-crystals of peptide 2 suitable for X-ray diffraction
analysis were obtained from methanol/chloroform (3:2 v/v)
mixtures. Our attempts to grow suitable single crystals of
model peptides 1 and 3, so far, remained unsuccessful. The
striking feature of crystal-state structure of peptide 2 is the
existence of four crystallographically independent mole-
cules, A, B, C and D conformers, in the asymmetric unit.
Figure 2 shows the molecular structures of the four conform-
The conformational analysis of substituted a,b- and a,g-
hybrid peptides provided many insights into the alternative
hydrogen bonded structural organisation of secondary and
supersecondary structures.[1,2] What would be the probable
conformation of a hybrid pep-
tide completely devoid of side-
chain functionality? To our
knowledge, no attempt has
been made to understand in-
trinsic folding/unfolding pro-
pensities
of
unsubstituted
hybrid peptides.[6] Therefore, in
view of our relevant research
interests to comprehend folding
behaviour of unsubstituted pep-
tides, we embarked on a re-
search programme aimed at
probing the conformational
characteristics of short linear
hybrid peptides generated from
ꢀ
ꢀ
non-proteinogenic
NH
ꢀ
ꢀ
ACHTUNGTRENNUNG(CH2)n CO
(n=1–3) type
amino acids (Figure 1). To ex-
plore the potential conforma-
tional ensemble of unsubstitut-
ed peptides, in a systematic ap-
proach, we synthesised three
variants of terminally blocked
model peptides of the type
ꢀ
ꢀ
ꢀ
Boc ACHTUNGTRENNUNG(CH2)n g-Abu NH2
(where n=1 (Gly: 1), 2 (b-Ala:
2), and 3 (g-Abu: 3) by solu-
tion-phase methods (see the
Supporting Information). In
this initial report, we deter-
Figure 2. An ORTEP presentation of the molecular structures of four crystallographically independent mole-
cules A, B, C and D of the model peptide Boc-b-Ala-g-Abu-NH2. The thermal ellipsoids are shown at the
ꢀ
40% probability level. Dashed lines indicate C H···O intramolecular hydrogen bonds, encompassing a non-co-
mined the crystal structure of a valent 14-membered ring-motif.
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R. Kishore and P. Venugopalan
ers are ꢃ5.6 ꢁ. The analogous Ca ···Ca
distances
i+3
Table 1. Selected backbone torsion angles [8] for Boc-b-Ala-g-Abu-NH2.
i
in b-turn structures, favouring peptide-chain rever-
Torsion angles
Definition
A
B
C
D
sal in globular proteins, polypeptides and peptides
C4-O1-C5-N1
O1-C5-N1-C6
C5-N1-C6-C7
N1-C6-C7-C8
C6-C7-C8-N2
C7-C8-N2-C9
C8-N2-C9-C10
N2-C9-C10-C11
C9-C10-C11-C12
C10-C11-C12-N3
C11-C12-N3-H
q0
w0
f1
q
178.1(5)
ꢀ177.4(4)
69.8(6)
169.5(4)
172.7(4)
68.9(6)
ꢀ178.2(4)
177.6(4)
ꢀ70.7(6)
ꢀ53.0(6)
131.0(4)
178.5(4)
132.7(4)
ꢀ69.6(6)
174.3(4)
137.2(5)
ꢀ179.5
ꢀ169.0(4)
ꢀ173.2(4) are usually ꢃ7.0 ꢁ.[10] The analysis also revealed
ꢀ69.4(6)
ꢀ55.8(5)
139.7(4)
177.3(4)
129.5(4)
ꢀ64.5(5)
173.6(4)
125.3(5)
ꢀ179.3
that the conformers A/C are relatively more com-
pact than B/D, the dC4···N3 distances are approxi-
mately 5.0 and 5.6 ꢁ, respectively. Also, the peptide
bonds in each conformer are orientated in opposite
directions. The question whether this is a general
feature of the folded b,g-hybrid peptide units, de-
serves further investigation.
Interestingly, the magnitudes of torsion angles in
all the conformers exhibit striking similarity. Never-
theless, taking into account the signs of torsion
53.6(5)
55.0(5)
y1
w1
f2
q1
q2
y2
w2
ꢀ130.8(4)
ꢀ176.8(4)
ꢀ133.4(5)
68.8(6)
ꢀ171.4(4)
ꢀ141.4(5)
ꢀ177.8
ꢀ137.8(4)
ꢀ175.8(4)
ꢀ133.5(5)
64.6(6)
ꢀ176.5(4)
ꢀ124.9(5)
177.4
ers, and Table 1 compares the main-chain torsion angles. In
all conformers the urethane moiety adopted a trans-trans ar-
rangement, torsion angles q1 ꢂw0 ꢂ ꢁ180ꢁ108, establishing
its orientation as type b.[8]
angles, these conformers could be grouped into two catego-
ries: 1) gauche+-gauche+-skewꢀ and skewꢀ-gauche+-transꢀ-
skewꢀ presented by the conformers A/B, and 2) gaucheꢀ-
gaucheꢀ-skew+ and skew+-gaucheꢀ-trans+-skew+ presented
by the conformers C/D. Figure 3 demonstrates remarkable
Interestingly, the analysis of main-chain torsion angles of
all the conformers reveals that the b-Ala and g-Abu resi-
dues adopt folded conformations, corresponding to gauche-
gauche-skew and skew-gauche-trans-skew orientations, re-
spectively. Also, the gauche orientation across the q and q1
torsion angles appears to be critical for promoting U-shaped
molecular structures in which in b-Ala and g-Abu residues
occupy the left-corner (or i+1) and right-corner (or i+2)
positions of the folded topology. Nevertheless, the confor-
mational restrictions imposed by the set of torsion angles
clearly precluded the formation of an amide–amide intramo-
lecular interaction. Unexpectedly, the enforced torsion
ꢀ
angles in each conformer disposition an unactivated C H
donor of the Boc group and the C-terminal C=O acceptor,
Figure 3. Superimposition of molecular structures of peptide Boc-b-Ala-
g-Abu-NH2 illustrating conformational similarities between conformers
A/B (left) and C/D (right), RMSD values being 0.342 and 0.366 ꢁ, re-
spectively. The superimposition was generated by using Mercury software
for only non-hydrogen atoms.
ꢀ
in all probability to fabricate an unconventional weak C
H···O intramolecular hydrogen bond, encompassing an un-
usual 14-membered ring motif. Employing more general
angle and distance criteria, the determined hydrogen bond
geometric parameters (Table 2) exhibited all the stereo-
ꢀ
chemical hallmarks of an effective C H···O hydrogen
conformational similarities between the two sets of conform-
ers, A/B and C/D. The structural disparity in the two sets
lies essentially in their handedness. Consequently, the analy-
sis allowed us to establish the existence of non-superimposa-
ble mirror-image relationships in a folded non-chiral one
component b,g-hybrid model system,[1h] perceived to be sta-
ꢀ
Table 2. The geometric parameters of the unconventional C H···O intra-
molecular hydrogen bonds for Boc-b-Ala-g-Abu-NH2.
Donor
D
Acceptor
A
Distance [ꢁ]
Angle [8]
ꢀ
D···A
H···A
D H···A
ꢀ
bilised by a weak C H···O intramolecular hydrogen bond.
ꢀ
C2 H (A)
O4
O4
O4
O4
3.721
3.777
3.822
3.940
2.872
2.957
3.388
3.531
148.0
144.2
109.8
108.3
Ever since Taylor and Kennard[7a] established unequivocal
existence and characterisation of different unconventional
hydrogen bonds, undoubted structural and/or functional im-
ꢀ
C3 H (B)
ꢀ
C3 H (C)
ꢀ
C1 H (D)
ꢀ
portance of C H···O interactions in a range of organic, bio-
organic and biological macromolecules have been docu-
bond.[7] The overall molecular topology displays close re-
semblance to the one described previously for b,a-hybrid
model peptides, mimicking a b-turn-like fold.[9] The compel-
ling evidence for the occurrence of tightly folded structures
was acquired by determining the shortest non-bonded dis-
tances between the N- and C-terminal groups. Expectedly,
the observed distances between the Boc and C-terminal
amide groups, that is, C4···N3 distances, in all the conform-
mented.[7,9,11] Even in small peptides, a C H···O hydrogen
ꢀ
bond is capable of exerting profound change(s) in torsion
angles; this testifies to its potential strength for stabilising
specific structural motifs.[1i,9,11f] Here, it must be noted that:
ꢀ
“the C H···O interaction is not really a van der Waals con-
tact but is primarily electrostatic; as its strength therefore falls
off much more slowly with distance. Therefore, even long
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Folding Propensity of an Unsubstituted b,g-Hybrid Peptide
FULL PAPER
ꢀ
Table 3. The geometric parameters of conventional N H···O intermolecular hydrogen
bonds for Boc-b-Ala-g-Abu-NH2.
C···O separations (about 4.00 ꢀ) have to be consid-
ered seriously.”[7b]
ꢀ
Donor D H Acceptor A Distance [ꢁ] Distance [ꢁ] Angle [8] Symmetry
ꢀ
The energetic contributions of C H···O hydrogen
ꢀ
dG
d
G
D H···A operation
bonds have appreciable magnitudes and are esti-
mated to be in the range of approximately 2.5–
Conformer A
ꢀ
N1 H
ꢀ
N2 H
O2D
O3D
O3A
O4D
2.892
3.073
3.008
2.808
2.136
2.228
2.160
1.956
146.51
167.23
168.72
170.30
[x+1/2,yꢀ1/2,z]
[x,y,z]
[x+1/2,y+1/2,z]
[x+1,y,z]
3.0 kcalmolꢀ1 in vacuo.[7f,12] The cohesive nature of
this interactions in 2 is suggested to be sufficiently
effective as primary structural determinant particu-
larly, in the absence of any other stabilising factor.
ACHTUNGTRENNUNG
N3-HA
ꢀ
N3 HB
ACHTUNGTRENNUNG
Conformer B
ꢀ
N1 H
ꢀ
N2 H
O2C
O3C
O3B
O4C
2.873
3.110
3.027
2.828
2.066
2.258
2.171
1.980
156.15
170.38
173.53
168.58
ACHTUNGTRNE[NUNG x,y,z]
ꢀ
Moreover, the C H···O hydrogen bond in each con-
[x+1/2,y+1/2,z]
[xꢀ1/2,y+1/2,z]
[xꢀ1/2,y+1/2,z]
ꢀ
former is formed by means of an unactivated C H
ꢀ
N3 HA
donor. In this regard, Desiraju and co-workers have
N3-HB
ꢀ
formerly emphasised that C H···O interactions can Conformer C
ꢀ
N1 H
O2B
O3B
O3C
O4B
2.869
3.088
3.005
2.814
2.120
2.235
2.154
1.963
145.32
171.05
170.46
170.17
[xꢀ1/2,yꢀ1/2,z]
ꢀ
be produced even by very feebly acidic C H groups
ꢀ
N2 H
ACHTUNGTRNE[NUNG x,y,z]
and not just activated ones.[7c,e] We, therefore, con-
ꢀ
N3 HA
[x+1/2,yꢀ1/2,z]
sider that the participation of an unactivated C-
ꢀ
N3 HB
ACHTUNGTRENNUNG
3
ꢀ
ꢀ
G
Conformer D
ꢀ
N1 H
ꢀ
N2 H
O2A
O3A
O3D
O4A
2.867
3.109
3.079
2.839
2.063
2.254
2.232
1.985
155.20
173.18
168.31
171.33
ACHTUNGTRNE[NUNG x,y,z]
drogen bond may represent another notable feature
of the crystal molecular structure. The lack of sta-
bilising force(s) generally results in unordered and/
or rather ill-defined structures.
[xꢀ1/2,y+1/2,z]
[xꢀ1/2,yꢀ1/2,z]
[xꢀ1/2,yꢀ1/2,z]
ꢀ
N3 HA
ꢀ
N3 HB
The observed molecular structures of 2, stabilised
ꢀ
by a weak C H···O intramolecular hydrogen bond,
apparently mimics a hydrogen bonded b-turn structure com-
prised of a-amino acids.[10] The f, y torsion angles of the
left- and right-corner residues determine the precise nature
of b-turn structures, that is, the b-turn types. Our analysis re-
veals that the left-corner b-Ala residue in molecules A/B
display the conformational characteristics of a d residue (f~
708, y~ꢀ1358) whereas, molecules C/D exhibit the features
of an l residue (f~ꢀ708, y~1358), constructive for type II’
and type II b-turn structures, respectively.[10] However, no
such correlations are perceived for the right-corner g-Abu
residue. Considering the magnitudes of f,y values across b-
Ala and g-Abu in conjunction with the orientation of central
peptide bond, the preferred U-shaped topology of 2 may be
recognised as being reminiscent of an amalgamated pseudo-
type II’–VIII’ (fi+1 ~708, yi+1 ~ꢀ1358, fi+2 ~ꢀ1358, yi+2
~
ꢀ
Figure 4. An illustration of N H···O intermolecular hydrogen bonds of
conformer A along with four symmetry related molecules observed in
ꢀ1358) or type II–VIII (fi+1 ~ꢀ708, yi+1 ~1358, fi+2 ~1358,
yi+2 ~1358) b-turn structure (Table 1 and Figure 3). To sub-
stantiate, in the crystal structure of Boc-b-Ala-Ac6c-OMe
(Ac6c=stereochemically constrained 1-aminocyclohexane-1-
carboxylic acid residue) the folded b-Ala residue (i.e., q or
m ~658) occupies the left-corner position of the pseudo-type
ꢀ
the crystal packing of Boc-b-Ala-g-Abu-NH2. Dashed lines indicate N
H···O hydrogen bonds. For the sake of clarity, the amide-NH groups in-
volved in intermolecular interactions is shown.
graphic ac plane, is shown in Figure 5. Along the a axis, the
molecules in each row interact in a pair-wise manner, that is,
in one row, the conformer A hydrogen bonds to molecule D
and vice versa (i.e., A:D pair) whereas, in the adjacent row,
molecule B hydrogen bonds to conformer C and vice versa
(i.e., B:C pair). There appears to be an estimated centro-
symmetric relationship between the conformers A/C, and B/
D (also see Table 1). Further, the b-Ala and g-Abu amide-
NH groups of all the conformers, that is, N1A–H, N2A–H;
N1B–H, N2B–H; N1C–H, N2C–H and N1D–H, N2D–H hy-
drogen bond with the urethane and b-Ala CO groups of the
symmetry related molecules D, C, B, and A, respectively.
Both the C-terminal amide-NH groups, N3–HA and N3–
HB, also take part in hydrogen bonding. One of the amide-
III (or III’) b-turn-like motif, stabilised by non-conventional
[9a]
ꢀ
weak C H···O intramolecular hydrogen bonds.
In the crystal, four independent conformers of 2 in the
asymmetric unit are primarily held together by conventional
amide–amide intermolecular interactions. All potential hy-
drogen-bond donor (i.e., amide-NH) and acceptor (i.e.,
ꢀ
amide-CO) groups of each molecule participate in N H···O
hydrogen bonding.[13] Table 3 summarises the determined
hydrogen bond geometric parameters. As illustrated in
ꢀ
Figure 4, the description of N H···O intermolecular interac-
tions is complete with the involvement of five symmetry re-
lated molecules. A perspective view of the crystal packing of
folded U-shaped molecules, as viewed along the crystallo-
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R. Kishore and P. Venugopalan
tion predominates in a relative-
ly non-polar solvent.
The analysis of the crystal
packing also revealed that inter-
molecular interactions are not
ꢀ
restricted to only N H···O hy-
drogen bonds. There exists a
more complex network of
weaker apolar interactions[7c,15]
vis-ꢂ-vis hydrophobic and/or
van der Waals contacts between
symmetry related molecules be-
longing to adjacent rows.
Figure 6 illustrates numerous
C···C short contacts between
Boc groups and methylene
units of b-Ala and g-Abu. As
recognised previously,[15] the ef-
fectual C···C distances in 2 also
ranged between approximately
3.80–4.20 ꢁ (Table 4). Howev-
er, there exists little experimen-
tal information concerning the
distance dependent effective-
ness of such hydrophobic con-
tacts. We presume that the ad-
Figure 5. The crystal packing of U-shaped molecular conformers of Boc-b-Ala-g-Abu-NH2, as viewed along
ꢀ
the c axis. The N H···O intermolecular hydrogen bonds are shown as dotted lines. All hydrogen atoms are
omitted for the sake of clarity.
NH groups, N3A–HA, N3B–HA, N3C–HA and N3D–HA,
interacts with the b-Ala CO group of their respective sym-
metry related molecules in the same row, whereas, the other
amide-NH, N3A–HB, N3B–HB, N3C–HB and N3D–HB,
hydrogen bonds with the g-Abu CO group of the conform-
ers D, C, B, and A, respectively
ditive nature of these interactions could be appreciable to
reinforce the crystal packing.
In general, proteinogenic amino acid side-chains undergo
rapid rotational motion that make peptide molecules more
ꢀ
ꢀ
flexible. Conversely, despite the fact that the CONH
(Figure 5 and Table 3). As a
result, the symmetry related
conformers in each row facili-
ꢀ
tate the construction of a N2
ꢀ
H···O3···H N3 type bifurcated
intermolecular hydrogen bond.
To ascertain that the crystal-
state conformation of the com-
pound is also the one that over-
whelmingly prevails in a solvent
of low polarity, the FT-IR ab-
sorption spectrum of 2 in dilute
CDCl3 solution (~1.0 mm) was
recorded.[14] As expected, the
observed IR spectrum in the in-
ꢀ
formative amide-A region (N
H stretching: 3500–3200 cmꢀ1)
exhibits no absorption band
below approximately 3400 cmꢀ1
(data not shown). The absence
of an absorption band below
3400 cmꢀ1, attributable to hy-
drogen
bonded
amide-NH
Figure 6. The crystal packing of U-shaped molecular conformers of Boc-b-Ala-g-Abu-NH2, as viewed along
the c axis. Thenon-covalent hydrophobic and/or van der Waals C···C contacts are shown by dotted lines. For
groups, confirms that the crys-
tal-state molecular conforma- the sake of clarity, all hydrogen atoms are omitted.
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Folding Propensity of an Unsubstituted b,g-Hybrid Peptide
FULL PAPER
Table 4. The determined C···C intermolecular distances in the crystal
packing of Boc-b-Ala-g-Abu-NH2.
plete absence of side-chains as well as amide–amide intra-
molecular interaction in 2, the structural role played by
Hydrophobic contacts
Distance [ꢁ] dACTHUGNETRNNU(G C···C)
ꢀ
weak C H···O intramolecular hydrogen bonds could be of
ꢀ
C1C C3D
4.125
3.970
4.069
3.916
3.954
3.875
3.944
4.067
3.849
3.888
4.128
paramount significance. The success of models in explaining
subtle, yet productive, structural variances in solid crystal-
line state is striking. In view of natural occurrence of the
two non-proteinogenic residues in bioactive molecules,[1i,4]
we believe that the characterisation of most probable molec-
ular conformations of the simplest b,g-hybrid dipeptide unit
may have wide structural implications for introducing novel
hydrogen bonded secondary structural mimics in designed
synthetic peptides.[2a]
ꢀ
C1B C6D
ꢀ
C2C C3D
ꢀ
C2D C6B
ꢀ
C3A C6C
ꢀ
C9B C9D
ꢀ
C9C C9D
ꢀ
C9B C9A
ꢀ
C9B C10A
ꢀ
C9C C10D
ꢀ
C9C C11D
ꢀ
ꢀ
ꢀ
ꢀ
(CH2)2 CONH
E
chains, peptide 2 exhibited multiple structural complexity,
that is, more than one hydrogen bonded molecular structure
in crystalline state. We, therefore, presume that the observed
solid-state conformations of 2 are likely to represent the
global energy minima of the b-Ala-g-Abu unit. Consequent-
ly, we conjecture that insertion of the b-Ala-g-Abu sequence
in short synthetic peptides, not influenced by any other
structure stabilising factor(s), may be expected to experi-
ence folded conformations. To emphasise, Karle, Balaram
and their colleagues have reported X-ray diffraction struc-
tures of two a-aminoisobutyric acid (Aib) containing oligo-
peptides with a centrally positioned b-Ala-g-Abu segmen-
t.[2a] In fact, the q and q1 torsion angles in these oligopepti-
des also preferred gauche orientations, although the precise
folding behaviour of the b-Ala-g-Abu segment, accommo-
dated in helical fold, is rather distorted (Table 5). From the
observed folding behaviour, it is plausible that an incidence
of the b-Ala-g-Abu unit in synthetic peptides could be effec-
tual and may be the method of choice for subtle modulation
of folded topological features confined to non-functionalised
b,g-hybrid sequence.
Conclusion
In summary, this study was an effort to establish the intrinsic
folding propensity of an unsubstituted b,g-hybrid peptide,
Boc-b-Ala-g-Abu-NH2. The model peptide adopted well-de-
fined folded conformations, mimicking a b-turn-like scaffold,
in crystalline state. The novel peptide chain reversal orient-
3
ꢀ
ed the unconventional C
G
groups to offer optimal spatial arrangement facilitating the
ꢀ
fabrication of a putative C H···O hydrogen bond as the
principal driving force for structure stabilisation. The varian-
ces in multiple conformers may further explain structural
complexity of the simplest b,g-hybrid peptide segment at the
molecular level. We also infer that the structures established
in the crystal state may correspond to the most preferred
conformation across the b-Ala-g-Abu unit. The presumed
structural and/or functional significance of b-Ala and g-Abu
moieties in bioactive molecules incline us to highlight that
analogous unsubstituted peptide units may have potential
therapeutic implications in peptide design and peptidomi-
metics research. Whether the non-functionalised b-Ala-g-
Abu peptide unit can actually be constructive for introduc-
ing more diverse folded topological features in short pepti-
des, merits further consideration. The structural explorations
of various correlated peptide sequences of varying chain
lengths are currently being pursued in our laboratory.
Crystal structure analysis of designed peptide molecules
has often provided unexpected observations. The construc-
tion of biologically relevant folded conformations in short
linear peptides, in principle, requires stereochemically con-
strained amino acid residues or chemical entities. Therefore,
the coexistence of multiple well-defined, folded conformers
in solid state, for the b,g-hybrid peptide represents an exam-
ple of the serendipitous observation. Despite intriguing fold-
ing-unfolding propensities associated with methylene units,
the adoption of biomimetic scaffoldings is clearly synchron-
ised by the gauche orientations of central torsion angles of
the b-Ala and g-Abu residues. We also assume that in com-
Experimental Section
All amino acids, reagents and deuterated NMR solvents were from
Sigma–Aldrich (St. Louis, Missouri, USA). All peptides were synthesised
by employing standard solution-phase methods.[16] The intermediates and
Table 5. Comparison of selected main-chain torsion angles [8] across the b-Ala-g-Abu unit in model peptides.[a]
Peptide
b-Ala
g-Abu
Ref.
f
q
y
f
q1
q2
y
Boc-b-Ala-g-Abu-NH2 (A)
Boc-b-Ala-g-Abu-NH2 (B)
Boc-b-Ala-g-Abu-NH2 (C)
Boc-b-Ala-g-Abu-NH2 (D)
69.8
68.9
ꢀ70.7
ꢀ69.4
ꢀ103.0
ꢀ103.0
53.6
55.0
ꢀ53.0
ꢀ55.8
76.0
ꢀ130.8
ꢀ137.8
131.0
139.7
162.0
ꢀ133.4
ꢀ133.5
132.7
68.8
64.6
ꢀ69.6
ꢀ64.5
58.0
ꢀ171.4
ꢀ176.5
174.16
173.6
66.0
ꢀ141.4
ꢀ124.9
137.2
this work
this work
this work
this work
[2a]
129.5
125.3
Boc-Leu-Aib-Val-b-Ala-g-Abu-Leu-Aib-Val-OMe[a]
Boc-Leu-Aib-Val-b-Ala-g-Abu-Leu-Aib-Val-Ala-Leu-Aib-OMe[a]
ꢀ108.0
ꢀ146.0
78.0
107.0
ꢀ121.0
63.0
57.0
ꢀ121.0
[2a]
[a] Boc, OMe and NH2 moieties are tert-butyloxycarbonyl, methylester and C-terminal amide groups, respectively.
Chem. Eur. J. 2013, 00, 0 – 0
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R. Kishore and P. Venugopalan
final peptides (1–3) were purified to homogeneity by silica-gel (60–120
mesh) column chromatography with 2.0–3.0% MeOH/CHCl3 mixtures as
eluents. The melting points reported are uncorrected. Aluminium sheet
coated with silica-gel (60F254: Merck) was used for determining Rf values.
The identity and purity of the covalent structures were ascertained by an-
Soc. Perkin Trans. 2 1992, 1233–1237; b) G. P. Dado, S. H. Gellman,
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Biomol. Chem. 2006, 4, 4214–4244 and references therein.
1
alytical one- (1D) and two-dimensional (2D) 1H$ H NMR correlation
spectroscopy (COSY) experiments.[17] Chemical-shift (d) values are re-
ported (ppm) with respect to the internal reference tetramethylsilane
1
(TMS) at 0.0 ppm for H NMR spectroscopy (for details, see the Support-
ing Information).
Boc-Gly-g-Abu-NH2 (1): M.p.=1358C; Rf =0.38 in 10% MeOH/CHCl3
mixture. 1H NMR (300 MHz, conc. ~8.0 mm in CDCl3): d=1.46 (9H, s,
(CH3)3), 1.87 (2H, m, g-Abu CbH2), 2.27 (2H, t, g-Abu CaH2), 3.36 (2H,
q, g-Abu CgH2), 3.77 (2H, d, Gly CaH2), 5.10 (1H, t, Gly NH), 5.38, 6.07
(2H, s1s2, amide-NH2), 6.47 ppm (1H, t, g-Abu NH).
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Boc-b-Ala-g-Abu-NH2 (2): M.p.=1428C; Rf =0.33 in 10% MeOH/CHCl3
mixture. 1H NMR (300 MHz, conc. ~5.0 mm in CDCl3), d=1.43 (9H, s,
(CH3)3), 1.85 (2H, m, g-Abu CbH2), 2.27 (2H, t, g-Abu CaH2), 2.41 (2H,
t, b-Ala CaH2), 3.33 (2H, m, g-Abu CgH2), 3.40 (2H, m, b-Ala CbH2),
5.17 (1H, t, b-Ala NH), 5.43, 6.17 (2H, s1s2, amide-NH2), 6.28 ppm (1H,
t, g-Abu NH).
Boc-g-Abu-g-Abu-NH2 (3): M.p.=818C, Rf =0.26 in 10% MeOH/CHCl3
mixture. 1H NMR (300 MHz, conc. ~6.0 mm in CDCl3): d=1.43 (9H, s,
(CH3)3), 1.80 (2H, m, g-Abu1 CbH2), 1.86 (2H, m, g-Abu2 CbH2), 2.22
(2H, t, g-Abu1 CaH2), 2.29 (2H, t, g-Abu2 CaH2), 3.16 (2H, q, g-Abu1
CgH2), 3.33 (2H, q, g-Abu2 CgH2), 4.82 (1H, t, Abu1 NH), 5.50, 6.50 (1H,
s1s2, amide-NH2), 6.61 ppm (1H, t, g-Abu2 NH).
X-ray diffraction: Colourless single crystals suitable for X-ray diffraction
experiments were grown from a solvent mixture of MeOH/CHCl3 (3:2 v/
v) by slow evaporation at room temperature. A good quality crystal of
the dimension (0.25ꢃ0.20ꢃ0.15 mm) was used for determination of the
cell parameters and crystal data collection. The X-ray diffraction intensi-
ties were collected at room temperature by using Siemens P4 diffractom-
eter equipped with MoKa radiation (l=0.71073 ꢁ) and highly oriented
graphite monochromator. A total of 6154 reflections were measured in
the 2qꢀq scan mode in the range 2.168ꢃqꢃ25.508. Crystal data:
C12H23N3O4, Mr =273.33; monoclinic, Cc; a=13.334 (2), b=13.360 (1),
c=33.824 (3); a=g=90.08, b=92.950(10); Z=16; V=6017.5(11) ꢁ3;
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1calcd =1.207 mgmꢀ13 and F
ACTHNUGRTENUNG(000)=2368. The observed intensity data were
corrected for Lorentzian and polarisation effects.
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The structure was solved by direct methods by using the SHELX-97
package and refined with the same programme suite.[18] A total of 686 pa-
rameters were used. All non-hydrogen atoms were refined anisotropically
and hydrogen atoms were included stereochemically in refinement as
riding model. The structure was refined by full matrix least-squares re-
finement (on F2) and converged to a final R value of 0.0475 [wR2=
0.1065, GOF=1.0631 for 5842 reflections [Iꢄ2s(I)]. A weighting scheme
2
of the form w=1/[s
2(F0 )+(aP)2 +bP] with a=0.0501, b=2.7973 was
used. The final difference map was featureless (D1max =0.207 eꢁꢀ3 and
D1max =ꢀ0.215 ꢁꢀ3).
CCDC-924477 (2) contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif.
Acknowledgements
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Folding Propensity of an Unsubstituted b,g-Hybrid Peptide
FULL PAPER
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Received: February 18, 2013
Published online: && &&, 0000
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!