Efficient access to enantiopure γ4-amino acids with proteinogenic side-chains and structural investigation of γ4- asn and γ4-ser in hybrid peptide helices

Article abstract of DOI:10.1002/chem.201302732

Hybrid peptides composed of α- and β-amino acids have recently emerged as new class of peptide foldamers. Comparatively, γ- and hybrid γ-peptides composed of γ⁴-amino acids are less studied than their β-counterparts. However, recent investigations reveal that γ⁴-amino acids have a higher propensity to fold into ordered helical structures. As amino acid side-chain functional groups play a crucial role in the biological context, the objective of this study was to investigate efficient synthesis of γ⁴-residues with functional proteinogenic side-chains and their structural analysis in hybrid-peptide sequences. Here, the efficient and enantiopure synthesis of various N- and C-terminal free-γ⁴-residues, starting from the benzyl esters (COOBzl) of N-Cbz-protected (E)-α,β-unsaturated γ-amino acids through multiple hydrogenolysis and double-bond reduction in a single-pot catalytic hydrogenation is reported. The crystal conformations of eight unprotected γ⁴-amino acids (γ⁴-Val, γ⁴-Leu, γ⁴-Ile, γ⁴-Thr(OtBu), γ⁴-Tyr, γ⁴-Asp(OtBu), γ⁴- Glu(OtBu), and γ-Aib) reveals that these amino acids adopted a helix favoring gauche conformations along the central C^γi￡ C^β bond. To study the behavior of γ⁴- residues with functional side chains in peptide sequences, two short hybrid γ-peptides P1 (Ac-Aib-γ⁴-Asn-Aib-γ⁴-Leu- Aib-γ⁴-Leu-CONH₂) and P2 (Ac-Aib- γ⁴-Ser-Aib-γ⁴-Val-Aib-γ⁴-Val- CONH₂) were designed, synthesized on solid phase, and their 12-helical conformation in single crystals were studied. Remarkably, the γ⁴-Asn residue in P1 facilitates the tetrameric helical aggregations through interhelical H bonding between the side-chain amide groups. Furthermore, the hydroxyl side-chain of γ⁴-Ser in P2 is involved in the interhelical H bonding with the backbone amide group. In addition, the analysis of 87 γ⁴-residues in peptide single-crystals reveal that the γ⁴-residues in 12-helices are more ordered as compared with the 10/12- and 12/14-helices. Copyright

Full text of DOI:10.1002/chem.201302732

DOI: 10.1002/chem.201302732
Efficient Access to Enantiopure g4-Amino Acids with Proteinogenic
Side-Chains and Structural Investigation of g4-Asn and g4-Ser in Hybrid
Peptide Helices
Sandip V. Jadhav, Rajkumar Misra, Sumeet K. Singh, and Hosahudya N. Gopi*[a]
Abstract: Hybrid peptides composed of
a- and b-amino acids have recently
emerged as new class of peptide fold-
(COOBzl) of N-Cbz-protected (E)-a,b-
unsaturated g-amino acids through
multiple hydrogenolysis and double-
bond reduction in a single-pot catalytic
hydrogenation is reported. The crystal
conformations of eight unprotected g4-
amino acids (g4-Val, g4-Leu, g4-Ile, g4-
Asn-Aib-g4-Leu-Aib-g4-Leu-CONH2)
and P2 (Ac-Aib-g4-Ser-Aib-g4-Val-Aib-
g4-Val-CONH2) were designed, synthe-
sized on solid phase, and their 12-heli-
cal conformation in single crystals were
studied. Remarkably, the g4-Asn resi-
due in P1 facilitates the tetrameric
ACHTUNGTRENNUNGamers. Comparatively, g- and hybrid g-
peptides composed of g4-amino acids
are less studied than their b-counter-
parts. However, recent investigations
reveal that g4-amino acids have
a higher propensity to fold into or-
dered helical structures. As amino acid
side-chain functional groups play a cru-
cial role in the biological context, the
objective of this study was to investi-
gate efficient synthesis of g4-residues
with functional proteinogenic side-
chains and their structural analysis in
hybrid-peptide sequences. Here, the ef-
ficient and enantiopure synthesis of
various N- and C-terminal free-g4-resi-
dues, starting from the benzyl esters
Thr
N
U
heliACTHNGUTRENNUcG al aggregations through interheli-
Glu
N
cal H bonding between the side-chain
amide groups. Furthermore, the hy-
droxyl side-chain of g4-Ser in P2 is in-
volved in the interhelical H bonding
with the backbone amide group. In ad-
dition, the analysis of 87 g4-residues in
peptide single-crystals reveal that the
g4-residues in 12-helices are more or-
dered as compared with the 10/12- and
12/14-helices.
these amino acids adopted a helix fa-
voring gauche conformations along the
g
b
ꢀ
central C
C bond. To study the be-
havior of g4-residues with functional
side chains in peptide sequences, two
short hybrid g-peptides P1 (Ac-Aib-g4-
Keywords: amino acids
· crystal
conformations · helical aggregation ·
hydrogenation · peptides
Introduction
12-heliACTHNGUTERNNcUG al structures composed of 1:1 alternating a- and g-
amino acids.[6] We recently reported the structural analogy
of the a,g-hybrid peptides containing g4-amino acids with re-
spect to the a-peptide helices and b-peptide 12-helix.[7] The
folding properties of a,g-hybrid peptides in both solution
and single crystals suggest that they can be used as mimetic
of b-peptide 12-helices without having stereochemically con-
strained and preorganized amino acids.[7a] However, thus far
only few g4-residues with hydrophobic side-chains (g4-Ala,
g4-Val, g4-Leu, and g4-Phe) have been utilized in the design
and synthesis of hybrid peptides. The g4-amino acids with
functional side-chains have not been explored in the design
of g-peptides probably due to the difficulties in the synthesis
of stereochemically pure and orthogonally protected g4-
amino acids. As amino acid side-chains play a pivotal role in
biomolecular interactions, we sought to investigate the mild,
racemization-free, and scalable protocol for the synthesis of
g4-residues containing side-chain functional groups and their
utility in the synthesis of g- and hybrid g-peptides. Herein,
we report the efficient and racemization-free protocol for
synthesis of free g4-amino acids, crystal conformations of
eight g4-amino acids having proteinogenic side-chains, solid-
phase synthesis and crystal conformations of a,g-hybrid
hexapeptide 12-helices comprised of g4-asparagine (g4-Asn)
The structural and functional properties of b- and hybrid b-
peptides have been extensively investigated over the last fif-
teen years.[1,2] The advantage of hybrid peptides over the ho-
mooligomers is that various ordered helical structures can
be designed by varying the position and sequence patterns
of the constituent amino acids. The structural diversity of
a,b-hybrid peptides have been exploited in the design of
biologically relevant peptidomimetics.[3] In addition, prelimi-
nary investigations by the groups of Seebach[4] and Hanes-
ACHTUNGTRENNUNG
sian[5] also reveal that the backbone flexible g4-peptides
have a general tendency to fold into helical conformations.
In continuation, the theoretical and experimental investiga-
tions on a,g-hybrid peptides reveal the formation of stable
[a] S. V. Jadhav, R. Misra, S. K. Singh, Dr. H. N. Gopi
Department of Chemistry
Indian Institute of Science Education and Research (IISER)
Dr. Homi Bhabha, Road, Pune-411008 (India)
Fax : (+91)20-2590-8186
E-mail: hn.gopi@iiserpune.ac.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302732.
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FULL PAPER
and g4-serine (g4-Ser) along with g4-leucine, and g4-valine.
Crystal structure analysis of free g4-amino acids reveals that,
with the exception of g4-Asp
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
g
b
ꢀ
ed gauche conformations along the C
C bond and anti
b
a
ꢀ
conformations along C C . Crystal conformations of hybrid
peptides yield an intriguing observation that the amide side-
chain of g4-Asn drives tetrameric helical oligomerization
through interhelical H bonding, which is very similar to that
observed in membrane proteins; whereas, the hydroxyl side-
chain of g4-Ser was directly involved in interhelical H bond-
ing with the backbone amide.
Scheme 1. A) Synthesis of g4-amino acids; i) Ph3P=CH-COOBzl, THF;
ii) Pd/C, H2, 10% CH3COOH in THF; B) local torsion angles of g4-resi-
dues.
Results and Discussion
Table 1. g4-Amino acids from Cbz-protected E-vinylogous amino benzyl
esters.
Synthesis and crystal conformations of g4-amino acids: As
a part of our investigation to understand the structural fea-
tures of a, g-hybrid peptides containing proteinogenic
amino acid side-chains, we sought to utilize the benzyl esters
of N-9-fluorenylmethoxycarbonyl (Fmoc)-protected a,b-un-
saturated g-amino acids as starting materials for the synthe-
sis of solid phase compatible N-Fmoc-g4-amino acids. The
pure Fmoc-protected E-vinylogous benzyl esters were sub-
jected to the catalytic hydrogenation for the concomitant re-
duction of double bonds as well as benzyl ester deprotec-
tion. During the process we isolated the byproduct 9-
methyl-9H-fluorene (see the Supporting Information),
which suggests that Fmoc deprotection occurs during the
process, leading to the decreased yield of final N-Fmoc-pro-
tected g4-amino acids. The lower yields and unexpected by-
product led us to search for a suitable protecting group
strategy for the synthesis of g4-amino acids. During the pro-
cess, we realized that the amino protecting group benzyloxy
carbonyl (Cbz) and benzyl ester can be removed at once by
using catalytic hydrogenation under neutral or mild acidic
conditions with concomitant reduction of the double bond.
We recently exploited this protocol for the synthesis of the
free g4-Phe.[7a] To understand the compatibility of this proto-
col to the other amino acids with functional side chains and
also to realize the racemization during the g4-amino acid
synthesis various N-Cbz-protected (E)-a,b-unsaturated
amino acid benzyl esters were synthesized by using the
Wittig reaction.[8] The schematic representation of the syn-
thesis of g4-amino acids starting from N-Cbz-amino alde-
hydes is shown in Scheme 1A. All N-Cbz-protected (E)-a,b-
unsaturated amino acid benzyl esters (Ia–l, Table 1) were
subjected to the catalytic hydrogenation in 10% acetic acid
in THF. The solvent conditions were optimized based on the
solubility of the free amino acids in acidic solution. As an-
ticipated the hydrogenolysis of both Cbz- and benzyl ester
along with the double bond reduction were achieved in
a single step and the g4-amino acids (IIa–l) were isolated as
acetate salts in moderate to good yields (60–90%). No cy-
clized products of g-residues were observed in the process
of catalytic hydrogenation from the E-vinylogous amino
No.
I[a]
II
Yield
[%]
a
b
c
d
e
f
g
h
i
Cbz-dgAla-OBzl
Cbz-dgVal-OBzl
Cbz-(D)dgVal-OBzl
Cbz-dgLeu-OBzl
Cbz-dgIle-OBzl
Cbz-dgSer
Cbz-dgThr
Cbz-dgAsp
Cbz-dgGlu(OtBu)-OBzl
H-g4-Ala-OH
H-g4-Val-OH
H-(D)g4-Val-OH
H-g4-Leu-OH
H-g4-Ile-OH
82
76
70
86
65
75
80
90
81
80
85
60
N
H-g4-Ser
H-g4-Thr
H-g4-Asp
H-g4-Glu
ACHTUNGTRENNUNG
G
ACHTUNGTRENNUNG
E
ACHTUNGTRENNUNG
N
ACHTUNGTRENNUNG
j
k
l
Cbz-dgTrp-OBzl
Cbz-dgTyr-OBzl
Cbz-dgAib-OBzl
H-g4-Trp-OH
H-g4-Tyr-OH
H-g-Aib-OH
[a] dg=a,b-Dehydro g-amino acid.
acids. Except for g4-Ala (IIa), (D) g4-Val (IIc), g4-Ser
ACHTUNGTRENNUNG(OtBu)
(IIf), and g4-Trp (IIj), we were able to obtain the single
crystals for all other eight g4-amino acids listed in Table 1
(IIb, IId, IIe, IIg, IIi, IIk, and IIl). The crystal conforma-
tions of g4-amino acids are shown in the Figure 1. The tor-
sional values were measured by introducing additional varia-
g
b
a
g
b
a
ꢀ
ꢀ
ꢀ
ꢀ ꢀ ꢀ
bles q1 (N C
C C ) and q2 (C C C C=O). It has been
observed that g4-residues adopt the gauche conformation
along q1 and q2 in helical peptides.[7,9–11] Here, crystal struc-
ture analysis of g-residues revealed that except g4-Asp, all
free g4-amino acids adopted a gauche conformation along q1
and an anti conformation along q2, which underlines the pro-
pensity of g4-residues towards helical organization. The de-
tailed crystallographic information of the g4-amino acids is
given in the Supporting Information. Furthermore, all the
free g4-amino acids were protected with Fmoc succinimide
(Fmoc-OSu)/Na2CO3 and isolated in a moderate to good
yields (data not shown).
Racemization studies: In addition, to understand the race-
mization during the synthesis of g4-amino acids, we coupled
Fmoc-g4-Ala obtained after the Fmoc-protection of g4-Ala,
with (ꢀ)-, (+)-, and (ꢁ)-a-methyl benzylamine by using
standard N,N-dicyclohexylcarbodiimide (DCC)/1-hydroxy-
benzotriazole (HOBt) coupling conditions. All three amide
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H. N. Gopi et al.
(ag)n-, (aga)n-, and (agg)n-hybrid peptide sequence helical
patterns.[9] Further, Gellman et al. reported the hybrid se-
quence of GCN4-pLI containing g4-Ala.[10] In addition the
Guichard group reported single-crystal conformations of
hybrid urea peptides containing g4-Phe, g4-Leu, and g4-Val
residues.[11] Recent crystal structure analysis of the a,g4-
hybrid peptides revealed that the orientation of the H bond-
ing in the hybrid peptides is similar to that of a-helices (a-
helix and 310-helix).[7,9] The a-helix is stabilized by the intra-
molecular H bonds between residues
i
to i+4
!
[CO(1) NH(5)], however, the NHs of the first three resi-
dues and CO groups of last three residues lack internal
H bonding. In contrast, hybrid peptide 12-helices are stabi-
lized by intramolecular H bonds between residues i to i+3
!
[CO(1) NH(4)] similar to the 310-helix, only two NHs at
the N termini and two CO groups at the C termini lack the
internal H bonding. Based on the protein structure analysis,
it is believed that amino acids such as asparagine and serine
at the N terminus may supply the H-bond partners for the
unpaired backbone CO and NH groups and induce the sta-
bility in the a-helices through side-chain-backbone interac-
tions.[12] This hypothesis was further verified using synthetic
model peptides by incorporating Asp, Asn, Ser, Gln, and so
on, at the N terminus.[13] In addition, Asn is also known for
oligomerization of coiled-coil peptides through intermolecu-
lar H bonding between the side-chain amide groups.[14] In
this regard, to understand the role of g4-Asn and g4-Ser, we
designed two model peptides P1 (Ac-Aib-g4-Asn-Aib-g4-
Leu-Aib-g4-Leu-CONH2) and P2 (Ac-Aib-g4-Ser-Aib-g4-
Val-Aib-g4-Val-CONH2) by incorporating g4-Asn and g4-Ser,
respectively, at the second positions of the hybrid hexapep-
Figure 1. Crystal conformations of g4-amino acids. The torsion angles
g
b
a
g
b
a
ꢀ
ꢀ
ꢀ
ꢀ ꢀ ꢀ
along the N C
C C and C C C C’ are given below the structures
(q’ indicates the torsion angles of the side chains).
tides. The g4-Asn(Trt) and g4-Ser
AHCTUNGTRENUNGN ACHTUNRTGENNUNG ACTHNUGTRENN(UGN OtBu) were synthesized as
shown in Scheme 1 and protected with the Fmoc group.
Peptides were synthesized on Knorr amide 4-methylbenz-
AHCTUNGTREGhNNUN ydrylamine (MBHA) resin by using the standard Fmoc-
chemistry protocol. The coupling reactions were mediated
by O-benzotriazole-N,N,N’,N’-tetramethyluronium hexa-
fluorophosphate (HBTU)/HOBt coupling conditions. The
HPLC-purified peptides were subjected for the crystalliza-
tion in a various solvent combinations.
Scheme 2. The (ꢀ)-, (+)- and (ꢁ)-a-methyl benzylamine derivatives of
Fmoc-g4-alanine.
derivatives (Scheme 2) were isolated and subjected to chiral
HPLC using a CHIRALPAK IA analytical column. The
HPLC profiles of all three amide derivatives are shown in
the Supporting Information. The Fmoc-g4-Ala derivative
with (ꢀ)-a-methyl benzylamine (A) eluted with a retention
time (tR) of 8.0 min, whereas the amide with (+)-a-methyl
benzylamine (B) eluted at tR =8.6 min. The Fmoc-g4-Ala de-
rivative with (ꢁ)-a-methyl benzylamine (C) gave two peaks
at tR =8.1 and 8.6 min corresponding to the (A) and (B),
respectively. The results indicate that the procedure we
adapted for the synthesis of g4-amino acids is free from
racemization.
Crystal structure analysis: Suitable X-ray quality crystals of
P1 and P2 were obtained from the slow evaporation of a so-
lution of aqueous methanol. The X-ray structures of the
hybrid peptides P1 and P2 are shown in Figure 2. As antici-
pated, both the peptides adopted 12-helical conformations
in single crystals. Single-crystal analysis of P1 reveals the
presence of four molecules in the asymmetric unit. The heli-
cal structure is stabilized by four intramolecular hydrogen
ꢀ
bonds between i and i+3 residues (C=O(i)···H NACTHNUTRGENUG(N i+3), in-
volved 12-atom ring H bonds). The peptides are fully hy-
drated with twenty water molecules. The top view of the
helices is shown in Figure 3A. Remarkably, the hydrophobic
groups projected away from the water molecules and the
polar head-groups containing amide side-chains are project-
ed inside in the crystal packing. All four peptide molecules
in the asymmetric unit are oriented antiparallel to one an-
Design, synthesis and crystal conformations of a/g4-hybrid
peptides containing polar amino acids: Previously we dem-
onstrated that the stable 12-helical conformations of various
a,g-hybrid peptides in single crystals containing g4-Phe.[7]
Recently, Balaram and colleagues utilized g4-Val to generate
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Efficient Access to Enantiopure g4-Amino Acids
FULL PAPER
been observed in the membrane proteins.[15] Water mole-
cules are mostly accumulated near the polar side-chain of
g4-Asn of P1 (Figure 3C and D). Analysis of the torsional
variables of g-residues, g4-Asn and g4-Leu, reveal that both
q1 and q2 adopted gauche+,gauche+ conformations, and
nicely accommodated into the helix. Interestingly, the C-ter-
minal amide group is not involved in the canonical intramo-
lecular H bonding, however, it is involved in the strong in-
termolecular H bonding with N-terminal amide of other
helix and water molecules.
Peptide P2 was synthesized by replacing g4-Asn with g4-
Ser, and g4-Leu with g4-Val. Crystal structure analysis de-
picts single molecule of P2 in the asymmetric unit. Analysis
of the crystal structure reveals that P2 also adopts a right-
handed helical conformation and the structure is stabilized
by the five consecutive intramolecular 12-membered
H bonds between the i and i+3 residues. Both C-terminal
amide-NH and N-terminal acetyl-CO groups are involved in
the intramolecular H bonding. Similar to P1, the torsional
variables q1 and q2 of the g-residues, g4-Ser and g4-Val,
adopted gauche+,gauche+ conformations, and accommodat-
ed into the helix. The polar N-terminal NHs and C-terminal
carbonyl groups involved in the intermolecular H bonds
with other helical peptides in a head-to-tail fashion mediat-
ed by a single water molecule. On substitution of g4-Asn
with g4-Ser, helical oligomerization through side-chain
amide H-bonding interactions were not observed. However,
the g4-Ser side-chain hydroxyl group is directly involved in
the intermolecular H bonding with the C-terminal back-
bone-amide group of other helical peptides. The antiparallel
sidewise interactions of P2 are shown in the Supporting In-
formation. In contrast to P1, peptide P2 is stabilized by five
consecutive intramolecular H bonds. The average torsion
angles of g4-residues in both the helical peptides are given in
the Table 2. The H-bond parameters and the torsional
Figure 2. X-ray structures of a,g4-hybrid peptides P1 and P2.
Table 2. Average torsion angles for g4-residues in P1 and P2.
Peptide
Residue
f
q1
q2
Y
P1
g4-Asn
g4-Leu
g4-Ser
g4-Val
ꢀ126
ꢀ125
ꢀ136
ꢀ129
52
52
55
53
61
62
58
60
ꢀ123
ꢀ118
ꢀ129
ꢀ119
P2
angles of a-residues are given in the Supporting Informa-
tion. Analysis of the torsional variables reveals that both f
(C’-N-Cg-Cb) and y (Cb-Ca-C’-N) torsional angles of g-resi-
dues adopted the anticlinal conformations with values of
approximately ꢀ1208, and q1 and q2 adopted the gauche+
Figure 3. A) Crystal packing of P1 depicting tetrameric helical aggrega-
tion; B) g4-asparagine side-chain amide-mediated interhelical H bonds in
the tetrameric aggregation; C) and D) channel of water molecules along
the g4-Asn side chains in the crystal packing.
AHCTUNGTREG(NNNU +608) conformations in both peptides. Furthermore, the a-
residue, Aib, displayed average f and y values (ꢀ60ꢁ3)
and (ꢀ40ꢁ5)8, respectively, in both the hybrid peptides.
These values are found to be consistent with the other a,g-
hybrid peptides containing 1:1 alternating a- and g4-amino
acids. Further, we analyzed the torsional variables of all g4-
residues so far reported in the crystal structures of hybrid
other. A more intriguing part of structure is that the side-
chain amide groups of g4-Asn in all four antiparallel helices
drives the helical oligomerization through interhelical
H bonding (Figure 3B). A similar type of interhelical hydro-
gen bonding, which regulate the strong interactions, has also
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H. N. Gopi et al.
Asn residues in the hybrid-peptide helix facilitates the heli-
cal aggregations through interhelical H bonding between the
g4-Asn amide side-chains. Further, the hydroxyl side-chain
of g4-Ser showed the intermolecular H bond with backbone
amide of another helix in the crystal packing. Analysis of
the torsional angles of g4-residues in the hybrid-peptide heli-
ces reveals the anticlinal conformations along the f and y,
whereas gauche+ conformations were revealed along q1 and
q2. Overall, the efficient synthesis of g4-amino acids, solid-
phase synthesis of a,g4-hybrid-peptide 12-helices containing
polar side-chains and their crystal conformations reported
here can be further utilized for the construction of g- and
hybrid g-peptides for their potential applications in the
design of peptidomimetics and biomaterials.
Experimental Section
General procedure for the Synthesis of g4-amino acids: The suspension
of activated Pd/C (20% by weight) and benzyl esters of N-Cbz-protected
vinylogous amino acid (4 mmol), which was synthesized using the report-
ed method, in 10% acetic acid in THF (20 mL) was stirred overnight at
room temperature in the presence of hydrogen. After completion of the
reaction, Pd/C was filtered through the bed of Celite and the filtrate was
evaporated to dryness under vacuum to obtain the corresponding free g4-
amino acids with a gummy consistency. The pure g4-amino acids were iso-
lated as a white powder after trituration with cold diethyl ether in moder-
ate to good yield.
Synthesis of hybrid peptides P1 and P2: Peptides P1 and P2 were synthe-
sized by manual solid-phase peptide synthesis on a Knorr Amide MBHA
resin by using Fmoc chemistry (0.1 mmol scale). Coupling reactions were
performed by using the HBTU/HOBt activation protocol. Fmoc depro-
tections were facilitated by using 20% piperidine in DMF. The N termi-
nus of the peptide was capped with an acetyl group. Peptides were
cleaved from the resin by using 95% trifluoroacetic acid, 2.5% water
and 2.5% triisopropyl silane cleavage mixture. Precipitation of the crude
products in cold diethyl ether and subsequent drying under reduced pres-
sure yielded crude powders. Reverse-phase HPLC purification (detector:
254 nm and 220 nm) on a C18 column (70–90% methanol in 40 min) at
a flow rate of 1.5 mLminꢀ1 was carried out to obtain the pure peptide.
Figure 4. A) A two-dimensional Ramachandran-type plot depicting f and
y torsion angles for g4-residues from the recently reported hybrid g-pep-
tide helices along with g4-residues from the present work. 87 g4-residues
which are participated in the helical conformations are only considered
for the analysis. The q1 and q2 torsion angles were kept constant as they
were adopted the gauche conformation; B) expanded view of the
bottom-left quadrant of the plot.
helices.[7,9–11] The two-dimensional Ramachandran-type plot
for f and y of all g4- residues including g4-residues in P1
and P2 is shown in Figure 4. Analysis reveals that the g4-res-
idues in C12/C14-helical structures are little scattered. How-
ever, g4-residues in C12-helical peptides were found to be
more ordered.
Crystal structure analysis: g4-Asp(OtBu): Crystals of g4-Asp
ACTHNUTRGENNUG ACHUTGNTREN(NUNG OtBu) were
grown by slow evaporation from a solution of aqueous methanol. A
single crystal, rectangular in shape (0.80ꢁ0.30ꢁ0.20 mm3), was mounted
on a loop. The X-ray data were collected at a temperature of 100(2) K on
a Bruker AXS SMART APEX CCD diffractometer using MoKa radiation
(l=0.71073 ꢂ), w-scans (2q=56.568), for a total number of 8874 inde-
pendent reflections. Space group P21; a=6.039(7), b=5.657(7), c=
17.257(19) ꢂ; a=g=908, b=91.628(18)8, V=589.3(12) ꢂ3, monoclinic
P; Z=2 for the chemical formula C10H17N1O4; 1calcd =1.213 mgmꢀ3; m=
Conclusion
0.093 mmꢀ1; F
ACTHNUTRGNE(NUG 000)=232. The structure was obtained by direct methods
using SHELXS-97. All non-hydrogen atoms were refined anisotropically.
The hydrogen atoms were fixed geometrically in the idealized position
and refined in the final cycle of refinement as riding over the atoms to
which they are bonded. The final R value was 0.0936 (wR2=0.2983) for
We have reported the mild and efficient synthesis of various
N- and C-terminal unprotected g4-amino acids. All g4-amino
acids were isolated in moderate to good yields. The crystal
conformations of g4-residues reveal that they adopt helix-fa-
2781 observed reflections (F0 ꢂ4s(jF0j)) and 141 variables; S=0.969.
T
The largest deference peak and hole were 0.581 and ꢀ0.360 eꢂꢀ3
,
g
b
a
ꢀ
ꢀ ꢀ
voring gauche conformations along the N C
C C bond
respectively.
g
b a
ꢀ
ꢀ ꢀ
(q1) and the anti conformation along the C
C
C
C’ (q2)
Peptide P1: Crystals of P1 were grown by slow evaporation from a solvent
composition of methanol/water (3:1). A single crystal, rectangular in
shape (0.80ꢁ0.30ꢁ0.20 mm3), was mounted on a loop. The X-ray data
were collected at 100(2) K temperature on a Bruker AXS SMART
APEX CCD diffractometer using MoKa radiation (l=0.71073 ꢂ), w-
scans (2q=56.568), for a total number of 8874 independent reflections.
Space group P21; a=15.997(2), b=34.214(4), c=18.3336(19) ꢂ; a=g=
bond. We further extended the design of a,g-hybrid peptides
by incorporating g4-amino acids containing side-chain func-
tional groups, which have not been explored in the design of
hybrid peptides. The hexapeptides P1 and P2 displayed 12-
helical conformations in single crystals. The insertion of g4-
16260
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ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 16256 – 16262

Efficient Access to Enantiopure g4-Amino Acids
FULL PAPER
908, b=111.790(2)8, V=9317.4(19) ꢂ3, monoclinic P; Z=2 for the
Jaun, Angew. Chem. 1999, 111, 1700–1703; Angew. Chem. Int. Ed.
1999, 38, 1595–1597; d) S. H. Choi, I. A. Guzei, L. C. Spencer, S. H.
Gellman, J. Am. Chem. Soc. 2010, 132, 13879–13885; e) L. Berlicki,
L. Pilsl, E. Wꢃber, I. M. Mꢄndity, C. Cabrele, T. A. Martinek, F.
Fꢅlçp, O. Reiser, Angew. Chem. 2012, 124, 2251–2255; Angew.
Chem. Int. Ed. 2012, 51, 2208–2212; f) B. Jagadeesh, A. Prabhakar,
G. D. Sarma, S. Chandrasekhar, G. Chandrashekar, M. S. Reddy, B.
Jagannadh, Chem. Commun. 2007, 371–373; g) G. V. M. Sharma, V.
Manohar, S. K. Dutta, V. Subash, A. C. Kunwar, J. Org. Chem. 2008,
73, 3689–3698; h) C. Fernandes, S. Faure, E. Pereira, V. Thery, V.
Declerck, R. Guillot, D. J. Aitken, Org. Lett. 2010, 12, 3606–3609;
i) D. Balamurugan, K. M. Muraleedharan, Chem. Eur. J. 2012, 18,
9516–9520.
chemical formula C36H66N8O8; 1calcd =1.168 mgmꢀ3
; ;
m=0.089 mmꢀ1
FACHTUNGTRENNUNG(000)=3536. The structure was obtained by direct methods using
SHELXS-97. All non-hydrogen atoms except C9, C90, C91, C127, and
C129 were refined anisotropically. The hydrogen atoms were fixed geo-
metrically in the idealized position and refined in the final cycle of re-
finement as riding over the atoms to which they are bonded. Hydrogen
atoms located on C128 oscillate even after higher refinement cycles,
which are responsible for increased shift to su ratio. The final R value
was 0.0879 (wR2=0.2337) for 31448 observed reflections (F0 ꢂ4s(jF0j))
R
and 2098 variables; S=0.960. The largest deference peak and hole were
1.050 and ꢀ0.656 eꢂꢀ3, respectively.
Peptide P2: Crystals of P2 were grown by slow evaporation from a solvent
composition of methanol/water (4:1). A single crystal, rectangular in
shape (0.60ꢁ0.40ꢁ0.20 mm3), was mounted on a loop. The X-ray data
were collected at 100(2) K on a Bruker AXS SMART APEX CCD dif-
fractometer using MoKa radiation (l=0.71073 ꢂ), w-scans (2q=56.808),
[3] a) W. S. Horne, L. M. Johnson, T. J. Ketas, P. J. Klasse, M. Lu, J. P.
Moore, S. H. Gellman, Proc. Natl. Acad. Sci. USA 2009, 106, 14751–
14756; b) H. S. Haase, K. J. Peterson-Kaufman, S. K. Lan Leven-
good, J. W. Checco, W. L. Murphy, S. H. Gellman, J. Am. Chem. Soc.
2012, 134, 7652–7655; c) W. S. Horne, J. L. Price, S. H. Gellman,
Proc. Natl. Acad. Sci. USA 2008, 105, 9151–9156; d) E. P. English,
R. S. Chumanov, S. H. Gellman, T. Compton, J. Biol. Chem. 2005,
281, 2661–2667; e) Y. Imamura, N. Watanabe, N. Umezawa, T. Iwat-
subo, N. Kato, T. Tomita, T. Higuchi, J. Am. Chem. Soc. 2009, 131,
7353–7359.
for
a total number of 19169 independent reflections. Space group
P212121; a=9.4835(18), b=14.598(3), c=27.319(5) ꢂ; a=b=g=908, V=
3782.1(13) ꢂ3, orthorhombic P; Z=4 for chemical formula C33H61N7O8;
1calcd =1.229 mgmꢀ3; m=0.085 mmꢀ1; F
ACTHNUTRGNEU(GN 000)=1520. The structure was ob-
tained by direct methods using SHELXS-97. All non-hydrogen atoms
were refined anisotropically. The hydrogen atoms were fixed geometrical-
ly in the idealized position and refined in the final cycle of refinement as
riding over the atoms to which they are bonded. The final R value was
[4] T. Hintermann, K. Gademann, B. Jaun, D. Seebach, Helv. Chim.
Acta 1998, 81, 893–1002.
[5] S. Hanessian, X. Luo, R. Schaum, S. Michnick, J. Am. Chem. Soc.
1998, 120, 8569–8570.
0.0693 (wR2=0.1504) for 9409 observed reflections (F0 ꢂ4s(jF0j)) and
T
454 variables; S=0.885. The largest deference peak and hole were 0.304
and ꢀ0.299 eꢂꢀ3, respectively.
[6] a) C. Baldauf, R. Gunther, H. J. Hofmann, J. Org. Chem. 2006, 71,
1200–1208; b) L. Guo, Y. Chi, A. M. Almeida, I. A. Guzei, B. K.
Parker, S. H. Gellman, J. Am. Chem. Soc. 2009, 131, 16018–16020;
c) P. G. Vasudev, S. Chatterjee, K. Ananda, N. Shamala, P. Balaram,
Angew. Chem. 2008, 120, 6530–6532; Angew. Chem. Int. Ed. 2008,
47, 6430–6432; d) S. Chatterjee, P. G. Vasudev, S. Raghothama, C.
Ramakrishnan, N. Shamala, P. Balaram, J. Am. Chem. Soc. 2009,
131, 5956–5965; e) G. V. M. Sharma, V. B. Jadhav, K. V. S. Ramak-
rishna, P. Jayaprakash, K. Narsimulu, V. Subash, A. C. Kunwar, J.
Am. Chem. Soc. 2006, 128, 14657–14668; f) F. Bouillꢆre, S. T. Lau-
rent, C. Kouklovsky, V. Alezra, Amino Acids 2011, 41, 687–707;
g) P. Schramm, G. V. M. Sharma, H. J. Hofmann, Peptide Science
2009, 94, 279–291; h) C. Baldauf, R. Gunther, H. J. Hofmann, Helv.
Chim. Acta 2012, 95, 2348–2383.
CCDC-955486 (P1), 955487 (P2) and 955482 (g4-Asp
ACTHNUTRGNEUNG(OtBu)) contain the
supplementary crystallographic data for this paper. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgements
We thank the Department of Science and Technology, Government of
India, for financial support. S.V.J. and R.M. are thankful to CSIR India
for a Senior Research Fellowship.
[7] a) S. V. Jadhav, A. Bandyopadhyay, H. N. Gopi, Org. Biomol. Chem.
2013, 11, 509–514; b) A. Bandyopadhyay, S. V. Jadhav, H. N. Gopi,
Chem. Commun. 2012, 48, 7170–7172; c) A. Bandyopadhyay, H. N.
Gopi, Org. Lett. 2012, 14, 2770–2773.
[1] a) D. Seebach, A. K. Beck, D. J. Bierbaum, Chem. Biodiversity 2004,
1, 1111–1239; b) D. Seebach, J. Gardiner, Acc. Chem. Res. 2008, 41,
1366–1375; c) W. S. Horne, S. H. Gellman, Acc. Chem. Res. 2008,
41, 1399–1408; d) R. P. Cheng, S. H. Gellman, W. F. DeGrado,
Chem. Rev. 2001, 101, 3219–3232; e) J. Venkatraman, S. C. Shankar-
amma, P. Balaram, Chem. Rev. 2001, 101, 3131–3152; f) P. G. Vasu-
dev, S. Chatterjee, N. Shamala, P. Balaram, Chem. Rev. 2011, 111,
657–687; g) J. L. Price, W. S. Horne, S. H. Gellman, J. Am. Chem.
Soc. 2010, 132, 12378–12387; h) I. V. Korendovych, S. J. Shandler,
G. L. Montalvo, W. F. DeGrado, Org. Lett. 2011, 13, 3474–3477;
i) G. V. M. Sharma, P. Nagendar, P. Jayaprakash, P. R. Krishna,
K. V. S. Ramakrishna, A. C. Kunwar, Angew. Chem. 2005, 117,
6028–6032; Angew. Chem. Int. Ed. 2005, 44, 5878–5882; j) J. A.
Kritzer, M. E. Hodsdon, A. Schepartz, J. Am. Chem. Soc. 2005, 127,
4118–4119; k) W. S. Horne, M. D. Boersma, M. A. Windsor, S. H.
Gellman, Angew. Chem. 2008, 120, 2895–2898; Angew. Chem. Int.
Ed. 2008, 47, 2853–2856; l) W. S. Horne, S. H. Gellman, J. Am.
Chem. Soc. 2011, 133, 10038–10041; m) M. A. Schmitt, S. H. Choi,
I. A. Guzei, S. H. Gellman, J. Am. Chem. Soc. 2006, 128, 4538–
4539; n) S. De Pol, C. Zorn, C. D. Klein, O. Zerbe, O. Reiser,
Angew. Chem. 2004, 116, 517–520; Angew. Chem. Int. Ed. 2004, 43,
511–514; o) R. S. Roy, I. L. Karle, S. Raghothama, P. Balaram, Proc.
Natl. Acad. Sci. USA 2004, 101, 16478–16482.
[8] a) S. M. Mali, A. Bandyopadhyay, S. V. Jadhav, M. G. Kumar, H. N.
Gopi, Org. Biomol. Chem. 2011, 9, 6566–6574; b) M. Hagihara, N. J.
Anthony, T. J. Stout, J. Clardy, S. L. Schreiber, J. Am. Chem. Soc.
1992, 114, 6568–6570; c) M. Smrcina, P. Majer, E. Majerova, T. A.
Guerassina, M. A. Eissenstat, Tetrahedron 1997, 53, 12867–12874;
d) V. Loukas, C. Noula, G. Kokotos, J. Pept. Sci. 2003, 9, 312–319.
[9] a) K. Basuroy, B. Dinesh, N. Shamala, P. Balaram, Angew. Chem.
2012, 124, 8866–8869; Angew. Chem. Int. Ed. 2012, 51, 8736–8739;
b) K. Basuroy, B. Dinesh, N. Shamala, P. Balaram, Angew. Chem.
2013, 125, 3218–3221; Angew. Chem. Int. Ed. 2013, 52, 3136–3139.
[10] Y. H. Shin, D. E. Mortenson, K. A. Satyshur, K. T. Forest, S. H.
Gellman, J. Am. Chem. Soc. 2013, 135, 8149–8152.
[11] N. Pendem, Y. R. Nelli, C. Douat, L. Fischer, M. Laguerre, E. Enni-
far, B. Kauffman, G. Guichard, Angew. Chem. 2013, 125, 4241–
4254; Angew. Chem. Int. Ed. 2013, 52, 4147–4151.
[12] a) J. S. Richardson, D. C. Richardson, Science 1988, 240, 1648–1652;
b) L. G. Presta, G. D. Rose, Science 1988, 240, 1632–1641; c) M. D.
Bruch, M. M. Dhingra, L. M. Gierasch, Proteins Struct. Funct.
Genet. 1991, 10, 130–139; d) W. Y. Wan, E. J. Milner-White, J. Mol.
Biol. 1999, 286, 1693–1694; e) P. G. Vasudev, M. Banerjee, C. Ram-
akrishnan, P. Balaram, Proteins Struct. Funct. Bioinf. 2012, 80, 991–
1002.
[2] a) S. H. Gellman, Acc. Chem. Res. 1998, 31, 173–180; b) W. S.
Horne, J. L. Price, J. L. Keck, S. H. Gellman, J. Am. Chem. Soc.
2007, 129, 4178–4180; c) D. Seebach, S. Abele, K. Gademann, B.
Chem. Eur. J. 2013, 19, 16256 – 16262
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
16261

H. N. Gopi et al.
[13] a) B. Forood, E. J. Feliciano, K. P. Nambiar, Proc. Natl. Acad. Sci.
USA 1993, 90, 838–842; b) A. Chakrabartty, A. J. Doig, R. L. Bald-
win, Proc. Natl. Acad. Sci. USA 1993, 90, 11332–11336.
[15] a) F. X. Zhou, M. J. Cocco, W. P. Russ, A. T. Brunger, D. M. Engel-
man, Nat. Struct. Biol. 2000, 7, 1085; b) C. Croma, H. Gratkowski,
J. D. Lear, W. F. DeGrado, Nat. Struct. Biol. 2000, 7, 161–166.
[14] a) J. D. Lear, H. Gratkowski, L. Adamian, J. Liang, W. F. Degrado,
Biochemistry 2003, 42, 6400–6407; b) K. Parthasarathy, X. Lin, S. M.
Tan, S. K. A. Law, J. Torres, Protein Sci. 2008, 17, 930–938; c) K. M.
Oshaben, R. Salari, D. R. McCaslin, L. T. Chong, W. S. Horne, Bio-
chemistry 2012, 51, 9581–9591.
Received: July 13, 2013
Published online: October 21, 2013
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Chem. Eur. J. 2013, 19, 16256 – 16262