Spiegelmeric 4R/S-hydroxy/amino-L/D-prolyl collagen peptides: conformation and morphology of self-assembled structures

Article abstract of DOI:10.1002/pep2.24140

The primary structure of collagen, the major protein in connective tissue of mammals, comprises of repeating triads [(_LPro-_LHyp-Gly)_n, P1, _LHyp being 4R-hydroxy-lProline)] in a single strand that adopts left-handed polyproline II type helix. Three such single stranded helices wind around each another and held together by interchain H-bonds to form right-handed triple helix. This manuscript reports on collagen derived from its mirror image triad [(_DPro-_DHyp-Gly)_n, P2, _DHyp being 4S-hydroxy-_DProline) and its 4-amino analogue (_DPro-_DAmp-Gly)_n P4, _DAmp being 4S-amino-_DProline that form corresponding spiegelmeric triplexes. The amino L-collagen peptide (_LPro-_LAmp-Gly)_n P3 and its D-analogue P4 show higher thermal stabilities compared to 4-hydroxy-lProline collagen peptides P1 and P2. The enantiomeric peptide pairs show mirror image CD profiles and identical thermal stability, with ionizable 4-amino group in P3 and P4 imparting pH dependent triplex stability. Upon cold mixing of the L- and D-collagen peptides, different morphological nanostructures arise from inter triplex peptide association. When the peptides are hot mixed (annealed), the inter peptide association occurs via interaction of single stranded peptide chains of opposite handedness leading to networked gel formation in P1 and P2, while the charged peptides P3 and P4 show more ordered nanofibers, different from the enantiomerically pure peptides. The nanocomposites of such chiral hybrid peptides may have not only interesting physicomorphology, but also biological properties that need exploration.

Full text of DOI:10.1002/pep2.24140

Received: 11 July 2019
Revised: 31 August 2019
Accepted: 23 September 2019
DOI: 10.1002/pep2.24140
F U L L P A P E R
Speigelmeric 4R/S-hydroxy/amino-L/D-prolyl collagen
peptides: conformation and morphology of self-assembled
structures
Shahaji H More1
| Krishna N Ganesh1,2
1Chemistry Department, Indian Institute of
Science Education and Research Pune, Pune,
India
Abstract
The primary structure of collagen, the major protein in connective tissue of mammals,
comprises of repeating triads [(LPro-LHyp-Gly)n, P1, LHyp being 4R-hydroxy-LProline)]
in a single strand that adopts left-handed polyproline II type helix. Three such single
stranded helices wind around each another and held together by interchain H-bonds
to form right-handed triple helix. This manuscript reports on collagen derived from its
mirror image triad [(DPro-DHyp-Gly)n, P2, DHyp being 4S-hydroxy-DProline) and its
4-amino analogue (DPro-DAmp-Gly)n P4, DAmp being 4S-amino-DProline that form
corresponding speigelmeric triplexes. The amino L-collagen peptide (LPro-LAmp-Gly)n
P3 and its D-analogue P4 show higher thermal stabilities compared to 4-hydroxy-
LProline collagen peptides P1 and P2. The enantiomeric peptide pairs show mirror
image CD profiles and identical thermal stability, with ionizable 4-amino group in P3
and P4 imparting pH dependent triplex stability. Upon cold mixing of the L- and D-
collagen peptides, different morphological nanostructures arise from inter triplex
peptide association. When the peptides are hot mixed (annealed), the inter peptide
association occurs via interaction of single stranded peptide chains of opposite hand-
edness leading to networked gel formation in P1 and P2, while the charged peptides
P3 and P4 show more ordered nanofibers, different from the enantiomerically pure
peptides. The nanocomposites of such chiral hybrid peptides may have not only
interesting physicomorphology, but also biological properties that need exploration.
2Department of Chemistry, Indian Institute of
Science Education and Research Tirupati,
Tirupati, Andhra Pradesh, India
Correspondence
Krishna N Ganesh, Department of Chemistry,
Indian Institute of Science Education and
Research Tirupati, Karkambadi Road,
Mangalam, Tirupati, Andhra Pradesh 517507,
India.
Email: kn.ganesh@iisertirupati.ac.in
K E Y W O R D S
collagen peptide, D/L peptides, morphology, speigelmer, triple helix
and proteins from proteases.[2–5] Longtime ago Linus Pauling surmised
1
| INTRODUCTION
that alternating D and L residues in polypeptide chains lead to β-ple-
ated-sheet structure,[6] but nature always prefers homochirality. Sev-
eral studies comprising of alternating L/D aminoacids in peptides have
been reported to result into interesting conformational
outcomes.[7–12] Inverting configuration of all L-amino acids into D-
amino acids in proteins result in their mirror image structure.[13] Physi-
cal mixing of mirror image (right and left-handed) peptides lead to inter
chain self assembly to form higher ordered structures.[14–17] Hydrogels
The biopolymers proteins and nucleic acids are homochiral with all
constituent monomers being of same chirality. Homochirality confers
well-defined structural and conformational features for bio-
macromolecules to be functional. Even though proteins usually com-
prise of L-amino acids exclusively, D-amino acids do often occur in
nature, ex., as constituents of cell wall of bacteria and in higher
organisms,[1] where they play an important role in protecting peptides
Peptide Science. 2019;e24140.
wileyonlinelibrary.com/peptidesci
© 2019 Wiley Periodicals, Inc.
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https://doi.org/10.1002/pep2.24140

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made from mixing of enantiomeric peptides are more rigid than the
hydrogels made from individual homochiral peptides.[18] Introduction of
D-amino acids in natural peptides and proteins not only enhances their
stability against proteases, but also improve their attributes as biomate-
rials. Peptides incorporating D-amino acids have also shown various ther-
apeutic effects such as antimicrobial activity, amyloidosis, drug
selectivity, hemostasis and improved drug delivery properties.[19]
and right-handed triple helices in solution led to formation of sheets
from interactions of complementary hydrophobic grooves of oppo-
site handed helices. In the crystal structure of collagen, 4R-hydroxyl
groups of LHyp project in one direction on the periphery of the tri-
plex cylinder.[40] The 4S-hydroxyl groups of DHyp in the enantio-
meric peptide should point in opposite direction and upon mixing,
the enantiomeric peptides may associate through H-bonds, in com-
parison to hydrophobic association seen in analogous enantiomeric
L/D-prolyl peptides. Study of enantiomeric, ionizable 4R/4S-amino
collagen peptides with LAmp and DAmp may further lead to pH
dependent variation in supramolecular assembly of derived racemic
triple helices. In this context, herein we present synthesis, compara-
tive CD and morphological studies of speigelmeric hydroxy and
amino collagen peptide pairs (LPro-LHyp-Gly)6 P1 / (DPro-DHyp-Gly)6
P2 and (LPro-LAmp-Gly)6 P3 / (DPro-DAmp-Gly)6 P4 (Figure 1).
Collagen is the most abundant structural protein found in the con-
nective tissue of mammals.[20] The primary structure of collagen com-
prises of repeating units of X-Y-Gly triad motif in a single strand that
adapts left-handed polyproline II type helix. Three such single stranded
helices intertwine around each another on a common axis, held
together by interchain H-bond to form right-handed triple helix.[21–23]
The imino acids in the X and Y positions in collagen are predominantly
L-proline (LPro) and 4R-hydroxy-L-proline (LHyp) respectively. The regio
(C4) and stereospecificity (4R) of hydroxyl group at Y-position in colla-
gen is proven to be necessary to provide the collagen triplex its
mechanical strength, broad resistance to the proteolytic enzyme and
thermal stability.[24] The replacement of 4R-OH function of LHyp at Y-
site in collagen peptide by other substituents such as F, CH3, SH and
N3 have led to collagen peptide variants with altered abilities to form
triplex. Inversion of stereochemistry of C4-substituent (R to S) or inter-
changing Pro and Hyp at X and Y-sites (LHyp-LPro-Gly)n resulted in
nonformation of triplex. The C4 substitutions essentially influence the
puckering mode (C4 endo/exo) of prolines and alter the cis-trans ratio of
N1-CO tertiary amide bond. We have previously shown that replacing
the nonionizable 4-OH group by ionizable 4-NH2 group as in 4R-
amino-L-proline (LAmp) at Y-site (LPro-LAmp-Gly)n significantly
enhances the thermal stability of 4R-amino prolyl collagen peptides in a
pH dependent manner.[25] The triplexes composed from 4R-amino
prolyl collagen peptide show peak stability at both acidic (pH 3.0) and
alkaline (pH 12.0) conditions and least stability around neutral pH 9.0.
This suggested that the 4R-NH2 group stabilizes the collagen triplex in
both protonated as well as free amino form.[25]
2
| MATERIALS AND METHODS
2.1 | General
All starting materials and reagents were of the commercially available
highest grade and used without further purification. Reactions were
monitored by thin-layer chromatography (TLC) using silica gel 60 F254
plates. Compounds on TLC were visualized by UV and by ninhydrin
spray. All column chromatography purifications were performed using
silica gel 100 to 200 particle size. 1H and 13C NMR spectra were
recorded on 400 MHz in methanol-d4 or CDCl3 and chemical shifts in
δ ppm are referenced to TMS. High-resolution mass spectra (HRMS)
were recorded in electrospray ionization-time of flight (ESI-TOF)
mode. MALDI-TOF mass spectra of all peptides were obtained in pos-
itive reflection mode using α-cyano-4-hydroxy cinnamic acid (CHCA)
or 2,5-dihydroxy benzoic acid (DHB) as a matrix. 4R-(OH)-D-proline
6 was synthesized from commercially available 4R-(OH)-L-proline
according to literature procedure.[41] 4R/S-O4(t-Bu)-N1-(Fmoc)-D-
proline (1, 2) and 4R/S-N4(Boc)-N1-(Fmoc)-D-proline (3, 4) were syn-
thesized from 4R-hydroxy-L-proline by sequential transformation of
functional groups, protection/ deprotection strategies as described
previously.[25,42–44]
The immunological and pathological problems associated with
natural collagen, for biological applications can perhaps be overcome
with synthetic collagen peptide analogs that form large assemblies
and so collagen inspired biomaterials have attracted much
attention.[26–28] Various strategies based on both covalent and
noncovalent modifications have been employed to induce supramo-
lecular assembly of synthetic collagen mimetic peptides.[29–39]
Nanda et al.[17] incorporated unsubstituted D-proline at X and Y
sites in collagen triad (DPro-DPro-Gly)n to generate left-handed triple
helix, the mirror image isomer of natural right handed triplex from
analogous L-Proline peptide (LPro-LPro-Gly)n. Mixing of such left
2.2 | Synthesis of compounds 2 to 14
2.2.1 | 4R-Hydroxy-N1(benzyloxycarbonyl)-D-
proline 7
A suspension of 6 (4 g, 30 mmol) in 10% aq. NaHCO3 (40 mL) was
cooled to 4 ꢀC to which Cbz-Cl (11 mL, 50% solution in toluene) was
FIGURE 1 Structure of L and D collagen
peptides P1 to P4

MORE AND GANESH
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added dropwise by addition funnel over a period of 30 minutes and
stirred for 12 hours at RT. Toluene was removed under reduced pres-
sure and the unreacted Cbz-Cl was extracted with hexane 3 to 4 times.
The aqueous layer was acidified to pH 3.0 with saturated KHSO4 and
extracted with ethyl acetate. The organic layer was washed with brine
solution, dried over anhydrous Na2SO4 and concentrated to yield
white sticky solid that was purified by silica gel chromatoghraphy to
yield compound 7. Yield: 6.5 g, (80%); (SiO2, 60% ethyl acetate/n-hex-
ane); 1H NMR (400 MHz, CDCl3, δ, ppm) 7.35 to 7.28 (m, 5H), 5.1
(ddd, J = 17.9, 12.5, 7.1 Hz, 2H), 4.46 to 4.31 (m, 2H), 3.61 to 3.45 (m,
2H), 2.18 (dd, J = 21.6, 9.0 Hz, 2H); 13C NMR (400 MHz, CDCl3, δ,
ppm): 37.6, 38.6, 55.1, 58.6, 67.7, 69.6, 70.9, 127.5, 128.1, 128.9,
136.2, 154.8, 155.3, 175.8; HRMS (ESI-TOF, m/z) [M + Na]+ calcu-
lated for C13H15NO5Na 288.0847, found 288.0850; Crystal structure
is deposited in the Cambridge crystallographic data centre, CCDC
1916283.
the compound 10, which was purified by silica gel chromatography.
Yield 3.1 g, (98%); (SiO2, 50% ethyl acetate/hexane); [α]D22 + 35.2ꢀ
(c 2.0, CH2Cl2); 1H NMR (400 MHz, CDCl3, δ, ppm) 7.38 to 7.26 (m,
5H), 5.22 to 4.96 (m, 2H), 4.55 to 4.31 (m, 2H), 3.83 to 3.47 (m, 5H),
2.32 (tdd, J = 14.4, 9.8, 4.6 Hz, 1H), 2.09 (dddd, J = 17.2, 9.3, 5.4,
1.0 Hz, 1H); 13C NMR (400 MHz, CDCl3, δ, ppm): 37.8, 38.7, 52.7,
53.0, 55.8, 57.8, 58.2, 67.4, 70.2, 71.2, 127.9, 128.0, 128.1, 128.5,
128.6, 136.3, 154.0, 154.9, 174.5; IR (Neat, cm−1) 3431, 2952, 1742,
1685, 1420, 1353, 1276, 1205, 1171, 1120; HRMS (ESI-TOF, m/z)
[M + Na]+ calculated for C14H17NO5Na 302.1004, observed
302.1004.
2.2.4 | 4S-O4(t-Butyl)-N1(benzyloxycarbonyl)-D-
proline methylester 11
A mixture of compound 10 (1.6 g, 5 mmol), silver oxide (Ag2O) (3.9 g,
17 mmol) and tertiary butyl bromide (3.2 g, 23 mmol) in cyclohexane
(50 mL) was stirred for 23 hours at room temperature. The resulting
suspension was filtered through celite and volatiles in the filtrate were
removed under reduced pressure to yield crude product that was puri-
fied to obtain 11. Yield 1.6 g, (80%); (SiO2, 15% ethyl acetate/hexane
elute); 1H NMR (400 MHz, CDCl3, δ, ppm) 7.39 to 7.23 (m, 5H), 5.20
to 4.98 (m, 2H), 4.35 (ddd, J = 22.1, 8.5, 5.6 Hz, 1H), 4.19 to 4.10
(m, 1H), 3.79 to 3.65 (m, 3H), 3.56 (s, 1H), 3.38 to 3.23 (m, 1H), 2.39
to 2.26 (m, 1H), 2.08 to 2.00 (m, 1H), 1.13 (d, J = 1.2 Hz, 9H); 13C
NMR (400 MHz, CDCl3, δ, ppm) 28.2, 37.9, 38.7, 52.0,52.2, 53.3,
53.6, 57.8, 67.1, 68.6, 69.4, 74.0, 127.9, 128.0, 128.4, 136.6, 154.3,
154.9, 172.5, 172.8; IR (Neat, cm−1) 2972, 1747, 1703, 1417, 1355,
1302, 1260, 1204, 1086; HRMS (ESI-TOF, m/z) [M + Na]+ calculated
for C18H25NO5Na 358.1630, found 358.1631.
2.2.2 | 4R-Hydroxy-N1(benzyloxycarbonyl)-D-
proline methylester 8
To a solution of 7 (13 g, 49 mmol) in anhydrous acetone (75 mL), and
anhydrous K2CO3 (16.9 g, 122 mmol), dimethylsulfate (5.6 mL,
58 mmol) was added. The reaction mixture was refluxed under nitro-
gen environment for 4 hours followed by concentration and work up
with ethyl acetate and washed with brine solution. The dried organic
layer was concentrated and the product was purified by silica gel
chromatography to afford compound 8 as colorless thick oil. Yield:
13.5 g, (98%); (SiO2, 30% ethyl acetate/hexane); [α]D22 + 36ꢀ (c 2.0,
CH2Cl2); 1H NMR (400 MHz, CDCl3, δ, ppm) 7.40 to 7.18 (m, 5H),
5.20 to 4.95 (m, 2H), 4.40 (ddd, J = 15.1, 11.7, 2.8 Hz, 2H), 3.78 to
3.53 (m, 5H), 2.30 (tdd, J = 14.1, 9.7, 4.6 Hz, 1H), 2.15 to 2.06 (m, 1H);
13C NMR (400 MHz, CDCl3, δ, ppm): 37.7, 39.0, 52.5, 56.0, 58.3, 67.4,
70.5, 71.4, 127.7, 128.5, 136.9, 154.9, 155.2, 174.9; IR (Neat, cm−1
)
2.2.5 | 4S-O4(t-Butyl)-N1(benzyloxycarbonyl)-D-
3456, 2953, 1748, 1697, 1424, 1360, 1277, 1210, 1122; HRMS (ESI-
TOF, m/z) [M + Na]+ C14H17NO5Na (calc) 302.1004, found 302.1006;
Crystal structure is deposited in the Cambridge crystallographic data
centre, CCDC 1916281.
proline 12
The ester 11 was subjected to hydrolysis using aq. LiOH (2 N) in THF
for 2 hours. THF was then removed under vacuum and the aqueous
layer was acidified with aq. HCl (2 N) to pH 2.0 and extracted with
ethyl acetate. The residue after evaporation of the solvent was puri-
fied by column chromatography to yield intermediate acid. Yield 1.5 g,
(98%). The acid was dissolved in methanol (20 mL) to which 10%
Pd/C (0.3 g) was added and suspension was stirred under an atmo-
sphere of H2. After completion of the reaction, the insoluble part was
removed by filtration. Removal of methanol in the filtrate under
reduced pressure gave the imino acid 12. 1H NMR (400 MHz, CDCl3,
δ, ppm) 7.41 to 7.25 (m, 5H), 5.23 to 5.03 (m, 2H), 4.45 to 4.14
(m, 2H), 3.72 to 3.60 (m, 1H), 3.40 (ddd, J = 42.2, 10.7, 3.1 Hz, 1H),
2.36 (dd, J = 8.9, 4.7 Hz, 1H), 2.17 to 2.06 (m, 1H), 1.16 (d,
J = 10.5 Hz, 9H); 13C NMR (400 MHz, CDCl3, δ, ppm): 28.1, 37.5,
38.3, 58.1, 67.4, 69.1, 69.7, 127.8, 128.2, 128.7, 136.5, 154.5, 154.7,
175.9; IR (Neat, cm−1) 2973, 1702, 1419, 1357, 1303, 1184; HRMS
(ESI-TOF, m/z) [M + Na]+ calculated for C17H23NO5Na 344.1473,
found 344.1472.
2.2.3 | 4S-Hydroxy-N1(benzyloxycarbonyl)-D-
proline methylester 10
To a suspension of 8 (4.4 g, 15 mmol) in dioxane (5 mL), DCC (3.9 g,
19 mmol) and CuCl (5 mg) was added. The mixture was stirred at
40 ꢀC to 50 ꢀC for 48 hours and dioxane was evaporated on vacuum.
To the residue, toluene (8 mL) and formic acid (1.1 g, 23 mmol) were
added and the reaction mixture was refluxed for 24 hours. The insolu-
ble solid was filtered and the filtrate was successively washed with
sat. aq. NaHCO3, brine, followed by concentration. The residue was
dissolved in THF (20 mL), treated with aq. 2 N NaOH at 10 ꢀC and
stirred at 25 ꢀC for 30 minutes. After acidifying the mixture to pH 2.0
with conc. HCl, the white suspension was extracted with ethyl ace-
tate. The dried organic layer was concentrated to get compound
9 (Yield 3 g, 71%). The procedure of compound 8 was repeated to get

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2.2.6 | 4S-O4(t-Butyl)-N1-
(fluorenylmethyloxycarbonyl)-D-proline 2
and reacted with (Boc)2O for 4 hours. After completion of the reac-
tion, the reaction mixture was concentrated and extracted with ethyl
acetate and purified by column chromatography to obtain 14. Yield,
2.0 g, (41%.); (SiO2, 17% ethyl acetate/hexane); [α]D25 + 15o (c, 2%,
MeOH); 1H NMR (400 MHz, CDCl3, δ, ppm) 7.38 to 7.25 (m, 5H),
5.11 (ddd, J = 46.5, 23.3, 10.3 Hz, 2H), 4.46 to 4.34 (m, 1H), 4.28 (s,
2H), 3.87 to 3.76 (m, 1H), 3.72 to 3.55 (m, 3H), 3.36 (ddd, J = 29.7,
11.1, 4.9 Hz, 1H), 2.27 to 2.10 (m, 2H), 1.42 (d, J = 2.5 Hz, 9H); 13C
NMR (400 MHz, CDCl3, δ, ppm): 28.0, 35.9, 36.8, 52.5, 57.8, 67.4,
127.7, 129.1, 136.4, 154.0, 155.7, 172.8;); IR (Neat, cm−1) 1698,
1509, 1421, 1357, 1213, 1166, 1124, 1079, 1015; HRMS (ESI-TOF,
m/z) [M + Na]+ calculated for C19H26N2O6Na 401.1688 found
401.1689.
Compound 12 (1.2 g, 6.5 mmol) was dissolved in water:dioxane, 1:1
(50 mL) to which was added 10% aq. Na2CO3 to bring the pH to 10.0
and cooled to 0 ꢀC. Fmoc-Cl (2 g, 7.7 mmol) was added portion-wise
for over 45 minutes while maintaining the temperature at 0 ꢀC for
4 hours. It was brought to room temperature and stirred for another
18 hours. The dioxane was removed under vacuum, the aqueous layer
extracted with diethyl ether to remove unreacted Fmoc-Cl. Water
layer was acidified with saturated KHSO4 to pH 2. Work up with ethyl
acetate gave crude product 2. Yield 2.1 g, (81%); (SiO2, 2% methanol/
dichloromethane); 1H NMR (400 MHz, CDCl3, δ, ppm) 7.80 to 7.25
(m, 8H), 5.15 (dt, J = 20.8, 10.5 Hz, 2H), 4.40 (dddd, J = 56.9, 26.9,
13.5, 8.9 Hz, 2H), 3.80 to 3.23 (m, 3H), 2.42 to 2.04 (m, 2H), 1.18 (dd,
J = 15.1, 4.7 Hz, 9H); 13C NMR (400 MHz, CDCl3, δ, ppm): 28.1, 37.2,
38.2, 52.9, 54.0, 54.6, 58.5, 67.6, 69.0, 69.7, 120.1, 125.0, 127.1,
127.8, 128.5, 136.1, 141.4, 174.2; IR (Neat, cm−1) 2972, 1704, 1426,
1358, 1214, 1091, 1015; HRMS (ESI-TOF, m/z) [M + Na]+ calculated
for C18H25NO5Na 432.1786, observed 432.1785.
2.2.9 | 4S-N4(t-Boc)-N1(Fmoc)-D-amino proline 4
Compound 14 (1 g, 4.3 mmol) was hydrolyzed in THF:water (1:1,
20 mL) using aq. LiOH (2 N) for 1 hour. THF was removed under
reduced pressure, washed aqueous layer with ethyl acetate (3 x
20 mL), acidified with aq. KHSO4, and extracted with ethyl acetate to
obtain crude product (acid). This was dissolved in dry methanol
(15 mL) and hydrogenated in the presence of 10% Pd/C (0.5 g) for
6 hours. The reaction mixture was filtered through celite. Removal of
methanol from the filtrate gave a white solid that was dissolved in
water:dioxane (1:1, 60 mL). This was treated with 10% aq. Na2CO3 at
0 ꢀC to bring to pH 9.0 and stirred for 15 minutes. Fmoc-Cl (1.4 g,
5.5 mmol) was added over a period of 30 minutes and stirred at 0 ꢀC
for 4 hours, and then overnight. After the completion of the reaction,
the mixture was acidification with aq. sat. KHSO4 to pH 4.0 and
followed by work up to obtain crude product, which was then purified
by silica gel chromatography to afford white solid of 14. Yield: 1 g,
(53%); (SiO2, 2% Methanol/CH2Cl2); [α]D25 + 7o (c, 2%, MeOH); 1H
NMR (400 MHz, CDCl3, δ, ppm) 7.73 (dd, J = 18.8, 7.5 Hz, 2H), 7.56
(q, J = 7.7 Hz, 2H), 7.45 to 7.22 (m, 4H), 6.07 (s, 2H), 4.77 (d,
J = 19.5 Hz, 1H), 4.40 to 4.07 (m, 3H), 3.81 (dd, J = 10.8, 6.7 Hz, 1H),
3.34 (dd, J = 10.8, 5.5 Hz, 1H), 2.44 to 2.12 (m, 2H), 1.45 (d, J = 14.2 Hz,
9H); 13C NMR (400 MHz, CDCl3, δ, ppm): 28.44, 29.72, 35.71, 37.03,
47.08, 49.27, 51.97, 55.00, 57.92, 68.01, 80.27, 120.07, 125.18,
127.21, 127.85, 141.38, 143.65, 154.07, 155.38, 172.01, 172.8;); IR
(cm−1) 2975, 1682, 1520, 1424, 1357, 1315, 1249, 1218, 1163,
1127, 1077; HRMS (ESI-TOF) (m/z) [M + Na]+ calculated for
2.2.7 | 4S-Azido-N1(benzyloxycarbonyl)-D-proline
methylester 13
A solution of compound 8 (4 g, 14 mmol) and triethylamine (3 mL,
21 mmol) in dry CH2Cl2 (50 mL) was cooled to 0 ꢀC under inert condi-
tions. Methanesulfonyl chloride (1.7 mL, 22 mmol) was added
dropwise over a period of 1 hour at 0 ꢀC. After completion of the
reaction, the mixture was extracted with ethyl acetate and concen-
trated under vacuum. The crude compound (5.1 g, 14 mmol) was dis-
solved in dry DMF (50 mL), NaN3 (10 g, 153 mmol) was added and
stirred at 60 ꢀC for 8 hours under nitrogen. DMF was removed under
reduced pressure and the residue was dissolved in water and
extracted with ethyl acetate. Organic layers were concentrated on
rotary evaporator to yield crude azide 13. Yield: 3.8 g, (98%); (SiO2,
20% ethyl acetate/hexane); [α]D25 + 45.2o (c, 2%, MeOH); 1H NMR
(400 MHz, CDCl3, δ, ppm) 7.40 to 7.25 (m, 5H), 5.22 to 4.98 (m, 2H),
4.52 to 4.38 (m, 1H), 4.24 to 4.15 (m, 1H), 3.79 to 3.70 (m, 3H), 3.64
(ddd, J = 16.4, 9.2, 6.5 Hz, 1H), 3.57 to 3.50 (m, 2H), 2.40 to 2.26
(m, 1H), 2.19 (qd, J = 7.3, 3.9 Hz, 1H); 13C NMR (400 MHz, CDCl3, δ,
ppm): 35.3, 36.7, 52.6, 57.5, 58.8, 59.4, 67.5, 127.7, 128.7, 136.3,
153.7, 154.5, 172.5; IR (Neat, cm−1) 2954, 2104, 1745, 1702, 1414,
1351, 1265, 1203, 1170, 1120; HRMS (ESI-TOF, m/z) [M + Na]+ cal-
culated for C14H16N4O4Na 327.1069 observed 327.1068.
C25H28N2NaO6Na 475.1844 found 475.1850.
2.3 | Solid-phase peptide synthesis
All peptides were synthesized manually in sintered glass vessel
equipped with stopcock using standard protocols.[25,42–44] Rink amide
resin with loading value 0.6 mmol/g was used using standard Fmoc
chemistry. The resin (100 mg) was functionalized with first monomer
by washing it with dry DCM (3 mL) followed by swelling with dry
DMF. The coupling reaction was carried out with in situ active ester
method, using HBTU as a coupling reagent, HOBt as a racemization
suppresser and DIPEA as a base catalyst. The completion of coupling
2.2.8 | 4S-N4(t-Boc)-N1-(benzyloxycarbonyl)-D-
proline methylester 14
The azide compound 13 (4 g, 12.5 mmol) in dry THF (40 mL) was
treated with PPh3 (5 g, 19 mmol) for overnight after which water
(4 mL) was added and stirring was continued for another 2 hours. The
crude amine product was obtained (3.6 g) after evaporation of THF.
The residue was dissolved in dioxane-water (1:1) containing NaHCO3

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5 of 11
reaction was followed by Kaiser's ninhydrin test. The resin bound
Fmoc group at every step was first deprotected using 20% piperidine
in DMF. This cycle was repeated for each coupling and after the final
coupling reaction with phenylalanine (used for estimating the synthe-
sized peptide by UV absorbance), the resin was capped with acetic
anhydride:pyridine (1:3). The synthesized peptide was cleaved from
the resin using TFA:TIPS (95:5) in DCM (8 mL). The filtrate after
removal of resin was dissolved in anhydrous methanol (0.4 mL) and
crude product peptide precipitated by addition of anhydrous diethyl
ether (20 mL).
dry at room temperature and then kept under ordinary light for
4 hours for complete drying and before imaging coated with gold. To
study the self assembly of mixed enantiomeric peptides, peptides in
triple-helix and in single stranded form were mixed peptide and kept
at 4 ꢀC for 4 days. In case of annealed peptides, samples were mixed
first and heated up to 90 ꢀC followed by gradually cooling to room
temperature.
2.7 | Atomic force microscopy (AFM)
Atomic force microscopy imaging was carried out on AFM instrument
using tapping mode cantilever auto-tuned for 190 KHz frequency.
The desired peptide sample (5 μL) was spotted on fresh mica allowed
to dry at room temperature followed by complete drying under an
ordinary lamp for 4 hours.
2.4 | Peptide purification and characterization
The crude peptides P1-P4 were purified by preparative RP-HPLC on
RP-4 (15 μm) column using gradient elution; Solvent A: 0.1% TFA in
95% H2O:CH3CN; Solvent B: 0.1% TFA in 50% H2O:CH3CN; gradient
0% to 100% B over 20 minutes and monitored at 260 nm. The struc-
tural integrity of purified peptides P1-P4 was confirmed by MALDI-
TOF mass spectrometry by using CHCA, DHB matrix. The retention
time in HPLC and MALDI-TOF data for peptides are shown in
Table 1.
3
| RESULTS AND DISCUSSION
3.1 | Synthesis of protected monomers 1 to 4
The protected monomer 4R-O4(tButyl)-N1(Fmoc)-LHyp (1) is commer-
cially available and 4R-N4H(Boc)-N1(Fmoc)-LAmp (3) needed for
assembling collagen L-peptides P1 and P3 was synthesized starting
from the commercial 4R-hydroxy-L-proline using reaction protocols as
described in earlier literature.[25,42–44] The protected monomers 4S-
O4(tButyl)-N1(Fmoc)-DHyp (2) and 4S-N4H(Boc)-N1(Fmoc)-DAmp (4)
necessary for synthesis of corresponding enantiomeric D-OH/NH2
collagen peptides (P2 and P4, Figure 2) were synthesized as shown in
Scheme 1.
2.5 | CD spectral studies
CD measurements were performed using a quartz cylindrical cuvette
of 1 cm path length and each spectra was an average of three scans
from 300 nm to 190 nm with a bandwidth 1 nm and a scan speed of
100 nm/min and a response time 1 second. The peptide samples were
dissolved in corresponding buffer and stored at 4 ꢀC for at least
24 hours prior to allow strand association to triple-helix. The tempera-
ture dependent of CD spectra was recorded in the range of 5 ꢀC to
90 ꢀC at increments of 5 ꢀC. Tm values were determined from mid-
points of sigmoidal plots of ellipticity at 225 nm vs temperature and
confirmed from the first derivative plots.
The 4R-hydroxy-D-proline 6 was synthesized from the commer-
cially available 4R-hydroxy-L-proline 5 by inversion of stereochemis-
try at C2 using known literature procedure by reaction with acetic
anhydride and hydrochloric acid.[41] The protection of N1 as in 7 was
achieved by reacting 6 with Cbz-Cl in the presence aq. NaHCO3,
which was followed by esterification using dimethyl sulfate to yield
the methyl ester 8. The inversion of stereochemistry of 4R-OH in
8 was done by its reaction with DCC in presence of catalytic CuCl
followed by treatment with formic acid and hydrolysis with aq. NaOH
to yield the desired 4S-N1(CBz)-DHyp acid 9. This was esterified to
compound 10 and the 4S-OH was protected as t-butyl ether to get
compound 11. This upon hydrolysis with aq. LiOH and deprotection
of N1-CBz group by hydrogenation over Pd yielded 4S-O4(tButyl)-
DPro acid 12. The protection of N1 as Fmoc derivative gave protected
monomer 2 needed for synthesis of 4S-OH-DPro collagen peptide P2.
2.6 | Field emission scanning electron microscopy
(FE-SEM)
FE-SEM imaging was performed using ULTRA PLUS electron micro-
scope operating at 30 kV. Calculated amounts of peptides taken in
desired water/buffer were drop cast on silicon wafer and allowed to
TABLE 1 HPLC Ret. time, MALDI-TOF data of peptides P1-P4
Ret.
Peptide
P1
time
Mol. formula
83H116N20NaO26Na
C83H116N20NaO26
Cal. Mass
1832.94
1849.05
1832.94
1849.05
1827.04
1827.04
Obs. mass
1832.90
1848.92
1833.01
1849.04
1826.55
1827.90
16.1
C
K
P2
16.3
C
C
C
C
83H116N20NaO26Na
83H116N20NaO26
83H122N26O20Na
83H122N26O20Na
K
P3
P4
15.0
15.1
FIGURE 2 Structure of L and D monomers 1 to 4

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SCHEME 1 A, Ac2O, Ac-OH, reflux,
6 hours. B, 2 N HCl, reflux, 3 hours, (73%). C, Cbz-
Cl, NaHCO3 (98%). D, DMS, K2CO3 (93%). E,
DCC, cat.CuCl. F, HCOOH. G, 2 N NaOH
(53%). H, DMS, K2CO3 (90%). I, t Bu-Br, Ag2O
(90%). J, 2 N LiOH. K, H2, Pd/C. L, Fmoc-Cl, 10%
Na2CO3 (50%)
L-peptides P1 and P3 and negative to positive band (Rn/p) in case of
D-peptides P2 and P4 was measured over a concentration range
25 to 500 μM (Figure S1, S2 and Table S1 ESI). The data indicated that
the CTC of peptides P1 to P4 are around 150 μM and hence all CD
studies were done at 300 μM to ensure that the peptides P1 to P4
are completely in triplex form (Figure 3). The 4R-OH/NH2-LPro pep-
tides (P1 and P3) show large negative CD band at 205 nm and a
small positive CD band around 225 nm characteristic of left-handed
PP II helix adopted by L-collagen. In comparison, CD spectra of
4S-OH/NH2-DPro peptides P2 and P4 show exact mirror images of
L-enantiomeric peptides P1 and P3; large positive CD band at 205 nm
and small negative band around 225 nm indicating the right-handed
PP-II helix adopted by D-collagen peptides P2 and P4. The CD spec-
tral data thus show that enantiomeric collagen peptide pairs P1-P2
and P3-P4 are exact mirror images of each other both in sign and
amplitude. However, the negative band of 4R-NH2-LPro peptide P3 at
200 nm was slightly lower than the mirror image positive band at
205 nm of enantiomeric 4S-NH2-DPro peptide P4, and the slight blue
shift of negative maxima at 200 nm in L-peptide P3 compared to
204 nm positive maxima of D-peptide P4 suggested some differences
in assembling properties of the enantiomeric peptides.
SCHE ME 2 A, MsCl, TEA, DCM. B, NaN3, DMF (80%). C, THF,
PPh3 H2O. D, (Boc)2O, NaHCO3 (50%). E, 2 N NaOH. F, H2, Pd/C. G,
Fmoc-Cl, 10% Na2CO3 (60%)
The 4R-OH-N1(CBz)-DPro methyl ester
8 was converted to
corresponding 4S-azide derivative 15 with inversion of stereochemis-
try at C4, by initial reaction with mesyl chloride in the presence of
NEt3 to obtain 4R-O-mesylate and subsequent reaction in situ with
NaN3 to yield the 4S-N3 15, which depicted characteristic IR fre-
quency for azide at 2104 cm−1. The 4S-N3 was reduced by Staudinger
reaction to obtain the 4S-NH2 compound that was in situ protected
as its Boc derivative to yield 4S-N4H(Boc)-DPro methyl ester 14.
Hydrolysis of ester with aq. LiOH, deprotection of N1-Cbz by hydro-
genation followed by protection of N1 as Fmoc derivative gave mono-
mer 4S-N4H(Boc)-N1(Fmoc)-DProline acid 4 required for synthesis of
4S-NH2-DPro collagen peptide P4 (Scheme 2).
The monomers (1-4) were individually incorporated into the
homochiral collagen peptide sequence (X-Y-Gly)6 at Y position (X = L/
D-proline) by solid phase peptide synthesis using standard Fmoc pro-
tocol on Rink resin. The coupling of desired iminoacid monomer into
peptides was followed by final reaction with N-protected Phe at the
N-terminus to enable accurate determination of peptide concentra-
tions by its UV absorbance at 257 nm ( = 195 M−1 cm−1).[45] Final
deprotection and end-capping of the peptides at N-terminus, cleavage
from resin, gave crude peptides P1 to P4, which were purified by RP-
C18 HPLC and characterized by MALDI-TOF and ESI mass
spectral data.
3.3 | CD Jobs plot of peptides P1 and P2
It is known in literature that on mixing of enantiomeric peptides
together in continuous variable ratios (100:0, 80:20, 60:40 etc.) leads
to effects on net helical content.[48] In order to determine the associa-
tion of oppositely handed triple helices, the enantiomeric peptides P1
and P2 were mixed together in different concentrations keeping the
final concentration constant at 400 μM. It is observed that such
mixing of enantiomeric peptides through continuous variation method
(Jobs plot), lead to changes in the triple-helical signals both at 202 and
224 nm (Figure S3 ESI). At equimolar concentrations of 200 μM of
each mixture of peptides, the CD bands at both 204 and 225 nm can-
celed out each other, leading to zero net ellipticity (Figure 4)
suggesting a of 1:1 for both P1:P2 and P3:P4 enantiomeric pairs. The
observed experimental CD intensities at 202/204 nm and
224/225 nm (for P1/P2 and P3/P4 peptides) intersect at 1:1 stoichio-
metric ratio, while the CD intensities calculated as weighted sum do
not show such intersection (Figure S4 ESI). This indicated that the
3.2 | CD spectroscopic studies
In aqueous solutions, collagen peptides show characteristics CD
signals of a weak positive band around 215 to 227 nm and a large
negative band around 200 nm.[46] In order to find out the critical
triple-helix concentration (CTC, the minimum concentration needed
to form triple helix) of the synthesized enantiomeric collagen peptides,
[47]
the ratio of the intensity of positive to negative band (Rp/n
)
for

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FIGURE 3 CD Spectra of peptide P1 to P4 at pH 7.2, concentration 300 μM. A, P1 (black), P2 (red). B, P3 (blue), P4 (green)
FIGURE 4 A, MRE Plot at 202 and 224 nm for peptide P1 to P2. B, MRE Plot at 204 and 225 nm for peptides P3 to P4 at pH 7.2
observed net intensities are not from isolated helical structures, but
from actual inter helical association of the enantiomeric peptides.
similar to that seen earlier for 4R-NH2-L-prolyl collagen pep-
tide P3.[25]
It is known that the triplex stability of 4R-OH-L-prolyl collagen
peptide P1 is invariant with pH due to nonionizable OH function,
while that of 4R-NH2-L-prolyl peptide P3 with ionizable NH2 function
is pH dependent.[25] Hence comparative Tms of the enantiomeric
4-NH2-collagen L/D-peptides P3 and P4 were determined as a func-
tion of pH in the range 3.0 to 12.0 (Table 2). The peptide 4S-NH2-
DPro collagen peptide P4 also exhibited pH dependence of its Tm,
higher at the extremes of pH (3.0 and 12.0) and minima at pH 9.0
(Figure S6 ESI). The variation profile was identical to that of 4R-NH2-
LPro collagen peptide P3 (Figure 5B) and no stability differences were
seen among the enantiomeric pair of peptides P3/P4 at acidic, neutral
or basic conditions. The reason for minima in Tm at pH 9.0 has been
attributed to pH dependent alteration in puckering modes of proline
ring in 4-NH2-prolyl peptides, based on the fully protonated form
3.4 | Thermal stability of 4R/S-OH/NH2-L/D-prolyl
collagen peptides
The comparative thermal stability of triplexes P1 to P4 were mea-
sured from temperature dependent CD spectra in the range of 5ꢀ to
90 ꢀC at concentration of 250 μM (above CTC of peptides) and
pH 7.2 by monitoring the positive CD band at 225 nm for P1/P2 and
negative band for peptides P3/P4 (Figure S5 ESI). The thermal dena-
turation profile for triplex to random coil transition for all peptides P1
to P4 was found to be sigmoidal in nature (Figure 5A) and the melting
temperatures (Tm) of the triplexes were accurately determined from
the minima of the derivative plots (Figure 5A, Inset). The CD-Tm of
enantiomeric 4(R/S)-OH-L/D-prolyl peptides P1/P2 were identical
(29.5 ꢀC 0.5 ꢀC), as was the case with the enantiomeric pair 4(R/S)-
NH2-L/D-prolyl peptides P3/P4 (54.5 ꢀC 0.5 ꢀC) at pH 7.0. The
4-NH2-prolyl collagen peptides P3/P4 exhibited higher triplex stability
(ΔTm ~ +25 ꢀC) than that of 4-OH-prolyl collagen peptides P1/P2,
+
(NH3 at acidic pH 3.0) and free amino form (NH2 at basic
pH 12.0).[25] While at extremes of pH the peptides will have uniform
puckering modes, at pH 9.0 equivalent to pKa of amine group, the
NH2 function is in 50% protonated form (NH3+) and 50% in free
amino form. Hence the peptides will have mixed puckering modes

8 of 11
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FIGURE 5 Thermal denaturation profiles of peptides P1 to P4 (250 μM) in 10 mM sodium phosphate buffer, pH 7.2, NaCl (10 mM)
monitored at 225 nm. A, normalized ellipticity θ, inset B, derivative plot of melting curves of peptides P1 to P4. C, Tms of peptides P3 and P4 as a
function of pH, Buffers: pH 3.0 (sodium acetate), pH 7.2 (sodium phosphate), and pHs 9.0 and 12.0 (borate)
TABLE 2 Melting Temperature (Tms in o C) P3, P4
Peptide
P3
pH 3.0
60
pH 7.2
55
pH 9.0
26
pH 12.0
49
P4
58
54
27
48.5
Note: Tms are accurate to 0.5 ꢀC; for buffer and sample details see
experimental.
along the peptide chain, leading to destabilization of triplex. The
kinetic effects may dominate the puckering changes as a function of
pH contributing more to observed stability effects.
3.5 | From triple helix conformation to self assembly
and morphology
FIGURE 6 A and B, FE-SEM images of 4-OH-L/D prolyl collagen
peptides P1 and P2. C and D, AFM of peptide P1 and P2 respectively
at 250 μM in water
The assembly of single stranded PPII conformation of collagen pep-
tides into a triplex is a manifestation of various interchain noncovalent
interactions such as H-bonding and van der Waals forces, modulated
by nature of C4-substitution on proline ring and the backbone of the
peptides.[47] Interestingly, enantiomeric recognition between
homochimeric unsubstituted prolyl peptides (LPro-LPro-Gly)10 and
(DPro-DPro-Gly)10 via corresponding triple-helices led to sheet like
structures.[17] Motivated by this observation, we studied enantiomeric
recognition between L- and D-4(R/S)-OH/NH2 collagen peptides that
may lead to self and cross-assembled structures. The morphology of
such assemblies was observed by field-emission scanning electron
microscopy (FE-SEM) and atomic force microscopy (AFM).
that the regular rice grain structures observed arise from self-
assembled triplex form.
When peptides P1 and P2 were mixed in 1:1 stoichiometry
above their CTC (250 μM) at ambient temperature in water, the
assembled fusion of the enantiomeric triplexes resulted in circular
concave cups of 1 to 2 μm diameter with 0.5 to 1 nm curvature as
seen in FESEM (Figure 7A). The size and shape were confirmed by
AFM image (Figure 7B) that also indicated height of ~4 nm
(Figure 7C). However, when the peptides P1 and P2 were mixed first
followed by heating to 80 ꢀC and cooled (annealed), the resultant
morphology was an irregular network and sticky structure of 3 nm
thickness, but with no defined pattern (Figure 7D-F). The concave
spherical nanoparticles obtained by mixing of enantiomeric peptides
as triplexes were lost upon annealing, indicating that the interaction
of enantiomeric peptides in single stranded form is nonspecific lead-
ing to heteromorphology.
The FE-SEM images (Figure 6A,B) of nanoparticles of 4-OH-L/D-
prolyl collagen peptides P1 and P2 at 250 μM concentration (above
CTC) in water show ellipsoid rice grain like structures with 300 to
400 nm size. The AFM images of the 4R/S-OH-L/D peptides P1 and
P2 (Figure 6C,D) corroborate well with this size and show vertical
thickness of 4 nm. The peptides at concentrations of 50 μM and
150 μM (below CTC) exhibited irregular nanospheres of size 60 to
70 nm and 120 to 200 nm respectively (Figure S7, S8 ESI), suggesting

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FIGURE 7 A and B, FE-SEM and AFM of triple helix mixing of P1 + P2. C, Line profile of AFM image B. D and E, FE-SEM and AFM of
annealed mixing of P1 + P2. F, Line profile of AFM image E. (Mixing studies at 250 μM each peptide.)
FIGURE 8 A and B, FE-SEM of peptide P3
and P4. C and D, AFM images of peptide P3 and
P4, respectively, in 1 mM concentration at pH 7.0
The analogous 4-NH2-L/D-prolyl peptides P3 and P4 at 1 mM
(above CTC) at neutral pH 7.2, individually formed nanospheres of
diameter around 200 nm with a hollow central cavity as seen by
FESEM images (Figures 8A,B). This was confirmed by AFM images
(Figure 8C,D) which also indicated height of nanoparticles to be 6 to
10 nm. Similar nanospheres of slightly larger size of 1 μm were
observed at pH 5.0; however, at pH 3.0, 9.0 and 12.0 the peptides
showed disordered nanostructures (Figure S9, S10 ESI). Thus the criti-
cal pH for formation of well-defined nanoparticles is the range pH 5.0
to 7.0, where the necessary factors of electrostatic, H-bonding and
hydrophobic interactions seem to be balanced well favoring self
assembly
nanofibrous sheets of size 1 to 2 μm length with concave surface and
sharp edges in FESEM images (Figure 9A), confirmed by AFM
(Figure 9B) also indicating thickness of ~6 to 8 nm (Figure 9C). Upon
mixing and annealing of 4-NH2-L/D prolyl collagen peptides at
pH 7.0, the racemic sample retained the nanosheet like morphology
but the thickness increased to 40 nm (Figure 9D-F). At pH 3.0, 5.0
and pH 7.0 the racemic mixture of peptides inter associated both in
triple helix as well as in single stranded forms to nanofibrous sheets of
size ~1 to 2 μm (Figure S11 ESI). However, at basic pHs of 9.0 and
12.0 the amino collagen peptides P3 and P4 exhibited disordered
nanostructures (Figure S11 ESI).
Simple mixing of enantiomeric peptides P3 and P4 (above CTC),
leads to formation of the individual nanospheres formed by kinetic
control (Figure 8A,B) that associate into thermodynamically more sta-
ble nanosheet structures (Figure 9A,B) by rearrangement of homo-
chiral triplexes into heterochiral triplex assemblies. After mixing
followed by heating and cooling, the melted nanosheets are initially
Upon mixing equimolar amounts of enantiomeric 4-NH2-L/D-
prolyl collagen peptides P3 and P4 at ambient temperatures to associ-
ate with one another in triple helix form at pH 7.0, the resultant
sample exhibited morphology, very different from that of individual
peptides. The inter assembly enantiomeric triplexes lead to longer

10 of 11
MORE AND GANESH
FIGURE 9 A, FE-SEM and B, AFM of triple helix mixture of P3 + P4. C, Line profile of AFM image B. D, FE-SEM and E, AFM of annealed
mixture of P3 + P4. F, Line profile of AFM image E. Mixing studies at 1 mM each peptide in pH 7.0
kinetically trapped into transient structures through changes in their
electrostatic and H-bonding interactions and finally into thermody-
namically more stable nanofiber structures (Figure 9D,E). This is con-
sistent with the kinetic and thermodynamic model postulated for
peptide self assemblies.[49] Studying the effects of annealing rate may
give more insights into formation of transient structures.
P3/P4 upon mixing and annealing still retained the short nanofibrous
structure, suggesting strong electrostatic/H-bonding interactions from
4-NH2 function dictates the assembly even in single stranded form
leading to well defined morphology. Whether the peptides exist as
single stranded PP-II helices or as triple helices affected their self-
assembling behavior. The self assembly of 4-substituted prolyl pep-
tides are distinctly different from the earlier reported enantiomeric
[17]
model peptide (LPro-LPro-Gly)10 and (DPro-DPro-Gly)10
.
These
results reinforce the role of chirality, the nature of 4-OH/NH2 substi-
tution on prolyl residues and the fine balance between the kinetic and
thermodynamic control of the self assembly process. The potential
applications of different peptide nanostructures are anticipated in
drug delivery, tissue engineering, wound healing, and as new surfac-
tants.[50,51] The introduction of chirality at molecular scale influences
self-assembled growth result in fibrillar networks of small organic mol-
ecules and lead to macroscopic manifestation in gel formation.[52,53]
The work presented here on speigelmeric collagen peptides, their con-
jugates and chimeric hybrid nanocomposites would serve as a step
towards designing collagen based hydrogels for wound healing and
related applications.[19,54]
4
| CONCLUSIONS
Peptides are well endowed for self assembly into a wide variety of
nanostructures, such as tubular structures, fibers, vesicles, and spheri-
cal and rod-coil structures. This is a consequence of their amide back-
bone geometry and functional side chains that orchestrate interaction
of the noncovalent electrostatic, H-bonding and van der Walls forces
to result in balanced hydrophobic / hydrophilic interactions to induce
various types of nanoassemblies. Here we have superimposed chiral-
ity as another dimension in exploring nanostructure formation by
4-OH/NH2-L/D-prolyl collagen peptides. The enantiomeric L/D colla-
gen peptides P1/P2 (4-OH-L/DPro peptides) and P3/P4 (4-NH2-L/
DPro peptides) show mirror image conformation profiles among each
pair, but identical CTC, triplex melting Tms and pH variation profile of
Tm for 4-amino collagen peptides P3/P4. The 4-OH-prolyl peptides
P1/P2 show ellipsoid rice grain like nanoparticles, while the 4-NH2-
prolyl peptides (P3/P4) formed hollow nanospheres. The mixing of
each enantiomeric peptide pair lead to 1:1 non covalent interpeptide
association with different morphologies. Upon cold mixing of each
enantiomeric peptide pairs in triplex form, the resultant was well-
formed circular concave nanocups for 4-OH peptides P1/P2 and
nanosheets with a concave interior for 4-NH2 peptides P3/P4. Thus,
the self assembly of oppositely handed intact triplex structures leads
to new morphological structures. When enantiomeric peptides P1/P2
were mixed after heating followed by annealing, the association of
enantiomeric peptides in single stranded form resulted in irregular
networked gel structures. In comparison, the 4-NH2-L/D-Pro peptides
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.
ORCID
Shahaji H More https://orcid.org/0000-0002-0042-4052
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How to cite this article: More SH, Ganesh KN. Speigelmeric
4R/S-hydroxy/amino-L/D-prolyl collagen peptides:
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Pept Sci. 2019;e24140. https://doi.org/10.1002/pep2.24140