Controlled synthesis of O-glycopolypeptide polymers and their molecular recognition by lectins

Article abstract of DOI:10.1021/bm201813s

The facile synthesis of high molecular weight water-soluble O-glycopolypeptide polymers by the ring-opening polymerization of their corresponding N-carboxyanhydride (NCA) in very high yield (overall yield > 70%) is reported. The per-acetylated-O-glycosylated lysine-NCA monomers, synthesized using stable glycosyl donors and a commercially available protected amino acid in very high yield, was polymerized using commercially available amine initiators. The synthesized water-soluble glycopolypeptides were found to be α-helical in aqueous solution. However, we were able to control the secondary conformation of the glycopolypeptides (α-helix vs nonhelical structures) by polymerizing racemic amino acid glyco NCAs. We have also investigated the binding of the glycopolypeptide poly(α-manno-O-lys) with the lectin Con-A using precipitation and hemagglutination assays as well as by isothermal titration calorimetry (ITC). The ITC results clearly show that the binding process is enthalpy driven for both α-helical and nonhelical structures, with negative entropic contribution. Binding stoichiometry for the glycopolypeptide poly(α-manno-O-lys) having a nonhelical structure was slightly higher as compared to the corresponding polypeptide which adopted an α-helical structure.

Full text of DOI:10.1021/bm201813s

Article
pubs.acs.org/Biomac
Controlled Synthesis of O-Glycopolypeptide Polymers and Their
Molecular Recognition by Lectins
Debasis Pati,† Ashif Y. Shaikh,† Soumen Das,† Pavan Kumar Nareddy,‡ Musti J Swamy,*,‡
Srinivas Hotha,*,§ and Sayam Sen Gupta*,†
†CReST, Chemical Engineering Division, National Chemical Laboratory (CSIR), Dr. Homi Bhabha Road, Pune-411 008, India
§Department of Chemistry, Indian Institute of Science Education and Research, Pune-411 021, India
‡School of Chemistry, University of Hyderabad, Hyderabad-500046, India
S
* Supporting Information
ABSTRACT: The facile synthesis of high molecular weight water-soluble O-
glycopolypeptide polymers by the ring-opening polymerization of their corresponding
N-carboxyanhydride (NCA) in very high yield (overall yield > 70%) is reported. The
per-acetylated-O-glycosylated lysine-NCA monomers, synthesized using stable glycosyl
donors and a commercially available protected amino acid in very high yield, was
polymerized using commercially available amine initiators. The synthesized water-
soluble glycopolypeptides were found to be α-helical in aqueous solution. However, we
were able to control the secondary conformation of the glycopolypeptides (α-helix vs nonhelical structures) by polymerizing
racemic amino acid glyco NCAs. We have also investigated the binding of the glycopolypeptide poly(α-manno-O-lys) with the
lectin Con-A using precipitation and hemagglutination assays as well as by isothermal titration calorimetry (ITC). The ITC
results clearly show that the binding process is enthalpy driven for both α-helical and nonhelical structures, with negative entropic
contribution. Binding stoichiometry for the glycopolypeptide poly(α-manno-O-lys) having a nonhelical structure was slightly
higher as compared to the corresponding polypeptide which adopted an α-helical structure.
However, a majority of these synthetic glycopolymers are
acrylate/acrylamide based and controlled radical polymer-
ization is used to synthesize polymers with controlled molecular
weight, glycosylation density, and position attributes that are
necessary for biological recognition processes. However, the
lack of biocompatibility of some of these polymers can render it
difficult for application in medicine such as drug delivery or
tissue engineering. On the other hand, glycopolypeptides
(glycopolymers with pendant carbohydrates on a polypeptide
backbone) not only mimic the molecular composition of
proteoglycans but also has the ability to fold into well-defined
secondary structures (e.g., helix).22,23 Therefore, it is desirable
to develop methodologies that afford easy and well-defined
synthetic glycopolypeptides.
Although well-defined polypeptides based on natural and
unnatural amino acids have been very successfully synthesized
by the ring-opening polymerization of their corresponding N-
carboxyanhydrides (NCA),24−29 the synthesis of glycopolypep-
tide still remains a major challenge.30,31 Synthesis of
glycopolypeptides by post polymerization modification of
synthetic polypeptides on the contrary has been more
successful and several methods have been reported re-
cently.32−36 We have recently reported the synthesis of the
per-O-benzoylated-D-glyco-L-lysine carbamate NCA from a
INTRODUCTION
■
Glycopolymers, synthetic polymers featuring pendant carbohy-
drate moieties, have been of particular interest to the field of
tissue engineering and drug delivery.1−12 This interest is
derived from the complex roles that carbohydrates play in vivo,
particularly in biomolecular recognition events such as
extracellular recognition, adhesion, cell growth regulation,
cancer cell metastasis, and inflammation.13,14 The key to the
recognition process is their interactions with carbohydrate-
binding protein receptors known as lectins.15,16 The interaction
between lectins and carbohydrates is weak; dissociation
constants, Kd, are typically 10−3 to 10−6 M, but may be greatly
enhanced through polyvalency. Because glycopolymers are
typically polyvalent, as they have several pendant carbohydrate
groups; they present a platform for which multiple copies of a
carbohydrate can be presented simultaneously, thus, enhancing
their affinity and selectivity for lectins many fold. Carbohydrate
recognizing receptors are found on many cell surfaces. An
excellent example is the asialoglycoprotein receptor (ASGP-R)
displayed on the hepatocyte cell surface that interacts uniquely
with galactose/N-acetyl-β-galactosamine containing carbohy-
drate ligands.17−20 Galactose containing synthetic linear
glycopolymers can therefore be used to guide hepatocyte
adhesion through this unique ASGP-R−carbohydrate inter-
action. This strategy has been used to design extracellular
matrices using galactose containing synthetic polymers for liver
tissue engineering.21 Similarly, the use of glycopolymers as
vehicles for therapeutics has also shown a lot of promise.8−12
Received: December 20, 2011
Revised: April 7, 2012
Published: April 12, 2012
© 2012 American Chemical Society
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3.13 (q, 2H, J = 6.1, 12.3 Hz), 3.98−4.16 (m, 2H), 4.30 (dd, 1H, J =
4.7, 12.5 Hz), 4.41 (m, 1H), 4.98 (t, 1H, J = 5.7 Hz), 5.10 (d, 2H, J =
1.5 Hz), 5.17 (d, 2H, J = 3.4 Hz), 5.23−5.43 (m, 4H), 6.01 (d, 1H, J =
1.4 Hz), 7.32−7.38 (m, 10H); 13C NMR (50.32 MHz, CDCl3): δ
20.5(3C), 20.6, 22.2, 28.8, 32.0, 40.6, 53.5, 61.9, 65.4, 66.8, 67.0, 68.4,
68.8, 70.0, 91.1, 127.9−128.5, 135.1, 136.1, 153.0, 155.9, 169.4, 169.6,
170.0, 170.1, 172.1; MALDI-TOF(m/z): Calcd for C36H44KN2O15,
783.2379; found, 783.2373.
stable glycosyl donor and a commercially available protected
amino acid in very high yield (overall yield > 70%).37 These
monomers underwent ring-opening polymerization using
simple primary amine initiators to form well-defined, high
molecular weight homo glycopolypeptides and diblock
coglycopolypeptides. However, attempts to synthesize the
fully deprotected water-soluble glycopolypeptide from these
polypeptides failed as we were unable to efficiently deprotect
the bulky benzoate groups. We hereby report a very efficient
synthesis fully water-soluble glycopolypeptides that were
synthesized from the ring-opening polymerization of per-O-
acetylated-D-glyco-L-lysine carbamate NCA monomers.
Although these glycopolypeptides are predominantly α-helical
in solution, we demonstrate that our synthetic methodology
allows us to alter their secondary structures from an α-helix to a
nonhelical structure. We have also investigated the binding of
these glycopolypeptides with the lectin Con A with the
objective of understanding the role of the secondary structure
of these polypeptides on Con A binding.
1
Compound 2b′. [α]D25 = +30.7 (c 1.0, CHCl3); H NMR (400.13
MHz, CDCl3): δ 1.16−1.92 (m, 6H), 1.92, 1.94, 2.08, 2.09 (4s, 12H),
3.13 (m, 2H), 3.94−4.00 (m, 1H), 4.03 (dd, 1H, J = 2.4, 12.3 Hz),
4.21 (dd, 1H, J = 4.5, 12.3 Hz), 4.33 (m, 2H), 4.99 (t, 1H, J = 5.7 Hz),
5.04 (d, 2H, J = 3.3 Hz), 5.10 (dd, 1H, J = 12.3, 23.8 Hz), 5.18−5.35
(m, 3H), 5.43 (d, 1H, J = 8.1 Hz), 6.01 (d, 1H, J = 1.8 Hz), 7.32−7.38
(m, 10H); 13C NMR (100.63 MHz, CDCl3): δ 20.5(2C), 20.6, 20.7,
22.2, 28.9, 32.1, 40.7, 53.6, 62.0, 65.5, 66.9, 67.1, 68.4, 68.8, 70.0, 91.2,
128.0−128.5, 135.2, 136.1, 153.0, 155.9, 169.4, 169.7, 170.0, 170.6,
172.1; MALDI-TOF(m/z): Calcd for C36H44KN2O15, 783.2379;
found, 783.2376.
25
1
Compound 2c. [α]D = −7.7 (c 1.0, CHCl3); H NMR (200.13
MHz, CDCl3): δ 1.30−1.89 (m, 6H), 1.95, 2.00, 2.03, 2.03, 2.05, 2.08,
2.14 (7s, 21H), 3.09 (q, 2H, J = 5.7, 12.4 Hz), 3.64−3.93 (m, 3H),
4.02−4.19 (m, 4H), 4.31−4.44 (m, 2H), 4.47 (s, 1H), 4.93 (dd, 2H, J
= 3.3, 10.3 Hz), 5.07 (dd, 1H, J = 3.7, 10.3 Hz), 5.09 (s, 2H), 5.12−
5.28 (m, 3H), 5.34 (d, 1H, J = 3.3 Hz), 5.41 (d, 1H, J = 8.3 Hz), 5.59
(d, 1H, J = 8.3 Hz), 7.32−7.39 (m, 10H); 13C NMR (50.32 MHz,
CDCl3): δ 20.4, 20.5(4C), 20.7(2C), 22.1, 28.8, 32.0, 40.4, 53.4, 60.7,
61.7, 66.5, 66.9, 67.1, 68.9, 70.4, 70.6, 70.9, 72.5, 73.1, 75.6, 92.5,
100.8, 127.9−128.6, 135.1, 136.1, 153.6, 155.9, 169.0, 169.5, 169.8,
170.0, 170.1, 170.3(2C), 172.1; MALDI-TOF (m/z): Calcd for
C48H60KN2O23, 1071.3224; found, 1071.3218.
EXPERIMENTAL SECTION
■
Materials and Methods. Propargyl 1,2-orthoesters of the
corresponding carbohydrates were prepared according to literature
procedure.38−40 CbzLys(Boc)OBn was synthesized using standard
literature procedure.41 HAuCl4, triphosgene, and azido-PEG-amine (n
= 11) were obtained from Aldrich and Polypure Inc. All other
chemicals used were obtained from Merck, India. Diethyl ether,
petroleum ether (60−80 °C), ethylacetate, dichloromethane, tetrahy-
drofuran, and dioxane were brought from Merck and dried by
conventional methods and stored in the glovebox. FT-IR spectra were
recorded on Perkin-Elmer FT-IR spectrum GX instrument by making
KBr pellets. Pellets were prepared by mixing 3 mg of sample with 97
General Procedure for the Glyco N-Carboxyanhydrides.
Hydrogenolysis of compounds 2a, 2b, 2b′, and 2c was carried out
using 10% Pd/C in MeOH/EtOAc (9:1) at 400 psi for 12 h. After
completion of the reaction, the reaction mixture was filtered and
concentrated under reduced pressure to afford per-O-acetylated-D-
galactose-L-lysine carbamate, per-O-acetylated-D-mannose-D\L-lysine
carbamate and per-O-acetylated-D-lactose-L-lysine carbamate in almost
quantitative yield. The resulting compounds were directly used for
NCA synthesis without any further purification.
Compounds 3a, 3b, and 3b′. To a solution of per-O-acetylated-D-
galactose-L-lysine carbamate or per-O-acetylated-D-mannose-L/D-lysine
carbamate (500 mg, 0.96 mmol) in freshly distilled out tetrahydrofur-
an (10 mL) was added accordingly a solution of triphosgene (142 mg,
0.480 mmol) in anhydrous tetrahydrofuran (2 mL) under argon and
the reaction mixture was heated to 50−55 °C. α-Pienene (0.228 mL,
1.44 mmol) was then added and the reaction mixture was allowed to
stir for an additional 1 h. The reaction mixture was then cooled to
room temperature and then poured into dry hexane (300 mL) to
afford a white precipitate; which was filtered off quickly and
crystallized two more times using a mixture of ethyl acetate and
petroleum ether. Finally, the white precipitate of glyco N-
carboxyanhydride 3a, 3b, and 3b′ obtained was dried under vacuum
and transferred into the glovebox. Final yield: 425 mg, 80%.
1
mg of KBr. H NMR spectra were recorded on Bruker Spectrometers
(200 MHz, 400 or 500 MHz). 13C NMR and DEPT spectra were
recorded on Bruker Spectrometer (50, 100, or 125 MHz) and reported
relative signals according to deuterated solvent used. HRMS data was
recorded on MALDI-TOF using 2,5-dihydroxybenzoic acid as solid
matrix. Size exclusion chromatography of the glycopolypeptides was
performed using an instrument equipped with Waters 590 pump with
a Spectra System RI-150 RI detector. Separations were effected by 105,
103, and 500 Å Phenomenex 5 μ columns using 0.1 M LiBr in DMF
eluent at 60 °C at the samples concentrations of 5 mg/mL. A constant
flow rate of 1 mL/min was maintained, and the instrument was
calibrated using polystyrene standards.
General Procedure for the Synthesis of Amino Acid Glycosyl
Carbamates (2a, 2b, 2b′, and 2c). To a solution of propargyl 1,2-
orthoester (0.1 mmol), CbzL\DLys(Boc)Obn and activated 4 Å
molecular sieves powder (50 mg) in anhydrous CH2Cl2 (5 mL) was
added HAuCl4 (10 mol %) under an argon atmosphere at room
temperature. The reaction mixture was stirred at room temperature for
the specified time and the reaction mixture was filtered and the filtrate
was concentrated in vacuo. The resulting residue was purified by silica
gel column chromatography using ethyl acetate-petroleum ether as the
mobile phase to afford the compounds 2a, 2b, 2b′, and 2c.
Compound 3c. To a solution per-O-acetylated-D-lactose-L-lysine
carbamate (500 mg, 0.618 mmol) in freshly distilled out
tetrahydrofuran (10 mL) was added a solution of triphosgene (91.70
mg, 0.309 mmol) in anhydrous tetrahydrofuran (2 mL) under argon
and the reaction mixture was heated to 50°-55 °C. α-Pienene (0.147
mL, 0.927 mmol) was then added and the reaction mixture was
allowed to stir for an additional 1 h. The reaction mixture was then
cooled to room temperature and then poured into dry hexane (300
mL) to afford a white precipitate; which was filtered off quickly and
crystallized two more times using a mixture of ethyl acetate and
petroleum ether. Finally, the white precipitate of glyco N-
carboxyanhydride (3c) was dried under vacuum and transferred into
the glovebox. Final yield: 410 mg, 80%.
25
1
Compound 2a. [α]D = +4.4 (c 1.0, CHCl3); H NMR (200.13
MHz, CDCl3): δ 1.15−1.91(m, 6H), 1.98, 2.03, 2.04, 2.12 (4s, 12H),
3.12 (td, 2H, J = 2.1, 6.5, 13.5 Hz), 3.98 (t, 1H, J = 6.5 Hz), 4.06 (dd,
1H, J = 7.7, 11.4 Hz), 4.11 (d, 1H, J = 1.0 Hz), 4.15 (d, 1H, J = 3.1
Hz), 4.39 (m, 1H), 5.11 (s, 2H), 5.05 (dd, 1H, J = 3.5, 10.4 Hz), 5.17
(d, 1H, J = 3.8 Hz), 5.29 (dd, 1H, J = 8.1, 10.4 Hz), 5.39 (d, 2H, J =
3.4 Hz), 5.62 (d, 1H, J = 8.2 Hz), 7.32−7.38 (m, 10H); 13C NMR
(50.32 MHz, CDCl3): δ 20.4(3C), 20.5, 22.0, 28.7, 31.8, 40.4, 53.5,
60.8, 66.7, 66.8, 66.9, 67.7, 70.6, 71.1, 93.0, 127.9−128.5, 135.1, 136.1,
153.7, 155.9, 169.4, 169.7, 170.0, 170.2, 172.0; MALDI-TOF (m/z):
Calcd for C36H44KN2O15, 783.2379; found, 783.2333.
Compound 3a. 1H NMR (400.13 MHz, CDCl3): δ 1.35−1.82 (m,
6H), 1.98, 2.03, 2.04, 2.14 (4s, 12H), 3.12 (td, 2H, J = 2.1, 6.5, 13.7
Hz), 4.04−4.20 (m, 3H), 4.33 (bs, 1H), 5.09 (dd, 1H, J = 3.3, 10.5
25
1
Compound 2b. [α]D = +22.1 (c 1.0, CHCl3); H NMR (200.13
MHz, CDCl3): δ 1.10−1.92 (m, 6H), 1.99, 2.02, 2.08, 2.17 (4s, 12H),
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Hz), 4.25 (dd, 1H, J = 8.5, 10.3 Hz), 5.30 (m, 1H), 5.42 (d, 1H, J = 3.1
Hz), 5.62 (d, 1H, J = 8.3 Hz), 7.14 (bs, 1H); 13C NMR (100.61 MHz,
CDCl3): δ 20.5, 20.6(2C), 20.7, 21.6, 28.8, 31.0, 40.3, 57.4, 60.9, 66.8,
68.0, 70.7, 71.4, 93.3, 152.4, 154.1, 169.8, 170.0(2C), 170.1, 170.5; FT-
IR (dioxane) 1785 and 1858 cm−1 νco (unsymmetrical stretching).
Compound 3b. 1H NMR (500.13 MHz, CD2Cl2): δ 1.35−1.88 (m,
6H), 1.99, 2.03, 2.07, 2.16 (4s, 12 Hz), 3.23 (q, 2H, J = 6.7, 12.6 Hz),
3.68 (t, 1H, J = 6.5 Hz), 4.08 (m, 1H), 4.13 (dd, 1H, J = 2.8, 12.2 Hz),
4.23 (dd, 1H, J = 4.6, 12.2 Hz), 4.35 (dd, 1H, J = 4.7, 6.9 Hz), 5.20−
5.35 (m, 3H), 5.94 (d, 1H, J = 1.7 Hz), 6.72 (bs, 1H); 13C NMR
(125.76 MHz, CD2Cl2): δ 20.8(2C), 20.9(2C), 22.2, 29.3, 31.6, 40.7,
57.9, 62.7, 66.1, 68.9, 69.3, 70.5, 91.7, 152.3, 153.8, 170.0, 170.3, 170.4,
170.6, 171.2; FT-IR (dioxane) 1785 and 1858 cm−1 νco (unsym-
metrical stretching).
CH2CH2O unit in initiator), 4.0−4.32 (m, 4H), 5.10−5.60 (m, 3H),
5.80−5.98 (m, 1H).
Polymer 5g. 1H NMR (400.13 MHz, CDCl3): δ 1.20−1.98(m,
6H), 1.99−2.16 (4s, 12 Hz), 3.10−3.40 (m, 2H), 3.62−3.68 (m, for
CH2CH2O unit in initiator), 4.0−4.32 (m, 4H), 5.10−5.60 (m, 3H),
5.80−5.98 (m, 1H).
1
Polymer 6a. H NMR (400.13 MHz, CDCl3): δ 1.15−1.92 (m,
6H), 1.94−2.12 (br, m, 21 Hz), 3.10−3.18 (m, 2H), 3.62−3.68 (m, for
CH2CH2O unit in initiator), 3.70−3.90 (m, 2H), 4.0−4.20 (m, 4H),
4.40−4.58 (m, 3H), 4.90−5.09 (m, 3H), 5.15−5.35 (m, 3H), 5.50−
5.60 (m, 1H).
Deprotection Procedure for the Glycopolypeptides. Hydra-
zine monohydrate (25 equiv) was added to the solutions of all the
acetyl-protected glycopolypeptides in methanol (10 mg/mL), and the
reactions were stirred for 7−8 h at room temperature. Reactions were
quenched by addition of acetone and then solvent was removed almost
completely under reduced pressure. The solid residues were dissolved
in deionized water and transferred to dialysis tubing (3.5 and 12 KDa
molecular weight cutoff according to polymer molecular weight). The
samples were dialyzed against deionized water for 3 days, with water
changes once every 2 h for the first day, and then thrice per day.
Dialyzed polymers were lyophilized to yield glycopolypeptides (4b, 4d,
5b, 5d, 5f, 5h, and 6b) as white fluffy solids (around 90% yield).
Polymer 4b. 1H NMR (400.13 MHz, D2O): δ 1.10−2.01 (m, 6H),
3.12 (m, 2H), 3.65−3.70 (m, for CH2CH2O unit in initiator), 3.58−
3.82 (m, 7H), 3.85−3.95 (m, 1H), 3.98−4.32 (m, 1H), 5.20−5.45 (m,
1H).
1
Compound 3b′. H NMR (400.13 MHz, CD2Cl2): δ 1.35−1.88
(m, 6H), 1.99, 2.03, 2.07, 2.16 (4s, 12 Hz), 3.23 (q, 2H, J = 6.7, 12.6
Hz), 3.68 (t, 1H, J = 6.5 Hz), 4.08 (m, 1H), 4.13 (m 1H), 4.23 (dd,
1H, J = 4.6, 12.2 Hz), 4.35 (dd, 1H, J = 4.7, 6.9 Hz), 5.20−5.35 (m,
3H), 5.96 (m, 1H), 7.2 (bs, 1H); 13C NMR (100.61 MHz, CD2Cl2): δ
20.8(2C), 20.9(2C), 22.2, 29.3, 31.6, 40.7, 57.9, 62.7, 66.1, 68.9, 69.3,
70.5, 91.7, 152.3, 153.8, 170.0, 170.3, 170.4, 170.6, 171.2; FT-IR
(dioxane) 1785 and 1858 cm−1 νco (unsymmetrical stretching).
Compound 3c. 1H NMR (500.13 MHz, CD2Cl2): δ 1.15−1.88 (m,
6H), 1.94, 2.01, 2.03, 2.04, 2.05, 2.10, 2.12 (7s, 21H), 3.18 (m, 2H),
3.75 (d, 1H, J = 7.9 Hz), 3.86 (t, 1H, J = 9.1 Hz), 3.91 (t, 1H, J = 5.9
Hz), 4.05−4.17 (m, 3H), 4.32 (t, 1H, J = 5.5 Hz), 4.50 (d, 1H, J = 8.0
Hz), 4.56 (d, 1H, J = 12.0 Hz), 4.97 (m, 2H), 5.05 (m, 1H), 5.14 (t,
1H, J = 7.4 Hz), 5.22 (t, 1H, J = 9.0 Hz), 5.35 (d, 1H, J = 2.7 Hz), 5.60
(d, 1H, J = 8.2 Hz), 6.65 (s, 1H); 13C NMR (125.76 MHz, CD2Cl2): δ
20.7(2C), 20.8(3C), 20.9, 21.0, 22.0, 29.3, 31.5, 40.5, 57.9, 61.3, 61.7,
67.1, 69.2, 70.7, 71.2, 71.2, 72.6, 73.7, 75.8, 93.1, 101.2, 152.2, 154.3,
169.4, 170.0, 170.1, 170.3, 170.4, 170.4, 170.6, 170.8; FT-IR (dioxane)
1785 and 1858 cm−1 νco (unsymmetrical stretching).
Polymer 4d. 1H NMR (400.13 MHz, D2O): δ 1.10−2.01 (m, 6H),
3.12(m, 2H), 3.65−3.70(m, for CH2CH2O unit in initiator), 3.58−
3.82 (m, 7H), 3.85−3.95 (m, 1H), 3.98−4.32 (m, 1H), 5.20−5.45 (m,
1H).
Polymer 5b. 1H NMR (400.13 MHz, D2O): δ 1.08−2.01 (m, 6H),
3.10−3.322 (m, 2H), 3.65−3.70 (m, for CH2CH2O unit in initiator),
3.70−3.93 (m, 7H), 3.85−3.95 (m, 1H), 3.98−4.32 (m, 1H), 5.70−
5.89 (m, 1H).
General Procedure for the Synthesis of Glycopolypeptides.
To a solution of glyco-L/D-lysine NCA (100 mg/mL) in dry dioxane
was added with “proton sponge” N,N′-tetramethylnapthalene (1.0
equiv to monomer; 1 M) as an additive and azido-PEG-amine (0.5 M)
as the initiator inside the glovebox. The progress of the polymerization
were monitored by FT-IR spectroscopy by comparing with the
intensity of the initial NCA’s anhydride stretching at 1785 and 1858
cm−1. The reactions generally completed within 36 h. Aliquotes were
removed after completion of polymerization for GPC analysis. Finally
the solvent was removed under reduced pressure from the reaction
mixture. The resulting residue was redissolved in dichloromethane and
then the polymer was precipitated out by addition of methanol. The
precipitated polymer was collected by centrifugation and dried to
afford white glycopolypeptides 4a, 4c, 5a, 5c, and 6a in almost 85−
90% yield.
Polymer 5d. 1H NMR (400.13 MHz, D2O): δ 1.08−2.01 (m, 6H),
3.10−3.322 (m, 2H), 3.65−3.70 (m, for CH2CH2O unit in initiator),
3.70−3.93 (m, 7H), 3.85−3.95 (m, 1H), 3.98−4.32 (m, 1H), 5.70−
5.89 (m, 1H).
Polymer 5h. 1H NMR (400.13 MHz, D2O): δ 1.08−2.01 (m, 6H),
3.10−3.322 (m, 2H), 3.65−3.70 (m, for CH2CH2O unit in initiator),
3.70−3.93 (m, 7H), 3.85−3.95 (m, 1H), 3.98−4.32 (m, 1H), 5.70−
5.89 (m, 1H).
Polymer 6b. 1H NMR (400.13 MHz, D2O): δ 1.15−1.88 (m, 6H),
3.10−3.18 (m, 2H), 3.46−3.54 (m, 2H), 3.65−3.70 (m, for CH2CH2O
unit in initiator), 3.55−4.0 (m, 11H), 4.35−4.47 (m, 1H), 5.35−5.48
(m, 1H).
Circular Dichroism Measurements. Aqueous solution of
glycopeptides 4b, 4d, 5b, 5d, 5f, 5h, and 6b were filtered through
0.22 μm syringe filters. CD (190−250 nm) spectra of the
glycopolypeptides (0.50 mg/mL in deionized water) were recorded
(JASCO CD SPECTROPOLARIMETER, Model Name J-815) in a
cuvette with a 1 mm path length. All the spectra were recorded for an
average of three scans and the spectra were reported as a function of
molar ellipticity [θ] versus wavelength. The molar ellipticity was
calculated using the standard formula, [θ] = (θ × 100 × Mw)/(C × l),
where θ = experimental ellipticity in millidegrees, Mw = average
molecular weight, C = concentration in mg/mL, and l = path length in
cm. The % α helicity was calculated by using the formula % α helicity
= (−[θ]222 nm + 3000)/ 39000.45
Precipitation Assay. Quantitative precipitations and analysis were
carried out by a method modified of Brewer,46 as adapted by
Keissling,47 Cloninger, and co-workers (Figure 2 SI).48
Hemagglutination Assay (HA assay). HA assays were
performed as has been described by Finn et al. (Figure 3 SI).49
Isothermal Titration Calorimetry. Calorimetric titrations were
performed on a VP-ITC isothermal titration calorimeter from
MicroCal (Northampton, MA), essentially as described earlier.50,51
Briefly, 5 μL aliquots of a 200−300 μM glycopolymer solution were
added at 7 min intervals via a rotating stirrer syringe to a 70 μM
For synthesis of polymers 5e and 5g, 3b (α-manno-O-L-lys NCA)
and 3b′ (α-manno-O-D-lys NCA) were mixed in equal proportions (by
weight) and then polymerization was carried out as has been described
above.42−44
1
Polymer 4a. H NMR (400.13 MHz, CDCl3): δ 1.30−1.97 (m,
6H), 1.98−2.14 (4s, 12H), 3.07−3.30 (m, 2H), 3.62−3.68 (m, for
CH2CH2O unit in initiator), 3.55−3.90 (m, 1H), 4.04−4.40 (m, 3H),
5.00−5.55 (m, 3H), 5.50−5.76 (m, 1H), 5.56−5.95 (amide H).
1
Polymer 4c. H NMR (400.13 MHz, CDCl3): δ 1.30−1.97 (m,
6H), 1.98−2.14 (4s, 12H), 3.07−3.30 (m, 2H), 3.62−3.68 (m, for
CH2CH2O unit in initiator), 3.55−3.90 (m, 1H), 4.04−4.40 (m, 3H),
5.00−5.55 (m, 3H), 5.50−5.76 (m, 1H), 5.56−5.95 (amide H).
1
Polymer 5a. H NMR (400.13 MHz, CDCl3): δ 1.20−1.98 (m,
6H), 1.99−2.16 (4s, 12 Hz), 3.10−3.40 (m, 2H), 3.62−3.68 (m, for
CH2CH2O unit in initiator), 4.0−4.32 (m, 4H), 5.10−5.60 (m, 3H),
5.80−5.98 (m, 1H).
1
Polymer 5c. H NMR (400.13 MHz, CDCl3): δ 1.20−1.98 (m,
6H), 1.99−2.16 (4s, 12 Hz), 3.10−3.40 (m, 2H), 3.62−3.68 (m, for
CH2CH2O unit in initiator), 4.0−4.32 (m, 4H), 5.10−5.60 (m, 3H),
5.80−5.98 (m, 1H).
1
Polymer 5e. H NMR (400.13 MHz, CDCl3): δ 1.20−1.98 (m,
6H), 1.99−2.16 (4s, 12 Hz), 3.10−3.40 (m, 2H), 3.62−3.68 (m, for
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Scheme 1. General Synthesis of Glycopolypeptides by the Ring-Opening Polymerization of Their Glycosylated Amino Acid
a
NCAs
a
(a) HAuCl4, CH2Cl2, rt, 0.5 h, 4Å MS Powder, Yield (90%); (b) Pd/C, H2, 400 psi, CH3OH, 12 h, Yield (>95%); (c) Triphosgene, THF, α-
pienene, 70 °C, Yield (80%); (d) Dioxane, Proton Sponge (1.0 equiv), 24 h, RT; (e) Methanol, Hydrazine hydrate (25 equiv), 6 h.
Table 1. Different Glycopolypeptides Synthesized by Our Methodology
solution of Con A (subunits) contained in a 1.445 mL sample cell.
Samples were dialyzed extensively against 50 mM Hepes buffer, pH 7.4
(containing 0.9 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 0.02% of
sodium azide) and degassed prior to loading into the cell. Because the
first injection was often found to be inaccurate, a 2 μL injection was
added first and the resultant point was deleted before the remaining
data were analyzed using the “one set of sites” binding model in
MicroCal Origin ITC analysis software, as described earlier.50,51 The
analysis yielded values of the following parameters: number of binding
sites (n), binding constant for the interaction (Kb), and enthalpy of
binding (ΔHb). From these values, free energy of binding (ΔG°b) and
entropy of binding (ΔSb) were calculated according to the following
basic thermodynamic equations:
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Table 2. Synthesis of Glycopolypeptides at RT
protected polymer
deprotected polymer
a
b
c
d
e
f
run No.
monomer (M)
β-galac-O-L-lys
M/I
M
polymer
Mn
Mw/Mn
DP
polymer
conformation
exp
1
2
3
4
5
6
7
30
50
30
50
30
50
30
15630
25670
15630
25670
15630
25670
24330
4a
4c
5a
5c
5e
5g
6a
21654
32698
19646
30690
18642
28180
28220
1.12
1.14
1.08
1.10
1.08
1.10
1.07
42
64
38
60
36
55
35
4b
4d
5b
5d
5f
α-helix
β-galac-O-L-lys
α-helix
α-manno-O-L-lys
α-manno-O-L-lys
Rac-α-manno-O-LD-lys
Rac-α-manno-O-LD-lys
β-lacto-O-L-lys
α-helix
α-helix
nonhelical
nonhelical
α-helix
5h
6b
a
b
c
M/I indicates monomer to initiator ratio. Expected molecular weight calculated from monomer/initiator. Number average molecular weight
d
calculated from NMR. Polydispersity index was estimated from GPC (DMF/0.1 M LiBr, 60 °C, RI) and calibrated with polystyrene standards.
e
f
1
Degree of polymerization from H NMR (DP). Secondary conformation was determined by CD spectroscopy in water.
ΔG°b = − RT ln Kb
(1)
(2)
CH2-CH2), with the proton peaks of the acetate group
(CH3CO-) present in the carbohydrate moiety (1.98−2.14
ppm). The Mn was estimated to be 21654 and 32698, while the
PDI was calculated to be 1.12 and 1.14 for 4a and 4c,
respectively. The slightly higher molecular weight that is
observed is probably due to incomplete initiation by azido-
PEG-NH2. In the same way, 3b (α-manno-O-L-lys NCA) was
polymerized using azido-PEG-NH2 (n = 11) as the initiator
(M/I = 30, 50) in dioxane (Table 1, runs 3 and 4). The
molecular weight distribution was again found to be reasonably
narrow (PDI 1.08 and 1.10, respectively), and the molecular
weights of the resulting polymers 5a and 5c were estimated to
be 19646 and 30690, respectively. Polymerization of 3c (β-
lacto-O-L-lys NCA), using azido-PEG-NH2 (n = 11) as the
initiator (M/I = 30) in dry dioxane (Scheme 1) afforded
polymer 6a. The molecular weight distribution observed from
GPC was monomodal, while the molecular weight and PDI
were calculated to be 28220 and 1.07, respectively. Although
per-O-acetate-D-lactose-O-L-serine NCA has been synthesized
before, their polymerization to the corresponding glycopoly-
peptide has not been reported.52 It should be noted that all the
molecular weights obtained were reasonably close to their
expected molecular weights. This is in contrast to transition
metal catalyzed polymerization of the C-linked glycopolypep-
tides reported recently, where the molecular weights obtained
were nearly three times that of the expected molecular
weights.30,31 All the glycopolypeptides obtained were then
deprotected by using NH2NH2·H2O in MeOH to remove the
acetyl groups present in the carbohydrate moiety. The
deprotected polymers were then purified by extensive dialysis
against deionized water to afford fully water-soluble glyco-
polypeptides 4b, 4d, 5b, 5d, 5f, 5h, and 6b (Table 2). The
complete removal of the acetyl groups was confirmed by the
ΔG°b = ΔHb − TΔSb
RESULTS
■
Synthesis of O-Glycopolypeptides. We have recently
reported the synthesis of per-O-benzoylated-D-glyco-L-lysine
carbamate by reaction of ε-Boc-protected CbzLysOBn and
propargyl 1,2-orthoester of per-O-benzoylated-glucose/man-
nose in the presence of HAuCl4/CH2Cl2/4 Å MS powder/
rt.38−40 The highlight of this reaction is the near quantitative
yield of the glycosidation step (>80%), which allows the
synthesis of their corresponding NCAs with an overall yield of
70%. The same methodology was used to synthesize per-O-
acetylated-D-glyco-L-lysine carbamate from their corresponding
propargyl 1,2-orthoester of per-O-acetylated carbohydrates.
Accordingly, glycosylation reaction between ε-Boc-protected
CbzLysOBn and propargyl 1,2-orthoester of galactose,
mannose, and lactose was conducted in the presence of
HAuCl4 and 4 Å molecular sieves powder in CH2Cl2 at room
temperature to afford the carbamates 2a, 2b, 2b′ (D-lysine), and
2c in around 80−90% yield (Scheme 1). Furthermore, we
continued our journey toward the glyco-NCA in two steps:
first, we subjected the glycoconjugates 2a, 2b, 2b′, and 2c to
hydrogenolysis using 10% Pd/C at 400 psi to obtain per-O-
acetylated-D-galactose-L-lysine carbamate, per-O-acetylated-D-
mannose-L-lysine carbamate, and per-O-acetylated-D-lactose-L-
lysine carbamate. They were then subsequently converted to
their corresponding NCAs 3a (β-galacto-O-L-lys), 3b (α-
manno-O-L-lys), 3b′ (α-manno-O-D-lys), and 3c (β-lactose-O-
L-lys) using triphosgene and α-pienene in 80% yield after three
crystallizations (Scheme 1). The purified NCAs 3a, 3b, 3b′, and
3c were thoroughly characterized by NMR and FT-IR
spectroscopic studies.
Polymerization of 3a(β-galac-O-lys NCA) was carried out in
the presence of 1.0 equiv of N,N′-tetramethylnapthalene
“proton sponge”, using azido-PEG-NH2 (n = 11) as the
initiator (M/I = 30, 50) in dry dioxane, as has been described
before (Scheme 1). The progress of the polymerization was
followed by monitoring the disappearance of the anhydride
stretch of the NCA ring at 1785 and 1858 cm−1, as was
observed by FT-IR. The resulting polymers 4a and 4c were
purified by reprecipitation and the structure was identified by
1H and 13C NMR (Figures 10−53 SI). The molecular weight
distribution observed from GPC was monomodal and found to
be reasonably narrow (Figure 6 SI). Its molar mass was
estimated from the relative intensity of the peak at 3.62−3.68
ppm due to characteristic protons present in the initiator (O-
1
absence of the acetyl protons in the H NMR of the water-
soluble glycopolypepitdes (Figure 10−53 SI).
All the glycopolypeptides above, were synthesized by the
polymerization of enantiomerically pure glyco-O-L-lysine
NCA’s to afford glycopeptides having a backbone composed
only of L-lysine. We were interested in synthesizing
glycopolypeptides which would have a backbone composed
of racemic DL-lysine. This would allow us to study the
properties of glycopolypeptides with a racemic peptide
backbone.42−44 To accomplish this, we synthesized α-manno-
O-L-lysine NCA (3b) and α-manno-O-D-lysine NCA (3b′) from
their corresponding L- or D-lysine amino acid glycol conjugates.
These NCAs (3b and 3b′) were mixed in equal proportions and
polymerized to afford polymers 5e and 5g. The acetate groups
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Figure 1. (A) Circular dichroism spectra of poly(β-gal-O-L-lys) 4d at 20 °C (0.5 mg/mL); (B) Circular dichroism spectra of poly(β-lacto-O-L-lys) 6b
at 20 °C (0.5 mg/mL); Inset: Percentage of α-helicity content (% helicity calculated using molar ellipticity at 222 nm) as a function of temperature.
of 5e and 5g were then deprotected to afford fully water-soluble
glycopolypeptide 5f and 5h.
To prove that the initiator was incorporated into the
polymer, the resultant polymers 4a and 5a were purified by
multiple reprecipitations and then characterized by NMR and
FT-IR. The FT-IR of 4a and 5a show sharp peak at 2110 cm−1
that is characteristic for the organo azide stretch (Figure 1 SI).
Usage of this bifunctional azido-PEG-amine initiator allows the
synthesis of end functionalized polymer 4a and 5a which was
further manipulated using Cu(I) catalyzed azide−alkyne “click
chemistry” with fluorecein alkyne (Scheme 1 SI).53 After click
reactions the azide intensity decreased by >80%. The fluorecein
labeled glycopolypeptides were deprotected by hydrazine
monohydrate in methanol to obtained water-soluble fluore-
cein-labeled glycopolypeptides, 7 and 8, respectively, the
polymers were characterized by 1H NMR (Figures 10−53
SI). These fluorecein-labeled glycopolypeptides can be used to
study their cellular internalization and trafficking as has been
shown before.54,55
Figure 2. Calorimetric titration of Con A with A [50-α-manno-O-L-
lys(OH)] 5d] and B [Rac-50-α-manno-O-LD-lys(OH) 5h] at 298 K.
Upper panels show the ITC raw data obtained from 20 automatic
injections of 5 μL aliquots of the glycopeptide (in the syringe) into
1.445 mL of Con A in the ITC cell. Lower panels show the integrated
heat of binding obtained from the raw data. See text for more details.
Conformation of Glycopolypeptides in Solution. The
conformation of the glycopolypeptides in solution was
investigated by circular dichroism (CD). The fully deprotected
polymer poly(glyco-O-lys) 4b, 4d, 5b, 5d, and 6b all of which
have a polypeptide backbone compose of enantiomerically pure
L-lysine, were found to be α-helical in water with a characteristic
minima at 208 and 222 nm (Figure 2; Figure 4 SI). For
example, poly(β-galacto-O-L-lys) polypeptide was found to be
70% helical in water at RT. The percentage helicity of 4d as a
function of temperature was also studied using CD. It was
found that the α-helical conformation got disrupted as the
temperature was increased from 0 to 70 °C. The polymer
regained its helicity completely upon cooling it back to 0 °C.
The percentage of helicity of these glycopolypeptides was
found to be dependent on the length of the glycopolypeptide.
For example, the percentage helicity of poly(α-manno-O-L-lys)
5d (60 mer) was 62%, while the % helicity of its corresponding
38 mer (5b) decreased to 30% at room temperature. The
conformation of the deprotected polypeptide poly(β-lacto-O-L-
lys) 6b was also studied as a function of temperature. This
polymer was also observed to be α-helical in water and the
helicity content was determined to be 55% at room
temperature (Figure 1).
containing blocks of L- and D-glyco amino acids would also give
no CD signal. To ascertain that no stereoselection took place
during our polymerization, we initiated polymerization of
racemic manno NCAs with a preincubated solution of 1:1
mixture of α-manno-O-L-lys NCA and N3-PEG-amine. The CD
of the reaction mixture was recorded at 50 and 100%
completion of the reaction and they were found to be identical.
This shows that the D- and L-glyco-NCAs are randomly
incorporated and no stereoselection occurred during the
polymerization (Figure 9 SI).
Interaction of Glycopolypeptides with Con A.
Precipitation and Hemagglutination Assay. Preliminary
studies on the binding of the mannose containing glyco-
polypeptides (5b, 5d, 5f, and 5h) with Con A was evaluated by
performing precipitation and hemagglutination assays. The
precipitation assay was performed to determine the number of
mannose units available in the glycopolypeptides (5b, 5d, 5f,
and 5h) for binding to each Con A lectin. In all experiments the
ratio of the mannose functionalized polypeptide to Con A
increased with increasing amount of the glycopolypeptide and
then remained fairly constant after a maximum had been
reached (Figure 2 SI). The stoichiometry of binding (number
of Con A tetramer per glycopolypeptide) was determined to be
4, 5, 3.5, and 3.5 for 5d, 5h, 5b, and 5f, respectively. The data
from the experiments suggest (Table 1 SI) that the number of
Glycopolypeptides 5f and 5h, where the polypeptide
backbone consisted of racemic DL-lysine, showed no helicity
(helicity <2% in water at RT, Figure 4 SI). This was expected
because a backbone having a racemic amino acid is not
expected to fold into an α-helix, as has been shown before.42−44
However, it must be noted that if stereoblock polymers
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a
Table 3. Thermodynamic Parameters for the Binding of Glycopolypeptides to Con A
b
conformation
polyvalency
(sugar)
c
d
polymer
(units)
n
Ka (M−1) × 10−6
ΔH (kcal/mol)
ΔS (cal/mol/K)
α-methyl mannopyranoside
1
0.0051 ( 0.0001)
2.44 ( 0.44)
4.13 ( 0.29)
10.21 ( 3.51)
9.11 ( 2.52)
−10.20 ( 0.07)
−81.20 ( 3.3)
−88.40 ( 2.7)
−95.50 ( 2.1)
−17.15 ( 0.25)
−243 ( 1.4)
−266 ( 5.7)
−288.5 ( 17.7)
−354.5 ( 3.5)
1
5b [30-α-manno-O-L-lys(OH)]
5f [Rac-30-α-manno-O-LD-lys(OH)]
5d [50-α-manno-O-L-lys(OH)]
5h [Rac-50-α-manno-O-LD-lys(OH)]
α-helix (38 units)
nonhelical (36 units)
α-helix (60 units)
nonhelical (55 units)
7.0 ( 0.5)
8.1 ( 0.7)
9.2 ( 0.1)
12
22
33
32
10.2 ( 0.6)
b
−115.0 ( 1.4)
a
c
Values shown are the average of two independent titrations. Determined from CD spectra in water. Degree of polymerization was determined by
d
1H NMR Number of Con-A subunit per glycopolypeptide
mannopyranoside units in the polypeptide bound per Con A
tetramer for a glycopolypeptide having a helical structure is
slightly higher than the corresponding glycopolypeptide with
no secondary structure (5d vs 5h and 5b vs 5f).
number of accessible sugar residues for binding. Finally, the
racemic glycopolypeptides were found to exhibit a slightly
higher binding stoichiometry as compared to the α-helical
counterparts, which may be due to their slightly longer lengths.
Hemagglutination assays (HA) were performed with
glycopolypeptides (5b, 5d, 5f, and 5h) to get preliminary
information regarding binding affinity,49 the use of HA to
measure inhibition of protein carbohydrate interactions is well
documented and gives us an essential entry-level comparison of
the different mannose containing glycopolypeptides synthe-
sized by us. When compared to the control monomer methyl
mannose, poly(α-manno-O-lys) glycopolypeptides 5b, 5d, 5f,
and 5h, showed an increase in binding affinity ranging from 15-
to 36-fold per mannose unit. This indicates an increase in
activity toward Con A of 1 order of magnitude per mannose
unit, which is suggestive of a glycoside clustering motif of all the
synthesized glycopolypeptides. Because the error associated
with the dose determination is a factor of 2, as dictated by the
2-fold dilutions of the assay, the polyvalencies of 5b, 5d, 5f, and
5h are the same within experimental error.
Isothermal Titration Calorimetry. Results of representative
calorimetric titrations obtained for the binding of glycopol-
ymers to Con A at 25 °C are shown in Figure 2. Figure 2A,B
correspond to the titration of Con A with glycopolypeptides 5d
and 5h, respectively. While the upper panels in these two
figures show the exothermic heat released upon binding at each
injection, the lower panels show plots of incremental heat
released as a function of the ligand/Con A subunit ratio.
Nonlinear least-squares fits of the data to one set of sites model
(shown as solid lines) indicate that the experimental data could
be described well by this model as judged by the high quality of
the fits. Similar high quality data were obtained for the titration
of Con A with glycopolypeptides 5b and 5f (Figure 4 SI). In
each case very similar data were obtained in duplicate
experiments and the average values of binding constants
(Kb), stoichiometry of binding (n), enthalpy of binding (ΔHb),
and entropy of binding (ΔSb) obtained from the calorimetric
titrations for the interaction of all the glycopolymers with Con
A are listed in Table 3.
DISCUSSION
■
Design of Glycopolypeptides for Lectin Binding
Study. We chose a set of four glycopolypeptides to probe
their binding to the lectin Con A and also probe the difference
between helical and nonhelical polypeptides. While glycopoly-
peptide 5h and 5d have the same structure and similar
molecular weights, their secondary structures are different. CD
measurements show 5d to be α-helical (62% helicity), whereas
the 5h shows no secondary structure (<2% helicity) at RT.
Similar glycopolypeptides 5b (30% α-helical) and 5f (non-
helical, <2% helicity) have similar molecular weights but differ
in their secondary structures. It is expected that a
predominantly α-helical polypeptide will be stiff, and good
binding is likely to be observed only if the distance between
two sugar units matches exactly to the distance between two
binding sites. This is unlikely in most cases as have been
observed by Kobayashi et al.56 where a rigid helical poly-
(glycosyl phenyl isocyanate) was observed to have very little
specific interactions with lectins, while the equivalent polymer
with a flexible phenylacrylamide showed good binding. On the
contrary, Kiick et al.57 have showed that a helical backbone in
polypeptides can be superior to coiled structures for binding to
lectin CT B5.57 Hence, conformational factors that determine
binding are complex. Because our synthetic methodology
allows the synthesis of polypeptides with the same structure
and molecular weight but with different secondary conforma-
tion (for example 5h and 5d), we can probe the effect of
secondary conformation of glycopolypeptides on lectin binding.
Lectin Binding of the Glycopolypeptides. ITC studies
show that there is very little difference in the number of Con A
monomer units bound per polypeptide for glycopolypeptides
having an α-helical conformation (5b, 5d) and with no
secondary structure (5f, 5h) of similar molecular weight. A
similar trend is also observed for the precipitation assay (Figure
2 SI), although the stoichiometry determined from the
precipitation assay is higher for all the glycopolypeptides used
in this study. Precipitation assays are semiquantitative and are
known to overestimate stoichiometry of binding, because a
macroscopic precipitation event could result from even partially
saturated binding events. To understand the stoichiometry for
Con A binding with the different glycopolypeptides, a
qualitative estimation of the length of the different
glycopolypeptides in solution would be useful. The three
limiting conditions for the length of the glycopolypeptides can
be assumed as (a) purely α-helical, (b) a coiled structure with
the freely jointed chain, and (c) a coiled structure with an
A comparison of the binding constants, stoichiometry, and
thermodynamic parameters obtained from the ITC studies
yielded a number of interesting features. First, the stoichiom-
etry of binding is higher for the longer glycopolymers (5d and
5h), but it does not increase in proportion to the increase in
chain length, which can be attributed to steric factors. More
interestingly, the binding constant increases by 2−4-fold for the
longer glycopeptides. Because even the shorter glycopeptides
(5b and 5f) already have a large number of covalently attached
mannose residues in close proximity, this additional increase in
binding affinity may be attributed to the increased statistical
probability of the binding event due to the increase in the
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extended chain. The length of the glycopolypeptide (n = 60)
under these limiting cases can be estimated to be 8.6, 7.9, and
21.7 nm (Figure 8 SI). This indicates that polypeptides with
purely α-helical and the freely jointed conformation can have a
similar length. Because racemic glycopolypeptides 5f and 5h
have a slightly higher binding stoichiometry than their helical
counterparts, they have a conformation that is probably in
between a freely jointed chain and an extended chain (more
toward a freely jointed chain). However, more structural studies
are required to justify this.
binding and entropy of binding are exactly compensated in the
binding of the glycopolypeptides to Con A. Such close
compensation of enthalpy and entropy of binding has been
observed for a large number of protein−ligand interactions
including lectin−carbohydrate interactions and this phenom-
enon has been attributed to the reorganization of water
structure around the binding site on the protein and the ligand.
A number of studies suggest that water molecules play an
important role in lectin−carbohydrate interaction.50,51,58−60
Indeed, ITC studies provided evidence indicating the
involvement of water molecules in the binding of manno-
oligosaccharides to Con A.61 In view of this, the enthalpy−
entropy compensation observed here can be explained in terms
of changes in water structure during the Con A/glycopolypep-
tide association.
The polyvalent effect observed for all the polypeptides were
low and only 1 order of magnitude higher (per mannose) than
their corresponding monomer α-methyl mannopyranoside.
Polyvalency arises from mainly two related but distinct terms:
(i) multivalent binding (the ability of one glycopolypeptide to
bind to multiple lectin binding sites) and (ii) glycoside
clustering (a ligand concentration effect). While the former
shows mild binding enhancements (typically 1 order of
magnitude), the later shows very large enhancements (two
orders and higher). Because our synthetic glycopolypeptides
only show small binding enhancements, we believe that this
effect is due to glycoside clustering and not due to multivalent
binding. It must be noted that both the ITC and HA assay
shows similar affinity data and polyvalency for all the
glycopolypeptides used in this study (Table 2 SI, Figure 3
SI). A closer look at the ITC data gives us an idea why two
glycopolypeptides with differing secondary structures have
similar binding constants. It is observed that the ΔHb values are
higher for the nonhelical polymers (5f and 5h) as compared to
the helical polymers (5b and 5d) for the same (comparable)
length, whereas the ΔSb values are higher for the helical
polymers. This can be rationalized in the following way. The
nonhelical polymers are more flexible than their helical
counterparts and hence bind better to the lectin, which results
in a larger enthalpy change. On the other hand, the higher ΔSb
values for the helical polymers are consistent with the entropy
penalty being less for binding to a rigid structure. However, it
should be noted that the differences in the enthalpy of binding
and entropy of binding for the helical versus nonhelical
polymers are relatively small and also partially compensate each
other, resulting in nearly comparable values of binding
constant.
CONCLUSIONS
■
We have reported a very easy three step synthesis of per-
acetylated-O-glycosylated lysine-NCA using a stable glycosyl
donor and a commercially available protected amino acid. The
highlight of the synthesis is that the key glycosylation step and
the subsequent deprotection reaction proceeds to completion
in near quantitative yield. The glycosylated NCAs were then
polymerized using commercially available simple amine
initiators to yield well-defined high molecular weight
glycopolypeptides in very high yields. We have also reported
the synthesis of poly(β-lacto-O-lys) glycopolypeptides having a
disaccharide lactose as the pendant side group which
demonstrates that this methodology can be extended to
synthesize glycopolypeptides having complex carbohydrates
on its side chains. Poly(β-galacto-O-lys) glycopolypeptide was
also synthesized. Because certain cancer cells like HepG2 cells
have receptors that bind specifically to β-galactose, we believe
these polymers can be used for drug delivery. The fully water-
soluble glycopolypeptides were found to be α-helical in
aqueous solution. However, we were able to control the
secondary conformation of the glycopolypeptides by polymer-
izing racemic amino acid glyco NCAs. The poly(α-manno-O-
lys) polypeptides synthesized by us also bind specifically to the
lectin Con A, and the binding affinity was found to be nearly
the same between polypeptides having enantiomerically pure L-
lysine and the corresponding DL-lysine backbone.
A careful examination of the ΔHb and ΔSb values for the
different glycopolypeptides (both helical and nonhelical)
suggested that the changes in enthalpy and entropy are
compensatory in nature. This is clearly seen in a plot of −ΔHb
versus −TΔSb shown in Figure 3. A linear least-squares fit of
the data yielded a slope of 0.99 indicating that the enthalpy of
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and spectral data for all NCA
monomers, glycopolypeptides, and fluorescently-labeled poly-
peptides. FT-IR, NMR spectra of polypeptides, precipitation
assay, hemagglutination assay, and additional ITC isotherms.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: mjswamy1@gmail.com; s.hotha@iiserpune.ac.in; ss.
sengupta@ncl.res.in.
■
Notes
The authors declare no competing financial interest.
Figure 3. Enthalpy−entropy compensation plot for the Con A−
glycopolypeptide interaction (A) [30-α-manno-O-L-lys(OH) 5b], (B)
[Rac-30-α-manno-O-LD-lys(OH) 5f], (C) [50-α-manno-O-L-lys(OH)
5d], and (D) [Rac-50-α-manno-O-LD-lys(OH) 5h].
ACKNOWLEDGMENTS
The authors acknowledge Dr. P. Rajmohanan for NMR
support, Mrs. D. Dhoble for help with GPC, and Mr. P. R.
■
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dx.doi.org/10.1021/bm201813s | Biomacromolecules 2012, 13, 1287−1295

Biomacromolecules
Article
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