Synthesis of Altrose Poly-amido-saccharides with β-N-(1→2)- d -amide Linkages: A Right-Handed Helical Conformation Engineered in at the Monomer Level

Article abstract of DOI:10.1021/jacs.7b07405

The design and synthesis of amide-linked saccharide oligomers and polymers, which are predisposed to fold into specific ordered secondary structures, is of significant interest. Herein, right-handed helical poly amido-saccharides (PASs) with β-N-(1→2)-d-amide linkages are synthesized by the anionic ring-opening polymerization of an altrose β-lactam monomer (alt-lactam). The right-handed helical conformation is engineered into the polymers by preinstalling the β configuration of the lactam ring in the monomer via the stereospecific [2+2] cycloaddition of trichloroacetyl isocyanate with a d-glycal possessing a 3-benzyloxy group oriented to the α-face of the pyranose. The tert-butylacetyl chloride initiated polymerization of the alt-lactam proceeds smoothly to afford stereoregular polymers with narrow dispersities. Birch reduction of the benzylated polymers gives water-soluble altrose PASs (alt-PASs) in high yields without degradation of the polymer backbone. Circular dichroism analysis shows the alt-PASs adopt a right-handed helical conformation in aqueous solutions. This secondary conformation is stable over a wide range of different conditions, such as pH (2.0 to 12.0), temperature (5 to 75 °C), ionic salts (2.0 M LiCl, NaCl, and KCl), as well as in the presence of protein denaturants (4.0 M urea and guanidinium chloride). Cytotoxicity studies reveal that the alt-PASs are nontoxic to HEK, HeLa, and NIH3T3 cells. The results showcase the ability to direct solution conformation of polymers through monomer design. This approach is especially well-suited and straightforward for PASs as the helical conformations formed result from constraints imposed by the relatively rigid and sterically bulky repeating units.

Full text of DOI:10.1021/jacs.7b07405

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Article
Synthesis of Altrose Poly-amido-saccharides with #-
N-(1#2)-D-amide Linkages: A Right-handed Helical
Conformation Engineered in at the Monomer Level
Ruiqing Xiao, Eric L Dane, Jialiu Zeng, Christopher James McKnight, and Mark W. Grinstaff
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07405 • Publication Date (Web): 13 Sep 2017
Downloaded from http://pubs.acs.org on September 14, 2017
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Synthesis of Altrose Poly-amido-saccharides with β-N-(1→2)-D-
amide Linkages: A Right-handed Helical Conformation Engineered in
at the Monomer Level
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Ruiqing Xiao,† Eric L. Dane,† Jialiu Zeng,‡ Christopher J. McKnight,|| and Mark W. Grinstaff †,‡,§,
*
†
‡
Department of Chemistry, and Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215,
United States
§
||
Department of Physiology and Biophysics, and Department of Medicine, Boston University School of Medicine, Boston,
Massachusetts 02118, United States
KEYWORDS: poly-amido-saccharide, polysaccharide, altrose, helical, biocompatible, ring-opening polymerization.
ABSTRACT: The design and synthesis of amideꢀlinked saccharide oligomers and polymers, which are predisposed to fold into
specific ordered secondary structures, is of significant interest. Herein, rightꢀhanded helical polyꢀamidoꢀsaccharides (PASs) with βꢀ
Nꢀ(1→2)ꢀDꢀamide linkages are synthesized by the anionic ringꢀopening polymerization of an altrose βꢀlactam monomer (altꢀ
lactam). The rightꢀhanded helical conformation is engineered into the polymer by preꢀinstalling the β configuration of the lactam
ring in the monomer via the stereospecific [2+2] cycloaddition of trichloroacetyl isocyanate with a Dꢀglycal possessing a 3ꢀ
benzyloxyl group oriented to the αꢀface of the pyranose. The tꢀbutylacetyl chloride initiated polymerization of the altꢀlactam proꢀ
ceeds smoothly to afford stereoregular polymers with narrow dispersities. Birch reduction of the benzylated polymers gives waterꢀ
soluble altrose PASs (altꢀPASs) in high yields without degradation of the polymer backbone. Circular dichroism analysis shows the
altꢀPASs adopt a rightꢀhanded helical conformation in aqueous solutions. This secondary conformation is stable over a wide range
of different conditions, such as pH (2.0 to 12.0), temperature (5 to 75 °C), ionic salts (2.0 M LiCl, NaCl, and KCl), as well as in the
presence of protein denaturants (4.0 M urea and guanidinium chloride). Cytotoxicity studies reveal that the altꢀPASs are nontoxic to
HEK, HeLa, and NIH3T3 cells. The results showcase the ability to direct solution conformation of polymers through monomer
design. This approach is especially wellꢀsuited and straightforward for PASs as the helical conformations formed result from conꢀ
straints imposed by the relatively rigid and sterically bulky repeating units.
INTRODUCTION
methods have been applied to the synthesis of polysacchaꢀ
rides.15ꢀ18 Among these methods, the most successful one is the
cationic ringꢀopening polymerization of anhydrosugars develꢀ
oped by Schuerch et al. in the 1960s and 1970s.19,20 This
method enables the synthesis of stereoregular αꢀ1,6ꢀlinked
polysaccharides with high degrees of polymerization (DP
>100),21ꢀ24 but is less effective in preparing wellꢀdefined polyꢀ
saccharides with other linkages, such as 1,4ꢀlinked and 1,2ꢀ
linked polysaccharides.25ꢀ30 Enzymatic polycondensation of
sugar fluorides and sugar oxazolines represents a promising
method as it affords polysaccharides with absolute control
over regioꢀ and stereospecificity.31ꢀ33 Although this approach
avoids the use of protecting groups, it requires expensive actiꢀ
vated monomers and currently suffers from a narrow scope of
transferaseꢀmediated glycosylations.34
Polysaccharides are structurally and compositionally diverse
biopolymers that perform many essential roles in biology and
medicine.1,2 Despite their importance, the synthesis of polyꢀ
saccharides lags behind that of proteins and nucleic acids due
to their structural complexity.3,4 Compared to proteins and
nucleic acids which are linear polymers connected by a specifꢀ
ic type of achiral linkage, polysaccharides can be branched
and possess different regioꢀ and stereoꢀtypes of linkages,5,6
and, thus, are challenging synthetic targets.7ꢀ10 Specifically, the
challenges in synthesizing wellꢀdefined polysaccharides arise
from: 1) defining the regioselectivity due to existence of mulꢀ
tiple hydroxyl groups with similar reactivity;11 2) controlling
the stereochemical outcome of glycosidic bond forming reacꢀ
tions;12,13 and 3) low efficiencies of glycosylation reactions.14
Various polycondensation and ringꢀopening polymerization
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The above challenges, and limited synthetic approaches to
Figure 2. Synthesis of the altꢀlactam monomer
1
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4
conventional polysaccharides, have inspired chemists to reꢀ
place the natural Oꢀglycosidic bonds with achiral linkages that
can be constructed with higher efficiency,35ꢀ43 such as amide,44ꢀ
50
56,57
carbonate,51ꢀ55 and phosphodiester
linkages. Recently,
the Grinstaff group described a methodology to synthesize
stereoregular polyꢀamidoꢀsaccharides (PASs) via the anionic
ringꢀopening polymerization of benzylated sugar βꢀlactams
followed by Birch reduction.58ꢀ60 These PASs possess a novel
main chain composed of either glucopyranose (glcꢀ) or galacꢀ
topyranose (galꢀ) repeating units joined by αꢀNꢀ(1→2)ꢀDꢀ
amide linkages, and adopt leftꢀhanded helical conformations.61
Our current interest lies in expanding the monomer library
for PASs and controlling polymer conformation a priori
through preꢀinstalled structural signals in the monomer. The
later is especially important, as the functions of biopolymers,
such as molecular recognition and catalysis, are largely atꢀ
tributable to their ability to fold and selfꢀassemble into specific
and wellꢀdefined conformations.62,63 Advances in chemical
synthesis and methodology have enabled the construction of
various foldamers that can mimic the secondary structures of
proteins and nucleic acids.64ꢀ66 However, the design and synꢀ
thesis of polymers that are predisposed to fold into a specified
secondary conformation remains challenging and is an active
area of research.67ꢀ70 Both the glcꢀ and galꢀPASs adopt similar
leftꢀhanded helical conformations because the chain extension
proceeds specifically via ringꢀopening of the 4ꢀmembered βꢀ
lactam which are cisꢀfused to the αꢀface of the pyranose rings
(Figure 1A).58,59 This lactam ring configuration is determined
by the stereochemically controlled [2+2] cycloaddition reacꢀ
tion between the glycal and chlorosulfonyl isocyanate (CSI):
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Reagents and conditions: (a) Ac2O, pyridine, 48 h, (93%). (b)
HBr, AcOH, DCM, 0−25 °C, 3 h (76%). (c) Zn, NMI, EtOAc,
reflux, 4h (72%). (d) K2CO3, MeOH, 12 h (100%). (e) NaH,
BnBr, DMF, 0−25 °C, 24 h (91%). (f) TCAI, MeCN, BnNH2,
8 h (42%).
conformational analysis of the deprotected βꢀNꢀ(1→2)ꢀDꢀ
linked altꢀPASs by circular dichroism (CD); and 4) cytotoxiciꢀ
ty studies.
RESULTS AND DISCUSSION
The monomer, altꢀlactam (1), was synthesized using estabꢀ
lished carbohydrate chemistry (Figure 2). Dꢀallopyranose (2),
the Cꢀ2 epimer of Dꢀaltropyranose, was used as the starting
material due to its commercial availability. Compound 2 was
converted into 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀallopyranosyl broꢀ
mide (3) through Oꢀacetylation followed by treatment with
hydrobromic acid in the presence of acetic anhydride.72 Reꢀ
ductive elimination of 3 was performed with zinc and Nꢀ
methylimidazole (NMI) in refluxing ethyl acetate to afford
3,4,6ꢀtriꢀOꢀacetylꢀDꢀaltral (4) as a white solid.73 Compound 4
was deacetylated, and subsequently benzylated by reaction
with benzyl bromide in the presence of sodium hydride in
N,Nꢀdimethylformamide (DMF), as the benzyl protecting
group is more stable under the cycloaddition and anionic ringꢀ
opening polymerization reaction conditions. The [2+2] cyꢀ
cloaddition of 3,4,6ꢀtriꢀOꢀbenzylꢀDꢀaltral (5) was attempted
initially using CSI, but only ringꢀopened byproducts were
obtained.71 However, the reaction of 5 with trichloroacetyl
isocyanate (TCAI) proceeded smoothly in acetonitrile at room
temperature, providing the altꢀlactam as a colorless viscous
liquid, after in situ removal of the Nꢀtrichloroacetyl group
using benzylamine.74,75 The structure of monomer 1 was charꢀ
acterized and confirmed by 1H NMR, 13C NMR, COSY,
HSQC, and HMBC combined with FTꢀIR spectroscopy, and
electrospray ionization mass spectrometry (ESIꢀMS) analyses
(SI).
Figure 1. Synthetic scheme for leftꢀ (A) and rightꢀhanded (B)
PASs
the isocyanate approaches the glycal molecule stereospecifiꢀ
cally anti with respect to the 3ꢀbenzyloxyl group which is oriꢀ
ented to the βꢀface of the pyranose rings.71 Based on this raꢀ
tionale, the stereospecific [2+2] cycloaddition between CSI
and a glycal with the 3ꢀbenzyloxyl group oriented to the αꢀ
face of the pyranose ring would yield a monomer with the βꢀ
lactam ring cisꢀfused to the βꢀface of the pyranose rings, and
we hypothesize that the anionic ringꢀopening polymerization
of such a monomer would afford a βꢀNꢀ(1→2)ꢀDꢀamide
linked PAS adopting a rightꢀhanded helical conformation
(Figure 1B). Herein, we describe such a rightꢀhanded PAS and
specifically report the: 1) stereospecific synthesis of an altrose
βꢀlactam monomer (altꢀlactam) and evaluation of the lactam
ringꢀopening reactivity; 2) anionic ringꢀopening polymerizaꢀ
tion of the altꢀlactam to give polymers with DP of 20ꢀ50; 3)
It has been reported that the polymerizability of bicyclic
sugar monomers is significantly affected by the configuration
patterns of the hydroxyl groups and steric interactions released
upon ringꢀopening of the anhydro ring.76,77 For example, 1,6ꢀ
anhydroꢀ2,3,4ꢀtriꢀOꢀbenzylꢀβꢀDꢀglucopyranose,78
ꢀ
mannopyranose,79 ꢀgalactopyranose,80 and ꢀallopyranose81
showed high reactivity for homopolymerization and copolyꢀ
merization. In contrast, 1,6ꢀanhydroꢀ2,3,4ꢀtriꢀOꢀbenzylꢀβꢀDꢀ
altropyranose exhibited negligible homopolymerizability, and
could only be copolymerized with 1,6ꢀanhydroꢀ2,3,4ꢀtriꢀOꢀ
benzylꢀβꢀDꢀglucopyranose.82 In order to evaluate the ringꢀ
opening ability of sugar βꢀlactams, the reactivity of the Nꢀtꢀ
butylacetyl derivative of altꢀlactam (6) and glcꢀlactam (7) was
determined, as the propagating chain ends are also an Nꢀ
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ization of monomer 1. For example, a less reactive related
1
2
Figure 4. Polymerization of the altꢀlactam and deprotection
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Reagents and conditions: (a) (CH3)3CH2COCl, LiHMDS, 3ꢀ6
h, 0°C. (b) Na, NH3 (l), −52 °C, 1 h.
monomer 8ꢀoxaꢀ6ꢀazabicyclo[3.2.1]octanꢀ7ꢀone, which has a
fiveꢀmembered lactam and a rate of aminolysis (k=0.83x10ꢀ4
L•molꢀ1•sꢀ1) less than that of 6,83 readily polymerized using its
Nꢀbenzoyl derivative as the initiator and potassium pyrroliꢀ
donate as the catalyst.85,86
The ringꢀopening polymerization of monomer 1 was first
performed with a monomerꢀtoꢀinitiator feed ratio ([M]/[I]) of
40:1 in anhydrous THF at
0
°C, using lithium
bis(trimethylsilyl)amide (LiHMDS) as the catalyst and tꢀ
butylacetyl chloride as the initiator (Figure 4).87 This initiator
was selected because the tꢀbutylacetyl group was stable under
both the polymerization and deprotection reaction conditions,
Figure 3. (A) Aminolysis reactions of Nꢀacyllactams 6 and 7
with nꢀpropylamine. (B) Kinetic analyses of the aminolysis
reactions in deuterated chloroform at 25 °C: a: concentration
of nꢀpropylamine: 0.30 mol/L; b: concentration of Nꢀ
acyllactams: 0.15 mol/L.
1
and would enable straightforward endꢀgroup analysis by H
NMR spectroscopy. After initiation of polymerization, aliꢀ
quots were taken from the reaction at predetermined time
points in order to monitor the monomer conversions and the
growth of the polymer chains as a function of time. The monꢀ
acyllactam type and the propagation proceeds via ringꢀopening
of this Nꢀacyllactam.83 The aminolysis reactions of 6 and 7
with nꢀpropylamine in deuterated chloroform at 25 °C were
monitored in real time by 1H NMR spectroscopy by comparing
the anomeric proton resonances of the reactants and correꢀ
sponding products. The apparent rate constants were calculatꢀ
ed by using the equation for secondꢀorder reactions. As shown
in Figure 3B, the value of k for the aminolysis reaction of 6
(k=1.09x10ꢀ4 L•molꢀ1•sꢀ1) is about one fifth of that for the ringꢀ
opening reaction of 7 (k=5.42x10ꢀ4 L•molꢀ1•sꢀ1), indicating its
relative lower ringꢀopening reactivity. This may be due to a
smaller ring strain for the altꢀlactam monomer compared to the
glcꢀlactam monomer, as observed for 1,6ꢀanhydroꢀ2,3,4ꢀtriꢀOꢀ
benzylꢀβꢀDꢀaltropyranose.84 Steric hin drance may also play
an important role, as the 5ꢀbenzyloxylmethyl group is oriented
on the same face with the fourꢀmembered βꢀlactam ring. Howꢀ
ever, the observed decrease in aminolysis rate for 6 in comparꢀ
ison to 7 does not necessarily preclude the successful polymerꢀ
1
omer conversions were determined using H NMR spectra by
comparing the integration of the anomeric protons from the
monomer (5.28 ppm) and the polymer (5.81 ppm). The numꢀ
berꢀaverage molecular weights (Mn) were calculated by comꢀ
parison of the integration of the tꢀbutyl protons (0.95 ppm)
originating from the initiator with the anomeric proton of the
polymer chain. As shown in Figure S1, monomers were not
completely consumed after four hours of reaction (81.5%).
This is in sharp contrast to the polymerization of the glcꢀ
lactam, in which monomer conversion reached to 100% within
five minutes with a [M]/[I] of 50:1.58 The Mn of the polymer
increased linearly with monomer conversion, and finally
reached to 17,230 g/mol, which corresponded to a DP of 37.2
(Figure S2).
In order to obtain altꢀPASs of different chain lengths, the
polymerization of 1 was conducted by varying the [M]/[I]
from 20:1 to 60:1 (Table 1). The reaction times were extended
Table 1. Polymerization of Monomer 1 at Different Monomer-to-Initiator ([M]/[I]) Ratios
b
c
Entry
[M]/[I]a
Time (h)
Mn(theo)
Mn(GPC)
Đd
DP(NMR)
Conv. (%)e
P1'
P2'
P3'
P4'
20:1
30:1
40:1
60:1
3
4
5
6
8460
8290
12950
17020
23640
1.12
1.15
1.16
1.27
21.5
30.6
38.7
53.6
91.0
88.9
86.8
79.6
12360
16060
22050
a All reactions were conducted in THF at 0°C, using tꢀbutylacetyl chloride as the initiator and LiHMDS as the catalyst. b Calculated
e
c
d
based on [M]/[I] ratios and monomer conversion, g/mol. Determined by THF GPC against polystyrene standards, g/mol. Mw/Mn.
Calculated from 1H NMR spectra.
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for high [M]/[I] ratios in order to achieve greater monomer
conversions. The resultant polymers (P1'ꢀP4') were purified by
repeated precipitation from dichloromethane into hexane and
then into methanol. In the 13C NMR spectra of the polymers,
all the carbon signals corresponding to sugar repeating units
appeared as single peaks (Figure S4), indicating that the polyꢀ
mers are stereoregular and epimerization did not occur at eiꢀ
ther the C1 or C2 position of the pyranose ring. The Mn values
the benzyl groups was confirmed by the disappearance of the
aromatic proton resonance at 7.05ꢀ7.37 ppm in H NMR specꢀ
1
1
2
3
4
5
6
7
8
tra, and by the disappearance the aromatic CꢀH stretch at
1
3000ꢀ3100 cmꢀ1 in IR spectra (Figure S7). The H NMR specꢀ
tra of these altꢀPASs show broad peaks (Figure S8), which is
in contrast to the wellꢀresolved signals and couplings of glcꢀ
PASs, possibly due to existence of different altropyranose ring
conformations.88ꢀ90 Increasing the temperature from 25 °C to
1
1
were determined by H NMR end group analysis and by gel
90 °C leads to the sharpening of the H NMR signals to some
extent (Figure S21). Based on GPC analysis, P1ꢀP4 have Mn
values ranging from 3730 to 8410 g/mol with narrow dispersiꢀ
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ties (Đ=1.21ꢀ1.36). The DP measured by H NMR and GPC
are in good agreement with the corresponding values for the
benzylated polymers (Table 2), indicating that the deprotection
process proceeded efficiently without noticeable degradation
of the polymer backbone. The MALDIꢀTOF spectrum of P1
showed a molecular weight range in good agreement with the
theoretical mass of the potassium adduct as well as the exꢀ
pected spacings of 189 amu between the species of different
DP. The repeated attempts to analyze the larger polymers (P2,
P3, and P4) were unsuccessful likely due to low ionization
efficiency of high molecular weight polysaccharides.91,92 Speꢀ
ͦͩ
cific optical rotations ([α] ) measured in water range from
͸
+6.7° for P1 to +10.6° for P4, consistent with the β configuraꢀ
tion of glycosidic linkages.93,94 The altꢀPASs are highly soluꢀ
ble in water. All the polymers were soluble at 50.0 mg/mL,
and the solutions were stable and remained clear through 7
days of observation (higher concentrations were not investiꢀ
gated). For comparison, the water solubility for the glcꢀPAS
with a DP of 50 was 2.0 mg/mL, and decreased over time due
to aggregation of the polymer chains.58
Figure 5. Linear dependence of Mn (filled circles) of polymer
P1'ꢀP4' on monomerꢀtoꢀinitiator ratio ([M]/[I]) and polydisꢀ
persities (open circles). The inset shows GPC elution curves
for P1'ꢀP4' (normalized LS intensity versus elution time).
permeation chromatography (GPC). GPC analysis showed that
all the polymers displayed a narrow monomodal molecular
weight distribution (Đ=1.12ꢀ1.27), and the Mn values inꢀ
creased linearly with stoichiometry (Figure 5). The Mn obꢀ
tained by 1H NMR and GPC were fairly close to the expected
values based on the [M]/[I] ratios, indicating the formation of
wellꢀdefined polymers. However, GPC monitoring of the
polymerization reaction with [M]/[I]=60:1 showed a slight
broadening of molecular weight distributions with extending
the reaction time (Figure S5), possibly due to undesired chainꢀ
transfer or chain termination reactions. The IR spectra of P1'ꢀ
P4' show strong amide I band at 1667 cmꢀ1 and amide II band
at 1495 cmꢀ1, which is consistent with the polyꢀβꢀpeptide naꢀ
ture of the polymer backbone (Figure S6). Polymers P1'ꢀP4'
are soluble in THF, chloroform, dichloromethane, DMF, and
dimethyl sulfoxide (DMSO).
To investigate the solution conformation of the altꢀPASs,
circular dichroism (CD) spectra of P1ꢀP4 were recorded in
water at room temperature. As shown in Figure 6, the CD
spectra of P1ꢀP4 display a minimum at 184 nm (mean residue
ellipticity (MRE)= ꢀ17,000 deg•cm2•dmolꢀ1) and a maximum
at 223 nm (MRE= 7,500 deg•cm2•dmolꢀ1), similar to the CD
spectra of the glcꢀPASs but with opposite sign, indicating that
the altꢀPASs adopt a rightꢀhanded helical conformation in
water.61 Interestingly, the CD spectra also display a small posiꢀ
tive absorption at 203 nm, which is attributable to the parallel
πꢀπ* exciton band.95 The CD spectrum of glcꢀPASs does not
show such a parallel πꢀπ* band, and the minimum is slightly
blueꢀshifted to 220 nm, probably due to the overlapping of the
nꢀπ* transition with the parallel πꢀπ* transition (Figure 6, Inꢀ
set).95 It was reported that selfꢀassociation of helices could also
lead to a characteristic band at 204 nm.96ꢀ98 However, this posꢀ
sibility was ruled out by the observation that there was no
change in the intensity of the CD spectra upon a 16ꢀfold diluꢀ
tion of P3 solution (0.80ꢀ0.050 M, Figure S9). Addition of
methanol (up to 75% by volume), which is known as a strong
The debenzylation of polymers P1'ꢀP4' was performed using
sodium metal in liquid ammonia at ꢀ52 °C followed by extenꢀ
sive dialysis against water. Lyophilization of the resultant
solutions afforded deprotected altꢀPASs (P1ꢀP4) as fluffy
white solids, which were characterized by NMR, IR, and
aqueous GPC against dextran standards. Complete removal of
Table. 2. Characterization of Debenzylated alt-PASs
a
b
d
̋̎
̊
Entry
Mn(theo)
Mn(GPC)
DP(GPC)
Đc
DP(NMR)
Yield (%)
[α] (°)
P1
P2
P3
P4
3460
5390
7180
9780
3730
5470
6560
8410
19.2
28.4
34.2
44.0
1.22
1.21
1.25
1.36
24.2
33.1
42.0
52.7
+6.7
+9.5
74.0
77.3
86.7
94.8
+8.6
+10.6
a Calculated based on the molecular weights of benzylated polymers, g/mol. b Determined by aqueous GPC against dextran standards,
g/mol. c Mw/Mn. d Calculated from 1H NMR spectra.
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not protected by intramolecular hydrogen bonds. This highly
structureꢀpromoting solvent for both αꢀ and βꢀpeptides,99ꢀ103
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Figure 8. Cytotoxicity study for polymer P3 against HEK,
HeLa, and NIH3T3 cell lines at different concentrations. All
experiments were done at n = 3, and error bars represent
standard deviation of the mean values.
Figure 6. CD spectra of the deprotected altꢀPASs in water.
The inset shows the CD spectrum of glcꢀPAS with a DP of 50.
did not increase the CD intensity of the 223 nm maximum
(Figure S10), suggesting that these polymers are fully folded
in water.104,105 The absolute MRE values of P1 at 184 nm and
223 nm are slightly smaller than those values for P2ꢀP4, posꢀ
sibly due to a larger contribution of lessꢀfixed terminal resiꢀ
dues.106
stabilized helical structure observed in the altꢀPASs is likely
due to the backbone constraints provided by the conformationꢀ
ally restricted pyranose rings.61,109ꢀ112
As a first step towards future use of these polymers in mediꢀ
cine, we evaluated the cytotoxicity of altꢀPASs against human
embryonic kidney (HEK293), human cervix adenocarcinoma
(HeLa), and mouse embryonic fibroblast (NIH3T3) cells in
vitro. The rationale for selecting these three cell lines was to
assess toxicity against: 1) cells originating from two different
species (human and mouse); 2) a human healthy, normal cell
line versus a cell line derived from a cancer cells (HEK293 vs.
HeLa); and 3) a cell line used in standard preꢀclinical FDA
biocompatibilty/safety testing (NIH3T3). The cells were incuꢀ
bated for 24 hours with various concentrations of P1ꢀP4. The
relative viabilities of polymerꢀtreated cells were measured by a
MTS cell proliferation assay and normalized to the values of
mediumꢀtreated control cells. As shown in Figure 8 and Figure
S16, the altꢀPASs are nonꢀcytotoxic to these cell lines at conꢀ
centrations up to 1000 ꢀg•mLꢀ1. The cell viabilities for the altꢀ
PASs are comparable to those for the glcꢀPASs (Figure S17),
indicating that the chain conformation of PASs does not have
an impact on cytotoxicity.
In conclusion, a strategy to synthesize polymers with a deꢀ
fined helical conformation (rightꢀ vs leftꢀhanded) in aqueous
solution is described by installing the structural cues in the
monomer for subsequent polymerization. Using this strategy,
the first synthesis of polyꢀamidoꢀsaccharides with βꢀNꢀ(1→2)ꢀ
Dꢀamide linkages is reported. Specifically, an altrose βꢀlactam
monomer is prepared efficiently by the stereospecific [2+2]
cycloaddition of trichloroacetyl isocynate with 3,4,6ꢀtriꢀOꢀ
benzylꢀDꢀaltral, which has a 3ꢀbenzyloxyl group oriented to
the αꢀface of the pyranose ring, giving a monomer with the βꢀ
lactam cisꢀfused to the βꢀface of the pyranose rings. Although
exhibiting lower reactivity compared to the glcꢀlactam monoꢀ
mer, the anionic ringꢀopening polymerization of this altꢀlactam
proceeds steoreospecifically to give wellꢀdefined polymers
with predictable molecular weights and narrow dispersities.
Deprotection of the benzylated polymers affords waterꢀsoluble
altꢀPASs which adopt a rightꢀhanded helical conformation in
aqueous solutions. This conformation is stable over a wide
range of different conditions, such as pH, temperature, ionic
Molecular dynamics simulations revealed that the amide
groups of the glcꢀPASs are oriented orthogonal to the helix
axis and interact predominantly with solvent water
molecules.61 In order to gain further insight on the conformaꢀ
tional stability of the altꢀPASs, the CD spectra of P3 in various
solutions were recorded (Figure S11ꢀ14). As shown in Figure
7, the CD spectra remained unchanged over a broad pH range
from 2.0 to 12.0 or in presence of high ionic strength (2.0 M
LiCl, NaCl and KCl).107 Introduction of 4.0 M urea or guaniꢀ
dinium chloride, two common protein denaturants, had no
effect on the CD intensity of P3.105 Heating from 5 to 75 °C
caused only a modest diminution of the intensity of the characꢀ
teristic maximum at 223 nm.108 These results indicate that the
secondary structure of the altꢀPASs is robust and interꢀresidue
hydrogen bond between peptide bonds is not the major conꢀ
tributor to conformational stability. This observation is conꢀ
sistent with the rapid amide proton exchange rate along the
PAS backbone (Figure S15). NH/ND exchange for P2 was
complete within 6 min, indicating that the amide protons are
Figure 7. CD spectra of polymer P3 under various condiꢀ
tions.
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salts, as well as in the presence of protein denaturants. Cytoꢀ
toxicity studies reveal that the altꢀPASs are nonꢀcytotoxic to
HEK, HeLa, and NIH3T3 cells.
(9) Wu, J.; Guo, Z. W. J Org Chem 2006, 71, 7067.
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1966, 21, 431.
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From a chemical design perspective, PASs bridge the comꢀ
positional space between polypeptides and polysaccharides,
and exhibit key characteristics of both classes of biopolymers.
Given the composition, chemical features of both peptides and
polysaccharides, and control over the solution conformation,
PASs are of interest for biomedical applications and we are
identifying such uses. One example would be for the encapsuꢀ
lation of cells and subsequent in vivo implantation for treatꢀ
ment of diabetes – polysaccharide have been extensively inꢀ
vestigated for this application with varying success.113 Finally,
studies are ongoing to further expand the repertoire of sugar βꢀ
lactam monomers that are amenable to this anionic ringꢀ
opening polymerization and to prepare new polymers that are
predisposed to fold into specific ordered secondary structures.
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures and data for all the compounds,
polymerization data, NMR, IR, and CD spectra of the polyꢀ
mers, as well as cell viability data. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*mgrin@bu.edu
ORCID IDs
Ruiqing Xiao: 0000ꢀ0001ꢀ7944ꢀ0508
Eric L. Dane: 0000ꢀ0002ꢀ4022ꢀ7264
Jialiu Zeng: 0000ꢀ0001ꢀ7802ꢀ1432
Christopher J. McKnight: 0000ꢀ0002ꢀ3237ꢀ0896
Mark W. Grinstaff: 0000ꢀ0002ꢀ5453ꢀ3668
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to thank Boston University for support
and Dr. Stacy L. Chin and Mr. William Blessing for the inꢀ
sightful discussions about circular dichroism.
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