Water-soluble aminocurdlan derivatives by chemoselective azide reduction using NaBH4

Article abstract of DOI:10.1016/j.carbpol.2014.12.020

Water-solubility can often enhance the utility of polysaccharide derivatives, for example in pharmaceutical and biomedical applications. Synthesis of water-soluble aminopolysaccharides, particularly those bearing other sensitive functional groups, can be a challenging endeavor. Curdlan is a bioactive β-1,3-glucan with considerable promise for biomedical applications. Aminocurdlans are intriguing target molecules for study of, for example, their interactions with the proteins that form tight junctions between enterocytes. Herein we report the preparation of two water-soluble 6-aminocurdlans starting from 6-bromo-6-deoxycurdlan. The 6-bromide was first displaced by nucleophilic substitution with sodium azide in dimethyl sulfoxide. The O-2 groups were acylated with hydrophilic oligo (ethylene oxide) esters, so as to enhance aqueous solubility. The resultant 6-azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan was then treated with excess sodium borohydride to reduce the azide; unexpectedly, the water-soluble product proved to be the amide, 6-trioxadecanamido-6-deoxycurdlan. Regioselectivity and degree of substitution (DS) of those derivatives were characterized by means of ¹H NMR, ¹³C NMR and FTIR-spectroscopy, elemental analysis, and titration. Alternatively, direct borohydride reduction of the parent 6-azido-6-deoxycurdlan afforded 6-amino-6-deoxycurdlan that was also water-soluble.

Full text of DOI:10.1016/j.carbpol.2014.12.020

Accepted Manuscript
Title: Water-soluble aminocurdlan derivatives by
chemoselective azide reduction using NaBH4
Author: Ruoran Zhang Kevin J. Edgar
PII:
S0144-8617(14)01203-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2014.12.020
CARP 9510
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Revised date:
Accepted date:
28-10-2014
15-12-2014
20-12-2014
Please cite this article as: Zhang, R., and Edgar, K. J.,Water-soluble aminocurdlan
derivatives by chemoselective azide reduction using NaBH4, Carbohydrate Polymers
(2014), http://dx.doi.org/10.1016/j.carbpol.2014.12.020
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1
2
Water-soluble aminocurdlan derivatives by chemoselective
azide reduction using NaBH4
a
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4
5
6
7
8
9
Ruoran Zhang , Kevin J. Edgara,b*
aMacromolecules and Interfaces Institute
bDepartment of Sustainable Biomaterials, College of Natural Resources and Environment
Virginia Tech, Blacksburg, VA 24061, United States
10 Abstract
11 Water-solubility can often enhance the utility of polysaccharide derivatives, for example in
12 pharmaceutical
and
biomedical
applications.
Synthesis
of
water-soluble
13 aminopolysaccharides, particularly those bearing other sensitive functional groups, can be a
14 challenging endeavor. Curdlan is a bioactive -1,3-glucan with considerable promise for
15 biomedical applications. Aminocurdlans are intriguing target molecules for study of, for
16 example, their interactions with the proteins that form tight junctions between enterocytes.
17 Herein we report the preparation of two water-soluble 6-aminocurdlans starting from 6-
18 bromo-6-deoxycurdlan. The 6-bromide was first displaced by nucleophilic substitution with
19 sodium azide in dimethyl sulfoxide. The O-2, 4 groups were acylated with hydrophilic
20 oligo(ethylene oxide) esters, so as to enhance aqueous solubility. The resultant 6-azido-6-
21 deoxy-2,4-di-O-trioxadecanoylcurdlan was then treated with excess sodium borohydride to
22 reduce the azide; unexpectedly, the water-soluble product proved to be the amide, 6-
23 trioxadecanamido-6-deoxycurdlan. Regioselectivity and degree of substitution (DS) of those
1
13
24 derivatives were characterized by means of H-, C- NMR and FTIR- spectroscopy,
25 elemental analysis, and titration. Alternatively, direct borohydride reduction of the parent 6-
26 azido-6-deoxycurdlan afforded 6-amino-6-deoxycurdlan that was also water-soluble.
27
28
29 Keywords: aminocurdlan; amidocurdlan; regioselectivity; sodium borohydride; water-soluble
30
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31
32 Highlights
33
34
35
o Highly regioselective bromination and azide displacement of curdlan at C-6
o Chemoselective reduction of azidocurdlan to amine by sodium borohydride
o Two water-soluble 6-aminocurdlans starting from 6-bromo-6-deoxycurdlan
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37
o Pathway to regiochemically defined 6-amino- and 6-amido-6-deoxycurdlan
derivatives
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63 1. Introduction
64 Natural polysaccharides are always hydrophilic; they contain many hydroxyl groups and in
65 some cases carboxylate, sulfate, amide and amino groups as well, providing a plethora of
66 hydrogen bond donor and acceptor groups for interaction with water molecules. However,
67 self-association through hydrogen bonding, and in some cases crystallization, prevents many
68 polysaccharides from dissolving in water; important examples of these phenomena include
69 cellulose, chitin, and curdlan. Considerable attention has been devoted to the study of water-
70 soluble derivatized polysaccharides in pharmaceutical and biomedical applications, such as in
71 drug delivery (Hassani, Hendra & Bouchemal, 2012; Henni-Silhadi et al., 2007; Liesiene &
72 Matulioniene, 2004), and for use as antimicrobial agents (Lee, Yeomans, Allen, Gross &
73 Kaplan, 1997; Roemhild, Wiegand, Hipler & Heinze, 2013). The natural polysaccharide
74 chitin and its conversion to partially N-deacetylated, cationic, more soluble chitosan
75 derivatives are exemplary; chitosan having been shown to possess for example the ability to
76 encapsulate anionic drugs, nucleic acids and other biological compounds (Ravi Kumar, 2000).
77 Chitosan can induce reversible opening of tight junctions between enterocytes that line the
78 gastrointestinal (GI) tract by interacting with the proteins that help form these junctions,
79 thereby increasing intestinal permeability and allowing paracellular absorption of otherwise
80 poorly absorbed drugs, such as large and hydrophilic proteins (Yeh et al., 2011). The
81 protonated amine groups of chitosan drive the necessary interactions with the tight junction
82 proteins; permanently charged N-peralkylated derivatives are even more effective (Sadeghi et
83 al., 2008). However, chitosan has its limitations for such uses. Its precursor, chitin, is isolated
84 from matrices that contain large amounts of shellfish proteins; quantitative separation is
85 essential since these proteins provoke severe immune responses in some individuals (Hajeb
86 & Selamat, 2012). Ranaldi et al. (Ranaldi, Marigliano, Vespignani, Perozzi & Sambuy, 2002)
87 have found that chitosan has an irreversible effect on tight junctions at the highest
88 concentration tested (0.01%), which could lead to degradation of the intestinal mucosal
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89 barrier function, permitting potentially toxic or allergenic molecules to enter the circulatory
90 system.
91
92 Recently researchers have explored amination of other polysaccharides including cellulose
93 (Fox & Edgar, 2012), curdlan (Zhang & Edgar, 2014b), amylose (Cimecioglu, Ball, Kaplan &
94 Huang, 1994), and pullulan (Pereira & Edgar, 2014) in an attempt to understand structure-
95 property relationships, and ultimately to synthesize cationic chitosan analogs that would
96 provide controlled interactions with proteins, for example short-term, reversible GI tight
97 junction opening. Many of these approaches employ highly regioselective C-6 bromination
98 of polysaccharides by triphenylphosphine in combination with a brominating agent (e.g. N-
99 bromosuccinimide (NBS)) (Furuhata, Koganei, Chang, Aoki & Sakamoto, 1992a). This
100 chemistry involves a double SN2 mechanism (Fox & Edgar, 2011) that provides essentially
101 perfect C-6 selectivity for polysaccharides (Furuhata, Chang, Aoki & Sakamoto, 1992;
102 Furuhata, Koganei, Chang, Aoki & Sakamoto, 1992b) that possess a C-6 primary alcohol;
103 this is perhaps the most regioselective reaction known in polysaccharide chemistry (Fox, Li,
104 Xu & Edgar, 2011). The 6-halo-6-deoxy polysaccharides are converted to azides by
105 nucleophilic displacement of bromide using a one-pot (CBr4/ LiN3/ Ph3P/ DMF) or two-step
106 (NBS/ Ph3P in DMAc/ LiBr; NaN3/ DMSO) approach. These approaches used the
107 remarkably chemoselective and versatile Staudinger reaction (Boger, Corcoran & Lehn, 1978;
108 Cimecioglu, Ball, Huang & Kaplan, 1997; Cimecioglu, Ball, Kaplan & Huang, 1994; Fox &
109 Edgar, 2012) to reduce azido precursors to the desired amines, even in the presence of ester
110 groups appended to the other polysaccharide OH groups. However, these Staudinger
111 products had solubility limitations; the isolated aminopolysaccharides were found to be
112 insoluble in water or any common organic solvent. Alternatively, in a case where there were
113 no other sensitive functional groups present, Matsui et al. (Matsui, Ishikawa, Kamitakahara,
114 Takano & Nakatsubo, 2005) applied sodium borohydride to reduce the azide group,
115 producing water-soluble 6-amino-6-deoxycellulose. While it is known that ester group
116 reduction by sodium borohydride is relatively slow in comparison with that of more easily
117 reduced groups like aldehydes and ketones, it is also known that sodium borohydride will
118 effectively reduce ester groups to primary alcohols under more forcing conditions, such as in
119 the presence of excess reagent (Brown & Rapoport, 1963). Rapoport observed (Brown &
120 Rapoport, 1963) “it is evident that esters are not resistant to reduction by sodium
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121 borohydride, although the rate of reduction is much slower than for aldehydes or ketones.”
122 Since reactions of polysaccharides are very frequently slower than those of analogous small
123 molecules, and do require more forcing conditions, one could not be confident about the
124 possibility of chemoselective reduction of azides in the presence of ester groups. Indeed, we
125 have found no such examples of chemoselective borohydride reductions of azides in the
126 presence of esters in the polysaccharide literature. Baumann et al. (Baumann, Liu & Faust,
127 2003; Liu & Baumann, 2002) described a clever approach to completely regioselectively
128 substituted 6-amino-6-deoxycellulose with DSC-6 1.0 via tosylcellulose (DStosyl = 2.02, DS6-tosyl
129 = 1.0). Nucleophilic displacement of tosyl groups by sodium azide at 50C occurred only at
130 C-6, while the tosyl groups at C-2/ 3 resisted displacement presumably due to the restricted
131 approach angles available to these secondary tosylates. Subsequent complete reduction of
132 azido groups to amino by use of lithium aluminium hydride (LiAlH4) was accompanied by
133 simultaneous reductive cleavage of the 2,3-O-tosyl groups, affording free cellulose hydroxyl
134 groups at C-2, 3.
135
136 Curdlan (-D-1,3-glucan) is a linear homopolysaccharide extracellularly produced by
137 Alcaligenes faecalis var. myxogenes (Harada & Harada, 1996; Zhang & Edgar, 2014a). Its
138 propensity to undergo thermal gelation has proven valuable in foods (Miwa, Nakao & Nara,
139 1993), including as a thickener, fat substitute, food additive, and other functions (Jin, Zhang,
140 Yin & Nishinari, 2006; Na, Park, Kim & Bae, 2000; Popescu, Pelin, Butnaru, Fundueanu &
141 Suflet, 2013). However, there are few reports in the literature describing how to prepare
142 water-soluble aminocurdlans for biomedical applications, regio- and/ or chemoselectively, or
143 otherwise. Our own previous work indicated that synthesis of 6-amino-6-deoxycurdlan and
144 its 2,4-O-diesters using the bromination/ azidation/ Staudinger reduction approach was
145 successful, but afforded derivatives with essentially no water solubility (Zhang & Edgar,
146 2014b).
147
148 The Heinze (Dorn, Pfeifer, Schlufter & Heinze, 2010; Heinze & Schaller, 2000), Zhang
149 (Zhou, Zhang, Okamura, Minoda & Miyamoto, 2001), and Edgar (Liu & Edgar, 2014)
150 groups have investigated the syntheses and water-solubility of cellulose trioxadecanoates
151 (TOD) with a range of DS values by different esterification approaches including reaction of
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152 cellulose with 3,6,9-trioxadecanoyl chloride (TODCl), 3,6,9-trioxadecanoic acid
153 (TODA)/ 1,1’-carbonyldiimidazole (CDI), TODA/ p-toluenesulfonyl chloride (TosCl) and
154 TODA/ oxalyl chloride/ DMF. We describe herein our attempts to prepare 6-amino-6-
155 deoxycurdlan derivatives, rendered water-soluble by incorporation of hydrophilic ester
156 (TOD) substituents, by using sodium borohydride to reduce their azido precursors, and
157 exploring aspects of regioselectivity and chemoselectivity of the preparative reactions
158 (Scheme 1). We also describe unexpected benefits from borohydride as opposed to
159 Staudinger reduction of the 6-azido-6-deoxy polysaccharides.
160
N3
Br
OH
NaN3
Ph3P/ NBS
O
O
O
HO
HO
HO
DMSO
O
DMAc/ LiBr
O
O
n
n
n
OH
H 3C
OH
OH
O
O
2
O
Cl
NaBH4
DMSO
DMAc, Pyr
N3
NH2
O
H 3C
O
O
O
O
O
HO
O
2
O
n
n
O
O
OH
O
NaBH4
DMSO
2
NHR
O
O
RO
H 3C
O
n
OR
O
R= H or
O
CH 3
2
O
161
162 Scheme 1. Synthetic scheme for 6-amino-6-deoxycurdlans.
163
164 2. Experimental
165 2.1 Materials
166 Curdlan (-(13)-glucan, (-C6H10O5-)n, DP~6173) was obtained from Wako Chemicals and
167 dried under vacuum at 40C overnight prior to use. Lithium bromide (LiBr, laboratory grade,
168 Fisher) and sodium azide (NaN3, 99%, Alfa Aesar) were dried under vacuum at 125C. N-
169 Bromosuccinimide (NBS, 99%, Acros) was recrystallized from boiling water and dried for
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170 two days under reduced pressure over anhydrous calcium chloride. N,N-Dimethylacetamide
171 (DMAc, reagent grade, Fisher) and dimethyl sulfoxide (DMSO, HPLC grade, Fisher) were
172 stored over 4 Å molecular sieves. Hydrochloric acid (HCl, 37%, ACS reagent, Fisher) was
173 used to make 1M HCl solution. 4-Dimethylaminopyridine (DMAP, Acros),
174 triphenylphosphine (Ph3P, 99%, Acros), pyridine (anhydrous, 99%, AcroSeal®), sodium
175 borohydride (NaBH4, 98+%, powder, Acros), 2-[2-(2-methoxyethoxy)ethoxy]acetic acid
176 (3,6,9-trioxadecanoic acid, TODA, technical grade, Sigma-Aldrich), acetic anhydride (Ac2O,
177 99+%, Sigma-Aldrich), sodium bicarbonate (reagent grade, Fisher) and regenerated cellulose
178 dialysis tubing (MW 3500, Fisher) were used as received.
179
180 2.2 Measurements
181 2.2.1 NMR and IR Spectroscopy
13
182 1H and C NMR spectra were obtained on a Bruker Avance II 500 MHz spectrometer in
183 CDCl3, DMSO-d6 or D2O at room temperature or 50 C, employing 32 and 15,000 scans,
184 respectively. Infrared spectroscopic analyses of samples as pressed KBr pellets were
185 performed on a Thermo Electron Nicolet 8700 instrument with 64 scans and 4 cm-1
186 resolution.
187
188 2.2.2 Ester saponification and back-titration
189 6-Azido-6-deoxy-O-trioxadecanoylcurdlan (238 mg, ~0.47 mmol) or its borohydride
190 reduction product (228 mg, ~0.71 mmol) was carefully weighed and charged into a 50 mL 3-
191 neck flask. An accurately measured volume of 0.1 N NaOH (20 mL) was added and stirred
192 vigorously in a closed system at room temperature for 24 h. The mixture was then titrated
193 with 0.1 N HCl solution. A potentiometric probe was inserted into the flask. Each
194 measurement was carried out by adding a 100 L aliquot of HCl solution, allowing the
195 mixture to stir for 20 seconds and then collecting the pH data point. The precipitate was
196 isolated by filtration, washed extensively with water, vacuum dried and then analyzed by
197 infrared spectroscopy.
198
199 DS calculation after saponification and back-titration:
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200
Eq. (1)
Eq. (2)
201 or
202
203 Where
(V1, V2 are the volumes (mL) of 0.1 N NaOH and 0.1 N HCl solution
204 used during the titration), and m (g) is the mass of added 6-azido or 6-amino curdlan
205 trioxadecanoate.
206
207 2.2.3 Elemental analysis (EA)
208 Amino curdlans were purified and vacuum dried at 40 C for 5 h prior to EA to determine
209 carbon, hydrogen and nitrogen contents, carried out by Micro Analysis Inc. using a Perkin
210 Elmer 2400 II analyzer. Carbon and nitrogen contents were determined by flask combustion
211 followed by ion chromatography.
212
213 2.3 Methods
214 2.3.1 Dissolution of curdlan in DMAc/ LiBr (referred to in examples as “standard cellulose
215 solution in DMAc/ LiCl” (Edgar, Arnold, Blount, Lawniczak & Lowman, 1995))
216 Dissolution of curdlan in DMAc/ LiBr was performed by a procedure reported in our
217 previous paper (Zhang & Edgar, 2014b). A mixture of dried curdlan (4.00 g, 24.7 mmol
218 AGU) and DMAc (150 mL) was kept at 165C for 26 min with vigorous stirring under
219 nitrogen. LiBr (36.00 g, 42.4 mmol) was added, and the mixture was stirred at 160C for 8
220 min. DMAc (40 mL) was distilled off to facilitate water removal. The slurry was allowed to
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221 cool to room temperature while being stirred continuously overnight, during which time
222 dissolution occurred to form a transparent solution.
223
224 2.3.2 Regioselective bromination and azide displacement of curdlan
225 Solutions of Ph3P (25.96 g, 4 eq per AGU) and NBS (17.58 g, 4 eq per AGU) were prepared,
226 each in 50 mL of dry DMAc. The Ph3P solution was added dropwise via a liquid addition
227 funnel to a solution of 4.00 g curdlan in DMAc/ LiBr prepared as described above, followed
228 by dropwise addition of the NBS solution. The resulting solution was then heated at 70C
229 for 1 h. The solution was cooled and then added slowly to 1 L of a 50:50 mixture of
230 methanol and deionized water, then filtered to recover the precipitate. The isolated product
231 was re-dissolved in DMSO and re-precipitated in ethanol, then the dissolution and
232 reprecipitation process was repeated. The product was dried under vacuum at 40C
233 overnight to yield 6-bromo-6-deoxycurdlan. 13C NMR (DMSO-d6): 103.2 (C-1), 84.9 (C-3),
234 74.4 (C-5), 73.6 (C-2), 70.1 (C-4), 34.6 (C-6-Br). Yield: 86%.
235
236 Dry 6-bromo-6-deoxycurdlan (1.00 g, 4.44 mmol) was dissolved in 25 mL of anhydrous
237 DMSO in a 100 mL flask. Then NaN3 (1.44 g, 5 eq per AGU) was added to the flask and
238 dissolved. The solution was heated to 80C and stirred for 24 h under nitrogen. The product
239 was isolated by cooling the reaction mixture, then pouring into 300 mL of deionized water.
240 The precipitate was collected by filtration. The product was re-dissolved in acetone and then
241 re-precipitated in deionized water, followed by filtration. The sample was dried under
13
242 vacuum at 40C overnight to yield 6-azido-6-deoxycurdlan. C NMR (DMSO-d6): 103.4
243 (C-1), 84.9 (C-3), 74.9 (C-5), 73.9 (C-2), 69.4 (C-4), 51.7 (C-6-N3). Yield: 92%.
244
245 2.3.3 Reaction of 6-azido-6-deoxycurdlan with 3,6,9-trioxadecanoyl chloride (TODCl)
246 TODA (71.2 g, 61.3 mL, 0.4 mol) was first weighed into a 250 mL three-neck round-bottom
247 flask equipped with a dropping funnel and a gas outlet connecting to a 5 M NaHCO3
248 solution. Thionyl chloride (31.2 mL, 0.44 mol) was added dropwise into the flask with
249 magnetic stirring at room temperature. It was stirred for 30 min and then heated to 70 C for
250 an additional 2 h until gas release stopped. The unreacted thionyl chloride was removed by
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251 rotary evaporation. The residual liquid (TODCl) was used for subsequent esterification
252 without further purification.
253
254 Dry 6-azido-6-deoxycurdlan (0.5 g, 2.67 mmol) was dissolved in 25 mL of DMAc in a 100
255 mL flask. Pyridine (2.15 mL, 10 eq per AGU) was injected all at once, then TODCl (4.6 mL,
256 10 eq per AGU) was added dropwise from an addition funnel at room temperature. The
257 mixture was heated to 80 C and stirred at that temperature for 24 h. The homogeneous
258 solution was cooled to room temperature and transferred to 3500 g/ mol MWCO dialysis
259 tubing (prewet with water) that was placed in a large beaker containing deionized water.
260 After 5 days of dialysis, the brown solution was then freeze-dried to yield 6-azido-6-deoxy-
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261 2,4-di-O-trioxadecanoyl-curdlan. C NMR (CDCl3):  169.1 (C=O), 100.1 (C-1), 72.9-67.2
262 (C-2~5 & C-8~14), 58.9 (C-16), 50.9 (C-6-N3). Yield: 78 %.
263
264 2.3.4 Peracetylation of 6-azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan
265 Dry 6-azido-6-deoxy-O-trioxadecanoylcurdlan (0.2 g) and DMAP (20 mg) were weighed into
266 a 50 mL round-bottom flask. Pyridine (0.32 mL, 10 eq per AGU) and acetic anhydride (0.74
267 mL, 20 eq per AGU) were then added dropwise to the solution. The mixture was stirred at
268 80C for 24 h and then cooled and added slowly to 200 mL deionized water. The crude
269 product was collected by filtration, and then re-dissolved in 10 mL chloroform. This solution
270 was added slowly with rapid stirring to 200 mL of ethanol. After filtration, the sample was
271 dried under vacuum at 40C.
272
273 2.3.5 Synthesis of 6-trioxadecanamido-6-deoxycurdlan using sodium borohydride from 6-
274 azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan
275 Dry 6-azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan (0.2 g) was dissolved in 20 mL of
276 DMSO in a 100 mL flask. Then NaBH4 (0.30 g, 20 eq per AGU) was added to the flask and
277 dissolved. The solution was heated to 100C and stirred for 24 h. It was cooled to 0C, then
278 HCl (1M) was added dropwise to the mixture at 0C in an ice bath until gas generation
279 ceased. The mixture was neutralized using saturated NaHCO3 solution and then transferred
280 to 3500 g/ mol MWCO dialysis tubing. After 3 days of dialysis, the colorless solution was
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13
281 then freeze-dried to yield 6-trioxadecanamido-6-deoxycurdlan. C NMR (D2O):  173.3
282 (C=O amide), 103.1 (C-1), 85.0-69.2 (C-2~5 & C-8~14), 58.6 (C-16), 41.6 (C-6-amide),
283 Yield: 79 %.
284
285 2.3.6 Synthesis of 6-amino-6-deoxycurdlan using sodium borohydride
286 Dry 6-azido-6-deoxycurdlan (0.2 g) was dissolved in 20 mL of DMSO in a 100 mL flask.
287 Then NaBH4 (0.81 g, 20 eq per AGU) was added to the flask and dissolved. The solution
288 was heated to 100C and stirred for 24 h. It was cooled to 0C, then HCl (1M) was added
289 dropwise to the mixture at 0C in an ice bath until gas generation ceased. The mixture was
290 neutralized using saturated NaHCO3 solution and then transferred to 3500 g/ mol MWCO
291 dialysis tubing. After 3 days of dialysis, the colorless solution was then freeze-dried to yield
13
292 6-amino-6-deoxycurdlan. C NMR (D2O): 6-amino-6-deoxycurdlan:105.4 (C-1), 86.1 (C-
293 3), 77.4 (C-5), 76.4 (C-2), 72.4 (C-4), 44.1 (C-6-NH2), Yield: 71%.
294
295 2.3.7 Synthesis of 6-acetamido-6-deoxycurdlan by sodium borohydride reduction of 6-azido-
296 6-deoxy-2,4-di-O-acetylcurdlan
297 Under nitrogen, dry 6-azido-6-deoxycurdlan (0.5 g, 2.67 mmol) and 4-
298 dimethylaminopyridine (DMAP, 20 mg) were weighed into a 50 mL round-bottom flask.
299 Pyridine (2.15 mL, 10 eq per AGU) and acetic anhydride (2.73 mL, 10 eq per AGU) were
◦
300 then added dropwise to the solution. The mixture was reacted at 80 C for 24h under
301 nitrogen. The homogeneous mixture was then added slowly to 200 mL deionized water. The
302 crude product was collected by filtration, and then re-dissolved in 10 mL chloroform. This
303 solution was added slowly with rapid stirring to 200 mL of ethanol. After filtration and
◦
304 washing with ethanol and water several times, the sample was dried under vacuum at 40 C
13
305 overnight to yield 6-azido-6-deoxy-2,4-di-O-acetyl-curdlan. C NMR (DMSO-d6): 169.6
306 (C=O), 99.4 (C-1), 78.0 (C-3), 72.1 (C-5), 71.1 (C-2), 68.5 (C-4), 50.5 (C-6-N3), 20.6 (CH3-
307 Ac), Yield: 97%.
308
309 Dry 6-azido-6-deoxy-2,4-di-O-acetylcurdlan (0.2 g, 0.74 mmol) was dissolved in 20 mL of
310 DMSO in a 100 mL flask. Then NaBH4 (0.56 g, 20 eq per AGU) was added to the flask and
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311 dissolved. The solution was heated to 100C and stirred for 24 h. It was cooled to 0C, then
312 HCl (1M) was added dropwise to the mixture at 0C in an ice bath until gas generation
313 ceased. The mixture was neutralized using saturated NaHCO3 solution and then transferred
314 to 3500 g/ mol MWCO dialysis tubing. After 3 days of dialysis, the colorless solution was
1
315 then freeze-dried to yield 6-acetamido-6-deoxycurdlan. H NMR (D2O):  3.87 - 3.30
316 (curdlan backbone protons), 2.03 (CH3-Ac), Yield: 72%.
317
318 3. Results and discussion
319 As we have previously reported (Zhang & Edgar, 2014b), curdlan can be brominated with
320 complete regioselectivity at C-6 and the resulting 6-bromo-6-deoxycurdlan can then be
321 reacted with NaN3 in DMSO at 80C to produce 6-azido-6-deoxycurdlan with a DSazide~1.0,
322 which can be easily esterified to produce 2,4-O-diester derivatives readily soluble in a range
323 of organic solvents. The azido curdlans have been found to be useful precursors for
324 regioselective synthesis of several new curdlan derivatives, including (O-acylated-)6-amino-6-
325 deoxycurdlan by Staudinger reduction, as well as a series of 6-amido-6-deoxy-2,4-di-O-acyl-
326 curdlans. Even though the mild Staudinger reaction chemoselectively reduces the 6-azide to
327 6-amine while leaving the esters at C-2/ 4 untouched, those aminodiester products showed
328 poor solubility in any common organic solvent once isolated from the reaction mixture. We
329 originally hypothesized that their insolubility could be attributed to intermolecular hydrogen
330 bonding between 6-amino groups, and/ or to the small amount of residual
331 triphenylphosphine-related impurities that could be seen in NMR spectra of the derivatives
332 (Cimecioglu, Ball, Kaplan & Huang, 1994; Fox & Edgar, 2012; Zhang & Edgar, 2014b).
333 Such poor solubility could be limiting with regard to applications as drug or gene delivery
334 agents, and especially with respect to oral protein delivery via tight-junction opening.
335
336 We planned to impart water solubility to 6-amino-6-deoxycurdlan derivatives by esterifying
337 the O-2/ 4 curdlan hydroxyl groups with 3,6,9-trioxodecanoate groups, by reacting 6-azido
338 curdlan with 3,6,9-trioxadecanoyl chloride in dimethylacetamide. Therefore, in spite of our
339 concerns about chemoselectivity and potentially competing ester reduction, we explored the
340 use of sodium borohydride as azide reducing agent in an attempt to circumvent these
341 solubility problems.
12
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342
343 3.1 Synthesis of 6-azido-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan
344 Acylation of 6-azido-6-deoxycurdlan with the appropriate acid chloride seemed like a
345 promising approach to the 2,4-di-O-trioxodecanoyl esters; therefore TODCl (Fig. S2) was
346 synthesized by reaction of TODA with thionyl chloride just prior to its use, then was reacted
347 with 6-azido-6-deoxycurdlan in DMAc in the presence of pyridine catalyst.
348
349 Identity and DS of the resulting 6-azido-6-deoxy-2,4-O-bis(trioxadecanoyl)curdlan were
350 established by FTIR and NMR spectroscopy, peracetylation, and saponification followed by
351 back-titration. FTIR analysis showed that, in comparison with the 6-azido starting material
352 (Fig. 5a), a characteristic ester carbonyl absorption at 1771 cm-1 was present (Fig. 1a),
353 indicating successful TOD ester introduction. Proton NMR is normally very useful for DS
354 determination of polysaccharide derivatives, but peaks for the trioxadecanoate methylene
355 groups at around 3.3-4.1 ppm overlap with the curdlan backbone proton resonances (3.7-4.8
356 ppm, Fig. 2) preventing DSTOD calculation by this method (unsurprisingly since all are
13
357 protons attached to carbons that also bear an electron-withdrawing oxygen atom). The C
358 NMR spectrum (Fig. 3a) contained a TOD carbonyl signal at  169.1 ppm and a single C-1
359 signal at 100.1 ppm. Resonances assigned to the carbons of the TOD moiety and the
360 curdlan C-2, 3, 4, 5 carbons are visible at  = 59.0 (C-16) and  = 65.7 – 73.8 (C-2 through 5,
361 C-8 through 14), with similar issues of signal overlap as observed in the 1H NMR. Therefore,
362 we treated the putative 6-azido-6-deoxy-2,4-O-bis(trioxadecanoyl)curdlan with acetic
363 anhydride in pyridine to yield products in which the proton NMR should show acetyl methyl
364 signals at 1.9-2.1 ppm if DSTOD < 2. However, no apparent acetyl methyl resonances were
365 observed (Fig. S3), indicating complete TOD esterification (DSTOD~2) at C-2/ 4 within the
366 1H NMR detection limits.
367
368 Alternatively, we employed saponification of 6-azido curdlan trioxadecanoate with aqueous
369 NaOH and back-titration of the excess base with HCl solution to determine DSTOD. This
370 approach has long been used for DS determination of polysaccharide esters (Freire, Silvestre,
371 Pascoal Neto & Rocha, 2005; Malm, Genung, Williams & Pile, 1944; Stojanović, Jeremić,
372 Jovanović & Lechner, 2005; Tihlarik & Pasteka, 1987). Complete trioxadecanoate hydrolysis
13
Page 13 of 23

373 under mild conditions (0.1 N aq NaOH, 25 C, 24 h) can be confirmed by FTIR (Fig. S4b),
374 in which the ester carbonyl band at 1771 cm-1 disappeared while the hydroxyl stretching
375 absorption near 3400 cm-1 increased. Aqueous HCl (0.1 N) was employed to back-titrate the
376 excess NaOH and the volume of added acid was plotted against the pH change (Fig. S5).
377 The peak at 10.8 mL indicated the equilibrium point that was used to calculate the DSTOD
=
378 1.89 by Eq. (1).
1771 cm-1 (C=O ester)
1a)
2105 cm-1 (N3)
1660 cm-1
(C=O amide)
O-H
1b)
-1
1547 cm
(N-H)
379
380 Figure 1. FTIR spectra of 1a) 6-azido-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan and 1b) 6-
381 trioxadecanamido-6-deoxycurdlan synthesized by NaBH4 reduction.
382
N3
O
H-16
RO
O
n
OR
O
R=
7
O
11
13
O
8
10
O
14
16
TMS
H-8~14
H-1~6/ 6'
curdlan backbone
CDCl3
383
384 Figure 2. 1H NMR spectrum of 6-azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan.
385
386 3.2 Borohydride reduction of 6-azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan
14
Page 14 of 23

387 Because of the solubility problems observed previously with the products of azide reduction
388 using the Staudinger reaction, we wished to pursue an alternative strategy. Borohydride is a
389 useful reagent for reduction of azides (Scriven & Turnbull, 1988), but as mentioned
390 previously we had concerns about partial or full concomitant reduction of the ester moieties
391 under the rather strong conditions that we thought would probably be needed to reduce
392 these polysaccharide azides. Indeed, preliminary experiments indicated that reaction with
393 sodium borohydride at 100C would be required for complete azide reduction. 6-Azido-6-
394 deoxy-2,4-di-O-trioxadecanoyl-curdlan was treated with 20 molar equivalent of sodium
395 borohydride at 100C for 24 h followed by dialysis and lyophilization. The product,
396 gratifyingly, did indeed have excellent water solubility. In Fig. 1b, the typical absorption of
397 azide around 2100 cm-1 was absent after reduction while N-H bending appeared at 1547 cm-1.
398 However, instead of the expected ester carbonyl absorptions, a carbonyl absorption was
399 observed at 1660 cm-1, an area that is typically associated with amides. No ester carbonyl
400 absorption was observed in the range of 1735 to 1750 cm-1, indicating no remaining
401 trioxadecanoate after reduction. Additionally, broadened absorptions in the range of 3300-
402 3500 cm-1 were assigned to O-H stretch of curdlan backbone hydroxyls resulting from ester
403 reductive cleavage. It was vital to confirm whether concomitant partial or complete
404 reduction of the TOD esters had occurred. More conclusive evidence of conversion to the
13
405 6-trioxadecanamido-6-deoxycurdlan is provided by the C NMR spectrum (Fig. 3b). The
406 chemical shift for C-6 has moved upfield from 50.9 to 41.6 ppm, in accord with spectral
407 changes observed with other polysaccharide azide to amide conversions (Pereira & Edgar,
408 2014; Zhang & Edgar, 2014b). More importantly, only one carbonyl resonance from the
409 TOD moiety was observed; it exhibited a downfield shift of about 4 ppm and changed from
410 the two carbonyl resonances of the starting diester ( 169) to a single resonance (173, Fig.
411 3). From this spectroscopic evidence, we hypothesize that under the harsh conditions
412 required to completely reduce the azide (100C, 24 h), the liberated amine reacted with one
413 or both of the 2,4-O-ester groups, with the result of acyl transfer to the more nucleophilic
414 nitrogen, and amide formation. Such O to N acyl transfers are well-known in organic
415 chemistry (Cao & Raleigh, 2010; Sohma et al., 2004) and even in carbohydrate chemistry
416 (Sproviero, 1973). Intramolecular acyl transfer from 4-O to 6-N is geometrically reasonable,
417 but we cannot rule out the possibility that at least some of the acyl transfer occurred
15
Page 15 of 23

418 intermolecularly. The excess sodium borohydride reductively cleaved the remaining TOD
419 esters at C-2/ 4, affording the observed amide product. Such acyl group migrations from O
420 to N have been observed by us before, for 6-amino-6-deoxycellulose 2,3-O-esters (Fox &
421 Edgar, 2012), and for the analogous derivatives of pullulan (Pereira & Edgar). However, in
422 those cases Staudinger reduction was used to reduce azide to amine, so that acyl migration
423 occurred from ester oxygen to a negatively charged nitrogen of the Staudinger ylide
424 intermediate. In order to confirm this hypothesis, we subjected 6-azido-6-deoxy-2,4-acetyl-
425 curdlan to the same NaBH4 reduction conditions (since the acetyl methyl resonates upfield
426 of the curdlan backbone hydrogens and is readily observed and quantified). IR and NMR
427 analyses (Fig. S7) of this reduction product show acetyl migration to form the amide and
428 reductive cleavage of the residual O-acetate esters, in exactly the same fashion observed for
429 the TOD esters. It is interesting to note that the 6-acetamido-6-deoxycurdlan product, an
430 isomer of chitin (which is insoluble in water and simple organic solvents), is soluble in water.
431 This sharp difference in properties is worthy of further exploration.
432
433 In order to quantify residual trioxadecanoate following borohydride reduction of the TOD
434 ester, the product was saponified with aqueous NaOH (0.1 N) followed by back-titration
435 using HCl (0.1 N). Two inflection points were evident in the titration curve (Fig. S6); the
436 first one for titration of excess NaOH, and the second for titration of the weakly basic 6-
437 NH2 groups. Based on the titrant consumed at the first inflection point (12.6 mL), DSTOD
438 was calculated to be 1.09 by Eq. (2). Combined with previous spectrometric evidence,
439 almost all ester groups had been reduced to hydroxyls during azide reduction (compare with
440 DSTOD of 1.89 in the starting 6-azido curdlan trioxadecanoate) and a new trioxadecanamide
441 was formed at C-6.
16
Page 16 of 23

3a)
N3
CDCl3
O
RO
R=
O
2~5 &
8~14
n
OR
O
7
16
O
11
13
O
8
10
O
14
16
6-N3

7
1
3b)
NHR
O
RO
O
n
OR
O
R= H or
O
CH 3
2
O
2~5 &
8~14
16
6-amide

1
DMSO
7
442
443
444
445 Figure 3. 13C NMR spectra of 3a) 6-azido-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan and 3b)
446 6-amino-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan.
447
448 3.3 Synthesis of water-soluble 6-amino-6-deoxycurdlan
449 Given these results, it was of interest to investigate the reduction of 6-azido-6-deoxycurdlan
450 to determine whether the poor water solubility previously observed for the parent amine (6-
451 amino-6-deoxycurdlan) could be improved by using borohydride rather than Staudinger
452 reduction. We employed a large excess of borohydride (20 eq NaBH4, 60C, 24 h) as used by
453 Matsui and co-workers, believing that it may be necessary to efficiently reduce recalcitrant
13
454 polysaccharide azides. In the product C NMR spectrum (Fig. 4a), residual azide could be
455 clearly observed as indicated by the azide-bearing C-6 resonance at 51 ppm. Partial
456 conversion to the desired amine product could be confirmed by appearance of a resonance
17
Page 17 of 23

457 for the amine-bearing C-6, shifted upfield to 44 ppm. In order to improve the extent of
458 reduction, we doubled the reaction time (48 h, Fig. S1b), but this was ineffective as the
459 DSamine decreased from previous 0.80 to 0.76 (determined by elemental analysis). Increases in
460 reaction temperature proved more useful; at 80C, we observed significant decrease in
461 intensity of the azide-bearing C-6 resonance at 51 ppm (Fig. S1a). Borohydride reduction at
462 100C gave complete disappearance of the azide peak, and appearance of a single C-6 peak
463 at 41 ppm, indicating complete reduction of the azide to the amine (Fig. 4b). No residual
464 Ph3P or Ph3PO peaks were observed (ca. 130 ppm), indicating complete removal of these
465 impurities left over from the bromination (and of course no such impurities are generated
466 during borohydride reduction, in contrast to the Staudinger reduction). Figure 5 shows the
467 FTIR spectra of 6-azido- and 6-amino-6-deoxycurdlan. The strong azide absorptions at 2107
468 cm-1 (Fig. 5a) present in the starting material are virtually completely eliminated in the
469 borohydride reduction product (6-amino-6-deoxycurdlan, Fig. 5b). The product absorption
470 at 1575 cm-1 is assigned to N-H bending of the free amine, confirming successful reduction.
471 6-Amino-6-deoxycurdlan prepared in this way is fully soluble in water (NMR spectra
472 measured in D2O); its DS(NH2) was calculated to be 0.95 by elemental analysis.
NH2
6
4
O
5
HO
2
O
3
1
6-NH2
(
n
OH
6-N3
(
4a)
No
Ph3P
& Ph3P=O
1
3
5 2 4
4b)
473
18
Page 18 of 23

474 Figure 4. 13C NMR spectra of 6-amino-6-deoxycurdlan: 4a) 60C, 24 h and 4b) 100C, 24 h.
475
2107 cm-1 (N3)
5a)
5b)
1575 cm-1 (N-H)
476
477 Figure 5. FTIR spectra of 5a) 6-azido-6-deoxycurdlan and 5b) 6-amino-6-deoxycurdlan
478 synthesized by NaBH4 reduction.
479
480 4. Conclusions
481 We have developed approaches to two water-soluble curdlan derivatives that are
482 characterized by very high regioselectivity. One approach involves appending hydrophilic,
483 uncharged 3,6,9-trioxodecanoate groups to the 2- and 4-OH groups of curdlan, by initial
484 bromination of curdlan, azide displacement of the bromide, and acylation of the 6-azido-6-
485 deoxycurdlan at both 2- and 4-OH groups, affording a water-soluble, regioselectively
486 substituted derivative. Interestingly, upon borohydride reduction of the 6-azido group to the
487 6-amine, acyl group migration occurred, along with concomitant reduction of residual ester
488 groups, providing the N-TOD amide, which is also water soluble. The ability to synthesize
489 water soluble 6-amido-6-deoxycurdlan derivatives in this way is of interest for the study of
490 the biological properties of these 6-substituted, 13 -linked analogues of natural chitin.
491
492 We were pleasantly surprised by the discovery that the parent polysaccharide, 6-amino-6-
493 deoxycurdlan, has good water solubility when synthesized by sodium borohydride reduction
494 of the corresponding azide, as opposed to the water insolubility observed for the Staudinger
19
Page 19 of 23

495 reduction product of the same azide. This is surprising given the close spectroscopic
496 similarity of the two products, differing only in the small quantity of arylphosphorus
497 containing impurities evident in the Staudinger product. Clearly borohydride reduction is
498 preferred in order to obtain the most soluble aminocurdlan products; it has the additional
499 benefits of being simpler and being compatible with easily handled solvents. Access to these
500 families of water-soluble, regioselectively substituted curdlan derivatives should permit
501 investigations of structure-property relationships for a variety of biomedical applications.
502 Studies of the properties of these water-soluble analogs of chitosan with regard to their
503 interactions with tight junction proteins are under way.
504
505 Acknowledgements We thank the Institute for Critical Technologies and Applied Science
506 (ICTAS) for financial support, and the Macromolecules and Interfaces Institute (MII) and
507 the Department of Sustainable Biomaterials at Virginia Tech for their facilities and
508 educational support. We thank the USDA for partial support of this work through Grant No.
509 2011-67009-20090.
510
13
511 Supporting Information C NMR spectra of 6-amino-6-deoxycurdlans prepared under
1
512 these conditions; 80C, 24 h and 60C, 48 h. H NMR spectra of 3,6,9-trioxadecanoic acid
513 (TODA), 3,6,9-trioxadecanoyl chloride (TODCl) and peracetylated 6-azido-6-deoxy-2,4-di-
514 O-trioxadecanoyl-curdlan. FTIR spectra of 6-azido-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan
1
515 and its residue after saponification. FTIR and H NMR spectra of 6-acetamido-6-
516 deoxycurdlan by sodium borohydride reduction of 6-azido-6-deoxy-2,4-acetyl-curdlan.
517 Titration curves of 6-azido-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan after saponification at
518 25C for 24 h: pH and dpH change vs. volume of added HCl (0.1 N, mL). Titration curve of
519 the product of reduction of 6-azido-6-deoxy-2,4-di-O-trioxadecanoyl-curdlan by sodium
520 borohydride, after saponification at 25C for 24 h: pH change vs. volume of added HCl (0.1
521 N, mL).
522
523
524
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