Regioselective benzylation of 2-deoxy-2-aminosugars using crown ethers: Application to a shortened synthesis of hyaluronic acid oligomers

Article abstract of DOI:10.1002/adsc.201400269

The combination of benzyl bromide, sodium hydroxide and 15-crown-5 in tetrahydrofuran is shown to be an efficient method for installing benzyl groups at both the 4- and 6-positions regioselectively directly from peracetylated N-trichloroacetyl-protected glucosamine and galactosamine. Application of this benzylation strategy proved to significantly shorten the synthetic route to hyaluronic acid tetra- and hexamers.

Full text of DOI:10.1002/adsc.201400269

FULL PAPERS
DOI: 10.1002/adsc.201400269
Regioselective Benzylation of 2-Deoxy-2-aminosugars using
Crown Ethers: Application to a Shortened Synthesis of
Hyaluronic Acid Oligomers
Chinmoy Mukherjee,a Lin Liu,b and Nicola L. B. Pohla,*
a
Department of Chemistry, Simon Hall, Indiana University, Bloomington, IN 47405-7003, USA
E-mail: npohl@indiana.edu
Department of Chemistry, Hach Hall, Iowa State University, Ames, Iowa 50011-3111, USA
b
Received: March 14, 2014; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201400269.
Abstract: The combination of benzyl bromide, cation of this benzylation strategy proved to signifi-
sodium hydroxide and 15-crown-5 in tetrahydrofuran cantly shorten the synthetic route to hyaluronic acid
is shown to be an efficient method for installing tetra- and hexamers.
benzyl groups at both the 4- and 6-positions regiose-
lectively directly from peracetylated N-trichloroace- Keywords: benzylation; carbohydrates; phase-trans-
tyl-protected glucosamine and galactosamine. Appli- fer catalysis; protecting groups; trichloroacetyl group
Introduction
ing lengthy multiple transformations and tedious sep-
arations, are essential to the practicability of a certain
In the synthesis of complex carbohydrates, the main synthetic route and thus are highly attractive. In the
focus lies logically on the stereoselective formation of past few years, increasing attention has been given to
glycosidic bonds; however, a significant portion of streamlining such protecting group manipulations.
effort in the development of a route for the chemical The groups of both Hung[7] and Beau[8] have reported
synthesis of oligosaccharides is unavoidably devoted highly selective 4,6-benzylidene acetal formation and
to protecting group manipulations.[1] The chosen pro- benzyl ether installations at the 3-position of mono-
tecting groups are much more than inert groups in saccharides using per-silylated substrates under Lewis
carbohydrates synthesis, since they can also have acid conditions to significantly shorten the steps
a profound impact on the reactivity in glycosylation needed to obtain certain building blocks. However,
of the substrate.[2] For example, we recently demon- due to the great diversity of the building blocks
strated that the protecting groups greatly influence needed for oligosaccharide synthesis, much creative
the reactivity of intermediates in the synthesis effort is still needed to enhance protecting group ma-
a carba-sugar.[3] The protecting groups on the donor nipulations. Efforts have also been done to optimize
and acceptors also play key roles in determining out- the protecting group manipulations on glucosamine.
comes in glycosylation reactions.[4] For example, we Hung[9] and Beau[10] with co-workers have reported
recently noted that a fluorous phosphate protecting one-pot syntheses of differently protected glucos-
group could change the stereochemical outcome of
ACHTUNGTRENaNUNG mine building blocks. We have recently reported
a glycosylation unexpectedly.[5] In addition, efforts to three new fluorous amine protecting groups appropri-
install a particular protecting group pattern can be ate for glucosamine building blocks.[11]
hampered by incompatibilities in conditions required
As part of our ongoing work on glycosaminogly-
to install different protecting groups sequentially. For cans, we specifically needed glucosamine and galac-
instance, the installation of multiple ether protecting tosamine acceptors having the 3-OH group unprotect-
groups, such as the often-used “permanent” benzyl ed. A hindered group at the 2-N of an amino sugar is
(Bn) ether,[6] can be challenging in the presence of known to exert hindrance on the incoming electro-
base-sensitive ester or amide bonds. This incompati- phile approaching to the 3-OH group; in the presence
bility can then lead to lengthy building block synthe- of a bulkier group, for example, a 2-N-phthaloyl[12] or
ses or even to completely changed synthetic plans. 2-N-TCP (N-tetrachlorophthaloyl) group,[13] glycosyla-
More effective protecting group manipulations, avoid- tion preferentially occurs at the 4-OH if both the 3-
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
ÞÞ
These are not the final page numbers!

FULL PAPERS
Chinmoy Mukherjee et al.
Figure 1. Comparison between the present work and a previously reported strategy[20] for the synthesis of 4,6-di-O-benzyl-2-
NHTCA-protected amino sugars using existing methods.
OH and 4-OH groups are free whereas similar reac- cleavage could be possible side reactions under com-
tions with smaller protecting groups – for example, monly utilized basic conditions like NaH or NaOH.[19]
N3 or NHAc (acetyl)[15] or Troc (2,2,2-trichloroethyl
In the few cases of benzylated NHTCA substrates,
[14]
carbonate)[16] – provide regioisomers. However, our the benzyl group was installed on 2-azido sugars,[21]
application required a protecting group at the 2-N and TCA was installed after azido reduction. This
that would exhibit both bulkiness and the flexibility strategy utilizes the azido group to circumvent the in-
to control reactions on the 3-OH site under certain compatibility problem, but adds additional steps to
conditions. To attempt to keep the 3-OH group free the route. In other cases, a lengthy synthesis was used
for chain extension reactions, we decided to probe the to install the benzyl ether groups on the 4- and 6- po-
possibility of a regioselective dibenzylation reaction – sitions.[20] (Figure 1) This route involves the benzyli-
preferentially at the 4-OH and 6-OH – by utilizing dene acetal formation at the 4,6 positions, installation
this known hindrance factor.
of a temporary protecting group at the 3-OH, selec-
Recently, the trichloroacetyl group (TCA) has tive benzylidene opening to give 4-OBn and a free
become a popular protecting group for amino sugars; primary hydroxy at C-6, followed by benzylation of
it has been used in the synthesis of hyaluronic acid, the 6-OH under non-basic conditions like silver oxide
chondroitin and other amino sugar-containing struc- or Dudley reagent.[22] In this route, it takes 6 steps to
tures as a participating group due to the fact that obtain the 4, 6-di-O-benzyl NHTCA substrates from
TCA could be either removed at the end of the syn- a 3,4,6-tri-O-acetyl-2-NHTCA substrate with a chosen
thesis under mild basic conditions[17] or transformed anomeric protecting group. Sometimes, due to incom-
to NHAc group under radical conditions.[18] We envi- patibilities amongst certain protecting groups, the 3-
sioned that a 2-NHTCA group might be a good O-protecting group needs to be swapped, thereby
choice as it is neither too small nor too big in size and leading to an even longer route. For example, in the
could provide the optimum masking group for pre- recently reported synthesis of an E. coli O111 O-spe-
venting the neighboring 3-OH from undergoing a sub- cific polysaccharide repeating unit, a 4,6-di-O-benzyl
sequent benzylation reaction. In our efforts towards NHTCA substrate with SEt as the anomeric protect-
the synthesis of various glycosaminoglycans, use of ing group and Fmoc (fluorenylmethyloxycarbonyl) as
the common benzyl ether as the permanent protecting the 3-O protecting group was synthesized in 7 steps
group at the 4- and 6- position of the N-trichloroace- from per-acetylated NHTCA substrate.[23] The steps
tyl-protected glucosamine or galactosamine made for the currently employed methods are long, and in
most sense. However, even though benzyl ether our hands, we obtained highly variable results using
groups have been so widely used, very much to our the reported non-basic benzylation methods on carbo-
surprise, there have only been a few reports using this hydrate substrates.
building block pattern. Most of the existing studies in-
The lack of good synthetic methods to generate 4,6-
volving NHTCA-GalN (galactosamine) or GlcN (glu- di-O-benzyl-protected NHTCA substrates seriously
cosamine) used either a 4,6-O-benzylidene or 4,6-O- hampered our syntheses of glycosaminoglycans using
di-tert-butylsilyl group to block these two hydroxy TCA groups. We decided to study if any improvement
groups. We envision the lack of usage of benzyl ether could be made to solve the problem. In a larger effort
groups at the 4,6 positions in the presence of NHTCA to significantly simplify the synthesis of carbohydrate
is due to the incompatibility between the basic condi- building blocks for our automated oligosaccharide
tions used to install benzyl ethers and the trichloro- synthesis platform,[24] we report herein the discovery
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
2
asc.wiley-vch.de
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!

FULL PAPERS
Regioselective Benzylation of 2-Deoxy-2-aminosugars using Crown Ethers
Table 1. Initial screening of benzylation conditions.
plication of this method to the synthesis of a hyaluron-
an hexasaccharide.
Results and Discussion
The most widely used method to install benzyl ethers
is to use NaH/BnBr in a polar aprotic solvent.[6]
Under certain circumstances, a low temperature ben-
zylation could be performed at the 3-OH of a benzyli-
dene-protected NHTCA-bearing carbohydrate sub-
strate; however, this method could not solve our
need, as the free 3-OH group would be difficult to
retain under stronger basic conditions, even at low
temperature. During the course of this work, new pro-
cedures to perform benzylation under acidic condi-
tions have been reported;[25] however, these reagents
also do not provide the regioselectivity needed. In
our efforts towards effective benzylation in the pres-
ence of the TCA group, we realized that the key to
successful benzylation on such substrates might be the
chemical stability difference between acyl groups and
Entry Base (equiv.)
PTC
Time Yield
1
2
3
4
5
6
7
KOH (6)
CsOH (6)
18-crown-6 20 h 21%
TBAI
–
20 h 18%
20 h <5%
BaO/Ba(OH)2 (6)
50 wt% NaOH-Al2O3 (12) 15-crown-5 20 h 16%
NaOH (6)
NaOH (9)
NaOH (12)
15-crown-5 20 h 68%
15-crown-5 6 h
15-crown-5 6 h
69%
80%
amide groups. By carefully choosing the reaction con- were cleaved and then benzyl bromide (3 equiv) was
ditions, we may have a chance of performing benzyla- added slowly to the resulted slurry under an inert at-
tion on hydroxy moieties or acyl-protected hydroxy mosphere. A non-polar spot appeared, based on TLC
groups without affecting the TCA group.
(thin layer chromatography) analysis, in a few hours
Benzylation reactions can be performed directly but no satisfactory progress of the reaction was seen
from acyl-protected sugar substrates using a nucleo- even after 20 h, as a large amount of unreacted polar
philic base.[26] In that case a one-pot, two-step reac- compound could be seen on TLC. Chromatographic
tion can be performed first by in situ deacylation, fol- separation yielded only 21% of that non-polar com-
lowed by benzylation. We decided to use compound pound (entry 1, Table 1). A careful nuclear magnetic
1, a 3,4,6-tri-O-acetylated 2-NHTCA-protected glu- resonance (NMR) study of the compound revealed
cosamine substrate with a methoxyphenol group at the formation of our expected 4,6-di-O-benzylated
the anomeric position, as the model compound to compound 2. The presence of four benzylic protons
study benzylation. Tetrahydrofuran (THF) was [at d=4.67, 4.58 (2 d, J=11.2 Hz each), 4.55, 4.47 (2
chosen as the solvent, for its lesser polarity might im- d, J=12.0 Hz each)] and a doublet for the NHTCA
1
prove selectivity.[27] Phase-transfer conditions have proton at d=7.04 (J=7.4 Hz) in the H NMR spec-
been used in benzylation reactions for a long time,[28] trum confirmed the incorporation of two benzyl
and allow the usage of milder bases in organic sol- groups without any base-catalyzed hydrolysis of the
vents. In many cases, phase-transfer conditions lead to amide bond or N-benzylation. Absence of any acetyl
selective benzylation on carbohydrate substrates.[29] group in the NMR spectra confirmed that a complete
However, the possibility of using such phase-transfer deacetylation reaction occurred prior to the benzyla-
conditions in benzylation reactions in the presence of tion reaction. The exact structure of the compound
an amide bond has not been thoroughly explored. In was established through a cross-peak correlation be-
1
our study, different bases with different phase-transfer tween H-3 and 3-OH protons in a H-1H COSY spec-
reagents in the presence of 3 equiv. of benzyl bromide trum (please see the Supporting Information).
were screened (Table 1).
Encouraged by this result, efforts were directed to-
Potassium hydroxide has been used earlier in ben- wards other available bases for regioselective benzyla-
zylation reactions directly on acylated sugars,[30] and tion. A combination of CsOH/TBAI (tetrabutylam-
therefore served as the starting point for our study. monium iodide) only gave an 18% yield of the diben-
The preliminary benzylation reaction of substrate zylated product under similar conditions, along with
1 was performed using a combination of BnBr/KOH triols from the starting materials in which the acetyl
in the presence of 18-crown-6, a phase-transfer cata- groups were cleaved by the treatment of base
lyst. Before addition of benzyl bromide, substrate (entry 2, Table 1). BaO/Ba(OH)2 has been reported to
1 was first treated with 6 equiv. of powdered KOH perform ether formation in the presence of an amide
and 18-crown-6 (0.05 equiv. of base) in anhydrous bond.[31] Using BaO/Ba(OH)2 only gave trace
THF for 1 hour. During this time all the acetyl groups amounts of the 4,6-di-O-benzyl product 2 (entry 3,
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
3
ÞÞ
These are not the final page numbers!

FULL PAPERS
Chinmoy Mukherjee et al.
Scheme 1. Selective benzylation on NHTCA-protected GlaN; R=4-methoxyphenyl.
Table 1). No tribenzylated product was isolated. considered for this purpose. From an N-trifluoroacetyl
[32]
When a mixed base of NaOH and Al2O3 was used (TFA) – which is smaller but more electronegative in
(entry 4, Table 1), in the presence of 15-crown-5, the comparison to the TCA group – protected glucos-
yield of 2 was only 16%. After getting unsatisfactory
amine substrate 6,[34] the dibenzylated product 7 was
ACHTUNGTRENNUNG
results with these bases, we turned our attention to isolated in 53% yield, along with 20% of the tetraben-
the inexpensive base sodium hydroxide. A BnBr/ zylated product 8. Compound 8 was likely formed
NaOH/TBAI combination had been reported by first by N-benzylation followed by amide hydrolysis.
Misra and co-workers for the benzylation of acylated The presence of four doublets from benzylic protons
non-amino sugar substrates.[26] In our case, a combina- at d=4.64, 4.57, 4.54 and 4.46, respectively and a dou-
tion of NaOH (6 equiv.), 15-crown-5 (0.05 equiv. of blet of NHTFA proton at d=6.63 (J=7.6 Hz) in
base) and benzyl bromide (3 equiv.) furnished 2 in 1H NMR and two quartets at d=157.8 (2JC,F
=
68% yield in 20 h (entry 5, Table 1). Delighted with 38.0 Hz, COCF3) and d=115.8 (1JC,F =287 Hz, CF3),
this result, we decided to proceed with NaOH/15- respectively, in 13C NMR supported the formation of
crown-5 to optimize the yield of the desired dibenzy- the dibenzylated product. The exact position of the
lated product. When the amount of NaOH was in- free hydroxy group was confirmed from a 1H-13C
creased to 9 equiv., the reaction gave a similar yield HMBC spectrum, as no correlation peaks between 3-
(69%) in only 6 h (entry 6, Table 1). This prompted us OH and H-3 protons were observed in the 1H-1H
to use more NaOH in the reaction. With 12 equiv. of COSY spectrum due to the appearance of a broad
NaOH, the reaction gave 2 in 80% yield in 6 h singlet for the 3-OH proton. The presence of correla-
(entry 7, Table 1). By using these optimized condi- tion peaks between the C-4 and C-6 carbons with
tions, we could synthesize 2 easily on a gram scale their adjacent benzylic protons and the absence of
from 1 in only one step, thereby eliminating the previ- any correlation peaks of benzylic protons with C-3 in
ously reported[20] six-step method (Figure 1).
1H-13C HMBC confirmed the formation of compound
Next, we applied these same conditions to a galac- 7 (please see the Supporting Information). The forma-
tosamine substrate. Interestingly, unlike the 4-position tion of tetrabenzylated product 8 was easily con-
epimer, glucosamine 1, the tribenzylated compound firmed through the presence of four benzyl groups
1
was also formed in moderate yield when the same and the absence of the NHTFA proton in H NMR as
conditions were applied to galactosamine 3. The di- well as absence of two quartets of the COCF3 group
benzylated product 4 was isolated in a 56% yield, in 13C NMR. As a TFA group is smaller than a TCA
along with 34% of the tribenzylated product 5. This moiety, comparatively less hindrance from the former
result reflects its more reactive character[33] as com- group is expected and therefore there is a greater pos-
pared to the analogous glucosamine derivative sibility of forming the tribenzylated compound under
(Scheme 1). When 2.2 equiv. of benzyl bromide were these conditions. Interestingly, no 3,4,6-tri-O-benzylat-
used, a slightly better yield of dibenzylated derivative ed product of Glc-NHTFA was formed; this may be
was found, though the reaction time was comparative- due to an electronic factor.[33] When compound 9[35]
ly much longer. As in the case of compound 2, the with an acetamido group was used, the tribenzylated
formation of dibenzylated 4 was confirmed through and tetrabenzylated compounds 10 and 11, respective-
the appearance of a correlation peak between the H-3 ly, were the major products, which were isolated in
1
and 3-OH in the H-1H COSY spectrum.
35% and 33% yields, respectively. To make the region
To study the role of the N-trichloroacetyl protecting more hindered, NHAc was replaced by an N-diacetyl
group in the observed selectivity, various other amino group. Treatment of compound 9 with isopropenyl
protecting groups were tested (Table 2). Both smaller acetate in the presence of acid catalyst at elevated
than trichloroacetyl and slightly hindered groups were temperature furnished compound 12 in 97% yield. A
4
asc.wiley-vch.de
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!

FULL PAPERS
Regioselective Benzylation of 2-Deoxy-2-aminosugars using Crown Ethers
Table 2. Scope for the phase-transfer benzylation method: different amino-protecting groups.[a]
[a]
Reaction time was 20 h for each example except compound 15.
Reaction time was 2 h.
[b]
similar benzylation reaction on 12 furnished tribenzy- diacetyl group did not survive the initial basic condi-
lated product 10 in 16% and tetrabenzylated product tions and thus directly transformed into NHAc to
11 in 49% yields as major products. Presumably, the behave similarly to compound 9.
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
5
ÞÞ
These are not the final page numbers!

FULL PAPERS
Chinmoy Mukherjee et al.
We also discovered that an azide group cannot a fully protected oligomer.[48] Recently, van der Marel
affect the selective benzylation. When a similar ben- and Codꢂe reported the automated solid-phase syn-
zylation reaction was carried out on compound 15, tri- thesis of hyaluronan oligosaccharides, obtaining a pen-
benzylated product 16 was obtained almost exclusive- tadecamer of hyaluronic acid.[49] Despite these efforts,
ly. When only 2.2 equiv. of BnBr were used, 16 was it is still a daunting task to perform the synthesis to
still isolated as the major product. An oxazoline obtain different lengths of hyaluronic acid fragments,
group in compound 17 also did not allow a selective partially due to the difficulties in synthesizing the re-
benzylation reaction. Tribenzylated compound 18 was quired building blocks on larger scales.
found as the major product even when 2.2 equiv. of
We envision that application of this phase-transfer
BnBr were used. Carbamate derivatives (19, 20) were benzylation reaction could afford a concise method
transformed into N-benzyl-2,3-trans-oxazolidinone for the production of building blocks of hyaluronic
19a under basic conditions.[36] Clearly, the TCA-pro- acid. When the donor and acceptor become larger,
tected nitrogen is key to the selectivity under these their reactivity decreases for steric and electronic rea-
conditions.
sons. Therefore, increasing the reactivity, especially
Based on the above studies, BnBr/NaOH/15-crown- for the uronic acid disaccharide acceptor, has become
5 in THF provides an efficient method to efficiently an important problem. To increase the reactivity of
create the desired protecting group pattern for a build- the acceptor, more electron-donating groups rather
ing block needed to make hyaluronic acid and related than electron-withdrawing groups should be installed
glycosaminoglycans. Hyaluronic acid (HA) is a linear, on the glucuronic building block. In the former syn-
unbranched repeating polymer of N-acetylglucosa- thesis of hyaluronic acids, 2-Bz,3-Bn-protected uronic
mine and d-glucuronic acid. Hyaluronic acid is dis- acid building blocks had been used several times to
tributed widely throughout connective, epithelial, and improve the reactivity of the block.[47b,50] We reasoned
neural tissues and is involved with multiple functions that if we could synthesize a disaccharide building
including cellular proliferation, cell-cell recognition, block with a uronic acid residue bearing 2,3-dibenzyl
and cell migration.[37] As the main function of this gly- protecting groups, the reactivity of the building block
cosaminoglycan family member was believed to be as might be further improved. 2-Bz,3-Bn-protected
a polymer required for the lubrication of joints, HA uronic acid building blocks could be synthesized fairly
received relatively little attention for years. However, easily; however, synthesizing a disaccharide building
more recently biological functions of shorter hyalur- block with a 2,3-dibenzyluronic acid residue was
onic acid fragments have been uncovered.[38] The deg- much more difficult and has not yet been reported.
radation products of hyaluronic acid, small oligosac- The benzyl group cannot be installed on the 2-posi-
charides with varying lengths, exhibit pro-angiogenic tion of the glucuronic building block prior to glycosy-
properties.[39] Short fragments of hyaluronic acid can lation since neighboring group participation from an
induce inflammatory responses in macrophages and acyl group helps form the required beta-glycosidic
dendritic cells in cases of tissue injury or skin trans- linkage. Therefore, a method to affect benzylation on
plant rejection.[40] Hyaluronic acid is also the main the disaccharide had to be developed to synthesize
component of the capsular polysaccharide of group A the disaccharide building block with a 2,3-dibenzyl-
Streptococci (Streptococcus pyogenes or GAS).[41] glucuronic acid residue. Our new benzylation method
Abundant production of hyaluronic acid in GAS is provides a unique strategy to install benzyl groups on
a key virulence determinant and is correlated with se- the 2 and 3-positions of the GlcA building block.
rious GAS infections.[42] As a result, low molecular
From the easily obtained allyl-protected per-acety-
weight hyaluronic acid is now being studied as a treat- lated NHTCA substrate 21,[48] in one pot using the
ment and prevention of infection and disease caused new benzylation reaction described above, the
by group A and group C Streptococci.[43]
GlcNHTCA building block 22 was obtained in good
Given the increasing biological interest in HA, yield (Scheme 2). The glycosylation between 21 and
there have been many reports on the synthesis of hya- 23,[51] the precursor of the GlcA unit, went well to
luronic acid fragment;[44] those syntheses mainly differ provide disaccharide 24 in high yield. The new benzy-
in the choice of protecting groups (especially on the lation reaction was performed on disaccharide 24 to
amino group), the choice of anomeric activating readily provide the desired tetrabenzylated disacchar-
group and the strategy of the oxidation.[45] In recent ide 25 in 72% yield. The benzylidene group was then
years, great advancements were made in this area. removed, and the primary hydroxy group was oxi-
For example, van der Marel and co-workers have re- dized and protected as the methyl ester. The 4-OH on
ported the synthesis of the tri-, penta-, and heptamer the GlcA unit was protected using a levulinate (Lev)
of hyaluronic acid.[46] Huang and co-workers have re- group to give the fully assembled hyaluronic acid di-
ported the synthesis of a dodecamer of hyaluronic
ACHTUNGTRENNUNG
acid.[44,47] Sleeman and Brꢁse reported the multi-gram
synthesis of a hyaluronic acid building block and ing routine steps. The corresponding tetrasaccharide
6
asc.wiley-vch.de
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!

FULL PAPERS
Regioselective Benzylation of 2-Deoxy-2-aminosugars using Crown Ethers
Scheme 2. Synthesis of the HA disaccharide building blocks using phase transfer benzylation reactions. Reaction conditions:
i) BnBr, NaOH, 15-crown-6, THF, room temperature, 8 h, 77%; ii) TMSOTf, CH2Cl2, 08C, 1 h, 98%; iii) BnBr, NaOH, 15-
crown-5, THF, room temperature, 36 h, 72%; iv) TFA, CH2Cl2-H2O, 0 8C to room temperature, 30 min, 92%; v) a) TEMPO,
BAIB, CH2Cl2-H2O (2:1), room temperature, 1 h, b) CH3I, K2CO3, DMF, room temperature, 2 h, 74% in two steps; vi) levu-
linic acid, DCC, DMAP, CH2Cl2, room temperature, 3 h, 96%; vii) a) [IrACHTUNGTRNENUG(COD)CAHTUNGTERNN(UGN PMePh2)2]PF6, THF then H2 (10–20 sec),
room temperature, 3 h, b) HgO, HgCl2, acetone-H2O (5:1, v/v), room temperature, 20 h, c) CCl3CN, DBU, CH2Cl2, 08C, 2 h,
67% in three steps.
Scheme 3. Synthesis of hyaluronic acid hexamer as its allyl glycoside. Reaction conditions: i) TMSOTf, CH2Cl2, 08C to room
temperature, 3 h, 70%; i) NH2NH2·H2O, allyl alcohol, pyridine-AcOH (4:1; v/v), 08C to room temperature, 2 h, 87%; iii) 29,
TMSOTf, CH2Cl2, 08C to room temperature, 1 h, 64%.
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
7
ÞÞ
These are not the final page numbers!

FULL PAPERS
Chinmoy Mukherjee et al.
Scheme 4. Synthesis of hyaluronic acid tetramer as its n-propyl glycoside. Reaction conditions: i) a) Bu3SnH, AIBN, benzene-
dimethylacetamide (4:1; v/v), 808C, 2 h, b) 1M aqueous LiOH, 30% H2O2, THF, H2O, room temperature, 16 h; then 4M
NaOH, MeOH, room temprature, 22 h; c) 20% Pd(OH)2-C, H2, MeOH-H2O (3:1, v/v), AcOH, room temperature, 22 h,
36% in three steps.
30 and hexasaccharide 32 were obtained in good yield while leaving a free 3-position hydroxy for chain ex-
after 2+2 and 2+4 glycosylations (Scheme 3). There tension or further elaboration. The N-trichloroacetyl
are several reports on the chemical synthesis of hya- group was shown to be the key to obtaining the di-
luronic acid oligomers where amino functionalities benzylated product. This new method then allowed
have been temporarily protected by trichloroacetyl a tetrabenzylated hyaluronic acid disaccharide build-
groups and the conversion of multiple NHTCA! ing block to be readily synthesized. Based on this tet-
NHAc groups was performed successfully, firstly by rabenzylated building block strategy, the tetramer and
deprotection of the TCA group in basic conditions, hexamer of hyaluronic acid were also easily made. We
followed by N-acetylation.[17b,c,49] Direct conversion of conclude that the greatly simplified method for selec-
NHTCA!NHAc was also reported by Brꢁse et al.[48] tive benzylation on trichloroacetyl-protected 2-deoxy-
They used Zn/AcOH conditions for this purpose; 2-amino sugars provides a convenient route to a previ-
however, yields were not satisfactory (only 15%) as ously under-utilized protecting group pattern not
they observed more of decomposition products. They readily available by existing synthetic methods, and
also tried the conversion with a tributyltin hydride/ should provide a nice alternative strategy in the syn-
AIBN-mediated radical reaction at elevated tempera- theses of a variety of glycosaminoglycans.
ture. Although the method was successful with a disac-
charide unit (one NHTCA group), only decomposi-
tion products were obtained when the approach was Experimental Section
applied to an HA tetrasaccharide. They reasoned that
Detailed experimental procedures and characterization data
of synthetic compounds 2--33 are given in Supporting Infor-
mation.
the b-1,4 glycosyl bond of the tetrasaccharide was
cleaved during the elevated temperature reaction.
Earlier, Hsieh-Wilson and co-workers performed the
conversion of NHTCA to NHAc successfully on a pre-
cursor of
a chondroitin sulfate tetramer using
Bu3SnH/AIBN.[17d] They used dimethylacetamide as
a co-solvent with benzene and performed the reaction
General Procedure for the Benzylation Reaction
at elevated temperature. We envisioned that a similar To a solution of the acetylated derivative (1 equiv.) in anhy-
drous THF (10 mLmmolꢀ1) was added powdered base
(12 equiv.) and 15-crown-5 (0.05 equiv. of base), respectively,
strategy could also be applied to hyaluronic acid
oligoACHTUNGTRENNUNGmers and thereby provide an additional method
and the reaction mixture was stirred at ambient temperature
under an argon atmosphere for 1 h. BnBr (3 equiv.) was
added to the slurry dropwise and the reaction mixture was
stirred for 2–20 h (required time for each reaction is given
on the Table 1 and Table 2). The reaction mixture was then
filtered through Celite and washed with dichloromethane;
the filtrate was concentrated under reduced pressure. The
crude mixture was diluted with dichloromethane and
to evaluate in the design of the global deprotection of
fully protected hyaluronic acid oligomers. Tetramer
30 was chosen as an experimental target to see wheth-
er these modified conditions would also be successful
with hyaluronic acid; gratifyingly, the NHTCA!
NHAc conversion was completed within two hours
(Scheme 4). This reaction, followed by saponification
and hydrogenolysis, respectively, nicely provided the washed with water and then brine. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. Alternatively, an easier and quicker work-up pro-
cedure was performed as follows. The reaction mixture was
neutralized with Dowexꢃ 50WX8 (H+), filtered and washed
with ethyl acetate. The combined filtrate was concentrated
under reduced pressure. The crude mixture was then puri-
fied through normal phase silica gel flash column chroma-
tography using a hexane and ethyl acetate solvent system as
the eluent to afford pure product. With this procedure, com-
pounds 2, 4, 5, 7, 8, 10, 11, 14, 16, 18, 19a and 22 were ob-
fully deprotected tetrasaccharide 33 in reasonable
overall yield.
Conclusions
To summarize, a combination of BnBr/NaOH/15-
crown-5 in THF is an efficient method for installing
benzyl groups at the 4- and 6-positions on acetylated
N-trichloroacetyl-protected 2-deoxy-2-amino sugars tained.
8
asc.wiley-vch.de
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!

FULL PAPERS
Regioselective Benzylation of 2-Deoxy-2-aminosugars using Crown Ethers
ˇ
´
ˇ
´
[19] a) A. V. Malkov, K. Vrankovꢅ, M. Cerny, P. Kocovsky,
J. Org. Chem. 2009, 74, 8425–8427; b) M. Prashad, D.
Har, B. Hu, H.-Y. Kim, O. Repic, T. J. Blacklock, Org.
Lett. 2003, 5, 125–128.
Acknowledgements
This work was supported in part by the National Institutes of
Health (1R01M090280).
[20] T. Horlacher, M. A. Oberli, D. B. Werz, L. Krçck, S.
Bufali, R. Mishra, J. Sobek, K. Simons, M. Hirashima,
T. Niki, P. H. Seeberger, ChemBioChem 2010, 11,
1563–1573.
References
[21] K. Pekari, D. Tailler, R. Weingart, R. R. Schmidt, J.
Org. Chem. 2001, 66, 7432–7442.
[22] K. W. C. Poon, G. B. Dudley, J. Org. Chem. 2006, 71,
3923–3927.
[23] O. Calin, S. Eller, H. S. Hahm, P. H. Seeberger, Chem.
Eur. J. 2013, 19, 3995–4002.
[24] a) F. A. Jaipuri, N. L. Pohl, Org. Biomol. Chem. 2008, 6,
2686–2691; b) N. L. Pohl, ACS Symp. Ser. 2008, 990,
272–287.
[1] R. Roychoudhury, N. L. B. Pohl, in: Modern Synthetic
Methods in Carbohydrate Chemistry, Wiley-VCH,
Weinheim, 2013, pp 221–239.
[2] S. Aubry, K. Sasaki, I. Sharma, D. Crich, in: Top. Curr.
Chem. Vol. 301, (Eds.: B. Fraser-Reid, J. Cristꢄbal Lꢄ-
pez), Springer Verlag, Berlin, Heidelberg, 2011,
pp 141–188.
[3] L. Liu, B. Abdel Motaal, M. Schmidt-Supprian, N. L. B.
Pohl, J. Org. Chem. 2012, 77, 1539–1546.
[25] a) K. Yamada, H. Fujita, M. Kunishima, Org. Lett.
2012, 14, 5026–5029; b) S. B. Tsabedze, D. E. K. Kabot-
so, N. L. B. Pohl, Tetrahedron Lett. 2013, 54, 6983–6985.
[26] S. K. Madhusudan, G. Agnihotri, D. S. Negi, A. K.
Misra, Carbohydr. Res. 2005, 340, 1373–1377.
[27] H. M. I. Osborn, V. A. Brome, L. M. Harwood, W. G.
Suthers, Carbohydr. Res. 2001, 332, 157–166.
[28] W. Szeja, I. Fokt, G. Grynkiewicz, Recl. Trav. Chim.
Pays-Bas 1989, 108, 224–226.
[29] a) K. Khanbabaee, K. Lçtzerich, M. Borges, M.
Großer, J. Prakt. Chem. 1999, 341, 159–166; b) D. Du-
breuil, J. Cleophax, A. Loupy, Carbohydr. Res. 1994,
252, 149–157.
[30] N. Girard, C. Rousseau, O. R. Martin, Tetrahedron
Lett. 2003, 44, 8971–8974.
[31] U. Schmid, H. Waldmann, Chem. Eur. J. 1998, 4, 494–
501.
[32] K. Sukata, Bull. Chem. Soc. Jpn. 1983, 56, 3306–3307.
[33] Z. Zhang, I. R. Ollmann, X.-S. Ye, R. Wischnat, T.
Baasov, C.-H. Wong, J. Am. Chem. Soc. 1999, 121, 734–
753.
[34] I. R. Greig, M. S. Macauley, I. H. Williams, D. J. Voca-
dlo, J. Am. Chem. Soc. 2009, 131, 13415–13422.
[35] R. Roy, F. Tropper, Synth. Commun. 1990, 20, 2097–
2102.
[4] J. Guo, X.-S. Ye, Molecules 2010, 15, 7235–7265.
[5] a) L. Liu, N. L. B. Pohl, Org. Lett. 2011, 13, 1824–1827;
b) L. Liu, N. L. B. Pohl, Carbohydr. Res. 2013, 369, 14–
24.
[6] A. Liptꢅk, A. Borbꢅs, I. Bajza, in: Comprehensive Gly-
coscience (Ed.: P. K. Johannis), Elsevier, Oxford, 2007,
pp 203–259.
[7] C.-C. Wang, J.-C. Lee, S.-Y. Luo, S. S. Kulkarni, Y.-W.
Huang, C.-C. Lee, K.-L. Chang, S.-C. Hung, Nature
2007, 446, 896–899.
[8] a) A. FranÅais, D. Urban, J.-M. Beau, Angew. Chem.
2007, 119, 8816–8819; Angew. Chem. Int. Ed. 2007, 46,
8662–8665; b) Y. Bourdreux, A. Lemetais, D. Urban, J.-
M. Beau, Chem. Commun. 2011, 47, 2146–2148.
[9] K.-L. Chang, M. M. L. Zulueta, X.-A. Lu, Y.-Q. Zhong,
S.-C. Hung, J. Org. Chem. 2010, 75, 7424–7427.
[10] G. Despras, D. Urban, B. Vauzeilles, J.-M. Beau, Chem.
Commun. 2014, 50, 1067–1069.
[11] R. Roychoudhury, N. L. B. Pohl, Org. Lett. 2014, 16,
1156–1159.
[12] a) J. Xia, J. L. Alderfer, R. D. Locke, C. F. Piskorz,
K. L. Matta, J. Org. Chem. 2003, 68, 2752–2759; b) Z.
Zhang, K. Niikura, X.-F. Huang, C.-H. Wong, Can. J.
Chem. 2002, 80, 1051–1054.
[13] U. Ellervik, G. Magnusson, J. Org. Chem. 1998, 63,
9314–9322.
[14] a) P. V. Nikrad, M. A. Kashem, K. B. Wlasichuk, G.
Alton, A. P. Venot, Carbohydr. Res. 1993, 250, 145–160;
b) A. Toepfer, R. R. Schmidt, J. Carbohydr. Chem.
1993, 12, 809–822.
[36] Y. Geng, L.-H. Zhang, X.-S. Ye, Tetrahedron 2008, 64,
4949–4958.
[37] a) J. Y. Lee, A. P. Spicer, Curr. Opin. Cell Biol. 2000,
12, 581–586; b) J. A. McDonald, T. D. Camenisch, Gly-
coconjugate J. 2002, 19, 331–339.
[38] R. Stern, Semin. Cancer Biol. 2008, 18, 275–280.
[39] A. Benitez, T. J. Yates, L. E. Lopez, W. H. Cerwinka,
A. A. Bakkar, V. B. Lokeshwar, Cancer Res. 2011, 71,
4085.
[15] A. Kameyama, H. Ishida, M. Kiso, A. Hasegawa, J.
Carbohydr. Chem. 1994, 13, 641–654.
[16] K. M. Koeller, C.-H. Wong, Chem. Eur. J. 2000, 6,
1243–1251.
[17] a) J. Dinkelaar, J. D. C. Codꢂe, L. J. van den Bos, H. S.
Overkleeft, G. A. van der Marel, J. Org. Chem. 2007,
72, 5737–5742; b) J. Dinkelaar, H. Gold, H. S. Over-
kleeft, J. D. C. Codꢂe, G. A. van der Marel, J. Org.
Chem. 2009, 74, 4208–4216; c) X. Lu, M. N. Kamat, L.
Huang, X. Huang, J. Org. Chem. 2009, 74, 7608–7617;
d) S. E. Tully, R. Mabon, C. I. Gama, S. M. Tsai, X.
Liu, L. C. Hsieh-Wilson, J. Am. Chem. Soc. 2004, 126,
7736–7737.
[40] R. Stern, A. A. Asari, K. N. Sugahara, Eur. J. Cell Biol.
2006, 85, 699–715.
[41] I. S. Roberts, Annu. Rev. Microbiol. 1996, 50, 285–315.
[42] a) I. Gryllos, H. J. Tran-Winkler, M.-F. Cheng, H.
Chung, R. Bolcome III, W. Lu, R. I. Lehrer, M. R.
Wessels, Proc. Natl. Acad. Sci. USA 2008, 105, 16755–
16760; b) M. R. Wessels, A. E. Moses, J. B. Goldberg,
T. J. DiCesare, Proc. Natl. Acad. Sci. USA 1991, 88,
8317–8321.
[18] D. B. Werz, A. Adibekian, P. H. Seeberger, Eur. J. Org.
Chem. 2007, 2007, 1976–1982.
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
9
ÞÞ
These are not the final page numbers!

FULL PAPERS
Chinmoy Mukherjee et al.
[43] F. Michon, S. Moore, M. Laude-Sharp, M. Blake,
(Baxter International Inc., USA; Baxter Healthcare
S.A.), Patent EP5310, 2002092131, 2002.
genthart, Tetrahedron Lett. 1993, 34, 7939–7942; k) T.
Slaghek, Y. Nakahara, T. Ogawa, Tetrahedron Lett.
1992, 33, 4971–4974.
[44] L. Huang, X. Lu, X. Huang, ACS Symp. Ser. 2008, 990,
29–53.
[46] a) J. Dinkelaar, J. D. C. Codee, L. J. Van den Bos, H. S.
Overkleeft, G. A. Van der Marel, J. Org. Chem. 2007,
72, 5737–5742; b) J. Dinkelaar, H. Gold, H. S. Over-
kleeft, J. D. C. Codee, G. A. van der Marel, J. Org.
Chem. 2009, 74, 4208–4216.
[47] a) L. Huang, X. Huang, Chem. Eur. J. 2007, 13, 529–
540; b) X. Lu, M. N. Kamat, L. Huang, X. Huang, J.
Org. Chem. 2009, 74, 7608–7617.
[48] M. Virlouvet, M. Gartner, K. Koroniak, J. P. Sleeman,
S. Brꢁse, Adv. Synth. Catal. 2010, 352, 2657–2662.
[49] M. T. C. Walvoort, A. G. Volbeda, N. R. M. Reintjens,
H. van den Elst, O. J. Plante, H. S. Overkleeft, G. A.
van der Marel, J. D. C. Codꢂe, Org. Lett. 2012, 14,
3776–3779.
[45] a) M. A. Serban, G. Yang, G. D. Prestwich, Biomaterials
2008, 29, 1388–1399; b) E. R. Palmacci, P. H. Seeberger,
Tetrahedron 2004, 60, 7755–7766; c) W. Jing, P. L.
DeAngelis, J. Biol. Chem. 2004, 279, 42345–42349;
d) S. L. Adamski-Werner, B. K. S. Yeung, L. A. Miller-
Deist, P. A. Petillo, Carbohydr. Res. 2004, 339, 1255–
1262; e) S. S. Iyer, S. M. Rele, S. Baskaran, E. L. Chai-
kof, Tetrahedron 2003, 59, 631–638; f) C. De Luca, M.
Lansing, F. Crescenzi, I. Martini, G.-J. Shen, M.
OꢆRegan, C.-H. Wong, Bioorg. Med. Chem. 1996, 4,
131–142; g) C. Coutant, J.-C. Jacquinet, J. Chem. Soc.
Perkin Trans. 1 1995, 1573–1581; h) T. M. Slaghek, T. K.
Hypponen, T. Ogawa, J. P. Kamerling, J. F. G. Vliegen-
thart, Tetrahedron: Asymmetry 1994, 5, 2291–2301;
i) M. B. Carter, P. A. Petillo, L. Anderson, L. E. Lerner,
Carbohydr. Res. 1994, 258, 299–306; j) T. M. Slaghek,
T. K. Hyppoenen, T. Ogawa, J. P. Kamerling, F. G. Vlie-
[50] Y. Zeng, Z. Wang, D. Whitfield, X. Huang, J. Org.
Chem. 2008, 73, 7952–7962.
[51] D. J. Lefeber, J. P. Kamerling, J. F. G. Vliegenthart,
Chem. Eur. J. 2001, 7, 4411–4421.
10
asc.wiley-vch.de
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 0000, 000, 0 – 0
ÝÝ
These are not the final page numbers!

FULL PAPERS
Regioselective Benzylation of 2-Deoxy-2-aminosugars using
Crown Ethers: Application to a Shortened Synthesis of
Hyaluronic Acid Oligomers
11
Adv. Synth. Catal. 2014, 356, 1 – 11
Chinmoy Mukherjee, Lin Liu, Nicola L. B. Pohl*
Adv. Synth. Catal. 0000, 000, 0 – 0
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
11
ÞÞ
These are not the final page numbers!