Integrating thin film microfluidics in developing a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases

Article abstract of DOI:10.1016/j.bmcl.2018.10.015

A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a potent inhibitor of α-N-acetylhexosaminidases, DGJNAc, has been developed.

Full text of DOI:10.1016/j.bmcl.2018.10.015

Accepted Manuscript
Integrating thin film microfluidics in developing a concise synthesis of
DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases
Siobhán S. Wills, Colin L. Raston, Keith A. Stubbs
PII:
S0960-894X(18)30805-9
https://doi.org/10.1016/j.bmcl.2018.10.015
BMCL 26074
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
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30 July 2018
8 October 2018
10 October 2018
Please cite this article as: Wills, S.S., Raston, C.L., Stubbs, K.A., Integrating thin film microfluidics in developing
a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.015
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Integrating thin film microfluidics in
developing a concise synthesis of DGJNAc: A
potent inhibitor of -N-
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acetylgalactosaminidases
Siobhán S. Wills, Colin L. Raston and Keith A. Stubbs

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Integrating thin film microfluidics in developing a concise synthesis of DGJNAc:
A potent inhibitor of -N-acetylgalctosaminidases
Siobhán S. Willsa, Colin L. Rastonb and Keith A. Stubbsa*
aSchool of Molecular Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia
bInstitute for Nanoscale Science and Technology, College of Science and Engineering, Flinders University, Sturt Road, Bedford Park, SA, 5042, Australia
Email:keith.stubbs@uwa.edu.au
ARTICLE INFO
ABSTRACT
Article history:
Received
A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a
potent inhibitor of -N-acetylhexosaminidases, DGJNAc, has been developed.
Revised
Accepted
2009 Elsevier Ltd. All rights reserved.
Available online
Keywords:
Inhibitors
Glycosidases
Flow Chemistry
Thin Film Microfluidics
Iminosugars
Carbohydrates are essential molecules for life, playing many
important roles in biological processes.1 Much of the
understanding of the roles of these biomolecules has been gained
through the use of tools to study the enzymes that process
glycosidic bonds. These bonds are linkages that connect
carbohydrates to not only other carbohydrates, but also to non-
carbohydrate motifs such as lipids and proteins.2
One specific class of enzymes that process glycosidic bonds is
the glycoside hydrolases (glycosidases) which act to cleave a
glycosidic bond with the aid of water.3-6 These enzymes are
abundant in nature and the underlying details of their catalytic
mechanisms are well established. Much of this detail has been
determined through the use of chemically synthesized molecules
which act to inhibit the enzyme and thus provide insight into
their function. An important class of inhibitors that has shown
great use is the iminosugars, which are classified by the
endocyclic oxygen being replaced with a nitrogen atom.7 These
Figure 1. (A) The chemical structure of DNJ as well as the premise for its
inhibitory potency. (B) Previous syntheses of the -N-acetylhexosaminidase
inhibitor DGJNAc, 1.
compounds are exemplified by 1-deoxynojirimycin (DNJ),8,9
a
molecule which has been demonstrated to be an excellent
inhibitor of glucosidases (Figure 1A).7 The rationale behind the
potency of not only DNJ, but iminosugars in general, lies with
the endocyclic nitrogen which when protonated is thought to
mimic some part of the charge formed at the putative transition
state of a glycosidase-catalysed reaction (Figure 1A).5
only been shown to process -N-acetylgalactosamine residues.
Despite the important utility of compound 1 for understanding
glycosidase catalysis10,11 and its potential use as a therapeutic,12
its synthesis has received limited attention.
Early syntheses of 1 used DNJ13,14 as a starting material. More
recently though, an asymmetric method starting from the
carbamate 215, which involves an air sensitive Pd-catalysed
allylic substitution, and a carbohydrate-based method starting
from D-glucuronolactone 3,16 along with a scalable synthesis,17
We were drawn to the related compound, 2-acetamido-1,5-
imino-1,2,5-trideoxy-D-galacitol (DGJNAc) 1, which has been
found to be one of the most potent inhibitors of -N-
acetylhexosaminidases. These are enzymes that, to date, have

have been realized (Figure 1B). Based on the length of these
syntheses, we were interested in exploring other synthetic
pathways. Another recent method used to access iminosugars,18-20
through a ulososide precursor, presented an appealing potential
route to 1. The pathway requires a 5,6-alkene 4, which upon
epoxidation in the presence of benzyl alcohol, gives the ulososide
5. Deacetylation under Zemplén conditions, followed by catalytic
hydrogenation, gives the in situ generated 1,5-dicarbonyl
compound 6. With the appropriate amine, 6 undergoes a double
reductive amination reaction to afford the desired iminosugar 7
(Figure 2).18-20
Figure 2. Generic synthetic methodology to prepare iminosugars utilizing a
ulososide.
Specifically, for the synthesis of 1 herein, the key intermediate
was the iodide 8 (Scheme 1). This compound could potentially be
prepared from methyl 2-azido-2-deoxy-β-D-galactopyranoside 9,
which is commercially available or can be synthesized from
literature procedures.21-24 Based on this, we commenced
investigations into realizing the synthesis of 8 from 9. We first
activated O6 through the use of a tosylate and subsequent
displacement with sodium iodide, and then acetylation gave the
iodide 8 (Scheme 1, Path A, conditions a)). Despite a good
overall conversion (90%), a substantial amount of an undesired
3,6-anhydro product, compound 10 was also formed (2:3 ratio of
8:10). This type of result has been observed for other types of
displacement reactions.24 With this result in hand we then
explored acetylation before iodide displacement, which would
then negate the formation of 10. However, after preparing the
tosylate 11, we established that upon treatment with sodium
iodide in large excess at high temperature for long periods, the
reaction did not go to completion, and only a modest yield of 8
resulted (Scheme 1, Path B). After these two results, we then
embarked on preparing 8 via a method used to prepare the
corresponding D-gluco derivatives, which involved first
preparing a 3-O-acetyl derivative.20 Thus, the azide 9 was
acetylated at C3, over 3 steps, to give the diol 12 (Scheme 1, Path
C). Activation of O6 through the use of a tosylate and subsequent
displacement with sodium iodide, in conjunction with acetylation
of the remaining hydroxyl groups, gave the iodide 8, in good
yield.
Scheme 1. Reagents: a) i. TsCl, C5H5N, CH2Cl2, rt, 4 h; ii. NaI, DMF, 100
°C, 6 h; iii. Ac2O, C5H5N, CH2Cl2, rt, 16 h, from 9 - 36% for 8, 54% for 10
over three steps, from 12 - 77% for 8 over three steps. b) i. TsCl, C5H5N
CH2Cl2, rt, 4 h; ii. Ac2O, C5H5N, CH2Cl2, rt, 16 h, 87% over two steps. c)
NaI, DMF, 100 °C, 16 h, 41% for 8. d) i. PhCH(OMe)2, CSA, CH3CN, rt, 2.5
h; ii. Ac2O, C5H5N, CH2Cl2, rt, 16 h; iii. AcOH:H2O (4:1), 80 °C, 3 h, 80%
over three steps. e) i. TsCl, C5H5N, CH2Cl2, rt, 4 h; ii. NaI, DMF, VFD-
mediated, 100 °C, 2 h; iii. Ac2O, C5H5N, CH2Cl2, rt, 2 h, 82% for 8, 9% for
10 over three steps.
Despite the success of forming the iodide 8 (Path C), the high
number of steps involved was undesirable, especially for gaining
access to large quantities of 1. We then decided to return our
attention to the first method which gave the iodide 8 in a one-pot
method, albeit disappointingly due to the formation of the
undesired acetate 10.
Microfluidic flow chemistry has seen an increase in use in
recent times as a method to produce molecules in an efficient and
timely manner, indeed in preparing compounds that are
inherently difficult using traditional batch processing.25-32 What
has aided researchers in utilizing the benefits of flow chemistry
has been the development of scaled-down flow chemistry
apparatus which allows for their use in laboratory settings.33-35
Figure 3. Reagents: a) i. TsCl, C5H5N, CH2Cl2, rt, 4 h, 90%. b). i. NaI, DMF, VFD-mediated, 100 °C, 2 h; ii. Ac2O, C5H5N, CH2Cl2, rt, 2 h, yields in figure. All
reactions were conducted in triplicate with the average shown. Errors were within ±2%.

One type of flow reactor that is receiving increased attention is
the vortex fluidic device (VFD, see Supporting Figure 1)36 which
takes advantage of angled vortex driven thin films as a medium
for controlling chemical reactions, amongst many other
applications.37
Advantages of using the VFD are that it, as do other flow
chemistry devices, allows for specific reagent quantities, surface
area of reaction and temperature to be controlled.35 Additional
advantages of the VFD are that (i) the reactions being studied are
carried out in a dynamic thin film, which is formed within a
rapidly rotating tube, and where reactions are not limited by
diffusion control, and (ii) the microfluidic platform does not
suffer from clogging, unlike in conventional channel-based
microfluidics. The thickness of the film in the VFD is controlled
by varying the rotational speed and the tilt angle of the glass
tube, and this allows for rapid heat transfer and uniform mixing
with no concentration gradients.37 Moreover, optimal rotational
speeds enhance both organic and enzymatic reaction outcomes
arising from pressure waves, as demonstrated for a growing
number of synthetic applications in the VFD.38-45
Scheme 2. Reagents: a) DBU, THF, reflux, 5 h, 94%. b) i. Me3P, Ac2O, H2O,
THF, 0 °C to rt, 2 h; ii. Ac2O, C5H5N, rt, 1 h, 72% over three steps. c) i.
mCPBA, BnOH, CH2Cl2, rt, 2 h; ii. NaOMe, MeOH, rt, 30 min.; iii.
NH4HCOO, Pd(OH)2/C, MeOH, H2O, rt, 48 h, 65% over three steps.
As expected, based on previous studies,18-20 only the D-galacto
configuration was observed.
In conclusion, a new synthesis of DGJNAc 1 is presented
where the use of a novel high shear thin film microfluidic flow
reactor allowed for the realization of an efficient preparation of a
key intermediate, adding to the limited knowledge base on using
the microfluidic platform to control chemical reactivity and
selectivity. This offers an efficient method for preparing
analogues of 1 through either modification of the acetamido
moiety or by using different amines in the reductive amination
step. In addition, this methodology also presents an opportunity
for using the VFD to mediate other troublesome carbohydrate-
based transformations. The success of this synthesis suggests that
this method could also be used to efficiently prepare amide
analogues of other epimers of DNJ.
Given the aforementioned disappointing yield observed for the
direct one-pot reaction to give 8 from 9, and the benefits and
remarkable applications of using the VFD in general, we were
motivated to explore the utility of the VFD for improving the
synthesis of the desired iodide 8. In the first instance, we treated
the tosylate 13 (0.2 mmol), which was prepared and purified
from 9, under the same conditions as were used in batch mode (5
equivalents of sodium iodide, in DMF at 100 °C) and used
standard operating parameters of the VFD, namely 4500 rpm
rotational speed and a tilt angle of 45° of a 20 mm OD
borosilicate glass tube (18.5 cm in length). This test reaction and
subsequent reactions were undertaken in the so called confined
mode of operation of the VFD for a finite volume of liquid held
in the glass tube, where the shear stress is also high, as in
continuous flow.36, 37
Acknowledgments
The authors wish to thank the Centre for Microscopy,
Characterisation and Analysis at The University of Western
Australia, which is supported by University, State and Federal
Government funding. C.L.R. thanks Flinders University, the
Government of South Australia, and both K.A.S. and C.L.R. also
thank the Australian Research Council for funding
(DP170100452). S.S.W. thanks the Australian Federal
Government and the University of Western Australia for an
Australian Postgraduate Award and the Bruce and Betty Green
Foundation for funding.
Gratifyingly, consumption of the tosylate 13 was observed
after only 2 hours, compared to 6 hours for Paths A-C. After
acetylation of the mixture, the ratio of the desired iodide 8 to the
3,6-anhydro compound 10 had dramatically shifted towards
favoring 8 (68:22). With this result in hand, we explored a range
of rotational speeds and tilt angles to try and further optimize the
formation of compound 8. This led to the optimal rotational
speed and tilt angle of 6000 rpm and 45° respectively,
corresponding to 85% yield of the iodide 8 (Figure 3). With this
improved outcome of the reaction in the VFD, we returned to the
starting material 9 and incorporated the VFD-mediated iodide
displacement component into the one-pot synthesis (Scheme 1,
Path A, conditions e)). Gratifyingly, we obtained a 82% yield of
the iodide 8 from the triol 9, along with a 9% yield of the 3,6-
anhydro compound 10.
Conflicts of interest
The authors declare no conflicts of interest.
A. Supplementary data
Supplementary figures, experimental details and characterization
of new compounds.
References and notes
Our attention then focused on moving forward to the goal of
preparing 1. The elimination reaction of 8 across the 5,6-bond
using DBU in THF gave the 5,6-alkene 14 in excellent yield
(Scheme 2). The alkene 14 was then converted to the acetamide
15 by reduction with trimethylphosphine, followed by treatment
with acetic anhydride. Of note is that other amide analogues
could be prepared using a variety of acyl anhydrides. The
acetamide 15 was then treated with 3-chloroperbenzoic acid in
the presence of benzyl alcohol followed by deprotection using a
one-pot method to give the presumed triol 16 as a mixture of
isomers. Treatment of 16 with ammonium formate, in the
presence of palladium hydroxide-on-carbon and hydrogen gas,
afforded DGJNAc 1 in 36% overall yield from 9.
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 A simple synthesis of DGJNAc has been
developed.
 Synthesis utilizes a thin film microfluidic
reactor for a problematic step.
 Synthetic route allows for large quantities of
DGJNAc to be prepared.