Chemoenzymatic synthesis of 3-deoxy-3-fluoro-l-fucose and its enzymatic incorporation into glycoconjugates

Article abstract of DOI:10.1039/d0cc02209h

The first synthesis of 3-deoxy-3-fluoro-l-fucose is presented, which employs ad- tol-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-l-fucitol. Enzymatic activation (FKP) and glycosylation using an α-1,2 and an α-1,3 fu

Full text of DOI:10.1039/d0cc02209h

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Vendeville, K. Hollingsworth, A. P. Mattey, T. Keenan, H. S. Chidwick, H. Ledru, K. Huonnic, K. Huang, M. E.
Light, N. J. Turner, J. Jimenez-Barbero, M. C. Galan, M. A. Fascione, S. L. Flitsch, W. B. Turnbull and B.
Linclau, Chem. Commun., 2020, DOI: 10.1039/D0CC02209H.
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DOI: 10.1039/D0CC02209H
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Chemoenzymatic synthesis of 3-deoxy-3-fluoro-L-fucose and its
enzymatic incorporation into glycoconjugates
Pablo Valverde,a,b,† Jean-Baptiste Vendeville,a,† Kristian Hollingsworth,c,† Ashley Mattey,d Tessa
Keenan,e Harriet Chidwick,e Helene Ledru,f Kler Huonnic,a Kun Huang,d Mark Light,a Nicholas
Turner,d Jesus Jimenez-Barbero,c,g,h M. Carmen Galan,f Martin A. Fascione,e Sabine Flitsch,d,* W.
Bruce Turnbullc,* and Bruno Linclaua,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The first synthesis of 3-deoxy-3-fluoro-L-fucose is presented, which
employs a D- to L-sugar translation strategy, and involves an
enzymatic oxidation of 3-deoxy-3-fluoro-L-fucitol. Enzymatic
activation (FKP) and glycosylation using an -1,2 and an -1,3
fucosyltransferase to obtain two fluorinated trisaccharides
demonstrates its potential as a novel versatile chemical probe in
glycobiology.
Figure 1. Fluorinated fucose derivatives
L-Fucose is a 6-deoxy hexose naturally occurring on N- and O-
linked glycans and in glycolipids present in a large variety of
different organisms.1, 2 It is the fifth most abundant sugar in
mammalian carbohydrates.3 The presence or absence of
fucosylation often crucially determines the structure of
biologically relevant antigens such as the histo blood groups and
2,3
Lewis-type motifs. Fucosylated glycans such as the Human
Milk Oligosaccharides (HMOs) play an important role in
development, and modifications in glycan fucosylation are
implicated in immunity, cancer, and many other important
biological events.4-7
Hence, the importance of this sugar has spurred the
development of L-fucose derivatives as probes or inhibitors to
investigate and manipulate fucose biosynthesis, and
8
fucosylation of glycans and glycoproteins.4,
Much used
inhibitors for enzymatic fucosylation include 2-deoxy-2-fluoro-
L-fucose 1 (2DFF, Chart 1)9,10 and 6,6,6-trifluoro-L-fucose 2,11, 12
Scheme 1. SN2-reactions at the 6-deoxysugar 3-position.
a. School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK.
b. CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia
Technology Park, 48160 Derio, Bizkaia, Spain.
although 6-fluoro-L-fucose can also be used.9 2DFF 1 is
commercially available, and a manufacturing process for 2 has
been reported.13 The methyl 4-deoxy-4-fluoro-l-fucose
anomers have been used as probes to investigate the DC-SIGN
binding epitope, an important receptor of the immune
system.14 Surprisingly, the synthesis of the remaining
deoxyfluorinated l-fucose, 3-deoxy-3-fluoro-L-fucose (3DFF) 3,
has not yet been reported.15
c. School of Chemistry and Astbury Centre for Structural Molecular Biology,
University of Leeds, Leeds, LS2 9JT.
d. Manchester Institute of Biotechnology (MIB), School of Chemistry, University of
Manchester, 131 Princess Street, M1 7DN, Manchester, UK.
e. Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK.
f. School of Chemistry, Cantock’s Close, University of Bristol, Bristol, BS8 1TS, UK.
g. Ikerbasque, Basque Foundation for Science, Maria Diaz de Haro 3, 48013 Bilbao,
Spain.
h. Department Organic Chemistry II, Faculty Science & Technology, EHU-UPV, Leioa,
Spain
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
The synthetic strategy for 3 presents some challenges: the
inversion of configuration (in the absence of neighboring group
participation) that takes place during deoxyfluorination of
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secondary alcohols is a well-known synthetic complication, To that end, cheap, commercially available methyl -D-
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especially when the required diastereomeric sugar is not glucoside 13 (Scheme 3) was selectiveDlyOIp: r1o0.t1e0c3t9e/Dd0aCtC0t2h2e096H-
available.16 This is especially so for L-sugars. In such cases, two- position, upon which treatment with DAST led to selective
step alcohol inversion prior to deoxyfluorination is required, conversion of the 4-OH group as reported by Card.24 Without
which is often not high-yielding, and all other alcohol groups prior anomeric deprotection, the methyl galactoside derivative
must be suitably protected. For 3DFF 3 synthesis, a suitably 14 was converted to the corresponding ring-opened
activated 6-deoxy-L-allose intermediate needs to be accessed. dithioacetal, using an adapted procedure from Redlich and
Unfortunately, Shimabukuro et al have reported that such an Kölln.25 The acidic conditions of this reaction also led to the
SN2 reaction is very challenging (Scheme 1).17 Even with an desired trityl hydrolysis, leading to 15. Unfortunately, all
excellent selenide nucleophile, attempted triflate displacement attempts to reduce the dithioacetal group led to the formation
in 5 mainly led to the elimination product 6. Speculating that of the desired fucitol derivative 16 together with an unidentified
the SN2 reaction may be stereoelectronically disfavoured as and inseparable byproduct.
predicted by the Richardson-Hough rules,18 and aiming to
hamper the elimination process, a less electron withdrawing 4-
OH protecting group and a cyclic protecting group at C1/2 were
installed. Now, SN2 reaction was observed in excellent yield, but
at the cost of a very long synthesis of the precursor 8. In fact, to
the best of our knowledge, deoxyfluorination to give 3-
fluorinated fucopyranose (or galactopyranose) products has not
yet been reported. In their recent syntheses of 3-deoxy3-fluoro-
D-fucosamine analogues,19 Kulkarni et al. circumvented the
issue by using via 3-OH inversion of the quinovose derivative 10
(equatorial 4-OR group), then deoxyfluorination to give the
fluorinated D-quinovosamine derivative 11, and finally followed
by installation of the stereochemistry at C4 by another inversion
to obtain the 3-fluorinated D-fucosamine derivative 12. Hence,
this requires multiple inversion steps, and as L-configured
quinovose is less available, this route is less suitable for the
synthesis of L-3.
OH
OTr
OH
OH
S
F
1) TrCl, py (97%)
SH
SH
O
O
HO
HO
S
HO
2) DAST, neat,
-10 ºC (40%)
HCl, CHCl3
(77 %)
OH
F
OH
HO
HO
OMe
OMe
13
15
14
OMe
1)
Raney-Ni, EtOH
OH
aq.
AcOH
p-TSA,
OH
acetone (66%)
selective
O
O
O
O
3
(79%)
oxidation
2) Raney-Ni
EtOH
(67%)
OH
F
OH
F
17
16
Scheme 2. Synthesis of 3-deoxy-3-fluoro-L-fucitol, with unsuccessful selective oxidation
of the primary alcohol.
After some experimentation, it was found that acetonide
protection of 15 to give 17 allowed for clean dithiane reduction,
leading to pure 16, albeit in moderate yield. Acetonide
hydrolysis then afforded pure 16 of which a crystal structure
could be obtained. Unfortunately, despite relatively close
precedence in the literature,26-28 all attempts to effect selective
oxidation at the primary position gave no result. TEMPO
oxidation proved inefficient in converting 3-deoxy-3-fluoro-L-
fucitol into 3, returning the starting material regardless of the
conditions used (NaOCl or trichloroisocyanuric acid (TCCA)
oxidant). The use of Dess-Martin periodinane did lead to
conversion, with NMR analysis indicating a mixture of cyclised
products, however, no anomeric signals could be detected
indicating overoxidation to lactone derivatives. It was not
possible to adjust reaction times to prevent overoxidation.
Selective hydrolysis of the terminal acetonide in 17 could also
not be achieved.
At this point it was decided to investigate an enzymatic
oxidation protocol, as opposed to developing a different
protecting group strategy. The Galactose Oxidase (GOase)
enzyme was identified as a possible solution. While GOase itself
is strictly limited to oxidizing the C6 hydroxyl of non-reducing
galactosides and a small number of aromatic primary alcohols,
previous engineering efforts have greatly expanded its
substrate scope to a large number of primary and secondary
alcohols.29, 30 In this enzymatic alcohol-oxidation protocol, H2O2
is produced which is converted back to O2 using catalase, whilst
Horse Radish peroxidase (HRP) is required for GOase activation.
Four easily expressed (E. coli) variants of GOase (M1, M3, F2, and
M3-5), selected to provide the greatest substrate scope, were
tested against 16 for activity (Table 1) using a coupled
colorimetric assay that measures the generated H2O2. In this
Sugar epoxides are frequently used for site-selective
fluorination. On a pyranose ring, these reactions are usually
very regioselective due to the stereo-electronically required
axial attack (Furst-Plattner rule).20 However, in L-fucose the
substituent at the 3-position is equatorial, ruling out this option
for the synthesis of 3.
As part of our studies in the use of fluorinated carbohydrates as
substrates for enzymatic glycosylations, a practical synthesis of
3DFF 3 was required. The synthetic route in Scheme 1 was
deemed unsuitable, given the number of steps involved. In
addition, the much reduced nucleophilicity of fluoride
compared to selenide was a potential complication that
discouraged us from proceeding this way. Driven by the desire
to start from a low-cost starting material, and to avoid the
requirement of prior inversion of a hydroxyl group in order to
arrive at the correct C–F configuration, a synthetic scheme
(Scheme 1) was devised that relied on a d- to l-sugar translation.
Hence, starting from a suitably protected glucoside 13,
fluorination and ring opening would lead to a 4-deoxy-4-fluoro-
d-galacto-configured intermediate A. Swapping C1 into C6 (D-
to L-conversion), followed by respective oxidation/reduction
then would lead to the desired fucose derivative 3. The
aldehyde reduction was envisaged via
a dithioacetal
intermediate. Hence, this strategy is a variation on Barros’ d-Gal
to L-Fuc synthesis.21, 22 Other chemical D- to L-sugar syntheses
include Okamoto’s synthesis of L-Gal from D-Gal,15 and Fleet’s
synthesis of L-Glc from D-Glc.23
2 | J. Name., 2012, 00, 1-3
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assay HRP utilizes H2O2 (generated from the GOase oxidation) inhibitor of fucosylation in vivo,9 3-deoxy fluorination would be
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to oxidize 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, expected to have less of an influence onDfOuIc: o10s.y1l0a3t9io/Dn0CreCa0c2t2i0o9nHs
ABTS) to a colored product.31 The variants displayed a wide and as such would be predicted to allow for fluorine
range of activities: the M1 variant was unable to oxidize the incorporation into glycoconjugates. First the formation of the
fluorinated fucitol, with M3 and M3-5 displaying very low specific required fucosyl donor, GDP-3-deoxy-3-fluorofucose 19
activity. However, the F2 variant provided significant activity (Scheme 4), was achieved using fucokinase/L-fucose-1-P-
useable for scale-up. This F2 variant was originally generated guanylyltransferase (FKP) from Bacteroides fragilis.34 This
from mutagenesis of the Arnold Group M1 variant,32 and is bifunctional enzyme converts 3-deoxy-3-fluorofucose 3 into 3-
known to be able to oxidize the primary hydroxyl group of a deoxy-3-fluorofucosyl phosphate 18, and then into GDP-3-
large panel of saccharides.29 Interestingly, the specific activity deoxy-3-fluorofucose 19 which could be confirmed by mass
of the F2-variant towards 16 is higher than its activity towards spectrometry. This demonstrates for the first time that 3-deoxy-
GlcNAc, which it was engineered towards.29
3-fluorofucose 3 is a substrate for FKP.
The synthesis of the Lewis x analogue 22 was then attempted
from the crude fucosyl donor 19 using an -1,3-
fucosyltransferase from H. Pylori.35 To our delight, the product
22 was isolated in 50% yield after purification showing that 3-
deoxy-3-fluorofucose 3 is successfully accepted as substrate. In
addition, the synthesis of the type 1 blood group antigen
analogue 23 from the fucosyl donor 19 was also successful using
a different fucosyltransferase (-1,2).36 As a result of limited
enzyme availability 20 times less enzyme was used, achieving an
8% yield.
Table 1. GOase variants screened against 25mM 3-Fluoro Fucitol using the HRP-ABTS
assay.
GOase variant
M1
M3
M3-5
F2
Specific activity
(µmol/min/mg)
0.1302
(±0.003)
0.0563
(±0.0004)
2.46
(±0.005)
-
Galactose
Oxidase F2,
Catalase
OH
OH
OH
O
HO
OH
Horse Radish
Peroxidase
pH 7.4
F
4-
F
OH
HO
P2O7
ATP
ADP
GTP
3
16
(64%, 0.6 mmol)
(59%, 2.1 mmol)
OGDP
OH
2-
OH
OH
OPO3
O
O
O
FKP
Mg2+
FKP
Mg2+
OH
F
F
HO
F
HO
Scheme 3. Successful synthesis and crystal structure of 3-deoxy-3-fluoro-l-fucose 3
3
18
19
HO
OH
HO
OH
Following
a successful analytical scale biotransformation
Galb(1,4)GlcNAcb(1,3)
azidopropane 20
O
O
O
showing full conversion (not shown), we were delighted to
observe, after overnight incubation at 25 mM substrate loading,
successful conversion of 3-deoxy-3-fluoro-L-fucitol to 3-deoxy-
3-fluorofucose 3 in good yield (Scheme 3). Pleasingly, sugar 3
was crystalline, and was found to crystallize as the -anomer.
In aqueous solution (D2O), 3 existed as a 1:2 mixture of anomers
in favor of the -anomer, which is essentially the same as the
ratio of the nonfluorinated fucose anomers.33 Analysis of the
coupling constants (Figure 1) indicated that both anomers exist
in the expected 1C4 conformation: the 3JH1-H2 value was large for
the -anomer and small for the -anomer, while both anomers
showed a large vicinal coupling between H2 and H3. The
equatorial position of the fluorine was confirmed by the small
3JF3-H4 values. Interestingly, a large long-range coupling between
the equatorial F3 and H1 was observed.
HO
O
N3
O
HO
NHAc
a1,3 fucosyltransferase
Mg2+
O
OH
F
22 (50%)
HO
OH
OH
HO
HO
Galb(1,3)GlcNAcb(1,3)
azidopropane 21
O
HO
O
O
N3
O
NHAc
a1,2 fucosyltransferase
Mg2+
O
O
OH
23 (8%)
F
HO
Scheme 4. Enzymatic synthesis of Lewis x analogue 22 and type 1 blood group
antigen analogue 23
In conclusion, we report the first synthesis of 3-deoxy-3-fluoro-
L-fucose 3 using a D- to L-sugar conversion strategy, in seven
steps starting from a cheap d-glucose starting material. Key
aspects of this synthesis are the avoidance of an alcohol
inversion operation prior to the deoxyfluorination step, and a
final regioselective enzymatic oxidation to the desired 3-deoxy-
3-fluoro-L-fucose 3 using a galactose oxidase variant. Contrary
to the 2-fluoroisomer 1, we have shown that 3 can be
successfully activated using the kinase FKP to achieve
conversion to the corresponding GDP-3-deoxy-3-fluoro-l-fucose
19, which furthermore is accepted by both an -1,3- and -1,2-
fucosyltransferase to lead to fluorinated Lewis x 22 and type 1
blood group antigen 23. This work demonstrates a novel
approach to deoxyfluorinated sugar synthesis, as well as the
potential of 3-deoxy-3-fluoro-L-fucose 3 as a metabolic probe
OH
H
9.8 Hz
H
H
H
OH
O
OH
OH
O
H
H
8.1 Hz
5.1 Hz
F
F
HO
H
HO
H
5.5 Hz
5.5 Hz
4.6 Hz
-anomer of 3
-anomer of 3
Figure 2. 1H NMR analysis of 3 in D2O
Next, it was investigated whether the incorporation of 3-deoxy-
3-fluorofucose 3 into glycoconjugates containing common
motifs found in human biology was possible. Contrary to the
well-studied 2-fluorination such as in compound 1, a known
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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13. M. M. Achmatowicz, J. G. Allen, M. M. Bio, M. D. Bartberger, C.
that is highly complementary to the widely used 2-fluoro isomer
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1.
14. P. Valverde, S. Delgado, J. D. Martínez, J.-B. Vendeville, J.
Malassis, B. Linclau, N.-C. Reichardt, F. J. Cañada, J. Jiménez-
Barbero and A. Ardá, ACS Chem. Biol., 2019, 14, 1660-1671.
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Conflicts of interest
“There are no conflicts to declare”.
Acknowledgement
We are grateful to T. Kaminski and M. E. Webb for their
contribution in expressing galactosyltransferase enzymes. This
project has been funded by the Industrial Biotechnology
Catalyst (Innovate UK, BBSRC, EPSRC, BB/M028941/1,
BB/M028747/1, BB/M028976, BB/M02487X/1) to support the
translation, development and commercialization of innovative
Industrial Biotechnology processes. BL also thanks the EPSRC
(EP/K039466/1), and SF thanks IBioIC, Prozomix Ltd. and Bio-
19. D. A. Williams, K. Pradhan, A. Paul, I. R. Olin, O. T. Tuck, K. D.
Moulton, S. S. Kulkarni and D. H. Dube, Chemical Science, 2020,
11, 1761-1774.
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shape
Ltd.,
RCUK
(BB/L013762/1;
BB/M027791/1;
BB/M028836/1) and ERC (788231-ProgrES-ERC-2017-ADG) for
funding. MCG thanks European Research Council (ERC-COG:
648239). JJB thanks the Agencia Estatal de Investigación (Spain)
(RTI2018-094751-B-C21 and SEV-2016-0644), and ERC (788143-
RECGLYCANMR-ERC-2017-ADG for funding, and the Ministerio
de Educación y Formación Profesional for a FPU fellowship
(14/06147) to PV
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