Synthesis of α-Deoxymono and Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases

Article abstract of DOI:10.1021/acs.joc.6b01485

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (Glc 1P) analogues have been synthesized. A promiscuity investigation of the thymidylyltransferase Cps2L and the guanidylyltansferase GDP-ManPP with these analogues showed that all were accepted b

Full text of DOI:10.1021/acs.joc.6b01485

Article
pubs.acs.org/joc
Synthesis of α‑Deoxymono and Difluorohexopyranosyl 1‑Phosphates
and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases
Jian-She Zhu,† Nicole E. McCormick,† Shannon C. Timmons,‡,§ and David L. Jakeman*,†,‡
†College of Pharmacy, Dalhousie University, 5968 College Street, Halifax, Nova Scotia B3H 3J5, Canada
‡Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
S
* Supporting Information
ABSTRACT: Eight fluorinated isosteric α-D-glucopyranosyl
1-phosphate (Glc 1P) analogues have been synthesized. A
promiscuity investigation of the thymidylyltransferase Cps2L
and the guanidylyltansferase GDP-ManPP with these ana-
logues showed that all were accepted by either enzyme, with
the exception of 1,6-diphosphate 6. Kinetic parameters were
determined for these analogues using a continuous coupled
assay. These data demonstrated the broad substrate promis-
cuity of Cps2L, with kcat/Km changes for monofluoro substitution at C-2, C-4, and C-6 and difluoro substitution at C-2 within
two orders of magnitude. In contrast, the kinetic analysis of GDP-ManPP was only possible with three out of eight analogues.
The pKa2 values of analogues (1−3) were determined by proton decoupled 31P and 19F NMR titration experiments.
Counterintuitively, the axial fluoro substituent in 3 did not change chemical shift upon titration, and there was no significant
increase in acidity for the difluoro analogue over the monofluoro analogues. No strong Brønsted linear free-energy correlations
were observed among all five substrates (1−3, Glc 1P, and Man 1P) for either enzyme-catalyzed reactions. However, Brønsted
correlations were observed among selected substrates, indicating that the acidity of the nucleophilic phosphate and the
configuration of the hexose each plays a significant role in determining the substrate specificity.
properties,9a,10 which is due to some unique features of the
INTRODUCTION
■
fluorine atom, including its high electronegativity, small size,
Sugar nucleotides are essential to many biological processes,
ability to form hydrogen bonds, and resistance to metabolic
including acting as sugar donors in the biosynthesis of oligo-
transformation.11 Furthermore, the preparation of unnatural
and polysaccharides by glycosyltransferases,1 mediating cell−
sugar nucleotides will complement one-pot multienzyme
cell communication,2 or as precursors of many deoxy-, amino-,
(OPME) chemo-enzymatic approaches toward the post-
or branched-chain sugars existing in many secondary
translational modification of proteins to help examine the
metabolites produced by microorganisms or plants.3 They are
roles of glycans in biology.12
often biosynthesized from sugar 1-phosphates by nucleotidylyl-
To expand our knowledge on the substrate specificity of
transferases, also known as pyrophosphorylases (Scheme 1).4
these nucleotidylyltransferases and in particular the effects of
The mechanism of nucleotidylyltransferases has been described
acidity upon substrate reactivity, a small family of isosteric α-D-
as ordered Bi−Bi where the nucleoside triphosphate binds first,
hexopyranosyl 1-phosphates with fluorine substituents on the
followed by the sugar phosphate. Upon completion of the
glucopyranose ring (Figure 1) were designed and synthesized.
nucleophilic attack of the sugar phosphate upon the nucleoside
Kinetic parameters were assessed using a continuous coupled
triphosphate, pyrophosphate is released, followed by the sugar
enzyme assay involving inorganic pyrophosphatase (IPP),
nucleotide.5
purine nucleoside phosphorylase (PNP), and xanthine oxidase
Cps2L6 and guanosine diphosphate-mannopyranose pyro-
(XO).13 The use of the continuous assay for both enzymes
phosphatase (GDP-ManPP),7 cloned from Streptococcus
pneumoniae and Salmonella enterica, respectively, are responsible
for catalyzing the synthesis of deoxythymidine diphosphate
glucopyranose (dTDP-Glc) and guanosine diphosphate-man-
permitted the collection of sufficient data for the construction
of Brønsted linear free-energy relationships to analyze reaction
rate data with measured pKa2 values.
nopyranose (GDP-Man). Exploring the substrate specificity of
RESULTS AND DISCUSSION
■
these enzymes is integral to providing access to a variety of
unnatural sugar nucleotides.1a,4a,8 Unnatural sugar nucleotides
Chemical Synthesis. The general strategy (Figure 2) used
to synthesize these fluorinated α-D-hexopyranosyl 1-phosphates
(1−8) included: (i) fluorine atom addition to monosaccharides
prepared via chemical or enzymatic synthesis have potential
applications as enzymes inhibitors9 for drug discovery and as
tools for providing insight into the structure and mechanism of
enzymes. Fluorinated sugar nucleotides have received increas-
ing attention since the C−F bond exhibits interesting biological
Received: June 21, 2016
© XXXX American Chemical Society
A
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Scheme 1. General Mechanism of a Nucleotidylyltransferase-Catalyzed Reaction
Figure 1. Isosteric α-D-glucopyranosyl 1-phosphate analogues prepared and evaluated.
Figure 2. Strategy for synthesis of fluorinated α-hexopyranosyl 1-phosphates.
using either electrophilic (Selectfluor) or nucleophilic (dieth-
ylaminosulfur trifluoride, DAST) fluorinating reagents, (ii)
selective hydrolysis of the anomeric acetate with NH4OAc in
DMF,14 followed by (iii) phosphorylation of the anomeric
hydroxyl group using diphenylphosphoryl chloride and n-BuLi
at low temperature, and finally (iv) global deprotection.15
The synthesis of difluorinated sugar 1-phosphate 3 (Scheme
2) commenced with preparation of fluoroglycal 9 from
commercially available tri-O-acetyl-D-glucal following published
procedures.16,17 Attempts to insert the second fluorine atom at
the C-2 position of 9 using Selectfluor in acetone-H2O failed
with the recovery of starting material. However, the expected
electrophilic fluorination of 9 with Selectfluor in nitromethane-
H2O produced 10 in 45% yield, furnishing exclusively the α
anomer.17 The coupling of 10 with diphenyl phosphoryl
chloride and n-BuLi at −78 °C provided diphenyl phosphate 11
as the α anomer predominant in 57% yield. The anomeric
configuration of 11 was unambiguously determined by the
Scheme 2. Synthesis of Compound 3
180 Hz, which is consistent for α-anomers.18 The resulting
diphenyl phosphate 11 was deprotected by hydrogenolysis
using Adam’s catalyst (PtO2) to remove phenyl substituents,
followed by hydrolysis of acetate protecting groups using Et3N-
H2O-MeOH (1:3:7) to give rise to compound 3 in a
quantitative yield. The identity of the two fluorine atoms in 3
1
was determined by a 2D H−19F HOESY experiment (Figure
1
magnitude of the heteronuclear coupling constant JC‑1,H‑1 of
3). The peak at −121.1 ppm corresponded to the fluorine in
B
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Figure 4. 1H−31P HMBC spectrum of compound 14 (500 MHz,
CDCl3).
1
Figure 3. 2D H−19F HOESY spectrum of compound 3 (500 MHz,
D2O).
osugars within 20−30 min. This result demonstrated the broad
substrate promiscuity of Cps2L. 2-Deoxy-α-D-glucopyranosyl 1-
phosphate (2-deoxyGlc 1P) has been demonstrated to be a
poor substrate for a homologous nucleotidylyltransferase.19 The
reason for this inability to transform 2-deoxyGlc 1P was
ascribed to the absence of interactions between the C-2
hydroxyl group and the nucleotidylyltransferase active site. If
the fluorine atom is an isostere of hydrogen, the turnover of 1−
3 might be considered surprising. However, since all three
compounds were accepted by Cps2L and effectively turned
over to the corresponding nucleotides, it is more consistent to
view the fluorine atom as isoelectronic to the hydroxyl group.
Thus, these C-2 fluorinated analogues (1−3) either structurally
resemble native Glc 1P, or sufficient binding interactions with
Cps2L at C-2 position are restored through the C-2 fluorine
atom serving as a hydrogen bonding acceptor.
Previously, GDP-ManPP has been shown to be capable of
tolerating a variety of substrates structurally related to Man 1P
including monoazidodeoxy, monomethoxy, and deoxy ana-
logues of Man 1P.7,20 In order to further probe its promiscuity,
all of the above synthetic fluorinated α-D-hexopyrannosyl 1-
phosphates, including both manno-configured and nonmanno-
configured substrates, were incubated with GDP-ManPP under
previously described assay conditions.7 As shown in Table 1,
when C-2 fluorinated substrates (1−3) were incubated with
GDP-ManPP, the reaction proceeded well with the GTP
conversion ranging from 93% to 98% within 2.5 h, which is
consistent with native Man 1P, demonstrating the tolerance of
GDP-ManPP for these structural changes. However, it was
found that other fluorinated analogues, including 4, 5, 7, and 8,
were poor substrates, as they required much longer reaction
times (Figure 5) to achieve similar levels of conversions.
The poorest substrate of GDP-manPP was 7, requiring 74 h
to achieve about 78% GTP conversion. The observed slow
conversion in the case of 4, 5, 7, and 8 could be attributed to
the equatorial position, while the peak at −125.2 ppm belonged
to the axial fluorine, as determined by the observation of a cross
1
peak between the axial fluorine and H-4 in the 2D H−19F
HOESY spectrum.
Phosphorylation of monosaccharides using diphenyl chlor-
ophosphate to prepare sugar 1-phosphates 1−8 resulted in the
isolation of only α-phosphorylated anomers after flash column
chromatography (see the Supporting Information). This
1
observation was consistent with H NMR analysis of crude
product mixtures which were absent of characteristic β-anomer
resonance signals between 5.5−6.5 ppm. It was also interesting
to find that, in the course of phosphorylation of 12, in addition
to the desired product 13, a 1,6-diphosphorylated side product
14 was also isolated in 7% yield (Scheme 3), whereas a similar
diphosphate product was not observed in the other
phosphorylation reactions. Both 13 and 14 were treated with
Adam’s catalyst followed by NaOMe in MeOH to afford 5 and
6 both in 90% yield, respectively.
1
The structure of 14 was confirmed by analysis of the H
NMR, COSY, and 2D 1H−31P HMBC spectroscopy.
Specifically, correlations between P-2 and H-5, P-2 and H-6
were observed (Figure 4), in addition to correlations between
P-1 and H-1, P-1 and H-2 in the 2D 1H−31P HMBC spectrum.
Substrate Evaluation with Cps2L and GDP-manPP
using HPLC. All of the synthetic α-D-deoxyfluorohexopyr-
anosyl 1-phosphates (1−8) as well as α-D-glucopyranosyl 1-
phosphate (Glc 1P) and α-D-mannopyranosyl 1-phosphate
(Man 1P) were subject to the enzymatic reaction screening
conditions as described previously.6 Product formation was
confirmed by HPLC and ESI-MS/MS analysis (see the
Supporting Information). Enzymatic assay results are summar-
ized in Table 1. It was found that Cps2L converted all
substrates almost as effectively as it did for the physiological
substrate Glc 1P, to provide corresponding dTDP-deoxyfluor-
Scheme 3. Formation of a 1,6-Diphosphorylated Product
C
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Table 1. HPLC Assay Results of Substrates with Cps2L and GDP-manPP
a
b
conv. (%)
incubation time (h)
Rt (min)
substrate
dTTP
GTP
dTTP
GTP
dTTP
GTP
1
2
3
4
5
6
7
8
94
93
95
93
94
95
98
93
87
79

78
94
94
98
0.33
0.33
0.33
0.5
2.5
2.5
2
5.61
5.62
5.85
5.84
5.90

6.01
6.00
6.14
6.11
5.81
48
33

74
8.5
1
0.5
c



93
95
97
92
0.5
6.14
5.76
5.47
5.49
6.14
6.10
6.03
5.92
0.5
d
Glc 1P
0.33
0.5
e
Man 1P
0.5
a
Percentage conversion = [AP/(AP + AT + AD)] × 100, where AP = the NDP-sugar product peak area, AT = the remaining NTP peak area, AD = the
b
hydrolysis product NDP peak area. The provided retention times are representative, as some variability in the retention times ( 0.2 min) were
c
observed due to instrument variability. Standard retention time: dTTP, 6.79 min; dTDP, 5.94 min; GTP, 6.82 min; GDP, 5.85 min. No desired
product formation under standard conditions. Reported 90% dTTP conversion.6 Reported above 90% GTP conversion.7
d
e
HPLC or ESI-MS, indicating the inability of these two enzymes
to accept 6.
Kinetic Analysis of Fluorinated Substrates (1−8) with
Cps2L and GDP-ManPP. To better understand how these
synthetic fluorinated isosteric analogues of Glc 1P interacted
with the two enzymes Cps2L and GDP-ManPP, kinetic
analyses were performed using a continuous coupled enzyme
assay.13 As shown in Table 2, the kinetic parameters of 8 with
Cps2L were shown to be the closest to those of Glc 1P,
indicating the C-6 hydroxyl group of Glc 1P makes the smallest
contribution to binding and catalysis with Cps2L, which is
consistent with the early reported results for this class of
enzyme.19 With regard to the three C-2 fluorinated analogues
(1−3), they displayed a 10- to 77-fold decrease in kcat and 2
orders of magnitude drop in kcat/Km in comparison to Glc 1P.
The presence of an axial fluoro substituent in 2 is less
detrimental to activity than the presence of an axial hydroxyl
group (Man 1P). The difluoro substitution at C-2 in 3 results
in tighter binding than either monofluoro analogue 1 or 2. The
difluoro analogue 3 was the least reactive, consistent with a
reduction in the second ionization constant (pKa2) of the
Figure 5. GDP-ManPP-catalyzed conversion of fluorohexopyranosyl
1-phosphates to GDP-Man derivatives: (blue solid square) 4, (green
“×”) 5, (red solid circle) 7, (orange solid triangle) 8.
GDP-ManPP preferring manno- over gluco-configured sub-
strates. Nevertheless, the presence of products over extended
incubation times demonstrates the potential for the chemo-
enzymatic synthesis of unnatural sugar nucleotides. As
expected, both Cps2L and GDP-ManPP failed to turn over
the 1,6-diphosphate 6, as no product signals were detected by
Table 2. Kinetic Data for Substrates with Enzyme Cps2L and GDP-ManPP
Cps2L
GDP-ManPP
kcat (s−1
substrates
Km (μM)
kcat (s−1
)
kcat/Km (mM−1 s−1
)
Km (μM)
)
kcat/Km (mM−1 s−1
)
1
2
3
4
5
7
8
267 23
243 30
97 15
2.3 0.1
0.94 0.07
8.6 4.3
3.9 2.3
3.3 1.4
523 73
187 20
(3.2 0.2) × 10−3
0.16 0.01
(8.9 0.6) × 10−3
(6.1 0.3) × 10−2
0.9 0.3
(8 3) × 10−3
0.31 0.02
1110 193
a
300 113
130 29
297 98
(1.1 0.2) × 10−3
0.57 0.05
(3.7 1.8) × 10−3
4.4 1.8
nd
nd
nd
nd
nd
nd
0.02 0.002
4.2 0.2
(6 2) × 10−2
80 30
nd
nd
nd
nd
52
9
nd
(7 0.4) × 10−2
1.65 0.18
nd
nd
b
b
b
Glc 1P
83 10
24
280
1304 150
165 40
nd
(5.7 2.5) × 10−2
Man 1P
632 108
1100 100
1100 330
0.49 0.04
0.08
3 × 10−4
0.8 0.4
7.2 × 10−2
3 × 10−4
10 4.5
nd
b
3NH2Glc 1P
b
3N3Glc 1P
nd
nd
nd
a
b
Not determined. Data previously reported.13
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anomeric phosphate and, therefore, rendering the phosphate
group less nucleophilic. Compound 5 has similar kinetic
parameters to those of the C-2 fluorinated counterparts,
whereas its C-4 epimer (7) was a demonstrably worse substrate.
It was found that 4 was the poorest Cps2L substrate among
these fluorinated analogues, as revealed by its almost 10- and
20-fold decrease in kcat and kcat/Km, respectively. This result is
in parallel to those of 3-deoxy-3-amino-α-D-glucopyranosyl 1-
phosphate (3NH2Glc 1P) and 3-deoxy-3-azido-α-D-glucopyr-
anosyl 1-phosphate (3N3Glc 1P), which were two very poor
substrates for Cps2L.13 Indeed, substitutions of the C-3
hydroxyl group with fluoro-, amino-, or azido- functionalities
all have a negative impact on Cps2L catalysis. The Km for
compound 4 was comparable to compound 1 and 2 and
significantly better than for 3NH2Glc 1P and 3N3Glc 1P. This
suggests that the fluorine replacement at C-3 position has a
small effect on the formation of the Michaelis−Menten
complex, but significantly affects the chemical transformation
to product, which indicates that 4 may be a potential inhibitor
of Cps2L.
Kinetic parameters of the five substrates (Man 1P, Glc 1P,
and 1−3) with GDP-ManPP were determined using the same
continuous coupled assay method as previously described for
Cps2L by measuring the release of pyrophosphate (Table 2).13
For the physiological substrate Man 1P, our calculated Km and
kcat data were not identical to those reported for the same
enzyme using an end point assay, which showed its Km and kcat
of 40 μM and 6 μM−1 s−1 respectively.7 This discrepancy
presumably arises as a result of the different assay method.
Nevertheless, the comparison between different substrate
analyses using the continuous coupled assay provides clear
insight into substrate specificity trends. Substitution of fluorine
for the C-2 hydroxyl group in Man 1P did not affect the Km,
but did result in a 10-fold decrease in both kcat and kcat/Km.
Introducing one additional fluorine to the C-2 position (i.e., 3),
however, led to approximately 6-, 200-, and 1300-fold decline in
Km, kcat, and kcat/Km, respectively, with respect to Man 1P.
Alteration of the C-2 hydroxyl group orientation from axial
(Man) to equatorial (Glc) resulted in binding affinity (Km),
turnover (kcat), and catalytic efficiency (kcat/Km) decreases of
approximately 1, 2, and 3 orders of magnitude, respectively. A
similar decrease was also observed in the kcat and kcat/Km of 1,
but surprisingly, its binding affinity did not decrease as much as
expected when compared with Man 1P. These results suggest
the indispensable engagement of the C-2 axial hydroxyl group
of Man 1P in binding with GDP-ManPP, which is consistent
with previously reported results7 and the observed sluggish
reaction rate when incubated with gluco-configured fluorinated
analogues including 4, 5, 7, and 8 for which we were unable to
characterize kinetically due to low turnover (Figure 4).
Table 3. NMR Determined pKa2 Values of 1−3, Man 1P, and
Glc 1P
pKa2
pKa2
determined by
determined by
reported
pKa2 value
average
a
compds
19F NMR
31P NMR
pKa2
b
1
2
3
5.90 0.03
6.12 0.02
5.78 0.01
5.73 0.02
6.10 0.02
5.69 0.02
5.69 0.02
6.11 0.02
5.74 0.02
5.71 0.02
6.08
c
Man
1P
6.08
c
Glc 1P 6.15
6.25 0.1
6.25 0.1
a
Average pKa2 refers to the average values determined by 19F and 31P
NMR. Values determined by 19F NMR,22 pKa2 determined by
b
c
potentiometric titration.21,23
substantially with each analogue. Compound 1, with the
equatorial fluoro-substituent, had only small increase in acidity
(0.2 pH units) relative to Glc 1P (Table 3). By contrast,
compound 3, with both axial and equatorial fluoro-substituents,
had a larger increase in acidity (0.4 pH units), relative to Glc
1P. Similar magnitude differences in acidity were not observed
between Man 1P, 2, and 3. Compound 3, was anticipated to be
the most acidic, yet it was only marginally more acidic than
compound 2. Furthermore, only the equatorial fluorine
resonance changed chemical shift (Figure 6) for compound
3, while the axial fluorine resonance remained unchanged upon
titration. By contrast, both the equatorial and axial fluorine
resonances changed chemical shift for compounds 1 and 2 (see
the Supporting Information). The reason behind this
phenomenon is not clear. It does, however, demonstrate a
limitation to the concept of providing additional inductively
electron-withdrawing substituents to increase acidity.
With both kcat/Km and pKa2 of compounds Glc 1P, Man 1P,
and 1−3 in hand, linear free-energy Brønsted plots comparing
turnover efficiency of Cps2L and GDP-ManPP with average
pKa2 values of these substrates were constructed (Figure 7).
There were no strong Brønsted correlations with all five
substrates and their pKa2 values in both Cps2L or GDP-
ManPP-catalyzed reactions, indicating that hexose configura-
tion is prioritized over negative charge recognition by both
enzymes. However, grouping the substrates into physiological
and nonphysiologically configured substrates for each enzyme
resulted in moderate to good correlations (Figure 7A,B).
Enzyme Cps2L with physiologically configured substrates (Glc
1P, 1, and 3) demonstrated a positive dependence on
nucleophile acidity (slope = 3.01, R2 = 0.69). Similarly, for
GDP-ManPP with physiologically configured substrates (Man
1P, 2, and 3) there was a positive dependence on nucleophile
acidity (slope = 6.2, R2 = 0.64). These trends are consistent
with a nucleotidylyltransferase mechanism, whereby the sugar
1-phosphate directly attacks the Pα of the nucleoside
triphosphate. The nonphysiologically configured substrates for
Cps2L, (Man 1P, 2, and 3) resulted in a negative dependence
on the nucleophile acidity (slope = −1.8, R2 = 0.97), while for
GDP-ManPP, the nonphysiologically configured substrates
(Glc 1P, 1, and 3) resulted in a positive dependence on
nucleophile acidity (slope = 2.3, R2 = 0.45). Thus, the two
enzymes affect catalysis with their nonphysiologically config-
ured substrates in a different manner.
The comparison between Cps2L and GDP-ManPP with
compounds (1−3) shows very different profiles. For Cps2L,
there is no significant difference between the catalytic efficiency
of 2 and 3, whereas for GDP-ManPP, the difluoro compound
(3) is an order of magnitude less reactive.
Acidity of Substrates and Determination of Linear
Free-Energy Relationships. The pKa2 values were readily
determined from the 31P and 19F chemical shift data for all
three C-2 fluorinated compounds, and the values were
consistent between each nucleus (Table 3). The pKa2 value
measured for Glc 1P was consistent with that obtained from
literature.21 All three synthesized compounds were more acidic
than Glc 1P, however, the magnitude of the changes varied
CONCLUSION
A small library of α-deoxyfluoro hexopyranosyl 1-phosphates
(1−8) have been accessed by a general chemical synthesis
■
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Figure 6. Sensitivity of two fluorine atoms in 3 to pH as measured by 19F NMR.
Figure 7. Brønsted linear free-energy plot of Cps2L and GDP-ManPP-catalyzed formation of sugar nucleotides as a function of phosphate
nucleophile pKa2. (A) Physiological substrate configuration for Cps2L (Glc 1P (blue solid diamond), 1 (blue solid triangle), 3(blue “×”), dashed
regression line) or GDP-ManPP (Man 1P (red solid square), 2 (red solid circle), 3 (red “×”), solid regression line); (B) Nonphysiological substrate
configuration for Cps2L (Man 1P (blue solid square), 2 (blue solid circle), 3 (blue “×”), dashed regression line) or GDP-ManPP (Glc 1P (red solid
diamond), 1 (red solid triangle), 3 (red “×”) solid regression line).
featuring the fluorine introduction, anomeric deacetylation,
phosphorylation, and global deprotection. These analogues
were then evaluated as substrates for the thymidylyltransferase
Cps2L and the guanidylyltansferase GDP-ManPP. Both
enzymes turned over all of the isosteric hexopyranosyl 1-
phosphates analogues provided sufficient incubation time, with
the exception of the 1,6-diphosphate 6. Kinetic analysis of these
analogues was performed using a continuous coupled assay.
Monofluoro substitutions at either C-2, C-4, or C-6 of Glc 1P
were well tolerated by Cps2L, whereas monofluoro substitution
at C-3 was significantly more adverse. Difluoro substitution at
C-2 maintains binding affinity in comparison to Glc 1P but
decreases turnover and catalytic efficiency approximately 10-
and 100-fold with Cps2L. Both HPLC assay and kinetic analysis
with GDP-ManPP demonstrated that the axial hydroxyl group
at the C-2 position of Man 1P is critical for GDP-ManPP
catalytic activity, and in contrast to Cps2L, replacement of axial
C-2 hydroxyl group with either a mono- or difluoro substituent
results in significantly lower turnover and efficiency of GDP-
ManPP. The axial fluoro substituent in the difluoro analogue 3
did not change chemical shift upon titration. The pKa2 values of
three C-2 fluoro-substituted analogues are surprisingly in the
order of 1 < 2 ≈ 3, which were measured by both 31P and 19F
NMR titrations. No strong Brønsted correlations among all five
substrates (1−3, Glc 1P, and Man 1P) were observed for either
Cps2L or GDP-ManPP-catalyzed reactions. However, moder-
ate correlations were observed among physiological configured
substrates for each enzyme, and a positive and a negative
correlation were found among the nonphysiological configured
substrates accepted by GDP-ManPP and Cps2L, respectively.
These correlations demonstrate that the Brønsted catalysis law
applies with selected substrates for each enzyme. The described
broad substrate promiscuity of these catalysts will enable the
preparation of building blocks for chemo-enzymatic synthesis
of unnatural glycans to address the needs of glycobiology.
F
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−13.51, {lit.24 19F{1H} NMR (377 MHz, CDCl3) δ −200.9, 31P NMR
(162 MHz, CDCl3) δ −12.9}.
EXPERIMENTAL SECTION
■
General Methods and Instrumentation: Chemical Synthesis.
All chemicals and reagents were purchased from commercial sources
and were used as received, unless otherwise noted. Syntheses that
required anhydrous conditions were performed under an inert
atmosphere of dried high-purity nitrogen. HPLC grade methanol
was employed where stated. Glassware was dried overnight in an oven
set at 120 °C and assembled under a stream of inert gas. Analytical
thin-layer chromatography (TLC) was performed using silica gel 60
F254 precoated glass plates; compound spots were visualized by
ultraviolet light at 254 and/or by charring after treatment with a
vanillin stain. Flash chromatography was performed using silica gel 60
(230−400 mesh). NMR spectra were recorded on either 300 or 500
MHz spectrometers. All 1H, 13C, 19F, and 31P chemical shifts are
reported in ppm using tetramethylsilane (0.00 ppm) or the solvent
signal [CDCl3 (1H 7.26 ppm; 13C 77.16 ppm); D2O (1H 4.79 ppm)]
as the internal reference or MeOD (13C 49.50 ppm in D2O) or 85%
aq. H3PO4 (31P 0.00 ppm) or CF3COOH (19F, −76 ppm) as an
external reference. Splitting patterns are indicated as follows: br, broad;
s, singlet; d, doublet; t, triplet; at, apparent triplet; q, quartet; m,
multiplet. All coupling constants (J) are reported in Hertz (Hz). The
ratios of counterion (Et3NH) in compounds 1−8 were determined by
the integration peaks of Et3N in their corresponding 1H NMR spectra.
High-resolution mass spectra were recorded using ion trap (ESI TOF)
spectrometers. For specific schemes for the synthesis of all
intermediates and final compounds, please see the Supporting
Information.
2-Deoxy-2-fluoro-α-D-glucopyranosyl 1-Phosphate (1). Following
general procedure C, (50 mg, 79% yield) of 1 was obtained as colorless
solid after lyophilization from 16 (76 mg, 0.14 mmol). 1H NMR (500
MHz, D2O) δ5.56 (dd, J = 6.5 and 3.0 Hz, 1H), 4.31 (dd, JH-2, F
=
49.5 and J = 9.0 Hz, 1H), 3.97−3.91 (m, 1H), 3.83−3.77 (m, 2H),
3.67 (dd, J = 12 and 4.5 Hz, 1H), 3.39 (t, J = 4.5 Hz, 1H), 3.11 (q, J =
7.5 Hz, 11 H), 1.20 (t, J = 7.5 Hz, 17 H). 19F{H} NMR (471 MHz,
D2O) δ −199.59, 31P{H} NMR (202 MHz, D2O) δ 0.57. {lit.22
19F{H} NMR (471 MHz, D2O) δ −200.20}.
2-Deoxy-2-fluoro-3,4,6-tri-O-acetyl-α-D-mannopyranosyl 1-Di-
phenylphosphate (18). 2-Deoxy-2-fluoro-3,4,6-tri-O-acetyl-α-D-man-
nopyranose 17 was prepared from 3,4,6-tri-O-acetyl-α-D-glucal over
three-steps according to a reported procedure.16 Following general
procedure B, 17 (308 mg, 1.0 mmol) was phosphorylated to yield 18.
After silica gel column chromatography by eluting with n-hexane/
EtOAc (3:1), 18 was obtained (318.6 mg, 59% yield) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 7.32−7.12 (m,10H), 5.98 (dt, J = 6.9
and 2.1 Hz, 1H, H-1), 5.37−5.16 (m, 2H), 4.73 (dt, JH‑2, F = 48.6 and J
= 2.1 Hz, H-2), 4.13 (dd, J = 12.6 and 4.5 Hz, H-6a), 4.02−3.98 (m,
1H), 3.87 (dd, J = 12.6 and 2.1 Hz, H-6b), 2.01−1.88 (m, 9H), 19F
NMR (282 MHz, CDCl3) δ −203.19, 31P NMR (122 MHz, CDCl3) δ
−14.19, {lit.25 1H NMR (300 MHz, CDCl3) δ 7.35−7.19 (m, 10H),
5.99 (ddd, J = 8.5, 7.1, and 2.2 Hz, 1H, H-1), 5.37 (ddd, J = 10.3, 10.2,
and 1.2 Hz, 1H, H-4), 5.25 (ddd, JH‑3, F = 27.2, J = 10.3 and 2.2 Hz,
1H, H-3), 4.74 (ddd, JH‑2, F = 48.9, J = 2.2 and 0.5 Hz, 1H, H-2), 4.13
(dd, J = 12.5 and 4.2 Hz, 1H, H-6), 4.04−3.98 (m, 1H, H-5), 3.88 (dd,
J = 12.5 and 1.4 Hz, 1H, H-6), 4.13 (dd, J = 12.5 and 4.2 Hz, 1H, H-
6), 2.09−1.95 (m, 9H)}.
General Procedure A: Selective Anomeric Deacetylation.
NH4OAc (12 mmol) was added to the solution of peracetated sugar (3
mmol) in anhydrous DMF (3 mL) and stirred for 2 days at rt. When
the reaction was deemed complete by TLC, the remaining NH4OAc
was filtered off, and the filtrate was concentrated to dryness.
2-Deoxy-2-fluoro-α-D-mannopyranosyl 1-Phosphate (2). Follow-
ing general procedure C, 2 (34 mg, 81% yield) was obtained as a
1
colorless solid after lyophilization from 18 (54 mg, 0.1 mmol). H
General Procedure B: Phosphorylation of Anomeric
Hydroxyl Group. n-BuLi (1.2 mmol, 2.5 M in hexane) was added
dropwise to the solution of sugars (1 mmol) in anhydrous THF (5
mL) at −78 °C under a N2 atmosphere. After stirring for 15 min,
diphenyl phosphoryl chloride (1.2 equiv) was added dropwise to the
above solution, and the reaction mixture was stirred for another 0.5 h
at the same temperature. TLC analysis indicated the complete
consumption of the starting material. The reaction was gradually
warmed to rt, quenched with aq sat. NH4Cl solution (2 mL), and
extracted with EtOAc (3 × 10 mL). The combined organic layer was
washed with aq sat. NaCl solution (3 mL), dried over MgSO4, filtered,
and concentrated in vacuo to afford the crude product.
General Procedure C: Global Deprotection. PtO2 (20 mol %)
was added to a diphenyl ester phosphate solution in EtOH (3 mL).
The resulting mixture was stirred overnight at rt under a H2
atmosphere (1 atm). Reactions were monitored by TLC; completed
reaction was quenched by filtering off the catalyst and concentrating
the reaction mixture under reduced pressure. The resulting crude
product was subject to 1H NMR analysis to confirm the completion of
the reaction before proceeding to the next step. Et3N (1 mL) was
added to the crude product residue in MeOH (2 mL), and the
resulting reaction mixture was concentrated under reduced pressure.
The resulting residue was dissolved in Et3N-H2O-MeOH (1:3:7 v/v/v,
5 mL) and stirred overnight at rt. Lastly, the reaction mixture was
concentrated in vacuo.
NMR (500 MHz, D2O) δ 5.53 (t, J = 6.5 Hz, 1H, H-1), 4.80 (m, 1H),
3.96 (ddd, JH‑2, F = 30.0, J = 10.0 and 2.5 Hz, 1H), 3.87−3.85 (m, 2H),
3.75 (dd, J = 12.5 and 5.5 Hz, 1H, H-6a), 3.70−3.66 (m, 1H), 3.19−
3.14 (m, 5H), 1.26−1.23 (m, 6.6H), 19F{1H} NMR (471 MHz, D2O)
δ −204.10, 31P{1H} NMR (202 MHz, MeOD) δ −2.75, {lit.25
1
tributylammonium salt, H NMR (300 MHz, D2O) δ 5.38 (ddd, J =
8.1 and 6.0 Hz, 1H, H-1), 4.59 (ddd, JH‑2, F = 48.7, J = 2.5 and 2.4 Hz,
1H, H-2), 3.76 (ddd, JH‑3, F = 30.8, J = 9.6 and 2.5 Hz, 1H, H-2), 3.48−
3.69 (m, 4H), 2.98−2.85 (m, 12H), 1.51−1.10 (m, 24H), 0.72 (m,
18H)}.
2-Deoxy-2,2-difluoro-3,4,6-tri-O-acetyl-α-D-arabino-hexopyrano-
syl 1-Diphenylphosphate (11). 2-Deoxy-2,2-difluoro-3,4,6-tri-O-ace-
tyl-α-D-arabinopyranose 10 was synthesized according to a reported
method.16,17 Following general procedure B, 10 (284 mg, 0.87 mmol)
was phosphorylated to yield 11. After silica gel column chromatog-
raphy eluting with n-hexane/EtOAc (4:1) and a second column
chromatography purification eluting with CH2Cl2/MeOH (400:1), 11
1
was obtained (278 mg, 57% yield) as colorless oil. H NMR (500
MHz, CDCl3) δ 7.43−7.25 (m, 10H), 5.93 (dd, J = 10.0 and 5.0 Hz,
1H, H-1), 5.60 (ddd, J = 15.0, 10.0, and 5.0 Hz, 1H, H-3), 5.32−5.25
(m, 1H, H-4), 4.18 (dd, J = 10.0 and 5.0 Hz, 1H, H-6), 4.10 (ddd, J =
10.5, 4.0, and 2.0 Hz, 1H, H-5), 3.92 (dd, J = 12.5 and 2.5 Hz, 1H, H-
6), 2.16 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H). 19F{1H} NMR (471 MHz,
CDCl3) δ −120.21 (d, JF, F = 259.1 Hz), −121.11 (d, JF, F = 259.1 Hz).
31P{1H} NMR (202 MHz, CDCl3) δ −14.16. 13C{1H} NMR (126
2-Deoxy-2-fluoro-3,4,6-tri-O-acetyl-α-D-glucopyranosyl 1-Diphe-
nylphosphate (16). 2-Deoxy-2-fluoro-3,4,6-tri-O-acetyl-α-D-glucopyr-
anose 15 was prepared from 3,4,6-tri-O-acetyl-α-D-glucal over three
steps according to a reported procedure.16 Following general
procedure B, 15 (500 mg, 1.62 mmol) was phosphorylated to yield
16. After silica gel column chromatography eluting with CH2Cl2/
MeOH (80:1), 16 was obtained (525 mg, 60% yield) as colorless oil.
1H NMR (500 MHz, CDCl3) δ 7.41−7.22 (m, 10H), 6.17−6.15 (m,
1H, H-1), 5.58 (q, J = 10.0 Hz, 1H), 5.09 (t, J = 10.0 Hz, 1H), 4.69−
4.58 (m, 1H, H-2), 4.17 (dd, J = 10.0 and 5.0 Hz, 1H), 4.04−4.02 (m,
1H), 3.85−3.82 (m, 1H), 2.10−2.01 (m, 9H), 19F{1H} NMR (471
MHz, CDCl3) δ −200.45, 31P {1H} NMR (202 MHz, CDCl3) δ
MHz, CDCl3) δ 170.6, 169.7, 169.2, 150.3, 150.2, 150.17, 150.1, 130.2,
130.16, 126.19, 126.16, 120.42, 120.38, 120.23, 120.2, 114.3 (dt, JC‑2, F
= 970.3, 47.1 Hz), 94.8, 94.6, 94.3, 70.4, 68.4 (t, JC, F = 75.4 Hz), 66.8
(d, JC, F = 23.6 Hz), 61.0, 20.7, 20.6, 20.5. HRMS (ESI+): found [M +
Na]+ 581.1003. C24H25F2NaO11P requires [M + Na]+ 581.0995.
2-Deoxy-2,2-difluoro-α-D-arabino-hexopyranosyl 1-Phosphate
(3). Following general procedure C, 3 (45 mg, 94% yield) was
obtained as colorless solid after lyophilization from 11 (60 mg, 0.11
mmol). 1H NMR (500 MHz, D2O) δ 5.41 (dd, J = 8.5 and 5.5 Hz, 1H,
H-1), 4.06 (ddd, JH, F = 32.0, J = 9.5 and 4.5 Hz, 1H), 3.93 (dd, J =
10.0 and 3.0 Hz, 1H), 3.84 (dd, J = 12.5 and 2.0 Hz, 1H), 3.74 (dd, J =
15.0 and 5.0 Hz, 1H), 3.59 (t, J = 9.5 Hz, 1H), 3.15 (q, J = 5.0 Hz,
G
DOI: 10.1021/acs.joc.6b01485
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Article
9H), 1.23 (t, J = 5.0 Hz, 14H). 19F{1H} NMR (471 MHz, D2O) δ
−121.01 (d, JF, F = 249.6 Hz), −125.22 (d, JF, F = 249.6 Hz). 31P{1H}
NMR (202 MHz, D2O) δ −0.26, 13C{1H} NMR (126 MHz, D2O) δ
92.3, 91.9, 91.7 (m, C-1), 72.6, 70.3 (t, JC‑2, F = 187.5 Hz, C-2), 68.3
(d, J = 7.5 Hz), 60.2, 46.7, 8.3. HRMS (ESI−): found [M − H]−
279.0084. C6H10F2O8P requires [M − H]− 279.0087.
−13.57. 13C{1H} NMR (125 MHz, CDCl3) δ 166.3, 165.8, 150.54,
150.48, 133.9, 133.8, 133.6, 130.3, 130.2, 130.13, 130.08, 129.8, 129.3,
128.8, 128.6, 126.0, 125.9, 120.3, 120.3, 120.3, 95.4 (d, J = 6.3 Hz, C-
1), 86.8 (d, JC‑4, F = 187.5 Hz, C-4), 70.6 (t, J = 7.5 Hz, C-3), 70.1 (d, J
= 6.3 Hz, C-2), 69.9 (d, J = 10.0 Hz, C-5), 62.1 (C-6). HRMS (ESI+):
found [M + Na]+ 749.1548. C39H32FNaO11P requires [M + Na]+
749.1558.
3-Deoxy-3-fluoro-2,4,6-tri-O-acetyl-α-D-glucopyranose (20). 3-
Deoxy-3-fluoro-1,2,4,6-tetra-O-acetyl-α-D-glucopyranose 19 was pre-
pared from diacetone glucose according to a published method.26
Following general procedure A, 19 (1.0 g, 2.86 mmol) was selectively
deacetylated to yield 20. After silica gel column chromatography
eluting with n-hexane/EtOAc (1.5:1), 20 was obtained (513 mg, 58%
1
Data for 14: H NMR (500 MHz, CDCl3) δ 8.05−8.03 (m, 2H),
7.90−7.88 (m, 2H), 7.60−7.13 (m, 27H), 6.24 (dt, J = 6.0 and 3.0 Hz,
1H, H-1), 6.12 (dt, J = 14.0 and 10.0 Hz, 1H, H-3), 5.22 (dt, J = 10.0
and 3.0 Hz, 1H, H-2), 4.77 (dt, JH‑4, F = 50.0 and J = 10.0 Hz, 1H, H-
4), 4.47−4.43 (m, 1H, H-6), 4.35 (dd, J = 10.0 and 5.0 Hz, 1H, H-6),
4.19−4.17 (m, 1H, H-5), 19F{1H} NMR (471 MHz, CDCl3) δ
−198.39. 31P{1H} NMR (202 MHz, CDCl3) δ −12.20, −13.86.
13C{1H} NMR (126 MHz, CDCl3) δ 165.6, 165.5, 150.6, 150.6, 150.4,
150.31, 150.25, 133.8, 133.7, 130.2, 130.1, 130.0, 129.1, 128.6, 128.6,
128.5, 128.4, 125.9, 125.8, 120.3, 120.2, 120.2, 120.14, 120.10, 95.1 (d,
J = 5.0 Hz), 85.4 (d, JC‑4, F = 187.5 Hz), 70.3 (t, J = 8.8 Hz), 70.0 (dd,
JC, F = 25.0 and J = 7.5 Hz), 69.7 (d, JC, F = 20.0 Hz), 65.5 (d, J = 5.0
Hz). HRMS (ESI+): found [M + Na]+ 877.1617. C44H37FNaO13P2
requires [M + Na]+ 877.1586.
1
yield) as light yellow oil. H NMR (500 MHz, CDCl3) (anomeric
mixture) δ 5.35 (m, 1H), 5.12−5.07 (m, 2H), 5.01 (m, 1H), 4.96−
4.90 (m, 0.4H), 4.86−4.85 (m, 1H), 4.84−4.82 (m, 0.5H), 4.77−4.73
(m, 0.5H), 4.16−4.01 (m, 4H), 2.05−2.00 (m, 12H), {lit.27 1H NMR
(300 MHz, CDCl3) (α-anomer) δ 5.45 (br, 1H), 5.10 (m, 2H), 4.64
(m, 1H), 4.16 (m, 3H), 2.13−2.08 (m, 9H)}.
3-Deoxy-3-fluoro-2,4,6-tri-O-acetyl-α-D-glucopyranosyl 1-diphe-
nylphosphate (21). Following the general procedure B, 20 (403
mg, 1.31 mmol) was phosphorylated to yield 21. After silica gel
column chromatography eluting with n-hexane/EtOAc (2:1), 21 was
4-Deoxy-4-fluoro-α-D-glucopyranosyl 1-Phosphate (5). PtO2 (40
mg, 200 mol %) was added to the solution of 13 (66 mg, 0.091 mmol)
in MeOH (3 mL). The resulting reaction mixture was stirred overnight
at rt under H2 atmosphere (balloon). When the reaction was complete
as determined by TLC, it was quenched by filtering off the catalyst and
concentrated under reduced pressure. The resulting crude product was
1
obtained (450 mg, 64% yield) as colorless oil. H NMR (500 MHz,
CDCl3) δ 7.37−7.19 (m, 10H), 6.10 (dt, J = 7.0 and 3.5 Hz, 1H, H-1),
5.32−5.25 (m, 1H), 5.12−5.07 (m, 1H), 4.84 (dt, JH‑3, F = 50.0 and J =
5.0 Hz, 1H, H-3), 4.18 (dd, J = 15.0 and 5.0 Hz, 1H), 4.04−4.02 (m,
1H), 3.94−3.92 (m, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H).
19F{1H} NMR (471 MHz, CDCl3) δ −200.81. 31P{1H} NMR (202
MHz, CDCl3) δ −13.98. 13C NMR (125 MHz, CDCl3) δ 170.4, 169.7,
169.1, 150.3, 150.2, 130.0, 129.9, 125.8, 120.2, 120.1, 120.0, 119.96,
95.2 (dd, J = 10.0 and 6.3 Hz, C-1), 88.6 (d, JC‑3, F = 188.8 Hz, C-3),
1
subject to H NMR analysis to further confirm the success of the
hydrogenolysis reaction before proceeding to the next step. Freshly
made NaOMe in MeOH (4 mL, 1.3 mmol/mL) was added to the
above product residue in MeOH (2 mL) and stirred 5 h at rt. Dowex
× 50 (H+ form) resin was added to neutralize the reaction mixture
(until pH 5) and then filtered off the resin. The filtrate was
immediately neutralized with Et3N (until pH about 8). The reaction
mixture was then concentrated in vacuo and lyophilized to give 5 (37.8
71.1 (dd, J = 18.0 and 7.3 Hz), 69.6 (d, JC, F = 67.5 Hz), 67.4 (d, JC, F
=
18.6 Hz), 60.9, 20.5, 20.3. HRMS (ESI+): found [M + Na]+ 563.1064.
C24H26FNaO11P requires [M + Na]+ 563.1089.
3-Deoxy-3-fluoro-α-D-glucopyranosyl 1-Phosphate (4). Following
general procedure C, 4 (67 mg, 100% yield) was obtained as a
1
mg, 90% yield) as colorless solid. H NMR (300 MHz, D2O) δ 5.44
1
(dt, J = 6.6 and 3.3 Hz, 1H, H-1), 4.33 (ddd, JH‑4, F = 51.0, J = 9.9 and
9.0 Hz, 1H, H-4), 4.05−3.94 (m, 2H), 3.84−3.71 (m, 2H), 3.57−3.52
(m, 1H), 3.16 (q, J = 7.2 Hz, 22H), 1.24 (s, J = 7.2 Hz, 34H), 19F{1H}
NMR (282 MHz, D2O) δ −198.34, 31P{1H} NMR (122 MHz, D2O) δ
−0.70, {lit.22 19F{1H} NMR (254 MHz, D2O) δ −199.07, 5 as a
bis(cyclohexylammonium) salt}.
4-Deoxy-4-fluoro-α-D-glucopyranosyl 1,6-Diphosphate (6). Fol-
lowing the same procedure as described for 5, 6 (31.4 mg, 90% yield)
was obtained as a colorless solid from 14 (67.8 mg, 0.079 mmol) after
purification using a LH20 column (eluted with H2O) and lyophilized.
1H NMR (300 MHz, D2O) δ 5.43 (dt, J = 6.6 and 3.3 Hz, 1H), 4.40
(dt, JH‑4, F = 50.7 and J = 9.6 Hz, 1H), 4.16−4.14 (m, 1H), 4.05−3.94
(m, 3H), 3.55 (dt, J = 7.2 and 2.7 Hz, 1H), 3.15 (q, J = 7.5 Hz, 6.7 H),
1.23 (t, 7.5 Hz, 10H), 19F NMR (471 MHz, D2O) δ −197.97, 31P
NMR (202 MHz, D2O) δ 1.17, 0.02, 13C NMR (125 MHz, D2O) δ
94.1 (d, J = 5.4 Hz), 88.8 (d, JC‑4, F = 179 Hz, C-4), 71.2 (d, J = 5.1
Hz), 71.1 (d, J = 3.9 Hz), 68.9 (dd, JC, F = 24.1 and J = 7.1 Hz), 63.0
(d, J = 4.1 Hz), 46.7, 8.3. HRMS (ESI+): found [M − H]− 340.9833.
C6H12FO11P2 requires [M − H]− 340.9844.
4-Deoxy-4-fluoro-1,2,3,6-tetra-O-acetyl-α-D-galactopyranose
(23). Methyl 4-deoxy-4-fluoro-6-trityl-α-D-galactopyranoside 22 was
synthesized from methyl-α-D-glucopyranoside according to a reported
method.29 Following a known procedure,30 to a solution of 22 (2.00 g,
4.57 mmol) in Ac2O (60 mL) was added dropwise concentrated
H2SO4 (0.6 mL) at rt. The resulting reaction mixture was stirred
overnight at rt. NaHCO3 (3 g) and aq. sat. NaHCO3 solution (20 mL)
were added to quench the reaction at 0 °C. The reaction mixture was
then extracted with CH2Cl2 (3 × 60 mL). The combined organic layer
was washed sequentially with water (10 mL), brine (10 mL), dried
over MgSO4, concentrated, and purified by silica gel column
chromatography using 2:1 n-hexane/EtOAc as an eluent to provide
23 (1.59 g, 87% yield) as a colorless oil. 1H NMR (500 MHz, CDCl3)
anomeric mixture:δ 6.40 (d, J = 5.0 Hz, 1H, H-1β), 5.72 (d, J = 10.0
Hz, 0.18H, H-1α), 5.41 (dd, J = 10.0 and 5.0 Hz, 1H, H-2β), 5.28
colorless solid after lyophilization from 21 (80 mg, 0.15 mmol). H
NMR (300 MHz, D2O) δ 5.44 (dt, J = 6.0 and 3.0 Hz, 1H, H-1), 4.55
(dt, JH‑3, F = 57.0 and J = 9.0 Hz, 1H, H-3), 3.85−3.62 (m, 5H), 3.12
(q, J = 6.0 Hz, 11H), 1.20 (t, J = 6.0 Hz, 17H). 19F{H} NMR (282
MHz, D2O) δ −198.86, 31P{H} NMR (122 MHz, D2O) δ −0.05,
{lit.26 19F{H} NMR (376 MHz, D2O) δ −200.5, 31P{H} NMR (162
MHz, D2O) δ −0.08}.
4-Deoxy-4-fluoro-2,3,6-tri-O-benzoyl-α-D-glucopyranose (12). 4-
Deoxy-4-fluoro-1-O-acetyl-2,3,6-tri-O-benzoyl-α-D-glucopyranose
(1.76 g, 3.28 mmol) was prepared according to the reported method,28
which was selectively deacetylated to yield 25, following general
procedure A. After silica gel column chromatography by eluting with n-
hexane/EtOAc (3:1), 12 was obtained (1.26 mg, 78% yield) as light
1
yellow solid. H NMR (500 MHz, DMSO-d6) anomeric mixture δ
8.09−7.86 (m, 9H), 7.71−7.46 (m, 14H), 6.01 (dt, J = 14.5 and 9.5
Hz, 1H), 5.96−5.93 (m, 0.27H), 5.51 (m, 1H), 5.25−5.20 (m, 1H),
5.06 (dt, JH‑4, F = 51.0 and J = 9.5 Hz, 1H), 4.69−4.65 (m, 1H), 4.59−
4.52 (m, 2H), 4.41−4.38 (m, 0.27H). 19F{1H} NMR (471 MHz,
DMSO-d6) anomeric mixture: δ −196.68, −199.12. {Lit.28 19F{1H}
NMR (282 MHz, CDCl3) δ −199.50}.
4-Deoxy-4-fluoro-2,3,6-tri-O-benzoyl-α-D-glucopyranosyl 1-Di-
phenylphosphate (13) and 4-Deoxy-4-fluoro-2,3,6-tri-O-benzoyl-
α-D-glucopyranosyl 1,6-Diphenylphosphate (14). Following general
procedure B, 12 (500 mg, 1.01 mmol) was phosphorylated to yield 13
and 14. After silica gel column chromatography eluting with n-hexane/
EtOAc (3.5:1) and a second column chromatography purification
eluting with n-hexane/acetone (4:1), 13 (300 mg, 55% yield) and 14
1
(61 mg, 7% yield) were obtained as colorless oils. Data for 13: H
NMR (500 MHz, CDCl3) δ 8.11−8.06 (m, 4H), 7.93−7.91 (m, 2H),
7.61−7.13 (m, 21H), 6.41 (dt, J = 6.0 and 3.0 Hz, 1H, H-1), 6.27 (dt, J
= 13.5 and 9.5 Hz, 1H, H-3), 5.48 (dt, J = 10.5 and 3.0 Hz, 1H, H-2),
4.94 (dt, JH‑4, F = 50.5 and J = 9.5 Hz, 1H, H-4), 4.59−4.58 (m, 2H, H-
6), 4.49 (ddd, J = 10.0, 7.0, and 3.0 Hz, 1H, H-5). 19F{1H} NMR (471
MHz, CDCl3) δ −197.69. 31P{1H} NMR (202 MHz, CDCl3) δ
H
DOI: 10.1021/acs.joc.6b01485
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Article
(ddd, J = 26.5, 10.5, and 5.0 Hz, 1H, H-3β), 5.03 (ddd, JH‑3α, F= 26.5, J
= 10.5 and 5.0 Hz, 0.18H, H-3α), 4.98 (dd, JH‑4β, F= 50.0 and J = 2.5
Hz, H-4β), 4.90 (dd, JH‑4α, F= 50.0 and J = 2.5 Hz, 0.18H, H-4α),
4.32−4.20 (m, 3.45H), 3.97 (dt, JH‑5α, F = 27.0 and J = 6.5 Hz, 0.18H,
H-5α), 2.17−2.04 (m, 15H). 19F{1H} NMR (471 MHz, CDCl3)
anomeric mixture: δ −217.03, −219.20. 13C{1H} NMR (126 MHz,
CDCl3) δ 170.5, 169.8, 168.9, 92.1, 89.8, 87.3, 86.6, 85.8, 85.1, 72.1,
72.0, 71.6, 71.4, 69.3, 69.2, 68.1, 68.0, 67.9, 66.3, 61.5, 61.5, 61.4, 61.4,
21.0, 20.9, 20.8, 20.8, 20.6. HRMS (ESI+): found [M + Na]+ 373.0900.
C14H19FNaO9 requires [M + Na]+ 373.0905.
Hz), 20.7, 20.7. HRMS (ESI+): found [M + Na]+ 331.0793.
C12H17FNaO8 requires [M + Na]+ 331.0800.
6-Deoxy-6-fluoro-2,3,4-tri-O-acetyl-α-D-glucopyranosyl 1-Diphe-
nylphosphate (28). Following general procedure B, 27 (343 mg, 1.11
mmol) was phosphorylated to yield 28. After silica gel column
chromatography eluting with n-Hexane/EtOAc (2.5:1), 28 was
obtained (344 mg, 57% yield) as a colorless oil. 1H NMR (500
MHz, CDCl3) δ 7.41−7.23 (m, 10H), 6.11 (dd, J = 7.0 and 3.5 Hz,
1H, H-1), 5.57 (t, J = 10.0 Hz, 1H), 5.18 (t, J = 10.0 Hz, 1H), 5.02 (dt,
J = 10.0 and 3.0 Hz, 1H), 4.38 (ddd, J = 14.5, 11.0, and 3.5 Hz, 1H),
4.29 (ddd, J = 14.5, 11.0, and 3.5 Hz, 1H), 4.10−4.02 (m, 1H), 2.07 (s,
3H), 2.05 (s, 3H), 1.85 (s, 3H). 19F{H} NMR (471 MHz, CDCl3) δ
−233.93. 31P{H} NMR (202 MHz, CDCl3) δ −13.80. 13C NMR (125
MHz, CDCl3) δ 170.0, 169.8, 169.3, 150.3, 150.2, 123.0, 129.9, 125.8,
4-Deoxy-4-fluoro-2,3,6-tri-O-acetyl-α-D-galactopyranose (24).
Following general procedure A, 23 (1.47 g, 4.2 mmol) was
deacetylated to yield 24. After silica gel column chromatography
eluting with n-hexane/EtOAc (1.3:1), 24 was obtained (779 mg, 52%
yield) as a colorless oil. 1H NMR (500 MHz, CDCl3) δ 5.54 (d, J = 3.5
Hz, 1H), 5.41−5.33 (m, 1H), 5.23 (dd, J = 10.5 and 3.5 Hz, 1H), 5.16
(dd, J = 10.0 and 8.5 Hz, 0.41H), 5.02 (ddd, JH, F = 25.0, J = 10.5, and
2.5 Hz, 0.41H), 5.00 (dd, JH‑4, F = 50.5 and J = 2.5 Hz, 1H), 4.89 (dd,
JH‑4, F = 50.0 and J = 2.5 Hz, 0.43H), 4.72−4.71 (m, 0.40H) 4.40−4.22
(m, 4H), 3.89 (dt, JH, F = 26.5 and J = 6.5 Hz, 0.54H), 2.15−2.04 (m,
14H). 19F{1H} NMR (471 MHz, CDCl3) anomeric mixture: δ
−216.84, −219.43, 13C{1H} NMR (126 MHz, CDCl3) δ 171.1, 170.9,
120.4, 120.4, 120.1, 120.1, 95.0 (d, J = 5.0 Hz, C-1), 80.4 (d, JC‑6, F
=
175.0 Hz, C-6), 70.4 (d, JC‑5, F = 18.8 Hz), 69. Seven (d, J = 17.5 Hz),
69.2, 67.2 (d, J = 2.5 Hz), 20.6, 20.5, 20.2. HRMS (ESI+): found [M +
Na]+ 563.1097. C24H26FNaO11P requires [M + Na]+ 563.1089.
6-Deoxy-6-fluoro-α-D-glucopyranosyl 1-Phosphate (8). Following
general procedure C, 8 (53 mg, 83% yield) was obtained as a colorless
solid after lyophilization from 28 (73 mg, 0.14 mmol). 1H NMR (500
MHz, D2O) δ 5.43 (dd, J = 7.0 and 3.5 Hz, 1H), 4.77−4.57 (m, 2H),
3.95 (dd, JH‑6, F = 30.5 and 10 Hz, 1H), 3.74 (t, J = 9.5 Hz, 1H), 3.51−
3.46 (m, 2H), 3.15 (q, J = 7.5 Hz, 12H), 1.23 (t, J = 7.5 Hz, 18H).
19F{H} NMR (471 MHz, D2O) δ −236.29. 31P{H} NMR (202 MHz,
D2O) δ 0.34. {Lit.22 19F{H} NMR (471 MHz, D2O) δ −237.48}
General Methods and Instrumentation: Enzymatic Analysis.
Cps2L, purine nucleoside phosphorylase (PNP), and GDP-ManPP
were overexpressed, isolated, and quantified as previously described.7
Microbial xanthine oxidase (XO) and recombinant inorganic
pyrophosphatase (IPP) were obtained from a supplier. IPP stock
solutions (0.1 EU/μL) were prepared in double distilled water; thawed
aliquots were kept in a fridge and were used for up to 1 month after
thawing. XO stock solutions (120 U/mL) were prepared in Tris·HCl
(pH 7.5, 25 mM) and stored at −30 °C; aliquots were used
immediately after thawing. Kinetic reactions were performed in 96-well
plates and were monitored using a UV spectrophotometer with
SoftMax Pro version 4.8. Nonlinear regression analysis was performed
using GraFit 5.0.4 Erithacus Software.
170.6, 170.5, 95.9, 90.7, 87.2 (d, JC‑4, F = 183.8 Hz), 86.2 (d, JC‑4, F
=
185.0 Hz), 77.5, 77.2, 77.0, 71.1, 71.0, 68.3, 68.1 (d, J = 17.5 Hz), 66.7
(d, JC, F = 18.8 Hz), 62.0 (d, J = 6.3 Hz), 61.8 (d, J = 5.0 Hz), 20.9,
20.8 HRMS (ESI+): found [M + Na]+ 331.0793. C12H17FNaO8
requires [M + Na]+ 331.0800.
4-Deoxy-4-fluoro-2,3,6-tri-O-acetyl-α-D-galactopyranosyl 1-Di-
phenylphosphate (25). Following general procedure B, 24 (779 mg,
2.53 mmol) was phosphorylated to yield 25. After silica gel column
chromatography by eluting with n-hexane/EtOAc (2.2:1) and further
purification eluting with n-hexane/acetone (3:1), 25 was obtained
1
(675 mg, 49% yield) as colorless oil. H NMR (500 MHz, CDCl3) δ
7.39−7.21 (m, 10H), 6.16 (dd, J = 6.0 and 2.5 Hz, 1H, H-1), 5.39−
5.30 (m, 2H), 4.98 (dd, JH‑4, F = 50.0 and 1.5 Hz, 1H, H-3), 4.30−4.16
(m, 3H), 2.15 (s, 3H), 1.98 (s, 3H), 1.89 (s, 3H). 19F{1H} NMR (471
MHz, CDCl3) δ −218.68. 31P{1H} NMR (202 MHz, CDCl3) δ
−13.67, 13C{1H} NMR (126 MHz, CDCl3) δ 170.3, 170.2, 169.9,
150.4, 150.4, 130.1, 123.0, 125.8, 120.4, 120.4, 120.2, 120.1, 95.7 (d, J
= 5.0 Hz, C-1), 86.5 (d, JC‑4, F = 185.0 Hz, C-3), 69.2 (d, JC, F = 18.8
Hz), 67.4 (d, J = 17.5 Hz), 66.9 (d, J = 6.3 Hz), 61.3 (d, J = 6.3 Hz),
20.8, 20.6, 20.4. HRMS (ESI+): found [M + Na]+ 563.1094.
C24H26FNaO11P requires [M + Na]+ 563.1089.
Analytical HPLC Assays. HPLC analysis of enzymatic reactions
was performed as previously described.6 A linear gradient from 90/10
A/B to 40/60 A/B over 8.0 min followed by a plateau at 40/60 A/B
over 2.0 min at 1.0 mL/min−1 was used, where A was an aqueous
buffer containing 12 mM Bu4NBr, 10 mM KH2PO4 (pH 4.0), and 5%
HPLC grade CH3CN and B is HPLC grade CH3CN.
4-Deoxy-4-fluoro-α-D-galactopyranosyl 1-Phosphate (7). Follow-
ing general procedure C, 7 (83 mg, 100% yield) was obtained as
1
colorless solid after lyophilization from 25 (96 mg, 0.18 mmol). H
IPP-PNP-XO Coupled Kinetic Assays with Cps2L. Stock
solutions containing Tris·HCl (pH 7.5, 25 mM), Glc 1P, or
fluorinated analogues (25−250 μM), dTTP (1 mM), MgCl2 (5.7
mM), inosine (1 mM), IPP (1.7 EU/mL), PNP (1 μM), and XO (1.5
EU/mL) were allowed to preincubate at room temperature for 5 min,
in order to consume contaminating Pi present in the solution. An
appropriate concentration of Cps2L was then added to initiate the
reaction. The initial reaction velocity was monitored spectrophoto-
metrically over 10 min at a wavelength of 290 nm. Rates were linear.
Observed initial kinetic rates were halved to account for the 2 equiv of
Pi derived from each PPi unit produced in the Cps2L reaction. Rates
were converted from absorbance units (mAu) to concentration units
(μM) using a phosphate standard curve. The standard curve was
generated using identical conditions to those described for kinetic
assays, except with variable Pi (10−100 mM NaH2PO4·H2O) instead
of sugar phosphate. λ290 values were taken once the reaction had
reached completion (after approximately 7 min) and plotted against
phosphate concentration producing data that were fit by linear
regression, providing a slope that was used as the conversion factor
between mAu and μM units.
NMR (500 MHz, D2O) δ 5.46 (dd, J = 7.0 and 3.5 Hz, 1H), 4.83 (dd,
JH‑4, F = 51.0 and J = 2.0 Hz, 1H), 4.15 (dt, JH, F = 31.5 and 6.0 Hz,
1H), 3.96 (dd, JH, F = 30.0 and 2.5 Hz, 1H), 3.76−3.74 (m, 1H), 3.71−
3.70 (m, 2H), 3.11 (q, J = 7.5 Hz, 12H), 1.20 (t, J = 7.5 Hz, 19H).
19F{1H} NMR (471 MHz, D2O) δ −219.69. 31P{1H} NMR (202
MHz, D2O) δ 0.59, 13C{1H} NMR (125 MHz, D2O) δ 94.8 (d, J = 5.0
Hz, C-1), 90.4 (d, JC‑4, F = 176.3 Hz, C-3), 70.3 (d, J = 17.5 Hz), 68.4
(d, J = 7.5 Hz), 68.0 (d, J = 8.8 Hz), 60.0 (d, J = 5.0 Hz), 46.7, 8.3.
{Lit.31 31P{1H} NMR (121 MHz, D2O) δ −0.22}.
6-Deoxy-6-fluoro-2,3,4-tri-O-acetyl-α-D-glucopyranose (27). Ac-
cording to a reported method,32,30 following general procedure A, 6-
deoxy-6-fluoro-1,2,3,4-tetra-O-acetyl-α-D-glucopyranose 26 (1.47 g, 4.2
mmol) was selectively deacetylated to yield 27. After silica gel column
chromatography eluting with n-hexane/EtOAc (1.3:1), 27 was
obtained (779 mg, 52% yield) as a colorless oil. 1H NMR (500
MHz, CDCl3) anomeric mixture: δ 5.54−5.51 (m, 1H), 5.45 (m,1H),
5.26−5.22 (m, 0.2H), 5.06−5.01 (m, 1H), 4.90−4.88 (m, 0.2H), 4.85
(dd, J = 10.0 and 3.5 Hz, 1H), 4.79 (t, J = 2.5 Hz, 0.2H), 4.54−4.33
(m, 4H), 4.28−4.20 (m, 1H), 3.80−3.73 (m, 0.3H), 2.06−2.00 (m,
11H). 19F{H} NMR (471 MHz, CDCl3) anomeric mixture: δ
−232.15, −232.70. 13C{1H} NMR (125 MHz, CDCl3) δ 170.6,
IPP-PNP-XO Coupled Kinetic Assays with GDP-ManPP. The
GDP-ManPP kinetic assay was performed exactly the same as
described in Cps2L, with stock solutions containing Tris·HCl (pH
7.5, 25 mM), Man 1P, or fluorinated analogues (25−250 μM), GTP
170.5, 167.0, 95.4, 90.0, 81.5 (d, JC‑6, F = 173.8 Hz), 81.3 (d, JC‑6, F
=
175.0 Hz), 72.9 (d, JC‑5, F = 62.5 Hz), 72.7 (d, JC‑4, F = 20.0 Hz), 71.2,
70.0, 68.3 (d, J = 6.3 Hz), 68.2 (d, J = 7.5 Hz), 68.0 (d, JC, F = 18.8
I
DOI: 10.1021/acs.joc.6b01485
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.6b01485.
■
S
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1H NMR, 31P{1H} NMR, 19F{1H} NMR and 13C{1H}
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AUTHOR INFORMATION
Corresponding Author
*E-mail: David.Jakeman@dal.ca.
■
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Present Address
§Department of Natural Sciences, Lawrence Technological
University, 21000 West Ten Mile Rd, Southfield, MI, 48075,
United States
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
(24) Salvado,
Boutureira, O. Org. Lett. 2015, 17, 2836.
́ ́
M.; Amgarten, B.; Castillon, S.; Bernardes, G. J. L.;
The Canadian Institutes for Health Research, the Natural
Sciences and Engineering Research Council of Canada, and
Glyconet are thanked for funding. We thank Todd L. Lowary
(Alberta Glycomics Centre and Department of Chemistry, the
University of Alberta, Edmonton) for the gift of the GDP-
ManPP plasmid.7
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DOI: 10.1021/acs.joc.6b01485
J. Org. Chem. XXXX, XXX, XXX−XXX