Studies on the substrate specificity of a GDP-mannose pyrophosphorylase from Salmonella enterica

Article abstract of DOI:10.3762/bjoc.8.136

A series of methoxy and deoxy derivatives of mannopyranose-1-phosphate (Manp-1P) were chemically synthesized, and their ability to be converted into the corresponding guanosine diphosphate mannopyranose (GDP-Manp) analogues by a pyrophosphorylase (GDP-ManPP) from Salmonella enterica was studied. Evaluation of methoxy analogues demonstrated that GDP-ManPP is intolerant of bulky substituents at the C-2, C-3, and C-4 positions, in turn suggesting that these positions are buried inside the enzyme active site. Additionally, both the 6-methoxy and 6-deoxy Manp-1P derivatives are good or moderate substrates for GDP-ManPP, thus indicating that the C-6 hydroxy group of the Manp-1P substrate is not required for binding to the enzyme. When taken into consideration with other previously published work, it appears that this enzyme has potential utility for the chemoenzymatic synthesis of GDP-Manp analogues, which are useful probes for studying enzymes that employ this sugar nucleotide as a substrate.

Full text of DOI:10.3762/bjoc.8.136

Studies on the substrate specificity of a
GDP-mannose pyrophosphorylase from
Salmonella enterica
Lu Zou, Ruixiang Blake Zheng and Todd L. Lowary*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 1219–1226.
Alberta Glycomics Centre and Department of Chemistry, The
University of Alberta, Edmonton, AB T6G 2G2, Canada
doi:10.3762/bjoc.8.136
Received: 29 May 2012
Accepted: 29 June 2012
Published: 01 August 2012
Email:
Todd L. Lowary* - tlowary@ualberta.ca
* Corresponding author
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
Keywords:
chemoenzymatic synthesis; kinetics; methylation; pyrophosphorylase;
sugar nucleotide
Guest Editor: T. K. Lindhorst
© 2012 Zou et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
A series of methoxy and deoxy derivatives of mannopyranose-1-phosphate (Manp-1P) were chemically synthesized, and their
ability to be converted into the corresponding guanosine diphosphate mannopyranose (GDP-Manp) analogues by a
pyrophosphorylase (GDP-ManPP) from Salmonella enterica was studied. Evaluation of methoxy analogues demonstrated that
GDP-ManPP is intolerant of bulky substituents at the C-2, C-3, and C-4 positions, in turn suggesting that these positions are buried
inside the enzyme active site. Additionally, both the 6-methoxy and 6-deoxy Manp-1P derivatives are good or moderate substrates
for GDP-ManPP, thus indicating that the C-6 hydroxy group of the Manp-1P substrate is not required for binding to the enzyme.
When taken into consideration with other previously published work, it appears that this enzyme has potential utility for the chemo-
enzymatic synthesis of GDP-Manp analogues, which are useful probes for studying enzymes that employ this sugar nucleotide as a
substrate.
Introduction
Modified sugar nucleotide analogues are valuable probes to preparation of a sugar 1-phosphate derivative followed by its
study glycosyltransferases and other enzymes that use these coupling to an activated nucleoside monophosphate to form the
activated glycosylating agents as substrates [1-5]. The syn- key pyrophosphate moiety (Figure 1A) [7]. In general, the yield
thesis of natural and non-natural sugar nucleotides is therefore a of this process is low, and the purification of the product can be
topic of continuing interest [6]. The classical method for tedious; hence, the development of new methods to prepare
chemically synthesizing sugar nucleotides involves the sugar nucleotides remains an area of active research [6].
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Figure 1: (A) Conventional approach for the chemical synthesis of sugar nucleotides from sugar 1-phosphates; (B) enzymatic conversion of sugar
1-phosphates into sugar nucleotides.
Although improved chemical methods have been developed from Salmonella enterica [23], which had previously
[8-13], another attractive strategy is to employ a chemo- been shown to have a relaxed specificity for the sugar 1-phos-
enzymatic approach, in which a synthetic sugar 1-phosphate phate moiety [24,25]. In particular, it has been shown
derivative is converted to the sugar nucleotide by a that the enzyme will accept mannopyranosyl 1-phosphate
pyrophosphorylase (Figure 1B) [14,15]. This approach is (Manp-1P) derivatives deoxygenated at C-2, C-3 and C-4 (1–3,
increasingly used for the synthesis of sugar nucleotides, but a Figure 2), as well as a substrate lacking the hydroxymethyl
limitation is that the specificity of the pyrophosphorylase must group at C-5 (4) [24]. A series monoazido derivatives (5–8)
be sufficiently broad to recognize the synthetic sugar 1-phos- were also shown to be substrates [25]. To further probe the
phate derivative. However, some of these enzymes have been potential of this enzyme for the chemoenzymatic synthesis of
demonstrated to have broad specificity, or can be engineered to modified GDP-Manp derivatives, we describe here the
have broad specificity, with regard to both the sugar 1-phos- preparation of all four singly methylated Manp-1P analogues
phate and nucleotide substrates [16-19].
9–12, as well as the 6-deoxy-Manp-1P derivative 13, and an
initial evaluation of their ability to serve as a substrate for
As part of a larger study on the specificity of mannosyltrans- S. enterica GDP-ManPP.
ferases involved in mycobacterial glycan biosynthesis [20-22],
Results and Discussion
we had the need for a panel of singly deoxygenated
and methylated guanosine diphosphosphate mannopyranose Synthesis of 2-methoxy derivative 9
(GDP-Man) derivatives. In developing a strategy for The synthesis of sugar 1-phosphate 9 containing a methyl group
the synthesis of these compounds, we chose to take advantage at O-2 commenced from 3-O-benzyl-4,6-O-benzylidene-α-D-
of a GDP-mannose pyrophosphorylase (GDP-ManPP) mannopyranoside 14 [26] as illustrated in Scheme 1. Methyl-
Figure 2: Structures of the Manp-1P derivatives (1–8) previously shown [24,25] to be substrates for S. enterica GDP-ManPP and analogues 9–13
studied in this paper.
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Beilstein J. Org. Chem. 2012, 8, 1219–1226.
Scheme 1: Reagents and conditions: (a) CH3I, NaH, DMF, 80%; (b) Ac2O–HOAc–H2SO4, 35:15:1, 81%; (c) EtSH, BF3·OEt2, CH2Cl2, 65%; (d)
HO-P(O)(OBn)2, NIS, AgOTf, CH2Cl2, 84%; (e) (i) H2, Pd(OH)2–C, toluene, Et3N, pyridine; (ii) CH3OH–H2O–Et3N, 5:2:1, 92%.
ation of the alcohol under standard conditions proceeded in Synthesis of 3-methoxy derivative 10
80% yield affording 15. The benzylidene protecting group was The preparation of the 3-methoxy Manp-1P analogue 10 fol-
cleaved, together with the methyl glycoside, by acetolysis lowed a route similar to that used for the synthesis of 9
giving the tetra-O-acetylated compound 16 in 81% yield. This (Scheme 2). Methyl 2-O-benzyl-4,6-O-benzylidene-α-D-
glycosyl acetate was converted to the corresponding thioglyco- mannopyranoside (19) [26] was first methylated giving 20 and
side (17), which was, in turn, coupled with dibenzyl phosphate then converted into glycosyl acetate 21 in 49% yield over the
under NIS–AgOTf activation conditions, providing compound two steps. Subsequent thioglycosylation provided a 52% yield
18 in 55% yield over two steps from 16. The anomeric stereo- of 22. The protected dibenzyl phosphate 23 was next formed by
chemistry in 18 was confirmed by the magnitude of the 1JC1,H1, the NIS–AgOTf promoted glycosylation of dibenzyl phosphate
which was 177.9 Hz, consistent with α-stereochemistry as with 22, which afforded the desired compound, 23, in 75%
described earlier by Timmons and Jakeman for rhamnopyra- yield. Hydrogenolysis of the benzyl groups and deacylation led
nosyl phosphates [27]. In the other phosphorylation reactions to the formation, in 67% yield, of Manp-1P derivative 10.
reported in this paper, the anomeric stereochemistry was deter-
mined in an analogous manner. Compound 18 was then depro- Synthesis of 4-methoxy derivative 11
tected in two steps, namely catalytic hydrogenolysis and then, As illustrated in Scheme 3, the synthesis of the 4-methoxy
without further purification, treatment with a mixture of Manp-1P analogue 11 started by treatment of methyl α-D-
CH3OH–H2O–Et3N 5:2:1 to remove the acetyl groups. This mannopyranoside (24) with trityl chloride in pyridine. The
series of reactions gave 2-methoxy Manp-1P analogue 9 in 92% product, 25, was then converted to the isopropylidene acetal 26
overall yield from 18.
in 65% overall yield from 24. The hydroxy group in 26 was
Scheme 2: Reagents and conditions: (a) CH3I, NaH, DMF, 76%; (b) Ac2O–HOAc–H2SO4, 35:15:1, 65%; (c) EtSH, BF3·OEt2, CH2Cl2, 52%; (d)
HO-P(O)(OBn)2, NIS, AgOTf, CH2Cl2, 75%; (e) (i) H2, Pd(OH)2–C, toluene, Et3N, pyridine; (ii) CH3OH–H2O–Et3N, 5:2:1, 67%.
Scheme 3: Reagents and conditions: (a) TrCl, DMAP, pyridine, 85%; (b) DMP, p-TsOH, 76%; (c) CH3I, NaH, DMF, 91%; (d) Ac2O–HOAc–H2SO4,
35:15:1, 55%; (e) EtSH, BF3·OEt2, CH2Cl2, 70%; (f) HO-P(O)(OBn)2, NIS, AgOTf, CH2Cl2, 80%; (g) (i) H2, Pd(OH)2–C, toluene, Et3N, pyridine; (ii)
CH3OH–H2O–Et3N, 5:2:1, 70%.
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methylated under standard conditions (CH3I, NaH) to give the The second route to 12 began with methyl 2,3,4-tri-O-benzoyl-
4-methoxy analogue 27 in 91% yield. Acetolysis of 27 to the α-D-mannopyranoside (37) [29] and is illustrated in Scheme 5.
corresponding glycosyl acetate 28, followed by reaction with Methylation of the free OH, even under mildly basic
ethanethiol and BF3·OEt2, yielded thioglycoside 29, in a modest conditions (e.g., Ag2O–CaSO4), led to significant amounts of
39% yield from 27 over two steps. This compound was then acyl group migration, and the desired product was obtained
converted to 11, in 56% yield, as outlined above, by successive in only 52% yield. Nevertheless, enough material was
phosphorylation and deprotection.
produced to move forward. Acetolysis conditions were used to
replace the methyl group at the anomeric center in 38 with an
acetyl group, resulting in a 96% yield of 39. Thioglycosylation,
Synthesis of 6-methoxy derivative 12
Two routes, differing in the choice of protecting groups, were followed by coupling of the resulting thioglycoside donor 40
explored to produce the 6-methoxy Manp-1P derivative 12 (obtained in 75% yield) with dibenzyl phosphate, gave phos-
(Scheme 4 and Scheme 5). In one route, the C-2, C-3, and C-4 phate 41 in a yield of 67% over the two steps. The 6-methoxy
hydroxy groups of the mannose residues were protected with Manp-1P analogue 12 was obtained by catalytic hydrogeno-
benzyl ethers and in the other they were protected with benzoyl lysis of the benzyl ethers followed by treatment with
esters. The overall yields of these two methods were 30% and CH3OH–H2O–Et3N 5:2:1 providing 12 in 85% yield over two
17%, respectively. In the first method (Scheme 4), the initial steps.
step was the conversion, in 78% yield, of the fully acetylated
thioglycoside 31 [28] into silyl ether 32 by treatment with Synthesis of 6-deoxy derivative 13
sodium methoxide and then tert-butyldiphenylchlorosilane in The synthesis of the 6-deoxy Manp-1P analogue 13 used an
DMF. Benzylation of 32 using benzyl bromide and sodium intermediate (37) prepared in the course of the synthesis of the
hydride gave 33 in 84% yield. The TBDPS group was then 6-methoxy analogue (Scheme 6). First, the hydroxy group of 37
cleaved and replaced with a methyl group to give the 6-methoxy was converted to the corresponding iodide in 65% yield, by
compound 35 in 72% yield over two steps. The protected using triphenylphospine and iodine. The product, 42, was then
dibenzyl phosphate 36 was formed in 70% yield by phosphory- subjected to acetolysis and catalytic hydrogenation, which gave
lation as described for the synthesis of 9–11. Catalytic 6-deoxy glycosyl acetate derivative 43 in 72% yield. The subse-
hydrogenolysis in the presence of NaHCO3 was used to cleave quent thioglycosylation, phosphorylation and deprotection steps
all the benzyl groups, which gave the 6-methoxy Manp-1P proceeded, as outlined above, to give the 6-deoxy Manp-1P 13
derivative 12 in 91% yield.
in 43% yield over four steps.
Scheme 4: Reagents and conditions: (a) (i) NaOCH3, CH3OH; (ii) TBDPSCl, imidazole, DMF, 78%; (b) BnBr, NaH, TBAI, 84%; (c) TBAF, THF, 83%;
(d) CH3I, NaH, DMF, 87%; (e) HO-P(O)(OBn)2, NIS, AgOTf, CH2Cl2, 70%; (f) H2, Pd(OH)2–C, NaHCO3, CH3OH, 91%.
Scheme 5: Reagents and conditions: (a) Ag2O, CaSO4, CH3I, 52%; (b) Ac2O–HOAc–H2SO4, 70:30:1, 96%; (c) EtSH, BF3·OEt2, CH2Cl2, 75%; (d)
HO-P(O)(OBn)2, NIS, AgOTf, CH2Cl2, 89%; (e) (i) H2, Pd(OH)2–C, toluene, Et3N, pyridine; (ii) CH3OH–H2O–Et3N, 5:2:1, 85%.
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Scheme 6: Reagents and conditions: (a) PPh3, imidazole, I2, 65%; (b) (i) Ac2O–HOAc–H2SO4, 35:15:1; (ii) Pd–C, H2, Et3N, EtOAc, 72%; (c) EtSH,
BF3·OEt2, CH2Cl2, 89%, α/β 4:1; (d) HO-P(O)(OBn)2, NIS, AgOTf, CH2Cl2, 67%; (e) (i) H2, Pd(OH)2–C, toluene, Et3N, pyridine;
(ii) CH3OH–H2O–Et3N, 5:2:1, 72%.
Evaluation of 9–13 as substrates for GDP-
observed (Figure S2 in Supporting Information File 1).
Man pyrophosphorylase
However, in the case of 11 and 9, a peak corresponding to GDP,
With 9–13 in hand, each was evaluated as a substrate for the resulting from hydrolysis of the GDP-sugar, was also observed,
S. enterica GDP-ManPP. Before doing that, the recombinant and, in the case of 9, a much smaller amount of the GDP-Manp
protein was produced and the natural substrate for the enzyme, analogue was produced. To confirm the identity of each GDP-
Manp-1P (46, Figure 3), was evaluated by incubation with the Manp analogue, the product peaks were isolated and analysed
enzyme and GTP. The reaction was monitored by HPLC by electrospray ionization mass spectrometry. For the reactions
(Figure S1 in Supporting Information File 1) and stopped when involving 9–12 a signal at m/z ≈ 618 was observed, as would be
the complete consumption of GTP was observed. Simultaneous expected for the [M − H]− ion of the methylated GDP-Man
with the loss of the GTP was the appearance of the signal for a derivatives (48–51, Figure 4). Similarly, for the reaction with
new product, which was found to elute at a retention time 13, a signal at m/z ≈ 588 was observed in the mass spectrum
similar to that for an authentic sample of GDP-Manp. The consistent with the 6-deoxy GDP-Man derivative 52.
product was isolated, and analysis by high-resolution electro-
Relative activity of Manp-1P analogues with
spray ionization mass spectrometry revealed an ion with m/z =
604.0691, which corresponds to the [M − H]− ion (calcd m/z = GDP-ManPP
604.0699) of GDP-Manp. After it was established that all five Manp-1P analogues could
serve as substrates for GDP-ManPP, the relative activity with
Having established that the enzyme GDP-ManPP was active, each was assessed. This was done by using an established
we carried out the same incubations for 9–13, and in all cases colorimetric activity assay, which relies on the detection of the
the corresponding GDP-Manp analogue peaks could be pyrophosphate (PPi, Figure 3) formed as a byproduct of the
Figure 3: Reaction catalyzed by GDP-ManPP.
Figure 4: Structure of modified GDP-Man derivatives 48–52 produced from 9–13.
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Beilstein J. Org. Chem. 2012, 8, 1219–1226.
Kinetic analysis of Manp-1P analogues with
enzymatic reaction [30]. As illustrated in Figure 5, all five syn-
thetic derivatives 9–13 were active as substrates, although at GDP-ManPP
lower levels than the parent compound 46. The 6-methoxy (12) To better understand how these 9–13 interact with GDP-
and 6-deoxy (13) analogues, demonstrated moderate to good ManPP, kinetic analyses were performed by using the colori-
relative activities, while the 2-methoxy (9), 3-methoxy (10), and metric activity assay mentioned above (Table 1). Both the
4-methoxy (11) compounds showed much lower activities. For 6-methoxy Manp-1P (12) and 6-deoxy Manp-1P (13) deriva-
example, the 2-methoxy, 3-methoxy, and 4-methoxy analogues tives bind relatively well to the enzyme, showing only a two- or
displayed a 6-, 14-, and 17-fold decrease relative to 46, respect- three-fold increase in KM, respectively, compared to the native
ively. Because both the 6-deoxy and 6-methoxy analogues (12 Manp-1P donor 46. The turnover rate of 6-methoxy analogue 12
and 13) showed relatively good activity it is likely that this is, however, much lower than the 6-deoxy counterpart (13) and
hydroxy group does not interact significantly with the enzyme. the natural substrate 46, as substantiated by a greater than
On the other hand, because the 2-methoxy, 3-methoxy, and 10-fold decrease in kcat. Taken together, these results suggest
4-methoxy compounds all showed a large decrease in activity, it that the C-6 hydroxy group does not engage in any critical
is likely that these positions are bound tightly in the active site hydrogen-bonding interactions and that a bulky substituent
of the enzyme. A graphical summary of the substrate speci- interferes with the rate of substrate turnover. The binding of the
ficity for GDP-ManPP is shown in Figure 6.
2-methoxy (9) and 4-methoxy (11) analogues is very weak
compared to the native substrate, as seen by the greater then
100-fold increase in KM; consequently, the turnover rates are
also low. The binding between 3-methoxy analogue 10 is
moderate, with only a five-fold increase in the observed KM, but
it shows an extremely low turnover rate. These results all
suggest that GDP-ManPP is not tolerant of bulky substituents at
the C-2, C-3, and C-4 positions, which is consistent with the
results obtained from their relative activity. It should be noted
that these trends are consistent with earlier studies of the
enzyme using deoxygenated or azido analogues [24,25].
Table 1: KM, kcat, and kcat/KM of GDP-ManPP kinetic studies.
compound
KM (μM)
kcat
kcat/KM
(min−1)
(min−1·μM−1)
Figure 5: Comparison of the relative activity of synthetic Manp-1P
analogues 9–13 for GDP-ManPP, with that of the parent compound 46.
Error bars represent the standard deviation of duplicate reactions.
9 (2-methoxy 4000 ± 1100 70 ± 11
analogue)
(2 ± 1) × 10−2
10 (3-methoxy 200 ± 72
5.2 ± 0.7 (2.6 ± 0.1) × 10−2
analogue)
11 (4-methoxy 3400 ± 870 31 ± 4.7 (9 ± 5) × 10−3
analogue)
12 (6-methoxy 120 ± 18
27 ± 1
0.23 ± 0.06
analogue)
13 (6-deoxy
analogue)
70 ± 13
40 ± 6
300 ± 13 4 ± 1
360 ± 16 9 ± 3
46 (Man-1P)
Conclusion
In this paper, we report the synthesis of a panel of methoxy and
deoxy analogues of Manp-1P. Five analogues, 9–13, in which
one of the hydroxy groups was methylated or deoxygenated
were generated by chemical synthesis, and the ability of these
compounds to be converted to the corresponding GDP-Manp
analogues by GDP-ManPP from S. enterica was evaluated. All
the derivatives acted as substrates for GDP-ManPP, but with
uniformly lower activity than the natural substrate Man-1P. The
Figure 6: Summary of the substrate specificity of GDP-ManPP. Data
from previous studies on the enzyme are also included as indicated
[24,25].
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7. Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G.
results suggest that the C-2, C-3, and C-4 hydroxy groups of
Manp-1P are bound within the active site of GDP-ManPP and
the addition of a methyl group at these positions is tolerated
very poorly. Conversely, the addition of a methyl group to, or
deoxygenation of, O-6 had a much smaller effect, suggesting
that this position protrudes from the active site, or is accommo-
dated in a pocket that can tolerate either of these modifications.
These results are consistent with earlier studies of this enzyme,
which were focused on deoxygenated and azido derivatives
[24,25]. Considered together, our studies and those published
previously suggest that this enzyme can be used to access deoxy
and azido derivatives of GDP-Man on a preparative scale, but
that the synthesis of analogues containing more sterically
demanding groups is likely to be only possible when the modifi-
cations are present on O-6.
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