Magnesium pyrophosphates in enzyme mimics of nucleotide synthases and kinases and in their prebiotic chemistry

Article abstract of DOI:10.1073/pnas.1516239112

Derivatives of ribosyl pyrophosphate have been synthesized, and examined with magnesium salts in the coupling of the ribose unit to various nucleophiles, including pyrazole and 2-chloroimidazole. Only with the magnesium salt present did they generate the ribosyl cation by binding to the leaving group and then couple the ribose derivative with nucleophiles. The role of magnesium salts in phosphorylation of methanol by ATP was also examined. Here a remarkable effect was seen: phosphorylation by ATP was slowed with low concentrations of Mg²⁺ but accelerated by higher concentrations. Related effects were also seen in the effect of Mg²⁺ on phosphorylation by ADP. The likely mechanisms explain these effects.

Full text of DOI:10.1073/pnas.1516239112

Magnesium pyrophosphates in enzyme mimics of
nucleotide synthases and kinases and in their
prebiotic chemistry
Purushothaman Gopinath, Vijayakumar Ramalingam, and Ronald Breslow1
Department of Chemistry, Columbia University, New York, NY 10027
Contributed by Ronald Breslow, August 15, 2015 (sent for review July 27, 2015); reviewed by Jacqueline K. Barton and Samuel H. Gellman)
Derivatives of ribosyl pyrophosphate have been synthesized, and
examined with magnesium salts in the coupling of the ribose unit
to various nucleophiles, including pyrazole and 2-chloroimidazole.
Only with the magnesium salt present did they generate the
ribosyl cation by binding to the leaving group and then couple the
ribose derivative with nucleophiles. The role of magnesium salts in
phosphorylation of methanol by ATP was also examined. Here a
remarkable effect was seen: phosphorylation by ATP was slowed
with low concentrations of Mg2+ but accelerated by higher
concentrations. Related effects were also seen in the effect of
Mg2+ on phosphorylation by ADP. The likely mechanisms explain
these effects.
The prebiotic synthesis of nucleotides can involve the direct
reaction of nucleobases with ribose, but in quite low yield (8).
Another proposed route involves an arabinose amino-oxazoline
intermediate that later inverts to ribonucleotide (9). Because the
phosphate esters and anhydrides dominate the living world (10),
and currently nucleotides are biosynthetically synthesized from
phosphoribosyl pyrophosphate, we believe that nucleotides could
have formed prebiotically by the reaction of ribose 1-pyrophos-
phate (RPP) or possibly the 5-phosphate derivative with the cor-
responding nucleobases in the presence of Mg2+ ions. D-Ribose
can be available in the prebiotic world, and there are many
credible ways in which phosphorylations could occur, with three
phosphorylations leading to 5-phosphoribosyl pyrophosphate.
We have examined such a nucleotide synthesis catalyzed by Mg2+
.
ATP ADP phosphoribosyl pyrophosphate nucleosides
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This is similar to the current biosynthetic pathways, but without
the enzymes.
nzyme mimics, lacking protein catalysts, can afford insight
into biochemical mechanisms because frequently the essential
We synthesized ribose with protecting groups on the 2-, 3-,
and 5-hydroxyl groups, so only the hydroxyl group on position 1
was available, and then synthesized protected ribose 1-phos-
phate (RP) and protected RPP as model compounds. To our
knowledge, there were no previous reports of the synthesis of
RPP derivatives nonenzymatically. For simple analogs we syn-
thesized 2,3,5-tribenzoylribose 1-pyrophosphate and 2,3,5-Tris-
phenylpropylribose 1-pyrophosphate.
E
chemical paths are similar, with the enzyme protein furnishing
greater velocity and selectivity. A good example from our own
work includes the mechanisms used when thiamine pyrophos-
phate is the coenzyme; thiamine itself can perform the essential
catalysis, but more slowly and less selectively than with the en-
zyme (1, 2). As another example, our mimics of ribonuclease use
the imidazole groups that are biochemically part of the enzyme
itself to catalyze cleavage of RNA (3, 4). We here describe
mimics of the enzymes that attach base groups to a ribose unit.
We also describe mimics of kinases that transfer a phosphate
group from ATP.
Synthesis of 2,3,5-Tribenzoylribose 1-Phosphate 5
1-O-Acetyl-2,3,5-Tris-O-benzoyl-β-D-ribofuranose 1, on treatment
with HBr-AcOH (11), afforded the corresponding ribosylbromide
2, which on reaction with dibenzyl phosphate 3 formed 2,3,5-
trisbenzoylribose dibenzylphosphate 4 (12). Compound 4 on re-
duction with Pd-C afforded the title compound 5 (Fig. 2).
The synthesis of nucleotides in modern biochemistry involves
5-phosphoribosyl pyrophosphate, formed by direct transfer of
the pyrophosphoryl group from ATP. This attaches to a nitrog-
enous base to generate a new carbon nitrogen bond by the dis-
placement of the pyrophosphate group with assistance by Mg2+
(Fig. 1). For purine nucleotides the nucleophile is an ammonia
molecule that is generated by cleaving an amide. This generates
ribosylamine-5-phosphate, and the rest of the base is constructed
around this amino group. For the synthesis of pyrimidine nu-
cleotides the nucleophile is orotic acid, which later undergoes
decarboxylation and other steps to generate the modern nucle-
otides. It seemed likely that binding to magnesium ions activates
the departure of the pyrophosphate leaving group. Simple binding
of pyrophosphate ions to Mg2+ is well studied (5).
Similarly a pyrophosphate group (ADP) is the leaving group in
ATP phosphorylations by ATP in kinase reactions (6). Some
studies have been reported on mimics for this process, and it
seemed likely that in this case also the departure of the ADP
leaving group would be assisted by binding to magnesium ions
(7). However, we now find that a more complex mechanism is
involved in the mimic, and thus likely in the prebiotic world and
in the modern enzymatic process itself. Of course the exact
structures now used in biochemistry may almost certainly have
evolved from much simpler structures on prebiotic earth, but the
same overall general chemistry is likely.
Significance
Nucleotides are biosynthesized from a ribose pyrophosphate
species, with enzymes and magnesium ions as catalysts. Mag-
nesium ions alone can perform this process with synthetic ri-
bose pyrophosphates, by binding to the pyrophosphate groups
and linking cyclic nitrogen bases to the ribose. In a kinase
mimic ATP transfers the terminal phosphate to receptors cat-
alyzed by magnesium ions, binding to pyrophosphate groups,
but magnesium ion at low concentrations inhibits the process,
and only at higher concentrations is it a catalyst. This affords
great insight into the mechanism of ATP phosphorylation, a
fundamental biological process. Our enzyme mimics could well
operate on prebiotic earth before enzymes were present. This
work strengthens the picture of how important components of
life could have been formed prebiotically.
Author contributions: P.G., V.R., and R.B. designed research; P.G. performed research;
P.G., V.R., and R.B. analyzed data; and P.G., V.R., and R.B. wrote the paper.
Reviewers: J.K.B., California Institute of Technology; and S.H.G., University of Wisconsin-
Madison.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. Email: rb33@columbia.edu.
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Fig. 1. Simple pathway for nucleotide synthesis. In modern biochemistry
the immediately bound nucleophile is further modified.
Fig. 3. Synthesis of 2,3,5-tribenzoylribose 1-pyrophosphate 9.
Fig. 4. Reaction of RP and RPP with methanol.
Fig. 2. Synthesis of 2,3,5-tribenzoylribose 1-phosphate 5.
(1 equiv, 0.1 M) at 60 °C for 40 h afforded 48% yield of the ribose-
imidazole derivative 21. Only a trace of compound 21 was
obtained in the absence of magnesium iodide (Table 2). We found
that the relatively weak base pyrazole and 2-chloroimidazole
added to the ribosyl unit but the more basic imidazole was not
incorporated, nor was simple ammonia. Apparently the basic
imidazole or ammonia binds to the magnesium ions, blocking
catalysis, and with an excess of magnesium the imidazole or
ammonia itself is bound and thus not a nucleophile.
Dibenzylphosphonate 6, on reaction with N-chlorosuccinimide,
afforded dibenzylphosphoryl chloride 7 (13), which on reaction
with previously synthesized ribosyl phosphate 5 afforded the cor-
responding ribosyl dibenzylpyrophosphate derivative 8. Compound
8 on reduction with Pd-C and triethylamine afforded the title
compound 9 (Fig. 3).
We examined the reaction of RP 5 and RPP 9 with and without
magnesium ions in methanol (used as solvent, Fig. 4). We found
that 1-methylriboside 10 was formed from ribosyl pyrophosphate
with MgCl2 in almost 50% yield in 48 h at room temperature
with no significant increase after longer times. No methylriboside
was obtained in the absence of magnesium ions (Table 1, entry 3
and 4). Under our conditions no methylriboside was obtained with
RP both with and without magnesium ions (Table 1, entry 1 and 2).
We also examined the formation of a C–N bond of a cyclic
nitrogen compound to the ribosyl pyrophosphate, but because the
benzoyl protecting groups reacted in the model study we synthe-
sized 2,3,5-Tris-phenylpropyl substituted RPP. Accordingly
D-ribose 11, on treatment with methanol and catalytic conc. sulfuric
acid furnished the methyl ribofuranoside 12 (14), which on treat-
ment with sodium hydride and phenylpropyl bromide 13 furnished
2,3,5-trisprotected methylribofuranoside, 14 (15). Compound 14 on
treatment with HBr-AcOH (16) gave the corresponding ribosyl-
bromide 15, which on reaction with dibenzyl phosphate 3 gave
ribose-dibenzylphosphate 16. Compound 16 on reduction with
Pd–C gave protected RP 17, which on reaction with dibenzyl-
phosphoryl chloride 7 yielded the corresponding ribosyl diben-
zylpyrophosphate derivative 18. Compound 18 on reduction with
Pd–C produced the title compound 19 (Fig. 5).
We found that compound 19 (1 equiv, 0.05 M), MgI2 (2 equiv,
0.1 M) in methanol at room temperature for 48 h afforded 78%
yield of methyl ribofuranoside 14 in the presence of MgI2 as
additive, whereas there was no reaction in the absence of MgI2
(used also with other nucleophiles as more soluble than MgCl2).
We examined the reaction with two amine nucleophiles, pyr-
azole and 2-chloroimidazole, in dimethylformamide, the best
solvent for all of the reactants (Fig. 6). The reaction of pyrazole
(2 equiv, 0.2 M) and magnesium iodide (4 equiv, 0.4 M) with ribosyl
pyrophosphate 19 (1 equiv, 0.1 M) at 60 °C for 40 h gave a 30%
yield of ribose-pyrazole derivative 20. The reaction gave no
desired product in the absence of magnesium iodide. Similarly,
the reaction of 2-chloroimidazole (8 equiv, 0.8 M) and magne-
sium iodide (4 equiv, 0.4 M) with ribosyl pyrophosphate 19
We also examined the possible role of Mg2+ in phosphoryla-
tions by ATP (adenosine triphosphate) 21 (Fig. 7). We performed
the reaction in 50% vol/vol of water/methanol (where MgCl2 is
quite soluble) and compared the yields of methyl phosphate with
various concentrations of added MgCl2 under the same condi-
tions. The results are listed in Table 3; the yield as a function of
time is shown in Table 4 and plotted in Fig. 8. The findings were
at first sight remarkable. Under our standard conditions the
presence of 10 equivalents of MgCl2 (0.25 M) led to a decrease in
the product formed, so Mg2+ was an inhibitor. With higher con-
centrations of Mg2+ there was an increase in product formed, so
here the Mg2+ furnished positive catalysis. These results are not
unexpected, considering the likely roles the magnesium ions play.
In ATP 22 phosphate #3, the terminal phosphate, is trans-
ferred as phosphate #1 and #2 are pyrophosphate leaving
groups, and this should be accelerated by Mg2+ binding of py-
rophosphates. However, the first Mg2+ would surely preferen-
tially bind to the pyrophosphate unit of phosphates #2 and #3.
Phosphate #3 will bind more strongly because it is a dianion,
the others are monoanions, and the result is that transfer of
phosphate #3 is inhibited because it will require breaking up
the magnesium-linked #2–#3 pyrophosphate group. Thus, the
Table 1. Reaction of RP and RPP in methanol with and without
MgCl2
Entry
Starting material
Additive
Yield, %
1
2
3
4
(OBz)3 RP (5)
(OBz)3 RP (5)
(OBz)3 RPP (9)
(OBz)3 RPP (9)
No additive
MgCl2
No additive
MgCl2
No reaction
Trace
No reaction
50
Compound 5 or 9 (1 equiv, 0.05 M) and MgCl2 (2 equiv, 0.1 M) for entry
2 and 4.
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Fig. 5. Synthesis of phenylpropyl substituted RPP 19 and its reaction with methanol using Mg2+ catalyst.
phosphate transfer is slowed. However, with increasing Mg2+
concentrations a second magnesium can then bind in the #1–#2
position, furnishing the driving force anticipated when a magne-
sium-bound pyrophosphate is the leaving group. At some point
this can apparently be enough to overcome the inhibition effect
from the binding at the first #2–#3 pyrophosphate site.
To add to this picture, we examined phosphorylation of meth-
anol by ADP, under the same conditions. As Table 5 shows, ADP
is a phosphorylating agent even without added Mg2+, and has this
role to some extent in modern enzyme-catalyzed biology, but it is
poorer than ATP (Table 3). Addition of Mg2+ has a significantly
smaller effect, and here there is also a decrease in yield at low
concentration when the chelate is present and then an increase
at higher concentrations. Its affinity for the first magnesium ion
should be less than for ATP, and at high concentrations a second
Mg2+ is bound. It does not have a pyrophosphate leaving group,
but breaking up a chelated magnesium pyrophosphate with added
magnesium ion could activate phosphorylation simply by par-
tially neutralizing the negative charges that make the phosphorus
less reactive.
Table 2. Reaction of RPP 19 with pyrazole and with 2-
chloroimidazole nucleophiles in the presence and absence of
magnesium iodide
Entry
Nucleophiles
Additive
Product
Yield, %
1*
2*
3†
4†
Pyrazole
Pyrazole
2-Chloroimidazole
2-Chloroimidazole
No additive
MgI2
No additive
MgI2
20
20
21
21
—
30
Trace
48
*Compound 19 (0.1 M), MgI2 (0.4 M), pyrazole (0.2 M), DMF (solvent).
†Compound 19 (0.1 M), MgI2 (0.4 M), 2-chloro imidazole (0.8 M), DMF
(solvent).
Fig. 6. Reaction of RPP 19 with pyrazole and with 2-chloroimidazole.
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Fig. 7. Structure of ATP with the phosphate residues numbered.
Fig. 8. Rate of methanol phosphorylation at 1.0 M MgCl2 concentration
with ATP (0.025 M) at 60 °C.
Table 3. Phosphorylation of methanol with ATP
Entry Phosphorylating agent Concentration of Mg2+, M Yield, %*
1
2
3
4
ATP.2Na
ATP.2Na
ATP.2Na
ATP.2Na
No MgCl2
0.25
1.0
36
23
32
1
1
1
Table 5. Phosphorylation of methanol with ADP
Entry Phosphorylating agent Concentration of Mg2+, M Yield, %*
2.0
50 0.5
1
2
3
4
ADP.2Na
ADP.2Na
ADP.2Na
ADP.2Na
No MgCl2
0.25
1.0
10.5 0.5
8.5 0.5
12.3 0.2
16.8 0.2
ATP (0.025 M), MgCl2, H2O:MeOH (1:1), 60 °C, 48 h.
*NMR yield.
2.0
ADP (0.025 M), MgCl2, H2O:MeOH (1:1), 60 °C, 48 h.
*NMR yield.
Table 4. Rate of methanol phosphorylation with ATP
Entry
Time, h
Yield, %*
1
2
3
4
5
12
24
36
48
60
8
How could enzymes play roles in these processes to accelerate
them? In both the systems binding of the substrates would of
course help, but with ATP we propose an extra effect. The
reason that the first magnesium ion is an inhibitor is that it should
spontaneously go to the 2–3 pyrophosphate group, but an enzyme
could bind it selectively to favor the magnesium 1–2 pyrophos-
phate structure. There the binding of the pyrophosphate leaving
group would be fully effective, as in our ribosyl pyrophosphate
systems. Thus, we propose that our results could be clues to
prebiotic chemistry where enzymes did not yet exist, and they
give insights into the modern enzyme systems for which they are
simple mimics.
15.6
23.7
30
36.2
ATP (0.025 M), MgCl2 (1 M), H2O:MeOH (1:1), 60 °C.
*NMR yield.
Conclusion
Here we see the other aspect of magnesium cation binding, ac-
tivating a phosphate group by simple charge neutralization. This
effect could also play a role with ATP, but we think binding the
1–2 pyrophosphate leaving group, as we invoked for ATP, is the
more likely explanation there.
ACKNOWLEDGMENTS. This work was supported by a grant from NASA.
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