The p: K a of Br?nsted acids controls their reactivity with diazo compounds

Article abstract of DOI:10.1039/c6cc03561b

We study the O-alkylation of phosphate groups by alkyl diazo compounds in a range of small molecules and biopolymers. We show that the relatively high pK_a of phosphate in comparison to the other naturally occurring Br?nsted acids can be exploited to control alkylation selectivity. We provide a simple protocol for chemical modification of some of the most important instances of phosphates in natural compounds including in small molecule metabolites, nucleic acids, and peptides.

Full text of DOI:10.1039/c6cc03561b

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The pK of Brønsted acids controls their reactivity
a
with diazo compounds†
Cite this:DOI: 10.1039/c6cc03561b
Na Fei, Basilius Sauter and Dennis Gillingham*
Received 28th April 2016,
Accepted 11th May 2016
DOI: 10.1039/c6cc03561b
www.rsc.org/chemcomm
We study the O-alkylation of phosphate groups by alkyl diazo
compounds in a range of small molecules and biopolymers. We
a
show that the relatively high pK of phosphate in comparison to
the other naturally occurring Brønsted acids can be exploited to
control alkylation selectivity. We provide a simple protocol for
chemical modification of some of the most important instances
of phosphates in natural compounds including in small molecule
metabolites, nucleic acids, and peptides.
The phosphate group and its esters are ubiquitous in Nature,
serving critical roles in nucleic acids, cell membranes, metabolism, Fig. 1 Diazo stability in water is controlled by its pKa (Path A); Brønsted
1
,2
signal transduction, and numerous secondary metabolites.
acids capable of protonating diazo compounds lead to O-alkylation (Path B).
Protein phosphorylation is the most important post-translation
modification of proteins, and yet, despite more than sixty years
3
of study, most instances remain poorly understood. It is a
We demonstrate here that pH control of the reaction medium
difficult problem to study: signal transduction networks are not only reduces acid-mediated hydrolysis of diazo compounds,
complicated and locating phosphorylation sites in proteins in time judged by diazo compound half-life, (see the ESI† page 5), but
4
–7
and space is analytically challenging.
Chemical methods also allows toggling of O-alkylation selectivity amongst Brønsted
to build or modify phosphate esters have been essential in acids (see Path B in Fig. 1).
studying their biology but the two main approaches have
Despite the importance of the phosphate esters and their known
limitations: phosphorus(III) chemistry is water-sensitive (although reactivity with diazo compounds no head-to-head comparison of
8
recent efforts are addressing this issue) and phosphate ester their reactivity with that of carboxylates or a detailed look at the
activation leads to side-reactions. For example, one challenge reactivity difference between phosphate mono- and diesters has been
9
,10
in selective inositol phosphate syntheses
is their proclivity attempted. There are a handful of cases of phosphate ester alkylation
1
1
15–21
for phosphate ester migration once they are activated. Diazo by diazo compounds.
based esterification would offer a solution to such problems were first modified by phenyldiazomethane in 1955, and caged
For example, nucleotide monophosphates
22
since it operates by O-alkylation rather than acyl transfer. Diazo inositol and nucleoside monophosphates are often made through
2
3,24
esterification is used often in organic synthesis and has reactions of phosphates with diazo compounds.
For example, a
25
recently been explored by the Raines lab as a labelling reaction diazo-substituted coumarin has been employed as a photocaging
in chemical biology. They have developed techniques for the reagent of DNA and RNA before delivery into zebrafish. While these
mild aqueous generation of diazo compounds and shown reports highlight the potential of phosphate alkylation, they did not
how these can be used for the reversible labelling of carboxylate explore the reaction scope, mechanism, or compare of reactions with
1
2
1
3,14
groups in proteins.
different Brønsted acids. In our own work on metal-catalysed DNA
26–28
and RNA alkylation by diazo compounds,
we sometimes found
low levels of phosphate alkylation in product streams. In this paper
we take a close look at phosphate monoester alkylation and compare
it to carboxylate and phosphate diester alkylation under neutral
pH aqueous conditions.
Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056, Basel,
Switzerland. E-mail: dennis.gillingham@unibas.ch
†
Electronic supplementary information (ESI) available: Detailed protocols,
assays, and characterization. See DOI: 10.1039/c6cc03561b
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For reactions of diazo compounds in water the pK
protonated form (the diazonium ion) is critical. If the conjugate of pK s for diazo compounds. Raines selected the pK of the
a a
a
of its the Brønsted acid. Unfortunately there has been no tabulation
13
13
base (1 in Fig. 1) is too basic it will be protonated quickly by water parent alkyl group as an expedient for diazo acidity in their
and decompose unselectively through hydrolysis (Path A in discussion of ester alkylation and found that diazo compounds
29
Fig. 1). When used to perform transformations in water diazo derived from fluorene or a-aryl amides delivered optimal alkylation
compounds akin to diazomethane must therefore be used in yields. The respective carbon acids of these have pK values in the
enormous excess since hydrolysis will always predominate (for mid-twenties. We elected to choose a different series of structures
a
2
9
example diazomethane has a pK
diazo compounds will not deprotonate the Brønsted acids and ethyl a-diazoacetate (not shown) were tested in a standard
required for Path B in Fig. 1) to initiate reaction – compounds reaction with CMP), but our results are qualitatively in agree-
a
of 10). If too weakly basic based on practical considerations (diazo compounds 1c, 1d,
(
in this class include diazomalonates and donor–acceptor sub- ment with those of the Raines lab. In our case the most effective
stituted diazo esters. These stable diazo compounds have long diazo compound was 1d, whose parent carbon acid has a pK of
a
31
lifetimes in water and hence can be used to perform aqueous approximately 25 in water. The more basic compound 1c also
metal-carbene reactions. Their weak basicity, however, renders gave product, but it was prone to rapid degradation; the resonance
them of little value in Brønsted acid alkylation. Quantifying the stabilized compound ethyl a-diazoacetate gave undetectable
middle ground between these two extremes is essential for levels of alkylation product.
controlling selectivity in diazo-type esterifications.
The reaction is general. We selected a range of biologically
The first step in a productive diazo-type O-alkylation is proto- relevant monophosphates and looked at their reaction with
30
nation by the Brønsted acid (see Path B in Fig. 1). It should then either diazo compounds 1c or 1d (see Table 1). The yields are
be possible to exploit pK differences between acids to control not high (21–74% depending on the substrate) but the approach’s
a
O-alkylation selectivity. To test this hypothesis we examined product simplicity and conciseness should override the practical dis-
ratios of CMP versus benzoic acid alkylation in aqueous buffer at advantage of moderate yields. In general we consider 1d the
different pHs using commercially available trimethylsilyldiazo- more valuable reagent because the ortho-nitrobenzyl (o-NO -Bn)
2
methane (1b in Fig. 2) as the diazo compound. Indeed at low pH adduct is photolabile, offering opportunities for photo-uncaging
benzoic acid alkylation is preferred, but as the pH is raised phosphate applications (see the ESI† pages 15 & 41 for photo-uncaging
32
alkylation begins to dominate (see Fig. 2). This is consistent examples with compounds 6g and 14). 1d has the additional
with benzoic acid being largely benzoate at pHs higher than six, advantage that it is more stable than most alkyl diazo compounds
while a large fraction of the phosphate remains protonated.
since the ortho-nitro group provides resonance stabilization of
Although 1b was a convenient, highly basic, diazo compound for the diazo anion.
preliminary hypothesis testing, its extreme reactivity (compara-
Modifications of phosphates within large biopolymers would be
ble to diazomethane) would limit its practical application. The a valuable application of diazo alkylation. While this reaction has
ideal diazo compound would have pK
b
that matches the pK
a
of previously been employed in the alkylation of nucleoside mono-
phosphates and nucleic acid oligomers, past work suffered from a
16,18
lack of selectivity, giving both mono- and diester alkylation.
The
exception is the work of the Friedman lab, who have shown that
terminal phosphates are strongly preferred over phosphate diesters
2
0,21
in oligonucleotide alkylation.
We propose that pH buffering in
is the decisive difference for
the vicinity of the Brønsted acid’s pK
a
selectivity. This is supported by the poor reactivity of the phosphate
diesters shown in the bottom two entries of Table 1 where less
than 5% conversion is observed – identical conditions lead to
clean alkylation of phosphate monoesters. There are cases where
phosphate diester alkylation has been used in practice, but an
1
9,25
enormous excess is always employed.
d we begin to see phosphate diester alkylation (data not
With 30 equivalents of
1
shown), but under these forcing conditions substantial amounts
of sulfonate alkylation of the MES buffer salt were also observed
(
see the ESI† page 9). The dramatic reactivity difference between
0
mono- and diesters augured well for a selective 5 -phosphate
alkylation in larger oligonucleotides.
Oligonucleotides bearing terminal phosphates are alkylated
selectively (Fig. 3). A simple hexanucleotide (8, see trace a in
0
Fig. 3) bearing a 5 -phosphate was treated with the diazo
compound 1d in varying amounts at pH 7 (see traces b–g).
Optimal conversion was observed at 20 equivalents of 1d with
further increases leading to little improvement. The reactions
a
Fig. 2 Competition experiments at varying pH support the role of pK in
reactions of Brønsted acids with trimethylsilyl diazomethane.
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Table 1 Alkylations of diverse naturally occurring monophosphates
0
Fig. 3 5 -Phosphates in DNA are alkylated cleanly by diazo compound 1d.
Conditions: the appropriate amount of 1d stock solution was added to a
1
00 mM solution of the oligonucleotide and aliquots were analysed by
HPLC at 30 minutes.
water (final pH 4.3) and treated directly with diazo, alkylation of
glutamic acid 531 (E531) is the major product (see entry 2).
Consistent with the pK s controlling selectivity, performing the
a
same reaction in pH 7 MOPS buffer switches the alkylation
a
Conditions: Diazo compound as a stock solution was added to a 50 mM
solution of the phosphate in 250 mM MES buffer. Reactions were
stopped when diazo compound was completely consumed as determined
by the disappearance of the orange colour.
Table 2 Selective phosphate alkylation in the C-terminal tail of proto-
oncogenic protein c-Src as examined by HPLC-MS analysis
are clean with most by-products derived from side-reactions of
the diazo compound with itself or water (see trace j for control
sample where 8 is not added to reaction) and not with the
backbone or the bases of the oligonucleotide (see trace h for
control experiment of 1d’s reaction with dephosphorylated 8).
Protein phosphorylation is a widespread post-translational
3
3
modification that regulates numerous functions. The ability
to selectively modify phosphates in a native protein would be a
Equiv. Major
Y527:
34
a
b
c
powerful tool for studying phosphorylation networks. Alkylation
to create products 6f and 6g in Table 1 already suggested that a
selective phosphate alkylation in a peptide or protein might be
feasible. Although whole proteins with a precisely defined phos-
phorylation state are difficult to obtain, many phosphorylated
model peptides are commercially available. Phosphorylation of
Y527 in the C-terminal region of c-Src causes this domain to fold
Entry Substrate pH 1d product % conv. (E524 + E531)
1
2
3
4
5
6
7
11
10
10
10
10
10
10
7
2
14
13
12
12
12
12
12
o5
n.a.
r1 : 9
d
4.3 20
40
7
7
7
7
7
2
3
5
10
15
43 (35) 13 : 1
52 (42) 15 : 1
68 (44) 11 : 1
75 (47) 11 : 1
95 (40) 11 : 1
35
over on its SH2 domain, holding the protein in an inactive state.
a
Run in 5 mM MOPS buffer at pH 7, pH 4.3 experiment is run in
b
A 13-mer phosphorylated peptide of the c-Src C-terminal domain unbuffered water and the pH is measured after the reaction. First
number is conversion of starting peptide; number in parenthesis refers
(residues 521–533, see 10 in Table 2 header) has therefore become
c
to amount of alkylated phosphate. Ratios are determined by HPLC
an important model peptide in studying Src signalling. If the
d
integration. A small side-product obscures phosphate alkylation hence
commercially available TFA salt of 10 is dissolved in unbuffered we can only report a lower limit (see the ESI, Fig. S9–S13 for details).
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preference to Y527 (cf. entries 2 & 3). Although there are three
carboxylates available for alkylation (C-terminus, E524 and
E531), phosphate alkylation is the major product along with a
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1
1
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1
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2
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