Identification of stable S-adenosylmethionine (SAM) analogues derivatised with bioorthogonal tags: Effect of ligands on the affinity of the E. coli methionine repressor, MetJ, for its operator DNA

Article abstract of DOI:10.1039/b816495a

The efficient synthesis of a range of stable SAM mimetics, and their ability to promote the binding of the E. coli methionine repressor (MetJ) to its operator DNA, is described.

Full text of DOI:10.1039/b816495a

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Identification of stable S-adenosylmethionine (SAM) analogues derivatised
with bioorthogonal tags: effect of ligands on the affinity of the E. coli
methionine repressor, MetJ, for its operator DNA†
Catherine Joce,a,b Jamie Caryl,b Peter G. Stockley,b Stuart Warrinera,b and Adam Nelson*a,b
Received 22nd September 2008, Accepted 8th December 2008
First published as an Advance Article on the web 19th December 2008
DOI: 10.1039/b816495a
The efficient synthesis of a range of stable SAM mimetics, and
their ability to promote the binding of the E. coli methionine
repressor (MetJ) to its operator DNA, is described.
gave the key amine intermediate 4.13 Our approach avoided
the synthesis of the 5¢-tosyl derivative of 3, which was prone
to cyclisation.14 Furthermore, the approach avoided the use of
methylamine in a high pressure reaction vessel, which was required
for conversion of the tosylate into 4.15 The amine 4 was reacted
with the mesylate 6, prepared from the corresponding aspartic
acid derivative,16 to give 5. Deprotection gave aza-SAM, 2, in 44%
yield (Scheme 1).
S-Adenosylmethionine or SAM, 1 (also known as AdoMet), is
a co-factor with a wide range of biological functions, including
methylation, regulation of gene expression in the bacterial me-
thionine biosynthetic pathway, catalysis of some biological radical
reactions1,2 and control of a number of riboswitch elements.3 In
total, SAM, 1, has been identified as a ligand for fifteen protein
superfamilies.1 The use of SAM, 1, to study these processes has
been hampered by its inherent instability.4,5 Thus, stable SAM
analogues, such as aza-SAM, 2, which is a titratable, isostructural
mimic, have been developed6 and shown to be useful tools in the
analysis of SAM-dependent processes.7,8
Here, we describe an improved synthesis of aza-SAM, 2, which
we have adapted to the preparation of a range of novel derivatives.
We have also determined the ability of the analogues to promote
the binding of the E. coli methionine repressor, the MetJ dimer,
to its operator DNA. MetJ represses at least nine genes in the
methionine biosynthetic pathway9 following activation by the non-
cooperative binding of two molecules of SAM, 1, which increases
the affinity of MetJ for its operator DNA by a factor of 100–
1000.2,8,10
Scheme 1 Improved synthesis of aza-SAM. Conditions: (a) MeNNsH,
DEAD, PPh3, THF, 18 h, 60%; (b) PhSH, Cs2CO3, MeCN, 18 h, 83%;
(c) 6, DIPEA, TBAI, MeCN, 65 ◦C, 13 h, 75%; (d) 2 M NaOH, MeCN,
3 h; (e) 5 M HCl, 1 h, 44% over two steps.
Fukuyama–Mitsunobu reaction11 of the protected adenosine
derivative12 3 with MeNNsH as the nucleophile gave the cor-
responding crystalline sulfonamide in 60% yield; deprotection
The terminal carboxyl group of SAM is exposed to solvent in
the structure of its complex with the MetJ dimer,2 suggesting that
it may be possible to attach a bioorthogonal tag at this position
(Fig. 1). In addition, we wanted to explore the effect of (a) removal
of the a-amino group in order to simplify the structure of aza-
SAM; and (b) quaternisation of the 5¢-tertiary amine in order to
determine the role of the charge.
The amide analogue 7 was prepared by aminolysis of the ester
5, followed by removal of the protecting groups (Scheme 2).
Similarly, alkylation of the amine 4 with methyl 4-iodobutyrate
aSchool of Chemistry, University of Leeds, Leeds, UK LS2 9JT, UK.
E-mail: a.s.nelson@leeds.ac.uk; Tel: +44 (0)113 3436502
bAstbury Centre for Structural Molecular Biology, University of Leeds,
Leeds, UK LS2 9JT
† Electronic supplementary information (ESI) available: Experimental,
NMR spectra of compounds, and materials and methods. See DOI:
10.1039/b816495a
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Table 1 EC50 values, i.e. the concentration of MetJ required to promote
half-maximal formation of its complex with the F-metC DNA, in the
presence and absence of ligands
Entry
Liganda
EC50/nM
96
650 40
910 10
1000 100
150 10
2900 20
1
2
3
4
5
6
SAM, 1
3
aza-SAM, 2
7
Fig. 1 Identification of a position for attaching a bioorthogonal tag.
8
9
no ligand
a The concentration of the ligands was 2 mM.
of modification of the SAM analogues on their relative ability to
promote complex formation.
Fitting the data to a sigmoidal binding model allowed extraction
of the EC50, the concentration of protein required to promote half-
maximal formation of the complex. Although the EC50 values
obtained (Table 1) allow the relative effect of the ligands to
be compared, the assay set-up may mean that the data has
little physiological relevance. The EC50 in the presence of 2 mM
SAM, 1, was 96
3 nM. In the absence of SAM, 1, the
affinity of MetJ for the DNA was about 30-fold lower (EC50
=
2900 20 nM). The effect of SAM was smaller than previously
reported estimates, which vary considerably.19,20 Aza-SAM, 2,
was a less effective ligand than SAM, 1, although it did still
promote complex formation (EC50 650 40 nM). The pKa of
aza-SAM, 2, has been shown to be 7.18,12 and so will only
be around 25% protonated under the assay conditions (pH 7.6
buffer).
Scheme 2 Conditions: (a) propylamine, 43 ◦C, 23 h, 99%; (b) 5 M HCl,
5 min, 91%; (c) methyl 4-iodobutyrate, DIPEA, MeCN, 70 ◦C, 6.5 h, 98%;
(d) propylamine, 47 ◦C, 5 d, 93%; (e) TFA, 1:1 MeOH–H2O, 5 min, 98%;
(f) MeI, 3:1 MeCN–H2O, 97%.
was followed by aminolysis with propylamine and deprotection
to yield the analogue 8 lacking the a-amino group. Treatment
of 8 with methyl iodide gave the corresponding quaternised
derivative 9.
The effects of the novel SAM analogues were also investigated.
Replacing the carboxylic acid of aza-SAM, 2, with the propyl
amide of 7 resulted in a higher EC50 (compare entry 3 with entry
2, Table 1) though removal of the a-amino functionality, as in 8,
did not have a significant effect (entry 4). However, quaternisation
of the tertiary amine of 8, to give 9, resulted in an EC50 that was
much lower than with aza-SAM, and comparable to that of SAM
itself (compare entry 5 with entry 1). Thus, amide formation and
removal of the a-amino group had a relatively small effect on
ligand function. However, the charge of the amine can be deduced
to be important for promoting the formation of the MetJ–DNA
complex. It was therefore decided to attach bioorthogonal tags
through an amide linkage formed from the terminal carboxyl
group.
Fluorescence anisotropy17 was used to measure the ability of
the SAM analogues to act as co-repressors. Two 18-base oligonu-
cleotides, one ofwhichhad acovalently-linked terminalfluorescein
group, were annealed to yield the fluorescently-labelled duplex
(F-metC). F-metC contained the shortest naturally occurring
operator sequence for MetJ: metC.18 MetJ was titrated into a
solution of 10 nM F-metC in 50 mM Trizma hydrochloride pH 7.6
and 100 mM KCl, in the presence or absence of 2 mM ligand, and
the increase in fluorescence anisotropy due to DNA binding was
recorded (Fig. 2). The assay thus allowed us to determine the effect
With this information in hand, we prepared ligands which were
functionalised with the bioorthogonal azide and cyclooctyne tags.
The preparation of these analogues was, however, problematic.
Alkylation of 4 with methyl 4-iodobutyrate was followed by
ester hydrolysis and amide formation with ethanolamine to yield
the amide 10 (Scheme 3). Activation of 10 by treatment with
methanesulfonic anhydride, however, triggered cyclisation to yield
the corresponding oxazoline; nonetheless, this intermediate could
be opened to yield the required azide 11 (yield: 56% from 10).
Similarly, coupling with 2-(cyclooct-2-ynoxy)-ethylamine did give
the required amide; however attempted deprotection resulted in
hydration of the strained alkyne. We therefore decided to delay the
formation of the cyclooctyne until after deprotection of the ligand.
Thus, 2-((Z)-2-bromocyclooct-2-enyloxy) ethylamine was reacted
Fig. 2 Cartoon illustrating the fluorescence anisotropy assay. Binding of
SAM molecules promotes formation of a SAM–F-metC–protein complex,
with two MetJ dimers bound to the 18 base-pair DNA duplex.
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Table 2 EC50 values, i.e. the concentration of MetJ required to promote
half-maximal formation of its complex with the F-metC DNA, in the
presence and absence of ligands
Entry
Liganda
EC50/nM
96
650 40
540 20
650 40
1
2
3
4
5
6
7
SAM, 1
3
aza-SAM, 2
12
14
15
154
65
6
3
16
no ligand
2900 20
a The concentration of the ligands was 2 mM.
the quaternised analogue, 16, that had been derivatised with the
cyclooctyne tag. Furthermore, 16 was a more effective ligand than
SAM itself (EC50 96 3 nM).
In summary, we have described an improved synthesis of aza-
SAM which we have adapted to the synthesis of a range of other
stable analogues of the co-factor SAM. We have investigated
the ability of these analogues to promote the binding of the
MetJ dimer to its DNA operator. In particular, we have shown
that quaternisation enhances the activity of aza-SAM analogues,
and that it is possible to append a bioorthogonal tag through
conversion of the terminal carboxyl group into an amide. SAM
analogues which have been derivatised with a bioorthogonal
tag may be useful chemical tools for isolating, identifying and
modulating other SAM-binding macromolecules such as methyl-
transferases, fluorinases, radical SAM proteins and riboswitch
domains.
Scheme 3 Conditions: (a) methyl 4-iodobutyrate, DIPEA, MeCN, 70 ◦C,
6.5 h, 98%; (b) NaOH, MeOH, 2 d, 57%; c) ethanolamine, PyBOP,
DIPEA, DMF, 30 min, 93%; (d) Ms2O, NEt3, CH2Cl2, 0 ◦C, 30 min
then NaN3, DMSO, 70 ◦C, 2 h, 56%; (e), 5 M HCl, 20 min, 94%;
(f) 2-((Z)-2-bromocyclooct-2-enyloxy) ethylamine, PyBOP, DIPEA, DMF,
30 min, 80%; (g) 5 M HCl, 6 min, 75%; (h) NaH, 1:1 DMF–THF, 30 min,
95%; (i) MeI, 3:1 MeCN–H2O, 15: 88%, 16: 86%.
Acknowledgements
We thank the Wellcome Trust, EPSRC and BBSRC for funding,
and Dr Bruce Turnbull for discussions.
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