534 | Nature | Vol 585 | 24 September 2020
Article
On-protein heterolytic reactivity. Halides (1 s, 1t,1u, 2b, 2ad, 1v, 1w)
are potentially powerful general 2e− electrophilic side chains and afford
a novel, on-protein heterolytic reaction platform for potential conjuga-
tion with off-protein nucleophiles. By varying the pH, the nucleophile
concentration and the halide, it was possible to selectively facilitate
intermolecular nucleophilic substitution at C–Hal (Fig. 2b), thereby
creating C–S, C–P and C–N bonds (creating, for example, methyllysine
PTMs and N 3 -bearing Anl) and even direct, Finkelstein-type halogen
exchange (Br → Cl or I → Cl), allowing further tuning of electrophile
reactivity. Therefore, this is a direct strategic inversion of common prac-
tice in the field of protein conjugation (using prevalent nucleophiles
in proteins such as Cys and Lys to target off-protein electrophiles).
On-protein homolytic reactivity. Mono- and difluoro-pySOOF side
chains (2ae, 2af) suggested immediate potential for generating
on-protein side-chain radicals (Fig. 2a). In agreement with the reactivity
of off-protein pySOOF, on-protein initiation of mono-fluoro-pySOOF
side chain 2ae failed. However, when histone-H3-pySOOF9 (containing
di-fluoro-pySOOF side chain 2af at site 9) was activated in the absence
of a co-reactant, a reduced product (H3-DfeGly9) was cleanly generated
(Fig. 2a and Supplementary Tables 23, 24), consistent with on-protein
carbon-centred radical initiation and Cγ–H bond formation.
To test the scope of this on-protein carbon-centred radical, we tar-
geted some more challenging Cγ–X bonds. First, Cγ–Cδ bonds were
formed using various off-protein, C=C radical acceptors (Fig. 2a, bottom17,700 17,8500 Mass100
%17,700 17,8500 Mass100
%17,700 17,8000 Mass100
%17,700 17,8000 Mass100
%17,700 17,8000 Mass100
%R Bpin
or or++or(i) (ii)HNOH 3 N
F
FF
FH
N
OHNOHistone eH3.1-Dha18 (F 2 )AcLys18 (F 2 )Lys18Calc.: 17,849 Da
Found: 17,849 DaCalc.: 17,787 Da
Found: 17,787 DaCalc.: 17,823 Da
Found: 17,823 DaCalc.: 17,782 Da
Found: 17,781 DaF
F(F 2 )Lys18H 3 NDhaSirt2Sirt2
deacylation
side-chain PTM
selectivity5
min30
min12 hH 3 NR =NHO
NHOR =N
HO
NH 3Sirt2 deacetylation
resolved by^19 F NMROverlayReaction
Fit(F 2 )AcLys18
Fit
(F 2 )Lys18
FitLys18AcLys18 BzLys18AcLys18 BzLys18R S NFOOFabSirt2
deacylation
diastereomer
selectivitycSBr SZn S
HSS HSKDM4A KDM4AHistone
eH3.1-Bhn4/9/27KDM4A
Histone eH3.1eH3.1-WT
eH3.1-Bhn4
eH3.1-Bhn9
eH3.1-Bhn27
KDM4A-KDM4ALadder(kDa)-Crosslink-eH3.1+ –––– + + + +- –––+ – – – +
–––+– – – + –- –+–– – + – –
–+––– + – – –
Histone eH3-Bhn9
Histone eH3-WT
No compoundBr B(OH) 2S 1728384962Proximity-driven
protein crosslinkingHistone
eH3.1-Bhn4/9/27Concentration of Zn(II) (μM)Time (s)1.0
0.8
0.6
0.4
0.2
0.0
0 2,000 4,000 6,000ZnSee
Extended Data
Fig. 11Tryptic digest
LC–MS/MSDetection of ejected Zn(II) SDS-PAGE con rmation of crosslinking10,000 15,000 20,000 25,0000 Mass100
%17,78710,000 15,000 20,000 25,0000 Mass100
%17,84910,000 15,000 20,000 25,0000 Mass100
%17,82310,000 15,000 20,000 25,0000 Mass100
%17,781(^19) F G (ppm) (^19) F G (ppm)
–102–101–100–99 –98 –97 –96 –101 –99 –97
17,700 17,850
0 Mass
100
%
D L
D D
D
L
Fig. 3 | Insertion of native, dif luoro-labelled and electrophile-containing
side chains into proteins provides insight into enzymes that
post-translationally modify them. a, The Sirt2 enzyme displays different
deacylation rates (via intact protein LC–MS monitoring) towards installed
acetyl (red) and benzoyl (blue) lysine on histone-eH3.1-K18 proteins, thereby
confirming both plasticity and selectivity towards acyl groups. Reagents and
conditions for installation: (i) histone-H3-Dha9 (66 μM), pinacol alkylboronate
(250 equiv.), catechol(100 equiv.), Ru(bpm) 3 Cl 2 (10 equiv.), NH 4 OAc (500 mM,
pH 6, 3 M GdnHCl), 50 W blue LED, RT, 1 h. b, The activity of Sirt2-deacetylation
can be directly and site-specifically monitored via^19 F NMR using a CγF 2 -
dif luoro tag on the γ carbon of installed Lys (green) and AcLys (light blue) side
chains. Reagents and conditions: (ii) histone-H3-Dha9 (66 μM), alkyl-pySOOF
(50 equiv.), FeSO 4 ·7 H 2 O (50 equiv.), Ru(bpy) 3 Cl 2 (2 equiv.), NH 4 OAc (500 mM,
pH 6, 3 M GdnHCl), 50 W blue LED, RT, 15 min, Mox = 15,838 Da (where Mox
denotes sulfoxide oxidation products of Met). Although four bonds away from
the site of PTM, the CγF 2 labels display sufficient sensitivity to the chemical
environment (δF perturbation) to allow direct simultaneous monitoring of
the chemo- and stereoselectivity of Sirt2 during processing (Extended Data
Fig. 9). c, Crosslinking between KDM4A and histone-eH3.1-Bhn4/9/27 traps
KDM4A-Zn-binding cysteines near the active site. Coomassie blue-stained
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE; right),
tryptic LC–MS/MS (bottom left; see also Extended Data Fig. 3c) and Zn(ii)
ejection (right) confirm crosslinking between KDM4A and histone-eH3.1-Bhn9
(see also Extended Data Fig. 11c). Zn(ii)-ejection rates: eH3-Bhn9, 9.27 ±
0.025 nM min−1; eH3-WT (wild type), 0.09 ± 0.006 nM min−1; 1u precursor,
0.805 ± 0.010 nM min−1; no compound, 0.87 ± 0.028 nM min−1; N = 3
independent experiments. Data plotted are averages ± s.d. (N = 3 technical
replic ate s), P < 0.0001, one-way ANOVA. See also Extended Data Fig. 11 for
further alkylator protein experiments.