Nature - USA (2020-09-24)

Nature | Vol 585 | 24 September 2020 | 533

groups for potential initiation. First, remarkably, halo-pySOOF reagents
pySOO–CF 2 –Hal (Hal, halogen; Fig. 2a) could be chemoselectively
activated to generate pySOO-F 2 C• radicals that allowed installation
of mono- and difluoro-pySOOF side chains (2ae, 2af) into proteins
(Fig. 2a). Second, BACED and pySOOF reagents bearing two distal

initiator groups allowed installation of a diverse range of side-chain

halides (F, Cl, Br, I; Fig. 2b), either with short alkyl linkers to the protein

backbone (1s, 1t, 1u, 2b, 2ad) or more complex substrate-mimetic side

chains (1v, 1w); these were installed without activation of the radical

precursor halide.

NH

O

S

X

F

N

O

O

NIS

F X

OO

NH

O

F

On-proteinF

radical

Iodo-pySOOF

On-protein

pySOOF

(iii)

NH

O

F

H F

NH

O

F

F

R

N

H O

F

SeF

NH

O

F

OF

N

HO

On-protein

polymerization

Dha

N

H O

F F

Diuoro-tagged

protein trapping

R R

n

Bpin NHAc

CO 2 H

NBoc

OAc OH

CO 2 Me

CO 2 Me

HO OH O NH 2

R

=

Hal Bpin

1,000 equiv.

Hal = Br, I

5 equiv.

N

H O

Hal

On-protein

Slow halogen exchange halogens

N 3 M NaCl, 64 h

H O

Cl

Calc.: 15,350 Da

Found: 15,349 Da

Calc.: 15,258 Da

Found: 15,258 Da

Hal = Br Hal = I

NH

O

Nu

Proximity-driven

crosslinking (see Fig. 3)

Nu

Interaction

partner

NH

O

N 3

NH

O

S

NH

O

NH

O

MenH3-nN

HO

(–O 2 C(CH 2 ) 2 ) 3 P

NaN 3

βME

TCEP

n = 1, 2, 3

Chloronorleucine, Cnl Bromonorleucine, Bnl Iodonorleucine, Inl

MenNH3-n

(i)

(ii)

*

** +

+ pySOOF (1 equiv.)

Crosslink

kDa

38

28

14

X = H, F Calc.: 15,355 Da

X = H Found: 15,355 Da

Lad

der

DhapySOOFDhaDha (1 equiv.)Dha (2 equiv.)Dha (4 equiv.)

TEMPO

a

b

*

**

Dimer

Dimer

N

H O

Dha-tagged

protein

(15,180 Da)

10,000 15,000 20,000

0 Mass

100

%

15,258

15,180

Hal = Cl

Calc.: 15,302 Da

Found: 15,302 Da

10,000 15,000 20,000

0 Mass

100

%

15,302

15,180

10,000 15,000 20,000

0 Mass

100

%

15,349

15,179

10,000^0 15,000 20,000Mass

100

%

15,355

Calc.: 15,373 Da

X = F Found: 15,373 Da

10,000^0 15,000 20,000Mass

100

%

15,373

No radical

acceptor

FeII (250 equiv.)

Calc.: 15,232 Da

Found: 15,231 Da

10,000 15,000 20,000

0 Mass

100

%

15,231

15,000^0 15,500 16,000Mass

100

%15,179

15,488

15,617

n = 1 15,746

n = 2

n = 3

n = 4

n = 5

Ph 2 Se 2

Ph

Calc.: 15,388 Da

Found: 15,387 Da

10,000 15,000 20,000

0 Mass

100

%

15,387

15,179

Calc.: 15,403 Da

Found: 15,403 Da

10,000^0 15,000 20,000Mass

100

%

15,403

15,179

NH

O

Dha-tagged

protein

(15,280 Da)

Fig. 2 | On-protein homolytic and heterolytic reactivity via installation of a
radical precursor and electrophile side chains. a,Utilization of iodo-pySOOF
allowed reductive installation of an on-protein pySOOF side chain, which is
itself a protein radical precursor. Both mono- and dif luoro-pySOOF side
chains were installed. Reagents and conditions: (i) histone-H3-Dha9 (66 μM),
iodo-pySOOF (5 equiv.), FeSO 4 ·7 H 2 O (20 equiv.), Ru(bpy) 3 Cl 2 (0.4 equiv.),
NH 4 OAc (500 mM, pH 6, 3 M GdnHCl), 50 W blue LED, RT, 15 min. Intact protein
liquid chromatography (LC)–MS (right, top). (iii) After activation under
standard conditions, the resulting on-protein radical allowed further protein
functionalization via on-protein homolytic bond-forming modes: polymerized
with various radical acceptors via C–C bond formation (right, bottom);
covalently trapped by another Dha-containing protein through C–C
bond-forming protein–protein crosslinking (left, bottom); quenched with
stable oxygen nitroxide radical to form C–O bonds (left, middle); used to
cleave diselenide (SePh) 2 to form C–Se bonds (centre, bottom); or reduced
(C–H bond formation) to DfeGly (right, middle). Typical reagents and
conditions: histone-H3-pySOOF9 (66 μM), substrate (10–250 equiv.),
FeSO 4 ·7 H 2 O (0–25 equiv.), Ru(bpy) 3 Cl 2 1–5 equiv.), NH 4 OAc (500 mM,

pH 6, 3 M GdnHCl), 50 W blue LED, RT, 15 min; see Supplementary

Tables 23–37 for details; residual Dha, 15,179 Da. TEMPOL, 4-hydroxy-

(2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl. b, Utilization of alkylhalide-

functionalized BACED reagent allowed oxidative installation with the C–Hal

bond unperturbed. This installs on-protein alkylhalide electrophile side

chains. This provided a further reaction platform for on-protein heterolytic

bond-forming modes. These on-protein alkylhalide electrophiles were reacted

through substitution with various small-molecule P, S, N and Hal nucleophiles

at higher concentrations, allowing diverse C–P, C–S, C–N and C–Hal bond

formation (residual Dha, 15,179 or 15,180 Da). The ability to install a range of

alkylhalide side chains (for example, chloro-(Cnl), bromo-(Bnl), iodo-(Inl)

norleucines; intact protein LC–MS, bottom left) also allowed protein–protein

crosslinking with interaction partners (see Fig. 3c). Reagents and conditions:

(ii) histone H3-Dha9 (66 μM), alkylboronic acid pinacol ester (1,000 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, 1–3 h. Calc., calculated; TCEP, tris(2-carboxyethyl)

phosphine; βME, betamercaptoethanol.