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Organoruthenium-catalyzed chemical protein
synthesis to elucidate the functions of epigenetic
modiﬁcations on heterochromatin factors†
Naoki Kamo, a Tomoya Kujirai, b Hitoshi Kurumizaka,
Gosuke Hayashi *c and Akimitsu Okamoto *adbHiroshi Murakami,c

The application of organometallic compounds for protein science has received attention. Recently, totalchemical protein synthesis using transition metal complexes has been developed to produce various
proteins bearing site-speciﬁc posttranslational modiﬁcations (PTMs). However, in general, signiﬁcantamounts of metal complexes were required to achieve chemical reactions of proteins bearing a large
number of nucleophilic functional groups. Moreover, syntheses of medium-size proteins (>20 kDa) were
plagued by time-consuming procedures due to cumbersome puriﬁcation and isolation steps, which
prevented access to variously decorated proteins. Here, we report a one-pot multiple peptide ligation
strategy assisted by an air-tolerant organoruthenium catalyst that showed more than 50-fold activity
over previous palladium complexes, leading to rapid and quantitative deprotection on a protein witha catalytic amount (20 mol%) of the metal complex even in the presence of excess thiol moieties.
Utilizing the organoruthenium catalyst, heterochromatin factors above 20 kDa, such as linker histone
H1.2 and heterochromatin protein 1a (HP1a), bearing site-speciﬁc PTMs including phosphorylation,ubiquitination, citrullination, and acetylation have been synthesized. The biochemical assays usingsynthetic proteins revealed that the citrullination at R53 in H1.2 resulted in the reduced electrostatic
Received 5th February 2021
Accepted 21st March 2021

interaction with DNA and the reduced binding aﬃnity to nucleosomes. Furthermore, we identiﬁed a key
phosphorylation region in HP1a to control its DNA-binding ability. The ruthenium chemistry developed

DOI: 10.1039/d1sc00731ahere will facilitate the preparation of a variety of biologically and medically signiﬁcant proteins containing

rsc.li/chemical-science

PTMs and non-natural amino acids.

Introduction
Synthetic methodology for homogeneously modied proteinshas been highly demanded to elucidate the biological signicance of posttranslational modications (PTMs) throughbiochemical and structural analysis. Total chemical protein
synthesis has streamlined the preparation of target proteinsbearing site-specic PTMs and functional molecules.1–5 For
example, histone proteins with various PTMs have been chemically synthesized to understand the roles of PTMs.6 In theprocess of chemical protein synthesis, a target protein is dividedaDepartment of Chemistry and Biotechnology, Graduate School of Engineering, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. E-mail:okamoto@chembio.t.u-tokyo.ac.jpbLaboratory of Chromatin Structure and Function, Institute for Quantitative
Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japanc
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya
University, Nagoya 464-8603, Japan. E-mail: hayashi@chembio.nagoya-u.ac.jpdResearch Center for Advanced Science and Technology, The University of Tokyo,
Meguro-ku, Tokyo 153-8904, Japan† Electronic supplementary
10.1039/d1sc00731a

information

5926 | Chem. Sci., 2021, 12, 5926–5937(ESI)

available.

See

DOI:

into several peptide segments that can be synthesized by solidphase peptide synthesis (SPPS).7 To assemble the peptide
segments, many researchers have employed native chemical
ligation (NCL) that utilizes the chemoselectivity between Nterminal cysteine (Cys) and C-terminal thioester to form
amide bonds between divided segments.8 However, assembly of
multiple peptide segments to obtain medium-size proteins (e.g.,>20 kDa) requires multiple reactions and purication steps,
which causes low overall yields and time-consuming works andtherefore limits the access to target proteins bearing various
patterns of PTMs. To address this issue, one-pot multiple
peptide ligation strategies to eliminate the intermediate purication steps, including a kinetically controlled strategy9–11 anda protecting group-removal strategy, have been developed,12
although these methods have been mainly exploited for theassembly of three segments to aﬀord small proteins.
Organometallic compounds have been applied for chemical
reactions on proteins, such as decaging13,14 and bioconjugation
utilizing Suzuki–Miyaura coupling,15 Cys arylation through C–S
reductive elimination using palladium (Pd) complexes,16 or
Tsuji–Trost allylations.17,18 Recently, Brik and coworkers paved
the way for the application of palladium (Pd) complexes for total© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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chemical protein synthesis.19–28 They exploited several protecting groups that were labile by specic Pd complexes for chemoselective masking and one-pot ligation of peptide
segments.21,24 Our group employed allyloxycarbonyl (alloc)
groups for the protection of N-terminal Cys, which were
removed with Pd/3,30 ,300 -phosphanetriyltris(benzenesulfonicacid) trisodium salt (TPPTS) complexes.29 The one-pot peptide
ligation using Pd/TPPTS complex allowed us to synthesize
histone H2AX eﬃciently. However, the repetitive metalmediated decaging reactions to ligate multiple peptide
segments necessitated considerable amounts of metal
complexes especially under NCL conditions containing an
excess amount of thiol compounds that signicantly degrade
metal complexes.29 Generally, decaging or cross-coupling reactions on protein surfaces under aqueous conditions require
excessive amounts of metal complexes13,14 probably because
nucleophilic functional groups on proteins such as thiolgroups30 would readily coordinate to metal complexes. Moreover, some metal complexes (e.g., Pd/TPPTS complexes) are very
unstable under aerobic conditions, and these organometallic
compounds required strict deoxygenated conditions. Furthermore, a high loading of transition metal complexes oen cause
protein aggregations,31 which interferes with the wholesynthetic procedure to obtain target proteins. To overcome
these problems, a highly active organometallic compound thatminimizes the metal loading has been demanded in chemical
protein synthesis to produce a variety of proteins of interest
with modications and accelerate biological research.
In this study, we focused on organoruthenium complexes toachieve decaging reactions on polypeptides in catalyticmanners. Aer our screening of metal complexes, we discovered
air-tolerant ruthenium (Ru) complexes that showed more than
50-fold catalytic activity compared with previous Pd complexes
for the decaging reaction and only 20 mol% of an organoruthenium catalyst allowed the deprotection of the alloc groups
on polypeptides under NCL conditions. The new one-pot
multiple peptide ligation strategy, including consecutive morethan ve reaction steps using the Ru catalyst, was applied forthe total chemical synthesis of linker histone H1.2 (size: 22 kDa)
and heterochromatin protein 1a (HP1a) (size: 22 kDa). Our new
streamlined synthetic method aﬀorded homogeneous two
kinds of H1.2s and six kinds of HP1a bearing diﬀerent patternsof PTMs including phosphorylation, ubiquitination, citrullination, and acetylation. Finally, we investigated the eﬀects of
those PTMs decorated in heterochromatin factors on chromatosome formations or the binding ability toward DNA to elucidate the functions of the epigenetic marks.

Results and discussion
Exploration of organoruthenium complexes
Under NCL conditions involving excess MPAA, the metal
complexes lost activity rapidly because of its sulfur poisoning
eﬀect. Therefore, it was important to increase the stability of themetal complexes under NCL conditions to achieve catalytic reactions on peptides or proteins. To accomplish this goal, we focusedon Ru complexes rather than Pd complexes. Electron-rich Pd (10© 2021 The Author(s). Published by the Royal Society of Chemistry

Chemical Scienceouter electrons) forms a strong bond with thiol moieties becauseof the p-back donation from Pd to the empty relatively low-energy
d orbitals of the sulfur.32 On the other hand, electron-decient Ru(8 outer electrons) favors ionic interactions to ll its d orbital anddoes not form a strong coordination bond with so nucleophiles.33 Moreover, electron-rich Pd is more sensitive to electronicmodication by sulfur poisoning, which causes signicantchanges in catalytic activity, than electron-poor Ru.33 When we
screened trivalent phosphine ligands for Pd, we could notdiscover more active ligands for the alloc removal than TPPTS(Fig. S1†). Although the Pd loading on proteins could be reducedby the addition of magnesium ion,21,34 these results suggested that
it would be diﬃcult to deprotect the alloc groups on peptides
under NCL conditions with Pd complexes in catalytic manners.
Meanwhile, some researchers reported that organoruthenium
complexes tolerated sulfur poisoning and catalyzed the allyltransfer to thiol moieties,35 but these Ru complexes had not been
applied for chemical reactions on biomolecules. To test whetherorganoruthenium complexes could sustain the catalytic activity
for the removal of the alloc group on a peptide under NCL conditions and air atmosphere, we prepared Cp*Ru(cod)Cl (Ru-1: Cp*¼ h5-pentamethylcyclopentadienyl, cod ¼ h4-1,5-cyclooctadiene),[Cp*Ru(QA)allyl]PF6 (Ru-2: QA ¼ 2-quinolinecarboxylate),[CpRu(QA)allyl]PF6 (Ru-3: Cp ¼ h5-cyclopentadienyl), [CpRu(QANMe2)allyl]PF6 (Ru-4: QA-NMe2 ¼ 4-(N,N-dimethylamino)-2quinolinecarboxylate) bearing N,O-bidentate ligand (Table 1).36–38
Next, we tested the alloc deprotection on peptide 1 with 10 mol%of metal complexes under NCL conditions (Table 1), and theconversion yields were calculated based on high-performanceliquid chromatography (HPLC). In previous research, 2 equiv. of
Pd/TPPTS complex was required to attain quantitative removal of
the alloc groups under NCL conditions.29 When the amount of the
Pd complex was reduced to 10 mol%, the conversion yield reached
to only 12% aer 3 h (Table 1, entry 1), indicating that the TON of
Pd/TPPTS complexes under NCL conditions was 1.2. In contrast,
Ru-1 and Ru-2 sustained the catalytic activity even in the presenceof a high concentration of MPAA (entries 2 and 3) under air
atmosphere, and quantitative removal of the alloc groups wasobserved aer 2 h upon the treatment with 10 mol% Ru-2 (entry
3). Furthermore, only 5 mol% of Ru-3 or Ru-4 rapidly catalyzed thealloc deprotection, and the reaction reached completion within
10 min (entries 6 and 7). We did not observe any side reactions,such as the transfer of the allyl groups to nucleophilic functionalgroups at side chains of peptide 1 (Fig. S2†). The slow reaction rateof Ru-2 compared to Ru-3 could be explained by the stericallyhindered Cp* disturbing nucleophilic attack of MPAA toward pallyl Ru complex, which was already reported.38 We should notethat the activity of the Ru catalysts diminished in the presence of
TCEP (entries 8–11); however, oxidized TCEP aer the reductionof the disulde bond of MPAA dimer did not aﬀect the activity of
Ru-4 (entry 12, Fig. S3†). We screened other Ru complexes for the
alloc deprotection under NCL conditions. We replaced the N,Oligand with an N,N-ligand, and synthesized [Cp(h2-bipy)(h3-allyl)
RuIV][PF6]2 (Ru-5: bipy ¼ bipyridine) and [Cp(h2-phen-NMe2)(h3allyl)RuIV][PF6]2 (Ru-6: phen ¼ phenanthroline).39 However, thecatalytic activities of Ru-5 and Ru-6 were much lower than those ofChem. Sci., 2021, 12, 5926–5937 | 5927

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Table 1

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Deprotection of the alloc group of peptide 1 with Pd or Ru complexes under NCL conditions

Conversion yieldsa [%]Entry

Metal complexes

Quantity (mol%)

Additive

10 min30 min

1h

2h

3h

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Pd/TPPTS
Ru-1
Ru-2
Ru-3
Ru-4
Ru-3
Ru-4
Ru-3
Ru-3
Ru-4
Ru-4
Ru-4
Ru-5
Ru-6
Ru-7
Ru-8
Ru-9
Ru-10
Ru-10
Ru-10

10
10
10
10
10
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5

—
—
—
—
—
—
—
TCEP (3 mM)
TCEP (10 mM)
TCEP (3 mM)
TCEP (10 mM)
TCEP (5 mM)b
—
—
—
—
—
—
TCEP (3 mM)
TCEP (10 mM)

11
28
41
>95
>95
>95
>95
65
<5
55
8
>95
<5
7
19
<5
18
93
50
<5

11
56
76
—
—
—
—
67
<5
67
9
—
<5
10
40
18
36
>95
61
<5

12
76
92
—
—
—
—
67
7
69
10
—
<5
14
60
47
53
—
66
11

12
86
>95
—
—
—
—
70
—
75
—
—
<5
17
76
64
73
—
68
—

12
94
—
—
—
—
—
72
—
78
—
—
6
17
80
71
76
—
68
—

a
The conversion yields were calculated from peak areas analyzed by HPLC (Fig. S2). b TCEP was completely consumed for the reduction of MPAA
disulde dimer before the addition of Ru-4 as shown in Fig. S3.

Ru-3 or Ru-4 (entries 13 and 14) probably because of those rigid
structures.
Previous research showed that the rate-determining step of
the alloc removal using Ru-3 or Ru-4 was the oxidative additionof the allyl groups toward Ru when strong nucleophiles, such as
thiol moieties, were employed.38 To accelerate the uncaging of
the alloc groups and further improve the stability under NCL
conditions, we focused on P,O-bidentate ligands to replace N,Oligands, and increase the electron density of Ru complexes.

5928 | Chem. Sci., 2021, 12, 5926–5937

Bruneau and coworkers reported Ru complexes coordinated byo-diphenylphosphino benzoate (o-DPPBz) or diphenylphosphinobenzene sulfonate (o-DPPBS).40 We synthesized [Cp*Ru(k2-oDPPBz)allyl]PF6 (Ru-7) and [Cp*Ru(k2-o-DPPBS)allyl]PF6 (Ru-8)(Table 1) and tested those catalytic activities. There was no
signicant diﬀerence between Ru-7 and Ru-8 (entries 15 and 16)
for the alloc deprotection of peptide 1 under NCL conditions. To
reduce the electron density of the phosphine ligand andenhance the nucleophilic attack of MPAA toward p-allyl Ru© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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complexes, we synthesized Ru complexes coordinated by oDPPBz bearing CF3 groups (Ru-9), but no improvement of theseRu complexes over Ru-7 was observed (entry 17). Considering
the eﬀective deprotection of the alloc group using Ru-3
compared to Ru-2 (entries 3 and 4), we newly synthesized[CpRu(k2-o-DPPBz)allyl]PF6 (Ru-10). The deprotection eﬃciency
was dramatically improved by replacing Cp* with Cp, and thereaction reached completion within 30 min (entry 18), although
the catalytic activity of Ru-10 was also reduced in the presence of
TCEP (entries 19 and 20). We concluded that organoruthenium
catalysts coordinated by Cp and X,O-bidentate (X ¼ N or P)
ligand eﬃciently catalyzed the removal of the alloc group under
NCL conditions.

Calculation of TON of Ru catalysts
We examined TON and turnover frequency (TOF) of Ru-3, Ru-4,
and Ru-10, which removed the alloc groups quantitatively with
5 mol% catalysts. We reduced the amount of each Ru complex
to 1 mol% and tested the removal of the alloc group of peptide 1
under NCL conditions (Fig. 1). Ru-3 (TOF: 19 min1) and Ru-4
(TOF: 20 min1) showed similar TOF value, but the TON of Ru-4
(TON: 70) was slightly higher compared to Ru-3 (TON: 56),
because of the NMe2 group in Ru-4, which increased the electron density of Ru and its stability against excess MPAA. The
reaction rate using Ru-10 (TOF: 6 min1) as a catalyst was slower
than Ru-3 or Ru-4 due to the steric hindrance of the P,Obidentate ligand of Ru-10, but Ru-10 sustained its catalytic
activity for a longer time because of its stability and showedsimilar catalytic activity (TON: 72) to that of Ru-4 and higher
than that of Ru-3. We further calculated the electrophilicity ofeach Ru catalyst based on density functional theory (DFT). The
lowest unoccupied molecular orbital (LUMO) level of o-DPPBz
was more than 0.5 eV higher than QA and QA-NMe2 (1.44 eV
vs. 2.15 eV vs. 1.94 eV) (Fig. S5†). This higher p-accepting
ability of QA or QA-NMe2 led to the higher electrophilicity of Ru3 or Ru-4 and the attack of MPAA toward p-allyl Ru complexes
became faster than Ru-10. To summarize, compared to theactivity of Pd/TPPTS complexes (TON: 1.2), Ru-4 and Ru-10
showed more than 50-fold catalytic activity, and catalyticallyremoved the alloc groups of peptides under aqueous conditions

Fig. 1 Calculation of TON and TOF of 1 mol% Ru-3, Ru-4, or Ru-10 forthe deprotection of the alloc group of peptide 1 (2 mM) in NCL buﬀer[Gn$HCl (6 M), NaH2PO4 (200 mM), MPAA (100 mM, 5000 equiv.
against the Ru catalyst) at pH 7.0]. The conversion yields underdiﬀerent conditions are shown in Table S1.†

© 2021 The Author(s). Published by the Royal Society of Chemistry

Chemical Scienceand even in the presence of 5000 equiv. of MPAA that normallypoisoned organometallic catalysts.
To further explore the diﬀerence between Ru-4 and Ru-10, we
added these Ru complexes to a solution containing peptide 1
without MPAA, a scavenger for p-allyl Ru complexes (Fig. S6†).
When 10 mol% of Ru-4 was tested, peptide 1 was completely
consumed within 30 min, and single or double allyl transfer tofunctional groups of peptides was observed. Based on MS/MS
analysis of each HPLC peak (Fig. S7†), it was found that theallyl groups were transferred to amine groups or thiol groups of
N-terminal Cys, not to other amino acids, such as Lys, Tyr, or Thr.
When 10 mol% of Ru-10 was examined, the starting material
remained for over 1 h reaction, and the allyl transfer to peptide 1
barely occurred (Fig. S6†). These results indicated that Ru-4 wasmore reactive with nucleophiles than Ru-10 because of its more
electrophilic p-allyl Ru complex, which presented the possibilityof side reactions, such as allyl transfer to the functional groups of
peptides via Ru-4. However, in the presence of excess MPAA (pKa
6.6),41 this strong nucleophile attacked p-allyl Ru complex before
peptides and suppressed the undesired reactions as observed bycomparing HPLC data shown in Fig. S2 and S6.†Inactivation by MPAA to form a binuclear Ru complex
To achieve one-pot peptide ligation by repeating the cycles of NCL
and decaging, the deactivation of metal complexes just before thesubsequent NCL was a critical step.29 Therefore, we investigatedthe deactivation of Ru-4 or Ru-10 under NCL conditions (Fig. S8†).
200 mol% of Pd/TPPTS complex against peptide 1 was rapidly
deactivated by 100 mM MPAA and lost activity within 10 min

Scheme 1

Simultaneous catalytic alloc deprotection and deactivationof Ru-4.

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Chemical Science(Table S2,† entry 1). In contrast, 10 mol% of Ru-4 and Ru-10
against peptide 1 showed stability under NCL conditions (entries
2 and 3), and Ru-10 was more stable than Ru-4 because of its bulky
P,O-bidentate ligand. We concluded that the greater stability ofthese Ru complexes over Pd complexes contributed to the catalytic
activity for the removal of the alloc groups of peptides. By NMR

Edge Article
and MALDI-TOF mass analysis, the half-sandwich Ru complex
reacted with MPAA to form a binuclear Ru complex,42 which might
be inactive for the alloc removal (Fig. S9 and S10†). Moreover, weobserved the detachment of the bidentate ligand from Ru throughthe ligand exchange with MPAA.

Chemical synthesis of linker histone H1.2 using the Ru catalyst. (A) Synthetic strategy. / represents the cleavage sites. (1) NCL condition:
peptides (2 mM), MPAA (100 mM) in denaturing buﬀer at pH 7.0, 37  C. Removal condition: Ru-4 (20 mol%), 37  C, 30 min. (2) Peptide (0.8 mM),
TCEP (300 mM), GSH (150 mM), VA-044 (20 mM) in denaturing buﬀer at pH 7.0, 37  C. (B) Reaction tracking of the one-pot ligation involving
catalytic amount of Ru-4 (20 mol%) for the alloc deprotection by analytical HPLC (gradient: 10–46% for 30 min) at 220 nm. Compounds 50 , 70 ,and 90 are the alloc-protected peptides 5, 7, and 9, respectively. # ¼ MPAA. * ¼ allyl-transferred MPAA. (a) 1st NCL (t ¼ 2 min). (b) 1st NCL (t ¼ 2 h).
(c) 1st deprotection. (d) 2nd NCL (t ¼ 2 h). (e) 2nd deprotection. (f) 3rd NCL (t ¼ 2 h). (g) 3rd deprotection. (h) 4th NCL (t ¼ 2 h). (C) HPLC proﬁle(left, gradient 20–50% for 30 min) and MALDI-TOF mass spectrum (right) of puriﬁed peptide 12. Calculated mass of 12 [M + H]+: 21 276.6; massfound [M + H]+: 21 277.8.
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To summarize, the alloc deprotection of peptides based on
the Tsuji–Trost reaction mechanism catalyzed by the Ru catalysts under NCL conditions proceeded very fast, and the reaction reached completion within 10 min. Along with the
deprotection, the Ru catalyst slowly lost the catalytic activity
through ligand exchange with MPAA to form a binuclear
complex, enabling the subsequent NCL with alloc-protected
peptides (Scheme 1). Newly synthesized Ru-10 coordinated by
P,O-ligands showed the highest catalytic activity and the greatest stability under NCL conditions among tested Ru complexes.
However, considering the facile deactivation by MPAA, we
decided to employ Ru-4 to attain total chemical synthesis ofproteins through one-pot multiple peptide ligation.

Chemical synthesis of histone H1.2 assisted by anorganoruthenium catalyst
To demonstrate the utility of Ru-4 catalyst for chemical protein
synthesis, we selected linker histone H1.2 as a target protein.
This protein interacts with nucleosomes to form chromatosomes and contributes to the formation of a higher-orderchromatin structure.43–46 Previous papers reported that various
PTMs can be decorated on H1.2, and some of these modications play roles in chromatin decondensation.47 However, it had
been challenging to achieve the chemical synthesis of H1.2
compared to core histone proteins (<140 amino acids), becauseof its large size (212 amino acids). Therefore, we aimed toestablish a synthetic method to obtain H1.2 through one-pot
multiple peptide ligation using Ru-4.48
As shown in Fig. 2A, Cys mutations for NCL reactions wereincorporated at Ala sites (A48C, A68C, A100C, A150C) and divided
into ve peptide segments of H1.2 (peptides 3, 4, 6, 8, and 10),which were synthesized by 9-uorenylmethyloxycarbonyl SPPS
(Fmoc-SPPS) (Fig. S11†). The N-terminal Cys of peptides 4, 6,
and 8 were protected by the alloc groups, and the one-pot vesegment ligation using Ru-4 was performed. All the NCL reactions and the removal of alloc groups were conducted in NCL
buﬀer (6.0 M guanidinium chloride, 200 mM NaH2PO4, 100 mM
MPAA) at 37  C.
Peptides 3 and 4 (1.1 equiv. relative to peptide 3) weredissolved in NCL buﬀer, and the NCL reaction reachedcompletion within 2 h to aﬀord peptide 5 (Fig. 2B). Ru-4 (0.2
equiv.) was added to the reaction solution, and the mixture
was stirred for 30 min to remove the alloc group and fully
deactivate Ru-4. Aer completion, a small amount of TCEP
solution was added to reduce disulde bonds and be
consumed completely to minimize the eﬀect on the next alloc
removal with Ru-4 (Fig. S3†). Powdered peptide 6 was thenadded to initiate the second NCL reaction. These operations
were repeated for the second deprotection and the third NCL
and deprotection (Fig. 2B).
Finally, powdered peptide 10 was added to initiate the fourth
NCL reaction. HPLC analysis showed a major peak corresponding to the desired ligated peptide 11 (Fig. 2B) aer sevenconsecutive reaction steps. The reaction product was puried by
HPLC, and 2.52 mg of peptide 11 was obtained in 23% yieldfrom 3 (Fig. S13†). Compared to the previous one-pot ligation© 2021 The Author(s). Published by the Royal Society of Chemistry

Chemical Science
method with Pd/TPPTS complex,29 the total amount of metal
complexes was reduced to 0.6 equiv. (Pd: more than 6.0 equiv.),
and strict deoxygenated conditions established by argon
bubbling were not required during the procedure. The mutated
Cys residues were converted into original Ala residues by freeradical desulfurization49 to aﬀord 1.34 mg of full-length H1.2
(12) almost quantitatively (Fig. 2C). Analysis by inductively
coupled plasma mass spectrometry (ICP-MS) showed that thequantity of Ru attached to synthesized H1.2 was only 0.000037%(w/w) (Table S3†), and the amount of metal was much lower
compared to the Pd content on previously synthesized histone
H2AX (0.00096%) produced by one-pot ligation using Pd/TPPTS
complexes.29
Next, we decided to incorporate PTMs into H1.2. As candidates of modications, we chose citrullination of 53rd Arg(R53Cit) and phosphorylation of 172nd Ser (S172ph). It was reported that the R53 site was modied to Cit by peptidyl arginine
deiminase 4 (PAD4), which regulates pluripotency, and thechromatin structure became decondensed and cells werereprogrammed.50 S172ph was observed specically during interphase and mitosis and might have an inuence on the stability ofchromatosome.51 Therefore, we tried to investigate the eﬀects ofthese PTMs on the binding aﬃnity of H1.2 for nucleosome bycreating homogeneously modied proteins. To synthesize
modied H1.2, we prepared a peptide segment of H1.2 bearing
R53Cit (8a) or S172ph (3a) by Fmoc-SPPS (Fig. S10†). According to
Fig. 2A, the divided peptide segments were assembled in a onepot manner with the Ru catalyst (Fig. S13†). Aer completionof all peptide ligations and purication, the Cys residues wereconverted into Ala residues. As a result, we accomplished thechemical synthesis of citrullinated H1.2 (12a) and phosphorylated H1.2 (12b) at mg scale (Fig. S14†). Thus, our facile and rapid
synthetic method helped the eﬃcient chemical synthesis of three
types of full-length H1.2.

Reconstitution of chromatosomes
We examined the formation of chromatosomes using our
synthetic H1.2 (Fig. 3A). The recombinant H1.2 or synthetic
H1.2 (12) was mixed with Nap1, a histone chaperone tosuppress aggregation during the reconstitution of chromatosomes,52 and each mixture was incubated at 37  C. The nucleosome solution was then added to each mixture to reconstitutechromatosomes. The synthetic H1.2 (12) formed chromatosomes in a similar way to recombinant H1.2 (Fig. S16†), suggesting that synthetic H1.2 was refolded properly forchromatosomes formations.
Next, we investigated the eﬀect of R53Cit or S172ph on thebinding aﬃnity for nucleosome by electrophoretic mobility
shi assay (EMSA) (Fig. 3B). There was a slight diﬀerence
between 12 and 12b with phosphorylation, but 12a with citrullination reduced its binding aﬃnity for nucleosomes, and its
conversion yield was reduced by approximately 30% compared
to nonmodied H1.2 (Fig. 3C). R53 interacts with the phosphatebackbone of the minor groove of linker DNA in chromatosome(Fig. 3D).53

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Gel band shift assay for reconstitution of chromatosomes. (A)Schematic representation of the reconstitution of chromatosomes. (B)
Representative gel image of the H1.2 binding assay. Increasing
amounts of wt H1.2, H1.2 bearing R53Cit, or H1.2 bearing S172ph (0mM: lanes 1, 6 and 11; 0.3 mM: lanes 2, 7 and 12; 0.45 mM: lanes 3, 8 and
13; 0.6 mM: lanes 4, 9 and 14; 0.7 mM: lanes 5, 10 and 15) were mixed
with nucleosomes (0.1 mM) in the presence of Nap1 (0.3 mM). Afterincubation at 37  C, the complexes were detected by nondenaturing
5% PAGE with ethidium bromide staining. (C) Quantiﬁcation of EMSAs
using synthetic H1.2s. (D) Position of R42 (red) of X. laevis histone
H1.0b, which is identical to R53 of human H1.2, in chromatosomes(PDB: 5NL0).53
Fig. 3

We concluded that H1.2 with R53Cit reduced its binding
ability toward nucleosomes because of the lack of electrostaticinteraction between Arg and DNA, which would contribute to
the relaxation of chromatin and reprogramming of cells.50 By
our research using homogeneously modied H1.2, the direct
eﬀects of PTMs of H1.2 were examined.
Chemical synthesis of HP1a with Ru-4
To broaden the applicability of the organoruthenium-catalyzed
chemical protein synthesis, we selected heterochromatinprotein 1a (HP1a) as a second target. This protein contains two
globular domains, a chromo domain (CD) and a chromo
shadow domain (CSD), which are connected by a hinge region5932 | Chem. Sci., 2021, 12, 5926–5937

Edge Article(HR).54–57 CD recognizes the trimethylation of Lys 9 within
histone H3 (H3K9me3), a hallmark of transcriptionally silenced
chromatin.58 CSD is a domain responsible for the selfdimerization of HP1a, and also provides the interface tointeract with proteins bearing the PXVXL/I binding motif, suchas shugoshin 1 (Sgo1).59 In the presence of H3K9me3, CDinteracts with this modication and CSD forms intermolecularbridging, which condenses chromatin to form transcriptionallysilenced states.60 HP1a was reported to be extensively decorated
with PTMs,61 and these PTMs aﬀected the interaction pattern,
distribution, and localization of HP1a.57 Therefore, the elucidation of the functions of PTMs on HP1a was essential tounderstand the behaviour of dynamic chromatin.
In accordance with Fig. 4A, Cys mutations for NCL reactions
were incorporated at Ala sites (A98C, A129C, A153C), and fulllength HP1a was divided into ve peptide segments (peptides
13, 14, 15, 16, 20), and each segment was synthesized by FmocSPPS (Fig. S17†). C-terminal Asp 58, bearing thioesters of
peptide 20, was protected by the allyl group to inhibit hydrolysisof thioesters and isomer formation via intramolecular cyclization.62,63 The N-terminal Cys of peptides 14 and 15 were protected with the alloc groups, and acetamidomethyl (acm)groups were introduced at N-terminal Cys of peptide 16 (C59)
and inner Cys of peptides 13 (C160). Furthermore, to improve thesolubility of peptide 14, we installed a solubilizing tag bearing
phenylacetamidomethyl (phacm) group at C133 site, which wasremoved in the same deprotection method as acm groups.22
Peptides 13, 14, 15, and 16 were assembled in a one-pot
manner using Ru-4 catalyst (Fig. 4B). The removal of the alloc
groups proceeded with only 20 mol% of Ru-4, and, aer three
NCL steps and two deprotection steps, the desired intermediate
peptide 17 was obtained in 35% isolated yield (Fig. S18 andS19†). Free-radical desulfurization was then performed toconvert mutated Cys residues into original Ala residues(Fig. S20†). To remove the acm groups and solubilizing tag, werst employed sodium tetrachloropalladate (Na2PdCl4).21,64 As
a result, aer the addition of this Pd complex (5.0 equiv. toward
peptide 18), some aggregates appeared in the reaction solution,
which led to low recovery rates aer HPLC purication(Fig. S21†). Therefore, we used silver acetate (AgOAc) instead of
Na2PdCl4.65 This time, aggregates were not observed aer theovernight reaction and the desired product 19 was isolated in
62% yield (Fig. S22†). Finally, peptide 20 was ligated withpeptide 19, and the reaction reached completion aer 2 h(Fig. 4C). When we successively added Pd/TPPTS complex toremove the ally group at Asp 58, some aggregates appeared
again. When we analyzed the aggregation of full-length HP1a in
the presence of Pd or Ru by dynamic light scattering (DLS), thecatalytic amount of Ru did not contribute to the formation of
the aggregates (Fig. S24†). Therefore, Ru-4 was used for deprotection of the allyl group, and the desired full-length HP1a (21)was obtained without aggregation in 53% isolated yield(Fig. 4D). The measurement of Ru content by ICP-MS showedthat 0.00011% of Ru was attached on 21 (Table S3†). The
circular dichroism spectrum of 21 was measured aer refolding(Fig. 4E), and the ellipticity around 208–218 nm of 21 was lower
than that of recombinant HP1a, indicating that the antiparallel© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Fig. 4 Chemical synthesis of HP1a using the Ru catalyst. (A) Synthetic strategy. / represents the cleavage cites. Red region represents chromodomain,underlined region represents hinge region, and blue region represents chromo shadow domain of HP1a. (1) NCL condition: peptides (2 mM), MPAA (100mM) in denaturing buﬀer at pH 7.0, 37  C. Removal condition: Ru-4 (20 mol%), 37  C, 30 min. (2) Peptide (0.8 mM), TCEP (300 mM), GSH (150 mM), VA044 (20 mM) in denaturing buﬀer at pH 7.0, 37  C, overnight. (3) Peptide (0.5 mM), AgOAc (30 mM), H2O/AcOH (1 : 1), 37  C, overnight. (4) NCL
condition: peptides (1.5 mM), MPAA (100 mM) in denaturing buﬀer at pH 7.0, 37  C. Removal condition: Ru-4 (20 mol%), 37  C, 30 min. (B) Reaction
tracking of the one-pot ligation with a catalytic amount of Ru-4 (20 mol%) for the alloc deprotection by analytical HPLC (gradient: 15–51% for 30 min) at
220 nm. (a) 1st NCL (t ¼ 4 h). (b) 3rd NCL after overnight reaction. (C) Reaction tracking of NCL between peptides 19 and 20, followed by the allyl removalwith Ru-4 (20 mol%) by analytical HPLC (gradient: 15–51% for 30 min) at 220 nm. (a) NCL (t ¼ 3 min). (b) NCL (t ¼ 1.5 h). (c) Allyl deprotection with Ru-4.
2100 ¼ 21 bearing allyl-protected Asp 58. (D) HPLC proﬁle (gradient 15–51% for 30 min) and MALDI-TOF mass spectrum of puriﬁed peptide 21. Calculated
mass of 21 [M + H]+: 22 225.7; mass found [M + H]+: 22 226.0. (E) CD spectrum of 21. (F) Chemically synthesized HP1a with PTMs in this study. 21: HP1awithout PTMs. 21a and 21b: HP1a with PTMs at N-terminus. 21c, 21d, and 21e: HP1a with PTMs at HR. 21f: HP1a with Ub at K154 at CSD.

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Chemical Scienceb sheet of 21 was more stabilized compared to commerciallyavailable recombinant HP1a. To summarize the chemical
synthesis of HP1a, it would be diﬃcult to apply Pd complexesfor HP1a due to the aggregation probably caused by intermolecular bridging of Cys of HP1a by Pd complexes and hydrophobic interactions among CSDs. These results emphasizedthat the choice of an adequate metal complex was crucial toproduce a target protein.

Preparation of a diversity of HP1a with diﬀerent patterns of
PTMs
We then tried to create HP1a bearing specic PTMs. First, we
focused on phosphorylations decorated at Ser residues of the Nterminal tail of HP1a (S11, S12, S13, S14),66 which increase its
binding aﬃnity toward H3K9me3 and disturb the interaction
between DNA and basic patches in HR of HP1a (residues 89–91
and residues 104–107).67 However, the eﬀect of the number ofphosphorylated Ser at the N-terminal tail had not been investigated. Moreover, it was reported that Ser residues of HR inmouse HP1a are phosphorylated in cells.66,68 S95ph mediated by
NDR1 kinase is required for mitotic progression and Sgo1
binding to mitotic centromeres,69 and Aurora B kinase also
mediates mitotic phosphorylation of HP1a at HR (mainly at
S92).70 To investigate the eﬀect of the phosphorylation at HR on
the DNA binding aﬃnity of HP1a, phosphorylated HP1a wasprepared by enzymatic methods.66,70 However, the possibility ofphosphorylations at other Ser sites by kinases could not be
excluded and homogeneous HP1a bearing site-specic phosphorylations were required to investigate the precise property ofeach phosphorylation site. On the other hand, although the
responsible enzymes had not been discovered, Lys 91 and Lys
106 in basic areas of HP1a were reported as decorated withacetylation,61 but the inuence of acetylation on DNA binding
was still unclear. To construct homogeneously phosphorylatedor acetylated HP1a, we newly synthesized peptides 15a, 16a,
16b, 16c, 20a, and 20b by Fmoc-SPPS (Fig. S17†). By following
the synthetic scheme shown in Fig. 4A, we constructed double
phosphorylated (S13ph, S14ph) (21a) and quadruple phosphorylated (S11ph, S12ph, S13ph, S14ph) (21b) HP1a at the Nterminus, singly phosphorylated (S95ph) (21c) and triple
phosphorylated (S92ph, S95ph, S97ph) (21d) HP1a at HR anddouble acetylated (K91ac, K106ac) (21e) HP1a at HR (Fig. 4F).
We also focused on the PTMs decorated at CSD of HP1a.
Ubiquitination (Ub) at K154 promotes the degradation of HP1athrough the autophagy pathway to enable eﬃcient DNA repair.71
K154 is located at the dimerization interface of CSDs, and we
hypothesized that steric hindrance of Ub might disturb the
dimerization of CSD and interaction with proteins bearing the
PXVXL/I binding motif to relax chromatin structures. To create
HP1a with Ub at K154, we synthesized peptides 22 and 23(Fig. S26†). Peptide 22, which contained the C-terminal regionof Ub linked by isopeptide linkage at K154 of HP1a, wasprepared in a similar way to previous research (Scheme S2†).72
To achieve one-pot synthesis by combining the convergent
method, peptide 24, bearing acyl hydrazide at its C-terminus,
was also synthesized. Peptides 14, 22, 23, and 16–24 were5934 | Chem. Sci., 2021, 12, 5926–5937

Edge Article
assembled in a one-pot manner using Ru-4 to obtain peptide 25(Fig. S27 and S28†), and, aer desulfurization followed by theremoval of the acm groups and the solubilizing tag (Fig. S29 andS30†), peptide 27 was ligated with peptide 20 followed by theallyl deprotection with Ru-4 to aﬀord HP1a ubiquitinated at
K154 (21f) (>30 kDa) (Fig. 4F and S31†). Finally, we obtained six
kinds of full-length HP1a bearing the diﬀerent patterns of
PTMs. We note that these proteins were rapidly prepared owing
to one-pot ligation of divided peptide segments using only
catalytic amounts of organoruthenium complexes.
Fluorescence anisotropy assay of synthesized HP1aAer refolding of chemically synthesized HP1a by dialysis, werst tested the interaction of the CD of HP1a with H3K9me3
peptide bearing uorescein at its N-terminus (residues 1–20) by
uorescence anisotropy assay. The binding aﬃnity of 21 [dissociation constant (Kd): 23 mM] was almost the same as that ofrecombinant HP1a (Kd: 19 mM) (Fig. 5A and S32†), indicating that
the CD of synthetic HP1a properly recognized the H3K9me3
mark. We further examined the binding of Sgo1 peptide (residues
446–466) bearing uorescein toward dimerized interfaces of
HP1a CSDs. 21 showed a similar Kd value (Kd: 0.47 mM) toward
Sgo1 peptide as the recombinant one (Kd: 0.51 mM). These results
indicated that our chemically synthesized HP1a was properly
refolded, and recognized H3K9me3 modication and PXVXL/I
binding motif through CD and dimerized CSDs, respectively.
Next, we investigated the inuence of Ub at K154 of HP1a on thebinding ability toward the Sgo1 peptide. The Kd value of 21f wasincreased to 1.87 mM, suggesting that the aﬃnity was reduced to
3.9-fold compared to canonical HP1a. We reasoned that Ub
modication might disturb the intermolecular dimerization of
HP1a CSDs probably because of the steric hindrance of Ub,
which was elucidated for the rst time by our study. It wasenvisaged that HP1a CSD might further reduce its aﬃnity toward
its CSD by being further attached with Ub chains.
Investigation of DNA binding ability of modied HP1a
To investigate the relationship between PTMs installed at the Nterminus, HR, or CSD and DNA-binding ability of HP1a, EMSAs
were performed using 193-bp DNA. In this assay, HP1a without
PTMs eﬃciently bound DNA, and its Kd value was 0.75 mM(Fig. 5B and C), which was consistent with the previous results.66
21a, containing doubly phosphorylated Ser residues at its Nterminus, showed a slightly higher Kd value compared tonormal HP1a (Kd: 1.32 mM), but 21b, containing quadruple
phosphorylation, signicantly lowered the DNA-binding abilityof HP1a (Kd: 4.67 mM). This result indicated that the number ofphosphorylated Ser residues at the N-terminus of HP1a had an
impact on its DNA-binding ability. We also evaluated the other
PTMs on HR or CSD. 21c, with double acetylation of Lys at HR,
slightly lowered its DNA-binding ability (Kd: 1.79 mM), and these
PTMs were found to be little inuential. Surprisingly, 21d and
21e with Ser phosphorylation at HR had little inuence on theaﬃnity of HP1a toward DNA [Kd: 0.95 mM (21d), Kd: 0.82 mM(21e)], although basic patches on HR of HP1a were the main
responsible sites for the interaction with DNA.67 We found that© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Chemical Science

Biochemical assays of chemically synthesized HP1as. (A) Fluorescence anisotropy assay. Binding of the CD of recombinant HP1a orsynthetic HP1a (21) with ﬂuorescein-label H3 peptide bearing K9me3 (red: CD of HP1a, blue: N-terminal tail of histone H3 with K9me3, PDB:
3FDT) and binding of the CSD of recombinant HP1a, 21, or 21f with ﬂuorescein-label Sgo1 peptide (red: CSD of HP1b, blue: Sgo1 peptide, PDB:
3Q6S). (B) EMSAs using chemically synthesized HP1a with PTMs. Representative gel images of the binding of HP1a toward DNA. Various
concentrations of HP1a were incubated with 193-bp DNA. The complexes were analyzed by 8% native-PAGE and visualized with SYBR Gold. (C)Quantiﬁcation of the EMSAs using HP1a with PTMs at b N-terminus, c HR or CSD. (D) DLS measurement of complexes composed of 193-bp DNA(50 nM) and modiﬁed HP1a (10 mM).
Fig. 5the binding aﬃnity of 21f with Ub at CSD was almost identicalto that of normal HP1a (Kd: 0.71 mM).
Next, we analyzed the particle size of complexes composed of
DNA and HP1a by DLS. HP1a without PTMs formed large particles with DNA, and its diameter was around 1000 nm (Fig. 5D).
21a also formed large particles around 1000 nm diameter with
DNA. However, 21b dramatically inhibited the formation ofcomplexes and we could not observe large particles, which would
be caused by the lower DNA-binding ability of 21b. AlthoughHP1a possesses an acidic patch at its N-terminal (residues 15–19),
HP1a has a higher binding ability toward DNA than other HP1
homologs. However, through the decoration of phosphorylationsat S11–S14, HP1a signicantly reduces its binding aﬃnity by theextended acidic patch. Moreover, the number and location ofphosphorylation were critical to control its DNA-binding ability,
which would aﬀect the behavior of HP1a in the nucleus. Thesesignicant discoveries in epigenetics were claried by H1.2s andHP1as with site-specic PTMs prepared through our new
synthetic method using the organoruthenium catalyst.

© 2021 The Author(s). Published by the Royal Society of Chemistry

Conclusions
We have developed a highly streamlined methodology for thechemical synthesis of various decorated proteins utilizing an
air-tolerant organoruthenium catalyst. Aer the screening oforganoruthenium complexes, we claried that Ru complexes
coordinated by Cp and X,O-bidentate (X ¼ N or P) ligand eﬃciently removed the alloc group in catalytic manners under NCL
conditions, and that the TONs of Ru-4 and Ru-10 exceeded 70
even in the presence of 5000 equiv. of MPAA, which could not be
achieved with other current metal complexes. The organoruthenium catalyst was applied for total chemical protein
synthesis to produce linker histone H1.2 and HP1a with twoand six diﬀerent modication patterns and our technology
aﬀorded a large protein above 30 kDa (ubiquitinated HP1a).
Especially for the synthesis of HP1a, by selecting appropriate
metal complexes, we prevented the generation of aggregates
during the synthetic procedure. Through the analysis of chromatosomes reconstituted by using three types of synthetic
H1.2s via EMSAs, we discovered that R53Cit modication

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contributed to the reduction of the binding aﬃnity of H1.2 fornucleosomes. Furthermore, we revealed that four consecutive
phosphorylations at S11–S14 of HP1a extended the acidic patchat its N-terminus, signicantly reduced its DNA-binding ability,and disrupted the formation of complexes with DNA. Withhomogeneously modied proteins synthesized by using the Ru
catalyst, the biological signicance of the epigenetic marks onheterochromatin factors was elucidated. We believe that thestreamlined chemical synthesis of proteins containing PTMs
and other functional molecules with an organoruthenium
catalyst would be a key for the elucidation of the mystery of
biological phenomena and the creation of new therapeuticproteins.

Author contributions
N. K. performed all experiments, conceived and designed the
project, and wrote the manuscript. T. K. performed reconstitution of chromatosomes. T. K., H. K., H. M. assisted in thewriting of the manuscript and ESI.† A. O. and G. H. supervised
the project and edited the manuscript.

Conﬂicts of interest
There are no conicts to declare.

Acknowledgements
This work was supported by the Japan Society for the Promotionof Science (JSPS) KAKENHI 18H03931, 18H05504, and19K22245 (A. O.), 18K05313, 19H05287, and 20H04704 and JST
PRESTO JPMJPR19K6 (G. H.). N. K. was supported by a Research
Fellow of the Japan Society for the Promotion of Science
19J13608. This work was also supported by JSPS KAKENHI
JP18H05534, and the Platform Project for Supporting Drug
Discovery and Life Science Research (BINDS) from Japan Agency
for Medical Research and Development (AMED) under Grant
Number JP20am0101076 and JST ERATO Grant Number
JPMJER1901 (H. K.) and AMED under Grant Numbers
20he0622010h0001 (H. M.). We thank Dr Nozomu Suzuki(Nagoya University) to kindly allow us to measure CD spectra ofrecombinant and synthetic HP1as. We also thank Dr Hiromi
Takiguchi and Mr Tomohiro Kato for the measurement of Ru
contents on synthetic proteins by using ICP-MS.

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