Chemical Protein Modification
withRhodium Metallopeptide
Catalysts
B4 Furuta
2018/02/10

1

Contents
1. Introduction
2. Advances in chemical protein modification
2-1. Ionic reaction
2-2. Radical reaction
2-3. Pericyclic reaction
2-4. Transition metal reaction

3. Rhodium metallopeptide
4. Conclusion
2

Contents
1. Introduction
2. Advances in chemical protein modification
2-1. Ionic reaction
2-2. Radical reaction
2-3. Pericyclic reaction
2-4. Transition metal reaction

3. Rhodium metallopeptide
4. Conclusion31. Chemical Protein Modification• Purpose• study effects of post-translational
protein modifications• interrogate and intervene biologicalsystems• confer desired properties (affinityprobes, fluorophores, reactive tags,
increasing the circulation halftime,…)Omar B, and Gonçalo J. L.B., Chem. Rev. 2015, 115, 2174
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1. Chemical Protein Modification• Requirements for the reactions
•
•
•
•
•
•
•

functional group tolerance or compatibility
selectivity (site- or regio-)
water as a reaction media
near neutral pH and room temperature (or up to 40℃)
high reaction rates and high reaction efficiency
low reactant concentrations
nontoxic reagents

Ellen M. S., and Carolyn R. B., Angew. Chem. Int. Ed. 2009, 48, 6974
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Contents
1. Introduction
2. Advances in chemical protein modification
2-1. Ionic reaction
2-2. Radical reaction
2-3. Pericyclic reaction
2-4. Transition metal reaction

3. Rhodium metallopeptide
4. Conclusion6

2-1. Ionic reaction• Classical methods have relied on reactions at nucleophilic amino acids.

(a) Amide Formation, (b) Urea and Thiourea Formation, (c) Reductive Amination, (d) Cys-Specific Disulfide
Exchange, (e) Alkylation, and (f) Conjugate Addition to a Representative Maleimide Michael Acceptor
Omar B, and Gonçalo J. L.B., Chem. Rev. 2015, 115, 21747

2-1. Ionic reaction• Electrophilic aromatic substitution is utilized in the modification of Tyr.

(a) Reaction with Diazonium Salts, (b) Three-Component Mannich Reaction, (c) Reaction
with Preformed Imines, and (d) Ene-type Reaction with Diazodicarboxylate ReagentsOmar B, and Gonçalo J. L.B., Chem. Rev. 2015, 115, 21748

2-2. Radical reaction• Radicallic C(sp3)-C(sp3) bond formation• tolerant of aqueous conditions• unreactive with the majority of the functionality
Free radical chemistry would be a powerful method
for protein chemistry.

CysDhaProf. Davis Prof. Park

Ser-P

Raphael H., and Jeffrey W. B., Science, 2016, 354, 553
Benjamin G. D.,et al., Science, 2016, 354, aag1465
Hee-Sung P., et al., Science, 2016, 354, 623
9

2-2. Radical reaction• Four complementary modes of elimination of cysteine to dehydroalanine

Benjamin Davis’smethod
Benjamin G. D., et al., Chem. Sci., 2011, 2, 166610

2-3. Pericyclic reaction1.

6π-azaelectrocyclization
LysFukase K., et al., ChemBioChem 2008, 9, 2392

2.

Ene-like reaction with triazoline-dione

Carlos F. B. III., et al., J. Am. Chem. Soc. 2010, 132, 1523

3.

Photoinduced cycloaddition of tetrazoles

Madhavan N., et al., Org. Biomol. Chem., 2015, 13, 3202
11
Qing L., et al., Angew. Chem. Int. Ed. 2008, 47, 2832

2-4. Transition metal reaction
1. Reductive alkylation reaction of Lys 2. Tsuji-Trost reaction
with Ir complex
with Pd (π-allylpalladium complex)

McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490.

3. Oxidative coupling reaction
with Au, Pd, …

Che, C.-M., et al., Chem. Commun. 2013, 49, 1428.

Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080.

4. C-H activation reactions
with Rh carbenoid, Pd complex,…

Ball, Z. T., et al., J. AM. CHEM. SOC. 2010, 132, 6660

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2-4. Transition metal reaction
1. Reductive alkylation reaction of Lys 2. Tsuji-Trost reaction
with Ir complex
with Pd (π-allylpalladium complex)

McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490.

3. Oxidative coupling reaction
with Au, Pd, …

Che, C.-M., et al., Chem. Commun. 2013, 49, 1428.

Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 1080.

4. C-H activation reactions
with Rh carbenoid, Pd complex,…

Ball, Z. T., et al., J. AM. CHEM. SOC. 2010, 132, 6660

13

Contents
1. Introduction
2. Advances in chemical protein modification
2-1. Ionic reaction
2-2. Radical reaction
2-3. Pericyclic reaction
2-4. Transition metal reaction

3. Rhodium metallopeptide
4. Conclusion
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3. Earliest examples of carbenoid chemistry for protein modification• Copper-carbenoid chemistry

Although mechanistic underpinnings ofthe reaction were unknown at the time,
it is reasonable that
this is catalytic O-H insertion reaction.

inactivated
Active-site carboxyl residue
Moore S. et al., J. Biol. Chem., 1966, 241, 4295
Delpierre G. R., Fruton J. S., Proc. Natl. Acad. Sci. U. S. A., 1966, 56, 1817

38 years later …

Rh(II) complexes promote catalytic X-H
insertion reactions for chemical biology.
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3. Selective Trp Modification with Rh carbenoids in aqueous solution• Preliminary study with model compound• HONH2 … a necessary additive(presumably by binding to the metalcenter and stabilizing the reactiveintermediates)• compound 7 … reaction with H2O• compound 8 … electrocyclization
pathway

Antos, J. M.; Francis, M. B., et al., J. Am. Chem. Soc. 2004, 126, 10256.

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3. Selective Trp Modification with Rh carbenoids in aqueous solution• Reaction with horse heart myoglobin

Rh2(OAc)4(+)

Rh2(OAc)4(-)

Clean conversion to the singly and doubly modified products
utilizing rhodium carbenoid species with very high selectivity.
• Problem
Using HONH2 causes the low pH of the reaction medium (<3.5)
The scope of this methodology is limited.
Antos, J. M.; Francis, M. B., et al., J. Am. Chem. Soc. 2004, 126, 10256.

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3. Overcoming the drawbacks• Coordination sites of rhodiumThis dramatic drop in catalytic
activity around pH 6.0 can be
explained by considering thepKa of HONH2 (pKa = 5.97) .

pH-dependent binding of HONH2 toRh2(OAc)4 in aqueous solution

This ligand exchange process effectively
deactivates the Rh catalyst.
Francis, M. B., et al., J. Am. Chem. Soc. 2009, 131, 6301. 18

3. Overcoming the drawbacks• Optimization of additives

Proposed binding of tBuNHOH with RH2(OAc)4 at pH 6.0

Francis, M. B., et al., J. Am. Chem. Soc. 2009, 131, 6301. 19

3. Overcoming the drawbacks• Addition of tryptophan containing tagsa single buried
Trp

FKBP
Melittincondition:for 15 h, at room temperature,
pH 6.0, add tBuNH2OH-HCl>40 %modification
more reactive Trp

A general protein labeling strategy with Trp was establishedbased on the expression of Trp-containing tags.
Francis, M. B., et al., J. Am. Chem. Soc. 2009, 131, 6301. 20

3. Different approach
However, in the Francis’s method, there are many points to be improved.
• require large amounts of Rh complex• modification is limited to Trp• may be necessary to denature with pH or temperature• conversions were only moderate

Different approach : Rhodium Metallopeptide• catalytic amount of Rh complex• modification of a variety of amino acid side-chain including unreactive ones
• site-selectivity controlled by catalyst• in a mild reation condition• high conversion
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3. Dawn of the metallopeptide chemistry• Contribution of zinc-liganding amino acid residues for α-helix formationin the zinc finger peptidehydrophobicinteraction
Cys10Zn
His27

Cys5
His23
H23G

H27G

Zinc finger peptide

By choosing an appropriate amino acid sequence, small metallopeptide
with the desired coordination geometry can be synthesized.
Nomura A., Sugiura Y., Inorg. Chem. 2002, 41, 3693
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3. Zachary Ball’s Chemistry• Zachary T. BallAssociate Professor
Department of Chemistry, Rice University

Research themes
1. Rhodium metallopeptides (synthesisand structure)
2. Rhodium metallopeptides thatinteract with proteins

3. Small-molecule catalysis
4. Copper catalysis and other research23

3. Concept of the rhodium metallopeptide
proximity-driven reactivity

molecular recognition
using coiled coildirhodium metallopeptide bound to side chain carboxylate• stable with respect to ligand exchange in water at pH ≤ 7• catalyze X-H and C-H insertion in waterBall, Z. T. et al., J. Am. Chem. Soc. 2010, 132, 6660
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3. Synthesis and structure of the rhodium metallopeptide• Reversible synthesis of a peptide-dirhodium adductafter purification by HPLCb) with KOAc
ZF + Rh crudeZF

In the presence of reactive amino acids, yield is low….
with Ac-His-NHMe (2 eq.) … > 50 % yieldwith Ac-Met-OMe (2 eq.) … ca. 40 % yield
Ball, Z. T. et al., Chem. Eur. J. 2009, 15, 8961
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3. Synthesis and structure of the rhodium metallopeptide• Structure of the rhodium metallopeptide
CD spectra

molecular dynamics calculations
a new negative peak
appearance of new turn structure

ZF
ZF-[Rh2(OAc)2]
complex ZF–[Rh2(OAc)2]

a strong negative peak disappears
disappearance of a random coil structure
analogous zinc-finger sequence

Dirhodium binding can be used to control the conformation of the boundpeptide.
Ball, Z. T. et al., Chem. Eur. J. 2009, 15, 8961
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3. Coiled coil interaction• Coiled coil … a simple and robust peptide assembly
Helical wheel diagram…
heptad repeat

H … hydrophobic
P … polarhydrophobic interfaces

e.g. E3/K3 pair

Derek N. W., et al., Faraday Discuss., 2009, 143, 1－+－+at the position of e and g,
K3 … lysine-rich
E3 … glutamate-richelectrostatic interaction
Ball, Z. T. et al., J. Am. Chem. Soc. 2010, 132, 6660
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3. Site-specific protein modification• comparison of rhodium metallopeptide to Rh2(OAc)4E3gW.

Only 2 mol % of K3a,eRh2 shows >103 rate enhancementcompared with 100 mol % of Rh2(OAc)4 due to substrate binding.
Ball, Z. T. et al., J. Am. Chem. Soc. 2010, 132, 6660

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3. Site-specific protein modification• Effect of the coiled coil interaction on the rate of modification

noncomplementary charges in
the flanking e and g positions

Trp residue at the opposite flank of
the coiled coil interface (position e)

noncomplementary charges in
the flanking e and g positionsb), c)

parallel > antiparallel

Ball, Z. T. et al., J. Am. Chem. Soc. 2010, 132, 6660

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3. Site-specific protein modification• Expansion of the scope of dirhodium-catalyzed side-chain modification

Wrandom E3gX

K3a,eRh2

Proximity-driven reactivity promotes thesite-specificity even in Tyr or Phe residue.

Unreactive residues (aliphatic residues)
can be modified with this rhodium
metallopeptide?
Ball, Z. T. et al., J. Am. Chem. Soc. 2010, 132, 6660

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3. Site-specific protein modification• Expansion of the scope of dirhodium-catalyzed side-chain modification>50% conversion for
X = Trp, Tyr, Phe, Asn,Gln, Asp, Glu, Arg, Cys(in order of reactivity)
comprising >40% of
natural protein space!

Ball, Z. T. et al., Chem. Sci., 2011, 2, 690
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3. Application of rhodium metallopeptide (1)Orthogonal protein modification in lysate
biotin-diazo reagent

high specificity and orthogonality !

1 : 1 mixture of lysates fromexpression of MBP-E3g2W
and GST-E3e2W
Ball, Z. T. et al., J. Am. Chem. Soc. 2012, 134, 10138

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3. Application of rhodium metallopeptide (2)SH3 domain site-selective modification
SH3 domain
protein (Fyn)SH3 binding peptide
VSL12(VSLARRPLPPLPP)
is used as ligand of Rh2• Improvement of efficiency and realization of useful functionalization①
Negative control doesn’t show +1
modification peak.

diazo 3 can be bound tofluorophore by Cu-mediatedazide-alkyne cycloaddition.
Non-selective reactivity diazo 2 is due tointeraction of the thiolester group with ametallocarbene intermediate.

Ball, Z. T. et al., Angew. Chem. Int. Ed. 2015, 54, 4587

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3. Application of rhodium metallopeptide (2)SH3 domain site-selective modification• Improvement of efficiency and realization of useful functionalization②
R5ERh → R5DRh … decrease conformational freedom (aspartate < gultamate)mixture of +1 mod and +2 modonly +1 modYes kinase Selectivity is retained
across of the othermembers of the Src SH3family.
 Selectivity is retainedeven in Yes SH3(containing buried SH3and Trp).

Ball, Z. T. et al., Angew. Chem. Int. Ed. 2015, 54, 4587

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3. Application of rhodium metallopeptide (2)SH3 domain site-selective modification• A wide range of Src-family SH3 domains modification

R5DRhL3ERh11LRh

Functionalization could be directed to three different sitesaround the peptide-binding sites.
Custom tailored-catalysts may deliver modification of virtually
any protein of interest, especially given advances in methods forligand discovery.
Ball, Z. T. et al., Angew. Chem. Int. Ed. 2015, 54, 4587

35

3. Application of rhodium metallopeptide (3)Identification of naphthalene-sulfonamide-binding site of STAT3… binding to SH2 domain(disrupting SH2-phosphotyrosine)or other site ?

Ball, Z. T. et al., Angew. Chem. Int. Ed. 2015, 54, 13085
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3. Application of rhodium metallopeptide (3)Identification of naphthalene-sulfonamide-binding site of STAT3

Conjugate to 3azide-7coumarin lane d, k … p-peptide doesn’t inhibit C188-9-Rh2 binding.
naphthalene sulfonamide doesn’t bind to SH2 domain.
 lane e, g, l … Other naphthalene sulfonamides inhibit C188-9-Rh2 binding.
Other naphthalene sulfonamides are bound to the same site.
Ball, Z. T. et al., Angew. Chem. Int. Ed. 2015, 54, 13085
37

3. Application of rhodium metallopeptide (3)Identification of naphthalene-sulfonamide-binding site of STAT3
Tryptic digestion of the modified STAT3LC-MS/MS

This Phe174
was identified
as the site ofmodification!

Structureof STAT3• Phe174 lies in a coiled coil domain.
One helix
turn away

This is consistent with the fact that the key residue in the CCD, apoint mutant of which exerted the largest effect on STAT3 activity
specially SH2 affinity, was Asp170.

Coiled coil region was identified as the targeting siteother than SH2.
Ball, Z. T. et al., Angew. Chem. Int. Ed. 2015, 54, 13085

38

3. Application of rhodium metallopeptide (4)Assessing intracellular fate of rhodium complex
In utilizing of rhodium complexes in living cells,• toxicity … inherently low (able to design with the proper ligand choice)• stability … limited stability under biologically relevant conditions (ligand exchange,irreversible reduction, …)• Improvement of the stability of rhodium complex

Ball, Z. T. et al., Chem. Commun., 2016, 52, 11685
39

3. Application of rhodium metallopeptide (4)Assessing intracellular fate of rhodium complex• Improvement of the stability of rhodium complex• faster decomposition in containing
surface-exposed thiols
Proposed mechanism

half-life of 1b with additive
3b was treated with aredox mediators.

• N-methylmaleimide (NMM),
a common thiol blocking
reagent, retards decompositionof 3b and slows the rate offluorescence turn-on.
3b (15 uM) in 1x PBS with GSH (10 mM)

Ball, Z. T. et al., Chem. Commun., 2016, 52, 11685

40

3. Application of rhodium metallopeptide (4)Assessing intracellular fate of rhodium complex• Improvement of the stability of rhodium complex

sterically demandingcarboxylate
1b ... maximally fluorescent within 30 min
2b … mostly dark after 30 min, but continued
to grow in brightness over time

Fluorescent image of cells (NIH 3T3)
Fluorescent probe (488 nm, green) is shown
overlaying cytosol stain (561 nm, red).

A long-lived rhodium
Fluorescence of 2b in MOL13-M cells
complex inside cells was(1 x 105) treated with 2b (30 mM, 0.5 h)aliquots taken at different times weresuccessfully prepared.
analyzed by flow cytometry.

Ball, Z. T. et al., Chem. Commun., 2016, 52, 11685

41

3. Application of rhodium metallopeptide (4)Assessing intracellular fate of rhodium complex• Improvement of the stability of rhodium complex
Improvement of cellular potency
to inhibit STAT3 activation(phosphorylation)!

• Intracellurar stability studies can be used
to optimize rhodium complex structure.

in vitro potency does not changeBall, Z. T. et al., Chem. Commun., 2016, 52, 11685

42

3. Application of rhodium metallopeptide (5)Antibody modification using hexa-rhodium metallopeptide
Assumed scheme

hexa-rhodium metallopeptide (triply metalated peptide)
singly metalated
peptides displays
much lower reactivity•

The reactivity of triplymetalated peptide may stemfrom synergetic action of thedirhodium cores.
••

one Rh acts as a catalytic siteother Rhs act as Lewis acids toanchor the catalyst at the bindingsite on the Fc region

Ball, Z. T. et al., J. Am. Chem. Soc. 2017, 139, 12617

43

3. Application of rhodium metallopeptide (5)Antibody modification using hexa-rhodium metallopeptide• Antibody’s functionalization using azide-alkyne cycloaddion①
Fluorescently labeling

SK-BR3 cells(Her2-overexpressing breast cancer cell line)

Herceptin-fluorescein
conjugate• localization of the fluorescein fluorescence on the surface of the cells (B),• absent in control experiment (E)Modified antibodies retain antigen-binding properties.
So, it can be used as a fluorescently labelled antibody.
Ball, Z. T. et al., J. Am. Chem. Soc. 2017, 139, 12617

44

3. Application of rhodium metallopeptide (5)Antibody modification using hexa-rhodium metallopeptide• Antibody’s functionalization using azide-alkyne cycloaddion②,③
Desthiobiotinylation for facile purification and Preparation of antibody-drug conjugates (ADCs)

3 : human IgG
4 : Herceptin•Chemiluminescence determined a 1:1 herceptin/ doxorubicin
ratio (DAR, drug antibody ratio).

•ADCs bind protein A with efficacy comparable to theunmodified antibodies.
Secondary antibodies show binding ability to F(ab’)2 of ADCs as
well as that of unmodified ones.

•Ball, Z. T. et al., J. Am. Chem. Soc. 2017, 139, 12617

45

3. Application of rhodium metallopeptide (5)Antibody modification using hexa-rhodium metallopeptide• Antibody’s functionalization using azide-alkyne cycloaddion②,③
Desthiobiotinylation for facile purification and Preparation of antibody-drug conjugates (ADCs)
SK-BR3 cells

3 : human IgG
4 : Herceptin•4-doxo retains affinity for both Her2 receptor and theantihuman antibodies.
the function of antibody can be maintainedin presence of modification when modified
site was chosen with tactful design.
Ball, Z. T. et al., J. Am. Chem. Soc. 2017, 139, 12617

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Contents
1. Introduction
2. Advances in chemical protein modification
2-1. Ionic reaction
2-2. Radical reaction
2-3. Pericyclic reaction
2-4. Transition metal reaction

3. Rhodium metallopeptide
4. Conclusion
47

4. Conclusion• Rhodium carbenoid is the approach of chemical modification of Trp
through C-H activation.
• Directing with the ligand peptide (rhodium metallopeptide) enables
catalytic and residue-selective reaction.
• Directing with the ligand peptide amino acids comprising >40% of
natural protein other than Trp. (over 50% conversion)• Many applications of rhodium metallopeptide are possible even incellular reaction or preparation of ADCs.

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