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Selective Metal-Site-Guided Arylation of Proteins Jens Willwacher, Ritu Raj, Shabaz Mohammed, and Benjamin G. Davis J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b04043 • Publication Date (Web): 23 Jun 2016 Downloaded from http://pubs.acs.org on June 27, 2016
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Journal of the American Chemical Society
Selective Metal-Site-Guided Arylation of Proteins Jens Willwacher, Ritu Raj, Shabaz Mohammed, Benjamin G. Davis* Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK Supporting Information Placeholder ABSTRACT: We describe palladium-mediated S-arylation that exploits natural metal-binding motifs to ensure high siteselectivity for a proximal reactive residue. This allows the chemical identification not only of proteins that bind metals but the environment of the metal-binding site itself through proteomic analysis of arylation sites. The transformation is easy to perform under standard conditions, does not require the isolation of a reactive Ar-Pd complex, is broad in scope and is applicable in cell lysates as well as to covalent inhibition/modulation of metaldependent enzymatic activity. Post-translational modification is nature’s method to decorate proteins (typically enzymatically) with structurally di1 verse functional ‘switches’ and recognition sites. Its advantage over the currently available chemical ‘toolbox’ for protein chemistry is its ability to carry out such reactions in a 2 highly site-selective manner. Whilst this is, in part, achieved via chemoselectivity, it is often also guided by mutual recognition of secondary structure in either the protein substrate or the modifying enzyme catalyst. This guides regioselection and enables selectivity for certain residues, often related to or guided by function. Whilst regioselectivity directed simply by accessibility has proved a potentially successful approach 3 in chemical protein modification, the ability to direct a protein-modifying chemical catalytic centre by virtue of functionally inherent motifs might allow regioselection guided by endogenous features in a manner that partially mimics nature’s approach. Here we show, as a proof-of-principle, a designed method for site-selective protein modification that appears to rely, at least in part, upon natural metal-binding. Several strategies for chemical protein modification have 4,5 been developed; a classic variant relies on (sometimes partial) chemoselectivity of certain natural side-chains (e.g. 6 lysine (Lys) / cysteine (Cys), Scheme 1a). For example, the amine/thiol groups of these residues can exhibit higher nucleophilicity towards some electrophiles. Most of these methods rely on solvent accessibility/exposure of particular Lys- or Cys-residues for regioselectivity, and often generate heterogeneous product mixtures. To reduce such heterogeneity in classical protein-conjugates, conceptually different site-selective modification strategies have emerged. These often rely on the incorporation of functional groups with beneficial reactivity (‘tags’) for improved chemoselection and hence regioselection, if reaction is complete. These include genetically-, enzymatically- and chemically- installed unnat7,8 ural amino acids (UAAs) with ‘bioorthogonal’ reactivity and enhanced chemoselectivity with an appropriate reagent 9,10 (Scheme 1b). Metal-mediated examples include azide11,12 13 alkyne ‘cycloadditions’, cross-couplings, and olefin cross14 metathesis. Elegant, prior, metal-guided covalent protein
15
modifications have typically exploited unnatural metalbinding peptide sequences at the termini of proteins, such as 16,17 18-20 21 oligo-aspartate, -histidine or -cysteine motifs. However, covalent protein modifications based on naturally oc22 curring motifs to achieve site-selectivity are rare. The need for programmed genetic incorporation of unnatural sequences at restricted locations prohibits their use as a tool for a priori identification of metal-binding proteins. We therefore set out to develop a conceptually different approach for siteselective protein modification by relying on endogenous metal-binding motifs (Scheme 1c). If successful, this would not only enable labeling of metal-binding proteins in vitro but also identification in cell lysates (e.g. through direct attachment of ‘pull-down handles’).
Scheme 1. Established strategies compared to metalsite guided protein modification. a Classical strategy: modification using unique reactivity pattern of natural amino acids SH
SH SH ligation
S S
S undesired oligo-modification
b Previous work: site-selective modification using chemoselectivity of unnatural amino acids
genetic incorporation of tag
chemoselective conjugation
c This work: metal-guiding motif allows site-selective modification of natural amino acids (plus functional mapping of endogenous sites and metal binding proteins) SH [M]
SH L L [M]
LL SH protein containing naturally occuring metal biniding motif (e.g. DxD)
S SH
:tag (may include linker)
guided site-selective modification : modification e.g. label
LL SH L : ligand residue
One form of common, naturally-occurring metal-binding motif is that found in enzyme active sites that enable metaldependent catalysis. We reasoned that these could direct a reactive metal complex into a given protein, thereby guiding selective reaction. Our proposed protein modification thus relied on two characteristic structural patterns: a) a metalbinding site that would steer the reactive complex; and b) a proximal, reactive amino-acid residue to react with the metal 23 complex resulting in covalent modification. Class A glycosyltransferases (GTs) are archetypal metal-dependent enzymes and we chose mannosylglycerate synthase (MGS), originally isolated from the thermophilic bacteria 24 Rhodothermus marinus, as a model system. MGS was the first mannosyltransferase to be fully structurally character25 26 ized and displays a well-examined metal binding profile. GT-A folds contain a common DxD sequence as a metal- 27 26 binding motif ; in MGS it is D100A101D102 (PDB 2BO6). We speculated that this could steer an M(II)-Aryl species generated in situ into the active site, allowing it to react with
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Scheme 2. Development, analysis and scope of guided site-selective, Pd-mediated, cysteine-arylation. a General reaction scheme (optimized conditions) NaO N N
b Representative LC-MS spectrum of crude reaction mixture (Ar = 2 ): ion series and deconvoluted spectra NMe 2 •Pd(OAc) 2 (80 eq)
NaO
2
ArI (500 eq) 35 mM TRIS, pH 7.6 5% MeCN or DMF 65°C, 4h
HS
S Ar
MGS (1.0 mg/mL) modified MGS : DXD metal binding site MW: 47514 MW: 47514 + Δm 233 c Major modified peptide identified by nanoLC-MS/MS: C(–S–2 )FAAIQSLQHE
S
MGS–2
O
NH 2
MGS
d Scope of site-selective cysteine S-arylation with respect to aryl iodide. *: 250 eq. of ArI were used O
H 2N 1 Δm calc: +76 found: +76 conversion: 83% 9 D5 Δm calc: +81 found: +79 conversion: 77% 15 N Δm calc: +77 found: +79 conversion*: 24%
2 O Δm calc: +119 found: +119 conversion: 88% 10
O
H 2N
3 Δm calc: +133 found: +134 conversion: 71%
O
MeO 4 Δm calc: +148 found: +147 conversion: 39%
11
12
13
MeO
HO 2C
N3
Δm calc: +106 found: +106 conversion: 40%
Δm calc: +120 found: +120 conversion*: 86%
Δm calc: +131 found: +132 conversion: 52%
16 HO HO
OH O OH
17 O
HO 5 Δm calc: +134 found: +135 conversion*: 83%
Δm calc: +268 found: +267 conversion: 25%
HO HO
a range of potential residues, depending on reactivity & 28 mechanism, including proximal Cys233 thiolate (Sch. 1c). We first investigated several transition metal pre13,29,30 catalysts in combination with iodobenzene for modify31 ing MGS-His6. Major emphasis was placed on palladium as a suitable metal mediator, as previous studies have shown its 13,32 versatility in aqueous Suzuki- and Sonogashira-coupling 33 reactions. After a screen of a variety of systems, Pd(OAc)2 ligated with the disodium salt of N,N-dimethyl-2-amino-4,6dihydroxypyrimidine (DM-ADHP) proved most reactive at 65°C and was used as a precatalyst (Scheme 2a). Other precatalysts such as Pd(OC(O)CF3)2, Pd(OAc)2, or other watersoluble palladium pre-catalysts were unreactive and using these only unmodified MGS was obtained (see SI). At ambient temperature or 37 °C, only unmodified protein was detected by LC-MS analysis (see SI). This unique performance of DM-ADHP as ligand suggested that at least one molecule of ligand was still attached to the Pd(II)-Aryl species during the reaction or was necessary to deliver the Pd-complex to the guiding site. Independently, Buchwald et al. have also elegantly shown that Pd-mediated cysteine S-arylation can 34 be rendered general by tuning the surrounding ligand. Under our optimized conditions, only a single product was observed in >85% conversion after 4 h (Scheme 2b). Consistent with our selectivity hypothesis, potential di- or oligomodified species were not detected by LC-MS despite other potential modifiable sites (e.g. Cys34, Cys209, Cys305); notably C305 is the most solvent exposed but does not arylate,
6 O 2N Δm calc: +121 found: +121 conversion: 75%
Δm calc: +101 found: +102 conversion: 82%
HN H
NH H
H N
S
O
NHAc
8 F 3C Δm calc: +144 found: +145 conversion: 43%
Δm calc: +309 found: +309 conversion: 19%
Δm calc: +331 found: +331 conversion: 26%
O 18
OH O
F Δm calc: +94 found: +95 conversion: 61%
O
14
N
7
HO HO
OH OH O
Δm calc: +268 found: +267 conversion: 10%
O further suggesting regioselectivity directed by protein structure and aryl-Pd species. Longer reaction times led only to loss of protein through degradation / precipitation pathways. The site of the modification was examined by LC-MS/MS analysis after in-gel digest with trypsin/endoproteinase GluC. This confirmed C233 as the major reaction site (Scheme 2c). Modified vs unmodified Cys sites were also established semiquantitatively through a carbamidomethylation strategy (see SI); this not only confirmed C233 as the primary reaction site but also highlighted the retained high reactivity of the Cys residues (which were unmodified by our metal-guided process) to non-directed Cys alkylation chemistry. To further test these sites of reactivity, we prepared mutants in which potentially reactive Cys were exchanged for Ala (C34A, C209A, C233A). The reaction outcome remained unchanged with the C34A or C209A (Table 1, entry 2-3), whereas no product (
