Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Nickel(II)-Promoted Amide N−H Arylation of Pyroglutamate− Histidine with Arylboronic Acid Reagents Kengo Hanaya, Mary K. Miller, and Zachary T. Ball* Department of Chemistry, Rice University, Houston, Texas 77005, United States
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S Supporting Information *
ABSTRACT: Small and simple bioorthogonal reactive handles that can be readily encoded by natural processes are important for bioconjugation. A rapid nickel-promoted N−H arylation of pyroglutamate−histidine sequences with 2-nitroarylboronic acids proceeds under mild aqueous conditions. Chemoselective activation of a lactam amide N−H within a peptide or protein provides a new approach to selective conjugation in polyamide structures.
U
nique bioorthogonal reactive handles are crucially important to modern biological sciences. Incorporating such handles into proteins provides access to complex biopolymer conjugates with atomic precision. Typically, bioorthogonal functionality is completely exogenous, such as selective incorporation of reactive azide, alkyne, 5-hydroxytryptophan,1 or aldehyde/ketone2,3 functional groups, some of which can be engineered into proteins by unnatural amino-acid incorporation. In contrast, naturally occurring and readily encodable moieties with biorthogonal reactivity are quite rare, despite the many benefits of a naturally encoded approach to bioorthogonal chemistry. In this letter, we describe a new nickel-promoted arylation of the pyroglutamate residue, pyroglutamate−histidine sequences in particular. Examples of biorthogonal reactivity with natural processes include sequence “tags” composed of many canonical amino acids.4−7 Several natural posttranslational modifications have potentially unique reactivity, but relatively few have been harnessed as general tools for protein chemistry. Some progress has been made to access the unique electrophile dehydroalanine as a residue in proteins,8 and selenocysteine is sufficiently rare9 that it can be considered a biorthogonal group. We recently observed10 selective copper-catalyzed reactivity at pyroglutamate−histidine sequences that led us to consider whether that dipeptide sequence could be viewed as a uniquely reactive handle. The pyroglutamate (Glp) residue occurs naturally in peptides and proteins and is the result of a condensation reaction at an N-terminal glutamine or glutamate to form a pyrrolidone (Figure 1). The biological function of pyroglutamine residues is poorly understood.11 Its formation is governed by cyclase enzymes, although nonenzymatic cyclization is also known.12 Pyroglutamate is found in diverse polypeptides, including antibodies,13 Aβ peptides,14 and © XXXX American Chemical Society
Figure 1. Histidine-directed C−N bond formation.
neuronal hormones.15 Recent studies have led to the development of expression systems for the selective and efficient production of pyroglutamate-containing proteins.16 As an N-terminal posttranslational modification, pyroglutamate would serve as a complementary target to existing N-terminal amine modification.17−21 From a chemical perspective, the pyroglutamate lactam structure appears to be a poor target for selective chemistry, given the ubiquity of amide bonds in peptides and proteins and the generally low reactivity of backbone amide bonds. Aqueous conditions for any arylation of amide N−H bonds are rare.22−24 For boronic acid reagents, Chan-Lam-type coupling with N−H bonds is well established for copper catalysts,25 but water-tolerant conditions are limited.26 Received: February 28, 2019
A
DOI: 10.1021/acs.orglett.9b00759 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Given these challenges, we were surprised to find that luteinizing hormone-releasing hormone (LHRH, 1), a peptide hormone with an N-terminal pyroglutamate−histidine sequence, reacts efficiently with 2-nitrophenylboronic acid (2a) in the presence of a simple nickel salt, providing an amide arylation product (Figure 2a−b and Figures S1−S5). Nickel
activate a specific N−H for productive chemical transformations. The reaction exhibited a significant pH dependence (Figure 2c). Under mild conditions in moderately basic aqueous solutions (pH 9.0−9.5), LHRH reacts within minutes under the developed conditions, but reactivity dropped significantly at pH 8.5 and below. This observation is in line with the sharp pH dependence observed for the formation of bis(amidate) nickel complexes at histidine sites.31 Fragmentation analysis by MS/MS verified the pyrrolidone N−H as the site of arylation (Figure 2e and Figure S2). Furthermore, the reaction with a simple tripeptide thyrotropin-releasing hormone (TRH, GlpHis-Pro) allowed product isolation and COSY NMR analysis, wherein disappearance of the pyroglutamate N−H was observed, and the α-proton of pyroglutamate shifted downfield (δ 4.06 → 4.78), without significant chemical shift changes to other protons (Figure S3). The reaction is quite rapid, with a half-life < 5 min under relevant conditions. To compare kinetic with other bioorthogonal reactions, we calculated an apparent second-order rate constant, kapp ∼ 1.7 M−1 s−1 (where kapp = k1/[2a]). By the definition of the terma substance increasing reaction rate without undergoing any chemical changeNi2+ is a catalyst in this transformation. Indeed, a substoichiometric Ni2+ (0.1 mM, 50 mol %) efficiently arylated peptide 1 with excellent conversion (>90%) after 30 min (Figure S4). Nonetheless, as with most metal-promoted bioconjugation reactions, stoichiometric concentrations of Ni2+ are probably most appropriate for effective reactions at biologically relevant substrate concentrations. We conducted a survey of boronic acid reagents and found a strict requirement for ortho π-conjugated, electron-deficient groups to produce the N-arylation product (2a−c and 2k−r in Figure 3; see also Figures S6 and S7). Reagents with acetyl (2b) or carboxamide (2c) substituents in the 2 position were reactive, although 2-nitro compounds had the highest activity. Compounds with other substitution patterns were unreactive (2d−f), as were ortho-substituted phenylboronic acids without a π-conjugated electron-deficient group (2g−j). Gratifyingly, the 2-nitrophenylboronic acid core allowed substitutions with a wide range of useful functionality and reactive handles (2k−r). The boronic acid scope of this Ni2+-promoted process differs significantly from Cu2+ N−H arylation,32 wherein ortho substitution is not tolerated and electron-rich arylboronic acids react efficiently. The Glp-His dipeptide is necessary for efficient coupling with 2-nitrophenylboronic acid reagents (Table 1). The hormone leuprolide (4) also reacts efficiently at its Glp-His terminus (entry 2 and Figure S8). Pyroglutamate (entry 3) or histidine (entries 5−7) alone are also unreactive. Proline− histidine located on the N-terminus, a structural analogue of Glp-His, resulted in low conversion (entry 4). Nonhistidinecontaining peptide did not afford detectable product (entry 8), and peptides with a disulfide bond were unreactive (entry 10), unlike reduced cysteine residues, which do react27 under these conditions. In a preliminary experiment, we sought to establish that the reactivity is compatible with more complex polypeptide structures (Figure 4). We chemically introduced a Glp-His tag (∼3.5 tags/molecule) into chymotrypsinogen A, as a model protein, via a reactive NHS ester (Figure S9). Notably, chymotrypsinogen A contains four disulfide linkages but no free cysteine thiols. The crude modification reaction with alkyne-functionalized 2-nitrophenylboronate 2q and tagged
Figure 2. (a) Modification of LHRH (1) with 2-nitrophenylboronic acid 2a. Reaction conditions: 1 (0.2 mM), boronic acid 2a (2 mM), and Ni(OAc)2 (0.2 mM) in N-methylmorpholine (NMM) buffer (10 mM, pH 8.0 and 8.5) or N,N′-diethylpiperazine (NEP) buffer (10 mM, pH 9.0 and 9.5) at 37 °C. (b) RP-HPLC analysis data of crude reaction mixtures after incubation at pH 9.0 for 0, 10, and 30 min. Numbers on peaks represent retention times. The fractions at 12.0 and 12.9 min correspond to 1 and 3a, respectively. (c) Kinetic data for reaction conversion by RP-HPLC analysis at varying pH. (d) MALDI-TOF MS spectrum for crude reaction mixture after incubation at pH 9.0 for 30 min. (e) Enlarged view of the MALDITOF MS/MS spectrum of 3a.
promoters of Chan−Lam reactivity are quite rare;27 we are aware of only a single report of N−H reactivity, which was reported to be incompatible with protic solvents.28 Compared to this previously reported reactivity, the chemistry we observe here is an example of a complete selectivity switch: Selective reactivity occurred at a (previously reported unreactive) secondary amide (pyroglutamate) in the presence of (previously reported reactive) primary amine side chains. This discovery complements related copper-catalyzed reactivity, for which ortho substitution is not tolerated.10 Furthermore, the significantly lower cellular toxicity of Ni2+ ions compared to that of Cu2+ ions29,30 could be important in biological applications. Taken together, copper- and nickelpromoted reactions point to a general role for pyroglutamate− histidine sequences to bind transition metals and also to B
DOI: 10.1021/acs.orglett.9b00759 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 4. Modification of Glp-His-tagged chymotrypsionogen A with 2q or 13 in the presence of Ni(OAc)2. (b) Gel images of modification reaction of Glp-His-tagged and native chymotrypsinogen A with 2q or 13 in the presence or absence of Ni(OAc)2. Reaction conditions: proteins (20 μM), boronic acid (2.0 mM), and Ni(OAc)2 (0.4 mM) in NEP buffer (10 mM, pH 9.0) at 37 °C for 30 min. (c) Reaction kinetics of modification of Glp-His-tagged (red squares) and native chymotrypsinogen A (blue triangles) with 2q, analyzed by chemical blotting. Reaction conditions: proteins (20 μM), 2q (2.0 mM), and Ni(OAc)2 (0.4 mM) in NEP buffer (10 mM, pH 9.0) at 37 °C for a given period. The plot represents the relative fluorescence intensity of bands at a certain time point compared with the intensity of Glp-His chymotrypsinogen A after 35 min (intensity = 1.0). (d) GFP expressed with Glp-His at the N-terminus. (e) Gel images of modification reaction of Glp-His GFP-GST with 2q or 13 in the presence or absence of Ni(OAc)2 (GST = glutathione S-transferase, GFP = green fluorescent protein). Free cysteines were capped using iodoacetamide. Reaction conditions: proteins (10 μM), boronic acid (2.0 mM), and Ni(OAc)2 (0.4 mM) in NEP buffer (50 mM, pH 9.0) at 37 °C for 1 h.
Figure 3. Scope of boronic acid. Conditions: LHRH (1) (0.2 mM), boronic acid 2a−r (2 mM), and Ni(OAc)2 (0.2 mM) in N,Ndiethylpiperazine (NEP) buffer (10 mM, pH 9.0) at 37 °C for 30 min. Relative conversion shown in the parentheses was assessed by RPHPLC analysis of the crude reaction mixture, unless otherwise indicated (“*” indicates conversion by MALDI-MS).
Table 1. Peptide Screensa entry
peptide
conv. (%)b
1 2 3 4 5 6 7 8 9 10
pEHWSYGLRPG-NH2 (1) pEHWSYlLRP-NHEt (4) pEQRLGNQWAVHLM-NH2 (5) H-PHPFHFFVYK-OH (6) H-WHWLQL-OH (7) Ac-SYSMQHFRWGKPVGKKR-OH (8) H-DRVYIHPFHL-OH (9) H-RPPGFSPFR-OH (10) Ac-RRWWCR-NH2 (11) H-AGCKNFFWKTFTSSC-OH (C3−C14) (12)
>99 98 −c 12 −c −c 2 −c >99 −c
study (Figure 4b, lanes 2, 3, and 5, and Figure S10). Native (untagged) chymotrypsinogen A showed no detectable alkyne incorporation (Figure 4b, lane 5). Modification kinetics were similar to the results from peptide studies. Maximal alkyne incorporation was achieved within minutes at 37 °C (Figure 4c and Figure S11). Pyroglutamate is readily incorporated into proteins of interest by E. coli expression systems,10,16 so we also examined a tagged glutathione GST−GLP fusion protein (Figure 4d,e).10 Again, we observed modification only in the presence of Ni2+ and the reactive boronic acid 2q. In the latter case, surface cysteine thiols were blocked (iodoacetamide) in order to avoid competing cysteine arylation. Although future work will be needed to validate the selectivity and generality of this method for protein substrates, this preliminary investigation hints that even quite complex and large molecularweight substrates are feasible. Nickel-promoted activation of pyroglutamate presumably proceeds via the formation of a tridentate, bis(amidate) complex (Figure 1) that resembles the well-studied amino-
a
Conditions: peptide (0.2 mM), 2a (2 mM), and Ni(OAc)2 (0.2 mM) in NEP buffer (10 mM, pH 9.0) at 37 °C for 30 min. bRelative conversion shown in the parentheses was assessed via MALDI-TOF MS of crude reaction mixture. cConversion
