Regioselective α-Peptide Bond Formation Through the Oxidation of

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Regioselective #-peptide bond formation through oxidation of amino thioacids Yasuhiro Kajihara, Ryo Okamoto, Takuya Haraguchi, Kota Nomura, Yuta Maki, and Masayuki Izumi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01239 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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Biochemistry

Regioselective α-Peptide Bond Formation Through Oxidation of Amino Thioacids Ryo Okamoto,†,‡ Takuya Haraguchi, Kota Nomura, Yuta Maki, †,‡ Masayuki Izumi, †,# Yasuhiro Kajihara†,‡* †Department

of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan. ‡:Project Research Center for Fundamental Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan ABSTRACT: Biological systems, including ribosomes and enzymes, produce peptides with extraordinary high speed and accuracy. On the other hand, a rational and regioselective α-peptide bond formation, without involving protecting groups, is difficult to achieve in chemical synthesis. In this study, α-amino thioacids were utilized for the generation of polypeptides without using any protecting groups. We found that an α-amino thioacid could oxidatively form a diaminoacyl–disulfide moiety and undergo a subsequent intramolecular S- to N-acyl transfer to form an α-peptide bond. Even the thioacid form of lysine, which has a free ε-amino group, generated a regioselective α-peptide bond. The oxidation of amino thioacids generated the oligomers of amino acids. Interestingly, this oligomerization reaction proceeded even in the presence of iron ore, a prebiotic element, thus suggesting a plausible prebiotic peptide bond forming reaction.

Formation of an α-peptide bond between the α-amino group of one α-amino acid and the carboxyl group of the other is an essential reaction for life. Biological systems use ribosomes and enzymes to achieve an endothermic dehydration process for the peptide bond formation. The sophisticated biological system can produce peptides with extraordinary high speed and accuracy. Significant advances have been made toward the chemical synthesis of peptides by utilizing coupling reagents and protected amino acids 1-2. The synthetic strategy for peptide bond is based on the activation of the carboxyl group of one amino acid, followed by the nucleophilic attack of the α-amine group of a second amino acid. This basic approach has enabled an efficient synthesis of peptides in conjunction with the solid phase synthesis 3. Advanced coupling reagents can efficiently activate amino acids and form peptide bonds at a high rate2. The recent synthetic technologies involving microwave and flow chemistry significantly promoted the basic coupling reactions and dramatically enhanced the synthetic speed 4-5. Despite recent advances in peptide chemistry, the use of protecting groups is still inevitable for the accurate construction of the peptide sequence. The bifunctional nature of amino acids makes the regioselective synthesis of peptides difficult under the general organic reaction conditions. Protecting groups control the reaction site accurately and allow us to construct the peptide sequence as intended. For example, when protecting groups are not used, coupling of amino acids with lysine can yield the required α-peptide bond along with the unnatural εpeptide bond. However, the use of protecting groups is generally disfavored for the synthesis of polypeptides, such as those consisting of over 50 amino acids, due to their intrinsic

hydrophobic nature. Moreover, the use of protecting groups for amino acids, which are small molecules, is atom economically disfavored. In this context, we have explored the rational reaction enabling the regioselective peptide bond formation without using any protecting groups. We focused on α-amino thioacids as potent amino acid precursors for the generation of polypeptides without utilizing protecting groups. Thioacids have a unique oxidation potential. This character allows amino thioacids to be oxidized, to yield the diaminoacyl-disulfide structures 6-10. Because the carbonyl carbon in the diacyl-disulfide moiety is highly electrophilic, we speculated that diaminoacyl-disulfide would undergo an intramolecular S- to N-acyl transfer and lead to the predominant formation of an α-peptide bond. Herein, we describe the unique oligomerization chemistry of α-amino thioacids via oxidation. We also describe the potential of this simple reaction in the prebiotic peptide bond formation. MATERIALS AND TYPICAL PROCEDURES Materials. Boc-Gly-OH, Boc-Ala-OH, Boc-Phe-OH, Boc-ValOH, Boc-Ser-(Bzl)-OH, Boc-Tyr(Bzl)-OH, 1-hydroxybenzotriazole (HOBt), were purchased from Peptide Institute Inc (Osaksa, Japan). Boc-Lys(Boc)-OH, (benzotriazol-1-yloxy) tripyrolidinophosphonium hexafluorophosphate (PyBOP), 2(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (WSCI·HCl) were purchased from Watanabe Chemical Industry (Hiroshima, Japan). Triisopropylsilane (TIPS), N,Ndiisopropylethylamine (DIEA), dimethyl sulfide (DMS) were

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purchased from Tokyo Chemical Industry. Trifluoroacetic acid (TFA), methanol (MeOH), N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), trifluoromethanesulfonic acid (TfOH), formic acid (FA), diethyl ether, urea, diethylamine (DEA), m-cresol, N,Ndimethyl-4-aminopyridine (DMAP), potassium hexacyanoferrate (Ⅲ), potassium hexacyanoferrate ( Ⅱ ) trihydrate, iron (Ⅲ) chloride hexahydrate, iron (Ⅱ) chloride tetrahydrate were purchased from Wako Pure Chemical (Osaka, Japan). Boc-Asn(Xan)-OH, Boc-Glu(OtBu)-OH, Fmoc-PheOH, Fmoc-Lys(Boc)-OH, were purchased from Merk. NMethyl-2-pyrrolidinone (NMP), guanidine hydrochloride (Gn·HCl), HPLC grade acetonitrile (MeCN) were purchased from Kanto Chemical Co. Inc (Tokyo, Japan). Triphenylmethanethiol (Trt-SH) was purchased from SigmaAldrich. Boc-Ser(tBu)-OH·DHCA was purchased from Fluka. Deuterium oxide (D2O), chloroform-d (CDCl3) dimethyl sulfoxide-d6 (DMSO-d6) were purchased from Cambridge Isotope Laboratories Inc (Tewksbury, MA). Analytical LC-MS. Liquid chromatography-mass spectrometry (LC-MS) analyses were performed on ThermoFisher Scientific UltiMate 3000 system equipped with on-line ESI-MS system (Bruker Daltonics amaZon ETD mass spectrometer). Cadenza CD-C18 (Imtakt corp., 2.0×50 mm or 2.0×100 mm) column was used with linear gradient of buffer A (0.1% aq. formic acid) and buffer B (90% aq. MeCN containing 0.09% FA) at the flow rate of 0.2 mL/min for chromatographic separations. Eluent was monitored by UV-absorbance at 218 nm and the on-line ESI-MS. For high-resolution mass analyses, Q-TOF-MS system (Bruker Daltonics compact mass spectrometer) was used as on-line MS system. Typical procedure for the homo-oligomerization of α-amino thioacids through oxidation. Phe-SH (0.4 mg, 2.2 μmol)* was dissolved in 0.2 M citrate buffer (54.0 μL, pH 1.8)** and added FeCl3•6H2O (3.0 mg, 11.0 μmol). The result of the reaction was analyzed by LCMS***. (*: In each experiment, Phe-SH was used with 0.4~1.0 mg, prepared from multi-milligram of Phe-SH; For example, Phe-SH (3.1 mg) was dissolved in 310 μL of 50 % MeCN solution, and 40 μL of this solution was separated and lyophilized to prepare 0.4 mg of Phe-SH; **: Citric buffer was purged with Ar gas for 5 min before using; ***: Analytical LC-MS was performed generally at 5 min and additionally at londger reaction time after quenching with 50 % aq. MeCN containing 0.1 % FA or sodium phosphate buffer (pH 7). For the investigation of pH dependency, homooligomerization reactions were performed as following; PheSH (0.1 mg, 0.55 μmol) was dissolved in 0.2 M citrate buffer (54 μL, pH was adjusted to 1.3, 1.5, 1.9, 2.0, 2.3 and 3.4). To the solution was added 5 fold excess of FeCl3 • 6H2O (2.75 μmol) by using 0.6 mg/μL FeCl3 • 6H2O solution (1.25 μL) dissolved in degassed water. The reaction was analyzed by LCMS after quenching with sodium phosphate buffer (pH 7). Typical procedure for the co-oligomerization of α-amino thioacids through oxidation. Phe-SH (0.55 μmol) and a different α-amino thioacid (2.76 μmol, 5 fold excess) were dissolved in degassed water (47.5 μL). To the solution was added FeCl3·6H2O solution (0.1 mg/μL, 7.5 μL) containing 5 fold excess of FeCl3·6H2O (2.76 μmol). The α-amino thioacids used with Phe-SH were Ala-SH (0.29 mg, 2.76 μmol), Ser-SH (0.33 mg, 2.76 μmol), Asn-SH (0.41 mg, 2.76 μmol), Glu-SH

(0.45 mg, 2.76 μmol) and Lys-SH (0.45 mg, 2.76 μmol). At each time point, an aliquot of the reaction mixture was quenched with sodium phosphate buffer (200 mM, pH 7) and subjected to analytical LCMS to monitor the reaction progress.

RESULTS AND DISCUSSION We initially carried out the oxidation reactions of the α-thioacid form of Phe (Phe-SH) and found that it led to unique oligomerization reactions (Figure 1). Phe-SH was synthesized in two steps from Boc-Phe-OH (Figure S1). For the formation of diacyl–disulfide bonds, we chose Fe(III) as an oxidizing agent. Oxidation of Phe-SH (10 mM) was conducted using FeCl3 (50 mM) in water and was monitored by liquid chromatography-mass spectrometry (LCMS). This reaction afforded the corresponding α-thioacid forms of oligomers including di to tetra phenylalanine within 5 min (Figure 1B). Tandem mass spectrometry revealed that several diaminoacyloligosulfide derivatives were also obtained as products (Figure S5). When the reaction time was increased to 24 h, the αthioacid forms of all the oligomers were hydrolyzed (Figure 1C). All the reactions could be monitored by mass spectrometry (Figure 1D, E).

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Biochemistry Figure 1. Oxidation of α-amino thioacid leads to unique oligomerization reaction under acidic pH solution. (A) Schematic representation of oligomerization of Phe-SH under the oxidation conditions. (B) Analytical LC-MS (Total ion chromatogram) after 5 min and (b) after 24 h. (D) MS data acquired from (B). (E) MS data acquired from (C). Hydrolyzed form and α-thioacid forms are indicated as –OH and –SH with the number of amino acids respectively (e. g. peptide consists of two phenylalanines depicted as 2F-OH or 2F-SH). DKP indicates diketopiperazine form of PheSH.

higher oxidation potency at a lower pH12. In contrast, the use of Fe(II) species, which is non-oxidative, did not induce the oligomerization of Phe-SH (Figure S3). These results suggested that the oligomerization of amino thioacids was triggered by the oxidation reactions.

Oxidation of Phe-SH led to the formation of a white precipitate during the course of the reaction (Figure 2A). We did not observe further extension of oligomers of Phe-SH by LC-MS after 5 min as shown in Figure 1. On the other hand, matrix assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOFMS) of the precipitate showed the ladder mass11 peaks corresponding to hepta- to dodecaphenylalanines (Figure 2B). This suggested that the oxidation of Phe-SH could generate longer oligophenylalanine derivatives that might be aggregated due to their intrinsic hydrophobicity. Figure 3. pH dependency of the oligomerization of Phe-SH. The amino acid sequences of oligo-Phe derivatives were confirmed by tandem mass spectrometry (Fig. S4 and S5). The efficiency of each reaction was estimated by the LC peak area intensities of the diphenylalanine derivatives as a fraction of Phe-SH at 4 h.

Figure 2. Oxidation of Phe-SH leads to oligomerization reaction under acidic aqueous solution. (A) Pictures of the reaction tubes during oxidation reaction of Phe-SH. White precipitate was observed within 2 h. (B) MALDI-TOF-MS spectrum of the precipitate yielded by the oxidation reaction of Phe-SH. m/z calcd for [M+Na]+ of 7F: 1070.5, 8F: 1217.6, 9F: 1364.6, 10F: 1511.7, 11F: 1658.8, 12F: 1805.8. e.g. Peptide consists of seven and eight phenylalanines depicted as 7F-OH and 8F-OH.

Intriguingly, the oligomerization reaction of Phe-SH was accelerated under acidic conditions (Figure 3). The pH dependence of oligomerization reactions was evaluated using FeCl3 (50 mM) and estimating the yield of diphenylalanine by LCMS. The oligomerization reaction was found to be more efficient at a lower pH, while almost no reaction was observed above pH 2.5. This pH dependence is consistent with the oxidation-reduction potentials of Fe(III) species, which shows

Next, we investigated the co-oligomerization of Phe-SH with several other α-amino thioacids (Figure 4). Phe-SH (10 mM) and a different α-amino thioacid (50 mM) were mixed in an aqueous FeCl3 solution (50 mM). This reaction afforded the cooligomers consisting of phenylalanine and the other amino acids as shown in Figure 4. Even hydrophilic amino acids such as lysine could result in the oligomer (Figure 4G). We also tested the co-oxidation of a tetrapeptide-α-thioacid and Phe-SH, which furnished oligo-Phe at the C-terminal of the tetrapeptide　 (Figure 5). Therefore, the oxidation of peptide-α-thioacids may be potentially involved in the peptide elongation. This result clearly showed that the conversion of polypeptide chains into the corresponding peptide-α-thioacid form could re-initiate the extension of their polypeptide length. Some amino acids have a carboxyl group or an amino group on their side chains. Regioselective extension of the α-peptide chain has not yet been achieved without specific catalytic systems such as ribosomes. In order to investigate the regioselectivity of the oligomerization of α-amino thioacids, we conducted co-oligomerization of Phe-SH and Lys-SH, which has an amino group at the ε-position. This reaction also afforded a mixture of various oligomers comprising Phe and Lys (Figure 4G and 6). In the pool of the Phe-Lys oligomers, a series of tripeptides comprising two Phe and one Lys were found to be well-separated upon LC analysis (Figure 6 i). The retention time for each of these was compared with that of the authentic tripeptides consisting of two Phe and one Lys. This analysis revealed that the oxidative co-oligomerization did not furnish isopeptides but exclusively generated native tripeptides. NMR analysis of one of the tripeptide (Phe-Phe-Lys) isolated from the co-oxidation reaction clearly indicated its α-peptide bond connection, which was also consistent with the LC analysis of the authentic peptides (Figure 7 and 6 ii-vi).

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Figure 6. LC profiles of co-oligomerization of phenylananine αthioacid with lysine α-thioacid. (i) synthetic crude sample obtained from the co-oxidation reaction of Phe-SH and Lys-SH, LC profile of authentic sample of (ii) FFK-OH, (iii) FKF-OH, (iv) KFF-OH, (v) FF(iso)K-OH, (vi) F(iso)KF-OH.

Figure 4. Co-oligomerization of α-amino thioacids by oxidation reaction. (A) Summary of the co-oligomerization between Phe-SH and other α-amino thioacids in separate reactions. The products are shown by one-letter style: e. g. peptide consists of a phenylalanine and a lysine depicted as FK-OH or FK-SH (B)-(G) MS spectra acquired from analytical LCMS of the co-oligomerization with other amino thioacids. (B) glycine thioacid; (C) alanine thioacid; (D) serine thioacid; (E) asparagine thioacid; (F) glutamic thioacid; (G) lysine thioacid. All data was acquired at 5 min by LCMS analysis.

Figure 7. NMR spectra of co-oligomerization of phenylananine αthioacid with lysine α-thioacid. (A) 1H NMR of synthetic FFK-OH (top) and authentic FFK-OH (bottom); (B) NOESY profile of synthetic FFK-OH. Asterisk indicates noise by the water suppression.

Figure 5. Extension of peptide thioacid with Phe-SH. A heteropolymeric form can be extended with an α-amino thioacid.

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Biochemistry

Figure 8. Proposed reaction mechanism of the oligomerization of amino thioacids triggered by oxidation reaction.

The proposed reaction mechanism for the oligomerization of α-amino thioacids includes two key steps: formation of a diacyl–disulfide bond and an intramolecular S- to N-acyl transfer reaction (Figure 8). Oxidation reaction (I) converts the α-amino thioacids into diacyl–disulfide derivatives (Figure 8 (a)-(c)). In these structures, the α-amino groups are preferentially acylated through a 6-membered-ring-type intramolecular S- to N-acyl transfer reaction (Figure 8 path A). The resultant peptide–dithioacid (d) is further oxidized by an αamino thioacid to give a diacyl-oligosulfide (e). Both (d) and (e) are subsequently converted into diacyl-disulfide structures (f) via disulfide exchange reactions (II) followed by oligomerization. Because of the intrinsic electrophilicity of diacyl–disulfide structures, we also proposed an alternative reaction pathway that involves a thiolysis reaction (III), which is a reminiscence of thiol-thioester exchanging reaction13, of diacyl–disulfide derivatives with the α-amino thioacids (Figure 8 path B). This pathway potentially yields thioanhydride structures (g) that form peptide bonds through intramolecular Sto N-acyl transfer reactions via the 5-membered ring type intermediates. Similarly, oligosulfide derivatives (d), (e), and (f) are also potentially converted into thioanhydride forms (i), resulting in oligomerization. The proposed intramolecular reaction mechanism explains the possibility of selective formation of α-peptide bonds. In a diaminoacyl–disulfide structure, ε-amino group of Lys could undergo an intramolecular S- to N-acyl transfer reaction via a strained 10-membered ring-type intermediate. This reaction is unfavorable, as opposed to the reaction pathway involving the α-amino groups; therefore, the isopeptide formation pathway could be excluded. Considering the reactivity of diaminoacyl–

disulfide and thioanhydride structures, intermolecular aminolysis is also a plausible route for the formation of α-amide bonds. However, the oxidation of Phe-SH and free Ala-OH did not give the co-oligomer of Phe and Ala but gave an oligomer of Phe, exclusively (Figure S13). This result strongly suggested that an intermolecular amide bond formation is unlikely in the oligomerization reaction of α-amino thioacids triggered by oxidation. Intermolecular rearrangements from the intermediates such as (d)-(f) could yield diketopiperazine (DKP) derivatives, which are simple cyclodipeptides. The DKP products are inactive under the oxidation conditions and thus could terminate the subsequent peptide bond formations. However, our result showed that the oxidation of α-amino thioacids proceeded with the oligomerization of amino acids. We speculated that the ring structures in the plausible transition state in path A and path B might be less strained or less hindered than that in the DKP formation. This might result in the unique oligomerization. The acidic reaction conditions might also contribute to the unique regioselectivity of the α-peptide bond formation. Because the reaction pH is acidic pH, the majority of amines exist in the non-nucleophilic ammonium form, which could suppress both inter- and intramolecular aminolysis. The intermediates such as (c) and (g) were able to the provide proximity effect that facilitated an intramolecular S- to N-acyl shift though the 5- or 6-membered ring type intermediate. Therefore, the residual nucleophilic α-amine, under acidic conditions, might preferentially result in the intramolecular peptide bond. This can further accelerate the intramolecular acyl-shift reaction via the equilibrium conditions between the free amine and the ammonium form of α-amines. We also studied whether the unique intramolecular reaction mechanism of peptide bond formation from α-amino thioacids could contribute to a chiral selection of peptides. In the transition state, the configuration of amino acids (L- and D-) or peptides could affect the kinetics of the intramolecular S- to Nacyl transfer reaction. To confirm this hypothesis, we carried out the co-oligomerization of L-Phe-SH and D-Phe-SH and estimated the yield of di-Phe-SH by LCMS. Consequently, no differences in product yields were observed among the optical isomers of di-Phe (Figure S14). Although we could not succeed in the analysis of all the oligo-Phe derivatives by LCMS, it is speculated that longer peptides that potentially acquire inherent secondary or tertiary structures might exhibit some chirality. Further precise experiments are necessary to investigate the chiral selectivity of the reaction system involving α-amino thioacids. The unique oligomerization reaction of α-amino thioacids through oxidation encouraged us to examine the reaction under a plausible prebiotic condition (Figure 9). On the primitive Earth, simple chemical reactions could have played an important role in the formation of peptide bonds in the absence of sophisticated biological systems14-16. α-Amino thioacids could have been generated by the reaction of amino acids with carbonyl sulfide in a prebiotic environment17-18. Therefore, we envisaged that the oxidation of α-amino thioacids was a reasonable reaction system that could lead to α-peptide bond formation under a prebiotic environment. In order to demonstrate the feasibility of the oligomerization reaction of αamino thioacids as a prebiotic peptide producing reaction, we performed the reaction in the presence of iron ore, which is considered a plausible element that provided Fe(III) in a

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prebiotic environment19. For setting an acidic prebiotic environment, we utilized sulfuric acid, which could be generated from sulfur dioxide, a volcanic gas component. Small pieces of iron ore were added to the solution containing α-amino thioacids dissolved in diluted H2SO4 (pH 1.2). As expected, this heterogeneous reaction system generated the oligomers of phenylalanine as well as the co-oligomers of Phe and Lys. We have identified tripeptides comprising two Phe and one Lys connected through α-peptide bonds as in the case of the reaction with the FeCl3 (Figure 6). Hence, we concluded that iron ore is an efficient promoter for the oxidation of α-amino thioacids that could work in a prebiotic peptide generating system. We also demonstrated the extension of peptide bond from peptide-αthioacid (Figure 5). This result also showed the possibility of prebiotic extension of peptides. The oligomerization of α-amino thioacids through oxidation might produce free peptides (peptide-OH). The peptide chain extension might be re-initiated through the conversion of peptide chains into the corresponding peptide-α-thioacids form by prebiotic conditions such as volcanic gas.

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CONCLUSION The oxidation of α-amino thioacids was found to be effective for the regioselective α-peptide bond formation. This was achieved by a plausible intramolecular reaction mechanism triggered by simple oxidation. Moreover, the oxidation of αamino thioacids resulted in the synthesis of oligomers of amino acids. This oligomerization occurred even in the combination of different α-amino thioacids including the thioacid form of Lys. Interestingly, the oligomerization of amino thioacids also proceeded in the presence of iron ore, a prebiotic element. Our results not only suggested that the oxidation of α-amino thioacids resulted in the synthesis of amino acid polymers but also indicated a plausible prebiotic peptide bond forming reaction.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. All experimental procedures, characterization, NMR spectra, and MS spectra (file type, PDF).

AUTHOR INFORMATION Corresponding Author [email protected]

Present Addresses #: Prof. Dr. Masayuki Izumi: Department of Chemistry and Biotechnology, Faculty of Science and Technology, Kochi University, 2-5-1 Akebonocho, Kochi, Kochi 780-8520, Japan., E-mail: [email protected]

ORCID Masayuki Izumi: 0000-0001-6486-9678 Ryo Okamoto: 0000-0001-9529-2525 Yuta Maki：0000-0002-5838-302X Yasuhiro Kajihara: 0000-0002-6656-2394

Funding Sources This work was also supported by financial support from the Japan Society for the Promotion of Science (KAKENHI Grant Number 17H01214).

REFERENCES Figure 9. Oligomerization of α-amino thioacids with iron ore and sulfuric acid. (A) LC profiles of the oligomerization of Phe-SH. (B) LC profiles of the co-oligomerization of Phe-SH with lysine thioacid. (C) LC profiles of the oligomerization of Phe-SH. (D) LC profiles of the co-oligomerization of Phe-SH with lysine αthioacid. (E-F) Control experiments under the same acidic solution without additive. (E) a oligomerization experiment without additive. (F) a co-oligomerization experiment without additive. Trace amounts of oligomerization were also observed in the control experiments. We speculate these are generated by air oxidation, which cannot exclude completely.

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