DOI:10.1002/cssc.201500318 Full Papers

C Nand N HBond Metathesis Reactions Mediated by CarbonÀ DioxideÀ Yehong Wang,[a] Jian Zhang,[a] Jing Liu,[a] Chaofeng Zhang,[a, b] Zhixin Zhang,[a] Jie Xu,*[a] Shutao Xu,[a] FangjunWang,[a] andFengWang*[a]

Herein, we report CO2-mediated metathesis reactions between ditions. The experimental data and in situ NMR and attenuated and DMF to synthesize . More than total reflectance IR spectroscopy measurements support the 20 amines, including primary,secondary,aromatic, and hetero- formation of the N-carbamic acid as an intermediate through cyclic amines,diamines, and amino acids, are converted to the the weak acid–base interaction between CO2 and the . corresponding formamideswith good-to-excellent conversions The metathesis reaction is driven by the formation of astable and selectivities under mild conditions. Thisstrategy employs , and areactionmechanism is proposed.

CO2 as amediator to activate the amine under metal-free con-

Introduction

The selective N-formylation of amines to formamidesisasignif- different to previous ones that required activated formylation icant reaction in organic,medicinal, andbiological chemistry.[1] reagents,metal-containing catalysts, and strong additives. In Formic acid[2] or in situ-generated formic acid,[3] DMF,[4] alde- addition, the reaction can be performed in air ( 400 ppm CO  2 hydes,[5] and their derivatives have been used as formylation in the atmosphere) with comparableresults. Moreover,ithas reagents.However,the majority of examples require high tem- broad substrate compatibility. peratures, water-sensitive reagents, or complex workups and, thus, are unsuitable for use with fine chemicals, pharmaceuti- cals, and molecules bearing diverse functional groups, such as proteins and thiophenes. Reactions induced by weak acid–base interactions play akey [6] role in living systems and in the chemical industry. For exam- Results and Discussion ple, CO2 generated from cellular respiration is expired in part through the reversible formation of acarbamate between CO2 First, we optimized the reaction temperature and time. The and the amino groups of .[7] Furthermore, the reac- temperature dependence of the conversion of benzylamine in tions of primary and secondary amines with CO2 are used to DMF under 1bar of CO2 gas is summarized in Figure S1. The captureCO2through the formation of azwitterion first and onset temperature for the reaction has been reported to be then acarbamate.[8] Encouraged by these previous studies, we more than 80 8Coreven as high as 1508C.[10] In this study,the proposed the possibility of utilizing the weak interactions be- onset temperature ( 40 8C, defined as the 30%conversion  tween CO and amines to functionalize N Hbonds. We report temperature) is clearly much lower.The conversion increased 2 À here the results on the N-formylation of amines mediated by with increasing reactiontemperature and reached 92%after CO through ametathesis reaction [Eq. (1)].Usually,C Nbond 4hat 1008C. The selectivity for N-benzylformamideisalso pre- 2 À activation requires metal catalysts.[9] This method is markedly sented in Figure S1. An increased reaction temperature of 608Corhigheroffered almost pure N-benzylformamide. The [a] Y. Wang, J. Zhang, J. Liu, C. Zhang, Z. Zhang, Prof. Dr.J.Xu, Dr.S.Xu, reactiondid not proceed under Ar gas. The reactioninair of- Dr.F.Wang, Prof. Dr.F.Wang fered comparable resultsin48h.In1973, Kraus reported that State Key Laboratory of (SKLC) the formylation of aliphatic amines occurred in pure DMF.[11] Dalian National Laboratoryfor Clean Energy(DNL) Up to 100 hwas required to complete the reaction. Our study Dalian Institute of Chemical Physics (DICP) Chinese Academy of Sciences revealed that the reactionconducted by Kraus might be medi- 457 Zhongshan Road, Dalian 116023 (PR China) ated by the slow absorption of atmospheric CO2 into the reac- E-mail:[email protected] tion mixture. [email protected] The progress of the reaction as monitored by GC is dis- [b] C. Zhang played in Figure 1. The formylation of benzylamine was con- University of the Chinese Academy of Sciences No.19A Yuquan Road, Beijing 100049 (PR China) ducted in DMF at 1008Cunder 1bar of CO2.The conversion of Supporting Information for this article is available on the WWW under benzylamine increased significantly to 86%in2hand then http://dx.doi.org/10.1002/cssc.201500318. slowed down and reached 93%in8hand > 99%in24h.

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Figure 1. Time-on-stream profile of the formylation of benzylamine to ben- zylformamide. Reactionconditions: benzylamine (1.5 mmol), DMF (2 mL),

1bar CO2,100 8C.

Through the whole reaction, the selectivity for N-benzylform- Figure 2. Relationship between amine conversion and pKa(N H) values. Reac- À was > 99%. tion conditions:amine (0.6 mmol), DMF (2 mL), 1bar CO2,1008C, 2h.

Areaction with isotopically labelled [D7]DMF was conducted to clarify the reaction route (Figure S2).The molecular ion (M+) thumb, it helps to determine which kind of amine can be con- peak of N-benzylformamide(normal m/z=135) increased to verted by this method. m/z=136 (labeled), and the M+ peak of dimethylamine (m/z= We then studied the substrate scope (Table 1). Sterically un- 45) increased accordingly to m/z=51 (Figures S3 and S4). We hindered linear aliphatic amines with chains of up to 12 13 also used CO2 instead of CO2 in this reaction system.There carbon atoms were converted to the corresponding forma- was no increase for the molecular ion peak of N-benzylforma- mides with >99 %selectivity and >99 %conversion(1–5); mide and it remained at m/z= 135. However,ifthe Catom these resultsare better than those reported previously.[10,14] Ali- 13 + came from CO2,the M peak of N-benzylformamide should cyclic cyclohexylamineand cyclopentylamine wereconverted have an m/z value of 136. The resultsconfirm that (1) ameta- satisfactorily to the corresponding formamides(4and 5). No thesis reactionoccurs between the amine and DMF,(2) DMF is conversion was observed for dipropylamine and diethylamine the formylation reagent, and (3) CO2 is amediator,not areac- (6 and 7). However,ahydroxy-terminated amine (8)and piperi- tant. In someother studies, although CO2 wasadded and for- dine (9)achieved 23 %and >99%conversion,respectively, mamides were products,the real reaction involved the hydro- with > 99% selectivity.These amines were different genationofCO2to formic acid, which functioned as the formy- at the terminal group. Steric as well as electronic and solvation [3h,i, 5a,12] lation reagent. In contrast, the CO2-mediated method effects might affect their activity.The two terminal hydroxy requires no metal catalystand no hydrogen. Moreover,itcan groups might form hydrogen bonds and, thus, expose more be used to prepare 13C-labeled formamides. space for the N Hbond activation. Piperidine, which could be À We then investigated the dependence of the initial amine viewed as amolecule in which the terminal groups are conversion (2 h) on the pKa(N H) values. The largerthe pKa(N H) bonded, achieved better results. In contrast, apiperidine for- À À value, the stronger the amine basicity.Aplot of pKa(N H) versus mamide was obtained with only 36%yield in aB(OCH2CF3)3– À conversion produces symmetric linear fits that intercept at the DMF system.[10] To show the practical application of the proce- maximum point of pKa(N H) = 9.9 (Figure 2). The left end point dure, agram-scale synthesis of dodecylamine(2.78 g) was per- À was determined by extending the left half line to y=0, which formed. The reaction offered 97 %isolated yield of dodecylfor- intercepted the x axis at pKa(N H) =8.6. The right end point was mamide (3). À determined by dipropylamine [pKa(N H) = 10.6].The experimen- Heterocyclic amines such as furfurylamine and 2-thiophene- À tal data showed that the metathesis reaction relies greatlyon methylamine were converted to formamides(10 and 11,re- the amine pKa(N H),and only amines within the pKa(N H) range spectively) with selectivities of > 99%. To the best of our À À 8.6–10.6were converted into formamides. This explains why knowledge,this methodrepresents arare example of the for- the reactions with aniline and some diamines weresluggish.[13] mylation of athiopheneamine because sulfur may poison The basicity of the amine affects the strength of the interaction most catalytic metal centers.Several benzylamines were con- between the Natom of the amine and the Catom of CO2: verted with excellent formamide selectivities (12–18). Aslight weak basicity [such as aniline, pKa(N H) <8.6] resultsinaninsta- substituent effect was evidenced by the lower conversion of À ble amine–CO2 adduct;strong basicity [pKa(N H) > 10.6] gener- the CF3-substituted benzylamine than for the F-substituted À ates an interaction that is too stable, and the CO2 is bound one. The less-nucleophilic aniline was inactive (19)because of strongly.The amines in Figure 2were selected after consider- its weak basicity (Figure 2). The formylation of the sterically un- ation of both the pKa(N H) and steric hindrance;therefore, some hindered hexane-1,6-diamine afforded 99%ofthe diforma- À amines fall out of this range. However,asasimple rule of mide (20). Aworse result than that for hexane-1,6-diamine was

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methods remains ahot topic.[16] In this study,wetested the la- Table 1. Substrate scope of the reaction.[a] beling of protein amine groupswith DMF through metathesis Structure Compound Conversion Selectivity reactions. We added alittle water to promote solubility but [%] [%] sacrificed some activity.After the reaction, both unreacted ar- 1 n=3 >99 >99[b] ginine (m/z=175) and formylated arginine (m/z=203) were 2 n=5 >99 >99 detected by means of matrix-assisted laser desorption/ioniza- 3 n=11 > 99 >99 tion–time of flight (MALDI-TOF)/TOF MS (Figure 3A). The reac- 4 >99 >99[b] tion was further quantified by LC–MS (Figure 3B)using an identicalamount of lysine as internal standard. After the reac- 5 >99 >99 tion, the relative intensity of arginine decreased greatly. The ar- 6 n=1trace – ginine conversion was 81%, as analyzed by LC–MS. 7 n=2trace – We further applied the method to more-complex amines from trypsin-digested bovine serum albumin (BSA). Trypsin is 8 23 >99 apancreatic serine endoproteasethat hydrolyzes peptide 9 >99 >99 bonds specifically at the carboxyl side of arginineand lysine residues and leads to two classes of peptides. One is the pep- 10 >99 >99 tide with the arginine residue at the carboxyl side, in which 11 94 >99 only the amine group of the N-terminus and the guanidine 12 X=H >99 >99 group of the arginine residue could be formylated, and the 13 X=p-Cl 95 94 other is the peptide with the lysine residue at the carboxyl 14 X=o-Cl 92 90 side, in which the amine group of the N-terminus and the 15 X=p-F 93 >99 lysine residue could be formylated. For the former,57% of the 16 X=p-CF3 96 68 amine groups of the N-terminus, 14%ofthe guanidine 17 91 >99[b] groups,and 0% of both were formylated (FigureS5). For the 18 98 98 later,these data were 67%, 72%, and 46 %, respectively.For

19 trace –

20 >99 >99 [c] 21 carried out in CO2 >99 72 [d] 22 catalyzed by CeO2 instead 70 4

of CO2 23[e] >99 68

24 >99 >99

[a] Reactionconditions: amine (1.5 mmol), DMF (2 mL), 100 8C, 24 h, 1bar

CO2 ;results were analyzed by GC and are presented as conversion/selec- tivity.[b] 48 h. [c]The remaining product was 1,4-diazepane-1-carbalde-

hyde (selectivity 28%). [d] Amine (1.5 mmol), DMF (2 mL), CeO2 (100 mg), 1508C, 24 h.[4k] 1,4-Diazepane-1-carbaldehyde selectivity 96%. [e] Pipera- zine-1-carbaldehyde selectivity 32%.

obtained for an alicyclic diamine with >99%conversion and 72%selectivity for the diformamide and 28%for the monofor- mamide (21). This might be due to the robuststructure of the alicyclic diamine. However,this method was still superiorto one with solid CeO2 catalyst, for which only 70%conversion and 4% of the diformamide was obtained (22).[4k] The formyla- tion of piperazine offered 68%diformamide and 32%mono- formamide (23). For dimethyl-substituted piperazine, monofor- mamide was obtained with >99%selectivity(24). Carbamylation reactions play important roles in multiple bio- logical processes, such as the transportation of aminoacids and protein synthesisinliving systems.[15] To date, the protein Figure 3. (A) MALDI-TOF results for arginine with a-cyano-4-hydroxycinnamic formylation methods employed use expensive labeling re- acid (CHCA)asthe matrix. The y axis for arginine was verticallyadjusted. agents and complex procedures,but the development of new (B) LC–MS results for the N-formylation of arginine.

ChemSusChem 2015, 8,2066 –2072 www.chemsuschem.org 2068 2015 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Papers histidine, 28 %ofthe side imidazole groups were formylated, which shows its inertness. The reactionwas governed by the pKa(N H) rule (Figure 2) and the effect of steric hindrance. Usual- À ly,acetylation labeling through chemical reactions can only be performed for the primary amino group on the protein or pep- tide N-terminus and the lysine side chain. In this study,the methodcan be performed not only on the N-terminus and lysine but also on the guanidine group of the side chain of ar- ginine. Although the CO2-mediated metathesis reactionisless efficient than the matureacetylation method, it provides apos- sible next-generation labeling strategy,especially foramines with coexisting functional groups sensitive to metal ions and strong acids or bases. In previous studies, DMF and its derivatives were activated by strongadditives such as HClO4,iodine, NaOMe, and RSO2Cl. Figure 5. Stackedinsitu FTIR spectrafor initial 30 min. Reactionconditions: benzylamine (3 mmol), DMF (24 mL), 1bar CO ,100 C. In our study,CO2is aweakly acidic molecule and cannot acti- 2 8 vate DMF.Webelieved CO2 would activate amines and con- firmed this through IR and NMR spectroscopy characteriza- for dipropylamine and d= 158 ppm for benzylamine were as- tions. signed to the carbonyl Catoms of the N-carbamic acids[Fig- The attenuated total reflectance (ATR)-IR spectra of benzyla- ure 6A and B].[17] The discernible low-field shifts of the C mine were measured before and after CO2 was bubbled atoms next to the Natoms from d=52–48 ppm for dipropyla- through it at room temperature (Figure 4A). The CO2 dissolved mine and from d= 46–44 ppm for benzylamine indicated the 1 in the benzylamine, as indicated by apeak at n˜ = 2340 cmÀ gain of electron density after the formation of the N-carbamic for the asymmetric stretchofCO2.Two peaks at n˜ = 1720 and acids. The chemical shift at 39 ppm for both amines was due 1 1648 cmÀ were assigned to the COOH vibration of the N-car- to the trace residueofnormal DMSO in the [D ]DMSO solvent. À 6 bamic acid.[17] No carbamate was formed, as indicated by the The presence of the N-carbamic acid was again confirmed by 1 [17] 1 absence of an absorption at n˜ 1570 cmÀ . For DMF with HNMR spectroscopy in [D ]DMSO. After the CO bubbling, the  6 2 CO bubblingatroom temperature (Figure 4B), the spectrum d(O H) of the N-carbamic acid appearedat7.7 ppm, whereas 2 À resembled that of pure DMF,which indicates that no interac- the d(C H) of dipropylamine at 2.4 ppm wasupshifted to À tion occurred between DMF and CO2.More detailed reaction 3.0 ppm (Figure 6C), which was assigned to the d(C H) of the [17] À information forbenzylamine, DMF,and CO2 was obtained by N-carbamic acid. After the bubbling of CO2 into benzyla- monitoring the initial 30 min reaction through ATR-IR spectros- mine, the d(C H) of the amine at 3.7 ppm shifted to 4.2 ppm, À copy (Figure 5). The bubbling of CO2 generated three peaks at which was indicative of d(C H) of the N-carbamic acid. The 1 À n˜ =1231, 1542, and 1720 cmÀ ,which indicated the formation newly formed d(O H) of the N-carbamic acid appeared at À of the N-carbamic acid. The formation of N-benzylformamide 8.7 ppm (Figure 6D). Signals at d=3.4 and 1.1 ppm wereat- 1 was evidenced by the peaks at n˜ = 1673 and 1382 cmÀ . tributed to ethanol contamination from the tube for CO2 bub- The formation of the N-carbamic acids of dipropylamine and bling. The signal at d=2.5 ppm originated from normal DMSO benzylamine after CO2 bubbling in [D6]DMSO was further char- in [D6]DMSO. acterized by 13CNMR spectroscopy.The signals at d=157 ppm We then studied the solventeffect by 13CNMR spectroscopy

(Figure 7). The addition of CO2 into amixture of benzylamine

and dipropylamine in [D6]DMSO and [D7]DMF at room temper- ature resulted in 100%conversion to the N-carbamic acids. This was illustrated by the presence of two new C=Osignals at

d< 160 ppm (159 and 158 ppm in [D6]DMSO, 160 and

159 ppm in [D7]DMF, respectively), in good agreement with the results in Figure 6. In contrast, only asingle signal appeared at

d= 163 ppm in CDCl3,but it was stronger than the signals re-

corded in [D6]DMSO and [D7]DMF.The value was larger than the normal shift of the Catoms of carbamic acids (less than 160 ppm). This signal originated from carbamate + [PhCH2NHCOOÀ (CH3CH2CH2)2NH2 ], the adduct of the amine [17] and CO2 in CDCl3;thus, only one signal was discerned.

The reaction of an amine and CO2 to form an N-carbamic 4 1 1 acid (CA) is rapid and has areaction rate k1 of 10 mÀ sÀ .The 1 1 1 [18] reversereaction is slow with k1’=10 mÀ sÀ . Previous stud- Figure 4. In situ ATR-IR spectraof(A) benzylamine and (B) DMF,flushedwith ies mentioned that the C Nbond might undergo an exchange À [19] Ar gas and in the presence of CO2. reactionofthe carboxylgroup with CO2. The formation of

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13 1 Figure 6. Cand HNMR spectroscopycharacterization of dipropylamine (A and C) andbenzylamine (B and D) in Ar or CO2.

CA was confirmed by 1HNMR, 13CNMR, and ATR-IR spectrosco- py.The key reaction is that the CA furtherreacts with DMF to

generate the formamide (FA) product and release CO2,which was also confirmed by IR and NMR spectroscopy.The reaction of DMF with CA may involveaC Nbond-metathesis transient À state. We isolated pure zwitterion (AC) and reacted it with DMF (Figure S6). As the reaction selectivity was >99 %, we used the FA production rate as the reaction 1 1 rate. The initial rate of 0LmolÀ sÀ might infer that AC was converted to CA during this period. Further,the reactionrates 6 1 1 decreased with time with an average rate k2 of 10À LmolÀ sÀ .

This rate was much smaller than k1;therefore step 2isthe rate-determining step. Therefore, the initial reactionquickly generated CA and then AC, as an amine reservoir (Figure 8). The FA was generated from CA and DMF.The addition of asmall amount of water ir- Figure 7. 13CNMR spectraofamixture of benzylamine and dipropylamine in reversibly hydrolyzedCAtocarbonate. Acontrol reaction in

CDCl3,[D6]DMSO, or [D7]DMF after CO2 bubbling. a1:1 volumetric ratio mixture of DMFand water at 1008Cfor

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gradual disappearance of the gel indicated the end of reaction, which was also tracked by GC (Agilent 7890A). The reaction solu- tion was cooled to room tempera- ture, and then asample of the re- action mixture ( 0.2 mL) was ana-  lyzed by GC–MS (Agilent 7890A- 5975C). In most cases, the reac- tion was very clean and, thus, the conversion of amine and the se- lectivity for formamide were ob- tained from the normalized GC in- tegration peak areas. General characterizations and Figure 8. The possible reaction mechanism. analyses: All liquid NMR spectros- copy experiments were performed with aBruker Avance III-400 spec-

trometer with [D6]DMSO, [D7]DMF, 1 13 4hgave 24 %conversion of benzylamine, in comparison with or CDCl3 as the deuterated solvent for locking. Hand CNMR 92%conversion in pure DMF.Moreover areaction of the sepa- spectroscopy experiments with proton decoupling were conducted rated carbonate with DMF generated no product. These facts at 100.62 MHz with 200 scans and a2srecycle delay.The chemical shifts were referenced to tetramethylsilane (TMS). In situ ATR-IR indicatethat carbonateisnot the reaction intermediate and its spectra were recorded with aMettler Toledo React IR 4000 spec- production is irreversible.The sterichindrance of the methyl trometer with aDiCom probe. The IR spectra were recorded with groups (R1,R2)connected to the Natom of the amine interrupt [benzylamine]=0.75m in DMF after CO2 degassing for at least the interaction of DMF with the amine and retard this reaction. 2min under continuous stirring. The metathesis product dimethylamine, as the leaving group Bovine serum albumin (BSA) digestion: The BSA was denatured of DMF,has apKa(N H) value of 10.6, which is large enough to with 8m /100 mm tetraethylammonium bromide (TEAB, À pH 8.0), reduced by 10 mm dithiothreitol at 608Cfor 1h,and alky- form astable carbamic acidproduct with CO2 under the reac- tion conditions and drive the reaction to the right. After the re- lated with 20 mm iodoacetamide in darkness at room temperature for 30 min, followed by dilution with 100 mm TEAB buffer (pH 8.0) action, an increaseintemperature or flushing with N would 2 and digestion with trypsin at 378Cfor 16 h. Finally,the BSA digests remove dimethylamine and CO2 gas;therefore, the reaction is were desalted with C18 solid-phase extraction (SPE) columns and clean andconvenient. lyophilized for use. MALDI-TOF MS analysis: The MALDI-TOFMSexperiments were performed using an AB Sciex 5800 MALDI-TOF/TOF mass spectrom- Conclusions eter (AB Sciex) equipped with apulsed Nd–YAG laser at 355 nm in reflective positive-ion mode. The sample ( 0.5 mL) and matrix We have reported the use of CO2 as amediatorinthe transfor- 1  (0.5 mL; 25 mgmLÀ 2,5-dihydroxybenzoic acid in 50%acetonitrile/ mation of amines to their corresponding formamidesinDMF. H2O) were spotted on the MALDI plate for MS analysis. The liquid The metathesis reactionisconducted without the use of any chromatography coupled with mass spectrometry/mass spectrom- metals.This will provide auseful methodfor the formylation etry (LC–MS/MS) experiments were performed using aThermo Q and protection of amine groups in asimple, green, and eco- Exactive mass spectrometer (Thermo) with ananospray ion source nomical way. and aU3000 RSLCnano system (Thermo). After lyophilization, the samples were redissolved with 0.1%formic acid solution and

loaded on aC18 capillary trap column (200 mmi.d.,4cm) packed Experimental Section with C18 AQ beads (5 mm, 120 Š, Daison) and separated by acapilla- ry analysis column (75 mmi.d.) with C18 AQ beads (3 mm, 120 Š, Methods: All chemicals were analytical grade and used as pur- Daison). The buffers used for the online analysis were 0.1%(v/v) chased without further purification. Most of the chemicals were formic acid in water and 0.1%(v/v)formic acid in acetonitrile, the 1 purchased from Aladdin Chemicals, except DMF,aniline, and dieth- flow rate was 300 nLminÀ for nanoflow LC–MS/MS analysis. Agra- ylamine, which were purchased from Tianjin Kemiou Chemical Re- dient from 5to35% (v/v)acetonitrile was achieved in 15 min for agent Co.,Ltd., n-butylamine and cyclohexylamine were purchased arginine samples and 90 min for BSA samples. The MS and MS/MS from Sinopharm Chemical Reagent Co.,Ltd.,and 2,6-dimethylpyr- spectra were collected by higher-energy collision-induced dissocia- azine was purchased from J&K chemicals. tion (HCD) at 28%energy in adata-dependent mode with one MS Catalytic reactions: The reactions were conducted in a50mL scan followed by 10 MS/MS scans. The RAWfiles collected by Xcali- glass batch reactor (heavy wall, maximum pressure:0.6 MPa, Syn- bur 2.1 were converted to MGF files by Proteome Discoverer thware Glass). Typically,benzylamine (1.5 mmol), DMF (2 mL), and (v1.2.0.208, Thermo) and searched with Mascot (version 2.3.0, astirring bar were placed in the reactor,which was then charged Matrix Science). Cysteine carboxamidomethylation was set as with CO2 gas (1 bar) from agas cylinder.The reaction solution astatic modification of 57.0215 Da, and formylation and methio- changed gradually into athick white gel over several minutes. The nine oxidation were set as variable modifications of 27.9949 Da reactor was sealed tightly with aTeflon stopper and then im- and 15.9949 Da, respectively.The mass tolerances were 10 ppm mersed in an oil bath preheated at the desired temperature. The and 0.5 Da for the parent and fragment ions, respectively.Amaxi-

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