Hydride transfer reactions via ion–neutral complex: fragmentation of protonated N‐benzylpiperidines and protonated N‐benzylpiperazines in mass spectrometry

An ion‐neutral complex (INC)‐mediated hydride transfer reaction was observed in the fragmentation of protonated N‐benzylpiperidines and protonated N‐benzylpiperazines in electrospray ionization mass spectrometry. Upon protonation at the nitrogen atom, these compounds initially dissociated to an INC consisting of [RC₆H₄CH₂]⁺ (R = substituent) and piperidine or piperazine. Although this INC was unstable, it did exist and was supported by both experiments and density functional theory (DFT) calculations. In the subsequent fragmentation, hydride transfer from the neutral partner to the cation species competed with the direct separation. The distribution of the two corresponding product ions was found to depend on the stabilization energy of this INC, and it was also approved by the study of substituent effects. For monosubstituted N‐benzylpiperidines, strong electron‐donating substituents favored the formation of [RC₆H₄CH₂]⁺, whereas strong electron‐withdrawing substituents favored the competing hydride transfer reaction leading to a loss of toluene. The logarithmic values of the abundance ratios of the two ions were well correlated with the nature of the substituents, or rather, the stabilization energy of this INC. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐phase fragmentation of the protonated benzyl ester of proline: intramolecular electrophilic substitution versus hydride transfer

In this study, the gas phase chemistry of the protonated benzyl esters of proline has been investigated by electrospray ionization mass spectrometry and theoretical calculation. Upon collisional activation, the protonated molecules undergo fragmentation reactions via three primary channels: (1) direct decomposition to the benzyl cation (m/z 91), (2) formation of an ion‐neutral complex of [benzyl cation + proline]⁺, followed by a hydride transfer to generate the protonated 4,5‐dihydro‐3H‐pyrrole‐2‐carboxylic acid (m/z 114), and (3) electrophilic attack at the amino by the transferring benzyl cation, and the subsequent migration of the activated amino proton leading to the simultaneous loss of (H₂O + CO). Interestingly, no hydrogen/deuterium exchange for the fragment ion m/z 114 occurs in the d ‐labeling experiments, indicating that the transferring hydride in path‐b comes from the methenyl hydrogen rather than the amino hydrogen. For para‐substituted benzyl esters, the presence of electron‐donating substituents significantly promotes the direct decomposition (path‐a), whereas the presence of electron‐withdrawing ones distinctively inhibits that channel. For the competing channels of path‐b and path‐c, the presence of electron‐donating substituents favors path‐b rather than path‐c, whereas the presence of electron‐withdrawing ones favors path‐c rather than path‐b. Copyright © 2013 John Wiley & Sons, Ltd.     

Gas phase retro‐Michael reaction resulting from dissociative protonation: fragmentation of protonated warfarin in mass spectrometry

A mass spectrometric study of protonated warfarin and its derivatives (compounds 1 to 5) has been performed. Losses of a substituted benzylideneacetone and a 4‐hydroxycoumarin have been observed as a result of retro‐Michael reaction. The added proton is initially localized between the two carbonyl oxygens through hydrogen bonding in the most thermodynamically favorable tautomer. Upon collisional activation, the added proton migrates to the C‐3 of 4‐hydroxycoumarin, which is called the dissociative protonation site, leading to the formation of the intermediate ion‐neutral complex (INC). Within the INC, further proton transfer gives rise to a proton‐bound complex. The cleavage of one hydrogen bond of the proton‐bound complex produces the protonated 4‐hydroxycoumarin, while the separation of the other hydrogen bond gives rise to the protonated benzylideneacetone. Theoretical calculations indicate that the 1, 5‐proton transfer pathway is most thermodynamically favorable and support the existence of the INC. Both substituent effect and the kinetic method were utilized for explaining the relative abundances of protonated 4‐hydroxycoumarin and protonated benzylideneacetone derivative. For monosubstituted warfarins, the electron‐donating substituents favor the generation of protonated substituted benzylideneacetone, whereas the electron‐withdrawing groups favor the formation of protonated 4‐hydroxycoumarin. Copyright © 2012 John Wiley & Sons, Ltd.     

Reversed Electron Apportionment in Mesolytic Cleavage: The Reduction of Benzyl Halides by SmI2

The paradigm that the cleavage of the radical anion of benzyl halides occurs in such a way that the negative charge ends up on the departing halide leaving behind a benzyl radical is well rooted in chemistry. By studying the kinetics of the reaction of substituted benzylbromides and chlorides with SmI₂ in THF it was found that substrates para‐substituted with electron‐withdrawing groups (CN and CO₂Me), which are capable of forming hydrogen bonds with a proton donor and coordinating to samarium cation, react in a reversed electron apportionment mode. Namely, the halide departs as a radical. This conclusion is based on the found convex Hammett plots, element effects, proton donor effects, and the effect of tosylate (OTs) as a leaving group. The latter does not tend to tolerate radical character on the oxygen atom. In the presence of a proton donor, the tolyl derivatives were the sole product, whereas in its absence, the coupling dimer was obtained by a S_N2 reaction of the benzyl anion on the neutral substrate. The data also suggest that for the para‐CN and CO₂Me derivatives in the presence of a proton donor, the first electron transfer is coupled with the proton transfer.     

Competitive benzyl cation transfer and proton transfer: collision‐induced mass spectrometric fragmentation of protonated N,N‐dibenzylaniline

Collision‐induced dissociation of protonated N ,N ‐dibenzylaniline was investigated by electrospray tandem mass spectrometry. Various fragmentation pathways were dominated by benzyl cation and proton transfer. Benzyl cation transfers from the initial site (nitrogen) to benzylic phenyl or aniline phenyl ring. The benzyl cations transfer to the two different sites, and both result in the benzene loss combined with 1,3‐H shift. In addition, after the benzyl cation transfers to the benzylic phenyl ring, 1,2‐H shift and 1,4‐H shift proceed competitively to trigger the diphenylmethane loss and aniline loss, respectively. Deuterium labeling experiments, substituent labeling experiments and density functional theory calculations were performed to support the proposed benzyl cation and proton transfer mechanism. Overall, this study enriches the knowledge of fragmentation mechanisms of protonated N ‐benzyl compounds. Copyright © 2017 John Wiley & Sons, Ltd.     

Synthesis and Properties of 2‐Phenylbenzoxazole‐Based Luminophores for in situ Photopolymerized Liquid‐Crystal Films

We report the synthesis of a series of blue‐emitting 2‐phenylbenzoxazoles (PBOs) substituted at either the 5‐ or 6‐position of the benzoxazole ring and the para‐position of the phenyl substituent. The thermal and optical properties of the materials can be rationalized in terms of the position of the substituent at the benzoxazole moiety and the electron‐withdrawing or electron‐donating character of the substituents. From the results, we conclude that the combination of an electron‐donating substituent at the benzoxazole fragment and an electron‐withdrawing one at the phenyl fragment has a more marked effect on the electronic properties of the aromatic PBO core than other possibilities. This particular combination gives luminophores that are suitable for optical applications on the basis of their high emission efficiency and photostability. In view of that, oriented films were prepared by in situ polymerization of a mixture of a liquid crystalline direactive matrix containing 5% (w/w) of the luminophore. The films exhibit linearly polarized emission.     

Synthesis and characterization of 1‐ and 2‐cinnamoyloxyacetonaphthones

The synthesis of 1‐ and 2‐cinnamoyloxyacetonaphthones was achieved in one step using hydroxyl acetonaphthones and substituted cinnamic acids in the presence of a catalytic amount of phosphoroxychloride. Structural characterization was accomplished using high‐resolution nuclear magnetic resonance (NMR) spectroscopy. Chemical shifts of the compounds were compared and the change in the chemical shifts relative to electron‐donating and ‐withdrawing groups is presented. Introduction of a thiophene ring instead of phenyl‐substituted analogs caused shielding of the olefinic proton. Copyright © 2010 John Wiley & Sons, Ltd.     

Amine‐blocked polyisocyanates. I. Synthesis of novel N‐methylaniline‐blocked polyisocyanates and deblocking studies using hot‐stage fourier transform infrared spectroscopy

A series of substituted N‐methylaniline‐blocked polyisocyanates based on 4,4′‐methylenebis(phenyl isocyanate) and poly(tetrahydrofuran) were prepared and characterized thoroughly with FTIR, ¹H NMR, and ¹³C NMR spectroscopy methods. Compared with unsubstituted N‐methylaniline, a blocking agent with an electron‐releasing substituent at the para position took a shorter time, whereas those with an electron‐releasing substituent at the ortho position or an electron‐withdrawing substituent at the ortho and para positions took longer times for the blocking reaction. The thermal dissociation reactions of blocked polyisocyanates were carried out with an FTIR spectrophotometer attached to hot‐stage accessories under dynamic and isothermal conditions. The dynamic method was used to determine the deblocking temperature, and the isothermal method was used to calculate the deblocking kinetics and activation parameters. The cure times of blocked polyisocyanates with hydroxyl‐terminated polybutadiene were also determined. The deblocking temperatures, the results of cure‐time studies, and the kinetic parameters revealed that the thermal dissociation of the N‐methylaniline‐blocked polyisocyanates was retarded by electron‐donating substituents and facilitated by electron‐withdrawing substituents. The action of N‐methylanilines as blocking agents for isocyanate was explained by the formation of a four‐center, intramolecularly hydrogen‐bonded ring structure during the thermal dissociation of the blocked polyisocyanates. The formation of such a hydrogen‐bonded ring structure was confirmed and supported by variable‐temperature ¹H NMR studies and entropy parameters, respectively. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 1557–1570, 2007     

N−H‐Type Excited‐State Proton Transfer in Compounds Possessing a Seven‐Membered‐Ring Intramolecular Hydrogen Bond

A series of compounds containing 5‐(2‐aminobenzylidene)‐2,3‐dimethyl‐3,5‐dihydro‐4H‐imidazol‐4‐one ( o ‐ABDI ) as the core chromophore with a seven‐membered‐ring N?H‐type intramolecular hydrogen bond have been synthesized and characterized. The acidity of the N?H proton and thus the hydrogen‐bond strength can be fine‐tuned by replacing one of the amino hydrogen atoms by a substituent R, the acidity increasing with increasing electron‐withdrawing strength of R, that is, in the order H<COCH₃<COPh<Tosyl<COCF₃. The tosyl and trifluoroacetyl derivatives undergo ultrafast, irreversible excited‐state intramolecular proton transfer (ESIPT) that results in proton‐transfer emission solely in the red region. Reversible ESIPT, and hence dual emission, involving the normal and proton‐transfer tautomers was resolved for the acetyl‐ and benzyl‐substituted counterparts. For o ‐ABDI , which has the weakest acidity, ESIPT is prohibited due to its highly endergonic reaction. The results clearly demonstrate the harnessing of ESIPT by modifying the proton acidity and hydrogen‐bonding strength in a seven‐membered‐ring intramolecular hydrogen‐bonding system. For all the compounds studied, the emission quantum yields are weak (ca. 10^?3) in dichloromethane, but strong in the solid form, ranging from 3.2 to 47.4 %.     

Chemoselectivity in the Reaction of 2‐Diazo‐3‐oxo‐3‐phenylpropanal with Aldehydes and Ketones

The chemoselectivity in the reaction of 2‐diazo‐3‐oxo‐3‐phenylpropanal ( 1 ) with aldehydes and ketones in the presence of Et₃N was investigated. The results indicate that 1 reacts with aromatic aldehydes with weak electron‐donating substituents and cyclic ketones under formation of 6‐phenyl‐4H‐1,3‐dioxin‐4‐one derivatives. However, it reacts with aromatic aldehydes with electron‐withdrawing substituents to yield 1,3‐diaryl‐3‐hydroxypropan‐1‐ones, accompanied by chalcone derivatives in some cases. It did not react with linear ketones, aliphatic aldehydes, and aromatic aldehydes with strong electron‐donating substituents. A mechanism for the formation of 1,3‐diaryl‐3‐hydroxypropan‐1‐ones and chalcone derivatives is proposed. We also tried to react 1 with other unsaturated compounds, including various olefins and nitriles, and cumulated unsaturated compounds, such as N,N′‐dialkylcarbodiimines, phenyl isocyanate, isothiocyanate, and CS₂. Only with N,N′‐dialkylcarbodiimines, the expected cycloaddition took place.     

Synthesis of 2‐amino‐4‐oxo‐5‐substitutedbenzylthiopyrrolo[2,3‐d]‐pyrimidines as potential inhibitors of thymidylate synthase

A series of ten novel 2‐amino‐4‐oxo‐5‐[(substitutedbenzyl)thio]pyrrolo[2,3‐d]pyrimidines 2‐11 were synthesized as potential inhibitors of thymidylate synthase and as antitumor agents. The analogues contain various electron withdrawing and electron donating substituents on the benzylsulfanyl ring of the side chains and were synthesized from the key intermediate 2‐amino‐4‐oxo‐6‐methylpyrrolo[2,3‐d]pyrimidine, 14 . Appropriately substituted benzyl mercaptans were appended to the 5‐position of 14 via an oxidative addition reaction using iodine, ethanol and water. The compounds were evaluated against human, Escherichia coli and Toxoplasma gondii thymidylate synthase and against human, Escherichia coli and Toxoplasma gondii dihydrofolate reductase. The most potent inhibitor, ( 6 ) which has a 4′‐methoxy substituent on the side chain, has an IC₅₀=25 μM against human thymidylate synthase. Contrary to analogues of general structure 1 , electron donating or electron withdrawing substituents on the side chain of 2‐11 had little or no influence on the human thymidylate synthase inhibitory activity.     

Solid‐State Emissive Aroyl‐S,N‐Ketene Acetals with Tunable Aggregation‐Induced Emission Characteristics

N‐Benzyl aroyl‐S,N‐ketene acetals can be readily synthesized by condensation of aroyl chlorides and N‐benzyl 2‐methyl benzothiazolium salts in good to excellent yields, yielding a library of 35 chromophores with bright solid‐state emission and aggregation‐induced emission characteristics. Varying the substituent from electron‐donating to electron‐withdrawing enables the tuning of the solid‐state emission color from deep blue to red.     

An experimental and computational investigation on the fragmentation behavior of enaminones in electrospray ionization mass spectrometry

The dissociation pathways of protonated enaminones with different substituents were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) in positive ion mode. In mass spectrometry of the enaminones, Ar? CO? CH?CH? N(CH₃)₂, the proton transfers from the thermodynamically favored site at the carbonyl oxygen to the dissociative protonation site at ipso‐position of the phenyl ring or the double bond carbon atom adjacent to the carbonyl leading to the loss of a benzene or elimination of C₄H₉N, respectively. And the hydrogen? deuterium (H/D) exchange between the added proton and the proton of the phenyl ring via a 1,4‐H shift followed by hydrogen ring‐walk was witnessed by the D‐labeling experiments. The elemental compositions of all the ions were confirmed by ultrahigh resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR‐MS/MS). The enaminones studied here were para‐monosubstituted on the phenyl ring and the electron‐donating groups were in favor of losing the benzene, whereas the electron‐attracting groups strongly favored the competing proton transfer reaction leading to the loss of C₄H₉N to form a benzoyl cation, Ar‐CO⁺. The abundance ratios of the two competitive product ions were relatively well‐correlated with the σ_p⁺ substituent constants. The mechanisms of these reactions were further investigated by density functional theory (DFT) calculations. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of Phenyl‐ and Methyl‐Substituents on p‐Phenylenediamine,an Electrochemical and Spectral Study

Two series of substituted p‐phenylenediamines have been studied for their electronic effects on redox potential and spectral properties. p‐Phenylenediamines and N,N,N′,N”‐tetramethyl‐p‐phenylenediamine substituted with different numbers of phenyl groups have been synthesized and their cyclic voltammograms have been obtained. The correlation between the substituent number and the redox potential appears linear. The slope reflects the additive effect of electron‐donating methyl and electron‐withdrawing phenyl groups. The absorption spectra of the cation radicals indicate that phenyl‐substituted ones have broad intervalence‐charge transfer bands. The p‐phenylenediamines exhibit different properties from triphenylamines in that the oxidized forms are more stable in CH₃CN then those in CH₂Cl₂. Some of the cation radicals or dications could undergo follow‐up chemical reactions and form products that are more easily oxidized.     

Gas-Phase Chemistry of Benzyl Cations in Dissociation of <Emphasis Type="BoldItalic">N</Emphasis>-Benzylammonium and <Emphasis Type="BoldItalic">N</Emphasis>-Benzyliminium Ions Studied by Mass Spectrometry

In this study, the fragmentation reactions of various N-benzylammonium and N-benzyliminium ions were investigated by electrospray ionization mass spectrometry. In general, the dissociation of N-benzylated cations generates benzyl cations easily. Formation of ion/neutral complex intermediates consisting of the benzyl cations and the neutral fragments was observed. The intra-complex reactions included electrophilic aromatic substitution, hydride transfer, electron transfer, proton transfer, and nucleophilic aromatic substitution. These five types of reactions almost covered all the potential reactivities of benzyl cations in chemical reactions. Benzyl cations are well-known as Lewis acid and electrophile in reactions, but the present study showed that the gas-phase reactivities of some suitably ring-substituted benzyl cations were far richer. The 4-methylbenzyl cation was found to react as a Brønsted acid, benzyl cations bearing a strong electron-withdrawing group were found to react as electron acceptors, and para-halogen-substituted benzyl cations could react as substrates for nucleophilic attack at the phenyl ring. The reactions of benzyl cations were also related to the neutral counterparts. For example, in electron transfer reaction, the neutral counterpart should have low ionization energy and in nucleophilic aromatic substitution reaction, the neutral counterpart should be piperazine or analogues. This study provided a panoramic view of the reactions of benzyl cations with neutral N-containing species in the gas phase.     

Triarylboranes with a 2‐Dimesitylboryl‐2’‐(N,N‐dimethylamino)biphenyl Core Unit: Structure–Property Correlations and Sensing Abilities to Discriminate Between F− and CN− Ions

A series of triarylboranes, in which different substituents are introduced at the para position of the dimethylamino group of a 2‐dimesitylboryl‐2’‐(N,N‐dimethylamino)biphenyl core unit, have been comprehensively investigated to explore the effect of structural modification on photophysical properties. The introduction of electron‐accepting substituents would facilitate the HOMO→LUMO charge transfer (CT) transition. In contrast, the intramolecular CT transition is significantly prohibited when electron‐donating substituents are incorporated. Notably, the HOMO→LUMO CT transition mainly consists of the transition from the electron‐donating amino group to an electron acceptor other than boryl when a strong electron acceptor such as the dicyanovinyl group is present. This dicyanovinyl‐substituted compound displays sensing abilities to discriminate fluoride and cyanide ions. In solution in THF, the fluoride ions first bind to the boron center, then attack the α‐carbon atom of the dicyanovinyl group, whereas the cyanide anion acts on the electron‐accepting centers in the reverse sequence. As a result, the absorption and emission change in different manners upon addition of fluoride and cyanide ions.     

NMR study of O and N,O‐substituted 8‐quinolinol derivatives

The ¹H and ¹³C NMR spectral study of several biologically active derivatives of 8‐quinolinol have been made through extensive NMR studies including homodecoupling and 2D‐NMR experiments such as COSY‐45°, NOESY, and HeteroCOSY. Electron donating resonance and electron withdrawing inductive effect of several groups showed marked changes in chemical shifts of nuclei at the seventh positions of O‐substituted quinolinols (2–15). Although in N‐alkyl, 8‐alkoxyquinolinium halides (16–21), ring A rightly showed low frequency chemical shift values. Copyright © 2013 John Wiley & Sons, Ltd.     

Substituent effects in trans‐p,p′‐disubstituted azobenzenes: X‐ray structures at 100 K and DFT‐calculated structures

The crystal and molecular structures of two para‐substituted azobenzenes with π‐electron‐donating –NEt₂ and π‐electron‐withdrawing –COOEt groups are reported, along with the effects of the substituents on the aromaticity of the benzene ring. The deformation of the aromatic ring around the –NEt₂ group in N,N,N′,N′‐tetraethyl‐4,4′‐(diazenediyl)dianiline, C₂₀H₂₈N₄, (I), may be caused by steric hindrance and the π‐electron‐donating effects of the amine group. In this structure, one of the amine N atoms demonstrates clear sp²‐hybridization and the other is slightly shifted from the plane of the surrounding atoms. The molecule of the second azobenzene, diethyl 4,4′‐(diazenediyl)dibenzoate, C₁₈H₁₈N₂O₄, (II), lies on a crystallographic inversion centre. Its geometry is normal and comparable with homologous compounds. Density functional theory (DFT) calculations were performed to analyse the changes in the geometry of the studied compounds in the crystalline state and for the isolated molecules. The most significant changes are observed in the values of the N=N—C—C torsion angles, which for the isolated molecules are close to 0.0°. The HOMA (harmonic oscillator model of aromaticity) index, calculated for the benzene ring, demonstrates a slight decrease of the aromaticity in (I) and no substantial changes in (II).     

The ESI CAD fragmentations of protonated 2,4,6‐tris(benzylamino)‐ and tris(benzyloxy)‐1,3,5‐triazines involve benzyl–benzyl interactions: a DFT study

The electrospray ionization collisionally activated dissociation (CAD) mass spectra of protonated 2,4,6‐tris(benzylamino)‐1,3,5‐triazine (1) and 2,4,6‐tris(benzyloxy)‐1,3,5‐triazine (6) show abundant product ion of m/z 181 (C₁₄H₁₃⁺). The likely structure for C₁₄H₁₃⁺ is α‐[2‐methylphenyl]benzyl cation, indicating that one of the benzyl groups must migrate to another prior to dissociation of the protonated molecule. The collision energy is high for the ‘N’ analog (1) but low for the ‘O’ analog (6) indicating that the fragmentation processes of 1 requires high energy. The other major fragmentations are [M + H‐toluene]⁺ and [M + H‐benzene]⁺ for compounds 1 and 6, respectively. The protonated 2,4,6‐tris(4‐methylbenzylamino)‐1,3,5‐triazine (4) exhibits competitive eliminations of p‐xylene and 3,6‐dimethylenecyclohexa‐1,4‐diene. Moreover, protonated 2,4,6‐tris(1‐phenylethylamino)‐1,3,5‐triazine (5) dissociates via three successive losses of styrene. Density functional theory (DFT) calculations indicate that an ion/neutral complex (INC) between benzyl cation and the rest of the molecule is unstable, but the protonated molecules of 1 and 6 rearrange to an intermediate by the migration of a benzyl group to the ring ‘N’. Subsequent shift of a second benzyl group generates an INC for the protonated molecule of 1 and its product ions can be explained from this intermediate. The shift of a second benzyl group to the ring carbon of the first benzyl group followed by an H‐shift from ring carbon to ‘O’ generates the key intermediate for the formation of the ion of m/z 181 from the protonated molecule of 6. The proposed mechanisms are supported by high resolution mass spectrometry data, deuterium‐labeling and CAD experiments combined with DFT calculations. Copyright © 2012 John Wiley & Sons, Ltd.     

1-萘甲酰苯胺和2-萘甲酰苯胺的分子内电荷转移研究

A series of 1-naphthanilides (1) and 2-naphthanilides (2) with varied substituents at the para- or meta-position of anilino phenyl ring were prepared and their absorption and fluorescence spectra in a nonpolar solvent cyclohexane were investigated. An abnormal long wavelength emission assigned to the charge transfer (CT) state was found for all of the prepared naphthanilides in cyclohexane. A linear free energy correlation between the CT emission energies and the Hammett constants of the substituent was found within series 1 and 2. The value of the linear slope with 1 (0.42 eV) was higher than that with 2 (0.32 eV) being close to that of the substituted benzanilides 3 (0.31 eV) The higher slope value suggested higher charge separation extent in the CT state of 1 than that of 2. It was found that the corresponding linear slope of anilino-substituted benzanilides remained unchanged when para-, meta-, ortho-, or ortho, ortho-methyls were introduced into the anilino moiety, which ruled out the possible contribution of the difference in the steric effect and the electron accepting ability of the naphthoyl acceptor in 1 and 2. Compared with the early reported N-substituted-benzoyl-aminonaphthalene derivatives 4 and 5, it was considered that 1-naphthoyl enhanced the charge transfer in 1 and the proximity of its ^1La and ^1Lb states was suggested to be responsible. It was shown that 1- and/or 2-substituted naphthalene cores acting as either electron acceptor (naphthoyl) or electron donor (aminonaphthalene) were different in not only electron accepting (donating) ability but also shaping the charge transfer pathway.