Direct β-selectivity of α,β-unsaturated γ-butyrolactam for asymmetric conjugate additions in an organocatalytic manner

The β-selective asymmetric addition of γ-butyrolactam with cyclic imino esters catalyzed by a bifunctional chiral tertiary amine has been developed, which provides an efficient access to optically active β-position functionalized pyrrolidin-2-one derivatives in both high yield and enantioselectivity (up to 78% yield and 95 : 5 er). This is the first catalytic method to access chiral β-functionalized pyrrolidin-2-one via a direct organocatalytic approach.

The asymmetric addition of γ-butyrolactam with cyclic imino esters catalyzed by (DHQD)₂AQN has been developed, which provides an access to β-position functionalized pyrrolidin-2-one derivatives in high levels yield and enantioselectivity.

Metal-free organocatalytic asymmetric transformations have successfully captured considerable enthusiasm of chemists as powerful methods for the synthesis of various kinds of useful chiral compounds ranging from the preparation of biologically important molecules through to novel materials.¹ Chiral pyrrolidin-2-ones have been recognized as important structural motifs that are frequently encountered in a variety of biologically active natural and synthetic compounds.² In particular, the β-position functionalized pyrrolidin-2-one backbones, which can serve as key synthetic precursors for inhibitory neurotransmitters γ-aminobutyric acids (GABA),³ selective GABA_B receptor agonists⁴ as well as antidepressant rolipram analogues,⁵ have attracted a great deal of attention. Therefore, the development of highly efficient, environmentally friendly and convenient asymmetric synthetic methods to access these versatile frameworks is particularly appealing.As a direct precursor to pyrrolidin-2-one derivatives, recently, α,β-unsaturated γ-butyrolactam has emerged as the most attractive reactant in asymmetric organometallic or organocatalytic reactions for the synthesis of chiral γ-position functionalized pyrrolidin-2-ones (Scheme 1). These elegant developments have been achieved in the research area of catalytic asymmetric vinylogous aldol,⁶ Mannich,⁷ Michael⁸ and annulation reactions⁹ in the presence of either metal catalysts or organocatalysts (a, Scheme 1). These well-developed catalytic asymmetric methods have been related to the γ-functionalized α,β-unsaturated γ-butyrolactam to date. However, in sharp contrast, the approaches toward introducing C-3 chirality at the β-position of butyrolactam through a direct catalytic manner are underdeveloped (b, Scheme 1)¹⁰ in spite of the fact that β-selective chiral functionalization of butyrolactam can directly build up α,β-functionalized pyrrolidin-2-one frameworks.Open in a separate windowScheme 1Different reactive position of α,β-unsaturated γ-butyrolactam in catalytic asymmetric reactions.So far, only a few metal-catalytic enantioselective β-selective functionalized reactions have been reported. For examples, a rhodium/diene complex catalyzed efficient asymmetric β-selective arylation^10a and alkenylation^10b have been reported by Lin group (a, Scheme 2). Procter and co-workers reported an efficient Cu(i)–NHC-catalyzed asymmetric silylation of unsaturated lactams (b, Scheme 2).^10c Despite these creative works, considerable challenges still exist in the catalytic asymmetric β-selective functionalization of γ-butyrolactam. First, the scope of nucleophiles is limited to arylboronic acids, potassium alkenyltrifluoroborates and PhMe₂SiBpin reagents. Second, the catalytic system and activation mode is restricted to metal/chiral ligands. To our knowledge, an efficient catalytic method to access chiral β-functionalized pyrrolidin-2-one via a direct organocatalytic approach has not yet been established. Therefore, the development of organocatalytic asymmetric β-selective functionalization of γ-butyrolactam are highly desirable. In conjunction with our continuing efforts in building upon chiral precedents by using chiral tertiary amine catalytic system,¹¹ we rationalized that the activated α,β-unsaturated γ-butyrolactam might serve as a β-position electron-deficient electrophile. This γ-butyrolactam may react with a properly designed electron-rich nucleophile to conduct an expected β-selective functionalized reaction of γ-butyrolactam under a bifunctional organocatalytic fashion, while avoiding the direct γ-selective vinylogous addition reaction or β,γ-selective annulation as outlined in Scheme 2. Herein we report the β-selective asymmetric addition of γ-butyrolactam with cyclic imino esters¹² catalyzed by a bifunctional chiral tertiary amine, which provides an efficient and facile access to optically active β-position functionalized pyrrolidin-2-one derivatives with both high diastereoselectivity and enantioselectivity.Open in a separate windowScheme 2β-Selective functionalization of γ-butyrolactam via metal- (previous work) or organo- (this work) catalytic approach.To begin our initial investigation, several bifunctional organocatalysts¹³ were firstly screened to evaluate their ability to promote the β-selective asymmetric addition of γ-butyrolactam 2a with cyclic imino ester 3a in the presence of 15 mol% of catalyst loading at room temperature in CH₂Cl₂ (entries 1–6,

Effect of C-terminus amidation of Aβ39–42 fragment derived peptides as potential inhibitors of Aβ aggregation

The C-terminus fragment (Val-Val-Ile-Ala) of amyloid-β is reported to inhibit the aggregation of the parent peptide. In an attempt to investigate the effect of sequential amino-acid scan and C-terminus amidation on the biological profile of the lead sequence, a series of tetrapeptides were synthesized using MW-SPPS. Peptide D-Phe-Val-Ile-Ala-NH₂ (12c) exhibited high protection against β-amyloid-mediated-neurotoxicity by inhibiting Aβ aggregation in the MTT cell viability and ThT-fluorescence assay. Circular dichroism studies illustrate the inability of Aβ₄₂ to form β-sheet in the presence of 12c, further confirmed by the absence of Aβ₄₂ fibrils in electron microscopy experiments. The peptide exhibits enhanced BBB permeation, no cytotoxicity along with prolonged proteolytic stability. In silico studies show that the peptide interacts with the key amino acids in Aβ, which potentiate its fibrillation, thereby arresting aggregation propensity. This structural class of designed scaffolds provides impetus towards the rational development of peptide-based-therapeutics for Alzheimer''s disease (AD).

Amidated C-terminal fragment, Aβ_39–42 derived non-cytotoxic β-sheet breaker peptides exhibit excellent potency, enhanced bioavailability and improved proteolytic stability.

First reported by Alois Alzheimer in 1906, Alzheimer''s Disease (AD) is a progressive, neurodegenerative disorder with an irreversible decline in memory and cognition.¹ It is commonly seen in elderly populations and is marked by two major histopathological hallmarks, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFT).² The number of patients suffering from AD has been increasing at an alarming rate. It is estimated that around 60 million patients will be suffering from AD by the end of 2020 and half of this patient population would require the care equivalent to that of a nursing home.³ Even after a century of its discovery, there has been no treatment that targets the pathophysiology of AD.⁴ The current treatment regimen includes acetylcholine-esterase inhibitors (AChEI) comprising of donepezil, rivastigmine and galantamine as well as N-methyl-d-aspartate (NMDA)-receptor antagonist, memantine. These provide only symptomatic relief to the patient. Most of the therapeutics that are currently being tested in clinical trials couldn''t proceed beyond phase II and phase III clinical trials due to lower efficacy in elderly patients, multiple side effects and limitations in their pharmacokinetic and pharmacodynamic profiles.^5–9 This has created challenges on the societal and economic upfront.Literature analysis reveals that the soluble oligomeric Aβ species is the culprit for neurotoxicity. It interrupts normal physiological functioning of the human brain. The central hydrophobic fragment Aβ_16–22 (KLVFFAE) and the C-terminus region fragment Aβ_31–42 (IIGLMVGGVVIA) are responsible for controlling the aggregation kinetics of the monomeric species, wherein the later still remains relatively less explored.^8,9 Our group is focused on development of peptidomimetic analogues for preventing Aβ aggregation. Studies on a complete peptide scan on the C-terminus region regions has already been published previously.^10–12 In an attempt to enhance the biological efficacy of the previously designed scaffolds,¹² we rationalized the use of sequential amino acid scan by modifying/replacing individual residues, as well as amide protection of the C-terminus on the lead tetrapeptide sequence (Val-Val-Ile-Ala) to enhance the proteolytic stability of the peptides.C-terminus amidated peptides were synthesized by microwave-assisted Fmoc-solid phase peptide synthesis protocol. Scheme 1 shows the general route for the synthesis of peptides employing Rink amide resin (detailed methodology is mentioned in the ESI, Section 1^†). These peptides were characterized using by analytical HPLC, ¹H and ¹³C NMR, APCI/ESI-MS and HRMS.Open in a separate windowScheme 1Synthesis of tetrapeptide 12c using MW-assisted Fmoc-solid phase peptide synthesis protocol employing Rink amide resin. Reaction conditions: (i) 20% piperidine in DMF (7 mL), MW (40 W), 60 °C, 2 cycles – 1.5 & 3 min; (ii) 2, 4, 6, 8 (4 equiv.), TBTU (4 equiv.), HoBt (4 equiv.), DIPEA (5 equiv.), DMF (3.5 mL), MW (40 W), 60 °C, 13.5 min; (iii) TFA : TIPS : H₂O (95 : 2.5 : 2.5), rt, 2.5 h; reaction monitoring was done by: UV measurement: Fmoc deprotection-dibenzofulvene adduct, Kaiser test: 1° amines; acetaldehyde test: 2° amines.Aggregation of Aβ₄₂ results in accumulation of toxic species, which interact with neurons and hinder their functioning, leading to loss of memory and cognition. Inhibiting the process of Aβ₄₂ aggregation would alleviate the neurotoxicity imparted in PC-12 cells; this was evaluated by MTT cell viability assay.¹³ Viability of untreated cells is considered 100%. Upon treatment with 2 μM of Aβ₄₂, only 74% cells were found to be viable. Out of the total tested peptides, seven tetrapeptides 12a, 12c, 12f, 13e, 14b, 15b and 15f showed complete inhibition of Aβ₄₂-induced toxicity by restoring the cell viability to 100%, at respective concentrations. Results for cell viability assay along with Thioflavin-T fluorescence assay for all the synthesized tetrapeptides has been summarized in No.Test peptide sequence^aMTT cell viability assayThT-fluorescence assayTest peptide concentration range (Aβ₄₂: test peptide)10 μM (1 : 5)4 μM (1 : 2)2 μM (1 : 1)10 μM (1 : 5)4 μM (1 : 2)2 μM (1 : 1)% viable cells^b% inhibition^d11Val-Val-Ile-Ala-OH (lead)93.690.578.966.260.633.911aVal-Val-Ile-Ala-NH₂82.289.386.452.152.758.812a d -Val-Val-Ile-Ala-NH₂81.6100.094.882.183.564.712b Phe-Val-Ile-Ala-NH₂92.195.796.277.666.251.912c d -Phe-Val-Ile-Ala-NH₂100.095.298.4100.0100.0100.012d d -Pro-Val-Ile-Ala-NH₂92.083.582.839.852.960.812e Nva-Val-Ile-Ala-NH₂83.486.174.170.351.977.212f Aib-Val-Ile-Ala-NH₂61.395.1100.093.866.064.512g Gly-Val-Ile-Ala-NH₂75.494.392.198.164.584.413aVal-d-Val-Ile-Ala-NH₂87.675.274.715.746.589.313bVal-d-Ile-Ile-Ala-NH₂94.692.890.349.435.165.613cVal-Pro-Ile-Ala-NH₂83.196.093.018.516.331.813dVal-Aib-Ile-Ala-NH₂100.093.075.145.350.052.113eVal-Phe-Ile-Ala-NH₂93.3100.079.357.261.7100.013fVal-d-Phe-Ile-Ala-NH₂99.792.994.262.566.275.814aVal-Val-d-Ile-Ala-NH₂69.676.579.721.825.349.614bVal-Val-Leu-Ala-NH₂86.3100.083.80.016.542.215aVal-Val-Ile-d-Ala-NH₂93.880.283.323.214.668.615bVal-Val-Ile-Aib-NH₂99.1100.071.935.142.024.915cVal-Val-Ile-Gly-NH₂90.588.580.75.431.862.715dVal-Val-Ile-Val-NH₂71.775.464.511.06.90.015eVal-Val-Ile-Leu-NH₂70.671.789.431.230.018.715fVal-Val-Ile-Ile-NH₂100.097.078.648.20.00.016a Pro-Pro-Ile-Ala-NH₂77.760.774.614.218.324.7Aβ₄₂74.05Control^c100.0Open in a separate window^aAmino acid residue modified within the tetrapeptide sequence is indicated in bold.^bCell viability studies were performed using MTT cell viability assay against PC-12 cells.^cThe percentage of untreated cells was considered 100% (positive control); percentage cell viability was calculated for the cells incubated along with Aβ₄₂ (2 μM) in absence (negative control) and presence of the test peptides in respective dose concentrations for 6 h. % of viable cells was calculated by the formula as 100 × [Aβ₄₂ + test peptide OD₅₇₀ − Aβ OD₅₇₀/control OD₅₇₀ − Aβ OD₅₇₀]. In a subset of triplicate wells, standard deviation values ranged 1.81–4.72.^dInhibition of Aβ₄₂ aggregation was calculated by Thioflavin-T fluorescence assay. % relative fluorescence units (% RFU) exhibited by Aβ fibrils were considered as 100%. ThT dye incubated alone was considered as control and % RFU units were computed when Aβ₄₂ was co-incubated with the test peptides for 24 h (λ_ex 440 nm, λ_em 485 nm). % inhibition of ThT fluorescence was calculated by using the formula: 100 × [100 − (Aβ₄₂ + test peptide RFU₄₈₅ − control RFU₄₈₅/Aβ₄₂ RFU₄₈₅ − control RFU₄₈₅)]. In a subset of triplicate wells, SD values ranged 1.22–4.83. Data for both the experiments was recorded for triplicate samples and the readings were averaged (<5% variation).The quantitative evaluation of β-sheet structures within the amyloid fibrils is accessed by the fluorescence of the Thioflavin-T dye.¹⁴ Inhibiting amyloid fibrillation would reduce or eliminate such an enhancement in fluorescence. This concept is utilized to evaluate compounds that would prevent Aβ from aggregating.^15–17 ThT fluorescence in the presence of Aβ₄₂ alone was considered 100% and % relative fluorescence unit (% RFU) values were calculated for Aβ₄₂ co-incubated with the respective inhibitor peptides. ThT incubated alone, exhibited % RFU of nearly 53.3% as compared to control solution without dye. Complete data of % inhibition of Aβ₄₂ by the test peptides has been summarized in 17 Out of all the tested peptides, peptides 12c and 13e showed minimal enhancement in ThT fluorescence when co-incubated with equimolar concentrations of Aβ₄₂. Peptides 12f and 12g showed >90% activity at five-fold excess dose concentrations. A comparative bar graph representation for the four most active peptides 12c, 12f, 12g and 13e has been depicted in Fig. 1A. To understand the % RFU values indicating the relative fluorescence of ThT and % inhibition exhibited by the most active test peptides 12c, 12f, 12g and 13e, values have been summarized in ESI, Tables S1 and S2.^† The observed RFU was close to that of the control wells where the dye incubated alone. Negligible increment of ThT fluorescence when the test peptides are co-incubated with Aβ₄₂ peptide clearly indicates the inhibition of fibrillation. It also provides further support to the inhibition of Aβ₄₂-induced neuronal toxicity as studied in the MTT assay. The % inhibition of Aβ₄₂ aggregation as depicted by the ThT fluorescence assay is in accordance with the % cell viability data obtained by the MTT assay. Exact correlation cannot be established between the two because of the differential behavior of Aβ₄₂ in the presence of a cellular environment as well as the treatment/incubation time for both the experiments.Open in a separate windowFig. 1Effect of most active test peptides on Aβ₄₂ aggregation: bar plots depicting the decrease in % RFU of ThT dye when Aβ₄₂ (2 μM) was co-incubated with test peptides at higher doses (A), and lower doses (B). Complete fluorescence was represented by the Aβ₄₂ peptide incubated along with the dye (black) and dye control (grey) represents the dye incubated alone. Subsequent bars represent Aβ peptide co-incubated with the varying concentrations of inhibitor peptides for 24 h. Significance values indicated with respect to the Aβ peptides, *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Dose dependent modulation of Aβ₄₂ aggregation-induced-neurotoxicity in PC-12 cells exhibited by the test peptide 12c (black). (D) Concentration dependent % inhibition on Aβ₄₂ aggregation mediated ThT fluorescence exhibited by the test peptide 12c (black). % inhibition of ThT fluorescence was calculated by using the formula: 100 × [100 − (Aβ₄₂ + test peptide RFU₄₈₅ − control RFU₄₈₅/Aβ₄₂ RFU₄₈₅ − control RFU₄₈₅)]. Readings (λ_ex 440 nm, λ_em 485 nm) was recorded for triplicate samples from three individual experiments and the readings were averaged (<5% variation). Error bars represent mean ± SD (n = 3). Data were analyzed by one-way anova test.Peptides 12c and 12f that exhibited >98% cell viability at the lowest tested concentration of 2 μM were then evaluated at lower dose concentrations of 1.0 μM, 0.5 μM and 0.1 μM, against 2 μM of Aβ₄₂ maintaining the ratios of 1 : 2, 1 : 4 and 1 : 20 (test peptide : Aβ₄₂), respectively. The graphical plot of dose dependent modulation of Aβ₄₂ aggregation-induced-neurotoxicity in PC-12 cells is depicted in Fig. 1C. Peptide 12c exhibited 78% inhibition of Aβ₄₂ even at a lowest tested dose of 0.1 μM. A graphical representation of the % decrease in RFU has been depicted in Fig. 1B and dose dependent % inhibition of Aβ₄₂ aggregation has been depicted in Fig. 1D. Excellent activities were exhibited when the test peptide is present in equimolar concentrations of Aβ₄₂, indicating 1 : 1 inhibition of the parent peptide.Upon the basis of careful analysis of data reported herewith and results published earlier,^10–12 a correlation between the amino acid residues within the tetrapeptide sequence and the exhibited activity was established. Replacement of the first residue, Val₃₉ with hydrophobic residues (Phe and D-Phe) exhibited >90% inhibition. The replacement of Val₄₀ with hydrophobic (Phe, D-Ile) or conformationally restricted amino acids (Pro, Aib) slightly enhanced the potency of the peptide. This holds true even in dual substitution at Val₃₉ and Val₄₀. Any replacement or modification yielded less active derivatives, indicating Ile₄₁ is critical for activity. Small analogous amino acid is preferred at the Ala₄₂ for retaining the activity. Amidation of the C-terminus results in enhancement of inhibition potential for some peptides. In some cases, a decrease in activity is also observed. A summary of the SAR is shown in Fig. 2. Understanding the sequential positioning of these residues would help us further develop derivatives with enhanced potency to inhibit Aβ₄₂ aggregation.Open in a separate windowFig. 2Derived structure–activity-relationship of tetrapeptides.It is reported that there are two species of Aβ i.e. Aβ₄₀ and Aβ₄₂, present in the diseased brain.^18–20 Evaluating the inhibitory activities of the test compounds on the aggregation of both the species would be of biological significance.²¹ Therefore, inhibitory potential of peptide 12c was evaluated on Aβ_40. The relative increase in the ThT fluorescence when Aβ₄₀ was incubated alone for 24 h was very less or similar to that of the control wells containing ThT (Fig. 3A). Further testing for 48 h and 72 h, respectively yielded no substantial results (ESI, Table S3^†).Open in a separate windowFig. 3Thioflavin-T fluorescence studies on Aβ species: % RFU exhibiting the effect of peptide 12c on aggregation of (A) Aβ₄₀ (5 μM) and (B) Aβ₄₀ & Aβ₄₂ mixture (5 μM) mediated ThT fluorescence. Complete fluorescence was represented by Aβ₄₀ and the 10 : 1 mixture of Aβ peptides incubated along with the dye (black) and dye incubated alone (grey). Subsequent bars represent the respective concentrations of inhibitor peptide 12c co-incubated with the corresponding Aβ peptides for 24 h. Readings (λ_ex 440 nm, λ_em 485 nm) was recorded for triplicate samples from three individual experiments and the readings were averaged (<5% variation). Error bars represent mean ± SD (n = 3). Data were analyzed by one-way anova test. Significance values indicated with respect to the Aβ peptides, *, p < 0.05; **, p < 0.01; ***, p < 0.001.The minimal increase in fluorescence could be attributed to the slower nucleation rate and longer lag phase in the kinetics of Aβ₄₀ fibril formation in comparison to Aβ_42.^22–24 Also, Aβ₄₀ is relatively less neurotoxic and its aggregation propensity enhances in the presence of Aβ₄₂, although the former is present in a ten-fold higher concentration. Thus, evaluation of the inhibitory activity of test peptide 12c on the mixtures of Aβ₄₀ : Aβ₄₂ in the ratio of 10 : 1 was performed. On incubation of a mixture of 5 μM of Aβ₄₀ and 0.5 μM of Aβ₄₂ (ratio of 10 : 1) the % relative increase in ThT fluorescence was comparatively higher than when Aβ₄₀ was incubated alone (Fig. 3B). When compared to that of Aβ₄₂ incubated alone as analyzed in the previous experiments, the fluorescence intensities were less. This gave us a clear indication that the aggregation propensity of Aβ₄₀ is amplified in the presence of 0.1 equimolar Aβ₄₂. On co-incubation of the test peptide 12c with the 5 μM mixture of Aβ₄₀ : Aβ₄₂ in the ratio of 10 : 1, substantial decrease in the fluorescence levels were observed (ESI, Table S4^†).As a prophylactic measurement, the potential ability of the test peptides to deform the aggregated Aβ₄₂ was investigated.^25,26 Monomeric Aβ₄₂ was pre-incubated for a period of 24 h and fluorescence was measured. As anticipated, there was a marked increase in the fluorescence due the fibril state of Aβ_42. Test peptide was added to each of the wells at the respective dose concentrations and readings were recorded at in a time dependent manner until 120 h of incubation. Time dependent % deformation of preformed fibrils in the presence of equimolar test peptide has been depicted in Fig. 4A. It could be observed that peptide 12c has the potential of deforming pre-aggregated Aβ₄₂ fibrils (>35%) until 48 h of incubation. Also, peptide 12c significantly reduced the fluorescence of Aβ₄₂ showing 57.5, 34.9 and 33.7% inhibition at 2, 1 and 0.5 μM, respectively after 24 h treatment. % inhibition and % RFU for time intervals of 24 h and 48 h has been provided in the ESI, Table S5.^†Open in a separate windowFig. 4Time dependent inhibition of Aβ₄₂: (A) % deformation or disaggregation of preformed Aβ₄₂ fibrils in presence of equimolar concentration of test peptide 12c as evaluated via ThT fluorescence assay. (B) Time and concentration dependent RFU comparison depicting the effect of individual tetrapeptide 12c on Aβ₄₂ mediated-ThT fluorescence. % inhibition of ThT fluorescence was calculated by using the formula: 100 × [100 − (Aβ₄₂ + test peptide RFU₄₈₅ − control RFU₄₈₅/Aβ₄₂ RFU₄₈₅ − control RFU₄₈₅)]. Readings (λ_ex 440 nm, λ_em 485 nm) was recorded for triplicate samples and the values were normalized to the ThT dye control and averaged (<5% variation). Data was interpreted from three individual experiments. Error bars represent mean ± SD (n = 3). Data were analyzed by one-way anova test.A time dependent ThT fluorescence assay was performed on the most active test peptide 12c at the similar concentrations of 2, 1 and 0.5 μM with Aβ₄₂ (2 μM) for a period of 7 days. Readings were recorded at regular time intervals of 24 h each. Fig. 4B shows the decrease in the RFU values when Aβ₄₂ was incubated in presence of peptide 12c. Aβ₄₂ on incubation alone with the ThT dye showed an enhancement in the fluorescence of about 57% that could be attributed to the aggregation of the Aβ₄₂ peptide. The fluorescence shown by the blank wells, wherein the dye incubated alone was considered as the control. Compared to the Aβ₄₂ sample, very low values of fluorescence were observed in the presence of peptide 12c. % inhibition of Aβ₄₂ aggregation exhibited by the test peptide has been summarized in ESI, Table S6.^† It can be summed that peptide 12c exhibits activity on preformed Aβ₄₂ fibrils as well as inhibits Aβ₄₂ aggregation until 120 h of treatment.ANS fluorescence assay was performed in complimentary to Thioflavin-T assay.^27–29 The effect of test peptide inhibiting the process of Aβ₄₂ aggregation as well as deformation of the preformed Aβ₄₂ fibrils was evaluated. The relative fluorescence for Aβ₄₂ fibrils was considered to be 100% and decrease in the % RFU when test peptides were co-incubated with Aβ₄₂ was computed. The fluorescence emitted by binding of ANS to the test peptide itself was subtracted as the test peptide is hydrophobic. The results for relative decrease in fluorescence obtained in both the sub-experiments have been summarized in Fig. 5A. Upon co-incubation of Aβ₄₂ and test peptide 12c in ratios 1 : 1 and 1 : 0.5, a marked decrease in the fluorescence intensity was observed, in comparison to that of the lowest tested concentration of 0.5 μM. These results are in accordance to the results seen in ThT fluorescence assay.Open in a separate windowFig. 5Additional fluorescence studies: (A) effect of varying concentration of tetrapeptide 12c on inhibition of Aβ₄₂ fibril formation (Set 1) and on pre-aggregated fibrils of Aβ (Set 2). Complete fluorescence was represented by the Aβ₄₂ (2 μM) incubated alone, monomeric (Set 1) and pre-aggregated t = 24 h (Set 2) and in the presence of respective concentrations of the test peptide 12c after 24 h. ANS dye incubated alone was considered as control and % RFU units for individual samples were computed by normalizing to the ANS dye control (λ_ex 480 nm, λ_em 535 nm). Subsequent bars represent the % RFU of the respective concentrations of the inhibitor peptide 12c co-incubated with the differential states of Aβ₄₂ peptide (2 μM) for 24 h. (B) Fluorescence spectrum showing effect of test peptide on Aβ₄₂ aggregation and its interaction with GUVs. Fluorescence of Aβ₄₂ alone at 0 h (green), 24 h (black); along with test peptides 12c (blue) after 24 h in the presence of GUVs (λ_ex 480 nm, λ_em 400–600 nm). ANS dye incubated alone was considered as control and relative FL. Intensities for individual samples were computed by normalizing to the ANS dye control. (C) Intrinsic tyrosine fluorescence of Aβ₄₂ during fibrillation and inhibition by test peptide 12c (λ_ex 260 nm, λ_em 280–410 nm). Fluorescence of 5 μM Aβ₄₂ (t = 0 h, green), 5 μM Aβ₄₂ incubated alone (t = 24 h, black), Aβ₄₂ co-incubated along with 5 μM of the test peptides, 12c (t = 24 h, blue). Readings was recorded for triplicate samples from three individual experiments and were averaged (<5% variation). Error bars represent mean ± SD (n = 3). Data were analyzed by one-way anova test.It is hypothesized that the aggregated soluble oligomeric form of Aβ₄₂ interacts with the neuronal membranes by hampering cellular processes and exhibiting neurotoxicity.⁷ The design of the experiment was similar to the MTT test conditions, wherein giant unilamellar vesicles (GUVs) with composition mimicking the rat neuronal myelin were prepared (ESI,^† Section 5.1) and interaction of Aβ was evaluated by ANS fluorescence measurements.^29–32 When Aβ₄₂ was just added to the prepared GUVs (t = 0 h), ANS showed a good emission spectrum from 450 to 550 nm. After 24 h incubation of Aβ with the vesicles, no emission band was seen, indicating the absence of hydrophobic binding domain, thus no fluorescence.This could be attributed that the hydrophobic region entered the vesicles, providing no binding site for the dye. Emission intensities obtained for the vesicles alone was considered as blank and was subtracted from the readings obtained for both the time points. To evaluate the effects of incubation of test peptides and its inhibitory effect on Aβ₄₂ aggregation, Aβ₄₂ and test peptide 12c along with the GUVs was incubated for 24 h at 37 °C and the relative change in the fluorescence was observed. Readings for the test peptide incubated alone with the vesicles at the similar concentration were subtracted the final readings so as to obtain a comparable result for Aβ. On co-incubation of Aβ₄₂ with 12c, two distinct observations were seen, primarily a visible emission spectrum and secondly a blue shift with a slight increase in the emission intensity (Fig. 5B). The emission spectrum indicates that the hydrophobic region was available for binding to ANS, thus indicating that Aβ was available in its monomeric form itself. The increase in emission intensity and the blue shift does suggest certain interactions between 12c and the full-length Aβ, which results into differential binding of ANS to the test peptide–Aβ complex (ESI, Fig. S2^†).Monitoring intrinsic Tyr fluorescence of Aβ₄₂ during fibril formation and interaction with most active test peptides was also studied. Tyr has significantly lower quantum yield than Trp and is usually only used as an intrinsic fluorescent probe in Trp-lacking proteins or peptides, since energy transfer to Trp residues usually quenches the Tyr fluorescence. Tyrosine shows a typical emission band at 305 nm, which is red shifted to 340 nm due to resonance of the phenol to phenolate ion. We hypothesized that in aggregated state of Aβ₄₂, stacking of phenolate ions of the tyrosine residues will produce slightly enhanced fluorescence in comparison to that of the monomeric or non-aggregated state. It could be clearly seen, in the overlay of fluorescence spectrum for Aβ₄₂ incubated alone for 0 h (green) and 24 h (black), as well as in presence of 12c (blue, Fig. 5C) that the monomeric state of Aβ₄₂ was retained in presence of the test peptides. A comparative bar plot analysis (ESI, Fig. S3^†) of the fluorescence response at 340 nm has been indicated to compare the change in observed fluorescence intensities.Since amyloid-β aggregation is preceded by the conformational transition towards increasing β-sheet structure, monitoring the content of β-sheet formation would therefore depict the effect of inhibitors on the aggregation of Aβ₄₂.^32,33 The effect of inhibitor peptides on the conformation of Aβ₄₂ was assessed via CD spectroscopy.^34–36 Spectra of equimolar concentrations of the individual test peptides were subtracted from the corresponding spectra of inhibitor peptides co-incubated Aβ₄₂. Predicted values of conformation are summarized in ESI, Table S7.^† When incubated alone, Aβ₄₂ exhibited a conformational transition from random coiling and turn to majorly β-sheet form. Initially, Aβ₄₂ peptide majorly comprised of 49.4% β-sheet conformation. At the end of 24 h incubation period, β-sheet content increased to 66.4%, with the α-helix content increasing from 6.3% to 17.0%. In the presence of inhibitor peptide 12c, β-sheet form completely vanished and turns and random coiling was seen to be present in 46.6 and 41.0% respectively. This clearly indicates that 12c inhibits β-sheet formation propensity of Aβ₄₂. The spectral curve obtained for Aβ₄₂ (t = 24 h) shows the presence of a positive maxima at 195 nm (black), clearly indicating the conformation of the peptide in the β-sheet form, is absent in the former that is, Aβ₄₂ (t = 0 h), which clearly exhibits a positive maxima at around 205 nm, indicating larger proportion of turn type conformation to be present. No definitive negative minima on the curve were visible at 217 nm, but the shallow curves in the expanded region were indicative of the presence of smaller proportions of α-helix conformations. In the presence of 12c reduction in β-sheet content can also be visualized by the complete absence of the positive maxima at 195 nm, wherein the spectrum follows the similar pattern to that of the Aβ₄₂ (t = 0 h). The prevention of conformational transition to β-sheet suggests the ability of 12c to inhibit the fibrillation process. The positive curve shifts more towards 200–205 nm, indicating a larger proportion of the peptide to be present in the turn form. These observations coincide to the predicted values by the Yang protocol. The effect of the presence of equimolar concentrations of inactive peptide 13a on the conformational changes on Aβ₄₂ was evaluated. A positive maxima at 195 nm is a clear indicative of higher proportions of β-sheet type of secondary structural conformation to be present. This indicates the inactiveness of the peptide 13a and its inability to prevent aggregation of Aβ₄₂. Self-aggregation potential of peptide 13a deters its potential to inhibit Aβ₄₂ aggregation.A compiled CD spectrum recorded depicting a relative comparison of peptides 12f and 13a, incubated for 24 h at 37 °C in the presence and absence of equimolar concentrations of Aβ₄₂ has been presented in Fig. 6. Spectra of test peptides incubated alone were subtracted to obtain the final spectra for comparing the conformational state of Aβ₄₂. A comparison of the individual CD spectrums of the test peptides incubated alone to that incubated in the presence of equimolar ratio of Aβ₄₂ has been summarized (ESI, Fig. S4^†).Open in a separate windowFig. 6Secondary structure analysis using CD: CD spectrum showing the conformational changes on Aβ₄₂ aggregation in the presence of active peptide 12c and inactive peptide 13a. Aβ₄₂ (10 μM) at 0 h (black) and 24 h (blue), co-incubated individually with equimolar ratios inhibitor peptide 12c (green) and inactive peptide 13a (red) for 24 h.To understand the process of inhibition of Aβ₄₂ in presence of 12c, mass fragmentation techniques were employed.³⁷ The MS spectrum of full-length Aβ₄₂ (10 μM) in the presence and absence of an equimolar amount of 12c was recorded. Fig. 7, shows the ESI spectrum for Aβ₄₂ incubated alone (A), along with 12c (B).Open in a separate windowFig. 7HRMS Analysis: ESI-MS for Aβ₄₂ (10 μM) incubated alone (A), in presence of equimolar ratios of test peptide 12c (B) for 24 h.The spectrum for Aβ₄₂ incubated alone shows a major peak at m/z 685.4357, which may be attributed to aggregated Aβ₄₂ depicting higher mass-to-charge ratio. Further peaks at m/z of 788.4373 [(Aβ₄₂)⁶⁺], 507.2713 [(Aβ_1–9)¹⁺], 958.3161 [(Aβ_10–42)⁷⁺] and 1308.0646 [(Aβ_1–11)¹⁺] represent the Aβ₄₂ monomer and its specific fragments, respectively. Hexameric form of Aβ₄₂ with m/z 2185.4363 [(6Aβ₄₂)¹³⁺] is also observed in the spectrum.³⁸ On comparing the spectrum obtained for Aβ₄₂ incubated along with 12c, and that of Aβ₄₂ incubated alone, additional signal peaks were seen. This suggested the occurrence of adduct between Aβ₄₂ and 12c, as these peaks were not analogous to the molecular weight of native Aβ₄₂ or test peptides themselves. Analyzing the interactions of 12c with that of Aβ₄₂, the mass spectrum shows a peak at m/z 471.7321 [(Aβ_12–42 + 12c)⁸⁺], depicting 1 : 1 covalent interaction with Aβ_12–42 fragment of Aβ₄₂. The spectrum also shows signal peaks corresponding to that of Aβ_42, seen in the previous spectrum. It was clear from the above spectrum that the test peptide interacts with the monomeric unit of the Aβ_42, thereby preventing its aggregation.Visual investigation of the effects of the peptide 12c, on the morphology and abundance of Aβ₄₂ fibrils was performed by high resolution transmission electron microscopy (HR-TEM).³⁹ Shapes and morphology of the fibrils were also examined using scanning transmission electron microscope (STEM).⁴⁰ Inactive peptide 13a was selected as a negative control. The control sample of Aβ₄₂ incubated alone at t = 0 h, where uniform distribution of smaller particles of Aβ was seen (Fig. 8A, HR-TEM and Fig. 8D, STEM).Open in a separate windowFig. 8Electron microscopy studies: HR-TEM and STEM images depicting the effects of active peptide 12c and inactive peptide 13a on the aggregation of Aβ₄₂. Aβ₄₂ (10 μM) was incubated alone t = 0 h (A and D), t = 24 h (B and E); with equimolar concentrations of inhibitor peptide 12c (C and F); inactive peptide 13a (H and K) as well as peptide 12c (G and J) and inactive peptide 13a (I and L) incubated alone, respectively. (Additional images have been provided in the ESI,^† Section 11.3).After an incubation span of 24 h, appearance of amyloid fibrils and an extensive network of long, straw-shaped fibrils were observed (Fig. 8B and E). In the presence of peptide 12c (Fig. 8C and F), only smaller particulate aggregates were seen, indicating complete inhibition of the amyloid fibrils. On co-incubation of Aβ₄₂ with the inactive peptide 13a, large aggregated structures (Fig. 8H and K) were observed. In order to visualize the aggregation of the peptide themselves, equimolar concentrations of peptides 12c and 13a were incubated alone under similar conditions and visualized. Very small granular structures were seen for the 12c (Fig. 8G and J) whereas slightly larger and patchy aggregates were seen for inactive peptide 13a (Fig. 8I and L).In order to evaluate and understand the biosafety and pharmacokinetic profile of the test peptides, cell-cytotoxicity studies employing PC-12 cells was performed. Test peptide were tested up to a highest tested concentration of 20 μM and none of the peptides exhibited undesirable cytotoxicity. Fig. 9A depicts a graphical representation of the % viable cells in presence of 20 μM concentration of peptide 12c.Open in a separate windowFig. 9Cytotoxicity and bioavailability study: (A) analysis of the cytotoxic effects of the peptide 12c (20 mM) on the viability of PC-12 cells evaluated using MTT cell viability assay. The percentage of untreated cells was considered 100% (positive control) and presence of the test peptides in respective dose concentration for 6 h. (B) BBB-permeability of peptide 12c in comparison to 11a, as determined by the PAMPA-BBB assay. P_e was calculated by using the formula V_dV_a/[(V_d + V_a)S_t] ln(1 − A_a/A_e), where V_d and V_a are the mean volumes of the donor and acceptor solutions, S is the surface area of the artificial membrane, t is the incubation time, and A_a and A_e are the UV absorbance of the acceptor well and the theoretical equilibrium absorbance, respectively. Data was recorded for triplicate samples in three individual experiments and the readings were averaged (<5% variation).A major challenge for peptide-based therapeutics is the BBB permeability and proteolytic stability against various enzymes within the body.⁴¹In vitro BBB penetration of the most active peptide 12c as well as the lead peptide 11a using parallel artificial membrane permeation assay (PAMPA-BBB) was performed following the previously reported protocols.^42–45 The UV/Vis absorptions of both the peptides was recorded after permeating through an artificial porcine polar brain lipid (PBL) membrane and the effective permeabilities (P_e) were calculated. As described in Fig. 9B, the P_e values of the most active peptide 12c was significantly higher than that of 11a, demonstrating enhanced permeability of the modified peptide.Trypsin is the one of the most notorious endopeptidases and cleaves the amide bond next to a charged cationic residue.⁴⁶ Hence, in order to evaluate whether the synthesized peptides have incorporated the proteolytic stability properties, trypsin and serum stability studies on the peptide 12c was performed. The peptide was incubated with 100-fold excess of trypsin and was subjected to analysis by RP-HPLC. Chromatograms depicted that the peptides exhibited intact integrity, having their retention time unaltered even after 24 h of trypsin treatment. It was observed that, there were no peaks seen before and after the main peak of the peptide indication no fragment and/or other intermediate formation. Superimposed HPLC chromatograms of time point''s intervals have been depicted in Fig. 10A.Open in a separate windowFig. 10Proteolytic stability study: (A) superimposed HPLC chromatograms of most active peptide 12c at time intervals of 0, 2, 4, 8, 12, 18 and 24 h after trypsin treatment; (B) graphical representation showing % degradation for peptide 12c on serum treatment. (C) Mass spectra for peptide 12c at 0, 12, 18 and 24 h of serum treatment. Analyzed by ACD-Mass Fragmenter tool. (D) Predicted susceptible cleavage sites for peptide 12c. Most susceptible peptide bond has been indicated in bold red.Serum stability assay following similar protocol was performed. The chromatograms depicted that the peptides exhibited intact integrity, having their retention time unaltered up to 12 h of serum treatment. The peaks obtained for the peptides at 18 h and 24 h of serum treatment, were comparatively smaller to that of the previous peaks indicating slight degradation of the peptide. Superimposed HPLC chromatogram for peptide 12c has been provided in ESI, Fig. S6.^†To calculate the rate of degradation of the peptide on serum treatment, analysis and comparison the area under the curve of the peak of the respective peptide at their specific time intervals.^47,48 A comparative analysis depicted that around 20% of the peptide is present after 24 h of serum treatment. Fig. 10B indicates the % degradation of the peptide in serum over a period of 24 h. The initial calculation of % peptide present in the sample aliquot is indicated in the ESI, Fig. S7.^† The extrapolated data helped us to determine the degradation rate of the peptide in serum (ESI, Table S8^†).^48–50 It can be concluded that approximately 50% of the peptide is stable until 12 h of serum treatment, following which it shows an decline in stability decreasing to about 22% until 24 h.In order to study the mode of degradation of the peptide and the susceptibility of the peptide towards peptide degradation, mass spectroscopy was employed. The samples analyzed for peptide content on RP-HPLC were further subjected to LCQ analysis.^51,52 At 0 h, the molecular ion peak (m/z 470) of the peptide corresponded to the molecular mass of the peptide itself. Sequential fragmentation pattern was minimal and a clear mass spectrum was seen. After 12 h, multiple fragmentation peaks were seen indicating that the peptide has undergone cleavage at multiple sites. ACD-Mass Fragmenter tool was used to analyze the specific fragmentation pattern. Fig. 10C summarizes the mass spectrums and shows the specific fragmentation pattern. Based on the fragmentation pattern for the tetrapeptide sequence, the most susceptible bonds that could easily undergo cleavage were identified. ACD Mass Fragmenter tool was used to understand the peptide fragmentation pattern. Fig. 10D depicts the structure of the peptide 12c indicating the most susceptible peptide bond. This understanding provides impetus in site specific modification in improving the stability of the peptides.Computationally understanding the binding mechanism and intra-residual interactions of the test peptides with the single monomeric as well as the proto-fibrillar unit of Aβ₄₂ would prove to be useful. Various structures for the both the forms of Aβ₄₂ are reported in the literature. A monomeric sequence bearing complete sequence of all 42 amino acids residues was used (PDB Id: 1IYT; ESI, Fig. S8^†). The structure comprises of α-helices and random coiling, similar to the data previously reported.⁵³ A proto-fibrillar unit comprising of 6 full length spatially arranged in a ‘S’ shaped manner recently reported by Colvin and co-workers⁵⁴ (PDB Id: 2NAO, ESI, Fig. S10^†) was used for understanding the interaction of the test peptides on the proto-fibrillar unit of Aβ. Reactive site analysis of the pre-optimized framework of the monomeric Aβ₄₂ showed that the hinge region is the most prone to aggregation due to the presence of reactive residues in that particular segment (Maestro, Bioluminate suite; ESI Fig. S9^†). To understand the interaction of the test peptide with the full-length Aβ₄₂, a grid incorporating the whole sequence was generated. Docking studies were performed with the peptides mentioned in this work along with a few molecules reported in literature^{10–12,55,56} for comparative analysis of the binding modes and interactions.^57–59 Docking, glide and residual interaction energy scores for the molecules selected from literature and test peptides from the current study are summarized in ESI (Tables S9 and S10^†). Although the analysis of the scores reveal that similar docking and glide scores were seen in both the cases. The energy of interaction of the test peptides with the monomeric unit proved to be a comparable factor to that of the literature reported ligands (ESI Table S11^†).Test peptides have shown to interact with Glu₁₁, His₁₃, His₁₄, Gln₁₅, Phe₁₉, Phe₂₀ and more specifically with Asp₂₃. These residues are involved to aid the aggregation of monomeric Aβ₄₂ into the fibrillar species, as also seen in reactive residue analysis (ESI, Fig. S11^†). Binding of the test peptides to these residues, block the free interaction of these residues to others, inhibiting their aggregation propensity. Ligand interactions of the most active test peptide 12c with the monomeric unit, their respective ligand interaction diagrams (2D and 3D) have been summarized in Fig. 11A and B. The interaction energies are in accordance to those exhibited by the standard ligands indicating similar kind of interactions and thus reinforcing the results of studies carried out in this work. A structural framework 2NAO-06 of proto-fibrillar Aβ₄₂ was rationalized to be the most optimal structure and was prepared for docking studies (ESI, Fig. S10^†). Since it is a proto-fibrillar unit, the most reactive sites for the binding were identified. SiteMap Analysis feature identified 5 ligand-binding sites (ESI, Fig. S12^†). The predicted sites did coincide with the predicted reactive residues, thus indicating certain interactions between those residues and the ligand to be feasible, which would inhibit the process of aggregation. To carry out docking studies, receptor grids at the predicted sites on the proto-fibrillar unit was generated. Since there has been no docking studies performed using 2NAO as the protein, validation of the use of 2NAO and the predicted sites, by docking standard ligands was performed (ESI, Tables S12 and S13^†). Analysis of the docking studies revealed SiteMap-2 to be the most plausible site for action of an inhibitor. The molecule can interact with the residues that aid in aggregation. This would block the further attachment of another monomeric unit to the site-recognition units on the proto-fibrillar structure. Subsequent interaction with the neighboring residues of both the chains destabilizes the preformed bonds, which hold the adjacent units together.Open in a separate windowFig. 11 In silico study: ligand interaction diagram showing interactions of the ligand with the residues of monomeric unit 1IYT-10 (A and B) and with the proto-fibrillar unit 2NAO-06 (C and D). 3D representation (Left) showed along with 2D representation (Right).Docking studies of the peptides presented in this work was carried out and docking scores and the residue interaction energies obtained for the set of synthesized tetrapeptides for SiteMap-2 has been summarized in ESI, Table S14.^† On careful analysis it can be seen that interaction with Met₃₅, Val₃₆, Gly₃₇ of chain D, as well as Glu₁₁. His₁₃, His₁₄ and Gln₁₅ of the neighboring chain A, show that the test peptide does interact with both the neighboring chains and especially with those amino acids that are solely responsible for maintaining the dimeric structure of the proto-fibrillar unit. To have a clear understanding of the interactions, ligand interaction diagrams for the test peptide 12c, depicting its interaction with the specific residues of the proto-fibrillar unit have been summarized in Fig. 11C and D.     

Simple and efficient synthesis of bicyclic enol-carbamates: access to brabantamides and their analogues

A novel synthetic approach towards the formation of the unusual bicyclic enol-carbamates, as found in brabantamides A–C, is reported. The bicyclic oxazolidinone framework was obtained in very good yield and with high E/Z selectivity from a readily available β-ketoester under mild reaction conditions using Tf₂O and 2-chloropyridine tandem. The major E isomer was used in the synthesis of the brabantamide A analogue.

A simple and short synthesis of bicyclic enol-carbamates with high E/Z selectivity and the synthesis of brabantamide A analogue are presented.

Brabantamides A–C (1–3) (Fig. 1) were first isolated in 2000 from the culture extracts of Pseudomonas fluorescens.¹ They displayed nanomolar inhibitory activity towards lipoprotein-associated phospholipase A₂ (Lp-PLA₂) and therefore they could be used in the treatment of the inflammatory diseases such as atherosclerosis.^1–3 It was found that the sugar moiety is not necessary for the biological activity against Lp-PLA₂. In contrast, deglycosylated brabantamides A and C showed improved inhibitory activity compared to their natural counterparts.³ Moreover, simplified brabantamide analogues with amide functional group and long alkyl side chains showed even higher inhibitory effect.⁴ Brabantamides A–C also exhibit significant activity against Gram-positive bacteria, fungi, and oomycetes.^5,6 A recent study confirmed that the bicyclic scaffold and the long lipophilic side chain are essential for the antibacterial activity.⁷Open in a separate windowFig. 1Structures of brabantamides A–C (1–3). Rha = rhamnose.Surprisingly, only five reports concerning the synthesis of the bicyclic oxazolidinone with an exocyclic double bond have been reported to date.In 2000, Pinto et al. prepared a series of analogues of brabantamide A where the enol-carbamate 5 was first obtained by direct iodocyclization from acetylene derivative 4 and then converted into key ester 6 by carbonylation of the corresponding vinyl iodide in the presence of 2-(trimethylsilyl)ethanol and PdCl₂ (Scheme 1a).⁴ Another synthesis of the related esters 9 has been reported by Snider et al. in 2006, employing Wittig reaction between stabilized ylides and bicyclic oxazolidindione 8 synthesized from l-proline 7 (Scheme 1b).⁸ Shortly after, bicyclic enol-carbamate 10 with an exocyclic methylene group was synthesized in one step from acetylene derivative 4 using gold(i) catalysts (Scheme 1c).⁹ Very recently, Witte et al. reported the synthesis of series of Z-analogues of brabantamides 12 by cyclization of β-ketoamides 11 using CDI (Scheme 1d).⁷ As a part of our ongoing research program aimed at the utilization of trifluoromethanesulfonic anhydride (Tf₂O)/2-halopyridine (2-XPy) tandem in the synthesis of bioactive natural products and their analogues, we envisioned that similar reaction conditions could be effectively used to form the bicyclic oxazolidinone framework as found in brabantamides.Open in a separate windowScheme 1Literature syntheses of bicyclic enol-carbamates and method proposed herein.The combination of Tf₂O/2-XPy was extensively and successfully used in the amide activation^10,11 as well as in generating isocyanate species from N-Boc and N-Cbz protected amines.¹² We anticipated that the N-Boc-protected β-ketoester 13 could react in its enolate form with in situ generated isocyanate ion by intramolecular 5-endo-dig cyclization to give bicyclic enol-cyclocarbamate 14 (Scheme 1e).At the start of our investigation, model β-ketoester 15, prepared from N-Boc-l-proline,⁷ was chosen as the model substrate to identify optimal reaction conditions (12c the initial using of 1.5 equivalents of Tf₂O and 3 equivalents of 2-ClPy led to a full conversion of the substrate 15 in 15 minutes (monitored by TLC) (13 Any variation of the amount of 2-ClPy did not have any positive impact on the reaction ( EntryTf₂OBaseTime (min)16a : 16b^aYield^b (%)11.5—60—–^c21.5Et₃N60—–^c31.5DMAP60—–^c41.5Pyridine60—–^c51.5^d2,6-Lutidine60—–^c61.5^dDBU6090 : 1041^e,f71.52-ClPy1593 : 753^e 8 1.1 2-ClPy 15 85 : 15 80 ^e 91.12-ClPy (1.5 equiv.)4085 : 1575^e101.12-ClPy (5 equiv.)1587 : 1364^e111.12-FPy1589 : 1171^e121.12-BrPy7086 : 1473^e131.12-IPy9086 : 1468^e14Witte''s protocol^gOvernight50 : 5036^eOpen in a separate window^aRatio determined by ¹H NMR of the crude reaction mixture.^bIsolated combined yield.^cTraces of products.^dReactions performed with 1.1 equiv. of Tf₂O did not lead to full conversion of ester 15.^eReactions were performed on 1 mmol of ester 15.^fReaction mixture contained a large amount of unidentified by-products.^gReaction conditions: (1) TMSOTf (2 equiv.), CH₂Cl₂, 0 °C, 1 h. (2) CDI (1.5 equiv.), CH₂Cl₂, 0 °C – rt, overnight.⁷It is noteworthy that the reaction can be performed on a gram scale without affecting the yield and both isomers are easily separable by FCC (see the ESI^†).The ¹H and ¹³C NMR data of the major E isomer 16a were consistent with those published previously.⁸ Possible racemization in the course of the reaction was dismissed based on the comparing specific optical rotation with the published data for 16a ([α]²²_D = −261.1 (c 1.01, MeOH); ref. 8: [α]²²_D = −207 (c 1.0, MeOH)). Most importantly, X-ray crystallographic analysis of 16a (Fig. 2; see the ESI^† for further details)¹⁴ confirmed its absolute configuration on the C-7a carbon atom.Open in a separate windowFig. 2Molecular structure of the enol-carbamate E-16a confirmed by X-ray crystallographic analysis.The minor Z isomer 16b was isolated for the first time as the pure compound and was fully characterized. Its structure was assigned on the basis of its ¹H, ¹³C, COSY, HSQC, and HMBC NMR spectra.A plausible mechanism of the cyclization of β-ketoester 15 was based upon previous works^12a–c and it is depicted in Scheme 2. Isocyanate cation II, as a key intermediate, can be formed directly from iminium triflate I (path A) or through the formation of carbamoyl triflate III with subsequent elimination of triflate ion spontaneously (path B). Ester enolate moiety IV then reacts as O-nucleophile via 5-endo-dig cyclization and leads predominantly to the formation of the enol-carbamate 16a.Open in a separate windowScheme 2Plausible mechanism of the cyclization β-ketoester 15.Next, the optimized conditions were briefly applied in the synthesis of the brabantamide A analogue 21 (Scheme 3). Starting β-ketoester 18 was synthetized in two steps in a 70% yield using both commercially available N-Boc-d-proline 17 and 2-(trimethylsilyl)ethanol. It ought to be mentioned that previously examined hydrolysis of the corresponding methyl ester 16a under acidic as well as basic conditions failed due to the instability of the bicyclic enol-carbamate.^3,8Open in a separate windowScheme 3Synthesis of the brabantamide A analogue 21.Subsequent cyclization of ester 18 using optimized reaction conditions afforded enol-carbamate 19 in 76% yield as a mixture of E and Z isomers in a ratio of 89 : 11. After isolation of the major isomer E-19a, it was treated with TBAF, providing free acid 20 in 89% yield. Finally, an amidation of 20 with tetradecylamine in the presence of EDCI gave amide 21 in moderate 40% yield. Both free acid 20 and amide 21 were fully characterized for the first time and their structures were assigned on the basis of its ¹H, ¹³C, COSY, HSQC, and HMBC NMR spectra. Moreover, their structures were unambiguously confirmed by X-ray crystallographic analysis (Fig. 3; see ESI^† for further details).¹⁴Open in a separate windowFig. 3Molecular structures of acid 20 (top) and amide 21 (bottom) confirmed by X-ray crystallographic analysis.     

Synthesis of enol phosphates directly from ketones via a modified one-pot Perkow reaction

A modified Perkow reaction, named Perkow-Shi reaction, was developed based on the one-pot α-tosyloxylation of ketones following by addition of P(iii)-reagents and 4 Å molecular sieves. Diversity of enol phosphates, as well as enol phosphonates, enol phosphinates, and enol phosphoramidates were synthesized in high yields directly from the ubiquitously available ketones instead of the unfavourable α-chloroketones under a mild and environmental friendly condition.

A modified Perkow reaction was developed based on the one-pot α-tosyloxylation of ketones following by addition of P(iii)-reagents and 4 Å molecular sieves.

Phosphoenols (P(O)–OC C) are the key structural motif of many agrochemicals and bioactive natural products (Fig. 1). For example, more than ten kinds of organophosphorus pesticides such as dichlorvos (DDVP), mevinphos and monocrotophos are typical enol phosphates (EPs).¹ The endogenetic phosphoenolpyruvate (PEP) plays a vital role in transformation of adenosine diphosphate (ADP) into adenosine triphosphate (ATP) in living organisms;² the naturally occurring cyclophostin,³ cyclipostins⁴ and salinipostins,⁵ characterized by a unique bicyclic core with a unusual seven-membered cyclic phosphoenol triester, are merging as a novel class of multi-target promising therapeutic candidates with diversity of biological properties; phosphaisocoumarins, a phosphorus analogue of isocoumarins, have displayed versatile and interesting biological activities.⁶Open in a separate windowFig. 1Selected examples of enol phosphates.Furthermore, as a kind of outstanding vinyl electrophiles and nucleophiles possessing high stability and accessibility, functionalized EPs are of great importance due to their versatile synthetic applications as EPs mainly have three active centres: the electrophilic phosphoryl group and enol oxygen-attached carbon atom, and the electrophilic carbon–carbon double bond, which makes them capable of participating in diverse type of phosphorylation and transition metal catalyzed cross-coupling reaction as well as cycloadditions leading to new P–O, C–O, C–H, C–C, C–X bonds formation (Fig. 2).Open in a separate windowFig. 2Representative reactions involving enol phosphates.In particular, the transesterification and acidolysis of EPs with cleavage of the enol linkage affording tris-substituted phosphates, mixed anhydrides namely acyl phosphates and pyrophosphates;⁷ and intramolecular olefin metathesis of terminal EPs affording new type of cyclic phosphoenols;⁸ and catalytic epoxidation of EPs with NaOCl affording phosphonyl epoxides;⁹ and Diels–Alder reaction of EPs with cyclodiene affording the corresponding adducts;¹⁰ and palladium catalyzed carbonylation of EPs with CO affording a range of enoates;¹¹ and reduction and alkylation of EPs with a variety of hydride transfer reagents affording alkenes widely exploited in total synthesis;¹² and Suzuki–Miyaura, Stille, Negishi, Heck, and related cross-coupling reactions of EPs affording more highly substituted alkenes;¹³ and also substitution by simple heteroatom nucleophiles affording the formation of alkenes with C-heteroatom (C–O, C–N, C–S, C–B, C–Sn, C–S) linkages,¹⁴ have attracted a lot of attentions as these functional groups are fundamental components of a large variety of natural products, versatile precursors, and important building blocks.To date, numerous methods to synthesize EPs have been reported which can be broadly categorized as follows: (1) the earliest recorded and still extensively used Perkow reaction between α-haloketones and phosphites;¹⁵ (2) the phosphorylation of in situ generated enolate of ketones by various kinds of metalation process;¹⁶ (3) other approaches such as hydrophosphoryloxylation of alkynes,¹⁷ tandem reaction of α-aryloxyacetophenones,¹⁸ and so on.¹⁹ However, whilst the classical Perkow reaction is much limited by the availability of the toxic and lachrymatory α-haloketones and the lack of effective regio- and stereocontrol, the non-Perkow methods always suffer from harsh reaction conditions including use of strong bases (mostly metallic) and unstable phosphorochloridates some of which are generally inconvenient to prepare such as P–C and/or P–N containing materials. Therefore, a more environmentally benign synthetic route to EPs is still much desirable.On the other hand, with respect to the known Perkow reaction mechanism,²⁰ we envisioned that substrates of ketones with a proper α-positioned leaving group (LG) instead of the unfavourable halogen should also probably be feasible of proceeding Perkow reaction. Nevertheless, to our best knowledge, only two attempts have been reported: one from Paleta that when treating the 3-trifluoroacyloxy quinolinedione with P(OEt)₃ in refluxing toluene for 7 h, a Perkow product was obtained in 36% yield;^21a the other from Denney that when stirring α-keto p-toluenesulfonates with phosphites in refluxing ether for several days, a mixture of unidentified vinylphosphates and methylphosphonates was obtained.^21b Herein we wish to report a modified one-pot Perkow reaction based on the α-tosyloxylation of ketones following by addition of P(iii)-reagents and 4 Å molecular sieves (Scheme 1).Open in a separate windowScheme 1Synthesis of enol phosphates.We initially selected (EtO)₃P and α-functionalized acetones as the test reaction. Solvent-free condition was first focused in view of its high regioselectivity.^15b As shown in EntryLGTime (h)Yield^b (%)1–OC(O)CH₃120^c2–OC(O)CF₃120^c3–OSO₂CH₃8874–OSO₂CF₃2865–OSO₂C₆H₄CH₃-p3956–OSO₂C₆H₄OCH₃-p12907–OSO₂C₆H₄F-p12888–OP(O)(OEt)₂120^cOpen in a separate window^aReaction conditions: 1 (1 mmol), 2 (1 mmol).^bIsolated yield.^cDetermined by ³¹P NMR.In light of the significant progress in α-tosyloxylation of ketones especially with those iodine(iii)-mediated reagents such as the most popular hydroxy(tosyloxy)-iodobenzene (HTIB),²² the readily accessible and economic tosylate was thus chosen as the prior α-LG for the next investigation.Inspired by the above achievements, we then turned into the possibility of the one-pot manipulation. Considering the convenience of monitoring, we then carried out the next experiments using aromatic acetophenone with HTIB following by the addition of (EtO)₃P. As shown in EntrySolvent T (°C)4a : HTIB : P(OEt)₃AdditiveConversion^b (%)3aa(EtO)₂PHO(EtO)₃PO1—401 : 1 : 1—2463132MeCN401 : 1 : 1—257053THF401 : 1 : 1—087134DCM401 : 1 : 1—494385EA401 : 1 : 1—4245136DCM401 : 1 : 14 Å MS84797DCM401.1 : 1 : 14 Å MS95328DCM251.1 : 1 : 14 Å MS99019^cDCM01.1 : 1 : 14 Å MS9721Open in a separate window^aReaction conditions: HTIB (1 mmol).^bDetermined by LC-MS and ³¹P NMR after 2 h reaction.^cReact for 5 h.Next, we take DCM as the solvent for optimization. According to the reaction mechanism, (EtO)₂PHO was supposed to be the hydrolysis product of (EtO)₃P by H₂O formed in the reaction system, whereas (EtO)₃PO should be the oxidation product of (EtO)₃P by the reactive hypervalent iodine reagents HTIB, which was verified by our following modifications experiments. As presumed, when adding equimolecular water-capturing 4 Å molecular sieves (MS) powder, the majority of (EtO)₂PHO were avoided of generation (iii) reagents possessing P–O, P–C, and/or P–N bonds as well as those cyclic ones gave the corresponding EPs in high yields. Various types of aromatic ketones with electron-donating or electron-withdrawing substituted groups especially those easily available hetero-aromatic ketones whose α-chloro species are generally commercially inaccessible, were all applied well to the present enolphosphorylation. In other words, our one-pot Perkow reaction methodology exhibited extreme tolerance in preparation of diversely structural enol phosphates, enol phosphonates, enol phosphinates, and enol phosphoramidates ( Open in a separate window^aReaction conditions: HTIB (1 mmol), 4 (1.1 mmol), P(iii) (1 mmol). Isolated yield.However, the reaction showed no preference of E/Z geometry in EP products. None of the conditions altering temperature, solvent and reaction time were found effective in controlling the stereochemistry and in most cases only mixtures of the E- and Z-isomers in nearly 1 : 1 ratio were obtained with a slightly decreased yields mainly due to the relatively low reactive α-tosyloxylated secondary carbons ( Open in a separate window^aReaction conditions: HTIB (1 mmol), 5 (1.1 mmol), P(OEt)3 (1 mmol). Isolated yield.To be further mentioned, for those asymmetric aliphatic ketones with two possible reactive sites, for example, butan-2-one, pentan-2-one and heptan-2-one, only mixed products were obtained with very low yields (about 10–20%). Moreover, the α-tosyloxylation was found to mainly occurred at the more substituted carbons of the asymmetric ketones, which is consistent with literature results.²³A plausible mechanism is shown in Scheme 2. Similar to the classical Perkow reaction, it should proceed through a pathway that the nucleophilic P(iii) atom of phosphite attacks the carbonyl C atom of in situ generated α-tosyloxylated ketone forming a triatomic heterocyclic intermediate which followed by the ring cleavage transforms into a phosphonium salt and then by the O-dealkylation gives the EP product. According to the nucleofugality model by Jaramillo,²⁴ the ω index of TsO (1.63 eV) is about two times of Cl (0.77 eV), suggesting TsO could be even more reactive than Br (0.90 eV) and I (1.34 eV) α-substituted ketones, both of which however have been proven to be less regioselective for Perkow reaction vs. Arbuzov reaction. It demonstrates that Perkow reaction may not just follow in agreement with the order of departure ability of the α-LGs from the substrate, which deserves further investigation.Open in a separate windowScheme 2Proposed reaction mechanism.In summary, we have developed a concise method for the synthesis of enol phosphates, as well as enol phosphonates, enol phosphinates, and enol phosphoramidates based on an modified Perkow reaction, herein named Perkow–Shi reaction, which employed the ubiquitously commercial ketones and the readily available α-tosyloxylation reagent HTIB by a kind of one-pot methodology. The E/Z-selectivity of the modified Perkow reaction may be achieved by the introduction of chirality on the α-tosyloxylated carbon, which are under investigation in our lab.     

A novel method for PEGylation of chitosan nanoparticles through photopolymerization

An ultrafast and convenient method for PEGylation of chitosan nanoparticles has been established through a photopolymerization reaction between the acrylate groups of PEG and methacrylated-chitosan nanoparticles. The nanoparticle characteristics under physiological pH conditions were optimized through altered PEG chain length, concentration and duration of UV exposure. The method developed here has potential for clinical translation of chitosan nanoparticles. It also allows for the scalable and fast synthesis of nanoparticles with colloidal stability.

An ultrafast and convenient method for PEGylation of chitosan nanoparticles has been established through a photopolymerization reaction between the acrylate groups of PEG and methacrylated-chitosan nanoparticles.

The latest progress regarding the framework of bioengineered nanoparticles has led to the development of new biodegradable nanoparticles for potential treatment purposes.^1–6 Recent methods in nanomedicine have leaned towards developing colloidally stable, biocompatible nanoparticles which can overcome biological barriers without causing any harm to living tissues. Accordingly, the surface charge and size optimization of such particles has gained great importance as they are major factors for the stability of the system.⁷The search for bioadhesive nanoparticles that have potential as clinically viable materials extends to a wide scope in the field. One such material is chitosan, which has an application range including food, cosmetics and biomedicine.^8–11 As this material is known for its biocompatible,¹² biodegradable¹³ and nontoxic nature, it becomes an expedient choice to be used for therapeutic purposes such as drug or gene delivery.^14–16 Moreover, the reaction of primary amines attached to chitosan backbone permit functionalization of this material with photosensitive groups. One of those functionalization is the addition reaction of the amino groups with methacrylic anhydride which produces photosensitive methacrylamide groups.^17,18 This modification of chitosan chains allows for photocrosslinking of this adhesive monomer in the presence of a photo initiator under UV light to form a gel.¹⁹Multiple methods are present to synthesize chitosan nanoparticles such as reverse micellar,²⁰ emulsification solvent diffusion,²¹ and ionotropic gelation method.^22,23 Chitosan nanoparticles have been synthesized at acidic pH due to the presence of negatively charged sodium tripolyphosphate (TPP) and cationic chitosan chains, within a short time at room temperature through ionic gelation.¹⁴ Majority of research that involves the synthesis of these nanoparticles consider the size and zeta potential of chitosan nanoparticles between pH 4.0 and 5.5, where these methods do not characterize the properties at physiological conditions regardless of this major applicability constraint.^24,25 To overcome this obstacle, we have developed a unique PEGylation approach which can be utilized for wide ranges of pH without compromising the solution stability of chitosan. For this reason, PEGylation may be considered as a critical route for advancing chitosan nanoparticles as biomedical tools and making the particle system colloidally stable.²⁶ There are various approaches used for the PEGylation of chitosan nanoparticles such as the PEGylation of chitosan chains prior to nanoparticle synthesis.^27,28 However, these existing methods used for direct chemical conjugation of PEG to chitosan are time consuming, require complex reactions and equipment. In our work, we address two fundamental issues for the utilization of chitosan nanoparticles in biomedicine: (i) optimization of chitosan nanoparticle properties at physiological pH, and (ii) development of a novel PEGylation method for chitosan nanoparticles for long term feasibility and large-scale synthesis.The novel method we introduce here for the PEGylation of chitosan overcomes the limitations associated with the use of chitosan nanoparticles in biomedicine. We prepare ionically crosslinked and PEGylated chitosan nanoparticles through simultaneous addition of photoinitiator with TPP and acrylate-PEG prior to UV light exposure at 365 nm, which promotes covalent bond formation between the acrylate groups of PEG and methacrylamide chitosan nanoparticles (CSMA) (Fig. 1). Methacrylamide chitosan was synthesized according to previously described protocol.^17–19 (ESI Experimental section 2^†). Synthesis of methacrylamide chitosan is a scalable one-step process and allows for the large amounts of product formation. Two groups of methacrylamide chitosan chains were prepared for the optimization of degree of methacrylation during nanoparticle formation: chitosan functionalized with (1) 50 μL and (2) 100 μL methacrylic anhydride (MA) per 30 mg of chitosan. Aggregation was observed inside the CSMA solution with the higher methacrylation degree (100 μL MA to 30 mg chitosan) for different groups at pH 4.7 (Table S1^†). Hence, we continued our experiments with the methacrylamide chitosan polymer that has lower degree of methacrylation. It was possible to obtain nanoparticles with high colloidal stability using the less methacrylated-chitosan polymer with sub 100 nm size and positive charge at pH 4.7 (Open in a separate windowFig. 1Overall strategy for the new method. (A) Synthesis of methacrylamide chitosan from chitosan. (B) Nanoparticle synthesis via ionic gelation method. Interaction between positively charged methacrylamide chitosan and negatively charged TPP crosslinker form nanoparticles. (C) PEGylation of methacrylamide chitosan nanoparticles. UV light triggers the reaction between methacrylic groups on nanoparticles and acrylate-PEG derivative in the presence of photoinitiator. PEGylated nanoparticles are obtained under optimized conditions.Size, PDI and zeta potential of different group of nanoparticles

Diastereoselective synthesis of new zwitterionic bicyclic lactams,scaffolds for construction of 2-substituted-4-hydroxy piperidine and its pipecolic acid derivatives

The synthesis of new chiral highly functionalized zwitterionic bicyclic lactams starting from acyclic β-enaminoesters derived from (R)-(−)-2-phenylglycinol is described. The key step involved an intramolecular non-classical Corey–Chaykovsky ring-closing reaction of the corresponding sulfonium salts derived from β-enaminoesters. This methodology permits the generation of two or three new stereogenic centers with high diastereoselectivity. The utility of these intermediates was demonstrated by the stereocontrolled total synthesis of cis-4-hydroxy-2-methyl piperidine and its corresponding pipecolic acid derivative.

The synthesis of new chiral highly functionalized zwitterionic bicyclic lactams starting from acyclic β-enaminoesters derived from (R)-(−)-2-phenylglycinol is described.

Stereocontrolled synthesis of polysubstituted piperidines is a very important field in organic synthesis due to the great variety of piperidine-moiety-containing drugs, making it the most prevalent nitrogen heterocycle in approved drugs;¹ therefore, their synthesis has been the focus of considerable attention.In this sense, chiral non-racemic bicyclic lactams derived from β-amino alcohols are commonly considered useful starting materials for preparing these substituted piperidine drugs.² One of the most effective methodologies for the synthesis of these bicyclic lactams is via an aza-annulation of β-enaminoesters³ derived from suitable β-amino alcohols.⁴We recently reported the preparation of new cyclic zwitterionic intermediates via an intramolecular non-classical Corey–Chaykovsky ring-closing reaction. Their utility was demonstrated by the synthesis of stereoisomers of σ¹-receptor agonists trozamicol⁵ or the synthesis of (+)- and (−)-Geissman–Waiss lactone (Scheme 1).⁶Open in a separate windowScheme 1Previous reports in the synthesis and utility of chiral cyclic zwitterionic intermediates.Considering this precedent and highlighting the utility of cyclic zwitterionic intermediates, we present a new versatile methodology to access new chiral zwitterionic non-racemic bicyclic lactams starting from acyclic β-aminoesters derived from (R)-(−)-2-phenylglycinol and its utility in the diastereoselective synthesis of 2-substituted-4-hydroxy piperidines.We commence our finding by synthesizing of a set of β-enaminoesters coming from (R)-(−)-2-phenylglycinol, which were prepared following the traditional methodologies, directly condensation of chiral amine with β-dicarbonyl compound⁷ or by addition to a corresponding alkyne.⁸ All enamino esters were obtained as an inseparable E : Z isomeric mixture and, these results are summarized below (Fig. 1).Open in a separate windowFig. 1β-Enamino esters derived from (R)-(−)-2-phenylglycinol.With a set of chiral β-enaminoesters in hand, next these compounds were condensed with bromo acetyl bromide,⁹ affording the desirable N-acyl oxazolidine with the formation of a new stereogenic center at the hemiaminal position in good to excellent diastereomeric ratio. The stereochemistry at the new stereogenic center of the major diastereoisomer was determined from its X-ray diffraction analysis of N-acyl oxazolidine 10 as (2R),¹⁰ therefore we assumed that this is the stereochemistry of the major diastereoisomer of each N-acyl oxazolidine (Scheme 2).Open in a separate windowScheme 2Synthesis of N-acyl oxazolidines. ^a Only the major diastereoisomer is shown. ^bN-acyl oxazolidines 12 and 14 were probed to be unstable therefore were employed without purification for the next reaction.Then, the diastereomeric mixture of 8 was selected as a model for preparing the desired zwitterionic chiral bicyclic lactam. To this end, 8 was treated with dimethyl sulfide in DCM,¹¹ delivering the corresponding sulfonium salt which was immediately subjected to the intramolecular non-classical Corey–Chaykovsky ring-closing reaction to avoid its conversion to the undesired thioether derivative.Firstly, sulfonium salt was treated with KOH (2 equiv.) in a mixture of CH₃CN : MeOH (9 : 1).⁶ Unfortunately, a mixture of expected zwitterion 22(a+b) and undesired pyridine-2-one 22c, was obtained. Therefore, screening experiments were performed. Gratifyingly, when the base was changed by K₂CO₃, the diastereomeric mixture of zwitterionic bicyclic lactams was exclusively obtained. ¹H-NMR analysis of the crude reaction revealed the presence of a diastereomeric mixture of bicyclic zwitterionic intermediates in a 73 : 27 dr. Two hemiaminal protons were assigned as a doublet of doublets at 5.40 ppm and 5.12 ppm with coupling constants J = 9.8, 5.3 Hz for the minor diastereoisomer and J = 11.4, 4.2 Hz for the major diastereoisomer. The assignment was confirmed by a ¹H–¹H COSY experiment ( EntryBaseGlobal yield [ratio 22(a+b)^b : 22c]1KOH (2.0 equiv.)95% [76 : 24]2KOH (1.0 equiv.)83% [82 : 18]3K₂CO₃ (1.2 equiv.)97% [100 : 0]Open in a separate window^aAll reactions were performed in CH₃CN : MeOH (9 : 1), for 4 hours at room temperature.^b22(a+b) was obtained in a 73 : 27 dr.The absolute configuration at the hemiaminal position of the zwitterionic bicyclic diastereomeric mixture was determined by its X-ray diffraction analysis. A single crystal was found to include two independent molecules in the asymmetric unit of a triclinic cell (space group P1 with Z′ = 2), one of which presenting a disorder on the chiral center C8a. Combining these features results in a mixture of disatereoisomers with a ratio refined to 77.5/22.5%,¹² close to that determined by NMR in solution, 73/27%. The determination of the absolute configuration in this crystal structure allowed the assignment for the major diastereoisomer 22a as (8aR) and the minor diastereoisomer 22b as (8aS) (Fig. 2).Open in a separate windowFig. 2X-ray structure of the diastereomeric mixture of chiral zwitterionic bicyclic intermediates 22(a+b).Once the optimal reaction conditions were established, the scope of our synthetic proposal was investigated. Chiral zwitterionic bicyclic intermediates containing alkyl or aryl substituents at the hemiaminal position were obtained in excellent chemical and stereochemical yields. Furthermore, the absolute configuration at the hemiaminal position was determined as (8aR) for the major diastereoisomer from the X-ray diffraction analysis of zwitterions 23a and 24a (Scheme 3).¹² In the case of 24a, orthorhombic single crystals feature three independent molecules in the asymmetric unit (space group P2₁2₁2₁, Z′ = 3), however, all display the same conformation and stereochemistry.Open in a separate windowScheme 3Intramolecular non-classical Corey–Chaykovsky ring-closing reaction for the synthesis of chiral zwitterionic bicyclic intermediates.To demonstrate the utility of these new chiral zwitterionic bicyclic compounds, the diastereoselective synthesis of 4-hydroxy piperidines was developed and 23a was selected as a model, therefore this compound was subjected to a desulfurization process to access the corresponding oxazolo[3,2-a]pyridine-5,7(6H)-dione 29. Catalytic hydrogenation was first attempted under various heterogeneous hydrogenation conditions. The use of Pd–C or RANEY®-Ni as a catalyst in acidic or neutral conditions led the oxazolo[3,2-a]pyridine-5,7(6H)-dione 29 in low chemical yield. The best result was obtained when reductive desulfurization was conducted using Zn (15 equiv.) in acetic acid, affording the compound 29 in 98% chemical yield (12 as in the starting material 23a.Screening experiments for desulfurization of zwitterion 23a^a

One-pot reductive amination of carbonyl compounds with nitro compounds over a Ni/NiO composite

Easily prepared Ni/NiO acts as a heterogeneous catalyst for the one-pot reductive amination of carbonyl compounds with nitroarenes to afford secondary amines with H₂ as a hydride source. This catalytic system does not require a special technique to avoid air-exposure, in contrast to the common heterogeneous Ni catalysts.

Easy-to-prepare Ni/NiO acts as an efficient heterogeneous catalyst for one-pot reductive amination of carbonyl compounds with nitroarenes.

Amines are among the most important organic compounds for the chemical, materials, pharmaceutical and agrochemical industries.¹ In particular, aromatic and heteroaromatic amines occupy a privileged position in medicinal chemistry,^1c,2 as exemplified in top-selling drugs such as atorvastatin, hydrochlorothiazide, furosemide and acetaminophen.³ The general methods to prepare aryl and heteroaryl amines are the reductive amination of carbonyl compounds,⁴ direct alkylation of amines with alkyl halides,⁵ Buchwald–Hartwig amination⁶ and Ullman-type C–N bond formation.⁷ These amination systems utilize aniline derivatives which are usually prepared in advance by hydrogenation of nitroarenes.⁸ Direct amine synthesis using nitroarenes is attractive because it eliminates the hydrogenation step, which saves time, energy and cost. Among the amination reactions, reductive amination has been actively investigated due to its high atom economy and ease of industrial application (Scheme 1);⁹ however, reductive amination has frequently been accompanied by unwanted side products due to the reduction of carbonyl compounds to alcohols and/or over-alkylation of product amines. One-pot reductive amination with nitro compounds has been achieved by precious metal catalysis.¹⁰ Recently, precious metal alloy nanoparticles were demonstrated to exhibit high catalytic performance for one-pot reductive amination with nitro compounds, even under mild reaction conditions.¹¹ In the context of economic efficiency and a ubiquitous element strategy, the replacement of precious metals with earth-abundant metals has gained much attention. Although some catalytic systems based on Fe,¹² Cu,¹³ Co ¹⁴ and Mo ¹⁵ have been reported using H₂ as a reductant, severe reaction conditions are typically required (Table S1, ESI^†). It is noteworthy that the nitrogen-doped carbon supported cobalt catalysts were reported to be active for one-pot reductive amination using nitroarenes using formic acid or CO/H₂O as a reductant though high temperature was required (Table S2, ESI^†).¹⁶Open in a separate windowScheme 1One-pot reductive amination using nitroarenes.To develop active catalysts for one-pot reductive amination using nitro compounds, we have focused on nickel catalysts, which are known as active catalysts for many transformation reactions.^17,18 The active species is typically metallic nickel, which is easily oxidized in air and becomes covered with NiO.¹⁹ Therefore, special techniques to avoid air-exposure (i.e., pre-reduction in the reaction vessel, glovebox) are required to achieve high catalytic performance for liquid phase reactions. Herein we report an easily prepared Ni/NiO composite as a heterogeneous catalyst for one-pot reductive amination using nitro compounds. This Ni catalyst can be handled under an air atmosphere, even though the supposedly active species, metallic nickel, is oxidized by air-exposure.Ni/NiO was prepared by the partial reduction of NiO with H₂ in the temperature range from 200 to 500 °C (Ni/NiO-X: X = reduction temperature). X-ray diffraction (XRD) patterns of the prepared Ni catalysts after exposure to air are summarized in Fig. 1(A). When NiO is treated in H₂ at 200 °C, the dominant phase produced is still nickel oxide. Metallic nickel was formed by reduction at ≥250 °C, which is consistent with the H₂-temperature programmed reduction (H₂-TPR) profile of NiO in which the H₂ consumption peak begins to increase at around 250 °C.²⁰ The ratio of Ni to NiO, determined by Rietveld analysis, increased with increasing reduction temperature and no peaks attributed to metallic nickel were evident for Ni/NiO-500 (†) and crystallite diameter estimated from (111) diffraction lines using Scherrer''s equation (Table S3, ESI^†). Fig. 1(C) shows an SEM image of Ni/NiO-300, where the particle size was estimated to be 0.5–3 μm, and the layered structure of NiO was maintained after the reduction treatment (see also Fig. S4, ESI^†).Open in a separate windowFig. 1(A) XRD patterns for Ni/NiO-X; (a) Ni/NiO-500, (b) Ni/NiO-400, (c) Ni/NiO-350, (d) Ni/NiO-300, (e) Ni/NiO-250, and (f) Ni/NiO-200 (♦: Ni, ○: NiO), and (B) SEM images of Ni/NiO-300.Specific surface areas and weight ratios of Ni to NiO

Asymmetric synthesis of polysubstituted chiral chromans via an organocatalytic oxa-Michael-nitro-Michael domino reaction

A catalytic asymmetric method for the synthesis of polysubstituted chromans via an oxa-Michael-nitro-Michael reaction has been developed. The squaramide-catalyzed domino reaction of 2-hydroxynitrostyrenes with trans-β-nitroolefins produced chiral chromans with excellent enantioselectivities (up to 99% ee), diastereoselectivities (up to >20 : 1 dr), and moderate to good yields (up to 82%).

A catalytic asymmetric method for the synthesis of polysubstituted chromans via an oxa-Michael-nitro-Michael domino reaction of 2-hydroxynitrostyrenes with trans-β-nitroolefins has been accomplished.

Chromans play an essential role in natural products and pharmaceutical molecules (Fig. 1).¹ Given the biological relevance and diverse applications of this indispensable structural motif, numerous valuable methods for building chiral chromans have been developed.² Rapid, direct, and highly atom-economical asymmetric strategies to construct optically active chromans are the first choice.Open in a separate windowFig. 1Selected biologically active compounds.In the past several years, considerable effort has been made to build chiral chroman derivatives with multichiral centers via asymmetric domino reactions³ catalyzed by aminocatalysts,⁴ thiourea organocatalysts⁵ squaramide organocatalysts,⁶ and other organocatalysts.⁷ Among these approaches, the addition to nitroolefins is a simple but highly efficient route to obtain chiral chromans containing nitro-group, and the nitro group can often lead to changes in chemical and physical properties.^{4d–h,4j,5b–d} In 2013, Zhu et al. reported the organocatalytic oxa-Michael-Michael cascade strategy for the construction of spiro [chroman/tetrahydroquinoline-3,3′-oxindole] scaffolds from 2-hydroxynitrostyrenes and N-Boc-protected methyleneindolinones using a squaramide-cinchona bifunctional catalyst.^6a Peng et al. disclosed the highly efficient synthesis of polysubstituted 4-amino-3-nitrobenzopyrans from 2-hydroxyaryl-substituted α-amido sulfones and nitroolefins mediated by chiral squaramides.^6b Furthermore, Yan and Wang''s group developed the squaramide-catalyzed cascade reaction of 2-hydroxychalcones with β-CF₃-nitroolefins to yield CF₃-containing heterocyclic compounds with a quaternary stereocenter.^6c Notably, a general strategy for the synthesis of chiral chromans bearing two nitro moieties was never reported, especially for 2-alkyl-substituted chromans. In 2003, an easy and efficient method for the synthesis of 3-nitrochromans via the reaction of 2-hydroxynitrostyrenes and trans-β-nitroolefins in the presence of DABCO was reported.⁸ Motivated by our previous work concerning the asymmetric synthesis of chromans,^4i,9 this paper presents a highly efficient asymmetric method for the synthesis of polysubstituted chiral chroman derivatives, especially 2-alkyl-substituted chiral types, from 2-hydroxynitrostyrenes and trans-β-nitroolefins using a chiral bifunctional squaramide organocatalyst.^10,11Given these considerations and previous work, the organocatalytic oxa-Michael-nitro-Michael reaction was performed with 2-hydroxynitrostyrene 1a and aliphatic trans-β-nitroolefin 2a as model substrates to examine the feasibility of our approach. Different parameters (5c,d An appropriate basic/nucleophilic moiety is critical to this kind of reaction. Under this consideration, chiral 1,2-diphenylethylenediamine, cyclohexanediamine, and quinine were selected as scaffolds. After a preliminary study, the quinine scaffold revealed excellent stereoinduction for the asymmetric synthesis of the chiral chroman 4a when paired with the squaramide unit ( EntryCatalystSolventYield^b (%)ee^c (%)dr^c13aCH₂Cl₂475>20 : 123bCH₂Cl₂TraceN.D.^dN.D.33cCH₂Cl₂42−39>20 : 143dCH₂Cl₂7380>20 : 1 5 3e CH ₂ Cl ₂ 78 92 >20 : 163fCH₂Cl₂6543>20 : 173gCH₂Cl₂7185>20 : 183hCH₂Cl₂6975>20 : 193iCH₂Cl₂7579>20 : 1103jCH₂Cl₂6758>20 : 1113kCH₂Cl₂6869>20 : 1123eCHCl₃6593>20 : 1133eClCH₂CH₂Cl7788>20 : 1143eEtOAc7157>20 : 1153eTHF32873 : 1163e1,4-DioxaneTraceN.D.N.D.173eEt₂OTraceN.D.N.D.183eCH₃CN6127>20 : 1193eToluene77818 : 1203eCyclohexane493810 : 1Open in a separate window^aAll the reactions were conducted with 1a (0.2 mmol), 2a (0.24 mmol), and solvent (2 mL) in the presence of 5 mol% organocatalyst 3 at room temperature with vigorous stirring for 24 h.^bIsolated yield of 4a.^cDetermined by chiral HPLC using an OJ–H column.^dN.D. = Not determined.The reaction scope was determined under the optimal conditions. To investigate the versatility of the catalytic system, we first explored the universality of 1-nitro-1-hexene 2a in this squaramide-catalyzed domino reaction. The reaction was tolerant to a range of substituents, such as OMe, OEt, Me, F, Cl, and Br, on the aromatic ring of 2-hydroxynitrostyrenes. The results revealed that the current transformation was a general and efficient strategy for the asymmetric synthesis of n-Bu group-substituted chiral chromans in the 2-position with three contiguous stereogenic centers. More specifically, when the substrates bore electron-donating groups (R¹ = OMe, OEt, Me) or electron-withdrawing groups (R¹ = F, Cl, Br) at the 6-, 7-, and 8-positions of the benzene ring, the target products achieved 68–82% yields with excellent diastereoselectivities (>20 : 1 dr) and enantioselectivities (71–99% ee). The targeted products with electron-donating groups resulted in high yields (Scheme 1, 4b, 4c, 4d, 4e, 4fversus4g, 4h, 4i, 4j). In particular, other aliphatic nitroolefins substituted by branched chain aliphatic group (R² = i-Pr) and cycloaliphatic group (R² = cyclohexyl) were further explored, and these compounds delivered products with good asymmetric inductions (99% and 89% ee, >20 : 1 dr).Open in a separate windowScheme 1Scope of 2-alkyl chromans.In-depth study of the current situation revealed that the enantioselectivities and diastereoselectivities of the chromans with 2-alkyl substituted groups were satisfactory. Therefore, further exploration of aromatic nitroolefins in the oxa-Michael-nitro-Michael domino reaction was necessary. Scheme 2 shows that nitroolefins incorporating electron-withdrawing groups and electron-donating groups at the aryl substituents in the 2-position could be successfully employed under these conditions, resulting in final adducts with high to excellent enantioselectivities (86–99% ee), high-to-excellent diastereoselectivities (up to >20 : 1 dr), and moderate yields (57–69%). In general, nitroolefins with OMe and Me as substituent R³ groups yielded products in excellent 95–99% ee (Scheme 2, 4n–4q, 4y), which were superior to nitroolefins incorporating electron-withdrawing groups (R³ = F, Cl, Br, NO₂). However, product 4t was an interesting special case with 99% ee. The absolute configuration of product 4s was determined to be (2S, 3S, 4S) by single-crystal X-ray diffraction analysis (Scheme 3).¹²Open in a separate windowScheme 2Scope of 2-aryl chromans.Open in a separate windowScheme 3Further investigation of the substrate scope.To further demonstrate the synthetic utility of this reaction, we tested trans-α-Me-β-nitroolefin. As listed in Scheme 4, trans-α-Me-β-nitroolefin was suitable for this reaction and afforded the product 5a with an all-carbon quaternary stereocenter in the 3-position in 59% yield, 91% ee, and 8 : 1 dr.Open in a separate windowScheme 4Proposed transition state for this reaction.On the basis of the X-ray crystallographic analysis of the absolute configuration of adduct 4s, we proposed a transition state model (Scheme 4). 2-Chloro-nitroolefin was activated well through the hydrogen-bonding interaction between the nitro group of 2-chloro-nitroolefin and the N–H of squaramide catalyst 3e. Meanwhile, 2-hydroxynitrostyrene 1a was activated through a hydrogen-bonding interaction between the hydroxyl group of 1a and the basic/nucleophilic moiety of 3e. Therefore, the hydroxyl group of 1a attacked the β-carbon of the activated 2-chloro-nitroolefin from the Si face under the control of the catalyst 3e. Subsequently, the α-carbon of activated 2-chloro-nitroolefin attacked the β-carbon of 1a from the Si face to yield the major stereoisomer of chiral chroman 4s with the configuration of (2S, 3S, 4S).In conclusion, the first enantioselective, organocatalytic oxa-Michael-nitro-Michael domino reaction of 2-hydroxynitrostyrenes with trans-β-nitroolefins was successfully demonstrated. The new domino reaction provided an easy and efficient approach to construct 2-alkyl-substituted chiral chroman derivatives bearing three contiguous stereogenic centers with two nitro moieties. This strategy was also suitable for the asymmetric synthesis of 2-aryl-substituted chiral derivatives. Furthermore, this methodology could be used to construct chiral chromans with an all-carbon quaternary stereocenter in the 3-position. Further applications of this organocatalytic system are ongoing in our laboratory.     

Azaheterocyclic diphenylmethanol chiral solvating agents for the NMR chiral discrimination of alpha-substituted carboxylic acids

A series of small-membered heterocycle probes, so-called azaheterocycle-containing diphenylmethanol chiral solvating agents (CSAs), have been developed for NMR enantiodiscrimination. These chiral sensors were readily synthesized were inexpensive and efficiently used for the chiral analysis of alpha-substituted carboxylic acids. The sensing method was operationally simple and the processing was straightforward. Notably, we propose (S)-aziridinyl diphenylmethanol as a promising CSA, which has excellent chiral discriminating properties and offers multiple detectable possibilities pertaining to the ¹H NMR signals of diagnostic split protons (including 25 examples, up to 0.194 ppm, 77.6 Hz). Its ability to detect the molecular recognition of fluorinated carboxylic acids were further investigated, with a good level of discrimination via the ¹⁹F NMR spectroscopic analysis. In addition, an accurate enantiomeric excess (ee) analysis of the p-methoxyl-mandelic acid with different optical compositions have been calculated based on the integration of well-separated proton signals.

Chiral azaheterocycle-containing diphenylmethanols with multiple hydrogen-bonding sites were described and used as NMR chiral solvating agents (CSAs). Highly resolved NMR spectra can be obtained directly in the NMR tube.

The detection and identification of chiral compounds have attracted considerable scientific attention due to their indispensable role in technological applications including pharmacology, biology, and modern chemistry for the last several decades.¹ So far, spectroscopic and chromatographic approaches are generally utilized for the enantioselective molecular recognition or quantitative analysis including enantio-discrimination, measurement of enantiomeric excess (ee) and assignment of absolute configuration.² Among them, the nuclear magnetic resonance (NMR) spectroscopy is one of the most convenient, versatile, and routinely available analytical techniques because it provides structural information in a multidimensional manner. For these NMR-based analytical methods, the use of diastereomers prepared from chiral solvating agents (CSAs) and guest compounds has become an efficient and operationally simple analytical approach for chiral discrimination.³ Compared with chiral derivatization agents (CDAs), CSAs form two in situ-formed diastereomeric complexes with analytes via non-covalent interactions, which can be tested immediately after mixing, avoiding chiral derivatization in advance. Over the last few decades, a number of CSAs have been developed, such as coordinative unsaturated chiral lanthanide or transition metal complexes,⁴ low molecular-weight synthetic Brønsted acids/bases,⁵ and supramolecular receptors,⁶ to generate anisochronous chemical shifts. Furthermore, some of them are obtained via synthesis and some from nature. It has been reported that enantiopure compounds/complexes are good NMR CSAs for the determination of the enantiomeric purity of different chiral substrates including alcohols or 1,2-diols, cyanohydrins, and hydroxy acids or amino acid derivatives.⁷ Although several CSAs are commercially available, the current methods possess several drawbacks such as line-broadening, narrow substrate scope, poor solubility, and poor resolution. Thus, the development of excellent CSAs with wide applications of the ¹H NMR chiral analysis is still highly desirable.Small-membered azaheterocycle derivatives have gained a lot of interest among organic and medicinal chemists for the synthesis of biological active nitrogen-containing compounds. Now, 2-substituted aziridines or azetidines have been already used as synthons for the synthesis of a wide range of compounds in stereoselective transformations, such as highly stereoselective addition or reduction, regioselective ring expansion reaction, and nucleophilic ring-opening polymerization.⁸ Their broad utility has made these aziridine, azetidine, and even pyrrolidine-based more specifically optically pure amino alcohols, a class of useful and attractive chiral ligands/catalysts in a number of catalytic asymmetric transformations, including the addition of transition metal-catalyzed and organocatalytic asymmetric processes.⁹ Among various N-heterocyclic amino alcohols, such as N-trityl aziridine carbinols,¹⁰ Ferrocenyl-substituted aziridinylalcohols,¹¹N-phenylethyl aziridine carbinols,¹²N-substituted azetidino alcohols,¹³ and azetidine-2-carboxamides,¹⁴ diphenylprolinols¹⁵ have received considerable attention and have been successfully applied to catalyze asymmetric reactions. Nevertheless, the literature concerning the guests of chiral analysis using a small-membered N-heterocyclic moiety and the study on enantiodiscrimination in isotropic solutions by NMR techniques are still not abundant. A few representative ones are the following: Rachwalski et al. explored chiral aziridine-mediated imines to evaluate their action as CSAs towards mandelic derivatives,¹⁶ and proline-derived diphenylprolinol had been successfully developed as an effective CSA for the analysis of chiral carboxylic acids.¹⁷ Therefore, the development of structurally simple efficient receptors for the enantioselective recognition of carboxylic acids is still highly desirable. Taking this fact into account and basing on our experience in the field of the synthesis of a different class of CSAs for the determination of enantiomer ratio, and the application of enantiodiscrimination.¹⁸ The suitably designed chiral heterocyclic amino alcohol derivatives might be a good candidate because of the existence of multiple H-bonding and the rigid ring of this candidate compounds. Thus, we specifically focus on the further applications of these 2-(diphenylhydroxy) azaheterocycles as chemical probes for the discrimination of carboxylic acids and the measurement of their ee ¹H NMR spectroscopy.The synthesis of chiral diphenylmethanol-containing N-heterocyclic amino alcohols, in general, was accomplished from commercially available methyl 1-aza-heterocycle-2-carboxylate in a two-step synthesis (the general synthetic procedures are illustrated in ESI Scheme S1^†). Four molecular structures of aza-heterocycle-containing diphenylmethanols 1–4 are shown in Fig. 1; in principle, these compounds are suitable candidates for H bonding receptors and they are often used by chemists as enantioselective catalysts in various reactions, and our finding that they are useful CSAs extend their utility.Open in a separate windowFig. 1Chiral aza-heterocycle-containing diphenylmethanols 1–4.We conducted the initial exploratory study using racemic analytes in chloroform-d as the most common solvent to determine their abilities to split the signals in the NMR analysis, and explored the binding properties of chiral aza-heterocycle-containing diphenylmethanols 1–4. The (S)-pyrrolidinyl diphenylmethanol 3 as CSA has been proved in our previous study,¹⁷ we had found that the suitable stoichiometries of the host–guest complex, the stoichiometric amounts of racemic mandelic acid and (S)-pyrrolidinyl diphenylmethanol 3 molar ratio of 1 : 1 was finally utilized to carry the application for the resolution of discrimination and measurement of carboxylic acids. Thus, equimolar amounts of all of the chiral aza-heterocycle-containing diphenylmethanols, relative to (±)-3,5-difluoro mandelic acid (MA), were found to be sufficient to discriminate between the corresponding enantiomers. The comparing results of these experiments are shown in †). Interestingly, the signal splitting was particularly significant with (S)-aziridinyl diphenylmethanol 1, achieving a value of 77.6 Hz (at 400 Hz) for methine proton (C^αH) signals. The preliminary results evidenced that CSA 1 can act as a more efficient chiral solvating agent towards MA because of its rigid structure of the aziridine ring. Enantiopure aziridinyl diphenylmethanol, i.e., ent-1 has also explored its efficacy in binding with racemic 3,5-difluoro-MA, a similar degree of splitting was obtained in the separated peaks (0.184 ppm, EntryAza-heterocycle-containing diphenylmethanolsProbe signal α-H ΔΔδ^a (ppm)1(S)-Aziridinyl diphenylmethanol 10.1942(S)-Azetidinyl diphenylmethanol 20.0463(S)-Pyrrolidinyl diphenylmethanol 30.0444(S)-Piperidinyl diphenylmethanol 40.0345(R)-Aziridinyl diphenylmethanol ent-10.184Open in a separate window^aΔΔδ = chemical shift non-equivalences for α-H signals.Having the most active (S)-aziridinyl diphenylmethanol 1 in hand, to define the binding interactions, NMR titration experiments were performed. The effect of concentration on the chemical shift differences (ΔΔδ) was examined by the gradual addition of CSA 1 to a solution of (rac)-4-MeO-MA (Fig. 2). Baseline resolution was achieved over a considerably wide concentration ratio of [(S)-aziridinyl diphenylmethanol 1]/[(rac)-4-MeO-MA] (ca. 0.25–4.0), with an upfield chemical shift (first upfield and then reach equilibrium). The largest ΔΔδ values of the methane proton were observed when 1 : 1 mixtures of host and guest were used, and it is worth pointing out that the split separation signals of the methoxy group of (rac)-4-MeO-MA are also observed (see ESI Fig. S16^†).Open in a separate windowFig. 2Part of the ¹H NMR spectra of (rac)-4-MeO-MA in the presence of different molar amounts of (S)-aziridinyl diphenylmethanol 1.The stoichiometries of the CSA and guests were further established by the ¹H NMR analysis using Job plots. Fig. 3 plots the chemical shift non-equivalencies (Δδ) of 4-MeO-MA enantiomers observed for the CSA 1 as a function of molar ratio. In most cases, the enantiomeric separation increases sharply as the molar ratio increases from 0 to about 0.5 and then steadily declines. The measured Δδ values represent the shift in the NMR signal of the α-H proton of (R)-4-MeO-MA in the presence of CSA. The X represents the molar fraction of the guest in the mixture. The Job plots of X × Δδ versus the molar fraction (X) of (R)- or (S)-4-MeO-MA in the mixture were obtained at 10 mM in CDCl₃, which showed a maximum at X = 0.5, and the best baseline resolution was both achieved with a 1 : 1 ratio of CSA 1/4-MeO-MA, as shown in Fig. 3. This further indicated that CSA 1 and the acid bind in a 1 : 1 complex under these conditions.Open in a separate windowFig. 3Job plots of (S)-aziridinyl diphenylmethanol 1 with (R)-4-MeO-MA and (S)-4-MeO-MA. Δδ stands for the chemical shift change of the α-H proton of (R)- and (S)-4-MeO-MA in the presence of (S)-aziridinyl diphenylmethanol 1. X stands for the molar fraction of the (S)-aziridinyl diphenylmethanol 1 (X = [(S)-aziridinyl diphenylmethanol 1]/[(S)-aziridinyl diphenylmethanol 1] + [4-MeO-MA]). The total concentration is 10 mM in CDCl₃.After obtaining the most active (S)-aziridinyl diphenylmethanol 1 and using the optimal conditions, in the next step of studies, the influence of various electron-donating and electron-accepting substituents in the racemic alpha-substituted carboxylic acids on the chiral recognition was examined and evaluated. All the experiments were carried out by adding a certain amount of solution of racemic carboxylic acid in CDCl₃ (10 mM) to a solution of (S)-aziridinyl diphenylmethanol 1 in CDCl₃ (10 mM). Immediately after each addition, the ¹H NMR spectrum was acquired in a 400 MHz spectrometer at room temperature. As shown in EntryCarboxylic acidsΔΔδ^bSpectra1 0.134, 53.6 2 0.137, 54.8 3 0.100, 40.0 4 0.148, 59.2 5 0.138, 55.2 6 0.194, 77.6 7 0.125, 50.0 8 0.121, 48.4 9 0.134, 53.6 10 0.118, 47.2 11 0.109, 43.6 12 0.050, 20.0 13 0.053, 21.2 14 0.111, 44.4 15 0.084, 33.6 16 0.068, 27.2 17 0.058, 23.2 18 0.079, 31.6 19 0.150, 60.0 20 0.101, 40.4 21 0.012, 4.8 22 0.104, 41.6 0.188, 75.2^d 23 0.039, 15.6 0.052, 20.8^c 24 0.041, 16.4^c 0.049, 19.6^d 25 0.029, 11.6^e Open in a separate window^aAll samples were prepared by mixing 1 : 1 of carboxylic acids (10 mM in CDCl₃) with (S)-CSA 1 in NMR tubes, and the spectra is recorded at 25 °C on a 400 MHz spectrometer.^bΔΔδ values in ppm and Hz for α-H are shown.^cChemical shift non-equivalences of the α-methoxy group.^dChemical shift non-equivalences of the α-methyl group.^eUse of 2 equiv. of (S)-CSA 1 for the dicarboxylic acid.In addition, we further evaluated the chiral discriminating abilities of (S)-aziridinyl diphenylcarbinol 1 for aliphatic 2-hydroxymonocarboxylic acids as guests, and these 2-hydroxycarboxylic acids are known for their relationship with particular human illnesses, especially but not only with inherited metabolic diseases.¹⁹ 2-hydroxy-3-methylbutyric acid (2-H-3-MBA), 2-hydroxyisocaproic acid (2-HICA), and 2-hydroxyoctanoic acid (2-HOA) are related to certain types of organic acidurias. For example, an increased amount of 2-HICA has been found in patients with the Zellweger syndrome, a congenital disorder.²⁰ The inspection of results presented in the fragments of ¹H NMR spectra (0.111 ppm, 44.4 Hz; 0.084 ppm, 33.6 Hz; 0.058 ppm, 23.2 Hz; 0.058 ppm, 23.2 Hz, and 0.079 ppm, 31.6 Hz, EntryCarboxylic acidsΔΔδSpectra1 0.076, 28.6 2 0.057, 21.5 3 0.054, 20.0 4 0.256, 96.2 5 0.056, 21.3^a 0.054, 20.4^b 6 0.068, 25.9^a 0.080, 30.2^b 7 0.859, 323.3 8 0.015, 5.6 Open in a separate window^aThe proton-decoupled ¹⁹F NMR spectra was obtained from o-substituted fluorine.^bThe proton-decoupled ¹⁹F NMR spectra was obtained from m-substituted fluorine.Now that (S)-aziridinyl diphenylcarbinol 1 had been established to demonstrate efficiently chiral discriminating abilities towards α-hydroxyl acids via¹H NMR spectroscopy, to further demonstrate the practical utility of (S)-aziridinyl diphenylcarbinol 1 as a CSA for enantiomeric determination. It allowed determining the enantiomeric composition of multiple nonracemic 4-MeO-MA samples by the simple integration of the split signals, and samples containing (R)-4-MeO-MA with 80, 60, 40, 20, 0, −20, −40, −60, −80 and −100% were prepared in the presence of (S)-aziridinyl diphenylcarbinol 1, and their ¹H NMR spectra were measured. Enantiomeric excesses of all samples were calculated based on the integration of the ¹H NMR signals of the α-H signal protons of the enantiomers of (R)- and (S)-4-MeO-MA. The overlaid partial ¹H NMR spectra and linear correlation between the theoretical (X) and observed ee% (Y) are shown in Fig. 4. The linear relationship between the NMR-determined values and those gravimetrically determined values is excellent with R² = 0.99995. The average absolute error in the ee measurement plotted in Fig. 4 is within 1%.Open in a separate windowFig. 4(a) Selected regions of the ¹H NMR spectra of different optical purities 4-MeO-MA samples (ee% = R% − S%) with (S)-aziridinyl diphenylcarbinol 1 in CDCl₃; (b) linear correlation between ee values determined by gravimetry (Y) and values were defined in terms of (R)-4-MeO-MA (X), R² = correlation coefficient.     

Enantioselective conjugate hydrosilylation of α,β-unsaturated ketones

Enantioselective conjugate hydrosilylation of β,β-disubstituted α,β-unsaturated ketones was realized. In the presence of a chiral picolinamide–sulfonate Lewis base catalyst, the reactions provided various chiral ketones bearing a chiral center at the β-position in up to quantitative yields with moderate enantioselectivities.

Enantioselective conjugate hydrosilylation of β,β-disubstituted α,β-unsaturated ketones was realized.

Chiral ketones are important intermediates for the synthesis of natural products or chiral drugs, and some themselves are useful chiral drugs.¹ Asymmetric conjugate reduction of α,β-unsaturated carbonyl compounds is an attractive and challenging transformation for the construction of chiral ketones. During the past few decades, many groups have devoted considerable efforts to this area and made great improvement, for instance, transition metal catalyzed asymmetric hydrogenation of α,β-unsaturated carbonyl compounds, including palladium,² iridium,³ copper,⁴ ruthenium,⁵ rhodium⁶ and cobalt.⁷ Besides, some organocatalyzed asymmetric conjugate transfer hydrogenations using Hantzsch ester⁸ or pinacolborane⁹ as the hydride source have also been reported. However, very few examples about chiral Lewis base catalyzed conjugate hydrosilylation of α,β-unsaturated carbonyl compounds have been reported and only chiral phosphine oxide Lewis base catalysts were used in these reactions (Scheme 1).¹⁰ Therefore, exploration of the application of other kinds of chiral Lewis base catalysts in this reaction is still highly desirable.Open in a separate windowScheme 1Enantioselective conjugate hydrosilylation of α,β-unsaturated ketones by chiral Lewis base catalysts.Recently, we developed a kind of easily accessible chiral picolinamide–sulfonate Lewis base catalysts and used them in asymmetric hydrosilylation of α-acyloxy-β-enamino esters,¹¹ one of them exhibiting excellent reactivity, diastereoselectivity and enantioselectivity. Herein we present the enantioselective conjugate hydrosilylation of β,β-disubstituted α,β-unsaturated ketones using this kind of Lewis base as catalysts, leading to various chiral ketones bearing a chiral center at β-position (Fig. 1).Open in a separate windowFig. 1Three typical chiral drugs containing chiral ketone moiety.First, various chiral Lewis base catalysts 2 were screened in the enantioselective hydrosilylation of (E)-1,3-diphenylbut-2-en-1-one 1a in acetonitrile at 0 °C. As shown in Fig. 2, l-piperazine-2-carboxylic acid derived N-formamide 2a,^12aR-(+)-tert-butylsulfinamide derived catalyst 2b^12b and picolinamide 2c^12c were found to be totally inactive for the reaction. Meanwhile picolinamide–tosylate catalyst 2d was highly active to afford the product with excellent yield in moderate ee value. Afterwards, several picolinamide–tosylate catalysts 2f–2h bearing electron-withdrawing group in 4-position of pyridine were employed in the reaction and 4-bromo picolinamide 2f delivered a slightly higher ee value. 5-Methoxy picolinamide 2i gave the product with the same ee value as that of 2d but in much lower yield. Lower enantioselectivities were observed with 4-phenyl picolinamide 2j and 3-methyl picolinamide 2k. When (R)-1-(2-aminonaphthalen-1-yl)naphthalen-2-ol derived catalyst 2l was used, no product was observed. When (1S,2R)-1-amino-2,3-dihydro-1H-inden-2-ol derived catalyst 2m gave the product in both poor yield and ee value. Moreover, two picolinamide–sulfonamide catalysts 2n and 2o were also used in the reaction and almost racemic products were obtained. Hence, 2f was determined as the optimal catalyst and was used through out our study.Open in a separate windowFig. 2Evaluation of the chiral Lewis base catalysts 2 in conjugate hydrosilylation of (E)-1,3-diphenylbut-2-en-1-one 1a. Unless otherwise specified, the reactions were carried out with 1a (0.1 mmol), trichlorosilane (0.2 mmol) and catalyst 2 (0.02 mmol) in 1 mL of acetonitrile at 0 °C for 24 hours. Isolated yield based on 1a. The ee values were determined by using chiral HPLC.Subsequently, the other reaction conditions were optimized. The results are summarized in Entry^aSolvent T (°C)Time [h]Yield^b [%]ee^c,d [%]1CH₃CN0247751 (R)2CH₃CH₂CN0248450 (R)3C₆H₅CN0248324 (R)4THF0249211 (R)51,4-Dioxane024635 (R)6CHCl₃0249225 (S)7CH₂Cl₂0249638ClCH₂CH₂Cl0249010 (R)9CCl₄0249535 (S)10Toluene0249555 (S)11Xylene024N.R.—12Mesitylene024N.R.—13C₆H₅CF₃0249531 (S)14Toluene−10489764 (S)15Toluene−20609265 (S)16Toluene−4072N.R.—Open in a separate window^aUnless otherwise specified, the reactions were carried out with 1a (0.1 mmol), trichlorosilane (0.2 mmol) and catalyst 2f (0.02 mmol) in 1 mL of solvent.^bIsolated yield based on 1a.^cThe ee values were determined by using chiral HPLC.^dThe absolute configuration of 3a was determined by comparison of the retention times of the two enantiomers on the stationary phase with those in the literatures.With the optimized conditions in hand, the scope and limitations of the reaction were explored. The results are summarized in Fig. 3. In the presence of 20 mol% of chiral Lewis base catalyst 2f and 2 equivalents of trichlorosilane, various α,β-unsaturated ketones were hydrosilylated. We first tested the effect of various 3-aryl groups of 1. 3-(4-Methoxy-phenyl) substrate 3e (Fig. 3) and 3-(naphthalen-2-yl) substrate 3i (Fig. 3) underwent the reaction to give the products with good yields in enantioselectivities close to 3a, while lower ee values were observed with 3b–3d, 3h, 3k and 3l (Fig. 3). No product was obtained with 3-(2-chloro-phenyl) substrate 3f and 3-(naphthalen-1-yl) substrate 3j, perhaps due to the high steric hindrance (Fig. 3). When 3-(2-methoxy-phenyl) substrate 3g was used, by prolonging the reaction time to 60 hours, the product was obtained with good yield but very poor enantioselection (Fig. 3). Trace amount of the product was detected with 3-(pyridin-2-yl) substrate 3m (Fig. 3). Next, some 3-phenyl-but-2-en-1-ones with different 1-aryl groups were also employed in the reaction. The 4-substituted or 3-substituted substrates delivered good yields of the products with similar or slightly lower enantioselectivities (Fig. 3), while much lower ee value was observed with 2-substituted substrate 3s (Fig. 3). For some other 3-alkyl chalcones, moderate to good yields and moderate ee values were obtained (Fig. 3), except for the bulkier tertiary butyl substituted 3w that gave trace amount of the product (Fig. 3). Reaction of 3y with two different 3,3-aryl groups afforded the product with moderate yield in very low enantioselectivity (Fig. 3). Finally, cyclic substrate 3z was subjected in the reaction and provided the product with excellent yield but in poor ee value (Fig. 3).Open in a separate windowFig. 3Substrate scope of the reaction. Unless otherwise specified, the reactions were carried out with 1 (0.1 mmol), trichlorosilane (0.2 mmol) and catalyst 2f (0.02 mmol) in 1 mL of toluene at −10 °C for 48 hours. Isolated yield based on 1. The ee values were determined by using chiral HPLC. The absolute configuration of 3a was determined by comparison of the retention times of the two enantiomers on the stationary phase with the literatures. The absolute configurations of other products were determined in analogy. ^aThe reaction time was 60 hours. ^bThe reaction time was 72 hours.Although detailed structural and mechanistic studies remain to be carried out, based on the absolute configuration of the product 3a, we propose a mechanism shown in Scheme 2. First, the nitrogen atom of the pyridine ring and the carbonyl oxygen atom of catalyst 2f are coordinated to Cl₃SiH to create an activated hydrosilylation species. Substrate 1a may approache the Cl₃SiH-catalyst complex to generate two transition states A and B. In transition state A, the N–H of catalyst 2f activates the carbonyl group of 1a through H-bonding. In addition, there could be π–π stackings between the two aromatic systems of the catalyst and the substrate. Then Si-face conjugate attack of the hydride to 1a generate (S)-product 3a. On the contrary, in transition state B through which (R)-product will be obtained, the carbonyl group of 1a can not be connected with the N–H of catalyst 2f. Thus the fact that (S)-enriched product 3a was obtained is consistent with the suggestion that the hydrosilylation predominantly proceeds through the pathway involving transition state A rather than transition state B.Open in a separate windowScheme 2A plausible reaction mechanism for hydrosilylation of 1a catalyzed by 2f.In conclusion, we have developed a facile, metal-free and mild enantioselective conjugate hydrosilylation of β,β-disubstituted α,β-unsaturated ketones. By using chiral picolinamide–sulfonate Lewis base as catalyst, the reactions provided various optically active ketones bearing a chiral center at β-position with moderate to good yields in moderate enantioselectivities. Comparing with the chiral phosphine oxide Lewis base catalysts, the chiral picolinamide–sulfonate is cheaper and easier accessible. The absolute configuration of one product was determined by comparison of the retention times of the two enantiomers on the stationary phase with those in the literature.     

Binding model-tuned room-temperature phosphorescence of the bromo-naphthol derivatives based on cyclodextrins

Herein, bromo-naphthol derivatives were synthesized to investigate the influence on their phosphorescence emission efficiency resulting from different binding models with cyclodextrins. And the results indicated that α-cyclodextrin could result in the highest phosphorescence emission efficiency, due to the tight encapsulation of the bromo-naphthol motif into the cavity.

Bromo-naphthol derivatives were synthesized to form host–guest complexation with cyclodextrins with different cavity sizes for the investigation of binding model-mediated room-temperature phosphorescence efficiency.

Organic room-temperature phosphorescence (RTP) materials have received widespread attention in the past decades owing to their potential application in anticounterfeiting,¹ biological imaging,^2,3 and optoelectronic materials.⁴ However, the relatively low intersystem crossing efficiency from singlet state to the triplet state and quenching of the triplet state by external oxygen and water traditionally make it difficult to obtain the RTP materials.^5–8 Therefore, to realize the RTP materials, much effort should be devoted on the enhancement of the intersystem crossing efficiency and shielding from the oxygen and water in the environment. Along with this line, variant methods have been developed, such as polymer matrix,^9–11 crystallization,^12,13 H-aggregation,¹⁴ and noncovalent interactions,^15–17 especially the host-guest interaction,^18–20 which could provide a relatively enclosed environment to stabilize the excited state of the organic molecules and shield them from the external substance. Even though there are many outstanding systems constructed by cucurbituril (CB)^21,22 and cyclodextrin (CD),^23,24 little research has been conducted to investigate the influence resulting from their binding model.CD, as a kind of macrocyclic molecule, has been widely investigated in the fabrication of functional materials.²⁵ Moreover, due to the hydrophobic cavities in this kind of macrocyclic molecule, the phosphors could be shielded from the external environment, and many RTP systems have been successfully constructed.^26–28 However, there are three kinds of CDs with different cavity sizes, which exhibit different binding affinities to the guest molecules. To the best of our knowledge, little research has been conducted to exploit the effects caused by their binding models on the RTP materials. Herein, two bromo-naphthol derivatives (4C and 6C) were synthesized to form host–guest complexation with α-CD, β-CD, and γ-CD (Scheme 1). Due to the different cavity size of these three host molecules, their binding models with bromo-naphthol derivatives were different, resulting in the different RTP properties. The α-CD could partially encapsulate the bromo-substituent motif, resulting in the highest phosphorescence quantum yield, as well as the longest phosphorescence lifetime. This research on tuning the RTP properties of phosphors by the binding models could provide a general guidance in the design of highly efficient RTP materials.Open in a separate windowScheme 1Schematic illustration of cyclodextrin-mediated RTP.The guest molecules of 4C and 6C were successfully synthesized according to synthesis route provided in the ESI^† (Scheme S1 and S2, ESI^†). Firstly, their host–guest properties with cyclodextrins were investigated by the ¹H NMR spectroscopy. As shown in Fig. 1, the protons H_1-6 of the aromatic groups in 4C exhibited obvious shift after the addition of cyclodextrins. Interestingly, different change of the chemical-shift was observed upon the addition of α-CD, β-CD, and γ-CD. The protons showed downfield-shift in the presence of α-CD and β-CD, and the broaden effect could be observed in the presence of α-CD, which might result from the relatively small size of α-CD, leading to the tight encapsulation of 4C into its cavity. And the protons exhibited the upfield-shift upon the addition of γ-CD, which might be caused by the π⋯π stacking interaction in the formed ternary host-guest complex. Moreover, 2D NOESY spectroscopy was employed to further investigate the binding models between 4C and cyclodextrins. As shown in Fig. S8^† in the ESI,^† the corresponding peaks assigned H_1-3 on 4C and H_c on the α-CD were obviously observed, indicating partial aromatic ring was encapsulated into the cavity of α-CD. And the signals between H_1-6 on 4C and H_c and H_e on the β-CD could be observed, indicating the aromatic ring was totally encapsulated into the cavity of β-CD (Fig. S9, ESI^†). However, the observed signals assigned to H_2,3,6 on 4C and C_c on γ-CD indicated the inclusion 4C into the cavity of γ-CD (Fig. S10, ESI^†), and the signals between H₂ and H₄ indicated the π⋯π stacking interaction between the aromatic rings. These results were consistent with the previous conclusions. Also similar phenomena could be observed in the systems of the compound 6C and cyclodextrins with different cavity sizes (Fig. S7, ESI^†). And the 2D NOESY spectra of 6C⊂α-CD, 6C⊂β-CD, and 6C⊂γ-CD also provided the same binding models between 6C and different cyclodextrins with cavity size.Open in a separate windowFig. 1 ¹H NMR (400 MHz, D₂O, 298 K) spectra of 4C (a) after adding 1.0 equivalent α-CD (b), β-CD (c), and γ-CD (d). ([4C] = [α-CD] = [β-CD] = [γ-CD] = 2 mM).Following, the binding stoichiometries between the 4C and cyclodextrins were measured by Job''s plot method using ¹H NMR spectroscopy. As shown in Fig. S14 in the ESI,^† it could be concluded the 1 : 1 binding ratio was determined between 4C and α-CD. Moreover, the binding stoichiometry between 4C and β-CD or γ-CD was determined to be 1 : 1 or 2 : 1 (Fig. S15 and S16, ESI^†), respectively. To determine the binding constants, titration experiments were carried out in aqueous solution containing constant concentration of 4C (2 mM) and varying concentration of cyclodextrins respectively. And the binding constants were calculated to be (2.3 ± 0.45) × 10³ M⁻¹ for 4C between α-CD (Fig. S17, ESI^†), (2.8 ± 0.84) × 10³ M⁻¹ for 4C between β-CD (Fig. S18, ESI^†), and (2.7 ± 0.39) × 10² M⁻¹ and (3.2 ± 0.72) × 10³ M⁻¹ for 4C between γ-CD (Fig. S19, ESI^†). Also due to the main difference between 4C and 6C was the length of alkyl chain, the binding sites between the 6C and cyclodextrins couldn''t be affected, which was also certificated by the ¹H NMR and 2D NOESY spectra (Fig. S7, S11, S12, and S13, ESI^†) between 6C and CDs (α-CD, β-CD and γ-CD), the binding stoichiometries and constants between 6C and CDs were similar with the above results.From the above results, it could be concluded the compound 4C could form the host–guest complexation with cyclodextrins, and due to the different size of the cavities, the binding models were different, which might result in different photoluminescence properties. Firstly, the UV-vis spectra of 4C in the absence or presence of cyclodextrins were recorded in Fig. 2a, from which the obvious increase in the absorption intensity could be observed, indicating the formation of host–guest complexation could affect its photoluminescence properties. Moreover, the decrease of the absorption intensity at 265 and 272 nm, and the increase in the intensity at 345 nm could be observed in the presence of γ-CD (Fig. S20, ESI^†), indicating the tightly stacking occurred between the aromatic rings of 4C, which was consistent with the previous 2D NOSEY spectroscopy results.Open in a separate windowFig. 2(a) UV-vis spectral changes in the aqueous solution of 4C (1.5 × 10⁻³ mM) upon adding equivalent α-CD and β-CD. (b) Solid state phosphorescence spectral changes of 4C, n_4C:n_α-CD = 1 : 1, n_4C:n_β-CD = 1 : 1 and n_4C:n_γ-CD = 1 : 1 (Ex: 280 nm, 298 K). (c)Pictures of 4C (a and e), n_4C:n_α-CD = 1 : 1 (b and f), n_4C:n_β-CD = 1 : 1 (c and g) and n_4C:n_γ-CD = 1 : 1 (d and h) under ambient light (a–d) and 365 nm lamp (e–h).Following, the host–guest complexation 4C⊂α-CD, 4C⊂β-CD, and 4C⊂γ-CD were successfully prepared through the grinding method.^18,19 Then its photoluminescence spectra were recorded, and presented as Fig. S24 in the ESI.^† From these spectra, the chromophore only emitted the blue fluorescence at 380 nm, which was consistent with the fluorescence spectrum in the aqueous solution (Fig. S21, ESI^†). This result also further certificated by the determination of its luminescence lifetime, from the decay curve, its lifetime was determined to be 0.77 ns. However, the addition of cyclodextrins could result in the appearance of the new emission peaks at 518 nm and 548 nm, and the average lifetimes at both 518 nm and 548 nm remarkably improved, indicating the formation of host–guest complexation could stabilize the triplet of 4C (†). This observation was also consistent with the photographs obtained under the UV light irradiation (365 nm). As shown in Fig. 2c, the solid of 4C emitted the violet luminescence, and the yellow luminescence could be observed from 4C⊂α-CD. However, the yellow luminescence was remarkably reduced in the complexation of 4C⊂β-CD, and 4C⊂γ-CD. This observation was also consistent with the previous results, the addition of α-CD could remarkably improve the phosphorescence intensity. Moreover, their quantum yields of luminescence were further determined. As being presented in τ _Phos at 548 nm ms⁻¹ τ _Phos at 518 nm ms⁻¹ τ _Fluo at 380 nm ns⁻¹ Φ _phos/% Φ _fluo/%4C0.480.780.770.560.29n_4C:n_α-CD = 1 : 15.896.923.4611.470.49n_4C:n_β-CD = 1 : 16.036.001.873.070.98n_4C:n_γ-CD = 1 : 13.943.971.325.730.58Open in a separate windowInterestingly, the similar phenomena could also be observed in the systems constructed by 6C (Fig. S25, S26, S32, and Table S1, S3, ESI^†). Therefore, a conclusion might be drawn that the cyclodextrins could improve the phosphorescence quantum yield, as well as prolong the decay time of phosphorescence. As is well known, the phosphor should exhibit more intensive photoluminescence in a more restricted circumstance, in these systems, the addition of cyclodextrins would encapsulate the phosphor into the cavity of the macrocyclic molecules. Moreover, different cyclodextrins exhibited different efficiency in the promotion of its photoluminescence properties. From the previous results obtained from the 2D NOESY spectroscopy, the partial aromatic rings containing the bromo unit was encapsulated into the cavity of α-CD, in this manner, the rotation of the guest molecule might be restricted, as well as the bromine atom was shielded from the outer environment, which would enhance the ISC and suppress the nonradiative relaxation, resulting in the promotion in the quantum yield and the prolongation of the lifetime. And in the host–guest complexation of 4C⊂β-CD, the entire aromatic ring was encapsulated into the cavity of β-CD due to the relatively larger cavity compared with α-CD. However, there would be more space for the rotation in the cavity, which could increase the nonradiative relaxation. This phenomenon also accounted for the relative lower quantum yield and lifetime of 4C⊂β-CD. And in the 4C⊂γ-CD, the π⋯π stacking was occurred in the cavity of the γ-CD, resulting in the further astrictions of the rotation of 4C even though in the largest cavity of cyclodextrins.To further confirm the host–guest interaction played a vital role in the promotion of quantum yields of phosphorescence. Compound 4C was employed as the model compound for the following experiments. The glucose (glu) was used as the model compound due to its being the repeating unit of the cyclodextrins. By using the same grinding method, the mixture of 4C@Glu was obtained. And from the obtained spectra (Fig. 3b), it was observed the addition of glucose could not remarkably improve the phosphorescence emission of 4C, which might result from the absence of the hydrophobic cavity to encapsulate the phosphors. Then the molar ratio of a-CD and 4C was further increased to certificate our envision. The obtained results were consistent with our anticipation, with the increase in the α-CD content, the phosphorescence emission intensity was enhanced. And the following determination of their phosphorescence quantum yields also further certificated the above results. And with the increase in the α-CD content, the quantum yield was increased from 11.47% to 20.24%, which might be caused by the incomplete complexation at the lower content of the α-CD. And the photographs of the solid under the UV light also provided the same results, in which the solid with the highest molar ratio between cyclodextrin and 4C presented the brightest yellow lights (Fig. S31, ESI^†). These phenomena also indicated the cavity of cyclodextrin played a vital role in improving its phosphorescence emission.Open in a separate windowFig. 3(a) UV-vis spectral changes in the aqueous solution of 4C (1.5 × 10⁻³ mM) upon adding different equivalent α-CD. (b) Solid state phosphorescence spectral changes of 4C, n_4C:n_α-CD = 1 : 1, n_4C:n_α-CD = 1 : 2, n_4C:n_α-CD = 1 : 4, n_4C:n_α-CD = 1 : 8, n_4C:n_glu = 1 : 6 (Ex: 280 nm, 298 K).To confirm the formation of host–guest complexation in the solid state, the Fourier transform infrared (FTIR) spectroscopy was employed. As shown in Fig. S33 in the ESI^†, the peak assigned to C–H stretching vibration at 3003 cm⁻¹, 2945 cm⁻¹, and 2867 cm⁻¹ could be obviously observed. Upon the addition of α-CD, these peaks broadened, and shifted to 2983 cm⁻¹ and 2916 cm⁻¹. This might be resulted from the formation inclusion complex between 4C and α-CD, which could decrease the distance between the C–H bond on the aromatic ring and the O atoms in the α-CD, resulting in the enhanced hydrogen bond interaction. Moreover, the XRD was also carried out to certificate the host-guest interaction in the solid state. From the obtained spectra (Fig. S34, ESI^†), the scattering signals of 4C changed upon the addition of α-CD, indicating the addition of α-CD could change the pattern model of 4C. Moreover, the distance of the aromatic rings was changed from 6.20 Å (2θ = 14.28°) to 6.75 Å (2θ = 13.10°) in the presence of α-CD. These results presented a solid evidence for the formation of host–guest complexation in the solid state.Herein, two bromo-naphthol derivatives were synthesized to form host–guest complexation with α-CD, β-CD, and γ-CD. Even though the binding affinities between the bromo-naphthol derivative and CDs were similar, their RTP efficiency was different. The host–guest complexation formed with α-CD presented the highest phosphorescence quantum yield, as well as the longest phosphorescence lifetime. In this complexation, the bromo-substituent motif was encapsulated into the cavity, therefore the exciting state could be stabilized, and shielded form the external environment, resulting in the enhancement in the RTP emission. This research on tuning the RTP properties of phosphors by the binding models could provide a general guidance in the design of highly efficient RTP materials.     

Diastereoselective synthesis of chroman bearing spirobenzofuranone scaffolds via oxa-Michael/1,6-conjugated addition of para-quinone methides with benzofuranone-type olefins

A simple and convenient cyclization of ortho-hydroxyphenyl-substituted para-quinone methides with benzofuran-2-one type active olefins via oxa-Michael/1,6-conjugated addition has been developed, which afforded an easy access to enriched functionalized chroman-spirobenzofuran-2-one scaffolds with good to excellent yields (up to 90%) and diastereoselectivities (up to >19 : 1 dr). This reaction provided an efficient method for constructing desired spirocyclic compounds combining both well-known heterocyclic pharmacophores chroman and benzofuran-2-one.

Highly diastereoselective synthesis of spirocyclic compounds combining both well-known heterocyclic pharmacophores chroman and benzofuran-2-one.

The chroman framework represents a privileged heterocyclic core commonly found within a wide variety of biologically active natural products¹ and synthetic compounds of medicinal interest (Fig. 1).² Owing to the wide application of these heterocyclic molecules, over the past few decades, numerous efforts have been devoted to the efficient synthesis of chroman nucleus motifs.^3,4 In particular, incorporating chroman into spiro-bridged and spiro-fused heterocyclic systems is appealing due to its fascinating molecular architecture and proven biological activity.⁵ Among the existing methods, the [4 + m] cycloaddition of para-quinone methides (p-QMs) is found to be an efficient pathway to access these valuable spirocyclic skeletons.⁶ For instance, the Enders group synthesized functionalized chromans with an oxindole motif by the asymmetric organocatalytic domino oxa-Michael/1,6-addition reaction.⁷ After that, the Hao,^8a Peng,^8b Shi,^8c,d Zhou,^8e Liang^8f and Wang^8g groups developed convenient methods to construct chromans bearing spirocyclic skeletons from p-QMs, respectively. Despite all these shining achievements, however, it is still very challenging to simply and conveniently construct chromans bearing quaternary carbon spirals for organic chemical or drug discovery among these [4 + m] cycloaddition reactions.Open in a separate windowFig. 1Representative chroman compounds.Benzofuran-2-(3H)-ones as one of the important oxygen-containing heterocycles that exist in a broad array of natural products⁹ and potential medicines.¹⁰ The streamlined synthesis of benzofuran-2-ones pose considerable challenge due to their quaternary carbon centers at the C-3 position,¹¹ especially those featuring relatively congested spirocyclic motifs represent challenging synthetic targets.^12,13 In our continuous interests in developing efficient method for the synthesis of spirocyclic compounds based on cyclization reaction,¹⁴ we wish to report a cycloaddition of para-quinone methides with benzofuranone derived olefins, affording the spiro-cycloadducts in good to excellent yields and diastereoselectivities. This cyclization features the simultaneous formation of chroman and spirobenzofuran-2-one skeletons in a single step (Scheme 1), which may be potentially applied as pharmaceutical agents.Open in a separate windowScheme 1Strategy for the synthesis of chroman-spirobenzofuran-2-one.We initiated our investigations with the readily available ortho-hydroxyphenyl-substituted para-quinone methides 1a and 3-benzylidenebenzofuran-2-one 2a in toluene at room temperature in the presence of base. Unfortunately, no desired chroman derivatives bearing spirobenzofuranone scaffolds 3a was isolated in the presence of 2 eq. Na₂CO₃ after stirring at room temperature for 48 h (entry 1, EntryBaseSolventYield^b (%)Dr^c1Na₂CO₃Toluene——2DMAPToluene——3K₂CO₃Toluene253 : 14CsFToluene525 : 15Cs₂CO₃Toluene706 : 16Cs₂CO₃THF68>19 : 17Cs₂CO₃Et₂O608 : 18Cs₂CO₃Dioxane537 : 19Cs₂CO₃CH₃CN50>19 : 110Cs₂CO₃DMF3115 : 111^dCs₂CO₃THF65>19 : 112^eCs₂CO₃THF75>19 : 113^fCs₂CO₃THF64>19 : 1Open in a separate window^aReaction conditions: p-QMs 1a (0.1 mmol), benzofuranones 2a (0.12 mmol) and base (0.2 mmol) in 2 mL of solvent for 6–48 h.^bIsolated yields.^cDetermined by crude ¹H NMR analysis.^dPerformed at 30 °C.^ePerformed at 10 °C.^fPerformed at 0 °C.With the optimized reaction conditions in hand, we explored the substrate scope of this cyclization reaction with a selection of benzofuranones. The results are shown in EntryR3Yield^b (%)Dr^c1C₆H₅3a75>19 : 122-ClC₆H₄3b69>19 : 132-BrC₆H₄3c6715 : 142-CH₃C₆H₄3d64>19 : 153-CH₃C₆H₄3e75>19 : 163-MeOC₆H₄3f72>19 : 173-NO₂C₆H₄3g71>19 : 183-BrC₆H₄3h75>19 : 194-CH₃C₆H₄3i7310 : 1104-MeOC₆H₄3j86>19 : 1114-CF₃C₆H₄3k8212 : 1124-NO₂C₆H₄3l8512 : 1134-ClC₆H₄3m83>19 : 1144-BrC₆H₄3n8112 : 1152-Naphthyl3o74>19 : 1163,4,5-(OMe)₃C₆H₂3p8812 : 1173-Pyridinyl3q79>19 : 1183-Thiophenyl3r80>19 : 1Open in a separate window^aReaction conditions: p-QMs 1a (0.1 mmol), benzofuranones 2 (0.12 mmol) and Cs₂CO₃ (0.2 mmol) in 2 mL of THF.^bIsolated yields for 4–48 h.^cDetermined by crude ¹H NMR analysis.Subsequently, the generality of this cyclization reaction was further evaluated through varying p-QMs 1. As shown in EntryR4Yield^b (%)Dr^c15-Cl4a73>19 : 125-Br4b65>19 : 135-CH₃4c90>19 : 145-OCH₃4d8010 : 154-CH₃4e72>19 : 164-OCH₃4f6610 : 1Open in a separate window^aReaction conditions: p-QMs 1 (0.1 mmol), benzofuranones 2a (0.12 mmol) and Cs₂CO₃ (0.2 mmol) in 2 mL of THF for 6–48 h.^bIsolated yields.^cDetermined by crude ¹H NMR analysis.The structure and relative configuration of 3a were determined by HRMS, NMR spectroscopy and single-crystal X-ray analysis.¹⁵ The relative configuration of other cycloadducts were tentatively assigned by analogy (Fig. 2 and see ESI^†).Open in a separate windowFig. 2X-ray crystal structure of 3a.     

A C1-symmetric N-heterocyclic carbene catalysed oxidative spiroannulation of isatin-derived enals: highly enantioselective synthesis of spirooxindole δ-lactones

A C₁-symmetric N-heterocyclic carbene (NHC)-catalysed activation of isatin-derived enals under oxidative conditions was achieved. The in situ generated α,β-unsaturated acyl azolium species was efficiently trapped by 1,3-dicarbonyl compounds via a Michael addition/spiroannualtion cascade, delivering a series of synthetically important spirooxindole δ-lactones with up to 96% enantioselectivity.

A NHC-catalysed spiroannulation of isatin-derived enals and 1,3-dicarbonyl compounds has been developed, enabling enantioselective synthesis of synthetically important spirooxindole δ-lactones.

Spirooxindoles are present in a wide variety of natural products and biologically active molecules.¹ Polycyclic spirooxindole scaffold with multiple stereocenters are particularly intriguing targets in organic synthesis owing to their structural complexity and potential pharmaceutical value.² For example, the spirooxindole δ-lactone constitutes the backbone of many natural products, such as trigolutes A–C (Fig. 1a).³ However, the catalytic asymmetric synthesis of this privileged scaffold in a highly stereocontrolled fashion is still a challenging task, and very limited protocols were established to this end.^3c,4 In this regard, the development of new, practical and efficient strategy for the highly enantioselective synthesis of spirooxindole δ-lactone scaffold is still in great demand.Open in a separate windowFig. 1(a) Examples of spirooxindole-containing natural products. (b) Assembly of spirooxindole δ-lactone scaffolds via C₁-symmetric NHC-catalysed spiroannulation (this work).Over the past decades, the ability of chiral N-heterocyclic carbenes (NHCs)⁵ toward activation of aldehyde and activated carboxylic acid derivatives through formation of transient acyl anion,⁶ enolate,⁷ homoenolate⁸ and acyl azolium⁹ intermediates was well-investigated for enantioselective C–C, C–O and C–N bond formations. Within the context, the oxidative NHC catalysis strategy enabled generation of acyl azolium from aldehydes via in situ oxidation, which provided new opportunities for reaction design and selectivity control, thus remarkably extending the scope of NHC catalysis.^10,11 However, despite fruitful achievements in the oxidative annulation of simple unsaturated aldehydes,¹² oxidative NHC catalysed addition/cyclization of β,β-disubstituted enals, such as isatin-derived enals, was a very attractive yet challenging topic with the concomitant formation of a congested quaternary center.¹³ Recently, we disclosed that C₁-symmetric biaryl-saturated imidazolium catalyst was a superior NHC for asymmetric oxidative annulation of α-aryl-substituted α,β-unsaturated aldehydes.¹⁴ With our ongoing interest in the development of practical methods toward asymmetric synthesis of chiral spirooxindole scaffolds,^4b,15 we attempted to explore the catalytic performance of C₁-symmetric NHC in oxidative activation of isatin-derived enals.¹⁶ The in situ generated isatin-derived α,β-unsaturated acyl azolium could be trapped by readily available 1,3-dicarbonyl compounds via a spiro-quaternary carbon-forming Michael addition/cyclization process, eventually delivering synthetically intriguing chiral spirooxindole δ-lactones (Fig. 1b).¹⁷Our investigation started with the reaction of isatin-derived α,β-unsaturated aldehyde 1a (a mixture of E and Z isomer) with 1,3-dicarbonyl compound 2a under the catalysis of C₁-symmetric diaryl-saturated imidazolium catalysts I–III.¹⁸ To our delight, the desired oxidative annulation product 3a and its regioisomer 3a'' (6 : 1 rr) were obtained in 23% yield and with high enantioselectivity (89% ee) when catalyst I/DBU combination were employed under the oxidation of 3,3′,5,5′-tetra-tert-butyldiphenoquinone in THF ( Entry^aCatBaseSolventTimeYield^b [%]Rr^cee^d [%]1IDBUTHF1 h236 : 1892IIDBUTHF1 h6710 : 1923IIIDBUTHF1 h657 : 1924IVDBUTHF4 h648 : 1395VDBUTHF10 min532 : 1596VIDBUTHF1 h273 : 167II t-BuOKTHF1 h626 : 1728IICsCO₃THF1 h629 : 1799IINaOAcTHF3 h555 : 15010IIDABCOTHF10 h525 : 18911IIDIPEATHF24 h575 : 18412IINEt₃THF24 h406 : 17713IIDBUToluene1 h7212 : 19114IIDBUCH₃CN1 h14ndnd15IIDBUEt₂O7 h407 : 17916IIDBUDioxane24 h254 : 19317IIDBUDCM1 h4911 : 16518^eIIDBUToluene1 h339 : 19119^fIIDBUToluene1.5 h7712 : 194Open in a separate window^aUnless otherwise mentioned, all reactions were performed using 1a (0.1 mmol) and 2a (0.2 mmol) in solvent (1.0 mL) in the presence of catalyst (20 mol%), base (20 mol%), and oxidant (0.2 mmol) at room temperature.^bCombined yields of 3a and 3a′.^cRr refers to ratio of 3a and 3a′, which is determined by ¹H NMR analysis.^dDetermined by HPLC analysis on a chiral column.^e10 mol% catalyst was used.^f0.1 mmol 1a and 0.1 mmol 2a were used.With the optimized reaction conditions in hand, the generality of the reaction was explored ( Entry^a R ¹/R² (2)3Yield^b [%]Rr^cEe^d [%]1Ph/Me (2a)3a7712 : 19424-BrC₆H₄/Me (2b)3b7514 : 19234-FC₆H₄/Me (2c)3c7814 : 19244-ClC₆H₄/Me (2d)3d6917 : 19354-NO₂C₆H₄/Me (2e)3e5018 : 18464-CF₃C₆H₄/Me (2f)3f6514 : 19074-MeC₆H₄/Me (2g)3g827 : 19584-t-BuC₆H₄/Me (2h)3h745 : 19493-MeOC₆H₄/Me (2i)3i7912 : 194103- MeC₆H₄/Me (2j)3j846 : 194113-BrC₆H₄/Me (2k)3k6916 : 192123-ClC₆H₄/Me (2l)3l7216 : 192133-FC₆H₄/Me (2m)3m7617 : 193143-NO₂C₆H₄/Me (2n)3n4519 : 183152-Thienyl/Me (2o)3o52>25 : 18316Me/Me (2p)3p58—9017^ePh/Me (2a)3q62>20 : 19418^fPh/Me (2a)3r7412 : 19419^gPh/Me (2a)3s528 : 19420Ph/Ph (2q)3t60—96Open in a separate window^aall reactions were performed using 1a (0.1 mmol) and 2 (0.1 mmol) in toluene (1.0 mL) in the presence of catalyst B (20 mol%), DBU (20 mol%), and oxidant (0.2 mmol) at room temperature.^bCombined yields of 3 and 3′.^cDetermined by ¹H NMR analysis.^dDetermined of the major isomer by HPLC analysis on a chiral column.^e1b was used.^f1c was used.^g1d was used.The absolute configuration of the product 3a was assigned to be S by X-ray crystallographic analysis (Cu target, see ESI^†).¹⁹ Based on our experimental results and previous literature reports, a proposed catalytic cycle is shown in Scheme 1. The free NHC catalyst, derived from precatalyst by in situ deprotonation, could react with isatin-derived unsaturated aldehyde 1a, giving rise to Breslow intermediate A, which could be converted to acyl azolium intermediate B through two-electron oxidation and deprotonation. Subsequent Re-face Michael attack of 2a-derived enolate to species B along with the proton transfer sequentially produced intermediates C and D. Intramolecular cyclization of D gave the product 3a and released the NHC catalyst to next catalytic cycle. Mechanistically, the formation of byproduct 3a′ arose from intramolecular annulation of intermediate D′, which was generated from Michael adduct Cvia undesired competing proton transfer step.²⁰Open in a separate windowScheme 1Proposed catalytic cycle.     

A novel binary flooding system of a biobased surfactant and hydrophobically associating polymer with ultralow interfacial tensions

The alkali free surfactant–polymer flooding system with ultralow interfacial tension is a challenge in enhanced oil recovery at present. A novel alkali free binary flooding system of a biobased zwitterionic surfactant and hydrophobically associating polymer with ultralow interfacial tension at a low surfactant dosage was studied in this paper.

A novel alkali free binary flooding system of a biobased zwitterionic surfactant and hydrophobically associating polymer with ultralow interfacial tension at a low surfactant dosage was studied in this paper.

Ternary alkali-surfactant–polymer (ASP) flooding, as one of the representative chemical methods, has been successfully applied in enhanced oil recovery (EOR) in last few decades.^1–4 However, the alkali in the ASP system simultaneously resulted in the reduction of reservoir permeability due to the dispersion and migration of clay and the alkaline scaling in formation.⁵ Alkali-free binary surfactant–polymer (SP) flooding with ultralow interfacial tension (≤10⁻³ mN m⁻¹) is a promising approach among tertiary oil recovery methods. It has received much attention from both the scientific and industrial communities since two-thirds of crude oil were still trapped in oil reservoirs after primary and secondary oil recovery.^1,6 Over the past few years, SP flooding has been conducted on modest scales and significantly improved EOR efficiency in Daqing Oilfield⁷ and Shengli Oilfield,⁸ China. In the absence of alkali, however, the most traditional surfactant–polymer flooding systems could not reach the required ultralow interfacial tension, and may consequently fail in EOR.^2,9,10In the present work, we developed an alkali free binary flooding system consisting of the biobased zwitterionic surfactant and hydrophobically associating polymer with ultralow interfacial tension. Zwitterionic surfactants usually have lower critical micelle concentration (CMC),¹¹ better water solubility¹² and satisfied interfacial tensions.¹³ The biobased zwitterionic surfactant (N-phenyloctade-canoicamidopropyl-N,N-dimethyl-carboxylbetaine, POAPMB) synthesized using renewable feedstock in our laboratory demonstrated excellent surface/interfacial activity and environmental biodegradability, and its CMC was 5.58 × 10⁻⁶ mol L⁻¹.¹⁴ The hydrophobically associating polymer (AP-P3) was synthesized in the laboratory as described in published literature.⁵ The structures of POAPMB and hydrophobically associating polymer were illustrated in Fig. 1. The interfacial tension (IFT), viscosity and phase behavior of the biobased zwitterionic surfactant/hydrophobically associating polymer binary system were investigated, which is, to the best of our knowledge, the first report about the SP flooding of alkali free biobased zwitterionic surfactant and hydrophobically associating polymer flooding system.Open in a separate windowFig. 1Structures of POAPMB and hydrophobically associating polymer.Unless otherwise specified, the POAPMB/AP-P3 system configured by Daqing simulated formation water (downhole temperature 45 °C) consisted of 0.50 g L⁻¹ POAPMB (∼0.95 mM) and 1.50 g L⁻¹ AP-P3 in the experiment. The interfacial tensions were measured by a spinning drop interfacial tensiometer at 45 °C. The IFT between simulated formation water and Daqing crude oil (the acid value: 0.06 mg KOH per g) was 9.9 mN m⁻¹ at 45 °C. The viscosity of POAPMB/AP-P3 system was measured by a rheometer at 45 °C.Dynamic interfacial tensions between Daqing crude oil and different concentrations of surfactant solutions alone and in the presence of 1.50 g L⁻¹ AP-P3 were showed in Fig. 2a and b (ESI Fig. 1a and b^†). The ultralow IFT could be achieved at a concentration as low as 0.05 g L⁻¹ (Fig. 2a and ESI Fig. 1a^†), which is fifty times lower than that of the sugar-based anionic-nonionic surfactant¹⁵ (ESI Table 11^†). Most of alkylbenzene sulfonates¹⁶ and propyl sulfobetaine¹⁷ can not achieve ultralow IFT without alkali.Open in a separate windowFig. 2The IFT between crude oil and different concentrations of POAPMB solutions (a) and in the presence of 1.50 g L⁻¹ AP-P3 (b) (n = 3).For the binary system POAPMB/AP-P3 (1.50 g L⁻¹) solution, the ultralow IFT was reached when the POAPMB concentrations ranged from 0.50 g L⁻¹ to 3.00 g L⁻¹ (Fig. 2b and ESI Fig. 1b^†), which implies that the POAPMB/AP-P3 system is promisingly applicable in tertiary oil recovery. The addition of the polymer increased minimum transient IFT (IFT_min), and it is possibly due to the formation of mixed micelle-like associations on the interface led to decline of interfacial free surfactant molecules and the lose adsorption.^6,9 In addition, it took more time to achieve the balance of IFT in the presence of the polymer. Compared with nonionic surfactant¹⁰ (ESI Table 11^†), POAPMB/AP-P3 binary system can effectively reduce interfacial tensions.In addition to the ultralow IFT, the performances of anti-dilution, stability, adsorption resistance, temperature adaptability, and salt tolerance also play an important role in SP flooding technology. For the anti-dilution, POAPMB/AP-P3 system achieved ultralow IFT when the solution was diluted to one eighth (†). For 3.00 g L⁻¹ POAPMB solution with 1.50 g L⁻¹ AP-P3, ultralow IFT could be achieved even if it was diluted to one fortieth (ESI Table 2^†).The minimum IFTs and the equilibrium IFTs between crude oil and POAPMB/AP-P3 system diluted (n = 3)

A straightforward synthesis of phenyl boronic acid (PBA) containing BODIPY dyes: new functional and modular fluorescent tools for the tethering of the glycan domain of antibodies

We report here on the efficient and straightforward synthesis of a series of modular and functional PBA-BODIPY dyes 1–4. They are an outstanding example of the efficient merge of the versatility of the 3,5-dichloro-BODIPY derivatives and the receptor-like ability of the PBA moiety. The potential bioanalytical applicability of these tools was assessed by measuring the binding to glycan chains of antibodies by a Quartz Crystal Microbalance (QCM).

PBA-BODIPY dyes as functional and modular fluorescent probes for the tethering of the glycan domain of mAbs.

High performance organic fluorophores (e.g. large molar extinction coefficient, high quantum yield, high photo-stability, large Stokes shift) represent a booming research topic, with a wide range of applications ranging from materials to life sciences.^1,2 In this framework, modular and functional probes have become highly sought-after and researchers'' attention is manly focused on the fine tuning of optical properties and new functionalities for highly versatile tethering. However, despite the huge amount of effort in this field, only few fluorophores meet all these theoretical claims.³ Moreover, the availability of convenient and few steps synthetic protocols associated with a good overall yield is one of the major bottlenecks that needs to be addressed for ensuring the successful wide applicability of these probes.⁴ A brilliant example of a versatile fluorophore is the UV-absorbing dye 4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene, known as BODIPY,⁵ which displays high quantum yield, tunable photo-physical properties and excellent photo-stability.^5,6 Strategic structural modifications of BODIPY''s molecular architecture offer an unparalleled opportunity to tune its spectroscopic features, and diverse successful functional and bioactive BODIPY-like probes have been proposed and some commercialized, so far.⁷ 3,5-Dichloro-BODIPY dyes are one of the most recent synthetic analogues whose potential versatility has been demonstrated by Dehaen and Boens.⁸ They showed that appropriate substituents at 3,5 positions of the pyrrole shifted the excitation/emission bands of the corresponding substituted BODIPY derivatives. Thus, the 3,5-dichloro-BODIPY dyes give access, by nucleophilic substitution, to a variety of symmetrical and non-symmetrical BODIPY with several applications including labeling, sensing, energy transfer cassette.^1e,8,9 Then, 3,5-dichloro-BODIPY derivatives have been directly employed for the selective detection, in in vitro models, of sulphur containing metabolites.¹⁰ Recently, the valuable properties of other BODIPY dyes have been used for saccharides detection by phenyl boronic acid (PBA) moiety introduced at the meso position of the BODIPY core.¹¹ Notably, the dynamic covalent interaction between boronic acid and saccharides has been studied since the pioneering work of Lorand et al.¹² and PBA ability to bind 1,2 and 1,3-cis-diols motifs of carbohydrates has been used for the development of synthetic ‘boron-lectins’,¹³ and lately for the fishing of glycoproteins from complex mixtures, for the site-oriented immobilization of antibodies and for biorthogonal conjugations.¹⁴ However, the few examples of PBA-BODIPY probes have been reported to date¹¹ and they are confined to 3,5-dimethyl-BODIPY derivatives that require specific protocols for the further functionalization.In this context, we report here a straightforward synthetic route to obtain the functional and modular PBA-containing dye 1 and the subsequent late-stage diversification of the architecture of 1. Therefore, we obtained a small family of PBA-BODIPY derivatives with a ‘traffic light’ emission range (Fig. 1) for which we have studied the optical properties in different solvents and in physiological media. Thus, the present approach aims at the outstanding merging of the versatility of the 3,5-dichloro-BODIPY dyes with the presence of a functional PBA at the meso position of the BODIPY core. Finally, we show an example of the formation of the boronate esters between the PBA-BODIPY 4 and the glycan chains of an antibody, i.e. anti-streptavidin monoclonal antibody, by using Quartz Crystal Microbalance.Open in a separate windowFig. 1(A) Structure of PBA-BODIPY derivatives 1–4. (B) Solution (1.0 mg mL⁻¹ in MeOH) of PBA-BODIPY derivatives (from the left to the right PDA-BODIPY 1, 4, 2, 3) (a) under white light and (b) under UV-light (λ_ex = 364 nm).The BODIPY-core is traditionally accessible following a few steps synthetic strategy,^8a,15 however, the main concerns are related to the low overall yields of some synthetic steps and to the small scale availability of BODIPY derivatives due to the unstable nature of pyrrole derivatives and/or of some synthetic intermediates.^15a,16 In this paper, the boron dipyrromethane dye 1 (Scheme 1) was prepared following the synthetic strategy usually reported for the synthesis of 3,5-dichloro-BODIPY dyes¹⁵ by using the commercially available pyrrole-based derivatives and p-substituted benzaldehyde as starting materials.Open in a separate windowScheme 1Synthesis of the PBA-BODIPY 1–4. (a) CF₃COOH, 0 °C → r.t., 15 minutes, 74%; (b) NCS, THF, −78 °C → −20 °C, 18 h; (c) DDQ, CH₂Cl₂, r.t., 16 h, 75% over two steps; (d) BF₃Et₂O, CH₂Cl₂, r.t., 2 h, 75%; (e) aniline, CH₂Cl₂, 60 °C, 48 h, 58%; (f) aniline, 140 °C, 0.5 h; (g) ethylendiamine, r.t., 45 minutes, 44%.At first, the meso-PBA substitute dipyrromethane 5 was prepared by acid-catalyzed condensation^8a,15a,b of commercially available 4-formylbenzeneboronic acid 6 with neat excess (25 eq.) pyrrole (Scheme 1). In general, the purification of dipyrromethane derivatives is not trivial,^15a,b,16 depending on substituents at the meso position, as previously reported.^8a,15b A mixture of side products, have been identified and fully characterized,^15b and some improvements on the purification steps have been reported^15a,b switching from flash chromatography to a bulb-to-bulb distillation followed by recrystallization protocols (yield 27–68%).^15b Here, we set up, a straightforward crystallization protocol for the isolation of the pure dipyrromethane 5 (74%) avoiding the low yielding flash chromatography and tedious bulb-to-bulb distillations elsewhere described.^15b We have also examined the effect related to the workup of the reaction media (pyrrole : 6, 25 : 1 ratio, catalytic TFA) on the yield of this synthetic step, by using diverse protocols to refine the formation of 5, observing an higher yield (74% vs. 50%) and an easier removal of side products,^15b by the addition of trimethylamine (see ESI^†) to neutralize the TFA, and the subsequent concentration to dryness of the reaction mixture. Notably, the use of pure 5 is critical for the overall yield of the two following synthetic steps. The chlorination of 5 (Scheme 1) with N-chlorosuccinimide (NCS) followed by oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) afforded the 3,5-disubstututed dipyrromethene 7 (75% over two steps). Finally, 7 was reacted with boron trifluoride diethyl etherate (BF₃–OEt₂) and N,N′-diisopropylethylamine (DIPEA) to give the green emitting PBA-BODIPY 1 (75%). Dye 1 is amenable to structural modifications at the 3,5 positions of the pyrrole moiety so we prepared a small family of dyes which showed the spectral shift in the absorption and emission bands depending on the substitution pattern (Scheme 1). To demonstrate the versatility of PBA-BODIPY 1 we selected aniline as nucleophile already studied in this kind of reactions.^9e,f Thus, the PBA-BODIPY 1 was reacted with an excess of aniline in dichloromethane to give PBA-BODIPY 2 (58%) as pink solid, according to earlier investigations showing that one of the two chlorine atoms is more prone to be involved in the nucleophilic substitution.^8,9 Therefore, the di-substitute PBA-BODIPY 3 has been obtained from 3,5-dichloro-BODIPY 1 by using more stringent experimental conditions (neat excess of aniline, 140 °C) as previous reported.^9f Finally, mono-substitute PBA-BODIPY 4 was prepared in order to have a functional group on the BODIPY-core useful for further functionalization of the dye for the following studies. Thus, 1 was reacted with a neat excess of 1,2-diaminoethane to afford the yellow emitting PBA-BODIPY 4 (44%).The spectroscopic properties of the PBA-BODIPY dyes 1–4 (Fig. 2, and S1–S11^†) were studied in methanol, dichloromethane, water and physiological PBS buffer (pH = 7.4). The spectra reported in the left side of Fig. 2, show the absorption pattern of known PBA-BODIPY derivatives 1–4.^8,9,17 In all tested media, the main absorption band attributed to the 0–0 band of the strong S₀ → S₁ transition is located around 510 nm for PBA-BODIPY 1 and 2 (Fig. 2A, and C) and 590 nm for PBA-BODIPY 3 (Fig. 2E). For PBA-BODIPY 4 (Fig. 2G) the S₀ → S₁ transition is not the main absorption and is observed around 490 nm. The data are summarized in Fig. 2, the normalized fluorescence spectra of PBA-BODIPY 1–4 are also reported. A reduced solvatochromic effect is observed as both the S₀ → S₁ absorption and the emission bands are slightly red-shifted in dichloromethane than in more polar media (e.g. MeOH or H₂O). The broader full width at half maximum as well as the shape of the absorption and fluorescence bands of asymmetric PBA-BODIPY dyes 2 and 4 are consistent with previously reported amino-containing dyes and they were attributed to the diverse contributes of the proposed Lewis structural formulas.^9b The molar extinction coefficient (ε) was assessed in the four different solvents, showing that the highest hyperchromic effect is observed in MeOH (†). Finally, the Stokes shift values result moderately low for the symmetric derivatives 1 and 3, while higher value are found for dyes 2 and 4, where symmetry is reduced. The S₁ → S₀ fluorescence quantum yields (Φ_f) of 1–4 have been measured in MeOH using Rhodamine 6G as standard reference for 1, 2 and 4 and DODCl for 3 (see ESI^†) in the same solvent. High values of Φ_f have been found for 1 and 3, 0.16 and 0.22 respectively, suggesting that the symmetric presence of bulky substituents on the indacene moiety can stiffen the molecule, promoting radiative decay processes towards the ground state. Moreover, the relatively small Stokes shift observed for both compounds point at a molecular structure that is not much affected by the electronic excitation. On the opposite, the large Stokes shifts measured for the dyes 2 and 4, indicate a non negligible molecular structure variations going from S₀ to S₁. The less rigid skeleton of these dyes can endorse thermal relaxation, lowering the respective Φ_f, as shown in Open in a separate windowFig. 2Normalized absorbance and fluorescence spectra of PBA-BODIPY 1–4 in MeOH (black line) physiological PBS buffer pH = 7.4 (green line), H₂O (blue line) and CH₂Cl₂ (red line). (A and B) PBA-BODIPY 1; (C and D) PBA-BODIPY 2; (E and F) PBA-BODIPY 3; (G and H) PBA-BODIPY 4.Absorption and fluorescence emission spectral data of PBA-BODIPY 1–4

Luminescent bis(benzo[d]thiazolyl)quinoxaline: facile synthesis,nucleic acid and protein BSA interaction,live-cell imaging,biopharmaceutical research and cancer theranostic application

A series of quinoxaline-2-hydroxyphenylbenzothiazole scaffolds were synthesized and characterized using NMR, UV, fluorescence spectroscopy and LCMS. These newly synthesized compounds were found to be cytotoxic in human epithelioid cervix carcinoma (HeLa) and human colon cancer cell lines (Caco-2). Selectivity of the compounds 7e and 7g are more than 9 fold higher in Caco-2 cells with respect to the normal cell line HEK-293. The most fluorescent compound 7e has displayed high cytoselectivity, significant cellular uptake in HeLa cells and strong binding efficacy with DNA and BSA. The most potent compound 7g has primarily classified as BCS class 4 and BDDCS class 4.

A series of luminescent bis(benzo[d]thiazolyl)quinoxalines have been synthesized and their fluorescence properties, anticancer potency, DNA and BSA interactions, cellular uptake, and metabolic stabilities are investigated.

Cancer is a leading cause of morbidity and mortality worldwide. Since it is intricate in nature it requires a multi-step process for diagnosis and treatment.¹ This impediment can be sorted out by theranostic drugs by which diagnosis and therapy are combined and implemented in a single agent. This facilitates the treatment as well as real-time monitoring of cells.^2,3 Heterocyclic drugs have voluminous biomedical applications.⁴ The drugs that are available for cancer treatment consist of one or more heterocyclic rings. Quinoxalines are a significant class among the heterocyclic drugs. They have attracted attention in pharmacology due to their wide scope in biological applications including as a well-known DNA intercalator.⁵ Quinoxaline is the key chemical component of many antibiotics such as echinomycin, bleomycin, actinomycin and clofazimine which are known to inhibit several Gram-positive bacteria and are also active towards various tumours.^6–8 Various anticancer drugs that incorporate quinoxaline as the core moiety had exhibited activity against solid tumours and are applicable in clinical trials.^9,10Similarly, benzothiazole possesses anticancer, antimicrobial, antidiabetic, anticonvulsant, anti-inflammatory, antiviral and anti-tubercular activities.¹¹ Benzothiazole conjugated compounds exhibited high potency in various cancer cell lines.¹² These compounds exhibited as a chelating metal ion. It chelates metal ion that is present in amyloids and it also arrests the accumulation of metals in amyloid fibrils.^13,14 Extended derivations of benzothiazoles are viable and it is applicable for cancer theranostics. For example, [Gd(DO3A-BTA)(H₂O)] a theranostic agent is employed in tumour specificity, confirmed by tracking MR images in cytosol and nuclei of MDA-MB-231, MCF-7, and SK-HEP-1 cancer cell lines.¹⁵ Recently our group has reported novel benzothiazole–quinoline conjugates as cancer theranostics.¹⁶ Furthermore, this type of conjugates is also developed as a novel human A₃ receptor antagonist.¹⁷ Moreover, benzothiazole conjugation with quinolones was applicable in sensing of Hg²⁺ in HeLa cells by fluorescence turnover.¹⁸ Novel fluorescent conjugates of 2-(benzothiazole-2-yl)-phenol (BTP) were also used in the selective detection of superoxide anions by ESIPT.¹⁹ Several benzothiazole conjugates were also reported in H₂O₂-responsive detection via theranostic probe for detection of endothelial injury.²⁰ Nowadays, scientists are more interested to design organic molecules which specifically target the proteins or nucleic acids like DNA to understand the drug metabolism, absorption, excretion and distribution and for getting a better perception of DNA–protein interactions.^21–24 In view of the significance of quinoxaline and benzothiazole in drug discovery, we have intrigued our attention on developing a novel pharmacophore in a single domain for cancer theranostic application (Fig. 1). Herein, we have developed a series of substituted quinoxaline in conjugation with well-known fluorophore 2-hydroxyphenylbenzothiazole by adopting an efficient methodology and their in vitro studies like DNA, BSA binding, cytotoxicity study, permeability, stability, and solubility study were done in detail.Open in a separate windowFig. 1Design of bis(benzo[d]thiazolyl)quinoxaline scaffold.Initially, an equal mole of 2-aminothiophenol (1) and 2-hydroxybenzaldehyde (2) were dissolved in ethanol and an adequate amount of silica gel was added to the mixture to prepare the slurry. The slurry was then air dried and subjected to a microwave oven at 490 watt (120 °C) for 15 min. The reaction was monitored by TLC using hexane/ethyl acetate (3 : 1) solvent system. After completion of the reaction, ethyl acetate was added to the solid support and the product was recovered by filtering the solution from the resin by Whatman filter paper. The solution is then transferred to the beaker and air dried. Subsequently, the solvent was reduced gradually and white needle-like crystals of benzothiazolylphenol (BTP) (3) was obtained with high yield (Scheme 1). Compound 3 was fully characterized by ¹H-NMR and ¹³C-NMR spectroscopy. The characteristic singlet OH peak at downfield around δ 12.56 ppm and aryl CH protons at δ 6.9–8.0 ppm was observed in ¹H-NMR.¹⁶ Similarly, another precursor quinoxaline dibromides (6a–g) were synthesized by dissolving the equivalent amount of 1,4-dibromobutane-2,3-dione (5) and phenylene-1,2-diamines (4a–g) in DCM stirring for 2 h at ambient temperature. The formation of the products with high yield was confirmed by TLC using hexane/ethyl acetate (3 : 1) solvent system. The solvent was evaporated and square shaped crystals of quinoxaline dibromide were obtained with high yield. The structures were confirmed by ¹H-NMR spectroscopy. In ¹H NMR the characteristic singlet methylene protons of compounds (6a–g) were observed in the upfield region at δ 4.9 ppm. The aromatic peaks of the quinoxaline ring were obtained at δ 7.7–8.0 ppm range. When electronegative halogen groups are substituted in quinoxaline ring at R₁ and R₂ position those peaks are shifted towards the more downfield region. To synthesize the quinoxaline–BTP conjugates (7a–g) quinoxaline derivatives and BTP (1 : 2 ratio) were dissolved in acetone followed by the slurry preparation using basic alumina. It is then air dried and subjected to microwave at 490 watts (120 °C) for 30 min. The progress of the reaction was monitored eventually by TLC using hexane/ethyl acetate (3 : 1) solvent system. After completion of the reaction, the compound was recovered by adding ethyl acetate to it and the alumina is filtered off using Whatman filter paper. The fine needle like crystals of compound 7a–7g was obtained by slow evaporation of ethylacetate (Scheme 1).Open in a separate windowScheme 1Synthetic scheme for bis(benzo[d]thiazolyl)quinoxaline analogues.The formation of these products was further confirmed by ¹H NMR, ¹³C-NMR and mass spectra. The characteristic singlet peak of aliphatic CH₂ protons was observed in the range of δ 5–6 ppm. Undoubtedly, the singlet peak of –OH was not found in the ¹H NMR. In ¹³C NMR the characteristic CH₂ peak is observed at δ 71 ppm. In the mass spectra, the characteristic (M + H)⁺ peaks of the compounds (7a–g) were observed which corresponded their molecular mass. The halogen groups containing compounds (7b, 7c, 7e, 7f) exhibited characteristic isotopic patterns in their mass spectra.To know the absorption and emission behaviour of these synthesized compounds (7a–g), a standard concentration (3 × 10⁻⁶ mol l⁻¹) in water has been used. In UV-vis spectra, λ_max of all these compounds are observed at 320–330 nm due to π–π* transition (Fig. S1a^†). The corresponding emission peaks are observed at 400–500 nm (Fig. S1b^†). The emission of quantum yield (Φ) was calculated for all of the compounds (7a–g) using eqn (1) (Table S1^†). We found that compound 7d and 7e exhibited the highest quantum yields (0.04) among all the other scaffolds.The stability of compounds in water, GSH, and MTT condition (10% DMSO in PBS buffer) was studied. Water is a key factor because it widens the application in the biological field. Mostly in cancer cells, GSH level are found higher and hence there are chances for cleavage of the compounds. The stability of complex 7e in water (pH 7.2) and GSH were investigated by UV-vis spectroscopy over a period of 20 h of time (Fig. S2a and b^†). There were no changes in absorbance and λ_max was observed with time which designated the stability of compound 7e in water and GSH. Likewise, in MTT condition, an insignificant change of absorbance and λ_max correspond to the stability of this compound even after 18 h (Fig. S2c^†).DNA is one of the key target for cancer therapy. The drug molecules bind with the DNA by three major modes (i) electrostatic interactions with the DNA double helix (ii) binding to grooves of DNA (iii) intercalation between the base pairs of DNA.²⁵ The titration in UV was performed by increasing concentration of DNA from 10–60 μM into a fixed drug concentration (20 μM). The intra-ligand absorption bands around 290–350 nm (π–π* transition) were used in observation of the interaction of DNA and compound 7e (Fig. S3a^†). There was a hypochromic shift with a slight blue shift in the wavelength of compound 7e was observed which concluded that complex 7e bind to DNA through intercalative binding mode.²⁶ Intrinsic binding constant K_b was calculated to be 6.9 × 10³ M⁻¹ using the eqn (2) and Fig. S3b.^† Ethidium bromide (EtBr) displacement assay is commonly used as a diagnostic technique to identify the intercalation ability of small molecules with DNA. Ethidium bromide, a nonspecific intercalator, gives a strong fluorescence while intercalating DNA base pairs. This enhanced fluorescence can be quenched by another chemical moiety which can competitively replace EtBr and binds to the same site.²⁷ A solution containing DNA bound ethidium bromide displayed intense fluorescence at λ 608 nm when excited at 485 nm. The original fluorescence intensity of EtBr–DNA complex is rapidly quenched by increasing the concentration of compound 7e (Fig. S3c^†). Stern–Volmer quenching constant (K_sv), apparent binding constant (K_app) and high intrinsic binding constant (K_b) for compound 7e were tabulated (†) which indicate the intercalative binding manner of this compound with DNA.DNA-binding parameter for compound 7e with CT-DNA

One-pot synthesis of α-aminophosphonates by yttrium-catalyzed Birum–Oleksyszyn reaction

For the first time, yttrium triflate was used as an efficient green catalyst for the synthesis of α-aminophosphonates through a one-pot three-component Birum–Oleksyszyn reaction. Under the action of this Lewis acid, enhancement of the yield and reaction chemoselectivity was provided by the achievement of an appropriate balance in the complex network of reactions.

For the first time, yttrium triflate was used as an efficient green catalyst for the synthesis of α-aminophosphonates through a one-pot three-component Birum–Oleksyszyn reaction.

Multicomponent reactions are commonly used to achieve molecular complexity. In particular, one-pot three-component reactions have been exploited for the synthesis of α-aminophosphonates. These organophosphorus compounds have attracted the attention of medicinal chemists due to their similarity to α-amino acids. They find application in agriculture as plant growth regulators¹ and herbicides,² in medicinal chemistry as antibacterial,^3,4 antiviral⁵ and antitumor agents,⁶ activity-based probes,⁷ and building blocks for peptides and proteins.⁸ Since the first preparation of α-aminophosphonates, reported in 1952 by Fields,⁹ various methods for their synthesis have been proposed.^10–16 Nowadays, one-pot three-component condensation of aldehyde, amine, and phosphite, catalyzed by an excess of acetic acid, is the most common method due to its simplicity and, in general, high yields of products.⁴ Application of Lewis acids as the catalyst instead of Brønsted acids was first reported in 1973 by Birum.¹⁷ The advantage of Lewis acids is their compatibility with acid-sensitive functional groups that, under typical conditions (glacial acetic acid) would be degraded.Condensation of aldehyde, carbamate, and triaryl phosphite has been named as Birum–Oleksyszyn reaction.¹⁸ Recently, a new biologically active α-aminophosphonate (UAMC-00050) was developed at the University of Antwerp.^19–21 This diarylphosphonate shows good inhibitory activity against urokinase plasminogen activator (uPA), an enzyme involved in several physiological processes, such as tissue remodeling.²² uPA can be also involved in the development of different diseases, for example, thrombolytic disorder,²³ cancer,²⁴ and eye diseases.²⁵ UAMC-00050 is currently under investigation for the treatment of irritable bowel syndrome²⁶ and dry eye disease.²¹ The key step of the synthesis of UAMC-00050, proposed by Joossens et al.,^19,21 involves Birum–Oleksyszyn reaction, catalyzed by Cu(OTf)₂ in acetonitrile, which, on a small scale, provides 20% yield of the product (Scheme 1). In current work, a new catalyst has been introduced for the Birum–Oleksyszyn reaction. For the first time, we report the use of yttrium salt in a one-pot three-component synthesis of α-aminophosphonates, which provides a remarkable improvement of the yield of product for the key step in the synthesis of UAMC-00050. The scope of the protocol has been demonstrated on a variety of aldehydes, phosphites, and carbamates.Open in a separate windowScheme 1Conditions for the preparation of intermediate 4 in the synthesis of UAMC-00050 (5).In the frame of the dry eye disease drug development (IT-DED3) project,²⁷ we have worked toward upscaling of the synthetic route and providing larger amounts of UAMC-00050 for more detailed research of its biological activity.During our attempts to upscale the key step to 3.0 g scale, we noticed a considerable decrease of the yield of product from 20% to 11%. In our view, the poor outcome of the process cannot be attributed to the Birum–Oleksyszyn reaction alone. The nature of substituents in the target molecule affects the overall efficiency of the transformation through both the main and a number of side reactions (Scheme 2). This three-component reaction involves unstable paracetamol phosphite 1, aliphatic aldehyde 2 bearing Boc-protected amino group, and benzyl carbamate 3 (Scheme 1). In general, aliphatic aldehydes are less reactive in Birum–Oleksyszyn reaction, which consequently requires a stronger catalyst or longer reaction time to achieve satisfactory yield of product.^15,28 The imine generated from the condensation of carbamate and aldehyde is highly reactive and can add a second molecule of carbamate forming aminal 7.²⁹ Compound 7 can participate in Arbuzov-type reactions,³⁰ if, as hypothesized, it is converted into reactive cation 13 by Brønsted²⁹ or Lewis acid.³¹ However, Lewis acid-catalyzed reactions of aminal 7 or its analogs and triaryl phosphites are not known.Open in a separate windowScheme 2Birum–Oleksyszyn reaction as the key step and possible side reactions in the synthesis of UAMC-00050.The presence of paracetamol moiety complicates the synthesis due to the lower stability of its phosphorus esters both in starting triaryl phosphite and in the formed α-aminophosphonate. In the reaction environment, they readily hydrolyze with one equivalent of water formed in the condensation of aldehyde 2 and the carbamate 3 generating diarylphosphite 9 and monoaryl side product 10.As previously reported, product 4 is stable in the presence of water.²¹ However, under the reaction conditions, hydrolysis proceeds with the help of the Lewis acid.³² With prolonged reaction time (i.e., 24 h) we noticed a decrease in the yield of product 4 almost in half compared to the yield obtained in the reaction performed for 4 h and formation of acid 10 as the major side product.Application of N-Boc-protected starting material is also challenging, since, in the presence of a Lewis acid catalyst, partial removal of the Boc group takes place and unprotected amino aldehyde 11 forms black-brown polymer 12 (Scheme 2).Improving the overall efficiency of the synthesis of α-aminophosphonate 4 requires simultaneous promotion of imine formation and Birum–Oleksyszyn reaction and suppression of unwanted hydrolysis and Boc group removal. In search for optimal conditions, we initiated screening of various acidic catalysts using equimolar ratio (1 : 1 : 1) of aldehyde 2, carbamate 3, and phosphite 1 with 10 mol% load of the catalyst in MeCN performing the reaction at room temperature for 4 h.¹⁹ Acetonitrile proved to be an optimal solvent for the preparation of 4, it is polar enough to dissolve all the starting materials, it is not a concern for the environment, like DCM, and it does not hydrolyze the product like protic solvents: MeOH, EtOH and H₂O.In EntryCatalystYield of product 4, %^bRatio 4/10Selectivity, %^c1AcOH^d00 : 10002TiCl₄844 : 66443ZrCl₄881 : 19814Cu(OTf)₂1176 : 24765BiCl₃1374 : 26746TfOH1382 : 18827Mg(OTf)₂1450 : 50508FeCl₃1568 : 32689LiOTf1585 : 158510Sc(OTf)₃1681 : 198111Et₂O·BF₃1676 : 247612Bi(NO₃)₃·5H₂O1976 : 247613Yb(OTf)₃2067 : 336714SnCl₄2257 : 435715ZnCl₂2281 : 198116La(OTf)₃2569 : 316917Bi(OTf)₃3176 : 247618AcOH3554 : 465419Y(OTf)₃4280 : 208020Y(OTf)₃^e1784 : 168421Y(OTf)₃^f3166 : 3466Open in a separate window^aReaction conditions: 1.0 equiv. of aldehyde 2, 1.0 equiv. of phosphite 1, 1.0 equiv. of benzyl carbamate 3 and 10 mol% of Lewis acid, anhydrous MeCN, under argon, RT, 4 h.^bIsolated yield.^cSelectivity expressed as a percent ratio of compounds 4 and 10.^dAcOH as a solvent.^e1.0 equiv. trimethyl orthoformate (TMOF).^f1.5 g of 4 Å MS.Although the yield of product 4 was low (13%), TfOH provided a much better stability of compound 4 toward the hydrolysis (82% of it survived vs. only 18% of the hydrolyzed 10). Lewis acids like TiCl₄, Cu(OTf)₂, ZnCl₂, and FeCl₃ are commonly used in three-component synthesis of α-aminophosphonates.^18,33–35 Triflate salts were included in the list because of their enhanced stability in the presence of water generated after the condensation of aldehyde 2 and carbamate 3.^36,37As reported in literature, phosphodiesters can be hydrolyzed by Lewis acids.^32,38 Paracetamol-containing phosphorus esters are much more sensitive to the presence of water compared to their alkyl analogs. Therefore, analysis of the catalyst efficiency can be carried out by using an additional parameter – selectivity for the formation of the target diester 4 compared to the proportion of the hydrolyzed monoester side product 10. This parameter is expressed as a percentage ratio of compounds 4 and 10, obtained from HPLC-UV assay with internal standard. Diagram with yield of product 4 on the horizontal axis and selectivity of the formation of compound 4 on the vertical axis is shown on Fig. 1.Open in a separate windowFig. 1Yield of product 4 (horizontal axis, percent) and selectivity of the formation of diaryl product 4 compared to monoaryl product 10 (vertical axis, percent ratio).For example, Lewis acids like TiCl₄, Mg(OTf)₂, and SnCl₄ hydrolyzed up to a half of diester 4 comprising the group with the lowest overall efficiency. On the other hand, LiOTf provided the best compatibility with diester 4 and the lowest degree of hydrolysis (85% of diester 4 preserved), but only a moderate yield of 15%. The two elements of group 1 and group 2 in catalysts LiOTf and Mg(OTf)₂ both provided low yield of product 4 (15% and 14%, respectively) but different selectivity, quite high for lithium (85%) and moderate for magnesium (50%).Three different salts of bismuth were tested, for all of which similar selectivity was registered (74–76%). However, quite large difference in yields of compound 4 was observed – 13% for BiCl₃, 19% for Bi(NO₃)₃·5H₂O, and 31% for Bi(OTf)₃. Eight triflate salts were screened and provided a wide range of yield and selectivity. Cu(OTf)₂ was the most ineffective in terms of yield, while Mg(OTf)₂ was the least selective toward the formation of product 4. Among four chloride salts, group 4 elements in catalysts TiCl₄ and ZrCl₄ ensured the same low yield of product 4 (8%) but differed in selectivity two-fold (44 and 81%, respectively). At the same time, SnCl₄ and ZnCl₂ afforded product 4 in three times higher yield (22%), but selectivity of SnCl₄ was much lower (57 vs. 81% for ZnCl₂). The poor performance of TiCl₄ and SnCl₄ can be explained by their hydrolysis with liberation of HCl under the reaction conditions. FeCl₃ was reported as an excellent catalyst for the synthesis of α-aminophosphonates from alkylphosphites,³⁹ but, in the case of triaryl phosphite 1, it was able to provide product 4 in only 15% yield with 68% selectivity.In addition, it was found that strong Lewis acids (e.g., TiCl₄ and BF₃) generate a large amount of a brown-black side product (polymer 12) formed by the removal of Boc protecting group and subsequent reaction of amine and aldehyde (Scheme 2).Less strong Lewis acids (e.g., Mg(OTf)₂ and LiOTf) generated significantly less polymer 12, however, most of aldehyde 2 was left unreacted providing poor conversion of the starting materials.An interesting comparison can be made between elements of group 3 – Y(OTf)₃ provided the highest yield (42%) and good selectivity (80%) of diaryl product 4, Sc(OTf)₃ showed similar selectivity (81%) but afforded much lower yield (16%) of product 4. Yb(OTf)₃ and La(OTf)₃ are located in the middle of Fig. 1, since they ensured formation of product 4 in 20 and 25% yield and with selectivity of 67 and 69%, respectively.The group of best-performing catalysts includes ZnCl₂, La(OTf)₃, Bi(OTf)₃, and Y(OTf)₃. On the scale of 3 g of target compound 4, the best performance was achieved with Y(OTf)₃, which provided the highest yield (42%), almost 4 times higher than in the case of Cu(OTf)₂ (11%). The stability of the paracetamol diester group was also one of the highest – only 20% of diester was hydrolyzed.This catalyst was used for elucidation of the behavior of aminal 7 in the reaction system. Y(OTf)₃-catalyzed reaction of aldehyde 2 and 2.0 equiv. of carbamate 3 after 2 h provided compound 7, which proved to be bench-stable and easily isolable. Aminal 7 was then reacted with tri(paracetamol) phosphite 1 under standard conditions in the presence of Y(OTf)₃. After 4 h, HPLC-UV assay with an internal standard showed only half amount of phosphonate 4 compared to that obtained in three-component Birum–Oleksyszyn reaction.According to these data, it can be concluded that aminal 7 can react with phosphite 1 but is twice less reactive than imine 6. Therefore, one-pot three-component reaction with in situ formation of imine is a preferable strategy for the synthesis of aryl α-aminophosphonates. Dehydrating agents like 4 Å MS and trimethyl orthoformate (TMOF) were not able to increase the yield over the standard protocol. A small increase in selectivity from 80% to 84% was noted when TMOF was used but with a much lower yield of 4 (17%).With the optimized conditions in hand, the efficiency of Y(OTf)₃ catalyst in the Birum–Oleksyszyn reaction was investigated using various aldehydes, carbamates, and aryl phosphites (Fig. 2). The highest yields of products were obtained for activated phosphites, for example, tris(p-methoxyphenyl) phosphite and the tris(p-acetamidophenyl) phosphite, combined with benzyl carbamate 3 and aromatic aldehydes (18, 22). The electron-donating substituent in phosphite facilitates the nucleophilic attack of phosphorus atom on the imine. Aliphatic aldehydes represented by t-butyl [4-(2-oxoethyl)phenyl]carbamate and 2-phenylacetaldehyde afforded lower yields (38–48%) of products (4, 29, 33) compared to the yields (72–92%) of products derived from benzaldehyde (14, 15, 17, 18). Among aromatic aldehydes, interesting results were obtained demonstrating how different halogen substituents in the para-position affect the yield of the respective product: 4-chlorobenzaldehyde (21) and 4-bromobenzaldehyde (26) provided higher yields (55 and 54%, respectively) compared to 4-fluorobenzaldehyde (20, 36%) and 4-iodobenzaldehyde (27, 25%). For product 27 the poor yield might be caused by its diminished solubility of starting aldehyde in MeCN. Compared to the standard conditions (application of AcOH) of Birum–Oleksyszyn reaction, the selected Lewis acid catalyst allows to perform the synthesis of α-aminophosphonates using acid-labile compounds, as demonstrated by the examples with t-butyl carbamate (34–36) and Boc-protected amine (4, 33). While yields of products 34 and 35 (43 and 41%, 34 and 35) were lower compared to those obtained for CbzNH₂ analogs (82 and 64%, 14 and 19), there were no elevated amounts of hydrolysis products. The lower conversion of starting materials might be explained by the higher steric hindrance of the tert-butyl group.¹⁸ Similarly, tri(o-tolyl)phosphite provided lower yields of products (34 and 26%, 16 and 25) when compared to the analogous reaction with triphenyl phosphite (82 and 55%, 14 and 21) and tri(p-tolyl)phosphite (75 and 43%, 15 and 24). These differences in yields also might be attributed to the increased steric hindrance near the phosphorus atom, which obstructs the nucleophilic attack of phosphite toward imine.Open in a separate windowFig. 2Scope of Birum–Oleksyszyn reaction catalyzed by Y(OTf)3 in anhydrous MeCN under argon. All the %yield presented is isolated yield.In general, the developed catalytic conditions using Y(OTf)₃ are well compatible with a variety of functional groups in both aldehyde and phosphite. When comparing series with benzaldehyde (15, 17, and 18) and 4-chlorobenzaldehyde (22, 23, and 24), the hydrolyzed side product was detected only in case of product 17 and 22 testifying the lower stability of the paracetamol ester.     

Nanoporous hybrid CuO/ZnO/carbon papers used as ultrasensitive non-enzymatic electrochemical sensors

In this research, we demonstrate a facile approach for the synthesis of a graphite-analogous layer-by-layer heterostructured CuO/ZnO/carbon paper using a graphene oxide paper as a sacrificial template. Cu²⁺ and Zn²⁺ were inserted into the interlayer of graphene oxide papers via physical absorption and electrostatic effects and then, the Mⁿ⁺-graphene oxide paper was annealed in air to generate 2D nanoporous CuO/ZnO nanosheets. Due to the graphene oxide template, the structure of the obtained CuO/ZnO nanosheets with an average size of ∼50 nm was duplicated from the graphene oxide paper, which displayed a layer-by-layer structure on the microscale. The papers composed of nanosheets had an average pore size of ∼10 nm. Moreover, the as-prepared CuO–ZnO papers displayed high hybridization on the nanoscale. More importantly, the thickness of the single-layer CuO/ZnO nanosheet was about 2 nm (3–4 layer atom thickness). The as-synthesized nano-hybrid material with a high specific surface area and conjunct bimodal pores could play key roles for providing a shorter diffusion path and rapid electrolyte transport, which could further facilitate electrochemical reactions by providing more active sites. As an electrode material, it displayed high performances as a non-enzymatic sensor for the detection of glucose with a low potential (0.3 V vs. SCE), high sensitivity (3.85 mA mM⁻¹ cm⁻²), wide linear range (5 μM to 3.325 mM), and low detection limit of 0.5 μM.

In this research, we demonstrate a facile approach for the synthesis of a graphite-analogous layer-by-layer heterostructured CuO/ZnO/carbon paper using a graphene oxide paper as a sacrificial template.

CuO/ZnO-based multifunctional nanohybrid materials are widely used in various applications, such as sensors,^1–5 photo degradation,^6,7 catalysis,⁸ energy storage,⁹ memory applications,¹⁰ and especially non-enzymatic sensors for the detection of glucose.^13,14 The heterostructured ZnO–CuO composite material has been extensively used for non-enzymatic sensing applications due to the extension of its electron depletion layer through the formation of a p–n junction.^11,12 Therefore, a number of studies have been devoted to the synthesis of CuO–ZnO hybrid materials,^12–18 such as three-dimensional porous ZnO–CuO hierarchical nanocomposites fabricated by coelectrospinning¹⁹ and flower-like CuO–ZnO heterostructured nanowire arrays on a mesh substrate obtained using a solution method.²⁰ Among them, 0D nanoparticles or 1D nanowires have been dominant for a long period. Recently, ultrathin two-dimensional (2D) nanostructured materials have attracted increasing attention due to their large surface-to-volume ratios and confined thickness on the nanometer scale and tunable nanoporous structure. They are expected to exhibit high performances for achieving superior catalytic, photovoltaic and electrochemical applications. Thus, 2D metal oxide (MO) nanostructures have been widely demonstrated in adsorption, catalysis, energy conversion and storage, and optoelectronic and biological applications.²¹ However, it is still very challenging to prepare large-scale 2D ultrathin and porous structured nanofilms via a simple pathway. To date, only a few cases have been successful in the synthesis of ultrathin 2D metal²² and metal oxide nanosheets^23,24 using graphene oxide (GO) as a template. Recently, our group used a GO paper as a template to obtain an ultralight single MO paper with tunable thickness and transparency.²⁵A graphene paper or film derived from various graphene-like materials such as GO, reduced graphene oxide (rGO) and pristine graphene has captured significant interest in a wide range of areas from physics,²⁶ materials science,²⁷ and chemistry²⁸ to environmental engineering.²⁹ In particular, the GO paper is widely used since it is prepared via assembling single-layer GO nanosheets, which are functionalized by abundant oxygen-containing groups and many defects on both their planes and edges. As a result, GO paper can capture ions and nanoparticles or covalently bind with other functional groups.³⁰ GO paper also displays a well-defined layer-by-layer porous nanostructure, with a d-spacing of about 1.2 nm, which allows ions to diffuse into the nanometer-scale pores of their interlayers.³¹ Most importantly, GO paper can be easily decomposed and removed by application of high temperature in an oxygen-rich atmosphere. Consequently, GO paper is one of the most desirable sacrificial templates for the synthesis of layer-by-layer, nanoporous and ultrathin 2D metal or metal oxide nanosheets.In this work, GO paper was prepared via the vacuum filtration of GO solution and was further used as a template for the synthesis of 2D CuO/ZnO/carbon paper on a large scale. Cu²⁺ and Zn²⁺ were loaded on the GO paper via electrostatic interaction and physical absorption between the oxygen groups and the desired cation. After removal of GO by heat, the metal oxide maintained the layer-by-layer, ultrathin and nanoporous structure. More interestingly, the as-prepared ZnO–CuO hybrid films displayed significantly improved performances as a non-enzymatic sensor for the detection of glucose.GO nanosheets was synthesized via a modified Hummer''s method according to our previous work.^32,33 Subsequently, some advanced techniques were applied to characterize the obtained GO, such as UV-vis spectroscopy (Fig. S1^†), AFM (Fig. S2^†) and XPS (Fig. S3A^†). All the results indicate a significant amount of oxygen-containing groups is present on the GO sheet surface, which significantly increased the sites for attaching positively charged molecular groups and nanoparticles on the GO sheet. In our case, Cu²⁺ and Zn²⁺ were successfully attached on the GO paper via the direct immersion of GO paper into the ion source solution, which was confirmed by XPS analysis (Fig. S4^†). In this process,²⁵ the metal ions were incorporated into the GO paper. After heat treatment, the obtained MO papers (as shown in the insert picture in Fig. 2, S5 and S6^†) mimicked the GO paper structure and the sample colour changed to white, black and black, respectively for ZnO/carbon, CuO/carbon, and CuO/ZnO/carbon.Open in a separate windowFig. 2SEM image of (A) CuO/ZnO/carbon and TEM images of (B and C) CuO/ZnO with different magnifications. The inset image of (A) is the picture of the CuO/ZnO/carbon paper, and the insert image of (C) is the diffraction pattern of the CuO/ZnO paper. (D) Detailed tapping mode AFM height images of CuO/ZnO and (E) line profile. The line profile was taken from the red lines marked in (D).The as-synthesized MO films were examined first by powder X-ray diffraction (Fig. 1). In the XRD pattern of ZnO, the definite line broadening of the XRD peaks indicates that the prepared material consisted of particles in the nanoscale range. From the XRD patterns analysis, we determined the peak intensity, position and width, and full-width at half-maximum (FWHM). The diffraction peaks located at 2θ = 31.7°, 34.4°, and 36.3° correspond to the (100), (002), and (101) planes, respectively, exhibiting the characteristic diffractions of a mixed-valence compound with a face-centred cubic structure (PDF #36-1451). The XRD pattern of CuO shows peaks at 2θ = 35.4°, 38.7°, and 48.7°, corresponding to the (002), (111), and (202) planes, respectively. All the diffraction peak can be readily indexed to the standard monoclinic phase (PDF #48-1548) without characteristic peaks assigned to possible impurities such as Cu₂O or Cu(OH)₂. In the case of CuO/ZnO, it displayed the diffraction peaks of both CuO and ZnO. It should be noted, in the XRD patterns of all the MO paper, the diffraction peck located at 2θ = 19.8° is attributed to the undestroyed graphene. XPS was also used to confirm that the CuO/ZnO hybrid materials were obtained (see Fig. S5^†). The binding energies of Cu 2p_3/2, Zn 2p_3/2, and O 1 s were identified at 933.63, 1021.9, and 529.67 eV, respectively. The high-resolution Cu and Zn scans indicate that both Cu and Zn were in the +2 oxidation state. The XPS measurements verify that the structures consist of CuO and ZnO, and furthermore, the atom ratio of Cu to Zn is about 1 : 1, suggesting that each cation has equal opportunity to diffuse in the interlayer GO paper. It should be mentioned that the carbon that appeared in Fig. S5A^† comes from the undestroyed graphene paper template.Open in a separate windowFig. 1XRD patterns of 2D nanoporous CuO/carbon, ZnO/carbon and hybrid CuO/ZnO/carbon papers.In general, MO is synthesized via chemical methods, where the shapes of most products are uncontrolled, and some further ligands are needed to prevent aggregation. Due to the fast grown process and no limit in growth direction, it is difficult to the control morphology of metal oxide. In our case, MO displayed ultrathin 2D nanosheets, with the size of about 20 nm and 50 nm corresponding to ZnO and CuO, respectively, according to TEM images shown in Fig. 2 and S6–S8.^† More interestingly, the TEM images suggest that the MO nanosheets connected each other, resulting in the formation of a continuous nanoporous network structure (the porous size ranged from about 5 nm to 10 nm). There are probable two reasons for the assembled nanoporous structure, one is that the MO nanosheets connected each other during the growth process, and the other is GO has lots defects and pores, which affected the growth of the MO nanosheets. As we expected, the BET surface area of the as-obtained CuO/ZnO/carbon hybrid material was estimated to be 80 m² g⁻¹ (Fig. S8^†), which was contributed by both the porous CuO/ZnO and undestroyed graphene, and the average pore size of ∼10 nm. The high-resolution TEM image of CuO/ZnO in Fig. S8^† shows lattice fringes with an interplanar spacing of 0.25 nm and 0.28 nm corresponding to the CuO 002 lattice face and ZnO 100 lattice face, respectively. The selected area electron diffraction (SAED) pattern in the inset in Fig. 2C is in good agreement with the XRD pattern and illustrates the polycrystalline structure of CuO/ZnO. The thickness of the nanosheets was measured via tapping mode AFM to be about 3 nm and 10 nm (Fig. 2D and S10^†) for ZnO and CuO, respectively. Furthermore, from the SEM images (Fig. 2A and B) of CuO/ZnO, the layer-by-layer structure is clearly seen on the microscale, which is similar with our previous report.²⁵ Moreover, the density of the 2D nanoporous CuO/ZnO nanosheet was calculated to be about 50 mg cm⁻², which is 10 times lighter than commercial CuO nanoparticles.To investigate the elemental of Cu and Zn distribution in the 2D nanoporous CuO/ZnO/carbon hybrid nanosheets, SEM and mapping analysis were performed for a single MO sheet. The SEM mapping images of the C, O, Cu and Zn elements are shown in Fig. 3. All the atoms were distributed uniformly in the nanocomposite, which suggests that a highly hybridized CuO/ZnO film was obtained. Both the high-resolution TEM image and FFT (Fig. S7C and D,^† respectively) indicate that the single CuO and ZnO nanosheets connected with each other on the nanoscale, which will provide huge chances for the continuous formation of p–n junction interfaces on the nanoscale, which is beneficial for facile electron transfer.Open in a separate windowFig. 3EDX mapping images of a single 2D nanoporous CuO/ZnO/carbon nanosheet. (A) Superimposed EDS image, (B)–(E) images of the elemental distributions, and (F) corresponding TEM image. Scale bar: 2 μm.More importantly, this type of layer-by-layer nanoporous CuO/ZnO/carbon nanosheet has a high surface area with conjunct bimodal pores, which can play key roles in providing shorter diffusion paths and rapid electrolyte transport, while providing more active sites for electrochemical reactions. The accurate detection of glucose is important for clinical diagnostics in diabetes control, analytical applications in biotechnology, and the food industry. Enzyme-based amperometric glucose biosensors can satisfy the requirements of clinical blood glucose level measurement. Glucose oxidase (GOx)-modified electrodes are the most common class of amperometric biosensors for glucose detection because GOx enables the catalytic oxidation of glucose with high sensitivity and selectivity. However, enzyme-modified electrodes have some disadvantages. For instance, the instability of the electrode and unsatisfactory reproducibility, complicated immobilization procedures, and expensive enzymes are easily deactivated. Thus, to address these issues, the development of non-enzymatic sensors with high sensitivity and stability, and interference-free glucose determination is very important. According to the XRD and XPS results, graphene was not completely removed, and thus graphene could still exist as a film coated with metal oxide surface or edge, which will be beneficial for electron transfer. Accordingly, the 2D CuO/ZnO nanosheets may exhibit good electrochemical performances as non-enzymatic sensors for the detection of glucose.Owing to their high specific area, 2D nanoporous nanosheets are promising materials for the fabrication of non-enzymatic glucose sensors. The cyclic voltammetry profiles of the 2D nanoporous CuO, ZnO and CuO/ZnO hybrid nanosheet-modified GCE electrodes in 40 mL 0.1 M KOH solution with and without 1 mM glucose were studied (Fig. 4). Obvious reduction peaks at around 0.6 V (vs. SCE) can be observed in the blank NaOH solution, which corresponds to the Cu(ii)/Cu(iii) redox couple according to previously reported studies (eqn (1)) (except the pure ZnO nanosheet electrode). After the injection of glucose, the Cu(iii) ion obtains an electron and acts as an electron delivery system, and electrons are transferred from glucose to the electrode, which leads to an increase in the peak current, and the reaction process is demonstrated in eqn (2). Comparing the CV without and with 1 mM glucose, the oxidation current dramatically increased, and the results suggest a kinetic limitation in the reaction between the redox sites of the CuO and CuO/ZnO modified-electrodes and glucose. Initially, the reaction potential was as low as 0.3 V (vs. SCE).CuO + OH⁻ → CuO(OH) + e⁻1CuO(OH) + glucose → CuO + gluconolactone + H₂O2Open in a separate windowFig. 4CVs of the (A) ZnO–CuO/carbon and (B) CuO/carbon papers measured in 0.1 M KOH. The dashed line and solid line indicate the electrolyte without and with 1 mM glucose, respectively. Scan rate 50 mV s⁻¹.To evaluate the electrochemical performance for the analysis of glucose, the non-enzymatic amperometric detection of glucose was performed at 0.6 V (vs. SCE) with the successive addition of glucose. Fig. 5A and B show the I–t curves of the different 2D porous ZnO, CuO and CuO/ZnO nanosheet-modified GCE electrodes in 0.1 M NaOH solution upon the successive addition of different concentrations of glucose. Both CuO and CuO/ZnO showed the typical steady-step response with very low noise, and remarkable increase in the oxidation current, which show the electrocatalytic activity of the modified electrodes towards the oxide of glucose. A linear relationship between the glucose concentration and catalytic current was found in the range of 5 μM to 3.325 mM at the CuO/ZnO/carbon-modified electrode (Fig. 5C), with a detection limit of 0.5 μM. The linear regression equation with a high correlation coefficient (R) of 0.998 was obtained, and the sensitivity was estimated to be 3850 μA cm⁻² mM⁻¹, which is over 3-fold higher than that for CuO (≈1000 μA cm⁻² mM⁻¹). It is understood that the formation of a p–n junction in the CuO/ZnO heterostructures results in the extension of the space charge region, which locally narrows the conducting channel for the charge carriers in ZnO, thus making the p–n junction more sensitive to glucose-induced charge transfer. The application of CuO/ZnO as a glucose sensor involves two types of sensing behaviours: adsorption-induced surface depletion and chemical conversion with a change in the electrical potential. It is also notable that the active area of CuO increased in the heterostructures.Open in a separate windowFig. 5Quantitative detection of glucose and selectivity analysis. (A) Full-range amperometric responses to the successive addition of glucose for three types of MO papers, (B) enlarged amperometric curves in the low-concentration region, (C) calibration curves, and (D) selectivity analysis by adding possible interfering species, while the successive addition of glucose was performed. Electrolyte 0.1 M KOH, and working electrode potential fixed at 0.6 V vs. (SCE).Since the 2D nanoporous CuO/ZnO acts as glucose oxidase, the electrochemical version of the Michaelis–Menten equation (eqn (3)) was used to evaluate the Michaelis constant (K_m).3where j_cat is the catalyst current obtained using the I–t curve, S is the glucose concentration, A is the surface area, n is number of electrons transferred, F is the Faraday constant, and K_cat is the apparent catalytic turnover rate. Considering n, F, A and K_cat as constants, eqn (3) could be simplified to eqn (4).4As shown in Fig. S10,^† the I–t curve could be fitted using eqn (4) with R = 0.999 and K_m is 5.4 mM. The apparent K_m is relatively low, close to that of a GOD-based sensor,³³ which suggests that the 2D CuO/ZnO/carbon displays good biocompatibility and has a high biological affinity to glucose.To further verify its sensing performance, some electrochemical characteristic tests were performed on the 2D porous CuO/ZnO/carbon nanosheet-based electrode. First, the reproducibility of the 2D porous CuO/ZnO nanosheet for nonenzymatic glucose sensing was evaluated. Three modified electrodes were prepared, and the relative standard deviation (RSD) was no more than 8.7% for the current response, indicating satisfactory electrode-to-electrode reproducibility. Additionally, the recovery was 97% with an RSD of 3.5% (S/N = 3) for 25 μM of glucose, demonstrating good intra-electrode reproducibility. To explore the specific detection of glucose in a real sample using the proposed approach based on the 2D nanoporous CuO/ZnO nanosheet-modified electrode, the I–t curve of the 2D CuO/ZnO nanosheet-modified GCE electrode was measured in 0.1 M NaOH solution upon the successive addition of 25 μM of glucose. Furthermore, the electrochemical response of common interfering species for 2D porous CuO/ZnO nanosheet-modified GCE electrode was examined, as shown in Fig. 5D. Some common interfering species in human serum, such as 250 μM uric acid (UA), ascorbic acid (AA), dopamine acid (DA), nicotinamide adenine dinucleotide (NADH), Mg²⁺, K⁺, Na⁺ and Ca²⁺, were chosen as the interfering species. As is clearly seen in Fig. 5D, the current responses of the interfering species has little effect compared to glucose, which indicated that 10 times higher concentration of interfering species than glucose has no response for the detection 25 μM glucose. Here, the good selectivity of the 2D nanoporous CuO/ZnO nanosheet against some reducing compounds, such as AA, can be ascribed to the obvious repulsion with the negatively charged CuO and ZnO (the isoelectric point of both is about 9.5) and the negatively charged AA (deprotonated) under highly basic conditions. The good selectivity of the 2D porous CuO/ZnO/carbon nanosheet electrode for glucose and against other interfering materials can be ascribed to the synergetic action of the well-designed 2D porous structure and high surface area, which provides the maximum number of active free paths to the glucose molecules and facilitates faster electron transfer. The results obtained with the proposed method were compared with other methods for the detection of glucose (Detection methodMaterialSensitivity (μA mM⁻¹ cm⁻²)LOD (mM)Linear range (mM)ReferenceElectrochemical biosensorsCuO PN22990.41 × 10⁻³0.005–0.825 34 Ni–SnOx/PANI/CuO13250.1301–10 35 CuO@C/D16500.5 × 10⁻³0.5 × 10⁻³ to 4 36 CuO-NPs1098.370.59 × 10⁻³5 × 10⁻³ to 0.6 37 CuO/GO/GCE262.520.69 × 10⁻³2.79 × 10⁻³ to 2.03 38 Nafion/GOx/ZnO/FcC11SH/Au0.0278—0.05–1.0 39 ZnO–CuO HNCs3066.40.21 × 10⁻³0.47 × 10⁻³ to 1.6 19 CuO–ZnO NRs/FTO2961.80.4 × 10⁻³0.001–8.45 40 ZnO–CuO core–shell spheres1217.41.677 × 10⁻³0.02–4.86 13 Plasmon-aided biosensorsHalf-rough Au/NiAu MNAs4831 × 10⁻³0.005–31 41 Photoelectrochemical sensorAu–NiO_1−x HNAs17771 × 10⁻³0.005–7 42 Photoelectrochemical sensorBiVO₄—0.13 × 10⁻³0–5 43 Electrochemical sensorsHybrid CuO/ZnO/carbon papers38500.5 × 10⁻³5 × 10⁻³ to 3.325This workOpen in a separate window