The authors greatly appreciate the financial support from the National Natural Science Foundation of China (No. 21708051 to X.C.).

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Chemicals and solvents used were of reagent grade and were used without further purification. All reactions involving air or moisture-sensitive reagents or intermediates were performed under an argon atmosphere.
1. Preparation of &#945;-Arylidiene Pyrazolinone Species
<ol>
	<li>Preparation of pyrazolones
	<ol>
		<li>Add 40 mL of glacial acetic acid to a 250 mL round-bottom flask from a graduated cylinder at room temperature. Stir the solution while adding hydrazine (1 equivalent, 1.58 mol/L) and methyl acetoacetate (1 equivalent, 1.58 mol/L). Equip the flask with a reflux condenser. 
		NOTE: This concentration is used because a lower concentration leads to a slower reaction rate.</li>
		<li>Heat the reaction flask to 120 &#176;C in an oil bath while stirring for 3 h. After cooling the reaction flask down to ambient temperature, remove the magnetic stir bar, using a stir bar retriever. Concentrate the reaction mixture, using a rotary evaporator at 60 &#176;C. Avoid spilling the reaction mixture because of negative pressure.</li>
		<li>Add 20 mL of deionized water to the reaction flask and transfer the solution into a separatory funnel. Extract the aqueous layer 3x with ethyl acetate (30 mL). Combine the organic layers in the separatory funnel and wash them 2x with brine (50 mL).</li>
		<li>Dry the combined organic layers over anhydrous sodium sulfate for 1 h and, then, remove the sodium sulfate by gravity filtration.</li>
		<li>Remove the solvent on a rotary evaporator at reduced pressure and at 35 &#176;C.</li>
		<li>After removing all the solvent, apply the pyrazolone species when performing section 4.</li>
	</ol>
	</li>
	<li>Preparation of &#945;-Arylidiene Pyrazolinones
	<ol>
		<li>Add pyrazolone (1 equivalent, 0.49 mol/L), benzaldehyde (1 equivalent, 0.49 mol/L), magnesium oxide (0.5 g, 0.6 equivalent), and a magnetic stir bar into an oven-dried 100 mL round-bottom flask under N2 atmosphere.</li>
		<li>Add anhydrous acetonitrile (40 mL) to the reaction flask, using an airtight syringe, and then, equip the flask with a reflux condenser. Heat the reaction flask to 120 &#176;C in an oil bath while stirring for 12 h.</li>
		<li>Monitor the progress of the reaction by thin layer chromatography (TLC), using petroleum ether:ethyl acetate (2:1 [v/v], retention factor Rf = 0.86) as an eluent.</li>
		<li>After the complete consumption of pyrazolone, cool the reaction flask down to room temperature. Filter off the magnesium oxide through a Celite plug.</li>
		<li>Remove the excess acetonitrile by using a rotary evaporator under reduced pressure and at 35 &#176;C. Purify the residue by column chromatography on silica gel eluting with petroleum ether:ethyl acetate (10:1 to 8:1 [v/v]) to provide the crude product.</li>
		<li>Add the crude product into a 100 mL Erlenmeyer flask equipped with a magnetic stir bar, and then, add a minimum volume of 95% ethanol. Place the flask on a hot plate and bring it to a gentle boil until the entire solid is just dissolved. Take the flask off the hot plate and cool it slowly without any agitation. 
		NOTE: When the mixture is cooled to room temperature, the corresponding &#945;-arylidiene pyrazolinone is formed as pure crystals.</li>
	</ol>
	</li>
</ol>
2. Synthesis of &#945;-imino &#947;-lactones species
<ol>
	<li>Add &#945;-amino-&#947;-butyrolactone hydrobromide (1 equivalent, 0.41 mol/L), magnesium sulfate (1 equivalent, 0.41 mol/L), triethylamine (1 equivalent, 0.41 mol/L), and a magnetic stir bar into an oven-dried 100 mL round-bottom flask under N2 atmosphere.</li>
	<li>Add 36 mL of anhydrous dichloromethane to the reaction flask, using an airtight syringe. Stir the reaction mixture at room temperature for 1 h. Add the corresponding thiophene-2-carbaldehyde (1.1 equivalent, 0.45 mol/L) to the solution and stir for another 12 h.</li>
	<li>Monitor the progress of the reaction by TLC, using petroleum ether:ethyl acetate (4:1 [v/v]) as an eluent until the complete consumption of the lactone species has occurred, and then, filter off the reaction mixture, using a filter paper with a pore size of 30&#8722;50 &#956;m.</li>
	<li>Add 5 mL of deionized water to the resulting mixture and separate the organic layer from the aqueous phase. Extract the aqueous phase 2x with dichloromethane (30 mL). Combine the organic layers in the separatory funnel and wash them 2x with brine (50 mL).</li>
	<li>Dry the combined organic layers over anhydrous sodium sulfate for 1 h, and then, remove the sodium sulfate by gravity filtration. Remove the solvent on a rotary evaporator at reduced pressure and at 35 &#176;C.</li>
	<li>After removing all the solvent, apply the &#945;-Imino &#947;-lactones species when performing section 4.</li>
</ol>
3. Synthesis of bifunctional squaramide catalyst C519
NOTE: For the synthesis of organocatalysts 5C, see Figure 2.
<ol>
	<li>Preparation of 3-((3,5-bis(trifluoromethyl)phenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (compound 1)
	<ol>
		<li>Add 3,4-dimethoxycyclobut-3-ene-1,2-dione (1 equivalent, 0.63 mol/L), 3,5-bis(trifluoromethyl)aniline (1.1 equivalent, 0.69 mol/L), 20 mL of methanol, and a magnetic stir bar into an oven-dried 100 mL round-bottom flask under N2 atmosphere.</li>
		<li>Stir the mixture at room temperature for 48 h. The formation of yellow precipitate is an indication that the reaction is taking place.</li>
		<li>Filter the reaction solution through a funnel fitted with filter paper and wash the solid product 3x with methanol (15 mL). Dry the yellow solid in vacuo overnight to afford the final products as yellow solid.</li>
	</ol>
	</li>
	<li>Synthesis of catalyst C5
	<ol>
		<li>Add 3-((3,5-bis(trifluoromethyl)phenyl)amino)-4-methoxycyclobut-3-ene-1,2-dione (compound 1; 1 equivalent, 0.2 mol/L) and (S)-(6-methoxyquinolin-4-yl)((1S,2R,4S,5R)-5-vinylquinuclidin-2-yl)methanamine (compound 2; 1 equivalent, 0.2 mol/L) and a magnetic stir bar into a 25 mL round-bottom flask under N2 atmosphere.</li>
		<li>Add anhydrous dichloromethane (5 mL), using an airtight syringe. Stir the mixture at room temperature for 48 h.</li>
		<li>Monitor the progress of the reaction by TLC, using dichloromethane:methanol (10:1 [v/v], Rf = 0.49) as an eluent. After the reaction is completed, concentrate the reaction mixture, using a rotary evaporator at 40 &#176;C.</li>
		<li>Purify the residue by column chromatography on silica gel eluting with dichloromethane:methanol (20:1 [v/v]) to provide the desired product.</li>
	</ol>
	</li>
</ol>
4. Asymmetric synthesis of bispirocyclic compounds
<ol>
	<li>Dry a 50 mL round-bottom reaction flask containing a magnetic stir bar. Remove the flask from the oven and cool it to room temperature by blowing on it with inert gas before use.</li>
	<li>Add &#945;-arylidiene pyrazolinone (1 mmol, 1 equivalent, 0.1 mol/L) and &#945;-imino &#947;-lactones (1.2 mmol, 1.2 equivalent, 0.12 mol/L) to the 50 mL round-bottom flask under N2 atmosphere.</li>
	<li>Add anhydrous ethyl ether (10 mL) to the reaction flask, using an airtight syringe. Then, add the corresponding organocatalyst (0.1 equivalent, 0.01 mol/L) to the solution and stir the reaction mixture at 40 &#176;C.</li>
	<li>Monitor the progress of the reaction by TLC, using petroleum ether:ethyl acetate (4:1 [v/v], Rf = 0.51) as an eluent. 
	NOTE: The spots of the starting materials and products were visualized using a hand-held 254 nm UV lamp.</li>
	<li>After the reaction was completed, concentrate the reaction mixture, using a rotary evaporator at 40 &#176;C.</li>
	<li>Purify the residue by column chromatography on silica gel eluting with petroleum ether:ethyl acetate (4:1 [v/v]) to provide the final product.</li>
	<li>Characterize the final product by 1H and 13C NMR spectra, using a 400 MHz NMR spectrometer. Determine the ee values of the product, using a chiral column.</li>
</ol>

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The authors have nothing to disclose.

The successful preparation of bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons is dependent on a number of factors.
The key step of this one-step asymmetric cycloaddition process is the synergistical activation of the &#945;-arylidiene pyrazolinone 1a and cyclic imino ester 2a by the bifunctional squaramide catalyst. It is achieved by the formation of multiple intermolecular hydrogen bonds between catalyst as a hydrogen-bond donor and two reaction substrates. Accordingly, with large steric hindrance, C5, out of all the hydrogen-bond donor bifunctional organocatalysts screened, exhibited the best stereoselectivity. The noted protocol uses 10 mol% catalyst in the model reaction. Besides, the requirement of high solubility of both substrates and catalyst is essential. As a result, the use of Et2O as the optimal solvent not only ensures that both substrates and catalyst are fully dissolved at room temperature but that they undergo a smooth cycloaddition with high yields and stereoselectivities as well. Notably, water in the reaction system would lead to poor stereoselectivity. In order to ensure a successful synthesis, it is critical to check the dryness of all of the reagents and solvent before starting the reaction.
The cycloaddition is compatible with a wide variety of substituted &#945;-arylidiene pyrazolinone. Specifically, substituents with a different aryl group on &#945;-arylidiene are well tolerated. Electron withdrawing aryl groups, due to their increased electrophilicity during 1,3-dipolar cycloaddition, are preferred in terms of yields and stereoselectivity. Also, substrates such as 3-position, replaced by ethyl, decyl, tert-butyl, and benzyl groups, and 1-position, functionalized by different electronic aryl rings, are highly tolerated. Moreover, substituents of the cyclic imino ester moiety on imino lactone with phenyl, thiophenyl, or naphthyl groups are also compatible with the reaction. It is notable that, in order to ensure a successful reaction, a small excess of imino lactone (1.2 equivalent) is required. In most cases, the concentrations of substrates are kept at a 0.1-0.12 mol/L scale in 1 mL of solvent. Depending on the types of substrates and catalysts, the one-step cycloaddition reaction may take 8-72 h at room temperature.
It is worth noting that this cycloaddition provided a high level of stereoselectivity when the R4 substituent of imino lactone 2 was a thiolphenyl, 5-methylthiophenyl, or 2-naphthyl group. However, when the R4 substituent was replaced with other alkyl substituents or heterocyclic substituents, either a low stereoselectivity or a low reaction yield was achieved.
In summary, the presented protocol allows the direct asymmetric construction of bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] using an efficient one-step organocatalytic 1,3-dipolar cycloaddition reaction in excellent yields and a high level of stereoselectivity. Moreover, this new methodology is compatible with two synthons bearing versatile functional groups and should be useful for the synthesis of diverse therapeutic agents with bispirocyclic scaffolds.

Chiral spirocyclic compounds found prevalent in natural products, chiral ligands and organometallic complexes have emerged as attractive synthetic targets due to their structural complexity and biological activity1,2,3. Specifically, bispirocyclic scaffolds, featured by three rings with two rigid spirocenters, are structural subunits in many natural products with important biological activities4,5. Consequently, the construction of compounds with stereocontrolled, optically pure bispirocyclic skeletons has drawn great attention over the last few decades. A large number of spirocyclic compounds and their derivatives have been synthesized successfully through organometallic approaches and organocatalytic approaches, for example, asymmetric cycloadditions such as 1,3-dipolar cycloadditions and Diels-Alder reactions6,7,8. However, these molecules are mostly monospirocyclic structures, while bispirocyclic structures are less reported on and limited to the construction of indole-based bispirocycles.
In order to obtain more structurally diverse bispirocyclic compounds, the versatility of cycloaddition synthons for the asymmetric construction of spirocyclic centers has been explored9,10,11. Especially with bifunctional squaramide organocatalysts, azomethine ylide12,13,14, such as &#945;-imino &#947;-lactones, and dipolarophiles, such as alkylidene pyrazolones15,16,17, are able to undergo a simple 1,3-dipolar cycloaddition to construct bispirocyclic skeletons with multiple stereocenters, making them the perfect spirocyclization synthons (Figure 1). After the optimization of the structure of organocatalyst and reaction solvent, this cycloaddition process efficiently affords the desired product with high yields and excellent enantio- and diastereoselectivity. Moreover, this reaction exhibits a relatively high structural tolerance on a broad scope of cycloaddition synthons with diverse functional groups18. This new method provides an efficient access to a variety of highly functionalized drug-like compounds with two quaternary spirocenters via a simple organocatalytic cycloaddition, shining lights on its application in the structural diversity-oriented synthesis of this intriguing class of compounds.

<table border="0" cellpadding="0" cellspacing="0" style="width:993px;" width="992">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Acetonitrile, anhydrous, 99.9%</td>
			<td style="width:213px;">Innochem (China)</td>
			<td style="width:213px;">A0080</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">&#945;-amino-&#947;-butyrolactone hydrobromide, 98%</td>
			<td style="width:213px;">Alfa Aesar</td>
			<td style="width:213px;">B23148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">3,5-bis(trifluoromethyl)aniline, 98+%</td>
			<td style="width:213px;">Adamas</td>
			<td style="width:213px;">48611B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Dichloromethane, 99.5%</td>
			<td style="width:213px;">Greagent&#160;</td>
			<td style="width:213px;">G81014H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">3,4-dimethoxycyclobut-3-ene-1,2-dione, 98+%</td>
			<td style="width:213px;">Leyan (China)</td>
			<td style="width:213px;">1062550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Ethanol, 99.5%</td>
			<td style="width:213px;">Greagent&#160;</td>
			<td style="width:213px;">G73537B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Ethyl acetate, 99.5%</td>
			<td style="width:213px;">Greagent&#160;</td>
			<td style="width:213px;">G23272L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Ethyl ether,anhydrous,99.5%</td>
			<td style="width:213px;">Greagent</td>
			<td style="width:213px;">G69159B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Ethyl 3-oxobutanoate, 98%</td>
			<td style="width:213px;">TCI</td>
			<td style="width:213px;">A0649</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">4-fluorobenzaldehyde, 98%</td>
			<td style="width:213px;">Innochem (China)</td>
			<td style="width:213px;">A24295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">&#160;Glacial acetic acid, 99.5%</td>
			<td style="width:213px;">Greagent&#160;</td>
			<td style="width:213px;">G73562B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Magnesium oxide, 99+%</td>
			<td style="width:213px;">Alfa Aesar</td>
			<td style="width:213px;">44733</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Magnesium sulfate, 98%</td>
			<td style="width:213px;">Greagent</td>
			<td style="width:213px;">G80872C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Methanol, 99.5%</td>
			<td style="width:213px;">Greagent</td>
			<td style="width:213px;">G75851A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Petroleum ether</td>
			<td style="width:213px;">Greagent&#160;</td>
			<td style="width:213px;">G84208D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Phenylhydrazine, 98%</td>
			<td style="width:213px;">Innochem (China)</td>
			<td style="width:213px;">A57671</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:361px;">(S)-(6-methoxyquinolin-4-yl)((1S,2R,4S,5R)-5-vinylquinuclidin-2-yl)methanamine</td>
			<td style="width:213px;">DAICEL Group</td>
			<td style="width:213px;">111240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Sodium sulfate,anhydrous,99%</td>
			<td style="width:213px;">Greagent</td>
			<td style="width:213px;">G82667A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Thiophene-2-carbaldehyde, 98%</td>
			<td style="width:213px;">J &#38; K scientific (China)</td>
			<td style="width:213px;">124605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Triethylamine, 99%</td>
			<td style="width:213px;">J &#38; k scientific (China)</td>
			<td style="width:213px;">432915</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient construction of drug-like bispirocyclic scaffolds via organocatalytic cycloadditions of &#945;-imino &#947;-lactones and alkylidene pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Various hydrogen-bond donor bifunctional organocatalysts were examined in the presence of organocatalysts in dichloromethane (DCM) at 25 &#176;C (Table 1). The representative synthetic process of organocatalysts is shown in Figure 1. The screening of different organocatalysts (Table 1, entries 1-6) resulted in C5 with excellent stereoselectivity (94% ee, &#62;20:1 d.r., entry 5) and the best yield (85% yield). A further optimization of the solvents (Table 1, entries 7-11) suggested that Et2O was preferable in this synthetic process.
Significantly, to examine the generality of the reaction, a variety of substituents of two spirocyclization synthons with different functional groups were tested successfully using the optimized model reaction conditions, resulting in the desired bispirocycles with good to excellent yields and stereoselectivity. The scope of the pyrazolinone 1a includes a replacement of the phenyl group on &#945;-arylidiene with a wide range of aryl, naphthyl, and thienyl groups, substrates with different substituents such as ethyl, decyl, tert-butyl, and benzyl group at 3-position, and substituents with different electronic properties on the aryl ring at 1-position. Besides, to explore the substrate scope of imino lactone 2a, the cyclic imino ester moiety was substituted with a 5-methylthiophenyl, a phenyl or 2-naphthyl group, providing bispirocycles&#160;with similar yields and considerable stereoselectivities.
The structure of bispirocyclic products was confirmed by 1H and 13C NMR spectroscopy. To characterize the optical purity and stereoselectivity of the stereoisomer products, the ee values were determined using chiral HPLC and the d.r. values were determined by 400 MHz 1H NMR. Reprehensive HPLC characterization results of compound 3e are given in Figure 3. To explore the structural relativity, X-ray crystallography was used to analyze 3e, revealing the absolute configuration of the product 3e as (5S,6R,7R,13R). The single-crystal structure of 3e is shown in Figure 4.&#160;CCDC 1590396 contains the crystallographic data of 3e, which can be obtained free of charge from the Cambridge Crystallographic Data Centre (www.ccdc.cam.ac.uk/data_request/cif).
For example, the characterization data for bispirocyclic product (3e) were as follows: Rf = 0.51 (4:1 [v/v], petroleum ether/EtOAc); 1H NMR (400 MHz, CDCl3) &#948; 7.55 (d, J = 7.2 Hz, 2H), 7.35-7.31 (m, 2H), 7.26-7.22 (m, 2H), 7.19-7.15 (m, 2H), 6.98-6.93 (m, 3H), 6.90-6.88 (m, 1H), 5.03 (d, J = 11.6 Hz, 1H), 4.57 (s, 1H), 4.46-4.40 (m, 1H), 4.09 (td, J = 8.8, 2.0 Hz, 1H), 3.80 (d, J = 11.6 Hz, 1H), 2.67-2.61 (m, 1H), 2.35-2.27 (m, 4H); 13C NMR (100 MHz, CDCl3) &#948; 178.8, 171.9, 163.9, 161.4, 158.5, 137.1, 136.0, 131.3, 131.2, 128.8, 127.0, 125.8, 125.4, 125.0, 119.7, 116.3, 116.1, 70.8, 67.4, 66.0, 64.1, 57.5, 34.6, 13.6; 19F NMR (376 MHz, CDCl3) &#948; 112.8. The ee value was determined by HPLC analysis, hexane/2-propanol 80/20, flow rate = 1.0 mL/min, 254 nm, tr = 8.63 min (major); HRMS (ESI) Calcd for C26H23N3O3SF+ [M+H]+ 476.1439, found 476.1446.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entry</td>
			<td>Catalyst</td>
			<td>Solvent</td>
			<td>Yield[a] (%)</td>
			<td>d.r.[c]</td>
			<td>ee[d] (%)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>C1</td>
			<td>DCM</td>
			<td>81</td>
			<td>&#62;20:1</td>
			<td>94</td>
		</tr>
		<tr>
			<td>2</td>
			<td>C2</td>
			<td>DCM</td>
			<td>82</td>
			<td>12:1</td>
			<td>90</td>
		</tr>
		<tr>
			<td>3</td>
			<td>C3</td>
			<td>DCM</td>
			<td>30</td>
			<td>9:1</td>
			<td>0</td>
		</tr>
		<tr>
			<td>4</td>
			<td>C4</td>
			<td>DCM</td>
			<td>78</td>
			<td>&#62;20:1</td>
			<td>71</td>
		</tr>
		<tr>
			<td>5</td>
			<td>C5</td>
			<td>DCM</td>
			<td>85</td>
			<td>&#62;20:1</td>
			<td>94</td>
		</tr>
		<tr>
			<td>6</td>
			<td>C6</td>
			<td>DCM</td>
			<td>70</td>
			<td>12:1</td>
			<td>93</td>
		</tr>
		<tr>
			<td>7</td>
			<td>C5</td>
			<td>Toluene</td>
			<td>79</td>
			<td>&#62;20:1</td>
			<td>95</td>
		</tr>
		<tr>
			<td>8</td>
			<td>C5</td>
			<td>THF</td>
			<td>73</td>
			<td>15:1</td>
			<td>89</td>
		</tr>
		<tr>
			<td>9</td>
			<td>C5</td>
			<td>CHCl3</td>
			<td>71</td>
			<td>&#62;20:1</td>
			<td>93</td>
		</tr>
		<tr>
			<td>10</td>
			<td>C5</td>
			<td>DCE</td>
			<td>81</td>
			<td>18:1</td>
			<td>91</td>
		</tr>
		<tr>
			<td>11</td>
			<td>C5</td>
			<td>Et2O</td>
			<td>88 (83[b])</td>
			<td>&#62;20:1</td>
			<td>98</td>
		</tr>
		<tr>
			<td colspan="6">[a]&#160;The yields were determined by&#160;1H NMR analysis of crude product using 4-iodoanisole as the internal standard. 
			[b]&#160;Isolated yield. 
			[c]&#160;Ratio is determined by&#160;1H NMR. 
			[d]&#160;Determined by chiral HPLC.</td>
		</tr>
	</tbody>
</table>
Table 1: Optimization of the reaction condition. The table has been modified from Chen et al.18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59155/59155fig1.jpg" /> 
Figure 1: Model reaction between 1a and 2a. Structures of bifunctional organocatalysts (C1-C6) are listed. This figure shows reactions performed with 1a (0.10 mmol), 2a (0.12 mmol), and catalyst (10 mol%) in solvent (1 mL) at room temperature for 8-72 h. For detailed experimental procedures, see the protocol. This figure has been modified from Chen et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59155/59155fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59155/59155fig2.jpg" /> 
Figure 2: Synthesis of organocatalyst 5C. The top panel is the synthesis of compound 1, and the bottom panel is the synthesis of 5C from compound 1 and compound 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59155/59155fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59155/59155fig3.jpg" /> 
Figure 3: HPLC spectra of racemic and chiral product 3e. The top panel is the HPLC spectrum of racemic product 3e, and the bottom panel is the HPLC spectrum of chiral 3e. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59155/59155fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59155/59155fig4.jpg" /> 
Figure 4: Single-crystal structure of 3e. The left structure is the single-crystal structure of 3e, and the right structure is 3e with the stereochemistry of each atom properly designated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59155/59155fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of Î±-Imino Î³-Lactones and Alkylidene Pyrazolones at JoVE.

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

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6.8K Views.  Lanzhou University. Enantiomerically enriched bispiro[γ-butyrolactone-pyrrolidin-4,4'-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Video: Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of α-Imino γ-Lactones and Alkylidene Pyrazolones

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of &#946;-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal &#946;-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (&#8804;1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.

This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

We present here a protocol to construct and validate models for nondestructive prediction of total sugar, total organic acid, and total anthocyanin content in individual blueberries by near-infrared spectroscopy.

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of α-Imino γ-Lactones and Alkylidene Pyrazolones

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