The present work has been supported by Vicerrector&#237;a de Investigaci&#243;n y Estudios de Posgrado from BUAP, Divulgation of Science, and Projects No. REOY-NAT14, 15, 16-G. HEAS-NAT17. RMG thanks CONACyT (Mexico) for scholarship 417887.

1. Reaction
<ol>
	<li>Place in a fume hood an opened round 100 mL flask with 0.5 mol (29.8 mL) glacial acetic acid and add 0.051 mol (5.82 mL) of 3,5-dimethylpyridine and 5 mL of H2O2 (35%). Keep the mixture reaction under constant magnetic stirring, at an inner temperature of 80 &#176;C for 5 h.</li>
	<li>After the reaction time, cool the flask to 24 &#176;C with ice (do not expose the acetic acid gases to the ice), and plug it to a high vacuum distillation unit for 90-120 min to remove excess acetic acid. 
	Caution: Do not use hot material. Wait until the glassware reaches a manageable temperature. This will also avoid vapors entering the top of the distillation unit.</li>
	<li>Add distilled water (10 mL) twice to ensure the removal of any trace of acetic acid and to concentrate the mixture much as possible.</li>
</ol>
2. Basicity Adjustment and Extraction
<ol>
	<li>Dissolve in bi-distillated water the isolated viscous and transparent product and use a potentiometer to adjust the pH to 10 with pure solid Na2CO3.</li>
	<li>Place carefully the solution in a 250 mL separation funnel and extract it 5 times with 250 mL of CHCl3 to improve the yield. Recover the organic layer and dry it over solid Na2SO4 for 30 min maximum, which will contain the product. If necessary, re-extract the aqueous phase with the desired amount of CHCl3. 
	Caution: CHCl3 may cause drowsiness and dizziness; handle with care and inside a fume hood.</li>
	<li>Remove the solvent under reduced pressure with a high vacuum distillation unit, until the formation of a very hygroscopic clear beige crystalline powder (70%).</li>
</ol>
3. Crystallization Process
<ol>
	<li>Dissolve 4.3 g of the crystalline powder in 50 mL of cold high performed liquid chromatography (HPLC) grade diethyl ether. Vacuum filter the solution to remove any trace of solid starting material or even dust. Pour the filtrate into a glass Petri dish, leaving it to slow evaporate at 4 &#176;C in a laboratory fridge.</li>
	<li>Ensure that after two days, clear colorless crystals are obtained. Then measure the melting point, which should be in the range of 310-311 K.</li>
</ol>
4. Analysis of 3,5-Lutidine N-oxide Dehydrate
<ol>
	<li>Remove the crystals that are formed, of prismatic shape and colorless, by decantation from the flask&#39;s walls for further X-ray analysis. If not immediately used, keep the crystals in diethyl ether to avoid crystal hydration.</li>
	<li>Dissolve 0.010 g of 3,5-lutidine N-oxide dehydrate in 0.4 mL of CDCl3 to perform NMR H1 and C13 analysis to prove the effectiveness of the procedure.</li>
</ol>

<ol>
	<li>Ochiai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Japanese+work+on+the+chemistry+of+pyridine+1-oxide+and+related+compounds.">Recent Japanese work on the chemistry of pyridine 1-oxide and related compounds.</a> J. Org. Chem. 18 (5), 534-551 (1953).</li><li>Solomons, T. W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organic Chemistry 2nd Edition. , 1110 (1976).</li><li>Albini, A., Pietra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Heterocyclic N-Oxides. , 328 (1991).</li><li>Koukal, P., Ulc, J., Necas, D., Kotora, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterocyclic+N.-Oxides.">Heterocyclic N.-Oxides.</a> Topics in Heterocyclic Chemistry. 53, 29-58 (2017).</li><li>Wen-Man, Z., Jian-Jun, D., Xu, J., Jun, X., Huan-Jian, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible-Light-Induced+C2+alkylation+of+pyridine+N.-oxides.">Visible-Light-Induced C2 alkylation of pyridine N.-oxides.</a> J. Org. Chem. 82 (4), 2059-2066 (2017).</li><li>Merino Garc&#237;a, M. R., R&#237;os-Merino, F. J., Bern&#232;s, S., Reyes-Ortega, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+3,5-dimethylpyridine+N-oxide+dihydrate.">Crystal structure of 3,5-dimethylpyridine N-oxide dihydrate.</a> Acta Cryst. 72 (12), 1687-1690 (2016).</li><li>Sarma, R., Karmakar, A., Baruah, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Oxides+in+Metal-Containing+Multicomponent+Molecular+Complexes.">N-Oxides in Metal-Containing Multicomponent Molecular Complexes.</a> Inorg. Chem. 47 (3), 763-765 (2008).</li><li>Youssif, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+trends+in+the+chemistry+of+pyridine+N-oxides.">Recent trends in the chemistry of pyridine N-oxides.</a> ARKIVOC. 2001, 242-268 (2001).</li><li>Chucholowski, A. W., Uhlendorf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base+catalyzed+rearrangement+of+5-cyanomethyl-2-isoxazolines;+novel+pathway+for+the+formation+of+2-aminopyridine+N-oxides.">Base catalyzed rearrangement of 5-cyanomethyl-2-isoxazolines; novel pathway for the formation of 2-aminopyridine N-oxides.</a> Tetrahedron Lett. 31 (14), 1949-1952 (1990).</li><li>Thellend, A., Battioni, P., Sanderson, W., Mansuy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+N-Heterocycles+by+H2O2+Catalyzed+by+a+Mn-Porphyrin:+An+Easy+Access+to+N-Oxides+Under+Mild+Conditions.">Oxidation of N-Heterocycles by H2O2 Catalyzed by a Mn-Porphyrin: An Easy Access to N-Oxides Under Mild Conditions.</a> Synthesis. 1997 (12), 1387-1388 (1997).</li><li>Cop&#233;ret, C., Adolfson, H., Tinh-Alfredo, V. K. h., Yudin, A. K., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+Efficient+Method+for+the+Preparation+of+Pyridine+N-Oxides.">A simple and Efficient Method for the Preparation of Pyridine N-Oxides.</a> J. Org. Chem. 63 (5), 1740-1741 (1998).</li><li>Ferrer, M., S&#225;nchez-Baeza, F., Messeguer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+preparation+of+amine+N-oxides+by+using+dioxiranes.">On the preparation of amine N-oxides by using dioxiranes.</a> Tetrahedron. 53 (46), 15877-15888 (1997).</li><li>Adam, W., Briviba, K., Duschek, F., Golsch, D., Kiefer, W., Sies, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+singlet+oxygen+in+the+deoxygenation+of+heteroarene+N-oxides+by+dimethyldioxirane.">Formation of singlet oxygen in the deoxygenation of heteroarene N-oxides by dimethyldioxirane.</a> J. Chem. Soc. Chem. Commun. 1995 (18), 1831-1832 (1995).</li><li>Murray, R. W., Singh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Facile+One-Step+Synthesis+of+C-Arylnitrones+Using+Dimethyldioxirane.">A Facile One-Step Synthesis of C-Arylnitrones Using Dimethyldioxirane.</a> J.Org.Chem. 55 (9), 2954-2957 (1990).</li><li>Kim, S. W., Um, T., Shin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Br&#248;nsted+acid-catalyzed+&#945;-halogenation+of+ynamides+from+halogenated+solvents+and+pyridine-N-oxides.">Br&#248;nsted acid-catalyzed &#945;-halogenation of ynamides from halogenated solvents and pyridine-N-oxides.</a> Chem. Commun. 53 (18), 2733-2736 (2017).</li><li>Campeau, L., Rousseaux, R., Fagnou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+solution+to+the+2-pyridyl+organometallic+cross-coupling+problem:+regioselective+catalytic+direct+arylation+of+pyridine+N-oxides.">A solution to the 2-pyridyl organometallic cross-coupling problem: regioselective catalytic direct arylation of pyridine N-oxides.</a> J. Am. Chem. Soc. 127 (51), 18020-18021 (2005).</li><li>Gang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-free+methylation+of+a+pyridine+N-oxide+C-H+bond+by+using+peroxides.">Metal-free methylation of a pyridine N-oxide C-H bond by using peroxides.</a> Org. Biomol. Chem. 13 (46), 11184-11188 (2015).</li><li>May, D., Nyman, M. J., Hampden-Smith, E. N., Duesler, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+reactivity+of+group+12+metal+thiocarboxylates+M(SOCR)2Lut2[M)+Cd,+Zn;+R+)+CH3,+C(CH3)3;+Lut+)+3,5-Dimethylpyridine+(Lutidine)].">Synthesis, characterization, and reactivity of group 12 metal thiocarboxylates M(SOCR)2Lut2[M) Cd, Zn; R ) CH3, C(CH3)3; Lut ) 3,5-Dimethylpyridine (Lutidine)].</a> Inorg. Chem. 36 (10), 2218-2224 (1997).</li><li>Cho, S. H., Hwang, S. J., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palladium-Catalyzed+C-H+Functionalization+of+Pyridine+N-Oxides:+Highly+Selective+Alkenylation+and+Direct+Arylation+with+Unactivated+Arenes.">Palladium-Catalyzed C-H Functionalization of Pyridine N-Oxides: Highly Selective Alkenylation and Direct Arylation with Unactivated Arenes.</a> J. Am. Chem. Soc. 130 (29), 9254-9256 (2008).</li><li>Ide, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin-crossover+between+high-spin+(S+=+5/2)+and+low-spin+(S+=+1/2)+states+in+six-coordinate+iron(III)+porphyrin+complexes+having+two+pyridine-N.+oxide+derivatives.">Spin-crossover between high-spin (S = 5/2) and low-spin (S = 1/2) states in six-coordinate iron(III) porphyrin complexes having two pyridine-N. oxide derivatives.</a> Dalton Trans. 46 (1), 242-249 (2017).</li><li>Drago, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physical Methods in Chemistry. , 750 (1977).</li><li>Cervantes-Mej&#237;a, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branched+Polyamines+Functionalized+with+Proposed+Reaction+Pathways+Based+on+1H-NMR,+Atomic+Absorption+and+IR+Spectroscopies.">Branched Polyamines Functionalized with Proposed Reaction Pathways Based on 1H-NMR, Atomic Absorption and IR Spectroscopies.</a> American Journal of Analytical Chemistry. 5 (16), 1090-1101 (2014).</li><li>Huheey, J. E., Keiter, E. A., Keiter, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inorganic Chemistry: Principles of Structure and Reactivity, 4th Edition. , 1023 (1997).</li><li>Rigaku, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CrysAlisPRO. , (2013).</li><li>Sheldrick, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHELXT+-+Integrated+space-group+and+crystal-structure+determination.">SHELXT - Integrated space-group and crystal-structure determination.</a> Acta Cryst. 71 (1), 3-8 (2015).</li><li>Sheldrick, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+refinement+with+SHELXL.">Crystal structure refinement with SHELXL.</a> Acta Cryst. 71 (1), 3-8 (2015).</li><li>Sheldrick, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+history+of+SHELX.">A short history of SHELX.</a> Acta Cryst. 64 (1), 112-122 (2008).</li><li>Macrae, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+CSD+2.0+-+new+features+for+the+visualization+and+investigation+of+crystal+structures.">Mercury CSD 2.0 - new features for the visualization and investigation of crystal structures.</a> J. Appl. Cryst. 41 (2), 466-470 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ChemBioDraw Ultra 13. , (2013).</li></ol>

All authors declare no conflict of interest.

The protocol presented here is a conventional method to link an oxygen atom to the nitrogen atom of the 3,5-lutidine as a functionalization method of substrates. This technique is also well established to yield X-ray suitable dehydrated crystals (Figure 5, pictures taken with a DSC-HX300 Cyber-shot Sony camera). As far as we are concerned, not many reports have described the production of such crystals16. Many compounds grow ideal crystals for X-ray analysis when they are chelated...

Nowadays, scientists around the globe have been investing resources into the development of new synthetic routes for the functionalization of aromatic groups, which are known for low reactivity front to addition reactions1,2,3. Pyridine, where a nitrogen atom substitutes a carbon atom, presents a similar chemical reactivity to analogue rings composed solely of carbon atoms3, and it usually undergoes a substitution mechanism rather than addition. N-oxides are distinctive by the presence of a donor bond between nitrogen and oxygen formed by the overlap of the nonbonding electron pair on the nitrogen with an empty orbital on the oxygen atom3. Particularly, pyridine N-oxides are Lewis bases, because their N-O moiety may act as an electron donor, and they may combine with Lewis acids forming the corresponding Lewis acid-base pairs. This property has an essential chemical consequence, because it can increase the nucleophilicity of the Lewis acids towards potential electrophiles and thus allow them to react under conditions where normally the reaction would not occur. Probably the most frequent use of such compounds is in various oxidation reactions where they act as oxidants4. Pyridine N-oxides and many of their ring-functionalized derivatives are recurrent molecules of biologically active and pharmacological agents5, and a clear spatial distribution by different spectroscopic tools has been established for some of them6,7. In research on attaching different groups to the pyridine ring, scientists have tested various methodologies to produce an easy and conventional method, since isoxazolines requires a catalytic amount of base such as DBU in boiling xylene to form 6-substituted-2-aminopyridine N-oxides8,9. A variety of pyridine derivatives were converted into their corresponding N-oxides in the presence of a catalytic amount of manganese tetrakis(2,6-diclorophenyl)porphyrin and ammonium acetate in CH2Cl2/CH3CN8,10. Other pyridines are oxidized to their oxides using H2O2 in the presence of catalytic amounts of methyltrioxorhenium8,11, or by the addition of excess dimethyldioxirane in CH2Cl2 at 0 &#176;C, which leads to the corresponding N-oxides8,12,13,14. Bis(trimethylsilyl)peroxide in the presence of trioxorhenium in CH2Cl2 has been used for the synthesis of pyridine N-oxides8,11. The synthesis of aminopyridine N-oxides involving acylation using Caro&#39;s acid (peroxomonosulfuric acid) has also been reported8. Nevertheless, the methodology reported here, and which uses part of the methodology reported by Ochiai1, provides very good results with the use of cheaper and accessible reagents, H2O2 and glacial acetic acid. This practice is more suitable for use in large scale preparations that act on tertiary amines, it produces good yields in a reaction that only requires 30% hydrogen peroxide and glacial acetic acid in a temperature between 70-80 &#176;C, and it uses a purification process that is available in most synthesis laboratories like distillation, without the use of catalyst or more expensive reagents1. The literature reports that other methodologies also frequently involve time frames from 10-24 h and temperatures above 100 &#176;C 4,8, and the yield of well-formed crystals for X-ray analyses is rarely reported.
Reactively, various N-oxide derivates are used to adequately activate the lutidine ring, in either a nucleophilic or electrophilic way. The nucleophilic or electrophilic factor is affected by the substituents. With the pyridine ring being the electron-withdrawing groups, the main factor is the nucleophilic characteristic1. The free N-oxide compounds are rarely isolated as suitable crystals for X-ray analysis due to the delocalized charge in the aromatic ring. However, the solvation factor is critical to stabilize the negative density of the oxygen15.

<table border="0" cellpadding="0" cellspacing="0" style="width:725px;" width="725">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">3,5-lutidine</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:119px;">L4206-500ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Fermont</td>
			<td>3015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hidrogen peroxide (35%)</td>
			<td>Sigma-Aldrich</td>
			<td>349887-500ML</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2CO3 anhydrous</td>
			<td>Productos Qu&#237;micos Monterrey</td>
			<td>1792</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2SO4 anhydrous</td>
			<td>Alfa reactivos</td>
			<td>25051-C</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CHCl3</td>
			<td>Fermont</td>
			<td>6205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl eter</td>
			<td>Mercury Chemist</td>
			<td>QME0309</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Distilled water</td>
			<td>Comercializadora Qu&#237;mica Poblana</td>
			<td>not-existent</td>
			<td></td>
		</tr>
	</tbody>
</table>

syntheses, crystallization, and spectroscopic characterization of 3,5-lutidine n-oxide dehydrate

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the methodology for non-substituted pyridines in 1957 in a 12 h process, but no X-ray suitable crystals were obtained. The substituted ring used in the methodology presented here clearly influenced the addition of water molecules into the asymmetric unit, which confers a different nucleophilic strength in 1. The X-ray suitable crystal compound 1 was possible due to the stabilization of the negative charge in the oxygen by the presence of two water molecules where the hydrogen atoms donate positive charge into the ring; such water molecules serve well to construct a supramolecular interaction. The hydrated molecules may be possible for the alkaline system that is reached by adjusting the pH to 10. Importantly, the double methyl substituted ring and a reaction time of 5 h, makes it a more versatile method and with wider chemical applications for future ring insertions.

The protocol is essentially an extension of Ochiai&#39;s technique1. However, lower temperature and less time are applied. This simple method can be used to obtain a versatile ligand, which is a substituted pyridine N-oxide derivate. To confirm the formation of 1, NMR 1H and 13C analysis are preferred to test the effectiveness of the procedure.
The chemical ...

Watch this Scientific Journal Video about Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate at JoVE.com

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

Herein, we report the synthesis and crystallization of 3,5-lutidine N-oxide dehydrate by a simple protocol that differs from the classical synthesis of pyridine N-oxide. This protocol utilizes different starting material and involves less reaction time to yield a new solvated supramolecular structure, which crystallizes under slow evaporation.

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the ...

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8.3K Views.  Benemérita Universidad Autónoma de Puebla. Herein, we report the synthesis and crystallization of 3,5-lutidine N-oxide dehydrate by a simple protocol that differs from the classical synthesis of pyridine N-oxide. This protocol utilizes different starting material and involves less reaction time to yield a new solvated supramolecular structure, which crystallizes under slow evaporation.

Video: Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and porous metal-dithiolene network in the highly ordered single crystalline state. Unlike the highly reactive free-standing thiols, which tend to decompose and complicate the crystallization of metal-thiolate open frameworks, the thioester reacts in situ to provide the thiol species, serving to mitigate the reaction between the mercaptan units and the metal centers, and to improve crystallization consequently. Specifically, the thioester was synthesized in a one-pot procedure: an aromatic bromide (hexabromotriphenylene) reacted with excess sodium thiomethoxide under vigorous conditions to first form the thioether intermediate product. The thioether was then demethylated by the excess thiomethoxide to provide the thiolate anion that was acylated to form the thioester product. The thioester was conveniently purified by standard column chromatography, and then used directly in the framework synthesis, wherein NaOH and ethylenediamine serve to revert in situ the thioester to the thiol linker for assembling the single-crystalline Pb(II)-dithiolene network. Compared with other methods for thiol synthesis (e.g., by cleaving alkyl thioether using sodium metal and liquid ammonia), the thioester synthesis here uses simple conditions and economical reagents. Moreover, the thioester product is stable and can be conveniently handled and stored. More importantly, in contrast to the generic difficulty in accessing crystalline metal-thiolate open frameworks, we demonstrate that using the thioester for in situ formation of the thiol linker greatly improves the crystallinity of the solid-state product. We intend to encourage broader research efforts on the technologically important metal-sulfur frameworks by disclosing the synthetic protocol for the thioester as well as the crystalline framework solid.

Here, we present a one-pot, transition-metal-free synthesis of thiols and thioesters from aromatic halides and sodium thiomethoxide, followed by the preparation of single crystals of a metal-dithiolene network using thiol species generated in situ from the more stable and tractable thioester.

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and ...

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and non-porous supports (polymer, ceramic, metal, carbon, and graphene). We developed a novel crystallization technique, which is termed the ENACT approach: the electrophoretic nuclei assembly for the crystallization of highly intergrown thin films (ENACT). This approach allows for a high density of heterogeneous nucleation of MOFs on a chosen substrate via the electrophoretic deposition (EPD) directly from the precursor sol. The growth of well-packed MOF nuclei leads to a highly intergrown polycrystalline MOF film. We show that this simple approach can be used for the synthesis of thin, intergrown zeolite imidazole framework (ZIF)-7 and ZIF-8 films. The resulting 500 nm-thick ZIF-8 membranes show a considerably high H2 permeance (8.3 x 10-6 mol m-2 s-1 Pa-1) and ideal gas selectivities (7.3 for H2/CO2, 15.5 for H2/N2, 16.2 for H2/CH4, and 2655 for H2/C3H8). An attractive performance for C3H6/C3H8 separation is also achieved (a C3H6 permeance of 9.9 x 10-8 mol m-2 s-1 Pa-1 and a C3H6/C3H8 ideal selectivity of 31.6 at 25 &#176;C). Overall, the ENACT process, owing to its simplicity, can be extended to synthesize intergrown thin films of a wide range of nanoporous crystalline materials.

A simple, reproducible, and versatile approach for the synthesis of intergrown, polycrystalline metal-organic framework membranes on a wide range of unmodified porous and non-porous supports is presented.

We report the synthesis of thin, highly intergrown, polycrystalline metal-organic framework (MOF) membranes on a wide range of unmodified porous and ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

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Chapters in this video

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

Synthesis and Characterization of Supramolecular Colloids

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Preparation and Characterization of C₆₀/Graphene Hybrid Nanostructures

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

Electrophoretic Crystallization of Ultrathin High-performance Metal-organic Framework Membranes

Synthesis and Characterization of Amphiphilic Gold Nanoparticles