Homonuclear COSY Research Articles

Analysis of the Alcohol Amine Mixtures by 1D Selective COSY

Proton nuclear magnetic resonance (¹H-NMR) is one of the most commonly used techniques for structure identification in the field of chemical warfare convention (CWC) because of its strong ability in aiding structure assignment of chemicals. However, due to the lack of separation function coupled with chromatograph, the greatest disadvantage of ¹H-NMR in the analysis of environmental and biological samples is that the resonances can be easily covered or interfered. One-dimensional NMR selective excitation technology can effectively remove background interference and reveal the hidden spectral peak information. In this paper, three schedule chemicals of Chemical Weapons Convention (CWC), triethanolamine, N-methyldiethanolamine and 2-diisopropylaminoethanol, were selected as model chemicals. Homonuclear 1D COSY NMR selective excitation analysis method was established with these chemicals to solve the problem of resonances covered in ¹H-NMR. Key experiment parameters of 1D COSY were optimized. The optimized experimental conditions of 1D COSY are as follows: pulse sequence is selcogp, shaped pulse is G3, relaxation delay of d1 is 4 s, the excitation width of <i>d<SUB>H</SUB></i> 0.91 (a), 2.51 (g), 2.62 (e) and 3.44 (d) is 39.16, 35.34, 20.00 and 26.03 Hz respectively. The established 1D COSY method was used to analyze 1~10 μg/mL alcohol amine mixtures with 2 mg/mL quinuclidinol as the background interference and the LOD was 5 μg/mL. This method is simple, fast and can wipe off the interference effectively in the ¹H NMR spectrum without proceeding complex sample pretreatment. 1D selective COSY reflects the more accurate position of hydrogen atom, and can be used as a reliable auxiliary technology for structural identification.

Sample Preparation and Data Analysis for NMR-Based Metabolomics.

NMR spectroscopy has become one of the preferred analytical techniques for metabolomics studies due to its inherent nondestructive nature, ability to identify and quantify metabolites simultaneously in a complex mixture, minimal sample preparation requirement, and high degree of experimental reproducibility. NMR-based metabolomics studies involve the measurement and multivariate statistical analysis of metabolites present in biological samples such as biofluids, stool/feces, intestinal content, tissue, and cell extracts by high-resolution NMR spectroscopy-the goal then is to identify and quantify metabolites and evaluate changes of metabolite concentrations in response to some perturbation. Here we describe methodologies for NMR sample preparation of biofluids (serum, saliva, and urine) and stool/feces, intestinal content, and tissues for NMR experiments including extraction of polar metabolites and application of NMR in metabolomics studies. One dimensional (1D) 1H NMR experiments with different variations such as pre-saturation, relaxation-edited, and diffusion-edited are routinely acquired for profiling and metabolite identification and quantification. 2D homonuclear 1H-1H TOCSY and COSY, 2D J-resolved, and heteronuclear 1H-13C HSQC and HMBC are also performed to assist with metabolite identification and quantification. The NMR data are then subjected to targeted and/or untargeted multivariate statistical analysis for biomarker discovery, clinical diagnosis, toxicological studies, molecular phenotyping, and functional genomics.

Synthesis of a novel bifunctional oxyammonium‐based ionic liquid: Application for the synthesis of pyrano[4,3‐b]pyrans and tetrahydrobenzo[b]pyrans

AbstractIn the present investigation, a novel bifunctional oxyammonium‐based ionic liquid, namely, 2,2′‐(ethane‐1,2‐diylbis[oxy])bis(ethan‐1‐aminium)‐2,2,2‐trifluoroacetate, was designed and synthesized. The structure of the titled ionic liquid was characterized using Fourier‐transform infrared spectroscopy (FT‐IR), proton nuclear magnetic resonance (1HNMR), carbon nuclear magnetic resonance (13CNMR), fluorine nuclear magnetic resonance (19FNMR), homonuclear COSY nuclear magnetic resonance (NMR), thermogravimetry (TG), derivative thermogravimetry (DTG) analysis, X‐ray diffraction patterns (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The described ionic liquid demonstrated robust catalytic performance in the synthesis of pyrano[4,3‐b]pyrans and tetrahydrobenzo[b]pyran derivatives. The ionic liquid presents a high potential of recycling and reusing capability in both types of model reactions.

Determination of Peptide Profile Consistency and Safety of Collagen Hydrolysates as Quality Attributes.

Collagen hydrolysates are dietary supplements used for nutritional and medical purposes. They are complex mixtures of low‐molecular‐weight peptides obtained from the enzymatic hydrolysis of collagen, which provide intrinsic batch‐to‐batch heterogeneity. In consequence, the quality of these products, which is related to the reproducibility of their mass distribution pattern, should be addressed. Here, we propose an analytical approach to determine the peptide pattern as a quality attribute of Colagenart®, a product containing collagen hydrolysate. In addition, we evaluated the safety by measuring the viability of two cell lines exposed to the product. The consistency of peptide distribution was determined using Size Exclusion Chromatography (SEC), Mass Spectrometry coupled to a reversed phase UPLC system (MS‐RP‐UPLC), and Shaped‐pulse off‐resonance water‐presaturation proton nuclear magnetic resonance spectrometry [1Hwater_presat NMR]. The mass distribution pattern determined by SEC was in a range from 1.35 to 17 kDa, and from 2 to 14 kDa by MS‐RP‐UPLC. [1Hwater_presat NMR] showed the detailed spin‐systems of the collagen hydrolysates components by global assignment of backbone Hα and NH, as well as side‐chain proton resonances. Additionally, short‐range intraresidue connectivity pathways of identified spin‐regions were obtained by a 2D homonuclear shift correlation Shaped‐pulse solvent suppression COSY scheme. Safety analysis of Colagenart® was evaluated in CaCo‐2 and HepG2 cells at 2.5 and 25 μg/mL and no negative effects were observed. The results demonstrated batch‐to‐batch reproducibility, which evinces the utility of this approach to establish the consistency of the quality attributes of collagen hydrolysates.Practical ApplicationWe propose state‐of‐the art analytical methodologies (SEC, MS, and NMR) to evaluate peptide profile and composition of collagen hydrolysates as quality attributes. These methodologies are suitable to be implemented for quality control purposes.

NMR-Based Metabolic Snapshot from Minibronchoalveolar Lavage Fluid: An Approach To Unfold Human Respiratory Metabolomics.

The utility of mini bronchoalveolar lavage (mBAL) and its applicability in metabolomics has not been explored in the field of human respiratory disease. mBAL, "an archetype" of the local lung environment, ensures a potent technique to get the snapshot of the epithelial lining fluid afflicted to human lung disorders. Characterization of the mBAL fluid has potential to help in elucidating the composition of the alveoli and airways in the diseased state, yielding diagnostic information on clinical applicability. In this study, one of the first attempts has been made to comprehensively assign and detect metabolites in mBAL fluid, extracted from human lungs, by the composite use of 800 MHz 1D and 2D NMR, J-resolved homonuclear spectroscopy, COSY, TOCSY, and heteronuclear HSQC correlation methods. A foremost all-inclusive sketch of the 50 metabolites has been corroborated and assigned, which can be a resourceful archive to further lung-directed metabolomics, prognosis, and diagnosis. Thus, NMR-based mBALF studies, as proposed in this article, will leverage many more prospective respiratory researches for routine clinical application and prove to be a viable approach to mirror the key predisposing factors contributing to the onset of lung disease.

Two-dimensional Nuclear Magnetic Resonance Spectroscopy with Parallel Acquisition of 1H-1H and 19F-19F Correlations

A pulse sequence was established to obtain the two-dimensional nuclear magnetic resonance (NMR) spectroscopy with parallel acquisition of 1H-1H and 19F-19F correlations (1H/19F PANSY-COSY) using a 600 MHz Agilent Direct Drive II NMR instrument equipped with a triple resonance HFX probe feeding 1H and 19F receiver channels independently. The results of homo-nuclear COSY experiments on 4,4,5,5,6,6,7,7,7-nonafluoro-1-heptanol provided simultaneously 1H-1H and 19F-19F correlation spectra, which were compared with the regular COSY spectra acquired under the same conditions. The 1H/19F PANSY-COSY data showed the same resolution and signal sensitivity as the regular 1H-1H COSY and 19F-19F COSY while saving 40% of the experiment time. To improve the resolution for 1H/19F PANSY-COSY signals along the indirectly detection dimension, a narrow spectral window was set up in favor of the 1H chemical shifts range. The resulted 19F-19F COSY signals were folded along the indirectly detection dimension, and the spectra quality was improved.

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Norditerpenoid Alkaloids from Delphinium flexuosum Bieb.

Delphiniflexine, a new norditerpenoid alkaloid, together with the three known norditerpenoid alkaloids methyllycaconitine, ajadine, and acoseptrigenine were isolated from the aerial parts of Delphinium flexuosum. The structure of delphiniflexine was established on the basis of 1H, 13C, DEPT, homonuclear 1H COSY, NOESY, HSQC, and HMBC NMR studies.

Diterpenoid Alkaloids of Aconitum vulparia Rchb.

From the roots of Aconitum vulparia Rchb., collected in Prüm (Germany), a new norditerpenoid alkaloid, named alexhumboldtine, has been isolated along with the known norditerpenoid alkaloids lappaconitine, anthranoyllycoctonine, lycoctonine, puberaconitine, ajacine, and septentriodine. The structure of alexhumboldtine was established on the basis of 1H, 13C, DEPT, homonuclear 1H COSY, NOESY, HSQC, and HMBC NMR studies. From the aerial parts of the plant another norditerpenoid alkaloid, aconorine, has been isolated.

New and old NMR experiments for the resonance assignment of complex oligosaccharides—application to a cyclodextrin derivative

The complete assignment of the (1)H and (13)C sugar resonances in mono-3,6-anhydro-heptakis(2,3-O-methyl)-hexakis(6-O-methyl)-β-cyclodextrin, an asymmetrically functionalized β-cyclodextrin, was carried out by means of 2D NMR experiments. The TOCSY and the homonuclear multiple relay COSY spectra provided most of the (1)H assignments. The multiplicity edited HSQC and a set of F(1) selective HSQC-TOCSY and multiple relay HSQC-COSY spectra gave access to most of the (13)C chemical shifts. The latter were fully and accurately determined by means of a pair of complementary, highly folded HSQC-TOCSY spectra. The TOCSY-ROESY and ROESY-TOCSY spectra yielded the sequential assignment of the sugar units. A high resolution F(1) selective F(1) decoupled version of the TOCSY-ROESY experiment was recorded.

The effect of ring size variation on the structure and stability of lanthanide(iii) complexes with crown ethers containing picolinate pendants

The coordination properties of the macrocyclic receptor N,N'-bis[(6-carboxy-2-pyridyl)methylene]-1,10-diaza-15-crown-5 (H(2)bp15c5) towards the lanthanide ions are reported. Thermodynamic stability constants were determined by pH-potentiometric titration at 25 °C in 0.1 M KCl. A smooth decrease in complex stability is observed upon decreasing the ionic radius of the Ln(III) ion from La [log K(LaL) = 12.52(2)] to Lu [log K(LuL) = 10.03(6)]. Luminescence lifetime measurements recorded on solutions of the Eu(III) and Tb(III) complexes confirm the absence of inner-sphere water molecules in these complexes. (1)H and (13)C NMR spectra of the complexes formed with the diamagnetic La(III) metal ion were obtained in D(2)O solution and assigned with the aid of HSQC and HMBC 2D heteronuclear experiments, as well as standard 2D homonuclear COSY and NOESY spectra. The (1)H NMR spectra of the paramagnetic Ce(III), Eu(III) and Yb(III) complex suggest nonadentate binding of the ligand to the metal ion. The syn conformation of the ligand in [Ln(bp15c5)](+) complexes implies the occurrence of two helicities, one associated with the layout of the picolinate pendant arms (absolute configuration Δ or Λ), and the other to the five five-membered chelate rings formed by the binding of the crown moiety (absolute configuration δ or λ). A detailed conformational analysis performed with the aid of DFT calculations (B3LYP model) indicates that the complexes adopt a Λ(λδ)(δδλ) [or Δ(δλ)(λλδ)] conformation in aqueous solution. Our calculations show that the interaction between the Ln(III) ion and several donor atoms of the crown moiety is weakened as the ionic radius of the metal ion decreases, in line with the decrease of complex stability observed on proceeding to the right across the lanthanide series.

Structure of a DNA Polymerase α-Primase Domain That Docks on the SV40 Helicase and Activates the Viral Primosome

DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

NMR and X-ray crystallographic studies of unsymmetrical 25,26;27,28-dibridged para-tert-butyl calix[4]arene bisphosphites with a large “through-space” P–P coupling

Three 25,26-bridged para-tert-butyl-calix[4]arene phosphites, 1–3, have been synthesized by linking two proximal phenolic oxygen atoms of para-tert-butyl-calix[4]arene to a P(OR) [R = 2,6-But2–4-Me-C6H2 (1), 2,6-Pri2-C6H3 (2) or 2,4-But2-C6H3 (3)] moiety. NMR spectroscopic data and X-ray crystallographic studies (1 and 2 only) show that the calixarene framework in 1 adopts a cone conformation, whereas in 2 and 3 it assumes a partial cone conformation. Unsymmetrical 25,26;27,28-dibridged para-tert-butyl-calix[4]arene bisphosphites 5–7 have been synthesized by the reaction of 25,26-bridged para-tert-butyl-calix[4]arene phosphite 1 with (R′O)PCl2 [R′ = 2,4-But2-C6H3 (5), 2,6-Pri2-C6H3 (6) or (1R,2S,5R)-(−)menthyl (7)]. NMR investigations (homonuclear 31P COSY) revealed an unprecedented “through-space” phosphorus–phosphorus coupling of 223–244 Hz for these unsymmetrical 25,26;27,28-dibridged para-tert-butyl-calix[4]arene bisphosphites. The molecular structure of unsymmetrical 25,26;27,28-bridged bisphosphite 5 was determined by X-ray crystallography; the two phosphorus atoms are in close proximity and the P⋯P distance (3.543(2) Å) is less than the sum of their van der Walls radii.

Apicin, a New Flavonoid from Artemisia apiacea.

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2,2′-Bipyridine,1,10-phenanthroline and 2,2′:6′,2″-terpyridine in gallium(III) chemistry: Complexes containing the [formula omitted] core

The reactions of Ga(NO 3) 3 · 9H 2O with 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen) and 2,2′:6′,2″-terpyridine (terpy) in alcohols afford the complexes [Ga 2(OH) 2(bpy) 4](NO 3) 4 ( 1), [Ga 2(OH) 2(phen) 4](NO 3) 4 ( 2) and [Ga 2(OH) 2(H 2O) 2(terpy) 2](NO 3) 4 ( 3), respectively, in good yields. The crystal structures of 1 · 1.3MeOH · 1.4H 2O, 2 · 4.5MeOH and 3 have been solved by single-crystal X-ray crystallography. The three complexes contain the { Ga III 2 ( μ -OH ) 2 } 4 + core. Four nitrogen atoms from two chelating bpy ( 1) or phen ( 2) complete distorted octahedral coordination at each metal centre. In 3 one tridentate chelating terpy and one aquo ligand complete six coordination at each Ga III atom. The crystal structure of 2 · 4.5MeOH is stabilized by hydrogen bonds and intermolecular π–π stacking interactions, while in 3 hydrogen bonds create a double-chain, ladder-like architecture. Characteristic IR data are discussed in terms of the nature of bonding and the structures of the three complexes. The 1H NMR spectra of complexes 2 and 3 suggest the presence of the free ligand and one solution species containing the coordinated ligand. The structure of the cation [Ca 2(OH) 2(bpy) 4] 4+ is retained in D 2O as deduced by the 2D homonuclear COSY map.

Apicin, A New Flavonoid from Artemisia apiacea

Artemisia species spreads widely in nature and are genus of the family Compositae consisting of more than 350 species. A. apiacea is distributed at wasteland and river beaches of Korea, Japan and China, and has been used as traditional medicine to treat eczema and jaundice. Investigations on the compounds from A. apiacea have revealed the presence of campesterol, stigmasterol, β-sitosterol, 7methoxycoumarin, 7,8-dimethoxycoumarin and 7,8-methylenedioxycoumarin, scopoletin, protocatechualdehyde and ethyl and methyl caffeates, daphnetin, 7-hydroxy-8-methoxycoumarin and 7-isopentenyloxy-8-methoxycoumarin, volatile constituents like α-pinene and artemisia ketone and artemicapin C, apigenin, daucosterol, cacticin, hyperin, α-amyrin, β-amyrin, β-sitosterol and 5,6,7-trimethoxycoumarin. During the course of our continued studies on the compounds from A. apiacea, a new flavonoid (1) was isolated and identified. Compound 1 is described here for the first time as a naturally occurring compound. Compound 1 was obtained as yellow crystals from MeOH. It responded positively to the Shinoda test. In the EI-MS of 1, the molecular ion peak showed at m/z 360 corresponding to the molecular formula C18H16O8. The characteristic fragment ion peaks at m/z 153 showed the retro Diels-Alder fragmentation of flavonoids. The IR spectrum of 1 showed absorption bands for hydroxyl at 3383 cm−1, α,β-unsaturated C=O at 1612 cm−1 and C-O at 1015 cm−1. In the H-NMR spectrum of 1, the typical flavonoid signals were observed. The singlets of aromatic 5-OH at δ 13.03 and three -OMe signals at δ 3.91, 3.80 and 3.71 were observed. The singlets of H-8, -2', -4', and -6' were observed at δ 6.95, 7.09, 6.55 and 7.44, respectively. Its C-NMR spectrum of 1 showed C=O at δ 183.1 and three -OMe at δ 57.3, 57.6 and 60.9. In the homonuclear COSY spectrum, the correlation of proton signals is not indicated. The assignments of the Hand CNMR signals derived hetero nuclear direct and long-range correlations on 1 are listed in Table 1. Accordingly, compound 1 was assigned as a new flavonoid and named apicin (3,5,3'-trihydroxy-6,7,5'-trimethoxyflavone).

Asymmetry in 13C−13C COSY Spectra Provides Information on Ligand Geometry in Paramagnetic Proteins

The relative intensity of Calpha-C' cross-peaks in homonuclear 13C COSY spectra depends on the relaxation properties of Calpha and C' spins, which, in the proximity of a paramagnetic center, are related to the metal-to-carbon distance. Their quantitative analysis has lead, for the cerium-substituted dicalcium protein, calbindin D9k, to the straightforward identification of peaks arising from metal-coordinating groups. The monodentate or bidentate metal binding mode of carboxylates was identified directly via NMR.

Identification of major metabolites in Aloe littoralis by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy.

Examination of the leaf exudate of the South African species Aloe littoralis by reversed-phase HPLC revealed the presence of two major metabolites. The identification of the two compounds without isolation was attempted by HPLC-NMR based on separation using a C18 column eluting with a deuterium oxide:acetonitrile solvent gradient and an inverse HPLC-NMR probe. For each compound, one-dimensional proton spectra, and two-dimensional homonuclear COSY and TOCSY, and heteronuclear HSQC and HMBC, spectra were collected. On the basis of the data obtained, the metabolites were characterised as 10-hydroxyaloin B and deacetyllittoraloin.

Norditerpenoid alkaloids from Delphinium species

From the aerial parts of four Delphinium species 11 known and 3 new norditerpenoid alkaloids have been isolated: from D. dissectum Huth: delavaine A/B, deoxylycoctonine, methyllycaconitine; new: 10-hydroxymethyllycaconitine; from D. excelsum Reichenb.: delcaroline, delectinine, delterine, methyllycaconitine; new: 10-hydroxymethyllycaconitine, 18- O-methyldelterine and 10-hydroxynudicaulidine; from D. grandiflorum L.: delcosine, deltatsine, grandiflorine, methyllycaconitine; from D. triste Fisch.: delcosine, macrocentridine, 14-dehydrodelcosine. The structures of the new alkaloids were established on the basis of MS, 1H, 13C, DEPT, homonuclear COSY, HMQC and HMBC NMR spectroscopic techniques.

Observation and characterization of intermolecular homonuclear single-quantum coherences in liquid nuclear magnetic resonance

Intermolecular zero-quantum and double-quantum coherences (iZQCs and iDQCs) are frequently discussed in literature since they may provide novel contrast mechanisms in magnetic resonance imaging and possibilities for high-resolution spectra in an inhomogeneous and unstable magnetic field. In a previous paper [J. Chem. Phys. 115, 10769 (2001)], we have studied both theoretically and experimentally the properties of iZQC and iDQC nuclear magnetic resonance (NMR) signals related to intermolecular dipolar interactions in two-component systems. In this paper, the investigation is extended to homonuclear intermolecular single-quantum coherences (iSQCs) from the second-order spin interactions, which have not been observed and studied previously. Selective excitation was used to suppress the strong conventional single-spin single-quantum signals. A combination of dipolar field treatment and Torrey equation was used to derive a general theoretical expression for the time evolution of spins with arbitrary flip angles of rf pulses. The expression was used to predict the optimal conditions for iSQCs among highly polarized spins in liquid. Dependence of the iSQC signals on the experimental parameters was measured and analyzed to verify the theoretical predictions. For the first time, signals from pure homonuclear two-spin iSQCs free of much larger conventional single-spin single-quantum signals, and intermolecular iSQC cross peaks in homonuclear pulsed-field gradient COSY experiments were observed and characterized, in one- and two-dimensional (1D and 2D) experiments, respectively. The use of coherence-selection gradients tilted at the magic angle results in the suppression of iSQC cross peaks. It provides strong evidence that the observed signals originate from distant dipolar interactions. Relaxation and diffusion properties of iSQCs in multiple-component samples were characterized and analyzed as well as the optimal rf flip angles. Theoretical and experimental results presented herein demonstrate that the signals from the homonuclear second-order iSQCs not only have a similar signal intensity as iZQCs or iDQCs, all of which are much stronger than that from three-spin iSQCs reported previously, but also provide spatial information related to dipolar correlation scales similar to iZQCs and iDQCs, which is not present in conventional SQC experiments. All 1D and 2D NMR experimental observations based on single- and multiple-component samples are in excellent agreement with the theoretical predictions. The quantitative study of iSQCs provides a better understanding of their unique mechanisms, and may find useful applications in NMR analyses such as sample purification and/or preparation of metabolites, biofluids, and natural compounds dissolved in nondeuterated solvents.