Trisulfide Bond Research Articles

Preparation of Disulfide/Trisulfide Core-Cross-Linked Polycarbonate Nanocarriers for Intracellular Reduction-Triggered Drug Release.

Polymeric nanocarriers have attracted significant attention in the field of anticancer drug delivery due to their unique advantages. However, designing nanocarriers that can maintain stability in the bloodstream while achieving specific drug release within tumor cells remains a major challenge. To address this issue, constructing reversible cross-linked polymeric nanocarriers that are sensitive to the intracellular reducible glutathione (GSH) characteristic of the tumor microenvironment is a promising strategy. Based on this, we designed and synthesized two novel six-membered bicyclic carbonate monomers containing disulfide (DSBC) and trisulfide (TSBC) bonds. Through a one-step ring-opening polymerization, a series of reduction-sensitive polycarbonate copolymers (i.e., PEG-PDSBC and PEG-PTSBC) were prepared, and doxorubicin (DOX)-loaded nanoparticles were fabricated using a nanoprecipitation method. The in vitro drug release behaviors of these nanoparticles were systematically investigated. The results showed that these polymers, due to the cross-linked structure formed by the ring-opening polymerization of their bicyclic monomers, could self-assemble into stable nanoparticles. Under different concentrations of glutathione, DOX-loaded PEG-PTSBC nanoparticles demonstrated faster drug release, indicating more optimized intracellular drug release properties. Further cytotoxicity experiments revealed that both types of blank nanoparticles exhibited good biocompatibility with the 4T1 and NIH-3T3 cells. Fluorescence microscopy and flow cytometry results further indicated that DOX-loaded PEG-PTSBC nanoparticles released more drugs in 4T1 cells, significantly inhibiting tumor cell growth compared with DOX-loaded PEG-PDSBC nanoparticles, with no noticeable difference in NIH-3T3 normal cells. In conclusion, this study suggests that trisulfide cross-linked polycarbonate-based nanocarriers hold promise as an anticancer drug delivery system that combines stability in the bloodstream with specific intracellular drug release, offering new insights for the development of novel, efficient, and safe anticancer nanomedicines.

Design of multifunctional theranostic nanoplatforms with glutathione-triggered H2S generation and H2S-activitated multi-modal therapy

Hydrogen sulfide (H2S) is a specific substance in the tumor microenvironment, and highly expressed H2S can be used to activate some agents for intelligent treatment of tumors. However, not all tumors contain overexpressed hydrogen sulfide. To address this obstacle, we report the design of theranostic nanoplatforms with glutathione-triggered H2S generation and H2S-triggered therapy activation functions, by using hollow mesoporous organosilica nanoparticles (HMON) to load Cu-HMME framework nanodots (CuHF) and garlic extract diallyl trisulfide (DATS). Such CuHF/DATS@HMON with sizes of about 110 nm exhibit obvious quenching fluorescence and is inactive. Importantly, when CuHF/DATS@HMON is mixed with glutathione (GSH) solution, the nucleophilic attack of GSH on trisulfide bonds (−S-S-S-) in DATS generates S-allyl glutathione disulfide (GSSA) and allyl perthiol (ASSH), with the latter releasing H2S upon reduction by GSH, conferring the overexpress of H2S as the first-cascade activation. Furthermore, endogenous H2S can react with CuHF to produce CuS nanoparticles with strong chemodynamic/photothermal effect and to release metal porphyrin ((Cu)HMME) with photodynamic effect, as the second-cascade activation. When CuHF/DATS@HMON is accumulated at the tumor site through EPR effect, DATS in the CuHF/DATS@HMON can react with endogenous glutathione (GSH) in cancer cells to in situ generate H2S, and then H2S can vulcanize CuHF to generate CuS nanoparticles and (Cu)HMME complexes, thus realizing chemokinetic, photodynamic and photothermal multi-mode therapy to inhibit tumor growth. At the same time, the tetrasulfide bonds (−S-S-S-S-) in HMON allow the material to be slowly broken down and metabolized. Therefore, this study not only provides a general design for constructing tumor-microenvironment-responsive agents, but also provides some insights for multi-modal therapy of cancers.

Industrial Distillation Fractions of Garlic Essential Oil, Design, Synthesis, and Antifungal Activity Evaluation of Aliphatic Substituted Trisulfide Derivatives.

Garlic oil has a wide range of biological activities, and its broad-spectrum activity against phytopathogenic fungi still has the potential to be explored. In this study, enzymatic treatment of garlic resulted in an increase of approximately 50 % in the yield of essential oil, a feasible GC-MS analytical program for garlic oil was provided. Vacuum fractionation of the volatile oil and determination of its inhibitory activity against 10 fungi demonstrated that garlic oil has good antifungal activity. The antifungal activity levels were ranked as diallyl trisulfide (S-3)>diallyl disulfide (S-2)>diallyl monosulfide (S-1), with an EC50 value of S-3 against Botrytis cinerea reached 8.16 mg/L. Following the structural modification of compound S-3, a series of derivatives, including compounds S-4~7, were synthesized and screened for their antifungal activity. The findings unequivocally demonstrated that the compound dimethyl trisulfide (S-4) exhibited exceptional antifungal activity. The EC50 of S-4 against Sclerotinia sclerotiorum reached 6.83 mg/L. SEM, In vivo experiments, and changes in mycelial nucleic acids, soluble proteins and soluble sugar leakage further confirmed its antifungal activity. The study indicated that the trisulfide bond structure was the key to good antifungal activity, which can be developed into a new type of green plant-derived fungicide for plant protection.

A Trisulfide Bond Containing Biodegradable Polymer Delivering Pt(IV) Prodrugs to Deplete Glutathione and Donate H2S to Boost Chemotherapy and Antitumor Immunity.

The clinical application of cisplatin (CisPt) is limited by its dose-dependent toxicity. To overcome this, we developed reduction-responsive nanoparticles (NP(3S)s) for the targeted delivery of a platinum(IV) (Pt(IV)) prodrug to improve efficacy and reduce the toxicity. NP(3S)s could release Pt(II) and hydrogen sulfide (H2S) upon encountering intracellular glutathione, leading to potent anticancer effects. Notably, NP(3S)s induced DNA damage and activated the STING pathway, which is a known promoter for T cell activation. Comparative RNA profiling revealed that NP(3S)s outperformed CisPt in enhancing T cell immunity, antitumor immunity, and oxidative stress pathways. In vivo experiments showed that NP(3S)s accumulated in tumors, promoting CD8+ T cell infiltration and boosting antitumor immunity. Furthermore, NP(3S)s exhibited robust in vivo anticancer efficacy while minimizing the CisPt-induced liver toxicity. Overall, the results indicate NP(3S)s hold great promise for clinical translation due to their low toxicity profile and potent anticancer activity.

Tetrasulfide bond boosts the anti-tumor efficacy of dimeric prodrug nanoassemblies

Dimeric prodrug nanoassemblies (DPNAs) stand out as promising strategies for improving the efficiency and safety of chemotherapeutic drugs. The success of trisulfide bonds (-SSS-) in DPNAs makes polysulfide bonds a worthwhile focus. Here, we explore the comprehensive role of tetrasulfide bonds (-SSSS-) in constructing superior DPNAs. Compared to trisulfide and disulfide bonds, tetrasulfide bonds endow DPNAs with superlative self-assembly stability, prolonged blood circulation, and high tumor accumulation. Notably, the ultra-high reduction responsivity of tetrasulfide bonds make DPNAs a highly selective "tumor bomb" that can be ignited by endogenous reducing agents in tumor cells. Furthermore, we present an "add fuel to the flames" strategy to intensify the reductive stress at tumor sites by replenishing exogenous reducing agents, making considerable progress in selective tumor inhibition. This work elucidates the crucial role of tetrasulfide bonds in establishing intelligent DPNAs, alongside the combination methodology, propelling DPNAs to new heights in potent cancer therapy.

Satellite-Type Sulfur Atom Distribution in Trithiocarbonate Bond-Bridged Dimeric Prodrug Nanoassemblies: Achieving Both Stability and Activatability.

Homodimeric prodrug nanoassemblies (HDPNs) hold promise for improving the delivery efficiency of chemo-drugs. However, the key challenge lies in designing rational chemical linkers that can simultaneously ensure the chemical stability, self-assembly stability, and site-specific activation of prodrugs. The "in series" increase in sulfur atoms, such as trisulfide bond, can improve the assembly stability of HDPNs to a certain extent, but limits the chemical stability of prodrugs. Herein, trithiocarbonate bond (─SC(S)S─), with a stable "satellite-type" distribution of sulfur atoms, is developed via the insertion of a central carbon atom in trisulfide bonds. ─SC(S)S─ bond effectively addresses the existing predicament of HDPNs by improving the chemical and self-assembly stability of homodimeric prodrugs while maintaining the on-demand bioactivation. Furthermore, ─SC(S)S─ bond inhibits antioxidant defense system, leading to up-regulation of the cellular ROS and apoptosis of tumor cells. These improvements of ─SC(S)S─ bond endow the HDPNs with in vivo longevity and tumor specificity, ultimately enhancing the therapeutic outcomes. ─SC(S)S─ bond is, therefore, promising for overcoming the bottleneck of HDPNs for efficient oncological therapy.

Trisulfide Bond-Mediated Molecular Phototheranostic Platform for "Activatable" NIR-II Imaging-Guided Enhanced Gas/Chemo-Hypothermal Photothermal Therapy.

Tumor microenvironment (TME)-triggered phototheranostic platform offers a feasible strategy to improve cancer diagnosis accuracy and minimize treatment side effects. Developing a stable and biocompatible molecular phototheranostic platform for TME-activated second near-infrared (NIR-II) fluorescence imaging-guided multimodal cascade therapy is a promising strategy for creating desirable anticancer agents. Herein, a new NIR-II fluorescence imaging-guided activatable molecular phototheranostic platform (IR-FEP-RGD-S-S-S-Fc) is presented for actively targeted tumor imaging and hydrogen sulfide (H2 S) gas-enhanced chemodynamic-hypothermal photothermal combined therapy (CDT/HPTT). It is revealed for the first time that the coupling distance between IR-FE and ferrocene is proportional to the photoinduced electron transfer (PET), and the aqueous environment is favorable for PET generation. The part of Cyclic-RGDfK (cRGDfk) peptides can target the tumor and benefit the endocytosis of nanoparticles. The high-concentration glutathione (GSH) in the TME will separate the fluorescence molecule and ferrocene by the GSH-sensitive trisulfide bond, realizing light-up NIR-II fluorescence imaging and a cascade of trimodal synergistic CDT/HPTT/gas therapy (GT). In addition, the accumulation of hydroxyl radicals (•OH) and down-regulation of glutathione peroxidase 4 (GPX4) can produce excessive harmful lipid hydroperoxides, ultimately leading to ferroptosis.

Next-generation cell line selection methodology leveraging data lakes, natural language generation and advanced data analytics.

Cell line development is an essential stage in biopharmaceutical development that often lies on the critical path. Failure to fully characterise the lead clone during initial screening can lead to lengthy project delays during scale-up, which can potentially compromise commercial manufacturing success. In this study, we propose a novel cell line development methodology, referenced as CLD 4, which involves four steps enabling autonomous data-driven selection of the lead clone. The first step involves the digitalisation of the process and storage of all available information within a structured data lake. The second step calculates a new metric referenced as the cell line manufacturability index (MI CL) quantifying the performance of each clone by considering the selection criteria relevant to productivity, growth and product quality. The third step implements machine learning (ML) to identify any potential risks associated with process operation and relevant critical quality attributes (CQAs). The final step of CLD 4 takes into account the available metadata and summaries all relevant statistics generated in steps 1-3 in an automated report utilising a natural language generation (NLG) algorithm. The CLD 4 methodology was implemented to select the lead clone of a recombinant Chinese hamster ovary (CHO) cell line producing high levels of an antibody-peptide fusion with a known product quality issue related to end-point trisulfide bond (TSB) concentration. CLD 4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD 4 embodies the core principles of Industry 4.0 and demonstrates the benefits of increased digitalisation, data lake integration, predictive analytics and autonomous report generation to enable more informed decision making.

Hybrid chalcogen bonds in prodrug nanoassemblies provides dual redox-responsivity in the tumor microenvironment

Sulfur bonds, especially trisulfide bond, have been found to ameliorate the self-assembly stability of homodimeric prodrug nanoassemblies and could trigger the sensitive reduction-responsive release of active drugs. However, the antitumor efficacy of homodimeric prodrug nanoassemblies with single reduction-responsivity may be restricted due to the heterogeneous tumor redox microenvironment. Herein, we replace the middle sulfur atom of trisulfide bond with an oxidizing tellurium atom or selenium atom to construct redox dual-responsive sulfur-tellurium-sulfur and sulfur-selenium-sulfur hybrid chalcogen bonds. The hybrid chalcogen bonds, especially the sulfur-tellurium-sulfur bond, exhibit ultrahigh dual-responsivity to both oxidation and reduction conditions, which could effectively address the heterogeneous tumor microenvironment. Moreover, the hybrid sulfur-tellurium-sulfur bond promotes the self-assembly of homodimeric prodrugs by providing strong intermolecular forces and sufficient steric hindrance. The above advantages of sulfur-tellurium-sulfur bridged homodimeric prodrug nanoassemblies result in the improved antitumor efficacy of docetaxel with satisfactory safety. The exploration of hybrid chalcogen bonds in drug delivery deepened insight into the development of prodrug-based chemotherapy to address tumor redox heterogeneity, thus enriching the design theory of prodrug-based nanomedicines.

Redox-responsive nano-micelles containing trisulfide bonds to enhance photodynamic efficacy of zinc naphthalocyanine

A novel amphiphilic polymer with the trisulfide bond was developed. The nano-micelles could be formed through the self-assembling of this polymer with hydrophobic zinc naphthalocyanine, a NIR-absorbing photosensitizer. The micelles showed unique rods, high drug loading, high stability, high sensitivity to GSH, and excellent biosafety. The singlet oxygen production quantum yield of micelles was obviously higher than that of free photosensitizers and micelles containing disulphide bonds in the simulated tumor environment. The phototoxicity of micelles in cancer cells was significant. The redox response drug-loaded micelle was promising to enhance the photodynamic efficacy of zinc naphthalocyanine in cancer cells.

Prodrug nanoassemblies bridged by Mono-/Di-/Tri-sulfide bonds: Exploration is for going further

Sulfur bonds, including thioether bond and disulfide bond, have been widely used to develop tumor redox-responsive drug delivery systems. Recently, trisulfide bond was also introduced into the construction of prodrug nanoassemblies. However, the integrated effects of these sulfur bonds on prodrug nanoassemblies remain elusive. Here, three paclitaxel homodimeric prodrugs are synthesized using thioether bond, disulfide bond or trisulfide bond as linkages. Interestingly, the sulfur bonds significantly changed the molecular assembly of prodrugs, thereby influencing the colloidal stability and in vivo destiny of prodrug nanoassemblies. More importantly, the trisulfide bond is found to be a novel redox dual-sensitive linkage, and the responsive mechanism is illustrated. The differences of thioether/disulfide/trisulfide bond are investigated in-depth, and their effect on the antitumor efficacy of prodrug nanoassemblies is also clarified. Our findings provide deep insights into the impacts of sulfur bonds on prodrug nanoassemblies, and highlight the potential of trisulfide bond for the development of redox dual-responsive nanomedicines.

Prodrug Nanoassemblies Bridged by Mono-/Di-/Tri-Sulfide Bonds: Exploration is for Going Further

Prodrug Nanoassemblies Bridged by Mono-/Di-/Tri-Sulfide Bonds: Exploration is for Going Further

Sulfane Sulfur Is a Strong Inducer of the Multiple Antibiotic Resistance Regulator MarR in Escherichia coli.

Sulfane sulfur, including persulfide and polysulfide, is produced from the metabolism of sulfur-containing organic compounds or from sulfide oxidation. It is a normal cellular component, participating in signaling. In bacteria, it modifies gene regulators to activate the expression of genes involved in sulfur metabolism. However, to determine whether sulfane sulfur is a common signal in bacteria, additional evidence is required. The ubiquitous multiple antibiotic resistance regulator (MarR) family of regulators controls the expression of numerous genes, but the intrinsic inducers are often elusive. Recently, two MarR family members, Pseudomonas aeruginosa MexR and Staphylococcus aureus MgrA, have been reported to sense sulfane sulfur. Here, we report that Escherichia coli MarR, the prototypical member of the family, also senses sulfane sulfur to form one or two disulfide or trisulfide bonds between two dimers. Although the tetramer with two disulfide bonds does not bind to its target DNA, our results suggest that the tetramer with one disulfide bond does bind to its target DNA, with reduced affinity. An MarR-repressed mKate reporter is strongly induced by polysulfide in E. coli. Further investigation is needed to determine whether sulfane sulfur is a common signal of the family members, but three members sense cellular sulfane sulfur to turn on antibiotic resistance genes. The findings offer additional support for a general signaling role of sulfane sulfur in bacteria.

Investigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.

Iron-doxorubicin prodrug loaded liposome nanogenerator programs multimodal ferroptosis for efficient cancer therapy

Ferroptosis is a new mode of cell death, which can be induced by Fenton reaction-mediated lipid peroxidation. However, the insufficient H2O2 and high GSH in tumor cells restrict the efficiency of Fenton reaction-dependent ferroptosis. Herein, a self-supplying lipid peroxide nanoreactor was developed to co-delivery of doxorubicin (DOX), iron and unsaturated lipid for efficient ferroptosis. By leveraging the coordination effect between DOX and Fe3+, trisulfide bond-bridged DOX dimeric prodrug was actively loaded into the core of the unsaturated lipids-rich liposome via iron ion gradient method. First, Fe3+could react with the overexpressed GSH in tumor cells, inducing the GSH depletion and Fe2+generation. Second, the cleavage of trisulfide bond could also consume GSH, and the released DOX induces the generation of H2O2, which would react with the generated Fe2+in step one to induce efficient Fenton reaction-dependent ferroptosis. Third, the formed Fe3+/Fe2+ couple could directly catalyze peroxidation of unsaturated lipids to boost Fenton reaction-independent ferroptosis. This iron-prodrug liposome nanoreactor precisely programs multimodal ferroptosis by integrating GSH depletion, ROS generation and lipid peroxidation, providing new sights for efficient cancer therapy.

Polyvinyl chloride: chemical modification and investigation of structural and thermal properties

ABSTRACT In this study, polyvinyl chloride (PVC) was largely dechlorinated using sodium trisulfide (Na2S3) and ethanol. During the process, we successfully incorporated trisulfide bonds into the PVC structure. The structural characteristics of the samples were identified by Fourier transform infrared (FTIR) spectroscopy. The morphological and thermal properties of the samples were studied using X-ray diffraction (XRD) and differential scanning calorimetry (DSC) and thermogravimetric analysis techniques (TGA), respectively. The amounts of chlorine and sulfur were measured by energy-dispersive X-ray spectroscopy (EDX). Also, proton nuclear magnetic resonance (1H NMR) was used to confirm the chemical structure. Moreover, the Na2S3 was characterized by Ultraviolet–visible-near infrared (UV-VIS-NIR) spectroscopy. The results showed that, with the addition of sulfur to the PVC, the crystallinity is partly reduced. The thermal behaviors of the samples showed that after modification, the glass transition temperature and melting temperature would occur at lower temperatures. In addition, the degradation of the sulfur samples included three DTGA peaks. Furthermore, the presence of sulfur caused a change in the degradation temperature and weight loss during these stages.

Trisulfide bond-mediated doxorubicin dimeric prodrug nanoassemblies with high drug loading, high self-assembly stability, and high tumor selectivity.

Rational design of nanoparticulate drug delivery systems (nano-DDS) for efficient cancer therapy is still a challenge, restricted by poor drug loading, poor stability, and poor tumor selectivity. Here, we report that simple insertion of a trisulfide bond can turn doxorubicin homodimeric prodrugs into self-assembled nanoparticles with three benefits: high drug loading (67.24%, w/w), high self-assembly stability, and high tumor selectivity. Compared with disulfide and thioether bonds, the trisulfide bond effectively promotes the self-assembly ability of doxorubicin homodimeric prodrugs, thereby improving the colloidal stability and in vivo fate of prodrug nanoassemblies. The trisulfide bond also shows higher glutathione sensitivity compared to the conventional disulfide bond, and this sensitivity enables efficient tumor-specific drug release. Therefore, trisulfide bond-bridged prodrug nanoassemblies exhibit high selective cytotoxicity on tumor cells compared with normal cells, notably reducing the systemic toxicity of doxorubicin. Our findings provide new insights into the design of advanced redox-sensitive nano-DDS for cancer therapy.

Degradation Urushiol Coating Composition Crosslinked by Diallyl Trisulfide

Urushiol (UR) was a kind of natural paint with a long history and excellent performance. Its high price restricts its development. For the sake of the sustainable development of UR, we prepared a new urushiol based coating composition which is of degradation. To be more specific, we develop a blend by diallyl trisulfide (TS) and urushiol, in which a free radical copolymerization process would take place to form a coating composition under UV irradiation. In this work, FT-IR was used to prove the reaction proces; the reversibly cross-linking properties and the rheological properties were also been proved; the basic mechanisms active in microstructure and the associated surface energy was elucidated; mechanical analysis and showed the mechanical property of coating composition. It was found that a copolymer formed by copolymerization of diallyl trisulfide and urushiol. The introduction of trisulfide bonds assists in achieving the reversibly cross-linking under extreme alkalinity (pH > 8). various trisulfide densities brought about different microstructures on the modified urushiol. The surface energy of coating composition had been enhanced after UV-curing and its increase was related to the phase inversion (represented by microstructure). What was more, the existence of flexible segments offered the coating composition with excellent flexibility (2 mm) and rheological properties (15 MPa.S), while the hardness of coating dropped to 2H.

Characterization of Ultraviolet Photoreactions in Therapeutic Peptides by Femtosecond Laser Catalysis and Mass Spectrometry.

Peptides and proteins have diverse ultraviolet (UV) photoreaction pathways that can be activated by the energy of the UV photons absorbed. Simple light sources such as lamps are conventionally used to study these photoreactions in solution. This work provides a proof of concept that femtosecond laser technology can function as a highly potent UV source in rapidly conducting UV photostability studies of peptides. Correspondingly, sufficient quantities of photoproducts were generated in 1 min or less, allowing for identification of known and new photomodifications in the therapeutic peptides somatostatin-14 and arginine vasopressin. Identical photoproducts were also generated with a conventional continuous source. The major modifications included N-formylkynurenine, a cross-link between Trp and Phe, a Tyr product with an NH3 loss, and disruption of an unstable disulfide bond into a complex mixture of a trisulfide bond and multiple scrambled dimeric products. In conclusion, femtosecond lasers are extremely useful to drive fast UV-induced reactions for high throughput screening of photostability and modifications in amino acid polymers.

33S nuclear quadrupole resonance study of dibenzyl disulfide toward understanding of cross-linked structures in rubber

Experimental and theoretical investigations of a sulfur-33 electric-field-gradient (EFG) tensor of disulfide bonds in 33S-labled dibenzyl disulfide have been presented. Temperature dependence of quadrupolar frequencies, νQ, is observed in the temperature range between 80 and 280 K, in which single peaks appear in all the 33S nuclear quadrupole resonance (NQR) spectra. Analysis of nutation echo 33S NQR spectra at 200 K yields the quadrupolar coupling constant, CQ value, of 46.8(6) MHz and the asymmetry parameter, ηQ, of 0.48(7). The orientation of the 33S EFG tensor of the disulfide is obtained by quantum chemical calculations: the largest EFG tensor component, VZZ, is approximately perpendicular to the molecular plane (C-S-S), and the smallest component, VXX, is approximately 41° off the C-S bond. Extensive quantum chemical calculations are systematically performed to investigate dependences of 33S EFG tensors on changes of torsion angles in disulfide and trisulfide bonds, indicating that analysis of νQ and CQ values potentially makes it possible to assign the secondary structures of cross-linking in a rubber.