Single Amino Group Research Articles

Reversible sulfur dioxide capture by amino acids containing a single amino group at low sulfur dioxide concentrations.

SO2, an air pollutant, is harmful to human health and causes air pollution; therefore, numerous studies have focused on the development of SO2 control technologies. Although limestone- and ammonia-based absorbents have been widely used in wet desulfurization, they are difficult to regenerate and do not enable the recycling of SO2, which is a useful resource. Recently, amino acids have attracted attention as reversible SO2 absorbents because they are eco-friendly and have excellent reactivity with SO2, as well as high regeneration performance. Glycine, L-alanine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, and 6-aminohexanoic acid were analyzed to investigate the relationship between SO2 absorption and the amino acid molecular structure using the simulated actual flue gas (200 ppmv SO2 + 13% CO2 in N2 balance). The SO2 absorption of amino acids (with the molecular structure of glycine and alkyl chains of various lengths) improved as the alkyl chain length increased, possibly owing to a decrease in the inductive effect in the molecular structure of the amino acid. Furthermore, 13C-nuclear magnetic resonance spectroscopy was conducted to analyze the SO2 absorption reaction mechanism (including the possible generation of irreversible species), and experiments involving a number of consecutive absorption-desorption cycles were used to confirm the reusability of the amino acids. The tested amino acids exhibited higher cyclic capacities compared to those of deep eutectic solvents and ionic liquids reported in the literature, thereby exhibiting excellent potential as SO2 absorbents. Thus, this study can guide the future design and development of eco-friendly SO2 absorbents.

Determination of Acyl-, O-, and N-Glucuronide Using Chemical Derivatization Coupled with Liquid Chromatography-High-Resolution Mass Spectrometry.

Glucuronidation is the most common phase II metabolic pathway to eliminate small molecule drugs from the body. However, determination of glucuronide structure is quite challenging by mass spectrometry due to its inability to generate structure-informative fragments about the site of glucuronidation. In this article, we describe a simple method to differentiate acyl-, O-, and N-glucuronides using chemical derivatization. The idea is that derivatization of acyl-, O-, or N-glucuronides of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry. The following two reactions were applied to specifically derivatize carboxyl and hydroxyl groups that are present on the aglycone and its glucuronide metabolite: Carboxyl groups were activated by thionyl chloride followed by esterification with ethanol. Hydroxyl groups were derivatized via silylation by 1-(trimethylsilyl)imidazole. The mass shift per derivatized carboxyl and hydroxyl group was +28.031 Da and +72.040 Da, respectively. This approach was successfully validated using commercial glucuronide standards, including benazepril acyl-glucuronides, raloxifene O-glucuronide, and silodosin O-glucuronide. In addition, this approach was applied to determine the type of glucuronide metabolites that were isolated from liver microsomal incubation, where alvimopan and diclofenac acyl-glucuronides; darunavir, haloperidol, and propranolol O-glucuronides; and darunavir N-glucuronide were identified. Lastly, this approach was successfully used to elucidate the definitive structure of a clinically observed metabolite, soticlestat O-glucuronide. In conclusion, a novel, efficient, and cost-effective approach was developed to determine acyl-, O-, and N-glucuronides using chemical derivatization coupled with liquid chromatography-high resolution mass spectrometry. SIGNIFICANCE STATEMENT: The method described in this study can differentiate acyl-, O-, and N-glucuronides and allow for elucidation of glucuronide structures when multiple glucuronidation possibilities exist. The type of glucuronidation information is particularly useful for a drug candidate containing carboxyl groups, which can form reactive acyl-glucuronides. Additionally, the method can potentially be used for definitive structure elucidation for a glucuronide with its aglycone containing a single carboxyl, hydroxyl, or amino group even when multiple types of functional groups are present for glucuronidation.

Improved anti-cancer effect of epidermal growth factor-gold nanoparticle conjugates by protein orientation through site-specific mutagenesis

ABSTRACT Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs.

Generation of luminescent silver nanodots in the presence of amino silane and sodium polyacrylate

Amino silane has been introduced to suppress the further growth of luminescent silver clusters (silver nanodots) to non-luminescent silver nanoparticles in the stabilization of the just-reduced silver clusters with sodium polyacrylate (PAA). The roles of the silver, silane, and sodium polyacrylate in the generation of such chromophores are still vague. We found that silver ions and the PAA chain formed chromophoric complexes in the aqueous solution. However, silver nanodots were generated only after chemical reduction. The silane with two or three amino groups promoted the generation of silver nanodots, but an increase in hydrophobicity of the silane, i.e., flanked by a single amino group, stimulated the formation of near-IR silver nanodot emitters. Nevertheless, the absorption spectrum of the reaction mixture did not superimpose in shape with the excitation spectrum of the silver nanodot, suggesting that there were multiple-species that caused strong absorptions in the visible and near-IR region. The pH of the solution strongly influenced the generation and stability of silver nanodots. Silver nanodots were generated efficiently when the reaction pH was between 6 and 8, whereas the already-formed silver nanodots were stable at the pH values between 6 and 10, suggesting that disruption of the silver nanodot protection by competitive species in the solution was slowed down due to the chelate effect of multiple bonding from the polymer chain. Our results suggest that the microenvironment of silver nanodots influences strongly the properties of silver nanodots.

Stabilization of Immobilized Lipases by Intense Intramolecular Cross-Linking of Their Surfaces by Using Aldehyde-Dextran Polymers.

Immobilized enzymes have a very large region that is not in contact with the support surface and this region could be the target of new stabilization strategies. The chemical amination of these regions plus further cross-linking with aldehyde-dextran polymers is proposed here as a strategy to increase the stability of immobilized enzymes. Aldehyde-dextran is not able to react with single amino groups but it reacts very rapidly with polyaminated surfaces. Three lipases—from Thermomyces lanuginosus (TLL), Rhizomucor miehiei (RML), and Candida antarctica B (CALB)—were immobilized using interfacial adsorption on the hydrophobic octyl-Sepharose support, chemically aminated, and cross-linked. Catalytic activities remained higher than 70% with regard to unmodified conjugates. The increase in the amination degree of the lipases together with the increase in the density of aldehyde groups in the dextran-aldehyde polymer promoted a higher number of cross-links. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of those conjugates demonstrates the major role of the intramolecular cross-linking on the stabilization of the enzymes. The highest stabilization was achieved by the modified RML immobilized on octyl-Sepharose, which was 250-fold more stable than the unmodified conjugate. The TLL and the CALB were 40-fold and 4-fold more stable than the unmodified conjugate.

What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin-Sensing Riboswitch.

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.

Self‐Assembled Fluorescent Organic Nanoparticles for Live‐Cell Imaging

Fluorescent, cell-permeable, organic nanoparticles based on self-assembled π-conjugated oligomers with high absorption cross-sections and high quantum yields have been developed. The nanoparticles are generated with a tuneable density of amino groups for charge-mediated cellular uptake by a straightforward self-assembly protocol, which allows for control over size and toxicity. The results show that a single amino group per ten oligomers is sufficient to achieve cellular uptake. The non-toxic nanoparticles are suitable for both one- and two-photon cellular imaging and flow cytometry, and undergo very efficient cellular uptake.

Spectroscopy of Protonated Tetraphenylporphyrins with Amino/Carbomethoxy Substituents: Hyperporphyrin Effects and Evidence for a Monoprotonated Porphyrin

Spectrophotometric titrations for a full series of para-amino/carbomethoxy-substituted tetraphenylporphyrins were carried out using methanesulfonic acid in DMSO to study the hyperporphyrin effect across different substitution patterns. The series included zero, one, two (cis and trans), three, and four amino groups, with the remaining para substituents carbomethoxy groups. With increasing numbers of aminophenyl groups, the relative basicity increased and the hyperporphyrin effect increased, marked by a strong red band and a split Soret band. The cis diamino derivative showed a stronger hyperporphyrin effect compared to the trans isomer, which can be explained based on simple resonance forms. For the monoamino derivative, an initial increase in the Soret band upon acid titration along with well-defined isosbestic points provided evidence for a monoprotonated porphyrin, distinct from the diprotonated and triprotonated states. The relative stability of this unusual intermediate is proposed to be due to charge delocalization of the first cation to the single amino group and destabilization of the second protonation by the electron-withdrawing carbomethoxy substituents.

DNA Reactivity Profile of trans‐Platinum Planar Amine Derivatives

New trans-platinum planar amines (TPAs) represent a family of platinum-based drugs with cytotoxicity equivalent to that of cisplatin, but with negligible cross-resistance. According to the substitution pattern around the metal center, distinct DNA adducts can be formed which yield various levels of cytotoxicity in cell lines. We compared the effects of leaving groups (Cl(-) versus formate or acetate) and amines (NH(3) versus aromatic heterocyclic planar systems) on the efficiency, kinetics, and mode of DNA platination. We show that the substitution of just a single amino group on the transplatin nucleus is optimal, with major effects on the kinetics of metal complex conversion into the reactive aquo species. Additionally, by monitoring TPA reactivity toward variable DNA structures, a lack of preference for double-stranded DNA in over single-stranded or G-quadruplex DNA was observed which is possibly related to steric effects of the planar amine groups. These properties can lead to a unique distribution of platination sites by TPA relative to the lead compound cisplatin, which may help to explain the unique cytotoxic profile of TPAs.

A pitfall of using 2‐[(2E)‐3‐(4‐tert‐butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile as a matrix in MALDI TOF MS: chemical adduction of matrix to analyte amino groups

2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) has been considered as an excellent matrix for matrix-assisted laser desorption/ionization (MALDI) of many types of synthetic compounds. However, it might provide troublesome results for compounds containing aliphatic primary or secondary amino groups. For these compounds, strong extra ion peaks with a mass difference of 184.1 Da were usually observed, which might falsely indicate the presence of some unknown impurities that were not detected by other matrices. On the basis of the possible mechanisms proposed, these extra ions are the products of nucleophilic reactions between analyte amino groups and DCTB molecules or radical cations. In these reactions, an amino group replaces the dicyanomethylene group of DCTB forming a matrix adduct via a -C=N-bond. An aliphatic primary amine could react easily with DCTB and the reaction could start once they are mixed in a MALDI solution. For an aliphatic secondary amine, on the other hand, the reaction most likely occurs in the gas phase. Protonation of amino groups by adding acid seems to be a useful way to stop DCTB adduction for compounds with one single amino group, but not for compounds with multiple amino groups. Unlike aliphatic primary or secondary amines, aliphatic tertiary amines and aromatic amines do not yield DCTB adducts. This is because tertiary amines do not have the required transferrable H-(N) atom to form an extra -C=N-bond, while aromatic amines are not sufficiently nucleophilic to attack DCTB. In view of the possible matrix adduction, care should be taken in MALDI time-of-flight mass spectrometry (TOF MS) when DCTB is used as the matrix for compounds containing amino group(s).

Study of ammonia molecule adsorbing on diamond (1 0 0) surface

The diamond (1 0 0) surface with amino terminations is investigated based on density function theory within the generalized gradient approximation. Our calculated negative electron affinity of diamond (1 0 0) surface with hydrogen termination provides a necessary condition for initiating radical reaction. The results display that the ammonia molecule can form stable C–N covalent bonds on the diamond surface. In addition, due to the lower adsorption energy of one amino group binding on diamond surface, single amino group (SAG) model is easy to be realized in experiment with the comparison of double amino group (DAG) model. The adsorbed ammonia molecule will induce acceptor-like gap states with little change of the valence and conduction band of diamond in SAG model. The adsorption mechanism in the formation of ammonia monolayer on H-terminated diamond (1 0 0) surface, and two possible adsorption structures (SAG and DAG) were especially studied.

Macromol. Rapid Commun. 3/2010

Front Cover: The picture presents the synthetic route to novel polyfunctional PEG-based polymers bearing a single terminal amino group and multiple hydroxyl groups. The lower pictures illustrate the tunable materials properties ranging from highly crystalline to amorphous depending on the amount of comonomer content. Further details can be found in the article by C. Mangold, F. Wurm, B. Obermeier, and H. Frey* on page 258.

Preparation, characterization and fluorescent imaging of multi-walled carbon nanotube–porphyrin conjugate

In order to track carbon nanotubes (CNTs) in vitro or in vivo, many technologies have already been applied. However, the potential toxic effects of technologies such as radio and quantum dot labeling are the major problems. Therefore, the need to search for a new kind of biocompatible material to label CNTs has become urgent. In this work, one kind of porphyrin derivative with a single amino group was synthesized and covalently conjugated to oxidized water-soluble multi-walled carbon nanotubes (MWCNTs). The physiochemical properties of the oxidized MWCNTs and the fluorescent characteristics of their conjugate were carefully investigated. The experimental results indicated that the carboxyl group was introduced to the oxidized MWCNTs, allowing reaction with the amino group of the porphyrin, while the tube-like structure was well retained. The conjugate of the amino porphyrin and the MWCNTs exhibited strong red fluorescence emissions at 670 nm and 730 nm when excited with the optimal excitation wavelength of 420 nm. An aqueous solution containing the conjugate was injected subcutaneously into mice and was then imaged in vivo and ex vivo. Results showed that the conjugate was mainly engulfed by macrophages under the skin, and most of the conjugates accumulated in subcutaneous connective mucosa and a small amount in the liver, over a long period.

Bacillus cereus strain 10-L-2 produces two arylamine N-acetyltransferases that transform 4-phenylenediamine into 4-aminoacetanilide

A bacterium, strain 10-L-2, that was isolated from soil and identified as Bacillus cereus grew well on medium containing 4-phenylenediamine and Polypepton. Strain 10-L-2 converted a wide variety of anilines, including 4-phenylenediamine, to their corresponding acetanilides. Growing cells acetylated a single amino group of 4-phenylenediamine to form 4-aminoacetanilide with a 97% molar yield, as shown by mass spectrometry and HPLC. Cell extracts exhibited arylamine N-acetyltransferase (NAT) activity toward 4-phenylenediamine. Two NATs, namely, NAT-a and NAT-b, were separated by DE52 column chromatography and were further purified and characterized. The subunit molecular masses of NAT-a and NAT-b were 31.0 and 27.5 kDa, respectively, as determined by SDS-PAGE analysis. The two enzymes had similar pH- and thermo-stabilities and were similarly affected by pH, temperature, and several reagents. The enzymes showed peak activity toward 5-aminosalicylic acid of the substrates tested, but they differed in substrate specificity. Only NAT-a had activity toward sulfamethazine. Although other wild-type bacterial cultures also synthesize NAT, the ability of strain 10-L-2 to convert and detoxify 4-phenylenediamine is much higher. This report provides the first evidence of two NATs in a eubacterium.

Use of Probes with Fluorescence Indicator Distributed throughout the Pharmacophore to Examine the Peptide Agonist-binding Environment of the Family B G Protein-coupled Secretin Receptor

Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mum guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.

Determination of carbofuran in water by homogeneous immunoassay using selectively conjugate mastoparan and terbium/dipicolinic acid fluorescent complex

Homogeneous immunoassay (LITRFIA) for carbofuran (CF) determination was performed using liposomes and mastoparan (Mast) conjugate as cytolitic agent. Mast was conjugated to the 5-(2-2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)-pentanoic acid (CPCF) both randomly and selectively to a single (V 1- or K 4-) amino-group. The conjugated compounds have been tested for the cytolytic activity on liposomes trapping Tb/citrate complex. Dipicolinic acid (DPA) was used as fluorescent chelating agent. The CPCF–V 1–Mast derivative (retaining almost the same lytic activity as Mast) was used in the immunoassay in competition with standard CF. Liposome lysis was proportional to the standard concentrations in a dynamic range between 10 pg and 10 ng. Assay has been performed for tap water analysis and for 10 real samples taken from an agricultural area to the south of Milan. Recovery in samples spiked with two different CF concentrations was between 92.5 and 105%.

Honeydew amino acids in relation to sugars and their role in the establishment of ant‐attendance hierarchy in eight species of aphids feeding on tansy (Tanacetum vulgare)

Abstract. The ratio of the concentration of honeydew total amino acids to total sugars in the honeydew of eight species of aphids, all feeding on tansy,Tanacetum vulgare(L.), was determined and correlated with honeydew production and ant‐attendance. The honeydew of the five ant‐attended aphid species [Metopeurum fuscoviride(Stroyan),Trama troglodytes(v. Hayd),Aphis vandergooti(Börner), Brachycardus cardui(L.),Aphis fabae(Scopoli)] was rich in total amino acids, ranging from 12.9 to 20.8 nmol µL−1compared with the unattended aphidMacrosiphoniella tanacetaria(Kalt.) with only 3 nmol µL−1. Asparagine, glutamine, glutamic acid and serine (all nonessential amino acids) were the predominant amino acids in the honeydew of all species. The total concentration of amino acids in the phloem sap of tansy was much higher (78.7 nmol µL−1) then in the honeydew samples, and the predominant amino acids were glutamate (34.3%) and threonine (17.7%). A somewhat unexpected result was the finding that those aphid species with the highest total amino acid concentration in the honeydew always had the highest concentration of sugars. The lowest amino acid–sugar combined value was 104–28.8 nmol µL−1in the non ant‐attended speciesM. tanacetaria, and the highest value was an average of 270–89.9 nmol µL−1for the three most intensely attended aphid speciesM. fuscoviride, A. vandergootiandT. troglodytes. There is no evidence that any single amino acid or group of amino acids in the honeydew acted as an attractant for ant‐attendance in these eight aphid species. The richness of the honeydew (rate of secretion × total concentration of sugars), along with the presence of the attractant sugar melezitose, comprised the critical factors determining the extent of ant‐attendance of the aphids feeding onT. vulgare. The high total amino acid concentration in sugar‐rich honeydews can be explained by the high flow‐through of nutrients in aphids that are particularly well attended by ants.

Modeling Noncompetitive Antagonism of a Nicotinic Acetylcholine Receptor

Models of closed and open channel pores of a muscle-type nicotinic acetylcholine receptor (nAChR) channel comprising M1 and M2 segments are presented. A model of the closed channel is proposed in which hydrophobic residues of the Equatorial Leucine ring screen the oxygen domain formed by the Serine ring, thereby preventing ion flux without completely occluding the pore. This model demonstrates a high similarity with the structure derived from a recent electron microscopy study. We propose that hydrophobic residues of the Equatorial Leucine ring are retracted when the pore is open. Our models provide a possible resolution of the nAChR gate controversy. We have also obtained explanations for the complex mechanisms underlying inhibition of nAChR by philanthotoxins (PhTXs). PhTX-343, containing a spermine moiety with a charge of +3, binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions. Both philanthotoxins may bind to a hydrophobic site located close to the external entrance of the pore in a region that includes residues associated with the regulation of desensitization.

Nitro and amino substitution within the A-ring of 5 H-8,9-dimethoxy-5-(2- N, N-dimethylaminoethyl)dibenzo[ c, h][1,6]naphthyridin-6-ones: influence on topoisomerase I-targeting activity and cytotoxicity

Recently, 5 H-8,9-dimethoxy-5-(2- N, N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[ c, h][1,6]naphthyridin-6-one, 1, was identified as a TOP1-targeting agent with pronounced antitumor activity. In the present study, the effect on activity of substituting a single nitro or amino group in the A-ring in lieu of the methylenedioxy moiety of 1 was evaluated. The presence of either a nitro or amino substituent at the 4-position had a pronounced adverse affect on both TOP1-targeting activity and cytotoxicity. To a lesser extent, derivatives with a nitro or amino substituent at the 1-position were also less active than 1. Replacement of the methylenedioxy moiety of 1 with either a nitro or amino substituent at either the 2- and 3-position did result in analogues with potent TOP1-targeting activity and cytotoxicity.

Heme cleavage with remarkable ease: paramagnetic intermediates formed by aerobic oxidation of a meso-amino-substituted iron porphyrin.

Hemes must be oxidatively stable to carry out their functions as biological oxidants, but introduction of a single amino group at a meso position of octaethylheme renders it extremely sensitive to ring opening by dioxygen. Exposure of a red pyridine (py) solution of diamagnetic (py)(2)Fe(II)(H(2)N-OEP) (1) (H(2)N-OEP is the dianion of meso-amino-octaethylporphyrin) to air results in the immediate formation of a green intermediate which is subsequently converted into a second species that has been crystallized and characterized by X-ray diffraction. This process is distinct from coupled oxidation, a model for biological heme cleavage, because it does not require a sacrificial reducing agent to initiate the process.