Cyanogen bromide: Difference between revisions
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{{Short description|Chemical compound (BrCN)}} |
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{{Chembox new |
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{{redirect-distinguish|CBrN|CBRN}} |
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| ImageFile = Cyanogen-bromide-3D-vdW.png |
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{{Chembox |
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| ImageSize = |
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|Watchedfields = changed |
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| IUPACName = Cyanic bromide |
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|verifiedrevid = 442343462 |
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| OtherNames = Bromine cyanide, Bromocyanide, Cyanobromide, Cyanogen bromide, Cyanogen monobromide, Carbononitridic bromide, Bromocyan, Bromocyanogen, Campilit, UN1889, CB |
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|ImageFile = Cyanogen-bromide-2D.png |
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| Section1 = {{Chembox Identifiers |
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|ImageFile_Ref = {{chemboximage|correct|??}} |
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| CASNo = 506-68-3 |
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|ImageSize = 100 |
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| EINECS = 208-051-2 |
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|ImageName = Skeletal formula of cyanogen bromide |
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| PubChem = 10476 |
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|ImageFile1 = Cyanogen-bromide-3D-vdW.png |
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| SMILES = BrC#N |
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|ImageFile1_Ref = {{chemboximage|correct|??}} |
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| InChI = 1/CBrN/c2-1-3 |
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|ImageSize1 = 100 |
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| RTECS = GT2100000 |
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|ImageName1 = Spacefill model of cyanogen bromide |
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}} |
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|PIN = Carbononitridic bromide<ref>{{Cite web|title=Cyanogen Bromide – Compound Summary |url=https://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=10476#x291|work=PubChem Compound|publisher=National Center for Biotechnology Information|access-date=4 June 2012|location=USA|date=26 March 2005|at=Identification}}</ref> |
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| Section2 = {{Chembox Properties |
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|OtherNames = {{Unbulleted list|Bromine cyanide<ref name="Merck">{{cite book |title=The Merck Index|edition=10th|year=1983|publisher=Merck & Co. |location=Rahway, NJ|page=385}}</ref>|Campilit<ref>{{cite web|url=http://www.chemindustry.com/chemicals/0308894.html|title=Campilit, CAS Number: 506-68-3|access-date=2013-03-14|archive-date=2023-03-20|archive-url=https://web.archive.org/web/20230320080339/https://www.chemindustry.com/chemicals/0308894.html|url-status=dead}}</ref>| |
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| Formula = CBrN |
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}} |
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| MolarMass = 105.92 g/mol |
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|Section1 = {{Chembox Identifiers |
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| Appearance = Colorless to white solid with pungent odor |
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|CASNo = 506-68-3 |
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| Density = 2.015 g/cm<sup>3</sup> |
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|CASNo_Ref = {{cascite|correct|CAS}} |
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| MeltingPt = 52 °C |
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|UNII_Ref = {{fdacite|correct|FDA}} |
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| BoilingPt = 61.4 °C |
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|UNII = OS382OHJ8P |
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| Solubility = Hydrolysis |
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|PubChem = 10476 |
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| VaporPressure = 13 kPa (20 °C)<br> |
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|ChemSpiderID = 10044 |
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16.2 kPa (25 °C) |
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|ChemSpiderID_Ref = {{chemspidercite|correct|chemspider}} |
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}} |
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|EINECS = 208-051-2 |
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| Section3 = {{Chembox Hazards |
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|UNNumber = 1889 |
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| MainHazards = Very toxic |
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|MeSHName = Cyanogen+Bromide |
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| FlashPt = |
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|RTECS = GT2100000 |
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| Autoignition = |
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|Beilstein = 1697296 |
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| NFPA-H = 4 |
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|SMILES = BrC#N |
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|StdInChI = 1S/CBrN/c2-1-3 |
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| NFPA-R = 1 |
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|StdInChI_Ref = {{stdinchicite|correct|chemspider}} |
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| NFPA-O = |
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|StdInChIKey = ATDGTVJJHBUTRL-UHFFFAOYSA-N |
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| RPhrases = {{R14}}, {{R26/27/28}}, {{R34}}, {{R36/37/38}} |
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|StdInChIKey_Ref = {{stdinchicite|correct|chemspider}} |
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| SPhrases = {{S28}}, {{S36/37}}, {{S39}}, {{S45}}, {{S60}}, {{S61}} |
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}} |
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| PEL = 5 mg/m<sup>3</sup> |
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|Section2 = {{Chembox Properties |
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}} |
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|Formula = BrCN |
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|MolarMass = 105.921 g mol<sup>−1</sup> |
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|Appearance = Colorless solid |
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|Density = 2.015 g mL<sup>−1</sup> |
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|MeltingPtC = 50 to 53 |
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|BoilingPtC = 61 to 62 |
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|Solubility = Reacts |
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|VaporPressure = 16.2 kPa |
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}} |
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|Section3 = {{Chembox Thermochemistry |
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|DeltaHf = 136.1–144.7 kJ mol<sup>−1</sup> |
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}} |
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|Section4 = {{Chembox Hazards |
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|GHSPictograms = {{GHS corrosion}} {{GHS skull and crossbones}} {{GHS environment}} |
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|GHSSignalWord = '''DANGER''' |
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|HPhrases = {{H-phrases|300|310|314|330|410}} |
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|PPhrases = {{P-phrases|260|273|280|284|302+350}} |
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|NFPA-H = 4 |
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|NFPA-F = 0 |
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|NFPA-R = 1 |
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|PEL = 5 mg m<sup>−3</sup> |
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}} |
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| Section5 = {{Chembox Related |
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| OtherFunction_label = alkanenitriles |
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| OtherFunction = {{Unbulleted list|[[Hydrogen cyanide]]|[[Thiocyanic acid]]|[[Cyanogen iodide]]|[[Cyanogen chloride]]|[[Cyanogen fluoride]]|[[Acetonitrile]]|[[Aminoacetonitrile]]|[[Glycolonitrile]]|[[Cyanogen]]}} |
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}} |
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}} |
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'''Cyanogen bromide''' is the [[inorganic compound]] with the [[chemical formula|formula]] (CN)Br or BrCN. It is a colorless solid that is widely used to modify [[biopolymer]]s, fragment [[protein]]s and [[peptide]]s (cuts the C-terminus of methionine), and synthesize other compounds. The compound is classified as a [[pseudohalogen]]. |
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'''Cyanogen bromide''' is a chemical primarily used to 'activate' [[biopolymers]] such as [[polysaccharides]] for [[chromatography]] columns or proteins for [[immunoassay]]s. Activating proteins allows for easy covalent coupling to other reagents under mild conditions. CNBr reacts electrophilically with the pendant [[hydroxyl]] groups of proteins and polysaccharides, forming imidocarbonate derivatives and cyanate esters. The imidocarbonate groups then react with amino groups belonging to reagents (such as proteins being passed through the column) with the formation of stable covalent linkages, which are generally broken in a controlled fashion by changing [[pH]] during elution of the column. |
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== Synthesis, basic properties, and structure == |
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Although this method is widely used for immobilization of proteins onto agarose resin, there are drawbacks to the technique: the covalent bond is relatively unstable at pH extremes and there is a consistent leakage of the immobilized ligand from the resin during use. Also, the ligand is attached to the agarose resin by an isourea bond, which is positively charged at neutral pH, and imparts ion-exchange properties to the resin.<ref>1</ref> Reductive amination is often used as an alternative method to covalently immobilize proteins onto agarose supports. |
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The [[carbon]] atom in cyanogen bromide is bonded to [[bromine]] by a single bond and to [[nitrogen]] by a [[triple bond]] (i.e. {{chem2|Br\sC\tN}}). The compound is linear and polar, but it does not spontaneously ionize in water. It dissolves in both water and polar [[organic solvent]]s. |
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Cyanogen bromide can be prepared by [[oxidation]] of [[sodium cyanide]] with [[bromine]], which proceeds in two steps via the intermediate [[cyanogen]] ({{chem2|(CN)2}}): |
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Cyanogen bromide is also used to fragment [[protein]]s and [[peptide]]s because it hydrolyzes peptide bonds exclusively at the C-terminus of methionine residues, unlike other peptide-cleaving [[protease]]s such as [[Factor Xa]]. This is useful for peptide mapping in [[proteomics]]. It is disfavored due to the [[toxicity]] of cyanogen bromide, both in neat (solid) and solution forms. However, very little cyanogen bromide is required to quantitatively cleave proteins and peptides. It is also cheaper than Factor Xa. |
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:<chem>2 NaCN + Br2 -> (CN)2 + 2 NaBr</chem> |
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:<chem>(CN)2 + Br2 -> 2 (CN)Br</chem> |
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The mechanism is as follows: |
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When refrigerated the material has an extended shelflife. Like some other cyanogen compounds, cyanogen bromide undergoes an exothermic trimerisation to [[cyanuric bromide]] ({{chem2|(BrCN)3}}). This reaction is catalyzed by traces of bromine, metal salts, acids and bases. For this reason, experimentalists avoid brownish samples.<ref name=EROS/> |
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[[Image:cyanogenbromide.png]] |
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:[[File:Cyanuric bromide synthesis.PNG|300px]] |
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Cyanogen bromide is strongly [[electrophilic]] and is attacked by the [[nucleophilic]] [[sulfur]] on the methionine. The resulting adduct is now most electrophilic at the gamma-carbon position, and so the methionine attacks this carbon, forming a five-membered ring. Another reason that a six-membered ring does not form, even though it is generally more thermodynamically stable, is that the five-membered ring is more [[reaction kinetics|kinetically]] favorable and, most importantly, that the resonance form of a double bond between the alpha carbon and adjacent nitrogen is more stable outside the ring as opposed to inside the ring. |
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Cyanogen bromide is [[hydrolyzed]] to form [[hydrogen cyanate]] and [[hydrobromic acid]]: <chem display=block>(CN)Br + H2O -> HOCN + HBr</chem> |
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Though [[Cysteine]] also has a nucleophilic sulfur atom, it is not cleaved because the cyanide adduct becomes rapidly deprotonated. The sulfur is thus uncharged, and the beta carbon of the cysteine is not electrophilic. Instead, the strongest electrophile is the cyanide nitrogen, which is most likely attacked by water, liberating toxic [[hydrogen cyanide]] gas and the original cysteine. Cyanylation techniques (under different conditions) have also been reported which cleave proteins and peptides with a clean C-terminus at cysteine selectively in the presence of methionine. |
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== Biochemical applications == |
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Generally, cyanogen bromide cleaves 90% of bonds except those that are methionine followed by [[Serine]] or [[Threonine]]. |
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The main uses of cyanogen bromide are to immobilize proteins, fragment proteins by cleaving [[peptide bond]]s, and synthesize [[cyanamide]]s and other molecules. |
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[[File:CNBr activated matrices reaction.PNG|thumb|left|150px|Cyanogen bromide activation method]] |
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[[Image:homoserine lactone mechanisms.png]] |
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=== Protein immobilization === |
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This is due to a side reaction that destroys the methionine without cleavage. This problem can be circumvented by reducing acid concentration because water is required for cleaving the iminolactone intermediate. Dilution also drives the kinetics toward addition of water across the imine as opposed to the hydroxyl from the serine or threonine reacting with the imine. |
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Cyanogen bromide is often used to immobilize proteins by coupling them to [[reagent]]s such as [[agarose]] for [[affinity chromatography]].<ref name="immobilized">{{cite book | title = Immobilized Affinity Ligand Techniques |author1=Hermanson, G. T. |author2=Mallia, A. K. |author3=Smith, P. K. | publisher = Academic Press | year = 1992 | isbn = 978-0-12-342330-6}}</ref> Because of its simplicity and mild [[pH]] conditions, cyanogen bromide activation is the most common method for preparing affinity gels. Cyanogen bromide is also often used because it reacts with the [[hydroxyl]] groups on agarose to form [[cyanate]] [[ester]]s and [[imidocarbonate]]s. These groups are reacted with [[primary amine]]s in order to couple the protein onto the agarose matrix, as shown in the figure. Because cyanate esters are more reactive than are cyclic imidocarbonates, the amine will react mostly with the ester, yielding [[isourea]] derivatives, and partially with the less reactive imidocarbonate, yielding substituted imidocarbonates.<ref name="sigma">{{cite web | url = http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/c9210pis.pdf | title = Cyanogen Bromide Activated Matrices | publisher = Sigma}} {{dead link|date=June 2020}}</ref> |
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The disadvantages of this approach include the toxicity of cyanogen bromide and its sensitivity to oxidation. Also, cyanogen bromide activation involves the attachment of a [[ligand]] to agarose by an isourea bond, which is positively charged at neutral pH and thus unstable. Consequently, isourea derivatives may act as weak [[anion exchanger]]s.<ref name="sigma"/>{{dead link|date=June 2020}} |
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G |
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=== Protein cleavage === |
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{{Reflist}} |
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Cyanogen bromide hydrolyzes [[peptide bond]]s at the C-terminus of [[methionine]] residues. This reaction is used to reduce the size of [[polypeptide]] segments for identification and [[protein sequencing|sequencing]]. |
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''Immobilized Affinity Ligand Techniques.'' Greg T. Hermanson, A. Krishna Mallia and Paul K. Smith. Academic Press, ''©'' 1992. |
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==== Mechanism ==== |
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"Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds." Raymond Kaiser and Lorraine Metzka, ''Analytical Biochemistry'', January 1999. Vol. 266, No. 1. pp. 1-8, 15 June 2007. http://www.ingentaconnect.com/content/ap/ab/1999/00000266/00000001/art02945 |
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[[File:CNBr5.png|thumb|left|150px|Cyanogen bromide peptide bond cleavage]] |
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The [[electron density]] in cyanogen bromide is shifted away from the carbon atom, making it unusually [[electrophilic]], and towards the more [[electronegative]] bromine and nitrogen. This leaves the carbon particularly vulnerable to attack by a [[nucleophile]], and the cleavage reaction begins with a substitution reaction in which bromine is ultimately replaced by the sulfur in methionine. This attack is followed by the formation of a five-membered ring as opposed to a six-membered ring, which would entail the formation of a [[double bond]] in the ring between nitrogen and carbon. This double bond would result in a rigid ring conformation, thereby destabilizing the molecule. Thus, the five-membered ring is formed so that the double bond is outside the ring, as shown in the figure. |
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== See also == |
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*[[Cyanogen]] |
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*[[Cyanogen chloride]] |
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Although the nucleophilic sulfur in methionine is responsible for attacking BrCN, the sulfur in [[cysteine]] does not behave similarly. If the sulfur in cysteine attacked cyanogen bromide, the bromide ion would deprotonate the cyanide [[adduct]], leaving the sulfur uncharged and the beta carbon of the cysteine not electrophilic. The strongest electrophile would then be the cyanide carbon, which, if attacked by water, would yield [[cyanic acid]] and the original cysteine. |
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==== Reaction conditions ==== |
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==External links== |
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Cleaving proteins with BrCN requires using a [[buffer solution|buffer]] such as 0.1M HCl ([[hydrochloric acid]]) or 70% ([[formic acid]]).<ref>{{cite journal |author1=Schroeder, W. A. |author2=Shelton, J. B. |author3=Shelton, J. R. | title = An Examination of Conditions for the Cleavage of Polypeptide Chains with Cyanogen Bromide | journal = Archives of Biochemistry and Biophysics | year = 1969 | volume = 130 | issue = 1 | pages = 551–556 | doi = 10.1016/0003-9861(69)90069-1 |pmid=5778667}}</ref> These are the most common buffers for cleavage. An advantage to HCl is that formic acid causes the formation of formyl esters, which complicates protein characterization. However, formic is still often used because it dissolves most proteins. Also, the oxidation of methionine to [[methionine sulfoxide]], which is inert to BrCN attack, occurs more readily in HCl than in formic acid, possibly because formic acid is a reducing acid. Alternative buffers for cleavage include [[guanidine]] or [[urea]] in HCl because of their ability to [[protein folding|unfold proteins]], thereby making methionine more accessible to BrCN.<ref name="kaiser">{{ cite journal |author1=Kaiser, R. |author2=Metzka, L. | title = Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds | journal = Analytical Biochemistry | year = 1999 | volume = 266 | issue = 1 | pages = 1–8 | doi = 10.1006/abio.1998.2945 | pmid=9887207}}</ref> |
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Water is required for normal peptide bond cleavage of the [[iminolactone]] intermediate. In formic acid, cleavage of Met-[[serine|Ser]] and Met-[[threonine|Thr]] bonds is enhanced with increased water concentration because these conditions favor the addition of water across the [[imine]] rather than reaction of the side chain hydroxyl with the imine. Lowered pH tends to increase cleavage rates by inhibiting methionine side chain oxidation.<ref name="kaiser" /> |
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*[http://www.thieme-connect.com/ejournals/pdf/synlett/doi/10.1055/s-2005-869872.pdf Usage of cyanogen bromide] |
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*[http://ptcl.chem.ox.ac.uk/MSDS/CY/cyanogen_bromide.html Safety MSDS data] |
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*[http://www.jtbaker.com/msds/englishhtml/C6600.htm MSDS at www.jtbaker.com] |
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*[http://www.jbc.org/cgi/reprint/173/2/503.pdf Thiamine and the cyanogen bromide reaction] |
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==== Side reactions ==== |
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When methionine is followed by [[serine]] or [[threonine]], side reactions can occur that destroy the methionine without peptide [[bond cleavage]]. Normally, once the iminolactone is formed (refer to figure), water and acid can react with the imine to cleave the peptide bond, forming a [[homoserine lactone]] and new C-terminal peptide. However, if the adjacent amino acid to methionine has a [[hydroxyl]] or [[sulfhydryl]] group, this group can react with the imine to form a homoserine without peptide bond cleavage.<ref name="kaiser" /> These two cases are shown in the figure. |
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== Organic synthesis == |
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[[Category:Bromides]] |
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Cyanogen bromide is a common reagent in [[organic synthesis]]. In most reactions, it acts as a source of electrophilic [[cyanide|cyanogen]] and nucleophilic [[bromide]]; carbocations preferentially attack the nitrogen atom.<ref name=EROS/> In the presence of a Lewis acid, it cyanidates [[arene]]s.<ref name=SynLett/> |
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[[Category:Cyanides]] |
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[[Category:Nitrogen compounds]] |
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[[Category:Nonmetal halides]] |
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BrCN converts [[alcohols]] to [[cyanates]]; [[amine]]s to [[cyanamide]]s or [[dicyanamide]]s.<ref name=EROS>{{cite encyclopedia|entry=Cyanogen Bromide|author1=Joel Morris|author2= Lajos Kovács|author3=Kouichi Ohe|encyclopedia=Encyclopedia of Reagents for Organic Synthesis|year=2015|doi=10.1002/047084289X.rc269.pub3|isbn=978-0471936237}}</ref> Excess BrCN continues the reaction to [[guanidine]]s; [[hydroxylamine]]s yield [[hydroxyguanidine]]s similarly.<ref name=SynLett/> |
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[[de:Bromcyanid]] |
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The cyanamides so formed [[umpolung|umpole]] the original amine, and tends to eliminate alkyl substituents. In the [[von Braun reaction]], tertiary amines react with cyanogen bromide to yield disubstituted cyanamides and an alkyl bromide.<ref name=SynLett>{{ cite journal | author = Kumar, V. | title = Cyanogen Bromide (CNBr) | journal = Synlett | year = 2005 | volume = 2005 | issue = 10 | pages = 1638–1639 | doi = 10.1055/s-2005-869872 | url = http://www.thieme-connect.com/ejournals/pdf/synlett/doi/10.1055/s-2005-869872.pdf | id = Art ID: V12705ST | doi-access = free }}</ref> That net reaction is similar to the [[Polonovski reaction|Polonovski elimination]], but does not require N-oxidation.<ref name=EROS/> |
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In bromocyanation, BrCN adds across multiple bonds to give a vicinal cyanobromide. Bromocyanated [[enol]]s spontaneously undergo a [[Darzens reaction|Darzens-like elimination]] to an epoxynitrile.<ref name=EROS/> |
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Cyanogen bromide is also a dehydrating agent, hydrolyzing to [[hydrogen bromide]] and [[cyanic acid]].<ref name=SynLett /> |
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The compound is used in the synthesis of [[4-Methylaminorex|4-methylaminorex]] ("ice") and [[viroxime]]. |
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== Toxicity, storage, and deactivation == |
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Cyanogen bromide can be stored under dry conditions at 2 to 8 °C for extended periods.<ref name="sigma" /> |
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Cyanogen bromide is volatile, and readily absorbed through the [[skin]] or [[gastrointestinal tract]]. Therefore, toxic exposure may occur by inhalation, physical contact, or ingestion. It is acutely toxic, causing a variety of [[nonspecific symptoms]]. Exposure to even small amounts may cause convulsions or death. LD<sub>50</sub> orally in rats is reported as 25–50 mg/kg.<ref name="NIH">{{cite web | url = http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+708 | title = Cyanogen Bromide HSDB 708 | work = HSDB | publisher = NIH / NLM | date = 2009-04-07}}</ref> |
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The recommended method to deactivate cyanogen bromide is with [[sodium hydroxide]] and [[bleach]].<ref>{{ cite journal |author1=Lunn, G. |author2=Sansone, E. B. | title = Destruction of Cyanogen Bromide and Inorganic Cyanides | journal = [[Analytical Biochemistry (journal)|Analytical Biochemistry]] | year = 1985 | volume = 147 | issue = 1 | pages = 245–250 | doi = 10.1016/0003-2697(85)90034-X | pmid = 4025821 |url=https://zenodo.org/record/1253784 }}</ref> The aqueous alkali hydroxide instantly hydrolyzes (CN)Br to alkali cyanide and bromide. The cyanide can then be oxidized by [[sodium hypochlorite|sodium]] or [[calcium hypochlorite]] to the less toxic cyanate ion. Deactivation is extremely [[exothermic]] and may be explosive.<ref name="NIH" /> |
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== References == |
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{{Reflist}} |
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== Further reading == |
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* {{ cite journal |author1=Gross, E. |author2=Witkop, B. | title = Nonenzymatic Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic Ribonuclease | journal = [[Journal of Biological Chemistry]] | year = 1962 | volume = 237 | issue = 6 | pages = 1856–1860 |doi=10.1016/S0021-9258(19)73948-9 | pmid = 13902203 | url = http://www.jbc.org/content/237/6/1856.full.pdf |doi-access=free }} |
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* {{ cite journal | author1 = Inglis, A. S. | author2 = Edman, P. | author-link2 = Pehr Edman | title = Mechanism of Cyanogen Bromide Reaction with Methionine in Peptides and Proteins | journal = [[Analytical Biochemistry (journal)|Analytical Biochemistry]] | year = 1970 | volume = 37 | issue = 1 | pages = 73–80 | doi = 10.1016/0003-2697(70)90259-9 | pmid = 5506566 }} |
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== External links == |
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* {{ cite web | url = http://hazard.com/msds/mf/baker/baker/files/c6600.htm | title = Cyanogen Bromide MSDS Number: C6600 | date = 1996-08-12 | publisher = J. T. Baker }} |
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* {{cite journal | author = Teeri, A. E. | title = Thiamine and the Cyanogen Bromide Reaction | journal = Journal of Biological Chemistry | year = 1948 | volume = 173 | issue = 2 | pages = 503–505 | doi = 10.1016/S0021-9258(18)57422-6 | pmid = 18910706 | doi-access = free }} |
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{{Chemical agents}} |
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{{Cyanides}} |
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[[Category:Bromine compounds]] |
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[[Category:Triatomic molecules]] |
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[[Category:Cyano compounds]] |
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[[Category:Nonmetal halides]] |
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[[Category:Blood agents]] |
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[[Category:Lachrymatory agents]] |
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[[Category:Pseudohalogens]] |
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Latest revision as of 13:24, 26 August 2024
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| Names | |
|---|---|
| Preferred IUPAC name
Carbononitridic bromide[3] | |
| Other names | |
| Identifiers | |
3D model (JSmol)
|
|
| 1697296 | |
| ChemSpider | |
| ECHA InfoCard | 100.007.320 |
| EC Number |
|
| MeSH | Cyanogen+Bromide |
PubChem CID
|
|
| RTECS number |
|
| UNII | |
| UN number | 1889 |
CompTox Dashboard (EPA)
|
|
| |
| |
| Properties | |
| BrCN | |
| Molar mass | 105.921 g mol−1 |
| Appearance | Colorless solid |
| Density | 2.015 g mL−1 |
| Melting point | 50 to 53 °C (122 to 127 °F; 323 to 326 K) |
| Boiling point | 61 to 62 °C (142 to 144 °F; 334 to 335 K) |
| Reacts | |
| Vapor pressure | 16.2 kPa |
| Thermochemistry | |
Std enthalpy of
formation (ΔfH⦵298) |
136.1–144.7 kJ mol−1 |
| Hazards | |
| GHS labelling: | |
  
| |
| Danger | |
| H300, H310, H314, H330, H410 | |
| P260, P273, P280, P284, P302+P350 | |
| NFPA 704 (fire diamond) | |
| NIOSH (US health exposure limits): | |
PEL (Permissible)
|
5 mg m−3 |
| Related compounds | |
Related alkanenitriles
|
|
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
| |
Cyanogen bromide is the inorganic compound with the formula (CN)Br or BrCN. It is a colorless solid that is widely used to modify biopolymers, fragment proteins and peptides (cuts the C-terminus of methionine), and synthesize other compounds. The compound is classified as a pseudohalogen.
Synthesis, basic properties, and structure
[edit]The carbon atom in cyanogen bromide is bonded to bromine by a single bond and to nitrogen by a triple bond (i.e. Br−C≡N). The compound is linear and polar, but it does not spontaneously ionize in water. It dissolves in both water and polar organic solvents.
Cyanogen bromide can be prepared by oxidation of sodium cyanide with bromine, which proceeds in two steps via the intermediate cyanogen ((CN)2):
When refrigerated the material has an extended shelflife. Like some other cyanogen compounds, cyanogen bromide undergoes an exothermic trimerisation to cyanuric bromide ((BrCN)3). This reaction is catalyzed by traces of bromine, metal salts, acids and bases. For this reason, experimentalists avoid brownish samples.[4]
Cyanogen bromide is hydrolyzed to form hydrogen cyanate and hydrobromic acid:
Biochemical applications
[edit]The main uses of cyanogen bromide are to immobilize proteins, fragment proteins by cleaving peptide bonds, and synthesize cyanamides and other molecules.
Protein immobilization
[edit]Cyanogen bromide is often used to immobilize proteins by coupling them to reagents such as agarose for affinity chromatography.[5] Because of its simplicity and mild pH conditions, cyanogen bromide activation is the most common method for preparing affinity gels. Cyanogen bromide is also often used because it reacts with the hydroxyl groups on agarose to form cyanate esters and imidocarbonates. These groups are reacted with primary amines in order to couple the protein onto the agarose matrix, as shown in the figure. Because cyanate esters are more reactive than are cyclic imidocarbonates, the amine will react mostly with the ester, yielding isourea derivatives, and partially with the less reactive imidocarbonate, yielding substituted imidocarbonates.[6]
The disadvantages of this approach include the toxicity of cyanogen bromide and its sensitivity to oxidation. Also, cyanogen bromide activation involves the attachment of a ligand to agarose by an isourea bond, which is positively charged at neutral pH and thus unstable. Consequently, isourea derivatives may act as weak anion exchangers.[6][dead link]
Protein cleavage
[edit]Cyanogen bromide hydrolyzes peptide bonds at the C-terminus of methionine residues. This reaction is used to reduce the size of polypeptide segments for identification and sequencing.
Mechanism
[edit]
The electron density in cyanogen bromide is shifted away from the carbon atom, making it unusually electrophilic, and towards the more electronegative bromine and nitrogen. This leaves the carbon particularly vulnerable to attack by a nucleophile, and the cleavage reaction begins with a substitution reaction in which bromine is ultimately replaced by the sulfur in methionine. This attack is followed by the formation of a five-membered ring as opposed to a six-membered ring, which would entail the formation of a double bond in the ring between nitrogen and carbon. This double bond would result in a rigid ring conformation, thereby destabilizing the molecule. Thus, the five-membered ring is formed so that the double bond is outside the ring, as shown in the figure.
Although the nucleophilic sulfur in methionine is responsible for attacking BrCN, the sulfur in cysteine does not behave similarly. If the sulfur in cysteine attacked cyanogen bromide, the bromide ion would deprotonate the cyanide adduct, leaving the sulfur uncharged and the beta carbon of the cysteine not electrophilic. The strongest electrophile would then be the cyanide carbon, which, if attacked by water, would yield cyanic acid and the original cysteine.
Reaction conditions
[edit]Cleaving proteins with BrCN requires using a buffer such as 0.1M HCl (hydrochloric acid) or 70% (formic acid).[7] These are the most common buffers for cleavage. An advantage to HCl is that formic acid causes the formation of formyl esters, which complicates protein characterization. However, formic is still often used because it dissolves most proteins. Also, the oxidation of methionine to methionine sulfoxide, which is inert to BrCN attack, occurs more readily in HCl than in formic acid, possibly because formic acid is a reducing acid. Alternative buffers for cleavage include guanidine or urea in HCl because of their ability to unfold proteins, thereby making methionine more accessible to BrCN.[8]
Water is required for normal peptide bond cleavage of the iminolactone intermediate. In formic acid, cleavage of Met-Ser and Met-Thr bonds is enhanced with increased water concentration because these conditions favor the addition of water across the imine rather than reaction of the side chain hydroxyl with the imine. Lowered pH tends to increase cleavage rates by inhibiting methionine side chain oxidation.[8]
Side reactions
[edit]When methionine is followed by serine or threonine, side reactions can occur that destroy the methionine without peptide bond cleavage. Normally, once the iminolactone is formed (refer to figure), water and acid can react with the imine to cleave the peptide bond, forming a homoserine lactone and new C-terminal peptide. However, if the adjacent amino acid to methionine has a hydroxyl or sulfhydryl group, this group can react with the imine to form a homoserine without peptide bond cleavage.[8] These two cases are shown in the figure.
Organic synthesis
[edit]Cyanogen bromide is a common reagent in organic synthesis. In most reactions, it acts as a source of electrophilic cyanogen and nucleophilic bromide; carbocations preferentially attack the nitrogen atom.[4] In the presence of a Lewis acid, it cyanidates arenes.[9]
BrCN converts alcohols to cyanates; amines to cyanamides or dicyanamides.[4] Excess BrCN continues the reaction to guanidines; hydroxylamines yield hydroxyguanidines similarly.[9]
The cyanamides so formed umpole the original amine, and tends to eliminate alkyl substituents. In the von Braun reaction, tertiary amines react with cyanogen bromide to yield disubstituted cyanamides and an alkyl bromide.[9] That net reaction is similar to the Polonovski elimination, but does not require N-oxidation.[4]
In bromocyanation, BrCN adds across multiple bonds to give a vicinal cyanobromide. Bromocyanated enols spontaneously undergo a Darzens-like elimination to an epoxynitrile.[4]
Cyanogen bromide is also a dehydrating agent, hydrolyzing to hydrogen bromide and cyanic acid.[9]
The compound is used in the synthesis of 4-methylaminorex ("ice") and viroxime.
Toxicity, storage, and deactivation
[edit]Cyanogen bromide can be stored under dry conditions at 2 to 8 °C for extended periods.[6]
Cyanogen bromide is volatile, and readily absorbed through the skin or gastrointestinal tract. Therefore, toxic exposure may occur by inhalation, physical contact, or ingestion. It is acutely toxic, causing a variety of nonspecific symptoms. Exposure to even small amounts may cause convulsions or death. LD50 orally in rats is reported as 25–50 mg/kg.[10]
The recommended method to deactivate cyanogen bromide is with sodium hydroxide and bleach.[11] The aqueous alkali hydroxide instantly hydrolyzes (CN)Br to alkali cyanide and bromide. The cyanide can then be oxidized by sodium or calcium hypochlorite to the less toxic cyanate ion. Deactivation is extremely exothermic and may be explosive.[10]
References
[edit]- ^ The Merck Index (10th ed.). Rahway, NJ: Merck & Co. 1983. p. 385.
- ^ "Campilit, CAS Number: 506-68-3". Archived from the original on 2023-03-20. Retrieved 2013-03-14.
- ^ "Cyanogen Bromide – Compound Summary". PubChem Compound. USA: National Center for Biotechnology Information. 26 March 2005. Identification. Retrieved 4 June 2012.
- ^ a b c d e Joel Morris; Lajos Kovács; Kouichi Ohe (2015). "Cyanogen Bromide". Encyclopedia of Reagents for Organic Synthesis. doi:10.1002/047084289X.rc269.pub3. ISBN 978-0471936237.
- ^ Hermanson, G. T.; Mallia, A. K.; Smith, P. K. (1992). Immobilized Affinity Ligand Techniques. Academic Press. ISBN 978-0-12-342330-6.
- ^ a b c "Cyanogen Bromide Activated Matrices" (PDF). Sigma. [dead link]
- ^ Schroeder, W. A.; Shelton, J. B.; Shelton, J. R. (1969). "An Examination of Conditions for the Cleavage of Polypeptide Chains with Cyanogen Bromide". Archives of Biochemistry and Biophysics. 130 (1): 551–556. doi:10.1016/0003-9861(69)90069-1. PMID 5778667.
- ^ a b c Kaiser, R.; Metzka, L. (1999). "Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds". Analytical Biochemistry. 266 (1): 1–8. doi:10.1006/abio.1998.2945. PMID 9887207.
- ^ a b c d Kumar, V. (2005). "Cyanogen Bromide (CNBr)" (PDF). Synlett. 2005 (10): 1638–1639. doi:10.1055/s-2005-869872. Art ID: V12705ST.
- ^ a b "Cyanogen Bromide HSDB 708". HSDB. NIH / NLM. 2009-04-07.
- ^ Lunn, G.; Sansone, E. B. (1985). "Destruction of Cyanogen Bromide and Inorganic Cyanides". Analytical Biochemistry. 147 (1): 245–250. doi:10.1016/0003-2697(85)90034-X. PMID 4025821.
Further reading
[edit]- Gross, E.; Witkop, B. (1962). "Nonenzymatic Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic Ribonuclease" (PDF). Journal of Biological Chemistry. 237 (6): 1856–1860. doi:10.1016/S0021-9258(19)73948-9. PMID 13902203.
- Inglis, A. S.; Edman, P. (1970). "Mechanism of Cyanogen Bromide Reaction with Methionine in Peptides and Proteins". Analytical Biochemistry. 37 (1): 73–80. doi:10.1016/0003-2697(70)90259-9. PMID 5506566.
External links
[edit]- "Cyanogen Bromide MSDS Number: C6600". J. T. Baker. 1996-08-12.
- Teeri, A. E. (1948). "Thiamine and the Cyanogen Bromide Reaction". Journal of Biological Chemistry. 173 (2): 503–505. doi:10.1016/S0021-9258(18)57422-6. PMID 18910706.