Cyanogen bromide: Difference between revisions

{{chembox

| Watchedfields = changed

| verifiedrevid = 401970390

| ImageFile = Cyanogen-bromide-2D.png

| ImageSize =

| ImageFile1 = Cyanogen-bromide-3D-vdW.png

| ImageSize1 =

| IUPACName = cyanobromane<br />bromidonitridocarbon

| OtherNames = Bromine cyanide, Bromocyanide, Cyanobromide, Carbononitridic bromide, Bromocyan, Bromocyanogen, Campilit, UN1889, CB

| Section1 = {{Chembox Identifiers

| ChemSpiderID_Ref = {{chemspidercite|correct|chemspider}}

| ChemSpiderID = 10044

| InChIKey = ATDGTVJJHBUTRL-UHFFFAOYAI

| StdInChI_Ref = {{stdinchicite|correct|chemspider}}

| StdInChI = 1S/CBrN/c2-1-3

| StdInChIKey_Ref = {{stdinchicite|correct|chemspider}}

| StdInChIKey = ATDGTVJJHBUTRL-UHFFFAOYSA-N

| CASNo = 506-68-3

| CASNo_Ref = {{cascite|correct|CAS}}

| EINECS = 208-051-2

| PubChem = 10476

| SMILES = BrC#N

| InChI = 1/CBrN/c2-1-3

| RTECS = GT2100000

}}

| Section2 = {{Chembox Properties

| C = 1

| Br = 1

| N = 1

| Appearance = Colorless to white solid with pungent odor

| Density = 2.015 g/cm³

| MeltingPtC = 52

| BoilingPtC = 61.4

| Solubility = Hydrolysis

| SolubleOther = soluble in [[alcohol]] and [[ether]] <ref>Pradyot Patnaik. ''Handbook of Inorganic Chemicals''. McGraw-Hill, 2002, ISBN 0070494398</ref>

| VaporPressure = 13 kPa (20 °C)<br />16.2 kPa (25 °C)

}}

| Section3 = {{Chembox Hazards

| MainHazards = Very toxic

| EUIndex = Not listed

| FlashPt =

| Autoignition =

| NFPA-H = 4

| NFPA-F = 0

| NFPA-R = 1

| NFPA-O =

| RPhrases =

| SPhrases =

| PEL = 5 mg/m³

}}

| Section8 = {{Chembox Related

| Function = cyanogen halides

| OtherFunctn = [[Cyanogen fluoride]]<br />[[Cyanogen chloride]]<br />[[Cyanogen iodide]]

| OtherCpds = [[Cyanogen]]

}}

'''Cyanogen bromide''' is a [[pseudohalogen]] [[Chemical compound|compound]] with the [[chemical formula|formula]] CNBr. It is a colorless solid that is widely used to modify [[biopolymer]]s, fragment [[protein]]s and [[peptide]]s, and synthesize other compounds.

==Synthesis, basic properties, and structure==

Although the formula is most commonly written CNBr, the [[carbon]] atom is actually bonded to [[bromine]] by a single bond and to [[nitrogen]] by a [[triple bond]] (i.e. Br&ndash;C≡N). The compound is linear and quite polar, but it does not spontaneously ionize in water. Therefore, it dissolves in both water and polar [[organic solvent]]s.

Cyanogen bromide can be prepared by [[oxidation]] of [[sodium cyanide]] with [[bromine]], which proceeds in two steps via the intermediate [[cyanogen]] ((CN)₂ or N≡C&ndash;C≡N).

:2 NaCN + Br₂ → (CN)₂ + 2 NaBr

:(CN)₂ + Br₂ → 2 BrCN

Cyanogen bromide is [[hydrolyzed]] by water to release [[hydrogen cyanide]] and [[hypobromous acid]]

:BrCN + H₂O → HCN + HOBr

==Biochemical applications==

[[Image:CNBr activated matrices reaction.PNG|thumb|left|150px|Cyanogen bromide activation method]]

===Protein immobilization===

Cyanogen bromide is often used to immobilize proteins by coupling them to [[reagent]]s such as [[agarose]] for [[affinity chromatography]].<ref name = "immobilized">''Immobilized Affinity Ligand Techniques.'' Greg T. Hermanson, A. Krishna Mallia and Paul K. Smith. Academic Press, ''©'' 1992.</ref> Because of its simplicity and mild [[pH]] conditions, cyanogen bromide activation is the most common method for preparing affinity gels. CNBr 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">[http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/c9210pis.pdf "Cyanogen Bromide Activated Matrices."] Sigma Product Information (Sigma).</ref>

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"/>

===Protein cleavage===

====Mechanism====

[[Image:CNBr5.png|thumb|left|150px|Cyanogen bromide peptide bond cleavage]]

In CNBr, the [[electron density]] 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 [[nucleophilic acyl 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 CNBr, 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 nitrogen, which, if attacked by water, would yield [[cyanic acid]] and the original cysteine.

====Reaction conditions====

Cleaving proteins with CNBr requires using a [[buffer solution|buffer]] such as 0.1M HCl ([[hydrochloric acid]]) or 70% ([[formic acid]]).<ref>Schroeder, W.A., Joan Balog Shelton, and J. Roger Shelton (1969). [http://proxy.library.upenn.edu:8111/science?_ob=ArticleURL&_udi=B6WB5-4DW2F04-T3&_user=489256&_coverDate=12%2F31%2F1969&_rdoc=69&_fmt=summary&_orig=browse&_srch=doc-info(%23toc%236701%231969%23998699999%23530622%23FLP%23display%23Volume)&_cdi=6701&_sort=d&_docanchor=&_ct=97&_acct=C000022721&_version=1&_urlVersion=0&_userid=489256&md5=20fea515d50a4100e64e0a7fc76fe570 "An Examination of Conditions for the Cleavage of Polypeptide Chains with Cyanogen Bromide."] Archives of Biochemistry and Biophysics 130 (1): 551-6.</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 CNBr 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 CNBr.<ref name = "kaiser">Kaiser, Raymond and Lorraine Metzka (1999). [http://www.ingentaconnect.com/content/ap/ab/1999/00000266/00000001/art02945 "Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds."] Analytical Biochemistry 266 (1): 1-8.</ref>

Note that 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"/>

====Side reactions====

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 N-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.

===Organic synthesis===

Cyanogen bromide is also widely used in [[organic synthesis]]. As stated earlier, the reagent is prone to attack by nucleophiles such as amines and alcohols because of the electrophilic carbon. In the synthesis of [[cyanamide]]s and [[dicyanamide]]s, primary and secondary amines react with CNBr to yield mono- and dialkylcyanamides, which can further react with amines and [[hydroxylamine]] to yield [[guanidine]]s and [[hydroxyguanidine]]s. In the [[von Braun reaction]], tertiary amines react with CNBr to yield disubstituted cyanamides and an alkyl bromide. CNBr can be used to prepare [[aryl]] [[nitrile]]s, nitriles, [[anhydride]]s, and [[cyanate]]s. It can also serve as a cleaving agent.<ref>Kumar, Vinod (2005).[http://www.thieme-connect.com/ejournals/pdf/synlett/doi/10.1055/s-2005-869872.pdf "Cyanogen Bromide (CNBr)."] Thieme 10 (1): 1638-9.</ref>

==Toxicity, storage, and deactivation==

Cyanogen bromide is moisture-sensitive but can be stored under dry conditions at 2 to 8 °C for extended periods of time.<ref name = "sigma"/>

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&nbsp;mg/kg.<ref name = "NIH">[http://dohs.ors.od.nih.gov/pdf/Cyanogen%20bromide.pdf "Cyanogen Bromide."] NIH Division of Occupational Health and Safety.</ref>

To deactivate CNBr in a solution not exceeding 60 g/L of CNBr (dilute if necessary), the recommended method is to add 1&nbsp;mol/L NaOH and 1&nbsp;mol/L NaOCl in volumes of ratio 1:1:2 (CNBr solution:NaOH:NaOCl).<ref>{{cite journal | author = Lunn, George and Eric B. Sansone | year = 1985 | title = Destruction of Cyanogen Bromide and Inorganic Cyanides | journal = [[Analytical Biochemistry (journal)|Analytical Biochemistry]] | doi = 10.1016/0003-2697(85)90034-X | volume = 147 | pages = 245 | pmid=4025821}}</ref> The aqueous alkali hydroxide instantly hydrolyzes CNBr to alkali cyanide and bromide. The cyanide can then be oxidized by [[sodium hypochlorite|sodium]] or [[calcium hypochlorite]] to the less toxic cyanate ion. Note that deactivation is extremely [[exothermic]] and may be explosive.<ref name = "NIH"/>

== See also ==

*[[Cyanogen]]

*[[Cyanogen chloride]]

==References==

{{reflist}}

==Further reading==

* {{Cite journal

| author = Erhard Gross; Bernhard Witkop

| title = Nonenzymatic Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic Ribonuclease.

| journal = [[Journal of Biological Chemistry]]

| volume = 237

| issue = 6

| pages = 1856–60

| date = June 1, 1962 | url = http://www.jbc.org/cgi/reprint/237/6/1856

| pmid = 13902203

}}

* {{Cite journal

| last1 = A.S. Inglis; [[Pehr Edman|P. Edman]]

| title = Mechanism of Cyanogen Bromide Reaction with Methionine in Peptides and Proteins.

| journal = [[Analytical Biochemistry (journal)|Analytical Biochemistry]]

| volume = 37

| issue = 1

| pages = 73–80

| year = 1970

| doi = 10.1016/0003-2697(70)90259-9

| first1 = A

| last2 = Edman

| first2 = P

| pmid=5506566}}

==External links==

*[http://ptcl.chem.ox.ac.uk/MSDS/CY/cyanogen_bromide.html Safety MSDS data]

*[http://hazard.com/msds/mf/baker/baker/files/c6600.htm MSDS at www.jtbaker.com]

*[http://www.jbc.org/cgi/reprint/173/2/503.pdf Thiamine and the cyanogen bromide reaction]

[[Category:Bromides]]

[[Category:Cyanides]]

[[Category:Inorganic nitrogen compounds]]

[[de:Bromcyan]]

[[he:ציאנוגן ברומיד]]

[[nl:Cyanogeenbromide]]

[[ja:臭化シアン]]

[[pl:Bromocyjan]]

[[zh:溴化氰]]