TCEP: Difference between revisions

{{cs1 config|name-list-style=vanc}}

{{for|the flame retardant chemical|tris(2-chloroethyl) phosphate}}

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| Watchedfields = changed

| verifiedrevid = 423494642

| ImageSize = 220px

| ImageAlt = Skeletal formula of TCEP

| ImageFile1 = TCEP-3D-spacefill.png

| ImageAlt1 = Space-filling model of the TCEP molecule

| PIN = 3,3′,3′′-Phosphanetriyltripropanoic acid

|Section1={{Chembox Identifiers

| CASNo_Ref = {{cascite|correct|??}}

| CASNo = 51805-45-9

| CASNo_Comment = (hydrochloride salt)

| UNII_Ref = {{fdacite|correct|FDA}}

| UNII = H49AAM893K

| PubChem = 119411

| ChemSpiderID_Ref = {{chemspidercite|changed|chemspider}}

| ChemSpiderID = 106653

| InChI = 1/C9H15O6P/c10-7(11)1-4-16(5-2-8(12)13)6-3-9(14)15/h1-6H2,(H,10,11)(H,12,13)(H,14,15)

| InChIKey = PZBFGYYEXUXCOF-UHFFFAOYAQ

| StdInChI_Ref = {{stdinchicite|changed|chemspider}}

| StdInChI = 1S/C9H15O6P/c10-7(11)1-4-16(5-2-8(12)13)6-3-9(14)15/h1-6H2,(H,10,11)(H,12,13)(H,14,15)

| StdInChIKey_Ref = {{stdinchicite|changed|chemspider}}

| StdInChIKey = PZBFGYYEXUXCOF-UHFFFAOYSA-N

| SMILES = OC(CCP(CCC(O)=O)CCC(O)=O)=O

|Section2={{Chembox Properties

| C=9 | H=15 | O=6 | P=1

| Appearance =

| Density =

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|Section3={{Chembox Hazards

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'''TCEP''' ('''tris(2-carboxyethyl)phosphine''') is a [[reducing agent]] frequently used in [[biochemistry]] and [[molecular biology]] applications. It is often prepared and used as a [[hydrochloride]] [[salt (chemistry)|salt]] (TCEP-HCl) with a molecular weight of 286.65 gram/mol. It is soluble in water and available as a stabilized solution at neutral pH and immobilized onto an [[agarose]] support to facilitate removal of the reducing agent.

Compared to the other two most common agents used for this purpose ([[dithiothreitol]] and [[2-Mercaptoethanol|β-mercaptoethanol]]), TCEP has the advantages of being odorless, a more powerful reducing agent, an irreversible reducing agent (in the sense that TCEP does not regenerate—the end product of TCEP-mediated disulfide cleavage is in fact two free thiols/cysteines), more hydrophilic, and more resistant to oxidation in air.<ref name=FT-242214>[http://www.interchim.fr/ft/2/242214.pdf TCEP technical information], from [[Interchim]]</ref> It also does not reduce metals used in [[immobilized metal affinity chromatography]].

TCEP is particularly useful when labeling cysteine residues with [[maleimide]]s. TCEP can keep the cysteines from forming di-sulfide bonds and, unlike [[dithiothreitol]] and [[2-Mercaptoethanol|β-mercaptoethanol]], it will not react as readily with the maleimide.<ref name=FT-242214 /> However, TCEP has been reported to react with maleimide under certain conditions.<ref>{{cite journal |author1=Shafer, D. E. |author2=Inman, J. K. |author3=Lees, A. | year = 2002 | title = Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with Maleimide and α-Haloacyl Groups: Anomalous Elution of TCEP by Gel Filtration | volume = 282 | pmid = 10860517 | issue = 1 | pages = 161–164 | doi = 10.1006/abio.2000.4609 | journal = Anal. Biochem.|s2cid=37825047 }}</ref><ref>{{cite journal |vauthors=Tyagarajan K, Pretzer E, Wiktorowicz JE | year = 2003 | title = Thiol-reactive dyes for fluorescence labeling of proteomic samples | volume = 24 | pmid = 12874870 | issue = 14 | pages = 2348–2358 | doi = 10.1002/elps.200305478 | journal = Electrophoresis| s2cid = 20446141 }}</ref>

TCEP is also used in the tissue homogenization process for RNA isolation.<ref>{{cite journal|pmid=14715294|year=2004|last1=Rhee|first1= S. S.|last2=Burke|first2=D. H.|title=Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry|journal=Anal. Biochem.|volume=325|issue=1|pages=137–143|doi=10.1016/j.ab.2003.10.019}}</ref>

For [[Ultraviolet–visible spectroscopy]] applications, TCEP is useful when it is important to avoid interfering absorbance from 250 to 285 nanometers which can occur with [[dithiothreitol]]. Dithiothreitol will slowly over time absorb more and more light in this spectrum as various redox reactions occur.

==History==

Reduction of biomolecules with [[trialkyphosphines]] received little attention for decades because historically available phosphines were extremely malodorous and/or insoluble in water.<ref name="Han1994">{{cite journal | last1=Han | first1=J.C. | last2=Han | first2=G.Y. | title=A Procedure for Quantitative Determination of Tris(2-Carboxyethyl)phosphine, an Odorless Reducing Agent More Stable and Effective Than Dithiothreitol | journal=Analytical Biochemistry | publisher=Elsevier BV | volume=220 | issue=1 | year=1994 | issn=0003-2697 | doi=10.1006/abio.1994.1290 | pages=5–10| pmid=7978256 }}</ref> In 1969, TCEP was reported as an oderless and water-soluble trialkyphosphine suitable for biochemical use,<ref name="Levison1969">{{cite journal | last1=Levison | first1=M. E. | last2=Josephson | first2=A. S. | last3=Kirschenbaum | first3=D. M. | title=Reduction of biological substances by water-soluble phosphines: Gamma-globulin (IgG) | journal=Experientia | publisher=Springer Science and Business Media LLC | volume=25 | issue=2 | year=1969 | issn=0014-4754 | doi=10.1007/bf01899076 | pages=126–127| pmid=4182166 | s2cid=20548859 }}</ref> however the potential use of TCEP for biochemical applications was almost totally ignored for decades. In 1991, Burns reported a new convenient synthetic procedure for TCEP,<ref name="Burns Butler Moran Whitesides 1991 pp. 2648–2650">{{cite journal | last1=Burns | first1=John A. | last2=Butler | first2=James C. | last3=Moran | first3=John | last4=Whitesides | first4=George M. | title=Selective reduction of disulfides by tris(2-carboxyethyl)phosphine | journal=The Journal of Organic Chemistry | publisher=American Chemical Society (ACS) | volume=56 | issue=8 | year=1991 | issn=0022-3263 | doi=10.1021/jo00008a014 | pages=2648–2650}}</ref> which set off TCEP becoming more widely available and marketed as a "new" reducing agent for biochemical use, & thus TCEP came into more widespread use throughout the 1990s.<ref name="Han1994"></ref>

==Reactions==

TCEPT will reduce a [[disulfide]]s to thiols in the presence of water:

:[[File:TCEP reaction reducing a disulfide bond.svg|TCEP reaction showing it reducing a disulfide bond]]

Some other side reactions have also been reported:

* Conversion of a [[cysteine]] residue into [[alanine]] in the presence of TCEP and heat (90˚C).<ref name="Wang2010">{{cite journal | last1=Wang | first1=Zhouxi | last2=Rejtar | first2=Tomas | last3=Zhou | first3=Zhaohui Sunny | last4=Karger | first4=Barry L. | title=Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry | journal=Rapid Communications in Mass Spectrometry | publisher=Wiley | volume=24 | issue=3 | date=2010-01-04 | issn=0951-4198 | doi=10.1002/rcm.4383 | pages=267–275| pmid=20049891 | pmc=2908508 }}</ref>

* Slow (but significant, 40% cleavage reported for two week storage at 4˚C) protein backbone cleavage at cysteine residues under mild conditions.<ref name="Liu2010">{{cite journal | last1=Liu | first1=Peiran | last2=O'Mara | first2=Brian W. | last3=Warrack | first3=Bethanne M. | last4=Wu | first4=Wei | last5=Huang | first5=Yunping | last6=Zhang | first6=Yihong | last7=Zhao | first7=Rulin | last8=Lin | first8=Mei | last9=Ackerman | first9=Michael S. | last10=Hocknell | first10=Peter K. | last11=Chen | first11=Guodong | last12=Tao | first12=Li | last13=Rieble | first13=Siegfried | last14=Wang | first14=Jack | last15=Wang-Iverson | first15=David B. | last16=Tymiak | first16=Adrienne A. | last17=Grace | first17=Michael J. | last18=Russell | first18=Reb J. | title=A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins | journal=Journal of the American Society for Mass Spectrometry | publisher=American Chemical Society (ACS) | volume=21 | issue=5 | date=2010-01-28 | issn=1044-0305 | doi=10.1016/j.jasms.2010.01.016 | pages=837–844| doi-access=free | pmid=20189823 }}</ref>

==Use in biological research==

TCEP is available from various chemical suppliers as the hydrochloride salt. When dissolved in water, TCEP-HCl is acidic. A reported preparation is a 0.5 [[Molar (unit)|M]] TCEP-HCl aqueous stock solution that is pH adjusted to near-neutral pH and stored frozen at -20˚C.<ref name="cytiva">{{cite web |title=Strategies for protein purification |url=https://cdn.cytivalifesciences.com/api/public/content/digi-15680-original |publisher=Cytiva |access-date=24 February 2023}}</ref> TCEP is reportedly less stable in phosphate buffers.<ref name="cytiva"></ref>

==See also==

* [[2-Mercaptoethanol]] (BME)

* [[Dithiothreitol]] (DTT)

* [[Dithiobutylamine]] (DTBA)

[[Category:Reducing agents]]