diazirine

A number of methods exist in the literature for the preparation of diazirines, which begin from a variety of reagents.{{Cite journal|url=https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.7b01561|doi=10.1021/acs.jmedchem.7b01561|title=Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis|year=2018|last1=Hill|first1=James R.|last2=Robertson|first2=Avril A. B.|journal=Journal of Medicinal Chemistry|volume=61|issue=16|pages=6945–6963|pmid=29683660}}

Generally, synthetic schemes that begin with ketones ({{chem2|>C\dO}}) involve conversion of the ketone with the desired substituents to diaziridines ({{chem2|>CN2H2}}). These diaziridines are then subsequently oxidized to form the desired diazirines.

Diaziridines can be prepared from ketones by oximation, followed by tosylation (or mesylation), and then finally by treatment with ammonia ({{chem2|NH3}}). Generally, oximation reactions are performed by reacting the ketone with hydroxylammonium chloride ({{chem2|NH3OH-Cl+}}) under heat in the presence of a base such as pyridine.{{Cite journal|title = New Fluorous Photoaffinity Labels (F-PAL) and Their Application in V-ATPase Inhibition Studies|journal = European Journal of Organic Chemistry|date = 2010-04-01|issn = 1099-0690|pages = 2176–2181|volume = 2010|issue = 11|doi = 10.1002/ejoc.200901463|first1 = Nadja|last1 = Burkard|first2 = Tobias|last2 = Bender|first3 = Johannes|last3 = Westmeier|first4 = Christin|last4 = Nardmann|first5 = Markus|last5 = Huss|first6 = Helmut|last6 = Wieczorek|first7 = Stephanie|last7 = Grond|first8 = Paultheo|last8 = von Zezschwitz}}{{Cite journal|title = Fluorous Aryldiazirine Photoaffinity Labeling Reagents|journal = Organic Letters|date = 2009-11-05|issn = 1523-7060|pages = 4882–4885|volume = 11|issue = 21|doi = 10.1021/ol901955y|first1 = Zhiquan|last1 = Song|first2 = Qisheng|last2 = Zhang|pmid=19807115}} Subsequent tosylation or mesylation of the alpha-substituted oxygen with tosyl or mesyl chloride in the presence of base yields the tosyl or mesyl oxime.{{Cite journal|title = Design and synthesis of an all-in-one 3-(1,1-difluoroprop-2-ynyl)-3H-diazirin-3-yl functional group for photo-affinity labeling|journal = Bioorganic & Medicinal Chemistry|date = 2009-08-01|pages = 5388–5395|volume = 17|issue = 15|doi = 10.1016/j.bmc.2009.06.048|first1 = Nag S.|last1 = Kumar|first2 = Robert N.|last2 = Young|pmid=19604700}} The final treatment of the tosyl or mesyl oxime with ammonia produces the diaziridine.{{Cite journal|title = Design and synthesis of biotin-tagged photoaffinity probes of jasmonates|journal = Bioorganic & Medicinal Chemistry|date = 2010-05-01|pages = 3012–3019|volume = 18|issue = 9|doi = 10.1016/j.bmc.2010.03.059|first1 = Min|last1 = Gu|first2 = Jianbin|last2 = Yan|first3 = Zhiyan|last3 = Bai|first4 = Yue-Ting|last4 = Chen|first5 = Wei|last5 = Lu|first6 = Jie|last6 = Tang|first7 = Liusheng|last7 = Duan|first8 = Daoxin|last8 = Xie|first9 = Fa-Jun|last9 = Nan|pmid=20395151}}

File:Screen Shot 2015-12-01 at 12.35.12 PM .png

Diaziridines can be also produced directly by the reaction of ketones with ammonia in the presence of an aminating agent such as a monochloramine or hydroxyl amine O-sulfonic acid.{{Cite journal|title = Synthesis and validation of a probe to identify quorum sensing receptors|journal = Chemical Communications|volume = |issue = 47|pages = 7378–7380|doi = 10.1039/b917507e|pmid = 20024234|first1 = Luba|last1 = Dubinsky|first2 = Lucja M.|last2 = Jarosz|first3 = Neri|last3 = Amara|first4 = Pnina|last4 = Krief|first5 = Vladimir V.|last5 = Kravchenko|first6 = Bastiaan P.|last6 = Krom|first7 = Michael M.|last7 = Meijler|date = 2009-11-24}}

Diaziridines can be oxidized to diazirines by a number of methods. These include oxidation by chromium-based reagents such as the Jones oxidation,{{Cite journal|title = Structure−Reactivity Relationships: Reactions of a 5-Substituted Aziadamantane in a Resorcin[4]arene-based Cavitand|journal = Organic Letters|date = 2010-01-15|issn = 1523-7060|pages = 332–335|volume = 12|issue = 2|doi = 10.1021/ol902667a|pmid = 20017550|first1 = Gerald|last1 = Wagner|first2 = Wolfgang|last2 = Knoll|first3 = Michael M.|last3 = Bobek|first4 = Lothar|last4 = Brecker|first5 = Hendrikus W. G.|last5 = van Herwijnen|first6 = Udo H.|last6 = Brinker}} oxidation by iodine and triethylamine, oxidation by silver oxide,{{Cite journal|title = Bi-3H-diazirin-3-yls as Precursors of Highly Strained Cycloalkynes|journal = Angewandte Chemie International Edition|date = 2006-01-01|issn = 1521-3773|pages = 309–311|volume = 45|issue = 2|doi = 10.1002/anie.200503124|first1 = Mohammad|last1 = Al-Omari|first2 = Klaus|last2 = Banert|first3 = Manfred|last3 = Hagedorn|pmid=16372311}} oxidation by oxalyl chloride, or even electrochemical oxidation on a platinum-titanium anode.{{Cite journal|title = Electrochemical synthesis of pentamethylenediazirine|journal = Russian Chemical Bulletin|date = 2006-11-01|issn = 1066-5285|pages = 2013–2015|volume = 55|issue = 11|doi = 10.1007/s11172-006-0544-0|first1 = M. D.|last1 = Vedenyapina|first2 = V. V.|last2 = Kuznetsov|first3 = E. A.|last3 = Nizhnikovskii|first4 = E. D.|last4 = Strel’tsova|first5 = N. N.|last5 = Makhova|first6 = M. I.|last6 = Struchkova|first7 = A. A.|last7 = Vedenyapin|s2cid = 97472127}}

File:Screen Shot 2015-12-01 at 7.51.23 PM.png

Diazirines can be alternatively formed in a one-pot process using the Graham reaction, starting from amidines.{{Cite journal|title = The Halogenation of Amidines. I. Synthesis of 3-Halo- and Other Negatively Substituted Diazirines1|journal = Journal of the American Chemical Society|date = 1965-10-01|issn = 0002-7863|pages = 4396–4397|volume = 87|issue = 19|doi = 10.1021/ja00947a040|first = W. H.|last = Graham}} This reaction yields a halogenated diazirine, whose halogen can be displaced by various nucleophilic reagents.{{Cite journal|title = Diazirines: Carbene Precursors Par Excellence|journal = Accounts of Chemical Research|date = 2006-02-09|issn = 0001-4842|pages = 267–272|volume = 39|issue = 4|doi = 10.1021/ar050155h|first = Robert A.|last = Moss|pmid = 16618094}}

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| image1 = Graham-reaction-2D-scheme.svg

| caption1 = The Graham reaction as a method of diazirine synthesis, where X = Cl or Br.

| image2 = Diazirine Exchange.svg

| caption2 = The diazirine exchange reaction using various anions and the counterion tetra-n-butylammonium.

}}

Upon irradiation with UV light, diazirines form reactive carbene species. The carbene may exist in the singlet form, in which the two free electrons occupy the same orbital, or the triplet form, with two unpaired electrons in different orbitals.

File:Diazirines decomposition.svg

The substituents on the diazirine affect which carbene species is generated upon irradiation and subsequent photolytic cleavage. Diazirine substituents that are electron donating in nature can donate electron density to the empty p-orbital of the carbene that will be formed, and hence can stabilize the singlet state. For example, phenyldiazirine produces phenylcarbene in the singlet carbene state{{Cite journal|title = Direct Observation of Carbene and Diazo Formation from Aryldiazirines by Ultrafast Infrared Spectroscopy|journal = Journal of the American Chemical Society|date = 2008-12-03|issn = 0002-7863|pages = 16134–16135|volume = 130|issue = 48|doi = 10.1021/ja805922b|first1 = Yunlong|last1 = Zhang|first2 = Gotard|last2 = Burdzinski|first3 = Jacek|last3 = Kubicki|first4 = Matthew S.|last4 = Platz|pmid=18998681}} whereas 3-chloro-3-[(4-nitrophenyl)methyl]diazirine or {{chem name|trifluoromethylphenyldiazirine}} produce the respective triplet carbene products.{{Cite journal|title = Ultrafast Dynamics of Isolated Phenylcarbenes Followed by Femtosecond Time-Resolved Velocity Map Imaging|journal = The Journal of Physical Chemistry A|date = 2009-04-02|issn = 1089-5639|pages = 3041–3050|volume = 113|issue = 13|doi = 10.1021/jp810974m|pmid = 19245233|first1 = Bastian|last1 = Noller|first2 = Lionel|last2 = Poisson|first3 = Raman|last3 = Maksimenka|first4 = Oliver|last4 = Gobert|first5 = Ingo|last5 = Fischer|first6 = J. M.|last6 = Mestdagh|bibcode = 2009JPCA..113.3041N}}{{Cite journal|title = The photoionisation of two phenylcarbenes and their diazirine precursors investigated using synchrotron radiation|journal = Physical Chemistry Chemical Physics|volume = 11|issue = 26|pages = 5384–5391|doi = 10.1039/b823269e|pmid = 19551206|first1 = Bastian|last1 = Noller|first2 = Patrick|last2 = Hemberger|first3 = Ingo|last3 = Fischer|first4 = Christian|last4 = Alcaraz|first5 = Gustavo A.|last5 = Garcia|first6 = Héloïse|last6 = Soldi-Lose|date = 2009-06-23|bibcode = 2009PCCP...11.5384N|url = https://zenodo.org/record/895863}}

Electron donating substituents can also encourage photoisomerization to the linear diazo compound,{{Cite journal |last=Korneev |first=Sergei M. |date=November 2011 |title=Valence Isomerization between Diazo Compounds and Diazirines |url=https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejoc.201100224 |journal=European Journal of Organic Chemistry |language=en |volume=2011 |issue=31 |pages=6153–6175 |doi=10.1002/ejoc.201100224 |issn=1434-193X}} rather than the singlet carbene, and hence these compounds are unfavorable for use in biological assays. On the other hand, {{chem name|trifluoroaryldiazirines}} in particular show favorable stability and photolytic qualities{{Cite journal|title = 3-Trifluoromethyl-3-phenyldiazirine. A new carbene generating group for photolabeling reagents|journal = The Journal of Biological Chemistry|date = 1980-04-25|issn = 0021-9258|pmid = 7364745|pages = 3313–3318|volume = 255|issue = 8|first1 = J.|last1 = Brunner|first2 = H.|last2 = Senn|first3 = F. M.|last3 = Richards|doi = 10.1016/S0021-9258(19)85701-0|doi-access = free}} and are most commonly used in biological applications. File:Screen Shot 2015-12-01 at 3.56.20 PM.pngCarbenes produced from diazirines are quickly quenched by reaction with water molecules,{{Cite journal|title = Influence of Solvent on Carbene Intersystem Crossing Rates|journal = Journal of the American Chemical Society|date = 2008-05-01|issn = 0002-7863|pages = 6604–6609|volume = 130|issue = 20|doi = 10.1021/ja711385t|first1 = Jin|last1 = Wang|first2 = Jacek|last2 = Kubicki|first3 = Huolei|last3 = Peng|first4 = Matthew S.|last4 = Platz|pmid=18433130}} and hence yields for photoreactive crosslinking assays are often low. Yet, as this feature minimizes unspecific labeling, it is actually an advantage of using diazirines.

Diazirines are often used as photoreactive crosslinking reagents, as the reactive carbenes they form upon irradiation with UV light can insert into C-H, N-H, and O-H bonds. This results in proximity-dependent labeling of other species with the diazirine containing compound. However, studies have found that diazirines have some pH dependence in labeling preferences, favoring acidic residues such as glutamate.{{Cite journal |last1=West |first1=Alexander V. |last2=Muncipinto |first2=Giovanni |last3=Wu |first3=Hung-Yi |last4=Huang |first4=Andrew C. |last5=Labenski |first5=Matthew T. |last6=Jones |first6=Lyn H. |last7=Woo |first7=Christina M. |date=2021-05-05 |title=Labeling Preferences of Diazirines with Protein Biomolecules |url=https://pubs.acs.org/doi/10.1021/jacs.1c02509 |journal=Journal of the American Chemical Society |language=en |volume=143 |issue=17 |pages=6691–6700 |doi=10.1021/jacs.1c02509 |pmid=33876925 |issn=0002-7863|pmc=11647638 }} Diazirine variants have been developed to reduce this bias.{{Cite journal |last1=West |first1=Alexander V. |last2=Amako |first2=Yuka |last3=Woo |first3=Christina M. |date=2022-11-23 |title=Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells |url=https://pubs.acs.org/doi/10.1021/jacs.2c08257 |journal=Journal of the American Chemical Society |language=en |volume=144 |issue=46 |pages=21174–21183 |doi=10.1021/jacs.2c08257 |pmid=36350779 |issn=0002-7863|pmc=11647570 }}

Diazirines are often preferred to other photoreactive crosslinking reagents due to their smaller size, longer irradiation wavelength, short period of irradiation required, and stability in the presence of various nucleophiles, and in both acidic and basic conditions.{{Cite journal|title = Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic frontier|journal = Current Topics in Medicinal Chemistry|date = 2002-03-01|issn = 1568-0266|pmid = 11944820|pages = 271–288|volume = 2|issue = 3|first1 = Yasumaru|last1 = Hatanaka|first2 = Yutaka|last2 = Sadakane|doi=10.2174/1568026023394182}} Benzophenones, which form reactive triplet carbonyl species upon irradiation, often require long periods of irradiation which can result in non-specific labeling, and moreover are often inert to various polar solvents.{{Cite journal|title = Benzophenone Photoprobes for Phosphoinositides, Peptides and Drugs|journal = Photochemistry and Photobiology|date = 1997-02-01|issn = 1751-1097|pages = 222–234|volume = 65|issue = 2|doi = 10.1111/j.1751-1097.1997.tb08548.x|first1 = Glenn D.|last1 = Prestwich|first2 = György|last2 = Dormán|first3 = John T.|last3 = Elliott|first4 = Dale M.|last4 = Marecak|first5 = Anu|last5 = Chaudhary|pmid = 9066302|s2cid = 12577596}} Aryl azides require a low wavelength of irradiation, which can damage the biological macromolecules under investigation.

Diazirines are widely used in receptor labeling studies. This is because diazirine-containing analogs of various ligands can be synthesized and incubated with their respective receptors, and then subsequently exposed to light to produce reactive carbenes. The carbene will covalently bond to residues in the binding site of the receptor. The carbene compound may include a bioorthogonal tag or handle by which the protein of interest can be isolated. The protein can then be digested and sequenced by mass spectrometry in order to identify which residues the carbene containing ligand is bound to, and hence the identity of the binding site in the receptor.

Examples of diazirines used in receptor labeling studies include:

• The discovery of a brassinosteroid receptor for brassinosteroid plant hormones by Kinoshita et al. Researchers used a plant hormone analog with a diazirine crosslinking moiety and a biotin tag for isolation to identity the new receptor.{{Cite journal|title = Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1|journal = Nature|pages = 167–171|volume = 433|issue = 7022|doi = 10.1038/nature03227|pmid = 15650741|first1 = Toshinori|last1 = Kinoshita|first2 = Ana|last2 = Caño-Delgado|first3 = Hideharu|last3 = Seto|first4 = Sayoko|last4 = Hiranuma|first5 = Shozo|last5 = Fujioka|first6 = Shigeo|last6 = Yoshida|first7 = Joanne|last7 = Chory|year = 2005|bibcode = 2005Natur.433..167K|s2cid = 4379617}} This study led to a number of similar studies conducted with regards to other plant hormones.

File:Brassinosteroid diazirine analog.jpg

• The discovery of novel non-CB1/CB2 cannabinoid receptors using anandamide analog probes containing a diazirine group by Balas et al.{{Cite journal|title = Total Synthesis of Photoactivatable or Fluorescent Anandamide Probes: Novel Bioactive Compounds with Angiogenic Activity|journal = Journal of Medicinal Chemistry|date = 2009-02-26|issn = 0022-2623|pages = 1005–1017|volume = 52|issue = 4|doi = 10.1021/jm8011382|first1 = Laurence|last1 = Balas|first2 = Thierry|last2 = Durand|first3 = Sattyabrata|last3 = Saha|first4 = Inneke|last4 = Johnson|first5 = Somnath|last5 = Mukhopadhyay|pmid = 19161308}}
• The binding cavity of the hypnotic agent propofol in the GABA_A receptor using a diazirine containing propofol analog.{{Cite journal|title = m-Azipropofol (AziPm) a Photoactive Analogue of the Intravenous General Anesthetic Propofol|journal = Journal of Medicinal Chemistry|date = 2010-08-12|issn = 0022-2623|pmc = 2917171|pmid = 20597506|pages = 5667–5675|volume = 53|issue = 15|doi = 10.1021/jm1004072|first1 = Michael A.|last1 = Hall|first2 = Jin|last2 = Xi|first3 = Chong|last3 = Lor|first4 = Shuiping|last4 = Dai|first5 = Robert|last5 = Pearce|first6 = William P.|last6 = Dailey|first7 = Roderic G.|last7 = Eckenhoff}}

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| image1 = Propofol.svg

| image2 = M-Azipropofol.png

| footer = Propofol (left) and m-azipropofol, a diazirine analog of it

}}

In a manner analogous to receptor labeling, diazirine containing compounds that are analogs of natural substrates have also been used to identify binding pockets of enzymes. Examples include:

• The synthesis of a diazirine containing analog of etoposide, a widely used cancer drug targeting topoisomerase II, which holds promise for the identification of the etoposide binding site.{{Cite journal|title = A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II|journal = Bioorganic & Medicinal Chemistry|date = 2010-01-15|pmc = 2818565|pmid = 20006518|pages = 830–838|volume = 18|issue = 2|doi = 10.1016/j.bmc.2009.11.048|first1 = Gaik-Lean|last1 = Chee|first2 = Jack C.|last2 = Yalowich|first3 = Andrew|last3 = Bodner|first4 = Xing|last4 = Wu|first5 = Brian B.|last5 = Hasinoff}}File:1-s2.0-S0968089611005062-gr27.png
• The discovery that caprolactam-type gamma-secretase inhibitors target the SPP subunit of the gamma-secretase, which has been implicated in Alzheimer's disease.{{Cite journal|title = Divergent Synthesis of Multifunctional Molecular Probes To Elucidate the Enzyme Specificity of Dipeptidic γ-Secretase Inhibitors|journal = ACS Chemical Biology|date = 2007-06-01|issn = 1554-8929|pages = 408–418|volume = 2|issue = 6|doi = 10.1021/cb700073y|pmid = 17530731|first1 = Haruhiko|last1 = Fuwa|first2 = Yasuko|last2 = Takahashi|first3 = Yu|last3 = Konno|first4 = Naoto|last4 = Watanabe|first5 = Hiroyuki|last5 = Miyashita|first6 = Makoto|last6 = Sasaki|first7 = Hideaki|last7 = Natsugari|first8 = Toshiyuki|last8 = Kan|first9 = Tohru|last9 = Fukuyama}}
• The finding that the O-GlcNAc post-translational modification mediates protein-protein interactions such as with 14-3-3 proteins.{{Cite journal |last1=Yu |first1=Seok-Ho |last2=Boyce |first2=Michael |last3=Wands |first3=Amberlyn M. |last4=Bond |first4=Michelle R. |last5=Bertozzi |first5=Carolyn R. |last6=Kohler |first6=Jennifer J. |date=2012-03-27 |title=Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners |journal=Proceedings of the National Academy of Sciences of the United States of America |volume=109 |issue=13 |pages=4834–4839 |doi=10.1073/pnas.1114356109 |doi-access=free |issn=1091-6490 |pmc=3323966 |pmid=22411826}}{{Cite journal |last1=Toleman |first1=Clifford A. |last2=Schumacher |first2=Maria A. |last3=Yu |first3=Seok-Ho |last4=Zeng |first4=Wenjie |last5=Cox |first5=Nathan J. |last6=Smith |first6=Timothy J. |last7=Soderblom |first7=Erik J. |last8=Wands |first8=Amberlyn M. |last9=Kohler |first9=Jennifer J. |last10=Boyce |first10=Michael |date=2021-05-20 |title=Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins |journal=Proceedings of the National Academy of Sciences of the United States of America |language=en |volume=115 |issue=23 |pages=5956–5961 |doi=10.1073/pnas.1722437115 |doi-access=free |pmc=6003352 |pmid=29784830}}

Diazirines have been used in photoaffinity labeling experiments involving nucleic acids as well. Examples include:

• Incorporation of a diazirine moiety on a nucleoside sugar in a DNA polymer to investigate interactions between the minor groove of DNA and DNA polymerases.{{Cite journal|title = A New Photoactive Building Block for Investigation of DNA Backbone Interactions: Photoaffinity Labeling of Human DNA Polymerase β|journal = ChemBioChem|date = 2006-12-04|issn = 1439-7633|pages = 1965–1969|volume = 7|issue = 12|doi = 10.1002/cbic.200600333|pmid = 17106908|first1 = Meike|last1 = Liebmann|first2 = Francesca|last2 = Di Pasquale|first3 = Andreas|last3 = Marx|s2cid = 22908416}}

File:1-s2.0-S0968089611005062-gr39.jpg

• Incorporation of a diazirine moiety on a nucleoside base in a DNA polymer to investigate the mode of DNA repair by proteins.{{Cite journal|title = Novel Diazirine-Containing DNA Photoaffinity Probes for the Investigation of DNA-Protein-Interactions|journal = ChemBioChem|date = 2009-01-05|issn = 1439-7633|pages = 109–118|volume = 10|issue = 1|doi = 10.1002/cbic.200800397|pmid = 19012292|first1 = Malte|last1 = Winnacker|first2 = Sascha|last2 = Breeger|first3 = Ralf|last3 = Strasser|first4 = Thomas|last4 = Carell|s2cid = 5605171}}File:1-s2.0-S0968089611005062-gr40.jpg

Diazirines have also been used to study protein lipid interactions, for example the interaction of various sphingolipids with proteins in vivo.{{Cite journal|title = Versatile Synthetic Method for Sphingolipids and Functionalized Sphingosine Derivatives via Olefin Cross Metathesis|journal = Organic Letters|date = 2006-11-01|issn = 1523-7060|pages = 5569–5572|volume = 8|issue = 24|doi = 10.1021/ol062258l|first1 = Tetsuya|last1 = Yamamoto|first2 = Hiroko|last2 = Hasegawa|first3 = Toshikazu|last3 = Hakogi|first4 = Shigeo|last4 = Katsumura|pmid=17107074}}

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Category:Nitrogen heterocycles

Category:Photochemistry