• Procedure for preparation of NPPOC chloride, a photolabile protecting group

phosphoramidite synthesis terms

The name nucleoside phosphoramidite comes from the phosphite group that has an NH2 instead of an OH group.

The "phosphate" group in normal nucleic acids is pentavalent, while here it is trivalent. The structure of amidophosphoric acid is present in pubchem [4].

[4] http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=211207

http://en.wikipedia.org/wiki/Ammonia_kinase

"Thus, the two substrates of this enzyme are ATP and NH3, whereas its two products are ADP and phosphoramide."

http://en.wiktionary.org/wiki/phosphoramide

phosphoramide = any derivative of phosphoric acid in which each hydroxy group has been replaced with an amino or substituted amino group; P(=O)(NR2)3

phosphoric acid = A colourless liquid, H3PO4, used in the manufacture of fertilizers, detergents and pharmaceuticals and as an additive in cola drinks.

phosphoramidite = derived from a phosphite by replacing a >P-O-R with a >P-N<R2 group

phosphite = any salt or ester of phosphorous acid phosphite = the anion PO33-, or the trivalent radical PO3

phosphorous acid = a yellowish hygroscopic solid, H3PO3; a mild reducing agent

ester = A compound most often formed by the condensation of an alcohol and an acid, with elimination of water. It contains the functional group carbon-oxygen double bond joined via carbon to another oxygen atom.

salt = One of the compounds formed from the reaction of an acid with a base, where a positive ion replaces a hydrogen of the acid.

H3PO4 = phosphoric acid H3PO3 = phosphorous acid

phosphite = ester of H3PO3; formed by condensation of an alcohol and an acid, with elimination of water. it contains the functional group carbon-oxygen double bond joined via carbon to another oxygen atom.

phosphoramidite = derived from an ester of H3PO3 by replacing a >P-O-R with a >P-N<R2 group. The phosphite group has an NH2 group instead of an OH group.

phosphoramidate = H2NO3P-2 phosphoramide = H6N3OP

phosphoramide = any derivative of phosphoric acid (H3PO4) in which each hydroxy group (OH) has been replaced with an amino (functional group with a basic nitrogen atom with a lone pair, plus either hydrogen atoms or some organic replacement) or substituted amino group; P(=O)(NR2)3

amino = Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines, trimethylamine and aniline;

pentaerythritol

http://en.wikipedia.org/wiki/Pentaerythritol

pentaerythritol = C(CH2OH)4

Preparation of pentaerythritol

[2] http://www.orgsyn.org/orgsyn/orgsyn/prepContent.asp?prep=CV1P0425

It can be prepared by condensation of acetaldehyde and formaldehyde in a basic environment.[2] The process occurs by successive aldol reactions followed by a Cannizzaro reaction. Impurities include dipentaerythritol and tripentaerythritol.[3]

2CH3CHO + 8 CH2O + Ca(OH)2 → 2C(CH2OH)4 + (HCOO)2Ca

[3] http://dx.doi.org/10.1021%2Fie50549a016

Cannizzaro reaction http://en.wikipedia.org/wiki/Cannizzaro_reaction

Aldol reactions http://en.wikipedia.org/wiki/Aldol_reaction

Acetaldehyde = CH3CHO http://en.wikipedia.org/wiki/Acetaldehyde

"Acetaldehyde (systematically: ethanal) is an organic chemical compound with the formula CH3CHO or MeCHO. It is a flammable liquid. Acetaldehyde occurs naturally in ripe fruit, coffee, and bread, and is produced by plants as part of their normal metabolism. It is popularly known as a chemical that causes hangovers.["

Formaldehyde = CH2O http://en.wikipedia.org/wiki/Formaldehyde

photoproduct: dimethoxyphenylbenzofuran

photolabile protecting groups

2-(3,4-methylenedioxy-6-nitrophenyl)propoxycarbonyl, MNPPOC

methylnitropiperonyl carbonate (MeNPOC) O-methyl-6-nitropiperonyloxycarbonyl

dimethoxybenzoin carbonate (DMB-carbonate) group

DMTr

2-(2-Nitrophenyl) propoxycarbonyl (NPPOC) third generation NPPOC chemistry Pfleiderer group

NPEOC = [2-(2-nitrophenyl)ethoxy]carbonyl

NPES = [2-(2-nitrophenyl)ethyl]sulfonyl

NVOC

"NPPOC-protected amino acids were found to be cleaved in the presence of UV light about twice as fast as the corresponding o-nitroveratryloxycarbonyl (NVOC)-protected amino acids."

"In the earlier studies by Fodor and co-workers[2.], [5.], [13], [14] and [15] amino acids were protected with the photolabile o-nitroveratryloxycarbonyl (NVOC) group, which was originally introduced by Patchornik et al. in 1970.[6.], [17] and [18] Unfortunately, the photolytic removal of NVOC is not very efficient, resulting in synthesis of low quality peptides. However, some improvement in the yield of photodeprotection has been reported by Holmes et al.,7 through use of the α-methyl-o-nitropiperonyloxycarbonyl (MeNPOC) group. More recently Beier and Hoheisel[8.], [21] and [22] have demonstrated that the efficiency of photolytic cleavage of 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC) protected nucleotides is significantly better than that for MeNPOC protected nucleotides. One difference between the NPPOC group and the NVOC and MeNPOC groups is that the former is a derivative of 2-(2-nitrophenyl)ethyl alcohol, whereas the latter two derive from 2-nitrobenzyl alcohol. The additional methylene group in the NPPOC group leads to a different photocleavage mechanism.9 In addition, the photodegradation products of the NVOC and MeNPOC groups include carbonyl compounds[10.], [25] and [26] which can react with amino groups and reduce stepwise synthetic yields.

Since the NPPOC group seems to be beneficial in oligonucleotide synthesis, we decided to investigate its utility in peptide synthesis, with the aim of using NPPOC protected amino acids for the synthesis of peptide microarrays. This communication describes the synthesis of several NPPOC-amino acids."

1. S.P.A Fodor, J.L Read, M.C Pirrung, L Stryer, A.T Lu and D Solas, Science 251 (1991), p. 767.

2. (a) C.P Holmes, C.L Adams, L.M Kochersperger, R.B Mortensen and L.A Aldwin, Biopolymers 37 (1995), p. 199. (b) Read, J. L.; Fodor, S. P. A.; Stryer, L.; Pirrung, M. C.; Hoeprich, P. D. US Patent, 2002, 6,420,169. (c) Pirrung, M. C.; Stryer, L.; Fodor, S. P. A.; Read, J. L. US Patent, 2002, 6,416,952.

Preparation of NPPOC chloride

Procedure for preparation of NPPOC chloride, a photolabile protecting group

1. Procedure for preparation of NPPOC chloride 3: To a solution of 2 (6 mmol) in anhydrous THF (5 mL) at 0°C, was added a solution of phosgene (20% in toluene, 9 mmol) over a period of 15 min with stirring under a nitrogen atmosphere. After 45 min, the ice bath was removed and stirring was continued at room temperature for 2 h. A stream of N2 was then bubbled through the solution for 1 h to remove the excess phosgene, after which the mixture was evaporated to dryness under vacuum to give compound 3 (99%, brown oil). 1H NMR (CDCl3, 400 MHz): δ/ppm 7.81 (d, J=8.0 Hz, 1H, Ar-H), 7.60 (t, J=7.4 Hz, 1H, Ar-H), 7.43 (d, J=7.6 Hz, 1H, Ar-H), 7.38 (t, J=7.6 Hz, 1H, Ar-H), 4.47 (d, J=6.4 Hz, 2H, CH2), 3.77 (m, 1H, CH), 1.39 (d, J=6.8 Hz, 3H, CH3); MS (CI+) m/z: 243.6 (M+H+).

2. Typical procedure for the preparation of NPPOC protected amino acids 4: Na2CO3 (2.2 mmol) was added to the solution of l-amino acid (1 mmol) in 10 mL water/1,4-dioxane (1:1) at 0°C, followed by the dropwise addition of 3 (1 mmol, in 1 mL THF). After 20 min the ice bath was removed and stirring was continued for 18–24 h. The reaction mixture was evaporated to dryness, 3 mL of water was added and the mixture was extracted with ethyl acetate (2×5 mL) to remove 3 or its hydrolysis product. The aqueous layer was acidified by addition of 5% HCl at 0°C and extracted with ethyl acetate (3×10 mL); the extracts were dried over Na2SO4 and concentrated at reduced pressure to give a glassy substance that, in most cases was essentially pure (free of by-products), based on spectroscopic measurements.

photolabile groups based on NPPOC moiety

New types of very efficient photolabile protecting groups based upon NPPOC moiety

The most common representatives featuring the anticipated photochemical lability are derived from o-nitrobenzyl alcohol (ˆ 2-nitrobenzene- methanol) [1] and its derivatives. It was shown that the quantum yield of photo- deprotection is strongly influenced by substituents at the phenyl moiety [5] and the CH2(a) group [6], which on substitution by a Me group at C(a) revealed a fivefold increase in quantum yield of the photocleavage. The o-nitrobenzyl function has been used since its discovery in 1901 [7] to protect hydroxy, amino, mercapto, carboxy, and carbonyl functions [1 ± 3] [8] and is well established in nucleic acid [9 ± 16], carbohy- drate [17], and peptide [4] [18 ± 20] chemistry. More recently, photolabile protection of the 5'-OH of 2'-deoxyribonucleoside 3'-phosphoramidites [5] [21 ± 23] and of the 3'-OH of the isomeric 2'-deoxyribonucleoside 5'-phosphoramidites [24] [25] has been em- ployed in the solid-phase synthesis of DNA probe arrays [26] [27].

Besides the o-nitrobenzyl group and its modified derivatives, other types of photolabile functions have been found such as the benzoine (ˆ 1,2-diphenyl-2'-hydroxyethanone) residue [22] [33] [34], the pyrenylmethyl group [35], and the [(9,10-dihydro-9,10-dioxoanthra- cen-2-yl)methoxy]carbonyl group and the cinnamyl esters [36] [37]. Their use, however, is limited to special applications, and the cleavage efficiences are only moderate.

1,5,6,7,

[1] V. N. R. Pillai, Synthesis 1980, 1. [5] A. C. Pease, D. Solas, E. J. Sullivan, T. M. Cronin, C. P. Holmes, S. P. A. Fodor, Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022. [6] E. Reichmanis, B. C. Smith, R. Gooden, J. Polymer Sci. 1985, 23, 1. [7] G. Ciamician, P. Silber, Ber. Deutsch. Chem. Ges. 1901, 34, 2040.

NPPOC

3‘-Nitrophenylpropyloxycarbonyl (NPPOC) Protecting Groups for High-Fidelity Automated 5‘ → 3‘ Photochemical DNA Synthesis

Two pyrimidine 5‘-phosphoramidites 1 and 2 (Chart 1) were prepared from the 5‘-tert-butyldiphenylsilylated nucleosides by treatment with NPPOC-Cl (generated from phosgene and the alcohol, 0 °C → rt, 12 h, pyridine, 88−92%), deprotection with triethylamine trihydrofluoride [ref 20] (10 equiv, THF, rt, 12 h, 91%), and treatment with β-cyanoethyl N,N-bis(diisopropylamino)chlorophosphine (i-Pr2EtN, 3 h, CH2Cl2, 0 °C → RT, 70−77%). The resulting amidites were purified by precipitation from methylene chloride/hexane.

[20] Pirrung, M. C.; Shuey, S. W.; Lever, D. C.; Fallon, L. Bioorg. Med. Chem. Lett. 1994, 4, 1345.

NPPOC-Cl

1. Procedure for preparation of NPPOC chloride (compound 3): To a solution of 2 (6 mmol) in anhydrous THF (5 mL) at 0°C, was added a solution of phosgene (20% in toluene, 9 mmol) over a period of 15 min with stirring under a nitrogen atmosphere. After 45 min, the ice bath was removed and stirring was continued at room temperature for 2 h. A stream of N2 was then bubbled through the solution for 1 h to remove the excess phosgene, after which the mixture was evaporated to dryness under vacuum to give compound 3 (99%, brown oil). 1H NMR (CDCl3, 400 MHz): δ/ppm 7.81 (d, J=8.0 Hz, 1H, Ar-H), 7.60 (t, J=7.4 Hz, 1H, Ar-H), 7.43 (d, J=7.6 Hz, 1H, Ar-H), 7.38 (t, J=7.6 Hz, 1H, Ar-H), 4.47 (d, J=6.4 Hz, 2H, CH2), 3.77 (m, 1H, CH), 1.39 (d, J=6.8 Hz, 3H, CH3); MS (CI+) m/z: 243.6 (M+H+).

tetrahydrofuran

THF = Tetrahydrofuran = C4H80

"About 200 million kilograms of tetrahydrofuran are produced annually.[1] The most widely used industrial process involves the acid-catalyzed dehydration of 1,4-butanediol. The butanediol is derived from carbonylation of acetylene followed by hydrogenation. Du Pont developed a process for producing THF by oxidizing n-butane to crude maleic anhydride followed by catalytic hydrogenation.[2] A third major industrial route entails hydroformylation of allyl alcohol followed by hydrogenation to the butanediol."

THF can also be synthesized by catalytic hydrogenation of furan derived from pentose. Although this method involves renewable resources, it is not widely practiced.[3]"

phosgene

phosgene = COCl2

http://en.wikipedia.org/wiki/Phosgene

"Upon ultraviolet radiation in the presence of oxygen, chloroform slowly converts into phosgene via a radical reaction. To suppress this photodegradation, chloroform is often stored in brown-tinted glass containers. Chlorinated compounds used to remove oils from metals may also react under the UV created in a welding arc to produce phosgene."

"Industrially, phosgene is produced by passing purified carbon monoxide and chlorine gas through a bed of porous activated carbon, which serves as a catalyst. The equation is described as follows:[2]

CO + Cl2 → COCl2 (ΔHrxn = −107.6kJ/mol)

The reaction is exothermic, therefore the reactor must be cooled. Typically, the reaction is conducted between 50 and 150 °C because above 200 °C, phosgene reverts to carbon monoxide and chlorine, Keq (300K) = 0.05M. Approximately 5000 tonnes were produced in 1989.

Because of safety issues, phosgene is almost always produced and consumed within the same plant and extraordinary measures are made to contain this toxic gas. It is listed on schedule 3 of the Chemical Weapons Convention: all production sites manufacturing more than 30 tonnes per year must be declared to the OPCW.[4] Although much less dangerous than most other chemical weapons (e.g. mustard gas), phosgene is still regarded as a viable chemical warfare agent because it is so easy to manufacture when compared to the production requirements of more technically advanced chemical weapons such as the first-generation nerve agent tabun."

toulene

http://en.wikipedia.org/wiki/Toluene

"The name toluene was derived from the older name toluol, which refers to tolu balsam, an aromatic extract from the tropical Colombian tree Myroxylon balsamum, from which it was first isolated. It was originally named by Jöns Jakob Berzelius."

L-amino acid

"The L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde is levorotary). "

"While L-amino acids represent the vast majority of amino acids found in proteins, D-amino acids are found in some proteins produced by exotic sea-dwelling organisms, such as cone snails.[9]"

blah

Solid-phase stereoselective synthesis of oligonucleoside phosphorothioates: The nucleoside bicyclic oxazaphospholidines as novel synthons

The nucleoside bicylic oxazaphospholidine derived from L-, or D-prolinol is a novel synthon with potential for solid-phase stereoselective synthesis of oligonucleoside phosphorothioates.

nucleosides

Nucleosides can be produced by de novo synthesis pathways, particularly in the liver, but they are more abundantly supplied via ingestion and digestion of nucleic acids in the diet, whereby nucleotidases break down nucleotides (such as the thymine nucleotide) into nucleosides (such as thymidine) and phosphate.

nucleotidase http://en.wikipedia.org/wiki/Nucleotidase

5'-nucleotidase:

• http://www.genenames.org/data/hgnc_data.php?match=NT5C
• http://www.genenames.org/data/hgnc_data.php?match=NT5C1A
• http://www.genenames.org/data/hgnc_data.php?match=NT5C1B
• http://www.genenames.org/data/hgnc_data.php?match=NT5C2
• http://www.genenames.org/data/hgnc_data.php?match=NT5C3

3'-nucleotidase:

• http://www.genenames.org/data/hgnc_data.php?match=NT3

"A nucleotidase is a hydrolytic enzyme that catalyzes the hydrolysis of a nucleotide into a nucleoside and a phosphate. For example, they convert adenosine monophosphate to adenosine, and guanosine monophosphate to guanosine. They have an important function in digestion in that they break down consumed nucleic acids."

"Soluble 5' nucleotidases are all known to belong to the haloacid dehalogenase superfamily of enzymes which are two domian proteins characterised by a modified Rossman fold as the core and variable cap or hood. The soluble forms are further subclassified based on the criterion mentioned above. mdN and cdN are mitochondrial and cytosolic 5'-3' pyrimidine nucleotidases. cN-I is a cytosolic nucleotidase(cN) characterized by its affinity towards AMP as its substrate.cN-II is identified by its affinity towards either IMP or GMP or both. cN-III is a pyrimidine 5' nucleotidase. 5' nucleotidases are involved in varied functions like cell-cell communication, nucleic acid repair, purine salvage pathway for the synthesis of nucleotides, signal transduction, membrane transport etc."

• Hunsucker, S. A., Mitchell, B. S. and Spychala, J. (2005). "The 5'-nucleotidases as regulators of nucleotide and drug metabolism". Pharmacol. Ther. 107, 1–30.

http://www.nlm.nih.gov/cgi/mesh/2009/MB_cgi?mode=&term=Nucleotidases