Assigning in vivo carbamylation and acetylation in human lens proteins using tandem mass spectrometry and database searching

In this paper, we show that ion trap mass spectrometers can differentiate acetylation and carbamylation modifications based on database search results for a lens protein sample. These types of modifications are difficult to distinguish on ion trap instruments because of their lower resolution and mass accuracy. The results were corroborated by using accurate mass information derived from MALDI TOF MS analysis of eluted peptides from a duplicate capillary RPLC separation. Tandem mass spectra of lysine carbamylated peptides were further verified by manual assignments of fragment ions and by the presence of characteristic fragment ions of carbamylated peptides. It was also observed that carbamylated peptides show a strong neutral loss of the carbamyl group in collision induced dissociation (CID), a feature that can be prognostic for carbamylation. In a lens tissue sample of a 67-year-old patient, 12 in vivo carbamylation sites were detected on 7 different lens proteins and 4 lysine acetylation sites were detected on 3 different lens proteins. Among the 12 in vivo carbamylation sites, 9 are novel in vivo carbamylation modification sites. Notably, in vivo carbamylation of γS crystallin, βA4 crystallin, βB1 crystallin, and βB2 crystallin observed in this study have never been reported before.     

Metabolism linked synthesis of fluorinated anti-tumour drug the fluorine - deuterium gambit

Metabolism studies of the known anti-tumour agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (C C N u), 4-[4-bis(2-chloroethyl)amino phenyl]butyric acid (chloroambucil) and [2-bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2 oxazaphosphorine-2-oxide (cyclophosphamide) and their polydeuterio derivatives have led to an understanding of their mode of action, deactivation and ultimate excretion.In an attempt to modify or halt these processes, with the object of improving drug design and potency we have synthesised analogues of these drugs with fluorinated substituents in strategic positions within the molecules as indicated by metabolism data.The synthesis of each set of derivatives will be described with emphasis on the overcoming of problems of isomer distribution.Biological test results will be presented.     

Synthesis and antiviral activities of carbocyclic oxetanocin analogues

9-Cyclobutyladenine (4a), cis- and trans-9-[3- (hydroxymethyl)cyclobutyl]adenine (4b) and 9-[3,3-bis(hydroxymethyl)cyclobutyl]adenine(4d) were prepared from the corresponding cyclobutylamine derivatives (1a, 1b and 1d). Guanine congeners (9a, cis- and trans-9b and 9d) and carbocyclic oxetanocin G (1',2'-trans-9f) were also prepared. Carbocyclic oxetanocin A(1',2'-trans-4f), the preparation of which we have already published, and G were found to be active against herpes simplex virus (type 1 and 2) in vitro, while cis-4b and cis-9b showed an in vitro antiretroviral activity against human immunodeficiency virus (type 1).     

Regio- and stereoselective synthesis of (E)-alkene trans-Xaa-Pro dipeptide mimetics utilizing organocopper-mediated anti-S(N)2' reactions

Proline dipeptides (Xaa-Pro) exist as an equilibrium mixture of cis- and trans-rotamers, which depends on the energy barriers for imide isomerization. This conformation mixture contributes to both structure and function of proline-containing peptides and proteins. Structural motifs resembling these cis- or trans-conformers have served as useful tools for elucidating contributions of proline residues in the physicochemical and biological profiles of structures which contain them. Among such motifs are alkene dipeptide isosteres which mimic cis- or trans-imide using (Z)- or (E)-alkene, respectively. In this report, the first regio- and stereoselective syntheses of (E)-alkene dipeptide isosteres (20, 31, and 35) corresponding to trans-proline dipeptides are described. Key to the synthesis of these mimetics is the anti-S(N)2' reaction of vinyl aziridines such as 15 or vinyl oxazolidinones such as 28 and 32 with organocopper reagents "RCu" (R = CH(2)SiMe(2)(Oi-Pr)). Reaction of cis-vinylaziridine 15 derived from L-serine with organocopper reagent gave a precursor of the trans-L-Ser-D-Pro type alkene isosteres 20, accompanied by an S(N)2 side product. One limitation with the use of such aziridine-mediated methodology is formation of the corresponding trans-aziridine 22, which leads to L-L type isosteres, that is unstable and obtainable only in low yield. On the other hand, both isomers of oxazolidinone derivatives can be easily obtained from N-Boc-protected amino alcohols. The reaction of trans- 28 or cis-oxazolidinone derivative 32 with organocopper reagents proceeds quantitatively with high regio- and diastereoselectivities in anti-S(N)2' fashion. Subsequent oxidative treatment of the newly introduced isopropoxydimethylsilylmethyl group yields trans-L-Ser-L-Pro 31 or trans-L-Ser-D-Pro type isosteres 35, respectively. Of note, synthesized isostere 31 can also be converted to trans-phosphoSer-Pro 42 and trans-Cys-Pro mimetics 44. The present synthetic methodology affords trans-Xaa-Pro alkene-type dipeptide isosteres in high yield with relatively simple manipulation.     

Biodistribution of A14C-and99mTc-labeled N-substituted nitrosourea (CCNU) in an animal tumor model

One formulation of¹⁴C labeled and another of^99mTc labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) were administered i.v. to tumor (glioma) bearing rats. The radiopharmacokinetics of¹⁴C-CCNU were followed up to 24 hours post injection. On a per organ basis the blood, liver, small bowel, kidney cortex and muscle contained most of the activity. Optimum tumor to organ ratios occurred at 4–12 hours. The^99mTc-CCNU biodistribution was determined at 4 hours and compared to^99mTc-NaTcO₄. Tumor capsule to brain (29.5) and to muscle (10.59) ratios suggest^99mTc-CCNU to be a potential tumor seeking agent. Funded in part by USPHS Grant RR-05486-12.     

Hydrolytic metal-mediated coupling of dialkylcyanamides at a Pt(IV) center giving a new family of diimino ligands

Addition of excess R(2)NCN to an aqueous solution of K(2)[PtCl(4)] led to the precipitation of [PtCl(2)(NCNR(2))(2)] (R(2) = Me(2) 1; Et(2) 2; C(5)H(10) 3; C(4)H(8)O, 4) in a cis/trans isomeric ratio which depends on temperature. Pure isomers cis-1-3 and trans-1-3 were separated by column chromatography on SiO(2), while trans-4 was obtained by recrystallization. Complexes cis-1-3 isomerize to trans-1-3 on heating in the solid phase at 110 degrees C; trans-1 has been characterized by X-ray crystallography. Chlorination of the platinum(II) complexes cis-1-3 and trans-1-4 gives the appropriate platinum(IV) complexes [PtCl(4)(NCNR(2))(2)] (cis-5-7 and trans-5-8). The compound cis-6 was also obtained by treatment of [PtCl(4)(NCMe)(2)] with neat Et(2)NCN. The platinum(IV) complex trans-[PtCl(4)(NCNMe(2))(2)] (trans-5) in a mixture of undried Et(2)O and CH(2)Cl(2) undergoes facile hydrolysis to give trans-[PtCl(4)[(H)=C(NMe(2))OH](2)] (9; X-ray structure has been determined). The hydrolysis went to another direction with the cis-[PtCl(4)(NCNR(2))(2)] (cis-5-7) which were converted to the metallacycles [PtCl(4)[NH=C(NR(2))OC(NR(2))=NH]] (11-13) due to the unprecedented hydrolytic coupling of the two adjacent dialkylcyanamide ligands giving a novel (for both coordination and organic chemistry) diimino linkage. Compounds 11-13 and also 14 (R(2) = C(4)H(8)O) were alternatively obtained by the reaction between cis-[PtCl(4)(MeCN)(2)] and neat undried NCNR(2). The structures of complexes 11, 13, and 14 were determined by X-ray single-crystal diffraction. All the platinum compounds were additionally characterized by elemental analyses, FAB mass-spectrometry, and IR and (1)H and (13)C[(1)H] NMR spectroscopies.     

Bis(acetylacetonato)ruthenium(II) complexes containing alkynyldiphenylphosphines. Formation and redox behaviour of [Ru(acac)2(Ph2PC triple bond CR)2] (R=H, Me, Ph) complexes and the binuclear complex cis-[{Ru(acac)2}2(micro-Ph2PC triple bond CPPh2)}2

Two equivalents of Ph(2)PC triple bond CR (R=H, Me, Ph) react with thf solutions of cis-[Ru(acac)(2)(eta(2)-alkene)(2)] (acac=acetylacetonato; alkene=C(2)H(4), 1; C(8)H(14), 2) at room temperature to yield the orange, air-stable compounds trans-[Ru(acac)(2)(Ph(2)PC triple bond CR)(2)] (R=H, trans-3; Me=trans-4; Ph, trans-5) in isolated yields of 60-98%. In refluxing chlorobenzene, trans-4 and trans-5 are converted into the yellow, air-stable compounds cis-[Ru(acac)(2)(Ph(2)PC triple bond CR)(2)] (R=Me, cis-4; Ph, cis-5), isolated in yields of ca. 65%. From the reaction of two equivalents of Ph(2)PC triple bond CPPh(2) with a thf solution of 2 an almost insoluble orange solid is formed, which is believed to be trans-[Ru(acac)(2)(micro-Ph(2)PC triple bond CPPh(2))](n) (trans-6). In refluxing chlorobenzene, the latter forms the air-stable, yellow, binuclear compound cis-[{Ru(acac)(2)(micro-Ph(2)PC triple bond CPPh(2))}(2)] (cis-6). Electrochemical studies indicate that cis-4 and cis-5 are harder to oxidise by ca. 300 mV than the corresponding trans-isomers and harder to oxidise by 80-120 mV than cis-[Ru(acac)(2)L(2)] (L=PPh(3), PPh(2)Me). Electrochemical studies of cis-6 show two reversible Ru(II/III) oxidation processes separated by 300 mV, the estimated comproportionation constant (K(c)) for the equilibrium cis-6(2+) + cis6 <=> 2(cis-6(+)) being ca. 10(5). However, UV-Vis spectra of cis-6(+) and cis-6(2+), generated electrochemically at -50 degrees C, indicate that cis-6(+) is a Robin-Day Class II mixed-valence system. Addition of one equivalent of AgPF(6) to trans-3 and trans-4 forms the green air-stable complexes trans-3 x PF(6) and trans-4 x PF(6), respectively, almost quantitatively. The structures of trans-4, cis-4, trans-4 x PF(6) and cis-6 have been confirmed by X-ray crystallography.     

Quantitative carbamylation as a stable isotopic labeling method for comparative proteomics

A method was developed that uses urea to both solublize and isotopically label biological samples for comparative proteomics. This approach uses either light or heavy urea ((12)CH(4)(14)N(2)O or (13)CH(4)(15)N(2)O, respectively) at a concentration of 8 M and a pH of 7 to dissolve the samples prior to digestion. After the sample is digested using standard proteomic protocols and dried, isotopic labeling is completed by resuspending the sample in a solution of 8 M urea at a pH of 8.5, using the same isotopic species of urea as used for digestion and incubating for 4 h at 80 degrees C. Under these conditions, carbamylation occurs only on the primary amines of the peptides. The effects of complete carbamylation on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) (collision-induced dissociation (CID)) were examined. Peptides that had a C-terminal carbamylated lysine residue were found to have a reduced intensity when viewed by MALDI-TOFMS. CID of a tryptic peptide that was carbamylated on both the N-terminus and the C-terminus was found to have a more uniform distribution of b- and y-ions, as well as prominent ions from loss of water. Reversed-phase chromatography coupled to ESI-MS/MS was used to identify and quantify the isotopically labeled standard proteins, bovine serum albumin (BSA), bovine transferrin, and bovine alpha-casein. Quantitative error between theoretical and observed data ranged from 1.7-10.0%. Relative standard deviations for protein quantitation ranged from 5.2-27.8% over a dynamic range from 0.1-10 (L/H). The development of a method utilizing urea-assisted carbamylation of lysines and N-termini to globally labeled samples for comparative proteomics may prove useful for samples that require a strong chaotrope prior to proteolysis.     

Relationship between the configurations of 2-phenyltetrahydrothiophenium 1-methylides and their rearrangement products

trans-2-Phenyltetrahydrothiophenium 1-methylide (trans-3), which is generated by fluoride ion-induced desilylation of trans-2-phenyl-1-[(trimethylsilyl)methyl]tetrahydrothiophenium salt (trans-2), gave a mixture of 1,4,5,10a-tetrahydro-3H-2-benzothiocine (4) ([2,3]sigmatropic rearrangement product) and 4-methylsulfanyl-1-phenyl-1-butene (5) (Hofmann elimination product). Ylide trans-3 cannot undergo [2,3]sigmatropic rearrangement because the ylide-carbon is too far from the phenyl group, and trans-3 would instead isomerize to cis-3. In this paper, we discuss the mechanism of the isomerization of trans-3 to cis-3.     

Synthesis of new dihydropyrazines with DNA strand-breakage activity

Treatment of 1,2-cyclohexanedione with 1,2-diamines, e.g. ethylenediamine and cis-(and trans-)1,2-diaminocyclohexane, caused [4+2] cyclocondensation to give the corresponding dihydropyrazine derivatives (compounds 1-6). They exhibited stronger DNA strand-breakage activity than that of dihydropyrazines, which has already been reported in previous papers.     

Conformations of the antitumor agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-urea in solution studied by 15N-NMR

A study of the Nnmr spectra of the anticancer agent 1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea (CCNU) employing both a natural abundance and selectively ¹⁵N enriched CCNU permitted assignment of ¹⁵N-resonance and its conformation relevant to Its mechanism of action.     

Synthesis of stereoisomeric derivatives of aminocyclohexanecarboxylic acids that contain the acyl moiety of p-[di-(2-chloroethyl)amino]phenylacetic acid

Conclusions The p-[di-(2-chloroethyl)amino]phenacetyl derivatives of the cis- and trans-4-aminocyclohexanecarboxylic acids and 4-aminocyclohexaneacetic acid were synthesized.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 1, pp. 192–194, January, 1977.     

Switch of electronic reactivity in fullerene C60: activation of three trans-4 positions via temporary saturation of the cis-1 positions

[Structure: see text] The regioselective functionalization of C60 with a trans-4,trans-4,trans-4 trisaddition pattern is not feasible directly. We have found an indirect approach taking advantage of the modified electronic reactivity of cis-1 bisadducts. The cis-1 addition pattern electronically activates three trans-4 C=C bonds on the opposite hemisphere of C60, allowing further highly regioselective additions at these positions. Thermal removal of the cis-1 blocking unit results in a trans-4,trans-4,trans-4 trisadduct with C3v symmetry.     

Preparative and structural studies on the carbonyl cyanides of iron, manganese, and ruthenium: fundamentals relevant to the hydrogenases

The reaction of cyanide, carbon monoxide, and ferrous derivatives led to the isolation of three products, trans- and cis-[Fe(CN)(4)(CO)(2)](2)(-) and [Fe(CN)(5)(CO)](3)(-), the first two of which were characterized by single-crystal X-ray diffraction. The new compounds show self-consistent IR, (13)C NMR, and mass spectroscopic properties. The reaction of trans-[Fe(CN)(4)(CO)(2)](2)(-) with Et(4)NCN gives [Fe(CN)(5)(CO)](3)(-) via a first-order (dissociative) pathway. The corresponding cyanation of cis-[Fe(CN)(4)(CO)(2)](2)(-), which is a minor product of the Fe(II)/CN(-)/CO reaction, does not proceed at measurable rates. Methylation of [Fe(CN)(5)(CO)](3)(-) gave exclusively cis-[Fe(CN)(4)(CNMe)(CO)](2)(-), demonstrating the enhanced nucleophilicity of CN(-) trans to CN(-) vs. CN(-) trans to CO. Methylation has an electronic effect similar to that of protonation as determined electrochemically. We also characterized [M(CN)(3)(CO)(3)](n)(-) for Ru (n = 1) and Mn (n = 2) derivatives. The Ru complex, which is new, was prepared by cyanation of a [RuCl(2)(CO)(3)](2) solution.     

Ring closure in 1,2-divinylcyclohexanes and isomerization to 3-methyl-3a,4,5,6,7,7a-hexahydro-1h-indenes catalyzed by titanocene hydride derivatives

Titanocene hydride derivatives induce the cyclization of 1,2-divinylcyclohexanes to trans- and cis-1-methylene-octahydro-1H-indene and their isomerization to trans- and cis-3-methyl-3a,4,5,6,7,7a-hexahydro-1H-indene.     

Synthesis of acetimino complexes of Pt(II) and Pt(IV) and the first heteronuclear mu-acetimido complexes

The reactions of [Ag(NH=CMe2)2]ClO4 with cis-[PtCl2L2] in a 1:1 molar ratio give cis-[PtCl(NH=CMe2)(PPh3)2]ClO4 (1cis) or cis-[PtCl(NH=CMe2)2(dmso)]ClO4 (2), and in 2:1 molar ratio, they produce [Pt(NH=CMe2)2L2](ClO4)2 [L = PPh3 (3), L2= tbbpy = 4,4'-di-tert-butyl-2,2'-dipyridyl (4)]. Complex 2 reacts with PPh3 (1:2) to give trans-[PtCl(NH=CMe2)(PPh3)2]ClO(4) (1trans). The two-step reaction of cis-[PtCl2(dmso)2], [Au(NH=CMe2)(PPh3)]ClO4, and PPh3 (1:1:1) gives [SP-4-3]-[PtCl(NH=CMe2)(dmso)(PPh3)]ClO4 (5). The reactions of complexes 2 and 4 with PhICl2 give the Pt(IV) derivatives [OC-6-13]-[PtCl3(NH=CMe2)(2)(dmso)]ClO4 (6) and [OC-6-13]-[PtCl2(NH=CMe2)2(dtbbpy)](ClO4)2 (7), respectively. Complexes 1cis and 1trans react with NaH and [AuCl(PPh3)] (1:10:1.2) to give cis- and trans-[PtCl{mu-N(AuPPh3)=CMe2}(PPh3)2]ClO4 (8cis and 8trans), respectively. The crystal structures of 4.0.5Et2O.0.5Me2CO and 6 have been determined; both exhibit pseudosymmetry.     

Intramolecular Friedel-Crafts-type reactions involving N-acyliminium ions derived from glycine templates

Enantiomerically pure 4-substituted 2-aralkyl-2,4-dihydro-1H-pyrazino[2,1-b]quinazoline-3,6-diones (1b-m) in which the alkyl chain is (CH(2))(n), n = 1-3, behave as glycine templates giving by treatment with [hydroxy(tosyloxy)iodo]benzene in ethyl acetate cis-1-tosyloxy derivatives. When these compounds contain electron-rich aryl substituents with n = 2, they spontaneously cyclize through intramolecular Friedel-Crafts-type diastereoselective reactions to give penta- or hexacyclic compounds. Otherwise, they give by solvolysis cis-1-alkoxy derivatives, which in a second step, may be cyclized in acid if n = 2, 3. All these reactions must occur through N-acyliminium species in S(N)1-like mechanisms. 1-Alkoxy-2-arylmethyl derivatives are reluctant to cyclize, giving trans-1-hydroxy compounds as the only isolated reaction products.     

Cobalt(III) complexes of monodentate N9-bound adeninate (ade-), [Co(ade-kappaN9)Cl(en)2]+ (en = 1,2-diaminoethane): syntheses, crystal structures, and protonation behaviors of the geometrical isomers

In acidic aqueous solution, a cobalt(III) complex containing monodentate N(9)-bound adeninate (ade(-)), cis-[Co(ade-kappaN(9))Cl(en)(2)]Cl (cis-[1]Cl), underwent protonation to the adeninate moiety without geometrical isomerization or decomposition of the Co(III) coordination sphere, and complexes of cis-[CoCl(Hade)(en)(2)]Cl(2) (cis-[2]Cl(2)) and cis-[Co(H(2)ade)Cl(en)(2)]Cl(3) (cis-[3]Cl(3)) could be isolated. The pK(a) values of the Hade and H(2)ade(+) complexes are 6.03(1) and 2.53(12), respectively, at 20 degrees C in 0.1 M aqueous NaCl. The single-crystal X-ray analyses of cis-[2]Cl(2).0.5H(2)O and cis-[3]Cl(2)(BF(4)).H(2)O revealed that protonation took place first at the adeninate N(7) and then at the N(1) atoms to form adenine tautomer (7H-Hade-kappaN(9)) and cationic adeninium (1H,7H-H(2)ade(+)-kappaN(9)) complexes, respectively. On the other hand, addition of NaOH to an aqueous solution of cis-[1]Cl afforded a mixture of geometrical isomers of the hydroxo-adeninato complex, cis- and trans-[Co(ade-kappaN(9))(OH)(en)(2)](+). The trans-isomer of chloro-adeninato complex trans-[Co(ade-kappaN(9))Cl(en)(2)]BF(4) (trans-[1]BF(4)) was synthesized by a reaction of cis-[2](BF(4))(2) and sodium methoxide in methanol. This isomer in acidic aqueous solution was also stable toward isomerization, affording the corresponding adenine tautomer and adeninium complexes (pK(a) = 5.21(1) and 2.48(9), respectively, at 20 degrees C in 0.1 M aqueous NaCl). The protonated product of trans-[Co(7H-Hade-kappaN(9))Cl(en)(2)](BF(4))(2).H(2)O (trans-[2](BF(4))(2).H(2)O) could also be characterized by X-ray analysis. Furthermore, the hydrogen-bonding interactions of the adeninate/adenine tautomer complexes cis-[1]BF(4), cis-[2](BF(4))(2), and trans-[2](BF(4))(2) with 1-cyclohexyluracil in acetonitrile-d(3) were investigated by (1)H NMR spectroscopy. The crystal structure of trans-[Co(ade)(H(2)O)(en)(2)]HPO(4).3H(2)O, which was obtained by a reaction of trans-[Co(ade)(OH)(en)(2)]BF(4) and NaH(2)PO(4), was also determined.     

Asymmetric allyl-metal bonding in substituted (eta3-allyl) palladium complexes: X-ray structures of cis- and trans-4-acetoxy-

Enantiomerically pure cis and trans isomers of 4-acetoxy-[eta3(1,2,3)-cyclohexenyl]palladium chloride dimers (cis-1 and trans-1) were prepared from enantiomerically pure trans-1-acetoxy-4-chloro-2-cyclohexene. X-ray analyses of these complexes show that in the trans complex (trans-1) the six-membered ring prefers a chair conformation, whereas in the cis complex (cis-1) the cyclohexenyl ring has a boat conformation. According to the X-ray structure of trans-1 the Pd-C3 bond is shorter than the other allylic terminal palladium-carbon bond (Pd-C1). On the other hand, in cis-1 the Pd-C3 and Pd-C1 bond lengths are identical within the experimental error. The calculated structures (B3PW91/LANL2DZ + P) of trans-1 and cis-1 also display differences in the allylpalladium bonding. The asymmetric allylpalladium bonding in trans-1 is explained on the basis of pi-sigma electronic interactions between the 4-acetoxy substituent and the allyl-metal moiety.     

Gas-chromatographic determination of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea in plasma after reaction with trifluoroacetic anhydride

The reaction of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (methyl-CCNU) with trifluoroacetic anhydride is reported. Gas-liquid chromatography of the resulting trifluoroacetyl derivative is applied to determine methyl-CCNU in plasma with a limit of detection of 1.5 μg ml^?1.