Age estimation in biopsy specimens of dentin

Determination of age at death on the basis of aspartic acid racemization in dentin is one of the most reproducible and accurate methods. In Germany, age estimation by this method has so far generally not been applied to living persons, since the extraction of a tooth exclusively for age estimation when it is not medically indicated is regarded as ethically and legally problematic. The development of a biopsy technique applicable to dentin took place against this background. Testing the technique and analysis of dentinal biopsy specimens revealed that the biopsy technique is a low-risk procedure that causes only minor discomfort to the affected person. It is readily practicable and facilitates standardized specimen removal. The relationship between the extent of aspartic acid racemization in dentinal biopsy specimens and age is very close, facilitating age estimation. A prerequisite for accurate results is the performance of biopsies under strictly standardized conditions. If this is guaranteed, age determination on the basis of aspartic acid racemization in dentinal biopsy specimens appears to be superior in precision to most other methods in living persons and can be used for all age groups.     

Peptide formation in the presence of metal ion protecting groups

A quantitative study of the degree of racemization induced by the [(NH₃)₅Co-(III)-] protecting group when bound to the C-terminal of the amino acids Leu, Phe, and His, as has been carried out. Racemization was determined by forming the diastereomeric cobalt dipeptides [(Leu)(AA)Co(III)(NH3)₅] where AA = L-Leu, L-Phe, and L-His; after cobalt removal (using NaBH₄), the peptide diastereomers were analyzed quantitatively using an amino acid analyzer. No racemization was observed within experimental error (0.3%) as a result of the substitution of the [(NH3)₅Co(III)-] group on the amino acids and peptides studied.     

STUDIES ON RACEMIZATION DURING COUPLINGS USING A SERIES OF MODEL TRIPEPTIDES INVOLVING ACTIVATED RESIDUES WITH UNFUNCTIONALIZED SIDE CHAINS

Racemization studies have been carried out using as model tests couplings of N-benzyloxycarbonylglycyl-l -χ with benzyl N^e-benzyloxycarbonyl-l -lysinate followed by deprotection and analysis for the diastereomeric tripeptides with an amino acid analyzer, for χ = alanine, leucine, phenylalanine, valine and isoleucine. The order of susceptibility to racemization of residues during these segment couplings depends on whether the solvent is polar or apolar, with isoleucyl and valyl, followed by phenylalanyl, being the most susceptible ones in polar solvents. The racemization depressing effect of some additives on carbodiimide-mediated couplings has been examined. Reconciliation of apparent discrepancies in data in the literature on the relative merits of l-hydroxy-benzotriazole and N-hydroxysuccinimide is made on the basis of the nature of the model tests used in acquiring the data.     

微波促进(R)-萘普生消旋反应

用微波辐射技术,以葡甲胺为催化剂、水为溶剂,于800W功率下回流反应100min,成功对(R)-萘普生进行消 旋,收率为95％.方法操作简单,反应时间短,后处理简便.     

Studies on the carbodiimide-mediated model couplings of Z-Pro-Leu-OH with benzoaza-15-crown-5

A series of model DCC-mediated couplings of Z-Pro-D-Leu-OH with 2,3-benzo-10-aza-1,4,7,13-tetraoxacyclopentadeca-2-ene were studied by high-performance liquid chromatography. It was found that racemization during preparation of N-(Z-Pro-Leu-)benzoaza-15-crown-5 is promoted by HOBT, an additive usually reducing this process.     

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO), a promising new reagent for racemization-free couplings

3-Dimethylphosphinothioyl-2(3H)-oxazolone (MPTO) was synthesized, and its ability to effect racemization-free couplings and cyclization of a peptide and its C-terminal epimer was examined. MPTO showed good reactivity in aprotic polar solvents such as N,N-dimethylformamide (DMF) and N-methylpyrrolidone. In reactivity MPTO resembles DPPA and dimethylphosphinothioyl azide (MPTA) previously developed by us, but it is much better than these reagents because the side reactions specific to the azide method could be avoided. In coupling of Z-Gly-Val-OH with H-Val-OMe in DMF at 0°C, no racemization was observed without use of racemization-suppressing additives. Slight racemization observed at room temperature could be completely suppressed by addition of HOBt but not by HOSu. The utility of MPTO was demonstrated by the synthesis of cyclo-(d -Trp-d -Glu(OBzl)-Ala-D-Val-Leu), an intermediate for an endothelin-binding inhibitor BE 18257A. In a comparative study using DPPA, MPTA and MPTO, no racemization was observed for MPTA or MPTO, while DPPA caused considerable racemization. When MPTO was used in the presence of HOBt rapid cyclization (3 h at RT) occurred to give the optically pure cyclic product.     

Kinetic study of racemization of aspartyl residues in model peptides of αA-crystallin

We have reported that two aspartyl (Asp-151 and Asp-58) residues in αA-crystallin in human eye lens were inverted to the D-isomer and isomerized to β-aspartyl residues with age. We report here the kinetics of the Asp racemization of three model peptides corresponding to fragments of αA-crystallin: IQTGLD¹⁵¹ATHAER (T18 peptide). TVLD⁵⁸SGISEVR (T6 peptide) and HFSPED⁸⁴LTVK (T10 peptide, as a control). The rate constants of the racemization of Asp residues in these peptides were measured at pH 7.0. at five temperatures: 50, 60, 70, 80 and 90 °C. From the Arrhenius equation, we estimated the activation energy (E) of racemization and the time required for the Asp D/L ratio to approximate to 1.0 (D/L ratio of Asp = 0.99) at body temperature. For the peptide T18, E=21.4 kcal/mol and t=13.5 yr. For the peptide T6, E= 26.8 kcal/mol and t= 49.5 yr. For the control peptide T10, E=28.3 kcal/mol and t= 78.1 yr. The racemization rate of Asp in these three peptides is parallel to that of Asp residues in αA-crystallin. The racemization rate of Asp in the T18 peptide was very rapid compared to that in the other peptides. This result also reflects the racemization rate in native αA-crystallin     

Protected amino acid tetrafluorophenyl esters for peptide synthesis

New 2,3,5,6-tetrafluorophenyl active esters of protected amino acids useful for peptide synthesis were prepared in high yield. For certain amino acid derivatives such as Boc-Pro-OH, Boc-Ile-OH, and Boc-Val-OH, their tetrafluorophenyl esters have significantly higher melting points than the corresponding pentafluorophenyl esters. Kinetic studies were carried out to compare the racemization rate constants (with triethylamine) and coupling rate constants (with valine methyl ester) of the tetrafluorophenyl and pentafluorophenyl esters of protected histidine and tyrosine. Results of the second-order kinetics showed similarly large k_coupling/k_racemization ratios for both tetra- and pentafluorophenyl esters. In particular, the use of 2,3,5,6-tetrafluorophenyl or pentafluorophenyl ester prevents extensive racemization of the N-tert.-butyloxycarbonyl-N^im-benzyl-histidine.     

Isolation and identification of C-terminal peptides of proteins

A method for the isolation and identification of C-terminal peptides of proteins has been developed. The procedure entails the racemization of the C-terminal amino acid by reaction of the N-trifluoroacetylated protein with acetic anhydride and pyridine. After deprotection, the protein is fragmented to yield a mixture of peptides, which in turn are digested with carboxypeptidases. All peptides are hydrolyzed to L-amino acids except the C-terminal peptide with the terminal D-amino-acid residue. It is resistant to the action of carboxypeptidases and is readily identified by peptide mapping. © Munksgaard 1995.