RESUMO
Organic synthesis often requires multiple steps where a functional group (FG) is concealed from reaction by a protecting group (PG). Common PGs include N-carbobenzyloxy (Cbz or Z) of amines and tert-butyloxycarbonyl (OtBu) of acids. An essential step is the removal of the PG, but this often requires excess reagents, extensive time and can have low % yield. An overarching goal of biocatalysis is to use "green" or "enzymatic" methods to catalyse chemical transformations. One under-utilised approach is the use of "deprotectase" biocatalysts to selectively remove PGs from various organic substrates. The advantage of this methodology is the exquisite selectivity of the biocatalyst to only act on its target, leaving other FGs and PGs untouched. A number of deprotectase biocatalysts have been reported but they are not commonly used in mainstream synthetic routes. This study describes the construction of a cascade to deprotect doubly-protected amino acids. The well known Bacillus BS2 esterase was used to remove the OtBu PG from various amino acid substrates. The more obscure Sphingomonas Cbz-ase (amidohydrolase) was screened with a range of N-Cbz-modified amino acid substrates. We then combined both the BS2 and Cbz-ase together for a 1 pot, 2 step deprotection of the model substrate CBz-L-Phe OtBu to produce the free L-Phe. We also provide some insight into the residues involved in substrate recognition and catalysis using docked ligands in the crystal structure of BS2. Similarly, a structural model of the Cbz-ase identifies a potential di-metal binding site and reveals conserved active site residues. This new biocatalytic cascade should be further explored for its application in chemical synthesis.
RESUMO
OBJECTIVE: To collect published data on spermatogonial quantity in the testes of healthy children and calculate the reference values of spermatogonial quantities throughout prepuberty. DESIGN: Systematic literature search in PubMed and EMBASE focusing on the number of spermatogonia per transverse tubular cross section (S/T) and spermatogonial density per cubic centimeter (cm3) of testicular volume (S/V) throughout prepuberty. SETTING: None. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Polynomial meta-regression analyses of S/T and S/V of healthy boys from the ages of 0 to 14 years. RESULT(S): We found six papers describing original quantitative data on S/T and S/V of healthy boys (total n = 334 and 62, respectively) that were suitable for meta-analysis. Polynomial meta-regression analyses of S/T and S/V demonstrated a clear pattern of spermatogonial quantity throughout prepubertal life. This consisted of a decline during the first 3 years of life, a gradual increase until the ages of 6 to 7 years, a plateau until the age of 11 years, and a sharp incline reaching pubertal numbers at 13 to 14 years of age. The association between S/T and S/V allowed us to perform S/T to S/V extrapolation, creating reference S/V (rS/V) values throughout prepubertal life from a cohort of 372 boys. CONCLUSION(S): Spermatogonial quantity varies during testicular development toward puberty. The values found in this study may serve as a baseline clinical reference to study the impact of diseases and adverse effects of gonadotoxic treatments on spermatogonial quantity in prepubertal testes. Spermatogonial quantity reference values may also help to evaluate the quality of testicular biopsy samples acquired for fertility preservation of prepubertal boys.