MODIFICATIONS OF SOLID PHASE PEPTIDE SYNTHESIS TO OBTAIN HOMOGENEOUS OLIGOPROLINES IN HIGH YIELD

When oligoprolines containing a [³H] proline in the second residue from the carboxyl terminus are synthesized by the standard solid phase method using stepwise addition of prolines there appears, in addition to the oligoprolines, a steady increase of ninhydrin-negative, but radioactive, impurities. These impurities were found to be N-trifluoroacetyl oligoprolines, and a substantial amount of diketopiperazine was present at the diproline stage. The trifluoroacetyl blockage of the N-termini was almost complete by the time 14 residues were added. To eliminate these side reactions, a block of Boc[³H]Pro₂ was coupled to prolyl-resin in order to avoid the diprolyl-resin stage, and 25% CF₃CO₂H in CHCl₃ was replaced by 4N HCl in dioxane as the deprotecting reagent. These changes eliminated the formation of diketopiperazine and trifluoroacetyl impurities, but still a steady increase of ninhydrin-negative, but radioactive, impurity was seen, reaching 22% after three couplings. These side reactions were traced to the neutralization step, and in order to avoid them it became necessary to eliminate the neutralization step, and replace it by incremental additions of Et₃N during the coupling step. As a result of these modifications, we now obtain homogeneous length oligoprolines upon cleavage from the resin, with no further purification, when analyzed by reversed-phase liquid chromatography.     

Neonatal Neutropenia Due to Maternal Isoimmunization

A family is reported with multiple cases of neonatal neutropenia. The firstchild died of severe infection, the second was asymptomatic, the third andthe fourth children had a benign course of agranulocytosis which lasted about10 weeks and was followed by spontaneous recovery. A potent leukoagglutininwas found in the maternal serum which agglutinated leukocytes obtained fromthe father and all three available children but failed to agglutinate themother’s own cells. Identical leukoagglutinin was found in the last baby’sserum; it disappeared after the baby recovered. The disorder is tentativelynamed "isoimmune neonatal neutropenia" and is considered to represent acondition analogous to erythroblastosis fetalis.

Submitted on May 4, 1959 Accepted on July 28, 1959     

A New Technic for Separation of Human Leukocytes

A simple technic for separation of human leukocytes is described. It utilizesdifferential sedimentation following red cell and platelet agglutination inducedby Polybrene. In this technic, lymphocytes are partially removed and over50 per cent of polymorphonuclears are recovered. The leukocyte/red cell ratiois about 5, if blood samples with normal leukocyte counts are used. By useof blood samples containing higher white cell counts this ratio may be increased. The separated cells proved to be highly viable as tested by phasemicroscopy, supravital staining and phagocytosis. There was no significantchange in oxygen uptake of leukemic cells in the presence of a relatively largePolybrene concentration.

Accepted on October 30, 1961