The convergent total synthesis of cryptophycins C and D is described. It has been shown that in both natural products the absolute configuration of the a-amino acid corresponds to the D-series. The structural assignment for cryptophycin C has been corrected to reflect this fact. Since the structure of cryptophycin A has been correlated to cryptophycin C, the chloro-0-methyltyrosine unit in cryptophycin A has the D-configuration.Cryptophycins are potent tumor-selective cytotoxins associated with the terrestrial blue-green algae Nostoc sp. GSV 224' and Nostoc sp. ATCC 53789.2 The major cytotoxin in each alga, cryptophycin A, shows excellent activity against solid tumors implanted in mice, including a drug-resistant tumor. Over 20 related cytotoxins are present in the GSV 224 strain as minor constituent^,'^^ and some of these compounds, e.g., cryptophycins B and C, have been isolated in sufficient amounts for in vivo e~aluation.~ In order to acquire adequate quantities of selected naturally-occurring cryptophycins and synthetic analogs for structure-activity relationship (SAR) studies, preclinical evaluation, and human clinical trials, we have designed a general synthesis. Cryptophycins C and D, as described in the original paper, were chosen to be the initial targets as they represented examples from both of the alleged L-and D-tyrosine series. We report here the total syntheses of cryptophycins C and D which (1) revise the structures of cryptophycins A and C to reflect the D-configuration for the a-amino acid unit as depicted in the structural drawings in this paper and (2) confirm the structures of cryptophycins B and D.Retrosynthetic analysis of the cryptophycins was straightforward: the structure is composed of four units (A-D, Figure 1); consequently several convergent approaches could be envisioned. The combination of two pairs of units (e.g., A-B and C-D) appeared to be optimally convergent. Since the success of the synthesis depended on the formation of a 16-membered depsipeptide from an acyclic precursor, a macrolactamization involving the amino group of unit C and the carboxylate of unit B appeared to be the best choice. The acyclic precursor to cryptophycin D would therefore be 1. This, in turn, suggested a disconnection into two fragments, one represented by 2 and composed of (S)-( -)-2-hydroxy-4-methylvaleric (L-leucic) acid (D) and (R)-3-amino-2-methylpropanoic acid (C) units, and the other by 3 and composed of O-methyl-D-tyrosine (B) and (2E,7E,5S,6R)-5-hydroxy-6-methyl-8-phenyloctadienoic acid (A) units. In the direction of the synthesis, @ Abstract published in Advance ACS Absfracts, February 15, 1995. (1) Trimurtulu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.; Corbett, T. H.; Valeriote, F. A.; Demchik, L. J. Am. Chem. SOC. 1994, 116, 4729-4731. (2) Schwartz, R. E.; Hirsch, C. F.; Sesin, D. F.; Flor, J. E.; Chartrain, M.; Fromtling, R. E.; Harris, G. H.; Salvatore, M. J.; Liesch, J. M.; Yudin, K. J. Ind. Microbiol. 1990, 5, 113-24. (3) Trimurtulu, G.; Ogino, J.; Heltzel, C. E.; Patterson, G...

These results suggest that blue LED-induced RD may be a useful animal model in which to study the pathogenesis of RD, including age-related macular degeneration, and to evaluate the effects of new therapeutic agents prior to clinical trials, where oxidative stress and inflammation are the underlying RD mechanisms.

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia. To obtain a better understanding of the protein networks involved in this phenomenon, we assessed global protein expression profiles in term placentas from scNT-derived and control animals. Proteomic analysis of term placentas from scNT-derived animals identified 43 proteins that were differentially expressed compared to control animals. Among them, 14-3-3 proteins and Annexin V, which are closely involved in the apoptotic signaling pathway, were significantly down- and up-regulated, respectively. Western blot analysis and immunohistochemistry indicated that down-regulation of 14-3-3 proteins in scNT-derived placentas induced apoptosis of cytotrophoblast cells via mitochondria-mediated apoptosis. Taken together, our results suggest that placental insufficiency in scNT-derived placentas may be due to apoptosis, induced in part by the down-regulation of 14-3-3 proteins and up-regulation of Annexin V. They also indicate that proteomic maps represent an important tool for future studies of placental insufficiency and pathology.

In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, five piglets were stillborn, 22 scNT piglets died suddenly within the first week of life, and 1 piglet died after 40 days. Twelve scNT piglets are still healthy. The birth weights of compromised scNT piglets in comparison with those of normal scNT piglets are significantly reduced (0.80 +/- 0.29 vs 1.27 +/- 0.30 kg, p < 0.05), in spite of longer gestation (114 versus 120 day). Significant findings from histological examinations showed that approximately 25% (7/28) of scNT piglets showed severe congestion of lung and liver or neutrophilic inflammation in brain indicating that unexpected phenotypes can appear as a result of somatic cell cloning. Two-dimensional gel electrophoresis experiments revealed changes in the responses of several detoxification-related proteins related to stress and inflammation and found significant alterations in myocardium-specific proteins, indicating hemodynamic disorder. scNT piglets that survived to adulthood did not show any abnormality except skin and hair color depigmentation. The present study suggests that cerebromeningitis and hemodynamic disorder are a major risk factor for sudden early death of scNT piglets. Although we cannot completely exclude the possibility that scNT piglets are susceptible to specific respiratory infections, our data suggests that the early death of scNT clones is due to cardiopulmonary functional abnormalities and cerebromeningitis.

BackgroundSomatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets.ResultsMicroscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC.ConclusionThese results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development.