the reaction mixture was brought to atmospheric pressure and the yellow solution was evaporated to a yellow oil. Recrystallization from THF/ toluene at -10 °C provided 0.3981 g (88%) of yellow crystalline 13: 'H NMR (THF-rfg) 7.55 (m, 35 H, PNP, Ph), 2.98, 2.80 (AB, 3/Hcch = 4.3 Hz, 1 , 1 H, HOC, HOC); IR (THF) r(CO) 2039 (w), 1895 (vs), 1864 (m), i-C(O) 1558 (w) cm"1.Acknowledgment. The financial support of this research by the National Science Foundation (Grant CHE 83-08281) is greatly appreciated. G. G. was the recipient of a DAAD/NATO Scholarship for which we are most grateful.

TMEDA (5.8 g, 7.55 mL, 0.5 mol) was added. The solution was chilled in an ice bath and n-butyllithium (31.4 mL of a 1.6 M solution in hexane, 0.05 mol) was added dropwise. The turbid solution was allowed to warm up to room temperature and then heated under reflux overnight (20 h). During this lithiation period a tannish-yellow precipitate formed. The mixture was again cooled in an ice bath and quenched by dropwise addition of a solution of dimethyl sulfate (6.94 g, 5.2 mL, 0.55 mL) in 50 mL of ether. The precipitate became more dense and turned white. After being stirred for 3 h, the mixture was poured over ice-water, the ether layer was separated, and the aqueous layer was extracted with ether. The combined ether extracts were washed with 2 N NH4OH, 2 N HC1, water, and brine and then dried (MgSQ4) and concentrated. The yellow residue was recrystallized from pentane: mp 168-169 °C;

The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.

No abstract

at 90 MHz were satisfactory. In all cases p-dimethoxybenzene was the internal standard. The quartets at 1.942 and 2.317 due to the H4 protons were used for 7a and 8a respectively. Doublets at 1.150 and at 1.087 due to the 5-CH3 group were used for 7b and 8b respectively. Quartets at 2.330 and at 1.966 (H4 protons) and singlets at 2.188 and 2.100 (CH3CO-) were used for 10, 9,11, and 8c, respectively. The multiplet at 5.49-5.10 (H; and H3) and the triplet at 2.56 (H4 protons) were used for 7d and 8d, respectively.Rate of Reaction of CAN with Carbonyl Compounds. The rate of disappearance of CAN in solutions containing the various carbonyl compounds were determined by iodometric analysis in the absence and in the presence of 1,3-butadiene. With acetone, the half-life time of CAN was 5 h in the presence and 17 h in the absence of 1,3-butadiene. Corresponding values for 2-butanone were 0.75 and 2.3 h. With ethyl acetoacetate the disappearance of CAN was almost instantaneous in the presence of 1,3-butadiene, whereas in the absence of diene only 58% of CAN was reduced after 30 min. With 3-methyl-2-butanone reduction of CAN was complete after 8 and 24 h, in the presence and in the absence of 1,3-butadiene, respectively.Acknowledgment. Thanks are due to the Italian National Council of Research (CNR) and the Ministero della Pubblica Istruzione for financial support.Registry No. 7a, 100431-93-4; 7b (isomer 1), 100431-95-6; 7b (isomer 2),