Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli. Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and 80 and to colicin M. In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M. Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues. Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M. Two of these regions were common among all four FhuA ligands. Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head. These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and 80 and colicin M.Phages T1, 80, and T5 bind to the FhuA (formerly TonA) outer membrane protein of Escherichia coli. FhuA is also required for the uptake of ferrichrome, the structurally analogous antibiotic albomycin, and colicin M. For transport of the latter compounds through the outer membrane, the proteins TonB, ExbB, and ExbD are required (6). After deletion of residues 322 to 355 of FhuA (FhuA ⌬322-355), uptake of ferrichrome and albomycin no longer required TonB and ExbBD (12). The uptake kinetics were typical of a diffusion process, in contrast to the TonB-and ExbBD-dependent uptake, which showed saturation kinetics and required energy provided by the electrochemical potential of the cytoplasmic membrane (2, 7). Cells expressing FhuA ⌬322-355 became sensitive to sodium dodecyl sulfate and bacitracin, in contrast to the resistance of cells expressing wild-type FhuA, indicating the formation of open channels by FhuA ⌬322-355. In black lipid membranes FhuA ⌬322-355 formed stable channels three times as large as the porin OmpF, while wild-type FhuA did not increase the conductance of KCl through artificial membranes (12). In a model predicting the transmembrane arrangement of the FhuA protein, residues 316 to 356 formed the largest loop at the cell surface (14). This segment of FhuA carrying an inserted C3 epitope of poliovirus reacted with anti-C3 antibodies, indicating exposure of the epitope at the E. coli cell surface (17). Since removal of this loop converted the FhuA transport protein into a diffusion channel, we proposed that the basic structure of FhuA is a channel which is closed by the loop formed by residues 316 to 356 (gating loop). Through interaction with the TonB-ExbBD complex, FhuA undergoes a conformational change in which the gating loop moves and opens the channel (3, 12).Cells expressing FhuA ⌬322-355 were also resistant to phages T1, 80, and T5 and were not killed by colicin M. Deletion of Asp-348 in the gating loop reduced T5 sensitiv...

COMMUNICATIONS 4a: 1 (473 mg, 1.8 mmol), 3 (430 mg, 1.8 mmol), and bis(triphenylphosphane1-(ethene)platinum (83 mg, 0.12 mmol, 6.2 mol%) in toluene (15 mL) were heated under reflux for 24 h. Afterwards, the precipitate was isolated by filtration, washed several times with THF and CH,Cl,, and dried in vacuum. Yield: 455 mg (50.6%)) m.p.: >310"C, 'HNMR (ZOOMHz, [D,]MeOH): 6 =6.5-6.9 (m); I3C NMR (50 MHz, [DJMeOH): 6 = 146.3,120.9, 116.4(C6H.,), CBnot observed; "BNMR (64 MHz, [DJMeOH): 6 = 34 (Av,,~ = 524 Hz), 18 (AvIj, = 58 Hz, C,H,02BOCD,); HR-MS (EI): m/z 500.1209 ( M + , calcd '2C,,1H,,'oB,'60,: 500.1217); correct C, H analysis. 2b, 2c: A suspension of 2a (785 mg, 1 mmol) in toluene (15 mL) and methyllithium (8 mL of a 1.5 M solution in diethyl ether, 12 mmol) was first stirred for 1.5 h at -5 "C and then for 8 h at 25 "C. After removing the insoluble components, a yellow solution of 2b was obtained which contained minor amounts of boranates. I I B 29 Hz); GC-MS: m/z ("A) 317 (100) [M+], 302 (17.4) [ M + -Me], 41 (92.4) [BMe,+]. An excess of pyridine was added to the yellow solution. Crystalline 2c precipitated from the resulting orange-red solution at room temperature. Yield: 296 mg(33%), m.p.: >250°C (decomp), 'HNMR (200 MHz, CDCI,): 6 = -0.3 to 0.3 (m, BMe), 5.8 -6.9 (m, C,H,), 7.0 (m, Py), 7.5 (m, Py), 8.2 (m, Py); 13C NMR (50MHz, CDCI,): 6 =13.0 (BMe, br), 114.6, 116.5, 118.2, 120.0, 153.6, 160.0 (C6H,), 123.7, 136.6, 149.6 (Py), 168.0 (CB, br); I l B NMR (64 MHz, CDCI,): 6 = 0.3 ( A V , ,~ = 204 Ht); MS (FD): m/z 982 [ M + -Py -4Mel. NMR (64 MHz): 6 = 6 (AVlj2 = 466 Hz), -17.4 (Av,,, = 87 Hz), -20.6 (AVlj2 =

No abstract

COMMUNICATIONS 23: The Fmoc compound 23 was prepared from 22 (0.32 g, 1 mmol) as described for 7. Crystallization from ethanol/ether. Yield 0.42 g (93 %); homogeneous (TLC system A); ESI-MS: m/i 448 [ M f ] , 470 [ M + Na]', 492 [ M + 2Na]+.

The 2,4,6-trimethoxybenzyl (Tmob) group is very suitable for temporary protection of the aminosulfonyl group of S-cysteine-and homo-S-cysteinesulfonamides. Both the introduction and removal of the Tmob-protecting group can be achieved in high yields. 2-Aminoethyl derivatization of the aminosulfonyl group of S-cysteine-and homo-S-cysteinesulfonamides has been carried out, and various derivatives useful for peptide synthesis with Z, Boc and Fmoc as N,-protecting groups have been prepared. In all cases, a racemic mixture of Scysteine-or homo-S-cysteinesulfonic acid has been used and subsequent enantiomer resolution has been achieved by treatment with Bacillus subtilis alkaline protease.Since a long time due to its structural similarity to asparagine, S-cysteinesulfonamide was suspected to have the ability to act as antagonist [']. Hence, several methods for the synthetic preparation of this sulfonamide have been reported by different authors, all based on oxidative chlorination of the disulfide bond in the cystine molecule followed by replacement of the chlorine atom in the sulfochloride by an amino g r o~p [~-~] .A common shortcoming of these methods is the high instability of the intermediate sulfochloride. The latter undergoes fast hydrolysis which is the reason for the low yields of the sulfonamide. Therefore, we began studies on S-cysteinesulfonamide derivatives and particular attention was payed to the stability of the respective sulfochloride intermediates. Our experiments showed that stable cysteine sulfochloride derivatives are obtained only if both the amino and the carboxy groups are blocked [6]. A convenient combination of protecting groups proved to be the N,-benzyloxycarbonyl and benzyl or ethyl ester group. For cysteine sulfochloride-containing peptides it was observed that stability is strongly dependent on the molecular mass of the peptide. If it is higher than 1000, the half-life is very shortr71.The investigation of the biological activity of S-cysteinesulfonamide derivatives with modified aminosulfonyl group[*] as well as S-cysteinesulfonamide-containing peptided9] demonstrated that the interest in those products is fully justified [lO]. In several cases, the peptides were obtained in significantly reduced yields due to side reactions of the unprotected aminosulfonyl group[' ' 1. Therefore, we had to find a suitable group for temporary protection of this function that would allow its utilization in peptide synthesis, including the solid-phase method. Our efforts were also directed to the preparation of suitably protected Scysteinesulfonamides (3-[(2,4,6-trimethoxybenzyl)aminosulfonyllalanine) and homo-S-cysteinesulfonamides (2-amino-4-[(2,4,6-trimethoxybenzyl)aminosulfonyl]butyric acid) to be used in solid-phase or conventional synthesis of biologically active compounds.The starting compounds were N,-benzyloxycarbonyl-protected sulfochlorides of S-cysteinesulfoni~[~-~~'~] and homo-S-cysteinesulfonic acid[l31 esters prepared by methods described in the literature. The aminosulfonyl group was prot...