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A new reagent, phthalyl-4-isothiocyanate, reacts specifically at the N-terminus of a peptide or protein with the production of a phthalic acid 4-thiocarbamyl derivative. The adjacent carboxyl groups of the phthalyl group serve as a strong binding site for lanthanides at pH 5 with a stability constant for praseodymium about 220 times that of a single carboxyl group.Reaction of phthalyl-4-isothiocyanate with a-amino groups of peptides at 25 "C in 2H,O at pH meter readings of 8.7 and 7.2 is complete in 15 min and 24 h respectively, without modification of histidine or tyrosine side chains. At the lower pH there is no reaction of the &-amino group of lysine. The phthalic acid 4-thiocarbamyl derivative of ribonuclease A is cyclised and cleaved by the normal Edman degradation in acid to produce the phthalic acid 4-thiohydantoin of lysine. The a-CH proton of lysine-1 in ribonuclease A produces a triplet nuclear magnetic resonance that has been assigned on the basis of its coupling constant, the pH dependence of its chemical shift (pK' is 7.6) and its disappearance on reaction of ribonuclease A with phthalyl-4-isothiocyanate.The reaction of phthalyl-4-isothiocyanate with a protein allows the introduction of a strong lanthanide binding site specifically at the N-terminus, as a starting point for structural studies using nuclear magnetic resonance spectroscopy.

On binding a paramagnetic broadening probe at the end of a random coil polypeptide chain, one obtains sequential broadening of the -CH resonances along the chain and, hence, can determine the sequence of the peptide. The method has been applied to tripeptides (0.25 mg of material being used), tetrapeptides and a hexapeptide under conditions such that gadolinium ions bind at the C-terminus or cupric ions at the N-terminus.Dimethylation of the lysine residues of lysozyrne and observation of the chemical shifts of the six dimethyl proton resonances as a function of pH allows the determination of their pK. From studies of selective broadening of the methyl resonances on addition of gadolinium ions and the known structure of the lysozyme-ion complex, it is possible to obtain the pK values of the lysine residues of lysozyme.Approaches to the determination of the structure in solution of proteins using paramagnetic broadening and shifting probes is discussed. Problems concerning the site of ion binding and the assignment of p.m.r. resonances arise when the crystal structure is unknown.The amount by which a proton magnetic resonance (p.m.r.) line is broadened by a paramagnetic probe with a long electronic relaxation time -e.g. gadolinium(III)-is dependent on the inverse sixth power of the distance between the nucleus giving rise to the resonance and the paramagnetic probe. Furthermore, if the paramagnetic probe is an ion which binds rapidly and reversibly at a specific site, then the width of the resonance line will progressively increase as the concentration of paramagnetic ion is increased until such time as the site is saturated. after which no further broadening will occur. On the binding of gadolinium ions at low concentration to lysozyme. there is preferential broadening of p.m.r. resonances that arise from protons which are closest to the binding 2 The binding site has been shown by x-ray studies3 to be located between glutamic acid 35 and aspartic acid 52. By the use of difference spectroscopy46, in this case the subtraction of a paramagnetically broadened spectrum from the spectrum of native lysozyme, it has been possible to observe the resonances from the side chains of valine 83

Several methods for the preparation of long-chain vicinal diketones are examined. The acyloin condensation, followed by oxidation, provides an excellent route to symmetrical compounds but is not satisfactoryfor unsymmetrical diketones. The latter are prepared (i) from dialkylacetylenes by semihydrogenation, hydroxylation, and oxidation with aqueous N-bromosuccinimide ; and (ii) from an a-acetoxy-acid chloride and the sodio-derivative of a bistetrahydropyranyl or dibenzyl alkylmalonate, followed by removal of the protecting groups and decarboxylation to give the a-acetoxy-ketone, which is then hydrolysed and oxidised.W. Rigby, J . Chem. SOC., 1951, 793.