Introduction of a Strong Binding Site for Lanthanides at the N-Terminus of Peptides and Ribonuclease A

Abstract

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.

A general method for highly selective crosslinking of unprotected polypeptides via pH-controlled modification of N-terminal .alpha.-amino groups

“…Percent yields of mono-and diacylated products were calculated as percentages of total product peptide absorbance; where percentages do not sum to 100, it indicates the corresponding amount of uncharacterized other peaks in the chromatogram. The table shows that, when reactions are not driven to complete loss of starting material, one obtains a range of selectivity ratios from 9 to 55, with most values at 18 or above.…”

“…Selective modification of a-amino groups by pH control has also been reported for other acylating agents, such as aryl isothiocyanates (9). Most attempts to selectively react the N-termini of polypeptides, however, have been with acetic anhydride.…”