Gas phase reactivity of carboxylates with N-hydroxysuccinimide esters.

N-hydroxysuccinimide (NHS) esters have been used for gas-phase conjugation reactions with peptides at nucleophilic sites, such as primary amines (N-terminus, ε-amine of lysine) or guanidines, by forming amide bonds through a nucleophilic attack on the carbonyl carbon. The carboxylate has recently been found to also be a reactive nucleophile capable of initiating a similar nucleophilic attack to form a labile anhydride bond. The fragile bond is easily cleaved, resulting in an oxygen transfer from the carboxylate-containing species to the reagent, nominally observed as a water transfer. This reactivity is shown for both peptides and non-peptidic species. Reagents isotopically labeled with O(18) were used to confirm reactivity. This constitutes an example of distinct differences in reactivity of carboxylates between the gas phase, where they are shown to be reactive, and the solution phase, where they are not regarded as reactive with NHS esters.

Solution versus gas-phase modification of peptide cations with NHS-ester reagents.

A comparison between solution and gas phase modification of primary amine sites in model peptide cations with N-hydroxysuccinimide (NHS) ester reagents is presented. In all peptides, the site of modification in solution was directed to the N-terminus by conducting reactions at pH=5, whereas for the same peptides, a lysine residue was preferentially modified in the gas phase. The difference in pKa values of the N-terminus and ε-amino group of the lysine allows for a degree of control over sites of protonation of the peptides in aqueous solution. With removal of the dielectric and multiple charging of the peptide ions in the gas phase, the accommodation of excess charge can affect the preferred sites of reaction. Interaction of the lone pair of the primary nitrogen with a proton reduces its nucleophilicity and, as a result, its reactivity towards NHS-esters. While no evidence for reaction of the N-terminus with sulfo-NHS-acetate was noted in the model peptide cations, a charge inversion experiment using bis[sulfosuccinimidyl] suberate, a cross-linking reagent with two sulfo-NHS-ester functionalities, showed modification of the N-terminus. Hence, an unprotonated N-terminus can serve as a nucleophile to displace NHS, which suggests that its lack of reactivity with the peptide cations is likely due to the participation of the N-terminus in solvating excess charge.

Rotational spectra of o-, m-, and p-cyanophenol and internal rotation of p-cyanophenol.

Rotational spectra of p-, m-, and o-cyanophenol have been measured in the range of 10.5-21 GHz and fit using Watson's A-reduction Hamiltonian coupled with nuclear quadrupole coupling interaction terms for the (14)N nuclei. Ab initio calculations at the MP2/6-311++G(d,p) and CCSD(T)/6-311++G(d,p) levels predict the cis conformers of m- and o-cyanophenol to be more stable than the corresponding trans conformers. A natural bond orbital analysis of the hydrogen bonding interaction in o- and m-cyanophenol revealed an intramolecular hydrogen bond that preferentially stabilizes the cis conformer of o-cyanophenol but there was no evidence of hydrogen bonding interactions in cis m-cyanophenol. We recorded 25 a- and b-type rotational transitions for cis o-cyanophenol; the rotational constants are A = 3053.758(2) MHz, B = 1511.2760(3) MHz, and C = 1010.7989(2) MHz. The trans conformer of o-cyanophenol was not observed. We recorded 14 a- and b-type rotational transitions for cis m-cyanophenol and 16 a- and b-type rotational transitions for trans m-cyanophenol. The rotational constants are A = 3408.9200(2) MHz, B = 1205.8269(2) MHz, and C = 890.6672(1) MHz and A = 3403.1196(3) MHz, B = 1208.4903(2) MHz, and C = 891.7241(2) MHz for the cis and trans species, respectively. Rotational transitions of the p-cyanophenol monomer are split due to the internal rotation of the hydroxyl group with respect to the aromatic ring. We recorded 25 a- and b-type rotational transitions for p-cyanophenol; the b-type transitions are split by 40 MHz. The rotational constants are A = 5612.96(2) MHz, B = 990.4283(6) MHz, and C = 841.9363(6) MHz. The ground state spitting DeltaE is 20.1608(6) MHz and the barrier to internal rotation, V(2), is 1413(2) cm(-1) from a fit of the rotational transitions to an internal axis system Hamiltonian. The barrier to internal rotation was modeled at the MP2/6-311++G(d,p) level and the effects of substituents on the phenolic ring and the barriers to internal rotation are discussed.