The unusual hydrogen-deuterium exchange of α-carbon protons in N-substituted glycine- containing peptides Remigiusz Bąchor, Bartosz Setner, Alicja Kluczyk, Piotr Stefanowicz and Zbigniew Szewczuk* Hydrogens connected to α-carbon (α-C) of amino acid residues are usually resistant to hydrogen-deuterium exchange (HDX) unless reaction conditions promote racemization. Although N-methylglycine (sarcosine) residue has been found in biologically active peptide such as cyclosporine, to the best of our knowledge, the HDX of α-C protons of this residue was not explored yet. Here, we presented a new and efficient methodology of α-C deuteration in sarcosine residues under basic aqueous conditions. The deuterons, introduced at α-C atom, do not undergo back-exchange in acidic aqueous solution. The electrospray ioniza- tion-MS and MS/MS experiments on proposed model peptides confirmed the HDX at α-C and revealed the unexpected hydro- gen scrambling in sarcosine-containing peptides. Although the observed HDX of α-C protons is only successful in N-acylglycine when the amide possesses a certain degree of alkylation, it offers a new approach to the analysis of sarcosine-containing peptides such as cyclosporine. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: HDX of peptides; ESI-MS/MS; sarcosine; hydrogen scrambling; cyclosporine Labile hydrogens in backbone and side-chain functional groups of peptides and proteins undergo exchange with protons of the solvent within a few minutes. [1] Hydrogens bound to carbon atoms are generally not exchangeable; however, under specific conditions, including pH-dependent and metal-dependent catal- ysis, it is possible to replace protons attached to α-carbon (α-C) by deuterons. Therefore, the exchange reactions involving the displacement of hydrogen attached to carbon by its heavier iso- topes are of interest for both mechanistic and product-orientated research. [2] The base-catalyzed, hydrogen-deuterium exchange (HDX) re- actions also provide a simple method for the exchange of acidic hydrogen atoms for deuterium by means of keto–enol equilib- ria. [3] The acidic hydrogen atoms bound to carbon in carbonyl compounds such as N-substituted acetamides and diketopiperazines are exchanged with high yield. [4] The reported HDX reaction involved the use of acetone-d 6 , triethylamine (TEA) or diazabicycloundec-7-ene and incubation at the temperature of 35–50 °C and was monitored via proton nuclear magnetic reso- nance ( 1 H NMR) spectroscopy. The acidity of α-C protons of amino acids and peptides is of interest because the correspond- ing enolates are key intermediates in nonenzymatic racemization during chemical synthesis of peptides and enzyme-catalyzed racemization during biochemical transformations. [5,4] Despite the importance of these enolates in chemistry and biology, there have been only few quantitative studies on their stability in water. The rate constants for their formation under extreme conditions have been determined in studies of α-hydrogen exchange or race- mization reactions of amino acids and peptides at high tempera- tures [6,7] or in the presence of high concentrations of acids [8] or bases. [9] Considerable attention is given to base-catalyzed methods for the preparation of α-deuterated amino acids. [10–12] The synthesis involves glycine derivatives, which are subjected to a basic HDX, whereas the desired side chain is inserted stereoselectively with the aid of chiral auxiliaries. [13] The differences in the acidity of α-C hydrogens of various amino acid derivatives have been extensively studied. Ho and coworkers [4,14] proved that α-C hydrogens in N-methylated ana- logs of cyclic dipeptides (diketopiperazines) are more acidic than these in unmethylated species. They also confirmed that the sub- stitution of the amino group with electron-withdrawing substitu- ents such as acetyl group facilitates the HDX of α-C hydrogens. Rios et al. found [5,15,16] that the acidity of the α-C hydrogen de- pends on the ionization state of the amino acid, moreover, the exhaustive N-methylation also affects the pKa of the α-C hydro- gens. The pKa determined for N-protonated glycine methyl ester is 21, whereas the value reported for betaine methyl ester is 18. Nowadays, compounds labeled by stable isotopes at carbon centers are of interest, as the deuterium-labeled compounds are commonly used as internal standards in MS for quantitative analy- sis and in elucidation of fragmentation mechanisms. Usually, it is easier and more cost-effective to obtain such compounds by HDX in the target molecule than by de novo synthesis. [10] As the devel- opment of methods for accurate protein quantitation is currently one of the most challenging areas in proteomic research, such stan- dards are needed. [17] It is worth noting that the MS signals derived * Correspondence to: Zbigniew Szewczuk, Faculty of Chemistry, University of Wroclaw, ul. F. Joliot-Curie 14, 50–383 Wroclaw, Poland. E-mail: zbigniew. szewczuk@chem.uni.wroc.pl Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, Wroclaw, Poland J. Mass Spectrom. 2014, 49, 43–49 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 15 July 2013 Revised: 7 November 2013 Accepted: 21 November 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3318 43