67 Rapid Communication Received: 22 October 2008 Revised: 10 November 2008 Accepted: 11 November 2008 Published online in Wiley Interscience: 17 December 2008 (www.interscience.com) DOI 10.1002/psc.1105 Building blocks for the synthesis of post-translationally modiﬁed glycated peptides and proteins Stefano Carganico, a,b Paolo Rovero, a,c Jose A. Halperin, d,e Anna Maria Papini a,b and Michael Chorev d,e* Growing interest in synthetic peptides carrying post-traslational modiﬁcations, in general, and the Amadori modiﬁcation in particular, raises the need for speciﬁc building blocks that can be used in stepwise peptide synthesis. Herein, we report the synthesis of N α -Fmoc-Lys-OH derivatives containing N ε -1-deoxyfructopyranosyl moiety. Copyright c  2008 European Peptide Society and John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article Keywords: glycation; Amadori rearrangement; Lys(N ε -1-deoxyfructopyranosyl); Amadori-containing building block; site-speciﬁc modiﬁcation; SPPS Glycation of proteins through non-enzymatic reactions between glucose or other reducing sugars and reactive amino groups represents one of the more abundant processes involved in post-translational modiﬁcation of proteins [1]. Spontaneous and reversible condensation of a reducing sugar and a free amino group of a protein forms an aldimine also known as the Schiff base that undergoes a rearrangement into the more stable ketoamine known also as the Amadori product [2]. In the case of glucose, the initially formed Schiff base rearranges into the more stable 1-deoxyfructopyranosyl moiety. Subsequent dehydration, condensation, fragmentation, oxidation, and cyclization reactions lead to the irreversible formation of advanced glycation end products (AGEs). This process leads to inactivation of proteins and is involved in pathologies such as senile cataract [3], arteriosc- lerosis [4], vascular complications of diabetes [5], dysfunction of skin collagen [6], and neurodegenerative diseases such as Alzheimer’s disease [7,8] and Parkinson disease [9]. Growing evidence suggests that glycation occurs preferentially at speciﬁc glycation motifs characterized by acidic amino acids, mainly glutamate and lysine residues that catalyze the glycation of nearby lysines [10,11]. Proximity to histidine either in the primary or in the secondary structure was also suggested to promote glycation of adjacent lysines [12,13]. Recent interest to fully characterize the glycation products and to use them as biomarkers and antigens for diagnosis and prognosis of disease, monitoring its progress and evaluation of the efﬁciency of therapy generated the need for glycated peptides representing the glycation motifs speciﬁcally modiﬁed by the 1-deoxyfructopyranosyl. Today, syntheses of site-speciﬁc Amadori-modiﬁed peptides are carried out on partially protected synthetic peptides in which only the lysyl residues designated for glycation are exposed while the rest are protected [14 – 17]. This approach involves orthogonal protection strategies and suffers from low yields and elaborated puriﬁcation schemes. Stepwise assembly of site-speciﬁc Amadori-modiﬁed peptides requires N α -protected-N ε -glycated-Lys building blocks and repre- sents a fully controlled and effective synthetic strategy. Herein, we report the synthesis, puriﬁcation, and characterization of N α - Fmoc, N ε -Boc, N ε -(1-deoxyfructopyranosyl)lysine building blocks needed for Fmoc-based solid phase synthesis of Amadori-modiﬁed peptides. This study offers a controlled side-speciﬁc introduction of N ε - Amadori-modiﬁed Lys residue into synthetic peptides during a stepwise assembly either in solution or in solid phase methodologies. This strategy will overcome three major problems associated with the modiﬁcation of already assembled peptides: (i) lack of site-speciﬁcity in the introduction of the modiﬁcation, (ii) need for elaborate orthogonal protection scheme in an effort to achieve site-speciﬁcity, and (iii) extremely low yields and complicated reaction mixtures due to side reactions following the direct thermal glycation. Adapting the conditions for generating Amadori peptides by direct thermal glycation in the presence of excess D-glucose [14] to the direct glycation of N α -Fmoc-lysine led to the synthesis of N α -Fmoc-Lys[N ε -1-deoxyfructopyranosyl)]-OH (1a) in 67% yield (Scheme 1, pathway A). Preliminary attempt to use ∗ Correspondence to: Michael Chorev, Laboratory for Translational Research, Harvard Medical School, One Kendall Square, Building 600, Cambridge, MA 02139, USA. E-mail: michael chorev@hms.harvard.edu a Laboratory of Peptide and Protein Chemistry and Biology, Polo Scientiﬁco e Tecnologico, University of Firenze, Sesto Fiorentino I-50019, Italy b Dipartimento di Chimica Organica, Polo Scientiﬁco e Tecnologico, University of Firenze, Via della Lastruccia 13, Sesto Fiorentino I-50019, Italy c Dipartimento di Scienze Farmaceutiche, University of Firenze, Via Ugo Schiff 3, Polo Scientiﬁco e Tecnologico, Sesto Fiorentino I-50019, Italy d Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA e Laboratory for Translational Research, Harvard Medical School, One Kendall Square, Building 600, Cambridge, MA 02139, USA J. Pept. Sci. 2009; 15: 67–71 Copyright c  2008 European Peptide Society and John Wiley & Sons, Ltd.