molecules Article Solid Phase Formylation of N-Terminus Peptides Anna Lucia Tornesello 1,*, Marina Sanseverino 2 and Franco Maria Buonaguro 3 1 CROM, Istituto Nazionale Tumori “Fondazione G. Pascale”—IRCCS, 80131 Napoli, Italy 2 Inbios Srl, via Pietro Castellino 111, 80131 Napoli, Italy; [email protected] 3 Molecular Biology and Viral Oncology Unit, Research Department, Istituto Nazionale Tumori “Fondazione G.Pascale”—IRCCS, 80131 Napoli, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-0825-1911-711 Academic Editor: Alessandro Palmieri Received: 26 April 2016; Accepted: 1 June 2016; Published: 4 June 2016 Abstract: Formylation of amino groups is a critical reaction involved in several biological processes including post-translational modification of histones. The addition of a formyl group (CHO) to the N-terminal end of a peptide chain generates biologically active molecules. N-formyl-peptides can be produced by different methods. We performed the N-formylation of two chemotactic hexapetides, Met1-Leu2-Lys3-Leu4-Ile5-Val6 and Met1-Met2-Tyr3-Ala4-Leu5-Phe6, carrying out the reaction directly on peptidyl-resin following pre-activation of formic acid with N,N-dicyclohexylcarbodiimmide (DCC) in liquid phase. The overnight incubation at 4 ˝C resulted in a significant increase in production yields of formylated peptides compared to the reaction performed at room temperature. The method is consistently effective, rapid, and inexpensive. Moreover, the synthetic strategy can be applied for the formylation of all primary amines at N-terminus of peptide chains or amino groups of lysine side-chains in solid phase. Keywords: formylation; peptide; solid phase synthesis; amines 1. Introduction Protein post-translational modifications by the covalent addition of functional groups to N-terminal amino acids greatly expand their biological properties in prokaryotic as well eukaryotic organisms. The most common reactions include acetylation, methylation, pyroglutammate formation, myristoylation, amidation, glycosyl phosphatidylinositol (GPI) attachment, and formylation [1]. N-formyl peptides derived from cleavage of bacterial and mitochondrial proteins have an important role in host defense against microbial agents for their ability to chemo attract phagocytic leukocytes to the site of infection or tissue damage [2]. Moreover, formylation is one of the many post-translational modifications occurring in histone protein which modulates chromatin conformation and gene activation [3]. In prokaryotic as well as eukaryotic cells, an increasing number of modifying enzymes have shown to contain formylation domains [4]. In organic synthesis, several methods for the formylation of amines have been developed mainly based on the dissolution of formylating reagents in liquid phase [5]. The main amine formylating reagents include chloroform [6], formic acid and derivatives [7], paraformaldehyde [8], methanol [9], carbon dioxide [10], and carbon monoxide [11]. Nevertheless, formic acid being inexpensive and easily available represents the preferential reagent to produce formylated molecules in high yields. The use of solid-supported reagents and microwave irradiation allows for the automation and acceleration the organic syntheses [12]. Recently, several chemical techniques have been published to convert amines into the corresponding N-formamides in good yields [5–17]. Waki and Meienhofer developed an efficient procedure for the preparation of Nα-formyl amino acid tert-butyl esters with minimal racemization to be utilized as starting material for peptide synthesis. The method is based on the Molecules 2016, 21, 736; doi:10.3390/molecules21060736 www.mdpi.com/journal/molecules Molecules 2016, 21, 736 2 of 7 Molecules 2016, 21, 736 2 of 7 mixing of formic acid and N,N-dicyclohexylcarbodiimmide (DCC) in the presence of chloroform to formchloroform the active to form formylating the active reagent, formylating which reagent, was added which to solutions was added of tertto solutions-butyl amino of tert acid-butyl esters amino [17 ]. However,acid esters a [17]. major However, issue in thea major reported issue chemicalin the reported technique chemical of formylation technique of remains formylation the removal remains of sidethe productsremoval of (i.e. side, urea) products from synthesized(i.e., urea) from molecules synthesized with lengthymolecule purificationss with lengthy and purifications reduction of and the reactionreduction yields. of the reaction yields. WeWe developed developed a a simple simple methodmethod forfor thethe N-formylationN-formylation of primary amines in in solid solid phase phase synthesis, synthesis, whichwhich affords affords highhigh quantitiesquantities ofof N-formylatedN-formylated peptides and and to to easily easily remove remove the the side side products products by by resinresin washings. washings. 2.2. Results and Discussion WeWe synthesized synthesized two peptides peptides (peptide (peptide “a” “a” and and peptide peptide “b”) “b”) corresponding corresponding to subunit to subunit 4 and 4 6 and of 6human of human mitochondrial mitochondrial NADH NADH dehy dehydrogenase,drogenase, respectively. respectively. The The formylated formylated sequences sequences act act as as chemoattractantschemoattractants forfor leukocytes,leukocytes, can trigger a dramatic increase in in the the phosphorylation phosphorylation levels levels of of ERK1/2,ERK1/2, andand are able to change the cytosolic calcium calcium concentration concentration in in promyelocytic promyelocytic HL-60 HL-60 cell cell line, line, stablystably expressing expressing eithereither FPR1FPR1 oror FPR2FPR2 [[18].18]. The synthesis of of peptide peptide “a” “a” is is illustrated illustrated in in Scheme Scheme 1.1 . TheThe protocol protocol isis basedbased onon thethe additionaddition ofof aa formylationformylation group to the N N-terminus-terminus of of a a peptidyl-resin peptidyl-resin followedfollowed by cleavage cleavage to to obtain obtain the thefinal final formylated formylated peptides peptides (Met1-Leu2-Lys3-Leu4-Ile5-Val6 (Met1-Leu2-Lys3-Leu4-Ile5-Val6 (MH+ (MH+= 744 a.m.u.) = 744 a.m.u.) and Met1-Met2-Tyr3-Ala4-Leu5-Phe6and Met1-Met2-Tyr3-Ala4-Leu5-Phe6 (MH+ (MH+= 803 a.m.u.)) = 803 a.m.u.)) illustrated illustrated in Figure in 1. Figure 1. a)Piperidine b) Fmoc-Ile-OH Fmoc-Val Peg-PS Fmoc-Ile-Val Peg-PS -Fmoc HBTU/DIPEA, DMF c) -Fmoc Piperidine d) Fmoc-AA-OH HBTU/DIPEA, DMF O e) HCOOH/DCC H-C NH-Met-Leu-Lys-Leu-Ile-Val Peg-PS NH2- Met-Leu-Lys-Leu-Ile-Val Peg-PS Diethyl ether f) TFA/TIS/H2O O H-C NH-Met-Leu-Lys-Leu-Ile-Val-OH SchemeScheme 1.1. Synthesis of of N N-formylated-formylated peptide: peptide: (a) (Fmoca) Fmoc deprotection deprotection of preloaded of preloaded resin resin with withthe first the firstamino amino acid acid(20% (20%piperidine piperidine in N,N in-dimethylformamideN,N-dimethylformamide (DMF) for (DMF) 10 min); for 10(b) min);amino ( bacid) amino coupling acid coupling(HBTU/Fmoc-AA-OH/DIPEA (HBTU/Fmoc-AA-OH/DIPEA (4:4:8), reaction (4:4:8), time reaction 20 min); time (c) 20Fmoc min); deprotection (c) Fmoc deprotection (as for step a); (as (d for) stepamino a); (acidd) amino coupling acid couplingas for step as for(b) stepfor all (b) amino for all aminoacids; acids;(e) formylation (e) formylation of the of peptidyl-resin the peptidyl-resin by byovernight overnight incubation incubation at 4 at°C 4with˝C a with formylating a formylating reagent reagent produced produced by incubati byon incubation of formic ofacid formic and acidN,N and-dicyclohexylcarbodiimmideN,N-dicyclohexylcarbodiimmide (DCC) in diethyl (DCC) inether diethyl at 0 °C ether for at4 h; 0 ˝(fC) Cleavage for 4 h; (fof) Cleavageformylated of peptides from the resin by 3 h incubation with a solution of TFA/TIS/H2O (95:5:5). See the formylated peptides from the resin by 3 h incubation with a solution of TFA/TIS/H2O (95:5:5). See the AbbreviationsAbbreviations section section for for definitions.definitions. Molecules 2016, 21, 736 3 of 7 Molecules 2016, 21, 736 3 of 7 Molecules 2016, 21, 736 3 of 7 FigureFigureFigure 1. 1.Structure 1. Structure Structure of ofof formylated formylated peptidepeptide peptide ( (aa )() aand and) and formylated formylated formylated peptide peptide peptide (b). (b ().b). TheThe formylatedThe formylated formylated peptides peptides peptides were werewere synthesized synthesized usingusing using a a standarda standard standard Fmoc-based Fmoc-based Fmoc-based solid solid solid phase phase phase peptide peptide peptide synthesissynthesissynthesis [ 19[19,20].,20 [19,20].]. In In particular,In particular, particular, the the the preloaded preloadedpreloaded Fmoc-Val-PEG-PSFm Fmoc-Val-PEG-PSoc-Val-PEG-PS resin resin resin was was was employed employed employed for peptide for for peptide peptide “a” “a” “a” and Fmoc-Phe-PEG-PS was employed for peptide “b”. The formylation reaction was performed after andand Fmoc-Phe-PEG-PS Fmoc-Phe-PEG-PS waswas employed for for peptide peptide “b”. “b”. The The formylation formylation reaction reaction was was performed performed after the complete assembly of the peptide chains on solid support by incubation with the formylating afterthe complete the complete assembly assembly of the ofpeptide the peptide chains on chains solid onsupport solid by support incubati byon incubation with the formylating with the reagent obtained, in liquid phase, by mixing formic acid with DCC in diethyl ether at reagent obtained, in liquid phase, by mixing formic acid with DCC in diethyl