Solid Phase Formylation of N-Terminus Peptides

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

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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.

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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 ether at formylating0 °C. The reagent formylating obtained, solution, in filtered liquid to phase, remove by the mixing side product formic urea acid and with concentrated DCC in by diethyl rotary ether ˝ at0 0°C.evaporator,C. The The formylating formylating was added solution, solution,to the filteredpeptidyl-resins filtered to remove to removewith theDIPEA theside (diisopropylethylamine) sideproduct product urea ureaand concentrated and in concentrated DMF (N by,N- rotary by rotaryevaporator,dimethylformamide). evaporator, was added was added Theto the reaction to peptidyl-resins the peptidyl-resinswas carried with out withDIPEAat 4 DIPEA°C (diisopropylethylamine)overnight. (diisopropylethylamine) Low temperatures in DMFwere in DMF (N,N- (Ndimethylformamide).,N-dimethylformamide).important for preventing The formic Thereaction reaction acid wasdecomposition. was carried carried ou The outt at outcome at 4 4 °C˝C overnight.of the N-formylation LowLow temperaturestemperatures reaction was were were importantimportantverified for for by preventing preventingthe Kaiser test. formic formic The acidfinal acid decomposition.step decomposition. consisted in the The The cleavage outcome outcome of the of of the formylated the N-formylation N-formylation peptide from reaction reaction the was was verifiedverifiedsolid by bysupport the the Kaiser Kaiser with test.an test. aqueous The The final final acidic step step solution consisted consisted cont inaining in the the cleavageTFA cleavage (trifluoroacetic of of the the formylated formylated acid) and peptide thepeptide addition from from the the solidsolidof support support TIS (triisopropylsilane) with with an an aqueous aqueous acidicas acidic scavengers solution solution containingto contpreventaining TFAby-product TFA (trifluoroacetic (trifluoroacetic formation acid) from acid) and electrophilic and the additionthe addition of intermediates present in the cleavage process. The final products were obtained in good yield TISof (triisopropylsilane)TIS (triisopropylsilane) as scavengers as scavengers to prevent to by-product prevent formationby-product from formation electrophilic from intermediates electrophilic (70%–75%) and in high purity after RP-HPLC purification. The analyses by HPLC and MALDI-Tof presentintermediates in the cleavage present process. in the Thecleavage finalproducts process. wereThe obtainedfinal products in good were yield obtained (70%–75%) in and good in highyield of purified products are reported in Figures 2 and 3, respectively. purity(70%–75%) after RP-HPLC and in high purification. purity after The RP-HPLC analyses purification. by HPLC and The MALDI-Tof analyses by of HPLC purified and products MALDI-Tof are reportedof purified in Figures products2 and are3 ,reported respectively. in Figures 2 and 3, respectively.

Figure 2. HPLC elution profile of purified peptides (a,b).

FigureFigure 2. 2.HPLC HPLC elution elution proﬁle profile of of puriﬁed purified peptides peptides (a (,ab,).b).

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FigureFigure 3. 3.Maldi-Tof Maldi-Tof analysis analysis of of peptide peptide ( a()a) (MH+ (MH+ = = 744 744 a.m.u) a.m.u) and and peptide peptide ( b(b)) (MH+ (MH+ == 803803 a.m.u.).a.m.u.).

DifferentDifferent temperatures temperatures were were used used in preliminary in prelimin reactionsary reactions in order in to identifyorder to optimal identify conditions. optimal Weconditions. found thatWe thefound temperature that the temperature has a significant has a effectsignificant on the effect formylating on the formylating reaction efficiency. reaction Reactionsefficiency. carriedReactions out carried between out 10between˝C and 10 room °C and temperature room temperature were much were fastermuch butfaster provided but provided very lowvery yields low yields (5% of (5% purified of purified product) product) in comparison in comparison to the to reactions the reactions performed performed at 4 ˝C at (yield 4 °C (yield 70%–75%). 70%– The75%). best The yields best were yields obtained were obtained by incubating by incubating formic acid formic with DCCacid atwith 0 ˝ CDCC to form at 0 the°C activeto form formylating the active reagentformylating and atreagent 4 ˝C to and perform at 4 °C the to formylation perform the of formylation N-terminus of peptides. N-terminus As reported peptides. in As the reported literature, in goodthe literature, yields of good formylation yields of (50%–90%) formylation have (50%–90%) been also have obtained beenin also liquid obtained phase in [5 ].liquid However, phase the[5]. synthesisHowever, in the liquid synthesis phase in is moreliquid laborious, phase is more requiring laborious, a higher requiring number a ofhigher steps. number Liquid phaseof steps. reactions Liquid havephase several reactions disadvantages have several such disadvantages as expensive such reagents, as expensive the formation reagents, of side the products,formation thermalof side instability,products, thermal and difficult instability, accessibility and difficult to reagents. accessib Chemicalility to modificationsreagents. Chemical in solid modifications phase facilitate in solid the entirephase procedurefacilitate the by theentire elimination procedure of tediousby the elimin purificationation of steps tedious that oftenpurifica contributetion steps to lowerthat often the yield.contribute Excess to oflower reagents the yield. are removed Excess of by reagents filtration. are removed by filtration. TheThe methodmethod that that we we have have developed developed is applicable is applicable in solid in phasesolid phase for the for formylation the formylation of all primary of all aminesprimary at amines N-terminus at N-terminus of peptide of chains peptide and chains for the and formylation for the formylation of amino groups of amino of lysine groups side-chains of lysine afterside-chains the selective after the removing selective of removing side-chain of protecting side-chain groups protecting on resins. groups on resins.

3.3. Materials Materials and Methods AllAll reagentreagent were were obtained obtained from from commercial commercial suppliers suppliers and were and used were without used further without puriﬁcation. further Preloadedpurification. resins Preloaded were purchased resins were from purchased Rapp Polymere from Rapp (Tuebingen, Polymere Germany), (Tuebingen, and protected Germany), amino and acidsprotected were amino purchased acids from were AGTC purchased Bioproducts from AGTC (East RidingBioproducts of Yorkshire, (East Riding UK). Formic of Yorkshire, acid and UK). all otherFormic chemicals acid and wasall other provided chemicals by Sigma-Aldrich was provided Srlby (Milano,Sigma-Aldrich Italy). Srl The (Milano, analytical Italy). and The preparative analytical reverseand preparative phase HPLC reverse (RP-HPLC) phase columnsHPLC (RP-HPLC were purchased) columns from Phenomenexwere purchased (Castelmaggiore, from Phenomenex Italy). Maldi-Tof(Castelmaggiore, spectral Italy). analyses Maldi-Tof were carriedspectral out anal onyses MALDI-Tof were carried Voyager-DE out on MALDI-Tof mass spectrometer Voyager-DE by Perspectivemass spectrometer Biosystems by Perspective (Framingham, Biosystems MA, USA). (Framingham, MA, USA).

3.1.3.1. PeptidesPeptides SynthesisSynthesis PeptidesPeptides Met1-Leu2-Lys3-Leu4-Ile5-Val6Met1-Leu2-Lys3-Leu4-Ile5-Val6 (a)(a) andand Met1-Met2-Tyr3-Ala4-Leu5-Phe6Met1-Met2-Tyr3-Ala4-Leu5-Phe6 (b)(b) werewere synthesizedsynthesized throughthrough solidsolid phasephase strategystrategy inin continuouscontinuous ﬂowflow withwith automaticautomatic synthesizersynthesizer SyroSyro byby MultisynthecMultisynthec using using the the Fmoc Fmoc (9-ﬂuorenylmethoxycarbonyl) (9-fluorenylmethoxycarbonyl) chemistry. chemistry. The The supports supports Fmoc-Val-PEG-PS Fmoc-Val-PEG- (resinPS (resin substitution: substitution: 0.23 0.23 mmol/g, mmol/g, from from Applied Applied Biosystem, Biosystem, (Foster (Foster City, City, California, California, CA, CA, USA) USA) andand Fmoc-Phe-PEG-PS (resin substitution: 0.20 mmol/g, from Applied Biosystem) allow for the obtainment of the peptides as acid at C-terminal. The synthesis was made on a 0.05-mmol scale for

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Fmoc-Phe-PEG-PS (resin substitution: 0.20 mmol/g, from Applied Biosystem) allow for the obtainment of the peptides as acid at C-terminal. The synthesis was made on a 0.05-mmol scale for each peptide, using as a protected amino acid Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Ala-OH, and Fmoc-Phe-OH. The automatic synthesis proceeded through Fmoc deprotection steps [piperidine:DMF, 0.2:1 (v/v)] and Fmoc-aminoacid coupling steps [Support:Fmoc-aminoacid:HBTU:DIEA, 1:4:4:8].

3.2. Formylation and Cleavage from the Solid Support The formylating reagent for each peptide was produced by incubation in a flask of formic acid (0.94 mL, 25 mmol) and DCC (2.57 g, 12.5 mmol) with diethyl ether (14.5 mL) at 0 ˝C for 4 h. This reagent was then filtered to remove DCU (dicyclohexylurea), and the total volume (about 16 mL) was reduced to 2 mL by evaporation under vacuum. The final solution was added to each peptidyl-resin in DMF (1 mL) with DIEA (20 microliters, 125 mmol), and the reaction tube was kept at 4 ˝C overnight to avoid formic acid decomposition. Completeness of formyl coupling reactions was monitored by the ninydrin test of Kaiser. The resin was washed consecutively with DMF (2 times), with dry DCM (dichloromethane) (3 times) and dried in vacuo. Each linear peptide was cleaved from the solid support, and amino acid side-chains were simultaneously deprotected by suspending the resin in 2 mL of a mixture of TFA:water:TIS [95:2.5:2.5 (v/v)] for three hours. The resin was then removed by filtration under a reduced pressure, and the filtrate poured into eight volumes of cold ether to achieve a good peptide precipitation. The suspensions were centrifuged, and the ether carefully decanted. After a further ether wash, the peptides were dissolved in 0.1% TFA (v/v) in water (3 mL) and then lyophilized.

3.3. Peptides Analysis and Purification Analytical RP-HPLC runs were carried out on a Shimadzu LC-10 ADVP (detector: SPDM) apparatus using a C12 column by Phenomenex (column 4.6 ˆ 150 mm; eluent A: 0.1%TFA in water; eluent B: 0.1%TFA in acetonitrile; gradient: from 5%B to 65%B in 20 min; flow 1.0 mL¨ min´1). Preparative RP-HPLC was carried out on a Shimadzu LC-8 apparatus equipped with an UV Shimadzu detector SPD-10AVP using a Phenomenex C12 column, 22 ˆ 250 mm with a flow rate of 20 mL¨ min´1 and with a linear gradient from 5%B to 65%B in 30 min. The main peaks of the analytical chromatogram, with Rt = 18.8 min for peptide “a” and with Rt = 20.6 min for peptide “b” were confirmed by MALDI-TOF spectrometry (peptide “a” (MH+ = 744 a.m.u.) and peptide “b” (MH+ = 803 a.m.u.).

4. Conclusions We developed a simple and efﬁcient method for N-formylation of peptides using the standard Fmoc-based solid phase peptide synthesis. This method affords good yields and high purity products in comparison with the liquid phase synthesis, which render the separation of the byproduct difﬁcult, and the formylation in acidic condition, which might cause some premature removal of the peptide from the resin. The formylation reaction performed directly on solid phase incubating the peptidyl-resin with the formylating reagent, obtained by reaction, in liquid phase, of formic acid with DCC in diethyl ether, is especially recommended because the reagents are easy to handle, inexpensive, commercially available, and upscalable for industrial processes.

Acknowledgments: A.L.T. is recipient of a postdoctoral fellowship granted by PO FSE 2007/2013 Decreto Regione Campania n. 134 del 18/05/2015. Author Contributions: A.L.T. and M.S. performed the experiments. Both authors edited and revised the manuscript under the supervision of F.M.B. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2016, 21, 736 6 of 7

Abbreviations The following abbreviations are used in this manuscript: DCC N,N-dicyclohexylcarbodiimmide DCM Dichloromethane DIPEA diisopropylethylamine; DMF N,N-dimethylformamide Fmoc 9-fluorenylmethoxycarbonyl HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate TFA trifluoroaceticacid TIS triisopropylsilane

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Sample Availability: Samples of the compounds are not available from the authors.

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