Synthesis of the β-amyloid fragment 5 RHDSGY 10 and its isomers

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The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The
    ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2008, Vol. 2, No. 3, pp. 288–292. © Pleiades Publishing, Ltd., 2008.Original Russian Text © E.Yu. Aleshina, N.V. Pyndyk, A.A. Moisa, M.A. Sanzhakov, O.N. Kharybin, E.N. Nikolaev, E.F. Kolesanova, 2008, published in BiomeditsinskayaKhimiya.  288  INTRODUCTIONAlzheimer’s disease is one of the most frequentcauses of dementia in eldery people. Senile plaquesaccumulated in the brain are the major histological hall-marks of Alzheimer’s disease; they mainly consist of   β  -amyloid peptide and its fragment (1–42) [1, 2]. Oneof hypotheses links plaque formation with isomeriza-tion of aspartic acid residues at the Zn-binding site of   β  -amyloid; this results in conformational changes of this protein followed by its aggregation [3, 4]. In thiscase β  -amyloid fragments containing isoaspartic acidresidue would serve as biomarkers of this disease andtherefore their detection would be applicable for earlydiagnostics of Alzheimer’s disease [5]. A β  -amyloidtryptic fragment, 5RHDSGY10, would be one of suchbiomarkers; it contains Asp7, which may epimerize inlarger peptide fragments and the whole amyloid [3, 4].The development of a method of mass spectrometricdetermination of this β  -amyloid fragment with Asp7 intwo epimeric forms required synthesis of correspond-ing test peptides RHDSGY and RH  β  -DSGY (  β  -D is anisoaspartic acid residue) and a reference peptideRHdSGY (d-D-Asp), in which D-Asp residue cannotundergo epimerization. It should be noted that theRHDSGY peptide may be characterized by high prob-ability of Asp epimerization into β  -Asp during synthe-sis via acid- or base-catalyzed aspartimide formation[6]. During synthesis of peptides containing the pairsAsp-Ser, Asp-Gly, Asp-Ala, Asp-Asp, Asp-Asn, whichemploys 9-fluorenyl methoxycarbonyl (Fmoc) groupsfor protection of [alpha]-amino groups of amino acidsthe Fmoc-group removal from the growing polypep-tide chain is achieved by means of such reagents asmorpholine and piperazine, which are less basic thantraditionally used 20% (v/v) piperidine (PIP) solution[7, 8]. However, these reagents are less effective inthe deblocking of the Fmoc-protected α  -aminogroups than PIP and total Fmoc-group removal isachieved by repeated deblocking reactions and/or useof more than a 5-fold excess of reagents [7]. Never-theless, this may not increase the resulting productyield and this also does not exclude appearance of side products [6, 8]. In this study we have synthesizedthe 5RHDSGY10 β  -amyloid fragment and two itsoptical isomers by means of solid-phase synthesis.These substances have been synthesized as amidesusing various reagents for deblocking of the Fmoc-protected α  -amino groups. This optimized synthesisof each peptide.MATERIALS AND METHODS   Reagents and solvents  . The following chemicalswere used in this study: N  α  -Fmoc-derivatives of L-amino acids and also D- and isoaspartic acids,1-hydroxybenzotriazole (HOBT) (Novabiochem, Swit-zerland), 1,8-diazabicyclo[5,4]undec-7-ene (DBU),pyrrolidine (PYR), 4-methyl-piperidine (4-MPIP),  Synthesis of the β  -Amyloid Fragment 5  RHDSGY   10  and Its Isomers  E. Yu. Aleshina*, N. V. Pyndyk, A. A. Moisa, M. A. Sanzhakov, O. N. Kharybin, E. N. Nikolaev, and E. F. Kolesanova  Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya ul. 10, Moscow, 119121 Russia; tel.: 246-33-75; fax: +7(495)245-08-57; e-mail:  Received July 5, 2007  Abstract  —The peptide RHDSGY, a fragment of the human β  -amyloid Zn-binding site, and its isomersRH(D-Asp)SGY and RH(  β  -Asp)SGY have been obtained as amides by means of solid-phase synthesis andanalyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGYpeptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(  β  -Asp)SGY (or RHDSGY during synthesis of RH(  β  -Asp)SGY) and RH(Asp-imide)SGY was solved via selection of individual reagents for removal of Fmoc groups from α  -aminogroups of the growing peptide chain.  Key words  : β  -amyloid, solid-phase peptide synthesis, β  -Asp, Fmoc-group removal.  DOI: 10.1134/S1990750808030098  *To whom correspondence should be addressed.  EXPERIMENTALSTUDIES   BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY   Vol. 2   No. 3   2008  SYNTHESIS OF β  -AMYLOID FRAGMENT 5  RHDSGY   10  AND ITS ISOMERS289  morpholine, 3,6-dioxa-1,8-octanedithiol (DODT), tri-fluoroacetic acid (TFA), N-methylpyrrolidone (NMP),isopropanol (IPA) (Acros Organics, Belgium),N,N'-diisopropyl carbodiimide (DIPC), PIP, triisopro-pyl silane (TIS) (Fluka, Switzerland), dichloromethane(DCM), methyl tert-butyl ether (ether) (Merck, Ger-many), petroleum ether, boiling point 40–70  °  C (Rea-khim, Russia). N,N-Dimethylformamide (DMF; Rea-khim) was purified as described [9] and kept overfreshly activated 0.4 nm molecular sieves.  Peptide synthesis  . Peptides were synthesized in thepeptide amide form (further defined as peptides) usingthe manual variant of the solid phase method by addingamino acids to C-end; the solid phase synthesis wascarried out on the type D-series Lantern Rink-amidepolyethylene pins (8 µ  mol scale) (Mimotopes, Austra-lia) [10]. Side groups of Ser and Tyr residues were pro-tected by tert-butyl group, His and Arg were protectedby trityl and by 2,2,4,6,7-pentamethyl-dihydrobenzo-furan-5-sulfonyl group, respectively; L-, D-, and iso-Asp were protected by the tert-butoxy group. Additionof Fmoc-protected amino acids to the growing peptidechain was carried out by the carbodiimide methodunder the following conditions: 120 µ  M Fmoc-aminoacid, 120 µ  M DIPC, 132 µ  M HOBT in DMF; 4 or14 h at 40  °  C. Fmoc-amino acid acylation was moni-tored by the bromophenol blue color test (5 µ  l of 0.01 M bromophenol blue in DMF per 1 ml of reac-tion medium).Peptide cleavage from the support with simulta-neous side (functional) group deblocking was carriedout for 4 h by adding 2 ml of the mixture TFA TFA-DODT-TIS-anisol-water (183 : 5 : 2 : 5 : 5 v/v) [11].The resultant mixture was evaporated in a rotor evapo-rator; the residue was reconstituted in 4 ml of ether-pethroleum ether (1 : 2, v/v). Suspension was cooled for30 min at –20  °  C and centrifuged. Supernatant wasremoved and the sediment was reconstituted in 2 ml of ether-pethroleum ether, cooled and centrifuged asabove. The resultant sediment was dissolved in 1 ml of the mixture acetic acid-acetonitrile-water (1 : 8 : 11).Solution was evaporated in the rotor evaporator and thesediment was then dried in a vacuum exicator for 2 daysover KOH.  Fmoc group removal from the growing peptidechain.  The following reagents were used to removeFmoc groups: (1) 20% (here and further these are vol-ume concentrations except specified; (2) PIP in DMF;20% PIP + 2% DBU in DMF; (3) 50% morpholine inDMF; (4) 6% (w/v) piperazine in DMF; (5) 20%4MPIP in DMF; (6) 20% 4MPIP + 2% DBU in DMF;(7) 20% PYR in DMF; (8) 20% PYR + 2% DBU inDMF. Incubation time was 20 min. During deprotectionof α  -amino groups of L-, D-, iso-aspartic acids solu-tions 2, 6, and 8 were replaced by 1, 5, and 7, respec-tively.   Analysis of products of peptide synthesis  . Final puri-fication involved only small proportion of resultantpreparations and so absolute yield of the purified prep-arations and determinations of their optical purity bychanges of rotation of the plane of polarized light werenot employed in this study.Analytical reverse phase high performance liquidchromatography (HPLC) was carried out using an Agi-lent 1100 Series HPLC system (Agilent Technologies,USA), a SB-C18 80A (150 ×  4.6 mm) column. Prepa-rations were eluted with buffer A (0.05% TFA in water)and buffer B (0.05% TFA in acetonitrile) using the fol-lowing mode: from 0 to 4 min—buffer A, from 4 to18 min—gradient of B in A from 0 to 100% at theelution rate of 0.5 ml/min. Eluate absorbance wasregistered at 214 nm. Samples were injected in thevolume of 20 µ  l.Mass spectrometric determination of products of peptide synthesis was carried out by the method of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) using aMicroflex mass spectrometer (Bruker Daltonics, Ger-many) and by the method of electrospray ionizationmass spectrometry using an Esquire mass spectrometer(Bruker Daltonics). α  -Cyano-4-hydroxycinnamic acidserved as matrix. Spectra of positively charged molec-ular ions were registered in the range of molecularmasses from 500 to 3000 Da.The presence of iso-Asp in peptides was determinedby Fourier transform ion cyclotron resonance massspectrometry [12], using an Apex-Qe FT Mass Spec-trometer (Bruker Daltonics); mass signal of iso-Aspcorresponded to the Arg-His + H   +  cleavage product(mass ion of 311.19).RESULTSWe employed a mini-scale synthesis of the β  -amy-loid fragment 5RHDSGY10 and its optical isomerRHdSGY and RHiso-DSGY as C-amide peptides usingpolyethylene pins with heads of complex geometricshape with a surface modified with 4-(2',4'-dimethox-yphenyl-Fmoc-aminomethyl)-phenoxymethyl (Fmoc-Rink amide). Each synthesis employed one pin. Duringsynthesis under standard conditions employing 20%piperidine solution for removal of Fmoc-groups fromthe growing peptide [10] the peptides RHdSGY andRHiso-DSGY were obtained as preparations contami-nated mainly with amides RHdSG and RHiso-DSG,which were detected by HPLC (Fig. 1) and MALDI-TOFand ESI mass spectrometry (m/e single charge ions of 567.99); they were also contaminated by shorter pep-tides. The RHiso-DSGY preparation contained a side-   290  BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY   Vol. 2   No. 3   2008  ALESHINA et al.  1400120010008006004002000  Resulting peptide   A 214  RHdSGY100%ACN  7006005004003002001000  Resulting peptideRHdSGY100%ACN  246810121416002468101214161840003500300025002000150010000  Resulting peptideRHiso-DSGY100%ACN  0246810121416187006005004003002001000  Resulting peptideRHiso-DSGY100%ACN  0246810121416500Time, min0000  Fig. 1.  HPLC of wet products 5  RHdSGY   10  (a, b) and   5  RHiso-DSGY   10  (c, d) synthesized using 20% PIP in DMF(v/v) for removal of Fmoc group; the SB-C18 80A (150 ×  4.6 mm) column (Agilent Technologies), gradient B in Afrom 0 to 100% (v/v) within 14 min.  reaction product, a peptide with aspartimide instead of   β  -Asp (in the MALDI-TOF spectrometer m/e of a sin-gle charge ion is 715.33; calculated m/e of 715.734);in addition an iso-Asp residue underwent significantepimerization to L-  α  -Asp. This was detected by anal-ysis of peptide preparation by the method of ioncyclotron resonance mass spectrometry by disappear-ance of a signal corresponding to the ion (Arg-His +H   +  ) with mass of 311.19, which is formed during ion-ization decay of the peptide with β  -Asp but not withL-  α  -Asp [12].The RHiso-DSGY and RHDSGY peptides are char-acterized by identical retention times during HPLC (seeFigs. 1 and 2) and so their separation represents a verydifficult task. For selection of conditions, which wouldresult in preparations of these peptides of higher puritywe have synthesized RHiso-DSGY and RHdSGY pep-tides using piperazine and morpholine as the reagentsfor removal of Fmoc-group. We have also tested theeffect of addition of 2% DBU (a stronger base usuallyemployed for removal of Fmoc-groups during peptidesynthesis with “hard” sequences [13, 14]) to 20% PIPin DMF on content of the resulting product in the unpu-rified preparation. However, table shows that additionof 2% DBU to 20% PIP had minor influence on the con-tent of the resulting product in preparations synthesizedas RHdSGY and RHiso-DSGY. In the case of RHdSGYsynthesis employment of piperazine and morpholinefor removal of Fmoc-groups decreased yield of theresulting peptide due to the increase in relative con-tent of contaminated shortened peptide RHdSG(according to HPLC data and mass spectrometry;data not shown). During synthesis of the RHiso-DSGY peptide using morpholine, piperazine or 2%DBU in 20% PIP in DMF for removal of Fmoc-groups yield of this heptapeptide was not increased(table), but the weaker bases, piperazine and morpho-line, caused significant decrease of aspartimide andsubsequent epimerization of iso-Asp residue. Thisresulted in the yield of the resulting peptide of higheroptical purity: in the mass spectrum of ion cyclotronresonance of the RHiso-DSGY peptide obtainedusing piperazine or morpholine there was a signalcorresponding to the ion (Arg-His + H   +  ) with m/e  of 311.19. The same signal was also detected in thespectrum of the RHiso-DSGY preparation purified bymeans of HPLC (Fig. 1d).  (a)(b)(c)(d)   BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY   Vol. 2   No. 3   2008  SYNTHESIS OF β  -AMYLOID FRAGMENT 5  RHDSGY   10  AND ITS ISOMERS291  The use of 2% DBU in 20% PIP in DMF as thereagent for removal of Fmoc group during synthesis of the RHDSGY peptide resulted in preparation withrather low yield of the resulting product (table andFig. 2a) contaminated with shorter peptides. In the caseof 20% PYR or 20% 4MPIP in DMF (in the absence orin the presence of 2% DBU) used for removal of Fmocgroups relative yield of the resulting product increased;the increase was especially notable with 20% 4MPIPwith 2% DBU (table, Fig. 2b). MALDI-TOF and ESImass-spectra of the RHDSGY preparations obtainedusing PYR and 4MPIP and their mixtures with DBUlacked signals corresponding to the RH(Asp-imide)SGY peptide as well as RH(Asp-pyrrolid-ide)SGY (calculated mass of a single charge ion of 786.844) and RH(Asp-4-methyl-piperidide)SGY (cal-culated mass of a single charge ion of 801.874). Ioncyclotron resonance mass spectrometry revealed lack of peptide with β  -Asp residue.DISCUSSIONAlthough 5RHDSGY10 and its optical isomers rep-resent rather short fragment of β  -amyloid there werecertain problems with their synthesis. The presence of the pair Asp-Ser in the natural fragment suggested thatunder standard conditions of solid phase synthesisemploying Fmoc-amino acids piperidine, the catalystfor removal of Fmoc groups from α  -amino groups of the growing peptide chain would also catalyze side-reaction of cyclization of Asp residue followed by for-mation of aspartimide [6–8]. Opening of aspartimidecycle induced by acid or base may result in epimeriza-tion of this residue with formation of β  -Asp. However,in the case of RHDSGY this reaction was negligible.However, in the case of synthesis of its isomerRHiso-DSGY this side reaction caused significantepimerization of β  -Asp into L-Asp. Only usingweaker bases, piperazine and morpholine, it was pos-sible to obtain the RHiso-DSGY peptide but of loweryield due to incomplete cleavage of Fmoc groupfrom the linker resin and growing peptide chain.During synthesis of the RHDSGY peptide the mainproblem consisted in incomplete cleavage of Fmoc-groups by the standard reagent, 20% PIP in DMIFand it was not solved by addition of the stronger baseDBU. Since PIP is in the list of precursors (List 4)and its purchase and use in laboratory practice arecomplicated the other cyclic secondary amines havebeen tested as the catalysts for removal of Fmocgroups. These are PYR and 4MPIP; in the presenceand in the absence of 2% DBU their basic propertiesinsignificantly differ from PIP (pKa values for PYRand PIP are 11.27 and 11.12, respectively). The bestresults were obtained using 4MPIP with 2% DBU forremoval of Fmoc groups. In 20% DMF PYR rapidly  180016001400120010008006004002000  Resulting peptideRHDSGY100%ACN  0   A 2140246810121416182040003500300025002000150010005000  Resulting peptideRHDSGY100%ACN  024681012141618207006005004003002001000  Resulting peptideRHDSGY100%ACN  02468101214161820Time, min00  Fig. 2.  HPLC of wet products 5  RHDSGY   10  synthesizedusing 2% DBU (v/v) in 20% PIP in DMF (v/v) (a), 2% DBU(v/v) in 20% in 4MPIP in DMF (v/v) (b, c) for removal of Fmoc group; the SB-C18 80A (150 ×  4.6 mm) column (Agi-lent Technologies), gradient B in A from to 100% (v/v)within 14 min.  (a)(b)(c)   292  BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY   Vol. 2   No. 3   2008  ALESHINA et al.  oxidized and therefore daily preparation of freshreagent was necessary.The RHdSGY peptide was obtained in satisfactoryquantity by means of the standard method; D-Asp didnot undergo aspartimide formation and subsequentepimerization into β  -Asp. Although we did not synthe-size this peptide by means of 4MPIP and 4MPIP +DBU it would be reasonable to suggest that under theseconditions yield of peptide with D-Asp as well as pep-tide with L-Asp would be increased.ACKNOWLEDGMENTSThis work was supported by Russian FoundationFor Basic Research (grant nos. 06-03-33033, 07-02-01482, 05-03-32870) and the Program of RussianAcademy of Medical Sciences “Proteomics for Medi-cine and Biotechnology.”REFERENCES  1.Clippingdale, A.B., Wade, J.D., and Barrow, C.J.,   J. Pept. Sci.  , 2001, vol. 7, no. 5, pp. 227–249.2.Hardy, J., and Selkoe, D.J., Science  , 2002,vol. 297(5580), pp. 353–356.3.Zirah, S., Kozin, S.A., Mazur, A.K., Blond, A., Chemi-nant, M., Segalas-Milazzo, I., Debey, P., and Rebuffat, S.,   J. Biol. Chem.  , 2006, vol. 281(4), pp. 2151–2161.4.Meyer-Luehmann, M., Coomaraswamy, J., Bolmont, T.,Kaeser, S., Schaefer, C., Kilger, E., Neuenschwander, A.,Abramowski, D., Frey, P., Jaton, A.L., Vigouret, J.M.,Paganetti, P., Walsh, D.M., Mathews, P.M., Ghiso, J.,Staufenbiel, M., Walker, L.C., and Jucker, M., Science  ,2006, vol. 313(5794), pp. 1781–1784.5.Galasko, D.,  J. Alzheimers Dis.  , 2005, vol. 8(4), pp. 339–346.6.Lloyd-Williams, P., Albericio, F., and Giralt, E., in  Chemical Approaches to the Synthesis of Peptides and Proteins  , New York: CRC Press LLC, 1997, p. 280.7.Wade, J.D., Mathieu, M.N., Macris, M., and Tregear, G.W.,   Letters in Peptide Science  , 2000, vol. 7(2), pp. 107–112(6).8.Cebrian, J., Domingo, V., and Reig, F.,  J. Peptide Res.  ,2003, vol. 62, pp. 238–244.9.Becker, H., Domschke, G., Fanghanel, E. et al., Organi-cum ( Russian translation), Moscow: Mir, 1992, vol. 2.10.SynPhase Technical Notes (STN) 002-1,, A., Benckhuijsen, W.E., de Koning, P.E., Val-entijn, A.R.P.M., and Drijfhout, J.W., Protein Pept. Lett. ,2002, vol. 9, pp. 379–385.12.Gonzalez, L.J., Shimizu, T., Satomi, Y., Betancourt, L.,Besada, V., Padron, G., Orlando, R., Shirasawa, T., Shi-monishi, Y., and Takao, T.,  Rapid Commun. Mass Spec-trom. , 2000, vol. 14(22), pp. 2092–2102.13.Hyde, C., Johnson, T., Owen, D., Quibell, M., and Shep-pard, R.C.,  Int. J. Pept. Protein Res. , 1994, vol. 43,pp. 431–440.14.Larsen, B.D. and Holm, A.,  Int. J. Pept. Protein Res. ,1994, vol. 43(1), pp. 1–9. Results of synthesis and characteristics of preparations of the 5 RHDSGY 10  peptide fragment of the human β -amyloid peptideand its optical isomersNo.SynthesizedpeptideReagent for Fmoc-group removal*Yield of unpuri-fied peptide, mgAsp/iso-Asp EpimerizationAspartimideThe content of the resulting product in the unpurified preparation, %1RHdSGY 20% PIP5.3–   1%312RHdSGY 20% PIP + 2% DBU 7.5–   1%25 3RHdSGY50% Morpholine5.9–   1%23 4RHdSGY6% Piperazine5.2–   1%265RHiso-DSGY20% PIP6.0Yes~2%20**6RHiso-DSGY20% PIP + 2% DBU5.8Yes<1%21**7RHiso-DSGY 50% Morpholine4.0No<1%218RHiso-DSGY6% Piperazine4.2No<1%269RHDSGY 20% PIP + 2% DBU6.5No<1%2710RHDSGY 20% PYR7.4No<1%5411RHDSGY 20% PYR + 2% DBU6.5No<1%4812RHDSGY20% 4MPIP9.3No<1%5113RHDSGY 20% 4MPIP + 2% DBU9.5No<1%65  * Reagent for Fmoc-group removal was dissolved in DMF, (% v/v).** Total content of RHiso-DSGY and RHDSGY.
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