Article信州大学農学部紀要 18(2): 183-190(1981)departmental bulletin pape

IZUMI,  Masayuki

KAYAHARA,  Hiroshi

TOMIDA,  Ichiro

WAKUI,  Kazuye

English

Shinshu University Institutional Repository

             Partial Hydrolysis of Oligopeptide, I.     ― Partial Hydrolysis of Two Tetrapeptides and Their Separation             Patterns of Fragments in Gas Chromatography           By Ichiro TOMIDA, Masayuki IZUMI, Kazuye WAKUl                          and Hiroshi KAYAHARA '          Laboratory of Biological Chemistry, Fac. Agric. Shinshu Univ.    In the sequencial analysis of oligopeptide by GLCi' (and also GLC/MS2') me-thod it is desirable that after its partial hydrolysis all the possible and reasonab}efragments are observed in such a harmonious amount ratio in the chromatogramas can be clearly deeected and identified. In this paper we selected twotetrapeptides, Z-Val-Ileu-Phe-Pro-OMe (1), which had once3) been reported to bethe part of the bitter peptide obtained frorn the partial hydrolysates'of casein, aRdZ-Phe-Pro-:Val-Ileu-OMe (3), which was the rearranged one of 1 iR view of com-paring the both, as model substaRces aRd investigated the separation patterns oftheir partial hydrolysates in GLC.Partial .Elydtolysis of Both the TetropePtides   Both tetrapeptides, 1 and 3, were treated with a conc. HCI solution in con-centration of lmg/ml at 500C for the time interval cited in Table I and II, respe-ctively. The hydrolysates were converted to N-Tfa-methyl ester derivatives andthen subjected to GLC-analysis in the same manner as described in the previouspapers. i,4) The individual peaks on each fractogram were identified to the respectivefragments in cornparison with the tR-va!ues of the standard Tfa-methyl esterderivatives (Table I and II).Results and Discussions   In the case of the compound (1), as can be seen from Flg. 1 and Table I, besldesall the possible fragments being deteced, several undesirable peaks were observedas fairly large peaks in all stages of hydrolysis. Tke height ratio of each peak forReceived September 30, 1981Abbreviation : DCCD, dicyclohexylcarbodi-imide ; HOBt, N-hydroxybenzotriazole ; GLC,gas liquicl chromatography ; MS, mass spectrometry ; Tfa, trifluoroacetyl ; Z, carboben-zoxy. Unless otherwise described, L-optical isomer Qi ehe amino acid can be assumectthroughout, except Gly.Table I. tR-Values of the Peaks and Their Assignmentin GLC-Analysisa) of the Partial Hydrolysates ofwere presented ln parenthesis).to the Corresponding Fragment1 (relative peak height ratios efSequenceseach peakHydrolysatesb)Peak No.1 2¢)3 4c)5 6¢) 7c)89lec)11HyL502,  tR[Min]Ratio [%] 4.714. 226. 233. 236. 440. 045. 254. 074. 0( 4>(15)( o)( o)(13>(24)(11)(l4)( 2)( 8)<IO)Hyl-5047  tR Ratio[Min] [%] 4. 423. 629. 021. 325. 432. 235. 739. 544. 653. 271, 3( 7>( 5)( 1)(IO)( 5)(15)( 7)(25)( 1)(15)(10)HyL50,2  tR Ratio[Min] [%] 4. 213. 718. 421. 633. 04e. o45. 554. 272. 0(26)( 4)(>1)(16)( 1)( 5)(>1>(40)( 1>( 1)( 6)Hyi-50ge  tR[Min]Ratio [%] 4. 314. 1l8. 021.232. 635. 939. 544. 853. 471. 4(32)( 1)( 3)(13)( o)( 5)< 2).(33)( 2)( 2)( 8)HyL5012o  tR Ratio[Min] [%] 4.013. 317.520. 532. 038. 644. 669. 0(46)( 1)( 3)(13)( e)( 4)( e)(3e)( 1)( o)( 2)Assignment tofragmentsequencesVal -Ileu6)Ileu-Phe6)Phe-ProVal-Ileu-Phe (4)Ileu-Phe-Pro (5)Val-Ileu-Phe-Pro (8)tR [)vSlin]standardofcomp.4. 518. 5   d)26. 339. 945. 875. 8a) Apparatus : a Hitachi Perkin-Elmer 063, column OV-17 (5%), lm × 3mm, oven temp. 160-2600120fmin programming and   then isothermal, injection temp. 3eOO, carrier gas N: O.95-1.e5 Kg/cm2 at the inlet, FFdetector, H2 O.6 Kglcm2 and air   l. 5Kg!cm2,b) For example, Hyi-5023 means the hydrolysates which was obtained by treating the compound 1 at 50e for 23hr.c) Unknown and u.ndesirable peal<sd) In this experiment, Tfa-Phe-Pro-OMe was not isolated as pure substance, but the neutral fraction from the reaction   mixture. was injected. It showed a single peak. Cf. Lit.6)pcoptde=-xeepttwaswasscaptasssNt)iEa.<s)oI. ToMiDA et al. : Partial Hydrolysis of Oligopeptide, I 185Partial Hydrolysates ofgR Z-Val-Ileu-Phe-Pro-OMekga   v  N   k  RbN   E.dRRg,acgefh tuN  ks -!i 8ijRA8iflksklmo 10 2e 30 40 50 60 70min%504e302010Fig. 1.lIgI t""":il N- r--.eml st,1,l Fvi twtN!l I FV ls liii,o i-'ell ii  Gas Chromatograrn of a Mixeure of Tfa-Peptide Methyl Estersobtained by Derivatization of an Acid Hydrolysate (12N HCI, 500C,3 days) of the Compound (i). (Oven temp. 160-2600120/min program-ming, other GLC conditions, cf. in Table I).eIXttl-Ilett  mex/:VXilrl-OIIeu-Phem Phe-Pro:-llXAii/;ii);{fxx.-.,,,l,ii.leNartial }lydrolysatesof Z-Phe-Pro-Val-Ileu-OMN:Bsele e:t "lg£N N lE e vNtse g5s･ig¢pt101 2 k>ett8tsA･81134 679RIIeu-PheSPro O 23 47 72 90 120hy    Cour$e ef hydrolysis  Relation between the RelativePeak Keight of the RespectiveFragment and the Course of Hy-drolysis of Z-Val-Ileu-Phe-Pro-OMe (1)10 2e 30 40Fig, 2.5emsRFig.3. Gas Chromatogram,of a Mixture     of Tfa-Peptide Methyl Esters ob-     tained by Derivatization of an Acid     Kydrolysate <12N HCi, 5eeC, 90hr)     of the Compound (3). (Oven temp.     150-2600120fmin programming,     other GLC conditions, s. in Tabie     I).thein a(Fig.total sum of all peaks iR each cltromatogram was estimated in Table I parenthesis and the values of the reasonable peaks were plotted on a 2.). As can be seen from this Fig, Val-Ileu and Val-IIett-Phe wereshown graphalways186Table            fiY･i･lJft(*ee*zz*elj ee18igee2e (1981)II. tR-Values of the Peaks and Their Assignment to the CorrespondingFragment Sequences in GLC-Analysisa) of the Partial llydrolysates of3(Relative peak height ratio of each peak for the sum of all ones isgiven in parenthesis> PartialPeak No.hydrolysates!23c)45c)67c)89c)Hy3-50geb)tR [Min] 5.910. 717-1823. 535-3736. 84248. 049Ratio [%]<29)( 1)( 1)(47)( 1)( 4)( 1)(16)( 2)Assignment tofragmentsequenceVal-Ileu6)Pro-Val (10)Phe-Prod)Pro-Val-Ileu (7)Phe-Pro-Val (6)tR [?t(Iin] ofstand.substance 5.811. 025. 839. 747. 2     a>, b), c) and d), s. the respective footnote in Table i.detectable as large peaks aRd this tendency increased as the hydrolysis proceeded.OR the otker ltand, in the case of the compound (3), although only one stage at90hr hydrolysis was analyzed as skown iR Table II and Fig. 3, Rot only Val-Ileubut also Phe-Pro were detected as large peaks of dipeptide and furthermore thelatter was larger than the former. It means apparently that Phe-Pro was producedin the more amount thaR Val-Ileu, for Pke-Pro with the larger tR-value must beobserved as relatively lower peak than VaNleu with the smaller tR-value. Phe--Pro-Vai was also produced in a relatively iarge amouRt. From these data it will beat least ciear that Val-Ileu ls always detectabie, but tkat Phe-Pro is stable onlywhen it is not located in the C-terminal region in the sequence,7) although it isnot simple to follow the relation between the increase and decrease of eachfragment during the course of hydrolysis exactly. In any case, it may be concludedthat the condition of hydrolysis under wkich only the reasonable peaks can beobserved in the average ratio of a peak heigkt is not easiiy found due to thepresumably complicated cyclic intermediates.     We have attempted a detailed study of the time course involved in the hope that the peak height of the standard Tfa-derivatives might be practically independent on the tR-values. It was revealed, however, that their peak heights decreased with the increase of tR-values, as can be seen in Fig. 4, but that this tendency was not regular for all the compounds. The relation factor of the amount to the peak area of the respective standard Tfa-derivative was also not always the same for ali the com- pounds.I. ToMmA et al. : Partial Hydrolysis of Oligopeptide, I 187Fig. 4.G-{!-fig8fiza£EQkes'<",scopa706050403e2e!oo ･o7654321o･[llgsgE69ua>S,i,.asmeeopa           10 20 30 40 50 60 70   . tR (min)'      ma Peak Area      == : Peak Height  Relatien between the Peak Height Ratios or Peak Area Ratiesand tR=Values of some Tfa-derivatives.1. Tfa-Pro-Val-, 2. -Ileu-Phe-, 3. -Pro-Val-Ileu-, 4. -Phe-Pro-Val- and 5. -Val-IIeu-Phe-OMe. (Each 3.00 mgof each substancewas weighted and they were mixed. The mixture was diselved inO. 5rnl MeOH+O. 5ml AcOEt and 2pt1 was injected to GLC. 160-260012"lmin programming condition. Apparatus and other conditions,cf. Table I.)Syntheses of the Two TetraPePtides and Some Standant 7117a-Methyl Ester Derivatives   The two tetrapeptides, 1 and 3, were synthesized according to the strategyshown in Fig. 5. Z was used for amino protection, removed by the action of 25%HBr/AcOH. The amino component was iiberated by the addition of triethyl amine.Coupling was throughout effected by means of DCCD/HOBt method. 5) Tfa--clerivativeswere synthesized from the corresponding Zrflerivatives in the same manner as des-cribed in the previous paper.6) Both Z-tetrapeptide rnethyl esters, 1 and 3, andall synthesized Tfa-tripeptide methyl esters, such as Tfa--:Val-Ileu-Phe-(4), -Ileu-Phe-Pro-(5),-Phe-Pro=VaH6), -Pro-Val-Ileu-(7) and -Val-IIeu-Phe-Pro-OMe (8) wereobtained as crystalline substance.EXPERIMENTAL SECTION   The general experimental methods and procedures were essentia}}y the sameand the used apparatus was also the same as described in the previous paper.4)AII me!ting points were not corrected. TLC: CMA (=chloroform/methanol/aceticacid (760:40:24 VoL )). Visualization, I2 or acridine. 4)1. Syntheses of the comPounds, 1-9:1, 2 and 3 were prepared according to thestrategy shown in Fig. 5. Z-derivatives were synthesized from two components se-Table !II. Yield and Properties of the Synthesized Compounds, ISNo.1.2.3.4.5.6.7.8.9.Synthesized compoundsZ-Val/Ileu-Phe-Pro-OMe(1)Z-ProlVal-Ileu-OIvfe (2)Z-PhelPyo-Val-Ileu-OMe(3)Tfa-Val-Ileu-Phe-OMe (4)d)Tfa-Ileu-Phe-Pro-OMe (5)Tfa-Phe-Pro-Val-OMe (6>Tfa-Pro-Val-Ileu-OMe (7)Tfa-Val-Ileu-Phe-Pro-OMe(8>Tfa-Pro=Val-OMe (9)e)Purificationa)AAABAAAA, CAYieldb)E%]7669573036609461Mp[eC]114-116149-151 7e-75232-233l95-197113-114. 5199-200201=2e3 81B3. 5Rf in TLC  ICMAe. 75O. 69O. 69O. 83O. 83O. 90O. 70e. 75O. 85[cr]2Dtc)[o ]-71.4-89.4-77. 9-6o.e-51.4-91.6.- so.eMolecular formula<molecular weight)C3`H4e07N4 (622;-8) -           t.t       'C2sH3,O,N3 (475. 6)             tttt tCs4H460,N, (622. s)Ce3H320sN3g3 (487. 5)           '   tt         .tCs3H3,O,N,F, (485.s)           'C22H2,O,N,F, (471.s)          ttt          tttCigH3eOsNaF3 (437. 5)      .t'     'C2sHsgOeN4F3 (584.'6)    '           'Ci3Hi,O,N,F, (324. 3)Elernental      canalysis HNCalcd. 65. 58-Found 65.34     63. I4     62. 94.     65.58     65. 68     56. 67     56.64     56. 90     57. 18     56. 06     56. 22     52. 17     51. 97     57.52     57. 73     48. 15     48. 267.45,7. 337. 847. 937. 457. 736. 626.716. 23-6. 35 ･5. 996. 046. 916. 696. 726.855. 915. 868. 999. 208. 848. 988. 998. 748. 628.778. 668. 418. 918. 849. 619. 559. 589.318. 648. 78a)b)c)d)e)A, ¢rystallization from AcOEtfn-hexane;B, sublimatien in vacuo; C, preparative TLC separatidn.' 'It was cencerned to the condensation reaction between the both components separated with the prime ln the case efZ-derivatives and to the corresponding Z-derivatives in the cases of Tfa-compounds. 1' ･ ･ ･.It was measured in MeOH (c :1.0) with an error within ! O.80.The starting material, Z-Val-Ileu-Phe-OMe, was obtained by condensing Z-:Val-OH with K-Ileu-Phe-OMe only in a lowyield (DCCD/HOBt : 15%, Mixed anhydride with EtOCOCI : 20%, mp 192-3a, but not enough analytically pure).In the previous paper6) it was obtained only as an oil..F-.oo,cota=-Xtwwaksstul'u'"ress,F,-lssssNQi,-uAptoooptwZZZ I. ToMmA et al. : Partial Hydrolysis of Oligepeptide, IVal Ileu Phe ProOMe ZOH HBr.HOMeOMeOMe (1)189zZiPheZZProZVa1 IleuOH HBr.KOH HBr.HOMeOMeOMeOMeOMe(2)(3>             Fig.5. Strategy for tlae syntheses of the tetrapeptidesparated by the prime in Table III by condensing them by DCCD/HOBt method. 5>Tfa-derivatives were prepared from tke corresponding Z-derivatives by treatingwith 25% HBr/AcOH, followed by transesterification with methyl trifluoroacetate. 6)Tlte data were su;nmarized IR the Table (Table III).2. Partial hydrolysis of tetrmpoptides, 1 and 3: The compound (1) (80mg) was treatedwith 80ml of conc. HCI solution at 500C and after each time ineerval cited inTable I each aliquot (15ml) of the solutioR was sampled. It was evaporated todryness in vacuo. 3 was also hydro!yzed analogously. The hydrolysates were convertedto Tfa-methyl ester derivatives and then analyzed by GLC in the same manner asdescribed in the previous paper.4) GLC conditions were footnoted under Table I.The data were summarized in Table I and II.                              REFERENCES1) F. Weygand, B. Kolb, A. Prox, M. A. Tilak und i. Tomida, HoPPe Seyler's Z. Physiol. Chem., 322, 38 (1960).2) H. Nau, Angew. Chem.,88, 74 (1976).3) T. Matoba, C. Nagayasu, R. Hayashi and T. Hata, Agric. Biol. Chem., 33, 1662 (1969). cf. N. Minamiura, Y. Matsumura, J. Ful<umoto ancl T. Yamamoto, Agric. Biol.  Chem., 36, 588 (1972).4) I. Tomida and H. Ukisu, Agric. Biol. Chem,, 44, 1941 (198e).5> W. K6nig und R. Geiger, Chem. BeL, 103, 788 (1970).6) I. Tomida, J. Ohashi, T. Tol<uda and M. Nakajima, IViPPon-IVbgei-Kagafeu-KZxishi, 39, 378, 391 (1965).7) Cf. The Organic Chemistj:y of PePtides, by Harry D. Law (1970 John Wiiey & Sons Ltd) p. 12--.190 儒州大学農学部紀要　第18巻第2号（1981）摘 要オリゴペプチドの部分的加水分解　Ⅰ―2つのテトラペプチドの部分的加水分解と，それらの開裂片のガスクロマトグラフにおける分離パターンについて富田一郎・泉正行・涌井和恵・茅原紘　　　　信州大学農学部　生物化学研究室　2つのテトラペプチド，Z～Va1－neu－Phe－Pro－QMe及びZ－PheTrQ－Va1－11eu℃Meを合成し，塩酸による加水分解を行なった。そして加水分解物をN一トリフルオロアセチルーメチルエステルに導き，ガスクμマトグラフで分離し，両者の分離パターンを比較した。また各々に特有なジー及びトリペプチドフラグメントを確認し，前志のテトラペプチドについては加水分解過程中の各フラグメントの消長についても観察した。そしてこの方法によりそれぞれのアミノ酸配列を確認しうることを示した。　なお，これらのテトラペプチドのうちの1つは，かつてカゼインの部分的加水分解物中の苦味ペプチドのシーケンスの一部であると報ぜられたものであるが，本論文はこのようなオリゴペプチドのシーケンスがガスクロマトグラフの方法によって迅速に区別，確認，さらに決定さえされることを示したものである。

Partial Hydrolysis of Oligopeptide, I. -Partial Hydrolysis of Two Tetrapeptides and Their Separation Patterns of Fragments in Gas Chromatography

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Partial Hydrolysis of Oligopeptide, I. -Partial Hydrolysis of Two Tetrapeptides and Their Separation Patterns of Fragments in Gas Chromatography

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