摘要:

1 162 J.C.S. Berkin IAmino-acids and Peptides. Part XXXVII. Trifluoroacetylation duringthe Coupling of t-Butoxycarbonylamino-acids with Peptide Esters in thePresence of Trifluoroacetate AnionBy George A. Fletcher, Miklos Low,and Geoffrey T. Young,*The Dyson Perrins Laboratory, Oxford University,Oxford OX1 3QYThe use of dicyclohexylcarbodi-imide to form peptide bonds in the presence of trifluoroacetate anion can causesubstantial trifluoroacetylation of the amino-component. The addition of 1 -hydroxybenzotriazole greatly reducesor eliminates this side-reaction.IT is common practice in peptide synthesis to prepare the evidence of formylation (and in a lesser degree, of acetyl-amino-component by the action of trifluoroacetic acid on ation) during the coupling of t-butoxycarbonylglycinet-butoxycarbonyl-amino-acid or -peptide esters, to pentachlorophenyl ester with prolyl-leucylglycine, in theliberate the free amino-group by the addition of a presence of the respective anions, and extensive formyl-tertiary amine, and to proceed with the coupling reaction ation under such conditions has been reported byin the presence of the trifluoroacetate so formed.We Schnabel, Klostermeyer, and Berndt.3Trifluoroacetylation during the coupling of peptide ester trifluoroacetates aCoupling Re- CrudebCarboxy-componentExpt. (mmol)1 Boc-Pro (4.0)2 Boc-Pro (2.0)3 Boc-Pro (2.0)4 Boc-Pro (0-75)5 Boc-Pro (1-5)6 Boc-Pro-OTcp (4.0)9 Boc-Phe (5.0)10 Boc-Pro (5-4)11 Boc-Pro (0.625)12 Boc-Pro (14.3)13 Boc-Pro-OTcp (3.75)14 Boc-Pro (6.35)15 Boc-Pro (0.80)16 Z-Pro (1.25)17 Boc-Pro (0.75)18 Boc-Pro-OPy (0.75) 619 Boc-Pro-SPy (0.75) j7 Boc-Gly (5.0)8 B O C - ~ U (5.0)Amino-component (mmol)Pro-Gly-OPic (0.5)Pro-Gly- OPic (0.2 5 )Pro-Gly-OPic (1.0)Pro-Gly-OPic (0.5)Pro-Gly-OPic (1.5)Pro-Gly-OPic (1-0) fPro-Gly-OPic (2.0)Pro-Gly-OPic (2.0)Pro-Gly-OPic (2.0)Phe-Arg(N0,)-OPic (2.1 7)Phe-Arg(N0,)-OPic (0.25)Phe-Arg(N0,)-OPic (10.9)Phe-Arg(N0,)-OPic (2.50)Cha-Arg(N0,)-OPic (2.1)Pro-Phe-OPic (0.32) hPro-Phe-OPic (0-50)Pro-Phe-OPic (0.50)Pro-Yhe-OPic (0.50)Pro-Phe-OPic (0.5)Anion present(mmol)CF,*CO,- (1.0)Xe,C*CO,- (0.5)Br- (0-5)CF,*C02- (2.0)CF,.CO,- (10-0)CF,.CO,- (3.0)CF,CO,- (2-0)CF,CO,- (20)CF,CO,- (20)CF,*CO,- (20)CF,*CO,- (4.34)Me,C*CO,- (1.0)Br- (0.50)CF,*CO,- (2 1 *8)CF,*CO,- (5.0)CF,.CO,- (4-2)CF,-CO,- (0-64)CF,.CO,- (1.0)CF,.CO,- (1.0)CF,CO,- (1.0)CF,*C02- (1.0)reagent action(mmol) timeDCCI (2.5) eDCCI (1-25) eDCCI (2.0)HOBt (2.0)DCCI (0.75)HOBt (0.75)DCCI (1.5)DCCI (3.0)DCCI (3.0)DCCI (3.0)DCCI (4.6)DCCI (0.375)2 hee4 weekseeeeeDCCI (14.2) 2 hHOBt (14.2)4 daysDCCI (4-45) eDCCI (0.48) eDCCI (0.75) eDCCI (0-75) 2 hHOBt (0.75)50 h50 hyield(%)8593968569889386898497s79084Trifluoroacetylderivative c (yield (yo)(No pivaloyl derivativedetected by t.1.c.)None detected by t.1.c.None detected by t.1.c.21% (F, 3.4%)5% (F, 0.8%)8% (F, 1.2%)9% (F, 1.4%)20% (F, 3.2%)None detected by t.1.c.26% (isolated)(No pivaloyl derivativedetected by t.1.c.)0*15Oj, (t.1.c.)0.150,/, (t.1.c.) CF,",23 "/o (isolated)None detected by t.1.c.0.1-0*20/, (CF,+)weak; F, 0%5% (F, 0.61%)3% (F, 0.32%)33% (F, 4.2%)or F analysis26% (F, 3.25%)a Further details are given in the Experimental section.Abbreviations follow the Tentative Rules of the I.U.P.A.C. Com-mission (reprinted in the Chemical Society Specialist Report on Amino-acids, Peptides, and Proteins, Vol. 4, 1972). Otherabbreviations are : Cha = /3-cyclohexylalanine; DCCI = dicyclohexylcarbodi-imide ; HOBt = 1-hydroxybenzotriazole ; Pic =4-picolyl ; Py = 2-pyridyl ; Spy = 2-pyridylthio : Tcp = 2,4,5-trichlorophenyl.Optically active amino-acids are of the L-series.The solvent for the reactions was dimethylformamide, except in Experiments 10 and 14 (see Experimental section). The per-centage yield is given only for those products containing relatively small amounts of trifluoroacetyl derivative. C When thefluorine analysis alone is given, the percentage yield of trifluoroacetyl derivative is calculated from this figure. d Part XXXYI(ref. 1). f This is the experiment described in Part XXXVI,' for the preparation ofauthentic protected tripeptide ester. g D. J. Schafer, G. T. Young, D. F. Elliott, and R. Wade, J . Chem. SOC. (C), 1971, 46.h R. Garner and G. T. Young, J . Chem. SOC. ( C ) , 1971, 50.e The reaction mixture was left overnight.i Ref. 5. We thank Dr.Morley for the sample. j Ref. 4.have found that in such cases trifluoroacetylation canbe a major side-reaction, particularly when dicyclo-hexylcarbodi-iniide is used to effect the coupling, thecarboxy-component being activated in the presence ofthe trifluoroacetate. The possibility was envisagedearlier by Steglich and Weygand,2 but no examples have,as far as we are aware, been reported, and we recordour experience here. One of us (If. L.) earlier obtained' Part XXXT'I, G. A. Fletcher and G. T. Young, J.C.S.,Pei'kiia I , 1972, 1867.The results are summarised in the Table. In twocases (Experiments 10 and 14) the trifluoroacetylpeptideester was isolated (after decomposition of the t-butoxy-carbonylpeptide ester by means of trifluoroacetic acid)and fully characterised; the molal percentages of tri-fluoroacetyl derivative in the whole isolated productswere 24.5 and 38%, respectively.In other cases, theF. Weygand and W. Steglich, 2. Natuiforsck., 1959,14b, 472.E. Schnabel, H. Klosterineyer, and H. Berndt, Anizalriz,1971, 749, 901973 1163amount of by-product was estimated by the fluorinecontent, with confirmation from semi-quantitative t .l.c.,and (in two cases) by mass spectrometry, the intensityof the peak at ?rz/e 69 (CF,+) being compared with thatfrom known mixtures of product and by-product. Inmost cases considerable excess of the carboxy-componentand of dicyclohexylcarbodi-imide over the amino-component was used, and this appears to favour tri-Auoroacetylation, but Experiment 5 shows that the useof stoicheiometric proportions of the reactants does noteliminate the side-reaction. It is to be expected that ahindered amino-component will encourage trifluoro-acetylation, but Experiments 10 and 14 show that it isnot necessary for a proline residue to be N-terminal.Similarly, the carboxy-component may be leucine orphenylalanine or even glycine.Benzyloxycarbonyl canreplace t-butoxycarbonyl (Experiment 16). The corre-sponding 2,4,5-trichlorophenyl esters gave little or noby-product (Experiments 6 and 13) but the highlyreactive 2-pyridyl esters *y5 and 2-pyridyl thioestersgave substantial amounts (Experiments 18 and 19) ;indeed, this side-reaction was first encountered in thislaboratory in the coupling of t-butoxycarbony1-L-proline 2-pyridyl thioester with L-leucylglycine 4-picolylester .Such a side-reaction could of course be countered bythe use of the free dipeptide ester or of a salt having amuch less nucleophilic anion.In a recent synthesis ofbradykinin' we used hydrogen chloride in dioxan toremove t-butoxycarbonyl groups and coupled, for ex-ample, t-butoxycarbonyl-L-proline with L-prolylglycyl-~-phenylalanyl-O-benzyl-~-seryl-~-prolyl-phenylalanyl-N( a)-nitro-L-arginine 4-picolyl ester by means of di-cyclohexylcarbodi-imide satisfactorily. But in somecases such deprotection reactions have been found to beincomplete, because of the precipitation of the hydro-chloride of the unchanged t-butoxycarbonylpeptide4-picolyl ester.s An alternative would be to replace thetrifluoroacetzite anion by a less nucleophilic anion such aspivalate, and in Experiments 2 and 11 no pivaloylationwas detected.However, we have found that in ourmodel reactions the dicyclohexylcarbodi-imide-l-hydr-oxybenzotriazole coupling procedure of Konig andGeiger very nearly eliminates this side reaction; inExperiments 3, 4, 12, and 17 only traces (ca. 0-15y0)of trifluoroacetyl derivatives were detected in the product,even when (Experiment 4) a large excess of triethyl-ammonium trifluoroacetate was present during thereaction.EXPERIMENTALThe general instructions in Part XXXVI apply. Thegeneral synthetic procedures described there were used forthe removal of the t-butoxycarbonyl group by means oftrifluoroacetic acid, for the liberation of the amino-compo-nent by means of triethylamine, and for coupling; allI<.Lloyd and G. T. Young, J . Chem. SOC. (C), 1971, 2890.A. S. Dutta and J . S. hlorley, J . Chem. SOC. (C). 1971, 2896.I<. Lloyd, D.Phi1. Thesis, Oxford University, 1969.protected peptide 4-picolyl esters were isolated by thecitric acid extraction procedure described there. In eachcase except Experiment 10 (described below) the couplingsolvent was dimethylformamide. The usual procedure forthe liberation of the amino-component from its trifluoro-acetate provides 2 molar proportions of trifluoroacetateanion. Authentic t-butoxycarbonyl-L-prolyl-L-prolylgly-cine 4-picolyl ester and t-butoxycarbonyl-L-prolyl-p-cyclo-hexyl-L-alanyl-N(o) -nitro-L-arginine Cpicolyl ester aredescribed in Part XXXV1,l and t-butoxycarbonyl-L-prolyl-L-phenylalanyl-N(o) -nitro-L-arginine 4-picolyl ester is de-scribed in Part XXXIL7 t-Butoxycarbonyl-L-prolyl-L-prolyl-L-phenylalanine 4-picolyl ester is new and is de-scribed below.Other coupling products were contaminatedwith the trifluoroacetyl by-product and were not purified.Further details of certain Experiments follow :The t-butoxycarbonyldipeptideester was treated with 2-8x-hydrogen bromide in acetic acidfor 25 min, the resulting dihydrobromide was precipitatedby ether, the free dipeptide ester was liberated by triethyl-amine in dimethylformamide, and the triethylammoniumpivalate was added. The absence of pivaloyldipeptideester in the product was confirmed by treating it with tri-fluoroacetic acid to destroy the t-butoxycarbonyl deriva-tive; again t.1.c.revealed only one component in each case.Experiment 10. t-Butoxycarbonyl-L-proline (5.4 mmol)was coupled with L-phenylalanyl-N(o)-nitro-L-arginine 4-picolyl ester (2.17 mmol; liberated in situ from the tri-fluoroacetate by triethylaniine in the usual way) by meansof dicyclohexylcarbodi-imide (4.6 mmol) in a mixture ofdichloromethane (10 nil) and dimethylformamide (2 ml) a troom temperature. After removal of the dicyclohexylurea,the filtrate was evaporated to dryness. The residue wasdissolved in ethyl acetate; the solution was washed withaqueous sodium hydrogen carbonate, dried, and evaporated.The residue was triturated with ether, giving the product(1-27 g) which t.1.c.showed to contain two main components[RF 0.31 and 0.39 (G3), the second spot being ninhydrin-negative]. The t-butoxycarbonyl group was removed inthe usual way by means of trifluoroacetic acid, giving theproduct (1.569 g ) , a portion (1.04 g) of which was dissolvedin ethyl acetate and aqueous 4% sodium hydrogen carbon-ate. The ethyl acetate layer was washed with water, dried,and evaporated; the residue (0-204 g , 0-367 mmol, 26%)was recrystallised from 50% ethanol, giving trifEzcoroacety1-L-(phenylaEanyE-N(w)-nitro-L-arginine 4-pCcolyl ester (0- 124 g) ofm.p. 118-121"; RF 0-25 (E4); 0-71 (Gl); 0-72 (H) (Found:C, 49.8; H, 4-7; N, 17-35. C2,H,,F,N,0, requires C, 49.8;H, 4.75; N, 17.7%). The sodium hydrogen carbonateextract was saturated with brine and extracted with amixture of n-butanol (50 ml), ethyl acetate (40 ml), anddichloromethane (10 ml) ; the extract was washed (brine),dried, and evaporated, giving amorphous prolylphenyl-alanyl-N(o)-nitroarginine 4-picolyl ester (0-626 g, 1.13mmol) (identical on t.1.c.with a sample prepared from thebis-trifluoroacetate) . The total isolated product thereforecontained 24.5 mol yo of trifluoroacetyl dipeptide ester.Trifluoroacetyl-L-phenylalanyl-N( w) -nitro-L-arginine4-picolyl ester was also prepared by dissolution of t-butoxy-carbonyl-L-phenylalanyl-N(o)-nitro-L-arginine 4-picolylester (0.45 mmol) in trifluoroacetic acid (1.0 ml) and (afterExperiments 2 and 11.D.J. Schafer, G. T. Young, D. F. Elliott, and R. Wade,&I. Low, unpublished work.W. Konig and R. Geiger, Chenz. B e y . , 1970, 103, 788.J . Chem. SOC. (C), 1971, 461164 J.C.S. Perkin I45 min) adding triethylamine (9-0 mmol), dimethylform-amide ( 1-5 ml) , and dicyclohexylcarbodi-imide (2.25 mmol) .Next day the product was isolated by the citric acidprocedure,l giving a 57 yo yield of trifluoroacetyldipeptideester, identical with the material reported above.The procedure described for Experiment10 gave crude product (1.15 g) from which was obtained0.488 mmol of trifluoroacetyldipeptide ester and 0.788mmol of prolyl-p-cyclohexylalanyl-ILT(w) -nitroarginine 4-picolyl ester ; the molal proportion of trifluoroacetyl by-product was therefore 38%, and the yield of by-product was23%.Tri~uoroacetyl-~-cyclo3zexyl-L-alanyl-N(~)-izitro-L-arginine 4-Picolyl ester had m.p 207-110", l i p 0.24 (E4);0.62 (G3) (Found: C, 49-8; H, 5 - 8 ; F, 10.3; hT, 17-3.C2,H,2F,N,06 requires C, 49.5; H, 5.6; F, 10.2; S, 17.6",).Experimeizt 1 7. t-Bzitoxycarbonyl-L-prolyZ-~-pYolyZ-L-pRenyZalanine 4-picolyl ester had [0llD2* - 62" (c 1-0 in EtOAc) ;RP 0.62 ( E 4 ) ; 0.70 (G3) (Found: C, 63-9; H, 6.9; N, 10.0.C30H3,N,06,H20 requires C, 63.7; H, 7.0; N, 9.9%).In these experiments the excessof active ester was destroyed a t the end of the reaction bythe addition of water.4Experiment 14.Experiments 18 and 19,Mass Spectrometric Detection of Trifluoroacetyl Derica-tives (By R. T. APLIN).-Spectra were recorded on an A.E.I.MS9 spectrometer set to give a resolution of a minimum of2500 (i.e. 400 p.p.m.) and the backgroixnd spectrum wasrecorded from mle 65 to $5. The sample was then intro-duced and the spectrum was recorded over the same massrange. The correct assignment of the CF3+ ion in the nz/e 60multiplet was achieved by peak enhancement with ' hepta-cosa ' (perfluorotributylamine) or (better) with ethyltrifluoroacetate. Common ions a t m/e 69 are CF,, 68.9952 ;C,H,NO, 69.0215; C,H,O, 69.0340; C,H,N, 69.05'iS ;C,H,, 69.0704. The limit of sensitivity of detection wasestablished by means of synthetic mixtures of trifluoro-acetyl derivative and pure product ; in each case, 0.1-0-20/,could be detected.Part of this work was carried out by Dr. M. Low, of theGedeon Richter Chemical Works, Budapest, Hungary,while in Oxford as a British Council visitor in 1970, and partby C. A. F., while holding an S.R.C.-C.A.P.S. studentship.[2/2431 Rcceived, 26th Octobev, 1972

ISSN:1472-7781    

DOI:10.1039/P19730001162

出版商:RSC    

年代:1973

数据来源: RSC