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Solid phase peptide synthesis: a simple esterification procedure
TetrahedronLetters No. 33, pp.3145-3148, 1971.
PergsmonPress. Printed in Great Britain.
SOLID PHASE PEPTIUB SINTBBSIS:A SIMPLE ESTBBIPICATIONPBOCBUUBB A. Marglin PockefellerUniversityand Divisionof Protein Chamistry Tufts Universi Schoo$of 19 July 1971) (Receivedin USA 17 June 1 71: "edisi?%%%~~%%%%%ie receive In solid phase peptide synthesis,the C-terminalamino acid is usually esterifiedto the chloromsthylresin by the procedureoriginallydevelopedby Merrifield,reflw with 0.9 equivalentstriethylaminein a suitablesolvent (1). Although several alternativeprocedures have been suggestedrecently (21, most of the peptidaswhich have been synthesizedby the solid phase method have been begun with the reflux procedure. Boc-Asn*,the C-terminal residue of the A-chain of insulin (3), undergoesside reactionswhen it is esterifiedunder raflux. In ethanol,significantamounts of Boc-Asp(OEt)-O-Resin are formed (4); in other refluxingsolvents (ethylacetate or dioxane),large amounts of Boc-Asp-O-F&sinare produced. I present here a simple method for esterifyingBoc-Asn tc the chloromsthylresin which allows completepreservationof the B-amide. In addition,this new method permits esterification of all the Boc-aminoacids testad to the readily availablechloromethylresin, and moreover it allows chargingthe resin
with
a desired amount of these amino acids in a
predictablefashion. In a typical experiment,2.0 g chloromethylated polystyrene-divinylbenzene resin is suspendedin 12 ml LMP (51 containing2.0 mmols Boc-Asn and 1.8 mmols Et3N. The mixturais shaken for 24 h at 25 C in a tightly stopperedvassal. It is then removed from the vessel, filtered,and the resin is washed with IMP, EtOH, WAC, EtOB and CH2C12 (about350 ml each g resin) and dried. A 50 mg aliquot is hydrolyeedin 6 gHCl/dioxane (6) for 24 h and the asparticacid liberatedis quantitatedon an amino acid analyzer (7) (TableI). To ascertainwhether the S-amideis indeed intact after the esterification,a 50 mg aliquot is treatedwith anhydrousSP (8) or with SBr/TPA (1). The amount of Asp relativeto Asn releasedby Ii!?or IiBrwas shown to reflect only the deamidation which has occurred during
3145
esterification,
and the quantities in the filtrate after release from the resin are determined
on the amino acid analyzer.
Less than 0.3% Asp was detected in Boc-Asn esterifications by the
new method, in contrast to Asp resulting from the reflux procedure
(15-505).
Boc-Asp(ONBz1)
gave about 6% conversion to Boc-Asp in the reflux procedure but was stable under the new conditions of 25O in DMF.
Boc-Gln and Boc-Glu(ONBsl1 were essentially stable (l-2% Glu) to
either procedure. Table I:
Resin (g)
Esterification of Boc-Asn-OH to chloromethyl resina in DMFb at 25O in the presence of 0.9 equivalents of Et3N.C Boc-Asn
(1~31)
Reaction time (h)
Result (nmIol/g)
2.0
1.0
24
0.11
2.0
2.0
24
0.20
2.0
1.0
72
0.16
2.0
2.0
72
0.32
1.0
1.0
24
0.15
1.0
2.0
24
0.20
1.0
4.0
24
0.32
a. 2% crosslinked, Cl - 2.0 mnol/g. b. All volumes 12.0 ml. For the l%-crosslinked resin, optimum results were obtained with 1.0 g resin/l0 ml DMF and multiples thereof. These volumes seem to be the minimum needed to provide homogeneous wetting of the resin. c. In DMF in the absence of Et3N, or in CB2C12 or EtOH with Et3N at room temperature, less than 0.01 mmol/g Boc-amino acid was esterified. Diisopropylethyl amine promotes esterification poorly (0.07 mmol/g even when a tenfold excess of the base is used).
Table I also illustrates that, under given reaction conditions, the esterified proportion of the amino acid is rather
constant.
In my hands this has allowed planning a desired degree
of substitution more precisely than under the reflux conditions. Extension of this method to some of the other EXX-amino acids is summarized in Table II. Boc-Met could not be esterified efficiently as such, apparently because of competing S-alkylation
(9).
l?oc-Methionine sulfoxide [Boc-Met(O) 1 was esterified successfully as
judged by amino acid analysis (6, 7, 101, Schiff base determination of the free, neutralized amino group (11). and direct sulfur analysis of the Boc-Met(O)-O-Resin
(12).
No.33
3147
Table If: Results of DMP esterifioationswith various Boc-aminoacids." Amount esterified (mmol/g)
Amino acid
Arg(NO2)
O.llb
Arg(Tos)
0.18
Gln
0.22
LYS(ZIC
0.22
Met(o)
0.24
Gly Phe
0.27
Ala
0.29
0.27
PrOC
0.31
Val
0.34
Leu
0.34
a. Pesin (2% crosslinked,Cl = 2.0 amol/g) 2.0 g, Boc-aminoacid 2.0 mmol, Et3N 1.8 mmol, DMP 12 ml, 24 h. b. Amount of estarificationcould be increasedto 0.18 mmol/g when 4.0 xrmls Boc-Arg(NO2)was used with 1.0 g chloromethylresin in 12 ml DMP. o. Esterifiedby V.A. Najjar, Divisionof ProteinChemistry,Tufts University School of Medicine.
To test the applicabilityof the new procedure for the synthesisof peptides,the A- and B-chainsof insulin were newly synthesized(1, 3). The C-terminaltetrapeptideof the B-chain [Boc-Thr(Bzl)-Pro-Lys(Z)-Ala+-Resin1 was examinedin detail at a sample substitutionof 0.38 mmol/g Boc-Ala. Cleavageby HBr/TFA-CH2C12(1): H-Thr-Pro-Lys-AladH(yield89%), homogeneous (13) by tic (Rfl 0.02, Rf2 0.141, paper electrophoresisat pH 5.0 @&
0.55),
cation-exchangechromatography(aminoacid analyzer,5 cm column,pH 5.25, sodium citrate 0.35 M: emergence20 ml, CLeu 1.04; less than 0.3% contaminationwith H-Pro-Lys-Ala-OH, which emerges at 27 ml elution in the same system). Cleavageby NH3/MsOH (14):H-Thr(Bzl)Pro-Lys(Z)-Ala-NH2(yield8101, homogeneousby tic (Rf' 0.33, Rf2 0.62) and electrophoresis @LYS
0.37). Tryptic digestionof the HP-deblocked(15)peptide-amideindicatedthat the
exclusiveN-terminuswas Ala-NH2
All these peptides gave the expected amino acid ratios
(7) after hydrolysisin 6 N,HCl for 20 h.
No.33
3148
+Akbreviations: Amino acid residuesand protectinggroups are abbreviatedas recommendedby IUPAC-IUB (J. Biol. Cbem. 241, 2491 (1966). Other abbreviations: DWF, dimethylformamide; TFA, trifluoroaceticacid; Boo-AzpKk?Bzl), Boc-Azparticacid-9-E-nitrobenzyl ester; Boc-Glu@~Bzl), Boc-Glutamicacid-y-e-nitrobenzyl ester. Acknowledgements: I am most grateful for the interest,conrments and suggestionsof R.B. Merrifieldin wnnection with this and my relatedwork. This researchwas supported in part by SpecialFellowship1-FG3-AM4047701, U.S. Public Health Service. REFERENCES 1. R.B. Merrifield,J. Amer. Chem. Sot. J. Biol. Chem. 246, 1922 (1971).
2,
2149
(1963);B. Gutte and R.B. Werrifield,
2. (a) M. Bodanszky and J.T. Sheehan,Chem. Ind. 1597 (1966); (b) G.I. Tesser end B.W.J. Ellenbrcek,in Peptides,Proceedingsof the 8th European Symposium,1966. NorthHolland,Amsterda, 1967, p. 124; (cl M.A. Tilzk and C.S. Xollinden,TetrahedronLett. 1297 (1968): (d) M.A. Tilak, TetrahedronLett. 6323 (1968); (8) L.C. Dorman and J. Love, J. Org. Chem. 34, 158 (196911 (f) P.G. Pietta and G.R. Marshall,Chem. Comannx.650 (1970); (g) A. Loffet, 2. Phys. Chem. 352, 4 (1971); (h) R.B. Rerrifield,Advan. En-l. 32, 221 (1969). 3. A. Marglin and R.B. Merrifield,J. Amer. Chem. Sot. x, 5051 (1966). 4. This alwholysia was first pointed out to me by Y. Shimonishi,DeutschesWollforschungsinstitut,Aachen, Germsny, 1966. RockefellerUniversity. LIRFis shaken on several successivedays with 5. A.R. Pfitchell, 4A molecularsieves (Matheson,Coleman,Bell) and then stored over the sieves, frcm which it is decantedbefore use. 6. G.R. Marshall and R.B. Werrifield,Biochemistry&, 2394 (1965). 7. D.H. Spackman,WE. Stein and S. Moore, Anal. Chem. 2, 1190 (1958). 8. J. Lenard and A.B. Robinson,J.
Amer.
Chem.
Sot.
89,
181 (1967).
9. P. Sieber and B. Iselin,Iielv.Chim. Acta 51, 622 (1968). 10. H.T. Eeutmann and J.T. Potts, Anal. Biochem.29, 175 (1969). 11. K. Esko, J. Karlsson,and J. Porath, Acta Chem. Stand. 2,
3342 (19681.
12. I thank Mr. T. Bella, RockefellerUniversity,for the sulfur analyses. system 1, @uOR:HOAc:H 0 = 4:l:l; system 2, r&BuOH:pyridine: 13. Thin-layerchromatography: HOAc:H20= 30:24:6:20. Paper electrophoresis:pyri .& ne-acetate,800 v, 90 min. was suspendedin anhydrousBeOH, which was saturatedwith 14. 400 mg of Boc-peptide-resin NR3 at 0 C. Tbe sealed suspensionwas stirred at 25 C for 40 h, cooled and opened, filteredandwasbedwith MeOHand DMP. The combinedfiltrateswere evaporated (rotary evaporator,bath temperature45 C, water aspirator)and the product was lyophilized from water. 15. S. Sakakibara,Y. Shimonishi,Y. Kishida,M. Okada and H. Sugihara,Bull. Chem. Sot. Japan 0, 2164 (1967).
PergsmonPress. Printed in Great Britain.
SOLID PHASE PEPTIUB SINTBBSIS:A SIMPLE ESTBBIPICATIONPBOCBUUBB A. Marglin PockefellerUniversityand Divisionof Protein Chamistry Tufts Universi Schoo$of 19 July 1971) (Receivedin USA 17 June 1 71: "edisi?%%%~~%%%%%ie receive In solid phase peptide synthesis,the C-terminalamino acid is usually esterifiedto the chloromsthylresin by the procedureoriginallydevelopedby Merrifield,reflw with 0.9 equivalentstriethylaminein a suitablesolvent (1). Although several alternativeprocedures have been suggestedrecently (21, most of the peptidaswhich have been synthesizedby the solid phase method have been begun with the reflux procedure. Boc-Asn*,the C-terminal residue of the A-chain of insulin (3), undergoesside reactionswhen it is esterifiedunder raflux. In ethanol,significantamounts of Boc-Asp(OEt)-O-Resin are formed (4); in other refluxingsolvents (ethylacetate or dioxane),large amounts of Boc-Asp-O-F&sinare produced. I present here a simple method for esterifyingBoc-Asn tc the chloromsthylresin which allows completepreservationof the B-amide. In addition,this new method permits esterification of all the Boc-aminoacids testad to the readily availablechloromethylresin, and moreover it allows chargingthe resin
with
a desired amount of these amino acids in a
predictablefashion. In a typical experiment,2.0 g chloromethylated polystyrene-divinylbenzene resin is suspendedin 12 ml LMP (51 containing2.0 mmols Boc-Asn and 1.8 mmols Et3N. The mixturais shaken for 24 h at 25 C in a tightly stopperedvassal. It is then removed from the vessel, filtered,and the resin is washed with IMP, EtOH, WAC, EtOB and CH2C12 (about350 ml each g resin) and dried. A 50 mg aliquot is hydrolyeedin 6 gHCl/dioxane (6) for 24 h and the asparticacid liberatedis quantitatedon an amino acid analyzer (7) (TableI). To ascertainwhether the S-amideis indeed intact after the esterification,a 50 mg aliquot is treatedwith anhydrousSP (8) or with SBr/TPA (1). The amount of Asp relativeto Asn releasedby Ii!?or IiBrwas shown to reflect only the deamidation which has occurred during
3145
esterification,
and the quantities in the filtrate after release from the resin are determined
on the amino acid analyzer.
Less than 0.3% Asp was detected in Boc-Asn esterifications by the
new method, in contrast to Asp resulting from the reflux procedure
(15-505).
Boc-Asp(ONBz1)
gave about 6% conversion to Boc-Asp in the reflux procedure but was stable under the new conditions of 25O in DMF.
Boc-Gln and Boc-Glu(ONBsl1 were essentially stable (l-2% Glu) to
either procedure. Table I:
Resin (g)
Esterification of Boc-Asn-OH to chloromethyl resina in DMFb at 25O in the presence of 0.9 equivalents of Et3N.C Boc-Asn
(1~31)
Reaction time (h)
Result (nmIol/g)
2.0
1.0
24
0.11
2.0
2.0
24
0.20
2.0
1.0
72
0.16
2.0
2.0
72
0.32
1.0
1.0
24
0.15
1.0
2.0
24
0.20
1.0
4.0
24
0.32
a. 2% crosslinked, Cl - 2.0 mnol/g. b. All volumes 12.0 ml. For the l%-crosslinked resin, optimum results were obtained with 1.0 g resin/l0 ml DMF and multiples thereof. These volumes seem to be the minimum needed to provide homogeneous wetting of the resin. c. In DMF in the absence of Et3N, or in CB2C12 or EtOH with Et3N at room temperature, less than 0.01 mmol/g Boc-amino acid was esterified. Diisopropylethyl amine promotes esterification poorly (0.07 mmol/g even when a tenfold excess of the base is used).
Table I also illustrates that, under given reaction conditions, the esterified proportion of the amino acid is rather
constant.
In my hands this has allowed planning a desired degree
of substitution more precisely than under the reflux conditions. Extension of this method to some of the other EXX-amino acids is summarized in Table II. Boc-Met could not be esterified efficiently as such, apparently because of competing S-alkylation
(9).
l?oc-Methionine sulfoxide [Boc-Met(O) 1 was esterified successfully as
judged by amino acid analysis (6, 7, 101, Schiff base determination of the free, neutralized amino group (11). and direct sulfur analysis of the Boc-Met(O)-O-Resin
(12).
No.33
3147
Table If: Results of DMP esterifioationswith various Boc-aminoacids." Amount esterified (mmol/g)
Amino acid
Arg(NO2)
O.llb
Arg(Tos)
0.18
Gln
0.22
LYS(ZIC
0.22
Met(o)
0.24
Gly Phe
0.27
Ala
0.29
0.27
PrOC
0.31
Val
0.34
Leu
0.34
a. Pesin (2% crosslinked,Cl = 2.0 amol/g) 2.0 g, Boc-aminoacid 2.0 mmol, Et3N 1.8 mmol, DMP 12 ml, 24 h. b. Amount of estarificationcould be increasedto 0.18 mmol/g when 4.0 xrmls Boc-Arg(NO2)was used with 1.0 g chloromethylresin in 12 ml DMP. o. Esterifiedby V.A. Najjar, Divisionof ProteinChemistry,Tufts University School of Medicine.
To test the applicabilityof the new procedure for the synthesisof peptides,the A- and B-chainsof insulin were newly synthesized(1, 3). The C-terminaltetrapeptideof the B-chain [Boc-Thr(Bzl)-Pro-Lys(Z)-Ala+-Resin1 was examinedin detail at a sample substitutionof 0.38 mmol/g Boc-Ala. Cleavageby HBr/TFA-CH2C12(1): H-Thr-Pro-Lys-AladH(yield89%), homogeneous (13) by tic (Rfl 0.02, Rf2 0.141, paper electrophoresisat pH 5.0 @&
0.55),
cation-exchangechromatography(aminoacid analyzer,5 cm column,pH 5.25, sodium citrate 0.35 M: emergence20 ml, CLeu 1.04; less than 0.3% contaminationwith H-Pro-Lys-Ala-OH, which emerges at 27 ml elution in the same system). Cleavageby NH3/MsOH (14):H-Thr(Bzl)Pro-Lys(Z)-Ala-NH2(yield8101, homogeneousby tic (Rf' 0.33, Rf2 0.62) and electrophoresis @LYS
0.37). Tryptic digestionof the HP-deblocked(15)peptide-amideindicatedthat the
exclusiveN-terminuswas Ala-NH2
All these peptides gave the expected amino acid ratios
(7) after hydrolysisin 6 N,HCl for 20 h.
No.33
3148
+Akbreviations: Amino acid residuesand protectinggroups are abbreviatedas recommendedby IUPAC-IUB (J. Biol. Cbem. 241, 2491 (1966). Other abbreviations: DWF, dimethylformamide; TFA, trifluoroaceticacid; Boo-AzpKk?Bzl), Boc-Azparticacid-9-E-nitrobenzyl ester; Boc-Glu@~Bzl), Boc-Glutamicacid-y-e-nitrobenzyl ester. Acknowledgements: I am most grateful for the interest,conrments and suggestionsof R.B. Merrifieldin wnnection with this and my relatedwork. This researchwas supported in part by SpecialFellowship1-FG3-AM4047701, U.S. Public Health Service. REFERENCES 1. R.B. Merrifield,J. Amer. Chem. Sot. J. Biol. Chem. 246, 1922 (1971).
2,
2149
(1963);B. Gutte and R.B. Werrifield,
2. (a) M. Bodanszky and J.T. Sheehan,Chem. Ind. 1597 (1966); (b) G.I. Tesser end B.W.J. Ellenbrcek,in Peptides,Proceedingsof the 8th European Symposium,1966. NorthHolland,Amsterda, 1967, p. 124; (cl M.A. Tilzk and C.S. Xollinden,TetrahedronLett. 1297 (1968): (d) M.A. Tilak, TetrahedronLett. 6323 (1968); (8) L.C. Dorman and J. Love, J. Org. Chem. 34, 158 (196911 (f) P.G. Pietta and G.R. Marshall,Chem. Comannx.650 (1970); (g) A. Loffet, 2. Phys. Chem. 352, 4 (1971); (h) R.B. Rerrifield,Advan. En-l. 32, 221 (1969). 3. A. Marglin and R.B. Merrifield,J. Amer. Chem. Sot. x, 5051 (1966). 4. This alwholysia was first pointed out to me by Y. Shimonishi,DeutschesWollforschungsinstitut,Aachen, Germsny, 1966. RockefellerUniversity. LIRFis shaken on several successivedays with 5. A.R. Pfitchell, 4A molecularsieves (Matheson,Coleman,Bell) and then stored over the sieves, frcm which it is decantedbefore use. 6. G.R. Marshall and R.B. Werrifield,Biochemistry&, 2394 (1965). 7. D.H. Spackman,WE. Stein and S. Moore, Anal. Chem. 2, 1190 (1958). 8. J. Lenard and A.B. Robinson,J.
Amer.
Chem.
Sot.
89,
181 (1967).
9. P. Sieber and B. Iselin,Iielv.Chim. Acta 51, 622 (1968). 10. H.T. Eeutmann and J.T. Potts, Anal. Biochem.29, 175 (1969). 11. K. Esko, J. Karlsson,and J. Porath, Acta Chem. Stand. 2,
3342 (19681.
12. I thank Mr. T. Bella, RockefellerUniversity,for the sulfur analyses. system 1, @uOR:HOAc:H 0 = 4:l:l; system 2, r&BuOH:pyridine: 13. Thin-layerchromatography: HOAc:H20= 30:24:6:20. Paper electrophoresis:pyri .& ne-acetate,800 v, 90 min. was suspendedin anhydrousBeOH, which was saturatedwith 14. 400 mg of Boc-peptide-resin NR3 at 0 C. Tbe sealed suspensionwas stirred at 25 C for 40 h, cooled and opened, filteredandwasbedwith MeOHand DMP. The combinedfiltrateswere evaporated (rotary evaporator,bath temperature45 C, water aspirator)and the product was lyophilized from water. 15. S. Sakakibara,Y. Shimonishi,Y. Kishida,M. Okada and H. Sugihara,Bull. Chem. Sot. Japan 0, 2164 (1967).