2282
nitrogen and evacuated, and 50 ml of HCl was added. The tube was sealed and warmed to 2 5 ’ ; after 2 hr, the spectrum was recorded using a Varian A-60 nmr spectrometer. The spectrum contained a pattern of sharp absorptions between 73 and 82 cps which is characteristic of a mixture of cis- and trmrs-2-butene, and broad, smooth absorption between 20 and 70 cps which is attributed to alkyl proton. The ratio of integrated absorptions was 1 :1.7.
Acknowledgments. The vpc technique was designed and all analyses were made by M ~~ ~ d. B. ~ ih ~ h Mr. W. M . Kipp and Mrs. Nancy Hillyard carried out the spectroscopic and calculations. Interpretations Of infrared Spectra were made by Miss Naomi E. Schlichter and by Professor R. c. Lord Of Massachusetts Institute of Technology.
Polypeptides with Known Repeating Sequence of Amino Acids. Synthesis of Poly-L-glutamyl-L-alanyl-L-glutamic Acid and Polyglycylglycyl-L-phenylalaninethrough Pentachlorophenyl Active Ester’ J. Kovacs, R. Giannotti, and A. Kapoor Contribution from the Department of Chemistry, St. John’s University, New York, New York. Received December 11,1965 Abstract: A general method for the synthesis of polypeptides with known repeating sequence of amino acids using pentachlorophenyl active esters was developed in this laboratory, and the applicability of this method is demonstrated by the synthesis of poly-Glu-Ala-Glu (XVIII) and poly-Gly-Gly-Phe (XVI). The following advantages in using active pentachlorophenyl esters were demonstrated experimentally: (a) pentachlorophenyl esters make a good combination with r-butyl and N-carbobenzoxy protecting groups when peptides with trifunctional amino acids are polymerized; in contrast with thiophenyl and p-nitrophenyl esters, the N-carbobenzoxy protecting group is selectively removed under controlled hydrogenation conditions leaving the pentachlorophenyl and t-butyl groups intact; (b) the resulting peptide pentachlorophenyl ester hydrochlorides as well as the intermediate active esters are easier to purify since they are frequently higher melting compounds than other active esters (Table I); (c) the very active tripeptide pentachlorophenyl esters polymerized fast in DMSO or DMF to high molecular weight polypeptides which proved to be in a practical sense (98 =I= 2 %) optically pure. Optically pure tripeptide active ester VI1 has been prepared by lengthening the peptide chain (a) from the N-terminal residue, (b) from the C-terminal residue through the azide, made from hydrazide XV, active esters, and (c) by coupling of N-carbobenzoxy-~-t-butyl-L-glutamyl-L-alanine with 7-r-butyl-L-glutamic acid pentachlorophenyl ester hydrochloride (XIII). This indicated that coupling through pentachlorophenyl esters yielded, after purification, optically pure tripeptide active esters. Highly purified HCI . HGlu(0Bu-r)-Ala-Glu(0Bu-t)-OPCP (VIII) and HC1’H-Gly-Gly-Phe-OPCP (XV) gave the corresponding polypeptides XVII and XVI in good yields. Removal of the t-butyl protecting groups with 90% trifluoroacetic acid afforded the water-soluble polypeptide XVIII which, after extensive dialysis, gave an approximate weight average molecular weight of 25,000 and a number average molecular weight of about 16,000. The ratio of M w / M , was 1.6, which is indicative of a not too heterogeneous polymer. The possibility of transpeptidation of a-glutamyl residues during the removal of t-butyl groups was investigated. Model a- and ytripeptides VI and XXXII were treated with trifluoroacetic acid, and it was found within 1 %accuracy that transpeptidation did not occur. The free poly-Glu-AlaGlu was not antigenic in contrast with the random polypeptide GlusoAlaro.
T
here has been a great demand for high molecular weight polypeptides with known repeating sequences of amino acids,2 especially for those which are water soluble and contain trifunctional amino acids. These polypeptides would be expected t o resemble proteins more closely than those consisting of only difunctional amino acids. I n 1958, Katchalski, in his excellent review,2a summarized the work pertaining t o (1) (a) A part of this work was presented at the IUPAC Meeting, Kyoto, April 1964, and communicated by J. I
OBu-t
I
OBu-t
+ Z-Glu-Ala-OPCP I
OBu-t 111
I1 111
KEta
+ HC1. H-Glu-OCH3
--f
OBu-t V
OBu-t IV V
NaOH
+2-Glu-Ala-Glu-OH I OBu-t VI
I OBU-t
Z-Glu-Ala-Glu-OPCP
,I
OBu-t
Z-Glu-Ala-Glu-0CH~
I
I
I,
noPCP ___f
DCC
H?-Pd ___f
1 mole HC1
OBU-t VI1 HC1. H-Glu-Ala-Glu-OPCP I
OPCP
=
I
OBu-t
I
-0-CsHjCls
(16) D. F. Elliot and D. W. Russel, Biochem. J . , 66, 49-P (1957). (17) Abbreviations for amino acids and peptides used in this paper are those recommended in “Proceedings of the 5th European Peptide Symposium on Peptides, Oxford, Sept 1962,” G.T. Young, Ed., The Macmillan Co., New York, N. Y.,1963.
/ May 20, 1966
2285
the same conditions. In case of the glutamic acid active ester derivative I, the for glutamic acid in the hydrolysate was $26.9' and that of the control was $26.8'. The untreated L-glutamic acid, however, gave [a],:5,, 33 ', which indicates that under the conditions of the hydrolysis some reaction must have taken place. It is known that during acid treatment L-glutamic acid is racemized slightly, and this must be one of the factors for the substantial drop of the specific rotation value in the control experiment. The fact that the [a]value for the hydrolysate was almost identical with that of the control led us to conclude that the glutamic acid residue in the active ester derivative underwent the same reactions as that in the control sample and, therefore, it was originally L in configuration. Goodman made similar observations in the case of aspartic acid peptide derivatives. l 9 The total hydrolysate of dipeptide active ester derivative I11 gave a [a]50jmp for combined (Glu Ala) of +19.7', and that for the control was +19.76'. The [~t]~:~,,, for the untreated (Glu Ala) in the same ratio was $26.2". Based on the same consideration discussed above, the optical purity of the dipeptide was 99.68%. However, if only the Ala residue racemized, the optical purity of Ala in the dipeptide was calculated to be 98.45%. Similar considerations allowed us to conclude that the tripeptide active ester derivative VI1 was practically free from racemized contaminations, Synthesis of N-Carbobenzoxyglycylglycyl-L-phenylalanine Pentachlorophenyl Ester (XIV). This tripeptide active ester was prepared in 78% yield from N-carbobenzoxyglycylglycyl-L-phenylalanineand pentachlorophenol as described above. The removal of the N-protecting group from XIV by hydrogenation in the presence of one equivalent of hydrogen chloride afforded glycylglycyl-L-phenylalanine pentachlorophenyl ester hydrochloride (XV), in 80 % yield. HOPCP Errm
alanyl-y-t-butyl-L-glutamic acid pentachlorophenyl ester hydrochloride (VIII) in 83 yield. Method B. N-Carbobenzoxy-L-alanine pentachlorophenyl ester (IX), which was prepared the usual way, was coupled with y-t-butyl-L-glutamic acid methyl ester (IV) in 75% yield. The resulting dipeptide methyl ester X, upan hydrogenation, gave XI, which was converted to tripeptide derivative V by coupling with pentachlorophenyl active ester I. This tripeptide derivative V was identical with that obtained through method A, including its specific rotation. The active ester derivative VI11 was obtained as described under method A. PiEta
+ HCI . H-Glu-OCH3 I
Z-Ala-OPCP
+
--f
OBu-t IV
IX
H2-Pd
+
Z-Ala-Glu-OCH3
+
1 mole HCI
I
OBU-t X
+
2-Glu-OPCP OBu-t
HCI . H-Ala-Glu-OCH3
______f
I
NEts
OBU-t XI 3 steps
-+ VI11
Z-Glu-Ala-Glu-OCHB
I
I
OBu-t
OBu-t V
Method C. Since the coupling of dipeptide derivative I11 with IV to give tripeptide V involved the use of an active ester which might have proceeded with some racemization concerning the alanine residue, this tripeptide was also prepared through the azide coupling procedure. Thus dipeptide methyl ester I1 was converted to the hydrazide XII, which after conversion to the azide was coupled with IV to give tripeptide V, having identical specific rotation with compound V, prepared under methods A and B. In addition the dipeptide azide obtained from XI1 was coupled directly to y-t-butyl-L-glutamic acid pentachlorophenyl ester hydrochloride (XIII). The tripeptide pentachlorophenyl active ester VI1 so obtained in 46% yield was identical with that obtained through method A, including the specific rotation. N?H4
Z-Glu-Ala-OCH3
__f
I
Z--Gly--Gly-PheOH
DCC
XV
I
HC1-AcOH
HC1' H-Glu-OCHa ABU-t
Z-Glu--Ala-N3
NEta
I
+Z-Glu-Ala-Glu-OCH3 I
OBU-t
OBU-t I
NEt3
I
OBU-t
V
HCI. H-Glu-OPCP
I
OBu-t L
XI11
+Z-Glu-Ala-Glu-OPCP I
I
OBu-t OBU-t VI1
Optical Purity of the Intermediate Peptides. The optical purity of the active esters I and I11 was investigated by determining the specific rotation of the total hydrolysate and comparing that with the specific rotation of the amino acid components treated under Kovacs, Giannotti, Kapoor
NEta DMSO -+
.
(-Gly-Gly-Phe-),
.
Polymerization of Tripeptide Active Ester Hydrochlorides. Many experiments were carried out to establish the optimal condition for polymerization; these data will be tabulated in the next paper in this series, Le., effect of solvent, temperature, concentration, catalyst, and time. Here the method which is considered to be the most satisfactory will be mentioned. Polymerization of a tripeptide active ester containing only difunctional amino acids, such as glycylglycyl-Lphenylalanine pentachlorophenyl ester hydrochloride (XV) to polypeptide XVI proceeded smoothly and raised fewer problems than that of the tripeptides with trifunctional amino acids. The main problems are the following. It is necessary to choose a solvent which keeps the initially formed polypeptide in solution in order to obtain high molecular weight polymers. This same solvent must also allow a high concentration of the
OBU-t XI1
I1
1 mole HC1
XIV HC1. H-Gly-Gly-Phe-OPCP
XVI
PiaNOz
OBu-r
-+
xv
--+
Z-Glu-Ala-NH-NH2
-+Z-Gly4ly-Phe-OPCP
(18) G. H. Wiltshire, Biochem. .I. 55, , 46 (1953). (19) M. Goodman and F. Boardman, J . A m . Chem. Soc., 85, 2483 (1963).
/
Polypeptides with Known Repeating Sequence of Amino Acids
2286
tripeptide active ester t o minimize cyclic peptide formation. It is essential to minimize or avoid racemization. In addition t o the foregoing considerations, in the presence of trifunctional amino acids, an important problem is t o eliminate or minimize undesired reactions of the side chain functional group. Specifically, here in the presence of glutamic acid, it is necessary t o avoid transpeptidation reactions. These problems will be discussed in connection with the polymerization of y-t-butyl-L-glutamyl-L-alanine-y-t-butyl-L-glutamic acid pentachlorophenyl ester hydrochloride (VIII). This active ester was polymerized in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) solution in the presence of triethylamine; in these solvents the polymerized product remains in solution long enough to allow the formation of fairly high molecular weight polypeptides. Great care was taken to use high purity solvents as well as purified triethylamine which was completely freed from primary and secondary amines in order t o avoid early termination of the polymerization reaction. 2o T o minimize cyclic peptide formation, the wellknown principle of concentration factor was applied. Even if a high concentration of tripeptide active ester hydrochloride VI11 is used, some cyclic peptide was expected t o be present. The majority of the cyclic peptides are probably cyclohexapeptide based on Schwyzer's elegant investigations. I n a solvent promoting hydrogen bonding, the tripeptide esters are associated in an antiparallel fashion. In this head-totail arrangement the cyclohexapeptides are easily formed. This doubling reaction is very favorable in the case of peptides with odd numbers of amino acids. These considerations are important guides in designing polymerizations. Thus a tetrapeptide active ester might be a better starting material for polymerization and such investigations are under way in this laboratory. Cyclic peptides are usually more soluble in organic solvents than the polymers.*' This property makes it possible to separate them at least partially from the polymer during the initial purification procedures. The tripeptide active ester hydrochloride VIII gave 59 poly-y-t-butyl-L-glutamyl-L-alanyl-y-t-butyl-~-glutamic acid (XVII) after removal of the more soluble fractions. The absence of the pentachlorophenyl ester peak in the infrared spectrum at 5.58 p is indicative of completion of the polyacylation reaction. Removal of the t-Butyl Protecting Group from the Polypeptide. The best method to remove the t-butyl ester group was that reported by Schwyzer22 using trifluoroacetic acid. The optimal conditions were established by systematic investigation^^^ and found to be the use of 90% trifluoroacetic acid at room temperature for 50 min. The yield under these conditions for the free poly-L-glutamyl-L-alanyl-L-glutamic acid (20) Triethylamine was purified with carbobenzoxyglycine p-nitrophenyl ester and, after several days of standing at room temperature, it was considered to be free of primary and secondary amines. Two distillations gave pure triethylamine. Dimethylformamide was purified similarly. (21) (a) R. Schwyzer, J. P. Carrion, B. Gorup, H. Nolting, and A. Tun-kyi, Helv. Chim. Acta, 47, 441 (1964); (b) R. Schwyzer and A. Tun-kyi, ibid., 45, 859 (1962); (c) R. Schwyzer and P. Lieber, ibid., 41, 2168 (1958). (22) R. Schwyzer, W. Rittel, H. Kappeler, and B. Iselin, Angew. Chem., 72, 915 (1960).
(23) Detailed investigations will be reported in the next paper in this
series.
Journal of the American Chemical Society
88:lO
1 May
(XVIII), from polyester XVII, is between 79 and 9 5 x after washing out the trifluoroacetic acid with ether. Again the completion of the reaction was established by the absence of the t-butyl ester peak in the infrared spectrum as well as by titration of the carboxyl groups. HCI .H-Glu-Ala-Glu-OPCP
I
I
OBU-t OBu-t VI11
KEts
-+ in DMSO or DhIF 90% FaCCOOH
r-Glu-Ala-Glu-
L
OBu-t
XVII
-+[Glu-Ala-Glu], 1 OBu-tj, 1
50min
XVIII
In order t o obtain a polymer with a narrow molecular weight distribution, an extensive dialysis was used. During this procedure it was expected that not only the small straight chain molecules would be removed, but also the small cyclic peptides. These were important considerations since the polymer was used as a protein model and was tested by Maurer for its antigenic properties.24 The fact that the ratio of the weight average molecular weight to number average molecular weight is about 1.6 and that the polymer sedimented under a fairly uniform single peak indicates that the above-mentioned aim was approached. Optical Purity of the Polypeptides. The polymerization of a tripeptide active ester involves the lengthening of the peptide chain at the carboxyl end. Therefore, racemization of the third, i.e., the C-activated amino acid residue-probably through an oxazoloneshould be first considered.25 In the cases of the two examples studied here, the L-phenylalanine and one of the two L-glutamic acid residues could be involved primarily in this racemization. In contrast to the synthesis of small peptides, such as the preparation of a tripeptide from a carbobenzoxy dipeptide active ester and an amino acid ester where the racemized product might be removed by crystallization, in polycondensation of tripeptide active esters the racemized amino acid residue will be permanently incorporated in the polypeptide chain. Therefore, the optical purity is of utmost importance. The optical purity of polyglycylglycyl-L-phenylalanine was calculated t o be 98 * 2z, by determining the optical activity of the total hydrolysate and comparing it with that of a control. The optical purity of poly-L-glutamyl-L-alanyl-L-glutamic acid was calculated to be 99.6 i 2%, and was determined as described for the tripeptide active ester VIJ. However, if only the C-terminal glutamic acid suffered racemization in the tripeptide active ester VI11 during the polymerization, then the optical purity of every second glutamyl residue would be 99.2 f 2%. In light of these results it was concluded that no significant racemization occurred during the polyacylation reactions. Presently only two coupling methods are known t o proceed without racemization, the azide procedure and coupling through 1-hydroxypiperidine esters. This latter method was developed by Young.26 How(24) P. Maurer, Seton Hall College of Medicine and Dentistry, Medical Center, Jersey City, N. J.; details of investigations will be reported elswehere. (25) M. Goodman and I
