640 Journal of Medicinal Chemistry, 1971, Vol. 14, No. 7
NOTES
mixt was dild with EtOH, and the ppts were collected by filtration and dried. Recrystn from Me2CO gave brown powder, mp 220' dec. Anal. (C17NtJVaOe) C, H, N.
Linear Polypeptides of a Known Primary Structure. Synthesis and Immunochemical Studies of Poly(0-methyl-L-tyrosyl-L-glutamyl~-alanylglycyl)glycine-l-1~C Ethyl Ester BRIANJ. JOHNSON* AND FRANCIS CHEN
Department of Chemistry, Tufts University, Medjord, Massachusetts 02155 Received January 26, 1571
A recent investigation of the immunochemical propFigure 1.--Precipitin curve: f, poly(Tyr-Glu-iila-CTly) z's. i t 2 erties of poly(Tyr-Gl~-Ala-Gly)Gly-l-~~C-OEt,~~2 has antisera;2 0 , poly(O-~Ie-Tyr-Glu-Ala-Gl~) u s . its antiiera. shown that the polypeptide is antigenic, eliciting antibodies in rabbits3 I n order t o ascertain the role of the Gly-I-14CEt, to their N e ethers still gave a molecule phenolic OH on the antigenicity of the polypeptide me that was antigenic. Thus, it has been concluded that wish to report the synthesis and immunochemical the presence of the phenolic OH groups is not a necesproperties of poly (O-Me-Tyr-Gl~-Ala-Gly)Gly-l-~~Csary prerequisite for antibody formation in this system. OEt (1). However, the equivalence points of the precipitin curves Chemistry.-The polymerizing unit 0-Me-Tyr-yof the two polymers are not the same. Thus it would tert-Bu-Glu-Ala-Gly-OCsC4. HC1 (4) and the necesappear that the antigenic determinates of the polypepsary intermediates for its preparation were synthesized tide 1 are different from those of the parent antigen, as detailed in the Experimental Section. The polypoly (Tyr-Glu- Ala-Gly) Gly-I - I 4 C-OEt . merization was performed at a reagent concentration of 100 mmoles/l. in the presence of a preformed monoExperimental Section mer since this has been shown to produce linear high molecular weight polypeptide^.^^^^^-^ Following the Melting points were taken with n LIel-Temp apparatus mid are uncorrected. Optical rotations were taken with R Carl Zeiss established procedure the insol polymer, poly(O-i\leprecision polarimeter. Tyr-~-tert-Bu-Glu-Ala-Gly)Gly-l-14C-OEt was pre2-0-Me-Tyr-r-tert-Bu-Glu-Ala-Gly M e t (2).-To a mixt of pared; from which the protecting tert-Bu groups were 17 g (58 mmoles) of Z-0-methyltyrosine in 400 ml of CHzC12 removed by the use of 90% F8CC02Hto yield polyand 16 g (58 mmoles) of pentachlorophenol was added 13.2 g (O-Me-Tyr-Gl~-Ala-Gly)Gly-l-~4C-OEt (1). After ex(64 mmoles) of N,S-dicyclohexylcarbodiimide. The soln was stirred at room temp for 12 hr, filtered, and then coricd in vacuo. tensive dialysis, the polymer was purified and fracThe residue was dissolved in EtOAc and washed with H20, tionated by passage through calibrated columns of XaHC03 soln, and H20, then dried (Na2S04)and coiicd in vacuo Sephadex G-lOO'O and Corning CPG 10-240 glass granto give the activated ester, 15 g ( 4 5 C l ) . To 10.5 g (18.2 mmoles) ules. By this means the mol wt of the polypeptide of this pentachlorophenyl ester in 250 ml of CH~CIY was added 6.5 g (17 mmoles) of y-tert-Bu-Glu-Ala-GIy-O;\le .HC1 aiid 1.73 was found to be a t least 1 X lo5. g (17 mmoles) of EtsS. The reaction mixt was stirred overnight Immunochemistry.-Two rabbits were immunized at room temp and then coiicd in cacuo. The residue was diswith poly(0-Me-Tyr-Glu-Ala-Gly) Gly-l-14C-OEt (1) , solved in EtOAc atid waslied with 1120, 10%. citric acid s o h , aiid using the same protocol as that previously d e ~ c r i b e d . ~ H20, then dried (Na2S04),and concd in vacuo. The crude prodiict was chromatographed oil Silicar-CC-7, eluted with CHCls and To aliquots of the pooled sera were added incremental crystd from EtOrlc-hexme to yield 7 g (63%)): nip 132-124'; amounts of the polypeptide 1. A precipitin reaction [ o ( I 3 l D -24.4' (c 1.04, DlIF). Anal. (C33Ha4;"rTaOlo) C, 1.1, N . was observed. The total amount of protein pptd was Z-O-Me-T~r-~,-tert-Bu-Glu-Ala.Gly-OCGCl~~ HCI (3).--A sohi S (Iijeldahl). From these estimated by analysis for J of 7 g (10.7 mmoles) of 2 in 250 ml of MeOH was treated with 10.7 results the precipitin curve shown in Figure 1 was obml of 1 N NaOH with stirring for 2 hr and then coricd under reduced pressure. The residue was flooded with HzO, ac,idified tained. For comparative purposes the precipitin with 1 HC1, and extd into E t 0 . 4 ~ . The EtOAc s o h was dried curve for poly(Tyr-Glu-Ala-Gly)Gly-2-14C-OEt3 is also (NazSO,) and concd under reduced pressure t o give the tetr:ishown. peptide free acid as an oil. To this material in 200 ml of CHzC12 L\7
Conclusions Conversion of the phenolic OH groups of the tyrosyl residues in the parent antigen, poly(Tyr-Glu-Ala-Gly)(1) B. J. Johnson and E. G. Trask, J . Chem. Soe. C, 2644 (1969). (2) B. J. Johnson, J . Pharm. Sei., 59, 1849 (1970). (3) B. J. Johsnon and E. G. Trask, i b i d . , 59, 724 (1970). (4) B. J. Johnson, J . M e d . Chem., 14, 488 (1971). (5) B. J. Johnson and E. G. Trask, i b d , 14, 251 (1971). (6) B. J. Johnson and D. S. Rea, C a n . J . Chem., 48, 2509 (1970) (7) B. J. Johnson and E. G. Trask, J . Chem. Soc. C, 2247 (1970). (8) B. J. Johnson, abid., C , 1412 (1969). (9) B. J. Johnson, ibid., C, 3008 (1968). (10) P. Andrews, Biochem. J . , 91, 222 (1964).
R was added 2.85 g (10.7 nimoles) of pent,achlorophenol and -5.0 (11.8 mmoles) of 1-cyclohexyl-3-(2-morpholinoethyl)cark,odiim~de metho-p-toluenesulfonate. The mixt was shaken for 48 lir at room temp. The solvent was removed in t~acuoand the residue was washed with H 2 0 aiid crystd from RIeOH to yield 3.6 g 137%): mD 202-205": [ a I a 1-21.2" ~ (c 1.09 DhIF). Anal. (C~H4iCkN4010) C, H, N. O-Me-Tyr-r-teit-Bu-Glu-Ala-Gly-OCsCl~~HC1 (4).--To a firie suspension of 3.5 g (3.93 mmoles) of the tetrapeptide peiitachlorophenyl ester 3 and 0.5 g of 10% Pd/C in 500 ml of XeOH was added 0.144 g (3.93 mmoles) of dry HC1 in AIeOH, and the suspeii;ion was hydrogenated for 2 hr. The reactioii mixt was filtered and the filtrate wai: concd. The re-idue wns crystd from LIe013___
t
Z = benzyloxycarbonyl
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 7 641
NOTES
EtzO to give 2.3 g (77%): mp 185"; [ a l a 1 ~ -0.55" (e 0.92, comparable to those of the cofactor forms of vitamin DMF). Anal. (CaoHasClsNiOs) C, H, N. Be (pyridoxal or pyridoxamine phosphates) we synPoly(O-Me-Tyr-Glu-Ala-G1y)Gly.l -14C-OEt(l).-To a soln of thesized the chloro ketone V (Scheme I) and have also 1 mg (7.6 mmoles) of glycine-l-14C-OEt.HCl (specific activity attempted to obtain the lower homologs of this compd. 3.15 mCi/mmole) and 1.33 g (15.1 mmoles) of Et3N in 5 in1 of The latter were considered as potential irreversible DMSO was slowly added a soln of 2.3 g (2.9 mmoles) of the polymerizing unit 4 in 15 ml of DMSO. The transfer vessels were inhibitors, primarily for enzymes that have nonphoswashed with 9.5 ml of DMSO which was added to the reaction phorylated forms of vitamin Be as substrates. mixt giving a final concn of 100 mmoles/l. The reaction mixt As the starting materials for the syntheses of the was shaken for 6 days and then centrifuged to yield the product a-chloro ketones, we used the homologous carboxylic which was washed with three 36-m1 portions of HzO and three 35-mi portions of Et,O and dried to give the blocked polymer. acids (I, VI, XIII),4 which were protected with an This material was diasolved in 50 ml of 90% FICCOZHand stirred a4,3-0-isopropylidene group. The higher homologs for 1hr, and then concd under reduced pressure to yield the crude (I and VI) could be readily converted to the correpolypeptide 1. This material was washed with EtzO, suspended sponding acid chlorides by carefully reacting the acids in 20 ml of H 2 0 , and dissolved by the addition of 1 N NaOH to with SOClz in C ~ H Bbut , we could not obtain an acid p H 7.8. The soln was dialyzed against distilled H20 for 12 hr, acidified to pH 2.5 with HC1, and dialyzed for 3 days. The chloride from the lowest homolog XI11 by this propptd polypeptide was collected by centrifugation and then lyocedure. Variations of reaction conditions with different philized to yield 0.3 g (247,): radioassay, 35.2 nCi/mg indicates chlorinating reagents did not give the desired result.6 ~* 47y0 incorporation of the radioactive label. Anal. ( C ~ O H Z ~ N ~ O Addition of the acid chlorides I1 and VI1 to a large HzO) C, H, N. Mol Wt Determination.--Calibrated columns of Sephadex excess of CHzNz in Et20 resulted in the formation of the G-100 (2.5 X 38.5 cm) and of Corning CPG 10-240 glass granules diazo ketones I11 and VIII. The higher homolog (2.0 X 28 cm) were employed for the mol wt detn. Using 0.1 I1 also gave an appreciable amt of the a-chloro ketone M NaC1-0.05 iM KHzPO4 corrected to pH 8.0 as eluent, 4.2 mg of IV, which was isolated; the mixt of I11 and IV, howpoly(O-?*le-Tyr-Glu-Ala-Gly)Gly-1-14C-OE t was passed through ever, gave the desired a-chloro ketone V on treatment each of these columns. The polypeptide was eluted from each column in a vol equiv to that corresponding to a mol w t of a t with HC1, as indicated by a positive Baker's test6 least 1 x 106. (a test for active halogen, applied as a tlc spray reagent), Immunachemical Results.-Two rabbits were treated a t weekly and by ir and nmr spectra. The lower homolog VIII, intervals with 500 pg of p~ly(O-?rle-Tyr-Glu-Ala-Gly)Gly-l-~~Con being similarly treated with HCl, gave a mixt of OEt 1. The first 2 weeks they were injected intradermally using complete Freunds adjuvant as suspension medium and the products, with a negative Baker's test. Treatment 3rd week they were injected sc. The injection on the 4th week of the diazo ketone VI11 with ethereal HC1 resulted in was done iv using buffered saline. Bleedings were conducted on the formation of the blocked a-chloro ketone IX, as the following week and the serum from each animal was found indicated by a positive Baker's tests. Mild hydrolysis to give a precipitin reaction with the antigenic polypeptide 1. of the protecting isopropylidene group gave exactly The preimmunized sera under the same conditions gave a negative precipitin reaction. The quantitative determination of the total the same mixt of products as was obtained directly precipitate was obtained by the addition of dils of poly(0-Mefrom the diazo ketone VI11 by treatment with HCl. Tyr-Gl~-Ala-Gly)Gly-I-~~C-OEt (1) to 2-ml samples of the The 2 products were separated by preparative tlc, pooled rabbit sera. The samples were inrubated a t 37" for 1 hr and the main product was shown to be the bicyclic hemiand then stood a t 4" for 48 hr. The ppts were collected by centrifugation, washed twice with buffered saline, and collected. acetal X of the a-chloro ketone, and was further charThe total amount of protein precipitated was estimated by analyacterized as the diacetyl derivative XII. The minor sis for N (Kjeldahl). From these results the precipitin curve component of the reaction mixt was probably formed shown in Figure 1 was obtained.
,
Acknowledgments.-This work was supported by a grant from the National Science Foundation.
Analogs of Vitamin B6w i t h Reactive Groups' W. KORYTNYK* AXD B . LACHMANN Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York Received iyovember 13, 1970
Pyridoxol analogs that have alkylating groups in appropriate positions may react irreversibly with cofactor sites of appropriate apoenzymes, or, more generally, with any of the receptor sites that are available for the various biologically active forms of vitamin BeS2j3I n order to obtain an analog with dimensions (1) Pyridoxine Chemistry. 25. (a) Preceding paper in this series: R. Korytnyk, H . Ahrens, and N. Angelino, Tetrahedron, 28, 5415 (1970); (b) a brief report of this study has appeared: 157th National Meeting of the American Chemical Society, Minneapolis, Minn., April 1969, M E D I 11. (2) For a review of the synthesis and biological activity of vitamin BB analogs see W. Korytnyk and M. Ikawa, Methods Enzymol., 18A, 524 (1970). (3) Two vitamin Be analogs that have bifunctional alkylating groups in the 5 position have been described earlier and found t o have weak antitumor
by dehydration of the major compd X, and it was indicated by nmr to be XI (see Experimental Section). The small amt of the sample precluded the further work necessary for making an unequivocal structural assignment. The a-chloro ketone V was tested as an inhibitor of several enzymes. It did not inhibit apotryptophanM ; but when the concn was raised to ase7v8a t 5 X 4X M , 99% of the enzyme activity was abolished. After being filtered through a Sephadex G-25 column,
effects: N~,N~-bis(Z-chloroethyl)-O'-methyliaopyridoxamine (methoxypyridoxyl N mustard) and N~,N~-bis(Z-ohloroethyl)-4-deoxyisopyridoxamine (4-deoxypyridoxyl N mustard) [C. C. Stock, 9. Buokley, K. Suguira, a n d C. P. Rhoads, Cancer Res. 11, 432 (1951)l. (4) (a) W. Korytnyk, E. J. Kris, and R. P. Singh, J. Ore. Chem., 29, 574 (1964); (b) W.Korytnyk, J. Med. Chem., 8 , 112 (1965); (0) W. Korytnyk, B. Paul, A. Bloch, and C. A. Nichol, ibid., 10, 345 (1967). (5) Attempted preparation of the acid chloride XIV from the acid XI11 according t o the method of I. Tomita, H. G. Brooks, and D. E. Metsler [ J . Heterocycl. Chem., 3, 178 (1966)l, gave the hydrochloride of the starting material XIII. Reaction with oxalyl chloride w a s also negative. Compare the unsuccessful efforts a t obtaining acid chlorides from pyridine carboxylic acids by E . Wenkert, F. Haglid, and S. L. Mueller, J. Oro. Chem., 34, 247 (1969). (6) B. R. Baker, D. V. Santi, J. K. Coward, H. S. Shapiro, and J. H. Jordaan, J. Heterocycl. Chem., 3, 434 (1966). (7) (a) Crystalline apotryptophanase was prepared according t o the method of 1%A. '. Newton, Y. Morino, and E . E . Snell, J . Biol. Chem., 240, 1211 (1965); (b) compd XV was synthesized by C. I w a t a a n d D. E. Metsler, J. Heterocycl. Chem., 4 , 319 (1969). (8) The assay method was t h a t of W. A. Newton and E. E. Snell, P r o c . Nat. Acad. Sei. U.S.,48, 1431 (1962).
