Garrison, Warren M.

Kland-English, Mathilde

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Lawrence Berkeley National LaboratoryRecent WorkTitleDEHYDROPEPTIDE PRODUCTION IN THE RADIOLYSIS OF SOLID PEPTIDES: A RADIOLYTIC SYNTHESIS OF PYRAZINE DERIVATIVES FROM CYCLIC DIPEPTIDESPermalinkhttps://escholarship.org/uc/item/8r32m0rmAuthorsKland-English, MathildeGarrison, Warren M.Publication Date1962-09-21eScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaUCRL-10186 University of California Ernest 0. Radiation lawrence l~boratory TWO-WEEK LOAN COPY This is a library Circulating Copy which may be borrowed for two weeks. For a personal retention copy, call Tech. Info. Dioision, Ext. 5545 Berkeley, California DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor the Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or the Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or the Regents of the University of California. UNIVERSITY OF CALIFORNIA Lawrence Radiation Laboratory . Berkeley, California Contract No. W~74o5-eng-48 UCRL-10186 DEHYDROPEPTIDE-"PROWCTION IN THE RADIOLYSIS: OF SOLID PEPTIDES: A RADIOLYTIC SYNTHESIS OF PYRAZINE DERIVATIVES FROM CYCLIC DIPEPTIDES Mathilde Kland-English and Warren M. Garrison September 21, 1962 UCRL-10186 DEHYDROPEPTIDE PRODUCTION IN THE RADIOLYSIS OF SOLID PEPTIDES: A RADIOLYTIC SYNTHESIS OF PYRAZINE DERIVATIVES FROM CYCLIC DIPEPTIDES. Mathilde Kland-English and Warren M. Garrison Lawrence Radiation Laboratory University of California, Berkeley, California September 21, 1962 Among the chemical changes observed on radiolysis of compounds ·containing the peptide,' bond are those attributed1 - 3 to the production of un-saturated linkages which may be represented in terms of the tautomeric structures, ~ON= C(CH2R)~ ~~ONH-C(=CHR)~. Compounds containing this type of chemical function have been designated dehydropeptides; such compounds are stable in water yet are readily hydrolyzed to yield ammonia and a carbonyl product on heating in dilute mineral acid, ~0N=C(C~2R) + 2H25 ~~OOH + NH3+ 4 . . RCH2CCh... The_ experimental evidence for production of d.ehyd.ropeptides in the radiolysis of proteins and simpler peptide sy terns has recently been reviewed. 5 Identification has been based primarily on stuoies of the formation of anu·nonia and carbonyl function during hydrolysis of the irracUated peptide system. For example, the hydrolytic production of ammonia and pyruvic acid from y-irradiated acetylalanine (solid, evacuated) has been found to parallel very closely the hydrolytic degradation of authentic acetyldehydroalanine. The cyclic dipeptides, such as glycine anhydride and alanine anhydride also show an analogous post-irradiation production of ammonia and a-keto acid during acid hydrolysis. 6 We note too that the dehydropeptide derivatives of these cyclic compounds represent isomeric forms of the 2,5-dihydroxypyrazines. Quite recently, Sorkin7 has given evidence that the dehydro d.erivative of glycylalanine anhydride, that is, 3-methylene-2,5-diketopiperazine (prepared '• - 2 - UCRL-10186 by the classical chemical method of Bergman and Miekely8 ), undergoes isomerization in alkaline solution to give 3-methyl-21 5-dihydroxypyrazine. We report here analogous evidence for the formation of pyrazine derivatives through r-radiolysis of the amino acid anhydrides followed by dissolution of the irradiated solids in alkaline solution. Glycylalanine anhydride was synthesized after the method of Fischer and Scheibler. 9 Glycine anhydride and alanine anhydride were obtained from the Nutritional Biochemicals Corp. The anhydrides were recrystallized three times from water, dried, and ground to a microcrystalline powder. Samples were evacuated on the vacuum line in pyrex ampoules for a minimum of 48 hours. After . 60 . 18 irradiation (Co r-rays, l. 7 x 10 ev/gm/min) 1 the ampoules were opened to the atmosphere and the contents were dissolved to a concentration of 0.1 M in 0,02 !! sodium hydroxide under nitrogen aeration. Complete dissolution required two to three' minutes. Changes in the absorption spectra of the oxygen-free solutionswere followed differentially (against a 0.1 M solution of unirradiated 'anhydride) over the range 300 ~ to 4oo ~. Solutions of irradiated glycylalanine anhydride (3-methyl~2,5-diketopiperazine) show a rapid increase in optical density with time over the " 300-4oO ~ range with ~max "" 370 ~· The spectrum is completely developed. 8 after about two hours. Authentic 3-methylene-21 5-diketopiperazine was found to exhibit almost identical spectrophotometric properties. The fully developed spectra obtained with i.rradiated glycylalanine anhydride and authentic 3-methylene-21 5-diketopiperazine in oxygen-free .02 !i sodium hydroxide solution are shown in Fig. l. Appearance of the absorption maximum at 370 lJll iS attributed to the formation of the dihydroxypyrazine nucleus (II) via the isomerization reaction: / ·· o=- C I CH.., H c. c " N --H \ ---+ - 3 -o:::C I UCRL-10186 f\\$ c· / " Ho-c \\ N ' H-N .C:::::o W-N C::.D N c-o\-\ ~ "c/ I \. H H c t H (To..\ (1l\ The absorption o~ect:rum or a.n authen.t1c C'.ibyc'troxy~yre.~ine (31 6-"imethyl) prepared after l<t'irs:oo.s and Spocyrri10 1G tnclu(!ed in Fig. l tor purpoaea ot co~1son. It iG note(! that the "iby('lro,.::ypyrE~~1nr.:s in alktUintJt sr.Jlutiou. e..re re.pi~.ly "est:royeel! C'.m t!:xpoaurt.~ t-) air. IISOle.tion of' 3-nlCthylene-8 1 5-(!1lr..etopi;>era.z1no (Ia, IIJ) from irrudiatet'J wna Wl\1!-.., a& the devulopin.g l1·~uid. Tha aut~mtic :oothylerw ~G:rivo.tive is readily' ~~teo't<ad at R.r ~ 0.4 Wl<1er ult:rco.violet 1llwait'IAt1onJtae irraditt.wd, slyoly(.l.la.nin~t a.o.l'.cy'dride also glvoe a atrongly abroorl..i1ng corq;;.onent &~.t thia Rr value. Tbo tvo ~as vcx-e- Ge~,arat<:iy elute(! ~nd tDtanrl.ned Bp~Ztct~.>photometrica.lly in oxygen .. :free 0.02 ! aocUum hydro:r.:.it'!eG The cbaract<9r1st1o growth in the 300 to 40o ~ region was obtl>~rvc<'! in both cr....eca an~ · tlw final curves vera eseent1ti.lly 1t'lonticu.1 to those given in Fig. 1. 1'.be )"1ald of )•metb.ylene-21 5• c't1lwto,t)!perad.M from gl;yoyloJ.a.n!ne unby~ri~.a at tbt) loveat dosoce stu~iet! (5 x. 1020 ov/f!}n) oorresponc"s to a G w . .iue of ""Q. 55· ,,, - 4 - UCRL-10106 The results obtained with alanine anhydride appear to be somewhat more complicated. In this case the development of.the absorption curve is accompanied by a gradual shift in ~ from 370 IIl!l to 360 Irlll• However, the . max · spectra obtained within 15 minutes of the time of dissolution coincide with that given by authentic 2,5-dihydroxy~3,6-dimenthylpyrazine. Solutions prepared from irradiated glycine anhydride show the character-istic dihydroxyprazine spectrum and on standing there is no detectable shift in ~ from 370 IIlll to shorter wavelengths. However, glycine anhydride differs max from both glycylalanine and alanine anhydrides in that the absorption spectrum shown in Fig. 1 is fully developed within the two to three minute period required for dissolution. This behavior may be interpreted as evidence for rapid isomer!-zation of dehydroglycine anhydride which must necessarily have only the endocyclic form as given by structure Ib. Finally we should like to co~nent on the fact that all three solid anhydrides develop a yellow coloration which is visually detectable at radiation dosages as low as ,.5 x 1019 ev/gm. The effect is most p·ronounced in the case of glycine anhydride. Coloration is not appreciably diminished by exposure to oxygen and remains visible in water extracts of the irradiated solids. This coloration then, cannot be ascribed to an optical absorption by free radicals or ionic transients. It is noted that. the authentic dihydroxypyrazines are· yellow in the solid state and~ although we have made only preliminary spectra-photometric studies of the irradiated solid anhydrides, the available evidence suggests that isomerization of dehydropeptides occurs to some extent in the solid state, · A mechanism for the radiation-induced isomerization of the ~0-ml~linkage in solid peptide systems has recently been described.5 A detailed report of the various aspects of the radiation chemistry of the cyclic dipeptides will be forthcoming. ... - 5 - UCRL-10186 REFERENCES l. M. E.-Jayko-andW.--M.-Ga-rrison, J, Chern. Phys., 25,1084 (1956). 2. A. Caputo and K. D:>se, z. Naturforschung. l2b, 172 (1957). 3· W. M. Garrison, M. E. Jayko and W. Bennett, Radiation Research 16, 483 (1962). 4. J. P. Greenstein and M. Winitz, Chemistry of the Amino Acids, John Wiley and 5. . 6. Sons Inc., 1961. W. M. Garrison and B. M. Weeks, Radiation Research 17, 000 (1962). . -M. Kland-English and W. M. Garrison, La,.,rrence Radiation Laboratory Report UCRL-8988 (Dec. 1959). 7. H. Sorkin, Isomerization of Methylene Diketopiperazines, Ph.D. Dissertation, Cornell University (1960). 8. M. Bergmann and A. Miekely, z. Physiol. Chern., 140, 128 (1924). 9. E. Fischer and H. Scheibler, Ann., 363, 136 (1908). 10. G; Karmas and R. E_. Spoerri, J. Am. Chern. Soc., ]2, 680 (1957). I~ .... 1/) c: Q) "C 0 (.) -Q. 0 -6-0.8 ~-o, I . J \ f 0.1 OL---~~--~-----L----~----~--~~--_j 330 350 Wave -length 370 (m,u.l 390 MU-28088 UCRL-10186 Fig. 1. Absorption spectra of irradiated cyclic dipeptides and authenic dehydropeptides in -02N sodium hyroxide 0, irradiateq glycine anhydride;O authenic 3-methylene-2,5-diketopiperazine; II authentic 2, 5··dihydroxy-3,6-dimethylpyrazine. This report was prepared as an account of Government sponsored work. Neither the United States, nor the Com-mission, nor any person acting on behalf of the Commission: A. Makes any warranty or representation, expressed or implied, with respect to the accuracy, completeness, or usefulness of the information contained in this report, or that the use of any information, appa-ratus, method, or process disclosed in this report may not infringe privately owned rights; or B. Assumes any liabilities with respect to the use of, or for damages resulting fiom the use of any infor-mation, apparatus, method, or process disclosed in this report. As used in the above, "person acting on behalf of the Commission" includes any employee or contractor of the Com-mission, or- employee of such contractor, to the extent that such employee or contractor of the Commission, or employee of such contractor prepares, disseminates, or provides access to, any information pursuant to his employment or contract with the Commission, or his employment with such contractor. I 

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DEHYDROPEPTIDE PRODUCTION IN THE RADIOLYSIS OF SOLID PEPTIDES: A RADIOLYTIC SYNTHESIS OF PYRAZINE DERIVATIVES FROM CYCLIC DIPEPTIDES

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DEHYDROPEPTIDE PRODUCTION IN THE RADIOLYSIS OF SOLID PEPTIDES: A RADIOLYTIC SYNTHESIS OF PYRAZINE DERIVATIVES FROM CYCLIC DIPEPTIDES

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