The reaction of the glucose-substituted imidazolidinone 1, structurally related to endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu), with acetone in the presence of acid catalyst gives 2,3-O-isopropylidene derivative 2 along with the isomeric monoacetal 3. Evidence is presented by using RP HPLC and NMR spectroscopy that, under the conditions of ketal formation, the peptidyl group migrated from the primary (2) to the adjacent secondary hydroxy group of the sugar moiety to give ester 3. The product distribution in the equilibrated reaction mixture (2 : 3 ≈ 1.5 : 1) indicated that 5-O-peptidyl derivative 2 is slightly more stable than ester 3. Peptidyl group migration in aqueous solution at pH = 5 shows slow 2 &#x2192; 3 rearrangement and markedly faster 3 &#x2192; 2 conversion

Maja Roščić

Štefica Horvat

English

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ISSN-0011-1643CCA-2681 Original Scientific PaperSynthesis and Peptidyl Group Migrationin Isopropylidene Acetals Derivedfrom the Glucose-substituted ImidazolidinoneRelated to Opioid Peptide Leucine-enkephalinMaja Ro{~i} and [tefica Horvat*Department of Organic Chemistry and Biochemistry, Ru|er Bo{kovi} Institute,P. O. Box 180, 10002 Zagreb, CroatiaReceived November 29, 1999; revised February 3, 2000; accepted February 23, 2000The reaction of the glucose-substituted imidazolidinone 1, structu-rally related to endogenous opioid pentapeptide leucine-enkephalin(Tyr-Gly-Gly-Phe-Leu), with acetone in the presence of acid cata-lyst gives 2,3-O-isopropylidene derivative 2 along with the isomericmonoacetal 3. Evidence is presented by using RP HPLC and NMRspectroscopy that, under the conditions of ketal formation, the pep-tidyl group migrated from the primary (2) to the adjacent secon-dary hydroxy group of the sugar moiety to give ester 3. The productdistribution in the equilibrated reaction mixture (2 : 3  1.5 : 1) in-dicated that 5-O-peptidyl derivative 2 is slightly more stable thanester 3. Peptidyl group migration in aqueous solution at pH  5shows slow 2  3 rearrangement and markedly faster 3  2 con-version.Key words: opioid, peptide, enkephalin, imidazolidinone, acetal, glu-cose.INTRODUCTIONIn earlier papers1–3 we found that intramolecular rearrangement of themonosaccharide ester in which D-glucose, D-mannose or D-galactose, is linked,through the C-6 hydroxy group, to the C-terminal carboxy group of the endo-genous opioid pentapeptide leucine-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH), in* Author to whom correspondence should be addressed. (E-mail: shorvat@rudjer.irb.hr)CROATICA CHEMICA ACTA CCACAA 73 (3) 773¿780 (2000)methanol as the solvent, resulted in the formation of an imidazolidinone inwhich C-1 of the glucose moiety forms a bridge between the -amino groupof the N-terminal tyrosine residue and the amide nitrogen of the Tyr-Glypeptide bond. Because of the new N, N'-acetal centre formed, the ring clo-sure in D-glucose-related ester resulted in imidazolidinone diastereoisomershaving cis and trans relative geometry of the substituents at the imida-zolidinone ring moiety.1,3 The imidazolidinone isomers were separated byRP HPLC. The major product, isolated in the yield of 35% and assumed tohave trans configuration of the 2- and 4-substituents with respect to theplane defined by the heterocyclic moiety, is the bicyclic imidazolidinone 1.The structural features of compound 1 resemble those of peptide- andcarbohydrate-containing macrocyclic compounds which have elicited muchinterest recently due to their potential to bind substrates in an enantiose-lective fashion and as efficient ion-transporters across model membranes.4To explore the reactivity of free hydroxy groups and utility for further syn-thetic transformations, herein we investigated the isopropylidenation ofimidazolidinone 1.RESULTS AND DISCUSSIONTreatment of the imidazolidinone 1 in acetone with catalytic amounts ofsulphuric acid under anhydrous conditions, for 4 h at room temperature, re-sulted in two new products which were separated by semipreparative RPHPLC to give isopropylidene derivatives 2 (20%) and 3 (24%) (Scheme 1).The structures of 2 and 3 were elucidated using 13C NMR spectroscopy. Theassignment of the signals (Table I) was made by comparison with those re-ported previously for the parent D-glucose-derived imidazolidinone 1,3 onthe basis of literature data for isopropylidene acetals5–8 and related acyclicsugar derivatives9 as well as by using 1H-13C COSY experiments. The 13Cchemical shifts of the isopropylidene acetal carbon atom and of the methylgroups clearly indicated the presence of only one five-membered 1,3-dioxo-lane ring in 2 and 3 (Table I). In accordance with detailed studies by Bucha-nan et al.,7,8 1,3-dioxolane structures gave an acetal carbon signal at  =110–112 and two methyl signals at  = 26–28 ppm whereas six-membereddioxane rings gave the acetal carbon at  = 99–101 and two methyl groupsat  = 29–30 and 19–20 ppm. Formation of cyclic acetals leads to a ratherlarge downfield shifts of the -carbon atoms,10 thus the 2',3'-position (seeScheme 1 for carbon atom numbering) of the isopropylidene group was de-duced from chemical shifts of the respective carbon atoms in compounds 2and 3. The obtained results are in agreement with the observed preferencefor 1,3-dioxolane ring formation in alditols from the hydroxy groups having774 M. RO[^I] AND [. HORVATPEPTIDYL GROUP MIGRATION 775OHOHNCHNHONHHNNHOOOOOCH2OHHOOH1+OHOHNCHNHONHHNNHOOOOOCH2OHOCO CH3CH32acetone[H2SO4]OHOHNHHNNHOOOOONCHNHOCH2OHOCO CH3CH33GlyPheLeu	'HHHOHOHOCO CH3CH3HONNNOOOOOHNCHNHOCH2OHf12345abcde1'2'3'4'5'4Scheme 1.776 M. RO[^I] AND [. HORVATTABLE I13C NMR data (DMSO-d6) of imidazolidinone compounds 1–4Residue Carbonatoma / ppm1b 2 3 4Imidazolidinone 2 73.7 73.3 74.2 73.9ring 4 59.3 60.6 59.3 59.3p-hydroxybenzyl a 34.3 35.7 34.6 34.7b 126.3 128.9 126.9 n.o.cc 130.5 130.1 130.6 130.4d 115.4 115.2 115.7 115.4e 156.6 156.1 156.8 156.6N1–CH2 f 43.2 43.1 44.0 42.9Sugar moiety 1' 67.2 69.1 65.2 66.92' 71.7 78.7 80.4 79.23' 72.3 76.5 74.3 76.44' 69.4 70.0 76.3 73.35' 66.8 64.1 60.7 63.3Gly  42.2 42.2 42.7 41.8Phe  54.9 54.4 53.9 53.8 37.2 37.6 37.4 37.8 137.9 138.0 137.6 138.8 129.3 129.3 129.6 129.5 128.5 128.5 128.8 128.3	 126.7 126.5 127.2 126.6Leu  50.8 51.7 52.4 50.4 39.6 39.7 39.7 39.8 24.2 24.1 24.4 24.4 21.4 21.3 21.8 21.4' 23.1 22.9 22.9 22.9Isopropylidene CH3 27.4 26.5 26.6CH3' 27.4 27.6 27.2C 109.2 111.2 109.4aSee Scheme 1 for carbon atom numbering. bData taken from Ref. 3. cn.o. = not observed.threo configuration,11 but interestingly, 1',2'-acetal of 1 was not detected inthe isopropylidene products. The reason for this behaviour is not clear atpresent. In the 13C NMR spectra of 3 downfield shift of the C-4' signal ac-companied by a corresponding upfield shift of the C-5' signal was observed.This indicated that acyl (peptidyl) group migration from O-5' to O-4' had oc-cured.Hydrolysis of the ester bond in either 2 or 3 (0.1 M NH4OH) gave aunique compound which was identified as 2',3'-O-isopropylidene derivative4 (Scheme 1). The 13C chemical shifts for 4 are summarized in Table I. Com-parison of the C-4' and C-5' chemical shifts of 2 and 3 with that of 4 demon-strated an upfield shift of the respective carbon atom in the deprotectedcompound 4 owing to the loss of the deshielding effect of the acyl group.Acyl migration to a neighbouring hydroxy group under acidic or basicconditions, via an ortho ester intermediate, has been well documented inthe partially acylated (usually acetylated or benzoylated) carbohydrates.12Acyl groups generally rearrange from secondary to primary positions. Togain better insight into the migratory tendency of the bulky peptidyl groupfrom the primary to the secondary hydroxy group, we next examined the re-lative amounts of the 5'-O- (2) and 4'-O-ester (3) present in the solution un-der the above conditions of the ketal formation. As evidenced by RP HPLCanalysis after 1 h, the reaction mixture contained 2 and 3 in a 5 : 1 ratiowhereas the product distribution obtained after 6 h, 2 : 3  1.5 : 1, wasfound to represent essentially the equilibrium mixture and was not signifi-cantly changed up to 72 h. From the relative proportions of 2 and 3 detectedin the acetalation mixture it is evident that at the beginning of the reactionthe starting compound 1 is rapidly converted into 2',3'-O-isopropylidene de-rivative 2. After 1 h, however, when 1 has been consumed, in the presence ofthe acid-catalyst, the amount of compound 3 started to increase indicatingpeptidyl residue migration from the primary to the adjacent secondary hy-droxy group. The product distribution in the equilibrated mixture indicatethat 5'-O-acyl derivative 2 is slightly more stable than 3. To investigate thereversibility of the peptidyl group migration, the isomerization of the 5'-O-(2) and 4'-O-isomer (3) was followed in aqueous solution (pH = 5) at ambienttemperature by using RP HPLC. Although isomerization of 2 is relativelyrapid in acetone containing acid-catalyst, water is less efficient in promot-ing the migration. Thus, 5'-O-acyl derivative 2 is isomerized into 3 to the ex-tent of only 14% after 8 days. However, the 4'-ester 3 is much more rapidlyisomerized by water. The mixture obtained after 48 h contains 36% of 2whereas the equilibrium mixture, attained in 8 days, contains 5'- and 4'-es-ters in the ratio  1 : 1, illustrating the migration from a secondary to a pri-mary hydroxy group in a polar solvent environment.PEPTIDYL GROUP MIGRATION 777EXPERIMENTALMelting points were determined on a Tottoli (Büchi) apparatus and are uncor-rected. Optical rotations were measured at room temperature using an Optical Ac-tivity LTD automatic AA-10 Polarimeter in a 1 dm cell; concentrations are given ing/100 mL. RP HPLC was performed on a Varian 9010 HPLC system with a Euro-spher 100 reversed-phase C-18 semipreparative column (250  8 mm I. D., 5 m) un-der isocratic conditions by using different concentrations of MeOH in 0.1% triflu-oroacetic acid (TFA), flow rate 1.1 mL. UV detection (Varian Model 9050 variable-wavelength UV-Vis detector) was performed at 280 nm. NMR spectra were recordedon a Varian Gemini spectrometer, operating at 300.1 MHz for 1H and 75.5 MHz for13C nuclei. All samples were measured in DMSO-d6 solution at 25° C in 5 mm NMRtubes. Chemical shifts, in ppm, are referred to TMS. Elemental analyses were car-ried out at the Microanalytical Laboratory, Ru|er Bo{kovi} Institute.cyclo-N-(2--5)-D-gluco-Pentitol-1-yl-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1  Oacetyl)glycyl-L-phenylalanyl-L-leucyl- (1)Compound 1 was obtained under the conditions described by Horvat et al.3 Inbrief, 6-O-(L-tyrosylglycylglycyl-L-phenylalanyl-L-leucyl)-D-glucopyranose13 was in-cubated in dry MeOH for 3 days at 60 °C. The solvent was evaporated and the resi-due was purified by semipreparative RP HPLC using 47% MeOH/0.1% TFA as theeluent to give the pure imidazolidinone 1 (yield 35%, major isomer, retention time:25.7 min) and the corresponding minor isomer (yield 10%, retention time: 24.4 min).Desalting on a Dowex 1X2, 200 (Ac) column (10  0.8 cm) and lyophilization gavepure 1 which was used in the subsequent experiments.cyclo-N-(2--5)-2,3-O-Isopropylidene-D-gluco-pentitol-1-yl-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1  Oacetyl)glycyl-L-phenylalanyl-L-leucyl- (2)andcyclo-N-(2--4)-2,3-O-Isopropylidene-D-gluco-pentitol-1-yl-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1  Oacetyl)glycyl-L-phenylalanyl-L-leucyl- (3)Bicyclic imidazolidinone 1 (40 mg, 0.06 mmol) was added at 0 °C to a solution ofacetone (4 mL) containing H2SO4 (0.01 mL). The solution was stirred under anhy-drous condition for 4 h at room temperature and the products precipitated by addi-tion of cold ether. The precipitate was centrifuged off and purified by semiprepara-tive RP HPLC using 52% MeOH/0.1% TFA as the eluent to give pure isopropylidenederivatives 2 (retention time: 24 min) and 3 (retention time: 22 min) which werecrystallized from MeOH / diisopropyl ether.Compound 2: 10 mg (20%), m.p. 140–150 °C (decomp.), D  –34° (c = 1 inMeOH). For 13C NMR data see Table I.Anal. Calcd. for C37H49N5O11 · CF3COOH (Mr = 853.84): C 54.86, H 5.90, N8.20%; found: C 54.99, H 5.92, N 8.04%.Compound 3: 12 mg (24%), m.p. 140–150 °C (decomp.), D  –50° (c = 1 inMeOH). For 13C NMR data see Table I.Anal. Calcd. for C37H49N5O11 · CF3COOH (Mr = 853.84): C 54.86, H 5.90, N8.20%; found: C 55.00, H 5.98, N 8.34%.778 M. RO[^I] AND [. HORVATN-2-(2,3-O-Isopropylidene-D-gluco-pentitol-1-yl)-4-(4-hydroxybenzyl)-5-oxoimidazolidin–1-ylacetylglycyl-L-phenylalanyl-L-leucine (4)Compound 2 (or 3) (24 mg, 0.028 mmol) was dissolved in 0.1 M NH4OH (5 mL)and stirred for 2 h at room temperature. The solvent was removed and the residuewas purified by RP HPLC using 52% MeOH/0.1% TFA as the eluent to afford pure 4(retention time: 15 min): 11 mg (52%), m.p. 125–135 °C (decomp.), D = –20° (c = 1in MeOH). For 13C NMR data see Table I.Anal. Calcd. for C37H51N5O12 (Mr = 757.83): C 58.64, H 6.78, N 9.24%; found: C58.70, H 6.99, N 9.38%.RP HPLC Analysis of the Peptidyl Group Migration in Compounds 2 and 3Isopropylidene derivatives 2 and 3 (1 mg) were each dissolved in water (1 mL)and kept at ambient temperature. The progress of the peptidyl group migration wasmonitored in aliquots removed every 24 h from the incubation mixtures, over a pe-riod of 8 days. The respective samples were directly analysed by RP HPLC by using52% MeOH /0.1% TFA as the eluent.Acknowledgements. – This work was supported by the Ministry of Science andTechnology of Croatia grant No. 00980704.REFERENCES1. [. Horvat, L. Varga-Defterdarovi}, M. Ro{~i}, and J. Horvat, J. Chem. Soc., Chem.Commun. (1998) 1663–1664.2. L. Varga-Defterdarovi}, D. Viki}-Topi}, and [. Horvat, J. Chem. Soc., PerkinTrans 1 (1999) 2829–2834.3. [. Horvat, M. Ro{~i}, and J. Horvat, Glycoconjugate J. 16 (1999) 391–398.4. D. Ranganathan, A. Thomas, V. Haridas, S. Kurur, K. P. Madhusudanan, R. Roy,A. C. Kunwar, and A. V. S. Sarma, J. Org. Chem. 64 (1999) 3620–3629; J. Dowden,P. D. Edwards, S. S. Flack, and J. D. Kihlburn, Chem.-Eur. J. 5 (1999) 79–89.5. D-P. Pham-Huu, M. Petru{ová, J. N. BeMiller, P. Köll, J. Kopf, and L. Petru{, Car-bohydr. Res. 306 (1998) 45–55.6. A. Awal, A. S. F. Boyd, J. G. Buchanan, and A. R. Edgar, Carbohydr. Res. 205(1990) 173–179.7. G. Aslani-Shotorbani, J. G. Buchanan, A. R. Edgar, and P. K. Shahidi, Carbohydr.Res. 136 (1985) 37–52.8. J. G. Buchanan, A. R. Edgar, D. I. Rawson, P. Shahidi, and R. H. Wightman, Car-bohydr. Res. 100 (1982) 75–86.9. C. Ortiz Mellet, J. L. Jiménez Blanco, J. M. García Fernández, and J. Fuentes, J.Carbohydr. Chem. 14 (1995) 1133–1152.10. K. Bock and C. Pedersen, Adv. Carbohydr. Chem. Biochem. 41 (1983) 27–66.11. D. M. Clode, Chem. Rev. 79 (1979) 491–513.12. A. H. Haines, Adv. Carbohydr. Chem. Biochem. 33 (1976) 11–109; T. Horrobin, C.H. Tran, and D. Crout, J. Chem. Soc., Perkin Trans. 1 (1998) 1069–1080; S. Khan,D. S. Teitz, and M. Jemal, Anal. Chem. 70 (1998) 1622–1628; S-K. Chung and Y-T.Chang, J. Chem. Soc., Chem. Commun. (1995) 13–14.13. [. Horvat, J. Horvat, D. Kantoci, and L. Varga, Tetrahedron 45 (1989) 4579–4584.PEPTIDYL GROUP MIGRATION 779SA@ETAKPriprava i premje{tanje peptidne skupine u izopropilidenskimacetalima dobivenima iz glukozom supstituiranog imidazolidinona,srodnoga opioidnom peptidu leucin-enkefalinuMaja Ro{~i} i [tefica HorvatReakcija glukozom supstituiranog imidazolidinona 1 (strukturno srodnoga endo-genom opioidnom pentapeptidu leucin-enkefalinu, Tyr-Gly-Gly-Phe-Leu) s acetonomu prisutnosti kiselog katalizatora rezultira nastajanjem 2,3-O-izopropilidenskog de-rivata 2 i izomernog monoacetala 3. Uporabom RP HPLC i NMR spektroskopije do-kazano je, pod uvjetima prire|ivanja ketala, premje{tanje peptidne skupine s pri-marne (2) na susjednu sekundarnu hidroksilnu skupinu u {e}ernom dijelu molekulekoje rezultira esterom 3. Raspodjela reakcijskih produkata u ravnote`noj smjesi (2 :3  1,5 : 1) pokazuje da je 5-O-peptidil-derivat 2 ne{to stabilniji od estera 3. Ispiti-vanje premje{tanja peptidne skupine u vodenoj otopini pri pH  5, pokazuje polaga-nu pregradnju 2  3 i znatno br`u pregradnju 3  2.780 M. RO[^I] AND [. HORVAT

Synthesis and Peptidyl Group Migration in Isopropylidene Acetals Derived from the Glucose-substituted Imidazolidinone Related to Opioid Peptide Leucine-enkephalin

Synthesis and Peptidyl Group Migration in Isopropylidene Acetals Derived from the Glucose-substituted Imidazolidinone Related to Opioid Peptide Leucine-enkephalin

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