Total Synthesis of Bleomycin A2 and Related Agents. 4. Synthesis of

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J. Am. Chem. SOC.1994,116, 5647-5656

5647

Total Synthesis of Bleomycin A2 and Related Agents. 4. Synthesis of the Disaccharide Subunit: 2 - 0 4 3-G-Carbamoy~-c~-D-mannopyranosy~)-L-gulopyranose and Completion of the Total Synthesis of Bleomycin A2 Dale L. Boger' and Takeshi Honda Contribution from the Department of Chemistry, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, California 92037 Received January 21, 1994'

Abstract:The chemical synthesis of 2-04 3-Ocarbamoyl-a-~-mannopyranosyl)-~-gulopyranose and its incorporation into a total synthesis of bleomycin A2 (1) readily adaptable to the preparation of structural analogs are detailed. Key strategic elements of the approach include diastereoselectivea-Oglycosidation of erythro- Na-CBZ-Nr-trityl-@-hydroxyL-histidinemethyl ester with the @-glycosyldiphenyl phosphate of hexaacetyl2-O( 3-Ocarbamoyl-cr-~-mannopyranosyl)L-gulopyranose to provide 4 (63%) with clean inversion of the glycosyl C1 stereochemistry. The subsequent convergent incorporation of 4 into bleomycin A2 (1) was accomplished through sequential couplings with tetrapeptide S (94%) and Nu-BOC-pyrimidoblamic acid (83%), both of which proceeded in exceptionally high yield without protection of the adorning functionality. The precursor disaccharides 2 and 3 were obtained in a diastereospecific glycosidation of benzyl 3,4,6-tri-Obenzyl-@-~-gulopyranoside (31) with the a-glycosyl diphenyl phosphate of 3-0-carbamoyl-2,4,6tri-Oacetyl-a-D-mannose (19), which proceeded in exceptionally good yield (93%) with net retention of the WDmannopyranosyl C1 stereochemistry. Both the suitably protected D-mannose and L-gulose derivatives 19 and 31 for incorporation into 1-4 were prepared from D-mannose with implementation of a regioselective equatorial C3 alcohol alkylation of the 2,3-Odibutylstannylene derived from 13 and 23. A tactically simple interconversion of a D-mannose to L-gulose derivative through inversion of the C5 stereochemistry by diastereoselectiveRh(1)-catalyzed hydroboration of 28 provided the key transformation for the preparation of 31.

Bleomycin A2 (1)' is the major constituent of a family of glycopeptideantitumor antibiotics2 that are thought to exert their biological effects through a metal-dependent oxidative cleavage of DNA3 or RNA4 in the presence of oxygen. In conjunction with efforts to define the structural features of 1contributing to its biological properties and sequence-selective cleavage of DNA2 herein we report a concise synthesis of the protected disaccharides 2 and 3,6 their incorporation into 4, and a total synthesis of bleomycin A2'.* (Figure 1). Inherent in thedesign of theapproach to 2 and 3 and its convergent incorporation into 1was the potential extension of the technology to structurally related analogs. Abstract published in Advance ACS Abstracts, May 15, 1994. (1) Takita, T.; Muraoka, Y.;Nakatani, T.; Fujii, A.; Umezawa, Y.; Naganawa, H.; Umezawa, H. J. Antibiot. 1978,31,801.Umezawa, H. Pure Appl. Chem. 1971,28,665. (2)Kane, S. A.; Hecht, S . M. Prog. Nucleic Acid Res. Mol. Biol., in press. Natrajan, A.; Hecht, S . M. In Molecular Aspects of Anticancer Drug-DNA Interactions; Neidle, S., Waring, M. J., Eds.; MacMillan Press: London, 1933;p 197.Ohno, M.; Otsuka, M. In Recent Progress in the Chemical Synthesis of Antibiotics; Lukas, G., Ohno, M., Eds.; Springer-Verlag: New York. 1990:D 387. Dedon. P. C.: Goldbern. I. H. Chem. Res. Toxicol. 1992. 5.31 i. Stub&, J.; Kozarich, J. W. Chem.%ev. 1987,87,1107.Hecht, S.M. Acc. Chem. Res. 1986.19, 383. (3)D'Andrea, A. D.; Haseltine, W. A. Proc. Narl. Acad. Sci. U.S.A. 1978, 75,3608.Takeahita, M.; Grollman, A. P.; Ohtsubo, E.; Ohsubo, H. Proc. Natl. Acad. Sci. U.S.A. 1978,75, 5983. (4)Magliozzo, R. S.; Peisach, J.; Cirolo, M. R. Mol. Pharmacol. 1989, 35,428.Holmes, C. E.; Carter, B. J.; Hecht, S . M. Biochemistry 1993,32, 4293. (5) (a) Boger, D. L.; Colletti, S. L.; Honda, T.; Menezes, R. F. J. Am. Chem. Soc., companion paper in this issue. (b) Boger, D. L.; Menezea, R. F. J. Org. Chem. 1992.57.4331. (c) Boger, D. L.; Honda, T.; Dang, Q. J. Am. Chem. Soc., companion paper in this issue. (d) Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S. L. J. Am. Chem. Soc., companion paper in this issue. (e) Boger, D. L.; Menem, R. F.; Dang, Q.J. Org. Chem. 1992,57, 4333.(f) Boger, D. L.; Menezes, R. F.; Dang, Q.; Yang, W. Bioorg. Med. Chem.Lett. 1992,2,261.(g) Boger, D. L.;Honda,T.; Menem, R. F.; Colletti, S. L.; Dang, Q.; Yang. W. J. Am. Chem. Soc. 1994,116,82.Boger, D. L.; Yang, W. Bioorg. Med. Chem. Lett. 1992,2,1649.Boger, D. L.; Dang, Q. J. Org. Chem. 1992,57,1631.(h) Boger, D. L.; Honda, T. TetrahedronLett. 1993,34, 1567. 0

Key strategic elements of the approach include a diastereoselective a-Oglycosidation of Nu-CBZ-Nr-trityl-erythr&@-hydroxy-L-histidine methyl ester (36)with the @-glycosyldiphenyl phosphate of hexaacetyl 2-0(3-Ocarbamoyl-a-~-mannopyranosy1)-L-gulopyranose with clean inversion of the glycosyl C 1 stereochemistry (63%) and the subsequent convergent incorporation of 4 into 1 through sequential couplings with tetrapeptide S (94%) and pyrimidoblamic acid (83%) (Scheme 1). In turn, the suitably protected disaccharides 2 and 3 were obtained in a diastereospecific glycosidation coupling of benzyl 3,4,6-tri-0 benzyl-@-L-gulopyranoside (31) with the a-glycosyl diphenyl phosphate of 3-Ocarbamoyl-2,4,6-tri-Oacetyl-a-~-manno(19, 93%), which proceeded with retention of the a-D-mannopyranosyl C1 stereochemistry predictably directed by C2 acetoxy neighboring-group participation. The suitably protected D-mannose and (6)(a) Katano, K.; Millar, A.; Pozsgay, V.; Primeau, J. L.; Hecht, S. M. J. Org. Chem. 1986,51,2927. (b) Minster, D. K.; Hecht, S . M. J. Org. Chem. 1978,43,3987.(c) Pozsgay, V.; Ohgi, T.; Hecht, S . M. J. Org. Chem. 1981,

46,3761.(d) Katano, K.; Chang, P A ;Millar, A.; Pozsgay, V.; Minster, D. K.;Ohgi,T.; Hecht, S. M. J. Org. Chem. 1985,50,5807.(e)Millar, A.; Kim, K. H.; Minster, D. K.; Ohgi, T.; Hecht, S . M. J. Org. Chem. 1986,51,189. (f) Tsuchiya, T.; Miyake, T.; Kageyama, S.; Umezawa, S.; Umezawa, H.; Takita, T. Tetrahedron Lett. 1981, 22, 1413. (7)Bleomycin A*: (a) Aoyagi, Y.;Katano, K.; Suguna, H; Primeau, J.; Chang, L.-H.; Hecht, S. M. J . Am. Chem. Soc. 1982,104,5337.(b) Takita, T.; Umezawa, Y.; Saito, S.;Morishima, H.; Naganawa, H.; Umezawa, H.; Tsuchiya,T.; Miyake, T.; Kageyama,S.;Umezawa, S.;Muraoka, Y.;Suzuki, M.; Otsuka, M.; Narita, M.; Kobayashi, S.; Ohno, M. Tetrahedron Lett. 1982,23,521.(c)Saito,S.;Umezawa.Y.;Yoshioka,T.;Takita,T.;Umezawa, H.; Muraoka, Y . J. Antibiot. 1983, 36,92. (8) Deglycobleomycin A2: Takita, T.; Umezawa, Y.;Saito, S.; Morishima, H.; Umezawa, H.; Muraoka, Y.;Suzuki, M.; Otsuka, M.; Kobayashi, S.; Ohno,M. TetrahedronLett.1981,22,671.Saito,S.;Umezawa,Y.;Morishima, H.; Takita, T.; Umezawa, H.; Narita, M.; Otsuka, M.; Kobayashi, S.; Ohno, M. TetrahedronLett. 1982.23.529.Aoyagi, Y.;Suguna, H.; Murugesan, N.; Ehrenfeld, G. M.; Chang, L.-H.; Ohgi, T.; Shekhani, M. S.; Kirkup, M. P.; Hecht, S . M. J. Am. Chem. Soc. 1982,104,5237.Boger, D. L.; Menezes, R. F.; Honda, T. Angew. Chem., Inr. Ed. Engl. 1993,32, 273. Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S . L. J. Am. Chem. Soc., companion paper in this issue.

OOO2-7863/94/1516-5647$04.50/00 1994 American Chemical Society

Boger and Honda

5648 J. Am. Chem. SOC.,Vo1. 116, No. 13, 1994

Scheme 2 R

T

L

H

q

:

HT CONHP

CONH,

5 R-H 6 R

-

9%

C02Et

NH,

Figure 1. Scheme 1

L-gulose

D-mannose

Bn/%(& B n d Bn

AO*

~ % f 0 , O M e

aminolysis of 6 (70-75%). Our efforts to employ this regioselective generation of 8 through aminolysis of the cyclic carbonates 5-7'O were not as successful as reported although this was not extensively investigated, In contrast to the report of Omoto, the aminolysis of 5 (liquidNH3,-78 O C , 1 h,-33 OC, 30min,91%) inourhands provided a 1:1 versus 9:l (quantitative) mixture of 8 and 9,1° the aminolysis of 6 (10%NH,-EtOH, 0 OC, 1 h) provided a 1 5 ratio of 8 and 9, and the aminolysis of 7 (10% NH3-EtOH, 0 OC, 1 h, 80%) provided a 1:6 ratio of 10 and 1111 (Scheme 2). The subtle experimental details responsible for the reversed regioselectivity in our hands were not investigated, but the observation of the predominant generation of the undesired 2-0-carbamoyla-&mannose derivatives proved analogousto the results disclosed in the report of Umezawa et al.7b The 2- versus 3-@carbamoyl derivatives(9 and 11versus 8 and 10) were readily distinguishable, since the proton adjacent to the carbamoyl group in the reaction products appears at a higher chemical shift with characteristic and diagnostic coupling constants. The C3-H of the 3-0carbamoyl derivative 8 was observed at 6 4.71 with coupling constants (dd, J = 2.8, 10.0 Hz) expected of an axial C3-H hydrogen. In contrast, the C2-H of the 2-Ocarbamoyl derivatives 9 and 11 was observed at 6 4.70 and 5.08 (dd, J = 1.6, 5.2 and 1.4,4.0 Hz, respectively) with coupling constants characteristic of an equatorial C2-H and with distinguishable and diagnostic coupling with the adjacent anomeric Cl-H (d, J = 1.6 and 1.4 Hz, respectively). In the development of an alternative direct route to a 3-0carbamoyl-a-D-mannosederivative requiring selective c 3 functionalization, we examined potential approaches based on established protocols for the selective alkylation or acylation of the readily available diol 13.12 In these studies, we found that thealkylationof the 2,3-O-dibutylstannylenederived from reaction

cBiqi H

OAc CONH,

0

30% unopt 1 5 (70-75% 8 )&

'""VOCH.

8

1:1

(> W%, 9:l)O

CPh,

L-gulosederivativesfor incorporationinto 24 were both prepared from D-mannose. The former was addressed through selective C3 functionalization of D-mannose employing a regioselective alkylation of the 2,3-Odibutylstannylene of 13'and was strategically designed to employ the intermediate 19 as the mannosederived glycosyl donor in which the C2 acetate serves the tactical function of controlling the stereochemistry of the glycosidation reaction. The L-gulosesubunit,which functionsfirst as a glycosyl acceptor and ultimately as a glycosyl donor, was also prepared from D-mannose. A second application of the selectivealkylation of an a-D-mannose 2,3-Odibutylstannylene for C3 functionalization was employed to access derivatives capable of selective C2 hydroxyl deprotection required of disaccharide coupling. A subsequent and tactically simple interconversion of a D-mannose to L-gulose derivative by inversion of the CS stereochemistry through implementation of a diastereoselectiveRh(1)-catalyzed hydroboration reaction of 28 provided the key conversion for the synthesis of 31. DMaMose Subunit. Initial efforts on the preparation of a suitably protected derivative of 3-Ocarbamoyl-a-~mannosewere based on the prior reports in which the regioselective aminolysis of a C2/C3 carbonate derivative of D4"nOSe was achieved selectively to provide the desired 3-Ocarbamoyl derivatives (Scheme 2). Notably, Omoto and co-workers9 described the aminolysis of 5 which selectively provided 8 versus 9 (9:1), and Hecht et al." have disclosed its similar preparation through (9) Omoto,S.; Takita, T.; Maeda, K.; Umezawa, S.Carbohydr. Res. 1973, 30, 239.

(10) Cyclic carbonate 7 was prepared by treatment of 13 with phosgene (1.1 equiv of Bu2Sn0, CHIOH, reflux, 1 h; 1.1 equiv of COClz, toluene, 0 OC, 30 min, 98%) or through acylation of thd 2,3-Odibutylstannylene (1.1 equiv of BuZSnO, CH,OH, reflux, 1 h) with di(2-pyridyl) carbonate (1.1 equiv, toluene, 25 'C, i2 h, 69%). For 7 1H NMR (CDCI,, 400 MHz) 6 7.30-7.50 (5H, m), 5.60 (lH, s), 5.04 (lH, s), 5.04 (lH, s), 4.86 (lH, dd, J = 7.2, 7.2 Hz), 4.71 (lH, d. J = 7.2 Hz), 3.80-4.00 (3H, m), 3.43 (3H, s). Acetal hydrolysis of 7 (1% HCI-CH,OH, 25 OC, 30 min, 62%) provided 5 1HNMR(CD30D,400MHz)64.92(1H,~),4.61(1H,dd,J= 10.0,lO.O Hz), 4.60 (1H. d, J = 10.0 Hz), 3.78 (lH, dd, J = 2.5, 12.0 Hz), 3.60 (lH, dd, J = 7.2, 12.0 Hz), 3.54 (lH, dd, J = 6.0, 10.0 Hz), 3.47 (lH, m). 3.34 (3H, s). Cyclic carbonate 6 was prepared as described in ref 9b. For 8: 1H NMR (DzO, 400 MHz) 6 4.71 (lH, dd, J 2.8, 10.0 Hz, C3-H), 4.55 (1H. d, J = 1.7 Hz, Cl-H), 3.87 (lH, dd, J = 1.7, 3.2 Hz), 3.40-3.80 (4H, m), 3.31(3H,s).For9 1HNMR(D20,400MHz)64.70(1H,dd,J= 1.6,5.2 Hz, C2-H), 4.61 (lH, d, J = 1.6 Hz, Cl-H), 3.40-3.80 (5H, m), 3.30 (3H, s). Methyl 2-Ocarbamoyl-6-Q(ethoxycarbonyl)-a-~-mannopyranoside was also isolated from studies of the aminolysis of 6 R/ 0.09 (Si02 65%EtOAchexane); IH NMR (CD,OD, 400 MHz) 6 4.71 (lH, dd, J = 1.7, 4.0 Hz, C2-H), 4.57 (lH, d, J 1.7 Hz, Cl-H), 4.31 (lH, dd, J 2.0, 12.0 Hz), 4.18 (lH,dd,J= 5.6, 12.0Hz),4.08 (2H,q,J= 7.2Hz), 3.72(1H,dd,J=4.0, 8.8 Hz), 3.50-3.65 (2H, m). 3.27 (3H, s), 1.18 (3H, J = 7.2 Hz). (1 1) For 11: R 0.01 (SiOz, 65%EtOAc-hexane); 1H NMR (CDCI,, 400 MHz) 6 7.20-7.56(5H, rn), 5.58 (lH, s, CHPh), 5.08 (lH, dd, J = 1.4.4.0 Hz, C2-H), 4.74 (lH, d, J = 1.4 Hz, Cl-H), 4.20-4.30 (2H, m), 3.60-4.00 (3H, m), 3.38 (3H, s). (12) Buchanan, J. G.; Schwarz, J. C. P. J . Chem. SOC.1962, 4770.

J. Am. Chem. SOC.,Vol. 116, No. 13, 1994 5649

Completion of the Total Synthesis of Bleomycin A2

9-

Scheme 3 H

T

870/0_

92 12

o*

-

91%

H PhCH(OMe), P d TsOH, DMF

Ptf

Scheme 4

H

- A

P

B

n

85%

PhCH(OMe), ptf TsOH, DMF L v o H B s F

Bu,SnO BnBr, DMF

-

13

M

86%

OAc

NH,

cat. H2S04

H ., Pd(OH),C, 97% 02NC6H40COC1’

l4

78 x 77%

L11 58RR--BHn G17 R CO2C6H4N0, I

Aqil;(’’”Z

y: __c

0~~ PhCHzNH,; ONH,

Her"

(pho)2p(o)c’

18

19

28

7%

30% HBr-HOAc

I

Bn

ONH, 20

(Ph3P),RhCI NaOH-H,O, 8%

BsF:

Aqo: 30

*

treatment of 18 with saturated HCl-CH2Clz (25 OC, 10 h, 67%), treatment with 30% HBr-HOAc (25 OC, 2 h, 79%) or sequential treatment with benzylamine (2.5 equiv, THF, 25 OC, 20 h, 78%) 21 and (PhO)zP(O)CI(l.3 equiv, -78 OC, 10min, 77%)16to provide thea-glycosyl chloride 21, [CY]”D +79 ( ~ 0 . 3CHCl3) , (litk [ a ] ” ~ of the diol 13 with BuzSnO (1.1 equiv, CH30H, reflux, 1 h)I3 +84 (c 0.5, CHCl3)), mp 126-128 O C (litk mp 132-134 “C), the with benzyl bromide (2 equiv, DMF, 100 OC, 30 min) cleanly a-glycosyl bromide 20, or the a-glycosyl diphenyl phosphate 19, provided 14 (91%, [ a l 2 ’ +37.3 ~ (C 0.25, EtOH) (lit13b,14 mp 140-142 OC, [aI2’D +25 (c 0.13, CHC13), respectively. +38 ( c 1, EtOH))) derived from exclusive alkylation of the L-Gulose Subunit. Past efforts to prepare a suitably protected equatorial C3 alcohol (Scheme 3). Attempts to introducethe C3 derivative of the L-gulose subunit of bleomycin A2 have employed carbamoyl group directly through acylation of the diol 13 or its a six-step synthesis from the rare L-gulose itself ( 3 4 % overall derived 2,3-@dibutylstannylenewith trimethylsilyl isocyanate, ~ i e l d ) ~and J ~ the successive head-to-tail inversion of a Dtrichloroacetyl isocyanate, or chlorosulfonyl isocyanate did not glucofuranosewith interconversion of the C1/C6 oxidation states. prove successful. Similarly, the diol-derived dibutylstannylene In the initial implementation of this latter approach, the key cleanly provided the corresponding cyclic carbonate 7 rather than mannose C3 carbamoylation was conducted with aminolysis of the C3 p-nitrophenylcarbonate upon reaction withp-nitrophenyl a disaccharide cyclic carbonate and provided predominantly the chloroformate(1.05 equiv, 1.2 equiv of Et3N, CH2C1~,25OC, 10 undesired 2-Ocarbamoyl(28%)versus thedesired 3-Ocarbamoyl h, 72%). derivative (1 Subsequent improvements in both these Single-step peracetylation of 14 (2% H2S04-Ac20, 25 OC, 20 approaches have been detailed.6d Complementary to the past min, 95%) cleanly provided the pentaacetate 15,” [a]”D +7.2 efforts, our approach to the L-gulose subunit was based on the (c0.2, CHC13). Notably, 15 wassecured in this approach in 75% use of a readily available @mannoseprecursor bearing the correct overall yield from @mannose, providing an improvement over its Cl-C4 stereochemistry anticipating the development of a prior preparationkand with an efficiency that effectivelycompetes tactically simple inversion of the C5 stereochemistry required for with the reported preparations of 18 and related agents based on interconversion of D-mannose to ~-gulose.17We were especially the aminolysis of 5 and 6.k.9 Following protocols introduced in attracted to this approach, since we could enlist the 2,3-O Hecht’s efforts, debenzylation of 15 was accomplished cleanly dibutylstannylene-mediated selective C3 versus C2 hydroxyl with use of Pearlman’s catalyst (H2,0.2 wt equiv of Pd(OH)2-C, alkylation for selectiveC2 functionalization required for eventual CH3OH, 25 OC, 10 h, 97%) and provided 16, [aI2’D +28 (c 0.23, disaccharide coupling. CHCl3). Unambiguous confirmation of the regioselectivity of Protection of the C4 and C6 hydroxyl groups of benzyl CY-Dthe selective benzylation of 13 was obtained from the *HNMR mannopyranoside (22)18 with formation of acetal 23 (1.0 equiv COSY spectrum of 16, which dearly indicated that the C2-H of PhCH(OMe)Z, catalytic TsOH, DMF, 50 OC, 3 h, 81%) coupled to the anomeric C1-H was shifted downfield consistent followed by selectiveC3 hydroxyl benzylation through alkylation with C2 versus C3 acetylation. Conversion of 16 to the of the intermediate 2,3-Odib~tylstannylene~~ (1.1 quiv of Buzp-nitrophenylcarbonate17 (4.0 equiv of C~CO~C~HINOZ, 2 equiv SnO, CH3OH, reflux, 1 h; 2.0 equiv of PhCHzBr, DMF, 100 OC, of DMAP, py, 40 OC, 2 h, 93%) followed by its controlled 30 min, 85%) provided 24 (Scheme 4). Notably, this clean aminolysis (10% NH3 in THF-CH2C12 (1:2), 25 OC, 1.5 h, 88%) provided 1,2,4,6-tetra-Oacetyl-3-Ocarbamoyl-a-D-mannopy- alkylation of the equatorial C3 alcohol permitted selective protection of the C2 hydroxyl group in a manner which ultimately ranOSe (18, [a]”D +39 (c 0.18, CHCl3)) in 70% overall yield for allows its selective deprotection prior to disaccharide coupling. the 4-5 steps from 13 and in 62% overall yield from @mannose. Acetylation of the C2 hydroxyl group (3.0 equiv of AczO, py, 25 Activation of 18 as a glycosyl donor was accomplished through OC, 3 h, 93%) followed by acetal hydrolysis of 25 (80% HOAcsat HCI-CH,CI, 67%

5%).6f77b

(1 3) (a) Auge,C.; David,S.; Veyrieres, A. J. Chem.Soc., Chem. Commun. 1976, 375. (b) Nashed, M. A. Carbohydr. Res. 1978,60,200. (c) Nashed, M. A.; Anderson, L. Carbohydr. Res. 1977, 56, 419. (d) Srivastava, V. K.; Schuerch,C. TetrahedronLett. 1979,3269. (e) Review: David,&; Hanessian, S. Tetrahedron 1985, 41, 643. (14) Bhattacharjee, S.S.; Gorin, P. A. J. Can. J. Chem. 1%9,47, 1195. (15) Ponpipom, M. M. Carbohydr. Res. 1977, 59, 311.

(16) Hashimoto,S.;Honda,T.;Ikegami,S.J. Chem.Soc..Chem.Commun. 1989, 685. (17) L-GII~Wpresently costs ca. S130-150/100 mg. For an alternative

synthesis of L-gulosestarting from D-mannose, see: Evans, M. E.; Parrish, F. W. Carbohydr. Res. 1973,28, 359. (18) Gorin, P. A. J.; Perlin, A. S.Can.J. Chem. 1961, 39, 2474.

5650 J. Am. Chem. Soc., Vol. 116, No. 13, 1994 Table 1 1.Cat.

Lgulose

D-mannose yield

entry 1

2 3 4 5 6

-

substrate

R = Me, RL= TBDMS,

R2 R3 CMQ R = Me, R1= TBDMS, R2 = Bn, R3 = Ac R = Me, R1= R'= R3 = Ac R = Me, R1 = Bn, R2 = R3 = Ac R = Bn, Rl = Bn, R2 = R3 = Ac R = Bn, R l = H, R2 = Bn, R3 = Ac

reductant

(%)

ratio

catecholborane nd

0100

catecholborane nd

5050

9-BBN catecholborane catecholborane catecholborane

37:63 95:5 95:5 98:2

85 78 nd 82

H20,60 "C, 1 h, 83%) provided the diol 26, [aI2'D +32 (c 0.2, CHClp). Satisfactory but lower yields for the acetal hydrolysis were observed when the deprotection reaction was conducted with 5% HCl-CHpOH (25 OC, 30 min, 62%). Confirmation of the selective C3 alcohol benzylation was derived from the 1H NMR of 25. Acetylation of the C2 alcohol provides a readily distinguishabledownfieldC2-H at 6 5.45, which exhibited coupling constants characteristic of an equatorial versus axial hydrogen (dd, J = 1.6, 2.8 Hz) and had a distinguishableand diagnostic coupling with the adjacent C1-H anomeric hydrogen (d, J = 1.6 Hz). This set the stage for inversion of the C5 stereochemistry with conversionfrom the D-mannose toL-guloseseries. Conversion of the primary alcohol 26 to the corresponding primary iodide 27 (1.2 equiv of PhpP, 1.2 equiv of 12, 1.5 equiv of imidazole, toluene, 100 "C, 3 h, 85%)*9followed by elimination of HI, which was best affected by treatment with AgF-pyZO (5.0 equiv, py, 25 "C, 3 h, 56%),pro~ided28.~1 Initialattempts toprotecttheC4alcohol as its benzyl ether at the stage of 27 were not successful but proved to be unnecessary, and alternative approaches to conducting the elimination of HI (e.g., 5.0 equiv of DBU, toluene, 90 "C, 8 h, 45%) proved less satisfactory. Rh(I)-catalyzed22 hydroboration-oxidation of 28 cleanly provided 29, +76 (c 0.2, CHClp), with inversion of the C5 stereochemistry in a reaction that proceeded with a diastereoselection of 250:l. Optimal conversions were obtained with fresh catalyst and upon oxidative workup under neutral rather than basic conditions (pH 7 phosphate buffer-50% aqueous H202) in order to avoid inadvertent acetate hydrolysis. This hydroboration reaction was examined in some detail and the observed diastereoselection was found to bedependent on thenatureof theallylicalcohol substituent (Table 1). A small allylic alcohol protecting group (CH2Ph) or the free allylic alcohol itself was found to favor near exclusive 8-face reagent delivery with clean introduction of the L-gulose C5 stereochemistry. Not surprisingly, only regeneration of the D-mannose C5 stereochemistrywas observed with 2,3-@acetonide derivatives and may be attributed to steric deceleration of 8-face reagent delivery. Thus, appropriate selection of the C4 allylic alcohol protecting group in conjunction with appropriate C2/C3 alcohol protecting groups permits clean access to the L-gulose or D-mannose series. Exhaustive benzylation of 29 (10 equiv of BnOC( =NH)CClp, catalytic CFpSOpH, CHzClthexane 1:2,25 (19) Garegg, P. J.;Samuelsson, B. J. Chem. Soc., Chem. Commun. 1979, 978. (20) Cheng, T. M.; Horton, D.; Weckerl, W. Corbohydr. Res. 1977,58, 139. (21) The bromide corresponding to 27 was also prepared (2.0 cquiv of PhlPand NBS, DMF, 50 OC, 45 min, 65%)but was not as effectivelyconverted to 28 through elimination of HBr. For benzyl 2-O-acetyl-3-Obenzyl6-bromo-6-deoxy-a-~-mannopyranoside: 'H NMR (CDCl3,400 MHz) 6 7.207.40 (10H. m),5.41 (IH, dd, J = 2.0,2.8 Hz), 4.93 (IH, d, J = 2.0 Hz), 4.80 (lH,d,J=l2.OHz),4.73(1H,d,J= 12.OHz),4.56(1H,d,J=12.0Hz), 4.41 (lH, d, J = 12.0 Hz), 3.70-3.90 (4H, m), 3.57 (IH, dd, J = 6.8, 10.8 Hz), 2.1 1 (3H, s). (22) Evans, D. A.; Fu, G. C.; Hoveyda, A. H. J. Am. Chem. Soc. 1992, 114, 6671.

(Pho)2POC"

L 34 R - P(O)(OPh), 3 R-H

"C, 1 h, 72%) followed by methanolysis of the C2 acetate of 30, [aI2'~ +70 (C 0.13, CHClp) (litac [ a ] " +72.7 ~ (C 1.4, CHClp)), provided benzyl 3,4,5-tri-Obenzyl-(3-~-gulopyranoside (31), [O~]"D +49 (C 0.12, CHClp) (litac [aI2'~ +48.3 (C 0.7, CHClp)), and a suitable L-gulose glycosyl acceptor for disaccharidecoupling. Dhccharide Formation: Preparationof 2 and 3. Disaccharide formation with 31 serving as the glycosyl acceptor and 19-21 serving as the glycosyldonors cleanly provided the key disaccharide 2, [aI2'D +38 ( c 0.05, CHCl3). Although the glycosidation reactions conducted with 20 (1.2 equiv of AgOTf, 3.0 equiv of TMU, CH2C12, 25 "C, 10-36 h, 7478%) and 21 provided 2 cleanly analogous to the report of Hecht," the use of the glycosyl diphenyl phosphate 19 (1.8 equiv of TMSOTf, 0 "C, CH2C12,lO min, 93%)16 provided 2 under milder reaction conditions (0 versus 25 "C) in much shorter reaction times (10 min versus 10-36 h) and in higher conversions (93% versus 7478%) (Scheme 5). Notably, the carbamoyl group could be taken through this glycosidationreaction without deliberateprotection or observation of competitive reactions. The exclusiveretention of the a-linkage stereochemistry may be attributed to neighboring-group participation of the mannopyranosyl C2' acetate, providing coupling with clean retention of the mannose C1' stereochemistry. This a-linkage was supported by lH NMR through observation of the Cl'-H anomeric proton of 2 at 6 4.76 with a coupling constant characteristic of an equatorial proton (d, J = 1.6 Hz). Debenzylation of 2 (0.1 wt equiv of Pd(OH)Z-C, H2, CHpOH, 25 "C, 15 h, 98%) followed by acetylation of 32 (10 equiv of Ac20, py, 25 "C, 10 h, 82%) provided 33, [ a ] 2 5+35.4 ~ (c 0.06, CHCL), and conducting these twosteps without the deliberate purification of 32 afforded 33in much higher conversions (98% for two steps). Activation of 3 as its @-glycosyldiphenyl phosphate 34,[(rI2'D +34 (c 0.17, CHClp), for coupling with a suitably protected 8-hydroxy-L-histidinederivative was accomplished by sequential treatment of 33with benzylamine (2.5 equiv, THF, 25 "C, 10-24 h, 8 1444%)and (PhO)zP(O)Cl( 1.5 equiv, -78 OC, 10min, 8 1%)16 with the intermediate generation of 3. Through this sequence the &stereochemistry of the L-gulopyranoside anomeric center was maintained and confirmed through observation of diagnostic Cl-H/CZ-H coupling constants ( J = 8.0 Hz) characteristic of an axial Cl-H coupled to the adjacent axial C2-H at 6 4.95 (lH, d , J = 8.0Hzfor2Cl-H),5.89(1H,d,J= 8.0Hzfor33Cl-H), 5.00(1H,d,J=8.0Hzfor3Cl-H),and5.71 (lH,dd,J=6.8, 8.0 Hz for 34 Cl-H).

Completion of the Total Synthesis of Bleomycin A2 Preparation of 4 and Incorporation into Bleomycin Ai. Key to the completion of the total synthesis of bleomycin A2 was the stage and manner by which the disaccharide was incorporated and the order of the assemblageof the subunits. In initial efforts, Umezawa and co-workers found that the glycosyl bromide of disaccharide 3 failed to couple with the free or fully protected aglycone itself but could be coupled with a fully protected pentapeptide S derivative albeit with generation of the desired 0-glycosidation product as a minor product.7b Competitive and preferential imidazole N-glycosidation was observed. Coupling of the glycosidated pentapeptideS with Nu-BOC-pyrimidoblamic acid and deprotection provided bleomycin A2 in low yield. They later modified their approach to 0-glycosidation of erythro-NaBOC-N*-Tos-j3-hydroxy-L-histidine methyl ester with the glycosyl bromide of disaccharide 3, which provided the desired a - 0 glycosidation product in modest yield (21%).7c Subsequent and sequential tetrapeptide S and Nu-BOC-pyrimidoblamic acid couplings followed by final deprotection provided bleomycin A2. This latter approach proved comparable to that initially described by Hecht7ain which the a-O-glycosidationof Na-BOC-N*-BOC0-hydroxy-L-histidine benzyl ester with the glycosyl bromide of N-acetyl-3 was accomplished in modest yield (20-25%). Sequential coupling of the 0-glycosidated histidine subunit with (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoate, demethyltripeptide S, and Nu-BOC-pyrimidoblamic acid followed by deprotectionprovideddemethylbleomycinAz. Notably, the Hecht approach employed the demethyl C-terminus, requiring penultimate S-methylation, a key 0-glycosidation coupling of the disaccharide with the protected j3-hydroxy-L-histidine subunit which proceeded in modest conversion (20-25%), and a linear sequential introduction of the pentanoic acid, tripeptide S,and pyrimidoblamic acid subunits. Our initial efforts at 0-glycosidation of Nu-BOC-Nr-trityl0-hydroxy-L-histidine methyl ester (35)Sa with the 0-glycosyl diphenyl phosphate 34 (1.5 equiv of TMSOTf, EtzO, 0 "C, 30 min) were not especially successful and provided a mixture of at least three components. Although this was not investigated in detail, Ne-BOC deprotection under the reaction conditions was observed. Consequently, the 0-glycosidation reaction was conducted with 34 and Nu-CBZ-N*-trityl-@-hydroxy-L-histidine methyl ester (36,5a4 equiv of TMSOTf, 2:l Et20-CH2Cl2,O OC, 20 min, 54-63%), thereby avoiding the competitive N-BOC deprotection, and provided the adduct 4 in excellent conversion as a 1 13: 1mixture of the desired a and undesired j3linked anomers (Scheme 6). In fact, the undesired j3 anomer was not unambiguously identified and only a trace amount (34%) of additional material containing the structural elements of both 34 and 36 was isolated. As such thediastereoselectivityof theglycosidation was estimated to be at least 13:l and may in fact be higher. Confirmation of the 0-glycosidation a-linkage arising from inversion of the disaccharide C1 stereochemistry was derived by IH NMRof 38through observation of the Cl-H anomericproton at 6 5.13 (d, J = 3.6 Hz) with a distinguishable coupling constant characteristic of an equatorial proton coupled to the adjacent axial C2-H at 6 3.99 (dd, J = 3.6, 3.6 Hz), which itself exhibits a diagnostic set of coupling constants with the adjacent C1 and C3 equatorial hydrogens. The surprisingly high and welcomed diastereoselectivity of this glycosidation reaction is especially noteworthy and may be a consequence of both the low reactivity of the glycosyl acceptor, which tends to favor formation of the most stable a anomer, and mechanistic elements characteristic of a glycosyl phosphate donor which favor reaction with inversion of the stereochemistry at the reacting anomeric center. Selective CBZ deprotection (0.35 wt equiv, 10% Pd-C, Hz, EtOAc-CH3OH 2: 1,25 OC, 5 h, 50%; 69% based on consumed 4) without competitive hydrogenolysis of the trityl protecting group followed by reprotection of the free amine of 37 (3.0 equiv of BOCz0,6.0 equiv of NaHC03, THF-H20 3:1,25 OC, 10 h, 94%) provided 38 suitably protected for sequential couplings with tetrapeptide S and WBOC-pyrimidoblamic acid. Exhaustive

J. Am. Chem. Soc., Vol. 116, No. 13, 1994 5651

-

1.63%

CBZ

FHO" 40

"Y

ester hydrolysis of 38 (0.1 N aqueous NaOH-CH30H 1:1,0 "C, 12 h, 78%) provided 39. Coupling of 39 with the free amine of tetrapeptide S (40,Sa 1.5 equiv, 2.0 equiv of DCC, 1.0 equiv of HOBt, 1.5 equiv of NaHCOp, DMF, 25 "C, 48 h, 94%) provided 41 in superb yield. Notably, this exceptionally effective coupling was conducted without deliberate protection of the disaccharide or tetrapeptide S hydroxyl groups and was conducted with the tetrapeptide S sulfonium salt installed in the substrate. Initial attempts to follow this same approach utilizing 47, in which the imidazole was protected as its N*-BOC derivative, were found to suffer deprotectionunder the ester hydrolysisconditions. Acidcatalyzed deprotection of 41 (20% TFA-CHzClz, 0 "C, 3 h, 95100%) served toremoveboth theBOCand tritylprotectinggroups cleanly without deglycosidation and provided 42. However, attempts to couple 42 with Na-BOC-pyrimidoblamic acid (1.O equiv of 44," 2.0 equiv of DCC, 1.0 equiv of HOBt, DMF, 25 OC, 36 h) in close albeit not identical analogy to the efforts disclosed by Hecht et al.7a provided only small amounts of N U BOC-bleomycin A2 (45) and provided principally the product derived from acylation of the free imidaz~le.~'Although it is possible that use of the free base of 42 or the deliberate addition of base to the coupling reaction mixture might improve this coupling of 42 with 44and that longer reaction times might result in imidazole to Nu-amine acyl transfer, the solution to this final

5652 J. Am. Chem. Soc., Vol. 116, No. 13, 1994 coupling reaction was more effectively addressed through the surprisingly selective deprotection of 41. Mild acid treatment of 41 (1 N HCl in 90% aqueous HOAc, 25 "C, 15 min) under conditions defined by Sieber and Rinikerz4cleanly provided 43 (82%)derived from removal of the BOC protectinggroup without deglycosidation or removal of the trityl protecting group. Subsequent coupling of 43 with Na-BOC-pyrimidoblamic acid (1 .O equiv of 44," 3 equiv of DCC, 1 equiv of HOBt, DMF, 25 "C, 48 h) cleanly provided Na-BOC-N*-tritylbleomycin A2 (46, 83%). Similar to the reaction of 39 with 40, this final coupling was conducted without protection of the disaccharide or tetrapeptide S hydroxyl groups with the terminal sulfonium salt intact in exceptionally good conversion. Final acid-catalyzed deprotection of 46 (20% TFA-CH2Cl2, 0 "C, 2.5 h) provided bleomycin A2 (1) identical in all compared respects with natural material (1H NMR, IR, MS, [ a ] ~ TLC, , HPLC).2s Concurrent with the successful incorporation of 4 into bleomycin A2 detailed in Scheme 6, we also examined the alternative order of couplings employingfirst Nu-BOC-pyrimidoblamicacid and finally tetrapeptide S. This latter approach, which proved less successful, was viewed initially as more attractive since the incorporation of the tetrapeptide S sulfonium salt would occur at the penultimate step potentially simplifying the purification of the preceding intermediates. Removal of the CBZ and trityl protecting groups of 4 (H2,0.8 wt equiv, 10%Pd-C, CHpOH, 25 "C, 8 h) in the presence of BOC2O (10 equiv) provided 47 (6066%)cleanly (Scheme 7). N-BOC deprotectionof47 (20%TFACH2C12,O "C, 2 h, 75% or TFA, 25 OC, 30 min, 78%) provided 48,and in contrast to the use of TFA, the attempted deprotection of 47 with HCl (4 M HCI-EtOAc) led to competitive deglycosidation. Like the attempted coupling of 42 with 44, coupling of 48with Na-BOC-pyrimidoblamicacid (44,Sc1.1 equiv of EDCI, 1.0-1.2 equiv of HOBt, DMF, 25 "C, 2-3 days, 6473%) did not provide 49, but rather preferential and clean imidazoleN-acylation was observed.26 However, coupling of 37 with Na-BOC-pyrimidoblamic acid (1.Oof equiv 44," 1.1 equiv of EDCI, 1.Oequiv of HOBt, DMF, 25 "C, 48 h, 58% unoptimized) cleanly provided 50. Exhaustive ester hydrolysis of 50 (1 5 equiv of LiOH, H20-CHsOH 1:1, 0 "C, 12 h, 50% unoptimized) followed by coupling of 51 with the free amine of tetrapeptide S 405. (1.5 equiv, 3.0 equiv of DCC, l,OequivofHOBt,DMF,25 "C,72 h)provided46(20%) identical to the material previously prepared albeit in modest conversions. The major product observed in several attempts proved to be dehydro-N@-BOC-N*-trityldeglycobleomycin A2 (40%)27derived from deglycosidation of 51 via elimination of the disaccharide

Boger and Honda scheme 7

OAc 49 R:H 50 R = CPh,

~

C

: / : $ ) -

O

N

UOH

50%

H d

H

~

-

DCC-HOB1 40 CPh,

20%

46

followed by coupling with 40. While the origin of this facile deglycosidation was not established, it is tempting to suggest that the increased acidity of the a-proton in the Nu-acyl (Le. 51) versus Nu-BOC/CBZ derivatives of the 8-hydroxy-L-histidine subunit including 39 upon carboxylate activation is sufficient to promote &elimination of the disaccharide. More importantly and more assuredly, these latter studies clearly illustrate that the order of assemblage of the bleomycin A2 subunits is critical and that the approach detailed in Scheme 6 is optimal. The extension of the work detailed herein to the preparation and evaluation of key structural analogs of bleomycin A2 is underway and will be reported in due course.

Experimental Section

(23) TheimidazoleN-acylationproductwasisolatedasa thinfilm(reverea Methyl 4 , 6 - O B e n z y U d e w - 3 - O b e n z y l - u - ~ ~ p ~(14). ide A phase C-18, 1&20% CH~OH-HZOgradient elution 88%): R 0.33 (SiOl, (13,0.35 suspensionof methyl 4,6-Obenzylidenca-~nnopyranoside~~ 109:l CH30H-10%aqueous CH~CO~NHclO?iaqutousN)40f+); IH NMR mmol, 100 mg) and dibutyltin oxide (0.38 mmol, 96 mg, 1.1 equiv) in (CDsOD, 400 MHz) 8 8.21 (lH, s), 8.10 (lH, I), 7.76 (IH, br s), 7.22 (lH, CH3OH (3 mL) was warmed at reflux for 1.5 h to providea clear solution, br s), 5.30 (lH, m), 5.15 (IH, m), 4.30 (lH, d, J = 4.5 Hz), 3.30420 (22H, m), 2.93 (6H, s), 2.10 (SH, m), 1.43 (9H, s), 1.05-1.15 (9H, m); IR (neat) cooled to 25 OC, and the solvent was removed under reduced pressure. um3361,1700,1650,1391,1157,903cm-~;FABMS(NBA) m/e1514(M+, The resulting methyl 4,6-0benzylidene-2,3-~(dibutylstannylcnc)-aC&9zNl70&). Acid-catalyzed deprotcction of the crude reaction product D-mannopyranoside was dried under vacuum for 6 h and taken up in (20% CF,COlH-CHzClz, 0 OC, 1.5 h) provided only small quantities of DMF (3 mL). The solution was treated with PhCHZBr (0.71 mmol, 120 bleomycin A2 and provided principally the deprotection product derived from mg, 2.0 cquiv) and warmed at 100 O C with stirring for 30 min. The the presumed imidazole N-acylation with pyrimidoblamic acid. (24) Sieber, P.; Riniker, B. Tetrohedron Lcrr. 1987, 28, 6031. cooled reaction mixture was poured into a two phase solution of EtOAc (25) Boger, D. L.; Menezes, R. F.; Yang, W. Bioorg. Med. Chem. Lcrr. (5 mL) and saturated aqueous NaHCOs (3 mL) with vigorous stirring. 1992.2.959, Freyder, C. P.; Zhou, W.; Doctach, P.W.; Marzilli, L. G. Prep. The organic layer was washed with saturated aqueous NaCl(3 mL) and Biochem. 1991,21, 257. dried (NaSO4). Chromatography (Si02,l X 6 cm, 20% EtOAc-hexane) (26) For the imidazole N-acylation product: a colorless foam; Rf 0.42 afforded 14 (120 mg, 131 mg theoretical, 911, typically 80-85%, 12.0 (SiOz, 20% CH3OH-CHCI3); (a]a,-6.7 (c0.06, CHsOH); IH NMR (CD3mmol scale) as a colorless syrup: Rf 0.30 (SiOz, 30% EtOAc-hexane); OD, 400 MHz) 6 7.71 (IH, s), 7.23 (1H. s), 5.15-5.30 (SH, m), 5.00 (IH, d , J - 1.6Hz),4.10-4.30(5H,m),4.03(lH,dd,J= 3.6,3.6Hz),3.95(1H, [ a I z 5+37.3 ~ (c 0.25, EtOH) (lit'* [ a ] " ~ +38 (c 1, EtOH)); 'H NMR m), 3.89 (lH, dd, J - 7.0, 12.8 Hz), 3.79 (3H, s), 3.59 (lH, m), 2.13 (3H, (CDC13,400 MHz) 6 7.20-7.70 (lOH, m), 5.61 (lH, s), 4.86 (lH, d, J s), 2.15 (3H, s), 2.09 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 1.97 (3H, s), 1.43 = 12.0 Hz), 4.76 (lH, d, J = 1.2 Hz), 4.73 (lH, d, J = 12.0 Hz), 4.28 (9H, I); IR (neat) um 3400, 1735, 1705, 1650, 1440, 1235, 1018 cm-1; (lH,dd,J=3.6,9.2Hz),4.09(1H,dd,J=9.2,9.2Hz),4.05(1H,dd, FABHRMS (NBA) m/e 1212.4462 (M+ + H, C~HssNllOurequires J = 1.2, 3.6 Hz), 3.80-3.95 (3H, m), 3.38 (3H, s). 1212.4465). (27) For debydr~N=-BOC-N'-trityldeglycoblmmycin Az: a colorlcasfoam; l,fq6-Tebr-Oacetyl-lObenzyl-a-~munnop~ (15). A soluRJ 0.44(Si@, 1091CH~OH-IO%aqueousCH~CO~N~lO?iaqueousN~tion of 2% HzS04-AczO (6 mL) was added to 14 (1.02 mmol, 380 mg), OH); 'H NMR (CDjOD, 400 MHz) 6 8.25 (lH, s), 8.14 (lH, s), 7.63 (lH, and the reaction mixture was stirred for 20 min at 25 O C . The reaction ~),7.28(1H,~),7.12-7.37(15H,~),6.94(1H,~),4.33(1H,d,J=4.0H~), 4.14(1H,m),4.08(2H,m),3.70(2H,m),3.60(3H,m),3.30-3.55(5H.m), mixture was poured into a two-layer solution of EtOAc (20 mL) and saturated aqueous NaHCO3 (10 mL). The organic layer was washed 2.97 (6H, s), 2.80 (2H, m), 2.65 (3H, m), 2.33 (3H, s), 2.18 (2H, m), 1.45 ~). (9H, s), 1.1&1.30 (9H, m); FABMS (NBA) m/e 1372(M+,C ~ ~ H ~ ~ N I ~ O I I SwithsaturatedaqueousNaC1 (lOmL),dried (NazSO,), andconccntrated

J. Am. Chem. SOC.,Vol. 116, No. 13. 1994 5653

Completion of the Total Synthesis of Bleomycin A2 in vacuo. Chromatography (Si02,3 X 6 cm, 20% EtOAc-hexane) gave 15" (405 mg, 446 mg theoretical, 91%; typically 80-95%, 1-1.5 mmol scale) as a syrup: Rf0.31 (Si@, 30% EtOAc-hexane); [a]% +7.2 (c 0.21, CHCl3), +4.8 (C 1.5, CHCl3); 'H NMR (CDCl3, 400 MHz) 6 7.10-7.40 (5H, m), 6.10 (lH, d, J = 2.4 Hz), 5.35 (lH, dd, J = 2.4, 3.6 Hz), 5.28 (lH, d d , J = 10.0, 10.0 Hz), 4.69 (lH, d, J = 12.4 Hz), 4.44 (lH, d, J = 12.4 Hz), 4.24 (lH, dd, J = 5.2, 12.4 Hz), 4.09 (lH, dd, J = 2.4, 12.4 Hz), 3.95 (lH, ddd, J = 2.4.5.2, 10.0 Hz), 3.87 (lH, dd, J = 3.6, 10.0 Hz), 2.17 (3H, s), 2.12 (3H, s), 2.09 (3H, s), 2.03 (3H, s); IR (CHCl3) ,,u 1750, 1733, 1519, 1419, 1217, 1043, 924 cm-I; FABHRMS (NBA-CsI) m/e 571.0565 (M+ Cs, C21H26010requires 571.0580). Conducting the reaction under identical conditions but for shorter reaction periods provided 15 in lower conversions (71-81%). 1 , 2 , 4 , 6 - T e m - O a c e t y l - a - ~ ~ y n a o(16). s e A solution of 15 (2.9 mmol, 1.27 g) in CH3OH (30 mL) was hydrogenated over 10% Pd(OH)& (240 me) under H2 (1 atm) at 25 "C for 10 h. The reaction mixture was filtered through a Celite pad and concentrated in vacuo. Chromatography (Si02,3 X 10 cm, 40% EtOAc-hexane) provided 16" (980 mg, 1.01 g theoretical, 97%;typically 97-99%) as a colorless syrup: RfO.10 (Si02, 50% EtOAc-hexane); [a]"D +28 (c 0.23, CHCI,); 'H NMR (CD,OD, 400 MHz) 6 5.93 (lH, d, J 1.6 Hz), 5.10 (lH, dd, J = 10.0, 10.0 Hz), 5.01 (lH, dd, J = 1.6, 3.6 Hz), 4.17 (lH, dd, J = 4.8, 12.4 Hz), 4.04 (lH, dd, J = 3.6, 10.0 Hz), 3.98 (lH, dd, J = 2.4, 12.4 Hz), 3.91 (lH, m), 2.09 (3H, s), 2.07 (3H, s), 2.03 (3H, s), 1.97 (3H,~);IR(CHCl~)u~3498,1742,1519,1419,1217,1151,1047,968, 924cm-I; FABHRMS (NBA-CsI) m/e481.0125 (M++ Cs, CZIHZOOIO requires 481.0111). 1,2,4,6-Tetra-Oacetyl-lO(@dtropbenyl)eub.moyI)-a-"opyr a m (17). A solution of 16 (8.64 mmol, 3.0 g) in pyridine (30 mL) in the presence of (N,N-dimethy1amino)pyridine (DMAP, 34.6 mmol, 4.3 g) was treated withp-nitrophenyl chloroformate (34.6 mmol, 7.0 8). The reaction mixture was stirred at 40 "C for 2 h and poured into a two-phase solution of EtOAc (30 mL) and H2O (10 mL) with vigorous stirring. The organic layer was washed with 10%aqueous HCl(3 X 10 mL), saturated aqueous NaHCO3 (10 mL), and saturated aqueous NaCl (10 mL), dried (Na2SO4), and concentrated in vacuo. Chromatography (Si02,3 X 10cm, 25%EtOAc-hexane) gave 1860 (3.8 g, 4.48 theoretical, 86%; typically 75-93%, 1 M . 3 mmol scale) as a syrup: Rf 0.58 (Si02, 50% EtOAc-hexane); [a]2'~+1.4 (c 0.21, CHCl3); 'H NMR (CDCl3, 400MHz)68.26(2H,m),7.41 (2H,m),6.16(1H,d,J=2.0Hz),5.48

+

vigorous stirring. The organic layer was washed with saturated aqueous NaCl(2 X 3 mL) and dried (Na2SO4). Chromatography (Si02,l X 15 cm,30% EtOAc-hexane in the prescncc of 5% Et3N) afforded 19 (656 mg, 865 mg theoretical, 77%) as a white solid mp 140-142 "C dcc; Rf 0.42 (Si02,60% EtOAc-hexane); [CrlZsD+25(c 0.13, CHCI3); 'H NMR (CDC13,400 MHz) 6 7.10-7.40 (lOH, m), 5.89 (lH, dd,J= 1.8,7.2 Hz), 5.25-5.40 (3H, m), 4.75 (2H, br s), 4.20 (lH, dd, J = 4.8,12.4 Hz), 4.10 (1H,m),3.93 (lH,dd,J= 2.0,12.4Hz),2.16(3H,s), 2.05 (3H,s), 1.98 (3H, s); "C NMR (CDCl3, 100 MHz) 6 170.5, 169.8, 169.5, 154.9, 130.0, 129.9, 125.8, 125.7, 120.2, 120.1, 120.0, 96.2, 70.7, 69.1, 69.0, 65.2, 61.6, 20.7, 20.6, 20.5; IR (CHCI3),,u 3364, 1718, 1598, 1462, 1220, 958 cm-l; FABHRMS (NBA-CsI) m/e 714.0366 (M+ Cs, C2sH~N013Prequires 714.0353).

+

S4,6-Tri-Oacetyl-l~rb."toopyRal chloride(21).

A solution of 18 (0.032 mmol, 12.4 mg) in CHzCl2 (3 mL) was treated with HCl(g) at -10 OC for 10 min. The reaction flask was stoppered tightly, and the reaction mixture was maintained at 25 "C for 10 h. The solution was concentrated in vacuo, and the residue was purified by flash chromatography (Si02, 1 X 2 cm, 25% EtOAc-hexane) to provide 21 (5.5 mg, 11 mg theoretical, 48%. typically 45-6796) as a colorless syrup, which wasdetermined not to becompletelystable to the chromatographic purification: Rf 0.42 (Si02,50% EtOAc-hexane); mp 126-128 OC (lit" +83.6 (C 0.5, mp 132-134 "c); [aI2'D +79 (C 0.3. CHCl3) (lit" [aIz5~ CHCI,)); 'H NMR (CDCl3,400 MHz) 6 5.93 (lH, d, J = 1.2 Hz), 5.46 (lH, dd, J = 3.2, 10.0 Hz), 5.34 (lH, dd, J = 1.2, 3.2 Hz), 5.27 (lH, dd, J = 10.0, 10.0 Hz), 4.60 (2H, br s), 4.25 (lH, dd, J = 12.0,5.0 Hz), 4.21 (lH, ddd, J = 10.0, 5.0, 2.0 Hz), 4.08 (lH, dd, J = 2.0, 12.0 Hz), 2.11 (3H, s), 2.04 (3H, s), 2.02 (3H, 8). Benzyl 4,6-OBenzylidene-a-D-mpyraBoside (23). A solution of 22'* (3.63 mmol, 972 mg) in DMF (8 mL) was treated with benzaldehyde dimethylacetal (3.63 mmol, 552 mg) and pTsOH (0.26 mmol, 50 mg) and warmed to 60 "C for 1 h under reduced prcssure (20 mm Hg) to remove the liberated CHIOH. The reaction mixture was poured into a two-phase solution of EtOAc (10 mL) and saturated aqueous NaHCO3 (8 mL) with vigorous stirring. The organic layer was washed with saturated aqueous NaCl (2 X 5 mL) and dried (Na2SOd). Chromatography (Si02,3 X 8 cm,30% EtOAc-hexane) afforded 23 (990 mg, 1.30 g theoretical, 76%; typically 76-81%, 40 mmol scale): Rf 0.28 (Si&, 50% EtOAc-hexane); [.I2+) +31.7 (c 0.23, CHCl3); 'H NMR (CDCl3, 400 MHz) 6 7.20-7.50 (lOH, m), 5.55 (lH, s), 4.92 (lH, s), 4.73 (lH, d, J = 12.0 Hz), 4.52 (lH, d, J = 12.0 Hz), 4.25 (lH, dd, J = 3.4, 9.5 Hz), 4.10 (lH, m), 4.05 (lH, m), 3.80-3.95 (3H, m); IR (lH,dd,J=2.0,3.2Hz),5.45(lH,dd,J=9.2,9.2Hz),5.22(1H,dd, (CHCl3) u,, 3430, 1519, 1418, 1217, 1028, 927 cm-I; FABHRMS J = 3.2,9.2 Hz), 4.33 (1H, dd, J = 4.8, 12.4 Hz), 4.16 (lH, dd, J 5 2.4, (NBA-CsI) m/e 491.0470 (M+ Cs, C d 2 2 0 6 requires 491.0471). 12.4 Hz), 4.09 (lH, m), 2.24 (3H, s), 2.18 (3H, s), 2.13 (3H, s), 2.11 Benzyl 4 , 6 - O B e n z y l l d e w - 3 - O b e s l z y l - a - ~ - ~ p y r a(24). ~i~ A (3H, s); IR (CHCl3) Y,, 1751, 1738, 1522, 1423, 1259, 1210, 1046, 23 (1.56 mmol, 560 mg) and BuzSnO (1.7 1 mmol, 426 mg, suspension of 1030, 926 cm-I; FABHRMS (NBA-CsI) m/e 646.0160 (M+ + Cs, 1.1 equiv) in CH3OH (10 mL) was warmed at reflux for 1.5 h to provide C21H23N014 requires 646.0173). a clear solution, cooled to 25 "C,and the solvent was removed under l,SqCTetra-Oacetyl-lOcarbamoyI-a-~monnopynaose(18). A reduced prcssure. The resulting benzyl 4,6-O-benzylidene-2,3-O(dibusolution of 17 (0.71 mmol, 360 mg) dissolved in CH2Cl2 (20 mL) was ty1stannylene)-a-D-mannopyranosidewas dried under vacuum for 6 h treated with a solution of 1O%NH3-THF (10 mL). Thecombinedsolution and taken up in DMF (8 mL). The solution was treated with PhCHzBr was stirred for 1.5 h at 25 "C and concentrated in vacuo. Flash (3.12 m o l , 533 mg, 2.0 equiv) and warmed at 100 OC with stirring for chromatography (Si02, 1 X 8 cm, 50% EtOAc-hexane) afforded 18" 30 min. The cooled reaction mixture was poured into a two-phase solution (240 mg, 276 mg theoretical, 88%) as a colorless syrup: Rf 0.10 (Si02, ofEtOAc(15mL) andsaturatedaqueousNaHCO3(10mL)withvigorous 50% EtOAc-hexane); [CY]25D+39 (c 0.18, CHCl3); 'H NMR (CDCl3, stirring. Theorganic layer was washed with saturated aqueous NaCl(l0 400 MHz) 6 6.10 (lH, d, J = 1.6 Hz), 5.34 (lH, dd, J = 9.6, 10.0 Hz), mL) anddried (NaS04). Chromatography (SiOz, 3 X 6cm, 25% EtOAc5.25-5.31 (2H, m), 4.71 (2H, br s), 4.31 (lH, dd, J = 4.8, 12.4 Hz), hexane) afforded 24 (597 mg, 700 mg theoretical, 85%) as a colorless 4.04-4.15 (2H, m), 2.18 (3H,s), 2.16 (3H, s), 2.10 (3H,s), 2.06 (3H, syrup: Rf0.25 (SiO2, 30% EtOAc-hexane); [aI2'D +53 (c 0.47, CHCl3); s); IR (CHCI,) urmX 3488, 1749, 1738, 1579, 1519, 1421, 1366, 1213, 'H NMR (CDCl3,400 MHz) S 7.20-7.60 (15H, m), 5.62 (lH, s), 4.97 1050, 973 cm-I; FABHRMS (NBA-CsI) m/e 524.0150 (M+ Cs, (lH,s),4.8?(1H,d,J= 12.0Hz),4.72(1H,d,J= 12.0Hz),4.71 (lH, C I ~ H ~ ~ Nrequires O I I 524.0169). d, J = 12.0 Hz), 4.52 (lH, d, J = 12.0 Hz), 4.27 (lH, m), 4.12 (2H, m), 546-TrCOacetyl-lOcubemoyl-a-D.muaopyr;rwclyl Diphenyl Phs3.80-4.05(3H,m);IR(CHC1~)u,,3462,1518,1371,1211,1041cm-1; phete (19). A solution of 18 (1.49 mmol, 550 mg) and benzylamine FABHRMS (NBA-CsI) m/e 581.0940 (M+ + Cs, C27H2806 requires (3.72 mmol, 380 mg, 2.5 equiv) in THF (3 mL) was stirred for 24 h at 58 1.0940). 25 "C and concentrated in vacuo. Chromatography (Si02, 1 X 12 cm, Benzyl 4,~O~nzyli~2-Oacetyl-~Obeslzyl-a-~-monnopy~ 50% EtOAc-hexane) gave 2,4,6-tri-Oacetyl-3-Ocarbamoyl-a-~-man-side (25). A solution of 24 (1.13 mmol, 510 mg) in pyridine ( 5 mL) was nopyranose (405 mg, 520 mg theoretical, 78%) as a colorless syrup: Rf treated with Ac2O (3.4 mmol, 346 mg, 3.0 equiv). The reaction mixture 0.25 (Si02,75% EtOAc-hexane): 'H NMR (CDCI,, 400 MHz) S 5.20was stirred at 25 "C for 4 h and poured into a two-phase solution of 5.40 (4H, m), 4.85 (2H, br s), 4.20-4.30 (2H, m), 4.15-4.20 (lH, m), EtOAc (15 mL) and H20 (8 mL) with vigorous stirring. The organic 2.15 (3H, s), 2.10 (3H, s), 2.07 (3H, s). layer was washed with 10% aqueous HCl(2 X 10 mL), saturated aqueous A solution of n-BuLi (1.8 mmol, 0.8 mL, 2.3 M in hexane) was added NaHCO3 (8 mL), and saturated aqueous NaCl(2 X 6 mL), dried (Na2to a stirred solution of 2,4,6-tri-Oacetyl-3-O-carbamoyl-a-~-mannopy- SO,), and concentrated in vacuo. Chromatography (Si02, 3 X 5 cm, 15% EtOAc-hexane) gave 25 (516 mg, 556 theoretical, 93%) as a syrup: ranose (1.49 mmol, 520 mg) in anhydrous THF (2.5 mL) at -78 "C. The resulting solution was stirred for 10 min at -78 "C before the addition Rf 0.76 (Si02, 60% EtOAc-hexane); [ a ] 2 S+29 ~ (c 0.55, CHCl3); 'H NMR(CDC13,400MHz) S7.20-7.60(15H,m), 5.64(1H,s),5.45 (lH, of diphenyl chlorophosphate (1.8 mmol, 0.37 mL, 483 mg). After stirring for 10 min at -78 OC, the reaction mixture was poured into a two-phase dd, J = 1.6, 2.8 Hz), 4.87 (lH, d, J = 1.6 Hz), 4.71 (2H, d, J = 12.0 Hz), 4.66 (lH, d, J = 12.0 Hz), 4.53 (lH, d, J = 12.0 Hz), 4.24 (lH, solution of EtOAc (8 mL) and saturated aqueous NaHCO3 (5 mL) with

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5654 J. Am. Chem. SOC.,Vol. 116, No. 13, 1994

Boger and Honda

dd, J = 2.8, 9.6 Hz), 4.054.10 (2H, m), 3.90 (lH, m), 3.85 (lH, dd, J s 9.6,IO.O Hz), 2.15 (3H, 8); IR (CHCl3) u- 3014,1728,1518,1422, 1371,121l,l041,922cm-'; FABHRMS (NBA-CsI) m/e 623.1050 (M+ Cs, C29H3007 requires 623.1046). Benzyl 2 - O A c e t y l - l O b e a z y l - a - ~ ~ p (26). y e A solution of 25 (0.81 mmol, 400 mg) in 80% HOAc-Hz0 (8 mL) was stirred for 1 h at 60 OC. The reaction mixture was poured into a two-phase solution of EtOAc (15 mL) and HzO (10 mL) with vigorousstirring. Theorganic layer was washed with saturated aqueous NaHCO3 (3 X 8 mL) and saturated aqueous NaCl (2 X 5 mL) and dried (NazSO4). Chromatography (SiOz,3 X 5 cm, 50% EtOAc-hexane) afforded 26 (270 mg, 325 mgthcoretical,83%)asasyrup:Rf 0.10(Si02,50%EtOAc-hexane);

127.7 (ZC), 127.5,98.6,76.7,73.3,72.7,70.9,70.1,69.7,63.3,20.9; IR (CHCl3) u- 3463,1734,1521,1420,1046,928,763 CIII-'; FABHRMS (NBA-CsI) m/e 535.0730 (M+ + Cs, CZZH2607 requires 535.0733). Benzyl 2 O A c e t y l - 3 , 4 , 6 M - O b e a z y l - 8 - ~ - g d e(30). A solution of 29 (1.78 mmol, 720 mg) in 30% CHzClrhexane ( 15 mL) was treated with benzyl trichloroacetimidate (17.8 mmol, 4.5 g, 10 equiv) and CF~SOIH(0.6 "01, 53 fiL, 90 mg, 0.3 equiv) at 25 OC. After 1 h at 25 OC, the reaction mixture was poured into a two-layer solution of EtOAc (15 mL) and saturated aqueous NaHCO3 (10 mL) with vigorous stimng. The organic layer was washed with saturated aqueous NaCl, dried (NazSOd), and concentrated in vacuo. Chromatography (SiOz, 3 X 10 cm, 5-1096 EtOAc-hexane gradient elution) afforded 30 (745 mg, [~]2S~+31.9(~0.19,CHCl~);'HNMR(CDCl~,400MH~)67.10-7.50 1.048 theoretical, 72%) as a syrup: Rf 0.48 (SiOz, 20% EtOAc-hexane); (lOH, m), 5.41 (1H, dd, J = 2.0,3.0 Hz), 4.91 (lH, d, J = 2.0 Hz), 4.74 [ a I z 5+70 ~ (C 0.13, CHCI3) (lit" [ a I Z 5+72.7 ~ (C 1.36, CHCl3)); 'H (lH, d, J = 12.0 Hz), 4.76 (lH, d, J = 12.0 Hz), 4.51 (lH, d, J = 12.0 NMR (CDCl3,400 MHz) 6 7.10-7.40 (2OH. m), 5.09 (lH, d, J = 3.2, Hz), 4.43 (lH, d, J = 12.0 Hz), 3.60-4.00 (SH, m), 2.11 (3H, m); IR 8.4 Hz). 4.95 (1H, d, J = 8.4 Hz),4.92 (lH, d, J = 12.0 Hz), 4.40-4.65 (CHC13)u,3450, 1728,1518,1472,1421,1376,1211,1041,928cm-~; (7H,m),4.16(1H,ddd,J= 12,6.4,6.4Hz),4.02(1H,dd,J=3.2,3.6 FABHRMS (NBA-CsI), m/e 535.0730 (M+ + Cs, CZZHZ607 requiea Hz).3.69(1H,dd,J= 6.5,9.6 Hz),3.64 (lH,dd,J= 6.4,9.6 Hz),3.53 535.0733). (lH, dd, J 1.2, 3.6 Hz), 2.01 (3H, 8); ' C NMR (CDCl3, 100 MHz) Benzyl 2 - O A c e t y l - l O b e a y l - d i o d o - 6 d ~ x y - a - ~ ~ ~6169.9,138.1,137.8(2C),137.6, ~ 128.8,128.4,128.3,128.2(2C),127.9 (27). A solution of 26 (4.9 mmol, 1.97 g), Ph3P (5.85 mmol, 1.53 g, 1.2 (2 C), 127.8, 127.7, 127.6, 127.5, 127.4, 97.7, 73.84, 73.75, 73.5, 73.3, equiv), and imidazole (7.4 mmol, 500 mg, 1.5 equiv) in toluene (30 mL) 72.5, 72.4, 71.1, 70.3, 68.8, 21.0; IR (CHC13) u- 1737, 1452, 1365, was treated with I2 (5.85 mmol, 1.48 g, 1.2 equiv) at 25 'C. The reaction 1230, 1077,910 cm-I; FABHRMS (NBA-CsI), m/e 715.1665 (M+ mixture was stirred for 3 hat 100 OC and poured intoa two-phase solution Cs, C&& requires 715.1672). of EtOAc (30mL) andsaturatedaqucousNaHCO3(20mL) withvigorous Benzyl 3,4,6-Tri-Obeayl-&bgulapymioside(31). A solution of 30 stirring. The organic layer was washed with saturated aqueous NaCl(2 (1.28 mmol, 745 mg) in CH3OH (30 mL) was treated with KzCO3 (0.86 X 10 mL) and dried (NazSO,). Chromatography (SiO2,3 X 8 cm, 17% mmol, 120 mg). The reaction mixture was stirred at 25 OC for 2 h and EtOAc-hexane) gave 27 (2.14 g, 2.52 g theoretical, 85%) as a syrup: poured intoa two-phasesolutionof EtOAc (40mL) andsaturatedaqueous Rf 0.45 (Si00 30% EtOAc-hexane); [ a I Z 5+21 ~ (c 1.07, CHCl3); IH NaCl (20 mL) with vigorous stirring. The organic layer was washed NMR (CDCl3,400 MHz) 6 7.20-7.50 (lOH, m), 5.40 (lH, dd, J = 1.8, with saturated aqueous NaCl (2 X 20 mL), dried (NazS04), and 3.2Hz),4.91 (lH,d,J=1.8Hz),4.84(lH,d,J= 12.0Hz),4.72(1H, concentrated in vacuo. Chromatography (SiOz, 3 X 7 cm, 10-15% d , J = 12.0Hz),4.57(1H,d,J= 12.0Hz),4.40(1H,d,J= 12.0Hz), EtOAc-hexane gradient elution) gave 31 (580 mg, 691 mg theoretical, 3.82 (lH, dd, J = 3.2,8.8 Hz), 3.70 (1H, ddd, J = 2.0,8.8,9.0 Hz), 3.60 84%) as a syrup, which could not be induced to crystallize: Rf 0.37 (2H, m), 3.32 (lH,dd,J= 8.0,9.0 Hz), 2.1 1 (3H, 9); W N M R (CDCl3, (sioz, 20% EtOAc-hexane); [ a I Z 5+49 ~ (C 0.12, CH&) (lit" [aIzS~ 100 MHz) 6 170.6, 137.8, 136.2, 128.6, 128.5, 128.4, 128.2, 128.17, +48.3 (~0.7,CHC13)); 'H NMR (CDC13,400 MHz) 6 7.10-7.40 (20H, 128.13,96.7,77.3,71.7,71.6,70.4,69.2,67.8,21.3,6.6;IR(CHCl~) Um), 4.95 (lH, d, J = 12.0 Hz), 4.72 (lH, d, J = 8.0 Hz), 4.40-4.60 (7H, 3410,1729,1518,1371,1248,121 1,1042,928 cm-I; FABHRMS (NBAm), 4.08 (IH, ddd,J= 1.2,6.4,6.4 Hz), 3.88 (lH,dd, J = 3.2,4.6 Hz), GI) m/e 644.9750 (M+ + Cs, C22Hz5061 requires 644.9750). 3.85 (lH, m), 3.69 (lH, dd, J = 6.4,9.6 Hz), 3.64 (lH, dd, J = 6.4,9.6 Benzyl 2 - ~ A c e t y l - l O b e a y l - 6 - d x ~ a - ~ ~ ~ x - 5 -Hz), ~ p y3.56 ~ -(lH, dd, J = 1.2, 3.6 Hz); I3C NMR (CDCl3, 100 MHz) 6 noside (28). A solution of 27 (13.6 mmol, 7.0 g) in pyridine (50 mL) 137.9,137.6,137.2,136.8,128.5,128.4 (4C), 128.3,128.2,128.1,127.9, was treated with AgF (68 mmol, 8.6 g, 5 equiv). The reaction mixture 127.8,127.7,127.6,100.0,75.9,73.6,73.44,73.36,72.8,72.3,70.6,69.5, was stirred at 25 OC for 3 h and poured into a two-phase solution of 68.8; IR (CHCI3) u,, 3405,1521,1441,1215,1092~m-~;FABHRMS EtOAc (100 mL) and Hz0 (50 mL) with vigorous stirring. The organic (NBA-CsI) m/e 673.1556 (M+ Cs, C34H3606 requires 673.1566). layer was washed with 10%aqueous HCI (3 X 40 mL), saturated aqueous 1.3,4,5-Tetra-Obenzyl-%O(54,dM-Oacetyl-3-OcrrbamoyI-a-~ NaHCO3 (30 mL), and saturated aqueous NaCl (2 X 30 mL), dried mrrmopyramsyl)-~-L-gulopyrrrnoside(2). A solution of 31 (0.07 mmol, (NazSOd), and concentrated in vacuo. Chromatography (SiOz,3 X 30 38 mg) and 19 (0.084 mmol,49 mg, 1.2 equiv) in dry CHzClz (1 mL) cm, 10% EtOAc-hexane) gave 28 (2.9 g, 5.2 g theoretical, 56%) as a was treated with TMSOTf (0.126 mmol, 25 pL, 30 mg, 1.8 equiv) at 0 - ' % . CHCl3); ~ syrup: Rf 0.37 (Sioz, 20% EtOAc-hexane); [ I X ] ~ ~ D(c0.3, OC. The reaction mixture was stirred at 0 OC for 10 min and poured into 1H NMR (CDCl3,400 MHz) Q 7.20-7.40 (lOH, m), 5.45 (lH, dd, J = a two-phase solution of EtOAc ( 5 mL) and saturated aqueous NaHCO3 1.8, 3.2 Hz), 4.95 (lH, d, J = 1.8 Hz), 4.91 (lH, dd, J = 1.0, 1.0 Hz), (3 mL) with vigorous stirring. The organic layer was washed with 4.84 (lH, d, J = 12.0 Hz), 4.77 (lH, dd, J = 1.0, 2.0 Hz), 4.71 (lH, saturated aqueous NaCl(2 X 2 mL), dried (NazSO4), and concentrated d , J = 12.0Hz),4.53(1H,d,J=12.0Hz),4.44(1H,d,J= 12.0Hz), invacuo. Chromatography (SiO2,l X lOcm, 25% EtOAc-hexane) gave 4.42(1H,m),3.83(1H,dd,J=3.2,10.0Hz),2.11 (3H,s);I3CNMR 2 (56 mg, 60 mg theoretical, 93%) as a syrup: Rf 0.38 (SiOz, 50% (CDCl3, 100 MHz) 6 170.6, 155.1, 137.3, 136.7, 128.6, 128.5, 128.2, EtOAc-hexane); [aIz5D+37.8 (c 0.12, CHCl3); 'H NMR (CDCI3,400 128.15, 128.12, 128.07,97.6,95.8,77.6,71.8,69.2,68.1,67.7,20.9;1R MHz) 6 7.10-7.50 (20H, m), 5.29 (1H, dd, J = 3.6,lO.O Hz), 5.23 (lH, (CHCl3) urmx 3430, 3018, 1724, 1519, 1422, 1211, 1045, 907 ~ m - l ; dd, J = 10.0, 10.0 Hz), 5.19 (lH, dd, J = 1.6, 3.6 Hz), 4.95 (lH, d, J FABHRMS (NBA-CsI) m/e 517.0630 (M+ + Cs, CZ2H2406 requires = 8.0 Hz), 4.93 (1H, d , J = 12.0 Hz), 4.76 (IH, d, J = 1.6 Hz), 4.30-4.70 5 17.0627). (7H, m), 4.30 (lH, m), 4.15 (lH, m), 3.94 (lH, dd, J = 3.2, 12.8 Hz), Benzyl 2-OAcetyl-lO-beayl-BcgulopyruK*ride (29). Catecholb 3.90 (lH, dd, J = 3.2.8.0 Hz), 3.80 (lH, dd, J = 3.2,3.2 Hz), 3.73 (lH, rane (8.72 mmol, 1.0 mL, 1.05 g, 4 equiv) was added to a mixture of 28 dd, J = 2.0, 14.8 Hz), 3.66 (lH, dd, J = 6.4, 12.8 Hz), 3.59 (lH, dd, (2.18 mmol, 840 mg) and Rh(PPhl),Cl (0.1 1 mmol, 106 mg, 0.05 equiv) J = 8.0,12.8 Hz), 3.52 (lH, dd, J = 1.2,3.2 Hz), 2.17 (3H, s), 2.02 (3H, in a solution of dry THF (18 mL) at -78 OC. The resulting solution was s), 1.98 (3H, 8); I3C NMR (CDCI,, 100 MHz) 6 170.6, 170.1, 169.9, warmed to 25 "C and stirred for 16 h. The reaction mixture was treated 155.0, 138.1, 137.7, 137.6, 137.4, 128.9 (2 C), 128.5, 128.44, 128.40, with 1:l EtOH-THF (4 mL), pH 7.0 buffer (4 mL, 0.05 M potassium 128.37, 127.28, 128.1, 128.0, 127.9, 127.7, 98.4, 94.3, 77.2, 74.2, 73.6, phosphate m o n o b a s i d i u m hydroxide), and then 50% aqueous H202 73.4,73.1,72.8,72.1,71.6,71.2,70.3,70.1,68.7,68.2,65.5,61.4,21.0, (4 mL) at 0 OC. The reaction mixture was stirred at 25 OC for 3 h and 20.8,20.7; IR (CHCl3) U- 3501,2957, 1743, 1602, 1363, 1263, 1230, poured into a two-phase solution of EtOAc (30 mL) and 30% aqueous 1091, 1047 cm-I; FABHRMS (NBA-CsI) m/e 1004.2433 (M+ Cs, NaHSO3 (20 mL) with vigorousstirring. The organic layer was washed C47H53N015 requires 1004.2470). with saturated aqueous NaHCO3 (20 mL) and saturated aqueous NaCl, 1,3,~dTetra-Oacetyl-2-0(2,4,6M-Oacetyl-lQc~rb.moyl-a-~ dried (NaSOd), and concentrated in vacuo. Chromatography (SiOz, 3 "opyraoosyl)-&L-gubpyraooside (33). A solution of 2 (0.61 "01, X 15 cm, 2540% EtOAc-hexanegradient elution) gave 29 (720mg, 878 530 mg) in CH3OH (40 mL) was hydrogenated over 10% Pd(0H)z-C mg theoretical, 82%) as a syrup: Rf 0.21 (SiOz, 60% EtOAc-hexane); (100 mg) under an atmosphere of H2 (1 atm) at 25 'C for 15 h. The [aIuD +75.9 (c 0.20, CHCl3); 'H NMR (CDCI,, 400 MHz) 6 7.10-7.40 reaction mixture was filtered through a Celite pad and concentrated in (lOH, m), 5.18 (lH, dd, J = 2.8.8.0 Hz), 5.00 (lH, d, J = 8.0 Hz), 4.91 vacuo. ( l H , d , J = 12.0Hz),4.66(1H,d,J= 12.0Hz),4.63(1H,d,J= 12.0 The residue containing 32 was dissolved in pyridine (20 mL) and Hz), 4.60 (lH, d, J = 12.0 Hz), 3.80-4.00 (SH, m), 2.03 (3H, s); 13C NMR (CDCl3, 100 MHz) 6 169.9, 137.8, 137.5, 128.4, 128.3, 127.9, treated with AczO (80 mmol, 810 mg, 10 equiv). The reaction mixture

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5656 J. Am. Chem. Soc., Vol. 116, No. 13, 1994

Boger and Honda

Rf0.33 (SiOz, 10% CH3OH-CHC13); [a]”D -1.7 (c 0.18, CHCl3); IH evaporated in vacuo to give an oily solid. The samplewas triturated with NMR (CDCl3.400 MHz) 6 8.02 (lH, s), 7.43 (lH, e), 6.00 (lH, d, J CHCl3 (2 X 0.5 mL). Chromatography (reverse-phase C-18, &lo% -8.OHz),5.05-5.30(5H,m),5.02(lH,d,J=1.6Hz),4.88(1H,m). CH3OH-H2Ogradient elution) afforded42(1.4 mg, 1.47 mg theoretical, 4.43 (lH, ddd, J = 1.2, 5.2, 5.2 Hz), 4.38 (lH, dd, J = 5.2, 12.8 Hz), 95%) as a thin film: Rf0.15 (SiOz, 109:l CH30H-l0% aqueous CHI4.16 (lH, m), 4.10 (lH, dd, J = 1.2, 12.8 Hz), 4.05 (lH, dd, J 3.6, COzNH4-lo% aquaws N&OH); [a]% +10 (c 0.03, CHIOH); lH 3.6 Hz), 4.01 (lH, m), 3.90 (lH, m), 3.70 (3H, I), 2.23 (3H, s), 2.15 NMR (CD,OD, 400 MHz) 6 8.22 (lH, s), 8.12 (lH, s), 7.67 (lH, br s), 7.13 (1H, br s), 4.60-5.00 (5H, m), 4.29 (lH, d, J = 4.5 Hz), 3.30(3, s), 2.12 (3H, s), 2.09 (3H, s), 2.08 (3H. s), 2.03 (3H, s), 1.62 (9H, e), 1.45 (9H, 8); IR (CHCl3) V- 3455, 3025, 1743, 1712, 1517, 1425, 4.20(22H, m), 2.93 (6H,s), 2.40 (lH,m), 2.16 (2H, t t , J = 7.0,7.0Hz), 1.15 (9H, m); IR (neat) phu 3333,1738,1707,1671,1548,1384,1133, 1220,1046,928 cm-l; FABHRMS (NBA-CsI) m/e 1137.2670 (M+ 1061 cm-’; FABHRMS (NBA) m/e 1107.3877 (M+, C ~ ~ H ~ N I O O I ~cs, S ~c42&0N4ou requires 1137.2652). rquires 1107.3797). ~84l~~~-aaccty1-2-0(54,6-~-01ecty~l0arbrmoyl14Aaido-[342’-(2””’-( (N(~”(R)-.~~”-Y’(S)-~Y&OXY-~”(S)-~a - ~ ~ p ~ y l ) - a - L - ~Methyl ~ yEster l (48). ~ L - ~ ~ y l p e a ~ y l ) - ~ - ~ y l ) ~ ) e ~ ) - 5The4compound ’ - ~ 47~ (0.004 ~ ~mmol, ~ ~4.2 mg) was treated with 20% TFAp y ~ ~ i m e t h y ~ o nrodidel-Wim (Mphenyhethyl) eryt/uw&[ 2”’-0 CHzClz (0.5mL), and the mixture was stirred at 0 OC for 2 h under Ar. (3””- ~carbamoy~-a-~-mannopyranosyl)-a-~-gnlopyr~n~yl]-~-~sThesolvent wasevaporatedinvacuotogiveanoilysolid. Chromatography tidhe (43). Compound 41 (0.003 “01.4.4 mg) was treated with 1 N (SiOz, 10-2046 CH~OH-CHZCI~ gradient elution) afforded 48 (2.6 mg, HCl in 90% aqueous HOAc (1.O mL), and the mixture was stirred at 25 3.3 mg theoretical, 78%) as a thin film: Rf0.35 (Si02,20% CHIOHOC for 15 min under Ar. The solvent was maporated in vacuo to give CHCI3); ‘H NMR (CDsOD, 400 MHz) 6 7.69 (1H, d, J 1.O Hz), 7.22 a solid. Chromatography (reversephase C-18,0-40% CH~OH-HZO (lH, d, J = 1.0 Hz), 5.24 (lH, dd, J = 3.6, 3.6 Hz), 5.21 (lH, d, J = gradient elution) afforded 43 (3.3 mg, 4.0 mg theoretical 83%) as a thin 8.0 Hz), 5.10 (2H, m), 5.07 (lH, dd, J = 3.2, 10.0 Hz), 4.98 (lH, d, J film: 4 0 . 2 0 (SiOz, 109:l CH,OH-10% aqueous CH3COzN&-lo% = 1.6 Hz), 4.90 (lH, dd, J = 1.2, 3.6 Hz), 4.47 (lH, d, J = 8.0 Hz), aqueous NH40H); [0rl2’D + 14.2 (c 0.02, CHIOH); ‘H NMR (CDIOD, 4.31 (lH, ddd, J = 1.2,6.4, 6.4 Hz), 4.15 (lH, dd, J = 6.4, 12.8 Hz), 400 MHz) 6 8.20 (lH, s), 8.1 1 (lH, s), 7.46 (lH, br s), 7.10-7.40 (15H. 4.07 (lH, dd, J = 1.2, 12.8 Hz), 4.05 (lH, dd, J = 3.6, 3.6 Hz), 4.05 m),7.05(1H,brs),4.80-5.10(5H,m),4.29(1H,d,J=4.3Hz),3.00- (lH, m), 3.90 (lH, dd, J = 6.0, 12.8 Hz), 3.72 (lH, dd, J = 7.0, 12.8 4.20 (22H, m), 2.93 (6H.s). 2.59 (lH, m),2.15 (2H, m), 1.15-1.30 (9H, Hz), 3.71 (3H, s), 2.07 (3H,s), 2.03 (3H,s), 1.99 (3H, s), 1.98 (3H, s), m); IR (neat) v,, 3410, 1700, 1652, 1480, 1210 cm-I; FABHRMS 1.97 (3H, s), 1.88 (3H, 8). (NBA) m/e 1350.4920 (M+ + H, Cs2H8lNloOlaS3 requires 1350.4971).

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-

w-((tart-Btlty~xy)erubonul)-l\b-[l-a~3(S)-[~~6-[8~d~

h b . ( ( I r r f - B U f y l O x y ) e l ) - W - ( ~ ~ Y ~ l ) ~ ~ A 1 ( 4 6 ) .W -( M p h e n y l m e t h y l ) - e r ~ ~ 83,4,6-M-[ &cetyl-2- 0 (2,4,6-M- 0

A solution of 44 (0.002 “01, 0.9 me) in DMF (20 pL) was treated 1.6 mg, 3.0 quiv), HOBt (0.002 sequentially with DCC (0.006 ”01, “01, 0.33 mg, 1.0 quiv), and 43 (0.002 “01, 2.8 mg, 1.0 equiv) dissolved in DMF (20 pL), and the mixture was stirred at 25 OC for 48 h under Ar. The crude mixture was dissolved in CH3OH (1 mL), and the insoluble inorganic salts were removed by centrifugation. The CHIOH solution was evaporated and the sample was triturated with CHCl3 (2 X 0.3 mL). Thesolid was purified by chromatography (reverse-phase C-18,10-20% CHaOH-HzO gradient elution) to afford 46 (2.9 mg, 3.5 mg theoretical, 83%)as a thin film: Rf 0.46 (SiOz, 109:l CH3OH-10% aqueous CH3COzNH4-10% aqueous N&OH); [a]25n-16.7 (c 0.01. CHIOH); ‘H NMR (CD3OD. 400 MHz) 6 8.20 (lH, s), 8.08 (lH, s), 7.00-7.40 (17H, m), 4.80-5.10 (5H, m), 4.20 (lH, m), 3.00-4.10 (26H, m), 2.93 (6H, s), 2.50-2.80 (5H, m), 2.28 (3H, s), 2.15 (2H. m), 1.42 (9H,s), 1.02-1.14 (9H,m); IR (neat) v-3430, 1705,1640,1480,1210 cm-I; FABMS (NBA) m/e 1757 (M+, C ~ ~ H I & J I ~ O ~ B ~ ) . Bleomych As (1). The compound 46 (0.0016 mmol, 2.8 mg) was treated with 20% TFA-CHzClz (0.5mL), and the mixture was stirred at 0 OC for 2.5 h under Ar before the solvent was evaporated in vacuo to give an oily solid. The sample was triturated with CHCl3 (0.2 mL), and the remaining solid was purified by preparative TLC (SiOz, CH3OH-10% aqueous CH3C02N&lO%aqueousN&OH, 1091) to afford 1(1.1 mg, 2.2 mg theoretical, 50%) as a thin film: Rf 0.43 (SiOz, 10:9 1 CH30H-10% aqueous CH3C@N&lO% a q u m N h O H ) ; [ a ] z ~ - l6 (C 0.03, 0.1 N HCl); ’H NMR (D20.400 MHz) 6 8.07 (lH, s), 7.87 (lH, s), 7.68 (lH, s), 7.10 (lH, s), 5.08 (2H, m), 4.87 (lH, d , J = 3.6

acetyl-%O e r r b r m o y l - a - D . n u n w p ~ y l ) - a - c p u l o p y r r tiayl methyl e - l ~ m ~ f l ( @ - B - -

Amide (50). A solution of 44 (0.0074 mmol,3.2 mg) in DMF (100 pL) was treated with 37 (0.0074 mmol, 7.7 me), HOBt (0.0090 mmol, 1.2 mg, 1.1 equiv), and EDCI (0.0090, 1.75 mg, 1.1 equiv), and the mixture was stirred under Ar at 25 *C for 48 h. The reaction mixture was concentrated in vacuo to give an oily solid. Chromatography (SiOz, 0.5 X 5 cm, 10-2096 CHJOH-CHCI~gradient elution) afforded 50 (6.2 mg, 10.7 mg theoretical, 58%) as a foam: RfO.18 (SiOz, 20% CH3OHCHCl3); [a]”~-13.8 (c0.13, CH3OH); IH NMR (CDsOD, 400 MHz) d 7.47 (8, lH), 7.00-7.50 (m,16H), 5.10-5.40 (m, 7H), 4.80 (m, 2H), 3.80-4.30 (m, 7H), 3.80 (5, 3H). 3.62 (m, 2H), 2.80 (m, 2H), 2.60 (m, lH), 2.45 (m, lH), 2.26 (s,3H), 2.10 (s,3H), 2.08 (s,3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.85 (s, 3H), 1.42 (s, 9H); IR (neat) v,, 3453,1762,1492, 1418, 1210 cm-l; FABHRMS (NBA) m/e 1454.5620 (M+ + H, CaH83NllO25 rquircs 1454.5640). ~-((tertButyloxy)~nyl)-~-[l-a~3(@-[~~qa~~ +( Mpkoylmethyl)-eqtbn+fl~2-O(%Of.rbrmoyI-a-~rrmnnopynnosyl)-a-~-gubpyrmosyll-L-histidyl Methyl E s t e r ~ y r h i d h - 2 - y l ~ p i o n %Y~I-(s)-B-~Amide (51). A solution of 50 (0.0026 “01, 3.7mg)inCH3OH(0.5mL) wascooledatOOCandtreatedwithaqueous 0.1 N NaOH (0.5 mL, 0.005 mmol, 20 quiv), and the reaction mixture was stirred at 0 OC for 12 h. After evaporation of the CH3OH under a Nzstream, the aqueous phase was neutralized to pH 7 with the addition of HOAc, and the mixture was concentrated under a Nz stream. Hz),4.78(1H,d,J=1.8Hz),4.63(1H,m),3.98(1H,d,J=4.5Hz), Chromatography (reversephase C-18, &SO% CH~OH-HZOgradient elution) afforded 51 (1.5 mg, 3.0 mg theoretical, 50%) as a foam: Rj 3.20-3.90 (22H, m), 3.10 (lH,m),2.80 (lH, m), 2.75 (6H,s), 2.50-2.70 0.21 (Si02, 4 l : l t-BuOH-H~O-HOAc); [aIuD +33.3 (C 0.16, CHI(4H, m), 2.40 (1H. m), 2.10 (2H, m), 1.90 (3H, s), 0.97 (3H. d, J = 6.0 OH); IH NMR (CDsOD, 400 MHz) d 6.90-7.40 (m, 17H), 4.80-5.30 Hz), 0.95 (3H, d, J = 6.0 Hz), 0.93 (3H, d, J = 6.0 Hz); IR (neat) u,, (m,5H),3.30-4.30(m,13H),2.80(m,2H),2.60(m,lH),2.35(m,lH), 3341,1723,1658,1406,1082,777cm-I; FABMS (NBA) m/e 1415 (M+, 2.31 (s, 3H), 1.43 (s,9H); IR (neat) v,, 3367, 1730, 1685, 1631,1509, CSSHE~NI~O~S~). 1395, 1200 cm-l; FABHRMS (NBA) m/e 1188.4815 (M+ H, Alternatively, the crude bleomycin A2 could be purified by reverse C55H~9N11019requires 1188.4849). phase chromatography (C- 18,0-20% CH~OH-HZOgradient elution) with greater rccovery (95%) but of a slightly lower purity. ~ P - ( ( t e t t - B n t y h x y ) a r b o n y l ) - W - ((t&butybxy)chomyl)Acknowkdgme!nt. We gratefully acknowledge the financial 8-[3,4,6-M-~.cetyl-2-0( 54,CM-08cetyl-3-Oerub.moyl-a-D.m;m- support of the National Institutes of Health (Grant CA42056) nopy”yl)-a-L-gubpymwyl~L-lWdineMethylEsta (47). A solution and the award of a Glaxo fellowship(T.H., 1993-94). We thank of 4 (0.007 mmol, 8.2 me) in CH3OH (2 mL) was hydrogenated over Q.Dang for conductingpreliminarystudiesof the Rh(I)-catalyzed 10% Pd-C (6 me) under Hz (1 atm) in the prcsencc of di-ferf-butyl hydroboration reaction (Table 1, entries 14), Dr. S.L. Colletti dicarbonate(O.O7mmol,15mg,lOequiv)at25OCfor 17h. Thereaction for supplies of 36,40,and 44, and Dr. T. W. Doyle (Bristolmixture was filtered through a Celite pad and concentrated in vacuo. Myers Squibb) for samplesof blenoxane from which an authentic gradient Chromatography (Si02, 1 X 10 cm, 1.5-2% CH~OH-CHZC~Z sample of bleomycin A2 was secured. elution) afforded 47 (4.2 mg, 7.0 mg theoretical, 60%) as a thin film:

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