Synthesis of Cytospolide Analogues and Late-State Diversification

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Synthesis of Cytospolide Analogues and Late-State Diversification Thereof Gunnar Ehrlich, and Christian B. W. Stark J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02999 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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The Journal of Organic Chemistry

Synthesis of Cytospolide Analogues and Late-State Diversification Thereof Gunnar Ehrlich* and Christian B. W. Stark* Department of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany [email protected] and [email protected] Table of Content/Abstract Graphic OH O

HO

OH O

HO O

R OH HO

O O

cytospolide D (natural product)

R

O

N N N

O

OH modified synthesis

O HO

O

Late-Stage Diversification

O

N N N R

cytospolide D analogue

R

HO OH

O

O

OH OH O

O O

O

O O

R

ABSTRACT The cytospolides are a novel group of fungal secondary metabolites first described in 2011. Although all 17 natural derivatives share the same C-14 polyketide backbone, they exhibit a fairly broad structural diversity regarding their oxygenation and acetylation pattern as well as macrolide structure, e.g. monocyclic nonanolide core or bicyclic ring systems with a bridging THF ring. In the present work, the prospects for an extension of the structural diversity of cytospolides have been investigated. Based on a previously established synthesis of cytospolide D, a modified route to a truncated analogue carrying an alkyne instead of the natural n-pentyl side chain has been developed. In a bioinspired approach the so-derived cytospolide D alkyne analogue has been further converted to bicyclic and THF ring containing derivatives with a different backbone architecture. Finally, Sonogashira couplings or Huisgen-Sharpless click reactions have been used for late-stage diversifications. Thus, a set 1

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of 15 novel and structurally divers natural product derivatives has been synthesized in a highly efficient manner. INTRODUCTION The group of cytospolide natural products A–Q (1–17) has been isolated by Zhang et al. from extracts of the endophytic fungus Cytospora sp. found on the evergreen shrub Ilex canariensis on Gomera Island.1,2 Structurally, this family of polyketides can be divided into monocyclic and bicyclic lactones. Cytospolides A–D (1–4) share the same nonanolide skeleton,3 but differ in the acetylation pattern of their hydroxy groups, while cytospolide E (5) is the C-2 epimer of cytospolide D (4) (Figure 1). The cytospolide members F–L (6–12) possess an additional hydroxy group in the pentyl side-chain and also show a different acetylation pattern. Cytospolides M–Q (13–17) can be regarded as higher oxygenated congeners of cytospolide D (4), J (10) and E (5). Cytospolides M (13) and N (14) display a bicyclic structure with a THF ring and are most likely derived through epoxidation of cytospolide D (4) and J (10), respectively, followed by transannular epoxide opening (Scheme 1).4 Cytospolide O (15) and P (16) are presumably the result of an oxa-Michael addition to the respective enones of D (4)

R1 O

R2

O

O

HO O

R4

O

R3

R1

R2

R3

OAc OH OAc OH OH OAc OAc OH OH OH OH

OH OAc OAc OH OH OH OH OH OH H H

H H H H OH OH OAc OAc H OH OAc

O O

R

cytospolide E (5)

cytospolides A-D and F-L

A (1) B (2) C (3) D (4) F (6) G (7) H (8) I (9) J (10) K (11) L (12)

OH OH

OH

cytospolides M and N M (13) R = H N (14) R = OAc

R4 H H H H H H H H OAc H H

O O

O O

cytospolide O (15) OAc O HO

O O

cytospolide P (16)

HO O O O

OH cytospolide Q (17)

Figure 1: Structures of cytospolides A–Q (1–17) 2


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and E (5) (structures of putative enone intermediates not shown in Figure 1; cf. Scheme 1). The -lactone Q (17) can be traced back to cytospolide M (13) by an intramolecular transesterification. Thus, cytospolide D (4) can be regarded as the key branching point of the biosynthesis and the main source for structural diversity in this class of natural products. Scheme 1: Supposed Biosynthetic Formation of Cytospolides O, M, and Q from Cytospolide D4 OH O

HO

O

oxidation to enone

O

HO O

O

intramolecular oxa-Michael addition

O

O O

O

cytospolide D (4)

cytospolide O (15)

19

epoxidation

O OH HO

8

O O 18

transannular epoxide opening

OH OH O

O

intramolecular transesterification

HO

O cytospolide M (13)

O O

O OH

cytospolide Q (17)

The biological activity of cytospolides is strongly dependent on their substitution pattern and stereochemistry. The absolute configuration at C-2 has a significant influence on their activity. While all of the monocyclic (2R) configured cytospolides are almost inactive against human cancer cell lines, the (2S) epimers E (5) and P (16) were shown to exhibit potent activity against A549, QGY, and U937 tumor cell lines.2 They induce an arrest of tumor cells in the G1 phase of the cell cycle. In the (2R) series on the other hand, bioactivity only arises when the 10-membered ring system is converted to a more rigid or less strained bicyclic architecture. Accordingly, THF derivatives cytospolide M (13) and N (14) as well as the lactone cytospolide Q (17) are cytotoxic with moderate activity against the A549 cell line. This bioactivity profile together with certain structural features, especially the 10-membered macrolactone3 with an (E) configured double bond, are some of the reasons why the cytospolides have attracted significant synthetic effort including some successful total syntheses.4-13 During our previous research in this area, we developed a synthesis of cytospolide D (4) and demonstrated its (putatively) biomimetic conversion to its more highly oxygenated congeners cytospolide M (13), O (15), and Q (17) (Scheme 1).4 However, the accessibility of cytospolide D (4) was somewhat limited due to the low yield of 21% for the crucial macrolactonization step.14,15 As part of our ongoing research on biologically active natural 3



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products16 and analogues thereof,17 we decided to investigate synthetic access to structurally diverse artificial derivatives of cytospolides. For this first generation of analogues, we focused on modifications of the side-chain to introduce hydrophilic functional groups or lipophilic residues or to modulate the steric demand. In addition, bioinspired late-stage scaffold modifications as well as synthetic access to natural and unnatural C-2 epimers was in the focus of this program. RESULTS AND DISCUSSION Given the broad structural diversity of the higher oxygenated cytospolides M (13), O (15) and Q (17) and their simple and straightforward access from cytospolide D (4)4 (Scheme 1), we were aiming for a synthesis of a modified cytospolide D analogue as a key compound to expand the structural diversity of this group of natural products. Ideally, a synthetic route would allow for modifications of the cytospolide D (4) backbone at a very late stage and in addition provide access to C-2 epimeric structures. Moreover, the synthesis should be arranged in a modular fashion so that the steps of modifications and oxidative conversion of cytospolide D analogues to analogues of cytospolide M, O and Q could be iteratively altered. For possible modifications of cytospolide D (4) we identified the pentyl side-chain as well as the alkenyl group (Scheme 2). In cytospolides generally the side-chain is either unsubstituted or hydroxylated in  or -1 position. A variation in the hydroxylation pattern or the chain length, however, is not easily feasible in case of an aliphatic chain and its modification would be required at an early stage of the synthesis. Instead, a truncated alkyne side-chain appeared useful as a handle for further functionalization by standard cross-coupling or cycloaddition reactions such as Sonogashira couplings18 or Sharpless-Huisgen click reactions19. Its reactivity can be regarded orthogonal to the endo-cyclic alkene moiety of the macrolide, so these reaction conditions are not affecting the alkene, while epoxidation of the olefin is not expected to affect the alkynyl side-chain. In addition, (terminal) alkynes can also be used in bio-conjugate chemistry20 such as approaches to identify cellular targets of natural products.21 Therefore, cytospolide alkyne derivatives themselves represent interesting target compounds. Finally, we presumed that the low steric demand of the ethynyl group and the higher reactivity of the propargylic alcohol might also favorably influence the macrolactonization. Based on this reasoning, we decided to replace the unbranched pentyl side-chain by an ethynyl moiety (Scheme 2).

4


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For modifications of the endo-cyclic alkene moiety, the oxygenation and biomimetic conversion of cytospolide D (4) to M (13), O (15), and Q (17) may serve as an inspiration (Scheme 1).4 For instance, instead of opening the epoxide by the C-8 OH group, leading to the cytospolide M skeleton, external nucleophiles might be probed for epoxide-opening. Similarly, after oxidation of the allylic alcohol, enone 25 might serve as a substrate for conjugate additions (Scheme 2). The intramolecular oxa-Michael addition of the free C-8 hydroxy group again would give rise to cytospolide O structures, but the addition of external nucleophiles might open up the way to e.g. C-2 epimers of cytospolide P analogues. As a prerequisite, all of these modifications should be practicable at a late stage of the synthesis. The preparation of cytospolide E analogues, on the other hand, would require an earlier amendment of the strategy in order to install the (2S)-configuration. In this case, a syn-aldol addition providing access to seco-acid 22 would be needed rather than the anti-aldol reaction (producing epimeric 23) of our original synthesis.4 Both approaches converge to the same ,-unsaturated aldehyde precursor 21 as the common starting material which in turn should be accessible from known chiral C5 aldehyde 204 (Scheme 2). Scheme 2: Strategy for Structural Modifications of Cytospolide D Backbone C-2 epimers

Positions for structural modifications conjugate addition (to the enone) OH HO

epoxidation, epoxide-opening

O

synthesis of C-2 epimers

O

TBSO O

cytospolide D (4) (natural product)

introduction of modified side-chains

O

O H PMBO 20

Wittig olefination

OPiv

H 21

Grignard addition

(2S)

OTBS

OH

22

OPMB

HO

modified hydroxy acids

OPMB

O

syn- or antialdol addition

OTBS

OH

23

OPMB

(2R)

HO

lactonization

Late-stage diversification OH

O HO

O

HO

O

O 25

O

cytospolide D analogue 24

conjugate addition cross coupling or cycloaddition epoxidation, epoxide-opening

Having identified cytospolide D analogue 24 and its hydroxy acid precursor 23 as intermediary target structures, we next modified our previously established synthetic route 5



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accordingly (Scheme 3). Thus, starting from -chiral aldehyde 204, installation of the truncated ethynyl side-chain was the first step. This Grignard addition proceeded smoothly and in a good yield of 87%, but with a low dr of 2:1 in favor of the undesired syndiastereomer 26b (Scheme 3). When pentylmagnesium bromide in diethyl ether was used during the total synthesis of natural cytospolide D (4)4, the diastereoselectivity was fully governed by the Cram-chelat pathway22 yielding the syn-diol with a dr > 95:5. As ethynylmagnesium bromide in contrast is not completely soluble in diethyl ether, THF had to be used which led to a decreased chelation and in turn a low dr of 2:1. However, both diastereomers were separable after repeated column chromatography and could be converted to the same stereochemically pure protected isomer 27. To this end, anti-diol 26a was directly protected as pivalate, while the syn-diol 26b was converted to the same pivalate via Mitsunobu esterification.23 As a protecting group for the propargylic alcohol we used the pivalate ester rather than an acetate4 as the latter underwent rapid acyl migration during the subsequent TBS deprotection (TBAF, THF). As expected, no acyl walk was observed for the pivalate ester when the proximal TBS ether was removed under standard conditions. The resulting primary alcohol 27 was then oxidized to aldehyde 58 using Dess-Martin periodinane.24 For the ensuing Wittig homologation of 58 to the ,-unsaturated aldehyde 21, the use of (triphenylphosphoranylidene)-acetaldehyde25 was not successful. Due to the low reactivity of this stabilized ylide prolonged heating up to 50°C for several days was necessary, during which significant decomposition occurred. Instead, reaction of 58 with ethyl (triphenylphosphoranylidene)-acetate yielded ester 28 in a good yield. This ester 28 was next reduced (DiBAl-H) in the presence of the pivalate protecting group to the allylic alcohol 59 which was finally re-oxidized to the ,-unsaturated aldehyde 21. For the subsequent aldol reaction, two diastereodivergent protocols are feasible. An anti-aldol reaction would set up the correct (2R,3R) configuration for the cytospolide D skeleton, while a syn-aldol reaction would open up the way to cytospolide E analogues. For the anti-aldol reaction we decided to make use of the well-established Paterson method26 which provided the desired addition product 30 in 81% yield (dr > 95:5; Scheme 3). The C-3 hydroxy group of aldol product 30 was protected as TBS ether. Next, the chiral auxiliary was removed by a sequence of ketone reduction and methanolysis of both the benzoate as well as the pivalate ester. Methanolysis of the benzoate proceeded rapidly but the cleavage of the pivalate required up to 3 days reaction time but eventually yielded 75% of the desired triol 6


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intermediate. Glycol cleavage27 of the vicinal diol moiety and oxidation of the resulting aldehyde using sodium chlorite28,29 furnished hydroxy acid 23, which was finally exposed to TBAF mediated TBS deprotection to produce dihydroxy acid 31 in 83% yield. Scheme 3: Synthesis of Epimeric Hydroxy Acids 22 and 23 and Dihydroxy Acids 31 and 34 O TBSO

H

OH

HC CMgBr, THF, 0°C, 1 h, then rt, 16 h

PMBO

(S)

TBSO

1) PivCl, iPr2NEt, DMAP, CH2Cl2, rt, 16 h HO 2) TBAF, THF, rt

(R)

PMBO

87%

83%

(5R) 26a

20

OPiv PMBO 27

1) PPh3, DEAD, PivOH, Et2O, 0°C, 2 h, then rt, 16 h

dr (26a : 26b) = 1:2 (5S) 26b

2) TBAF, THF, rt, 16 h 71%

1) Dess-Martin periodinane, CH2Cl2, rt

O

2) Ph3P=CHCO2Et, CH2Cl2, rt, 16 h

O

29

O

1) DiBAl-H, THF, -78°C, 3 h, 95% 2) Dess-Martin periodinane, CH2Cl2, rt, 30 min, 94%

PMBO

i) Cy2BCl, Me2NEt, Et2O, -78°C 30 min, BzO then 0°C 4 h

OPiv

H

28

80%

BzO

OPiv

EtO

PMBO 21

O

OH

ii) 21, -78°C 15 min, then -20°C for 16 h 81%

OPiv PMBO 30

1) TBSCl, imidazole, CH2Cl2, rt, 16 h, 88% 2) i) NaBH4, MeOH, ii) NaOMe, 16 h, 75%

O

OTBS

OH

(2R)

HO

3) NaIO4 on silica, CH2Cl2, rt, 4 h 4) NaClO2, 2-Me-2-butene, t BuOH, sat. NaH2PO4, rt, 30 min, 88%

PMBO 23 TBAF, THF, rt, 5 h 83% O

OH

OH

(2R)

HO

PMBO 31

O O 32

O N Bn

i) Bu2BOTf, Et3N, CH2Cl2, -78°C, 15 min, then 0°C, 30 min ii) 21, -78°C, 15 min, then 0°C, 1 h 93%

O O

O

OH

OPiv

N PMBO Bn

1) TBSCl, imidazole, CH2Cl2, rt, 16 h, 95% 2) LiOH, THF/H2O2, rt, 3 d, 81%

O

OTBS

OH

(2S)

HO

PMBO

33

22 TBAF, THF, rt, 16 h 79% O HO

OH

OH

(2S)

PMBO 34

For the synthesis of the (2S) isomer, common aldehyde 21 was subjected to an Evans aldol reaction.30 Again, the C-3 hydroxy group was protected as TBS ether (61) and the chiral auxiliary was removed by hydrolysis using LiOH in H2O2/THF. The hydrolysis of the oxazolidinone moiety proceeded rapidly, but hydrolysis of the pivalate again was sluggish and required 3 days reaction time. However, an acceptable overall yield of 81% was achieved. 7



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When the unprotected aldol product 33 was subjected to LiOH in H2O2/THF, a considerable proportion of retro-aldol processes was observed. Altogether, the yields and the number of steps for the synthesis of the novel truncated open-chain cytospolide analogues are similar to those of our previous synthesis of the hydroxy acid carrying the natural n-pentyl side-chain. Having synthesized both C-2 epimeric hydroxy acids 23 and 22, we next investigated the crucial macrolactonization.14 During our previous synthesis of cytospolide D (4) it turned out that with a protecting group on C-3 OH, no 10-membered macrolide was formed. Only with a free hydroxy group at C-3 and using Shiina´s reagent,31 the desired macrolide was obtained in 21% yield.4 For the modified hydroxy acid 23 we observed a similar outcome (Scheme 4). Thus, lactonization of 23 carrying a TBS protecting group at C-3, no 10-membered macrolide could

be

isolated

irrespective

of

the

lactonization

method,

i.e.

Yamaguchi

macrolactonization32 or Shiina’s method31. In contrast, when the dihydroxy acid 31 was used as a starting material, the desired macrolide 35 was formed in a good yield of 50% and sidereactions such as dehydrative decarboxylation4 through intermediary -lactones were not observed. The increased yield for the lactonization step, compared to 21% of the cytospolide D synthesis, may be attributed to the significantly lower steric hindrance of the propargylic

Scheme 4: Macrolactonization of seco-Acids to Form 10-membered Macrolides O

OTBS

OH

method A Yamaguchi lactonization

HO 23

O

OH

OPMB

Shiina lactonization

OH

HO 31

O

OTBS

OPMB

OH

HO 22

O

OH

MNBA, DMAP, toluene, add. over 12 h, then 6 h at rt 50%

OH

OH PMBO

O

DDQ, CH2Cl2/ pH 6.7 buffer, rt 95%

OH O

HO

O 35

MNBA, DMAP, toluene, add. over 8 h, then 16 h at rt

OPMB

HO 34

no macrolide, only dimeric and polymeric structures

method B

no macrolide, only dimeric and polymeric structures

O 24

NO2 O

O

NO2

O

MNBA, DMAP, toluene, add. over 8 h, then 6 h at rt

no macrolides

2-methyl-6-nitrobenzoic anhydride (MNBA)

OPMB

8


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alcohol and its higher reactivity. However, the epimeric hydroxy acids 22 and 34 showed a different behavior under these conditions. Despite substantial effort, neither the TBS protected hydroxy acid 22 nor the dihydroxy acid 34 gave any reasonable amount of the desired 10membered lactone. Hence, access to the cytospolide E backbone proved not successful following this synthetic strategy. Similar to our findings during the synthesis of cytospolide D (4)4, the PMB protected macrolide 35 showed a second set of NMR signals of about 10% intensity. This set of signals is not caused by impurities and is largely independent from the nature of the side-chain (npentyl or ethynyl). When macrolactone 35 was subjected to PMB deprotection with DDQ33 the truncated cytospolide D analogue 24 was isolated in 95% yield (Scheme 4). Again, NMR analysis of ethynyl-derivative 24 showed the presence of a second set of signals although at lower intensity of about 5%. For both compounds 35 and 24, this second set of signals may be attributed to the presence of a minor conformer. Likewise, in the original publication by Zhang et al.1 DFT-calculations of a simplified cytospolide A (1) analogue (C-9 methyl instead of C-9 pentyl) in CHCl3 solution also revealed the existence of a group of three structurally very similar conformers with a major conformer (87.7%) and two minor conformers populated with about 2%. With the core structure of cytospolide D analogues (24) in hand, we next focused on its derivatization (Scheme 5). In principle, the Sonogashira reaction18 allows for the coupling of the terminal alkyne with bromo/iodoarenes or -alkenes. In these cases, the alkynyl side-chain would be elongated with larger lipophilic substituents. However, the so-obtained eneyne or areneyne motifs are conformationally very rigid and the extended -system may influence the stability of the propargylic lactone. Initially, optimal conditions for Sonogashira couplings were investigated. Due to the insolubility of the starting material 24 in a mixture of toluene and iPr2NEt, CH2Cl2 was used instead of the commonly employed toluene. When 24 was reacted with iodobenzene (36) or p-iodonitrobenzene (37) in the presence of 0.5 eq. CuI and Pd(PPh3)2Cl2, the reaction proceeded within a few hours at 35°C. In both cases, formation of a more polar byproduct was observed, the structure of which could not be elucidated. The yields of the desired coupling products 39 and 40 are in the same range with around 30%. The Sonogashira coupling of 24 with an electron-rich iodoarene such as p-iodophenol lead to a very low yield of the coupling product and the reaction with 1,2-dibromoethene did not yield any coupling product at all. When allyl bromide (38) was used as organohalide, the coupling 9



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reaction proceeded more rapidly and the yield of coupling product 41 was significantly higher with 52%. It is worth noting that compound 41 is a hexadehydro-analogue of cytospolide D (4). Now the unfunctionalized native n-pentyl side chain is polyunsaturated with plenty of options for further modifications. In all Sonogashira couplings, formation of the unknown polar side product was observed. It appears that its production increased steadily over time and longer reaction times required in case of iodoarenes lead to an increased amount of this degradation product. Moreover, we found that derivatives 39–41 are sensitive in solution and slow decomposition was observed within a couple of days. In an attempt to couple 24 with (E)-4-iodo-3-buten-1-ol (49) the obtained enyne was even too unstable for further purification. On the other hand, parent macrolide 24 is well stable in CDCl3 even when left at room temperature for extended periods of time. Scheme 5: Late-Stage Derivatization of 24 OH

OH Pd(PPh3)2Cl2, CuI, 36-38

O

HO

i

Pr2NEt, CH2Cl2 35°C, 2-4 h

O 24

O R

Br

I 36

O2N

O

HO

38 I

39 R =

32%

37

40 R =

NO2 31%

41 R =

52%

OH Cu(OAc)2, Na2S2O4, 42-44 24

HO

MeOH/H2O, rt, 16 h N3

N3

42

CO2t Bu 43

44

R

O N N N

O

45 R =

37%

46 R =

CO2t Bu 43%

47 R =

26%

N3

Next, the incorporation of 1,2,3-triazoles at the position of the side-chain by a SharplessHuisgen click reaction was investigated.19 Substituted 1,2,3-triazoles are often used in drug design to mimic the electronic properties of an amide group.34 With regard to cytospolide D analogue 24, formation of a 1,2,3-triazole would resemble the introduction of a carboxamide at position C-9. A further substitution of the triazole with lipohilic or hydrophilic residues can 10


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easily be achieved by varying the organic azide. In particular, triazole 46 with a tertbutyloxycarbonylmethylene substituent may be regarded as an amide mimic of the “cytospolide D C-9 carboxylic acid” and Gly-OtBu. When 24 was subjected to azides 42–44, the corresponding triazoles 45–47 were isolated in moderate yields of about 30–40% (Scheme 5). Having established a route to a first set of cytospolide D analogues with an intact 10membered lactone ring, we next explored the conversion of key intermediate 24 into the bicyclic cytospolide M analogue 48 (Scheme 6). Thus, epoxidation of 24 and transannular epoxide opening under slightly acidic conditions produced oxa-bicycle 48 in a very good yield of 90% over two steps. In order to further broaden the structural diversity, we also tried to open the epoxide with nucleophiles other than the internal C-8 OH group. To prevent the transannular epoxide opening by C-8 OH, the PMB protected macrolide 35 was epoxidized under identical conditions as for the unprotected derivative 24 (data not shown). For the following epoxide opening, basic reaction conditions were deemed to also provoke lactone opening. Therefore, methanol and a small amount of CSA were added aiming for an intermolecular epoxide opening under acidic conditions. However, even with the PMB protected C-8 OH again an intramolecular reaction occurred with concomitant loss of the PMB-group. Bicycle 48 was obtained as the sole product. This result underscores a proximity driven strong preference for the transannular epoxide opening even in the presence of a large excess of external nucleophiles. It has however to be noted that a different protecting group at C-8 OH may well prevent intramolecular attack of the epoxide and allow for the introduction of external nucleophiles in future studies. Next, modification of the ethynyl side-chain of bicyclic cytospolide M analogue was exemplary demonstrated by Sonogashira coupling of 48 with allyl bromide (38) and (E)-4-iodo-3-buten-1-ol (49). In both cases the yield of 70% was significantly higher compared to the coupling using 24 as the starting material. It seems that even though a 9-membered lactone is still a highly strained compound, relieving the strain of the (E) double bond and closing the THF ring increased the stability of the propargylic lactone favorably so that even with the enyne side-chain in 51 no degradation was observed. We then explored the biomimetic transesterification of bicyclic [6.2.1]-lactones 50 and 51 to cytospolide Q analogues with a neighboring butenolide-THF-system. Both lactones were opened with (CH3)3SiOK35 followed by addition of a small amount of water and CSA to support closure of the -lactone ring. In both cases, butyrolactones 52 and 53 were isolated in 11



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low yields accompanied by another compound, which is assumed to be the C-2 epimer. Again, allylated compounds 50 and 52 are hexadehydro-derivatives of cytospolide M (14) and Q (17), respectively. Scheme 6: Late-Stage Derivatizations of Bicyclic Cytospolides OH HO

O

mCPBA, CH2Cl2, -15°C, 16 h,

OH OH O

O

then CSA, -15°C, 1 h 90%

O

O

i

O

Pr2NEt, CH2Cl2, 35°C, 1-2 h

O 48

24

OH OH

Pd(PPh3)2Cl2, CuI, 38, 49

O R

Br 38 I

50 R =

OH 49

70%

51 R =

OH 70%

(CH3)3SiOK, THF, rt, 30 min, then CSA, H2O, rt, 3-4 h R

HO

52 R =

O O

OH

53 R =

28% OH 25%

O

OH PMBO

O

Dess-Martin periodinane

35

CH2Cl2, rt, 30 min 78%

O

O PMBO

O O

1) DDQ, CH2Cl2/ pH 6.7 buffer, rt, 67%

73% PMBO

O

O

2) CSA, CH2Cl2, rt, 3 d, 60%

54

CH2(CO2Et)2, KOtBu, THF, rt, 1h

O

O 56

Cu(OAc)2, Na2S2O4, t BuO2CCH2N3 (43) MeOH/H2O, rt, 16 h 61%

O O

O

O N N N 57 CO2tBu

CH(CO2Et)2 O O O

dr 1:1

55

For the final part of the present project, the conjugate addition of external nucleophiles to the enone moiety was studied (Scheme 6). After oxidation of the PMB protected macrolide 35, the resulting enone 54 was subjected to a malonate addition. Initially, we were assuming that the conformation of the macrocyclic ring would at least partially shield one hemisphere of the double bond leading to a preferential attack of the nucleophile from the exo-face of the macrocycle.36 However, although the malonate addition proceeded in a good yield of 73%, Michael product 55 was obtained with a dr of 1:1 and both diastereomers proved to be inseparable by column chromatography. Similarly, the reaction of 55 with ammonia only provided an inseparable 1:1 mixture of diastereomers (not shown in Scheme 6). The absence of any diastereoselectivity, and correspondingly the absence of macrocyclic stereocontrol,36 12


Page 13 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


suggests a more or less in-plane orientation of the double bond in macrolide 54. As the addition of external nucleophiles proved to be rather unselective, the intramolecular conjugate addition was next investigated. Deprotection of the PMB ether under buffered standard conditions proceeded with moderate efficiency. The ensuing transannular oxa-Michael addition was supported by addition of CSA and provided the bicyclic -ketolactone 56 in 60% yield after 3 days reaction time at room temperature. Finally, the late-stage functionalization of the ethynyl moiety was exemplary shown by cycloaddition of tert-butyl azidoacetate (43). Triazole 57 was isolated in a good yield of 61%. CONCLUSION In summary, we were able to synthesize a first set of analogues of the cytospolide group of natural products. The initial idea to introduce a truncated alkyne side-chain as precursor for further derivatizations proved effective. Fully functionalized seco-acid intermediates (22, 23, 31, and 34) with their 5 stereoelements were available in only 10-13 steps and excellent overall yields of up to 37% (from known aldehyde 204). Key steps involved a Mitsunobu inversion, a Wittig homologation and a Paterson or Evans aldol reaction, respectively. The macrolactonization results for 22, 23, 31, and 34 were similar to previous findings from our total synthesis of cytospolide D (4).4 The TBS protected hydroxy acids 22 and 23 provided no 10-membered macrolide. Only with a free C-3 OH group in dihydroxy acid 31 the formation of macrolide 35 was observed. It is worth noting that the propargylic alcohol also favorably affected the yield of the macrolactonization. The significantly better yield of 50% compared to 21% for the dihydroxy acid carrying the natural n-pentyl side-chain can be explained by its higher reactivity and reduced steric hindrance. In addition, the cyclisation reaction is now quite robust regarding the addition rate of the dihydroxy acid and side-reactions such as dehydrative decarboxylation leading to dienes have not been observed. Unfortunately, the cyclisation of the C-2 epimeric dihydroxy acid 34 proved unsuccessful so the attempted generation of the cytospolide E backbone failed. Similar to the proposed biosynthesis, skeletal diversity could be effected using transannular cyclizations (intramolecular epoxide opening or Michael addition) or ring rearrangements. The use of external nucleophiles to open an intermediary epoxide or add to an enone intermediate were somewhat less effective. Finally, the alkyne moiety could be modified at a very late stage of the synthesis using Sonogashira cross-coupling reactions or azide cycloadditions. Moderate to good yields were obtained in these reactions, in part depending 13



Page 14 of 42

on ring strain and stability of both the stating material and the product. Best results were achieved for compounds with a [6.2.1]-oxabicyclic core structure related to cytospolides M and O. In this manner, a set of 15 densely functionalized cytospolide analogues were synthesized. The same reaction principle as exemplified in this account may be used to produce a larger library of structurally highly divers analogues and investigate their pharmacological potential in some more detail.

14


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EXPERIMENTAL SECTION General Techniques. All reagents were used as purchased from commercial suppliers. Solvents were purified by conventional methods prior to use. Reactions were monitored by thin layer chromatography using Machery-Nagel pre-coated TLC-sheets ALUGRAM® Xtra SIL G/UV254 and visualized with potassium permanganate [(2.4 g KMnO4, 16 g K2CO3, 4 mL NaOH (5 %), 196 mL H2O)] or ceric ammonium molybdate [(phosphomolybdic acid (5 g), Ce(SO4)2·2 H2O (2 g), H2SO4 conc (12 mL), H2O (188 mL)]. Chromatographic purification was performed as flash chromatography on Fluka silica gel 60 (particle size 0.040-0.063 mm). Yields refer to chromatographically purified and spectroscopically pure compounds. NMR spectra were recorded on a Bruker AV-400 (operating at 400 MHz for 1H and 101 MHz for 13C

acquisitions), a Bruker AV-500 (operating at 500 MHz for 1H and 126 MHz for

13C

acquisitions) or a Bruker AV-600 (operating at 600 MHz for 1H and 151 MHz for

13C

acquisitions). Chemical shifts  are reported in ppm with the solvent resonance as the internal standard: chloroform-d1: 7.26 (1H-NMR), 77.16 (13C-NMR); methanol-d4: 3.31 (1H-NMR), 49.00 (13C-NMR); acetone-d6: 2.05 (1H-NMR), 206.26 (13C-NMR). Coupling constants J are given in Hertz (Hz). Multiplicities are classified as follows: s = singlet, d = doublet, t = triplet, q = quartet, qui = quintet and combinations thereof, or m = multiplet or br = broad signal. Two-dimensional NMR (H−COSY, HSQC, HMBC) were used for the assignment of all resonance signals. For simplicity, the numbering of the carbon atoms of a given structure does not necessarily follow IUPAC rules. The chosen numbering system for each compound can be found in the SI. High resolution mass spectra were obtained on an Agilent 6224 ESI-TOF. IR spectra were recorded on a Bruker ALPHA FT-IR Platinum ATR. Wave numbers ṽ are reported in reciprocal centimeters (cm-1). Optical rotation data were measured with a Krüss Optronic P8000 at 598 nm using a 100 mm path-length cell in the solvent, at the concentration and temperature indicated. Melting Points were measured with a Büchi Melting Point M-565 and are uncorrected. All compounds were named according to IUPAC rules. (3R,4S)-7-(tert-Butyldimethylsilyl)oxy-3-hydroxy-4-(p-methoxybenzyl)oxy-hept-1-yne (26a) and

(3S,4S)-7-(tert-Butyldimethylsilyl)oxy-3-hydroxy-4-(p-methoxybenzyl)oxy-hept-1-yne

(26b) To a solution of ethynylmagnesium bromide (1.2 eq, 16.4 mL, 8.19 mmol, 0.5 M in THF) at 0°C was added a solution of aldehyde 2037 (2.40 g, 6.83 mmol) in THF (13 mL) over 15 min. After complete addition, the solution was stirred for 1 h at 0°C and then allowed to warm to room temperature overnight. Sat. NH4Cl-solution was added until the magnesium 15



Page 16 of 42

hydroxide was dissolved. The organic layer was separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Column chromatography (silica gel, hexanes/EtOAc 3:1) yielded the diastereomeric alcohols 26a and 26b (2.24 g, 5.92 mmol, 87%) as a colorless oil in a 1:2 mixture. Repeated column chromatography (silica gel, toluene/EtOAc 8:1) allowed for almost complete separation of both diastereomers (1.48 g of 26b, 0.64 g of 26a and 0.11 g of a mixed fraction). Analytical data for 26a: []23D = -5.1 (c 1.10, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.6 Hz, 2H, H-10), 6.88 (d, J = 8.7 Hz, 2H, H-11), 4.60 (d, J = 11.3 Hz, 1H, H8a), 4.56 (d, J = 11.3 Hz, 1H, H-8b), 4.47 (dd, J = 3.7, 2.2 Hz, 1H, H-3), 3.80 (s, 3H, H-13), 3.61 (t, J = 6.3 Hz, 2H, H-7), 3.54 (ddd, J = 7.3, 5.6, 3.7 Hz, 1H, H-4), 2.46 (d, J = 2.2 Hz, 1H, H-1), 1.78–1.72 (m, 2H, H-5), 1.69–1.62 (m, 1H, H-6a), 1.54 (ddq, J = 12.9, 9.5, 6.4 Hz, 1H, H-6b), 0.89 (s, 9H, (CH3)3CSi), 0.04 (s, 6H, (CH3)2Si); 13C{1H} NMR (151 MHz, CDCl3) δ 159.5 (C-12), 130.2 (C-9), 129.7 (C-10), 114.0 (C-11), 82.0 (C-2), 80.8 (C-4), 74.5 (C-1), 72.2 (C-8), 64.0 (C-3), 63.1 (C-7), 55.4 (C-13), 28.9 (C-6), 26.5 (C-5), 26.1 ((CH3)3CSi), 18.5 ((CH3)3CSi), -5.2 ((CH3)2Si) ppm; IR (ATR) ṽ 3436 (w, br), 3308 (w, br), 2952 (m), 2928 (m), 2883 (m), 2856 (m), 1612 (m), 1513 (s), 1463 (m), 1301 (m), 1246 (ss), 1173 (ss, br), 1034 (ss) cm-1; HRMS (ESI) m/z calcd for C21H34NaO4Si+ [M+Na]+ 401.2119, found 401.2127. Analytical data for 26b: []19D = +6.2 (c 1.17, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 8.6 Hz, 2H, H-10), 6.88 (d, J = 8.6 Hz, 2H, H-11), 4.65 (d, J = 11.0 Hz, 1H, H-8a), 4.61 (d, J = 11.0 Hz, 1H, H-8b), 4.31 (dd, J = 5.6, 2.2 Hz, 1H, H-3), 3.80 (s, 3H, H-13), 3.61 (t, J = 6.1 Hz, 2H, H-7), 3.58–3.55 (m, 1H, H-4), 2.65 (s, br, 1H, OH), 2.47 (d, J = 2.2 Hz, 1H, H-1), 1.82–1.72 (m, 1H, H-6a), 1.71–1.51 (m, 3H, H-6b, H-7), 0.90 (s, 9H, (CH3)3CSi), 0.05 (s, 6H, (CH3)2Si); 13C{1H} NMR (126 MHz, CDCl3) δ 159.5 (C-12), 130.3 (C-9), 129.8 (C-10), 114.0 (C-11), 83.0 (C-2), 81.4 (C-4), 74.0 (C-1), 72.9 (C-8), 64.5 (C-3), 63.1 (C-7), 55.4 (C-13), 28.5 (C-5), 27.4 (C-6), 26.1 ((CH3)3CSi), 18.5 ((CH3)3CSi), -5.2 ((CH3)2Si) ppm; IR (ATR) ṽ 3428 (w, br), 3308 (w), 2952 (m), 2929 (m), 2884 (w), 2856 (m), 1612 (m), 1513 (s), 1463 (m), 1246 (s), 1173 (m), 1069 (ss), 1034 (s) cm-1. HRMS (ESI) m/z calcd for C21H34NaO4Si+ [M+Na]+ 401.2119, found 401.2122. (4S,5R)-4-(p-Methoxybenzyl)oxy-5-pivaloyloxyhep-6-yn-1-ol (27) From 26a: To a solution of alcohol 26a (0.68 g, 1.80 mmol) in CH2Cl2 (15 mL) was added ethyldiisopropylamine (3 eq, 0.92 mL, 0.70 g, 5.40 mmol), pivaloyl chloride (2 eq, 0.44 mL, 0.44 g, 3.60 mmol) and DMAP (0.1 eq, 22 mg, 0.18 mmol). After stirring overnight at room temperature, sat. 16


Page 17 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NaHCO3-solution was added to the brownish solution and stirred for 10 min. The organic layer was separated and the aqueous layer was extracted twice with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The brownish crude product was purified by column chromatography (silica gel, hexanes/EtOAc 10:1) to give the TBS protected pivalate as a colorless oil (0.79 g, 1.70 mmol, 95%). TBS deprotection: The TBS ether was dissolved in THF (10 mL) and TBAF-solution (1.2 eq, 2.0 mL, 2.0 mmol, 1.0 M in THF) was added. After complete conversion, water and EtOAc were added. The organic layer was separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. After purification by column chromatography (silica gel, hexanes/EtOAc 2:1) alcohol 27 was obtained as a colorless oil (0.52 g, 1.50 mmol, 88%, 83% from 26a). From 26b: To a solution of alcohol 26b (1.52 g, 4.02 mmol) in diethyl ether (25 mL) at 0°C were added triphenylphosphine (1.0 eq, 1.05 g, 4.02 mmol), pivalic acid (1.0 eq, 0.41 g, 4.02 mmol) and a solution of DEAD (1.0 eq, 4.02 mmol, 1.74 ml, 40% in toluene). After being stirred at 0°C for 2 h, the same amount of reagents was added again and the turbid reaction suspension was stirred overnight and allowed to warm to room temperature. Then, the suspension was filtered through a pad of silica gel, which was thoroughly rinsed with diethyl ether. The filtrate was concentrated in vacuo and purified by column chromatography (hexanes/EtOAc 20:1). The TBS protected pivalate was obtained as a colorless oil (1.60 g, ca. 3.4 mmol), but still contained some minor impurities. This slightly impure material was directly subjected to the subsequent deprotection step. TBS deprotection: The TBS ether was dissolved in THF (20 mL) and TBAF-solution (1.2 eq, 4.2 mL, 4.2 mmol, 1.0 M in THF) was added. After stirring overnight, water and EtOAc were added. The organic layer was separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. After column chromatography (silica gel, hexanes/EtOAc 2:1) alcohol 27 was isolated as a colorless oil (1.00 g, 2.86 mmol, 71% from 26b): []22D = -69.3 (c 1.15, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.26 (d, J = 8.6 Hz, 2H; H-10), 6.86 (d, J = 8.6 Hz, 2H, H-11), 5.66 (dd, J = 3.3, 2.3 Hz, 1H, H-5), 4.72 (d, J = 10.9 Hz, 1H, H-8a), 4.41 (d, J = 10.9 Hz, 1H, H-8b), 3.79 (s, 3H, H-13), 3.63–3.58 (m, 3H, H-1, H-4), 2.47 (d, J = 2.3 Hz, 1H, H-7), 1.87– 1.56 (m, 6H, H-5, H-6, OH, H2O), 1.23 (s, 9H, H-16);

13C{1H}

NMR (101 MHz, CDCl3) δ

177.4 (C-14), 159.5 (C-12), 130.0 (C-9), 129.9 (C-10), 113.9 (C-11), 79.6 (C-4), 79.1 (C-6), 17



Page 18 of 42

75.1 (C-7), 72.2 (C-8), 64.6 (C-5), 62.7 (C-1), 55.4 (C-13), 39.0 (C-15), 29.1 (C-6), 27.2 (C5), 27.2 (C-16) ppm; IR (ATR) ṽ 3412 (w, br), 3282 (w), 2956 (m), 2935 (m), 1730 (s), 1612 (m), 1513 (s), 1478 (m), 1460 (m), 1278 (m), 1246 (ss), 1144 (ss), 1054 (s), 1030 (ss) cm-1; HRMS (ESI) m/z calcd for C20H28NaO5+ [M+Na]+ 371.1829, found 371.1834. (4S,5R)-4-(p-Methoxybenzyl)oxy-5-pivaloyloxyhep-6-ynal (58) To a solution of alcohol 27 (0.997 g, 2.861 mmol) in CH2Cl2 (20 mL) was added Dess-Martin periodinane (1.3 eq, 1.58 g, 3.72 mmol). The solution became rapidly turbid and stirring was continued until full conversion was monitored by TLC. The suspension was concentrated in vacuo at room temperature and the residue was suspended in CH2Cl2 and filtered through a plug of silica, which was repeatedly rinsed with CH2Cl2. The filtrate was concentrated again to a residual volume of about 10 mL and the solution of the crude aldehyde 58 was directly used for the subsequent Wittig reaction. Analytical data for a purified sample of 58 (silica gel, hexanes/EtOAc 3:1): []21D = -80.8 (c 1.09, CHCl3); 1H NMR (400 MHz, CDCl3) δ 9.67 (t, J = 1.5 Hz, 1H, H-1), 7.23 (d, J = 8.6 Hz, 2H, H-10), 6.86 (d, J = 8.7 Hz, 2H, H-11), 5.67 (dd, J = 3.3, 2.3 Hz, 1H, H-5), 4.68 (d, J = 10.9 Hz, 1H, H-8a), 4.34 (d, J = 10.9 Hz, 1H, H-8b), 3.79 (s, 3H, H-13), 3.61 (dt, J = 9.6, 3.3 Hz, 1H, H-4), 2.57–2.50 (m, 1H, H-2a), 2.49 (d, J = 2.3 Hz, 1H, H-7), 2.48–2.40 (m, 1H, H-2b, 2.07 (dtd, J = 14.4, 7.2, 3.3 Hz, 1H, H-3a), 1.95 (ddt, J = 14.3, 9.6, 7.0 Hz, 1H, H-3b), 1.23 (s, 9H, H-16);

13C{1H}

NMR (101 MHz, CDCl3) δ

202.0 (C-1), 177.2 (C-14), 159.5 (C-12), 130.0 (C-10), 129.8 (C-9), 113.9 (C-11), 78.8 (C-6), 78.5 (C-4), 75.3 (C-7), 72.1 (C-8), 64.0 (C-5), 55.4 (C-13), 40.2 (C-2), 39.0 (C-15), 27.2 (C16), 23.3 (C-3) ppm; IR (ATR) ṽ 3278 (w), 2968 (m), 2936 (w), 2872 (w), 2837 (w), 2724 (w), 1725 (ss), 1612 (m), 1513 (s), 1478 (m), 1461 (m), 1277 (m), 1246 (s), 1142 (ss), 1031 (ss) cm-1; HRMS (ESI) m/z calcd for C20H26NaO5+ [M+Na]+ 369.1672, found 369.1675. Ethyl (3E,6S,7R)-6-(p-methoxybenzyl)oxy-7-pivaloyloxynon-3-en-8-ynoate (28) To the solution of the crude aldehyde 58 in CH2Cl2 (ca. 10 mL) was added ethyl (triphenylphosphoranylidene)-acetate (2 eq calc. for 27, 2.0 g, 5.72 mmol) and the reaction mixture was stirred overnight. The solution was then directly concentrated in vacuo and the residue was purified by column chromatography (silica gel, hexanes/EtOAc 8:1) to give ester 28 as a colorless oil (0.953 g, 2.288 mmol, 80% from 27): []21D = -68.5 (c 1.11, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.24 (d, J = 8.5 Hz, 2H, H-12), 6.90 (dt, J = 7.4, 6.5 Hz, 1H, H-3), 6.87 (d, J = 8.5 Hz, 2H, H-13), 5.77 (dt, J = 15.7, 1.6 Hz, 1H, H-2), 5.66 (dd, J = 3.2, 2.3 Hz, 1H, H-7), 4.70 (d, J = 11.0 Hz, 1H, H-10a), 4.37 (d, J = 11.0 Hz, 1H, H-10b), 4.17 (q, J = 7.2 18


Page 19 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hz, 2H, H-19), 3.80 (s, 3H, H-15), 3.57 (dt, J = 7.9, 3.6 Hz, 1H, H-6), 2.48 (d, J = 2.2 Hz, 1H, H-9), 2.37 (ddtd, J = 14.6, 8.1, 6.2, 1.7 Hz, 1H, H-4a), 2.23–2.15 (m, 1H, H-4b), 1.86–1.75 (m, 2H, H-5), 1.28 (t, J = 7.2 Hz, 3H, H-20), 1.23 (s, 9H, H-18); 13C{1H} NMR (151 MHz, CDCl3) δ 177.3 (C-16), 166.7 (C-1), 159.5 (C-14), 148.3 (C-3), 130.0 (C-11), 129.9 (C-12), 121.9 (C-2), 113.9 (C-13), 79.0 (C-8), 78.7 (C-6), 75.2 (C-9), 72.2 (C-10), 64.3 (C-7), 60.3 (C-19), 55.4 (C-15), 39.0 (C-17), 29.0 (C-5), 28.4 (C-4), 27.2 (C-18), 14.4 (C-20) ppm; IR (ATR) ṽ 3279 (w), 2975 (m), 2935 (w), 2872 (w), 2837 (w), 1717 (ss), 1653 (m), 1612 (m), 1513 (s), 1478 (m), 1462 (m), 1367 (m), 1247 (ss), 1204 (m), 1142 (ss), 1096 (w), 1032 (ss) cm-1; HRMS (ESI) m/z calcd for C24H32NaO6+ [M+Na]+ 439.2091, found 439.2094. (3E,6S,7R)-6-(p-Methoxybenzyl)oxy-7-pivaloyloxynon-3-en-8-yn-1-ol (59) Ester 28 (0.878 g, 2.108 mmol) was dissolved in THF (10 mL) and cooled to -78°C with dry ice/acetone. Then, a solution of DiBAl-H (2.5 eq, 4.4 mL, 5.27 mmol, 1.2 M in toluene) was added over 15 min. As full conversion was not reached after stirring for 2 h at -78°C, additional reagent (DiBAl-H-solution 1.2 M in toluene, 0.5 mL, 0.6 mmol) was added. After stirring for an additional 1 h at -78°C, acetone (1 mL) was added and the solution was warmed up to 0°C. Water was added and the biphasic mixture was vigorously stirred until precipitation of aluminum hydroxide started. Then, a few drops of hydrochloric acid were added until the aluminum hydroxide was dissolved. The organic layer was separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, hexanes/EtOAc 2:1) yielded alcohol 59 as a colorless oil (0.749 g, 2.000 mmol, 95%): []19D = -65.1 (c 1.22, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.26 (d, J = 8.7 Hz, 2H, H-12), 6.87 (d, J = 8.6 Hz, 2H, H-13), 5.66–5.56 (m, 2H, H-2, H-3), 4.70 (d, J = 11.1 Hz, 1H, H-10a), 4.40 (d, J = 11.1 Hz, 1H, H-10b), 4.05 (d, J = 4.0 Hz; 2H, H-1), 3.80 (s, 3H, H-15), 3.58 (td, J = 6.3, 3.3 Hz, 1H, H-6), 2.46 (d, J = 2.2 Hz, 1H, H-9), 2.28–2.19 (m, 1H, H-4a), 2.12–2.01 (m, 1H, H-4b), 1.79–1.73 (m, 2H, H-5), 1.47 (s, br, 1H, OH), 1.23 (s, 9H, H-18);

13C{1H}

NMR (126 MHz, CDCl3) δ 177.4 (C-16), 159.5 (C-14), 132.2 (C-3), 130.3 (C-11), 129.9 (C2), 129.8 (C-12), 113.9 (C-13), 79.1 (C-8), 78.8 (C-6), 75.0 (C-9), 72.2 (C-10), 64.8 (C-7), 63.8 (C-1), 55.4 (C-15), 39.0 (C-17), 30.2 (C-5), 28.4 (C-4), 27.2 (C-18) ppm; IR (ATR) ṽ 3340 (br, w), 3286 (w), 2935 (m), 2871 (w), 1731 (s), 1612 (m), 1513 (s), 1478 (m), 1461 (m), 1278 (m), 1246 (ss), 1143 (ss), 1110 (m), 1089 (m), 1031 (ss), 970 (s) cm-1; HRMS (ESI) m/z calcd for C22H30NaO5+ [M+Na]+ 397.1985, found 397.1990. 19



Page 20 of 42

(3E,6S,7R)-6-(p-Methoxybenzyl)oxy-7-pivaloyloxynon-3-en-8-ynal (21) Alcohol 59 (0.735 g, 1.963 mmol) was dissolved in CH2Cl2 (20 mL) and Dess-Martin periodinane (1.3 eq, 1.08 g, 2.55 mmol) was added. The solution became rapidly turbid and full conversion was determined after 30 min by TLC. The suspension was directly concentrated at rt and subjected to column chromatography (silica gel, hexanes/EtOAc 4:1). Aldehyde 21 was obtained as a colorless oil (0.684 g, 1.836 mmol, 94%): []20D = -72.6 (c 0.98, CHCl3); 1H NMR (500 MHz, CDCl3) δ 9.45 (d, J = 7.8 Hz, 1H, H-1), 7.23 (d, J = 8.5 Hz, 2H, H-12), 6.87 (d, J = 8.5 Hz, 2H, H-13), 6.75 (dt, J = 15.6, 6.6 Hz, 1H, H-3), 6.05 (ddt, J = 15.7, 7.8, 1.6 Hz, 1H, H-2), 5.69 (dd, J = 3.1, 2.3 Hz, 1H, H-7), 4.71 (d, J = 11.0 Hz, 1H, H-10a), 4.36 (d, J = 11.1 Hz, 1H, H-10b), 3.80 (s, 3H, H-15), 3.58 (dt, J = 9.0, 3.4 Hz, 1H, H-6), 2.53–2.45 (m, 1H, H-4a) 2.49 (d, J = 2.2 Hz, 1H, H-9), 2.34 (ddtd, J = 15.6, 8.6, 7.1, 1.4 Hz, 1H, H-4b), 1.93–1.83 (m, 2H, H-5), 1.24 (s, 9H, H-18); 13C{1H} NMR (126 MHz, CDCl3) δ 194.0 (C-1), 177.3 (C-16), 159.6 (C-14), 157.8 (C-3), 133.3 (C-2), 130.0 (C-12), 129.8 (C-11), 114.0 (C-13), 78.9 (C-8), 78.5 (C-6), 75.3 (C-9), 72.1 (C-10), 64.1 (C-7), 55.4 (C-15), 39.0 (C-17), 29.0 (C-4), 28.8 (C5), 27.2 (C-17) ppm; IR (ATR) ṽ 3272 (w), 2959 (m), 2934 (m), 2871 (w), 2737 (w), 1732 (s), 1686 (ss), 1612 (m), 1513 (s), 1478 (m), 1461 (m), 1277 (m), 1247 (ss), 1138 (ss), 1031 (ss), 975 (s) cm-1; HRMS (ESI) m/z calcd for C22H28NaO5+ [M+Na]+ 395.1829, found 395.1831. (2S,4R,5R,6E,10S,11R)-2-Benzoyloxy-5-hydroxy-10-(p-methoxybenzyl)oxy-3-methyl-11pivaloyloxytridec-6-en-12-yn-3-one (30) (2S)-2-Benzoyloxy-3-pentanone38 (29) (1.5 eq, 0.570 g, 2.764 mmol) was dissolved in diethyl ether (25 mL) and cooled to -78°C. Then chlorodicyclohexylborane (1.5 eq, 2.8 mL, 2.8 mmol, 1 M in hexanes) and Nethyldimethylamine (2.5 eq, 0.50 mL, 0.34g, 4.63 mmol) were added. After stirring at -78°C for 30 min, the solution was stirred for further 4 h at 0°C. During this time, large amounts of a white precipitate were formed. Then the suspension was again cooled to -78°C and a solution of aldehyde 29 (0.684 g, 1.836 mmol) in diethyl ether (5 mL) was added. The reaction mixture was kept at -78°C for 30 min and then stored at -20°C overnight. After this time water was added at 0°C and the biphasic solution was stirred for 15 min. The organic layer was separated and the aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. During a first chromatographic separation process (hexanes/EtOAc 4:1) excessive ketone 29 could be reisolated. Asecond column chromatography (silica gel, hexanes/EtOAc 3:1) gave aldol product 30 as a sticky, highly viscous residue (0.862 g, 1.490 mmol, 81%): []19D = -25.7 (c 1.22, 20


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CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.08 (d, J = 6.8 Hz, 2H, H-26), 7.58 (tt, J = 7.4, 1.3 Hz, 1H, H-28), 7.46 (t, J = 7.8 Hz, 2H, H-27), 7.26 (d, J = 8.6 Hz, 2H, H-16), 6.87 (d, J = 8.5 Hz, 2H, H-17), 5.67–5.60 (m, 2H, H-6, H-11), 5.43 (q, J = 7.1 Hz, 1H, H-2), 5.40 (dd, J = 15.5, 7.5 Hz, 1H, H-7), 4.70 (d, J = 11.1 Hz, 1H, H-14a), 4.39 (d, J = 11.1 Hz, 1H, H-14b), 4.19 (t, J = 7.9 Hz, 1H, H-5), 3.79 (s, 3H, H-19), 3.56 (ddd, J = 7.9, 4.9, 3.2 Hz, 1H, H-10), 2.87 (qui, J = 7.3 Hz, 1H, H-4), 2.46 (d, J = 2.3 Hz, 1H, H-13), 2.29–2.16 (m, 2H, H-8a, OH), 2.09-2.01 (m, 1H, H-8b), 1.78–1.72 (m, 2H, H-9), 1.56 (d, J = 7.0 Hz, 3H, H-1), 1.23 (s, 9H, H-22), 1.14 (d, J = 7.2 Hz, 3H, H-23); 13C{1H} NMR (126 MHz, CDCl3) δ 211.2 (C-3), 177.3 (C-20), 166.0 (C-24), 159.5 (C-18), 133.5 (C-6), 133.4 (C-28), 131.1 (C-7), 130.2 (C-25), 129.9 (C-26), 129.8 (C-16), 129.7 (C-15), 128.6 (C-27), 114.0 (C-17), 79.1 (C-12), 78.8 (C10), 75.1 (C-5), 75.0 (C-2), 72.2 (C-14), 64.7 (C-11), 55.4 (C-19), 48.3 (C-4), 39.0 (C-21), 30.2 (C-9), 28.4 (C-8), 27.2 (C-22), 15.8 (C-1), 14.6 (C-23) ppm; IR (ATR) ṽ 3521 (br, w), 3276 (w), 2973 (m), 2936 (m), 2874 (w), 1718 (ss), 1513 (m), 1452 (m), 1267 (s), 1249 (s), 1146 (s), 1115 (s), 1070 (m), 1032 (s) cm-1; HRMS (ESI) m/z calcd for C34H42NaO8+ [M+Na]+ 601.2772, found 601.2797. (2S,4R,5R,6E,10S,11R)-2-Benzoyloxy-5-(tert-butyldimethylsilyl)oxy-3-methyl-10-(pmethoxybenzyl)oxy-11-pivaloyloxytridec-6-en-12-yn-3-one (60) To a solution of alcohol 30 (0.862 g, 1.490 mmol) in CH2Cl2 (10 mL) was added imidazole (2.0 eq, 0.20 g, 2.98 mmol). After complete dissolution of imidazole, TBSCl (1.0 eq, 0.22 g, 1.49 mmol) was added and the reaction mixture was stirred at room temperature. After 1 h, an additional amount of imidazole (2.0 eq, 0.20 g, 2.98 mmol) and TBSCl (1.0 eq, 0.22 g, 1.49 mmol) were added. As the conversion was not complete after 3 h, a third portion of imidazole (2.0 eq, 0.20 g, 2.98 mmol) and TBSCl (1.0 eq, 0.22 g, 1.49 mmol) were added and the reaction mixture was stirred overnight. The white suspension was diluted with CH2Cl2 and water was added. The organic layer was separated and the aqueous layer was extracted twice with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, hexanes/EtOAc 15:1) yielded the TBS protected aldol product 60 as a highly viscous, colorless oil (0.906 g, 1.31 mmol, 88%): []20D = -35.4 (c 1.30, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 7.1 Hz, 2H, H-26), 7.57 (t, J = 7.4 Hz, 1H, H-28), 7.45 (t, J = 7.7 Hz, 2H, H-27), 7.26 (d, J = 8.6 Hz, 2H, H-16), 6.87 (d, J = 8.6 Hz, 2H, H-17), 5.62 (dd, J = 3.2, 2.2 Hz, 1H, H-11), 5.52 (dt, J = 15.4, 6.6 Hz, 1H, H-7), 5.41 (q, J = 6.9 Hz, 1H, H-2), 5.29 (dd, J = 15.6, 8.4 Hz, 1H, H-6), 4.72 (d, J = 11.0 Hz, 1H, H-14a), 4.40 (d, J = 11.0 Hz, 1H, H-14b), 4.22 (t, J = 8.7 Hz, 1H, H-5), 3.80 (s, 3H, H21



Page 22 of 42

19), 3.59 (ddd, J = 8.0, 5.0, 3.4 Hz, 1H, H-10), 2.86 (dq, J = 9.3, 7.0 Hz, 1H, H-4), 2.46 (d, J = 2.3 Hz, 1H, H-13), 2.22 (dq, J = 13.5, 6.8 Hz, 1H, H-8a), 2.05 (dq, J = 14.9, 8.3 Hz, 1H, H8b), 1.79–1.70 (m, 2H, H-9), 1.52 (d, J = 7.0 Hz, 3H, H-1), 1.23 (s, 9H, H-22), 1.00 (d, J = 7.1 Hz, 3H, H-23), 0.82 (s, 9H, (CH3)3CSi), -0.03 (s, 3H, (CH3)2Si), -0.03 (s, 3H, (CH3)2Si); 13C{1H}

NMR (101 MHz, CDCl3) δ 209.4 (C-3), 177.3 (C-20), 165.8 (C-24), 159.5 (C-18),

133.3 (C-28), 132.8 (C-6), 132.0 (C-7), 130.2 (C-15), 129.9 (C-17), 129.8 (C-25), 128.5 (C27), 113.9 (C-17), 79.1 (C-12), 78.9 (C-10), 76.4 (C-5), 75.4 (C-2), 75.0 (C-13), 72.3 (C-14), 64.7 (C-11), 55.4 (C-19), 49.0 (C-4), 38.9 (C-21), 30.3 (C-9), 28.2 (C-8), 27.2 (C-22), 26.0 ((CH3)3CSi), 18.1 ((CH3)3CSi), 15.3 (C-1), 14.5 (C-23), -3.9 ((CH3)2Si), -4.6 ((CH3)2Si) ppm; IR (ATR) ṽ 3270 (w), 2954 (m), 2932 (m), 2856 (w), 1720 (ss), 1513 (m), 1452 (m), 1249 (ss), 1145 (s), 1114 (s), 1067 (s), 1035 (s), 1002 (s) cm-1; HRMS (ESI) m/z calcd for C40H56NaO8Si+ [M+Na]+ 715.3637, found 715.3635. (2R,3R,4E,8S,9R)-3-(tert-Butyldimethylsilyl)oxy-9-hydroxy-8-(p-methoxybenzyl)oxy-2methylundec-4-en-10-ynoic acid (23) The TBS protected aldol product 60 (0.476 g, 0.687 mmol) was dissolved in methanol (10 mL) and two portions of NaBH4 (1 eq, 26 mg, 0.69 mmol) were added within 20 min. After stirring for 30 min sodium metal (40 mg) was added and the solution was stirred at room temperature overnight. The solvent was mostly removed in vacuo and the residue was diluted with EtOAc and water. The organic layer was separated and the aqueous layer was extracted three times with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. After column chromatography the triol was isolated as a mixture of diastereomes (silica gel, 0.261 g, 0.515 mmol, 75%). Next, the triol (0.261 g, 0.515 mmol) was dissolved in CH2Cl2 (6 mL) and NaIO4, immobilized on silica, (2 eq, 1.71 g, 1.12 mmol, c = 0.65 mmol/g) was added. The suspension was vigorously stirred for 4 h and then filtered through cotton wool. The silica was repeatedly rinsed with CH2Cl2 and the filtrate, containing the aldehyde, was concentrated in vacuo. The residue was dissolved in tBuOH (7 mL) and NaH2PO4-solution (7 M, 4 mL) was added. Then 2-methyl-2butene (30 eq, 1.6 mL, 15.4 mmol) was added, followed by NaClO2 (10 eq, 0.58 g, c = 80%, 5.15 mmol). The slightly greenish biphasic solution was vigorously stirred for 30 min. Then water (10 mL) and CH2Cl2 (10 mL) were added. The organic layer was separated and the aqueous layer was extracted three times with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The carboxylic acid 23 (0.215 g, 0.451 mmol, 88%) was obtained as a colorless oil after purification by column chromatography (silica gel, hexanes/EtOAc 2:1 + 1% trimethylamine): []20D = -11.4 (c 1.09, CHCl3); 1H 22


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NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.6 Hz, 2H, H-15), 6.89 (d, J = 8.6 Hz, 2H, H-16), 5.59 (dt, J = 15.5, 6.7 Hz, 1H, H-5), 5.35 (dd, J = 15.4, 7.6 Hz, 1H, H-4), 4.59 (d, J = 11.4 Hz, 1H, H-13a), 4.55 (d, J = 11.3 Hz, 1H, H-13b), 4.50 (dd, J = 3.7, 2.3 Hz, 1H, H-9), 4.18 (t, J = 7.5 Hz, 1H, H-3), 3.80 (s, 3H; H-18), 3.52 (td, J = 6.6, 3.6 Hz, 1H, H-8), 2.52 (qui, J = 7.2 Hz, 1H, H-2), 2.48 (d, J = 2.2 Hz, 1H, H-11), 2.19 (dq, J = 14.1, 7.1 Hz, 1H, H-6a), 2.07 (dq, J = 14.8, 7.4 Hz, 1H, H-6b), 1.76 (td, J = 7.7, 5.7 Hz, 2H, H-7), 1.07 (d, J = 7.0 Hz, 3H, H12), 0.86 (s, 9H, (CH3)3CSi), 0.05 (s, 3H, (CH3)2Si), 0.02 (s, 3H, (CH3)2Si);

13C{1H}

NMR

(101 MHz, CDCl3) δ 180.0 (C-1), 159.6 (C-17), 133.0 (C-5), 131.0 (C-4), 130.1 (C-14), 129.8 (C-15), 114.1 (C-16), 81.7 (C-10), 80.4 (C-8), 76.0 (C-3), 74.7 (C-11), 72.4 (C-13), 63.8 (C9), 55.4 (C-18), 47.1 (C-2), 29.5 (C-7), 28.2 (C-6), 25.8 ((CH3)3CSi), 18.2 ((CH3)3CSi), 13.6 (C-12), -3.8 ((CH3)2Si), -5.0 ((CH3)2Si) ppm; IR (ATR) ṽ 3290 (w, br), 2952 (m), 2930 (m), 2885 (w), 2856 (m), 1708 (s), 1612 (m), 1513 (s), 1461 (m), 1247 (ss), 1174 (m), 1096 (s), 1059 (ss), 1034 (ss) cm-1; HRMS (ESI) m/z calcd for C26H40NaO6Si+ [M+Na]+ 499.2486, found 499.2481. (2R,3R,4E,8S,9R)-3,9-Dihydroxy-8-(p-methoxybenzyl)oxy-2-methylundec-4-en-10-ynoic acid (31) The hydroxy acid 23 (0.215 g, 0.451 mmol) was dissolved in THF (4 mL) and TBAF-solution (1.5 eq, 0.677 mmol, 0.68 mL, 1.0 M in THF) was added. The solution was stirred for 5 h and water and EtOAc were added. The organic layer was separated and the aqueous layer was extracted three times with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, CH2Cl2/MeOH 10:1 + 1% HOAc) yielded the dihydroxy acid 31 as a colorless oil (0.135 g, 0.372 mmol, 83%): []22D = -13.5 (c 1.09, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.6 Hz, 2H, H-15), 6.88 (d, J = 8.6 Hz, 2H, H-16), 5.68 (dt, J = 15.4, 6.7 Hz, 1H, H-5), 5.42 (dd, J = 15.4, 7.3 Hz, 1H, H-4), 4.60 (d, J = 11.3 Hz, 1H, H-13a), 4.54 (d, J = 11.4 Hz, 1H, H-13b), 4.49 (dd, J = 3.7, 2.2 Hz, 1H, H-9), 4.14 (t, J = 7.4 Hz, 1H, H-3), 3.80 (s, 3H, H-18), 3.51 (ddd, J = 7.2, 5.4, 3.5 Hz, 1H, H-8), 2.54 (qui, J = 7.2 Hz, 1H, H-2), 2.49 (d, J = 2.2 Hz, 1H, H-11), 2.21 (dq, J = 13.0, 6.6 Hz, 1H, H-6a), 2.07 (dq, J = 15.2, 7.7 Hz, 1H, H6b), 1.79–1.72 (m, 2H, H-7), 1.15 (d, J = 7.2 Hz, 3H, H-12);

13C{1H}

NMR (126 MHz,

CDCl3) δ 179.9 (C-1), 159.6 (C-17), 133.8 (C-5), 130.4 (C-4), 130.1 (C-14), 129.9 (C-15), 114.1 (C-16), 81.9 (C-10), 80.3 (C-8), 74.8 (C-11), 74.6 (C-3), 72.4 (C-13), 63.9 (C-9), 55.4 (C-18), 45.5 (C-2), 29.5 (C-7), 28.3 (C-6), 14.1 (C-12) ppm; IR (ATR) ṽ 3397 (br, m), 3289 (m), 2927 (m), 1708 (s), 1611 (m), 1512 (s), 1458 (m), 1302 (m), 1245 (ss), 1174 (m), 1066 23



Page 24 of 42

(m), 1029 (ss) cm-1; HRMS (ESI) m/z calcd for C20H26NaO6+ [M+Na]+ 385.1622, found 385.1629. 4-((S)-Benzyl)-3-((2S,3R,4E,8S,9R)-3-hydroxy-8-(p-methoxybenzyl)oxy-2-methyl-1-oxo-9(pivaloyloxy)-undec-4-en-10-yn-1-yl)-oxazolidin-2-one (33) To a solution of (4S)-4benzyloxazolidin-2-one (32) (1.2 eq, 0.514 g, 2.203 mmol) in CH2Cl2 (10 mL) were added at -78°C n-Bu2BOTf (1.2 eq, 2.2 mL, 2.2 mmol, 1.0 M in CH2Cl2) and triethylamine (1.4 eq, 0.36 mL, 0.26 g, 2.57 mmol). The solution was stirred at -78°C for 15 min and then at 0°C for 30 min. Then the solution was cooled again to -78°C and aldehyde 21 (0.684 g, 1.84 mmol), dissolved in CH2Cl2 (6 mL), was slowly added. The stirred solution was kept at -78°C for 15 min and then at 0°C for 1 h. The reaction was quenched by addition of pH 6.8 buffer-solution (10 mL) and H2O2 (6 mL) and the mixture was intensively stirred at 0°C for 20 min. The organic layer was separated and the aqueous layer was extracted twice with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, hexanes/EtOAc 2:1) yielded the aldol product 33 (1.030 g, 1.700 mmol, 93%) as a highly viscous residue: []21D = -8.5 (c 1.29, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.38 (dd, J = 8.0, 6.6 Hz, 2H, H-28), 7.35–7.31 (m, 3H, H-27, H-29), 7.25 (d, J = 6.8 Hz, 2H, H-15), 6.93 (d, J = 8.6 Hz, 2H, H-16), 5.75 (dt, J = 16.0, 6.8 Hz, 1H, H-5), 5.68 (t, J = 2.7 Hz, 1H, H-9), 5.52 (dd, J = 15.5, 6.2 Hz, 1H, H-4), 4.77–4.71 (m, 1H, H-24), 4.76 (d, J = 11.1 Hz, 1H, H-13a), 4.47 (t, J = 5.1 Hz, 1H, H-3), 4.46 (d, J = 11.2 Hz, 1H, H-13b), 4.25 (dd, J = 16.4, 9.1 Hz, 1H, H-23a), 4.22 (dd, J = 9.1, 3.0 Hz, 1H, H-23b), 3.90 (qd, J = 7.0, 3.8 Hz, 1H, H-2), 3.85 (s, 3H, H-18), 3.64 (ddd, J = 8.1, 4.9, 3.2 Hz, 1H, H-8), 3.30 (dd, J = 13.5, 3.4 Hz, 1H, H-25a), 2.84 (dd, J = 13.5, 9.4 Hz, 1H, H25b), 2.50 (d, J = 2.2 Hz, 1H, H-11), 2.30 (dq, J = 13.9, 6.8 Hz, 1H, H-6a), 2.12 (dq, J = 15.1, 7.7 Hz, 1H, H-6b), 1.84–1.78 (m, 2H, H-7), 1.30–1.28 (m, 3H, H-12) 1.28 (s, 9H, H-21); 13C{1H}


135.2 (C-26), 132.4 (C-5), 130.2 (C-14), 129.9 (C-4), 129.8 (C-27), 129.5 (C-15), 129.1 (C28), 127.5 (C-29), 113.9 (C-16), 79.1 (C-10), 78.9 (C-8), 75.0 (C-11), 72.7 (C-3), 72.2 (C-13), 66.3 (C-23), 64.8 (C-9), 55.4 (C-18), 55.3 (C-24), 42.9 (C-2), 38.9 (C-20), 37.9 (C-25), 30.3 (C-7), 28.4 (C-7), 27.2 (C-21), 11.4 (C-12) ppm; IR (ATR) ṽ 3522 (br, w), 3278 (w), 2971 (w), 2935 (w), 2873 (w), 1777 (ss), 1731 (s), 1696 (m), 1513 (m), 1384 (s), 1363 (m), 1281 (m), 1246 (ss), 1209 (ss), 1146 (ss), 1110 (m), 1032 (s) cm-1; HRMS (ESI) m/z calcd for C35H43NNaO8+ [M+Na]+ 628.2881, found 628.2898. 24


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4-((S)-Benzyl)-3-((2S,3R,4E,8S,9R)-3-(tert-Butyldimethylsilyl)oxy-8-(p-methoxybenzyl)oxy2-methyl-1-oxo-9-(pivaloyloxy)-undec-4-en-10-yn-1-yl)-oxazolidin-2-one (61) The aldol product 33 (1.030 g, 1.700 mmol) was dissolved in CH2Cl2 (12 mL) and imidazole (4 eq, 0.46 g, 6.80 mmol) and TBSCl (3 eq, 0.77 g, 5.10 mmol) were added. Rapidly large amounts of a white precipitate were formed. The suspension was stirred at room temperature overnight and MeOH (1 mL) was added. The reaction mixture was stirred for further 5 min and sat. NaHCO3-solution was added. The organic layer was separated and the aqueous layer was extracted twice with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification was performed by column chromatography (silica gel, hexanes/EtOAc 5:1) to give the TBS protected aldol product 61 (1.157 g, 1.607 mmol, 95%) as a colorless, viscous oil: []25D = +1.0 (c 0.96, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.33 (t, J = 7.1 Hz, 2H, H-28), 7.29–7.24 (m, 1H, H-29), 7.26 (d, J = 8.6 Hz, 2H, H-15), 7.21 (d, J = 6.6 Hz, 2H, H-27), 6.87 (d, J = 8.7 Hz, 2H, H-16), 5.61 (dd, J = 3.3, 2.2 Hz, 1H, H-9), 5.595.52 (m, 1H, H-5), 5.48 (dd, J = 15.6, 6.6 Hz, 1H; H-4), 4.71 (d, J = 10.9 Hz, 1H; H-13a), 4.57 (ddt, J = 9.8, 6.3, 3.0 Hz, 1H, H-24), 4.39 (d, J = 10.9 Hz, 1H, H-13b), 4.27 (t, J = 6.4 Hz, 1H, H-3), 4.15–4.08 (m, 2H, H-23), 3.97 (qui, J = 6.8 Hz, 1H, H-2), 3.79 (s, 3H, H-18), 3.59 (ddd, J = 7.9, 4.6, 3.3 Hz, 1H, H-8), 3.27 (dd, J = 13.4, 3.2 Hz, 1H, H-25a), 2.76 (dd, J = 13.4, 9.6 Hz, 1H, H-25b), 2.41 (d, J = 2.2 Hz, 1H; H-11), 2.19 (dq, J = 13.9, 6.5 Hz, 1H, H6a), 2.04 (tt, J = 14.6, 6.7 Hz, 1H, H-6b), 1.75–1.57 (m, 2H, H-7), 1.22 (s, 9H, H-21), 1.19 (d, J = 6.8 Hz, 3H, H-12), 0.87 (s, 9H, (CH3)3CSi), 0.01 (s, 3H, (CH3)2Si), -0.01 (s, 3H, (CH3)2Si);

13C{1H}

NMR (101 MHz, CDCl3) δ 177.3 (C-19), 174.9 (C-1), 159.4 (C-17),

153.3 (C-22), 135.5 (C-26), 131.9 (C-4), 131.5 (C-5), 130.2 (C-14), 129.7 (C-15/C-27), 129.6 (C-15/C-17), 129.1 (C-28), 127.4 (C-29), 113.9 (C-16), 79.1 (C-8/C-10), 79.1 (C-8/C-10), 75.1 (C-3/C-11), 75.0 (C-3/C-11), 72.4 (C-13), 66.0 (C-23), 64.8 (C-9), 55.8 (C-24), 55.4 (C18), 44.4 (C-2), 38.9 (C-20), 37.9 (C-25), 30.5 (C-7), 28.2 (C-6), 27.2 (C-21), 25.9 ((CH3)3CSi), 18.2 ((CH3)3CSi), 12.8 (C-12), -4.0 ((CH3)2Si), -4.9 ((CH3)2Si) ppm; IR (ATR) ṽ 3248 (w), 2955 (m), 2931 (m), 2856 (m), 1778 (ss), 1733 (s), 1697 (s), 1513 (m), 1460 (m), 1379 (s), 1361 (s), 1247 (ss), 1208 (s), 1143 (ss), 1107 (ss), 1032 (ss), 970 (s) cm-1; HRMS (ESI) m/z calcd for C41H57NNaO8Si+ [M+Na]+ 742.3746, found 742.3755. (2S,3R,4E,8S,9R)-3-(tert-Butyldimethylsilyl)oxy-9-hydroxy-8-(p-methoxybenzyl)oxy-2methyl-undeca-4-en-10-ynoic acid (22) To a solution of the TBS protected aldol product 61 (0.294 g, 0.408 mmol) in THF (8 mL) and water (3 mL), LiOH (6 eq, 59 mg, 2.5 mmol) and H2O2 (10 eq, 0.5 mL, 4.1 mmol, c = 30%) were added. The solution was vigorously stirred at 25



Page 26 of 42

room temperature. Hydrolysis of the auxiliary was observed to proceed within 6 h, but the cleavage of the pivalate required prolonged stirring for 3 d. Then water and EtOAc were added and the organic layer was separated. The aqueous layer was extracted twice with EtOAc and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, hexanes/EtOAc 2:1) yielded the hydroxy acid 22 (0.157 g, 0.329 mmol, 81%) as a colorless oil: []22D = -1.3 (c 1.14, CHCl3); 1H

NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.7 Hz, 2H; H-15), 6.89 (d, J = 8.7 Hz, 2H, H-16),

5.60 (dt, J = 15.6, 6.8 Hz, 1H, H-5), 5.41 (dd, J = 15.4, 7.5 Hz, 1H, H-4), 4.58 (d, J = 11.3 Hz, 1H, H-13a), 4.54 (d, J = 11.4 Hz, 1H, H-13b), 4.49 (dd, J = 3.7, 2.2 Hz, 1H, H-9), 4.28 (dd, J = 7.1, 5.4 Hz, 1H, H-3), 3.80 (s, 3H, H-18), 3.50 (ddd, J = 7.3, 5.6, 3.5 Hz, 1H, H-8), 2.58 (qd, J = 7.0, 5.1 Hz, 1H, H-2), 2.48 (d, J = 2.3 Hz, 1H, H-11), 2.17 (dt, J = 14.8, 7.3 Hz, 1H, H-6a), 2.06 (dq, J = 14.4, 7.4 Hz, 1H, H-6b), 1.75 (tq, J = 10.2, 8.0 Hz, 2H, H-7), 1.11 (d, J = 7.0 Hz, 3H, H-12), 0.89 (s, 9H, (CH3)3CSi), 0.08 (s, 3H, (CH3)2Si), 0.05 (s, 3H, (CH3)2Si); 13C{1H}


129.8 (C-4), 129.8 (C-15), 114.1 (C-16), 81.7 (C-10), 80.3 (C-8), 75.4 (C-3), 74.8 (C-11), 72.4 (C-13), 63.8 (C-9), 55.4 (C-18), 46.1 (C-2), 29.5 (C-7), 28.2 (C-6), 25.9 ((CH3)3CSi), 18.2 ((CH3)3CSi), 12.1 (C-12), -4.0 ((CH3)2Si), -4.9 ((CH3)2Si) ppm; IR (ATR) ṽ 3290 (w, br), 2952 (m), 2930 (m), 2885 (w), 2856 (m), 1708 (s), 1612 (m), 1513 (s), 1461 (m), 1247 (ss), 1174 (m), 1096 (m), 1059 (ss), 1034 (ss), 973 (m) cm-1; HRMS (ESI) m/z calcd for C26H40NaO6Si+ [M+Na]+ 499.2486, found 499.2481. (2S,3R,4E,8S,9R)-3,9-Dihydroxy-8-(p-methoxybenzyl)oxy-2-methyl-undeca-4-en-10-ynoic acid (34) To a solution of the TBS protected hydroxy acid 22 (0.157 g, 0.329 mmol) in THF (5 mL) was added TBAF-solution (1.3 eq, 0.43 mL, 0.43 mmol, 1.0 M in THF). The solution was stirred overnight at room temperature. Then water and EtOAc were added and the organic layer was separated. The aqueous layer was extracted three times with EtOAc and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:2 + 1% HOAc) to give the dihydroxy acid 34 (94 mg, 0.26 mmol, 79%) as a colorless oil: []21D = -10.3 (c 1.20, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.4 Hz, 2H, H-15), 6.88 (d, J = 8.5 Hz, 2H, H-16), 5.67 (dt, J = 14.8, 6.7 Hz, 1H, H-5), 5.45 (ddt, J = 15.4, 6.6, 1.5 Hz, 1H, H-4), 4.60 (d, J = 11.3 Hz, 1H, H-13a), 4.53 (d, J = 11.3 Hz, 1H, H-13b), 4.48 (dd, J = 3.7, 2.2 Hz, 1H, H-9), 4.31 (t, J = 5.5 Hz, 1H, H-3), 3.79 (s, 3H, H-18), 3.50 (td, J = 6.4, 3.7 Hz, 1H, H-8), 26


Page 27 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.63 (qd, J = 7.1, 4.3 Hz, 1H, H-2), 2.49 (d, J = 2.2 Hz, 1H, H-11), 2.19 (dq, J = 14.0, 7.0 Hz, 1H, H-6a), 2.07 (dq, J = 15.0, 7.6 Hz, 1H, H-6b), 1.75 (q, J = 7.6 Hz, 2H, H-7), 1.14 (d, J = 7.2 Hz, 3H, H-12); 13C{1H} NMR (126 MHz, CDCl3) δ 179.3 (C-1), 159.5 (C-17), 133.2 (C5), 130.0 (C-14), 129.9 (C-15), 129.4 (C-4), 114.1 (C-16), 81.9 (C-10), 80.3 (C-8), 74.8 (C11), 73.3 (C-3), 72.4 (C-13), 63.8 (C-9), 55.4 (C-18), 44.8 (C-2), 29.5 (C-7), 28.2 (C-6), 11.6 (C-12) ppm; IR (ATR) ṽ 3397 (br, m), 3289 (m), 2927 (m), 1708 (s), 1611 (m), 1512 (s), 1458 (m), 1302 (m), 1245 (ss), 1174 (m), 1066 (m), 1029 (ss), 972 (m) cm-1; HRMS (ESI) m/z calcd for C20H26NaO6+ [M+Na]+ 385.1622, found 385.1629. (3R,4R,9S,10R,E)-10-Ethynyl-4-hydroxy-9-(p-methoxybenzyl)oxy-3-methyl-3,4,7,8,9,19hexahydro-2H-oxecin-2-one (35) DMAP (6 eq, 0.54 g, 4.43 mmol) was dissolved in toluene (220 mL, c = 0.02 M) and 2-methyl-6-nitrobenzoic anhydride (1.2 eq, 0.305 g, 0.877 mmol) was added. The solution was stirred at room temperature until most of the anhydride was dissolved. The dihydroxy acid 31 (0.268 g, 0.739 mmol) was dissolved in CH2Cl2 (3 mL) and the solution was diluted with toluene (180 mL, c = 0.004 M). Then the solution of acid 31 was slowly added over a period of 12 h to the solution of DMAP and anhydride. During the addition the formation of a crystalline precipitate was observed. After completion of addition the solution was stirred for additional 6 h at room temperature and then the solvents (toluene and CH2Cl2) was removed in vacuo. The residue was dissolved in CH2Cl2 (as little as possible) and subjected to column chromatography (silica gel, hexanes/EtOAc 2:1). Macrolide 35 started to crystallize in some eluent fractions. Macrolide 35 (0.128 g, 0.370 mmol, 50%) was obtained as a pale white, readily crystallizing solid. The 1H NMR revealed the existence of two conformers: []24D = -114.9 (c 1.26, CHCl3); mp = 133–134°C; 1H NMR (600 MHz, CDCl3) δ 7.30 (d, J = 8.6 Hz, 2H, H-15), 6.88 (d, J = 8.6 Hz, 2H, H-16), 5.65– 5.58 (m, 2H, H-4, H-5), 5.26 (dd, J = 8.1, 2.2 Hz, 1H, H-9), 4.65 (d, J = 10.9 Hz, 1H, H-13a), 4.54 (d, J = 10.9 Hz, 1H, H-13b), 4.33 (d, J = 3.0 Hz, 1H, H-3), 3.80 (s, 3H, H-18), 3.61 (td, J = 8.0, 1.7 Hz, 1H, H-8), 2.73 (qd, J = 7.0, 3.2 Hz, 1H, H-2), 2.48 (d, J = 2.2 Hz, 1H, H-11), 2.37–2.30 (m, 1H, H-6a), 2.11–2.03 (m, 3H, H-6b, H-7a, OH), 1.84 (dddd, J = 15.1, 9.4, 7.4, 2.1 Hz, 1H, H-7b), 1.30 (d, J = 7.0 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) major conformer δ 172.9 (C-1), 159.4 (C-17), 133.9 (C-4), 130.1 (C-14), 129.6 (C-15), 127.1 (C-5), 113.9 (C-16), 80.9 (C-10), 80.3 (C-8), 73.9 (C-11), 72.5 (C-3/C-13), 72.4 (C-3/C-13), 66.4 (C-9), 55.4 (C-18), 46.9 (C-2), 34.5 (C-7), 27.0 (C-6), 12.5 (C-12); minor conformer δ 173.6, 159.3, 134.3, 133.1, 130.2, 129.4, 79.8, 79.4, 75.0, 72.4, 71.3, 64.0, 47.6, 29.7, 26.9, 11.6 ppm; IR (ATR) ṽ 3509 (br, w), 3282 (w), 2929 (m), 2854 (w), 1736 (s), 1612 (m), 1513 (s), 27



Page 28 of 42

1457 (m), 1441 (m), 1364 (m), 1301 (m), 1246 (ss), 1158 (ss), 1096 (s), 1072 (s), 1032 (s), 1000 (m), 978 (m) cm-1; HRMS (ESI) m/z calcd for C20H24NaO5+ [M+Na]+ 367.1516, found 367.1523. (3R,4R,9S,10R,E)-10-Ethynyl-4,9-dihydroxy-3-methyl-3,4,7,8,9,19-hexahydro-2H-oxecin2-one (24) To a solution of the PMB protected macrolide 35 (0.125 g, 0.364 mmol) in CH2Cl2 (6 mL) were added pH 6.7 buffer-solution (4 mL) and DDQ (1.5 eq, 0.124 g, 0.546 mmol). The initially orange-colored biphasic reaction mixture turned rapidly into deep green and after 30 min the color changed to brownish. After complete conversion was detected (TLC), the reaction mixture was poured into sat. NaHCO3-solution (4 mL) and Na2SO3 (20 mg) was added to reduce excessive amounts of DDQ. The organic layer was separated and the aqueous layer was extracted three times with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The brownish residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:1) yielding macrolide 24 as a colorless, rapidly crystallizing solid (78 mg, 0.349 mmol, 95%): []24D = -178.9 (c 2.05, CHCl3); mp = 141– 143°C; 1H NMR (600 MHz, CDCl3) δ 5.60 (ddd, J = 15.9, 2.9, 1.3 Hz, 1H, H-4), 5.52 (dddd, J = 15.7, 9.7, 4.2, 1.4 Hz, 1H, H-5), 5.24 (dd, J = 8.2, 2.2 Hz, 1H, H-9), 4.34 (dp, J = 3.2, 1.6 Hz, 1H, H-3), 3.69 (td, J = 8.4, 1.5 Hz, 1H, H-8), 2.72 (qd, J = 7.0, 3.3 Hz, 1H, H-2), 2.61 (d, J = 2.2 Hz, 1H, H-11), 2.43 (dddt, J = 11.9, 7.7, 4.0, 1.8 Hz, 1H, H-6a), 2.19–2.11 (m, 2H, H6b, H-7a), 1.70 (dddd, J = 15.4, 11.6, 8.6, 2.1 Hz, 1H, H-7b), 1.30 (d, J = 7.0 Hz, 3H, H-12); 13C{1H}

NMR (151 MHz, CDCl3) δ 172.6 (C-1), 133.6 (C-4), 127.4 (C-5), 79.7 (C-10), 76.3

(C-11), 73.2 (C-8), 72.3 (C-3), 68.2 (C-9), 46.7 (C-2), 37.3 (C-7), 29.3 (C-6), 12.4 (C-12) ppm; IR (ATR) ṽ 3471 (s), 3258 (m), 2928 (m), 2883 (w), 2852 (w), 1725 (ss), 1377 (m), 1246 (m), 1168 (ss), 1098 (s), 1000 (m), 982 (s) cm-1; HRMS (ESI) m/z calcd for C12H16NaO4+ [M+Na]+ 247.0941, found 247.0937. (3R,4R,9S,10R,E)-4,9-Dihydroxy-3-methyl-10-(phenylethynyl)-3,4,7,8,9,10-hexahydro-2Hoxecin-2-one (39) To a solution of alkyne 24 (5.5 mg, 25 µmol) in CH2Cl2 (0.5 mL) were added ethyldiisopropylamine (0.5 mL), iodobenzene (36) (2 eq, 4.9 µL, 9.0 mg, 44 µmol), CuI (0.5 eq, 2.4 mg, 13 µmol) and Pd(PPh3)2Cl2 (1 mg). The suspension was vigorously stirred and warmed up to 35°C, at which the color changed from yellow-orange to brownish. After full conversion was monitored (TLC) the solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:1). The substituted alkyne 39 was obtained as a pale white solid (2.4 mg, 8.0 µmol, 32%): []20D = 28


Page 29 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


205.4 (c 0.35, CHCl3); mp = 155–157 °C; 1H NMR (600 MHz, CDCl3) δ 7.46 (dd, J = 8.2, 1.5 Hz, 2H, H-14), 7.37-7.30 (m, 3H, H-15, H-16), 5.64 (ddd, J = 15.8, 3.0, 1.3 Hz, 1H, H-4), 5.56 (dddd, J = 15.7, 10.1, 4.1, 1.4 Hz, 1H, H-5), 5.51 (d, J = 8.1 Hz, 1H, H-9), 4.35 (s, br, 1H, H-3), 3.75 (t, J = 8.2 Hz, 1H, H-8), 2.76 (qd, J = 7.0, 3.2 Hz, 1H, H-2), 2.49–2.44 (m, 1H, H-6a), 2,37 (s, br, 1H, OH), 2.24–2.14 (m, 2H, H-6b, H-7a), 2.01 (s, br, 1H, OH), 1.75 (dddd, J = 14.9, 11.2, 8.7, 2.1 Hz, 1H, H-7b), 1.55 (s, br, 1H, OH), 1.33 (d, J = 7.0 Hz, 3H); 13C{1H} NMR (151 MHz, CDCl3) δ 172.7 (C-1), 133.5 (C-4), 132.2 (C-14), 129.3 (C-16), 128.5 (C15), 127.5 (C-5), 121.5 (C-13), 88.3 (C-11), 84.3 (C-10), 73.6 (C-8), 72.3 (C-3), 69.2 (C-9), 46.8 (C-2), 37.3 (C-7), 29.7 (C-6), 12.5 (C-12) ppm; IR (ATR) ṽ 3466 (br, m), 2927 (m), 2854 (w), 1831 (w), 1731 (ss), 1162 (ss), 1098 (s), 1067 (m), 982 (m) cm-1; HRMS (ESI) m/z calcd for C18H20NaO4+ [M+Na]+ 323.1254, found 323.1256. (3R,4R,9S,10R,E)-4,9-Dihydroxy-3-methyl-10-(p-nitrophenylethynyl)-3,4,7,8,9,10hexahydro-2H-oxecin-2-one (40) To a solution of alkyne 24 (13.7 mg, 61.1 µmol) in CH2Cl2 (0.7 ml) were added ethyldiisopropylamine (0.5 mL), p-iodonitrobenzene (37) (3 eq, 45 mg, 0.183 mmol), CuI (0.5 eq, 5.8 mg, 31 µmol) and Pd(PPh3)2Cl2 (2 mg). The suspension was vigorously stirred and warmed up to 35°C, where the color changed from yellow-orange to brownish. As alkyne 24 and the p-nitrophenyl substituted alkyne 40 share the same Rf-value, monitoring the reaction via TLC was not possible. However, in this case the conversion was complete after 4 h. The solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, hexanes /EtOAc, 1:1), yielding the p-nitrophenyl substituted alkyne 40 (6.5 mg, 19 µmol, 31%) as an orange solid: []20D = -170.6 (c 0.65, CHCl3); mp = 142–143 °C; 1H NMR (600 MHz, CDCl3) δ 8.19 (d, J = 8.8 Hz, 2H, H-15), 7.61 (d, J = 8.8 Hz, 2H, H-14), 5.65 (ddd, J = 15.8, 2.9, 1.2 Hz, 1H, H-4), 5.58 (dddd, J = 15.6, 9.7, 4.1, 1.4 Hz, 1H, H-5), 5.50 (d, J = 8.1 Hz, 1H, H-9), 4.37 (s, 1H, H-3), 3.81 (td, J = 8.3, 1.4 Hz, 1H, H-8), 2.76 (qd, J = 7.0, 3.2 Hz, 1H, H-2), 2,50–2.44 (m, 1H, H-6a), 2.25– 2.16 (m, 2H, H-6b, H-7a), 1.82–1.75 (m, 1H, H-7b), 1.33 (d, J = 7.0 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 172.6 (C-1), 147.8 (C-16), 133.7 (C-4), 133.0 (C-14), 128.5 (C13), 127.4 (C-5), 123.7 (C-15), 89.8 (C-10), 85.7 (C-11), 73.5 (C-8), 72.3 (C-3), 68.8 (C-9), 46.8 (C-2), 37.6 (C-7), 29.3 (C-6), 12.5 (C-12); IR (ATR) ṽ 3466 (br, m), 3106 (w), 2933 (w), 2853 (w), 1735 (m), 1594 (m), 1518 (s), 1344 (ss), 1159 (s), 1098 (m) cm-1; HRMS (ESI) m/z calcd for C18H19NNaO6+ [M+Na]+ 368.1105, found 368.1104.

29



Page 30 of 42

(3R,4R,9S,10R,E)-4,9-Dihydroxy-3-methyl-10-(pent-4-en-1-yn-1-yl)-3,4,7,8,9,10hexahydro-2H-oxecin-2-one (41) To a solution of alkyne 24 (10.0 mg, 44.6 µmol) in CH2Cl2 (0.7 mL) were added ethyldiisopropylamine (0.5 mL), allyl bromide (38) (4 eq, 16 µL, 22 mg, 0.18 mmol), CuI (0.5 eq, 4.4 mg, 23 µmol) and Pd(PPh3)2Cl2 (2 mg). The suspension was vigorously stirred and warmed up to 35C. The color changed from yellow to orange and full conversion was monitored after 2 h. The solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2:1) yielding the substituted alkyne 41 as a colorless oil (6.1 mg, 23 µmol, 52%): []20D = -80.2 (c 0.59, CHCl3); 1H NMR (600 MHz, CDCl3) δ 5.78 (ddt, J = 17.0, 10.0, 5.4 Hz, 1H, H-13), 5.60 (ddd, J = 15.8, 3.0, 1.3 Hz, 1H, H-4), 5.51 (dddd, J = 15.8, 10.0, 4.1, 1.4 Hz, 1H, H-5), 5.31–5.26 (m, 2H, H-9, H14(Z)), 5.13 (dq, J = 10.0, 1.6 Hz, 1H, H-14(E)), 4.33 (dqui, J = 3.1, 1.7 Hz, 1H, H-3), 3.62 (dt, J = 8.4 Hz, 1.4 Hz, 1H, H-8), 3.03 (dq, J = 5.5, 1.9 Hz, 2H, H-12), 2.72 (qd, J = 7.0, 3.2 Hz, 1H, H-2), 2.46–2.41 (m, 1H, H-6a), 2.19–2.10 (m, 2H, H-6b, H-7a), 1.68 (dddd, J = 15.3, 11.6, 8.7, 2.1 Hz, 1H, H-7b), 1.31 (d, J = 7.0 Hz, 3H, H-15);

13C{1H}

NMR (151 MHz,

CDCl3) δ 172.8 (C-1), 133.5 (C-4), 131.6 (C-13), 127.5 (C-5), 117.0 (C-14), 86.3 (C-11), 78.0 (C-10), 73.5 (C-8), 72.3 (C-3), 69.0 (C-9), 46.7 (C-2), 37.2 (C-7), 29.7 (C-6), 23.2 (C-12), 12.5 (C-15) ppm; IR (ATR) ṽ 3467 (s, br), 2924 (ss), 2853 (m), 1735 (ss), 1166 (s), 1099 (m), 1020 (m) cm-1; HRMS (ESI) m/z calcd for C15H20NaO4+ [M+Na]+ 287.1254, found 287.1257. (3R,4R,9S,10R,E)-10-(1-Benzyl-1H-1,2,3-triazol-4-yl)-4,9-dihydroxy-3-methyl-3,4,7,8,9,10hexahydro-2H-oxecin-2-one (45) To a solution of alkyne 24 (10.3 mg, 45.9 µmol) in MeOH (1 mL) were added water (1 mL), benzylazide (42)39 (excess, 20 µL), Cu(OAc)2 (0.3 eq, 2.5 mg, 14 µmol) and Na2S2O4 (4 mg). The initial color changed from light blue to intensive yellow. The reaction mixture was vigorously stirred for 12 h and then diluted with water and EtOAc. The organic layer was separated and the aqueous layer was extracted with EtOAc three times. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:2) yielding triazole 45 (6.0 mg, 17 µmol, 37%) as a colorless solid: []19D = -65.0 (c 0.6, CHCl3); mp = 176–178 °C; 1H NMR (600 MHz, CDCl3) δ 7.52 (s, 1H, H-11), 7.40–7.35 (m, 3H, H-16, H-17), 7.27 (dd, J = 7.4, 2.2 Hz, 2H, H-15), 5.71–5.63 (m, 2H, H-4, H-5), 5.58 (d, J = 8.7 Hz, 1H, H-9), 5.55 (d, J = 14.8 Hz, 1H, H-13a), 5.45 (d, J = 14.7 Hz, 1H, H-13b), 1.93–1.86 (m, 1H), 4.39 (t, J = 7.7 Hz, 1H, H-8), 4.36–4.34 (m, 1H, H-3), 2.68 (qd, J = 7.0, 3.2 Hz, 1H, H-2), 2.50–2.44 (m, 1H, H-6a), 2.29–2.22 (m, 2H, H-6b, H-7a), 1.93–1.86 (m, 30


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1H, H-7b), 1.22 (d, J = 7.0 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 173.7 (C-1), 146.4 (C-10), 134.1 (C-14), 133.9 (C-4), 129.4 (C-16), 129.1 (C-17), 128.4 (C-15), 127.4 (C5), 124.5 (C-11), 72.5 (C-3), 72.2 (C-8), 71.2 (C-9), 54.6 (C-13), 46.8 (C-2), 37.7 (C-7), 28.1 (low int. only visible in HSQC, C-6), 12.5 (C-12) ppm; IR (ATR) ṽ 3416 (br, m), 3144 (w), 3033 (w), 2925 (s), 2853 (m), 2249 (w), 1829 (w), 1730 (ss), 1456 (m), 1365 (m), 1224 (m), 1164 (ss), 1099 (s), 1055 (m) cm-1; HRMS (ESI) m/z calcd for C19H24N3O4+ [M+H]+ 358.1761, found 358.1769. (3R,4R,9S,10R,E)-10-(1-(tert-Butyloxycarbonylmethyl)-1H-1,2,3-triazol-4-yl)-4,9dihydroxy-3-methyl-3,4,7,8,9,10-hexahydro-2H-oxecin-2-one (46) To a solution of alkyne 24 (9.7 mg, 44 µmol) in MeOH (1 mL) were added water (1 mL), tert-butyl azidoacetate (43)40 (excess, 40 µL), Cu(OAc)2 (0.3 eq, 2.5 mg, 14 µmol) and Na2S2O4 (4 mg). The initial color changed from light blue to intensive yellow. The reaction mixture was vigorously stirred for 12 h and then diluted with water and EtOAc. The organic layer was separated and the aqueous layer was extracted three times with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:2) yielding triazole 46 (7.2 mg, 19 µmol, 43%) as a colorless oil: []19D = -76.8 (c 0.69, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.72 (s, 1H, H-11), 5.73–5.68 (m, 2H, H-4, H-5), 5.63 (d, J = 8.7 Hz, 1H, H-9), 5.06 (d, J = 17.4 Hz, 1H, H-13a), 5.01 (d, J = 17.4 Hz, 1H, H-13b), 4.41 (t, J = 7.8 Hz, 1H, H-8), 4.37 (d, J = 2.9 Hz, 1H, H-3), 2.71 (qd, J = 7.0, 3.2 Hz, 1H, H-2), 2.51–2.45 (m, 1H, H-6a), 2.30– 2.24 (m, 2H, H6b, H-7a), 1.96–1.90 (m, 1H, H-7b), 1.47 (s, 9H, H-16), 1.24 (d, J = 7.0 Hz, 3H, H-12); 13C{1H}


127.4 (C-5), 126.0 (C-11), 84.2 (C-15), 72.6 (C-3), 72.1 (C-8), 71.1 (C-9), 51.8 (C-13), 46.8 (C-2), 37.6 (C-7), 28.1 (C-16 and C-6), 12.5 (C-12) ppm; IR (ATR) ṽ 3431 (br, w), 3147 (w), 2978 (w), 2930 (w), 1735 (ss), 1369 (s), 1235 (s), 1155 (ss), 1099 (s), 1055 (m), 996 (m) cm-1; HRMS (ESI) m/z calcd for C18H28N3O6+ [M+H]+ 382.1973, found 382.1979. (3R,4R,9S,10R,E)-10-(1-(But-3-en-1-yl)-1H-1,2,3-triazol-4-yl)-4,9-dihydroxy-3-methyl3,4,7,8,9,10-hexahydro-2H-oxecin-2-one (47) To a solution of alkyne 24 (10.6 mg, 47.3 µmol) in MeOH (1 mL) were added water (1 mL), 3-butenyl azide (44)41 (crude solution, 100 µL), Cu(OAc)2 (0.3 eq, 2.5 mg, 14 µmol) and Na2S2O4 (4 mg). The initial color changed from light blue to intensive yellow. The reaction mixture was vigorously stirred for 12 h and then diluted with water and EtOAc. The organic layer was separated and the aqueous layer was 31



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extracted three times with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:2) to give triazole 47 (4.0 mg, 23 µmol, 26 %) as a colorless oil: []19D = -93.3 (c 0.40, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.59 (s, 1H, H-11), 5.75 (ddt, J = 17.1, 10.3, 6.8 Hz, 1H, H-15), 5.70–5.68 (m, 2H, H-4, H-5), 5.60 (d, J = 8.7 Hz, 1H, H-9), 5.12–5.06 (m, 2H, H-16), 4.46–4.35 (m, 4H, H-13, H-3, H-8), 2.70 (tt, J = 7.1, 3.6 Hz, 1H, H2), 2.66 (dd, J = 7.0, 1.4 Hz, 2H, H-14), 2.51–2.46 (m, 1H, H-6a), 2.30–2.24 (m, 2H, H-6b, H-7a), 1.92 (ddt, J = 13.7, 11.0, 5.4 Hz, 1H, H-7b), 1.24 (d, J = 7.0 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 173.7 (C-1), 145.9 (C-10), 133.9 (C-4), 133.0 (C-15), 127.5 (C-5), 124.7 (C-11), 118.9 (C-16), 72.6 (C-3), 72.1 (C-8), 71.2 (C-9), 50.1 (C-13), 46.8 (C-2), 37.7 (C-7), 34.4 (C-14), 28.2 (low int., C-6), 12.5 (C-12) ppm; IR (ATR) ṽ 3427 (br, s), 3143 (w), 3078 (w), 2925 (m), 2853 (w), 1732 (s), 1165 (ss), 1100 (m), 1059 (m) cm-1; HRMS (ESI) m/z calcd for C16H24N3O4+ [M+H]+ 322.1761, found 322.1760. (1S,2R,5R,6S,7S,8R)-2-Ethynyl-6,7-dihydroxy-5-methyl-3,11-dioxabicyclo[6.2.1]undecan4-one (48) To a solution of alkyne 24 (21.1 mg, 94.1 µmol) in CH2Cl2 (3 mL) mCPBA (1.5 eq, 24 mg, 0.14 mmol) was added at -20°C and the solution was stirred overnight at -15°C. Then, CSA (3 mg) was added and the solution was stirred for further 1 h at -15°C. After TLC showed complete conversion, sat. NaHCO3-solution was added and stirring was continued for 5 min. The organic layer was separated and the aqueous layer was extracted three times with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification of the residue by column chromatography (silica gel, CH2Cl2/MeOH 20:1) yielded the bicyclic lactone 48 (20.2 mg, 84.8 µmol, 90%) as a colorless solid: []24D = -83.3 (c 0.52, CHCl3); mP = 184–186°C, 1H NMR (600 MHz, CDCl3) δ 6.01 (dd, J = 4.3, 2.3 Hz, 1H, H-9), 4.23 (ddd, J = 8.4, 6.6, 4.2 Hz, 1H, H-8), 4.14 (dd, J = 9.2, 6.8 Hz, 1H, H-5), 3.90 (d, J = 2.8 Hz, 1H, H-3), 3.60 (d, J = 9.2 Hz, 1H, H-4), 2.81 (qd, J = 7.0, 2.8 Hz, 1H, H-2), 2.52 (dd, J = 2.4, 0.7 Hz, 1H, H-11), 2.16– 1.99 (m, 6H, H-6, H-7, OH), 1.30 (d, J = 6.8 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 173.4 (C-1), 81.8 (2C, C-3. C-5), 80.4 (C-8), 77.7 (C-10), 76.1 (C-4/C-11), 76.1 (C-4/C-11), 64.6 (C-9), 45.4 (C-2), 29.4 (C-6), 25.5 (C-7), 14.4 (C-12) ppm; IR (ATR) ṽ 3389 (s, br), 3292 (m), 2981 (w), 2936 (m), 2853 (w), 1741 (ss), 1181 (m), 1159 (s), 1082 (s), 1066 (s), 1022 (m), 1005 (m), 982 (m) cm-1; HRMS (ESI) m/z calcd for C12H16NaO5+ [M+Na]+ 263.0890, found 263.0893.

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(1S,2R,5R,6S,7S,8R)-2-(Pent-4-en-1-yn-1-yl)-6,7-dihydroxy-5-methyl-3,11dioxabicyclo[6.2.1]undecan-4-one (50) To a solution of alkyne 48 (9.8 mg, 41µmol) in CH2Cl2 (0.7 ml) were added ethyldiisopropylamine (0.5 mL), allyl bromide (38) (4 eq, 14 µL, 20 mg, 0.16 mmol), CuI (0.5 eq, 3.9 mg, 21 µmol) and Pd(PPh3)2Cl2 (1 mg). The suspension was vigorously stirred and warmed up to 35°C, where the color changed from yellow to orange. Full conversion of starting material was observed after 2 h and the solvents were removed in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:3) to give the substituted alkyne 50 as a colorless solid (8.1 mg, 29 µmol, 70%): []27D = -84.9 (c 0.81, CHCl3); mP = 174–175°C; 1H NMR (600 MHz, CDCl3) δ 6.04 (dt, J = 4.2, 2.0 Hz, 1H, H-9), 5.79 (ddt, J = 17.0, 10.3, 5.3 Hz, 1H, H-13), 5.30 (dq, J = 17.0, 1.8 Hz, 1H, H-14z), 5.14 (dq, J = 10.0, 1.6 Hz, 1H, H-14E), 4.21 (ddd, J = 8.2, 7.1, 4.3 Hz, 1H, H-8), 4.13 (dd, J = 9.1, 6.8 Hz, 1H, H-5), 3.89 (d, J = 2.7 Hz, 1H, H-3), 3.60 (d, J = 9.1 Hz, 1H, H-4), 3.02 (dq, J = 5.5, 1.9 Hz, 2H, H-12), 2.81 (qd, J = 6.9, 2.8 Hz, 1H, H-2), 2.12–2.07 (m, 3H, H-7, H-6a), 2.06–1.86 (m, 4H, H-6b, OH, H2O), 1.28 (d, J = 6.9 Hz, 3H, H-15); 13C{1H}

NMR (151 MHz, CDCl3) δ 173.6 (C-1), 131.7 (C-13), 116.8 (C-14), 85.4 C-11),

81.8 (C-3), 81.7 (C-5), 80.9 (C-8), 76.3 (C-10), 76.2 (C-4), 65.3 (C-9), 45.4 (C-2), 29.4 (C-6), 25.5 (C-7), 23.1 (C-12), 14.4 (C-15) ppm; IR (ATR) ṽ 3388 (s, br), 2972 (w), 2938 (w), 2905 (w), 1737 (ss), 1185 (m), 1163 (s), 1114 (m), 1080 (m), 1064 (s), 1007 (m), 986 (m), 972 (m) cm-1; HRMS (ESI) m/z calcd for C15H20NaO5+ [M+Na]+ 303.1203, found 303.1196. (1S,2R,5R,6S,7S,8R)-

6,7-Dihydroxy-2-((E)-6-hydroxyhex-3-en-1-yn-1-yl)-5-methyl-3,11-

dioxabicyclo[6.2.1]undecan-4-one (51) To a solution of alkyne 48 (13.3 mg, 55.8 µmol) in CH2Cl2 (1 mL) were added ethyldiisopropylamine (0.5 mL), (E)-4-iodobut-3-en-1-ol (49)42 (excess, 30 µL), CuI (0.5 eq, 5.3 mg, 28 µmol) and Pd(PPh3)2Cl2 (2 mg). The suspension was vigorously stirred and warmed up to 35°C. The initially intensive yellow color changed to orange and full conversion of starting material was observed after 1 h. The solvents were removed in vacuo and the residue was purified by column chromatography (silica gel, EtOAc). The substituted alkyne 51 was obtained as colorless solid (11.5 mg, 39 µmol, 70%), which was poorly soluble in most solvents except MeOH: []27D = -124.4 (c 1.11, MeOH); mP = 188–189°C; 1H NMR (600 MHz, CD3OD) δ 6.25 (dt, J = 16.0, 7.2 Hz, 1H, H-13), 6.03 (dd, J = 4.4, 1.9 Hz, 1H, H-9), 5.65 (dq, J = 15.9, 1.7 Hz, 1H, H-12), 4.20–4.16 (m, 2H, H-5, H-8), 3.88 (d, J = 3.1 Hz, 1H, H-3), 3.65 (d, J = 9.4 Hz, 1H, H-4), 3.63 (t, J = 6.5 Hz, 2H, H15), 2.74 (qd, J = 6.9, 3.1 Hz, 1H, H-2), 2.37 (qd, J = 6.5, 1.6 Hz, 2H, H-14), 2.11–2.04 (m, 33



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3H, H-7, H-6a), 2.01–1.95 8m, 1H, H-6), 1.25 (d, J = 7.0 Hz, 3H, H-16); 13C{1H} NMR (151 MHz, CD3OD) δ 175.0 (C-1), 144.3 (C-13), 111.4 (C-12), 86.9 (C-11), 82.7 (C-10), 82.1 (C5), 81.7 (C-3), 81.4 (C-8), 75.9 (C-4), 66.3 (C-9), 61.8 (C-15), 46.0 (C-2), 37.3 (C-14), 30.1 (C-6), 26.4 (C-7), 14.7 (C-16) ppm; IR (ATR) ṽ 3280 (br, s), 2933 (m), 1736 (ss), 1171 (m), 1159 (m), 1064 (m) cm-1; HRMS (ESI) m/z calcd for C16H22NaO6+ [M+Na]+ 333.1309, found 333.1324. (2S,2´R,3S,4R,5´S)-3-Hydroxy-5´-((R)-1-hydroxyhex-5-en-2-yn-1-yl)-4-methylhexahydro[2,2´-bifuran]-5(2H)-one (52) Lactone 50 (5.1 mg, 18 mmol) was dissolved in THF (2 mL) and (CH3)3SiOK (1.5 eq, 3.5 mg, 27 µmol) was added to the stirred solution at rt. The solution became rapidly turbid and the color changed to slightly orange. After 30 min complete conversion of the starting material was observed. Then water (0.1 mL) and CSA (5 mg) were added and the solution was stirred at room temperature. The formation of the five-membered lactone 52 and another, slightly less polar byproduct, was detected by TLC (CH2Cl2/EtOAc 3:1) and shown to be complete after 3 h. The solution was diluted with water and EtOAc and the organic layer was separated. The aqueous layer was extracted three times with EtOAc and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, CH2Cl2/EtOAc 3:1) allowed for the separation of both compounds, yielding lactone 52 (1.4 mg, 5 µmol, 28%) as a colorless oil and the slightly less polar byproduct (1.1 mg). Lactone 52 was found to decompose in solution upon standing for 2 days. Analytical data for 52 are: []27D = -4.5 (c 0.10, CHCl3); 1H

NMR (600 MHz, CDCl3) δ 5.81 (ddt, J = 15.5, 10.2, 5.3 Hz, 1H, H-13), 5.30 (dt, J = 16.9,

1.6 Hz, 1H, H-14Z), 5.14 (dt, J = 10.1, 1.6 Hz, 1H, H-14E), 4.55 (dd, J = 7.0, 3.0 Hz, 1H, H3), 4.52 (d, J = 1.8 Hz, 1H; H-9), 4.18 (dd, J = 7.7, 3.0 Hz, 1H, H-4), 4.13 (td, J = 7.0, 3.2 Hz, 1H, H-8), 4.00 (td, J = 7.2, 4.9 Hz, 1H, H-5), 3.01 (dt, J = 5.4, 1.7 Hz, 2H, H-12), 2.84 (qui, J = 7.4 Hz, 1H, H-2), 2.13-2.09 (m, 1H, H-6a), 2.06–1.99 (m, 3H, H-6b, H-7), 1.65 (s, br, 4H, OH, H2O), 1.26 (d, J = 7.6 Hz, 3H, H-15); 13C{1H} NMR (151 MHz, CDCl3) δ 178.1 (C-1), 132.2 (C-13), 116.7 (C-14), 85.7 (C-4), 84.1 (C-11), 82.7 (C-8), 80.0 (C-10), 79.4 (C-5), 71.2 (C-3), 64.3 (C-9), 39.3 (C-2), 28.9 (C-6), 25.4 (C-7), 23.2 (C-12), 8.9 (C-15) ppm; IR (ATR) ṽ 3421 (br, s), 2980 (w), 2922 (m), 2854 (w), 1754 (ss), 1456 (m), 1418 (m), 1380 (m), 1347 (m), 1182 (s), 1138 (m), 1099 (m), 1032 (s), 985 (s) cm-1; HRMS (ESI) m/z calcd for C15H20NaO5+ [M+Na]+ 303.1203, found 303.1199.

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(2S,2´R,3S,4R,5´S)-3-Hydroxy-5´-((1R)-1,7-dihydroxyhept-4-en-2-yn-1-yl)-4methylhexahydro-[2,2´-bifuran]-5(2H)-one (53) Lactone 51 (5.6 mg, 18 mmol) was dissolved in THF (2 mL) and (CH3)3SiOK (1.5 eq, 3.5 mg, 27 µmol) was added to the stirred solution at rt. The solution became rapidly turbid and the color changed to slightly orange. After 30 min complete conversion of the starting material was observed. Then water (0.1 mL) and CSA (5 mg) were added and the solution was stirred at room temperature. The formation of the five-membered lactone 53 and another, slightly less polar byproduct, was detected by TLC (CH2Cl2/MeOH 10:1) and shown to be complete after 4 h. The solution was diluted with water and EtOAc and the organic layer was separated. The aqueous layer was extracted three times with EtOAc and the combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, CH2Cl2/MeOH 20:1) yielded lactone 53 (1.4 mg, 4.5 µmol, 25%) as a colorless oil and the slightly less polar byproduct (1.2 mg). Lactone 53 proved to be hardly soluble in most solvents except MeOH. Beginning decomposition was observed upon standing for 2 days in solution. Analytical data for 53 are: []22D = +97.5 (c 0.16, CH3OH); 1H NMR (600 MHz, CD3OD) δ 6.19 (dt, J = 15.9, 7.2 Hz, 1H, H-13), 5.64 (dq, J = 15.9, 1.6 Hz, 1H, H-12), 4.52 (d, J = 6.2 Hz, 1H, H-3), 4.44 (dd, J = 4.0, 1.8 Hz, 1H, H-9), 4.34 (d, J = 4.3 Hz, 1H, H-4), 4.11 (ddd, J = 8.8, 6.2, 4.3 Hz, 1H, H-5), 4.06 (td, J = 6.6, 6.2, 4.0 Hz, 2H, H-8), 3.62 (t, J = 6.5 Hz, 2H, H-15), 3.09 (qd, J = 7.4, 6.1 Hz, 1H, H-2), 2.36 (qd, J = 6.6, 1.6 Hz, 2H, H-14), 2.10–2.04 (m, 2H, H-6a, H7a), 2.03–1.98 (m, 1H, H-7b), 1.91–1.86 (m, 1H, H-6b), 1.18 (d, J = 7.5 Hz, 3H, H-16); 13C{1H}

NMR (151 MHz, CD3OD) δ 181.7 (C-1), 142.9 (C-13), 112.0 (C-12), 89.6 (C-4),

87.7 (C-10), 84.8 (C-11), 83.9 (C-8), 80.4 (C-5), 70.9 (C-3), 65.6 (C-9), 62.0 (C-15), 40.8 (C2), 37.3 (C-14), 28.3 (C-6), 27.4 (C-7), 8.6 (C-16) ppm; IR (ATR) ṽ 3348 (ss, br), 2927 (s), 2883 (s), 1762 (ss), 1651 (m), 1577 (ss), 1448 (m), 1413 (s), 1378 (m), 1316 (s), 1179 (s), 1089 (s), 1040 (ss), 987 (m) cm-1; HRMS (ESI) m/z calcd for C16H22NaO6+ [M+Na]+ 333.1309, found 333.1303. (3R,9S,10R,E)-10-Ethynyl-4-hydroxy-9-(p-methoxybenzyl)oxy-3-methyl-3,4,7,8,9,19hexahydro-2H-oxecin-2,4-dione (54) The PMB protected macrolide 35 (39.2 mg, 0.114 mmol) was dissolved in CH2Cl2 (2 mL) and Dess-Martin periodinane (1.5 eq, 72 mg, 0.17 mmol) was added. The suspension was stirred at room temperature until complete conversion was monitored. Then the solvent was removed in vacuo at room temperature and the residue was purified by column chromatography (hexanes/EtOAc 4:1). Enone 54 was isolated as a colorless oil (30.4 mg, 88.8 µmol, 78%): []21D = +124.1 (c 1.70, CHCl3); 1H NMR (600

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MHz, CDCl3) δ 7.16 (d, J = 8.6 Hz, 2H, H-15), 6.92–6.83 (m, broad, 1H, H-5), 6.86 (d, J = 8.6 Hz, 2H, H-16), 5.99 (t, J = 2.4 Hz, 1H, H-9), 5.69 (d, broad, J = 16.6 Hz, 1H, H-4), 4.62 (d, J = 11.0 Hz, 1H, H-13a), 4.27 (d, J = 11.0 Hz, 1H, H-13b), 4.01 (s, br, 1H, H-2), 3.83 (dd, J = 9.2, 1.6 Hz, 1H, H-8), 3.80 (s, 3H, H-18), 2.56 (d, J = 2.4 Hz, 1H, H-11), 2.44 (qd, J = 11.9, 4.0 Hz, 1H, H-6a), 2.34–2.29 (m, 1H, H-6b), 2.24 (dddd, J = 15.6, 11.8, 4.0, 1.0 Hz, 1H, H-7a), 2.15 (ddt, J = 14.3, 9.0, 4.4 Hz, 1H, H-7b), 1.33 (d, J = 6.7 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 196.4 (C-3), 170.4 (C-1), 159.4 (C-17), 151.2 (broad, low intensity, C-5), 129.8 (C-14), 129.3 (C-15), 127.0 (broad, low intensity, C-4), 114.0 (C-16), 79.7 (C-8), 79.0 (C-10), 75.9 (C-11), 71.4 (C-13), 64.5 (C-9), 55.4 (C-18), 53.5 (C-2), 28.0 (C-6), 27.3 (broad, low intensity, C-7), 10.6 (C-12) ppm; IR (ATR) ṽ 3271 (w), 2938 (w), 2865 (w), 1740 (ss), 1688 (ss), 1513 (s), 1328 (m), 1300 (m), 1246 (ss), 1204 (m), 1176 (m), 1133 (s), 1116 (s), 1032 (m), 976 (m) cm-1; HRMS (ESI) m/z calcd for C20H22NaO5+ [M+Na]+ 365.1359, found 365.1347. (1S,2R,5R,8R)-2-Ethynyl-5-methyl-3,11-dioxabicyclo[6.2.1]undecan-4,6-dione (56) To a solution of the PMB protected enone 54 (23.4 mg, 68.3 µmol) in CH2Cl2 (3 mL) were added pH 6.7 buffer-solution (1 mL) and DDQ (2 eq, 31 mg, 0.14 mmol). The biphasic reaction mixture was intensively stirred at room temperature. When complete conversion was monitored by TLC, sat. NaHCO3-solution and Na2SO3 (10 mg) were added. After further stirring for 5 min, the organic layer was separated and the aqueous layer was extracted three times with CH2Cl2. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (hexanes/EtOAc 3:1) to give the deprotected enone as a colorless oil (10.2 mg, 45.9 µmol, 67%). The enone (10.2 mg, 45.9 µmol) was then dissolved in CH2Cl2 (2 mL) and CSA (1 eq, 11 mg, 46 µmol) was added. The solution was stirred at room temperature for 3 d, until complete conversion was detected (TLC). The solution was diluted with CH2Cl2, sat. NaHCO3-solution was added and the organic layer was separated. The aqueous layer was extracted three times with CH2Cl2, dried over MgSO4, filtered and concentrated in vacuo. Purification by column chromatography (silica gel, hexanes/EtOAc 3:1) yielded the bicyclic lactone 56 as a colorless, readily crystallizing solid (6.1 mg, 27 µmol, 60%): []22D = -46.2 (c 0.65, CHCl3); mP = 176– 177 °C; 1H NMR (600 MHz, CDCl3) δ 6.00 (dd, J = 4.4, 2.3 Hz, 1H, H-9), 4.58 (ddd, J = 11.1, 6.9, 4.4 Hz, 1H, H-5), 4.26 (ddd, J = 8.8, 7.3, 4.4 Hz, 1H, H-8), 3.77 (q, J = 6.7 Hz, 1H, H-2), 3.22 (dd, J = 11.8, 11.0 Hz, 1H, H-4a), 2.56 (d, J = 2.3 Hz, 1H, H-11), 2.36 (dd, J = 11.9, 4.3 Hz, 1H, H-4b), 2.34–2.29 (m, 1H, H-7a), 2.20 (dtd, J = 13.1, 7.6, 1.5 Hz, 1H, H-7b), 36


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2.02 (tdd, J = 12.3, 7.8, 7.1 Hz, 1H, H-6a), 1.79 (dd, J = 12.5, 7.2 Hz, 1H, H-6b), 1.26 (d, J = 6.7 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 202.7 (C-3), 169.1 (C-1), 80.1 (C-8), 78.5 (C-5), 77.7 (C-10), 76.3 (C-11), 65.2 (C-9), 57.0 (C-2), 47.1 (C-4), 30.9 (C-6), 26.2 (C7), 10.4 (C-12) ppm; IR (ATR) ṽ 3252 (ss), 2977 (w), 2962 (w), 2949 (w), 2935 (w), 2914 (w), 2876 (w), 1737 (ss), 1708 (ss), 1372 (m), 1357 (m), 1241 (m), 1204 (s), 1186 (s), 1164 (m), 1117 (s), 1086 (m), 1065 (m), 1042 (m) cm-1; HRMS (ESI) m/z calcd for C12H14NaO4+ [M+Na]+ 245.0784, found 245.0784. (1S,2R,5R,8R)-2-(1-(tert-Butyloxycarbonylmethyl)-1H-1,2,3-triazol-4-yl)-5-methyl-3,11dioxabicyclo[6.2.1]undecan-4,6-dione (57) To a solution of alkyne 56 (6.0 mg, 27 µmol) in tBuOH

(0.5 mL) were added water (0.5 mL), tert-butyl azidoacetate (43)34 (excess, 50 µL),

Cu(OAc)2 (0.4 eq, 2.0 mg, 11 µmol) and Na2S2O4 (4 mg). The initial color changed from light blue to intensive yellow. The reaction mixture was vigorously stirred for 16 h and then diluted with water and EtOAc. The organic layer was separated and the aqueous layer was extracted with EtOAc three times. The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc 1:1 to 1:2) yielding triazole 57 (6.2 mg, 16 µmol, 61%) as a colorless solid: []23D = -50.0 (c 0.59, CHCl3); mP = 170–172 °C; 1H NMR (600 MHz, CDCl3) δ 7.73 (s, 1H, H-11), 6.61 (d, J = 4.2 Hz, 1H, H-9), 5.10 (d, J = 17.5 Hz, 1H, H-13a), 5.06 (d, J = 17.5 Hz, 1H, H-13b), 4.63 (ddd, J = 12.4, 6.4, 2.9 Hz, 1H, H-5), 4.59 (ddd, J = 8.7, 5.7, 3.5 Hz, 1H, H8), 3.83 (q, J = 6.7 Hz, 1H, H-2), 3.24 (dd, J = 11.8, 10.7 Hz, 1H, H-4a), 2.41 (dd, J = 11.9, 4.4 Hz, 1H, H-4b), 2.20–2.13 (m, 1H, H-7a), 2.05–1.95 (m, 2H, H-6a, H-7b), 1.77 (dd, J = 11.2, 7.0 Hz, 1H, H-6b), 1.50 (s, 9H, H-16), 1.29 (d, J = 6.7 Hz, 3H, H-12); 13C{1H} NMR (151 MHz, CDCl3) δ 203.2 (C-3), 169.8 (C-1), 165.2 (C-14), 144.2 (C-10), 123.5 (C-11), 84.2 (C-15), 80.8 (C-8), 78.0 (C-5), 70.4 (C-9), 57.2 (C-2), 51.7 (C-13), 47.3 (C-4), 31.1 (C-6), 28.2 (C-16), 25.6 (C-7), 10.6 (C-12) ppm; IR (ATR) ṽ 3144 (w), 3103 (w), 2956 (m), 2874 (w), 1749 (ss), 1714 (s), 1459 (m), 1366 (m), 1237 (s), 1204 (m), 1180 (s), 1156 (ss), 1111 (ss), 1088 (m), 1065 (m), 1056 (m), 1041 (m) cm-1; HRMS (ESI) m/z calcd for C18H26N3O6+ [M+H]+ 380.1816, found 380.1806. SUPPORTING INFORMATION AVAILABILITY Determination of relative stereochemistry, copies of 1H and

13C

NMR for all compounds

including the numbering system for allocation of protons and carbon atoms is available free of charge at: 37



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ACKNOWLEDGEMENT We wish to thank the Fonds der Chemischen Industrie for financial support of this research. REFERENCES 1

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For the synthesis of 20 please refer to ref. 4.

38

This reagent was prepared according to: Paterson, I.; Steadman, V. A.; McLeod, M. D.; Trieselmann, T.

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Benzyl azide (42) was prepared as follows: Sodium azide (72 mg, 1.1 mmol) was suspended in DMSO (2 mL)

and benzyl bromide (0.171 g, 1.0 mmol) was added. Sodium azide was dissolved after a while and the solution was stirred overnight. Then water and diethyl ether were added and the organic layer was separated. The aqueous layer was extracted once with diethyl ether and the combined organic layers were dried over MgSO4, filtered and concentrated at 30 mbar. The obtained crude benzyl azide (42) was used without further purification. 40

tert-Butyl azidoacetate (43) was prepared as follows: Sodium azide (72 mg, 1.1 mmol) was suspended in

DMSO (2 mL) and tert-butyl bromoacetate (0.195 mg, 1.0 mmol) was added. Sodium azide was dissolved after a while and the solution was stirred overnight. Then water and diethyl ether were added and the organic layer was separated. The aqueous layer was extracted once with diethyl ether and the combined organic layers were dried over MgSO4, filtered and concentrated at 100 mbar. The crude solution of tert-butyl azidoacetate (43) was used without further purification. 41

3-Butenyl azide (44) was prepared as follows: Sodium azide (71 mg, 1.1 mmol) was suspended in DMSO (2

mL) and 4-bromo-1-butene (0.135 mg, 1.0 mmol) was added. Sodium azide was dissolved within 1 h and the solution turned slightly orange. After stirring overnight, water and diethyl ether were added. The organic layer was separated and the aqueous layer was extracted once with diethyl ether. The combined organic layers were dried over MgSO4, filtered and concentrated at 500 mbar. The crude solution of 3-butenyl azide (44) was used without further purification.

41



42

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(E)-4-iodobut-3-en-1-ol (49) was prepared according to: Chong, J. M.; Heuft, M. A. Hydroalumination of 3-

butyn-1-ol: Application to a stereoselective synthesis of (3E,5Z)-3,5-dodecadienyl acetate, the sex pheromone of the leaf roller. Tetrahedron 1999, 55, 14243–14250.

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Synthesis of Cytospolide Analogues and Late-State Diversification

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