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Synthesis of 3-C-Branched Kdo Analogs via Sonogashira Coupling of 3-Iodo Kdo Glycal with Terminal Alkynes Wenjing Fan, Yan Chen, Qixin Lou, Liqin Zhuang, and You Yang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00356 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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The Journal of Organic Chemistry

Synthesis of 3-C-Branched Kdo Analogs via Sonogashira Coupling of 3-Iodo Kdo Glycal with Terminal Alkynes Wenjing Fan, Yan Chen, Qixin Lou, Liqin Zhuang and You Yang* Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Supporting Information Placeholder OAc

AcO AcO

O AcO

COOEt

+ R

1) Pd(OAc)2, Cs2CO3, DMF, rt 2) H2, Pd(OH)2/C, EtOH, rt 3) NaOH, dioxane, MeOH, H2O, rt

HO R HO HO O COOH HO

I

ABSTRACT: A highly efficient approach for the synthesis of 3-C-branched mono- and di-Kdo enyne analogs is developed for the first time based on Sonogashira coupling of terminal alkynes with 3-iodo Kdo glycal obtained by NIS/TMSOTf-promoted one-step reaction from peracetylated Kdo ethyl ester. Further transformation of 3-C-branched mono- and di-Kdo enyne analogs by asymmetric hydrogenation and saponification provided 2-deoxy-β-carboxyl Kdo analogs in a stereocontrolled mode.

In the family of 2-keto-3-deoxy sugar acids, 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) is often found as an essential component of the inner core structures of lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria.1-3 In addition to the synthesis of Kdo-containing oligosaccharides for the development of carbohydrate-based vaccines,4,5 creation of Kdo analogs as potential therapeutic agents serves as another strategy combating antibiotic-resistant bacteria.6 From a biosynthetic point of view, assembly of LPS is critical for the survival of bacteria, and involves the Kdo metabolism via multiple enzymatic steps for subsequent attachment of Kdo to lipid A.6 Based on well-founded biochemical and NMR spectroscopic data, only a few types of Kdo analogs such as 2deoxy-β-Kdo and its 8-amino derivatives have been found as CMP-Kdo synthetase (CKS) inhibitors, while the 2-deoxy-αKdo counterparts are not active.6-8 In view of the potential application of 2-deoxy-β-Kdo analogs as CKS inhibitors, the 2-deoxy-α-Kdo analogs are often utilized as important precursors for preparation of the corresponding β-Kdo products via the Burke′s epimerization protocol.8d,8e Due to the enzymatic and chemical stability of C–C bonds, C-branched sugars have been extensively studied as sugar mimetics for exploring their biological application as inhibitors of carbohydrate-processing enzymes and modulators of biological processes.9-11 Furthermore, they can serve as important chiral synthons for constructing naturally occurring antibiotics, macrolides, and polysaccharides.12 Considering the potential use of new types of Kdo analogs for glycobiological research and complex natural product synthesis, assembly of structurally diverse C-branched Kdo analogs is very attractive. However, to the best of our knowledge, C-modification of

Kdo at the non-anomeric branching points to produce 3-Cbranched Kdo analogs has never been reported. In contrast to the synthesis of 2-C-branched aldoses,10 stereocontrolled synthesis of 3-C-branched mono- and di-Kdo analogs might be quite elusive considering the absence of a hydroxyl group at the C-3 position and the presence of an electron-withdrawing carboxylic group at the C-1 position. Specificly, there is still no effective method for the direct formation of 3-iodo Kdo glycal from peracetylated Kdo esters. Furthermore, the couplings between 3-iodo Kdo glycal and coupling partners as well as the asymmetric hydrogenation of the resulting double bonds remain uncertain due to electronic and steric effects of the unique eight-carbon acidic keto-sugar skeleton. Here, we report a highly efficient method to access the 3-C-branched mono- and di-Kdo enyne analogs based on Sonogashira coupling of 3-iodo Kdo glycal with a range of terminal alkynes and dialkynes. Moreover, asymmetric hydrogenation of the resulting Kdo enyne analogs over Pearlman′s catalyst followed by saponification can be effectively carried out to provide the 2-deoxy-β-carboxyl Kdo analogs in a stereospecific manner. With respect to 2-iodo glycals, they were often employed as substrates of Heck, Sonogashira, and Suzuki-Miyaura coupling for the synthesis of 2-C-branched aldoses, and usually prepared by one-step reaction of glycals with NIS/AgNO3 or two-step transformations involving treatment of glycals with iodonium ion in aqueous medium followed by elimination with Ph2SO/Tf2O.10 Thus, we envisioned that the desired tetraO-acetyl-3-iodo Kdo glycal 3 could be synthesized by activation of Kdo glycal 2 with NIS/AgNO3. In the beginning, Kdo glycal 2 was readily prepared by the known TMSOTf (0.2 eq.)-catalyzed elimination reaction of 2,4,5,7,8-penta-O-acetyl

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Kdo ethyl ester 1 at room temperature in 88% yield (Scheme 1).13 As expected, exposure of Kdo glycal 2 to NIS (2.8 eq.) and AgNO3 (0.8 eq.) in acetonitrile at 60 °C gave 3-iodo Kdo glycal 3 in good yield (80%). Moreover, we found that an even higher yield (83%) of compound 3 was obtained by replacing AgNO3 with TMSOTf under the similar conditions, although almost no reaction occurred when other Lewis acids such as BF3‧OEt2 and AgOTf were used for this reaction. Since TMSOTf was the common promoter for the preparation of Kdo glycal 2 and 3-iodo Kdo glycal 3, it was envisaged that one-step synthesis of 3-iodo Kdo glycal 3 could be realized from Kdo ethyl ester 1 via the glycal intermediate 2 using NIS/TMSOTf as the promoter. Indeed, treatment of 1 with NIS (2.8 eq.) and AgNO3 (0.8 eq.) in acetonitrile at 60 °C resulted in a very clean reaction, providing 3-iodo Kdo glycal 3 in excellent yield (90%). Scheme 1. Efficient preparation of 3-iodo Kdo glycal 3.

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acetylene 6a in DMF at room temperature underwent a smooth reaction, affording 3-C-branched Kdo enyne 7a in 83% yield. Similarly, terminal alkynes with different alkyl chain lengths (1-hexyne 6b, 1-dodecyne 6c, and 1-octadecyne 6d) were effectively coupled with compound 3 to provide 3-C-branched Kdo enynes 7b-7d in good yields (66–84%). Pd(OAc)2catalyzed Sonogashira coupling of 2-methyl-1-buten-3-yne 6e with 3 gave the corresponding 3-C-branched Kdo enyne 7e in 81% yield, without affecting the terminal double bond in the branching chain. Moreover, when terminal alkynes bearing ester, amide, and phenyl groups (methyl 5-hexynoate 6f, diethyl 2-propynylmalonate 6g, 2-phenylethylacetylene 6h, N(4-pentynyl)phthalimide 6i) were utilized as coupling partners, the coupled Kdo enynes 7f-7i could also be obtained in high yields (79–85%). Intriguingly, Sonogashira coupling of terminal alkyne-containing steroid hormone drug levonorgestrel14 6j with 3-iodo Kdo glycal 3 proceeded smoothly to afford Kdolevonorgestrel enyne conjugate 7j in 78% yield, paving a new way for the synthesis of carbohydrate-modified drugs. Scheme 3. Sonogashira coupling of 3-iodo Kdo glycal 3 with terminal alkynes 6a-6j. OAc

AcO AcO

Pd(OAc)2 (0.05 eq.), Cs2CO3 (1.5 eq.), DMF, rt

O AcO

COOEt

+ R

OAc

AcO AcO

O AcO

COOEt

I

3

6a-6j

7a-7j R

AcO AcO

OAc

AcO AcO

To procure the 3-C-branched Kdo analogs, Pd(OAc)2catalyzed Heck coupling of 3-iodo Kdo glycal 3 with methyl acrylate 4 was first attempted (Scheme 2). Although the synthesis of 2-C-branched diene analogs by Heck coupling of 2iodo glucal or 2-iodo galactal with methyl acrylate could be efficiently achieved,10c the coupling reaction of 3-iodo Kdo glycal 3 with 4 under the catalysis of Pd(OAc)2 (0.3 eq.) in the presence of PPh3 (0.5 eq.) and Et3N (2 eq.) in DMF at 90 °C led to an incomplete reaction, in which the 3-C-branched Kdo diene 5 was isolated in only 53% yield and the starting material 3 was recovered in 37% yield. This result might be attributed to the influence of electronic effect of the C-1 carboxylic group in the Kdo unit. Scheme 2. Heck coupling of 3-iodo Kdo glycal 3 with methyl acrylate 4.

COOEt

OAc

AcO AcO O

O AcO

OAc

AcO AcO

O

AcO

COOEt

O

AcO

COOEt

AcO

COOEt

C10H21 7a (83%)

AcO AcO

OAc

AcO AcO

AcO AcO O

O

7d (78%)

OAc

AcO AcO

COOEt

COOEt

OAc O

O

AcO

AcO

C16H33

7c (66%)

7b (84%) OAc

OAc

AcO

COOEt

AcO

COOEt

EtOOC COOEt

MeOOC 7e (81%)

AcO AcO

7g (85%)

7f (81%)

7h (79%)

OAc OAc

O AcO

COOEt

OAc

OH O H

H H

O

OAc OAc

COOEt H

O

N O

7j (78%)

7i (80%)

Next, we explored Sonogashira coupling of 3-iodo Kdo glycal 3 with terminal alkynes to produce the 3-C-branched Kdo enynes (Scheme 3). Employing Pd(OAc)2 (0.05 eq.) as catalyst and Cs2CO3 (1.5 eq.) as base,10d copper-free Sonogashira coupling of 3-iodo Kdo glycal 3 with cyclopropyl

To demonstrate the feasibility of Sonogashira coupling of terminal dialkynes with 3-iodo Kdo glycal 3, dialkynes 8a-8c with different alkyl chain lengths were selected for accessing the 3-C-branched di-Kdo enynes (Scheme 4). By tuning the reaction parameters, Sonogashira coupling reactions of excess


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amount of compound 3 (3 eq.) with 1,5-hexadiyne 8a, 1,7octadiyne 8b, and 1,9-decadiyne 8c under the promotion of Pd(OAc)2 (0.3 eq.) and Cs2CO3 (5 eq.) in DMF at room temperature gave di-Kdo enynes 9a-9c in 36–78% yields, respectively, although a minor amount of mono-Kdo enynes could also be detected. With the extension of alkyl chain lengths between the terminal triple bonds in 8a-8c, the coupling yields of 9a-9c were found to be significantly improved, indicating that the steric hindrance between the two Kdo residues in the di-Kdo enynes 9a-9c might have a profound influence on the outcome of the coupling reactions. Scheme 4. Sonogashira coupling of 3-iodo Kdo glycal 3 with terminal dialkynes 8a-8c.

With the 3-C-branched mono- and di-Kdo enynes in hand, we then turned our attention to the hydrogenation of the C＝C and C≡C bonds on the enyne skeletons.15 Exhilaratingly, asymmetric hydrogenation of mono-Kdo enynes 7c, 7d, and 7f-7h over Pearlman′s catalyst for overnight favored complete

reduction of C≡C bonds and cis-addition to the C＝C bonds from the bottom face probably arising from the inherent stereochemistry of the Kdo scaffolds, providing 2-deoxy-βcarboxyl Kdo analogs 10-14 as the only products in good to excellent yields (78–97%; Scheme 5).16 The newly forming stereocenters of 10-14 were unambiguously confirmed based on the coupling constant of H-3 and H-4 (JH-3,H-4 < 6.0 Hz) and the observed NOE effects (H-2/H-4, H-2/H-6; see Experimental Section and Supporting Information for details). Following this reaction procedure, di-Kdo enyne 9b was also subjected to the substrate-controlled asymmetric hydrogenation to afford 2-deoxy-β-carboxyl Kdo disaccharide analog 15 in an excellent 92% yield. As demonstrated in the structure assignment of compounds 10-14, the structure of 15 was also determined by the analysis of 1H, 13C, and 2D NMR spectra (see Experimental Section and Supporting Information for details). Scheme 6. Synthesis of 3-C-branched Kdo analogs 16-19.

Scheme 5. Asymmetric hydrogenation of 3-C-branched Kdo enynes 7c, 7d, 7f-7h, and 9b.



AcO AcO

OAc

O

O AcO

HO

COOEt

7d, 9b R

COOH

16, 17 R

NaOH, dioxane, MeOH, H2O, rt

HO R HO HO O COOH HO

AcO R AcO AcO O COOEt AcO

18, 19

11, 15 HO

OH

HO HO

OH

HO

OH

HO

HO

O

O HO

HO

COOH

COOH

HOOC

OH O OH OH OH

C16H33 16 (75%) HO HO HO HO C16H33 HO HO O COOH HO 18 (88%)

O

HOOC

OH O OH

HO

COOH

19 (92%)

OH OH 17 (90%)

Finally, deprotection of the 3-C-branched Kdo enynes (7d, 9b) and the 2-deoxy-β-carboxyl Kdo analogs (11, 15) was performed (Scheme 6). Saponification of all ester groups in 7d and 9b under the basic conditions (NaOH, dioxane, MeOH, H2O, rt) afforded 3-C-branched Kdo enyne analogs 16 and 17 in good yields (75% for 16; 90% for 17), without the formation of the bicyclic byproducts that could be generated from the attack of the C-1 carboxylic group on the C≡C bond. Removal of all ester groups in 11 and 15 under the similar conditions furnished 2-deoxy-β-carboxyl Kdo analogs 18 and 19 in excellent yields (88% for 18; 92% for 19). In summary, we have developed a facile and efficient method for the synthesis of a series of 3-C-branched mono- and diKdo enyne analogs via Sonogashira coupling of 3-iodo Kdo glycal with terminal alkynes and dialkynes. One-step synthesis of 3-iodo Kdo glycal from peracetylated Kdo ethyl ester in the presence of NIS/TMSOTf was achieved in excellent yield. A three-step reaction sequence involving Sonogashira coupling, asymmetric hydrogenation and saponification was described for efficient construction of the 2-deoxy-β-carboxyl Kdo analogs in a stereospecific fashion. The present method serves as basis for further synthesis of the corresponding 3-C-branched 2-deoxy-β-Kdo products as potential enzyme inhibitors.

EXPERIMENTAL SECTION General Information. All reactions were performed with anhydrous solvents in oven-dried glassware with magnetic stirring under argon or nitrogen unless otherwise stated. The chemicals were used as supplied except where noted. Acetonitrile (CH3CN) and N,N-dimethylformaide (DMF) were dried over freshly activated 4Å molecular sieves. Analytical thin layer chromatography (TLC) was conducted on precoated plates of silica gel (0.25-0.3 mm, Shanghai, China). The TLC plates were visualized by exposure to UV light or by staining with a sulfuric acid-ethanol solution. Silica gel column chro-

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matography was performed on silica gel AR (100-200 mesh, Shanghai, China). NMR spectra were recorded with a Bruker Avance III 400 or Bruker Avance III 500 spectrometer. The 1H and 13C NMR spectra were calibrated against the residual proton and carbon signals of the solvents as internal references (CDCl3:‧δH = 7.26 ppm and δC = 77.2 ppm; CD3OD:‧δH = 3.31 ppm and δC = 49.0 ppm; D2O: δH = 4.79). Multiplicities are quoted as singlet (s), broad singlet (br s), doublet (d), triplet (t), quartet (q), doublet of doublets (dd), doublet of doublet of doublets (ddd) or multiplet (m). Optical rotations (OR) were measured with a Rudolph Research Analytical Autopol I automatic polarimeter. IR spectra were recorded on a Nicolet 6700 Fourier transform infrared spectrometer and are reported in wave numbers (cm–1). High-resolution mass spectra were recorded on ESI-TOF spectrometers. 3-Iodo Kdo glycal 3. NIS (159 mg, 0.706 mmol, 2.8 eq.) and TMSOTf (38 µL, 0.202 mmol, 0.8 eq.) were added to a solution of 2,4,5,7,8-penta-O-acetyl Kdo ethyl ester 1 (120 mg, 0.252 mmol, 1 eq.) in anhydrous CH3CN (6 mL) at room temperature under argon. The mixture was stirred at 60 °C for overnight and then quenched with saturated aqueous NaHCO3, diluted with CH2Cl2, and washed with brine and 10% aqueous Na2S2O3. The organic layer was dried over Na2SO4 and filtered. Concentration in vacuo and purification by silica gel column chromatography (petroleum ether/EtOAc: 7/1) afforded 3 (123 mg, 90%) as a pale yellow syrup: [α]25D = +45.8 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.66 (dd, J = 0.8, 4.4 Hz, 1 H), 5.43 (dd, J = 0.8, 4.8 Hz, 1 H), 5.23 (ddd, J = 2.4, 3.6, 9.6 Hz, 1 H, H-7), 4.52 (dd, J = 2.0, 12.4 Hz, 1 H, H-8a), 4.42 (d-like, J = 9.6 Hz, 1 H, H-6), 4.29 (q, J = 7.2 Hz, 2 H), 4.16 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.10 (s, 3 H), 2.08 (s, 6 H), 2.03 (s, 3 H), 1.35 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 169.8, 169.6, 161.4, 146.2, 74.6, 73.7, 68.3, 67.1, 62.5, 62.4, 61.9, 20.9, 20.8, 20.7, 14.1; HRMS (ESI) m/z calcd for C18H23O11INa [M + Na]+ 565.0183, found 565.0184. IR (film) 2961, 2921, 1751, 1372, 1225 cm–1. 3-C-Branched Kdo diene 5. To a stirred mixture of 3-iodo Kdo glycal 3 (68 mg, 0.125 mmol, 1 eq.), Pd(OAc)2 (8.5 mg, 0.038 mmol, 0.3 eq.), Et3N (35 µL, 0.25 mmol, 2 eq.) and PPh3 (16 mg, 0.063 mmol, 0.5 eq.) in anhydrous DMF (2.5 mL) at room temperature, was added dropwise methyl acrylate 4 (45 µL, 0.50 mmol, 4 eq.) under argon. After the reaction was stirred at 90 °C for overnight, the mixture was diluted with deionized water and then extracted with CH2Cl2. The organic layer was dried over Na2SO4 and filtered. Concentration in vacuo and purification by silica gel column chromatography (petroleum ether/EtOAc: 6/1) provided 5 (33 mg, 53%) as a colorless syrup: [α]25D = +59.0 (c 0.63, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 16.4 Hz, 1 H), 5.95 (d, J = 4.8 Hz, 1 H), 5.64 (d, J = 16.4 Hz, 1 H), 5.53 (d, J = 5.2 Hz, 1 H), 5.31 (ddd, J = 2.4, 3.6, 9.6 Hz, 1 H, H-7), 4.54 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.33 (m, 3 H), 4.19 (dd, J = 4.0, 12.4 Hz, 1 H, H8b), 3.73 (s, 3 H), 2.09 (s, 3 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.37 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.5, 170.3, 169.6, 167.1, 161.2, 147.8, 137.4, 119.7, 114.4, 73.3, 67.1, 63.9, 62.6, 61.9, 61.8, 51.9, 20.9, 20.8, 20.7, 14.2; HRMS (ESI) m/z calcd for C22H28O13Na [M + Na]+ 523.1428, found 523.1426. IR (film) 3558, 2972, 2923, 1751, 1375, 1227 cm–1. General Procedure for the Synthesis of 3-C-Branched Kdo Enynes 7a-7j. To a stirred mixture of 3-iodo Kdo glycal


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3 (1 eq.), Pd(OAc)2 (0.05 eq.), and Cs2CO3 (1.5 eq.) in anhydrous DMF at room temperature, was added dropwise terminal alkyne (1.5 eq.) under argon. After the reaction was stirred at room temperature for overnight, concentrated aqueous solution of ammonium hydroxide was added. The mixture was extracted with ethyl acetate and then washed with deionized water. The organic layer was dried over Na2SO4 and filtered,. Concentration in vacuo and purification by silica gel column chromatography provided the coupled Kdo enyne. 7a (83% yield, colorless syrup): [α] 25D = +30.3 (c 0.6, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.71 (d, J = 4.0 Hz, 1 H), 5.42 (d, J = 4.4 Hz, 1 H), 5.23 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.56 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.28 (m, 3 H), 4.20 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.07 (s, 3 H), 2.06 (s, 3 H), 2.05 (s, 3 H), 2.02 (s, 3 H), 1.39 (m, 1 H), 1.33 (t, J = 7.2 Hz, 3 H), 0.84 (m, 2 H), 0.71 (m, 2 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 170.1, 169.6, 160.9, 147.4, 104.5, 103.7, 73.2, 68.9, 67.3, 65.8, 61.9, 61.8, 61.2, 20.9, 20.8, 20.7, 20.6, 14.3, 9.1, 0.9; HRMS (ESI) m/z calcd for C23H28O11Na [M + Na]+ 503.1529, found 503.1528. IR (film) 2976, 2935, 2221, 1751, 1374, 1226 cm–1. 7b (84% yield, pale yellow syrup): [α]25D = +54.7 (c 0.87, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.74 (d, J = 4.4 Hz, 1 H), 5.44 (d, J = 4.8 Hz, 1 H), 5.24 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.55 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.29 (m, 3 H), 4.20 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.36 (t, J = 6.8 Hz, 2 H), 2.08 (s, 3 H), 2.07 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.50 (m, 2 H), 1.41 (m, 2 H), 1.32 (t, J = 7.2 Hz, 3 H), 0.89 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.7, 170.5, 170.1, 169.7, 160.9, 147.4, 104.3, 100.5, 73.6, 73.2, 67.3, 65.8, 62.0, 61.8, 61.2, 30.7, 22.1, 20.9, 20.8, 20.7, 20.6, 19.7, 14.3, 13.7; HRMS (ESI) m/z calcd for C24H32O11Na [M + Na]+ 519.1842, found 519.1846. IR (film) 2969, 2923, 1753, 1373, 1225 cm–1. 7c (66% yield, colorless syrup): [α] 25D = +47.0 (c 0.56, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.73 (d, J = 4.4 Hz, 1 H), 5.44 (dd, J = 0.8, 4.4 Hz, 1 H), 5.24 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.56 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.29 (m, 3 H), 4.20 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.35 (t, J = 7.2 Hz, 2 H), 2.08 (s, 3 H), 2.07 (s, 3 H), 2.04 (s, 3 H), 2.03 (s, 3 H), 1.51 (m, 2 H), 1.33 (t, J = 7.2 Hz, 3 H), 1.24 (m, 14 H), 0.86 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.5, 170.1, 169.6, 160.9, 147.4, 104.3, 100.6, 73.5, 73.2, 67.3, 65.8, 62.0, 61.8, 61.2, 32.0, 29.7, 29.6, 29.5, 29.3, 29.1, 28.8, 22.8, 20.9, 20.8, 20.7, 20.6, 20.1, 14.3; HRMS (ESI) m/z calcd for C30H44O11Na [M + Na]+ 603.2781, found 603.2777. IR (film) 2927, 2856, 1753, 1373, 1227 cm–1. 7d (78% yield, colorless syrup): [α] 25D = +37.3 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.73 (d, J = 4.4 Hz, 1 H), 5.43 (d, J = 4.4 Hz, 1 H), 5.24 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.55 (dd, J = 2.0, 12.4 Hz, 1 H, H-8a), 4.28 (m, 3 H), 4.20 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.35 (t, J = 7.2 Hz, 2 H), 2.08 (s, 3 H), 2.06 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.51 (m, 2 H), 1.32 (t, J = 7.2 Hz, 3 H), 1.24 (m, 26 H), 0.86 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 170.0, 169.6, 160.9, 147.4, 104.3, 100.5, 73.5, 73.2, 67.3, 65.8, 61.9, 61.8, 61.2, 32.1, 29.8, 29.7, 29.5, 29.3, 29.1, 28.7, 22.8, 20.9, 20.8, 20.7, 20.6, 20.1, 14.3, 14.2; HRMS (ESI) m/z calcd for C36H56O11Na [M + Na]+ 687.3720, found 687.3727. IR (film) 2924, 2853, 1754, 1373, 1226 cm–1.

7e (81% yield, colorless syrup): [α] 25D = +73.0 (c 0.33, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.80 (d, J = 4.8 Hz, 1 H), 5.48 (d, J = 4.4 Hz, 1 H), 5.27 (m, 3 H), 4.58 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.35 (d-like, J = 9.6 Hz, 1 H, H-6), 4.30 (q, J = 7.2 Hz, 2 H), 4.22 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.10 (s, 3 H), 2.08 (s, 3 H), 2.07 (s, 3 H), 2.04 (s, 3 H), 1.35 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.7, 170.4, 170.0, 169.6, 160.7, 147.6, 126.8, 122.8, 103.7, 99.9, 81.8, 73.4, 67.3, 65.6, 62.0, 61.9, 61.0, 23.2, 20.9, 20.8, 20.7, 20.6, 14.3; HRMS (ESI) m/z calcd for C23H28O11Na [M + Na]+ 503.1529, found 503.1524. IR (film) 2974, 2924, 1752, 1375, 1228 cm–1. 7f (81% yield, colorless syrup): [α]25D = +50.0 (c 0.7, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.72 (d, J = 4.4 Hz, 1 H), 5.44 (d, J = 4.4 Hz, 1 H), 5.23 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.55 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.29 (m, 3 H), 4.20 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 3.66 (s, 3 H), 2.44 (m, 4 H), 2.09 (s, 3 H), 2.07 (s, 3 H), 2.05 (s, 3 H), 2.03 (s, 3 H), 1.84 (m, 2 H), 1.32 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 173.6, 170.7, 170.5, 170.1, 169.7, 160.8, 147.7, 103.9, 98.7, 74.5, 73.2, 67.3, 65.8, 61.9, 61.8, 61.1, 51.8, 32.8, 23.9, 20.9, 20.8, 20.7, 20.6, 19.5, 14.2; HRMS (ESI) m/z calcd for C25H32O13Na [M + Na]+ 563.1741, found 563.1722. 7g (85% yield, pale yellow syrup): [α]25D = +43.3 (c 0.73, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.68 (d, J = 4.4 Hz, 1 H), 5.42 (d, J = 4.8 Hz, 1 H), 5.22 (ddd, J = 2.4, 3.6, 9.6 Hz, 1 H, H-7), 4.54 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.31–4.16 (m, 8 H), 3.53 (t, J = 7.6 Hz, 1 H), 2.94 (d, J = 7.6 Hz, 2 H), 2.08 (s, 3 H), 2.07 (s, 6 H), 2.02 (s, 3 H), 1.33 (t, J = 7.2 Hz, 3 H), 1.26 (t, J = 6.8 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 170.2, 169.6, 167.9, 160.6, 148.2, 103.4, 95.1, 75.3, 73.3, 67.2, 65.5, 62.0, 61.9, 61.0, 51.3, 20.9, 20.8, 20.7, 20.6, 19.9, 14.2, 14.1; HRMS (ESI) m/z calcd for C28H36O15Na [M + Na]+ 635.1952, found 635.1951. 7h (79% yield, colorless syrup): [α] 25D = +45.2 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.30–7.19 (m, 5 H), 5.72 (d, J = 4.4 Hz, 1 H), 5.45 (d, J = 4.4 Hz, 1 H), 5.25 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.57 (dd, J = 2.0, 12.4 Hz, 1 H, H-8a), 4.34–4.26 (m, 3 H), 4.21 (dd, J = 4.0, 12.4 Hz, 1 H, H8b), 2.85 (t, J = 7.6 Hz, 2 H), 2.66 (t, J = 7.6 Hz, 2 H), 2.09 (s, 3 H), 2.08 (s, 3 H), 2.03 (s, 3 H), 1.98 (s, 3 H), 1.32 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 170.1, 169.6, 160.8, 147.6, 140.5, 128.6, 128.4, 126.5, 104.0, 99.3, 74.1, 73.2, 67.3, 65.7, 61.9, 61.8, 61.1, 35.0, 22.1, 20.9, 20.8, 20.7, 20.6, 14.2; HRMS (ESI) m/z calcd for C28H32O11Na [M + Na]+ 567.1842, found 567.1838. IR (film) 2965, 2923, 1747, 1719, 1373, 1243, 1227, 1201 cm–1. 7i (80% yield, colorless syrup): [α] 25D = +36.0 (c 0.83, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.81 (m, 2 H), 7.70 (m, 2 H), 5.68 (d, J = 4.4 Hz, 1 H), 5.43 (d, J = 4.4 Hz, 1 H), 5.22 (ddd, J = 2.4, 4.0, 9.6 Hz, 1 H, H-7), 4.54 (dd, J = 2.0, 12.4 Hz, 1 H, H-8a), 4.27 (m, 3 H), 4.19 (dd, J = 4.0, 12.4 Hz, 1 H, H8b), 3.76 (t, J = 6.8 Hz, 2 H), 2.44 (t, J = 7.2 Hz, 2 H), 2.08 (s, 3 H), 2.07 (s, 3 H), 2.06 (s, 3 H), 2.02 (s, 3 H), 1.91 (m, 2 H), 1.31 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 170.1, 169.6, 168.4, 160.7, 147.7, 134.1, 132.2, 123.4, 103.9, 98.3, 74.3, 73.2, 67.2, 65.7, 61.9, 61.8, 61.1, 37.3, 27.6, 20.9, 20.8, 20.7, 20.6, 17.9, 14.2; HRMS (ESI) m/z calcd for C31H33O13NNa [M + Na]+ 650.1850, found 650.1869.



7j (78% yield, colorless syrup): [α]25D = –1.9 (c 0.88, CHCl3); H NMR (400 MHz, CDCl3) δ 5.82 (s, 1 H), 5.77 (d-like, J = 4.4 Hz, 1 H), 5.44 (d, J = 4.8 Hz, 1 H), 5.24 (ddd, J = 2.4, 3.6, 9.6 Hz, 1 H, H-7), 4.55 (dd, J = 2.0, 12.4 Hz, 1 H, H-8a), 4.32 (d-like, J = 9.6 Hz, 1 H, H-6), 4.24 (q, J = 7.2 Hz, 2 H), 4.20 (dd, J = 4.0, 12.4 Hz, 1 H, H-8b), 2.49–2.38 (m, 2 H), 2.32– 2.21 (m, 4 H), 2.16–1.96 (m, 16 H), 1.88–1.80 (m, 2 H), 1.66– 1.36 (m, 7 H), 1.32 (t, J = 7.2 Hz, 3 H), 1.08 (m, 2 H), 0.99 (t, J = 7.2 Hz, 3 H), 0.85 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 200.0, 170.6, 170.4, 170.1, 169.6, 166.7, 160.5, 148.1, 124.8, 103.4, 102.4, 82.3, 79.6, 73.4, 67.2, 65.5, 62.0, 61.9, 61.0, 50.9, 49.1, 48.4, 42.6, 41.0, 39.6, 36.7, 35.7, 30.9, 28.7, 26.8, 20.9, 20.8, 20.7, 19.1, 14.3, 9.7; HRMS (ESI) m/z calcd for C39H50O13Na [M + Na]+ 749.3149, found 749.3152. IR (KBr) 2959, 2930, 2873, 2854, 1754, 1669, 1619, 1373, 1226 cm–1. General Procedure for the Synthesis of 3-C-Branched Di-Kdo Enynes 9a-9c. To a stirred mixture of 3-iodo Kdo glycal 3 (3 eq.), Pd(OAc)2 (0.3 eq.), and Cs2CO3 (5 eq.) in anhydrous DMF at room temperature, was added dialkyne (1 eq.) under argon. After the reaction was stirred at room temperature for overnight, concentrated aqueous solution of ammonium hydroxide was added. The mixture was extracted with ethyl acetate and then washed with deionized water. The organic layer was dried over Na2SO4 and filtered. Concentration in vacuo and purification by silica gel column chromatography provided the coupled di-Kdo enyne. 9a (36% yield, pale yellow syrup): [α]25D = +56.2 (c 0.43, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.75 (d, J = 4.4 Hz, 2 H), 5.44 (d, J = 4.8 Hz, 2 H), 5.25 (ddd, J = 2.4, 3.6, 9.6 Hz, 2 H, H-7), 4.56 (dd, J = 2.0, 12.4 Hz, 2 H, H-8a), 4.36 (d-like, J = 9.6 Hz, 2 H), 4.27 (q, J = 7.2 Hz, 4 H), 4.21 (dd, J = 4.0, 12.4 Hz, 2 H, H-8b), 2.60 (s, 4 H), 2.08 (s, 12 H), 2.03 (s, 12 H), 1.33 (t, J = 7.2 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 170.7, 170.4, 170.1, 169.6, 160.7, 147.8, 103.9, 97.6, 74.8, 73.3, 67.3, 65.7, 62.0, 61.9, 61.1, 20.9, 20.8, 20.7, 20.6, 20.2, 14.3; HRMS (ESI) m/z calcd for C42H50O22Na [M + Na]+ 929.2691, found 929.2700. 9b (66% yield, pale yellow syrup): [α]25D = +57.1 (c 0.47, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.72 (d, J = 4.4 Hz, 2 H), 5.43 (d, J = 4.4 Hz, 2 H), 5.24 (ddd, J = 2.4, 4.0, 9.6 Hz, 2 H, H-7), 4.55 (dd, J = 2.4, 12.4 Hz, 2 H, H-8a), 4.33–4.24 (m, 6 H), 4.20 (dd, J = 4.0, 12.0 Hz, 2 H, H-8b), 2.39 (m, 4 H), 2.09 (s, 6 H), 2.07 (s, 6 H), 2.04 (s, 6 H), 2.03 (s, 6 H), 1.63 (m, 4 H), 1.32 (t, J = 7.2 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 170.6, 170.4, 170.0, 169.6, 160.8, 147.6, 104.0, 99.4, 74.0, 73.2, 67.2, 65.7, 61.9, 61.8, 61.1, 27.6, 20.9, 20.8, 20.7, 20.6, 19.5, 14.2; HRMS (ESI) m/z calcd for C44H54O22Na [M + Na]+ 957.3004, found 957.3006. IR (film) 2974, 2923, 1752, 1375, 1226 cm–1. 9c (78% yield, pale yellow syrup): [α]25D = +47.0 (c 0.47, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.73 (d, J = 4.8 Hz, 2 H), 5.43 (d, J = 4.8 Hz, 2 H), 5.25 (ddd, J = 2.8, 4.0, 9.6 Hz, 2 H, H-7), 4.56 (dd, J = 2.0, 12.4 Hz, 2 H, H-8a), 4.32–4.25 (m, 6 H), 4.21 (dd, J = 4.0, 12.4 Hz, 2 H, H-8b), 2.36 (t, J = 7.2 Hz, 4 H), 2.09 (s, 6 H), 2.07 (s, 6 H), 2.04 (s, 6 H), 2.03 (s, 6 H), 1.52 (m, 4 H), 1.38 (m, 4 H), 1.33 (t, J = 7.2 Hz, 6 H); 13C NMR (100 MHz, CDCl3) δ 170.7, 170.5, 170.0, 169.7, 160.9, 147.5, 104.3, 100.2, 73.7, 73.2, 67.3, 65.8, 62.0, 61.8, 61.2, 28.6, 20.9, 20.8, 20.7, 20.6, 20.0, 14.3; HRMS (ESI) m/z calcd for C46H58O22Na [M + Na]+ 985.3317, found 985.3318. 1

Page 6 of 9

General Procedure for the Synthesis of 2-Deoxy-β βCarboxyl Kdo Analogs 10-15. A mixture of Kdo enyne and Pd(OH)2/C in ethanol was stirred under an atmosphere of H2 at room temperature for overnight. Filtration, concentration in vacuo and elution through silica gel column chromatography provided 2-deoxy-β-carboxyl Kdo analog. 10 (85% yield, colorless syrup): [α]25D = –7.3 (c 0.4, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.27 (m, 2 H, H-5, H-7), 5.10 (dd, J = 3.6, 5.2 Hz, 1 H, H-4), 4.55 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.26–4.15 (m, 3 H, H-8b, OCH2), 4.11 (d, J = 2.0 Hz, 1 H, H-2), 3.78 (dd, J = 1.2, 9.6 Hz, 1 H, H-6), 2.31 (m, 1 H, H3), 2.06 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.96 (s, 3 H), 1.72 (m, 1 H), 1.54 (m, 1 H), 1.29 (t, J = 7.2 Hz, 3 H), 1.23 (m, 20 H), 0.87 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.8, 170.4, 170.1, 169.8, 168.9, 78.4, 75.1, 71.5, 67.6, 65.5, 62.5, 61.4, 39.7, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 24.6, 22.9, 21.0, 20.9, 20.8, 14.3; HRMS (ESI) m/z calcd for C30H50O11Na [M + Na]+ 609.3251, found 609.3252. IR (KBr) 2924, 2854, 1749, 1371, 1244, 1220 cm–1. 11 (97% yield, colorless syrup): [α]25D = –6.4 (c 0.73, CHCl3); 1 H NMR (500 MHz, CDCl3) δ 5.26 (m, 2 H, H-5, H-7), 5.11 (dd, J = 3.5, 5.0 Hz, 1 H, H-4), 4.55 (dd, J = 2.0, 12.0 Hz, 1 H, H-8a), 4.26–4.16 (m, 3 H, H-8b, OCH2), 4.11 (d, J = 2.0 Hz, 1 H, H-2), 3.78 (dd, J = 1.5, 10.0 Hz, 1 H, H-6), 2.32 (m, 1 H, H-3), 2.06 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.96 (s, 3 H), 1.73 (m, 1 H), 1.54 (m, 1 H), 1.29 (t, J = 7.0 Hz, 3 H), 1.24 (m, 32 H), 0.87 (t, J = 7.0 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.7, 170.4, 170.0, 169.7, 168.9, 78.3, 75.1, 71.5, 67.6, 65.4, 62.4, 61.4, 39.7, 32.0, 29.9, 29.8, 29.7, 29.6, 29.5, 29.4, 24.6, 22.8, 21.0, 20.9, 20.8, 20.7, 14.3, 14.2; HRMS (ESI) m/z calcd for C36H62O11Na [M + Na]+ 693.4190, found 693.4188. IR (KBr) 2923, 2851, 1750, 1371, 1244, 1222 cm–1. 12 (78% yield, colorless syrup): [α]25D = –6.4 (c 0.43, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.25 (m, 2 H, H-5, H-7), 5.10 (dd, J = 3.2, 5.2 Hz, 1 H, H-4), 4.54 (dd, J = 2.8, 12.4 Hz, 1 H, H-8a), 4.26–4.14 (m, 3 H, H-8b, OCH2), 4.10 (d, J = 2.0 Hz, 1 H, H-2), 3.77 (dd, J = 1.6, 9.6 Hz, 1 H, H-6), 3.64 (s, 3 H), 2.30 (m, 1 H, H-3), 2.26 (t, J = 7.2 Hz, 2 H), 2.05 (s, 3 H), 2.03 (s, 3 H), 2.02 (s, 3 H), 1.95 (s, 3 H), 1.74 (m, 1 H), 1.56 (m, 3 H), 1.29 (t, J = 7.2 Hz, 3 H), 1.24 (m, 4 H); 13C NMR (100 MHz, CDCl3) δ 174.2, 170.8, 170.4, 170.0, 169.8, 168.9, 78.3, 75.1, 71.4, 67.6, 65.4, 62.4, 61.5, 51.7, 39.6, 34.1, 29.5, 29.1, 24.9, 24.4, 21.0, 20.9, 20.8, 20.7, 14.3; HRMS (ESI) m/z calcd for C25H38O13Na [M + Na]+ 569.2210, found 569.2202. 13 (88% yield, colorless syrup): [α]25D = –5.4 (c 0.65, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 5.24 (m, 2 H, H-5, H-7), 5.10 (dd, J = 3.2, 5.2 Hz, 1 H, H-4), 4.54 (dd, J = 2.4, 12.4 Hz, 1 H, H-8a), 4.26–4.12 (m, 7 H, H-8b, OCH2), 4.10 (d, J = 2.4 Hz, 1 H, H-2), 3.77 (dd, J = 1.6, 10.0 Hz, 1 H, H-6), 3.23 (t, J = 7.2 Hz, 1 H), 2.29 (m, 1 H, H-3), 2.05 (s, 3 H), 2.04 (s, 3 H), 2.01 (s, 3 H), 1.95 (s, 3 H), 1.79 (m, 3 H), 1.56 (m, 1 H), 1.30–1.22 (m, 11 H); 13C NMR (100 MHz, CDCl3) δ 170.8, 170.4, 170.0, 169.8, 169.5, 169.4, 168.7, 78.2, 75.1, 71.2, 67.5, 65.4, 62.4, 61.6, 61.5, 61.4, 51.9, 39.5, 29.0, 27.1, 24.4, 20.9, 20.8, 20.7, 14.3, 14.2; HRMS (ESI) m/z calcd for C28H42O15Na [M + Na]+ 641.2421, found 641.2419. IR (film) 2974, 2923, 1737, 1370, 1218 cm–1. 14 (85% yield, colorless syrup): [α]25D = –4.6 (c 0.5, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 7.27–7.12 (m, 5 H), 5.26 (m, 2 H, H-5, H-7), 5.10 (dd, J = 3.6, 5.2 Hz, 1 H, H-4), 4.55 (dd, J


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= 2.4, 12.4 Hz, 1 H, H-8a), 4.16 (m, 2 H), 4.10 (d, J = 2.4 Hz, 1 H, H-2), 4.05 (m, 1 H), 3.77 (dd, J = 1.2, 9.6 Hz, 1 H, H-6), 2.56 (t, J = 7.2 Hz, 2 H), 2.31 (m, 1 H, H-3), 2.06 (s, 3 H), 2.00 (s, 3 H), 1.99 (s, 3 H), 1.96 (s, 3 H), 1.77 (m, 1 H), 1.56 (m, 3 H), 1.25–1.17 (m, 5 H); 13C NMR (100 MHz, CDCl3) δ 170.8, 170.4, 170.0, 169.8, 168.8, 142.4, 128.5, 128.4, 125.9, 78.3, 75.1, 71.4, 67.6, 65.4, 62.4, 61.4, 39.7, 35.9, 31.8, 28.9, 24.6, 21.0, 20.9, 20.8, 20.7, 14.2; HRMS (ESI) m/z calcd for C28H38O11Na [M + Na]+ 573.2312, found 573.2315. IR (KBr) 2926, 2855, 1746, 1370, 1242, 1219 cm–1. 15 (92% yield, colorless syrup): [α]25D = –6.6 (c 0.48, CHCl3); 1 H NMR (500 MHz, CDCl3) δ 5.25 (m, 4 H, H-5, H-7), 5.10 (dd, J = 3.5, 5.0 Hz, 2 H, H-4), 4.55 (dd, J = 2.5, 12.5 Hz, 2 H, H-8a), 4.25–4.15 (m, 6 H, H-8b, OCH2), 4.10 (d, J = 1.5 Hz, 2 H, H-2), 3.77 (d-like, J = 9.5 Hz, 2 H, H-6), 2.30 (m, 2 H, H3), 2.05 (s, 6 H), 2.03 (s, 6 H), 2.02 (s, 6 H), 1.95 (s, 6 H), 1.70 (m, 2 H), 1.54 (m, 2 H), 1.28 (t, J = 7.5 Hz, 6 H), 1.18 (m, 12 H); 13C NMR (100 MHz, CDCl3) δ 170.8, 170.4, 170.0, 169.8, 168.9, 78.3, 75.1, 71.5, 67.6, 65.4, 62.5, 61.4, 39.7, 30.1, 29.7, 29.5, 24.6, 21.0, 20.9, 20.8, 14.3; HRMS (ESI) m/z calcd for C44H66O22Na [M + Na]+ 969.3943, found 969.3951. 3-C-Branched Kdo enyne analog 16. A solution of compound 7d (19 mg, 0.028 mmol) in dioxane, methanol and 10 M aq NaOH (1/1/2, v/v/v, 2 mL) was stirred at room temperature for 24 h. After TLC indicated complete consumption of the starting material, the reaction was diluted with methanol and then neutralized with Amberlite IR120 H+ resin. The mixture was filtered, and the filtrate was concentrated in vacuo. The resulting residue was purified by reverse phase C-18 column (MeOH) to provide 16 (10 mg, 75%) as a pale yellow syrup: [α]25D = +25.8 (c 0.27, MeOH); 1H NMR (400 MHz, CD3OD) δ 4.26 (d, J = 4.4 Hz, 1 H), 4.09 (d, J = 4.4 Hz, 1 H), 3.91 (m, 1 H), 3.84 (d-like, J = 8.4 Hz, 1 H), 3.74 (m, 2 H), 2.32 (t, J = 6.8 Hz, 2 H), 1.54 (m, 2 H), 1.41 (m, 2 H), 1.29 (m, 24 H), 0.90 (t, J = 6.8 Hz, 3 H); 13C NMR (100 MHz, CD3OD) δ 155.9, 99.1, 95.0, 78.1, 76.9, 70.7, 68.2, 65.4, 63.7, 33.1, 30.9, 30.8, 30.7, 30.5, 30.4, 30.2, 30.1, 23.7, 20.8, 14.4; HRMS (ESI) m/z calcd for C26H44O7Na [M + Na]+ 491.2985, found 491.2987. IR (KBr) 3437, 2920, 2851, 1727, 1696, 1598, 1467, 1402, 1385, 1194 cm–1. 3-C-Branched Kdo enyne analog 17. Compound 9b (84 mg, 0.09 mmol) was added to a solution of dioxane, methanol and 1.5 M aq NaOH (1/1/1, v/v/v, 3 mL). After the reaction was stirred at room temperature for 8 h, the mixture was diluted with methanol and then neutralized with Amberlite IR120 H+ resin. Filtration and concentration in vacuo gave a residue, which was purified by Sephadex LH-20 column (H2O) to provide 17 (44 mg, 90%) as a colorless syrup: [α]25D = +16.0 (c 0.67, H2O); 1H NMR (400 MHz, D2O) δ 4.40 (d, J = 4.0 Hz, 2 H), 4.15 (d, J = 4.4 Hz, 2 H), 3.91 (m, 4 H), 3.82 (d-like, J = 12.0 Hz, 2 H), 3.74 (dd, J = 4.0, 12.8 Hz, 2 H), 2.39 (br s, 4 H), 1.65 (br s, 4 H); 13C NMR (100 MHz, D2O) δ 169.1, 152.9, 99.9, 96.7, 76.2, 75.8, 68.8, 66.5, 63.1, 62.2, 27.1, 18.6; HRMS (ESI) m/z calcd for C24H30O14Na [M + Na]+ 565.1533, found 565.1539. IR (KBr) 3384, 2933, 1703, 1593, 1404, 1328, 1146 cm–1. 2-Deoxy-β-carboxyl Kdo analog 18. Compound 11 (68 mg, 0.1 mmol) was added to a solution of dioxane, methanol and 1.5 M aq NaOH (1/1/1, v/v/v, 4.2 mL). After the reaction was stirred at room temperature for 8 h, the mixture was diluted with methanol and then neutralized with Amberlite IR120 H+

resin. Filtration, concentration in vacuo, and purification by Sephadex LH-20 column (CH2Cl2/MeOH: 1/1) provided 18 (42 mg, 88%) as a colorless syrup: [α] 25D = –3.2 (c 0.43, MeOH); 1H NMR (500 MHz, CD3OD) δ 3.94 (m, 1 H), 3.91 (br s, 1 H), 3.85 (br s, 1 H), 3.75 (m, 3 H), 3.33 (m, 1 H), 2.13 (br s, 1 H), 1.82 (m, 1 H), 1.45 (m, 1 H), 1.28 (m, 32 H), 0.90 (t, J = 7.0 Hz, 3 H); 13C NMR (125 MHz, CD3OD) δ 177.5, 81.5, 80.2, 73.5, 71.6, 69.6, 64.7, 44.0, 33.1, 31.9, 31.7, 31.0, 30.9, 30.8, 30.5, 25.4, 23.7, 14.5; HRMS (ESI) m/z calcd for C26H50O7Na [M + Na]+ 497.3454, found 497.3455. IR (KBr) 3409, 2919, 2851, 1602, 1467, 1424, 1085 cm–1. 2-Deoxy-β-carboxyl Kdo analog 19. Compound 15 (41 mg, 0.043 mmol) was added to a solution of dioxane, methanol and 1.5 M aq NaOH (1/1/1, v/v/v, 3 mL). After the reaction was stirred at room temperature for 8 h, the mixture was diluted with methanol and then neutralized with Amberlite IR120 H+ resin. Filtration, concentration in vacuo, and purification by Sephadex LH-20 column (H2O) provided 19 (22 mg, 92%) as a colorless syrup: [α]25D = –0.6 (c 0.38, H2O); 1H NMR (400 MHz, D2O) δ 3.93–3.86 (m, 6 H), 3.83 (dd, J = 2.4, 12.0 Hz, 2 H), 3.80 (br s, 2 H), 3.70 (dd, J = 5.6, 12.0 Hz, 2 H), 3.33 (d, J = 7.6 Hz, 2 H), 2.09 (m, 2 H), 1.63 (m, 2 H), 1.34–1.21 (m, 14 H); 13C NMR (100 MHz, D2O) δ 178.1, 80.0, 77.5, 71.8, 69.2, 67.3, 63.1, 42.0, 29.9, 29.4, 28.6, 23.5; HRMS (ESI) m/z calcd for C24H42O14Na [M + Na]+ 577.2472, found 577.2471. IR (KBr) 3409, 2925, 2855, 1606, 1423, 1090 cm–1.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ NMR spectra for new compounds (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID You Yang: 0000-0003-4438-2162 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support from the National Thousand Young Talents Program (Y100-4Q-1701, YC0140103) and the Shanghai Pujiang Program (15PJ1401500) is gratefully acknowledged.

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