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Synthesis of ABO histo-blood group type I and II antigens
Carbohydrate Research 345 (2010) 2305–2322
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Synthesis of ABO histo-blood group type I and II antigens Peter J. Meloncelli, Todd L. Lowary ⇑ Department of Chemistry and Alberta Ingenuity Centre for Carbohydrate Science, Gunning-Lemieux Chemistry Centre, University of Alberta, Edmonton, AB, Canada T6G 2G2
a r t i c l e
i n f o
Article history: Received 25 June 2010 Received in revised form 13 August 2010 Accepted 17 August 2010 Available online 16 September 2010 Keywords: ABO histo-blood group ABO type I ABO type II Chemical synthesis Trichloroacetimidate 7-Octen-1-yl
a b s t r a c t The ABO histo-blood group system is one of the most clinically important antigen families. As part of our overall goal to prepare the entire set of the A, B and H type I–VI antigens for a range of biochemical investigations, we report herein the synthesis of the type I and II antigens with a 7-octen-1-yl aglycone. This linker was chosen to facilitate not only the future conjugation of the antigens to a protein or solid support but also the synthesis of the H type I and II octyl glycosides for enzyme kinetic studies. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The ABO histo-blood group system has long been of interest to the biological and medical sciences as these antigens are one of the most important clinical considerations for both transfusion and transplantation.1,2 This family of antigens consists of three oligosaccharide epitopes (1–3, Fig. 1), the structures of which were elucidated by Morgan and Watkins in 1957.3 The H(O) antigen was shown to be a disaccharide consisting of L-fucose and D-galactose (3, Fig. 1), while the A and the B antigens (1 and 2) were shown to be trisaccharides containing the disaccharide H antigen with an additional N-acetyl-galactosamine or a galactose residue, respectively. It was later shown by Tuppy and Staudenbauer that individuals who are of the A histo-blood group possess an N-acetyl-galactosaminyl transferase (GTA) capable of adding N-acetyl galactosamine to the 3-OH group of the H antigen galactose moiety.4 On the other hand, individuals of the B histo-blood group possess a galactosyl transferase (GTB) that adds galactose to this hydroxyl group. The genetic basis of the ABO histo-blood group system was discovered in the 1990s, when the genes responsible for coding GTA and GTB were identified.5–7 The ABO antigens can be further divided into six subtypes, type I–VI (Table 1).8 The type I–IV and VI antigens are present either as glycoproteins, glycolipids, or as free oligosaccharides in humans.1 After the publication of our synthesis of the ABO type V and VI histo-blood group antigens,9 it came to our attention that the origin of
⇑ Corresponding author. Tel.: +1 780 492 1861; fax: +1 780 492 7705. E-mail address: [email protected] (T.L. Lowary). 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.08.012
the nomenclature of the type V and VI antigens was not clear.10 Although a bit tangential to the main goal of this paper, we describe briefly this ambiguity, as a concise description does not appear to be present elsewhere in the literature. The first report of a type VI antigen structure from human sources was achieved in 1956 by Kuhn and co-workers, who isolated the H type VI antigen as the free oligosaccharide in appreciable quantities from human milk.11 Subsequently, a type VI antigen from tissue samples was reported by Briemer and co-workers in 1982.12 This glycolipid, named A-4 by Briemer, contained what was in the future termed the A type VI antigen, linked to ceramide. The origin of the type V antigen proved more difficult to uncover. The first mention of this structure was by Mollicone and co-workers in 1990.8 The authors obtained synthetic samples of the type V and VI antigens from Chembiomed, a now defunct commercial entity, and this is presumably the origin of this nomenclature. To the best of our knowledge, no ABH type V structure has been isolated from a mammalian source. A recent review by Yamamoto on the ABO histo-blood group system makes reference to the type I–IV and type VI antigens, excluding the type V antigen.1 This exclusion of the type V antigen was likely based on the lack of evidence for existence of ABH type V structures in humans. This is surprising because Hakomori and Stellner reported the isolation of a glycolipid from human erythrocytes that contained the type V (b-D-Galp-(1?3)-b-D-Gal) backbone at the terminal end.13 This structure could easily be converted into the H type V antigen using the same fucosyl transferase responsible for the biosynthesis of the H type I–IV antigens present on erythrocytes. Based on this chronology, the nomenclature assignment of the type V and VI antigens should be reversed; however, as the nomenclature is well
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HO
HO
OH O HO
OH
HO
O
OR
OH O
O HO
O
HO OH
HO
O AcHN
OH O HO
OR
HO OR
O
O
O
O OH
O
O
OH
OH OH
OH
OH HO
HO
HO A Antigen 1
B Antigen
H Antigen
2
3
Figure 1. Structures of the A, B and H(O) antigens. R = Glycoprotein, glycolipid or free oligosaccharide.
Table 1 Carbohydrate moieties responsible for the six subtypes (I–VI) of the ABO histo-blood group antigens Type Type Type Type Type Type Type
Carbohydrate I II III IV V VI
ABO-b-(1?3)-b-D-GlcpNAc ABO-b-(1?4)-b-D-GlcpNAc ABO-b-(1?3)-a-D-GalpNAc ABO-b-(1?3)-b-D-GalpNAc ABO-b-(1?3)-b-D-Galp ABO-b-(1?4)-b-D-Glcp
established in multiple sources we have adhered to the conventional naming of the type I–VI antigens. The overall goal of our synthetic endeavour is to prepare all known ABO type I–VI histo-blood group antigens. We hope that with all of the antigens in hand, we will be able to conduct a range of studies on the biological importance of these sub-types. An example of such an investigation is quantitatively determining the difference between the A1 and A2 blood groups. It has been shown that individuals of the A1 subtype have a higher density of A antigen on their erythrocytes than individuals of the A2 subtype.14 Further work by Clausen and co-workers has demonstrated that this difference can be attributed to the fact that GTA1 is able to transfer N-acetyl-galactosamine onto the H type I–IV structures present on erythrocytes, while GTA2 is only able to affect transfer onto the H type I and type II structures.15 These pioneering studies were conducted in a qualitative manner via immunostaining. Access to synthetic samples of these antigens would enable more detailed quantitative kinetics assays to be conducted. Herein we report the synthesis of the ABO type I and II histoblood group antigens (4–9, Fig. 2) attached to an octen-1-yl aglycone. To date, the synthesis of the ABO type I and II antigens has received more interest than the type III–VI structures. The chemical synthesis of all three type I antigens has been reported by Paulsen and Kolárˇ16 as well as by Bovin and co-workers.17,18 The chemoenzymatic synthesis of the A type I and II tetrasaccharides was reported by Palcic and co-workers,19 and more recently Wang and co-workers have developed a chemoenzymatic synthesis of the B type I antigen.20 Interestingly, the latter study did not use the human glycosyltransferases, but rather three enzymes present in Escherichia coli O86, which produces a B type III mimetic in its lipopolysaccharide O-chain. The synthesis of the type II antigens has been more limited, most notably the chemical synthesis of the ABO type II structures was achieved by Paulsen and co-workers21,22 as well as by Bovin and co-workers.23
The A1 and A2 blood groups should not be confused with the A type I and type II structures. The latter refers to distinct chemical entities while the former refers to subclasses of the A blood type, which are characterized by different densities of the antigen on erythrocytes.
2. Results and discussion To prepare 4–9 we chose a linear chemical synthesis, similar to the strategy employed in our recently reported synthesis of the type V and VI antigens.9 This strategy would enable an expedited synthesis of all three of the type I and II antigens in sufficient quantities for a range of investigations. A block synthesis, such as that reported by Bovin and co-workers,17 would not enable access to the H structures. We chose to employ a 7-octen-1-yl aglycone for two reasons. Firstly, it would enable ready access to the octyl glycoside, via catalytic hydrogenation, for enzyme kinetics studies.24 Secondly, it would enable facile preparation of glycoconjugates. We have demonstrated that a thiol-ene ‘click reaction’25 can be used to introduce a reactive linker that allows direct conjugation of carbohydrates to silica-based surfaces.26 2.1. Synthesis of type I antigens The synthesis of the type I ABO histo-blood group antigens commenced from the known27 and easily prepared trichloroacetimidate donor 10 (Scheme 1). Glycosylation with the 7-octen-1-ol acceptor afforded the 7-octen-1-yl glycoside 11 as a mixture of anomers (a/b, 11:41). In the absence of a C2 participating group, the stereochemical outcome for this glycosylation was dependent on the anomeric configuration of the trichloroacetimidate. To ensure a high ratio of the desired b-anomer, a trichloroacetimidate donor with predominately the a stereochemistry was required. Presumably, the mechanism for the formation of the b-glycoside involves an SN2-type process.28 Introduction of the aglycone in the presence of an azido protecting group was not trivial when compared to other amine protecting groups such as N-phthalimide; however, the azido group can be converted into an amine under milder conditions than the phthalimide. Deacetylation with sodium methoxide, followed by introduction of a benzylidene acetal, enabled isolation of the corresponding b and a glycosides, 13 and 14, respectively, in moderate yield, with the desired b anomer being the predominant product. The 1H NMR spectrum of the a anomer 13 showed an anomeric proton signal appearing at 4.90 ppm as a doublet with a coupling of 3.6 Hz, while the spectrum of the b anomer 14 had an anomeric proton signal at 4.42 ppm as a doublet with a coupling of 8.0 Hz. Introduction of the galactose core residue was achieved by glycosylation of 13 with trichloroacetimidate 15 affording disaccharide 16 (Scheme 2). To facilitate removal of trichloroacetimidate-related impurities, a deacetylation was conducted under Zemplén conditions to afford diol 17 in moderate yield (63%) over two steps. Introduction of the pivaloyl ester selectively at the 30 position of 17 was achieved with complete regioselectivity using trimethylacetyl chloride in pyridine in excellent yield (89%). Next, introduction of the a-L-fucopyranosyl moiety was achieved through the use of b-L-fucopyranosyl trichloroacetimidate 19
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HO
HO
OH O
OH
OH O
OH O HO
HO HO
OH
AcHN
O
NHAc
O O
O
OH 5 A Type I
OH
4 B Type I
OH HO
HO
O
O
O
NHAc
O
OH O
OH O HO
HO
O
O
OH O
OH HO
HO
OH O
OH O HO
O
O
HO
OH O
HO
NHAc
O
OH O
O HO
O
O NHAc
O
O
OH
OH
6 H Type I
OH HO
HO
OH
OH
HO HO
HO
HO
OH O
OH
O
O HO
O
O O NHAc
O
O OH 8 B Type II
HO
OH
O
NHAc OH
OH O
AcHN
O
O HO
O
OH O
HO
OH
O O
7 H Type II
OH
OH HO
OH 9 A Type II
Figure 2. Structure of ABO histo-blood group type I and II antigen targets.
OAc O
AcO AcO
NH CCl3
O
N3
(a)
AcO AcO
OAc O N3
10
O
11 (b) O
Ph
O
O
O
HO N3 13 O
Ph
(c)
OH O N3
O
O
HO HO
O
12
HO N3 14
O
Scheme 1. Reagents: (a) HO(CH2)6CH@CH2, 4 Å MS, TMSOTf, CH2Cl2; (b) NaOCH3, CH3OH; (c) PhCH(OCH3)2, p-TsOH, DMF, (a/b, 11:41) 52% (three steps).
following the methodology of Schmidt and co-workers.29 Treatment of 18 with trichloroacetimidate 19 under standard glycosylation conditions afforded trisaccharide 20 in good yield (80%) and with excellent stereoselectivity. The anomeric proton of the fucopyranoside of 20 showed an anomeric signal at 5.41 ppm as a doublet with a coupling constant of 1.5 Hz. Removal of the pivaloate ester from 20 required forcing conditions (lithium methoxide in methanol at reflux) and prolonged reaction times (seven days); however, the reaction afforded alcohol 21 in good yield. A similar observation was made in our synthesis of the type V and VI antigens. The decision to continue using this protecting group as op-
posed to others that might be removed more efficiently was based on the fact that the pivaloate ester could be installed and removed in good yield and with minimal by-products.9 Access to the H type I antigen was achieved first by conversion of the azide moiety into an N-acetyl group using thiolacetic acid in pyridine to afford 22 (Scheme 3). Surprisingly, some acetylation of the alcohol in 22 was observed during this reaction. The formation of this by-product was not, however, problematic as Zemplén deacetylation easily converted the O-acetylated compound into 22. Next, global deprotection was achieved using a Birch reduction resulting in the conversion of 22 into 6 in 92% yield. For investiga-
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
O
Ph
O
O
O
HO N3
Ph
13
Ph O
Ph
O
O
O
(a)
O
O
Ph
(b) AcO
O
N3
HO 18
16 R = Ac (c)
17 R = H
CCl3
O
O
PivO
N3
OR
O AcO
O
O O
O
O
RO
O
Ph
O
O O
O
NH
15
O OBn
O
NH (d)
OBn BnO
Ph
CCl3
19
Ph O
O
O
O O
Ph
(e)
O
OBn
OBn OBn
21
BnO
N3
O
O OBn
O
O
PivO
N3
O
O
O O
O
O
HO
O
Ph
O
O O
20
BnO
Scheme 2. Reagents: (a) TMSOTf, 4 Å MS, CH2Cl2; (b) NaOCH3, CH3OH, 63% (two steps); (c) (CH3)3CCOCl, pyridine, 89%; (d) TMSOTf, 4 Å MS, Et2O, CH2Cl2, 80%; (e) LiOCH3, CH3OH, 82%.
Ph
Ph O
O
O
O
O
Ph
(a)
N3
O OBn
O
O
HO
NHAc
O
O OBn
O
O O
O
O
HO
O
Ph
O
O O
O OBn
21
22
OBn
BnO
BnO
(b)
HO
OH O
OH O HO
O(CH2)7CH3
O
HO
HO
NHAc
O
O HO (c)
23
NHAc
O O
OH HO
O
O
HO
O OH
OH O
OH
OH OH HO
6
Scheme 3. Reagents: (a) (i) CH3COSH, pyridine; (ii) NaOCH3, CH3OH, 62%; (b) NH3, Na, CH3OH, THF, 92%; (c) H2, 10% Pd–C, THF, H2O, 83%.
tions involving conjugation of the H type I antigen to a support, the 7-octen-1-yl glycoside 6 was desired; however, other studies, such as the examination of GTA and GTB kinetics, required the octyl glycoside. To this end, catalytic reduction of the alkene moiety of 6 afforded octyl glycoside 23 in 83% yield. Access to the A type I antigen was achieved first by glycosylation of 21 with trichloroacetimidate 24 to afford tetrasaccharide 25 in good (92%) yield (Scheme 4). The stereochemistry of the newly formed glycosidic linkage was confirmed using 1H NMR
spectroscopy; the anomeric proton of the 2-azido-2-deoxy-a-Dgalactopyranoside appeared at 5.88 ppm as a doublet with a coupling of 3.2 Hz. Conversion of the two azides into N-acetyl groups using thiolacetic acid in pyridine afforded 26. Deacetylation under Zemplén conditions afforded 27 and subsequent complete deprotection using dissolving metal reduction afforded 5. Overall, tetrasaccharide 5 was obtained in 69% yield from 25. The B type I antigen could be obtained first by glycosylation of 21 with trichloroacetimidate 28 affording tetrasaccharide 29 in
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 Ph O O O
Ph
O
O O HO
Ph
O
O
AcO
N3
O O
(a)
O
Ph N3
21
BnO
O
AcO
OBn OBn
O
OAc O
O
O O
O
O
O
N3
O AcO
O
OAc O O
AcO N3
OBn 25
OBn
CCl3
BnO
NH
24
(b) Ph HO
RO
OH O
OH
OH O
OH O HO
HO AcHN
O
O
O
(d)
O O
Ph
RO
O
O O
AcHN
O
O
O
NHAc
O
O
OR O
NHAc
O O
O
OBn
OH OBn
OH BnO
HO
26 R = Ac
5
(c)
27 R = H
Scheme 4. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 92%; (b) CH3COSH, pyridine, 74%; (c) NaOCH3, CH3OH, 94%; (d) NH3, Na, CH3OH, THF, 88%.
Ph O O O
Ph
O
O O HO
Ph
O
O
BnO
N3
O O
(a)
O
Ph O O
BnO
21
BnO
O O
BnO
OBn OBn
OBn O
O
O
O
N3
O BnO
O
O
OBn O
OBn O
BnO OBn 28
OBn
CCl3
29
BnO
NH (b) Ph
HO
OH O
BnO OH
OH O
OH O HO
HO HO
O
O
O O
(c)
OBn O
O O O O
BnO
4
O NHAc
O O
OH HO
O
O
O
NHAc
O OH
O
Ph
BnO
OBn OBn BnO
30
Scheme 5. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 73%; (b) CH3COSH, pyridine, 65%; (c) NH3, Na, CH3OH, THF, 94%.
moderate yield (Scheme 5). Conversion of the azide into an N-acetyl group using thiolacetic acid in pyridine afforded 30, under conditions analogous to those used in the preparation of 22. Other
techniques do exist to convert an azide into an N-acetyl group, and several were explored including a Staudinger reaction followed by acetylation;30 however, none were found to be as
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
type II antigen 7 with the 7-octen-1-yl glycoside moiety intact. Reduction of part of this material by hydrogenation afforded, in 96% yield, the octyl glycoside 40 for use in enzymatic studies. The B type II antigen was accessed first by treatment of 38 with the trichloroacetimidate donor 28 under standard conditions (Scheme 9). Unfortunately, complete purification of 41 was not possible; therefore, the unequivocal confirmation of the stereochemistry was not possible until after the conversion of 41 into 42 using thiolacetic acid in pyridine. In 1H NMR spectrum of 42, the anomeric signal for the a-galactopyranosyl linkage appeared at 5.36 ppm as a doublet with a coupling constant of 3.6 Hz, as expected for this stereochemistry. Final global deprotection of 42 upon treatment with sodium and liquid ammonia in THF afforded 8 in high yield (96%). Finally, access to the A type II antigen was achieved in an analogous manner to its A type I counterpart (Scheme 10). Glycosylation of 38 with trichloroacetimidate 24 afforded tetrasaccharide 43; subsequent reduction–acetylation of the two azides using thiolacetic acid in pyridine afforded 44. Confirmation of the stereochemistry of the glycosidic linkage was achieved using 1H NMR spectroscopy. The anomeric proton of the newly formed glycosidic linkage appeared as a doublet at 5.09 ppm with a 3.7 Hz coupling constant. Deacetylation of 44 to provide 45 followed by complete deprotection resulted in the preparation of the A type II antigen 9 in excellent yield (88% yield over the two steps).
efficient as the tandem reduction–acetylation process induced by treatment with thiolacetic acid. Final deprotection of 30 using a Birch reduction afforded the B type I antigen 4 in 94% yield. In our synthesis of the type V and VI antigens,9 the yield for deprotection reactions of this type on similar substrates was at times low (60–70%). These lower yields were caused by precipitation of the starting material in liquid ammonia, leading to incomplete removal of the protecting groups. In the course of the synthesis of the targets reported here, we found that increasing the volume of the THF used to dissolve the starting material successfully circumvented this problem. 2.2. Synthesis of type II antigens The type I and II antigens are quite similar in structure. The only difference is that the galactose core residue is linked to the reducing-end GlcNAc moiety by either a b-(1?3) or b-(1?4) linkage in the type I and II antigens, respectively. This led us to exploit the reducing end acceptor 13, which was used in the type I synthesis, and putting it to use in the preparation of the type II antigens (Scheme 6). Protection of 13 using standard benzylation conditions afforded 31 in excellent yield, and then selective ring opening of the benzylidene acetal using trifluoroacetic acid and triethylsilane resulted in formation of the 4-OH derivative 32. Confirmation of the regiochemistry of the ring opening was achieved by acetylation (acetic anhydride and DMAP in pyridine) and observance of a downfield shift of H-4 in the 1H NMR spectrum from 3.63 ppm in 32 to 5.05–4.91 ppm in 33. Next, treatment of glycosyl acceptor 32 with trichloroacetimidate 15 using a TMSOTf promoter system yielded an impure disaccharide 34. Contamination of 34 with trichloroacetimidate-derived by-products necessitated the conversion of diacetate 34 into diol 35 to facilitate purification (Scheme 7). Protection of the 30 position of the galactose core was again achieved using trimethylacetyl chloride in pyridine to yield 36. Glycosylation using the b-L-fucopyranosyl trichloroacetimidate 19 afforded the trisaccharide in excellent yield (94%) and with complete stereoselectivity. The anomeric proton of the a-fucopyranoside of 20 showed an anomeric signal at 5.41 ppm as a doublet with a coupling constant of 3.8 Hz, consistent with a stereochemistry. Removal of the pivaloate ester was achieved using refluxing lithium methoxide in methanol; again, forcing conditions were needed, similar to those required to prepare 21. Given the structural similarities between the type I and II antigens, the deprotection strategies would be expected to be analogous and this was indeed the case. The conversion of the azido group in 38 to an N-acetyl functionality using thiolacetic acid afforded 39 in good (88%) yield (Scheme 8). Interestingly, in contrast to the preparation of the type I counterpart 22 (see Scheme 3 above), no acetylation of the alcohol on 39 was observed. Exhaustive deprotection was achieved in an analogous manner to the type I counterpart: Birch reduction of 39 afforded a 75% yield of the H
O
Ph
Ph
O
O
(a) O
HO N3 13
2.3. NMR analysis of 4–9 With the ABO type I and II antigens 4–9 in hand, we conducted detailed NMR studies to probe if there are any conformational differences between the structures. Unequivocally assigning proton resonances in deprotected oligosaccharides using traditional 1D and 2D techniques is difficult due to spectral overlap. To overcome this problem for the ring protons in 4–9, a gradient-enhanced chemical shift selective filtering (ge-CSSF) technique was used. In particular, 1D-ge-CSSF-TOCSY experiments were carried out to provide coupling constants and to assign chemical shifts.31,32 The use of this experiment enables the straightforward analysis of the 1H NMR spectra of each ring individually. In addition to analyzing any differences between the type I and II structures, the measurement of 1D-ge-CSSF-TOCSY enabled complete assignment of all 1H NMR chemical shifts for 4–9. The main limitation of this approach is that signal transfer throughout each ring is dependent on the size of the coupling constants. In the case of both galactose and fucose, when a ring is irradiated at H1, signal transfer from H3 to H4 is significantly reduced (due to 3JH3,H4 <3.5 Hz), and transfer to H5 and H6 does not occur. In most cases this problem could be circumvented by irradiation of the H5 or H6 resonance and then allowing the magnetization to transfer the opposite way around the spin system. The chemical shift data from these studies are shown in Table 2, and the associated coupling constants are shown in Table 3. The
OBn
O O BnO
O
(b) O
N3
HO BnO
O O N3 32
31 OBn
(c)
O
AcO BnO
O N3 33
Scheme 6. Reagents: (a) BnBr, NaH, DMF, 98%; (b) Et3SiH, CF3COOH, 4 Å MS, CH2Cl2 83%; (c) Ac2O, DMAP, pyridine, 85%.
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 OBn O
HO BnO
O
Ph
Ph
N3 32
O
O
O
Ph
O
(a)
O
OBn
O RO
O
RO
PivO
O
O
O BnO
HO
O
N3
O AcO AcO
OBn
O
O
O BnO
(b) O
36
34 R = Ac (c)
35 R = H
CCl3
N3
NH
15
NH O (d)
CCl3
OBn BnO
Ph
O OBn
19
Ph O
O
O
O OBn
O HO O
PivO
(e)
O
OBn
O N3
O
OBn
OBn OBn
38
BnO
O
O BnO
O
N3
O
OBn
O
O
O BnO
37
BnO
Scheme 7. Reagents: (a) TMSOTf, 4 Å MS, CH2Cl2; (b) NaOCH3, CH3OH, 78% (two steps); (c) (CH3)3CCOCl, pyridine, 88%; (d) TMSOTf, 4 Å MS, Et2O, 94%; (e) LiOCH3, CH3OH, 83%.
Ph
Ph
O
O O
O
HO
(a)
O
O BnO
O
HO
O
O OBn
OBn
OBn
38
BnO
O NHAc
N3
OBn
O BnO
O
O
O
OBn
O
OBn
O
39
BnO
(b)
HO
HO
OH
HO O
OH
OH
O O HO
OH
O (c)
O
HO
O(CH2)7CH3
O
O HO
NHAc
O NHAc
O
O OH
OH
O
OH
40
HO
OH
7
HO
Scheme 8. Reagents: (a) CH3COSH, pyridine, 88%; (b) Na, NH3, CH3OH, THF, 75%; (c) H2, 10% Pd–C, CH3OH, 96%.
residue assignments are shown in Figure 3. In all cases only the coupling constants for the ring protons were analysed. A signal is referred to as a multiplet only in instances where the coupling constant could not be determined, either due to second-order coupling or where inadequate signal transfer in the 1D-ge-CSSF-TOCSY (outlined above) resulted in individual proton resonances not being able to be identified. In cases where insufficient information was obtained using 1D-ge-CSSF-TOCSY, additional techniques such as 1 H–1H COSY, HSQC and HMBC were employed to aid in the identification of proton resonances. Not surprisingly, the NMR spectra of
all six structures showed significant similarity; little deviation was observed between the spectra of any of the compounds with respect to both chemical shift (Table 2) and coupling constants (Table 3). 2.4. Conclusions In summary, we report the linear syntheses of the A, B and H type I and II histo-blood group antigens (4–9) attached to either a 7-octen-1-yl or an octyl aglycone. Key to the synthesis was the
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 Ph O O Ph
OBn
O HO
O
O BnO
O
BnO
N3
O
(a)
O
OBn O
O
O
BnO
OBn
OBn
O
BnO O
OBn
38
BnO
O
O BnO
O
O N3
O BnO
OBn
OBn O
41
OBn BnO O
BnO OBn
CCl3 NH
28
(b)
Ph HO
BnO
OH O
OH
OH
HO HO
O
O HO
O
O
BnO
OH
O
O
OBn O
(c)
O
BnO
O
O NHAc
O O
O OBn
OH HO
O BnO
NHAc
O OH
OBn
O O
8
OBn BnO
42
Scheme 9. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 75%; (b) CH3COSH, pyridine, 91%; (c) Na, NH3, CH3OH, THF, 96%.
preparation of the two reducing-end disaccharides 13 and 32, which served as the core upon which the rest of the structures were built. In addition, approaches developed in the synthesis of the type V and VI antigen synthesis9 were adapted to the synthesis of the type I and II antigens. Use of these methods enabled the targets to be obtained with a high level of efficiency. Most notably, the preparation of the 7-octen-1-yl b-D-glucopyranoside 11 was efficiently prepared without anchimeric assistance. In addition, the installation of the a-L-fucopyranosyl linkage was entirely stereoselective, taking advantage of the excellent stereoselectivity of the trichloroacetimidate methodology.29 An NMR study was conducted on each of the type I and II ABO antigens (4–9). Based upon the chemical shift and coupling constant data, it was clear that the reducing-end residue has little to no impact on the conformation of the canonical di- and trisaccharide blood group antigens at the non-reducing end of the molecule. 3. Experimental
performed on Silica Gel 60 (40–60 lm). Iatrobeads refer to a beaded silica gel 6RS-8060, which is manufactured by Iatron Laboratories (Tokyo). C18 silica gel (35–70 lm) was manufactured by Toronto Research Chemicals. Optical rotations were measured at 22 ± 2 °C and are in units of deg dm1 cm3 g1; in all cases the concentrations are in the units g/100 mL. 1H NMR spectra were recorded at 400 and 500 MHz, and chemical shifts were referenced to the peak for TMS (0.0 ppm, CDCl3) or CD3OD (3.30 ppm, CD3OD). For final compounds, analysis was conducted using 1D-ge-CSSFTOCSY31,32 at 600 MHz, to identify individual proton resonances. Samples for 1D-ge-CSSF-TOCSY experiments were prepared in CD3OD at a concentration of 21 mg/mL, and the experiments were carried out at 300 K. 13C NMR (APT) spectra were recorded at 125 or 100 MHz, and 13C chemical shifts were referenced to the peak for internal CDCl3 (77.1 ppm, CDCl3) or CD3OD (49.0, CD3OD). All spectra were recorded in CDCl3 unless specified otherwise. Melting points were measured using a Perkin–Elmer Thermal Analysis. Electrospray-ionization mass spectra were recorded on samples suspended in mixtures of THF with CH3OH and added NaCl.
3.1. General methods All reagents were purchased from commercial sources and were used without further purification, unless otherwise stated. Reaction solvents were purchased and were used without purification; dry solvents were purified by successive passage through columns of alumina and copper under nitrogen. All reactions were carried out at room temperature under a positive pressure of argon, unless otherwise stated. Thin layer chromatography (TLC) was performed on E. Merck Silica Gel 60 F254 aluminium-backed plates that were stained by heating (>200 °C) with either p-anisaldehyde in 5% sulfuric acid in EtOH or 10% ammonium molybdate in 10% sulfuric acid. Unless otherwise indicated, all column chromatography was
3.2. 7-Octen-1-yl 2-acetamido-2-deoxy-3-O-(2-O-(a-Lfucopyranosyl)-3-O-(a-D-galactopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (4) Redistilled liquid ammonia (15 mL) was collected in a flask cooled to 78 °C and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 30 (67 mg, 0.042 mmol) in THF (4 mL) and CH3OH (15 lL, 0.37 mmol) was added dropwise, and the solution was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 Ph O O
Ph OBn
O HO O
AcO
O
O BnO
O N3
O OBn
O
AcO
(a)
OBn
O N3
38
OBn
O
OAc O
O
O
O BnO
O
BnO
O N3
O OBn AcO
43
OBn
OAc O
BnO O
AcO N3
CCl3 NH
24
(b)
Ph HO
OH O
RO OH
OH
HO
OH
O AcHN O O
O HO
OR O
O O
RO (d)
O
OBn
O AcHN
O
O O
O
O BnO
O
NHAc
NHAc
O
O
OH
OBn
OH HO
9
OBn BnO R = Ac 44 R = H 45
(c)
Scheme 10. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 94%; (b) CH3COSH, pyridine, 76%; (c) NaOCH3, CH3OH, 92%; (d) Na, NH3, CH3OH, THF, 96%.
to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 4 (31 mg, 94%) as a colourless oil. [a] +69.0 (c 1.3, MeOH); 1H NMR: see Tables 2 and 3; 13C NMR (175 MHz): dC 173.2 (C@O), 140.0 (CH2@CH), 114.7 (CH2@CH), 103.6 (C1), 102.1 (C10 ), 100.5 (C100 ), 96.2 (C1000 ), 80.2, 79.9, 77.6, 76.2, 74.5, 73.6, 73.2, 71.4, 71.2 (2C), 70.6, 70.0, 69.8 (C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C400 , C4000 , C5, C50 , C5000 ), 70.7 (CH@CH2(CH2)5CH2O), 67.4 (C500 ), 65.7 (C40 ), 63.4, 62.7, 62.5 (C6, C60 , C6000 ), 55.9 (C2), 34.8 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O), 30.1 (CH@CH2 (CH2)5CH2O), 29.9 (CH@CH2(CH2)5CH2O), 26.9 (CH@CH2(CH2)5 CH2O), 23.2 (CH3C@O), 16.5 (C600 ). ESIMS: m/z Calcd [C34H59NO20]Na+: 824.3523. Found: 824.3522. 3.3. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2-deoxya-D-galactopyranosyl)-2-O-(a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (5) Redistilled liquid ammonia (20 mL) was collected in a flask cooled to 78 °C and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 27 (58 mg, 0.045 mmol) in THF (4 mL) and CH3OH (9.1 lL, 0.225 mmol) was added dropwise, and the solution was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+), and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 5 (33.5 mg, 88%) as a colourless oil. [a] +27.1 (c 0.2, H2O); 1H NMR: see Tables 2 and 3; 13C NMR (125 MHz, D2O): dC 175.7 (C@O), 174.5 (C@O), 141.2 (CH2@CH), 114.9 (CH2@CH), 102.8, 100.8, 100.0 (C1, C10 , C100 ), 92.1 (C1000 ), 78.3, 76.33, 76.27, 75.7, 74.7, 72.7, 71.8, 70.6, 69.7, 69.4, 68.53,
68.50, 67.5, 63.8 (C20 , C200 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C50 , C500 , C5000 ), 71.5 (CH@CH2(CH2)5CH2O), 62.3, 62.1, 61.6 (C6, C60 , C6000 ), 55.6 (C2), 50.5 (C2000 ), 34.0 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 29.0 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2 (CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 23.2 (CH3C@O), 22.8 (CH3C@O), 16.1 (C600 ). ESIMS: m/z Calcd [C36H62N2O20]Na+: 865.3788. Found: 865.3788.
3.4. 7-Octen-1-yl 2-acetamido-2-deoxy-3-O-(2-O-(a-Lfucopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (6) Redistilled liquid ammonia (15 mL) was collected in a flask cooled to 78 °C and treated with sodium until the blue colour persisted. A solution of trisaccharide 22 (130 mg, 0.120 mmol) in THF (4 mL) and CH3OH (24 lL, 0.60 mmol) was added dropwise, and the solution was stirred (78 °C, 2 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected trisaccharide 6 as a colourless oil (70 mg, 92%). [a] 62.7 (c 1.0, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125 MHz, CD3OD): dC 173.1 (C@O), 140.1 (CH2@CH), 114.8 (CH2@CH), 103.6 (C1), 102.2 (C10 ) 101.2 (C100 ), 80.3, 78.0, 77.7, 77.0, 75.6, 73.6, 71.4, 70.8, 70.5, 70.3, 67.6 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C50 , C500 ), 70.7 (CH@CH2(CH2)5CH2O), 62.7, 62.6 (C6, C60 ), 56.1 (C2), 34.9 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2 (CH2)5CH2O), 30.2 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5 CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.2 (CH3C@O), 16.6 (C600 ). ESIMS: m/z Calcd [C28H49NO15]Na+: 662.2994. Found: 662.3000.
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
Table 2 1 H Assignments (ppm) of the ABH type I–II antigens 4–9, recorded at 600 MHz with CD3OD as solvent Proton
A Type 1 5
A Type II 9
B Type 1 4
B Type II 8
000
H1 H2000 H3000 H4000 H5000 H6000 H6000
5.15 4.29 3.85 3.87 4.20 3.75 3.68
(d) (dd) (dd) (d) (dd) (dd) (dd)
5.15 4.32 3.82 3.89 4.16 3.76 3.68
5.14 3.83 3.81 3.89 4.16 3.73 3.68
(d) (dd) (dd) (dd) (dd) (dd) (dd)
5.15 (d) 3.84 (dd) 3.78 (dd) 3.9 (d) 4.14–4.10 (m) 3.76–3.71 (m) 3.70–3.66 (m)
H100 H200 H300 H400 H500 H600
5.23 3.69 3.65 3.72 4.36 1.19
(s) (dd) (dd) (d) (q) (d)
S.33 (d) 3.72 (dd) 3.68 (dd) 3.69 (d) 4.32 (q) 1.21 (d)
5.21 (d) 3.7 (dd) 3.65 (dd) 3.71 (d) 4.45 (q) 1.18 (d)
H10 H20 H30 H40 H50 H60 H60
4.57 (d) 3.91 (dd) 3.86 (dd) 4.09 (d) 3.49 (dd) 3.77–3.73 (m) 3.66–3.65 (m)
4.52 (d) 3.99 (dd) 3.90 (dd) 4.09 (d) 3.29–3.25 (m) 3.81–3.77 (m) 3.72–3.66 (m)
H1 H2 H3 H4 H5 H6 H6
4.25–4.20 3.86–3.79 3.86–3.79 3.41–3.37 3.32–3.28 3.78–3.74 3.69–3.66
(m) (m) (m) (m) (m) (m) (m)
4.39 (d) 3.71 (dd) 3.69 (dd) 3.59 (dd) 3.28 (dd) 3.90 (d) 3.78(d)
Other CH2O CH2O CH@CH2 CH@CH2 CH@CH2 (CH2)5 (CH2)5 (CH2)5 CH3CO CH3CO
3.91–3.87 3.44–3.40 4.99–4.95 4.91–4.89 5.84–5.76 2.08–2.01 1.57–1.47 1.42–1.26 1.99 (s) 1.99 (s)
(m) (m) (m) (m) (dddd) (m) (m) (m)
3.86–3.83 3.45–3.41 5.00–4.94 4.92–4.88 5.84–5.76 2.07–2.01 1.58–1.48 1.42–1.27 2.00 (s) 1.96 (s)
(d) (dd) (dd) (d) (dd) (dd) (dd)
(m) (dt) (m) (m) (dddd) (m) (m) (m)
H Type 1 6
H Type II 7
5.32 (m) 3.73 (m) 3.69–3.66 (m) 3.69–3.66 (m) 4.29 (q) 1.20 (d)
5.20 (d) 3.73 (dd) 3.68 (dd) 3.71–3.69 (m) 4.29 (q) 1.19 (d)
5.21 (d) 3.77–3.71 (m) 3.77–3.71 (m) 3.66(d) 4.17 (q) 1.20 (d)
4.60–4.56 (m) 3.93–3.87 (m) 3.93–3.87 (m) 4.10 (s) 3.54 (dd) 3.79 (dd) 3.68 (dd)
4.53 (d) 3.98 (dd) 3.94 (dd) 4.12 (d) 3.60–3.54 (m) 3.79 (dd) 3.67 (dd)
4.54–4.51 3.71–3.65 3.71–3.65 3.80–3.78 3.52 (dd) 3.67–3.64 3.77–3.74
(m) (m) (m) (m) (m) (m)
4.50–4.44 3.75–3.68 3.75–3.68 3.78(d) 3.54 (dd) 3.65–3.61 3.75–3.73
4.25–4.19 3.86–3.77 3.86–3.77 3.41–3.37 3.31–3.28 3.93–3.83 3.76–3.64
(m) (m) (m) (m) (m) (m) (m)
4.36 (d) 3.70 (dd) 3.59 (dd) 3.66 (dd) 3.30–3.26 (m) 3.90 (dd) 3.77 (dd)
4.28–4.22 3.84–3.78 3.84–3.78 3.42–3.38 3.32–3.28 3.74–3.65 3.92–3.84
(m) (m) (m) (m) (m) (m) (m)
4.37 (d) 3.72 (dd) 3.59 (dd) 3.69 (dd) 3.33–3.29 (m) 3.89 (dd) 3.83 (dd)
3.92–3.83 3.46–3.37 4.99–4.95 4.92–4.89 5.84–5.75 2.07–2.01 1.57–1.46 1.42–1.27 1.99 (s)
(m) (m) (m) (m) (dddd) (m) (m) (m)
3.91–3.83 3.48–3.43 5.00–4.95 4.93–4.88 5.84–5.76 2.08–2.01 1.58–1.49 1.43–1.25 1.95 (s)
3.45–3.37 3.91–3.83 4.99–4.93 4.92–4.88 5.84–5.75 2.07–2.00 1.58–1.46 1.41–1.26 1.97 (s)
(m) (m) (m) (m) (dddd) (m) (m) (m)
3.47–3.42 3.91–3.82 4.99–4.94 4.92–4.88 5.83–5.75 2.07–2.01 1.58–1.48 1.41–1.28 1.96 (s)
(m) (dt) (m) (m) (dddd) (m) (m) (m)
(m) (m) (m)
(m) (m)
(m) (dt) (m) (m) (dddd) (m) (m) (m)
Table 3 2 JH,H,3JH,H (Hz) of the ABH type I–II antigens 4–9, recorded at 600 MHz with CD3OD as solvent Coupling
A Type I 5
A Type II 9
B Type I 4
B Type II 8
3
JH-l000 ,H-2000 3 JH-2000 ,H-3000 3 JH-3000 ,H-4000 3 JH-4000 ,H-5000 3 JH-5000 ,H-6a000 3 JH-5000 ,H-6b 2 JH-6a000 ,H-6b000
3.8 10.8 3.4 0 7.6 4.1 11.7
3.7 11.0 3.0 0 7.2 4.5 11.5
3.5 10.2 3.1 0.8 7.3 4.3 11.4
3.8 10.1 2.9 m rr m m
3
JH-l00 ,H-200 JH-200 ,H-300 3 JH-300 ,H-400 3 JH-500 ,H-600
3.9 10.5 3.1 6.5
3.9 9.5 3.3 6.6
4.1 9.3 3.3 6,6
3
JH-l0 ,H-20 JH-20 ,H-30 3 JH-30 ,H-40 3 JH-50 ,H-6a0 3 JH-50 ,H-6b0 2 JH-6a0 ,H-6b0
7.3 9.9 3.1 7.9 4.3 m
7.7 9.8 3.0 rr m rr
3
JH-l,H-2 JH-2,H-3 3 JH-3,H-4 3 JH-4,H-5 3 JH-5,H-6a 3 JH-5,H-6b 2 JH-6a,H-6b
m m m m m m m
CH2O CH@CH2
m 17.1, 10.2, 6.7, 6.7
3
3
3
H Type I 6
H Type II 7
3.9 9.9 m 6.6
3.8 10.0 3.1 6.6
3.3 m 2.8 6.6
m m 0 7.8 4.4 11.4
7.4 9.7 2.7 7.3 3.9 11.6
m m m 7.7 4.3 m
8.4 9.3 8.9 9.8 4.9 0 11.2
m m m m m m m
8.4 10.4 8.8 9.2 5.0 1.8 11.9
m m m m m m rr
8.4 10.5 8.8 10.4 4.4 1.9 12.0
9.7, 6.6 17.0, 10.2, 6.7, 6,7
m 17.0, 10.2, 6.7, 6.7
9.7, 6.6 17.0, 10.2, 6.7, 6.7
m 17.0, 10.2, 6.7, 6.7
9.6, 6.6 17.0, 10.2, 6.7, 6.7
4.0 7.6 4.0
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
HO
OH O
OH
OH
HO
O
HO O
O O H1′ HO
H1′′′ O OH HO
H1′′ OH 8 B Type II
OH O O NHAc H1
Figure 3. Nomenclature system used for 4–9, using the B type II antigen 8 for illustrative purposes. This nomenclature is used for the data presented in Tables 2 and 3.
3.5. 7-Octen-1-yl 2-acetamido-2-deoxy-4-O-(2-O-(a-Lfucopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (7) Redistilled liquid ammonia (20 mL) was collected in a flask cooled to (78 °C) and treated with sodium until the blue colour persisted. A solution of trisaccharide 39 (185 mg, 0.157 mmol) in THF (4 mL) and CH3OH (38 lL, 0.94 mmol) was added dropwise, and the mixture was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected trisaccharide 7 (75 mg, 75%) as a colourless oil. [a] 78.9 (c 0.4, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125.7 MHz, CD3OD): dC 178.4 (C@O), 140.1 (CH2@CH), 114.8 (CH2@CH), 102.9, 102.6 (C1, C10 ), 101.9 (C100 ), 79.1, 78.3, 77.1, 77.0, 75.3, 74.1, 73.6, 71.7, 70.8, 70.7 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C50 ), 70.7 (CH@CH2(CH2)5CH2O), 68.3 (C500 ), 62.6, 61.7 (C6, C60 ), 56.8 (C2), 34.8 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O), 30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.0 (CH3CO), 16.8 (C600 ). ESIMS: m/z Calcd [C28H49NO15]Na+: 662.2994. Found: 662.2985. 3.6. 7-Octen-1-yl 2-acetamido-2-deoxy-4-O-(2-O-(a-Lfucopyranosyl)-3-O-(a-D-galactopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (8) Redistilled liquid ammonia (20 mL) was collected in a flask cooled to (78 °C) and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 42 (244 mg, 0.144 mmol) in THF (4 mL) and CH3OH (58 lL, 1.44 mmol) was added dropwise, and the mixture was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 8 (111 mg, 96%) as a colourless oil. [a] 28.9 (c 0.5, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125.7 MHz, CD3OD): dC 173.4 (C@O), 140.1 (CH2@CH), 114.8 (CH2@CH), 102.8, 102.2 (C1, C10 ), 100.3 (C100 ), 96.1 (C1000 ), 79.8, 78.6, 77.1, 76.7, 74.1, 73.8, 73.6, 73.1, 71.8, 71.4, 71.3, 70.0, 69.9, 67.6, 65.8 (C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C50 , C500 , C5000 ), 70.7 (CH@CH2(CH2)5CH2O), 63.3, 62.6, 61.8 (C6, C60 , C6000 ), 56.8 (C2), 34.8 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5 CH2O), 30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.0 (CH3CO), 16.6 (C600 ). ESIMS: m/z Calcd [C34H59NO20]Na+: 824.3523. Found: 824.3518. 3.7. 7-Octen-1-yl 2-acetamido-4-O-(3-O-(2-acetamido-2-deoxy-
a-D-galactopyranosyl)-2-O-(a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (9) A solution of tetrasaccharide 44 (520 mg, 0.345 mmol) in CH3OH (20 mL) was treated with a catalytic amount of NaOCH3
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in CH3OH, and the solution was stirred for 2 h. The solution was then neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to flash chromatography (CH2Cl2–CH3OH 10:1) to afford triol 45 (440 mg, 92%) as a colourless oil. Redistilled liquid ammonia (20 mL) was collected in a flask cooled to (78 °C) and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 45 (221 mg, 0.160 mmol) in THF (4 mL) and CH3OH (39 lL, 0.961 mmol) was added dropwise, and the mixture was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 9 (129 mg, 96%) as a colourless oil. [a] +5.8 (c 1.6, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125.7 MHz, CD3OD): dC 174.4 (C@O), 173.3 (C@O), 140.0 (CH2@CH), 114.8 (CH2@CH), 104.2 (C10 ), 100.5 (C100 ), 98.1 (C1), 93.9 (C1000 ), 77.6, 77.4, 76.2, 75.8, 73.4, 72.7, 71.8, 71.6, 70.6, 70.1, 70.0, 69.88 (C20 , C200 , C3, C30 , C300 , C3000 , C4, C400 , C4000 , C5, C50 , C5000 ), 68.94 (CH@CH2(CH2)5CH2O), 68.4 (C500 ), 64.9 (C40 ), 63.4, 62.8, 62.5 (C6, C60 , C6000 ), 51.4, 50.9 (C2, C2000 ), 34.9 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O), 30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.2 (CH@CH2(CH2)5CH2O), 22.9 (CH3CO), 22.8 (CH3CO), 16.9 (C600 ). ESIMS: m/z Calcd [C36H62NO20]Na+: 865.3788. Found: 865.3783. 3.8. 7-Octen-1-yl 2-azido-4,6-O-benzylidene-2-deoxy-b-Dglucopyranoside (13) and 7-Octen-1-yl 2-Azido-4,6-Obenzylidene-2-deoxy-a-D-glucopyranoside (14) A stirred solution of trichloroacetimidate27 10 (8.69 g, 18.3 mmol) and 7-octen-1-ol (2.82 g, 22.0 mmol) in dry CH2Cl2 (50 mL) was treated with 4 Å molecular sieves (3.5 g), and the mixture was stirred (rt, 1 h). The mixture was cooled (30 °C), treated with TMSOTf (300 lL) and allowed to slowly warm to 0 °C. The mixture was neutralized by the addition of Et3N (1 mL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:3) to give an inseparable a/b mixture 11, which was used immediately in the subsequent step. The oil was taken up in CH3OH (80 mL) and treated with a catalytic amount of NaOCH3 in CH3OH, and the solution was stirred (rt, 1 h). The solution was then neutralized with Amberlite IR 120 (H+), and the mixture was filtered; concentration followed by flash chromatography (EtOAc–hexanes 2:1) yielded triol 12 (3.32 g) as an inseparable a/b mixture. A solution of triol 12 (3.32 g, 10.5 mmol) in dry DMF (20 mL) was then treated with benzaldehyde dimethyl acetal (2.13 g, 14.0 mmol) and p-TsOH (100 mg), and the solution was stirred (50 °C, 4 h). The solution was treated with Et3N (1 mL) and concentrated, and the residue was subjected to flash chromatography (EtOAc–hexanes 1:3) to firstly afford the b glycoside 13 as a colourless oil (3.05 g, 41%). [a] 38.4 (c 0.4, CH2Cl2); Rf 0.18 (EtOAc–hexanes 7:3); 1H NMR (500 MHz): dH 7.52–7.35 (5H, m, Ph), 5.87–5.76 (1H, m, CH@CH2), 5.54 (1H, s, PhCH), 5.04–4.92 (2H, m, CH@CH2), 4.42 (1H, d, J1,2 = 8.0 Hz, H1), 4.34 (1H, dd, J6,6 = 10.3 Hz, J5,6 = 5.0 Hz, H6), 3.97–3.89 (1H, m, CH@CH2(CH2)5CH2O), 3.79 (1H, dd, J6,6 = J5,6 = 10.3 Hz, H6), 3.69–3.51 (3H, m, H3, H4, CH@CH2(CH2)5CH2O), 3.45–3.35 (2H, m, H2, H5), 2.69 (1H, br s, OH), 2.10–1.99 (2H, m, CH@CH2(CH2)5CH2O), 1.73–1.56 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.25 (6H, m, CH@CH2(CH2)5CH2O). 13 C NMR (125 MHz): dC 139.0 (CH@CH2), 136.8 (Ph), 129.4 (Ph), 128.4 (Ph), 126.2 (Ph), 114.3 (CH@CH2), 102.7, 102.0 (PhCH, C1), 80.6, 72.0, 66.5, 66.2 (C2, C3, C4, C5), 70.7 (CH@CH2(CH2)5CH2O), 68.5 (C6), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 28.78 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C21H29N3O5]Na+:
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
426.2000. Found: 426.2002. Further elution yielded a glycoside 14 as a colourless oil (0.8 g, 9%). [a] +80.3 (c 0.7, CH2Cl2); Rf 0.16 (EtOAc–hexanes 7:3); 1H NMR (500 MHz): dH 7.41–7.37 (5H, m, Ph), 5.87–5.77 (1H, m, CH@CH2), 5.55 (s, PhCH), 5.04– 4.93 (2H, m, CH@CH2), 4.90 (1H, d, J1,2 = 3.6 Hz, H1), 4.28 (1H, dd, J6,6 = 10.2 Hz, J5,6 = 4.9 Hz, H6), 4.23 (1H, dd, J2,3 = 10.0 Hz, J3,4 = 9.6 Hz, H3), 3.87 (1H, ddd, J5,6 = 9.9 Hz, J4,5 = 9.9 Hz, J5,6 = 4.9 Hz, H5), 3.77–3.71 (2H, m, H6, CH@CH2(CH2)5CH2O), 3.54–3.46 (2H, m, H4, CH@CH2(CH2)5CH2O), 3.23 (1H, dd, J2,3 = 10.0 Hz, J1,2 = 3.6 Hz, H2), 2.76 (1H, br s, OH), 2.09–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.70–1.62 (2H, m, CH@CH2(CH2)5 CH2O), 1.46–1.33 (6H, m, CH@CH2(CH2)5CH2O). 13C NMR (125 MHz): dC 139.0 (CH@CH2), 136.9 (Ph), 129.5 (Ph), 128.4 (Ph), 126.4 (Ph), 114.3 (CH@CH2), 102.1 (PhCH), 98.6 (C1), 82.0 (C4), 68.9, 68.8 (C6, CH@CH2(CH2)5CH2O), 68.7 (C3), 63.1, 62.4 (C2, C5), 33.7 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 28.78 (CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C21H29N3O5]Na+: 426.2000. Found: 426.1995.
3.9. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-O-pivaloylb-D-galactopyranosyl)-2-deoxy-b-D-glucopyranoside (18) A solution of acceptor 13 (2.3 g, 5.7 mmol) in dry CH2Cl2 (20 mL) was stirred over 4 Å molecular sieves (1.5 g), and the mixture was stirred (rt, 1 h). The solution was then cooled (40 °C) and treated with TMSOTf (0.2 mL), followed by the dropwise addition of trichloroacetimidate 1533 (8.0 g, 16 mmol). The mixture was allowed to warm to 0 °C, and it was then neutralized by the addition of Et3N (2 mL). Concentration and flash chromatography (EtOAc–hexanes 1:1) of the residue afforded a colourless oil that was immediately used in the next step. The colourless oil was taken up in CH3OH (100 mL), treated with a solution of NaOCH3 in CH3OH and stirred (rt, 3 h). The solution was neutralized with Amberlite IR 120 (H+), filtered and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford diol 17 as a colourless oil (2.3 g, 63%). A solution of 17 (2.20 g, 3.37 mmol) in dry pyridine (30 mL) was treated with trimethylacetyl chloride (600 mg, 5 mmol), and the solution was stirred (rt, 30 min). Further trimethylacetyl chloride (600 mg, 5 mmol) was added, and the solution was stirred (rt, 30 min). The solution was then concentrated and subjected to flash chromatography (EtOAc–hexanes 2:3) to afford 18 as a colourless oil (2.2 g, 89%). [a] +5.8 (c 0.1, CH2Cl2); Rf 0.75 (EtOAc–hexanes 2:3); 1H NMR (500 MHz): dH 7.52–7.46 (4H, m, Ph), 7.38–7.30 (6H, m, Ph), 5.86–5.76 (1H, m, CH2@CH), 5.54 (1H, s, PhCH), 5.46 (1H, s, PhCH), 5.04–4.92 (2H, m, CH2@CH), 4.78 (1H, dd, J20 ,30 = 9.5 Hz, J30 ,40 = 3.6 Hz, H30 ), 4.49 (1H, d, J10 ,20 = 7.9 Hz, H10 ), 4.47 (1H, d, J1,2 = 8.0 Hz, H1), 4.37–4.29 (2H, m, H40 , H6), 4.17 (1H, d, J60 ,60 = 12.1 Hz, H60 ), 4.05 (1H, dd, J20 ,30 = 9.5 Hz, J10 ,20 = 8.2 Hz, H20 ), 3.96–3.88 (2H, m, H60 , CH@CH2(CH2)5CH2O), 3.80 (1H, dd, J6,6 = J5,6 = 10.1 Hz, H6), 3.77– 3.72 (2H, m, H3, H4), 3.62–3.49 (2H, m, H2, CH@CH2(CH2)5CH2O), 3.46–3.35 (1H, m, H5), 3.33–3.29 (1H, m, H50 ), 3.02–2.96 (1H, br s, OH), 2.11–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.72–1.60 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.30 (6H, m, CH@CH2(CH2)5CH2O), 1.22 (9H, s, (CH3)3C). 13C NMR (125 MHz): dC 178.3 (C@O), 139.0 (CH2@CH), 137.9 (Ph), 137.0 (Ph), 129.1 (Ph), 128.7 (Ph), 128.2 (Ph), 128.0 (Ph), 126.02 (Ph), 125.96 (Ph), 114.3 (CH2@CH), 104.5 (C10 ), 102.7 (C1), 101.4 (PhCH), 100.5 (PhCH), 79.9, 79.8 (C3, C4), 73.21, 73.20 (C30 , C40 ), 70.8 (CH@CH2(CH2)5CH2O), 69.1 (C20 ), 68.8, 68.5 (C6, C60 ), 67.0 (C50 ), 66.3 (C5), 65.5 (C2), 39.0 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.80 (CH@CH2(CH2)5CH2O), 28.77 (CH@CH2(CH2)5CH2O), 27.1 ((CH3)3C), 25.7 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C44H59N3O13]Na+: 760.3416. Found: 760.3415.
3.10. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-Opivaloyl-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (20) A solution of acceptor 18 (415 mg, 0.563 mmol) in dry Et2O– CH2Cl2 (90:10, 20 mL) was stirred over 4 Å molecular sieves (rt, 1 h). The solution was then cooled (10 °C), treated with TMSOTf (20 lL), followed by dropwise addition of trichloroacetimidate29 19 (1.02 g, 13.8 mmol) in dry Et2O (15 mL). The mixture was treated with Et3N (0.5 mL) and filtered, and the resulting residue was subjected to flash chromatography (EtOAc–hexanes 1:3) to yield trisaccharide 20 as a colourless oil (510 mg, 80%). [a] 20.7 (c 0.2, CH2Cl2); Rf 0.59 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.55–7.22 (25H, m, Ph), 5.87–5.77 (1H, m, CH2@CH), 5.41 (1H, d, J100 ,200 = 1.5 Hz, H100 ), 5.48 (1H, s, PhCH), 5.37 (1H, s, PhCH), 5.04– 4.92 (4H, m, H30 , PhCH2, CH2@CH), 4.79, 4.74 (2H, AB, J = 11.5 Hz, PhCH2), 4.76 (1H, d, J10 ,20 = 8.1 Hz, H10 ), 4.69 (1H, A of AB, J = 11.7 Hz, PhCH2), 4.79, 4.63 (2H, AB, J = 11.5 Hz, PhCH2), 4.51 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.42 (1H, d, J1,2 = 7.7 Hz, H1), 4.34– 4.29 (2H, m, H6, H60 ), 4.24 (1H, d, J10 ,20 = J20 ,30 = 8.5, H20 ), 4.13–4.07 (3H, m, H200 , H300 , H400 ), 3.97–3.91 (1H, m, CH@CH2(CH2)5CH2O), 3.83–3.70 (5H, m, H3, H4, H40 , H6, H60 ), 3.63–3.56 (1H, m, CH@CH2(CH2)5CH2O), 3.43–3.35 (2H, m, H2, H5), 3.04–2.99 (1H, m, H50 ), 2.11–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.73–1.63 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.31 (6H, m, CH@CH2(CH2)5CH2O), 1.20 (3H, d, J500 ,600 = 6.3 Hz, H600 ), 1.08 (9H, s, (CH3)3C). 13C NMR (125 MHz): dC 177.9 (C@O), 139.01 (Ph), 138.98 (CH2@CH), 138.6 (Ph), 138.4 (Ph), 137.6 (Ph), 136.8 (Ph), 129.2 (Ph), 128.7 (Ph), 128.5 (Ph), 128.4 (Ph), 128.33 (Ph), 128.28 (Ph), 128.2 (Ph), 128.0 (2C, Ph), 127.6 (Ph), 127.5 (Ph), 127.44 (Ph), 127.38 (Ph), 126.2 (2C, Ph), 114.3 (CH2@CH), 102.8 (C1), 101.05 (C10 ), 101.7 (PhCH), 100.8 (PhCH), 96.8 (C100 ), 79.9, 79.7, 78.0, 77.5, 76.55, 76.52 (C3, C30 , C300 , C4, C40 , C400 ), 75.0 (PhCH2), 73.5 (PhCH2), 72.9 (PhCH2), 72.6, 70.1 (C20 , C200 ), 70.7 (CH@CH2(CH2)5CH2O), 68.8, 68.6 (C6, C60 ), 66.5 (C500 ), 66.4, 65.9, 65.7 (C2, C5, C50 ), 38.8 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.83 (CH@CH2(CH2)5CH2O), 28.78 (CH@CH2(CH2)5CH2O), 27.0 ((CH3)3C), 25.8 (CH@CH2(CH2)5CH2O), 16.9 (C600 ). ESIMS: m/z Calcd [C66H79N3O15]Na+: 1176.5403. Found: 1176.5402. 3.11. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-2-O-(2,3,4tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2deoxy-b-D-glucopyranoside (21) A solution of pivaloyl ester 20 (1.456 g, 1.26 mmol) in CH3OH (150 mL) was treated with catalytic LiOCH3 (100 mg), and the solution was heated at reflux (7 d). The solution was then concentrated, extracted with EtOAc (400 mL) and washed with satd aq NaHCO3 and brine. The organic extract was then dried, concentrated and subjected to flash chromatography (EtOAc–hexanes 3:7) to afford alcohol 21 as a colourless oil (1.10 g, 82%). [a] 20.7 (c 0.1, CH2Cl2); Rf 0.26 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.63–7.51 (4H, m, Ph), 7.42–7.19 (21H, m, Ph), 5.89–5.79 (1H, m, CH2@CH), 5.57 (1H, s, PhCH), 5.54 (1H, s, PhCH), 5.33 (1H, s, H100 ), 5.06–4.94 (3H, m, PhCH2, CH2@CH), 4.85 (1H, A of AB, J = 11.5 Hz, PhCH2), 4.84–4.74 (3H, m, PhCH2), 4.68 (1H, d, J10 ,20 = 7.1 Hz, H1´), 4.66 (1H, A of AB, J = 11.1 Hz PhCH2), 4.42 (1H, d, J1,2 = 8.2 Hz, H1), 4.35 (1H, dd, J6,6 = 10.5 Hz, J5,6 = 4.8 Hz, H6), 4.31 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.23 (1H, d, J60 ,60 = 12.4 Hz, H60 ), 4.18 (1H, d, J30 ,40 = 3.2 Hz, H40 ), 4.13–4.06 (2H, m, H200 , H300 ), 3.98–3.90 (3H, m, H20 , H6, CH@CH2(CH2)5CH2O), 3.86–3.79 (3H, m, H400 , H60 , OH), 3.78–3.72 (2H, m, H3, H30 ), 3.68 (1H, dd, J3,4 = 9.0 Hz, J4,5 = 9.0 Hz, H4), 3.63–3.58 (1H, m, CH@CH2(CH2)5CH2O), 3.44 (1H, dd, J1,2 = 8.2 Hz, J2,3 = 8.2 Hz, H2), 3.41–3.36 (1H, m, H5), 3.26 (1H, s, H50 ), 2.12–2.04 (2H, m, CH@CH2(CH2)5CH2O), 1.78–1.56 (2H, m, CH@CH2(CH2)5CH2O),
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
1.49–1.32 (6H, m, CH@CH2(CH2)5CH2O), 1.24 (d, 3H, J500 ,600 = 6.3 Hz, H600 ). 13C NMR (125 MHz): dC 139.0 (CH2@CH), 138.8 (Ph), 138.7 (Ph), 137.9 (Ph), 137.8 (Ph), 137.1 (Ph), 129.0 (Ph), 128.7 (Ph), 128.41 (Ph), 128.38 (Ph), 128.36 (Ph), 128.23 (2C, Ph), 128.19 (Ph), 128.1 (Ph), 127.8 (Ph), 127.54 (Ph), 127.46 (Ph), 127.4 (Ph), 126.9 (Ph), 126.1 (Ph), 114.3 (CH2@CH), 102.9 (C1), 101.7, 101.2, 100.9 (3C, PhCH, C10 ), 99.41 (C100 ), 79.9, 78.8, 78.0, 77.8, 77.1, 76.5, 75.5 (C20 , C200 , C3, C30 , C300 , C4, C40 ), 75.0 (PhCH2), 74.0 (PhCH2), 73.8 (C400 ), 72.7 (PhCH2), 70.7 (CH@CH2(CH2)5CH2O), 69.0, 68.5 (C6, C60 ), 66.8, 66.74, 66.70, 66.6 (C2, C5, C50 , C500 ), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.83 (CH@CH2(CH2)5CH2O), 28.80 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2 (CH2)5CH2O), 17.04 (C600 ). ESIMS: m/z Calcd [C61H71N3O14]Na+: 1092.4828. Found: 1092.4823. 3.12. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-2-O(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2deoxy-b-D-glucopyranoside (22) A solution of azide 21 (250 mg, 0.234 mmol) in dry pyridine (2 mL) was treated with CH3COSH (4 mL), and the solution was stirred (rt, 8d). The mixture was filtered, concentrated and subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford firstly unreacted azide 21 (55 mg, 22%). Further elution yielded the desired compound 22 contaminated with some acetylated alcohol. The resulting oil was taken up in MeOH (15 mL) and treated with a catalytic amount of NaOMe in MeOH, and the solution was stirred (rt, 12 h). The solution was neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford 22 (155 mg, 62%) as a colourless oil. [a] 22.2 (c 0.1, CH2Cl2); Rf 0.81 (CH2Cl2–MeOH, 9:1); 1 H NMR (500 MHz): dH 7.61–7.54 (4H, m, Ph), 7.39–7.15 (19H, m, Ph), 7.07–7.01 (2H, m, Ph), 5.85–5.76 (1H, m, CH2@CH), 5.58 (1H, s, PhCH), 5.56 (1H, s, PhCH), 5.11 (1H, d, J100 ,200 = 3.0 Hz, H100 ), 5.03–4.93 (3H, m, PhCH2, CH2@CH), 4.90 (1H, d, J1,2 = 8.2 Hz, H1), 4.79–4.67 (4H, m, PhCH2), 4.63 (1H, A of AB, J = 11.5 Hz, PhCH2), 4.39 (1H, d, J10 ,20 = 7.6 Hz, H10 ), 4.36 (1H, dd, J6,6 = 10.4 Hz, J5,6 = 4.9 Hz, H6), 4.28–4.20 (2H, m, H3, H40 ), 4.15–4.04 (4H, m, H200 , H300 , H500 , H60 ), 3.97–3.88 (3H, m, H20 , H60 , OH), 3.87–3.78 (2H, m, H6, CH@CH2(CH2)5CH2O), 3.70 (1H, dd, J3,4 = J4,5 = 9.2 Hz, H4), 3.66–3.62 (2H, m, H30 , H400 ), 3.41–3.53 (3H, m, H2, H5, CH@CH2(CH2)5CH2O), 3.16–3.14 (m, H50 ), 1.85 (3H, s, CH3C@O), 2.07–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.59–1.48 (2H, m, CH@CH2(CH2)5CH2O), 1.40–1.25 (6H, m, CH@CH2(CH2)5CH2O), 1.22 (d, 3H, J500 ,600 = 6.2 Hz, H600 ). 13C NMR (125 MHz): dC 170.6 (C@O). 139.0 (CH2@CH), 138.5 (Ph), 138.4 (Ph), 138.1 (Ph), 137.5 (Ph), 136.8 (Ph), 129.0 (Ph), 128.9 (Ph), 128.6 (Ph), 128.44 (Ph), 128.41 (Ph), 128.3 (Ph), 128.2 (Ph), 128.15 (Ph), 128.07 (Ph), 128.1 (Ph), 127.7 (Ph), 127.6 (Ph), 127.4 (Ph), 126.7 (Ph), 126.1 (Ph), 114.2 (CH2@CH), 102.82 (C10 ), 101.5, 101.3, 101.0 (3C, C1, PhCH), 99.0 (C100 ), 80.2, 79..0, 78.9, 77.5, 77.3, 77.2, 74.5, 72.4 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 ), 74.8, 73.1 (3C, PhCH2), 69.9, 69.1, 68.8 (C6, C60 , CH@CH2(CH2)5CH2O), 68.6, 66.3 (3C, C5, C50 , C500 ), 57.55 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2 (CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5 CH2O), 25.7 (CH@CH2(CH2)5CH2O), 23.1 (CH3C@O), 17.0 (C600 ). ESIMS: m/z Calcd [C63H75NO15]Na+: 1108.5029. Found: 1108.5029. 3.13. 7-Octyl 2-acetamido-2-deoxy-3-O-(2-O-(a-Lfucopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (23) Octenyl glycoside 6 (82 mg, 0.128 mmol) was then taken up in THF–H2O (1:1, 10 mL), treated with Pd–C (10%, 15 mg) and stirred under a H2 atmosphere (48 h). The mixture was then filtered and concentrated to afford the fully deprotected trisaccharide 23 as a
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colourless oil (68 mg, 83%). [a] 15.1 (c 0.1, H2O); 1H NMR (500 MHz, D2O): dH 5.19 (1H, d, J100 ,200 = 4.0 Hz, H100 ), 4.65 (1H, d, J1,2 = 7.7 Hz, H1), 4.41 (1H, d, J10 ,20 = 8.8 Hz, H10 ), 4.32 (1H, q, J500 ,600 = 6.6 Hz, H500 ), 4.01–3.45 (17H, m, H2, H20 , H200 , H3, H30 , H300 , H4, H40 , H400 , H5, H50 , H6, H60 , CH3(CH2)6CH2O), 2.06 (3H, s, CH3C@O), 1.60–1.48 (2H, m, CH3(CH2)6CH2O), 1.37–1.26 (10H, m, CH3(CH2)6CH2O), 1.24 (3H, d, 3H, J500 ,600 = 6.6 Hz, H600 ), 0.87 (3H, t, J = 7.0 Hz, CH3(CH2)6CH2O). 13C NMR (125 MHz, D2O): dC 175.0 (C@O), 103.4, 101.7, 101.0 (C1, C10 , C100 ), 78.9, 78.2, 76.9, 76.6, 75.0, 73.3, 70.9, 70.7, 70.3, 69.6, 68.0 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C50 , C500 ), 72.1 (CH3(CH2)6CH2O), 62.7, 62.3 (C6, C60 ), 56.4 (C2), 32.7 (CH3(CH2)6CH2O), 30.1 (CH3(CH2)6CH2O), 30.0 (CH3(CH2)6CH2O), 29.9 (CH3(CH2)6CH2O), 26.6 (CH3(CH2)6CH2O), 23.5 (CH3(CH2)6CH2O), 16.8 (C600 ), 14.9 (CH3(CH2)6CH2O). ESIMS: m/z Calcd [C28H51NO15]Na+: 664.3151. Found: 664.3160.
3.14. 7-Octen-1-yl 2-azido-3-O-(3-O-(2-azido-2-deoxy-3,4,6tetra-O-acetyl-a-D-galactopyranosyl)-4,6-O-benzylidene-2-O(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2deoxy-b-D-glucopyranoside (25) A solution of acceptor 21 (621 mg, 0.58 mmol) and the trichloroacetimidate (823 mg, 1.74 mmol) in dry Et2O (15 mL) was treated with 4 Å molecular sieves (rt, 1 h). The mixture was cooled (20 °C) and treated with TMSOTf (10 lL, 0.058 mmol) and allowed to warm (0 °C). The mixture was treated with Et3N (200 lL), filtered, concentrated and subjected to flash chromatography (EtOAc–CH2Cl2 3:97) to afford tetrasaccharide 25 as a colourless oil (737 mg, 92%). [a] +8.9 (c 0.1, CH2Cl2); Rf 0.25 (EtOAc–hexanes 2:3); 1H NMR (500 MHz): dH 7.55–7.20 (25H, m, Ph), 5.87–5.77 (1H, m, CH2@CH), 5.53 (1H, d, J100 ,200 = 2.5 Hz, H100 ), 5.51 (1H, s, PhCH), 5.49 (1H, s, PhCH), 5.28 (1H, d, J1000 ,2000 = 3.2 Hz, H1000 ), 5.20 (1H, dd, J2000 ,3000 = 11.0 Hz, J3000 ,4000 = 2.9 Hz, H3000 ), 5.18–5.12 (2H, m, PhCH2, H4000 ), 5.04–4.93 (3H, m, PhCH2, CH2@CH), 4.90 (1H, A of AB, J = 11.9 Hz, PhCH2), 4.75 (2H, s, PhCH2), 4.67 (1H, d, J10 ,20 = 7.9 Hz, H10 ), 4.63 (1H, A of AB, J = 11.9 Hz, PhCH2), 4.52 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.46 (1H, d, J1,2 = 8.0 Hz, H1), 4.33 (1H, dd, J6,6 = 10.5 Hz, J5,6 = 4.7 Hz, H6), 4.28–4.25 (1H, m, H40 ), 4.22–4.12 (5H, m, H20 , H200 , H300 , H5000 , H60 ), 3.88 (1H, d, J60 ,60 = 12.4 Hz, H60 ), 3.84–3.74 (5H, m, H30 , H4, H400 , H6, H6000 ), 3.70 (1H, dd, J2,3 = J3,4 = 9.2 Hz, H3), 3.99– 3.92 (1H, m, CH@CH2(CH2)5CH2O), 3.65–3.60 (1H, m, CH@CH2(CH2)5CH2O), 3.55 (1H, dd, J2000 ,3000 = 11.0 Hz, J1000 ,2000 = 3.2 Hz, H2000 ), 3.50–3.37 (2H, m, H2, H5), 3.22 (1H, dd, J6000 ,6000 = 11.5 Hz, J5000 ,6000 = 3.5 Hz, H6000 ), 3.10–3.07 (1H, m, H50 ), 2.10–2.06 (2H, m, CH@CH2(CH2)5CH2O), 2.09 (3H, s, CH3C@O), 2.09 (3H, s, CH3C@O), 1.94 (3H, s, CH3C@O), 1.77–1.55 (2H, m, CH@CH2(CH2)5CH2O), 1.47–1.35 (6H, m, CH@CH2(CH2)5CH2O), 1.22 (3H, d, J500 ,600 = 6.3 Hz, H600 ). 13C NMR (125 MHz): dC 170.3 (C@O), 169.7 (C@O), 169.4 (C@O), 139.4 (Ph), 139.0 (Ph), 138.81 (Ph), 138.79 (CH2@CH), 137.6 (Ph), 137.0 (Ph), 129.0 (Ph), 128.7 (Ph), 128.3 (Ph), 128.24 (Ph), 128.16 (Ph), 128.1 (Ph), 128.0 (Ph), 127.45 (Ph), 127.42 (Ph), 127.37 (Ph), 127.3 (Ph), 127.2 (Ph), 126.2 (Ph), 126.1 (Ph), 114.3 (CH2@CH), 102.9 (C1), 101.4, 101.2, 100.7 (3C, C10 , PhCH), 97.9 (C100 ), 94.1 (C1000 ), 80.7 (C20 ), 79.7 (C3), 74.9 (PhCH2), 74.0 (PhCH2), 72.5 (PhCH2), 77.9, 77.8, 77.3, 76.0, 72.0 (C200 , C30 , C300 , C4, C400 ), 72.0 (C40 ), 70.8 (CH@CH2(CH2)5CH2O), 69.1, 68.6 (C6, C60 ), 68.8, 68.0 (C3000 ,C4000 ), 67.6 (C5000 ), 66.7, 66.4, 66.11, 66.09 (C2, C5, C50 , C500 ), 62.7 (C6000 ), 57.9 (C2000 ), 33.7 (CH@CH2 (CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.82 (CH@CH2(CH2)5 CH2O), 28.80 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 20.7 (CH3C@O), 20.62 (CH3C@O), 20.58 (CH3C@O), 16.9 (C600 ). ESIMS: m/z Calcd [C71H84N5O19]Na+: 1405.5738. Found: 1405.5740.
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
3.15. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2deoxy-3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-4,6-Obenzylidene-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (26) A solution of tetrasaccharide 25 (355 mg, 0.257 mmol) in pyridine (2 mL) was treated with AcSH (4 mL), and the solution was stirred (14 d). The mixture was filtered, concentrated and subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford 26 as a colourless oil (270 mg, 74%). [a] 8.9 (c 0.2, CH2Cl2); Rf 0.76 (MeOH–CH2Cl2 1:9); 1H NMR (500 MHz): dH 7.55–7.23, 7.20–7.10 (25H, m, Ph). 6.78 (1H, d, J = 7.3 Hz, NH), 5.85–5.76 (1H, m, CH2@CH), 5.50 (1H, s, PhCH), 5.35 (1H, s, PhCH), 5.43 (1H, d, J3000 ,4000 = 2.1 Hz, H4000 ), 5.27 (1H, d, J100 ,200 = 4.1 Hz, H100 ), 5.20 (1H, d, J = 9.8 Hz, NH000 ), 5.05–4.92 (7H, m, H1000 , H3000 , PhCH2, CH2@CH), 4.72 (1H, A of AB, J = 11.6 Hz, PhCH2), 4.67 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.60 (1H, A of AB, J = 11.1 Hz, PhCH2), 4.54– 4.46 (3H, m, H1, H2000 , H5000 ), 4.38 (1H, d, J10 ,20 = 8.2 Hz, H10 ), 4.32 (1H, dd, J6,6 = 10.7 Hz, J5,6 = 4.8 Hz, H6), 4.13 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.11–3.92, 3.89–3.74 (14H, 2m, H2, H20 , H200 , H3, H300 , H4, H40 , H400 , H6, H60 , H6000 , CH@CH2(CH2)5CH2O), 3.69 (1H, d, J60 ,60 = 12.6 Hz, H60 ), 3.62 (1H, dd, J20 ,30 = 9.6 Hz, J30 ,40 = 3.8 Hz, H30 ), 3.50–3.41 (2H, m, H5, CH@CH2(CH2)5CH2O), 2.76 (s, H50 ), 2.12 (3H, s, CH3C@O), 2.07–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.94 (3H, s, CH3C@O), 1.85 (3H, s, CH3C@O), 1.71 (3H, s, CH3C@O), 1.60–1.51 (2H, m, CH@CH2(CH2)5CH2O), 1.40–1.28 (2H, m, CH@CH2(CH2)5CH2O), 1.27 (3H, s, CH3C@O), 1.22 (3H, d, J500 ,600 = 6.3 Hz, H600 ). 13C NMR (125 MHz): dC 170.6 (C@O), 170.3 (C@O), 170.2 (C@O), 170.0 (C@O), 169.8 (C@O), 139.7 (Ph), 139.1 (CH2@CH), 138.7 (Ph), 138.6 (Ph), 137.7 (Ph), 137.3 (Ph), 129.5 (Ph), 129.4 (Ph), 128.4 (Ph), 128.3 (2C, Ph), 128.21(Ph), 128.19 (Ph), 128.0 (Ph), 127.9 (Ph), 127.7 (Ph), 127.6 (Ph), 127.3 (Ph), 127.1 (Ph), 126.6 (Ph), 126.2 (Ph), 114.2 (CH2@CH), 102.7, 102.5, 101.9, 101.5 (4C, C1, C10 , PhCH), 98.2, 92.3 (C100 , C1000 ), 80.9, 80.0, 77.9, 77.5, 77.4, 76.5 (C20 , C200 , C3, C300 , C4, C400 ), 75.0 (PhCH2), 74.1 (PhCH2), 73.4 (C30 ), 73.0 (PhCH2), 70.2 (C40 ), 69.7, 69.1, 69.0 (C6, C60 , CH@CH2(CH2)5CH2O), 68.7, 67.7, 67.3, 66.8, 66.6, 65.5 (C3000 , C4000 , C5, C50 , C500 , C5000 ), 62.4 (C6000 ), 55.2 (C2), 46.9 (C2000 ), 33.7 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 28.84 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 25.6 (CH@CH2(CH2)5CH2O), 22.9 (CH3C@O), 22.5 (CH3C@O), 20.69 (CH3C@O), 20.66 (CH3C@O), 20.6 (CH3C@O), 17.3 (C600 ). ESIMS: m/z Calcd [C77H94N2O23]Na+: 1437.6140. Found: 1437.6138. 3.16. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2deoxy-a-D-galactopyranosyl)-4,6-O-benzylidene-2-O-(2,3,4-triO-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2-deoxy-bD-glucopyranoside (27) A solution of triacetate 26 (225 mg, 0.160 mmol) in CH3OH was treated with a catalytic amount of NaOCH3 in CH3OH, and the solution was stirred (2 h). The solution was neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford triol 27 as a colourless oil (192 mg, 94%). [a] 12.9 (c 0.3, CH3OH); Rf 0.55 (CH3OH–CH2Cl2 1:9); 1H NMR (500 MHz, CD3OD): dH 7.57–7.18 (25H, m, Ph), 5.85–5.73 (1H, m, CH2@CH), 5.60 (1H, s, PhCH), 5.52 (1H, s, PhCH), 5.29 (1H, d, J100 ,200 = 3.8 Hz, H100 ), 5.05 (1H, d, J1000 ,2000 = 3.3 Hz, H1000 ), 5.00–4.88 (4H, m, PhCH2, CH2@CH), 4.77 (1H, A of AB, J = 12.5 Hz, PhCH2), 4.69 (1H, A of AB, J = 10.5 Hz, PhCH2), 4.67 (1H, d, J1,2 = 6.7 Hz, H1), 4.83, 4.60 (2H, AB, J = 10.8 Hz, PhCH2), 4.49–4.35 (3H, m, H10 , H500 , H6), 4.33–4.06, 4.00–3.90, 3.87–3.79 (16H, 3 m, H2, H20 , H200 , H2000 , H3, H300 , H3000 , H40 , H400 , H4000 , H5000 , H6, H60 , CH@CH2(CH2)5CH2O), 3.73 (1H, dd, J3,4 = J4,5 = 9.2 Hz, H4), 3.58–3.53 (1H, m, H6000 ), 3.52–3.41 (3H, m, H3´, H5, CH@CH2(CH2)5CH2O), 3.28–3.22 (1H, m, H6000 ), 3.22–3.18 (1H, m,
H50 ), 2.06–1.99 (2H, m, CH@CH2(CH2)5CH2O), 1.85 (3H, s, CH3C@O), 1.60–1.50 (2H, m, CH@CH2(CH2)5CH2O), 1.48 (3H, s, CH3C@O), 1.41–1.28 (6H, m, CH@CH2(CH2)5CH2O), 1.28 (1H, d, J500 ,600 = 6.4 Hz, H600 ). 13C NMR (125 MHz): dC 172.5 (C@O), 171.6 (C@O), 139.2 (Ph), 138.8 (Ph), 138.7 (Ph), 138.6 (CH2@CH), 138.3 (Ph), 137.6 (Ph), 128.6 (Ph), 128.3 (Ph), 128.0 (Ph), 127.9 (Ph), 127.84 (2C, Ph), 128.78 (Ph), 127.7 (Ph), 127.2 (2C, Ph), 127.0 (Ph), 126.8 (Ph), 126.4 (Ph), 126.0 (2C, Ph), 113.4 (CH2@CH), 102.5, 101.3, 101.1, 100.9 (4C, C1, C10 , PhCH), 98.0 (C100 ), 91.8 (C1000 ), 80.1 (C4), 74.9 (PhCH2), 72.7 (PhCH2), 71.9 (PhCH2), 79.7, 77.7, 76.28, 76.27, 75.3, 73.5, 71.4, 70.5 (C20 , C200 , C3, C300 , C3000 , C40 , C400 , C4000 ), 69.4 (C30 ), 69.6, 68.8, 68.3 (C6, C60 , CH@CH2(CH2)5CH2O), 68.2, 66.8, 66.4, 66.0 (C5, C50 , C500 , C5000 ), 62.0 (C6000 ), 55.4 (C2), 49.7 (C2000 ), 33.4 (CH@CH2(CH2)5CH2O), 29.2 (CH@CH2(CH2)5CH2O), 28.7 (CH@CH2 (CH2)5CH2O), 28.5 (CH@CH2(CH2)5CH2O), 25.5 (CH@CH2(CH2)5 CH2O), 22.0 (CH3C@O), 21.0 (CH3C@O), 15.8 (C600 ). ESIMS: m/z Calcd [C71H88N2O20]Na+: 1311.5823. Found: 1311.5819. 3.17. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-3-O(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-2-O-(2,3,4-tri-Obenzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2-deoxy-b-Dglucopyranoside (30) A solution of acceptor 21 (400 mg, 0.37 mmol) and trichloroacetimidate 2834 (770 mg, 1.12 mmol) in dry Et2O (15 mL) was treated with 4 Å molecular sieves (rt, 1 h). The mixture was cooled (20 °C) and treated with TMSOTf (10 lL, 0.058 mmol) and allowed to warm (0 °C). The mixture was treated with Et3N (200 lL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford tetrasaccharide 29 as a colourless oil (440 mg, 73%). A solution of tetrasaccharide (350 mg, 0.220 mmol) in dry pyridine (4 mL) was treated with AcSH (2 mL) and stirred (rt, 8 d). Concentration followed by flash chromatography (EtOAc–CH2Cl2 3:7) afforded firstly unreacted 29 (70 mg, 20%). Further elution afforded the desired 30 as a colourless oil (230 mg, 65%). [a] 16.0 (c 0.6, CH2Cl2); Rf 0.57 (EtOAc–hexanes 1:1); 1H NMR (500 MHz): dH 7.55–7.48 (4H, m, Ph), 7.38–7.12 (33H, m, Ph), 7.10–6.97 (8H, m, Ph), 6.79 (1H, d, J = 7.4 Hz, NH), 5.85–5.75 (1H, m, CH2@CH), 5.52 (1H, s, PhCH), 5.47 (1H, s, PhCH), 5.31 (1H, d, J1000 ,2000 = 4.0 Hz, H1000 ), 5.23 (1H, d, J100 ,200 = 3.5 Hz, H100 ), 5.02–4.92 (2H, m, CH2@CH), 4.88 (1H, A of AB, J = 11.8 Hz, PhCH2), 4.83 (1H, A of AB, J = 11.3 Hz, PhCH2), 4.68 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.61–4.51 (4H, m, H1, PhCH2), 4.49–4.29 (9H, m, H10 , H6, PhCH2), 4.23 (1H, A of AB, J = 11.8 Hz, PhCH2), 4.19 (1H, d, J30 ,40 = 3.3 Hz, H40 ), 4.15 (1H, dd, J200 ,300 = 10.0 Hz, J300 ,400 = 2.6 Hz, H300 ), 4.10–3.95 (5H, m, H20 , H200 , H3, H500 , H60 ), 3.92 (1H, dd, J2000 ,3000 = 10.0 Hz, J1000 ,2000 = 4.0 Hz, H2000 ), 3.88– 3.70 (9H, m, H2, H30 , H3000 , H400 , H4000 , H6, H60 , H6000 , CH@CH2 (CH2)5CH2O), 3.56–3.40 (4H, m, H4, H5, H6000 , CH@CH2(CH2)5CH2O), 3.30 (1H, d, J = 1.8 Hz, H5000 ), 2.80 (1H, s, H50 ), 2.08–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.72 (3H, s, CH3C@O), 1.61–1.49 (2H, m, CH@CH2(CH2)5CH2O), 1.41–1.23 (6H, m, CH@CH2(CH2)5CH2O), 1.16 (3H, d, J500 ,600 = 6.4 Hz, H600 ). 13C NMR (125 MHz): dC 170.4 (C@O), 139.1 (CH2@CH), 139.0 (Ph), 138.8 (Ph), 138.7 (Ph), 138.64 (Ph), 138.58 (Ph), 138.3 (Ph), 128.2 (Ph), 137.6 (Ph), 137.3 (Ph), 129.3 (Ph), 128.9 (Ph), 128.5 (Ph), 128.44 (Ph), 128.43 (2C, Ph), 128.40 (2C, Ph), 128.34 (Ph), 128.27 (Ph), 128.22 (Ph), 128.17 (Ph), 128.12 (Ph), 128.10 (Ph), 128.04 (Ph), 127.98 (Ph), 127.94 (Ph), 127.89 (Ph), 127.87 (Ph), 127.82 (Ph), 127.78 (2C, Ph), 127.67 (2C, Ph), 127.65 (Ph), 127.61 (Ph), 127.5 (Ph), 127.42 (Ph), 127.38 (Ph), 127.1 (Ph), 127.05 (2C, Ph), 127.02 (2C, Ph), 126.95 (Ph), 126.4 (Ph), 126.2 (Ph), 114.2 (CH2@CH), 102.9, 102.4, 101.9, 101.0 (4C, C1, C10 , PhCH), 97.6 (C1000 ), 92.4 (C100 ), 80.9, 79.5, 78.3, 77.8, 77.3, 76.5, 71.0, 69.5 (12C, C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5), 74.8 (PhCH2), 74.4 (PhCH2), 73.6 (2C, PhCH2), 72.82 (PhCH2), 72.78 (PhCH2), 71.6 (PhCH2), 69.9, 69.6, 69. 2, 69.1 (C6, C60 , C6000 , CH@CH2(CH2)5CH2O), 67.4, 66.6, 65.8 (C50 , C500 , C5000 ),
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
2319
55.5 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.84 (CH@CH2(CH2)5CH2O), 28.82 (CH@CH2(CH2)5CH2O), 25.6 (CH@CH2(CH2)5CH2O), 23.0 (CH3C@O), 17.1 (C600 ). ESIMS: m/z Calcd [C97H109NO20]Na+: 1630.7435. Found: 1630.7455.
65.8 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.85 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C28H37N3O5]Na+: 518.2625. Found: 518.2621.
3.18. 7-Octen-1-yl 2-azido-3-O-benzyl-4,6-O-benzylidene-2deoxy-b-D-glucopyranoside (31)
3.20. 7-Octen-1-yl 4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxyb-D-glucopyranoside (33)
A stirred solution of alcohol 13 (7.11 g, 17.6 mmol) in dry DMF (50 mL) was cooled (10 °C), the solution was treated with BnBr (4.20 mL, 35.3 mmol) and NaH (60%, 960 mg, 24.0 mmol) and allowed to warm (rt, 1 h). The mixture was then treated with CH3OH (1 mL) and partially concentrated; the residue was taken up in EtOAc (250 mL) and washed with water and brine. The organic extract was dried, concentrated and then subjected to flash chromatography (EtOAc–hexanes 2:3) to afford benzyl ether 31 as a white crystalline solid (8.50 g, 98%). Mp 53–55 °C; [a] 70.8 (c 1.5, CH2Cl2); Rf 0.81 (EtOAc–hexanes 3:7). 1H NMR (500 MHz): dH 7.52–7.47 (2H, m, Ph), 7.43–7.28 (8H, m, Ph), 5.87–5.78 (1H, m, CH@CH2), 5.58 (1H, s, PhCH), 5.05–4.99 (1H, m, CH@CH2), 4.97–4.94 (1H, m, CH@CH2), 4.93, 4.81 (2H, AB, J = 11.3 Hz, PhCH2), 5.01 (1H, d, J1,2 = 8.2 Hz, H1), 4.35 (1H, dd, J6,6 = 10.7 Hz, J5,6 = 4.9 Hz, H6), 3.92 (1H, ddd, J = 9.4 Hz, 6.9 Hz, 6.9 Hz, CH@CH2(CH2)5CH2O), 3.81 (1H, dd, J6,6 = 10.7 Hz, J5,6 = 10.3 Hz, H6), 3.72 (1H, dd, J3,4 = 9.2 Hz, J4,5 = 9.2 Hz, H4), 3.61–3.52 (2H, m, H3, CH@CH2(CH2)5CH2O), 3.46 (1H, dd, J2,3 = 9.5 Hz, J1,2 = 8.2 Hz, H2), 3.39 (1H, ddd, J5,6 = 10.3 Hz, J4,5 = 9.2 Hz, J5,6 = 4.9 Hz, H5), 2.10–2.03 (2H, m, CH@CH2(CH2)5 CH2O), 1.72–1.61 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13C NMR (125.7 MHz): dC 139.0 (CH@CH2), 137.9 (Ph), 137.1 (Ph), 129.0 (Ph), 128.3 (Ph), 128.2 (Ph), 128.1 (Ph), 127.8 (Ph), 126.0 (Ph), 114.2 (CH@CH2), 102.6 (C1), 101.3 (PhCH), 81.5 (C4), 78.9 (C3), 74.8 (PhCH2), 70.6 (C6), 68.6 (CH@CH2(CH2)5CH2O), 66.3, 66.1 (C2, C5), 33.6 (CH@CH2(CH2)5 CH2O), 29.35 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 28.7 (CH@CH2(CH2)5CH2O), 25.7 (CH@CH2(CH2)5CH2O), ESIMS: m/z Calcd [C28H35N3O5]Na+: 516.2469. Found: 516.2467.
A solution of alcohol 32 (130 mg, 0.262 mmol) in pyridine (5 mL) was treated with Ac2O (1 mL) and DMAP (5 mg), and the solution was stirred (2 h). The excess Ac2O was quenched by the addition of CH3OH (2 mL), and the solution was concentrated. The residue was subjected to flash chromatography (EtOAc–hexanes 1:2) to afford 33 as a colourless oil (120 mg, 85%). [a] 18.3 (c 1.1, CH2Cl2); Rf 0.64 (EtOAc–hexanes 3:7). 1H NMR (400 MHz): dH 7.39–7.26 (10H, m, Ph), 5.89–5.77 (1H, m, CH@CH2), 5.05–4.91 (3H, m, H4, CH@CH2), 4.83, 4.63 (2H, AB, J = 10.1 Hz, PhCH2), 4.53 (2H, s, PhCH2), 4.31 (1H, d, J1,2 = 7.8 Hz, H1), 3.95 (1H, ddd, J = 9.4 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.60–3.37 (6H, m, H2, H3, H5, H6, CH@CH2(CH2)5CH2O), 2.11–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.86 (3H, s, CH3C@O), 1.72–1.63 (2H, m, CH@CH2(CH2)5CH2O), 1.49–1.30 (6H, m, CH@CH2(CH2)5CH2O); 13 C NMR (100.3 MHz): dC 167.12 (C@O), 136.5 (CH@CH2), 135.31 (Ph), 135.29 (Ph), 125.9 (Ph), 125.8 (Ph), 125.4 (Ph), 125.34 (Ph), 125.30 (Ph), 125.2 (Ph), 111.7 (CH@CH2), 99.6 (C1), 77.8 (C3), 72.3 (PhCH2), 71.1 (PhCH2), 71.0, 68.4 (C4, C5), 67.9, 67.1 (C6, CH@CH2(CH2)5CH2O), 63.5 (C2), 31.2 (CH@CH2(CH2)5CH2O), 27.0 (CH@CH2(CH2)5CH2O), 26.33 (CH@CH2(CH2)5CH2O), 26.30 (CH@CH2(CH2)5CH2O), 23.3 (CH@CH2(CH2)5CH2O), 18.3 (CH3C@O). ESIMS: m/z Calcd [C30H39N3O6]Na+: 560.2731. Found: 560.2733.
3.19. 7-Octen-1-yl 2-azido-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (32) A solution of benzylidene acetal 31 (9.5 g, 19.3 mmol) in CH2Cl2 (200 mL) was treated with 4 Å molecular sieves (5 g), and the mixture was stirred (rt, 1 h). The mixture was cooled (0 °C) and treated with Et3SiH (15.4 mL, 96.7 mmol) and CF3COOH (7.45 mL, 96.7 mmol), and the mixture was allowed to slowly warm (rt, 2 h). The mixture was then neutralized with Et3N (10 mL), filtered and then diluted with CH2Cl2 (500 mL). The organic extract was washed with water and brine, dried and then filtered. Concentration followed by flash chromatography (EtOAc–hexanes 1:2) afforded alcohol 32 as a colourless oil (7.95 g, 83%). [a] 27.3 (c 1.1, CH2Cl2); Rf 0.54 (EtOAc–hexanes 3:7). 1H NMR (500 MHz): dH 7.43–7.23 (10H, m, Ph), 5.87–5.77 (1H, m, CH@CH2), 5.03–4.98 (1H, m, CH@CH2), 4.96–4.93 (1H, m, CH@CH2), 4.92, 4.78 (2H, AB, J = 11.0 Hz, PhCH2), 4.61, 4.58 (2H, AB, J = 12.6 Hz, PhCH2), 4.29 (1H, d, J1,2 = 8.5 Hz, H1), 3.91 (1H, ddd, J = 9.4 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.77–3.70 (2H, m, H6), 3.63 (1H, ddd, J3,4 = 9.3 Hz, J4,4 = 9.3 Hz, J = 2.3 Hz, H4), 3.54 (1H, ddd, J = 9.4 Hz, 6.8 Hz, 6.8 Hz, CH@CH2(CH2)5CH2O), 3.44–3.39 (1H, m, H5), 3.38 (1H, dd, J2,3 = 10.1 Hz, J1,2 = 8.5 Hz, H2), 3.25 (1H, dd, J2,3 = 10.1 Hz, J3,4 = 9.3 Hz, H3), 2.67 (1H, d, J = 2.3 Hz, OH), 2.10– 2.02 (2H, m, CH@CH2(CH2)5CH2O), 1.69–1.61 (2H, m, CH@CH2(CH2)5CH2O), 1.45–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13 C NMR (125.7 MHz): dC 139.1 (CH@CH2), 138.2 (Ph), 137.7 (Ph), 128.6 (Ph), 128.5 (Ph), 128.1 (Ph), 128.0 (Ph), 127.8 (Ph), 127.7(Ph), 114.2 (CH@CH2), 102.2 (C1), 82.5 (C3), 75.0 (PhCH2), 73.9 (C5), 73.7 (PhCH2), 72.1 (C4), 70.3 (C6, CH@CH2(CH2)5CH2O),
3.21. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-b-Dgalactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (35) A solution of acceptor 32 (1.32 g, 2.67 mmol) in dry CH2Cl2 (20 mL) was stirred over 4 Å molecular sieves (1.5 g), and the mixture was stirred (rt, 1 h). The solution was then cooled (40 °C), treated with TMSOTf (0.2 mL), followed by dropwise addition of trichloroacetimidate 1532 (3.3 g, 6.7 mmol), and then the mixture was allowed to warm (0 °C). The mixture was neutralized with Et3N (2 mL), concentrated and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford a colourless oil that was immediately used in the next step. The colourless oil was taken up in CH3OH (100 mL), treated with a solution of NaOCH3 in CH3OH and stirred (rt, 3 h). The solution was neutralized with Amberlite IR 120 (H+), filtered and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford diol 35 as a colourless oil (1.54 g, 78%). [a] 37.5 (c 0.8, CH2Cl2); Rf 0.28 (EtOAc–hexanes 1:1); 1H NMR (500 MHz): dH 7.51–7.44 (4H, m, Ph), 7.39–7.21 (11H, m, Ph), 5.86–5.78 (1H, m, CH2@CH), 5.47 (1H, s, PhCH), 5.06, 4.89 (2H, AB, J = 12.2 Hz, PhCH2), 5.04–4.99 (1H, m, CH2@CH), 4.97– 4.93 (1H, m, CH2@CH), 4.71, 4.57 (2H, AB, J = 12.2 Hz, PhCH2), 4.52 (1H, d, J10 ,20 = 7.8 Hz, H10 ), 4.41 (1H, d, J1,2 = 7.5 Hz, H1), 4.26 (1H, d, J60 ,60 = 12.5 Hz, H60 ), 4.06–4.00 (3H, m, H4, H40 , H6), 3.92 (1H, J = 9.4 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.83–3.76 (2H, m, H6, H60 ), 3.67–3.61 (1H, m, H20 ), 3.58–3.51 (1H, m, CH@CH2(CH2)5CH2O), 3.49–3.41 (5H, m, H2, H3, H30 , H5, OH), 2.94 (1H, s, H50 ), 2.50 (1H, d, J = 8.8, OH), 2.11–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.72–1.61 (2H, m, CH@CH2(CH2)5CH2O), 1.49–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13C NMR (125.7 MHz): dC 139.1 (CH2@CH), 138.5 (Ph), 137.72 (Ph), 137.67 (Ph), 129.1 (Ph), 128.4 (Ph), 128.3 (Ph), 128.22 (Ph), 128.17 (Ph), 127.9 (Ph), 127.8 (Ph), 127.7 (Ph), 126.4 (Ph), 114.2 (CH2@CH), 103.3 (PhCH), 102.3 (C1), 101.3 (C10 ), 82.0 (C3), 77.4 (C4), 75.3 (PhCH2), 75.1, 74.5, 72.8, 72.4 (C20 , C30 , C40 , C5), 73.4 (PhCH2), 70.3
2320
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(CH@CH2(CH2)5CH2O), 68.9 (C60 ), 68.2 (C6), 66.7, 66.4 (C2, C50 ), 33.7, (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2 (CH2)5CH2O). ESIMS: m/z Calcd [C41H51N3O10]Na+: 768.3467. Found: 768.3466. 3.22. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-3-Opivaloyl-b-D-galactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (36) A solution of diol 35 (970 mg, 1.30 mmol) in dry pyridine (10 mL) was treated with trimethylacetyl chloride (230 lL, 1.8 mmol), and the solution was stirred (rt, 2 h). The solution was concentrated, and the residue subjected to flash chromatography (EtOAc–hexanes 1:3) to afford 36 as a colourless oil (949 mg, 88%). [a] 16.8 (c 0.4, CH2Cl2); Rf 0.59 (EtOAc–hexanes 3:7); 1H NMR (400 MHz): dH 7.49–7.23 (15H, m, Ph), 5.90–5.77 (1H, m, CH2@CH), 5.44 (1H, s, PhCH), 5.06–4.89 (4H, m, PhCH2, CH2@CH), 4.73, 4.58 (2H, AB, J = 12.2 Hz, PhCH2), 4.66 (1H, dd, J20 ,30 = 10.2 Hz, J30 ,40 = 3.7 Hz, H30 ), 4.62 (1H, d, J10 ,20 = 7.8 Hz, H10 ), 4.26 (1H, J1,2 = 7.8 Hz, H1), 4.20 (1H, d, J30 ,40 = 3.7 Hz, H40 ), 4.08–3.86 (5H, m, H20 , H4, H6, H60 , CH@CH2(CH2)5CH2O), 3.79 (1H, dd, J60 ,60 = 4.3 Hz, J50 ,60 = 2.0 Hz, H60 ), 3.76 (1H, dd, J6,6 = 5.3 Hz, J5,6 = 2.0 Hz, H6), 3.55 (1H, ddd, J = 9.4 Hz, J = 6.8 Hz, J = 6.8 Hz, CH@CH2(CH2)5CH2O), 3.48–3.40 (3H, m, H2, H3, H5), 3.28 (1H, br s, OH), 2.90 (1H, s, H50 ), 2.12–2.02 (2H, m, CH@CH2(CH2)5CH2O), 1.75–1.64 (2H, m, CH@CH2(CH2)5CH2O), 1.51–1.33 (6H, m, CH@CH2(CH2)5CH2O), 1.24 (9H, s, (CH3)3C) 13C NMR (100 MHz): dC 178.6 (C@O), 139.3 (CH2@CH), 138.8 (Ph), 138.1 (Ph), 137.7 (Ph), 128.9 (Ph), 128.7 (Ph), 128.5 (Ph), 128.4 (Ph), 128.2 (Ph), 128.1 (Ph), 127.6 (Ph), 127.5 (Ph), 126.2 (Ph), 114.5 (CH2@CH), 103.9 (C10 ), 102.6 (C1), 100.6 (PhCH), 82.5 (C3), 77.7 (C4), 75.3 (PhCH2), 74.6, 73.7, 73.4, 69.7 (C20 , C30 , C40 , C5), 73.9 (PhCH2), 70.5 (CH@CH2(CH2)5CH2O), 69.0, 68.7 (C6, C60 ), 66.8, 66.6 (C2, C50 ), 39.2 (CH3)3C), 33.9 (CH@CH2(CH2)5CH2O), 29.7 (CH@CH2 (CH2)5CH2O), 29.1 (CH@CH2(CH2)5CH2O), 29.0 (CH@CH2(CH2)5 CH2O), 27.3 (CH3)3C), 26.0 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C46H59N3O11]Na+: 852.4042. Found: 852.4034. 3.23. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-3-Opivaloyl-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (37) A solution of alcohol 36 (920 mg, 1.11 mmol) in dry Et2O (10 mL) was treated with 4 Å molecular sieves (500 mg), and the mixture was stirred (rt, 1 h). The mixture was then cooled (10 °C), treated with TMSOTf (50 lL), followed by dropwise addition of trichloroacetimidate 1929 (1.87 g, 3.30 mmol) in dry Et2O (5 mL). The mixture was treated with Et3N (0.5 mL), filtered and subjected to flash chromatography (EtOAc–hexanes 1:3) to yield trisaccharide 37 as a colourless oil (1.30 g, 94%). [a] 66.7 (c 0.2, CH2Cl2); Rf 0.5 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.52–7.16 (30H, m, Ph), 5.88–5.79 (1H, m, CH2@CH), 5.45 (1H, s, PhCH), 5.41 (1H, d, J100 ,200 = 3.8 Hz, H100 ), 5.17 (1H, A of AB, J = 10.4 Hz, PhCH2), 5.05– 4.94 (3H, m, CH2@CH, PhCH2), 4.81 (1H, A of AB, J = 10.4 Hz, PhCH2), 4.80 (1H, dd, J20 ,30 = 10.2 Hz, J30 ,40 = 3.7 Hz, H30 ), 4.77–4.57 (6H, m, PhCH2), 4.44 (1H, d, J10 ,20 = 7.9 Hz, H10 ), 4.38 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.33 (1H, d, J30 ,40 = 4.0 Hz, H40 ), 4.28 (1H, dd, J60 ,60 = 12.4 Hz, J50 ,60 = 1.0 Hz, H60 ), 4.22–4.15 (3H, m, H1, H20 , H500 ), 4.13–4.05 (2H, m, H200 , H4), 3.96–3.90 (2H, m, H60 , CH@CH2(CH2)5CH2O), 3.83–3.76 (2H, m, H300 , H6), 3.68 (1H, d, J30 ,40 = 2.1 Hz, H400 ), 3.60–3.51 (2H, m, H6, CH@CH2(CH2)5CH2O), 3.39 (1H, dd, J2,3 = 9.8 Hz, J1,2 = 8.2 Hz, H2), 3.21 (1H, dd, J2,3 = 9.8 Hz, J2,3 = 9.0 Hz, H3), 3.13–3.09 (1H, m, H5), 3.02 (1H, s, H50 ), 2.11–2.04 (2H, m, CH@CH2(CH2)5CH2O), 1.74–1.63 (2H, m,
CH@CH2(CH2)5CH2O), 1.49–1.32 (6H, m, CH@CH2(CH2)5CH2O), 1.16 (3H, d, J500 ,600 = 6.5 Hz, H600 ), 1.13 (9H, s, (CH3)3C); 13C NMR (125.7 MHz): dC 178.0 (C@O), 139.1 (CH2@CH), 138.7 (Ph), 138.61 (Ph), 138.58 (Ph), 138.98 (Ph), 138.96 (Ph), 137.9 (Ph), 129.1 (Ph), 128.8 (Ph), 128.5 (Ph), 128.42 (Ph), 128.39 (Ph), 128.33 (Ph), 128.29 (Ph), 128.2 (Ph), 128.0 (Ph), 127.9 (Ph), 127.8 (Ph), 127.6 (Ph), 127.54 (Ph), 127.46 (Ph), 127.4 (Ph), 127.2 (Ph), 126.2 (Ph), 114.3 (CH2@CH), 102.1 (C1), 100.9, 100.7 (C10 , PhCH), 97.2 (C100 ), 81.2 (C3), 79.2 (C300 ), 77.5 (C50 ), 76.6, 75.7, 75.6, 75.3, 72.8, 71.3 (C20 , C200 , C30 , C4, C40 , C400 ), 75.8 (PhCH2), 74.8 (PhCH2), 73.4 (PhCH2), 73.2 (PhCH2), 72.5 (PhCH2), 70.2 (CH@CH2(CH2)5CH2O), 68.7, 67.8 (C6, C60 ), 66.6, 66.3, 65.8 (C2, C5, C500 ), 38.9 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2 (CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 27.1 ((CH3)3C), 25.8 (CH@CH2(CH2)5CH2O), 16.7 (C600 ). ESIMS: m/z Calcd [C73H87N3O15]Na+: 1268.6029. Found: 1268.6027. 3.24. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-2-O-(2,3,4tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-3,6-diO-benzyl-2-deoxy-b-D-glucopyranoside (38) A solution of trisaccharide 37 (651 mg, 0.522 mmol) in CH3OH (50 mL) was treated with a catalytic amount of LiOCH3 (100 mg), and the solution heated at reflux (7 d). The solution was neutralized with Amberlite IR 120 (H+), filtered and subjected to flash chromatography (EtOAc–hexanes 1:2) to firstly afford unreacted 37 (60 mg, 9%). Further elution (EtOAc–hexanes 1:1) afforded alcohol 38 (503 mg, 83%) as a colourless oil. [a] 75.8 (c 0.3, CH2Cl2); Rf 0.19 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.57–7.53 (4H, m, Ph), 7.41–7.06 (26H, m, Ph), 5.87–5.77 (1H, m, CH2@CH), 5.59 (1H, s, PhCH), 5.14, 4.61 (2H, AB, J = 10.4 Hz, PhCH2), 5.03–4.92 (2H, m, CH2@CH), 5.01, 4.66 (2H, AB, J = 11.7 Hz, PhCH2), 4.95 (1H, d, J100 ,200 = 3.7 Hz, H100 ), 4.82, 4.74 (2H, AB, J = 11.3 Hz, PhCH2), 4.80, 4.72 (2H, AB, J = 12.0 Hz, PhCH2), 4.69, 4.40 (2H, AB, J = 11.3 Hz, PhCH2), 4.40 (1H, d, J10 ,20 = 8.1 Hz, H10 ), 4.32 (1H, d, J60 ,60 = 12.0 Hz, H60 ), 4.17–4.11 (3H, m, H1, H40 , OH), 4.08–4.03 (2H, m, H200 , H4), 3.99 (1H, dd, J60 ,60 = 12.0 Hz, J50 ,60 = 1.3 Hz, H60 ), 3.92–3.84 (4H, m, H5, H500 , H6, CH@CH2(CH2)5CH2O), 3.79 (1H, dd, J20 ,30 = 9.8 Hz, J10 ,20 = 8.1 Hz, H20 ), 3.64–3.55 (3H, m, H30 , H400 , H6), 3.49 (1H, ddd, J = 9.4 Hz, 6.8 Hz, 6.8 Hz, CH@CH2(CH2)5CH2O), 3.37 (1H, dd, J2,3 = 9.9 Hz, J1,2 = 8.2 Hz, H2), 3.27–3.19 (2H, m, H3, H300 ), 3.12 (1H, s, H50 ), 2.10–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.70–1.60 (2H, m, CH@CH2(CH2)5CH2O), 1.45–1.29 (6H, m, CH@CH2(CH2)5CH2O), 1.07 (3H, d, J500 ,600 = 6.5 Hz, H600 ); 13C NMR (125.7 MHz): dC 139.1 (CH2@CH), 138.6 (Ph), 138.5 (Ph), 138.13 (Ph), 138.11 (Ph), 138.08 (Ph), 137.3 (Ph), 129.2 (2C, Ph), 128.9 (Ph), 128.8 (2C, Ph), 128.4 (2C, Ph), 128.34 (Ph), 128.31 (Ph), 128.26 (Ph), 128.2 (Ph), 128.1 (2C, Ph), 127.74 (Ph), 127.65 (Ph), 127.5 (Ph), 126.5 (Ph), 114.2 (CH2@CH), 102.0 (C1), 101.4 (C10 ), 101.3 (PhCH), 99.6 (C100 ), 81.1 (C3), 79.0 (C5), 78.5 (C20 ), 77.5, 77.3, 75.9, 75.7, 75.0, 72.6 (C200 , C30 , C300 , C4, C4´, C400 ), 75.8 (PhCH2), 74.8 (PhCH2), 74.3 (PhCH2), 73.4 (PhCH2), 73.0 (PhCH2), 70.1 (CH@CH2(CH2)5CH2O), 69.0, 67.9 (C6, C60 ), 67.5, 66.6, 65.8 (C2, C50 , C500 ), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 16.8 (C600 ). ESIMS: m/z Calcd [C68H79N3O14]Na+: 1184.5454. Found: 1184.5441. 3.25. 7-Octen-1-yl 2-acetamido-4-O-(4,6-O-benzylidene-2-O(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)3,6-di-O-benzyl-2-deoxy-b-D-glucopyranoside (39) A solution of trisaccharide 38 (500 mg, 0.431 mmol) in dry pyridine (6 mL) was treated with AcSH (3 mL), and the solution was stirred (rt, 8 d). The mixture was concentrated and then subjected to flash chromatography (CH3OH–CH2Cl2 1:9) to afford 39
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
as a colourless oil (426 mg, 84%). [a] 54.5 (c 0.4, CH2Cl2); Rf 0.30 (EtOAc–hexanes 1:1); 1H NMR (500 MHz): dH 7.53–7.47 (4H, m, Ph), 7.40–7.10 (26H, m, Ph), 5.84–5.74 (1H, m, CH2@CH), 5.71 (1H, d, J = 7.1 Hz, NH), 5.55 (1H, s, PhCH), 5.03 (1H, d, J100 ,200 = 3.5 Hz, H100 ), 5.01–4.91 (4H, m, CH2@CH, PhCH2), 4.83, 4.71 (2H, AB, J = 11.6 Hz, PhCH2), 4.78 (1H, A of AB, J = 12.4 Hz, PhCH2), 4.76 (1H, d, J1,2 = 8.2 Hz, H1), 4.73 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.67–4.59 (3H, m, PhCH2), 4.43–4.39 (2H, m, H10 , PhCH2), 4.26 (1H, d, J60 ,60 = 12.0 Hz, H60 ), 4.14 (1H, d, J30 ,40 = 3.5 Hz, H40 ), 4.07 (1H, dd, J200 ,300 = 10.1, J100 ,200 = 3.5 Hz, H200 ), 4.03 (1H, dd, J3,4 = J4,5 7.5 Hz, H4) 4.00–3.92 (3H, m, H3, H500 , H60 ), 3.90 (1H, dd, J200 ,300 = 10.1 Hz, J300 ,400 = 2.6 Hz, H300 ) 3.86–3.75 (3H, m, H20 , H6, CH@CH2(CH2)5CH2O), 3.72 (1H, dd, J6,6 = 10.5 Hz, J5,6 = 3.3 Hz, H6), 3.66–3.60 (2H, m, H30 , H400 ) 3.56–3.46 (2H, m, H2, H5), 3.45–3.38 (1H, m, CH@CH2(CH2)5CH2O), 3.16 (1H, s, H50 ), 2.06–1.98 (2H, m, CH@CH2(CH2)5CH2O), 1.84 (3H, s, CH3CO), 1.59–1.49 (2H, m, CH@CH2(CH2)5CH2O), 1.41–1.23 (6H, m, CH@CH2(CH2)5CH2O), 1.13 (3H, d, J500 ,600 = 6.4 Hz, H600 ); 13C NMR (125.7 MHz): dC 170.1 (C@O), 139.1 (CH2@CH), 138.8 (Ph), 138.62 (Ph), 138.58 (Ph), 138.3 (Ph), 138.0 (Ph), 137.5 (Ph), 129.0 (Ph), 128.7 (Ph), 128.6 (Ph), 128.4 (Ph), 128.3 (Ph), 128.24 (Ph), 128.22 (Ph), 128.18 (Ph), 128.1 (Ph), 128.0 (Ph), 127.7 (Ph), 127.59 (Ph), 127.57 (Ph), 127.5 (Ph), 127.4 (Ph), 126.5 (Ph), 114.2 (CH2@CH), 101.3 (PhCH), 101.0 (C10 ), 99.85, 99.78 (C1, C100 ), 79.1 (C300 ), 78.8 (C20 ), 77.6, 77.4 (C200 , C400 ), 76.1, 75.6, 72.9 (4C, C3, C30 , C4, C500 ), 74.8 (PhCH2), 74.7 (C40 ), 74.1 (2C, PhCH2), 73.4 (PhCH2), 72.8 (PhCH2), 69.4, 69.0, 68.8 (C6, C60 , CH@CH2(CH2)5CH2O), 67.6, 66.5 (C5, C50 ), 55.0 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (2C, CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 23.4 (CH3CO), 16.8 (C600 ). ESIMS: m/z Calcd [C70H83NO15]Na+: 1200.5655. Found: 1200.5661. 3.26. 7-Octyl 2-acetamido-4-O-(2-O-(a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (40) A solution of trisaccharide 7 (145 mg, 0.227 mmol) was taken up in CH3OH, treated with Pd–C (10%, 20 mg) and subjected to a H2 atmosphere (rt, 1 d). The mixture was then filtered and concentrated to afford octyl glycoside 40 as a colourless oil (140 mg, 96%). [a] 106.1 (c 0.5, CH3OH); 1H NMR (500 MHz, CD3OD): dH 5.21 (1H, d, J100 ,200 = 3.2 Hz, H100 ), 4.48 (1H, d, J10 ,20 = 7.1 Hz, H10 ), 4.37 (1H, d, J1,2 = 8.4 Hz, H1), 4.17 (1H, q, J500 ,600 = 6.5 Hz, H500 ), 3.93–3.80, 3.79– 3.53, 3.34–3.30 (16H, 3 m, H2, H20 , H200 , H3, H30 , H300 , H4, H40 , H400 , H5, H50 , H6, H60 , CH3(CH2)6CH2O), 3.48–3.41 (1H, m, CH3(CH2)6CH2O), 1.96 (3H, s, CH3CO), 1.57–1.48 (2H, m, CH3(CH2)6CH2O)), 1.37–1.24 (10H, m, CH3(CH2)6CH2O), 1.20 (3H, d, J500 ,600 = 6.5 Hz, H600 ), 0.89 (3H, t, J = 6.9 Hz, CH3(CH2)6CH2O). 13C NMR (125.7 MHz, CD3OD): dC 173.4 (C@O), 102.9, 102.6 (C1, C10 ), 101.9 (C100 ), 79.1, 78.3, 77.1, 77.0, 75.3, 74.1, 73.6, 71.7, 70.8, 70.70 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C500 ), 70.7 (CH3(CH2)6CH2O), 68.3 (C50 ), 62.6, 61.7 (C6, C60 ), 56.8 (C2), 33.0 (CH3(CH2)6CH2O), 30.7 (CH3(CH2)6CH2O), 30.5 (2C, CH3(CH2)6CH2O), 27.2 (CH3(CH2)6CH2O), 23.7 (CH3(CH2)6CH2O), 23.0 (CH3CO), 16.8 (C600 ), 14.4 (CH3(CH2)6CH2O). ESIMS: m/z Calcd [C28H51NO15]Na+: 664.3151. Found: 664.3156. 3.27. 7-Octen-1-yl 2-acetamido-4-O-(4,6-O-benzylidene-3-O(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-2-O-(2,3,4-tri-Obenzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-3,6-di-Obenzyl-2-deoxy-b-D-glucopyranoside (42) A solution of acceptor 38 (1.22 g, 1.05 mmol) in dry Et2O (40 mL) was treated with 4 Å molecular sieves (1 g), and the mixture was stirred (rt, 1 h). The mixture was then cooled (10 °C) and treated with TMSOTf (50 lL, 0.29 mmol); trichloroacetimidate
2321
2834 (1.49 g, 3.15 mmol) in dry Et2O (15 mL) was then added dropwise, and the mixture was allowed to stand (20 min). The mixture was neutralized with Et3N (0.5 mL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:4) to afford the nearly pure tetrasaccharide 41 (1.33 g, 75%) as a colourless oil. A solution of tetrasaccharide 41 in dry pyridine (6 mL) was treated with AcSH (3 mL), and the solution was stirred (rt, 2 d). The solution was then concentrated and subjected to flash chromatography (CH3OH–CH2Cl2 1:9) to afford 42 (510 mg, 91%) as a colourless oil. [a] 7.8 (c 0.4, CH2Cl2); Rf 0.50 (CH3OH–CH2Cl2 1:20); 1H NMR (500 MHz): dH 7.42–7.09 (48H, m, Ph), 7.03–6.98 (2H, m, Ph), 5.87–5.76 (2H, m, CH2@CH, NH), 5.62 (1H, d, J100 ,200 = 3.6 Hz, H100 ), 5.38 (1H, s, PhCH), 5.36 (1H, d, J1000 ,2000 = 3.6 Hz, H1000 ), 5.02–4.97 (2H, m, CH2@CH, PhCH2), 4.96–4.92 (2H, m, CH2@CH, PhCH2), 4.87–4.79 (4H, m, H1, PhCH2), 4.72 (1H, A of AB, J = 12.3 Hz, PhCH2), 4.66–4.57 (6H, m, PhCH2), 4.52 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.47–4.30 (9H, m, H10 , H500 , H60 , PhCH2), 4.18–4.12 (2H, m, H20 , H40 ), 4.08–3.91 (6H, m, H200 , H2000 , H3, H300 , H4, H6), 3.87– 3.76 (5H, m, H30 , H3000 , H5000 , H60 , CH@CH2(CH2)5CH2O), 3.71 (1H, s, H400 ), 3.59–3.54 (1H, m, H5), 3.53–3.40 (5H, m, H2, H4000 , H6, H6000 , CH@CH2(CH2)5CH2O), 3.16–3.10 (1H, m, H6000 ), 2.92 (1H, s, H50 ), 2.08–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.84 (3H, s, CH3CO), 1.63–1.53 (2H, m, CH@CH2(CH2)5CH2O), 1.41–1.23 (9H, m, H600 , CH@CH2(CH2)5CH2O). 13C NMR (125.7 MHz): dC 170.1 (C@O), 139.2 (Ph), 139.04 (CH2@CH), 138.99 (Ph), 138.96 (Ph), 138.76 (Ph), 138.72 (Ph), 138.66 (Ph), 138.5 (Ph), 138.4 (Ph), 138.3 (Ph), 137.8 (Ph), 128.8 (Ph), 128,6 (Ph), 128.4 (Ph), 128.34 (Ph), 128.32 (Ph), 128.25 (Ph), 128.21 (Ph), 128.18 (Ph), 128.08 (Ph), 128.06 (Ph), 127.98 (Ph), 127.65 (Ph), 127.59 (Ph), 127.5 (Ph), 127.45 (Ph), 127.40 (Ph), 127.37 (Ph), 127.24 (Ph), 127.15 (Ph), 127.07 (Ph), 126.3 (Ph), 114.2 (CH2@CH), 101.5, 101.1 (PhCH, C10 ), 99.8 (C1), 97.8 (C100 ), 92.8 (C1000 ), 80.0, 78.1, 78.0, 77.2, 76.32, 76.26, 76.1, 75.5, 75.3, 72.4, 72.2, 70.4 (13C, C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C5000 ), 75.0 (PhCH2), 74.5 (PhCH2), 74.2 (PhCH2), 73.5 (PhCH2), 73.4 (PhCH2), 73.2 (PhCH2), 72.8 (PhCH2), 72.3 (PhCH2), 71.3 (PhCH2), 69.8, 69.5, 69.1, 69.0 (C6, C60 , C6000 , CH@CH2(CH2)5CH2O), 66.5 (C500 ), 66.2 (C50 ), 54.9 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.6 (CH@CH2(CH2)5CH2O), 28.91 (CH@CH2 (CH2)5CH2O), 28.89 (CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5 CH2O), 23.5 (CH3CO), 16.9 (C600 ). ESIMS: m/z Calcd [C104H117NO20]Na+: 1722.8061. Found: 1722.8063.
3.28. 7-Octen-1-yl 2-acetamido-4-O-(3-O-(2-acetamido-2deoxy-3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-4,6-Obenzylidene-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (44) A solution of acceptor 38 (1.17 g, 1.01 mmol) in dry Et2O (45 mL) was treated with 4Å molecular sieves (1 g) and the mixture was stirred (rt, 1 h). The mixture was then cooled (10 °C), treated with TMSOTf (50 lL, 0.29 mmol); trichloroacetimidate 2435 (1.43 g, 0.965 mmol) in dry Et2O (15 mL) was then added dropwise and the mixture was allowed to stand (20 min). The mixture was neutralized with Et3N (0.5 mL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:3) to afford the nearly pure tetrasaccharide as a colourless oil (1.21 g, 94%). The residue was taken up in pyridine (6 mL) and treated with AcSH (3 mL) and the solution was stirred (7 d). The solution was concentrated and subjected to flash chromatography (CH2Cl2–CH3OH, 10:1) to afford 44 as a colourless oil (530 mg, 76%). Rf 0.65 (CH3OH–CH2Cl2 1:9); 1H NMR (500 MHz): dH 7.44–7.18 (30H, m, Ph), 5.85–5.73 (2H, m, CH2@CH, NH), 5.45 (1H, d, J100 ,200 = 3.9 Hz, H100 ), 5.41 (1H, s, PhCH), 5.38 (1H, J = 9.8 Hz, NH), 5.20 (1H, A of AB, J = 12.3 Hz, PhCH2), 5.12 (1H, A of AB, J = 11.1 Hz, PhCH2),
2322
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
5.09 (1H, d, J1000 ,2000 = 3.7 Hz, H1000 ), 5.04–4.90 (7H, m, H1, H3000 , H4000 , CH2@CH, PhCH2), 4.71–4.55 (8H, m, H10 , H2000 , PhCH2), 4.45–4.38 (1H, m, H500 ), 4.32–4.06 (7H, m, H20 , H200 , H3, H4, H40 , H5000 , H60 ), 4.01–3.97 (1H, m, H60 ), 3.94–3.81 (4H, m, H30 , H300 , H6, CH@CH2(CH2)5CH2O), 3.80–3.72 (2H, m, H400 , H6), 3.67 (1H, dd, J6000 ,6000 = 11.5 Hz, J5000 ,6000 = 7.6 Hz, H6000 ), 3.52–3.41 (2H, m, H5, CH@CH2(CH2)5CH2O), 3.33–3.26 (1H, m, H2), 3.20 (1H, s, H50 ), 3.09 (1H, dd, J6000 ,6000 = 11.5 Hz, J5000 ,6000 = 3.7 Hz, H6000 ), 2.10–1.99 (5H, m, CH3CO, CH@CH2(CH2)5CH2O), 1.94 (3H, s, CH3CO), 1.87 (3H, s, CH3CO), 1.42 (3H, s, CH3CO), 1.64–1.55 (2H, m, CH@CH2 (CH2)5CH2O), 1.41–1.24 (12H, m, CH3CO, H600 , CH@CH2(CH2)5 CH2O). 13C NMR (125.7 MHz): dC 170.34 (2C, C@O), 170.32 (C@O), 170.1 (C@O), 170.0 (C@O), 139.4 (Ph), 139.0 (CH2@CH), 138.7 (Ph), 138.70 (Ph), 138.67 (Ph), 128.3 (Ph), 138.2 (Ph), 137.6 (Ph), 129.1 (Ph), 128.8 (Ph), 128.4 (Ph), 128.31 (Ph), 128.27 (Ph), 128.2 (Ph), 128.1 (Ph), 128.04 (Ph), 128.02 (Ph), 127.8 (Ph), 127.61 (Ph), 127.57 (Ph), 127.51 (Ph), 127.47 (Ph), 127.4 (Ph), 127.31 (Ph), 126.5 (Ph), 126.2 (Ph), 114.3 (CH2@CH), 101.2, 101.0 (PhCH, C10 ), 99.7 (C1), 98.6 (C100 ), 92.1 (C1000 ), 80.1, 77.7, 77.5, 77.0, 75.8, 75.3, 71.8, 70.4, 69.7, 69.0, 67.0, 66.2 (13C, C20 , C200 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C500 , C5000 ), 74.9 (PhCH2), 73.7 (PhCH2), 73.4 (PhCH2), 73.3 (PhCH2), 72.1 (PhCH2), 68.7, 68.4, 67.4 (C6, C60 , CH@CH2(CH2)5CH2O), 67.7 (C50 ), 63.0 (C6000 ), 56.6 (C2), 46.5 (C2000 ), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (2C, CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5CH2O), 23.5 (CH3CO), 22.6 (CH3CO), 20.69 (CH3CO), 20.66 (CH3CO), 20.65 (CH3CO), 16.8 (C600 ). ESIMS: m/z Calcd [C84H102NO23]Na+: 1529.6766. Found: 1529.6744. Acknowledgements Funding was provided by a Canadian Institutes of Health Research (CIHR) team grant (RMF 92091), a Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR) CHRP grant (CHRPJ35094608), and the Alberta Ingenuity Centre for Carbohydrate Science. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.carres.2010.08.012.
References 1. Yamamoto, F. Immunohematology 2004, 20, 3–22. 2. West, L. J.; Pollock-Barziv, S. M.; Dipchand, A. I.; Lee, K. J.; Cardella, C. J.; Benson, L. N.; Rebeyka, I. M.; Coles, J. G. N. Eng. J. Med. 2001, 344, 793–800. 3. Watkins, W. M.; Morgan, W. T. J. Nature 1957, 180, 1038–1040. 4. Tuppy, H.; Staudenbauer, W. L. Nature 1966, 210, 316–317. 5. Yamamoto, F.; Clausen, H.; White, T.; Marken, J.; Hakomori, S. Nature 1990, 345, 229–233. 6. Yamamoto, F.; McNeill, P. D.; Hakomori, S. Glycobiology 1995, 5, 51–58. 7. Yamamoto, F.; Marken, J.; Tsuji, T.; White, T.; Clausen, H.; Hakomori, S. J. Biol. Chem. 1990, 265, 1146–1151. 8. Mollicone, R.; Gibaud, A.; François, A.; Ratcliffe, M.; Oriol, R. Eur. J. Biochem. 1990, 191, 169–176. 9. Meloncelli, P. J.; Lowary, T. L. Aust. J. Chem. 2009, 62, 558–574. 10. Palcic, M. M. Personal Communication, 2010. 11. Kuhn, R.; Baer, H. H.; Gauhe, A. Chem. Ber. 1956, 89, 2513. 12. Breimer, M. E.; Hansson, G. C.; Karlsson, K.-A.; Leffler, H. J. Biol. Chem. 1982, 257, 906–912. 13. Stellner, K.; Hakomori, S. J. Biol. Chem. 1974, 249, 1022–1025. 14. Mäkelä, O.; Ruoslahti, E.; Ehnholm, C. J. Immunol. 1969, 102, 763–771. 15. Clausen, H.; Levery, S. B.; Nudelman, E.; Tsuchiya, S.; Hakomori, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1199–1203. ˇ . Angew. Chem., Int. Ed. Engl. 1978, 17, 771. 16. Paulsen, H.; Kolárˇ, C 17. Korchagina, E. Y.; Ryzhov, I. M.; Byrgazov, K. A.; Popova, I. S.; Pokrovsky, S. N.; Bovin, N. V. Mendeleev Commun. 2009, 19, 152–154. 18. Bovin, N. V.; Khorlin, A. Y. Bioorg. Khim. 1984, 10, 853–860. 19. Compston, C. A.; Condon, C.; Hanna, H. R.; Mazid, M. A. Carbohydr. Res. 1993, 239, 167–176. 20. Yi, W.; Shao, J.; Zhu, L.; Li, M.; Singh, M.; Lu, Y.; Lin, S.; Li, H.; Ryu, K.; Shen, J.; Guo, H.; Yao, Q.; Bush, C. A.; Wang, P. G. J. Am. Chem. Soc. 2005, 127, 2040–2041. ˇ . Tetrahedron Lett. 1979, 20, 2881–2884. 21. Paulsen, H.; Kolárˇ, C ˇ . Chem. Ber. 1981, 114, 306–321. 22. Paulsen, H.; Kolárˇ, C 23. Pazynina, G. V.; Tyrtysh, T. V.; Bovin, N. V. Mendeleev Commun. 2002, 12, 143– 145. 24. Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, O. Glycoconjugate J. 1988, 5, 49–63. 25. Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995–8997. 26. Wright, V. W.; Cooper, A. M.; Meloncelli, P. J.; Harris, K. D.; West, L. J.; Lowary, T. L.; Buriak, J. M., in preparation. 27. Rele, S. M.; Iyer, S. S.; Baskaran, S.; Chaikof, E. L. J. Org. Chem. 2004, 69, 9159– 9170. 28. Ali, I. A. I. Novel Trichloroacetimidates and their Reactions; Cuvillier: Göttingen, 2004. 29. Schmidt, R. R.; Toepfer, A. J. Carbohydr. Chem. 1993, 12, 809–822. 30. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981, 37, 437– 472. 31. Robinson, P. T.; Pham, T. N.; Uhrín, D. J. Magn. Reson. 2004, 170, 97–103. 32. Duncan, S. J.; Lewis, R.; Bernstein, M. A.; Sandor, P. Magn. Reson. Chem. 2007, 45, 283–288. 33. Figueroa-Pérez, S.; Vérez-Bencomo, V. Carbohydr. Res. 1999, 317, 29–38. 34. Wegmann, B.; Schmidt, R. R. J. Carbohydr. Chem. 1987, 6, 357–375. 35. Gerhard, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826–1847.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Synthesis of ABO histo-blood group type I and II antigens Peter J. Meloncelli, Todd L. Lowary ⇑ Department of Chemistry and Alberta Ingenuity Centre for Carbohydrate Science, Gunning-Lemieux Chemistry Centre, University of Alberta, Edmonton, AB, Canada T6G 2G2
a r t i c l e
i n f o
Article history: Received 25 June 2010 Received in revised form 13 August 2010 Accepted 17 August 2010 Available online 16 September 2010 Keywords: ABO histo-blood group ABO type I ABO type II Chemical synthesis Trichloroacetimidate 7-Octen-1-yl
a b s t r a c t The ABO histo-blood group system is one of the most clinically important antigen families. As part of our overall goal to prepare the entire set of the A, B and H type I–VI antigens for a range of biochemical investigations, we report herein the synthesis of the type I and II antigens with a 7-octen-1-yl aglycone. This linker was chosen to facilitate not only the future conjugation of the antigens to a protein or solid support but also the synthesis of the H type I and II octyl glycosides for enzyme kinetic studies. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The ABO histo-blood group system has long been of interest to the biological and medical sciences as these antigens are one of the most important clinical considerations for both transfusion and transplantation.1,2 This family of antigens consists of three oligosaccharide epitopes (1–3, Fig. 1), the structures of which were elucidated by Morgan and Watkins in 1957.3 The H(O) antigen was shown to be a disaccharide consisting of L-fucose and D-galactose (3, Fig. 1), while the A and the B antigens (1 and 2) were shown to be trisaccharides containing the disaccharide H antigen with an additional N-acetyl-galactosamine or a galactose residue, respectively. It was later shown by Tuppy and Staudenbauer that individuals who are of the A histo-blood group possess an N-acetyl-galactosaminyl transferase (GTA) capable of adding N-acetyl galactosamine to the 3-OH group of the H antigen galactose moiety.4 On the other hand, individuals of the B histo-blood group possess a galactosyl transferase (GTB) that adds galactose to this hydroxyl group. The genetic basis of the ABO histo-blood group system was discovered in the 1990s, when the genes responsible for coding GTA and GTB were identified.5–7 The ABO antigens can be further divided into six subtypes, type I–VI (Table 1).8 The type I–IV and VI antigens are present either as glycoproteins, glycolipids, or as free oligosaccharides in humans.1 After the publication of our synthesis of the ABO type V and VI histo-blood group antigens,9 it came to our attention that the origin of
⇑ Corresponding author. Tel.: +1 780 492 1861; fax: +1 780 492 7705. E-mail address: [email protected] (T.L. Lowary). 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.08.012
the nomenclature of the type V and VI antigens was not clear.10 Although a bit tangential to the main goal of this paper, we describe briefly this ambiguity, as a concise description does not appear to be present elsewhere in the literature. The first report of a type VI antigen structure from human sources was achieved in 1956 by Kuhn and co-workers, who isolated the H type VI antigen as the free oligosaccharide in appreciable quantities from human milk.11 Subsequently, a type VI antigen from tissue samples was reported by Briemer and co-workers in 1982.12 This glycolipid, named A-4 by Briemer, contained what was in the future termed the A type VI antigen, linked to ceramide. The origin of the type V antigen proved more difficult to uncover. The first mention of this structure was by Mollicone and co-workers in 1990.8 The authors obtained synthetic samples of the type V and VI antigens from Chembiomed, a now defunct commercial entity, and this is presumably the origin of this nomenclature. To the best of our knowledge, no ABH type V structure has been isolated from a mammalian source. A recent review by Yamamoto on the ABO histo-blood group system makes reference to the type I–IV and type VI antigens, excluding the type V antigen.1 This exclusion of the type V antigen was likely based on the lack of evidence for existence of ABH type V structures in humans. This is surprising because Hakomori and Stellner reported the isolation of a glycolipid from human erythrocytes that contained the type V (b-D-Galp-(1?3)-b-D-Gal) backbone at the terminal end.13 This structure could easily be converted into the H type V antigen using the same fucosyl transferase responsible for the biosynthesis of the H type I–IV antigens present on erythrocytes. Based on this chronology, the nomenclature assignment of the type V and VI antigens should be reversed; however, as the nomenclature is well
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HO
HO
OH O HO
OH
HO
O
OR
OH O
O HO
O
HO OH
HO
O AcHN
OH O HO
OR
HO OR
O
O
O
O OH
O
O
OH
OH OH
OH
OH HO
HO
HO A Antigen 1
B Antigen
H Antigen
2
3
Figure 1. Structures of the A, B and H(O) antigens. R = Glycoprotein, glycolipid or free oligosaccharide.
Table 1 Carbohydrate moieties responsible for the six subtypes (I–VI) of the ABO histo-blood group antigens Type Type Type Type Type Type Type
Carbohydrate I II III IV V VI
ABO-b-(1?3)-b-D-GlcpNAc ABO-b-(1?4)-b-D-GlcpNAc ABO-b-(1?3)-a-D-GalpNAc ABO-b-(1?3)-b-D-GalpNAc ABO-b-(1?3)-b-D-Galp ABO-b-(1?4)-b-D-Glcp
established in multiple sources we have adhered to the conventional naming of the type I–VI antigens. The overall goal of our synthetic endeavour is to prepare all known ABO type I–VI histo-blood group antigens. We hope that with all of the antigens in hand, we will be able to conduct a range of studies on the biological importance of these sub-types. An example of such an investigation is quantitatively determining the difference between the A1 and A2 blood groups. It has been shown that individuals of the A1 subtype have a higher density of A antigen on their erythrocytes than individuals of the A2 subtype.14 Further work by Clausen and co-workers has demonstrated that this difference can be attributed to the fact that GTA1 is able to transfer N-acetyl-galactosamine onto the H type I–IV structures present on erythrocytes, while GTA2 is only able to affect transfer onto the H type I and type II structures.15 These pioneering studies were conducted in a qualitative manner via immunostaining. Access to synthetic samples of these antigens would enable more detailed quantitative kinetics assays to be conducted. Herein we report the synthesis of the ABO type I and II histoblood group antigens (4–9, Fig. 2) attached to an octen-1-yl aglycone. To date, the synthesis of the ABO type I and II antigens has received more interest than the type III–VI structures. The chemical synthesis of all three type I antigens has been reported by Paulsen and Kolárˇ16 as well as by Bovin and co-workers.17,18 The chemoenzymatic synthesis of the A type I and II tetrasaccharides was reported by Palcic and co-workers,19 and more recently Wang and co-workers have developed a chemoenzymatic synthesis of the B type I antigen.20 Interestingly, the latter study did not use the human glycosyltransferases, but rather three enzymes present in Escherichia coli O86, which produces a B type III mimetic in its lipopolysaccharide O-chain. The synthesis of the type II antigens has been more limited, most notably the chemical synthesis of the ABO type II structures was achieved by Paulsen and co-workers21,22 as well as by Bovin and co-workers.23
The A1 and A2 blood groups should not be confused with the A type I and type II structures. The latter refers to distinct chemical entities while the former refers to subclasses of the A blood type, which are characterized by different densities of the antigen on erythrocytes.
2. Results and discussion To prepare 4–9 we chose a linear chemical synthesis, similar to the strategy employed in our recently reported synthesis of the type V and VI antigens.9 This strategy would enable an expedited synthesis of all three of the type I and II antigens in sufficient quantities for a range of investigations. A block synthesis, such as that reported by Bovin and co-workers,17 would not enable access to the H structures. We chose to employ a 7-octen-1-yl aglycone for two reasons. Firstly, it would enable ready access to the octyl glycoside, via catalytic hydrogenation, for enzyme kinetics studies.24 Secondly, it would enable facile preparation of glycoconjugates. We have demonstrated that a thiol-ene ‘click reaction’25 can be used to introduce a reactive linker that allows direct conjugation of carbohydrates to silica-based surfaces.26 2.1. Synthesis of type I antigens The synthesis of the type I ABO histo-blood group antigens commenced from the known27 and easily prepared trichloroacetimidate donor 10 (Scheme 1). Glycosylation with the 7-octen-1-ol acceptor afforded the 7-octen-1-yl glycoside 11 as a mixture of anomers (a/b, 11:41). In the absence of a C2 participating group, the stereochemical outcome for this glycosylation was dependent on the anomeric configuration of the trichloroacetimidate. To ensure a high ratio of the desired b-anomer, a trichloroacetimidate donor with predominately the a stereochemistry was required. Presumably, the mechanism for the formation of the b-glycoside involves an SN2-type process.28 Introduction of the aglycone in the presence of an azido protecting group was not trivial when compared to other amine protecting groups such as N-phthalimide; however, the azido group can be converted into an amine under milder conditions than the phthalimide. Deacetylation with sodium methoxide, followed by introduction of a benzylidene acetal, enabled isolation of the corresponding b and a glycosides, 13 and 14, respectively, in moderate yield, with the desired b anomer being the predominant product. The 1H NMR spectrum of the a anomer 13 showed an anomeric proton signal appearing at 4.90 ppm as a doublet with a coupling of 3.6 Hz, while the spectrum of the b anomer 14 had an anomeric proton signal at 4.42 ppm as a doublet with a coupling of 8.0 Hz. Introduction of the galactose core residue was achieved by glycosylation of 13 with trichloroacetimidate 15 affording disaccharide 16 (Scheme 2). To facilitate removal of trichloroacetimidate-related impurities, a deacetylation was conducted under Zemplén conditions to afford diol 17 in moderate yield (63%) over two steps. Introduction of the pivaloyl ester selectively at the 30 position of 17 was achieved with complete regioselectivity using trimethylacetyl chloride in pyridine in excellent yield (89%). Next, introduction of the a-L-fucopyranosyl moiety was achieved through the use of b-L-fucopyranosyl trichloroacetimidate 19
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HO
HO
OH O
OH
OH O
OH O HO
HO HO
OH
AcHN
O
NHAc
O O
O
OH 5 A Type I
OH
4 B Type I
OH HO
HO
O
O
O
NHAc
O
OH O
OH O HO
HO
O
O
OH O
OH HO
HO
OH O
OH O HO
O
O
HO
OH O
HO
NHAc
O
OH O
O HO
O
O NHAc
O
O
OH
OH
6 H Type I
OH HO
HO
OH
OH
HO HO
HO
HO
OH O
OH
O
O HO
O
O O NHAc
O
O OH 8 B Type II
HO
OH
O
NHAc OH
OH O
AcHN
O
O HO
O
OH O
HO
OH
O O
7 H Type II
OH
OH HO
OH 9 A Type II
Figure 2. Structure of ABO histo-blood group type I and II antigen targets.
OAc O
AcO AcO
NH CCl3
O
N3
(a)
AcO AcO
OAc O N3
10
O
11 (b) O
Ph
O
O
O
HO N3 13 O
Ph
(c)
OH O N3
O
O
HO HO
O
12
HO N3 14
O
Scheme 1. Reagents: (a) HO(CH2)6CH@CH2, 4 Å MS, TMSOTf, CH2Cl2; (b) NaOCH3, CH3OH; (c) PhCH(OCH3)2, p-TsOH, DMF, (a/b, 11:41) 52% (three steps).
following the methodology of Schmidt and co-workers.29 Treatment of 18 with trichloroacetimidate 19 under standard glycosylation conditions afforded trisaccharide 20 in good yield (80%) and with excellent stereoselectivity. The anomeric proton of the fucopyranoside of 20 showed an anomeric signal at 5.41 ppm as a doublet with a coupling constant of 1.5 Hz. Removal of the pivaloate ester from 20 required forcing conditions (lithium methoxide in methanol at reflux) and prolonged reaction times (seven days); however, the reaction afforded alcohol 21 in good yield. A similar observation was made in our synthesis of the type V and VI antigens. The decision to continue using this protecting group as op-
posed to others that might be removed more efficiently was based on the fact that the pivaloate ester could be installed and removed in good yield and with minimal by-products.9 Access to the H type I antigen was achieved first by conversion of the azide moiety into an N-acetyl group using thiolacetic acid in pyridine to afford 22 (Scheme 3). Surprisingly, some acetylation of the alcohol in 22 was observed during this reaction. The formation of this by-product was not, however, problematic as Zemplén deacetylation easily converted the O-acetylated compound into 22. Next, global deprotection was achieved using a Birch reduction resulting in the conversion of 22 into 6 in 92% yield. For investiga-
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
O
Ph
O
O
O
HO N3
Ph
13
Ph O
Ph
O
O
O
(a)
O
O
Ph
(b) AcO
O
N3
HO 18
16 R = Ac (c)
17 R = H
CCl3
O
O
PivO
N3
OR
O AcO
O
O O
O
O
RO
O
Ph
O
O O
O
NH
15
O OBn
O
NH (d)
OBn BnO
Ph
CCl3
19
Ph O
O
O
O O
Ph
(e)
O
OBn
OBn OBn
21
BnO
N3
O
O OBn
O
O
PivO
N3
O
O
O O
O
O
HO
O
Ph
O
O O
20
BnO
Scheme 2. Reagents: (a) TMSOTf, 4 Å MS, CH2Cl2; (b) NaOCH3, CH3OH, 63% (two steps); (c) (CH3)3CCOCl, pyridine, 89%; (d) TMSOTf, 4 Å MS, Et2O, CH2Cl2, 80%; (e) LiOCH3, CH3OH, 82%.
Ph
Ph O
O
O
O
O
Ph
(a)
N3
O OBn
O
O
HO
NHAc
O
O OBn
O
O O
O
O
HO
O
Ph
O
O O
O OBn
21
22
OBn
BnO
BnO
(b)
HO
OH O
OH O HO
O(CH2)7CH3
O
HO
HO
NHAc
O
O HO (c)
23
NHAc
O O
OH HO
O
O
HO
O OH
OH O
OH
OH OH HO
6
Scheme 3. Reagents: (a) (i) CH3COSH, pyridine; (ii) NaOCH3, CH3OH, 62%; (b) NH3, Na, CH3OH, THF, 92%; (c) H2, 10% Pd–C, THF, H2O, 83%.
tions involving conjugation of the H type I antigen to a support, the 7-octen-1-yl glycoside 6 was desired; however, other studies, such as the examination of GTA and GTB kinetics, required the octyl glycoside. To this end, catalytic reduction of the alkene moiety of 6 afforded octyl glycoside 23 in 83% yield. Access to the A type I antigen was achieved first by glycosylation of 21 with trichloroacetimidate 24 to afford tetrasaccharide 25 in good (92%) yield (Scheme 4). The stereochemistry of the newly formed glycosidic linkage was confirmed using 1H NMR
spectroscopy; the anomeric proton of the 2-azido-2-deoxy-a-Dgalactopyranoside appeared at 5.88 ppm as a doublet with a coupling of 3.2 Hz. Conversion of the two azides into N-acetyl groups using thiolacetic acid in pyridine afforded 26. Deacetylation under Zemplén conditions afforded 27 and subsequent complete deprotection using dissolving metal reduction afforded 5. Overall, tetrasaccharide 5 was obtained in 69% yield from 25. The B type I antigen could be obtained first by glycosylation of 21 with trichloroacetimidate 28 affording tetrasaccharide 29 in
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 Ph O O O
Ph
O
O O HO
Ph
O
O
AcO
N3
O O
(a)
O
Ph N3
21
BnO
O
AcO
OBn OBn
O
OAc O
O
O O
O
O
O
N3
O AcO
O
OAc O O
AcO N3
OBn 25
OBn
CCl3
BnO
NH
24
(b) Ph HO
RO
OH O
OH
OH O
OH O HO
HO AcHN
O
O
O
(d)
O O
Ph
RO
O
O O
AcHN
O
O
O
NHAc
O
O
OR O
NHAc
O O
O
OBn
OH OBn
OH BnO
HO
26 R = Ac
5
(c)
27 R = H
Scheme 4. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 92%; (b) CH3COSH, pyridine, 74%; (c) NaOCH3, CH3OH, 94%; (d) NH3, Na, CH3OH, THF, 88%.
Ph O O O
Ph
O
O O HO
Ph
O
O
BnO
N3
O O
(a)
O
Ph O O
BnO
21
BnO
O O
BnO
OBn OBn
OBn O
O
O
O
N3
O BnO
O
O
OBn O
OBn O
BnO OBn 28
OBn
CCl3
29
BnO
NH (b) Ph
HO
OH O
BnO OH
OH O
OH O HO
HO HO
O
O
O O
(c)
OBn O
O O O O
BnO
4
O NHAc
O O
OH HO
O
O
O
NHAc
O OH
O
Ph
BnO
OBn OBn BnO
30
Scheme 5. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 73%; (b) CH3COSH, pyridine, 65%; (c) NH3, Na, CH3OH, THF, 94%.
moderate yield (Scheme 5). Conversion of the azide into an N-acetyl group using thiolacetic acid in pyridine afforded 30, under conditions analogous to those used in the preparation of 22. Other
techniques do exist to convert an azide into an N-acetyl group, and several were explored including a Staudinger reaction followed by acetylation;30 however, none were found to be as
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
type II antigen 7 with the 7-octen-1-yl glycoside moiety intact. Reduction of part of this material by hydrogenation afforded, in 96% yield, the octyl glycoside 40 for use in enzymatic studies. The B type II antigen was accessed first by treatment of 38 with the trichloroacetimidate donor 28 under standard conditions (Scheme 9). Unfortunately, complete purification of 41 was not possible; therefore, the unequivocal confirmation of the stereochemistry was not possible until after the conversion of 41 into 42 using thiolacetic acid in pyridine. In 1H NMR spectrum of 42, the anomeric signal for the a-galactopyranosyl linkage appeared at 5.36 ppm as a doublet with a coupling constant of 3.6 Hz, as expected for this stereochemistry. Final global deprotection of 42 upon treatment with sodium and liquid ammonia in THF afforded 8 in high yield (96%). Finally, access to the A type II antigen was achieved in an analogous manner to its A type I counterpart (Scheme 10). Glycosylation of 38 with trichloroacetimidate 24 afforded tetrasaccharide 43; subsequent reduction–acetylation of the two azides using thiolacetic acid in pyridine afforded 44. Confirmation of the stereochemistry of the glycosidic linkage was achieved using 1H NMR spectroscopy. The anomeric proton of the newly formed glycosidic linkage appeared as a doublet at 5.09 ppm with a 3.7 Hz coupling constant. Deacetylation of 44 to provide 45 followed by complete deprotection resulted in the preparation of the A type II antigen 9 in excellent yield (88% yield over the two steps).
efficient as the tandem reduction–acetylation process induced by treatment with thiolacetic acid. Final deprotection of 30 using a Birch reduction afforded the B type I antigen 4 in 94% yield. In our synthesis of the type V and VI antigens,9 the yield for deprotection reactions of this type on similar substrates was at times low (60–70%). These lower yields were caused by precipitation of the starting material in liquid ammonia, leading to incomplete removal of the protecting groups. In the course of the synthesis of the targets reported here, we found that increasing the volume of the THF used to dissolve the starting material successfully circumvented this problem. 2.2. Synthesis of type II antigens The type I and II antigens are quite similar in structure. The only difference is that the galactose core residue is linked to the reducing-end GlcNAc moiety by either a b-(1?3) or b-(1?4) linkage in the type I and II antigens, respectively. This led us to exploit the reducing end acceptor 13, which was used in the type I synthesis, and putting it to use in the preparation of the type II antigens (Scheme 6). Protection of 13 using standard benzylation conditions afforded 31 in excellent yield, and then selective ring opening of the benzylidene acetal using trifluoroacetic acid and triethylsilane resulted in formation of the 4-OH derivative 32. Confirmation of the regiochemistry of the ring opening was achieved by acetylation (acetic anhydride and DMAP in pyridine) and observance of a downfield shift of H-4 in the 1H NMR spectrum from 3.63 ppm in 32 to 5.05–4.91 ppm in 33. Next, treatment of glycosyl acceptor 32 with trichloroacetimidate 15 using a TMSOTf promoter system yielded an impure disaccharide 34. Contamination of 34 with trichloroacetimidate-derived by-products necessitated the conversion of diacetate 34 into diol 35 to facilitate purification (Scheme 7). Protection of the 30 position of the galactose core was again achieved using trimethylacetyl chloride in pyridine to yield 36. Glycosylation using the b-L-fucopyranosyl trichloroacetimidate 19 afforded the trisaccharide in excellent yield (94%) and with complete stereoselectivity. The anomeric proton of the a-fucopyranoside of 20 showed an anomeric signal at 5.41 ppm as a doublet with a coupling constant of 3.8 Hz, consistent with a stereochemistry. Removal of the pivaloate ester was achieved using refluxing lithium methoxide in methanol; again, forcing conditions were needed, similar to those required to prepare 21. Given the structural similarities between the type I and II antigens, the deprotection strategies would be expected to be analogous and this was indeed the case. The conversion of the azido group in 38 to an N-acetyl functionality using thiolacetic acid afforded 39 in good (88%) yield (Scheme 8). Interestingly, in contrast to the preparation of the type I counterpart 22 (see Scheme 3 above), no acetylation of the alcohol on 39 was observed. Exhaustive deprotection was achieved in an analogous manner to the type I counterpart: Birch reduction of 39 afforded a 75% yield of the H
O
Ph
Ph
O
O
(a) O
HO N3 13
2.3. NMR analysis of 4–9 With the ABO type I and II antigens 4–9 in hand, we conducted detailed NMR studies to probe if there are any conformational differences between the structures. Unequivocally assigning proton resonances in deprotected oligosaccharides using traditional 1D and 2D techniques is difficult due to spectral overlap. To overcome this problem for the ring protons in 4–9, a gradient-enhanced chemical shift selective filtering (ge-CSSF) technique was used. In particular, 1D-ge-CSSF-TOCSY experiments were carried out to provide coupling constants and to assign chemical shifts.31,32 The use of this experiment enables the straightforward analysis of the 1H NMR spectra of each ring individually. In addition to analyzing any differences between the type I and II structures, the measurement of 1D-ge-CSSF-TOCSY enabled complete assignment of all 1H NMR chemical shifts for 4–9. The main limitation of this approach is that signal transfer throughout each ring is dependent on the size of the coupling constants. In the case of both galactose and fucose, when a ring is irradiated at H1, signal transfer from H3 to H4 is significantly reduced (due to 3JH3,H4 <3.5 Hz), and transfer to H5 and H6 does not occur. In most cases this problem could be circumvented by irradiation of the H5 or H6 resonance and then allowing the magnetization to transfer the opposite way around the spin system. The chemical shift data from these studies are shown in Table 2, and the associated coupling constants are shown in Table 3. The
OBn
O O BnO
O
(b) O
N3
HO BnO
O O N3 32
31 OBn
(c)
O
AcO BnO
O N3 33
Scheme 6. Reagents: (a) BnBr, NaH, DMF, 98%; (b) Et3SiH, CF3COOH, 4 Å MS, CH2Cl2 83%; (c) Ac2O, DMAP, pyridine, 85%.
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 OBn O
HO BnO
O
Ph
Ph
N3 32
O
O
O
Ph
O
(a)
O
OBn
O RO
O
RO
PivO
O
O
O BnO
HO
O
N3
O AcO AcO
OBn
O
O
O BnO
(b) O
36
34 R = Ac (c)
35 R = H
CCl3
N3
NH
15
NH O (d)
CCl3
OBn BnO
Ph
O OBn
19
Ph O
O
O
O OBn
O HO O
PivO
(e)
O
OBn
O N3
O
OBn
OBn OBn
38
BnO
O
O BnO
O
N3
O
OBn
O
O
O BnO
37
BnO
Scheme 7. Reagents: (a) TMSOTf, 4 Å MS, CH2Cl2; (b) NaOCH3, CH3OH, 78% (two steps); (c) (CH3)3CCOCl, pyridine, 88%; (d) TMSOTf, 4 Å MS, Et2O, 94%; (e) LiOCH3, CH3OH, 83%.
Ph
Ph
O
O O
O
HO
(a)
O
O BnO
O
HO
O
O OBn
OBn
OBn
38
BnO
O NHAc
N3
OBn
O BnO
O
O
O
OBn
O
OBn
O
39
BnO
(b)
HO
HO
OH
HO O
OH
OH
O O HO
OH
O (c)
O
HO
O(CH2)7CH3
O
O HO
NHAc
O NHAc
O
O OH
OH
O
OH
40
HO
OH
7
HO
Scheme 8. Reagents: (a) CH3COSH, pyridine, 88%; (b) Na, NH3, CH3OH, THF, 75%; (c) H2, 10% Pd–C, CH3OH, 96%.
residue assignments are shown in Figure 3. In all cases only the coupling constants for the ring protons were analysed. A signal is referred to as a multiplet only in instances where the coupling constant could not be determined, either due to second-order coupling or where inadequate signal transfer in the 1D-ge-CSSF-TOCSY (outlined above) resulted in individual proton resonances not being able to be identified. In cases where insufficient information was obtained using 1D-ge-CSSF-TOCSY, additional techniques such as 1 H–1H COSY, HSQC and HMBC were employed to aid in the identification of proton resonances. Not surprisingly, the NMR spectra of
all six structures showed significant similarity; little deviation was observed between the spectra of any of the compounds with respect to both chemical shift (Table 2) and coupling constants (Table 3). 2.4. Conclusions In summary, we report the linear syntheses of the A, B and H type I and II histo-blood group antigens (4–9) attached to either a 7-octen-1-yl or an octyl aglycone. Key to the synthesis was the
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 Ph O O Ph
OBn
O HO
O
O BnO
O
BnO
N3
O
(a)
O
OBn O
O
O
BnO
OBn
OBn
O
BnO O
OBn
38
BnO
O
O BnO
O
O N3
O BnO
OBn
OBn O
41
OBn BnO O
BnO OBn
CCl3 NH
28
(b)
Ph HO
BnO
OH O
OH
OH
HO HO
O
O HO
O
O
BnO
OH
O
O
OBn O
(c)
O
BnO
O
O NHAc
O O
O OBn
OH HO
O BnO
NHAc
O OH
OBn
O O
8
OBn BnO
42
Scheme 9. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 75%; (b) CH3COSH, pyridine, 91%; (c) Na, NH3, CH3OH, THF, 96%.
preparation of the two reducing-end disaccharides 13 and 32, which served as the core upon which the rest of the structures were built. In addition, approaches developed in the synthesis of the type V and VI antigen synthesis9 were adapted to the synthesis of the type I and II antigens. Use of these methods enabled the targets to be obtained with a high level of efficiency. Most notably, the preparation of the 7-octen-1-yl b-D-glucopyranoside 11 was efficiently prepared without anchimeric assistance. In addition, the installation of the a-L-fucopyranosyl linkage was entirely stereoselective, taking advantage of the excellent stereoselectivity of the trichloroacetimidate methodology.29 An NMR study was conducted on each of the type I and II ABO antigens (4–9). Based upon the chemical shift and coupling constant data, it was clear that the reducing-end residue has little to no impact on the conformation of the canonical di- and trisaccharide blood group antigens at the non-reducing end of the molecule. 3. Experimental
performed on Silica Gel 60 (40–60 lm). Iatrobeads refer to a beaded silica gel 6RS-8060, which is manufactured by Iatron Laboratories (Tokyo). C18 silica gel (35–70 lm) was manufactured by Toronto Research Chemicals. Optical rotations were measured at 22 ± 2 °C and are in units of deg dm1 cm3 g1; in all cases the concentrations are in the units g/100 mL. 1H NMR spectra were recorded at 400 and 500 MHz, and chemical shifts were referenced to the peak for TMS (0.0 ppm, CDCl3) or CD3OD (3.30 ppm, CD3OD). For final compounds, analysis was conducted using 1D-ge-CSSFTOCSY31,32 at 600 MHz, to identify individual proton resonances. Samples for 1D-ge-CSSF-TOCSY experiments were prepared in CD3OD at a concentration of 21 mg/mL, and the experiments were carried out at 300 K. 13C NMR (APT) spectra were recorded at 125 or 100 MHz, and 13C chemical shifts were referenced to the peak for internal CDCl3 (77.1 ppm, CDCl3) or CD3OD (49.0, CD3OD). All spectra were recorded in CDCl3 unless specified otherwise. Melting points were measured using a Perkin–Elmer Thermal Analysis. Electrospray-ionization mass spectra were recorded on samples suspended in mixtures of THF with CH3OH and added NaCl.
3.1. General methods All reagents were purchased from commercial sources and were used without further purification, unless otherwise stated. Reaction solvents were purchased and were used without purification; dry solvents were purified by successive passage through columns of alumina and copper under nitrogen. All reactions were carried out at room temperature under a positive pressure of argon, unless otherwise stated. Thin layer chromatography (TLC) was performed on E. Merck Silica Gel 60 F254 aluminium-backed plates that were stained by heating (>200 °C) with either p-anisaldehyde in 5% sulfuric acid in EtOH or 10% ammonium molybdate in 10% sulfuric acid. Unless otherwise indicated, all column chromatography was
3.2. 7-Octen-1-yl 2-acetamido-2-deoxy-3-O-(2-O-(a-Lfucopyranosyl)-3-O-(a-D-galactopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (4) Redistilled liquid ammonia (15 mL) was collected in a flask cooled to 78 °C and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 30 (67 mg, 0.042 mmol) in THF (4 mL) and CH3OH (15 lL, 0.37 mmol) was added dropwise, and the solution was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322 Ph O O
Ph OBn
O HO O
AcO
O
O BnO
O N3
O OBn
O
AcO
(a)
OBn
O N3
38
OBn
O
OAc O
O
O
O BnO
O
BnO
O N3
O OBn AcO
43
OBn
OAc O
BnO O
AcO N3
CCl3 NH
24
(b)
Ph HO
OH O
RO OH
OH
HO
OH
O AcHN O O
O HO
OR O
O O
RO (d)
O
OBn
O AcHN
O
O O
O
O BnO
O
NHAc
NHAc
O
O
OH
OBn
OH HO
9
OBn BnO R = Ac 44 R = H 45
(c)
Scheme 10. Reagents: (a) TMSOTf, 4 Å MS, Et2O, 94%; (b) CH3COSH, pyridine, 76%; (c) NaOCH3, CH3OH, 92%; (d) Na, NH3, CH3OH, THF, 96%.
to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 4 (31 mg, 94%) as a colourless oil. [a] +69.0 (c 1.3, MeOH); 1H NMR: see Tables 2 and 3; 13C NMR (175 MHz): dC 173.2 (C@O), 140.0 (CH2@CH), 114.7 (CH2@CH), 103.6 (C1), 102.1 (C10 ), 100.5 (C100 ), 96.2 (C1000 ), 80.2, 79.9, 77.6, 76.2, 74.5, 73.6, 73.2, 71.4, 71.2 (2C), 70.6, 70.0, 69.8 (C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C400 , C4000 , C5, C50 , C5000 ), 70.7 (CH@CH2(CH2)5CH2O), 67.4 (C500 ), 65.7 (C40 ), 63.4, 62.7, 62.5 (C6, C60 , C6000 ), 55.9 (C2), 34.8 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O), 30.1 (CH@CH2 (CH2)5CH2O), 29.9 (CH@CH2(CH2)5CH2O), 26.9 (CH@CH2(CH2)5 CH2O), 23.2 (CH3C@O), 16.5 (C600 ). ESIMS: m/z Calcd [C34H59NO20]Na+: 824.3523. Found: 824.3522. 3.3. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2-deoxya-D-galactopyranosyl)-2-O-(a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (5) Redistilled liquid ammonia (20 mL) was collected in a flask cooled to 78 °C and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 27 (58 mg, 0.045 mmol) in THF (4 mL) and CH3OH (9.1 lL, 0.225 mmol) was added dropwise, and the solution was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+), and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 5 (33.5 mg, 88%) as a colourless oil. [a] +27.1 (c 0.2, H2O); 1H NMR: see Tables 2 and 3; 13C NMR (125 MHz, D2O): dC 175.7 (C@O), 174.5 (C@O), 141.2 (CH2@CH), 114.9 (CH2@CH), 102.8, 100.8, 100.0 (C1, C10 , C100 ), 92.1 (C1000 ), 78.3, 76.33, 76.27, 75.7, 74.7, 72.7, 71.8, 70.6, 69.7, 69.4, 68.53,
68.50, 67.5, 63.8 (C20 , C200 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C50 , C500 , C5000 ), 71.5 (CH@CH2(CH2)5CH2O), 62.3, 62.1, 61.6 (C6, C60 , C6000 ), 55.6 (C2), 50.5 (C2000 ), 34.0 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 29.0 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2 (CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 23.2 (CH3C@O), 22.8 (CH3C@O), 16.1 (C600 ). ESIMS: m/z Calcd [C36H62N2O20]Na+: 865.3788. Found: 865.3788.
3.4. 7-Octen-1-yl 2-acetamido-2-deoxy-3-O-(2-O-(a-Lfucopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (6) Redistilled liquid ammonia (15 mL) was collected in a flask cooled to 78 °C and treated with sodium until the blue colour persisted. A solution of trisaccharide 22 (130 mg, 0.120 mmol) in THF (4 mL) and CH3OH (24 lL, 0.60 mmol) was added dropwise, and the solution was stirred (78 °C, 2 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected trisaccharide 6 as a colourless oil (70 mg, 92%). [a] 62.7 (c 1.0, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125 MHz, CD3OD): dC 173.1 (C@O), 140.1 (CH2@CH), 114.8 (CH2@CH), 103.6 (C1), 102.2 (C10 ) 101.2 (C100 ), 80.3, 78.0, 77.7, 77.0, 75.6, 73.6, 71.4, 70.8, 70.5, 70.3, 67.6 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C50 , C500 ), 70.7 (CH@CH2(CH2)5CH2O), 62.7, 62.6 (C6, C60 ), 56.1 (C2), 34.9 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2 (CH2)5CH2O), 30.2 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5 CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.2 (CH3C@O), 16.6 (C600 ). ESIMS: m/z Calcd [C28H49NO15]Na+: 662.2994. Found: 662.3000.
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P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
Table 2 1 H Assignments (ppm) of the ABH type I–II antigens 4–9, recorded at 600 MHz with CD3OD as solvent Proton
A Type 1 5
A Type II 9
B Type 1 4
B Type II 8
000
H1 H2000 H3000 H4000 H5000 H6000 H6000
5.15 4.29 3.85 3.87 4.20 3.75 3.68
(d) (dd) (dd) (d) (dd) (dd) (dd)
5.15 4.32 3.82 3.89 4.16 3.76 3.68
5.14 3.83 3.81 3.89 4.16 3.73 3.68
(d) (dd) (dd) (dd) (dd) (dd) (dd)
5.15 (d) 3.84 (dd) 3.78 (dd) 3.9 (d) 4.14–4.10 (m) 3.76–3.71 (m) 3.70–3.66 (m)
H100 H200 H300 H400 H500 H600
5.23 3.69 3.65 3.72 4.36 1.19
(s) (dd) (dd) (d) (q) (d)
S.33 (d) 3.72 (dd) 3.68 (dd) 3.69 (d) 4.32 (q) 1.21 (d)
5.21 (d) 3.7 (dd) 3.65 (dd) 3.71 (d) 4.45 (q) 1.18 (d)
H10 H20 H30 H40 H50 H60 H60
4.57 (d) 3.91 (dd) 3.86 (dd) 4.09 (d) 3.49 (dd) 3.77–3.73 (m) 3.66–3.65 (m)
4.52 (d) 3.99 (dd) 3.90 (dd) 4.09 (d) 3.29–3.25 (m) 3.81–3.77 (m) 3.72–3.66 (m)
H1 H2 H3 H4 H5 H6 H6
4.25–4.20 3.86–3.79 3.86–3.79 3.41–3.37 3.32–3.28 3.78–3.74 3.69–3.66
(m) (m) (m) (m) (m) (m) (m)
4.39 (d) 3.71 (dd) 3.69 (dd) 3.59 (dd) 3.28 (dd) 3.90 (d) 3.78(d)
Other CH2O CH2O CH@CH2 CH@CH2 CH@CH2 (CH2)5 (CH2)5 (CH2)5 CH3CO CH3CO
3.91–3.87 3.44–3.40 4.99–4.95 4.91–4.89 5.84–5.76 2.08–2.01 1.57–1.47 1.42–1.26 1.99 (s) 1.99 (s)
(m) (m) (m) (m) (dddd) (m) (m) (m)
3.86–3.83 3.45–3.41 5.00–4.94 4.92–4.88 5.84–5.76 2.07–2.01 1.58–1.48 1.42–1.27 2.00 (s) 1.96 (s)
(d) (dd) (dd) (d) (dd) (dd) (dd)
(m) (dt) (m) (m) (dddd) (m) (m) (m)
H Type 1 6
H Type II 7
5.32 (m) 3.73 (m) 3.69–3.66 (m) 3.69–3.66 (m) 4.29 (q) 1.20 (d)
5.20 (d) 3.73 (dd) 3.68 (dd) 3.71–3.69 (m) 4.29 (q) 1.19 (d)
5.21 (d) 3.77–3.71 (m) 3.77–3.71 (m) 3.66(d) 4.17 (q) 1.20 (d)
4.60–4.56 (m) 3.93–3.87 (m) 3.93–3.87 (m) 4.10 (s) 3.54 (dd) 3.79 (dd) 3.68 (dd)
4.53 (d) 3.98 (dd) 3.94 (dd) 4.12 (d) 3.60–3.54 (m) 3.79 (dd) 3.67 (dd)
4.54–4.51 3.71–3.65 3.71–3.65 3.80–3.78 3.52 (dd) 3.67–3.64 3.77–3.74
(m) (m) (m) (m) (m) (m)
4.50–4.44 3.75–3.68 3.75–3.68 3.78(d) 3.54 (dd) 3.65–3.61 3.75–3.73
4.25–4.19 3.86–3.77 3.86–3.77 3.41–3.37 3.31–3.28 3.93–3.83 3.76–3.64
(m) (m) (m) (m) (m) (m) (m)
4.36 (d) 3.70 (dd) 3.59 (dd) 3.66 (dd) 3.30–3.26 (m) 3.90 (dd) 3.77 (dd)
4.28–4.22 3.84–3.78 3.84–3.78 3.42–3.38 3.32–3.28 3.74–3.65 3.92–3.84
(m) (m) (m) (m) (m) (m) (m)
4.37 (d) 3.72 (dd) 3.59 (dd) 3.69 (dd) 3.33–3.29 (m) 3.89 (dd) 3.83 (dd)
3.92–3.83 3.46–3.37 4.99–4.95 4.92–4.89 5.84–5.75 2.07–2.01 1.57–1.46 1.42–1.27 1.99 (s)
(m) (m) (m) (m) (dddd) (m) (m) (m)
3.91–3.83 3.48–3.43 5.00–4.95 4.93–4.88 5.84–5.76 2.08–2.01 1.58–1.49 1.43–1.25 1.95 (s)
3.45–3.37 3.91–3.83 4.99–4.93 4.92–4.88 5.84–5.75 2.07–2.00 1.58–1.46 1.41–1.26 1.97 (s)
(m) (m) (m) (m) (dddd) (m) (m) (m)
3.47–3.42 3.91–3.82 4.99–4.94 4.92–4.88 5.83–5.75 2.07–2.01 1.58–1.48 1.41–1.28 1.96 (s)
(m) (dt) (m) (m) (dddd) (m) (m) (m)
(m) (m) (m)
(m) (m)
(m) (dt) (m) (m) (dddd) (m) (m) (m)
Table 3 2 JH,H,3JH,H (Hz) of the ABH type I–II antigens 4–9, recorded at 600 MHz with CD3OD as solvent Coupling
A Type I 5
A Type II 9
B Type I 4
B Type II 8
3
JH-l000 ,H-2000 3 JH-2000 ,H-3000 3 JH-3000 ,H-4000 3 JH-4000 ,H-5000 3 JH-5000 ,H-6a000 3 JH-5000 ,H-6b 2 JH-6a000 ,H-6b000
3.8 10.8 3.4 0 7.6 4.1 11.7
3.7 11.0 3.0 0 7.2 4.5 11.5
3.5 10.2 3.1 0.8 7.3 4.3 11.4
3.8 10.1 2.9 m rr m m
3
JH-l00 ,H-200 JH-200 ,H-300 3 JH-300 ,H-400 3 JH-500 ,H-600
3.9 10.5 3.1 6.5
3.9 9.5 3.3 6.6
4.1 9.3 3.3 6,6
3
JH-l0 ,H-20 JH-20 ,H-30 3 JH-30 ,H-40 3 JH-50 ,H-6a0 3 JH-50 ,H-6b0 2 JH-6a0 ,H-6b0
7.3 9.9 3.1 7.9 4.3 m
7.7 9.8 3.0 rr m rr
3
JH-l,H-2 JH-2,H-3 3 JH-3,H-4 3 JH-4,H-5 3 JH-5,H-6a 3 JH-5,H-6b 2 JH-6a,H-6b
m m m m m m m
CH2O CH@CH2
m 17.1, 10.2, 6.7, 6.7
3
3
3
H Type I 6
H Type II 7
3.9 9.9 m 6.6
3.8 10.0 3.1 6.6
3.3 m 2.8 6.6
m m 0 7.8 4.4 11.4
7.4 9.7 2.7 7.3 3.9 11.6
m m m 7.7 4.3 m
8.4 9.3 8.9 9.8 4.9 0 11.2
m m m m m m m
8.4 10.4 8.8 9.2 5.0 1.8 11.9
m m m m m m rr
8.4 10.5 8.8 10.4 4.4 1.9 12.0
9.7, 6.6 17.0, 10.2, 6.7, 6,7
m 17.0, 10.2, 6.7, 6.7
9.7, 6.6 17.0, 10.2, 6.7, 6.7
m 17.0, 10.2, 6.7, 6.7
9.6, 6.6 17.0, 10.2, 6.7, 6.7
4.0 7.6 4.0
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
HO
OH O
OH
OH
HO
O
HO O
O O H1′ HO
H1′′′ O OH HO
H1′′ OH 8 B Type II
OH O O NHAc H1
Figure 3. Nomenclature system used for 4–9, using the B type II antigen 8 for illustrative purposes. This nomenclature is used for the data presented in Tables 2 and 3.
3.5. 7-Octen-1-yl 2-acetamido-2-deoxy-4-O-(2-O-(a-Lfucopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (7) Redistilled liquid ammonia (20 mL) was collected in a flask cooled to (78 °C) and treated with sodium until the blue colour persisted. A solution of trisaccharide 39 (185 mg, 0.157 mmol) in THF (4 mL) and CH3OH (38 lL, 0.94 mmol) was added dropwise, and the mixture was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected trisaccharide 7 (75 mg, 75%) as a colourless oil. [a] 78.9 (c 0.4, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125.7 MHz, CD3OD): dC 178.4 (C@O), 140.1 (CH2@CH), 114.8 (CH2@CH), 102.9, 102.6 (C1, C10 ), 101.9 (C100 ), 79.1, 78.3, 77.1, 77.0, 75.3, 74.1, 73.6, 71.7, 70.8, 70.7 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C50 ), 70.7 (CH@CH2(CH2)5CH2O), 68.3 (C500 ), 62.6, 61.7 (C6, C60 ), 56.8 (C2), 34.8 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O), 30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.0 (CH3CO), 16.8 (C600 ). ESIMS: m/z Calcd [C28H49NO15]Na+: 662.2994. Found: 662.2985. 3.6. 7-Octen-1-yl 2-acetamido-2-deoxy-4-O-(2-O-(a-Lfucopyranosyl)-3-O-(a-D-galactopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (8) Redistilled liquid ammonia (20 mL) was collected in a flask cooled to (78 °C) and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 42 (244 mg, 0.144 mmol) in THF (4 mL) and CH3OH (58 lL, 1.44 mmol) was added dropwise, and the mixture was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 8 (111 mg, 96%) as a colourless oil. [a] 28.9 (c 0.5, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125.7 MHz, CD3OD): dC 173.4 (C@O), 140.1 (CH2@CH), 114.8 (CH2@CH), 102.8, 102.2 (C1, C10 ), 100.3 (C100 ), 96.1 (C1000 ), 79.8, 78.6, 77.1, 76.7, 74.1, 73.8, 73.6, 73.1, 71.8, 71.4, 71.3, 70.0, 69.9, 67.6, 65.8 (C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C50 , C500 , C5000 ), 70.7 (CH@CH2(CH2)5CH2O), 63.3, 62.6, 61.8 (C6, C60 , C6000 ), 56.8 (C2), 34.8 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5 CH2O), 30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.0 (CH@CH2(CH2)5CH2O), 23.0 (CH3CO), 16.6 (C600 ). ESIMS: m/z Calcd [C34H59NO20]Na+: 824.3523. Found: 824.3518. 3.7. 7-Octen-1-yl 2-acetamido-4-O-(3-O-(2-acetamido-2-deoxy-
a-D-galactopyranosyl)-2-O-(a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (9) A solution of tetrasaccharide 44 (520 mg, 0.345 mmol) in CH3OH (20 mL) was treated with a catalytic amount of NaOCH3
2315
in CH3OH, and the solution was stirred for 2 h. The solution was then neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to flash chromatography (CH2Cl2–CH3OH 10:1) to afford triol 45 (440 mg, 92%) as a colourless oil. Redistilled liquid ammonia (20 mL) was collected in a flask cooled to (78 °C) and treated with sodium until the blue colour persisted. A solution of tetrasaccharide 45 (221 mg, 0.160 mmol) in THF (4 mL) and CH3OH (39 lL, 0.961 mmol) was added dropwise, and the mixture was stirred (78 °C, 1 h). The reaction was then quenched by the addition of CH3OH (4 mL), and ammonia was evaporated. The solution was taken up in CH3OH (100 mL), neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to C18 chromatography (CH3OH–H2O 1:1) to afford the fully deprotected tetrasaccharide 9 (129 mg, 96%) as a colourless oil. [a] +5.8 (c 1.6, CH3OH); 1H NMR: see Tables 2 and 3; 13C NMR (125.7 MHz, CD3OD): dC 174.4 (C@O), 173.3 (C@O), 140.0 (CH2@CH), 114.8 (CH2@CH), 104.2 (C10 ), 100.5 (C100 ), 98.1 (C1), 93.9 (C1000 ), 77.6, 77.4, 76.2, 75.8, 73.4, 72.7, 71.8, 71.6, 70.6, 70.1, 70.0, 69.88 (C20 , C200 , C3, C30 , C300 , C3000 , C4, C400 , C4000 , C5, C50 , C5000 ), 68.94 (CH@CH2(CH2)5CH2O), 68.4 (C500 ), 64.9 (C40 ), 63.4, 62.8, 62.5 (C6, C60 , C6000 ), 51.4, 50.9 (C2, C2000 ), 34.9 (CH@CH2(CH2)5CH2O), 30.6 (CH@CH2(CH2)5CH2O), 30.1 (CH@CH2(CH2)5CH2O), 30.0 (CH@CH2(CH2)5CH2O), 27.2 (CH@CH2(CH2)5CH2O), 22.9 (CH3CO), 22.8 (CH3CO), 16.9 (C600 ). ESIMS: m/z Calcd [C36H62NO20]Na+: 865.3788. Found: 865.3783. 3.8. 7-Octen-1-yl 2-azido-4,6-O-benzylidene-2-deoxy-b-Dglucopyranoside (13) and 7-Octen-1-yl 2-Azido-4,6-Obenzylidene-2-deoxy-a-D-glucopyranoside (14) A stirred solution of trichloroacetimidate27 10 (8.69 g, 18.3 mmol) and 7-octen-1-ol (2.82 g, 22.0 mmol) in dry CH2Cl2 (50 mL) was treated with 4 Å molecular sieves (3.5 g), and the mixture was stirred (rt, 1 h). The mixture was cooled (30 °C), treated with TMSOTf (300 lL) and allowed to slowly warm to 0 °C. The mixture was neutralized by the addition of Et3N (1 mL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:3) to give an inseparable a/b mixture 11, which was used immediately in the subsequent step. The oil was taken up in CH3OH (80 mL) and treated with a catalytic amount of NaOCH3 in CH3OH, and the solution was stirred (rt, 1 h). The solution was then neutralized with Amberlite IR 120 (H+), and the mixture was filtered; concentration followed by flash chromatography (EtOAc–hexanes 2:1) yielded triol 12 (3.32 g) as an inseparable a/b mixture. A solution of triol 12 (3.32 g, 10.5 mmol) in dry DMF (20 mL) was then treated with benzaldehyde dimethyl acetal (2.13 g, 14.0 mmol) and p-TsOH (100 mg), and the solution was stirred (50 °C, 4 h). The solution was treated with Et3N (1 mL) and concentrated, and the residue was subjected to flash chromatography (EtOAc–hexanes 1:3) to firstly afford the b glycoside 13 as a colourless oil (3.05 g, 41%). [a] 38.4 (c 0.4, CH2Cl2); Rf 0.18 (EtOAc–hexanes 7:3); 1H NMR (500 MHz): dH 7.52–7.35 (5H, m, Ph), 5.87–5.76 (1H, m, CH@CH2), 5.54 (1H, s, PhCH), 5.04–4.92 (2H, m, CH@CH2), 4.42 (1H, d, J1,2 = 8.0 Hz, H1), 4.34 (1H, dd, J6,6 = 10.3 Hz, J5,6 = 5.0 Hz, H6), 3.97–3.89 (1H, m, CH@CH2(CH2)5CH2O), 3.79 (1H, dd, J6,6 = J5,6 = 10.3 Hz, H6), 3.69–3.51 (3H, m, H3, H4, CH@CH2(CH2)5CH2O), 3.45–3.35 (2H, m, H2, H5), 2.69 (1H, br s, OH), 2.10–1.99 (2H, m, CH@CH2(CH2)5CH2O), 1.73–1.56 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.25 (6H, m, CH@CH2(CH2)5CH2O). 13 C NMR (125 MHz): dC 139.0 (CH@CH2), 136.8 (Ph), 129.4 (Ph), 128.4 (Ph), 126.2 (Ph), 114.3 (CH@CH2), 102.7, 102.0 (PhCH, C1), 80.6, 72.0, 66.5, 66.2 (C2, C3, C4, C5), 70.7 (CH@CH2(CH2)5CH2O), 68.5 (C6), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 28.78 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C21H29N3O5]Na+:
2316
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
426.2000. Found: 426.2002. Further elution yielded a glycoside 14 as a colourless oil (0.8 g, 9%). [a] +80.3 (c 0.7, CH2Cl2); Rf 0.16 (EtOAc–hexanes 7:3); 1H NMR (500 MHz): dH 7.41–7.37 (5H, m, Ph), 5.87–5.77 (1H, m, CH@CH2), 5.55 (s, PhCH), 5.04– 4.93 (2H, m, CH@CH2), 4.90 (1H, d, J1,2 = 3.6 Hz, H1), 4.28 (1H, dd, J6,6 = 10.2 Hz, J5,6 = 4.9 Hz, H6), 4.23 (1H, dd, J2,3 = 10.0 Hz, J3,4 = 9.6 Hz, H3), 3.87 (1H, ddd, J5,6 = 9.9 Hz, J4,5 = 9.9 Hz, J5,6 = 4.9 Hz, H5), 3.77–3.71 (2H, m, H6, CH@CH2(CH2)5CH2O), 3.54–3.46 (2H, m, H4, CH@CH2(CH2)5CH2O), 3.23 (1H, dd, J2,3 = 10.0 Hz, J1,2 = 3.6 Hz, H2), 2.76 (1H, br s, OH), 2.09–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.70–1.62 (2H, m, CH@CH2(CH2)5 CH2O), 1.46–1.33 (6H, m, CH@CH2(CH2)5CH2O). 13C NMR (125 MHz): dC 139.0 (CH@CH2), 136.9 (Ph), 129.5 (Ph), 128.4 (Ph), 126.4 (Ph), 114.3 (CH@CH2), 102.1 (PhCH), 98.6 (C1), 82.0 (C4), 68.9, 68.8 (C6, CH@CH2(CH2)5CH2O), 68.7 (C3), 63.1, 62.4 (C2, C5), 33.7 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 28.78 (CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C21H29N3O5]Na+: 426.2000. Found: 426.1995.
3.9. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-O-pivaloylb-D-galactopyranosyl)-2-deoxy-b-D-glucopyranoside (18) A solution of acceptor 13 (2.3 g, 5.7 mmol) in dry CH2Cl2 (20 mL) was stirred over 4 Å molecular sieves (1.5 g), and the mixture was stirred (rt, 1 h). The solution was then cooled (40 °C) and treated with TMSOTf (0.2 mL), followed by the dropwise addition of trichloroacetimidate 1533 (8.0 g, 16 mmol). The mixture was allowed to warm to 0 °C, and it was then neutralized by the addition of Et3N (2 mL). Concentration and flash chromatography (EtOAc–hexanes 1:1) of the residue afforded a colourless oil that was immediately used in the next step. The colourless oil was taken up in CH3OH (100 mL), treated with a solution of NaOCH3 in CH3OH and stirred (rt, 3 h). The solution was neutralized with Amberlite IR 120 (H+), filtered and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford diol 17 as a colourless oil (2.3 g, 63%). A solution of 17 (2.20 g, 3.37 mmol) in dry pyridine (30 mL) was treated with trimethylacetyl chloride (600 mg, 5 mmol), and the solution was stirred (rt, 30 min). Further trimethylacetyl chloride (600 mg, 5 mmol) was added, and the solution was stirred (rt, 30 min). The solution was then concentrated and subjected to flash chromatography (EtOAc–hexanes 2:3) to afford 18 as a colourless oil (2.2 g, 89%). [a] +5.8 (c 0.1, CH2Cl2); Rf 0.75 (EtOAc–hexanes 2:3); 1H NMR (500 MHz): dH 7.52–7.46 (4H, m, Ph), 7.38–7.30 (6H, m, Ph), 5.86–5.76 (1H, m, CH2@CH), 5.54 (1H, s, PhCH), 5.46 (1H, s, PhCH), 5.04–4.92 (2H, m, CH2@CH), 4.78 (1H, dd, J20 ,30 = 9.5 Hz, J30 ,40 = 3.6 Hz, H30 ), 4.49 (1H, d, J10 ,20 = 7.9 Hz, H10 ), 4.47 (1H, d, J1,2 = 8.0 Hz, H1), 4.37–4.29 (2H, m, H40 , H6), 4.17 (1H, d, J60 ,60 = 12.1 Hz, H60 ), 4.05 (1H, dd, J20 ,30 = 9.5 Hz, J10 ,20 = 8.2 Hz, H20 ), 3.96–3.88 (2H, m, H60 , CH@CH2(CH2)5CH2O), 3.80 (1H, dd, J6,6 = J5,6 = 10.1 Hz, H6), 3.77– 3.72 (2H, m, H3, H4), 3.62–3.49 (2H, m, H2, CH@CH2(CH2)5CH2O), 3.46–3.35 (1H, m, H5), 3.33–3.29 (1H, m, H50 ), 3.02–2.96 (1H, br s, OH), 2.11–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.72–1.60 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.30 (6H, m, CH@CH2(CH2)5CH2O), 1.22 (9H, s, (CH3)3C). 13C NMR (125 MHz): dC 178.3 (C@O), 139.0 (CH2@CH), 137.9 (Ph), 137.0 (Ph), 129.1 (Ph), 128.7 (Ph), 128.2 (Ph), 128.0 (Ph), 126.02 (Ph), 125.96 (Ph), 114.3 (CH2@CH), 104.5 (C10 ), 102.7 (C1), 101.4 (PhCH), 100.5 (PhCH), 79.9, 79.8 (C3, C4), 73.21, 73.20 (C30 , C40 ), 70.8 (CH@CH2(CH2)5CH2O), 69.1 (C20 ), 68.8, 68.5 (C6, C60 ), 67.0 (C50 ), 66.3 (C5), 65.5 (C2), 39.0 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.80 (CH@CH2(CH2)5CH2O), 28.77 (CH@CH2(CH2)5CH2O), 27.1 ((CH3)3C), 25.7 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C44H59N3O13]Na+: 760.3416. Found: 760.3415.
3.10. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-3-Opivaloyl-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (20) A solution of acceptor 18 (415 mg, 0.563 mmol) in dry Et2O– CH2Cl2 (90:10, 20 mL) was stirred over 4 Å molecular sieves (rt, 1 h). The solution was then cooled (10 °C), treated with TMSOTf (20 lL), followed by dropwise addition of trichloroacetimidate29 19 (1.02 g, 13.8 mmol) in dry Et2O (15 mL). The mixture was treated with Et3N (0.5 mL) and filtered, and the resulting residue was subjected to flash chromatography (EtOAc–hexanes 1:3) to yield trisaccharide 20 as a colourless oil (510 mg, 80%). [a] 20.7 (c 0.2, CH2Cl2); Rf 0.59 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.55–7.22 (25H, m, Ph), 5.87–5.77 (1H, m, CH2@CH), 5.41 (1H, d, J100 ,200 = 1.5 Hz, H100 ), 5.48 (1H, s, PhCH), 5.37 (1H, s, PhCH), 5.04– 4.92 (4H, m, H30 , PhCH2, CH2@CH), 4.79, 4.74 (2H, AB, J = 11.5 Hz, PhCH2), 4.76 (1H, d, J10 ,20 = 8.1 Hz, H10 ), 4.69 (1H, A of AB, J = 11.7 Hz, PhCH2), 4.79, 4.63 (2H, AB, J = 11.5 Hz, PhCH2), 4.51 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.42 (1H, d, J1,2 = 7.7 Hz, H1), 4.34– 4.29 (2H, m, H6, H60 ), 4.24 (1H, d, J10 ,20 = J20 ,30 = 8.5, H20 ), 4.13–4.07 (3H, m, H200 , H300 , H400 ), 3.97–3.91 (1H, m, CH@CH2(CH2)5CH2O), 3.83–3.70 (5H, m, H3, H4, H40 , H6, H60 ), 3.63–3.56 (1H, m, CH@CH2(CH2)5CH2O), 3.43–3.35 (2H, m, H2, H5), 3.04–2.99 (1H, m, H50 ), 2.11–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.73–1.63 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.31 (6H, m, CH@CH2(CH2)5CH2O), 1.20 (3H, d, J500 ,600 = 6.3 Hz, H600 ), 1.08 (9H, s, (CH3)3C). 13C NMR (125 MHz): dC 177.9 (C@O), 139.01 (Ph), 138.98 (CH2@CH), 138.6 (Ph), 138.4 (Ph), 137.6 (Ph), 136.8 (Ph), 129.2 (Ph), 128.7 (Ph), 128.5 (Ph), 128.4 (Ph), 128.33 (Ph), 128.28 (Ph), 128.2 (Ph), 128.0 (2C, Ph), 127.6 (Ph), 127.5 (Ph), 127.44 (Ph), 127.38 (Ph), 126.2 (2C, Ph), 114.3 (CH2@CH), 102.8 (C1), 101.05 (C10 ), 101.7 (PhCH), 100.8 (PhCH), 96.8 (C100 ), 79.9, 79.7, 78.0, 77.5, 76.55, 76.52 (C3, C30 , C300 , C4, C40 , C400 ), 75.0 (PhCH2), 73.5 (PhCH2), 72.9 (PhCH2), 72.6, 70.1 (C20 , C200 ), 70.7 (CH@CH2(CH2)5CH2O), 68.8, 68.6 (C6, C60 ), 66.5 (C500 ), 66.4, 65.9, 65.7 (C2, C5, C50 ), 38.8 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.83 (CH@CH2(CH2)5CH2O), 28.78 (CH@CH2(CH2)5CH2O), 27.0 ((CH3)3C), 25.8 (CH@CH2(CH2)5CH2O), 16.9 (C600 ). ESIMS: m/z Calcd [C66H79N3O15]Na+: 1176.5403. Found: 1176.5402. 3.11. 7-Octen-1-yl 2-azido-3-O-(4,6-O-benzylidene-2-O-(2,3,4tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2deoxy-b-D-glucopyranoside (21) A solution of pivaloyl ester 20 (1.456 g, 1.26 mmol) in CH3OH (150 mL) was treated with catalytic LiOCH3 (100 mg), and the solution was heated at reflux (7 d). The solution was then concentrated, extracted with EtOAc (400 mL) and washed with satd aq NaHCO3 and brine. The organic extract was then dried, concentrated and subjected to flash chromatography (EtOAc–hexanes 3:7) to afford alcohol 21 as a colourless oil (1.10 g, 82%). [a] 20.7 (c 0.1, CH2Cl2); Rf 0.26 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.63–7.51 (4H, m, Ph), 7.42–7.19 (21H, m, Ph), 5.89–5.79 (1H, m, CH2@CH), 5.57 (1H, s, PhCH), 5.54 (1H, s, PhCH), 5.33 (1H, s, H100 ), 5.06–4.94 (3H, m, PhCH2, CH2@CH), 4.85 (1H, A of AB, J = 11.5 Hz, PhCH2), 4.84–4.74 (3H, m, PhCH2), 4.68 (1H, d, J10 ,20 = 7.1 Hz, H1´), 4.66 (1H, A of AB, J = 11.1 Hz PhCH2), 4.42 (1H, d, J1,2 = 8.2 Hz, H1), 4.35 (1H, dd, J6,6 = 10.5 Hz, J5,6 = 4.8 Hz, H6), 4.31 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.23 (1H, d, J60 ,60 = 12.4 Hz, H60 ), 4.18 (1H, d, J30 ,40 = 3.2 Hz, H40 ), 4.13–4.06 (2H, m, H200 , H300 ), 3.98–3.90 (3H, m, H20 , H6, CH@CH2(CH2)5CH2O), 3.86–3.79 (3H, m, H400 , H60 , OH), 3.78–3.72 (2H, m, H3, H30 ), 3.68 (1H, dd, J3,4 = 9.0 Hz, J4,5 = 9.0 Hz, H4), 3.63–3.58 (1H, m, CH@CH2(CH2)5CH2O), 3.44 (1H, dd, J1,2 = 8.2 Hz, J2,3 = 8.2 Hz, H2), 3.41–3.36 (1H, m, H5), 3.26 (1H, s, H50 ), 2.12–2.04 (2H, m, CH@CH2(CH2)5CH2O), 1.78–1.56 (2H, m, CH@CH2(CH2)5CH2O),
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
1.49–1.32 (6H, m, CH@CH2(CH2)5CH2O), 1.24 (d, 3H, J500 ,600 = 6.3 Hz, H600 ). 13C NMR (125 MHz): dC 139.0 (CH2@CH), 138.8 (Ph), 138.7 (Ph), 137.9 (Ph), 137.8 (Ph), 137.1 (Ph), 129.0 (Ph), 128.7 (Ph), 128.41 (Ph), 128.38 (Ph), 128.36 (Ph), 128.23 (2C, Ph), 128.19 (Ph), 128.1 (Ph), 127.8 (Ph), 127.54 (Ph), 127.46 (Ph), 127.4 (Ph), 126.9 (Ph), 126.1 (Ph), 114.3 (CH2@CH), 102.9 (C1), 101.7, 101.2, 100.9 (3C, PhCH, C10 ), 99.41 (C100 ), 79.9, 78.8, 78.0, 77.8, 77.1, 76.5, 75.5 (C20 , C200 , C3, C30 , C300 , C4, C40 ), 75.0 (PhCH2), 74.0 (PhCH2), 73.8 (C400 ), 72.7 (PhCH2), 70.7 (CH@CH2(CH2)5CH2O), 69.0, 68.5 (C6, C60 ), 66.8, 66.74, 66.70, 66.6 (C2, C5, C50 , C500 ), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.83 (CH@CH2(CH2)5CH2O), 28.80 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2 (CH2)5CH2O), 17.04 (C600 ). ESIMS: m/z Calcd [C61H71N3O14]Na+: 1092.4828. Found: 1092.4823. 3.12. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-2-O(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2deoxy-b-D-glucopyranoside (22) A solution of azide 21 (250 mg, 0.234 mmol) in dry pyridine (2 mL) was treated with CH3COSH (4 mL), and the solution was stirred (rt, 8d). The mixture was filtered, concentrated and subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford firstly unreacted azide 21 (55 mg, 22%). Further elution yielded the desired compound 22 contaminated with some acetylated alcohol. The resulting oil was taken up in MeOH (15 mL) and treated with a catalytic amount of NaOMe in MeOH, and the solution was stirred (rt, 12 h). The solution was neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford 22 (155 mg, 62%) as a colourless oil. [a] 22.2 (c 0.1, CH2Cl2); Rf 0.81 (CH2Cl2–MeOH, 9:1); 1 H NMR (500 MHz): dH 7.61–7.54 (4H, m, Ph), 7.39–7.15 (19H, m, Ph), 7.07–7.01 (2H, m, Ph), 5.85–5.76 (1H, m, CH2@CH), 5.58 (1H, s, PhCH), 5.56 (1H, s, PhCH), 5.11 (1H, d, J100 ,200 = 3.0 Hz, H100 ), 5.03–4.93 (3H, m, PhCH2, CH2@CH), 4.90 (1H, d, J1,2 = 8.2 Hz, H1), 4.79–4.67 (4H, m, PhCH2), 4.63 (1H, A of AB, J = 11.5 Hz, PhCH2), 4.39 (1H, d, J10 ,20 = 7.6 Hz, H10 ), 4.36 (1H, dd, J6,6 = 10.4 Hz, J5,6 = 4.9 Hz, H6), 4.28–4.20 (2H, m, H3, H40 ), 4.15–4.04 (4H, m, H200 , H300 , H500 , H60 ), 3.97–3.88 (3H, m, H20 , H60 , OH), 3.87–3.78 (2H, m, H6, CH@CH2(CH2)5CH2O), 3.70 (1H, dd, J3,4 = J4,5 = 9.2 Hz, H4), 3.66–3.62 (2H, m, H30 , H400 ), 3.41–3.53 (3H, m, H2, H5, CH@CH2(CH2)5CH2O), 3.16–3.14 (m, H50 ), 1.85 (3H, s, CH3C@O), 2.07–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.59–1.48 (2H, m, CH@CH2(CH2)5CH2O), 1.40–1.25 (6H, m, CH@CH2(CH2)5CH2O), 1.22 (d, 3H, J500 ,600 = 6.2 Hz, H600 ). 13C NMR (125 MHz): dC 170.6 (C@O). 139.0 (CH2@CH), 138.5 (Ph), 138.4 (Ph), 138.1 (Ph), 137.5 (Ph), 136.8 (Ph), 129.0 (Ph), 128.9 (Ph), 128.6 (Ph), 128.44 (Ph), 128.41 (Ph), 128.3 (Ph), 128.2 (Ph), 128.15 (Ph), 128.07 (Ph), 128.1 (Ph), 127.7 (Ph), 127.6 (Ph), 127.4 (Ph), 126.7 (Ph), 126.1 (Ph), 114.2 (CH2@CH), 102.82 (C10 ), 101.5, 101.3, 101.0 (3C, C1, PhCH), 99.0 (C100 ), 80.2, 79..0, 78.9, 77.5, 77.3, 77.2, 74.5, 72.4 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 ), 74.8, 73.1 (3C, PhCH2), 69.9, 69.1, 68.8 (C6, C60 , CH@CH2(CH2)5CH2O), 68.6, 66.3 (3C, C5, C50 , C500 ), 57.55 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2 (CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5 CH2O), 25.7 (CH@CH2(CH2)5CH2O), 23.1 (CH3C@O), 17.0 (C600 ). ESIMS: m/z Calcd [C63H75NO15]Na+: 1108.5029. Found: 1108.5029. 3.13. 7-Octyl 2-acetamido-2-deoxy-3-O-(2-O-(a-Lfucopyranosyl)-b-D-galactopyranosyl)-b-D-glucopyranoside (23) Octenyl glycoside 6 (82 mg, 0.128 mmol) was then taken up in THF–H2O (1:1, 10 mL), treated with Pd–C (10%, 15 mg) and stirred under a H2 atmosphere (48 h). The mixture was then filtered and concentrated to afford the fully deprotected trisaccharide 23 as a
2317
colourless oil (68 mg, 83%). [a] 15.1 (c 0.1, H2O); 1H NMR (500 MHz, D2O): dH 5.19 (1H, d, J100 ,200 = 4.0 Hz, H100 ), 4.65 (1H, d, J1,2 = 7.7 Hz, H1), 4.41 (1H, d, J10 ,20 = 8.8 Hz, H10 ), 4.32 (1H, q, J500 ,600 = 6.6 Hz, H500 ), 4.01–3.45 (17H, m, H2, H20 , H200 , H3, H30 , H300 , H4, H40 , H400 , H5, H50 , H6, H60 , CH3(CH2)6CH2O), 2.06 (3H, s, CH3C@O), 1.60–1.48 (2H, m, CH3(CH2)6CH2O), 1.37–1.26 (10H, m, CH3(CH2)6CH2O), 1.24 (3H, d, 3H, J500 ,600 = 6.6 Hz, H600 ), 0.87 (3H, t, J = 7.0 Hz, CH3(CH2)6CH2O). 13C NMR (125 MHz, D2O): dC 175.0 (C@O), 103.4, 101.7, 101.0 (C1, C10 , C100 ), 78.9, 78.2, 76.9, 76.6, 75.0, 73.3, 70.9, 70.7, 70.3, 69.6, 68.0 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C50 , C500 ), 72.1 (CH3(CH2)6CH2O), 62.7, 62.3 (C6, C60 ), 56.4 (C2), 32.7 (CH3(CH2)6CH2O), 30.1 (CH3(CH2)6CH2O), 30.0 (CH3(CH2)6CH2O), 29.9 (CH3(CH2)6CH2O), 26.6 (CH3(CH2)6CH2O), 23.5 (CH3(CH2)6CH2O), 16.8 (C600 ), 14.9 (CH3(CH2)6CH2O). ESIMS: m/z Calcd [C28H51NO15]Na+: 664.3151. Found: 664.3160.
3.14. 7-Octen-1-yl 2-azido-3-O-(3-O-(2-azido-2-deoxy-3,4,6tetra-O-acetyl-a-D-galactopyranosyl)-4,6-O-benzylidene-2-O(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2deoxy-b-D-glucopyranoside (25) A solution of acceptor 21 (621 mg, 0.58 mmol) and the trichloroacetimidate (823 mg, 1.74 mmol) in dry Et2O (15 mL) was treated with 4 Å molecular sieves (rt, 1 h). The mixture was cooled (20 °C) and treated with TMSOTf (10 lL, 0.058 mmol) and allowed to warm (0 °C). The mixture was treated with Et3N (200 lL), filtered, concentrated and subjected to flash chromatography (EtOAc–CH2Cl2 3:97) to afford tetrasaccharide 25 as a colourless oil (737 mg, 92%). [a] +8.9 (c 0.1, CH2Cl2); Rf 0.25 (EtOAc–hexanes 2:3); 1H NMR (500 MHz): dH 7.55–7.20 (25H, m, Ph), 5.87–5.77 (1H, m, CH2@CH), 5.53 (1H, d, J100 ,200 = 2.5 Hz, H100 ), 5.51 (1H, s, PhCH), 5.49 (1H, s, PhCH), 5.28 (1H, d, J1000 ,2000 = 3.2 Hz, H1000 ), 5.20 (1H, dd, J2000 ,3000 = 11.0 Hz, J3000 ,4000 = 2.9 Hz, H3000 ), 5.18–5.12 (2H, m, PhCH2, H4000 ), 5.04–4.93 (3H, m, PhCH2, CH2@CH), 4.90 (1H, A of AB, J = 11.9 Hz, PhCH2), 4.75 (2H, s, PhCH2), 4.67 (1H, d, J10 ,20 = 7.9 Hz, H10 ), 4.63 (1H, A of AB, J = 11.9 Hz, PhCH2), 4.52 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.46 (1H, d, J1,2 = 8.0 Hz, H1), 4.33 (1H, dd, J6,6 = 10.5 Hz, J5,6 = 4.7 Hz, H6), 4.28–4.25 (1H, m, H40 ), 4.22–4.12 (5H, m, H20 , H200 , H300 , H5000 , H60 ), 3.88 (1H, d, J60 ,60 = 12.4 Hz, H60 ), 3.84–3.74 (5H, m, H30 , H4, H400 , H6, H6000 ), 3.70 (1H, dd, J2,3 = J3,4 = 9.2 Hz, H3), 3.99– 3.92 (1H, m, CH@CH2(CH2)5CH2O), 3.65–3.60 (1H, m, CH@CH2(CH2)5CH2O), 3.55 (1H, dd, J2000 ,3000 = 11.0 Hz, J1000 ,2000 = 3.2 Hz, H2000 ), 3.50–3.37 (2H, m, H2, H5), 3.22 (1H, dd, J6000 ,6000 = 11.5 Hz, J5000 ,6000 = 3.5 Hz, H6000 ), 3.10–3.07 (1H, m, H50 ), 2.10–2.06 (2H, m, CH@CH2(CH2)5CH2O), 2.09 (3H, s, CH3C@O), 2.09 (3H, s, CH3C@O), 1.94 (3H, s, CH3C@O), 1.77–1.55 (2H, m, CH@CH2(CH2)5CH2O), 1.47–1.35 (6H, m, CH@CH2(CH2)5CH2O), 1.22 (3H, d, J500 ,600 = 6.3 Hz, H600 ). 13C NMR (125 MHz): dC 170.3 (C@O), 169.7 (C@O), 169.4 (C@O), 139.4 (Ph), 139.0 (Ph), 138.81 (Ph), 138.79 (CH2@CH), 137.6 (Ph), 137.0 (Ph), 129.0 (Ph), 128.7 (Ph), 128.3 (Ph), 128.24 (Ph), 128.16 (Ph), 128.1 (Ph), 128.0 (Ph), 127.45 (Ph), 127.42 (Ph), 127.37 (Ph), 127.3 (Ph), 127.2 (Ph), 126.2 (Ph), 126.1 (Ph), 114.3 (CH2@CH), 102.9 (C1), 101.4, 101.2, 100.7 (3C, C10 , PhCH), 97.9 (C100 ), 94.1 (C1000 ), 80.7 (C20 ), 79.7 (C3), 74.9 (PhCH2), 74.0 (PhCH2), 72.5 (PhCH2), 77.9, 77.8, 77.3, 76.0, 72.0 (C200 , C30 , C300 , C4, C400 ), 72.0 (C40 ), 70.8 (CH@CH2(CH2)5CH2O), 69.1, 68.6 (C6, C60 ), 68.8, 68.0 (C3000 ,C4000 ), 67.6 (C5000 ), 66.7, 66.4, 66.11, 66.09 (C2, C5, C50 , C500 ), 62.7 (C6000 ), 57.9 (C2000 ), 33.7 (CH@CH2 (CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.82 (CH@CH2(CH2)5 CH2O), 28.80 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 20.7 (CH3C@O), 20.62 (CH3C@O), 20.58 (CH3C@O), 16.9 (C600 ). ESIMS: m/z Calcd [C71H84N5O19]Na+: 1405.5738. Found: 1405.5740.
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3.15. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2deoxy-3,4,6-tetra-O-acetyl-a-D-galactopyranosyl)-4,6-Obenzylidene-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (26) A solution of tetrasaccharide 25 (355 mg, 0.257 mmol) in pyridine (2 mL) was treated with AcSH (4 mL), and the solution was stirred (14 d). The mixture was filtered, concentrated and subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford 26 as a colourless oil (270 mg, 74%). [a] 8.9 (c 0.2, CH2Cl2); Rf 0.76 (MeOH–CH2Cl2 1:9); 1H NMR (500 MHz): dH 7.55–7.23, 7.20–7.10 (25H, m, Ph). 6.78 (1H, d, J = 7.3 Hz, NH), 5.85–5.76 (1H, m, CH2@CH), 5.50 (1H, s, PhCH), 5.35 (1H, s, PhCH), 5.43 (1H, d, J3000 ,4000 = 2.1 Hz, H4000 ), 5.27 (1H, d, J100 ,200 = 4.1 Hz, H100 ), 5.20 (1H, d, J = 9.8 Hz, NH000 ), 5.05–4.92 (7H, m, H1000 , H3000 , PhCH2, CH2@CH), 4.72 (1H, A of AB, J = 11.6 Hz, PhCH2), 4.67 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.60 (1H, A of AB, J = 11.1 Hz, PhCH2), 4.54– 4.46 (3H, m, H1, H2000 , H5000 ), 4.38 (1H, d, J10 ,20 = 8.2 Hz, H10 ), 4.32 (1H, dd, J6,6 = 10.7 Hz, J5,6 = 4.8 Hz, H6), 4.13 (1H, q, J500 ,600 = 6.3 Hz, H500 ), 4.11–3.92, 3.89–3.74 (14H, 2m, H2, H20 , H200 , H3, H300 , H4, H40 , H400 , H6, H60 , H6000 , CH@CH2(CH2)5CH2O), 3.69 (1H, d, J60 ,60 = 12.6 Hz, H60 ), 3.62 (1H, dd, J20 ,30 = 9.6 Hz, J30 ,40 = 3.8 Hz, H30 ), 3.50–3.41 (2H, m, H5, CH@CH2(CH2)5CH2O), 2.76 (s, H50 ), 2.12 (3H, s, CH3C@O), 2.07–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.94 (3H, s, CH3C@O), 1.85 (3H, s, CH3C@O), 1.71 (3H, s, CH3C@O), 1.60–1.51 (2H, m, CH@CH2(CH2)5CH2O), 1.40–1.28 (2H, m, CH@CH2(CH2)5CH2O), 1.27 (3H, s, CH3C@O), 1.22 (3H, d, J500 ,600 = 6.3 Hz, H600 ). 13C NMR (125 MHz): dC 170.6 (C@O), 170.3 (C@O), 170.2 (C@O), 170.0 (C@O), 169.8 (C@O), 139.7 (Ph), 139.1 (CH2@CH), 138.7 (Ph), 138.6 (Ph), 137.7 (Ph), 137.3 (Ph), 129.5 (Ph), 129.4 (Ph), 128.4 (Ph), 128.3 (2C, Ph), 128.21(Ph), 128.19 (Ph), 128.0 (Ph), 127.9 (Ph), 127.7 (Ph), 127.6 (Ph), 127.3 (Ph), 127.1 (Ph), 126.6 (Ph), 126.2 (Ph), 114.2 (CH2@CH), 102.7, 102.5, 101.9, 101.5 (4C, C1, C10 , PhCH), 98.2, 92.3 (C100 , C1000 ), 80.9, 80.0, 77.9, 77.5, 77.4, 76.5 (C20 , C200 , C3, C300 , C4, C400 ), 75.0 (PhCH2), 74.1 (PhCH2), 73.4 (C30 ), 73.0 (PhCH2), 70.2 (C40 ), 69.7, 69.1, 69.0 (C6, C60 , CH@CH2(CH2)5CH2O), 68.7, 67.7, 67.3, 66.8, 66.6, 65.5 (C3000 , C4000 , C5, C50 , C500 , C5000 ), 62.4 (C6000 ), 55.2 (C2), 46.9 (C2000 ), 33.7 (CH@CH2(CH2)5CH2O), 29.4 (CH@CH2(CH2)5CH2O), 28.84 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 25.6 (CH@CH2(CH2)5CH2O), 22.9 (CH3C@O), 22.5 (CH3C@O), 20.69 (CH3C@O), 20.66 (CH3C@O), 20.6 (CH3C@O), 17.3 (C600 ). ESIMS: m/z Calcd [C77H94N2O23]Na+: 1437.6140. Found: 1437.6138. 3.16. 7-Octen-1-yl 2-acetamido-3-O-(3-O-(2-acetamido-2deoxy-a-D-galactopyranosyl)-4,6-O-benzylidene-2-O-(2,3,4-triO-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2-deoxy-bD-glucopyranoside (27) A solution of triacetate 26 (225 mg, 0.160 mmol) in CH3OH was treated with a catalytic amount of NaOCH3 in CH3OH, and the solution was stirred (2 h). The solution was neutralized with Amberlite IR 120 (H+) and filtered, and the residue was subjected to flash chromatography (EtOAc–CH2Cl2 1:1) to afford triol 27 as a colourless oil (192 mg, 94%). [a] 12.9 (c 0.3, CH3OH); Rf 0.55 (CH3OH–CH2Cl2 1:9); 1H NMR (500 MHz, CD3OD): dH 7.57–7.18 (25H, m, Ph), 5.85–5.73 (1H, m, CH2@CH), 5.60 (1H, s, PhCH), 5.52 (1H, s, PhCH), 5.29 (1H, d, J100 ,200 = 3.8 Hz, H100 ), 5.05 (1H, d, J1000 ,2000 = 3.3 Hz, H1000 ), 5.00–4.88 (4H, m, PhCH2, CH2@CH), 4.77 (1H, A of AB, J = 12.5 Hz, PhCH2), 4.69 (1H, A of AB, J = 10.5 Hz, PhCH2), 4.67 (1H, d, J1,2 = 6.7 Hz, H1), 4.83, 4.60 (2H, AB, J = 10.8 Hz, PhCH2), 4.49–4.35 (3H, m, H10 , H500 , H6), 4.33–4.06, 4.00–3.90, 3.87–3.79 (16H, 3 m, H2, H20 , H200 , H2000 , H3, H300 , H3000 , H40 , H400 , H4000 , H5000 , H6, H60 , CH@CH2(CH2)5CH2O), 3.73 (1H, dd, J3,4 = J4,5 = 9.2 Hz, H4), 3.58–3.53 (1H, m, H6000 ), 3.52–3.41 (3H, m, H3´, H5, CH@CH2(CH2)5CH2O), 3.28–3.22 (1H, m, H6000 ), 3.22–3.18 (1H, m,
H50 ), 2.06–1.99 (2H, m, CH@CH2(CH2)5CH2O), 1.85 (3H, s, CH3C@O), 1.60–1.50 (2H, m, CH@CH2(CH2)5CH2O), 1.48 (3H, s, CH3C@O), 1.41–1.28 (6H, m, CH@CH2(CH2)5CH2O), 1.28 (1H, d, J500 ,600 = 6.4 Hz, H600 ). 13C NMR (125 MHz): dC 172.5 (C@O), 171.6 (C@O), 139.2 (Ph), 138.8 (Ph), 138.7 (Ph), 138.6 (CH2@CH), 138.3 (Ph), 137.6 (Ph), 128.6 (Ph), 128.3 (Ph), 128.0 (Ph), 127.9 (Ph), 127.84 (2C, Ph), 128.78 (Ph), 127.7 (Ph), 127.2 (2C, Ph), 127.0 (Ph), 126.8 (Ph), 126.4 (Ph), 126.0 (2C, Ph), 113.4 (CH2@CH), 102.5, 101.3, 101.1, 100.9 (4C, C1, C10 , PhCH), 98.0 (C100 ), 91.8 (C1000 ), 80.1 (C4), 74.9 (PhCH2), 72.7 (PhCH2), 71.9 (PhCH2), 79.7, 77.7, 76.28, 76.27, 75.3, 73.5, 71.4, 70.5 (C20 , C200 , C3, C300 , C3000 , C40 , C400 , C4000 ), 69.4 (C30 ), 69.6, 68.8, 68.3 (C6, C60 , CH@CH2(CH2)5CH2O), 68.2, 66.8, 66.4, 66.0 (C5, C50 , C500 , C5000 ), 62.0 (C6000 ), 55.4 (C2), 49.7 (C2000 ), 33.4 (CH@CH2(CH2)5CH2O), 29.2 (CH@CH2(CH2)5CH2O), 28.7 (CH@CH2 (CH2)5CH2O), 28.5 (CH@CH2(CH2)5CH2O), 25.5 (CH@CH2(CH2)5 CH2O), 22.0 (CH3C@O), 21.0 (CH3C@O), 15.8 (C600 ). ESIMS: m/z Calcd [C71H88N2O20]Na+: 1311.5823. Found: 1311.5819. 3.17. 7-Octen-1-yl 2-acetamido-3-O-(4,6-O-benzylidene-3-O(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-2-O-(2,3,4-tri-Obenzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-2-deoxy-b-Dglucopyranoside (30) A solution of acceptor 21 (400 mg, 0.37 mmol) and trichloroacetimidate 2834 (770 mg, 1.12 mmol) in dry Et2O (15 mL) was treated with 4 Å molecular sieves (rt, 1 h). The mixture was cooled (20 °C) and treated with TMSOTf (10 lL, 0.058 mmol) and allowed to warm (0 °C). The mixture was treated with Et3N (200 lL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford tetrasaccharide 29 as a colourless oil (440 mg, 73%). A solution of tetrasaccharide (350 mg, 0.220 mmol) in dry pyridine (4 mL) was treated with AcSH (2 mL) and stirred (rt, 8 d). Concentration followed by flash chromatography (EtOAc–CH2Cl2 3:7) afforded firstly unreacted 29 (70 mg, 20%). Further elution afforded the desired 30 as a colourless oil (230 mg, 65%). [a] 16.0 (c 0.6, CH2Cl2); Rf 0.57 (EtOAc–hexanes 1:1); 1H NMR (500 MHz): dH 7.55–7.48 (4H, m, Ph), 7.38–7.12 (33H, m, Ph), 7.10–6.97 (8H, m, Ph), 6.79 (1H, d, J = 7.4 Hz, NH), 5.85–5.75 (1H, m, CH2@CH), 5.52 (1H, s, PhCH), 5.47 (1H, s, PhCH), 5.31 (1H, d, J1000 ,2000 = 4.0 Hz, H1000 ), 5.23 (1H, d, J100 ,200 = 3.5 Hz, H100 ), 5.02–4.92 (2H, m, CH2@CH), 4.88 (1H, A of AB, J = 11.8 Hz, PhCH2), 4.83 (1H, A of AB, J = 11.3 Hz, PhCH2), 4.68 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.61–4.51 (4H, m, H1, PhCH2), 4.49–4.29 (9H, m, H10 , H6, PhCH2), 4.23 (1H, A of AB, J = 11.8 Hz, PhCH2), 4.19 (1H, d, J30 ,40 = 3.3 Hz, H40 ), 4.15 (1H, dd, J200 ,300 = 10.0 Hz, J300 ,400 = 2.6 Hz, H300 ), 4.10–3.95 (5H, m, H20 , H200 , H3, H500 , H60 ), 3.92 (1H, dd, J2000 ,3000 = 10.0 Hz, J1000 ,2000 = 4.0 Hz, H2000 ), 3.88– 3.70 (9H, m, H2, H30 , H3000 , H400 , H4000 , H6, H60 , H6000 , CH@CH2 (CH2)5CH2O), 3.56–3.40 (4H, m, H4, H5, H6000 , CH@CH2(CH2)5CH2O), 3.30 (1H, d, J = 1.8 Hz, H5000 ), 2.80 (1H, s, H50 ), 2.08–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.72 (3H, s, CH3C@O), 1.61–1.49 (2H, m, CH@CH2(CH2)5CH2O), 1.41–1.23 (6H, m, CH@CH2(CH2)5CH2O), 1.16 (3H, d, J500 ,600 = 6.4 Hz, H600 ). 13C NMR (125 MHz): dC 170.4 (C@O), 139.1 (CH2@CH), 139.0 (Ph), 138.8 (Ph), 138.7 (Ph), 138.64 (Ph), 138.58 (Ph), 138.3 (Ph), 128.2 (Ph), 137.6 (Ph), 137.3 (Ph), 129.3 (Ph), 128.9 (Ph), 128.5 (Ph), 128.44 (Ph), 128.43 (2C, Ph), 128.40 (2C, Ph), 128.34 (Ph), 128.27 (Ph), 128.22 (Ph), 128.17 (Ph), 128.12 (Ph), 128.10 (Ph), 128.04 (Ph), 127.98 (Ph), 127.94 (Ph), 127.89 (Ph), 127.87 (Ph), 127.82 (Ph), 127.78 (2C, Ph), 127.67 (2C, Ph), 127.65 (Ph), 127.61 (Ph), 127.5 (Ph), 127.42 (Ph), 127.38 (Ph), 127.1 (Ph), 127.05 (2C, Ph), 127.02 (2C, Ph), 126.95 (Ph), 126.4 (Ph), 126.2 (Ph), 114.2 (CH2@CH), 102.9, 102.4, 101.9, 101.0 (4C, C1, C10 , PhCH), 97.6 (C1000 ), 92.4 (C100 ), 80.9, 79.5, 78.3, 77.8, 77.3, 76.5, 71.0, 69.5 (12C, C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5), 74.8 (PhCH2), 74.4 (PhCH2), 73.6 (2C, PhCH2), 72.82 (PhCH2), 72.78 (PhCH2), 71.6 (PhCH2), 69.9, 69.6, 69. 2, 69.1 (C6, C60 , C6000 , CH@CH2(CH2)5CH2O), 67.4, 66.6, 65.8 (C50 , C500 , C5000 ),
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
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55.5 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.84 (CH@CH2(CH2)5CH2O), 28.82 (CH@CH2(CH2)5CH2O), 25.6 (CH@CH2(CH2)5CH2O), 23.0 (CH3C@O), 17.1 (C600 ). ESIMS: m/z Calcd [C97H109NO20]Na+: 1630.7435. Found: 1630.7455.
65.8 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.85 (CH@CH2(CH2)5CH2O), 28.81 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C28H37N3O5]Na+: 518.2625. Found: 518.2621.
3.18. 7-Octen-1-yl 2-azido-3-O-benzyl-4,6-O-benzylidene-2deoxy-b-D-glucopyranoside (31)
3.20. 7-Octen-1-yl 4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxyb-D-glucopyranoside (33)
A stirred solution of alcohol 13 (7.11 g, 17.6 mmol) in dry DMF (50 mL) was cooled (10 °C), the solution was treated with BnBr (4.20 mL, 35.3 mmol) and NaH (60%, 960 mg, 24.0 mmol) and allowed to warm (rt, 1 h). The mixture was then treated with CH3OH (1 mL) and partially concentrated; the residue was taken up in EtOAc (250 mL) and washed with water and brine. The organic extract was dried, concentrated and then subjected to flash chromatography (EtOAc–hexanes 2:3) to afford benzyl ether 31 as a white crystalline solid (8.50 g, 98%). Mp 53–55 °C; [a] 70.8 (c 1.5, CH2Cl2); Rf 0.81 (EtOAc–hexanes 3:7). 1H NMR (500 MHz): dH 7.52–7.47 (2H, m, Ph), 7.43–7.28 (8H, m, Ph), 5.87–5.78 (1H, m, CH@CH2), 5.58 (1H, s, PhCH), 5.05–4.99 (1H, m, CH@CH2), 4.97–4.94 (1H, m, CH@CH2), 4.93, 4.81 (2H, AB, J = 11.3 Hz, PhCH2), 5.01 (1H, d, J1,2 = 8.2 Hz, H1), 4.35 (1H, dd, J6,6 = 10.7 Hz, J5,6 = 4.9 Hz, H6), 3.92 (1H, ddd, J = 9.4 Hz, 6.9 Hz, 6.9 Hz, CH@CH2(CH2)5CH2O), 3.81 (1H, dd, J6,6 = 10.7 Hz, J5,6 = 10.3 Hz, H6), 3.72 (1H, dd, J3,4 = 9.2 Hz, J4,5 = 9.2 Hz, H4), 3.61–3.52 (2H, m, H3, CH@CH2(CH2)5CH2O), 3.46 (1H, dd, J2,3 = 9.5 Hz, J1,2 = 8.2 Hz, H2), 3.39 (1H, ddd, J5,6 = 10.3 Hz, J4,5 = 9.2 Hz, J5,6 = 4.9 Hz, H5), 2.10–2.03 (2H, m, CH@CH2(CH2)5 CH2O), 1.72–1.61 (2H, m, CH@CH2(CH2)5CH2O), 1.46–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13C NMR (125.7 MHz): dC 139.0 (CH@CH2), 137.9 (Ph), 137.1 (Ph), 129.0 (Ph), 128.3 (Ph), 128.2 (Ph), 128.1 (Ph), 127.8 (Ph), 126.0 (Ph), 114.2 (CH@CH2), 102.6 (C1), 101.3 (PhCH), 81.5 (C4), 78.9 (C3), 74.8 (PhCH2), 70.6 (C6), 68.6 (CH@CH2(CH2)5CH2O), 66.3, 66.1 (C2, C5), 33.6 (CH@CH2(CH2)5 CH2O), 29.35 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 28.7 (CH@CH2(CH2)5CH2O), 25.7 (CH@CH2(CH2)5CH2O), ESIMS: m/z Calcd [C28H35N3O5]Na+: 516.2469. Found: 516.2467.
A solution of alcohol 32 (130 mg, 0.262 mmol) in pyridine (5 mL) was treated with Ac2O (1 mL) and DMAP (5 mg), and the solution was stirred (2 h). The excess Ac2O was quenched by the addition of CH3OH (2 mL), and the solution was concentrated. The residue was subjected to flash chromatography (EtOAc–hexanes 1:2) to afford 33 as a colourless oil (120 mg, 85%). [a] 18.3 (c 1.1, CH2Cl2); Rf 0.64 (EtOAc–hexanes 3:7). 1H NMR (400 MHz): dH 7.39–7.26 (10H, m, Ph), 5.89–5.77 (1H, m, CH@CH2), 5.05–4.91 (3H, m, H4, CH@CH2), 4.83, 4.63 (2H, AB, J = 10.1 Hz, PhCH2), 4.53 (2H, s, PhCH2), 4.31 (1H, d, J1,2 = 7.8 Hz, H1), 3.95 (1H, ddd, J = 9.4 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.60–3.37 (6H, m, H2, H3, H5, H6, CH@CH2(CH2)5CH2O), 2.11–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.86 (3H, s, CH3C@O), 1.72–1.63 (2H, m, CH@CH2(CH2)5CH2O), 1.49–1.30 (6H, m, CH@CH2(CH2)5CH2O); 13 C NMR (100.3 MHz): dC 167.12 (C@O), 136.5 (CH@CH2), 135.31 (Ph), 135.29 (Ph), 125.9 (Ph), 125.8 (Ph), 125.4 (Ph), 125.34 (Ph), 125.30 (Ph), 125.2 (Ph), 111.7 (CH@CH2), 99.6 (C1), 77.8 (C3), 72.3 (PhCH2), 71.1 (PhCH2), 71.0, 68.4 (C4, C5), 67.9, 67.1 (C6, CH@CH2(CH2)5CH2O), 63.5 (C2), 31.2 (CH@CH2(CH2)5CH2O), 27.0 (CH@CH2(CH2)5CH2O), 26.33 (CH@CH2(CH2)5CH2O), 26.30 (CH@CH2(CH2)5CH2O), 23.3 (CH@CH2(CH2)5CH2O), 18.3 (CH3C@O). ESIMS: m/z Calcd [C30H39N3O6]Na+: 560.2731. Found: 560.2733.
3.19. 7-Octen-1-yl 2-azido-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (32) A solution of benzylidene acetal 31 (9.5 g, 19.3 mmol) in CH2Cl2 (200 mL) was treated with 4 Å molecular sieves (5 g), and the mixture was stirred (rt, 1 h). The mixture was cooled (0 °C) and treated with Et3SiH (15.4 mL, 96.7 mmol) and CF3COOH (7.45 mL, 96.7 mmol), and the mixture was allowed to slowly warm (rt, 2 h). The mixture was then neutralized with Et3N (10 mL), filtered and then diluted with CH2Cl2 (500 mL). The organic extract was washed with water and brine, dried and then filtered. Concentration followed by flash chromatography (EtOAc–hexanes 1:2) afforded alcohol 32 as a colourless oil (7.95 g, 83%). [a] 27.3 (c 1.1, CH2Cl2); Rf 0.54 (EtOAc–hexanes 3:7). 1H NMR (500 MHz): dH 7.43–7.23 (10H, m, Ph), 5.87–5.77 (1H, m, CH@CH2), 5.03–4.98 (1H, m, CH@CH2), 4.96–4.93 (1H, m, CH@CH2), 4.92, 4.78 (2H, AB, J = 11.0 Hz, PhCH2), 4.61, 4.58 (2H, AB, J = 12.6 Hz, PhCH2), 4.29 (1H, d, J1,2 = 8.5 Hz, H1), 3.91 (1H, ddd, J = 9.4 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.77–3.70 (2H, m, H6), 3.63 (1H, ddd, J3,4 = 9.3 Hz, J4,4 = 9.3 Hz, J = 2.3 Hz, H4), 3.54 (1H, ddd, J = 9.4 Hz, 6.8 Hz, 6.8 Hz, CH@CH2(CH2)5CH2O), 3.44–3.39 (1H, m, H5), 3.38 (1H, dd, J2,3 = 10.1 Hz, J1,2 = 8.5 Hz, H2), 3.25 (1H, dd, J2,3 = 10.1 Hz, J3,4 = 9.3 Hz, H3), 2.67 (1H, d, J = 2.3 Hz, OH), 2.10– 2.02 (2H, m, CH@CH2(CH2)5CH2O), 1.69–1.61 (2H, m, CH@CH2(CH2)5CH2O), 1.45–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13 C NMR (125.7 MHz): dC 139.1 (CH@CH2), 138.2 (Ph), 137.7 (Ph), 128.6 (Ph), 128.5 (Ph), 128.1 (Ph), 128.0 (Ph), 127.8 (Ph), 127.7(Ph), 114.2 (CH@CH2), 102.2 (C1), 82.5 (C3), 75.0 (PhCH2), 73.9 (C5), 73.7 (PhCH2), 72.1 (C4), 70.3 (C6, CH@CH2(CH2)5CH2O),
3.21. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-b-Dgalactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (35) A solution of acceptor 32 (1.32 g, 2.67 mmol) in dry CH2Cl2 (20 mL) was stirred over 4 Å molecular sieves (1.5 g), and the mixture was stirred (rt, 1 h). The solution was then cooled (40 °C), treated with TMSOTf (0.2 mL), followed by dropwise addition of trichloroacetimidate 1532 (3.3 g, 6.7 mmol), and then the mixture was allowed to warm (0 °C). The mixture was neutralized with Et3N (2 mL), concentrated and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford a colourless oil that was immediately used in the next step. The colourless oil was taken up in CH3OH (100 mL), treated with a solution of NaOCH3 in CH3OH and stirred (rt, 3 h). The solution was neutralized with Amberlite IR 120 (H+), filtered and subjected to flash chromatography (EtOAc–hexanes 1:1) to afford diol 35 as a colourless oil (1.54 g, 78%). [a] 37.5 (c 0.8, CH2Cl2); Rf 0.28 (EtOAc–hexanes 1:1); 1H NMR (500 MHz): dH 7.51–7.44 (4H, m, Ph), 7.39–7.21 (11H, m, Ph), 5.86–5.78 (1H, m, CH2@CH), 5.47 (1H, s, PhCH), 5.06, 4.89 (2H, AB, J = 12.2 Hz, PhCH2), 5.04–4.99 (1H, m, CH2@CH), 4.97– 4.93 (1H, m, CH2@CH), 4.71, 4.57 (2H, AB, J = 12.2 Hz, PhCH2), 4.52 (1H, d, J10 ,20 = 7.8 Hz, H10 ), 4.41 (1H, d, J1,2 = 7.5 Hz, H1), 4.26 (1H, d, J60 ,60 = 12.5 Hz, H60 ), 4.06–4.00 (3H, m, H4, H40 , H6), 3.92 (1H, J = 9.4 Hz, 6.5 Hz, 6.5 Hz, CH@CH2(CH2)5CH2O), 3.83–3.76 (2H, m, H6, H60 ), 3.67–3.61 (1H, m, H20 ), 3.58–3.51 (1H, m, CH@CH2(CH2)5CH2O), 3.49–3.41 (5H, m, H2, H3, H30 , H5, OH), 2.94 (1H, s, H50 ), 2.50 (1H, d, J = 8.8, OH), 2.11–2.03 (2H, m, CH@CH2(CH2)5CH2O), 1.72–1.61 (2H, m, CH@CH2(CH2)5CH2O), 1.49–1.31 (6H, m, CH@CH2(CH2)5CH2O); 13C NMR (125.7 MHz): dC 139.1 (CH2@CH), 138.5 (Ph), 137.72 (Ph), 137.67 (Ph), 129.1 (Ph), 128.4 (Ph), 128.3 (Ph), 128.22 (Ph), 128.17 (Ph), 127.9 (Ph), 127.8 (Ph), 127.7 (Ph), 126.4 (Ph), 114.2 (CH2@CH), 103.3 (PhCH), 102.3 (C1), 101.3 (C10 ), 82.0 (C3), 77.4 (C4), 75.3 (PhCH2), 75.1, 74.5, 72.8, 72.4 (C20 , C30 , C40 , C5), 73.4 (PhCH2), 70.3
2320
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(CH@CH2(CH2)5CH2O), 68.9 (C60 ), 68.2 (C6), 66.7, 66.4 (C2, C50 ), 33.7, (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2 (CH2)5CH2O). ESIMS: m/z Calcd [C41H51N3O10]Na+: 768.3467. Found: 768.3466. 3.22. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-3-Opivaloyl-b-D-galactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (36) A solution of diol 35 (970 mg, 1.30 mmol) in dry pyridine (10 mL) was treated with trimethylacetyl chloride (230 lL, 1.8 mmol), and the solution was stirred (rt, 2 h). The solution was concentrated, and the residue subjected to flash chromatography (EtOAc–hexanes 1:3) to afford 36 as a colourless oil (949 mg, 88%). [a] 16.8 (c 0.4, CH2Cl2); Rf 0.59 (EtOAc–hexanes 3:7); 1H NMR (400 MHz): dH 7.49–7.23 (15H, m, Ph), 5.90–5.77 (1H, m, CH2@CH), 5.44 (1H, s, PhCH), 5.06–4.89 (4H, m, PhCH2, CH2@CH), 4.73, 4.58 (2H, AB, J = 12.2 Hz, PhCH2), 4.66 (1H, dd, J20 ,30 = 10.2 Hz, J30 ,40 = 3.7 Hz, H30 ), 4.62 (1H, d, J10 ,20 = 7.8 Hz, H10 ), 4.26 (1H, J1,2 = 7.8 Hz, H1), 4.20 (1H, d, J30 ,40 = 3.7 Hz, H40 ), 4.08–3.86 (5H, m, H20 , H4, H6, H60 , CH@CH2(CH2)5CH2O), 3.79 (1H, dd, J60 ,60 = 4.3 Hz, J50 ,60 = 2.0 Hz, H60 ), 3.76 (1H, dd, J6,6 = 5.3 Hz, J5,6 = 2.0 Hz, H6), 3.55 (1H, ddd, J = 9.4 Hz, J = 6.8 Hz, J = 6.8 Hz, CH@CH2(CH2)5CH2O), 3.48–3.40 (3H, m, H2, H3, H5), 3.28 (1H, br s, OH), 2.90 (1H, s, H50 ), 2.12–2.02 (2H, m, CH@CH2(CH2)5CH2O), 1.75–1.64 (2H, m, CH@CH2(CH2)5CH2O), 1.51–1.33 (6H, m, CH@CH2(CH2)5CH2O), 1.24 (9H, s, (CH3)3C) 13C NMR (100 MHz): dC 178.6 (C@O), 139.3 (CH2@CH), 138.8 (Ph), 138.1 (Ph), 137.7 (Ph), 128.9 (Ph), 128.7 (Ph), 128.5 (Ph), 128.4 (Ph), 128.2 (Ph), 128.1 (Ph), 127.6 (Ph), 127.5 (Ph), 126.2 (Ph), 114.5 (CH2@CH), 103.9 (C10 ), 102.6 (C1), 100.6 (PhCH), 82.5 (C3), 77.7 (C4), 75.3 (PhCH2), 74.6, 73.7, 73.4, 69.7 (C20 , C30 , C40 , C5), 73.9 (PhCH2), 70.5 (CH@CH2(CH2)5CH2O), 69.0, 68.7 (C6, C60 ), 66.8, 66.6 (C2, C50 ), 39.2 (CH3)3C), 33.9 (CH@CH2(CH2)5CH2O), 29.7 (CH@CH2 (CH2)5CH2O), 29.1 (CH@CH2(CH2)5CH2O), 29.0 (CH@CH2(CH2)5 CH2O), 27.3 (CH3)3C), 26.0 (CH@CH2(CH2)5CH2O). ESIMS: m/z Calcd [C46H59N3O11]Na+: 852.4042. Found: 852.4034. 3.23. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-3-Opivaloyl-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (37) A solution of alcohol 36 (920 mg, 1.11 mmol) in dry Et2O (10 mL) was treated with 4 Å molecular sieves (500 mg), and the mixture was stirred (rt, 1 h). The mixture was then cooled (10 °C), treated with TMSOTf (50 lL), followed by dropwise addition of trichloroacetimidate 1929 (1.87 g, 3.30 mmol) in dry Et2O (5 mL). The mixture was treated with Et3N (0.5 mL), filtered and subjected to flash chromatography (EtOAc–hexanes 1:3) to yield trisaccharide 37 as a colourless oil (1.30 g, 94%). [a] 66.7 (c 0.2, CH2Cl2); Rf 0.5 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.52–7.16 (30H, m, Ph), 5.88–5.79 (1H, m, CH2@CH), 5.45 (1H, s, PhCH), 5.41 (1H, d, J100 ,200 = 3.8 Hz, H100 ), 5.17 (1H, A of AB, J = 10.4 Hz, PhCH2), 5.05– 4.94 (3H, m, CH2@CH, PhCH2), 4.81 (1H, A of AB, J = 10.4 Hz, PhCH2), 4.80 (1H, dd, J20 ,30 = 10.2 Hz, J30 ,40 = 3.7 Hz, H30 ), 4.77–4.57 (6H, m, PhCH2), 4.44 (1H, d, J10 ,20 = 7.9 Hz, H10 ), 4.38 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.33 (1H, d, J30 ,40 = 4.0 Hz, H40 ), 4.28 (1H, dd, J60 ,60 = 12.4 Hz, J50 ,60 = 1.0 Hz, H60 ), 4.22–4.15 (3H, m, H1, H20 , H500 ), 4.13–4.05 (2H, m, H200 , H4), 3.96–3.90 (2H, m, H60 , CH@CH2(CH2)5CH2O), 3.83–3.76 (2H, m, H300 , H6), 3.68 (1H, d, J30 ,40 = 2.1 Hz, H400 ), 3.60–3.51 (2H, m, H6, CH@CH2(CH2)5CH2O), 3.39 (1H, dd, J2,3 = 9.8 Hz, J1,2 = 8.2 Hz, H2), 3.21 (1H, dd, J2,3 = 9.8 Hz, J2,3 = 9.0 Hz, H3), 3.13–3.09 (1H, m, H5), 3.02 (1H, s, H50 ), 2.11–2.04 (2H, m, CH@CH2(CH2)5CH2O), 1.74–1.63 (2H, m,
CH@CH2(CH2)5CH2O), 1.49–1.32 (6H, m, CH@CH2(CH2)5CH2O), 1.16 (3H, d, J500 ,600 = 6.5 Hz, H600 ), 1.13 (9H, s, (CH3)3C); 13C NMR (125.7 MHz): dC 178.0 (C@O), 139.1 (CH2@CH), 138.7 (Ph), 138.61 (Ph), 138.58 (Ph), 138.98 (Ph), 138.96 (Ph), 137.9 (Ph), 129.1 (Ph), 128.8 (Ph), 128.5 (Ph), 128.42 (Ph), 128.39 (Ph), 128.33 (Ph), 128.29 (Ph), 128.2 (Ph), 128.0 (Ph), 127.9 (Ph), 127.8 (Ph), 127.6 (Ph), 127.54 (Ph), 127.46 (Ph), 127.4 (Ph), 127.2 (Ph), 126.2 (Ph), 114.3 (CH2@CH), 102.1 (C1), 100.9, 100.7 (C10 , PhCH), 97.2 (C100 ), 81.2 (C3), 79.2 (C300 ), 77.5 (C50 ), 76.6, 75.7, 75.6, 75.3, 72.8, 71.3 (C20 , C200 , C30 , C4, C40 , C400 ), 75.8 (PhCH2), 74.8 (PhCH2), 73.4 (PhCH2), 73.2 (PhCH2), 72.5 (PhCH2), 70.2 (CH@CH2(CH2)5CH2O), 68.7, 67.8 (C6, C60 ), 66.6, 66.3, 65.8 (C2, C5, C500 ), 38.9 ((CH3)3C), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2 (CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 27.1 ((CH3)3C), 25.8 (CH@CH2(CH2)5CH2O), 16.7 (C600 ). ESIMS: m/z Calcd [C73H87N3O15]Na+: 1268.6029. Found: 1268.6027. 3.24. 7-Octen-1-yl 2-azido-4-O-(4,6-O-benzylidene-2-O-(2,3,4tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-3,6-diO-benzyl-2-deoxy-b-D-glucopyranoside (38) A solution of trisaccharide 37 (651 mg, 0.522 mmol) in CH3OH (50 mL) was treated with a catalytic amount of LiOCH3 (100 mg), and the solution heated at reflux (7 d). The solution was neutralized with Amberlite IR 120 (H+), filtered and subjected to flash chromatography (EtOAc–hexanes 1:2) to firstly afford unreacted 37 (60 mg, 9%). Further elution (EtOAc–hexanes 1:1) afforded alcohol 38 (503 mg, 83%) as a colourless oil. [a] 75.8 (c 0.3, CH2Cl2); Rf 0.19 (EtOAc–hexanes 3:7); 1H NMR (500 MHz): dH 7.57–7.53 (4H, m, Ph), 7.41–7.06 (26H, m, Ph), 5.87–5.77 (1H, m, CH2@CH), 5.59 (1H, s, PhCH), 5.14, 4.61 (2H, AB, J = 10.4 Hz, PhCH2), 5.03–4.92 (2H, m, CH2@CH), 5.01, 4.66 (2H, AB, J = 11.7 Hz, PhCH2), 4.95 (1H, d, J100 ,200 = 3.7 Hz, H100 ), 4.82, 4.74 (2H, AB, J = 11.3 Hz, PhCH2), 4.80, 4.72 (2H, AB, J = 12.0 Hz, PhCH2), 4.69, 4.40 (2H, AB, J = 11.3 Hz, PhCH2), 4.40 (1H, d, J10 ,20 = 8.1 Hz, H10 ), 4.32 (1H, d, J60 ,60 = 12.0 Hz, H60 ), 4.17–4.11 (3H, m, H1, H40 , OH), 4.08–4.03 (2H, m, H200 , H4), 3.99 (1H, dd, J60 ,60 = 12.0 Hz, J50 ,60 = 1.3 Hz, H60 ), 3.92–3.84 (4H, m, H5, H500 , H6, CH@CH2(CH2)5CH2O), 3.79 (1H, dd, J20 ,30 = 9.8 Hz, J10 ,20 = 8.1 Hz, H20 ), 3.64–3.55 (3H, m, H30 , H400 , H6), 3.49 (1H, ddd, J = 9.4 Hz, 6.8 Hz, 6.8 Hz, CH@CH2(CH2)5CH2O), 3.37 (1H, dd, J2,3 = 9.9 Hz, J1,2 = 8.2 Hz, H2), 3.27–3.19 (2H, m, H3, H300 ), 3.12 (1H, s, H50 ), 2.10–2.01 (2H, m, CH@CH2(CH2)5CH2O), 1.70–1.60 (2H, m, CH@CH2(CH2)5CH2O), 1.45–1.29 (6H, m, CH@CH2(CH2)5CH2O), 1.07 (3H, d, J500 ,600 = 6.5 Hz, H600 ); 13C NMR (125.7 MHz): dC 139.1 (CH2@CH), 138.6 (Ph), 138.5 (Ph), 138.13 (Ph), 138.11 (Ph), 138.08 (Ph), 137.3 (Ph), 129.2 (2C, Ph), 128.9 (Ph), 128.8 (2C, Ph), 128.4 (2C, Ph), 128.34 (Ph), 128.31 (Ph), 128.26 (Ph), 128.2 (Ph), 128.1 (2C, Ph), 127.74 (Ph), 127.65 (Ph), 127.5 (Ph), 126.5 (Ph), 114.2 (CH2@CH), 102.0 (C1), 101.4 (C10 ), 101.3 (PhCH), 99.6 (C100 ), 81.1 (C3), 79.0 (C5), 78.5 (C20 ), 77.5, 77.3, 75.9, 75.7, 75.0, 72.6 (C200 , C30 , C300 , C4, C4´, C400 ), 75.8 (PhCH2), 74.8 (PhCH2), 74.3 (PhCH2), 73.4 (PhCH2), 73.0 (PhCH2), 70.1 (CH@CH2(CH2)5CH2O), 69.0, 67.9 (C6, C60 ), 67.5, 66.6, 65.8 (C2, C50 , C500 ), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (CH@CH2(CH2)5CH2O), 28.8 (CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 16.8 (C600 ). ESIMS: m/z Calcd [C68H79N3O14]Na+: 1184.5454. Found: 1184.5441. 3.25. 7-Octen-1-yl 2-acetamido-4-O-(4,6-O-benzylidene-2-O(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)3,6-di-O-benzyl-2-deoxy-b-D-glucopyranoside (39) A solution of trisaccharide 38 (500 mg, 0.431 mmol) in dry pyridine (6 mL) was treated with AcSH (3 mL), and the solution was stirred (rt, 8 d). The mixture was concentrated and then subjected to flash chromatography (CH3OH–CH2Cl2 1:9) to afford 39
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
as a colourless oil (426 mg, 84%). [a] 54.5 (c 0.4, CH2Cl2); Rf 0.30 (EtOAc–hexanes 1:1); 1H NMR (500 MHz): dH 7.53–7.47 (4H, m, Ph), 7.40–7.10 (26H, m, Ph), 5.84–5.74 (1H, m, CH2@CH), 5.71 (1H, d, J = 7.1 Hz, NH), 5.55 (1H, s, PhCH), 5.03 (1H, d, J100 ,200 = 3.5 Hz, H100 ), 5.01–4.91 (4H, m, CH2@CH, PhCH2), 4.83, 4.71 (2H, AB, J = 11.6 Hz, PhCH2), 4.78 (1H, A of AB, J = 12.4 Hz, PhCH2), 4.76 (1H, d, J1,2 = 8.2 Hz, H1), 4.73 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.67–4.59 (3H, m, PhCH2), 4.43–4.39 (2H, m, H10 , PhCH2), 4.26 (1H, d, J60 ,60 = 12.0 Hz, H60 ), 4.14 (1H, d, J30 ,40 = 3.5 Hz, H40 ), 4.07 (1H, dd, J200 ,300 = 10.1, J100 ,200 = 3.5 Hz, H200 ), 4.03 (1H, dd, J3,4 = J4,5 7.5 Hz, H4) 4.00–3.92 (3H, m, H3, H500 , H60 ), 3.90 (1H, dd, J200 ,300 = 10.1 Hz, J300 ,400 = 2.6 Hz, H300 ) 3.86–3.75 (3H, m, H20 , H6, CH@CH2(CH2)5CH2O), 3.72 (1H, dd, J6,6 = 10.5 Hz, J5,6 = 3.3 Hz, H6), 3.66–3.60 (2H, m, H30 , H400 ) 3.56–3.46 (2H, m, H2, H5), 3.45–3.38 (1H, m, CH@CH2(CH2)5CH2O), 3.16 (1H, s, H50 ), 2.06–1.98 (2H, m, CH@CH2(CH2)5CH2O), 1.84 (3H, s, CH3CO), 1.59–1.49 (2H, m, CH@CH2(CH2)5CH2O), 1.41–1.23 (6H, m, CH@CH2(CH2)5CH2O), 1.13 (3H, d, J500 ,600 = 6.4 Hz, H600 ); 13C NMR (125.7 MHz): dC 170.1 (C@O), 139.1 (CH2@CH), 138.8 (Ph), 138.62 (Ph), 138.58 (Ph), 138.3 (Ph), 138.0 (Ph), 137.5 (Ph), 129.0 (Ph), 128.7 (Ph), 128.6 (Ph), 128.4 (Ph), 128.3 (Ph), 128.24 (Ph), 128.22 (Ph), 128.18 (Ph), 128.1 (Ph), 128.0 (Ph), 127.7 (Ph), 127.59 (Ph), 127.57 (Ph), 127.5 (Ph), 127.4 (Ph), 126.5 (Ph), 114.2 (CH2@CH), 101.3 (PhCH), 101.0 (C10 ), 99.85, 99.78 (C1, C100 ), 79.1 (C300 ), 78.8 (C20 ), 77.6, 77.4 (C200 , C400 ), 76.1, 75.6, 72.9 (4C, C3, C30 , C4, C500 ), 74.8 (PhCH2), 74.7 (C40 ), 74.1 (2C, PhCH2), 73.4 (PhCH2), 72.8 (PhCH2), 69.4, 69.0, 68.8 (C6, C60 , CH@CH2(CH2)5CH2O), 67.6, 66.5 (C5, C50 ), 55.0 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (2C, CH@CH2(CH2)5CH2O), 25.8 (CH@CH2(CH2)5CH2O), 23.4 (CH3CO), 16.8 (C600 ). ESIMS: m/z Calcd [C70H83NO15]Na+: 1200.5655. Found: 1200.5661. 3.26. 7-Octyl 2-acetamido-4-O-(2-O-(a-L-fucopyranosyl)-b-Dgalactopyranosyl)-2-deoxy-b-D-glucopyranoside (40) A solution of trisaccharide 7 (145 mg, 0.227 mmol) was taken up in CH3OH, treated with Pd–C (10%, 20 mg) and subjected to a H2 atmosphere (rt, 1 d). The mixture was then filtered and concentrated to afford octyl glycoside 40 as a colourless oil (140 mg, 96%). [a] 106.1 (c 0.5, CH3OH); 1H NMR (500 MHz, CD3OD): dH 5.21 (1H, d, J100 ,200 = 3.2 Hz, H100 ), 4.48 (1H, d, J10 ,20 = 7.1 Hz, H10 ), 4.37 (1H, d, J1,2 = 8.4 Hz, H1), 4.17 (1H, q, J500 ,600 = 6.5 Hz, H500 ), 3.93–3.80, 3.79– 3.53, 3.34–3.30 (16H, 3 m, H2, H20 , H200 , H3, H30 , H300 , H4, H40 , H400 , H5, H50 , H6, H60 , CH3(CH2)6CH2O), 3.48–3.41 (1H, m, CH3(CH2)6CH2O), 1.96 (3H, s, CH3CO), 1.57–1.48 (2H, m, CH3(CH2)6CH2O)), 1.37–1.24 (10H, m, CH3(CH2)6CH2O), 1.20 (3H, d, J500 ,600 = 6.5 Hz, H600 ), 0.89 (3H, t, J = 6.9 Hz, CH3(CH2)6CH2O). 13C NMR (125.7 MHz, CD3OD): dC 173.4 (C@O), 102.9, 102.6 (C1, C10 ), 101.9 (C100 ), 79.1, 78.3, 77.1, 77.0, 75.3, 74.1, 73.6, 71.7, 70.8, 70.70 (C20 , C200 , C3, C30 , C300 , C4, C40 , C400 , C5, C500 ), 70.7 (CH3(CH2)6CH2O), 68.3 (C50 ), 62.6, 61.7 (C6, C60 ), 56.8 (C2), 33.0 (CH3(CH2)6CH2O), 30.7 (CH3(CH2)6CH2O), 30.5 (2C, CH3(CH2)6CH2O), 27.2 (CH3(CH2)6CH2O), 23.7 (CH3(CH2)6CH2O), 23.0 (CH3CO), 16.8 (C600 ), 14.4 (CH3(CH2)6CH2O). ESIMS: m/z Calcd [C28H51NO15]Na+: 664.3151. Found: 664.3156. 3.27. 7-Octen-1-yl 2-acetamido-4-O-(4,6-O-benzylidene-3-O(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-2-O-(2,3,4-tri-Obenzyl-a-L-fucopyranosyl)-b-D-galactopyranosyl)-3,6-di-Obenzyl-2-deoxy-b-D-glucopyranoside (42) A solution of acceptor 38 (1.22 g, 1.05 mmol) in dry Et2O (40 mL) was treated with 4 Å molecular sieves (1 g), and the mixture was stirred (rt, 1 h). The mixture was then cooled (10 °C) and treated with TMSOTf (50 lL, 0.29 mmol); trichloroacetimidate
2321
2834 (1.49 g, 3.15 mmol) in dry Et2O (15 mL) was then added dropwise, and the mixture was allowed to stand (20 min). The mixture was neutralized with Et3N (0.5 mL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:4) to afford the nearly pure tetrasaccharide 41 (1.33 g, 75%) as a colourless oil. A solution of tetrasaccharide 41 in dry pyridine (6 mL) was treated with AcSH (3 mL), and the solution was stirred (rt, 2 d). The solution was then concentrated and subjected to flash chromatography (CH3OH–CH2Cl2 1:9) to afford 42 (510 mg, 91%) as a colourless oil. [a] 7.8 (c 0.4, CH2Cl2); Rf 0.50 (CH3OH–CH2Cl2 1:20); 1H NMR (500 MHz): dH 7.42–7.09 (48H, m, Ph), 7.03–6.98 (2H, m, Ph), 5.87–5.76 (2H, m, CH2@CH, NH), 5.62 (1H, d, J100 ,200 = 3.6 Hz, H100 ), 5.38 (1H, s, PhCH), 5.36 (1H, d, J1000 ,2000 = 3.6 Hz, H1000 ), 5.02–4.97 (2H, m, CH2@CH, PhCH2), 4.96–4.92 (2H, m, CH2@CH, PhCH2), 4.87–4.79 (4H, m, H1, PhCH2), 4.72 (1H, A of AB, J = 12.3 Hz, PhCH2), 4.66–4.57 (6H, m, PhCH2), 4.52 (1H, A of AB, J = 12.0 Hz, PhCH2), 4.47–4.30 (9H, m, H10 , H500 , H60 , PhCH2), 4.18–4.12 (2H, m, H20 , H40 ), 4.08–3.91 (6H, m, H200 , H2000 , H3, H300 , H4, H6), 3.87– 3.76 (5H, m, H30 , H3000 , H5000 , H60 , CH@CH2(CH2)5CH2O), 3.71 (1H, s, H400 ), 3.59–3.54 (1H, m, H5), 3.53–3.40 (5H, m, H2, H4000 , H6, H6000 , CH@CH2(CH2)5CH2O), 3.16–3.10 (1H, m, H6000 ), 2.92 (1H, s, H50 ), 2.08–2.00 (2H, m, CH@CH2(CH2)5CH2O), 1.84 (3H, s, CH3CO), 1.63–1.53 (2H, m, CH@CH2(CH2)5CH2O), 1.41–1.23 (9H, m, H600 , CH@CH2(CH2)5CH2O). 13C NMR (125.7 MHz): dC 170.1 (C@O), 139.2 (Ph), 139.04 (CH2@CH), 138.99 (Ph), 138.96 (Ph), 138.76 (Ph), 138.72 (Ph), 138.66 (Ph), 138.5 (Ph), 138.4 (Ph), 138.3 (Ph), 137.8 (Ph), 128.8 (Ph), 128,6 (Ph), 128.4 (Ph), 128.34 (Ph), 128.32 (Ph), 128.25 (Ph), 128.21 (Ph), 128.18 (Ph), 128.08 (Ph), 128.06 (Ph), 127.98 (Ph), 127.65 (Ph), 127.59 (Ph), 127.5 (Ph), 127.45 (Ph), 127.40 (Ph), 127.37 (Ph), 127.24 (Ph), 127.15 (Ph), 127.07 (Ph), 126.3 (Ph), 114.2 (CH2@CH), 101.5, 101.1 (PhCH, C10 ), 99.8 (C1), 97.8 (C100 ), 92.8 (C1000 ), 80.0, 78.1, 78.0, 77.2, 76.32, 76.26, 76.1, 75.5, 75.3, 72.4, 72.2, 70.4 (13C, C20 , C200 , C2000 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C5000 ), 75.0 (PhCH2), 74.5 (PhCH2), 74.2 (PhCH2), 73.5 (PhCH2), 73.4 (PhCH2), 73.2 (PhCH2), 72.8 (PhCH2), 72.3 (PhCH2), 71.3 (PhCH2), 69.8, 69.5, 69.1, 69.0 (C6, C60 , C6000 , CH@CH2(CH2)5CH2O), 66.5 (C500 ), 66.2 (C50 ), 54.9 (C2), 33.7 (CH@CH2(CH2)5CH2O), 29.6 (CH@CH2(CH2)5CH2O), 28.91 (CH@CH2 (CH2)5CH2O), 28.89 (CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5 CH2O), 23.5 (CH3CO), 16.9 (C600 ). ESIMS: m/z Calcd [C104H117NO20]Na+: 1722.8061. Found: 1722.8063.
3.28. 7-Octen-1-yl 2-acetamido-4-O-(3-O-(2-acetamido-2deoxy-3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-4,6-Obenzylidene-2-O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-b-Dgalactopyranosyl)-3,6-di-O-benzyl-2-deoxy-b-Dglucopyranoside (44) A solution of acceptor 38 (1.17 g, 1.01 mmol) in dry Et2O (45 mL) was treated with 4Å molecular sieves (1 g) and the mixture was stirred (rt, 1 h). The mixture was then cooled (10 °C), treated with TMSOTf (50 lL, 0.29 mmol); trichloroacetimidate 2435 (1.43 g, 0.965 mmol) in dry Et2O (15 mL) was then added dropwise and the mixture was allowed to stand (20 min). The mixture was neutralized with Et3N (0.5 mL), filtered, concentrated and subjected to flash chromatography (EtOAc–hexanes 1:3) to afford the nearly pure tetrasaccharide as a colourless oil (1.21 g, 94%). The residue was taken up in pyridine (6 mL) and treated with AcSH (3 mL) and the solution was stirred (7 d). The solution was concentrated and subjected to flash chromatography (CH2Cl2–CH3OH, 10:1) to afford 44 as a colourless oil (530 mg, 76%). Rf 0.65 (CH3OH–CH2Cl2 1:9); 1H NMR (500 MHz): dH 7.44–7.18 (30H, m, Ph), 5.85–5.73 (2H, m, CH2@CH, NH), 5.45 (1H, d, J100 ,200 = 3.9 Hz, H100 ), 5.41 (1H, s, PhCH), 5.38 (1H, J = 9.8 Hz, NH), 5.20 (1H, A of AB, J = 12.3 Hz, PhCH2), 5.12 (1H, A of AB, J = 11.1 Hz, PhCH2),
2322
P. J. Meloncelli, T. L. Lowary / Carbohydrate Research 345 (2010) 2305–2322
5.09 (1H, d, J1000 ,2000 = 3.7 Hz, H1000 ), 5.04–4.90 (7H, m, H1, H3000 , H4000 , CH2@CH, PhCH2), 4.71–4.55 (8H, m, H10 , H2000 , PhCH2), 4.45–4.38 (1H, m, H500 ), 4.32–4.06 (7H, m, H20 , H200 , H3, H4, H40 , H5000 , H60 ), 4.01–3.97 (1H, m, H60 ), 3.94–3.81 (4H, m, H30 , H300 , H6, CH@CH2(CH2)5CH2O), 3.80–3.72 (2H, m, H400 , H6), 3.67 (1H, dd, J6000 ,6000 = 11.5 Hz, J5000 ,6000 = 7.6 Hz, H6000 ), 3.52–3.41 (2H, m, H5, CH@CH2(CH2)5CH2O), 3.33–3.26 (1H, m, H2), 3.20 (1H, s, H50 ), 3.09 (1H, dd, J6000 ,6000 = 11.5 Hz, J5000 ,6000 = 3.7 Hz, H6000 ), 2.10–1.99 (5H, m, CH3CO, CH@CH2(CH2)5CH2O), 1.94 (3H, s, CH3CO), 1.87 (3H, s, CH3CO), 1.42 (3H, s, CH3CO), 1.64–1.55 (2H, m, CH@CH2 (CH2)5CH2O), 1.41–1.24 (12H, m, CH3CO, H600 , CH@CH2(CH2)5 CH2O). 13C NMR (125.7 MHz): dC 170.34 (2C, C@O), 170.32 (C@O), 170.1 (C@O), 170.0 (C@O), 139.4 (Ph), 139.0 (CH2@CH), 138.7 (Ph), 138.70 (Ph), 138.67 (Ph), 128.3 (Ph), 138.2 (Ph), 137.6 (Ph), 129.1 (Ph), 128.8 (Ph), 128.4 (Ph), 128.31 (Ph), 128.27 (Ph), 128.2 (Ph), 128.1 (Ph), 128.04 (Ph), 128.02 (Ph), 127.8 (Ph), 127.61 (Ph), 127.57 (Ph), 127.51 (Ph), 127.47 (Ph), 127.4 (Ph), 127.31 (Ph), 126.5 (Ph), 126.2 (Ph), 114.3 (CH2@CH), 101.2, 101.0 (PhCH, C10 ), 99.7 (C1), 98.6 (C100 ), 92.1 (C1000 ), 80.1, 77.7, 77.5, 77.0, 75.8, 75.3, 71.8, 70.4, 69.7, 69.0, 67.0, 66.2 (13C, C20 , C200 , C3, C30 , C300 , C3000 , C4, C40 , C400 , C4000 , C5, C500 , C5000 ), 74.9 (PhCH2), 73.7 (PhCH2), 73.4 (PhCH2), 73.3 (PhCH2), 72.1 (PhCH2), 68.7, 68.4, 67.4 (C6, C60 , CH@CH2(CH2)5CH2O), 67.7 (C50 ), 63.0 (C6000 ), 56.6 (C2), 46.5 (C2000 ), 33.7 (CH@CH2(CH2)5CH2O), 29.5 (CH@CH2(CH2)5CH2O), 28.9 (2C, CH@CH2(CH2)5CH2O), 25.9 (CH@CH2(CH2)5CH2O), 23.5 (CH3CO), 22.6 (CH3CO), 20.69 (CH3CO), 20.66 (CH3CO), 20.65 (CH3CO), 16.8 (C600 ). ESIMS: m/z Calcd [C84H102NO23]Na+: 1529.6766. Found: 1529.6744. Acknowledgements Funding was provided by a Canadian Institutes of Health Research (CIHR) team grant (RMF 92091), a Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR) CHRP grant (CHRPJ35094608), and the Alberta Ingenuity Centre for Carbohydrate Science. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.carres.2010.08.012.
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