β-Mannosidase and β-hexosaminidase inhibitors: synthesis of 1,2-bis-epi-valienamine and 1-epi-2-acetamido-2-deoxy-valienamine from d-mannose

DOI: 10.1016/j.tetasy.2009.02.016

Source and publish data:

Tetrahedron Asymmetry p. 795 - 807 (2009)

Update date:2022-08-02

Topics:

Authors:

Ramstadius, Clinton

Hekmat, Omid

Eriksson, Lars

Stalbrand, Henrik

Cumpstey, Ian

Read Full Text PDF DownLoad Join now for total 90,000,000 free articles

Article abstract of DOI:10.1016/j.tetasy.2009.02.016

A partially protected C-5{double bond, long}C-5a unsaturated carbasugar with α-lyxo configuration is synthesised in five steps and 26% overall yield from a known mannose-derived hemiacetal, using ring-closing metathesis as a key step. This carbasugar is c

Full text of DOI:10.1016/j.tetasy.2009.02.016

Tetrahedron: Asymmetry 20 (2009) 795–807

Contents lists available at ScienceDirect

Tetrahedron: Asymmetry

journal homepage: w ww.elsevier.com/locate/tetasy

b-Mannosidase and b-hexosaminidase inhibitors: synthesis

of 1,2-bis-epi-valienamine and 1-epi-2-acetamido-2-deoxy-

valienamine from ^D-mannose

Clinton Ramstadius ^a, Omid Hekmat ^b, Lars Eriksson ^c, Henrik Stålbrand ^b, Ian Cumpstey ^a,

^aDepartment of Organic Chemistry, Stockholm University, Arrhenius Laboratory, 106 91 Stockholm, Sweden

^bDepartment of Biochemistry, Lund University, Centre for Chemistry and Chemical Engineering, Box 124, 221 00 Lund, Sweden

^cDivision of Structural Chemistry, Stockholm University, Arrhenius Laboratory, 106 91 Stockholm, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history:

A partially protected C-5@C-5a unsaturated carbasugar with

steps and 26% overall yield from a known mannose-derived hemiacetal, using ring-closing metathesis

as a key step. This carbasugar is converted into valienamine derivatives with b-lyxo (i.e., corresponding

-lyxo conﬁguration is synthesised in ﬁve

Received 2 February 2009

Accepted 11 February 2009

Available online 22 April 2009

to b-manno at C-1–C-4),

a-lyxo (i.e., corresponding to a-manno at C-1–C-4) and b-2-acetamido-2-

deoxy-xylo (i.e., corresponding to b-GlcNAc at C-1–C-4) conﬁgurations. This is the ﬁrst report of the syn-

thesis of the b-lyxo compound, 1,2-bis-epi-valienamine, which was found to inhibit Cellulomonas ﬁmi b-

mannosidase (CfMan2A) with K_i140 lM. We report the crystal structures of three protected C-5@C-5a

Special edition of Tetrahedron: Asymmetry

in honour of Professor George Fleet’s 65th

birthday

unsaturated carbasugars with lyxo conﬁguration.

1. Introduction

NH₂

Valienamine 1 (Fig. 1) is an unsaturated carbahexopyranose

that has the same conﬁguration at C-1–C-4 as -glucose (i.e., a-

xylo conﬁguration), occurring in nature as a component of larger

Valienamine 1

1,2-bis-epi-valienamine

structures, for example, acarbose^1aor the validomycins.^1bIt has

moderate activity as an inhibitor of

a-glucosidases (IC₅₀18 lM–

1 mM),²while larger oligosaccharide analogues containing an N-

substituted valienamine moiety may have higher inhibitory activity

(e.g., acarbose, K_i260 nM against sucrase¹). Some epimers of valien-

amine as well as N-substituted variants have been synthesised in at-

tempts to inhibit other glycosidases, with varying degrees of

success.³For example, N-octyl 1-epi-valienamine has been shown

to inhibit b-glucocerebrosidase (IC₅₀30 nM),⁴N-octyl 1,4-bis-epi-

valienamine has been shown to inhibit bovine liver b-galactosidase

(IC₅₀870 nM)⁵and recently, 2-acetamido-2-deoxy-1-epi-valien-

amine (i.e., corresponding to b-GlcNAc conﬁguration at C-1–C-4)

has been shown to inhibit various b-hexosaminidases.⁶

α-glucoside

-mannoside

Figure 1.

Glycosidases have been classiﬁed into families based on primary

sequence and structural similarities.^àb-Mannosidases are retaining

exo-glycosidases that hydrolyse b-mannosidic linkages, and are

classiﬁed as belonging to glycosidase families 1, 2 or 5. Some microbial

b-mannosidases^7,8are part of the hydrolytic enzyme systems that

hydrolyse mannans and heteromannans, major components of

hemicellulose.⁹The active site architecture may vary among b-man-

nosidases, as shown by the two high-resolution crystal structures that

* Corresponding author. Tel.: +46 (0)8 16 2481; fax: +46 (0)8 15 4908.

E-mail address: cumpstey@organ.su.se (I. Cumpstey).

Valienamine and other carbocyclic derivatives are treated as carbasugars and

numbered as such (see IUPAC recommendations, Carbohydr. Res. 1997, 297, 1–90; Ref.

3; Yamagishi, K.; Ogawa, S. Liebigs Ann. 1995, 279–284); numbering is shown in

Figure 1. Hence, the carbocyclic derivatives with C-1–C-4 conﬁgurational match with

-mannose have the a-lyxo conﬁguration; C-1–C-4 conﬁgurational match with b-

mannose have the b-lyxo conﬁguration (e.g., 1,2-bis-epi-valienamine 2); C-1–C-4

conﬁgurational match with b-glucose have the b-xylo conﬁguration (e.g., 2-acetam-

ido-2-deoxy-1-epi-valienamine 27); Open-chain derivatives are numbered as shown

in Scheme 1.

Carbohydrate Active Enzymes (CAZy) database (http://www.cazy.org/). See:

Coutinho, P. M.; Henrissat, B. In Recent Advances in Carbohydrate Bioengineering,

Gilbert, H. J., Davies, G., Henrissat, B., Svensson, B., Eds.; The Royal Society of

Chemistry: Cambridge, 1999, pp 3–12.

doi:10.1016/j.tetasy.2009.02.016

796

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

have been published.¹⁰The family 2 b-mannosidase of the human gut

bacterium Bacteroides thetaiotaomicron has an active site formed by

several protein domains,^10awhereas the family 5 b-mannosidase of

the soil bacterium Cellvibrio mixtus has an active site within a single

domain,^10bas do endo-b-1,4-mannanases of the same family.¹¹To fur-

ther investigate catalytic mechanisms and enzyme–carbohydrate

interactions of b-mannosidases,^7,8we became interested in potential

inhibitors of these enzymes.¹²It has been proposed that several b-

mannoside hydrolases of families 2, 5 and 26 operate with a boat-like

(B_2,5) substrate conformation at the transition state,¹³a suggestion

that has been further strengthened by very recent work with a number

of monosaccharide-mimicking inhibitors.¹⁴Of the various b-mannos-

idase inhibitors that have been reported in the literature, some have

been described as transition-state analogues where a six-membered

ring has a ﬁxed or low-energy conformation resembling a postulated

transition state in the cleavage of a b-mannoside linkage,¹⁵while for

others (e.g., polyhydroxylated pyrrolidines), the resemblance to

b-mannose is less obvious.¹⁶We undertook to synthesise the as yet

unknown 1,2-bis-epi-valienamine 2 (Fig. 1; i.e., corresponding to a

b-manno conﬁguration at C-1–C-4, whose potential biological rele-

vance has been noted³) and test for b-mannosidase inhibition.

Conversion of carbohydrates to unsaturated carbocycles has

been described before using, for example, an intramolecular Wads-

worth–Emmons reaction¹⁷or ring-closing metathesis.^18,19We

planned the synthesis around a ring-closing metathesis reaction

that would form the cyclohexene with the trisubstituted C-5@C-

5a double bond. The required polyhydroxylated diene should be

accessible from mannose, which necessarily has the required con-

ﬁguration at C-2–C-4. Introduction of the two alkenes could then

be achieved by vinyl Grignard addition and Wittig methylenation;

related approaches to carbocyclic carbohydrate derivatives have

been reported before.

2. Results and discussion

Hemiacetal 3 is synthesised from mannose in six steps on a

multigram scale without the need for chromatography, following

literature procedures.²³Vinyl Grignard addition gave the triols

4a,b as an inseparable diastereomeric mixture (4a:4b, 3:1, Scheme

1). Protection with an isopropylidene acetal (2,2-dimethoxypro-

pane, DMF, CSA, rt; heating to 70 °C did not change the product dis-

tribution) gave the required ﬁve-ring compound 5a as the major

component of an inseparable mixture (5a:5b:6, 3.8:1.2:1).²¹Oxida-

tion to the ketones 7a,b was followed by Wittig methylenation to

give the inseparable dienes 8a,b. Before attempting metathesis,

the isopropylidene protection was removed to give diastereomers

9a and 9b, which could now easily be separated by ﬂash

chromatography.

Treatment of diene 9a with the Grubbs’ second generation

ruthenium complex gave variable yields (22–76%) of carbocycle

10 along with unidentiﬁed by-products. The diol 9a was pro-

tected as a diacetate 11, which was ring-closed by Grubbs’ second

generation complex in toluene at 60 °C to give carbocycle 12 in

consistently good yield (82%, Scheme 2). The Hoveyda–Grubbs

second generation ruthenium complex gave good cyclisation re-

sults with both the diacetate (11?12; 87–96%) and the diol

(9a?10; 81%) under the same conditions. The minor diastereo-

mer 9b was protected as its diacetate 13, and treatment with

Hoveyda–Grubbs second generation complex in toluene at 60 °C

gave the carbocycle 14 (Scheme 2). The acetates were removed

from 12 and 14 by methanolysis to give the respective carbocyclic

diols 10 and 15.

To reach 1,2-bis-epi-valienamine 2 from the carbocyclic diol 10,

it was necessary to introduce nitrogen at C-1 with inversion of con-

ﬁguration. We ﬁrst attempted this under Mitsunobu conditions,²⁴

but treatment with triphenylphosphine, DIAD and phthalimide in

THF gave no nitrogen substitution, and the epoxide 16 was identi-

ﬁed as the major product (42%). Neither did a different nitrogen

source, diphenylphosphoryl azide (DPPA) give any substitution,

but a better yield of the epoxide 16 (71%, Scheme 3) was obtained.

Starting from a partially protected mannose hemiacetal 3 with

OH-2 free has several potential beneﬁts:^19jﬁrstly, we may expect

a diastereoselective Grignard addition arising from a chelate ef-

fect;²⁰secondly, the product of Grignard addition would contain

a 1,2-diol that it should be possible to selectively protect leaving

OH-5 free²¹and ﬁnally, it should be possible to access GlcNAc

valienamine analogues^6,22by S_N2 substitution of OH-2 of the car-

bocyclic product with a nitrogen nucleophile.

Epoxide opening with azide gave the known

a-lyxo conﬁgured

derivative 17, which has been converted into 2-epi-valienamine

and its peracetate 18 by Shing.²⁵

BnO

OBn

5a,b

BnO

^1aOH

BnO

(iii)

(i)

(ii)

BnO

OBn

BnO

OBn

7a,b

OBn

4a,b

BnO

OBn

(iv)

BnO

(v)

OBn

8a,b

Scheme 1. Reagents and conditions: (i) vinylmagnesium bromide, THF, 70 °C, 82% (4a:4b, 3:1); (ii) 2,2-dimethoxypropane, DMF, CSA, 88% (5a:5b:6, 3.8:1.2:1); (iii) oxalyl

chloride, DMSO, CH₂Cl₂, ꢀ78 °C, then Et₃N, ꢀ78 °C?rt, 81% (7a:7b, 3.3:1); (iv) ^tBuOK, Ph₃PCH₃Br, toluene, 80%; (v) AcOH, H₂O, HCl (1 M), 80 °C; 9a, 69%; 9b, 19%.

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

797

OAc

BnO

(i)

BnO

(i)

BnO

OBn

(ii)

OAc

BnO

(iii)

OBn

Scheme 2. Reagents and conditions: (i) Ac₂O, py, DMAP; 11, 92%; 13, 93%; (ii) Hoveyda–Grubbs second generation complex, toluene, 60 °C; 12, 87%; 10, 81%; 14 88%; (iii)

NaOMe, MeOH; 10, 88%; 15, 95%.

converted into its diimidate 19 (Scheme 4). Treatment of the diim-

BnO

(i)

BnO

idate with a Lewis acid at low temperature resulted in the forma-

tion of a major product. This material could be isolated and gave

NMR data consistent with the oxazoline, but it was somewhat

unstable during the work-up, and the hydrolysis product, the

amide 20, was also isolated. Adding water to the reaction mixture

after the complete consumption of diimidate 19 resulted in hydro-

lysis of the ﬁrst-formed oxazoline to give the amide 20.

OBn

(ii)

Subjection of the trichloroacetamide 20 to Birch conditions

cleaved the benzyl ethers and reduced the trichloroacetamide

group to an acetamide (?21). Peracetylation of 21 gave the penta-

acetate 22. A minor by-product from the Birch reduction was iden-

tiﬁed as the amine 21a, presumably formed by incomplete

reduction of the CCl₃group and nucleophilic substitution of one

chloride by ammonia. Peracetylation of this compound gave 22a

(9% from 19, Fig. 2). The acetamide in 21 was cleaved with LiOH

to give the 1,2-bis-epi-valienamine 2; the peracetate 22 could also

be completely deprotected to give 2 under the same conditions.

The C-1 stereochemistry of the carbocycles could be assigned

using ³J_1,2¹H–¹H coupling constants according to Shing:²⁷the car-

bocycles derived from the major diastereomer from the Grignard

NHAc

OAc

N₃

AcO

BnO

(iii)

OAc

OBn

Scheme 3. Reagents and conditions: (i) DPPA, PPh₃, THF, 0 °C?rt, 71%; (ii) NaN₃,

NH₄Cl, H₂O, MeOH, THF, 0 °C, 77%; (iii) Ref. 25.

We therefore tried a different approach: Danishefsky has con-

verted the allylic alcohol of a trans 1,2-diol into a cis amino alcohol,

with nitrogen introduced at the allylic position, by elimination of a

trichloroacetamide from a diimidate, a ﬁve-membered intermedi-

ate oxazoline ring ensuring cis stereochemistry in the product.²⁶

We attempted the analogous reaction on our system: diol 10 was

addition (i.e., 12, 10) were assigned with

a-lyxo conﬁguration

(J_1,27–8 Hz); those derived from the minor diastereomer from

Grignard addition (i.e., 14, 15) were assigned b-lyxo stereochemis-

try (J_1,2ca. 2 Hz). This treatment implies that

a-lyxo compounds

CCl₃

BnO

CCl₃

(i)

(ii)

BnO

CCl₃

OBn

(v)

CCl₃

NHAc

OAc

NH₂

NHAc

AcO

(iv)

(vi)

(iii)

BnO

OAc

OBn

Scheme 4. Reagents and conditions: (i) Cl₃CCN, DBU, CH₂Cl₂, 99%; (ii) BF₃ꢁOEt₂, CH₂Cl₂, ꢀ78 °C, then H₂O, rt; (iii) Na, NH_3(l), THF, ꢀ78 °C, 83% from 19; (iv) LiOH, H₂O, 33%; (v)

Ac₂O, pyridine, 61% from 19; (vi) LiOH, THF, H₂O, 77%.

798

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

monosaccharide D-mannose (substrate analogue) suggests that 2

NHAc

NH₂

binds to the active site of CfMan2A more strongly than to just a

monosaccharide. To put the inhibitory potency of the monosaccha-

ride-mimicking inhibitor 2 into context, we compare its K_iagainst

CfMan2A with the K_ivalues of other reported monosaccharide-

mimicking inhibitors against the related GH family 2 b-mannosi-

dase BtMan2A: the K_ivalue of 2 is two orders of magnitude lower

than that of deoxymannojirimycin (K_i= 33 mM), but on the other

hand, two orders of magnitude higher than that of noeuromycin

AcO

OAc

22a

21a

Figure 2.

(K_i= 1 l

M).¹⁴

have a ³H₂solution conformation in which H-1 and H-2 have a

pseudoaxial–axial relationship. We solved crystal structures of

three compounds: 15, 18 and 22 (Fig. 3), conﬁrming this stereo-

chemical assignment and conﬁrming that the major product of

the Grignard addition was that predicted from a chelation-con-

trolled model; that epoxide-opening occurred with inversion of

conﬁguration at C-1 to give the 1,2-trans product; and that the

oxazoline route indeed gave a 1,2-cis arrangement of the substitu-

ents on the carbocyclic ring. In both of the crystal structures of the

protected b-lyxo conﬁgured unsaturated carbasugars 15 and 22,

In conclusion, a metathesis approach may be used to transform

-mannose into a versatile carbocyclic diol 10, which may then be

converted into both

a- and b-mannose- and b-GlcNAc-mimicking

C-5@C-5a unsaturated carbasugars. Work to further demonstrate

the utility of this approach by the synthesis of carbasugar-contain-

ing pseudodisaccharides is in progress, and the results will be re-

ported in due course. 1,2-bis-epi-Valienamine 2, synthesised here

for the ﬁrst time, does indeed act as a b-mannosidase inhibitor,

and fails to inhibit a b-glucosidase. Further work is necessary to

understand the structural basis for inhibition.

the carbocyclic rings have ²H₃conformations. The

a-lyxo com-

pound 18 in contrast, crystallises with the carbocycle in a ³H₂con-

formation. The conformation of the carbasugars can have

important consequences for the biological activity, although the

bound conformation is not always identical to unbound solution

or solid-phase conformations.^6,28

3. Experimental

3.1. General methods

Melting points were measured using a Gallenkamp melting

point apparatus and are uncorrected. Proton nuclear magnetic res-

onance (¹H) spectra were recorded on Bruker Avance II 500

(500 MHz), Bruker Avance II 400 (400 MHz) or Varian Mercury

400 (400 MHz) spectrometers; multiplicities are quoted as singlet

(s), doublet (d), doublet of doublets (dd), triplet (t), apparent triplet

(at), doublet of apparent triplets (dat) or multiplet (m). Carbon nu-

clear magnetic resonance (¹³C) spectra were recorded on Bruker

Avance II 500 (125 MHz), Bruker Avance II 400 (100 MHz) or Var-

ian Mercury 400 (100 MHz) spectrometers. ¹H and ¹³C spectra

and ¹³C multiplicities were assigned using COSY, HSQC and DEPT

experiments. All chemical shifts are quoted on the d-scale in parts

per million (ppm). Residual solvent signals or TMS were used as an

internal reference. Low- and high-resolution (HRMS) electrospray

(ESI) mass spectra were recorded using a Bruker Microtof instru-

ment. Infra-red spectra were recorded on a Perkin–Elmer Spectrum

One FT-IR spectrometer using the thin ﬁlm method on NaCl plates.

Optical rotations were measured on a Perkin–Elmer 241 polarime-

ter with a path length of 1 dm; concentrations are given in g/

100 mL. Thin layer chromatography (TLC) was carried out on

Merck Kieselgel sheets, pre-coated with 60F₂₅₄silica. Plates were

visualised with UV light and developed using 10% sulfuric acid,

or an ammonium molybdate (10% w/v) and cerium(IV) sulfate

(2% w/v) solution in 10% sulfuric acid. Flash column chromatogra-

We also demonstrate that nitrogen can be introduced at C-2 of

the lyxo conﬁgured cyclohexenes with inversion of conﬁguration to

give GlcNAc analogues. Trichloroacetamide 20 was triﬂated and

azide introduced at C-2 to give the b-xylo conﬁgured azide 23

(Scheme 5). This was converted into the b-GlcNAc valienamine

27 recently synthesised by Stubbs et al.⁶as follows: A protecting

group swap at N-1 gave the Boc derivative 24. Subjection of this

compound to Birch conditions cleaved the benzyl ethers and re-

duced the azide, and acetylation of the crude reaction mixture gave

the tetraacetate 25. The ester protection was removed with NaOMe

in MeOH to give 26, and then the Boc protection was cleaved with

HCl to give the GlcNAc valienamine 27 as its HCl salt.

The glycosidase inhibitory effects of GlcNAc analogue 27 against

various b-hexosaminidases have already been investigated.⁶We

evaluated the inhibitory effect of 1,2-bis-epi-valienamine 2 towards

b-mannosidases by incubating it separately with a glycosidase fam-

ily 2 b-mannosidase (recombinant Cellulomonas ﬁmi b-mannosi-

dase; CfMan2A)^8aand also a b-glucosidase (Aspergillus niger b-

glucosidase; AnGlu) under steady-state conditions. The CfMan2A-

catalysed initial rates of hydrolysis of pNPMan were decreased in

the presence of 1,2-bis-epi-valienamine 2 at lower than 1.0 mM con-

centrations, hence we carried out full steady-state inhibition analy-

sis to evaluate the inhibition pattern and the inhibition constant K_i.

The Lineweaver-Burk double-reciprocal plot (Fig. 4A) shows a pat-

tern consistent with a reversible competitive inhibition scheme²⁹

suggesting that b-manno-valienamine 2 serves as a competitive

inhibitor of CfMan2A with respect to pNPMan substrate. The disso-

phy was carried out on silica gel (35–70 lm, Grace). Grubbs’ sec-

ond generation complex (CAS No.: 246047-72-3) was bought

from Sigma Aldrich and coated in wax (complex 12% by weight)

before use.³⁰Hoveyda–Grubbs second generation complex (CAS

No.: 301224-40-8) was bought from Sigma Aldrich and used as

supplied. Dichloromethane was distilled from calcium hydride.

Diethyl ether and THF were distilled from sodium benzophenone

ketyl radical. Toluene was distilled from sodium. Reactions per-

formed under an atmosphere of nitrogen or argon were maintained

by an inﬂated balloon.

ciation constant K_iwas determined to be 140 30 lM from a replot

of apparent K_m(K^app) values versus 1,2-bis-epi-valienamine 2 con-

centrations (Fig. 4B). In contrast, the AnGlu-catalysed initial rates

of hydrolysis of pNPGlc were unchanged in the presence of up to

2.0 mM 1,2-bis-epi-valienamine 2 (data not shown) suggesting that

2 may be a b-mannosidase-speciﬁc inhibitor.

We also compared the effect of 1,2-bis-epi-valienamine 2 on the

hydrolytic activity of CfMan2A to that of the substrate analogue

mannose. The CfMan2A-catalysed hydrolysis of pNPMan was not

inhibited by -mannose at concentrations up to 1.0 mM and indeed

the overall initial rates slightly increased in the presence of -man-

D-

3.1.1. (2R,3R,4R,5R,6RS)-1,3,4-Tri-O-benzyl-1,2,3,4,5,

6-hexahydroxy-oct-7-ene 4a,b

Hemiacetal 3 (6.95 g, 15.4 mmol) was dissolved in THF (30 mL,

freshly distilled) under Ar. Vinylmagnesium bromide (0.7 M in

THF, 92 mL, 64.4 mmol) was added and the yellow reaction mix-

nose (data not shown), possibly due to transglycosylation in addi-

tion to hydrolysis.^8cThe inhibition of CfMan2A by 2 and not by the

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

799

Figure 3. X-ray structures drawn as thermal ellipsoids at 50% probability and showing crystallographic atom numbering scheme. (a) Compound 15; (b) compound 22 and (c)

compound 18.

ture was warmed to 65 °C and left at reﬂux for 20 h. TLC (toluene/

EtOAc, 1:2) indicated the formation of a major product (R_f0.7) and

the presence of some starting material (R_f0.5). The reaction was

quenched by addition of ammonium chloride (25 mL, satd aq) at

0 °C and diluted with EtOAc (100 mL). The solution was washed

with water (3 ꢂ 100 mL) and brine (2 ꢂ 100 mL). The aqueous

phases were combined and extracted with EtOAc (3 ꢂ 50 mL).

The organic phases were combined, dried (Na₂SO₄), ﬁltered and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (pentane/EtOAc, 2:1?1:1) to yield recovered

800

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

Selected data for minor diastereomer 4b: d_H(500 MHz, CDCl₃)

2.51 (1H, d, J_OH,15.0 Hz, OH-1), 3.01 (1H, d, J_OH,55.5 Hz, OH-5),

3.04 (1H, d, J_OH,24.5 Hz, OH-2); d_C(125 MHz, CDCl₃) 70.6 (d, C-5),

71.3 (t, C-6), 73.3, 73.6 (2 ꢂ t, 2 ꢂ PhCH₂), 74.1 (d, C-1), 117.7 (t,

C-1b).

CCl₃

BnO

(i)

N₃

OBn

3.1.2. (2R,3R,4S,5R,6RS)-1,3,4-Tri-O-benzyl-5,6-O-isopropylidene-

1,2,3,4,5,6-hexahydroxy-oct-7-ene 5a,b and (2R,3S,4S,5R,6S)-1,3,4-

tri-O-benzyl-2,5-O-isopropylidene-1,2,3,4,5,6-hexahydroxy-oct-7-

ene 6

(ii)

Triols 4a,b (6.05 g, 12.6 mmol) were dissolved in DMF (60 mL)

under Ar, and 2,2-dimethoxypropane (12.5 mL, 101 mmol) was

added. Camphorsulfonic acid (2.8 g, 12.1 mmol) was added and

the reaction mixture was stirred at rt for 2 h. TLC (toluene/EtOAc,

3:1) indicated the formation of a major product (R_f0.6) and a min-

or product (R_f0.5), and the complete consumption of starting

material (R_f0.1). The reaction was quenched by addition of NEt₃

until the pH was raised from 2 to 8. The reaction mixture was di-

luted with EtOAc (100 mL) and washed with brine (3 ꢂ 80 mL).

The brine fractions were combined and extracted with EtOAc

(2 ꢂ 50 mL). The combined organic phases were dried (Na₂SO₄), ﬁl-

tered and concentrated in vacuo. The crude product was puriﬁed

by ﬂash column chromatography (pentane/EtOAc, 5:1?3:1) to

yield an inseparable mixture of acetals 5a,b and 6 (5.78 g, 88%)

NHBoc

NHAc

NHBoc

N₃

AcO

BnO

(iii)

OAc

OBn

(iv)

NHBoc

NHAc

NH₂

(v)

NHAc

as a colourless oil;

_max/cm^ꢀ13480 (br, OH); m/z (ESI⁺) 541

(M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₃₂H₃₈O₆Na (M+Na⁺)

541.2561; found 541.2572.

Scheme 5. Reagents and conditions: (i) Tf₂O, py, CH₂Cl₂, 0 °C; then NaN₃, DMF,

70 °C, 63% from 19; (ii) KOH, PrOH, 40 °C; then Boc₂O, CH₂Cl₂, 68%; (iii) Na, NH_3(l)

THF, ꢀ78 °C; then Ac₂O, py, 87%; (iv) NaOMe, MeOH, 79%; (v) HCl, H₂O, 93%.

Selected data for major diastereomer 5a: d_H(500 MHz, CDCl₃)

1.40, 1.44 (6H, 2 ꢂ s, C(CH₃)₂), 2.60 (1H, d, J_OH,55.5 Hz, OH-5),

3.56 (1H, dd, J_6,69.5 Hz, J_5,65.5 Hz, H-6), 3.66 (1H, dd, J_5,63.0 Hz,

H-6⁰), 3.75 (1H, dd, J_4,58.0 Hz, J_3,43.0 Hz, H-4), 3.94 (1H, dd, J_2,3

7.5 Hz, H-3), 3.99 (1H, m, H-5), 4.08 (1H, at, J 7.8 Hz, H-2), 4.42

(1H, at, J 7.0 Hz, H-1), 5.16 (1H, d, J_1a,1b10.5 Hz, H-1b), 5.36 (1H,

starting material 3 (381 mg, 5%) and triols 4a,b (6.07 g, 82%, insep-

arable diastereomeric mixture, 4a:4b, 2.9:1) as a colourless oil;

HRMS (ESI⁺) calcd for C₂₉H₃₄O₆Na (M+Na⁺) 501.2248; found

501.2230.

Selected data for major diastereomer 4a: d_H(500 MHz, CDCl₃)

2.21 (1H, d, J_OH,16.9 Hz, OH-1), 2.91 (1H, d, J_OH,25.5 Hz, OH-2),

2.94 (1H, d, J_OH,55.0 Hz, OH-5), 3.81 (1H, m, H-2), 4.30 (1H, ddd,

d, J_1a,1b17.1 Hz, H-1b⁰), 5.91 (1H, ddd, J_1,1a6.4 Hz, H-1a), 7.24–

7.36 (15H, m, Ar-H); d_C(125 MHz, CDCl₃) 27.1, 27.3 (2 ꢂ q,

C(CH₃)₂), 109.0 (s, C(CH₃)₂ﬁve-ring), 117.6 (t, C-1b), 136.3 (d, C-

1a), 138.0, 138.2, 138.5 (3 ꢂ s, 3 ꢂ Ar-C).

Selected data for minor diastereomer 5b: d_H(500 MHz, CDCl₃)

1.36, 1.51 (6H, 2 ꢂ s, C(CH₃)₂), 2.58 (1H, OH-5), 6.00 (1H, ddd,

J_1,21.5 Hz, J_1,1a5.1 Hz, H-1), 5.22 (1H, obs dat, J_1a,1b10.5 Hz, J_1b,1b

1.5 Hz, H-1b), 5.33 (1H, obs dat, J_1a,1b17.5 Hz, H-1b⁰), 5.90 (1H,

J_1,1a7.7 Hz, J_1a,1b10.4 Hz, J_1a,1b17.1 Hz, H-1a); d_C(125 MHz, CDCl₃)

25.8, 28.3 (2 ꢂ q, C(CH₃)₂), 108.6 (s, C(CH₃)₂ﬁve-ring), 118.4 (t, C-

obs ddd, H-1a), 7.21–7.31 (15H, m, Ar-H); d_C(125 MHz, CDCl₃)

70.4 (d, C-5), 71.2 (t, C-6), 71.5 (d, C-1), 72.9 (d, C-2), 73.7, 73.9,

74.0 (3 ꢂ t, 3 ꢂ PhCH₂), 77.5, 78.0 (2 ꢂ d, C-3, C-4), 116.2 (t, C-

1b), 128.0, 128.1, 128.2, 128.5, 128.6, 128.6, 128.6, 128.7 (8 ꢂ d,

Ar-CH), 136.8, 137.6, 137.7, 138.0 (4 ꢂ s, Ar-C), 138.3 (d, C-1a);

m/z (ESI⁺) 501 (M+Na⁺, 100%).

1b), 135.1 (d, C-1a).

Selected data for regioisomer 6: d_H(500 MHz, CDCl₃) 1.28, 1.34

(6H, 2 ꢂ s, C(CH₃)₂), 2.14 (1H, d, J_OH,110.5 Hz, OH-1); d_C

(125 MHz, CDCl₃) 24.6, 25.2 (2 ꢂ q, C(CH₃)₂), 100.8 (s, C(CH₃)₂se-

ven-ring), 115.4 (t, C-1b).

0.5

300

250

200

150

100

0.4

0.3

0.2

0.1

-0.20 -0.05 0.10 0.25 0.40 0.55 0.70

[I] (mM)

-14

-9

-4

1 / [pNPMan] (mM^-1)

Figure 4. Inhibition of the CfMan2A-catalysed hydrolysis of pNPMan by b-manno-valienamine 2: (A) Lineweaver-Burk double-reciprocal plot showing a competitive

inhibition pattern. b-manno-valienamine concentrations were 0 (j), 100 (N), 450 (ꢀ) and 600 (d) l

M. The lines are the linear regression ﬁts of data. (B) Replot of K^a_m^ppas a

function of b-manno-valienamine concentration [I]. The line is the linear regression ﬁt of data. K_ivalue was determined according to the relationship: K^app¼ K_mꢁ ð1 þ ½Iꢃ=K_iÞ.

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

801

3.1.3. (3S,4R,5R,6RS)-1,3,4-Tri-O-benzyl-5,6-O-isopropylidene-

1,3,4,5,6-pentahydroxy-oct-7-ene-2-one 7a,b

(125 MHz, CDCl₃) 27.0, 27.1 (2 ꢂ q, 2 ꢂ CH₃), 70.2 (t, C-6), 71.0,

72.7, 75.4 (3 ꢂ t, 3 ꢂ PhCH₂), 79.0 (d, C-1), 80.0 (d, C-2), 80.8 (d,

C-3), 81.6 (d, C-4), 108.6 (s, C(CH₃)₂ﬁve-ring), 117.4 (t, C-5a),

118.2 (t, C-1b), 127.6, 127.6, 127.7, 127.7, 128.2, 128.3, 128.3,

128.3, 128.5 (9 ꢂ d, Ar-CH), 136.7 (d, C-1a), 138.3, 138.3, 138.6

(3 ꢂ s, 3 ꢂ Ar-C), 142.3 (s, C-5).

Oxalyl chloride (3.6 mL, 42 mmol) was dissolved in CH₂Cl₂

(40 mL, freshly distilled) under Ar at ꢀ78 °C. DMSO (6.4 mL,

90 mmol) was dissolved in CH₂Cl₂(20 mL, freshly distilled) under

Ar at ꢀ78 °C and then transferred to the oxalyl chloride solution

by cannula. After 45 min, a solution of alcohols 5a,b and 6

(5.67 g, 10.9 mmol) in CH₂Cl₂(30 + 10 + 10 mL) was transferred

to the reaction vessel at ꢀ78 °C. After 1.5 h, NEt₃(16 mL,

114.8 mmol) was added to the reaction mixture. After an addi-

tional 1 h 15 min, TLC (toluene/EtOAc, 3:1) indicated the complete

consumption of starting material (R_f0.6) and the formation of a

major product (R_f0.7). The reaction mixture was diluted with

CH₂Cl₂(150 mL), transferred to a separatory funnel and washed

with brine (3 ꢂ 100 mL). The aqueous phases were combined and

extracted with CH₂Cl₂(2 ꢂ 50 mL). The organic phases were com-

bined, dried (Na₂SO₄), ﬁltered and concentrated in vacuo, to yield

the crude product (6.8 g) as a slightly yellow oil. The crude product

was puriﬁed by ﬂash column chromatography (toluene?toluene/

EtOAc, 10:1?8:1) to yield the ketones 7a,b (4.57 g, 81%, insepara-

Selected data for minor diastereomer 8b: d_H(500 MHz, CDCl₃)

1.32, 1.45 (6H, 2 ꢂ s, 2 ꢂ CH₃), 3.79 (1H, dd, J_3,43.6 Hz, J_2,37.8 Hz,

H-3), 4.27 (1H, d, H-4), 6.00 (1H, ddd, J_1,1a7.5 Hz, J_1a,1b10.0 Hz,

J_1a,1b17.0 Hz, H-1a); d_C(125 MHz, CDCl₃) 25.5, 28.1 (2 ꢂ q,

2 ꢂ CH₃), 108.3 (s, C(CH₃)₂), 135.3 (d, C-1a), 138.3, 138.5, 138.7

(3 ꢂ s, 3 ꢂ Ar-C), 143.1 (s, C-5).

3.1.5. (3R,4R,5R,6S)-1,3,4-Tri-O-benzyl-1,3,4,5,6-pentahydroxy-

2-methylene-oct-7-ene 9a and (3R,4R,5R,6R)-1,3,4-tri-O-benzyl-

1,3,4,5,6-pentahydroxy-2-methylene-oct-7-ene 9b

Acetals 8a,b (3.59 g, 6.98 mmol) were suspended in AcOH

(45 mL) and water (15 mL), and HCl (1 M aq, 1 mL) was added,

and the resulting suspension was heated to 80 °C. After 1 h, TLC

(pentane/EtOAc, 4:1) indicated the complete consumption of start-

ing material (R_f0.7) and the formation of a major product (R_f0.1).

The heating was turned off and the reaction mixture concentrated

in vacuo by co-evaporation of solvent with toluene. The crude

product (3.66 g) was puriﬁed by ﬂash column chromatography

(pentane/EtOAc, 4:1?1:1) to yield 9b (635 mg, 19%) as a colourless

ble diastereomers, 7a:7b, 3.3:1) as a yellow oil; m

_max/cm^ꢀ11730 (s,

C@O); m/z (ESI⁺) 539 (M+Na⁺,100%); HRMS (ESI⁺) calcd for

C₃₂H₃₆O₆Na (M+Na⁺) 539.2404; found 539.2420.

Selected data for major diastereomer 7a: d_H(500 MHz, CDCl₃)

1.38, 1.40 (6H, 2 ꢂ s, 2 ꢂ CH₃), 4.00–4.04 (2H, m, H-2, H-4), 4.24,

4.36 (2H, ABq, J_6,618.5 Hz, H-6, H-6⁰), 4.27 (1H, d, J_3,43.5 Hz, H-

oil; ½a ²_D³

ꢃ

¼ ꢀ0:3 (c 1.0, CHCl₃);

_max/cm^ꢀ13436 (br, OH); d_H

(400 MHz, CDCl₃) 2.75 (1H, d, J_OH,14.4 Hz, OH-1), 3.03 (1H, d,

3), 4.36 (1H, m, H-1), 5.19 (1H, d, J_1a,1b10.5 Hz, H-1b), 5.36 (1H,

obs d, J_1a,1b17.3 Hz, H-1b⁰), 5.88 (1H, ddd, J_1,1a6.5 Hz, H-1a),

J_OH,24.8 Hz, OH-2), 3.67 (1H, dd, J_3,44.3 Hz, J_2,36.6 Hz, H-3), 3.80

(1H, m, H-2), 4.00, 4.12 (2H, ABq, J_6,612.6 Hz, H-6, H-6⁰), 4.18

7.15–7.34 (15H, m, Ar-H); d_C(125 MHz, CDCl₃) 27.1, 27.1 (2 ꢂ q,

2 ꢂ CH₃), 73.3, 74.7, 74.7 (3 ꢂ t, 3 ꢂ PhCH₂), 74.8 (t, C-6), 79.0 (d,

C-2), 80.1 (d, C-1), 81.9 (d, C-4), 84.2 (d, C-3), 109.3 (s, C(CH₃)₂

ﬁve-ring), 118.0 (t, C-1b), 135.9 (d, C-1a), 137.0, 137.3, 137.3

(3 ꢂ s, Ar-C), 208.8 (s, C@O).

(1H, m, H-1), 4.34 (1H, d, J 11.6 Hz, PhCHH⁰), 4.38 (1H, d, H-4),

4.45–4.54 (3H, m, J_AB11.8 Hz, PhCHH⁰, PhCH₂), 4.61–4.65 (2H, m,

2 ꢂ PhCHH⁰), 5.24 (1H, dat, J 1.5 Hz, J_1a,1b10.6 Hz, H-1b), 5.31

(1H, dat, J 1.5 Hz, J_1a,1b17.2 Hz, H-1b⁰), 5.52 (1H, s, H-5a), 5.56

Selected data for minor diastereomer 7b: d_H(500 MHz, CDCl₃)

1.35, 1.47 (6H, 2 ꢂ s, 2 ꢂ CH₃), 5.23 (1H, br d, J_1a,1b10.5 Hz, H-

(1H, d, J 1.6 Hz, H-5a⁰), 5.90 (1H, ddd, J_1,1a6.4 Hz, H-1a), 7.22–

7.35 (15H, m, Ar-H); d_C(100 MHz, CDCl₃) 71.2, 72.7, 73.7 (3 ꢂ t,

3 ꢂ PhCH₂), 71.2 (t, C-6), 73.5 (d, C-2), 73.8 (d, C-1), 80.0 (d, C-4),

80.6 (d, C-3), 116.8 (t, C-5a), 116.9 (t, C-1b), 127.8, 127.8, 128.0,

128.1, 128.3, 128.4, 128.5, 128.6 (8 ꢂ d, Ar-CH), 137.0 (d, C-1a),

137.6, 137.8, 138.0 (3 ꢂ s, 3 ꢂ Ar-C), 142.2 (s, C-5). m/z (ESI⁺) 497

(M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₃₀H₃₄O₅Na (MNa⁺)

497.2298; found 497.2310.

1b), 5.94 (1H, ddd, J_1,1a7.0 Hz, J_1a,1b17.5 Hz, H-1a); d_C(125 MHz,

CDCl₃) 25.7, 28.1 (2 ꢂ q, 2 ꢂ CH₃), 108.7 (s, C(CH₃)₂) ﬁve-ring),

118.1 (t, C-1b), 134.1 (d, C-1a), 209.3 (s, C@O).

3.1.4. (3R,4S,5R,6RS)-1,3,4-Tri-O-benzyl-5,6-O-isopropylidene-

1,3,4,5,6-pentahydroxy-2-methylene-oct-7-ene 8a,b

Triphenylmethylphosphonium bromide (18.7 g, 52 mmol) was

suspended in freshly distilled toluene (80 mL) under Ar. After

20 min, potassium tert-butoxide (5.6 g, 50 mmol) was added, and

the resulting suspension was heated to 80 °C. After a few minutes,

the suspension turned intensely yellow. After a further 2 h 40 min,

the suspension containing the ylid was allowed to cool to rt, and

ketones 7a,b (4.51 g, 8.73 mmol) were dissolved in toluene (freshly

distilled, 40 + 10 + 10 mL) and added to the ylid by cannula. After

45 min, TLC (toluene/EtOAc, 6:1) indicated the complete consump-

tion of starting material (R_f0.5) and the formation of a major prod-

uct (R_f0.6). The reaction mixture was diluted with toluene (80 mL),

ﬁltered through Celite and concentrated in vacuo. The crude prod-

uct (15 g) was puriﬁed by ﬂash column chromatography (pentane/

EtOAc, 20:1?10:1) to yield dienes 8a,b (3.61 g, 80%, inseparable

diastereomeric mixture, 8a:8b, 3.3:1) as a yellow oil m/z (ESI⁺)

537 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₃₃H₃₈O₅Na (M+Na⁺)

537.2611; found 537.2612.

And 9a (2.32 g, 69%) as a colourless oil; ½a _D²³

¼ ꢀ22:9 (c 1.0,

ꢃ

CHCl₃); m

_max/cm^ꢀ13431 (br, OH); d_H(400 MHz, CDCl₃) 2.62 (1H,

d, J_OH,16.4 Hz, OH-1), 2.99 (1H, d, J_OH,25.2 Hz, OH-2), 3.68 (1H,

ddd, J_2,36.7 Hz, J_1,22.0 Hz, H-2), 3.79 (1H, dd, J_3,45.0 Hz, H-3),

4.02, 4.11 (2H, ABq, J_6,612.4 Hz, H-6, H-6⁰), 4.26–4.30 (2H, m, H-

1, H-4), 4.34, 4.63 (2H, ABq, J_AB12.2 Hz, PhCH₂), 4.49, 4.55 (2H,

ABq, J_AB11.6 Hz, PhCH₂), 4.60, 4.70 (2H, ABq, J_AB11.0 Hz, PhCH₂),

5.17 (1H, dat, J 1.6 Hz, J_1a,1b10.5 Hz, H-1b), 5.28 (1H, dat, J

1.6 Hz, J_1a,1b17.2 Hz, H-1b⁰), 5.41 (1H, s, H-5a), 5.49 (1H, d, J

1.2 Hz, H-5a⁰), 5.86 (1H, ddd, J_1,1a5.0 Hz, H-1a), 7.25–7.37 (15H,

m, Ar-H). d_C(100 MHz, CDCl₃) 71.1 (t, C-6), 71.2, 72.8, 74.8 (3 ꢂ t,

3 ꢂ PhCH₂), 71.7 (d, C-1), 72.6 (d, C-2), 80.3 (d, C-4), 80.7 (d, C-3),

115.8 (t, C-1b), 116.7 (t, C-5a), 127.8, 127.8, 128.1, 128.1, 128.3,

128.4, 128.6, 128.6 (8 ꢂ d, Ar-CH), 137.7, 137.9, 138.2 (3 ꢂ s,

3 ꢂ Ar-C), 138.3 (d, C-1a), 142.3 (s, C-5). m/z (ESI⁺) 497 (M+Na⁺,

100%); HRMS (ESI⁺) calcd for C₃₀H₃₄O₅Na (MNa⁺) 497.2298; found

497.2307.

Selected data for major diastereomer 8a: d_H(500 MHz, CDCl₃)

1.35, 1.38 (6H, 2 ꢂ s, 2 ꢂ CH₃), 3.89 (1H, dd, J_3,46.5 Hz, J_2,34.9 Hz,

H-3), 3.99 (1H, dd, J_1,27.8 Hz, H-2), 4.03–4.08 (2H, m, H-4, H-6),

3.1.6. 3,4,6-Tri-O-benzyl-5a-carba-a-D-lyxo-hex-5(5a)-

enopyranose 10

4.13 (1H, d, J_6,613.0 Hz, H-6⁰), 4.35 (1H, d, J 11.5 Hz, PhCHH⁰),

Method 1: Cyclisation of unprotected diol. Diene 9a (24 mg,

0.05 mmol) was dissolved in toluene (2 mL, freshly distilled) under

Ar and heated to 60 °C. Hoveyda–Grubbs’ second generation com-

plex (3 mg, 0.005 mmol) was added. After 50 min, TLC (toluene/

EtOAc, 1:1) indicated the complete consumption of starting

4.48–4.57 (4H, m, PhCH₂, PhCHH⁰, H-1), 4.67, 4.76 (2H, ABq, J_AB

11.0 Hz, PhCH₂), 5.14 (1H, d, J_1a,1b10.5 Hz, H-1b), 5.29–5.32 (2H,

m, H-5a, H-1b⁰), 5.46 (1H, d, J_5a,5a1.0 Hz, H-5a⁰), 5.80 (1H, ddd,

J_1,1a7.1 Hz, J_1a,1b17.1 Hz, H-1a), 7.22–7.36 (15H, m, Ar-H); d_C

802

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

material (R_f0.7) and the formation of a major product (R_f0.2). The

reaction mixture was concentrated in vacuo and the residue puri-

ﬁed by ﬂash column chromatography (toluene?toluene/EtOAc,

6:1?1:1) to yield carbocycle 10 (18 mg, 81%) as a colourless oil;

TLC indicated the consumption of starting material and the forma-

tion of product. The reaction mixture was concentrated in vacuo

and the residue puriﬁed by ﬂash column chromatography (pen-

tane/EtOAc, 6:1?4:1) to yield starting material 11 (256 mg, 10%),

a ²_D³

ꢃ

¼ ꢀ15:3 (c 1.0, CHCl₃);

_max/cm^ꢀ13394 (br, OH); d_H

and carbocycle 12 (2.05 g, 87%) as a colourless oil; ½a _D²³

¼ þ63:9

ꢃ

(500 MHz, CDCl₃) 2.57 (1H, br d, J_OH,15.5 Hz, OH-1), 2.62 (1H, br

(c 1.0, CHCl₃); m

_max/cm^ꢀ11741 (s, C@O); d_H(500 MHz, CDCl₃)

d, J_OH,27.6 Hz, OH-2), 3.81 (1H, dat, J 7.8 Hz, J_2,32.8 Hz, H-2), 3.85

2.04, 2.05 (6H, 2 ꢂ s, 2 ꢂ CH₃), 3.89, 4.15 (2H, ABq, J_6,613.0 Hz,

H-6, H-6⁰), 4.02 (1H, dd, J_2,32.3 Hz, J_3,44.2 Hz, H-3), 4.07 (1H, d,

H-4), 4.37, 4.48 (2H, ABq, J_AB11.5 Hz, PhCH₂), 4.48, 4.65 (2H,

ABq, J_AB11.3 Hz, PhCH₂), 4.54, 4.60 (2H, ABq, J_AB12.0 Hz, PhCH₂),

5.36 (1H, dd, J_1,27.4 Hz, H-2), 5.64 (1H, d, H-1), 5.78 (1H, m, H-

5a), 7.27–7.35 (15H, m, Ar-H); d_C(125 MHz, CDCl₃) 21.2, 21.2

(2 ꢂ q, 2 ꢂ CH₃), 69.2 (d, C-1), 70.3 (t, C-6), 71.3 (d, C-2), 72.0,

72.8, 73.9 (3 ꢂ t, PhCH₂), 74.0 (d, C-4), 75.6 (d, C-3), 124.4 (d, C-

5a), 127.8, 127.9, 128.0, 128.1, 128.1, 128.2, 128.4, 128.5, 128.6

(9 ꢂ d, Ar-CH), 137.5 (s, C-5), 138.0, 138.2 (2 ꢂ s, Ar-C), 170.5,

170.6 (2 ꢂ s, 2 ꢂ C@O); m/z (ESI⁺) 569 (M+K⁺, 14), 553 (M+Na⁺,

100%); HRMS (ESI⁺) calcd for C₃₂H₃₄O₇Na (M+Na⁺) 553.2197; found

553.2210.

(1H, d, J_6,612.0 Hz, H-6), 3.88 (1H, at, J 3.0 Hz, H-3), 4.04 (1H, d,

J_3,42.5 Hz, H-4), 4.11 (1H, dat, J 1.4 Hz, H-6⁰), 4.32–4.34 (2H, m,

H-1, PhCHH⁰), 4.44, 4.55 (2H, ABq, J_AB11.5 Hz, PhCH₂), 4.45 (1H,

d, J 11.5 Hz, PhCHH⁰), 4.55, 4.58 (2H, ABq, J_AB12.0 Hz, PhCH₂),

5.79 (1H, m, H-5a), 7.22–7.33 (15H, m, Ar-H); d_C(125 MHz, CDCl₃)

69.7 (d, C-1), 70.4 (t, C-6), 71.8, 72.7, 73.5 (3 ꢂ t, 3 ꢂ PhCH₂), 72.7

(d, C-2), 73.6 (d, C-4), 78.0 (d, C-3), 127.7, 127.9, 127.9, 128.0,

128.1, 128.4, 128.5, 128.6, 128.6 (9 ꢂ d, Ar-CH), 129.6 (d, C-5a),

134.2 (s, C-5), 137.8, 138.1, 138.2 (3 ꢂ s, 3 ꢂ Ar-C); m/z (ESI⁺) 469

(M+Na⁺, 100%), HRMS (ESI⁺) calcd for C₂₈H₃₀O₅Na (M+Na⁺)

469.1985; found 469.1991.

Method 2: Deacetylation of carbocycle 12. Diacetate 12 (2.01 g,

3.80 mmol) was dissolved in MeOH (25 mL) under Ar. Sodium

(40 mg, 1.7 mmol) was dissolved in MeOH (5 mL) and added to

the reaction mixture. After 1 h, TLC (toluene/EtOAc, 1:1) indicated

the complete consumption of starting material (R_f0.8) and the for-

mation of a major product (R_f0.4). The reaction mixture was con-

centrated in vacuo by co-evaporation with toluene and the residue

puriﬁed by ﬂash column chromatography (pentane/EtOAc,

6:1?EtOAc) to yield diol 10 (1.50 g, 88%) identical to that de-

scribed above.

3.1.9. (3R,4S,5R,6R)-5,6-Di-O-acetyl-1,3,4-tri-O-benzyl-pentahy-

droxy-2-methylene-oct-7-ene 13

Diol 9b (620 mg, 1.31 mmol) was dissolved in acetic anhydride

(3 mL, 32 mmol), and pyridine (3 mL, 37 mmol) and dimethylami-

nopyridine (25 mg, 0.2 mmol) were added. After 6 h, TLC (pentane/

EtOAc, 4:1) indicated the complete consumption of starting mate-

rial (R_f0.1) and the formation of a major product (R_f0.5). The reac-

tion mixture was concentrated in vacuo by co-evaporation with

toluene. The residue was puriﬁed using ﬂash column chromatogra-

phy (pentane/EtOAc, 6:1?EtOAc) to yield diacetate 13 (677 mg,

3.1.7. (3R,4S,5R,6S)-5,6-Di-O-acetyl-1,3,4-tri-O-benzyl-pentahy-

droxy-2-methylene-oct-7-ene 11

93%) as a yellow oil; ½a _D²²

ꢃ

¼ ꢀ12:9 (c 1.0, CHCl₃);

_max/cm^ꢀ11744

Diol 9a (2.32 g, 4.89 mmol) was dissolved in acetic anhydride

(20 mL, 212 mmol), and pyridine (20 mL, 248 mmol) and dimethyl-

aminopyridine (50 mg, 0.4 mmol) were added. After 2.5 h, TLC

(pentane/EtOAc, 4:1) indicated the complete consumption of start-

ing material (R_f0.1) and the formation of a major product (R_f0.6).

The reaction mixture was concentrated in vacuo by co-evaporation

with toluene. The residue was puriﬁed by ﬂash column chromatog-

raphy (pentane/EtOAc, 7:1?4:1) to yield diacetate 11 (2.52 g, 92%)

(s, C@O); d_H(500 MHz, CDCl₃) 1.88, 1.95 (6H, 2 ꢂ s, 2 ꢂ CH₃),

3.79 (1H, at, J 6.3 Hz, H-3), 4.07, 4.16 (2H, ABq, J_6,613.3 Hz, H-6,

H-6⁰), 4.11 (1H, d, J_3,45.5 Hz, H-4), 4.29 (1H, d, J 11.5 Hz, PhCHH⁰),

4.51–4.57 (3H, m, PhCHH⁰, PhCH₂), 4.62, 4.77 (2H, ABq, J_AB11.3 Hz,

PhCH₂), 5.18–5.25 (2H, m, H-1b, H-1b⁰), 5.31 (1H, dd, J_2,36.0 Hz, J_1,2

4.3 Hz, H-2), 5.35, 5.48 (2H, 2 ꢂ s, H-5a, H-5a⁰), 5.65 (1H, dd, J_1,1a

6.7 Hz, H-1), 5.85 (1H, ddd, J_1a,1b10.6 Hz, J_1a,1b17.4 Hz, H-1a),

7.24–7.35 (15H, m, Ar-H); d_C(125 MHz, CDCl₃) 21.0, 21.1 (2 ꢂ q,

2 ꢂ CH₃), 70.4 (t, C-6), 70.9, 74.2 (2 ꢂ t, 2 ꢂ PhCH₂), 72.8 (d, t, C-

2, PhCH₂), 73.7 (d, C-1), 79.2 (d, C-3), 81.3 (d, C-4), 116.6 (t, C-

5a), 119.2 (t, C-1b), 127.6, 127.7, 127.7, 127.7, 127.9, 128.3,

128.4, 128.4, 128.5 (9 ꢂ d, Ar-CH), 132.6 (d, C-1a), 138.0, 138.4,

138.4 (3 ꢂ s, 3 ꢂ Ar-C), 142.1 (s, C-5), 169.7, 169.7 (2 ꢂ s,

2 ꢂ C@O); m/z (ESI⁺) 581 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for

C₃₄H₃₈O₇Na (M+Na⁺) 581.2510; found 581.2520.

as a yellow oil; ½a _D²³

ꢃ

¼ ꢀ28:3 (c 1.0, CHCl₃);

_max/cm^ꢀ11745 (s,

C@O); d_H(500 MHz, CDCl₃) 1.93, 2.03 (6H, 2 ꢂ s, 2 ꢂ CH₃), 3.84

(1H, dd, J_3,45.4 Hz, J_2,36.9 Hz, H-3), 4.08, 4.18 (2H, ABq, J_6,6

13.0 Hz, H-6, H-6⁰), 4.10 (1H, d, H-4), 4.28, 4.51 (2H, ABq, J_AB

11.0 Hz, PhCH₂), 4.42, 4.69 (2H, ABq, J_AB10.5 Hz, PhCH₂), 4.54,

4.57 (2H, ABq, J_AB12.3 Hz, PhCH₂), 5.14–5.20 (2H, m, H-1b, H-

1b⁰), 5.29 (1H, dd, J_1,22.5 Hz, H-2), 5.38 (1H, s, H-5a), 5.49 (1H, s,

H-5a⁰), 5.69–5.76 (2H, m, H-1, H-1a), 7.23–7.35 (15H, m, Ar-H);

d_C(125 MHz, CDCl₃) 21.0, 21.1 (2 ꢂ q, 2 ꢂ CH₃), 70.4 (t, C-6),

71.1, 72.8, 75.1 (3 ꢂ t, 3 ꢂ PhCH₂), 72.1 (d, C-2), 72.7 (d, C-1),

78.6 (d, C-3), 81.2 (d, C-4), 116.4 (t, C-5a), 117.1 (t, C-1b), 127.6,

127.7, 127.8, 128.4, 128.5 (5 ꢂ d, Ar-CH), 133.4 (d, C-1a), 137.9,

138.4 (2 ꢂ s, Ar-C), 142.2 (s, C-5), 169.7, 169.8 (2 ꢂ s, 2 ꢂ C@O);

m/z (ESI⁺) 597 (M+K⁺, 12), 581 (M+Na⁺, 100%); HRMS (ESI⁺) calcd

for C₃₄H₃₈O₇Na (M+Na⁺) 581.2510; found 581.2524.

3.1.10. 1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-5a-carba-b-D-lyxo-hex-

5(5a)-enopyranose 14

Protected diene 13 (650 mg, 1.16 mmol) was dissolved in

freshly distilled toluene (5 mL) under Ar. Hoveyda–Grubbs’ second

generation complex (12 mg, 0.02 mmol, 2 mol %) was added and

the yellow-green reaction mixture was heated to 60 °C. After 1 h,

TLC (pentane/EtOAc, 3:1) indicated the formation of a major prod-

uct (R_f0.5) and the presence of starting material (R_f0.7). Two addi-

tional portions of Hoveyda–Grubbs’ second generation complex

(8 + 3 mg, 0.02 mmol, 2 mol %) were added to the reaction mixture

over the course of 4 h, after which time TLC analysis indicated the

consumption of starting material and the formation of product. The

reaction mixture was then concentrated in vacuo and the residue

puriﬁed by ﬂash column chromatography (pentane/EtOAc,

5:1?3:1) to yield carbocycle 14 (545 mg, 88%) as a yellow oil;

3.1.8. 1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-5a-carba-a-D-lyxo-hex-

5(5a)-enopyranose 12

Protected diene 11 (2.48 g, 4.44 mmol) was dissolved in freshly

distilled toluene (40 mL) under Ar. Hoveyda–Grubbs’ second gen-

eration complex (30 mg, 0.05 mmol, 1 mol %) was added and the

yellow-green reaction mixture was heated to 60 °C. After 15 h,

TLC (pentane/EtOAc, 4:1) indicated the formation of a major prod-

uct (R_f0.5) and the presence of starting material (R_f0.6). Three

additional portions of Hoveyda–Grubbs’ second generation com-

plex (10 + 10 + 15 mg, 0.06 mmol, 1.3 mol %) were added to the

reaction mixture over the course of 5 h, and after a further 4 h,

a ²_D³

ꢃ

¼ ꢀ75:1 (c 1.0 CHCl₃);

_max/cm^ꢀ11745 (s, C@O); d_H

(500 MHz, CDCl₃) 2.05, 2.11 (6H, 2 ꢂ s, 2 ꢂ CH₃), 3.88 (1H, dd, J_2,3

2.0 Hz, J_3,47.5 Hz, H-3), 3.97, 4.27 (2H, ABq, J_6,612.5 Hz, H-6, H-

6⁰), 4.38 (1H, br d, H-4), 4.46, 4.55 (2H, ABq, J_AB12.0 Hz, PhCH₂),

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

803

4.53, 4.75 (2H, ABq, J_AB11.5 Hz, PhCH₂), 4.66, 4.84 (2H, ABq, J_AB

11.0 Hz, PhCH₂), 5.54 (1H, m, H-1), 5.62 (1H, m, H-5a), 5.82 (1H,

m, H-2), 7.25–7.34 (15H, m, Ar-H); d_C(125 MHz, CDCl₃) 21.0,

21.1 (2 ꢂ q, 2 ꢂ CH₃), 68.3 (d, C-1), 68.7 (d, C-2), 69.9 (t, C-6),

71.7, 72.3, 75.3 (3 ꢂ t, 3 ꢂ PhCH₂), 76.6 (d, C-4), 79.7 (d, C-3),

122.5 (d, C-5a), 127.8, 127.8, 127.8, 128.0, 128.3, 128.5, 128.5,

128.6 (8 ꢂ d, Ar-CH), 137.6, 138.2, 138.5, 138.6 (4 ꢂ s, 3 ꢂ Ar-C,

C-5), 170.3, 170.7 (2 ꢂ s, 2 ꢂ C@O); m/z (ESI⁺) 569 (M+K⁺, 26),

553 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₃₂H₃₄O₇Na (M+Na⁺)

553.2197; found 553.2206.

solution of epoxide 16 (36 mg, 0.08 mmol) in THF (2 ꢂ 0.75 mL)

was added by cannula. After 14 h, TLC (toluene/EtOAc, 3:1) indi-

cated the formation of a major product (R_f0.6) and the complete

consumption of starting material (R_f0.7). The reaction was

quenched by addition of NaHCO₃(satd aq, 20 mL) and extracted

with EtOAc (20 mL). The organic phase was washed with NaHCO₃

(satd aq, 2 ꢂ 20 mL) and the combined aqueous fractions were ex-

tracted with EtOAc (2 ꢂ 20 mL). The combined organic phases

were dried (Na₂SO₄), ﬁltered and concentrated in vacuo. The resi-

due was puriﬁed by ﬂash column chromatography (pentane/EtOAc,

10:1?6:1?4:1) to give the azide 17 (32 mg, 77%) as a colourless

3.1.11. 3,4,6-Tri-O-benzyl-5a-carba-b-

-lyxo-hex-5(5a)-

oil; ½a ²_D³

ꢃ

¼ þ50:5 (c 1.0, CHCl₃) (lit.²⁵+36 (c, 1.3 in CHCl₃)); m_max

enopyra-nose 15

cm^ꢀ12100 (s, N₃); d_H(400 MHz, CDCl₃) 2.22 (1H, d, J_OH,27.8 Hz,

OH-2), 3.87–3.95 (3H, m, H-2, H-3, H-6), 4.05 (1H, d, J_3,42.9 Hz,

Diacetate 14 (520 mg, 0.98 mmol) was dissolved in MeOH

(7 mL) under Ar. Sodium (16 mg, 0.7 mmol) was dissolved in

MeOH (3 mL) and added to the reaction mixture. After 1 h, TLC

(pentane/EtOAc, 3:1) indicated the complete consumption of start-

ing material (R_f0.5) and the formation of a major product (R_f0.1).

The reaction mixture was concentrated in vacuo by co-evaporation

with toluene and the residue puriﬁed by ﬂash column chromatog-

raphy (pentane/EtOAc, 1:1?1:2) to yield diol 15 (415 mg, 95%) as

white crystals, mp 85–86 °C (EtOAc/pentane), (lit.²⁵79–85 °C).

H-4), 4.10 (1H, dat, J 1.5 Hz, J_6,612.4 Hz, H-6⁰), 4.15 (1H, br d, J_1,2

8.0 Hz, H-1), 4.36, 4.48 (2H, ABq, J_AB11.8 Hz, PhCH₂), 4.47 (1H, d,

J 11.8 Hz, PhCHH⁰), 4.53–4.62 (3H, m, 2 ꢂ PhCH₂, PhCHH⁰), 5.74

(1H, m, H-5a), 7.23–7.36 (15H, m, Ar-H); d_C(100 MHz, CDCl₃)

61.3 (d, C-1), 70.3 (t, C-6), 71.0 (d, C-2), 72.1, 72.8, 73.7 (3 ꢂ t,

3 ꢂ PhCH₂), 73.3 (d, C-4), 78.0 (d, C-3), 124.5 (d, C-5a), 127.8,

127.9, 128.0, 128.2, 128.2, 128.3, 128.5, 128.6, 128.7 (9 ꢂ d, Ar-

CH), 136.6 (s, C-5), 137.8, 137.9, 138.2 (3 ꢂ s, Ar-C); m/z (ESI⁺)

510 (M+K⁺, 20), 494 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for

C₂₈H₂₉N₃O₄Na 494.2050 (M+Na⁺); found 494.2027.

a ²_D³

ꢃ

¼ ꢀ69:1 (c 1.0, CHCl₃) (lit.²⁵ꢀ75.7);

_max/cm^ꢀ13417 (br s,

OH); d_H(500 MHz, CDCl₃) 2.64 (1H, d, J_OH,110.9 Hz, OH-1), 3.12

(1H, d, J_OH,28.4 Hz, OH-2), 3.89, 4.13 (2H, ABq, J_6,612.5 Hz, H-6,

H-6⁰), 3.92 (1H, ddd, J 1.0 Hz, J_2,32.0 Hz, J_3,43.6 Hz, H-3), 4.03–

4.06 (2H, m, H-1, H-2), 4.16 (1H, d, H-4), 4.36, 4.48 (2H, ABq, J_AB

11.9 Hz, PhCH₂), 4.45, 4.59 (2H, ABq, J_AB11.4 Hz, PhCH₂), 4.54,

4.57 (2H, ABq, J_AB11.7 Hz, PhCH₂), 5.99 (1H, d, J_1,5a4.2 Hz, H-5a),

7.22–7.34 (15H, m, Ar-H); d_C(125 MHz, CDCl₃) 66.7, 67.3 (2 ꢂ d,

C-1, C-2), 70.5 (t, C-6), 72.0, 73.3, 73.5 (3 ꢂ t, 3 ꢂ PhCH₂), 73.8 (d,

C-4), 79.8 (d, C-3), 127.7, 127.7, 128.0, 128.2, 128.3, 128.3, 128.3,

128.4, 128.5, 128.7 (10 ꢂ d, Ar-CH, C-5a), 135.6 (s, C-5), 137.6,

137.9, 138.1 (3 ꢂ s, 3 ꢂ Ar-C); m/z (ESI⁺) 485 (M+K⁺, 54), 469

(M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₂₈H₃₀O₅Na (M+Na⁺)

469.1985; found 469.1987.

3.1.14. 1,2-Di-O-trichloroacetimidoyl-3,4,6-tri-O-benzyl-5a-

carba-

Diol 10 (375 mg, 0.84 mmol) was dissolved in freshly distilled

CH₂Cl₂(15 mL) under Ar. Trichloroacetonitrile (420 L, 4.2 mmol)

and DBU (150 L, 1.0 mmol) were added, and the reaction mixture

a-D-lyxo-hex-5(5a)-enopyranose 19

turned from colourless to orange. After 1 h 20 min, the reaction

mixture turned dark brown and TLC (toluene/EtOAc, 5:1) indicated

the complete consumption of starting material (R_f0.1) and the for-

mation of a major product (R_f0.8). The reaction mixture was con-

centrated in vacuo and the residue puriﬁed by ﬂash column

chromatography (toluene with 1% Et₃N) to yield diimidate 19

(610 mg, 0.83 mmol, 99%) as a colourless oil; ½a _D²³

¼ þ38:1 (c 1.0,

ꢃ

3.1.12. 1,2-Anhydro-3,4,6-tri-O-benzyl-5a-carba-b-

-lyxo-hex-

CHCl₃); m

_max/cm^ꢀ13341 (s, N–H), 1662 (s, C@N); d_H(500 MHz,

CDCl₃) 3.92, 4.22 (2H, ABq, J_6,612.7 Hz, H-6, H-6⁰), 4.15 (1H, d,

J_3,43.6 Hz, H-4), 4.35–4.37 (2H, m, H-3, PhCHH⁰), 4.45, 4.64 (2H,

ABq, J_AB11.1 Hz, PhCH₂), 4.51 (1H, d, J 11.8 Hz, PhCHH⁰), 4.57,

4.79 (2H, ABq, J_AB11.9 Hz, PhCH₂), 5.75 (1H, d, J_1,27.6 Hz, H-2),

5.96 (1H, m, H-5a), 6.04 (1H, d, H-1), 7.28–7.35 (15H, m, Ar-H),

8.44, 8.54 (2H, 2 ꢂ s, 2 ꢂ NH); d_C(125 MHz, CDCl₃) 70.3 (t, C-6),

71.8, 73.3, 73.7 (3 ꢂ t, 3 ꢂ PhCH₂), 73.9 (d, C-1), 74.4 (d, C-3, C-

4), 76.5 (d, C-2), 91.4, 91.4 (2 ꢂ s, 2 ꢂ CCl₃), 123.6 (d, C-5a),

127.7, 127.9, 128.0, 128.1, 128.1, 128.5, 128.5, 128.6, 128.6

(9 ꢂ d, Ar-CH), 137.6 (s, C-5), 137.9, 138.1, 138.3 (3 ꢂ s, 3 ꢂ Ar-

C), 162.0, 162.2 (2 ꢂ s, 2 ꢂ C@N); m/z (ESI⁺) Isotope distribution

M+Na⁺: 762.0 (11), 761.0 (34), 760.0 (28), 759.0 (84), 758.0 (39),

757.0 (100), 756.0 (19), 755.0 (47). C₃₂H₃₀O₇N₂Cl₆Na requires

762.0 (12), 761.0 (34), 760.0 (28), 759.0 (79), 758.0 (35), 757.0

(100), 756.0 (19), 755.0 (52%); HRMS (ESI⁺) calcd for C₃₂H₃₀O₅N_2-

Cl₆Na (M+Na⁺) 755.0178; found 755.0199.

5(5a)-enopyranose 16

Triphenylphosphine (85 mg, 0.32 mmol) was dissolved in THF

(2 mL) and cooled to 0 °C. DIAD (70 L, 0.36 mmol) was added and

the reaction mixture was left for 25 min before DPPA (325 L,

1.5 mmol) and diol 10 (41 mg, 0.09 mmol, dissolved in 1 + 1 mL

THF) were added and the cooling was removed. After 17 h, TLC (pen-

tane/EtOAc, 1:1) indicated the formation of a major product (R_f0.8)

and the presence of some starting material (R_f0.1). The reaction mix-

ture was concentrated in vacuo and the residue puriﬁed by ﬂash col-

umnchromatography(pentane/EtOAc, 4:1?2:1)toyieldepoxide16

(28 mg, 71%) as a colourless oil; ½a _D²²

¼ þ21:5 (c 1.0, CHCl₃); d_H

ꢃ

(400 MHz, CDCl₃) 3.41 (1H, at, J 4.2 Hz, H-1), 3.57 (1H, dd, J_1,2

4.4 Hz, J_2,31.2 Hz, H-2), 3.96 (1H, dd, J_3,47.6 Hz, H-3), 4.05 (1H, d,

J_6,613.6 Hz, H-6), 4.18 (1H, dat, J 1.5 Hz, H-6⁰), 4.26 (1H, dat, J

1.2 Hz, H-4), 4.47, 4.52 (2H, ABq, J_AB12.0 Hz, PhCH₂), 4.69, 4.87

(2H, ABq, J_AB10.8 Hz, PhCH₂), 4.82, 4.86(2H, ABq, J_AB11.8 Hz, PhCH₂),

6.02 (1H, m, H-5a), 7.25–7.43 (15H, m, Ar-H); d_C(100 MHz, CDCl₃)

49.1 (d, C-1), 52.6 (d, C-2), 69.3 (t, C-6), 72.2, 72.4, 75.5 (3 ꢂ t,

3 ꢂ PhCH₂), 77.6 (d, C-4), 81.5 (d, C-3), 119.4 (d, C-5a), 127.7,

127.7, 127.7, 127.8, 127.9, 128.1, 128.4, 128.5 (8 ꢂ d, Ar-CH),

138.2, 138.3, 138.5 (3 ꢂ s, 3 ꢂ Ar-C), 144.0 (s, C-5); m/z (ESI⁺) 451

(M+Na⁺, 12), 469 (M+H₂O+Na⁺, 100%); HRMS (ESI⁺) calcd For

C₂₈H₂₈O₄Na (M+Na⁺) 451.1880; found 451.1902.

3.1.15. 3,4,6-Tri-O-benzyl-1-deoxy-1-trichloroacetamido-5a-

carba-b-D-lyxo-hex-5(5a)-enopyranose 20

Diimidate 19 (582 mg, 0.79 mmol) was dissolved in freshly dis-

tilled CH₂Cl₂(15 mL) under Ar and cooled to ꢀ78 °C. Boron triﬂuor-

ide diethyletherate (200 lL, 1.6 mmol) was added, and after 2 h

30 min, TLC (toluene/EtOAc, 4:1) indicated the presence of starting

material (R_f0.8) and the formation of a major product (R_f0.6) and a

minor product (R_f0.3). Further boron triﬂuoride diethyletherate

3.1.13. 1-Azido-3,4,6-tri-O-benzyl-1-deoxy-5a-carba-a-D-lyxo-

hex-5(5a)-enopyranose 17

(50 lL, 0.4 mmol) was added, and after a further 1 h 45 min, TLC

NaN₃(26 mg, 0.40 mmol) and NH₄Cl (9 mg, 0.17 mmol) were

indicated the complete consumption of starting material (R_f0.8)

dissolved in MeOH (3 mL) and H₂O (0.4 mL) and cooled to 0 °C. A

and the formation of major (R_f0.6) and minor (R_f0.3) products.

804

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

The reaction mixture was allowed to warm to rt and in this process

it changed from colourless to yellow. Water (150 L, 8.2 mmol)

Data for minor compound 21a: d_H(500 MHz, D₂O) 3.79–3.81

(3H, m, H-3, CH₂), 4.10–4.15 (2H, m, H-2, H-6), 4.19 (1H, d, J_6,6

was added, and after 20 min, TLC indicated the complete conver-

sion of the product with R_f0.6 to that with R_f0.3. The reaction

was quenched by addition of NaHCO₃(10 mL, satd aq). The reac-

tion mixture was diluted with EtOAc (30 mL), transferred to a

separatory funnel and washed with NaHCO₃(3 ꢂ 30 mL, satd aq).

The aqueous fractions were combined and extracted with EtOAc

(2 ꢂ 30 mL). The combined organic phases were dried (Na₂SO₄), ﬁl-

tered and concentrated in vacuo. The residue was puriﬁed twice by

ﬂash column chromatography; ﬁrst (pentane/EtOAc, 5:1?1:1) and

then (CH₂Cl₂/ether, 15:1) to yield amide 20 contaminated with tri-

14.0 Hz, H-6⁰), 4.27 (1H, d, J_3,47.5 Hz, H-4), 4.70 (1H, m, H-1),

5.48 (1H, m, H-5a); m/z (ESI⁺) 255 (M+Na⁺, 100), 233 (M+H⁺,

96%); HRMS (ESI⁺) calcd for C₉H₁₇N₂O₅233.1132 (M+H⁺); found

233.1120. Calcd for C₉H₁₆N₂O₅Na 255.0951 (M+Na⁺); found

255.0940.

The minor product was acetylated as follows: 21a (8 mg, con-

taining salts) was suspended in Ac₂O and pyridine at rt and left

for 5 days. The reaction mixture was diluted with EtOAc (20 mL)

and washed with 1 M HCl (3 ꢂ 10 mL). The combined aqueous

phases were extracted with EtOAc (2 ꢂ 10 mL) and the combined

organic phases washed with NaHCO₃(satd aq, 2 ꢂ 10 mL), dried

(Na₂SO₄), ﬁltered and concentrated in vacuo. The crude product

was puriﬁed by ﬂash column chromatography (EtOAc) to yield

22a (7 mg, 0.02 mmol) as a thin yellow ﬁlm. d_H(500 MHz, CDCl₃)

2.02, 2.03, 2.07, 2.07, 2.16 (15H, 5 ꢂ s, 5 ꢂ CH₃), 3.76 (1H, dd,

chloroacetamide (473 mg), as a yellow oil; m

_max/cm^ꢀ13385 (br, N–

H), 1710 (s, C@O); d_H(500 MHz, CDCl₃) 2.52 (1H, d, J_OH,26.9 Hz,

OH-2), 3.91–3.94 (2H, m, H-3 or H-4 and H-6), 4.15 (1H, dat, J

1.3 Hz, J_6,612.5 Hz, H-6⁰), 4.24 (1H, d, J_3,43.6 Hz, H-3 or H-4),

4.27 (1H, ddd, J 5.0 Hz, J 2.1 Hz, H-2), 4.40 (1H, d, J 11.8 Hz,

PhCHH⁰), 4.50–4.54 (3H, PhCHH⁰, 2 ꢂ PhCHH⁰), 4.59 (1H, d, J

11.0 Hz, PhCHH⁰), 4.64–4.68 (2H, m, H-1, PhCHH⁰), 5.80 (1H, d,

J_1,5a4.6 Hz, H-5a), 7.24–7.35 (15H, m, Ar-H), 7.56 (1H, d, J_1,NH

9.3 Hz, NH); d_C(125 MHz, CDCl₃) 49.1 (d, C-1), 66.4 (d, C-2), 70.3

(t, C-6), 72.3, 73.3, 73.7 (3 ꢂ t, 3 ꢂ PhCH₂), 73.5 (d, C-4), 79.6 (d,

C-3), 92.6 (s, CCl₃), 124.4 (d, C-5a), 127.9, 128.2, 128.3, 128.5,

128.5, 128.5, 128.6, 128.7 (8 ꢂ d, Ar-CH), 136.9, 137.2, 137.7,

137.9 (4 ꢂ s, C-5, 3 ꢂ Ar-C), 162.4 (s, C@O); m/z (ESI⁺) Isotope dis-

tribution M+Na⁺: 616.1 (31), 615.1 (31), 614.1 (100), 613.1 (33),

612.1 (97). 759.0 (84), 758.0 (39), 757.0 (100), 756.0 (19), 755.0

(47). C₃₀H₃₀O₅NCl₃Na requires 616.1 (31), 615.1 (32), 614.1 (97),

613.1 (33), 612.1 (100%). HRMS (ESI⁺) calcd for C₃₀H₃₀O₅NCl₃Na

(M+Na⁺) 612.1082; found 612.1062.

J_{CHH ,NH}5.5 Hz, J_gem15.5 Hz, CHH⁰), 3.96 (1H, dd, J_{CHH ,NH}5.5 Hz,

CHH⁰), 4.40, 4.68 (2H, ABq, J_6,613.3 Hz, H-6, H-6⁰), 5.03 (1H, m,

H-1), 5.18 (1H, dd, J_2,32.5 Hz, J_3,47.5 Hz, H-3), 5.49 (1H, m, H-2),

5.69 (1H, m, H-5a), 5.76 (1H, d, H-4), 6.39 (1H, at, J 5.3 Hz, NH),

6.73 (1H, d, J_NH,19.5 Hz, NH-1); d_H(125 MHz, CDCl₃) 20.9, 20.9,

21.0, 23.0 (4 ꢂ q, CH₃), 44.2 (t, CH₂), 46.3 (d, C-1), 63.2 (t, C-6),

68.0 (d, C-4), 69.5 (d, C-2), 71.9 (d, C-3), 127.5 (d, C-5a), 133.0 (s,

C-5), 169.0, 170.0, 170.3, 170.5, 170.5, 171.4 (6 ꢂ s, 6 ꢂ C@O); m/

z (ESI⁺) 465.2 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₁₉H₂₆N₂O₁₀Na

(M+Na⁺) 465.1485; found 465.1533.

3.1.17. 1,2-Bis-epi-valienamine 2

Method 1: Deprotection of acetamide 21. Acetamide 21 (40 mg,

0.18 mmol) was dissolved in water (4 mL, Milli-Q), LiOH (22 mg,

0.9 mmol) was added, and the reaction mixture was heated to

70 °C. After 1.5 h, TLC (MeOH/CHCl₃/NH₄OH (25%, aq), 5:5:1) indi-

cated the presence of mostly starting material (R_f0.6) and the for-

mation of small amounts of a product (R_f0.1). Further LiOH (15 mg,

0.6 mmol) was added, and after a further 18 h, TLC indicated the

presence of small amounts of starting material and the formation

of a major product (R_f0.1). Further LiOH (7 mg, 0.3 mmol) was

added and the reaction mixture stirred at 70 °C for another 7 h un-

til TLC indicated almost complete consumption of starting material

(R_f0.6) and the formation of a major product, which stained with

ninhydrin (R_f0.1). The reaction mixture was concentrated in vacuo

by co-evaporation with toluene. The crude yellow residue (67 mg)

was suspended in MeOH (2.5 mL) and ﬁltered through a plug of

cotton wool to remove salts. The procedure was repeated twice

(2 ꢂ 1 mL, MeOH). The yellow residue (51 mg) was puriﬁed by

ﬂash column chromatography (MeOH/CHCl₃/NH₄OH (25%, aq),

5:5:1) to yield impure material (13 mg) as a pale yellow ﬁlm. This

material was puriﬁed by reverse phase ﬂash column chromatogra-

phy (Waters Sep-Pak C18) eluting with water, to yield 2 (12 mg,

27% over 3 steps from 19) as a pale yellow solid. To ensure repro-

ducible NMR data, AcOH was added to form the acetate salt before

analysis: d_H(500 MHz, D₂O) 2.05 (s, CH₃), 3.87 (1H, dd, J_2,32.1 Hz,

J_3,46.4 Hz, H-3), 4.10 (1H, m, H-1), 4.20–4.22 (3H, m, H-2, H-6, H-

6⁰), 4.28 (1H, br d, H-4), 5.70 (1H, m, H-5a); d_C(125 MHz, D₂O) 21.0

(q, CH₃), 49.7 (d, C-1), 61.7 (t, C-6), 67.4 (d, C-2), 68.9 (d, C-4), 73.7

(d, C-3), 117.4 (d, C-5a), 143.4 (s, C-5), 177.5 (s, C@O); m/z (ESI⁺)

198 (M+Na⁺, 73), 176 (M+H⁺, 100%); HRMS (ESI⁺) calcd for

C₇H₁₄NO₄(M+H⁺) 176.0917; found 176.0923. Calcd for C₇H₁₃NO_4-

Na (M+Na⁺) 198.0737; found 198.0741.

NMR data for the oxazoline intermediate: d_H(400 MHz, CDCl₃)

3.94 (1H, d, J_6,612.4 Hz, H-6), 3.98 (1H, dd, J_2,33.9 Hz, J_3,45.2 Hz,

H-3), 4.04 (1H, d, H-4), 4.12 (1H, dat, J 1.7 Hz, H-6⁰), 4.38 (1H, d, J

12.4 Hz, PhCHH⁰), 4.43–4.45 (3H, m, PhCH₂, PhCHH⁰), 4.54, 4.75

(2H, ABq, J_AB12.4 Hz, PhCH₂), 4.82 (1H, dat, J_1,29.3 Hz, J 1.6 Hz,

H-1), 5.28 (1H, dd, H-2), 6.15 (1H, m, H-5a), 7.15–7.34 (15H, m,

Ar-H); d_C(100 MHz, CDCl₃) 64.5 (d, C-1), 71.3 (t, C-6), 71.9, 73.7,

73.9 (3 ꢂ t, 3 ꢂ PhCH₂), 72.8 (d, C-4), 73.8 (d, C-3), 81.1 (d, C-2),

94.4 (s, CCl₃), 124.3 (d, C-5a), 127.7, 127.8, 128.0, 128.1, 128.1,

128.2, 128.5, 128.5, 128.6 (9 ꢂ d, Ar-CH), 136.6 (s, C-5), 137.8,

137.9, 138.2 (3 ꢂ s, 3 ꢂ Ar-C), 162.9 (s, C@N).

3.1.16. 1-Acetamido-1-deoxy-5a-carba-b-D-lyxo-hex-5(5a)-enopy-

ranose 21

Amide 20 (131 mg, 0.22 mmol) was dissolved in THF (freshly

distilled, 5 mL) under Ar and cooled to ꢀ78 °C. Ammonia (ca.

15 mL) was condensed into the reaction ﬂask and sodium was

added (115 mg, 5 mmol). After a few minutes, the reaction mixture

turned dark blue. After 50 min, the reaction mixture turned yellow,

and more sodium (18 mg, 0.8 mmol) was added, after which the

blue colour reappeared. After another 30 min, the reaction mixture

was quenched by addition of solid NH₄Cl (312 mg, 5.8 mmol) and

left to warm to rt in order for the ammonia to evaporate. The crude

residue was puriﬁed by ﬂash column chromatography (MeOH/

CHCl₃/NH₄OH (25%, aq), 5:5:1), to give major 21 (67 mg, R_f0.6)

and minor 21a (8 mg, R_f0.2) products. The major product was puri-

ﬁed from salts by repeating the ﬂash column chromatography

(MeOH/CHCl₃/NH₄OH (25%, aq), 5:5:1) yielding 21 (40 mg, 83%

from 19) as a pale yellow solid. d_H(500 MHz, D₂O) 2.05 (3H, s,

CH₃), 3.82 (1H, dd, J_2,32.2 Hz, J_3,47.9 Hz, H-3), 4.12 (1H, ddd, J_2,5a

1.5 Hz, J_1,23.7 Hz, H-2), 4.16 (1H, m, H-6), 4.23 (1H, m, H-6⁰),

4.31 (1H, d, H-4), 4.65 (1H, m, H-1), 5.50 (1H, m, H-5a); d_C

(125 MHz, D₂O) 22.0 (q, CH₃), 49.2 (d, C-1), 61.4 (t, C-6), 69.1 (d,

C-4), 70.6 (d, C-2), 74.0 (d, C-3), 122.1 (d, C-5a), 139.3 (s, C-5),

173.8 (s, C@O); m/z (ESI⁺) 240 (M+Na⁺, 100%); HRMS (ESI⁺) calcd

for C₉H₁₅NO₅Na (M+Na⁺) 240.0842; found 240.0839.

Method 2: Deprotection of pentaacetate 22. Pentaacetate 22

(17 mg, 0.04 mmol) was dissolved in THF (1 mL), and water

(1 mL) was added. LiOH (19 mg, 0.8 mmol) was added and the

reaction mixture was heated to 70 °C. After 15 h, TLC (MeOH/

CHCl₃/NH₄OH (25%, aq), 5:5:1) indicated the complete consump-

tion of starting material (R_f0.9) and the formation of a major prod-

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

805

uct (R_f0.1), which stained with ninhydrin. The reaction mixture

was concentrated in vacuo and the crude product puriﬁed by ﬂash

column chromatography (MeOH/CHCl₃/NH₄OH (25%, aq), 5:5:1) to

yield the free base 2 (6 mg, 77%) as a pale yellow ﬁlm. Addition of

AcOH to form the acetate salt gave NMR spectral data identical to

that described above.

were dried (Na₂SO₄), ﬁltered and concentrated in vacuo. The resi-

due was puriﬁed by ﬂash column chromatography (pentane/EtOAc,

5:1?4:1) to yield azide 23 (98 mg, 0.16 mmol, 63% from 19) as a

yellow oil; ½a ²_D³

ꢃ

¼ ꢀ97:8 (c 1.0, CHCl₃);

_max/cm^ꢀ13328 (br, N–

H), 2110 (s, N₃), 1696 (s, C@O); d_H(500 MHz, CDCl₃) 3.75 (1H,

dd, J_1,26.3 Hz, J_2,37.7 Hz, H-2), 3.85 (1H, dd, J_3,45.3 Hz, H-3), 3.94

(1H, d, J_6,612.1 Hz, H-6), 4.19–4.24 (2H, m, H-4, H-6⁰), 4.45–4.52

3.1.18. 1-Acetamido-2,3,4,6-tetra-O-acetyl-1-deoxy-5a-carba-b-

(3H, m, H-1, PhCH₂), 4.65–4.70 (3H, m, PhCHH⁰, PhCH₂), 4.74 (1H,

d, J 11.3 Hz, PhCHH⁰), 5.70 (1H, m, H-5a), 6.98 (1H, d, J_NH,19.0 Hz,

NH), 7.26–7.36 (15H, m, Ar-H); d_C(100 MHz, CDCl₃) 50.6 (d, C-1),

62.7 (d, C-2), 70.1 (t, C-6), 72.8, 74.5, 74.7 (3 ꢂ t, 3 ꢂ PhCH₂), 75.7

(d, C-4), 79.9 (d, C-3), 92.4 (s, CCl₃), 122.7 (d, C-5a), 128.0, 128.0,

128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 128.7 (9 ꢂ d, Ar-CH),

137.1, 137.8, 137.9, 138.4 (4 ꢂ s, 3 ꢂ Ar-C, C-5), 161.6 (s, C@O);

m/z (ESI⁺) Isotope distribution M+Na⁺: 641.1 (28), 640.1 (28),

639.1 (100), 638.1 (30), 637.1 (100). C₃₀H₂₉O₄N₄Cl₃Na requires

641.1 (31), 640.1 (32), 639.1 (96), 638.1 (33), 637.1 (100%); HRMS

(ESI⁺) calcd for C₃₀H₂₉Cl₃N₄O₄Na (M+Na⁺) 637.1147; found

637.1123.

-lyxo-hex-5(5a)-enopyranose 22

Trichloroacetamide 20 (50 mg, 0.08 mmol) was dissolved in

THF (freshly distilled, 3 mL) under Ar and cooled to ꢀ78 °C. Ammo-

nia (ca. 10 mL) was condensed into the reaction ﬂask and sodium

was added (50 mg, 2.2 mmol). After a few minutes, the reaction

mixture turned dark blue. After a further 10 min, the reaction mix-

ture turned yellow and further sodium (30 mg, 1.3 mmol) was

added, after which the blue colour reformed. After a further

20 min, the reaction was quenched by addition of NH₄Cl

(390 mg, 7.3 mmol) and left to warm to rt for the ammonia to

evaporate.

The solid residue, crude 21, was suspended in pyridine (1 mL,

12 mmol) and acetic anhydride (1 mL, 11 mmol) and left for 5 h.

The reaction mixture was diluted with EtOAc and washed with

1 M HCl (3 ꢂ 10 mL). The aqueous phases were extracted with

EtOAc (2 ꢂ 10 mL) and the combined organic phases were washed

with satd aq NaHCO₃(2 ꢂ 10 mL), dried (MgSO₄), ﬁltered and con-

centrated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (toluene/EtOAc, 1:2) to yield 22 (20 mg, 61%) as a

yellow oil, which was recrystallised to give white crystals, mp 177–

178 °C (EtOAc/pentane); d_H(400 MHz, CDCl₃) 1.99, 2.04, 2.08, 2.08,

3.1.20. 2-Azido-3,4,6-tri-O-benzyl-1-(tert-butyloxycarbonylami-

no)-1,2-dideoxy-5a-carba-b-D-xylo-hex-5(5a)-enopyranose 24

Trichloroacetamide 23 (70 mg, 0.11 mmol) was dissolved in i-

PrOH (3 mL) under Ar. KOH (38 mg, 0.7 mmol) was added, and

the reaction mixture was heated to 40 °C. After 17.5 h, TLC (tolu-

ene/EtOAc, 1:1) indicated the formation of a major product (R_f

0.1) and the presence of starting material (R_f0.95). Further KOH

(22 mg, 0.4 mmol) was added, and the reaction mixture was stirred

at 40 °C for a further 1 h 50 min. The solvent was then removed by

co-evaporation with toluene, and the residue was suspended in

2.14 (15H, 5 ꢂ s, 5 ꢂ CH₃), 4.43, 4.66 (2H, ABq, J_6,613.4 Hz, H-6, H-

6⁰), 5.09 (1H, m, H-1), 5.20 (1H, dd, J_2,32.1 Hz, J_3,46.8 Hz, H-3), 5.51

(1H, dd, J_1,24.4 Hz, H-2), 5.67–5.74 (3H, m, H-4, H-5a, NH); d_C

(100 MHz, CDCl₃) 20.9, 20.9, 20.9, 21.0, 23.3 (5 ꢂ q, 5 ꢂ CH₃), 45.9

(d, C-1), 63.3 (t, C-6), 67.8 (d, C-4), 69.6 (d, C-2), 71.9 (d, C-3),

128.0 (d, C-5a), 132.7 (s, C-5), 169.5, 169.7, 170.2, 170.2, 170.5

(5 ꢂ s, 5 ꢂ C@O); m/z (ESI⁺) 408 (M+Na⁺, 100%); HRMS (ESI⁺) calcd

for C₁₇H₂₃NO₉Na 408.1265; found 408.1265.

CH₂Cl₂(5 mL) under Ar. Di-tert-butyl dicarbonate (240 lL,

1.0 mmol) was added, and after 2.5 h, TLC (toluene/EtOAc, 1:1)

indicated the formation of a major product (R_f0.9). The reaction

mixture was concentrated in vacuo and the residue puriﬁed by

ﬂash column chromatography (toluene?toluene/EtOAc, 10:1)

yielding 24 (44 mg, 68%) as a white solid; ½a _D²³

¼ ꢀ98:1 (c 1.0,

ꢃ

CHCl₃); m

_max/cm^ꢀ13342 (br, N–H), 2107 (s, N₃), 1691 (br, C@O);

d_H(500 MHz, CDCl₃) 1.47 (9H, s, C(CH₃)₃), 3.53 (1H, at, J 9.3 Hz,

3.1.19. 2-Azido-3,4,6-tri-O-benzyl-1,2-dideoxy-1-trichloroacety-

H-2), 3.73 (1H, dd, J_2,310.0 Hz, J_3,47.7 Hz, H-3), 3.88 (1H, d, J_6,6

lamido-5a-carba-b-

Amide 20 (153 mg, 0.26 mmol, containing traces of trichloroac-

etamide) was dissolved in CH₂Cl₂(5 mL, freshly distilled) under Ar

and cooled to 0 °C. Pyridine (65

anesulfonic anhydride (130 L, 0.8 mmol) were added, and the col-

ourless solution instantly turned yellow. After 1 h 15 min, TLC

(toluene/EtOAc, 5:1) indicated the formation of a major product

(R_f0.8) and the presence of starting material (R_f0.3). Further pyr-

-xylo-hex-5(5a)-enopyranose 23

11.5 Hz, H-6), 4.17–4.27 (3H, m, H-1, H-4, H-6⁰), 4.46, 4.49 (2H,

ABq, J_AB12.0 Hz, PhCH₂), 4.66–4.68 (2H, m, NH, PhCHH⁰), 4.77,

4.88 (2H, ABq, J_AB10.8 Hz, PhCH₂), 4.78 (1H, d, J 10.5 Hz, PhCHH⁰),

5.60 (1H, s, H-5a), 7.25–7.38 (15H, m, Ar-H); d_C(125 MHz, CDCl₃)

28.5 (q, C(CH₃)₃), 51.8 (d, C-1), 66.2 (d, C-2), 70.1 (t, C-6), 72.7,

74.8, 75.4 (3 ꢂ t, 3 ꢂ PhCH₂), 78.8 (d, C-4), 80.3 (s, C(CH₃)₃), 82.6

(d, C-3), 126.2 (d, C-5a), 128.0, 128.0, 128.2, 128.3, 128.4, 128.5,

128.6, 128.7, 128.8 (9 ꢂ d, Ar-CH), 137.0, 137.8, 138.1, 138.2

(4 ꢂ s, 3 ꢂ Ar-C, C-5), 155.3 (s, C@O); m/z (ESI⁺) 609 (M+K⁺, 20),

593 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for C₃₃H₃₈N₄O₅Na

(M+Na⁺) 593.2734; found 593.2735.

lL, 0.8 mmol) and triﬂuorometh-

idine (50

lL, 0.6 mmol) and triﬂuoromethanesulfonic anhydride

(100 L, 0.6 mmol) were added, and the reaction mixture changed

from yellow to orange-brown. After 20 min, TLC (toluene/EtOAc,

5:1) indicated the formation of a major product (R_f0.8) and the

complete consumption of starting material (R_f0.3). The reaction

mixture was diluted with CH₂Cl₂(30 mL) and washed with ice-

water (3 ꢂ 30 mL). The aqueous phases were combined and ex-

tracted with CH₂Cl₂(2 ꢂ 30 mL). The combined organic phases

were dried (Na₂SO₄), ﬁltered and concentrated in vacuo to yield

crude triﬂate (227 mg) as a yellow oil, which was used without fur-

ther puriﬁcation.

Crude triﬂate (227 mg, 0.26 mmol) was dissolved in DMF (5 mL)

under Ar. Sodium azide (170 mg, 2.6 mmol) was added, and the

reaction mixture was heated to 70 °C. After 9.5 h, TLC (toluene/

EtOAc, 5:1) indicated the formation of a major product (R_f0.6)

and the complete consumption of starting material (R_f0.7). The

reaction mixture was diluted with CH₂Cl₂(40 mL) and washed

with brine (3 ꢂ 30 mL). The combined aqueous phases were ex-

tracted with CH₂Cl₂(2 ꢂ 30 mL) and the combined organic phases

3.1.21. 2-Acetamido-3,4,6-tri-O-acetyl-1-(tert-butyloxycarbo-

nylamino)-1,2-dideoxy-5a-carba-b-D-xylo-hex-5(5a)-

enopyranose 25

Azide 24 (15 mg, 0.026 mmol) was dissolved in THF (1 mL) un-

der Ar and cooled to ꢀ78 °C. Ammonia (5 mL) was condensed into

the ﬂask and sodium (13 mg) was added, after which the reaction

mixture turned dark blue within a couple of minutes. Over the next

45 min, sodium (10 mg + 6 mg, in total 1.3 mmol) was added twice

in order to maintain the blue colour of the reaction mixture, after

which the reaction was quenched by addition of solid NH₄Cl

(67 mg, 1.3 mmol). The ammonia was allowed to evaporate, and

the reaction mixture was concentrated in vacuo. The residue was

suspended in acetic anhydride (1 mL) and pyridine (1 mL). After

3 h 15 min, TLC (toluene/EtOAc, 1:2) indicated formation of a ma-

jor product (R_f0.1), a by-product (R_f0) and the complete consump-

806

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

tion of starting azide 24 (R_f0.9). The reaction was quenched by the

addition of HCl (1 M aq, 20 mL) and extracted with EtOAc (20 mL).

The organic phase was washed with HCl (1 M aq, 2 ꢂ 20 mL) and

the aqueous phases were extracted with EtOAc (2 ꢂ 20 mL). The

combined organic phases were then washed with NaHCO₃(satd

aq, 3 ꢂ 20 mL), dried (Na₂SO₄), ﬁltered and concentrated in vacuo.

The residue was puriﬁed by ﬂash column chromatography (tolu-

ene/EtOAc, 1:2?1:3, with 1% NEt₃) to yield 25 (10 mg, 87%) as a

from Megazyme (Bray, Ireland). Each enzyme was dialysed into

the respective assay buffer. Both enzyme preparations appeared

as single bands when analysed by SDS–PAGE (Coomassie staining)

using a 6 cm 4–12% gradient gel (Invitrogen Life Sciences, Carlsbad,

CA, USA). Protein concentrations were determined using the Micro

BCA^TMProtein Assay Reagent Kit with BSA standards (Pierce, Rock-

ford, IL, USA). All other chemicals were obtained from Sigma Al-

drich Chemical Co. (Schnelldorf, Germany) and were of reagent

grade.

white solid; ½a ²_D³

ꢃ

¼ ꢀ65:6 (c 0.7, CHCl₃);

_max/cm^ꢀ13363 (s, N–

H), 3263 (s, N–H), 1748 (s, C@O), 1688 (s, C@O), 1653 (s, C@O);

d_H(500 MHz, CDCl₃) 1.42 (9H, s, C(CH₃)₃), 1.94, 2.04, 2.05, 2.06

(12H, 4 ꢂ s, 4 ꢂ C(O)CH₃), 4.25 (1H, aq, J 10.3 Hz, H-2), 4.36, 4.66

3.3. Steady-state initial velocity kinetics

(2H, ABq, J_6,612.8 Hz, H-6, H-6⁰), 4.42 (1H, at, J 9.5 Hz, H-1), 4.91

All kinetic experiments were performed using 1 cm path length

cuvettes in a Varian CARY 4000 spectrophotometer attached to a

temperature control unit. CfMan2A initial velocity assays were car-

ried out in 50 mM sodium phosphate buffer, pH 7.0 at 25 °C. The

enzyme and substrate concentrations in the assays were

(1H, d, J_NH,19.5 Hz, NH), 5.11 (1H, dd, J_2,311.5 Hz, J_3,47.8 Hz, H-

3), 5.80 (1H, s, H-5a), 5.84 (1H, d, H-4), 5.89 (1H, d, J_NH,29.5 Hz,

NH); d_C(125 MHz, CDCl₃) 20.8, 20.8, 20.9, 23.3 (4 ꢂ q,

4 ꢂ C(O)CH₃), 28.4 (q, C(CH₃)₃), 52.0 (d, C-1), 53.9 (d, C-2), 62.9

(t, C-6), 70.6 (d, C-4), 73.4 (d, C-3), 80.4 (s, C(CH₃)₃), 130.3 (d, C-

5a), 132.8 (s, C-5), 156.2 (s, C@O), 170.0, 170.6, 171.0, 171.5

(4 ꢂ s, 4 ꢂ C@O); m/z 465 (M+Na⁺, 100%); HRMS (ESI⁺) calcd for

C₂₀H₃₀N₂O₉Na (M+Na⁺) 465.1844; found 465.1862.

1.53 l

g mL^–1CfMan2A and 0.05–0.40 mM para-nitrophenyl b-

mannoside (pNPMan) at each of a series of the 1,2-bis-epi-valien-

amine inhibitor concentrations of 0.00, 0.10, 0.45 and 0.60 mM.

pNPMan concentrations lower than 0.5 mM were used in this

study to avoid complications arising from substrate inhibition at

higher concentrations as reported previously.^8cThe enzyme and

the inhibitor in the assay buffer were pre-incubated at 25 °C for

10 min before addition of the substrate. CfMan2A-catalysed initial

rates of hydrolysis of pNPMan were determined by following the

increase in the absorbance at 400 nm due to the release of para-

nitrophenolate into solution. The initial velocity curves (V_ovs [S])

were hyperbolic with respect to substrate concentration and ﬁtted

to the standard Michaelis–Menten equation using nonlinear

regression with the program GraphPad Prism 4 (GraphPad Soft-

ware, San Diego, CA, USA). The initial velocity data were also plot-

ted in the form of a Lineweaver-Burk double-reciprocal plot (1/V_o

vs 1/[S]) using linear regression with the same software. The values

3.1.22. 2-Acetamido-1-(tert-butyloxycarbonylamino)-1,

2-dideoxy-5a-carba-b-D-xylo-hex-5(5a)-enopyranose 26

Tetraacetate 25 (14 mg, 0.03 mmol) was dissolved in MeOH

(1.5 mL). Sodium (8 mg, 0.17 mmol) was dissolved in MeOH

(0.5 mL) and added to the reaction mixture. After 1 h, TLC (tolu-

ene/EtOAc, 1:1) indicated the complete consumption of starting

material (R_f0.2) and the formation of a major product (R_f0). The

reaction mixture was neutralised by the addition of Dowex

50WX8 (H⁺) resin. After ﬁltration, the reaction mixture was con-

centrated in vacuo and the residue was puriﬁed by ﬂash column

chromatography (MeOH/EtOAc, 1:4) to yield triol 26 (8 mg, 79%)

as a colourless oil; d_H(500 MHz, D₂O) 1.41 (9H, s, C(CH₃)₃), 2.01

(3H, s, C(O)CH₃), 3.66 (1H, dd, J_2,311.3 Hz, J_3,48.2 Hz, H-3), 3.89

of apparent K_m(K^app) were then determined from the x-intercepts

(1H, at, J 10.5 Hz, H-2), 4.10 (1H, d, J_6,613.5 Hz, H-6), 4.18–4.23

of the double-reciprocal plot, replotted versus inhibitor concentra-

tions and ﬁtted using linear regression with the same software.

(2H, m, H-1, H-6⁰), 4.27 (1H, d, H-4), 5.55 (1H, s, H-5a); d_C

(125 MHz, D₂O) 22.2 (q, C(O)CH₃), 27.6 (q, C(CH₃)₃), 51.8 (d, C-1),

55.2 (d, C-2), 61.1 (t, C-6), 72.0 (d, C-4), 73.9 (d, C-3), 81.2 (s,

C(CH₃)₃), 124.3 (d, C-5a), 139.0 (s, C-5), 157.6 (s, C@O), 174.6 (s,

C@O).

Possible inhibition of CfMan2A activity by

tested in the assay buffer at 25 °C. The enzyme and substrate con-

centrations in the assays were 1.53

g mL^ꢀ1CfMan2A and

0.40 mM pNPMan at each of a series of the inhibitor ( -mannose)

concentrations of 0.00, 0.50 and 1.00 mM. CfMan2A and -man-

D-mannose was also

3.1.23. 1-epi-2-Acetamido-2-deoxy-valienamine hydrochloride 27

Boc-protected compound 26 (8 mg, 0.025 mmol) was dissolved

in water (1.5 mL), and HCl (12 M, 1.0 mL) was added dropwise.

After 15 min, TLC (EtOAc/MeOH, 1:2) indicated the complete con-

sumption of starting material (R_f0.9) and the formation of a major

product (R_f0.3). The reaction mixture was concentrated in vacuo

and the residue puriﬁed twice by reverse phase ﬂash column chro-

matography (Waters Sep-Pak aminopropyl and C18 cartridges) to

yield the GlcNAc analogue 27 (5.9 mg, 93%) as a pale yellow pow-

nose in the assay buffer were pre-incubated at 25 °C for 10 min be-

fore addition of the substrate.

AnGlu initial velocity assays were carried out in 50 mM sodium

acetate buffer, pH 4.0 at 40 °C. The enzyme and substrate concen-

trations in the assays were 6.33 l

g mL^ꢀ1AnGlu and 1.00 mM para-

nitrophenyl b-glucoside (pNPGlc) at each of a series of the 1,2-bis-

epi-valienamine inhibitor concentrations of 0.00, 0.60 and

2.00 mM. The enzyme and the inhibitor in the assay buffer were

pre-incubated at 40 °C for 15 min before addition of the substrate.

AnGlu-catalysed initial rates of hydrolysis of pNPGlc were deter-

mined by following the increase in the absorbance at 400 nm

due to the release of para-nitrophenolate into solution.

der; ½a ²_D³

ꢃ

¼ ꢀ65:6 (c 0.7, H₂O); (lit.⁶ꢀ59.6); d_H(500 MHz, D₂O)

2.09 (3H, s, C(O)CH₃), 3.72 (1H, dd, J_2,310.9 Hz, J_3,48.0 Hz, H-3),

4.04 (1H, m, H-1), 4.12 (1H, dd, J_1,29.8 Hz, H-2), 4.20, 4.27 (2H,

ABq, J_6,614.8 Hz, H-6, H-6⁰), 4.32 (1H, d, H-4), 5.69 (1H, m, H-

5a); d_C(125 MHz, D₂O) 22.2 (q, C(O)CH₃), 52.5 (d, C-1), 52.6 (d,

C-2), 60.7 (t, C-6), 71.6 (d, C-4), 72.8 (d, C-3), 117.0 (d, C-5a),

143.6 (s, C-5), 175.5 (s, C@O); m/z (ESI⁺) 239 (M+Na⁺, 100), 217

(M+H⁺, 54%); HRMS (ESI⁺) calcd for C₉H₁₇N₂O₄217.1183 (M+H⁺);

found 217.1180. Calcd for C₉H₁₆N₂O₄Na 239.1002 (M+Na⁺); found

239.0999.

3.4. X-ray data collection, structure solution and reﬁnement

Crystals for diffraction were grown by slow diffusion of pentane

into a EtOAc solution of the compound for each of 15, 22 and 18.

The resulting crystals were measured with an Oxford Diffraction

Excalibur-II

using Mo K

diffractometer equipped with a sapphire-3 CCD

(k = 0.71073 Å) radiation (DIAMOND (Version 2.1e, Crys-

3.2. Enzyme assay: General materials and methods

tal Impact, Germany), ^{CRYSALIS CCD}and CRYSALIS RED (Version 1.170, Ox-

ford Diffraction Ltd, UK)). All measurements were made at ambient

temperature. The structures were solved using standard direct

methods using ^SHELXS-97 and reﬁned with full-matrix least-square

Recombinant C. ﬁmi b-mannosidase (CfMan2A),^8a(Lot. 30901)

and A. niger b-glucosidase (AnGlu), (Lot. 90301) were purchased

C. Ramstadius et al. / Tetrahedron: Asymmetry 20 (2009) 795–807

807

Table 1

2. Chen, X.; Fan, Y.; Zheng, Y.; Shen, Y. Chem. Rev. 2003, 103, 1955–1977.

3. Ogawa, S.; Kanto, M.; Suzuki, Y. Mini-Rev. Med. Chem. 2007, 7, 679–691.

Crystal data for compounds 15, 18 and 22

4. Ogawa, S.; Ashiura, M.; Uchida, C.; Watanabe, S.; Yamazaki, C.; Yamagishi, K.;

Inokuchi, J.-i. Bioorg. Med. Chem. Lett. 1996, 6, 929–932.

Compound

5. Ogawa, S.; Sakata, Y.; Ito, N.; Watanabe, M.; Kabayama, K.; Itoh, M.; Korenaga,

T. Bioorg. Med. Chem. 2004, 12, 995–1002.

6. Scafﬁdi, A.; Stubbs, K. A.; Dennis, R. J.; Taylor, E. J.; Davies, G. J.; Vocadlo, D. J.;

Stick, R. V. Org. Biomol. Chem. 2007, 5, 3013–3019.

7. Ademark, P.; de Fries, R. P.; Hägglund, P.; Stålbrand, H.; Visser, J. Eur. J. Biochem.

2001, 268, 2982–2990.

8. (a) Stoll, D.; Stålbrand, H.; Warren, R. A. J. Appl. Environ. Microbiol. 1999, 65,

2598–2605; (b) Stoll, D.; He, S.; Withers, S. G.; Warren, R. A. J. Biochem. J. 2000,

351, 833–838; (c) Zechel, D. L.; Reid, S. P.; Stoll, D.; Nashiru, O.; Warren, R. A. J.;

Withers, S. G. Biochemistry 2003, 42, 7195–7204.

Formula

C₂₈H₃₀O₅

446.52

293(2)

P22₁2₁(18)

5.8194(3)

18.9944(10)

22.2329(11)

2457.5(2)

1.207

C₁₇H₂₃NO₉

385.36

293(2)

C₁₇H₂₃NO₉

385.36

293(2)

P2₁2₁2₁(19)

7.93420(10)

11.4114(2)

22.9755(4)

Formula weight

Temperature (K)

Space group

a (Å)

b (Å)

c (Å)

P2₁(4)

9.1983(2)

8.5360(2)

12.8687(3)

98.731(2)

998.70(4)

1.281

0.105

12,985, 3251,

2381

0.0375

250

(°)

b (°)

9. Lundqvist, J.; Jacobs, A.; Palm, M.; Zacchi, G.; Dahlman, O.; Stålbrand, H.

Carbohydr. Polym. 2003, 51, 203–211.

(°)

V (Å³)

2080.21(6)

1.230

0.100

39,175, 7001,

2680

10. (a) Tailford, L. E.; Money, V. A.; Smith, N. L.; Dumon, C.; Davies, G. J.; Gilbert, H.

J. J. Biol. Chem. 2007, 282, 11291–11299; (b) Dias, F. M.; Vincent, F.; Pell, G.;

Prates, J. A.; Centeno, M. S.; Tailford, L. E.; Ferreira, L. M. A.; Fontes, C. M. G. A.;

Davies, G. J.; Gilbert, H. J. J. Biol. Chem. 2004, 279, 25516–25517.

11. (a) Sabini, E.; Schubert, H.; Murshudov, G.; Wilson, K. S.; Siika-aho, M.; Penttilä,

M. Acta Crystallogr., Sect. D 2000, 56, 3–13; (b) Larsson, A. M.; Anderson, L.; Xu,

B.; Munoz, I. G.; Uson, I.; Janson, J.-C.; Stålbrand, H.; Ståhlberg, J. J. Mol. Biol.

2006, 357, 1500–1510.

Density

(mm^ꢀ1

)

0.082

15,542, 4337,

1237

0.1091

265

N_meas, N_unique, N_obs

R_int

N_par

0.0503

249

wR₂(all), R₁(all)

wR₂(obs), R₁(obs)

Completeness, h

Residual density (e/

Å³)

0.1293, 0.2014

0.1043, 0.0514

0.996, 25.03

ꢀ0.199 to 0.288

0.0856, 0.0590

0.0809, 0.0384

0.995, 25.03

ꢀ0.112 to 0.142

0.1097, 0.1230

0.1025, 0.0436

0.965, 32.25

ꢀ0.182 to 0.247

12. (a) Borges de Melo, E.; da Silveira Gomes, A.; Carvalho, I. Tetrahedron 2006, 62,

10277–10302; (b) Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev.

2002, 102, 515–553.

13. (a) Ducros, V. M.-A.; Zechel, D. L.; Murshudov, G. N.; Gilbert, H. J.; Szabo,

L.; Stoll, D.; Withers, S. G.; Davies, G. J. Angew. Chem., Int. Ed. 2002, 41,

2824–2827; (b) Davies, G. J.; Ducros, V. M.-A.; Varrot, A.; Zechel, D. L.

Biochem. Soc. Trans. 2003, 31, 523–527; (c) Money, V. A.; Smith, N. L.;

Scafﬁdi, A.; Stick, R. V.; Gilbert, H. J.; Davies, G. J. Angew. Chem., Int. Ed.

2006, 45, 5136–5140.

methods using SHELXL-97.³¹Compound 15 was the weakest scat-

terer of the three compounds, a fact that can describe the rather

high internal R-value for this compound. However, this is mainly

a result of the large amount of weak reﬂections. No extra absorp-

tion correction except the scaling applied by the diffractometer

software was applied due to the low value of the linear absorption

coefﬁcient. The absolute conﬁguration was set from the known

14. Tailford, L. E.; Offen, W. A.; Smith, N. L.; Dumon, C.; Morland, C.; Gratien, J.;

Heck, M.-P.; Stick, R. V.; Bleriot, Y.; Vasella, A.; Gilbert, H. J.; Davies, G. J. Nat.

Chem. Biol. 2008, 4, 306–312.

15. (a) Böhm, M.; Lorthiois, E.; Meyyappan, M.; Vasella, A. Helv. Chim. Acta 2003,

86, 3787–3817; (b) Terinek, M.; Vasella, A. Helv. Chim. Acta 2003, 86, 3482–

3509; (c) Heck, M.-P.; Vincent, S. P.; Murray, B. W.; Bellamy, F.; Wong, C.-H.;

Mioskowski, C. J. Am. Chem. Soc. 2004, 126, 1971–1979; (d) Amorim, L.; Diaz,

D.; Calle-Jimenez, L. P.; Jimenez-Barbero, J.; Sinay, P.; Bleriot, Y. Tetrahedron

Lett. 2006, 47, 8887–8891.

16. (a) Molyneux, R. J.; Pan, Y. T.; Tropea, J. E.; Elbein, A. D.; Lawyer, C. H.; Hughes,

D. J.; Fleet, G. W. J. J. Nat. Prod. 1993, 56, 1256–1364; (b) Yoda, H.; Shimojo, T.;

Takabe, K. Tetrahedron Lett. 1999, 40, 1335–1336.

17. Fleet, G. W. J.; Shing, T. K. M. J. Chem. Soc., Chem. Commun. 1983, 849–850.

18. (a) Madsen, R. Eur. J. Org. Chem. 2007, 399–415; (b) Arjona, O.; Gomez, A. M.;

Lopez, J. C.; Plumet, J. Chem. Rev. 2007, 107, 1919–2036.

19. For metathesis approaches to carbocycles unsaturated between C-5 and C-5a

(carbapyranoses) or C-4 and C-4a (carbafuranoses), see: (a) Callam, C. S.;

Lowary, T. L. Org. Lett. 2000, 2, 167–169; (b) Callam, C. S.; Lowary, T. L. J. Org.

Chem. 2001, 66, 8961–8972; (c) Seepersaud, M.; Al-Abed, Y. Tetrahedron Lett.

2000, 41, 7801–7803; (d) Gillaizeau, I.; Charamon, S.; Agrofoglio, L. A.

Tetrahedron Lett. 2001, 42, 8817–8819; (e) Whalen, L. J.; Halcomb, R. L. Org.

Lett. 2004, 6, 3221–3224; (f) Chang, Y.-K.; Lee, B.-Y.; Kim, D. J.; Lee, G. S.; Jeon,

H. B.; Kim, K. S. J. Org. Chem. 2005, 70, 3299–3302; (g) Cho, J. H.; Bernard, D. L.;

Sidwell, R. W.; Kern, E. R.; Chu, C. K. J. Med. Chem. 2006, 49, 1140–1148; (h)

Wang, Y.; Bennet, A. J. Org. Biomol. Chem. 2007, 5, 1731–1738; (i) Cumpstey, I.

Tetrahedron Lett. 2005, 46, 6257–6259; (j) Frigell, J.; Cumpstey, I. Tetrahedron

Lett. 2007, 48, 9073–9076; (k) Cumpstey, I.; Gehrke, S.; Erfan, S.; Cribiu, R.

Carbohydr. Res. 2008, 343, 1675–1692.

20. Cram, D. J.; Kopecky, K. R. J. Am. Chem. Soc. 1959, 81, 2748–2755.

21. Grindley, T. B.; Cote, C. J. P.; Wickramage, C. Carbohydr. Res. 1985, 140, 215–

238.

22. Ogawa, S.; Tsunoda, H. Liebigs Ann. Chem. 1993, 755–769.

23. (a) Shaffer, K. J.; Taylor, C. M. Org. Lett. 2006, 8, 3959–3962; (b) Millar, A.; Kim,

K. H.; Minster, D. K.; Ohgi, T.; Hecht, S. M. J. Org. Chem. 1986, 51, 189–196.

24. Bach, R. D.; Coddens, B. A.; Wolber, G. J. J. Org. Chem. 1986, 51, 1030–

1033.

chirality of the starting material, D-mannose.

The rings deﬁned by C-1–C-2–C-3–C-4–C-5a can be described by

the Cremer–Pople parameters³²for each compound, calculated

using the ^PLATONprogram.³³For 15: Q(2) = 0.408(5) Å, Q(3) =

ꢀ0.328(5) Å, u(2) = 252.5(7)°, Puckering Amplitude (Q) = 0.524(5)

Å, h = 128.8(5)°, u = 252.5(7)°. For 18: Q(2) = 0.384(2) Å, Q(3) =

0.325(2) Å, u(2) = 92.3(4)°, Puckering Amplitude (Q) = 0.504(2) Å,

h = 49.8(2)°, u = 92.3(4)°. For 22: Q(2) = 0.3768(17) Å, Q(3) =

ꢀ0.3146(17) Å,

u(2) = 263.8(3)°,

Puckering

Amplitude

(Q) = 0.4909(17) Å, h = 129.9(2)°, u = 263.8(3)°. The ring in 18 is de-

scribed with the half-chair pucker descriptor (Table 1).

Crystallographic data (excluding structure factors) for the struc-

tures in this paper have been deposited with the Cambridge Crys-

tallographic Data Centre as supplementary publication numbers

CCDC 718023 (15), CCDC 718024 (22) and CCDC 718025 (18). Cop-

ies of the data can be obtained, free of charge, on application to

CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 (0)1223

336033 or e-mail: deposit@ccdc.cam.ac.uk).

Acknowledgements

25. Shing, T. K. M.; Li, T. Y.; Kok, S. H.-L. J. Org. Chem. 1999, 64, 1941–1946.

26. (a) Link, J. T.; Gallant, M.; Danishefsky, S. J. J. Am. Chem. Soc. 1993, 115, 3182–

3183; (b) Link, J. T.; Raghavan, S.; Danishefsky, S. J. J. Am. Chem. Soc. 1995, 117,

552–553.

27. Kok, S. H.-L.; Shing, T. K. M. Tetrahedron Lett. 2000, 41, 6865–6868.

28. Kuntz, D. A.; Tarling, C. A.; Withers, S. G.; Rose, D. R. Biochemistry 2008, 47,

10058–10068.

Financial support from the Swedish research council (Vet-

enskapsrådet) is gratefully acknowledged (I.C. and H.S.). H.S.

thanks the Crafoordska stiftelsen for a mobility grant that sup-

ported O.H. I.C. is supported by an Ivar Bendixson grant.

29. Segel, I. H. Enzyme Kinetics; John Wiley & Sons: New York, 1975.

30. Taber, D. F.; Frankowski, K. J. J. Org. Chem. 2003, 68, 6048–6049.

31. Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112–122.

32. Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354–1358.

33. Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7–13.

References

1. (a) Schmidt, D. D.; Frommer, W.; Junge, B.; Müller, L.; Wingender, W.;

Truscheit, E.; Schäfer, D. Naturwissenschaften 1977, 64, 535–536; (b) Horii, S.;

Kameda, Y. J. Chem. Soc., Chem. Commun. 1972, 747–748.

Products guided by the article

Product name:C₃₁H₂₉Cl₃F₃NO₇S

Cas No:1169701-69-2

R&D Labs maybe for 1169701-69-2

Weifang Adde Economic And Trade Co.,LTD.

Contact:86-536-8885548

Address:Room 1402,Wanda Plaza B Block,No.958,Yuanfei Road,Kuiwen District
NINGBO YINLIANG FOREIGN TRADE CO., LTD.

Contact:+86-574-87834016 / 87898057

Address:23F NO.666JINYU ROAD,YINGZHOU DISTRICT,NINGBO.CHINA
Jiangsu Jiuri Chemical Co.,Ltd.

Contact:+86-519-82118868

Address:Tianwang Town, Jurong City, Jiangsu Province, China
Tianjin Dongchang Fine Chemical Industry Co., Ltd.

Contact:+86-22-29894595

Address:Economic Developing Zone, Ji County, Tianjin, China
Naturalin Bio-Resource Co., Ltd

website:http://www.naturalin.com

Contact:+86-0731-84430651

Address:B1-402, Lu-Valley Enterprise Square.No.27 Wenxuan Road. Lu-Valley Hi-Tech District.

Relevant to this article

The first bioreversible prodrug of metformin with improved lipophilicity and enhanced intestinal absorption

Doi:10.1021/jm900274q
(2009)
Kinetics of the base hydrolysis of iron (II) complexes with pyridyl-quinolyl Schiff base ligands in aqueous and aqueous/methanol binary mixtures

Doi:10.1007/s13738-015-0622-z
(2015)
Synthesis and spectroscopic studies of charge transfer complexes between chloranilic acid and some heterocyclic amines in ethanol

Doi:10.1016/j.molstruc.2009.03.025
(2009)
A simple and green approach for the synthesis of tetrahydrobenzo[a]- xanthen-11-one derivatives using tetrabutyl ammonium fluoride in water

Doi:10.1055/s-0028-1088214
(2009)
Bis-14-membered ring diketal diamines: Synthesis and evaluation of their anti-HIV and anti-tumoral activities

Doi:10.1016/j.ejmech.2009.03.008
(2009)
Deaminoteicoplanin and its derivatives. Synthesis, antibacterial activity, and binding strength to Ac-D-Ala-D-Ala

Doi:10.1021/jm00122a007
(1989)

Article Doi

β-Mannosidase and β-hexosaminidase inhibitors: synthesis of 1,2-bis-epi-valienamine and 1-epi-2-acetamido-2-deoxy-valienamine from d-mannose

DOI: 10.1016/j.tetasy.2009.02.016

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.tetasy.2009.02.016

Full text of DOI:10.1016/j.tetasy.2009.02.016

Products guided by the article

R&D Labs maybe for 1169701-69-2

Relevant to this article

Hot Product