Synthesis and biological evaluation of RON-neoglycosides as tumor cytotoxins

DOI: 10.1016/j.carres.2011.09.019

Source and publish data:

Carbohydrate Research p. 2663 - 2676 (2011)

Update date:2022-08-03

Topics:

Authors:

Langenhan, Joseph M.

Endo, Matthew M.

Engle, Jeffrey M.

Fukumoto, Liane L.

Rogalsky, Derek R.

Slevin, Lauren K.

Fay, Lindsay R.

Lucker, Ryan W.

Rohlfing, James R.

Smith, Kyle R.

Tjaden, Anja E.

Werner, Halina M.

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Article abstract of DOI:10.1016/j.carres.2011.09.019

Cardenolides such as digitoxin have been shown to inhibit cancer cell growth, to reduce cancer metastasis, and to induce apoptosis in tumor cells. Among the most potent digitoxin-based cytotoxins identified to date are MeON-neoglycosides generated via oxyamine neoglycosylation. Here, we report our studies of oxyamine neoglycosylation aimed at facilitating the elucidation of linkage-diversified digitoxin neoglycoside structure-activity relationships. We identified conditions suitable for the convenient synthesis of digitoxin neoglycosides and found that sugar structure, rather than RON-glycosidic linkage, exerts the strongest influence on neoglycoside yield and stereochemistry. We synthesized a library of digitoxin neoglycosides and assessed their cytotoxicity against eight human cancer cell lines. Consistent with previous findings, our data show that the structure of RON-neoglycosidic linkages influences both the potency and selectivity of digitoxin neoglycosides.

Full text of DOI:10.1016/j.carres.2011.09.019

Carbohydrate Research 346 (2011) 2663–2676

Contents lists available at SciVerse ScienceDirect

Carbohydrate Research

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

Synthesis and biological evaluation of RON-neoglycosides as tumor cytotoxins

⇑

Joseph M. Langenhan , Matthew M. Endo, Jeffrey M. Engle, Liane L. Fukumoto, Derek R. Rogalsky,

Lauren K. Slevin, Lindsay R. Fay, Ryan W. Lucker, James R. Rohlﬁng, Kyle R. Smith, Anja E. Tjaden,

Halina M. Werner

Department of Chemistry, Seattle University, Seattle, WA 98122, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

Cardenolides such as digitoxin have been shown to inhibit cancer cell growth, to reduce cancer metasta-

sis, and to induce apoptosis in tumor cells. Among the most potent digitoxin-based cytotoxins identiﬁed

to date are MeON-neoglycosides generated via oxyamine neoglycosylation. Here, we report our studies of

oxyamine neoglycosylation aimed at facilitating the elucidation of linkage-diversiﬁed digitoxin neoglyco-

side structure–activity relationships. We identiﬁed conditions suitable for the convenient synthesis of

digitoxin neoglycosides and found that sugar structure, rather than RON-glycosidic linkage, exerts the

strongest inﬂuence on neoglycoside yield and stereochemistry. We synthesized a library of digitoxin neo-

glycosides and assessed their cytotoxicity against eight human cancer cell lines. Consistent with previous

ﬁndings, our data show that the structure of RON-neoglycosidic linkages inﬂuences both the potency and

selectivity of digitoxin neoglycosides.

Received 15 July 2011

Received in revised form 13 September

2011

Accepted 19 September 2011

Available online 29 September 2011

Keywords:

Glycoconjugate

Glycosylalkoxylamines

Hydrolysis rates

Cardenolides

Digitoxin

1. Introduction

CH₃

Cardiac glycosides have been used for several centuries as drugs

to treat congestive heart failure and arrhythmias.¹However, more

recently, cardenolides such as digitoxin (1, Fig. 1) have been shown

to inhibit cancer cell growth, to reduce cancer metastasis, and to

induce apoptosis in tumor cells.^2–10Thus, the receptor of cardiac

glycosides, Na⁺/K⁺-ATPase,¹¹is receiving increasing attention as a

novel target for cancer chemotherapy.¹²The primary role of Na⁺/

K⁺-ATPase is to maintain an electrochemical gradient across the

plasma membrane of eukaryotes by transporting sodium ions out

of cells and potassium ions into cells. The resulting Na⁺gradient

plays a role in osmotic regulation and drives secondary transport

processes ranging from nutrient intake to Na⁺/Ca²⁺exchange. How-

ever, a growing body of evidence suggests that a subset of Na⁺/K⁺-

ATPase, likely localized within plasma membrane caveolae, plays a

role in cell signaling instead of ion transport.¹³Inhibition of this

population by cardiac glycosides activates the non-receptor

tyrosine kinase Src,¹⁴leading to a number of downstream effects

that can inﬂuence the development and progression of cancers.

These effects include the transactivation of EGRF and activation

of the Ras/MAPK signaling cascade,^14b,15an increase of reactive

oxygen species in mitochondria,¹⁶the regulation of caveolin-1

H₃C

H O

Digitoxin (1)

Figure 1. Digitoxin, a cardiac glycoside, is receiving increasing attention for its

activity against human cancer cells.

trafﬁcking,¹⁷the modulation of the structure of cell–cell tight junc-

tions,¹⁸and apoptosis.^{2–9,10a–c}

As researchers work to elucidate the complex mechanisms of ac-

tion associated with the anticancer activities of cardiac glycosides,

structure–activity relationship (SAR) studies have identiﬁed several

structural features of digitoxin derivatives that are critical to Na⁺/

K⁺-ATPase inhibition and cytotoxicity.^9,10,19The presence of the car-

bohydrate moiety is critical; cardiac glycosides are invariably better

Na⁺/K⁺-ATPase inhibitors than the corresponding aglycons.^9c,19The

cytotoxicity of both O-glycosidic and MeON-neoglycosidic analogs

of digitoxin is dependent on carbohydrate stereochemistry^10aand

on saccharide chain length,^9a,10bwith monosaccharide derivatives

displaying the most potent activities.

Among the most potent digitoxin-based cytotoxins identiﬁed to

date are L-riboside and L

-xyloside MeON-neoglycosides^9cgenerated

via oxyamine neoglycosylation (Fig. 2), a chemoselective glycosyl-

ation methodology that employs unprotected, unactivated

⇑

Corresponding author.

E-mail address: langenha@seattleu.edu (J.M. Langenhan).

doi:10.1016/j.carres.2011.09.019

2664

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

CH₃

OMe

3: X = OH, Y = H

4: X = H, Y = OH

OMe

DMF/AcOH (3:1)

40 ^o

5: Digitoxin β-

-riboside (X = OH, Y = H)

-xyloside (X = H, Y = OH)

6a: Digitoxin β-

Figure 2.

-Riboside and

-xyloside MeON-neoglycosides generated via oxyamine neoglycosylation.

Table 1

reducing sugars to form the corresponding closed ring neoglyco-

sides in good yields.^9,20–23The fact that these molecules are active,

despite containing non-natural MeON-neoglycosidic linkages, led

us to realize that glycosidic linkages could represent a third point

of diversity to optimize cardiac glycosides, complementing the

modiﬁcation of aglycons and sugars. Although this additional site

of diversiﬁcation has received little attention, we recently showed

that oxyamine linkages can be modiﬁed to identify structural vari-

ants with enhanced tumor selectivities.^9b

Effect of reaction conditions on percent conversion of neoglycosylation

solvent

40^oC, 2 days

Here, we report our studies of oxyamine neoglycosylation

aimed at facilitating the further elucidation of digitoxin neoglyco-

side structure–activity relationships. First, we explored new oxy-

amine neoglycosylation reaction conditions, as well as the

inﬂuence different oxyamine structures have on RON-neoglycoside

yield, stereochemistry, and stability. Finally, we synthesized and

evaluated an expanded library of digitoxin cytotoxins bearing

RON-neoglycosidic linkages.

Entry

Solvent

Conversion^a(%)

DMF/AcOH (3:1)

Pyridine/AcOH (3:1)

Acetone/AcOH (3:1)

THF/AcOH (3:1)

AcOH

MeOH^b

EtOH^b

iPrOH^b

2. Results and discussion

MeOH/CHCl₃(9:1)^b

MeOH/CHCl₃(4:1)^b

MeOH/CHCl₃(1:1)^b

2.1. Oxyamine neoglycosylation optimization

As estimated by LC–MS.

With 1 eq AcOH.

Many conditions for oxyamine neoglycosylation have been pub-

lished;^9,20–23most take place in either aqueous acidic buffers or or-

ganic solvents containing acetic acid. Previous efforts to generate

digitoxin analogs via oxyamine neoglycosylation successfully em-

ployed DMF/AcOH (3:1),^9c,20,21but we have found that the difﬁcul-

ties associated with evaporating DMF from crude reaction

mixtures signiﬁcantly diminish our ability to generate neoglyco-

side libraries efﬁciently. Thus, we set out to determine the compat-

ibility of lower boiling solvent mixtures with oxyamine

neoglycosylation. We conducted this study using a simple, achiral

secondary oxyamine (7a) that we generated from inexpensive

starting materials (Fig. 3).

2.2. RON-neoglycoside yield and stereochemistry

Because of the expense associated with generating digitoxin

oxyamines, we decided to employ model aglycons to study sys-

tematically the inﬂuence of oxyamine structure on RON-neoglyco-

side yield, stereochemistry, and stability. Model secondary

oxyamine aglycons 7a–f were synthesized from p-tolualdehyde

(9) by a simple reductive oxyamination strategy as shown in Fig-

ure 3. These oxyamines were reacted with

D-glucose, D-galactose,

Methoxyamine 7a was treated with D-glucose (1.1 equiv) for

-mannose, N-acetylglucosamine, and N-acetylgalactosamine un-

two days in 10 different solvent mixtures that contained either a

molar equivalent of AcOH or AcOH as a cosolvent; percent conver-

sions were estimated using LC–MS (Table 1). We were gratiﬁed to

ﬁnd that all conditions we examined provided good to excellent

percent conversions to neoglucoside 8a. Although conditions

employing AcOH as a cosolvent generally provided the highest

yields, it was simpler to evaporate solvents from reaction mixtures

containing only a single molar equivalent of AcOH. Because digi-

toxin oxyamines 2 were sparingly soluble in alcoholic solvents,

somewhat soluble in MeOH/CHCl₃(4:1), and readily soluble in

MeOH/CHCl₃(9:1), we chose the latter conditions for our subse-

quent studies.

der the optimized conditions discussed above to form RON-neogly-

cosides 8a–y (Table 2).

For a given sugar, oxyamine structure appeared not to signiﬁ-

cantly inﬂuence yield until a steric threshold was reached; nearly

all of the tested oxyamine neoglycosylations proceeded with good

to excellent yields, the chief exception being those involving steri-

cally bulky oxyamine 7d (R = t-Bu). Interestingly, among the gly-

cosylated O-tert-butylhydroxylamines, the mannoside provided a

signiﬁcantly higher yield (46%) than the other glycosides (17–

19%). It is possible that the axial O-2 group of the mannoside does

not interact as strongly with the tert-butyl group as does the equa-

torial O-2 of the other derivatives. In accord with previously pub-

lished work,²⁰isomer ratios varied dramatically as a function of

the sugar, with glucose derivatives affording b-pyranose products,

galactose derivatives affording b-pyranose/b-furanose mixtures,

1. RONH₂*HCl (10a-f),

R 7a:

R = Me (56 %)

pyr., MeOH

7b: R = Et (47 %)

7c:

7d:

R = i -Pr (50 %)

R = t-Bu (28 %)

2. Me₃N*BH₃,

10 % aq. HCl,

EtOH

and D-mannose affording b-pyranose/a-pyranose/a-furanose mix-

7e: R = allyl (48 %)

tures. Attempts to resolve neoglycoside product mixtures are

known to fail due to rapid equilibration between product

isomers.^9c

7f:

R = Bn (54 %)

Figure 3. Synthesis of model aglycons.

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

2665

Table 2

Effect of aglycon and sugar on percent yield and glycoside stereochemistry

7a-f

MeOH/CHCl₃(9:1)

AcOH, 40^oC, 2 days

n = 0, 1

8a-y

Entry

Substrate

Sugar

Neoglycoside

Yield (%)

b-Pyr/a-pyr/b-fur/a

-fur^a

i-Pr

t-Bu

Allyl

i-Pr

t-Bu

Allyl

i-Pr

t-Bu

Allyl

i-Pr

t-Bu

Allyl

Glc

GlcNAc

Gal

GalNAc

Man

100:0:0:0

n.d.^b

100:0:0:0

96:0:4:0

71:0:29:0

63:0:37:0

n.d.^b

89:0:11:0

59:0:41:0

41:0:59:0

59:0:41:0

61:0:39:0

n.d.^b

64:0:36:0

49:0:51:0

37:41:0:21

39:39:0:22

39:40:0:21

n.d.^b

7^c

40:39:0:21

39:40:0:21

Estimated by ¹H NMR in CD₃OD.

Not determined due to signal overlap.

Performed in 2%M aq NH₄OAc (pH 4.5) since GlcNAc was not soluble in MeOH/CHCl₃.

Oxyamine neoglycosylation is thought to proceed by a mecha-

in glucose derivatives the exclusive formation of pyranosides with

b-anomeric stereochemistry is easily rationalized. Both possible

glucofuranoside anomers experience unfavorable steric interac-

tions between the O-3 and C-5 groups (Fig. 4). In contrast, b-gluco-

pyranosides contain no axial substituents and therefore little

nism analogous to Fischer glycosidation, involving an intermediate

oxyimminium ion.²⁰The isomer ratios observed in Fisher glycosi-

dations reﬂect a balance between the steric inﬂuence of monosac-

charide substituents and electronic inﬂuences including the

anomeric effect; the outcomes of oxyamine neoglycosylations

can be understood using the same considerations. For instance,

strain; the

a-glucopyranoside anomer contains a single axial sub-

stituent and evidently oxyamines 7 do not impose a signiﬁcant

β-pyranosides

α-pyranosides

β-furanosides

α-furanosides

glucoside

isomers

major

galactoside

isomers

major

minor

mannoside

isomers

major

minor

Figure 4. The possible isomers arising from oxyamine neoglycosylation. Those observed are shown in black; those not detected are shown in gray. Double headed arrows

illustrate unfavorable steric interactions between the O-3 and C-5 groups. The favorably opposed C-1–C-2 dipole in -mannopyranosides is also illustrated.

2666

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

Table 3

enough anomeric effect to offset the strain this arrangement would

introduce. b-Galactopyranoside isomers were predominant in

galactoside product mixtures. However, because of the 3,4-erythro

conﬁguration of galactose, b-galactofuranosides do not experience

the unfavorable steric interactions between the O-3 and C-5 groups

experienced by glucoside derivatives. Thus, furanosides make a

signiﬁcant contribution to the equilibrium mixture; 1,2-trans

Pseudo-ﬁrst order hydrolysis rates and half-lives for 8a–f at pH 3.0^a

Entry

Neoglycoside

k_obs(s^À1

)

t_1/2(h)

i-Pr

t-Bu

Allyl

1.3 Â 10⁶

1.6 Â 10⁶

1.2 Â 10⁶

1.1 Â 10⁶

0.6 Â 10⁶

0.5 Â 10⁶

10.7

9.1

11.6

13.6

25.5

31.8

b-anomer is favored relative to 1,2-cis

a-anomer, presumably for

steric reasons and consistent with an insigniﬁcant oxyamine

anomeric effect. While the mixture of three isomers observed in

A sample of each neoglycoside (2 mM) in 20 mM NaH₂PO₄(pH 3.0) with 2%

DMSO at 25 °C was analyzed by HPLC approximately every 3 h for 2.5 days.

mannosides is more challenging to rationalize, the mixture of

and b pyranoside anomers in mannosides is consistent with a bal-

ance between steric and electronic factors where additional 1,3-

diaxial interactions and gauche interactions suffered by the

2.4. Library synthesis and biological evaluation

a-mannopyranoside anomer are offset by a favorably opposed

C-1–C-2 dipole and possibly a modest anomeric effect. -Manno-

Having optimized reaction conditions and improved our under-

standing of oxyamine glycosylation outcomes as a function of agly-

con structure, we set out to further elucidate digitoxin

neoglycoside structure–activity relationships by modifying glyco-

sidic linkages. Our previous work suggested that i-PrON-neoglyco-

sides displayed enhanced cancer cell line selectivity relative to

MeON-neoglycosides; however, our prior study probed cytotoxicity

toward only four cell lines.^9bThus, we synthesized an expanded li-

brary of digitoxin neoglycosides that included three i-PrON-neogly-

coside derivatives (sec-Bu-, c-Pent-, c-HexON-neoglycosides) and

assessed their cytotoxicity against eight human cancer cell lines.

Digitoxin (1) was simultaneously hydrolyzed and oxidized un-

der acidic conditions to provide digitoxigenone (Fig. 6). Subse-

quent treatment with alkoxyamines 10b–h provided oxime

ethers that were reduced with tert-butylamine-borane to provide

separable ꢀ1:1 mixtures of the desired C-3 b stereoisomers (2b–

furanoside also makes a contribution to the equilibrium, despite

unfavorable steric interactions between the O-3 and C-5 groups.

Unlike sugar structure, oxyamine structure did not appear to

inﬂuence the isomeric outcome of oxyamine neoglycosylations sig-

niﬁcantly in most cases. However, oxyamine structure did appear

to exert a subtle inﬂuence on the pyranose/furanose equilibrium

in galactosides. Methoxy-, ethoxy, and isopropoxy-amine derived

galactosides provided 4%, 29%, and 37% of b-furanose, respectively.

2.3. RON-neoglycoside hydrolytic stability

Few groups have investigated the stability of glycosylated oxy-

amines in aqueous solution,^9c,24and rates of hydrolysis as a func-

tion of RON-neoglycoside structure have not been studied. Thus,

we measured the rates of hydrolysis of neoglycosides 8a–f at pH

3.0 and 7.0 (Fig. 5, Table 3). Pseudo-ﬁrst order rate constants and

half-lives for hydrolysis were calculated from semi-logarithmic

plots of the hydrolysis traces. Consistent with previous results^9c,24

and an acid-catalyzed hydrolysis mechanism, we found that 8a–f

were completely stable under neutral conditions (data not shown)

and that they hydrolyzed with half-lives ranging from 9 to 32 h at

pH 3.0. While half-lives for neoglycosides 8e and 8f were approx-

imately twice as long as those for neoglycosides 8a–d we observed

no systematic variation in hydrolysis rate as a function of oxy-

amine structure.

h) and undesired C-3

then reacted with

stereoisomers. The C-3 b aglycons were

L-xylose under our optimized conditions

(Table 2). In contrast to the good yields observed in our model

studies (Table 2), neoglycosides 6b–f were produced in low yields.

Steric environment differences are a likely explanation for these

results. The oxyamine nitrogen in model aglycons 7a–f is attached

to a primary carbon; in contrast, the oxyamine nitrogen in agly-

cons 2a–h is attached to a secondary carbon. Despite low yields,

excellent stereoselectivities (100% b-pyranoside) were observed,

and sufﬁcient quantities of 6b–f were obtained to evaluate biolog-

ical activity (see Fig. 7).

We assessed the activity of neoglycosides 6a–h in cytotoxicity

assays on eight human cancer cell lines representing a range of tu-

mor types including basal epithelial, prostate, liver, colon, breast,

brain, and ovarian cancers. Consistent with previous ﬁndings,^9b

our data show that the structure of RON-neoglycosidic linkages

inﬂuences both the potency and selectivity of digitoxin neoglyco-

sides. Digitoxin displayed potent cytotoxicity toward ﬁve of the

eight cell lines tested (IC₅₀= 10–60 nM). Like digitoxin (1),

MeON-neoglycoside 6a was relatively potent and non-selective.

Neoglycosides 6b–h were signiﬁcantly less potent cytotoxins than

MeON-neoglycoside 6a. However, i-PrON-neoglycoside 6c and

i-PrON-neoglycoside analogs 6f–h appeared to display modestly

enhanced selectivity relative to digitoxin (1) and other neoglyco-

side derivatives. For example, i-PrON-neoglycoside 6c was two

times more potent against NCI/ADR-RES cells (IC₅₀= 120 10 nM)

than any other cell line tested.^9bTo the best of our knowledge,

we are the ﬁrst group to observe such cell line selectivity resulting

from a simple structural modiﬁcations to a glycosidic linkage. This

ﬁnding is intriguing since NCI/ADR-RES cells have high levels of P-

glycoprotein expression that confer multi-drug resistance.²⁵Since

many cardiac glycosides are substrates for P-glycoprotein, a possi-

ble explanation for such tumor speciﬁcity is that 6c may no longer

be a P-glycoprotein substrate.²⁶Interestingly, i-PrON-neoglycoside

derivatives 6f–h also displayed signiﬁcant cell line selectivity, but

Figure 5. Hydrolysis of neoglycosides 8a–f in 20 mM NaH₂PO₄(pH 3.0) with 2%

DMSO at 25 °C. Peak areas at 220 nM were used to estimate the neoglycoside/

aglycon ratio, which is reported as percent neoglycoside remaining [A_neoglycoside

(A_neoglycoside+ A_aglycon)].

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

2667

1. CrO₃, H₂SO₄,

acetone, H₂O

2. RONH₂*HCl (10b-h),

pyr, MeOH

R = Me^a

R = Bn (37 %)^b

2a:

2b:

2e:

2f:

R = Et (44 %)^b

R = s-Bu (33 %)^b

R = c-Pent (28 %)

2c: R = i -Pr (32 %)^b2g:

digitoxin (1)

3. tBuNH₂*BH₃,

10 % aq. HCl,

EtOH

R = t-Bu (25 %)^b

2h: R = c-Hex (14 %)^b

2d:

R = Me^a

6a:

6b:

R = Et (36 %)

6c: R = i -Pr (14 %)

6d:

6e:

R = t-Bu (0 %)

R = Bn (33 %)

-xylose (4)

MeOH/CHCl₃

AcOH (1 eq.)

40 ^oC

6f: R = s-Bu (10 %)

6g:

6h:

R = c-Pent (23 %)

R = c-Hex (17 %)

Figure 6. Synthesis of digitoxin neoglycosides. ^aSynthesized as previously described.^{9c b}Isolated yield for the desired C-3 b-isomer.

100

A549: lung epithelial adenocarcinoma

Du145: prostate carcinoma

Hep 3B: hepatocellular carcinoma

HT-29: colorectal adenocarcinoma

MCF-7: breast adenocarcinoma

MDA-MB-231: breast adenocarcinoma

SF268: glioblastoma

NCI/ADR-RES: multi-drug resistant ovarian carcinoma

Figure 7. Summary of IC₅₀data from cytotoxicity assays. Reciprocal IC₅₀values are displayed for clarity; standard errors are depicted with error bars. The IC₅₀values of 6a–h

against MCF-7 and MDA-MB-231 cells was >10 M. The IC₅₀of 1 against MCF-7, MDA-MB-231, and SF268 cells was >0.5 M.

against A549 cells rather than against NCI/ADR-RES cells. For

example, c-Hex-neoglycoside 6h was approximately three times

more potent against A549 cells than the other seven cell lines

(IC₅₀= 145 3 nM). Since A549 cells express a relatively high level

display isoform speciﬁcity could be considered as targeted

therapeutics.

3. Conclusions

of the a

1 subunit of Na⁺/K⁺-ATPase,²⁷it is possible that neoglyco-

sides 6f–h display a degree of selectivity toward this isoform. Be-

cause Na+/K+-ATPase isoforms are expressed in a tissue speciﬁc

manner and since isoform expression differences have been noted

between normal tissues and cancerous tissues,²⁷cardenolides that

Traditional chemical glycosylation strategies are often under-

mined by elaborate protecting and activating group schemes. Oxy-

amine neoglycosylation has emerged as powerful tool that

addresses this shortcoming.²⁰We became interested in determining

2668

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

if modiﬁed RON-neoglycosidic linkages produced by oxyamine

neoglycosylation could inﬂuence the biological activities of analogs

of digitoxin.^9bAs a preliminary step toward this goal, we initiated a

model study to optimize oxyamine neoglycosylation reaction con-

ditions and then set out to assess the inﬂuence a simple steric ser-

ies of oxyamine structures has on RON-neoglycoside yield,

stereochemistry, and stability. We identiﬁed conditions suitable

for the convenient synthesis of digitoxin neoglycosides and found

that sugar structure, rather than RON-glycosidic linkage, exerts

the strongest inﬂuence on neoglycoside yield and stereochemistry.

To further elucidate digitoxin neoglycoside structure–activity

relationships, we synthesized an expanded library of digitoxin neo-

glycosides that included three i-PrON-neoglycoside derivatives

(sec-Bu-, c-Pent-, c-HexON-neoglycosides) and we assessed their

cytotoxicity against eight human cancer cell lines. Consistent with

previous ﬁndings,^9bour data show that the structure of RON-

neoglycosidic linkages inﬂuences both the potency and selectivity

of digitoxin neoglycosides.

ratio, which is reported as percent neoglycoside remaining

[A_neoglycoside/(A_neoglycoside+ A_aglycon)]. Pseudo-ﬁrst order rate con-

stants and half-lives for hydrolysis were calculated from semi-log-

arithmic plots of the hydrolysis traces.

4.4. Cytotoxicity assays

The product solutions were concentrated, weighed, and dis-

solved in DMSO to make 30 mM stock solutions. All cell lines were

maintained as previously reported.¹Cells were harvested by tryp-

sinization using 0.25% trypsin and 0.1% EDTA and then counted in a

Cellometer Auto T4 cell counter (Nexcelom, Inc.), before dilution

for assay plating. Cell plating, compound handling and assay set

up were performed as previously reported¹except the cells were

plated in 50 lL volumes in 384-well clear bottom, tissue culture

plates (Corning-Costar, Inc.). Compounds were added from the

384-well compound stock plates at a 1:100 dilution using a Biomek

FX liquid handler equipped with a 384 channel head (Beckman

Coulter, Inc.). Cell titer-glo reagent (15 lL) (Promega Corporation,

Inc.) was added and incubated for 10 min at room temperature

with gentle agitation to lyse the cells. Each plate was read for lumi-

nescence. The IC₅₀value for each compound represents at least

four replicates of dose–response experiments conducted over six

concentrations at two-fold dilutions. Within each experiment, per-

cent inhibition values at each concentration were expressed as a

percentage of the maximum luminescence signal observed for a

0 nM control. IC₅₀values were determined using XLFit 4.0 as previ-

ously reported (see Supplementary data for IC₅₀values).^9c

4. Experimental

4.1. General methods

Proton nuclear magnetic resonance (¹H NMR) spectra were re-

corded in deuterated solvents on a Bruker Avance III 400 MHz

spectrometer. Chemical shifts are reported in parts per million

(ppm, d) relative to tetramethylsilane (0.00) for d-chloroform, or

the residual protic solvent peak for other solvents. ¹H NMR split-

ting patterns with observed ﬁrst order coupling are designated as

singlet (s), doublet (d), triplet (t), or quartet (q). Splitting patterns

that could not be interpreted or easily visualized are designated as

multiplet (m). Carbon nuclear magnetic resonance (¹³C NMR) spec-

tra were recorded on a Bruker Avance III 400 MHz spectrometer.

Mass spectra (MS) were obtained using electrospray ionization

(ESI). Commercially available reagents and solvents were used

without further puriﬁcation. Analytical thin layer chromatography

(TLC) was carried out on TLC plates pre-coated with Silica Gel 60

(250 lm layer thickness). Visualization was accomplished using

either a UV lamp or potassium permanganate stain (2 g KMnO₄,

13.3 g K₂CO₃, 2 mL 2 M NaOH, 200 mL H₂O). Flash column chroma-

tography was performed on 40–60 lm silica gel (230–400 mesh).

Solvent mixtures used for TLC and ﬂash column chromatography

are reported in v/v ratios.

4.5. Synthesis

4.5.1. O-Methyl-N-(4-methylbenzyl)hydroxylamine (7a)

p-Tolualdehyde (4.6 mL, 38.9 mmol), methoxyamine hydro-

chloride (4.55 g, 54.5 mmol), and pyridine (6.9 mL, 85.6 mmol)

were dissolved in CH₂Cl₂(60 mL) and the resulting solution was

stirred overnight. The reaction mixture was washed with 1 M

HCl, brine, dried over MgSO₄, ﬁltered, and then concentrated. The

mixture of oxime diastereomers was suspended in ethanol

(19 mL) and cooled to 0 °C. Borane pyridine complex (19.5 mL,

155.6 mmol) was added, followed by the dropwise addition 20%

aq HCl (39 mL). The reaction mixture was stirred overnight at room

temperature. Na₂CO₃was added until gas evolution ceased, and

the mixture was concentrated. The resulting residue was dissolved

in CH₂Cl₂, washed with water, satd aq NaHCO₃, and brine, dried

over MgSO₄, ﬁltered, and concentrated. The crude product mixture

was puriﬁed by SiO₂column chromatography eluting with 1:7

EtOAc/hexane to provide 7a (TLC R_f= 0.42 in 1:4 EtOAc/hexane)

as a volatile oil (3.29 g, 56% yield). ¹H NMR (CDCl₃, 400 MHz) d

7.25–7.13 (m, 4H), 5.67 (s, 1H), 4.01 (s, 2H), 3.51 (s, 3H), 2.34 (s,

3H); ¹³C NMR (CDCl₃, 100 MHz): d 148.6, 140.0, 129.4, 126.9,

61.9, 21.5; ESI-MS m/z (M+H) calculated for C₉H₁₄NO 152.1, ob-

served 152.1.

4.2. Percent conversion estimates

LCMS percent conversion estimates used reverse phase HPLC on

a Zorbax Eclipse XDB-C8 column (4.6 Â 150 mm; Agilent Technol-

ogies) with a ﬂow rate of 0.8 mL/min and a linear gradient of 45%

CH₃OH/H₂O to 85% CH₃OH/H₂O over 20 min and ESI. Percent con-

version was estimated by dividing the sum of the peak areas at

220 nm of peaks corresponding to the desired product mass by

the total area of all peaks eluting between 5 and 22 min.

4.5.2. O-Ethyl-N-(4-methylbenzyl)hydroxylamine (7b)

p-Tolualdehyde (1.0 mL, 8.2 mmol), ethoxyamine hydrochloride

(1.15 mg, 11.8 mmol), and pyridine (1.5 mL, 18.0 mmol) were dis-

solved in CH₂Cl₂(18 mL) and the resulting solution was stirred for

1 h. The reaction mixture was washed with 1 M HCl, 1 M CuSO₄,

and brine, then dried over MgSO₄, ﬁltered, and concentrated. The

mixture of oxime diastereomers was suspended in ethanol

(18 mL) and cooled to 0 °C. Borane trimethylamine complex

(1.64 g, 22.5 mmol) was added, followed by the dropwise addition

10% aq HCl (18 mL). The reaction mixture was stirred 3 h at room

temperature then diluted in CH₂Cl₂, washed with water and brine,

dried over MgSO₄, ﬁltered, and concentrated. The crude product

mixture was puriﬁed by SiO₂column chromatography eluting with

4.3. Neoglycoside hydrolysis

The hydrolytic degradation of neoglycosides 8 was monitored

by reverse phase HPLC on a Zorbax Eclipse XDB-C8 column

(4.6 Â 150 mm; Agilent Technologies) with a ﬂow rate of 0.8 mL/

min and a linear gradient of 4.5% CH₃OH/H₂O to 95.5% CH₃OH/

H₂O over 60 min. At t = 0, a solution of neoglycoside (2 mM) in

20 mM NaH₂PO₄buffer (pH 3) with 2% DMSO or a solution of neo-

glycoside (2 mM) in 20 mM K₂HPO₄buffer (pH 7) with 2% DMSO

was immediately injected onto the HPLC column at 25 °C. Peak

areas at 220 nM were used to estimate the neoglycoside/aglycon

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

2669

1:4 EtOAc/hexane to provide 7b (TLC R_f= 0.45 in 1:9 EtOAc/hex-

ane) as an oil (632.0 mg, 47% yield). ¹H NMR (CDCl₃, 400 MHz) d

7.24–7.12 (m, 4H), 5.54 (s, 1H), 4.00 (s, 2H), 3.70 (t, 2H, J = 7.0),

2.33 (s, 3H), 1.14 (t, 3H, J = 7.0); ¹³C NMR (CDCl₃, 100 MHz): d

137.1, 134.4, 129.1, 128.9, 69.2, 56.3, 21.1, 14.2; ESI-MS m/z

(M+H) calculated for C₁₀H₁₆NO 166.1, observed 166.1.

(m, 2H), 4.02 (s, 2H), 2.33 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d

137.1, 134.6, 134.5, 129.1, 129.0, 117.6, 75.1, 56.3, 21.2, ESI-MS

m/z (M+H) calculated for C₁₁H₁₆NO 177.1, observed 177.1.

4.5.6. O-Benzyl-N-(4-methylbenzyl)hydroxylamine (7f)

p-Tolualdehyde (0.5 mL, 4.2 mmol), O-benzylhydroxylamine

hydrochloride (94.5 mg, 5.9 mmol), and pyridine (0.75 mL,

9.3 mmol) were dissolved in CH₂Cl₂(10 mL) and the resulting solu-

tion was stirred 24 h. The reaction mixture was washed with 1 M

HCl, 1 M CuSO₄(2Â), and brine, then dried over MgSO₄, ﬁltered,

and concentrated. The mixture of oxime diastereomers was sus-

pended in ethanol (12 mL) and cooled to 0 °C. Borane trimethyl

complex (1.02 mg, 13.9 mmol) was added, followed by the drop-

wise addition 10% aq HCl (11.4 mL). The reaction mixture was stir-

red 2.5 h at room temperature then Na₂CO₃was added until gas

evolution ceased. The mixture was diluted in CH₂Cl₂, washed with

water and brine, dried over MgSO₄, ﬁltered, and concentrated. The

crude product mixture was puriﬁed by SiO₂column chromatogra-

phy eluting with 12:1 hexanes/EtOAc to provide 7f (TLC R_f= 0.33 in

12:1 hexanes/EtOAc) as an oil (584.5 mg, 54% yield). ¹H NMR

(CDCl₃, 400 MHz) d 7.34–7.25 (m, 5H), 7.25–7.13 (m, 4H), 5.69 (s,

1H), 4.66 (s, 2H), 4.01 (s, 2H), 2.33 (s, 3H); ESI-MS m/z (M+H) cal-

culated for C₁₅H₁₈NO 227.1, observed 227.1.

4.5.3. N-(4-Methylbenzyl)-O-iso-propylhydroxylamine (7c)

p-Tolualdehyde (0.77 mL, 6.4 mmol), iso-propoxyamine hydro-

chloride (1.0 g, 8.96 mmol), and pyridine (1.2 mL, 14.1 mmol) were

dissolved in CH₂Cl₂(14 mL) and the resulting solution was stirred

90 min. The reaction mixture was washed with 1 M HCl, 1 M

CuSO₄, and brine, then dried over MgSO₄, ﬁltered, and concen-

trated. The mixture of oxime diastereomers was suspended in eth-

anol (16.3 mL) and cooled to 0 °C. Borane trimethyl complex

(1.45 g, 19.8 mmol) was added, followed by the dropwise addition

of 10% aq HCl (16.3 mL). The reaction mixture was stirred over-

night at room temperature then diluted in CH₂Cl₂, washed with

water and brine, dried over MgSO₄, ﬁltered, and concentrated.

The crude product mixture was puriﬁed by SiO₂column chroma-

tography eluting with 1:4 EtOAc/hexane to provide 7c (TLC

R_f= 0.61 in 1:4 EtOAc/hexane) as an oil (572.0 mg, 50% yield). ¹H

NMR (CDCl₃, 400 MHz) d 7.25–7.12 (m, 4H), 5.37 (s, 1H), 3.98 (s,

2H), 3.81 (sept, 1H, J = 6.2), 2.33 (s, 3H), 1.12 (t, 6H, J = 6.2); ¹³C

NMR (CDCl₃, 100 MHz): d 137.2, 134.7, 129.2, 74.9, 56.9, 21.5,

21.3; ESI-MS m/z (M+H) calculated for C₁₁H₁₈NO 180.1, observed

180.1.

4.5.7. General procedure for the synthesis of glycosylated

oxyamines 8

Aglycon 7 (1 equiv) and sugar (1.1 equiv) were added to a glass

vial equipped with a stirring ﬂea and then were dissolved in 9:1

MeOH/CHCl₃(0.1 mL/mmol). AcOH was added (1 equiv) and the

reaction mixture was stirred at 40 °C for 2 days. Silica gel

(125 mg) was added and the crude reaction mixture was concen-

trated then puriﬁed via SiO₂column chromatography. NMR spectra

were assigned via gCOSY experiments and comparison to pub-

lished chemical shift values and coupling constants.²⁰

4.5.4. O-tert-Butyl-N-(4-methylbenzyl)hydroxylamine (7d)

p-Tolualdehyde (1.5 mL, 12.3 mmol), tert-butoxyamine hydro-

chloride (2.23 g, 17.8 mmol), and pyridine (2.25 mL, 27.9 mmol)

were dissolved in CH₂Cl₂(28 mL) and the resulting solution was

stirred 90 min. The reaction mixture was washed with 1 M HCl,

1 M CuSO₄, and brine, then dried over MgSO₄, ﬁltered, and concen-

trated. The crude oxime was suspended in ethanol (22 mL) and

cooled to 0 °C. Borane trimethyl complex (2.30 g, 26.4 mmol) was

added, followed by the dropwise addition 10% aq HCl (22 mL).

The reaction mixture was stirred 6 h at room temperature then di-

luted in CH₂Cl₂, washed with water and brine, dried over MgSO₄,

ﬁltered, and concentrated. The crude product mixture was puriﬁed

by SiO₂column chromatography eluting with 99:1 toluene/EtOAc

to provide 7d (TLC R_f= 0.34 in 99:1 toluene/EtOAc) as an oil

(428.0 mg, 28% yield). ¹H NMR (CDCl₃, 400 MHz) d 7.26–7.12 (m,

4H), 4.94 (s, 1H), 3.94 (s, 2H), 2.33 (s, 3H), 1.17 (s, 9H); ¹³C NMR

(CDCl₃, 100 MHz): d 136.1, 133.3, 128.2, 128.0, 76.1, 56.5, 25.8,

20.2; ESI-MS m/z (M+H) calculated for C₁₂H₂₀NO 194.2, observed

194.2.

4.5.7.1.

syl)hydroxylamine (8a).

(28.6 mg, 0.189 mmol),

AcOH (10.8

O-Methyl-N-(4-methylbenzyl)-N-(b-D-glucopyrano-

Via the general procedure, 7a

-glucose (37.5 mg, 0.208 mmol), and

L, 0.189 mmol) were reacted. The crude product mix-

ture was puriﬁed by SiO₂column chromatography eluting with

10:1 CH₂Cl₂/MeOH to afford 8a (TLC R_f= 0.26 in 10:1 CH₂Cl₂/

MeOH) as a white powder (39.8 mg, 71% yield). ¹H NMR (MeOD-

d₄, 400 MHz) d 7.30 (A of AB, 2H, J = 8.2), 7.11 (B of AB, 2H,

J = 7.7), 4.12 (A of AB, 1H, J = 12.4), 3.99 (B of AB, 1H, J = 12.3),

3.89 (d, 1H, J = 8.7, H-1), 3.87 (A of ABX, 1H, J = 12.0, 3.9, H-6),

3.67 (B of ABX, 1H, J = 12.0, 15.2, H-6’), 3.50 (m, 1H, H-2), 3.40 (s,

3H), 3.29 (m, 2H, H-3, H-4), 3.13 (m, 1H, H-5), 2.30 (s, 3H). ¹³C

NMR (MeOD-d₄, 100 MHz): d 136.7, 133.8, 129.5, 128.3, 91.5,

78.1, 78.0, 70.0, 69.7, 61.3, 61.0, 55.7, 19.7. HRMS m/z (M+H) calcu-

lated for C₁₅H₂₄NO₆314.16014, observed 314.15932.

4.5.5. O-Allyl-N-(4-methylbenzyl)hydroxylamine (7e)

p-Tolualdehyde (0.5 mL, 4.2 mmol), O-allylhydroxylamine

hydrochloride (67.5 mg, 5.9 mmol), and pyridine (0.75 mL,

9.3 mmol) were dissolved in CH₂Cl₂(10 mL) and the resulting solu-

tion was stirred 30 min. The reaction mixture was washed with

1 M HCl, 1 M CuSO₄(2Â), and brine, then dried over MgSO₄, ﬁl-

tered, and concentrated. The mixture of oxime diastereomers

was suspended in ethanol (8 mL) and cooled to 0 °C. Borane tri-

methyl complex (68.3 mg, 9.4 mmol) was added, followed by the

dropwise addition of 10% aq HCl (8 mL). The reaction mixture

was stirred 2.5 h at room temperature then Na₂CO₃was added un-

til gas evolution ceased. The mixture was diluted in CH₂Cl₂,

washed with water and brine, dried over MgSO₄, ﬁltered, and con-

centrated. The crude product mixture was puriﬁed by SiO₂column

chromatography eluting with 9:1 hexanes/EtOAc to provide 7e

(TLC R_f= 0.32 in 4:1 hexanes/EtOAc) as an oil (503.0 mg, 48% yield).

¹H NMR (CDCl₃, 400 MHz) d 7.26–7.13 (m, 4H), 5.91 (ddt, 1H,

J = 17.3, 10.4, 5.8), 5.69 (s, 1H), 5.25 (m, 1H), 5.17 (m, 1H), 4.16

4.5.7.2.

syl)hydroxylamine (8b).

(23.6 mg, 0.138 mmol),

AcOH (7.9

O-Ethyl-N-(4-methylbenzyl)-N-(b-D-glucopyrano-

Via the general procedure, 7b

D-glucose (27.3 mg, 0.151 mmol), and

L, 0.138 mmol) were reacted. The crude product mix-

ture was puriﬁed by SiO₂column chromatography eluting with

10:1 CH₂Cl₂/MeOH to afford 8b (TLC R_f= 0.33 in 10:1 CH₂Cl₂/

MeOH) as

white powder (37.0 mg, 82% yield). ¹H NMR

(MeOD-d₄, 400 MHz) d 7.31 (A of AB, 2H, J = 7.9), 7.13 (B of AB,

2H, J = 7.9), 4.12 (A of AB, 1H, J = 12.4), 4.02 (B of AB, 1H,

J = 12.4), 3.91 (d, 1H, J = 8.9, H-1), 3.89 (A of ABX, 1H, J = 12.0,

2.2, H-6), 3.72 (B of ABX, 1H, J = 12.0, 5.4, H-6’), 3.70 (A of

ABX₃, 1H, J = 7.2, 3.8), 3.58 (B of ABX₃, 1H, J = 7.2, 3.8), 3.50 (m,

1H, H-2), 3.30 (m, 2H, H-3, H-4), 3.14 (m, 1H, H-5), 2.32 (s, 3H),

1.01 (t, 3H, J = 7.0). ¹³C NMR (MeOD-d₄, 100 MHz): d 138.8,

138.2, 135.4, 131.1, 129.9, 93.2, 79.6, 71.6, 71.2, 70.8, 62.8, 57.7,

2670

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

21.2, 14.1. ESI-MS m/z (M+H) calculated for C₁₆H₂₆NO₆328.2, ob-

served 328.2.

0.135 mmol) were dissolved in 0.18 mL of 2 M aq ammonium ace-

tate buffer (pH 4.5) and stirred at 40 °C for 72 h. The mixture was

concentrated and 1 mL of toluene was added. After concentrating

again, the crude reaction mixture was puriﬁed by SiO₂column

chromatography eluting with 8:1:1 EtOAc/MeOH/H₂O to afford

8g (TLC R_f= 0.32 in 8:1:1 EtOAc/MeOH/H₂O) as a white powder

(22.3 mg, 47% yield). ¹H NMR (MeOD-d₄, 400 MHz) d 7.25 (m,

2H), 7.12 (m, 2H), 4.15 (d, 1H, J = 9.9, H-1), 4.13–3.97 (m, 2H),

3.98 (m, 1H, H-2), 3.91 (m, 1H, H-6), 3.73 (m, 1H, H-6’), 3.39–

3.30 (m, 2H, H-3, H-4), 3.26 (s, 3H), 3.18 (m, 1H, H-5), 2.31 (s,

3H), 2.00 (s, 3H). ¹³C NMR (MeOD-d₄, 100 MHz): d 176.5, 141.3,

138.9, 134.2, 133.1, 95.0, 83.1, 80.9, 75.0, 66.2, 65.2, 59.3, 57.1,

26.3, 24.4. ESI-MS m/z (M+H) calculated for C₁₇H₂₇N₂O₆355.2, ob-

served 355.2.

4.5.7.3. N-(4-Methylbenzyl)-N-(b-D-glucopyranosyl)-O-iso-pro-

pylhydroxylamine (8c).

Via the general procedure, 7c

(35.0 mg, 0.195 mmol),

D-glucose (38.7 mg, 0.215 mmol), and

AcOH (11.0 lL, 0.195 mmol) were reacted. The crude product mix-

ture was puriﬁed by SiO₂column chromatography eluting with

10:1 CH₂Cl₂/MeOH to afford 8c (TLC R_f= 0.37 in 10:1 CH₂Cl₂/

MeOH) as a white powder (48.6 mg, 73% yield). ¹H NMR (MeOD-

d₄, 400 MHz) d 7.28 (A of AB, 2H, J = 7.6), 7.11 (B of AB, 2H,

J = 7.7), 4.07 (q, 2H, J = 12.6), 3.89 (d, 1H, J = 9.4, H-1), 3.88 (m,

1H, H-6), 3.88 (m, 1H), 3.70 (m, 1H, H-6’), 3.46 (t, 1H, J = 8.7, H-

2), 3.29 (m, 2H, H-3, H-4), 3.09 (m, 1H, H-5), 2.30 (s, 3H), 1.15 (d,

3H, J = 5.9), 1.01 (d, 3H, J = 5.6). ¹³C NMR (MeOD-d₄, 100 MHz): d

136.6, 133.7, 129.6, 129.4, 128.3, 91.5, 78.0, 75.0, 70.1, 69.6, 61.3,

56.0, 20.3, 20.0, 19.7. ESI-MS m/z (M+H) calculated for C₁₇H₂₈NO₆

342.2, observed 342.2.

4.5.7.8. O-Methyl-N-(4-methylbenzyl)-N-(b-D-galactosyl)hydrox-

ylamine (8h).

Via the general procedure, 7a (28.4 mg,

0.188 mmol),

D-galactose (37.2 mg, 0.207 mmol), and AcOH

(10.7 lL, 0.188 mmol) were reacted. The crude product mixture

4.5.7.4. O-tert-Butyl-N-(4-methylbenzyl)-N-(

ylamine (8d). Via the general procedure, 7d (22.5 mg,

0.116 mmol), -glucose (23.1 mg, 0.128 mmol), and AcOH (6.6 L,

0.116 mmol) were reacted. The crude product mixture was puriﬁed

by SiO₂column chromatography eluting with 10:1 CH₂Cl₂/MeOH

to afford 8d (TLC R_f= 0.23 in 10:1 CH₂Cl₂/MeOH) as a white powder

(8.0 mg, 19% yield). ¹H NMR (MeOD-d₄, 400 MHz) d 7.29 (m, 2H),

7.12 (m, 2H), 4.45–2.87 (m, 5H), 4.15 (m, 1H), 3.89 (m, 1H), 2.32

(s, 3H), 1.49–0.85 (m, 9H). ESI-MS m/z (M+H) calculated for

-glucosyl)-hydrox-

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8h (TLC R_f= 0.27 in 10:1 CH₂Cl₂/MeOH)

as a white powder (48.2 mg, 82% yield). The product comprised

an inseparable mixture of b-pyranoside (96%) and b-furanoside

(4%) isomers. ESI-MS m/z (M+H) calculated for C₁₅H₂₄NO₆314.2,

observed 314.2. O-Methyl-N-(4-methylbenzyl)-N-(b-D-galactopyr-

anosyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.32 (A of

AB, 2H, J = 8.2), 7.12 (B of AB, 2H, J = 7.8), 4.13 (A of AB, 1H,

J = 12.7), 4.01 (B of AB, 1H, J = 12.7), 3.90 (d, 1H, J = 9.0, H-1),

3.85–3.71 (m, 4H, H-2, H-4, H-6, H-6’), 3.44–3.33 (m, 2H, H-3, H-

5), 3.40 (s, 3H), 2.33 (s, 3H). ¹³C NMR (MeOD-d₄, 100 MHz): d

138.1, 135.6, 131.1, 129.9, 93.8, 78.5, 76.5, 70.6, 69.0, 62.7, 62.4,

C₁₈H₃₀NO₆356.2, observed 356.2.

4.5.7.5. O-Allyl-N-(4-methylbenzyl)-N-(b-

hydroxylamine (8e). Via the general procedure, 7e (28.8 mg,

0.162 mmol), -glucose (32.2 mg, 0.179 mmol), and AcOH (9.3 L,

D-glucopyranosyl)-

56.9, 21.2. O-Methyl-N-(4-methylbenzyl)-N-(b-D-galactofurano-

syl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.28 (A of AB,

2H, J = 8.2), 7.12 (B of AB, 2H, J = 7.8), 4.45 (d, 1H, J = 5.3, H-1),

4.26 (dd, 1H, J = 7.9, 5.3, H-2), 4.13 (A of AB, 1H, J = 12.7), 4.01 (B

of AB, 1H, J = 12.7), 4.09 (dd, 1H, J = 8.4, 6.8, H-3), 4.05–3.55 (m,

4H, H-4, H-5, H-6, H-6’), 3.40 (s, 3H), 2.33 (s, 3H).

0.162 mmol) were reacted. The crude product mixture was puriﬁed

by SiO₂column chromatography eluting with 10:1 CH₂Cl₂/MeOH

to afford 8e (TLC R_f= 0.47 in 10:1 CH₂Cl₂/MeOH) as a white powder

(29.5 mg, 54% yield). ¹H NMR (MeOD-d₄, 400 MHz) d 7.31 (A of AB,

2H, J = 8.1), 7.14 (B of AB, 2H, J = 7.8), 5.79 (ddt, 1H, J = 17.4, 10.3,

6.1), 5.16 (ddt, 1H, J = 17.4, 1.6, 1.6), 5.10 (m, 1H), 4.16 (A of AB,

1H, J = 12.4), 4.16 (m, 1H), 4.04 (B of AB, 1H, J = 12.4), 4.02 (ddd,

1H, J = 11.5, 6.4, 1.1), 3.93 (d, 1H, J = 12.3, H-1), 3.89 (A of ABX,

1H, J = 12.2, 2.2, H-6), 3.72 (B of ABX, 1H, J = 12.2, 5.3, H-6’), 3.53

(m, 1H, H-2), 3.31 (m, 2H, H-3, H-4), 3.15 (m, 1H, H-5), 2.34 (s,

3H). ¹³C NMR (MeOD-d₄, 100 MHz): d 138.3, 135.3, 134.8, 131.2,

129.9, 119.0, 93.2, 79.7, 79.5, 77.1, 71.6, 71.2, 62.8, 57.8, 21.2.

ESI-MS m/z (M+H) calculated for C₁₇H₂₆NO₆340.2, observed 340.2.

4.5.7.9. O-Ethyl-N-(4-methylbenzyl)-N-(b-

amine (8i). Via the general procedure, 7b (14.1 mg, 0.085 mmol),

-galactose (16.9 mg, 0.094 mmol), and AcOH (5.0 L, 0.085 mmol)

D-galactosyl)hydroxyl-

were reacted. The crude product mixture was puriﬁed by SiO₂col-

umn chromatography eluting with 10:1 CH₂Cl₂/MeOH to afford 8i

(TLC R_f= 0.30 in 10:1 CH₂Cl₂/MeOH) as a white powder (24.8 mg,

88% yield). The product comprised an inseparable mixture of b-

pyranoside (71%) and b-furanoside (29%) isomers. ESI-MS m/z

(M+H) calculated for C₁₆H₂₆NO₆328.2, observed 328.2. O-Ethyl-

4.5.7.6. O-Benzyl-N-(4-methylbenzyl)-N-(b-

hydroxylamine (8f). Via the general procedure, 7f (23.4 mg,

0.103 mmol), -glucose (20.4 mg, 0.113 mmol), and AcOH (6.0 L,

D-glucopyranosyl)-

N-(4-methylbenzyl)-N-(b-D

-galactopyranosyl)hydroxylamine: ¹H

NMR (MeOD-d₄, 400 MHz) d 7.31 (A of AB, 2H, J = 8.0), 7.12 (B of

AB, 2H, J = 7.8), 4.12 (A of AB, 1H, J = 12.3), 4.01 (B of AB, 1H,

J = 12.6), 3.91 (d, 1H, J = 9.0, H-1), 3.87–3.70 (m, 4H, H-2, H-4, H-

6, H-6’), 3.70–3.40 (m, 2H), 3.45–3.34 (m, 2H, H-3, H-5), 2.32 (s,

3H), 1.00 (t, 3H, J = 7.0). ¹³C NMR (MeOD-d₄, 100 MHz): d 138.1,

135.9, 131.1, 129.8, 94.1, 83.1, 78.5, 76.5, 70.7, 69.1, 62.8, 57.2,

0.103 mmol) were reacted. The crude product mixture was puriﬁed

by SiO₂column chromatography eluting with 10:1 CH₂Cl₂/MeOH

to afford 8f (TLC R_f= 0.42 in 10:1 CH₂Cl₂/MeOH) as a white powder

(26.7 mg, 67% yield). ¹H NMR (MeOD-d₄, 400 MHz) d 7.34 (m, 1H),

7.29 (m, 3H), 7.18 (m, 4H), 4.65 (A of AB, 1H, J = 9.7), 4.46 (B of AB,

1H, J = 9.7), 4.16 (A of AB, 1H, J = 12.4), 4.05 (B of AB, 1H, J = 12.4),

4.00 (d, 1H, J = 9.0, H-1), 3.86 (A of ABX, 1H, J = 12.1, 2.2, H-6), 3.70

(B of ABX, 1H, J = 12.1, 5.1, H-6’), 3.65 (m, 1H, H-2), 3.31 (m, 2H, H-

3, H-4), 3.15 (m, 1H, H-5), 2.35 (s, 3H). ¹³C NMR (MeOD-d₄,

100 MHz): d 138.4, 137.7, 135.4, 131.3, 130.7, 129.9, 129.4, 129.3,

93.3, 79.63, 79.58, 78.1, 71.6, 71.1, 62.7, 57.8, 21.3. ESI-MS m/z

(M+H) calculated for C₂₁H₂₈NO₆390.2, observed 390.2.

21.2, 14.1. O-Ethyl-N-(4-methylbenzyl)-N-(b-D-galactofurano-

syl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.27 (A of AB,

2H, J = 8.0), 7.12 (B of AB, 2H, J = 7.8), 4.46 (d, 1H, J = 5.7, H-1),

4.25 (dd, 1H, J = 6.9, 5.8, H-2), 4.15–3.99 (m, 2H), 4.08 (m, 1H, H-

3), 3.95–3.34 (m, 6H), 2.32 (s, 3H), 0.97 (t, 3H, J = 7.0). ¹³C NMR

(MeOD-d₄, 100 MHz): d 138.1, 135.9, 131.0, 129.8, 98.7, 83.1,

78.8, 77.4, 73.0, 71.0, 64.6, 58.3, 21.2, 14.1.

4.5.7.10.

hydroxylamine (8j).

0.264 mmol), -galactose (52.3 mg, 0.290 mmol), and AcOH

(15 L, 0.264 mmol) were reacted. The crude product mixture

N-(4-Methylbenzyl)-N-(b-

D-galactosyl)-O-iso-propyl-

4.5.7.7.

deoxy-b-

O-Methyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

-glucosyl)hydroxylamine (8g). Aglycon 7a (21.0 mg,

-glucose (29.8 mg,

Via the general procedure, 7c (47.3 mg,

0.139 mmol) and 2-aminoacetyl-2-deoxy-

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

2671

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8j (TLC R_f= 0.33 in 10:1 CH₂Cl₂/MeOH) as

a white powder (74.9 mg, 87% yield). The product comprised an

inseparable mixture of b-pyranoside (63%) and b-furanoside

(37%) isomers. ESI-MS m/z (M+H) calculated for C₁₇H₂₈NO₆342.2,

anosyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.35–7.15

(m, 9H), 4.60 (A of AB, 1H, J =9.6), 4.51 (B of AB, 1H, J = 9.6), 4.15

(A of AB, 1H, J = 12.1), 4.07 (A of AB, 1H, J = 12.1), 3.99 (d, 1H,

J = 8.9, H-1), 3.91 (m, 1H, H-2), 3.81 (m, 2H, H-4, H-6), 3.70 (m,

1H, H-6’), 3.43 (m, 2H, H-3, H-5), 2.34 (s, 3H). ¹³C NMR (CDCl₃,

100 MHz): d 138.2, 138.0, 135.6, 131.3, 130.5, 129.9, 129.3, 94.1,

78.6, 77.8, 76.5, 70.8, 69.1, 62.8, 57.2, 21.2. O-Benzyl-N-(4-methyl-

observed 342.2. N-(4-Methylbenzyl)-N-(b-D-galactopyranosyl)-O-

iso-propylhydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.31 (A

of AB, 2H, J = 7.2), 7.12 (B of AB, 2H, J = 5.8), 4.13–3.34 (m, 10H,

H-1–6), 2.31 (s, 3H), 1.16 (d, 3H, J = 5.8), 0.96 (d, 3H, J = 4.5). ¹³C

NMR (MeOD-d₄, 100 MHz): d 138.1, 135.7, 131.3, 129.9, 94.6,

83.0, 78.5, 76.5, 70.6, 62.9, 58.9, 54.9, 21.9, 21.6, 21.3. N-(4-Meth-

benzyl)-N-(b-D

-galactofuranosyl)hydroxylamine: ¹H NMR (MeOD-

d₄, 400 MHz) d ¹H NMR (MeOD-d₄, 400 MHz) d 7.35–7.15 (m,

9H), 4.56 (m, 2H), 4.53 (m, 1H, H-1), 4.38 (m, 1H, H-2), 4.13 (m,

1H, H-3), 4.11 (m, 2H), 3.91 (m, 1H, H-4), 3.86–3.37 (m, 3H, H-5,

H-6, H-6’), 2.34 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): d 138.2,

138.0, 135.8, 131.2, 130.3, 129.9, 129.2, 98.9, 83.0, 78.7, 78.3,

77.3, 72.0, 69.1, 64.5, 58.5, 21.2.

ylbenzyl)-N-(b-D-galactofuranosyl)-O-iso-propylhydroxylamine:

¹H NMR (MeOD-d₄, 400 MHz) d 7.27 (A of AB, 2H, J = 7.4), 7.12 (B of

AB, 2H, J = 5.8), 4.47 (d, 1H, J = 5.6, H-1), 4.30 (dd, 1H, J = 6.6, 6.6, H-

2), 4.13–3.34 (m, 8H, H-3–6), 2.31 (s, 3H), 1.08 (d, 3H, J = 6.0), 1.05

(d, 3H, J = 5.7). ¹³C NMR (MeOD-d₄, 100 MHz): d 138.1, 135.7,

131.0, 129.8, 98.2, 83.0, 78.5, 77.4, 76.1, 72.2, 64.7, 58.1, 21.9,

21.8, 21.3.

4.5.7.14.

deoxy-b-

O-Methyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

D-galactosyl)hydroxylamine (8n). Via the general pro-

cedure, 7a (23.0 mg, 0.152 mmol), 2-aminoacetyl-2-deoxy-

D-gal-

actose (37.0 mg, 0.167 mmol), and AcOH (8.7 L, 0.152 mmol)

4.5.7.11.

hydroxylamine (8k). Via the general procedure, 7d (51.2 mg,

0.265 mmol), -galactose (52.5 mg, 0.291 mmol), and AcOH

(15.2 L, 0.285 mmol) were reacted. The crude product mixture

O-tert-Butyl-N-(4-methylbenzyl)-N-(D-galactosyl)-

were reacted. The crude product mixture was puriﬁed by SiO₂col-

umn chromatography eluting with 10:1 CH₂Cl₂/MeOH to afford 8n

(TLC R_f= 0.32 in 10:1 CH₂Cl₂/MeOH) as a white powder (42.1 mg,

78% yield). The product comprised an inseparable mixture of

b-furanoside (59%) and b-pyranoside (41%) isomers. ESI-MS m/z

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8k (TLC R_f= 0.35 in 10:1 CH₂Cl₂/MeOH)

(M+H) calculated for

₁₇H₂₇N₂O₆355.2, observed 355.2. O-

-galac-

white powder (18.3 mg, 19% yield). ¹H NMR (CDCl₃,

Methyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-

400 MHz) d 7.18 (m, 4H), 4.69–2.89 (m, 9H), 2.33 (s, 3H), 1.48–

0.99 (m, 9H). ESI-MS m/z (M+H) calculated for C₁₈H₃₀NO₆356.2,

observed 356.2.

tofuranosyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.26

(m, 2H), 7.11 (m, 2H), 4.69 (dd, 1H, J = 8.2, 6.9, H-2), 4.43 (d, 1H,

J = 6.9, H-1), 4.16–3.82 (m, 7H, H-3–6), 3.36 (s, 3H), 2.31 (s, 3H),

1.98 (s, 3H). O-Methyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

4.5.7.12. O-Allyl-N-(4-methylbenzyl)-N-(b-D-galactosyl)hydrox-

deoxy-b-D

-galactopyranosyl)hydroxylamine: ¹H NMR (MeOD-d₄,

ylamine (8l).

Via the general procedure, 7e (23.7 mg,

400 MHz) d 7.26 (m, 2H), 7.11 (m, 2H), 4.26–3.81 (m, 2H), 4.23

(m, 1H, H-2), 4.14 (d, 1H, J = 9.8, H-1), 3.84 (m, 1H, H-6), 3.83 (m,

1H, H-4), 3.74 (m, 1H, H-6’), 3.50 (dd, 1H, J = 10.1, 3.2, H-3), 3.43

(m, 1H, H-5), 3.23 (s, 3H), 2.31 (s, 3H), 2.01 (s, 3H).

0.134 mmol),

D-galactose (26.5 mg, 0.147 mmol), and AcOH

(7.7 lL, 0.134 mmol) were reacted. The crude product mixture

was puriﬁed by SiO₂column chromatography eluting with 5:1

CH₂Cl₂/MeOH to afford 8l (TLC R_f= 0.75 in 5:1 CH₂Cl₂/MeOH) as

a white powder (21.5 mg, 47% yield). The product comprised an

inseparable mixture of b-pyranoside (89%) and b-furanoside

(11%) isomers. ESI-MS m/z (M+H) calculated for C₁₇H₂₆NO₆340.2,

4.5.7.15.

deoxy-b-

O-Ethyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

-galactosyl)hydroxylamine (8o). Via the general pro-

-gal-

L, 0.089 mmol)

cedure, 7b (14.7 mg, 0.089 mmol), 2-aminoacetyl-2-deoxy-

actose (21.6 mg, 0.098 mmol), and AcOH (5.0

observed 340.2. O-Allyl-N-(4-methylbenzyl)-N-(b-D-galactopyran-

osyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d ¹H NMR

(MeOD-d₄, 400 MHz) d 7.31 (A of AB, 2H, J = 7.8), 7.12 (B of AB,

2H, J = 8.0), 5.79 (ddt, 1H, J = 17.2, 10.4, 6.4), 5.15 (ddd, 1H,

J = 17.3, 3.0, 1.3), 5.08 (m, 1H), 4.15 (A of AB, 1H, J = 12.8), 4.04 (B

of AB, 1H, J = 12.8), 4.07 (m, 2H), 3.95–3.39 (m, 7H, H-1–6), 2.32

(s, 3H). ¹³C NMR (MeOD-d₄, 100 MHz): d 138.2, 135.6, 135.0,

131.1, 129.9, 118.6, 94.1, 78.6, 76.9, 76.5, 70.6, 69.1, 62.8, 57.2,

were reacted. The crude product mixture was puriﬁed by SiO₂col-

umn chromatography eluting with 10:1 CH₂Cl₂/MeOH to afford 8o

(TLC R_f= 0.22 in 10:1 CH₂Cl₂/MeOH) as a white powder (25.1 mg,

77% yield). The product comprised an inseparable mixture of b-

pyranoside (59%) and b-furanoside (41%) isomers. ESI-MS m/z

(M+H) calculated for C₁₈H₂₉N2O₆369.2, observed 369.2. O-Ethyl-

N-(4-methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-D-galactopyran-

21.2. O-Allyl-N-(4-methylbenzyl)-N-(b-D-galactofuranosyl)hydrox-

osyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.25 (m, 2H),

7.11 (m, 2H), 4.26–3.40 (m, 11H, H-1–6), 2.31 (s, 3H, 2.01 (s, 3H),

0.89 (t, 3H, J = 7.1). O-Ethyl-N-(4-methylbenzyl)-N-(2-aminoace-

ylamine: ¹H NMR (MeOD-d₄, 400 MHz) d ¹H NMR (MeOD-d₄,

400 MHz) d 7.28 (A of AB, 2H, J = 8.2), 7.23 (B of AB, 2H, J = 7.9),

5.87 (ddt, 1H, J = 17.3, 10.4, 5.8), 5.21 (ddd, 1H, J = 17.2, 3.4, 1.6),

5.12–5.02 (m, 1H), 4.47 (d, 1H, J = 5.7, H-1), 4.28 (dd, 1H, J = 6.9,

5.3, H-2), 4.08 (m, 1H, H-3), 4.16–3.39 (m, 8H), 2.32 (s, 3H). ¹³C

NMR (MeOD-d₄, 100 MHz): d 138.1, 135.7, 134.9, 131.0, 130.2,

117.6, 94.2, 79.5, 77.4, 77.1, 70.6, 69.1, 62.8, 56.7, 21.2.

tyl-2-deoxy-b-

-galactofuranosyl)hydroxylamine:

¹H

NMR

(MeOD-d₄, 400 MHz) d 7.25 (m, 2H), 7.11 (m, 2H), 4.67 (dd, 1H,

J = 8.2, 6.9, H-2), 4.46 (d, 1H, J = 6.9, H-1), 4.26–3.40 (m, 9H), 2.31

(s, 3H), 1.98 (s, 3H), 0.98 (t, 3H, J = 7.1).

4.5.7.16.

N-(4-Methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-

4.5.7.13.

hydroxylamine (8m).

0.114 mmol), -galactose (22.6 mg, 0.125 mmol), and AcOH

(6.5 L, 0.114 mmol) were reacted. The crude product mixture

O-Benzyl-N-(4-methylbenzyl)-N-(b-

D-galactosyl)-

galactosyl)-O-iso-propylhydroxylamine (8p).

procedure, 7c (26.1 mg, 0.146 mmol), 2-aminoacetyl-2-deoxy-

galactose (35.4 mg, 0.160 mmol), and AcOH (8.3 lL, 0.146 mmol)

Via the general

Via the general procedure, 7f (26.0 mg,

D-

were reacted. The crude product mixture was puriﬁed by SiO₂

column chromatography eluting with 10:1 CH₂Cl₂/MeOH to afford

8p (TLC R_f= 0.32 and 0.22 in 10:1 CH₂Cl₂/MeOH) as a white powder

(35.7 mg, 64% yield). The product comprised an inseparable mix-

ture of b-pyranoside (61%) and b-furanoside (39%) isomers. ESI-

MS m/z (M+H) calculated for C₁₉H₃₁N₂O₆383.2, observed 383.2.

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8m (TLC R_f= 0.55 in 10:1 CH₂Cl₂/MeOH)

as a white powder (26.2 mg, 59% yield). The product comprised

an inseparable mixture of b-pyranoside (59%) and b-furanoside

(41%) isomers. ESI-MS m/z (M+H) calculated for C₂₁H₂₈NO₆390.2,

observed 390.2. O-Benzyl-N-(4-methylbenzyl)-N-(b-D-galactopyr-

2672

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

O-N-(4-Methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-

-galactopyr-

J = 8.4, 7.2, H-2), 4.50 (d, 1H, J = 7.1, H-1), 4.11 (m, 1H, H-3),

4.39–3.60 (m, 8H), 2.34 (s, 3H), 2.00 (s, 3H).

anosyl)-O-iso-propylhydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz)

d 7.26 (m, 2H), 7.10 (m, 2H), 4.25–4.10 (m, 2H, H-1, H-2), 4.10–3.69

(m, 2H), 3.88 (m, 1H, H-6), 3.85 (m, 1H, H-4), 3.71 (m, 1H, H-6’),

3.55 (dd, 1H, J = 9.6, 3.0, H-3), 3.47 (dd, 1H, J = 7.0, 5.0, H-5), 3.28

(m, 1H), 2.31 (s, 3H), 2.03 (s, 3H), 1.01 (d, 3H, J = 5.8), 0.68 (d,

4.5.7.20. O-Methyl-N-(4-methylbenzyl)-N-(D-mannosyl)hydrox-

ylamine (8t).

Via the general procedure, 7a (22.0 mg,

0.146 mmol),

-mannose (29.0 mg, 0.160 mmol), and AcOH

3H, J = 6.0). N-(4-Methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-

(8.3 lL, 0.146 mmol) were reacted. The crude product mixture

galactofuranosyl)-O-iso-propylhydroxylamine: ¹H NMR (MeOD-

d₄, 400 MHz) d 7.26 (m, 2H), 7.10 (m, 2H), 4.67 (dd, 1H, J = 8.0,

7.1, H-2), 4.49 (d, 1H, J = 7.1, H-1), 4.05 (m, 1H, H-3), 4.25–3.58

(m, 6H), 3.78 (m, 1H), 2.30 (s, 3H), 1.97 (s, 3H), 1.07 (d, 3H,

J = 6.0), 1.03 (d, 3H, J = 6.2).

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8t (TLC R_f= 0.48 in 10:1 CH₂Cl₂/MeOH) as

a white powder (41.6 mg, 91% yield). The product comprised an

inseparable mixture of

and -furanoside (21%) isomers. ESI-MS m/z (M+H) calculated for

₁₅H₂₄NO₆314.2, observed 314.1. O-Methyl-N-(4-methylbenzyl)-

N-(

-mannopyranosyl)hydroxylamine: ¹H NMR (MeOD-d₄,

a-pyranoside (41%), b-pyranoside (37%),

4.5.7.17. O-tert-Butyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

a-D

deoxy-b-

procedure, 7d (49.8 mg, 0.258 mmol), 2-aminoacetyl-2-deoxy-

galactose (62.7 mg, 0.283 mmol), and AcOH (14.8 L, 0.258 mmol)

were reacted. The crude product mixture was puriﬁed by SiO₂

column chromatography eluting with 10:1 CH₂Cl₂/MeOH to af-

ford 8q (TLC R_f= 0.27 in 10:1 CH₂Cl₂/MeOH) as a white powder

(17.7 mg, 17% yield). ¹H NMR (MeOD-d₄, 400 MHz) d 7.16 (m,

2H), 6.98 (m, 2H), 4.65–3.39 (m, 9H), 2.20 (s, 3H), 1.26–0.69 (m,

9H). ESI-MS m/z (M+H) calculated for C₂₀H₃₃N₂O₆397.2, observed

397.2.

-galactosyl)-hydroxylamine (8q).

Via the general

400 MHz) d 7.20 (m, 4H), 4.04 (d, 1H, J = 3.0, H-1), 3.61 (m, 1H,

H-3), 3.41 (dd, 1H, J = 9.3, 3.0, H-2), 4.31–3.19 (m, 6H), 3.23 (s,

3H), 3.22 (m, 1H), 2.32 (s, 3H). O-Methyl-N-(4-methylbenzyl)-N-

(b-

-mannopyranosyl)hydroxylamine:

¹H

NMR

(MeOD-d₄,

400 MHz) d ¹H NMR (MeOD-d₄, 400 MHz) d 7.20 (m, 4H), 4.27 (d,

1H, J = 12.8, H-1), 3.88 (m, 1H, H-2), 4.41–3.52 (m, 7H), 3.33 (s,

3H), 2.32 (s, 3H). O-Methyl-N-(4-methylbenzyl)-N-(a-D-mannofur-

anosyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d ¹H NMR

(MeOD-d₄, 400 MHz) d 7.20 (m, 4H), 4.54 (d, 1H, J = 6.6, H-1),

4.35 (dd, 1H, J = 6.6, 4.6, H-2), 4.24–3.53 (m, 7H), 3.34 (s, 3H),

2.32 (s, 3H).

4.5.7.18.

deoxy-b-

O-Allyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

-galactosyl)hydroxylamine (8r). Via the general

4.5.7.21. O-Ethyl-N-(4-methylbenzyl)-N-(D-mannosyl)hydroxyl-

procedure, 7e (21.8 mg, 0.123 mmol), 2-aminoacetyl-2-deoxy-

amine (8u).

Via the general procedure, 7b (15.2 mg,

galactose (29.9 mg, 0.135 mmol), and AcOH (7.0 L, 0.123 mmol)

0.092 mmol),

D-mannose (18.2 mg, 0.101 mmol), and AcOH

were reacted. The crude product mixture was puriﬁed by SiO₂col-

umn chromatography eluting with 10:1 CH₂Cl₂/MeOH to afford 8r

(TLC R_f= 0.27 and 0.17 in 10:1 CH₂Cl₂/MeOH) as a white powder

(32.4 mg, 69% yield). The product comprised an inseparable mix-

ture of b-pyranoside (64%) and b-furanoside (36%) isomers. ESI-

MS m/z (M+H) calculated for C₁₉H₂₉N₂O₆381.2, observed 381.2.

(5.0 lL, 0.092 mmol) were reacted. The crude product mixture

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8u (TLC R_f= 0.27 in 10:1 CH₂Cl₂/MeOH)

as a white powder (26.3 mg, 88% yield). The product comprised

an inseparable mixture of

(39%), and -furanoside (22%) isomers. ESI-MS m/z (M+H) calcu-

lated for C₁₆H₂₆NO₆328.2, observed 328.2. O-Ethyl-N-(4-methyl-

benzyl)-N-( -mannopyranosyl)hydroxylamine: NMR

¹H

a-pyranoside (39%), b-pyranoside

O-Allyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-D-galac-

topyranosyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d 7.26

(m, 2H), 7.11 (m, 2H), 5.67 (ddt, 1H, J = 17.0, 10.2, 6.5), 5.19–5.01

(m, 2H), 4.27–4.14 (m, 2H, H-1, H-2), 4.14–3.63 (m, 4H), 3.86 (m,

1H, H-6), 3.85 (m, 1H, H-4), 3.73 (dd, 1H, J = 11.4, 5.0, H-6’), 3.51

(dd, 1H, J = 9.7, 3.2, H-3), 3.44 (m, 1H, H-5), 2.32 (s, 3H), 2.01 (s,

a-D

(MeOD-d₄, 400 MHz) d 7.27 (m, 2H), 7.12 (m, 2H), 4.29–3.28 (m,

4H), 4.04 (d, 1H, J = 3.0, H-1), 3.75 (m, 1H, H-6), 3.62 (m, 3H, H-3,

H-4, H-6’), 3.39 (dd, 1H, J = 9.4, 3.1, H-2), 3.21 (ddd, 1H, J = 8.2,

5.9, 2.3, H-5), 2.32 (s, 3H), 0.99–0.90 (m, 3H). O-Ethyl-N-(4-meth-

3H). O-Allyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-deoxy-b-

ylbenzyl)-N-(b-

-mannopyranosyl)hydroxylamine:

¹H

NMR

galactofuranosyl)hydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz) d

7.26 (m, 2H), 7.11 (m, 2H), 5.77 (ddt, 1H, J = 17.3, 10.5, 6.2),

5.19–5.01 (m, 2H), 4.70 (dd, 1H, J = 8.2, 7.0, H-2), 4.46 (d, 1H,

J = 6.9, H-1), 4.09 (m, 1H, H-3), 4.27–3.63 (m, 8H), 2.32 (s, 3H),

1.97 (s, 3H).

(MeOD-d₄, 400 MHz) d 7.27 (m, 2H), 7.12 (m, 2H), 4.27 (d, 1H,

J = 13.1), 4.24–3.28 (m, 10H), 2.32 (s, 3H), 0.99–0.90 (m, 3H). O-

Ethyl-N-(4-methylbenzyl)-N-(a-D-mannofuranosyl)hydroxylamine:

¹H NMR (MeOD-d₄, 400 MHz) d 7.27 (m, 2H), 7.12 (m, 2H), 4.54 (d,

1H, J = 6.4, H-1), 4.34 (dd, 1H, J = 6.3, 4.6, H-2), 4.24–3.28 (m, 9H),

2.32 (s, 3H), 0.99–0.90 (m, 3H).

4.5.7.19.

deoxy-b-

O-Benzyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-

-galactosyl)hydroxylamine (8s). Via the general

4.5.7.22. N-(4-Methylbenzyl)-N-(D-mannosyl)-O-iso-propylhydr-

procedure, 7f (23.2 mg, 0.102 mmol), 2-aminoacetyl-2-deoxy-

oxylamine (8v).

0.092 mmol),

(5.0 lL, 0.092 mmol) were reacted. The crude product mixture

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8v (TLC R_f= 0.30 in 10:1 CH₂Cl₂/MeOH)

as a white powder (26.3 mg, 88% yield). The product comprised

Via the general procedure, 7c (15.2 mg,

-mannose (18.2 mg, 0.101 mmol), and AcOH

galactose (24.8 mg, 0.112 mmol), and AcOH (5.8 L, 0.102 mmol)

were reacted. The crude product mixture was puriﬁed by SiO₂

column chromatography eluting with 10:1 CH₂Cl₂/MeOH to af-

ford 8s (TLC R_f= 0.36 in 10:1 CH₂Cl₂/MeOH) as a white powder

(26.9 mg, 61% yield). The product comprised an inseparable

mixture of b-pyranoside (49%) and b-furanoside (51%) isomers.

ESI-MS m/z (M+H) calculated for C₂₃H₃₁N₂O₆431.2, observed

431.2. O-Benzyl-N-(4-methylbenzyl)-N-(2-aminoacetyl-2-deoxy-

an inseparable mixture of

(39%), and -furanoside (22%) isomers. ESI-MS m/z (M+H) calcu-

lated for C₁₇H₂₈NO₆342.2, observed 342.2. N-(4-Methylbenzyl)-

N-(

-mannopyranosyl)-O-iso-propylhydroxylamine: ¹H NMR

a-pyranoside (39%), b-pyranoside

D-galactopyranosyl)hydroxylamine:

¹H

NMR

(MeOD-d₄,

a-D

400 MHz) d 7.32–7.10 (m, 9H), 4.66 (d, 1H, J = 9.3, H-1), 4.30

(m, 1H, H-2), 4.40–3.59 (m, 4H), 3.84 (m, 2H, H-4, H-6), 3.71

(m, 1H, H-6’), 3.51 (dd, 1H, J = 10.1, 3.1, H-3), 3.44 (m, 1H,

H-5), 2.34 (s, 3H), 2.06 (s, 3H). O-Benzyl-N-(4-methylbenzyl)-

(MeOD-d₄, 400 MHz) d 7.27 (m, 2H), 7.11 (m, 2H), 4.14–3.57 (m,

3H), 4.03 (d, 1H, J = 3.1, H-1), 3.75 (m, 1H, H-6), 3.59 (m, 3H, H-3,

H-4, H-6’), 3.34 (dd, 1H, J = 9.4, 3.1, H-2), 3.15 (ddd, 1H, J = 9.5,

6.1, 2.2, H-5), 2.31 (s, 3H), 1.11–0.95 (m, 6H). N-(4-Methylben-

N-(2-aminoacetyl-2-deoxy-b-

-galactofuranosyl)hydroxylamine:

zyl)-N-(b-

D-mannopyranosyl)-O-iso-propylhydroxylamine:

¹H

¹H NMR (MeOD-d₄, 400 MHz) d 7.32–7.10 (m, 9H), 4.84 (dd, 1H,

NMR (MeOD-d₄, 400 MHz) d 7.27 (m, 2H), 7.11 (m, 2H), 4.28 (d,

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

2673

1H, J = 13.6, H-1), 4.04 (m, 1H, H-2), 4.14–3.57 (m, 8H), 2.31 (s, 3H),

1.11–0.95 (m, 6H). N-(4-Methylbenzyl)-N-( -mannofuranosyl)-

O-iso-propylhydroxylamine: ¹H NMR (MeOD-d₄, 400 MHz)

4.52 (d, 1H, J = 6.8, H-1), 4.37 (dd, 1H, J = 6.5, 4.6, H-2), 4.20 (dd,

1H, J = 4.6, 2.4, H-3), 4.14–3.57 (m, 7H), 2.31 (s, 3H), 1.11–0.95

(m, 6H).

4.5.8. General procedure for generation of aglycons 2

-D

Digitoxigenone^9c(9.48 g, 25.5 mmol) was dissolved in methanol

(2.2 mL/mmol) and pyridine (2.2 equiv). Oxyamine hydrochloride

(1.6 equiv) was added, and the solution was stirred for 2.5 h then

concentrated. The resulting residue was dissolved in CH₂Cl₂and

washed with 1 M HCl, brine, dried over MgSO₄, ﬁltered, and then

concentrated. The mixture of oxime diastereomers (1 equiv) was

suspended in ethanol (2.9 mL/mmol) and cooled to 0 °C. Borane

tert-butylamine complex (3.3 equiv) was added, followed by the

dropwise addition 10% aq HCl (2.7 mL/mmol). The reaction mix-

ture was stirred at 0 °C for 2.5 h. After this time, Na₂CO₃was added

until gas evolution ceased, and the mixture was partitioned be-

tween water and CHCl₃. The organic layer was washed with brine,

dried over MgSO₄, ﬁltered, and concentrated. The resulting diaste-

reomeric mixture was resolved via SiO₂column chromatography.

4.5.7.23.

O-tert-Butyl-N-(4-methylbenzyl)-N-(D-mannosyl)-

hydroxylamine (8w).

Via the general procedure, 7d

(21.7 mg, 0.112 mmol),

D-mannose (22.2 mg, 0.123 mmol), and

AcOH (6.4 lL, 0.112 mmol) were reacted. The crude product mix-

ture was puriﬁed by SiO₂column chromatography eluting with

10:1 CH₂Cl₂/MeOH to afford 8w (TLC R_f= 0.47 in 10:1 CH₂Cl₂/

MeOH) as a white powder (18.4 mg, 46% yield). ¹H NMR (MeOD-

d₄, 400 MHz) d 7.28–6.95 (m, 4H), 3.52–2.80 (m, 9H), 2.21 (s,

3H), 1.36–0.74 (m, 9H). ESI-MS m/z (M+H) calculated for

4.5.8.1. 3b-Ethoxyaminodigitoxigenin 2b.

Via the general

C₁₈H₃₀NO₆356.2, observed 356.2.

procedure, digitoxigenone (200 mg, 0.537 mmol) was converted

to a mixture of ethoxyamine diastereomers. The mixture was puri-

ﬁed by SiO₂column chromatography eluting with 1:1 EtOAc/hex-

ane to elute 2b (b-isomer) (TLC R_f= 0.24 in 1:1 EtOAc/hexane)

4.5.7.24. O-Allyl-N-(4-methylbenzyl)-N-(

-mannosyl)hydroxyl-

amine (8x).

Via the general procedure, 7e (29.9 mg,

0.169 mmol),

-mannose (33.4 mg, 0.185 mmol), and AcOH

and then with 3:2 EtOAc/hexane to elute the undesired

a-isomer

(9.7 lL, 0.169 mmol) were reacted. The crude product mixture

(TLC R_f= 0.07 in 1:1 EtOAc/hexane). Aglycon 2b was obtained as

a foam (76.5 mg, 44% yield). ¹H NMR (CDCl₃, 300 MHz) d 5.87 (m,

1H), 5.01 (A of ABX, 1H, J = 18.1, 1.2), 4.82 (B of ABX, 1H, J = 18.1,

1.7), 3.73 (q, 2H), 3.24 (br s, 1H), 2.79 (m, 1H), 2.15 (m, 2H), 1.85

(m, 3H), 1.73–1.22 (m, 17H), 1.17 (t, 3H), 0.94 (s, 3H), 0.87 (s,

3H); ¹³C NMR (CDCl₃, 100 MHz): d 174.9, 174.6, 117.5, 85.5, 73.5,

69.6, 55.0, 50.9, 49.6, 41.8, 40.0, 36.6, 35.6, 35.5, 33.1, 30.4, 28.7,

26.9, 26.6, 25.3, 23.7, 21.2, 21.0, 15.8, 14.3; ESI-MS m/z (M+H) cal-

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8x (TLC R_f= 0.40 in 10:1 CH₂Cl₂/MeOH)

as a white powder (48.5 mg, 85% yield). The product comprised

an inseparable mixture of

(40%), and -furanoside (21%) isomers. ESI-MS m/z (M+H) calcu-

lated for C₁₇H₂₆NO₆340.2, observed 340.2. O-Allyl-N-(4-methyl-

benzyl)-N-( -mannopyranosyl)hydroxylamine: NMR

¹H

a-pyranoside (39%), b-pyranoside

-D

(MeOD-d₄, 400 MHz) d 7.29 (m, 2H), 7.12 (m, 2H), 5.80–5.62 (m,

1H), 5.14–5.03 (m, 2H), 4.06 (m, 1H, H-1), 3.76 (m, 1H, H-6), 3.62

(m, 3H, H-3, H-4, H-6’), 4.12–3.60 (m, 4H), 3.40 (dd, 1H, J = 9.1,

2.8, H-2), 3.22 (ddd, 1H, J = 9.6, 5.8, 2.2, H-5), 2.32 (s, 3H). O-Al-

culated for C₂₅H₄₀NO₄418.3, observed 418.3. The undesired a-iso-

mer was obtained as a foam (69.5 mg, 40% yield). ¹H NMR (CDCl₃,

300 MHz) d 5.87 (s, 1H), 5.00 (A of ABX, 1H, J = 18.1, 1.4), 4.82 (B of

ABX, 1H, J = 18.1, 1.5), 3.74 (q, 2H), 2.91 (m, 1H), 2.77 (m, 1H), 2.16

(m, 2H), 1.85 (m, 3H), 1.74–1.22 (m, 17H), 1.18 (t, 3H), 0.94 (s, 3H),

0.87 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d 174.9, 174.7, 117.7, 85.6,

77.4, 73.6, 70.1, 60.6, 51.1, 49.7, 42.1, 41.7, 40.1, 36.4, 35.6, 35.3,

33.4, 31.3, 27.3, 27.0, 25.6, 23.6, 21.7, 21.1,15.9, 14.4.

lyl-N-(4-methylbenzyl)-N-(b-D-mannopyranosyl)hydroxylamine:

¹H NMR (MeOD-d₄, 400 MHz) d 7.29 (m, 2H), 7.12 (m, 2H), 5.80–

5.62 (m, 1H), 5.14–5.03 (m, 2H), 4.29 (d, 1H, J = 13.2, H-1), 3.92

(m, 1H, H-2), 4.25–3.60 (m, 9H), 2.32 (s, 3H). O-Allyl-N-(4-methyl-

benzyl)-N-(a-D

-mannofuranosyl)hydroxylamine: ¹H NMR (MeOD-

d₄, 400 MHz) d 7.29 (m, 2H), 7.12 (m, 2H), 5.80–5.62 (m, 1H),

5.14–5.03 (m, 2H), 4.56 (d, 1H, J = 6.6, H-1), 4.37 (dd, 1H, J = 6.4,

4.6, H-2), 4.25–3.60 (m, 9H), 2.32 (s, 3H).

4.5.8.2. 3b-iso-Propoxyaminodigitoxigenin 2c.

Via the gen-

eral procedure, digitoxigenone (200 mg, 0.537 mmol) was con-

verted to a mixture of iso-propoxyamine diastereomers. The

mixture was puriﬁed by SiO₂column chromatography eluting with

2:3 EtOAc/hexane to elute 2c (b-isomer) (TLC R_f= 0.30 in 2:3

4.5.7.25. O-Benzyl-N-(4-methylbenzyl)-N-(D-mannosyl)hydrox-

ylamine (8y).

Via the general procedure, 7f (29.5 mg,

EtOAc/hexane) and then the undesired

a-isomer (TLC R_f= 0.16 in

0.130 mmol),

-mannose (25.7 mg, 0.143 mmol), and AcOH

2:3 EtOAc/hexane). Aglycon 2c was obtained as a foam (62.5 mg,

32% yield). ¹H NMR (CDCl₃, 300 MHz) d 5.87 (m, 1H), 5.01 (A of

ABX, 1H, J = 18.0, 1.3), 4.91 (B of ABX, 1H, J = 18.0, 1.6), 3.81 (sept,

1H), 3.20 (br s, 1H), 2.77 (m, 1H), 2.16 (m, 2H), 1.86 (m, 3H), 1.73–

1.22 (m, 17H), 1.14 (d, 6H), 0.93 (s, 3H), 0.87 (s, 3H); ¹³C NMR

(CDCl₃, 100 MHz): d 175.0, 174.7, 117.7, 85.7, 77.4, 74.8, 73.6,

55.2, 51.1, 49.8, 42.0, 40.2, 36.8, 35.8, 35.6, 33.3, 30.6, 29.0, 27.0,

26.8, 23.9, 21.5, 21.3, 21.2, 15.9; ESI-MS m/z (M+H) calculated for

(7.4 lL, 0.130 mmol) were reacted. The crude product mixture

was puriﬁed by SiO₂column chromatography eluting with 10:1

CH₂Cl₂/MeOH to afford 8y (TLC R_f= 0.39 in 10:1 CH₂Cl₂/MeOH)

as a white powder (44.4 mg, 88% yield). The product comprised

an inseparable mixture of

(39%), and -furanoside (21%) isomers. ESI-MS m/z (M+H) calcu-

lated for C₂₁H₂₈NO₆390.2, observed 390.2. O-Benzyl-N-(4-meth-

ylbenzyl)-N-(

-mannopyranosyl)hydroxylamine: ¹H NMR

a-pyranoside (40%), b-pyranoside

-D

C₂₆H₄₂NO₄432.3, observed 432.4. The undesired a-isomer was ob-

tained as a foam (66.9 mg, 34% yield). ¹H NMR (CDCl₃, 300 MHz) d

5.87 (s, 1H), 5.00 (A of ABX, 1H, J = 18.1, 1.6), 4.81 (B of ABX, 1H,

J = 18.1, 1.7), 3.82 (sept, 1H), 2.87 (m, 1H), 2.77 (m, 1H), 2.15 (m,

2H), 1.84 (m, 3H), 1.74–1.22 (m, 17H), 1.15 (d, 6H), 0.93 (s, 3H),

0.87 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d 174.7, 174.6, 117.6,

85.5, 77.3, 75.2, 73.5, 60.5, 50.9, 49.6, 42.0, 41.6, 40.0, 36.3, 35.5,

35.3, 33.2, 31.3, 27.2, 26.9, 25.6, 23.5, 21.6, 21.4, 20.9, 15.8.

(MeOD-d₄, 400 MHz) d 7.33 (m, 2H), 7.25 (m, 3H), 7.16 (m, 2H),

7.11 (m, 2H), 4.53–3.58 (m, 4H), 4.11 (d, 1H, J = 3.0, H-1), 3.76

(m, 1H, H-6), 3.63 (m, 3H, H-3, H-4, H-6’), 3.40 (dd, 1H, J = 9.4,

3.2, H-2), 3.23 (ddd, 1H, J = 9.6, 5.8, 2.2, H-5), 2.34 (s, 3H). O-Ben-

zyl-N-(4-methylbenzyl)-N-(b-D-mannopyranosyl)hydroxylamine:

¹H NMR (MeOD-d₄, 400 MHz) d 7.33 (m, 2H), 7.25 (m, 3H), 7.16

(m, 2H), 7.11 (m, 2H), 4.53–3.58 (m, 9H), 4.31 (d, 1H, J = 13.6,

H-1), 3.94 (m, 1H, H-2), 2.34 (s, 3H). O-Benzyl-N-(4-methylben-

zyl)-N-(a-D

-mannofuranosyl)hydroxylamine: ¹H NMR (MeOD-d₄,

4.5.8.3. 3b-tert-Butoxyaminodigitoxigenin 2d.

eral procedure, digitoxigenone (200 mg, 0.537 mmol) was con-

verted to mixture of tert-butoxyamine diastereomers. The

mixture was puriﬁed by SiO₂column chromatography eluting with

Via the gen-

400 MHz) d 7.33 (m, 2H), 7.25 (m, 3H), 7.16 (m, 2H), 7.11 (m,

2H), 4.62 (d, 1H, J = 6.0, H-1), 4.45 (m, 1H, H-2), 4.53–3.58 (m,

9H), 2.34 (s, 3H).

2674

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

1:4 EtOAc/toluene to elute 2d (b-isomer) (TLC R_f= 0.19 in 1:4

EtOAc/toluene) and then the undesired -isomer (TLC R_f= 0.14 in

36.2, 35.4, 35.3, 35.2, 33.2, 31.2, 28.2, 27.1, 26.8, 25.61, 25.57,

23.5, 21.6, 20.9, 18.8, 15.7, 10.0, 9.9.

1:4 EtOAc/toluene). Aglycon 2d was obtained as a white solid

(37.2 mg, 25% yield). ¹H NMR (CDCl₃, 300 MHz) d 5.87 (br t, 1H),

5.00 (A of ABX, 1H, J = 17.9, 1.3), 4.81 (B of ABX, 1H, J = 17.9, 1.7),

3.12 (br s, 1H), 2.78 (m, 1H), 2.16 (m, 2H), 1.86 (m, 3H), 1.73–

1.22 (m, 17H), 1.18 (s, 9H), 0.93 (s, 3H), 0.87 (s, 3H); ¹³C NMR

(CDCl₃, 100 MHz): d 174.8, 174.7, 117.8, 85.8, 76.5, 73.6, 55.2,

51.1, 49.8, 42.1, 40.2, 36.9, 35.8, 35.6, 33.4, 30.8, 29.0, 27.2, 27.1,

26.8, 24.0, 23.1, 21.4, 21.3, 15.9; ESI-MS m/z (M+H) calculated for

4.5.8.6. 3b-Cyclopentoxyaminodigitoxigenin 2g.

Via the

general procedure, digitoxigenone (92 mg, 0.247 mmol) was con-

verted to a mixture of cyclopentoxyamine diastereomers. The mix-

ture was puriﬁed by SiO₂column chromatography eluting with 2:3

EtOAc/hexane to elute 2g (b-isomer) (TLC R_f= 0.25 in 2:3 EtOAc/

hexane) and then the undesired

a-isomer (TLC R_f= 0.11 in 2:3

EtOAc/hexane). Aglycon 2g was obtained as a foam (31.7 mg, 28%

yield). ¹H NMR (CDCl₃, 400 MHz) d 5.87 (m, 1H), 5.00 (A of ABX,

1H, J = 18.1, 1.7), 4.82 (B of ABX, 1H, J = 18.1, 1.8), 4.17 (m, 1H),

3.21 (s, 1H), 2.79 (m, 1H), 2.15 (m, 2H), 1.85 (m, 3H), 1.77–1.16

(m, 26H), 0.93 (s, 3H), 0.87 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d

174.7, 174.6, 117.6, 85.6, 84.6, 73.5, 55.0, 50.9, 49.6, 41.8, 40.0,

36.6, 35.6, 35.5, 33.2, 31.7, 30.5, 29.7, 28.8, 26.9, 26.6, 23.8,

23.61, 23.58, 22.9, 21.2, 21.0, 15.8. ESI-MS m/z (M+H) calculated

C₂₇H₄₄NO₄446.3, observed 446.4. The undesired a-isomer was ob-

tained as a white solid (83.8 mg, 57% yield). ¹H NMR (CDCl₃,

300 MHz) d 5.87 (s, 1H), 5.00 (A of ABX, 1H, J = 18.0, 1.3), 4.81 (B

of ABX, 1H, J = 18.0, 1.4), 4.71 (br s 1H), 2.77 (m, 2H), 2.15 (m,

2H), 1.92–1.22 (m, 20H), 1.17 (s, 9H), 0.93 (s, 3H), 0.87 (s, 3H);

¹³C NMR (CDCl₃, 100 MHz): d 174.9, 174.7, 117.7, 85.6, 77.4, 76.4,

73.6, 60.5, 51.1, 49.7, 42.1, 41.9, 40.1, 36.4, 35.6, 35.5, 31.7, 31.7,

27.3, 27.1, 26.0, 23.7, 21.7, 21.1, 15.9.

for C₂₈H₄₄NO₄458.3, observed 458.3. The undesired

a-isomer

was obtained as a white powder (31.2 mg, 28% yield). ¹H NMR

(CDCl₃, 400 MHz) d 5.87 (m, 1H), 4.99 (m, 1H), 4.81 (m, 2H), 4.20

(m, 1H), 2.88 (m, 1H), 2.78 (m, 1H), 2.15 (m, 2H), 1.92–1.00 (m,

29H), 0.93 (s, 3H), 0.87 (s, 3H).

4.5.8.4. 3b-Benzyloxyaminodigitoxigenin 2e.

Via the general

procedure, digitoxigenone (200 mg, 0.537 mmol) was converted to

a mixture of benzyloxyamine diastereomers. The mixture was

puriﬁed by SiO₂column chromatography eluting with 2:3 EtOAc/

toluene to elute 2e (b-isomer) (TLC R_f= 0.24 in 2:3 EtOAc/hexane)

4.5.8.7. 3b-Cyclohexoxyaminodigitoxigenin 2h.

Via the gen-

and then the undesired

-isomer (TLC R_f= 0.13 in 2:3 EtOAc/hex-

eral procedure, digitoxigenone (101.4 mg, 0.272 mmol) was con-

verted to a mixture of cyclohexoxyamine diastereomers. The

mixture was puriﬁed by SiO₂column chromatography eluting with

2:3 EtOAc/hexane to elute 2h (b-isomer) (TLC R_f= 0.27 in 2:3

ane). Aglycon 2e was obtained as a foam (77.5 mg, 37% yield). ¹H

NMR (CDCl₃, 400 MHz) d 7.31 (m, 5H), 5.86 (s, 1H), 5.43 (br s,

1H), 5.00 (A of ABX, 1H, J = 18.1, 1.4), 4.81 (B of ABX, 1H, J = 18.1,

1.8), 4.70 (s, 2H), 3.29 (s, 1H), 2.77 (m, 1H), 2.15 (m, 2H), 1.85

(m, 3H), 1.74–1.22 (m, 17H), 0.94 (s, 3H), 0.86 (s, 3H); ¹³C NMR

(CDCl₃, 100 MHz): d 175.0, 174.7, 138.2, 128.6, 128.4, 127.9,

117.7, 85.6, 73.6, 55.1, 51.1, 49.8, 41.9, 40.1, 36.7, 35.8, 35.6,

33.3, 30.5, 28.8, 27.0, 26.7, 23.9, 23.0, 21.3, 21.2, 15.9; ESI-MS m/

z (M+H) calculated for C₃₀H₄₂NO₄480.3, observed 480.4. The unde-

EtOAc/hexane) and then the undesired

a-isomer (TLC R_f= 0.16 in

2:3 EtOAc/hexane). Aglycon 2h was obtained as a foam (17.6 mg,

14% yield). ¹H NMR (CDCl₃, 400 MHz) d 5.88 (m, 1H), 5.00 (A of

ABX, 1H, J = 18.2, 1.6), 4.82 (B of ABX, 1H, J = 18.2, 1.8), 3.49 (m,

1H), 3.21 (s, 1H), 2.79 (m, 1H), 2.17 (m, 2H), 1.98–1.16 (m, 31H),

0.93 (s, 3H), 0.87 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d 174.63,

174.6, 117.7, 85.6, 80.6, 73.5, 55.1, 50.9, 49.6, 41.9, 40.0, 36.6,

35.6, 35.5, 33.2, 32.2, 31.7, 30.5, 28.8, 26.9, 26.6, 25.9, 24.21,

24.18, 23.8, 22.9, 21.2, 21.0, 15.8. ESI-MS m/z (M+H) calculated

sired

a-isomer was obtained as a white powder (73.1 mg, 35%

yield). ¹H NMR (CDCl₃, 400 MHz) d 7.32 (m, 5H), 5.87 (br t, 1H),

5.48 (br s, 1H), 4.99 (A of ABX, 1H, J = 18.1, 1.4), 4.81 (B of ABX,

1H, J = 18.1, 1.8), 4.73 (s, 2H), 2.96 (m, 1H), 2.77 (m, 1H), 2.15 (m,

2H), 1.84 (m, 3H), 1.86–1.00 (m, 17H), 0.93 (s, 3H), 0.86 (s, 3H);

¹³C NMR (CDCl₃, 100 MHz): d 174.8, 174.7, 138.0, 128.5, 128.4,

128.0, 117.7, 85.6, 77.1, 73.6, 60.6, 51.1, 49.7, 42.1, 41.7, 40.1,

36.4, 35.6, 35.4, 33.4, 31.2, 27.3, 27.0, 25.6, 23.6, 21.7, 21.1, 15.9.

for C₂₉H₄₆NO₄472.3, observed 472.3. The undesired

a-isomer

was obtained as a white powder (14.2 mg, 12% yield). ¹H NMR

(CDCl₃, 400 MHz) d 6.02 (m, 1H), 5.91 (s, 1H), 4.93 (m, 2H), 4.10

(m, 1H), 2.73 (m, 2H), 2.03 (m, 2H), 1.85–1.01 (m, 31H), 0.86 (s,

3H), 0.76 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d 176.4, 173.9,

116.2, 83.7, 79.5, 73.1, 59.9, 59.7, 50.2, 49.4, 41.3, 41.1, 35.4,

35.1, 32.2, 31.3, 30.5, 27.0, 26.3, 25.6, 25.0, 23.6, 23.4, 21.3, 20.8,

20.6, 15.7, 14.1.

4.5.8.5. 3b-(2-Butyloxyamino)digitoxigenin 2f.

eral procedure, digitoxigenone (890 mg, 0.239 mmol) was con-

verted to mixture of 2-butyloxyamine diastereomers. The

Via the gen-

mixture was puriﬁed by SiO₂column chromatography eluting with

4.5.9. General procedure for generation of neoglycosides 6

2:3 EtOAc/hexane to elute 2f (3b-isomer) (TLC R_f= 0.37 in 2:3

Aglycon 2 (1 equiv) and L-xylose (1.1 equiv) were added to a

EtOAc/hexane) and then the undesired 3

in 2:3 EtOAc/hexane). Aglycon 2f was obtained as

-isomer (TLC R_f= 0.23

glass vial equipped with a stirring ﬂea and then were dissolved

in 9:1 MeOH/CHCl₃(5.6 mL/mmol). AcOH was added (1 equiv)

and the reaction mixture was stirred at 40 °C for 4 days. The crude

reaction mixture was concentrated, then suspended in 2% MeOH in

CHCl₃. The crude suspension was puriﬁed on a disposable SiO₂so-

lid-phase extraction column. Three treatments with 2 mL of 3%

MeOH/CH₂Cl₂eluted unreacted aglycon and ﬁve treatments with

2 mL of 5% MeOH/CH₂Cl₂eluted product. After puriﬁcation, only

a single product spot was observed by TLC.

foam

(33.8 mg, 33% yield). ¹H NMR (CDCl₃, 400 MHz) d 5.88 (m, 1H),

5.00 (A of ABX, 1H, J = 18.1, 1.6), 4.81 (B of ABX, 1H, J = 18.1, 1.7),

3.59 (dq, 1H, J = 12.6, 6.0), 3.22 (m, 1H), 2.79 (m, 1H), 2.15 (m,

2H), 1.77–1.16 (m, 18H), 1.13 (d, 3H, J = 6.4), 0.93 (s, 3H), 0.90 (t,

3H, J = 7.7), 0.87 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): d 174.6,

174.5, 117.7, 85.6, 80.0, 73.4, 55.0, 55.0, 50.9, 49.6, 41.9, 40.0,

36.6, 35.6, 35.5, 33.2, 30.5, 28.8, 28.7, 28.2, 28.1, 26.9, 26.6, 23.8,

23.0, 22.9, 21.2, 21.0, 18.8, 15.8, 9.98, 9.95. ESI-MS m/z (M+H) cal-

culated for C₂₇H₄₄NO₄446.3, observed 446.3. The undesired 3

4.5.9.1. 3b-Ethoxyaminodigitoxigen-b-

Via the general procedure, 2b (10.7 mg, 0.026 mmol),

(4.2 mg, 0.029 mmol), and AcOH (1.1 L, 0.026 mmol) were re-

acted to provide 6b as a white powder after puriﬁcation (5.1 mg,

36%). (TLC R_f= 0.53 in 10% MeOH/CH₂Cl₂). ¹H NMR (DMSO-d₆,

400 MHz) d 5.91 (s, 1H), 4.99–4.87 (m, 4H), 4.48 (m, 1H), 4.10 (s,

1H), 3.91 (m, 1H), 3.87 (d, 1H, J = 7.8), 3.68 (dd, 1H, J = 11.1, 5.2),

3.59 (m, 1H), 3.48–3.09 (m, 5H), 2.92 (t, 1H, J = 10.9), 2.73 (m,

-xylopyranoside (6b).

isomer was obtained as a white powder (45.2 mg, 45% yield). ¹H

NMR (CDCl₃, 400 MHz) d 5.87 (m, 1H), 4.99 (A of ABX, 1H,

J = 18.0, 1.5), 4.81 (B of ABX, 1H, J = 18.0, 1.7), 3.60 (dq, 1H,

J = 12.3, 6.2), 2.88 (m, 1H), 2.77 (m, 1H), 2.15 (m, 2H), 1.84 (m,

3H), 1.86–1.00 (m, 17H), 1.13 (d, 3H, J = 5.5), 0.93 (s, 3H), 0.91

(m, 3H), 0.87 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): d 174.6, 174.5,

117.6, 85.6, 80.44, 80.40, 73.4, 60.5, 50.9, 49.5, 42.0, 41.6, 39.9,

L-xylose

J. M. Langenhan et al. / Carbohydrate Research 346 (2011) 2663–2676

2675

1H), 2.05 (m, 2H), 1.93–1.05 (m, 18H), 0.99 (t, 3H, J = 6.2), 0.88 (s,

3H), 0.76 (s, 3H); HRMS m/z (M+H) calculated for C₃₀H₄₈NO₈

550.3380, observed 550.3371.

Sadilek, Loren Kruse, and Dale Whittington for MS assistance.

J.M.L. was supported by the Camille and Henry Dreyfus Founda-

tion, the Research Corporation, the MJ Murdock Charitable Trust

and Seattle University.

4.5.9.2.

(6c).

3b-iso-Propoxyaminodigitoxigen-b-

Via the general procedure, 2c (13.1 mg, 0.030 mmol),

L, 0.030 mmol) were

D-xylopyranoside

Supplementary data

xylose (5.0 mg, 0.033 mmol), and AcOH (1.7

reacted to provide 6c as a white powder after puriﬁcation (2.4 mg,

14%). (TLC R_f= 0.42 in 10% MeOH/CH₂Cl₂). ¹H NMR (DMSO-d₆,

400 MHz) d 5.91 (s, 1H), 4.99–4.87 (m, 4H), 4.48 (m, 1H), 4.09 (s,

1H), 3.98 (m, 1H), 3.89 (d, 1H, J = 8.5), 3.69 (m, 1H), 3.48–3.09

(m, 5H), 2.91 (t, 1H, J = 10.7), 2.73 (m, 1H), 2.05 (m, 2H), 1.86–

1.05 (m, 18H), 1.07 (d, 6H, J = 6.2), 0.88 (s, 3H), 0.76 (s, 3H); HRMS

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.carres.2011.09.019.

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(6e).

3b-sec-Butoxyaminodigitoxigen-b-

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L, 0.043 mmol) were

D-xylopyranoside

xylose (7.0 mg, 0.047 mmol), and AcOH (2.5

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3.92–3.08 (m, 6H), 2.91 (m, 1H), 2.72 (m, 1H), 2.04 (m, 2H),

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(6f).

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4.11 (s, 1H), 3.87 (d, 1H, J = 9.0), 3.68 (dd, 1H, J = 11.0, 5.2), 3.52–

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D-xylopyranoside

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Acknowledgments

We thank Noël Peters (Keck-UWCCC Small Molecule Screening

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(Seattle University), Gary Girdaukus (Univ. of Wisconsin), Martin

2676

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