Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity

www.elsevier.nl/locate/carres

Carbohydrate Research 327 (2000) 223–260

Synthesis of single- and double-chain ﬂuorocarbon and

hydrocarbon galactosyl amphiphiles and their

anti-HIV-1 activity

Barbara Faroux-Corlay ^a, Laurence Clary ^a, Catherine Gadras ^a, Djilali Hammache ^b,

Jacques Greiner ^a, Catherine Santaella ^a, Anne-Marie Aubertin ^c, Pierre Vierling ^a,*,

Jacques Fantini ^b

^aLaboratoire de Chimie Bioorganique, ESA 6001 CNRS, Uni6ersite´ de Nice Sophia-Antipolis,

Faculte´ des Sciences, Parc Valrose, F-06108 Nice, France

^bLaboratoire de Biochimie et Biologie de la Nutrition, ESA 6033 CNRS, Faculte´ des Sciences de St Je´roˆme,

F-13397 Marseille, France

^cINSERM U74, Institut de Virologie, F-67000 Strasbourg, France

Received 10 November 1999; accepted 11 January 2000

Abstract

Galactosylceramide (GalCer) is an alternative receptor allowing HIV-1 entry into CD4(−)/GalCer(+) cells. This

glycosphingolipid recognizes the V3 loop of HIV gp120, which plays a key role in the fusion of the HIV envelope

and cellular membrane. To inhibit HIV uptake and infection, we designed and synthesized analogs of GalCer. These

amphiphiles and bolaamphiphiles consist of single and double hydrocarbon and/or ﬂuorocarbon chain b-linked to

galactose and galactosamine. They derive from serine (GalSer), cysteine (GalCys), and ethanolamine (GalAE). The

anti-HIV activity and cytotoxicity of these galactolipids were evaluated in vitro on CEM-SS (a CD4(+) cell line),

HT-29, a CD4(−) cell line expressing high levels of GalCer receptor, and/or HT29 genetically modiﬁed to express

CD4. GalSer and GalAE derivatives, tested in aqueous medium or as part of liposome preparation, showed moderate

anti-HIV-1 activities (IC₅₀in the 20–220 mM range), whereas none of the GalCys derivatives was found to be active.

Moreover, only some of these anti-HIV active analogs inhibited the binding of [³H]suramin (a polysulfonyl

compound which displays a high afﬁnity for the V3 loop) to SPC3, a synthetic peptide which contains the conserved

GPGRAF region of the V3 loop. Our results most likely indicate that the neutralization of the virion through

masking of this conserved V3 loop region is not the only mechanism involved in the HIV-1 antiviral activity of our

Keywords: Galactolipid; Galactosylceramide; Fluorocompound; Liposome; HIV; Gp120

1. Introduction

the human CD4(+) macrophages, monocytes

or lymphocytes T4, by the binding of its envel-

ope glycoprotein gp120 to the cellular CD4

receptor and then to a coreceptor, such as

mainly CCR5 or CXCR4 which are seven-

transmembrane proteins of the chemokine re-

ceptor family (for recent reviews, see Refs.

[1–4]). HIV can also infect, in vitro, CD4(−)

The human immunodeﬁciency virus (HIV),

which causes AIDS, initiates the infection of

* Corresponding author. Tel.: +33-492-076143; fax: +33-

492-076151.

E-mail address: vierling@unice.fr (P. Vierling).

PII: S0008-6215(00)00055-0

224

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

neural and colon epithelial cells. Galactosyl-

ceramide (GalCer) (Scheme 1) has been pro-

posed as an alternative HIV receptor for the

infection of these CD4(−) cells [5]. Their

infection proceeds through gp120/GalCer in-

teractions which involve the third variable do-

main (V3) of gp120 [6,7], although a region in

the C2 domain has also been suggested to be

important [8]. It has been further established

that co-expression of CXCR4 on CD4(−)

colon epithelial cells is necessary for their

infection by HIV, raising the possibility that

chemokine receptors may function as co-re-

ceptors for HIV entry into CD4(−)/Gal-

Cer(+) cells [5]. Galactosylceramide sulfate

(or sulfatide), which is the natural sulfated

derivative of GalCer, has in the past also been

proposed as an alternative receptor for HIV

[5]. However, the sulfatide has been recently

shown to inhibit HIV-1 entry into CD4(−)/

CXCR4(+) cells (the sulfatide mediates

gp120 binding, but is not able to initiate the

fusion event) [9].

The V3 loop also plays a fundamental role

in infection after the virus has bound to CD4

and contains determinants for cell tropism

(for a review, see Ref. [2]). The V3 domain is

indeed involved in the association of the CD4/

gp120 complex with the chemokine co-recep-

tors. This association is required for

conformational changes of gp41 (the

transmembrane protein of the viral envelope)

that mediate fusion of the viral membrane

with the target cell membrane, hence virus

entry into the cells. In addition, gp120 is also

expressed at the surface of HIV-infected cells,

indicating that the gp120 is a valuable target

for the development of site-directed anti-HIV

therapies. Ligands that are efﬁciently recog-

nized by the viral or cellular gp120 could

indeed be both (i) efﬁcient competitors for the

gp120/CD4(or GalCer)/co-receptor interac-

tions, thus blocking acute and/or chronic HIV

infection, and preventing the propagation of

the virus, and (ii) targeting components for

anti-HIV drug carrier systems directed either

to HIV or to HIV-infected cells.

The potential of galactosphingolipids to in-

hibit HIV uptake and infection has recently

instigated the syntheses of various galacto-

lipids and the examination of their biological

(anti-inﬂuenza virus and anti-HIV) activities

[10–15]. As part of our contribution to this

ﬁeld, we reported in a preliminary communi-

cation the synthesis of some GalCer analogs

deriving from b-galactosylated

D

,

L

- and -ser-

L

ine and possessing ﬂuorocarbon and/or hydro-

carbon chains (compounds of the GalSer

series in Scheme 2) [16]. We have now ex-

tended this series to: (i) new

ine derivatives; (iii) -cysteine derivatives

(compounds of GalCys series in Scheme 2);

(iv) -serine and -cysteine galactosaminyl

L

- and (ii)

D

-ser-

L

derivatives (compounds of Gal(NHAc)Ser,

Gal(NH₂)Cys and Gal(NHAc)Cys series); (v)

L

-cysteine sulfated galactosaminyl analogs

(Gal(NHAc)(Sul)Cys

series),

and

(vi)

ethanolamine-based galactosylated (bola)am-

phiphiles (compounds of the GalAE and Gal-

BAE series). We describe here their synthesis

together with the results of biological testings,

including their anti-HIV-1 activity in various

CD4(+), CD4(−) or GalCer(+) cell cul-

tures and their ability to interact with SPC3, a

synthetic peptide which is a mimic of the V3

loop (this assay proved useful to select analogs

that recognize the V3 loop, a good correlation

being generally found between the afﬁnity for

the V3 loop of a given analog and its anti-

HIV activity) [14,17].

The highly ﬂuorinated galactosylated

(bola)amphiphiles were more particularly de-

signed for their enhanced hydrophobic and

lipophobic character resulting from the pres-

ence of the ﬂuorocarbon chains [18]. These

compounds should exhibit a stronger tendency

to self-organize and to form galactosyl-rich

clusters and domains or patches when incor-

porated within conventional membranes, thus

increasing the interactions with gp120 [19,20].

They may further serve as components for the

Scheme 1. Chemical structure of galactosylceramide (GalCer)

and of its sulfated analog.

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

225

Scheme 2. Chemical structure and code name of the GalCer analogs reported in this study.

formulation of targeted ﬂuorinated liposomes,

which are attractive drug carrier and delivery

systems [21], owing to their extended blood

circulation times [22].

The anomeric b-conﬁguration of galactosyl

was selected for all galactosylated derivatives

reported here, as this conﬁguration seems to

be essential for anti-HIV activity [23–25]. The

hydrophobic chains have been connected to

the sugar moiety through serine in order to

mimic the ceramide part of GalCer. Although

close analogs of the GalSer and Gal(N-

HAc)Ser compounds have been reported by

other groups [12,13], they differ by the nature

and length of the hydrophobic chains con-

nected to serine. Supported by the anti-HIV

activity found for C-glycosides (i.e., the car-

bohydrate moiety and the hydrophobic

residue are linked together through a CꢀC

bond) [10] and in order to avoid the in vivo

enzymatic hydrolysis of glycosides, which is

expected to reduce their potential anti-HIV

activity, we designed the more stable S-

glycosides deriving from cysteine. Modiﬁca-

tions on the sugar moiety, which were found

to increase anti-HIV activity in other series of

glycolipids [10–15], were also performed.

These consist of the replacement of the 2-OH

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B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

for an amino or acetamido group, and the

sulfation of the OH of the galactosyl residue

[9,13].

plied for the synthesis of the single-chain

I-Gal(NHAc)Ser derivatives (Scheme 9);

3. Alternatively, some double-chain cysteine

galactolipids, e.g., compounds II-GalCys

(Scheme 5, path B), II-Gal(NH₂)Cys and

II-Gal(NHAc)Cys (Scheme 10), were pre-

pared using a combination of strategy (i)

and (ii), e.g., glycosylation of a single-

chain amido cysteine derivative, the second

lipophilic chain being introduced in a fol-

lowing step.

2. Results and discussion

Synthesis.—For the preparation of the

galactosylated derivatives shown in Scheme 2,

three general synthetic approaches were

considered:

1. O- or S-Glycosylation of serine, amido-

ethanol or cysteine building blocks con-

taining one or two hydrophobic chains.

This approach was applied for the synthe-

sis of the monoamido I-GalSer (Scheme 3),

I-GalCys (Scheme 5, path A) and I-Gal(N-

HAc)Cys (Scheme 9) series, of the diamido

II-GalSer series (Scheme 4) and of the

II-GalAE and II-GalBAE derivatives

(Schemes 7 and 8);

2. Coupling of appropriately protected

aminoacid and sugar, then successive con-

jugation of a hydrophobic chain on each

of the aminoacid functions using conven-

tional reactions in peptide chemistry. This

approach, which has beneﬁted from the

recent development of efﬁcient methods

for the synthesis of glycosylated amino

acids as building blocks [26,27], was ap-

GalSer deri6ati6es. The synthesis of the

monoamido and diamido long-chain serine b-

galactosides I-GalSer and II-GalSer is pre-

sented in Schemes 3 and 4, respectively. It ﬁrst

requires the preparation of the aglycones 1

and 7. The monoamido

derivatives 1 were obtained in one step (50–

80% yield range) starting from Fmoc- -,

Fmoc- - or Fmoc- -serine, respectively. The

D,

L

-,

L

- and

D-serine

D L

,

L

D

condensation of these Fmoc-derivatives with

tetradecyl-, hexadecyl- or 11-(F-hexyl)-undecyl-

amine was performed in the presence of DCC

(or EDC)–HOBt and NEt(iPr)₂(in order to

avoid Fmoc-deprotection) [28]. The mixed

hydrocarbon–ﬂuorocarbon double-chain 7a

and 7b aglycones and the ﬂuorocarbon–

ﬂuorocarbon double-chain 7c one were syn-

thesized in four steps starting from O-ben-

zyl-N-Boc-

L

- or

D

, -serine (Scheme 4) [29].

L

Scheme 3. Synthetic route to the hydrocarbon and ﬂuorocarbon single-chain I-GalSer derivatives. (i) R¹NH₂–DCC–HOBt–

DMF; (ii) 2,3,4,6-tetra-O-acetyl-a- -galactopyranosyl trichloroacetimidate [or GalOC(ꢁNH)CCl₃]–TMSOTf–CH₂Cl₂; (iii) mor-

pholine–CHCl₃; (iv) 2:1:1 MeOH–Et₃N–water; (v) MeONa–MeOH.

D

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

227

Scheme 4. Synthetic route to the hydrocarbon and ﬂuorocarbon double-chain II-GalSer derivatives. (i) R¹NH₂–DCC–HOBt–

DMF; (ii) CF₃CO₂H; (iii) R²COCl–Et₃N–CHCl₃; (iv) H₂–Pd–C, AcOH–MeOH; (v) GalOC(ꢁNH)CCl₃–TMSOTf–CH₂Cl₂; (vi)

2:1:1 MeOH–Et₃N–water; (vii) 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide, Ag₂CO₃–I₂–CHCl₃; (viii) HgBr₂–MeNO₂.

Scheme 5. Synthetic route to the hydrocarbon single-chain I-GalCys(L) and double-chain II-GalCys(L) derivatives. (i) 1,2,3,4,6-

penta-O-acetyl-b-D-galactopyranose, BF₃·Et₂O–CH₂Cl₂; (ii) morpholine–CHCl₃for 10a; (iii) H₂–Pd–C, MeOH for 10b; (iv)

2:1:1 MeOH–Et₃N–water; (v) R²CO₂H–DCC–HOBt–DMF.

Condensation of this protected aminoacid

derivative with tetradecyl-, hexadecyl- or 11-

(F-butyl)undecyl-amine in the presence of

DCC–HOBt, then Boc-deprotection gave 6a–

c, respectively. Acylation of these latter com-

pounds with the appropriate perﬂuoroalkyl-

ated acid chloride and further hydrogenolysis

of the benzyl group afforded the diamido

serines 7a–c in almost 60% overall yields.

Galactosylation of aglycones 1 and 7 was

best performed using the Schmidt method

(Schemes 3 and 4, respectively) [30,31]. Thus,

the b-galactosides 2 and 8 were obtained in

yields ranging from 30 to 65% by reacting 1

228

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

ture for close analogs [43,44]: the anomeric

H-1 proton resonance of compounds 2 and 8

is a doublet centered at ꢀ4.5 ppm (J_1,27.8

Hz), in agreement with a b-conﬁguration, un-

equivocally conﬁrmed by the anomeric ¹³C-1

resonance at ꢀ101.5 ppm (a a-conﬁguration

is characterized by a coupling of 2–3 Hz and

an anomeric ¹³C-1 resonance at 96–97 ppm).

The diamidoserine b-galactosides II-GalSer

were obtained in 80–90% yields after acetyl

deprotection of 8 in a 2:1:1 MeOH–NEt₃–

water mixture [45]. By contrast, the deprotec-

tion of the serine amino group and of the

galactose moiety of 2 to produce the

monoamido-serine b-galactosides (I-GalSer)

was most delicate. This deprotection, using a

procedure similar to that described in litera-

ture [46,47], was best performed, in terms of

efﬁciency and reproducibility, in two steps

which consist ﬁrst of the Fmoc cleavage using

morpholine [28], then the deacetylation of 3

thus produced with 2:1:1 MeOH–NEt₃–water

(overall yields ꢀ65%). We also found out

that puriﬁcation of 3 prior to deacetylation

was essential for a clean reaction. Both the

amino and galactose deprotection steps have

to be carried out very rapidly (in about 1 h) in

order to avoid degradation and b-elimination.

This is related to the low stability of the

O-glycosidic bond in basic media for gly-

copeptide with a serine residue [48] due to the

acidity of the serine CH bond. By contrast,

our attempts to deprotect 2a in one step with

2:1:1 MeOH–NEt₃–water were not successful.

Under these conditions, the deprotection took

several hours for completion and led to a

complex mixture consisting of the expected

I-GalSer[C14] galactoside along with N-tetra-

decyl-2-amino-2-propenamide (4a) and N-tetra-

decyl-2-iminopropanamide (4a%), as a result of

b-elimination. Compounds 4a and 4a% were

further obtained almost quantitatively when

the acetyldeprotection was performed with

MeONa–MeOH on the Fmoc-deprotected 3a

(Scheme 3).

and 7 with GalOC(ꢁNH)CCl₃[32,33] and a

catalytic amount of TMSOTf, respectively.

This method was preferred to the Koenigs–

Knorr glycosylation reaction [34]. Indeed, the

condensation of

D

, -serine derivative 7a with

L

‘GalBr’ in the usual Koenigs–Knorr condi-

tions (Scheme 4, step vii), gave mainly the

13

1,2-orthoester 5a (anomeric C-1 and quater-

nary ¹³C resonance at 98 and 121 ppm, respec-

tively) and the expected b-galactoside 8a. The

orthoester 5a, when heated in nitromethane

with a catalytic amount of HgBr₂[34,35], was

almost quantitatively converted into 8a (64%

overall yield for the glycosylation). Among the

glycosylation methods tested, our attempts to

prepare the b-galactosides by condensing the

serine derivatives with 1,2,3,4,6-penta-O-

acetyl- -galactopyranose in the presence of a

D

Lewis acid such as SnCl₄or BF₃·Et₂O failed

[36–38].

One drawback of the galactosylation which

was encountered using the Schmidt method

was the acetylation of the aglycones 1 and 7

(up to 20%), besides the formation of the

expected galactosides 2 and 8. Acetyl transfers

from acetyl-protected sugars to the aglycone

have often been reported to compete with

glycosylation [34,39,40]. To account for this

side-reaction, a reaction mechanism involving

an orthoester intermediate has been postu-

lated [40]. This is supported here by the for-

mation of the orthoester when the

galactosylation was performed in the condi-

tions of the Koenigs–Knorr reaction (vide

supra).

The low to moderate yields by which the

b-galactosides 2 and 8 were obtained could

also be due to the deactivation of the hydroxyl

of serine resulting from the formation of an

intramolecular hydrogen bond with the amide

function. This accounts for the poor accepting

properties of ceramides, N-acylsphingosines

[41], serine and threonine derivatives [42] in

the glycosidation reaction. In line with these

results, a low reactivity has also been shown

for the diamidoserine compounds 7 in the

course of their phosphorylation [29].

1

13

The H and C NMR spectra of the depro-

tected I-GalSer and II-GalSer are in full

agreement with the proposed structures. Their

anomeric carbon resonances are found in the

102.8–105.3 ppm range, consistent with the

The structure of the peracetylated galac-

1

tosides was established by H and ¹³C NMR,

the spectral assignments being ascertained by

comparison with the data reported in litera-

b-conﬁguration. The

D

,

L

-serine galactosyl

derivatives were obtained as their diastereoiso-

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

229

meric mixture. This is well reﬂected by their

¹³C NMR spectrum which shows two ¹³C

resonances for several carbons (e.g., C-1b:

galactosyl-protected cysteine intermediate 11.

The double-chain galactocysteine derivative 12

was obtained in 80% yield by condensing te-

tradecanoic acid with 11 using DCC–HOBt.

Deacetylation of 11 and 12 was performed

with 2:1:1 MeOH–NEt₃–water (from 60–75%

yield), as described for the single- and double-

chain GalSer derivatives.

105.3 and 104.9 for I-GalSer[C14](

D

,L)). By

contrast, the ¹³C NMR spectra of the pure

I-GalSer[C14]( or I-GalSer[C14](

D

)

L

)

diastereoisomers display a single resonance for

each carbon, indicating that no racemization

has occurred during the deprotection step of

these derivatives.

The key single-chain galactosyl-protected

cysteine synthon 11 was best obtained using a

two-step procedure consisting into the galacto-

sylation of the N-Fmoc-protected amido-cys-

teine 9a followed by Fmoc-deprotection. The

galactosylation of 9a was performed in 93%

GalCys deri6ati6es. The synthesis of the

monoamido and diamido long-chain

L

-cys-

) and II-Gal-

), respectively) is presented in Scheme 5.

teine galactosides (I-GalCys(

L

Cys(

L

The strategy used for the synthesis of the

cysteine derivatives differs substantially from

that described above for the preparation of

the serine analogs. It also allows the prepara-

tion, from an unique single-chain derivative,

of a larger range of double-chain compounds

differing by the length and nature of the sec-

ond hydrophobic chain.

yield with 1,2,3,4,6-penta-O-acetyl- -galac-

D

topyranose in the presence of BF₃·Et₂O, illus-

trating that thiols readily condense on

peracetylated glycopyranoses in the presence

of Lewis acids, in contrast to alcohols. The

Fmoc deprotection of 10a thus obtained was

achieved with morpholine in ꢀ80% yield. A

second route for the synthesis of 11 starting

from the benzyloxycarbonyl-(or Z)-protected

derivative 9b was also tested. Although the

galactosylated Z-derivative 10b could be read-

ily obtained (78% yield), the Z-deprotection

by catalytic hydrogenolysis failed however. In-

deed, hydrogenolysis of 10b led to a complex

mixture in which we could identify the b-elim-

ination products 4a and 4a%.

Both the monoamido I-GalCys(

amido II-GalCys( ) derivatives have been pre-

pared from the common single-chain

L

) and di-

L

Concerning thiols 9a,b (Scheme 6), they

were prepared in two steps starting from

Fmoc- or benzyloxycarbonyl-protected

L

-

cystine 13a,b, respectively. Coupling of tetra-

decylamine with 13a,b using DCC–HOBt

(90% yield), then reduction of the disulﬁde

14a,b by action of Zn–AcOH (90% yield for

14a) [49], or of dithiothreitol (70% yield for

14b) [50] afforded thiol 9a,b. Alternatively,

compound 9a was also prepared starting from

S-trityl and N-Fmoc protected -cysteine 15,

L

as shown in Scheme 6. Unfortunately, the

trityl-deprotection of 16 with triﬂuoroacetic

acid [51] was most difﬁcult to achieve. Com-

pound 9a could however be obtained from 16

with acceptable yields (63%) by action of

iodine [52], then reduction of the disulﬁde 14a

1

Scheme 6. Synthetic pathway for the monoamido-L-cysteine

thus obtained. The H NMR signal of the

derivatives 9. (i) R¹NH₂–DCC–HOBt–DMF; (ii) I₂, MeOH;

(iii) Zn–AcOH for X=Fmoc; dithiothreitol for X=Z; (iv)

CF₃CO₂H.

anomeric proton at 4.3 ppm (J_1,29.3 Hz) and

13

the C NMR resonance of the anomeric car-

230

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

Scheme 7. Synthetic route to the hydrocarbon and ﬂuorocarbon double-chain galactosyl-amidoethanol II-GalAE derivatives. (i)

PhCH₂Cl–NaOH–n-Bu₄N⁺HSO₄⁻–water–CH₂Cl₂; (ii) CF₃CO₂H then HCl; (iii) RCOCl–Et₃N–CH₂Cl₂; (iv) LiAlH₄–THF; (v)

R²COCl–Et₃N–CH₂Cl₂; (vi) H₂–Pd–C, AcOH–MeOH; (vii) GalOC(ꢁNH)CCl₃–TMSOTf–CHCl₃, −10 °C; (viii) 2:1:1

MeOH–Et₃N–water.

Scheme 8. Synthetic route to the bolaform hydrocarbon–hydrocarbon and hydrocarbon–ﬂuorocarbon galactosyl-amidoethanol

II-GalBAE(OH) and (bis)-galactosyl-amidoethanol II-GalBAE(Gal) derivatives. (i) Cl(O)C(CH₂)₂₂C(O)Cl–Et₃N–CH₂Cl₂; (ii)

LiAlH₄–THF; (iii) R²COCl–Et₃N–CH₂Cl₂; (iv) H₂–Pd–C–EtOH; (v) GalOC(ꢁNH)CCl₃–TMSOTf–CHCl₃, −10 °C; (vi)

MeOH–MeONa.

bon at 86–87 ppm conﬁrmed the b-conﬁgura-

tion of the S-glycosidic bond.

GalAE and GalBAE deri6ati6es. The synthe-

ses of the double-chain galactosyl amphiphiles

II-GalAE and bolaamphiphiles II-Gal-

BAE(OH) deriving from aminoethanol are

presented in Schemes 7 and 8, respectively.

They were obtained in 35 and 22% overall

yields by galactosylation of aglycones 22 and

27 using the Schmidt method, then deacetyla-

tion of the resulting compounds 23 and 28

with MeONa–MeOH, respectively. The di-

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

231

20 with 7-(perﬂuorooctyl)heptanoyl chloride

afforded 21 (83%). Acylation of diamine 25

with 5-(perﬂuorohexyl)pentanoyl chloride or

dodecanoyl chloride afforded 26a (90%) or

26b (98%), respectively. O-Benzyl deprotec-

tion by hydrogenolysis of 21 and 26 afforded

almost quantitatively building blocks 22 and

27. High hydrogen pressure (40 atm) was nec-

essary for the benzyl cleavage in the case of

the bolaform compounds 26. For some inter-

galactosylated compounds II-GalBAE(Gal)

were formed as by-products in the course of

the bolaamphiphile II-GalBAE(OH) syn-

theses.

The presence of two equivalent hydroxyl

functions in 27 and the very low solubility of

the ﬂuorocarbon–hydrocarbon 27b made

their mono-galactosylation difﬁcult to control.

We proceeded in a stochastic way, hence using

stoichiometric quantities of the two reagents

(1:1 molar ratio). A multi-step synthesis, in-

cluding protection of one of the two hydroxyl

functions, galactosylation, then deprotection,

would probably not have given better results.

It should be noted that the reaction mixtures

after glycosylation were complex and difﬁcult

to purify. Formation of 28 was accompanied

by that of the di-galactosylated compounds 29

(up to 8% in the case of 29b), as expected, and

1

mediates, both the H and ¹³C NMR spectra

shows the doubling of some signals, which

indicates the presence of two conformational

isomers due to the amide bond [53].

Single-chain I-Gal(NHAc)Ser and I-Gal(N-

HAc)Cys deri6ati6es. The preparation of the

single-chain N-acetylated galactosamine com-

pounds derived from serine and cysteine is

depicted in Scheme 9. Their syntheses have

been performed starting with the N-allyloxy-

carbonyl (Aloc)-protected galactosamine 32.

The N-Aloc approach for the synthesis of

by

2,3,4,6-tetra-O-acetyl- -galactopyranose

D

(which is also formed during the synthesis of

23). It was most difﬁcult to separate this latter

compound from 23 and 28. However, it was

much easier to purify their respective II-

GalAE and II-GalBAE(OH) derivatives after

deacetylation.

2-amino-b- -galactosides was chosen for its

D

efﬁciency in glycosylation reactions, its b-

selectivity [54], and the possibilities of chemo-

selective cleavage of the NH–Aloc function

either into the acetamido group or into the

free amino group under very smooth condi-

tions [55,56] (possibilities which were used for

the synthesis of the double-chain II-Gal(N-

HAc)Cys and II-Gal(NH₂)Cys compounds,

vide infra). Stereocontrolled syntheses of 1,2-

trans glycosaminides by electrophilic activa-

tion of an anomeric leaving group have indeed

been achieved through participation of the

C-2 amido substituent, such as NH–Aloc,

-acetyl, -chloroacetyl, or -trichloroacetyl

[34,54].

¹H and ¹³C NMR analyses of 23, 28, 29,

and of their respective deprotected II-GalAE

and II-GalBAE analogs are in full agreement

with the proposed structures. These analyses

showed more particularly a b-conﬁguration

for the anomeric O-glycosidic bond (doublet

at 4.39 ppm for the anomeric proton with a

coupling constant J_1,2of 7.8 Hz for 28, a

single resonance for the anomeric carbon in

the 101.5–103.8 ppm range [43]).

The aglycone 22 and bolaform aglycones 27

were prepared in six steps from commercial

N-(Boc)ethanolamine 17 with overall yields in

the 20–30% range (Schemes 7 and 8, respec-

tively). After O-benzyl-protection of 17 then

N-Boc-deprotection, the O-benzyl-ethanol-

amine 18 thus obtained was condensed with

palmitoyl chloride or a,v-tetracosanoyl

dichloride giving 19 and 24, respectively.

These amides were reduced into amine 20 and

a,v-tetracosanediamine 25, respectively. The

reduction was extremely slow for 24 owing to

its low solubility in THF. The preparation of

diamine 25 needed therefore two successive

reductions for completion. Acylation of amine

The key NH–Aloc–galactopyranose syn-

thon 32 was prepared in four steps (40% over-

all yield) from -galactosamine hydrochloride,

D

applying the method used for the synthesis of

its glucose analog [57]. The b-anomeric

conﬁguration of the resulting galactoside 32

1

was established by H NMR (presence of a

doublet for the anomeric proton at 5.69 ppm

with J_1,28.7 Hz), in agreement with the litera-

ture [58].

The b-galactosamine serine derivative 33

was obtained in ꢀ40% yield by reacting 32

with N-Fmoc- -serine in the presence of

L

232

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

Our attempts to obtain the fully deprotected

I-Gal(NH₂)Cys[C14]( ) analog of I-Gal(N-

HAc)Cys[C14]( ) from compound 34b were

BF₃·Et₂O. Conventional conjugation of te-

tradecylamine to the free acid function of 33

gave the single-chain derivative 34a, which

was converted in three steps into I-Gal(N-

L

unsuccessful. Simultaneous deprotection of

Aloc and Fmoc using Pd(PPh₃)₄–morpholine

[55,59], then acetyl cleavage with 2:1:1

MeOH–NEt₃–water led to a complex mixture

from which one could isolate I-Gal(N-

HAc)Ser[C14]( ) (53% overall yield). These

L

steps consisted successively in the Aloc–Ac

exchange with Pd(PPh₃)₄–Bu₃SnH–Ac₂O [55],

Fmoc cleavage with morpholine, then deacety-

lation with 2:1:1 MeOH–NEt₃–water.

HAc)Cys[C14]( ), indicating that an acetyl

L

transfer has occurred.

The single-chain I-Gal(NHAc)Cys[C14]( )

L

Double-chain II-Gal(NH₂)Cys and II-

Gal(NHAc)Cys deri6ati6es. The synthesis of

the double-chain II-Gal(NH₂)Cys and II-

Gal(NHAc)Cys derivatives is illustrated in

Scheme 10. These derivatives were prepared in

four steps starting from the same key single-

chain compound 34b in ꢀ60% overall yields.

After Fmoc-deprotection, the resulting 37 was

acylated with lauric or myristic acid using

EDC–HOBt. Deacetylation of 38 thus ob-

tained by action of MeOH–NEt₃(10:1 ratio),

then Aloc cleavage in 39 with Pd(PPh₃)₂Cl₂in

the presence of Bu₃SnH–water [55,56] yielded

derivatives II-Gal(NH₂)Cys. This sequence of

deprotection has been applied in order to

avoid any possibility of O- to N-acetyl migra-

tion (see preceding section). On the other

hand, an Aloc–Ac exchange in 38 promoted

analog was obtained using a very similar syn-

thetic pathway (56% yield from 32). The

unique modiﬁcation with the precedent strat-

egy lies in the glycosylation step which was

performed with 32 onto the pre-formed single-

chain amido N-Fmoc-

¹H and ¹³C NMR analyses of I-Gal(N-

HAc)Ser[C14]( ) and I-Gal(NHAc)Cys[C14]-

) were consistent with the b-conﬁguration of

L

-cysteine derivative 9a.

L

(L

the O- and S-glycosidic bond. This is more

particularly conﬁrmed by the anomeric proton

resonance which appears as a doublet at ꢀ4.4

ppm with a large coupling constant (J_1,28.1

and 10.3 Hz, respectively), and by the

anomeric ¹³C resonance which is located at

101.8 and 83.9 ppm for the serine and cysteine

derivative, respectively.

Scheme 9. Synthetic route to the single-chain

L

-serine I-Gal(NHAc)Ser and

L

-cysteine I-Gal(NHAc)Cys 2-acetamido-b-

D

-galacto-

-serine,

syl derivatives. (i) p-MeOPhCHO, NaOH; (ii) Ac₂O, pyridine; (iii) 5 N HCl; (iv) allyl chloroformate; (v) N-Fmoc-

L

BF₃·Et₂O–CH₂Cl₂; (vi) R¹NH₂–EDC–HOBt–DMF; (vii) 9a, BF₃·Et₂O; (viii) Pd(Ph₃)₄–Bu₃SnH–Ac₂O; (ix) morpholine; (x)

2:1:1 MeOH–Et₃N–water.

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

233

Scheme 10. Synthetic route to the double-chain cysteine galactosyl-amido II-Gal(NH₂)Cys, galactosyl-acetamido II-Gal(N-

HAc)Cys and sulfogalactosyl-acetamido II-Gal(NHAc)(Sul)Cys derivatives. (i) morpholine; (ii) R²CO₂H–EDC–HOBt–DMF; (iii)

1:10 Et₃N–MeOH; (iv) Pd(Ph₃)₂Cl₂–Bu₃SnH–water; (v) Pd(Ph₃)₂Cl₂–Bu₃SnH–Ac₂O; (vi) SO₃·pyridine, DMF.

by the action of Pd(PPh₃)₂Cl₂–Bu₃SnH–Ac₂O

[55], then deacetylation of 40 with MeOH–

NEt₃(10:1 ratio), afforded the II-Gal(N-

HAc)Cys derivatives.

The H and C NMR data collected on the

galactosamine II-Gal(NHAc)Cys and II-

Gal(NH₂)Cys derivatives, are also in full

agreement with the proposed structures and,

more particularly, with the anomeric b-

conﬁguration of the S-glycosidic bond.

Double-chain II-Gal(NHAc)(Sul)Cys[C14]-

[C12] deri6ati6e. The O-sulfation of the N-

acetylated II-Gal(NHAc)Cys[C14][C12] deri-

vative was achieved with the sulfur trioxide–

pyridine complex in DMF [60]. After treat-

ment with Na₂CO₃, and chromatography on

silica gel, II-Gal(NHAc)(Sul)Cys[C14][C12], as

its Na⁺salt, was isolated in 60% yield. Its

structure was unambiguously attested to by

¹H and ¹³C NMR, 2D (¹H–¹H) COSY, and

ESIMS. The presence of the sulfate groups

was conﬁrmed by ESIMS (which indicated the

presence three sulfate groups) and by TLC

analysis (positive test with Azure A, which is a

speciﬁc reagent for sulfated glycolipids [61]). It

was noticed that the O-sulfation of II-Gal(N-

HAc)Cys was accompanied by a downﬁeld

shift of the signals corresponding to the

CHꢀO protons and carbons by about 1–1.5

and 4–6 ppm, respectively.

1

13

Biological e6aluation.—The aim of this

study was to design and synthesize analogs of

GalCer that were expected to bind to the V3

loop of HIV-1 gp120 and thus to block HIV-1

infection. The anti-HIV activity and cytotoxic-

ity of the GalCer analogs was evaluated in

vitro on CEM-SS, a CD4(+) cell line, HT-29,

a CD4(−) cell line expressing high levels of

GalCer receptor, and/or HT29 genetically

modiﬁed to express CD4. Some of these

analogs were also evaluated for their ability to

inhibit the binding of [³H]suramin (a polysul-

fonyl compound having high afﬁnity for the

V3 loop) to SPC3, a synthetic V3 peptide

(eight GPGRAF motifs radially branched on

uncharged poly-Lys core matrix). This peptide

has been found to be an inhibitor of HIV-1

infection in both CD4(+) and CD4(−) cells

[17,62]. Owing to their extremely low solubil-

234

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

ity in water, some single chain I-GalSer and I-

GalCys and the double-chain II-GalSer, II-

GalCys and II-GalAE derivatives were also

tested as 2:1 PL–CH liposomal formulations.

The results are collected in Table 1.

A speciﬁc anti-HIV activity, although mod-

erate, was measured for some single-chain I-

GalSer derivatives and some II-GalSer and

II-GalAE liposomal formulations on CEM-SS

cells (IC₅₀from 40 to 60 mM) and/or on Gal-

Cer(+)/CD4(−) or CD4(+) HT29 cells

(IC₅₀from less than 20 to 216 mM). For com-

parison, an IC₅₀of 47 mM on HT29 CD4(−)

cells has been reported for a soluble analog of

GalCer which was also able to inhibit HIV-1

induced cell fusion as well as entry in CD4(+)

cells [14], and suramin displays an IC₅₀of 38

mM on HT29 CD4(−) cells [63]. Most of the

active compounds also proved to be well toler-

ated by the cells, at least over the concentra-

tion range investigated: the CC₅₀on CEM-SS

were higher than 100 mM and inhibition on

HT29 was not associated with any toxicity as

evidenced by the XTT assay [14].

It should be noted that a similar antiviral

activity of the galactolipid-based formulations

Table 1

Anti-HIV-1 activity and cytotoxicity of the GalCer analogs

Compound

Form

CEM-SS

HT29 GalCer(+)

CD4(−)

[³H]suramin/

SPC3 inhibition

[nm(SD)] ^a

CD4(+)

IC₅₀(mM) CC₅₀(mM) IC₅₀(mM)

IC₅₀(mM)

I-GalSer[C14](

I-GalSer[C16](

D

,

)

L

)

aq

60

\100

60

\100

75

216

\1080

216

43

D

n.i. ^e

L

)

50

I-GalSer[F6C11](

D

,

L

)

aq

40

\100

F1 [110(30)]

aq

\110 ^b

n.i. ^f

I-Gal(NHAc)Ser[C14](

L

)

\10 ^b

II-GalSer[C16][F6C11](

L

)

,

F1 [80(30)]

F1 [75(25)]

F1 [50(20)]

F2 [90(25)]

F2 [120(40)]

F2 [450(100)]

F2 [500(100]

F2 [350(150)]

aq

F2 [160(40)]

F2 [160(50)]

F2 [130(40)]

[180(50)]

32

36

20 (IC₇₀)

II-GalSer[C14][F4C11](

D

L

)

36

20 (IC₄₀)

II-GalSer[F4C11][F6C11](

L

d

c

I-GalCys[C14](

L

)

27

30

d

I-Gal(NHAc)Cys[C14](

L

)

d

II-GalCys[C14][C14](

L

)

\25 ^b

18

d

II-Gal(NHAc)Cys[C14][C12](

II-Gal(NH₂)Cys[C14][C12](

II-Gal(NHAc)(Sul)Cys[C14][C12](

II-GalBAE[C24][F6C5]OH ^d

II-GalBAE[C24][C12]OH ^d

II-GalAE[C16][F8C7] ^d

L

)

d

L

)

22

L

)

\10 ^b

h

i

26

28

j

24

22

2

c

PL–CH (2:1 molar ratio)

66 ^k

270BIC₅₀B540 ^k270BIC₅₀B540 ^kn.i. ^g

^aaq: aqueous solution; F1 and F2: PL–CH–glycolipid liposome formulation 2:1:0.6 and 2:1:0.3 molar ratio, respectively. In

brackets: the mean size in nm of the liposomes with the associated standard deviation (SD), as measured by light scattering.

^bNot soluble or not dispersible as PL–CH-based liposomes at higher concentration.

^cNo speciﬁc anti-HIV activity was detected up to the corresponding CC₅₀concentration.

^dWhen tested as aqueous solution, no activity was detected for a concentration of up to 10 mM.

^en.i.: no inhibition detected up to 170 mM.

^fn.i.: no inhibition detected up to 110 mM.

^gn.i.: no inhibition detected up to 550 mM of PL–CH 2:1.

^hA maximum of 40% of inhibition is measured for a concentration ]21 mM.

ⁱA maximum of 20% of inhibition is measured for a concentration ]30 mM.

^jNo speciﬁc activity could be detected for this galactolipid, its liposomal formulation being as active as the PL–CH controls.

^kThese values correspond to the PL concentration.

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

235

not inhibited the binding of suramin to SPC3

(see below).

on the CD4(+) and CD4(−) HT29 cells is

found. This result is not unexpected if the

putative mechanism of action of the galacto-

lipids consists indeed in the masking of the V3

loop, the V3 loop being also involved in the

fusion process between the HIV-1 particle and

the plasma membrane of the CD4(+) HT29

cells (through interaction with the CXCR4

chemokine receptor expressed by these cells

[5,9]). Thus, our data suggest that the galac-

tolipids may affect HIV-1 infection by two

distinct mechanisms: (i) prevention of GalCer-

mediated HIV-1 attachment to the surface of

CD4(−)/GalCer(+) cells (see also discussion

below) and (ii) post-binding inhibition of

HIV-1 entry into CD4(+) cells.

None of the b-S-galactosyl analogs (Gal-

Cys series) of GalCer, even when formulated

as liposomes, was found to exhibit an anti-

HIV activity, these formulations being rather

cytotoxic. These derivatives were designed

more particularly in view of the higher

afﬁnity of a b-c-galactosyl analog of GalCer

for the HIV-1 gp120, as compared with its

b-O-galactosyl homologue, which was at-

tributed to improved resistance to both

chemical and enzymatic deglycosylation [10].

The lack of antiviral activity of these b-S-

galactosyl analogs shows that it is likely that

steric factors and/or glycosyl conformation

changes resulting from the O/C/S replace-

ment [66] are almost as important for speciﬁc

gp120 (V3) recognition, if this constitutes the

mechanism of action of all of the GalCer

analogs reported in this study (vide infra).

Concerning structure–activity relation-

ships, our results obtained in the GalSer and

GalCys series indicate that replacing one hy-

droxyl for a NHAc or a NH₂group on the

galactose did not signiﬁcantly increase nor

induce a speciﬁc anti-HIV activity. Neither

could we obtain active GalCys compounds by

substituting all the hydroxyles of II-Gal(N-

HAc)Cys[C14][C12] for a sulfate group as in

II-Gal(NHAc)(Sul)Cys[C14][C12]. This result

contrasts with that obtained in an analogous

Gal(NHAc)Ser series for which sulfation sub-

stantially increased anti-HIV activity [13].

Attention was also paid to the demonstra-

tion that the anti-HIV activity found for

some of the GalCer analogs described here

was related to the masking of the V3 loop, as

reported elsewhere for other GalCer analogs

[14]. Among the anti-HIV active compounds,

only II-GalAE[C16][F8C7] and I-GalSer-

It is also noticeable that the II-GalSer and

II-GalAE liposomal formulations display a

signiﬁcantly higher anti-HIV activity on the

HT-29 cells than the aqueous I-GalSer solu-

tions. Furthermore, in a given series, the most

active anti-HIV compounds or formulations

were those containing ﬂuorinated chains or

components, respectively. This can be at-

tributed to the formation of galactosyl-rich

aggregates or domains which are more fa-

vored in the case of the highly ﬂuorinated

derivatives, owing to their higher hydro-

phobicity and lipophobicity. This in turn

is expected to enhance their interaction

with gp120, hence decreasing cellular infec-

tion [64,65]. Indeed, the nonlinear relation-

ships found between the GalCer con-

centration and its binding to gp120 suggested

a degree of cooperativity at higher GalCer

concentration raising the possibility that

gp120 preferentially or exclusively binds to

glycolipid-rich domains in the liposomal bi-

layer [64].

Concerning the impact of the serine

conﬁguration (

D

or

activity, our results, which show a compar-

able activity of the racemic mixture and

enantiomer of I-GalSer[C14] and no activity

for the enantiomer, suggest that the

L

isomer) on anti-HIV

[C14](

D

,L) were able to prevent the binding

L

of [³H]suramin to SPC3 in a dose-dependent

manner (IC₅₀of 2 and 43 mM, respectively).

By contrast, II-GalAE[C24][F6C5]OH and II-

GalAE[C24][C12]OH, which are structurally

closely related to II-GalAE[C16][F8C7], inter-

fered with SPC3 recognition very weakly and

in a dose-saturable manner. Moreover, and

very surprisingly, the anti-HIV active I-

D

L

enantiomer should be the active derivative in

the racemic mixture (one should underscore

that the

as that of sphingosine in the natural GalCer).

Surprisingly, the -stereoisomer did not show

a higher activity than the -mixture, and did

L

-conﬁguration of serine is the same

L

D,L

236

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

biochem, N-Fmoc-

bonyl- -cystine (N-Z-

-serine from Bachem and N-Fmoc-

from Propeptide.

N-Fmoc- -serine was prepared by reac-

tion between -serine and Fmoc–Cl accord-

ing to Ref. [67]. 11-(Perﬂuorobutyl)-

undecylamine and 11-(perﬂuorohexyl)unde-

cylamine were prepared according to Ref. [29],

and 11-(perﬂuorobutyl)undecanoyl chloride

and 11-(perﬂuorohexyl)undecanoyl chloride to

Ref. [68]. 5-(Perﬂuorohexyl)pentanoic or 7-

(perﬂuorooctyl)heptanoic acids were prepared

from perﬂuorohexyl iodide (Elf-Atochem) and

ethyl 4-pentenoate, or from perﬂuorooctyl io-

dide (Elf-Atochem) and 6-heptenoic acid, re-

spectively [68]. Tetracosanedioyl dichloride,

L

-cystine, N-benzyloxycar-

-cystine) and N-Fmoc-

-serine

GalSer[C14](

L

),

L

-stereoisomer of I-GalSer-

[C14](

D

,L

), and I-GalSer[F6C11](

D

,

L

) a ﬂuori-

L

nated analog of I-GalSer[C14](

D L

,

), did not

D

L

inhibit the binding of [³H]suramin to SPC3.

These results most likely indicate that the

neutralization of the virion through masking

of the highly conserved GPGRAF region of

the V3 loop is not the only mechanism in-

volved in the HIV-1 antiviral activity of our

GalCer analogs. The binding to a site of the

V3 loop that does not involve the conserved

motif as well as interference with other steps

of viral replication are likely to occur. Further

studies, including inhibition of binding to re-

combinant gp120 (in the presence or not of

speciﬁc antibodies directed against V3 epi-

topes), inhibition of syncytia formation, etc.,

are necessary to fully understand our puzzling

results and the mechanism of action of the

different anti-HIV-1 active GalCer analogs de-

scribed here.

D L

,

D L

,

dodecanoyl

chloride,

5-(perﬂuorohexyl)-

pentanoyl chloride and 7-(perﬂuorooctyl)-

heptanoyl chloride [68] were prepared from

their respective acids following conventional

procedure using SOCl₂. 2,3,4,6-Tetra-O-

acetyl-b-

pared from 1,2,3,4,6-penta-O-acetyl-b-

galactopyranose and 33% HBr–AcOH. The

-galactopyranosyl

trichloroacetimidate [GalOC(ꢁNH)CCl₃] was

obtained by 1-O-deacetylation of 1,2,3,4,6-

D

-galactopyranosyl bromide was pre-

3. Experimental

D

-

Chemical section

2,3,4,6-tetra-O-acetyl-b-

D

General. Unless indicated otherwise, the re-

actions were performed under anhyd N₂using

dry solvents and reagents. Anhydrous solvents

were prepared by standard methods.

Dicyclohexylcarbodiimide (DCC), 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDC), N-Boc-ethanolamine,

tetracosanedioic acid, 4-pentenoic acid, 6-hep-

tenoic acid, tetradecylamine, hexadecylamine,

penta-O-acetyl-b- -galactopyranose by hy-

D

drazine acetate [69] followed by condensation

with trichloroacetonitrile in the presence of

DBU [32,33]. N-Fmoc-3-O-b-

syl- -serine was prepared according to Refs.

[37,38].

D-galactopyrano-

L

Column chromatography puriﬁcations were

carried out on Silica Gel 60 (E. Merck, 70-230

mesh). The purity of all new compounds was

checked by thin-layer chromatography (TLC),

NMR and elemental analysis. TLC analyses

were performed on precoated Silica Gel F₂₅₄

plates (E. Merck) with detection by UV and

by charring with 50% methanol–sulfuric acid

solution, KMnO₄, ninhydrin, Dragendorff’s

reagents (Sigma), or 5,5%-dithiobis(2-nitroben-

zoic acid) (Aldrich). Optical rotations were

measured with a Perkin–Elmer 141 polarime-

ter (1-dm cell) at 589 nm. Melting points,

determined with a Reichert apparatus, are un-

corrected. IR spectra were recorded on a

Bruker FT-IFS 45 spectrometer as KBr discs

1,2,3,4,6-penta-O-acetyl-b- -galactopyranose,

D

hydrazine acetate, 1,8-diazabicyclo[5.4.0]-

undec-7-ene (DBU), trimethylsilyl triﬂuoro-

methanesulfonate (TMSOTf), 9-ﬂuorenyl-

methyl chloroformate (Fmoc-Cl), 1-hydroxy-

benzotriazole (HOBt), p-anisaldehyde, allyl

chloroformate, tributyltin hydride, sulfur tri-

oxide pyridine complex, dithiothreitol,

galactosamine hydrochloride, tetrakis-

D-

(triphenylphosphine)palladium(0) [Pd(PPh₃)₄],

dichlorobis(triphenylphosphine)palladium(II)

[Pd(PPh₃)₂Cl₂] and trichloroacetonitrile were

purchased from Aldrich, N-Boc-O-benzyl-

D,

L

-serine from Fluka, and boron triﬂuoride

diethyl etherate from Sigma. N-Fmoc-3-S-

trityl- -cysteine was purchased from Nova-

L

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

237

for the crystalline samples and as ﬁlms for the

solution of 1a(

D

, ) (1.15 g, 2.2 mmol), Gal-

L

1

neat liquids. H, ¹³C, and ¹⁹F NMR spectra

OC(ꢁNH)CCl₃(1.41 g, 2.9 mmol) in CH₂Cl₂

,

were recorded with a Bruker AC 200 spec-

trometer at 200, 50.3, and 188.3 MHz, respec-

tively. Chemical shifts (l) are given in ppm

relative to the signal (i) for internal reference

(60 mL) containing 5 g of 4 A molecular

sieves. The mixture was stirred for 6 h, diluted

with CH₂Cl₂, ﬁltered through Celite, washed

with saturated NaHCO₃then with brine. The

organic phase was dried with Na₂SO₄, ﬁltered,

and evaporated under diminished pressure.

Puriﬁcation by silica gel chromatography

1

Me₄Si or indirectly to CHCl₃(l 7.27) for H,

(ii) for internal reference Me₄Si or indirectly

to CDCl₃(l 76.9) for ¹³C, (iii) to internal

reference CFCl₃for ¹⁹F. Elemental analyses

were performed by the Service Central de

Microanalyse du CNRS. Electrospray mass

analyses, positive or negative mode, were ef-

fected on a ADP220 Bellingham and Stanley

apparatus.

(CHCl₃) gave 3-O-(2,3,4,6-tetra-O-acetyl-b-

D

-

galactopyranosyl)-N^a-Fmoc-

D,L

-serine tetra-

decylamide (2a( ,L), 0.54 g, 67%) as a white

D

solid [¹³C NMR (CDCl₃–CD₃OD): C-1b at

101.1 ppm].

Step 2 (one-step Fmoc- and acetyl-deprotec-

Synthesis of the single-chain I-GalSer

deri6ati6es

tion): a suspension of 2a(

D

,L) (0.12 g, 0.14

mmol) in 5:1.5:1.5 MeOH–Et₃N–water was

stirred at 35 °C for 15 h. The solvents were

evaporated under diminished pressure and the

residue was puriﬁed by chromatography

(CHCl₃to 1:3 CHCl₃–MeOH) giving a

diastereoisomeric mixture (94%) of I-

3-O-(i-

decylamide (I-GalSer[C14](

D

-Galactopyranosyl)-

D

, -serine tetra-

L

D,L

)). A solution of

DCC (1.39 g, 6.7 mmol) in DMF (25 mL) was

added dropwise at 0 °C to a solution of Fmoc-

D

, -serine (2.0 g, 6.1 mmol), tetrade-

L

cylamine (1.3 g, 6.1 mmol), and of HOBt (0.84

g, 6.2 mmol) in DMF (70 mL). The reaction

mixture was stirred at 0 °C for 30 min, then at

rt for 6 h. After evaporation of DMF under

diminished pressure, the residue was chro-

matographed on silica gel (CHCl₃) yielding

GalSer[C14](

D

, ): R_f0.09 (7:3 CHCl₃–

L

1

MeOH). H NMR (CD₃OD): l 4.30 (d, J 7.0

Hz, 1 H, H-1 Gal), 4.20–2.90 (m, 9 H, H-2–6

Gal and OCH₂CH), 3.20 (t, J 7.0 Hz, 2 H,

NHCH₂), 1.80–1.30 (m, 24 H, (CH₂)₁₂), 1.00

13

(t, J 7.0 Hz, 3 H, CH₃). C NMR (CD₃OD):

N-Fmoc-

48%), 1a(

D

,

L

-serine tetradecylamide (1.51 g,

l 172.6 and 172.3 (C(O)NH), 105.3 and 104.9

(C-1b Gal), 76.8 (C-5 Gal), 74.7 and 74.6 (C-3

Gal), 72.3 and 72.2 (C-2 Gal), 71.9 and 71.7

(OCH₂), 70.1 (C-4 Gal), 62.4 (C-6 Gal), 55.8

and 55.5 (OCH₂CH), 40.4 (NHCH₂), 32.9

(CH₂CH₂CH₃), 30.6, 30.5, 30.3, and 30.2

[(CH₂)₉], 27.9 (NHCH₂CH₂), 23.6 (CH₂CH₃),

14.3 (CH₃)

D

,L) as a white solid. R_f0.42 (49:1

CHCl₃–MeOH). IR (w cm⁻¹, KBr): 3440

(OH), 1700 (CꢁO carbamate), 1650 (CꢁO

1

amide). H NMR (CDCl₃–CD₃OD): l 7.60

(d, J 8.0 Hz, 2 H, Fmoc), 7.50 (d, J 8.0 Hz, 2

H, Fmoc), 7.35–7.10 (m, 4 H, Fmoc), 4.30 (d,

J 7.0 Hz, 2 H, C(O)OCH₂), 4.20–4.00 (m, 2

H, C(O)OCH₂CH and HOCH₂CH), 3.75 and

3.55 (AB part of an ABX system, J_AB11.1,

J_AX5.6 Hz, 2 H, CH₂OH), 3.20 (t, J 7.0 Hz, 2

H, NHCH₂), 1.70–1.00 [m, 24 H, (CH₂)₁₂],

0.85 (t, J 7.0 Hz, 3 H, CH₃). ¹³C NMR

(CDCl₃–CD₃OD): l 170.8 [C(O)NH], 157.0

[NHC(O)O], 141.3 and 143.7 (C, Fmoc),

127.8, 127.1, 125.0 and 120.0 (CH, Fmoc),

67.3 [C(O)OCH₂], 62.7 (CH₂OH), 55.5

(HOCH₂CH), 47.1 (C(O)OCH₂CH), 39.7

(NHCH₂), 33.9, 31.9, 29.7, 29.6, 29.5, 29.4,

29.3, 26.9, 25.6, and 24.9 [(CH₂)₁₁], 22.7

(CH₂CH₃), 14.1 (CH₃).

3-O-(i-

decylamide (I-GalSer[C14](

ine tetradecylamide (1a( )) was prepared in

a similar way using Fmoc- -serine with 77%

D

-Galactopyranosyl)-

L

-serine tetra-

L

)). N-Fmoc-

L-ser-

L

yield. [R_f0.42 (49:1 CHCl₃–MeOH); mp

1

122 °C: H and ¹³C NMR (CDCl₃–CD₃OD):

identical to those of 1a(

Then, the galactosylation procedure as de-

scribed above in Step 1, when applied to

D

,L), respectively].

1a(

yield) and 3-O-acetyl-N-Fmoc-

decylamide as a by-product (25% yield). 2a(

L), afforded 2a(

L

) as a white solid (67%

-serine tetra-

):

L

R_f0.5 (4:1 CH₂Cl₂–Et₂O). [h]_D+5.7°

(c 1.1; 4:1 CHCl₃–MeOH). IR (w cm⁻¹, KBr):

1750 (CꢁO ester), 1655 (CꢁO amide). ¹H

Step 1 (galactosylation): TMSOTf (0.55

mL, 2.2 mmol) was added dropwise at rt to a

238

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

NHCH₂), 2.08, 2.00, 1.98, 1.91,[(all s, all 3 H,

CH₃C(O)], 1.70 (bs, 2 H, NH₂), 1.50–1.10 [m,

24 H, (CH₂)₁₂], 0.81 (t, J 6.6 Hz, 3 H, CH₃).

171.0

[C(O)NH], 170.5, 170.2, 170.1, and 169.7

[CH₃C(O)], 101.3 (C-1b Gal), 71.9

NMR (CDCl₃–CD₃OD): l 7.68 (d, J 8.0 Hz,

2 H, Fmoc), 7.55 (d, J 8.0 Hz, 2 H, Fmoc),

7.35–7.20 (m, 4 H, Fmoc), 5.24 (bd, J_3,43.2

Hz, 1 H, H-4 Gal), 5.00 (dd, J_1,27.4, J_2,310.4

Hz, 1 H, H-2 Gal), 4.92 (dd, J_2,310.4, J_3,43.2

Hz, 1 H, H-3 Gal), 4.40 (d, J 7.4 Hz, 1 H, H-1

Gal), 4.30–3.60 [m, 9 H, H-5–6 Gal and

OCH₂CH and NHC(O)OCH₂CH], 3.15 (m, 2

H, NHCH₂), 2.06, 1.97, 1.94, and 1.91 [all s,

12 H, CH₃C(O)], 1.50–1.10 [m, 24 H,

(CH₂)₁₂], 0.80 (t, J 7.0 Hz, 3 H, CH₃). ¹³C

NMR (CDCl₃): l 170.8 [C(O)NH], 170.4,

170.3, 169.9 and 169.4 [CH₃C(O)], 156.3

[NHC(O)O], 143.7 and 141.3 (C, Fmoc),

127.1, 127.8, 124.9 and 120.0 (CH, Fmoc),

101.5 (C-1b Gal), 70.9 and 70.8 (C-3-5 Gal),

69.6 (OCH₂CH), 68.7 (C-2 Gal), 67.1 (C-4

Gal), 66.9 [C(O)OCH₂], 61.3 (C-6 Gal), 54.2

(OCH₂CH), 47.1 [C(O)OCH₂CH], 39.7

(NHCH₂), 31.9 (CH₂CH₂CH₃), 29.7, 29.6,

29.5, 29.3, 29.2 and 29.1 [(CH₂)₉], 26.8

(NHCH₂CH₂), 22.6 (CH₂CH₃), 20.54, 20.45,

20.41, 20.38 [CH₃C(O)], 13.9 (CH₂CH₃).

¹³C NMR (CDCl₃–CD₃OD):

l

(OCH₂CH), 71.0 (C-3 Gal), 70.8 (C-5 Gal),

68.9 (C-2 Gal), 67.1 (C-4 Gal), 61.3 (C-6 Gal),

55.0 (OCH₂CH), 39.4 (NHCH₂), 32.0

(CH₂CH₂CH₃), 29.9, 29.7, 29.7, 29.6, 29.5,

29.4, and 29.3 [(CH₂)₉], 27.0 (NHCH₂CH₂),

22.8 (CH₂CH₃), 20.9, 20.8, 20.7, and 20.6

[CH₃C(O)], 14.2 (CH₃).

The O-acetyl-deprotection, when performed

on 3a( ) as described above in Step 2, af-

L

forded, after chromatography (CHCl₃to 1:1

CHCl₃–MeOH), I-GalSer[C14]( ) as a white

L

solid (98% yield). R_f0.5 (76:21:3 CHCl₃–

MeOH–water). [h]_D0° (c 0.77; MeOH). mp

1

180 °C. H NMR (CDCl₃–CD₃OD): l 4.05–

3.40 (m, 10 H, H-1–6 Gal and OCH₂CH),

3.15 (t, J 6.8 Hz, 2 H, CH₂NH), 1.65–1.10 [m,

24 H, (CH₂)₁₂], 0.85 (t, J 6.6 Hz, 3 H, CH₃).

3-O-Acetyl-N-Fmoc-

L

-serine

tetradecyl-

¹³C NMR (CDCl₃–CD₃OD):

l

170.8

amide: R_f0.6 (4:1 CH₂Cl₂–Et₂O). ¹³C NMR

(CDCl₃): l 171.0 [C(O)NH], 168.6 [CH₃C(O)],

156.2 [NHC(O)O], 143.7 and 141.4 (C, Fmoc),

127.2, 127.9, 125.1, and 120.1 (CH, Fmoc),

64.3 (OCH₂CH), 67.5 [C(O)OCH₂], 49.5

(OCH₂CH), 47.2 [C(O)OCH₂CH], 39.9

(NHCH₂), 32.0 (CH₂CH₂CH₃), 29.7, 29.6,

29.5, 29.4, and 29.3 [(CH₂)₉], 26.9 (NHCH₂-

CH₂), 22.8 (CH₂CH₃), 20.9 [CH₃C(O)], 14.2

(CH₃).

[C(O)NH], 102.8 (C-1b Gal), 74.8 (C-5 Gal),

73.0 (C-3 Gal), 70.7 (C-2 Gal), 70.1 (OCH₂),

68.7 (C-4 Gal), 61.1 (C-6 Gal), 54.0

(OCH₂CH), 39.4 (NHCH₂), 31.6 (CH₂CH₂-

CH₃), 29.4, 29.3, 29.0, and 28.9 [(CH₂)₉], 26.7

(NHCH₂CH₂), 22.3 (CH₂CH₃), 13.6 (CH₃).

Anal. Calcd for C₂₃H₄₆N₂O₇·3/2H₂O (489.65):

C, 56.42; H, 10.09; N, 5.72. Found: C, 56.19;

H, 10.01; N, 5.88.

Note: a mixture of isomers 4a and 4a%

(quantitative yields) was obtained when the

2a( ) (150 mg, 0.17 mmol) and morpholine

L

(1.15 mL) in CHCl₃(2 mL) were stirred at rt

for 7 h (Fmoc-deprotection). After evapora-

tion of the solvent, the residue was puriﬁed by

chromatography (CH₂Cl₂to 1:1 CH₂Cl₂–

deprotection was performed from 2a(

mixture of 2:1:1 MeOH–Et₃N–water at 35 °C

for 12 h, or when reacting 2a( ) with morpho-

L

) in a

L

line in DMF, then with a catalytic amount of

NaOMe in MeOH. ¹³C NMR (CDCl₃) of 4a

and 4a%: l 167.8 [C(O)NH, 4a%], 161.0

[C(O)NH, 4a], 132.6 (CꢁNH, 4a%), 131.0

(CH₂ꢁC, 4a), 128.9 (CH₂ꢁC, 4a), 38.9

(NHCH₂), 32.0 (CH₂CH₂CH₃), 30.4, 29.9,

29.8, 29.5, and 29.1 [(CH₂)₁₀], 22.8 (CH₂CH₃),

14.2 (CH₃), 11.0 (CH₃, 4a%).

Et₂O) giving 3-O-(2,3,4,6-tetra-O-acetyl-b-

galactopyranosyl)- -serine tetradecylamide

(3a( ), 76 mg, 76%) as a white solid. R_f0.6

D-

L

1

(23:2 CHCl₃–MeOH). H NMR (CDCl₃): l

7.34 (t, J 5.6 Hz, 1 H, NHCH₂), 5.33 (bd, J_3,4

3.2 Hz, 1 H, H-4 Gal), 5.12 (dd, J_1,27.8, J_2,3

10.4 Hz, 1 H, H-2 Gal), 4.95 (dd, J_2,310.4, J_3,4

3.2 Hz, 1 H, H-3 Gal), 4.48 (d, J 7.8 Hz, 1 H,

H-1 Gal), 4.07 (m, 2 H, H-6,6% Gal), 3.86 (m,

2 H, H-5 Gal and OCH_aH_b), 3.77 (bt, J 8.2

Hz, 1 H, OCH_aH_b), 3.48 (dd, J 8.2, J 4.4 Hz,

1 H, OCH₂CH), 3.15 (q, J 6.6 Hz, 2 H,

3-O-(i-

decylamide (I-GalSer[C14](

ine tetradecylamide, 1a( ) was prepared in a

similar way using Fmoc- -serine with 50%

yield [R_f0.68 (9:1 CHCl₃–MeOH): ¹H and ¹³C

D

-Galactopyranosyl)-

D

-serine tetra-

D

)). N-Fmoc-

D-ser-

D

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

239

NMR (CDCl₃–CD₃OD): identical to those of

1a( ), respectively]. Then, the three-step

procedure described for the preparation of

I-GalSer[C14]( ), when applied to 1a( ), gave

successively 2a( ) as an oil (puriﬁcation by

chromatography with CH₂Cl₂to 99:1

The procedure described for the preparation

of 1a( ) when applied to a solution of DCC

(0.56 g, 2.7 mmol) in CHCl₃(10 mL) and a

solution of Fmoc- -serine (0.81 g, 2.5

D L

,

D,L

L

D

D,L

D

mmol), 11-(F-hexyl)undecylamine hydrochlo-

ride (1.3 g, 2.5 mmol), NEt(iPr)₂(0.86 mL, 5

mmol), and HOBt (0.34 g, 2.55 mmol) in

CH₂Cl₂–Et₂O; 20%), 3a(

D

) (80%) as a white

) (85%) as a white

solid, then I-GalSer[C14](

D

CHCl₃(80 mL) afforded N-Fmoc-

11-(F-hexyl)undecylamide (1.60 g, 83%)

(1c( )) as a white solid. R_f0.23 (24:1

D

,

L-serine

solid.

2a(

) [R_f0.24 (24:1 CHCl₃–MeOH)]: H and

¹³C NMR (CDCl₃) spectra are identical to

those of 2a( ) and 3a( ), respectively.

I-GalSer[C14]( ): R_f0.36 (76:21:3 CHCl₃–

D

) [R_f0.40 (4:1 CH₂Cl₂–Et₂O)] and

D,L

1

3a(D

CHCl₃–MeOH). H NMR (CDCl₃–CD₃OD):

l 7.70 (d, J 8.0 Hz, 2 H, Fmoc), 7.50 (d, J 8.0

Hz, 2 H, Fmoc), 7.45–7.15 (m, 4 H, Fmoc),

4.30 (d, J 6.5 Hz, 2 H, C(O)OCH₂), 4.15 (t, J

6.5 Hz, 1 H, C(O)OCH₂CH), 4.10 (m, 1 H,

OCH₂CH), 3.85 and 3.60 (AB part of an ABX

system, J_AB11.2, J_AX5.7, J_BX4.5 Hz, 2 H,

CH₂OH), 3.15 (t, J 7.0 Hz, 2 H, CH₂NH),

2.00 (t, J 8.0, J_HF19.0 Hz, 2 H, CH₂CF₂),

1.70–1.10 [m, 18 H, (CH₂)₉]. ¹³C NMR

(CDCl₃–CD₃OD): l 170.7 [C(O)NH], 156.7

[NHC(O)O], 143.6 and 141.2 (C, Fmoc),

127.6, 127.0, 124.8, and 119.8 (CH, Fmoc),

67.1 (C(O)OCH₂), 62.3 (CH₂OH), 55.9

(OCH₂CH), 47.0 [C(O)OCH₂CH], 39.5

(NHCH₂), 30.7 (t, J_CF22 Hz, CH₂CF₂), 29.3,

29.2, 29.1, 29.0, and 28.9 [(CH₂)₇], 26.7

L

D

MeOH–water). [h]_D+3.6° (c 0.67; MeOH).

¹H NMR (CD₃OD): l 4.20 and 4.02 (AB part

of an ABX spectrum, J_AB6.0, J_AX9.0 Hz, 2

H, OCH₂CH), 3.80–3.30 (m, 8 H, H-1–6 Gal

and OCH₂CH), 3.16 (t, J 7.0 Hz, 2 H,

NHCH₂), 1.65–1.45 (m, 2 H, NHCH₂CH₂),

1.45–1.10 [m, 22 H, (CH₂)₁₁], 0.85 (t, J 6.0 Hz,

3 H, CH₃). ¹³C NMR (CD₃OD): l 170.0

[C(O)NH], 105.3 (C-1b Gal), 76.7 (C-5 Gal),

74.6 (C-3 Gal), 72.3 (C-2 Gal), 70.1 (C-4 Gal),

68.9 (OCH₂CH), 62.4 (C-6 Gal), 55.8

(OCH₂CH), 40.4 (NHCH₂), 32.9 (CH₂CH₂-

CH₃), 30.7, 30.6, 30.5, 30.3 and 30.2 [(CH₂)₉],

27.9 (NHCH₂CH₂), 23.5 (CH₂CH₃), 14.2

(CH₃). Anal. Calcd for C₂₃H₄₆N₂O₇·3/2H₂O

(489.65): C, 56.42; H, 10.09; N, 5.72. Found:

C, 56.54; H, 10.15; N, 5.85.

(NHCH₂CH₂),

19.9

(t,

J_CF

4

Hz,

CH₂CH₂CF₂). Then, the two-step procedure

described for the preparation of I-

GalSer[C14](

gave successively 3-O-(2,3,4,6-tetra-O-acetyl-

b- -galactopyranosyl)-N-Fmoc- -serine 11-

(F-hexyl)undecylamide, 2c( ) (68% yield)

D

,L

), when applied to 1c(

D

,L

),

3-O-(i-

decylamide (I-GalSer[C16](

serine hexadecylamide (1b( )) was prepared in

) using hexadecyl-

D

-Galactopyranosyl)-

L

-serine hexa-

L

)). N-Fmoc- -

L

D

L

D L

,

a similar way as 1a(

D L

,

[¹³C NMR (CDCl₃–CD₃OD): C-1b at 100.1

1

amine. H NMR (CDCl₃–CD₃OD) spectrum

and 100.3 ppm], and I-GalSer[F6C11](

a white solid (40% yield).

D

,L) as

from l 7.70–1.90 ppm identical to that of

1a(

D

, ) then l 1.50–1.10 [m, 28 H, (CH₂)₁₄],

L

Alternatively, the Fmoc- and acetyl-depro-

) was also performed as fol-

) (0.08 g, 0.07 mmol) in 2:1

DMF-morpholine (2.25 mL) was stirred until

the total disappearance of 2c( ). After evap-

0.80 (t, J 6.0 Hz, 3 H, CH₃). Then, the three-

tection of 2c(

lows: 2c(

D,L

step procedure described for the preparation

D L

,

of I-GalSer[C14](

gave I-GalSer[C16](

NMR (CDCl₃–CD₃OD): spectrum from l

4.10–3.15 identical to that of I-GalSer[C14](

L

), when applied to 1b( ),

L

1

L

) in 40% overall yield. H

D L

,

oration to dryness, methanolic 1 M MeONa

was added dropwise to the residue in anhyd

MeOH and stirred at rt for 20 min. Puriﬁca-

tion by chromatography (CHCl₃to 1:1

CHCl₃–MeOH) of the residue obtained after

solvent evaporation afforded I-GalSer-

L

)

then l 1.65–1.10 [m, 28 H, (CH₂)₁₄], 0.85 (t, J

6.0 Hz, 3 H, CH₃). ¹³C NMR (CDCl₃–

CD₃OD), spectrum identical to that of I-

GalSer[C14]( ). Anal. Calcd for C₂₅H₅₀-

L

N₂O₇·2H₂O (526.71): C, 57.01; H, 10.33; N,

5.32. Found: C, 57.12; H, 9.98; N, 5.21.

[F6C11](

D

, ) (45 mg, 85%): R_f0.07 (7:3

L

1

CHCl₃–MeOH). H NMR (CDCl₃–CD₃OD):

3-O-(i-

D

-Galactopyranosyl)-

D

,

L

-serine 11-

l 4.31 and 4.30 (d, J 7.0 Hz, 1 H, H-1 Gal),

(F-hexyl)undecylamide (I-GalSer[F6C11](

D,L

)).

240

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

127.8 (CH, para Ph), 127.6 and 128.3 (CH,

4.20–3.40 (m,

9

H, H-2–6 Gal and

ortho and meta Ph), 80.1 [C(CH₃)₃], 73.3

(PhCH₂), 69.8 (CH₂O), 53.8 (OCH₂CH), 39.4

(NHCH₂), 31.8 (CH₂CH₂CH₃), 29.5, 29.4,

29.2, and 29.1 [(CH₂)₉], 28.1 [(CH₃)₃C], 26.7

(CH₂CH₂N), 22.5 (CH₂CH₃), 14.0 (CH₃).

OCH₂CH), 3.25 (t, J 7.0 Hz, 2 H, NHCH₂),

2.10 (tt, J_HF18.0, J 8.0 Hz, 2 H, CH₂CF₂),

1.80–1.20 [m, 18 H, (CH₂)₉]. ¹³C NMR

(CDCl₃–CD₃OD):

l

172.2 and 172.3

[C(O)NH], 102.9 and 103.3 (C-1 Gal), 74.7

(C-5 Gal), 72.8 (C-3 Gal), 70.7 and 71.1

(OCH₂), 70.4 and 70.5 C-2 Gal), 68.3 (C-4

Gal), 60.6 (C-6 Gal), 53.9 and 54.3

(OCH₂CH), 38.7 (NHCH₂), 30.0 (t, J_CF22.3

Hz, CH₂CF₂), 28.7, 28.5, 28.4, and 28.2

[(CH₂)₇], 26.1 (NHCH₂CH₂), 19.3 (t, J_CF3.5

Hz, CH₂CH₂CF₂). ¹⁹F NMR (CDCl₃–

CD₃OD): l −80.5 (3 F, CF₃), −113.8 (2 F,

CF₂CH₂), −121.2, −122.2, and −122.8

[3×2 F (CF₂)₃CF₂CH₂], −125.5 (2 F,

CF₃CF₂). Anal. Calcd for C₂₆H₃₉F₁₃N₂O₇·

1H₂O (756.58): C, 41.28; H, 5.46; N, 3.70.

Found: C, 41.33; H, 5.28; N, 3.05.

A solution of N-Boc-O-benzyl-

D

, -serine

L

tetradecylamide (3.0 g, 6.1 mmol) in

CF₃CO₂H (10 mL) was stirred at rt for 1 h.

The reaction mixture was evaporated to dry-

ness under diminished pressure. The residue

was dissolved in CHCl₃, washed with 10%

Na₂CO₃, dried over Na₂SO₄, ﬁltered and evap-

orated to dryness under diminished pressure,

affording 6a(

D

, ) (2.4 g, 100%) as a white

L

solid: R_f0.32 (49:1 CHCl₃–MeOH). IR (w

cm⁻¹, ﬁlm): 1639 cm⁻¹(CꢁO).

N-[11-(F-Butyl)undecanoyl]-

D

, -serine tetra-

L

decylamide (7a(

D

,L

)): Step 1: a solution of

Synthesis of the double-chain II-GalSer

deri6ati6es

6a( ) (2.0 g, 5.1 mmol) and triethylamine

D L

,

(0.9 mL) in CHCl₃(25 mL) was added drop-

wise at rt to a solution of 11-(F-butyl)-

undecanoyl chloride (2.5 g, 6.1 mmol) in

CHCl₃(30 mL). The reaction mixture was

stirred at rt for 24 h. After evaporation to

dryness, the residue was chromatographed on

silica gel (CHCl₃) giving N-[11-(F-butyl)-

Synthesis of aglycones 7a–c. The synthesis

and characterization of compounds 6b(

L

),

) starting from N-Boc-O-

-serine have already been described in

6c(

L

), 7b(

L

) and 7c(

L

benzyl-

L

Ref. [29].

O-Benzyl-

D

,

L-serine tetradecylamide (6a(

D

,L)):

A solution of DCC (1.9 g, 9.3 mmol) in DMF

(25 mL) was added dropwise at 0 °C to a

solution of N-Boc-O-benzyl-

undecanoyl]-O-benzyl-

D

, -serine tetradecyl-

L

amide (3.2 g, 81%) as a white solid: R_f0.71

1

D

,

L-serine (2.5 g,

(49:1 CHCl₃–MeOH). H NMR (CDCl₃): l

8.5 mmol), Et₃N (1.2 mL, 8.5 mmol), tetrade-

cylamine (1.8 g, 8.5 mmol), and HOBt (1.2 g,

8.6 mmol) in DMF (100 mL). The reaction

mixture was stirred at 0 °C for 30 min, then at

50 °C for 20 h. After evaporation of DMF

under diminished pressure, the residue was

chromatographed on silica gel (CHCl₃) giving

7.30 (m, 5 H, Ph), 6.85 (t, J 6.0 Hz, 1 H,

CH₂NH), 6.50 (d, J 6.7 Hz, 1 H, CHNH),

4.60–4.50 (m, 3 H, OCH₂Ph and OCH₂CH),

3.75 and 3.50 (AB part of an ABX system, J_AB

9.2, J_AX8.0, J_BX4.3 Hz, 2 H, CH₂OBn), 3.20

(m, 2 H, NHCH₂), 2.20 [t, J 7.2 Hz, 2 H,

CH₂C(O)], 2.00 (tt, J 7.5, J_HF18.0 Hz, 2 H,

CH₂CF₂), 1.70–1.10 [m, 40 H, (CH₂)₈and

(CH₂)₁₂], 0.85 (t, J 7.0 Hz, 3 H, CH₃). ¹³C

NMR (CDCl₃): l 173.3 [C(O)CH₂], 170.0

[CHC(O)], 137.5 (C, Ph), 128.0 (CH para Ph),

128.6 and 127.8 (CH, ortho and meta Ph),

73.5 (PhCH₂O), 69.6 (CH₂OBn), 52.2

(OCH₂CH), 39.7 (NHCH₂), 36.6 (CH₂CO),

32.0 (CH₂CH₂CH₃), 30.4 (t, J_CF22 Hz,

CH₂CF₂), 29.7, 29.6, 29.5, 29.3, and 29.1

[(CH₂)₉and (CH₂)₆], 26.9 (CH₂CH₂N), 25.6

(CH₂CH₂CO), 22.7 (CH₂CH₃), 20.0 (t, J_CF4.0

Hz, CH₂CH₂CF₂), 14.1 (CH₃).

N-Boc-O-benzyl-

D

,L-serine

tetradecylamide (3.0 g, 72%) of as a white

solid [R_f0.86 (49:1 CHCl₃–MeOH)]. IR (w

1

cm⁻¹, ﬁlm): 1713 and 1659 cm⁻¹(CꢁO). H

NMR (CDCl₃): l 7.35 (m, 5 H, Ph), 6.45 (t, 1

H, CH₂NH), 5.45 (m, 1 H, CHNH), 4.60 and

4.50 (AB system, J 11.7 Hz, 2 H, OCH₂Ph),

4.25 (m, 1 H, OCH₂CH), 3.90 and 3.55 (AB

part of an ABX system, J_AB9.2, J_AX6.6, J_BX

3.9 Hz, 2 H, CH₂OBn), 3.25 (td, J 6.2, J 6.5

Hz, 2 H, NHCH₂), 1.50 [s, 9 H, (CH₃)₃C],

1.40–1.20 [m, 24 H, (CH₂)₁₂], 0.85 (t, J 6.8 Hz,

3 H, CH₃). ¹³C NMR (CDCl₃): l 169.9

[C(O)NH], 155.3 [OC(O)NH], 137.3 (C, Ph),

Step 2: H₂(1 L/min) was bubbled through a

suspension of N-[11-(F-butyl)undecanoyl]-O-

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

241

monitoring shows the disappearance of the

singlet of the orthoester methyl proton at 1.6

ppm). After concentration and chromatog-

raphy (99:1 CHCl₃–MeOH), diastereoiso-

benzyl-

D

, -serine tetradecylamide (3.1 g, 4.0

L

mmol) in MeOH (40 mL) and AcOH (10 mL)

at 50 °C and containing Pd/C (0.31 g, 10%

w/w). After cooling, the mixture was diluted

with CHCl₃, ﬁltered through Celite. The or-

ganic phase was washed with water, dried

over Na₂SO₄, ﬁltered and evaporated to dry-

ness under diminished pressure affording com-

meric b-anomer 8a(

D

,L) (0.8 g, 64%) was ob-

tained as a white solid [¹³C NMR (CDCl₃–

CD₃OD): C-1b at 101.6 and 102.2 ppm].

Step 2: acetyl-deprotection of 8a(

D,L).

8a(

D

,L) (0.35 g, 0.35 mmol) was solubilized in

pound 7a(

D

,L) (2.8 g, 100%) as a white solid:

R_f0.25 (49:1 CHCl₃–MeOH). ¹H NMR

(CDCl₃): l 7.15 (m, 1 H, CH₂NH), 6.85 (d, J

7.1 Hz, 1 H, CHNH), 4.45 (m, 1 H,

OCH₂CH), 4.05 and 3.50 (AB part of an ABX

system, J_AB11.5, J_AX4.7, J_BX3.0 Hz, 2 H,

CH₂OH), 3.20 (td, J 6.7, J 6.3 Hz, 2 H,

NHCH₂), 2.20 [t, J 7.2 Hz, 2 H, CH₂C(O)],

2.00 (tt, J 7.5, J_HF18.0 Hz, 2 H, CH₂CF₂),

1.70–1.10 [m, 40 H, (CH₂)₈and (CH₂)₁₂], 0.85

2:1:1 MeOH–Et₃N–water mixture (10 mL).

The reaction mixture was stirred at 30 °C for

15 h until total disappearance of the starting

compound (TLC monitoring). After evapora-

tion and chromatography (CHCl₃to 3:7

CHCl₃–MeOH)

of

the

residue,

II-

GalSer[C14][F4C11](

D L

,

) (130 mg, 45%) was

obtained as a white solid: R_f0.51 (17:3

CHCl₃–MeOH). H NMR (CDCl₃–CD₃OD):

1

l 4.55 (t, J 5.1 Hz, 1 H, OCH₂CH), 4.50–3.40

(m, 9 H, H-1–6 Gal and OCH₂), 3.20 (t, J 7.2

Hz, 2 H, NHCH₂), 2.20 [t, J 7.1 Hz, 2 H,

CH₂C(O)], 2.10 (tt, J_HF17.9, J 8.1 Hz, 2 H,

CH₂CF₂), 1.70–1.10 [m, 40 H, (CH₂)₁₂and

(CH₂)₈], 0.81 (t, J 7.3 Hz, 3 H, CH₃). ¹³C

NMR (CDCl₃–CD₃OD): l 174.4 and 174.2

[NHC(O)CH₂], 170.1 and 170.0 [C(O)-

NHCH₂], 103.8 and 103.3 (C-1b Gal), 75.0

and 74.7 (C-5 Gal), 73.3 (C-3 Gal), 71.0 (C-2

Gal), 69.0 (C-4 Gal), 69.3 and 68.8 (OCH₂),

61.7 and 61.4 (C-6 Gal), 52.8 and 52.5

(OCH₂CH), 39.6 (NHCH₂), 36.1 and 36.0

[CH₂C(O)], 31.8 (CH₂CH₂CH₃), 30.6 (t, J_CF

22 Hz, CH₂CF₂), 28.9–30.1 (CH₂)₆and

(CH₂)₉, 26.8 (CH₂CH₂NH), 25.4 [CH₂CH₂-

C(O)], 22.5 (CH₂CH₃), 19.9 (t, J_CF4.0 Hz,

CH₂CH₂CF₂), 13.9 (CH₃). ¹⁹F NMR (CDCl₃–

13

(t, J 7.0 Hz, 3 H, CH₃). C NMR (CDCl₃): l

174.4 [NHC(O)CH₂], 171.0 [CHC(O)NH],

63.1 (CH₂OH), 53.9 (OCH₂CH), 39.6

(NHCH₂), 36.5 (CH₂CO), 32.0 (CH₂CH₂-

CH₃), 30.8 (t, J_CF22 Hz, CH₂CF₂), 29.7, 29.6,

29.4, 29.3, and 29.1 [(CH₂)₉and (CH₂)₆], 27.0

(CH₂CH₂NH), 25.7 (CH₂CH₂CO), 22.7

(CH₂CH₃), 20.1 (t, J_CF4.0 Hz, CH₂CH₂CF₂),

19

14.1 (CH₃). F NMR (CDCl₃): l −80.9 (3 F,

CF₃), −114.1 (2 F, CF₂CH₂), −124.0 (2 F,

CF₂CF₂CH₂), −125.6 (2 F, CF₂CF₃).

Synthesis of the II-GalSer deri6ati6es

N-[11-(F-Butyl)undecanoyl]-O-i-

pyranosyl- -serine tetradecylamide

GalSer[C14][F4C11]( )): Step 1: galactosyla-

tion of 7a( ). A solution of 2,3,4,6-

tetra-O-acetyl-a- -galactopyranosyl bromide

(0.7 g, 1.7 mmol) in anhyd CHCl₃(15 mL)

was added dropwise to 7a( ) (1.2 g, 1.7

D-galacto-

D L

,

(II-

D,L

D L

,

D

CD₃OD): identical to that of 7a(

D

,L). Anal.

D,L

Calcd for C₃₈H₆₅F₉N₂O₈·1/2H₂O (857.92): C,

53.20; H, 7.75; N, 3.27. Found: C, 53.15; H,

7.75; N, 3.38.

mmol), Ag₂CO₃(0.66 g, 2.4 mmol), I₂(43 mg)

and of 4 A molecular sieves (3 g) in CHCl₃(40

,

mL). The suspension was shaken in the dark

at rt for 7 days, then ﬁltered through Celite

which was washed with CHCl₃(100 mL). The

combined ﬁltrates were then concentrated un-

der diminished pressure. Column chromatog-

raphy (3:2 CH₂Cl₂–hexane) of the residue led

to a solid (1.2 g, 1.2 mmol) consisting in the b

N-[11-(F-Hexyl)undecanoyl]-O-i-

pyranosyl- -serine hexadecylamide

GalSer[C16][F6C11]( )): When applied to

7b( ) the galactosylation procedure described

for the synthesis of I-GalSer[C14]( ) afforded,

after silica gel chromatography [CHCl₃to 49:1

D

-galacto-

L

(II-

L

D

CHCl₃–MeOH)], pure b-anomer 8b( ) as a

L

white solid (60% yield) [¹³C NMR (CDCl₃–

anomer 8a(

Conversion of the orthoester 5a(

8a( ) was performed by reﬂuxing the ob-

D

,

L

) and the orthoester 5a(

D

,

L

).

D,L

) into b-

CD₃OD): C-1b at 101.1 ppm]. Deacetylation

D L

,

of 8b( ) in a 5:1.5:1.5 MeOH–Et₃N–water

L

tained solid with HgBr₂(21 mg, 0.06 mmol) of

mixture at 30 °C for 15 h and chromatog-

in anhyd nitromethane for 8 h (¹H NMR

raphy (CHCl₃to 3:7 CHCl₃–MeOH) gave II-

242

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

F, CF₂CH₂), −122.5, −123.4, −124.1, and

GalSer[C16][F6C11]( ) as a white solid (90%

L

yield): R_f0.51 (17:3 CHCl₃–MeOH). [h]_D

+

−125.2 [4×2 F, (CF₂)₃CF₂CH₂and

CF₂CF₂CH₂], −126.6 (4 F, CF₃CF₂). Anal.

Calcd for C₄₁H₅₈F₂₂N₂O₈·2H₂O (1160.91): C;

42.42; H, 5.38; N, 2.41. Found: C, 42.35; H,

5.29; N, 2.25.

1

3.4° (c 0.97, 4:1 CHCl₃–MeOH). H NMR

(CDCl₃–CD₃OD): l 4.60 (t, J 6.1 Hz, 1 H,

OCH₂CH), 4.25 (d, J 6.5 Hz, 1 H, H-1 Gal),

4.05 (dd, J 10.2, J 5.1 Hz, 1 H, H-3 or 4 or 5

Gal), 4.00–3.40 (m, 7 H, H-2 Gal, H-6 Gal,

two of H-3–5 Gal, OCH₂), 3.20 (t, J 7.0 Hz, 2

H, NHCH₂), 2.20 (t, J 8.3 Hz, 2 H, CH₂CO),

2.00 (tt, 2 H, J_HF18.2, J 8.1 Hz, CH₂CF₂),

1.70–1.10 [m, 44 H, (CH₂)₁₄and (CH₂)₈,], 0.85

Synthesis of the single-chain I-GalCys and

double-chain II-GalCys deri6ati6es

Synthesis of aglycones 9

N-Benzyloxycarbonyl- -cysteine tetradecyl-

L

amide (9b): Tetradecylamine (1.68 g, 7.90

mmol) and HOBt (1.06 g, 7.85 mmol) was

13

(t, 3 H, CH₃). C NMR (CDCl₃–CD₃OD): l

174.2 [CH₂C(O)NH], 170.0 [C(O)NHCH₂],

103.8 (C-1b Gal), 74.8 (C-5 Gal), 73.3 (C-3

Gal), 71.1 (C-2 Gal), 69.6 (OCH₂), 69.3 (C-4

Gal), 62.0 (C-6 Gal), 52.8 (OCH₂CH), 39.7

(NHCH₂), 36.2 (CH₂CO), 31.8 (CH₂CH₂-

CH₃), 30.8 (t, J_CF22 Hz, CH₂CF₂), 29.6 to

29.0 [(CH₂)₆and (CH₂)₁₁], 26.8 (CH₂CH₂NH),

25.5 (CH₂CH₂CO), 22.6 (CH₂CH₃), 20.0 (t,

J_CF4.0 Hz, CH₂CH₂CF₂), 13.9 (CH₃). ¹⁹F

NMR (CDCl₃–CD₃OD): identical to that of

added at 0 °C to a solution of N-Z- -cysteine

L

13b (2.0 g, 3.93 mmol) in DMF (8 mL). After

15 min of stirring, DCC (1.63 g, 7.93 mmol)

was added to the mixture and the solution was

stirred at rt for 1 h. The solvent was removed

under diminished pressure and the residue

taken up in CHCl₃and ﬁltered. The solution

was successively washed with 5% KHSO₄(2×

30 mL), water (2×30 mL), 10% NaHCO₃

(2×30 mL) and water (3×30 mL). After

drying over Na₂SO₄and ﬁltration, the solvent

was evaporated and the residue puriﬁed by

chromatography (49:1 CHCl₃–MeOH) giving

I-GalSer[F6C11](

D

,L). Anal. Calcd for

C₄₂H₆₉F₁₃N₂O₈·3/2H₂O (1004.01): C, 50.24; H,

7.23; N, 2.79. Found: C, 50.30; H, 7.16; N,

2.70.

N,N%-bis(benzyloxycarbonyl)- -cystine bis(tetra-

L

N-[11-(F-Hexyl)undecanoyl]-O-i-

pyranosyl- -serine 11-(F-butyl)-undecylamide

(II-GalSer[F4C11][F6C11]( )): When applied

to 7c( ) the galactosylation procedure de-

scribed for the synthesis of I-GalSer[C14](

afforded pure b anomer 8c( ) as a white solid

(30% yield) [¹³C NMR (CD₃OD): C-1b at

101.1 ppm]. Deacetylation of 8c( ) in a

D-galacto-

decylamide) (14b, 3.0 g, 85%) as a white solid:

R_f0.8 (49:1 CHCl₃–MeOH). mp 164 °C. IR (w

cm⁻¹, KBr): 1690 (CꢁO carbamate), 1650

L

1

(CꢁO amide). H NMR (CDCl₃–CD₃OD): l

D

)

7.30 (bs, 10 H, Ph), 5.04 (s, 4 H, OCH₂), 4.67

(m, 2 H, SCH₂CH), 3.10 (m, 4 H, NHCH₂),

2.90 (m, 4 H, SCH₂), 1.50–1.04 [m, 48 H,

(CH₂)₁₂], 0.80 (t, J 6.2 Hz, 6 H, CH₃). ¹³C

NMR (CDCl₃–CD₃OD): l 169.7 [C(O)NH],

155.7 [NHC(O)O], 136.0 (C, Ph), 128.6 and

128.3 (CH, ortho and meta Ph), 127.6 (CH,

para Ph), 67.3 (OCH₂), 55.4 (SCH₂CH), 39.9

(NHCH₂), 32.0 (CH₂CH₂CH₃), 29.8, 29.7,

29.6, 29.5 [(CH₂)₉and SCH₂], 27.1

(NHCH₂CH₂), 22.8 (CH₂CH₃), 14.2 (CH₃).

Dithiothreitol (0.72 g, 6.93 mmol) was

added to a solution of 14b (2.0 g, 2.27 mmol)

and Et₃N (1 mL, 7.2 mmol) in CHCl₃(80

mL). After stirring at rt for 24 h, the solution

was washed with 5% KHSO₄(3×30 mL),

then water (3×30 mL). After drying over

Na₂SO₄and ﬁltration, the solvent was evapo-

rated and the residue puriﬁed by chromatog-

raphy (CHCl₃) giving 9b (1.4 g, 70%) as a

L

5:1.5:1.5 MeOH–Et₃N–water mixture at

30 °C for 15 h and chromatography (CHCl₃to

4:1

CHCl₃–MeOH)

gave

II-GalSer-

[F4C11][F6C11](

L

) (60% yield): R_f0.52 (17:3

CHCl₃–MeOH). [h]_D+2.1° (c, 0.98; 4:1

1

CHCl₃–MeOH). H NMR (CDCl₃–CD₃OD):

from 4.60 to 2.20 ppm identical to that of

II-GalSer[C16][F6C11]( ), then 2.00 (tt, J_HF

L

18.2, J 8.1 Hz, 4 H, CH₂CF₂), 1.70–1.10 [m,

13

34 H, (CH₂)₉and (CH₂)₈]. C NMR (CDCl₃–

CD₃OD): from 174.2 to 36.2 ppm identical to

that of II-GalSer[C16][F6C11]( ), then 30.7

L

and 30.8 (t, t, J_CF22.3 Hz, CH₂CF₂), 29.6–

29.0 [(CH₂)₅and (CH₂)₇], 26.8 (CH₂CH₂NH),

25.5 (CH₂CH₂CO), 20.0 (t, J_CF3.5 Hz,

19

CH₂CH₂CF₂). F NMR (CDCl₃–CD₃OD): l

−81.4 and −81.6 (2×3 F, CF₃), −115.1 (4

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

243

156.0 [NHC(O)O], 144.5 (C, Ph), 143.8, 143.7

and 141.4 (C, Fmoc), 129.7 (CH, ortho Ph),

128.1 (CH, meta Ph), 127.8 and 127.1 (CH,

Fmoc), 127.0 (CH, para Ph), 125.1 and 120.1

(CH, Fmoc), 67.4 [C(O)OCH₂], 67.1 [C(Ph)₃],

54.2 (SCH₂CH), 47.2 [C(O)OCH₂CH], 39.7

(NHCH₂), 34.2 (CH₂S), 32.0 (CH₂CH₂CH₃),

29.8, 29.7, 29.6, 29.4, 29.3 and 26.9 [(CH₂)₁₀],

22.8 (CH₂CH₃), 14.2 (CH₃).

white solid: R_f0.7 (49:1 CHCl₃–MeOH). mp

113 °C. IR (w cm⁻¹, KBr): 1665 (CꢁO carba-

mate), 1650 (CꢁO amide), 695 (CꢀS). ¹H

NMR (CDCl₃): l 7.23 (bs, 5 H, Ph), 6.37 (bs,

1 H, NHCH₂), 5.78 (d, J 8.3 Hz, 1 H,

CHNH), 5.04 (s, 2 H, OCH₂), 4.30 (m, 1 H,

SCH₂CH), 3.10 (td, J 8.3, J 6.8 Hz, 2 H,

NHCH₂), 3.00 (m, 1 H, SCH_aH_b), 2.66 (m,1

H, SCH_aH_b), 1.60–1.10 [m, 24 H, (CH₂)₁₂],

0.80 (t, J 6.2 Hz, 3 H, CH₃). ¹³C NMR

Compound 16 (163 mg, 0.20 mmol) was

stirred in TFA (4 mL) for 5 h. After evapora-

tion, ether was added and the organic phase

was washed successively with water, aq

NaHCO₃(8%), and water until neutrality. Af-

ter drying over MgSO₄and ﬁltration, the sol-

vent was evaporated and the residue puriﬁed

by chromatography (19:1 CH₂Cl₂–Et₂O) giv-

ing 9a (33 mg, 30%) as a white solid: R_f0.45

(CDCl₃):

l

169.7

[C(O)NH],

156.0

[NHC(O)O], 136.1 (C, Ph), 128.7 and 128.4

(CH, ortho and meta Ph), 128.2 (CH, para

Ph) 67.4 (OCH₂), 57.0 (SCH₂CH), 39.9

(NHCH₂), 32.0 (CH₂CH₂CH₃), 30.0, 29.9,

29.8, 29.7, 29.6, 29.5, 29.4, 27.2 [(CH₂)₉and

SCH₂], 27.0 (NHCH₂CH₂), 22.8 (CH₂CH₃),

14.2 (CH₃).

N-(Fmoc)-

L

-cysteine tetradecylamide (9a):

-cysteine

1

(19:1 CH₂Cl₂–Et₂O). mp 140 °C. H NMR

Method 1: from N-Fmoc-S-trityl-

L

(CDCl₃): l 7.78 (d, J 6.9 Hz, 2 H, Fmoc), 7.59

(d, J 7.2 Hz, 2 H, Fmoc), 7.33 (m, 4 H,

Fmoc), 6.34 (bs, 1 H, NHCH₂), 5.80 [bd, J 7.9

Hz, 1 H, NHC(O)O], 4.47 [m, 2 H,

C(O)OCH₂], 4.38 (m, 1 H, SCH₂CH), 4.22 [t,

J 6.4 Hz, 1 H, C(O)OCH₂CH], 3.25 (dt, J 6.6,

J 5.9 Hz, 2 H, NHCH₂), 3.12 (m, 1 H,

SCH_aH_b), 2.70 (m, 1 H, SCH_aH_b), 1.82 (s, 1

H, SH), 1.50 (m, 2 H, NHCH₂CH₂), 1.26 [m,

22 H, (CH₂)₁₁CH₃], 0.90 (t, J 6.4 Hz, 3 H,

(15). DCC (166 mg, 0.80 mmol) was added to

a stirred solution of 15 (469 mg, 0.80 mmol),

HOBt (108 mg, 0.80 mmol) and tetradecyl-

amine (172 mg, 0.806 mmol) in anhyd DMF

(6 mL). After 12 h, the mixture was ﬁltered,

the solvent removed under diminished pres-

sure, and the residue taken up in CHCl₃and

ﬁltered. The solution was washed with 8%

NaHCO₃, water, 5% KHSO₄then water. The

organic phase was dried over MgSO₄, then

ﬁltered. After evaporation and chromatog-

raphy of the residue (7:3 CH₂Cl₂–Et₂O), N-

13

CH₃). C NMR (CDCl₃): l 169.3 [C(O)NH],

156.1 [NHC(O)O], 143.7, 141.4 (C, Fmoc),

127.9, 127.1, 125.0 and 120.1 (CH, Fmoc),

67.2 [C(O)OCH₂], 56.3 (SCH₂CH), 47.2

Fmoc-3-S-trityl-

L

-cysteine

tetradecylamide

(16) was obtained as a white solid (571 mg,

80%): R_f0.13 (CH₂Cl₂); 0.45 (19:1 CH₂Cl₂–

Et₂O). [h]_D24+8.3° (c 1.2; CHCl₃). mp 47–

49 °C. IR (w cm⁻¹, KBr): 3415 and 3300

(NH), 1700 (CꢁO carbamate), 1660 (CꢁO

amide). ¹H NMR (CDCl₃): l 7.78 (dd, J 7.2, J

3.0 Hz, 2 H, Fmoc), 7.59 (d, J 7.2 Hz, 2 H,

Fmoc), 7.45 (m, 7 H, Tr), 7.26 (m, 12 H, Tr),

5.88 (t, J 5.7 Hz, 1 H, NHCH₂), 5.21 (d, J 7.9

Hz, 1 H, NHC(O)O), 4.41 (d, J 6.7 Hz, 2 H,

C(O)OCH₂), 4.21 (t, J 6.7 Hz, 1 H,

C(O)OCH₂CH), 3.84 (ddd, J 7.9, J 7.4, J 5.7

Hz, 1 H, SCH₂CH), 3.18 (dt, J 6.6, J 5.7 Hz,

2 H, NHCH₂), 2.73 (dd, J 13.1, J 7.4 Hz, 1 H,

SCH_aH_b), 2.63 (dd, J 13.1, J 5.7 Hz, 1 H,

SCH_aH_b), 1.89 (m, 2 H, NHCH₂CH₂), 1.30

(m, 22 H, (CH₂)₁₁CH₃), 0.93 (t, J 6.7 Hz, 3 H,

CH₃). ¹³C NMR (CDCl₃): l 169.9 [C(O)NH],

[C(O)OCH₂CH],

39.8

(NHCH₂),

32.0

(CH₂CH₂CH₃), 29.7, 29.6, 29.5, 29.4, 29.3,

27.0, 26.9 [(CH₂)₁₀and HSCH₂], 22.7

(CH₂CH₃), 14.2 (CH₃).

Method 2: from N-Fmoc-cystine 13a. The

procedure described for the synthesis of 9b

from 13b when applied to 13a, afforded N-

Fmoc- -cystine bis(tetradecylamide) (14a,

L

90%). Compound 14a was also obtained in

63% yield by stirring, for 30 min at rt, 16a and

iodine in MeOH followed by addition of a 1

N aq sodium thiosulfate solution, extraction

with CHCl₃, washing with water, then drying

on MgSO₄, evaporation and chromatography:

R_f0.55 (19:1 CH₂Cl₂–Et₂O). mp 193–195 °C.

IR (w cm⁻¹, KBr): 1695 (CO carbamate), 1660

1

(CONH). H NMR (CDCl₃): l 7.68 (d, J 7.2

244

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

169.6 [CH₃C(O) and C(O)NH], 156.0

[NHC(O)O], 143.7, 141.4, 141.3 (C, Fmoc),

127.9, 127.2, 125.1, 120.1 (CH, Fmoc), 85.6

(C-1b Gal) 75.1, 71.6, 67.0, 66.0 (C-2–5 Gal),

67.5 [C(O)OCH₂], 62.0 (C-6 Gal), 54.6

(SCH₂CH), 47.2 [C(O)OCH₂CH], 39.8

(NHCH₂), 34.2 (SCH₂), 32.0 (CH₂CH₂CH₃),

29.8, 29.7, 29.6, 29.5, 29.4, and 29.3 [(CH₂)₉],

26.9 (CH₂CH₂N), 22.7 (CH₂CH₃), 20.8 and

20.6 [CH₃C(O)], 14.2 (CH₂CH₃).

Hz, 2 H, Fmoc), 7.52 (d, J 7.0 Hz, 2 H,

Fmoc), 7.33 (m, 4 H, Fmoc), 5.96 [d, J 7.9 Hz,

1 H, NHC(O)O], 4.87 (bs, 1 H, NHCH₂),

4.45–4.15 [m, 4 H, C(O)OCH₂CH and

SCH₂CH], 3.15 (m, 2 H, NHCH₂), 2.95 (m, 2

H, SCH₂), 1.50–1.10 [m, 24 H, (CH₂)₁₂CH₃],

0.89 (t, J 6.3 Hz, 3 H, CH₃). ¹³C NMR

(CDCl₃):

l

169.7

[C(O)NH],

156.0

[NHC(O)O], 143.7, 141.4 (C, Fmoc), 127.9,

127.1, 125.0 and 120.1 (CH, Fmoc), 67.8

[C(O)OCH₂], 57.8 (SCH₂CH), 47.2 [C(O)-

OCH₂CH], 39.2 (NHCH₂), 32.1 (CH₂CH₂-

CH₃), 29.8, 29.7, 29.6, 29.5, 29.4, 29.2 [(CH₂)₉

and SCH₂], 27.1 (NHCH₂CH₂), 22.7 (CH₂-

CH₃), 14.3 (CH₃).

3-S-(2,3,4,6-Tetra-O-acetyl-i-

D

-galacto-

pyranosyl)-N-benzyloxycarbonyl- -cysteine tetra-

L

decylamide (10b): The above procedure de-

scribed for the synthesis of 10a when applied

to 9b, gave 10b as an oily compound (78%): R_f

1

Four portions of Zn (1.7 g) were added over

6 h to 14a (2.7 g) in AcOH (30 mL) at 60 °C.

The mixture was then diluted with CHCl₃and

ﬁltered on Celite and washed with water until

neutrality. The crude product was puriﬁed by

chromatography (CHCl₃) giving 9a (2.5 g,

90%).

0.45 (9:1 CH₂Cl₂–Et₂O). H NMR (CDCl₃): l

7.27 (bs, 5 H, Ph), 6.52 (bs, 1 H, NHCH₂),

6.03 [d, J 7.1 Hz, 1 H, NHC(O)O], 5.36 (bs, 1

H, H-4 Gal), 5.22–4.96 (m, 2 H, H-2-3 Gal),

5.02 [s, 2 H, NHC(O)OCH₂], 4.55 (d, J 9.7

Hz, 1 H, H-1 Gal), 4.32–4.03 (m, 7 H, H-5–6

Gal, C(O)OCH₂CH and SCH₂CH), 3.17 (m, 3

H, CH₂NH and SCH_aH_b), 2.79 (m, 1 H,

SCH_aH_b), 2.03, 1.98, 1.91, and 1.89 [all s, all 3

H, CH₃C(O)], 1.40 (bs, 2 H, NHCH₂CH₂),

1.15 [m, 22 H, (CH₂)₁₁], 0.80 (t, J 6.5 Hz, 3 H,

CH₃). ¹³C NMR (CDCl₃): l 170.4, 170.1,

169.9, 169.7, 169.6 [CH₃C(O) and C(O)NH],

156.0 [NHC(O)O], 136.3 (C, Ph), 128.6 and

128.2 (CH, ortho and meta Ph), 128.2 (CH,

para Ph) 85.3 (C-1b Gal), 75.1, 71.6, 67.5 and

66.8 (GalC-2–5), 67.1 [C(O)OCH₂], 62.0 (C-6

Gal), 54.5 (SCH₂CH), 39.7 (NHCH₂), 33.9

(SCH₂), 32.0 (CH₂CH₂CH₃), 29.8, 29.7, 29.6,

29.5, 29.4, and 29.3 [(CH₂)₉], 26.9

(CH₂CH₂N), 22.7 (CH₂CH₃), 20.8 and 20.6

[CH₃C(O)], 14.2 (CH₂CH₃).

Synthesis of I-GalCys[C14](

3-S-(2,3,4,6-Tetra-O-acetyl-i-

pyranosyl)-N-Fmoc- -cysteine tetradecylamide

(10a): A solution of 9a (0.21 g, 0.40 mmol),

1,2,3,4,6 - penta -O - acetyl - - galactopyranose

L

)

D

-galacto-

L

D

(0.19 g, 0.48 mmol) and BF₃·Et₂O (250 mL, 2

mmol) in dry CH₂Cl₂(5 mL) was stirred at rt

for 24 h under anhyd N₂. The mixture was

then diluted with CH₂Cl₂, washed with 10%

NaHCO₃, then water until neutrality. After

drying over Na₂SO₄and ﬁltration, the solvent

was evaporated and the residue puriﬁed by

chromatography (CH₂Cl₂) giving 10a (0.32 g)

as an oily compound (93%): R_f0.40 (9:1

CH₂Cl₂–Et₂O). [h]_D24+9.5° (c 0.67; CHCl₃).

¹H NMR (CDCl₃): l 7.67 (d, J 7.2 Hz, 2 H,

Fmoc), 7.51 (d, J 7.0 Hz, 2 H, Fmoc), 7.33

(m, 4 H, Fmoc), 6.53 (bs, 1 H, NHCH₂), 6.07

[d, J 7.3 Hz, 1 H, NHC(O)O], 5.34 (bd, 1 H,

H-4 Gal), 5.18 (dd, J_2,39.9 and J_1,29.7 Hz, 1

H, H-2 Gal), 5.02 (dd, J_2,39.9, J_3,42.9 Hz, 1

H, H-3 Gal), 4.55 (d, J 9.7 Hz, 1 H, H-1 Gal),

4.30–3.95 [m, 7 H, H-5–6 Gal, C(O)OCH₂CH

and SCH₂CH], 3.16 (m, 3 H, CH₂NH and

SCH_aH_b), 2.82 (m, 1 H, SCH_aH_b), 2.02, 1.95,

1.89, and 1.87 [all s, all 3 H, CH₃C(O)], 1.40

(bs, 2 H, NHCH₂CH₂), 1.15 [m, 22 H,

(CH₂)₁₁], 0.82 (t, J 6.1 Hz, 3 H, CH₃). ¹³C

NMR (CDCl₃): l 170.4, 170.1, 169.9, 169.7,

3-S-(2,3,4,6-Tetra-O-acetyl-i-

pyranosyl)- -cysteine tetradecylamide (11):

D

-galacto-

L

Compound 10a (155 mg, 0.18 mmol) and

morpholine (1.2 mL, 14 mmol) in CHCl₃(2.4

mL) was stirred at rt for 8 h. After evapora-

tion of the solvent, the crude product was

puriﬁed by chromatography on silica gel (1:1

CH₂Cl₂–Et₂O) giving 11 (90 mg, 78%) as an

oily compound: R_f0.05 (4:1 CH₂Cl₂–Et₂O).

1

[h]_D24−11.3° (c 0.79; CHCl₃). H NMR

(CDCl₃): l 7.38 (t, J 5.6 Hz, 1 H, NHCH₂),

5.37 (d, J_3,43.2 Hz, 1 H, H-4 Gal), 5.16 (dd,

J_2,39.9, J_1,29.8 Hz, 1 H, H-2 Gal), 5.00 (dd,

J_2,39.9 Hz, 1 H, H-3 Gal), 4.57 (d, J 9.8 Hz,

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

245

DMF (2 mL). The resulting mixture was

stirred at rt overnight. After evaporation of

the solvent, the residue was taken up in CHCl₃

and ﬁltered. The solution was successively

washed with 5% KHSO₄, water, 10%

NaHCO₃, then water until neutrality. After

drying over Na₂SO₄and ﬁltration, the solvent

was evaporated and the residue puriﬁed by

chromatography (CHCl₃) giving N-tetra-

1 H, H-1 Gal), 4.20–3.90 (m, 3 H, H-5–6

Gal), 3.44 (ddd, J 3.9, J 8.0 Hz, 1 H,

SCH₂CH), 3.27 (dd, J 13.9, J 3.8 Hz, 2 H,

SCH_aCH_b), 3.22 (m, 2 H, CH₂NH), 2.88 (dd,

J 13.9, J 8.0 Hz, 1 H, SCH_aH_b), 1.91, 2.00 and

2.10 [all s, 12 H, CH₃C(O)], 1.85 (bs, 2 H,

NH₂), 1.10–1.50 [m, 24 H, (CH₂)₁₂], 0.80 (t, J

6.4 Hz, 3 H, CH₃). ¹³C NMR (CDCl₃): l

171.4 [C(O)NH], 170.6, 170.2, 170.0, and

169.7 [CH₃C(O)], 84.4 (C-1 Gal), 74.8, 71.4,

67.4, and 67.2 (C-2–5 Gal), 61.7 (C-6 Gal),

54.7 (SCH₂CH), 39.6 (NHCH₂), 35.5 (CH₂S),

32.0 (CH₂CH₂CH₃), 29.7, 29.6, and 29.4

[(CH₂)₉], 27.1 (CH₂CH₂NH), 22.8 (CH₂CH₃),

20.8, 20.7, and 20.6 [CH₃C(O)], 14.2 (CH₃).

decanoyl-3-S-(2,3,4,6-tetra-O-acetyl-b-

D-galac-

topyranosyl)-

L

-cysteine tetradecylamide (12,

120 mg, 80%) as a white solid: R_f0.40 (49:1

1

CHCl₃–MeOH). H NMR (CDCl₃–CD₃OD):

l 5.39 (bs, H-4 Gal, 1 H), 5.08 (m, 2 H, H-2-3

Gal), 4.67 (d, J 8.6 Hz, 1 H, H-1 Gal), 4.08

(m, 4 H, SCH₂CH and H-5–6 Gal), 3.14 (m, 3

H, NHCH₂and SCH_aCH_b), 2.87 (m, 1 H,

3-S-(i-

D

-Galactopyranosyl)-

L

-cysteine tetra-

decylamide (I-GalCys[C14](

L

)): A solution

of 11 (90 mg, 0.14 mmol) in 2:1:1 MeOH–

Et₃N–water (2.5 mL) was stirred at rt for 1 h.

After evaporation of the solvents, the crude

product was puriﬁed by chromatography on

silica gel (4:1 to 3:2 CHCl₃–MeOH) giving

SCH_aCH_b), 2.15 [t,

NHC(O)CH₂], 2.10, 1.99, 1.98, and 1.92 [all s,

H, CH₃C(O)], 1.50–1.30 [m, H,

J

7.1 Hz,

2

H,

3

4

NHCH₂CH₂and NHC(O)CH₂CH₂], 1.30–

1.10 [m, 42 H, (CH₂)₁₁CH₃and (CH₂)₁₀CH₃],

0.80 (t, J 6.6 Hz, 6 H, CH₃CH₂). ¹³C NMR

(CDCl₃–CD₃OD): l 174.1, 170.7, 170.4,

170.2, 170.1, and 169.9 [C(O)NH and

CH₃C(O)], 84.6 (C-1 Gal), 74.5, 71.7, 67.5,

and 67.2 (C-2–5 Gal), 61.7 (C-6 Gal), 52.7

(SCH₂CH), 39.4 (NHCH₂), 36.0 [NHC(O)-

CH₂], 32.8 (SCH₂), 31.7 (CH₂CH₂CH₃), 29.4,

29.1, and 29.0 [(CH₂)₉CH₂CH₂CH₃and

(CH₂)₈CH₂CH₂CH₃], 26.7 (NHCH₂CH₂), 25.5

[NHC(O)CH₂CH₂], 22.4 (CH₂CH₃), 20.2 and

20.1 [CH₃C(O)], 13.6 (CH₃).

I-GalCys[C14]( ) (40 mg, 60%) as a white

L

powder: R_f0.25 (76:21:3 CHCl₃–MeOH–wa-

1

ter). [h]_D−7.3° (c 0.80; MeOH). H NMR

(CDCl₃–CD₃OD): l 4.33 (d, J 9.3 Hz, 1 H,

H-1 Gal), 3.89 (bd, J 2.8 Hz, 1 H, H-4 Gal),

3.86–3.56 (m, 5 H, SCH₂CH, H-2 Gal and

H-5–6 Gal), 3.48 (dd, J_2,39.1, J_3,43.2 Hz, 1 H,

H-3 Gal), 3.21 (t, J 6.8 Hz, 2 H, NHCH₂),

3.12 (dd, J 14.2, J 5.3 Hz, 1 H, SCH_aH_b), 2.83

(dd, J 14.2, J 8.2 Hz, 1 H, SCH_aH_b), 1.70–

1.50 (m, 2 H, NHCH₂CH₂), 1.40–1.20 [m, 22

13

H, (CH₂)₁₁], 0.89 (t, J 6.6 Hz, 3 H, CH₃). C

A solution of 12 (120 mg, 0.14 mmol) in a

2:1:1 MeOH–Et₃N–water (2.5 mL) was then

stirred at rt for 6 h. After evaporation to

dryness under diminished pressure, the crude

residue was puriﬁed by chromatography

(CHCl₃to 3:2 CHCl₃–MeOH) giving II-Gal-

NMR (CDCl₃–CD₃OD): l 172.9 [C(O)NH],

86.0 (C-1b Gal), 79.2 (C-5 Gal), 74.6 (C-3

Gal), 69.5 (C-2 Gal), 69.2 (C-4 Gal), 61.7 (C-6

Gal), 54.7 (SCH₂CH), 39.4 (NHCH₂), 34.7

(SCH₂), 31.7 (CH₂CH₂CH₃), 29.4 and 29.1

[(CH₂)₉CH₂CH₂CH₃], 26.8 (NHCH₂CH₂),

22.4 (CH₂CH₃), 13.7 (CH₃). Anal. Calcd for

C₂₃H₄₆N₂O₆S·1/2H₂O (487.69): C; 56.64; H,

9.71; N, 5.74; S 6.57. Found: C, 56.23; H,

9.44; N, 6.44.

Cys[C14][C14]( ) (70 mg, 73%) of as a white

L

solid: R_f0.71 (76:21:3:CHCl₃–MeOH–water).

1

[h]_D−7.1° (c 0.80; 4:1 CHCl₃–MeOH). H

NMR (CDCl₃–CD₃OD): l 4.56 (dd, J 6.5 Hz,

1 H, SCH₂CH), 4.31 (d, J 9.1 Hz, 1 H, H-1

Gal), 3.85–3.46 (m, 5 H, H-2 Gal and H4-6

Gal), 3.41 (dd, J_2,39.2, J_3,42.9 Hz, 1 H, H-3

Gal), 3.15 (t, J 7.1 Hz, 2 H, NHCH₂), 2.82 (m,

2 H, SCH₂), 2.15 [t, J 7.5 Hz, 2 H, C(O)CH₂],

Synthesis of II-GalCys deri6ati6es

N-Tetradecanoyl-3-S-(i-

D

-galactopyran-

tetradecylamide (II-Gal-

)): DCC (40 mg, 0.19 mmol)

osyl)-

L

-cysteine

Cys[C14][C14](

L

was added at 0 °C to a mixture of 11 (115 mg,

0.178 mmol), tetradecanoic acid (45 mg, 0.19

mmol), and HOBt (27 mg, 0.19 mmol) in

1.60–1.40 [m,

4

H, NHCH₂CH₂and

C(O)CH₂CH₂], 1.30–1.10 [m, 42 H, (CH₂)₁₁

13

and (CH₂)₁₀], 0.80 (t, J 6.0 Hz, 6 H, CH₃). C

246

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

129.4 and 128.9 (CH, ortho and meta Ph),

128.8 (CH, para Ph), 74.1 (PhCH₂), 66.7

(OCH₂), 40.6 (CH₂N).

NMR (CDCl₃–CD₃OD): l 174.3 and 170.5

[C(O)NH], 87.2 (C-1 Gal), 79.3, 74.8, 70.2,

and 69.4 (C-2–5 Gal), 62.1 (C-6 Gal), 53.5

(SCH₂CH), 39.6 (NHCH₂), 36.2 [C(O)CH₂],

33.2 (SCH₂), 31.8 (CH₂CH₂CH₃), 29.6, 29.4,

29.2, and 29.1 [(CH₂)₉CH₂CH₂CH₃and

(CH₂)₈CH₂CH₂CH₃], 26.8 (NHCH₂CH₂), 25.6

[C(O)CH₂CH₂], 22.6 (CH₂CH₃), 13.8 (CH₃).

Anal. Calcd for C₃₇H₇₂N₂O₇S·H₂O (707.07):

C, 62.85; H, 10.55; N, 3.96; S, 4.54. Found: C,

62.65; H, 10.45; N, 3.97; S, 4.39.

Step 3: hexadecanoyl chloride (8.9 g, 32

mmol) in CHCl₃(25 mL) was added dropwise

at rt to

a

mixture of 2-(benzyloxy)-

ethylamine·HCl (3.2 g, 17 mmol) and NEt₃

(7.3 mL, 52 mmol) in CHCl₃(50 mL). The

mixture was further stirred for 48 h, then

evaporated to dryness. The crude residue was

chromatographed on silica gel (9:1 CHCl₃–

Synthesis of the II-GalAE and II-GalBAE

deri6ati6es

hexane)

giving

N-[2-(benzyloxy)ethyl]-

hexadecylamide (19, 5.2 g, 78%) as a white

powder: R_f0.7 (49:1 CHCl₃–MeOH). IR (w

N-[2-(i-

(hexadecyl)-7-(F-octyl)-heptanamide

GalAE[C16][F8C7]). Step 1:

D

-Galactopyranosyloxy)ethyl]-N-

1

(II-

N-Boc-

cm⁻¹, KBr): 3450 (NH), 1653 (CꢁO). H

NMR (CDCl₃): l 7.26 (m, 5 H, Ph), 4.42 (s, 2

ethanolamine (15.1 g, 94 mmol) and benzyl

chloride (17.8 g, 1.5 eq, 141 mmol) in CH₂Cl₂

(40 mL) were added to an aq NaOH (10 eq,

37.8 g) solution (60 mL) and Bu₄NHSO₄(0.07

eq, 2.26 g). The mixture was reﬂuxed under

vigorous stirring for 24 h. The organic phase

was then washed until neutrality and dried

over Na₂SO₄. After ﬁltration, evaporation and

chromatography (hexane), 2-benzyloxy-N-

Boc-ethylamine (17, 23 g, 98%) as a white

solid was obtained: R_f0.32 (7:3 hexane–

H, OCH₂Ph), 3.50–3.30 (m,

4

H,

NCH₂CH₂O), 2.06 [t, J 7.2 Hz, 2 H,

CH₂C(O)], 1.70–1.45 [m, 2 H, CH₂CH₂C(O)],

1.32–1.12 [m, 24 H, (CH₂)₁₂], 0.79 (t, J 6.1 Hz,

13

3 H, CH₃). C NMR (CDCl₃): l 173.2 (CO),

137.9 (C, Ph), 128.5 and 127.8 (CH, ortho and

meta Ph), 127.8 (CH, para Ph), 73.1

(OCH₂Ph), 69.1 (CH₂OBn), 39.2 [CH₂C(O)],

36.8 (NCH₂), 31.9 (CH₂CH₂CH₃), 29.7, 29.6,

29.5, 29.4, and 29.3 [(CH₂)₁₀], 25.7

[CH₂CH₂C(O)], 22.7 (CH₂CH₃), 14.1 (CH₃).

Step 4: compound 19 (5.2 g, 13 mmol) in

THF (25 mL) was added dropwise to LiAlH₄

(1.6 g, 43 mmol) suspended in 75 mL of Et₂O

at 0 °C. After stirring at rt for 48 h and

hydrolysis with a 1:1 THF–water solution, the

organic phase was washed with water until

1

Et₂O). H NMR (CDCl₃): l 7.24, (m, 5 H,

Ph), 4.98 (bs, 1 H, NH), 4.40 (s, 2 H,

OCH₂Ph), 3.42 (t, J 5.1 Hz, 2 H, CH₂CH₂O),

3.21 (m, 2 H, CH₂N), 1.35 [s, 9 H, (CH₃)₃].

¹³C NMR (CDCl₃): l 156.0 (CO), 138.2 (C,

Ph), 128.6 and 127.9 (CH, ortho and meta

Ph), 127.8 (CH, para Ph), 80.0 [C(CH₃)₃], 73.2

(OCH₂Ph), 69.4 (CH₂CH₂O), 41.0 (CH₂N),

28.6 (CH₃).

neutrality,

giving

N-[2-(benzyloxy)ethyl]-

hexadecylamine (20, 4.9 g, 100%) as a white

1

solid: R_f0.1 (49:1 CHCl₃–MeOH). H NMR

Step 2: compound 17 (23 g, 91.6 mmol) and

TFA (20 mL) were stirred overnight at rt.

After evaporation, the residue was dissolved

in CH₂Cl₂, washed with 10% Na₂CO₃. The aq

phase was extracted several times with

CH₂Cl₂. The combined organic phases were

dried over Na₂SO₄, ﬁltered and evaporated to

dryness under diminished pressure. The crude

2-(benzyloxy)ethylamine dissolved in Et₂O

(200 mL) was precipitated as its HCl salt 18

(10.4 g 61%) with 2 mL of concentrated HCl

(CD₃OD): l 7.36 (m, 5 H, Ph), 4.56 (s, 2 H,

OCH₂Ph), 3.66 (t, J 5.1 Hz, 2 H, CH₂OBn),

2.90 (t, J 5.1 Hz, 2 H, OCH₂CH₂N), 2.68 [t, J

7.2 Hz, 2 H, NCH₂(CH₂)₁₄], 1.32 [m, 28 H

(CH₂)₁₄], 0.93 (t, J 6.1 Hz, 3 H, CH₃). ¹³C

NMR (CD₃OD): l 139.4 (C, Ph), 129.9 and

128.6 (CH, ortho and meta Ph), 128.8 (CH,

para Ph), 74.0 (OCH₂Ph), 68.9 (CH₂OBn),

47.6 and 50.1 (NCH₂), 33.0 (CH₂CH₂CH₃),

30.7, 30.6, 30.5, 30.4, and 29.5 [CH₂)₁₁], 28.1

(CH₂CH₂CH₂N), 23.6 (CH₂CH₃), 14.4 (CH₃).

Step 5: 7-(F-octyl)heptanoyl chloride (9.6 g,

17 mmol) in CHCl₃(10 mL) was added drop-

wise at rt to a mixture of 20 (4.9 g, 13 mmol)

and NEt₃(5.5 mL, 39 mmol) in CHCl₃(50

1

(37% w/v): H NMR (CD₃OD): l 7.34 (m, 5

H, Ph), 4.59 (s, 2 H, OCH₂Ph), 3.68 (t, J 5.1

Hz, 2 H, CH₂CH₂O), 3.14 (t, J 5.1 Hz, 2 H

13

CH₂N). C NMR (CD₃OD): l 138.8 (C, Ph),

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

247

21. Anal. Calcd for C₃₃H₅₀F₁₇NO₂(815.744): C,

48.58; H, 6.17; N, 1.71. Found: C, 48.56; H,

6.02; N, 1.97.

mL). The resulting mixture was stirred for 48

h, then evaporated to dryness. The crude

residue was chromatographed on silica gel

(CHCl₃) giving N-hexadecyl-N-[2-(benzyl-

oxy)ethyl]-7-(F-octyl)heptylamide (21, 9.9 g,

83%) as a white solid: R_f0.7 (49:1 CHCl₃–

Step 7: the galactosylation procedure de-

scribed for the preparation of 2a( ), when

L

applied to 22 (1.0 g, 1 eq, 1.23 mmol) of

GalOC(ꢁNH)CCl₃(0.76 g, 1.25 eq, 1.54

mmol), and TMSOTf (0.06 mL, 0.33 mmol),

afforded after workup and chromatography,

1

MeOH). IR (w cm⁻¹, KBr): 1649 (CꢁO). H

NMR (CDCl₃): l 7.23 (m, 5 H, Ph), 4.44 and

4.43 (s, s, 2 H, OCH₂Ph), 3.49 and 3.47 (2 t, J

4.4 Hz, 4 H, CH₂NCH₂), 3.25 (bt, J 5.1 Hz, 2

H, CH₂OBn), 2.24 and 2.25 [2 t, J 6.0 Hz, 2

H, CH₂C(O)], 2.16–1.76 (m, 2 H, CH₂CF₂),

1.58–1.16 [m, 36 H, (CH₂)₄and (CH₂)₁₄], 0.80

N-[2-(2,3,4,6-tetra-O-acetyl-b- -galactopyran-

D

osyloxy)ethyl] - N - (hexadecyl) - 7 - (F - octyl)-

heptylamide (23, 1.3 g). Deacetylation of 23

(1.3 g, 1.13 mmol) in 2:1:1 MeOH–Et₃N–wa-

ter and workup as described for the prepara-

13

(t, J 6.1 Hz, 3 H, CH₃). C NMR (CDCl₃): l

tion of I-GalSer[C14](

L

)

gave II-Gal-

172.9 and 172.7 (CO), 138.3 (C, Ph), 128.4

and 127.6 (CH, ortho and meta Ph), 127.8

(CH, para Ph), 73.4 and 73.1 (OCH₂Ph), 68.8

and 68.3 (CH₂OBn), 49.4, 47.6, 46.3, and 46.1

(CH₂NCH₂), 32.9 and 32.8 [CH₂C(O)], 30.9

(CH₂CH₂CH₃), 29.7, 29.6, 29.4, 29.3, 29.1,

29.0, 27.7, 27.0, and 26.9 [(CH₂)₄and (CH₂)₁₃],

25.1 [CH₂CH₂C(O)], 22.7 (CH₂CH₃), 20.1 (t,

J_CF5 Hz, CH₂CH₂CF₂), 14.1 (CH₃). ¹⁹F NMR

(CDCl₃): l −81.3 (3 F, CF₃), −114.9 (2 F,

CF₂CH₂), −122.4 (2 F, CF₂CF₂CH₂), −

123.3 (2 F, CF₂g CH₂), −124.1 (2 F, CF₂d

CH₂), −126.7 (2 F, CF₂CF₃).

AE[C16][F8C7] (0.38 g, 34%): ¹H NMR

(CDCl₃): l 4.80–2.95 (m, 13 H, OCH₂CH₂N,

CH₂N, H-1–6 Gal), 2.23 [t, J 7.2 Hz, 2 H,

CH₂C(O)], 1.98 (m, 2H, CH₂CF₂), 1.51 [m, 4

H, CH₂CH₂C(O), CH₂CH₂CH₂N], 1.30–1.17

[m, 32 H, CH₃(CH₂)₁₃, (CH₂)₃CH₂CH₂C(O)],

0.80 (t, J 6.4 Hz, 3 H, CH₃). ¹³C NMR

(CDCl₃): l 174.0 (CO), 103.8 (C-1 Gal), 74.8

(C-5 Gal), 73.9 (C-3 Gal), 71.3 (C-2 Gal), 68.9

(C-4 Gal), 66.8 (OCH₂CH₂N), 61.5 (C-6 Gal),

48.8 (OCH₂CH₂N), 45.7 (CH₂CH₂CH₂N),

33.1 [CH₂C(O)], 32.0 (CH₂CH₂CH₃), 30.9 (t,

J_CF

22.9

Hz,

CH₂CF₂),

29.8–29.1

Step 6: hydrogen was bubbled through a

suspension of Pd/C (0.9 g. 10% w/v) and 21

(9.9 g) in MeOH (30 mL) and AcOH (10 mL)

for 4 h. After ﬁltration, evaporation and chro-

matography (99:1 CHCl₃–MeOH), N-hexade-

cyl-N-[2-hydroxyethyl]-7-(F-octyl)heptylamide

(22, 7.9 g, 88%), as a white solid was obtained:

[(CH₂)₁₁CH₂CH₂N and (CH₂)₂CH₂CH₂CF₂],

27.0 (CH₂CH₂CH₂N), 25.2 [CH₂CH₂C(O)],

22.8 (CH₃CH₂), 20.2 (bs, CH₂CH₂CF₂), 14.1

19

(CH₃). F NMR (CDCl₃): identical to that of

21. Anal. Calcd for C₃₉H₆₀F₁₇NO₇(977.888): C,

47.90; H, 6.18; N, 1.43. Found: C, 47.45; H,

6.14; N, 1.58.

R_f0.2 (49:1 CHCl₃–MeOH). IR (w cm⁻¹

,

1

KBr): 1647 (CꢁO). H NMR (CDCl₃): l 3.71

(m, 2 H, CH₂OH), 3.48 (t, J 5.2 Hz, 2 H,

NCH₂CH₂O), 3.22 (t, J 6.9 Hz, 2 H,

NCH₂CH₂CH₂), 2.31 [t, J 7.2 Hz, 2 H,

CH₂C(O)], 2.10 (2 t, J 8.2, J_HF16.5 Hz, 2 H,

CH₂CF₂), 1.65–1.48 (m, 4 H, CH₂CH₂CO and

CH₂CH₂CF₂), 1.48–1.15 [m, 32 H, (CH₂)₂and

(CH₂)₁₄], 0.80 (t, J 6.1 Hz, 3 H, CH₃). ¹³C

NMR (CDCl₃): l 62.9 (CH₂OH), 49.7 and

50.1 (CH₂NCH₂), 32.9 [CH₂C(O)], 31.9

(CH₂CH₂CH₃), 30.8 (t, J_CF24 Hz, CH₂CF₂),

29.7, 29.6, 29 5, 29.3, 29.0, 28.9, 27.0, 26.8,

and 25.0 [(CH₂)₂and (CH₂)₁₃], 22.6

(CH₂CH₃), 20.0 (t, J_CF5 Hz, CH₂CH₂CF₂),

14.0 (CH₃). ¹⁹F (CDCl₃): identical to that of

N¹-[2-(i- -Galactopyranosyloxy)ethyl]-N²⁴-

D

(2 - hydroxyethyl) - N¹,N²⁴- bis[5 - (F - hexyl)-

pentanoyl]-1,24-tetracosanediamide (II-GalBAE-

[C24][F6C5](OH)) and N¹,N²⁴-bis[2-(i-

-g-

D

alactopyranosyloxy)ethyl] - N¹,N²⁴- bis[5 - (F-

hexyl)pentanoyl]-1,24-tetracosanediamide (II-

GalBAE[C24][F6C5](Gal)). Step 1: tetra-

cosanedioyl dichloride (1.82 g, 4.2 mmol),

2-(benzyloxy)ethylammonium chloride (18, 1.9

g, 10.4 mmol) and Et₃N (5.2 mL, 37.6 mmol)

in anhyd CH₂Cl₂(100 mL) were stirred at

50 °C for 48 h. The organic phase was then

washed with water until neutrality, dried over

Na₂SO₄, ﬁltered and evaporated to dryness

under diminished pressure. After chromatog-

248

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

(26b, 1.24 g, 90%) as a white powder: R_f0.26

raphy (49:1 CHCl₃–MeOH) of the residue,

N¹,N²⁴- bis[2 - (benzyloxy)ethyl] - 1,24 - tetra-

cosanediamide (24, 1.96 g, 71%) was obtained

as a white solid: R_f0.56 (49:1 CHCl₃–MeOH).

IR (w cm⁻¹, KBr): 3320 (NH), 1640 (CꢁO). ¹H

NMR (CDCl₃): l 7.26 (m, 10 H, Ph), 5.70 (m,

2 H, NH), 4.44 (s, 4 H, OCH₂Ph), 3.41 and

3.49 (2m, 2×4 H, NCH₂CH₂), 2.08 [t, J 8.0

1

(49:1 CHCl₃–MeOH). H NMR (CDCl₃): l

7.23 (m, 10 H, Ph), 4.42 and 4.43 (2s, 2 H, 2

H, OCH₂Ph), 3.49 (m, 8 H, OCH₂CH₂N), 3.24

(t, J 7.3 Hz, 4 H, CH₂CH₂CH₂N), 2.29 (m, 4

H, CH₂CO), 2.00 (m, 4 H, CH₂CF₂), 1.59 (m,

12 H, CH₂CH₂CO, CH₂CH₂CF₂, CH₂CH₂-

CH₂N), 1.18 [bs, 40 H, (CH₂)₂₀]. ¹³C NMR

(CDCl₃): l 171.9 and 172.3 (CO), 137.8 and

138.3 (C, Ph), 128.3 and 128.5 (CH, meta Ph),

127.9 (CH, ortho Ph), 127.6 (CH, para Ph),

73.1 and 73.5 (PhCH₂O), 68.1 and 68.8

(CH₂O), 47.6 and 49.3 (OCH₂CH₂N), 46.1

and 46.3 (CH₂N), 32.5 and 32.6 (CH₂CO),

30.9 (t, J_CF22 Hz, CH₂CF₂), 27.7 and 29.1–

29.7 [(CH₂)₂₀], 26.9 and 27.0 (CH₂CH₂N), 24.7

Hz,

4

H, CH₂C(O)], 1.52 [m,

4

H,

CH₂CH₂C(O)], 1.18 [s, 36 H, (CH₂)18]. ¹³C

NMR (CDCl₃): l 173.2 (CO), 138.0 (C, Ph),

128.5 and 127.9 (CH, ortho and meta Ph),

127.8 (CH, para Ph), 73.2 (OCH₂Ph), 69.1

(CH₂O), 39.3 (CH₂NH), 36.9 (CH₂CO), 29.73,

29.67, 29.5, 29.4, and 29.3 [(CH₂)18], 25.8

(CH₂CH₂CO).

(CH₂CH₂CO),

20.1

[t,

J

3.7

Hz,

Step 2: a mixture of 24 (3.24 g, 4.87 mmol)

and LiAlH₄(1.9 g) in anhyd THF (75 mL)

was reﬂuxed for 7 days. After hydrolysis with

a 1:1 THF–water solution, ﬁltration and ex-

traction with CHCl₃, the solvents were evapo-

rated. The crude product N¹,N²⁴-bis-

[2-(benzyloxy)ethyl]-1,24-tetracosanediamine

(25) was then precipitated from CHCl₃as its

bis-(HCl) salt (2.35 g, 68%). Characterization

was performed on a aliquot of 25 puriﬁed by

chromatography (1:1 CHCl₃–CH₃OH): R_f

CH₂CH₂CF₂). ¹⁹F NMR (CDCl₃): l −81.3 (6

F, CF₃), −114.8 (4 F, CF₂CH₂), −122.4,

−123.4 and −124.0 [3×4 F, (CF₂)₃], −

126.6 (4 F, CF₂CF₃).

Step 4: compound 26b (6 g, 4.16 mmol) and

Pd/C (0.6 g, 10% w/w) in EtOH (50 mL) were

stirred under 40 atm H₂for 21 days. After

usual workup, N¹,N²⁴-bis[2-hydroxyethyl]-N¹,

N²⁴- bis[5 - (F - hexyl)pentanoyl] - 1,24 - tetra-

cosanediamine (27b, 4.19 g, 80%) was ob-

tained: IR (w cm⁻¹, KBr): 3400 (OH), 1610

1

(CꢁO). H NMR (CDCl₃): l 3.68 (t, 4 H,

0.28 (1:1 CHCl₃–MeOH). H (CDCl₃): 7.27

CH₂OH), 3.45 (t, J 4.9 Hz, 4 H, OCH₂CH₂N),

3.20 (t, J 7.7 Hz, 4 H, CH₂N), 2.32 (m, 4 H,

CH₂CO), 2.03 (m, 4 H, CH₂CF₂), 1.57 (m, 12

H, CH₂CH₂CF₂, CH₂CH₂CH₂N, CH₂CH₂-

[m, 10 H, 2Ph], 4.46 [s, 4 H, 2(OCH₂Ph)], 3.53

[t, 4 H, 2(OCH₂CH₂NH)], 2.74 [t, J 5.2 Hz, 4

H, 2(OCH₂CH₂NH)], 2.52 [t, J 7.2 Hz, 4 H,

2(CH₂NH)], 1.64 [m, 2 H, 2(NH)], 1.41 [m, 4

H, 2(CH₂CH₂CH₂N)], 1.18 [s, 40 H, (CH₂)₂₀].

¹³C (CDCl₃): 137.9 [2(C, Ph)], 128.4 and 127.8

[2(CH, ortho and meta Ph)], 127.7 [2(CH,

13

CO), 1.18 [m, (CH₂)₂₀, 40 H]. C NMR (CD-

Cl₃): l 174.2 (CO), 62.5 (CH₂OH), 50.0 (O-

CH₂CH₂N), 49.7 (CH₂N), 32.6 (CH₂CO),

30.9 (t, J_CF22 Hz, CH₂CF₂), 29.1 to 29.7

[(CH₂)₂₀], 26.9 (CH₂CH₂N), 24.7 (CH₂CH₂-

CO), 20.1 (t, J 3.5 Hz, CH₂CH₂CF₂). ¹⁹F

NMR (CDCl₃): identical to that of 26b. Anal.

Calcd for C₅₀H₇₄F₂₆N₂O₄(1261.114): C, 47.62;

H, 5.91; N, 2.22. Found: C, 47.34; H, 5.77; N,

2.08.

ortho

Ph)],

73.2

[2(OCH₂Ph)],

69.7

[2(OCH₂CH₂NH)], 50.0 [2(OCH₂CH₂NH)],

49.5 [2(CH₂N)], 30.2, 29.7, and 29.6 [(CH₂)₂₀],

27.4 [2(CH₂CH₂NH)].

Step 3: 5-(F-hexyl)pentanoyl chloride (1.3 g,

2.9 mmol) were added to a mixture of 25 (0.68

g, 0.96 mmol) as its HCl salt, and Et₃N (3 mL,

21.8 mmol) in anhyd CH₂Cl₂(50 mL). The

resulting solution was stirred at 60 °C for 3

days. The organic phase was then washed with

water until neutrality, dried over Na₂SO₄,

ﬁltered and evaporated to dryness. Puriﬁca-

tion by chromatography (CHCl₃to 1:1

CHCl₃–MeOH) of the residue afforded

N¹,N²⁴-bis[2-(benzyloxy)ethyl]-N¹,N²⁴-bis[5-

(F - hexyl)pentanoyl] - 1,24 - tetracosanediamine

Step 5: the procedure described for the syn-

thesis of 2a( ) when applied to 27b, afforded,

D

after workup and chromatography (CHCl₃to

49:1 CHCl₃–MeOH), N¹-[2-(2,3,4,6-tetra-O-

acetyl-b-

-galactopyranosyloxy)ethyl]-N¹,N²⁴-

D

bis[5-(F-hexyl)pentanoyl]-N²⁴-(2-hydroxyethyl)-

1,24-tetracosanediamide (28b, 34%), and a

mixture

of

N¹,N²⁴-bis[2-(2,3,4,6-tetra-O-

acetyl-b-

D

-galactopyranosyoxyl)ethyl]-N¹,N²⁴-

bis[5 - (F - hexyl)pentanoyl] - 1,24 - tetracosane-

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

249

F, 34.71; N, 1.97. Found: C, 47.59; H, 6.13; F,

32.58; N, 1.74.

diamide (29b) and 2,3,4,6-tetra-O-acetyl- -

D

galactopyranose, as white solids.

II-GalBAE[C24][F6C5](Gal): ¹H NMR

(CDCl₃–CD₃OD): l 4.10 (d, J_1,24 Hz, 1 H,

H-1 Gal), 4.00–3.00 [m, 24 H, 2(OCH₂CH₂N,

CH₂CH₂CH₂N, H-2–6 Gal)], 2.24 [m, 4 H,

2(CH₂CO)], 1.97 [m, 4 H, 2(CH₂CF₂)], 1.51

[m, 12 H, 2(CH₂CH₂N, CH₂CH₂CF₂,

CH₂CH₂CO)], 1.11 [m, 40 H, (CH₂)₂₀]. ¹³C

(CDCl₃–CD₃OD): 173.3 (CO), 103.2 (C-1

Gal), 74.3 (C-5 Gal), 73.3 (C-3 Gal), 70.9 (C-2

Gal), 68.9 (C-4 Gal), 66.6 (OCH₂CH₂N), 61.2

(C-6 Gal), 48.5 (OCH₂CH₂N) partially hin-

dered by CD₃OD], 45.5 (CH₂N), 32.4

(CH₂CO), 30.5 (t, J_CF21 Hz, CH₂CF₂), 29.5–

29.2 [(CH₂)₂₀], 26.6 (CH₂CH₂N), 24.5

(CH₂CH₂CO), 19.9 (t, J_CF3.5 Hz, CH₂CH₂-

CF₂). ¹⁹F NMR (CDCl₃–CD₃OD): identical

to that of 26b.

1

Compound 28b: H NMR (CDCl₃): l 5.33

(d, J 3.2 Hz, 1 H, H-4 Gal), 5.17–4.90 (m, 2

H, H-2-3 Gal), 4.39 (d, J 7.8 Hz, 1 H, H-1

Gal), 4.20–3.85 (m, 5 H, GalOCH₂CH₂and

H-5–6 Gal), 3.68 (t, J 5 Hz, 2 H, CH₂OH),

3.42 (m, 4 H, OCH₂CH₂N), 3.20 (t, J 7.4 Hz,

4 H, CH₂N), 2.28 [m, 4 H, CH₂C(O)], 2.11–

1.91 (m, 16 H, CH₃, CH₂CF₂), 1.63 [m, 12 H,

CH₂CH₂CH₂N, CH₂CH₂CF₂, CH₂CH₂C(O)],

13

1.18 [bs, 40 H, (CH₂)₂₀]. C NMR (CDCl₃): l

174.3 and 172.1 [C(O)N], 170.4, 170.2, and

170.0 [C(O)CH₃], 101.5 (C-1 Gal), 68.5 (C-3

Gal), 68.2 (C-5 Gal), 67.3 (C-2 Gal), 66.1 (C-4

Gal), 62.6 (CH₂CH₂OH), 61.8 (C-6 Gal), 61.3

(GalOCH₂CH₂), 50.0 (NCH₂CH₂OH), 49.6

(CH₂NCH₂CH₂OH), 48.0 (GalOCH₂CH₂),

46.2 (CH₂NCH₂CH₂OGal), 32.6 (CH₂CO),

30.8 (t, J_CF22 Hz, CH₂CF₂), 29.7 to 29.0

N¹-[2-(i-

(2-hydroxyethyl)-N¹,N²⁴-bis(dodecanoyl)-1,24-

tetracosanediamide (II-GalBAE[C24][C12]-

D

-Galactopyranosyloxy)ethyl]-N²⁴-

[(CH₂)₂₀],

26.8

(CH₂CH₂N),

24.7

(CH₂CH₂CO), 20.8, 20.6, and 20.5 (CH₃), 20.1

(t, J_CF3.5 Hz, CH₂CH₂CF₂). ¹⁹F NMR

(CDCl₃): identical to that of 26b.

(OH)). Step 1: the procedure described for the

synthesis of 26b when applied to 25 (3.5 g, 1

eq, 4.93 mmol) as its HCl salt, dodecanoyl

chloride (3.8 g, 3.4 eq, 17.4 mmol) and Et₃N

(10 mL, 72 mmol) in anhyd CHCl₃(150 mL)

gave N¹,N²⁴-bis[2-(benzyloxy)ethyl]-N¹,N²⁴-

bis(dodecanoyl)-1,24-tetracosanediamine (26a,

Step 6: deacetylation of 28b and of the

mixture containing 29b was performed with 1

M MeONa–MeOH at rt overnight. After

evaporation, the crude residues were chro-

matographed twice on silica gel (CHCl₃to 1:1

CHCl₃–MeOH) affording II-GalBAE[C24]-

[F6C5](OH) (60%) and II-GalBAE[C24]-

[F6C5](Gal) as white powders, respectively.

II-GalBAE[C24][F6C5](OH): ¹H NMR

(CDCl₃): l 4.42–3.16 (m, 24 H, CH₂N,

GalOCH₂CH₂N, HOCH₂CH₂N, H-1–6 Gal

and OH), 2.31 [m, 4 H, CH₂C(O)], 2.02 (m, 4

H, CH₂CF₂), 1.57 (m, 12 H, CH₂CH₂N,

CH₂CH₂CF₂, CH₂CH₂CO), 1.18 [m, 40 H,

(CH₂)₂₀]. ¹³C NMR (CDCl₃): l 174.2 and

174.3 (CO), 103.8 (C-1 Gal), 74.6 (C-5 Gal),

73.8 (C-3 Gal), 71.3 (C-2 Gal), 69.1

(GalOCH₂CH₂N), 66.8 (C-4 Gal), 62.2

(NCH₂CH₂OH), 61.8 (C-6 Gal), 49.9

(NCH₂CH₂OH), 49.6 (CH₂NCH₂CH₂OH),

48.6 (GalOCH₂CH₂N), 45.7 (CH₂NCH₂-

CH₂OGal), 32.6 (CH₂CO), 30.9 (t, J CF 22

Hz, CH₂CF₂), 29.7–29.1 [(CH₂)₂₀], 26.8

(CH₂CH₂N), 24.7 (CH₂CH₂CO), 20.1 [t, J_CF

3.7 Hz, CH₂CH₂CF₂). ¹⁹F NMR (CDCl₃):

identical to that of 26b. Anal. Calcd for

C₅₆H₈₄F₂₆N₂O₉(1423.258): C, 47.26; H, 5.95;

1

4.8 g, 98%) as a white powder: H NMR

(CDCl₃): l 7.26 (m, 10 H, Ph), 4.43 and 4.44

(2s, 2 H, 2 H, OCH₂Ph), 3.49 (m, 4 H,

OCH₂CH₂N), 3.47 (t, J 13.5 Hz, 4 H,

OCH₂CH₂N), 3.24 (t, J 7.6 Hz, 4 H, CH₂N),

2.25 and 2.22 [2t, J ꢀ8.0 Hz, 2×2 H,

CH₂CO), 1.51 (m,

8

H, CH₂CH₂CO,

CH₂CH₂CH₂N), 1.18 (bs, 72 H, (CH₂)₂₀and

13

(CH₂)₈CH₃), 0.81 (t, J 6.4 Hz, 6 H, CH₃). C

NMR (CDCl₃): l 173.3 and 173.4 (CO), 137.9

and 138.5 (C, Ph), 128.5, 128.6 and 127.9

(CH, ortho and meta Ph), 127.7 (CH, para

Ph), 73.3 and 73.6 (OCH₂Ph), 68.5 and 69.0

(OCH₂CH₂N), 47.7 and 49.6 (OCH₂CH₂N),

46.2 and 46.5 (CH₂N), 33.2 and 33.4

(CH₂CO), 32.1 (CH₂CH₂CH₃), 27.9 and

29.3–29.9 [(CH₂)₂₀and CH₃CH₂CH₂(CH₂)₆],

27.0 and 27.2 (CH₂CH₂N), 25.6 (CH₂CH₂-

CO), 22.8 (CH₃CH₂)], 14.3 (CH₃).

Step 2: the procedure described for the syn-

thesis of 27b, when applied to 26a (4.83 g, 4.82

mmol), gave after 2 days of reaction and usual

250

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

1

workup, N¹,N²⁴-bis(2-hydroxyethyl)-N¹,N²⁴-

bis(dodecanoyl)-1,24-tetracosanediamine (27a,

3.96 g, 100%): IR (w cm⁻¹, KBr): 3390 (OH),

1610 (CꢁO). ¹H NMR (CDCl₃): l 3.69 (t, 4 H,

a white powder: H NMR (CDCl₃): l 4.27–

3.23 (m, 19 H, CH₂N, NCH₂CH₂OH,

GalOCH₂CH₂N, HOCH₂CH₂N, H-1–6 Gal),

2.29 (m, 4 H, CH₂CO), 1.60 (m, 8 H,

CH₂CH₂CO, CH₂CH₂CH₂N), 1.26 (m, 72 H

(CH₂)₂₀and (CH₂)₈CH₃), 0.88 (t, J 6.4 Hz, 6

H, CH₃). ¹³C NMR (CDCl₃): l 174.5 and

175.7 (CO), 103.6 (C-1 Gal), 74.8 (C-5 Gal),

73.9 (C-3 Gal), 71.6 (C-2 Gal), 69.4 (C-4 Gal),

67.2 (GalOCH₂CH₂N), 62.8 (NCH₂CH₂OH),

62.5 (C-6 Gal), 50.2 (HOCH₂CH₂N), 49.9

(CH₂NCH₂CH₂OH), 48.6 (GalOCH₂CH₂N),

45.7 (CH₂NCH₂CH₂OGal), 33.3 [2(CH₂CO)],

32.0 (CH₂CH₂CH₃), 29.8–29.2 [(CH₂)₂₀and

CH₃CH₂CH₂(CH₂)₆], 26.9 (CH₂CH₂CH₂N),

25.5 (CH₂CH₂CO), 22.8 (CH₃CH₂), 14.2

(CH₃). Anal. Calcd for C₅₈H₁₁₄N₂O₉

(983.563): C, 70.83; H, 11.68; N, 2.85. Found:

C, 70.60; H, 11.64; N, 2.65.

CH₂OH), 3.45 (t,

J

4.0 Hz,

4

H,

NCH₂CH₂OH), 3.20 (t, J 7.6 Hz, 4 H, CH₂N),

2.26 (t, J 7.5 Hz, 4 H, CH₂CO), 1.53 (m, 8 H,

CH₂CH₂CO, CH₂CH₂CH₂N), 1.19 [m, 72 H,

(CH₂)₂₀, (CH₂)₈CH₃], 0.81 (t, J 6.4 Hz, 6 H,

CH₃). ¹³C NMR (CDCl₃): l 173.0 (CO), 63.1

(CH₂OH), 50.3 (HOCH₂CH₂N), 49.9 (CH₂N),

33.3 (CH₂CO), 32.0 (CH₂CH₂CH₃), 29.8–29.2

[(CH₂)₂₀and (CH₃CH₂CH₂(CH₂)₆)], 26.9

(CH₂CH₂N),

25.5

(CH₂CH₂CO),

22.8

(CH₃CH₂)], 14.2 (CH₃). Anal. Calcd for

C₅₂H₁₀₄N₂O₄(821.42): C, 76.04; H, 12.76; N,

3.41. Found: C, 75.55; H, 12.85; N, 3.74.

Step 3: the galactosylation procedure de-

scribed for the synthesis of 2a(

D

) when applied

to 27a, afforded N¹-[2-(2,3,4,6-tetra-O-acetyl-

Synthesis of the Gal(NHAc) single-chain

deri6ati6es

3-O-(2-Acetamido-2-deoxy-i-

anosyl)- -serine tetradecylamide (I-Gal(N-

HAc)Ser[C14]( )). Step 1: p-anisaldehyde (5.6

mL, 46 mmol) was added to a solution of

-galactosamine hydrochloride 30 (1.0 g, 4.6

b-

-galactopyranosyloxy)ethyl]-N²⁴-(2-hydro-

D

xyethyl)-N¹,N²⁴-bis(dodecanoyl)-1,24-tetra-

cosanediamide (28a) contaminated by 2,3,4,6-

D

-galactopyr-

L

tetra-O-acetyl-

D

-galactopyranose. An aliquot

L

was puriﬁed by chromatography for analysis.

¹H NMR (CDCl₃): l 5.28 (m, 1 H, H-4 Gal),

5.10–4.80 (m, 2 H, H-2-3 Gal), 4.35 (d, J 7.9

Hz, ¹H, H-1 Gal), 4.10–3.75 (m, 5 H,

GalOCH₂CH₂and H-5–6 Gal), 3.64 (t, J 5.1

D

mmol) in 1 N NaOH (4.7 mL). After stirring

vigorously for 2 h at rt, the crystalline mate-

rial was ﬁltered off, washed with ice–water,

EtOH and Et₂O, and dried, giving N-

Hz,

2

H, CH₂OH), 3.38 (m,

4

H,

NCH₂CH₂O), 3.16 (m, 4 H, CH₂N), 2.25 (t, J

(p-methoxybenzylidene)-2-amino-2-deoxy- -

D

7.7 Hz, 4 H, CH₂CO), 2.05–1.87 (m, 12 H,

CH₃CO), 1.49 (m,

galactopyranose (1.0 g, 73%). This compound

was then allowed to react with Ac₂O (2.0 mL,

21 mmol) in pyridine at 0 °C for 1 h, then at

rt overnight. The reaction was poured in ice

water and the precipitate was ﬁltered and

dried, affording N-(p-methoxybenzylidene)-

8

H, CH₂CH₂CO,

CH₂CH₂CH₂N), 1.14 [m, 72 H, (CH₂)₂₀,

(CH₂)₈CH₃)], 0.77 (t, J 6.7 Hz, 6 H, CH₃CH₂).

¹³C NMR (CDCl₃): l 175.6 (CO), 170.3

(COCH₃), 101.6 (C-1 Gal), 70.8 (C-3 Gal),

69.5 (C-5 Gal), 67.5 (C-2 Gal), 66.1 (C-4 Gal),

62.9 (CH₂OH), 61.8 (GalOCH₂CH₂), 61.3 (C-

1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-b- -

D

galactopyranose (31, 1.2 g, 75%) R_f0.65 (24:1

1

6

Gal),

50.2

(HOCH₂CH₂N),

49.9

CHCl₃–MeOH): H NMR (CDCl₃): l 8.14 (s,

(CH₂NCH₂CH₂OH), 49.6 (GalOCH₂CH₂N),

46.1 (CH₂NCH₂CH₂OGal), 33.2 (CH₂CO),

32.0 (CH₂CH₂CH₃), 29.2–29.8 and 27.1

[(CH₂)₂₀and 2CH₃CH₂CH₂(CH₂)₆], 26.9

(CH₂CH₂N), 25.5 (CH₂CH₂CO), 22.8 (CH₃-

CH₂), 20.9–20.6 (CH₃CO), 14.2 (CH₃CH₂).

Step 4: the deacetylation procedure used for

1 H, NꢁCH), 7.59 (m, 2 H, Ph), 6.84 (m, 2 H,

Ph), 5.85 (d, J 8.2 Hz, 1 H, H-1b Gal), 5.38

(d, J 3.2 Hz, 1 H, H-4 Gal), 5.18 (dd, J_2,310.4,

J_3,43.2 Hz, 1 H, H-3 Gal), 4.11 (m, 3 H,

H-5–6 Gal), 3.77 (s, 3 H, OCH₃), 3.53

(dd, J_2,310.4, J_1,28.2 Hz, 1 H, H-2 Gal), 2.10,

1.99, 1.96 and 1.82 (all s, 12 H, CH₃). ¹³C

NMR (CDCl₃): l 170.5, 170.2, 169.8 and

168.9 [CH₃C(O)], 164.5 [C(O)Me], 162.4

(CCHꢁN), 130.3 (CH, Ph), 128.6 (CHꢁN),

114.2 (CH, Ph), 93.7 (C-1b Gal), 71.9, 71.7,

the

preparation

of

II-GalBAE[C24]-

[F6C5](OH), when applied to 28a, gave, after

chromatography (CHCl₃to 1:1 CHCl₃–

MeOH), II-GalBAE[C24][C12](OH) (22%) as

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

251

(acetone-d₆): l 7.84 (d, J 6.9 Hz, 2 H, Fmoc),

7.75 (d, J 7.2 Hz, 2 H, Fmoc), 7.50–7.25 (m,

4 H, Fmoc), 6.65 (m, 1 H, NH-Fmoc), 5.90

(m, 2 H, OCH₂CHꢁCH₂and NH–Aloc), 5.36

(bs, 1 H, H-4 Gal), 5.30–5.10 (m, 3 H,

OCH₂CHꢁCH₂, H-3 Gal), 4.80 (bs, 1 H, H-1

Gal), 4.70 (bs, 2 H, OCH₂CHꢁCH₂), 4.60–

3.80 [m, 10 H, H-2 Gal, H-5–6 Gal,

OCH₂CHNH and C(O)OCH₂CHFmoc], 2.11,

2.09, and 1.94 [all s, all 3 H, CH₃C(O)]. ¹³C

68.9 and 66.1 (C-2–5 Gal), 61.5 (C-6 Gal),

55.6 (OCH₃), 20.9, 20.8 and 20.6 [CH₃C(O)].

Step 2: the latter derivative 31 (1.1 g, 2.36

mmol) was dissolved in acetone (20 mL) and 5

N HCl (0.43 mL) was added. The reaction

mixture was reﬂuxed for 15 min, the precipi-

tate was ﬁltered and washed with Et₂O, yield-

ing

b-

1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-

D

-galactopyranose hydrochloride (0.80 g,

85%). This compound (0.40 g, 1.0 mmol) was

then reacted with NaHCO₃(0.17 g) in water

(10 mL). After 10 min of stirring, allyl chloro-

formate (0.15 mL, 1.4 mmol) in CHCl₃(5 mL)

was added and the reaction mixture was

stirred for 1 h at rt. The aq layer was ex-

tracted twice with CHCl₃, the combined

CHCl₃solutions were washed with water. Af-

ter drying with Na₂SO₄, the solution was

ﬁltered and evaporated to dryness under di-

minished pressure. The residue was recrystal-

lized from Et₂O giving 1,3,4,6-tetra-

O-acetyl-2-allyloxycarbonylamino-2-deoxy-b-

NMR (acetone-d₆):

l

170.6 and 170.3

[CH₃CO and COOH], 157.3 [NHC(O)O],

145.1, 145.0 and 142.0 (C, Fmoc), 134.0

(OCH₂CHꢁCH₂), 128.4, 128.0, 126.2 and

120.7 (CH, Fmoc), 117.2 (OCH₂CHꢁCH₂),

102.7 (C-1 Gal), 71.3 (C-5 Gal), 70.5 (C-3 Gal

and OCH₂CHNH), 67.6 and 67.5 [C-4 Gal

and C(O)OCH₂Fmoc], 66.1 (OCH₂CHꢁCH₂),

61.8 (-6 Gal), 56.0 [CHC(O)OH], 52.9 (C-2

Gal), 47.8 [C(O)OCH₂CH], 20.5 [CH₃C(O)].

Step 5: a solution containing 33 (90 mg,

0.128 mmol), tetradecylamine (27 mg, 0.126

mmol), EDC (27 mg, 0.14 mmol), and HOBt

(17 mg, 0.126 mmol) in DMF (5 mL) was

stirred at 0 °C for 1 h, then at rt overnight.

After evaporation of DMF under diminished

pressure, the residue was taken up in CHCl₃

and successively washed with 5% KHSO₄, 8%

NaHCO₃, then water until neutrality. After

drying over Na₂SO₄and ﬁltration, the solvent

was evaporated and the residue puriﬁed by

recrystallization in Et₂O giving 3-O-(3,4,6-tri-

O-acetyl-2-allyloxycarbonylamino-2-deoxy-b-

D

-galactopyranose (32, 0.40 g, 85%) as a white

1

solid: R_f0.64 (24:1 CHCl₃–MeOH). H NMR

(CDCl₃): l 5.81 (m, 1 H, CHꢁCH₂), 5.69 (d, J

8.7 Hz, 1 H, H-1b Gal), 5.55 (d, J 9.6 Hz, 1

H, NH), 5.33 (d, J 3.0 Hz, 1 H, H-4 Gal), 5.13

(m, 3 H, CHꢁCH₂and H-3 Gal), 4.50 (bd, 2

H, OCH₂CHꢁ), 4.15-3.95 (m, 4 H, H-2 Gal,

H-5 Gal, and H-6–6% Gal), 2.10, 2.06, 1.97,

and 1.94 (all s, 12 H, CH₃). ¹³C NMR

(CDCl₃): l 170.5, 170.4, and 169.5 [CH₃C(O)],

156.1 [NHC(O)], 132.8 (CHꢁCH₂), 117.5

(CHꢁCH₂), 93.0 (C-1b Gal), 71.7 (C-3 Gal or

C-5 Gal), 70.6 (C-3 Gal or C-5 Gal), 66.7 (C-4

Gal), 65.7 (OCH₂CHꢁ), 61.6 (C-6 Gal), 51.4

(C-2 Gal), 20.9 and 20.7 [CH₃C(O)].

D

-galactopyranosyl)-N-Fmoc- -serine tetra-

L

decylamide (34a, 90 mg, 79%) as a white solid:

R_f0.40 (49:1 CHCl₃–MeOH). ¹H NMR

(CDCl₃): l 7.70 (d, J 7.2 Hz, 2 H, Fmoc), 7.52

(d, J 7.1 Hz, 2 H, Fmoc), 7.35–7.20 (m, 4 H,

Fmoc), 6.40 (bs, 1 H, NHCH₂), 5.85–5.65 (m,

2 H, OCH₂CHꢁCH₂and NH–Fmoc), 5.28

(bd, J 2.9 Hz, 1 H, H-4 Gal), 5.24-5.06 (m, 3

H, NHC(O)OCH₂CHꢁCH₂), 5.01 (dd, J_2,3

11.2, J_3,43.2 Hz, 1 H, H-3 Gal), 4.88 (d, J 8.6

Hz, 1 H, H-1 Gal), 4.60–3.60 [m, 12 H, H-2

Gal, H-5–6 Gal, OCH₂CH, OCH₂CHꢁCH₂,

and C(O)OCH₂CH], 3.15 (m, 2 H, NHCH₂),

2.07, 1.96, and 1.94 [all s, all 3 H, CH₃C(O)],

1.55–1.35 (m, 2 H, NHCH₂CH₂), 1.35–1.10

[m, 22 H, (CH₂)₁₁CH₃], 0.81 (t, J 5.2 Hz, 3 H,

CH₂CH₃]. ¹³C NMR (CDCl₃): l 170.6, 170.2

Step 3: 32 (150 mg, 0.348 mmol), N^a-Fmoc-

-serine (140 mg, 0.427 mmol), and BF₃·Et₂O

L

(0.14 mL, 1.13 mmol) in CH₂Cl₂(6 mL) were

stirred overnight at rt. After adding CH₂Cl₂

(20 mL) and ﬁltration over Celite, the organic

phase was washed with 1 N HCl, then with

water until neutrality. After drying over

Na₂SO₄and ﬁltration, the solvent was evapo-

rated and the residue puriﬁed by chromatog-

raphy (99:1 to 19:1 CHCl₃–MeOH) giving

3-O-(3,4,6-tri-O-acetyl-2-allyloxycarbonyl-

amino - 2 - deoxy - b -

Fmoc- -serine (33, 100 mg, 41%) as a white

solid: R_f0.30 (9:1 CHCl₃–MeOH). H NMR

D

- galactopyranosyl) - N-

L

1

252

B. Faroux-Corlay et al. / Carbohydrate Research 327 (2000) 223–260

of the solvent, the residue was puriﬁed by

chromatography (24:1 to 9:1 CHCl₃–MeOH)

giving 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-

and 169.2 [CH₃C(O) and C(O)NH], 156.1

[NHC(O)O], 143.9 and 141.5 (C, Fmoc), 132.6

(OCH₂CHꢁCH₂), 127.9, 127.2, 125.2 and

120.2 (CH, Fmoc), 117.8 (OCH₂CHꢁCH₂),

102.5 (C-1 Gal), 71.2 (C-5 Gal), 70.2 (C-3

Gal), 69.9 (OCH₂CHNH), 67.2 [C(O)OCH₂-

Fmoc], 66.8 (C-4 Gal), 66.0 (OCH₂CHꢁCH₂),

61.6 (C-6 Gal), 54.2 [CHC(O)NH], 52.5 (C-2

Gal), 47.3 [C(O)OCH₂CH], 39.9 (NHCH₂),

32.0 (CH₂CH₂CH₃), 29.8, 29.6, and 29.4

[(CH₂)₉CH₂CH₂CH₃], 27.0 (NHCH₂CH₂),

22.8 (CH₂CH₃), 20.7 [CH₃C(O)], 14.2 (CH₃).

Step 6: to a solution of 34a (90 mg, 0.10

mmol) in CH₂Cl₂(2 mL), Ac₂O (23 mL, 0.25

mmol), Pd(PPh₃)₄(3 mg, 2.6×10⁻³mmol),

and Bu₃SnH (27 mL, 0.10 mmol) were added

successively. The mixture was stirred for 1 h at

rt. After ﬁltration and evaporation of the sol-

vent, the crude was recrystallized from Et₂O

affording 3-O-(2-acetamido-3,4,6-tri-O-acetyl-

deoxy-b-

D

-galactopyranosyl)- -serine tetrade-

L

cylamide (36a, 35 mg, 90%) as a white solid:

R_f0.16 (24:1 CHCl₃–MeOH). ¹H NMR

(CDCl₃): l 7.38 (t, 1 H, NHCH₂), 6.13 [d, J

8.6 Hz, 1 H, NHC(O)CH₃], 5.28 (bd, J 2.9

Hz, 1 H, H-4 Gal), 5.08 (dd, J_2,311.2, J_3,43.3

Hz, 1 H, H-3 Gal), 4.56 (d, J_1,28.4 Hz, 1 H,

H-1 Gal), 4.15–4.01 (m, 3 H, H-2 Gal, H-6

Gal), 3.90-3.60 (m, 3 H, H-5 Gal and OCH₂),

3.50 (dd, J 7.6, J 4.7 Hz, 1 H, OCH₂CH), 3.16

(dt, J 6.3, J 5.8 Hz, 2 H, NHCH₂), 2.08 [s, 3

H, CH₃C(O)NH], 2.02 (s, 2 H, NH₂), 1.98,

and 1.93 [all s, all 3 H, CH₃C(O)], 1.89 [s, 3 H,

CH₃C(O)NH], 1.50–1.30 (m, 2 H, NHCH₂-

CH₂), 1.30–1.10 [m, 22 H, (CH₂)₁₁CH₃], 0.82

(t, J 6.5 Hz, 3 H, CH₂CH₃]. ¹³C NMR

(CDCl₃): l 172.2 [C(O)NH], 170.7, 170.5, and

170.3 [CH₃C(O)], 101.7 (C-1 Gal), 72.0

(OCH₂), 71.0 (C-5 Gal), 70.4 (C-3 Gal), 66.8

(C-4 Gal), 61.6 (C-6 Gal), 55.3 (OCH₂CH),

50.9 (C-2 Gal), 39.4 (NHCH₂), 32.0

(CH₂CH₂CH₃), 29.7, 29.6, and 29.4 [(CH₂)₉-

CH₂CH₂CH₃], 27.0 (NHCH₂CH₂), 23.7

[CH₃C(O)NH], 22.7 (CH₂CH₃), 20.7 [CH₃-

C(O)], 14.2 (CH₃CH₂).

2-deoxy-b-

D

-galactopyranosyl)-N-Fmoc- -

L

serine tetradecylamide (35a, 60 mg, 70%) as a

1

white solid: R_f0.25 (49:1 CHCl₃–MeOH): H

NMR (CDCl₃): l 7.70 (d, J 7.1 Hz, 2 H,

Fmoc), 7.53 (d, J 7.1 Hz, 2 H, Fmoc), 7.37–

7.19 (m, 4 H, Fmoc), 6.47 (bs, 1 H, NHCH₂),

5.83 (bs, 2H, NHC(O)CH₃and NH–Fmoc),

5.27 (bd, J 2.7 Hz, 1 H, H-4 Gal), 5.06 (dd,

J_2,311.2, J_3,43.2 Hz, 1 H, H-3 Gal), 4.60–3.80

[m, 10 H, H-1-2 Gal, H-5–6 Gal, OCH₂, and

C(O)OCH₂CH], 3.66 (m, 1 H, OCH₂CH),

3.16 (dt, J 6.2, J 5.7 Hz, 2H, NHCH₂), 2.07,

1.95, and 1.93 [all s, all 3 H, CH₃C(O)O], 1.82

[s, 3 H, CH₃C(O)NH], 1.60–1.10 [m, 24 H,

(CH₂)₁₂CH₃], 0.82 (t, J 6.5 Hz, 3 H, CH₂CH₃].

¹³C NMR (CDCl₃): l 170.9 and 170.8

[C(O)NH], 170.5, 170.2, and 169.3 [CH₃C(O)-

O], 156.2 [NHC(O)O], 143.8 and 141.4 (C,

Fmoc), 127.9, 127.2, 125.1 and 120.1 (CH,

Fmoc), 102.2 (C-1 Gal), 71.2 (C-5 Gal), 70.2

(C-3 Gal), 69.3 (OCH₂), 67.1 [C(O)OCH₂],

66.7 (C-4 Gal), 61.6 (C-6 Gal), 54.2

(OCH₂CH), 50.9 (C-2 Gal), 47.3 (C(O)OCH₂-

CH), 39.9 (NHCH₂), 32.0 (CH₂CH₂CH₃),

29.8, 29.7, 29.5, and 29.4 [(CH₂)₉CH₂-

CH₂CH₃], 27.0 (NHCH₂CH₂), 23.4 [CH₃C-

(O)NH], 22.8 (CH₂CH₃), 20.7 [CH₃C(O)],

14.2 (CH₃).

Step 8: the O-deacetylation of 36a (34 mg,

0.054 mmol) was performed in a 2:1:1

MeOH–Et₃N–water mixture (0.72 mL) at rt

for 1 h. After evaporation of the solvents, the

crude residue was puriﬁed by chromatography

(9:1 to 7:3 CHCl₃–MeOH) affording I-Gal(N-

HAc)Ser[C14]( ) (23 mg, 85%) as a white

L

solid: R_f0.40 (7:3 CHCl₃–MeOH). ESIMS:

1

m/z=504.5 [M+H]⁺, 526.5 [M+Na]⁺. H

NMR (CDCl₃–CD₃OD): l 4.41 (d, J 8.1 Hz,

1 H, H-1 Gal), 4.00–3.50 (m, 9 H, OCH₂CH,

H-2–6 Gal), 3.20 (t, J 6.8 Hz, 2 H, NHCH₂),

2.02 [s, 3 H, CH₃C(O)NH], 1.60–1.40 (m, 2

H, NHCH₂CH₂), 1.40–1.10 [m, 22 H,

(CH₂)₁₁CH₃], 0.88 (t, J 6.5 Hz, 3 H, CH₂CH₃].

¹³C NMR (CDCl₃–CD₃OD):

l

172.0

[C(O)NH], 100.8 (C-1 Gal), 74.8 (C-5 Gal),

71.8 (C-3 Gal), 70.2 (OCH₂), 68.1 (C-4 Gal),

61.2 (C-6 Gal), 53.9 (OCH₂CH), 52.5 (C-2

Gal), 39.3 (NHCH₂), 31.5 (CH₂CH₂CH₃),

29.2, 29.1, 28.9, and 28.8 [(CH₂)₉-

CH₂CH₂CH₃], 26.5 (NHCH₂CH₂), 22.2

[CH₃C(O)NH], 22.1 [CH₂CH₃], 13.5 (CH₃).

Step 7: compound 35a (50 mg, 0.058 mmol)

and morpholine (0.52 mL) in DMF (1 mL)

were stirred at rt for 1 h 30. After evaporation

Article Doi

Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity

DOI: 10.1016/S0008-6215(00)00055-0

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