Carbohydrate Research 381 (2013) 146–152

Contents lists available at ScienceDirect

Carbohydrate Research

journal homepage: www.elsevier .com/locate /carres

Note

Stereoselective a-glycosylation of C(6)-hydroxyl myo-inositolsvia nickel catalysis—application to the synthesis of GPI anchorpseudo-oligosaccharides

0008-6215/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.carres.2013.09.006

⇑ Corresponding author.E-mail address: hien-nguyen@uiowa.edu (H.M. Nguyen).

Matthew S. McConnell, Enoch A. Mensah, Hien M. Nguyen ⇑Department of Chemistry, University of Iowa, Iowa City, IA 52242, United States

a r t i c l e i n f o

Article history:Received 29 July 2013Received in revised form 6 September 2013Accepted 14 September 2013Available online 21 September 2013

Keywords:Pseudosaccharides1,2-cis-2-Amino glycosidic bondsNickel catalysisN-Substituted benzylidene groupsInositols

a b s t r a c t

Glycosylphosphatidyl inositol (GPI) anchors play a key role in many eukaryotic biological pathways.Stereoselective synthesis of GPI anchor analogues have proven to be critical for probing the biosynthesis,structure, and biological properties of these compounds. Challenges that have emerged from these effortsinclude the preparation of the selectively protected myo-inositol building blocks and the stereoselectiveconstruction of glucosamine a-linked myo-inositol containing pseudodisaccharide units. Herein, wedescribe the effectiveness of the cationic nickel(II) catalyst, Ni(4-F-PhCN)4(OTf)2, at promoting selectiveformation of 1,2-cis-2-amino glycosidic bonds between the C(2)-N-substituted benzylideneamino tri-haloacetimidate donors and C(6)-hydroxyl myo-inositol acceptors. This catalytic coupling process allowsrapid access to pseudosaccharides of GPI anchors in good yields and with excellent levels of a-selectivity(a:b = 10:1–20:1). In stark contrast, activation of trichloroacetimidate donors containing the C(2)-N-substituted benzylidene group with TMSOTf and BF3

.OEt2 provided the desired pseudodisaccharides asa 1:1 mixture of a- and b-isomers.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The GPI anchor (1, Fig. 1) is a glycophospholipid which is post-translationally tethered to a protein for attachment to a cell mem-brane.1 Proteins containing the GPI anchor are functionally diverseand play a pivotal role in biochemical processes such as signaltransduction, prion disease pathogenesis, immune response, thepathobiology of trypanosomal parasites, and cancer metastasis.2

All GPI structures share the basic core that includes aphosphoethanolamine linker, a tetrasaccharide attached to theC(6)-position of myo-inositol, and phospholipid tail.3 The glycancore of the GPI anchor family can be further modified with specificsugar, lipid, and phosphoethanolamine groups. While studies haveshown that the main function of the GPI anchor is to bind proteinsto a cell surface, the relationship between structural diversity andbiological function is poorly understood.4 Synthetic analogues ofthe GPI anchor could provide a useful insight into thisrelationship.5

Despite remarkable advances in the syntheses of GPI anchors,6,7

their analogues,5 and other bioactive compounds containing myo-inositol unit,8 there are several key challenges that remain. Firstis the stereoselective construction of the 1,2-cis-2-amino glycosidicbond between the glucosamine unit and myo-inositol moiety.6–8

Current approaches provide this type of pseudodisaccharide withmodest to excellent a-selectivity. Another challenge involves thesynthesis of myo-inositol moiety with differential protectinggroups at C(6)-hydroxyl centers. Several approaches toward thesynthesis of C(6)-hydroxyl myo-inositols have been reported inan effort to generate the GPI anchor.9,10 For GPI anchor synthesis,the second challenge involves the formation of the a-glycosidiclinkages which connect the mannose units without the use ofC(2)-acyl neighboring group participation in the glycosyl donorunit. This is essential, as the basic conditions employed to removethe acyl groups could potentially cleave the ester bonds of the lipidchains attached to the myo-inositol.

Our goal is to develop a reliable and operationally simpleglycosylation procedure that provides alternative approaches to avariety of pseudosaccharides of GPI anchors and their analogues.Herein, we present a cationic nickel(II) catalyst, Ni(4-F-PhCN)4

(OTf)2, that efficiently promotes the a-selective pseudosaccharideformation from the C(2)-N-benzylideneamino trihaloacetimidatedonors and C(6)-hydroxyl myo-inositols.

2. Results and discussion

We previously reported a suitable model system to screen anumber of C(2)-N-substituted benzylidene D-glucosamine donors2–5 with the sterically hindered inositol acceptor 611b (Fig. 2a)under cationic nickel conditions. The most promising substrate

Table 1Studies with C(6)-hydroxyl perbenzylated myo-inositola

O

NAcO

AcO

O CCl3

NH

AcO 10 mol %Ni(4-F-PhCN)4(OTf)2,CH2Cl2, 25 oC, 3 h

X

O

NAcO

AcOAcO

19 - 22HO

BnO

BnO OBnOBn

OBn14

OBnO

BnO OBnOBn

OBnX

Entry Donors Donor/acceptor Products Yieldb (%) a:bc

1 15: X = 4-F 1/1.3 19 72 11:12 16: X = 4-CF3 1/1.3 20 78 11:13 15: X = 4-F 1.3/1 19 67 10:14 16: X = 4-CF3 1.3/1 20 65 13:15 17: X = 2-F 1/1.3 21 66 15:16 18: X = 2-CF3 1/1.3 22 79 15:1

a All reactions were run at 0.2 M with 10 mol % catalyst at 35 �C.b Isolated yield.c 1H NMR ratio.

O

H2NHO

HOO

OH

HOHO

O

OHO

HO

O

HO

OOH

HOHO

O

O P

O

OO-

HN Protein

O

O PO

O-

OC18H37O

C21H35

O

C15H31O

1 OO

O OHOH

OH

6

GPI Anchor

Figure 1. Structure of GPI anchor.

M. S. McConnell et al. / Carbohydrate Research 381 (2013) 146–152 147

identified was the N-para-fluorobenzylideneamino donor 4, whichunderwent glycosylation to provide the desired pseudodisaccha-ride 9 in 72% yield and solely as the a-isomer. Here, we furtherinvestigate the ability of Ni(4-F-PhCN)4(OTf)2 as an effective cata-lyst to promote the glycosylation of C(6)-hydroxyl inositol accep-tors 129a,d (Fig. 2b) with trihaloacetimidate donors 11. Thepurpose of the current studies is to explore the reaction on inositolsubstrates that could be further functionalized so that the fattyacid and phospholipid tail can be selectively installed at both theC(1)- and C(2)-positions of the inositol unit of pseudodisaccharides13 (Fig. 2b).

Given our previous studies with nickel-catalyzed glycosyla-tions,11 it was prudent to investigate the electronic properties ofthe C(2)-N-substituted benzylidene group and their effects on yieldand a-selectivity in D-glucosamine trichloroacetimidate donors.Our studies have shown that electron-withdrawing substituentson the benzylidene group increase the yield and a-selectivity ofthe coupling products.11c To simplify our investigations, we ini-tially tested the efficacy of the cationic nickel catalyst, Ni(4-F-PhCN)4(OTf)2, in promoting the coupling of C(6)-hydroxyl

HOO

O OBnOBn

OBn

MeMe

6

1

O

NAcO

AcO

O CCl3

NH

AcO

X

+ Ni(

CH

2: X = OMe3: X = H4: X = F5: X = CF3

6

Previous work:

Current work:

HOR''O

R'O OBnOBn

OBn

O

NO R2

NR1

X

(RO)n

11: R1=H, R2 = CCl3 or R1 = Ph, R2 = CF3

+

12

Ni(

(a)

(b)

Figure 2. Previous work

perbenzylated myo-inositol 1412,13 (Table 1) with four differentC(2)-benzylidene D-glucosamine trichloroacetimidate donors 15–18 at varying donor/acceptor ratios. Overall, the desired pseudodi-saccharides 19–22 were isolated in good yield (65–79%) and withhigh a-selectivity (a:b = 10:1–15:1). The 2-trifluoromethyl-N-ben-zylidene 18 showed the highest reactivity and a-selectivity (entry6), affording pseudodisaccharide 22 in 79% yield as a 15:1 a:b ratio.The 2-fluoro-N-benzylidene substrate 17 (entry 5) showed similara-selectivity (a:b 15:1) but with decreased yield (66%). While notideal, the 4-substituted benzylidene donors 15 and 16 (entries 1and 2) reacted with C(6)-hydroxyl myo-inositol acceptor 14 to pro-vide the coupling products 19 and 20 in good yield (72–78%) andwith high a-selectivity (a:b 11:1). Use of excess donor 16 relativeto acceptor 14 (entry 4) was shown to increase a-selectivity(11:1 ? 13:1) but decrease yield (72% ? 65%). On the other hand,excess equivalents of donor 15 (entry 3) only led to a decrease inboth a-selectivity (11:1 ? 10:1) and yield (78% ? 67%).

Since the 2-trifluoromethyl-benzylidene donor 18 (Table 1, en-try 6) showed the highest a-anomeric selectivity and yield, itseemed practical to use this glycosyl donor for further studies.Unfortunately, the a-trichloroacetimidate of 18 is the minor prod-uct from the reaction of hemiacetal and trichloroacetonitrile

5 mol %4-F-PhCN)4(OTf)2

2Cl2, 25oC, 4-7 h

O

NAcO

AcOAcO

X

O O

O OBnOBn

OBn

MeMe

7: X = OMe8: X = H9: X = F10: X = CF3

67% (α only)64% (α only)72% (α only)62% (α only)

O

N OR''O

R'O OBnOBn

OBnX

(RO)n6

131 2

4-F-PhCN)4(OTf)2

versus current work.

O

NAcO

AcO

O CF3

N

AcO 10 mol %Ni(4-F-PhCN)4(OTf)2,CH2Cl2, 35 oC, 12 h

HOBnO

BnO OBnOBn

OBn14

CF3Ph

NO REACTION

23

Scheme 1. Attempted coupling with N-phenyl trifluoroacetimidate 23.

148 M. S. McConnell et al. / Carbohydrate Research 381 (2013) 146–152

(a:b = 1:3). Since the b-isomer of 18 is unreactive under nickel con-ditions,11b we looked into utilizing N-phenyl trifluoroacetimidatedonors.14 We have established that a mixture of a- and b-isomerof 2-substituted benzylidene N-phenyl trifluoroacetimidate donorseffectively reacts with a wide variety of glycosyl acceptors underour nickel conditions.11c Unfortunately, the glycosylation of inosi-tol 14 with N-phenyl trifluoroacetimidate 23 (Scheme 1) did notproceed, even at elevated temperature, increased catalyst loading,and extended reaction times. We hypothesized that this may bedue to steric encumbrance surrounding the C(6)-hydroxyl groupin perbenzylated myo-inositol 14.

Since the 4-trifluoromethyl benzylidene 16 gave the next high-est a-selectivity (Table 1, entry 4), this directing group was em-ployed to examine the substrate scope of various donors reactingwith other myo-inositols 25 and 26 (Table 2). When the C(6)-hy-droxyl tetrabenzylated myo-inositol 25 bearing a C(1)-p-methoxy-benzyl ether (Table 2, entry 1) was glycosylated with

Table 2Substrate scopea

O

NAcO

RO

O CCl3NH

AcO10 mol % CatalyCH2Cl2, 25 - 35o

F3C HOR1O

R2O OBnOB

OBn25: R1 = PMB, R2 =26: R1 = R2 = Ac

16, 23, 24

Entry Donors Acceptors Catalyst

1

O

N

AcOAcO

AcO

O

F3CNH

CCl3

16

25 Ni(4-F-P

2 16 26 Ni(4-F-P

3

O

N

AcOAcO

AcO

O

NPh

CF3

23CF3

26 Ni(4-F-P

4 16 26 BF3�OEt5 16 26 TMSOTf

6

OAcOAcO

AcO OAc

O

N

OAcO

O

F3C

NH

CCl324

AcO

26 Ni(4-F-P

a All reactions were run at 0.2 M with 10 mol % catalyst. All trichloroacetimidate donoperformed at 35 �C.

b Isolated yield.c 1H NMR ratio.

trichloroacetimidate 16, a similar yield (71%) and a-selectivity(a:b = 10:1) was observed when compared with the coupling ofC(6)-hydroxyl perbenzylated inositol 14 with donor 16 (Table 1,entry 2). Given this result we infer that inositol 25 would reactin a similar manner as acceptor 14; both too sterically hinderedto undergo glycosylation with N-phenyl trifluoroacetimidate sub-strates. Gratifyingly, myo-inositol 26 (Table 2, entry 2) showed animprovement in yield (71% ? 79%) and a-selectivity(10:1 ? 12:1) when coupled with glycosyl donor 16. We hypothe-size that this improvement may be due to a reduction in stericcrowding around the C(6)-hydroxyl group of myo-inositol resultingfrom the acetyl groups incorporated at the C(1)- and C(2)-positionsin inositol 26 in lieu of the C(1)-PMB ether and C(2)-benzyl ether ininositol 25. With that in mind, we decided to attempt coupling ofinositol 26 with a a/b-mixture of the N-phenyl trifluoracetimidatedonor 23 (entry 3). To our excitement, after 18 h of reaction time,the desired pseudodisaccharide 29 was isolated in 72% yield andwith a:b = 10:1.

To evaluate the unique properties of the cationic nickel catalyst,Ni(4-F-PhCN)4(OTf)2, to efficiently promote the a-selective glyco-sylation with myo-inositol acceptors, control experiments wereperformed with traditional Lewis acids, BF3�OEt2,15 and TMSOTf16

(Table 2, entries 4 and 5). In these reactions, the desired pseudodi-saccharide 28 (entries 4 and 5) was obtained in much lower yield(79% ? 55%) and a-selectivity (12:1 ? 1:1) when compared to theglycosylation performed with the nickel catalyst (entry 2), Ni(4-F-PhCN)4(OTf)2. Pseudotrisaccharide 30 obtained in entry 6

st,C

O

NAcO

AcOAcO

27 - 30n

OR1O

R2O OBnOBn

OBn

Bn,

F3C

Products Time (h) Yieldb (%) a:bc

hCN)4(OTf)2 27 4 71 10:1

hCN)4(OTf)2 28 4 79 12:1

hCN)4(OTf)2 29 18 72 10:1

2 28 4 40 1:128 4 55 1:1

hCN)4(OTf)2 30 4 40 11:1

rs were conducted at 25 �C, except for N-phenyl trifluoroacetimidate 23 which was

M. S. McConnell et al. / Carbohydrate Research 381 (2013) 146–152 149

highlights the efficacy of the nickel-catalyzed glycosylation meth-od. Accordingly, Ni(4-F-PhCN)4(OTf)2 was applied to the morechallenging mannose-a(1,4)-glucosamine disaccharide trichloro-acetimidate 24, chosen for its similar connectivity to that foundin GPI anchor 1 (Fig. 1). Although the glycosylation provided thecorresponding pseudotrisaccharide 30 in only moderate yield(40%), a high a-selectivity (11:1) was still observed in the couplingprocess.

To validate the ability of the C(2)-N-substituted benzylidene toserve as an effective protecting group in oligosaccharide synthesisand to demonstrate the utility of our pseudodisaccharides as valu-able building blocks in GPI anchor synthesis, N-phenyl trifluoro-acetimidate 31 (Scheme 2), with labile TES protecting group atthe C(4) position, was investigated in the glycosylation of C(6)-hy-droxyl myo-inositol 26. The coupling process proceeded smoothly,providing pseudodisaccharide 32 in 61% yield and with 11:1 a:bselectivity. The TES group in 32 was subsequently removed usingTBAF (Scheme 2) to reveal the free C(4)-hydroxyl group of pseudo-disaccharide 33. This acceptor 33 was then used in a glycosylationwith tetrabenzylated D-mannose trichloroacetimidate 34 in thepresence of TMSOTf as a catalyst to afford a-pseudotrisaccharide35 exclusively in 47% yield over two steps, highlighting the versa-tility of the N-substituted benzylidene protecting group under var-ious coupling conditions. The benzylidene group can be easilyremoved in less than 5 min with HCl and acetone.11c

Overall, the results obtained in Table 2 and Scheme 2 demon-strate the efficacy of our nickel catalyst, Ni(4-F-PhCN)4(OTf)2, topromote the high-yielding and a-selective coupling of a number

BnOBnO

BnO

20 mCH2

35 (56%) 3

O

N

AcO

AcO

CF3

OAcO

AcO OBnOBn

OBn

OBnOBnO

BnO OBn

O

O

N

TESOAcO

AcO

CF3

O

NPh

CF3

31

Ni(4-F-CH2

HOAcO

A

Scheme 2. Another approach to GP

CH2Cl2, 25 oC,12 h

HOAllylO

PMBO OBnOBn

OBn

Glycosyl Donor 110 mol% Ni(4-F-PhCN)

36 X

CH2Cl2, 25 oC,12 h

HOAllylO

PMBO OBn OBn

OBn

Glycosyl Donor 10 mol % Ni(4-F-PhCN)

36X

Scheme 3. Attempted glycosy

of C(6)-hydroxyl myo-inositols with monosaccharide and disac-charide trihaloacetimidate donors bearing the C(2)-N-substitutedbenzylidene groups. To further explore the scope and limitationunder nickel catalysis, the coupling of inositol acceptor 36 bear-ing the C(1)-allyl and C(2)-PMB groups with both donors 16 and23 (Scheme 3) were examined. Unfortunately, inositol 36 did notperform well with Ni(4-F-PhCN)4(OTf)2. Pseudodisaccharides 37and 38 were obtained in very poor yield and selectivity alongwith other byproducts. In fact, we were unable to fully analyzeand characterize compounds 37 and 38.

We have established that the b-isomer of C(2)-N-substituted-benzylideneamino trichloroacetimidate donors would not reactwith alcohol acceptor in the presence of Ni(4-F-PhCN)4(OTf)2,11b

and the a-orientation of the trichloroacetimidate leaving group isimportant for the ionization and subsequent glycosylation to oc-cur. Based on these previous studies, we performed a controlexperiment containing a mixture of b-isomer of N-phenyl trifluoro-acetimidate (23b) and 10 mol % of Ni(4-F-PhCN)4(OTf)2 (Scheme 4)in CH2Cl2 in the absence of an inositol acceptor to determinewhether 23b undergoes isomerization to its corresponding a-iso-mer 23a prior to the coupling event. The reaction mixture wasmonitored by 1H NMR at 10-minute intervals. The b-isomer 23b,whose anomeric proton resonance resided at d 6.05 pm, did notconvert to the corresponding a-isomer 23a (anomeric proton res-onance at d 6.74).17 As the reaction progressed, the b-isomer 23btransformed into the undesired glycal product 39 (anomeric protonresonance at d 7.28). This appeared to be the major product after80 min. These results suggest that N-phenyl trifluoroacetimidate

O

N

AcOTESO

AcO

CF3

TBAF, AcOH/THF,pH=7

OOBn

O

NH

CCl3

OAcO

AcO OBnOBn

OBn

3261% (α:β=11:1)

O

N

AcOHOAcO

CF3

OAcO

AcO OBnOBn

OBn

33 (84%)

ol% TMSOTf,Cl2, 0 oC, 3 h

4

10 mol %PhCN)4(OTf)2,Cl2, 25 - 35oC

cO OBnOBn

OBn26

I anchor pseudotrisaccharide.

O

NAcO

AcOAcO

37

OAllylO

PMBO OBnOBn

OBn

64(OTf)2

F3C

O

NAcO

AcOAcO

38

OAllylO

PMBO OBnOBn

OBn

234(OTf)2

CF3

lation of myo-inositol 36.

CH2Cl2, 35oC, 80 min

O

NAcO

AcO O CF3

N

AcO

CF3

Ph

10 mol %Ni(4-F-PhCN)4(OTf)2,

23β

H

O

NAcO

AcO

O CF3

N

AcO

CF3Ph

23α

H

NOT OBSERVED

O

N

AcOAcOAcO

CF3

39

H

Scheme 4. Attempted isomerization of b-isomer to a-isomer.

150 M. S. McConnell et al. / Carbohydrate Research 381 (2013) 146–152

substrates may react via a different pathway compared to trichlo-roacetimidate donors under nickel conditions.

3. Conclusion

In summary, we have developed an efficient strategy for thepreparation of pseudodisaccharides that are applicable to thesynthesis of GPI anchor analogues. We have addressed a majorhurdle that is commonly encountered in the synthesis of thesecompounds by achieving high a-selectivity in the glycosylationstep to form the major pseudosaccharide moieties. The cationicnickel catalyst, Ni(4-F-PhCN)4(OTf)2, acts as an effective activat-ing reagent for promoting the coupling of myo-inositols with avariety of trichloroacetimidate and N-phenyl trifluoroacetimidatedonors bearing the C(2)-N-substituted benzylidene group. Thedesired 1,2-cis-2-amino pseudosaccharides were obtained inmoderate to good yields and excellent a-selectivity where thebest results were obtained from less sterically encumbered inos-itols. In addition, both a- and b-isomers of N-phenyl trifluoroace-timidates are viable glycosyl donors in the nickel-catalyzedreaction, an important finding for increasing the overall effi-ciency. In contrast, TMSOTf and BF3�OEt2 coupling of myo-inosi-tols with the N-substituted benzylidene donors providedpseudodisaccharides as a 1:1 mixture of a- and b-isomers. Lastly,the synthesis of the GPI anchor pseudotrisaccharides highlightsthe utility of the C(2)-N-benzylidine functionality to serve notonly as a directing group in nickel-catalyzed glycosylation, butalso as a stable protecting group under various conditions.

4. Experimental section

4.1. General methods

All reactions were performed in oven-dried Schlenk flasks fittedwith glass stoppers under a positive pressure of argon. Organicsolutions were concentrated by rotary evaporation below 40 �Cat 25 torr. Analytical thin-layer chromatography (TLC) was rou-tinely used to monitor the progress of the reactions and performedusing pre-coated glass plates with 230–400 mesh silica gel impreg-nated with a fluorescent indicator (250 nm). Visualization wasachieved using UV light, iodine, or ceric ammonium molybdate.Flash chromatography was performed and employed 230–400mesh silica gel. Dichloromethane were distilled from calcium hy-dride under an argon atmosphere at 760 torr. All of the nickel cat-alysts were prepared according to our procedure.11b All otherchemicals were obtained from commercial vendors and used with-out further purification. All proton (1H) nuclear magnetic reso-nance spectra were recorded on 400 MHz spectrometer. Allcarbon (13C) nuclear magnetic resonance spectra were recordedon 100 MHz NMR spectrometer. Chemical shifts are expressed inparts per million (d scale) downfield from tetramethylsilane andare referenced to the residual proton in the NMR solvent (CDCl3:d 7.26 ppm, d 77.00 ppm). Data are presented as follows: chemicalshift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,m = multiplet, and br s = broad singlet), integration, and coupling

constant in hertz (Hz). Infrared (IR) spectra were reported incm�1. High resolution TOF mass spectrometry utilizing electro-spray ionization in positive mode or electron ionization was per-formed to confirm the identity of the compounds.

4.2. General glycosylation with trichloroacetimidate donorsusing Ni(4-F-PhCN)4(OTf)2

4.2.1. 3,4,6-Tri-O-acetyl-2-deoxy-2-p-fluorobenzylidene-amino-a-D-glucopyranosyl-(1?6)-1,2,3,4,5-penta-O-benzyl-myo-inositol (19)

An oven dried, argon flushed 10 mL Schlenk flask was chargedwith 15 (83.4 mg, 0.15 mmol, 1.0 equiv), 14 (123 mg, 0.195 mmol,1.3 equiv), and dichloromethane (1 mL). A preformed solution ofNi(4-F-PhCN)4(OTf)2, which was generated in situ from Ni(4-F-PhCN)4Cl2 (9.22 mg, 0.015 mmol, 10 mol %) and AgOTf (7.70 mg,0.03 mmol, 20 mol %) in dichloromethane (0.5 mL) was then addedto the solution. The reaction mixture was stirred at room temper-ature. When the reaction was complete as monitored by TLC (hex-ane/ethyl acetate = 3/1), the reaction mixture was filtered,evaporated, and purified by flash chromatography on silica gel(hexane/ethyl acetate = 3/1 ? 3/2 with 1% Et3N) to give 19 as a yel-low semisolid: 104 mg, 78%, 11:1 a:b; ½a�24

D 36.7 (c 1.0, CHCl3); 1HNMR (CDCl3, 400 MHz): d = 7.93 (s, 1H, N@CH), 7.78–7.74 (m, 0.2H,Ar), 7.55–7.52 (m, 2H, Ar), 7.43–7.27 (m, 16H, Ar), 7.23–7.13 (m,9H, Ar), 7.03 (t, J = 11.0 Hz, 2H, Ar), 6.87 (d, J = 8.5 Hz, 2H, Ar),5.72–5.68 (m, 2H, H-10, H-40), 5.24 (d, J = 14.5 Hz, 1H, CH2), 5.04–4.99 (m, 2H, CH2), 4.88 (dd, J = 14.5, 5.0 Hz, 2H, CH2), 4.80 (d,J = 8.5 Hz, 2H, CH2), 4.64 (d, J = 8.5 Hz, 2H, CH2), 4.46 (d,J = 13.0 Hz, 1H, CH2), 4.39–4.34 (m, 2H, H-30, H-6a0), 4.24–4.18(m, 2H, H-6b0, H-6), 4.00 (t, J = 3.0 Hz, 1H, H-2), 3.65–3.59 (m,3H, H-50, H-1, H-5), 3.51–3.42 (m, 3H, H-20, H-3, H-4), 2.04 (s, 3H,CH3), 1.90 (s, 3H, CH3), 1.84 (s, 3H, CH3). 13C NMR (CDCl3,100 MHz): d = 170.6, 169.8, 169.7 (C@O), 162.5 (N@C), 138.7,138.6, 138.5, 138.3, 137.8, 131.8, 131.8, 130.4, 130.3, 129.1,128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7,127.6, 127.4, 127.0, 126.0, 125.4, 125.3, 115.6, 115.4 (Ar), 99.0(C-10), 82.1 (C-40), 82.0 (C-30), 81.2 (C-60), 80.9 (C-6), 75.9, 75.6,75.4, 74.3, 73.4 (CH2), 72.9 (C-1), 72.7 (C-5), 71.7 (C-50), 71.5 (C-2), 68.3 (C-4), 67.1 (C-3), 61.4 (C-20), 20.8 (CH3), 20.7 (CH3). IR (film,cm�1): m = 3063, 3030, 2869, 1744, 1644, 1601, 1508, 1454, 1363,1227, 1125, 1048, 1021. HRMS (ESI): calcd for C60H63 NO13F(M+H): 1024.4283; found: 1024.4290.

4.3. General glycosylation with N-phenyl trifluoroacetimidatedonors using Ni(4-F-PhCN)4(OTf)2

4.3.1. 3,4,6-Tri-O-acetyl-2-deoxy-2-o-trifluoromethylbenzylideneamino-a-D-glucopyranosyl-(1?6)-1,2-O-di-acetyl-3,4,5-tri-O-benzyl-myo-inositol (29)

A 10 mL oven-dried Schlenk flask was charged with donor 23(53.1 mg, 0.084 mmol, 1.5 equiv), inositol acceptor 26 (30.0 mg,0.056 mmol, 1.0 equiv), and CH2Cl2 (0.5 mL). Then 0.5 mL of pre-formed solution of Ni(4-F-PhCN)4(OTf)2, which was generatedin situ from a reaction of Ni(4-F-PhCN)4Cl2 (3.4 mg, 0.00561 mmol,

M. S. McConnell et al. / Carbohydrate Research 381 (2013) 146–152 151

10 mol %) and AgOTf (2.9 mg, 0.0112 mmol, 20 mol %) in CH2Cl2 for30 min, was added to the solution. The resulting mixture was stir-red at 35 �C overnight. When the reaction was complete as moni-tored by TLC (toluene/acetonitrile = 4/1), the mixture waspurified by silica gel flash chromatography (hexane/ethyl ace-tate = 5/1 ? 3/1 ? 3/2 ? 1/1 with 1% Et3N) to afford disaccharide29 as a semisolid. 72%, 10:1 a:b; ½a�24

D 67.4 (c 1.0, CHCl3); 1HNMR (CDCl3, 500 MHz): d = 8.69 (d, J = 2 Hz, 1H, N@CH), 8.59 (d,J = 8.0 Hz, 1H, Ar), 7.66 (d, J = 7.5 Hz, 1H, Ar), 7.60 (t, J = 7.5 Hz,1H, Ar), 7.51 (t, J = 7.5 Hz, 1H, Ar) 7.40–7.30 (m, 15H, Ar), 5.75 (t,J = 10.0 Hz, 1H, H-40), 5.64 (t, J = 10.0 Hz, 1H, H-30), 5.58 (t,J = 10.0 Hz, 0.1H, H-40), 5.16–5.11 (m, 2H, H-10, H-1), 5.04 (d,J = 11.0 Hz, 1H, CH2), 4.96 (d, J = 10.5 Hz, 1H, CH2), 4.92–4.88 (m,2H, CH2), 4.83 (d, J = 12.0 Hz, 1H, CH2), 4.74 (d, J = 12.0 Hz, 1H,CH2), 4.67 (d, J = 11.0, 1H, H-60), 4.63 (dt, J = 10.5, 2.5 Hz, 1H,H-60), 4.41 (t, J = 2.5 Hz, 1H, H-2), 4.30 (t, J = 10.0 Hz, 1H, H-6),3.93 (dd, J = 12.5, 3.0 Hz, 1H, H-5), 3.70 (dd, J = 10.0, 3.5 Hz, 1H,H-50), 3.64 (dd, J = 12.5, 2.0 Hz, 1H, H-3), 3.57–3.55 (m, 2H, H-4,H-20), 2.06 (s, 3H, CH3), 2.04 (s, 3H, CH3), 1.91 (s, 3H, CH3), 1.86(s, 3H, CH3), 1.76 (s, 3H, CH3). 13C NMR (CDCl3, 125 MHz):d = 170.6, 170.0, 169.9, 169.7, 169.1 (C@O), 160.8 (N@C), 132.7,130.5, 130.2, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9,127.9, 127.7, 127.6, 127.4, 127.2 (Ar), 125.1, 125.0 (CF3), 99.5(C-10), 81.7 (C-40), 80.7 (C-30), 78.6 (C-1), 75.9 (C-2), 75.4 (C-60),73.6, 73.5, 72.8 (CH2), 72.1 (C-6), 72.0 (C-50), 70.8 (C-5), 68.6(C-4), 67.7 (C-3), 61.4 (C-20), 29.7, 20.8, 20.4 (CH3). IR (film,cm�1): m = 3032, 2952, 2926, 2859, 1753, 1643, 1455, 1367, 1315,1239, 1123, 1044. HRMS (ESI): calcd for C51H55 NO15F3 (M+H):978.3524; found: 978.3549.

4.4. Preparation of 2,3,4,6-tetra-O-benzyl-a-D-mannopyranosyl-(1?4)-3,6-di-O-acetyl-2-deoxy-2-o-trifluoromethylbenzylideneamino-a-D-glucopyranosyl-(1?6)-1,2-O-di-acetyl-3,4,5-tri-O-benzyl-myo-inositol (35)

A 10 mL oven-dried Schlenk flask was charged with donor 34(10.0 mg, 0.015 mmol, 2.0 equiv), pseudodisaccharide acceptor33 (7.0 mg, 0.0075 mmol, 1.0 equiv), crushed 4 Å molecular sieves(10 mg), and CH2Cl2 (0.5 mL). The reaction mixture was stirred for1 h at room temperature. The mixture was cooled to 0 �C andTMSOTf (0.27 lL, 0.0075 mmol, 0.2 equiv.) was added. The reac-tion was stirred at 0 �C for 4 h. Triethylamine (0.5 mL) was addedto quench the reaction. The reaction mixture was filtered andconcentrated. The crude product was purified by flash chromatog-raphy on silica gel (hexane/ethyl acetate = 5/1 ? 3/1 ? 1/1 with1% Et3N) to afford pseudotrisaccharide 35 (6.1 mg, 56%) as a yel-low oil. 1H NMR (CDCl3, 500 MHz): d = 8.64 (d, J = 2.5 Hz, 1H,N@CH), 8.57 (d, J = 8.0 Hz, 1H, Ar), 7.65 (d, J = 7.5 Hz, 1H, Ar),7.59 (t, 7.5 Hz, 1H, Ar), 7.51 (t, J = 8.0 Hz, 1H, Ar), 7.37–7.24 (m,40H, Ar), 5.74–5.69 (m, 2H, H-10, H-30), 5.11 (d, J = 2.0 Hz, 1H,CH2), 5.08 (d, J = 4.0 Hz, 1H, CH2) 5.05–4.86 (m, 6H, CH2), 4.78–4.77 (m, 3H, CH2, H-100), 4.70–4.64 (m, 5H, CH2, H-1), 4.51–4.46(m, 3H, CH2, H-60), 4.38 (t, J = 2.0 Hz, 1H, H-2), 4.30 (t, J = 9.5 Hz,1H, H-40), 4.15 (t, J = 9.5 Hz, 1H, H-6), 4.02 (dd, J = 12.5, 2.0 Hz,1H, H-60), 3.91 (t, J = 9.5 Hz, 1H, H-40), 3.87–3.85 (m, 2H, H-60,H-30), 3.65 (d, J = 7.5 Hz, 1H, H-5), 3.58–3.53 (m, 4H, H-200, H-3,H-4, H-60), 3.46–3.43 (m, 2H, H-20, H-50), 2.02 (s, 3H, CH3), 1.83(s, 3H, CH3), 1.75 (s, 3H, CH3), 1.72 (s, 3H, CH3). 13C NMR (CDCl3,125 MHz): d = 170.5, 169.9, 169.1, 169.0 (C@O), 160.1 (N@C),138.6, 138.5, 138.5, 138.5, 138.4, 138.3, 137.4, 133.1, 132.8,130.9, 130.5, 130.3, 128.8, 128.7, 128.4, 128.4, 128.3, 128.3,128.3, 128.2, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.6,127.5, 127.5, 127.4, 127.4, 127.4, 127.3 (Ar), 125.0, 125.0 (CF3),100.9 (C-10), 99.4 (C-1’’), 81.5 (C-30), 80.6 (C-1), 79.5 (C-2), 78.6(C-40), 76.2 (C-60), 75.9 (C-6), 75.7, 75.3, 74.4, 73.5, 73.4, 73.2,73.2 (CH2), 73.0 (C-50), 72.8 (C-2’’), 72.1 (C-3), 72.0 (C-3’’), 71.9

(C-5), 70.6 (C-4’’), 68.7 (C-5’’), 68.6 (C-4), 68.2 (C-6’’), 62.8 (C-20), 38.7, 30.4, 29.7, 28.9, 26.5, 23.8, 23.0, 22.7, 20.9, 20.8, 20.6,20.4 (CH3). IR (film, cm�1): m = 3088, 3066, 3033, 2928, 2854,1752, 1497, 1455, 1367, 1315, 1238, 1224, 1163, 1038. HRMS(ESI): calcd for C83H87NO19F3 (M+H) 1458.5824; found:1458.5841.

Acknowledgements

We would like to thank Ms. Kathleen White for the preparationof imidate donor 24 and inositol 25. M.S.M. thanks the GraduateCollege for a Summer Research Fellowship. Funding of this workby the University of Iowa and National Science Foundation (CHEM1106082) is gratefully acknowledged.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.carres.2013.09.006.

References

1. Goel, M.; Azev, V. N.; d’Alarcao, M. Future Med. Chem. 2010, 23, 95.2. (a) Nosjean, O.; Briolay, A.; Roux, B. Biochim. Biophys. Acta 1997, 1331, 153; (b)

Chesebro, B.; Trifilo, M.; Race, R.; Meade-White, K.; Teng, C.; LaCasse, R.;Raymond, L.; Favara, C.; Baron, G.; Priola, S.; Caughey, B.; Masliah, E.; Oldstone,M. Science 2005, 308, 1435.

3. Low, M.; Hoessli, D. C.; Ilangumaran, S. In GPI-Anchored Membrane Proteins andCarbohydrates; Landes Company: Austin, TX, 1999; pp 1–14.

4. (a) van den Berg, C. W.; Cinek, T.; Hallett, M. B.; Horejsi, V.; Morgan, B. P. J. CellBiol. 1995, 131, 669; (b) Zhang, F.; Schmidt, W. G.; Hou, T.; Williams, A. F.;Jacobson, K. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5231.

5. Representative examples of GPI analogues: (a) Paulick, M. G.; Wise, A. R.;Forstner, M. B.; Groves, J. T.; Bertozzi, C. R. J. Am. Chem. Soc. 2007, 129, 11543;(b) Paulick, M. G.; Forstner, M. B.; Groves, J. T.; Bertozzi, C. R. Proc. Natl. Acad.Sci. U.S.A. 2007, 104, 20332; (c) Swarts, B. M.; Guo, Z. Chem. Sci. 2011, 2, 342; (d)Tsai, Y.-H.; Liu, X.; Seeberger, P. H. Angew. Chem., Int. Ed. 2012, 51, 11438.

6. Representative examples of the syntheses of GPI anchors: (a) Murakata, C.;Ogawa, T. Carbohydr. Res. 1992, 235, 95; (b) Mayer, T. G.; Kratzer, B.; Schmidt, R.R. Angew. Chem., Int. Ed. 1994, 33, 2177; (c) Lu, J.; Jayaprakash, K. N.; Schlueter,U.; Fraser-Reid, B. J. Am. Chem. Soc. 2004, 126, 7540; (d) Baeschlin, D. K.;Chaperon, A. R.; Green, L. G.; Hahn, M. G.; Ince, S. J.; Ley, S. V. Chem. Eur. J. 2000,6, 172; (e) Schofield, L.; Hewitt, M. C.; Evans, K.; Siomos, M.-A.; Seeberger, P. H.Nature 2002, 418, 785; (f) Liu, X.; Kwon, Y.-U.; Seeberger, P. H. J. Am. Chem. Soc.2005, 127, 5004; (g) Xue, J.; Guo, Z. J. Am. Chem. Soc. 2003, 125, 16334; (h) Xue,J.; Shao, N.; Guo, Z. J. Org. Chem. 2003, 68, 4020; (i) Wu, X.; Guo, Z. Org. Lett.2007, 9, 4311; (j) Swarts, B. M.; Guo, Z. J. Am. Chem. Soc. 2010, 132, 6648; (k)Srinivas, B.; Swarts, B. M. Chem. Eur. J. 2012, 18, 1194; (l) Tsai, Y.-H.; Goetze, S.;Vilotijevic, I.; Grube, M.; Silva, D. V.; Seeberger, P. H. Chem. Sci. 2013, 4, 468; (m)Yashunsky, D. V.; Borodkin, V. S.; Ferguson, M. A. J.; Nikolaev, A. V. Angew.Chem., Int. Ed. 2006, 45, 468–474.

7. For review of the syntheses and characterizations of GPI anchors, see: (a) Guo,Z.; Bishop, L. Eur. J. Org. Chem. 2004, 3585; (b) Swarts, B. M.; Guo, Z. Adv.Carbohydr. Chem. Biochem. 2012, 67, 137; (c) Homans, S. W.; Ferguson, M. A. J.;Dwek, R. A.; Rademacher, T. W.; Anand, R.; Williams, A. F. Nature 1988, 333,269.

8. (a) Knapp, S.; Gonzalez, S.; Myers, D. S.; Eckman, L. L.; Bewley, C. A. Org. Lett.2002, 4, 4337; (b) Nicholas, G. M.; Eckman, L. L.; Kovac, P.; Otero-Quintero, S.;Bewley, C. A. Bioorg. Med. Chem. 2003, 11, 2641; (c) Knapp, S.; Amorelli, B.;Darout, E.; Ventocilla, C. C.; Goldman, L. M.; Huhn, R. A.; Minnihan, E. C. J.Carbohydr. Chem. 2005, 24, 103; (d) Metaferia, B. B.; Fetterolf, B. J.; Shazad-ul-Hussan, S.; Moravec, M.; Smith, J. A.; Ray, S.; Gutierrez-Lugo, M.-T.; Bewley, C.A. J. Med. Chem. 2007, 50, 6326; (e) Rawat, M.; Johnson, C.; Cadiz, V.; Av-Gay, Y.Biochem. Biophys. Res. Commun. 2007, 63, 71; (f) Slattegard, R.; Gammon, D. W.;Oscarson, S. Carbohydr. Res. 1943, 2007, 342; (g) Metaferia, B. B.; Ray, S.; Smith,J. A.; Bewley, C. A. Bioorg. Med. Chem. Lett. 2007, 17, 444; (h) Stewart, M. J. G.;Jothivasan, V. K.; Rowan, A. S.; Wagg, J.; Hamilton, C. J. Org. Biomol. Chem. 2008,6, 385; (i) Lamprecht, D. A.; Muneri, N. O.; Eastwood, H.; Naidoo, K. J.; Strauss,E.; Jardine, A. Org. Biomol. Chem. 2012, 10, 5278.

9. (a) Guo, Z.; Bishop, L. Eur. J. Org. Chem. 2004, 3585; (b) Lopez-Prados, J.; Martin-Lomas, M. J. Carbohydr. Chem. 2005, 24, 393; (c) Conrad, R. M.; Grogan, M. J.;Bertozzi, C. R. Org. Lett. 2002, 4, 1359; (d) Yu, F.; Guo, Z. Bioorg. Med. Chem. Lett.2009, 3852.

10. Ferrier, R. J.; Middleton, S. Chem. Rev. 1993, 93, 2779.11. (a) Mensah, E. N.; Nguyen, H. M. J. Am. Chem. Soc. 2009, 131, 8778; (b) Mensah,

E. N.; Yu, F.; Nguyen, H. M. J. Am. Chem. Soc. 2010, 132, 14288; (c) Yu, F.;Nguyen, H. M. J. Org. Chem. 2012, 77, 7330; (d) McConnell, M. S.; Yu, F.; Nguyen,H. M. Chem. Commun. 2013, 4313.

152 M. S. McConnell et al. / Carbohydrate Research 381 (2013) 146–152

12. (a) Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J. J. Chem. Soc., Perkin Trans. 11993, 2945; (b) Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W. J. Org. Chem.1994, 59, 4805; (c) Roemer, S.; Stadler, C.; Rudolf, M. T.; Jastorff, B.; Schultz, C. J.Chem. Soc., Perkin Trans. 1 1996, 1683.

13. (a) Kornienko, A.; Turner, D. I.; Jaworek, C. H.; d’Alarcao, M. Tetrahedron:Asymmetry 1998, 9, 2783; (b) Anikumar, G. N.; Jia, Z. J.; Kraehmer, R.; Fraser-Reid, B. J. Chem. Soc., Perkin. Trans. 1 1999, 3591; (c) Saikam, V.; Raghupathy, R.;Yadav, M.; Gannedi, V.; Singh, P. P.; Qazi, N. A.; Sawant, S. D.; Vishwakarma, R.A. Tetrahedron Lett. 2011, 52, 4277.

14. Representative examples of glycosyl N-phenyl trifluoroacetimidate donors: (a)Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405; (b) Cai, S.; Yu, B. Org. Lett. 2003,

5, 3827; (c) Huchel, U.; Tiwari, P.; Schmidt, R. R. J. Carbohydr. Chem. 2010, 29,61.

15. (a) Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. 1980, 19, 731; (b) Schmidt, R.R.; Michel, J. J. Carbohydr. Chem. 1985, 4, 141; (c) Zimmermann, P.; Bommer, R.;Bär, T.; Schmidt, R. R. J. Carbohydr. Chem. 1988, 7, 435.

16. (a) Schmidt, R. R. Angew. Chem., Int. Ed. 1986, 25, 212; (b) Schmidt, R. R.;Behrendt, M.; Toepfer, A. Synlett 1990, 694; (c) Schaubach, R.; Hemberger, J.;Kinzy, W. Liebigs Ann. Chem. 1991, 607; (d) Paulsen, H.; Wilkens, R.; Reck, F.;Brockhausen, I. Liebigs Ann. Chem. 1992, 1303; (e) Kulkarni, S. S.; Hung, S.-C.Lett. Org. Chem. 2005, 2, 670.

17. See the Supplementary data for 1H NMR spectral analysis.