C-Glycosyl amino acids through hydroboration-cross-coupling of exo-glycals and their application in automated solid-phase synthesis

FULL PAPER

DOI: 10.1002/chem.201300150

C-Glycosyl Amino Acids through Hydroboration–Cross-Coupling of

exo-Glycals and Their Application in Automated Solid-Phase Synthesis

Stefan Koch,^[a]Dieter Schollmeyer,^[a]Holger Lçwe,^{[a, b]}and Horst Kunz*^[a]

Abstract: O-Glycosylation is one of the

most important post-translational mod-

ifications of proteins. The attachment

of carbohydrates to the peptide back-

bone influences the conformation as

well as the solubility of the conjugates

and can even be essential for binding

to specific ligands in cell–cell interac-

tions or for active transport over mem-

branes. This makes glycopeptides an in-

teresting class of compounds for medi-

cal applications. To enhance the long-

term availability of these molecules in

vivo, the stabilization of the glycosidic

bond between the amino acid residue

and the carbohydrate is of interest. The

described modular approach affords b-

linked C-glycosyl amino acids by a se-

quence of Petasis olefination of glyco-

nolactones, stereoselective hydrobora-

tion and a mild B-alkyl-Suzuki cou-

pling reaction. The coupling products

were transformed to C-glycosyl amino

acid building-blocks suitable for solid-

phase synthesis and successfully incor-

porated into a partial sequence of the

tumor-associated MUC1-glycopeptide.

The resulting C-glycopeptides are can-

didates for the development of long-

term stable mimics of O-glycopeptide

vaccines.

Keywords: amino acids · anticancer

vaccines · C-glycosides · cross-cou-

pling · hydroboration · Suzuki cou-

pling

Introduction

The epithelium is one of the four major tissue types in the

human organism. It covers inner and outer surfaces of an or-

ganism and includes, for example, the epidermis as an outer

and the endothelium as an inner coating. Endothelial cells

are always found on interfaces and, therefore, exposed to

elevated external stress in comparison to the rather protect-

ed connective tissue below. To enhance the resistance of the

epithelial tissue, the cell membranes are covered with an ad-

ditional protecting film of densely packed carbohydrate con-

taining molecules, named glycocalyx^[1](Scheme 1).

Scheme 1. Membrane of an epithelial cell (schematic).

These glycosylated cell-surface molecules, in particular

glycoproteins, reach out far into the extracellular space. As

a consequence, they are not only involved in the protection

of the cell surface, but also take part in specific interactions

with external components and other cells as shown, for ex-

ample, in the reactions of the ABO blood-group system.^[2]

The knowledge of the carbohydrate structures involved in

a certain biological process enables for new approaches with

tailor-made glycosylated drugs. This strategy is followed, for

example, in recent research activities concerning cancer dis-

eases. During the past two decades cell biological and bio-

chemical investigations have shown that normal epithelial

cells and epithelial tumor cells are very different with re-

spect to the glycosylation patterns of their membrane glyco-

proteins.^[3,4]These differences between normal and malig-

nant cells can be exploited for the development of an immu-

nization directed against tumor cells.^[5]Actually, immuniza-

tion of mice with synthetic vaccines consisting of synthetic

glycopeptide partial structures of the tumor-associated

mucin MUC1 linked to Tetanus Toxoid induced strong

immune responses and IgG antibodies, which efficiently

bound to MCF-7 breast tumor cells.^[6]Another field for the

exploration of glycopeptide drugs is the therapy of chronic

inflammation by selectine inhibitors based on Sialyl-Lewis^x

as a lead structure.^[7]In the context of pain therapy, O-glyco-

sylated enkephaline analogues show prolonged life times in

the blood stream and an enhanced penetration of the blood

[a] Dr. S. Koch, Dr. D. Schollmeyer, Prof. Dr. H. Lçwe,

Prof. Dr. H. Kunz

Institut fꢀr Organische Chemie

Johannes Gutenberg-Universitꢁt Mainz

Duesbergweg 10-14, 55128 Mainz (Germany)

Fax : (+49) 6131-39-24786

E-mail: hokunz@uni-mainz.de

[b] Prof. Dr. H. Lçwe

IMM Institut fꢀr Mikrotechnik Mainz

Carl Zeiss-Strasse 18–20, 55129 Mainz (Germany)

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/chem.201300150.

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brain barrier probably due to active transport by glucose-

transporters.^[8]

These examples give evidence that the glycosylation of

peptides in many cases offers an enormous potential with

regard to medical applications. A drawback of glycosylated

drugs concerns their limited long-term stability due to deg-

radation by glycosidases, in particular endo-glycosidases.

Therefore, research activities aim at the stabilization of the

acetal-type linkage between carbohydrate and peptide por-

tions of glycopeptides. Substitution of hydroxyl for fluorine

substituents within the saccharide can inhibit the degrada-

tion by glycosidases.^[9]Another approach includes the ex-

change of O-glycoside for S-glycoside bonds.^[10]The use of

carbasugars^[11]in which the ring-oxygen is replaced by a

methylene group is also possible. A reliably stabilized link-

age between the saccharide and peptide parts is achieved if

C-glycosyl amino acids derived from proteinogenic amino

acids are incorporated in the sequences of glycopeptides

(Scheme 2).

Scheme 3. Retrosynthetic scheme for the synthesis of C-glycosyl amino

acid building-blocks.

chloroacetimidate with allyl alcohol.^[21]After deacetylation

and purification by flash-chromatography, the product was

converted to a mixture of the exo/endo-3,4-benzylidene-ac-

tetals 2, which were separable by flash-chromatography. Re-

markably, these epimers show quite different optical rota-

tion values. Treatment of both isomers with sodium hydride

and benzyl bromide in dimethylformamide (DMF) gave the

fully protected b-galactosides 3. The corresponding a-allyl

galactoside 6 was obtained from galactose 4 through a one-

pot isopropylidenation, 6-O-benzylation, and Fischer glyco-

sylation of allyl alcohol to afford the a-allyl galactoside 5.

Different from our expectation, its reaction with dimethoxy-

toluene did not selectively give the endo acetal. Subsequent

benzylation of the mixture gave compounds 6 (Scheme 4).

Scheme 2. Schematic presentation of alternatives to the native O-glyco-

side linkage in glycopeptides.

The synthesis of C-glycosyl amino acids has been descri-

bed in a number of reports,^[12]most of them concern C-gly-

cosyl serines,^[13]obtained by auxiliary controlled reactions,^[14]

cross-metathesis processes^[15]or Wittig- and Horner olefina-

tion reactions.^[16]A few syntheses of C-glycosyl threonine^[17]

or C-glycosyl tyrosine^[18]derivatives are known. However,

there is no general procedure in the literature to access C-

glycoside analogues of the three usually O-glycosylated

amino acids.

We here describe a general modular synthesis of C-glyco-

syl amino acids that is based on a stereoselective hydrobora-

tion of exo-glycals and the cross-coupling of the formed or-

ganoboranes with sp²-hybridized halides derived from the

Scheme 4. Syntheses of the exo- and endo-isomers of allyl-3,4-O-benzyli-

denegalactopyranosides.

natural amino acids in

a

B-alkyl-Suzuki reaction

(Scheme 3). As a prerequisite for the realization of the out-

lined strategy, the sugar lactones have to be prepared. These

glyconolactones are converted to the exo-glycals by a micro-

wave-supported Petasis olefination in a flow reactor.^[19]

A mixture of exo/endo allyl glycosides 3 was isomerized

under catalysis with 1,5-cycloctadienyl-bis(methyldiphenyl-

phosphine)-iridium hexafluorophosphate (method A),^[22]

whereas isomerization of the corresponding a-allyl glalacto-

side 6 was performed by using KOtBu in DMSO (method

B).^[23]The intermediate propenyl galactosides formed by

both methods (Scheme 5) were hydrolyzed by treatment

with iodine in weakly basic solution to give 7. These reduc-

ing sugars 7 were oxidized by Dess–Martin periodinane to

give the exo/endo isomers of the corresponding galactono-

lactone 8, which can be separated by flash chromatography.

To obtain the 4,6-benzylidene-2,3-di-O-benzyl-galactono-

lactone 10, the known allyl galactopyranoside^[23a]9 was sub-

Results and Discussion

To demonstrate the scope of the modular synthesis of C-gly-

cosyl amino acids, saccharide components containing acid-

sensitive acetalic protecting groups were subjected to the

envisioned process. Starting from 2,3,4-tri-O-acetyl-6-O-

benzyl-d-galactose 1,^[20]the corresponding allyl b-galactopyr-

anoside was synthesized through the treatment of the tri-

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C-Glycosyl Amino Acids

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eters with small quantities of the valuable substrates before

larger amounts of the products are prepared by simply run-

ning the flow reactor for a prolonged time. Within a few mi-

nutes of residence time inside the reaction zone, the rather

labile cyclic enol ethers can be obtained in moderate to

good yields after flash chromatography.

For the planned coupling reactions, l-phenylalanine was

converted to 4-iodo-phenylalanine,^[29]before the tert-buty-

loxycarbonyl (Boc) group and the tert-butyl ester^[30]were in-

troduced to form 21 (Scheme 8). Boc-protected l-serine and

Scheme 5. Synthesis of exo- and endo-3,4-O-benzylidene-galactonolac-

tones 8.

Scheme 6. Synthesis of the glyconolactones.

jected to a one pot deallylation according to method B fol-

lowed by oxidation with DMSO/acetic anhydride^[24]

(Scheme 6). In an analogous way the known allyl 4,6-O-ben-

zylidene-2,3-di-O-benzyl-a-d-glucopyrano-side^[25]was deally-

lated to afford 4,6-O-benzylidene-2,3-di-O-benzyl-a-d-gluco-

pyranose 12^[26]which was oxidized to give the gluconolac-

tone 13. This compound should not be stored in solution for

a long time, because it is prone to isomerize to the isomers

of the 5-ring-lactone 14 (Scheme 6).

Scheme 8. Syntheses of Boc-protected aryl and alkenyl amino acid build-

ing blocks.

Another lactone l-fuconolactone^[27]16 was prepared from

thiofucoside^[28]15 by hydrolysis and subsequent oxidation.

As previously reported,^[19]all these glyconolactones 8, 10,

13, and 16 were transformed to the corresponding exo-gly-

cals by using a microwave-assisted Petasis olefination under

continuous flow conditions (Scheme 7). This procedure elim-

inates the volume-limitation of the microwave batch proce-

dure and allows a facile optimization of the reaction param-

l-threonine also were transformed to their tert-butyl esters

by treatment with a solution of tert-butanol, cylcohexylcar-

bodiimide and cat. CuCl in dichloromethane.^[30]Boc-serine

tert-butyl ester was mesylated and treated with 1,8-

diazabicycloACTHNUGTRNEUNG[5.4.0]undec-7-ene (DBU) to give the dehydroa-

lanine derivative which was subjected to reaction with N-

bromo-succinimide (NBS) to afford the Boc-protected b-

bromo dehydroalanine tert-butyl ester^[31]22 (Scheme 8). The

corresponding Boc-threonine tert-butyl ester^[30]had to be

treated with thionyl chloride to give a cyclic sulfamidate,^[32]

which upon treatment with DBU afforded the amino-dehy-

drobutyric acid derivative. Its treatment with NBS gave the

separable E- and Z-isomers of Boc-b-bromo-2,4-dehydroa-

minobutyric acid tert-butyl ester 23 (Scheme 8). In the

¹H NMR spectrum of Z-isomer 23a, proton signals of the

NH- and CH₃group are shifted downfield.

At first, the Suzuki coupling reactions were performed

under conditions that had been described in the literature^[32]

for Suzuki reactions of exo-glycals. The exo-glycal was treat-

ed with 9-BBN in THF (Scheme 9). After completion of the

Scheme 7. exo-Glycals from glyconolactones obtained by microwave-sup-

ported Petasis reaction in a continuous flow reactor.^[19]

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H. Kunz et al.

high stereoselectivity of the hy-

droboration can be explained

by a stabilizing anomeric effect

in the transition state closely re-

lated to the intermediate glyco-

syl cation, which is formed

upon attack of the borane at

the enol ether double bond

(Scheme 11b).

We concluded from the suc-

cessful use of K₂CO₃in these

À

C C coupling reactions that a

B-alkyl-Suzuki reaction can be

Scheme 9. C-Glycosyl amino acids by B-alkyl Suzuki coupling under conditions reported in the literature.^[32]

hydroboration step (monitoring by TLC), aqueous K₃PO₄-

solution as the alkaline component, the [bis(diphenylphos-

phino)ferrocene]palladium catalyst,^[33]DMF for homogeni-

zation and the amino acid derived coupling component 21,

22, or 23 were added. Under these conditions, the coupling

products 24–27 (Scheme 9) were obtained, albeit in low

yields. The isolated amounts allowed for the characterization

of the compounds, but not for further chemical transforma-

Scheme 11. X-ray structure (left) of 30 (CCDC-No. 920035), and explana-

tion for the stereoselective formation of the b-anomers (in the case of

20) during the hydroboration step (right).

tions.

Systematic optimization of both the hydroboration and

the coupling step resulted in substantial improvement of the

yield of these coupling processes. The decisive change con-

sisted of the use of dry K₂CO₃as the base and the subse-

quent slow addition of water to the reaction mixture instead

of employing the stronger alkaline aqueous K₃PO₄solution

(Scheme 10). Under these modified conditions, good yields

of the C-glycosyl amino acid derivatives 28–31, up to 86%

in the case of C-galactosyl-derivative 30, were obtained

(Scheme 10).

achieved under thus mild reaction conditions, which should

not affect fluorenyl-9-methoxycarbonyl (Fmoc) groups in

amino acid coupling components. The required halogen-aryl

and halogen-alkenyl building blocks (Scheme 12) were pre-

pared in a few steps.

C-Glycosyl amino acid 30 was obtained in crystalline

form. Its X-ray analysis unambiguously confirmed the b-

configuration of the C-glycosyl linkage (Scheme 11a). The

Scheme 12. Preparation of the Fmoc-protected building-blocks for the

coupling procedure.

Actually, the stability of the Fmoc group under the out-

lined optimized coupling conditions is sufficient, so that the

B-alkyl Suzuki coupling with exo-glycals 17a, 18, 19, and 20

to give the Fmoc-protected C-glycosyl amino acid deriva-

tives proceeded faster than the removal of the protecting

group (Scheme 13). Nevertheless, the reaction needs careful

monitoring by TLC and must be stopped by addition of

acetic acid before substantial formation of dibenzofulvene is

detected.

Especially the coupling products 34–37 of tyrosine-ana-

logues were obtained in good yields (Scheme 13), whereas

the yield of the isolated serine analogue 38 remained low. In

Scheme 10. B-alkyl-Suzuki coupling under optimized conditions using

K₂CO₃as the base.

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ly, compound 36, after acidolytic cleavage of the benzyli-

dene group, was subjected to O-acetylation and careful sap-

onification of the methyl ester (Scheme 14). In contrast to

procedures described in the literature,^[33]the selective cleav-

age of the methyl ester in the presence of the base-labile

Fmoc group was not achieved by slow addition of LiOH-sol-

ution. Therefore, the Fmoc group had to be re-introduced

by treating with FmocOSu to give the building-block 42 in a

yield of 82% (Scheme 14).

Oligosaccharide-containing C-glycosyl amino acids are ac-

cessible from the described monosaccharide precursors

through selective functionalization (Scheme 15).

Scheme 13. B-Alkyl Suzuki cross-coupling of the Fmoc-halogenalkenyl

amino acids.

this case, the removal of the Fmoc group from 33, as well as

from 38, is faster than from the phenylalanine derivatives, as

can be concluded from TLC monitoring. Neither variation

of the amount of base nor use of the more reactive iodo de-

rivative 33b resulted in an increased yield (Scheme 13).

Stereoselective hydrogenation of the dehydro amino acid

double bond of 33 by using (R)-Tol-BINAP as the ligand for

the rhodium-catalyzed reaction gave a single diastereomer

of 40 (Scheme 13).

Scheme 15. Strategy for further functionalization of the initial coupling

products including a regioselective opening of a benzylidene acetal under

flow conditions.

Adopting the model described by Imamoto^[34]for the in-

terpretation of the stereoselective hydrogenation of dehydro

amino acids, molecule 40 is assumed to have R configura-

tion. However, experimental clarification still needs to be

achieved.

For application of the C-glycosyl amino acids in solid-

phase peptide synthesis according to the automated Fmoc-

protocol, protecting group manipulations had to be carried

out. The exchange of protecting groups on N-Boc-protected

C-glycosyl amino acids was performed as is shown for com-

pound 28 in Scheme 14. After hydrogenolysis of the protect-

ing groups in the carbohydrate moiety, O-acetylation and

subsequent treatment with trifluoroacetic acid (see the Ex-

perimental Section), the N-Fmoc-group was introduced by

Scheme 16. Experimental setup for regioselective opening of benzylidene

acetals under continuous flow conditions.

For example, the regioselective opening of the benzyli-

dene acetals of the C-glucosyl-tyrosine or -serine derivatives

(36 and 40, respectively, Scheme 15) was performed under

flow conditions by using a Microglas interdigital mixer to

assure instant mixing of the two liquid phases (Scheme 16).

This setup again enabled a step-by-step optimization of the

reaction parameters with small quantities of the carbohy-

drate substrates and eliminated the problem of scale-up for

larger amounts. As the reaction

using

O-(fluorenyl-methoxycarbonyl)-N-hydroxy-succini-

mide (FmocOSu) to afford the C-glucosyl-tyrosine building-

block 41 for solid-phase synthesis (Scheme 14). Alternative-

can be conducted at 08C, which

means a much higher tempera-

ture than commonly reported in

literature, the reaction runs

very fast and is complete within

3.75 min. The flow procedure in

this case is useful to ensure a

reliable control over the resi-

dence time under the harsh

Scheme 14. Transformation of the coupling products into building blocks for solid-phase peptide synthesis.

acidic conditions, as observed

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H. Kunz et al.

previously by Fukase.^[35]The obtained products 43 and 44

were subjected to Schmidt glycosylation^[21a]by using 6-O-

benzyl-2,3,4-tri-O-acetyl-galactosyl-trichloroacetimidate^[20,21]

45 to give the C-lactosyl-tyrosine 45 and serine derivative 46

in high yield (Scheme 15).

The C-lactosyl amino acids also are directly accessible by

the use of a lactose-derived exo-glycal in the Suzuki cou-

pling (Scheme 17). The required lactone 47 obtained from

glycosylated tandem repeat glycopeptides of the tumor-asso-

ciated MUC1 (Scheme 18). C-Glycosyl-tyrosines were

chosen since they are known to have l-configuration, where-

as d-amino acids could cause undesired immunological ef-

fects. For incorporation of the C-glycosyl amino acids into

the peptide sequences no changes of the reaction parame-

ters were necessary compared to syntheses of the O-glycosy-

lated derivatives. The solid phase synthesis was performed

by using Tentagel R-resin pre-

loaded through the trityl-

anchor with Fmoc-alanine.^[5a,6,9b]

The Fmoc-protected amino

acids (10 equiv) were coupled

with

O-benzotriazol-1-yl-

N,N,N’,N’-tetramethyluronium-

hexafluorophosphate (HBTU)/

1-hydroxy-benzotriazole

(HOBt) within 20 min, whereas

the C-glycosyl amino acids 39

and 42 (1.0–1.3 equiv) as well

as the terminal spacer-amino

acid were coupled after activa-

tion with O-azobenzotriazol-1-

yl-N,N,N’,N’-tetramethyluroni-

um-hexafluorophosphate

(HATU)/1-hydroxy-7-azabenzo-

triazole (HOAt) within 5 h of

Vortex shaking (Scheme 18).

The amino acids Ala¹⁹and

Gly¹⁶, whose couplings might be

Scheme 17. Syntheses of the C-lactosyl-amino acids 50 and 52.

hindered due to steric reasons,

were treated in a double-cou-

hepta-O-benzyl-d-lactose^[36]was converted to the corre-

sponding exo-glycal 48 under the elaborated microwave con-

ditions^[19]in good yield (Scheme 17). It was used in the se-

quence of hydroboration and Suzuki cross-coupling under

the described conditions. C-Lactosyl-tyrosine coupling prod-

uct 49 was isolated in 68% yield (Scheme 17).

pling using HBTU/HOBt. After the incorporation of the N-

terminal spacer amino acid,^[38]the terminal Fmoc-group was

removed and the resin was transferred to a Merrifield reac-

tor (Scheme 18). Treatment with trifluoroacetic acid (TFA),

triisopropylsilane (TIS) and water accomplished the detach-

ment of the C-glycopeptides from the resin and concomitant

removal of all amino acid protecting groups (Scheme 18).

Preparative reversed phase HPLC of the crude C-glycopep-

tides gave the pure glycopeptides 54 (27%) and 55 (38%;

Scheme 18). Removal of the protecting groups of the carbo-

hydrate portions was achieved according to proven method-

s.^[5a,6,37]The pure target C-glycopeptides MUC1(22)-

Tyr¹⁷cFuc (56) and MUC1(22)Tyr¹⁷cGlc (57) were isolated

after preparative RP-HPLC (Scheme 18). Preliminary inves-

tigations show that these C-glycopeptides are recognized by

antisera induced in mice through synthetic MUC1-glycopep-

tide vaccines^[6,9b]and, thus, stimulate further immunological

studies.

The conversion of 49 to its Fmoc-protected building-block

50 was achieved by hydrogenation, O-acetylation, acidolytic

removal of the tert-butyl groups and introduction of the N-

Fmoc group (Scheme 17). The hydroboration, followed by

Suzuki coupling with the cis isomer of dehydro aminobutyric

acid derivative 23a, gave the C-lactosyl conjugate 51 in

74% yield (Scheme 17). In contrast to the hydrogenation of

the serine derivative 38, a diastereoselective hydrogenation

of 51 was not achieved. Heterogenous catalysis by using

Pd(OH)₂/C led to the formation of two diastereomers of the

lactosyl threonine derivative, which after transformation to

the Fmoc-protected C-lactosyl threonine building-block 52

can be separated by preparative HPLC (Scheme 17). The

chromatographic purification also allowed for the isolation

of two side-products 53a and 53b (Scheme 17) in which the

3-O-acetyl-group of the glucose moiety is missing as can be

concluded from the two-dimensional NMR spectra.

Conclusion

A modular synthesis of b-C-glycosyl amino acids derived

from serine, threonine, and tyrosine has been developed.

The key steps of this approach consist of the Petasis olefina-

Two of the Fmoc C-glycosyl amino acid building-blocks

were used in automated solid-phase syntheses to prepare C-

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Scheme 18. Solid-phase synthesis of the C-glycosylated MUC1-peptides (55 and 56) and subsequent deprotection to MUC1(22)Tyr¹⁷cFuc (58) and

MUC1(22)Tyr¹⁷cGlc (59).

tion of fully-protected carbohydrate lactones to give exo-gly-

cals, which subsequently are subjected to a reaction se-

quence of hydroboration and alkyl Suzuki cross-coupling.

This strategy was successfully applied to the coupling of

exo-glycals derived from various monosaccharides as well as

from lactose to alkenyl and aryl halides derived from the

amino acids to give the corresponding C-glycosyl amino acid

derivatives. Further functionalization of these coupling

products was achieved by regioselective opening of benzyli-

dene acetals. This step was beneficially carried out under

continuous flow conditions allowing for an easy optimization

and simple scale-up of the reaction. C-Glycosyl tyrosine

building-blocks were incorporated into glycopeptide sequen-

ces of the tumor-associated mucin MUC1 by solid-phase

synthesis. The obtained C-glycosyl glycopeptides are inter-

esting candidates for the utilization as long-term stable

mimics of natural tumor-associated glycopeptide antigens in

synthetic vaccines.

Experimental Section

General remarks: Solvents for moisture-sensitive reactions were distilled

and dried by standard procedures. Glycosylations were performed in

flame-dried glassware under argon. DMF (amine-free, for peptide syn-

thesis) and N-methylpyrrolidone (NMP) were purchased from Roth,

Karlsruhe, Germany, and Ac₂O and pyridine in p.a. quality from Acros

Chemicals. Reagents were purchased in the highest available commercial

quality and were used as supplied. Exceptions are noted. Fmoc-protected

amino acids were purchased from Orpegen Peptide Chemicals, Heidel-

berg, Germany. For solid-phase syntheses, alanine pre-loaded Tentagel R

resin (Rapp Polymere, Tꢀbingen, Germany) was employed. Reactions

were monitored by TLC with pre-coated silica gel (60 F₂₅₄) aluminium

plates (Merck KGaA, Darmstadt, Germany). Flash chromatography was

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performed with silica gel (230–400 mesh) from Merck. RP-HPLC analy-

ses were performed with a JASCO-HPLC system and a Phenomenex

phase was separated and the aquaeous phase was extracted three times

with ethyl acetate. The combined organic extracts were dried over

MgSO₄and the solvent was removed in vacuo to give quite pure N-tert-

butyloxycarbonyl-4-iodo-l-phenylalanine (7.32 g, 18.7 mmol, 89%) as a

yellow amorphous solid. tert-Butanol (6.05 g, 81.6 mmol), dicyclohexyl-

carbodiimide (DCC, 16.16 g, 78.3 mmol) and copper(I) chloride (135 mg,

1.36 mmol) were then stirred under exclusion of light for 3 days. The mix-

ture was then diluted with dry dichlororomethane (25 mL), and a solu-

tion of N-tert-butyloxycarbonyl-4-iodo-l-phenylalanine (7.2 g, 18.4 mmol)

in dry dichlororomethane (50 mL) was added. After 3 days, N,N’-dicyclo-

hexylurea was filtered off and silica gel was added to the filtrate. The

fine powder obtained after removal of the solvent in vacuo was subjected

to flash-chromatography (petroleum ether/ethyl acetate 10:1) to yield

pure 21 (5.28 g, 11.8 mmol, 64%) as a pale yellow viscous oil. R_f=0.43

(cyclohexane/ethyl acetate 9:1); [a]²_D³= +33.4 (c=1.0, CHCl₃); ¹H NMR

Luna-C18(2) (250ꢃ4.6 mm, 10 mm) column at a flow rate of 1 mL min^À1

.

Preparative HPLC separations were carried out with a JASCO-HPLC

System and and

a Phenomenex Luna-C18(2) (250ꢃ30 mm, 10 mm)

column at a flow rate of 20 mL min^À1. H₂O/MeCN mixtures were used as

solvents; if required TFA (0.1 %) was added. H, ¹³C and 2D NMR spec-

1

tra were recorded with a Bruker AC-300, a Bruker AM-400, a Bruker

AV-400, or a Bruker DRX 600 spectrometer. The chemical shifts are re-

ported in ppm relative to the signal of the deuterated solvent. Multiplici-

ties are given as: s (singlet), d (doublet), t (triplet), m_c(centered multip-

let), m (multiplet), and q (quartet). The index “b” indicates a broad

signal. Assignment of proton and carbon signals was achieved by means

of additional COSY, TOCSY, HSQC and HMBC experiments when

noted. The signals of the saccharide portions were denoted as follows: d-

glucosamine (GlcN), l-Fucose (Fuc), d-galactose (Gal), d-glucose (Glc)

and amino acid (AS). ESI and HR-ESI mass spectra were recorded with

3

[¹H-¹H-COSY] (400 MHz, CDCl₃): d=7.58 (d, 2H, J=8.2 Hz, H_Ar), 6.90

3

(d, 2H, J=8.2 Hz, H_Ar), 5.02 (d, 1H, J_NH;Ha=7.9 Hz, NH), 4.40 (m_c, 1H,

H^a), 3.01 (dd, 1H, ²J=13.9 Hz, ³J=6.2 Hz, H^ba), 2.94 (dd, 1H, ²J=

13.9 Hz, ³J=6.0 Hz, H^bb), 1.40, 1.39 ppm (2ꢃs, 18H, CH₃-Boc and CH₃-

tBu); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=170.54 (COOtBu),

154.90 (C=O-Boc), 137.20 (C_m), 136.04 (C_ipso), 131.40 (C_o), 92.14 (C_p),

82.21 (C_q, tBu), 79.66 (C_q, Boc), 54.49 (C^a), 37.91 (C^b), 28.20, 27.85 ppm

(CH₃, Boc and tBu).

a

Micromass Q TOF Ultima-3 spectrometer. Optical rotations were

measured at 546 and 578 nm with a PerkinElmer polarimeter 241. Micro-

wawe reactions were conducted in a CEM Discover microwave oven

using 10 mL vials. The interdigital glass mixer was provided by Mikroglas

Chemtech GmbH (Mainz, Germany).

Allyl 6-O-benzyl-3,4-O-endo,exo-benzylidene-b-d-galactopyranoside (2),

Allyl 2,6-di-O-benzyl-3,4-O-exo-benzylidene-b-d-galactopyranoside (3a),

allyl 2,6-di-O-benzyl-3,4-O-endo-benzylidene-b-d-galactopyranoside (3b),

allyl 6-O-benzyl-a-d-galactopyranoside (5), and allyl 6-O-benzyl-3,4-O-

endo,exo-benzylidene-a-d-galactopyranoside (6) were synthesized and

characterized as described in the Supporting Information.

(Z)-N-(tert-Butyloxycarbonyl)-b-bromo-a,b-dehydroalanine

tert-butyl

ester (22): l-Serine (7.00 g, 66.6 mmol) was dissolved in a solution of 1m

NaOH (125 mL) and 1,4-dioxane (63 mL) and cooled to 08C. Next, di-

tert-butyldicarbonate (Boc₂O) (16.3 g, 74.7 mmol, 1.1 equiv) was added.

The mixture was then stirred for 12 h at room temperature. Dioxane was

removed in vacuo and the aqueous solution extracted with diethyl ether

(100 m). The aqueous phase was adjusted to pH 7 by addition of 2n HCl.

Ethyl acetate was then added. Under vigorous stirring, the pH was then

adjusted to 2–2.5 with 2n HCl. The organic layer was separated and the

aqueous solution extracted three times with ethyl acetate. The combined

organic extracts were dried over MgSO₄and the solvent was removed in

vacuo to give N-tert-butyloxycarbonyl-l-serine (13.0 g, 63.1 mmol, 95%)

General procedures for the selective removal of anomeric allyl protecting

group are also given in the Supporting Information.

2,6-Di-O-benzyl-3,4-O-endo,exo-benzylidene-d-galactose (7): prepared

through one of the anomeric deprotection procedures. R_f=0.23 (cyclo-

hexane/ethyl acetate 2:1); R_f=0.11 (cyclohexane/ethyl acetate 3:1); MS

(ESI): m/z: 471.20 ([M]⁺, calcd: 471.18).

The syntheses of 2,6-Di-O-benzyl-3,4-O-endo,exo-benzylidene-d-galacto-

no-(1,5)-lactone (8), 2,3-Di-O-benzyl-4,6-O-benzylidene-d-galactono-

(1,5)-lactone (10), 2,3-Di-O-benzyl-4,6-O-benzylidene-d-glucono-(1,5)-

lactone (11), and 2,3,4-Tri-O-benzyl-l-fuconolactone (16) through oxida-

tion of the corresponding monosaccharides deprotected at the anomeric

function are also described in the Supporting Information.

as

a yellow oil. tert-Butanol (18.8 g, 254 mmol, 4.3 equiv), 40.2 g

(195 mmol, 2.2 equiv) of dicyclohexylcarbodiimide (DCC) and 563 mg

(5.7 mmol) of copper(I)-chloride were stirred under exclusion of light for

5

days. The mixture was then diluted with dry dichlororomethane

(120 mL), and solution of N-tert-butyloxycarbonyl-l-serine (12.0 g,

a

58.5 mmol) was added in dry dichlorormethane (80 mL) at 08C. The stir-

ring continued at room temperature for 1 h. N,N’-Dicyclohexylurea was

filtered off and the filtrate washed three times with a solution of saturat-

ed NaHCO₃(160 mL) and three times with brine (80 mL). The organic

layer was dried over MgSO₄, the solvent removed in vacuo, and the re-

maining oil subjected to flash chromatography (cyclohexane/ethyl acetate

4:1 to 2:1) to give 10.8 g (41.4 mmol, 71%) N-tert-butyloxycarbonyl-l-

serine-tert-butylester as a colorless oil; [a]²_D³= +60.0 (c=1.0, CHCl₃).

This product was dissolved in dichloromethane (220 mL) and triethyla-

mine (7.83 mL, 55.8 mmol, 1.3 equiv) was added. After addition of meth-

anesulfonyl chloride (4.3 mL, 55.7 mmol, 1.3 equiv), the reaction mixture

was stirred overnight, then diluted with ethyl acetate (220 mL) and

washed twice with water and once with a solution of saturated NaHCO₃.

After drying of the organic phase over MgSO₄the solvent was removed

in vacuo and the residue taken up in dry dichloromethane (150 mL) and

treated with DBU (7.6 mL, 50.8 mmol, 1.2 equiv) at room temperature

for 2 h. After dilution with dichloromethane (200 mL), it was sequentially

washed twice with a solution of 1m KHSO₄solution (200 mL) and once

with brine. The organic layer was dried over MgSO₄, the solvent removed

in vacuo and the residue subjected to flash chromatography (cyclohex-

ane/ethyl acetate 3:1) to give 8.72 g (35.8 mmol, 87%) N-(tert-butyloxy-

carbonyl)-a,b-dehydroalanine-tert-butylester as a yellow oil. R_f=0.57 (cy-

clohexane/ethyl acetate 3:1); ¹H NMR [¹H-¹H-COSY] (400 MHz,

CDCl₃): d=7.04 (s_b, 1H, NH-AS), 6.06 (s_b, 1H, H^ba-AS), 5.63 (d, 1H,

²J=1.3 Hz, H^bb -AS), 1.51, 1.47 ppm (2ꢃs, 18H, CH₃-Boc and CH₃-tBu).

N-Bromosuccinimide (6.79 g, 38.3 mmol, 1.1 equiv) was added to a solu-

2,3-Di-O-benzyl-5,6-O-benzylidene-d-glucono-(1,4)-lactone (14): Separa-

tion and characterization of the two isomers was successful after olefina-

tion of this lactones to the corresponding exo-glycals as described in

ref. [19].

N-tert-Butyloxycarbonyl-4-iodo-l-phenylalanine tert-butyl ester (21): l-

Phenylalanine (8.00 g, 48.4 mmol) and iodine (4.9 g, 19.3 gmmol,

0.40 equiv) were suspended in glacial acetic acid (50 mL). Potassium

iodate (2.3 g, 10.7 mmol, 0.22 equiv) and conc. sulfuric acid (6 mL) were

added. The reaction mixture was heated to 808C for 40 min. After cool-

ing to room temperature potassium periodate (0.5 g) were added, and the

mixture was again heated to 808C for a few minutes, during which time

the color of the flask content turned from red to light-orange. The sol-

vent was then removed in vacuo and water (100 mL) was added to the

residue. The aqueous phase was washed with diethyl ether (100 mL) and

dichlororomethane (100 mL) before it was stirred with activated charcoal

for several min and filtered. The filtrate was neutralized by addition of

KOH solution which led to precipitation of the iodated product. The

solid was filtered off and washed twice with water (100 mL) and once

with ethanol (50 mL). Recrystallization from acetic acid (150 mL) yielded

the intermediate product, which was co-evaporated twice with toluene to

remove residual acetic acid. The obtained 4-iodo-l-phenylalanine (6.14 g,

21.1 mmol, 44) was dissolved in a mixture of water (60 mL), 1,4-dioxane

(60 mL) and KOH (3.3 g, 58.8 mmol, 2.8 equiv). Di-tert-butyldicarbonate

(Boc₂O) (7.7 g, 35.2 mmol, 1.7 equiv) was added and the mixture stirred

for 22 h. Dioxane was removed in vacuo and the aqueous solution ex-

tracted with diethyl ether. By addition of 2n HCl the aqueous phase was

adjusted to a pH value of 7. Ethyl acetate was then added, and under vig-

orous stirring, the pH was adjusted to 2–2.5 with 2n HCl. The organic

tion

of

N-(tert-butyloxycarbonyl)-a,b-dehydroalanine-tert-butylester

(8.72 g, 35.8 mmol) in dichloromethane (130 mL). After stirring for 17 h,

(10.1 mL, 72.0 mmol, 2.0 equiv) of triethylamine were added and stirring

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C-Glycosyl Amino Acids

FULL PAPER

was continued for 3 h. The mixture was then diluted with dichlorome-

thane (260 mL). The organic phase was washed with water (200 mL) and

brine (250 mL). After drying the organic layer over MgSO₄and evapora-

tion of the solvent in vacuo the crude product was subjected to flash-

chromatography (cyclohexane/ethyl acetate 25:1) to yield 22 as a color-

less amorphous solid. R_f=0.17 (cyclohexane/ethyl acetate 25:1);

¹H NMR (400 MHz, CDCl₃): d=6.79 (s_b, 1H, H^b,), 6.13 (s_b, 1H, NH,),

1.49, 1.47 ppm (2ꢃs, 18H, CH₃, Boc and,tBu); ¹³C NMR [BB, DEPT]

(100.6 MHz, CDCl₃): d=161.15 (COOtBu), 151.78 (C=O, Boc), 133.74

(C^a), 108.41 (C^b), 82.77 (C_q, tBu), 81.30 (C_q, Boc), 28.06, 27.86 ppm (CH₃,

Boc and,tBu).

tained as a colorless amorphous solid. R_f=0.51 (cyclohexane/ethyl ace-

1

tate 2:1); H NMR (400 MHz, CDCl₃): d=5.95 (s_b, 1H, NH), 2.36 (s, 3H,

CH₃), 1.52, 1.45 (2ꢃs, 18H, CH₃, Boc and CH₃, tBu); ¹³C NMR [BB,

DEPT] (100.6 MHz, CDCl₃): d=162.82 (COOtBu), 152.66 (C=O-Boc),

127.39 (C^b), 120.46 (C^a), 82.62 (C_q, tBu), 81.07 (C_q, Boc), 28.12, 27.92

(CH₃, Boc and tBu), 25.67 ppm (CH₃).

General procedure for the formation of the C-glycoside linkage: Hydro-

boration: The exo-glycal was dissolved in dry dichloromethane and trans-

ferred to a Schlenk flask. The solvent was removed in vacuo and the re-

maining substance dried in high vacuum for 30 min, then set under an

argon atmosphere and cooled in a bath of liquid nitrogen until beginning

the condensation of argon. Cooling was removed immediately and the

N-tert-Butyloxycarbonyl-b-bromo-a,b-dehydro-2-aminobutyric acid tert-

butyl ester (23): Di-tert-butyldicarbonate (Boc₂O) was added to l-threo-

nine (6.20 g, 52.0 mmol) in a solution of 1m NaOH (150 mL) and 1,4-di-

oxane (75 mL, 16.8 g, 76.7 mmol, 1.5 equiv). The mixture was then stirred

for 3 days. Dioxane was removed in vacuo and the aqueous solution was

extracted with diethyl ether. By addition of 2n HCl, the pH was adjusted

to 2.5, and the solution extracted three times with a total volume of

500 mL of ethyl acetate. The combined organic layers were dried over

MgSO₄, the solvent was removed in vacuo and the resulting colorless oil

directly used in the next step. tert-Butanol (16.0 g, 216 mmol), dicyclohex-

ylcarbodiimide (DCC) (35.0 g, 105 mmol), and 380 mg (3.84 mmol) of

copper(I) chloride were stirred under exclusion of light for 3 days. The

mixture was diluted with dry dichloromethane (100 mL). A solution of

the crude N-tert-butyloxycarbonyl-l-threonine (11.6 g, max. 52.0 mmol)

in dry dichloromethane (150 mL) was added at room temperature. After

2.5 h, N,N’-dicyclohexylurea was filtered off, silica gel was added to the

filtrate and the solvent removed in vacuo. The remaining powder was

subjected to flash chromatography (cyclohexane/ethyl acetate 7:1) to give

N-tert-butyloxycarbonyl-l-threonine-tert-butylester (11.6 g, 42.1 mmol,

81%) as a viscous colorless oil. R_f=0.29 (toluene/ethyl acetate 4:1); R_f=

0.32 (cyclohexane/ethyl acetate 2:1); [a]²_D³=À27.8 (c=1.0, MeOH). The

formed N-tert-butyloxycarbonyl-l-threonine tert-butyl ester (11.2 g,

40.7 mmol) was dissolved in dichloromethane, cooled to À788C and

treated with triethylamine (113 mL, 82.4 g, 20 equiv). Slow addition of

thionyl chloride (4.43 mL, 7.26 g, 61.1 mmol, 1.5 equiv) was followed by

stirring at À788C for 2 h. The solvent and excess reagents were removed

in vacuo and the residue was co-distilled twice with toluene. The crude

sulfamidate was subjected to flash chromatography (petroleum ether/

ethyl acetate 10:1) to give a yellow oil (10.2 g), which was taken up in di-

corresponding amount of 0.5m 9-borabicycloACTHNUTRGNE[NUG 3.3.1]nonane (9-BBN) solu-

tion in tetrahydrfuran was added dropwise. The resulting suspension was

placed on a shaker and allowed to slowly warm up to room temperature.

The respective reaction time for the hydroboration step is given for each

substance.

Cross-coupling: In another Schlenk flask the amino acid, the base (3m

aq. K₃PO₄-solution or dry K₂CO₃, respectively) and the catalyst [PdCl₂-

AHCTUNGTERGUN(NN dppf)CH₂Cl₂] were mixed. The mixture was degassed by three cycles of

gentle evacuation and flushing with argon. The given volume of degassed

DMF was added. Directly afterwards, the hydroboration solution was

transferred to the cross-coupling reaction flask and the given amount of

water was added dropwise (only if dry K₂CO₃is used as base). The reac-

tion is monitored by TLC. The respective reaction time for the cross-cou-

pling step is given for each substance as well as the concrete work-up

procedure.

(Z)-N-(tert-Butyloxycarbonyl)-g-[exo-2,6-di-O-benzyl-3,4-O-benzylidene-

b-d-galactopyranosyl]-a,b-dehydro-2-aminobutyric acid tert-butyl ester

(24): Synthesis according to the general procedure, except for the follow-

ing deviations: the exo-glycal was first dissolved in THF (2 mL). 9-BBN

was added at 08C and, after warming to room temperature, the hydrobo-

ration-mixture was heated to 608C for 90 min. Hydroboration: 17a

(68 mg, 0.153 mmol); 0.5m 9-BBN solution (2.0 mL, 1.00 mmol,

6.5 equiv) in THF; reaction time: 2 h (thereof 1.5 h at 608C); cross-cou-

pling: 22 (60 mg, 0.186 mmol, 1.22 equiv); 3mK₃PO₄solution (0.1 mL,

0.30 mmol, 1.96 equiv); [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂]; (10 mg, 8 mol%); DMF

(0.3 mL); reaction time: 20 h. For the work-up, water was added and the

mixture was extracted four times with dichloromethane. The combined

organic extracts were dried over MgSO₄, the solvent was removed in

vacuo and the residue subjected to flash chromatography (cyclohexane/

ethylacetate 20:1 to 10:1) to yield 24 (10 mg, 14.5 mmol, 8%) as a color-

chloromethane (200 mL). After addition of 1,8-diazabicycloACTHNUTRGENUGN[5.4.0]undec-

7-ene (DBU) (3 mL, 20.1 mmol, 0.5 equiv) the mixture was stirred for

20 min. An equal volume of water was added and the pH adjusted to 4

by addition of citric acid. The organic phase was separated and the aque-

ous layer extracted twice with 100 mL of dichloromethane. The combined

organic extracts were dried over MgSO₄and the solvent was removed in

vacuo to give N-tert-butyloxycarbonyl-a,b-dehydro-2-aminobutyric acid

tert-butyl ester (8.40 g, 32.6 mmol, 80%) as a viscous yellow oil. R_f=0.50

(cyclohexane/ethylacetate 2:1); R_f=0.32 (petroleum ether/ethyl acetate

less amorphous solid. R_f=0.48 (cyclohexane/ethylacetate 2:1); [a]_D²³

=

À4.2 (c=1.0, CHCl₃); MS (ESI): m/z: 710.31 ([M+Na]⁺, calcd: 710.81);

¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=7.48–7.24 (m, 15H, H_Ar

,

Ph), 6.52 (s_b, 1H, NH), 6.45 (t_b, 1H, ³J=7.5 Hz, =CH), 6.07 (s, 1H, CH-

Ph), 4.95 (d, 1H, ²J=11.4 Hz, CH₂, Bn), 4.72 (d, 1H, ²J=11.5 Hz, CH₂,

Bn), 4.67 (d, 1H, ²J=12.1 Hz, CH₂, Bn), 4.54 (dd, 1H, ³J_H3;H2=6.1 Hz,

1

³J_H3;H4=5.6 Hz, H3, Gal), 4.51 (d, 1H, ²J=12.0 Hz, CH₂, Bn), 4.23 (dd,

10:1); H NMR (400 MHz, CDCl₃): d=6.53 (q, 1H, ³J=7.2 Hz, H^b), 6.03

3

1H, ³J_H4;H3=5.5 Hz, ³J_H4;H5=1.9 Hz, H4, Gal), 3.88 (dt, 1H, J_H5;H6a

=

(s_b, 1H, NH), 1.75 (d, 3H, ³J=7.2 Hz, CH₃), 1.46, 1.44 ppm (2ꢃs, 18H,

CH₃, Boc and tBu). ¹³C NMR [BB, DEPT] (100.6 MHz, CDCl₃): d=

163.95 (COOtBu), 153.02 (C=O, Boc), 130.07 (C^b), 127.69 (C^a), 81.35 (C_q,

tBu), 80.06 (C_q, Boc), 28.14, 27.97 (CH₃, Boc and tBu), 14.25 ppm (CH₃-

AS). N-Bromosuccinimide (6.32 g, 35.5 mmol, 1.1 equiv) ware added to a

solution of N-tert-butyloxycarbonyl-a,b-dehydro-2-aminobutyric acid tert-

butyl ester (8.30 g, 32.3 mmol) in dichloromethane (150 mL). After stir-

ring for 16 h, triethylamine (8.96 mL, 64.5 mmol, 2.0 equiv) was added

and the stirring was continued for 20 min. Silica gel was added, the sol-

vent was removed in vacuo, and the crude product was subjected to flash

chromatography (cyclohexane/ethyl acetate 20:1) to give the two separat-

ed isomers of 22. The Z isomer 23a (5.49 g, 16.3 mmol, 51%) was ob-

tained as a colorless amorphous solid. R_f=0.59 (cyclohexane/ethyl ace-

3

³J_H5;H6b=6.0 Hz, J_H5;H4=1.8 Hz, H5, Gal), 3.74 (d, 2H, ³J=6.0 Hz, H6ab,

Gal), 3.45 (dd, 1H, ³J_H2;H1=9.4 Hz, ³J_H2;H3=6.6 Hz, H2, Gal), 3.36 (m_c,

2

3

1H, H1, Gal), 2.79 (ddd, 1H, J=15.1 Hz, J_Hexo;a;=CH=7.4 Hz, J_Hexo;a;H1

=

2.4 Hz, CH_2a, exo), 2.42 (ddd, 1H, ²J=15.2 Hz, ³J_Hexo;b;=CH;=7.4 Hz,

³J_Hexo;b;H1=8.0 Hz, CH_2b, exo), 1.49, 1.47 ppm (2ꢃs, 18H, CH₃, Boc and

tBu); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=163.83 (COOtBu),

153.28 (C=O, Boc), 138.71, 137.98, 137.59 (C_ipso, Ph), 129.16, 128.40,

128.38, 128.35, 128.32, 128.31, 127.87, 127.66, 127.55, 126.19 (C_o;m;p, Ph,

C^b, C^a), 103.31 (CH-Ph), 81.28 (C_q, tBu), 80.55 (C3-Gal), 80.04 (C_q, Boc),

77.10 (C1-Gal), 76.16 (C2, Gal), 75.55 (C5, Gal), 74.15 (C4, Gal), 73.52,

72.67 (CH₂, Bn), 69.63 (C6, Gal), 30.82 (CH₂-exo), 28.19, 27.96 ppm

(CH₃, Boc and tBu).

1

(Z)-N-(tert-Butyloxycarbonyl)-g-[2,3-di-O-benzyl-4,6-O-benzylidene-b-d-

glucopyranosyl]-a,b-dehydro-2-aminobutyric acid tert-butyl ester (25):

Synthesis according to the general procedure, except for the following

deviations: the exo-glycal was first dissolved in THF (2 mL); 9-BBN was

added at 08C and then allowed to warm to room temperature. Hydrobo-

ration: 19 (158 mg, 0.355 mmol); 9-BBN solution (2.0 mL, 1.00 mmol,

tate 2:1); H NMR (400 MHz, CDCl₃): d=6.21 (s_b, 1H, NH), 2.50 (s, 3H,

CH₃), 1.51, 1.46 ppm (2ꢃs, 18H, CH₃, Boc and tBu); ¹³C NMR [BB,

DEPT] (100.6 MHz, CDCl₃): d=161.44 (COOtBu), 152.29 (C=O, Boc),

128.72 (C^b), 82.58 (C_q, tBu), 81.01 (C_q, Boc), 28.11, 27.88 (CH₃, Boc and

CH₃, tBu), 24.30 ppm (CH₃); The signal of C^acould not be detected due

to low intensity. The E isomer 23b (4.10 g, 12.2 mmol, 38%) was ob-

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H. Kunz et al.

2.8 equiv) of 0.5m in THF; reaction time: 20 h; cross-coupling: 22

(129 mg, 0.400 mmol, 1.13 equiv); 3mK₃PO₄solution (0.23 mL,

0.69 mmol, 1.95 equiv); [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂] (20 mg, 7 mol%); DMF

724.35); ¹H NMR [¹H-¹H-COSY] (400 MHz, CDCl₃): d=7.52–7.46 (m,

2H, H_Ar, Ph), 7.42–7.26 (m, 13H, H_Ar, Ph), 6.77 (s_b, 1H, NH), 5.59 (s,

1H, CH-Ph), 5.01 (d, 1H, ²J=11.2 Hz, CH₂, Bn), 4.97 (d, 1H, ²J=

11.1 Hz, CH₂, Bn), 4.79 (d, 1H, ²J=11.2 Hz, CH₂, Bn), 4.66 (d, 1H, ²J=

(0.6 mL); reaction time: 48 h. For the work-up, citric acid (250 mL) in

water (3 mL) were added. After stirring for 5 min, water (13 mL) and di-

chloromethane were added (15 mL). The mixture was neutralized by ad-

dition of sat. NaHCO₃solution, before ethanolamine (0.5 mL) were

added. The organic layer was separated and the aqueous phase extracted

three times with dichloromethane. The combined organic extracts were

dried over MgSO₄and the solvent was removed in vacuo. The remaining

residue was subjected to flash chromatography (cyclohexane/ethylacetate

10:1 to 2:1). The crude product was purified by preparative TLC (cyclo-

hexane/ethylacetate 5:2) to give 25 (14 mg, 20.4 mmol, 6%) as a colorless

2

3

11.0 Hz, CH₂, Bn), 4.35 (dd, 1H, J=10.3 Hz, J=4.8 Hz, H6a, Glc), 3.84

(t, 1H, ³J_H3;H2=³J_H3;H4=8.8 Hz, H3, Glc), 3.76–3.64 (m, 2H, H4, Glc

{3.72}, H6b, Glc {3.68}), 3.49 (m_c, 1H, H1, Glc), 3.41 (m_c, 1H, H5, Glc),

3

3.35 (dd, 1H, J_H2;H1=9.2 Hz, J_H2;H3=8.6 Hz, H2, Glc), 2.42–2.33 (m, 2H,

CH₂-exo), 1.94 (s, 3H, CH₃), 1.51, 1.46 ppm (2ꢃs, 18H, CH₃, Boc and

tBu); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=164.26 (COOtBu),

153.50 (C=O, Boc), 138.39, 137.73, 137.24 (C_ipso, Ph), 132.90 (C^b), 128.97,

128.50, 128.40, 128.26, 128.13, 128.06, 128.00, 127.73 (C_o;m;p, Ph), 126.99

(C^a), 125.97 (C_o;m;p, Ph), 101.16 (CH-Ph), 83.09 (C3, Glc), 82.23 (C4,

Glc), 81.09 (C2, Glc), 80.74 (C_q, tBu), 79.96 (C1, Glc), 79.70 (C_q, Boc),

75.32, 75.01 (CH₂, Bn), 70.08 (C5, Glc), 68.61 (C6, Glc), 37.06 (CH₂-exo),

28.25, 28.01 (CH₃, Boc and tBu), 19.20 ppm (CH₃).

amorphous solid. R_f=0.48 (cyclohexane/ethylacetate 2:1); [a]²_D³=À20.0

1

(c=0.5, CHCl₃); H NMR (400 MHz, CDCl₃): d=7.53–7.47 (m, 2H, H_Ar

,

3

Ph), 7.42–7.27 (m, 13H, H_Ar, Ph), 6.41 (t_b, 1H, J=7.0 Hz, =CH), 6.23 (s_b,

1H, NH), 5.59 (s, 1H, CH-Ph), 5.00 (d, 1H, ²J=11.1 Hz, CH₂, Bn), 4.92

(d, 1H, ²J=10.6 Hz, CH₂, Bn), 4.79 (d, 1H, ²J=11.1 Hz, CH₂, Bn), 4.60

(d, 1H, ²J=10.7 Hz, CH₂, Bn), 4.35 (dd, 1H, ²J=10.4 Hz, ³J=5.0 Hz,

(Z)-N-(tert-Butyloxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-b-

d-glucopyranosyl]-a,b-dehydrovaline tert-butyl ester (Z-27): Synthesis ac-

cording to the general procedure, except for the following details: the

exo-glycal was first dissolved in 2 mL of THF; 9-BBN was added at 08C.

Hydroboration: 19 (158 mg, 0.355 mmol); 0.5m 9-BBN solution (2.0 mL,

1.00 mmol, 2.8 equiv) in THF; reaction time: 20 h; cross-coupling: 23a

(128 mg, 0.381 mmol, 1.07 equiv); 3mK₃PO₄-solution (0.23 mL,

H6a, Glc), 3.83 (t, 1H, ³J_H3;H2=³J_H3;H4=9.0 Hz, H3, Glc), 3.75–3.64 (m,

3

2H, H4-Glc, H6b, Glc), 3.53 (m_c, 1H, H1, Glc), 3.44 (dt, 1H, J_H5;H6a

=

5.0 Hz, ³J_H5;H6b=³J_H5;H4=9.8 Hz, H5, Glc), 3.35 (dd, 1H, ³J_H2;H1=9.4 Hz,

³J_H2;H3=8.6 Hz, H2, Glc), 2.76–2.67 (m, 1H, CH_2a-exo), 2.42 (mc, 1H,

CH_2b-exo), 1.50,1.44 ppm (2ꢃs, 18H, CH₃-Boc and tBu).

0.69 mmol, 1.95 equiv); [PdCl₂ACHTNUGRTNEUNG(dppf)CH₂Cl₂] (20 mg , 7 mol%) DMF

N-(tert-Butyloxycarbonyl)-C-[exo-2,6-di-O-benzyl-3,4-O-benzylidene-b-

d-galactopyranosyl]-l-tyrosine tert-butyl ester (26): Synthesis according

to the general procedure, except for the following deviations: 9-BBN was

added at room temperature and then heated to 808C for 90 min. Hydro-

boration: 17a (45 mg, 0.101 mmol); 0.5m 9-BBN solution (2.0 mL,

1.00 mmol, 9.90 equiv) in THF; reaction time: 2 h (thereof 1.5 h at

808C); cross-coupling: 21 (110 mg, 0.246 mmol, 2.26 equiv); 3m K₃PO₄-

(0.6 mL); reaction time: 48 h. For the work-up, citric acid (250 mL) in

water (3 mL) were added. After stirring for 5 min, water (13 mL) and di-

chloromethane (15 mL) were added. The mixture was neutralized by ad-

dition of sat. NaHCO₃solution before ethanolamine (0.5 mL) were

added. The organic layer was separated and the aqueous phase extracted

three times with dichloromethane. The combined organic extracts were

dried over MgSO₄and the solvent was removed in vacuo. The remaining

residue was subjected to flash chromatography (petroleum ether/ethyl

acetate 10:1 to 2:1). The crude product was purified by preparative TLC

(cyclohexane/ethyl acetate 5:2) to give 6 mg (8.5 mmol, 2%) of (Z)-27 as

a colorless amorphous solid. R_f=0.60 (cyclohexane/ethyl acetate 2:1);

¹H NMR (400 MHz, CDCl₃): d=7.50–7.44 (m, 2H, H_Ar, Ph), 7.40–7.24

(m, 13H, H_Ar, Ph), 5.65 (s_b, 1H, NH), 5.54 (s, 1H, CH-Ph), 4.97 (d, 1H,

²J=11.1 Hz, CH₂, Bn), 4.94 (d, 1H, ²J=10.8 Hz, CH₂, Bn), 4.76 (d, 1H,

²J=11.2 Hz, CH₂, Bn), 4.62 (d, 1H, ²J=10.9 Hz, CH₂, Bn), 4.26 (dd, 1H,

²J=10.4 Hz, ³J=5.0 Hz, H6a, Glc), 3.81 (t, 1H, ³J_H3;H2=³J_H3;H4=9.0 Hz,

H3, Glc), 3.63 (t, 2H, ³J_H4;H3=³J_H4;H5=9.6 Hz, H4, Glc and ²J=³J=

solution (0.1 mL, 0.30 mmol, 2.97 equiv); [PdCl₂ACHTNUTRGNE(UGN dppf)CH₂Cl₂] (7 mg,

8 mol%); DMF (0.2 mL); reaction time: 20 h. For the work-up water and

dichloromethane were added and the organic layer was separated. The

aqueous phase was extracted three times with dichloromethane, the com-

bined organic extracts were dried over MgSO₄and the solvent was re-

moved in vacuo. The residue was subjected to flash-chromatography (pe-

troleum ether/ethylacetate 10:1 to 2:1). The crude product was purified

by preparative TLC (cyclohexane/ethyl acetate 3:1) to afford 26 (20 mg,

26.1 mmol, 26%) as a colorless amorphous solid. R_f=0.32 (petroleum

ether/ethyl acetate 2:1); ¹H NMR (400 MHz, CDCl₃): d=7.38–7.05 (m,

17H, H_Ar, Ph, H_Ar, Tyr), 6.93 (d, 2H, ³J=7.9 Hz, H_Ar, Tyr), 5.95 (s, 1H,

9.6 Hz, H6b, Glc), 3.56 (dt, 1H, ³J_H1;Hexo;a=3.2 Hz, ³J_H1;Hexo;b=³J_H1;H2

=

3

CH-Ph), 4.86 (d, 1H, J_NH;Ha=8.2 Hz, NH), 4.82 (d, 1H, ²J=11.6 Hz,

9.5 Hz, H1, Glc), 3.37–3.26 (m, 2H, H5, Glc {3.33}, H2, Glc {3.30}), 2.94

(d_b, 1H, ²J=13.5 Hz, CH_2a-exo), 2.72 (dd, 1H, ²J=13.5 Hz, ³J=9.8 Hz,

CH_2b-exo), 1.77 (s, 3H, CH₃), 1.45, 1.43 ppm (2ꢃs, 18H, CH₃, Boc and

tBu); MS (ESI): m/z: 724.32 ([M+Na]⁺, calcd: 724.35).

CH₂, Bn), 4.57 (d, 1H, ²J=11.6 Hz, CH₂, Bn), 4.44 (m_c, 1H, H3, Gal),

2

4.38 (d, 1H, J=12.0 Hz, CH₂, Bn), 4.35–4.26 (m, 2H, CH₂, Bn, H^a, Tyr),

4.13 (dd, 1H, ³J_H4;H3=5.5 Hz, ³J_H4;H5=1.6 Hz, H4, Gal), 3.70–3.54 (m,

3H, H6a, Gal, H6b, Gal, H5, Gal), 3.39–3.30 (m, 2H, H1, Gal, H2, Gal),

N-(tert-Butyloxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-b-d-glu-

copyranosyl]-l-tyrosine tert-butyl ester (28): Synthesis according to the

general procedure, except for the following deviations: 9-BBN was added

at 08C. An additional volume of THF (3 mL) was used to rinse the hy-

droboration flask and add this amount to the cross-coupling mixture. Hy-

droboration: 19 (256 mg, 0.576 mmol); 0.5m 9-BBN solution (3.0 mL,

1.50 mmol, 2.60 equiv) in THF; reaction time: 2.5 h; cross-coupling: 21

(308 mg, 0.689 mmol, 1.20 equiv); K₂CO₃(450 mg, 3.26 mmol,

2

3.09 (d_b, 1H, J=14.4 Hz, CH_2a-exo), 2.88 (m_c, 2H, 2ꢃH^b, Tyr), 2.65–2.55

(m, 1H, CH₂b-exo), 1.30, 1.27 ppm (2ꢃs, 18H, CH₃, Boc and tBu).

(E)-N-(tert-Butyloxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-b-

d-glucopyranosyl]-a,b-dehydrovaline tert-butyl ester (E-27): Synthesis ac-

cording to the general procedure, except for the following deviations: the

exo-glycal is first dissolved in THF (2 mL); 9-BBN was added at 08C.

Hydroboration: 19 (158 mg, 0.355 mmol); 9-BBN solution (0.5m, 2.0 mL,

1.00 mmol, 2.8 equiv) in THF; reaction time: 20 h; cross-coupling: 23b

(133 mg, 0.396 mmol, 1.14 equiv); 3mK₃PO₄solution (0.23 mL,

5.65 equiv); [PdCl₂ACHTNUGTRNEG(UN dppf)CH₂Cl₂] (15 mg, 3 mol%); DMF (6.0 mL);

water (0.2 mL); reaction time: 20 h. For work-up, water and dichlorome-

thane were added. The organic layer was separated and the aqueous

phase extracted three times with dichloromethane. The combined organic

extracts were dried over MgSO₄and the solvent was removed in vacuo.

The remaining residue was purified by flash-chromatography (petroleum

ether/ethyl acetate 4:1 to 2:1) to afford 28 (294 mg, 0.384 mmol, 67%) as

a colorless amorphous solid. R_f=0.49 (cyclohexane/ethyl acetate 2:1);

[a]²_D³= +1.7 (c=1.0, CHCl₃); ¹H NMR [1H1H-COSY] (400 MHz,

CDCl₃): d=7.51–7.47 (m, 2H, H_Ar, Ph), 7.42–7.27 (m, 13H, H_Ar, Ph),

0.69 mmol, 1.95 equiv); [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂] (20 mg, 7 mol%); DMF

(0.6 mL); reaction time: 48 h. For the work-up, citric acid (250 mL) in

water (3 mL) were added. After stirring for 5 min, water (13 mL) and di-

chloromethane (15 mL) were added. The mixture was neutralized by ad-

dition of sat. NaHCO₃solution before ethanolamine (0.5 mL) was added.

The organic layer was separated and the aqueous phase extracted three

times with dichloromethane. The combined organic extracts were dried

over MgSO₄and the solvent was removed in vacuo. The remaining resi-

due was subjected to flash-chromatography (petroleum ether/ethyl ace-

tate 10:1 to 2:1). The crude product was purified by preparative TLC (cy-

clohexane/ethyl acetate 5:2) to yield 15 mg (21.0 mmol, 6%) of (E)-27 as

a colorless amorphous solid. R_f=0.49 (cyclohexane/ethyl acetate 2:1);

[a]²_D³=À0.2 (c=0.5, CHCl₃); MS (ESI): m/z: 724.32 ([M+Na]⁺, calcd:

3

7.13 (d, 2H, J=8.1 Hz, H_Ar, Tyr), 7.08 (d, 2H, J=8.1 Hz, H_Ar, Tyr), 5.57

(s, 1H, CH-Ph), 5.04–4.98 (m, 2H, CH₂, Bn), 4.79 (d, 1H, ²J=11.2 Hz,

CH₂, Bn), 4.67 (d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.44 (m_c, 1H, H^a), 4.26

2

3

(dd, 1H, J=10.4 Hz, J=5.0 Hz, H6a, Glc), 3.87 (t, 1H, J_H3;H2=³J_H3;H4

=

8.8 Hz, H3, Glc), 3.73–3.63 (m, 2H, H6b, Glc {3.70}, H4, Glc {3.67}), 3.59

&

10

&

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Chem. Eur. J. 0000, 00, 0 – 0

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C-Glycosyl Amino Acids

FULL PAPER

3

(dt_b, 1H, J_H1;H2=³J_H1;Hexo;b=9.1 Hz, J_H1;Hexo;a=1.9 Hz H1-Glc), 3.39–3.30

(m, 2H, H2-Glc {3.36}, H5, Glc {3.34}), 3.14 (dd, 1H, ²J=14.3 Hz, ³J=

1.8 Hz, CH_2a-exo), 3.07–2.96 (m, 2H, 2ꢃH^b), 2.65 (dd, 1H, ²J=14.4 Hz,

³J=8.7 Hz, CH_2b-exo), 1.43, 1.39 ppm (2ꢃs,18H, CH₃, Boc and tBu);

¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=171.02 (COOtBu),

THF; reaction time: 2 h; cross-coupling: 23a (74 mg, 0.220 mmol,

1.25 equiv); K₂CO₃(73 mg, 0.528 mmol, 3.0 equiv); [PdCl₂ACTHNUGTRNEUNG(dppf)CH₂Cl₂]

(14 mg, 10 mol%); DMF (1.5 mL); water (0.1 mL); reaction time: 2 h.

For work-up, water and dichloromethane were added. The organic phase

was separated and the aqueous phase extracted three times with di-

chloromethane. The combined organic extracts were dried over MgSO₄

and the solvent was removed, in vacuo. The remaining residue was sub-

jected to flash chromatography (cyclohexane/ethyl acetate 5:1 to 4:1) to

yield 30 (106 mg, 0.151 mmol, 86%) as a colorless amorphous solid. R_f=

0.23 (cyclohexane/ethyl acetate 4:1); [a]²_D³= +37.2 (c=1.0, CHCl₃);

¹H NMR [¹H¹H-COSY, NOESY] (400 MHz, CDCl₃): d=7.60–7.56 (m,

2H, H_Ar, Ph), 7.43–7.26 (m, 13H, H_Ar, Ph), 5.49 (s, 1H, CH-Ph), 5.01 (d,

1H, ²J=11.0 Hz, CH₂, Bn), 4.79 (d, 1H, ²J=12.1 Hz, CH₂, Bn), 4.73 (d,

1H, ²J=12.1 Hz, CH₂, Bn), 4.65 (d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.26 (d_b,

1H, ²J=12.9 Hz, H6a, Gal), 4.23 (d, 1H, ³J_H4;H3=3.3 Hz, H4, Gal), 3.97

155.07 (C=O, Boc), 138.47, 138.17, 137.40 (C_ipso, Ph), 136.84, 134.25 (C_ipso

,

Tyr), 129.53, 129.29 (C_o;m, Tyr), 128.88, 128.47, 128.38, 128.23, 128.10,

127.92, 127.82, 127.69, 125.95 (_Co;m;p, Ph), 101.05 (CH-Ph), 83.37 (C3,

Glc), 82.56 (C4-Glc), 81.89 (C_q-tBu), 81.09 (C2-Glc), 80.49 (C1-Glc),

79.61 (C_q-Boc), 75.33, 75.01 (CH₂, Bn), 70.03 (C5, Glc), 68.91 (C6, Glc),

54.78 (C^a,Tyr), 38.19 (C^b, Tyr), 37.69 (CH₂-exo), 28.30, 27.92 ppm (CH₃,

Boc and tBu); MS (ESI): m/z: 788.43 ([M+Na]⁺, calcd: 788.38).

(Z)-N-(tert-Butyloxycarbonyl)-g-[exo-2,6-di-O-benzyl-3,4-O-benzylidene-

b-d-galactopyranosyl]-a,b-dehydrovaline tert-butyl ester (31b): Synthesis

according to the general procedure. Hydroboration: 17a (90 mg

(0.202 mmol); 0.5m 9-BBN solution (1.33 mL, 0.665 mmol, 3.28 equiv) in

THF; reaction time: 5 h; cross-coupling: 23a (75 mg, 0.223 mmol,

2

3

(d_b, 1H, J=12.7 Hz, H6b, Gal), 3.75 (t, 1H, J_H2;H1=³J_H2;H3=9.5 Hz, H2,

Gal), 3.64 (dd, 1H, ³J_H3;H2=9.4 Hz, ³J_H3;H4=3.5 Hz, H3, Gal), 3.75 (dt,

1H, ³J_H1;H2=³J_H1;Hexo;a=9.5 Hz, ³J_H1;Hexo;b=1.3 Hz, H1, Gal), 3.31 (s_b, 1H,

G5, Gal), 2.68 (dd, 1H, ²J=13.1 Hz, ³J=9.8 Hz, CH_2a-exo), 2.45 (d_b, 1H,

²J=13.1 Hz, CH_2b-exo), 1.98 (s, 3H, CH₃), 1.53 (s, 9H, CH₃, Boc),

1.42 ppm (s, 9H, CH₃, tBu); ¹³C NMR [BB, HSQC, HMBC] (100.6 MHz,

CDCl₃): d=164.55 (COOtBu), 153.86 (C=O, Boc), 138.19, 138.17, 137.99

(C_ipso, Ph), 132.48 (C^b), 128.90, 128.39, 128.38, 128.09, 127.98, 127.76,

127.73, 127.68 (C_o;m;p, Ph), 127.44 (C^a), 126.48 (C_o;m;p, Ph), 101.30 (CH-

Ph), 81.64 (C3, Gal), 80.75 (C_q, tBu), 79.31 (C_q, Boc), 78.62 (C1, Gal),

77.57 (C2, Gal), 75.39 (CH₂, Bn), 73.54 (C4, Gal), 71.07 (CH₂, Bn), 69.55

(C6, Gal), 69.25 (C5, Gal), 37.19 (CH₂-exo), 28.17, 27.99 (CH₃, Boc and

tBu), 19.06 ppm (CH₃); MS (ESI): m/z: 724.35 ([M+Na]⁺, calcd:

724.35); HRMS (ESI) m/z calcd for C₄₁H₅₁NO₉: 724.3462 [M+Na]⁺;

found: 724.3469; X-ray analysis: CCDC-920035 contains the supplemen-

tary crystallographic data for this paper. These data can be obtained free

of charge from The Cambridge Crystallographic Data Centre via

www.ccdc.cam.ac.uk/data_request/cif.

1.10 equiv); K₂CO₃(60 mg, 0.434 mmol, 2.15 equiv); [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂]

(15 mg, 9 mol%); DMF (1.5 mL); water (0.1 mL); reaction time: 4 h. For

the work-up, water and dichloromethane were added. A pH of 7 was ad-

justed by addition of 2n hydrochloric acid. After phase separation the

aqueous phase was extracted four times with dichloromethane. The com-

bined organic solutions were dried over MgSO₄and the solvent was re-

moved in vacuo. The remaining residue was subjected to flash chroma-

tography (cyclohexane/ethyl acetate 10:1) to yield 28 (68 mg, 96.9 mmol,

48%) as a colorless amorphous solid. R_f=0.40 (petroleum ether/ethyl

acetate 3:1); [a]_D²³=À23.1 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY]

(400 MHz, CDCl₃): d=7.47–7.22 (m, 15H, H_Ar, Ph), 6.09 (s, 1H, CH-Ph),

4.96 (d, 1H, ²J=11.7 Hz, CH₂, Bn), 4.74 (d, 1H, ²J=11.7 Hz, CH₂, Bn),

4.68 (d, 1H, ²J=12.0 Hz, CH₂, Bn), 4.51 (t, 1H, ³J_H3;H2=³J_H3;H4=6.2 Hz,

H3, Gal), 4.44 (d, 1H, ²J=12.1 Hz, CH₂, Bn), 4.18 (dd, 1H, J_H4;H3

=

3

5.5 Hz, ³J_H4;H5=1.7 Hz, H4, Gal), 3.83 (m_c, 1H, H5, Gal), 3.74–3.62 (m,

2H, H6ab, Gal), 3.42 (dd, 1H, ³J_H2;H1=9.3 Hz, ³J_H2;H3=6.7 Hz, H2, Gal),

3.29 (m_c, 1H, H1, Gal), 2.47 (d_b, 2H, ³J=5.3 Hz, CH₂-exo), 1.97 (s, 3H,

CH₃), 1.48, 1.42 ppm (2ꢃs, 18H, CH₃, Boc and tBu).

N-(tert-Butyloxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-b-d-gal-

actopyranosyl]-l-tyrosine tert-butyl ester (29): Synthesis according to the

general procedure. Hydroboration: 18 (78 mg, 0.176 mmol); 0.5m 9-BBN

solution (1.06 mL, 0.530 mmol, 3.0 equiv) in THF; reaction time: 2 h;

cross-coupling: 21 (98 mg, 0.219 mmol, 1.24 equiv); K₂CO₃(73 mg,

(E)-N-(tert-Butyloxycarbonyl)-g-[exo-2,6-di-O-benzyl-3,4-O-benzylidene-

b-d-galactopyranosyl]-a,b-dehydrovaline tert-butyl ester (31a): Synthesis

according to the general procedure. Hydroboration: 17a (90 mg,

0.202 mmol); 0.5m 9-BBN solution (1.33 mL, 0.665 mmol, 3.28 equiv) in

THF; reaction time: 5 h; cross-coupling: 23b (75 mg, 0.223 mmol,

0.528 mmol, 3.0 equiv); [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂] (14 mg, 10 mol%); DMF

(1.5 mL); water (0.1 mL); reaction time: 2 h. For the work-up, water and

dichloromethane were added. The organic phase was separated and the

aqueous phase extracted three times with dichloromethane. The com-

bined organic extracts were dried over MgSO₄and the solvent was re-

moved in vacuo. The remaining residue was subjected to flash chroma-

tography (cyclohexane/ethyl acetate 5:1 to 4:1) to afford 29 (98 mg,

0.128 mmol, 73%) as a colorless amorphous solid. R_f=0.25 (cyclohexane/

1.10 equiv); K₂CO₃(60 mg, 0.434 mmol, 2.15 equiv); [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂]

(15 mg, 9 mol%); DMF (1.5 mL); water (0.2 mL); reaction time: 4 h. For

work-up, water and dichloromethane were added. A pH of 7 was adjust-

ed by addition of 2n hydrochloric acid. The organic phase was separated,

extracted four times with dichloromethane, and the combined organic ex-

tracts were dried over MgSO₄. The solvent was removed in vacuo and

the remaining residue subjected to flash chromatography (cyclohexane/

ethyl acetate 10:1) to give 28 (70 mg, 0.100 mmol, 49%) as a colorless

amorphous solid. R_f=0.30 (petroleum ether/ethyl acetate 3:1); ¹H NMR

1

ethyl acetate 1:1); [a]_D²³= +24.0 (c=1.0, CHCl₃); H NMR [¹H¹H-COSY]

(400 MHz, CDCl₃): d=7.59–7.53 (m, 2H, H_Ar, Ph), 7.44–7.26 (m, 15H,

H_Ar, Ph, H_Ar, Tyr), 7.08 (d, 2H, ³J=8.1 Hz, H_Ar, Tyr), 5.49 (s, 1H, CH-

Ph), 5.03 (d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.77 (d, 1H, ²J=12.1 Hz, CH₂,

Bn), 4.72 (d, 1H, ²J=12.2 Hz, CH₂, Bn), 4.64 (d, 1H, ²J=11.0 Hz, CH₂,

Bn), 4.46 (mc, 1H, H^a, Tyr), 4.24 (dd, 1H, ²J=12.2 Hz, ³J=1.2 Hz, H6a,

Gal), 4.20 (d, 1H, ³J_H4;H3=2.9 Hz, H4, Gal), 3.95 (dd, 1H, ²J=12.2 Hz,

[¹H¹H-COSY, NOESY] (400 MHz, CDCl₃): d=7.50–7.27 (m, 15H, H_Ar

,

Ph), 6.07 (s, 1H, CH-Ph), 5.67 (s_b, 1H, NH), 4.97 (d, 1H, ²J=11.6 Hz,

CH₂, Bn), 4.73 (d, 1H, ²J=11.6 Hz, CH₂, Bn), 4.63–4.50 (m, 3H, CH₂,

Bn, H3, Gal), 4.26 (dd, 1H, ³J_H4;H3=5.5 Hz, ³J_H4;H5=1.8 Hz, H4, Gal),

3.84 (m_c, 1H, H5, Gal), 3.79–3.70 (m, 2H, H6ab Gal), 3.53 (m_c, 1H, H1,

Gal), 3.44 (dd, 1H, ³J_H2;H1=9.1 Hz, ³J_H2;H3=6.5 Hz, H2, Gal), 3.23 (d_b,

3

³J=1.6 Hz, H6b, Gal), 3.76 (t, 1H, J_H2;H1=³J_H2;H3=9.2 Hz, H2, Gal), 3.67

(dd, 1H, ³J_H3;H2=9.2 Hz, ³J_H3;H4=3.5 Hz, H3, Gal), 3.49 (m_c, 1H, H1,

Gal), 3.26–3.19 (m, 2H, CH_2a-exo, H5, Gal), 3.06–3.02 (m, 2H, 2ꢃH^b),

2.87 (dd, 1H, ²J=14.3 Hz, ³J=8.4 Hz, CH_2b-exo), 1.44, 1.40 ppm (2ꢃs,

18H, CH₃, Boc and tBu); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃):

d=171.01 (COOtBu), 155.08 (C=O, Boc), 138.59, 138.25, 138.02, 137.52

(C_ipso, Ph, C_ipso, Tyr), 133.89 (C_ipso, Tyr), 129.84, 129.14 (C_o;m, Tyr), 128.91,

128.37, 128.34, 128.14, 128.11, 127.89, 127.72, 127.66, 127.62, 126.44,

126.40 (C_o;m;p, Ph), 101.20 (CH-Ph), 81.94 (C3, Gal), 81.86 (C_q, tBu),

79.94 (C1-Gal), 79.55 (C_q-Boc), 77.22 (C2-Gal), 75.26 (CH₂-Bn), 73.76

(C4-Gal), 71.02 (CH₂, Bn), 69.69 (C6, Gal), 69.16 (C5, Gal), 54.75 (C^a,

Tyr), 38.03 (C^b, Tyr), 37.39 (CH₂-exo), 28.27, 27.90 (CH₃, Boc and tBu);

MS (ESI): m/z: 788.38 ([M+Na]⁺, calcd: 788.38); HR-MS (ESI): m/z

calcd for C₄₆H₅₅NO₉: 788.3775 [M+Na]⁺; found: 788.3765.

1H, ²J=13.1 Hz, CH_2a-exo), 2.57 (dd, 1H, J=13.9 Hz, J=6.5 Hz, CH_2b

-

2

3

exo), 1.86 (s, 3H, CH₃), 1.50, 1.48 ppm (2ꢃs, 18H, CH₃-Boc and tBu);

¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=164.06 (COOtBu),

153.47 (C=O, Boc), 138.78, 138.24, 137.91 (C_ipso, Ph), 129.12 (C^b), 128.37,

128.27, 128.24, 128.18, 127.63, 127.43, 126.23 (C_o;m;p, Ph), 125.16 (C^a),

103.31 (CH-Ph), 81.07 (C_q, tBu), 80.57 (C3, Gal), 79.78 (C_q, Boc), 77.33,

77.25 (C1-Gal, C2, Gal), 75.58 (C5, Gal), 74.22 (C4, Gal), 73.43, 72.46

(CH₂, Bn), 69.86 (C6, Gal), 36.56 (CH₂-exo), 28.21, 27.98 (CH₃, Boc and

tBu), 20.05 ppm (CH₃).

(Z)-N-(tert-Butyloxycarbonyl)-g-[2,3-di-O-benzyl-4,6-O-benzylidene-b-d-

galactopyranosyl]-a,b-dehydrovaline tert-butyl ester (30): Synthesis ac-

cording to the general procedure. Hydroboration: 18 (78 mg,

0.176 mmol); 0.5m 9-BBN solution (1.06 mL, 0.530 mmol, 3.0 equiv) in

N-Fluorenyl-9-methoxycarbonyl-4-iodo-l-phenylalanine methyl ester

(32): l-Phenylalanine (16.0 g, 96.9 mmol) was transformed to 4-iodo-l-

Chem. Eur. J. 2013, 00, 0 – 0

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H. Kunz et al.

phenylalanine as described above and the obtained crude product sus-

pended in methanol (250 mL). The suspension was cooled to 08C and thi-

onylchloride (6 mL, 82.7 mmol) was added. Stirring was continued for 1 h

before the pale-yellow mixture was gently heated at reflux for 2 h. The

solvent and excess of reagent were removed in vacuo. The residue was

co-evaporated once with methanol and dissolved in hot methanol

(50 mL). Addition of diethyl ether (250 mL) precipitated 4-iodo-l-phe-

nylalanine-methylester (13.8 g, 40.4 mmol, 42%) as a fine powder, which

was dried in vacuo. [a]²_D³= +12.8 (c=1.0, MeOH); ¹H NMR (400 MHz,

MeOD): d=7.50 (d, 2H, ³J=8.0 Hz, H_Ar), 6.84 (d, 2H, ³J=8.1 Hz, H_Ar),

4.02 (m_c, 1H, H^a), 3.61 (s, 3H, OCH₃), 3.06–3.00 ppm (m, 2H, 2ꢃH^b);

¹³C NMR [BB] (100.6 MHz, CDCl₃): d=168.47 (COOMe), 137.74 (C_m),

133.02 (C_ipso), 130.98 (C_o), 93.03 (C_p), 62.83 (OCH₃), 53.37 (C^a),

35.26 ppm (C^b). This substance (3.00 g, 8.78 mmol) was dissolved in 1,4-

dioxane (8 mL) and water (17 mL). Na₂CO₃(1.70 g, 16.0 mmol,

1.8 equiv) was added and the clear mixture cooled to 08C. FmocOSu

(3.25 g, 9.63 mmol, 1.1 equiv) in 1,4-dioxane (18 mL) was added, the cool-

ing removed and the stirring continued for 2 h. After the addition of ace-

tone (20 mL) the mixture was stirred overnight at room temperature.

Water and dichloromethane were added and the organic layer was sepa-

rated. The aqueous phase was extracted twice with dichloromethane and

the solvent was removed in vacuo from the combined organic solutions.

The residue was co-evaporated twice with toluene and then dissolved in

ethyl acetate (250 mL) heated at reflux. Addition of petroleum ether

(160 mL) led to precipitation of the product as a white powder, which

was dried in high vacuum to give 32 (3.68 g, 6.98 mmol, 79%). R_f=0.54

(cyclohexane/ethyl acetate 1:1); [a]²_D³= +10.1 (c=1.0, CHCl₃); ¹H NMR

20.2 mmol, 1.2 equiv) was slowly added and the solution stirred for

10 min before 5.28 mL (38.0 mmol, 2.2 equiv) of triethylamine were in-

troduced through a syringe. Stirring was continued overnight at room

temperature. An equivalent volume of water was added and the organic

layer separated. The aqueous phase was extracted twice with dichlorome-

thane and the combined organic extractives were dried over MgSO₄. The

solvent was removed in vacuo and the residue subjected to flash-chroma-

tography (cyclohexane/ethyl acetate 5:1) to afford the dehydro amino

acid (5.26 g, 16.3 mmol, 93%) as a colorless solid. R_f=0.39 (cyclohexane/

ethyl acetate 5:1); MS (ESI): m/z: 346.12 ([M+Na]⁺, calcd: 346.11);

¹H NMR (400 MHz, CDCl₃): d=7.79 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc),

3

7.61 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.42 (t, 2H, J=7.3 Hz, H_Ar, Fmoc),

7.37–7.31 (m, 2H, H_Ar, Fmoc), 7.28 (s_b, 1H, NH), 6.26 (s_b, 1H, H^ba), 5.81

(s, 1H, H^bb), 4.47 (d, 2H, ³J=7.0 Hz, CH₂, Fmoc), 4.27 (t_b, 1H, ³J=

6.9 Hz, CH, Fmoc), 3.86 ppm (s, 3H, OCH₃); ¹³C NMR [BB, DEPT]

(100.6 MHz, CDCl₃): d=164.23 (COOMe), 153.13 (C=O, Fmoc), 143.57

(C1a, Fmoc, C8a, Fmoc), 141.27 (C4a, Fmoc, C5a, Fmoc), 130.88 (C^a),

127.77 (C3, Fmoc, C6, Fmoc), 127.09 (C2, Fmoc, C7, Fmoc), 124.95 (C1,

Fmoc, C8, Fmoc), 120.02 (C4, Fmoc, C5, Fmoc), 106.18 (C^b), 67.12 (CH₂,

Fmoc), 52.96 (OCH₃), 46.91 ppm (CH, Fmoc); MS (ESI): m/z: 364.15

([M+Na]⁺, calcd: 364.12). This dehydro alanine (2.04 g, 6.31 mmol) was

dissolved in dry dichloromethane and cooled to À788C. A solution of

bromine (4.5 g) in dry dichloromethane (8.8 mL) was added dropwise

through a syringe until the red color remained. Stirring was continued at

this temperature for additional 15 min before triethylamine (0.9 mL,

6.49 mmol, 1.0 equiv) in dichloromethane (2.1 mL) was added. Stirring at

À788C was continued for 1 h. Cooling was removed and brine was added

at room temperature. By addition of 2n hydrochloric acid a pH of 1 was

adjusted, the organic layer was separated and the aqueous phase extract-

ed four times with dichloromethane. Silica gel was added, the solvent re-

moved in vacuo and the remaining powder subjected to flash chromatog-

raphy (cyclohexane/ethyl acetate 10:1 to 5:1 to 2:1) to give 33a (2.22 g,

5.52 mmol, 88%) as a colorless amorphous solid. R_f=0.36 (cyclohexane/

ethyl acetate 2:1); ¹H NMR (400 MHz, CDCl₃): d=7.78 (d, 2H, ³J=

3

(400 MHz, CDCl₃): d=7.78 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.63–7.53 (m,

4H, H_Ar, Fmoc), 7.42 (t, 2H, ³J=7.4 Hz, H_Ar, Fmoc), 7.35–7.30 (m, 2H,

3

³J=7.4 Hz, H_Ar), 6.82 (d, 2H, ³J=8.1 Hz, H_Ar), 5.27 (d, 2H, J_NH;Ha

=

8.1 Hz, NH), 4.65 (m_c, 1H, H^a), 4.48 (dd, 1H, ²J=10.6 Hz, ³J=7.1 Hz,

2

3

CH_2a, Fmoc), 4.37 (dd, 1H, J=10.6 Hz, J=6.8 Hz, CH_2b, Fmoc), 4.21 (t,

1H, ³J=6.8 Hz, CH, Fmoc), 3.73 (s, 3H, OCH₃), 3.09 (dd, 1H, ²J=

13.9 Hz, ³J=5.7 Hz, H^ba), 3.02 ppm (dd, 1H, ²J=13.9 Hz, ³J=5.9 Hz,

H^bb); ¹³C NMR [BB, DEPT] (100.6 MHz, CDCl₃): d=171.58 (COOMe),

155.43 (C=O, Fmoc), 143.74, 143.58 (C1a, Fmoc, C8a, Fmoc), 141.30

(C4a, Fmoc, C5a, Fmoc), 137.61 (C_m), 135.33 (C_ipso), 131.25 (C_o), 127.72

(C3, Fmoc, C6, Fmoc), 127.04 (C2, Fmoc, C7, Fmoc), 125.01, 124.94 (C1,

Fmoc, C8-Fmoc), 119.99, 119.96 (C4, Fmoc, C5, Fmoc), 92.67 (C_p, Ph),

66.83 (CH₂, Fmoc), 54.46 (C^a), 52.46 (OCH₃), 47.09 (CH, Fmoc),

37.64 ppm (C^b).

3

7.5 Hz, H_Ar, Fmoc), 7.62 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.42 (t, 2H, J=

7.4 Hz, H_Ar, Fmoc), 7.36–7.30 (m, 2H, H_Ar, Fmoc), 7.15 (s_b, 1H, NH),

6.54 (s_b, 1H, H^b), 4.48 (d, 2H, ³J=7.2 Hz, CH₂, Fmoc), 4.27 (t_b, 1H, ³J=

7.1 Hz, CH, Fmoc), 3.81 ppm (s, 3H, OCH₃); ¹³C NMR [BB, HSQC]

(100.6 MHz, CDCl₃): d=162.32 (COOMe), 152.85 (C=O, Fmoc), 143.39

(C1a, Fmoc, C8a, Fmoc), 141.18 (C4a, Fmoc, C5a, Fmoc), 131.82 (C^a),

127.69 (C3, Fmoc, C6, Fmoc), 127.01 (C2, Fmoc, C7, Fmoc), 124.95 (C1,

Fmoc, C8, Fmoc), 119.91 (C4, Fmoc, C5, Fmoc), 112.84 (C^b), 67.76 (CH₂,

Fmoc), 52.81 (OCH₃), 46.81 ppm (CH, Fmoc); MS (ESI): m/z: 424.02

([M+Na]⁺, calcd: 424.02);

(Z)-N-(Fluoren-9-ylmethoxycarbonyl)-b-bromo-a,b-dehydroalanine

methyl ester (33a): l-Serine (8.00 g, 76.1 mmol) was dissolved in metha-

nol (75 mL), and thionylchloride (5.5 mL, 75.8 mmol, 1.0 equiv) were

added. The mixture was heated at reflux for 4 h and thereafter concen-

trated in vacuo to 1/5 of its volume. Toluene was added and the solvent

evaporated in vacuo. The obtained crude l-serine-methyl ester, a white

powder, was dissolved in NaHCO₃solution (14.8 g in 160 mL water) and

cooled in an ice bath. FmocOSu (24.0 g, 71.2 mmol, 1.0 equiv) in 1,4-di-

oxane (160 mL) was added within 30 min and stirring was continued

overnight. The solution was extracted four times with ethyl acetate

(100 mL) and the combined organic extracts were sequentially washed

with 0.2n hydrochloric acid (80 mL) and brine. After drying the organic

layer over MgSO₄the solvent was removed in vacuo to quantitatively

give the Fmoc-protected serine-methyl ester as a colorless solid. R_f=0.21

(cyclohexane/ethyl acetate 1:1); [a]²_D³= +7.1 (c=1.0, CHCl₃); ¹H NMR

(Z)-N-(Fluoren-9-ylmethoxycarbonyl)-b-iodo-a,b-dehydroalanine methyl

ester (33b): In a sealed microwave flask, a mixture of N-(fluoren-9-ylme-

thoxycarbonyl)-a,b-dehydroalanine methyl ester (400 mg, 1.20 mmol) in

THF (4.7 mL), K₂CO₃(264 mg, 1.44 mmol, 1.2 equiv) and iodine

(360 mg, 1.42 mmol, 1.2 equiv) were heated to 808C for 160 min. Water

was added and the solution saturated with NaCl, extracted three times

with dichloromethane, and the combined organic extracts were dried

over MgSO₄. After addition of silica gel the solvent was removed in

vacuo, and the residue subjected to flash chromatography (cyclohexane/

ethyl acetate 5:1 to 2:1) to yield 33b (90 mg, 0.200 mmol, 17%) as a col-

orless amorphous solid. R_f=0.35 (cyclohexane/ethyl acetate 12:5);

¹H NMR (400 MHz, CDCl₃): d=7.77 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc),

7.61 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.44–7.39 (m, 3H, H_Ar, Fmoc, H^b),

3

(400 MHz, CDCl₃): d=7.75 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.63–7.56 (m,

3

2H, H_Ar, Fmoc), 7.39 (t, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.30 (t, 2H, ³J=

7.36–7.30 (m, 2H, H_Ar, Fmoc), 6.42 (s_b, 1H, NH), 4.47 (d, 2H, J=7.2 Hz,

CH₂, Fmoc), 4.28 (t_b, 1H, ³J=7.2 Hz, CH, Fmoc), 3.80 ppm (s, 3H,

OCH₃); ¹³C NMR [BB, HSQC, HMBC] (100.6 MHz, CDCl₃): d=161.55

(COOMe), 152.87 (C=O, Fmoc), 143.42 (C1a, Fmoc, C8a, Fmoc), 141.23

(C4a, Fmoc, C5a, Fmoc), 136.69 (C^a), 127.75 (C3, Fmoc, C6, Fmoc),

127.08 (C2, Fmoc, C7, Fmoc), 125.03 (C1, Fmoc, C8, Fmoc), 119.98 (C4,

Fmoc, C5, Fmoc), 87.79 (C^b), 67.83 (CH₂, Fmoc), 52.98 (OCH₃),

46.86 ppm (CH, Fmoc); MS (ESI): m/z: 471.99 ([M+Na]⁺, calcd:

472.00).

3

7.4 Hz, H_Ar, Fmoc), 5.98 (d, 1H, J_NH;Ha=8.0 Hz, NH), 4.49–4.34 (m, 3H,

CH₂, Fmoc, H^a), 4.21 (t_b, 1H, ³J=6.9 Hz, CH, Fmoc), 3.98 (d_b, 1H, ²J=

10.4 Hz, H^ba, Ser), 3.88 (d_b, 1H, ²J=10.5 Hz, H^bb, Ser), 3.75 (s, 3H,

OCH₃), 3.00 ppm (s_b, 1H, OH); ¹³C NMR [BB, DEPT] (100.6 MHz,

CDCl₃): d=171.07 (COOMe), 156.25 (C=O, Fmoc), 143.53 (C1a, Fmoc,

C8a, Fmoc), 141.17 (C4a, Fmoc, C5a, Fmoc), 127.62 (C3, Fmoc, C6,

Fmoc), 126.97 (C2, Fmoc, C7, Fmoc), 124.96 (C1, Fmoc, C8, Fmoc),

119.88 (C4, Fmoc, C5, Fmoc), 66.91 (CH₂-Fmoc), 62.92 (C^b-Ser), 55.93

(C^a-Ser), 52.60 (OCH₃), 46.94 ppm (CH-Fmoc). Fmoc-protected serine-

methyl ester (6.00 g, 17.6 mmol) was dissolved in 200 mL of dichlorome-

thane and cooled to 08C. Methansulfonic acid chloride (1.56 mL,

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-

b-d-glucopyranosyl]-l-tyrosine methyl ester (36): Synthesis according to

the general procedure. Hydroboration: 19 (370 mg, 0.832 mmol), 0.5m 9-

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C-Glycosyl Amino Acids

FULL PAPER

BBN solution (5.0 mL, 2.50 mmol, 3.00 equiv) in THF; reaction time: 1 h

15 min; cross-coupling: 32 (439 mg, 0.832 mmol, 1.00 equiv); K₂CO₃

(345 mg, 2.50 mmol, 3.00 equiv), [PdCl₂ACTHNUTRGNEN(UG dppf)CH₂Cl₂] (68 mg, 10 mol%),

66.82 (CH₂, Fmoc), 54.71 (C^a), 52.24 (OCH₃), 47.10 (CH, Fmoc), 37.75

(C^b, Tyr), 37.41 (CH₂-exo), 17.16 ppm (C6, Fuc); MS (ESI): m/z: 854.37

([M+Na]⁺, calcd: 854.37).

DMF (6.0 mL); water (1.5 mL); reaction time: 20 h. For the work-up,

water and dichloromethane were added. The organic phase was separat-

ed and the aqueous phase extracted three times with dichloromethane.

The combined organic extracts were dried over MgSO₄and the solvent

was removed in vacuo. The remaining residue was subjected to flash-

chromatography (cyclohexane/ethyl acetate 4:1 to 2:1) to give 520 mg

(0.615 mmol, 74%) of 36 as a pale yellow amorphous solid. R_f=0.25 (pe-

troleum ether/ethyl acetate 2:1); MS (ESI): m/z: 868.45 ([M+Na]⁺,

calcd: 868.35); [a]_D²³= +7.0 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY]

(400 MHz, CDCl₃): d=7.77 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.57 (t, 2H,

³J=7.9 Hz, H_Ar, Fmoc), 7.51–7.46 (m, 2H, H_Ar, Fmoc, H_Ar, Ph), 7.43–7.27

N-(Fluoren-9-ylmethoxycarbonyl)-C-[exo-2,6-di-O-benzyl-3,4-O-benzyli-

dene-b-d-galactopyranosyl]-l-tyrosine methyl ester (34): Synthesis ac-

cording to the general procedure. Hydroboration: 17a (90 mg,

0.202 mmol); 0.5m 9-BBN solution (1.33 mL, 0.665 mmol, 3.3 equiv) in

THF; reaction time: 5 h; cross-coupling: 32 (118 mg, 0.224 mmol,

1.11 equiv) K₂CO₃(60 mg, 0.434 mmol, 2.15 equiv); [PdCl₂ACTHNUGTRNEUNG(dppf)CH₂Cl₂]

(15 mg, 9 mol%); DMF (1.5 mL); water (0.2 mL); reaction time: 16 h.

For the work-up, water and dichloromethane were added and a pH of 7

was adjusted by the addition of 2n hydrochloric acid. The organic phase

was separated and the aqueous phase extracted four times with dichloro-

methane. The combined organic extracts were dried over MgSO₄and the

solvent was removed in vacuo and high vacuum. The remaining residue

was subjected to flash chromatography (cyclohexane/ethyl acetate 15:2)

to yield 34 (72 mg, 85 mmol, 42%) as a colorless amorphous solid. R_f=

0.17 (petroleum ether/ethyl acetate 3:1); [a]²_D³= +5.6 (c=1.0, CHCl₃);

3

(m, 17H, H_Ar, Fmoc, H_Ar, Ph), 7.15 (d, 2H, J=7.9 Hz, H_Ar, Tyr), 7.01 (d,

3

2H, ³J=7.9 Hz, H_Ar, Tyr), 5.57 (s, 1H, CH-Ph), 5.27 (d, 1H, J_NH;Ha

=

8.3 Hz, NH), 5.02 (d, 1H, ²J=11.1 Hz, CH₂, Bn), 5.01 (d, 1H, ²J=

11.2 Hz, CH₂, Bn), 4.79 (d, 1H, J=11.2 Hz, CH₂, Bn), 4.72–4.63 (m, 2H,

2

H^a{4.68}, CH₂, Bn), 4.44 (dd, 1H, ²J=10.6 Hz, ³J=7.3 Hz, CH_2a, Fmoc),

4.35 (dd, 1H, ²J=10.6 Hz, ³J=6.9 Hz, CH_2b, Fmoc), 4.26 (dd, 1H, ²J=

10.4 Hz, ³J=5.0 Hz, H6a, Glc), 4.22 (t, 1H, ³J=7.1 Hz, CH, Fmoc), 3.86

(t, 1H, ³J_H3;H2=³J_H3;H4=8.8 Hz, H3, Glc), 3.74 (s, 3H, OCH₃), 3.72–3.63

(m, 2H, H6b, Glc {3.69}, H4, Glc {3.66}), 3.59 (mc, 1H, H1, Glc), 3.38–

3.29 (m, 2H, H2, Glc, H5, Glc), 3.17–3.04 (m, 3H, CH_2a-exo, 2ꢃH^b),

2.66 ppm (dd, 1H, ²J=14.4 Hz, ³J=8.6 Hz, CH_2b-exo); ¹³C NMR [BB,

HSQC] (100.6 MHz, CDCl₃): d=171.98 (COOMe), 155.52 (C=O, Fmoc),

143.82, 143.70 (C1a, Fmoc, C8a, Fmoc), 141.28 (C4a, Fmoc, C5a, Fmoc),

138.45, 138.17, 137.42 (C_ipso, Ph), 137.26, 133.52 (C_ipso, Tyr), 129.83, 129.09

(C_o;m, Tyr), 128.87, 128.47, 128.39, 128.21, 128.11, 127.90, 127.83, 127.70,

127.04, 125.96 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph),

125.10, 125.05 (C1, Fmoc, C8, Fmoc), 119.98 (C4, Fmoc, C5, Fmoc),

101.03 (CH-Ph), 83.35 (C3, Glc), 82.53 (C4, Glc), 81.01 (C2, Glc), 80.36

(C1, Glc), 75.29, 75.01 (CH₂, Bn), 70.02 (C5, Glc), 68.88 (C6, Glc), 66.94

(CH₂, Fmoc), 54.72 (C^a), 52.34 (OCH₃), 47.11 (CH, Fmoc), 37.81 (C^b),

37.68 ppm (CH₂-exo).

¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=7.79 (d, 2H, ³J=7.5 Hz,

3

H_Ar, Fmoc), 7.58 (t, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.51–7.21 (m, 21H, H_Ar

,

Fmoc, H_Ar, Ph), 7.02 (d, 2H, ³J=7.9 Hz, H_Ar, Tyr), 6.11 (s, 1H, CH-Ph),

5.31 (d, 1H, J_NH;Ha=8.3 Hz, NH), 4.98 (d, 1H, ²J=11.6 Hz, CH₂, Bn),

3

4.72 (d, 1H, J=11.6 Hz, CH₂, Bn), 4.69 (m_c, 1H, H^a), 4.60–4.56 (m, 1H,

2

H3, Gal), 4.50 (d, 1H, J=11.9 Hz, CH₂, Bn), 4.48–4.41 (m, 2H, CH₂, Bn,

CH_2a, Fmoc), 4.36 (dd, 1H, ²J=10.6 Hz, ³J=7.0 Hz, CH_2b, Fmoc), 4.26

(dd, 1H, ³J_H4;H3=5.6 Hz, ³J_H4;H5=1.6 Hz, H4, Gal), 4.23 (t_b, 1H, ³J=

7.1 Hz, CH, Fmoc), 3.83–3.68 (m, 6H, H5, Gal {3.79}, H6a, Gal, H6b,

Gal {3.75}, OCH₃{3.73}), 3.51–3.47 (m, 2H, H2-Gal, H1, Gal), 3.24 (d_b,

1H, ²J=13.9 Hz, CH_2a-exo), 3.13 (dd, 1H, ²J=13.9 Hz, ³J=5.6 Hz, H^ba),

3.07 (dd, 1H, ²J=13.8 Hz, ³J=6.1 Hz, H^bb), 2.75 ppm (dd, 1H, ²J=

14.3 Hz, ³J=8.0 Hz, CH_2b-exo); ¹³C NMR [BB, HSQC] (100.6 MHz,

CDCl₃): d=171.97 (COOMe), 155.50 (C=O, Fmoc), 143.78, 143.70 (C1a,

Fmoc, C8a, Fmoc), 141.23 (C4a, Fmoc, C5a, Fmoc), 138.82, 138.15,

137.83 (C_ipso, Ph), 137.48, 133.40 (C_ipso, Tyr), 129.86, 129.14 (C_o;m, Tyr),

129.02, 128.40, 128.38, 128.21, 128.17, 127.81, 127.65, 127.50, 127.46,

127.00, 126.21 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph),

125.08, 125.02 (C1, Fmoc, C8, Fmoc), 119.92 (C4, Fmoc, C5, Fmoc),

103.35 (CH-Ph), 80.58 (C3, Gal), 78.62 (C1, Gal), 76.19 (C2, Gal), 75.59

(C5, Gal), 74.27 (C4, Gal), 73.34, 72.51 (CH₂, Bn), 69.71 (C6, Gal), 66.90

(CH₂, Fmoc), 54.72 (C^a), 52.28 (OCH₃), 47.06 (CH, Fmoc), 37.82,

37.79 ppm (C^b, CH₂-exo); MS (ESI): m/z: 868.35 ([M+Na]⁺, calcd:

868.35).

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,4-tri-O-benzyl-b-l-fucopyrano-

syl]-l-tyrosine methyl ester (37): Synthesis according to the general pro-

cedure. Hydroboration: 20 (440 mg, 1.022 mmol), 0.5m 9-BBN solution

(6.1 mL, 3.05 mmol, 3.0 equiv) in THF; reaction time: 2.5 h; cross-cou-

pling: 32 (539 mg, 1.02 mmol, 1.00 equiv), K₂CO₃(283 mg, 2.04 mmol,

2.00 equiv); [PdCl₂ACHTUNGTRENNUNG(dppf)CH₂Cl₂] (84 mg, 10 mol%) DMF (6.0 mL);

water (2.0 mL); reaction time: 3.5 h. For the work-up, water and di-

chloromethane were added and a pH of 7 was adjusted by addition of

acetic acid. The organic phase was separated and the aqueous phase ex-

tracted three times with dichloromethane. The combined organic extracts

were dried over MgSO₄and the solvent was removed in vacuo. The re-

maining residue was subjected to flash chromatography (cyclohexane/

ethyl acetate 6:1 to 5:1) to give 36 (468 mg, 0.563 mmol, 55%) as a color-

less amorphous solid. R_f=0.10 (cyclohexane/ethyl acetate 5:1); [a]²_D³= +

12.0 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-

b-d-galactopyranosyl]-l-tyrosine methyl ester (35): Synthesis according

to the general procedure. Hydroboration: 18 (78 mg, 0.176 mmol); 0.5m

9-BBN solution (1.06 mL, 0.530 mmol, 3.0 equiv) in THF; reaction time:

2 h; cross-coupling: 32 (116 mg, 0.220 mmol, 1.25 equiv); K₂CO₃(73 mg,

0.528 mmol, 3.0 equiv), [PdCl₂ACHTNUGTRNEUNG(dppf)CH₂Cl₂] (14 mg, 10 mol%); DMF

(1.5 mL), water (0.1 mL); reaction time: 2 h. For the work-up, water and

dichloromethane were added. The organic phase was separated and the

aqueous phase extracted three times with dichloromethane. The com-

bined organic solutions were dried over MgSO₄and the solvent was re-

moved in vacuo and high vacuum. The remaining residue was subjected

to flash chromatography (petroleum ether/ethyl acetate 4:1 to 2:1) to

yield 35 (115 mg, 0.136 mmol, 77%) as a colorless amorphous solid. R_f=

0.38 (petroleum ether/ethyl acetate 1:1); ¹H NMR [¹H¹H-COSY]

3

7.80 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.61 (d, 2H, J=7.5 Hz, H_Ar, Fmoc),

7.46–7.30 (m, 19, H_Ar, Fmoc, H_Ar, Ph), 7.26 (d, 2H, ³J=8.0 Hz, H_ArTyr),

3

7.00 (d, 2H, J=7.9 Hz, H_Ar, Tyr), 5.31 (d, 1H, J_NH;Ha=8.3 Hz, NH), 5.05

(d, 1H, ²J=10.9 Hz, CH₂, Bn), 5.04 (d, 1H, ²J=10.9 Hz, CH₂, Bn), 4.84–

4.66 (m, 5H, 4ꢃCH₂, Bn, H^a{4.69}), 4.48 (dd, 1H, ²J=10.5 Hz, ³J=

2

3

7.2 Hz, CH_2a, Fmoc), 4.38 (dd, 1H, J=10.6 Hz, J=6.9 Hz, CH_2b, Fmoc),

4.24 (t, 1H, ³J=7.0 Hz, CH, Fmoc), 3.79 (t, 1H, ³J_H2;H1=³J_H2;H3=9.3 Hz,

3

(400 MHz, CDCl₃): d=7.77 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.63–7.53 (m,

3

H2, Fuc), 3.75 (s, 3H, OCH₃), 3.69 (d, 1H, J_H4;H3=2.3 Hz, H4, Fuc), 3.64

4H, H_Ar,Fmoc), 7.47–7.27 (m, 19H, H_Ar, Fmoc, H_Ar-Tyr), 7.03 (d, 2H,

3

³J=7.9 Hz, H_Ar, Tyr), 5.49 (s, 1H, CH-Ph), 5.33 (d, 1H, J_NH;Ha=8.4 Hz,

(dd, 1H, J_H3;H2=9.3 Hz, J_H3;H4=2.6 Hz, H3, Fuc), 3.45–3.33 (m, 2H, H1,

Fuc, H5, Fuc), 3.18–3.10 (m, 3H, 2ꢃH^b, CH_2a-exo), 2.78 (dd, 1H, ²J=

14.3 Hz, ³J=9.4 Hz, CH_2b-exo), 1.14 ppm (d, 3H, ³J=6.3 Hz, H6,-Fuc);

¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=171.94 (COOMe), 155.49

(C=O, Fmoc), 143.81, 143.67 (C1a, Fmoc, C8a, Fmoc), 141.24 (C4a,

Fmoc, C5a, Fmoc), 138.58, 138.50, 138.44, 138.34 (C_ipso, Ph, C_ipso, Tyr),

133.01 (C_ipso, Tyr), 129.76, 128.87 (C_o;m, Tyr), 128.41, 128.37, 128.19,

128.13, 128.02, 127.65, 127.55, 127.52, 127.00 (C3, Fmoc, C6, Fmoc, C2,

Fmoc, C7, Fmoc, C_o;m;p, Ph), 125.07, 125.00 (C1, Fmoc, C8, Fmoc), 119.91

(C4, Fmoc, C5, Fmoc), 85.37 (C3, Fuc), 80.56 (C1, Fuc), 78.54 (C2, Fuc),

76.24 (C4, Fuc), 75.31, 74.40 (CH₂, Bn), 73.97 (C5, Fuc), 72.33 (CH₂, Bn),

NH), 5.06 (d, 1H, ²J=10.9 Hz, CH₂, Bn), 4.79 (d, 1H, ²J=12.2 Hz, CH₂,

Bn), 4.73 (d, 1H, ²J=12.2 Hz, CH₂, Bn), 4.70 (m_c, 1H, H^a), 4.66 (d, 1H,

²J=11.0 Hz, CH₂, Bn), 4.47 (dd, 1H, ²J=10.4 Hz, ³J=7.2 Hz, CH_2a,

2

3

Fmoc), 4.34 (dd, 1H, J=10.6 Hz, J=7.1 Hz, CH_2b, Fmoc), 4.26–4.16 (m,

3

3H, CH, Fmoc, H4, Gal, H6a, Gal), 3.90 (t, 1H, ²J=12.2 Hz, J=1.6 Hz,

H6b, Gal), 3.76 (t, 1H, ³J_H2;H1=³J_H2;H3=9.3 Hz, H2, Gal), 3.75 (s, 3H,

3

OCH₃), 3.66 (dd, 1H, J_H3;H2=9.2 Hz, ³J_H3;H4=3.5 Hz, H3, Gal), 3.47 (m_c,

2

1H, H1, Gal), 3.22 (d_b, 1H, J=14.0 Hz, CH_2a-exo), 3.19–3.06 (m, 3H, 2ꢃ

2

3

H^b, H5, Gal), 2.89 (dd, 1H, J=14.0 Hz, J=8.5 Hz, CH_2b-exo); ¹³C NMR

[BB, HSQC] (100.6 MHz, CDCl₃): d=171.98 (COOMe), 155.51 (C=O,

Chem. Eur. J. 2013, 00, 0 – 0

ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

&

13

&

ÞÞ

These are not the final page numbers!

H. Kunz et al.

Fmoc), 143.72, 143.69 (C1a, Fmoc, C8a, Fmoc), 141.21, 141.18 (C4a,

Fmoc, C5a, Fmoc), 138.61, 138.25, 138.04 (C_ipso, Ph), 137.93, 133.19 (C_ipso

Tyr), 130.09, 128.89 (C_o;m, Tyr), 128.34, 128.12, 127.83, 127.70, 127.66,

127.60, 127.02, 126.42 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p

Ph), 125.11, 125.00 (C1, Fmoc, C8, Fmoc), 119.89 (C4, Fmoc, C5, Fmoc),

101.13 (CH-Ph), 81.86 (C3, Gal), 79.79 (C1, Gal), 77.17 (C2, Gal), 75.18

(CH₂, Bn), 73.70 (C4, Gal), 70.95 (CH₂, Bn), 69.64 (C6, Gal), 69.10 (C5,

Gal), 66.88 (CH₂, Fmoc), 54.69 (C^a), 52.28 (OCH₃), 47.03 (CH, Fmoc),

37.79 (C^b), 37.36 ppm (CH₂-exo); MS (ESI): m/z: 863.40 ([M+Na]⁺,

calcd: 863.37);

clohexane/ethyl acetate 1:1, then in cyclohexane/ethyl acetate/acetic acid

15:5:1) to give 39 (185 mg, 0.226 mmol, 82%) as a colorless amorphous

solid. R_f=0.13 (cyclohexane/ethyl acetate/acetic acid 15:5:1); [a]²_D³= +

20.3 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=

7.72–7.67 (m, 2H, H_Ar, Fmoc), 7.54–7.50 (m, 2H, H_Ar, Fmoc), 7.37–7.23

(m, 19H, H_Ar, Fmoc, H_Ar, Ph), 7.18–7.15 (m, 2H, H_Ar, Tyr), 7.06–7.12 (m,

2H, H_Ar, Tyr), 4.96 (d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.93 (d, 1H, ²J=

11.0 Hz, CH₂, Bn), 4.74–4.62 (m, 4H, CH₂, Bn), 4.54 (m, 1H, H^a), 4.38

(dd, 1H, ²J=10.4 Hz, ³J=7.0 Hz, CH_2a, Fmoc), 4.23 (dd, 1H, ²J=

,

10.4 Hz, ³J=7.0 Hz, CH_2b, Fmoc), 4.11 (t, 1H, ³J=7.0 Hz, CH, Fmoc),

3

3.70 (t, 1H, ³J_H2;H1=³J_H2;H3=9.4 Hz, H2, Fuc), 3.62 (d, 1H, J_H4;H3

=

(Z)-N-(Fluoren-9-ylmethoxycarbonyl)-g-[2,3-di-O-benzyl-4,6-O-benzyli-

dene-b-d-glucopyranosyl]-a,b-dehydro-2-aminobutyric acid methyl ester

(38)

2.7 Hz, H4, Fuc), 3.56 (dd, 1H, ³J_H3;H2=9.6 Hz, J_H3;H4=2.7 Hz, H3, Fuc),

3

3.33 (t_b, 1H, J_H1;H2=³J_H1;Hexo;b=9.2 Hz, H1, Fuc), 3.28 (m_c, 1H, H5, Fuc),

3

3.13 (dd, 1H, ²J=13.9 Hz, ³J=5.2 Hz, H^ba), 3.0 (d_b, 1H, ²J=14.3 Hz,

Variant A: Synthesis according to the general procedure. Hydroboration:

19 (257 mg, 0.578 mmol); 0.5m 9-BBN solution (3.47 mL, 1.73 mmol,

3.0 equiv) in THF; reaction time: 1 h; cross-coupling: 33a (220 mg,

0.547 mmol, 0.95 equiv) K₂CO₃(160 mg, 1.16 mmol, 2.0 equiv); [PdCl₂-

ACHTUNGTRENNUNG(dppf)CH₂Cl₂] (47 mg, 10 mol%) DMF (3.5 mL); water (1.0 mL); reac-

tion time: 45 min; yield after work-up: 151 mg (0.197 mmol, 34%).

2

3

2

CH_2a-exo), 3.00 (dd, 1H, J=13.9 Hz, J=6.8 Hz, H^bb), 2.66 (dd, 1H, J=

14.3 Hz, ³J=9.4 Hz, CH_2b-exo), 1.05 ppm (d, 3H, ³J=6.3 Hz, H6, Fuc);

¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=173.41 (COOH), 155.83

(C=O, Fmoc), 143.45, 143.38 (C1a, Fmoc, C8a, Fmoc), 140.87 (C4a,

Fmoc, C5a, Fmoc), 138.08, 137.96, 137.93, 137.77 (C_ipso, Ph, C_ipso, Tyr),

133.46 (C_ipso, Tyr), 129.18, 128.72, 128.61, 128.10, 128.07, 128.04, 127.84,

127.76, 127.41, 127.33, 127.25, 126.69 (C_o;m, Tyr, C3, Fmoc, C6, Fmoc, C2,

Fmoc, C7, Fmoc, C_o;m;p, Ph), 124.77, 124.68 (C1, Fmoc, C8, Fmoc), 119.53

(C4, Fmoc, C5, Fmoc), 84.75 (C3, Fuc), 80.24 (C1, Fuc), 78.25 (C2, Fuc),

76.19 (C4, Fuc), 75.01, 74.35 (CH₂, Bn), 73.67 (C5, Fuc), 71.94 (CH₂, Bn),

66.45 (CH₂, Fmoc), 54.50 (C^a), 46.72 (CH, Fmoc), 37.07, 36.97 (C^b, CH₂-

exo), 16.63 ppm (C6, Fuc); MS (ESI): m/z: 840.35 ([M+Na]⁺, calcd:

840.35); HR-MS (ESI): m/z calcd for C₅₂H₅₁NO₈: 840.3512 [M+Na]⁺;

found: 840.3516.

Variant B: Synthesis according to the general procedure. Hydroboration:

19 (103 mg, 0.232 mmol); 0.5m 9-BBN solution (1.40 mL, 0.700 mmol,

3.0 equiv) in THF; reaction time: 4 h; cross-coupling: 33b (90 mg

(0.200 mmol, 0.865 equiv) K₂CO₃(64 mg, 0.463 mmol, 2.00 equiv);

[PdCl₂ACHTUNGTRENNUNG(dppf)CH₂Cl₂] (19 mg, 10 mol%) DMF (2.0 mL); water (0.5 mL);

reaction time: 1 h; yield after work-up: 63 mg (82.1 mmol, 35%).

For the work-up, water and dichloromethane were added and a pH of 7

was adjusted by addition of acetic acid. The organic phase was separated

and the aqueous phase extracted four times with dichloromethane. The

combined organic extracts were dried over MgSO₄and the solvent was

removed in vacuo and high vacuum. The remaining residue was subjected

to flash chromatography (petroleum ether/ethyl acetate 4:1 to 2:1) to

afford 38 as a colorless amorphous solid. R_f=0.29 (petroleum ether/ethyl

acetate 2:1); [a]_D²³=À32.9 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY]

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3-di-O-benzyl-4,6-O-benzylidene-

b-d-glucopyranosyl]-serine methyl ester (40): Compound 36 (402 mg,

0.524 mmol) was dissolved in THF (20 mL). Bis(cycloocta-1,5-diene)rho-

dium(I)-tetrafluoroborate

([Rh

(cod)₂]BF₄)

(40 mg,

98.5 mmol,

0.188 equiv) and (R)-Tol-BINAP (100 mg, 0.147 mmol, 0.281 equiv) were

added. After degassing with a stream of argon the hydrogenation was

carried out under a hydrogen-pressure of 30 bar in an autoclave. After

24 h the reaction mixture was transferred into a flask, silica gel was

added and the solvent removed in vacuo. The remaining powder was sub-

jected to flash chromatography (cyclohexane/ethyl acetate 2:1) to give 40

(341 mg, 0.443 mmol, 85%) as a colorless amorphous solid. R_f=0.40 (cy-

clohexane/ethyl acetate 2:1); [a]²_D³=À19.9 (c=1.0, CHCl₃); ¹H NMR

3

(400 MHz, CDCl₃): d=7.79 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.60 (d_b, 2H,

³J=7.0 Hz, H_Ar, Fmoc), 7.55–7.49 (m, 2H, H_Ar), 7.46–7.22 (m, 17H, H_Ar),

6.58 (t, 1H, ³J=7.3 Hz, =CH), 5.60 (s, 1H, CH-Ph), 5.03 (d, 1H, ²J=

11.2 Hz, CH₂, Bn), 4.95 (d, 1H, ²J=10.9 Hz, CH₂, Bn), 4.81 (d, 1H, ²J=

11.2 Hz, CH₂, Bn), 4.62 (d, 1H, ²J=10.9 Hz, CH₂, Bn), 4.44 (d, 2H, ³J=

7.0 Hz, CH₂, Fmoc), 4.34 (dd, 1H, ²J=10.4 Hz, ³J=5.0 Hz, H6a, Glc),

3

4.24 (t_b, 1H, J=6.5 Hz, CH, Fmoc), 3.86 (t_b, 1H, J_H3;H2=³J_H3;H4=8.8 Hz,

H3, Glc), 3.79 (s, 3H, OCH₃), 3.73–3.66 (m, 2H, H6b, Glc, H4, Glc), 3.56

(ddd, 1H, ³J_H1;H2=9.8 Hz, ³J_H1;Hexo;a=3.3 Hz, ³J_H1;Hexo;b=7.5 Hz, H1, Glc),

3.45 (dt, 1H, ³J_H5;H4=³J_H5;H6b=9.9 Hz, ³J_H5;H6a=5.0 Hz, H5, Glc), 3.37

(dd, 1H, ³J_H2;H1=9.4 Hz, ³J_H2;H3=8.5 Hz, H2, Glc), 2.72 (ddd, 1H, ²J=

16.0 Hz, ³J_Hexo;a;=CH=7.0 Hz, ³J_Hexo;a;H1=3.1 Hz, CH_2a-exo), 2.42 ppm (dt,

1H, ²J=15.6 Hz, ³J_Hexo;b;=CH=³J_Hexo;b;H1=7.5 Hz, CH_2b-exo). ¹³C NMR

[BB, HSQC] (100.6 MHz, CDCl₃): d=164.69 (COOMe), 153.80 (C=O,

Fmoc), 143.69, 143.62 (C1a, Fmoc, C8a, Fmoc), 141.30 (C4a, Fmoc, C5a,

Fmoc), 138.33, 137.76, 137.27 (C_ipso, Ph), 131.12 (=CH), 128.95, 128.43,

128.40, 128.25, 128.10, 128.08, 127.89, 127.74, 127.07, 125.94 (C3, Fmoc,

C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p-Ph), 125.04, 125.03 (C1, Fmoc, C8,

Fmoc), 119.99 (C4, Fmoc, C5, Fmoc), 101.10 (CH-Ph), 83.00 (C3, Glc),

82.33 (C4, Glc), 80.57 (C2 Glc), 78.83 (C1, Glc), 75.35, 75.03 (CH₂, Bn),

70.23 (C5, Glc), 68.70 (C6, Glc), 67.27 (CH₂, Fmoc), 52.46 (OCH₃), 47.04

(CH, Fmoc), 30.56 ppm (CH₂-exo); MS (ESI): m/z: 790.34 ([M+Na]⁺,

calcd: 790.30);

[¹H¹H-COSY] (400 MHz, CDCl₃): d=7.81 (d, 2H, ³J=7.5 Hz, H_Ar

-

Fmoc), 7.68–7.62 (m, 2H, H_Ar-Fmoc), 7.57–7.53 (m, 2H, H_Ar, Fmoc),

7.47–7.28 (m, 17H, H_Ar, Fmoc, H_Ar, Ph), 5.59 (s, 1H, CH-Ph), 5.37 (d,

3

1H, J_NH;Ha=8.3 Hz, NH), 5.04 (d, 1H, ²J=11.2 Hz,CH₂, Bn), 4.98 (d,

1H, ²J=11.0 Hz, CH₂, Bn), 4.84 (d, 1H, ²J=11.2 Hz, CH₂, Bn), 4.66 (d,

1H, ²J=10.9 Hz, CH₂, Bn), 4.51 (dd, 1H, ²J=10.6 Hz, ³J=6.9 Hz, CH_2a,

2

3

Fmoc), 4.42 (dd, 1H, J=10.6 Hz, J=6.9 Hz, CH_2b, Fmoc), 4.39–4.31 (m,

2H, H6a, Glc {4.34}, H^a{4.37}), 4.27(t_b, 1H, ³J=6.8 Hz, CH, Fmoc), 3.86

(t, 1H, ³J_H3;H2=³J_H3;H4=8.7 Hz, H3, Glc), 3.75 (s, 3H, OCH₃), 3.72–3.64

(m, 2H, H6b, Glc {3.69}, H4, Glc {3.67}), 3.47–3.35 (m, 2H, H1, Glc

{3.39}, H5, Glc {3.42}), 3.31 (t, 1H, ³J_H2;H1=³J_H2;H3=8.8 Hz, H2, Glc),

1.99–1.79 (m, 3H, CH_2a-exo {1.92}, H^ba {1.91}, H^bb {1.85}), 1.53–1.42 ppm

(m, 1H, CH_2b-exo); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=

172.74 (COOMe), 155.89 (C=O, Fmoc), 143.82, 143.69 (C1a, Fmoc, C8a,

Fmoc), 141.24, 141.22 (C4a, Fmoc, C5a, Fmoc), 138.39, 137.86, 137.32

(C_ipso, Ph), 128.85, 128.39, 128.32, 128.18, 128.12, 128.06, 128.01, 127.87,

127.64, 127.00, 125.90 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p

,

Ph), 124.99, 124.94 (C1, Fmoc, C8, Fmoc), 119.94 (C4, Fmoc, C5 Fmoc),

100.99 (CH-Ph), 83.08 (C3, Glc), 82.39 (C4, Glc), 80.88 (C2, Glc), 79.06

(C1, Glc), 75.33, 74.92 (CH₂, Bn), 70.14 (C5, Glc), 68.73 (C6, Glc), 66.79

(CH₂, Fmoc), 53.66 (C^a), 52.31 (OCH₃), 47.08 (CH, Fmoc), 28.13 (C^b),

27.48 ppm (CH₂-exo); MS (ESI): m/z: 792.35 ([M+Na]⁺, calcd: 792.31);

HR-MS (ESI): m/z calcd for C₄₇H₄₇NO₉: 792.3149 [M+Na]⁺; found:

792.317.

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,4-tri-O-benzyl-b-l-fucopyrano-

syl]-l-tyrosine (39): Compound 28 (229 mg, 0.275 mmol) was dissolved in

THF (16 mL) and cooled to 08C. A 0.2m solution of LiOH (1.93 mL,

0.386 mmol, 1.4 equiv) diluted with water (6 mL) was slowly added over-

night by using a syringe pump. As the reaction remained incomplete, a

solution of 0.2m LiOH (2.83 mL, 0.566 mmol, 2.1 equiv) was added until

the complete disappearance of 28 was observed. The pH of the reaction

mixture was between 8 and 9, when methanol (5 mL) and fluoren-9-yl-

methyl-succinimidyl-carbonate (Fmoc-OSu) (106 mg, 0.314 mmol,

1.1 equiv) were added. After 30 min, the pH was adjusted to 5 by addi-

tion of acetic acid. Silica gel was added and the solvent removed in

vacuo. The remaining powder was subjected to flash chromatography (cy-

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,4,6-tetra-O-acetyl-b-d-glucopyr-

anosyl]-l-tyrosine (41): Compound 28 (156 mg, 0.204 mmol) was dis-

solved in ethanol (10 mL). The solution was degassed before Pd(OH)₂/C

(15–20 wt%, 40 mg) was added and the mixture was stirred under a hy-

drogen atmosphere for 28 h. After filtration and washing with ethanol,

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C-Glycosyl Amino Acids

FULL PAPER

3

the solvent was removed and the residue dried in vacuo. Pyridine (5 mL)

and acetic anhydride (2.5 mL) were added and the solution was stirred

for 5 h before the excess of reagents was removed in vacuo and high

vacuum. The remaining oil was co-evaoporated three times with toluene

and dried in high vacuum to give the crude intermediate (103 mg,

0.155 mmol, 76%) as a viscous oil. R_f=0.23 (cyclohexane/ethyl acetate

3:2); ¹H NMR (400 MHz, CDCl₃): d=7.14–7.01 (m, 4H, H_Ar, Tyr), 5.12

H_Ar, Tyr), 7.05 (d, 2H, J=7.9 Hz, H_Ar, Tyr), 5.38 (d, 1H, J_NH;Ha=8.4 Hz,

NH), 5.05 (t, 1H, ³J_H4;H3=³J_H4;H5=9.7 Hz, H4, Glc), 4.93 (d, 1H, ²J=

11.0 Hz, CH₂, Bn), 4.86 (d, 1H, ²J=11.3 Hz, CH₂, Bn), 4.72 (d, 1H, ²J=

11.4 Hz, CH₂, Bn), 4.71 (m_c, 1H, H^a), 4.68 (d, 1H, J=11.1 Hz, CH₂, Bn),

2

4.47 (dd, 1H, ³J=7.1 Hz, ²J=10.5 Hz, CH_2a, Fmoc), 4.34 (dd, 1H, ³J=

2

7.2 Hz, J=10.6 Hz, CH_2b, Fmoc), 4.26–4.19 (m, 2H, H6a, Glc {4.23}, CH,

Fmoc {4.21}), 3.97 (dd, 1H, ²J=12.1 Hz, ³J=2.3 Hz, H6b, Glc), 3.77 (s,

3H, OCH₃), 3.68 (t, 1H, ³J_H3;H2=³J_H3;H4=9.0 Hz, H3, Glc), 3.46–3.34 (m,

3H, H1, Glc {3.41}, H2, Glc {3.38}, H5, Glc {3.36}), 3.18 (dd, 1H, ²J=

(t, 1H, ³J_H3;H2=³J_H3;H4=9.4 Hz, H3, Glc), 5.01 (t, 1H, ³J_H4;H3=³J_H4;H5

=

9.7 Hz, H4, Glc), 4.89 (t, 1H, ³J_H2;H1=³J_H2;H3=9.6 Hz, H2, Glc), 4.38 (q,

1H, ³J_Ha;NH=³J_Ha;Hb=6.8 Hz, H^a), 4.19 (dd, 1H, ²J=12.2 Hz, ³J=5.2 Hz,

H6b, Glc), 3.99 (dd, 1H, ²J=12.2 Hz, ³J=2.1 Hz, H6b, Glc), 3.65–3.50

(m, 2H, H1, Glc, H5, Glc), 3.00–2.92 (m, 2H, 2ꢃH^b), 2.76–2.67 (m, 2H,

CH₂-exo), 2.00, 1.97, 1.95, 1.93 (4ꢃs, 12H, CH₃, Ac), 1.37, 1.36 ppm (2ꢃs,

18H, CH₃, Boc and tBu); ¹³C NMR [BB, DEPT] (100.6 MHz, CDCl₃):

d=170.90, 170.58, 170.35, 169.62, 169.42 (COOtBu, C=O, Ac), 155.01

(C=O, Boc), 135.55, 134.52 (C_ipso, Tyr), 128.93, 128.13 (C_o;m, Tyr), 81.91

(C_q, tBu), 79.56 (C_q, Boc), 78.23 (C1, Glc), 75.38 (C5, Glc), 74.29 (C3,

Glc), 71.85 (C2, Glc), 68.51 (C4, Glc), 62.09 (C6, Glc), 54.70 (C^a), 37.90

(C^b), 37.38 (CH₂-exo), 28.21, 27.86 (CH₃, Boc and tBu), 21.90, 20.69,

20.54 ppm (4ꢃCH₃, Ac). The intermediate (103 mg, 0.155 mmol) was

mixed with trifluoroacetic acid (8 mL) and water (1 mL). After stirring

for 3.5 h, toluene (25 mL) were added and all solvents removed in vacuo.

The remaining oil was co-distilled three times with toluene and then dis-

solved in dichloromethane (20 mL). By the addition of N-ethyldiisopro-

pylamine the pH was adjusted to 9–9.5 and of fluoren-9-ylmethyl-succini-

midylcarbonate (Fmoc-OSu) (64 mg, 0.190 mmol, 1.2 equiv) was added.

The stirring was continued overnight. By addition of acetic acid a pH of

6 was adjusted, silica gel added and the solvent removed in vacuo. The

remaining powder was subjected to flash chromatography (dichlorome-

thane/ethanol/acetic acid 300:15:7.5 to 200:2:5) to yield 41 (50 mg,

0.068 mmol, 44%) as a light yellow amorphous solid. R_f=0.39 (dichloro-

methane/ethanol/acetic acid=20:1:0.5); [a]²_D³= +10.1 (c=1.0, CHCl₃);

13.9 Hz, J=5.5 Hz, H^ba), 3.13–3.05 (m, 2H, CH_2a-exo {3.11}, H^bb {3.08}),

3

2.68 (dd, 1H, ²J=14.3 Hz, ³J=8.3 Hz, CH_2b-exo), 2.04 (s, 3H, CH₃, Ac),

1.96 ppm (s, 3H, CH₃, Ac); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃):

d=171.90, 170.59, 169.63 (C=O, Ac, COOMe), 155.44 (C=O, Fmoc),

143.67 (C1a, Fmoc, C8a, Fmoc), 141.15 (C4a, Fmoc, C5a, Fmoc), 137.99,

137.82, 137.24 (C_ipso, Ph, C_ipso, Tyr), 133.49 (C_ipso, Tyr), 129.74, 128.91

(C_o;m, Tyr), 128.42, 128.37, 127.83, 127.68, 127.65, 127.60, 126.95 (C3,

Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph), 125.06, 124.95 (C1,

Fmoc, C8, Fmoc), 119.87 (C4, Fmoc, C5, Fmoc), 84.46 (C3 Glc), 81.28

(C2, Glc), 79.87 (C1, Glc), 75.45 (C5, Glc), 75.20, 75.06 (CH₂, Bn), 70.48

(C4, Glc), 66.82 (CH₂, Fmoc), 62.54 (C6, Glc), 54.63 (C^a), 52.22 (OCH₃),

46.99 (CH, Fmoc), 37.70 (C^b), 37.04 (CH₂-exo), 20.71, 20.65 ppm (CH₃,

Ac); MS (ESI): m/z: 864.39 ([M+Na]⁺, calcd: 864.34); HR-MS (ESI):

m/z calcd for C₅₀H₅₁NO₁₁: 864.3360 [M+Na]⁺; found: 864.3354. This in-

termediate (240 mg, 0.285 mmol) was dissolved in THF (10 mL) and

cooled to 08C. Over a period of 9 h, a solution of 0.2m LiOH (2.85 mL,

0.570 mmol, 2.0 equiv) was added by using a syringe pump. Stirring was

continued at room temperature for 14 h. The solution was neutralized

with 2n hydrochloric acid, the solvent removed in vacuo, and the remain-

ing residue co-evaporated with toluene. Dichloromethane (10 mL) and

FmocOSu (96 mg, 0.285 mmol, 1.0 equiv) were added. With N-ethyldiiso-

propylamine, a pH of 9 was adjusted and maintained for 8 h. The reac-

tion mixture was brought to a pH of 4.5 by addition of acetic acid, the

solvent was removed in vacuo and the remaining crude product subjected

to flash chromatography (cyclohexane/ethyl acetate 1:1, then cyclohex-

ane/ethyl acetate/acetic acid 10:10:0.5). To remove traces of silica gel, the

product dissolved in dichloromethane and filtered through a syringe filter

to give 42 (119 mg, 0.144 mmol, 50%) as a colorless amorphous solid.

R_f=0.23 (cyclohexane/ethyl acetate/acetic acid 10:10:0.5); [a]²_D³= +9.4

(c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=7.78 (d,

¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=7.79–7.76 (m, 2H, H_Ar

Fmoc), 7.55 (d, 1H, J=7.5 Hz, H_Ar, Fmoc), 7.49 (d, 1H, J=7.5 Hz, H_Ar

,

3

Fmoc), 7.43–7.36 (m, 2H, H_Ar, Fmoc), 7.34–7.26 (m, 2H, H_Ar, Fmoc),

3

7.15–7.06 (m, 4H, H_Ar, Tyr), 5.32 (d, 1H, J_NH;Ha=8.2 Hz, NH,), 5.11 (t,

1H, ³J_H3;H2=³J_H3;H4=9.3 Hz, H3, Glc), 5.00 (t, 1H, ³J_H4;H3=³J_H4;H5

=

9.7 Hz, H4, Glc), 4.89 (t, 1H, ³J_H2;H1=³J_H2;H3=9.5 Hz, H2, Glc), 4.71 (q,

1H, ³J_Ha;NH=³J_Ha;Hb=6.9 Hz, H^a), 4.43 (dd, 1H, ²J=10.6 Hz, ³J=7.0 Hz,

CH_2a, Fmoc), 4.27 (dd, 1H, ²J=10.4 Hz, ³J=7.5 Hz, CH_2b, Fmoc), 4.20–

4.11 (m, 2H, CH, Fmoc, H6a, Glc), 3.96 (dd, 1H, ²J=12.1 Hz, ³J=

1.6 Hz, H6b, Glc), 3.50–3.43 (m, 1H, H1, Glc), 3.42–3.35 (m, 1H, H5,

Glc), 3.21 (dd, 1H, ²J=14.0 Hz, ³J=5.3 Hz, H^ba), 3.06 (dd, 1H, ²J=

14.0 Hz, ³J=7.0 Hz, H^bb), 2.78–2.67 (m, 2H, CH₂-exo), 2.02, 2.00, 1.99,

1.96 ppm (4ꢃs, 12H, CH₃, Ac); ¹³C NMR [BB, HSQC] (100.6 MHz,

CDCl₃): d=175.31, 170.83, 170.46, 169.78, 169.53 (COOH, C=O, Ac),

155.74 (C=O, Fmoc), 143.68, 143.56 (C1a, Fmoc, C8a, Fmoc), 141.21

3

2H, J=7.5 Hz, H_Ar, Fmoc), 7.59 (d, 1H, J=7.4 Hz, H_Ar, Fmoc), 7.53 (d,

1H, ³J=7.4 Hz, H_Ar, Fmoc), 7.46–7.29 (m, 14H, H_Ar, Fmoc, H_Ar, Ph),

3

7.21 (d, 2H, J=7.9 Hz, H_Ar, Tyr), 7.13 (d, 2H, J=7.9 Hz, H_Ar, Tyr), 5.45

(d, 1H, J_NH;Ha=8.0 Hz, NH), 5.05 (t, 1H, ³J_H4;H3=³J_H4;H5=9.7 Hz, H4,

3

Glc), 4.99–4.90 (m, 2H, CH₂, Bn), 4.86 (d, 1H, ²J=11.5 Hz, CH₂, Bn),

4.78–4.64 (m, 2H, CH₂, Bn, H^a), 4.47–4.39 (m, 1H, CH_2a, Fmoc), 4.36–

4.28 (m, 1H, CH_2b, Fmoc), 4.24–4.12 (m, 2H, CH, Fmoc, H6a, Glc), 3.97

(d_b, 1H, ²J=11.4 Hz, H6b, Glc), 3.66 (t, 1H, ³J_H3;H2=³J_H3;H4=8.6 Hz, H3,

Glc), 3.43–3.21 (m, 3H, H1-Glc, H2 Glc, H5, Glc), 3.14–3.03 (m, 3H, 2ꢃ

H^b, CH_2a-exo), 2.66 (dd, 1H, ²J=13.4 Hz, ³J=7.5 Hz, CH_2b-exo), 2.04 (s,

3H, CH₃, Ac), 1.96 ppm (s, 3H, CH₃, Ac); ¹³C NMR [BB] (100.6 MHz,

CDCl₃): d=170.98, 169.81 (C=O, Ac), 155.78 (C=O, Fmoc), 143.65,

143.59 (C1a, Fmoc, C8a, Fmoc), 141.15 (C4a, Fmoc, C5a, Fmoc), 137.97,

137.78, 137.25 (C_ipso, Ph, C_ipso, Tyr), 133.58 (C_ipso, Tyr), 129.78, 128.54

(C_o;m, Tyr), 128.45, 128.40, 127.94, 127.89, 127.83, 127.70, 127.64, 127.00

(C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph), 125.12, 124.99

(C1, Fmoc, C8, Fmoc), 119.89 (C4, Fmoc, C5, Fmoc), 84.40 (C3, Glc),

81.38 (C2, Glc), 79.88 (C1, Glc), 75.39 (C5, Glc), 75.22, 75.11 (CH₂, Bn),

70.51 (C4, Glc), 66.98 (CH₂, Fmoc), 62.60 (C6-Glc), 54.60 (C^a), 46.95

(CH, Fmoc), 37.33 (C^b), 37.08 (CH₂-exo), 20.71, 20.67 ppm (CH₃, Ac);

MS (ESI): m/z: 850.43 ([M+Na]⁺, calcd: 850.32).

(C4a, Fmoc, C5a, Fmoc), 136.05, 133.84 (C_ipso, Tyr), 129.70, 129.19 (C_o;m

,

Tyr), 127.74 (C3, Fmoc, C6, Fmoc), 127.04 (C2, Fmoc, C7, Fmoc), 125.14,

125.00 (C1, Fmoc, C8, Fmoc), 120.03 (C4-Fmoc, C5-Fmoc), 78.07 (C1-

Glc), 75.30 (C5-Glc), 74.30 (C3-Glc), 71.91 (C2, Glc), 68.53 (C4, Glc),

67.08 (CH₂, Fmoc), 62.13 (C6, Glc), 54.50 (C^a), 47.00 (CH, Fmoc), 37.46

(C^b), 37.39 (CH₂-exo), 20.71, 20.63, 20.59 ppm (4ꢃCH₃, Ac); MS (ESI):

m/z: 754.23 ([M+Na]⁺, calcd: 754.25); HR-MS (ESI): m/z calcd for

C₃₉H₄₁NO₁₃: 754.2476 [M+Na]⁺; found: 754.2490.

N-(Fluoren-9-ylmethoxycarbonyl)-C-[4,6-di-O-acetyl-2,3-di-O-benzyl-b-

d-glucopyranosyl]-l-tyrosine (42): The acidolytic removal of the benzyli-

dene acetal occurred as a side reaction during the opening of benzylidene

acetal 36 to 43 in the presence of moisture. The isolated side product

(220 mg, 0.290 mmol) was taken up in pyridine (4 mL) and acetic anhy-

dride (2 mL). After stirring for 2.5 h, the excess reagents was removed in

vacuo and high vacuum before the residue was co-evaporated three times

with toluene. The crude product was subjected to flash chromatography

(cyclohexane/ethyl acetate 2:1 to 3:2) to give the acetylated intermediate

(242 mg, 0.287 mmol, 99%) as a colorless amorphous solid. R_f=0.28 (cy-

clohexane/ethyl acetate 3:2); [a]²_D³= +7.1 (c=1.0, CHCl₃); ¹H NMR

Experimental setup for the benzylidene acetal-opening under flow condi-

tions: Two solutions (A/B on the first channel and C on the second) were

delivered by using syringe pumps through PTFE-capillaries (OD=

3.18 mm, ID=1.76 mm) with a Mikroglas Interdigital mixer. Thereafter

the reaction mixture runs through a residence unit of PTFE tubing (total

volume 3.0 mL) before it was quenched by dropping into a stirred satu-

rated NaHCO₃solution. The entire device was cooled to 08C.

[¹H¹H-COSY] (400 MHz, CDCl₃): d=7.79 (d, 2H, ³J=7.5 Hz, H_Ar

,

3

Fmoc), 7.61 (d, 1H, J=7.4 Hz, H_Ar, Fmoc), 7.56 (d, 1H, J=7.4 Hz, H_Ar

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,6-tri-O-benzyl-b-d-glucopyrano-

syl]-l-tyrosine methyl ester (43): The following solutions were prepared:

Fmoc), 7.46–7.29 (m, 14H, H_Ar, Fmoc, H_Ar, Ph), 7.21 (d, 2H, ³J=7.9 Hz,

Chem. Eur. J. 2013, 00, 0 – 0

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H. Kunz et al.

A: 36 (406 mg, 0.480 mmol), absol. dichloromethane (5 mL), triethylsi-

lane (2.0 mL, 12.5 mmol, 22.5 equiv); B: absol. dichloromethane (5 mL),

triethylsilane (2.0 mL, 12.5 mmol); C: absol. dichloromethane (20 mL),

trifluoroacetic acid (10 mL, 130 mmol). For conditioning, 1.5 mL of B

and C were pumped at 0.4 mLmin^À1; then the solutions A and C were

pumped at 0.4 mLmin^À1until A was depleted; the reaction was complet-

ed by pumping the rest of B and C before it was rinsed with dichlorome-

thane (20 mL) on both channels. For the work up procedure, the neutral-

ized two-phase mixture was diluted with water and neutralized with

acetic acid. The organic layer was separated and the aqueous layer was

extracted three times with dichloromethane. The combined organic

layers were dried over MgSO₄, silica gel was added, and the solvent re-

moved in vacuo. The remaining powder was subjected to flash chroma-

tography (petroleum ether/ethyl acetate 2:1) to give 43 (337 mg,

0.397 mmol, 83%) as a colorless viscous oil. R_f=0.18 (petroleum ether/

ethyl acetate 2:1); [a]_D²³= +7.0 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY]

(400 MHz, CDCl₃): d=7.80 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.61 (d, 1H,

³J=7.5 Hz, H_Ar, Fmoc), 7.58 (d, 1H, ³J=7.4 Hz, H_Ar, Fmoc), 7.47–7.29

Glc), 70.31 (C6, Glc), 66.83 (CH₂, Fmoc), 53.75 (C^a), 52.28 (OCH₃), 47.06

(CH, Fmoc), 28.17 (C^b), 27.39 ppm (CH₂-exo); MS (ESI): m/z: 794.32

([M+Na]⁺, calcd: 794.33); HR-MS (ESI): m/z calcd for C₄₇H₄₉NO₉:

794.3305 [M+Na]⁺; found: 794.3311.

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,6-tri-O-benzyl-4-O-(2,3,4-tri-O-

acetyl-6-O-benzyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-l-tyrosine

methyl ester (45): Molecular sieves (3 ꢄ, 1.3 g) were freshly dried by

heating in vacuo in a Schlenk flask and set under vacuum. In another

flask, compound 43 (337 mg, 0.397 mmol) and the galactosyl trichloroace-

timidate^[20,21](301 mg, 0.596 mmol, 1.5 equiv) were dissolved in dry di-

chloromethane, the solvent was removed and the remaining solid mixture

dried in high vacuum for 30 min. Under argon the carbohydrate mixture

was dissolved in dry dichloromethane (20 mL) and transferred to the

Schlenk flask containing the molecular sieves. After stirring for 30 min,

the mixture was cooled to À458C. Trimethylsilyltrifluoromethansulfonate

(TMSOTf) (0.2 mL of a solution of 0.05 mL) was then added to the clear

solution in dry dichloromethane (0.95 mL). After 10 min no more tri-

chloroacetimidate was present as was monitored by TLC analysis. To

complete the conversion, the cooling was removed and stirring continued

until the solution was clear. At this point, an additional 100 mg

(0.198 mmol, 0.50 equiv) of galactosyl trichloroacetimidate^[20,21]was

added followed by the addition of a solution of TMSOTf (0.1 mL) in di-

chloromethane. After 10 min, saturated NaHCO₃solution (100 mL) was

added. At room temperature, the organic layer was separated and the

aqueous layer extracted three times with dichloromethane. The combined

organic extracts were dried over MgSO₄, silica gel was added and the sol-

vent removed in vacuo. The remaining powder was subjected to flash

chromatography (petroleum ether/ethyl acetate 2:1) to obtain 45

(407 mg, 0.332 mmol, 84%) as a colorless amorphous solid. R_f=0.21 (pe-

troleum ether/ethyl acetate 3:2); [a]²_D³=À1.3 (c=1.0, CHCl₃); ¹H NMR

3

(m, 19H, H_Ar, Fmoc, H_Ar, Ph), 7.23 (d, 2H, J=7.8 Hz, H_Ar, Tyr), 7.03 (d,

3

2H, J=7.8 Hz, H_Ar, Tyr), 5.35 (d, 1H, J_NH;Ha=8.3 Hz, NH), 4.99 (d, 1H,

²J=11.0 Hz, CH₂, Bn), 4.96 (d, 1H, ²J=11.7 Hz, CH₂, Bn), 4.92 (d, 1H,

²J=11.4 Hz, CH₂, Bn), 4.71 (d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.56 (d, 1H,

²J=12.1 Hz, CH₂, Bn), 4.51 (d, 1H, ²J=12.2 Hz, CH₂, Bn), 4.46 (dd, 1H,

²J=10.7 Hz, ³J=7.3 Hz, CH_2a, Fmoc), 4.37 (dd, 1H, ²J=10.6 Hz, ³J=

7.1 Hz, CH_2b, Fmoc), 4.24 (t, 1H, ³J=7.1 Hz, CH, Fmoc), 3.75 (s, 3H,

OCH₃), 3.74–3.66 (m, 3H, H4, Glc {3.70}, H6ab, Glc {3.68}), 3.60 (t, 1H,

3

³J_H3;H2=³J_H3;H4=8.8 Hz, H3, Glc), 3.51 (t_b, 1H, J_H1;H2=³J_H1;Hexo;b=9.2 Hz,

H1, Glc), 3.34 (m_c, 2H, H2, Glc, H5-Glc), 3.20–3.05 (m, 3H, CH_2a-exo

{3.16}, H^ba -Tyr {3.12}, H^bb -Tyr {3.10}), 2.77 (s, 4-OH, Glc), 2.69 ppm

(dd, 1H, ²J=14.0 Hz, ³J=8.7 Hz, CH_2b-exo); ¹³C NMR [BB, HSQC]

(100.6 MHz, CDCl₃): d=171.95 (COOMe), 155.48 (C=O, Fmoc), 143.74,

143.66 (C1a, Fmoc, C8a, Fmoc), 141.20 (C4a, Fmoc, C5a, Fmoc), 138.59,

[¹H¹H-COSY] (400 MHz, CDCl₃): d=7.76 (d, 2H, ³J=7.5 Hz, H_Ar

Fmoc), 7.56 (t, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.43–7.27 (m, 20H, H_Ar

,

Fmoc, H_Ar-Ph), 7.24–7.13 (m, 6H, H_Ar, Ph, H_Ar, Tyr), 6.98 (d, 2H, ³J=

7.9 Hz, H_Ar, Tyr), 5.40 (d, 1H, ³J_H4;H3=3.5 Hz, H4, Gal), 5.24 (d, 1H,

138.08, 137.86 (C_ipso, Ph), 137.61, 133.34 (C_ipso, Tyr), 129.75, 128.95 (C_o;m

,

Tyr), 128.47, 128.43, 128.30, 127.83, 127.79, 127.72, 127.63, 127.55, 126.98

(C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph), 125.06, 124.99

(C1, Fmoc, C8, Fmoc), 119.91 (C4, Fmoc, C5, Fmoc), 86.76 (C3, Glc),

81.24 (C2, Glc), 79.85 (C1, Glc), 77.58 (C5, Glc), 75.20, 75.00, 73.48

(CH₂, Bn), 72.73 (C4, Glc), 70.43 (C6, Glc), 66.88 (CH₂, Fmoc), 54.68

(C^a), 52.25 (OCH₃), 37.72 (C^b), 37.39 ppm (CH₂-exo); MS (ESI): m/z:

870.37 ([M+Na]⁺, calcd: 870.37); HRMS (ESI): m/z calcd for

C₅₃H₅₃NO₉: 870.3618 [M+Na]⁺; found: 870.3600.

³J_NH;Ha=8.3 Hz, NH), 5.11–5.05 (m, 2H, H2, Gal {5.09}, CH₂, Bn), 4.97

3

(d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.84 (dd, 1H, ³J_H3;H2=10.4 Hz, J_H3;H4

=

3.5 Hz, H3, Gal), 4.71–4.60 (m, 4H, 3ꢃCH₂, Bn {4.69, 4.68, 4.63}, H^a

{4.66}), 4.56 (d, 1H, ³J_H1;H2=8.0 Hz, H1, Gal), 4.49–4.31 (m, 4H, 2ꢃCH₂,

Fmoc {4.42–4.34}, 2ꢃCH₂, Bn {4.47–4.40}), 4.24–4.14 (m, 2H, CH, Fmoc,

CH₂, Bn), 3.97 (t, 1H, ³J_H4;H3=³J_H4;H5=9.5 Hz, H4, Glc), 3.73 (s, 3H,

OCH₃), 3.69–3.54 (m, 4H, H5, Gal {3.58}, H3, Glc {3.59}, H6a, Glc {3.64},

H6b, Glc {3.62}), 3.39 (t, 1H, ³J_H1;H2=³J_H1;Hexo;b=8.5 Hz, H1, Glc), 3.31–

3.21 (m, 3H, H6a, Gal {3.27}, H2, Glc {3.27}, H6b, Gal {3.23}), 3.18 (m_c,

1H, H5, Glc), 3.11–3.01 (m, 3H, 2ꢃH^b{3.08}, CH_2a-exo {3.08}), 2.65 (dd,

1H, ²J=14.3 Hz, ³J=8.9 Hz, CH_2b-exo), 1.99, 1.96, 1.93 ppm (3ꢃs, 9H,

CH₃, Ac); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=171.99,

170.09, 170.03, 169.27 (C=O, Ac, COOMe), 155.54 (C=O, Fmoc), 143.82,

143.70 (C1a, Fmoc, C8a, Fmoc), 141.28 (C4a, Fmoc, C5a, Fmoc), 139.04,

138.33, 137.79, 137.89 (C_ipso, Ph), 137.76, 133.36 (C_ipso, Tyr), 129.86, 129.02

(C_o;m, Tyr), 128.51, 128.46, 128.40, 128.02, 127.97, 127.90, 127.78, 127.73,

127.31, 127.05 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph),

125.09, 125.07 (C,1 Fmoc, C8, Fmoc), 120.00 (C4, Fmoc, C5-Fmoc),

100.00 (C1-Gal), 85.15 (C3-Glc), 81.09 (C2-Glc), 80.19 (C1-Glc), 78.74

(C5, Glc), 76.50 (C4, Glc), 75.25, 75.15, 73.52, 73.29 (CH₂, Bn), 71.72 (C5,

Gal), 71.25 (C3, Gal), 69.76 (C2, Gal), 67.79 (C6, Glc), 67.33 (C4, Gal),

66.99 (CH₂, Fmoc), 66.47 (C6, Gal), 54.75 (C^a), 52.35 (OCH₃), 47.11

(CH, Fmoc), 37.79 (C^b), 37.51 (CH₂-exo), 20.76, 20.63, 20.61 ppm (CH₃,

Ac); MS (ESI): m/z: 1248.51 ([M+Na]⁺, calcd: 1248.49); HR-MS (ESI):

m/z calcd for C₇₂H₇₅NO₁₇:1248.4933 [M+Na]⁺; found: 1248.4933.

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,6-tri-O-benzyl-b-d-glucopyrano-

syl]-serine methyl ester (44): Identical procedure as for the synthesis of

43 by using the following solutions: A: 40 (340 mg, 0.442 mmol), absol.

dichloromethane (5 mL), triethylsilane (2.0 mL, 12.5 mmol, 22.5 equiv);

B: absol. dichloromethane (5 mL), triethylsilane (2.0 mL, 12.5 mmol); C:

absol. dichloromethane (20 mL), trifluoroacetic acid (10 mL, 130 mmol).

After flash chromatography, compound 44 (289 mg, 0.374 mmol, 85%)

was isolated as a colorless amorphous solid. R_f=0.15 (petroleum ether/

ethyl acetate 2:1); [a]_D²³=À15.2 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY]

3

(400 MHz, CDCl₃): d=7.79 (d, 2H, J=7.5 Hz, H_Ar, Fmoc), 7.66–7.60 (m,

2H, H_Ar, Fmoc), 7.46–7.27 (m, 19H, H_Ar, Fmoc, H_Ar-Ph), 5.44 (d, 1H,

³J_NH;Ha=8.2 Hz, NH), 4.95 (d, 1H, ²J=11.4 Hz, CH₂, Bn), 4.90 (d, 1H,

²J=10.8 Hz, CH₂, Bn), 4.88 (d, 1H, ²J=11.3 Hz, CH₂, Bn), 4.64 (d, 1H,

²J=11.0 Hz, CH₂, Bn), 4.59 (d, 1H, ²J=12.1 Hz, CH₂, Bn), 4.54 (d, 1H,

²J=12.0 Hz, CH₂, Bn), 4.45 (dd, 1H, ²J=10.6 Hz, ³J=7.1 Hz, CH_2a,

Fmoc), 4.42–4.32 (m, 2H, CH_2b, Fmoc {4.40}, H^a, Ser {4.36}), 4.24 (t, 1H,

³J=7.0 Hz, CH, Fmoc), 3.78–3.61 (m, 6H, OCH₃{3.73}, H6a, Glc, H6b,

3

Glc {3.70}, H4, Glc {3.66}), 3.55 (t, 1H, J_H3;H2=³J_H3;H4=8.4 Hz, H3, Glc),

3.439 (m_c, 1H, H5, Glc), 3.31–3.20 (m, 2H, H1, Glc {3.28}, H2, Glc

{3.25}), 2.67 (d, 1H, ³J=2.1 Hz, 4-OH), 1.99–1.80 (m, 3H, CH_2a-exo, 2ꢃ

H^b, Ser), 1.53–1.41 ppm (m, 1H, CH_2b-exo); ¹³C NMR [BB, HSQC]

(100.6 MHz, CDCl₃): d=172.79 (COOMe), 155.93 (C=O, Fmoc), 143.85,

143.72 (C1a, Fmoc, C8a, Fmoc), 141.22 (C4a, Fmoc, C5a, Fmoc), 138.55,

137.84, 137.73 (C_ipso, Ph), 128.56, 128.52, 128.43, 128.36, 128.32, 128.05,

128.00, 127.89, 127.83, 127.78, 127.74, 127.69, 127.64, 127.00 (C3, Fmoc,

C6, Fmoc, C2, Fmoc, C7, Fmoc, C_o;m;p, Ph), 125.05, 125.00 (C1, Fmoc, C8,

Fmoc), 119.92 (C4, Fmoc, C5, Fmoc), 86.56 (C3, Glc), 81.26 (C2, Glc),

78.38 (C1, Glc), 77.65 (C5, Glc), 75.20, 75.08, 73.51 (CH₂, Bn), 72.47 (C4,

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,6-tri-O-benzyl-4-O-(2,3,4-tri-O-

acetyl-6-O-benzyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-serine

methyl ester (46): Molecular sieves (3 ꢄ, 1.3 g) were freshly dried by

heating in vacuo in a Schlenk flask and set under vacuum. In another

flask, compound 44 (375 mg, 0.486 mmol) and galactosyl trichloroacetimi-

date^[20,21](368 mg, 0.729 mmol, 1.5 equiv) were dissolved in dry dichloro-

methane, the solvent was removed and the remaining solid dried in high

vacuum for 30 min. Under argon the solid was dissolved in dry dichloro-

methane (20 mL) and transferred to the Schlenk flask containing the mo-

lecular sieves. After stirring for 30 min the mixture was cooled to À458C.

&

16

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C-Glycosyl Amino Acids

FULL PAPER

Trimethylsilyltrifluormethanesulfonate (TMSOTf) (0.15 mL of a solution

of 0.05 mL) in dry dichloromethane (0.95 mL) was then added to the

clear solution,. After 5 min, the solution turned cloudy indicating com-

plete conversion (TLC monitoring). A solution of saturated NaHCO₃

(100 mL) was added, and the organic layer was separated and the aque-

ous layer extracted three times with dichloromethane. The combined or-

ganic layers were dried over MgSO₄, silica gel was added and the solvent

removed in vacuo. The remaining powder was subjected to flash chroma-

tography (petroleum ether/ethyl acetate 3:2) give 46 (532 mg,

0.463 mmol, 95%) as a colorless amorphous solid. R_f=0.18 (petroleum

ether/ethyl acetate 3:2); [a]²_D³=À15.4 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-

127.78, 127.72, 127.68, 127.63, 127.60, 127.51, 127.46, 127.41, 127.39,

127.17 (C_o;m;p, Ph), 103.27 (C1, Gal), 94.24 (CH₂-exo), 82.58 (C3, Glc),

82.36 (C3, Gal), 79.67 (C2, Gal), 77.91 (C2, Glc), 77.77 (C5, Glc), 76.90

(C4, Glc), 75.16, 74.65 (CH₂, Bn), 73.65, 73.53, 73.38, 73.13, 73.02 (CH₂,

Bn, C4, Gal, C5, Gal), 72.62, 72.24 (CH₂, Bn), 68.28 (C6, Glc), 68.21 ppm

(C6, Gal); MS (ESI): m/z: 991.46 ([M+Na]⁺, calcd: 991.44); HRMS

(ESI): m/z calcd for C₆₂H₆₄O₁₀: 991.4397 [M+Na]⁺; found: 991.4398.

N-(tert-Butyloxycarbonyl)-C-[2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-

benzyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-l-tyrosine

tert-butyl

ester (49): Synthesis according to the general procedure. Hydroboration:

48 (1.10 g, 1.13 mmol); 0.5m 9-BBN solution (6.78 mL, 3.39 mmol,

3.0 equiv) in THF; reaction time: 3 h; cross-coupling: 21 (632 mg,

1.41 mmol, 1.25 equiv), K₂CO₃(468 mg, 3.39 mmol, 3.0 equiv); [PdCl₂-

COSY, TOCSY] (400 MHz, CDCl₃): d=7.78 (d, 2H, ³J=7.5 Hz, H_Ar

,

Fmoc), 7.66–7.60 (m, 2H, H_Ar, Fmoc), 7.45–7.15 (m, 24H, H_Ar, Fmoc,

3

H_Ar, Ph), 5.42 (d, 1H, ³J_H4;H3=3.3 Hz, H4, Gal), 5.39 (d, 1H, J_NH;Ha

=

AHCTUNGTERGN(NNU dppf)CH₂Cl₂] (92 mg, 10 mol%) DMF (6.0 mL), water (1.0 mL); reac-

8.3 Hz, NH), 5.14–5.05 (m, 2H, H2, Gal {5.10}, CH₂, Bn), 4.91 (d, 1H,

tion time: 30 min. For the work-up, water and dichloromethane were

added. The organic layer was separated and the aqueous phase extracted

three times with dichloromethane. The combined organic extracts were

dried over MgSO₄and the solvent was removed in vacuo. The remaining

residue was subjected to flash chromatography (cyclohexane/ethyl ace-

tate 10:1 to 6:1) to obtain 49 (988 mg, 0.765 mmol, 68%) as a colorless

amorphous solid. R_f=0.34 (cyclohexane/ethyl acetate 4:1); [a]²_D³= +10.3

(c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY, TOCSY] (400 MHz, CDCl₃):

d=7.49–7.26 (m, 35H, H_ArPh), 7.23 (d, 2H, ³J=7.5 Hz, H_Ar,Tyr), 7.15

(d, 2H, ³J=8.0 Hz, H_Ar, Tyr), 5.30 (d, 1H, ²J=10.5 Hz, CH₂, Bn), 5.17–

5.02 (m, 3H, NH, 2ꢃCH₂, Bn), 4.90 (m, 2H,CH₂, Bn), 4.82–4.72 (m, 4H,

CH₂, Bn), 4.65 (d, 1H, ²J=11.5 Hz, CH₂, Bn), 4.64 (d, 1H, ²J=12.4 Hz,

²J=11.0 Hz, CH₂, Bn), 4.84 (dd, 1H, ³J_H3;H2=10.4 Hz, ³J_H3;H4=3.5 Hz,

H3, Gal), 4.75 (d, 1H, ²J=12.1 Hz, CH₂, Bn), 4.71 (d, 1H, ²J=10.6 Hz,

3

CH₂, Bn), 4.60 (d, 1H, ²J=11.0 Hz, CH₂, Bn), 4.55 (d, 1H, J_H1;H2

=

8.0 Hz, H1, Gal), 4.51–4.32 (m, 5H, CH_2a, Fmoc {4.43}, CH_2b, Fmoc

{4.37}, H^a{4.36}, 2ꢃCH₂, Bn), 4.28–4.18 (m, 2H, CH, Fmoc {4.24}, CH₂,

Bn), 3.98 (t, 1H, ³J_H4;H3=³J_H4;H5=9.4 Hz, H4, Glc), 3.75–3.65 (m, 5H,

OCH₃{3.72}, H6a, Glc {3.69}, H6b, Glc {3.68}), 3.61–3.54 (m, 2H, H5,

Gal {3.58}, H3, Glc {3.57}), 3.33–3.17 (m, 5H, H6a, Gal {3.28}, H5, Glc

{3.25}, H6b, Gal {3.25}, H2, Glc {3.21}, H1, Glc {3.20}), 2.01, 1.99, 1.97

(3ꢃs, 9H, CH₃, Ac), 1.92–1.81 (m, 3H, 2ꢃH^b, CH_2a-exo), 1.51–1.38 ppm

(m, 1H, CH_2b-exo); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=

172.84, 170.05, 169.97, 169.22 (C=O, Ac, COOMe), 155.93 (C=O, Fmoc),

143.85, 143.73 (C1a, Fmoc, C8a, Fmoc), 141.24 (C4a, Fmoc, C5a, Fmoc),

138.99, 138.06, 137.72, 137.61 (C_ipso, Ph), 128.54, 128.38, 128.35, 128.06,

127.96, 127.81, 127.69, 127.66, 127.25, 127.02 (C3, Fmoc, C6, Fmoc, C2,

Fmoc, C7, Fmoc, C_o;m;p, Ph), 125.10, 125.02 (C1, Fmoc, C8, Fmoc), 119.94

(C4, Fmoc, C5, Fmoc), 99.93 (C1, Gal), 84.94 (C3, Glc), 80.95 (C2, Glc),

78.66 (C5, Glc), 78.58 (C1-Glc), 76.40 (C4, Glc), 75.17, 75.14, 73.54, 73.25

(CH₂, Bn), 71.70 (C5, Gal), 71.18 (C3, Gal), 69.84 (C2, Gal), 67.64 (C6,

Glc), 67.27 (C4, Gal), 66.94 (CH₂, Fmoc), 66.45 (C6, Gal), 53.68 (C^a),

52.29 (OCH₃), 47.08 (CH, Fmoc), 28.19 (C^b), 27.35 (CH₂-exo), 20.73,

20.57, 20.56 ppm (CH₃, Ac); MS (ESI): m/z: 1172.42 ([M+Na]⁺, calcd:

1172.46); HR-MS (ESI): m/z calcd for C₆₆H₇₁NO₁₇: 1172.4620 [M+Na]⁺;

found: 1172.4579.

CH₂, Bn), 4.58 (d, 1H, ³J_H1;H2=7.7 Hz, H1, Gal), 4.55–4.44 (m, 3H, H^a,

3

2ꢃCH₂, Bn), 4.35 (d, 1H, ²J=11.8 Hz, CH₂, Bn), 4.11 (t, 1H, J_H4;H3

=

³J_H4;H5=9.4 Hz, H4, Glc), 4.03 (d, 1H, ³J_H4;H3=2.8 Hz, H4, Gal), 3.94–

3.85 (m, 2H, H2, Gal, H6a, Glc), 3.78–3.70 (m, 2H, H6b, Glc, H3, Glc),

3.61 (t, 1H, ²J=³J=7.2 Hz, H6a, Gal), 3.57–3.44 (m, 4H, H1, Glc, H5,

Gal, H3, Gal, H6b, Gal), 3.42–3.33 (m, 2H, H2, Glc, H5, Glc), 3.20 (d_b,

1H, ²J=13.4 Hz, CH_2a-exo), 3.11 (m_c, 2H, 2ꢃH^b), 2.77 (t, 1H, ²J=

14.3 Hz, ³J=8.7 Hz, CH_2b-exo), 1.52, 1.46 ppm (2ꢃs, 18H, CH₃, Boc and

tBu); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=170.96 (COOtBu),

154.98 (C=O, Boc), 139.05, 138.93, 138.59, 138.42, 138.38, 138.00 (C_ipso

,

Ph), 137.32, 133.87 (C_ipso, Thr), 129.51, 129.04 (C_o;m, Thr), 128.29, 128.23,

128.19, 128.09, 128.05, 127.99, 127.93, 127.85, 127.77, 127.70, 127.67,

127.57, 127.52, 127.36, 127.25, 127.15, 126.95 (C_o;m;p, Ph), 102.58 (C1,

Gal), 85.44 (C3, Glc), 82.33 (C3, Gal), 81.68 (C_q, tBu), 80.92 (C2, Glc),

79.99, 79.88 (C1, Glc, C2, Gal), 79.42 (C_q, Boc), 79.05 (C5-Glc), 76.68

(C4, Glc), 75.15, 75.09, 74.99, 74.54 (CH₂, Bn), 73.58 (C4, Gal), 73.25

(CH₂, Bn), 72.87 (C5, Gal, CH₂, Bn), 72.47 (CH₂, Bn), 68.09 (C6, Glc),

67.94 (C6, Gal), 54.72 (C^a), 38.01 (C^b), 37.43 (CH₂-exo), 28.19, 27.78 ppm

(CH₃, Boc and tBu).

4-O-(2,3,4,6-Tetra-O-benzyl-b-d-galactopyranosyl)-2,3,6-tri-O-benzyl-1,5-

d-gluconolactone (47): For the preparation from d-lactose see the Sup-

porting Information.

4-O-(2,3,4,6-Tetra-O-benzyl-b-d-galactopyranosyl)-2,3,6-tri-O-benzyl-1-

deoxy-1-methylidene-d-glucopyranose (48): Olefination of lactone 47 to

the corresponding exo-glycals 48 as described in ref. [19]. After comple-

tion of the reaction, silica gel was added and the solvent is removed in

vacuo. The remaining powder was subjected to flash chromatography (cy-

clohexane/ethyl acetate 20:1 to 5:1). The reaction was conducted as fol-

lows. Microwave batch reaction: microwave power: 200 W; temperature:

658C; pressure: max. 14 bar; reaction time: 30 min. Yield: 47 (1.02 g,

1.05 mmol). Yield: 48 (647 mg (0.695 mmol, 66%) as a slightly yellow oil.

Continuous flow procedure: microwave power: 50 W; flow rate:

1.0 mLmin^À1; residence time: 8.5 min. Starting material: 47 (917 mg,

0.944 mmol); yield: 48 (600 mg, 0.619 mmol, 66%) as a pale yellow oil.

R_f=0.41 (cyclohexane/ethyl acetate 4:1); [a]²_D³= +11.1 (c=1.0, CHCl₃);

¹H NMR [¹H¹H-COSY, TOCSY] (400 MHz, CDCl₃): d=7.43–7.14 (m,

35H, H_Ar, Ph), 5.00 (d, 1H, ²J=11.4 Hz, CH₂, Bn), 4.91 (d, 1H, ²J=

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-

O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-l-tyrosine

(50):

Compound 21 (988 mg, 0.765 mmol) was dissolved in THF (10 mL), etha-

nol (5 mL), water (1 mL) and acetic acid (1 mL). The mixture was de-

gassed with a constant stream of argon for 10 min. A spatula tip of

Pd(OH)₂/C (15–20 wt%) was added and the mixture degassed for anoth-

er 10 min. Hydrogenation was performed at room temperature in an au-

toclave (20 bar) for 5 days. The reaction mixture was then transferred to

a flask and the solvent removed in vacuo. After co-evaporation with tol-

uene/ethanol (1:1), pyridine (40 mL) and acetic anhydride (20 mL) were

added, and stirring was continued for 2 days. Excess of reagents were re-

moved in vacuo and the remaining solid was co-evaporated twice with

toluene. The crude product was subjected to flash chromatography (pe-

troleum ether/ethyl acetate 5:2 to 1:1) to yield 269 mg (0.282 mmol,

37%) of the acetylated intermediate as a pale-yellow amorphous solid.

R_f=0.23 (petroleum ether/ethyl acetate 1:1); [a]²_D³= +9.2 (c=1.0,

CHCl₃); ¹H NMR [¹H¹H-COSY, TOCSY] (400 MHz, CDCl₃): d=7.11–

2

11.3 Hz, CH₂, Bn), 4.81 (d, 1H, J=11.2 Hz, CH₂, Bn), 4.78–4.57 (m, 9H,

3

CH₂, Bn, CH_2a-exo {4.74}), 4.56 (s_b, 1H, CH_2b-exo), 4.45 (d, 1H, J_H1;H2

=

7.7 Hz, H1, Gal), 4.42 (d, 1H, ²J=12.1 Hz, CH₂, Bn), 4.38 (d, 1H, ²J=

11.9 Hz, CH₂, Bn), 4.30 (d, 1H, ²J=11.8 Hz, CH₂, Bn), 4.10 (dd, 1H,

³J_H4;H3=6.7 Hz, ³J_H4;H5=9.9 Hz, H4, Glc), 3.94 (d, 1H, ³J_H4;H3=3.9 Hz,

H4, Gal), 3.93 (s_b, 1H, H2, Glc), 3.89–3.82 (m, 2H, H6a, Glc, H5, Glc),

3.79 (d_b, 1H, ³J_H3;H4=6.8 Hz, H3, Glc), 3.77 (dd, 1H, ³J_H2;H1=3.5 Hz,

³J_H2;H3=7.8 Hz, H2, Gal), 3.74–3.69 (m_c, 1H, H6b, Glc), 3.60 (t, 1H, ²J=

³J=8.3 Hz, H6a, Gal), 3.47–3.37 ppm (m, 3H, H6b, Gal, H5 Gal, H3,

Gal); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=155.83 (C1, Glc),

138.91, 138.79, 138.69, 138.46, 138.17, 138.02, 137.97 (C_ipso, Ph), 128.36,

128.33, 128.25, 128.21, 128.17, 128.15, 128.04, 127.86, 127.82, 127.80,

3

7.01 (m, 4H, H_Ar, Tyr), 5.33 (dd, 1H, J_H4;H3=3.3 Hz, J_H4;H5=0.7 Hz, H4,

Gal), 5.15 (t, 1H, ³J_H3;H2=³J_H3;H4=9.2 Hz, H3, Glc), 5.09 (dd, 1H,

³J_H2;H1=7.9 Hz, ³J_H2;H3=10.4 Hz, H2, Gal), 4.96 (d, 1H, J_NH;Ha=8.1 Hz,

3

NH), 4.94 (dd, 1H, ³J_H3;H2=10.4 Hz, ³J_H3;H4=3.4 Hz, H3, Gal), 4.82 (t,

1H, ³J_H2;H1=³J_H2;H3=9.6 Hz, H2, Glc), 4.46 (d, 1H, ³J_H1;H2=7.9 Hz, H1,

Gal), 4.43–4.37 (m, 2H, H6a, Glc, H^a), 4.16–4.02 (m, 3H, H6ab, Gal,

H6b, Glc), 3.85 (t_b, 1H, ³J_H5;H6b=³J_H5;H6a=6.8 Hz, H5, Gal), 3.72 (t, 1H,

Chem. Eur. J. 2013, 00, 0 – 0

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www.chemeurj.org

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H. Kunz et al.

³J_H4;H3=³J_H4;H5=9.5 Hz, H4, Glc), 3.62–3–54 (m, 1H, H1, Glc), 3.51–3.45

(m, 1H, H5, Glc), 3.07–2.92 (m, 2H, 2ꢃH^b), 2.78–2.64 (m, 2H, CH₂-exo),

2.13, 2.05, 2.04, 2.02, 2.01, 1.97, 1.95 (7ꢃs, 21H, CH₃, Ac), 1.41, 1.40 ppm

(2ꢃs, 18H, CH₃, Boc, tBu); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃):

d=170.93, 170.33, 170.14, 170.06, 169.97, 169.93, 169.02 (COOtBu, C=O,

11.1 Hz, CH₂, Bn), 4.79 (s, 2H, CH₂, Bn), 4.73–4.71 (m, 2H, CH₂, Bn),

4.70–4.60 (m, 3H, CH₂, Bn), 4.57 (d, 1H, ²J=11.5 Hz, CH₂, Bn), 4.44–

4.38 (m, 3H, H1, Gal {4.40}, CH₂, Bn), 4.28 (d, 1H, ²J=11.9 Hz, CH₂,

3

Bn), 4.03 (t, 1H, ³J_H4;H3=³J_H4;H5=9.5 Hz, H4, Glc), 3.91 (d, 1H, J_H4;H3

=

2.5 Hz, H4, Gal), 3.83 (dd, 1H, ²J=10.7 Hz, ³J=3.5 Hz, H6a, Glc), 3.76

(dd, 1H, ³J_H2;H1=7.8 Hz, ³J_H2;H3=9.7 Hz, H2, Gal), 3.64–3.55 (m, 2H,

H6b, Glc, H3, Glc), 3.54–3.47 (m, 1H, H6a, Gal), 3.43–3.35 (m, 4H, H6b,

Gal {3.41}, H5-Gal {3.39}, H3, Gal {3.38}, H1, Glc {3.34}), 3.35–3.27 (m,

1H, H5, Glc), 3.24 (t, 1H, ³J_H2;H1=³J_H2;H3=9.1 Hz, H2, Glc), 2.46 (dd,

Ac), 155.06 (C=O, Boc), 135.59, 134.52 (C_ipso, Thr), 129.41, 129.33 (C_o;m

,

Thr), 101.04 (C1, Gal), 81.97 (C_q, tBu), 79.64 (C_q, Boc), 77.95 (C1, Glc),

77.20 (C4, Glc), 76.34 (C5, Glc), 74.38 (C3, Glc), 72.19 (C2, Glc), 70.94

(C3, Gal), 70.59 (C5, Gal), 69.07 (C2, Gal), 66.56 (C4, Gal), 62.11 (C6,

Glc), 60.74 (C6, Gal), 54.76 (C^a), 37.93 (C^b), 37.35 (CH₂-exo), 28.27,

27.93 (CH₃, Boc, tBu), 20.83, 20.81, 20.70, 20.60, 20.58, 20.56, 20.47 ppm

(7ꢃCH₃, Ac); MS (ESI): m/z: 953.39 ([M+Na]⁺, calcd: 953.39). Tri-

fluoroacetic acid (10 mL) was added to the acetylated intermediate

(269 mg, 0.282 mmol) in dichloromethane (10 mL). Stirring was contin-

ued for 2 h. Solvent and acid were removed in vacuo with aqueous KOH

solution in the receiving flask. The remaining solid was co-evaporated

three times with toluene and dissolved in acetone (14 mL) and water

(5 mL). Fluoren-9-ylmethyl-succinimidylcarbonate (Fmoc-OSu) (120 mg,

0.355 mmol, 1.3 equiv) and NaHCO₃(88 mg, 1.05 mmol, 3.7 equiv) were

added. Stirring was continued at room temperature for 30 min. Acetic

acid (0.3 mL) was added, the solvent removed in vacuo and the remain-

ing solid co-evaporated twice with toluene/ethanol and once with tol-

uene. The crude product was subjected to flash chromatography (petrole-

um ether/ethyl acetate 1:1 to 1:2, then ethyl acetate +5% acetic acid) to

yield 50 (249 mg, 0.244 mmol, 87%) as a colorless amorphous solid. R_f=

0.10 (petroleum ether/ethyl acetate/acetic acid 10:10:0.3); [a]²_D³= +17.0

(c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY, TOCSY] (400 MHz, CDCl₃):

d=7.79–7.70 (m, 2H, H_Ar, Fmoc), 7.57–7.48 (m, 2H, H_Ar, Fmoc), 7.42–

7.35 (m, 2H, H_Ar, Fmoc), 7.30–7.25 (m, 2H, H_Ar, Fmoc), 7.10–7.04 (m,

1H, ²J=13.1 Hz, ³J=9.9 Hz, CH_2a-exo), 2.32 (d_b, 1H, ²J=12.6 Hz, CH_2b

-

exo), 1.96 (s, 3H, CH₃), 1.53, 1.42 ppm (2ꢃs, 18H, CH₃, Boc and tBu);

¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=164.45 (COOtBu),

153.90 (C=O, Boc), 139.08, 138.97, 138.73, 138.50, 138.21, 138.18, 138.07

(C_ipso, Ph), 133.33 (C^b), 128.43, 128.36, 128.31, 128.14, 128.11, 128.00,

127.95, 127.84, 127.75, 127.72, 127.64, 127.62, 127.54, 127.47, 127.35,

127.30, 127.08 (C_o;m;pPh, C^a-AS), 102.65 (C1, Gal), 85.16 (C3, Glc), 82.33

(C3, Gal), 80.95 (C_q, tBu), 80.70 (C2, Glc), 79.82 (C2, Gal), 79.52 (C_q,

Boc), 78.76 (C1, Glc), 78.48 (C5, Glc), 76.32 (C4, Glc), 75.21, 74.99, 74.62

(CH₂, Bn), 73.72 (C4, Gal), 73.41 (CH₂, Bn), 73.23 (C5, Gal), 72.58 (CH₂,

Bn), 68.26 (C6, Glc, C6, Gal), 37.17 (CH₂-exo), 28.27, 28.01 (CH₃, Boc

and tBu), 19.24 ppm (CH₃).

N-(Fluoren-9-ylmethoxycarbonyl)-C-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-

O-acetyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-threonine

(52):

Compound 51 (590 mg, 0.481 mmol) was dissolved in THF (10 mL), etha-

nol (5 mL) and acetic acid (1 mL). The mixture was degassed with a con-

stant stream of argon for 5 min. A spatula tip of Pd(OH)₂/C (15–

20 wt%) was added and the mixture degassed for another 5 min. Hydro-

genated was carried out at room temperature in an autoclave (25 bar) for

6 days. The reaction mixture was transferred to a flask and the solvent re-

moved in vacuo. After co-evaporation with toluene/ethanol (1:1, twice)

and toluene, pyridine (20 mL) and acetic anhydride (10 mL) were added.

Stirring was continued for 24 h. The excess reagents were removed in

vacuo and the remaining solid was co-evaporated twice with toluene. The

crude product was subjected to flash chromatography (petroleum ether/

ethyl acetate 3:2 to 1:1) to yield the acetylated intermediate (365 mg,

max. 0.409 mmol, 85%) as a pale-yellow amorphous solid. The product

consists mainly of the two diastereomers of the product and to a small

amount of a mixture of two diastereomers lacking an acetyl group at po-

sition 3 of the glucose-part. Trifluoroacetic acid (8 mL), triethylsilane

(1 mL) and water (1 mL) were added to the mixture of diastereomers in

chloroform (40 mL). After stirring for 5 h, solvent and acid were re-

moved in vacuo with aqueous KOH solution in the receiving flask. The

remaining solid was co-evaporated twice with toluene and taken up in

acetone (25 mL) and water (3 mL). FmocOSu (206 mg, 0.611 mmol,

1.5 equiv) and a spatula tip of NaHCO₃were added and stirring was con-

tinued at room temperature for 30 min. Additional water (3 mL) and an-

other spatula tip of NaHCO₃were added to adjust a pH of 8. After a few

min, acetic acid (5 mL) and toluene (20 mL) were added, the solvent was

removed in vacuo and the remaining solid co-evaporated twice with tol-

uene/ethanol and once with toluene. The crude product was subjected to

flash chromatography (petroleum ether/ethyl acetate 1:1 to 1:2, then

ethyl acetate +5% acetic acid) to give mainly 52 (377 mg, max.

0.321 mmol, 78%) as a colorless amorphous solid. The two diastereomers

of 52a and 52b as well as the two diastereomers 53a and 53b with the

missing acetyl group can be separated by preparative RP-HPLC (Phe-

nomenex Luna, MeCN/H₂O, (50:50) to (65:35), 60 min). 52a: colorless,

amorphous solid; analytical HPLC: t_R=30.1 min (Phenomenex Luna,

MeCN/H₂O, (50:50) to (65:35), 60 min, l=214 nm); preparative HPLC:

t_R=48.7 min (Phenomenex Luna, MeCN/H₂O, (50:50) to (65:35), 60 min,

l=214 nm); [a]²_D³=À17.0 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY,

TOCSY] (400 MHz, CDCl₃): d=7.76 (d, 2H, ³J=7.4 Hz, H_Ar, Fmoc),

7.61–7.55 (m, 2H, H_Ar, Fmoc), 7.40 (d, 2H, ³J=7.3 Hz, H_Ar, Fmoc), 7.30

3

4H, H_Ar, Tyr), 5.42 (d, 1H, J_NH;Ha=8.2 Hz, NH), 5.33 (d, 1H, J_H4;H3

=

3.3 Hz, H4, Gal), 5.15–5.06 (m, 2H, H3, Glc, H2, Gal), 4.96 (dd, 1H,

³J_H3;H2=10.4 Hz, ³J_H3;H4=3.4 Hz, H3, Gal), 4.80 (t, 1H, ³J_H2;H1=³J_H2;H3

=

3

9.5 Hz, H2, Glc), 4.70–4.62 (m, 1H, H^a), 4.46 (d, 1H, J_H1;H2=7.9 Hz, H1,

Gal), 4.43–4.25 (m, 4H, 2ꢃCH₂, Fmoc, CH, Fmoc, H6a, Glc), 4.17–4.02

3

(m, 3H, H6b, Glc, H6ab, Gal), 3.85 (t, 1H, J_H5;H6a=³J_H5;H6b=6.8 Hz, H5,

Gal), 3.69 (t, 1H, ³J_H4;H3=³J_H4;H5=9.5 Hz, H4, Glc), 3.55–3.47 (m, 1H,

H1, Glc), 3.42–3.36 (m, 1H, H5, Glc), 3.18 (dd, 1H, ²J=13.9 Hz, ³J=

4.9 Hz, H^ba), 3.05 (dd, 1H, ²J=13.8 Hz, ³J=6.5 Hz, H^bb), 2.75–2.63 (m,

2H, CH₂-exo), 2.14, 2.08, 2.03, 2.01, 1.96 ppm (5ꢃs, 21H, CH₃, Ac);

¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=176.21, 174.82, 170.53,

170.39, 170.18, 170.09, 170.02, 169.06 (COOH, C=O, Ac), 155.72 (C=O,

Fmoc), 143.61, 143.55 (C1a, Fmoc, C8a, Fmoc), 141.13 (C4a, Fmoc, C5a,

Fmoc), 135.80, 133.87 (C_ipso, Tyr), 129.63, 129.09 (C_o;m, Tyr), 127.68,

127.65 (C3, Fmoc, C6, Fmoc), 126.95 (C2, Fmoc, C7, Fmoc), 125.02,

124.93 (C1, Fmoc, C8, Fmoc), 119.93 (C4, Fmoc, C5, Fmoc), 100.88 (C1,

Gal), 77.61 (C1, Glc), 76.53 (C4, Glc), 76.18 (C5, Glc), 74.28 (C3, Glc),

72.07 (C2, Glc), 70.88 (C3, Gal), 70.45 (C5, Gal), 69.02 (C2, Gal), 66.97

(CH₂, Fmoc), 66.56 (C4, Gal), 62.05 (C6, Glc), 60.73 (C6, Gal), 54.48

(C^a), 46.93 (CH, Fmoc), 37.30, 37.21 (C^b, CH₂-exo), 20.72, 20.70, 20.58,

20.48, 20.39 ppm (7ꢃCH₃, Ac); MS (ESI): m/z: 1042.33 ([M+Na]⁺,

calcd: 1042.35).

(Z)-N-(tert-Butyloxycarbonyl)-C-[2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-

benzyl-b-d-galactopyranosyl)-b-d-glucopyranosyl]-a,b-dehydrovaline tert-

butyl ester (51)

Synthesis according to the general procedure: Hydroboration: 48

(673 mg, 0.695 mmol); 0.5m 9-BBN solution (4.17 mL, 2.08 mmol,

3.0 equiv) in THF; reaction time: 3 h; cross-coupling: 23a (292 mg,

0.868 mmol, 1.25 equiv); K₂CO₃; (288 mg, 2.08 mmol, 3.0 equiv); [PdCl₂-

ACHTUNGTRENNUNG(dppf)CH₂Cl₂] (57 mg, 10 mol%), DMF (5.0 mL); water (0.5 mL); reac-

tion time: 75 min. For the work-up, water and dichloromethane were

added. The organic layer was separated and the aqueous phase extracted

three times with dichloromethane. The combined organic layers were

dried over MgSO₄and the solvent was removed in vacuo. The remaining

residue was subjected to flash chromatography (petroleum ether/ethyl

acetate 6:1 to 4:1) to afford 51 (635 mg, 0.518 mmol, 75%) as a colorless

amorphous solid. R_f=0.25 (petroleum ether/ethyl acetate 4:1); [a]²_D³= +

25.9 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY, TOCSY, NOESY]

(400 MHz, CDCl₃): d=7.42–7.11 (m, 35H, H_Ar, Ph), 5.20 (d, 1H, ²J=

10.5 Hz, CH₂, Bn), 5.01 (d, 1H, ²J=11.5 Hz, CH₂, Bn), 4.98 (d, 1H, ²J=

3

(d, 2H, ³J=7.3 Hz, H_Ar, Fmoc), 5.47 (d, 1H, J_NH;Ha=8.7 Hz, NH), 5.34

(d, 1H, ³J_H4;H3=3.2 Hz, H4, Gal), 5.18–5.08 (m, 2H, H3, Glc {5.15}, H2,

3

Gal {5.11}), 4.95 (dd, 1H, J_H3;H2=10.4 Hz, J_H3;H4=3.3 Hz, H3, Gal), 4.74

(t, 1H, ³J_H2;H1=³J_H2;H3=9.4 Hz, H2, Glc), 4.52–4.36 (m, 5H H1, Gal

{4.47}, CH_2a, Fmoc {4.46}, H^a{4.45}, CH_2b, Fmoc {4.42}, H6a, Glc {4.40}),

4.22 (t, 1H, ³J=6.8 Hz, CH, Fmoc), 4.16–3.97 (m, 3H, H6a, Gal, H6b,

Gal {4.14–4.07}, H6b, Glc {4.02}), 3.86 (t, 1H, ³J_H5;H6a=³J_H5;H6b=6.7 Hz,

&

18

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ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ

These are not the final page numbers!

C-Glycosyl Amino Acids

FULL PAPER

H5, Gal), 3.70 (t, 1H, ³J_H4;H3=³J_H4;H5=9.3 Hz, H4, Glc), 3.50–3.34 (m,

2H, H1, Glc {3.39}, H5, Glc {3.43}), 2.33–2.25 (m, 1H, H^b), 2.15, 2.05,

2.03, 2.03, 2.02, 1.97, 1.96 (7ꢃs, 21H, CH₃, Ac), 1.68–1.53 (m, 1H, CH_2a-

exo), 1.37–1.30 (m_c, 1H, CH_2b-exo), 0.94 ppm (d, 3H, ³J=6.4 Hz, CH₃,

Thr); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=175.10, 170.68,

170.42, 170.19, 170.11, 170.00, 169.14 (C=O, Ac, COOH), 156.33 (C=O,

Fmoc), 143.75, 143.64 (C1a, Fmoc, C8a, Fmoc), 141.27, 141.25 (C4a,

Fmoc, C5a, Fmoc), 128.98, 128.18, 127.78, 127.75, 127.05 (C3, Fmoc, C6,

Fmoc, C2, Fmoc, C7, Fmoc), 125.25, 124.93 (C1, Fmoc, C8, Fmoc),

120.03, 120.01 (C4, Fmoc, C5, Fmoc), 101.07 (C1, Gal), 76.62 (C4, Glc),

76.45 (C5, Glc), 75.82 (C1, Glc), 74.24 (C3, Glc), 72.47 (C2, Glc), 70.96

(C3, Gal), 70.56 (C5, Gal), 69.09 (C2, Gal), 66.94 (CH₂, Fmoc), 66.60

(C4, Gal), 62.40 (C6, Glc), 60.79 (C6, Gal), 57.89 (C^a), 47.08 (CH,

Fmoc), 34.72 (CH₂-exo), 32.57 (C^b), 20.81, 20.64, 20.60, 20.48 (CH₃, Ac),

15.35 ppm (CH₃, Thr); MS (ESI): m/z: 980.32 ([M+Na]⁺, calcd: 980.32);

HR-MS (ESI): m/z: 980.3167 ([M+Na]⁺, calcd for C₄₆H₅₅NO₂₁:

980.3164). 52b: colorless, amorphous solid; analytical HPLC: t_R=

28.2 min (Phenomenex Luna, MeCN/H₂O, (50:50) to (65:35), 60 min, l=

214 nm); preparative HPLC: t_R=45.9 min (Phenomenex Luna, MeCN/

H₂O, (50:50) to (65:35), 60 min, l=214 nm); [a]²_D³= +0.4 (c=1.0,

143.73 (C1a, Fmoc, C8a, Fmoc), 141.43, 141.38 (C4a, Fmoc, C5a, Fmoc),

127.83, 127.78, 127.11, 126.98 (C3, Fmoc, C6, Fmoc, C2, Fmoc, C7,

Fmoc), 124.79, 124.77 (C1, Fmoc, C8, Fmoc), 120.14, 120.03 (C4, Fmoc,

C5, Fmoc), 101.90 (C1, Gal), 83.01 (C4, Glc), 75.95 (C5, Glc), 75.04 (C1,

Glc), 74.60 (C3, Glc), 73.23 (C2, Glc), 71.36 (C5, Gal), 70.90 (C3, Gal),

68.61 (C2, Gal), 66.83 (C4, Gal), 66.54 (CH₂, Fmoc), 63.16 (C6, Glc),

61.84 (C6, Gal), 57.97 (C^a), 47.16 (CH, Fmoc), 34.66 (CH₂-exo), 33.69

(C^b), 20.83, 20.63, 20.60, 20.48, 20.37 (CH₃, Ac), 16.88 ppm (CH₃, Thr);

MS (ESI): m/z: 938.30 ([M+Na]⁺, calcd: 938.31). 53b: colorless, amor-

phous solid; analytical HPLC: t_R=22.2 min (Phenomenex Luna, MeCN/

H₂O, (50:50) to (65:35), 60 min, l=214 nm); preparative HPLC: t_R=

37.9 min (Phenomenex Luna, MeCN/H₂O, (50:50) to (65:35), 60 min, l=

214 nm); [a]²_D³= +9.2 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY]

(400 MHz, CDCl₃): d=7.77 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.59 (t, 2H,

³J=7.8 Hz, H_Ar, Fmoc), 7.41 (t, 2H, ³J=7.3 Hz, H_Ar, Fmoc), 7.35–7.28

3

(m, 2H, H_Ar, Fmoc), 5.37 (s_b, 1H, H4, Gal), 5.24 (dd, 1H, J_H2;H1=8.1 Hz,

³J_H2;H3=10.2 Hz, H2, Gal), 5.00 (d_b, 1H, ³J_H3;H2=10.2 Hz, H3, Gal), 4.73

(t, 1H, ³J_H2;H1=³J_H2;H3=8.9 Hz, H2, Glc), 4.57–4.48 (m, 2H, H1, Gal

{4.54}, H^a{4.52}), 4.43 (dd, 1H, ²J=10.1 Hz, ³J=7.4 Hz, CH_2a, Fmoc),

4.35 (dd, 1H, ²J=10.6 Hz, ³J=7.2 Hz, CH_2b, Fmoc), 4.32–4.20 (m, 2H,

H6a, Glc {4.30}, CH, Fmoc {4.23}), 4.17–4.06 (m, 2H, H6a, Gal, H6b,

Gal), 4.04–3.97 (m, 2H, H5, Gal {4.01}, H6b, Glc {4.00}), 3.73 (t, 1H,

³J_H3;H2=³J_H3;H4=8.1 Hz, H3, Glc), 3.61–3.52 (m, 2H, H1, Glc, H5, Glc),

3.51–3.43 (m, 1H, H4, Glc), 2.46 (s_b, 1H, H^b), 2.16, 2.08, 2.07, 2.03, 1.98,

1.95 (6ꢃs, 18H, CH₃, Ac), 1.63–1.53 (m, 1H, CH_2a-exo), 1.44–1.35 (m,

1H, CH_2b-exo), 0.92 ppm (d, 3H, ³J=6.7 Hz, CH₃, Thr); ¹³C NMR [BB,

HSQC] (100.6 MHz, CDCl₃): d=175.27, 170.94, 170.51, 170.06, 169.99,

169.50 (C=O, Ac, COOH), 156.47 (C=O, Fmoc), 143.60 (C1a, Fmoc, C8a,

Fmoc), 141.27, 141.25 (C4a, Fmoc, C5a, Fmoc), 127.80, 127.10 (C3, Fmoc,

C6, Fmoc, C2, Fmoc, C7, Fmoc), 125.04, 124.96 (C1, Fmoc, C8, Fmoc),

120.05 (C4, Fmoc, C5, Fmoc), 101.97 (C1, Gal), 83.18 (C4, Glc), 75.31

(C5, Glc), 74.66 (C1, Glc), 74.37 (C3, Glc), 73.45 (C2, Glc), 71.37 (C5,

Gal), 70.83 (C3, Gal), 68.64 (C2, Gal), 67.26 (CH₂, Fmoc), 66.77 (C4,

Gal), 63.03 (C6, Glc), 61.72 (C6, Gal), 55.79 (C^a), 47.06 (CH, Fmoc),

34.68 (CH₂-exo), 31.61 (C^b), 20.74, 20.59, 20.47, 20.38 (CH₃, Ac),

15.72 ppm (CH₃, Thr); MS (ESI): m/z: 938.29 ([M+Na]⁺, calcd: 938.31).

1

CHCl₃); H NMR [¹H¹H-COSY, TOCSY] (400 MHz, CDCl₃): d=7.76 (d,

2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.59 (t, 2H, ³J=7.3 Hz, H_Ar, Fmoc), 7.40 (t,

2H, ³J=7.4 Hz, H_Ar, Fmoc), 7.35–7.28 (m, 2H, H_Ar, Fmoc), 5.37 (d, 1H,

³J_NH;Ha=8.9 Hz, NH), 5.34 (d, 1H, ³J_H4;H3=3.1 Hz, H4, Gal), 5.19 (t, 1H,

3

³J_H3;H2=³J_H3;H4=9.0 Hz, H3, Glc), 5.11 (dd, 1H, ³J_H2;H1=8.0 Hz, J_H2;H3

=

10.4 Hz, H2, Gal), 4.95 (dd, 1H, ³J_H3;H2=10.4 Hz, ³J_H3;H4=3.4 Hz, H3,

Gal), 4.75 (t, 1H, J_H2;H1=³J_H2;H3=9.5 Hz, H2, Glc), 4.56–4.38 (m, 4H, H^a

3

{4.51}, H1, Gal {4.47}, H6a, Glc {4.44}, CH_2a, Fmoc {4.42}), 4.34 (dd, 1H,

²J=10.5 Hz, ³J=7.4 Hz, CH_2b, Fmoc), 4.22 (t, 1H, ³J=7.0 Hz, CH,

Fmoc), 4.15–4.01 (m, 3H, H6a, Gal, H6b, Gal, H6b, Glc), 3.86 (t, 1H,

³J_H5;H6a=³J_H5;H6b=7.0 Hz, H5, Gal), 3.71 (t, 1H, ³J_H4;H3=³J_H4;H5=9.4 Hz,

H4-Glc), 3.66–3.57 (m, 2H, H1-Glc, H5-Glc), 2.45 (s_b, 1H, H^b), 2.08,

2.05, 2.03, 2.01, 1.96, 1.90 (6ꢃs, 21H, CH₃, Ac), 1.62–1.51 (m, 1H, CH_2a-

exo), 1.42–1.31 (m, 1H, CH_2b-exo), 0.90 ppm (d, 3H, ³J=6.4 Hz, CH₃,

Thr); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=175.52, 170.73,

170.41, 170.21, 170.12, 169.86, 169.07 (C=O, Ac, COOH), 156.48 (C=O,

Fmoc), 143.71, 143.53 (C1a, Fmoc, C8a, Fmoc), 141.26, 141.23 (C4a,

Fmoc, C5a, Fmoc), 128.99, 128.18, 127.79, 127.08 (C3, Fmoc, C6, Fmoc,

C2, Fmoc, C7, Fmoc), 125.03, 124.95 (C1, Fmoc, C8, Fmoc), 120.02 (C4,

Fmoc, C5, Fmoc), 100.94 (C1, Gal), 76.73 (C4, Glc), 76.52 (C5, Glc),

74.90 (C1, Glc), 74.08 (C3, Glc), 72.70 (C2, Glc), 70.97 (C3, Gal), 70.60

(C5, Gal), 69.06 (C2, Gal), 67.25 (CH₂, Fmoc), 66.62 (C4, Gal), 62.51

(C6, Glc), 60.82 (C6, Gal), 55.78 (C^a), 47.06 (CH, Fmoc), 34.84 (CH₂-

exo), 31.59 (C^b), 20.81, 20.73, 20.61, 20.58, 20.50 ppm (CH₃, Ac), 15.61

(CH₃, Thr); MS (ESI): m/z: 980.32 ([M+Na]⁺, calcd: 980.32); HRMS

(ESI): m/z calcd for C₄₆H₅₅NO₂₁: 980.3164 [M+Na]⁺; found: 980.3167.

Side products with missing acetyl group: N-(Fluoren-9-ylmethoxycarbon-

yl)-C-[2,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl)-

b-d-glucopyranosyl]-threonine; 53a: colorless, amorphous solid; analyti-

cal HPLC: t_R=25.2 min (Phenomenex Luna, MeCN/H₂O, (50:50) to

(65:35), 60 min, l=214 nm); preparative HPLC: t_R=41.9 min (Phenom-

Amino-4,7,10-trioxadodecanylamido-N-l-prolyl-l-alanyl-l-histidyl-l-

glycyl-l-valyl-l-threonyl-l-seryl-l-alanyl-l-prolyl-l-aspartyl-l-threonyl-l-

arginyl-l-prolyl-l-alanyl-l-prolyl-l-glycyl-C-[2,3,4-tri-O-benzyl-b-l-fuco-

pyranosyl]-l-tyrosyl-l-threonyl-l-alanyl-l-prolyl-l-prolyl-l-alanine (54):

The synthesis was carried out in an Applied Biosystems ABI 433A pep-

tide synthesizer starting with Tentagel R resin (563 mg) pre-loaded with

Fmoc-Ala-O-Trt (0.09 mmol, loading 0.16 mmolg^À1). For the coupling re-

actions, the amino acids Fmoc-Ala-OH, Fmoc-Arg

Asp-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Ser-

(tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Val-OH were employed. In

ACHTUNGTREN(NGNU Pmc)-OH, Fmoc-

AHCTUNGTRENNUNG

A

ACHTUNGTRENNUNG

every coupling cycle, the N-terminal Fmoc group was removed by treat-

ment of the resin with a solution of piperidine (20%) in NMP for at least

3ꢃ2.5 min. The coupling of Fmoc amino acids (1 mmol or 10 equiv based

on the loaded resin) was carried out with HBTU (1 mmol), HOBt

(1 mmol), and diisopropylethylamine (DIPEA, 2 mmol) in DMF (20–

30 min vortex). After every coupling step, unreacted amino groups were

capped by treatment with a mixture of Ac₂O (0.5m), DIPEA (0.125m),

and HOBt (0.015m) in NMP (10 min vortex). After the coupling of the

first two proline-building blocks, Ala⁴was added in a double coupling

step. Coupling of C-glycoside 39 (87 mg, 0.106 mmol, 1.18 equiv based on

the loaded resin) was performed by using HATU (48.3 mg, 0.127 mmol,

1.20 equiv with respect to 39), HOAt (17.4 mg, 0.128 mmol, 1.21 equiv)

and N-methylmorpholine (NMM, 30 mL, 0.273 mmol, 2.57 equiv) for acti-

vation (8 h, vortex). The following glycine-building block was added in a

double coupling step. After coupling of the remaining amino acids by the

standard procedure, a triethylene glycol spacer was added by using N-

enex Luna, MeCN/H₂O, (50:50) to (65:35), 60 min, l=214 nm); [a]_D²³

=

À1.1 (c=1.0, CHCl₃); ¹H NMR [¹H¹H-COSY] (400 MHz, CDCl₃): d=

7.77 (d, 2H, ³J=7.5 Hz, H_Ar, Fmoc), 7.61–7.56 (m, 2H, H_Ar-Fmoc), 7.40

(t, 2H, ³J=7.4 Hz, H_Ar-Fmoc), 7.31 (t, 2H, ³J=7.4 Hz, H_Ar, Fmoc), 5.65

3

(d, 1H, J_NH;Ha=8.9 Hz, NH), 5.37 (d, 1H, J_H4;H3=3.3 Hz, H4, Gal), 5.27

(dd, 1H, ³J_H2;H1=8.4 Hz, ³J_H2;H3=10.2 Hz, H2, Gal), 5.01 (dd, 1H,

³J_H3;H2=10.4 Hz, ³J_H3;H4=3.4 Hz, H3, Gal), 4.66 (t, 1H, ³J_H2;H1=³J_H2;H3

=

9.6 Hz, H2, Glc), 4.59–4.52 (m, 2H, H1, Gal {4.57}, CH_2a, Fmoc {4.55}),

4.43 (dd, 1H, ²J=10.6 Hz, ³J=6.1 Hz, CH_2b, Fmoc), 4.38–4.33 (m_c, 1H,

H^a), 4.22 (m_c, 1H, CH, Fmoc), 4.18–4.08 (m, 3H, H6a, Glc {4.15}, H6a,

Gal, H6b, Gal

3.85 (dd, 1H, ²J=11.5 Hz, ³J=6.5 Hz, H6b, Glc), 3.66 (t, 1H, J_H3;H2

ACHTUNGTRENNUNG

{4.12}), 4.00 (t, 1H, ³J_H5;H6a=³J_H5;H6b=6.4 Hz, H5, Gal),

3

(9H-Fluoren-9-yl)-methoxycarbonylamido-4,7,10-trioxadecanic

acid^[38]

=

³J_H3;H4=8.7 Hz, H3, Glc), 3.38 (t, 1H, ³J_H4;H3=³J_H4;H5=8.8 Hz, H4, Glc),

3.29–3.17 (m, 2H, H1, Glc {3.24}, H5, Glc {3.21}), 2.26–2.18 (m, 1H, H^b),

2.17, 2.10, 2.05, 2.03, 1.99, 1.87 (6ꢃs, 18H, CH₃, Ac), 1.62–1.51 (m, 1H,

CH_2a-exo), 1.40–1.30 (m, 1H, CH_2b-exo), 1.01 ppm (d, 3H, ³J=6.7 Hz,

CH₃, Thr); ¹³C NMR [BB, HSQC] (100.6 MHz, CDCl₃): d=170.92,

170.57, 170.06, 169.97 (C=OAc, COOH), 156.24 (C=O, Fmoc), 143.88,

(444 mg, 0.100 mmol, 1.00 equiv based on the loaded resin) by using

HATU (456 mg, 1.20 mmol, 1.20 equiv), HOAt (164 mg, 1.20 mmol,

1.20 equiv), and NMM (0.28 mL, 2.50 mmol, 2.5 equiv) for activation

(8 h, vortex). The N-terminal Fmoc group was removed by using piperi-

dine (20%) in NMP. The peptide was detached from the resin with simul-

taneous removal of all side-chain protecting groups by shaking with TFA

Chem. Eur. J. 2013, 00, 0 – 0

ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

&

19

&

ÞÞ

These are not the final page numbers!

H. Kunz et al.

(10 mL), TIS (1 mL) and H₂O (1 mL) for 3 h. The solution was filtered,

the resin washed with TFA (4ꢃ2 mL) and toluene (12 mL) were added.

The liquid phase was evaporated in vacuo and the remaining solid co-

evaporated three times with toluene/methanol (5:3). After drying in high

vacuum, the crude product (284 mg) was taken up in methanol (1.5 mL)

and dropped into ice-cold diethyl ether (20 mL) under stirring. The pre-

cipitate was centrifugated (3000 rpm, 5 min) and the supernatant solution

was decanted. The colorless solid was dissolved in methanol (5 mL) and

transferred to a flask. Toluene (5 mL) was added, the solvents were re-

moved in vacuo and the remaining colorless amorphous solid was dried

in high vacuum. Purification by preparative RP-HPLC (Phenomenex

Luna, MeCN/H₂O+0.1% TFA, (5:95) to (70:30), 60 min) afford C-glyco-

peptide 54 (57 mg, 20.8 mmol, 23%) as a colorless amorphous solid. Ana-

lytical HPLC: t_R=36.1 min (Phenomenex Luna, MeCN/H₂O+0.1%

TFA, (5:95) to (65:35), 60 min, l=214 nm); preparative HPLC: t_R=

41.5 min (Phenomenex Luna, MeCN/H₂O+0.1% TFA, (5:95) to (70:30),

7.19–7.13 (m, 4H, H_Ar, Tyr), 4.98 (d, 1H, ²J=11.8 Hz, CH₂, Bn), 4.82 (d,

1H, ²J=11.7 Hz, CH₂, Bn), 4.72–4.58 (m, 9H, H^a-Tyr {4.69}, H^a-His

{4.69}, H^a-Arg {4.69}, CH₂, Bn {4.65}, H^a-Asp {4.67}, CH₂, Bn {4.60}, H^a-

Ala {4.65, 4.63, 4.61}), 4.48–4.18 (m, 16H, H^a-Ser {4.46}, H^a-Pro {4.45,

4.41, 4.40, 4.37, 4.36, 4.33}, H^a-Thr {4.44, 4.37, 4.34}, H^a-Ala {4.36}, H^a-

Val {4.29}, H^a-Ala {4.27}, H^b-Thr {4.27, 4.23, 4.19}), 4.06–3.91 (m, 4H,

H^aa-Gly¹{4.03}, H^aa-Gly²{3.93}, H4, Fuc {3.93}, H^a-Gly1 {3.91}), 3.89–

3.55 (m, 31H, H^ba-Ser {3.87}, 4ꢃH^d-Pro {3.79}, H^ba-Ser {3.79}, 2ꢃCH₂O,

spacer {3.78}, 2ꢃCH₂O, spacer {3.72}, H3, Fuc {3.60}, 4ꢃH^d-Pro {3.68},

H^ab-Gly²{3.67}, 8ꢃCH₂O, spacer {3.69–3.62}, H2, Fuc {3.57}, 4ꢃH^d-Pro

{3.62}), 3.44 (m, 1H, H5, Fuc), 3.38–3.33 (m, 2H, H1, Fuc {3.35}, H^ba-His

{3.35}), 3.25–3.11 (m, 6H, H^bb-His {3.21}, 2ꢃH^d-Arg {3.22–3.18}, H^ba-Tyr

{3.18}, 2ꢃCH₂, spacer {3.13}), 3.07 (d_b, 1H, ²J=14.4 Hz, CH_2a-exo), 3.04–

2.85 (m, 3H, H^bb-Tyr {3.02}, H^ba-Asp {2.92}, 1ꢃCH₂, spacer {2.90}), 2.76–

2.66 (m, 2H, H^bb-Asp, 1ꢃCH₂, spacer), 2.62 (dd, 1H, ²J=14.4 Hz, ³J=

9.4 Hz, CH_2b-exo), 2.33–2.14 (m, 7H, 6ꢃH^b-Pro {2.29–2.15}), H^b-Val

{2.17}), 2.13–1.66 (m, 22H, 12ꢃH^g-Pro {2.12–1.92}, 6ꢃH^b-Pro {2.05–1.91},

H^ba-Arg {1.86}, H^bb-Arg {1.75}, 2ꢃH^g-Arg {1.69}), 1.44–1.30 (m, 15H,

H^b-Ala {1.41, 1.40, 1.37, 1.36, 1.35}), 1.22–1.14 (m, 12H, H^g-Thr {1.20,

1.19, 1.15}, H6abc-Fuc {1.19}), 1.01–0.93 ppm (m, 6H, H^ga-Val {1.01},

H^gb-Val {0.99}); ¹³C NMR [BB, HSQC, HMBC] (150.9 MHz, MeOD):

d=174.53, 173.75, 173.73, 173.13, 173.11, 172.97, 172.64, 172.56, 172.51,

172.29, 171.92, 171.75, 171.54, 171.26, 171.06, 170.94, 170.86, 170.80,

170.68, 170.37, 170.31, 170.08 (C=O), 157.11 (C=NH), 138.65, 138.47

(C_ipso, Ph), 137.87, 134.59 (C_ipso, Tyr), 133.72 (C^g-His), 129.16, 128.72

(C_o;m, Tyr), 128.00, 127.78, 127.72, 127.35, 127.31 (C^e-His, C_o;m;p, Ph),

117.64 (C^d-His), 83.50 (C3, Fuc), 80.12 (C1, Fuc), 77.87 (C2, Fuc), 74.73

(CH₂, Bn), 73.78 (C5, Fuc), 70.37 (CH₂, Bn), 70.08, 69.99, 69.82, 69.72

(CH₂O, spacer), 68.64 (C4, Fuc), 67.22, 67.09, 66.89 (C^b-Thr), 66.48, 66.29

(CH₂O, spacer), 61.55 (C^b-Ser), 61.19, 60.93, 60.24, 59.98, 59.82, 59.56

(C^a-Pro), 58.87 (C^a-Val), 58.51, 58.44, 58.40 (C^a-Thr), 55.30 (C^a-Ser),

55.19 (C^a-Tyr), 52.24 (C^a-His), 50.64 (C^a-Arg), 50.40 (C^a-Asp), 49.61,

48.17 (C^a-Ala), 48.09–47.05 (3ꢃC^a-Ala, 6ꢃC^d-Pro), 42.21, 41.95 (C^a-Gly),

40.71 (C^d-Arg), 39.20 (CH₂, spacer), 37.37 (CH₂-exo), 36.97 (C^b-Tyr),

34.35 (C^b-Asp), 33.92 (CH₂, spacer), 30.16 (C^b-Val), 29.49, 29.31, 29.08,

29.03, 28.66 (6ꢃC^b-Pro), 27.93 (C^b-Arg), 26.58 (C^b-His), 24.82, 24.72,

24.61, 24.56, 24.48, 24.40 (C^g-Pro), 24.25 (C^g-Arg), 18.89, 18.79, 18.52 (C^g-

Thr), 18.45 (C^ga-Val), 17.43 (C^gb-Val), 16.10 (C6, Fuc), 15.90, 15.85,

60 min, l=214 nm); [a]_D²³=À84.0 (c=1.0, MeOH); ¹H NMR [¹H¹H-

3

COSY, TOCSY, ROESY] (400 MHz, MeOD): d=8.79 (d, 1H, J_He;Hd

=

1.3 Hz, H^e-His), 7.44–7.37 (m, 5H, H^g-His {7.42}, H_Ar, Ph), 7.36–7.25 (m,

8H, H_Ar, Ph), 7.17–7.08 (m, 7H, H_Ar-Ph, H_Ar, Tyr), 4.98–4.91 (m, 2H,

CH_2,Bn), 4.81 (d, 1H, J=11.7 Hz, CH₂, Bn), 4.71–4.56 (m, 10H, H^a-Tyr

2

{4.69}, H^a-His {4.68}, H^a-Arg {4.68}, CH₂-Bn {4.68}, H^a-Asp {4.66}, CH₂,

Bn {4.66}, CH₂, Bn {4.64}, H^a-Ala {4.62, 4.61, 4.59}), 4.50–4.15 (m, 16H,

H^a-Ser {4.45}, H^a-Pro {4.43, 4.40, 4.39, 4.38, 4.35, 4.33}, H^a-Thr {4.42, 4.36,

4.34}, H^a-Ala {4.34}, H^a-Val {4.27}, H^a-Ala {4.26}, H^b-Thr {4.25, 4.23,

4.17}), 4.06–3.87 (m, 3H, H^aa -Gly¹{4.03}, H^aa -Gly²{3.93}, H^ab -Gly1

{3.91}), 3.86–3.53 (m, 31H, H4, Fuc {3.84}, H^b-Ser {3.84}, 4ꢃH^d-Pro

{3.77}, H^bb-Ser {3.77}, 1ꢃCH₂O, spacer {3.76}, 3ꢃCH₂O, spacer {3.71–

3.69}, H3, Fuc {3.67}, 4ꢃH^d-Pro {3.67}, H^ab -Gly²{3.67}, 8ꢃCH₂O, spacer

3

{3.66–3.60}, H2, Fuc {3.63}, 4ꢃH^d-Pro {3.61}), 3.44 (q_b, 1H, J_H5;H6abc

=

6.5 Hz, H5, Fuc), 3.40–3.28 (m, 2H, H1, Fuc {3.36}, H^ba-His {3.34}), 3.24–

3.09 (m, 6H, H^bb-His {3.19}, 2ꢃH^d-Arg {3.17}, H^ba-Tyr {3.15}, 2ꢃCH₂,

spacer {3.12}), 3.06 (d_b, 1H, ²J=14.2 Hz, CH_2a-exo), 3.04–2.83 (m, 3H,

H^bb-Tyr {2.99}, H^ba-Asp {2.88}, 1ꢃCH₂, spacer {2.88}), 2.72–2.64 (m, 2H,

H^bb-Asp {2.68}, 1ꢃCH₂, spacer {2.67}), 2.59 (dd, 1H, ²J=14.5 Hz, ³J=

9.2 Hz, CH_2b-exo), 2.29–2.11 (m, 7H, 6ꢃH^b-Pro {2.23–2.13}), H^b-Val

{2.15}), 2.12–1.59 (m, 22H, 12ꢃH^g-Pro {2.07–1.89}, 6ꢃH^b-Pro {2.01–1.87},

H^ba -Arg {1.84}, H^bb -Arg {1.71}, 2ꢃH^g-Arg {1.65}), 1.42–1.26 (m, 15H,

H^b-Ala {1.39, 1.37, 1.34, 1.33, 1.32}), 1.21–1.09 (m, 12H, H^g-Thr {1.18,

1.14, 1.13}, H6, Fuc {1.13}), 1.01–0.93 (m, 6H, H^ga-Val {0.98}, H^gb-

Val ppm {0.97}); ¹³C NMR [BB, HSQC, HMBC] (100.6 MHz, MeOD):

d=174.55, 173.87, 173.83, 173.79, 173.21, 172.89, 172.68, 172.60, 172.58,

172.34, 172.00, 171.79, 171.71, 171.32, 171.17, 171.14, 170.98, 170.91,

170.89, 170.75, 170.44, 170.37, 170.15 (C=O), 157.20 (C=NH), 138.82,

138.66, 138.59 (C_ipso, Ph), 137.88, 134.65 (C_ipso, Tyr), 133.75 (C^g-His),

129.23 (C_o;m, Tyr), 128.79 (C^e-His), 128.15, 128.08, 128.04, 127.97, 127.91,

127.88, 127.60, 127.44, 127.39 (C_o;m;p, Ph), 117.66 (C^d-His), 84.86 (C3-

Fuc), 80.36 (C1, Fuc), 78.31 (C2, Fuc), 77.26 (C4, Fuc), 74.95, 74.83 (CH₂,

Bn), 73.98 (C5, Fuc), 71.75 (CH_2,Bn), 70.14, 70.05, 69.88, 69.79 (CH₂O,

spacer), 67.30, 67.22, 66.96 (C^b-Thr), 66.54, 66.37 (CH₂O, spacer), 61.64

(C^b-Ser), 61.30, 60.97, 60.42, 60.06, 59.90, 59.67 (C^a-Pro), 58.94 (C^a-Val),

58.64, 58.54, 58.46 (C^a-Thr), 55.38 (C^a-Ser), 55.25 (C^a-Tyr), 52.36 (C^a-

His), 50.70 (C^a-Arg), 50.48 (C^a-Asp), 49.75 (C^a-Ala), 48.25–47.05 (4ꢃC^a-

Ala, 6ꢃC^d-Pro), 42.33, 42.12 (C^a-Gly), 40.79 (C^d-Arg), 39.28 (CH₂,

spacer), 37.33 (CH₂-exo), 37.06 (C^b-Tyr), 34.45 (C^b-Asp), 34.01 (CH₂,

spacer), 30.22 (C^b-Val), 29.54, 29.37, 29.15, 29.08, 27.96 (6ꢃC^b-Pro), 28.00

(C^b-Arg), 26.58 (C^b-His), 24.87, 24.80, 24.68, 24.62, 24.55, 24.47 (C^g-Pro),

24.33 (C^g-Arg), 18.97, 18.87, 18.60 (C^g-Thr), 18.53 (C^ga-Val), 17.54 (C^gb-

Val), 16.29 (C6, Fuc), 16.19, 15.92, 15.83, 15.75, 15.34 ppm (C^b-Ala); MS

(ESI): m/z: 1374.78 ([M+2H]²⁺, calcd: 1374.70); HRMS (ESI): m/z

calcd for C₁₃₁H₁₉₀N₂₈O₃₇: 1374.7002 [M+2H]²⁺; found: 1374.7195. Be-

sides the desired product, a small amount of a side product (10 mg,

3.8 mmol, 4%) was isolated with a missing benzyl group at OH-3. Color-

less amorphous solid; analytical HPLC: t_R=28.0 min (Phenomenex

Luna, MeCN/H₂O+0.1% TFA, (5:95) to (65:35), 60 min, l=214 nm);

preparative HPLC: t_R=34.5 min (Phenomenex Luna, MeCN/H₂O+

0.1% TFA, (5:95) to (70:30), 60 min, l=214 nm); [a]²_D³=À74.3 (c=1.0,

CHCl₃); ¹H NMR [¹H¹H-COSY, TOCSY, ROESY] (600 MHz, MeOD):

d=8.79 (s_b, 1H, H^e-His), 7.45–7.28 (m, 11H, H^g-His {7.42}, H_Ar, Ph),

15.70, 15.63, 15.23 ppm (C^b-Ala); MS (ESI): m/z: 1329.75 ([M+2H]²⁺

,

calcd: 1329.67);

Amino-4,7,10-trioxa-dodecanylamido-N-l-prolyl-l-alanyl-l-histidyl-l-

glycyl-l-valyl-l-threonyl-l-seryl-l-alanyl-l-prolyl-l-aspartyl-l-threonyl-l-

arginyl-l-prolyl-l-alanyl-l-prolyl-l-glycyl-C-[4,6-di-O-acetyl-2,3-di-O-

benzyl-b-d-glucopyranosyl]-l-tyrosyl-l-threonyl-l-alanyl-l-prolyl-l-

prolyl-l-alanine (55): The synthesis was carried out as described for 55

starting from Tentagel R resin (563 mg) pre-loaded with Fmoc-Ala-O-Trt

(0.09 mmol, loading 0.16 mmolg^À1). Coupling of C-glycoside 42 (104 mg,

0.126 mmol, 1.26 equiv based on the loaded resin) was performed using

HATU (57.5 mg, 0.151 mmol, 1.20 equiv with respect to 42), HOAt

(20.7 mg, 0.152 mmol, 1.21 equiv) and NMM (35 mL, 0.318 mmol,

2.52 equiv) for activation (8 h, vortex). Final purification by preparative

RP-HPLC (Phenomenex Luna, MeCN/H₂O+0.1% TFA, (5:95) to

(70:30), 60 min) afforded C-glycopeptide 55 (94 mg, 34.3 mmol, 38%) as

a colorless amorphous solid. Analytical HPLC: t_R=30.3 min (Phenomen-

ex Luna, MeCN/H₂O+0.1% TFA, (5:95) to (70:30), 60 min, l=214 nm);

preparative HPLC: t_R=38.3 min (Phenomenex Luna, MeCN/H₂O+

0.1% TFA, (5:95) to (70:30), 60 min, l=214 nm); [a]²_D³=À92.8 (c=1.0,

MeOH); ¹H NMR [¹H¹H-COSY, ROESY] (400 MHz, MeOD): d=8.80

3

(d, 1H, J_He;Hd=1.3 Hz, H^e-His), 7.44–7.25 (m, 11H, H^g-His {7.42}, H_Ar

,

Ph), 7.21–7.12 (m, 4H, H_Ar, Tyr), 4.96–4.85 (m, 2H, H4, GlcACHTNUGTRENUNG{4.93}, CH₂,

Bn {4.88}), 4.80 (d, 1H, ²J=11.4 Hz, CH₂, Bn), 4.75–4.55 (m, 9H, H^a-Tyr

{4.72}, H^a-His {4.69}, CH₂, Bn {4.69}, H^a-Arg {4.68}, CH₂, Bn {4.66}, H^a-

Asp {4.66}, H^a-Ala {4.65, 4.64, 4.60}), 4.52–4.17 (m, 17H, H^a-Ser {4.46},

H^a-Pro {4.44, 4.43, 4.41, 4.39, 4.38, 4.35}, H^a-Thr {4.43, 4.38, 4.35}, H^a-Ala

{4.35}, H^a-Val {4.29}, H^a-Ala {4.27}, H6a, Glc {4.20}, H^b-Thr {4.25, 4.22,

4.19}), 4.06–3.90 (m, 4H, H^aa-Gly¹{4.03}, H^aa-Gly²{3.94}, H6b, Glc

{3.93}, H^ab-Gly¹{3.93}), 4.88–3.57 (m, 28H, H^ba-Ser {3.86}, 4ꢃH^d-Pro

{3.78}, H^ba-Ser {3.78}, 1ꢃCH₂O, spacer {3.78}, 3ꢃCH₂O, spacer {3.72–

3.69}, H3, Glc {3.67}, 4ꢃH^d-Pro {3.66}, H^ab-Gly²{3.66}, 8ꢃCH₂O, spacer

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C-Glycosyl Amino Acids

FULL PAPER

{3.67–3.60}, 4ꢃH^d-Pro {3.62}), 3.56–3.47 (m, 2H, H5, Glc {3.53}, H1, Glc

{3.51}), 3.40–3.29 (m, 2H, H^ba-His {3.36}, H2, Glc {3.32}), 3.25–2.86 (m,

10H, H^bb-His {3.20}, 2ꢃH^d-Arg {3.19}, H^ba-Tyr {3.18}, 2ꢃCH₂, spacer

{3.12}, CH_2a-exo {3.06}, H^bb-Tyr {2.99}, H^ba-Asp {2.89}, 1ꢃCH₂, spacer

(m, 12H, H^g-Thr {1.18, 1.16, 1.13}, H6, Fuc {1.15}), 1.01–0.95 ppm (m,

6H, H^ga-Val {0.98}, H^gb-Val {0.97}); ¹³C NMR [BB, HSQC, HMBC]

(150.9 MHz, MeOD): d=174.53, 173.83, 173.80, 173.20, 172.85, 172.68,

172.59, 172.54, 172.35, 171.98, 171.77, 171.70, 171.30, 171.15, 171.12,

170.97, 170.87, 170.86, 170.72, 170.43, 170.38, 170.14 (C=O), 157.14 (C=

NH), 138.08, 134.60 (C_ipso, Tyr), 133.75 (C^g-His), 129.24 (C_o;m, Tyr), 128.73

(C^e-His), 117.64 (C^d-His), 81.13 (C1, Fuc), 75.22 (C3, Fuc), 74.00 (C5,

Fuc), 72.32 (C4, Fuc), 70.97, 70.10, 70.01, 69.84 (CH₂O, spacer), 69.74

(C2, Fuc), 67.29, 67.21, 66.95 (C^b-Thr), 66.50, 66.32 (CH₂O, spacer), 61.61

(C^b-Ser), 61.27, 60.98, 60.40, 60.02, 59.86, 59.58 (C^a-Pro), 58.89 (C^a-Val),

58.56, 58.49, 58.44 (C^a-Thr), 55.34 (C^a-Ser), 55.22 (C^a-Tyr), 52.32 (C^a-

His), 50.68 (C^a-Arg), 50.44 (C^a-Asp), 49.71 (C^a-Ala), 48.12–47.20 (4ꢃC^a-

Ala, 6ꢃC^d-Pro), 42.28, 42.08 (C^a-Gly), 40.74 (C^d-Arg), 39.23 (CH₂,

spacer), 37.18 (CH₂-exo), 37.03 (C^b-Tyr), 34.50 (C^b-Asp), 34.40 (CH₂,

spacer), 30.23 (C^b-Val), 29.52, 29.36, 29.13, 29.07 (6ꢃC^b-Pro), 27.98 (C^b-

Arg), 26.56 (C^b-His), 24.87, 24.77, 24.67, 24.60, 24.52, 24.44 (C^g-Pro),

24.30 (C^g-Arg), 18.94, 18.85, 18.56 (C^g-Thr), 18.50 (C^ga-Val), 17.52 (C^gb-

Val), 16.13 (C6, Fuc), 15.89, 15.87, 15.77, 15.70, 15.30 ppm (C^b-Ala); MS

(ESI): m/z: 1239.65 ([M+2H]²⁺, calcd: 1239.63); HRMS (ESI): m/z

calcd for C₁₁₀H₁₇₂N₂₈O₃₇: 1239.6298 [M+2H]²⁺; found: 1239.6283.

{2.89}), 2.72–2.60 (m, 3H, H^bb-Asp {2.68}, 1ꢃCH₂, spacer {2.68}), CH_2b

-

exo {2.63}), 2.30–1.80 (m, 32H, 6ꢃH^b-Pro {2.26–2.14}, H^b-Val {2.15}, 12ꢃ

H^g-Pro {2.10–1.91}, CH₃, Ac {1.99, 1.94}, 6ꢃH^b-Pro {2.03–1.89}, H^ba-Arg

{1.86}), 1.78–1.68 (m, 3H, H^bb-Arg {1.73}, 2ꢃH^g-Arg {1.69}), 1.42–1.24

(m, 15H, H^b-Ala {1.40, 1.39, 1.35, 1.34, 1.33}), 1.21–1.10 (m, 9H, H^g-Thr

{1.19, 1.18, 1.16}), 1.02–0.96 ppm (m, 6H, H^ga-Val {0.99}, H^gb-Val {0.98});

¹³C NMR [BB, HSQC, HMBC] (100.6 MHz, MeOD): d=174.51, 173.87,

173.85, 173.72, 173.20, 172.85, 172.66, 172.59, 172.57, 172.33, 171.97,

171.77, 171.74, 171.31, 171.18, 171.10, 170.98, 170.89, 170.75, 170.47,

170.44, 170.36, 170.14 (C=O), 157.20 (C=NH), 138.42, 138.36 (C_ipso, Ph),

136.94, 135.05 (C_ipso, Tyr), 133.76 (C^g-His), 129.40, 129.25 (C_o;m, Tyr),

128.90 (C^e-His), 128.17, 128.13, 127.64, 127.62, 127.54, 127.47 (C_o;m;p, Ph),

117.65 (C^d-His), 84.42 (C3, Glc), 81.32 (C2, Glc), 79.78 (C1, Glc), 75.43

(C5, Glc), 75.00, 74.68 (CH₂, Bn), 70.87 (C4, Glc), 70.13, 70.04, 69.87,

69.78 (CH₂O, spacer), 67.30, 67.23, 66.97 (C^b-Thr), 66.53, 66.36 (CH₂O,

spacer), 62.55 (C6, Glc), 61.66 (C^b-Ser), 61.31, 61.01, 60.45, 60.25, 60.05,

59.90 (C^a-Pro), 59.63 (C^a-Val), 58.91, 58.63, 58.48 (C^a-Thr), 55.37 (C^a-

Ser), 55.23 (C^a-Tyr), 52.38 (C^a-His), 50.70 (C^a-Arg), 50.49 (C^a-Asp),

49.76 (C^a-Ala), 48.26–47.02 (4ꢃC^a-Ala, 6ꢃC^d-Pro), 42.33, 42.14 (C^a-Gly),

40.78 (C^d-Arg), 39.27 (CH₂, spacer), 37.10, 36.99 (CH₂-exo, C^b-Tyr), 34.58

(C^b-Asp), 34.44 (CH₂, spacer), 30.24 (C^b-Val), 29.53, 29.39, 29.16, 29.08

(6ꢃC^b-Pro), 27.99 (C^b-Arg), 26.58 (C^b-His), 24.87, 24.80, 24.70, 24.62,

24.54, 24.47 (C^g-Pro), 24.33 (C^g-Arg), 19.66, 19.61 (CH₃, Ac), 18.97, 18.91,

18.59 (C^g-Thr), 18.53 (C^ga-Val), 17.55 (C^gb-Val), 16.19, 15.90, 15.80,

Amino-4,7,10-trioxa-dodecanylamido-N-l-prolyl-l-alanyl-l-histidyl-l-

glycyl-l-valyl-l-threonyl-l-seryl-l-alanyl-l-prolyl-l-aspartyl-l-threonyl-l-

arginyl-l-prolyl-l-alanyl-l-prolyl-l-glycyl-C-[b-d-glucosyl]-l-tyrosyl-l-

threonyl-l-alanyl-l-prolyl-l-prolyl-l-alanine (57): C-Glycopeptide 55

(94 mg, 34.3 mmol) was dissolved in methanol (15 mL) and acetic acid

(1 mL). The mixture was degassed with a constant stream of argon for

10 min. A spatula tip of Pd(OH)₂/C (15–20 wt%) was added and the mix-

ture degassed for another 5 min. The mixture was hydrogenated at room

temperature in an autoclave (15 bar) for 5 days. The reaction mixture

was then filtered and the residue washed with methanol+0.1% TFA,

water+0.1% TFA and a mixture thereof. The solvents were removed in

vacuo and the remaining crude product was taken up in methanol

(35 mL). A pH value of 9.5 was adjusted by addition of sodium methox-

ide solution. The reaction mixture was stirred for 21 h, neutralized by ad-

dition acetic acid 1 mL) and the solvent was removed in vacuo. The re-

maining crude product was co-evaporated with methanol/toluene (2:15)

and with toluene and then purified by RP-HPLC (Phenomenex Luna,

MeCN/H₂O+0.1% TFA, (5:95) to (35:65), 60 min) to yield C-glycopep-

tide 57 (76 mg, 30.5 mmol, 89%) as a colorless amorphous solid. Analyti-

cal HPLC: t_R=13.3 min (Phenomenex Luna, MeCN/H₂O+0.1% TFA,

(5:95) to (40:60), 60 min, l=214 nm); preparative HPLC: t_R=29.9 min

(Phenomenex Luna, MeCN/H₂O + 0.1% TFA, (5:95) to (35:65), 60 min,

l=214 nm); [a]²_D³=À87.9 (c=1.0, MeOH); ¹H NMR [¹H¹H-COSY,

ROESY] (600 MHz, MeOD): d=8.79 (s_b, 1H, H^e-His), 7.42 (s, 1H, H^g-

15.34 ppm (5ꢃC^b-Ala); MS (ESI): m/z: 1379.77 ([M+2H]²⁺

1379.68); HRMS (ESI): m/z calcd for C₁₂₈H₁₈₈N₂₈O₄₀: 1379.6847; found:

1379.6985 ([M+2H]²⁺

, calcd:

.

Amino-4,7,10-trioxa-dodecanylamido-N-l-prolyl-l-alanyl-l-histidyl-l-

glycyl-l-valyl-l-threonyl-l-seryl-l-alanyl-l-prolyl-l-aspartyl-l-threonyl-l-

arginyl-l-prolyl-l-alanyl-l-prolyl-l-glycyl-C-[b-d-fucopyranosyl]-l-tyro-

syl-l-threonyl-l-alanyl-l-prolyl-l-prolyl-l-alanine (56): The combined

product fractions of 54 were dissolved in of methanol (15 mL), ethyl ace-

tate (1 mL) and acetic acid (1 mL). The mixture was degassed with a con-

stant stream of argon for 10 min. A spatula tip of Pd(OH)₂/C (15–

20 wt%) was added and the mixture degassed for another 5 min. The

mixture is hydrogenated at room temperature in an autoclave (15 bar)

for 5 days. The reaction mixture was then filtered and the residue was

washed with methanol+0.1% TFA, water+0.1% TFA and a mixture

thereof. The solvents were removed in vacuo and the remaining crude

product was purified by RP-HPLC (Phenomenex Luna, MeCN/H₂O+

0.1% TFA, (5:95) to (35:65), 60 min) to afford C-glycopeptide 56 (55 mg,

22.2 mmol, 90%) as a colorless amorphous solid. Analytical HPLC: t_R=

13.0 min (Phenomenex Luna, MeCN/H₂O+0.1% TFA, (5:95) to (40:60),

60 min, l=214 nm); preparative HPLC: t_R=30.4 min (Phenomenex

Luna, MeCN/H₂O+0.1% TFA, (5:95) to (35:65), 60 min, l=214 nm);

[a]²_D³=À84.2 (c=1.0, MeOH); ¹H NMR [¹H¹H-COSY, ROESY]

(600 MHz, MeOD): d=8.79 (s_b, 1H, H^e-His), 7.42 (s, 1H, H^g-His), 7.18

(d, 2H, ³J=8.0 Hz, H_Ar, Tyr), 7.13 (d, 2H, ³J=8.1 Hz, H_Ar, Tyr), 4.71–

4.56 (m, 7H, H^a-Tyr {4.69}, H^a-His {4.67}, H^a-Arg {4.67}, H^a-Asp {4.65},

H^a-Ala {4.64, 4.61, 4.58}), 4.46–4.30 (m, 11H, H^a-Ser {4.44}, H^a-Pro {4.43,

4.42, 4.38, 4.37, 4.35, 4.33}, H^a-Thr {4.42, 4.35, 4.33}, H^a-Ala {4.33}), 4.29–

4.15 (m, 5H, H^a-Val {4.27}, H^a-Ala {4.25}, H^b-Thr {4.24, 4.20, 4.17}), 4.05–

3.89 (m, 3H, H^aa-Gly¹{4.02}, H^aa-Gly²{3.98}, H^ab-Gly¹{3.92}), 3.86–3.57

(m, 28H, H^ba-Ser {3.84}, 4ꢃH^d-Pro {3.78}, H^bb-Ser {3.77}, 2ꢃCH₂O,

spacer {3.75}, 2ꢃCH₂O, spacer {3.70}, 4ꢃH^d-Pro {3.66}, H^ab-Gly²{3.67},

8ꢃCH₂O, spacer {3.66–3.59}, 4ꢃH^d-Pro {3.63}, H4, Fuc {3.62}), 3.47–3.37

(m, 3H, H2, Fuc {3.40}, H3, Fuc {3.43}, H5, Fuc {3.44}), 3.37–3.32 (m,

1H, H^ba-His), 3.26–3.09 (m, 8H, H1, Fuc {3.23}, H^bb-His {3.19}, 2ꢃH^d-

Arg {3.18}, H^ba -Tyr {3.16}, 2ꢃCH₂-spacer {3.12}, CH_2a-exo {3.11}), 3.01

(m_c, 1H, H^bb-Tyr), 2.95–2.83 (m, 2H, H^ba-Asp {2.88}, 1ꢃCH₂, spacer

{2.88}), 2.73–2.63 (m, 2H, H^bb-Asp {2.68}, 1ꢃCH₂, spacer {2.68}), 2.60

(dd, 1H, ²J=14.5 Hz, ³J=9.5 Hz, CH_2b-exo), 2.29–1.81 (m, 26H, 6ꢃH^b-

Pro {2.24–2.15}, H^b-Val {2.15}, 12ꢃH^g-Pro {2.11–1.93}, 6ꢃH^b-Pro {2.02–

1.91}, H^ba-Arg {1.85}), 1.76–1.64 (m, 3H, H^bb-Arg {1.71}, 2ꢃH^g-Arg

{1.66}), 1.42–1.27 (m, 15H, H^b-Ala {1.39, 1.38, 1.35, 1.34, 1.33}), 1.20–1.11

His), 7.21 (d, 2H, ³J=7.7 Hz, H_Ar, Tyr), 7.13 (d, 2H, ³J=7.6 Hz, H_Ar

,

Tyr), 4.76–4.58 (m, 7H, H^a-Tyr {4.72}, H^a-His {4.71}, H^a-Arg {4.68}, H^a-

Asp {4.66}, H^a-Ala {4.66, 4.65, 4.61}), 4.54–4.17 (m, 16H, H^a-Ser {4.47},

H^a-Pro {4.46, 4.45, 4.43, 4.41, 4.37, 4.36}, H^a-Thr {4.46, 4.39, 4.37}, H^a-Ala

{4.36}, H^a-Val {4.31}, H^a-Ala {4.28}, H^b-Thr {4.27, 4.23, 4.19}), 4.07–3.93

(m, 3H, H^aa-Gly¹{4.05}, H^aa-Gly²{3.98}, H^ab -Gly¹{3.95}), 3.89–3.60 (m,

29H, H^ba-Ser {3.87}, 4ꢃH^d-Pro {3.81}, H^bb-Ser {3.79}, H6a, Glc {3.78}, 2ꢃ

CH₂O, spacer {3.78}, 2ꢃCH₂O, spacer {3.73}, 4ꢃH^d-Pro {3.69}, H^ab-Gly2

{3.70}, 8ꢃCH₂O, spacer {3.69–3.61}, 4ꢃH^d-Pro {3.64}, H6b, Glc {3.64}):

3.40–3.32 (m, 3H, H^ba-His {3.38}, H1, Glc {3.37} H3, Glc {3.36}), 3.26 (t,

1H, ³J_H4;H3=³J_H4;H5=9.3 Hz, H4, Glc), 3.25–3.13 (m, 8H, H^bb-His {3.22},

2ꢃH^d-Arg {3.20}, H^ba-Tyr {3.20}, CH_2a-exo {3.15}, H5, Glc {3.15}, 2ꢃCH₂,

spacer {3.15}), 3.08 (t, 1H, ³J_H2;H1=³J_H2;H3=9.1 Hz, H2, Glc), 3.03 (m_c,

1H, H^bb-Tyr), 2.98–2.86 (m, 2H, H^ba-Asp {2.92}, 1ꢃCH₂, spacer {2.92}),

2.76–2.66 (m, 3H, H^bb-Asp {2.71}, 1ꢃCH₂, spacer {2.71}, CH_2b-exo

{2.68}), 2.33–1.84 (m, 26H, 6ꢃH^b-Pro {2.26–2.17}, H^b-Val {2.17}, 12ꢃH^g-

Pro {2.12–1.95}, 6ꢃH^b-Pro {2.04–1.93}, H^ba-Arg {1.87}), 1.79–1.67 (m, 3H,

H^bb-Arg {1.76}, 2ꢃH^g-Arg {1.69}), 1.44–1.30 (m, 15H, H^b-Ala {1.41, 1.40,

1.38, 1.37, 1.35}), 1.24–1.14 (m, 9H, H^g-Thr {1.20, 1.19, 1.17}), 1.04–

0.96 ppm (m, 6H, H^ga-Val {1.01}, H^gb-Val {0.99}); ¹³C NMR [BB, HSQC,

HMBC] (150.9 MHz, MeOD): d=174.53, 173.83, 173.80, 173.77, 173.18,

172.85, 172.66, 172.57, 172.51, 172.49, 172.32, 171.94, 171.75, 171.67,

171.29, 171.12, 171.09, 170.94, 170.82, 170.68, 170.40, 170.34, 170.11 (C=

O), 157.11 (C=NH), 137.40, 134.68 (C_ipso, Tyr), 133.71 (C^g-His), 129.44

(C_o;m, Tyr), 129.16 (C^e-His), 128.71 (C_o;m, Tyr), 117.60 (C^d-His), 80.22 (C1-

Chem. Eur. J. 2013, 00, 0 – 0

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H. Kunz et al.

Glc), 80.05 (C5, Glc), 78.46 (C3, Glc), 73.49 (C2, Glc), 70.48 (C4, Glc),

70.06, 69.97, 69.80, 69.70 (CH₂O, spacer), 67.28, 67.19, 66.93 (C^b-Thr),

66.47, 66.29 (CH₂O, spacer), 61.60 (C^b-Ser), 61.21 (C6, Glc), 60.93, 60.36,

60.18, 59.99, 59.84 (6ꢃC^a-Pro), 59.52 (C^a-Val), 58.84, 58.54, 58.42 (C^a-

Thr), 55.30 (C^a-Ser), 55.18 (C^a-Tyr), 52.28 (C^a-His), 50.68 (C^a-Arg), 50.40

(C^a-Asp), 49.68 (C^a-Ala), 48.12–47.20 (4ꢃC^a-Ala, 6ꢃC^d-Pro), 42.24,

42.06 (C^a-Gly), 40.71 (C^d-Arg), 39.20 (CH₂, spacer), 37.05 (CH₂-exo),

36.97 (C^b-Tyr), 34.53 (C^b-Asp), 34.36 (CH₂, spacer), 30.22 (C^b-Val), 29.50,

29.33, 29.11, 29.05 (6ꢃC^b-Pro), 27.95 (C^b-Arg), 26.53 (C^b-His), 24.84,

24.74, 24.66, 24.58, 24.50, 24.42 (C^g-Pro), 24.27 (C^g-Arg), 18.92, 18.82,

18.54 (C^g-Thr), 18.48 (C^ga-Val), 17.49 (C^gb-Val), 16.11, 15.88, 15.76, 15.70,

[14] P. Arya, R. N. Ben, H. Qin, Tetrahedron Lett. 1998 , 39 , 6131.

[15] a) A. Dondoni, A. Marra, P. P. Giovannini, J. Chem. Soc. Perkin

Trans. 1 2001 , 2380; b) E. Nolen, A. Kurish, J. Potter, L. Donahue,

M. Orlando, Org. Lett. 2005 , 7 , 3383.

[16] a) C. R. Bertozzi, P. D. Hoeprich, M. D. Bednarski, J. Org. Chem.

1992 , 57 , 6092; b) T. Fuchs, R. R. Schmidt, Synthesis 1998, 753;

c) S. D. Debenham, J. Cossrow, E. J. Toone, J. Org. Chem. 1999 , 64 ,

9153.

[17] a) T. Gustafsson, M. Saxin, J. Kihlberg, J. Org. Chem. 2003 , 68 ,

2506; b) N. Bragnier, R. Guillot, M.-C. Scherrmann, Org. Biomol.

Chem. 2009 , 7 , 3918.

[18] A. J. Pearce, S. Ramaya, S. N. Thorn, G. B. Bloomberg, D. S. Walter,

T. Gallagher, J. Org. Chem. 1999 , 64 , 5453.

15.30 ppm (C^b-Ala); MS (ESI): m/z: 1247.49 ([M+2H]²⁺

,

calcd:

1247.63).

[19] S. Koch, H. Lçwe, H. Kunz, Synlett 2011, 1978.

[20] H. Herzner, J. Eberling, M. Schultz, J. Zimmer, H. Kunz, J. Carbo-

hydr. Chem. 1998 , 17 , 759.

Acknowledgements

[21] a) R. R. Schmidt, Pure Appl. Chem. 1989 , 61 , 1257; b) W. Stahl, U.

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Received: January 15, 2013

Published online: && &&, 2013

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www.chemeurj.org

ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 0000, 00, 0 – 0

ÝÝ

These are not the final page numbers!

C-Glycosyl Amino Acids

FULL PAPER

Amino Acids

S. Koch, D. Schollmeyer, H. Lçwe,

H. Kunz* ...................... &&&& — &&&&

C-Glycosyl Amino Acids through

Hydroboration–Cross-Coupling of exo-

Glycals and Their Application in

Automated Solid-Phase Synthesis

Manufacturing mimics: C-Glycosyl

amino acids, stable mimics of the natu-

rally occurring O-glycosyl amino acids,

are stereoselectively accessible from

exo-glycals by using a strategy based

on a cascade of hydroboration reaction

and Suzuki coupling (see figure). After

protecting-group manipulations, C-gly-

cosyl amino acid building-blocks were

used in solid-phase syntheses of C-gly-

copeptides sequences of tumor-associ-

ated mucine MUC1, which are recog-

nized by antibodies induced in mice

through synthetic MUC1-O-glycopep-

tide vaccines.

Chem. Eur. J. 2013, 00, 0 – 0

ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

&

23

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ÞÞ

These are not the final page numbers!

Article Doi

C-Glycosyl amino acids through hydroboration-cross-coupling of exo-glycals and their application in automated solid-phase synthesis

DOI: 10.1002/chem.201300150

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