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O- and N-Glycopeptides: Synthesis of Selectively Deprotected Building Blocks

O- and N-Glycopeptides: Synthesis of Selectively Deprotected Building Blocks

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Kunz



266



signals involved in

within multicellular organisms. in particular, they are recognition

of the cell growth,

n

regulatio

,

adhesion

cell

in

example,

for

intercellular commun ication,

l elucidation of

infectious processes, and immunological differentiations [2]. For structura

carbohydrate

specified

exactly

of

tides

glycopep

model

s,

these biological selection processe

and peptide structure (e.g., type 1 and 2 are of interest.

--HN-CH -CO--·

I

HC-R



.- HN -'iH - CO - Xaa - Ser(Thr)-



OH



~

0



HO.

HO



CH2



I



6=0



ACHN 0



I

NH



HO



NHAc



if!jo



2 R = H, CHa



OH



HO



ted and used as

Until the late 1970s 0- and N-glycosylated amino acids were construc

carbohyd rate and

standards for structural elucidations of the linkage regions between

tides demande d the

peptide parts of glycoproteins [3,4]. However, the synthesis of glycopep

[5]. This holds true, in

development of versatile and selective protecting group techniques

which themselves

particular, for glycopeptides with complex oligosaccharide portions,

has to be paid to the

must be formed in laborious multistep syntheses [6,7]. Much attention

glycosidic and interglycosidic bond present in all natural glycoproteins. The acetal-type

O-glycosyl serine and

for

and,

acids

strong

to

sensitive

ly

potential

are

bonds

ic

saccharid

d and selective

threonine derivatives (e.g., of 2), also sensitive to bases. Therefore, controlle

acids and

amino

glycosyl

tional

polyfunc

the

of

group

l

functiona

one

only

of

deblocking

of

synthesis

the

Because

.

synthesis

tide

glycopeptides is a critical problem in glycopep

[6,7] and is

reviews

of

number

a

in

described

been

has

es

glycosid

and

es

oligo saccharid

g group techpresented in succeeding chapters, this contribution is focused on protectin

.

chemistry

tide

niques in glycopep

demonstrated

A major progress in glycopeptide synthesis was achieved when it was

ly reselective

be

can

group

(Fmoc)

l

ycarbony

ylmethox

9-fluoren

that the N~-terminal

This holds true for

moved from glycopeptides using the weak base morpholine (pKa 8.3).

lyzed ~-elimination

O-glycosyl serine and threonine esters, which are sensitive to base-cata

of the carbohydrate [10].

.. ,

,~

o•

I



Bzl



~



-Q



Jl.. HN-CH.C OO-CH2 0

\



A



7H - CHa

AcHN



OH



HN-CH-C OO-Bzl

I

yH-CHa



morpholine



AcHN



..



0



. .

The Fmoc group is also useful in the synthesis of N- g I~copeptl.des contaimng the N4_

th N I

glycosyl asparagine structure 1 B

. ecaubse e ~g ycosyl amide bond is relatively stable to

acids and bases the Fmoc

b

group can e combine d with

,

I anum er of carboxyl-protecting

groups in the synthesis of glycopeptides (e g th e b

ten-butyl ester [16],

as well as polymeric benzyl- [14] and ~Ii' I_t enzy ester [15], the

in solid-phase

anchors

ester

[17,18]

ype

.y

ms'

proble

synthesis). However

eptide s th . I'f sensitive gly, a n s e even In N-glycop

.

yn esis,

.

cosidic bonds (e.g ., fucosl'de bo nd s) are present III

.

the gl

y.cane portion [19].

Combinations of the benzylox carbon I

l ester [20] and

ten-buty

the

WIth

y .(~) group

of the tert-butyloxycarbonyl (Boc(

ter [21] have

benzyles

ic)

(polymer

th.e

Wit

gr~~p

gl

in

successfully been applied

The sensitivity of the Boc group

toward the acidic conditions of g~~:~~:~i~nsyn~esls.

ons

and the o~ten.sluggishly proceeding

heterogeneous hydrogenation are unfavorabl:~~~ a

applicatIOn of these protecting

general

I

all

th

and

[22]

ester

allyl

The

.

group strategies

group [23] were proved to be

bonYI

y.~xYCar

e

.

d

stable under conditions usually appr

tide synthesis. Nevertheglycopep

d~d

e

pepn

~n

cl:U

groups

g

protectin

allylic

these

less,

and N-glycopeptides

[22,24]

0om

re:v~

:

[25] under practically neutral conditions

allyl transfer by using

ed

-catalyz

a~lUm(O)

P

Yl'

all

the

trap

ly

irreversib

that

iles

nucleoph

y IC moiety,



.

Z



Z-HN-CH -COOH



I



BZIO~O\.



THF I morpholine

100m temp., 30 min.



5



CH2



BzIO~

OBzl



~



6



97%



The efficiency and chemoselectivity of this meth

od was demonstrated in syntheses of

glycopeptides that contain the biologi all .

c y Important, but rather sensitive fucoside linkage

[19,26-2 8].

molecules

Enzymatic reactions provide new tools in the synthesis of polyfunctional



ko;jJ-O~.

OAc



AcO



0



3



HO



OH



Fmoc- Sar- OH



_hO-q!}



pH 7, 37"C



7



AcO



OAc



8



HOm



HOm

HO



267



Hep

~



Fmoc



-



Protecting Groups In Glycopeptide Synthesis



4



quantitative



(e.g., pentaBecause O-glycosylation can also be accomplished with active esters

technique provides a

Fmoc

the

,

threonine

and

serine

Fmoc

of

[Il]

esters)

nyl

fluorophe

reich antigen glycogeneral method for the synthesis of glycopeptides. Thomsen -Frieden

ve amounts

preparati

in

method

this

by

obtained

been

have

proteins

neoglyco

peptides and

technique

Fmoc

this

anchors,

ester

benzyl

[12]. In combination with acid-labile polymeric

.

[11,13,14]

tides

glycopep

of

s

synthese

se

solid-pha

to

was applied



60%



AcO OAc



AcO~\.



-AcNHl



Z-: ;



Thr-Ala- OHap



#/



Aco



AcO



Lipase M



o



OAc



9



76%



Kunz



268



activity selectively

such as glycopeptides. For example, lipases exhibiting no protease

the ester-protecting

s,

condition

these

Under

[29].

esters

heptyl

tide

glycopep

e

hydrolyz

protection, and the

groups in the carbohydrate portion, the peptide bond, the amino

d.

unaffecte

remain

bonds

c

glycosidi

stability of

The choice of protecting groups also has all important effect on the

protecting

of

cleavage

c

acidolyti

If

acids.

toward

bonds

c

glycosidi

intersaccharidic and

partial or

of

ment

establish

the

tides,

groups is necessary during the synthesis of glycopep

For the

[19,26].

favorable

is

units

e

saccharid

the

within

n

complete acyl-type protectio

es, glycosyl

construction of the O-glycosidic linkages to serine and threonine derivativ

triflate [30], is

halides and promotion by mercury or silver salts, in particular by silver

alactose donors

generally efficient. Mucin-type glycopeptides are accessible using 2-azidog

demonstrated to be

[6]. Glycosyl trichloroacetimidates [7,22] and thioglycosides were also

ilic activation

useful donors in the synthesis of O-glycopeptides. Furthermore, electroph

access

General

[31].

synthesis

tide

glycopep

in

ve

perspecti

g

of glycals offers an interestin

are reduced to glyto N-glycopeptides is attained through glycosyl azides [4,25-28 ] that

these being seleccosylamines, and the latter condensed with aspartic acid derivatives,

ne derivatives

lucosarni

N-acetylg

vely,

Alternati

group.

yl

13-carbox

the

at

d

unblocke

tively

glycosylnding

correspo

the

form

to

e

carbonat

react directly with ammonium hydrogen

[14].

amines



Protectin g Groups in Glycope ptide Synthes is

Fmoc- HN-CjH- COOBzJ



BZO~~

BzO ----,'.,;;::l



OBzBr



Fmoc-HN -CjH-COO BzI



11



rHo,

OH



BZO~ O"



AgOTf



BzO~



as well as 0- and

Glycopeptides contain many functional groups of different reactivity

of the applied

lectivity

chemose

and

N-glycosidic bonds. Therefore, the compatibility

efficient

chapter,

this

In

.

synthesis

tide

glycopep

in

ite

prerequis

ntal

reactions is a fundame

d by examples

and generally applicable methods and their combinations will be illustrate

[5,8,9].



A.



The Fluoren ylmetho xycarbo nyl Group



proved an efficient

The Fmoc group, which is very useful in peptide synthesis [32], has

amino function is

tool in glycopeptide chemistry [10]. Because the Fmoc protection of the

g groups and

protectin

-type

tert-butyl

with

combined

be

can

it

rather stable to acids,

acidic milieu.

exposed to stereoselective glycosylations that require a more or less

l-Ci-DGlycosylation of the Fmoc serine benzyl ester 11 with 2,3,4-tri-0-benzoy

13-xylosyl serine

xylopyranosyl bromide 12 promoted by silver triflate (30) afforded the

Fmoc technique in

conjugate 13 in high yield [10]. The efficiency and compatibility of the

t with morpholine

glycopeptide synthesis was demonstrated on substrate 13 [10]. Treatmen

[33]) resulted in

de

formami

dimethyl

or

[18]

1:1

ethane

(neat or diluted with dichlorom

hydrogenation

whereas

14,

give

to

group

Fmoc

the

of

removal

selective

and

ive

quantitat

lectivity of

chemose

The

15.

in

resulting

function

selectively deblocked the carboxylic

14 and 15

blocks

Building

17.

tide

glycopep

the

for

shown

also

was

ion

deprotect

the amino

glycoconstruct

to

extension

chain

erminal

COOH-t

can be used for alternative NH z- or

n from the carboprotectio

O-acyl

the

of

l

Remova

.

sequence

desired

a

of

peptides

for the formation

hydrate portion is achieved under weakly basic conditions, as is shown

of 16 [10].



C~



6



(M"2N)zCO



..



BZO~O"



1387%



12



Fmoc-HN -



Hii'd



OBz



1H-COOH

CH

6z



BzO~

OBz 15 92%



H·HN-CHC OOBzJ



I



C~



6

BzO~



BZO~O"



r



Fmoc- Asn-le
~



OBz



1. Ho,IPd



14 R =Bz, R' = BzJ, 98%



2. HzNN~1

CHaOH --16 R = H, R' = H, 82%



~OEI

I



COCEt

(EEDQ)



17 98% (slep 2)



The Fmoclte rt-Butyl Ester Combin ation

.

b

The Frnoc group can also be combined with th

utyl es~er In O-glycopeptide synthesis

[16,34]. The Fmoc serine and threonine ~ - u y esters

free 13- hydroxyl groups are

WIth

er

. d

. try Th

require as starting materials for this che mrs

cse compounds (e.g., the threonine

.

B.



II. METHODS



269



t: i:



Fmoc-HN -9H-COO H



Fmoc- HN-C,H-C OOtBu

C1H-CHa )

CH- cHa

~

>-. ----- 1...

I

OH'

OH



tBu-OH + DCC + Cu(I)CI (3d at 200C)



18



3



:rt"

~



HN - 9H-COO lBu

fH-CH a



Na 0



AC0Rf:j

AcO



.



biotin



OAc



23 overall yield 93%



21 quan!.



Kunz



270



amino acid with a

derivative 18) become readily accessible by treatment of the Fmoc

[34]. Glycosylachloride

coppeul)

and

ide,

carbodiim

nol,

tert-buta

of

mixture

d

prereacte

«-anome r of

desired

the

mainly

yielded

19

donor

tion of 18 using the thioglycoside

graphy. In the 0'- or,

chromato

flash

by

obtained

were

anomers

pure

The

[34].

20

conjugate

using formic

alternatively l3-anomer of 20, the tert-butyl ester group is selectively cleaved

v/v).

(1:1

ane

lorometh

acid/dich

cetic

trifluoroa

s

acid or anhydrou

ine to give 22

Alternatively, the Fmoc group is selectively eliminated with morphol

in solution as

synthesis

tide

glycopep

tending

chain-ex

of

basis

the

are

[34]. Both reactions

d 22 often

compoun

in

group

amino

well as on solid phases [11,12,14]. Because the free

nt

subseque

,

solution)

in

least

(at

ation

l3-elimin

or

transfer

acetyl

N

~

gives rise to 0

biotin,

with

22

of

ation

Condens

peptide condensation should be carried out immediately.

e 23 containing a

using a water-soluble carbodiimide (WSC) yielded the biotinyl conjugat

label (biotin) as well as a photoactivatable group (azide).



Protectin g Groups In Glycope ptide Synthes is



D. The Allyloxy carbony l Groups

.

I A

In this chapter the efficiency of the all lox c b

ot~c~in~ group is

illustrated in the synthesis of N-glYCop~PtiIesarl~ny ( l~c)harruno-pr

dic h~alge of

~~~~~~

d:n;

co:;::;:n

the

of

tion

N-~Iycopeptid.es is formed by condensa

- -acety g ucosarrune derivative with a N-protected as artie id

According to ~s

principle, the chitobiosyl azide 30 selec~ve:y ;~~ro~~:::~te~t~r [4].

was coupled with

[26]

6

.

"

d

[35]

31

.

O-benzylated «-fucosyl bromide

.

un er m SItuanomenzation conditttons

[36] to give

[26].

32

azide

yl

chitobios

l

o-fucosy

the branched

Bzro

CH



3



BZI



~



Br



,



Fmoc- HN- CH- COOAII



Fmoc- HN - CH - COOAII

I



24



yH- CH3



.



OH

N



Sr



X 0

AcOF t;!

AcO



OAc



AeofFl

AeO



OAe



31

Et4WBr"

- _ _...........



OH



AeO~N3



AcO~O



Boc- [25,28], or

The allyl ester [22] can be selectively removed in the presence of Z- [22],

glycosylated

was

24

ester

allyl

threonine

Fmoc

Fmoc amino protection [24]. For example,

[6] to give the

2S

bromide

syl

topyrano

'-D-galac

-deoxY-0

-acetyl-2

,4,6-tri-O

with 2-azido-3

ation of its

conjugate 26 in high yield [24]. Separation of the o-anome r and transform



I



CH



AcO~o~O.



Allyl Ester Protect ing Group



CH-CH3



BzlO



OBzl



0



AcNH



C.



271



32,

C 33,



.



Afoc.-Asp-OtBu 34



\



CH-CH3



BzlO

(EEOQ)



0



AeHN



0



AcO



OAe



3



28



trioctylp hosphine 2-azido moiety to the acetamido group using either thioacetic acid or

removal of the

Selective

[24J.

27

element

l

structura

antigen

acetic acid afforded the Tn

(see earlier discusFmoc group was achieved quantitatively by treatment with morpholine

should immediately

sion). Again, the amino-deblocked compound 28 is not very stable and

tion.

condensa

further

for

be used

talyzed

The alternative selective cleavage of the allyl ester was achieved by Pd(O)-ca

weak base must be

very

a

ne,

morpholi

to

sensitive

is

group

Fmoc

the

Because

transfer.

allyl

, and its use as a

used as the allyl-trapping nucleophile. N-Methyl aniline was favorable

yield.

high

a

in

29

acid

the

to

rise

gave

ile

nucleoph

r

scavenge



BzI



N~ /COOtBu



~



AcNH



AcO<= O

OBzl



AcOF t;!



29 93%



~o.Jl

0



CH



,



o



OBzl



0



AcO~O~o.



I



Ac0Ft;!

OAc



OEt

I

COOEt



H2N-CH-CO OAII



I



AcO



~

VlNA



morpholine



Fmoc-H N-CH-C OOH

\

CH-CH3



74%

95%



g



33



AcHN



X ee N3



X ~ NH2



.'

I

Subsequent hydrogenolysis of the azido rou

cata yzed by Raney nickel (washed to

neutral reaction) furnished the glycos I . ;3

:ith N-allyloxycarb?nyl asp~i~ acid a-tert-butyl est:r ~I~as ~cc~~;~:~~~nu~~:3

g thyl 2-ethoxy-I,2dihydroqumolme-I-carboxylate (EEDQ) [37] t . th

e fully protected N-glycosyl aspar0 give

h

agine derivative 3S [26] S"I

. al

. IITIl ar to t e allyl ester the NH t

~-.ermm allyloxycarbonyl

(Aloe) group can be removed under racticallv neu

as was demonstrated on fucosyl chitobiose asparagi~e conju:a~e~~~I~~~~~tIons [23],



27 X ~ NHAc, 94%

\



0



AeNH



Aloe

[



OBzl



0



OBzl



26 X ~ N3 , 80%, ( l : ~ ~ 10: 1

(CeHI7h PI AcOH



25



~



A:O~~~X



H2/Raney-N i



30



BZI



AcNH



AeNH



OBzl



3



BzlO



BZI



~

0



CH

3



AeNH



H2N.... ,COOtBu

CH

I

yH2



OBzl



yH



2



C~



I



AeO ~NH

AeNH



35, 75%



C-O

~O

0.-



AeO ~O



Aeo~_- -.O AcO

OBzl



CH

I



NH

AcNH



36, 91%



.

.

The Pd(O)-catalyzed transfer of the all I moie

morpholine [22],

or N,N' -dimethyl barbituric acid [27] result~. th ty to dimedons [23],

cleavage of the

selective

ly

complete

tie

I~

pr

other

s

numerou

Aloe group, whereas the

o c ng groups and the glycosidic bonds,



tide Synt hesi s

Prot ectin g Grou ps in Glyc opep



Kunz



273



272



this selective

unaffected [19,26]. It is obvious, that

including the fucoside bond, remained

tides (e.g.,

opep

glyc

g

ndin

dema

of

esis

for the synth

and mild method is a promising tool

ast, the

contr

In

[38].

)

problematic 13-mannoside bond

core N-glycopeptides containing the

g the

oyin

destr

out

with

s

ition

cond

c

ed under acidi

tert-butyl ester of 35 cannot be cleav

o-fucoside bond.



'_-HN&O~O.(\....a -O~N~NH~\



39



0



ion

Stab ility in the Sac cha ride Port

E. Esta blish ing Suff icie nt Acid

esis are to become

ed in peptide and solid-phase synth

If the acidolytic reactions often appli

aride portions of

sacch

the

of

, a general stabilization

adaptable to glycopeptide chemistry

and O-acetylps

grou

o

amid

Acet

.

shed

mpli

be acco

glycopeptides toward acids must

ence on the

influ

g

lizin

stabi

ates exhibit an effective

protecting groups in the carbohydr

ction is of

prote

type

acylfor

-type

ether

of

ange

glycosidic bonds. Therefore, the exch

chitobiose

syl

fuco

for

n

de synthesis, as is show

importance for a versatile glycopepti

derivative 37 [19,26J.



a



OMpm



CH3

2. hydrazine hydrate

..



idine

Ae~o~QN3 3.~O/pyr

O~

Ae

a

AeO

NPht



0



AeNH



OAe



I



Aeo~oAg



0rv



a



-l



Jl



N

4

CH3



AeNH



a



a



...







CH



0ey 0Non

/VN~COOH



O~3N H A



NH

YA NH



0 ; .COOtBu







.i



a



(cH;,)



NH



N



a

3

HN yNH -Mt r

HN

I



:



CH3



a



y> 95% (HPLC)

40 overall yield (18 steps ): 95%, purit



~O



AeNH

Ae or- J-l "o

AeO~6 AeO

OAe



DIC,HOB~



t' IPh:J PJ4Pd(0),Morpholine,DMFJDMSO''CH2 CI2



AeO



AeO~AeOAe



CH300C~-O

~MPm

CH3



.

t



C V ",

H3y N



HOB~ DMF; AC20fPyridine



DMF; AC20

.' .

O,ID.

DMF' Ac2~.

HOB'

DlC

H

yndm e

J>ro.O

Fmoc3eq

DMFlMorpholine 1:1:



H



a



_



/CH~12flFA}



"



Mpm



~



o.oH,IDlC,

DMFlMorpholine 1:1; 3eq(Boc-J>r

Hunig Base; 4eq Fmoc-Aia-OH,



H



N3



AeNH



38 overall yield 72%



37



nce of the

be selectively cleaved, even in the prese

The methoxybenzyl ether in 37 can

thaloyl

N-ph

the

ntly,

eque

Subs

te.

nitra

m

ammoniu

ps are

anomeric azide, by oxidation with eerie

grou

oxyl

hydr

the

and the amino as well as

group is removed with hydrazine,

ragine

Aspa

esis.

synth

de

pepti

glyco

in

now be used

tive

acetylated to give 38, which can

selec

a

to

cted

subje

aride side chain can be

conjugates carrying this type of sacch

6J.

[19,2

bond

side

fuco

the

ting

affec

s without

acidolysis of tert-butyl esters and ether



ase Syn thes is of Glyc ope ptid es

F. Ally lic Anc hors in the Soli d-Ph

efficiency of the

de synthesis and, in particular, the

To illustrate the progress in glycopepti

opeptides on a

glyc

e

n-typ

muci

of

esis

synth

se

-pha

allylic-protecting method, the solid

stability of the

the

to

g

Owin

[33J.

or 39 is outlined

polymeric support with an allylic anch

Boc group can be

the

as

well

as

c

Fmo

the

s,

ition

cond

allylic ester to both acidic and basic

is the ability to

. One advantage of this versatility

used for temporary amino protection

tide to avoid

dipep

d

linke

merpoly

the

of

level

on the

switch from Fmoc to Boc protection

ed out by

carri

de

pepti

glyco

re, the release of the

diketopiperazine formation. Furthermo

titatively,

quan

st

almo

eeds

proc

ne

anili

ethyl

to N-m

paliadium(O)-catalyzed allyltransfer

40 in an

tide

apep

onon

groups, to yield the glyc

and without affecting other protecting

to highrding

(acco

95%

than

more

of

y

and a purit

overall yield (relative to 39) of 95%

y detivel

selec

is

h

whic

(HPLC)]. Compound 40,

performance liquid chromatography

tions.

ensa

cond

ent

fragm

in

used

be

ely

immediat

blocked at the carboxyl group, can



cting7groJ u P hni

her with the verIn conclusion, the selective-prote

ak tee niques [5,8,9J toget

[6

esi

synth

.

aride

sacch

tid

oligo

lex gly

satile methods of

, m e comp

SIS

. copep es available, which

I hi

.

ti

ti

inv

ry

plina

disci

inter

in

est

processes. The

are of mter

mi es Itgha IOns 0 biological selection

choice of compatible methods. deter Illes e Success of syntheses f these po Iyfunctional

~

th d

I 1

sele ti

mo ecu es. On the basis of the elaboratedf ~ ~e me 0 s, the chemical synthesis of glyconts

amou

ve

arati

ified structure.

peptides delivers prep

o mo e compounds of exactly spec



/iI.



S*

EXPERIMENTAL PROCEDURE



A.



~~hOgOnal Deprotection of



rs

Fmo c D-g lyco pep tide Benzyl Este



arbonyl)_0_(2,3,4_tri_ _

N-(9 -Flu o renylmethoxyc

0 benzoyl-(3-D-xylopyranosyl)_L_serine

13

r

Este

Benz yl



'.

esulfonate (7 71

dlchloromethane

To a solution of silver trifluoromethan

. g: 30 mmol) in drylrn

.

dr

d

adde

is

h

dark

the

in

C

fl

9

(100 mL) at -40°

opwise a. solutIOnof - uoreny et oxycarbonyl

.

1) 2 3 4

senne benzyl ester 11 (8.45 g' 20 mm 0 , , , -tn-O-benzoyl -a:-D : x y1?pyranosy I bromide

12 (11.14 g, 21.2 mrnol) and tetramethyl urea (3 65 3

oromethane (l00

tempe t . thg, 1.~ ~ol? III dlchl off and washed

mL). After 18 h of stirring at room

ed

filter

IS

e

pitar

preci

.e

ure,

r~

with dichloromethane (200 rnl.) . Th e organic solution ISwashed ith

WI water (200.mL ), 1%

KH

r, dried with N

C03 solution (twice 200 mL) and wate

concentrated In vacuo.

and

4'

llzSO

from th I

The crude product is recrystallized

(If the reaction was not

e.

hexan

te-n~ceta

Yt

gel

complete, chromatography on silica

mended). Yield:

(e 13JnC~~~ne/ethanoI9:1 is recom ol 26:1).

15 g (87%); mp 136°C, [a:]o -27. 80

3)' R f 0.64 (toluene/ethan

. ,



6;.



at 220C.

'Opti cal rotations were measured



Kunz



275



Protecting Groups in Glycopeptide Synthesis



274

h F

G up' General Procedure

Removal of t e moc

I cos I e tide ester (1 mmol) is stirred in

Yan;(rl)P for 30 min. After addition of

The protected O-glycosyl ammo ~cld ed~tehrlor 0 gth

. (10 L) or morpholine-: IC oromernane u.

.

.'

h d with diluted aqueous acid (citric acid or

morpholme

m

100

mL)

the

solution

IS

was

e

.

dichlorometh ane (

.'

4 50 mL) dried with N"-SO , and concentrated in

mL)

and

WIth

water

(

x

,

-.

4

h

50

4

.,

in 2 5 mL of ethyl acetate. During chromatograp Y

HC I p H ,

vac~~. The crude pro?uct IS dlssOI;t~~r eth-l-acetate (2:1), N_(9_fluorenylmethyl)morp~0­

on silica gel (50 g) WIth petrolum d bl ked

'no acid or peptide ester is eluted WIth

line is eluted. Subsequently, the e oc e 1 arm nosyl)-L-serine benzyl ester 14: yield,

.-O-benzoyl-13- D - xy opyra

.

3 4 T no.

-414 (c 0.5, CH OH). N_(L_Asparagmyl-L-leucyl-)methanol. 0- (2 ,,0.63 g (98%); mp 55 C, [la]o

. l)-L-serine b~nzyl ester 17: yield, 0.85 g (98%);

0_(2,3,4,-0-benzoyl-13-D-xy opyranosy

amorphous: [a]o -42.3° (c 0.5; CHPH).



ro...



_



Removal of the Benzyl Ester



2 3 4 t . 0 benzoyl-ll-D-xylopyranosyl)N_(9_Fluorenylmethoxycarbon~I)-0-(, l' - tn- 13- (lOg 1 2 mmol) is stirred in



0 xylosyl senne benzy es er

.,.

F

d bi t d to hydrogenolysis for 18 h under

t

temperature an su ~ec e

mL)

.

h

al (02 g 5%) as the catalyst. The educt 13

methanol (40

a ro?m

atmospheric pressure using pal~ad~~m-~ a;o nd the ;olvent evaporated in vacuo. If the

dissolves slowly. The ca~alyst IS. tere om' a atography (TLC), it is dissolved in 2 mLof

residue is not pure accordmg to thin-layer c hom a short colunm of silia gel 60. The bygrap

Yth°nl cetate; the product 15 with methanol:

ethyl acetate and purified by chromat°

d ith petroleum eth er-e y a

,

1

products are e ute WI °

12 60 ( 0 3 CH OH)' R 0.64 (toluene-ethanol, 1:2).

c.,

3

'f

yield: 0.85 (92%); mp 109 C, [a]o - .



r.-senue 15. The moe



Removal of the Carboxyl and O-Acyl Protection

.

10 ranosyl)-L-serine. A solution of xylosyl ser0-(2,3,4- Trl-O-benzoyl-ll-D~( inP~ethanol (20 ml.) is hydrogenated for 18 h using

ine benzyl ester 14 (0.5 g, 0.78 mmol)

1 st. The catalyst is filtered off, and the solvent

palladium-char:oal (5%, 0.1 g) as:~ c~:~J 041 g (95%), mp 130°_133°C; [a1 o -29.2

evaporated to gIve the pure produ . Y 'h' 1 ater 13'5'24).

R 048 ( thyl acetate-met ano -w

. . .

1

(c 0.6, CHPH); f '

e

. 16 To a solution of O-benzoyl protected xylosy

O_(ll_D_Xylopyrano~yl)-L:en~e(20 mL) is added at room temperature hydrazine

o

b TLC (ethyl acetate-methanol-water 13:5:2.4)

serine (0.23 g, 0.4 mmol) In ~e

hydrate (100%, 20 mL). Momton~gth' Y30 . After 40 min acetone (50 mL) is added to

ti t be complete WI m

rmn,

,

" 1 h th

proves the reac on 0

. (which is volatile) After stirring ror

, e

t

th h d . e to the acetone azine w ·

transfonn e y razm.

he resi d

is stirred with ethyl acetate to extrac

solution is concentrated In vacuo, ~ e r~~ ue (860/<) mp 2150_2180C (decomposition);



:m



impurities and gi~e a pu~e prodU~~~I~~d(C O.~~O), ::ported [39] [a]o _12° (c 1.0, HzO)·

reported [39] 230 -235 C, [a1o

.



B.



Orthogonal Deprotection of Fmoc D-Glycopeptide tert-Butyl

Esters



b

l)-L-threonine tert-butyl ester 18 [34]

N_(9_Fluo renylmethoxycar ony b dii id (928 g 045 mol) tert-butanol (43.4 g, 0.58

. ,.

f 1 3 di clohexyl car 0 nrm e

A mixture 0 , - ICy.

001 mol) is stirred in a tightly sealed flask at room

mol) and copper(!) chloride (1 g, .



temperature in the dark. After 5 days, dry dichloromethane (300 mL) is added. A solution of

Fmoc threonine (47.16 g, 0.138 mol) in dry dichloromethane (300 ml.) is added dropwise to

the stirred mixture. After stirring for 4 h (TLC monitoring, toluene-ethanol 10:1), the

mixture is filtered, and the filtrate concentrated in vacuo to about 150 mL. Newly precipitated dicyclohexyl urea is again removed by filtration. The filtrate is diluted with dichloromethane to a volume of 500 ml., washed with saturated NaHC0 3 solution (3 x 100 mL). In

cases of unsatisfactory phase separation, the aqueous layer is re-extracted with dichloromethane. The organic layer is dried with MgS0 4 and the solvent evaporated in vacuo. The

remainder is dissolved in a small volume of ethyl acetate, kept at - 28°C for some hours and

filtered once more to remove any urea. The filtrate was concentrated in vacuo and the

residue purified by flash chromatography on silica gel (500 g, 0.043-0.06 mrn; E. Merck,

Darmstadt, Germany) in petroleum ether-ethyl acetate (2:1), and subsequent recrystallization from ether-petroleum ether: yield, 42 g (77%); mp 74°C, reported [161 83°C; [a1 o

-9.5 (c 1.05, CHCI 3) ; reported [l61 [a1 o 9.0° (c 1.15, CHCI 3) .

Other N-protected threonine derivatives as well as N-protected serine derivatives

can be converted to the corresponding tert-butyl esters using this method [34].



N-(9-Fluorenylmethoxycarbonyl-O-(3.4.6-tri-O-acetyl-2-azido-2-deoxy-a-Dgalactopyranosylt-t.-threonine tert-Butyl Ester 20 [341

Under careful exclusion of moisture and oxygen, a solution of ethyl I-thio-3,4,6-tri-0acetyl-2-deoxy-2-azido-13-D-galactopyranoside [40] (375 mg, 1 mmol) and Fmoc-ThrOtBu 18 (600 mg, 1.5 mmol) in dry toluene (10 mL) is stirred witlt powdered 4-A molecular

sieves (500 mg) for 1 h at 20°e. After cooling to 5°C, a solution of dimethyl(methyltltio)sulfonium tetrafluoroborate [411 (520 mg, 2 mmol) in dry dichloromethane (10 mL) is

added. After 1 h at 5°C and 16 h at 25°C, ethyl-diisopropylamine (130 mg, 1 mmol) is added.

The mixture is stirred for 1 h, filtered, concentrated in vacuo, and toluene (10 mL) is distilled

off in vacuo from the residue. Purification by flash-chromatography in toluene-acetone

(9:2) gives the mixture of anomers: yield: 520 mg (86%), cxI13 = 3.1. Separation of the

anomers is carried out by flash chromatography in dichloromethane-acetone 100:3 on

silica gel (200 g): a-anomer 20: yield, 380 mg (63%); [a]o 84.6° (c 1, CHCI3 ; reported [16]

[a1 o 69.3° (c I, CHCI 3) .

13 Anomer: N-(9-Fluorenylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy13-D-galactopyranosyl)-L-threonine tert-Butyl Ester: yield, 115 mg (19%), [a1 o -8.0° (c 1,

CHCI 3) ·

N-(9-Fluorenylmethoxycarbonyl)-O-(3.4.6-tri-O-acetyl-2-azido-2-deoXY-13-Dgalactopyranosyl)-L-threonine 21

To Fmoc O-glycosyl threonine tert-butyl ester 20 (290 mg, 0.4 mmol) dissolved in dry

dichloromethane (5 mL) is added dry trifluoroacetic acid (3 mL) at O°C. The mixture is

allowed to warm to room temperature, and the reaction is monitored by TLC (dichloromethane-ethanol 10:1). After 3 h, the conversion is complete. The solvent is evaporated in

vacuo. Toluene (10 mL) is codistilled in vacuo from the remainder. Small amounts of an

unpolar impurity are separated by flash chromatography on silica gel (20 g) in dichloromethane-ethanol (15:1): yield 230 mg (90%); [a1 o 69.7° (c 1, CHCI3) ;

0.32 (CH2CI2ethanol 10:1).



s,



Kunz



277



Protecting Groups in Glycopeptide Synthesis



276



Removal of the Fmoc Group and Labeling with Biotin

0-(3,4,6- Tri_0_acetyl-2-azido-2-deoxy-a_D_galactopyranosyl)-L-threonine tertButyl Ester 22. A solution of Fmoc-Thr(aAc3GaIN3)-OtBu 20 (215 mg, 0.3 mmol) in

freshly distilled morpholine (2 mL) is stirred at room temperature for 20 min. After

concentration in vacuo and codistillation with toluene (twice 3 mL) in vacuo, 22 is obtained



quantitatively. It is immediately used in the following reaction.

N_(D_Biotinyl)_0_(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D_galactopyranosyl)-Lthreonine tert-Butyl Ester 23 [34], A mixture of D-biotin (150 mg, 0.6 mmol),

l_ethyl_3_(3_dimethylaminopropyl)-carbodiimide (EDC: 580 mg, 3 mmol), and I-hydroxybenzotriazol (HOBt: 540 mg, 4 mmol) in dimethylformamide (DMF: 2 mL) is stirred

e

under exclusion of moisture at 22°C. After 45 min, the biotin is dissolved, and a solution of

freshly prepared glycosyl threonine ester 22 (0.3 mmol, preceding procedure) in dichloromethane (2 ml.) is added at O°C. After stirring for 16 h at room temperature, the solvent is

evaporated in vacuo, the remainder dissolved in dichloromethane (50 ml.), extracted with

ice-cold 0.2 N HCI (3 x 25 ml.), water (25 ml.), and saturated NaHC0 3 solution (2 x

25 mL), dried with MgS0 and concentrated in vacuo. purification by flash chromatogra4,

phy on silica gel (20 g) in dichloromethane-ethanol (25:1) yields 23; 200 mg (93%); [01.]0

96.50 (c I, CHCI



3);



e, 0.29



(toluene-acetone 4:1).



C. The Fmoc-Allyl Ester combination [24]

N_(Fluorenylmethoxycarbonyl)-L-threonine Allyl Ester 24

To a solution of L-threonine allyl ester hydrochloride [22,42] (6.0 g, 30.6 mrnol, the

corresponding hydrotrifluoroacetate or hydrotoluenesulfonate can also be used) in saturated NaHC0 solution (100 mL) and dioxane (100 mL) is added dropwise at O°Ca solution

3

of 9-f1uorenylmethyl

chloroformate (10.8 g, 41.8 mmol) in dioxane (50 mL). After stirring

for 24 h, the solvent is evaporated in vacuo, the remainder dissolved in ethyl acetate (200

ml.), washed with 0.5 NHCI, saturated NaHC0 3 solution, and water (each 100 mL), dried

with MgS0 and concentrated in vacuo. The crude product is subjected to chromatography

4, (300 g) in petroleum ether-ethyl acetate (4:1), and the obtained product

on silica gel

is recrystallized from ethyl acetate-petroleum ether to give pure 24: yield, 11.2 g (96%);

mp 980-100°C; [01.]0 -17 .2° (c 1, dimethylformamide); R, 0.33 (petroleum ether-ethyl



[3. anomers (a/[3 20:1), 5.8 g (79%). Repeated flash ch

.

YIeld,4.5 g (61%); mp 590-610C , [01. ] +667°

1 c~omatOgraPhY

gives the a anomer

26:

CI3); RfOAO (CH

. (c,

2CI 2-acetone 45:1).

0

N-(9-Fluorenylmethoxycarbonyl)-0-(2-acetamido-34 6-t '-0galactopyranosylt-u-threonine Allyl Ester 27

"n

acetyl-Zsdeoxy-cc-t»



To a solution of Fmoc- Thr(Ac GalN ) OAll

added at 00C acetic acid (1 ~ a;d26 (4.0 g, 5.8 ~ol) in dry THF (50 mL) is

(304 mL, 704 mmol) in THF (10 ~L) A.:tubs;tue~tly, .a solu~lOn of tri-n-octylphosphine

complete the N-acetylation the remai~der~r ti ' tde ~hxture. IS concentrated in vacuo. To

..

10 mL

'

ISS rre WIt acetic anhydrid (2 mL)

) for 6 h, concentrated in vacuo. Toluene 3 X

...

-pyndme

( .

residue, which is subsequently purified b fI h( h 20 mL) ISdistilled in vacuo from the

1 1

y as c romatography on sili

th I

n Sl ca. ge (00 g) in

petro Ieum ether-ethyl acetate ~

~ e y acetate. The product .

acetate-petroleum ether: yield, 3.9 g (94%)' m 66 6 ° . IS recrystalli;ed from ethyl

R, 0.56 (ethyl acetate).

' p

- 8 C, [aJ o +36.3 (c I, CHCl 3);



~



The selective removal of the Fmoc rou from

.

.

..

Sections IILA and IILB for its removal S.g '1 p

27 IS camed out m strict analogy to

and must be subjected to further react'. I~I ar tO .product 22, the obtained 28 is not stable

IOn imme d lately.

N-(9-Fluorenylmethoxycarbonyl)-0-(2-acetamido-3

-'"

galactopyranosyl)-L-threonine 29

,4,6 trt O-acetyl-2-deoxy-a-D-



To a solution of Fmoc-Thr(Ac 3GaINAc)-OAll 27 2

.

under argon atmosphere is added N-methyl aniline (6.5 g, 2.8 mmol) in ~ry THF (20 ml.)

0.043 mmol; 1.5 mol%)

\50 mg,

the dark for 2 h and then concentrated in

Fl

aurumrur. The mixture IS stirred in

in ethyl acetate ~ methanol and recrystal1~:~~~~ fr~Sh chromatography on silica gel (60 g)

acetate-petroleum ether gives 29 as cryst I' . Id m methanol-petroleum ether and ethyl

a

[16] amorphous, obtained by a different pr sJle ,1.8 g (96%); mp 114°-116°C, reported

0c(CH

ureC)I;

[16] [OI.J +65.00 (c 1045, CDCI ). R 0 46

[aC]o +75.8° (c 0.5, CHPH), reported

3' f .

2 2- HpH 1:1).

o



tetrakiS(triPhenYIPhOSPhine)Pal~~~ and(Oa)catalytl~ amo~nt



D. Removal of the Allyloxycarbonyl Group [19,23,26]



°



acetate 2:1).



gIUCOpymnOSYI)-3-0-acetyl-6-0-(2,3,4-tr:~-be~ - 1~b~nZYI-2-deoxy-[3-D-



N_(9_Fluorenylmethoxycarbonyl)-0-(3,4,6-tri-o-acetyl-2-azido-2-deoxy-a-D-



Tetraethylammonium bromide (4



galactopyranosyl)-L-threonine Allyl Ester 26

Under argon atmosphere and exclusion oflight and moisture, a mixture of Fmoc- Thr-OAll

24 (4.9 g, 12.9 mmol), Ag

(4.0 g, 14.5 mmol), and powdered 4-A molecular sieves

2C0 3

(2 g) in dry toluene (60 ml.) and dichloromethane (90 ml.) is stirred for 1 h. At room

temperature, AgCIC0 (004 g, 1.9 mmol) is added. After 20 min, a solution of 3,4,6-tri-04

acetyl_2_azido_2_deoxy-a-D-galactopyranosyl bromide [43] 25 (4.2 g, 10.7 rnmol) in

toluene (90 mL)_dichloromethane (90 mL) is added dropwise within 1 h. After 24-40 h at

room temperature (TLC monitoring; dichloromethane-acetone 45:1), dichloromethane

(100 mL) is added. The mixture is filtered through Hyflow, and the filtrate is extracted with

saturated NaHC0 solution (2 x 100 mL) and water. The organic layer is dried with MgS0 4

3

and the solvent evaporated

in vacuo. purification by flash chromatography on silica gel (100

g) in dichloromethane ~ dichloromethane-methanol (250:1) yields a mixture of the a and



dimethylformamide-dichloromethane (2'1) f a 3r sle:es (6 ~) were stirred in 15 mL of

I f '

or 0 min Usmg a yri

23 4

b

enzy -a-D- ucopyranosyl bromide [35] 31 (3

6

'.

s nnge, ,,-tri-O(5 mL) was added dropwise After 4 da

th g,. mmol) dissolved in dichloromethane

.

ys, e mixture was filtered thr

hC .

s.u bsequently was washed with dichloromethane (300

elite, which

I'

mL). The combmed organic solunons were extracted with 1 M KHCO

.

3 so utron (3 x 100 ml.) dri d ith M

' e WI

gS04' concentrate d m vacuo, and the remainder was dried' hi h

in Ig vacuo Chromat

h (100

I) .

I

ge m petro eum ether-ethyl acetate 2:1

di h i '

ograp y

g silica

recrystallization from dichloromethane dii ~ IC oromethane-methanol 20:1, and by

[01.]0 -38.650 (c 0.5 chloroform. R 0-4 (1ICsoHPCroIPYMIe ther gave 32: 1.5 g (74%); mp 175°C'

,

,

f .

3- eOH 10:1).

'



2-Acetamido-4-0-(2-acetamido-34-di-0glucopyranosyl Azide 32



I6



zy a L-fucopyranosyl)-2-deoxy-[3-D-



.

acetyl-6-0-benzYI-2-deoxy-[3-D-gl~~O19 mmol)i 2-acetanudo-4-0-(2-acetamido-3,4-di-Oazide [26] 30 (1.25 g, 1.88 mol) and !!a~~~~:~~-o~acetYI-2-deOXy-[3-D-gIUCOPyranOSYI



~ug



Kunz



279



Protecting Groups In Glycopeptide Synthesis



278

2_Acetamido_4_0_(2_acetamido-3,4-di-O-acetyl-6-0-benzyl-2-deoxy-f3-ooglucOpyranosyl)_3_0_acetyl-6-0-(2,3,4-tri-O-benzyl-a-L-!ucopyranosyl)-2-deoxy-f3glucopyranosylamine 33

The trisaccharide azide 32 (I g, 0.92 mmol) was dissolved in methanol (20 ml.). After

addition of Raney nickel (200 mg; E. Merck, Darmstadt, Germany), which was washed ten

times with water, hydrogenation was performed for 3 h. After filtration and concentration in

vacuo the glycosylamine 33 was obtained: yield, 927 mg (95%); [a]D -24.8° (c 0.5,



CH



);

2CI2



R f 0.2 (CHCL 3-MeOH 10:1).



N_Allyloxycarbonyl-aspartic acid a-tert-Butyl Ester 34

To a solution ofl-0-tert-butyl aspartate [44] (3 g, 15.9 mmol) and KHC0 3 (3 g, 32 mmo1) in

water (50 mL) was added at O°C allyl chloroformate (1.7 ml, 16 mmol; E. Merck,

Darmstadt, Germany). After stirring for 1 h, the mixture was extracted with diethyl ether

(100 mL). The aqueous layer was acidified to pH 2 using 1 M HCl and extracted with diethyl

ether (4 x 50 mL). These ether solutions were combined, dried with MgS04· The solvent

was evaporated in vacuo to give 34 as an oil; 3.87 g (89%), [a]D +21° (c 1, CH 2CI 2) ; RfO.25



E.



Exchan~e of Et~er-type for Ester-type Protection in the

Saccharide Portion: Generation of Stability Against Acids [19,26]



°



2-Acetamido-4-0-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-Q.-o-gluc

I) 3

acetyl-ti-Cr-tcc-t-f)

I

...

opyranosy - - a-i-jucopyranosy )-2-deoxy-f3-o-glycopyranosyl Azide



To a solution of 2-acetamido-4-0-(3,4,6-tri-O-acet 1-2-deox - ..

pyranosyl)-3-0-acetyl-6-0-[2 3 4-tri 0 (4 meth Yb

I Y 2 phthalimido-Bvn-glucoQ

I

"

- - oxy enzy )-a-L-fucopyranosy] 2 d

...-n-gammoni

ucopyranosyl. azide 3[26] 37 (II

g 0 .907 mmol) In

. ace tonitril

eoxy.,

orntn e-water (9:1)- is- added

eerie



complete (ab:: ;I:a~~ g, 5.~7 ~?l), and the mixture is stirred until the reaction is

nitrile (30 mL) and c'

t m~m ~nng In CHCI3-CH30H 10:1). After addition of aceto..

oncen ration In vacuo, the remainderis subjected to chromat

on

SIlica

gel (50 g) in dichloromethane-rnethancl Stl.l

CHCl

fCH

. ~ 6'1

.. Th e cru d e pr od uct (Rography

0 15 .

30H 10:1), containing inorganic material stemming from th

idizi

f'

I.n

3

used for further conversion.

e OXI ZIng reagent IS



~



~~~~~~~g~;4;~-~2.-c:;etami10-3,4,6-tri-o-acetyl-2-deoXY-13-o-glycopyranosyl)-3-0[26]

, , - n- -acety -a-L-fucopyranosyl)-2-deoxy-f3-o-glucopyranosyl Azide 38



(petroleum ether-ethyl acetate 2:1).

N2_(Allyloxycarbonyl)_N4-(2-acetamido-4-0-(2-acetamido-3,4_di_O_acetyl-6-0-benzyl-2deoxy_f3_o_glucopyranosyl)-3-0-acetyl-6-0-(2,3,4-tri-O-benzyl-a-L-!ucopyranosyl)-2deoxy_13_o_glucopyranOSyl)-L-aSparagine tert-Butyl Ester 35 [26]

The trisaccharide amine 33 (0.5 g, 0.47 mmol), Aloc-Asp-OtBu 34 (0.2 g, 0.73 mmol), and

EEDQ [37] (0.6 g, 2.4 mmol) were stirred in dimethylformamide (5 mL). After 3 days the

solvent was distilled off in high vacuo. The remainder was purified by flash chromatography on silica gel (60 g) in petroleum ether-ethyl acetate 2:1 ~ dichloromethane-methanol

50:1 to give 35: 466 mg (75%), [a]D -28.4° (c 0.75, CH 2CI2) ; RfO.38 (CHCI3-MeOH 10:1).

The corresponding a-anomer was isolated by this chromatography as a by-product:

N2_(Allyloxycarbonyl)_N4-(2-acetamido-4-0-(2-acetarnido-3,4-di_O_acetyl_6_0_benzyl-2deOXy_f3_0_g1ucopyranosyl)-3-0-acetyl-6-0-(2,3,4-0-benzyl-a_L_fucopyranosyl)-2-deoxya_o_glucopyranosyl)-L-aSparagine tert-butyl ester: yield, 62 mg (10%); R, 0.42 (CHCI3-



The cmd~ .product obtained in the preceding experiment is dissolved in ethanol

..

(40 mL).

after addition of hydrazine hydrate (100% 10 mL) th e I '

(removal of the phthaloyl group). Acetone (30 mL i~ ad so utron IS s~rred ~t 80°C for I h

tthe rmxture IS

in vacuo. Codistillation with acetone (30 mL) is



!epeate~~~~d



co~centrated



~zi::d~~ remainder is drie~ ~nder high vacuum and diS~~~V~;~~~~:~~~:~:ti:s ~~

t~uene ~~~'~~;U;~. Aft~ s~m3~g for.18 h, concentration in vacuo, and codistillation with

T

I

, e cru e

IS punfied by flash chromatogra h



Elution with acetone and then with dichloromethane-methanoI3&1 y .on siuca ge (70 g).

yield: 581 mg (72%); mp 137°C' [a] -725° ( 025 CH CI2); . yields pure Sdroverall

10:1).

'

D



c.,

2

R, 0.4 (CHCI3-CH30H



REFERENCES



MeOH 10:1).

N4_f2_Acetamido-4-0-(2-acetamido-3,4-di-O-acetyl-6-0-benzyl-2-deoxy-f3-ooglucopyranosyl)_3_0_acetyl-6-0-(2,3,4-tri-O-benzyl-a-L-!ucopyranosyl)-2-deoXY-13glucopyranosyl]-L-aSparagine tert-Butyl Ester 36

To a solution of N2_allyloxycarbonyl_N4_[2_acetarnido-4-0-(2-acetarnido-3,4-di-O-acetyl6_0_benzyl_2_deoxy-f3-0-glucOpyranosyl)-3-0-acetyl-6-0-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)_2_deOxy-f3-o-g1ucOpyranosyl]-L-asparagine tert-butyl ester [19,26] 35 [400 mg;

0.305 mmol; [a]D -28.4° (c 0.75, CH 2Cl2] and 5,5_dimethyl-cyclohexane-1,3-dion (dimedone, I g, 7.1 mmol) in oxygen-free tetrahydrofuran (15 mL) is added tetrakis(triphenyl2

phosphine)-paliadium(O) (50 mg, 0.043 mmol) under argon atmosphere. After stirring for

h in the dark, the solvent is evaporated in vacuo and the remainder dissolved in dichloromethane (200 mL). The organic layer is washed with I N KHC03 solution (50 mL) and

dried with MgS0 After evaporation of the solvent in vacuo, the crude product is purified

4. on silica gel (40 g) in dichloromethane-methanoI20:1 ~ 15:1 to give

by chromatography

pure 36: yield: 341 mg (91%), [a]D 37.4° (c 0.5, CH 2CI2) ; R f 0.35 (CHCL 3-CH30H 10:1).



R. L'

J. Ivatt,

0" GIycoproteins,

. PIenum Press, New York 1984

21. H

d ed. The Biology ."

. 218:

. IS

an N Sharon Protem glycosylation. StructuraI and functional aspects,

,.

I (1993).'

Eur: J. Biochern.



3. J. Martinez, A. A. Pavia, and F. Winternitz Synthese d'un 0- I

.

I'

. g ycopeptide par allongementde la

chain peptidique da cote N-terminal d'un ' I

and references cited therein.

g ycosy aminoacide, Carbohydr. Res. 50: 148, (1976)

4. H. G. Garg and R. W: Jeanloz, The synthesis of 2-acetamido-3 4 5 .

oxycarbonyl)-L-aspart-I- and 4-0 I] 2 de

' , -tn-O-acetyl-N-[N-(benzyl437 (1972), and references cited ~e~e~n. oxy-Bvo-glucopyranosylamine, Carbohydr. Res. 23:

5. H. Kunz, Synthesis of glycopeptides. Partial structures of biolo .

..

Angew. Chern. Int. Ed. Eng!. 26:294 (1987).

gical recognition components,

6. H. Paulsen, Progress in the chemical synthesis of com I

li

.

Ed. Engl. 21:155 (1982).

p ex 0 igosaccharides, Angew. Chern. Int.

7.



~. R. Schmidt, New methods of glycoside and oligosaccharid

Ch

e syntheses-are there alternatives to the Koenigs-Knorr method A

,ngew.

ern. Int. Ed. Eng!. 25:212 (1986).



280



Kunz



H. Kunz, Glycopeptides of biological interest. A challenge for chemical synthesis, Pure App!.

Chern. 65:1223 (1993).

9. H. G. Garg, K. von dem Bruch, and H. Kunz, Developments in the synthesis of glycopeptides

containing glycosyl iasparagine, L-serine and L-threonine, Adv. Carbohydr. Chern. Biochem.

50:277 (1994).

10. P. Schultheiss-Reimann and H. Kunz, O-Glycopeptide synthesis using 9-f\uorenylmethoxycarbonyl (Fmoc)-protected synthetic units, Angew. Chern. Int. Ed. Eng!. 22:62 (1983).

S. Peter, T. Bielefeldt, M. Meldal, K. Bock, and H. Paulsen, Multiple-column solid-phase

II.

glycopeptide synthesis, J. Chern. Soc. Perkin Trans 1:1163 (1992).

.

12. H. Kunz and S. Bimbach, Synthesis of O-glycopeptides of the tumor associated TN- and

'f-antigen type and their binding to bovine serum albumin, Angew. Chern. Int. Ed. Engl. 25:360

8.



13.

14.



15.



(1986).

B. Luning, T. Norberg, and J. Tejbrant, Synthesis of mono- and disaccharide amino-acid

derivatives for use in solid phase peptide synthesis, Glycoconjugate J. 6:5 (1989).

L. Otvos, Jr., L. Urge, M. Hollosi, K. Wroblewski, G. Graczy, G. D. Fasman, and J. Thurin,

Automated solid-phase synthesis of N-glycoproteins antennae into T cell epitopic peptides,



Tetrahedron Lett. 31:5889 (1990).

H. Kunz and P. Schultheiss-Reimann, unpublished; P. Schultheiss-Reimann, Glycopeptidsynthese mit der 9-Fluorenylmethoxycarbonyl-Schutzgruppe, Dissertation, Universitiit Mainz,



Germany, 1984.

16. H. Paulsen and K. Adermann, Synthese von O-Glycopeptid-Sequenzen des N-Terminus von

Interleukin, Liebigs Ann. Chern. p. 751 (1989).

.

17. H. Kunz and B. Dombo, Solid-phase synthesis of peptides and glycopeptides on polymenc

support with allylic anchor groups, Angew. Chern. Int. ~d. En.gl. ?7:711 (1988).

.

18. W. Kosch, J. Marz, and H. Kunz, Synthesis of glycopeptide derivatives of peptide T on a solidphase using an allylic linkage, React. Polym. 22:181 (1994).. . ,

.

19. H. Kunz and C. Unverzagt, Protecting group-dependent stability of intersaccharide bondssynthesis of a fucosyl-chitobiose glycopeptide, Angew. Chern. Int', Ed. Eng!. 27:1697 (19~8).

20. V. Bencomo and P. Sinay, Synthesis of M and N active glycopeptldes. Part of the N-terrrunal

region of human glycophorin A, Glycoconjugate J. 1:5 (1984).

.

.

21. S. Lavielle, N. C. Ling, R. Saltmann, and R. Guillemin, Synthesis of a glycotnpeptlde ~nd

glycosomatostatin containing the 3_0_(2_acetamido_2_deoxy_(3_D_glucopyranosyl)-L_senne

22.



residue, Carbohydr. Res. 89:229 (1981).

H. Kunz and H. Waldmann, The allyl group as mildly and selectively removable carboxyprotecting group for the synthesis of labile O-glycopeptides, Angew. Chern. Int. Ed. Engl. 23:71



23.



(1984).

H. Kunz and C. Unverzagt, The allyloxycarbonyl (Aloe) moiety-conversion of an unsuitable

into a valuable amino protecting group for peptide synthesis, Angew. Chern. Int. Ed. Engl.



24.



23:436 (1984).

M. Ciommer and H. Kunz, Synthesis of glycopeptides with partial structure of human glycophorin using the f1uorenylmethoxycarbonylJallyl ester protecting group combination, Synlett p.



25.



26.

27.



28.



593 (1991).

H. Kunz, H. Waldmann, and J. Marz, Synthese von N-Glycopeptid-Partialstrukturen der Verknupfungsregion sowohl der Transmembran-Neuraminidase eines Influenza-Virus als auch des

Faktors B des menschlichen Komplementsystems, Liebigs Ann. Chern. p. 45 (1989).

C. Unverzagt and H. Kunz, Synthesis of glycopeptides and neoglycopeptides containing the

fucosylated linkage region of N-glycoproteins, Bioorg. Med. Chern.. 2:118.9 (199~).

H. Kunz and J. Marz, Synthesis of glycopeptides with LeWIS' antlg~n Side cham and HIV

peptide T sequence using the trichloroethoxycarbonalJallyl ester protecting group combination,



Synlett p. 591 (1992).

. '

. x

.

.

K. von dem Bruch and H. Kunz, Synthesis of N-glycopeptlde elusters With LeWIS antigen Side

chains and their coupling to carrier proteins, Angew. Chern. Int. Ed. Eng!. 33:101 (1994).



Protecting Groups in Glycopeptide Synthesis

29.

30.

31.

32.



281



P. Braun, H . .waldmann, ~nd H. Kunz, Chemoenzymatic synthesis of glycopeptides carrying the

tumor associated TN antigen structure, Bioorg. Med. Chern. 1:197 (1993).

S. Hanessian and J. Banoub, Chemistry of the glycosidic linkage. An efficient synthesis of

1,2-trans-di-saccharides, Carbohydr. Res. 53:C13, (1977).

S. J. Danishefsky, K. F. McClure, J. T. Randolph and R. B. Ruggeri, A strategy for the solidphase synthesis of oligosaccharides, Science 260: 1307 (1993).

L. A. Carpino and G. Y. Han, The 9-f\uorenylmethoxycarbonyl amino-protecting group, J. Org.

Chern. 37:3404 (1972).



33.



O. Seitz and H. Kunz, A novel allylic anchor for solid-phase synthesis. Synthesis of protected

and unprotected O-glycosylated mucin-type glycopeptides, Angew. Chern. Int. Ed. Engl 34'803

(1995~

. .



34.



H. Kunz. ~nd G. Braum, unpublished results; G. Braum, Synthese von Glycopeptiden mit

photo~tlvlerb~en Gruppen nn Saccharidteil, Dissertation, Universitat Mainz, Germany, 1991.

M. Dejter-Juszinsky and H. M. Flowers, Koenigs-Knorr reaction. II-synthesis of an n-Lhnked disaccharide from tri-O-benzyl-L-fucopyranosyl bromide, Carbohydr. Res. 18'219

(1971).

.



35.



36.

37.

38.

39.



40.

41.

42.

43.

44.



R. U. Lemieux and J. 1. Haymi, The mechanism of the anomerization of the tetro-O-acetyl-oglucosyl chlorides, Can. J. Chern. 43:2162 (1965).

B. Belleau and G. Malek, A new convenient reagent for peptide synthesis, J. Arn. Chern. Soc.

90: 1651 (1968).

W. Gunther an~ H. Kunz, Synthesis of a (3-mannosyl-chitobiose-asparagine conjugate-s-a

central core region umt of the N-glycoproteins, Angew. Chern. Int. Ed. Engl. 29: 1050 (1990).

J; M. L~combe, A. A. Pavia, and R. M. Rocheville, Un nouvel agent de glycosylation:

I Anhydnde tnfluoromethanesulfonique. Synthese des n et (3O-glycosyl-L-serine -t-threonine

et -i.-hydroxyproline, Can. J. Chern. 59:473 (1981).

'

H. Paulsen,

Rauwald, and U. Weichert, Glyeosydation of oligosaccharide thioglycosides to

O-glycoprotem segments, Liebigs Ann. Chern. p. 75 (1986).

~. Fugedi. and P. J. Garegg, A novel promoter for the efficient construction of 1,2-trans linkages

in glycoside synthesis, using thioglycosides as gIycosyl donors, Carbohydr. Res. 149:C9 (1986).

H. ~aldmann and H. ~unz, AJlylester als selektiv abspaltbare Carboxylschutzgruppen in der

Peptid- und Glycopeptidsynthese, Liebigs Ann. Chern. p. 1712 (1983).

H. Paulsen and M. Paal, Blocksynthese von O-Glycopeptiden und anderen T-Antigen Strukturen, Carbohydr. Res. 135:71 (1984).



r.:.



R. W. Roeske, Preparation of r-butyl esters of free amino acids, J. Org. Chern. 28: 1251 (1963).



12

Oligosaccharide Synthesis with

Trichloroacetimidates

Richard R. Schmidt and Karl-Heinz lung

Universitst Konstanz, Konstanz, Germany



I.



II.



III.



Introduction

A. Activation through anomeric oxygen-exchange reactions

B. Activation through retention of the anomeric oxygen

The Trichloroacetimidate Method

A. Trichloroacetimidate formation (activation step)

B. Glycosylation reactions (glycosylation step)

Experimental Procedures

A. Synthesis of O-glycosyl trichloroacetimidates

B. Glycosylation reactions with O-glycosyl

trichloroacetimidates

References and Notes



283

285

286

289

289

290

296

296

298

308



I. INTRODUCTION

Glycoside synthesis is a very common reaction in nature, thus providing a great variety of

compounds, such as various types of oligosaccharides, or glycoconjugates with lipids

(glycolipids), with proteins (glycoproteins or proteoglycans), and with many other naturally occurring compounds. The important biological implications of the attachment of

sugar moieties and especially of complex oligosaccharide structures to an aglycon are only

now becoming more and more obvious, thus creating a growing general interest in the field

[1-7]. The great structural variety available to sugars [la] complicates the synthesis of

glycosides and, even more so, of complex oligosaccharide structures. It has only recently

become possible to develop methods for the synthesis of such complex compounds [1-4].

The results obtained in this endeavor are summarized in Scheme 1, which will be shortly

discussed; however, the main emphasis in this chapter will be devoted to the tri-



283



Schmidt and Jung



284



Oligosaccharide Synthesis with Trichloroacetimidates



285



chloroacetimidate method, which has become a very competitive, widely applicable procedure for glycoside bond formation [1,4].

The methods for glycoside bond formation developed thus far usually consist of an

activation of the anomeric center (activation step); this activated species is then used to

release the glycos' I donor, with the help of a promoter or a catalyst, to generate the

glycosidic bond to e acceptor (glycosylation step). In principle, for the activation step,

two different appr aches are employed: (I) activation through anomeric oxygen-exchange

reactions and (2) ctivation through retention of the anomeric oxygen [1]. Obviously, this

basic difference has wide-ranging implications as will be discussed later. The latter method

has been essentially developed in our laboratory [1).



A.



f



Ql

II:



~





E



Ql



.c

(J



en



Activation Through Anomeric Oxygen-Exchange Reactions



The Fischer-Helfericb Method

Still the simplest glycosylation procedure available is the Fischer-Helfericli method [1,8]

(see Scheme 1, A); it consists of a convenient direct anomeric oxygen-exchange reaction

in a cyclic hemiacetal following mechanistically a typical acid-catalyzed acetal formation

reaction. This method has great merits in the synthesis of simple alkyl glycosides for which

an excess glycosyl acceptor can be employed, thereby inhibiting self-condensation of

unprotected sugar moieties and thus also acting as a solvent for the starting materials and

the product(s). However, because ofthese limitations and because of the reversibility of this

method, it has not gained importance in the synthesis of complex oligosaccharides and

glycoconjugates. For this, irreversible methods are required, which generally can be gained

by preactivation of the anomeric center, thereby generating strong glycosyl donor properties in the presence of mild promoters or catalysts.

The Koenigs-Knorr Method



One of these methods that leads to strong glycosyl donor properties in the activated species

is exchange of the anomeric hydroxyl group by bromine and chlorine, respectively, in the

activation step (Koenigs-Knorr method [9], see Scheme 1, B). Thus an o:-haloether is

generated that can be readily activated in the glycosylation step by halophilic promoters,

typically heavy-metal salts, thus resulting in irreversible glycosyl transfer to the acceptor.

This method is the basis of a very valuable technique for the synthesis of complex

oligosaccharides and glycoconjugates, which has been extensively reviewed [1-4]. It has

been continually developed and widely applied (e.g., for the synthesis of 1,2-transglycosides by the silver triflate tetramethylurea method) [10]. In spite of the generality

of the method, the requirement of at least an equimolar amount (often up to 4 eq) of metal

salt as promoter (frequently incorrectly termed a "catalyst") and problems concerning the

disposal of waste material (e.g., mercury salts) could be limiting factors for large-scale

preparations. Therefore, alternative methods are of great interest.

Methods Related to the Koenigs-Knorr Method



Other anomeric oxygen-exchange reactions in the activation step have been recently quite

extensively investigated. Thus, closely related to the classic Koenigs-Knorr method is the

introduction of fluorine as the leaving group (see Scheme 1, B; X = F) [3,11-13] which,

owing to the stability of the C-F bond, leads to much more stable glycosyl donor

intermediates. Because of the difference in halophilicity of this element compared with



Schmidt and Jung



286



bromine and chlorine, further promoter systems, besides silver salts, were found useful in

glycosylation reactions [14). However, because of the generally lower glycosyl donor

properties [Id] and because also at least equimolar amounts of promoter are required, these

intermediates generally have no real advantages over the corresponding glycosyl bromides

or chlorides.

Anomeric oxygen-exchange reactions by thio groups (see Scheme 1, B; X = SR) have

recently attracted considerable attention for the generation of glycosol donors [2,15).

Thioglycosides, thus obtained, offer sufficient temporary protection of the anomeric center,

thereby enabling various ensuing chemical modifications of the glycosyl donor without

affecting the anomeric center. Additionally, they present several alternative possibilities for

the generation of glycosyl donor properties. Besides various thiophilic heavy-metal salts

[16J, which also exhibit the foregoing disadvantages, iodonium, bromonium, and chloronium ions are also highly thiophilic; however, with counterions, such as bromide and

chloride, a subsequent Koenigs-Knorr type reaction is encountered [15,17). Therefore, a

poor nucleophile is required as counterion of the halonium ion, for instance use of N-iodoor N-bromosuccinimide, enabling direct reaction with the acceptor as nucleophile. However, owing to the strong activation required for thioglycosides, often low a,~-selectivities

are obtained for nonneighboring group-assisted reactions [16d,18) and, especially, for

sugars with lower glycosyl donor properties. Because of the high nucleophilicity of the

sulfur atom in thioglycosides, methyl trifluoromethanesulfonate (methyl triflate) was successfully employed for their activation [19). However, again low a,~-selectivities, the

health hazard of this reagent, the requirement of at least equimolar amounts, and possible

formation of methylation products and other nucleophilic centers (for instance, amide

groups or the hydroxy group of the acceptor) are major disadvantages of this procedure.

Consequently, commercially available dimethyl (methylthio)sulfonium triflate (DMTST),

readily obtained from dimethyl disulfide and methyl triflate, was extensively used as a

thiophilic reagent because it seems to give better results than methyl triflate [IOJ. However,

the basic drawbacks of the overall method are also associated with this promoter system.



B.



Activation Through Retention of the Anomeric Oxygen



Oligosaccharide Synthesis with Trichloroacetlmidates



sugars, thereby simply generating at first an anomeric oxide structure from a pyranose or a

furanose (see Scheme 1, C and D) [IJ.

Anomeric O-Aklylation Method



Alkylation of the ano~~.c oxide, generated by base addition to pyranoses or furanoses

should directly and irre rsibly lead to glycosides. This process was termed by us "anomeric O-alkylation met od" [IJ (see Scheme 1, C). In the beginning, this process was

considered unlikely to f Ifill all of the requirements for glycoside and saccharide synthesis.

Even when all remaining functional groups are blocked by protecting groups, the ringchain tautomerism between the anomeric forms and the open-chain form (Scheme 2)

W



OR



XR~OH

Z



I



Base



~

W



OR



X~~

RO



Z 08



A



~R'X



W



~



X

RO



OR 9

W OR

0-----'"0

...- X

0

Z H



B



W-Z=OR,H

R • Bn, Ac, H, etc.



The first step should consist of an activation of the anomeric center under

formation of a stable glycosyl donor (activation step).

2. The second step (glycosylation step) should consist of a sterically uniform, highyielding glycosyl transfer to the acceptor; however, by a truly catalytic process,

where diastereocontrol may be derived from the glycosyl donor anomeric configuration (by inversion or retention), by anchimeric assistance, by influence of the

solvent, by thermodynamics, or by any other effects.

1.



Because only simple means meeting these requirements will lead to a generally

accepted method, we decided to investigate, instead of acid activation, base activation of



R~oe

Z



C



~R'X

~-Glycopyranoside



a-Glycopyranoslde



The requirements for glycoside synthesis-high chemical and stereochemical yield, applicability to large-scale preparations, with avoidance of large amounts of waste materials, by

having a glycosyl transfer from the activated intermediate through a catalytic processwere not effectively met by any of the methods described in the foregoing for the synthesis

of complex oligosaccharides and glycoconjugates. However, the general strategy for

glycoside bond formation seems to be correct:



287



Scheme 2



already gives three sites for attack of the alkylating agent. In addition, base-catalyzed

elimination in the open-chain form of the sugar could become an important side reaction.

Therefore, the yield, the regioselectivity, and the stereose!ectivity of the anomeric O-alkylation was not expected to be outstanding.

Surprisingly, no studies employing this simple method for the synthesis of complex

glycosides and glycoconjugates had been reported before our work. Only a few scattered

examples with simple alkylating agents, for instance, excess of methyl iodide or dimethyl

sulfate, have been found [l a]. However, in our hands, direct anomeric O-alkylation of

O-benzyl-, O-acyl-, or O-alkylidene-protected sugars in the presence of a base and triflates

(R-X =R-OTf) of various primary and secondary alcohols, including sugars as alkylating

agents, has become a very convenient method for glycoside bond formation [1,21). Also

O-unprotected sugars and less reactive alkylating agents have recently been successfully

employed in this reaction, furnishing directly, and often with very high anomeric stereocontrol, the desired glycosidic products [22J. Potential decomposition reactions, partie-



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