Use of fluorobenzoyl protective groups in synthesis of glycopeptides: β-elimination of O-linked carbohydrates is suppressed

Article abstract of DOI:10.1021/jo001584q

Fluorobenzoyl groups have been investigated as alternatives to acetyl and benzoyl protective groups in carbohydrate and glycopeptide synthesis. D-Glucose and lactose were protected with different fluorobenzoyl groups and then converted into glycosyl bromides in high yields (>80% over two steps). Glycosylation of protected derivatives of serine with these donors gave 1,2-trans glycosides in good yields (～60-70%) and excellent stereoselectivity without formation of ortho esters. The resulting glycosylated amino acid building blocks were then used in solid-phase synthesis of two model O-linked glycopeptides known to be unusually sensitive to β-elimination on base-catalyzed deacylation. When either a 3-fluoro- or a 2,5-difluorobenzoyl group was used for protection of each of the two model glycopeptides the extent of β-elimination decreased from 80% to 10% and from 50% to 0%, respectively, as compared to when using the ordinary benzoyl group. Fluorobenzoyl groups thus combine the advantages of the benzoyl group in formation of glycosidic bonds (i.e., high stereoselectivity and low levels of ortho ester formation) with the ease of removal characteristic of the acetyl group.

Full text of DOI:10.1021/jo001584q

J . Org. Chem. 2001, 66, 2957-2965
2957
Use of F lu or oben zoyl P r otective Gr ou p s in Syn th esis of
Glycop ep tid es: â-Elim in a tion of O-Lin k ed Ca r boh yd r a tes Is
Su p p r essed †
Petter Sjo¨lin and J an Kihlberg*
Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden
jan.kihlberg@chem.umu.se
Received November 6, 2000
Fluorobenzoyl groups have been investigated as alternatives to acetyl and benzoyl protective groups
in carbohydrate and glycopeptide synthesis. D-Glucose and lactose were protected with different
fluorobenzoyl groups and then converted into glycosyl bromides in high yields (>80% over two
steps). Glycosylation of protected derivatives of serine with these donors gave 1,2-trans glycosides
in good yields (∼60-70%) and excellent stereoselectivity without formation of ortho esters. The
resulting glycosylated amino acid building blocks were then used in solid-phase synthesis of two
model O-linked glycopeptides known to be unusually sensitive to â-elimination on base-catalyzed
deacylation. When either a 3-fluoro- or a 2,5-difluorobenzoyl group was used for protection of each
of the two model glycopeptides the extent of â-elimination decreased from 80% to 10% and from
50% to 0%, respectively, as compared to when using the ordinary benzoyl group. Fluorobenzoyl
groups thus combine the advantages of the benzoyl group in formation of glycosidic bonds (i.e.,
high stereoselectivity and low levels of ortho ester formation) with the ease of removal characteristic
of the acetyl group.
In tr od u ction
benzylethers,which are deprotected byhydrogenolysis,15-18
or in two acid-catalyzed steps,19 is another alternative
that has found some use. However, in most syntheses of
glycopeptides the hydroxyl groups of the carbohydrate
are protected as acetyl- or benzoyl esters. These have the
advantage of stabilizing the O-glycosidic bonds against
acid-catalyzed degradation during cleavage from the
solid-phase,20,21 which is especially important for deoxy-
sugars such as fucose.20,22,23
The choice of protective groups is crucial in order to
succeed in synthesis of glycopeptides.1-5 Today most
glycopeptides are prepared on solid-phase according to
the Fmoc-protocol.6,7 This strategy utilizes the base-labile
Fmoc-group8 for NR-protection of the amino acids in
combination with side chain protective groups that are
removed during cleavage from the solid phase with
trifluoroacetic acid (TFA). As demonstrated recently the
hydroxyl groups of the carbohydrate moieties can also
be protected with groups that are cleaved by TFA, such
as silyl- and methoxybenzyl groups or acetals.9-14 Use of
A disadvantage with using acetyl or benzoyl protective
groups for carbohydrate moieties in glycopeptides is the
need for a base-catalyzed deprotection step. This can
cause â-elimination of carbohydrates linked to serine or
threonine, as well as epimerization of peptide R-stereo-
centers. In the case of removal of O-acetyl groups these
side-reactions occur only as rare exceptions,23,24 since very
mild conditions may be used for deprotection (e.g., 6 mM
methanolic sodium methoxide).25 O-Benzoyl groups may
* To whom correspondence should be addressed.
† Dedicated to Professor J oachim Thiem on the occasion of his 60th
birthday.
(1) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1990, 29, 823-839.
(2) Meldal, M. In Neoglycoconjugates: Preparation and applications;
Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, 1994; pp 145-
198.
(3) Kihlberg, J .; Elofsson, M. Curr. Med. Chem. 1997, 4, 79-110.
(4) Kihlberg, J . In Fmoc solid-phase peptide synthesis: A practical
approach; Chan, W. C., White, P. D., Eds.; Oxford University Press:
Oxford, 2000; pp 195-213.
(15) Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996,
292, 71-81.
(16) Guo, Z.-W.; Nakahara, Y.; Nakahara, Y.; Ogawa, T. Carbohydr.
Res. 1997, 303, 373-377.
(5) Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem. Rev. 2000,
100, 4495-4537.
(17) Guo, Z.-W.; Nakahara, Y.; Nakahara, Y.; Ogawa, T. Angew.
Chem., Int. Ed. Engl. 1997, 36, 1464-1466.
(18) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Carbohydr. Res.
1998, 309, 287-296.
(19) Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Tetrahedron
Lett. 1997, 38, 7211-7214.
(20) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1988,
27, 1697-1699.
(21) Urge, L.; J ackson, D. C.; Gorbics, L.; Wroblewski, K.; Graczyk,
G.; Otvos, L., J r. Tetrahedron 1994, 50, 2373-2390.
(22) Unverzagt, C.; Kunz, H. Bioorg. Med. Chem. 1994, 2, 1189-
1201.
(6) Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J .; Sheppard, R.
C.; Williams, B. J . J . Chem. Soc., Chem. Commun. 1978, 537-539.
(7) Chang, C.-D.; Meienhofer, J . Int. J . Peptide Protein Res. 1978,
11, 246-249.
(8) Carpino, L. A.; Han, G. Y. J . Org. Chem. 1972, 37, 3404-3409.
(9) Christiansen-Brams, I.; J ansson, A. M.; Meldal, M.; Breddam,
K.; Bock, K. Bioorg. Med. Chem. 1994, 2, 1153-1167.
(10) Elofsson, M.; Salvador, L. A.; Kihlberg, J . Tetrahedron 1997,
53, 369-390.
(11) Broddefalk, J .; Bergquist, K.-E.; Kihlberg, J . Tetrahedron 1998,
54, 12047-12070.
(12) Broddefalk, J .; Ba¨cklund, J .; Almqvist, F.; J ohansson, M.;
Holmdahl, R.; Kihlberg, J . J . Am. Chem. Soc. 1998, 120, 7676-7683.
(13) Holm, B.; Linse, S.; Kihlberg, J . Tetrahedron 1998, 54, 11995-
12006.
(14) Broddefalk, J .; Forsgren, M.; Sethson, I.; Kihlberg, J . J . Org.
Chem. 1999, 64, 8948-8953.
(23) Peters, S.; Lowary, T. L.; Hindsgaul, O.; Meldal, M.; Bock, K.
J . Chem. Soc., Perkin Trans. 1 1995, 3017-3022.
(24) Sjo¨lin, P.; George, S. K.; Bergquist, K.-E.; Roy, S.; Svensson,
A.; Kihlberg, J . J . Chem. Soc., Perkin Trans. 1 1999, 1731-1742.
(25) Sjo¨lin, P.; Elofsson, M.; Kihlberg, J . J . Org. Chem. 1996, 61,
560-565.
10.1021/jo001584q CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/12/2001

2958 J . Org. Chem., Vol. 66, No. 9, 2001
Sjo¨lin and Kihlberg
Sch em e 1
require higher concentration of base for removal (i.e.,
∼100 mM methanolic sodium methoxide), which leads
to that â-elimination,24,26 and epimerization of R-stereo-
centers27 can occur more frequently. Unfortunately, the
acetyl group has some disadvantages, as compared to the
benzoyl group which prevents its exclusive use in glyco-
peptide synthesis. These are related to side reactions that
may occur in glycoside synthesis, e.g., in glycosylation
of serine or threonine. Thus, formation of ortho esters,
an insufficient neighboring group effect that leads to a
mixture of R- and â-glycosides, and migration of the acyl
group from the glycosyl donor to the hydroxyl group being
glycosylated are common when acetates are used as
protective groups.28,29
Parameters such as the solvent, structural features of
the glycopeptide,24 and the nature of the base also
influence the outcome of base-catalyzed deprotection of
glycopeptides. For instance, use of water as solvent has
been found to give enhanced amounts of side-products,
as compared to deacylation in organic solvents.25 Am-
monia,27 sodium methoxide, hydrazine hydrate30, or
cyanide ion are all being used as bases, with methanolic
sodium methoxide and ammonia being the most popular.
Hydrazine hydrate and cyanide ion allow mild conditions
for deprotection, but the fact that hydrazine is a sus-
pected carcinogen and cyanide ion is very toxic may
explain their limited use. In addition, hydrazine has been
reported to attack C-terminal amides and the side-chains
of asparagine thereby giving hydrazides.23
Deprotection of the carbohydrate hydroxyl groups is
the last step in the synthesis of glycopeptides and the
protective group pattern must therefore be chosen care-
fully before embarking on the synthesis. Since acetyl and
benzoyl groups have different advantages in synthesis
of O-linked glycopeptides, i.e., ease of removal for acetates
and improved properties during formation of glycosidic
bonds for benzoates, it would be of general interest if
these advantages could be combined in an alternative
acyl protective group. We now show that certain fluori-
nated benzoyl groups appear to fulfill this requirement
and allow substantial improvements in preparation of
two glycopeptides that are difficult to prepare and
undergo large amounts of â-elimination during base-
catalyzed deprotection.
blood coagulation and fibrinolysis.32 Interestingly, at-
tempts to remove O-benzoyl protective groups from
synthetic glycopeptides having a â-glucosyl serine moiety
were found to be accompanied by â-elimination.26 Syn-
thesis of glucosylated tripeptide 3, in which the tendency
for â-elimination is enhanced by N-methylation of the
glycosylated serine, was therefore chosen as a first model
for evaluation of novel acyl protective groups in the
present study.
Glycodecapeptide 6 (Scheme 1) is derived from residues
52-61 of hen egg lysozyme (HEL) and has previously
been prepared24 from glycosylated serine 4 as part of an
investigation of how T cells respond to glycopeptides.33
In the synthesis of 6, a substantial amount of â-elimina-
tion (∼50%) could not be avoided during removal of the
benzoyl protective groups from 5.24 In this case several
attempts to prepare a building block corresponding to 4,
which carried acetyl instead of benzoyl protective groups,
gave very low yields due to formation of a mixture of R-
and â-glycosides as well as to formation of ortho esters.34
Synthesis of glycopeptide 6 was therefore chosen as a
second model for evaluation of alternative protective
groups.
Selection a n d P r elim in a r y Eva lu a tion of Su bsti-
tu ted O-Ben zoyl P r otective Gr ou p s. Introduction of
electron-withdrawing substituents on the benzoyl group,
such as halogen atoms, carbonyl, or nitro groups, should
facilitate deprotection under basic conditions. It is less
clear how such substituents would influence the proper-
ties of the benzoyl group when employed as the partici-
pating group in glycoside synthesis. The σ-values35 of 4-
and 3-fluorobenzoic acid (0.06 and 0.34, respectively)
reveal that the electron withdrawing effect from a
Resu lts a n d Discu ssion
Selection of Difficu lt Glycop ep tid es a s Mod el
System s. We recently found that â-elimination could not
be avoided during deacetylation of the N-methylated
glycopeptide 1 to give 2 (Scheme 1).31 Glycopeptide 2 is
exceptionally sensitive to â-elimination since the N-
methyl group attached to the glycosylated serine prevents
formation of a protective aza-enolate on treatment with
base.31 Glycoproteins or glycopeptides found in nature do
not contain galactose linked directly to serine, but the
â-glucosyl serine linkage is found in proteins involved in
(26) Reimer, K. B.; Meldal, M.; Kusumoto, S.; Fukase, K.; Bock, K.
J . Chem. Soc., Perkin Trans. 1 1993, 925-932.
(27) Paulsen, H.; Schultz, M.; Klamann, J .-D.; Waller, B.; Paal, M.
Liebigs Ann. Chem. 1985, 2028-2048.
(28) Banoub, J .; Bundle, D. R. Can. J . Chem. 1979, 57, 2091-2097.
(29) Garegg, P. J .; Konradsson, P.; Kvarnstro¨m, I.; Norberg, T.;
Svensson, S. C. T.; Wigilius, B. Acta Chem. Scand. 1985, B 39, 569-
577.
(32) Nishimura, H.; Takao, T.; Hase, S.; Shimonishi, Y.; Iwanaga,
S. J . Biol. Chem. 1992, 267, 17520-17525.
(33) Deck, M. B.; Sjo¨lin, P.; Unanue, E. R.; Kihlberg, J . J . Immunol.
1999, 162, 4740-4744.
(34) Elofsson, M.; Broddefalk, J .; Ekberg, T.; Kihlberg, J . Carbohydr.
Res. 1994, 258, 123-133.
(35) McDaniel, D. H.; Brown, H. C. J . Org. Chem. 1958, 23, 420-
427.
(30) Schultheiss-Reimann, P.; Kunz, H. Angew. Chem. Suppl. 1983,
39-46.
(31) Sjo¨lin, P.; Kihlberg, J . Tetrahedron Lett. 2000, 41, 4435-4439.

Fluorobenzoyl Protective Groups
J . Org. Chem., Vol. 66, No. 9, 2001 2959
Ta ble 1. Syn th esis of â-D-Glu cosyl Ser in e Bu ild in g
Block s in Wh ich th e Glu cose Moiety Is P r otected w ith
Ben zoyl or Differ en t F lu or oben zoyl Gr ou p s
and from the three methyl fluorobenzoates formed on
treatment with sodium methoxide, it was possible to
monitor the progress of the deprotection. This showed
that the 3-fluorobenzoyl group was most easily removed
followed by the 2-fluoro- and finally the 4-fluorobenzoyl
group. In addition, studies monitored by TLC revealed
that perbenzoate 8 was deprotected somewhat slower
than 4-fluorobenzoylated 11. These results prompted a
continued examination of fluorobenzoyl protective groups
in glycopeptide synthesis. It was also decided to include
the 2,5-difluorobenzoyl group, which combines the sub-
stitution pattern of the two most reactive monofluoroben-
zoates, in this investigation. Therefore, D-glucose was
protected by treatment with 2,5-difluorobenzoyl chloride,
as in the preparation of 8-11, to give 12.
P r ep a r a t ion of â-D-Glu cosyl Ser in e Bu ild in g
Block s w h ich Ca r r y F lu or ob en zoyl P r ot ect ive
Gr ou p s. Fluorobenzoyl groups must not only allow facile
removal in order to be of use in synthesis of glycopeptides;
suitable glycosyl donors, e.g., glycosyl halides, in which
the hydroxyl groups are protected with fluorobenzoyl
groups must also be readily accessible. Furthermore, the
glycosyl donors should allow amino acids to be glycosy-
lated in high yields thereby giving building blocks that
can be used for assembly of glycopeptides using standard
techniques.
step Ca
(R′ ) H)
step Ca
(R′ ) Me)
R
step Aa
step Ba
Bz
8: 82%
9: 94%
10: 83%
11: 90%
12: 91%
13: 97%b
14: 93%
15: 96%
16: 89%
17: 95%
18: 59%
19: 74%
20: 71%
21: 51%
22: 58%
23: 91%
2-FBz
3-FBz
4-FBz
2,5-diFBz
24: 87%
25: 86%
26: 70%
a
Yields were determined after purification by flash column
chromatography. Not purified by chromatography.
b
Perbenzoates 8-12 were transformed into glycosyl
donors by treatment with hydrogen bromide in a mixture
of acetic acid and acetic anhydride at 50-70 °C, which
gave bromosugars 13-17 in 89-97% yields (Table 1).
J ust as for benzoylated glucosyl bromide 13, the fluo-
robenzoylated bromosugars 14-17 could be purified by
chromatography on silica gel without degradation and
are stable at room temperature. Bromosugars 13-17
were then used to glycosylate NR-(fluoren-9-ylmethoxy-
carbonyl)-L-serine pentafluorophenyl ester (Fmoc-Ser-
OPfp)42 under promotion by silver triflate in dichlo-
romethane at temperatures ranging from -30 °C to room
temperature.26 In our hands, this gave the glucosylated
serine building blocks 18-22 in 51-74% yields. The
yields thus showed some variation depending on the type
of fluorobenzoyl group being used, but did not differ
substantially from those obtained when the ordinary
benzoyl group was employed. The nature of the fluo-
robenzoyl protective group did, however, influence the
reactivity of the glycosyl donor to a somewhat larger
extent. For instance, the 3-fluorobenzoylated glucosyl
bromide 15 did not react with Fmoc-Ser-OPfp at -30 °C,
but gave 20 in 71% yield when allowed to attain room
temperature. In contrast, the 2-fluorobenzoylated bro-
mosugar 14 reacted smoothly at -30 °C to give building
block 19 in an almost identical yield. Importantly,
formation of R-glycosides and ortho esters corresponding
to 19-22 were not observed in the glycosylations employ-
ing fluorobenzoylated 14-17, nor was any benzoyl mi-
gration to serine detected. The fluorobenzoyl groups thus
perform equally well as, or even better than, the ordinary
benzoyl group in syntheses of bromosugars and in
preparation of glycosylated amino acids.
fluorine substituent may be large and depends on its
location on the benzoyl group. In addition, the electron
withdrawing effect is accumulated in di- and polyfluori-
nated benzoates. These observations, together with the
commercial availability of a large number of fluorinated
benzoyl chlorides and the chemical inertness of organof-
luorine compounds, focused our attention on evaluation
of fluorinated benzoyl protective groups. An additional
advantage with such protective groups is that 19F NMR
spectroscopy allows selective monitoring of reactions
performed in solution or in solid-phase.36-38 The proper-
ties of benzyl ether protective groups are often tuned by
different substituents but, surprisingly enough, this
concept has not been developed for benzoyl esters.39-41
A preliminary evaluation of fluorobenzoyl protective
groups was obtained by comparing the rate of deprotec-
tion of glucose carrying 2-, 3-, and 4-fluorobenzoyl protec-
tive groups with that of perbenzoylated glucose 8. To
allow this study D-glucose (7) was protected by treatment
with benzoyl chloride, or with 2-, 3-, or 4-monofluoroben-
zoyl chloride, to give the fully protected derivatives 8-11
as R/â-mixtures (Table 1). Fluorobenzoates 9-11 were
then mixed, dissolved in MeOH-d4, and introduced in an
NMR tube before sodium methoxide was added. After
mixing briefly 19F NMR spectra were recorded every two
minutes at room temperature. Since the resonances for
fluorobenzoates 9-11 are well separated from each other,
(36) Shapiro, M. J .; Kumaravel, G.; Petter, R. C.; Beveridge, R.
Tetrahedron Lett. 1996, 37, 4671-4674.
(37) Svensson, A.; Fex, T.; Kihlberg, J . Tetrahedron Lett. 1996, 37,
7649-7652.
(38) Svensson, A.; Bergquist, K.-A.; Fex, T.; Kihlberg, J . Tetrahedron
Lett. 1998, 39, 7193-7196.
Syn th esis a n d Dep r otection of F lu or oben zoy-
la ted Mod el Glycotr ip ep tid es. The influence of dif-
ferent fluorobenzoyl protective groups on the extent of
â-elimination during base-catalyzed deprotection was
first evaluated in preparation of model glycopeptide 3,
(39) Greene, T. W.; Wuts, P. G. M. Protective groups in organic
synthesis, 3rd ed.; J ohn Wiley & Sons: New York, 1999.
(40) Hanessian and co-workers (Lou, B.; Reddy, G. V.; Wang, H.;
Hanessian, S. In Preparative Carbohydrate Chemistry; Hanessian, S.,
Ed.; Marcell Dekker: 1997; pp 389-430.) have briefly described the
use of the 4-fluorobenzoyl protective group without developing this
concept further.
(41) Kocienski, P. J . Protecting Groups; Georg Thieme Verlag:
Stuttgart, 1994.
(42) Kisfaludy, L.; Scho¨n, I. Synthesis 1983, 325-327.

2960 J . Org. Chem., Vol. 66, No. 9, 2001
Sjo¨lin and Kihlberg
Ta ble 2. In flu en ce of th e Typ e of Ben zoyl Gr ou p Used
for P r otection of Glycop ep tid es 27-30 on th e Am ou n t of
â-Elim in a tion Obta in ed on Dea cyla tion w ith Meth a n olic
Sod iu m Meth oxid e
compared to when using benzoyl protection. In this
context it should also be pointed out that â-elimination
could not be avoided on deprotection of the structurally
related, O-acetylated glycopeptide 1 (cf. Scheme 1).31
Syn th esis a n d Dep r otection of F lu or oben zoy-
la ted Mod el Glycod eca p ep tid es. The 4-fluoro- and 2,5-
difluorobenzoyl groups were chosen for use in attempts
to reduce the extent of â-elimination encountered during
deprotection of 5 to give glycodecapeptide 6 (Scheme 1).
These two fluorobenzoyl groups show the lowest and
highest rates of base-catalyzed cleavage and therefore
provide information on the limits of â-elimination that
can be expected for the different fluorobenzoates, when
employed in a more complex glycopeptide such as 6.
Synthesis of the fluorobenzoylated glycopeptides started
with protection of lactose (32) by reaction with 4-fluo-
robenzoyl chloride and 2,5-difluorobenzoyl chloride, re-
spectively, to give derivatives 33 and 34 in high yields
(Table 3). Treatment of 33 and 34 with HBr in a mixture
of acetic acid and acetic anhydride gave bromosugars 35
and 36, which were then used to glycosylate Fmoc-Ser-
OPfp. By using silver triflate as the promotor, building
blocks 38 and 39 were obtained in 59% and 64% yields,
respectively, which should be compared with 72% re-
ported for 37 that carries ordinary benzoyl groups.34 It
is important to note that, in contrast to when acetyl
groups were employed for protection of the lactosyl
bromide, ortho ester formation or formation of R/â-
mixtures was not detected in the synthesis of 38 and 39.
Use of building blocks 38 and 39 in solid-phase synthesis,
under conditions reported previously,24 then gave glyco-
peptides 40 and 41 (Table 4), which have the carbohy-
drate hydroxyl groups protected as 4-fluoro- and 2,5-
difluorobenzoyl esters, respectively. Glycopeptide 5, which
carries benzoyl protective groups, was prepared as de-
scribed previously.24
Glycopeptides 5, 40 and 41 were treated with 20 mM
LiOH in methanol in order to remove the protecting
groups from the carbohydrate moiety and the progress
of the reactions were monitored with analytical reversed-
phase HPLC.46 The benzoylated glycopeptide 5 was not
fully deprotected even after 8 h of treatment with meth-
anolic LiOH and ∼50% of the glycopeptide had under-
gone â-elimination to give 42.24,47 For glycopeptide 40,
which has 4-fluorobenzoyl protective groups, a small im-
provement was obtained but â-elimination still amounted
to ∼40% when deprotection was complete after 8 h. In
sharp contrast, deprotection of glycopeptide 41, which
carries 2,5-difluorobenzoyl groups, had reached comple-
tion in less than 30 min with no sign of â-elimination.
This allowed glycopeptide 6 to be prepared in 41% yield
by deprotection of 41, as compared to 22% from benzoy-
lated 5.24
compound
R
reaction time
amount of 31a
27
28
29
30
Bz
7 h
1 h
3 h
1 h
∼80%
∼20%
∼40%
∼10%
3-FBz
4-FBz
2,5-diFBz
a
The reactions were allowed to proceed until all of the protected
glycopeptide had been consumed. The extent of â-elimination to
give 31 was then determined by integration of peaks in chromato-
grams obtained by analytical reversed-phase HPLC.
in which the glycosylated serine is N-methylated (Table
2). To allow this investigation NR-(fluoren-9-ylmethoxy-
carbonyl)-NR-methyl-L-serine pentafluorophenyl ester
(Fmoc-NMe-Ser-OPfp)31 was glycosylated with benzoy-
lated bromosugar 13, 3- and 4-monofluorobenzoylated 15
and 16, as well as the 2,5-difluorobenzoylated 17. This
gave building blocks 23-26 in 70-91% yields when silver
triflate was used as promoter under the same conditions
as described for synthesis of 18-22 (Table 1). The
2-fluorobenzoyl group has a cleavage rate between the
3- and 4-fluorobenzoyl groups and was therefore not
included in this study. Building blocks 23-26 were then
used in assembly of glycopeptides 27-30 on a TentaGel
S NH2 resin functionalized with the Rink linker.43,44 The
use of 1-hydroxy-7-azabenzotriazole45 (HOAt) in combi-
nation with N,N-diisopropylcarbodiimide as coupling
reagent and 6 equiv of Fmoc-Ala-OH in the final coupling
step was found to be important in order to give 27-30
in acceptable yields (48-68%). Glycopeptides 27-30 were
then subjected to treatment with methanolic sodium
methoxide (6 mM) in separate experiments. The low
concentration of sodium methoxide was chosen in order
to allow the progress of the deacylations to be reliably
monitored by analytical reversed-phase HPLC. The reac-
tions were allowed to progress until all of the protected
glycopeptides had been consumed, after which the extent
of â-elimination was determined by analytical reversed-
phase HPLC. Deprotection of the benzoylated 27 resulted
in predominant â-elimination (∼80%) to give 3131 (Table
2), the formation of which was reduced to ∼40% for
4-fluorobenzoylated 29. â-Elimination was further re-
duced for the 3-fluorobenzoylated 28 (∼20%), whereas the
2,5-difluorobenzoylated 30 gave an even better result
(∼10% of 31). Thus, protection of the very base-labile
glycopeptide 3 with either of the latter two fluorobenzoyl
protective groups allowed the levels of â-elimination
during deprotection to be decreased substantially, as
Benzoylated glycopeptide 5 and the 2,5-difluoroben-
zoylated 41 were also deprotected under milder condi-
tions with saturated methanolic ammonia.27 Glycopeptide
5 was not fully deprotected even after 48 h and ∼50%
had undergone â-elimination. In comparison, 41 was fully
deprotected in less than 2 h with no sign of â-elimination.
(46) Deprotection with LiOH in methanol gave HPLC chromato-
grams with sharper peaks as compared to when methanolic sodium
methoxide was employed, explaining the use of LiOH in this study.
The time required to reach completion of the reactions was, however,
the same for LiOH as for sodium methoxide.
(43) Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790.
(44) Bernatowicz, M. S.; Daniels, S. B.; Ko¨ster, H. Tetrahedron Lett.
1989, 30, 4645-4648.
(47) The structure of the â-eliminated peptide 42 was confirmed by
(45) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-4398.
FABMS [calcd 1133 (M + H+), found 1133].

Fluorobenzoyl Protective Groups
J . Org. Chem., Vol. 66, No. 9, 2001 2961
Ta ble 3. Syn th esis of â-D-La ctosyl Ser in e Bu ild in g
Block s in Wh ich th e La ctose Moiety Is P r otected w ith
Ben zoyl or F lu or oben zoyl Gr ou p s
a negative charge on O-1 which occurs during â-elimina-
tion. A similar effect could apply to glycopeptides con-
taining carbohydrates with an acetamido group at C-2,
which may give a protective “aza-enolate” upon treat-
ment with base. Finally, it should be mentioned that
epimerization of peptide R-stereocenters was not observed
when 41 was deprotectied with methanolic ammonia
during 24 h.
Con clu sion s. We have demonstrated that fluoroben-
zoyl groups are useful as protective groups in synthesis
of O-linked glycopeptides. J ust as for the ordinary acetyl
and benzoyl group the fluorobenzoyl groups were readily
attached to carbohydrates and allowed preparation of
glycosyl donors in high yields. The fluorobenzoylated
donors performed equally well in synthesis of glycosy-
lated amino acids as when benzoyl groups were employed
but better than when using acetyl groups. 1,2-Trans
glycosides were thus obtained in high yields without
formation of ortho esters or R-glycosides: side-products
which are common when using acetyl protective groups.
The rate of base-catalyzed deprotection of fluorobenzoy-
lated glucose was found to depend on the number of
fluorine atoms and on their position(s) on the benzoyl
group giving the following order of decreasing deprotec-
tion rates: 2,5-difluorof 3-fluorof 2-fluorof 4-fluo-
robenzoyl. Consequently, base-catalyzed deprotection of
O-linked glycopeptides carrying 3-fluoro- or 2,5-difluo-
robenzoyl groups was found to proceed substantially
faster than when using the benzoyl group. Protection
with either of these two fluorobenzoyl groups also allowed
large reductions in the extent of â-elimination on depro-
tection of two base-labile glycopeptides which carried
carbohydrates O-linked to serine. It thus appears that
fluorobenzoyl groups combine the advantages of the
benzoyl group in formation of glycosidic bonds with the
ease of removal characteristic for the acetyl group. Use
of fluorobenzoyl protecting groups should prove to be
valuable in the synthesis of O-linked glycopeptides which
are prone to undergo â-elimination and for glycopeptides
which carry large carbohydrates that require prolonged
reaction times in the final deacylation step.
R
step Aa
step Ba
step Ca
Bz
37: 72%b
38: 59%
39: 64%
4-FBz
2,5-diFBz
33: 99%
34: 97%
35: 91%
36: 86%
a
Yields were determined after purification by flash column
chromatography. Cf. Reference 34.
b
Ta ble 4. In flu en ce of th e Typ e of Ben zoyl Gr ou p Used
for P r otection of Glycop ep tid es 5, 41, a n d 42 on th e
Exten t of â-Elim in a tion d u r in g Ba se-Ca ta lyzed
Dea cyla tion
Exp er im en ta l Section
Gen er a l P r oced u r es. All reactions were carried out under
an inert atmosphere with dry solvents under anhydrous
conditions, unless otherwise stated. CH2Cl2 was distilled from
calcium hydride. MeOH and pyridine were dried over 3 and 4
Å molecular sieves, respectively. DMF was distilled and then
sequentially dried over 3 Å molecular sieves. TLC was
performed on Silica Gel 60 F254 (Merck) with detection by UV
light and charring with aqueous sulfuric acid. Flash column
chromatography was performed on silica gel (Matrex, 60 Å,
35-70 µm, Grace Amicon) with distilled solvents. Organic
solutions were dried over Na2SO4 before being concentrated.
1,2,3,4,6-Penta-O-benzoyl-D-glucopyranose48 (8) and 2,3,4,6-
tetra-O-benzoyl-R-D-glucopyranosyl bromide48 (13) were pre-
pared as described below for compounds 9 and 14 and had data
in agreement with the cited reference. NR-(Fluoren-9-yl-
methoxycarbonyl)-L-serine pentafluorophenyl ester42 and NR-
(fluoren-9-ylmethoxycarbonyl)-NR-methyl-L-serine pentafluo-
rophenyl ester31 were prepared as described in the cited
references.
compound
R
base
reaction time amount of 42a
5
40
41
41
Bz
4-FBz
2,5-diFBz LiOH
2,5-diFBz NH3
LiOH
LiOH
8 h
8 h
<0.5 h
∼50%
∼40%
0%
2 h
0%
a
The reactions were allowed to proceed until all of the protected
glycopeptides had been consumed. The extent of â-elimination to
give 42 was then determined by integration of peaks in chromato-
grams obtained by analytical reversed-phase HPLC.
Interestingly, when deprotection of 41 with methanolic
ammonia was allowed to continue for 24 h, no sign of
â-elimination to give 42 was observed. This reveals that
a deprotected glycopeptide is substantially more stable
against â-elimination than a fully protected or a partially
deprotected one. A probable explanation for this observa-
tion is that the hydroxyl groups of the carbohydrate
moiety, once deprotected, become partially ionized under
the basic conditions used for deprotection. The resulting
partial negative charge on the carbohydrate, especially
when located at O-2, should prevent the development of
The 1H NMR spectra were recorded at 400 MHz for solutions
in CDCl3 [residual CHCl3 (δH 7.27) as internal standard], CD3-
OD [residual CD2HOD (δH 3.31) as internal standard], or
(48) Ness, R. K.; Fletcher, H. G.; Hudson, C. S. J . Am. Chem. Soc.
1950, 72, 2200-2205.

2962 J . Org. Chem., Vol. 66, No. 9, 2001
Sjo¨lin and Kihlberg
acetone-d6 [residual acetone-d5 (δH 2.05) as internal standard]
at 300 K. First-order chemical shifts and coupling constants
were obtained from one-dimensional spectra, whereas proton
resonances were assigned from COSY49 experiments. Reso-
nances for aromatic protons and resonances that could not be
assigned are not reported. Ions for positive FABMS were
produced by a beam of Xenon atoms (6 keV) from a matrix of
glycerol and thioglycerol. In the amino acid analyses, glutamine
and asparagine were determined as glutamic acid and aspartic
acid, respectively. Analytical and preparative HPLC separa-
tions were performed on Kromasil C-8 columns (100 Å, 50 µm,
4.6 or 20 × 250 mm, respectively) with flow rates of 1.5 and
11 mL/min, respectively, using the solvent systems A (aqueous
0.1% TFA) and B (0.1% TFA in MeCN) as eluants. UV
detection was at 214 nm.
1,2,3,4,6-P en ta-O-(2-flu or oben zoyl)-D-glu copyr an ose (9).
D-Glucose (300 mg, 1.67 mmol) and 4-(dimethylamino)pyridine
(40 mg, 0.33 mmol) were dissolved in pyridine (5 mL), after
which 2-fluorobenzoyl chloride (1.50 mL, 12.6 mmol) was
added droppwise over 15 min. The solution was stirred for 18
h and then methanol (2 mL) was added. After additional
stirring for 1 h the solution was diluted with CH2Cl2 (70 mL)
and washed with water (100 mL). The aqueous phase was
extracted with CH2Cl2 (50 mL) and the combined organic
phases were dried and concentrated. Flash cromatography of
the residue on silica gel with heptane-EtOAc (3:1 f 2:1) gave
9 (1.23 g, 94%): 1H NMR (CDCl3) R/â-ratio 4.7:1, δ (R-anomer)
6.89 (d, 1 H, J ) 3.6 Hz, H-1), 6.27 (t, 1 H, J ) 10.0 Hz, H-3),
5.85 (t, 1 H, J ) 9.7 Hz, H-4), 5.66 (dd, 1 H, J ) 3.6, 10.2 Hz,
H-2) 4.55-4.70 (m, 3 H, H-5 and H-6), δ (â-anomer) 5.99 (t, 1
H, J ) 9.4 Hz, H-3); HRMS (FAB) calcd for C41H27F5NaO11
813.1372 (M + Na+), found 813.1367.
mL). The organic phase was dried and concentrated, and the
residue was purified by flash column chromatography on silica
gel with heptane-EtOAc (4:1 f 2:1) to give 14 (517 mg,
93%): [R]D +109 (c 0.5, CHCl3); H NMR (CDCl3) δ 6.88 (d,
1 H, J ) 4.0 Hz, H-1), 6.23 (t, 1 H, J ) 9.8 Hz, H-3), 5.82 (t,
1 H, J ) 9.8 Hz, H-4), 5.30 (dd, 1 H, J ) 4.1, 10.0 Hz, H-2),
4.70 (m, 1 H, H-5), 4.55-4.70 (m, 2 H, H-6); HRMS (FAB) calcd
for C34H23BrF4NaO9 753.0360 (M + Na+), found 753.034. Anal.
Calcd for C34H23BrF4O9: C, 55.8; H, 3.2. Found: C, 56.0; H,
3.3.
20
1
2,3,4,6-Tet r a -O-(3-flu or ob en zoyl)-R-D-glu cop yr a n osyl
Br om id e (15). Compound 10 (700 mg, 0.89 mmol) was treated
and purified as in the synthesis of 14 to give 15 (620 mg,
20
1
96%): [R]D +112 (c 0.5, CHCl3); H NMR (CDCl3) δ 6.84 (d,
1 H, J ) 4.1 Hz, H-1), 6.21 (t, 1 H, J ) 9.8 Hz, H-3), 5.78 (t,
1 H, J ) 10.0 Hz, H-4), 5.33 (dd, 1 H, J ) 4.1, 9.9 Hz, H-2),
4.73 (m, 1 H, H-5), 4.66 (dd, 1 H, J ) 2.8, 12.6 Hz, H-6),
4.53 (dd, 1 H, J ) 4.3, 12.6 Hz, H-6); HRMS (FAB) calcd for
C
34H23BrF4NaO9 753.0360 (M + Na+), found 753.0346. Anal.
Calcd for C34H23BrF4O9: C, 55.8; H, 3.2. Found: C, 55.9; H,
3.4.
2,3,4,6-Tet r a -O-(4-flu or ob en zoyl)-R-D-glu cop yr a n osyl
Br om id e (16). Compound 11 (800 mg, 1.01 mmol) was treated
and purified as in the synthesis of 14 to give 16 (660 mg,
20
1
89%): [R]D +107 (c 0.5, CHCl3); H NMR (CDCl3) δ 6.83 (d,
1 H, J ) 4.0 Hz, H-1), 6.19 (t, 1 H, J ) 9.8 Hz, H-3), 5.76
(t, 1 H, J ) 10.0 Hz, H-4), 5.31 (dd, 1 H, J ) 4.0, 10.0 Hz,
H-2), 4.71 (m, 1 H, H-5), 4.65 (dd, 1 H, J ) 2.6, 12.6 Hz, H-6),
4.50 (dd, 1 H, J ) 4.4, 12.6 Hz, H-6); HRMS (FAB) calcd for
C
34H23BrF4NaO9 753.0360 (M + Na+), found 753.0346. Anal.
Calcd for C34H23BrF4O9: C, 55.8; H, 3.2. Found: C, 56.0; H,
3.4.
2,3,4,6-Tetr a -O-(2,5-d iflu or oben zoyl)-R-D-glu cop yr a n o-
syl Br om id e (17). Compound 12 (664 mg, 0.75 mmol) was
treated and purified as in the synthesis of 14 to give 17 (573
1,2,3,4,6-P en t a -O-(3-flu or ob en zoyl)-D-glu cop yr a n ose
(10). Compound 10 was synthesized from D-glucose (300 mg,
1.67 mmol) as described for 9, using 3-fluorobenzoyl chloride
as acylating agent. Purification by flash column chromatog-
raphy gave 10 (1.09 g, 83%): 1H NMR (CDCl3) R/â-ratio 6.5:1,
δ (R-anomer) 6.84 (d, 1 H, J ) 3.8 Hz, H-1), 6.23 (t, 1 H, J )
10.0 Hz, H-3), 5.82 (t, 1 H, J ) 9.9 Hz, H-4), 5.67 (dd, 1 H, J
) 3.8, 10.2 Hz, H-2), 4.50-4.65 (m, 3 H, H-5 and H-6), δ (â-
anomer) 6.00 (t, 1 H, J ) 9.5 Hz, H-3); HRMS (FAB) calcd for
mg, 95%): [R]20 +100 (c 0.5, CHCl3); 1H NMR (CDCl3) δ 6.85
D
(d, 1 H, J ) 4.1 Hz, H-1), 6.17 (t, 1 H, J ) 9.8 Hz, H-3), 5.75
(t, 1 H, J ) 9.7 Hz, H-4), 5.29 (dd, 1 H, J ) 4.1, 9.9 Hz, H-2),
4.68 (m, 1 H, H-5), 4.60-4.65 (m, 2 H, H-6); HRMS (FAB) calcd
for C34H19BrF8NaO9 824.9983 (M + Na+), found 824.9979.
Anal. Calcd for C34H19BrF8O9: C, 50.8; H, 2.4. Found: C, 51.0;
H, 2.5.
C
41H27F5NaO11 813.1372 (M + Na+), found 813.1384.
NR-(F lu or en -9-ylm et h oxyca r b on yl)-3-O-[2,3,4,6-t et r a -
O-(2-flu or ob en zoyl)-â-D-glu cop yr a n osyl]-L-ser in e P en -
taflu or oph en yl Ester (19). NR-(Fluoren-9-ylmethoxycarbonyl)-
L-serine pentafluorophenyl ester42 (Fmoc-Ser-OPfp, 120 mg,
0.24 mmol), silver trifluoromethanesulfonate (105 mg, 0.41
mmol), and powdered molecular sieves (3 Å, 300 mg) in
CH2Cl2 (10 mL) were protected from light and cooled to -30
°C for 30 min. Bromosugar 14 (250 mg, 0.34 mmol) in CH2Cl2
(5 mL) was then added dropwise and the mixture was stirred
for 2 h at -30 °C before being allowed to attain room tem-
perature during 3 h. The mixture was diluted with CH2Cl2
(80 mL), filtered (Hyflo SuperCel), and washed with saturated
aqueous NaHCO3 (100 mL). The aqueous phase was extracted
with CH2Cl2 (50 mL) and the combined organic phases were
dried and concentrated. The residue was purified by flash
1,2,3,4,6-P en t a -O-(4-flu or ob en zoyl)-D-glu cop yr a n ose
(11). Compound 11 was synthesized from D-glucose (300 mg,
1.67 mmol) as described for 9, using 4-fluorobenzoyl chloride
as acylating agent. Purification by flash column chromatog-
raphy gave 11 (1.19 g, 90%): 1H NMR (CDCl3) R/â-ratio 1.3:1,
δ (R-anomer) 6.82 (d, 1 H, J ) 3.7 Hz, H-1), 6.23 (t, 1 H, J )
10.0 Hz, H-3), 5.75-5.85 (m, 1 H, H-4), 5.66 (dd, 1 H, J ) 3.8,
10.3 Hz, H-2), δ (â-anomer) 6.26 (d, 1 H, J ) 8.1 Hz, H-1),
5.99 (t, 1 H, J ) 9.6 Hz, H-3), 5.75-5.85 (m, 1 H, H-4); HRMS
(FAB) calcd for C41H27F5NaO11 813.1372 (M + Na+), found
813.1381.
1,2,3,4,6-P en t a -O-(2,5-d iflu or ob en zoyl)-D-glu cop yr a -
n ose (12). Compound 12 was synthesized from D-glucose (300
mg, 1.67 mmol) as described for 9, using 2,5-difluorobenzoyl
chloride as acylating agent. Purification by flash column
chromatography gave 12 (1.33 g, 91%): 1H NMR (CDCl3) R/â-
ratio 2.0:1, δ (R-anomer) 6.86 (d, 1 H, J ) 3.6 Hz, H-1), 6.17
(t, 1 H, J ) 9.9 Hz, H-3), 5.70-5.80 (m, 1 H, H-4), 5.63 (dd, 1
H, J ) 3.6, 10.2 Hz, H-2), δ (â-anomer) 6.24 (d, 1 H, J ) 7.9
Hz, H-1), 5.93 (t, 1 H, J ) 9.2 Hz, H-3), 5.70-5.80 (m, 2 H,
H-2 and H-4); HRMS (FAB) calcd for C41H22F10NaO11 903.0900
(M + Na+), found 903.0920.
chromatography on silica gel with toluene-CH3CN (30:1 f
1
25:1) to give 19 (205 mg, 74%): [R]20 -0.4 (c 0.5, CHCl3); H
D
NMR (CDCl3) δ 5.92 (t, 1 H, J ) 9.6 Hz, H-3), 5.82 (d, 1 H, J
) 8.4 Hz, Ser-NH), 5.69 (t, 1 H, J ) 9.7 Hz, H-4), 5.53 (dd, 1
H, J ) 8.0, 9.6 Hz, H-2), 4.90-4.95 (m, 1 H, Ser-HR), 4.90 (d,
1 H, J ) 8.0 Hz, H-1), 4.50-4.60 (m, 1 H, Ser-Hâ), 4.15-4.20
(m, 1 H, H-5), 4.08 (dd, 1 H, J ) 3.3, 10.3 Hz, Ser-Hâ); HRMS
(FAB) calcd for C58H38F9NaNO14 1166.2047 (M + Na+), found
1166.2036. Anal. Calcd for C58H38F9NO14: C, 60.9; H, 3.4; N,
1.2. Found: C, 61.4; H, 3.5; N, 1.3.
2,3,4,6-Tet r a -O-(2-flu or ob en zoyl)-R-D-glu cop yr a n osyl
Br om id e (14). Compound 9 (600 mg, 0.76 mmol) was dis-
solved in a mixture of acetic acid (6 mL) and acetic anhydride
(2.25 mL), after which HBr in acetic acid (33%, 6 mL) was
added. The solution was heated to 50 °C and stirred for 4.5 h.
It was then diluted with CH2Cl2 (70 mL) and washed with
water (100 mL) followed by saturated aqueous NaHCO3 (100
NR-(F lu or en -9-ylm et h oxyca r b on yl)-3-O-[2,3,4,6-t et r a -
O-(3-flu or ob en zoyl)-â-D-glu cop yr a n osyl]-L-ser in e P en -
t a flu or op h en yl E st er (20). Bromosugar 15 (250 mg, 0.34
mmol) was reacted with Fmoc-Ser-OPfp (120 mg, 0.24 mmol)
at room temperature for 7 h as described for 19. The crude
product was purified by flash chromatography on silica gel
with toluene-CH3CN (30:1 f 25:1) followed by heptane-
(49) Derome, A. E.; Williamson, M. P. J . Magn. Reson. 1990, 88,
177-185.
EtOAc (3:1) to give 20 (196 mg, 71%): [R]20 -0.7 (c 0.4,
D

Fluorobenzoyl Protective Groups
J . Org. Chem., Vol. 66, No. 9, 2001 2963
CHCl3); 1H NMR (CDCl3) δ 5.90 (t, 1 H, J ) 9.7 Hz, H-3), 5.60-
5.70 (m, 2 H, Ser-NH and H-4), 5.50 (dd, 1 H, J ) 8.0, 9.6 Hz,
H-2), 4.85-4.90 (m, 2 H, Ser-HR and H-1), 4.45-4.55 (m, 1 H,
Ser-Hâ), 4.10-4.20 (m, 1 H, H-5), 4.05 (dd, 1 H, J ) 3.5, 10.3
Hz, Ser-Hâ); HRMS (FAB) calcd for C58H38F9NaNO14 1166.2047
1180.2233. Anal. Calcd for C59H40F9NO14: C, 61.2; H, 3.5; N,
1.2. Found: C, 61.2; H, 3.5; N, 1.3.
N R-(F lu or e n -9-ylm e t h oxyca r b on yl)-N R-m e t h yl-3-O-
[2,3,4,6-tetr a -O-(4-flu or oben zoyl)-â-D-glu cop yr a n osyl]-L-
ser in e P en ta flu or op h en yl Ester (25). Bromosugar 16 (377
mg, 0.52 mmol) was reacted with NR-(fluoren-9-ylmethoxycar-
bonyl)-NR-methyl-L-serine pentafluorophenyl ester (184 mg,
0.36 mmol) at -30 °C for 1.5 h, as described for 19. The crude
product was purified by flash chromatography on silica gel
with toluene-CH3CN (30:1 f 25:1) followed by heptane-
EtOAc (5:1 to 3:1) to give 25 (362 mg, 86%): [R]20D -6.7 (c 0.4,
CHCl3); 1H NMR (CDCl3) rotamer ratio, ∼2:1, major rotamer
) δ 5.84 (t, 1 H, J ) 9.7 Hz, H-3), 5.64 (t, 1 H, J ) 9.7 Hz,
H-4), 5.50 (dd, 1 H, J ) 7.9, 9.7 Hz, H-2), 4.96 (d, 1 H, J ) 7.9
Hz, H-1), 4.10-4.20 (m, 1 H, H-5), 2.96 (s, 3 H, N-Me); minor
rotamer ) δ 5.82 (t, 1 H, J ) 9.6 Hz, H-3), 5.63 (t, 1 H, J )
9.7 Hz, H-4), 5,43 (dd, 1 H, J ) 8.0, 9.7 Hz, H-2), 4.68 (d, 1 H,
J ) 7.7 Hz, H-1), 4.06 (m, 1 H, H-5), 2.90 (s, 3 H, N-Me); HRMS
(FAB) calcd for C59H40F9NaNO14 1180.2203 (M + Na+), found
1180.2212. Anal. Calcd for C59H40F9NO14: C, 61.2; H, 3.5; N,
1.2. Found: C, 61.2; H, 3.7; N, 1.2.
(M + Na+), found 1166.2068. Anal. Calcd for C58H38F9NO14
:
C, 60.9; H, 3.4; N, 1.2. Found: C, 60.5; H, 3.5; N, 1.2.
NR-(F lu or en -9-ylm et h oxyca r b on yl)-3-O-[2,3,4,6-t et r a -
O-(4-flu or ob en zoyl)-â-D-glu cop yr a n osyl]-L-ser in e P en -
t a flu or op h en yl E st er (21). Bromosugar 16 (500 mg, 0.68
mmol) was reacted with Fmoc-Ser-OPfp (240 mg, 0.49 mmol)
as described for 19. The crude product was purified by flash
chromatography on silica gel with toluene-CH3CN (30:1 f
25:1) followed by heptane-EtOAc (3:1) to give 21 (282 mg,
51%): [R]D -0.7 (c 0.4, CHCl3); 1H NMR (CDCl3) δ 5.86 (t, 1
20
H, J ) 9.7 Hz, H-3), 5.64 (t, 1 H, J ) 9.8 Hz, H-4), 5.63 (d, 1
H, J ) 8.2 Hz, Ser-NH), 5.46 (dd, 1 H, J ) 7.9, 9.7 Hz, H-2),
4.86 (m, 1 H, Ser-HR), 4.82 (d, 1 H, J ) 7.8 Hz, H-1), 4.40-
4.50 (m, 1 H, Ser-Hâ), 4.10-4.20 (m, 1 H, H-5), 4.03 (dd, 1 H,
J ) 3.4, 10.3 Hz, Ser-Hâ); HRMS (FAB) calcd for C58H38F9-
NaNO14 1166.2047 (M + Na+), found 1166.2019. Anal. Calcd
for C58H38F9NO14: C, 60.9; H, 3.4; N, 1.2. Found: C, 60.5; H,
3.3; N, 1.2.
N R-(F lu or e n -9-ylm e t h oxyca r b on yl)-N R-m e t h yl-3-O-
[2,3,4,6-tetr a-O-(2,5-diflu or oben zoyl)-â-D-glu copyr an osyl]-
L-ser in e P en ta flu or op h en yl Ester (26). Bromosugar 17
(275 mg, 0.34 mmol) was reacted with NR-(fluoren-9-yl-
methoxycarbonyl)-NR-methyl-L-serine pentafluorophenyl ester
(123 mg, 0.24 mmol) at -30 °C for 3 h, as described for 19.
The crude product was purified by flash chromatography on
silica gel with toluene-CH3CN (30:1 f 25:1) followed by
NR-(F lu or en -9-ylm et h oxyca r b on yl)-3-O-[2,3,4,6-t et r a -
O-(2,5-diflu or oben zoyl)-â-D-glu copyr an osyl]-L-ser in e P en -
t a flu or op h en yl E st er (22). Bromosugar 17 (225 mg, 0.28
mmol) was reacted with Fmoc-Ser-OPfp (100 mg, 0.20 mmol)
as described for 19. The crude product was purified by flash
chromatography on silica gel with toluene-CH3CN (30:1 f
25:1) followed by heptane-EtOAc (3:1) to give 22 (143 mg,
heptane-EtOAc (3:1) to give 26 (209 mg, 70%): [R]20 -4.1 (c
D
58%): [R]20 -2.8 (c 0.5, CHCl3); 1H NMR (CDCl3) δ 5.85 (t, 1
0.4, CHCl3); 1H NMR (CDCl3) rotamer ratio, ∼2:1, major
rotamer ) δ 5.84 (t, 1 H, J ) 9.6 Hz, H-3), 5.63 (t, 1 H, J )
9.6 Hz, H-4), 5.49 (dd, 1 H, J ) 7.9, 9.6 Hz, H-2), 4.95 (d, 1 H,
J ) 7.9 Hz, H-1), 4.10-4.20 (m, 1 H, H-5), 3.02 (s, 3 H, N-Me);
minor rotamer ) δ 5.75-5.85 (m, 1 H, H-3), 5.59 (t, 1 H, J )
9.6 Hz, H-4), 5.39 (br t, 1 H, J ) 8.9 Hz, H-2), 4.50-4.60 (m,
1 H, H-1), 3.97 (m, 1 H, H-5), 2.92 (s, 3 H, N-Me); HRMS (FAB)
D
H, J ) 9.5 Hz, H-3), 5.73 (d, 1 H, J ) 8.3 Hz, Ser-NH), 5.64 (t,
1 H, J ) 9.7 Hz, H-4), 5.47 (dd, 1 H, J ) 8.1, 9.7 Hz, H-2),
4.93 (m, 1 H, Ser-HR), 4.90 (d, 1 H, J ) 8.0 Hz, H-1), 4.50-
4.55 (m, 1 H, Ser-Hâ), 4.10-4.15 (m, 1 H, H-5), 4.07 (dd, 1 H,
J ) 3.4, 10.3 Hz, Ser-Hâ); HRMS (FAB) calcd for C58H34F13
-
NaNO14 1238.1670 (M + Na+), found 1238.1665. Anal. Calcd
for C58H34F13NO14: C, 57.3; H, 2.8; N, 1.2. Found: C, 57.3; H,
3.1; N, 1.1.
calcd for
C
59H36F13NaNO14 1252.1826 (M + Na+), found
1252.1855. Anal. Calcd for C59H36F13NO14: C, 57.6; H, 3.0; N,
1.1. Found: C, 57.8; H, 3.1; N, 1.2.
N R-(F lu or e n -9-ylm e t h oxyca r b on yl)-N R-m e t h yl-3-O-
(2,3,4,6-tetr a-O-ben zoyl-â-D-glu copyr an osyl)-L-ser in e P en -
t a flu or op h en yl E st er (23). Bromosugar 13 (340 mg, 0.52
mmol) was reacted with NR-(fluoren-9-ylmethoxycarbonyl)-NR-
methyl-L-serine pentafluorophenyl ester (184 mg, 0.36 mmol)
at -30 °C for 3 h, as described for 19. The crude product was
purified by flash chromatography on silica gel with toluene-
CH3CN (30:1 f 25:1) followed by heptane-EtOAc (5:1) to give
23 (361 mg, 91%): [R]20D +3.4 (c 0.4, CHCl3); 1H NMR (CDCl3)
rotamer ratio, ∼2:1, major rotamer ) δ 5.93 (t, 1 H, J ) 9.7
Hz, H-3), 5.70 (t, 1 H, J ) 9.7 Hz, H-4), 5.57 (dd, 1 H, J ) 7.9,
9.7 Hz, H-2), 4.98 (d, 1 H, J ) 7.9 Hz, H-1), 4.15-4.25 (m, 1
H, H-5), 2.97 (s, 3 H, N-Me); minor rotamer ) δ 5.91 (t, 1 H,
J ) 9.7 Hz, H-3), 5.68 (t, 1 H, J ) 9.2 Hz, H-4), 5,49 (dd, 1 H,
J ) 8.1, 9.7 Hz, H-2), 4.60-4.70 (m, 1 H, H-1), 4.05-4.10 (m,
1 H, H-5), 2.88 (s, 3 H, N-Me); HRMS (FAB) calcd for C59H44F5-
NaNO14 1108.2580 (M + Na+), found 1108.2593. Anal. Calcd
for C59H44F5NO14: C, 65.3; H, 4.1; N, 1.3. Found: C, 65.4; H,
4.4; N, 1.2.
NR-Acetyl-L-a la n yl-NR-m eth yl-3-O-(2,3,4,6-tetr a -O-ben -
zoyl-â-D-glu cop yr a n osyl)-L-ser yl-L-p h en yla la n in e Am id e
(27). Glycopeptide 27 was synthesized manually in a mechani-
TM
cally agitated reactor on a Tentagel S NH2 resin (80 µmol)
functionalized with the Rink amide linker, p-[R-(fluoren-9-
ylmethoxyformamido)-2,4-dimethoxybenzyl]phenoxyacetic acid.43
NR-Fmoc-Phe-OH (0.24 mmol) was coupled as a 1-benzotri-
azolyl ester, prepared in situ by reaction with 1-hydroxyben-
zotriazole (HOBt, 0.36 mmol) and 1,3-diisopropylcarbodiimide
(DIC, 0.23 mmol) in DMF (1.5 mL). The glycosylated serine
derivative 23 (0.12 mmol) was coupled in the presence of HOBt
(0.36 mmol). NR-Fmoc-Ala-OH (0.48 mmol) was coupled with
DIC (0.46 mmol) in the presence of HOAt (0.72 mmol). The
couplings were monitored by addition of Bromophonol Blue50
(200 nmol) to the reactor. Cleavage of the Fmoc-group was
performed using 20% piperidine in DMF (4 min flow) and
washing was done with DMF. After the final Fmoc cleavage
the N-terminus was acetylated by the addition of acetic
anhydride (0.4 mL) in DMF (1.5 mL). The glycopeptide resin
was washed with DMF and dichloromethane and then dried
under vacuum. Cleavage from the resin was affected with
CF3CO2H-H2O (9:1) and glycopeptide 27 was obtained in 48%
yield after purification by flash chromatography on silica gel
with EtOAc-MeOH-HOAc (16:1:1) followed by reversed-
phase HPLC. Glycopeptide 27 had the following characteris-
tics: 1H NMR (acetone-d6) major rotamer ) δ Ala 7.85-7.95
(m, 1 H, NH), 4.75 (t, 1 H, J ) 6.6 Hz, H-R), 1.21 (d, 3 H, J )
6.9 Hz, Me); Ser 5.13 (dd, 1 H, J ) 4.1, 10.3 Hz, H-R), 4.13 (t,
1 H, J ) 10.6 Hz, H-â), 4.01 (dd, 1 H, J ) 4.1, 10.7 Hz, H-â);
Phe 8.44 (d, 1 H, J ) 8.6 Hz, NH), 4.45-4.55 (m, 1 H, H-R),
3.28 (dd, 1 H, J ) 3.6, 13.9 Hz, H-â), 2.91 (dd, 1 H, J ) 11.5,
N R-(F lu or e n -9-ylm e t h oxyca r b on yl)-N R-m e t h yl-3-O-
[2,3,4,6-tetr a -O-(3-flu or oben zoyl)-â-D-glu cop yr a n osyl]-L-
ser in e P en ta flu or op h en yl Ester (24). Bromosugar 15 (250
mg, 0.34 mmol) was reacted with NR-(fluoren-9-ylmethoxycar-
bonyl)-NR-methyl-L-serine pentafluorophenyl ester (123 mg,
0.24 mmol) at 0 °C for 2.5 h, as described for 19. The crude
product was purified by flash chromatography on silica gel
with toluene-CH3CN (30:1 f 25:1) followed by heptane-
EtOAc (3:1) to give 24 (244 mg, 87%): [R]20 -5.1 (c 0.4,
D
CHCl3); 1H NMR (CDCl3) rotamer ratio, ∼2:1, major rotamer
) δ 5.88 (t, 1 H, J ) 9.6 Hz, H-3), 5.67 (t, 1 H, J ) 9.8 Hz,
H-4), 5.53 (dd, 1 H, J ) 8.1, 9.6 Hz, H-2), 4.98 (d, 1 H, J ) 7.6
Hz, H-1), 4.15-4.25 (m, 1 H, H-5), 2.98 (s, 3 H, N-Me); minor
rotamer ) δ 5.80-5.90 (m, 1 H, H-3), 5.64 (t, 1 H, J ) 10.1
Hz, H-4), 5,45 (br t, 1 H, J ) 8.8 Hz, H-2), 4.60-4.70 (m, 1 H,
H-1), 4.06 (m, 1 H, H-5), 2.90 (s, 3 H, N-Me); HRMS (FAB)
(50) Flegel, M.; Sheppard, R. C. J . Chem. Soc., Chem. Commun.
1990, 536-538.
calcd for
C
59H40F9NaNO14 1180.2203 (M + Na+), found

2964 J . Org. Chem., Vol. 66, No. 9, 2001
Sjo¨lin and Kihlberg
13.9 Hz, H-â); Glc 5.98 (t, 1 H, J ) 9.6 Hz, H-3), 5.69 (t, 1 H,
J ) 9.7 Hz, H-4), 5.44 (dd, 1 H, J ) 8.1, 9.7 Hz, H-2), 5.25 (d,
1 H, J ) 8.0 Hz, H-1), 4.50-4.60 (m, 1 H, H-5); minor rotamer
) δ Ala 1.15 (d, 3 H, J ) 6.8 Hz, Me); Ser 4.80 (m, 1 H, H-R),
4.26 (dd, 1 H, J ) 8.6, 10.6 Hz, H-â); Phe 3.16 (dd, 1 H, J )
5.1, 14.2 Hz, H-â); Glc 6.01 (t, 1 H, J ) 9.6 Hz, H-3), 5.75 (t,
1 H, J ) 9.7 Hz, H-4), 5.24 (d, 1 H, J ) 7.9 Hz, H-1). MS (TOF
ES+) calcd for C53H52N4O14 957.4 (M + H+), found 957.5.
NR-Acet yl-L-a la n yl-NR-m et h yl-3-O-[2,3,4,6-t et r a -O-(3-
flu or oben zoyl)-â-D-glu cop yr a n osyl]-L-ser yl-L-p h en yla la -
n in e Am id e (28). Glycopeptide 28 was prepared from Fmoc-
Phe-OH, Fmoc-Ala-OH, and compound 24 as described for 27.
Purification, using the same conditions as for 27, gave 28 in
68% yield. Glycopeptide 28 had the following characteristics:
1H NMR (acetone-d6) major rotamer ) δ Ala 7.75-7.85 (m, 1
H, NH), 4.74 (t, 1 H, J ) 6.6 Hz, H-R), 1.19 (d, 3 H, J ) 6.9
Hz, Me); Ser 5.12 (dd, 1 H, J ) 4.2, 10.2 Hz, H-R), 4.12 (t, 1 H,
J ) 10.5 Hz, H-â), 4.02 (dd, 1 H, J ) 4.1, 10.6 Hz, H-â); Phe
8.39 (d, 1 H, J ) 8.5 Hz, NH), 4.45-4.55 (m, 1 H, H-R), 3.27
(dd, 1 H, J ) 3.6, 13.9 Hz, H-â), 2.90 (dd, 1 H, J ) 11.5, 14.0
Hz, H-â); Glc 5.98 (t, 1 H, J ) 9.5 Hz, H-3), 5.75 (t, 1 H, J )
9.6 Hz, H-4), 5.45 (dd, 1 H, J ) 8.1, 9.6 Hz, H-2), 5.27 (d, 1 H,
J ) 8.0 Hz, H-1), 4.45-4.55 (m, 1 H, H-5); minor rotamer ) δ
Ala 1.14 (d, 3 H, J ) 6.9 Hz, Me); Ser 4.80-4.85 (m, 1 H, H-R),
4.20-4.30 (m, 1 H, H-â); Phe 3.16 (dd, 1 H, J ) 5.3, 14.2 Hz,
H-â), 2.90-3.00 (m, 1 H, H-â); Glc 6.00-6.05 (m, 1 H, H-3),
5.80 (t, 1 H, J ) 9.6 Hz, H-4), 5.24 (d, 1 H, J ) 8.0 Hz, H-1).
MS (TOF ES+) calcd for C53H48F4N4O14 1029.3 (M + H+), found
1029.4.
27-30. The protected glycopeptide (20 mg) was dissolved in
MeOH (20 mL), after which methanolic sodium methoxide (0.6
mL, 0.2 M) was added. The solution was stirred at room
temperature and samples (each 50 µL) were taken from the
solution. After addition of HOAc (50 µL) the samples were
concentrated, dissolved in acetonitrile-water (1:1, 100 µL), and
then analyzed with analytical reversed-phase HPLC using a
linear gradient of 100% A to 100% B in 30 min. When all
starting material had been converted to a mixture of 3 and
31, the deprotection was quenched by addition of HOAc (200
µL) which gave pH ∼4-5. After concentration the residue was
purified with reversed-phase HPLC using a linear gradient of
100% A to 100% B in 60 min to give 3 and 31.
Compound 3 had: 1H NMR (MeOH-d4) major rotamer ) δ
Ala 4.90-4.80 (m, 1 H, H-R), 1.34 (d, 3 H, J ) 6.9 Hz, Me);
Ser 5.22 (dd, 1 H, J ) 4.4, 9.5 Hz, H-R), 3.90-4.05 (m, 2 H,
H-â,â′); Phe 8.49 (d, 1 H, J ) 8.3 Hz, NH), 4.65-4.55 (m, 1 H,
H-R), 3.00-2.90 (m, 1 H, H-â); Glc 4.27 (d, 1 H, J ) 7.8 Hz,
H-1), 3.08 (dd, 1 H, J ) 7.8, 9.1 Hz, H-2); minor rotamer ) δ
Ala 4.70 (q, 1 H, J ) 6.9 Hz, H-R), 1.23 (d, 3 H, J ) 7.0 Hz,
Me); Ser 5.13 (t, 1 H, J ) 6.9 Hz, H-R), 4.05-3.90 (m, 2 H,
H-â,â′); Phe 7.93 (d, 1 H, J ) 7.8 Hz, NH), 4.65-4.55 (m, 1 H,
H-R), 3.00-2.90 (m, 1 H, H-â); Glc 4.29 (d, 1 H, J ) 8.5 Hz,
H-1), 3.13 (dd, 1 H, J ) 7.9, 9.1 Hz, H-2); HRMS (FAB) calcd
for C24H36O10NaN4 563.2329 (M + Na+), found 563.2328.
Compound 31 had 1H NMR data in agreement with data
reported previously.31
1,2,3,6-Tet r a -O-(4-flu or ob en zoyl)-4-O-[2,3,4,6-t et r a -O-
(4-flu or ob e n zoyl)-â-D-ga la ct op yr a n osyl]-D-glu cop yr a -
n ose (33). Reaction of D-lactose (1.00 g, 2.92 mmol) with
4-fluorobenzoyl chloride and purification, as described for 9,
gave 33 (3.82 g, 99%). Compound 33 had the following
characteristics: 1H NMR (CDCl3) R-anomer δ 6.72 (d, 1 H, J
) 3.8 Hz, H-1), 6.15 (m, 1 H, H-3), 5.65-5.75 (m, 1 H, H-2′),
5.58 (dd, 1 H, J ) 3.8, 10.3 Hz, H-2), 5.40 (dd, 1 H, J ) 3.2,
10.4 Hz, H-3′), 4.94 (d, 1 H, J ) 7.9 Hz, H-1′), 4.25-4.35 (m,
1 H, H-4), 4.05-4.15 (m, 1 H, H-5); â-anomer δ 6.13 (d, 1 H,
J ) 8.2 Hz, H-1), 5.91 (t, 1 H, J ) 9.3 Hz, H-3), 5.75 (d, 1 H,
J ) 3.3 Hz, H-4′), 5.65-5.75 (m, 2 H, H-2,2′), 5.41 (dd, 1 H, J
) 3.4, 10.4 Hz, H-3′), 4.90 (d, 1 H, J ) 7.9 Hz, H-1′), 4.25-
4.35 (m, 1 H, H-4), 4.05-4.15 (m, 1 H, H-5); HRMS (FAB) calcd
for C68H46F8NaO19 1341.2404 (M + Na+), found 1341.2421.
1,2,3,6-Tetr a -O-(2,5-d iflu or oben zoyl)-4-O-[2,3,4,6-tetr a -
O-(2,5-d iflu or oben zoyl)-â-D-ga la ctop yr a n osyl]-D-glu cop y-
r a n ose (34). Reaction of lactose (250 mg, 0.73 mmol) with 2,5-
difluorobenzoyl chloride at 70 °C, as described for 9, and
purification gave 34 (1.03 g, 97%). Compound 34 had the
following characteristics: 1H NMR (CDCl3) R-anomer δ 6.73
(d, 1 H, J ) 3.7 Hz, H-1), 6.08 (t, 1 H, J ) 9.8 Hz, H-3), 5.71
(dd, 1 H, J ) 8.0, 10.3 Hz, H-2′), 5.45-5.50 (m, 2 H, H-2 and
H-3′), 5.09 (d, 1 H, J ) 8.0 Hz, H-1′), 4.48 (t, 1 H, J ) 9.7 Hz,
H-4), 4.30-4.40 (m, 1 H, H-5); â-anomer δ 6.10 (d, 1 H, J )
7.7 Hz, H-1), 5.84 (t, 1 H, J ) 8.9 Hz, H-3), 5.80 (d, 1 H, J )
3.2 Hz, H-4′), 5.70 (dd, 1 H, J ) 8.0, 10.3 Hz, H-2′), 5.65 (dd,
1 H, J ) 7.8, 8.8 Hz, H-2), 5.48 (dd, 1 H, J ) 3.3, 10.4 Hz,
H-3′), 5.05 (d, 1 H, J ) 8.0 Hz, H-1′), 4.55 (t, 1 H, J ) 9.5 Hz,
NR-Acet yl-L-a la n yl-NR-m et h yl-3-O-[2,3,4,6-t et r a -O-(4-
flu or oben zoyl-â-D-glu cop yr a n osyl]-L-ser yl-L-p h en yla la -
n in e Am id e (29). Glycopeptide 29 was prepared from Fmoc-
Phe-OH, Fmoc-Ala-OH, and compound 25 as described for 27.
Purification, using the same conditions as for 27, gave 29 in
64% yield. Glycopeptide 29 had the following characteristics:
1H NMR (acetone-d6) major rotamer ) δ Ala 7.89 (d, 1 H, J )
5.9 Hz, NH), 4.74 (t, 1 H, J ) 6.6 Hz, H-R), 1.19 (d, 3 H, J )
6.9 Hz, Me); Ser 5.12 (dd, 1 H, J ) 4.2, 10.3 Hz, H-R), 4.11 (t,
1 H, J ) 10.5 Hz, H-â), 4.00 (dd, 1 H, J ) 4.1, 10.7 Hz, H-â);
Phe 8.44 (d, 1 H, J ) 8.7 Hz, NH), 4.45-4.55 (m, 1 H, H-R),
3.23 (dd, 1 H, J ) 3.5, 14.0 Hz, H-â), 2.90 (dd, 1 H, J ) 11.6,
13.9 Hz, H-â); Glc 5.93 (t, 1 H, J ) 9.5 Hz, H-3), 5.66 (t, 1 H,
J ) 9.7 Hz, H-4), 5.40 (dd, 1 H, J ) 8.0, 9.6 Hz, H-2), 5.24 (d,
1 H, J ) 8.0 Hz, H-1), 4.50-4.60 (m, 1 H, H-5); minor rotamer
) δ Ala 1.14 (d, 3 H, J ) 6.7 Hz, Me); Ser 4.82 (dd, 1 H, J )
5.4, 8.2 Hz, H-R), 4.21 (dd, 1 H, J ) 8.3, 10.5 Hz, H-â); Phe
3.16 (dd, 1 H, J ) 5.3, 14.2 Hz, H-â); Glc 5.96 (t, 1 H, J ) 9.7
Hz, H-3), 5.72 (t, 1 H, J ) 9.6 Hz, H-4), 5.22 (d, 1 H, J ) 7.9
Hz, H-1). MS (TOF ES+) calcd for C53H48F4N4O14 1029.3 (M +
H+), found 1029.4.
N R-Ac e t y l-L -a la n y l-N R-m e t h y l-3-O -[2,3,4,6-t e t r a -O -
(2,5-d iflu o r o b e n zo y l-â-D -g lu c o p y r a n o s y l]-L -s e r y l-L -
p h en yla la n in e Am id e (30). Glycopeptide 30 was prepared
from Fmoc-Phe-OH, Fmoc-Ala-OH and compound 26 as de-
scribed for 27. Purification, using the same conditions as for
27, gave 30 in 48% yield. Glycopeptide 30 had the following
1
characteristics: H NMR (acetone-d6) major rotamer ) δ Ala
H-4), 4.05-4.10 (m, 1 H, H-5); HRMS (FAB) calcd for C68H38F16
-
7.84 (d, 1 H, J ) 5.9 Hz, NH), 4.77 (t, 1 H, J ) 6.6 Hz, H-R),
1.22 (d, 3 H, J ) 6.9 Hz, Me); Ser 5.15 (dd, 1 H, J ) 4.4, 10.0
Hz, H-R), 4.11 (t, 1 H, J ) 10.7 Hz, H-â), 4.04 (dd, 1 H, J )
4.3, 10.6 Hz, H-â); Phe 8.38 (d, 1 H, J ) 8.7 Hz, NH), 4.45-
4.55 (m, 1 H, H-R), 3.26 (dd, 1 H, J ) 3.5, 13.9 Hz, H-â), 2.91
(dd, 1 H, J ) 11.3, 14.0 Hz, H-â); Glc 5.93 (t, 1 H, J ) 9.5 Hz,
H-3), 5.72 (t, 1 H, J ) 9.6 Hz, H-4), 5.42 (dd, 1 H, J ) 8.1, 9.5
Hz, H-2), 5.22 (d, 1 H, J ) 8.0 Hz, H-1), 4.55-4.65 (m, 1 H,
H-5); minor rotamer ) δ Ala 1.15 (d, 3 H, J ) 6.8 Hz, Me);
Ser 4.88 (dd, 1 H, J ) 5.6, 8.6 Hz, H-R), 4.22 (dd, 1 H, J ) 8.7,
10.2 Hz, H-â); Phe 3.17 (dd, 1 H, J ) 5.1, 14.0 Hz, H-â), 2.95-
3.00 (m, 1 H, H-â); Glc 5.96 (t, 1 H, J ) 9.5 Hz, H-3), 5.74 (t,
1 H, J ) 9.8 Hz, H-4), 5.19 (d, 1 H, J ) 8.1 Hz, H-1). MS (TOF
ES+) calcd for C53H44F8N4O14 1101.3 (M + H+), found 1101.4.
NR-Acet yl-L-a la n yl-3-O-â-D-glu cop yr a n osyl-L-ser yl-L-
p h en yla la n in e Am id e (3) a n d NR-a cetyl-L-a la n yl-(2-a m i-
n op r op -2-en oyl)-L-p h en yla la n in e Am id e (31). The follow-
ing general procedure was used for deprotection of glycopeptides
NaO19 1485.1650 (M + Na+), found 1485.1635.
2,3,6-Tr i-O-(4-flu or oben zoyl)-4-O-[2,3,4,6-tetr a -O-(4-flu -
or ob en zoyl)-â-D-ga la ct op yr a n osyl]-R-D-glu cop yr a n osyl
Br om id e (35). Compound 33 (1.00 g, 0.76 mmol) was treated
at room temperature for 4 h, as descibed for 14, and purified
1
to give 35 (870 mg, 91%): [R]20 +99 (c 0.4, CHCl3); H NMR
D
(400 MHz, CDCl3) δ 6.70 (d, 1 H, J ) 4.1 Hz, H-1), 6.09 (t, 1
H, J ) 9.6 Hz, H-3), 5.74 (d, 1 H, J ) 3.4 Hz, H-4′), 5.68 (dd,
1 H, J ) 7.9, 10.4 Hz, H-2′), 5.39 (dd, 1 H, J ) 3.4, 10.4 Hz,
H-3′), 5.23 (dd, 1 H, J ) 4.1, 9.9 Hz, H-2), 4.93 (d, 1 H, J ) 7.9
Hz, H-1′), 4.59 (dd, 1 H, J ) 3.8, 12.5 Hz, H-6), 4.53 (dd, 1 H,
J ) 2.0, 12.4 Hz, H-6), 4.44 (m, 1 H, H-5), 4.24 (t, 1 H, J ) 9.7
Hz, H-4); HRMS (FAB) calcd for C61H42BrF7NaO17 1281.1392
(M + Na+), found 1281.1410. Anal. Calcd for C61H42BrF7O17
C, 58.2; H, 3.4. Found: C, 57.9; H, 3.6.
:
2,3,6-Tr i-O-(2,5-d iflu or ob en zoyl)-4-O-[2,3,4,6-t et r a -O-
(2,5-d iflu or oben zoyl)-â-D-ga la ctop yr a n osyl]-R-D-glu cop y-
r a n osyl Br om id e (36). Compound 34 (900 mg, 0.62 mmol)

Fluorobenzoyl Protective Groups
J . Org. Chem., Vol. 66, No. 9, 2001 2965
was treated at 50 °C for 2 h, as described for 14, and purified
Am id e (40). Synthesis was performed on a TentaGel S NH2
resin (30 µmol) as described previously,24 followed by purifica-
tion by preparative reversed phase HPLC, to give 40 (32 mg,
46% based on resin capacity). Glycopeptide 40 had the follow-
ing characteristics: MS (TOF ES+) calcd for C109H121F7N16O34
2330.8 (M + 2H+), found 2331.0.
1
to give 36 (732 mg, 86%): [R]20 +86 (c 0.4, CHCl3); H NMR
D
(CDCl3) δ 6.73 (d, 1 H, J ) 4.1 Hz, H-1), 6.06 (t, 1 H, J ) 9.5
Hz, H-3), 5.80 (d, 1 H, J ) 3.4 Hz, H-4′), 5.70 (dd, 1 H, J )
8.0, 10.2 Hz, H-2′), 5.47 (dd, 1 H, J ) 3.4, 10.3 Hz, H-3′), 5.15
(dd, 1 H, J ) 4.1, 10.0 Hz, H-2), 5.08 (d, 1 H, J ) 8.0 Hz, H-1′),
4.46 (t, 1 H, J ) 9.5 Hz, H-4); HRMS (FAB) calcd for C61H35
-
L-R-Asp a r tyl-L-tyr osylglycyl-L-isoleu cyl-O-{2,3,6-tr i-O-
(2,5-diflu or oben zoyl)-4-O-[2,3,4,6-tetr a-O-(2,5-diflu or oben -
zoyl)-â-D-ga la ctop yr a n osyl]-â-D-glu cop yr a n osyl}-L-ser yl-
L -g lu t a m i n y l-L -i s o le u c y l-L -a s p a r a g i n y l-L -s e r y l-L -
a r gin in e Am id e (41). Synthesis was performed on a TentaGel
S NH2 resin (40 µmol) as described previously,24 followed by
purification by preparative reversed-phase HPLC, to give 41
(60 mg, 61% based on resin capacity). Glycopeptide 41 had
the following characteristics: MS (TOF ES+) calcd for
BrF14NaO17 1407.0732 (M + Na+), found 1407.0713. Anal.
Calcd for C61H35BrF14O17: C, 52.9; H, 2.6. Found: C, 52.8; H,
2.7.
NR-(F lu or en -9-ylm eth oxyca r bon yl)-3-O-{2,3,6-tr i-O-(4-
flu or oben zoyl)-4-O-[2,3,4,6-tetr a -O-(4-flu or oben zoyl)-â-D-
galactopyr an osyl]-â-D-glu copyr an osyl}-L-ser in e P en taflu -
or op h en yl Ester (38). Bromosugar 35 (156 mg, 0.12 mmol)
was reacted with Fmoc-Ser-OPfp (50 mg, 0.10 mmol) as
described for 19. The crude product was purified by repeated
flash chromatography on silica gel with heptane-EtOAc (4:1
C
109H114F14N16O34 2456.7 (M + 2H+), found 2457.0.
Gen er a l P r oced u r e for th e Dea cyla tion of Glycop ep -
to 2:1) to give 38 (100 mg, 59%): [R]20 +22 (c 0.4, CHCl3); 1H
D
tid es 5, 40, a n d 41. The time-course of deacylation of the
glycopeptides was first investigated on an analytical scale
using methanolic lithium hydroxide or ammonia as base in
the following manner: (a) Methanolic lithium hydroxide (0.1
mL, 0.2 M) was added to a solution of each of glycopeptides 5,
40, and 41 (1 mg) in MeOH (0.9 mL). The solutions were
stirred at room temperature and samples (each 50 µL) were
taken from the solutions. After addition of HOAc (50 µL) the
samples were concentrated. The residues were dissolved in
acetonitrile-water (1:1, 100 µL) and then analyzed with
analytical reversed-phase HPLC (100% A to 100% B over 30
min). (b) Glycopeptides 5, 40, and 41 (1 mg) were also dissolved
in saturated methanolic ammonia (1.5 mL) and the reactions
were monitored in the same way.
On a preparative scale glycopeptide 41 (10 mg) was dis-
solved in methanolic lithium hydroxide (10 mL, 0.02 M). The
solution was stirred for 1 h before HOAc (200 µL) was added.
After concentration the residue was purified with preparative
reversed-phase HPLC using a linear gradient of 100% A to
100% B in 60 min to give 6 (4.8 mg, 41% based on resin
capacity and peptide content). Compound 6 had MS (FAB) and
1H NMR data in agreement with those reported previously.24
NMR (CDCl3) δ 5.76 (t, 1 H, J ) 9.4 Hz, H-3), 5.72 (d, 1 H, J
) 3.4 Hz, H-4′), 5.67 (dd, 1 H, J ) 7.9, 10.4 Hz, H-2′), 5.52 (d,
1 H, J ) 8.2 Hz, Ser-NH), 5.38 (dd, 1 H, J ) 3.4, 10.3 Hz,
H-3′), 5.34 (dd, 1 H, J ) 7.5, 9.6 Hz, H-2), 4.86 (d, 1 H, J ) 7.9
Hz, H-1′), 4.82 (m, 1 H, Ser-HR), 4.67 (d, 1 H, J ) 7.8 Hz,
H-1), 4.35-4.45 (m, 1 H, Ser-Hâ), 4.10-4.20 (m, 1 H, H-4),
3.85-3.95 (m, 1 H, Ser-Hâ); HRMS (FAB) calcd for C85H57F12
-
NaNO22 1694.3079 (M + Na+), found 1694.3110. Anal. Calcd
for C85H57F12NO22: C, 61.1; H, 3.4; N, 0.8. Found: C, 61.0; H,
3.7; N, 0.9
NR-(Flu or en -9-ylm eth oxycar bon yl)-3-O-{2,3,6-tr i-O-(2,5-
diflu or oben zoyl)-4-O-[2,3,4,6-tetr a-O-(2,5-diflu or oben zoyl)-
â-D-ga la ctop yr a n osyl]-â-D-glu cop yr a n osyl}-L-ser in e P en -
t a flu or op h en yl E st er (39). Bromosugar 36 (352 mg, 0.28
mmol) was reacted with Fmoc-Ser-OPfp (100 mg, 0.20 mmol)
as described for 19 at 0 °C for 2 h. The crude product was
purified by flash chromatography on silica gel with toluene-
CH3CN (30:1 f 25:1) to give building block 39 (232 mg, 64%):
[R]20 +20 (c 0.4, CHCl3); 1H NMR (CDCl3) δ 5.81 (d, 1 H, J )
D
3.3 Hz, H-4′), 5.77 (t, 1 H, J ) 9.5 Hz, H-3), 5.72 (d, 1 H, J )
8.4 Hz, Ser-NH), 5.71 (dd, 1 H, J ) 7.9, 10.3 Hz, H-2′), 5.50
(dd, 1 H, J ) 3.4, 10.3 Hz, H-3′), 5.38 (dd, 1 H, J ) 8.1, 9.5
Hz, H-2), 5.05 (d, 1 H, J ) 8.0 Hz, H-1′), 4.90 (m, 1 H, Ser-
HR), 4.78 (d, 1 H, J ) 7.9 Hz, H-1), 4.48 (dd, 1 H, J ) 2.9, 10.2
Hz, Ser-Hâ), 4.35-4.45 (m, 1 H, H-4), 3.90-4.00 (m, 1 H, Ser-
Hâ); HRMS (FAB) calcd for C85H50F19NaNO22 1820.2419 (M
+ Na+), found 1820.2410. Anal. Calcd for C85H50F19NO22: C,
56.8; H, 2.8; N, 0.8. Found: C, 56.7; H, 3.0; N, 0.8.
Ack n ow led gm en t. This work was supported by the
Swedish Natural Science Research Council.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
spectra for compounds 9-12, 27-30, 3, 33, and 34. This
material is available free of charge via the Internet at
http://pubs.acs.org.
L-R-Asp a r tyl-L-tyr osylglycyl-L-isoleu cyl-O-{2,3,6-tr i-O-
(4-flu or oben zoyl)-4-O-[2,3,4,6-tetr a -O-(4-flu or oben zoyl)-
â-D-ga la ct op yr a n osyl]-â-D-glu cop yr a n osyl}-L-se r yl-L-
glu ta m in yl-L-isoleu cyl-L-a sp a r a gin yl-L-ser yl-L-a r gin in e
J O001584Q