Novel type of rigid C-linked glycosylacetylene-phenylalanine building blocks for combinatorial synthesis of C-linked glycopeptides

Article abstract of DOI:10.1021/jo980517h

C-linked 3- and 4-(glycosylacetylene)-Fmoc-phenylalanines were synthesized as rigid building blocks for synthesis of libraries of neoglycopeptide templates. Perbenzylated 1,5-galactonolactone, 1,5- gluconolactone, and 1,5-mannonolactone were reacted with cerium TMS-acetylide and the products reduced to the C-glycosylacetylene-TMS derivatives. The gluco and galacto configurations yielded exclusively the β-C-glycoside, whereas the mannonolactone gave a mixture of α-C- and β-C-anomer. The products were subjected to acetolysis and TMS cleavage. The resulting peracetylated glycosylacetylenes were cross-coupled with the (R,S)-N(α)- acetyl-3- and 4-iodophenylalanine O-methyl esters by Pd(0) catalysis in piperidine to give the eight possible C-linked glycosyl amino acid building blocks 33-40 as diastereomeric pairs. These were O-deacetylated. As an example of further conversion into neoglycopeptide templates the N-acetyl group of the 4-linked galacto-diastereomeric pair 43 was hydrolyzed selectively by acylase 1 and separated from the (R)stereoisomer. The product was converted to 4-(β-C-galactosylacetylene)(N(α)-Fmoc-)-L-phenylalanine (52) by reaction with Fmoc-OSu. Acid hydrolysis of galactosyl acetylene phenyl alanine 43 at elevated temperature afforded the conversion of the acetylene to the 1'-oxo derivative which was also N(α)-Fmoc protected. The building blocks were used in the glycopeptide synthesis of three neoglycopeptide templates 54, 55, and 56 known to bind to murine MHC class II E(k) and to present the glycan for interaction with the T-cell receptor. This template has previously been employed to elicit a carbohydrate specific T- cell response.

Full text of DOI:10.1021/jo980517h

J . Org. Chem. 1998, 63, 9657-9668
9657
Novel Typ e of Rigid C-Lin k ed Glycosyla cetylen e-P h en yla la n in e
Bu ild in g Block s for Com bin a tor ia l Syn th esis of C-lin k ed
Glycop ep tid es
Todd Lowary,† Morten Meldal,*,† Arnim Helmboldt,† Andrea Vasella,‡ and Klaus Bock†
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark, and
Institute of Organic Chemistry, ETH-Center, Universita¨tstrasse 16, CH-8092 Zu¨rich, Switzerland
Received March 18, 1998
C-linked 3- and 4-(glycosylacetylene)-Fmoc-phenylalanines were synthesized as rigid building
blocks for synthesis of libraries of neoglycopeptide templates. Perbenzylated 1,5-galactonolactone,
1,5- gluconolactone, and 1,5-mannonolactone were reacted with cerium TMS-acetylide and the
products reduced to the C-glycosylacetylene-TMS derivatives. The gluco and galacto configurations
yielded exclusively the â-C-glycoside, whereas the mannonolactone gave a mixture of R-C- and
â-C-anomer. The products were subjected to acetolysis and TMS cleavage. The resulting per-
acetylated glycosylacetylenes were cross-coupled with the (R,S)-NR-acetyl-3- and 4-iodophenylalanine
O-methyl esters by Pd(0) catalysis in piperidine to give the eight possible C-linked glycosyl amino
acid building blocks 33-40 as diastereomeric pairs. These were O-deacetylated. As an example of
further conversion into neoglycopeptide templates the N-acetyl group of the 4-linked galacto-
diastereomeric pair 43 was hydrolyzed selectively by acylase 1 and separated from the (R)-
stereoisomer. The product was converted to 4-(â-C-galactosylacetylene)(NR-Fmoc-)-L-phenylalanine
(52) by reaction with Fmoc-OSu. Acid hydrolysis of galactosyl acetylene phenyl alanine 43 at
elevated temperature afforded the conversion of the acetylene to the 1′-oxo derivative which was
also NR-Fmoc protected. The building blocks were used in the glycopeptide synthesis of three
neoglycopeptide templates 54, 55, and 56 known to bind to murine MHC class II Ek and to present
the glycan for interaction with the T-cell receptor. This template has previously been employed to
elicit a carbohydrate specific T-cell response.
The structural diversity of N- and O-linked oligosac-
charides correlates with their role in cellular communica-
tion and regulatory functions. This has recently been
highlighted by the discovery of the ubiquitous family of
the galactose binding galectins that are involved in such
important processes as development, metastasis, and
apoptosis.1 Similarly, the mannose 6-phosphate receptor
is involved in intracellular protein sorting and binds to
glycopeptide templates with high affinities.2 The key role
played by the recognition of carbohydrate epitopes by E-,
L-, and P-selectins in the process of cell-cell adhesion
and inflammation is well recognized, and the identifica-
tion of the SLex tetrasaccharide as the ligand for selectin
binding has generated enormous interest in the design
and synthesis of carbohydrate-based inhibitors of SLex-
selectin binding as novel therapeutic agents. As a rule,
the natural oligosaccharide ligands require a lengthy and
costly synthesis and are not accessible in sufficiently
large amounts. This has led to considerable efforts toward
developing carbohydrate mimics. In a particularly at-
tractive class of such mimics a large portion of the
oligosaccharide is replaced by non-carbohydrate and
particularly by peptide scaffolds.3 As a wide range of
amino acids are commercially available, this approach
offers great potential for the preparation of glycopeptide
libraries. It is also attractive, because several glycopep-
tides mimicking the oligosaccharide have displayed rela-
tively higher affinities to their receptors,2,4 while oli-
gosaccharides often bind with relatively low affinities.
However, while small glycopeptides are an attractive
alternative to complex oligosaccharides, glycopeptides
containing naturally occurring (O- or N-linked) carbohy-
drate-amino acid linkages could be anticipated to be poor
therapeutics. They are metabolically unstable and pos-
sess a relatively short biological half-life. The delivery
of such polar compounds into the cell across the lipid
bilayer is generally difficult. Finally, while the flexibility
associated with the linkage between sugar and peptide
is not necessarily a negative attribute, decreased flex-
ibility should improve the interaction with receptors or
enzymes by reducing the entropy of binding. Indeed,
considerable effort has been allocated to the synthesis of
conformationally rigid oligosaccharide analogues for the
investigation of carbohydrate-protein binding.5
We have envisaged the preparation of a new class of
glycosyl-amino acid building blocks comprised of glyco-
sylacetylenes C-linked to aromatic amino acids of the
general type 1 (Schemes 1 and 2). Over the last years,
(3) Meldal, M.; Christiansen-Brams, I.; Christensen, M. K.; Mou-
ritsen, S.; Bock, K. In Complex carbohydrates in drug research.
Structural and functional aspects; Bock, K., Clausen, H., Eds.; Munks-
gaard: Copenhagen: 1994; pp 153-165.
(4) Uchiyama, T.; Woltering, T. J .; Wong, W.; Lin, C. C.; Kajimoto,
T.; Takebayashi, M.; Weitz, S. G.; Asakura, T.; Noda, M.; Wong, C.-H.
Bioorg. Med. Chem. 1997, 4, 1149-1165.
(5) Franzyk, H.; Christensen, M. K.; J ørgensen, M.; Meldal, M.;
Cordes, H.; Mouritsen, S.; Bock, K. Bioorg. Med. Chem. 1997, 5, 21-
40.
* Corresponding author. Phone: (45) 33275301. Fax: 45 33274708.
E-mail: MPM@CRC.DK.
† Carlsberg Laboratory.
‡ ETH-Center.
(1) Pace, K. E.; Baum, L. G. Trends Glycosci. Glycotechnol. 1997, 9,
21-29.
(2) Christensen, M. K.; Meldal, M.; Bock, K.; Cordes, H.; Mouritsen,
S.; Elsner, H. J . Chem. Soc., Perkin Trans. 1 1994, 1299-1310.
10.1021/jo980517h CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/08/1998

9658 J . Org. Chem., Vol. 63, No. 26, 1998
Lowary et al.
Sch em e 1
Sch em e 2
stable. The acetylene bridge between the sugar and
amino acid is less polar than the normal ether or amide
linkage of O- or N-glycopeptides and may confer improved
membrane transport properties. This bridge is also rigid,
so that acetylenoglycopeptides should present the sac-
charide moieties in a much less flexible fashion than
naturally occurring glycopeptides.
However, the most intriguing feature of these mol-
ecules is linked to the chemical versatility of the acety-
lene functionality and to the many ways in which 1 can
be converted into a number of other building blocks
(Scheme 1). There is thus enormous potential for creating
molecular diversity from these monomers. Acetylenes can
be hydrated to ketones (A), oxidized into R,â diketones
(B), 21 reduced to either (E)- or (Z)-alkenes (C and D),22,23
converted to cyclohexadienes by Diels-Alder reaction
with dienes (E),24 and transformed into R,â unsaturated
(6) Nakahara, Y.; Iijima, H.; Ogawa, T. Synth. Oligosaccharides;
ACS Symposium Series 560; American Chemical Society: Washington,
DC, 1994; pp 249-266.
(7) Meldal, M. Curr. Opin. Struct. Biol. 1994, 4, 710-718.
(8) Meldal, M. In Neoglycoconjugates: preparation and application;
Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego: CA, 1994; pp
145-198.
(9) Meldal, M.; Bock, K. Glycoconjugate J . 1994, 11, 59-63.
(10) Meldal, M.; Hilaire, P. M. St. Curr. Opin. Biol. Chem. 1997, in
press
(11) Norberg, T.; Lu¨ning, B.; Tejbrant, J . Methods Enzymol. 1995,
247, 87-106.
efficient methods for the synthesis of O- and N-linked
glycopeptides have been reported.6-11 In the most suc-
cessful approaches glycopeptide synthesis is preceded by
the preparation of the required glycosyl-amino acid
building blocks that are incorporated into the peptide
chain using standard peptide synthesis protocols. Re-
cently, one of us has published several syntheses of
oligomers of C-glycosylacetylenes.12-20 The monomers
possess a number of attractive features which may
contribute to solve many of the above-mentioned prob-
lems associated with N- or O-linked glycopeptides. Simi-
lar to other C-glycosides, acetylenoglycopeptides should
be tolerant of both the acidic and basic conditions
normally used for glycopeptide synthesis, and their
carbohydrate-protein linkages should be metabolically
(12) Alzeer, J .; Vasella, A. Helv. Chim. Acta 1995, 78, 177-193.
(13) Alzeer, J .; Cai, C.; Vasella, A. Helv. Chim. Acta 1995, 78, 242-
264.
(14) Cai, C.; Vasella, A. Helvetica Chimica Acta 1995, 78, 732-
757.
(15) Alzeer, J .; Vasella, A. Helv. Chim. Acta 1995, 78, 1219-1237.
(16) Cai, C.; Vasella, A. Helv. Chim. Acta 1995, 78, 2053-2064.
(17) Cai, C.; Vasella, A. Helv. Chim. Acta 1996, 79, 255-268.
(18) Bu¨rli, R.; Vasella, A. Helv. Chim. Acta 1996, 79, 1159-1168.
(19) Ernst, A.; Vasella, A. Helv. Chim. Acta 1996, 79, 1279-1294.
(20) Xu, J .; Egger, A.; Bernet, B.; Vasella, A. Helv. Chim. Acta 1996,
79, 2004-2012.
(21) Srinivasan, N. S.; Lee, D. G. J . Org. Chem. 1979, 44, 1574-
1580.
(22) McEwan, A. B.; Guttieri, M. J .; Maier, W. F.; Laine, Y.; Shvo,
Y. J . Org. Chem. 1983, 48, 4436-4438.
(23) Ulan, J . G.; Maier, W. F.; Smith, R. M. J . Org. Chem. 1987, 52,
3132-3142.

Combinatorial Synthesis of C-Linked Glycopeptides
J . Org. Chem., Vol. 63, No. 26, 1998 9659
aldehydes by rhodium-catalyzed hydroformylation (F).25
Many of these products could be further elaborated, e.g.
by reductive amination of the ketones, R,â diketones, or
R,â unsaturated aldehydes or by Michael addition to the
R,â unsaturated aldehydes. As presented in Schemes 1
and 2 an indefinitely large number of glycopeptides can
be envisaged resulting from a combination of the large
number of conceivable glycosylacetylene-amino acid
building blocks with the large number of subsequent syn-
thetic manipulations. Related to this is a recent report20
on a Bergman cyclization of 1,2-bis(2′-glucosylalkynyl)-
benzenes to provide 2,3-diglucosylated naphthalenes.
Reported here is the preparation of building blocks 33-
38 and 41-53 where glucosyl, galactosyl, and mannosyl
residues are â-linked via the ethynediyl bridge to the
meta or para position of phenylalanine (Figure 1 and
Scheme 6). The preparation of the conjugates 39 and 40,
where phenylalanine is linked to an R-mannosylacetylene
is also described. The preparation of the building blocks
involves the reductive introduction of the “anomeric”
ethynyl moiety, resolution of the racemic phenylalanines,
alkynyl-aryl coupling,26-28 and appropriate protection/
deprotection of the glycosyl and phenylalanyl moieties.
For the incorporation of these glycosyl-amino acid build-
ing blocks into glycopeptides, we planned to activate the
carboxyl group in the presence of the free hydroxyl groups
using TBTU.29
We also report the synthesis of C-linked glycosyl-
acetylene-conjugated analogues of the mouse hemoglobin-
derived decapeptide Hb(67-76), VITAFNEGLK substi-
tuted at position 72, binding well to the MHC Class II
Ek molecule, and possibly eliciting a carbohydrate specific
T-cell response. These conjugates were synthesized by
multiple-column peptide synthesis (MCPS) employing the
building blocks 52 and 53 (Scheme 6). Previous studies
suggest that T-cells may recognize glycans that are in
direct contact with the T-cell receptor when displayed on
the Hb(67-76) epitope.
Syn th esis of th e Glycosyla cetylen es. The glucosyl-
acetylenes 2 and 3 were prepared as reported.12 Similarly,
we synthesized the galactosylacetylenes 4 and 5, as
illustrated in Scheme 3. The known lactone 630 was
converted in 91% yield to the ketose 7 by reaction with
cerium TMS-acetylide. The hemiacetal was reduced by
treatment with triethylsilane and boron trifluoride ether-
ate to provide the â-linked C-glycoside 8 (71%). None of
the corresponding R-anomer could be isolated; however,
traces (4%, product characterized only by NMR) of the
TES derivative 9 were formed by silyl exchange. The
alkyne was desilylated with sodium hydroxide, yielding
99% of the benzylated alkyne 4. However, unlike the
glucose case,12 where the benzyl groups could be removed
within a few hours by acetolysis (acetic anhydride/O-
(trimethylsilyl)trifluoromethanesulfonate), debenzylation
(24) Bastide, J .; Henri-Rosseau, O. In The chemistry of the triple
bond; Wiley: New York, 1978; pp 447-552.
(25) J ohnson, J . R.; Cuny, G. D.; Buchwald, S. L. Angew. Chem.,
Intl. Ed. Engl. 1995, 34, 1760-1763.
(26) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett.
1975, 16, 4467-4470.
(27) Takahashi, S.; Kuroyama, Y.; Sonogashira, K.; Hagihara, N.
Synthesis 1980, 627-630.
(28) Sonogashira, K. In Comprehensive organic synthesis; Trost, B.
M., Ed.; Pergamon: Oxford, U.K., 1991; pp 521-549.
(29) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahe-
dron Lett. 1989, 30, 1927-1930.
(30) Lewis, M. D.; Cha, J . K.; Kishi, Y. J . Am. Chem. Soc. 1982,
104, 4976-4978.
F igu r e 1.

9660 J . Org. Chem., Vol. 63, No. 26, 1998
Lowary et al.
Sch em e 3
Sch em e 4
resulted in both the R- and â-C-mannosides 13 and 15,
respectively. Chromatography provided pure 13 in 55%
yield, while 15 was contaminated by a small amount of
the TES derivative 14. Treatment of the pure â-anomer
13 with sodium hydroxide provided a 98% yield of 16.
Similar treatment of the mixture of 14 and 15 followed
by chromatographic separation afforded pure 17 in 22%
yield from ketose 12. Acetolysis of the perbenzylated acet-
ylenes 16 and 17 provided the tetraacetates 18 and 19
in 72% and 61% yields, respectively. Again, acetolysis was
protracted; the byproduct being the 3-O-benzyl-2,4,6-tri-
acetate. It appears that, at the least for glycosylacety-
lenes, acetolysis of the equatorial benzyloxy group of a
pair of vicinal cis benzyloxy groups proceeds much more
slowly.
Syn th esis of Ar om a tic Am in o Acid Der iva tives.
Racemic mixtures of the 3-bromo-N-acetylphenylalanine
methyl ester 20,31 the 4-bromo-N-acetylphenylalanine
methyl ester 21,31 and the 4-O-triflate of N-acetyl-L-
tyrosine methyl ester 2232 were prepared as described
previously. Commercially available racemic 4-iodophen-
ylalanine 23 was acetylated and esterified with dimethoxy-
propane and hydrogen chloride to yield 97% of racemic
24 (Scheme 4). The 3-iodo-N-acetylphenylalanine methyl
ester was prepared by condensation of 3-nitrobenzalde-
hyde (25) with N-acetylglycine (26) followed by treatment
of the azalactone 27 with methanol to provide the
crystalline enamide 28 in an overall yield of 27%.
Hydrogenation of 28 in the presence of Pd/C yielded 96%
of the 3-aminophenylalanine derivative 29 that was
transformed into the iodo analogue 30 by diazotization
and treatment with potassium iodide (48%).
of the galacto-isomer was very sluggish, and extended
reaction times were required for complete conversion.
Shorter reaction times resulted in large amounts of the
3-O-benzyl derivative 10. Nevertheless, after 3 days the
desired tetraacetate 5 was obtained in 81% yield. At-
tempts to shorten the reaction by heating resulted in
decomposition, leading to large amounts of tarry byprod-
ucts that complicated the purification.
For the synthesis of the R- and â-mannosylacetylenes
16-29 (Scheme 3), we treated the mannopyranosyl lac-
tone 1130 similarly to 6 to yield 93% of the ketose 12.
However, whereas the reduction of the glucose and ga-
lactose acetylenes with triethylsilane and boron trifluo-
ride etherate gave only the â-anomers, reduction of 12
(31) Burk, M. J .; Feaster, J . E.; Nugent, W. A.; Harlow, R. L. J .
Am. Chem. Soc. 1993, 115, 10125-10138.

Combinatorial Synthesis of C-Linked Glycopeptides
J . Org. Chem., Vol. 63, No. 26, 1998 9661
Sch em e 5
O-(trimethylsilyl)trifluoromethanesulfonate led to de-
composition. However, cross coupling of the acetylated
alkyne 3 with 24 proceeded equally well, the only
problem being that during the course of the reaction the
acetoxy groups were partially cleaved. The crude product
was thus acetylated prior to purification. The acetylated
glycosylacetylenes 3, 5, 18, and 19 were then coupled
with the iodinated phenylalanine derivatives 24 and 30
leading to the products 33-40 (Figure 1) in yields
between 69% and 91%. Deacetylation with sodium meth-
oxide and methanol was followed by saponification of the
methyl ester with sodium hydroxide to give 41-48, each
as pairs of diastereoisomers, in yields of over 95%.
Nr-Deacetylation of th e Glycosyl Acetylen e Am in o
Acid s. Two methods for the NR-deacetylation of the free
acids 41-48 were tested. Nonenzymatic deacetylation
requires forcing conditions,34 but the glycosylacetylene
amino acid conjugates possessing a carboxyl group may
well be substrates for deacetylating enzymes that would
ensure mild conditions. Also, enzymes should be selective
for the configuration of the amino acid moiety and
facilitate the isolation of one enantiomerically and dias-
tereoisomerically pure amino acid.
Ch em ica l Dea cetyla tion . Deacetylation of the ga-
lactose derived acetamide 43 was optimized (Scheme 6).
Initially, 43 was hydrolyzed in 1 N HCl at 90 °C. TLC
and analytical RP-HPLC showed the slow formation of
two major and one minor product, all with shorter
retention times on RP-HPLC. After neutralization, the
products were isolated by semiprepartaive RP-HPLC and
characterized by NMR and ES-MS or MALDI-TOF-MS.
The expected (racemic) amine 49 was obtained in 29%
yield. The faster migrating 51 was obtained in 43% yield.
It showed a higher mass (by 18 units) than 49. For an
unambiguous characterization, 51 was peracetylated
under standard conditions. The NMR spectrum of the
resulting tetraacetate showed the anomeric proton as a
ddd at a low field, evidencing the formation of a ketone
by regioselective hydration of the alkyne. A minor product
eluting last must be the acetamido ketone corresponding
to 51, as evidenced by a peak in the MALDI-TOF-MS at
412.7 and a ddd for H-3 at 3.80 ppm. The hydration/N-
deacetylation ratio was not influenced by the reaction
temperature, as shown by further experiments. Extended
reaction times lead to a quantitative transformation to
51. This unexpected hydration is preparatively useful and
illustrates the versatility of the acetylene functionality.
En zym a tic Dea cetyla tion . The stereoisomerically
pure phenylalanines were prepared in two steps by
enzymatic resolution35 of the mixture R-R,S-stereoiso-
meric acetamides followed by Fmoc protection. The
enzymatic deacetylation of the acetamide 43 was per-
formed with the acylase I from Aspergillus melleus,
available from Sigma, at 30 °C in phosphate buffer at
pH 7.5 instead of pH 8 as indicated by Sigma. This
enzyme has been demonstrated to be highly selectively
for the L-configuration of the R-carbon atom and hydro-
lyze only N-acetylated amino acids with the L-configu-
ration. The deacetylation was conveniently followed by
HPLC. After 24 h the intensity of the HPLC peak for
product and starting material were equal and constant.
Cr oss-Cou p lin g. Initial attempts at cross-coupling
involved the reaction of the bromo derivative 20 and the
benzylated glucosylacetylene 2. However, the use of
standard conditions (triethylamine, copper iodide, (Ph3)2-
PdCl2, room temperature) resulted in an 82% yield of the
known butadiyne 3112 (Scheme 5) and none of the desired
cross-coupled product 32. Since yields in these cross-
coupling reactions have been reported to be very sensitive
to the solvent,33 a number of organic bases (triethylamine,
N-ethylmorpholine, piperidine, and N,N,N′,N′-tetra-
methylethylenediamine) were investigated as solvents,
without positive result. Similarly, replacement of the
catalyst with (Ph3)4Pd gave none of the cross-coupled
product, nor did increasing the reaction temperature (up
to 120 °C in N,N,N′,N′-tetramethylethylenediamine).
Similarly, attempted coupling of the glucosylacetylene 2
and the tyrosine triflate 22 under a variety of conditions
led only to the butadiyne 31.
In contrast, the reaction between 2 and the 4-iodophen-
ylalanine 24 at room temperature in diethylamine with
copper iodide and (Ph3)4Pd gave a 20% yield of the desired
cross-coupled products 32 (Scheme 5). The diastereomeric
pair was most conveniently separated later by enzymatic
differentiation as described below. Encouraged by this
result, we optimized the reaction conditions. Best results
were obtained by heating the iodide 24 and the acetylene
2 at 80 °C in the presence of copper iodide and (Ph3)4Pd
in piperidine. Under these conditions, we obtained 32 in
a 81% yield. Piperidine has recently been reported20 to
be the solvent of choice for the cross-coupling of 2 with
other iodobenzene derivatives. Unfortunately, debenzy-
lation by treatment of 32 with acetic anhydride and
(34) Greene, T. W.; Wuts, P. G. M. In Protective groups in organic
synthesis; Greene, T. W., Ed.; J ohn Wiley & Sons Inc.: New York, 1991;
pp 309-405.
(35) Chenault, H. K.; Dahmer, J .; Whitesides, G. M. J . Am. Chem.
Soc. 1997, 111, 6354-6364.
(32) Petrakis, K. S.; Nagabhushan, T. L. J . Am. Chem. Soc. 1987,
109, 2831-2833.
(33) Alami, M.; Ferri, F.; Linstrumelle, G. Tetrahedron Lett. 1993,
34, 6403-6406.

9662 J . Org. Chem., Vol. 63, No. 26, 1998
Lowary et al.
Sch em e 6
Preparative HPLC and hydrolysis of the methyl ester
provided 41% of 50.
amino acids were coupled as Pfp esters. The glycosy-
lacetylene amino acids were coupled for 3 h after activa-
tion with TBTU. The resin was washed thoroughly with
DMF after Fmoc removal and after each acylation step.
Acidolytic cleavage and deprotection of the peptide with
95% aqueous TFA gave the desired crude glycosylacety-
lene-containing peptide 54 which was then purified by
semipreparative RP-HPLC and fully characterized by ES-
MS and by 1D- and 2D-1H NMR spectroscopy. The
ketones 53 yielded the peptides 55 and 56 which could
easily be separated by HPLC and were similarly char-
acterized.
P r otection of th e Bu ild in g Block w ith F m oc. The
mixtures of the diastereoisomeric amino acid conjugates
49 and 51 and the diastereomerically pure amino acid
conjugate 50 were transformed into the Fmoc derivatives
using Fmoc-OSu under standard conditions. The acids
precipitated upon acidification of the reaction mixture.
Product remaining in the supernatant was isolated by
preparative RP-HPLC.
P ep tid e Syn th esis. The peptide syntheses were per-
formed on custom-made PEGA resin.36,37 The resin was
derivatized by a hydroxymethyl phenoxyacetic acid
(HMPA) linker. The linker was coupled to the resin by
activation with O-(benzotriazol-1-yl)-N,N,N,N-tetra-
methyluronium tetrafluoroborate (TBTU) and 4-ethyl-
morpholine (NEM). The first amino acid, lysine, was
coupled as Fmoc-Lys(Boc)-COOH by activation with
1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT) and
N-methylimidazol.38 Fmoc cleavages were effected by
treatment with 20% piperidine in DMF. The successive
Con clu sion
A range of eight glycosylacetylene amino acid building
blocks containing C-linked glycan with a configuration
corresponding to Glc, Gal, and Man have been synthe-
sized. The building blocks were formed by the palladium-
(0)-catalyzed cross-coupling reactions of glycosylacety-
lenes with iodophenylalanines. In the Glc- and Gal-
containing building blocks the C-glycosides were â-linked
(36) Meldal, M. Tetrahedron Lett. 1992, 33, 3077-3080.
(37) Auzanneau, F. I.; Meldal, M.; Bock, K. J . Peptide Sci. 1995, 1,
31-44.
(38) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett.
1990, 31, 1701-1704.

Combinatorial Synthesis of C-Linked Glycopeptides
J . Org. Chem., Vol. 63, No. 26, 1998 9663
CH3CN/CH2Cl2 (25 mL) and cooled to -10 °C. Separately, two
stock solutions were prepared: solution A, Et3SiH (1.4 mL) in
2:1 CH3CN/CH2Cl2 (6 mL); solution B, BF3OEt2 (0.56 mL) in
CH3CN (4 mL). To the solution of the ketose was added first
solution A (4.65 mL, 5.6 mmol Et3SiH) followed by solution B
(2.9 mL, 5.3 mmol BF3OEt) and the reaction allowed to stir at
-10 °C for 1 h, before being diluted with EtOAc and washed
with water and a saturated NaCl solution. After drying (Na2-
SO4) and evaporation of the solvent the residue was chromato-
graphed (9:1 petroleum ether/EtOAc) to give the product 8 as
an oil: Rf 0.52 (6:1 petroleum ether/EtOAc); [R]D -11.6° (c 0.9,
whereas the Man was R- and â-linked. It has been
demonstrated that these building blocks can be obtained
in the diastereomerically pure form by enzymatic resolu-
tion of the N-acetate followed by Fmoc protection. It was
possible to perform chemical modification of the acetylene
by hydrolysis to the ketone. The different building blocks
were compatible with the conditions of Fmoc-based
peptide synthesis and have been used in the synthesis
of C-linked glycopeptide T-cell antigens. The building
blocks can be employed in the synthesis of C-linked,
spaced, and templated glycopeptide libraries.3
1
CHCl3); selected H NMR (250 MHz, CDCl3) δ 4.10 (1 H), 3.61
(1 H), 3.57 (1 H), 4.04 (1 H), 4.10 (1 H), 3.68 (2 H); 13C NMR
δ 102.55, 90.50, 70.62, 77.21, 83.26, 73.71, 79.02, 68.37.
Complete and assigned NMR data are presented in the
Supporting Information Tables 1 and 2. Anal. Calcd for
Exp er im en ta l Section
Gen er a l Meth od s. Optical rotations were measured at 22
( 2 °C. Flash chromatography was performed on Silica Gel
60, 40-63 µM. Acylase I from Aspergillus melleus was
purchased from Sigma as lyophilized powder. Suitable pro-
tected NR-Fmoc amino acid-OPfp esters were purchased from
Bachem (Switzerland) and NovaBiochem (Switzerland). PEGA
resin was custom synthesized at Carlsberg. All commercial
reagents were used as supplied and HPLC grade solvents
(LabScan, Dublin) were used for chromatography. 1H NMR
C
39H44O5Si (620.86): C, 75.45; H, 7.14. Found: C, 75.20; H,
7.09.
3,7-An h ydr o-4,5,6,8-tetr a-O-ben zyl-1,1,2,2-tetr adeh ydr o-
1,2-D-glycer o-L-m a n n ooctitol (4). TMS derivative 8 (2.69 g,
4.33 mmol) was dissolved in 5:1 CH3OH/CH2Cl2 (150 mL), 1
N NaOH (8 mL) was added, and the reaction was stirred for
1 h before being neutralized by the addition of 1 N HCl (8 mL).
The solvent was then evaporated and the residue partitioned
between EtOAc and water. The organic layer was washed with
water and a saturated solution of NaCl, before being dried
(Na2SO4) and evaporated to give the product 4 (2.35 g, 99%)
as an oil that solidified upon standing: Rf 0.15 (9:1 petroleum
1
chemical shifts were assigned on the basis of 1H-1H and H-
13C correlation spectroscopy, and in cases of spectral overlap
the shifts were derived from the center of the cross-peaks in
1
1
the appropriate 2D experiment. For H NMR spectra recorded
ether/EtOAc): [R]D +5.9° (c 0.8, CHCl3); selected H NMR (250
in CD3COOD-H2O the HOAc signal at 2.03 ppm was used as
an internal reference. For the assignment of signals 1H-1H-
COSY, 1H-1H double quantum filtered phase sensitive COSY
and NOE in rotating frame (ROESY) spectroscopy experiments
were used. MALDI-TOF MS was performed using a matrix of
R-cyano-4-hydroxycinnamic acid. Unless otherwise stated, all
reactions were carried out at room temperature. Organic
solution were dried (Na2SO4) prior to concentration under
vacuum at <40 °C (bath). Microanalyses were carried out by
the microananlytical laboratory at the University of Copen-
hagen. Analytical, semipreparative, and preparative reverse
phase HPLC separations were performed using analytical
RCM (8 × 100 mm), Delta PAK (15 um, 300 Å, 25 × 200 mm),
or 50 × 300 mm radial pressure C-18 columns with flow rates
of 1, 10, and 20 cm3 min-1, respectively. Detection was at 215
and 280 nm. Solvent system: A, 0.1% TFA in water; B, 0.1%
TFA in 90% acetonitrile-10% water.
MHz, CDCl3) δ 2.68(1 H), 4.20 (1 H), 3.73 (1 H), 3.70 (1 H),
4.15 (1 H), 4.21(1 H), 3.77 (2 H); 13C NMR δ 73.72, 79.02, 70.00,
77.32, 83.36, 73.68, 78.78, 68.55. Complete and assigned NMR
data are presented in the Supporting Information Tables 1 and
2. Anal. Calcd for C36H36O5 (548.68): C, 78.81; H, 6.61.
Found: C, 78.44; H, 6.67.
3,7-An h ydr o-4,5,6,8-tetr a-O-acetyl-1,1,2,2-tetr adeh ydr o-
1,2-D-glycer o-L-m a n n ooctitol (5). Benzylated derivative 4
(2.35 g, 4.28 mmol) was dissolved in Ac2O (100 mL) and then
cooled to 0 °C before TMSOTf (5.5 mL, 28.5 mmol) was added.
The reaction was then warmed to room temperature and
stirred for 72 h, before the dark brown solution was cooled to
0 °C and quenched by the cautious addition of a solution of
NaHCO3. Dilution of the reaction mixture with EtOAc was
followed by washing of the organic layer with a solution of
NaHCO3, water, and a saturated solution of NaCl. After drying
(Na2SO4), the organic layer was evaporated to provide a dark
brown oil that was chromatographed (2:1 petroleum ether/
EtOAc), yielding the product 5 (1.25 g, 81%) as light yellow
solid: Rf 0.28 (2:1 petroleum ether/EtOAc); [R]D +26.6° (c 1.1,
4,5,6,8-Tetr a-O-ben zyl-1,1,2,2-tetr adeh ydr o-1,2-dideoxy-
1-C-(tr im eth ylsilyl)-D-galactooct-3-u lopyr an ose (7). CeCl3‚
7H2O (1.043 g, 2.80 mmol) was heated under vacuum at 120
°C for 1 h and then 140 °C for 1 h. The flask was cooled to 0
°C and flushed with argon, and then freshly distilled THF (10
mL) was added. The suspension was allowed to stir at room
temperature for 2 h and then cooled to -78 °C. In a separate
flask a solution of Li-TMS acetylene was prepared by dis-
solving TMS acetylene (0.5 mL, 3.54 mmol) in THF (4 mL)
and then cooling the mixture to -78 °C before adding 1.6 M
n-BuLi in hexane (2 mL). After being stirred for 45 min at
-78 °C, the CeCl3 suspension was treated with the freshly
prepared acetylene solution (5 mL). The resulting yellow slurry
was allowed to stir at -78 °C for 30 min before 2,3,4,6-tetra-
O-benzylgalactopyranosyl lactone1 6 (758 mg, 1.40 mmol) in
THF (10 mL) was added dropwise. After being stirred for 2 h
while warming to room temperature the reaction was diluted
with EtOAc and then washed with 0.1 HCl, water, and a
saturated NaCl solution. The organic layer was then dried
(Na2SO4) and evaporated to provide an oil that was chromato-
graphed (6:1 petroleum ether/EtOAc) to give the product 7,
as an anomeric mixture (810 mg, 91%) of an oil, Rf 0.16 (6:1
1
CHCl3); selected H NMR (250 MHz, CDCl3) δ 2.51(1 H), 4.17
(1 H), 5.39 (1 H), 4.98 (1 H), 5.40 (1 H), 3.89 (1 H), 4.11 (2 H);
13C NMR δ 77.82, 75.61, 68.79, 68.16, 71.34, 67.19, 74.51,
61.50. Complete and assigned NMR data are presented in the
Supporting Information Tables 1 and 2. Anal. Calcd for
C
16H20O9 (356.33): C, 53.93; H, 5.66. Found: C, 53.92; H, 5.48.
4,5,6,8-Tetr a-O-ben zyl-1,1,2,2-tetr adeh ydr o-1,2-dideoxy-
1-C-(tr im eth ylsilyl)-D-m an n ooct-3-u lopyr an ose (12). CeCl3‚
7H2O (1.10 g, 2.95 mmol) was heated under vacuum at 120
°C for 1 h and then 140 °C for 1 h. The flask was then cooled
to 0 °C and flushed with argon, and then freshly distilled THF
(10 mL) was added. The suspension was allowed to stir at room
temperature for 2 h and then cooled to -78 °C. In a separate
flask a solution of Li-TMS acetylene was prepared by dis-
solving TMS acetylene (0.5 mL, 3.54 mmol) in THF (4 mL)
and then cooling the mixture to -78 °C before adding 1.6 M
n-BuLi in hexane (2 mL). After being stirred for 45 min at
-78 °C, the CeCl3 suspension was treated with the freshly
prepared acetylene solution (5.25 mL). The resulting yellow
slurry was allowed to stir at -78 °C for 30 min before 2,3,4,6-
tetra-O-benzylmannopyranosyl lactone1 (11) (791 mg, 1.47
mmol) in THF (10 mL) was added dropwise. After being stirred
for 2 h while warming to room temperature, the reaction was
diluted with EtOAc and then washed with 0.1 HCl, water, and
a saturated NaCl solution. The organic layer was then dried
(Na2SO4) and evaporated to provide an oil that was chromato-
1
petroleum ether/EtOAc). H NMR (250 MHz, CDCl3): δ 6.9-
7.5 (m, 20 H, Ph), 0.015, -0.005 (combined 9H, Si(CH3)3). Anal.
Calcd for
Found: C, 72.52; H, 7.19.
C
39H44O6Si‚1/2H2O (645.87): C, 72.53; H, 7.02.
3,7-An h ydr o-4,5,6,8-tetr a-O-ben zyl-1,1,2,2-tetr adeh ydr o-
1,2-d id eoxy-1-C-(tr im eth ylsilyl)-D-glycer o-L-m a n n oocti-
tol (8). Ketose 7 (860 mg, 1.35 mmol) was dissolved in 7:3

9664 J . Org. Chem., Vol. 63, No. 26, 1998
Lowary et al.
graphed (6:1 petroleum ether/EtOAc) to give, as an anomeric
brown oil that was chromatographed (2:1 petroleum ether/
EtOAc), yielding the product 18 (1.65 g, 72%) as light yellow
solid: Rf 0.25 (2:1 petroleum ether/EtOAc); [R]D -41.5° (c 1.0,
mixture, the product 12 (870 mg, 93%) as an oil: Rf 0.26 (6:1
1
petroleum ether/EtOAc); H NMR (250 MHz, CDCl3) δ 6.9-
1
7.5 (m, 20 H, Ph), 0.002, -0.01 (combined 9H, Si (Si(CH3)3).
Anal. Calcd for C39H44O6Si (636.86): C, 73.55; H, 6.96.
Found: C, 72.10; H, 7.04.
CHCl3); selected H NMR (250 MHz, CDCl3) δ 2.51 (1 H), 4.45
(1 H), 5.52 (1 H), 5.06 (1 H), 5.25 (1 H), 3.67 (1 H), 4.25 (1 H),
4.14 (1 H); 13C NMR δ 75.42, 76.81, 68.02, 69.44, 71.41, 65.50,
76.49, 62.59. Complete and assigned NMR data are presented
in the Supporting Information Tables 1 and 2. Anal. Calcd for
C16H20O9 (356.33): C, 53.93; H, 5.66. Found: C, 53.86; H, 5.45.
3,7-An h ydr o-4,5,6,8-tetr a-O-acetyl-1,1,2,2-tetr adeh ydr o-
1,2-D-glycer o-L-ta looctitol (19). Benzylated derivative 17
(164 mg, 0.3 mmol) was dissolved in Ac2O (10 mL) and cooled
to 0 °C, and TMSOTf (0.4 mL, 2.07 mmol) was added. The
reaction was warmed to room temperature, stirred for 30 h,
and then worked up as described for 18. Chromatography (2:1
petroleum ether/EtOAc) yielded the product 19 (65 mg, 61%)
as light yellow solid: Rf 0.21 (2:1 petroleum ether/EtOAc); [R]D
+30.5° (c 0.8, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ
2.76 (1 H), 4.76 (1 H), 5.32 (1 H), 5.46 (1 H), 5.26 (1 H), 4.17
(1 H), 4.29 (1 H), 4.11 (1 H); 13C NMR δ 79.15, 75.98, 66.93,
70.91, 69.28, 65.91, 71.59, 62.30. Complete and assigned NMR
data are presented in the Supporting Information Tables 1 and
2. Anal. Calcd for C16H20O9 (356.33): C, 53.93; H, 5.66.
Found: C, 53.75; H, 5.62.
3,7-An h ydr o-4,5,6,8-tetr a-O-ben zyl-1,1,2,2-tetr adeh ydr o-
1,2-d id eoxy-1-C-(tr im eth ylsilyl)-D-glycer o-D-ga la ctoocti-
tol (13) a n d 3,7-An h yd r o-4,5,6,8-tetr a -O-ben zyl-1,1,2,2-
t et r a d eh yd r o-1,2-d id eoxy-D-glycer o-D-t a looct it ol (17).
Ketose 12 (870 mg, 1.37 mmol) was dissolved in 7:3 CH3CN/
CH2Cl2 (25 mL) and cooled to -10 °C. Separately, two stock
solutions were prepared: solution A, Et3SiH (1.4 mL) in 2:1
CH3CN/CH2Cl2 (6 mL); solution B, BF3OEt (0.56 mL) in CH3-
CN (4 mL). To the solution of the ketose was added first
solution A (4.7 mL, 5.6 mmol Et3SiH) followed by solution B
(3.0 mL, 5.3 mmol BF3OEt). The reaction was allowed to stir
at -10 °C for 1 h, before being diluted with EtOAc and washed
with water and a saturated NaCl solution. After drying (Na2-
SO4) and evaporation of the solvent the residue was chromato-
graphed (9:1 petroleum ether/EtOAc) to provide two products.
The more polar product, Rf 0.27 (6:1 petroleum ether/EtOAc),
was the â-TMS acetylene 13 (465 mg, 55%) obtained as an oil
that solidified upon standing: [R]D -39.2° (c 0.4, CHCl3);
selected 1H NMR (250 MHz, CDCl3) δ 4.01 (1 H), 3.86 (1 H),
3.38 (1 H), 3.76 (1 H), 3.30 (1 H), 3.63 (1 H), 3.65 (1 H); 13C
NMR δ 101.69, 90.87, 69.56, 75.79, 83.34, 74.49, 79.87, 69.34.
Complete and assigned NMR data are presented in the
Supporting Information Tables 1 and 2. Anal. Calcd for
N-Acetyl-4-iod o-(DL)-p h en yla la n in e Meth yl Ester (24).
4-Iodo-(DL)-phenylalanine (23) (100 mg, 0.343 mmol) was
suspended in CH3OH (10 mL), and then Ac2O (65 µL, 0.689
mmol) and NaHCO3 (62 mg, 0.74 mmol) were added. After
being stirred for 20 min the solution became clear, and after
45 min the reaction was complete. The solution was then
evaporated to give a residue that was immediately resus-
pended in 2,2-dimethoxypropane (10 mL). Concentrated HCl
(350 µL) was added, and after being stirred for 4 h the reaction
was evaporated. Chromatography (9:1 CH2Cl2/CH3OH) gave
the product 24 (116 mg, 97%) as a white solid: Rf 0.74 (19:1
C
39H44O5Si (620.86): C, 75.45; H, 7.14. Found: C, 74.98; H,
7.12.
The less polar product, Rf 0.35 (9:1 petroleum ether/EtOAc),
was a mixture of the R-TMS acetylene 15 and the â-TES
acetylene 14. This impure product was then dissolved in 5:1
CH3OH/CH2Cl2 (12 mL), and 1 N NaOH (0.6 mL) was added.
After being stirred for 1 h the reaction was neutralized by the
addition of 1 N HCl (0.6 mL), the solvent evaporated, and the
residue partitioned between EtOAc and water. The organic
layer was washed with water and a saturated solution of NaCl,
before being dried (Na2SO4) and evaporated to give the
partially deprotected product 17 (186 mg, 22%, 2 steps) as an
oil: Rf 0.23 (6:1 petroleum ether/EtOAc); [R]D +23.5° (c 0.9,
1
CH2Cl2/CH3OH); H NMR (CDCl3, 250 MHz) δ 7.641 (d, 2 H,
3
3J 8.2 Hz, H-2, H-6), 6.897 (d, 2 H, J 8.2 Hz, H-3, H-5), 5.936
(d, 1 H, J CH,NH 7.0 Hz, NH), 4.894 (ddd, 1 H, J CH,NH 7.0, J R,â
5.9, J R,â′ 5.5 Hz, H-R), 3.759 (s, 3 H, OCH3), 3.145 (dd, 1 H,
J R,â 5.9, J â,â′ 13.9 Hz, H-â), 3.085 (dd, 1 H, J R,â 5.5, J â,â′ 13.9
Hz, H-â′), 2.021 (s, 3 H, NHCOCH3); 13C NMR (CDCl3, 62.5
MHz) δ 171.810, 169.555 (CdO), 137.615 (C-2, C-6), 135.511
(C-4), 131.204 (C-3, C-5), 92.603 (C-1), 52.892 (C-R), 52.413
(OCH3), 37.355 (C-â), 23.116 (NHCOCH3). Anal. Calcd for
1
CHCl3); selected H NMR (250 MHz, CDCl3) δ 2.41 (1 H), 4.73
(1 H), 3.75 (1 H), 3.94 (1 H), 3.89 (2 H), 3.71 (1 H), 3.64 (1 H);
13C NMR δ 74.71, 80.68, 68.96, 75.82, 83.33, 74.58, 79.93,
69.36. Complete and assigned NMR data are presented in the
Supporting Information Tables 1 and 2. Anal. Calcd for
C
12H14INO3 (347.15): C, 41.52; H, 4.04; N, 4.03. Found: C,
41.45; H, 3.99; N, 4.02.
Meth yl 3′-n itr o-r-a ceta m id ocin n a m a te (28). N-Acetyl-
glycine 26 (5.24 g, 44.7 mmol), 3-nitrobenzaldehyde (25) (10
g, 66.2 mmol), and sodium acetate (2.71 g, 33.1 mmol) were
suspended in Ac2O (30 mL), and the reaction was heated at
reflux for 1 h. During the course of the reaction the solution
turned dark brown and, upon cooling to room temperature,
solidified. The yellow-brown solid obtained was then suspended
in ice water, filtered off, and washed extensively with cold
water before being dissolved in CH2Cl2. After drying (Na2SO4),
the organic solution was evaporated to yield 27 as a yellow-
brown solid. This product was not purified further but was
instead immediately dissolved in CH3OH (200 mL), and the
solution was heated at reflux for 9 h. Evaporation of the
solvent provided a brown oil that was chromatographed (19:1
CH2Cl2/CH3OH) to give a slightly colored product. Dissolution
of this product in CH3OH and then decolorization with
activated carbon gave the product 28 (4.3 g, 27%) as a yellow-
C
36H36O5 (548.68): C, 78.81; H, 6.61. Found: C, 78.39; H, 6.85.
3,7-An h ydr o-4,5,6,8-tetr a-O-ben zyl-1,1,2,2-tetr adeh ydr o-
1,2-D-glycer o-L-ga la ctooctitol (16). TMS derivative 13 (4.08
g, 6.57 mmol) was dissolved in 5:1 CH3OH/CH2Cl2 (150 mL);
then 1 N NaOH (10 mL) was added and the reaction stirred
for 1 h before being neutralized by the addition of 1 N HCl
(10 mL). The solvent was then evaporated and the residue
partitioned between EtOAc and water. Washing of the organic
layer with water and a saturated solution of NaCl was followed
by drying (Na2SO4) and evaporation of the solvent to give the
product 16 (3.54 g, 98%) as an oil that solidified upon
standing: Rf 0.15 (6:1 petroleum ether/EtOAc); [R]D -29.1° (c
1
0.5, CHCl3); selected H NMR (250 MHz, CDCl3) δ 2.38 (1 H),
4.05 (1 H), 3.88 (1 H), 3.46 (1 H), 3.84 (1 H), 3.68 (1 H), 3.63
1
(1 H). Complete and assigned H NMR data are presented in
the Supporting Information Tables 1 and 2. Anal. Calcd for
1
C
36H36O5 (548.68): C, 78.81; H, 6.61. Found: C, 78.10; H, 6.81.
white solid, pure by H NMR and TLC. The product could be
3,7-An h ydr o-4,5,6,8-tetr a-O-acetyl-1,1,2,2-tetr adeh ydr o-
recrystallized from EtOAc: Rf 0.22 (1:1 petroleum ether/
EtOAc); H NMR (DMSO-d6, 250 MHz) δ 9.839 (s, 1 H, NH),
1
1,2-D-glycer o-L-ga la ctooctitol (18). Benzylated derivative 16
(3.54 g, 6.45 mmol) was dissolved in Ac2O (100 mL) and then
cooled to 0 °C before TMSOTf (8 mL, 41.4 mmol) was added.
The reaction was then warmed to room temperature and
stirred for 72 h, before the dark brown solution was cooled to
0 °C and quenched by the cautious addition of a solution of
NaHCO3. Dilution of the reaction mixture with EtOAc was
followed by washing of the organic layer with a solution of
NaHCO3, water, and a saturated solution of NaCl. After drying
(Na2SO4), the organic layer was evaporated to provide a dark
8.467 (s, 1 H, H-2), 8.220 (dd, 1 H, 4J 1.9, 3J 8.0 Hz, H-6),
8.046 (d, 1 H, 3J 8.0 Hz, H-4), 7.726 (t, 1 H, 3J 8.0, H-5), 7.315
(s, 1 H, H-â), 3.756 (s, 3 H, OCH3), 2.031 (s, 3 H, NHCOCH3);
13C NMR (DMSO-d6, 62.5 MHz) δ 170.210, 166.046 (CdO),
148.714 (C-1), 136.836 (C-4), 136.073 (C-3), 130.967 (C-5),
129.486 (C-â), 128.917 (C-R), 124.630 (C-2), 124.369 (C-4),
53.241 (OCH3), 23.281 (NHCOCH3). Anal. Cald. for C12H12N2O5
(264.24): C, 54.55; H, 4.58; N, 10.60. Found: C, 54.50; H, 4.58;
N, 10.60.

Combinatorial Synthesis of C-Linked Glycopeptides
J . Org. Chem., Vol. 63, No. 26, 1998 9665
N-Acetyl 3-a m in o-(DL)-p h en yla la n in e Meth yl Ester
Aceta te Sa lt (29). Cinnamate 28 (4.2 g, 15.9 mmol) was
dissolved in acetic acid (250 mL) and then 10% Pd/C (1.5 g)
added. The reaction was allowed to stir for 20 h under H2,
before the catalyst was filtered away and the solvent evapo-
MHz, CDCl3) δ 4.41 (1 H), 5.18 (1 H), 5.16 (1 H), 5.06 (1 H),
3.74 (1 H), 4.28 (1 H), 4.15 (1 H), 7.34 (2 H), 7.04 (2 H), 4.86
(1 H), 3.17 (1 H), 3.05 (1 H); 13C NMR δ 86.48, 82.21, 69.71,
68.53, 71.41, 67.34, 74.45, 61.57, 120.36, 132.11 (2 C), 129.20
(2 C), 137.13, 52.92, 37.75. Complete and assigned NMR data
are presented in the Supporting Information Tables 3 and 4.
Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86;
N, 2.40. Found: C, 57.31; H, 5.52; N, 2.39.
1
rated to yield the product 25 (4.5 g, 96%) as a brown oil. H
NMR showed the product as pure: Rf 0.24 (19:1 CH2Cl2/CH3-
1
OH); H NMR (CDCl3, 250 MHz) δ 7.058 (t, 1 H, 3J 7.7 Hz,
H-5), 6.573 (m, 1 H, H-4), 6.481 (m, 1 H, H-6), 6.458 (m, 1 H,
H-2), 6.110 (d, 1 H, J CH,NH 7.7 Hz, NH), 4.823 (ddd, 1 H, J CH,NH
7.7, J R,â 5.8, J R,â′ 6.1 Hz, H-R), 3.713 (s, 3 H, OCH3), 3.034 (dd,
1 H, J R,â 5.8, J â,â′ 13.8 Hz, H-â), 2.963 (dd, 1 H, J R,â 6.1, J â,â′
13.8 Hz, H-â′), 1.957 (s, 3 H, NHCOCH3); 13C NMR (CDCl3,
62.5 MHz) δ 172.149, 169.728 (CdO), 146.141 (C-1), 136.982
(C-3), 129.426 (C-5), 119.506 (C-6), 115.969 (C-2), 114.080 (C-
4), 52.992 (C-R), 52.206 (OCH3), 37.667 (C-â), 23.000 (NH-
COCH3).
N-Acetyl 4-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
tetr a d eh yd r o-1,2-D-glycer o-D-ga la ctooctityl)-DL-p h en yl-
a la n in e m eth yl ester (37): yield 68.7 mg, 91%; 1H NMR (250
MHz, CDCl3) δ 4.67 (1 H), 5.60 (1 H), 5.12 (1 H), 5.29 (1 H),
3.72 (1 H), 4.30 (1 H), 4.16 (1 H), 7.11 (2 H), 7.03 (2 H), 4.86
(1 H), 3.15 (1 H), 3.05 (1 H); 13C NMR δ 86.53, 82.29, 69.04,
69.82, 71.52, 65.66, 76.40, 62.65, 120.34, 132.03 (2 C), 129.21
(2 C), 137.11, 52.90, 37.74. Complete and assigned NMR data
are presented in the Supporting Information Tables 3 and 4.
Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86;
N, 2.40. Found: C, 57.35; H, 5.88; N, 2.42.
N-Acetyl-3-iod o-(DL)-p h en yla la n in e Meth yl Ester (30).
N-Acetyl-3-amino-(DL)-phenylalanine methyl ester (29) (15.2
g, 51.7 mmol) was dissolved in 1 N HCl (130 mL and the
solution cooled to 0 °C. To the reaction was added, in 2 mL
portions, NaNO2 (4.71 g, 68.3 mmol) as a cold solution in water
(150 mL). During the course of the addition the temperature
of the mixture was kept below 5 °C by the addition of ice to
the reation mixture. After the last aliquot of NaNO2 solution
was added, the reaction was allowed to stir for 30 min at 0-5
°C before KI (21.5 g, 129.5 mmol) was added, in 3 g portions.
The reaction immediately turned purple, and gas evolution
occurred. The flask was then removed from the ice bath and
was heated at 80 °C for 1 h. After cooling of the flash to room
temperature, ethyl acetate was added and then the aqueous
layer was washed with ethyl acetate until no product was
observed in the organic layer. The combined organic extracts
were then dried (Na2SO4), and the solvent was evaporated.
Chromatography (19:1 CH2Cl2/CH3OH) gave the product as a
yellow solid. Recrystallization from EtOAc/petroleum ether
gave the product 30 (8.6 g, 48%) as white crystals: Rf 0.44
(19:1 CH2Cl2/CH3OH); 1H NMR (CDCl3, 250 MHz) δ 7.589 (m,
1 H, H-6), 7.667 (s, 1 H, H-2), 7.087 (m, 1 H, H-4), 7.026 (t, 1
H, 3J 7.6 Hz, H-5), 6.025 (d, 1 H, J CH,NH 6.2 Hz, NH), 4.855
(ddd, 1 H, J CH,NH 6.2, J R,â 5.8, J R,â′ 5.7 Hz, H-R), 3.740 (s, 3 H,
OCH3), 3.109 (dd, 1 H, J R,â 5.8, J â,â′ 13.9 Hz, H-â), 3.019 (dd,
1 H, J R,â 5.7, J â,â′ 13.9 Hz, H-â′), 2.010 (s, 3 H, NHCOCH3);
13C NMR (CDCl3, 62.5 MHz) δ 171.747, 169.619 (CdO),
138.338 (C-2), 136.147 (C-6), 136.145 (C.3), 130.205 (C-5),
128.408 (C-4), 94.375 (C-1), 53.038 (C-R), 52.415 (OCH3),
37.352 (C-â), 23.079 (NHCOCH3). Anal. Calcd for C12H14INO3
(347.15): C, 41.52; H, 4.04; N, 4.03. Found: C, 42.02; H, 3.84;
N, 4.06.
N-Acetyl 4-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
t et r a d eh yd r o-1,2-D-glycer o-D-t a looct it yl)-DL-p h en yla la -
1
n in e m eth yl ester (39): yield 52.1 mg, 69%; H NMR (250
MHz, CDCl3) δ 4.97 (1 H), 5.42 (1 H), 5.54 (1 H), 5.32 (1 H),
4.24 (1 H), 4.32 (1H), 4.15 (1 H), 7.41 (2 H), 7.08 (2 H), 4.87 (1
H), 3.19 (1 H), 3.09 (1 H); 13C NMR δ 90.24, 81.17, 67.70, 71.20,
69.56, 65.99, 71.58, 62.39, 119.95, 132.11 (2 C), 129.33 (2 C),
137.49, 52.94, 37.74. Complete and assigned NMR data are
presented in the Supporting Information Tables 3 and 4. Anal.
Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N,
2.40. Found: C, 57.11; H, 5.56; N, 2.40.
N-Acetyl 3-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
tetr a d eh yd r o-1,2-D-glycer o-D-gu looctityl)-DL-p h en yla la -
1
n in e m eth yl ester (34): yield 65.6 mg, 87%; H NMR (250
MHz, CDCl3) δ 4.40 (1 H), 5.21 (1 H), 5.25 (1 H), 5.12 (1 H),
3.74 (1 H), 4.27 (1 H), 4.15 (1 H), 7.15 (1 H), 7.08 (1 H), 7.23,
(1 H) 7.30 (1 H), 4.86 (1 H), 3.13 (1 H), 3.04 (1 H); 13C NMR δ
86.84, 82.94, 69.34, 73.47, 71.19, 68.15, 75.94, 62.02, 121.78,
132.67, 136.23, 130.05, 128.56, 130.77, 52.96, 37.54. Anal.
Calcd for C28H33NO12‚H2O (593.58): C, 56.66; H, 5.94; N, 2.36.
Found: C, 56.95; H, 5.59; N, 2.37.
N-Acetyl-3-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
t et r a d eh yd r o-1,2-D-glycer o-D-m a n n ooct it yl)-DL-p h en yl-
a la n in e m eth yl ester (36): yield 61 mg, 81%; 1H NMR (250
MHz, CDCl3) δ 4.38 (1 H), 5.47 (1 H), 5.05 (1 H), 5.44 (1 H),
3.95 (1 H), 4.14 (1 H), 4.14 (1 H), 7.17 (1 H), 7.07 (1 H), 7.22,
(1 H) 7.31 (1 H), 4.85 (1 H), 3.11 (1 H), 3.03 (1 H); 13C NMR δ
86.49, 83.15, 69.67, 68.47, 71.36, 67.32, 74.45, 61.55, 121.80,
132.61, 136.19, 129.96, 128.50, 130.73, 52.92, 37.51. Anal.
Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N,
2.40. Found: C, 57.51; H, 5.62; N, 2.38.
N-Acetyl-3-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
tetr a d eh yd r o-1,2-D-glycer o-D-ga la ctooctityl)-DL-p h en yl-
a la n in e m eth yl ester (38): yield 52 mg, 69%; 1H NMR (250
MHz, CDCl3) δ 4.66 (1 H), 5.60 (1 H), 5.11 (1 H), 5.29 (1 H),
3.72 (1 H), 4.29 (1 H), 4.16 (1 H), 7.12 (1 H), 7.07 (1 H), 7.22,
(1 H) 7.28 (1 H), 4.46 (1 H), 3.14 (1 H), 3.03 (1 H); 13C NMR δ
86.56, 82.27, 69.03, 69.80, 71.51, 65.65, 76.41, 62.63, 121.78,
132.55, 136.23, 129.95, 128.54, 130.67, 52.93, 37.53. Anal.
Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N,
2.40. Found: C, 57.39; H, 5.64; N, 2.36.
Gen er a l P r oced u r e for P d -Cou p lin g Rea ction s. The
acetylene (55 mg, 0.15 mmol) and the iodide (45 mg, 0.13
mmol) were dissolved in piperidine (2 mL) and the flask purged
with argon while heating to 80 °C. Following the simultaneous
addition of (Ph3P)4Pd (7.5 mg, 6.5 µmol) and CuI (2.5 mg, 13
µmol) the reaction was stirred at 80 °C for 1 h before being
cooled and evaporated. The residue was then redissolved in
1:1 acetic anhydride/pyridine (5 mL) and stirred overnight, and
the reaction was quenched by cooling the solution to 0 °C and
adding methanol (3 mL). Evaporation of the solvent gave a
brown oil that was chromatographed (EtOAc) to give the
coupled product, Rf 0.45 (EtOAc).
N-Acetyl-3-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
t et r a d eh yd r o-1,2-D-glycer o-D-t a looct it yl)-DL-p h en yla la -
N-Acetyl 4-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
tetr a d eh yd r o-1,2-D-glycer o-D-gu looctityl)-DL-p h en yla la -
1
n in e m eth yl ester (40): yield 53.5 mg, 71%; H NMR (250
1
n in e m eth yl ester (33): yield 61 mg, 81%; selected H NMR
MHz, CDCl3) δ 4.97 (1 H), 5.42 (1 H), 5.53 (1 H), 5.32 (1 H),
4.23 (1 H), 4.33 (1 H), 4.15 (1 H), 7.20 (1 H), 7.12 (1 H), 7.27,
(1 H) 7.38 (1 H), 4.86 (1 H), 3.15 (1 H), 3.05 (1 H); 13C NMR δ
90.26, 81.15, 67.68, 71.18, 69.53, 65.98, 71.62, 62.40, 121.39,
132.65, 136.45, 130.23, 128.65, 130.74, 53.02, 37.52. Anal.
Calcd for C28H33NO12 (575.57): C, 58.43; H, 5.78; N, 2.43.
Found: C, 58.08; H, 5.66; N, 2.43.
Gen er a l P r oced u r e for Dea cyla tion Rea ction s. The
coupled product (ca. 1 mmol) was dissolved in CH3OH (20 mL),
a small piece of sodium was added, and the reaction stirred
for 2 h and evaporated. The residue was redissolved in water
(20 mL), stirred for an additional 2 h, and then brought to pH
(250 MHz, CDCl3) δ 4.41 (1 H), 5.18 (1 H), 5.16 (1 H), 5.06 (1
H), 3.74 (1 H), 4.28 (1 H), 4.15 (1 H), 7.34 (2 H), 7.04 (2 H),
4.86 (1 H), 3.17 (1 H), 3.05 (1 H); 13C NMR δ 86.79, 82.96,
69.35, 73.49, 71.21, 68.15, 75.91, 62.03, 120.33, 132.13 (2 C),
129.23 (2 C), 137.17, 52.93, 37.77. Complete and assigned NMR
data are presented in the Supporting Information Tables 3 and
4. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H,
5.86; N, 2.40. Found: C, 57.29; H, 5.62; N, 2.44.
N-Acetyl 4-C-(3,7-a n h yd r o-4,5,6,8-tetr a -O-a cetyl-1,1,2,2-
t et r a d eh yd r o-1,2-D-glycer o-D-m a n n ooct it yl)-DL-p h en yl-
a la n in e m eth yl ester (35): yield 68.5 mg, 91%; 1H NMR (250

9666 J . Org. Chem., Vol. 63, No. 26, 1998
Lowary et al.
2-3 by the addition of IR 120 (H+) resin. After filtration of
the resin the solution was evaporated to give the product, pure
by NMR.
h yd r o-1-oxo-1,2-D-glycer o-D-ga la ctooctit-1-yl)-DL-p h en yl-
a la n in e (51). Meth od 1: Ch em ica l Dea cetyla tion . The
N-acetyl derivative 43 (10 mg, 330 µmol) was dissolved in 30
mL of 1 N HCl and the solution stirred at 90 °C. The reaction
was followed by analysis of 10 µL samples on analytical RP-
HPLC using a linear gradient 0-50% B over 40 min. Two fast
eluting products were formed. After 6 h the starting material
had disappeared and the reaction mixture was cooled to room
temperature and neutralized by addition of 5% NaOH solution.
Preparative RP-HPLC using a linear gradient 0-30% B over
90 min gave the product 49 (33.7 mg, 29%): 1H NMR (250
MHz, CDCl3) δ 4.24 (1 H), 3.77 (1 H), 3.64 (1 H), 3.98 (1 H),
3.73 (3 H), 7.54 (2 H), 7.31 (2 H), 4.28 (1 H), 3.34 (1 H), 3.02
(1 H); 13C NMR δ 86.33, 85.88, 71.37, 71.08, 73.74, 69.24, 79.41,
61.55, 121.25, 132.88 (2 C) 129.97 (2 C), 135.78, 54.55, 35.99.
ES-MS: (M + H)+, m/z 352.2; C17H21NO7 requires M, 351.355.
Furthermore, the compound 51 (52.0 mg, 43%) was isolated:
1H NMR (250 MHz, CDCl3) δ 3.48 (1 H), 3.23 (1 H), 3.80 (1
H), 3.51 (1 H), 3.58 (1 H), 3.90 (1 H), 3.57 (1 H), 3.59 (2 H),
7.92 (2 H), 7.40 (2 H), 4.26 (1 H), 3.35 (1 H), 3.22 (1 H); 13C
NMR δ 202.49, 171.99, 41.73, 76.52, 71.02, 74.31, 69.52, 78.96,
61.54, 141.01, 130.22 (2 C) 129.53 (2 C), 136.28, 54.49, 35.10.
ES-MS: found for (M + H)+, m/e 370.5; C17H23NO8 requires
M, 369.371.
3-C-(3,7-An h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-glycer o-D-
ga la ctooctityl)- L-p h en yla la n in e (50). Meth od 2: Acyla se-
Ca ta lyzed Rea ction . The N-acetyl derivative 43 (102 mg, 259
µmol) was dissolved in 10 mL of PBS buffer (pH ) 7.5). Acylase
I (326 mg, 163 units) was added. The mixture was stirred for
a total of 48 h, during which the pH was kept at 7.5 by addition
of a saturated NaHCO3 solution. The reaction was followed
by analytical RP-HPLC of 5 µL samples of the reaction mixture
using a gradient buffer A f buffer B 100:0 f 50:50 over 40
min. When no evolution was observed anymore, the mixture
was acidified by addition of 1 N HCl solution and filtered.
Purification by preparative RP-HPLC yielded a fraction of
impure starting material (72.4 mg) and 58.7 mg (62%) of pure
product 50: [R]D +5.8° (c 2.0, H2O, pH ) 1); ES-MS (M + H)+
m/z 352.1, C17H21NO7 requires M, 351.355; 1H NMR (250 MHz,
CDCl3) δ 4.24 (1 H), 3.76 (1 H), 3.64 (1 H), 3.98 (1 H), 3.72 (3
H), 7.54 (2 H), 7.31 (2 H), 4.35 (1 H), 3.36 (1 H), 3.22 (1 H);
13C NMR δ 86.28, 85.89, 71.35, 71.06, 73.73, 69.23, 79.40,
61.53, 121.32, 132.88 (2 C) 129.97 (2 C), 135.55, 54.18, 35.83.
Nr-(F lu r or en -9-ylm eth oxyca r bon yl)-4-C-(3,7-a n h yd r o-
1,1,2,2-tetr a d eh yd r o-1,2-D-glycer o-D-ga la ctooctityl)p h en -
yla la n in e (52, DL Mixtu r e). The glycosyl amino acid 49 (30
mg, 85 µmol) was dissolved in 5 mL of water, and the pH was
adjusted to 10 by addition of a 1 M solution of K2CO3. Fmoc-
OSu (32 mg, 94 µmol) was added as solution in 2 mL of
acetone. After stirring of the reaction overnight TLC (15:5:2.5
DCM/MeOH/AcOH) and analytical C-18 HPLC showed com-
plete conversion of the stating material. The reaction mixture
was acidified to pH ) 2 by addition of 1 N HCl thereby
precipitating the Fmoc derivative. The solution was extracted
4 times with EtOAc, and the organic extracts were combined,
dried over MgSO4, and evaporated. Semipreparative C18-
HPLC purification yielded 25.1 mg of 52 (DL form, 47%). ES-
MS: found for (M + Na)+, m/e 596.2; C32H31NO9 requires M,
573.598.
N-Acetyl 4-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-gu looctityl)-DL-p h en yla la n in e (41). The conju-
gate 33 (641 mg, 1.11 mmol) yielded the product 41 (425 mg,
97%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.34 (1 H), 3.51
(1 H), 3.57 (1 H), 3.56 (1 H), 3.53 (1 H), 3.94 (1 H), 3.74 (1 H),
7.50 (2 H), 7.28 (2 H), 4,63 (1 H), 3.24 (1 H), 3.01 (1 H), 1.95
(3 H); 13C NMR δ 89.96, 85.21, 71.01, 69.91, 77.03, 73.77, 80.28,
61.23, 120.18, 132.35 (2 C), 129.76 (2 C), 138.65, 54.67, 37.17.
N-Acetyl-4-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-m a n n ooctityl)-DL-p h en yla la n in e (43). The con-
jugate 35 (730 mg, 1.27 mmol) yielded the product 43 (485
mg, 97%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.27 (1 H),
3.81 (1 H), 3.68 (1 H), 4.02 (1 H), 3.43 (1 H), 3.58 (2 H), 7.52
(2 H), 7.29 (2 H), 4,64 (1 H), 3.25 (1 H), 3.02 (1 H), 1.95 (3 H);
13C NMR δ 86.60, 85.45, 71.43, 71.08, 73.74, 69.24, 79.36,
61.51, 120.26, 132.36 (2 C), 129.76 (2 C), 138.51, 54.55, 37.10.
N-Acetyl 4-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-ga la ctooctityl)-DL-p h en yla la n in e (45). The con-
jugate 37 (640 mg, 1.11 mmol) yielded the product 45 (420
mg, 96%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.51 (1 H),
4.10 (1 H), 3.59 (1 H), 3.66 (1 H), 3.30 (1 H), 3.94 (1 H), 3.76
(1 H), 7.50 (2 H), 7.29 (2 H), 4.52 (1 H), 3.24 (1 H), 3.02 (1 H),
1.95 (3 H); 13C NMR δ 86.71, 84.71, 70.50, 71.72, 73.75, 66.96,
80.55, 61.48, 120.36, 132.31 (2 C), 129.74 (2 C), 138.35, 54.35,
37.00.
N-Acetyl 4-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-ta looctityl)-DL-p h en yla la n in e (47). The conju-
gate 39 (272 mg, 0.47 mmol) yielded the product 47 (183 mg,
98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.97 (1 H), 4.18
(1 H), 4.09 (1 H), 3.71 (1 H), 3.88 (1 H), 3.86 (1 H), 3.81 (1 H),
7.48 (2 H), 7.27 (2 H), 4,60 (1 H), 3.24 (1 H), 2.99 (1 H), 1.94
(3 H); 13C NMR δ 89.75, 83.08, 70.11, 72.32, 71.54, 67.33, 76.02,
61.33, 120.05, 132.18 (2 C), 129.75 (2 C), 138.81, 54.97, 37.31.
N-Acetyl-3-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-gu looctityl)-DL-p h en yla la n in e (42). The conju-
gate 34 (647 mg, 1.12 mmol) yielded the product 42 (435 mg,
98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.35 (1 H), 3.49
(1 H), 3.54 (1 H), 3.53 (1 H), 3.50 (1 H), 3.95 (1 H), 3.75 (1 H),
7.42 (1 H), 7.46 (1 H), 7.38, (1 H) 7.32 (1 H), 4.59 (1 H), 3.23
(1 H), 2.98 (1 H), 1.95 (3 H); 13C NMR δ 86.96, 85.02, 71.00,
77.04, 73.76, 69.91, 80.30, 61.23, 121.68, 132.80, 137.88,
130.76, 129.19, 130.55, 55.09, 37.15.
N-Acetyl-3-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-m a n n ooctityl)-DL-p h en yla la n in e (44). The con-
jugate 36 (651 mg, 1.13 mmol) yielded the product 44 (435
mg, 98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.28 (1 H),
3.86 (1 H), 3.68 (1 H), 4.02 (1 H), 3.76 (1 H), 3.79 (2 H), 7.45
(1 H), 7.49 (1 H), 7.40, (1 H) 7.36 (1 H), 4.66 (1 H), 3.24 (1 H),
3.01 (1 H), 1.96 (3 H); 13C NMR δ 86.54, 85.50, 71.40, 71.07,
73.75, 69.24, 79.38, 61.55, 121.87, 132.79, 137.46, 130.87,
129.23, 130.47, 54.34, 36.77.
N-Acetyl-3-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-ga la ctooctityl)-DL-p h en yla la n in e (46). The con-
jugate 38 (675 mg, 1.17 mmol) yielded the product 46 (447
mg, 97%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.66 (1 H),
4.11 (1 H), 3.79 (1 H), 3.66 (1 H), 3.46 (1 H), 3.95 (1 H), 3.76
(1 H), 7.47 (1 H), 7.41 (1 H), 7.43, (1 H) 7.36 (1 H), 4.63 (1 H),
3.23 (1 H), 3.00 (1 H), 1.96 (3 H); 13C NMR δ 86.70, 84.71,
70.49, 71.71, 73.75, 66.96 80.57, 61.49, 121.91, 132.72, 137.63,
130.77, 129.21, 130.44, 54.65, 36.92. ES-MS: found (M + H)+,
m/e 394.3; C19H23NO8 requires M, 393.39.
Nr-(F lu r or en -9-ylm eth oxyca r bon yl)-4-C-(3,7-a n h yd r o-
1,1,2,2-tetr adeh ydr o-1,2-D-glycer o-D-galactooctityl)-L-ph en -
yla la n in e (52). The amino acid derivative 50 (45 mg, 129
µmol) was dissolved in 10 mL of water, and the pH was
adjusted to 10 by dropwise addition of a 1 M solution of K2-
CO3. A solution of Fmoc-OSu (48 mg, 142 µmol) in 2 mL of
acetone was added. The reaction mixture was stirred at room
temperature, and every few hours the pH was readjusted to
10 by addition of 1 M K2CO3. After 12 h TLC (15:5:2.5 or 15:
5:2.5 DCM/MeOH/AcOH) showed that the reaction was fin-
ished. The reaction mixture was diluted to 20 mL and washed
with 10 mL of diethyl ether. The water phase was transferred
to two centrifuge tubes and under agitation acidified to pH 2
by addition of 1 N HCl. After centrifugation at 7000 r/min for
10 min the supernatant was decanted from the white precipi-
tate. The precipitate was dispersed in water and collected by
N-Acetyl-3-C-(3,7-a n h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-
glycer o-D-ta looctityl)-DL-p h en yla la n in e (48). The conju-
gate 40 (272 mg, 0.47 mmol) yielded the product 48 (182 mg,
98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.97 (1 H), 4.19
(1 H), 4.09 (1 H), 3.71 (1 H), 3.97 (1 H), 3.96 (1 H), 3.81 (1 H),
7.42 (1 H), 7.45 (1 H), 7.38, (1 H) 7.32 (1 H), 4.61 (1 H), 3.22
(1 H), 2.97 (1 H), 1.95 (3 H); 13C NMR δ 89.72, 83.16, 70.09,
72.31, 71.56, 67.32, 76.06, 61.35 121.68, 132.66, 137.68, 130.62,
129.22, 130.53, 54.68, 36.94.
4-C-(3,7-An h yd r o-1,1,2,2-tetr a d eh yd r o-1,2-D-glycer o-D-
ga la ctooctityl)-DL-p h en yla la n in e (49) a n d 4-C-(3,7-An -

Combinatorial Synthesis of C-Linked Glycopeptides
J . Org. Chem., Vol. 63, No. 26, 1998 9667
Ta ble 1. 1H NMR Da ta (δ w ith Hz in P a r en th eses) th e
O-lin k ed Glycop ep tid es 54-56 Mea su r ed in CD3COOD/
D2O (1:1) a t 600 MHz a t 300 K
centrifugation. This was repeated twice. The precipitate was
lyophilized to yield 55.8 mg of white product. Product remain-
ing in the supernatant and washings was recovered by running
a preparative RP-HPLC of the combined concentrated solu-
tions, and after lyophilization additional (7.71 mg) white power
could be obtained (85% total yield of 52). The two fractions
were identical as analyzed by NMR spectroscopy and mass
spectrometry: [R]D +6.5° (c 0.8, DMF); 1H NMR (250 MHz,
CDCl3) δ 3.99 (1 H), 3.58 (1 H), 3.30 (1 H), 3.71 (1 H), 3.40 (1
H), 3.50 (2H), 7.2-7.6 (4 H), 4.20 (1 H), 3.11 (1 H), 2.90 (1 H);
13C NMR δ 89.29, 84.72, 72.14, 71.42, 75.11, 69.45, 80.11,
61.59, 121-136 (6 C), 56.05, 37.12; ES-MS found for (M + Na)+
596.2, C32H31NO9 requires M, 573.598.
54
55
56
Val
3.94
2.23
1.00
0.99
R-H
3.95
2.24
1.01
1.00
3.95
2.23
1.01
1.00
â-H
γ-H3
γ-H3
Ile
NH
R-H
â-H
8.38 (8.0)
4.34
1.84
8.38 (8.0)
4.32
1.84
8.38 (8.0)
4.40
1.85
Nr-(F lu r or en -9-ylm eth oxyca r bon yl)-3-C-(3,7-a n h yd r o-
1-oxo-1,2-D-glycero-D-galactooctityl)-DL-phenylalanine (53).
The amino acid derivative 51 (52 mg, 141 µmol) was dissolved
in 10 mL of water, and the pH was adjusted to 10 by dropwise
addition of a 1 M solution of K2CO3. A solution of Fmoc-OSu
(53 mg, 157 µmol) in 2 mL of acetone was added. The reaction
mixture was stirred at room temperature, and every few hours
the pH was readjusted to 10 by addition of 1 M K2CO3. After
20 h analytical HPLC showed that the reaction was finished.
The reaction was worked up as for compound 52. Product
remaining in the supernatant and washings was recovered by
preparative HPLC. A total of 66.6 mg (77%) of white product
53 was obtained: 1H NMR (250 MHz, CDCl3) δ 3.27 (1 H),
3.10 (1 H), 3.66 (1 H), 3.35 (1 H), 3.29 (1 H), 3.73 (1 H), 3.27
(1 H), 3.45 (1 H), 3.33 (1 H), 7.35-7.95 (4 H), 4.24 (1 H), 3.17
(1 H), 2.95 (1 H); 13C NMR δ 198.81, 173.91, 42.22, 77.38, 71.37,
75.53, 69.43, 79.63, 61.16, 141.55, 129-130 (4 C), 136.29, 55.88,
37.13; ES-MS (M + Na)+ m/z 596.2, C32H31NO9 requires M,
573.598.
γ-H2
γ-H3
δ-H3
1.51/1.16
0.90
0.85
1.51/1.17
0.87
0.84
1.53/1.18
0.88
0.84
Thr
NH
8.12 (8.0)
4.40
4.22
8.11 (7.5)
4.40
4.21
8.27 (8.5)
4.30
4.30
R-H
â-H
γ-H3
1.15
1.13
1.16
Ala
NH
R-H
â-H3
8.00 (6.5)
4.32
1.28
8.00 (6.0)
4.31
1.22
8.12 (5.5)
4.30
1.24
Phe
NH
R-H
â-H2
Phenyl
7.27 meta
7.21 para
7.84 (7.0)
4.59
3.02/2.94
7.12 ortho
7.26 meta
7.22 para
7.89 (7.0)
4.57
3.00/2.92
7.11 ortho
7.28 meta
7.24 para
7.89 (7.0)
4.58
3.03/2.87
7.12 ortho
P r ep a r a tion of th e P EGA Resin . Syntheses of the pep-
tides were performed in DMF using PEGA resin prepared in
the laboratory.36,37 The resin (5 g, loading 0.2 mmol/g) was
packed into a 50 cm3 disposable syringe fitted with a Teflon
filter. The syringe was connected to a suction flask through a
Teflon tube with a manual 2-way Teflon valve, and the resin
was swelled in DMF, treated with 20% piperidine in DMF for
10 min, and washed 6 times with DMF. The resin was
derivatized with the ((hydroxymethyl)phenoxy)acetic acid
(HMPA linker). The HMPA linker (455 mg, 2.5 mmol), O-(1H-
benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluo-
roborate (TBTU) (750 mg, 2.37 mmol), and 4-ethylmorpholine
(315 µL, 2.5 mmol) were dissolved in DMF, and after 10 min
the solution was added to the resin and left to react overnight.
The resin was washed carefully with DMF and dichlo-
romethane. NR-Fmoc-Lys(Boc)-COOH (1.75 mg, 3.75 mmol)
and N-methylimidazole (231 mg, 223 µL, 2.81 mmol) were
dissolved in dichloromethane (20 cm3), and 1-(mesitylenesulfo-
nyl)-3-nitro-1,2,4-triazole (MSNT) (1.11 g, 3.71 mmol) was
added. After 5 min the solution was added to the resin and
allowed to react for 3 h. The resin was washed thoroughly with
dichloromethane, DMF, and diethyl ether and dried in vacuo.
Gen er a l P r oced u r e for th e SP P S of P ep tid es. Deriva-
tized PEGA resin (0.2 mmol/g capacity, 0.66 equiv compared
to the amount of glycosylated building block employed) was
transferred into a syringe equipped with a Teflon filter. Apart
from the glycosyl-amino acids all amino acids used were NR-
Fmoc-amino acid OPfp (pentafluorophenyl) esters. NR-Fmoc
deprotection was effected by successive 2 and 20 min treat-
ments of the resin with 20% piperidine in DMF. The washing
procedure (6 times with DMF) was repeated after each
coupling/Fmoc deprotection step. Each NR-Fmoc-amino acid
OPfp ester (3 equiv) and Dhbt-OH (3,4-dihydro-3-hydroxy-4-
oxo-1,2,3-benzotriazine) (1 equiv) were dissolved in DMF. The
solutions were added to the resins and then left for a minimum
of 3 h. After each coupling step the resin was washed, the NR-
Fmoc group removed, and the resin washed as described above.
In the case of the glycosyl building blocks 52 and 53 only 1.5
equiv was employed. These were preactivated in DMF by
adding 0.95 equiv of TBTU and 1.6 equiv of NEM for 20 min.
These solutions were then transferred to the resins and
allowed to react for 3 h. In the case of the synthesis using
glycosyl-amino acid 53, the resin was washed and the free
Phe (Conjugate)
8.02 (8.0)
4.66
NH
R-H
â-Ha
â-Hb
7.97 (8.0)
4.61
3.12
8.06 (7.0)
4.63
3.10
3.22
3.09
3.04
3.07
Glu
8.04 (7.5)
4.34
2.10/1.96
2.39
NH
8.01 (7.5)
4.34
2.11/1.95
2.41
8.25 (7.5)
4.37
1.93/1.78
2.03
R-H
â-H2
γ-H2
Gly
8.07 (6.0)
3.94
NH
R-H2
7.92 (6.0)
3.96
8.23 (6.0)
3.95
Leu
7.85 (7.0)
4.41
1.60
1.60
NH
7.84 (7.5)
4.43
1.61
1.61
0.90/0.85
7.90 (7.0)
4.42
1.60
1.60
0.91/0.85
R-H
â-H2
γ-H
δ-H3
0.90/0.85
Lys
8.12 (7.5)
4.42
1.90/1.75
1.43
1.68
NH
R-H
â-H
γ-H
δ-H
ꢀ-H
8.11 (8.0)
4.43
1.92/1.77
1.45
1.69
3.02
8.09 (8.0)
4.40
1.89/1.75
1.43
1.68
3.01
3.02
7.46
NH2
7.46
7.45
Gal-Acetylene
2-Ha
2-Hb
3-H
4-H
5-H
6-H
7-H
8-Ha
8-Hb
2-H′, H-6′
3-H′, H-5′
3.51 (1.0)
3.49 (2.0)
3.28 (17.0, 9.0) 3.31 (17.0, 9.0)
4.24 (9.5)
3.84 (9.5)
3.68 (3.5)
4.04
3.71
3.81 (7.5)
3.86
3.63
3.66
3.99
3.66
3.69
3.88
3.64
3.68 (3.0)
4.00
3.68
3.70
3.61
7.97
7.36
3.76 (12.0, 4.5) 3.64
7.43
7.21
7.95
7.38
amino groups in excess were capped by treatment of the resin
with a 20% solution of Ac2O in DMF for 20 min. The resin

9668 J . Org. Chem., Vol. 63, No. 26, 1998
Lowary et al.
was washed as usual, and the peptide synthesis was continued
using OPfp esters. After cleavage of the final NR-Fmoc group
the resin was washed with DMF, CH2Cl2, and ether and dried.
Cleavage of the glycopeptides was achieved by treatment with
95% TFA for 2 h. The resin was filtered off and washed 4 times
with 95% TFA. The solution was concentrated and the glyco-
peptides precipitated by addition of ether. The crude glyco-
peptides were dissolved in water-TFA and analyzed by RP-
HPLC using a linear gradient from 0 to 100% B over 50 min.
Purification by semipreparative RP-HPLC using a linear
gradient from 3 to 50% B over 70 min gave the pure glyco-
peptides. Using the building block 52 (L-epimer) the pure
glycopeptide 54 (45.6 mg, 57%) (ES-MS: (M + H)+, m/z 1310.9;
H)+, m/z 1328.9, C63H97N11O20 requires M, 1328.52), which
could be easily separated by RP-HPLC. The capping of free
amino functions had avoided the formation of the shorted
sequence VITAFEGLK. The 1H NMR spectra of 54-56 were
all assigned by 2D-NMR spectroscopy, and the data are
presented in Table 1.
Ack n ow led gm en t. This work was supported with
a grant (T.L.) from the Mitzutani Foundation.
Su p p or tin g In for m a tion Ava ila ble: Tables S1-S12 of
the fully assigned 1H and 13C NMR data for compounds 4, 5,
8, 13, 16-19, and 33-53 (12 pages). This material is contained
in libraries on microfiche, immediately follows this article in
the microfilm version of the journal, and can be ordered from
the ACS; see any current masthead page for ordering
information.
C
63H97N11O20 requires M, 1310.51) was obtained. Synthesis
with the DL-mixture of 52 obtained from 49 gave the mixture
of the two epimers of 54 in impure form after preparative RP-
HPLC.
The building block 53 gave the two epimeric peptides 55
(29.6 mg, 34%) (ES-MS: (M + H)+, m/z 1329.0; C63H97N11O20
requires M, 1328.52) and 56 (28.7 mg, 33%) (ES-MS: (M +
J O980517H