Gel-phase 19F NMR spectral quality for resins commonly used in solid-phase organic synthesis; a study of peptide solid-phase glycosylations

Article abstract of DOI:10.1039/b404802d

The spectroscopic properties for seven different commercial resins used in solid-phase synthesis were investigated with F NMR spectroscopy. A fluorine-labeled dipeptide was synthesized on each resin, and the resolution of the ¹⁹F resonances in CDCl₃. DMSO-d₆, benzene-d₆ and CD₃OD were measured with a conventional NMR spectrometer, i.e. without using magic angle spinning. In general, resins containing polyethylene glycol) chains (ArgoGel, TentaGel and PEGA) were found to be favorable for the ¹⁹F NMR spectral quality. Three serine containing tri-, penta-. and heptapeptides were then prepared on an ArgoGel resin functionalized with a fluorine-labeled linker. The resin bound peptides were glycosylated utilizing a thiogalactoside glycosyl donor carrying fluorine-labeled protective groups. Monitoring of the glycosylations with gel-phase ¹⁹F NMR spectroscopy allowed each glycopeptide to be formed in ～80% yield, using a minimal amount of glycosyl donor (3 × 2 equivalents). In addition, it was found that the glycosylation yields were independent of peptide length.

Full text of DOI:10.1039/b404802d

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19
Gel-phase F NMR spectral quality for resins commonly used
in solid-phase organic synthesis; a study of peptide solid-phase
glycosylations
Mickael Mogemark,a Frida Gårdmo,a Tobias Tengel,a Jan Kihlberg*a,b and Mikael Elofsson*a
a Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden
b AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden.
E-mail: mikael.elofsson@chem.umu.se; jan.kihlberg@chem.umu.se
Received 31st March 2004, Accepted 7th May 2004
First published as an Advance Article on the web 25th May 2004
The spectroscopic properties for seven different commercial resins used in solid-phase synthesis were investigated
with 19F NMR spectroscopy. A fluorine-labeled dipeptide was synthesized on each resin, and the resolution of the 19
F
resonances in CDCl3, DMSO-d6, benzene-d6 and CD3OD were measured with a conventional NMR spectrometer, i.e.
without using magic angle spinning. In general, resins containing poly(ethylene glycol) chains (ArgoGel, TentaGel
and PEGA) were found to be favorable for the 19F NMR spectral quality. Three serine containing tri-, penta-, and
heptapeptides were then prepared on an ArgoGel resin functionalized with a fluorine-labeled linker. The resin bound
peptides were glycosylated utilizing a thiogalactoside glycosyl donor carrying fluorine-labeled protective groups.
Monitoring of the glycosylations with gel-phase 19F NMR spectroscopy allowed each glycopeptide to be formed
in ∼80% yield, using a minimal amount of glycosyl donor (3 × 2 equivalents). In addition, it was found that the
glycosylation yields were independent of peptide length.
tivity (the natural abundance of 19F is 100%), and distribution
Introduction
of 19F resonances over a wide spectral range as a result of the
Since the pioneering work of Merrifield,1 solid-phase synthesis
high polarizability of the 19F nucleus. Thus, analysis of
reactions directly on the solid phase by using a fluorinated
has been widely applied for preparation of oligopeptides2–5 and
oligonucleotides6 on an individual basis or in the form of
linker as an internal standard in combination with fluorinated
libraries.7,8 In recent years the introduction of combinatorial
building blocks,43,48 or building blocks carrying fluorine-labeled
chemistry and parallel synthesis has renewed the interest
protective groups,44,50,51 provides easily interpreted spectra.
in solid-phase synthesis of, for instance, oligosaccharides,9–13
These contain both quantitative and qualitative information,
glycopeptides14–17 and especially for parallel synthesis of small
including yield and stereoselectivity, and are obtained in a
“drug-like” molecules.7,8,18–21 In essence, the advantages with
couple of minutes with a conventional NMR spectrometer.
solid-phase synthesis compared to traditional solution-phase
Although solid-phase synthesis is in common use in organic
synthesis is that excess of reagents can be used to drive reac-
chemistry, the understanding of the physicochemical and
tions to completion, and that reagents and soluble by-products
spectroscopic properties of the resins used in synthesis remains
can simply be removed by filtration and washing of the poly-
1
somewhat limited.53–56 HR-MAS H NMR spectroscopy has
meric support. These properties render solid-phase synthesis
been used to study the spectrum quality for commercial
suitable for automation, which is important for speeding up the
resins loaded with aspartic acid,30 and in a comparison of
discovery process of new biologically active compounds.22–24
PEG-grafted resins and PEG-cross-linked polymers.57 At
A major limitation when performing reactions on solid phase is
the difficulties in analyzing the outcome of complex reactions
when the product is still attached to the polymeric support. In
order to avoid problematic purifications of the final product, it
present, no other NMR techniques have been used to examine
the spectroscopic properties of commonly used resins.
In view of the advantages that gel-phase 19F NMR spectro-
scopy provides for monitoring reactions directly on the solid
should be possible to analyze formation and accumulation of
support, this article describes: 1) a study of the influence of
side-products in each synthetic step.
resin structure, tether length, and solvent upon the 19F NMR
IR spectroscopy25 and MALDI-TOF mass spectrometry in
spectral quality for seven commercially available and com-
monly used resins and, 2) a study of O-glycosylation of
resin-bound peptides of varying lengths with a thiogalactoside
donor.
combination with a color test have in some cases been success-
fully employed for quantification of solid-phase reactions.26
Ideally, analytical NMR spectroscopic methods employed
in solution-phase synthesis would constitute a powerful tool
for monitoring solid-phase synthesis directly on the solid
Results and discussion
support.27 Therefore approaches such as high resolution
magic-angle-spinning (HR-MAS) NMR spectroscopy and
gated decoupling 13C NMR spectroscopy have been utilized to
quantify reactions directly on the solid support.28–32 However,
these methods are somewhat restricted by cost and the require-
ment of specialized equipment. Identification of products may
also be problematic if the reactions are incomplete or if large
amounts of by-products have been formed. To address this
issue, 19F NMR spectroscopy has been developed as a versatile
and straightforward method for monitoring solid-phase
synthesis.33–52 19F NMR spectroscopy has several advantages,
including the lack of interfering background signals, high sensi-
In order to study the influence of the resin on the spectral
quality, a N-acylated dipeptide was synthesized on seven
commercially available amino-functionalized polymeric
supports, i.e. polystyrene (1), ArgoPore (2), ArgoGel (3),
TentaGel (4), NovaGel (5), PEGA (6) and SPAR-50 (7) (Fig. 1).
The 19F NMR spectrum of each peptide-resin was then
recorded in four different solvents; CDCl3, DMSO-d6, benzene-
d6 and CD3OD (Fig. 2). The resin-bound dipeptides 1–7 all
contained four fluorine atoms as analytical markers which
allowed comparison of the spectral qualities.58 The two fluorine
resonances from the terminal o,p-difluorobenzoyl group
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 7 7 0 – 1 7 7 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Table 1 Mean line widths (Hz) of the two fluorine resonances derived
from the terminal o,p-difluorobenzoyl group of resins 1–7a
Resin
CDCl3
DMSO-d6
Benzene-d6
CD3OD
Polystyrene 1
ArgoPore 2
ArgoGel 3
TentaGel 4
NovaGel 5
PEGA 6
nm
nm
37
27
nm
38
78
nm
40
39
81
35
148
nm
nm
48
nm
nm
100
102
nm
80
40
nm
116
nm
SPAR-50 7
nm
244
a Mean value of the line widths (in Hz) measured at half peak height.
nm = not measured.
Fig. 1 Fluorinated dipeptides 1–7 prepared on seven commercial
amino functionalized resins.
appeared between Ϫ103 and Ϫ106 ppm and between Ϫ108 and
Ϫ109 ppm, respectively. The 19F signals derived from the two
p-FPhe residues appeared at Ϫ116 ppm. Comparison of the
spectral quality for resins 1–7, included evaluation of the
separation between p-FPhe signals and the determination of
the 19F NMR line widths of the two fluorine signals derived
from the terminal o,p-difluorobenzoyl group (Table 1).
With polystyrene resin 1, which has 1% of divinyl benzene
(DVB) cross-linking, a 19F NMR spectrum of moderate quality
could be generated only in DMSO-d6 (Fig. 2). This classical
Merrifield resin is frequently used for solid-phase synthesis due
to its mechanical stability and high loading capacity. In general,
the spectroscopic and physicochemical properties of this
“gel-type” resin are highly dependent on the degree of DVB
cross-linking,59 in which a low degree of cross-linking favors the
molecular mobility of the resin, and thus improves the spectral
quality. This resin is available with 1 or 2% of DVB cross-
linking to ensure adequate mechanical stability, swelling, and
diffusion rate of the reagents in a wide range of solvents.56,59,60
Although the spectral quality of the polystyrene resin is
moderate, it has been successfully employed to monitor SNAr
reactions34,61 and formation of tertiary amines46 directly on the
resin with gel-phase 19F NMR spectroscopy. The more rigid
ArgoPore resin 2, which is based on a highly cross-linked
macroporous polystyrene framework, did not generate an
acceptable spectrum in any of the solvents (Fig. 2). The
regional molecular mobility of this resin is restrained by the
high DVB cross-linking, and thus the quality of the 19F NMR
spectra is low.
The ArgoGel resin 3 and TentaGel resin 4 produced high
quality 19F NMR spectra in CDCl3, DMSO-d6 and benzene-d6,
whereas use of CD3OD resulted in spectra of moderate quality
(Fig. 2). ArgoGel and TentaGel resins are polystyrene based
resins that are grafted with poly(ethylene glycol) chains (PEG)
on the hydrophobic core. The polystyrene core provides the
resins with mechanical stability whereas the flexible PEG grafts
results in excellent swelling properties in a wide variety of
solvents. This type of resin swells well in both polar and non-
polar solvents, and importantly, the organic moiety attached
to the resins possesses a high molecular mobility. As a con-
sequence both spin lattice relaxation and the chemical shift
anisotropy are reduced and spectra with high quality are
obtained in a wide variety of solvents.30 The line-widths of the
Fig. 2 19F NMR spectra of resins 1–7 recorded in CDCl3, DMSO-d6, benzene-d6 and CD3OD.
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Scheme 1 Solid-phase synthesis of peptide resins 9–11 on resin 8. Reagents and conditions: i) Fmoc-Ala-OH, Fmoc-Phe-OH or Fmoc-Leu-OH
(4 equiv.), MSNT (4 equiv), MeIm (3 or 4 equiv.), CH2Cl2, RT. ii) Peptide synthesis according to the Fmoc protocol. MSNT = 1-(mesitylenesulfonyl)-
3-nitro-1,2,4-triazole, MeIm = 1-methylimidazole.
two 19F signals that originate from the terminal o,p-difluoro-
benzyl group revealed that the best spectra for both resins were
obtained in CDCl3, closely followed by DMSO-d6, benzene-d6
and CD3OD (Table 1). Moreover, the 19F resonances from the
two p-FPhe residues were completely separated in both CDCl3
and benzene-d6. After a closer inspection, it could be concluded
that the TentaGel resin 4 generally displayed a somewhat
narrower line width than the ArgoGel resin 3 (Table 1).
which used HR-MAS 1H NMR spectroscopy to investigate the
spectral quality of commercial resins.30 These results imply that
the influence from the PEG tether is the most important factor
affecting the spectral quality, closely followed by the rigidity of
the resin structure, i.e. the degree of cross-linking.
Thus, the ArgoGel and TentaGel resins have good qualities
for monitoring solid-phase organic synthesis with gel-phase 19
F
NMR spectroscopy. In addition, PEG containing resins exhibit
excellent swelling properties, and high reaction rates for polar
and ionic reagents.56,57,60 Therefore we chose to evaluate solid-
phase peptide glycosylations with the ArgoGel resin, since this
NovaGel is also a polystyrene PEG grafted copolymer that
exhibits a loading capacity similar to the Merrifield resin. In
addition, this resin has similar swelling characteristics as the
TentaGel and ArgoGel resins. However, the location of the
functional groups of the NovaGel resin is not at the end of the
PEG chain, as for the TentaGel and ArgoGel resins, instead
they are situated on the hydrophobic polystyrene core, just as
for the Merrifield resin. Hence, the organic moiety attached to
the resin will not possess the same molecular mobility as the
TentaGel and Argogel resins, and accordingly, the spectral
quality of the NovaGel resin 5 was in general low (Fig. 2).
resin displays better loading capacity (0.4 to 0.5 mmol gϪ1
)
than TentaGel (0.2 to 0.3 mmol gϪ1),62 which is synthetically
advantageous.
At present the most reliable method for synthesis of glyco-
peptides, is by solid-phase stepwise assembly of amino acids
and glycosylated amino acid building blocks.14–16 However, a
strategy based on attachment of a carbohydrate moiety to free
hydroxyl groups of serine, threonine, tyrosine, hydroxyproline
or hydroxylysine residues in a preformed peptide is very
appealing, since it offers possibilities to create diversity using
different glycosyl donors at a late stage in the synthesis.
Attempts to find protocols towards direct O-glycosylation of
resin bound peptides are fairly rare and initial attempts using
polystyrene as solid support gave the target glycopeptides in
very low yields.63–65 However, a few successful glycosylations on
a polyoxyethylene-polyoxypropylene resin (POEPOP) by using
trichloroacetimidates as glycosyl donor have been reported.66,67
The difficulties encountered in glycosylation of peptides have
been suggested to be due to the low solubility of peptides under
the conditions used for glycosylation and/or adsorption of the
Lewis acid by the peptide-amide functionalities.68
In an effort to find conditions for glycosylation of resin-
bound peptides, tripeptide 9, pentapeptide 10 and heptapeptide
11 were prepared on the linker-loaded ArgoGel resin 8 (Scheme
1). A fluorinated “Wang-type” linker was used to fulfil the
analytical requirements and the 19F signal derived from linker
served as an internal standard throughout the syntheses.36 In
addition, the linker-peptide bond is essentially stable under the
conditions used in glycosylations and peptide synthesis, but is
readily cleaved under nucleophilic conditions or by acidolysis
with trifluoroacetic acid (TFA).36,38 Thioglycoside 13 (Scheme 2)
was employed as glycosyl donor, since thioglycosides can
be conveniently activated by soft Lewis acids or converted
to other glycosyl donors.69 Thiogalactoside building block 13
was prepared from 4,6-O-m-fluorobenzylidene protected
thiocresyl galactoside 1251 in 98% yield by treatment with
p-fluorobenzoyl chloride in pyridine (Scheme 2). Thioglycosides
have recently been successfully employed in solid-phase
However, recording the spectrum in DMSO-d6, produced a 19
F
NMR spectrum of moderate quality comparable to that of
polystyrene resin 1 (Table 1).
The polymeric supports PEGA and SPAR-50 are acrylamide
based polymers that mainly have found uses within peptide
synthesis and for enzymatic applications, due to their excellent
swelling properties in both polar and non-polar solvents. The
mechanical stability of this type of resin is however somewhat
low since it lacks the stabilizing polystyrene core. PEGA is a
poly-acrylamide support that is cross-linked with PEG chains,
which gives the resin good spectroscopic and swelling properties
in a wide range of solvents. As expected, the PEGA polymer 6
indeed produced 19F NMR spectra of high resolution in both
CDCl3 and DMSO (Fig. 2, Table 1). However, four major
resonances arises from one of the p-FPhe residues in the spec-
trum, these presumably originates from different stereoisomers
of the secondary amine functionality and inhomogeneous
distribution of the functional sites. Thus, only the p-FPhe that
is attached to the resin-amino functionality is affected due to its
close proximity to the stereocentre. Furthermore, just as for
TentaGel and ArgoGel, the spectral quality decreased when
recording in CD3OD. The spectral quality for resin 6 was also
poor when using benzene-d6 as solvent. SPAR-50 is a poly-
acrylamide polymer with large pore size, which is particularly
suitable for enzymatic applications since the large pores
facilitate the entrance of the enzyme into the polymer matrix.
In contrast to PEGA, this polymer does not contain PEG
tethers, and as expected, the molecular mobility of this polymer
is low compared to PEGA. Hence, 19F NMR spectra of low
quality were obtained. This is consistent with a previous study,
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was thwarted and well resolved 19F NMR spectra were obtained
both for the resin bound peptides 10 and 11 and glycopeptides
15 and 16 (Fig. 3). These results strongly imply that aggregation
of the resin bound peptides is not pivotal for the outcome of
the glycosylation, when the reaction is carried out with a thio-
glycoside under activation by NIS and TfOH. The yields of the
glycopeptide 14–16 were all the same after each consecutive
glycosylation, although the glycosylations were performed in
CH2Cl2, which is a less polar solvent than CDCl3. Initially,
saponification with aqueous LiOH in THF was used to cleave
glycopeptide 14 from the solid phase. Careful monitoring of the
cleavage of resin 14 with 19F NMR spectroscopy and LC-MS
analysis of the reaction mixture revealed that the hydrolysis was
slow and that β-elimination of the carbohydrate moiety from
the peptide was a severe problem. This was circumvented by
acidolysis of resin 14–16 with TFA at 60 ЊC,38 which resulted in
cleavage of the glycopeptides from the linker with simultaneous
removal of the benzylidene group (Scheme 3). Subsequently,
the remaining p-fluorobenzoyl groups were saponified with
a catalytic amount of LiOH (20 mM) in methanol without
β-elimination.75 This gave glycopeptides 17, 18 and 19 in 27, 26
and 29% overall yields, respectively, based on the initial loading
capacity of the amino ArgoGel resin (0.45 mmol gϪ1). The
β-galactosidic linkages of the isolated glycopeptides 17–19 were
confirmed by the coupling constant between the H1 and H2 in
Scheme 2 Preparation of thiogalactoside donor 13. Reagents and
conditions: i) 12, pFBzCl (2.6 equiv.), pyridine, RT.
glycosylations,44,50,51,70–72 and it has been shown that N-iodo-
succinimide (NIS) in combination with a catalytic amount
of trifluoromethanesulfonic acid (TfOH) is an excellent pro-
moter system.73,74 Furthermore, in a previous study NIS was
found to be superior to dimethyl(methylthio)sulfonium triflate
(DMTST).44
The parallel solid-phase glycosylations of peptides 9–11 with
thioglycoside 13 (2 equivalents) were carried out in CH2Cl2
under promotion by NIS and a catalytic amount of TfOH for
two hours at room temperature (Scheme 3). Comparison of the
integrals of the 19F NMR signals from the carbohydrate pro-
tective groups with the 19F resonances derived from the linker
and the N-terminal p-fluorobenzoyl amide group of resins
14–16 revealed ∼30% glycosylation of all the three peptides.
Repeating the glycosylation once under identical conditions
increased the yield to ∼60% and after a third glycosylation the
glycopeptides 14–16 were formed in ∼80% yields (Fig. 3). More-
over, since the 19F signals from each protective group appeared
as a uniform singlet, it was assumed that only the desired
β-galactosidic linkage was formed. Interestingly, the resin
bound tripeptide 9 and glycosylated tripeptide 14 gave high
quality 19F NMR spectra in CDCl3, whereas the penta- and
heptapeptides 10 and 11 and the corresponding glycopeptides
15 and 16 furnished inadequate spectra in both CDCl3 and
benzene-d6. This observation was assumed to originate from
aggregation of these resin bound peptides and glycopeptides
due to the low polarity of CDCl3 and benzene-d6.68 However,
when the solvent was changed to DMSO-d6, the aggregation
the galactoside moieties (3J1, ≈ 7 Hz). However, coupling of
2
the first amino acid of each of the three peptides with resin 8
resulted in ∼20 to 35% epimerization, as determined by 1H
NMR spectroscopy of the isolated glycopeptides 17–19. The
epimerization could not be detected in the gel-phase 19F NMR
spectra, probably due to that the distance between the fluorine
atom in the linker and the stereogenic center of the amino acid
is too large. This indicates that proximity between a stereo-
center and a fluorine label is required to obtain stereochemical
information.
In conclusion, the 19F NMR spectral quality for seven
commercial resins were examined in four frequently used NMR
solvents. It was found that flexible PEG-tethers grafted on a
Scheme 3 Solid-phase glycosylations of peptides 9–11 with thiogalactoside 13 followed by cleavage of glycopeptides 14–16 from the solid support
and removal of the carbohydrate protective groups. Reagents and conditions: i) 13 (2 equiv.), NIS (2 equiv.), TfOH (0.2 equiv.), CH2Cl2, RT,
the reactions were repeated twice. ii) TFA/H2O (9/1, v/v), 60 ЊC. iii) LiOH (20 mM in H2O/MeOH, 1/4, v/v), RT. NIS = N-iodosuccinimide,
TfOH = trifluoromethanesulfonic acid.
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groups in combination with a fluorinated linker, both the
anomeric purity and yield of the peptide glycosylations could
be extracted from the 19F NMR spectra.
Experimental
General methods and materials
All reactions were carried out at room temperature under an
inert nitrogen atmosphere using dry, freshly distilled solvents.
CH2Cl2 was distilled from calcium hydride. Organic solutions
were dried over Na2SO4 before being concentrated. TLC was
performed on Silica Gel F254 (Merck) and detection was carried
out by examination under UV light and by charring with 10%
sulfuric acid. Flash column chromatography was performed
on Silica Gel (Matrex, 60 Å, 35–70 µm, Grace Amicon).
Preparative HPLC separations were performed on a Beckman
System Gold HPLC, using a Kromasil C-8 column (250 × 20
mm, 5 µm, 100 Å) with a flow rate of 11 mL minϪ1 and detec-
tion at 214 nm. Analytical HPLC was performed on a Beckman
System Gold HPLC, using a Kromasil C-8 column (250 × 4.6
mm, 5 µm, 100 Å) with a flow rate of 1.5 mL minϪ1 and detec-
tion at 214 nm. 1H and 13C NMR spectra were recorded with a
Bruker DRX-400 or ARX-500 spectrometer for solutions in
CDCl3 [residual CHCl3 (δH 7.26 ppm), CDCl3 (δC 77.0 ppm) as
internal standard], [D6]DMSO [residual [D5]DMSO (δH 2.50
ppm), [D6]DMSO (δC 39.51 ppm) as internal standard], or
CD3OD [residual CD2HOD (δH 3.35 ppm), CD3OD (δC 49.0
ppm) as internal standard] at 300 K. First order chemical shifts
and coupling constants were determined from one-dimensional
spectra and proton resonances were assigned from COSY,
TOCSY, NOESY and HETCOR experiments. Proton reson-
ances that could not be assigned are not reported. Proton
resonances from the minor isomeric by-product of glycopeptide
17–19 are not reported. 13C resonances that originated from the
fluorinated protective groups were split by JC–F coupling and
therefore signals downfield from 110.0 ppm are not reported.
Proton decoupled gel-phase 19F NMR spectra were recorded
with a Bruker DRX-400 spectrometer for resin suspensions in
CDCl3[CFCl3 (δF 0.00 ppm) as internal standard] at 300 K. Two
peaks appear in the spectra at around 0.00 ppm, one resonance
originates from CFCl3 inside the polymer the other resonance
from CFCl3 outside the polymer. The peak with the highest
chemical shift was used as internal standard. Optical rotation
was measured with a Perkin-Elmer 343 polarimeter and is given
in 10Ϫ1 deg cm2 Ϫ1. Mass spectra for glycopeptide 18 was
g
recorded on a Waters Micromass ZQ using positive electrospray
ionization (ESϩ). High resolution mass spectra were recorded
on a JEOL SX102 A mass spectrometer. Ions for FABMS were
produced by a beam of xenon atoms (6 keV) from a matrix of
glycerol and thioglycerol.
Resin 1–7
Fig. 3 19F NMR spectra of resin bound glycopeptides 14–16 after
three identical glycosylations; (a) 90% conversion of resin 9, (b) 80%
conversion of resin 10, (c) 80% conversion of resin 11.
Peptide syntheses were performed manually in a mechanically
agitated reactor on commercial amino functionalized resins
with Fmoc-p-fluorophenylalanine and o,p-difluorobenzoic acid
as building blocks and N,NЈ-diisopropylcarbodiimide and
1-hydroxybenzotriazole according to previously reported pro-
cedures to give resins 1–7;76 resin 1 had: 19F NMR (376 MHz,
CDCl3) δ Ϫ103, Ϫ109, Ϫ116; 19F NMR (376 MHz, DMSO-d6)
δ Ϫ105.6, Ϫ108.4, Ϫ116.5; resin 2 had: 19F NMR (376 MHz,
DMSO-d6) δ Ϫ106, Ϫ117; resin 3 had: 19F NMR (376 MHz,
CDCl3) δ Ϫ103.7, Ϫ108.9, Ϫ116.0, Ϫ116.4; 19F NMR (376
MHz, DMSO-d6) δ Ϫ105.7, Ϫ108.4, Ϫ116.5, Ϫ116.6; 19F NMR
hydrophobic polystyrene core, or cross-linked into a poly-
acrylamide polymer, were essential to obtain good NMR
spectroscopic properties. Glycosylations of resin-bound tri-,
penta- and heptapeptides on an ArgoGel resin were
successfully performed by employing a thiogalactoside glycosyl
donor protected with fluorine-labeled protective groups. In this
study it was found that neither the length nor aggregation of the
peptides influenced the yield of the glycosylations. Analysis of
the resin bound glycopeptides with gel-phase 19F NMR
spectroscopy, revealed that the yields could be increased to
∼80% after three identical glycosylations that consumed in
all six equivalents of glycosyl donor. Moreover, by using a
saccharide building block carrying fluorine-labeled protective
(376 MHz, benzene-d6) δ Ϫ104.9, Ϫ108.3, Ϫ116.3, Ϫ116.6; 19
F
NMR (376 MHz, CD3OD) δ Ϫ104.7, Ϫ108.7, Ϫ116.5; resin 4
had: 19F NMR (376 MHz, CDCl3) δ Ϫ103.7, Ϫ108.9, Ϫ116.0,
Ϫ116.4; 19F NMR (376 MHz, DMSO-d6) δ Ϫ105.7, Ϫ108.4,
Ϫ116.5, Ϫ116.6; 19F NMR (376 MHz, benzene-d6) δ Ϫ104.9,
Ϫ108.4, Ϫ116.3, Ϫ116.7; 19F NMR (376 MHz, CD3OD)
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δ Ϫ104.8, Ϫ108.7, Ϫ116.6; resin 5 had: 19F NMR (376 MHz,
CDCl3) δ Ϫ103, Ϫ109, Ϫ116; 19F NMR (376 MHz, DMSO-d6)
δ Ϫ105.6, Ϫ108.4, Ϫ116.6; 19F NMR (376 MHz, benzene-d6)
δ Ϫ104, Ϫ116; 19F NMR (376 MHz, CD3OD) δ Ϫ104, Ϫ108,
Ϫ116; resin 6 had: 19F NMR (376 MHz, CDCl3) δ Ϫ103.5,
Ϫ108.9, Ϫ115.9, Ϫ116.2, Ϫ116.3, Ϫ116.4, Ϫ116.4; 19F NMR
(376 MHz, DMSO-d6) δ Ϫ105.6, Ϫ108.3, Ϫ116.4, Ϫ116.5; 19F
NMR (376 MHz, benzene-d6) δ Ϫ104.8, Ϫ108.3, Ϫ116.3; 19F
NMR (376 MHz, CD3OD) δ Ϫ104.7, Ϫ108.5, Ϫ116.5; resin 7
had: 19F NMR (376 MHz, CDCl3) δ Ϫ103, Ϫ109, Ϫ116;
19F NMR (376 MHz, DMSO-d6) δ Ϫ105.5, Ϫ108.3, Ϫ116.4;
19F NMR (376 MHz, CD3OD) δ Ϫ104.6, Ϫ108.6, Ϫ116.3.
The procedure was repeated twice to give resin 14 in 87% yield;
19F NMR (376 MHz, CDCl3) δ Ϫ104.8, Ϫ105.1, Ϫ108.3,
Ϫ113.5, Ϫ115.3.
Resin 15
Resin 10 was treated as described for resin 14 to give resin 15 in
80% yield; 19F NMR (376 MHz, DMSO-d6) δ Ϫ104.8, Ϫ105.2,
Ϫ109.0, Ϫ113.0, Ϫ115.6.
Resin 16
Resin 11 was treated as described for resin 14 to give resin 16 in
84% yield; 19F NMR (376 MHz, DMSO-d6) δ Ϫ104.7, Ϫ105.1,
Ϫ109.0, Ϫ112.9, Ϫ115.5.
Resin 8
N,NЈ-Diisopropylcarbodiimide (0.24 mL, 1.55 mmol) was
added to a solution of pentafluorophenol (0.43 g, 2.32 mmol) in
EtOAc (30 mL) at 0 ЊC. After 30 min, a solution of 3-fluoro-4-
(hydroxymethyl)phenoxyacetic acid36 (0.31 g, 1.55 mmol) in
EtOAc (40 mL) was added and the solution was stirred for 1 h
and subsequently added to the ArgoGel-NH2 resin (1.80 g,
0.77 mmol). After agitation for 16 h at room temperature
the N-acylation was complete according to monitoring with
bromophenol blue (0.19 mL, 2.0 mM in DMF). The resin was
washed with EtOAc, MeOH and THF (5 × 10 mL each) to give
resin 8; 19F NMR (376 MHz, CDCl3) δ Ϫ117.2.
4-Fluorobenzoyl-glycyl-O-(ꢀ-D-galactopyranosyl)-L-seryl-L-
valine (17)
A solution of trifluoroacetic acid in H2O (9 : 1, 12 mL) was
added to resin 14 (43 µmol) and the mixture was heated to 60 ЊC
without stirring. After 3 h the resin was removed by filtration
and washed with HOAc (2 × 5 mL). The filtrate was evapor-
ated, whereafter HOAc (10 mL) was added and the solution
was lyophilized. The residue was purified with reversed-phase
HPLC (gradient: 10
100% MeCN in H2O, both containing
0.1% TFA, during 60 min) and lyophilized. The resulting color-
less solid was dissolved with MeOH (3.2 mL) and aqueous
LiOH (0.8 mL, 0.1 M) was added drop-wise (5 min). After 1 h
the solution was neutralized with HOAc, evaporated, diluted
with HOAc (5 mL) and lyophilized. The residue was purified
Resin 9–11
MSNT (0.16 g, 0.54 mmol) and the first amino acid (0.53
mmol) were dissolved in CH2Cl2 (3.0 mL), and added to
ArgoGel resin 8 (0.13 mmol), functionalized with a fluorinated
linker.36 Methyl imidazole (31 µL, 0.39 mmol) was added and
the mixture was mechanically agitated during 14 h. The resin
was washed with CH2Cl2, DMF and CH2Cl2 (3 × 5 mL each),
whereafter the resin was subjected to peptide synthesis accord-
ing to previous reported procedures to give resin 9, 10 and 11;76
resin 9 had: 19F NMR (376 MHz, CDCl3) δ Ϫ108.4, Ϫ115.2;
resin 10 had: 19F NMR (376 MHz, DMSO-d6) δ Ϫ109.5,
Ϫ116.1; resin 11 had: 19F NMR (376 MHz, DMSO-d6)
δ Ϫ109.0, Ϫ115.5.
with reversed-phase HPLC (gradient: 0
100% MeCN in
H2O, both containing 0.1% TFA, during 60 min) and lyophil-
ized to provide 17 (5.1 mg, 22%) as a colorless solid; 1H NMR
(500 MHz, DMSO-d6) δ 7.95 (2H, m, ArH ), 7.31 (2H, t, J = 8.7
Hz, ArH ), Gly: 8.80 (1H, dd, J = 6.4 Hz, NH), 3.93 (2H, m,
αH), Ser: 8.16 (1H, d, J = 7.8 Hz, NH), 4.63 (1H, m, αH), 3.95
(1H, m, βH), 3.63 (1H, m, βH), Val: 7.92 (1H, d, J = 7.5 Hz,
NH), 4.13 (1H, m, αH), 2.06 (1H, m, βH), 0.89 (6H, m, γCH3),
Gal: 4.73 (1H, d, J = 7.3 Hz, H-1), 3.67 (1H, m, H-3), 3.58 (1H,
m, H-4), 3.56 (1H, m, H-2); HRMS (FAB) calcd for C23H31-
FN3Na2O11 590.1733 m/z (M ϩ Na)ϩ, observed 590.1730.
4-Methylphenyl 2,3-di-O-(4-fluorobenzoyl)-4,6-O-(3-fluoro-
benzylidene)-1-thio-ꢀ-D-galactopyranoside (13)
4-Fluorobenzoyl-L-alaninyl-glycyl-O-(ꢀ-D-galactopyranosyl)-L-
seryl-L-valinyl-L-phenylalanine (18)
Compound 1251 (0.63 g, 1.61 mmol) was dissolved in pyridine
(7.0 mL) and 4-fluorobenzoyl chloride (0.49 mL, 4.17 mmol)
was added drop-wise over 5 min. The solution was stirred for 16
h at room temperature and then diluted with CH2Cl2 (100 mL),
washed with sat. aqueous NaHCO3 (2 × 50 mL) and H2O
(50 mL). The organic phase was concentrated and residual
pyridine was removed by co-concentration with toluene. The
resulting brown oil was purified by flash column chromato-
Resin 15 (43 µmol) was treated as described for 17 to give 18
(8.7 mg, 26%) as a colorless solid; 1H NMR (500 MHz, DMSO-
d6) δ 7.98 (2H, dd, J = 5.5, 9.0 Hz, ArH ), 7.31 (2H, m, ArH ),
Ala: 8.56 (1H, d, J = 7.6 Hz, NH), 4.52 (1H, q, J = 7.3 Hz, αH),
1.34 (3H, d, J = 7.3 Hz, βH), Gly: 8.18 (1H, d, J = 6.7 Hz, NH),
3.76 (2H, d, J = 6.7 Hz, αH), Ser: 8.48 (1H, d, J = 8.1 Hz, NH),
4.54 (1H, m, αH), 3.55 (2H, d, J = 6.4 Hz βH), Val: 7.87 (1H, d,
J = 8.8 Hz, NH), 4.20 (1H, dd, J = 6.4, 8.8 Hz, αH), 1.99 (1H, m,
βH), 0.83 (3H, d, J = 6.4 Hz, γCH3), 0.77 (3H, d, J = 6.4 Hz,
γCH3), Phe: 8.18 (1H, d, J 7.6 Hz, NH), 4.41 (1H, m, αH), 3.04
(1H, m, βH), 2.88 (1H, m, βH), 7.26 (2H, d, J = 7.7 Hz, ArH ),
7.21 (3H, m, ArH ), Gal: 4.93 (1H, d, J = 6.8 Hz, H-1), 4.02 (1H,
H-4), 3.91 (1H, H-5 or H-6), 3.76 (1H, t, J = 10.3, 6.3 Hz, H-2),
3.65 (1H, H-5 or H-6), 3.60 (1H, H-3), 3.54 (1H, H-5 or H-6);
MS (ESϩ) calcd for C35H46FN5O13 763.3 m/z (M)ϩ, observed
763.3.
graphy (heptane/EtOAc, 5 : 1
4 : 1) to give 13 (1.00 g, 98%)
1
as a colorless foam; [α]20 ϩ28 (c 0.37, CHCl3); H NMR (400
D
MHz, CDCl3) δ 8.04–7.91 (4H, m, ArH ), 7.51–7.48 (2H, d,
J = 8.1 Hz, ArH ), 7.35–6.96 (10H, m, ArH ), 5.67 (1H, t, J = 9.9
Hz, H-2), 5.48 (1H, s, 3-FPhCHO2), 5.33 (1H, dd, J = 10.0, 3.4
Hz, H-3), 4.89 (1H, d, J = 9.8 Hz, H-1), 4.55 (1H, d, J = 3.2 Hz,
H-4), 4.45 (1H, dd, J = 12.4, 1.5 Hz, H-6), 4.08 (1H, dd,
J = 12.4, 1.5 Hz, H-6), 3.75 (1H, s, H-5), 2.35 (3H, s, SPhMe);
13C NMR (100 MHz, CDCl3) δ 100.0, 85.0, 74.1, 73.6, 69.7,
69.1, 67.2, 21.2; 19F NMR (376 MHz, CDCl3) δ Ϫ113.4,
Ϫ105.5, Ϫ105.1; HRMS (FAB) calcd for C57H59F5NO18
659.1322 m/z (M ϩ Na)ϩ, observed 659.1328.
4-Fluorobenzoyl-L-phenylalaninyl-L-alaninyl-glycyl-O-(ꢀ-D-
galactopyranosyl)-L-seryl-L-valinyl-L-phenylalaninyl-L-leucine
(19)
Resin 14
Resin 16 (11 µmol) was treated as described for 17 to give 19
(3.0 mg, 27%) as a colorless solid; 1H NMR (500 MHz, DMSO-
d6) δ 7.85 (2H, m, ArH ), 7.26 (2H, m, ArH ), Phe: 8.58 (1H, d,
J = 8.25 Hz, NH), 4.72 (1H, dd, J = 6.4, 10.2 Hz, αH), 3.15 (1H,
m, βH), 2.96 (1H, m, βH), 7.24 (3H, m, ArH ), 7.16 (2H, d,
J = 7.3 Hz, ArH ), Ala: 8.27 (1H, d, J = 6.4 Hz, NH), 4.34 (1H,
TfOH (0.77 µL, 8.6 µmol) was added to a suspension of resin 9
(43 µmol), NIS (19 mg, 85 µmol) and 13 (55 mg, 86 µmol) in
CH2Cl2 (3.0 mL) in the absence of light. After 3 h agitation at
room temperature, the resin was washed with CH2Cl2, THF,
20% piperidine in DMF, DMF and CH2Cl2 (5 × 3 mL each).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 7 7 0 – 1 7 7 6
1775

View Article Online
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J. Kihlberg, J. Chem. Soc., Perkin Trans. 1, 1999, 1731–1742.
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dq, J = 6.4, 12.9 Hz, αH), 1.27 (3H, d, J = 6.9 Hz, βH), Gly: 8.05
(1H, d, J = 9.0 Hz, NH), 3.80 (1H, m, αH), 3.73 (1H, m, αH),
Ser: 8.03 (1H, d, J = 8.2 Hz, NH), 4.54 (1H, m, αH), 3.88 (1H,
m, βH), 3.54 (1H, m, βH), Val 7.65 (1H, d, J = 8.7 Hz, NH),
4.14 (1H, m, αH), 1.90 (1H, m, βH), 0.74 (6H, d, J = 6.4 Hz,
γCH3), Phe 8.05 (1H, d, J = 6.5 Hz, NH), 4.57 (1H, m, αH), 3.06
(1H, m, βH), 2.77 (1H, m, βH), 7.35 (2H, d, J = 7.7 Hz, ArH ),
7.24 (3H, m, ArH ), Leu: 8.11 (1H, d, J = 9.6 Hz, NH), 4.22
(1H, m, αH), 1.51 (2H, dd, J = 8.7, 13.1 Hz, βH), 1.62 (1H, m,
γH), 0.88 (3H, d, J = 6.5 Hz, δCH3), 0.82 (3H, d, J = 6.5 Hz,
δCH3), Gal: 4.70 (1H, d, J = 6.4 Hz, H-1), 4.13 (1H, H-4), 3.63
(1H, H-5 or H-6), 3.51 (1H, H-5 or H-6), 3.34 (1H, H-3), 3.30
(2H, H-2 and H-5 or H-6); HRMS (FAB) calcd for C50H65-
FN7Na2O15 1068.4313 m/z (M ϩ Na)ϩ, observed 1068.4324.
Acknowledgements
44 M. Mogemark, M. Elofsson and J. Kihlberg, Org. Lett., 2001, 3,
This work was funded by grants from the Swedish Research
Council and the Göran Gustafsson Foundation for Research in
Natural Sciences and Medicine.
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 7 7 0 – 1 7 7 6
1776