Synthesis of carbohydrate-functionalised sequence-defined oligo(amidoamine)s by photochemical thiol-ene coupling in a continuous flow reactor

Article abstract of DOI:10.1002/chem.201203927

Poly/oligo(amidoamine)s (PAAs) have recently been recognised for their potential as well-defined scaffolds for multiple carbohydrate presentation and as multivalent ligands. Herein, we report two complimentary strategies for the preparation of such sequence-defined carbohydrate-functionalised PAAs that use photochemical thiol-ene coupling (TEC) as an alternative to the established azide-alkyne cycloaddition ( click ) reaction. In the first approach, PAAs that contained multiple olefins were synthesised on a solid support from a new building block and subsequent conjugation with unprotected thio-carbohydrates. Alternatively, a pre-functionalised building block was prepared by using TEC and assembled on a solid support to provide a carbohydrate-functionalised PAA. Both methods rely on the use of a continuous flow photoreactor for the TEC reactions. This system is highly efficient, owing to its short path length, and requires no additional radical initiator. Performing the reactions at 254 nm in Teflon AF-2400 tubing provides a highly efficient TEC procedure for carbohydrate conjugation, as demonstrated in the reactions of O-allyl glycosides with thiols. This method allowed the complete functionalisation of all of the reactive sites on the PAA backbone in a single step, thereby obtaining a defined homogeneous sequence. Furthermore, reaction at 366 nm in FEP tubing in the flow reactor enabled the large-scale synthesis of an fluorenylmethyloxycarbonyl (Fmoc)-protected glycosylated building block, which was shown to be suitable for solid-phase synthesis and will also allow heterogeneous sequence control of different carbohydrates along the oligomeric backbone. These developments enable the synthesis of sequence-defined carbohydrate-functionalised PAAs with potential biological applications. Sweet flow chemistry: Monodisperse, sequence-defined glycooligomers were obtained through a combination of automated solid-phase synthesis and carbohydrate conjugation in a flow photoreactor by using thiol-ene chemistry (see figure). Copyright

Full text of DOI:10.1002/chem.201203927

DOI: 10.1002/chem.201203927
Synthesis of Carbohydrate-Functionalised Sequence-Defined
ꢀ
Oligo(amidoamine)s by Photochemical Thiol Ene Coupling in a Continuous
Flow Reactor
Felix Wojcik,[a, b] Alexander G. OꢀBrien,[a, b] Sebastian Gçtze,[a, b]
Peter H. Seeberger,*[a, b] and Laura Hartmann*[a, b]
Abstract:
Poly/oligo(amidoamine)s
using TEC and assembled on a solid
support to provide carbohydrate-
alisation of all of the reactive sites on
the PAA backbone in a single step,
(PAAs) have recently been recognised
for their potential as well-defined scaf-
folds for multiple carbohydrate presen-
tation and as multivalent ligands.
Herein, we report two complimentary
strategies for the preparation of such
sequence-defined carbohydrate-func-
tionalised PAAs that use photochemi-
a
functionalised PAA. Both methods rely
on the use of a continuous flow photo-
reactor for the TEC reactions. This
system is highly efficient, owing to its
short path length, and requires no addi-
tional radical initiator. Performing the
reactions at 254 nm in Teflon AF-2400
tubing provides a highly efficient TEC
procedure for carbohydrate conjuga-
tion, as demonstrated in the reactions
of O-allyl glycosides with thiols. This
method allowed the complete function-
thereby obtaining a defined ho
neous sequence. Furthermore, reaction
at 366 nm in FEP tubing in the flow re-
actor enabled the large-scale synthesis
of an fluorenylmethyloxycarbonyl
ACHTUNGTRENNUNGmoACHTUNGTRENNUNGge-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
(Fmoc)-protected glycosylated building
block, which was shown to be suitable
for solid-phase synthesis and will also
allow heterogeneous sequence control
of different carbohydrates along the
oligomeric backbone. These develop-
ments enable the synthesis of se-
quence-defined carbohydrate-function-
alised PAAs with potential biological
applications.
ꢀ
cal thiol ene coupling (TEC) as an al-
ternative to the established azide–
alkyne cycloaddition (“click”) reaction.
In the first approach, PAAs that con-
tained multiple olefins were synthe-
ACHTUNGTRENNUNGsised on a solid support from a new
building block and subsequent conjuga-
tion with unprotected thio-carbohy-
drates. Alternatively, a pre-functional-
ised building block was prepared by
Keywords: carbohydrates · flow re-
actors · photochemistry · polymers ·
solid-phase synthesis
Introduction
immune response or receptor interaction.[2] Precise control
of the spatial orientation and distance between carbohydrate
groups along a scaffold is desirable for promoting multiva-
lent interactions and for controlling the binding affinity and
selectivity of the multivalent ligands.[3] As a consequence of
efforts to develop such synthetic carbohydrate ligands and
to understand the underlying structure–activity relationships
(SARs), various strategies for the presentation of carbohy-
drates have been explored.[4] Recently, we demonstrated the
use of solid-phase synthesis for the preparation of monodis-
perse sequence-defined glycopolymer segments that were
based on poly/oligo(amidoamine) (PAA) substructures.[5]
These compounds belong to the class of “precision poly-
mers”, a new class of macromolecular systems that combine
the advantages of biopolymer scaffolds, such as peptides or
DNA,[6] with the advantages of synthetic scaffolds, such as
polymers[7] or dendrimers,[8] because they are highly defined,
versatile in their structure (linear or branched), and biocom-
patible, with a decreased risk of inherent immunogenicity.[9]
In a previous study, we observed a strong dependence of
binding affinity on the number and spacing of the sugar li-
gands along the oligomeric backbone, thus indicating that
monodisperse, sequence-defined scaffolds have a good po-
ꢀ
ꢀ
Carbohydrate protein, carbohydrate DNA, and carbohy-
ꢀ
drate carbohydrate interactions underpin many biological
processes and are crucial to understanding immunology.[1]
The weak nature of these interactions requires multiple pre-
sentation of carbohydrate moieties to elicit the desired
[a] F. Wojcik,+ Dr. A. G. OꢀBrien,+ S. Gçtze, Prof. Dr. P. H. Seeberger,
Dr. L. Hartmann
Department of Biomolecular Systems
Max Planck Institute of Colloids and Interfaces
Am Mꢁhlenberg 1, 14476 Potsdam (Germany)
[b] F. Wojcik,+ Dr. A. G. OꢀBrien,+ S. Gçtze, Prof. Dr. P. H. Seeberger,
Dr. L. Hartmann
Institute for Chemistry and Biochemistry
Freie Universitꢂt Berlin
Arnimallee 22, 14195 Berlin (Germany)
E-mail: peter.seeberger@mpikg.mpg.de
laura.hartmann@mpikg.mpg.de
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203927.
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Chem. Eur. J. 2013, 19, 3090 – 3098

FULL PAPER
tential for the presentation of new synthetic carbohydrate li-
gands. Herein, mannose moieties were reacted with alkyne-
presenting oligomers through copper-catalysed azide–alkyne
cycloaddition.[10] Although this approach is highly suitable
for the solid-phase synthesis of glycopolymer segments, the
triazole linkage is potentially immunogenic,[11] thus limiting
the application of this method in the targeting and stimula-
tion of immune cells.
carbohydrates, or 2) a pre-functionalised building block was
prepared that was then used to construct the same structure
but with the potential to access heterogeneous carbohy-
drate-functionalisation patterns. Herein, we discuss the rela-
tive merits of these strategies. We report two new non-natu-
ral amino-acid building blocks, based on a diamine and a
diacid unit, and their coupling on a solid support by using
an automated standard peptide synthesiser and established
SPPS procedures. The recently described tert-butoxycarbon-
yl (Boc)-protected building block BDS[16] (Boc-protected di-
ethylenetriamine succinic acid, 2) was modified for this
work to contain the pent-4-enamide moiety, which is highly
active towards TEC,[17] thus giving the new DDS (double-
bond-presenting diethylenetriamine succinic acid) building
block.[18] Our method also required the development of con-
ditions for efficient TEC that would reliably conjugate all of
the olefins that were spaced along the scaffold but could
also be used to prepare functionalised building blocks on a
large scale for solid-supported synthesis. We considered that
a continuous flow reactor would be ideal in this case,[19] be-
cause such systems allow precise control over the reaction
conditions. Efficient irradiation of the sample was achieved
by virtue of a sub-millimetre path length and the products
were removed as they were formed, thereby minimising un-
wanted side-reactions. These processes were simple to scale
up by driving the reactor for an extended period of time.[20]
Thus, we also report the development and testing of two
dedicated continuous flow photoreactor systems for per-
forming TEC in a flow and their application in the synthesis
of carbohydrate-functionalised PAAs.
One of our long-term goals is to apply the solid-phase
synthesis of such precision polymers[9] to the synthesis of
heteromultivalent ligands that present different sugar li-
gands at different positions along the backbone. By using a
classical polymer approach, until now, heteromultivalent sys-
tems have only been obtained in a strongly alternating,
block, or random fashion.[7] The synthesis of sequence-con-
trolled heteromultivalent structures is expected to give
more-detailed insight into the multivalent binding modes of
such artificial ligands, as well as enable the design of ligands
with not only increased affinity but also improved selectivi-
ty. The most-straightforward synthetic strategy towards such
heteromultivalent systems would be the use of glycosylated
building blocks during solid-phase synthesis. However, gly-
cosylated building blocks must be obtainable in large quan-
tities and in high quality to be suitable for chain assembly
on a solid support. The complicated purification steps that
are required to provide Cu-free material after solution-
phase conjugation prompted us to explore alternative chem-
istry to the commonly used azide–alkyne cycloaddition re-
ACHTUNGTRENNUNGaction. Thus, we sought to couple carbohydrates to PAAs by
ꢀ
using thiol ene chemistry (TEC), which would result in a
non-immunogenic linkage[11a] with a functional group that
would remain inert during solid-phase synthesis, thereby al-
lowing the introduction of carbohydrates at the building
block level or by functionalisation of the fully assembled
oligomer. TEC, that is the anti-Markovnikov addition of a
thermally or photolytically generated thiyl radical to an
olefin,[12] has been widely used in the development of chemi-
cal tools for the study of biological processes,[13] in particular
those that involve the presentation of carbohydrates. Prece-
dent for the use of TEC in the
Results and Discussion
Regardless of the approach used, building blocks for the
synthesis of carbohydrate-functionalised PAAs have to fulfil
certain criteria: They must be soluble in an appropriate cou-
pling solvent, such as DMF or N-methyl-2-pyrrolidone
(NMP), and must be accessible on a multigram scale with
synthesis of glycoconjugate vac-
cines was established by the
groups of Kunz, Dondoni, and
Davis.[11a,14] The photochemical
variant of TEC, which is widely
employed for its tolerance of
biomolecular functionalities,[15]
was chosen to demonstrate this
approach.
Therefore, we explored two
complementary strategies for
the synthesis of carbohydrate-
functionalised PAAs by using
TEC (Scheme 1): 1) Olefin-pre-
senting oligomers were pre-
pared on a solid phase and sub-
sequently conjugated with thio- Scheme 1. Complementary strategies for the preparation of carbohydrate-functionalised PAA.
Chem. Eur. J. 2013, 19, 3090 – 3098
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P. H. Seeberger, L. Hartmann et al.
high purity. Furthermore, decreasing the number of requisite
chromatographic purification steps is desirable. Thus, we
began by developing large-scale syntheses of olefin-present-
ing building block DDS (5) and spacer building block BDS
(2) from differentially protected diethylenetriamine (1),
itself prepared according to our recently published proce-
dure (Scheme 2).[16] Thus, treatment of compound 1 with
solid-phase coupling of the DDS (5) and BDS (2) building
blocks allowed a defined distribution of olefins along the
backbone, depending on the coupling sequence, to allow a
fine tuning of potential multivalent carbohydrate receptor
interactions.[5] Firstly, five DDS building blocks were assem-
bled on a solid support, thereby giving pentamer 6 in 90%
yield after TFA cleavage from the resin and precipitation
with Et2O. Furthermore, no isomerisation/transacylation was
observed, even under prolonged piperidine exposure during
the solid-phase synthesis.[21] Next, oligomer 7, which consist-
ed of five DDS building blocks, each spaced with one BDS
building block, was prepared in 90% yield and 87% purity
(by HPLC, integration of the UV signal at 214 nm). Finally
15-mer 8, which consisted of five DDS building blocks, each
spaced with two BDS building blocks, was obtained accord-
ing to the same coupling procedure in similarly high yield
and purity (see the Supporting Information).
Next, our attention turned to the functionalisation of
oligomers 6–8 by photochemical TEC. The design for the
flow reactor was based on that of Booker-Milburn[22] and
consisted of loops of transparent tubing wrapped around a
suitable light source and cooling jacket (see the Supporting
ꢀ
Information). The rate of the thiol ene reaction is wave-
length dependent and occurs more rapidly at 254 nm than at
366 nm.[23] Therefore, we envisaged that a reactor that was
constructed of a material that was spectroscopically trans-
parent at 254 nm would allow the treatment of water-soluble
Scheme 2. Synthesis of DDS and BDS building blocks for solid-phase
coupling.
4-pentenoyl chloride and TEA
gave amide 3, which contained
the required olefin functionality
for TEC. Exchange of the tri-
fluoroacetic acid (TFA) pro-
tecting group of compound 3
for an Fmoc group afforded
carbamate 4. Acidic trityl de-
protection and treatment of the
free amine intermediate with
succinic anhydride installed the
diacid spacer unit to give 25 g
of pure DDS (5) in 50% over-
all yield after final recrystallisa-
tion (>98% purity by HPLC,
integration of the UV-signal at
214 nm). No intermediate chro-
matographic purification steps
were required.
With scalable syntheses of
building blocks 2 and 5 estab-
lished, olefin-bearing PAAs
were assembled by automated
solid-phase synthesis under
O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium
hexafluorophosphate (HATU)
Scheme 3. Synthesis of sequence-defined PAAs on a solid support; purity was determined by RP-HPLC (inte-
activation (Scheme 3). The gration of the UV signal at 214 nm).
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Synthesis of Carbohydrate-Functionalised Sequence-Defined Oligo(amidoamine)s
FULL PAPER
carbohydrates, PAA scaffolds, and similar compounds with-
out the need for a radical initiator. Because fluorinated eth-
ylene polymer (FEP) tubing decomposes at this wavelength,
Teflon AF-2400,[24,25] which is transparent in the deep-UV
region, was chosen as the tubing material and was wrapped
around a quartz cooling jacket that contained a In/Hg lamp
(100 W, Figure 1). Teflon AF-2400 had not previously been
Figure 2. Plot of residence time versus conversion for the TEC of a-O-
allyl galactose with l-cysteine in water, as determined by RP-HPLC/MS
with evaporation light-scattering detection (ELSD).
below). Diminished reactivity was observed (8% conversion
by reverse-phase (RP) HPLC, tres =5 min).
Figure 1. Schematic diagram of the photoreactor for TEC: A) Syringe
pump; B) syringes that contain the thiol and olefin reactants; C) ETFE
T-union; D) PTFE inlet line (1/16“ OD, 0.3 mm ID); E) quartz cooling
jacket; F) 100 W NNIQ In/Hg lamp; G) power supply; H) cryostat filled
with ultrapure water; I) loop of Teflon AF-2400 tubing (1700 mL, 0.6 mm
ID, 0.8 mm OD); J) PEEK ”Microtight“ union; K) loop of Teflon AF-
2400 tubing (566 mL, 0.6 mm ID, 0.8 mm OD); L) PTFE outlet line (1/
16” OD, 0.3 mm ID); M) collection vessel; N) safety casing.
The reactions of O-allyl glycosides 9a–9e showed little re-
lationship between carbohydrate structure and reactivity[27]
under these conditions. The reactions were repeated at 0.1m
to afford coupling products 10a–10e with complete conver-
sion and in good yields (Table 1). NMR analysis of the
Table 1. Reaction of O-allyl glycosides 9a–9e with thiols at 254 nm.
used as a material for preparative photochemistry, yet it has
been employed as a membrane for the continuous addition
of gases in flow reactors.[24b] A temperature of 258C was
maintained for all experiments with a cryostat by using ul-
trapure water as a coolant. Separate solutions of the thiol
and olefin components were delivered by syringe pump and
mixed with a T-union. All optimisation reactions were per-
formed by using a loop of Teflon AF-2400 (566 mL), whilst a
2266 mL loop was used for scale-up. Although the reaction is
tolerant towards oxygen,[15a] the solvents were degassed
before use.
To assess the reactor prior to its use in the conjugation of
PAAs, water-soluble O-allyl glycosides 9a–9e were reacted
with l-cysteine in water to give amino acids 10a–10e.[26]
Plots of residence time versus conversion were obtained at
various concentrations for the reaction of O-allyl galactose
(9a) with l-cysteine (Figure 2).
Almost-complete conversion was observed within a resi-
dence time of 15 min, even at the lowest concentration stud-
ied. No thermal background reaction was observed at 0.1m
(tres =15 min). To assess the importance of the wavelength
(254 nm), the reaction was repeated by using a loop of FEP
tubing that was wrapped around a cooled medium-pressure
Hg lamp (400 W, lmax =366 nm) with a Pyrex filter (see
Entry
Glycoside
Thiol
Product
Yield [%]
1
2
3
4
5
6
a-Gal (9a)
b-Gal (9b)
a-Glu (9c)
b-Glu (9d)
a-Man (9e)
a-Gal (9a)
l-cysteine
l-cysteine
l-cysteine
l-cysteine
l-cysteine
10a
10b
10c
10d
10e
10 f
77[a]
70[a]
80[a]
71[a]
75[a]
74
HSCH2CH2OH
1
[a] Yield determined by H NMR spectroscopy of the crude mixture with
maleic acid as an internal standard; analytical samples were obtained as
N-Fmoc derivatives.
crude mixtures with maleic acid as an internal standard sug-
gested that some decomposition occurred under these condi-
tions. The products were highly water-soluble and, therefore,
were purified as their N-Fmoc derivatives (11a–11e) by
preparative HPLC. This process was not limited to cysteine;
2-mercaptoethanol reacted with a-O-allyl galactose 9a in
equivalent yield.
Next, continuous-flow TEC conditions were applied di-
rectly to the multiple conjugation of oligomers 6–8 with gly-
cosides 12 and 13. For initial optimisation, the reaction of 6
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P. H. Seeberger, L. Hartmann et al.
(pentamer) with b-thioglucose sodium salt (12) under acidic
conditions was studied by using the 566 mL loop (see
above). The moderate water-solubility of pentamer 6 neces-
sitated a diluting of the reaction concentration to 0.025m.
This was compensated for by use of a larger quantity of the
thiol component (2.0 equiv per olefin). Complete conjuga-
tion of all five olefin moieties was observed by HPLC-MS
within a residence time of 10 min (Figure 3). The entire re-
actor output was collected, lyophilised, and purified by pre-
pentameric structure of compound 6 was relatively unaffect-
ed (<15% oxidation by HPLC-MS), S oxidation (Fig-
ure 3m+O)[28] was significantly more pronounced during the
conjugation of larger structures 7 and 8 to give compounds
16 and 17, respectively. However, treatment of the crude
conjugate with TMSBr/HSCH2CH2SH/DMF (TMS=
trimethACHTNUTRGNEyUNG lsilyl) afforded the reduced homogenous product in
moderate yield after preparative HPLC (Table 2, PAAs 16
and 17).[29,30]
A
N
tive HPLC. HPLC-MS peaks that corresponded to the
By using the previously described conjugation approach
in combination with a 254 nm flow reactor (Scheme 1), we
observed complete functionalisation whilst consuming
2 equivalents of the thiol-bearing carbohydrate structure per
olefin. Nevertheless, this approach only allows the synthesis
of a homogenous carbohydrate sequence. To obtain hetero-
geneous carbohydrate-functionalised PAAs, we explored the
alternative approach for the synthesis of functionalised-
PAAs by using a DDS building
addition of between one and five carbohydrate moieties
were observed upon shortening the residence time
(Figure 3). Pentamer 6 reacted in a similar manner to a-
thioACHTUNGTRENNUNGethyl-mannose 13, thereby affording oligomer 15. Next,
these conditions were used for the conjugation of oligomers
7 and 8 with a-thioethyl-mannose 13. Oxidation of the sul-
fide product occurred under these conditions. Although the
block (5) that had been pre-
functionalised with protected b-
thioglucose by TEC. Large-
scale access to this carbohy-
drate-functionalised
building
block was required for solid-
phase coupling and, therefore, a
continuous-flow procedure was
sought.
Decomposition
of
DDS
building block 5 under 254 nm
irradiation, presumably owing
to the presence of the Fmoc
protecting group, necessitated a
change in the reactor design
and the use of a longer wave-
length. This effect could also be
expected for peptide sequences
that contain aromatic groups,
such tryptophan or tyrosine;
thus, for conjugation in the
presence of such peptide seg-
ments, reaction at 366 nm
would also be advisable. The
modified photoreactor consist-
ed of loops of FEP tubing
wrapped around
a Pyrex-fil-
tered, medium-pressure Hg
lamp (400 W, lmax =366 nm)
that was cooled to 258C by
using a cryostat (see the Sup-
porting Information). Continu-
ous delivery of the reagents was
enabled by replacement of the
syringe pump with a Vapourtec
R2 two-channel pump module.
TEC of DDS (5), which was
stable at 366 nm, with acetyl
protected b-thioglucose (18)[14a]
Figure 3. RP-HPLC analysis of oligomer 6 (MeCN/water, 5–95%, within 30 min) and (crude) multiple TEC in
flow with b-GlcSNa (12, MeCN/water, 5–95%, within 30 min) and a-HSEt-Man (13, MeCN/water, 5–50%,
within 30 min), thereby giving carbohydrate-functionalised PAAs 14 and 15, respectively.
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Synthesis of Carbohydrate-Functionalised Sequence-Defined Oligo(amidoamine)s
Table 2. Overview of the carbohydrate-functionalised oligo(amidoamine)s; complete
FULL PAPER
sesses a higher molecular weight than the BDS and
DDS building blocks, glycosylated building block
19 retained a high coupling efficiency. Pentamer 14
was prepared with HATU activation by using a
single coupling procedure. Deacetylation with
NaOMe/MeOH, cleavage from the resin with TFA/
triisopropylsilyl (TIS)/water, and precipitation from
the cleavage solution with Et2O afforded oligomer
14 in high yield and purity (as determined by RP-
HPLC, see the Supporting Information).
conversion was obtained in all cases.
Product PAA
Thiol component
MS (MALDI) m/z
calcd[a]
found
Yield after
RP-HPLC [%]
14
15
16
17
6
6
7
8
b-GlcSNa (12)
2356.01 [M+Na]+ 2356.13
61
45
33
30
a-HSEt-Man (13) 2576.14 [M+Na]+ 2576.26
a-HSEt-Man (13) 3544.77 [M+Na]+ 3544.62
a-HSEt-Man (13) 4448.37 [M+H]+
4447.98
[a] The most-abundant ion adduct is shown here; detailed MS (MALDI) analysis and
spectra can be found in the Experimental Section and the Supporting Information.
Evaluation of the benefits and drawbacks of
these strategies for the synthesis of carbohydrate-
was optimised by using a 2.14 mL reactor loop. The reac-
tants were poorly soluble in MeOH and in MeOH/water
mixtures, thereby limiting the reaction concentration; both
components were highly soluble in either THF or AcOH/
THF (10% v/v). AcOH increased the solubility of DDS (5)
in THF and ensured Fmoc stability during the TEC. Al-
though an increased residence time (30 min) was required
compared to experiments at 254 nm, high conversion (98%)
was obtained without the need for an additional photoinitia-
tor, by using 0.2m solutions of DDS (5) and acetyl-protected
b-thioglucose (18). Mixing of the solutions at an equal rate
gave an overall 0.1m reaction concentration. To increase
productivity, a 10 mL loop was fitted; however, a lower con-
version was observed (81%), which was attributed to
uneven irradiation of the increased area of tubing along the
functionalised PAAs must take into account the yield-limit-
ing nature of the solid-phase coupling step. Functionalisa-
tion of an olefin-presenting scaffold only requires a small
excess of the thiol-bearing carbohydrate and, therefore, is
preferable when only low quantities of the complex carbo-
hydrates are available. Homogenously glycosylated scaffolds
are exclusively obtained. Conversely, the solid-phase assem-
bly of a carbohydrate-functionalised building block allows a
defined sequence of carbohydrates to be constructed along
the PAA scaffold. Although a far greater excess of the car-
bohydrate is consumed during the solid-phase synthesis, this
approach is appropriate for simpler carbohydrates that are
available on a larger scale and, in particular, if the presenta-
tion of different carbohydrates is required.
ꢀ
arc of the lamp. Nevertheless, under these conditions, GlcS
DDS (19) was obtained in 71% yield after column chroma-
tography, thus corresponding to a throughput of the func-
tionalised building block of 29.6 g (in 24 h). Subsequently,
Conclusion
In summary, we have demonstrated two complementary
routes for the synthesis of carbohydrate-functionalised
PAAs via solid phase synthesis and TEC. Scalable syntheses
ꢀ
oligomer 14 was prepared from GlcS DDS 19 on a solid
support (Scheme 4) with HATU activation. Although it pos-
Scheme 4. Synthesis of carbohydrate-functionalised PAAs by using glycosylated building blocks for chain assembly on a solid support.
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P. H. Seeberger, L. Hartmann et al.
1H NMR spectroscopy in D2O (relaxation delay: 5 s) by using maleic acid
as an internal standard.
of olefin-presenting or carbohydrate-functionalised building
blocks were developed that provide large quantities of mate-
rial required for solid phase synthesis. An efficient continu-
ous flow photoreactor was developed for the photochemical
TEC that can be performed in a variety of solvents at either
254 or 366 nm without an additional radical initiator. After
evaluation of this system using the reaction of O-allyl glyco-
sides with thiols, PAA scaffolds of varying length, each pre-
senting five olefins were conjugated in flow at 254 nm
(Teflon AF-2400 tubing) with b-thioglucose and a-thioethyl-
mannose. Partial S-oxidation during conjugation of larger
scaffolds 7 and 8 was overcome by reductive work up of the
crude mixture. Reconfiguration of the flow reactor allowed
the scalable functionalisation of DDS building block 5 with
b-thioglucose 18 at 366 nm (FEP tubing), which was coupled
on solid phase to give carbohydrate-functionalised PAA 14.
Presented together, these techniques allow the synthesis of
monodisperse, sequence-defined carbohydrate functional-
ised PAAs with a non-immunogenic linkage between the
scaffold and the carbohydrate as well as the potential for
the solid phase assembly of heteromultivalent systems. The
choice of technique depends on UV absorptions properties
of the reactants, the amount of carbohydrate available and
the desired application. Studies into the biological applica-
tions of these functionalised sequence-defined scaffolds as
an antigen presenting platform and investigations of carbo-
hydrate-protein interactions by using variations of the car-
bohydrate functionalisation patterns are ongoing.
General procedure C: To the freeze-dried crude material from general
procedure
(0.5 mL),
B
(30 mg, 0.088 mmol, 1.0 equiv) were added acetone
(9H-fluoren-9-yl)methyl-(2,5-dioxopyrrolidin-1-yl)carbonate
(36 mg, 0.105 mmol, 1.2 equiv), and sodium bicarbonate (11 mg,
0.132 mmol, 1.5 equiv). After stirring at RT for 12 h, the reaction mixture
was taken up in water (10 mL), washed with CH2Cl2 (3ꢄ10 mL), and the
aqueous phase was concentrated by freeze-drying under reduced pres-
sure. Purification by preparative HPLC (Nucleodur C18 Pyramid,
MeCN/water/TFA, 10:90:0.1–50:50:0.1, 30 min) gave glycoamino acids 11
as white solids.
Selected procedure for the reaction of O-allyl glycosides with thiols at
254 nm:
A solution of a-O-allyl galactose 9a (100 mg, 0.454 mmol,
1.0 equiv) in water (2.27 mL) was reacted with a solution of l-cysteine
(66 mg, 0.545 mmol, 1.2 equiv) in water (2.24 mL) according to general
procedure B and concentrated by freeze-drying under reduced pressure
to give compound 10a (168 mg, 71% purity, 77% yield) as an off-white
solid. A sample of the crude mixture (30 mg) was reacted according to
general procedure C. Purification by preparative HPLC (Nucleodur C18
Pyramid column, MeCN/water 20–50%, within 30 min, retention time:
26.1 min) afforded an analytical sample of compound 11a as a white
solid. M.p. 69–718C; ½aꢁ2D0 =+64.4 (c=0.25 in MeOH); 1H NMR
(400 MHz, CD3OD): d=7.82 (d, J=7.5 Hz, 2H), 7.71 (d, J=7.5 Hz, 2H),
7.41 (t, J=7.5 Hz, 2H), 7.41 (t, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H),
4.82 (d, J=3.0 Hz, 1H), 4.42–4.33 (m, 3H), 4.27 (t, J=7.0 Hz, 1H), 3.90
(br s, 1H), 3.86–3.80 (m, 2H), 3.78–3.76 (m, 2H), 3.72 (dd, J=6.5, 3.0 Hz,
2H), 3.54 (dt, J=10.0, 6.0 Hz, 1H), 3.07 (dd, J=14.0, 4.0 Hz, 1H), 2.88
(dd, J=14.0, 8.0 Hz, 1H), 2.73 (t, J=7.5 Hz, 2H), 1.98–1.87 ppm (m,
2H); 13C NMR (100 MHz, [D6]DMSO): d=171.9, 155.3, 143.9, 143.9,
140.7, 127.6, 127.1, 125.3, 120.1, 98.8, 71.2, 69.6, 68.7, 68.5, 65.50, 65.45,
60.36, 55.0, 46.7, 34.2, 29.4, 28.3 ppm; IR (film): n˜ =3221, 2926, 1694,
1586, 1145, 1031, 972, 738 cmꢀ1; MS (ESI): m/z: 1149 [M2+Na]+, 586
[M+Na]+, 440, 179; HRMS (ESI): m/z calcd for C27H33NO10S: 586.1723
[M+Na]+; found: 586.1716.
Selected procedures for oligomer functionalisation by TEC under a con-
tinuous flow: H2N-[GlcS DDS]5-CONH2 (14): By using a photochemical
flow reactor (set up according to the Supporting Information, Figures S1
and S3) that was fitted with a loop of Teflon AF2400 tubing (566 mL), a
solution of 1-thio-b-d-glucose sodium salt hydrate (12, 81 mg, 345 mmol,
10.0 equiv) in water (900 mL) was reacted with a solution of oligomer 6
(50 mg, 34 mmol, 1.0 equiv) in water (900 mL) and AcOH (20 mL; resi-
dence time: 10 min, flow rate: 28.3 mLminꢀ1 per syringe). The reactor
output was lyophilised and purified by preparative HPLC (Waters
XBridge Prep C18, MeCN/water, 5–50%, within 30 min) to give glucose-
functionalised oligomer 14 (49 mg, 61% yield) as a hygroscopic white
Experimental Section
General procedures for the reactions of O-allyl glycosides with thiols at
254 nm: General procedure A, for the optimisation of the reaction: A pho-
toreactor was set up according to the diagram in the Supporting Informa-
tion, Figure S1, with a loop of Teflon AF-2400 polymer tubing (566 mL,
0.8 mm outer diameter (OD), 0.6 mm internal diameter (ID)) wrapped
around a quartz cooling jacket that contained a NNIQ low-pressure In/
Hg lamp. A recirculating chiller (Julabo FL-460, filled with spectroscop-
ACHTUNGTRENNUNG
1
powder. H NMR (400 MHz, D2O): d=4.52 (d, J=10.0 Hz, 5H), 3.89 (d,
J=11.5 Hz, 5H), 3.73–3.16 (m, 65H), 2.88–2.65 (m, 10H), 2.55–2.37 (m,
30H), 1.66 ppm (br s, 20H); MS (MALDI TOF): m/z calcd for
C95H168N16O40S5Na: 2356.01 [M+Na]+; found: 2356.13 (monoisotopic);
RP-HPLC: MeCN/water 5–25%, 60 min, tr =16.9 min.
A
A
ACHTUNGTRENNUNG
eluted from the reactor, an aliquot of the output solution was collected
and analysed directly by HPLC/MS to assess the fractional conversion of
the allyl glycoside (9) into glycoamino acid 10.
H2N-[ManEtS DDS-BDS]5-CONH2 (16): By using a photochemical flow
reactor (set up according to the Supporting Information, Figure S1) that
was fitted with a loop of Teflon AF2400 tubing (566 mL), a solution of
a-thioethyl-mannose (13, 33 mg, 137 mmol, 10.0 equiv) in water (300 mL)
General procedure B: A photoreactor was set up according to the dia-
gram in the Supporting Information, Figure S1, with a loop of Teflon AF-
2400 polymer tubing (2266 mL, 0.8 mm OD, 0.6 mm ID) wrapped around
a quartz cooling jacket that contained a NNIQ low-pressure In/Hg lamp.
A recirculating chiller (Julabo FL-460, filled with spectroscopically pure
water as a coolant) was used to maintain a reactor temperature of 258C.
By using a syringe pump (Harvard PHD2000), separate solutions of the
thiol (0.545 mmol, 1.2 equiv) and the allyl glycoside (100 mg, 0.454 mmol,
1.0 equiv) in degassed water (2.27 mL) were mixed through a T-mixer at
was reacted with oligomer
7 (32 mg, 14 mmol, 1.0 equiv) in water
(300 mL) and AcOH (8 mL; residence time: 10 min, flow rate:
28.3 mLminꢀ1 per syringe). The reactor output was lyophilised and dis-
solved in a solution of DMF (4 mL), TMSBr (200 mL), and ethanedithiol
(120 mL). After shaking for 35 min, the crude product was precipitated
with ice-cold Et2O (20 mL) and washed once with Et2O (20 mL). The
crude product was dissolved in water (1 mL) and purified by preparative
HPLC (Waters XBridge Prep C18, MeCN/water, 5–30%, within 30 min)
to give mannose-functionalised PAA 16 (16 mg, 33% yield) as a white,
hygroscopic powder. 1H NMR (400 MHz, D2O): d=4.91 (s, 5H), 3.98–
3.63 (m, 40H), 3.60–3.09 (m, 84H), 2.88–2.77 (m, 10H), 2.71–2.37 (m,
60H), 1.65 ppm (br s, 20H); MS (MALDI TOF): m/z calcd for
a rate of 113 mLminꢀ1 (per syringe, combined flow rate: 226 mLminꢀ1
,
residence time: 10 min) and passed into the photoreactor. After discard-
ing the first reactor volume of output, the entire product solution was
collected and freeze-dried under reduced pressure to afford the crude
products. The yields of the glycoamino acids were determined by
3096
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ꢃ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 3090 – 3098

Synthesis of Carbohydrate-Functionalised Sequence-Defined Oligo(amidoamine)s
FULL PAPER
C147H268N32O55S5Na: 3544.77 [M+Na]+; found: 3544.62 (monoisotopic);
RP-HPLC: MeCN/water 5–30%, 60 min, tr =26.7 min.
Opin. Chem. Biol. 2002, 6, 742; c) Y. M. Chabre, R. Roy, Curr. Top.
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ꢀ
Synthesis of GlcS DDS (19) referring to a 366 nm flow reactor: By using
a photochemical flow reactor (set up according to the Supporting Infor-
mation, Figures S2 and S4) that was fitted with a loop of FEP tubing
(10 mL),
a solution of acetyl-protected b-thioglucose 18 (3.45 g,
9.46 mmol, 1.5 equiv) in THF (31 mL) was allowed to react with DDS 5
(3.2 g, 6.3 mmol, 1.0 equiv) in THF (27.9 mL) and AcOH (3.1 mL; resi-
dence time: 30 min, flow rate: 167 mLminꢀ1 per syringe). The reactor
output was concentrated under reduced pressure and purified by column
chromatography on silica gel (CH2Cl2/MeOH, 15:1,+1% AcOH) to give
GlcS DDS (19, 3.9 g, 71%) as a white foam. ½aꢁ2D0 =ꢀ10.33 (c=0.25 in
ꢀ
MeOH); 1H NMR (600 MHz, [D6]DMSO): d=12.03 (br s, 1H), 7.87 (d,
J=7.5 Hz, 2H), 7.68–7.60 (m, 2H), 7.41–7.38 (m, 2H), 7.33–7.29 (m,
2H), 5.25 (dt, J=9.5, 5.0 Hz, 1H), 4.91–4.84 (m, 2H), 4.80 (dt, J=9.5,
3.5 Hz, 1H), 4.28 (dd, J=18.0, 7.0 Hz, 2H), 4.18 (t, J=6.8 Hz, 1H), 4.14–
4.08 (m, 1H), 4.01–3.91 (m, 2H), 3.32–3.05 (m, 8H), 2.66–2.53 (m, 2H),
2.42–2.37 (m, 2H), 2.30–2.22 (m, 4H), 2.00–1.90 (m, 12H), 1.52 ppm
(br s, 4H); 13C NMR (150 MHz, [D6]DMSO, mixture of rotamers): d=
173.8, 173.7, 172.1, 172.0, 171.3, 171.0, 170.0, 170.0, 169.5, 169.2, 169.0,
169.0, 156.2, 156.1, 143.9, 143.8, 140.7, 127.6, 127.0, 125.1, 125.0, 120.1,
82.0, 74.3, 73.0, 69.8, 68.2, 65.4, 65.3, 61.9, 47.1, 47.0, 46.7, 46.7, 45.3, 45.0,
38.2, 37.5, 36.6, 31.5, 31.4, 30.0, 30.0, 29.3, 29.3, 29.1, 29.0, 29.0, 28.9, 23.9,
20.5, 20.4, 20.4, 20.4, 20.3, 20.3 ppm; IR (film): n˜ =2944, 1749, 1631,
1037 cmꢀ1
; HRMS (ESI): m/z: calcd for C42H53N3O15SNa: 894.3095
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Acknowledgements
The authors thank Dr. Helmut Schlaad for his support and helpful discus-
sions and the Max Planck Society, the German Research Foundation
(DFG, Emmy Noether program HA5950/1-1), and AstraZeneca (UK)
for generous funding. Vapourtec (http://www.vapourtec.co.uk) is kindly
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Received: November 2, 2012
Published online: January 16, 2013
3098
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Chem. Eur. J. 2013, 19, 3090 – 3098