| TentaGel S : | "S" means Standard resin, applicable to a large number of applications, useful in batch and flow through systems. |
| TentaGel R : | a special suited resin for research purpose synthesis. The resin shows an increased swelling volume but is less pressure resistant. Well suited for large peptides and difficult sequences. |
| TentaGel HL: | this highloaded version of TentaGel combines a significant higher capacity with the advantages of TentaGel resins. |
| TentaGel MB: | TentaGel Macrobeads are highlighted by extraordinary large particle diameters and high capacities based on the TentaGel technology and designed for single bead synthesis and single bead analysis. |
| TentaGel N : | this resin type is designed for automated large scale oligonucleotide synthesis. |
| TentaGel J : | this resin type has been developed for polymeric immunoconjugates. |
| TentaGel M : | the microspherical shape of 10,20,30 µm of this TentaGel and it's monodispersity allows applications in automated sorters, for creating huge libraries, high speed synthesis etc. |
| TentaGel B : | describes bifunctional TentaGel resins, where the reactive sites on the outer surface of the bead is orthogonally protected to the reactive sites located in the internal volume of the bead and hybrid resins for sequentional cleavage. |
Figure 1: Chemical architecture of TentaGel resins
TentaGel resins are grafted copolymers consisting of a low crosslinked polystyrene matrix on which polyethylene glycol (PEG or POE) is grafted. As PEG is a "cameleon type" polymer with hydrophobic and hydrophilic properties, the graft copolymer shows modified physico chemical properties. There are in principle two ways to introduce PEG onto the modified polystyrene matrix. The simplest immobilization procedure is to couple PEG via one of its terminal hydroxyl groups to chloromethylated polystyrene according to the classical ether synthesis or to use other bifunctional PEG's for coupling onto the solid support. However, when long chain PEG's (>800 dalton) are used, the yields of this approach are unsatisfactory. Moreover, the homo-bifunctional PEG's react to a large extent at both ends to give cyclic polyethylene glycols and additional crosslinking which can not be controlled. Consequently the number of free functions will be reduced with concomitant lowering of the capacity of the graft copolymer. We have found that by means of anionic graft copolymerization setting up the PEG step by step directly on the matrix, PEG chains of molecular masses up to 20 kilodalton have been immobilized on functionalized crosslinked polystyrenes. Graft copolymers with PEG chains of about 2000-3000 dalton proved to be optimal in respect of kinetic rates, mobility, swelling and resin capacity. As there is no procedure to get monodisperse PEG with more than 10 ethylene oxide units by any polymerization techniques, there is theoretically no way to introduce monodisperse PEG chains with more than 10 ethylene oxide units to the resin or to get monodisperse PEG by direct polymerization onto the polystyrene backbone (monodisperse is defined as: PEG without any molecularweight distribution). Our process guarantees a very narrow molecular weight distribution of the grafted PEG spacers. These graft copolymers are pressure stable and can be used in batch processes as well as under continuous flow conditions. Figure 1 illustrates the chemical architecture of the tentacle polymer. The copolymer contains about 50 - 70 % PEG (w/w). Therefore, the properties of these polymers are highly dominated by the properties of PEG and no longer by the polystyrene matrix.
This new physico chemical behavior is especially shown by the very consistent and almost solvent-independent swelling of the graft copolymer in comparison to 1% crosslinked polystyrene.
Table 1 summarizes the swelling of TentaGel resins in comparison to polystyrene.
|
Solvent |
Water |
MeOH |
EtOH |
CH2Cl2 |
Toluene |
DMF |
MeCN |
THF |
Dioxane |
Ether |
DMSO |
|
Polystyrene 1% DVB |
-- |
1.6 |
1.7 |
8.3 |
8.5 |
5.6 |
3.2 |
8.8 |
7.8 |
4.0 |
|
|
Polystyrene 2% DVB |
-- |
1.6 |
1.7 |
6 |
5.2 |
3.2 |
1.9 |
5.6 |
5.4 |
|
|
|
TentaGel S 0.25-0.3 mmol/g |
3.6 |
3.6 |
2.9 |
6.3 |
4.8 |
4.7 |
4.2 |
5.0 |
5.4 |
1.9 |
3.9 |
|
TentaGel HL 0.4-0.6 mmol/g |
3.1 |
3.6 |
3.5 |
5.7 |
4.1 |
4.6 |
3.9 |
4.2 |
4.8 |
2.4 |
|
|
TentaGel macrobeads 0.25-0.35 mmol/g |
4.0 |
5.25 |
3.9 |
4.9 |
6.2 |
6.6 |
5.9 |
6.5 |
6.9 |
2.1 |
|
|
HypoGel 400 0.6-0.8 mmol/g |
1.8 |
2.9 |
2.8 |
6.9 |
5.5 |
5.2 |
4.6 |
5.3 |
5.6 |
2.6 |
|
Table 1: swelling volume [ml/g] of polystyrene (1% crosslinked, dry volume: 1.6 ml/g) and TentaGel resins (dry volume 1.7 ml/g).
The PEG-graft copolymer swells in all solvents which dissolve PEG and, on the other hand, swelling is negligible in solvents which do not dissolve PEG, e.g. aliphatic hydro-carbons and diethylether. This broad range of usable good swelling solvents allows the use of TentaGel resins in almost all solvent systems. Thus, after a reaction e.g. in methylene chloride, a solvent gradient can be used, e.g. from methylene chloride via tetrahydrofurane to aqueous systems, and then back from this hydrophilic system via a gradient to pure organic solvent systems, e.g. from water via tetrahydrofurane or ethanol to toluene.
The reactive sites which are located at the end of the spacer arms behave kinetically like in solution. This could be demonstrated by kinetic measurements where the coupling constants of the Boc-Gly-ONp active ester to both, low molecular mass compounds and graftcopolymer, are in the same order of magnitude. Because of the flexibility and the good solvation properties of the PEG-tentacles polymer supported reactions could be performed on the solid support under quasi homogeneous conditions.
The 13C NMR relaxation time measurements (T1) also indicate the high flexibility of the PEG-spacers and the reactive sites at the end of the spacer. The NMR signals show a very narrow linewidth which is comparable to that of small molecules. This physicochemical property of the resin allows the use of 13C NMR and MAS 1H NMR spectroscopic techniques for investigations of the resin, for analysis of resin-bound molecules and resin functionality.
Setting up a chemical library or peptide library by the "one bead one compound" approach it is essential to know the number of beads which are available within a certain amount of resin as well as the capacity of single beads. Table 2 summarizes some particle sizes and correlates them to the corresponding capacity of a single bead. The calculations are based on a typical loading of TentaGel beads which are in the range of 0.25 - 0.3 mmol/g. For analytical characterization at least 5 pmol of resin-bound peptide are needed for sequencing on a bead. In order to estimate the optimum resin quantity for the library which can be handled economically one has to take into account the bead sizes and bead capacities. In respect to homogeneity of diffusion process and kinetic rates as well as for single bead analysis and single bead quantification, all our beads show a very narrow size distribution.
|
resin |
size [µm] |
beads/g |
capacity/bead |
|
TentaGel NH2 |
750 µm |
4.62 x 103 |
65 nmol |
|
TentaGel NH2 |
500 µm |
1.5 x 104 |
19 nmol |
|
TentaGel NH2 |
300 µm |
6.4 x 104 |
4 nmol |
|
TentaGel NH2 |
200 µm |
2.15 x 105 |
1.3 nmol |
|
TentaGel NH2 |
130 µm |
8.87 x 105 |
280 - 330 pmol |
|
TentaGel NH2 |
90 µm |
2.86 x 106 |
80 - 100 pmol |
|
TentaGel M NH2 |
35 µm |
4.55 x 107 |
5.5 pmol |
|
TentaGel M NH2 |
20 µm |
2.4 x 108 |
1.0 pmol |
|
TentaGel M NH2 |
10 µm |
1.95 x 109 |
0.13 pmol |
There are several types of TentaGel resins available showing tailored properties dependent on their application:
TentaGel S resins:
the PEG spacer is attached to the polystyrene backbone via an alkyl linkage. This linkage is not sensitive to acids or bases. This type of resin is a standard type of resin used for peptide synthesis, solid phase organic synthesis or combinatorial chemistry.
TentaGel PAP resin:
the PEG is attached to the polystyrene backbone via a benzyl ether linkage. This benzyl ether linkage is sensitive to harsh acid conditions like 100 % TFA or mixtures of TFA/TMSBr.
These specially tailored resins are used for immunization procedures or for synthesizing PEG modified derivatives (PEG Attached Products). Using harsh acid conditions, the PEG spacer is cleaved together with the synthesized compound from the solid support to get soluble PEG modified compounds by applying solid phase conditions ( e.g. PEG modified peptides).
TentaGel N resins:
the PEG spacer is attached to the polystyrene backbone via a benzyl ether linkage. These tailored resins are used in oligonucleotide chemistry for small and large scale oligonucleotide synthesis. In comparison to CPG glass the capacity is increased by a factor of 10.
As TentaGel resins are copolymers composed from polystyrene and polyethylene glycol, chemical and physico chemical properties of both base polymers have to be taken in account.
PEG itself is a hygroscopic polymer. It is known from literature that PEG esters are not very stable and easily hydrolyzed. Dependent on the storage conditions and storage time, PEG itself can be oxidized along the polyether chain to form peroxides or esters. Consequently, acid treatment or treatment with bases hydrolyzes the formed PEG - esters which result in a small amount of "PEG - leakage". This leakage can be noticed by MS or NMR as PEG signals and impurities in the final product. This chemical behavior is true to all PEG´s - and PEG based polymers.
TentaGel S - Preactivated Resins
TentaGel S resins with handles
TentaGel S for Peptide Synthesis