High shear solid phase synthesis

Abstract

The present disclosure relates to solid phase synthesis of organic molecules and particularly to highly efficient methods for synthesizing polymers, such as peptides, nucleotides or saccharides, employing solid phase synthesis.

Claims

1. A method for performing at least one cycle of solid phase synthesis of an organic molecule, the method comprising the steps of: i. providing a reactor comprising a reaction chamber and a stirring apparatus, wherein the stirring apparatus comprises an impeller having at least two blades rotatable about an axis; ii. inserting beads of functionalized polymeric resin and at least one solvent into the reactor to provide a reaction mixture, wherein the reaction mixture is in contact with the rotatable blades; iii. inserting at least one reactant into the reaction chamber; iv. spinning the impeller for a period of time, at a rotational rate of at least 600 rounds per minute (rpm), while maintaining a shear rate of at least 3.Math.10.sup.3 sec.sup.−1; thereby performing at least one cycle of the solid phase synthesis of the organic molecule.

2. The method of claim 1, wherein the stirring apparatus is a mechanical stirrer and spinning of the impeller is performed at a rotational rate of 600 to 1400 rounds per minute, maintaining a shear rate of at least 3.Math.10.sup.3 sec.sup.−1.

3. The method of claim 2, wherein the spinning of the impeller comprises maintaining shear stress of at least 1.5 N/m.sup.2 in the reaction mixture.

4. The method of claim 1, wherein the stirring apparatus is a homogenizer and spinning of the impeller is performed at a rotational rate of 5,000-30,000 rounds per minutes, maintaining a shear rate of at least 1.Math.10.sup.6 sec.sup.−1.

5. The method of claim 4, wherein the homogenizer is a rotor-stator homogenizer.

6. The method of claim 1, wherein the functionalized beads of polymeric resin have particle size of 20-200 μm.

7. The method of claim 1, wherein the organic molecule is a polymer comprising a molecule selected from the group consisting of a peptide chain, a nucleotide chain and a sugar.

8. The method of claim 1, wherein the organic molecule comprises a peptide chain.

9. The method of claim 7, for performing a cycle in the solid phase synthesis of a polymeric organic molecule, wherein the method comprises the steps of: (a) providing a reactor comprising a reaction chamber and a stirring apparatus comprising an impeller having at least two blades rotatable about an axis; (b) inserting beads of functionalized polymeric resin and at least one solvent into the reactor to provide a reaction mixture, wherein the reaction mixture is in contact with the rotatable blades; (c) inserting at least one protected monomeric organic molecule and at least one coupling agent into the reaction chamber and spinning the impeller, thereby forming a coupling product of the protected monomeric organic molecule and the resin; (d) washing excess of the protected monomeric organic molecule; and (e) inserting at least one deprotecting reagent into the reaction chamber and spinning the impeller, thereby removing at least one protecting group from the coupling product, forming a coupling product of the deprotected monomeric organic molecule and the resin, thereby completing a cycle in the solid phase synthesis of a polymeric organic molecule; wherein the spinning of the impeller in at least one of steps (c) and (e) is performed for a period of time, at a rotational rate of at least 600 rounds per minute, while maintaining a shear rate of at least 3.Math.10.sup.3 sec.sup.−1, optionally wherein steps (c) to (e) are repeated a plurality of cycles.

10. The method of claim 9, wherein both steps (c) and (e) are performed for a period of time, at a rotational rate of at least 600 rounds per minute, while maintaining a shear rate of at least 3.Math.10.sup.3 sec.sup.−1.

11. The method of claim 7, wherein the at least one reactant is selected from the group consisting of: a deprotection agent, a coupling agent, and a protected monomeric organic molecule.

12. The method of claim 7, wherein the at least one reactant comprises a coupling agent and a protected monomeric organic molecule, thereby performing at least one coupling cycle of the solid phase synthesis of the organic molecule.

13. The method of claim 12, wherein the protected monomeric organic molecule is an α-N-protected amino acid.

14. The method of claim 7, wherein the reactant comprises at least one deprotecting reagent.

15. The method of claim 1, further comprising the steps of washing the reaction mixture and filtering the beads of polymeric resin.

16. The method of claim 1, further comprising repeating steps (ii) to (iv) at least one more time thereby performing at least one additional cycle of the solid phase synthesis of the organic molecule.

17. The method of claim 16, comprising at least two steps of coupling of an amino acid to the resin and at least two steps of removal of protecting group.

18. The method of claim 7, further comprising the step of cleavage of the polymeric organic molecule from the polymeric resin.

19. The method of claim 1, comprising an initial step of swelling the beads of polymeric resin in at least one solvent.

20. The method of claim 19, wherein swelling the beads of polymeric resin comprises mixing the beads of polymeric resin for a specified period of time in the solvent, at a rotational rate of at least 600 rounds per minute and maintaining shear rate of at least 3.Math.10.sup.3 sec.sup.−1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Examples illustrative of embodiments are described below with reference to figures attached hereto. In the figures, identical structures, elements or parts that appear in more than one figure are generally labeled with a same numeral in all the figures in which they appear. Alternatively, elements or parts that appear in more than one figure may be labeled with different numerals in the different figures in which they appear. Dimensions of components and features shown in the figures are generally chosen for convenience and clarity of presentation and are not necessarily shown in scale. The figures are listed below.

(2) FIG. 1A is a photograph of an High Sheer SPS (HS-SPS) apparatus according to some embodiments.

(3) FIG. 1B is a close-up photograph of an HS-SPS apparatus according to some embodiments.

(4) FIG. 2 is a photograph of a magnetic stirrer apparatus.

(5) FIG. 3 is a photograph of a homogenizer stirrer apparatus (Apparatus 3).

(6) FIG. 4A is a photograph of TentaGel resin beads before being rotated in water in HS-SPS Apparatus 1—a stirrer consisting of a small impeller with three 3.8 cm long blades spinning upwards at an angle of 32.2°.

(7) FIG. 4B is a close-up photograph of TentaGel resin beads after 30 minutes of being rotated in water in HS-SPS Apparatus 1.

(8) FIG. 4C is a photograph of TentaGel resin beads after 30 minutes of being rotated in water in HS-SPS Apparatus 1.

(9) FIG. 5A is a photograph of TentaGel resin beads before being rotated in water in HS-SPS Apparatus 1.

(10) FIG. 5B is a photograph of TentaGel resin beads after 42 seconds of being rotated in water in HS-SPS Apparatus 1.

(11) FIG. 5C is a photograph of TentaGel resin beads after 1.1 minutes of being rotated in water in HS-SPS Apparatus 1.

(12) FIG. 5D is a photograph of TentaGel resin beads after two minutes of being rotated in water in HS-SPS Apparatus 1.

(13) FIG. 5E is a photograph of TentaGel resin beads after three minutes of being rotated in water in HS-SPS Apparatus 1.

(14) FIG. 5F is a photograph of TentaGel resin beads after four hours of being rotated in water in HS-SPS Apparatus 1.

(15) FIG. 6A is a photograph of TentaGel resin beads before being rotated in water in a magnetic stirrer.

(16) FIG. 6B is a photograph of TentaGel resin beads after two hours of being rotated in water in a magnetic stirrer.

(17) FIG. 6C is a photograph of TentaGel resin beads after 18 hours of being rotated in water in a magnetic stirrer.

(18) FIG. 7A is a photograph of TentaGel resin beads before being rotated in NMP in HS-SPS Apparatus 2—A BOLA-mini impeller stirrer with three 5 cm long blades (h=35 cm spinning downwards at an angle of 41.1°.

(19) FIG. 7B is a photograph of TentaGel resin beads after 24 hours of being rotated in NMP in HS-SPS Apparatus 2.

(20) FIG. 8A is a photograph of TentaGel resin beads without solvent.

(21) FIG. 8B is a photograph of TentaGel resin beads immediately after contact with NMP.

(22) FIG. 8C is a photograph of TentaGel resin beads after 30 minutes of being rotated in NMP in HS-SPS Apparatus 2.

(23) FIG. 9A is a photograph of TentaGel resin beads before being rotated in NMP in HS-SPS Apparatus 2.

(24) FIG. 9B is a photograph of TentaGel resin beads after 30 seconds of being rotated in NMP in HS-SPS Apparatus 2.

(25) FIG. 9C is a photograph of TentaGel resin beads after one minute of being rotated in NMP in HS-SPS Apparatus 2.

(26) FIG. 9D is a photograph of TentaGel resin beads after 1.3 minutes of being rotated in NMP in HS-SPS Apparatus 2.

(27) FIG. 9E is a photograph of TentaGel resin beads after two minutes of being rotated in NMP in HS-SPS Apparatus 2.

(28) FIG. 9F is a photograph of TentaGel resin beads after 2.3 minutes of being rotated in NMP in HS-SPS Apparatus 2.

(29) FIG. 9G is a photograph of TentaGel resin beads after three minutes of being rotated in NMP in HS-SPS Apparatus 2.

(30) FIG. 9H is a photograph of TentaGel resin beads after four minutes of being rotated in NMP in HS-SPS Apparatus 2.

(31) FIG. 10 is a graph showing the % progression of the Fmoc cleavage for a mechanical stirrer apparatus employed at 700 rpm with 5% piperidine (full circles); a shaker apparatus with 5% piperidine (empty triangles); a shaker apparatus with 20% piperidine solution (full triangles); a mechanical stirrer apparatus employed at 100 rpm with 5% piperidine (empty circles); and an immobilized reactor apparatus without mixing, with 5% piperidine (empty squares). The error is the standard deviation between three independent experiments conducted.

DETAILED DESCRIPTION OF THE INVENTION

(32) In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

(33) The methods of the present invention are suitable of synthesis of any organic molecule which can be synthesized on a solid support. This includes both polymeric molecules such as peptides and polynucleotides. The methods are particularly suitable for molecules produced using multiple synthesis steps and requiring orthogonal protection of reactive groups.

(34) The methods of the present invention provide at least one improvement in synthesis parameters, including but not limited to synthesis time, synthesis yield, reduction of side product formation, and reduction of racemization rate.

(35) Without wishing to be bound to any mechanism of action, these improvements may be due to applying high shear force during synthesis steps, elimination of accumulation of reactants or intermediates in the reaction mixture, and maintaining of reaction mixtures having improved homogeneity.

(36) Some of the mixing apparatuses that may be employed according to the present invention were not previously used or disclosed for organic synthesis. These include for example homogenizers (rotor-stator and other type of homogenizers), that are commonly used in biology for distraction of tissues, for example.

(37) A rotor-stator homogenizer employs a high speed, tightly fitted rotor inside a toothed stator. The samples to be homogenized are drawn into the center of the rotor having been mixed, accelerated and pressed through the narrow gap between the rotor and stator.

(38) Basic understanding of hydrodynamic energies, as well as shear and compressive stress and forces applied to the reaction media could be critical to the success of scale up synthesis processes. Nevertheless, up till now very few investigation were made on the influence of the hydrodynamic parameters on the yield and side reactions in SPS. Some important hydrodynamic parameters include ‘shear rate’, ‘shear stress’, and ‘shear force’.

(39) The term ‘shear rate’, measured in inverse seconds (SI unit) refers to rate at which a progressive shearing deformation is applied to a material. As used herein, shear rate refers to the rate at which the deformation is applied to the polymeric resin beads within the reaction mixture while being mixed. Generally, the shear rate for a fluid flowing between two parallel plates, one moving at a constant speed and the other one stationary is defined by:
{dot over (γ)}=v/h′
wherein γ is the shear rate; v is the velocity of the moving plate (in sec.sup.−1); and h is the distance between the two parallel plates. For the simple shear case, it is just a gradient of velocity in a flowing material.

(40) In stirred tanks, the following correlations were derived [Perez et al. Chem Eng. J. 124, 2006, 1; and Metzner and Otto AIChE Journal, 3, 1957, 3]:

(41) $\gamma =\frac{P}{\tau .Math.V}\gamma =k_{i}N$
wherein k.sub.i is an impeller constant; N is the agitation speed, (i.e. the rotational speed of the impeller) measured in sec.sup.−1; τ is the shear stress measured in pascal (newton per square meter); P is the power input (in Watts), which depends on the torque of the impeller and on its rotational speed (N); and V is the volume of the fluid in the tank.

(42) The term ‘shear stress’, measured in inverse seconds refers to a component of stress coplanar with a material cross section. As used herein, shear stress refers to the component of stress applied on the polymeric beads, which is coplanar with their cross section. Generally, shear stress arises from the force vector component parallel to the cross section.

(43) Being a measure of stress, shear stress is measured in force per unit area (in SI units: N/m.sup.2)
τ=F/A′
wherein τ is the shear stress; F is the force applied; and A is the cross-sectional area of material with area parallel to the applied force vector (i.e. cross-sectional area of the beads).

(44) With reference to other hydrodynamic parameters, shear stress can also be derived from shear rate by: τ={dot over (γ)}μ where μ is the dynamic viscosity of the fluid.

(45) The term ‘viscosity’ refers to a hydrodynamic property of a fluid depicting its measure of resistance to gradual deformation by shear stress or tensile stress. Viscosity arises from collisions between neighboring particles in the fluid that are moving at different velocities. As used herein, viscosity refers to the viscosity of the reaction mixture comprising the solvent, the beads and other added reagents.

(46) The term ‘shear force’ refers to a force acting in a direction parallel to a surface or to a planar cross section of a body. As used herein, shear force refers to the force acting in a direction parallel to a surface or to a planar cross section of the polymeric beads while being mixed. Shear force, Fs, can be derived from shear stress as it consists of the integrated shear stress (τ) over the surface area (A) of a body.
Fs=∫.sub.AτdA

(47) Being heterogeneous reactions, (i.e. where the protected amino acids are in solution, whereas the resin is not) the reactions performed in the SPS cycle are diffusion controlled reactions highly affected by stirring or agitation. As described above gentle mixing methods are routinely employed in the large scale production of peptides (e.g. vortex, nitrogen stream, rotary evaporator rotor, agitation by rocking), such that a low shear stress over the polymeric beads is maintained thereby avoiding damage thereof. In contrast with the gentle mixing approach in solid phase synthesis, mixing methods used in the production of small molecules in solution are frequently performed in high rpm.

(48) The influence of the typically employed gentle mixing in SPS is not well documented and characterized. There are two main parameters that could be critical in the steps of the SPS cycle (i.e. coupling of protected building block; deprotection and washing). First, compressive and shear stress applied to the resin beads could cause them to break and as a consequence the isolation process will be tedious due to slow filtration and possible blockage of the filter. Second, the rates of the coupling and the deprotection reactions and the washing steps might be influenced by breaking up of the beads. Moreover, a secondary parameter stem from the interaction between the solid phase (resin beads) and the liquid phase. The better distribution of the resin beads, caused by circular flow rate of the beads in the media, could increase the mass transfer between the liquid bulk to the solid surface and thus accelerate the reaction. In specific reactions, it could decrease the generation of impurities and improve the impurity profile of the final product.

(49) While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced be interpreted to include all such modifications, additions and sub-combinations as are within their true spirit and scope.

EXAMPLES

Example 1—Resin Beads Stability Check

(50) Equipment and Configuration

(51) In order to characterize and optimize the process for industrial application, the influence of the equipment operation and geometry, namely the hydrodynamics of the system, on the process was estimated. The study evaluated the stability of polymeric resin beads consequent to mixing, and compared three mixing methods: (i) A mixing method including an HS-SPS apparatus consistent with some embodiments of the invention (FIGS. 1A and 1B). (ii) A mixing method using a magnetic stirrer (FIG. 2). (iii) A mixing method using a homogenizer (FIG. 3).

(52) The HS-SPS apparatus consisted of a reactor including a 250 mL beaker flask (6.8 cm diameter and 10 cm height) equipped with a mechanical stirrer and a high shear stress equipment. The reactor also included 4 baffles of 0.6 cm thickness each. Two mechanical stirrers were examined: 1. A stirrer consisting of a small impeller with three 3.8 cm long blades spinning upwards at an angle of 32.2°. The apparatus comprising this stirrer is referred as the ‘HS-SPS Apparatus 1’ or ‘Apparatus 1’ hereinafter. 2. A BOLA-mini impeller stirrer with three 5 cm long blades and impeller height of 35 cm, spinning downwards at an angle of 41.1°. The apparatus comprising this stirrer is referred as the ‘HS-SPS Apparatus 2’ or ‘Apparatus 2’ hereinafter.

(53) This kind of equipment, in different configurations, is widely used in the fine chemical and Active Pharmaceutical Ingredient (API) industry at all volume levels, although similar apparatuses have been considered unfitting for processes employing solid phase synthesis in general and solid phase peptide synthesis in particular.

(54) The magnetic stirrer apparatus consisted of a 250 ml round bottom flask and an oval or octagonal shaped magnetic stirrer. The magnetic stirrer's geometrics:

(55) Length=25 mm;

(56) Width=10 mm;

(57) Perimeter=43 mm;

(58) Diameter=12 mm;

(59) Weight=6.6189 g;

(60) Distance between parallel stripes=5 mm.

(61) The homogenizer apparatus of FIG. 3 (referred as the ‘HS-SPS Apparatus 3’ or ‘Apparatus 3’) consisted of a 250 mL beaker flask (6.8 cm diameter and 10 cm height) equipped with a Polytron PT 6100,PT-DA3020/ZEC homogenizer including geometrics as follows:

(62) Homogenizer diameter=2 cm;

(63) Round window diameter=1 cm;

(64) Number of teeth=14;

(65) Number of windows=14;

(66) Height of window (from bottom)=0.3 cm;

(67) Width of window=0.2 cm

(68) Z(window)=0.8 cm

(69) Two types of PS-DVB (polystyrene-divinylbenzene) based resins were used: (i) a traditional PS-1% DVB (a polystyrene consisting of 1% divinylbenzene monomers) chloromethylated resin (Merrifield resin); (ii) Rink Amide Tentagel resin (a modified PS-1% DVB polymer grafted with ethylene oxide monomers, including a Rink Amide linker). Specifically, this resin was ‘TentaGel HL RAM, 12 023’ purchased from RAPP POLYMER having a capacity of 0.4 mmol/g and particle size of 75 μm.

(70) Mechanical stabilities of the resins were tested in both directions. Shear stress was applied to a mixture of the two resins in N-Methylpyrrolidone (NMP) and water solvents, for different time periods. The concentration of beads in the solvents were set to 10% w/w, 12.5 g beads in 125 g of solvent.

(71) The stability of the resin beads was checked under a high power microscope to reveal any damage. The microscope that was used to view and measure the beads is a Zeiss scope A.1 AxioCam iCc3 microscope.

(72) Results

(73) First, the HS-SPS Apparatus 1 was examined with water as solvent and the TentaGel resin. Spinning the impeller at 1450 rpm resulted in a turbid solution due to air entrance to the vessel. Only at about 600-700 rpm, the solution didn't appear turbid and no vortex was created so the maximal impeller rotation speed was set at that speed.

(74) The beads were rotated in the mechanic stirrer of the HS-SPS Apparatus 1, in water for 24 hours at room temperature. Samples were taken every 30 minutes for the first 8 hours, and a final sample was taken after 24 hours. As can be seen in FIG. 4 the diameter of the beads remained substantially unchanged. Thus, in order to test the beads' swelling, the kinetics was tested under the microscope, without any stirring, at small time intervals.

(75) As seen in FIGS. 5A-F, the beads remained in a flawless round shape, before and after being stirred for 24 hours in the HS-SPS Apparatus 1. Moreover, the swelling of the beads took place as expected (Table 1).

(76) TABLE-US-00001 TABLE 1 Swelling of TentaGel beads in water hours in HS-SPS Apparatus 1 Time (min) Beads' average diameter (μm) 0 95.45 42 sec 102.12 1.1 106.74 2 107.08 3 106.77 4 107.74

(77) In the next stage of the experiment, after the stirring of the beads in water for 24 hours, their durability towards stirring under high shear stress was tested in the homogenizer apparatus. In the experiment, half of the initial solution used in the HS-SPS Apparatus 1 experiment was stirred for five more minutes at 23,000 rpm in the homogenizer apparatus, resulting in only few of the beads damaged in shape.

(78) Next, the durability of the beads, which were stirred by the HS-SPS Apparatus 1 and the homogenizer apparatus, was examined using the magnetic stirrer apparatus. After 24 hours and after one week of stirring, a sample was taken and observed under the microscope.

(79) The procedure above conducted with TentaGel beads in water with HS-SPS Apparatus 1, was repeated with TentaGel beads in NMP with HS-SPS Apparatus 2; and with polystyrene beads in NMP HS-SPS Apparatus 2 (polystyrene in water is irrelevant, due to this polymer poor swelling in aqueous conditions). In all cases, the shear stress was measured using a dynamometer (Dynamometer FH 10 from PCE instruments). Table 2 summarizes the stabilities of the different beads corresponding to the stirring time, the apparatus and stresses employed.

(80) TABLE-US-00002 TABLE 2 Sensitivity of various resins toward shear stress and operation rate. Stability Mechanical Stirrer Magnetic Stirrer Homogenizer Water.sup.1 NMP.sup.2 Water NMP Water NMP TentaGel 24 h Stable Not stable After 15 min 5 min Stable above 24 h After 2-3 2 hour Stable Stable Days not stable Merrifield Not 1 week Not After Not 5 min resin relevant* Stable relevant* 2 hour relevant* Stable. no stable Compressive Not Not 15.6 N/m.sup.2 21.1 N/m.sup.2 Not Not stress relevant* relevant* relevant* relevant* Shear 1.84 N/m.sup.2 2.4 N/m.sup.2   8 N/m.sup.2   11 N/m.sup.2 1540 N/m.sup.2 2042 N/m.sup.2 stress .sup.1With HS-SPS Apparatus 1; .sup.2With HS-SPS Apparatus 2.

(81) Table 2 and FIGS. 7A-7B reveal that, surprisingly, even at high shear rate operations with the HS-SPS Apparatus or with the homogenizer apparatus, the resin beads were not damaged. The only configuration that damaged the resin beads was the employment of the magnetic stirrer apparatus. FIGS. 6A-C shows the structural difference of the resin before and after stirring in the magnetic stirrer apparatus. It is apparent that the beads are significantly damage in structure after 2 hours of stirring in the magnetic stirrer apparatus (FIG. 6B), whereas after 18 hours in the same conditions a complete smearing of the beads is observed, which practically led to their disappearance (FIG. 6C). Unexpectedly, these finding were in complete contrast to the results under similar conditions with HS-SPS Apparatus 1 and HS-SPS Apparatus 2. As shown in Table 2, both TentaGel beads and Merrifield resin beads remained stable over 24 hours of high shear rate stirring in the different solvents. For example, stirring Tentagel beads with NMP solvent in HS-SPS Apparatus 2, did not have visible effect on the structure of the beads, as can be inferred upon comparison of microscope inspections before (FIG. 7A) and after (FIG. 7B) stirring in the in HS-SPS Apparatus 2.

(82) Without wishing to be bound by any theory or mechanism, the unexpected variance between magnetic and mechanical stirring stems from the different levels of compressive stress applied by each apparatus. The HS-SPS Apparatus is applying mainly shear stress to the media at different levels. Similarly, the magnetic stirring apparatus is applying the same kind of shear stress. However, because the magnetic bar agitator is located at the bottom of the flask, significant amounts of compressive stress are also applied. The direction of the stress tension may be the main reason for the destruction of the resin in magnetic stirred vessels.

Example 2—Resin Beads Swelling Examination

(83) Swelling of the dry resin beads is performed in the initial stages of solid phase peptide synthesis processes. NMP, DMF (dimethylformamide) and DCM (dichloromethane) are considered good swelling solvents for polystyrene-divinylbenzene based resins and are routinely used in SPSS processes, although TentaGel resins are known to also swell to some extent in water. The swelling is a direct function of the interaction and compatibility of the solvent properties with the resin and the operation conditions in the equipment. In the frequently used shaker reaction vessels the swelling step is typically complete within one to two hours.

(84) Swelling kinetic of the polymeric beads was tested in an agitated tank, by adding the beads to a solvent in the tank and stirring at 600-700 rpm for two hours. Much like in the stability evaluation experiments, swelling kinetics of TentaGel bead were evaluated in water with HS-SPS Apparatus 1, and in NMP solvent with HS-SPS Apparatus 2, whereas Merrifield resin beads bead were evaluated only in NMP with HS-SPS Apparatus 2.

(85) Samples were taken every 30 seconds and examined under the microscope. For each sample, the mean diameter of the beads was measured by the microscope's software. Since the beads swelled at a short time in NMP it was decided to test also swelling without agitation under the microscope in small time intervals to observe the effect of the solvent itself in real time. A photograph was taken every 30 seconds for the first two minutes and then every minute for the remaining two minutes. This procedure was conducted for the same samples mentioned above. Diameter growing of the beads was measured under two types of operations at different times: (1) directly under the microscope by adding resin to a solvent film without agitation, and (2) in the mechanically stirred device with high shear stress equipment. The results are shown in Table 3.

(86) TABLE-US-00003 TABLE 3 Swelling kinetics of two resins. Solvent Water NMP Resin No agitation With agitation No agitation With agitation Tentagel 0 min 105 μm  1 min 106 μm 0.5 min   150 μm  1 min 4 min 107 μm 10 min 4 min 150 μm 10 min 30 min 120 μm 30 min 300 μm Merrifield NR NR 0.5 min  50 μm resin  2 hour  90 μm  2 hour 140 μm NR—not relevant

(87) It was found that immediately upon contact of both two resins with NMP, they swells to about half of their maximal extent. When immersed in NMP, Tentagel swells to a diameter of about 150 μm within few seconds, whereas upon stirring in HS-SPS Apparatus 2, it swells to 300 μm diameter within 30 minutes. FIG. 8 shows the starting beads before contact with NMP (FIG. 8A); the same beads immediately after contact with NMP (FIG. 8B); and the beads after 30 minutes of mechanical stirring in NMP in HS-SPS Apparatus 2 (FIG. 8C). No damage to the resins was observed in this study as can be further concluded from FIGS. 9A-H. The beads' average diameter as function of time increased as expected (Table 4).

(88) TABLE-US-00004 TABLE 4 Swelling of TenraGel resin in NMP with HS-SPS Apparatus. Time Beads' average diameter (min) (μm) 0 104.51 0.5 157.19 1 156.81 1.3 154.79 2 152.39 2.3 153.37 3 151.04 4 152.83 30 300 μm

(89) From the results above it is concluded that short time swelling can be employed to the resin, thereby reducing the total time of synthesis.

Example 3—Coupling and Racemization Examination

(90) As mentioned, peptide synthesis is usually conducted in the solid phase by employing a shaker apparatus. In order to demonstrate the feasibility of the High Shear Solid Phase Peptide Synthesis (HS-SPPS) method, a tripeptide synthesis was used as a model in which the two methods were compared. The selected transformation was a coupling reaction of an amino-protected amino acid to a dipeptide connected to a resin, thus forming a resin comprising a tripeptide. Specifically, the model peptide to be synthesized was Fmoc-L-His-Phe-Gly-NH.sub.2. Therefore, the model reaction consisted of coupling the free amine residue of a resin comprising a glycine (Gly) and phenylalanine (Phe) units (H.sub.2N-Phe-Gly-Resin) with the carboxyl residue of a histidine unit protected with trityl (triphenylmethyl-Ph.sub.3C) on its heterocyclic nitrogen and with Fmoc (Fluorenylmethyloxycarbonyl) on its primary amine (Fmoc-His(Trt)OH; N.sub.α—Fmoc-N.sub.(im)-trityl-L-histidine; CAS NO 109425-51-6). It is noted that the coupling of Fmoc-His(Trt)OH to H.sub.2N-Phe-Gly-Resin, using coupling agents, such as DIPC (N,N′-diisopropyl carbodiimide), DCC (N,N′-dicyclohexylcarbodiimide) and EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) without additives or base, is known to cause racemization and leads to reduced yield of the tripeptide. In other words, the coupling of Fmoc-L-His(Trt)OH is considered challenging mainly due to enhanced formation of the diastereomeric peptide side product containing D-Histidine.

(91) In order to monitor the rate of the coupling reaction and the degree of racemization, the two pure diastreomeric peptides (i.e. Fmoc-L-His-Phe-Gly-NH.sub.2 and Fmoc-D-His-Phe-Gly-NH.sub.2), were separately prepared using known coupling methods. The two pure peptides were examined in HPLC. The examination revealed two separate peaks, which also don't overlap with the HPLC signals of the starting materials, thus allowing the follow-up of both conversion and racemization of the tripeptide product at the same time.

(92) In the experiment, the coupling of Fmoc-His(Trt)OH to H.sub.2N-Phe-Gly-Resin was tested in the presence of EDC in NMP solvent (without base or additives) using (i) a traditional shaker apparatus, and (ii) the HS-SPS Apparatus 2. Thereafter, aliquots were taken for “small cleavage” (as described in Falb et al. The Journal of Peptide Research (1999) 53, 507-517) of the product from the resin and samples were examined for degrees of conversion and racemization using HPLC. The results are summarized in Table 5.

(93) TABLE-US-00005 TABLE 5 Degrees of conversion and racemization in the coupling of Fmoc-His(Trt)OH to H.sub.2N-Phe-Gly-Resin, without additives, using shaker and HS-SPPS conditions. Reaction Mixing Addition Time % % No. system of EDC [minutes] conversion Disomer additives 1 (i) HS-SPS gradual.sup.1 30 39.4 5.2 — Apparatus 2 1 (ii) HS-SPS gradual.sup.1 90 70.6 8.5 — Apparatus 2 2 shaker at once.sup.2 90 0.00 — — 3 shaker at once.sup.3 90 61.0 44.0  — .sup.1EDC was added gradually by micro syringe. .sup.2The coupling did not progress and a symmetrical histidine anhydride was formed. .sup.3Addition of histidine anhydride at once to the resin suspension.

(94) From Table 5 it is evident that Reaction 2, in which EDC was added to a mixture of His(Trt)OH and H.sub.2N-Phe-Gly-Resin in NMP, did not progress to provide any of the target tripeptide within 90 minutes.

(95) Due to the fact that product was not forming in Reaction 2, the symmetrical protected histidine anhydride was prepared separately and was added to a suspension of the H.sub.2N-Phe-Gly-Resin in NMP (Reaction 3). This method allowed 67% conversion after 1.5 hours of reaction, albeit with almost a half of the product being the undesired D diastereomer racemization product.

(96) Surprisingly, under similar reaction conditions of mixing the reactants with EDC in NMP, stirring in an HS-SPS Apparatus, instead of shaking, provided 70% conversion with only 8.5% of the D diastreomer (Reaction 1). Without wishing to be bound to any mechanism, it is suggested that gradual addition of the EDC, which is possible in the method of the present invention, but not in the other method, resulted in more efficient and faster coupling reaction and reduced racemization and other side reactions. This possibly due to elimination of accumulation of reactants or intermediates in the reaction mixture. The fact that in both under HS-SPS conditions and the shaker the conversion was the same (58.4%) and it did not reach 100% reflects the known fact that the reaction of the active ester 4 with the peptide-resin is diffusion controlled and therefore cannot be accelerated by stirring.

(97) A similar experiment was conducted with the addition of HOBt (hydroxybenzotriazole) as an additive using HS-SPS Apparatus 2 (Reaction 4) and using a shaker (Reaction 5). The reactions were monitored and the results are provided in Table 6.

(98) TABLE-US-00006 TABLE 6 Degrees of conversion and racemization in the coupling of Fmoc- His(Trt)OH to H.sub.2N-Phe-Gly-Resin, with additives, using shaker and HS-SPPS conditions. Reaction Mixing Time % D Conversion No. system [minutes] isomer % Yield % 4 (i) HS-SPS 0 0 0.0 0.0 Apparatus 2.sup.1 4 (ii) HS-SPS 5 0 0.0 0.0 Apparatus 2.sup.1 4 (iii) HS-SPS 15 0 12.4 12.4 Apparatus 2.sup.1 4 (iv) HS-SPS 30 0 37.5 37.5 Apparatus 2.sup.1 4 (v) HS-SPS 45 0 53.0 53.0 Apparatus 2.sup.1 4 (vi) HS-SPS 60 0 58.4 58.4 Apparatus 2.sup.1 5 Shaker.sup.2 , 60 min 5.5 55.3 52.2 .sup.1DIC was added gradually for 5 minutes by micro syringe. .sup.2The DIC added at once and isomer with D-Histidine formed.

(99) From Table 6 it is evident that Reaction 6, in which EDC and HOBt were added to a mixture of His(Trt)OH and H.sub.2N-Phe-Gly-Resin in NMP in a shaker, proceeded to form the product within 90 minutes, but with 5.5% racemization.

(100) Surprisingly, under similar reaction conditions of mixing the reactants with EDC an HOBtin NMP, stirring in an HS-SPS Apparatus, instead of shaking, provided 60% conversion with no D diastreomer witnessed (Reaction 4).

Example 4—Cleavage of Protecting Group

(101) To evaluate the effect of the mixing method on reaction rates occurring on the solid support, cleavage reactions of the primary amine protecting group FMOC were monitored. Fmoc removal typically takes place multiple times during peptide elongation in Fmoc based SPPS and is performed with large amounts of piperidine (20% v/v) in DMF or in NMP. Piperidine has been associated with acute and chronic health effects including eye and skin irritations and damage to mucous membranes. Thus, huge toxic piperidine waste is being produced both in research and in industrial facilities.

(102) Accordingly, Fmoc cleavage progression was monitored under different conditions by spectrophotometrically quantifying the amount of dibenzofulvene liberated. The rate of Fmoc removal from Fmoc Rink amide resin was determined using 5%-20% piperidine solution in NMP in (i) a regular shaker, (ii) a mechanical stirrer at 100 RPM; (iii) a mechanical stirrer at 700 RPM; and (iv) an immobilized reactor. The amount of dibenzofulvene was quantified and normalized compared to the Fmoc content of the resin measured by an Fmoc quantification test.

(103) FIG. 10 is a graph showing the % progression of the Fmoc cleavage (as determined by dibenzofulvene formation) vs. time, for the following apparatus configurations, and amount of piperidine: mechanical stirrer apparatus employed at 700 rpm with 5% piperidine (full circles); shaker apparatus with 5% piperidine (empty triangles); shaker apparatus with 20% piperidine solution (full triangles); mechanical stirrer apparatus employed at 100 rpm with 5% piperidine (empty circles); immobilized reactor apparatus without mixing, with 5% piperidine (empty squares). The error is the standard deviation between three independent experiments conducted.

(104) As seen in FIG. 10, the UV measurements for formation of dibenzofulvene showed that 95% Fmoc removal was achieved in 20% piperidine/NMP after 10 min reaction in a shaker (full triangles), but only 60% cleavage was obtained after 10 minutes with 5% piperidine/NMP using the same apparatus (empty triangles). Strikingly, the cleavage profiles achieved with 5% piperidine/NMP with two separate mechanical stirring apparatuses (700—full circles; and 100 rpm—empty circles) were almost identical to that achieved with 20% piperidine/NMP in the shaker with both high and low rpm rates of mechanical stirring. As a control, Fmoc removal in immobilized reactor with 5% piperidine/NMP was performed. The amount of Fmoc removed was only 20% after 20 min of reaction, which is significantly lower than the amount removed with overhead stirring (empty squares). Without wishing to be bound by any theory or mechanism of action, it is assumed that the permeation of reagents inside the solid support depends on the mixing properties of the solution. The experiment proved that the reaction rate with a mechanical stirrer using the 5% solution is much faster than using the same solution with a shaker. It is suggested that the higher diffusion rate achieved by mechanical stirring allowed for the local concentration of piperidine to remain high while rapidly dispersing the dibenzofulvene product. Thus, stirring has a combined effect in which both the diffusion to the beads and the permeation inside the beads are increased.

(105) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.