WO1996040734A1

WO1996040734A1 - Process for cleavage of blocked peptide resins using liquid hydrogen fluoride

Info

Publication number: WO1996040734A1
Application number: PCT/US1996/007289
Authority: WO
Inventors: Henry M. Franzen
Original assignee: Immulogic Pharmaceutical Corporation
Priority date: 1995-06-07
Filing date: 1996-05-22
Publication date: 1996-12-19
Also published as: AU5866496A

Abstract

A process is disclosed for HF cleavage of blocked peptides in which a scavenger compound is dissolved in liquid HF and the mixture cooled before addition to the peptide resin, with temperature inside the reaction vessel being closely monitored over the reaction period.

Description

Process for cleavage of bl ocked peptide rei sns using l iquid hydrogen fl uoride

The present invention relates generally to improvements in the preparation of polypeptide chains, more particularly to an improved process for using hydrogen fluoride and a scavenger to remove protective blocking groups from the peptide resins resulting from conventional peptide synthesis techniques.

BACKGROUND OF THE INVENTION

The synthesis of polypeptide chains has long posed formidable technical difficulties. In general, the synthesis of the peptide bond between two amino acids poses no special problems by itself. The difficulty lies in the fact that the common reagents required to form peptide bonds can also react with other functional groups not involved in the peptide linkage, such as the free amino group of the NIVterminal residue, the free carboxyl group of the COOH-terminal residue, or certain R groups, e.g., the thiol group of cysteine. One approach to this familiar problem has been to "block" or shield these vulnerable groups by appropriate preliminary reactions in order to protect these groups from the reagents during the peptide synthesis step. After the peptide bond has been formed, however, the protective blocking groups must be removed to yield the free peptide.

A number of more of less successful approaches to these peptide synthesis problems have been developed. One recognized breakthrough in this important field was the pioneering work of R.B. Merrifield in developing an automatically programmed machine that performs, with high yields, the many repetitive chemical steps for adding each amino acid to the growing polypeptide chain. A thorough discussion of the Merrifield process and related peptide synthesis techniques appears in John M. Stewart and Janis D. Young's book "Solid Phase Peptide Synthesis" (2nd ed.- 1984) published by Pierce Chemical Company, which book is incorporated herein by reference. Portions of the following description are excerpted from this reference volume.

In the Merrifield process, a programmed sequence of reactions takes place in a single

reaction chamber, with reagents from reservoirs automatically added by means of measuring pumps. The apparatus employs exactly timed reaction periods at each step and uses a method called the solid-phase peptide synthesis (SPPS) technique for separating the desired reaction product from by-products in high yield.

The synthesis of a peptide chain in one illustrative example is begun by attaching the carboxyl group of the COOH-terminal amino acid residue of the chain to be built to an insoluble resin particle large enough to be easily separated from a liquid phase by filtration. The next amino acid to be introduced, following the blocking of its amino group, is allowed to react with the free amino group of the COOH-terminal residue in the presence of the condensing agent dicyclohexylcarbodiimide. This forms an amino-blocked dipeptide that is covalently attached to the insoluble resin particle via the COOH-terminal carboxyl group. The amino-blocking group is then removed by acidification; it decomposes into the gaseous products carbon dioxide and isobutylene. These steps are repeated many times. The entire polypeptide chain is built with the COOH-terminal residue anchored to the solid resin particle. Finally, after the entire blocked peptide has been assembled on the polymer support, a different type of reagent is applied to cleave the peptide from the polymer. The blocking groups which have protected side-chain functional groups must also be removed, and usually are chosen so that they can be removed simultaneously with cleavage of the peptide from the resin. The most popular reagent for cleavage of peptides from the resin at the end of the synthesis is anhydrous hydrogen fluoride. Of all the cleavage procedures used until now, this appears to be the most versatile and least harmful to a wide variety of peptides. Thus, following completion of assembly of the desired blocked peptide on the resin, the peptide- resin is treated with anhydrous hydrogen fluoride (HF) to cleave the benzyl ester linking the peptide to the resin in order to liberate the free peptide. Side-chain functional groups of amino acids are usually blocked during the synthesis by benzyl-derived blocking groups, which are also cleaved from the peptide simultaneously with its removal from the polymer support. The free peptide is then extracted from the resin with a suitable solvent, purified,

and characterized. But, cleavage of blocked peptides using HF has led to still another set of problems.

First, HF is a toxic, corrosive gas (boiling point 19°C), and it must always be used with adequate ventilation, such as a fume hood. Since it attacks glass very rapidly, with an exothermic reaction, all equipment for handling HF must be made exclusively of plastic, such as Teflon-Kel-F or polytetrafluoroethylene (PTFE), or non-corrosive metal. A commercially available plastic vacuum line for handling HF allows all of the operations necessary for successful SPPS cleavage reactions to be carried out without any hazard to operators. This commercial apparatus also allows transfer of HF under vacuum; this is particularly important for removal of HF at the end of the cleavage reaction. HF cleavage is generally carried out at 0°C for about 30 minutes. These conditions will generally cleave the peptide effectively from the resin and remove all side-chain blocking groups. Several side reactions have been shown to be temperature dependent and are much worse at elevated temperatures. Since many of the side-chain blocking groups are removed at significantly lower temperatures by HF, some investigators prefer to do cleavage at a lower temperature, or at least a preliminary cleavage at a lower temperature followed by cleavage at 0°C. This may give a significant improvement in results, particularly if the peptide is large and contains many residues with susceptible side-chains.

When side-chain blocking groups are cleaved by acidolysis, reactive carbonium (or nitronium) ions are formed which can attack easily alkylatable residues in the peptide. Benzyl and t-butyl carbonium ions, for example, can readily alky late methionine, cysteine, tyrosine and tryptophan residues. These destructive alkylations can be largely prevented if a large excess of a suitable nucleophilic scavenger is included in the HF reaction mixture. Anisole has been most frequently used for this purpose, but other nucleophiles may be even more effective. Resorcinol, thioanisole, dimethylsulfide, ethylmethylsulfide, methionine, 1,2- dithioethane, and indole have all been used for this purpose, with favorable results. Many investigators use a mixture of several scavengers simultaneously, with apparently improved results in the case of very sensitive peptides. In some more recent work, peptide-resins have been cleaved successfully by use of a low concentration of HF in a large amount of dimethylsulfide or other nucleophilic scavenger reagent. Thus, peptides synthesized according to the Merrifield procedure with benzyl-like protecting groups would conventionally be mixed with a scavenger and placed in a Teflon or PTFE reaction vessel equipped with a magnetic stirring bar. The mixture would be cooled with dry ice in ethanol for five minutes or longer. After evacuation of the reaction vessel, anhydrous HF vapor would be distilled into the reaction vessel from a nearby HF reservoir heated sufficiently to promote the HF distillation. Such an arrangement is illustrated, for example, in Figure 2-7 at page 86 of the Stewart and Young book referred to above. The reaction vessel would then be placed in an ice bath and allowed to react for about one hour at about 0°C. HF is then evaporated from the reaction vessel, and the peptide resin is washed with ether before extracting the peptide in conventional fashion such as with dilute acetic acid.

The foregoing procedure suffers in practice from a number of problems. First, because the temperature inside the reaction vessel following HF addition cannot be readily monitored nor controlled with the conventional equipment arranged in the conventional configuration, the reaction vessel temperature may easily and unknowingly rise above 0°C and thereby cause some unnecessary and undesirable degradation of the peptide. The conventional equipment as described above simply is not designed to accommodate an internal probe to monitor temperature inside the reaction vessel. Because of the use of vapor- phase HF in this conventional procedure, the reaction vessel must be kept tightly sealed.

The undesired temperature rise in the reaction vessel is due both to the condensation of relatively warm, vapor-phase HF coming from the heated reservoir as well as the exothermic nature of the reaction itself. Although the reaction vessel may be located in the dry ice-ethanol cooling bath, as described above, the Teflon or Teflon-like materials which comprise the reaction vessel are thermal insulators. As a result, heat transfer through the walls of the reaction vessel is slow. Tests have shown that it can take thirty minutes or longer to cool the contents of the reaction vessel to -60 °C using the cooling bath.

Second, it has been found that in practice it is difficult to evenly mix the scavenger compound with the peptide resin prior to the HF addition. If a solid-phase scavenger, such as p-cresol, is used, it will freeze and prevent homogenous mixing during the initial HF condensation into the reaction vessel. As a result, the local concentrations of scavenger inside the reaction vessel will differ. Some portions of the peptide resin more distant from the scavenger will be directly hit by HF upon initial HF addition and thereby suffer more undesired side reactions. These and other problems with and limitations of the prior art are overcome with the improved hydrogen fluoride cleavage process of this invention.

OBJECTIVES OF THE INVENTION

Accordingly, a principal object of this invention is to provide a process for hydrogen fluoride cleavage of blocked peptides that promotes more thorough mixing of the ingredients and facilitates better monitoring and control of temperature inside the reaction chamber. It is a specific object of this invention to provide a peptide cleavage process that utilizes liquid-phase HF.

It is also an object of this invention to provide a peptide cleavage process wherein temperature inside the reaction chamber is monitored and held within relatively narrow tolerances below about 0°C. Another object of this invention is to provide a peptide cleavage process that can be readily carried out in a batch or semi-continuous unit operation.

Specifically, it is an object of this invention to provide a process wherein a suitable scavenger compound is dissolved in liquid HF and the mixture, at a desired temperature, is then added to the blocked peptide to insure substantially homogenous temperature and chemical composition conditions throughout the interior of the reaction chamber.

Other objects and advantages of the present invention will in part be obvious and will in part appear hereinafter. The invention accordingly comprises the methods and processes, involving the several steps and the relation and order of one or more of such steps with respect to each of the others and to the apparatus exemplified in the following detailed disclosure and as illustrated by the drawing, and the scope of the application of which will be indicated in the claims. SUMMARY OF THE INVENTION

The HF cleavage process of this invention generally comprises dissolving a suitable scavenger compound in liquid HF and cooling the mixture to an appropriate temperature prior to adding the mixture to a peptide resin contained in a reaction vessel. The temperature inside the reaction vessel is closely monitored and held within a relatively narrow range below about 0°C. Numerous process efficiencies and economies, and an improved peptide product, are thereby realized.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 is a schematic process flowchart of an HF blocked peptide cleavage process in accordance with this invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The process of this invention broadly comprises premixing a suitable scavenger compound with liquid HF and cooling that solution to a desired temperature prior to adding the HF-scavenger solution to blocked peptide resin contained in a reaction vessel. Referring to Fig. 1, a suitable scavenger is added either continuously or in batch, for example via feed 10, to a mixing reservoir 50, which may initially be empty or else already contain liquid HF. Reservoir 50 is initially maintained at a temperature of about 0°C or below utilizing appropriate cooling means, such as immersion in a first cooling bath (not shown) or the like, or alternatively by cold-air flow. Reservoir 50 may also optionally be provided with an internal temperature probe 55 extending through a sealed connection in the top of reservoir 50 to monitor the temperature of the materials inside reservoir 50. Such a probe 55 can be useful in one batch operation embodiment of this invention as hereinafter described. The portion of probe 55 that will be immersed in HF or exposed to HF vapor of course must be protected, such as with a coating of impervious material such as a tetrafluoroethylene resin like Teflon or Teflon-like plastic. Reservoir 50 may comprise a fluorocarbon polymer plastic, such as Teflon, or a similar tetrafluoroethylene resin, or another material impervious to HF yet also able to maintain its strength and resiliency at low temperatures down to -60 °C or below for example some types of stainless steel. Similar material requirements apply to reservoir 52 and reaction vessel 54 as hereinafter described. The scavenger may comprise any suitable scavenger compound or mixtures of two or more such compounds as appropriate, depending in part on the nature of the peptide resin and the blocking groups to be cleaved. Some well-known scavengers useful in the HF cleavage process of this invention include anisole, p-cresol, p-thiocresol, resorcinol, thioanisole, dimethylsulfide, ethylmethylsulfide, methionine, l,2-dithioethane, indole, and mixtures thereof. The scavenger can be added in the solid phase to reservoir 50, or alternatively it can be heated to melting and added as a liquid. A mix of several scavengers may result in a liquid mixture.

Next, if reservoir 50 did not initially contain liquid HF, an HF stream 20 from an HF tank or cylinder 40 is passed through flow control means, such as valve 22, and condensed into reservoir 50 as liquid HF, where it is mixed with the scavenger. Alternatively, reservoir

50 may initially contain HF to which the scavenger is added. The ratio of scavenger to HF in reservoir 50 may range from about 1 gram of scavenger to about 1 milliliter HF (defined herein as a 1:1 ratio) to about 1 gram of scavenger to about 100 milliliters HF (defined herein as a 1: 100 ratio). The preferred range for scavenger/HF in reservoir 50 is about a 1:10 to 1:30 ratio, as herein defined. The scavenger-HF mixture is then gently agitated by appropriate mixing means to facilitate dissolution of the scavenger in the liquid HF. The mixing means for reservoir 50 may, in one embodiment, comprise a magnetic stirring bar

51 inside reservoir 50 activated by external means or, in alternative embodiments, a motor- powered mechanical stirrer extending through a sealed connection in the top of reservoir 50 or inert gas bubbling means, such as nitrogen, may be used for agitation. Said mixing means must of course be protected by a fluorocarbon polymer plastic such as a Teflon or Teflon-like plastic coating to prevent HF contact with a metallic core. The selection of scavenger for use in this invention will, therefore, in part be dictated by the solubility of the scavenger in liquid HF at temperatures of about 0°C or below. Although the exact temperature at which the scavenger dissolution step of this invention is carried out is not critical, provided it is below the 19°C boiling point of HF, it will be appreciated by those skilled in the art that dissolution of scavenger in liquid HF proceeds more quickly at a higher temperature. On the other hand, the scavenger-HF mixture must then be cooled prior to being mixed with blocked peptide resin. The higher the temperature at which the dissolution step is carried out, the longer it will subsequently take to cool the mixture to the desired temperature. Optimizing the temperature conditions at this stage of the process, however, can be determined by routine experimentation for any given scavenger and relative scavenger/HF proportions.

Once the scavenger is dissolved in the liquid HF, the mixture is then cooled to below 0°C, preferably to a temperature of about -30°C to -60°C or lower. The purpose of this cooling step is to insure that, upon adding the HF solution to the peptide resin, the heat generated by the exothermic reaction does not raise the temperamre in any part of the reaction vessel above about 0°C. This cooling step may be carried out in the same reservoir 50 where the scavenger was dissolved in liquid HF if the process is being run as a batch operation. In this case, reservoir 50 would be immersed in a second, colder cooling bath (not shown) for a period of time sufficient to cool the scavenger-HF mixture to the desired temperamre. Alternatively, the temperature of first cooling bath may be reduced, or the temperature of the cold air flow stream may be lowered to accomplish the same result. For this embodiment, temperamre probe 55 would be useful in monitoring the cooling of the scavenger-HF mixture. Alternatively, to facilitate a semi-continuous operation, the scavenger-HF mixture can be withdrawn from reservoir 50 by suitable means, such as a vacuum pump and syphon tube combination (not shown), as fluid stream 60, passed through flow control means, such as valve 62, and transferred into cooling reservoir 52 maintained at a temperature of about - 30 °C to -60 °C utilizing appropriate cooling means such as immersion in a cooling bath (not shown), for example of dry ice in ethanol. Reservoir 52 may also optionally be provided with an internal temperamre probe 57 extending through a sealed connection in the top of reservoir 52 to monitor the temperature of the materials inside reservoir 52. Similar to probe 55, at least that portion of probe 57 that will be immersed in HF or exposed to HF vapor must be protected, such as with a fluorocarbon polymer plastic coating. In this embodiment of the invention, as soon as mixing reservoir 50 has been evacuated, additional HF and scavenger can be introduced for mixing a fresh batch of solution.

In the meantime, a batch of blocked peptide resin is introduced to reaction vessel 54, for example via feed 12. Reaction vessel 54 is provided with an internal temperature probe 59 extending through a sealed connection in the top of reaction vessel 54. Similar to probes

55 and 57, at least that portion of probe 59 that will be immersed in HF or exposed to HF vapor must be protected, such as with a fluorocarbon polymer plastic coating. Reaction vessel 54 is further provided with appropriate mixing means to agitate the contents of vessel 54. In a preferred embodiment, the mixing means comprises a magnetic stirring bar 56 inside reaction vessel 54 and activated by external means (not shown). The metallic core of stirring bar 56 is coated with fluorocarbon polymer plastic to prevent contact with HF. In an alternative embodiment, the mixing means may comprise a motor-powered, plastic-coated mechanical stirrer extending through a sealed connection in the top of reaction vessel 54, or inert gas bubbling means, such as nitrogen, may be used for agitation inside reaction vessel 54. Reaction vessel 54 is initially maintained at a temperamre of about -0°C to -60°C, preferably about -15 °C to -30 °C, utilizing appropriate cooling means such as immersion in a cooling bath (not shown), for example of dry ice in ethanol, or alternatively a cold air flow. When the scavenger-HF mixmre in reservoir 52 (or reservoir 50) has been cooled to the desired temperamre of about -30°C to -60°C, that mixmre is withdrawn from reservoir 52 by suitable means as fluid stream 70, passed through flow control means, such as valve 72, and transferred relatively quickly into reaction vessel 54 where it is mixed with the blocked peptide resin. One efficient technique for carrying out this transfer is the use of a syphon tube (not shown) that extends from the bottom of reservoir 52 and connects outside reservoir 52 to tubing going to valve 72. The syphon could be actuated either by applying a vacuum to reaction vessel 54 in coordination with air or nitrogen flow to reservoir 52 or, alternatively, by over-pressuring reservoir 52 such as with a nitrogen stream. Upon opening valve 72, the liquid contents in reservoir 52 would be quickly transferred to reaction vessel 54. The ratio of peptide resin to scavenger-HF solution in reaction vessel 54 may range from about 1 gram of peptide resin to about 2 milliliters of scavenger-HF solution (defined herein as a 1:2 ratio) to about 1 gram of peptide resin to about 10 milliliters of scavenger-HF solution (defined herein as a 1:10 ratio). The preferred range for peptide/ scavenger-HF solution in reaction vessel 54 is about a 1:4 to 1:7 ratio, as herein defined. The temperamre in reaction vessel 54 is then raised (or allowed to rise) to about 0°C to -20°C, preferably about -3°C to -10°C, and the reaction is allowed to proceed until substantial completion, typically about one-half to two hours. When the reaction is complete, excess HF and reaction product gases are removed from reaction vessel 54 as by-product stream 80, and free peptide is thereafter extracted and recovered through conventional techniques. The following example is illustrative of the process of this invention.

Example Utilizing a batch apparatus arrangement instead of that illustrated schematically in Fig. 1, 1 g. of the scavenger p-cresol is added to an empty mixing reservoir, and 10 ml. of HF is then condensed from an HF tank into the mixing reservoir. The p-cresol is allowed to dissolve by gentle mixing or agitation in the reservoir. When the dissolution of the p-cresol is completed, the reservoir is cooled to about -50°C. The reservoir is equipped with a PTFE- covered temperamre probe to monitor the internal temperature and a PTFE-coated magnetic stirring bar. A reaction vessel is then loaded with 1 g. of a blocked peptide resin, pre-cooled to about -10 °C and connected in line to a syphon mbe extending from the bottom of the reservoir.

At this point, the reaction vessel is evacuated with a vacuum pump so as to create at least a partial vacuum and a nitrogen flow to the reservoir is initiated. When the valve in the line connecting the interior of the mixing reservoir to the reaction vessel is opened, the cresol-HF mixmre is quickly siphoned into the reaction vessel. The reaction vessel is also equipped with a PTFE-coated magnetic stirring bar which is used to constantly stir the mixmre. In addition, the reaction vessel is equipped with a PTFE-covered temperamre probe used to monitor the temperamre of the contents of the reaction vessel. Temperamre in the reaction vessel is increased to about -3°C, where it is maintained as the reaction is allowed to proceed for one hour. At the end of this time, vacuum is again applied to the reaction vessel for about one hour to remove HF and reaction product gases leaving peptide residue in the bottom of the reaction vessel. This residue is then washed with ether or ethylacetate several times before being dryed. The final desired product is then extracted with dilute acetic acid and lyophilized by conventional techniques to yield a relatively large quantity (relative to the quantities of starting materials) of a high quality free peptide product as a fluffy powder.

The foregoing example illustrates that the HF cleavage process of this invention can be carried out in a laboratory or commercial setting more efficiently and effectively than conventional processes to yield higher proportions of a higher quality peptide product as compared with the prior art processes.

Since certain changes may be made in the above-described process without departing from the scope of the invention herein involved, it is intended that all matter contained in the above description shall be interpreted in an illustrative and not in a limiting sense.

Having described the invention, what I claim is:

Claims

1. A process for cleavage of a blocked peptide resin comprising the following steps: (a) dissolving a scavenger material in liquid hydrogen fluoride to form a solution; (b) cooling said solution to a temperamre of about 0°C to -60 °C; (c) mixing the cooled solution with said blocked peptide resin under agitation conditions to form a substantially homogenous reaction mixmre; and (d) monitoring the temperamre of said reaction mixmre to maintain a temperature range of about 0°C to -20 °C until the cleavage reaction is substantially complete.

2. Process of claim 1 wherein said scavenger material is selected from the group consisting of anisole, p-cresol, p-thiocresol, resorcinol, thioanisole, dimethylsulfide, ethylmethylsulfide, methionine, 1,2-dithioethane, indole, and mixtures thereof.

3. Process of claim 1 wherein said scavenger material is p-cresol.

4. Process of claim 1 wherein the proportion of scavenger material to liquid hydrogen fluoride in step (a) ranges from about a 1: 1 to 1: 100 ratio.

5. Process of claim 1 wherein the proportion of scavenger material to liquid hydrogen fluoride in step (a) ranges from about a 1:10 to 1:30 ratio.

6. Process of claim 1 wherein step (a) is carried out in a sealed container comprising a fluorocarbon polymer plastic or stainless steel.

7. Process of claim 6 wherein said sealed container comprises mixing means.

8. Process of claim 6 wherein said sealed container comprises internal temperamre monitoring means.

9. Process of claim 1 wherein step (b) comprises cooling said solution to about -30°C to -60°C.

10. Process of claim 1 wherein said blocked peptide resin is cooled to a temperamre of about 0°C to -60°C before being mixed with said cooled solution in step (c).

11. Process of claim 1 wherein step (c) is carried out in a sealed reaction vessel comprising a fluorocarbon polymer plastic or stainless steel.

12. Process of claim 11 wherein said reaction vessel comprises mixing means.

13. Process of claim 11 wherein said reaction vessel comprises internal temperamre momtoring means.

14. Process of claim 1 wherein the proportion of blocked peptide resin to cooled solution in step (c) ranges from about a 1:2 to 1:10 ratio.

15. Process of claim 1 wherein the proportion of blocked peptide resin to cooled solution in step (c) ranges from about a 1:4 to 1:7 ratio.

16. Process of claim 1 wherein said reaction mixmre in step (d) is maintained at a temperamre of about -3°C to -10°C until the reaction is substantially complete.

17. Process of claim 1 wherein said reaction in step (d) is allowed to run for about one-half to two hours.

18. Process for cleavage of a blocked peptide resin comprising the following steps :

(a) dissolving a scavenger material in liquid hydrogen fluoride in a sealed fluorocarbon polymer plastic or stainless steel container equipped with internal temperamre monitoring means to form a solution in a ratio of about 1 : 10 to 1 :30 of scavenger to hydrogen fluoride;

(b) cooling said solution to a temperamre of about -30°C to -60°C; (c) syphoning the cooled solution into a sealed fluorocarbon polymer plastic reaction vessel equipped with internal temperamre monitoring means, said reaction vessel containing said peptide resin pre-cooled to a temperamre of about 0°C to -60 °C in a ratio of about 1 :4 to 1 :7 of peptide resin relative to the volume of said cooled solution; and (d) agitating the reaction mixmre in said reaction vessel while maintaining the temperamre of the reaction mixmre between about -3°C to - 10°C for a period of about 1/2 to 2 hours.