BE683574A - - Google Patents

Description

  

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 having as object: "Controlled synthesis processes of polyeptides and polypptiden thus produced"

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 li ;. P) "'::" w'1.te àn, r; nt4, J. ,, is relative to a new proceed POt ,!' 7. syntli13a; by. stages, controlled of polypetidB da proteins. The present invention also relates to new products of interest in
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 such syntheses. More particularly still, the
 EMI2.3
 pr6J = nto invention relates to a process for
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 -pr, l -, ';:'. (''; xspà. 'at eric.ar.a of polypeptides in the course of duqî, z, 1 (, (1>' I1ul tiplen 8'ta.dee sequential are performed
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 without Fr;> lr # of ept..e8 ¯ntel'm. intermediates.

     c polypeptides form a large class of
 EMI2.6
 001'1 \, "'' J .Sdw,; 3 ,,, including the dipeptides, the triP0p ''.,. -; 1.;: - 1; 7''1 (H) t¯des and the peptides superior in J. ':' C1. "; 1r '! 1> r; 1, .... j, n () 3, .. 'j U aa' '! Have linked to each other by li ".f' '; 1.!; J. ro:!, d "1 r8ias peptides>. A very W :: '; 11): "0,, 1),., -,.". R "O; .1.Y have;:,'! 81) 16s by hydrolysis '1;', C, -: 'w ¯' '"';,".; '\ r.:z some polypeptides 1; 1: = 1 <. '; 6 chemically. Many poi; r #;> 7-; Ti- (1, .. ,, '. 4cul.>. T.re relatively low cor; e YT ...

     âC, ¯'.t- up to 6 to 8 amino acids are equal: J'ü1 .; ->, "Lua proteins are largely '.>'. - 'f \'" .; "r '\ ntenen usually a' 1!":} '1'! "'r'" i-j: de zeg: # <5 ;; # e amino acids linked to one E 'pG pe: ptide8 are of interest not only 0 "-" "': .. tI" ': base for protein synthesis but é =, 1. = ; nt because some of them are the-
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 rapeutiqucs'snt actives They are also useful for the study and the analysis of proteins. One of the best

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 Ways of studying protein structures involves partial hydrolysis of the protein followed by isolation and analysis of the polypeptide fragments produced. These methods give only limited information regarding structure-function relationships and requirements for polypeptides and proteins.

   Exquisite information of this nature could be obtained by synthesizing polypeptides in a gradual and controlled manner.



   Moreover, these studies as they were carried out do not give any insight into the mode of action of enzymes, hormones and other proteins with important bodily functions. These methods also do not provide information which would allow the preparation of interesting variants of natural proteins nor any information which would allow the preparation of therapeutic agents specifically designed to interact with natural proteins in an interesting way. ,
One of the most difficult problems in modern biology is to gain new insight into the mode of action of enzymes,

   hormones and other physiologically activating proteins relatively little is known about the relationship between chemical structure and biological activity of these / important substances. For example, information is lacking as to sites of specific activity. in enzymes, for reasons of specificity and functions of sections of enzymes which seem to have no apparent role in enzymatic reactions. No single enzyme has ever been chemically synthesized.

   The methods available so far are totally inadequate to solve the problems posed

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 by the relations existing between the ohimic structure and the biological eotivity, since they cannot be structured without requiring human labor extending over many years, to prepare the large number of very diverse emulogical products of substances, physiologically active preparations existing in nature, which solve these problems. would be necessary in order to be able to know how to treat diseases could be reduced to a more rational basis.



   Although a number of synthetic methods have been devised, no ideal method for the synthesis of polypeptidea has heretofore been discovered. An interesting synthetic process has not been invented which allows the formation of successive peptide bonds in a growing peptide chain and which avoids the often difficult stage of selective elimination of the protective group without the need for it. isolate the intermediates at each successive step. The methods which have been used are tedious, expensive and time consuming.

   For the formation of a single dipeptide by reaction between two different amino acids, the best prior art methods require the blocking of the amino group of one amino acid and the carboxyl group of the other so that a reaction selective occurs so as to give only the desired dipetide of the four potential products. In addition, the carboxyl group which is to react generally requires activation by transformation into an acid halide or some other modification which is more reactivates than the carboxyl group. The two molecules are then condensed to form the dipeptide and finally

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 the blocking groups are removed.

   The reactions * exi-
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 at! lt by conspuent (1) the setting in a.oa of the groups blozueuro, (8) the activation of the group C4 "hci * ylet zij the cond, ercxe. ' , f), 'cn3Yreent blocker groups.



  All the -romedium must be repeated to add just one t: ''. 111r (; p (dde opp2men.re to the dipeptide and repeat again to '7'Je.,. ::; again to add the suooes amino acid segments - sîfae In addition, in order to prepare polypeptides suitable for an et'¯ ".IQ ¯.: i'P <real full .1'f: naoignemen% s, it is usual eRt n cez3sairg lt <Eter the raoemeation of esino acids and peptides i # i during the reactions so as to prâ3: .re:; p :::,: r'.u - ':; > pure r4u-îtluement.

   When this problem of emissivity is considered with the fact that the overall yields on peptide synthesis are extremely low and with the fact that there are more than 1026 possible polypeptides containing only 20 different amino acid segments, it is not surprising that despite the recognized importance of these products, very few large polypeptides and simple proteins have actually been prepared. The products that were prepared took several years to complete. Several specialist groups have worked on insulin all over the world and its synthesis was the result of ten years of effort on the part of one of these groups of researchers,.

   A process to which we paid attention oonsi.-
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 maple. for the unregulated synthesis of homopolymers such as polyalanine and polyglutamic acid of varying molecular weights and for the unregulated synthesis of heteropolymers with randomly arranged amino acid segments in the N process -oarboxy anhydride is called in
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 present brief proceeded to NCA.

   These r6aotinne of tOIVZ4-

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 The results were carried out in organic solvents using basic catalyst amounts the products prepared look like; su.p. .oie3..e has the 'natural proteins in -I which 0 XOloerne cflr% of their physical properties but practically
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 of no value for the study of the physical and physiological roles of proteins. In addition, the methods are not applicable to the preparation of heteropeptides of known structure and molecular weight containing amino acids specifically defined in pre-positions.
 EMI6.3
 determined in the peptide chain.



   Other researchers, because of the ease with which homopeptides are formed, have thought that the
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 NOA process could be applied to the stepwise, controlled synthesis of heteropeptides. In fact, early attempts were made to adapt the process to the synthesis of low molecular weight heteropeptides in organic solvents. The method has never been used in aqueous media for the stepwise, controlled synthesis of heteropeptides in which two or more amino acids were added in successive steps.
 EMI6.5
 with a growing peptide chain.

   Indeed, the conclusion of those who had suggested such successive reactions was that the stepwise synthesis of polypeptides by the NCA process was impossible because the relative proportions of the main reaction and the side reactions could not even occur. be settled.



   It has now been found that it is possible to use
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 Read the process to prepare, het4ropfip% ides. In this process, a starting acidic amine is first transformed

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 me to an N-carboxy amino acid anhydride. This trans-
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 formatioR 0Y advantageea. It blocks the amino group and activates in mcmo tGdl '! ,,'. ' . ',, (eg carboxyl.

   The anhydride is enauite aie '' '': ;; '. ; vo, ct.o: ú ': <:; :) '"" another amino acid or.:; l'1, t: 1Ac to form' Jl1: 1-011; -: bo%:, / dipcptidc or an SUp9ur peptide which turns into the desired compound by of carboxylation, Dani organic solvents. in the presence of a base '! 1.C' ";;; 11 <J dfoarboxylation takes place so easily that the r6u- ',:')): 1 "1 cannot be controlled or regulated with the result that polymers and others are produced.
 EMI7.2
 unwanted fw = mr;%, 0 # has found that such an uncontrolled reaction or n 1. replication: can be avoided in water.



   The use of aqueous media is ideally suited for the stepwise, controlled synthesis of polypeptides for a number of reasons. One of the main reasons is that amino acids and peptides are generally soluble in aqueous media and not in organic solvents because they are soluble.
 EMI7.3
 form dee cspb: i.onl {c '': and that, in the case of peptides, they #: "t: 'ncnt hydrophiloa amide groups.



  In addition, SOl "ln:;. ', 6 -3r ,, é: .ques often promote racemization during peptide synthesis and can damage higher peptides. It is also unwelcome to note that peptides and proteins found in nature are mostly found in aqueous media.



   The control of the NCA process in aqueous media is so difficult that it has never been tried.
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 to carry out the stepwise synthesis of h ± t4ropoptidos in such media. The main factors contributing to

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 the difficulty of the control are (1) polymerization, (2) over-reaction, (3) hydrolysis of NOA and (4) interference with the reaction by the released carbon dioxide. The
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 "'J. ;; nJô: r: t :: \ :::' 0n and the over-reaction comes from the. De- = .r,: ac,.;: An of the compound N-Carboxy intermediai- ui . c 1.

    > d, ùz; that the product is obtained from comes available JO1: '.' ':' 1:; 1; (): r in competition with the starting material ohoi- 3 la; reaction with the reagent of the type N-cap'boxy #, n> 1, - ôo even, the hydrolysis of l starting anhydride ein> <1C '"incomplete faction due to insufficient <1.:iiy9é,; 1st start. The carbon dioxide liberated from any of the intermediates or% react preferably with the starting material and inactivate the latter because of the incompleteness of the reaction.



   Another difficulty which arises in adapting the NCA process to stepwise synthesis is really another aspect of the problem. This aspect is the difficulty of purification. Unless each successive step in a peptide synthesis is carried out so as to control or regulate the polymerization, the incompleteness of the reaction and the over-reaction, the reaction medium becomes contaminated with undesirable by-products including products. Reactions of higher or lower molecular weight, particularly those differing from the desired peptide by only one two amino acids.



  The contamination can also come from the production of peptides having the same molecular weight but a bad sequence compared to that of the peptide. The physical and chemical properties of these new products are so similar to those of the desired product that the separation becomes difficult. and often imposed

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 a1ble. It is evident that certain contaminants may be tolerated, particularly those due to the different physical and nical properties. The insoluble polymers are readily available. tightened by filtration.



  Despite the discouraging results obtained until pFësaRi and they.> Oncl, Jsi> Jn3 unfavorable drawn from these results we have now discovered that the N0A p (n: (j. 'Tr¯ used for "' 1:> 'c¯ ": .. r simply and quickly-
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 in tJ.C éa, 1 mie in ¯ of peptide, by step and regulated with ') ;, f:; n!; l; i "-, iùn intermediate peptides produced 0.e iiani' ' .. * '' - "t. vs- ', <.r produced peptides
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 such as .'axytc3ne, ra v. "? 9p '" c.'3' <.na, angiotensin or brady; in, ine aiR'L 's3 proteinaceous substances such as indrén' orto + '0p. and insulin.

   In addition, the invention pertains to: '' '= :; esnt to realize the synthesis of peptides ot of aftersproteins including enzymes and n'i; = ¯' f ze, '' - '' '.. 1.'I!'! J '' re produced by the procedures used J3qtè present pr <ce;> 11. = à .lfl se 1e In the present invention for the synthesis r4g #, é? -1. ('Ti'¯': pt1df't of their drifts having a m1lmum Q:

   The x amide bonds in the palm chain is naraatôria4 in that an amino compound is reacted as the starting material.
 EMI9.4
 selected from the group formed by amino acids peptides and their derivatives containing a free amino group, with an N-carboxy amino acid anhydride or a derivative thereof, by bringing the reagents into contact in an aqueous medium under controlled conditions so that the only group has, at least present in concentration

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 appreciable in reactive form during the reaction
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 is the amino group of the amino acid, dv r <; 1,;.; D5 or derivatives thereof which must r4ag - c pc. Form the desired product to any amino group present including those of the unreacted product peptide.

   Under the carefully regulated conditions of the present invention,
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 the reaction between the anhydride and the amin4. acid, pep ", '1.1' or its derivative proceeds at each step of the synthesis with minimal formation of by-products which could interfere with the following reaction of the anhydride or
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 with the purification of the desired product, Under the prescribed conditions, the intermediate N-carboxy compound is prevented from deoarbo: ¯J: .i.6 or, when decarboxylation takes place, the resulting amino group is protected by protonatlort until to 0: that substantially the material of step 6 has reacted with the anhydride.

   The rr; a.ltat of this careful adjustment is that the reaction with the highest rate is the glue which produces the desired compound and that the pro-
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 ductian of undesirable side-products., in particular (Joux resulting from an 8ur-re.otion and a poly- .e: s¯, was kept within limits which do not inter- '; " ': JlI, 1j pao with the stage SUbséqUentf1e the synthesis réRl6e.



  An2,, t, the 4Fctnfiooi% ion of the intermediate N-oarboxy and do 01. anhydride which does not degrade the peptide product thus formed can then be reacted with another anhydride under analogous reaction conditions without isolate intermediaries. The invention is'
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 yields which normally reach 95-98% and which are often essentially quantitative as well as in the production of the aTCO dipeptide.

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 preparation of polypeptides and proteins: of predetermined structureec.



   The desired summary, that is to say the formation of a new peptic bond is obtained by putting
The anhydride and the starting material in contact in phases advantageously separated by the addition of the solid anhydride to an aqueous solution of the starting material, at a rate under the controlled conditions of temperature, / of mixing; and hydrogen ion concentration, although the only free amino group present in reactive form in reactive concentration is the amino group which is to join during the formation of the peptide bond, the amino group of the product thus formed. being protected.



   In the reaction mixture for the preparation of a peptide one or more amino groups may be present in addition to the amino group which is to react so as to form the desired product. These can include, for example, additional basic groups on the anhydride such as an omega amino group of lysine, the guan dino group of arginine or the imido group of tryptophan. They can also include the amino groups of the amino acid or of the peptide used as a starting material. The amino group of the product, i.e. the amino group formed by the decarboxylation of the intermediate carbamate, can also be taken into consideration.

   Any of these groups are potentially capable of interfering with the course of the desired reaction by competing with the starting reagent, namely N-carboxy anhydride. It is therefore evident that the concentration of these

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   :, 3oc potentially interfering in the form d-, -. ,,:. as free amines should be kept to a minimum.



   For the sake of simplicity, the description is limited to only two amino groups, A and B.



  Amino group A is the group which must react to form the desired product. The amino group B is the group
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 : 0: t1011 interfering. Several situations can: produce according to the relative basicity of the two groups If B is a more powerful base than A, the reaction can be affected at a pH at which the greater part of B is protected by protonation and the greater part The wax of A is not protonated and therefore reactive. However, the pH must be high enough to stabilize the oarbamate.

   For the same reason that the amino group of the formed product generated by decarboxylation of the oarbamate is much more basic than the amino group of the starting material, the reaction should be carried out at a pH, such as the amino group of the product. and amino group B are protected by protonation, however amino group A is not,
If the A and B groups are of substantially similar basicity, then it is generally not possible to increase the concentration of amino group A relative to amino group B by adjusting the pH and amino group B should be. protected by a blocking group, advantageously a carbobenzoxy group.

   The pH of the reaction, however, should always be chosen so that the potentially competing amino groups are protected by protonation.

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   If A is a much stronger base than B, the reaction is carried out at a high pH, for example 9
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 It * In oonditicns, the 4 is found in concoentrai:, 'J! \ D!.'. Bstall'.iialle in the form of free amine ,, Marne -; r F r.¯, .t 1 { if: E '.' OU11 '? 3. or ' <j-- free amine, the product: tO "cr.0;", r; rt1 the prod "' <1Asire obtained by reaction with 1 ";!,; Y & 3J f.1roii'10 au In 1,; z drux d'J" ':' 't1i - :, t's case, the pH must be
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 su.ff:!. aam # .e;: -.-, j1, "'' '1: rou :::, tt:' - -er the co, rbamate. The pH must 0h'e orôis;:, - '' ': m' '' '1' 'sri:' - ". .1 ')] 1 below about i 1.



  Oepc'llt.1:.: N: b :, le M *.,; du -; 1 ".: '? itic given a.F.deaw. =,". ", a concentration of the ions
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 hydroxyl es sffâ. : 5 .. = z = t ri t el .: 'r1- ?! so that a competitive rearrangement with the anhydride can occur,
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 In oj.,. à it is not necessary to protect the B amino group. CG This is often the case with po7> y- h5¯1 ;; 1 at poic. high ecular or with a group: 9 ')! Ï (the element is not reactive due to the presence of ateric factors.



   The reaction is carried out at a protective pH.



  The term "pH embraces the area of concentration.
 EMI13.5
 tration of hydrocp'jne ions in which the amino group which is to react is ± 1 appreciable concentration in reactive form, that is to say in the form of a group of free amino reactive with the acylating agents, the potentially il1terféJ Kroupea: antej are protected, the oarba. mate intermediate is stabilized or the amino group obtained by its decarboxylation is protonated. the aforementioned expression also includes the adjustment of the pH

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 EMI14.1
 so that the hydrolysis of EcboX1 anhydride <y 1- no help is minimized.



  There is a> 11 prot6o: .. nl.t: r 'Qp4': 4 lice each reaction .. According to r1a: ':: Oli) ±; .. t. J, i; ioul1à:, (!, 08 pH can be between 4 and i.: R reactions in which the carbamate erc the protective group, the optimum protective pH is found in otté aloal1nff1 + is usually between A and 10, 5. Preferably 10, ce + t3s5 In the case of peptide reagents, the protective pH to achieve substantially complete action may be lower than that cut for amino acid reagents. Due to the fact that the group & Q1ùl0 is prot3: d by prat? .ic, the optimum protective pH is t :: "'' J: .ty ,.

   On the acid side ;, usually between about 4 and 6, This determined, the reaction should be carried out in con- allions in which the pH is adjusted as narrow-
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 'slow as much as possible from optimum protective pH. The variations in pH can be followed by a rapid electro-) 0 6ponoe and it is advisable to adjust the pH by automatic titration or by external addition of a neutralizer, Even taking these precautions,
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 : 1.1 cao !; ''; <* '?. green difficult because of the coiled speed do lt, rCL,'.! '. Qn to maintain the pH exactly at the value of an optimal protective pH.

   Some variation from the optimum protective pH is acceptable. However, it is preferable that the pH does not fluctuate more than 0.2 units from the optimum protective pH although a variation.
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 "t going up to 0.5 units of the protective pH then Gtre tolerated ,.

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   Test reactions can be used to determine the corrective pH as well as the optimum conditions for the concentration, temperature, time and molar excess of anhydride which should be used to process. the method of the present invention. For example, in the preparation of a polypeptide. test reactions can be performed with aliquots of the reaction mixture in order to prepare the next upper member of the series at a number of pHs, concentrations, temperatures, reaction times and excess molar of different anhydride.

   The results are evaluated by determination of the color of ninhydrin using conventional thin-layer chromatography and paper chromatography methods with various combinations of solvents such as n-butanol-acetic acid-water, dry butanol-acetic acid-. water, pyridine-isoamyl alcohol-water, phenol-water and isoamyl alcohol-water. The product can be local using any of certain staining reagents, for example, ninhydrin staining. It is also convenient to use techniques using radioactive tracers to analyze reactions of. test.

   These techniques allow easy determination of the R-value for each new product and provide a suitable method for estimating product yield and percent contamination by by-products.



   For example, for the preparation of a high molecular weight polypeptide using techniques

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 Each new radioactive material can be taken to the next stage in the synthesis and based on radioactive measurements, the yield in this next stage under various reaction conditions can be achieved. for this reason, the radioactive anhydrides of the N-carboxy amino acid of each of the common amino acids
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 e *, laughs ¯ '? ia are prepared and used in tests * 71 '1 ", n1 M of reaction products suitable for the 0c -1.%'. where the test is 1.0 mm reaotife per 15 l, '1ng aqueous .

   Of particular interest is to establish a series of compounds labeled for each arnica acid labeled with a C14 carbon, deuterium and with the two isotopic elements. These isotopes can be measured differentially by known techniques.



  This factor allows the exact identification of the product.



   The reaction is very fast and is noticeably complete in a minute or less. It is seldom necessary to continue the reaction for more than five minutes. If desired, the course of the reaction can be followed by subjecting aliquots of the reaction medium to the known hydroxamic test and the reaction can be continued until that the test is negative indicating that substantially all of the N-carboxy anhydride has reacted.



   Among the critical control conditions for the present invention, it should be noted the discovery of the applicant that by carrying out each reaction under conditions of intimate mixing, for example.
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 (, '.r, 10 we agitation to produce turbulence, side reactions may be in effect.

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 largely eliminated, which often results in
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 iue the yields are essential3rnt quantitative. In addition, '; .1]' .. rapid mixing ensures that the N ... oarboxy anhydride does not react in locally depleted areas.
 EMI17.2
 in út1rG starting Day hydrolyze or polymerize.



  Such undesirable side reactions are substantial for periods longer than those specified above. Likewise, the quick mix shortens the pd: ao. This results in the rapid dissolution of the N-carboxy anhydride the release of the carbamate produces 9, an undesirable x4: ai an a0o'1r leading to both an over-reaction and inactivation of the starting material,
The reaction is normally carried out at a temperature below 10 C, preferably between -5 C and 5 C. This is an important aspect of the reaction.
 EMI17.3
 glage nevsa.re the setting 'HI1.H 1.'8 of the present invention. The value of the 11-jau piu diciitme with a decreasing% temperature.

   At 6 C the pK value of water is
 EMI17.4
 of 10.1; & It appears that, operating around 0 0, the concentration of. hydroxyl ions is reduced with a concurrent increase in the concentration of hydrogen ions. This important feature of adjusting or controlling the reaction helps to minimize hydrolysis of the starting N-carboxy anhydride and decarboxylation of the intermediate oarbamate.



   Equimolar amounts of the reagents can often be used. When the NCA is partially consumed by hydrolysis, it is often lent-

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 The optimum excess can be determined by test reactions, however, to use an excess of anhydride to achieve a complete conversion of the starting amino acid or peptide from the starting product. The degree of excess which can vary within wide limits will suitably vary depending on such factors as the nature of the anhydride, the molecular weight and the basicity of the peptide or equivalent reagent.

   As the molecular weight of the peptide produced increases, the use of excess anhydride becomes less of a problem because the chemical and physical properties of the desired product differ appreciably from those resulting from the reaction. hydrolysis of the excess anhydride, so that isolation and purification of the produced polypeptide can easily be carried out.



   A particular advantage of the process of the present invention is the ease of removal or elimination of the protecting group. Thus, when the reaction is carried out at a pH such that the product is protected by sretenation, the deoarboxylation takes place spontaneously.



  A carbamate which is stable under alkaline conditions is isly decarboxylated by reducing the pH by the addition of acids.



   When the intermediate requires deoarboxylation, this is best carried out by adjusting the concentration of hydrogen ions in the reaction medium until an acidic pH is obtained, for example, a pH ranging from
 EMI18.1
 goes . 3 to about 5. Deoarboxylation can also be carried out by allowing the mixtures to stand, while slightly heating them or by freeze-drying.

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  However, these are not the preferred methods because of the time they require. The bubbling of an inert gas such as nitrogen through the mixture during deoarboxylation facilitates the complete removal of carbon dioxide as this compound is formed.
After decarboxylation, an additional amino acid or derivative thereof can be added to the peptide chain by repeating the above procedure.
There are a number of methods that can be employed to aid in pH adjustment.



  For example, the reaction can be carried out in a buffer. Any of the buffer systems can be used, the choice of a particular buffer being indicated by the protective pH determined during the test reaction. Borate and phosphate buffers can be used.



  Potassium borate is particularly useful for maintaining an alkaline pH.



   The reaction can be carried out in a mangesum hydroxide solution to maintain a pH of about 10.



   Barium hydroxide is a suitable base for adjusting the pH since barium ions can be easily removed from the reaction mixture by precipitation with an acid such as sulfuric acid. An advantage of this process is that a large proportion of the inorganic salts is removed, although these compounds do not accumulate in various successive stages, which could complicate the purification of the final product.



   The external addition of a base, for example

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 of sodium or potassium hydroxide, can be made to maintain an accurate control during a reaction, for example, when the change in pH is monitored using a pH meter. Often such an addition will be used in conjunction with a buffer substance.



   When operating under the aforementioned conditions, polypeptides of pre-determined structure can be prepared by successive reactions of amino acid, a p tide or a derivative / of these compounds with N-oarboxy. an -dride, yes, by decarboxylating the N-oar-boxy aimi compound obtained if it proves necessary and by repeating the reaction without isolation of the products formed as much as is necessary in the original reaction medium until the reaction medium. desired polypeptide having chain length / is obtained. An N-carboxy anhydride of one peptide can also be reacted with another peptide in order to obtain high molecular weight polypeptides of precisely known structure.



   Normally the polypeptide will be soluble under acidic and basic conditions since it is amphoteric. However, it may happen that the structure of the product formed is such that it precipitates under the conditions of the reaction or upon subsequent adjustment. of pH. In such a case it is often good practice to isolate the product by filtration and to carry out the subsequent reactions in fresh aqueous media.



   It often happens that peptides containing a high proportion of amino acids with aromatic groups such as phenylalanine or tyrosine precipitate from the acidic solution after decarboxylation. In some cases,

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   carbamates of polypeptides are insoluble in the basic media in which they are formed and precipitate;

     therefore These salts can be separated by filtration and decarboxylated in a fresh aqueous acid before the subsequent neaction with another anhydride as already described previously, the process according to the present invention allows to obtain the desired products of 'excellent purity with high yield and giving only minimal accumulation of side products. The side products which are formed are generally sufficiently different in their physical and chemical properties little? do not appreciably interfere with the successive reactions. It is, however, advisable to separate the desired intermediates from time to time, for example 'after ten or fifteen reactions.';

  . It often happens that the structure of the prepared peptide is such that it can be separated very easily from the accumulated by-products.



  For example, a peptide containing arginine or another basic amino acid could be easily purified by contact with a cationic ion exchange resin, for example a carboxylic resin such as Amberlite IRC 50 on the acidic oyol. a pH at which the peptide and by-product are selectively absorbed and eluted.



  The eluate containing the peptide can be used as a reaction medium for the subsequent reaction with another anhydride. Analogous procedures can be used with peptides containing amino dicarboxylic acids.

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  Intermediaries and end products
 EMI22.1
 prepared by the process according to the preoento;.> ven% ion can be isolated from any suitable material, for example by crystalliaation, by extraction as a salt, for example e; r \ c of ammonium sulfate or by adjusting the pH to the point where the product precipitates. Separative chromatography using silica gel, ion exchange resins or
 EMI22.2
 chromatographic tanimolecular type, such as crosslinked dextrans available in various forms under the trade name "Sephadex" from the company
 EMI22.3
 A.3, gharaese. Uppsala, Sweden.

   A suitable method is to prepare the final product with a "processing function" or "carrier function", such as a strongly basic group such as the basic group on
 EMI22.4
 arginine readily reacts with an ion exchange resin or other reagent to separate the desired product from the by-products not containing arginine. The "carrier function" is then removed. For example, if the desired product is a hexapeptide, a
 EMI22.5
 > = 5;% app% ià, with 1targinine as the carboxyl-terminal amino acid. Arginine is strongly basic and it results in that the heptapeptide is easily absorbed on a cationic ion exchange resin so as to
 EMI22.6
 achieve separation of by-products.

   The heptapeptide is then eluted and subjected to the action of oarboxypeptidase-B which is an enzyme which selectively removes @ginine to give the desired hexapeptide.



   The arginine "carrier function" is also of interest for the preparation of polypeptides in

  <Desc / Clms Page number 23>

 in which an excess of N-oarboxy anhydride is used during the preparation. If in an earlier stage in the reaction the N-carboxy annhydride or arginine is added to the polypeptide chain or if the arginine is the amino acid with a terminal oarboxy group, the polypeptide containing the artinie thus obtained will possess physical properties. and chemical which permit easy separation of any of the unwanted byproducts, especially those arising from use, of excess anhydride in one or more of the reaction stages.



   The product can be prepared as described above and, if desired, the carboxy-terminal arginine can be removed with carboxypoptidass-B,
One method which is of particular interest for the isolation of the products is a capillary flow chromatography technique. The method is analogous to large scale thin layer chromatography. An effort has been made to reproduce as closely as possible the conditions of thin lying ohromatography, using only larger amounts of reagents. We maintain the
The same ratio of absorbent to amino acid or peptide.



   In this process, a column is filled with silica gel, cellulose or other absorbent material. A solution containing the peptide is contacted with the lower part of the column and allowed to grow. It is observed that the Rf values of the constituents of the reaction mixture are substantially identical to the Rf values for the same constituents, determined by thin layer chromatography.

  <Desc / Clms Page number 24>

      



  The appropriate part of the column containing the desired product, as previously verified by small strain methods, is then simply cut off and eluted.



  Any other reagent can also be sprayed onto the surface of the minhydrin column which will cause the color to flash to locate the products.
 EMI24.1
 



  1 is then segmented. the layer of ourtace Qol '"-'? (" word scraped off and the product is eluted from the remaining part of the process section of the present invention is applicable to acidic amines and peptides, both natural and synthetic, which are in the form of dextro and levo It can be used with compounds having functional groups other than amino and arboxyl groups of the desired peptide bond such as tyrolaine, arginine, threonine, glutamic acid and the like.



  It is also applicable to derivatives in which these additional functional groups are blocked by a group which is easily removed after the formation of the peptide, such as for example the N-carboxy anhydrides of im-benzyl histidine, trifluoroacetyl serine or tetra-hydropyranyl-. tyrosine.

   The term "derivative" as used herein refers to amides, esters and other functional modifications of the arboxylic group of the amino acid or peptide and amino acid homologues and analogs in which a or several hydrogen atoms are substituted, for example p, - a lower alkyl group, such as x-methylleucine or α-fluoro glycine. These latter compounds are of particular interest for structure-active analysis.

  <Desc / Clms Page number 25>

 found in physiologically active proteins such as hormones and to prepare these antagonistic substances for such active proteins for various therapeutic purposes.

   The method of the present invention can
 EMI25.1
 4be: '5ili? Ë for: xr physiologically retive derivatives of thialcohols, primary and secondary amines and compounds obtained from acids containing
 EMI25.2
 phosphorus, such as the phraphates esters.



  A! Jal1 "': - 6 of their structure, some of the amino acids require conrided ur = a" 1 = n social in the process of the present iv ± ntin.



  The nitildazole side of histidine N-carboxy anhydride bit 3 '': - iio, 4g before the reaction by the formation of a QJ im-be! \ Zyl derivative which can be removed from the peptide by hydrofenacion or by action The imidazole group of this amino acid does not interfere with subsequent ions with anhydride when t,. the peptide is fora '.



   The hydre yl group in serine and threonin anhydrides is appropriately bluated.
 EMI25.3
 By forming trifluoroacetyl or trichloroacetyl derivatives of the respective N-carboxy anhydrides, these groups are particularly important since they are hydrolitically removed during the formation of the peptide. The hydroxyl groups can be protected by the formation of ether, for example, benzyl ether, tetrahydropyanyl ether or other compounds such as acetals or celais.



   There is a feature of the present invention which can be unexpectedly used.

  <Desc / Clms Page number 26>

 
 EMI26.1
 the anhydrides of aspartic and 7.utam., without
 EMI26.2
 need to protect their tsr :, z.x.



  Cystine which is a di h: "1 araino &. Acid with symmetrical β-amino carboxylic acid moieties on either side of the Y bond is usable in the present invention in a remarkable manner.
 EMI26.3
 to produce two identical polypeptides containing>: 1. <1, ...: or oyet61ne, an amino acid containing only: a sulfhydryl group. Dane oe proceeds, a sumetric di-N-oarboxy anhydride of cystine is reacted with a
 EMI26.4
 1 starting peptide '] amino acid or a gold derivative thereof. , produce a symmetrical product with at least one peptide lidJ5n on each o8td of the \) 18u.1f'1.: I '; bond.



  For example, cystine di-anhydride can be reacted with alanine to produce bis (alanyl) oystine. Then the chain growth is continued as described above, for example by step reactions with the anhydrides of leuoine and de.
 EMI26.5
 valine do so as to obtain bis (leucyl-valyl- a. aiay, 4 tize. When a polypeptide having the etruc- ', 1J.r meieoulaire has been prepared the compound is reduced by example with hydrogen sulfide, thioSlyolium aldehyde or an equivalent reducing agent in order to cleave the disulfide bond so as to form two identical molecules of the desired polypeptides each containing oystin.

   In the example above
 EMI26.6
 tioned the product would be louoyl-valyl-alany; -oyotéînet
The novel process of the present invention is readily adaptable to the preparation of a series of long-chain protein analogues, such as

  <Desc / Clms Page number 27>

 enzymes or hormones in which an amino acid in the middle of '. string varies, For example, if the
10th, amino acid / of a polypeptide containing 20 amino acids varies so as to prepare analogs in which all other amino acids are conserved in the same sequence, one should first prepare a series of peptides containing; 9 amino acids and a second series containing 10 amino acids.

   The partners of the first series will be converted to various decapeptides by reaction with N-carboxy amino acid anhydrides of various amino acids. Each of the decapeptides so produced can be coupled with the decapeptides of the second series to produce polypeptides containing 20 amino acids in which the only variable is a single amino acid in the middle of the chain. Such coupling can be effected by conventional coupling methods / example by the arbobenzoxy method.



   An additional and important advantage of the present invention is that the process can be carried out in such a way that the racemization is substantially completely controlled. The optical purity of the products according to the present invention can be easily determined, for example, by subjecting them to the hydrolytic action of a specific enzyme, such as leucine aminopaptidase which attacks only one optical isomer,

   
No discussion has been made so far regarding the use of thio analogs of N-oarboxy amine acid anhydrides in which ether oxygen is replaced by sulfur for stepwise synthesis.

  <Desc / Clms Page number 28>

 
 EMI28.1
 ld'tropeptide9. These analogues were not <'> Neither will L184e in the unregulated synthesis of homo- l. ,, ptideae of substantial molecular weight. an important aspect of the present invention lies in the discovery that these thio analogs can be advantageously used for the stepwise synthesis,
 EMI28.2
 regulated dlhdtdropeptidese The compounds can be represented 130: '- ;; 6 ::, by the following structural formula:
 EMI28.3
 where R is an amino acid residue.

   These are substituted thiazolid-2,5-diones and may be referred to as amino acid N-thioanhydrides. For convenience they are referred to herein as "M anhydrides. -thocarboxy amino acid and the process for their synthesis will be called the "TCA" process,
According to this feature of the present in-
 EMI28.4
 In particular, a starting amino acid is first converted to an h-thiooarboxy amino acid anhydride. This anhydride is then reacted with another amino acid or peptide to form a! -thiooarboxy amino acid or peptide. a superior peptide which transforms it into a compound you,

   read by dathiooarboxylationt It has been found that when the process conditions more fully described
 EMI28.5
 ci., doosouet it is possible to regulate these reactions so as to produce the desired compounds with a product.

  <Desc / Clms Page number 29>

 Minimal interference with by-products, As with the NOA process, the TCA process is carried out in aqueous media.



   The TOA method can be used to achieve simple and rapid stepwise, controlled peptide synthesis in water, with or without isolation of the intermediate peptides produced to obtain polypeptides, such as oxytocin, vasporessione, angiotensin or bradykinin and proteinaceous substances, such as adrenooortotropin and insulin. In addition, the invention also allows the synthesis of higher proteins and peptides including enzymes and hormones which could not be produced by the methods used heretofore.



  The TCA process according to the present invention. for the controlled synthesis of peptides and derivatives thereof is characterized by the reaction of an amino compound serving as depar material selected from the group consisting of amino acids, peptides and their derivatives containing a free amino group , with an N-thiooarboxy mino acid anhydride or a derivative thereof by contacting the reactants in an aqueous medium under controlled conditions such that the only amino group present in appreciable concentration in reactive form during the reaction is the amino group of the amino acid, peptide or derivative which can react to form the desired product,

   any other amino group present therein includes those in the product peptide as formed which cannot react. Under the carefully controlled conditions of the present invention, the reaction between thioanhydri-

  <Desc / Clms Page number 30>

 de and the amino acid, the peptide or their derivatives proceeds at each stage of the synthesis with minimal formation of by-products which might interfere with the reaction to the next thioanhydride or the purification of the desired product.

   Under the set conditions, the N-thiocarboxy compound is prevented from undergoing dethiccarboxylatino or, if dethiooarboxylation takes place, the resilient amino group is protected by protonation until substantially all of the starting material has reacted. with thioanhydrides The result of this careful control is that the reaction which proceeds with the greatest rate is that which produces the desired compound and that the production of unwanted side products especially those from over-reaction and polymerization is kept within limits which do not interfere with the subsequent stages of controlled synthesis.

   After decomposition of the N-thiocarboxy intermediate or any other unreacted thioanhydride, the. The product peptide thus formed can then be reacted with another thioanhydride under analogous reaction conditions without isolation of the intermediates.



  The invention is applicable to the preparation of dipeptides in yields which are normally 95-98% and which are often substantially quantitative, as well as to the preparation of polypeptides and proteins of predetermined structure.



     There is an optimum protective pH for each reaction of the TCA process. Depending on the particular reaction, this pH can be between 4 and 10.0. For reactions where the thocarbamaqte group is the protecting group,

  <Desc / Clms Page number 31>

   the optimum material power is on the alkaline side. orid-
 EMI31.1
 nairemcnt of 0 "i"): ', ..i the case of peptide reagents the pi protcctcu? .po.5. -:. '. t "t ') It 3'13ibly completes T'eu *, being supposedly lower than that used for reagents of the amino acid type.

   If the product is one in which the amino group is protected by protonation, the optimum protective pH on the acid side is usually between 4 and 6. This once determined the reaction should be carried out under conditions in which the pH is adjusted from as close as possible to the optimum pH. Some pH variation from optimal & protective / pH is acceptable. Preferably, however, the pH cannot vary by more than 0.2 units of the optimum protective pH although variations up to
0.5 units can be tolerated. The optimum pH can be determined using techniques of the NCA method.



   The pH can be monitored and adjusted using the methods described above in connection with the NCA method.



   One advantage of the TCA process as compared to the NCA process is that the control or control techniques required in the first-mentioned process are less demanding and less difficult to perform, for example. In the NCA process, the reaction is normally completed within a minute. Because of this rapid reaction it is often difficult to follow or regulate or control the changes in the concentration of hydrogen ions during the reaction.



   Rather, the TCA reaction can be carried out over a period of between 10 minutes and about 4 hours depending on the temperatures used and the identity of the specific reagents among other factors.

  <Desc / Clms Page number 32>

 



  The bare TCA process is carried out in oondi-
 EMI32.1
 ,, '') 1: 1. <0fl ±. ca2, intimate using the same prooéàés> a. <r ducsits about the N'CA process to achieve or. "0 \ '. 11": more advantageous The temperature limits used in the
 EMI32.2
 Drooúd6 an BOA are wider than in the
 EMI32.3
 r, usually vary from about 0'0 to about. <o <"'n' ''. on c. ',' ;. e somewhat higher temperatures w te" ... eroo 'J8d1 tee can be used without r', 1¯.'lio1e.ble blind. It is often convenient to perform
 EMI32.4
 reaction at room temperature, that is to say between about 80 ° and 30 ° C.

   The possibility of obtaining produced
 EMI32.5
 at room temperature without undue accumulation of side products is a particular advantage of the TCA process.



   If one of the reactants is an amino acid, the preferred reaction conditions range from 0 C to 25 C over a period of about 10 to 30 minutes. If the reagent is glycine, the preferred reaction time is reduced by a factor of 10, i.e. the
 EMI32.6
 period extends in this case from 1 to. minutes.

   Peptides are often somewhat slower to react and the preferred conditions are in this case about 15 minutes.
 EMI32.7
 tes at 4 hours and at a temperature between approximately 0 C and 25 C.
 EMI32.8
 Equimolar amounts of reagents can often be used * When thioanhydride is absent
 EMI32.9
 : i) 11eot consumed by the hydrolysis, it is often preferable to use it in excess in order to obtain a trana- du
 EMI32.10
 , forma% complete ion of the starting amino acid or 7-peptide to product.

   The degree of excess can vary between wide

  <Desc / Clms Page number 33>

 limits according to the same factors as those described above about the NCA procedure
 EMI33.1
 5. dthioearooxyiation is carried out using 1 '8t:.' '; - procéàia and by applying the same prin- cîren as those employed and applied in the decarboxy- .s.vi.t by the proceeda to the NOI, After the . dêthiaaarbozyle- tt.1 ", an additional anino acid or one of its derivatives: 'can be added the pptid1que chain by repeating the 6p'3 mode.' ': sire described ie,. ^ sur.



  When operating the deorite conditions. above we can prepare polypeptides of predetermined structure, by suooessive reactions of an amino acid, a peptide or: ', 3j> 4A: "e of these compounds with the chosen N-thiocarboxy anhydride, then operating a dethiocarboxyltien of the N-thiooarbamate obtained, if it proves necessary, and by repeating the reaction without isolation of the products formed as often as
 EMI33.2
 this is neo3aire dt, "the: / the original reaction until the ': il'od'l1,1;

   polypeptide having the desired structure and chain l; 5l1, ctu,.; t are obtained * An N-thiooarbox1 anhydride of one peptide can be reacted with another peptide to obtain molecular hair polypeptides high structure known with precision.



   Isolation procedures and techniques applied
 EMI33.3
 The NCA process can also be used for the TCA process.



   A feature of the TCA process is that the thioanhydrides of aspartic and glutamic acids and histidine can be unexpectedly used without protection being necessary.

  <Desc / Clms Page number 34>

 their additional functional groups,
 EMI34.1
 The 4-thioarboxy anhydrides .¯,, r * no acid used in the processes of the present intention are prepared by esting an alkyl thionourethane derivative or
 EMI34.2
 an amino acid, for example, by treatment with a trihalide of, phoephoretel,, eu, letr.bromuxe of phots phore. thionourethane is prepared by reaction between an amino acid and a dialkyl or dialkaryl xanthate, preferably the first compound.

   The process can be represented as follows:
 EMI34.3
 
 EMI34.4
 In loe -, quatîone which precedes, R this an annuity of amino acid and the 8ubsti $ uanto R and Rg which can They may be identical or different, have interior alkyl groups or aralkyl groups containing, for example, up to B carbon atoms.
 EMI34.5
 the following preparations of N-thiccarboxy amino anhydrides should be oonaid ± n & 6 as illustrative of the preparation of the desired components.

  <Desc / Clms Page number 35>

 
 EMI35.1
 



  , 'A? IN 1 éihil $'? Fl-é '±', "$.>.> Àéïné! ': 1Q ......" 4 "c :: -'" 00-0 0.X 1 - - -if; ' ; i '. -.,. ".., r 'r1 t) 34 J 2 ml"',>? <.,, i;>? ii.li - # i. '' '.. :: <. <l ..., .. '. 'h,:; .. t jc taa- 4' X:. ' <i. "i #; 1 = '"' '' .-:, ¯.,; iU. '>, Ut JO. we tî-4oute 1 o # icv: 1 'j;' -; ': xii "J <.; t;> 'It in 40 m "of metha-
 EMI35.2
 ? t.Jl, ri '' :; ': Y'3 ,, -, ne, \ two phases is heated to about 50 0 ac ¯: = slc è. forming a single phase system with d, aem2t of zethyl meroaptan. The solution is held at 50 0 pentean4-. about 90 minutes and then concentrated from zaniérs to ('aaer the greater part
 EMI35.3
 ethanol. The residue is diluted with 100 ml of water and washed twice with 100 ml fractions of ac-
 EMI35.4
 ethyl tate so as to drive out the xanthate having
 EMI35.5
 not reacted.

   The aqueous layer is added to 200 ml of ethyl acetate containing 34 ml of hydrochloric acid and an additional 50 ml of water is added. By this treatment, the potassium salt of N-thiocarbalkoxy leuoine is
 EMI35.6
 transformed into 1 '': the àé = iné and extracted in the organic layer. The oouc3r '' '' ',' '' ee is washed with ethyl acetate eupplêC1ent '\ ire (, t the combined organic layers are washed twice with @ fmotiono 40 ml dytine solution saturated with chloride of sodium, adhere to the wall of anhydrous sodium sulfate and filtered The filtrate is c0ncen% 14 under reduced pressure to give a viscous oil which cracks on standing.

   The product can be prepared by taking it up in hot benzene and precipitating it by the addition of hexant. To transform the N-thloo8rbetho% 1 leuoine thus prepared into N-thooarbo% 1 louolnes anhydride

  <Desc / Clms Page number 36>

 
 EMI36.1
 'fl #lC 5 this product is taken up in 20 ml of)': 0 ';' ('\' ,;; '! * 1l of; b,' x "ora of phosphorus are added *:," 'i ,VS "( , <,, heated to about bzz while acting so much.



  , '.' , c * 3 n8 \ 11 h cooled to crystallize the - ',, 3, <= -> ;. 0'Bt recovered by filtration, washed with - ".iio.r <. '. 7'oi <1 and: 1 -: - water and dried ..
 EMI36.2
 



  ; pAA'0 '! P 2 ... znhvdride of 1-thioaarboxwl! groin
A total of 60 g of glycine are taken up in 16 ml n and 75 ml of ethanolic potassium hydroxide (11.7 N and then 97.5 g of xanthate are then added.
 EMI36.3
 of diméÜl1e. The reaction mixture is kept at about 23 ° C for 2 hours. The mixture is then heated up to about 45 C for half an hour and oonenorced for reduced pressure so as to expel the most.
 EMI36.4
 large part of ethanol * The concentrate is diluted with 180 ml of water and extracted with two fractions of 100 ml of ether so as to remove the xanthate which has not reacted.

   The aqueous alkaline layer is covered with 150 ml of ethyl acetate and then 73 ml of 12 N hydrochloric acid is added together with 70 ml of water. The aqueous layer is separated and again extracted with 100 ml of ethyl acetate. Lea organic layers united
 EMI36.5
 are washed twice with 50 ml portions of saturated sodium chloride solution and dried over sodium sulfate.

     The drying agent is separated by filtration and the filtrate is concentrated under pressure.
 EMI36.6
 71- fr'ibla so as to give methyl% hion0ur '% ha- ne glycine which is purified by taking up this product in ethyl Ó 150 ml of aceta / and filtering and precipitating the product.

  <Desc / Clms Page number 37>

 the desired result by the addition of 350 ml of hexane.



   Methyl thionourethar- glycine (8g) as well
 EMI37.1
 prepared this resumption 4ans 40 ml of tetrahydrofuran and 691 1 G tribromiare of phosphorus are added slowly cependriit that the temperature is maintained between 25 and: 3500 7te C, 7? : angel réaatioxa3, is suddenly cooled in SOC ml dftz * .ie aqueous solution of lu of aodiulli bicarbonate and 100 ml of ethyl aoetato.

   The layers organi- i &quot; 0 o: a. Coiled with L.3C ai fractions, all aolvticn saturated with eod1um chloride. r'1élHh CU: t 'elu calfate an? 1; .r4 = ,: # sodium, filtered and the solvent is then ohas' è of the filtrate? Joua preeaion little ôlsiJée of m! 1.0:' f.'e 1, J :,>. ,, àr - <'desired product. The product is purified by taking it up in 150 ml of benzene, filtering it and removing it. precipitating it by adding 150 ml of hexane to the yarn%; ,,;.
 EMI37.2
 



  Lj.; JZ1: llil ......! H! ¯! ¯: 22Z¯E! L !!!!!! 'A total of 5 µg of ethyl thionourethane, phenylalanine is prepared as described in
 EMI37.3
 preparation 2 in r snp5.a o.nt the glyoine with an equivalent amount of phenylalanine and this component is taken up in 25 ml of benzene. The mixture is cooled to about 3 ° C. and treated dropwise. drop under a nitrogen atmosphere with a solution of 0.95 ml of phosphorus tribromide in 5 ml of benzene. After addition of the phosphorus tribromide solution, the mixture is stirred for a further 20 minutes at the same temperature, oepen-
 EMI37.4
 as long as a suspension containing the thioanbydride does not form.



  This suspension is added to a mixture of 50 ml

  <Desc / Clms Page number 38>

 
 EMI38.1
 ethyl aoetate and 50 ml of water. The gel layer is separated and washed with an additional fraction of water. It is then dried over anhydrous sodium Pu, filtered and the filtrate is brought to water. sicoity under reduced pressure. The residue is taken up in benzene and the
 EMI38.2
 desired product is precipitated by the addition of oyoloher. ,! \\: 'It is purified by reoristallieation in ben2gna ob oyolohexane.



   Various modifications can be made to the basic procedures to take account of certain groups.
 EMI38.3
 For example, the hydroxyl group on tyrosine may be protépd by tetrahydropyranyl groups * ": /. pr!) - teotoursè The aliphatic hydrox group on threonine and serine can be protested by trifluoroacetyl or trichloroaoetyl groups4 Dioarboxylic acids aspartic and glumatic acid can be employed in the form of half-esters, omega groups
 EMI38.4
 .:;:., rbo1 <: yle being esterified.

   The omega-amino-diaminoid group such as lysine can be protected by alooxy carbonyl m aralooxyoarbonyl groups, such as methoxycarbonyl or benzoxyoarbonyl radicals. The process of the present invention can also be used in combination or together with the method. NCA process as illustrated in the examples.



   It was found that according to another aspect of the
 EMI38.5
 For the present invention, the hydroxyl groups on aliphatic 1-oarboxy or 1P-thioaarboxy amino acids, i.e., hydroxyl groups which are of an alcoholic rather than a phenolic character, can be blocked with of

  <Desc / Clms Page number 39>

 
 EMI39.1
 r.'t;,; t ',; t-. îi ,, .. c'3. .'5, '' a r .1 - :::. :: "" "1l 1s '' Jp'ome ltltl.C? / :, ixtC. ',) ....' 9èt! .O ':::' 1. - '' ''".,., 'lsi>, more particularly Ü01 (.a T;' t'f2 ¯friu b <': i.Cil ... IF "0' Cti'r'.9 'jL:? fo' 3 (,, -" - 1: j "]. n0a cor. ',.' a!.;. .f t- '... i. c': <'uT:' 9SH feels
 EMI39.2
 1t} & I "00 J.tt runs: le la r? .Ctic7i cru3rt.on.

   The trihaloaoetyl anhydrides N-carboxy amino acid and the anhydrides of N-thiocarboxy amino aoidea are new
 EMI39.3
 calves C;) t '! flooés.



  Compounds are prepared by reaction between the selected trihaloaoetylating agent and N-arboxy or N-thiocarboxy amir..0 L'Q d6 11, "" oxyU as appropriate.



  Trihaloac6ty * -. C., E.3, 11, 't': J: ahydrides and their halides, in particular chloride or bromide, can be used. It is better to use lea
 EMI39.4
 the corresponding anhydride8 as the trihaloacetylation agent for the trifluoroactyl derivatives and the acid chloride for the trichloroacetyl derivatives and since it has been found that these methods give the
 EMI39.5
 best rondec-. 3.

   In a ,,, n ± :, 1 <; n typical effeotée according to the present invention, carboxy anhydride or thiooar-
 EMI39.6
 boxy anhydride of aoino acid having an alcoholic hydroxyl group is taken up in a solvent or a mixture of inert, anhydrous reaction solvents, for example, an ether, in particular a cyclic ether, such as and
 EMI39.7
 zyfdioxene or tetrahydrofuran, 71'ant of trihaloacetylation is then added.

   The ptef4renco reaction is carried out at room temperature c'e-h-dirw at a temperature between about 20 C and about 30 C, although somewhat higher or somewhat lower temperatures can be used without.

  <Desc / Clms Page number 40>

 
 EMI40.1
 ': J' .61, ieîcïablea The reaotiorrheability period may vary ''; t:.: - 3 rather wide limits depending mainly]; ¯1 it <.n.iôoec: ture chosen and the reactivity of the group 'f.'? ': ylo 1: protect and the trihaloaoetylation reagent n! HY1. :::. ;; 1..o.:; cte duration of The reaction can vary, for example, n2, tr ,, f from about one to sixty hours, with a period ranging from E 1 (3 four hours being preferred for the double purpose. 1 ". Good yields and periods of reaction pta. <ii> q, 'Jt acceptable.

   Although equimolar amounts. '. * B -, 4.îf can be used one uses norma- Il .m4 t ::::: i) s, for example, ---------- a molar exoess going up to 10%, of the trihaloacetylation agent in order to obtain a reaction, as complete as possible. An inert atmosphere such as nitrogen can be employed to minimize side reactions, but it is not necessary to practice so.



   The product can be isolated by any of the suitable methods. A process which is of particular interest is to remove the solvent under reduced pressure and obtain the desired product as a residue.



   Although of particular interest for the preparation of derivatives of amino acids which are normally found in association with animal tissues, such as serine and threonine, the reaction is not, however, limited thereto. It can be used with anything. - So much ease to prepare "unnatural" amo-acium analogs such as α-hydroxynorvaline, 1 '-haidroxynoralie and others such as those described herein.

  <Desc / Clms Page number 41>

 
 EMI41.1
 



  The .pr.o4uite 'rrs can be sorted using only i, w¯ "a.'" Y'N8,0 of iY9rB het'leptide8 by the
 EMI41.2
 precede ", lll; .jc <; oeéa: 'ns': <: fl13 examples. ;. on.desire 1] n¯fi, r.: .. "F" J .. "." eRT used for preparation é, fbPi ;; -., .g, u) # 1: 1 :, a ; 3 a.ac, l3x $ 41, evé. such as N; p -.¯ = wn and pr 9; ; .6 # nine by polymerization dwi T. c? Ni ... '":;. Iq er" A base., From such [) ooàpµJ4 ;. are db, 'l' '!'. t3. ': 7f3I1Ct.1 like horn ,, poi <Fr uc <:% 1 <Jc =. ;; ". L'4". 'U.'? E:. ". 'Rot <physical r14téù .e 2'JtUâà5% EZ .a, lû, ;; - ''. e 1.

    Yô p>.? 5 "t9 Se: '. C * ras, a =: the present in'! 'Ntion the groups hyeTcxyl? Hi" ..;, t .. :: fi ... hydroxy acids) -de .3 or -'- j -u 010Q s, ÎX. = d. .. ¯ i -u '¯iy1 ailyl, from Tele- Fsnoe des, zr>; u% és 45.:"...-.E,,nyl 1 ".> Er silyl such; that los group '"a.3" zy ..:., yl. 0 found that these groups are at 'J:, t 002'.tCUrr-, i: .ß sibrated during, xa 9i an of';?. Tion .. '' '' inlydrides of 0-tri - alkyl oi lyl Îi-ov.rlJox3. amîno '..' has been new
 EMI41.3
 compounds and enter 4 years 1.1 ;! within the scope of the present invention.



  Prepaid oven <= 1, es "" "." I1.A'r: .. l'J.X composed of the present: Lnveo1'l u.n h â o; rnre of tri-alkyl ei1y1, from mA 'n1è1.i. appropria, the ch ']>) the \ u'. 'e or the broaiire, is reacted c ,,, 7ec the anhydr the 1? - <; = rbo, ry amine hydroxy acid! the selected at an elevated temperature in the presence of an organic base. The reaction can be represented by the following ion: Biontrant, by way of example, the prepared. 0-Trimethylilyl-N-carboxy serine arhydride ion.

  <Desc / Clms Page number 42>

 
 EMI42.1
 



   As shown, the reaction is a reversible reaction if the equilibrium is such that the equilibrium mixture normally contains large starting amounts. The reaction can be forced to produce the compound trimethylsilcsx by the presence of a base which neutralizes the halogenated acid as it is formed. Preferably, the salt formed is insoluble in the reaction mixture, although it can be removed by filtration. It cannot be expected that the reaction proceeds in this manner since it is well known that N-oarboxy amino acid anhydrides polymerize rapidly, even in the presence of traces of alkalis, in particular in solvents. organic.

   For this reason, weak organic bases, in particular bases enoted with a tertiary nitrogen atom such as pyridine, lutidine, collidine and tri-lower alkyl amines, such as trimethylamine or triethylamine are. employees. Particularly preferred weak base and 4-methyl thiazole.



   With bases such as pyridine having a pH value of 5.2 or more, the basic reagent is added after the main reagents have been mixed. The base should be added with good agitation to avoid local excess. base which could initiate the polymerization of the anhydride.

  <Desc / Clms Page number 43>

 
 EMI43.1
 r; r '. u t, 1, -, - * - I ^ ment the sufficient quantity der base for 2, * a..e: 1 '= u. t '1 \ .. - "elm, With weak bases ràEa ± 41; t 1el + a; y # rs of iX ae 2, Q such that 4-m4thl1' 1a = ce.

   T., distribute these benefits to other workers at any time. halide
In can use an excess of tri-alkyl as
 EMI43.2
 aolvan +, ", 'it is that it is liquid at the temperature raat. It is preferable to use an inert ergmic 801- vant as the reaction medium. Among these solvents there may be mentioned, for example, the solvents. oxygenated in particular ethers such as tetrahydrofuran, dioxane, ethyl or butyl ether, or solvents of the hydrocarbon type, in particular aromatic solvents, such as benzene or toluene.



   The reaction is carried out at a low temperature, for example between about -5 C and about 5 C. It is generally complete in a relatively short period, for example in a period of between 15 minutes and one hour, depending on the quantities of reagent used.



   Usually equimolar amounts of reagent are used to avoid contamination of the desired product. However, a slight molar excess for example, a 5% molar excess of each reagent can be employed without affecting the detrimental den product.



   Although particularly useful for the preparation of derivatives of amino acids which are normally found in association with animal tissues, such as erine, threonine, hydroxyproline, tyrosine and halogen derivatives of tire

  <Desc / Clms Page number 44>

 
 EMI44.1
 ¯ t1f that the $, 5-àibrono- or 515-di10d0% y> osih% -, '(0n tl * and however not limited. It can
 EMI44.2
 ......, 'it "? ix" 9 with just as much ease for pre-. - # <# G6riva analogs of unnatural taminoaoids% * 'r'. '#G 1 [. 8-hydroxyleuoine, 1 '-hydnoxynorvaline, "'." ,, 1 "::, x; 'norval'ine and others.



  Prepared products can be used for the preparation of various heteropeptides as shown in the Examples. If desired, they can also be used for the preparation.
 EMI44.3
 paratio; '' high molecular weight homopolymers, such as polyrin and polythreonine by polymerization in an organic solvent in the presence of a base,
According to a further aspect of the present invention the hydroxyl groups on aromatic N-carboxy6 or N-thiooarboxy amino anhydrides,
 EMI44.4
 ie the hydroxyl groups are phenolic rather than alcoholic in character, are blocked by a tetrahydropyranyl group.

   It has been found that this group is spontaneously and concurrently released during the deoarboxylation or dethiaoarboxplat3an reaction. The new tetrahydropyranyl anhydrides J-oarbX7. amino acid and the N-thio oarboxy amino acid anhydrides of such aromatic amine acids are within the scope of the present invention.
 EMI44.5
 



  It has been found that although phenolic goups h1-droxyl substantially interfere with the coupling reaction in which tyrosine or the like
 EMI44.6
 ect linked to an amino acid or a peptide in a growing polypeptide chain, they do not generate ma-

  <Desc / Clms Page number 45>

 
 EMI45.1
 Appreciable reactions of ooputi.oua 8ube.- quentes in which amino acids 8uppl'men- his :. added to the chain.



   The new compounds according to the present in-
 EMI45.2
 vention is made pi -epep by reaction between i1- 3dihydr appropriate rar 6 .; The anhydride / of 3 - os.rboxy or ± -thio-oarboxy amino: .te in the presence of an oata3.eeur acid, in a typical reaction carried out in the preparation of the invention, 1-carboxy or .- th1oarboZ7 anhyin1àof'e tyrosine is repeated in Qih, ttx rhymes against a ratalytic amount of acids.

    be both as a reagent and as a reactive medium. mpe- the Product this insoluble * Therefore the mixture is simply added, preferably at room temperature, for example between 25 C and
 EMI45.3
 , r5''G until oe / 'neib1ement ltanti (retention of the starting material is dissolved, the product has been sealed by any oc: r: venab1e process.



  Solvents, particularly cyclic ether type solvents, such as dioxan or trab74ro-furan, can be used if desired. Solvents are generally used in cases where an equimolar amount or only a slight excess,
 EMI45.4
 for example a 10% molar excess of dihydropyran is used.

   Since an excess of dihydropyran helps to obtain the best results, it is preferable to avoid the use of solvents and to carry out the reaction using dihydrcpyran both as a reagent and as a reaction medium.

  <Desc / Clms Page number 46>

 
 EMI46.1
 the reaction temperature should preferably not exceed esaai3.cmt 40 * 0, at t --mute the tendency of the dihydropyran to and polywriner to teupd. high erasures. The faction period can vary within wide limits, for example, between 10 and 60 hours.

   Teoemmb- appreciably lower than 2000 should not be amiaer. At tMe & tare M & - biante which is the temperature prdfdr4ti, the Té) MjL <m is generally completed in 30 to 50 hours * the acid type catalyst <ex1; pif'mono a catalyst of the organic acid type such as IgteJAe para-tolueneaulfonique, sultoniqmg acid. or 1 corresponding rulfonyl halidee, ea partlmaler sulfonyl chlorides. Inorganic acids, in particular mineral acids, such as sulfuric or hydrochloric acids can atM wtili <4a The chosen catalyst is used in qmaatitea eatayti <a <Mt, for example, in quantities between ea.vir <! & 40 to about 100 mg per g of anhydride or thioonWà1iàe.



  The preferred amount from the point of view of obtaining
 EMI46.2
 economic efficiency is between
 EMI46.3
 ut 60 mag per g of amino acid derivatives <To. at the end of the reaction period the product can be isolated by chromatography if desired <However, the preferred method is to dilute Reaction Mixture # 1 by the addition of a precipitating liquid such as a hydrocarbon or a mixture of hydrocarbons containing up to 8 carbon atoms. . Petroleum ether
 EMI46.4
 is suitable for this purpose, although an aromatic hydrocarbon
 EMI46.5
 such as benzene can also be used.

  <Desc / Clms Page number 47>

 



  The reaction is suitable for the preparation of
 EMI47.1
 polypeptides co. tyrosine and other aromatic amino acids, such as 3,5-dibromotyrvina, and 5-diiodotyrvina.



  The products prepared can be used for the preparation of a large number of thetropoptidon according to the methods illustrated by the examples. If desired they can also be used for the preparation.
 EMI47.2
 preparation of high molecular weight homopolymers such as polytyroeine by polymerization in an organic solvent containing a base. Such polymeric compounds are used on a large scale as model compounds for the study of properties. physical structures of protein-like structures.



   Although most of the tetrahydropyranylic segment is removed during the
 EMI47.3
 decarboxylation or dethiocarboxylation which, aâmma illustrated sweats. take place under acidic conditions # it is not essential that the state be completed during the first copulation resation.

   This reaction can just as easily be terminated at
 EMI47.4
 During copulation reactions that occur frequently ... In fact, under certain conditions, it may even be preferable for it to be paused in this way. ? ar example, in
 EMI47.5
 In the case of preparing a d6capeptide in which tyrosine is the second segment of the chain, considerable reaction time can be saved if the decarboxylation or dethiooarboxylation is carried out as rapidly as possible.

   During this period, the greater part / fraction titrated

  <Desc / Clms Page number 48>

 
 EMI48.1
 b.droranylic will be removed, but the amount which at this point will remove you, during subsequent population reactions. An additional novel compound which is within the scope of the present invention is N- acid anhydride. aapartic carboxy which is prepared by reaction with aspartic acid and at least an equimo-
 EMI48.2
 ln0 '3s phosgene at a temperature oomprise between 15 0 and approx on 30 C and which is isolated by a process, in the (here product and transferred to another solvent at point,

  higher boiling which is possible with the inert reaction solvent before the solvent originates.
 EMI48.3
 ginel s1t completely eliminated. Oe01. And: 4a111é by adding a large amount of the second solvent to the reaction medium at the end of the reaction period
 EMI48.4
 and by cooling the reaction mixture until substantially all the entybridity of the original solvent has been removed, so as to leave the product still dissolved in the second solvent. The second solvent is normally a poorer solvent for anhydri-
 EMI48.5
 of 1-carboxy aspartic acid and is generally less polar than the reaction solvent.

   The product is precise
 EMI48.6
 pit of the second solvent by adding a miscible liquid in which the product is insoluble. In the preferred method, sufficient liquid precipitant is added to make the mixture slightly cloudy. The mixture is then stirred until crystals
 EMI48.7
 of product is formed, after which additional precipitate is added, the original crystals serve as starter or growth crystals and the product

  <Desc / Clms Page number 49>

   thus obtained is of particularly high purity.



     It is also desirable to precipitate the final product from a relatively dilute solution, since a more highly purified product can be obtained from a dilute solution.



   Any of a number of pairs of organic solvents can be used as the reaction solvent and the transfer solvent. The reaction solvent is inert to the reaction and boils at a lower temperature under normal conditions than the transfer solvent and preferably has better solvent capacity for reactants and products than the solvent. Suitable reaction solvents include ethers containing up to about 8 carbon atoms, although ethers of low molecular weight, such as ethyl ether and propyl ether are available. preferred because they have lower boiling points.

   Particularly suitable are ethyl esters such as dioxane and tetrahydrofuran. Non-cyclic ethers, particularly low molecular weight ethers, often contain traces of alcohols and peroxides. Since these chemicals can cause undesirable side reactions to occur, they must be removed before using the solvents, so as to render the solvent inert to the reaction.



   Preferred transfer solvents are aliphatic esters containing up to about six carbon atoms since these products combine the characteristics of generally possessing a eu- boiling point.

  <Desc / Clms Page number 50>

 higher than that of ether and to be generally of))) good solvents for reagents and produces them than ethers, while having the property of being msic- able to ethers. Pairs of reaction solvents and suitable transfer agents are, for example, tetra-drofuran-ethyl acetate, dioxane-acetate -b-butyl, ethyl ether-ethyl acetate and propyl ether propyl acetate.



   The precipitating liquid should be miscible with the transfer solvent and the N-carboxy aspartic acid anhydride should be substantially insoluble in that liquid. Preferred precipitants are cycloalocans, liquid alkanes and mixtures thereof containing up to about 10 carbon atoms. Hexane, oyolohexane, octane, isoootane, nonane and the mixture of these compounds optionally containing other aloans or oyoloaloanes constitute typical precipitation liquids,
In the process of the present invention;

     The aspartic acid is reacted with phosgene in the reaction solvent at a temperature of from about 150 to about 30 ° 0, preferably at room temperature, i.e. at room temperature. 25 to 30 C in the chosen reaction medium. To obtain the best results, at least an equimolar amount of phosgene is used, although it is preferable to use an excess of phosgene.



  A molar excess of this compound of 200 percent and even more can be used without having an adverse effect. At the end of the reaction period, which varies with the amounts of reagents but which is generally between about 10 and about 120 minutes, the solvent

  <Desc / Clms Page number 51>

 
 EMI51.1
 transfer is added and the ooly = tu ramais are distilled Bous Ni13sion reduced * until k and the antierate of the reaction solvent is ohaamaa, The faction product can then be considered 00 ... being transferred to the tranaafert solvent.

   The product
 EMI51.2
 can then be precipitated by the addition of the liquida
 EMI51.3
 precipitant, 1 but, preferably, the solution is alleviated by the addition of a larger quantity of transfer solvent before operating the precipitation. the amounts of solvents and strong liquid used vary with their identity.

   Gem4ral ..- at,
 EMI51.4
 the amount of transfer solvent will be at least equal
 EMI51.5
 to the volume of the reaction solvent and this amount can be up to five times the volume of the solvent da ra <.otiom, although the relative amounts of solvent * powering vary appreciably between these limits *. 8. the boiling point of the transfer solvent would have been significantly higher than that of the riaotiv% 4 solvent. However, large amounts of the trtMIA solvent! f% p8UY could be used, since more than that of the solvents at point d lower boiling p & a., .. "rtm1i in the distillate.

   If the boiling points are relatively close to each other, at 8 & Yair, per bzz to 5 k 10., more tratmatic solvent should be used. <tnaf * st ut t be added in one tois, in which case 8in8ibl. 1% p = tibrati of the reaction solvent will be removed in dilution.

   If we added the solvent for tran * hO ¯on two several fractionsst the reaction solvent is used without, in two or more stages of d, ati, 3.aion, 3n none

  <Desc / Clms Page number 52>

   water The distillation / should be continued until all or most of the solvents have been removed, since under these conditions the desired product will usually continue to react to form gummy products, probably of a poly- mother.



   The amount of precipitating liquid used will vary with the identity of the transfer solvent, the particular precipitating liquid chosen and the concentration of the product in the transfer solvent. Generally it is preferable to precipitate the product from dilute solutions containing up to about 2% by weight solute, as purer products are obtained from dilute solutions. However, more concentrated solutions can be used, if desired.



   The optimum effective amounts of solvents and precipitating liquids for specific solvent pairs and for precipitation can readily be determined by test reactions. For example, various amounts of transfer solvent can be added to fixed amounts of reaction solvent and the mixture can be distilled under reduced pressure until the mixture no longer meets the tests for the solvent! reaction.

   Various amounts of precipitating liquid can be added to the transfer solvent containing N-carboxy sapartic acid anhydride and the amount and quality of the precipitated product can be tested to determine the most effective amount.

  <Desc / Clms Page number 53>

 
 EMI53.1
 ficnoe of <tg <mt of 4 <diB! aam JieB 8eJ.wm'it8 it 'A pr.-d.; t1 uc3. also e'Ay <! -! - <B! flue a! f! im. 410 idxïitmaw I; s4 1 té dfl peets pnrthti t pr a & BRjjRtM 'Un on re & eti & B 1neru, pr B181t ce: D.11 $ 3)) (iI- tee for m1n1m1 & n 1- r6artlun ..nrt6a1f't <08tt auwmg "- xe noting oepeztmt pu * aar. E.



  Itaat tomé la tJ.u11.l'4.1toI '8'ft'D IMIXMUM: n ......



  .-left'!. .... tranefMTa * in a: r3, ti. "1J ...... 1J81I8 .. a: nhyr.d.!" '&. T. a1ldM> * 1: 111.t'Qe..tl dtutt! D8I; "" 4. irc-Uter gi 1 1L-O'Cfa, <l1t Zw 'p, rMen1be ttk8. lady produces J'lls11> 1.mt ': J1't ptELBaodL apat 1 * 2ama dt "dtokllv 1.'t8D &l; rtl. ti 4 * frt a 4 & eewwert tU le teai%% Ill. 1 from lu bzz par de c' ass Ao l1t ... r1rt.ut pure .. de l'n.U8 -t1ta "41 U1.no nOcbtbu. # aurd. t obte-, a la t da .. 1.1 ......



  81 and from 1.ú .. c- prodilte 4 x4oU- 4 & -alestet 9b! T <M'v < <t <M lex r4a0U- ..att..108Lt; ... syUtri. t. 1.8: ¯the ass ... Mr ...



  Another pelt 441 la pr4s "4 mit: Lm omk- seme 1.1Rnydrlde de jf-cbo <MtpM'e% mw 1. ' My.



  I-m3.rb.ox, - glatanin and the prooü- for the. 3 .. of oes oom: pcs. 088 compoe48 are bl.t4re8l88D.1ia for the preparation of,., 1; 14 .. oGl1tse: a1 de l '.. pareg1Ja8 ft da la: g111tas1ne or at the liver this * two 81114 .. M .., - u .
 EMI53.2
 acid,
 EMI53.3
 M-carboxy ..,. 4 \ 1ae .1; do ¯ glutamine has not yet been prepared 4year * the 1ieo1m1 ... that earlier, the pro & 1 Ola8i.q, ue by leq 1

  <Desc / Clms Page number 54>

 
 EMI54.1
 the N-oarboxy amiao anhydrides have been prepared both for the reaction of an amino acid with 3M phossnw and for the desired products. , ¯¯ ¯, ¯ ¯ We discovered according to the <MaLt <e lavmtîm that the desired products can be drawn # µpq6 * # * o 4o õpsç% n4enen% s by, reaction between ri'aex da Y. phoaphore and Ilasparagite or ï. <ïtaadjM, to e <m-. - <. "* '" -t.- # <... "y ..,. <, fs, E <. # g. ... t. <.

     .. dition that, .the function a-asno44e âanclàt. e l 'e a rua .. po.tlatear you q the benzoxyoarbonyle zozo. the protector group is o3rai * yes <of the reaction. It was more î pdz4 of hole r that phosphorus tribromide this 4apabl * 4'offoo% * 1 the desired oyolization a-foc ellaini14n 6o & 8 <tZ'c <mtw of the blocking group in particular peÀa 40 at a functional non-protected amide goeup, we would have p t '& tt <a & to a considerable impediment of the% ,, Îaotï têtitdâ M operating in this way, 3app.9, oat. ooutout 4t 1oaa of phosphorus tribromide in the preparation year. of these valuable anhydrides was purely and inoperable due to the fact that other <1 '*' - io6é =;

  a% ion, such as phosphorus pentachloride and 1 chloride of hiouyl, nc give paa..3.ea âuta ymaut'o These oompséSt as it is well known posed similar activities to stick phosphorus tribromide in many chemical reactions <. , ¯,, ¯ ¯ ,, ¯ As suitable blocking groups which should be used, to protect the c-aaino group from the accounts: 'starting, there are 108.8 groups alooxyoarbonyl and "'" ..... aralkoxyoarbonyl containing, of suitably up to 8 carbon atoms such as methoiw- radicals

  <Desc / Clms Page number 55>

   oarbonyl, ethoxycarbonyl, propoxyoarbonyl, iso-amyloxycarbonyl, benzoxycarbonyl and substituted benzoxyoarbonyl, such as methyl, ethyl, methoxy, ethoxy, chloro or bromobenzoxyoarbonyl radicals;

   in particular when these substituents are in the para position in the benzyl part of the benzoxyoarbonyl group.



   The reaction between asparagine or protected glutamine, for example, benzyloxyoarbonyl asparagine or glutamine, and phosphorus tribromide is carried out by mixing the reagents in an organic solvent inert to the reaction or in a mixture of such solvents. , preferably a cyclic ether, such as dioxane or tetrahydrofuran at a temperature of from about 15 to about 30 ° C. for a period of from about 2 to about 24 hours. It is preferred, for reasons of convenience. , to carry out the reaction at room temperature, namely between 25 and 30 ° 0 while stirring the reagents in a selected solvent medium for a period ranging from 2 to 8 hours.

   Although a slight excess, i.e. a slight 10% polar excess of the tribromide can be used, it is preferable to use equimolar amounts of the reagents, since under these conditions the danger of occurring. side reactions is monk great. An inert atmosphere, for example a nitrogen atmosphere can help to minimize side reactions, however this nitrogen atmosphere is not essential.
At the end of the reaction period, the products are isolated by chromatography on silica gel using increasingly polar solvents for the elution.

  <Desc / Clms Page number 56>

 



   As previously described, the novel compound is suitable for the preparation of dipeptides and polypeptides, including high molecular weight heteropeptides. The methods of forming peptides are illustrated in the examples.



   Another aspect of the present invention by which hydroxylated amino acid peptides can be prepared relates to novel amino acid chelates
 EMI56.1
 hydro: "71és. More particularly the invention relates to metallic or oh61ated α-amino acids which are substituted by a hydroxyl group. These compounds are useful for the preparation of anhydrous N-oarboxy of these. amino acids, more particularly, the N-oarboxy anhydrides of tyrosine, threonine, serine and hydroxyproline. The preparation of these anhydrides is within the scope of the present invention. The N-oarboxy anhydrides, in turn, are suitable for the syn-
 EMI56.2
 thbut of etheropeptides and other proteinaceous substances. containing these amino acids.



   A suitable process for the preparation
 EMI56.3
 of anhyr1de. of N-carboxy amino acid consists of reacting the acidic amine / with phosgene in an organic solvent.
 EMI56.4
 The reaction is usually inert for life with respect to the reaction, usually the reaction is carried out by taking up the amino acid in the reaction mixture at a temperature of between about 2500 and 10000. This process, although suitable for the preparation. The anhydride, # e X-carboxy amino acids in which there are no other functional groups such as the α-amino group and the carboxyl group, is ¯, ¯ generally unsatisfactory otherwise. function groups

  <Desc / Clms Page number 57>

   .nels which are reactive with phosgene are substituted on the Ó-amino acid.

   Hydroxyl groups such as present on serine, threonine and similar amino acids are particularly troublesome from a point of view given the high degree of activity of the hydroxy group. Therefore, it has long been used. It is customary in practice to protect the hydroxyl group before carrying out the phosgenation reaction. Oeoi results in an undesirable decrease in yield since the blocking group must later be removed in a separate reaction.



   It has now been found that according to the present invention it is possible to prepare anhydrides of
N-car / xoy amino acid of serine and the like without the need to block the hydroxyl group. This desirable result is obtained by forming a metal or oelate complex of the selected amino acid, for example serine silver chelate, prior to phosgenation. In the ohelate, the amino group of the amino acid is apparently activated so that when the chelate is exposed to the phlsegen, the N-arboxy anhydride is formed with minimal interference from the hydroxyl group.

   A particular advantage of this process is that the hydroxyl group in the N-carboxy anhydride thus obtained is free so that it can be protected by groups such as the trimethylsilyl group or the trifluoroacetyl group. These groups, as described above, are cleaved during the formation of peptides under the conditions described above, but are too labile to resist the phosgenation reaction.

   It is thus possible to prepare

  <Desc / Clms Page number 58>

 peptides without the need to perform a re. further action to remove the proteotor group of a radioal hydroxyl using anhydrous N-carboxyls prepared by the process of the present inventions
According to the preferred method of the present invention, salts. Metals or transition metal bases are reacted with the selected hydroxy amino acid and the chelate thus obtained is phosgenated to give the desired N-arboxy amino acid anhydride.

   Transition metals which include copper, silver, gold, of group Ib of the periodic table of the elements; cadmium and mercury from group IIb of the periodic table of the elements; thorium from group IIIb of the periodic table of the elements; and iron, ruthenium, osmium, cobalt, rodium, iridium, nickel, palladium and platinum of group VIII co-ordinating with nitrogen so as to give rise to the formation of ohelate .



   The reaction is preferably carried out in an aqueous medium using metal salts or bases which are at least partially soluble in water.



   Salts or bases of the aforementioned metals can be used in the form of hydroxides, oxides, sulfates, chlorides; nitrates, carbonates; acetates; perchlorates or the like. Copper, silver and meroure are particularly preferred because they are readily found and because they form salts which are extremely soluble in common phosgenation solvents. These salts can easily be reacted with the medium / phosgenation by filtration. Silver oxide is particularly suitable since the

  <Desc / Clms Page number 59>

 Silver chloride is extremely insoluble in the phcsgeation medium.



   Copper chelates with hydroxylated amino acids in the form of cupric ions, as well as cuprous salts can be used in the process of the present invention since they readily acididize in the cupric form under the conditions of the process. reaction.



   Mercury is used in the form of the mercuric compound. The mercury chloride which forms during the reaction of phosgenation is soluble in the reaction medium. This salt is easily reduced to meroureux chloride by the addition of an approximate equimolar amount of metallic meroure. The merous chloride is insoluble and is easily removed by filtration.



   The chelate is formed by adding the selected chelating agent to the aqueous medium containing the hydroxylated amino acid at a relatively low temperature of up to about 25 C. It is preferable to carry out the reaction at around 0 C. at about 10 ° C since the lower temperatures minimize the formation of an unwanted colloidal by-product which can be observed to form during the addition of the chelating agent.



  The reaction is continued at the selected temperature, preferably with stirring, until substantially all of the loosening agent is dissolved. The reaction is best controlled using equimolar amounts of chelating agents and amino acid, although somewhat less or somewhat greater amounts may be used, i.e., for example, amounts. by 5% above or below the equivalent amount. Yield is not necessarily reduced by

  <Desc / Clms Page number 60>

 using too little ohelating agent.

   The purification problem may be unnecessarily complicated in obtaining a compensating increase in yield by the use of too much chelating agent.



   At the end of the reaction period, the chelate can be recovered by collecting the precipitate formed by the addition of an organic solvent miscible with water,
 EMI60.1
 Preferably a lower aloanol, such as methanol in the vctional medium.



   The phosgenation is carried out in an organic medium which is inert towards the reaction, preferably an oxygenated solvent comprising ethers such as
 EMI60.2
 tetrah drofuranel di-n-but 1 or dioxane.Ze chelate. , to /% é% rahydrofuran, al-n-bu% ylÀ # dioxane. The chelate,. for example, the silver salt is taken up in the organic liquid and the reaction is carried out in a suitable manner, passing an equivalent molar amount of phosgene in the mixture at a rate such that too rapid an increase is prevented of the temperature.



  The duration depends on the quantities of reagents used.



  An excess of phosgene should be avoided in such a way as to
 EMI60.3
 exploit the possibility of reaction with hydroxyl groups. As soon as all of the phosgene has been added, the reaction mixture can be heated for about half an hour to about 2 hours. An inert atmosphere, such as nitrogen is preferably used to minimize side reactions. The desired product can be isolated by freeze drying after removing the metal halide which forms.

  <Desc / Clms Page number 61>

 



   The products formed according to the process of the present invention are useful for the preparation of peptides as illustrated by the examples,
Although the invention has been described as being particularly useful for the preparation of amino acid derivatives which are normally found in association with animal tissues such as serine, threonine, hydroxyproline, tyrosine and halogenated derivatives tele tyrosine as 3,5-dibromo- and 3,5-diiodotyrosine, however, it is not limited thereto. It can be used with just as much ease to prepare analogous derivatives of amino acids " non-natural "such as ss-hydroxyleucine Ó-hydroxynorvaline, Y-hydroxy-norvaline and the like.

     aspect '' Another aspect of the present invention relates to the processes for the preparation of peptides containing basic amino acids and to the new compounds used for these preparations. More particularly, it relates to the preparation of novel N-oarboxy halomerouric anhydrides of basic amino acids, such as arginine, histidine, lysine and ornithine and to the use of. these compounds for the preparation of peptides.



   The synthesis of peptides containing basic amino acids, such as lysine, histidine and arginine has posed a particularly difficult problem because of the presence of the additional functional groups. Several methods have been described to avoid the difficulty. In most of them..the group. additional functional is prevented from entering the peptide formation reaction by protecting it by

  <Desc / Clms Page number 62>

 an easily removable group. The imido part of hiidine, for example, can be blocked with a benzyl group which is then removed by hydrogenation or other reducing process.

   Although this procedure may be acceptable for simple peptides containing only a few amino acid segments, it often poses problems with more complex peptides where the conditions required for removal of the bonzyl group or other. protective group are often so stringent that other functional groups are adversely affected *
The adaptation of the process to NOA to basic amino acids is often troublesome mainly because of the fact that the additional functional group reacts during the formation of anhydride.

   It is usually better to protect the additional basic group so as to avoid loss of yield in peptide formation, loss due to the reaction between these groups and a molecule / anhydride. in preparing lysly-phenylanine by reaction between anhydride of
N-carboxy-lyeine and phenylalanine, the yield may be reduced by reaction between one anhydride molecule and the (-amino / group of another anhydride moleolina unless the -amino group is protected.

   Usual protecting groups such as the benzyloxy-oarbonyl group or. its analogs and homologues are, however, unsatisfactory because of the difficulty of introducing and removing the protecting groups and the fact that the yields which are often obtained in these reactions are low.

  <Desc / Clms Page number 63>

 



   According to the present invention, basic amino acid N-oarboxy anhydrides can be readily prepared and used in the NCA synthesis. In the process, a basic amino acid halomerouric salt is first reacted with chosgene to prepare an N-carboxy balomerouic anhydride, such as N-oarboxyarginine halomerouric anhydride.

   in which the additional functional group is protected by an XHg group in which
X is halogen are then reacted with an amino acid under intermediate controlled aqueous alkaline conditions (so as to produce oarbamates / which are decarboxylated by the addition of acid, thereby to give peptides.



   Mercury ions in the solution are removed by precipitation with hydrogen sulfide.



     This will be more easily understood by referring to the following reaction sequence which illustrates the invention as applied to the preparation of lyslyl histidine,
 EMI63.1
 

  <Desc / Clms Page number 64>

 
 EMI64.1
 
In the aforementioned series of equations an ethylene diamine tetracetic acid (EDTA) buffer

     is used as an illustration of the class of buffers which can be employed to relate the hydrogen ion concentration of the reaction mixture to the course of the

  <Desc / Clms Page number 65>

 copulation reaction *
The halomerouric salt is first prepared
 EMI65.1
 by reacting the amino acid and '*? wt mol equivalents of T% ho.3.odnide mercuric in aqueous alkali * Well% It other mercuric halides may be ui: 1.i:; s, it is preferable to use merouric chloride because of its superior lubility in comparison with other morcuric halides. The reaction normally effootitates with mixing;

   reactants in aqueous aloali at a pH of about 8 to 9 at a temperature of about room temperature that is
 EMI65.2
 Say 25 to 3500 and about,.,.,. ', 75%, allowing the reaction to proceed, preferably with stirring, until substantial amounts of product are formed.



  The reaction time varies with temperature, amounts of reagents and other factors, but in most cases a reaction period ranging from
 EMI65.3
 about one to three hours are: "'.': ia.taiaantee. The pH can be kept within desired limits with alkali metal carbonates, particularly alkali metal carbonates, such as sodium or sodium carbonate. lithium.



  At hydrogen ion concentrations appreciably within 8, the yield of desired salts containing two mercury ions is considerably reduced. At concentrations of hydrogen ions above pH 9, hydroxyl ions interfere with the course of the reaction.



     For convenience, the salts thus prepared will be referred to herein as "salts.
 EMI65.4
 halomerouriqueem,

  <Desc / Clms Page number 66>

 
 EMI66.1
 The halomercuric salts are converted to halomerouric N-oarboxy anhydrides by re; c + # on with phosgene in a pclairo organic solvent. to prefer- 08; a ketone containing, for example, up to eight carbon atoms. Acetone is preferred because it is readily available. The reaction is preferably carried out
 EMI66.2
 at a low temperature, i.e. between approx. <> 10 ° C. and about 10 ° C. by passing phosgene preferably, with stirring, through a mixture of the anhydride in a chosen solvent.

   Temperatures somewhat above or below these limits can be employed if desired. The new compounds as well
 EMI66.3
 The products may exist as polymeric compounds, but may react as a monomer> 38 and may be represented by formulas such as Formula II mentioned above.



   At the end of the reaction period, which is normally between about one and four hours, the mixture contains the merouric halide and the new
 EMI66.4
 N-oarboxy amino basic halomerouric acid anhydride in solution. Merouric chloride is reduced to sea salt
 EMI66.5
 t, 1: r:; soluble E <J "C '' '': - 7Z7q: ui-can then be separated together with the oxob of metallic mercury by filtration or by any other suitable method. The desired product can then be isolated by removing the solvent. , preferably, by distillation under reduced pressure.



   The anhydride, thus prepared, can be trnas-
 EMI66.6
 ? adorned with a peptide, including dipeptides and p11peptides, by reaction with an amino acid or a

  <Desc / Clms Page number 67>

 peptide / at a low temperature, preferably between
 EMI67.1
 about 09 and 10DC in an aqueous alkaline medium over a reaction period of from about 30 seconds to about five minutes with good stirring. The intermediate carbamate which forms is then deoarboxylated pre-
 EMI67.2
 férexca, by the addition of acid, so as to reduce the 3rd pH of the medium to about two to oinq.



   The carbamate is formed under alkaline conditions, preferably at a pH between about 8 and 10.5 * The desired alkalinity can be maintained using any of the known basic, solid or liquid reagents, such as. than magnesium oxide or
 EMI67.3
 It is often necessary to add an additional basic-reagent during the reaction so as to maintain the desired alkalinity. Various buffers can be used. The besic reagent, however, must be included. one which does not react with the merouric ion present in the reaction mixture to pre-
 EMI67.4
 oipitate a merouric salt.

   For this reason, borate and phosphata buffers do not remember, except when used in small amounts in combination with other buffers. A buffer system which has proved to be particularly interesting for fine
 EMI67.5
 of the present invention is zaza, o'eat-d1re ethylene diaminettrdbetic acid. This buffer is prepared by taking up EDTA in water, usually until. one molar concentration and then added euf-
 EMI67.6
 Using the alkali metal hydroxide to adjust the µg to that desired. The buffer can be used along with small amounts of borate buffer if desired.

  <Desc / Clms Page number 68>

 



   After deoarboxylation, the reaction mixture mainly removes the desired product, i.e. the basic amino acid containing the peptide and the mercic ion, which can be combined with the buffer to form a soluble salt. The merouric ion is removed from the reaction mixture by precipitation as the sulphide. This is suitably done by passing hydrogen sulaide through the solution.



   The precipitated mercuric sulfide is preferably filtered off.



   As desired, the peptide can be recovered chromatographically from solution, or it can be recovered by freeze drying or any other suitable method. The mixture can also be made alkaline by the addition of a buffer or other base and the peptide prepared according to the present invention can be transformed into a higher peptide. These two alternatives are illustrated in the examples.



   The process is applicable to all basic amino acids, whether natural or not. It is applicable to both L and D forms of acids and to raoemic mixtures. It does not affect the symmetry of the molecule at all, ¯¯ ¯ ¯, ¯
Another feature of the present invention is the preparation of addition salts with N-carboxy arginine / anhydride / and N-carboxy histidine anhydride.

   More particularly, the invention relating to ohloridrates and bromidrates of N-carboxy arginine anhydride and N-carboxy histidine anhydride, The addition salts with acids

  <Desc / Clms Page number 69>

 prepared according to the present invention are useful for the preparation of peptides containing argine or histidine or these two amino acids at the same time.



   Previous attempts to prepare acid addition salts of N-arboxy arginine anhydride have not been successful. The conventional process in which the N-arboxy amino acid anhydrides are formed by reacting a mino acid with phosgene is generally unsatisfactory with arginine / state that virtually no desired product is formed. Bromidrate and N-arboxy histidine anhydride hydrochloride are new compounds.



   It has been found that the desired products can be prepared with good yields by reaction with halogenating agents and arginia or histidine, provided that the Ó-aimno el'amino aid function is blocked with a blocking agent. that it is sterically free and able to take a more positive fourarge by donating a pair of electrons during the reaction.



   The halogenating agents used in the process of the present invention encompass a known class of halogenating agents of the type generally employed for the preparation of acid chlorides and acid bromides. They include, for example, thionyl chloride and bromide, phosphorus pentaohloride and pentabromide and the corresponding trihalides.

   Among these compounds, it is preferred to use phosphorus tribromide for reasons which will be explained in more detail below in this specification,

  <Desc / Clms Page number 70>

 
Blocking agents which may be employed include alkoxycarbonyl and aralkoxyoarbonyl groups, advantageously containing, for example, up to 8 atoms.
 EMI70.1
 The carbon atoms and which are 100% precluded due to the fact that the carbon atom of the alooxy or aralkoxy group which is attached to an oxygen atom is substituted by at least two hydrogen atoms. In addition to the fact that
 EMI70.2
 this atom) of oarboneyd. must be eterically free, the group must be one capable of becoming more posi-
 EMI70.3
 tif during the reactions giving a pair of eleotr es.



  The preferred carboxyc & rbonyl group is%>: radioal melhnxy-oarbonyl. gives that it is most easily polylated, although other alooxyoarbonyl groups can also be used. Typical alooxyoarbonyl groups which are suitable as blocking agents herein.
 EMI70.4
 invention are, for example, ethoxycarbonyl radicals; = ropo # yocrbonylo, isobutoxycarbonyl, or ieoamy11xyoar-;) onyl. The preferred aralooxyoarbonyl group is the Ocnqycrbony radical although other aralooxyoarbo-syl'3, 211 groups especially the benzoxycarbonyl aubsti- groups. 'u'j, such as methyl- or ethylbenzoxycarbony-lc groups; w6thoxy- or ethoxybenzoxycarbonyleg or chlorine- or bromobr-Áyoarbonye can also be employed.



  Leu substituents on the benzyl part exert their most favorable electronic effects if they are in the para position, although this is not essential.
The desired blocking groups are derived from the corresponding ohlo- @@@ Ce or bromides, for example chloride.
 EMI70.5
 do benzylox1oarbonyl or methoxyoarbonyl bromide.

  <Desc / Clms Page number 71>

 



  If a chloride is used, a hydrochloride is obtained,
 EMI71.1
 The bromides tl01L ':: 1t' t, 3e bromidratest p-nitrobenzyloxycarbonyl chloride analogues in which the substituent on the benzyl group exerts a strong inductive effect cannot be used
 EMI71.2
 as sources of blocking groups for the present invention.

   exact
Although the course of the reaction by which the compounds of the present invention are prepared is not fully understood, it is believed that the reaction takes place with the aid of an intermediate which in the case of blocked arginine by a benzyloxyoarbonyl radical can be represented by the following structure
 EMI71.3
 
The positive charge which indicates an electron deficiency in the ring structure is neutralized by loss of electrons from the benzyl group which separates as an oarbonium ion and is, in turn neutralized.
 EMI71.4
 by a halide ion released by the dthalogenatïon agent.



  This mechanism is corroborated by the fact that benzyl chloride is released during the reaction. This mechanism also helps to explain why the. blocking group must be eterically free and capable

  <Desc / Clms Page number 72>

 to donate electrons. Unless the group is sterically hindered, the halide ion is prevented from approaching the Ó carbon atom. The positive charge on the cyclic part cannot be neutralized unless the group helps electrons.

   The lack of utility of the p-nitrobenzyloxycarbonyl group in the process of the present invention is also explained by the proposed mechanism. Due to the strong inductive effect or nitro group, the blocking group does not give its elec- tr. consider the cyclic part,
The reaction can be carried out with or without a solvent. Suitable solvents include solvents of the aromatic hydrocarbon type containing, for example, up to 9 carbon atoms, such as benzene. or toluene. Cyclic ethers containing up to 8 carbon atoms, in particular solvents of the cyclic ether type, such as dioxane or tetrahydroofuran are of particular interest.

   With halogenating agents such as thionyl chloride or thionyl bromide, an excess of the reagent can be used as a solvent.



   In a preferred aspect of the present invention, phosphorus tribromide is used as the halogenating agent and tetrahydrofuran is used as the solvent Aveo this combination of the amino acid reacts with the halogenating agent, forms an intermediate. soluble and the desired product precipitates from solution. The reaction is carried out at room temperature, i.e. between 25 and 35 ° 0 without the need to adjust the temperature.

  <Desc / Clms Page number 73>

 



   The reaction can be carried out between wide temperature limits, for example, between about room temperature and about 100 C. The preferred reaction temperature is based on such factors as the activity of the halogenating agent, the temperature. particulate solvent, if one is used, and the amount of reagents,
Normally the reaction is very fast.



  * Reaction time will vary with factors such as those listed above.



   For reasons of ease, it is preferable to carry out the reaction as quickly as possible, for example, over a period of about 2 to 15 minutes, although the reaction may extend over a period of up to a period of time. hour and even more.



  , ¯ Although equimolar amounts of reagents can be used, it is preferable to use an excess of the halogenating agent in order to obtain as high a yield as possible. Normally at least a $ 10 molar excess of halogenating agent is used, it being understood that the use of an excess of fifty times the halogenating agent or even more is often useful. mentioned above, the halogenating agent can be used as a solvent.



  With thionyl chloride or thionyl bromide the yield can often be improved by carrying out the two-stage reaction. In the first stage, the reaction is carried out in a solvent such as benzene.



  At the end of the reaction period, the solvent and the excess halogenating agent are removed. The residue is pounded and the reaction is repeated omitting the solvent.

  <Desc / Clms Page number 74>

 



  It is believed that in the first stage the product forms a coating on the starting amino acid which prevents the reaction from continuing. Pounding breaks down the degradation so that the reaction ends in the second stage.



   The following non-limiting examples illustrate the invention.



    EXAMPLE ') .- valyl-alanyla-alyanyl-prolyl-phenylalanyl-arginein. -
A mixture of 4PO mg (2 moles) of arginine hydrochloride in 20 ml of aqueous potassium borate, one serving as a buffer oontaining 5 g of ice to a pH 11 is cooled to. 0 ° C. and 386 mg (2.01 mole) of solid N-arboxy phenylalanine anhydride are then added thereto with rapid stirring. The mixture is mixed for one minute and the pH is adjusted to 3 with concentrated sulfuric acid at 0 ° C. while nitrogen is bubbled through the mixture for about 10 minutes. The phenylalanylarginine produced is not produced. is not isolated.



   The pH of the mixture is adjusted to 9.56 by the addition of concentrated potassium hydroxide and the mixture is cooled to 0 C. 5 g of ice are added then 285 mg (2.02 m mo) d N-carboxy proline anhydride and the mixture is mixed for three minutes. The decarboxylation is carried out as described above and the product is not isolated.



   The above process is repeated at pH 10 with the addition of 235 mg (2.04 moles) of N-carboxyanine anhydride, with mixing for one minute. The alanyl-alanyl-prolyl-phenyl-lanyl-arginien which is obtained by deoarboxy- the% ion is not isolated.



   The same process is repeated at pH 10 with stirring.

  <Desc / Clms Page number 75>

 for one minute using 235 mg (2 m moles) of main N-carbexy-alanine anhydride to obtain alanyl-alanyl-propyl-phenylalanyl-arginian which is not isolated.



   Repeating the process at pH 10 with 294 mg (2.05 moles) of N-arboxy valine anhydride gives valyl-alanyl-alanyl-prolyl-phenylalanyl-arginine.



   The pH of the solution thus obtained (46 ml) is adjusted with aqueous sodium hydroxide to
7.5 and the solution is introduced into a column of
30 ml of IRC 50 resin on the acid cycle contained in a column with an internal diameter of 1.5 µm, using a feed rate of 1.0 ml / min. The column is washed with 200 ml of water at a flow rate of 2 ml / min and is eluted.
75 ml of 0.25 N sulfuric acid, followed by 75 ml of acid containing 1.0 N sulfuric acid. The eluate / peptide containing the arginine is collected in 25 ml fractions at a flow rate of
1 ml / min.



   Unit samples of all fractions are reported as spots for circular ohromatography in the secondary butanol / water / acetic acid system, 6/3/1. With a solvent front at approximately
6 cm, eluate fractions 3, 4, 5 and 6 show powerful ninhydrin positive bands at an Rf of 0.39 indicating the presence of the desired hexapeptide, (Rf values of product and reagents are presle. - clearly determined by thin layer chromatography),
These fractions are combined, concentrated under vacuum, the pH is adjusted to 7.5 with an aqueous solution of hy-

  <Desc / Clms Page number 76>

 sodium droxide and then freeze dried.



  The freeze-dried solid is extracted with four fractions of 10 ml of methanol and the methanol is removed in vacuo. The residue is dissolved in 6 ml of water and the pH is adjusted to 3.0 with aqueous sulfuric acid. and the product is precipitated by the addition of 25 ml of metinol, this addition being followed by the addition of 55 ml of ethanol. The suspension is refrigerated until the next day and filtered to give the desired product as produced by the analysis of amino acid Spinoo after hydrolysis.



   Below is a list of amino acids which are used in the liver in the acid form and in the form of N-oarboxy anhydrides in the process of the present invention to produce various polypeptides. An abbreviation is assigned to each. amino acids and in the following list, products which are prepared by the process of the present invention are identified by these abbreviations.



   In the second line, the product which is identified as GLY-ALA-LEU-ILEU-VAL-PHE is glyvl-alanyl-leucyl-isoleuoyl-valyl-phenylanine. It is prepared like each of the successive products, by the process illustrated above. The optimum conditions with regard to the concentration of the reactants, the pH, the temperature and the reaction time are determined by test reactions in which.

   small aliquots of the reagents are reacted before carrying out the reaction on a large scale the reactions are and-

  <Desc / Clms Page number 77>

 
 EMI77.1
 temperature, a pH value of 4 to 11, generally using an excess of the anhydride at low temperatures.
 EMI77.2
 enters ..SC 1540 in a blender. The temperature is maintained by internal or external cooling or by internal and external cooling,., ¯,
The following acid anhydrides are used
 EMI77.3
 as trifluoroacetylic derivatives, serine, threonine, p-hydroxy leuoine, Y-hydroxy norvaline.



  The following acids anhydrides are used
 EMI77.4
 sés as S-benzylic derivatives: diete1 and -thicl norvaline, penoillamine.



  Tyroain-1-X-carboxy anhydride is used in the form of its t4tahydrofurenic derivative.



   The omega amine / groups on lysine and ornithine are blocked by an arbobenzyloxy group which is removed from the final product by hydrogenation. Other blocking groups are also removed at the end of the reaction.



  1 Glycine -GLY il * Histidine - HIS
 EMI77.5
 2. itlanine -.LA 12. Serine - OBR 5. Leucine -LEU 13. Threonine - ter 4. Ieoleucine-IZEÜ 14. Proline - prao 5, Saline -VAL 15. Oyste1ne - 0Y8 'il
 EMI77.6
 6. Phenylalanine -PHE 16. Cystine -CYST 7. Tyrosine- TYR 17, Methionine - MET 8, Tryptophan -TRY 18. Aspartic acid ASP
 EMI77.7
 9, Arginine -AGN 19. Glutamicx3ZU Acid 10. Lysine - LYS 20. Asparagine- ASN

  <Desc / Clms Page number 78>

 
 EMI78.1
 21, Glutamine - t # ?, N '4. p-hydroxy norvaline -µ0E, ìAe 22, adipic α-amino-α; AD 35.

   Penioiline - UNM 23. a-amino pimel:! Q14a-o:, PI 3. 6 - thiol norvaline, pTh, 7AL 24, a-amino inniqae-, 8V 37, a-amino phenylaoetique
 EMI78.2
  DAD
 EMI78.3
 25. a-amino ebapqae- a, au '
 EMI78.4
 38. 0-ohloro phenylala-
 EMI78.5
 26. g-methyl aeparti: fe-! 3, M, ASP nine - 0-C1-PIE 27. g-methyl glutam3que-M, tD0 39, yurfurylalanine -! 'AU 28 * 090-dimethyl aspar- 40. Thienylalanine - .AADA -tique -M,! 4, ASP 41. a-napthylalanine -OLNALA
 EMI78.6
 29. Ornithine - GOLD! , 42, 3-thionaphthenyla-
 EMI78.7
 30, a-amino i8obutyrjqut, IBU lanine - TNALA Il 31. -amino diethyl acetiqADA 43. 3-aoumaronylalanine
 EMI78.8
 
 EMI78.9
 32. α-amino diisopropyl COUPALA. aoet- a, ADIA 44. ± -methyl glycine -nom, 33, α-amino methyl isopropy1 GZY j acetic - a, AEIA 45. E-methyl alanine HM, ALA
 EMI78.10
 
 EMI78.11
 46. 1P-methyl leuoine NM,
 EMI78.12
 LEU
 EMI78.13
 47.

   E-methyl% yro8ine -nui TYR By the process described above, the following hezapeptides are obtained GàY-AU-IL3U-iAà-WOE-TYBI TRY. . <LJN-LYX-XISB3R-TERa PRO-CYBT-fET-ASP Gi1-A8N; GLN-ata AD- #, I-o:! BU-o:, SE-, M, ASJ M, GLV-pM, ASP-ORN-ot-IBU-ADA-o:, ADIA! S-AEIA-, vAZ-PEI-xh, YAZ-a, APA-0-CD-.PI3E; , 'ATA-TIAIA- a, ALA-1 \ fALA-COU, ALA-NM, GLY; MN, ALA-NM, LBUtNH, TYR-5LY-A? 11-hEQ; di-LEU7VAL. (OYS-bis, ALi.) SER-tihü-'AiA-1IM, ATA- A8N-GLNI and THR-MET-ORN-SER-a, PI-ABP.
 EMI78.14
 



  EXAMPLE 2.-
 EMI78.15
 Ae axt 1- 1 a 1- 1 a 1-leuoine
 EMI78.16
 A mixture of 2 m moles of leuoine in 20 ml

  <Desc / Clms Page number 79>

 aqueous potassium borate as a buffer at pH 10
 EMI79.1
 is cooled to -5 * 0 and 2.05 moles of solid% -oarboxy glycine anhydride are added? under rapid stirring. The reaction is continued for 40 seconds and the pH is adjusted to 5 with sulfuric acid entering at 0 ° C. while a stream of nitrogen is passed through the mixture for about 10 minutes.
 EMI79.2
 



  ....., ........ the process is repeated at pH 10 without the need to isolate the glycine leucine intermediate produced using 2.02 moles of N-oar- anhydride.
 EMI79.3
 boxy glycine so as to produce glyoyl-glyoyl-leuoine which is not isolated.
 EMI79.4
 



  ..v .. The pH of the mixture is adjusted to 8.5 by the addition .. of. bydl'oxyd ... de .., potas81um concentrate and 2.1 moles. aspartic h-oarboxy anhydride are added at t 0 with rapid stirring. After 30 seconds the pH of the mixture
 EMI79.5
 is adjusted to 4 however a stream of nitrogen is passed through the mixture.

   The desired tetrapeptido forms and
 EMI79.6
 is isolated by allication with ammonium sulfate * By replacing the aspartic N-arboxy anhydride with the appropriate anhydride by repeating the aforementioned process, the following compounds are prepared:
 EMI79.7
 G1utamyl-glyoyl-glyoyl-leuoine Glutamyl-glyoyl-glyoyl-leuo1ne Aspaqaginyl-glycyl-glyoyl-leucine XtxPL '' 3. -! l ± 2l!: !! l!: E! l !!!!:
A mixture of 3 m moles of phenylalanine in 30 ml of aqueous potassium borate as a buffer containing 5 g of ice at a pH of 10.5 is cooled to 30 ° C.

  <Desc / Clms Page number 80>

 and 3.2 moles of solid N-arboxy leucine anhydride are added with rapid stirring.

   The reaction is continued for 30 seconds and the pH is adjusted to 3 h with concentrated sulfuric acid at 0 C,
 EMI80.1
 however that one passes a current of azotan8 the m61ancc. The product which is leuoyl-pbenylalanine is not olole.



   The pH of the mixture is adjusted to 10.0 by the Ci% concentrated potassium hydroxide ion and
 EMI80.2
 34- B of anhydrous tetrahydropyranyl tyrosine-.N..oarboxy was added with rapid stirring while the temperature was maintained at about 0 ° C. The reaction. is continued for one minute and the de-
 EMI80.3
 orboxyla..tion is carried out by adjusting the pH to 3 with sulfuric acid% The mixture is allowed to return to ambient temperature and stored for one hour so as to remove the protecting groups. The desired product is isolated by chromatography on a silica gel column.



   The same compound is prepared by repeating the
 EMI80.4
 process described above using 6thOX: r'h .: rltyro-B1no-N-oarbox: r-anhl "", EXEHPLB 4.



  !!! l1: 1! 2l!:! l!: 2l !!!!!:
A mixture of 2 moles of cystine in 30 ml of aqueous potassium borate as a buffer at pH 10 is maintained at about 10.0, while adding
 EMI80.5
 4.1 moles of N-arboxy.alanine anhydride with rapid stirring. The reaction is continued for 2 minutes and the pH is adjusted to 3 aveo of dihydrochloric acid.

  <Desc / Clms Page number 81>

 
 EMI81.1
 read at 290 # however that one passes a running itatote dama the mixture> pcodu nn81 obtained which is eyatyl-bis-alanine is not w â cx, The pH of the melunge is adjusted to. 10.2. It is re: rt'I) / J: 7. juiq.i '/ 1, O'C and we add 4.2 m moles of an- 3ayr, do of N ... car'oox'f 1. ncine, FOUS rapid stirring.

   The reaction takes about one minute and the deoarboxylation is carried out by adjusting the pH to 315, 5%; that the cl: 1 faî lw, .1a.,. e,.,. "1 stream of nitrogen in the mixture, la, d: L-" 4, tttuyl (cy., 3 intermediate is not isolated, mati is treated at pH 10 with 4.3 mol of anhydride, N-o ', \ y',; ox; y valine at 0 "0, Bous rapid stirring and <? capb'3'l '' ''; with sulfuric acid at pH 3 so as to give di-valyl-leuoyloystyl-bia.-a2anine) which is removed from the reaction mixture by aalifioa% j.oi; .



  The product is reduced by hydrogen sulfide to give 1 ", v" yl-leucyl-alanyl-oY8 tine.



  11Ey,;.? L. ';; Sl5S <5.-.'... ëSXë2.i: 22i52. '. From the g1: ,, yl-r <lyoyl-leucine and prepared by Belon the procedure described in Example 2 and the reaction mixture 1a containing 2 and 3 pure treated 2.5 moles of ome8a-carbobenzox7-N-carboxy lysine anhydride 0 * 0 with rapid stirring for a period of 40 ne-oondeat. The decarboxylation is carried out with aulfuric acid in the presence of nitrogen at pH 4.

   N-oarbobenzoxy-
 EMI81.2
 produced is separated from the
 EMI81.3
 mixture by salification and recovered by filtration *

  <Desc / Clms Page number 82>

 
 EMI82.1
 The arbobenzoxy group is removed by hydrogenation at atmospheric pressure, t, x room temperature with an equal weight of a solution in carbon acetic acid supporting 10% palladium, and the product is isolated by expelling the solvent. vacuum after
 EMI82.2
 i.5.trat.on of catalyst 5XBtSFIiB 6 - 3: X2X3: '* ËÉ5X3: Ï **' '5i ** To an Ediange of 4 m moles of leuoine in 20ml of water at 060 we add 3.1 ta moles of ± -oarboxy phenylair nyl qutor maintains the pH at 10.5 by adding .ntorra.tv. to of bee hydroxide, ryum powder.

   The reaction is tersain within one minute, the p3 is added to 6.5% by the addition of 10% eulfuric acid. The barium sulphate which precipitates is separated by filtration.
 EMI82.3
 % ion and, the filtrate containing phenylalanyl leuoine is% t'4i% 60 per 3.2 moles dtanbydride of N-oarboxy glycine "5 * C <ot a lot5 pH is maintained using 1 # ro # t; rd; do barium. The decarboxylation is carried out in the $ H at 3.5 with concentrated sulfuric acid; The desired product is isolated by freezing drying, the same product is obtained if the reaction is
 EMI82.4
 However, dilute aqueous sodium hydroxide is added in place of solid barium hydroxide.



  Obviously, no barium sulphate is formed so that the filtration step can be omitted. The product is recovered by first passing the mixture through a carbon column. The peptide is absorbed and then eluted with a mixture of acetic acid
 EMI82.5
 and water. it is then re-cut from Iléluat by drying

  <Desc / Clms Page number 83>

 by freezing.



    EXAMPLE '7.-
 EMI83.1
 IP.meth, leual -lo:. d, s ï vev .-. oI ,. ieoleuaine A mixture containing 4 m moles dm g.yay, .glyoy ,. glyol1-glyoyl-1sQleucine in 30 ml of aqueous potassium borate serving as a pFI buffer, 5 is cooled to about 000 and 4p3 moles of anhydrous N-methyl leuoine N-oarboxy are added to the acus. rapid stirring. The mixture is rapidly cooled for 30 seconds and the pH is adjusted with concentrated sulfuric acid to 3, so as to effect the deoarboxylation in the presence of nitrogen. The desired product is isolated by passing the solution through. carboxylic resin and then eluting with ammonium hydroxide solution.



   EXAMPLES.-
 EMI83.2
 ! 2l!: !!! l!: !!! l: !!! l!: E2! L!: Ehl! Al! =! S !!
The procedure of Example 1 is repeated and fractions 3, 4, 5 and 6 obtained from the ohro- column.
 EMI83.3
 matographic coa nan7.a va3y1-alanyl-alany.-pro3.yl-phd nylalanyl-arginine are combined and conoentréon up to a volume of 20 ml. This mixture is cooled to 0 C and added 1.5 m mol of N-carboxy leucine anhydride, however the pH is maintained at 10.5 by titration
 EMI83.4
 automatic with dilute potassium hydroxide. The reaction is completed in 90 seconds, the deoarboxylation is carried out in the usual way with acid.
 EMI83.5
 concentrated sulfuric acid. Heptapoptide produces * and isolates.



  , The identical product * and obtained by isolation and purification of Yalyl-alanyl-proll1-h & nylalan1-arginine and the aubadquente reaction with 1.5 mole of J-Qrbox1-1.uoine anhydride in borate of potassium

  <Desc / Clms Page number 84>

 buffer at pH 10.5 to 0 0 / within a minute's space followed by decarboxylation by adjusting the pH to 3.3 with concentrated ulfuric acid. It is isolated chromatographically using the procedure of Example 1.



    EXAMPLE 9 - encyl-alanyl-glycyl-prolyl-phenylalanyl-argine.



   A total of 3.48 g of arginine is taken up in
 EMI84.1
 Of potassium borate one molar serving as ts.Gpor \ r "pH aat adjusted to 5 with sulfuric acid and nitrogen so as to ensure removal of carbon dioxide. is then adjusted until
11 With dilute aqueous potassium hydroxide the mixture Y ge anhydride is cooled to. 0 and 3.84 g of / N-carboxy-phenylalanyl were added with stirring at high speed. (two drops of oaprylated alcohol are added to the mixture to inhibit foaming during the reaction).

   At the end of the minute, the pH of the reaction mixture is 10.9 * The intermediate N-oarboxy-phenylalanyl-arginine is deoarboxylated by adjusting the pH to 3 with sulfuric acid so as to produce sulfuric acid. phenylalanyl-arginine which is not isolated.



   The following table summarizes ---- the reaction conditions under which subsequent reactions are carried out to obtain the final product. The various amino acids and peptides are identified by the same abbreviations as those used in Example 1.
 EMI84.2
 

  <Desc / Clms Page number 85>

 
 EMI85.1
 pE Ter- pH -00 B [S. \ ¯¯¯¯, initial ± t. Durôo final lI Product
 EMI85.2
 1Í'O 2.82 g 9.5 000 1 8.9 3 PRO-PRE-AGI GIY 212 g 11 oec 1 10.4 3 OELY-oeFt0-vOE-A0h
 EMI85.3
 
 <tb> \ LA <SEP> 2.30 <SEP> 10 <SEP> 0 C <SEP> 1 <SEP> 9.6 <SEP> 3 <SEP> ALA-GLY-PRO-PHE
 <tb> ASN
 <tb>
 
 EMI85.4
 LEU ', 14 10 0 fl 1 9.8 7 lEU-ALA-GLY-mo
 EMI85.5
 
 <tb> PHE-ANG
 <tb>
 
The optimum pH in each case is determined by test reactions.
 EMI85.6
 



  The pK of the mixture is adjusted to 7 and the mixture is hand filtered. From an olar solution, from which the desired product is isolated. In the last step, the decarboxylation takes place at a pH of 7 by standing.



   A total of 335 ml of the clear solution as well
 EMI85.7
 obtained is diluted by 1a: u Up to a volume of 1340 ml. The pE of the solution thus obtained is do 713. It is introduced into a column of 400 ml of IRO 50 resin on the acid cycle contained? danr @@ column with a diameter
 EMI85.8
 interior ', 3 om at an introduction rate of 15 ml / min.



  The column is washed pi., 3800 μl of esm at an introduction rate of 20-2 $ m1 / min, The resin is then eluted with sulfuric acid art at a rate of 15 mifmin, 1 L 'Eluat is collected in raot: .ons of 250 mlp Fractions 3, 4e 5 and 6 and 7 are combined so as to give two product eompoe.tea.4kag, uc composite product, *% ¯ adjusted to pH?, 5 with sodium hydroxide solution and freeze-dried 4. Each solid is extracted with oong41a% igne, with 4 x 100.1 l of methanol and the methanol is removed in vacuo. The residues are dissolved in water and dried again by freezing.

  <Desc / Clms Page number 86>

 



  The combined residues are dissolved in 50 ml of water, the
 EMI86.1
 fold is adjusted to 3.4 with Act Juii,> .: Oique a% sol sulfate of the hexapeptide precipitated with one liter of ethanol.



   The product is converted into a free peptide by dissolving the salt in water, adding sufficient
 EMI86.2
 Barium hydroxide starch, to precipitate the entire sulfate in the form of barium sulfate, filtering and freezing the filtrate.



   A sample of the product is hydrolyzed and the hydrolysis mixture is analyzed to give the following results;
Thin Report
 EMI86.3
 Mierogra aid = eo / mg. , damino.¯ & ci-'e ¯¯¯ .¯¯.¯¯¯¯¯ .¯; orv6 '", t ho i, îe
 EMI86.4
 AM 1113 0.98 1 rivu 0.99 lyol 1:
 EMI86.5
 
 <tb> PRO <SEP> 1.19 <SEP> 0.95 <SEP> 1
 <tb>
 
 EMI86.6
 MY 1``03 1.10 1 AM 1.09 0.92
 EMI86.7
 
 <tb>
 <tb>
 <tb>
 
 EMI86.8
 7, '- 1.06 1, OS 1
 EMI86.9
 ¯ ¯ gìhéfl, ¯, 1.0, z% c; .a, l.etri2loZ-ro.l-hérealanrZ- âri'riilielw The procedure of the prieddent example is repeated on a somewhat larger scale to pre - Parer 5-carboxy roy3 .-. phnylalaz :: y2.argia.ne which is d6oarboxylated at PR 3. About 900 MI of the solution thus obtained are adjusted until pH 7 with 1! sulfuric acid ov -liluted until ' to 10 liters with water.

   The solution is passed through a column of. 3 liters of

  <Desc / Clms Page number 87>

 of IRO 50 resin on the acid cycle and washed with 17.5 liters of water. The column is eluted with 7 liters of acid
 EMI87.1
 sulfuric acid, 0.5N and separated into a 4 liter fraction and a 3 liter fraction. The 4 liter fraction is adjusted to pH 7 with aqueous sodium hydroxide, freeze dried and the residue is extracted with 15 liters of anhydrous methanol. The solution is concentrated to dryness in vacuo, and the residue is dissolved in water.



  The tripeptide is precipitated to form salt sulfate by adjusting the pH to 5 with sulfuric acid, in addition
 EMI87.2
 both an equal volume of methanol and a double volume of d-thanol and cooling.



   This example illustrates aspects of this. invention, when, because of the ease of isolation,
 EMI87.3
 an intermediate tripeptide is isolated during the preparation of a higher peptide.



   The following table describes the process by which 1808 g of prolyl-phenylalanyl-arginine sulfate is
 EMI87.4
 processed in the desired hexptide, PH -00 10.1 Initial weight 7th ,, np4 Duration ²nal fold 2 Product ........-- L4- z .... ¯., ... 1
 EMI87.5
 GLY '399 mg 10 oor i 9.6 3 oly-I> RO-PHB-
 EMI87.6
 
 <tb> ARG
 <tb>
 
 EMI87.7
 AZA 460 mg 10 Or () 1 9t5 3 ALA-GLY-PRO- THE-ARG LEO 634 mg 10 0'0 1 9.6 3 ZEU-AI $ A-GLY PAO-PHE-AR3
 EMI87.8
 The product isolated chromatographically.



  E; F: rPI; E ll, - q '.olrl- - SXB2ïBÉ3ï3: 53ïl''SES.B2'
A toal of 4.21 g of arginian hydrochloride is taken up in 200 cc of a buffer of the po- borate type.

  <Desc / Clms Page number 88>

 tassium, the pH is reduced to 3.0, while nitrogen is bubbled through the mixture to remove carbon dioxide and this pH is then adjusted to 11.0 by the addition of aqueous potassium hydroxide * The mixture is stirred rapidly in a blender for one minute.



  The final pH is 10.7 and the pH is finally adjusted to 3 with sulfuric acid to effect the decarboxy- on $
The pH is then increased to 10 with the addition of potassium hydroxide and the process is repeated with 2 g of N-carboxy proline anhydride. The final pH was 9.85 and then adjusted to 3.0 for deoarboxylation.



   The process is repeated with 2.22 g of anhydride - oarboxy glyoine at an initial pH of 9.5. The final pH is 9.1. It is adjusted to 7% sulfuric acid and the mixture is then allowed to stand for about 16 hours at a time to achieve decarboxylation.



   The reaction mixture is diluted tenfold to a total volume of 10 liters and then passed through an 800 ml column of IRC 50 over 18 hours. The column is washed with 4 liters of water at a flow rate of 15 ml / min. The column is eluted with 35N sulfuric acid and the eluate is collected in fractions of 300 ml. The fractions give a positive test. ninhydrin and freeze dried and the solids combined and extracted with methanol until the extracts no longer gave a ninhydrin test.

   The extracts which give a positive ninhydrin test are combined and evaporated to

  <Desc / Clms Page number 89>

 dryness under vacuum. The residue is taken up in the minimum quantity of sulfuric acid diluted to pH 4t5 and precipitated
 EMI89.1
 by the addition of 5 volumes of mdh, u'01, followed by the addition of 1C volumes of ethanol. The precipitate is collected by filtration, dissolved in water and precipitated with the r2moo qualities of methanol and ethanol. The precipitated product which is glyoyl-prolyl-phenylanalyl-arginine is isolated and washed with methanol and ether.
 EMI89.2
 



  'EC'; Tr '12 - 2ysEyiëysyly2ïsy22j.i' A total of 19 I mdas of glycine is distributed in 200 ml of a buffer of the potassium borate type at pH
 EMI89.3
 11, cooled to 060 and 19 mYoles of N-carboxy glycine anhydride are added.



   The mixture is stirred rapidly for one minute and the pH is adjusted to 3 so as to operate the decarboxylation. The pH is then brought to 11 / with potassium hydroxide and the process is repeated with
19 ', 5 moles of N-arboxy glycine anhydride.



   To the solution containing glyoyl-glyoyl-glyoine at pH 11, 22 moles of anhydride are added.
 EMI89.4
 8-Benzyl N-oarboxy oystene and 50 g of ice, and the mixture is stirred for one minute. The final pH is 10.2.



  It is adjusted to 5.5 with sulfuric acid and the mixture is passed through a 100 ml carbon column. The column is washed with 200 ml of water and
 EMI89.5
 then with 800 ml of 5% acetic acid The elution is carried out with 5 aoetic acid in 50% acetone The eluate is evaporated in vacuo so as to give the desired product as a residue. product is roaris- tallized in a mixture of acetone and water,

  <Desc / Clms Page number 90>

 
 EMI90.1
 3XtYPI2 13.-, xanrl-al.rn1-rlanl-a.an..Qin To a solution of 60 m oolf. "Of glycine in 4- liters of sodium borate â.111psl. Rit 10 and to Ü1 we add 80 moles of 1; -oarboxy alanine anhydride while stirring rapidly.

   After 2 minutes the pH is
 EMI90.2
 lowered juoquth 3 to effect c, decarboxylation. while a stream of nitrogen is passed through the mixture. The pH is brought to 10 with a 50% aqueous solution of sodium hydroxide and the reaction is repeated. The process is repeated two more times * After 1 final decarboxylation at pH 3, a precipitate forms
 EMI90.3
 which is r1otfllieé by dissolving sodium hydroxide o.quou :: à, :: 1 11 and lowering the pH to 5.5. The identity of the compound is established by elemental analysis and by hycrolysis of Amina Acid Spinco.



  EIEt! PLE 14 or - Sheep α-cort3aotropne-Bnthee Your sheep α-cortiootrogen structure is countered below.
 EMI90.4
 

  <Desc / Clms Page number 91>

 
 EMI91.1
   00 compound is prepared by the methods described in the preceding examples, using the conditions
 EMI91.2
 d6tin1es in .lt8 tables 1, 2, 3 and 4 below which show the preparation of the various segments described below.
 EMI91.3
 above, In the first stage of the synthesis of the txahydr ether:,.;, icic of N-carboxy tyrosine anhydride coupled with adrine under the conditions indicated in stage 1 of Table I, the sequence is continued as indicated in Ion tables.

  <Desc / Clms Page number 92>

 
 EMI92.1
 



  Tlk3Ti? ÀV I.- ± ± 2E !!!! 2¯! ¯ !!!! ¯! - {',:. 4:' '' '' '¯:' J1I. Excess pH Duration pu \ J ¯Li n. : fl.J.i> 4 ODlployed from en deoar .. copula- minu- boxyla- @ tion tes 1 ion
 EMI92.2
 
 <tb> TYN2 <SEP> 5 <SEP> 10.0 <SEP> 2 <SEP> 3
 <tb>
 
 EMI92.3
 3 <3 :; 25 1 0, 2 1 "'2 z7 9.8 = i 1.ù ib" 10 ge8' FiJ 10 10.6 é. ù *% zut3 10.2 2 The product in which the histidine is prey
 EMI92.4
 x4e pa =: Q action of the im-benzyl derivative, wat isolated using the IRO 50 resin.



  Lo Table II shows the conditions for the r:; Rr2 t, oa of peptide A in pqptiàe AB.



  Qé2Ié% àīiI, =? S5-ÊÊ-.SiâS-5 - ËSEE-Ë2i N 0Onài- BOA r: '1! pH Duration pH of the tioao used in de-staging the ad copu- min- boxy- ruac- tion t: ton .¯¯ ¯¯. ¯¯ # .... fL #
 EMI92.5
 
 <tb> 1 <SEP> III <SEP> TRY <SEP> 30 <SEP> 10.2 <SEP> 2 <SEP> 5
 <tb>
 <tb>
 <tb> 2 <SEP> III <SEP> GLY <SEP> 5 <SEP> 10.1 <SEP> 2 <SEP> 5
 <tb>
 <tb>
 <tb> 3 <SEP> III <SEP> LYSA <SEP> 7 <SEP> 9.9 <SEP> 2 <SEP> 5
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 4 <SEP> III <SEP> PRO <SEP> 10 <SEP> 10.0 <SEP> 2 <SEP> 25
 <tb>
 <tb>
 <tb> 5 <SEP>]: he <SEP> VAL <SEP> 10 <SEP> 10.2 <SEP> 3 <SEP> 3
 <tb>
 <tb>
 <tb>
 <tb> III <SEP> GLY <SEP> 10 <SEP> 10.4 <SEP> 3 <SEP> 3
 <tb>
 <tb>
 <tb> 7 <SEP> III <SEP> LYGA <SEP> 15 <SEP> 10.4 <SEP> 1 <SEP> 3
 <tb>
 
 EMI92.6
 1 LYS -. 15 10.4 1 3 p III 1.113 20 9t8 3 13 Ili 4t:

  G 25 10.0 .1

  <Desc / Clms Page number 93>

 
 EMI93.1
 The peptide, B is transformed into ABC peptide according to the methods defined in 1. ¯: 2ea, z, Itf. Amino terminal group re 1;?: Lysine represented ùt4 J 1, ez tblec.ux 2 t 3 is protected by a group ex'b '.a E The gro1J: 1! hydroxyl in tyrosine dQn5;: lr Jans the tabi - '3 is protected by a group% str * - "Y ::: v, yral) .11e The hydroxyl rump of serine in t8.bleo.J. 4' is ": 'J:' o": 6, :) '6th by t1 ..' "triiluoro-aoetylel troop! Lee gl'o't: es protectora are removed from the ooura dse 'r4> o "ocpulation lions.



  'JM3JIRÛ Q.- - "" ± - "' -; 1111111-" '' '--- j ", .- J.;.'; 1;" ¯ .. "" - Oj -, j; --- ", * ----- ,, ';' -...-------., No Condi- fIA 7" .. ". É, # R Duration pE e: te.de tions Ëif3, .V é3: 'Èna.o'ct (l en de d4car- da la - "at.o tee boxyla- µéâôtation tes% ion t1011 u ---- w y ... r ... wrwa, e., .., .ar
 EMI93.2
 1 1; "': 1"; 9, e Y TAL- 7 3 3 1 .. 1ìX 10 H "f: 2 2 1 TAI. 10, 2 2 9YR, .0,2
 EMI93.3
 
 <tb>
 <tb>
 
 EMI93.4
 1, .f 20 10.4 1 ALÀ; 0 10! 2 1 G: L1 '10 10.2 1 t'x2u' 15 10.4 1 10 1 18 15 10? 4 1 3 11 1 A8? 20 10.6 ¯ 3
 EMI93.5
 This peptido is then transformed into an LBOD product according to the procedures shown in Table 4.

  <Desc / Clms Page number 94>

 



  BABLAU 4, -
 EMI94.1
 EÉESS2S-S.-ESBÊË-.32 -. SâES-.i '-.î JSS.L Ne Oond1 .. NOA txoàs pH Hard <;; pH stage tione employee employee do en de do la c, zo, - minu- 4'oarréao- lation tes boxylation ¯¯¯ ¯¯¯ ¯¯% ion
 EMI94.2
 
 <tb> 1 <SEP> I <SEP> GLU <SEP> 5 <SEP> 10.2 <SEP> 2 <SEP> 5
 <tb>
 
 EMI94.3
 2 1 A '4 5 10, 4 2 5
 EMI94.4
 
 <tb> 3 <SEP> I <SEP> WILL <SEP> 7 <SEP> la, 4 <SEP> 1
 <tb>
 
 EMI94.5
 4 1 GL1f 8 10.2 rut
 EMI94.6
 
 <tb> 5 <SEP> I <SEP> TO <SEP> 3 <SEP> 9.8
 <tb>
 <tb> 6 <SEP> 1 <SEP> PHE <SEP> 10 <SEP> lottery <SEP> 1 <SEP> 5
 <tb>
 <tb>
 <tb> 7 <SEP> I <SEP> DTP <SEP> 10 <SEP> 10.2 <SEP> 1
 <tb>
 <tb>
 <tb> S <SEP> I <SEP> THEM <SEP> 10 <SEP> 10.6 <SEP> 3 <SEP> 5
 <tb>
 
 EMI94.7
 1 GLU 15 10.2
 EMI94.8
 
 <tb> 10 <SEP> I <SEP> PHE <SEP> 20 <SEP> 10,

  4
 <tb>
 
The product is isolated using RC 50 resin
 EMI94.9
 The o8, benzoxr and benzyl groups are <li'- 't <1-? T 2eeL'uotion with sodium in ammonia * j, ..; ¯ gol "I19 ± @; - Ut ±! 2¯!.! 2! ZE! E !!!:! 1 ' Illustrates the process by which arcà: nini,>> .1¯anlev4e of a peptide using oatboxypàp% iàaso-2. ,,
Three mixtures are prepared as follows 100 cc of buffer (0.1 m in sodium chloride
 EMI94.10
  <:; b 0.2 m in trihydroxymethyl aadno methane) at pH 7.5 (2) 8.5 Ng of-leucyl-alanyl-glycyl-prolyl sulfate

  <Desc / Clms Page number 95>

 
 EMI95.1
 ph6nyQialanyl-ar6inine in 3 oo of the same buffer, (3) 10 oc of mt5L "" d '= n; / me by diluting 0.015 oo of a mixture of oal'bo.: n ....,: p .i . "the, ee:

  B containing 10 mg of enzyme per ml with the same buffer.



   The mixtures are mixed as follows;
2 tsp of mixture (1)
1 cc of mixture (2) 1 cc of mixture (3) and incubated at 75 C for one hour.
 EMI95.2
 



  ..the mixture is then subjected to an ohromiogra.- phy in silica silica thin oouohes, and two spots are observed, one is arginine. The other is the pentapeptide,
When the reaction is carried out on a preparative scale, the sautions are mixed in a mette report and the inoubation mixture is dried by freezing,
 EMI95.3
 extracted with methanol, the methanol solution is concentrated jusuâ cicoité ', the residue is taken up in water and the prodl:' e:;:;. \ 'o1é of the aqueous solution by chromatography 8U' d "" . raineIRC 50 $$, 1; J¯1 '],', i 14 'Yaye, n1-la, rcl3.hr elnn.' arinna
This example illustrates the preparation of a murqué peptide.
 EMI95.4
 



  A mixture is prepared containing 0.92 mg of 0'4 primed arginine (0.5 millicurie) in 0.01 hour hydrochloric acid and 421 mg of unlabeled arginine in 20 ml of sodium borate. potnoR1um1 M pie buffer 10t5 * A 0.2 & 1 aliquot is removed.

  <Desc / Clms Page number 96>

 
 EMI96.1
 to al'.ngo 401 mg (a molar excess of 5%) are added .. 7.! = ce.roo :: cy phenylalanine anhydride in a fraction .r rapid mixing at a temperature of 0 to 2-0, the mixing being continued for one minute, while one
 EMI96.2
 control the pH between 10.5 and 10.4 by the addition of hydro-Xyo.9 Cc potassium 5Nt At the end of the reaction period, a drop of oapryl alcohol is added as agent. '. 11; "" "'.. :: r.:e.

   The pH is then reduced to 3.5 by the addition of concentrated sulfuric acid, and the mixture is passed through with water for ten minutes. PH
 EMI96.3
 is eleOE: 1 to 10.2 with potassium hydroxide concentrated and a 0.1 ml aliquot of the reaction mixture containing prolyl (014) arginine is removed.



   To the mixture thus obtained, 300.5 mg (6.5 molar excess) of proline N-carboxy anhydride is added with rapid stirring at a temperature of 0 to 20. The reaction is continued for one minute while it is being carried out. maintains the pH at 10.2 - 10.1 with 5 N potassium hydroxide. Decarboxylation is carried out at pH 3.0
 EMI96.4
 of the number described above. The pH is then brought to 10.2 with concentrated potassium hydroxide and a 0.1 ml aliquot containing glyoyl-prolyl-
 EMI96.5
 phenyleLlanyl - '(0 "') arginine is removed for analysis., ¯ ...



  . The. t6traJ? pid is -converted into% -oarboxy pap- tide of alanyl-glycyl-prolyl-phenylalanyl- (0 ') arginine' by the addition. 248 mg (8% molar excess) of N-oarboxy
 EMI96.6
 13.l'1 '- ..:!:' Td 'of alanine to the reaction mixture at 0-2 C soue act:' x5, on fast. The reaction is continued for one minute while maintaining the pH 10.2-10.0 with

  <Desc / Clms Page number 97>

 5N potassium hydroxide and N-carboxy peptide
 EMI97.1
 93t deoarboxylated at PR 3 of the manita described above.



  The pH is brought to 10.2 with aqueous concentrated potassium hydroxide and a 0.1 ml aliquot containing the desired compound is removed for analysis.



   340 mg are then added to the reaction mixture.
 EMI97.2
 n-oarboxyvaline anhydride with rapid stirring at 0-2 C The reaction is continued for two minutes while the pH is maintained between 1, 5 and 10.0
 EMI97.3
 with hydroryde de .otaai't'11 52. The pH is then adjusted to 6.5 with concentrated Z \ 1) .ful'ic acid and a 0.2 ml aliquot containing the radioactive hexapeptide. is removed for anaoyae purposes.



   Each of the aliquots collected
 EMI97.4
 The above is toraphed on paper using the butanol-acetic acid-water system in the ratio of 4t1: 5. The chromatograms are completed with a radioactivity band tester. The results indicate that each of the desired reactions proceeds in greater than 85% yield so as to give a product which is not. not significantly contaminated by side reactions.
 EMI97.5
 



  The lécotionnel mixture is dried by freezing, the residue is extracted three times with fractions of 150 m7. of methanol and the combined fractions are concentrated to vacuo at 40 ° C. The residue is taken up in 2 ml of water and 1/4 of the solution is diluted.
 EMI97.6
 up to 1 liter.

   The diluted solution is chrmto8rapée on 200 ml of an oarboxylic ion exchange column.

  <Desc / Clms Page number 98>

   (OG-50 sold by the company ROME and HAAS. Philadelphia; diameter 2.2 cm; height: 55 cm.) On the hydrogen cycle, The column is fed by the soluiton at a flow rate of 2.5 ml per minute and it is then washed to collect two liters of water. The column is eluted with 0.01N sulfuric acid * The effluent stream and adjusted for the C14 isotope using an anthracene column, initially fed at a flow rate of 0.2ml per minute.

   The feed rate is gradually increased to 0.6 ml per minute over 20 hours and the rate is kept constant until the end of the test. The eluent is collected in 24 ml fractions and the hairy product is obtained from fractions 25 to 72 by adjusting the pH to 9.5 with 50% sodium hydroxide and freeze drying. The residue is extracted with methanol, the extract is concentrated to vacuo at 40 ° C. The residue is dissolved in a small amount of water and the pH adjusted to 3.5 with hydrochloric acid. concentrated.

   The solution is dried by freezing to give the desired product which is recrietallized in a mixture of water and ethanol. EXAMPLE 17.-
A total of 3 mM of phenylalanine amide is taken up in 30 ml of potassium borate buffer at pH 10.2.



  The solution is cooled to 0 ° C. and 3 mM of N-arboxy aspartic acid anhydride are added, however, which is stirred rapidly in a mixer. The reaction is carried out for one minute and the pH is adjusted to 4 with oonoentric sulfuric acid. however, a stream of nitrogen is passed through the mixture.

  <Desc / Clms Page number 99>

 
 EMI99.1
 at;. 3 ", r ± .i.3.LÉ; K pU 4, the concentration of
 EMI99.2
 ions, r;, -. =. ¯. <'":: -', o at. 'pH C', 3, '1' Cidd.'i0T1, (4'JC.3 '.' f3 'Tt'5 do J'; wE?:?: ' ->. 7. = t, " <t.fl:. # Ei e3t again (I.n refreshed JUGqu'11. oee 1 "1; .n # .s =. '..1; #; rt, i anhydri-; l; <> n¯I1 '"1ht'1)',. '; j.' :: h ,. i '; - ,,," r,.,'> 1 a, jf tunt .. 'l.t.'L4SiÂflY;

   
 EMI99.3
 At 1.:i. f:! R J minutes the pB is adjusted up to 4 with? ' ai.c3 sulfuric concentrate and an oourax12; the maole is bubbled through the mixture for 15 minutes.



  1) The reoipity which forms during deaarboxylation is 3isrou. adjusting the pH to 10.2 with ps: zm hydroxide. '') '' '' entr't The solution is cooled to 100 and 4.6 mM of anhydride of -CaiboX9 'tryptophan with rapid stirring. The fold is maintained at 10.2 by the internal addition of aqueous potassium hydroxide. The mixture is clarified by filtration and the pH of the filtrate is adjusted to 2.0 by the addition of concentrated hydrochloric acid.



   The solids are separated by filtration.
 EMI99.4
 the solution (in * '- on 50 f'! 1) is chro: cxto; raffia on a column of 1.000 - of 0 ('.:, Aox G-25 (pcrleo fines- dian- tiret7,5 cm, length 10000) The column is developed with deionized water while 100 ml fractions are collected.

   Fractions Nos. 39 and 40 contain the tripeptide back to unreacted methonyl-aar, nrtyl-phnyl-alanyl amide. Other minou- fractions.
 EMI99.5
 rea of undetermined structure are rscu <3illios in fractions 41 to 59, The desired product is obtained from frac-
 EMI99.6
 tiona combined 60 to 64, by adjusting the original pH (7.4) to 2 with concentrated hydrochloric acid, this operation being followed by freezing drying,

  <Desc / Clms Page number 100>

 
 EMI100.1
 ', :: 1 .'- /: n': S "é ot the optical integrity of the product are eta-, 1'-" '' "the i ''. 1: -" - Jr1YtHJ Spinco and by enzymatic hydrolysis ":> tc o 1 :: '. 1 # Mcin> amino-peptidaaef a specific enzyme --'-; -,: ,, 0':

   '""' () f,! ilà!. 's,! I analyze Spincc establishes that the' \: iii ;;: " <- iî'5ré ::; we presented in # the proportions 1.> fli;.:. .:.] 14 J1 :: ', I'G enzymatic hydrolysis is ana-' .'7 !. . ;; ixà. <'Ü!, T)! :::, tcl: hi3 in thin layers using>: = # 1 = # 1> iicél # t <not used as a test reagent. On the ohromato- E ¯ ¯ ',. ¯.: ¯> ta of the spots corresponding to each of ':.,.: Tn: A'} c; of 46çat% and it ntyf3. no oorrespon- 'stain:. -i..t intermediate products {n1 of stain corresponding to the fruit. These last two factors are the most improt etc.

   If any optical inversion had taken place in the reaction, one or% had to find products or intermediates or all of these substances at the same time on the chromatogram. The reason is that the enzymatic hydrolysis would stop at the invert amino acid, which is the cause of the optical specificity of the enzyme.



   EXAMPLE 18 Use of miscible and immiscible solvents An apparatus formed of a glass tube is used.



   It consists of a horizontal section which is dilated, with a bulb-shaped segment located in the center.



   A section of glass tube leading to a beoher in a mixture of galace and salt is integral with the enlarged section and communicates with it. At both ends of the horizontal section are sections extending towards the top equipped with to hold syringes hypo- of solutions q .. The syringes contain / previously cooled reagents in miscible or im-

  <Desc / Clms Page number 101>

 
 EMI101.1
 miaoâ.b7.ee, The syringes are actuated simultaneously so that the solutions reach the enlarged part ssnai'.à: cr4t at the same time.

   Due to their speed, the k 1 <±?;! a :: cause turbulence in the. active zone '3i although the reactants are intimately mixed = 63. 3z => 4action takes place and the N-carboxy peptide flows through the down tube into the beaker. After about
 EMI101.2
 2 minutes, the decarboxylation is carried out in the beaker with the addition of concentrated hydrochloric acid with rapid stirring while nitrogen is passed through the mixture.
 EMI101.3
 



  Dan3 mie tel13, ztperio; ce 85 mg (oye44 mà6) dian.- hydride of N-carboxy phfplalr.i '} in 2 m # da dioxane and 18 ml of water, previously cooled to about 30 C are introduced a serine and 42 mg (0.40 mM) of serine in 20 ml of potassium borate are introduced into the other
 EMI101.4
 syringe. The $ o3utionsare amc;, 'es together in the reaotiory and are allowed to flow into 3.a beaker.



  At the end of 90 seconds the product is decarboxylated in beohey at pH 3 with concentrated hydrochloric acid. with rapid stirring, however, a continuous stream of nitrogen is bubbled through the mixture for 15 minutes.
 EMI101.5
 nutes. The mixture is then dried by oonslation and the residue is extracted with methanol * Its methanolic solution is chromated. 8 in a column of silica gel and the column is developed with a mixture of 4 g: de.n-butanol-acid acetic - water.

   The first fractions contain phenylalanine, the middle fractions contain the desired product the last fractions contain unreacted serine.

  <Desc / Clms Page number 102>

 
 EMI102.1
 In a similar experiment, the
 EMI102.2
 dioxane with ethyl acetate: rt'è8 d: w .. "Barlatiari the layer with ethyl acetate is separated and the product is re-formed from the aqueous layer oonne in the above process.



  EXB'TLE 19.- lriny3-p: oly3.-pxoly3-gxyoyl-p? Ény.alany, --¯¯¯lt: E2tlt: E! L !! l!: E6 !! l: - # -SËEXBE2YlEÉ5X3: Së5ïlSES53.! .- 1?, 9.fYl-t'rol; y: l-nhénx.c, lanYl-arg: tnine.u. '15.75 g of arginine hydrochloride (75 are dieaoua in 2.62 l of sodium borate buffer at pH 10, the pE cat adjusted to 3 with sulfuriqv-oonoentric acid and bubbled with l The pH is brought to 10.2 with 1 sodium hydroxide at 50 and the solution is cooled to 0.0.



  400 g of slaoe are added, this addition being followed by the addition of S2.5 mM of K-arboxy phenyl-n1a anhydride dissolved in 200 ml of acetone. After stirring for one minute, the solution is acidified at day 5 (')'; E 3 is bubbled with nitrogen for 10 minutes.
 EMI102.3
 



  1 ..: t then adjusted to. 9.5 and 15.9 gases (fi 2, 5 ldi of an-.:.? Ú. 'T ": e 5-oarboxy proline dane 150 ml of acetone are
 EMI102.4
 ijoi;, t4: 1 <3.; C \ of 400, of ice in order to "maintain the.. A'j (: '¯" u.1.', ", Q 0.0, the solution is rigorously stirred pdr..av Mac ninqo, ot deoarboxylated as described above. The product is not isolated. The process is repeated, L, p13 with the addition of 34 g (150 mg of anhydride of 111'.taroaadtyl-1-aa, rboxy serine in 150 ml of aoeto.

   After 3 admixture for one minute, the pH is adjusted to 7 and frozen. The total solids are extracted.

  <Desc / Clms Page number 103>

 
 EMI103.1
 he '.:. ''. ' -.¯- - * with methanol and methanol extracts C :. ' ,. 1- of gel da oilioe et amenda, fJ1oo1tÓ. ! '1 z * 1 le # .i, u d t "added to Ululoolon-
 EMI103.2
 of silica gel prepared dane of 7.'iavpropanol. The column is developed with methanol, 80 parts! water, 18 parts, ammonia, 2 parts. The desired tetrapeptide is isolated from fractions 18 to 21 and the structure is confiscated by Spinoo analysis.
 EMI103.3
 



  Pher.ylaJ.anyl-8érrl-prolYl-phenrlalan1-arini.



   1.8 g of seryl-prolyl-phenylalanyl arginine (3.56 mM) is dissolved in 125 ml of sodium borate buffer at pH 9.5, cooled to 0 C ot 70 g of ice are added so as to maintain temperature. 755 mg (3.92mM) of N-aarboxy phenylalanine anhydride are dissolved in acetone and added to the buffered solution with vigorous stirring. The pH is adjusted to. 7 and the total reaction is introduced into a column of 40 ml of carbon, The column is washed in 500 ml of water and
 EMI103.4
 euba6q, use it with ammonium hydroxide until the fraotî-iii zonates a ninhydrin negative whole.

   The fractions which contain the desired material are dried by freezing. The solid residue is ohromatographed on silica gel. The column is expanded
 EMI103.5
 pée with seo-butanol, 6 partiont # water # 5 partial. acetal acid ,. Part 1, Fractions 6-9 contain the desired pehtaetida. Structure is confirmed by 8pinio analysis.



  Prol¯yl-prolyl-l'çi, ne 1,5 (20mK) of glycine is dissolved in 250 ml of sodium borate buffer at 10 pfft and then cooled to 0 '(. 50 g of glaofotto audi-

  <Desc / Clms Page number 104>

 
 EMI104.1
 \. '"-Ant followed by the addition of 2.82 g (20mX) of anhy-; O -carboxy pro line in 20 ml of acetone. The reaction then vigorously for one minute and the> 1î ': an: lowered to 3 with concentrated sulfuric acid Nitrogen is bubbled through the mixture
 EMI104.2
 reaction for 10 minutes and the pH is adjusted to
 EMI104.3
 4r by the addition of 50% sodium hydroxide, the solution. When cooled to 0009, 50 g of ice and 3 '(22mM) of N-carboxyproline anhydride in 25% star are added.

   After mixing for 1 minute the pH is bitter. to 7 and the mixture is freeze dried.



  The product is desalted by extracting it three times with 25 ml of methanol and the methanolic extracts are adsorbed on IRO-50 resin and developed with a mixture of acetic acid and water * The product is oristal- read in a mixture of water and acetone, 2.5 g (9.3 mM) of the isolate, namely prolyl-prolyl-glyoine are dis-
 EMI104.4
 aouandams of 2t5Xt sodium hydroxide so that the pg of the solution is 10 and cooled to 0 -5 * 0 1t75 g (10.4 tai) of orbob8n.oxl chloride is added dropwise with vigorous stirring The temperature is maintained at 0 ° C. and the fold is reduced by the addition of 2.5N sodium hydroxide.

   After the pH has remained constant, the reaction is stirred for an additional 30 minutes The reaction is extracted for 2
 EMI104.5
 times 10 Ni of manibro k ether remove the excess of diacid chloride and one proceeds eo8uita W¯une odit1ça- iox. jt 1quau Congo Red Paper aveo de lucide oblor- h) drique 205N * An oil has separated and is crystallized in chloroform,

  <Desc / Clms Page number 105>

 
 EMI105.1
 2185 g (7mK) of oarbobenzoxy prolyl-prolyl-glycine are dissolved in 15 ml of dioxane and 800 (7 m) of hydroxy-aucoinimide are added to the solution, 1.4 g (7 mX) of '- dicyo. ohexeyloarbadümida is added and the solution is refrigerated overnight.

   The dioyolhe-xylurea is separated by filtration and washed * with dioxane. The filtrate is concentrated to give an oil which is
 EMI105.2
 oristallized. in a mixture of ethyl acetate and r1trv \ etber, A solution of 652 mg (1mK) of p.iap.pt1de and 168 mg (3 '? M) 18 sodium bicarbonate in 2 m2 of water is prepared. To this solution is added 50 mg (1mK) of the N-hydroxyBUoai ester: '' *: i4iq.ue 'u oarbobeuaQX1 prolyl-prolyl-glyoine dissolved in 3 ml of ethanol.

   The reaction mixture is oonaerv6 at room temperature overnight. After acidification to pH 2 with hydrochloric acid, the ethanol is removed under vacuum in a manner.
 EMI105.3
 re to precipitate a buil,, The oil is separated from the aqueous layer, discussed in ethyl acetate, washed with water, dried over magnesium sulfate and concentrated to dryness so as to give 565 mc of ca: rbobenzoxî, -octapeptide 485c! µ of cartobenxoxr-oatapapti, of (0, ° 62matz are dissolved in 10 ml of glacial acetic acid and 500 mg of charcoal supporting 10% palladium serving as a catalyst are added.

   The compound is hydrogenated
 EMI105.4
 under a pressure of about 2t kg per cm for 2 hours, The catalyst is separated and the solution is concentrated to give an oil, Ohromatography in

  <Desc / Clms Page number 106>

 Thin lines showed that the product is still the protected peptide. The oil is redoubled in 30 ml of glacial acetic acid and another 500 mg of charcoal supporting 10% palladium is added.

   The re-
 EMI106.1
 The action is then hydrogenated when the next day is applied to a pressure of about 2.8 kg per eu 24 The catalyst is separated and the reaction is oonentrated to s.oo3 t so as to give ibn mg of pxclyl prc3.y ,, yoï .. phenrla, anri.sr1-prolyi.plaaayla, any, axgiz3ne unprotected, 48 mg of octapeptide are dissolved in 1) 8 91 of a buffer at pH 10 and the pH is adjusted to 9 # 5 to 000, To this solution is added 0.3 ml of 1-carboxy arginine anhydride in dimethylfoimanida.> Amount of 101 added and 100% excess.

     After mixing for one minute the pH is adjusted to 6.3 and the reaction mixture
 EMI106.2
 is axtnBo% ion aU m4thanoµÙt¯ to ± ¯ will have an oolonne to # repair extraction with mdthanonl-t poured onto a column of renal containing IRC-50 and the nonapeptide eluted with 25-50% acetic acid in some water.



   After freezing drying, fractions
 EMI106.3
 . -15..u 179 mg of bradykinin are iaoléa, this aubetan- ce being; lisxoutēdané 7 ml of methanol, centrifuged to er. separate some insoluble parts, concentrated at room temperature to a small volume, and a centrifuged amorphous solid, washed with 1! ether and. dried.
 EMI106.4
 empty. Spinoo amino acid analysis confirms the atruo-
 EMI106.5
 '' "'re bradykinin.

  <Desc / Clms Page number 107>

 



    EXAMPLE 20, -
 EMI107.1
 ib LI y! M2- --- le 2 y! :: y 2lzl: & l y 2in-e- A Lee melaiigfi is added containing 2.02 mM of phenylalanine N-oarboxyanhydride and 2 mM of glycyl-leucyl-valyl-glyoine in a fraction to 20 ml of a rapidly stirred aqueous solution of potassium borate serving as a buffer at pH 9 and 0 C. The mixture is stirred for 45 seconds and the pH is adjusted to 4 with concentrated sulfuric acid at 0 C. However, a stream of nitrogen is passed through the mixture for about 10 minutes.
 EMI107.2
 



  The product is recovered by freeze drying. This operation being followed by the extraction of the residue with methanol. The methanolic solution is developed
 EMI107.3
 lopped with a 4tlt5 n-butanol-acetic acid-water system.



   EXAMPLE 21.-
 EMI107.4
 l! l!:! l1: i1: El1! 1 !! ¯: A mixture of 3 r, moles of D-ph6nylalan'.ne in 30 ml of aqueous sodium borate buffer containing a layer of folded ice 109.e was cooled to 30 and then 3.2 moles of N-carboxyl leuoine anhydride were added rapidly, the addition being carried out with rapid stirring.
The reaction is continued for 30 seconds and the pH is adjusted to 3 with concentrated sulfuric aid at 0 C while passing one oor.
 EMI107.5
 nitrogen flow through the mixture t The product is leu- .phen11alanine which is not isolated,

  <Desc / Clms Page number 108>

 The pH of the mixture is adjusted to 10.0 by
 EMI108.1
 addition of concentrated potassium hydroxide and a-,;

  Then 3.4 moles of N-oarboxy tetra- ..ropyranyl-tyroeineteoride anhydride is then stirred rapidly, however, the temperature is kept at about 000. is continued for one minute and the deoarboxyla-
 EMI108.2
 "'' is carried out by adjusting the pH to 3 with sulfuric acid. The mixture is allowed to return to ambient tGm63 ro 'and kept thus for one hour from.;,: ...' to be removed the protecting group The desired product is isolated by chromatography on a column of silica gel.



   EXAMPLE 22, -.
 EMI108.3
 



  P- (3t4'-dihydroxyphenyl) -L-alanyl-phenylalanyl-glycyl- --- ¯¯¯ !! 2l!: !!! l *: l2 !! ¯¯ ----------- -------------
The product of Example 21 (2.02 mM) is taken up in 30 ml of potassium borate buffer / at pH 10.



   The solution is cooled to 0 C and added with 3 mM
 EMI108.4
 N-Oarboxy-p- (2,4-dihydroxyphenyl) -li-alanine di-tetrabydropyranylic ether (DOPA) while stirring rapidly in a blender. The reaction is continued for one minute and the pH is adjusted to 4 with concentrated sulfuric acid while a stream of nitrogen is passed through the mixture. The passage of nitrogen is continued for 20 minutes. The mixture is then allowed to come back to room temperature and thus stored for one hour. The product is isolated
 EMI108.5
 T ""? '"ChI' ..J: '1atography.

  <Desc / Clms Page number 109>

 



    EXAMPLE 23.-
 EMI109.1
 ! 2! 2l1: Y! L1: 5! L2l!: FE! IE !! ' A mixture of 3 mM epinephrine in 30 ml
 EMI109.2
 of .bor .: r; = of aqueous potassium buffer containing 5 g of ice at pH 10 is cooled to 0 C and 3.01 mM of N-carboxy glyoine anhydride is added, the addition being carried out with rapid stirring The reaction is continued for 40 seconds and the pH is reduced to 4 with 2 ° C sulfuric acid while nitrogen is bubbled through the mixture for 20 minutes.
 EMI109.3
 your. The product which is glycyl-epinephrine is not isolated.



  Table 1 ... above describes the process by which glycyl-epinephrine is transformed into the final product,
Tempe- -CO2 NOA mM pH rature Duration pH
 EMI109.4
 VAL 3.1 10 200 1 min. 3 ILEU 3.4 10.5 0 C 45 eeo. 4
The product is isolated by ohromatography, EXAMPLE 24.-
 EMI109.5
 .! l2l: 1! l!: !!! l! ::! Ehl !!!! A mixture of 2.0 mM α-zaphthylamine in 30 ml of a tam% on aoetic acid-sodium aoltate mixture at pH 4.6 is cooled to 5C and added with 2.0mlVt of N-oarboxy valyl.an3iydride with rapid stirring. The valyl-a-naphthylamà.ne oarbamate which forms deoarbqxy- le spontaneously. The mixture is flushed with nitrogen for 20 minutes.

   The pH is then adjusted to 10 by the addition of aqueous sodium hydroxide and 2.3 mM of N-arboxy leuoine anhydride is added with rapid stirring at a temperature of 2 C. The pH is then added. adjusted up to 3

  <Desc / Clms Page number 110>

 with sulfuric acid so as to de-
 EMI110.1
 oarboxylatîou and the mixture is swept with nitrogen for 15 minutes. At the end of this period the pH is adjusted to 9.5 with sodium hydroxide and 2.9 mM of N-carboxy glycine anhydride is added with stirring.
 EMI110.2
 rapid reaction to 360, The pH is then brought to 3 with concentrated sulfuric acid and the mixture is swept with nitrogen for 20 minutes.

   The product is isolated by chromatography on a column of silica gel,
 EMI110.3
 EX: PIE C 5. -! lt: 6! l2l1: s! ll1:! 2! Has a mixture of 2 mmoles of leuoine in 20 ml
 EMI110.4
 of aqueous potassium borate buffer at pH 10 25 0, 2.05 moles of solid N-thiooarboxy glycine anhydride are added with rapid stirring. The reaction is continued
 EMI110.5
 for one minute and the pH is adjusted to 5 with concentrated sulfuric acid in order to obtain d4thioarboxylation, while passing a stream of nitrogen b. through the mixture for about 10 minutes d,.> W.xo to produce glyoyl-leuoine which is not 3 r .c; ..



  The process is repeated at pH 10 through 0 minutes of a 0 minute reaction using 2.02 m N-mol of thiooarboxy glycine anhydride so as to obtain glyoyl-glyoyl-1 uoino which is not isolated. ,
The pH of the mixture is adjusted to 9.5 by the addition of concentrated potassium hydroxide and the mixture is obtained.
 EMI110.6
 Then 2.2 moles of 2540 N-thioaarbaxy phenylalanine anhydride with rapid stirring.

   After 30minutemp the pH is adjusted to 4, while we do

  <Desc / Clms Page number 111>

 pass nitrogen through the mixture. The tetrapeptide
 EMI111.1
 desired is formed and isolated parea11tioat1Qn with ammonium sulfate.
 EMI111.2
 By replacing N-th1ooarboxy phenylalanine anhydride with the appropriate anhydride and repeating the above-mentioned procedure, the following compounds are obtained!
 EMI111.3
 valyl-glyoyl - glyoyl-leuoire Isoleuoyl-glyoyl. 61yoyl-leuoino Alanyl-glycyl-glycyl-leuoine
 EMI111.4
 1!: XE: 'PLE 26.- T ros 1-leuo 1- hen alanine. l2l!: 2l !:

  Er !!
To a mixture of 3 moles of phenylalanine in 30 ml of aqueous potassium borate buffer at solid pH 10 is added 3.2 moles of N-tahiocarboxy leucine anhydride. with rapid stirring, the temperature reached 40 ° C. and kept at this value for 10 minutes. The mixture is then cooled and the pH is adjusted to 3 with
 EMI111.5
 000 concentrated sulfuric acid while passing 1 stream of nitrogen through the mixture.



  Euoy7.-pheny3.aanine is not isolated.



   The pH of the mixture is adjusted to 10.0 by the addition of concentrated potassium hydroxide and the mixture is added.
 EMI111.6
 then 3t4 m moles of t'trahydropyranyl-tlro8in.-.- th1o carboxy anhydride all rapidly stirring while
 EMI111.7
 The temperature is maintained at about 25 ° C. The reaction is continued for 20 minutes and the etchiorxboxylation is carried out by adjusting the pH to 3 with sulfuric acid. The mixture allowed to stand for an hour in order to remove the protective group

  <Desc / Clms Page number 112>

 The desired product is isolated by ohromatography on a
 EMI112.1
 

  * gel column of 81110e.



   The same compound is prepared by repeating the procedure as above using e-
 EMI112.2
 1ho = "y ±% hyl% yrosine-N-oarboxy anhydride.



  EXAMPLE 27 Iyi15y3: & 5yi2ys155.



   A mixture of 2 m moles of cystine in 30 ml of aqueous potassium beta buffered at pH 10 is maintained.
 EMI112.3
 ti ^, -. u, <- 'Y1ron 10 01 while adding with rapid stirring 4.1 moles of N-thiooarboxy anhydride
 EMI112.4
 alanine. The reaction is continued for 15 minutes and the 22 is adjusted to 3 with hydrochloric acid diluted to 2 ° 0 while passing a stream of nitrogen through the mixture. The product thus obtained, which is (alanyl) oystine is not isolated.



   The pH of the mixture is adjusted to 10.2. The mixture is cooled to 0 ° C. and 4.2 moles of N-thiooarboxy leuoine anhydride are added thereto while stirring rapidly. The reaction is continued for a period of time.
 EMI112.5
 for one hour and the dethooarboxylation is carried out by adjusting the pH to 3.5 o while passing
 EMI112.6
 a stream of nitrogen through the mixture. The bi8 (leuoyl-alanyl) oyatin intermediate is not isolated, but is treated with 4 # 3 m moles of lth.oa * ba valine anhydride at 2500 while stirring rapidly and the dethioonrb5xyiakiQn is carried out with sulfuric acid at pH d ":;

   will deny obtaining the b1e (valyl ... ltuoyl-alanyl) oyst1ne which is separated by ealifioation of the reaction mixture

  <Desc / Clms Page number 113>

 The product is reduced by hydro-
 EMI113.1
 gene so as to obtain valyl-leuoyl-alanyl-07Itein.



  EXAMPLE 28 ÉS 2. Glcyl-g11011-1uolne is prepared by the procedure of Example 25, and the reaction mixture containing it is treated with 2.5 moles of solid anhydride.
 EMI113.2
 dl {-OarbObenZox1-J-thiooarbox1 lysinev to C3 Q under rapid agitation within 2 hours. t of thiooarboxylate and ett'ect ,, 4e with sulfuric acid in nitrogen pr40enàa at pH 4.

   The product which is -oarbob.nloX1 lairg.royl.-g.3y. luß: .r.s is extracted by. & litio & ton from the reaction mixture and recovered by filtrations. The arbobanaxy group is removed by hydrogenation and the product is isolated by elimination of the wol. Both pressure reduced by filtration of the satalyst with niokel Raney, EXAMPLE 29.-
 EMI113.3
 ! l ± l: Ehl1l!:! 2 !! A solution of 40 moles of leuonia in 400 ml of buffered boric acid is adjusted to pH 10.5 at 2 C with 50% sodium hydroxide in a mixture.
 EMI113.4
 geur under nitrogen atmosphere. To the stirred solution was rapidly added 35 cl of di N-arboxy phenyl anhydride, alan, while the pH was maintained at 50% by the addition of 50% sodium hydroxide. .

   The mixture is,
 EMI113.5
 filtered and the colorless filtrate is acidified to pH 5.4 with 50% sulfuric acid while nitrogen is bubbled through the solution. The phenylalanyl-leucine produced precipitates and is recovered by filtration *

  <Desc / Clms Page number 114>

 
 EMI114.1
 A mixture of 2 mM phenylalanyl-leuoime in 10 mM boric acid 0.45 m buffer is adjusted to pH 9.5 to
 EMI114.2
 400 by the addition of 50% sodium hydroxide to a nitrogen atmosphere.

   2.1 ml of N-thiooarboxy glycine anhydride are added to the csi% mixture, while the pH is maintained at 9.5 by the addition of 50% sodium hydride, After stirring for 10 minutes at about 4 C the mixture
 EMI114.3
 The gas is filtered and the pH adjusted to 5.5 with 50% sulfuric acid, while nitrogen is bubbled through the mixture.

   The desired product which is
 EMI114.4
 glycyl-phdnylalanyl-leucine is recovered by filtration. fltXAMPLE .'0 Jo: Sl2l: E2! l: EE! l !! el:!: i !!! 'A total of 2.0 ml of arg1 hydrochloride is taken up in 200 cc of buffered potassium borate, the pH is reduced to 3.0, while nitrogen is bubbled through the gas. Mainly mixture to remove carbon dioxide therefrom and the pH is then adjusted to 10.5 by the addition of aqueous potassium hydroxide. The mixture
 EMI114.5
 and cooled to 000 and 25 g of g3.aoa o3t% are added <; addition being followed by the addition of 3.82 g of N-thiooarboxy phenylalanine danhydride. The mixture is stirred rapidly in a mixer for 2 hours.

   The pH is adjusted to 3 with sulfuric acid so as to achieve la.déthiooarboxylati9n ... po is then raised to 10 by addi-. tion of potassium hydroxide and the process is repeated at 25 C during a reaction period of 30 minutes
 EMI114.6
 with 2.02 mM N-thiocarboxy proline anhydride. the pH is adjusted to 3.0 with sulfuric acid to effect dethiooarboxylation.

  <Desc / Clms Page number 115>

 



   The process is repeated with 2.02 mM of N-thiooarboxy glyoine anhydride at 20 C and at an initial pH of 9.5 for 15 minutes of reaction. The pH is adjusted to 7 with sulfuric acid and the mixture is allowed to stand for approximately 16 hours to effect the dethiooarboxylation.



   The reaction mixture is diluted 10-fold to a total of 10 liters / and is passed through an 800 ml column of IRO-50 resin over 18 hours. The column is washed with 4 liters of water at a flow rate of 15 ml / min. The column is eluded with 0.35N sulfuric acid and the eluate is collected in 300 ml fractions.

   The fractions giving a positive for ninhydrin are freeze-dried, the solids which they contain are combined and extracted with methanol until the extracts no longer give a test with ninhydrin. The extracts which give a positive test with ninhydrin are combined and evaporated to dryness in vacuo.

   The residue is taken up in the minimum quantity of sulfuric acid. dilsed to pH 4.5 and a precipitate is formed by the addition of 5 volumes of methanol, this addition being followed by the addition of 10 volumes of ethanol. The precipitate is collected by filtration, dissolved in water. water and again precipitated with the marls quantities of methanol and ethanol. The precipitated product which is glycyl-prolyl-phenylalanyl-arginine is isolated and washed with methanol and ether.



   EXAMPLE 31. -
Bradykinin.



   The following table illustrates the process by-

  <Desc / Clms Page number 116>

 
 EMI116.1
 which the following nonapeptide arginyl-prolyl-prolyl-glyoyl-phenylalanyl-adryl-prolyl-phenylalanyl-arginine is produced according to the method of the present invention. The table gives the sequence step by step by which N-arboxy anhydrides or N-thiooarboxy anhydrides are successively added to give the desired product. The réao-
 EMI116.2
 initial tion o'offeolue between 1 mM of arginine and 1 mM of. anhydride N-oarboxy phenylalanine in 10 ml of a mid.
 EMI116.3
 place. ^: a.aoux buffered with boric acid at pH la, 5.



  The pH of the reaction is maintained by the addition of aqueous sodium oxide at 505 during the reaction period. The intermediate prolyl-phenylalanyl-arginine is isolated after the final decarboxylation at pH 3 by first drying the mixture by freezing when the residue is taken up in methanol. The
 EMI116.4
 methanolic solution is chromatographed on a column of silica gel and the desired tripeptide eluted with 20% aqueous methanol containing sufficient acetic acid for the pH to be 5. This tripeptide is trans-
 EMI116.5
 formed into the desired norapeptide by successive reactions without isolating the intermediates.

   In the first reaction, one mole of tripeptide in 5 moles of water is reacted in an equivalent amount of N-thiooarboxy serine anhydride at pH 9.0 and at temperature. of 4 ° C in a reaction period of 30 minutes. The conditions for the second reactions are given in the table, reactions in which the pH is adjusted using sodium barium hydroxide. The dethiooarboxylation is carried out at the end of each reaction with oonoentric sulfuric acid.

  <Desc / Clms Page number 117>

 



  The barium sulfate which precipitates is filtered off before proceeding with the next reaction. The end product of this series of reactions is a solution of soluble bradykinin sulphate which is converted to the free base and isolated by ohromatography on IRO-50, a reagent.
 EMI117.1
 Sulfonic acid type ion exchange sine by gradual elution with aqueous acetic acid.



     TABLE 1-
 EMI117.2
 l9Ca- Substrate Reagent 00na * pH Tem- Duration of, #. ¯¯¯¯¯ ¯¯¯¯. sword, -
 EMI117.3
 1 Phe NOA Arg. 1 tn1t / 1Oml 10.5 0.0 1 min.



  2 no FOA l'he-arg imm / lomi 10,0 000 1 min 3 Ser TC! i lmU / 5ml 9tO. 0 30 min 4 Phe TOA 1mK / 5m, 9.5 î5 0 1 hour 5 Gly TOA at 1mM / 5ml 9PO 4 0 10 min 6 Pro TCA at 1mK / 5ml 9.0 2500 30min 7 Pro TOA at 1mM / 5ml 9, 0 2, .0 30 min 8 Arg TCIs, 1mMi 5m4 9t5 25! 1 hour @ Substrates the previously prepared peptide.



     EXEMPTA 32.-
 EMI117.4
 (1'Yavl-leua .- hn la..an 1-. C1 i ar .n.,.



   This example illustrates the example of preparation of a labeled peptide.
 EMI117.5
 



  A mixture is prepared containing t?, R9 mg of uniformly marinated arginine. , in i 4 (0, milliourie) dane 5.0 ml of ah3orhydr3.qua. 4.01 acid and 421 mg of unlabeled arginine, in 20 ml of 1 M potassium borate buffer at pH 10p5o. an aliquot of 02 nil. To the mixture is added a 5% molar excess of the 1-t1ooarboxy phenylalanine anhydride in a fraction while

  <Desc / Clms Page number 118>

 mixing rapidly at a temperature of 25-30 C, mixing being continued for 15 minutes while the pH is maintained between 10 and 10.5 by the addition of 5N potassium hydroxide. At the end of the reaction period, a drop of caprylic alocol is added.
 EMI118.1
 as an anti-foaming agent.

   The pH is then reduced to 3.5 by the addition of concentrated sulfuric acid and nitrogen is passed through the mixture.
 EMI118.2
 for 10 minutes. The pH is brought to 10.2 by concentrated potassium hydroxide and an aliquot of
 EMI118.3
 0.1 ml of the reaction mixture containing phenyl-alanyt (C14) arginine is removed.



   To the mixture thus obtained, a moderate excess is added.
 EMI118.4
 Leuoine N-thiocarboxy anhydride 6.5 layer, with rapid stirring, at a temperature between 25 and 3000. The reaction is continued for 20 minutes, while the pH is maintained at 9 - 9.5. With 5N potassium hydroxide The dethiooarboxylation is carried out at pH 3.0 as described above.



  The pH is then raised to 10.2 with concentrated potassium hydroxide and an aliquot is removed.
 EMI118.5
 of 0.1 ml containing leuayl-phenylalanyl- (0 ') argi- Rine for analysis.



  The tripeptid is converted into a glyoyl-leuoyl-phenylalanyl- (0) arginine 3-thiooarboxy peptide by the addition of an 8fi molar excess of the 1-thiooarboxy an glycine hydride to the reaction mixture at 1500 and under rapid aid,, The reaction is continued for 1. ' zinu% e ,, while maintaining the pH at 9t6-10 av, Ja of 51N potassium hydroxide and and h-thioa1-

  <Desc / Clms Page number 119>

 
 EMI119.1
 bi ry t1d oct d6orboJ14 to pi 3 oomco described ol- des8ue. lx; 1 "'1 *' to 10, ea of potassium hydroxide - \! lH'1 ... 'r <, r,: .. - remove an aliquot of mi nrntoenan% le; wapùo4 d <! i-4 & de8 t'ïno d'ana- 1;

   -fl. "
 EMI119.2
 Each of the above aliquots r $ OR * ilIÎ4 * is chromatographed on pap1 using the jutanol-acetic acid-water system in the ratio itself. Vantt 41li5 The chromatograms are examined with a radioactivity band tester . The results indicate that each of the desired reactions proceeded in a yield greater than 85% of the time.
 EMI119.3
 re giving a product that is not seriously affected by side reactions.



   The reaction mixture is dried by freezing, the residue is extracted 3 times with portions of 150 ml of methanol and the combined fractions are oonoentréea to dryness under vacuum at 40 ° C. The residue is taken up in? ml of water and 1/4 of the solution is diluted
 EMI119.4
 up to 1 liter. This dilute solution is an ehromatographic exchange d phie eur 200 ml of an oo2ozm oarboxylic ions (CG-50 sold by the company ROHX and Baase of Philadelphia; diameter t 2.2 cm; height 155 cm) on the oyol.
 EMI119.5
 hydrogen .ha column is fed with the solution at a rate of 2.5 ml per minute and is then washed so as to collect 2 liters of water.

   The column is eluded with 0.01 N sulfuric acid. The effluent stream. And adjusted for the isotope C14 by
 EMI119.6
 using an anthracene column, initially fed at a flow rate of 0.2 ml per minute. The flow deali.

  <Desc / Clms Page number 120>

 
 EMI120.1
 



  '1'10 was gradually brought to 0.6 ml per minute:,;,' "'> me period of 20 hours and the flow this hand ....



  , r. iur, r; as long as 8 until the end of the eseai. Lf41tat is 24 ml. -: 1'L '. he fractions / and the product desires this obtained .i. e:. "'smt the fractions having the content ieotopi"' q :: 10,:; psr40 in tetrapeptide by adjusting the p7 to 9 f 5 .v'o l! aqueous sodium hydroxide to 50 and removing by 1 <, - '' ', tone. The residue is extracted with methanol.



  Itext '.0; is concentrated to empty BOUS at .40.0.



  The ses is dissolved in a small amount of water,
 EMI120.2
 and the 1. iit adjusted to. 3.5 With 6.1-aqueous hydrochloric acid, the solution is dried by freezing to give the product which is reorista11ized in a mixture of water and ethanol.



   EXAMPLE 33.-
 EMI120.3
 !!!! l:! l!: !! l!: 6! ll:! 2! 2 !! To a mixture containing 4 mM dlalanyl-leuoyl-glycyl-isoleuoine in 30 ml of aqueous potassium borate buffer at pH 9.5, 4.3 mM of α-thiooarboxy histidine hydrochloride is added with rapid stirring, however. that the temperature is maintained at 15 ° C. The mixture is maintained at this temperature for 20 minutes and the temperature, .and then brought back to about 3 ° C.
 EMI120.4
 ph'eot adjusted to 3% so as to effect the dthioaarboxylation and is purged with nitrogen. The pH is adjusted to 7 and the desired product is isolated by chromatography on silica gel.
 EMI120.5
 EXEMPT 4.- E1 0 1-hist, d I- 1 a 1- hn lalan, -leuoine.



  ! llh !!!! dl! = S! l2l!: Eh! l !!! l!: !! 2! ..



  A solution of 40 mM of leuoin in 400 ml

  <Desc / Clms Page number 121>

 boric acid 0.45 ± buffer is adjusted to pH
 EMI121.1
 10e5 to 200 with sodium hydroxide solution in a mixer under nitrogen atmosphere. 1 to the stirred solution 35 mM of N- anhydride is quickly added.
 EMI121.2
 oarboxy phenylalaninn, while maintaining the piu 10.5 by adding sodium hydroxide to 505.



  The mixture is filtered and the colorless filtrate is cooled, with sulfuric acid, 09, uaqu, a pX of 5t while nitrogen is bubbled through the solution. The phJnylalanyl-leuU5na produced precipitates and is recovered by filtration.



  2UY3Bà, 15, -, ¯, 3-caxrox an'hvdrd da tr.'luoraaotl-xir 'A total of 3.93 g dt (N-crbOX'1 oerinesot anhydride added, 200 ml of dioxane frafohomont purified this addition being followed by the addition of 5 ml of trifluoroacetic onhydride The solution is stirred at about 25 ° C. for 1.75 hours and concentrated under reduced pressure The residual oil is taken up in dioxane and the solvent. is driven off again under reduced pressure so as to obtain the desired product in the form of a residue.
 EMI121.3
 



  The trifluoroacetyl N-carboxv anhydrides of threonine, hydroxyproline, P-phenyladrinee, P-7hy-droxyleuoine, α-hydrozynorvaline, and Y-hydroxynorvaline are prepared analogously by replacing the N-oarboxy anhydride of serine with an equivalent amount of the appropriate amino acid N-carboxy anhydride.

  <Desc / Clms Page number 122>

 
 EMI122.1
 



  EXAMPLE 6 ...:. i: 2âE22ï-SE2Ëâ5-â2-E22S-Ë5É-.5Êj.



  A total of 3t93 g of N-carboxy serine anhydride is dissolved in 20a 3. ml of freshly distilled dioxane, this addition being followed by the addition of 4 ml of triohloroaoetyl chloride. The mixture is stirred for 16 hours at approximately 25 C. The mixture is filtered and the filtrate is concentrated under reduced pressure. The residual oil is taken up in fresh dioxane and again concentrated under reduced pressure so as to give the mixture. desired product as a residue.
 EMI122.2
 



  Threonine, hydroxyproline, wphenylBérine, 5-hydroxyloucinte a-hyçroxynorralne and Y-hydroxyRvrva'-line tmioh.oroaoetyl N-carboxy anhydrides are prepared analogously by replacing N-carboxy anhydride. of serine by an equivalent quantity
 EMI122.3
 slow of the appropriate X-carboxy amino acid anhydride.



  EXAMPLE .¯37.- 2-.thiooarbox anhydride of tri12uoroaoet 1 erine.



  A total of 84 g of serine is taken up in 75 ml
 EMI122.4
 ,) Ithan9l containing 18 ml of water and 68 ml of po hydroxide; Bsium 1117 4, To the mixture is added under a nitrogen atmosphere. and while stirring, 97.5 g of ethyl methyl xanthate while maintaining the temperature at approx.
 EMI122.5
 ron 2500 - 30 0 using a cooling bath.



  The reaction is continued at this temperature for two hours and then it is heated to 45 ° 0 and
 EMI122.6
 It was held for 0.5 hour at this temperature with continuous stirring; ê ... p: l; 8 .rande. part of the alcohol is driven off under reduced pressure and 180 ml of water are added.

  <Desc / Clms Page number 123>

 



  The mixture is then extracted twice with fractions d: 100 ml of ether so as to drive out the unreacted xanthate. the alkaline aqueous layer is reopened with 100 ml of ethyl acetate and 73 ml of 12N hydrochloric acid / then 70 ml of water are added.



  The mixture is shaken and the organic layer is separated.



  The aqueous layer is again extracted with 100 ml of ethyl acetate and the combined organic coats are washed twice with 50 ml portions of saturated sodium chloride solution. The organic layer is separated, dried over sodium sulfate and concentrated under reduced pressure so as to precipitate the desired aerine methyl thionourethane.



   A total of 8 g of methyl thinourethane .erine is taken up in 40 ml of tetrahydrofuan and 6.1 ml of phosphorus tribromide are added while stirring, however, while the temperature is maintained at 0 ° -5 ° C. - The reaction mixture is suddenly cooled in 200 ml of 10% aqueous sodium bicarbonate and extracted. three times with 100 ml portions of ethyl acetate *
The combined organic layers are washed twice with 50 ml portions of a concentrated sodium chloride solution and dried over sodium sulfate.



   N-thicoarboxy serine anhydride is obtained by removing the solvent under reduced pressure.



   A total of 5.8 g of the product prepared above is added to 200 ml of freshly distilled dioxane, this addition being followed by the addition of 5 ml of trifluoroaoetic anhydride. The solution is stirred at about 25 ° C. for 2 hours and concentrated under reduced pressure. The

  <Desc / Clms Page number 124>

 The residue is taken up in 100 ml of fresh dioxane and again concentrated under reduced pressure to give the desired product as a residue.



   The trifluoroaoetyl N-thiooarboxy anhydrides of
 EMI124.1
 thr6cnine, hydroxyproline, p-phenylaerine, µ-ho- droxyleuoine, α-hydroxynorvaline and Y-hydroxynorva- @ are prepared analogously by replacing the serire with an equivalent amount of the amino acid ap- own.
 EMI124.2
 



  BIEMPLB 18.- -t: iooarbox anhdride-detriohloroaoit3.-edrine.



  5.8 g of N-thiooarboxy serine anhydride are taken up in 200 ml of freshly distilled dioxane and 4 ml of trichloroacetyl chloride are added. The mixture is stirred for 8 hours, filtered and the filtrate is concentrated under reduced pressure to give the desired product as a residue.
 EMI124.3
 



  The triohioroaoétyl h-% hiooarboxy anhydrides of threonine, hydroxyproline, -phenylsin, P-by-. α-hydroxynorvaline droxyleuoin and Y-hydroxynorvaline droxyleuoin are prepared analogously by replacing serine N-thiooarboxy anhydride with an equivalent amount of the appropriate amino acid anhydride.
 EMI124.4
 ph $ 3.w l: E2! l! = E ±! l !! 1! l!: !! S !!! =.



  A total of 15.75 g of arginine hydroxyohloride are dissolved in 2.62 liters of a buffer solution of sodium f4trnb..tate at pH 10. The pH is adjusted to 3 with this concentrated sulfuric acid and nitrogen is bubbled through the solution for 10 minutes. The pH is

  <Desc / Clms Page number 125>

 brought to 10.2 aveo of 50% aqueous sodium hydroxide and the solution is cooled to 0 C. To the mixture is added
400 g of ice then 82.5 millimoles of N-carboxy anhydride elanine $ phenyl / dissolved in 200 ml of acetone.

   The mixture is stirred vigorously at this temperature for one minute and acidified with concentrated sulfuric acid to pH 3 while nitrogen is bubbled through the mixture for 10 minutes. is then adjusted to
9.5 with 50% aqueous sodium hydroxide and 400 g of ice and then 112.5 mm of proline N-carboxy anhydride in 150 ml of aoetone are added. The solution is stirred vigorously at 0 C for one minute and boxylated deodorant as described above. The process is repeated at pH 9.3 with the addition of 15.0 millimoles of trfluoroacetyl serine N-carboxy anhydride in 150 ml of acetone.

   After mixing for one minute, the pH is adjusted to 7 and the mixture is freeze-dried. The silica gel containing the adsorbed product is then added to a column of silica gel previously prepared in isporpanol. The column is developed with a methanol: water system; Ammonia, 80: 18: 2 The desired tetrapeptide free of the trifluoroacetyl group is isolated.



   The compounds)? The following are prepared in an analogous manner using the trifluocracetyl and trichloroacetyl derivative of the appropriate N-oarboxy or N-tio-oarboxy amino acid anhydride. In each case the product is isolated free from the protective hydroxy group. With N-thiocatboxy compounds, the coupling pH is 8.8 instead of 9.3.

  <Desc / Clms Page number 126>

 
 EMI126.1
 



  1'hronyl-proly.-phnylale.ny .. asgi.nine ', hydroxypralyl-praxy.-pYzér.y.a.sxy3.-arginïee 0-phenyleeryl-prolyl-phenylalanyl-arginine.



  .-hydroxyleuoyl -? ^: ^ a3.yl-phrzy.als.ny, -s.rginiue -hydroxynorvalyl-prolyl-phenylalanyl-arginin.



  Y-hydroxynorva1yl-prolyl-phenylalanyl-argin1ne EXAMPLE 40.- Q = !!! 1hl !! l! =! 2 !!:! = 2È2! L¯l !!! A total of 4.5 ml (35.1 m M) of trimethy3.ohlo. ro-silane is slowly added to 5.1 g (35t2 m X) of threonine N-oarboxy anhydride in 65 ml of tetrahydro.



  dry uranium under a nitrogen atmosphere, while the temperature is maintained at about 2 C.
 EMI126.2
 Slowly add 2.98 ml (34 m M) of dry 4-methyl-thiazole. The hydrochloride salt of the base precipitates from the solution during the addition. The reaction mixture is then brought to room temperature and the precipitate is separated by pressure filtration of astatine. The solvent is removed from the filtrate under reduced pressure and the residue is taken up in 35 ml of ethyl acetate kept at about 10 ° C. for 16 hours. The solution
 EMI126.3
 y, x, eante this reduced volume and the solvent is chaD '' '; under vacuum.

   The product which precipitates is gold: 1stAl bound by trituration with hexane and recovered by filtration under nitrogen pressure.
 EMI126.4
 



  The Nwearboxy anhydride O-tri4thy18ilyl threonine, the B-oarboxy anhydrid- dtO-tr1opylsilyl ... threonine, the '-carbaxy anhydride of 0-tri-n- "butylailyl thr4-"': hl, are prepared in an analogous manner " from N-carboxy threonine anhydride from tipiethyl-chlorouilanet from triropylbromo81lane, and from tri-n-butyl-
 EMI126.5
 br4moi..e respectively.

  <Desc / Clms Page number 127>

 
 EMI127.1
 



  EXAMPLE¯41 "± = gµg ± ± µ ± ¯Jµjgz ± gi ± f¯µ ± = ± jgfjgzfgz) ¯gµ ± j A total of 3.60 ml (26.8 a5 of trimdthyi, -oh4- rosilane is added under nitrogen atmosphere at 3.75 g (28.6 mM) n-carboxy serine anhydride in 65 ml of dry tetrahydrofuran while the temperature is maintained at about 0 C. To the mixture is added 2 , 51 ml (28.6 mM) of anhydrous 4-methyl-thiazole at the same temperature.
 EMI127.2
 The reaction mixture is left to stand until it is stirred to room temperature, and then for 2 hours.

   It is filtered and the filtrate is concentrated in vacuo so as to give an oily residue which is taken up in 30 ml of ethyl acetate. 100 ml of herane are quickly added, the mixture is left at approximately room temperature. for two hours and the supernatant oouohe is decanted. The settled layer is concentrated
 EMI127.3
 up to 80%, the residual product is purified by taking it up in hexane, filtering it and then washing it with additional h vane.
 EMI127.4
 



  O-triethyl + 10-oxy anhydride. serine, le, a-oarboxy anhydride of t? -tr.propulaily7. serine and W-oarboX1 O-tri-n-hexl1a1111 anhydride are prepared in 8im1aJ: ement of l-oarbox1 serine anhydride and tri '' 4th1lchloo8ilan, tripropl1bromoilan and r - heX1-. ohlorosilano respectively.

   The X-oarboxy anhydride 4tQ7 trimethylailyl hydrox1prolin.t prepared from MmUre ana-, logue using the Ii-oarboxy anhydride of hydroxypro11- ne and # r.rthylo3loroari.sna as a motiérov 4. start,

  <Desc / Clms Page number 128>

 
 EMI128.1
 ¯ EXAMPLE 42, àt,: ± 2:! ¯! Hl !!! ¯2: !!! l !! l¯l2 !!! A total of 7.36 ml (53.6 mM) of trimethyloblol eot added to 11.2 g (53.6 mM) N-arboxy anhydride 1. ,: coe "4.ne in 100 ml of ethyl acetate seo containing 1.3Q µ (53e6 mX) of 4-methyl-thiazole at -500, The mixture
 EMI128.2
 reaction is allowed to return to room temperature
 EMI128.3
 ant that it is agitated and that it is kept at
 EMI128.4
 rat ".>: SBbiante for one hour. It is filtered, oonoen- tr ,,, e, reduced pressure and the product is re-cut according to the pro le Example 41.
 EMI128.5
 



  N-oarboxy O-trimethylilyl 3,5 "dibronol: yroeine and N-oarboxy, O-trimethyl-ailyl anhydride ,, - diiodotyro8ine are prepared analogously with N-oarboxy anhydride 3. , 5-dibromotyroain.e and de, 5. d., Odttyroeine respectively. HC; '3'PI 4, ¯-¯!: 2! È2l¯l !! ¯Q: !! l !!!! l! ¯ : El !!!!! A total of 4.5 ml (35.1 ml) of trîmethylohlorouilano is added under a nitrogen atmosphere to 10.9 g ('5.1.mq) of p-phenyleerine in 150 ml of benzene however As long as the temperature is maintained at about 500 with rapid stirring, to this mixture is added 2.84 ml (35r1trI
 EMI128.6
 of pyridine at the same temperature, we constantly stir
 EMI128.7
 te Reaction is allowed to return µ the temperature
 EMI128.8
 ambient and filtered. The filtrate is concentrated and the
 EMI128.9
 residue taken up in 50 ml of ether.

   The mixture is con- Drv6 for about 20 hours about 10009 the island layer is the concentrated J. Separated sludge reduced preeaion do 1brQ to precipitate the product which is purified.

  <Desc / Clms Page number 129>

 by trituration with hexane and recovered by filtration.
 EMI129.1
 



  O-trimethylolyl-hydroxy-succin N-carboxy anhydride O-triethyl.ailyl p-hydroxyvaline N-carboxy anhydride and O-tr.n.bu-ty.sily N-arboxy anhydride. a-hydroxynorvaline are prepared analogously with the appropriate tri-loweralkyl-ohloro-silane
 EMI129.2
 β-hydroxyvaline, -hydoxa11ne and -hydroxyleucine, -hydroxyvaine} 7, and α-hy- hydroxynorvaine pootively. The same products are prepared in analogous fashion to the removal of material. \ a18 replacing the P / r1dinar an amount 4qui- 'kî.J. ± .48 of tridthylamire.



  EX? "'M: ¯44t- EÉ52yB) !. lâssi
A solution containing 145 mg of phenylalanine in 10 ml of water and 10 ml of 0.9M brick acid in a blender is adjusted to pH 10.5 with sodium hydroxide.
 EMI129.3
 sodium auquex at 5C and 326 mg r. 0¯ tri-methyl threonine N-carboxy anhydride are added in fractions over a period of about 3 minutes acu "nitrogen atmosphere and with rapid stirring. The mixture is kept at about 500 and, the pH is kept constant by the mixture. dropwise addition of a total of 0.75 ml of 2.5N aqueous sodium hydroxide during the reaction period.

   The pH is in- ....., it further reduced to 3 by the addition of sulfuric acid, The temperature is allowed to rise to 30 C and the mixture is maintained at this temperature for 5 minutes so as to effect deoarboxylation.



     A small sample of the reaction mixture

  <Desc / Clms Page number 130>

 
 EMI130.1
 is subjected to 3.'elaotrophorea on paper at pH 9.5 in 0.1 molar sodium tetraborate at 600 volte for 3 hours and threonyl-phenylalanine identified by comparison of its mobilized with a sample
 EMI130.2
 genuine product in identical oonditiona.



   The remainder of the reaction mixture is adjusted to pH 6.5 with 2.5N aqueous hydrochloric acid and freeze dried. The residue is extracted with methanol, filtered and the filtrate is evaporated to dryness. the residue is taken up in a small quantity of water and filtered. The filtrate is evaporated to dryness and the residue is triturated with methanols.
 EMI130.3
 lut i or .. methc -:,:>: '1that is chromatograph on carbon deactivates and eludes, first with water (19 trac-
 EMI130.4
 of 2 ml) and then with acetic acid 1k 5! water (aoetone 47.5 ml; water 47.5 ml;

   aodti- '.o 9 s'l) The product is obtained by combining the pulls 21.ih 26 each containing about 2 ml and drying by number:' "1, ong 3X $ l $? 1345 * t er Z- hën lalanine.-.



  A solution containing 330 mg of Phenylalanine, 10 cl of water and 10 ml of 9.9 molar boric acid, a mixture is adjusted to H 10.5 with aqueous sodium hydroxide at 50 and 406.6 mg. , of N-oe: rbqxy trimethylsilyl anhydride are added in fractions.en@lleepace of 3 minutes, stirring rapidly, under a nitrogen atmosphere,
 EMI130.5
 rrndant Qu11ton maintains the temperature at 3 0.

   The pH kept constant by the dropwise addition of a total of 0.85 ml of 2.5N aqueous hydroxide. Part ali.

  <Desc / Clms Page number 131>

 Quote is dissolved in a 10: 1: 3 mixture of butanol-acetic acid-cau and subjected to thin layer ohromatography on ally gel. The product is identified by comparing its mobility in the system with that of an authentic sample.



   EXAMPLE 46.-
 EMI131.1
 :! 2l: 2l: Q:! L !!!: l!: !! ± 1! 2l:! H! 2 !!!:
A mixture of 10 ml of water and 10 ml of 0.9 molar boric acid in a mixer is adjusted to pH 10 with 50% aqueous sodium hydroxide and 1.081 g
 EMI131.2
 of mdt.onyâ.0-bonayâ; ry.-c: leuolthrozins are added The pH is then adjusted to the space with additional aqueous sodium hydroxide and 478 mg
 EMI131.3
 of triaethylilyl threonine X-oarboxy anhydride are added in portions while stirring rapidly in a space of gentle minutes, while maintaining the
 EMI131.4
 temperature around 300.

   During the addition a total of 1 ml of 2 # 51 aqueous sodium hydroxide is added so that mgintr - constant br. The mixture is diluted up to five times #onolume with water, filtered, the filtrate is washed and the pH of the combined filtrate and washings is adjusted to 6.7 with aqueous sulfuric acid. at 50%. An amorphous solid precipitates and is
 EMI131.5
 identified as the desired product by electrophoresis on pH 9.5 paper on 0.1 sodium tetraborate
 EMI131.6
 molar at 600 volts for fi, 5 hours.



  . ? The S 47 t-.



  !! l2l!:! lE2! l: !! ± l!: E! l !!!!!
A mixture of 3 mM of phenylalanine, 30 ml of aqueous potassium borate buffer at pH 10.5 has re

  <Desc / Clms Page number 132>

 
 EMI132.1
 you1 to 300 and 3.2 mM of solid leuoine 1-oarboxy anhydride is added, with rapid stirring. The réao-
 EMI132.2
 . fl; m is continued for 30 seconds and the pH is adjusted to 0. sulfuric acid concentrated to 000, however, which - nitrogen is passed through the mixture the pi <, i-ilcat not isolated.



  The fold of the mixture is adjusted to 10.0 by stad- concentrated potassium hydroxide and 3.4 stad- of N-. Triethylsylyl tyrosine anhydride is added rapidly, while the reaction is maintained at about 0 ° C. The reaction is continued.
 EMI132.3
 p4: f: .. nt "-le Biauta and the pH is adjusted up to 3 with l * & f" i '"b ..: uriq, ue so as to achieve ddoarbola t: .l' 11.e The product is not isolated * he pu of the mixture is adjusted to 10.2 by adw ditiox. concentrated potassium hydroxide and 6 mM Vtv d;

   wveF '.', '. "f ,, s,.' r., glycine anhydride are added while at: S, 1; 1.i%: t ': 1pidament, while maintaining the tom- p:> .; x. about 3 * 0 and the pE at 10.0 to 10.2 by the addition of 1 - 2 N n drop of sodium hydroxide = .11 - the reaction 6 V \ on, ca% e by drop of Bodiu hydroxide. The reaction is 1.J ## i, = ivis laying about 2 minutes and the deoar- bo - '"'? z tc: was carried out by adjusting the pH to 3 $ 2 with dz 1- 1 sulfuric acid and allowing the mixture to heat up to room temperature. The mixture is left at rcp :. about an hour and clarifies by filtration. The desired product is obtained by passing through to a. boi lo; .. 1 '; 1.: e reaction on a carbon column.
 EMI132.4
 



  ': 0 ojt adsorbed and it is% "then elected" with a -, au and acetic acid & 5. It is recovered (.1- lt.:".1 .: by sdohage-by freezing.

  <Desc / Clms Page number 133>

 
 EMI133.1
 EXPL3 $ 9. : 2EP.2! L¯! ÀlS !! ¯! ¯! EEElE! L¯! LE ± !! A total of 1 g of tyrox3, n6 N-arboxy anhydride is distributed in 20 ml of dihydropyran (containing 60 mg of p-tolueneaulfonyl chloride) and the mixture is stirred at rowing 25 ° for 48 hours. At the end of this period, most of the anhydride is dissolved indicating that the reaction is substantially
 EMI133.2
 complete. The reaction mixture is diluted with the addition of 30 ml of petroleum ether and the desired product precipitates,
 EMI133.3
 2X2 %% LE # = ¯ i-carbaxf nh tx3ds of tetrh dro ran 1 YEosne.



  !: ± ± 2! ¯! L !! ¯g! ¯! E2l ± 2El ±! L! ¯! L2 !! E! A total of 1 g of 1-carbaxy tyrosine anhydride and an equivalent molar amount of dihydropyran are taken up in 20 ml of dioxane containing 40 mg of p-toluere Bulfoniqlan acid, and the mixture is stirred. at 35 0 for 10 hours. At the end of this period, the solution is concentrated to half the volume under reduced pressure brought back to the original volume with ethyl acetate.
 EMI133.4
 and then added with 0.25 g of s11ioe gel.

   The mixture is then filtered, concentrated to half a volume and the desired product is precipitated by addition of petroleum ether. '
 EMI133.5
 EXAMPLE 51t-!: 2! ± Ê2! L¯ !! ¯! ¯ !!! hl22l! L¯l2 !!! i- 'A total of 1 g of 3-carboxy tyro- eine anhydride and a equivalent molar amount of dihydropyr6.n $ are taken up in 20 ml of deotrahydrofuran containing 60 mg

  <Desc / Clms Page number 134>

 
 EMI134.1
 of p-tolu.niultùl1: fl. chloride, and the mixture is anilu-6 h 20 0 for 60 hours.

   At the end of this period, the mat solution, concentrated to half a volume or reduced, brought to its original volume with ethyl acetate and added with 0.25 g of ni- gel.
 EMI134.2
 The mixture is then filtered and concentrated until
 EMI134.3
 half a volume and the desired product is precipitated by 1 ad-
 EMI134.4
 petroleum ether edition.
 EMI134.5
 



  The following compost are prepared of the analogous number by replacing the tyrosine derivative with u, equivalent amount of the appropriate depazt compound, tetraiayàropyxanyl x-arboxy anhydride, 5wdiromotyrosino; or.3aa: y tetrahydropyranyl anhydride 3, -di.ado- 'tYl'o0ino; : SXE !! PL1 !: 52 t-: h ± ± È2! Z¯! Hl !!! ¯2! ¯ !!! 2l2l: l! ¯! L! 2 !!! 1
 EMI134.6
 A total of 145 g of tyrosine is included in 75
 EMI134.7
 ml of etiol containing 18 ml of water and 68 ml of hydroxide in pt <3ium il, 7 N. To the mixture is added, under atmo8pb-
 EMI134.8
 ,, 1t; It, .., e and while stirring, 97.5 g of ethyl xanthata. -'-: rl (1 c, ndant that. one maintains the temperature at in-
 EMI134.9
 viron SC -.30 "0 using a cooling bath.



  The is continued at this temperature for two hours and then heated to 4500 and the temperature is maintained for ot5 hour 8upp14mentair6 while continuing stirring. Most of Itacool is sung under reduced pressure and 180 mil cont added. the mixture is then extracted twice with de4fractions of 100 ml of ether so

  <Desc / Clms Page number 135>

 to remove the xanthate which has not reacted The alkaline aqueous layer is covered with 100 ml of ethyl acetate and 73 ml of 12N hydrochloric acid, then 70 ml of water are added. The mixture is shaken. and the organic layer is separated.

   The aqueous layer is again extracted with 100 ml of ethyl acetate and the combined organic layers are washed twice with 50 ml fractions of a saturated sodium chloride solution. The organic layer is separated, dried over sodium sulfate and concentrated under reduced pressure so as to precipitate the desired methyl thionourethane tyrosine.



   A total of 8 g of methyl thionourethane tyrosine is taken up in 40 ml of tetrahydrofuran and 6.1 ml of phosphorus tribromide are added while stirring while maintaining the temperature between 0 and 5 C. The reaction mixture is abruptly cooled in 200 ml of 10% aqueous sodium bicarbonate and extracted three times with 100 ml portions of ethyl acetate. The combined organic layers are washed twice with 50 ml portions of ethyl acetate. a concentrated solution of sodium chloride and dried over due to sodium salt @ The desired product, namely N-thioarboxy tyrosine anhydride, is obtained by removing the solvent under reduced pressure,
A total of 5,

  8 g of the product prepared above is converted into the desired compound in dihydropyran by the method described in Example 50.



   The 3,5-dibromotyroins and 3,5-diiodotyrosine tetrahydropyrunyl N-thicoarxoy anhydrides are pre-

  <Desc / Clms Page number 136>

 similarly trimmed by replacing tyrosine
 EMI136.1
 rare an equivalent amount of the appropriate amino acid.



  EIEr, tPLE 5 '.- gzg ± ± z) = gj ±) z1 = ± gµz) µjgzé = µggé%, =
A total of 15.75 g of arginine hydrochloride is dissolved in 2.62 liters of a buffer solution of
 EMI136.2
 % sodium etraborate at pH 10. The pH is adjusted to 3 with concentrated sulfuric acid and es is bubbled through the solution for 10 minutes.
 EMI136.3
 ± 1J.i ± '1 to 10.2 with aqueous sodium hydroxide at 50 ... the solution cooled down to 0 C. To this mixture is added 400 g of ice, this addition being followed by 'addition of 82.5 millimoles of N-carboxy anhydride
 EMI136.4
 pht (nylalanine dissolved in 200 ml of aoetone.

   The mixture is stirred vigorously at this temperature for one minute and acidified with concentrated sulfuric acid to pH 3, while nitrogen is bubbled through the mixture for 10 minutes. The pH is then adjusted to 9.5 with 50% aqueous sodium hydroxide and 400 g of ice and then 112.5 mmoles of N-oarboxy anhydride of proline in 150 ml of acetone are added. The solution is stirred vigorously for one minute at
 EMI136.5
 090 and ddoarboxylated as described above. The process is repeated at pH 9.3 with the addition of 15 moles of 3vnrbozy tetrahydropyranyl tyrosine anhydride in 150 ml of aoetone.

   After mixing for one minute the pH is adjusted to 7 and the mixture is dried with oonsela- 'iOtl 1) 8 rds3du is taken up in methanol and the product' 'Jorb6 from the solution on 8il101 gel.

  <Desc / Clms Page number 137>

 



  The silica gel with the adsorbed product is then added to a column of silica gel previously prepared.
 EMI137.1
 in Ilisopropanol, the oolonne is developed with a methylanC0 ffiethano1: water: ammonia, 80! 18! 2} and the desired tetrapeptide is ibolefree of the tetrahydropyranyl group.



  The following compounds are prepared analogously using the tetrahydropyranyl derivative of the appropriate N-arboxy or N-thiooarooxyamino acid anhydride. In each case the product is isolated free from the protective tetrahydropyranyl group. Aven them
 EMI137.2
 Composed of 3-thiocarboxy, the coupling pH is 8.8 instead of 9.3 and the reaction period is 40 minutes.
 EMI137.3
 , 5-d3.bxotaotyroeyi..praxy "-p'hnyla.anyl-arg.nina 3, 5-düodutyro syi-prol.yl--phny3.a.anyl-argirz.ne EX.! PLE fiv 2yÊlâ2-.iËSÊ2 <.S: 2ÊE2S-5BES.



   A total of 5g of dry aspartic acid is taken up in 200 ml of tetrahydrofuran and bubbled
 EMI137.4
 phosgee through the mixture at about 2500 for 20 minutes. The mixture is then left to stand for 10 minutes and 300 ml of ethyl acetate arec.



  The solution is concentrated to a total volume of about 40 ml and a further 60 ml of ethyl acetate is added. A small amount of precipitate is separated by filtration under a nitrogen atmosphere and 85 ml of hexane are added. The slightly cloudy solution is stirred for about 10 minutes and then 120
 EMI137.5
 additional ml, hexane with constant stirring.



  Stirring is continued for a further 5 minutes.

  <Desc / Clms Page number 138>

   tairea and the precipitated N-carboxy aspartic acid anhydride is collected by filtration. The product is washed
 EMI138.1
 with a 3% 2 mixture of hexane-ethyl asetate and dried with nitrogen gas.
 EMI138.2
 



  The following table summarizes the results of further examples for the preparation of 4Bpartic N-arboxy acid anhydride. In all cases. 5 g of tuapartic acid in 200 ml / of reaction solvent are used.



  In example 2, an equimolar amount of phosgene
 EMI138.3
 tot used The molar oxoei of pboogbne used in the following examples is given in the table.



  The tmpdratur6 rdaotio = it in example 2 is' of bzz0 * 0 it in example 3 it is 15 * 0, All the other examples were carried out at the temperature
 EMI138.4
 ab1ante ..
 EMI138.5
 ¯SplYantd97iï'anBfM7 '
 EMI138.6
 
 <tb> Phos- <SEP> Hairy <SEP> Identity <SEP> Hairy <SEP> Liqui-
 <tb>
 
 EMI138.7
 r.x.

   Golvant gene told me to pre- ml. nal cipi- .¯¯¯¯¯¯ ¯¯¯¯¯¯¯¯. ml. -ercant 55 dioxahne - 200 ButYl aoéta- 40 oyolo- hexane 56 t6tva- 50 300 Ethyl aceta- 100 'oyolo-
 EMI138.8
 
 <tb> funanne <SEP> you <SEP> hexane
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> @ <SEP> Dioxane <SEP> 100 <SEP> 200 <SEP> Butyl <SEP> aodta- <SEP> 100 <SEP>.

    <SEP> hexane
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> you
 <tb>
 <tb>
 <tb> 38 <SEP> Ether <SEP> 200 <SEP> 200 <SEP> Ethyl <SEP> augusta- <SEP> 40 <SEP> pentane
 <tb>
 <tb>
 <tb>
 <tb> ethyli- <SEP> you
 <tb>
 <tb>
 <tb> that
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> 59 <SEP> Ether <SEP> 100 <SEP> 1000 <SEP> Proyl <SEP> Aug- <SEP> 100 <SEP> iaooo-
 <tb>
 <tb>
 <tb>
 <tb> propyli- <SEP> tate <SEP> tane
 <tb>
 Original mixture concentrated under reduced pressure to about 40 ml of total volume and diluted by adding about 60 ml of transfer solvent.
The identity of N-carboxy acid anhydride as-

  <Desc / Clms Page number 139>

 
 EMI139.1
 partique is established by conversion to ieoaepe:

  ragina. This reaction is carried out by dissolving 1.0 mM of the product in 2 ml of dioxane freshly distilled from sodium and adding the solution thus obtained dropwise to a cold, saturated solution of anhydrous ammonia in dioxane. An amorphous precipitate forms immediately. The reaction mixture is taken up jvsqutà sicoité under reduced pressure and the
 EMI139.2
 product is subjected to chromatography in layers min "oes on silica gel.

   The product is then tested for each of the iaopropanoli eystemeolvanta iaopropanoli acid leap aodc, 7: 2: 1S pyridine! 4'ethyl acetate: acetic aoid! water, 5t5tlt3i and acetic n-butanoliaoid) water, 10! l! 3t using authentic asparagine samples
 EMI139.3
 and isoaaparagino as a witness * and we see. elder! that it consists essentially of pure i8oa.paragine which establishes that the starting compound is substantially pure N-carboxy aspartic acid anhydride.



  * EXAMPLE 60.-
 EMI139.4
 hl !! ¯! ¯!: ±! 2! l¯ !! E !!! S !! - A total of 2.0 g of benzoxyoarbonyï asparagine, is taken up in 20 ml of dioxane and adds 0.24 ml of phosphorus tribromide while stirring at about 25 C.



  The mixture is stirred for 18 hours and then passed through a 50 ml column of silia gel prepared with a 1: 1 mixture of methylene chloride and dicxane, then dioxane. After addition of the reaction mixture to the column, it is developed with successive fractions of 50 ml of methylene chloride (8 fractions);

   a 1: 1 mixture of acetone and methylene chloride (4 fractions) and acetone (4 fractions) * The

  <Desc / Clms Page number 140>

 desired product is obtained by removing the solvent from the last two fractions under reduced pressure. The said is recrystallized by taking it up in acetone, heating moderately and concentrating it in a nitrogen mixture until the solution is cloudy.



  The product is then precipitated by the slow addition of hexene and with cooling and stirring.



   The procedure described above is repeated with methoxycarbonyl asparagine and 4-methylbdenzoxycar @@@ sparagine to obtain the same compound.



   EXAMPLE 61. -
N-carboxy glutamine anhydride.



   A total of 2 g of beznoxycarbonyl gultamine is repeated in 20 ml of dioxane at 25 ° C. and 0.24 ml of phosphorus tribromide are added. The mixture is stirred for 4 hours and the product is isolated by chromotography in a 200 ml column of silica gel prepared with petroleum ether and developed as follows! 200 ml of methylene chloride
4 x 100 ml of a 1: 1 mixture of aoetone and methylene chloride
4 x100 ml of a 3: 1 mixture of acetone and methylene chloride
4 x 100 ml of acetone.



   The estisole product enchassantle the solvent of the 7 doornières fractions under reduced pressure.



   The same product is obtained by replacing the bans cxycebaronyl glutamine with an equivalent amount of ethoxycarboyl glutamine and by stirring; / for 24 hours.

  <Desc / Clms Page number 141>

 res and also with an equivalent amount of 4-
 EMI141.1
 ohlorobenzoxyoarbonyi glutamiae and shaking 3000 for 4 hours. The isolation process is the same as that described above,
EXAMPLE 62.-
 EMI141.2
 !!;! l!: !!!!! l
A total of 1.0 g of N-carboxyglutamine anhydride is taken up in 25 ml of sodium borate buffer at pH 10 and cooled to about 0 C.

   To the mixture, 0.5 g of alanine is added with rapid stirring.
 EMI141.3
 minutes of stirring, the fold is adjusted to 3t5 with concentrated sulfuric acid and nitrogen is bubbled through the mixture to facilitate removal of the anhydride. The pH is then brought to 5.7 with aliquots of sodium hydroxide and the product is isolated by obronatography using a sulfuric ion exchange resin (Dowex 50-X2; sold by the Dow Chemioal Company, Midland, Miohigan) on oyol hydrogen The product is eluted with ammonium hydroxide.
 EMI141.4
 



  In the ohromatographic process, the reaction mixture is passed through a column of 800 ml of resin. The column is eluted with ammonium hydroxide pH 11.0 at a rate of 60 ml per minute and the product is collected in fractions of 500 ml. * the desired product is obtained by combining and freezing the 17th and 18th fractions *
The process is repeated to prepare luta-miynl-leucine by replacing the alanine with an equivalent amount of leuoin.

   In this box the desired product is

  <Desc / Clms Page number 142>

 recovered by screwing in and freezing the 15th and 16th fractions
 EMI142.1
 iaapaxag.nyl..phany3.a, .ar: .e is prepared in the analogous manner using equivalent amounts.
 EMI142.2
 tes of N-carboxy asparagine and phn.a.7.an.ne BXEHPLË 63.- µ ± zz) = g) gjµggz) = 6) zz) = ± gfzgµ] gz = éfygµj =
To a mixture of 4.0 mM of leuoine in 20 ml of water at 0 C is added 3.1 moles of N-oarboxy anhydride
 EMI142.3
 However, the pH is maintained at 10.5 by intermittent hearing with powdery barium hydroxide. 1: 1'60.0 '\'; ' <P Jo is over in a minute,.

   The pH is adjusted to 3.5 by the addition of 10% sulfuric acid. The barium sulphate which precipitates is separated by
 EMI142.4
 filtration and the filtrate containing the phenylalanylZu .. cine is treated with 3.3 mM of carboxy glycine anhydride
 EMI142.5
 h -33 and urî5il of 10.5 is maintained. using barium oxyxide. Deoarboxylation is carried out by adding p3 to 3t5 with sulfuric acid <m <:% ...;:, Jr, 1 and the barium sulfate which precipitates is separated J'l li: .t: 1: ation.

   The pH of the filtrate is adjusted to 10.0 with sodium dc -.1 hydromide at 40 and 4 ml of 1-oarboxy anbydrate rp tline are added to 0 * 0 while stirring rapidly. After two minutes, the pH is adjusted to 5.3 by the addition of concentrated sulfuric acid and nitrogen is bubbled through the mixture for 3 minutes
 EMI142.6
 The pi cat then adjuet6 to 10 av of hydroxide; : dium 4o # and the mixture is cooled down to 2 '! ....



  Then added 4.0 MX of loarboX1 glycine anhydride with rapid stirring. After one minute, the pH is

  <Desc / Clms Page number 143>

 
 EMI143.1
 r4à; iL # to,; o <,> <to i by the addition of sulfuric acid
 EMI143.2
 and we do ')! ': .. .... aZ0te through the melanoma penàan1J 5 min.itz, 1 ,; -, x /; '' 6 h 7 with hydroxy-
 EMI143.3
 de- of sodium and the mdla:, ^ ¯ is a (: CI1,: by freezingt The solid n4: .idiAei is m Wr ;. three times with \, Î: '6'thml: 3: and the methanol extracts combined are bitter / 1cit. the desired product is obtained further from @ @ is purified by chromatography, eg. -64.
 EMI143.4
 



  2: iËÎ-.3lËëË5Î-ÊS-ë2-
A total of 13.00 g of serine is dissolved in 500 ml of distilled water and 14.33 g of freshly prepared silver oxide is added. The mixture is stirred at the ce
 EMI143.5
 room temperature juaqu 1 silver oxide n / a d18.01ve.



  During the reaction, a brown colloidal material forms and is separated by filtration through diatomaceous earth. The filtrate thus obtained is treated with methanol at room temperature until it becomes cloudy.
 EMI143.6
 while waving it around. desired product separates and is reov; 6c "mir filtration.



  Y? "', 6.- sisarent. Of thx6ozrine. Silver chelate of thrdonine.



   A total of 11.9 g of threoin is dissolved in 25 ml of water and 11.6 g of freshly prepared silver oxide is added over 10 minutes. The mixture is stirred for an additional hour while all
 EMI143.7
 the oxide is dissolved.

   The resulting mixture which contains an occlial gel is stirred for one hour with activated charcoal and filtered. The filtrate is cooled in

  <Desc / Clms Page number 144>

 
 EMI144.1
 ,. = ;. 1. t1.t 'gln.ae and 270 ml of methanol are added at "t, v4ron an hour and a half to the aliquo- ...>. =>: ± 4 of Solange and scrape so induce - = n -, o. 16 The mixture contains crystals -: 'i: .é and is then reintroduced into the #, t the whole is cooled for about <'. Slow stirring at 0 (the desired product is recovered by filtration. The following compounds are prepared from the appropriate hydroxylated amino acid in such a manner as to prepare them.



  Salt used for
 EMI144.2
 Co - n, .. = ', ri its preparation ,, th3.ata 1 cobalt-aerine Cobalt sulphate Ohelate # follow-tyroaine aoetate cupric 0h41ate de ouivre-3,5-dibromo-tyroslne aoétate cupric Copper chelate-3p5 -diodotYrosine bromide ouprous Chelate d, C 1, -4 Tre - hydroxyproline sulfite oupreux Gold ch41ate-0-hydroxyloucine gold cyanide
 EMI144.3
 
 EMI144.4
 Meroure-p-hydroxynorvaline chelate merouric cyanide Meroure-Y-hydroxynorvaline sulphate chelate Merouric niokel-serine chelate nickel bromide Niokel-tyrosine chelate nickel iodide platinum serine platinum tetrachloride chelate. silver thrâone.¯ .... silver carbonate ZXE: 7PL'S 66.- n: 2! 2! l¯! l! ¯! ¯ !!!! A total of 21f20, & of the product as deorit at ,. l 'Oxomple 64 is suspended, dane 25Q oa 4 # 4io-.
 EMI144.5
 



  . , '' lct - .. nt distilled under a nitrogen atmosphere and, 0.1 aola fii; :: L ', ..; t:, t'!, - and r..hné9 If. pass in- 1I1! ... oua 1.1; -
 EMI144.6
 Catien;:,: k: -the mixture heats up straight to the heart.

  <Desc / Clms Page number 145>

 of the addition and at the end of the addition it was heated to about 70 ° C. and kept at this temperature for about 1 hour. The mixture is cooled and the precipitated argot chloride is separated by filtration.



  The filtrate is washed with additional benzene and the filtrate and the washings are combined and dried by freezing to obtain the desired product.
 EMI145.1
 



  ± tt'.hT3 1] ,, -!: ±! 2 ±! Z¯h! ± !! ¯! ¯a! A total of 3% of the product prepared in Example 65 is minus 100 ml of dioxane! And a proportion (.qumoled phC3gn.Q is fine to bubble through the mixture in l '). 7 1/2 minutes space.



  During the addition of the phosgene, the temperature spontaneously rose to about 40 ° C. The mixture is then stirred for one hour, filtered so as to remove the silver chloride therefrom and the filtrate is dried by freezing.



  The residue is taken up in 30 ml of ethyl acetate and is precipitated by the addition of 70 ml of hexanes The mixture
 EMI145.2
 ge is old! until about 10% overnight and the desired product is recovered by filtration.



   The following compounds are prepared analogously using the chelates formed by the procedure described in Example 65 ...
 EMI145.3
 



  Tyrosine 7orboxy.anhydrid; 3,5-diiodotyrosine N-oarboxy anhydride; N7oarxy-anhydridēd, 5-4bro.oy-rosin; N-oarboxy hydroxyprolint anhydride -aetrbo; w-oydroxynor-valine O-hydroxyleueinet N-oarboxy anhydride; Y-Hydroxynorvalin-aarboxlα-ahydride.

  <Desc / Clms Page number 146>

 
 EMI146.1
 



  25ùiPZ 68.- 11-chloromerouri histidine N-oarbo a.rah drida.- ..... w ..... ¯ ...... ¯¯ .... ¯., ¯¯.¯¯¯ ..., ........ ¯.¯ ......



     A total of 15.52 g of L-hietidine is added to an aqueous solution of 15.0 g of a mixture of potassium bicarbonate and potassium carbonate in one liter of water. To this mixture is added, with rapid stirring, 54.30 g of merouric chloride with slight heating. The temperature is allowed to rise to about 65 ° C. and stirring is continued while the reaction mixture cools to the temperature.
 EMI146.2
 ambient. 140 product which is N-obloromerouri hi8ti dine precipitates and is collected by filtration.



   A total of 29.44 g of the product thus prepared
 EMI146.3
 out 31tû yànàin% approximately 1 hour and a half in 100 ml of august zÉho do reanière to form a fine suspension. 100 ml or 6L of aoetone were added and the mixture was cooled in an ice bath, however it passed through an excess of pho8gn. during, "'1.:.."10 rJ1 ::: iodine about 40 minutes. The ice is separated
 EMI146.4
 :. 1 '.'. the 3rd day. are added to the mixture.

   The precipitate is separated by filtration and the filtrate is
 EMI146.5
 '116 nouo little pressure' raised by mqniµ3, at ¯phassor the solvent 'j8t give the desired product in the form of, rd'du4 {;; product is purified by taking it up in 100ml of nthyl-ethyl oetone from which it precipitates by cooling. The precipitate is collected, washed with a small amount of cold methyZ. Ethyl oetoxide, then with benzene.

   From the product eupple- "" = # r.irfl were obtained by the careful addition of benzene to the mother liquor of methyl ethl ketone *

  <Desc / Clms Page number 147>

 
The compounds below are prepared in analogous manner, in each case the starting amino acid is converted to a halomercuric salt by reacting 0.1 mole of the starting amino acid with 0.2 mole.
 EMI147.1
 of merouric chloride or of merouric bromide in an aqueous medium at pH 8-9 according to the procedure described above. The halomercuric salt is isolated and converted into anhydride in acetone or methyl ethyl ketone.



  The first two compounds are prepared at 0 C and the last five compounds at 10 C.
 EMI147.2
 



  1-obloromerouri N-carboxy anhydride arginine N-oarboxy anhydride of N.-oh3.oromeraur. lysine N-oarboxy anhydride of P-ahloramerour3. ornithLne N-oarboxy N-bromomerouri arginine anhydride
 EMI147.3
 N-carboxy li-bromomereuri anhydride lysine N-carboxy N-bromomercuri anhydride crinthine N-carboxy N-bromonmercuir histidine anhydride
 EMI147.4
 ls: 7CFt3p'L '69.-' t.-alanine Un, to% 1:

    d- 90 mu of alanine is dissolved Danish ml of 0.2 molar EDTA tanpon at pH 10 and 1.292 8. of T oarboxy anhydride of N-ohloromerouri histidine are added rapidly at 0 C while stirring.! at the end of minutes the pH of the mixture is reduced to 2.5 by adding concentrated sulfuric acid. Hydrogen sulfide is bubbled through the mixture in order to precipitate the mercury and the precipitate this separated by filtration. .



  The filtrate is purged of hydrogen sulfide and other gases with nitrogen and is diluted to a volume of 50 ml. The pH is adjusted to about 8 by the addition

  <Desc / Clms Page number 148>

 sodium hydroxide dilute The solution is desalted and passed through a carbon column.

   The product is isolated by chromatography by passage through a
 EMI148.1
 colo = e of sillee gel and eluted by a ls9 mixture 1. ' c -: -: c: io.que-methanol. t'f-EITPIE 70, - 5.XiSl3: 5X3: iÊX XiS3: XSï2l 2l55.L A total of 368 mg of alanyl-leuoyl-glyoyl- isolsi 1L, '03t sepria in 100 ml of water containing
 EMI148.2
 : 'j. t n gas 8 '! , 68 g of N-oarboxy anhydride
 EMI148.3
 il-o100 '. curi lysine is added at 3 o with r.pio * stirring. at the end of '0 seconds, the pH of the mixture is 4'J.t ¯. ;, uf â 2 per, addition, diacid sulfuric con- oontrÓ. Cw1 bubbles hydrogen sulfide through the mixture so as to precipitate the mercury foruo from the sulfide, which is separated by filtration. Nitrogen is bubbled through the filtrate so as to have 1? putçor of ul! ere8iel hydrogen and carbon dioxide.

   The filtrate is then diluted up to! 150 µl and the pH is adjusted to 8.5 by the addition of dilute sodium hydroxide. The solution is desalted by passing it over an oarbon column and the desired product is isolated by chromatography in a silica column.



   EXAMPLE 71. -
 EMI148.4
 ! 2l!: 6! Lt: 6! Ll!: E! L !!! l!: !!!
To a mixture of 4.0 mM of leuoine in 20 ml of at 0 C was added 3.1 moles of -car- anhydride.
 EMI148.5
 by 5x3nylalr.nine while maintaining the pH at 10.5 by careful addition of barium hydroxide powder. The reaction is completed in one minute. We adjust

  <Desc / Clms Page number 149>

 
 EMI149.1
 the pH to 3.5 by the addition of sulfuric acid 1 1.



  1 ;; .inlàt3 of barium which precipitates: 3% separated by filtration -t the filtrate containing the pnJrlal & n11-1euc1n. this za.,. ' :: ', 2 DZ of: N-arboX1i11cine-. -j "' 0"> t at pH 10.5 maintained using hyoxide / barium * The decarboxylation is carried out by; jug6ï ' The pH to 3.5 with 8ulturic acid, concentrated, and the barium sulfate which precipitated was 8pary by i: ..tratio? The pH of the filtrate was adjusted to 9.5 with ED '. buffer and 1.15 g of l-oarbo% 1 H'xam4març anhydride :. arp.:-+1'l! ne was added to 5''0 without rapid shaking. At the end of 2 minutes, the pH is yellowed to 2 by the addition of concentrated sulfuric acid.



  Hydrogen sulfide is bubbled through the mixture to precipitate the mercury as
 EMI149.2
 of sulphide which (t separated by filtrasion t The filtrate is purged with nitrogen and the pH is adjusted to 10 with
 EMI149.3
 40% hydroxide. The mixture is cooled to 0 ° 0 and 3.5 ml of K-arboxy glycine anhydride are added while stirring rapidly. After one minute the pH reduced to 3 by the addition. sulfuric acid and nitrogen bubbled through the mixture for 5 minutes. The pH is adjusted to 7 and the mixture is freeze dried.

   The residual solid is extracted three times with methanol and the methanolic extracts are adsorbed on silica gel and taken up to dryness. Silica gel then added to a co-
 EMI149.4
 lonne of silica gel prepared in isopropanolt The column is developed with a methanol mixture! to the.
 EMI149.5
 ammonia-80! l8! 2 in order to isolate the desired ezuauit.

  <Desc / Clms Page number 150>

 
 EMI150.1
 



  1: "'; 11'1115 12.- ± t2El !! ¯:! L! ¯! ¯!: 2! 2 ±! ϯ! 6 !!!! A 30 ug of Yda benyloxycarbony7, .- argi.n .ne dane 0.25 of beznene, 1 cc of thionyl chloride is added and the mixture is stirred.It is heated in a steam bath for about 30 seconds while scraping it off.
 EMI150.2
 and 0n â. ' : 3.tnt The mixture is cooled down to temperature and the benzene and tblonyl chloride are decanted $ * The residue is washed twice with benzene, a liver with ether. oil
 EMI150.3
 and: A vacuum formed to give the hydrochloride i; * ydr.d of N-carboxy arginine.



  The bronhydrate salt is prepared in a similar manner, la9cnt the thionyl chloride with one, u.t.t equivalent of thionyl bromide.



  1-oarboxy anhydride hydrochloride); liai! - dina and the hydrobromide of this compound are obtained from ma- ,, free enalongue.



   EXAMPLE 73. -
 EMI150.4
 212hlS:! ¯! L !! ¯! ¯!: 2! È2! L¯! S !!!
A mixture containing 4 g of benzyl- is prepared,
 EMI150.5
 # é; irhon; 1 ar oinine7aupension dane, about 5 cc of bcn: :): l to Q 17 oc of t3iory chloride, are added to Le m33: .Ro 0t QB; t at room temperature during z2 bouret and .exce of thionyï chloride is deoante <The residue is washed first with 50 cc of benzene and then with 50% of hexane.



   The washed residue is dried and we empty it at time.
 EMI150.6
 r :: 1; IU anbiant and 20 00 of ae thionyï lion chloride added. The mixture is heated to about 80 C.

  <Desc / Clms Page number 151>

 in the steam bath, the excess thionyl chloride is removed and the residue is washed successively with
 EMI151.1
 50ec portions of benzine and 4.lhoxanee The p'-oarboxy arginine anhydride hydrochloride which is obtained purified by dissolving it in 10 cc of diaethrl form-m1det by centrifuging the mixture, decanting and precipitating by ' the addition of 30 cc of ethyl acetate in oen - ;. i;:. t again and decanting the dorantt EXE: .fPLE 1t '"± l ±! ¯! l !! ¯¯ !:

  = !! 2! L¯Ë !!!!!! At 4 m1llimole of benzyloxyoarbon11histi41n. in 5 cc of tetrahydroturan, 8 Billiaolew of phosphorus tribromide is added and the mixture is stirred at room temperature. After about 5 minutes a solution is obtained. Stirring is continued for a further 5 minutes and the β -carboxy histidine anhydride hydrobromide precipitates and is collected by filtration.
 EMI151.2
 1-Carboxy biatidine anhydride hydrochloride is prepared analogously by replacing the phospheve tribromide with an equivalent amount of phosphorus trichloride.
 EMI151.3
 



  ! 1: I:. "RP!. 'E 75- Eë2XBE2ïBÉ ïS' '.



   This example illustrates the use of N-carboxy arginine anhydride hydrochloride to prepare the tri-
 EMI151.4
 arginl1-prQ111-phenl1a1anine peptide.



  A total of 1.68 81l1i801.8 stra.arhphénrls3s, .. nine # and dissolved in 70% of Lappish potato borate containing 60 g of ice at PU 9.5 and 4 milli-moles of hydrochloride were added. aarboxt arginine anhydride

  <Desc / Clms Page number 152>

 
 EMI152.1
 Dim4tbyl tormami4. Lanse, .. t, 3, d about 3% for 8800 nde. and the fl. aj ;; J;% 4 to 5.6 by the addition of 8ulturic acid in order
 EMI152.2
 a: ao11er the intermediate à-oarbanato. Lt w'18lg0;.; .. c..1: 'ihfio coloane de oarbone 25 ml ot la.



  ,, -1 ,. ..i = "'â É is washed by 300 00 water. Ille ost -'." <M.: ¯ ":" 1: s.v9c of 50 aqueous loetone containing elastic A. The acetone-acid mixture. aoetic ..> i :: psessioa reduced from monibro to give. which '9i3t repria in the minimum quantity of mh0n in13que of 9t1. This solution is adsorbed to? a lioness of silica gel eluted With the same sol- cc.
 EMI152.3
 The desired product is obtained by combining the fractions 5 - 9 and evaporating the solvent therefrom under reduced pressure.



   EXAMPLE 76. 0
 EMI152.4
 üstidl-hdnlalanïne.



  This example illustrates the use of β-arboxy histidine essaiaihydride brominate for the preparation of a dipeptide.
 EMI152.5
 To a total of 1 millimole of phenylalahine in 10 cc of potassium borate buffer at pH 9.5 is added
 EMI152.6
 1 milliQo1e of N-arboxy histidine anhydride hydrochloride and the mixture is stirred for about 1 minute at 0 0. The intermediate oarbase is arboxed by the addition of a sufficient amount of sulfuric acid.
 EMI152.7
 louse? adjLt0r the pù to 3, while we pass from
 EMI152.8
 " <1 = 1> through the mixture, the final product of airl,.:. L: olé by cliroratographio.

 

Claims (1)

CLAIMS.- EMI153.1 ---------------- 1.- Process for the controlled synthesis of compounds containing an amide bond, characterized in that a starting amino compound containing a group is reacted. EMI153.2 pu & 3! i: .o owning ru. auins a hydrogen atom which can be replaced by a reagent selected from the groups formed by the anhydrides of N-thiocarboxy amine acid and their derivatives. 2. - Process according to claim 1, suitable for the controlled and stepwise synthesis of peptides and their derivatives, characterized in that the starting amino compound is chosen from the groups formed by amino acids, peptides and their. derivatives. 3.- Method according to one of claims 1 and 2, characterized in that the reaction is carried out by contacting the starting amino compound and the reactants in an aqueous medium under controlled conditions so that the reaction having the speed whichever is greater is that which produces said compound containing an amide bond. 4. A process according to claim 3, characterized in that the pH is maintained at loto-1015 and in that the mixture is subjected to sufficient agitation to produce turbulence. 5. A process according to claim 4, charac- terized in that the reagent consists of the anhydrides of N-oarboxy amino acid, the anhydrides. EMI153.3 of 'wth.acarbaxy amine acid and derivatives of said oarboxy and thicarboxy anhydrides. <Desc / Clms Page number 154> 6. A process according to claim 5, characterized in that, in the case of using anhydrides of N-carboxy amino acids or their derivatives as reactants, the conditions set are such that the pH is maintained at 10.0 - 105, while the reaction mixture is stirred sufficiently to produce turbulence. 7. A process according to claim 5, characterized in that the reagent this chosen purmi N-carboxy aspartic acid anhydride, N-carboxy glutamic acid anhydride, acid anhydride. N-thiocrboxy appartic, N-thiocarboxy glubatic acid anhydride and N-thicoarboxy histidine anhydride, 8.- Process according to one or the other of the preceding claimants, characterized in that it actually has a buffer for adjusting the pH. 9. The method of claim 8d, wherein the buffer is a borate buffer. 10. A process according to either of the preceding re- ications, characterized in that the pH is adjusted to the addition of an alkaline reagent during the reaction. 11.- Process for the preparation of N-carboxy anhydride of α-hydroxylated amino acids, characterized in that the said amino acids are reacted in an aqueous medium with a reagent chosen from bases and transition metals. and in that the compounds thus obtained are reacted with phosgenes <Desc / Clms Page number 155> 12. - Process according to claim 11, oa.-, raoterized in that the amino acid is serine, threonine, hydroxyproline or tyrosine and in that the reagent is silver oxide. 13.- Process for preparing 0-tri- derivatives EMI155.1 3, lower-silyl of I-oarboxy anhydrides of α-amino hydroxy acids, characterized in that a tri-lower alkyl halosilane is reacted with an N-oarboxy anhydride of an α-amino acid hydroxylated to a low temperature in the presence of a sufficient quantity of a weak organic base to neutralize the halogen acid formed during the reaction. 14. A process according to claim 13, characterized in that the silyl derivative is an O-tri-methyl silyl derivative and in that the amino acid is erine, threonine or tyrosine. 15.- Process for the preparation of hydrate and hydrochloride of N-carboxy arginine anhydrides and EMI155.2 anhydride of 1., '. rbox; r histidine, caraotirlai in oe which is reacted' - 'halogenating agent with the corresponding amino acid in which the c-amino group is protected by an alooxy carbonyl group or carbonyl aralooxy which is eterically free and capable of donating an electron pair during the reaction. 16. A process according to claim 15, characterized in that the halogenating agent has phoephoric tribromide or thionyl chloride. 17.- Process for the preparation of a compound EMI155.3 tetrabydropyranyl of a 1-oerboxy or 1-thiooarboxy of an anhydridemino aromatic acid hldrox11l, oarao% 'rioé <Desc / Clms Page number 156> EMI156.1 and reacting the 1-carboxy or N-thiocarboX1 anhydride with dihydropyran at a temperature of between about 20 and about 40 ° C. 18. A process according to claim 17, characterized in that the amino acid is tyrosine; la3,5-di- EMI156.2 iodotyrosine or 395-dibomotyrosinet 19.- Process for the preparation of a compound selected from the group consisting of N-arboxy asparagine anhydride and N-carboxy glutamine ant 'wrinkle, characterized in that it is produced up to a molar excess of 10% of phosphorus tribromide with oorreepondant amino acid amide in which the a-amino group is protected by an alkayxcarbonyl or aralooxyoarbonyl group containing up to 8 carbon atoms in an organic solvent inert to -vis of the reaction at a temperature between about 15 0 and about 30 C for a period between about 2 and about 24 hours. 20.- A method according to claim 19, in that EMI156.3 % 4rie4 / T'lGiTC of amino acid and I-benzyloxyoarbonyl aspargine or N-benzyloxyoarbonyl glutamine. EMI156.4 21.- Process for the preparation of halomerouri fi-oarboxy anhydrides of amino acids b8iquearaotri.,. in that a halomerouric salt of a basic acid amino is reacted with phosgene at a temperature of between about 0 and 10 C. 22.- A method according to claim 21, charac- EMI156.5 terial where halomerouric salt is salt c. risk of arginine, lysine, ornithine or histidine. <Desc / Clms Page number 157> 23.- Process for the preparation of peptides containing a basic amino acid, characterized in that one reacts a halomercuri N-carboxy anhydride of a basic amiho acid with an amino acid or a peptide at a temperature between about 0 and about 10 0 for a period of from about 30 seconds to about 5 minutes in an aqueous medium at a pH of between about 8 and about 10.5 so as to form a carbamenyte intermediate and to decarboxylor the said carbamte. 24.- Process according to claim 23, characterized in that the halomerouri N-carboxy anhydride is chlormercuri N-carboxy anhydride of arginine, lysine, ornithine or histidine. 25.- A process for the production and isolation of N-carboxy aspartic acid anhydride, characterized in that aspartic acid is reacted with phosgene under substantially anhydrous conditions at a temperature. between about 15 C and about 30 C in an organic solvent inert to the reaction, adding an anhydrous transfer solvent which is miscible with the reaction solvent and boils at a temperature higher than this reaction solvent, in that the mixture thus obtained is titrated under reduced pressure until substantially all of the reaction solvent has been removed so as to produce a solution of N-carboxy aspartic acid anhydride in said transfer solvent and in that said N-arboxy aspartic acid anhydride is precipitated in said transfer solvent by the addition of a liquid / precipitant which is poor <Desc / Clms Page number 158> with the transferate solvent in which N-chroboxy aspartic anhydride is insoluble. 26, - Metal chelates of amino acids hydrogenated with transition metals. EMI158.1 27.- Chutâtes m6ta113.qaoa of amino acids ho droxyleo with transition metals of groups Ib, âlb, 11 "i VIII of the periodic system of elements 28, - Silver chelates of serine, threonine, EMI158.2 d h; rdroxypra3.ine and tyrosine. 29.- O-trie, lower yl-silyl derivative of it-aarboyy anhydride. α-amino hydroxy acids. 30.- 0-trimethylilyl-deprived of N-carboxy anhydrides of serine, threonine, tyrosine and hy- EMI158.3 droxypro3.ixs. 31.- Beta trihaloacetyl of Y-carboxy and β-thiooarboxy anhydrides of α-amino acids aliphati- g.:xos hydrosylea in which the hYdroXY1 group is alcoholic and the halogen atoms have an atomic weight inferior to 36. EMI158.4 32.- Trifluoroacetyl esters and trichloro- ".'sd.; Yl: .que3 of i-oarboxy and β-thiooarboxy anhydrides of erine and threonine. 33.- Addition salts of the hydrochloride and hydrobromide type of 1-carboxy histidine anhydride ... EMI158.5 4.-TetrabydroPyan111quel derivatives of N-oar ... boxy and N-thiooarbox1 anhydrides of #-amino hydroxyl aromatic acids. J5; - Tetrahyropranyl derivative8 of N-'oarboxy and of N-thiocarboxy anhydrides of tyrosine; from 3.5- EMI158.6 diiodotyrosine and 3,5-dibromotyrosines <Desc / Clms Page number 159> 36.- N-carboxy asparagine anhydride and N-carboxy glutamine anhydride. 37.- Halomercuri N-oarboxy anhydrides of basic amino acids. 38.- Chloromercuri N-carboxy anhydrides of arginine, histidine, lysine and ornithine. 39.- N-cargoxy aspartic acid anhydride, 30.- N-thiocarboxy amino hydroxy acids anhydrides. EMI159.1 41.- Proceeds and oompoa4J, in substance, as described above, in particular in the examples *