Peptide hydrazides

Much more important than these reactions, however, are the reactions of CDI and its analogues with carboxylic acids, leading to AAacylazoles, from which (by acyl transfer) esters, amides, peptides, hydrazides, hydroxamic acids, as well as anhydrides and various C-acylation products may be obtained. The potential of these and other reactions will be shown in the following chapters. In most of these reactions it is not necessary to isolate the intermediate AAacylazoles. Instead, in the normal procedure the appropriate nucleophile reactant (an alcohol in the ester synthesis, or an amino acid in the peptide synthesis) is added to a solution of an AAacylimidazole, formed by reaction of a carboxylic acid with CDI. Thus, CDI and its analogues offer an especially convenient vehicle for activation of... 

K Hofmann, A Lindenmann, MZ Magee, NH Khan. Studies on polypeptides III. Novel routes to a-amino acid and peptide hydrazides. (protected hydrazides). J Am... 

JK Chang, M Shimuzu, S-S Wang. Fully automated synthesis of fully protected peptide hydrazides on recycling hydroxymethyl resin. J Org Chem 41, 3255, 1976. 

S-S Wang, ID Kulesha, DP Winter, R Makofske, R Kutny, J Meienhofer. Preparation of protected peptide hydrazides from the acids and hydrazine by dicyclohexylcarbo-diimide-hydroxybenzotriazole coupling. Int J Pept Prot Res 11, 297, 1978. 

Indeed, there were those who described the azide coupling method as racemization-free. [15l However, this viewpoint proved to be overly optimistic. In 1970, Sieber reported that during a synthesis of calcitonin M by the azide method, significant epimerization occurred during two of the segment condensation steps in one of these reactions 40% of the epimerized product was observed. 16 There is a crucial detail in the experimental procedure here. The workers used tert-butyl nitrite to convert a peptide hydrazide into a peptide azide, but did not isolate the azide as was typical for research at that time. Instead, they neutralized the active intermediate in situ with DIPEA and added the amino segment for acylation. This demonstrates another important theme in the control of epimerization, the presence of a tertiary amine in the reaction mixture, even if only as a neutralization equivalent, can result in the formation of epimerized products. Indeed, most observations of racemization during... 

Reaction of an isocyanate with an amino acid or peptide hydrazide affords products with a central azaamino acid residue 3,10,19 20 (Scheme 3). 

Monofunctional polyethyleneglycol supports containing benzyloxycarbonylhydra-zide, p-benzyloxybenzyloxycarbonylhydrazide and t-butyloxycarbonylhydrazide anchoring groups, 22, 23 and 24 respectively have been developed recently for the liquid phase synthesis of protected peptide hydrazides 199). 

General procedure lor azide coupling. Protected peptide hydrazide 1 (26 mmol) in DMF (84 mL) at -2CrC was treated with 3.35N NO in dloxan (70 mmol). t-Butyl nitme (3 85 mL) (or NaNOa in water) was added at -15°C and the mixture was maintained at -10"C tor 10 mia Tbe amino component 2 (18 irwnol) in DMF (320 mL) was added dropwise at -15"C, then ethyidiisopropylamlne (70 mmol) and the mixture kept at 0 C for 24 h. During the First 6 h, 0.5 mL of the base was added ever hour Finally the product 3 was precipitated in ice-cold 1 % AcOH (1800 mL). 

It has also been used to create a third dimension in protection schemes such as for peptide hydrazide protection in the synthesis of ribonuclease A by Yajima and Fujii.P Due to its full stability to HF treatments it is used for the SPPS synthesis of minimally protected peptide segments for assembly of protein sequences in a manner similar to the 4-pyr-idylmethoxycarbonyl group (iNoc, see Section 2.1.1.1.1.2)f l which is also stable to HF, but cleaved with Zn dust in acetic acid.P ... 

N -Protected anoino acid or peptide hydrazides are prepared from the corresponding N -protected amino adds or peptides and monosubstituted hydrazine by any of the conventional coupling methods (see Section 3.1.1.4). 

Scheme 1 Different Routes to Peptide Hydrazides and Peptide Azides...

In general the synthesis of suitably protected amino acid and peptide hydrazides from the corresponding alkyl or aryl esters by reaction with hydrazine hydrate is carried out in alcohols or DMF at ambient temperature.P However, with C-terminal branched amino acids such as valine or isoleucine, or larger peptides, more vigorous conditions are required, i.e. higher temperature (25-80°C) and longer reaction times. To avoid formation of symmetrical bis(A-acyl-aminoacyl)hydrazides an excess of hydrazine hydrate or hydrazine is recommended, which also enhances the reaction rates. In this context, the solvent is known to play an important role, with alcohols being more appropriate than DMFP 0 or solvent mixtures such as dioxane/methanol.f l The use of hydrazine hydrate without any solvent has also been reported. ... 

Peptide Hydrazides from Activated Carboxylic Acids... 

Peptide hydrazides are readily obtained by hydrazinolysis of suitably protected peptides linked to the resin via a benzyl ester.Alternative to the hydroxymethyl ester linkage of the Merrifield resins, l-methyl-2-oxo-phenylethyl ester,2-nitrobenzyl ester O or 4-meth-oxybenzyl ester type linkages are also utilized.b - Recently, fuUy protected hydrazides were obtained in good yields on 2-methoxy-4-alkoxybenzyl alcohol resin (Sasrin ) by hydrazinolysis with 20% anhydrous hydrazine in DMF using DMA as solvent the reaction was significantly slower, but with less side products. 

Table 1 Protection Schemes for Synthesis of Peptides with N -Protected Peptide Hydrazides...

The first A-acylannino acid azides were synthesized according to procedures developed by Curtius.01 The reactions were carried out under acidic conditions, such as aqueous acetic acid, hydrochloric add or mixtures of both. Low temperature solutions of sodium nitrite were used as oxidizing agents.h l These older procedures have been comprehensively de-scribedh and are presently used mainly in the preparation of reagents like Boc-Nj (see Section 2.1.1). Since the sodium nitrite procedure when applied to peptide hydrazides, is cumbersome and readily accompanied by side reactions, alternative methods were developed by Honzl and Rudinger which are based on the use of alkyl nitrites. With these... 

It was shown that amide formation can be suppressed by the appropriate choice of reaction conditions, such as homogeneous solutions, e.g. in DMF, DMSO, HMPA, high acidity and low temperatures (-30 to -5°C). If the peptide hydrazide contains acid-labile protecting groups, e.g. trityl, 2-(4-biphenyl)propyloxycarbonyl, tert-butyloxycarbonyl, tert-butyl esters and ethers, the temperature has to be kept below -20 °C. Generally, tert-butyl or butyl nitrite is used as the organic nitrite, and for sterically hindered peptides amyl nitrite is employed. The time required for full conversion of the hydrazide into the azide may vary between 5 to 30 min and can be monitored by spray reagents (see experimental procedure below). 

In the maximum protection procedure, the protected peptide hydrazide is used as a carboxy component for the construction of large peptides. As described in Sections 4.1.1.1.1.4 and 4.1.2, protected peptide hydrazides are derived from the corresponding alkyl esters by treatment with hydrazine hydrate. Another method for the synthesis of protected peptide hydrazides is by conversion from substituted hydrazides. 

Synthesis of a Protected Peptide Hydrazide from the Alkyl Ester... 

As shown in Scheme 21, a protected tetrapeptide hydrazide corresponding to sequence 6-9 of bovine neurotensin was prepared. Z(OMe)-Arg(Tos)-Arg(Tos)-OMe was prepared by the DCC procedure. After treatment of this dipeptide with TFA, the resulting amine was coupled with Z(OMe)-Lys(Z)-Pro-OPcp to give Z(OMe)-Lys(Z)-Pro-Arg(Tos)-Arg-(Tos)-OMe (25). This methyl ester was treated with hydrazine hydrate to give the desired protected peptide hydrazide 26. 

In the case of the protected peptide alkyl ester containing Arg(N02), Asp(OtBu), Asp(OBzl), or Glu(OBzl), treatment with hydrazine hydrate results in side reactions, such as hydrazinolysis of side-chain functional groups. In order to avoid such side reactions, it is preferable to synthesize the protected peptide hydrazide from the corresponding substituted hydrazide. The representative substituted hydrazides for R C(0)NH-CHR -C(0)-NHNH-X are X = Z,b°3] c(0)OTrt,[i° l Boc,[i l Troc,[i l C(0)0CH2-4-pyridyl,b°d and C(0)OCMc2-CCl3.[ l... 

Protected peptides are cleaved from the resin with N-hydroxypiperidine (HOPip), followed by treatment with zinc in AcOH to afford the free acidJ Resin-bound peptides can be cleaved from the oxime resin using the tetrabutylammonium salts of side-chain protected amino acids to directly provide the a-amino-protected (Boc), side-chain-protected pep-tide.[ 3o- 32] Peptide esters can be obtained from peptidyl oxime resin by treating with amino acid estersJ Peptide hydrazides are derived from peptidyl oxime resins on treatment with anhydrous hydrazine.b Protected peptide amides can be also derived from peptidyl oxime resins on treatment with ammoniaJ l Cyclic peptide acids as carboxy components were successfully prepared by this procedure through the Pac ester. 

Photolabile a-halophenacyl resins (lo-lq) have been used to form an acid-stable anchor to the carboxyl group of amino acids prior to peptide elongation [97]. Cleavage of the final products was achieved by irradiation with light of wavelength 350 nm. Related linkers that differ in the mode of attachment to the resin have been reported [98-100]. Carboxylic acids can also be released by treatment of these supports with base [101] or cyanide ion [102] and peptide hydrazides have been released by treatment with hydrazine [97,103]. 

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