Letters in Peptide Science, 9: 153–165, 2002. KLUWER/ESCOM © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
153
Review Article
Heterogeneous catalytic transfer hydrogenation in peptide synthesis D. Channe Gowda∗ & K. Abiraj Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, India (∗ Author for correspondence; E-mail:
[email protected], Fax: 091 0821 421263/518835) Received 30 October 2002; accepted in revised form 25 December 2002
Key words: catalyst, deprotection, hydrogen donor, peptide synthesis, protecting groups, solid phase peptide synthesis Abbreviations: Boc, tert-butyloxycarbonyl; Bom, benzyloxymethyl; BrZ, bromobenzyloxycarbonyl; Bzl, benzyl; Cbz, carbobenzoxy; 2-ClZ, 2-chlorobenzyloxycarbonyl; 2,6-Cl2 Bzl, 2,6-dichlorobenzyl; CTH, Catalytic Transfer Hydrogenation; Fmoc, 9-fluorenylmethyloxycarbonyl; OBzl, benzyl ester; OBut , tert-butyl ester; OcHx, cyclohexyl ester; OEt, ethyl ester; OMe, methyl ester; PMZ, p-methoxybenzyloxycarbonyl; Z, benzyloxycarbonyl. Introduction The early pioneering work by Braude on CTH for the reduction of organic compounds was ignored because of poor yields and long reaction times [1]. Later an immense work has been done in the field of CTH with increased yield and rapidity using different catalysts and hydrogen donors making this method as a vital tool for the reduction of various functionalities [2–11] such as alkenes, alkynes, arenes, nitroalkenes, nitroarenes, azo compounds, carbonyls, nitriles, azides, oximes, etc. Chemoselectivity, which is often a necessary criterion for organic synthesis can be achieved by varying the reaction conditions, catalysts and hydrogen donor during the reduction of above functionalities using CTH. Rapid and selective removal of protecting groups under moderate reaction conditions is often a necessary step in the area of peptide chemistry. A number of reagents have been developed for this purpose. Of all the methods available for the cleavage of hydrogenolysable protecting groups in peptide synthesis, heterogeneous catalytic transfer hydrogenation has been relatively underutilized. The reason for this lack of popularity of transfer hydrogenation has been the very successful exploitation of catalytic hydrogenation method, which is potentially less damaging to peptide by preventing strong acidic and alkaline conditions for which the peptides are very sensitive. Catalytic hydrogenation using molecular hydrogen, a gas of low
molecular weight and therefore diffusibility, is easily ignited and presents considerable hazards, particularly on large scale. In comparison with catalytic hydrogenation or with other methods of deblocking, catalytic transfer hydrogenation (CTH) has real and potential advantages both in solution and solid phase such as; (i) low cost, (ii) rapidity, (iii) mild conditions, usually avoiding strong acid or base, (iv) selectivity, (v) simple operation and work up and (vi) broad applicability. The use of CTH in peptide synthesis was first accomplished by Jackson and Johnstone [12]. Earlier to this, a few scattered references to the use of catalytic transfer hydrogenation of benzyl groups have appeared [13] as, for example the conversion of benzyl chloride to toluene [1]. However the reaction has not been investigated in a general sense and there appear to be no reports of its use in peptide chemistry, where benzyloxycarbonyl is a common protecting group removable by strong acid [14] or catalytic hydrogenation [15]. In these studies 10% Pd-C catalyst and cyclohexene as hydrogen donor, a catalyst-substrate ratio of 1:1 by weight was used but later work with a catalystsubstrate ratio of 1:5 showed only a slight diminution in the rate of deprotection (Scheme 1). For Z-protected amino acids and peptides, complete deprotection was achieved in 15 min, at 65 ◦ C, the reaction being monitored by one or more of TLC, GLC and 1 H-NMR. The deprotection of 4-methoxybenzyloxycarbonyl group as monitored by GLC was completed in less than 5 min. Hydrogenolysis of Z-Leu-Ala-OBzl removed
154 Table 1. Heterogeneous catalytic transfer hydrogenolysis of protected amino acids and peptides Entry
1 2
Protected amino acid or peptide
Catalystf
Donorg
Reaction time (hr)
Temp (◦ C)
Product
Yield (%)
Ref
Z-Ser(OBut )-OMe Z-Phe-OH
A A A A A A A A A A A A A A A A A A A A A A B A A B B B B B A B B
X X Z X X X X X X X X X X X X X X X X X X X X Z X X X Z X Y Z X X
0.25 0.25 0.75 0.25 0.25 0.25 0.25 0.08 0.03 0.25 0.07 0.25 0.25 0.25 0.25 0.25 0.25 2.0 1.5 1.5 2.0 1.5 0.75 0.75 24.0 6.0 3.0 0.75 3.0 1.0 0.75 1.0 6.0
65 65 25 65 65 65 65 65 65 65 65 65 65 65 65 65 65 70 70 70 70 70 70 25 70 70 70 25 70 50 25 70 70
H-Ser(OBut )-OMe H-Phe-OH
90 90 99 90 90 90 90 100 >90 100 100 100 100 100 0 0 0 80 90 84 93 95 95 88 77/88 91.5 97 100 93 96 100 90 83
12 12 20 12 12 12 12 12 12 12 12 12 12 12 12 12 12 16 16 16 16 16 16 20 16 16 16 20 16 19 20 16 16
B B B B B B B B B A A B A A A B A B
Y Y Y Y Y Y Y Y Y Z Z Z Z Z Z Z Z Z
1.5 8.0 1.0 0.5 0.5 0.5 1.5 4.0 1.5 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 20
50 50 50 50 50 50 50 50 50 25 25 25 25 25 25 25 25 25
B
Z
20
25
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Z-Phe-NH2 Z-Pro-OH Z-Tyr-OMe Z-Val-OMe PMZ-Val-OMe PMZ-Leu-OMe Z-Leu-OMe Z-Leu-Ala-OBzl Z-Val-Phe-OMe Z-Phe-Leu-Gly-OMe Z-Pro-Phe-Leu-Gly-OMea Z-Gly-Leu2 -Gly2 -OEtb Z-Met-OMe Z-Ala-Met-OMe Z-Gly-Gly-OH Z-Ser-Gly-OBzl Z-Pro-Val-Gly-OEt Z-Ala-Asp(OBzl)-Ser-Gly-OHc Boc-Lys(Z)-OH
22
Boc-Phe-Arg(NO2 )-Trp-Gly-OHc
23
Boc-His(N im -Bzl)-OH
24
Boc-Tyr(Bzl)-OH
25 26
Z-β -Ala-Tyr-Ser-Met-OMe H-Asn-Glu(OBzl)-Glu(OBzl)-GlyLeu-Phe-Gly-Gly-Arg(NO2 )-OBzlc Z-Leu-OBut Boc-Ser(Bzl)-OH Boc-Arg(NO2 )-Leu-OBut Z-Arg(NO2 )-Gly-NH2 Z-Phe-Leu-OBut Z-Ser-Phe-Leu-OBut Z-Trp-Leu-OBut Z-Gln-Trp-Leu-OBut Z-Val-Gln-Trp-Leu-OBut Z-Ala-OH Z-Ser-OBzl Z-Met-OH Z-Gly-Pro-OH Boc-Phe-Gln-OBzl Z-Lys(Boc)-Thr(But )-OMe Z-Arg(NO2 )-Pro-Pro-OButd Z-Lys(Boc)-Asn-Phe-Phe-OMee Boc-Ile-Ile-Lys(Z)-Asn-Ala-Tyr(Bzl)Lys(Z)-Lys(Z)-Gly-Glu(OBzl)-OBzld Boc-Tyr (Bzl)-Lys(Z)-Lys(Z)-GlyGlu(OBzl)-OBzle
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
a Catalyst plus dry HCl. b Catalyst in 10% AcOH. c Acetic acid added until dissolution. d Reaction carried out in glacial acetic acid. e Reaction carried out in dimethylacetamide. f A = 10% Pd/C; B = Pd black. g X = Cyclohexene; Y = Hydrazine; Z = 1,4-Cyclohexadiene.
H-Phe-NH2 H-Pro-OH H-Tyr-OMe H-Val-OMe H-Val-OMe H-Leu-OMe H-Leu-OMe H-Leu-Ala-OH H-Val-Phe-OMe H-Phe-Leu-Gly-OMe H-Pro-Phe-Leu-Gly-OMe Starting material Starting material Toluene in small quantity H-Gly-Gly-OH H-Ser-Gly-OH H-Pro-Val-Gly-OEt H-Ala-Asp-Ser-Gly-OH Boc-Lys-OH
Boc-Phe-Arg-Trp-Gly-OH Boc-His-OH Boc-Tyr-OH
β -Ala-Tyr-Ser-Met-OMe H-Asn-Glu-Glu-Gly-Leu-PheGly-Gly-Arg-OH H-Leu-OBut Boc-Ser-OH Boc-Arg-Leu-OBut H-Arg-Gly-NH2 H-Phe-Leu-OBut H-Ser-Phe-Leu-OBut H-Trp-Leu-OBut H-Gln-Trp-Leu-OBut H-Val-Gln-Trp-Leu-OBut H-Ala-OH H-Ser-OH H-Met-OH H-Gly-Pro-OH Boc-Phe-Gln-OH H-Lys(Boc)-Thr(But )-OMe H-Arg-Pro-Pro-OBut H-Lys(Boc)-Asn-Phe-Phe-OMe Boc-Ile-Ile-Lys-Asn-Ala-TyrLys-Lys-Gly-Glu-OH Boc-Tyr-Lys-Lys-Gly-Glu-OH
90 60 90 95 95 95 95 90 95 95 99 83 99 84 99 99 85 79
19 19 19 19 19 19 19 19 19 20 20 20 20 20 20 20 20 20
95
20
155
Scheme 1.
Scheme 2.
both the N−benzyloxycarbonyl and the C-terminal benzyl group in 4 min. No evidence of racemization was observed. The amino acids and peptides deprotected by this method are shown in Table 1 (entries 1−16). In a later study, the effectiveness of CTH using Pd/C with cyclohexene for removal of several commonly used protecting groups was confirmed [16]. Thus, protecting groups like N−benzyloxycarbonyl, C-terminal benzyl ester, nitro of nitroarginine, N im benzyl of N im -benzylhistidine, and benzyl ether of O−benzyltyrosine or serine are easily removed by refluxing (70 ◦ C) an ethanolic solution of the protected peptide with cyclohexene in the presence of palladium-charcoal or palladium black. The use of freshly prepared palladium black facilitated the removal of the benzyl group from protected histidyl compounds and of a nitro group from arginyl residues. The time required for this deprotection is much less than that for the usual catalytic hydrogenation and the product is of greater purity. Dissolution difficulties were partially overcome by using acetic acid as the solvent. The relevant data concerning these investigations are listed in Table 1 (entries 17−26). Further, the significance of this system was established by deblocking biologically active peptides. Bradykinin, Boc-Arg(NO2)-Pro-Pro-Gly-PheSer(Bzl)-Pro-Phe-Arg(NO2)-OMe was deblocked to Boc-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OMe by
using cyclohexene as hydrogen donor and palladium as heterogeneous catalyst [17]. The efficacy of hydrazine, as a hydrogen donor for the reduction of aromatic nitro compounds [18] in presence of palladium catalyst prompted Anwer et al. [19] to use hydrazine, instead of cyclohexene, as a hydrogen donor in the catalytic transfer hydrogenation of protected peptides (Scheme 2; Table 1: entries 27−35). The obvious advantage of this system is that the hydrogenolysis can be effected at lower temperature (50 ◦ C). Thus, when cyclohexene and hydrazine are employed as hydrogen donors, temperatures of 70 and 50 ◦ C, respectively, have to be maintained during hydrogenolysis, and this requirement is a disadvantage if the products are heat-sensitive peptides. Several other donors have been investigated to reduce the reaction temperature. 1,4-Cyclohexadiene, which is more easily dehydrogenated than cyclohexene, can be used to carry out catalytic transfer hydrogenation at room temperature in the presence of 10% palladium on charcoal [20]. Under these conditions, removal of benzyloxycarbonyl, benzyl ester and tyrosine benzyl ether protecting groups was completed in two hours and good yields of analytically pure amino acids and peptides were obtained directly. They have also been investigated that more efficient palladium black catalyst was required for cleavage of the N im - benzyl group of histidine, N g -nitro of nitroarginine and benzyl ether
156 Table 2. Catalytic transfer hydrogenolysis of protected amino acids and peptides using formic acid as hydrogen donor at room temperature Protected amino acid or peptide
Catalystc
Reaction time (min)
Product
Yield (%)
Ref
1
Z-Phe-Met-NH2 Boc-Trp-Ser(Bzl)-Tyr-OMea Boc-Ala-Tyr(Bzl)-Gly-Leu-OEta
4
Boc-Leu-Phe-Gly-Gly-Arg(NO2 )-OBzla
5
Z-Lys-OH
6
Z-Gly-Gly-OH
7 8
Z-Phe-Phe-OEt Z-Gly-Arg(NO2 )-OH
60 10 180 180 120 600 180 10 3 10 5 10 300 120 600 10 5 10 – 3 3 3 3 3 5 3 360 10 8 8 5 120 10 120
H-Phe-Met-NH2 ·HCOOH
2 3
B A B B A B A B A B A B B A B B A B B A A A A A A A A A A A A A A A
87.5 82 81 80 85 76 83 80 92 90 90 97 86 84 81 98 95 92 – 95 92 95 95 89 88 92 92 90 85 86 90 84 80 88
21 33 21 21 33 21 33 22 33 22 33 22 22 33 22 22 33 22 22 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33
Entry
9 10
Z-Lys(N −Bzl)-OH Boc-Asp(OBzl)-OH
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Z-Met-Gly-OEt Z-Cys(S-Bzl)-Phe-OEt Z-Gly-OH Z-Pro-OH Z-Phe-OH Z-Ala-OH Z-Met-OH Z-Leu-OBut Boc-Lys(Z)-OHa Boc-Lys(2-ClZ)-OHa Boc-Glu(OBzl)-OH Boc-Tyr(Bzl)-OH Boc-Ser(Bzl)-OH Boc-Thr(Bzl)-OH Boc-His(Bom)-OH Z-Phe-Leu-OBut Boc-Ala-Ser(Bzl)-Tyr-OMea
H-Trp-Ser-Tyr-OMe·HCOOH H-Ala-Tyr-Gly-Leu-OEt·HCOOH H-Leu-Phe-Gly-Gly-Arg-OH·2HCOOH H-Lys-OH·2HCOOH H-Gly-Gly-OH·HCOOH H-Phe-Phe-OEt·HCOOH H-Gly-Arg-OH·2HCOOH H-Lys-OH·HCOOH Boc-Asp-OHb H-Met-Gly-OEt·HCOOH Incomplete reaction H-Gly-OH·HCOOH H-Pro-OH·HCOOH H-Phe-OH·HCOOH H-Ala-OH·HCOOH H-Met-OH·HCOOH H-Leu-OBut ·HCOOH H-Lys-OH·2HCOOH H-Lys-OH·2HCOOH Boc-Glu-OHb Boc-Tyr-OHb Boc-Ser-OHb Boc-Thr-OHb Boc-His-OH·HCOOH H-Phe-Leu-OBut ·HCOOH H-Ala-Ser-Tyr-OMe·HCOOH
a 98−100% Formic acid is used. b Isolated as dicyclohexylammonium salt. c A = 10% Pd/C; B = Pd black.
groups from serine and threonine at room temperature (Table 1: entries 2, 21, 23, 24, 36−45). The high cost of 1,4-cyclohexadiene restricted its general use as hydrogen donor even though it is more efficient for deprotection at room temperature. CTH emerged as a potent tool for hydrogenolysis of protected peptides with the introduction of formic acid, as a hydrogen donor, which makes the deprotection more inexpensive and rapid at room temperature [21–23]. The procedure is simple, more rapid and the products are purer (Table 2: entries 1−12). In a
typical experiment the protected peptide is taken up in 85% formic acid and stirred with freshly prepared palladium black at room temperature. After completion of hydrogenolysis, the catalyst was filtered off and the formic acid is removed in vacuum. Further, the reaction rate is greatly enhanced by the addition of a small quantity of sodium formate, but its presence renders the purification of the product more complicated. It is well known that formic acid removes Boc- group from protected peptide, but 85% formic acid does not remove it completely. For the
157
Scheme 3.
complete removal of Boc- group 98−100% formic acid has been used [21]. The successful exploitation of formic acid catalytic transfer hydrogenation for the synthesis of substance P, H-Arg-Pro-Lys-Pro-GlnGln-Phe-phe-Gly-Leu-Met-NH2 [24] and analogues of LHRH [25, 26] is documented. Mery et al. [26] found that, this deprotection method induces reduction of the tryptophan residue and epimerization at the histidine level if the reaction carried out for long time. A further refinement of the CTH system using ammonium formate, as the hydrogen donor and palladium on charcoal as the heterogeneous catalyst was reported by Anwer and Spatola [27]. Ammonium formate has been successfully used at several stages in the synthesis of leucine-enkephalin for the reductive cleavage of benzyloxycarbonyl group (Scheme 3). The protecting groups removed included the benzyloxycarbonyl, benzyl ethers, nitro and benzyl ester, in view of the neutral condition employed. Such common but acid-labile amine protecting groups as the Boc group were unaffected by this treatment. The use of 10% palladium on charcoal/ammonium formate makes this a rapid, low cost alternative to palladium black and reduces the work-up to a simple filtration and (optional) extraction/ lyophilization operation. Most of the reactions are complete in less than 1 min as monitored by the disappearance of starting material and concomitant formation of products via reverse phase high-pressure liquid chromatography methods. The arginine N g nitro group was cleanly removed in less than 5 min, while benzyl ethers (serine and threonine) were deprotected in 30 and 90 min, respectively, although in the later case an acidic medium was required for the reactions to proceed to completion (Table 3: entries 1−14). The use of deuterated ammonium formate as the transfer agent in the CTH process when performed in a deuterated solvent medium results in the predominant formation of the labeled product. It was demonstrated that deuterated ammonium formateCTH is a convenient and rapid method for D labeling
of p−chloro-phenylalanine containing peptides [28]. Thus, DCO2 ND4 -CTH of [D-Ala2 , p-ClPhe4 ]-Leuenkephalin in 80% CD3 COOD/D2 O yielded [D-Ala2, Phe (4D)4 ]-Leu-enkephalin (Scheme 4). The versatility of ammonium formate as hydrogen donor for catalytic hydrogen transfer reductions in organic synthesis is reviewed [29]. A further elaboration in the use of ammonium formate as a donor and palladium on carbon for deblocking of protected peptides has been reported [30]. The removal of 2-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyl, 2-bromobenzyloxycarbonyl and phenacyl ester groups from amino acids, peptides and polymers has been demonstrated (Table 3: entries 15−18). The relevance of this system was confirmed by deblocking biologically active peptides. Leucineenkephalin, Boc-Tyr(2,6-Cl2Bzl)-Gly-Gly-Phe-LeuOBut was deblocked to Boc-Tyr-Gly-Gly-Phe-LeuOBut within 15 min. To further address the issue of its applicability to large and more complex peptides and proteins, high molecular weight elastic protein-based polymers were deblocked. This method of deblocking gave a yield of about 87% of poly[(GVGVP)9,(GYGVP)] as against 52% of yield obtained when HF was used for deblocking [31]. The yields of poly(GKGIP) and poly[(GVGIP)4, (GKGIP)] are 89 and 87% as compared to 67 and 68%, respectively [32]. Although ammonium formate is the most effective donor for deblocking protected peptides, it poses complications during purification of the water-soluble products. The utility of formic acid, an excellent solvent for most peptides and peptide derivatives has been reported [33] for the cleavage of N α benzyloxycarbonyl, N ε -2-chlorobenzyloxycarbonyl, C-terminal benzyl ester, O-benzyl ether of O-benzyltyrosine and -serine or -threonine, nitro of nitroarginine and N im -benzyloxymethyl of histidine using 10% palldium on carbon (Table 2: entries 1, 3−6, 8, 10, 13−27) instead of high cost Pd black [21].
158 Table 3. Catalytic transfer hydrogenolysis of protected amino acids and peptides using ammonium formate as hydrogen donor at room temperature Protected amino acid or peptide
Catalyste
Reaction time (hr)
Product
Yield (%)
Ref
1
Boc-Asp(β-OBzl)-OH Boc-Glu(γ -OBzl)-OH
3
Boc-Tyr(Bzl)-OH
4
Boc-Ser(Bzl)-OHb
5
Boc-Thr(Bzl)-OHb
6
Boc-Lys(Z)-OH
7
Z-Phe-OH
8
Z-Trp-OH
0.05 2.0 0.05 2.0 0.16 2.0 0.5 3.0 1.5 3.0 0.08 1.5 0.08 1.5 0.08 1.5 0.08 0.08 2.0 0.08 0.08 0.08 0.08 0.16 0.25 0.16 0.16 1.5 3.0 3.0 2.0 3.0 1.0 2.0 2.5 24 24
Boc-Asp-OHa
2
A C A C A C A C A C A C A C A C A A C A A A A A A A A C C C C C C C C C C
98 95 81 95 85 89 70 88 90 90 95 95 95 92 95 92 98 89 89 98 98 98 92 95 90 92 95 92 89 94 88 89 93 90 89 – –
27 37 27 37 27 37 27 37 27 37 27 37 27 37 27 37 27 27 37 27 27 27 27 30 30 30 30 37 37 37 37 37 37 37 37 37 37
Entry
a b c d
9 10
Z-Phenylalaninol Boc-Arg(NO2 )-Leu-OBut
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Z-Trp-Leu-OBut Z-Leu- OBut Z-Phe-Leu-OBut Z-Gly-Gly-Phe-Leu-OBut Boc-Lys(2-ClZ)-OH Boc-Tyr(2,6-Cl2 Bzl)-OH Boc-Tyr(2-BrZ)-OH Boc-Pro-OPa Z-Gly-OH Boc-Tyr(2-BrZ)-OH Boc-Lys(2-ClZ)-OH Boc-His(Z)-OH Boc-His(Bom)-OH Boc-Gly-Val-Gly-Val-Pro-OBzl Boc-Arg(NO2 )-Pro-Arg(NO2 )-Pro-OBzl Boc-D-Lys(2-ClZ)-Pro-Arg(NO2 )Pro-OBzl Fmoc-Gly-OH Fmoc-Phe-OH
Boc-Glu-OHa Boc-Tyr-OH Boc-Ser-OHa Boc-Thr-OHa Boc-Lys-OH H-Phe-OH H-Trp-OH L-Phenylalaninol H-Arg-Leu-OH·2CF3 COOHc H-Trp-Leu-OBut H-Leu- OBut H-Phe-Leu- OBut H-Gly-Gly-Phe-Leu- OBut Boc-Lys-OH Boc-Tyr-OH Boc-Tyr-OH Boc-Pro-OH H-Gly-OH Boc-Tyr-OH Boc-Lys-OH Boc-His-OH Boc-His-OH Boc-Gly-Val-Gly-Val-Pro-OH Boc-Arg-Pro-Arg-Pro-OH Boc-D-Lys-Pro-Arg-Pro-OH Fmoc-Gly-OHd Fmoc-Phe-OHd
Isolated as dicyclohexylammonium salt. The reaction medium was a 1:1 mixture of CH3 OH/AcOH. CF3 COOH was used. Starting material was recovered almost quantitatively. e A = 10% Pd/C; C = Magnesium.
Exploration of hydrazinium monoformate, as a hydrogen donor for catalytic hydrogen transfer reductions of nitro [34] and nitrile [9] functionalities initiates to find its application as a donor for the removal of some commonly used protecting groups in peptide synthesis using palladium on carbon as cata-
lyst [35]. Hydrazinium monoformate, a more effective donor than hydrazine and formic acid was prepared by neutralizing slowly, equal moles of hydrazine hydrate and 85% formic acid in an ice water bath, with constant stirring. The Boc and base labile protecting
159
Scheme 4.
groups were completely stable under these conditions (Table 4: entries 1−21). The CTH in peptide synthesis is mainly centered on the use of expensive palladium on carbon and palladium black as catalysts. More recently, magnesium powder catalyzed transfer hydrogenation for the removal of some commonly used protecting groups in peptide synthesis has been reported [36, 37]. Two hydrogen donors such as hydrazinium monoformate [36] (Table 4: entries 1−21) and ammonium formate [37] (Table 3: entries 1−8, 10, 19−28) were successfully used for deblocking peptide-protecting groups using magnesium powder as catalyst (Scheme 5). These two systems are equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis. The protecting groups that have been successfully removed include the N α benzyloxycarbonyl, N ε -benzyloxycarbonyl, and N ε 2-chlorobenzyloxycarbonyl of lysine, a C-terminal benzyl ester, the O-benzyl ether of O-benzyl-tyrosine, serine or threonine, nitro group of nitro-arginine, 2,6dichlorobenzyl and bromobenzyloxycarbonyl ether of tyrosine, benzyloxymethyl and benzyloxycarbonyl of
histidine. Under these experimental conditions the Boc and Fmoc groups appears to be quite stable. Recently, microwave-assisted CTH for the rapid deprotection of N−Cbz and N−Bzl derivatives has been reported [38]. Thus, amino groups, protected as Cbz or Bzl, can be deprotected in few minutes by microwave-assisted-transfer hydrogenation with Pd/C in i−PrOH as the solvent and HCOONH4 as the hydrogen donor (Scheme 6; Table 5: entries 1−9). This method is particularly suitable for the synthesis of short peptides and can also be carried out on supported molecules.
CTH in Solid Phase Peptide Synthesis: The introduction of solid phase method by Merrifield [39] in 1963 heralded a new era in the area of peptide synthesis. The speed and ease with which this new technique could be employed, together with its adaptability to automation has resulted in the synthesis of a large variety of biologically active peptides and their analogues.
160 Table 4. Catalytic transfer hydrogenolysis of protected amino acids and peptides using hydrazinium monoformate as hydrogen donor at room temperature Entry
Protected amino acid or peptide
Product
Reaction timea (hr)
Reference [35] Catalystd Yield (%)
Reference [36] Catalystd Yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Z-Gly-OH Z-Phe-OH Z-Trp-OH Boc-Thr(OBzl)-OH Boc-Ser(OBzl)-OH Boc-Asp(OBzl)-OH Boc-Asp(OcHx)-OH Boc-Glu(OBzl)-OH Boc- Glu(OcHx)-OH Boc-Tyr(OBzl)-OH Boc-Tyr (2-BrZ)-OH Boc-Lys(Z)-OH Boc-Lys(2-ClZ)-OH Boc-His(Z)-OH Boc-His(Bom)-OH Fmoc-Gly-OH Fmoc-Phe-OH Z-Phe-Leu-OBut Boc-Gly-Val-Gly-Val-Pro-OBzl Boc-Arg(NO2 )-Pro-Arg(NO2 )Pro-OBzl Boc-D-Lys(2-ClZ)-Pro-Arg(NO2 ) Pro-OBzl
H-Gly-OH H-Phe-OH H-Trp-OH Boc-Thr-OHb Boc-Ser-OHb Boc-Asp-OHb Boc-Asp-OHb Boc-Glu-OHb Boc-Glu-OHb Boc-Tyr-OH Boc-Tyr-OH Boc-Lys-OH Boc-Lys-OH Boc-His-OH Boc-His-OH Fmoc-Gly-OHc Fmoc-Phe-OHc Phe-Leu- OBut Boc-Gly-Val-Gly-Val-Pro-OH Boc-Arg-Pro-Arg-Pro-OH
0.5 0.5 0.5 1.0 2.0 1.0 2.0 1.0 2.0 0.5 2.0 0.5 1.5 1.0 2.0 24 24 0.75 0.5 2.0
A A A A A A A A A A A A A A A A A A A A
90 93 93 91 90 96 90 89 91 88 91 92 92 91 92 – – 88 88 92
C C C C C C C C C C C C C C C C C C C C
92 92 92 90 88 95 93 95 94 89 89 95 94 88 89 – – 90 93 90
Boc-Lys-Pro-Arg-Pro-OH
2.5
A
91
C
89
21 a b c d
Time required by both the catalysts to complete the reaction is almost same. Isolated as dicyclohexylammonium salt. Starting material was recovered almost quantitatively. A = 10% Pd/C; C = Magnesium.
Scheme 5.
Scheme 6.
161 Table 5. Microwave-assisted deprotection of different N −Cbz and N −Bzl derivatives [38]
a Yields determined on the product, recovered after filtration and evaporation of the solvent. b These products were deprotected using a microwave organic synthesis apparatus doing a single cycle of 20 min with 30 W of power. c Yields of product isolated by column chromatography.
162
Scheme 7.
Scheme 8.
The basic principle of the Merrifield procedure involves building of the peptide chain on a solid support that is insoluble in the solvents that are commonly used for peptide synthesis. Thus, the growing peptide chain will be attached to the insoluble support; consequently the excess reagents, byproducts and the side products that are insoluble can be removed by filtration. The solid support of choice is chloro-methylated co-polystyrene-2% divinylbenzene. The first amino acid is attached to this resin via a benzyl ester linkage by reaction of the carboxyl group of the N-acylamino acid with the chloromethyl group on the resin. The Nacyl group employed for the protection of the α-amino group of the amino acid, to be useful in solid phase peptide synthesis, must permit its quantitative removal during deprotection. Further, complete stability of the polymer-peptide bond as well as the side-chain protecting groups is essential under the conditions of deprotection. This has generally been achieved by us-
ing a mild acid-labile protecting group such as Boc for the temporary α-amino protection and benzyl-type protection for the semipermanent side-chain protection. The peptide-resin linkage is also of the benzyl ester type. Thus, Boc-benzyl combination has been the basis for the solid phase synthesis of a wide range of peptides. Success in peptide synthesis depends not so much on the methods of activation and coupling as on the availability of an array of selectively removable protecting groups, particularly side chain protecting groups and cleavage of peptide from resin. A nearly universal problem in heterogeneous support systems for solid phase peptide synthesis [40] involves the final step of the synthesis in which the desired product is detached from the resin. While acid-based treatments are most commonly employed (HF [41], HBr-TFA [42], CF3 SO3 H-TFA [43] and B{O2 CCF3 }3 [44]), these procedures are often deleterious when acid-sensitive
163
Scheme 9.
functionalities are present. The native lysozyme, when exposed to conditions for anhydrous HF cleavage, lost 87% of its biological activity [45]. The reasons for this are yet unclear but are at least partly related to N→O migrations with serine moieties and other carbocation – mediated side-reaction products [46−48]. Numerous alternatives to the most commonly employed Merrifield techniques have been proposed including changes in the type of support used (from polystyrene based to polyamides [49, 50] or polyhalocarbons [51]), liquid phase methods [52], liquid-solid phase systems [53], multidetachable links [54], photolabile links [55], and various types of transesterification reactions for removal of the peptides under more gentle conditions [56, 57]. In order to overcome these difficulties, it has long been felt that a new strategy in the selection of protecting groups and peptide-resin linkages, which would avoid repeated acidolytic procedures, is necessary. This can best be achieved by employing a base-labile protecting group for N α -amino protection while using mild acid-labile protecting groups (tBu base groups) for side-chain protection only so that acid treatment can be limited to a single final step. Among the several known base-labile protecting groups, the Fmoc [58, 59] appears most attractive because of its favorable properties that permit its use in solid phase synthesis. Even that single mild acid treatment can be avoided by the use of Fmoc group for the temporary α-amino protection, benzyl type protection for the semipermanent side-chain protection and benzyl-ester type peptide resin linkages. Thus, the final cleavage of peptide from resin as well as deblocking of side chain protecting groups can be achieved by catalytic transfer hydrogenation. The earliest report of hydrogenolysis of peptide resin benzyl esters using conventional hydrogenation techniques was by Schlatter et al. [60]. Thus Boc-protected (Val5 )-enkephalin was obtained in 71%
yield by treating the peptide resin with hydrogen gas at 40 ◦ C and 60 psi for 24 hr in the presence of palladium black generated in situ as catalyst. This technique was further applied to the synthesis of biologically active compound, Leu-enkephalin [61]. The CTH procedure has been applied for the simultaneous deprotection and release of nonapeptide from polystyrene resin [62]. N α -Boc-N G ,N G’ -dinitro-Obenzyl-bradykinin resin was subjected to hydrogenolysis at atmospheric pressure, 70 ◦ C (bath temperature) for 4 hr in presence of palladium black generated in situ from palladium (II) acetate to yield Boc-brandykinin in 60% yield (Scheme 7). Utility of 1,4-cyclohexadiene, as hydrogen donor to cleave peptide from polystyrene resin by catalytic transfer hydrogenolysis using palladium black, generated in situ, as catalyst was demonstrated [63]. For the solid-phase synthesis of thymosin α1 , an acetyl octacosapeptide, in which for the first time no treatment with acid was employed [64]. Final cleavage of the peptide from a substituted benzhydrylamine resin was carried out by catalytic transfer hydrogenolysis using 1,4-cyclohexadiene as donor and palladium black, generated in situ, as catalyst (Scheme 8). Exploitation of ammonium formate, as hydrogen donor for the cleavage, and concomitant deprotection of the peptide from polymer resin support makes this catalytic transfer hydrogenolysis process extremely rapid even under ambient conditions of temperature and pressure in a neutral medium [65, 66]. This method was used for the quantitative cleavage of Leuenkephalin from a 1% divinyl benzene cross-linked polystyrene resin (Scheme 9). Identical procedure also worn for the cleavage of the pentadecapeptide fragment, an ACTH analogue, from Merrifield resin [66].
164 Conclusion The heterogeneous catalytic transfer hydrogenation is very versatile, selective, cost-effective, mild and rapid method for deprotection of protected peptides and also for cleavage of peptides from resin support. Although, it has been suggested that the conventional hydrogenation technique is apparently not suitable for longer peptides [40], recent advances in CTH methods predicted their potential use in such applications. CTH has potential in combinatorial chemistry for the selective deblocking of benzyl type protecting groups. In addition, CTH is an efficient technique for the deprotection and cleavage of even reasonably lengthy peptide sequences in short reaction times under exceptionally mild conditions in spite of the dual heterogeneity of catalyst and polymer support. The flexibility and selectivity offered by this technique should be compatible with the synthesis by fragment condensation of large peptides and possibly, small protein sequences, using suitably protected fragments.
Acknowledgement The authors wish to thank University Grants Commission, New Delhi, India for financial support.
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