Indi an Journal of Chemistry Vol. 4 1 B, May 2002, pp. 1028-1031

Coupling in the absence of a tertiary base: A method for the deprotonation of hydrochloride salts of peptide esters to free amino peptide esters t

Subramanyam J Tantry, K Ananda & V V Sliresh Babll*

Departme nt of Stud ies in Chemistry, Cen tral College Campus, Bangalorc University, Bangalore 560 00 I. India

Received 24 April 200 I ; accepred (revised) 25 Ocrober 200 I

The deprotonation of hydrochl oride salts of peptide esters is accompli shcd using ac ti vated zinc dust. The reaction is neat and quantitative. Addition of a tertiary base is eliminated during the coupling. The free amino peptide esters have been

isolated in good yield and purity . Thi s method is extended for the synthesis of l3-casomorphin (Tyr-Pro-Phe-Pro-Gly). During its synthesis, all the intermediate free amino pept ide esters have been isolated and characterized.

A classical approach in peptide synthesis is the protection of a-amino group with the acid sensitive t- butyloxycarbonyl (Boc) group introduced by Carpino. It is stable to catalytic hydrogenation, catalytic transfer hydrogenation , Na in liquid NH) and is cleaved by anhydrous HCl in an organic solvent like ethyl acetate or THF, trifluoroacetic acid in CH2Ch, 98% HCOOH, 10% H2S04 in dioxane, BF3.OEt2, etc. After its removal, it results in the formation of salts of peptide esters. Such protonated amino components formed have to be deprotonated before the coupling of the next amino acid. t-4 Thus treatment with a tertiary base for the in situ deprotonation of HCI salt of peptide ester is mandatory. In synthesis carried out in solution, it is usual practice to add the acylating agent to a mixture of the protonated amine and a tertiary base. This procedure, however, has several drawbacks: at any given time only a fraction of the amino component is available for coupling, the hydroxyl and amide protons are abstracted and the resulting anions initiate several side-reactions such as O-acylation, succinimide and glutarimide formation, diketo­piperazine ring closure and racemization.5-10 It is also reported that the presence of salts of tertiary amines increases the degree of racemization in most of the methods of the formation of peptide bonds. II In DCC mediated coupling reactions, the formation of substantial amounts of N-acylurea has also been observed. 12 Therefore, it is desirable to eliminate the use of tertiary base in the coupling reaction.

t Dedicaled to Professor K M Sivanandai ah on the occasion of his 70th birth anniversary.

Alternatively, the free amino components were also obtained by using several alcoholates (MeONa, EtONa) and phenolates (sodium pentachlorophenol ate) which in some cases also lead to the formation of N­acylurea. 13 Deprotonation of peptide ester salts employing an aqueous NaHCO) wash requires extraction of the resulting free amino peptide ester using an organic solvent. This is tedious and does not permit the quantitative collection of the free amine. In some instances, this also results in racemization. 14.15

In this context, Bodanszky and hi s co-workers rec­ommended several alternatives to circumvent the use of a base. These include the use of Nps, Trt and Bpoc groups which can be removed by using HOBt or pen­tachlorophenol and conversion of formate salts of amino components using tetrazole salts, etc. Recently our group demonstrated the synthesis of peptides em­ploying Fmoc-amino acid chlorides and Fmoc-/Boc­/Z-amino acid fluorides mediated by zinc dust. 16 This method is extended for the synthesis of dialkylamjno acids as wel!.17 The utility of zinc dust for the intro­duction of Z- and Fmoc-groups employing Z-Cl and Fmoc-Cl, respectively into amino acids was also demonstrated. 18. 19 Its use in the conversion of hydro­chloride salts of amino acid ester into the correspond­ing free amino acid esters is also explored. 20 This pa­per deals with the application of zinc dust for the de­protonation of HCl salts of peptide esters which per­mits the isolation of free amino peptide esters in Boc­chemistry.

It is now found that the deprotonation of hydrochloride salts of peptide esters can be accomplished using activated zinc dust. A suspension of the protonated peptide ester in CH2Cl2 or THF was

SURESH BABU el al. : COUPLING IN THE ABSENCE OF A TERTIARY BASE 1029

stilTed with zinc dust. The reaction was monitored by TLC and IR. The deprotonation was complete in about 5 min. After the reaction, the free amino peptide ester dissolved completely in CH2CI2. The pH of the solution was also found to be neutral. The IR analysis clearly indicated a sharp doublet at 3260 cm'l which confirms complete deprotonation. After filtration of ZnCh and unreacted zinc dust, the solution containing the free amino peptide ester can be used directly in the coupling or evaporation of the solvent led to its isolation . The coupling was carried out using DCC/HOBt method employing free amino acid esters as amino components. No tertiary base was added . The deprotection of Boc group was carried out by using 3N HCI-ethyl acetate. The list of free amino di-, tri- and tetrapeptide esters synthesized and their physical constants are given in the Table I . The HPLC profile of the pure free dipeptide (H-Ala-Ala­OMe) is given in Figure 1. During this process, zinc oxidizes to Zn2

+ along with the liberation of hydrogen gas. Then Zn2

+ converts to its chloride which precipitates .

This method is further extended for the synthesis of p-casomorphin (Tyr-Pro-Phe-Pro-Gly) which was accomplished using the Boc group for N-protection and the DCC/HOBt method for coupling. All the intermediate HCl salts of peptide esters were deprotonated using zinc dust and the resulting pept ide

Table I - Characterization data of peptide esters

SI No. Peptide esters··b m.p. TLC [al ~ (0C) Rr B (c=I, CHCI))

I H-Gly-Val-OBzl 202-05 0.38 -1 2.8

2 H-Pro-Gly-OB zl gum 0.34 -60.5

3 H-Ala-Ala-OMe 126-28 0.39 -35.0

4 H-Phe-lIe-OMe 101-02 0.45 - 13.0 (OMF)

5 H-Ser(Bzl)-Gly-OEt 116-17 0.4 1 +13 .9

6 H-Pro- Phe-G ly-OBzl 188-90 0.29 -30.8

7 H-Gly-Phe-Leu-OBzl 168-70 0.38 +9 .5

8 H-Gly-Phe-Leu-OMe 108-09 0.44 - 10.0 (OMF)

9 H -Pro-Trp-Leu-OMe 227-28 0.5 1 -40.6 (OM F)

10 H-Ser-Al a-Ala-OEt 150-52 0.49 -20.0 (OM F)

II H-Pro- Val-Gly-OEt 127-28 0.58 -74

12 H-Gly-Leu-Val-OMe 115-17 0.51 -50

13 H. Val-Tyr-I1e-OMe 146-48 0.46 -0.4

14 H-Pro-Ser(Bzl)-Gly-OEt 162-63 0.6 1 -31.1

15 H-Pro-Pro-Pro-Tyr-OMe 226-28 0.61 -85.0 (OMF)

a All the free peptide esters were charac terized usi ng IR . b All the free peptide esters gave sati sfactorily I H NMR spectra.

4 .56

Figure 1 - Analytical RP-HPLC of H-Ala-Ala-OMe : Waters C-18 deltapak column (3.9 x 300mm, 15J,L); flow rate , I mLimin ; eluant aceetonitrile - 0.1 % TFA and H20 (65 : 35 ; isocratic ; monitoring at 220 nm).

8 .36

Figure 2 - Analytical RP-HPLC of pure (3-casomorphin : Waters C-18 deJtapak column (3.9 x 300mm, 15J,L); flow rate, ImLimin; e luant acetonitrile - 0.1 % TFA and H20 (65 : 35 ; isocrat ic ; monitoring at 220 nm).

esters were isolated and characterized (the HPLC data are given in Figure 2) . The synthetic p-casomorphin exhibited the biological activity (opiate like properties tested by guinea pig ileum assay) simi lar to that of the natural molecule.21 Thus, activated zinc dust can be used instead of an organic tertiary amine for the deprotonation of the HCl salts of peptide esters. It is a simple, efficient and alternative method. The resulting free peptide esters, similar to Fmoc-/4-AMP chemistry22, can be. isolated in good yield and purity.

Thus the long known demand of peptide chemists, i.e. the recommendation to use free amines rather than a combination of amine salt and tertiary base as an amino component during coupling can be met easily by this s imple technique.

Experimental Section Melting points were determined by using capillary

tubes and are uncorrected. TLC analysis was carried on precoated si lica gel plates using solvent systems : (A) CHCb: methanol: acetic acid (40:2:1; v/v/v), (B ) n-butanol : acetic acid: water (4:1:1; v/v/v) and (C) CHCb : methanol (9: 1; v/v) and the Rr values are designated as Rr A, Rr B, RrC; respectively. IR spectra were recorded on a Nicolet model Impact 400D FT­IR spectrometer (KBr pellets, 3 cm· 1 resoluti on).

1030 INDIAN J. CHEM., SEC B, MA Y 2002

Optical rotations were measured with an automatic AA-lO Polarimeter (Optical Acti vity, U.K.). IH NMR spectra were recorded on a Bruker ACF 200 MHz spectrometer using TMS as an internal standard. RP­HPLC was carried out using a Waters LC-3000 system consisti ng of a 484 tunable absorbance U.V. detector and a Millipore 745 data module. Amino acid methyl and ethyl esters were prepared us ing the methanol/SOCh procedure. The coupling was carried out by the DCC/HOB t method. 22 Deprotection of Boc-group was carried out using 3 N HCl - ethy l acetate following reported procedures ?4 Zinc dust was activated by pretreatment with IN HCl which was thoroughly washed with water and acetone and dried prior to use.

Deprotonation of hydrochloride salts of peptide esters : General procedure. To a suspension of amino ac id ester hydrochlori de salt (1 mmole) in THF 00 mL) was added zinc du st (1 00 mg) in one portion. The mixture was stirred fo r 5-10 min at r.t. After complet ion of the reaction, it was filtered , evaporated ill vacuo and precipitated using dry ether to obtain free ami no peptide ester as a crystalline solid.

Coupling by the DCC/HOBt method : General procedure23

. To an ice-cold solution of Boc-amino acid (10 mmoles) in dry THF (10 mL) was added DCC (2.0 g, 10 mmoles) and HOBt (1.35 g, 10 mmoles). A solution of free amino acid ester (10.2 mmoles) in THF (10 mL) was then added and the reaction mixture was stirred for 2-3 hr. After the completion of the reaction, the precipitated dicyclohexylurea was filtered and evaporated the solvent in vacuo. It was dissolved in CH2Cl2 and washed with IN HCl (3 x 10 mL), 10% NaHC03 (3 x 10 mL) and then with water, dried over anhydrous Na2S04 and evaporated to an oil which was recrystallized using CH2Cl2 / n-hexane.

Deprotection of Boc- group General procedure. The Boc- peptide ester (2 mmoles) was dissolved in 3 N HCI- ethyl acetate (5 mL) and stirred for 30 min. After the completion of the reaction, the solution was removed in vacuo and the oil was triturated with ether to get the peptide ester hydrochloride salt as a crystalline solid.

Synthesis of ~-casomorphin Boc-Pro-Gly-OMe (I). To an ice-cold solution of

Boc-Pro (2 .1 g, 10 mmoles) in dry THF (10 mL) was added DCC (2.0 g, 10 mmoles) and HOBt (1.35 g, 10 mmoles). A solution of H-Gly-OMe (0.89 g, 10

mmoles) in THF (10 mL) was then added and stirred to yield 2.S g (90%) of the peptide I as a foam; Rr A,

0.80; [a ] ~ - \ 6.0 (c=l , CHCI3).

H-Pro-Gly-OMe (II). Boc-Pro-Gly-OMe (I, 2.5 g, 9 mmoles) was dissolved in I N HCI- ethyl acetate (20 mL) and stirred for 30 mi n. After complet ion of the reaction the solution was evaporated in vacuo and the resulting oi l was tri tu rated with ether to get the peptide ester hydrochloride salt. Further, it was deprotonated using zinc dust following the general procedure to yield 1.4 g (86%) of the peptide II, m.p.

174-77 DC; Rr A, 0.61; Rr B, 0.56; [al ~ - 30.6 (c= I,

CHCb); IR : 3128, 3211 cm- I. Boc-Phe-Pro-Gly-OMe (III). To an ice-cold

solution of Boc-Phe (2.0 g, 7.7 mmoles) in dry THF (10 mL) was added DCC ( 1.5 g, 7.7 mmoles) and HOSt (1.0 g, 7.7 mmoles). A solution of H-Pro··Gly-OMe (11, 1.4 g, 7.7 mmoles) in THF (10 mL) was added and stirred to yield 2.9 g (87%) of the peptide III; m.p., 153-55DC; Rr

A, 0.76, [aJ ~ - 29.3 (c=l , CHCh).

H-Phe-Pro-Gly-OMe (IV). Boc-Phe-Pro-Gly­OMe (III, 2.9 g, 6.75 mmoles) was dissolved in HC1-ethyl acetate (20 mL) and stirred for 30 min . After completion of the reaction the solution was evaporated in vacuo and the resulting oil was triturated with ether to get the peptide ester hydrochloride salt. Further, it was deprotonated using zinc dust following the general procedure to yield 1.8 g (81 %) of the peptide IV as a foam; Rr A, 0.63 ; Rr B,

0.61; [a] ~ - 40.1 (c=l, CHCI3); IR; 3219, 3240 cm- I.

Boc-Pro-Phe-Pro-Gly-OMe (V). To an ice cold solution of Boc-Pro (1.15 g, 5.5 mmoles) in dry THF (8 mL) was added DCC (1.1 g. 5.5 mmoles) and HOBt (0.74 g, 5.5 mmoles). A solution of H-Phe-Pro­Gly-OMe (IV 1.8 g, 5.5 mmoles) in THF (10 mL) was added and stirred to yield 2.3 g (80%) of the

peptide V; m.p. 158-59DC; Rr A, 0.86; [a] ~ -12.8

(c=l, CHCI3) .

H-Pro-Phe-Pro-Gly-OMe (VI). Boc-Pro-Phe-Pro­Gly-OMe (V, 2.3 g, 4.37 rnmoles) was dissolved in HC1-ethyl acetate (15 mL) and stirred for 30 min. After completion of the reaction the solution was evaporated in vacuo and the resulting oil was triturated with ether to get the peptide ester hydrochloride salt. Further, it was deprotonated using zinc dust following the general procedure to yield 1.5 g (80%) of peptide

VI; m.p. 168-69DC; Rr A, 0.58; Rr B, 0.54; [a] ~ -46.9

(c=l, CHCI3); IR: 3310, 3109 cm- I.

S UR ESH BABU et al. : COUPLI NG IN TH E ABSENCE OF A TERT IARY BASE 103 1

Boc-Tyr(Bul)-Pro-Phc-Pro-Gly-OMc (VII). To an ice-cold solu tion of Boc-Tyr(Bul) (1.0 g, 3.5 mmoles) in dry THF (10 mL) was added DCC (0 .7 g, 3.5 mmoles) and HOBt (0.47 g, 3.5 mmoles). A solution of H-Pro-Phe-Pro-Gly-OMe (VI, 1.5 g, 3.5 mmoles) in THF (10 mL) was added and sti rred to yield 2.1 g (84%) of the peptide VII ; m.p. 184-86°C;

RrA, 0.74; [a] ~ -30.1 (c=l, CHCI3).

H-Tyr-Pro-Phe-Pro-Gly-OMe (VIII). Boc-Tyr(Bul)-Pro-Phe-Pro-Gly-OMe (VII, 2.0 g, 2.9 mmoles) was dissolved in HCI-ethyl acetate (15 mL) and stirred for 30 min . After completion of the reaction the solution was evaporated in vacuo and the resulting oil was triturated with ether to get the peptide ester hydrochloride salt. Further, it was deprotonated using zinc dust following the general procedure to yield 1.3 g (77%) of the peptide VIII;

m.p., ISO-51 °C; Rr A, 0.60; Rr B, 0.42; [a] ~-60.2 (c=l, CHCI3); IR. : 32 11 ,3 198 em· l

.

Tyr-Pro-Phe-Pro-Gly (IX): A solution of peptide ester (VIII , 1.18 g, 2 mmoles) in methanol (25 mL) and IN NaOH (5 mL) was stirred for 3 hr by maintaining the pH between 8 and 9. After the completion of the reaction the solution was neutralized using IN HCI and the precipitated solid was filtered and dried to get 0 .60 g (50%) of the free

peptide IX; m.p. lSI-53°C; [a] ~ -48.5 (c=0.5, DMF)

[Reported2s : m.p. 152-54°C; [a] ~ -48 (c=0.5 ,

DMF)]; Rr B 0.40; Rr C, 0.42. Anal. Found: C, 62.29; H, 6.02; N, 12.28. Calc. for C30H3SNs07 (578) : C, 62.34; H, 6.10; N, 12.17%. Rh 8.36 [(Waters C-18 deltapak column (3 .9 x 300 mm, IS )..I.); using as the eluant acetonitrile - 0. 1 % trifluoroacetic acid (TFA) and water (65:35; isocratic, flow rate I mLimin, monitoring at 220 nm).

Acknowledgement

We thank Prof. B S Sheshadri for useful discussions. We are grateful to the Department of Science and Technology, Govt. of India for financial assistance. We also thank Jupiter Orga Ltd., Hyderabad for partial financial support. One of the

authors (K A) thanks the CSIR, New Delh i for the award of SRF.

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