Inr. J. Peptide Protein Kes. 38, 1991, 25-31
Sulfonation of arginine residues as side reaction in Fmoc-peptide synthesis ANNETTE G. BECK-SICKINGER’ , GERD SCHNORRENBERQ, J d R G METZGER’ and GUNTHER JUNG’
‘Institute for Organic Chemistry, University of Tiibingen, Tiibingen, ’Boehringer Ingelheim KG, Medicinal Chemistry Department, Ingelheim. FRG
Received 19 September 1990, accepted for publication 14 January 1991 Several arginine-rich peptides containing the C-terminus of neuropeptide Y (NPY) were prepared by solid phase peptide synthesis using Fmoc chemistry and cleaved from the resin with trifluoroacetic acid (TFA). The products were characterized by fast atom bombardment-MS, LC-thermospray-MS,ion spray-MS/MS, and Edman degradation.The side products could be identified as peptides with sulfonated arginine residues resulting from an unexpected cleavage of Mtr or Pmc protecting groups. The degree of sulfonation depended on the choice and composition of the cleavage solution. Several scavenger mixtures were used and a mixture of thioanisole/thiocresolwas found to be the most efficient for suppressing sulfonation. Furthermore treatment with the enzyme arylsulfate-sulfohydrolase desulfonated the peptides yielding the correct sequence. Key words: arginine side-chain protection; Fmoc-strategy; neuropeptide Y; peptide synthesis
tecting groups like Mtr (3) or Pmc (4) are not easily removable and require treatment with strong acids for complete removal. Alkylation of tryptophan (5) and sulfonation of tyrosine (6) during deprotection of Arg(Mtr) and Arg(Pmc) containing peptides were reported recently. The removal of the nitro group, the “classical.” protective group of arginine, affords an additional hydrogenation step which is not easily applicable for multiple peptide synthesis or large scale procedures. We observed in crude products containing two or more arginine residues peptidic byproducts up to 30%. The peptides were synthesized by solid phase method using Fmoc strategy and the Mtr protecting group for arginine. In the case of neuropeptide .Y Abbreviation: Aca, &-aminocaproic acid; Boc, tert.-butyloxycaranalogues (Fig. I), such as NPY 1-33-36 (H-YRQbonyl; BHA, benzhydrylamine; tBu, terr.-butyl; CID, collision RY-NH,) or NPY I -4-Aca-25-36 (H-YPSK-Acainduced dissociation; DMF, dimethylformamide; FAB-MS, fast RHYIN LITRQRY-NH,), these byproducts did not atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxy- exhibit any receptor binding or biological activity, carbonyl; HOBt, I-hydroxybenzotriazole; IS-MS, ion spray mass whereas the correct peptide NPY 1-4-Aca-25-36 is spectrometry; LC-MS, liquid chromatography mass spectrometry; highly active in several bioassays (7, 8). This led us to Mtr, 4-methoxy-2,3,5-trimethylbenzenesuIfonyl; NPY, neuropeptide Y; Pmc, 2,2,5,7,8-pentamethylchromansulfonyl;PS-DVB, the conclusion that a modification of the C-terminal tetrapeptide had taken place as this part of the molpolystyrene-(1 %)divinylbenzene, PTH, phenylthiohydantoin; ecule is responsible for receptor binding (I). Here we TBTU, hydroxybenzotriazol-1-yl-oxy-bis-(dimethylamino)-uroniumtetrafluoroborate; Tmob, 2.4.6-trimethoxybenzyl; TFA, trifluoro-, report on the identification of arginine modifying byacetic acid; Trt, trityl. products using FAB-MS, LC-MS, IS-MS, IS-MS/MS Arginine, the guanidino side-chain containing residue is the strongest basic amino acid in peptides and proteins and often involved in the ligand-protein interactions of immunoglobulins and hormone receptors. In neuropeptide Y, a peptide amide containing 36 amino acids (Fig. 1) with strong vasoconstrictive and neuromodulative activity, two arginine residues in the C-terminal part of the molecule are directly involved in the hormone-receptor-interaction (1). Despite numerous synthetic efforts arginine still causes many problems in peptide synthesis even in Fmoc-strategy. The use of unprotected arginine residues leads to intramolecular cyclisation (2), pro-
25
Pro-NPY 36-39
and Edman degradation. Different cleavage conditions to suppress the formation of these byproducts and an enzymic method to convert the byproducts to the desired peptides are investigated. MATERIALS A N D METHODS
Pept ide synthesis Neuropeptide Y analogues were synthesized on an automatic peptide synthesizer (430A, Applied Biosystems, Weiterstadt) using Fmoc-strategy on pbenzyloxy-benzylalcohol-PS-DVB (for peptide acids) or on 5-(4'-aminomethyl-3',5'-dimethoxy-phenoxy)valeryl-alanyl-PS-DVB (for peptide amides) (9). Fmoc-amino acids (1 equiv.) were activated with 'TBTU (1 equiv.), HOBt (1 equiv.) and diisopropylethylamine (1.5 equiv.) in 4-fold excess. The side-chain protection was as follows: His(Trt), Lys(Boc), Asn (Tmob), Gln(Tmob), Ser(tBu), Thr(tBu) and Tyr (tBu) (Nova Biochem, Sandhausen). Arginine guanidino groups were protected with either Pmc (Nova Biochem, Sandhausen) or Mtr (Bachem, Heidelberg). The synthesis was performed in DMF with single coupling procedures. Deprotection of the Fmoc-group was carried out with 25% piperidine in D M F two times within IOmin. After the final N,-deprotection the peptides were cleaved from the resin with either TFA/ thioanisole/thiocresol (9:0.5:0.5)or with TFA/thioanisole (9: 1) or with TFA/anisole (9: 1) within 4 h. TFA was removed in ~acuo,the peptides were redissolved in acetic acid, precipitated with diethyl ether, collected by centrifugation, and lyophilized from water. Variation of cleavage conditions Various mixtures of scavengers and TFA (Table 3) were added to samples of side-chain protected, polymer bound NPY 1-4-Aca-25-36 (1 00 mg). After 3 h the resin was filtered off and the crude products precipitated with diethyl ether. The peptides were analyzed by HPLC using a gradient 15% B to 60% B within 30min (see below). Peaks were identified by IS-MS.
YGKR
FIGURE I Amino acid sequence of neuropeptide Y and of the synthesized analogues.
preparative 25 x 250 mm); flow rate 1 mL/min and 12 mL/min at room temperature, detection at 220 nm and gradient system (I) 15% B to 60% B within 30min, (11) 20% B to 60% B within 30min, or Dynamax C,, reversed phase column, 3 p (50 x 21.4mm) and gradient (111) 5% B to 60% B within IOmin with A: water/TFA 1OO:O.l and B: acetontrile/ TFA 1OO:O.l. Amino acid analysis Peptide samples (200 nmol) were dissolved in 6 N HCl containing phenol (10%) and hydrolyzed in sealed tubes for 24h at 110". Amino acids were detected quantitatively as trifluoroacetyl-amino acid-n-propylesters on Chirasil-Val [lo] by gas chromatography, a method which simultaneously gives the enantiomer composition. Mass spectrometry Fast atom bombardment mass spectra were measured on a Finnigan MAT 90 and a Varian MAT 711 A mass spectrometer at a source temperature of 30" using an argon gun. The matrix was glycerol or 4ni trobenzylalcohol. Liquid chromatography-mass spectrometry was performed on a HPLC H P 109OL connected with the HP thermospray MS-system 5988A (Hewlett-Packard, Waldbronn). 0.05 N ammonium acetatelacetonitrile/ TFA 53:47:0.1 was used as isocratic solvent system. Ion spray mass spectra and daughter ion mass spectra were recorded on a API 111 triple quadrupole mass spectrometer equipped with an IonSprayTM interface (Sciex, Toronto). Samples were dissolved in acetonitrile/l% TFA and introduced into the ion spray source at a flow rate of 5pL/min using the solvent delivery system ABI 140 A (Applied Biosystems, Weiterstadt). Argon was used as collision gas for MSIMS.
Edman degradation Automated Edman degradation was performed in a pulsed-liquid protein sequencer 417 A equipped with HPLC analysis an on-line microbore phenylthiohydantoin (PTH)Preparative and analytical HPLC were carried out on amino acid analyzer 120 A (Applied Biosystems, a Waters 600 multisolvent delivery system, combined Weiterstadt). All reagents and solvents were purwith Waters 712 WISP autoinjector and Waters 990 chased from Applied Biosystems. A glass fiber filter photodiode array detector or on a Gilson autoprep activated with TFA was coated with 1 mg of Biobrene M303 combined with a MI16 UV detector. The fol- Plus prior to adding of the sample. Sequencing was lowing separation systems were used: Nucleosil C,* carried out using standard programmes BEGIN-1 and reversed phase column, 5 p (analytical 4.6 x 250 mm, NORMAL-I (Applied Biosystems). 26
Arginine side-chain protection TABLE 1 Synthesis of NP Y 1-4-Aca-2s-36: effects of different deprotection mixtures on product composition Arginine protecting group A B C D
Scavenger in TFA for cleavage thioanisole thioanisole/thiocresol (1 : 1) thioanisole thioanisole/thiocresol (1 : I)
Mtr Mtr Pmc Pmc
Desired product (YO)
Z sulfonated products (%)
I: Mtr/Pmcproducts (YO)
21.1
17 8 15.2 5
46 79 42 88
Sulfonation of arginine residues as side reaction in Fmoc-peptide synthesis ANNETTE G. BECK-SICKINGER’ , GERD SCHNORRENBERQ, J d R G METZGER’ and GUNTHER JUNG’
‘Institute for Organic Chemistry, University of Tiibingen, Tiibingen, ’Boehringer Ingelheim KG, Medicinal Chemistry Department, Ingelheim. FRG
Received 19 September 1990, accepted for publication 14 January 1991 Several arginine-rich peptides containing the C-terminus of neuropeptide Y (NPY) were prepared by solid phase peptide synthesis using Fmoc chemistry and cleaved from the resin with trifluoroacetic acid (TFA). The products were characterized by fast atom bombardment-MS, LC-thermospray-MS,ion spray-MS/MS, and Edman degradation.The side products could be identified as peptides with sulfonated arginine residues resulting from an unexpected cleavage of Mtr or Pmc protecting groups. The degree of sulfonation depended on the choice and composition of the cleavage solution. Several scavenger mixtures were used and a mixture of thioanisole/thiocresolwas found to be the most efficient for suppressing sulfonation. Furthermore treatment with the enzyme arylsulfate-sulfohydrolase desulfonated the peptides yielding the correct sequence. Key words: arginine side-chain protection; Fmoc-strategy; neuropeptide Y; peptide synthesis
tecting groups like Mtr (3) or Pmc (4) are not easily removable and require treatment with strong acids for complete removal. Alkylation of tryptophan (5) and sulfonation of tyrosine (6) during deprotection of Arg(Mtr) and Arg(Pmc) containing peptides were reported recently. The removal of the nitro group, the “classical.” protective group of arginine, affords an additional hydrogenation step which is not easily applicable for multiple peptide synthesis or large scale procedures. We observed in crude products containing two or more arginine residues peptidic byproducts up to 30%. The peptides were synthesized by solid phase method using Fmoc strategy and the Mtr protecting group for arginine. In the case of neuropeptide .Y Abbreviation: Aca, &-aminocaproic acid; Boc, tert.-butyloxycaranalogues (Fig. I), such as NPY 1-33-36 (H-YRQbonyl; BHA, benzhydrylamine; tBu, terr.-butyl; CID, collision RY-NH,) or NPY I -4-Aca-25-36 (H-YPSK-Acainduced dissociation; DMF, dimethylformamide; FAB-MS, fast RHYIN LITRQRY-NH,), these byproducts did not atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxy- exhibit any receptor binding or biological activity, carbonyl; HOBt, I-hydroxybenzotriazole; IS-MS, ion spray mass whereas the correct peptide NPY 1-4-Aca-25-36 is spectrometry; LC-MS, liquid chromatography mass spectrometry; highly active in several bioassays (7, 8). This led us to Mtr, 4-methoxy-2,3,5-trimethylbenzenesuIfonyl; NPY, neuropeptide Y; Pmc, 2,2,5,7,8-pentamethylchromansulfonyl;PS-DVB, the conclusion that a modification of the C-terminal tetrapeptide had taken place as this part of the molpolystyrene-(1 %)divinylbenzene, PTH, phenylthiohydantoin; ecule is responsible for receptor binding (I). Here we TBTU, hydroxybenzotriazol-1-yl-oxy-bis-(dimethylamino)-uroniumtetrafluoroborate; Tmob, 2.4.6-trimethoxybenzyl; TFA, trifluoro-, report on the identification of arginine modifying byacetic acid; Trt, trityl. products using FAB-MS, LC-MS, IS-MS, IS-MS/MS Arginine, the guanidino side-chain containing residue is the strongest basic amino acid in peptides and proteins and often involved in the ligand-protein interactions of immunoglobulins and hormone receptors. In neuropeptide Y, a peptide amide containing 36 amino acids (Fig. 1) with strong vasoconstrictive and neuromodulative activity, two arginine residues in the C-terminal part of the molecule are directly involved in the hormone-receptor-interaction (1). Despite numerous synthetic efforts arginine still causes many problems in peptide synthesis even in Fmoc-strategy. The use of unprotected arginine residues leads to intramolecular cyclisation (2), pro-
25
Pro-NPY 36-39
and Edman degradation. Different cleavage conditions to suppress the formation of these byproducts and an enzymic method to convert the byproducts to the desired peptides are investigated. MATERIALS A N D METHODS
Pept ide synthesis Neuropeptide Y analogues were synthesized on an automatic peptide synthesizer (430A, Applied Biosystems, Weiterstadt) using Fmoc-strategy on pbenzyloxy-benzylalcohol-PS-DVB (for peptide acids) or on 5-(4'-aminomethyl-3',5'-dimethoxy-phenoxy)valeryl-alanyl-PS-DVB (for peptide amides) (9). Fmoc-amino acids (1 equiv.) were activated with 'TBTU (1 equiv.), HOBt (1 equiv.) and diisopropylethylamine (1.5 equiv.) in 4-fold excess. The side-chain protection was as follows: His(Trt), Lys(Boc), Asn (Tmob), Gln(Tmob), Ser(tBu), Thr(tBu) and Tyr (tBu) (Nova Biochem, Sandhausen). Arginine guanidino groups were protected with either Pmc (Nova Biochem, Sandhausen) or Mtr (Bachem, Heidelberg). The synthesis was performed in DMF with single coupling procedures. Deprotection of the Fmoc-group was carried out with 25% piperidine in D M F two times within IOmin. After the final N,-deprotection the peptides were cleaved from the resin with either TFA/ thioanisole/thiocresol (9:0.5:0.5)or with TFA/thioanisole (9: 1) or with TFA/anisole (9: 1) within 4 h. TFA was removed in ~acuo,the peptides were redissolved in acetic acid, precipitated with diethyl ether, collected by centrifugation, and lyophilized from water. Variation of cleavage conditions Various mixtures of scavengers and TFA (Table 3) were added to samples of side-chain protected, polymer bound NPY 1-4-Aca-25-36 (1 00 mg). After 3 h the resin was filtered off and the crude products precipitated with diethyl ether. The peptides were analyzed by HPLC using a gradient 15% B to 60% B within 30min (see below). Peaks were identified by IS-MS.
YGKR
FIGURE I Amino acid sequence of neuropeptide Y and of the synthesized analogues.
preparative 25 x 250 mm); flow rate 1 mL/min and 12 mL/min at room temperature, detection at 220 nm and gradient system (I) 15% B to 60% B within 30min, (11) 20% B to 60% B within 30min, or Dynamax C,, reversed phase column, 3 p (50 x 21.4mm) and gradient (111) 5% B to 60% B within IOmin with A: water/TFA 1OO:O.l and B: acetontrile/ TFA 1OO:O.l. Amino acid analysis Peptide samples (200 nmol) were dissolved in 6 N HCl containing phenol (10%) and hydrolyzed in sealed tubes for 24h at 110". Amino acids were detected quantitatively as trifluoroacetyl-amino acid-n-propylesters on Chirasil-Val [lo] by gas chromatography, a method which simultaneously gives the enantiomer composition. Mass spectrometry Fast atom bombardment mass spectra were measured on a Finnigan MAT 90 and a Varian MAT 711 A mass spectrometer at a source temperature of 30" using an argon gun. The matrix was glycerol or 4ni trobenzylalcohol. Liquid chromatography-mass spectrometry was performed on a HPLC H P 109OL connected with the HP thermospray MS-system 5988A (Hewlett-Packard, Waldbronn). 0.05 N ammonium acetatelacetonitrile/ TFA 53:47:0.1 was used as isocratic solvent system. Ion spray mass spectra and daughter ion mass spectra were recorded on a API 111 triple quadrupole mass spectrometer equipped with an IonSprayTM interface (Sciex, Toronto). Samples were dissolved in acetonitrile/l% TFA and introduced into the ion spray source at a flow rate of 5pL/min using the solvent delivery system ABI 140 A (Applied Biosystems, Weiterstadt). Argon was used as collision gas for MSIMS.
Edman degradation Automated Edman degradation was performed in a pulsed-liquid protein sequencer 417 A equipped with HPLC analysis an on-line microbore phenylthiohydantoin (PTH)Preparative and analytical HPLC were carried out on amino acid analyzer 120 A (Applied Biosystems, a Waters 600 multisolvent delivery system, combined Weiterstadt). All reagents and solvents were purwith Waters 712 WISP autoinjector and Waters 990 chased from Applied Biosystems. A glass fiber filter photodiode array detector or on a Gilson autoprep activated with TFA was coated with 1 mg of Biobrene M303 combined with a MI16 UV detector. The fol- Plus prior to adding of the sample. Sequencing was lowing separation systems were used: Nucleosil C,* carried out using standard programmes BEGIN-1 and reversed phase column, 5 p (analytical 4.6 x 250 mm, NORMAL-I (Applied Biosystems). 26
Arginine side-chain protection TABLE 1 Synthesis of NP Y 1-4-Aca-2s-36: effects of different deprotection mixtures on product composition Arginine protecting group A B C D
Scavenger in TFA for cleavage thioanisole thioanisole/thiocresol (1 : 1) thioanisole thioanisole/thiocresol (1 : I)
Mtr Mtr Pmc Pmc
Desired product (YO)
Z sulfonated products (%)
I: Mtr/Pmcproducts (YO)
21.1
17 8 15.2 5
46 79 42 88
