tnt. J. Peptide Protein Res. 7, 1975, 295-305 Published by Munksgaard Copenhagen Denmark N o part may be reprodudd by any pr&s without written permission from the author@
THE METHYLSULFONYLETHYLOXYCARBONYL G R O U P , A N E W A N D VERSATILE A M I N O PROTECTIVE F U N C T I O N G. I. TESSER and I. C. BALVERT-GEERS
Department of Organic Chemistry, R.C. University, Nijmegen, the Netherlands
Received 7 September 1974
A new amino protecting group, the methylsulfonylethyloxycarbonyl(Msc) group, is described which combines well with other familiar groups (benzyloxycarbonyl, t-butyloxycarbonyl) in peptide syntheses. Its main characteristics are an extreme acid stability, a high base lability and a high polarity which .enhances solubility in polar solvents including water. The group resists catalytic hydrogenolysis but does not poison the catalyst. It has good crystallizing properties. Application in peptide synthesis is exemplified in the synthesis of Msc-Phe-Arg-Trp-Cly-OMe.HC1. Deprotection of the rnasked tetrapeptide was accomplished within 5 sec with a 1.0 N solution of base (OH- and OCH,-). A reaction scheme for the cleavage of the group is suggested.
The application of the principle of 8-elimination in the design of protecting functions is not new. The usefulness in peptide synthesis has already been demonstrated by Kadar & Stirling (1). Rydon (2), Wunsch (3), and Carpino (4). From our department, an application in solid phase synthesis was announced in a brief note ( 5 ) and worked out by Buis (6). Meanwhile, the same concept has been examined by Merrifield (7). In this paper, we report on general procedures for the introduction of the methylsulfonylethyloxycarbonyl group (Msc*) as an amino protective function, removal of which is also based on P-elimination. The group was developed in order to arrive at a protective group which enhances solubility in polar solvents of intermediates and products in peptide syntheses in solution. The Msc-group combines several interesting and remarkable properties which distinguish it from other protecting groups. For that reason the group may be a n important, new expedient in the synthesis of peptides.
* Abbreviation suggested by Prof. Dr. R. Schwyzer, in accordance with the general convention of the IUPAC-IUB commission for nomenclature (8).
METHODS AND RESULTS
Introduction of the Msc-group in amino compounds The parent compound of the Msc-group, 2-methylsulfonylethanol (I), can readily be obtained from the commerically available 2methylmercaptoethanol by catalytic oxidation with hydrogen peroxide and sodium tungstate (9). The product (I) is a crystalline solid, having limited solubility in organic solvents. It decomposes on distillation, even in high uacuo, by elimination of water, but can be crystallized from isopropyl alcohol. Several suitable reagents (11-V, Fig. 1.) for the preparation of Msc-amino derivatives could be derived from I. Treatment of the alcohol with p-nitrophenyl chloroformate in pyridine, yields the well crystallizing methylsulfonylethyl p-nitrophenyl carbonate (Msc-ONp, 11). An excess of phosgene in tetrahydrofuran solution converts I into the crystalline chloroformate (Msc-CI, 111). Treatment of this compound with hydroxy compounds in the presence of a tertiary amine can be used to obtain mixed carbonates (IVa and b) derived from hydroxy compounds which in themselves do not readily give a chloroformate. Methylsulfonylethyloxy295
G. I. TESSER AND I. C. BALVERT-GEERS
+
CH3SOZCHaCHzOH CICOOCbH4N02-p-
CH~SO~CHZCH~OCOOC~,H~NOZ-~
p-NOnCsHdOH COCIZ
/
I 1 CH~SOZCHZCHzOCOCl -HOY,->
/
I1
/Co-cr
CHJSOZCHZCHZOCOOY
1Va: Y
I11
=
-N
\
CO--CH,
/c o*
\
co
V FIGURE 1 Synthesis of reagents (11-V)for the introduction of an Msc-group.
carbonyl azide (Msc-N,, V) can also be prepared in nearly quantitative yield from 111 and sodium azide. Most of the amino acids react rapidly with Msc-ONp, and this reagent was used for the preparation of all but one of the Msc-derivatives given in.Table 1. To that aim, a suspension or solution of an amino acid in an acetonitrile/ water mixture was stirred for some time with equivalent amounts of the mixed carbonate and triethylamine. The reaction products remained in solution and the Msc-derivatives were separated from p-nitrophenol, after evaporation of acetonitrile, by extraction with ether at pH 5. In some instances (Msc-Phe, Msc-Leu, MscVal) the product crystallized from the water phase on further acidification. Msc-derivatives, having a moderate solubility in water, could be isolated by extraction with ethyl acetate. In all other cases where the Msc-derivatives had a high solubility in water, the aqueous solution containing the product as the triethylammonium salt was filtered through a strongly acidic ionexchange resin and the Msc-derivatives were
296
isolated by evaporation. Some Msc-derivatives crystallized spontaneously; if not, they were converted into dicyclohexylammonium salts which crystallize from iso-propyl alcohol. Msc-azide (V) and especially the mixed carbonates IVa and b are valuable reagents for the introduction of the Msc-group into compounds with a low reactivity, e.g., S-tritylcysteine or peptides having this residue in N terminal position (10). Msc-CI (111) has been used for the acylation of the &-aminofunction in the copper chelate of lysine. Stability of Msc-deriuatices The stability of Msc-protected amino compounds was investigated under the conditions required for removal of other protective groups. Samples of a solution of N"-tert-butyloxycarbonyl N"-methylsulfonylethyloxycarbonyl-L iysine (Boc-Lys(Msc)-OH) in 90% trifluoroacetic acid, kept at room temperature during timeintervals from 5 min to 2 days, were evaporated and analyzed by thin layer chromatography. In all cases, only one spot, coinciding with that
-
-
a
The symbol Aca is used to designate &-aminocaproic acid.
85 126 155 113 171 156 105-110 150 130 128
Acid Acid DCA salt Acid DCA salt DCA salt Acid DCA salt Acid Acid
95% 78% 74% 84% 80% 75% 69% 95% 75% 66%
-
171 176 151 150 172 167 94 175 2 17-224
83 180
M.p.
Acid DCA salt oil DCA salt DCA salt DCA salt DCA salt DCA salt DCA salt Acid DCA salt Zwitterion
Isolated as
75% 71% 95% 77% 85% 73% 82% 100% 96% 76% 82% 78%
-
Proce- Yield dure
Msc-AM-OH" A Msc-Ala-OH A Msc-Arg-OH A Msc-Asn-OH A MSC-ASP-OH A Msc-Gln-OH A MSC-Glu-OH A Msc-Glu(OBut)-OH B M~c-Gly-0H A Msc-Ile-OH A Msc-Leu-OH B H-Lys(Msc)-OH see text B~c-LYs( Msc)-OH B Z-Lys(MsC)-OH B Msc-Met-OH A B Msc-Phe-OH Msc-Ro-OH A Msc-!%-OH A Msc-Thr-OH A MSC-TWOH B B Msc-Tyr(Bu')-OH Msc-Val-OH A
Mcs-derivative
1% in water
_
~
45.44 50.22 52.47 49.52 56.48 52.27 35.68 60.42 52.70 40.44
45.2 50.2 52.3 49.5 56.8 52.7 35.5 60.5 52.9 40.6
51.3 58.6 52.4 60.0 56.5 53.0 42.6 57.1 40.5
51.81 59.50 52.80 60.06 56.16 53.18 42.69 57.1 1 40.53
-
42.9 54.2
-
Analysis N
_
7.12 6.09 8.39 5.43 8.58 8.31 5.61 7.89 6.50 6.41
8.05 9.20 8.23 9.32 8.67 8.43 6.81 9.15 6.80
6.81 8.62
_
7.1 6.1 8.3 5.4 8.6 8.3 5.5 7.7 6.5 6.5
7.07 6.51 5.83 4.44 6.27 6.42 5.20 7.83 3.60 5.24
9.06 6.51 8.80 6.37 5.25 6.89 4.98 6.06 9.45
7.9 9.2 8.2 9.4 8.7 8.4 6.8 9.2 6.5
-
4.98 6.66
7.0 8.7
7.0 6.4 5.8 4.4 6.3 6.3 5.0 7.8 3.6 5.1
8.8 6.2 8.6 6.3 5.3 6.8 5.0 6.0 9.4
_
5.0 6.5
Found Calc. Found Calc. Found
42.49 54.26
Calc.
H
1% in 1N HCI 2% in EtOAc 1% in AcOH.
_
+4.3 +4.2; -7.Zb -2.6 -29.6 -8.7 + 1.6 -5.4 +4.0; +10.3' -0.07; -2.2'
+ 3.9 + 2.5 +7.8 + 3.0 -1.1 + 8.0 + 5.8 -6.0; -9.6b + 13.5' + 3.6d
-
c = 1% in MeOH
C
TABLE 1 Synthetic, physical and analytic data of Msc-amino acids
8.09 7.45 13.34 10.17 7.18 7.34 11.91 5.97 8.28 12.00
11.40 7.62 6.92 4.96 6.71 4.86 6.00 7.89 11.40 6.93 10.82
Calc.
S
8.2 7.3 13.2 10.2 7.3 7.1 11.9 6.0 8.3 12.0
6.9 5.0 6.7 4.7 6.0 7.7 11.1 6.8 10.8
-
11.4 7.8
Found
G. I. TESSER AND 1. C. BALVERT-GEERS
of H-Lys(Msc)-OH, could be detected with ninhydrin as well as with chlorineitolidine. Even after 2 weeks, no trace of free iysine could be found in the solution, demonstrating that the Mscamino group is completely resistant towards 90% TFA [cf. (1 I)]. Very high acid-stability was also observed during cleavage of the benzyloxycarbonyl group from Z-Lys(Msc)-OH in aqueous 12 N hydrochloric acid for 1 h at 40". The residue obtained after evaporation of the solvent over moist sodium hydroxide pellets was dissolved in water and subjected to thin layer chromatography. It appeared that the starting compound had completely disappeared, and that only about 2% free lysine was present in the product, H-Lys(Msc)-OH. Cleavage of the Z-group from ZLys(Msc)-OH by hydrogenation in acetic acid with palladiumlcharcoal (10%) as the catalyst also proceeded with complete preservation of the Msc-group, giving quantitatively H-Lys (Msc)-OH. Alkaline reagents decompose Msc-amino compounds. On dissolution of Msc-Phe-OH in piperidine, a precipitate gradually forms. Thin layer chromatography of the residue, obtained by evaporation of the base after 6 h, revealed that extensive decomposition had occurred and phenylalanine had been formed. In the procedure of Carpino (4) L-phenylalanine was set free in 30 min. The sensitivity towards strong bases is very high. When a solution of Msc-Phe-OH in I N NaOH was immediately neutralized with hydrochloric acid, L-phenylalanine appeared to be the sole amino compound left in the solution. Deprotection of Msc-amino compounds The most suited reagent for the complete and rapid removal of Msc-groups from amino acid or peptide derivatives was found to be the mixture dioxan-methanol-4N NaOH (7.5: 2.25: 0.25) being 0.1N in base (OH- and OCH,-). From a rather large number of experiments the following results may be mentioned. From the pentapeptide derivative, Msc-Val-Lys(Boc)-Val-TyrPro-OBu', the highly selective cleavage of the Msc-group was complete within a few seconds; no by-products could be detected in the reaction mixture. The same result was obtained with MscGlu(0Bu')-OH. tert-Butyl esters of dipeptides
298
containing the Msc-group (e.g., Msc-Gly-LeuOBu') show, however, simultaneous hydrolysis of the ester function during Msc-cleavage. The hydrolysis is much more pronounced with dipeptide esters having a C-terminal glycine residue (Msc-Leu-Gly-OBu', Msc-Ala-Gly-OBu', MscTrp-Gly-OBu'). However, in neither of these cases was formation of a diketopiperazine observed. Cleavage of the Msc-group from the tetrapeptide amide derivative, Msc-Val-Gly-AlaPro-NH,, occurred again without any lysis of the amide bond. An interesting observation was made in the removal of the Msc-group from Msc-Phe-ArgTrp-Gly-OMe. Although the expected product, H-Phe-Arg-Trp-Gly-OH is insoluble in alkali, no precipitate was formed on treatment with the basic cleavage reagent. It arose only after acidification, followed by removal of carbon dioxide and addition of a base. The remainder of the Msc-group on cleavage from Msc-amines with the dioxan-methanolsodium hydroxide mixture is always methylsulfonylethyl methyl ether [cf. (1211. In some experiments the ether was isolated from the reaction mixture and identified by mass spectro=oPY. The results of the cleavage experiments may be explained by the reaction scheme, given in Fig. 2. The elimination of the protecting group is ascribed to the EIcB-mechanism(13), resulting in the loss of methylsulfonylethylene and leaving a negatively charged, soluble carbamate which is stable under the reaction conditions. It is converted into the amino hydrochloride by acidification and elimination of carbon dioxide. The free amine then precipitates on further addition of a base. Application of the Msc-group in peptide synthesis The use of the new protecting group is exemplified by the synthesis of H-Phe-Arg-Trp-Gly-OH (Fig. 3). The tetrapeptide constitutes an intermediate in the synthesis of ACTH (14). Application of the Msc-group is attractive in this case, because removal of other current protective groups (Z, Boc) from tryptophyl peptides always leads to traces of colored by-products as a consequence of oxidative deterioration of the indole nucleus in acidic medium. Moreover the occurrence of carbonium ions in strongly acidic media may
-
THE METHYLSULFONYLETHYLOXYCARBONYL GROUP
Dioxan/MeOH CH~SOzCH~CHzOCO-NH-R
CHJSOZCH- CHZOCONH-R
I
B-
I
CH3S02CH = CH2 + -0OFNH-R (soluble)
I
CHsSOZCH2CHa0CH3
I H+
HOOCNH-R
FIGURE 2 Removal of Msc-groups from Msc-aminocompounds.
+
Msc-Phe-OH
H O W N O 2
C/
/
Msc-Phe-ONp +- H-Arg-OH
Msc-Phe-Arg-OH (VI11)
i
Z-Tr p-G l y-0Me
H-Trp-Gly-OMe.HC1
(1x1
DCC/HOBt 100%
Msc-Phe-Arg-Trp-Gly-0Me.HCI
1
(X) Deprotection go:/,
H-Phe-Arg-Trp-Gly-OH
(XI)
FIGURE 3 Synthesis of a tetrapeptide using the Msc-group for the N-terminal protection.
299
0.I. TESSER AND 1. C. BALVERT-GEERS
lead to side-products by alkylation of the indole group. The tetrapeptide was prepared by fragment condensation because removal of the Msc-group at an earlier stage is precluded by the hydrolysis of the C-terminal ester function. The requisite acetylating dipeptide derivative Msc-Phe-ArgOH (VIII) was readily obtained from MscPhe-ONp (VI) and arginine. The compound crystallized from water. The residue after evaporation of the mother liquor was hydrolyzed with 6 N hydrochloric acid and on cooling deposited optically pure Msc-L-Phe-OH, thus excluding any racemization up to this stage. Condensation of VIlI and tryptophylglycine methyl ester hydrochloride (IX) using dicyclohexylcarbodiimide and N-hydroxybenzotriazole yielded quantitatively the Msc-tetrapeptide ester mono-hydrochloride X as a water soluble compound. Enzymatic hydrolysis of the crude compound with trypsin gave a digest containing VIII, IX and the diketopiperazine from IX, but no trace of X. The end-product XI was obtained in 90% yield by treatment of X with the alkaline reagent specified in the previous section. The reaction time for the removal of the Msc function was less than 5 sec. DISCUSSION
The characteristic chemical properties of Mscamino compounds provide several interesting applications in peptide synthesis as well as in other fields of synthetic chemistry. The excellent acid stability of Msc-derivatives as in N'-protected-W-methylsulfonylethyloxycarbonyllysines (e.g., Boc-Lys(Msc)-OH or ZLys(Msc)-OH) has been explored during the synthesis of a-MSH analogues (15). The high polarity of the Msc-group results in an enhanced solubility of protected peptides in polar solvents, which facilitates purification and processing of such derivatives in polar or even aqueous media. The introduction of Msc-groups into insulin has already been carried out and led to products of increased solubility, as compared with other protective groups (16). The group can be removed in the homogeneous phase, giving a volatile and highly soluble ether in the presence of methanol. These properties meet the criteria of a group
300
that can be applied to the semi synthesis of larger, protein resembling compounds; this is now under investigation. From the high acid stability it may be concluded, that the group will serve well in solid phase peptide synthesis (cf. 11). In combination with the 2-hydroxyethylsulfonylmethyl substituted styrene-divinylbenzene copolymer (5,6,7), unmasked lysyl peptides can be obtained in one operation. In other fields, in which amines or peptides have to be attached uniformly to large carrier molecules (e.g., gels in affinity chromatography or in the preparation of antigenic compounds) the Msc-group is being investigated. Apart from protein chemistry, protection of amino groups with the Msc-function has been applied to acid-sensitive but base-stable compounds such as amino acetals. The easily crystallizable Msc-amino acetals undergo acid catalyzed transacetalizations in alcoholic solution, yielding new amino acetals on deprotection (17). EXPERIMENTAL PROCEDURES
2-Methylsulfonylethanol (I).92.1 g (1 mole) of 2 hydroxyethyl-methyl sulfide and 1 0 0 ml of water were placed in a three-necked roundbottomed flask equipped with a stirrer, thermometer, and a dropping funnel. A solution of 1 g of sodium tungstate dihydrate in 50 ml of water was slowly added with stirring. Then, 100 ml (110 g, 1 mole) of aqueous hydrogen peroxide containing 9.05 mole per kg (by titration) was added dropwise to the well stirred clear solution at a rate which maintained the ensuing exothermic reaction at about 60". A second 100 ml portion was added in the same way but the reaction temperature was now allowed to rise to about 80".After completion of the addition, the reaction mixture was left for 1 h at ambient temperature, then heated again to 50" and treated with 250 mg of palladium on barium sulfate to decompose the remaining hydrogen peroxide. After the evolution of oxygen had ceased, the solution was filtered to remove the palladium catalyst. For removal of sodium tungstate the filtrate was passed through two columns containing 20 ml of Dowex 1 x 2 (OH cycle; 200400 mesh) and 20 ml of Dowex 50 x 2 (H-cycle, 200400 mesh), respectively. The neutral filtrate was concentrated in uacuo. After two evaporations with ethanol and one with isopropyl alcohol a
THE METHYLSULFONYLETHYLOXYCARBONYL GROUP
colorless oil remained that solidified overnight in the refrigerator or directly upon seeding. Yield 125 g (quantitative). The product cannot be distilled without decomposition. Even through a "short-way" distillation in high vacuo the distillate contained methylsulfonylethylene and water (NMR in hexadeutero acetone). The compound can be crystallized, however, from isopropyl alcohol (m.p. 29"). The 3 3 dinitrobenzoate was purified by recrystallization from acetic acid (m.p. 139"). Anal. calcd. for CIOHIONIOIS(318.26): C, 37.74; H, 3.17; N, 8.80; S, 10.07. Found: C, 37.7; H, 3.1; N, 8.7; S, 10.1. The alcohol is very soluble in water, methanol, ethanol, tetrahydrofuran and pyridine. Intermediate solubility is found in isopropyl alcohol and ethyl acetate. Ether, methylene chloride chloroform and benzene do not dissolve the cort.lpound.
Afier about 2 h the outlet valve was connected to a water aspirator and the solution was evaporated to dryness. A pale yellow-colored oil remained, which was dissolved in tetrahydrofuran (15 ml) and cooled in ice. The product crystallized upon addition of 25 ml of dry ethyl ether giving 14.9 g (80%)of white, nearly odorless, and analytically pure crystals. M.p. 4949.5". Anal. calcd. for C4HX104S (186.62): C, 25.97; H, 3.74; S, 17.18; C1, 19.00. Found: C, 25.8; H, 3.8; S, 17.3; CI, 19.1. The chloroformate dissolves quickly in tetrahydrofuran, dioxan and acetone, but has limited solubility in ethyl acetate and methylene chloride, and is insoluble in ether, toluene and petroleum ether. On exposure to moist air, I11 is converted into methylsulfonylethyl carbonate melting at 116-1 17" (from methanol). Anal. calcd. for C7HI4SZO7:C, 30.65; H, 5.14; S, 23.37. Found: C, 30.8; H, 5.1; S, 23.3.
Methylsulfonylethyl succinimido carbonate (MscMethylsulfonylethylp-nitrophenyl carbonate (Msc- ONSu W a ) .To a solution of 5.75 g (50 mmoles) ONp, 11).80.5 g of I (648 mmoles)were dissolved of N-hydroxysuccinimide in 50 ml of dry, freshly in absolute pyridine (200 ml). The solution was distilled acetonitrile, 7 ml (50 mmoles) of tricooled at 0" and 118.9 g (590 mmoles) of p - ethylamine were added, and the mixture was nitrophenyl chloroformate were added with stirr- cooled to 0". A solution of 9.35 g (50 mmoles) ing. The mixture was left for 5 h a t room tem- of I11 in 50 ml of the same solvent was added perature and then concentrated in vacuo. The dropwise with stirring. A precipitate (Et,N.HCI) thick syrup was poured into 1 liter of 1.5 N hydro- that gradually formed, was filtered after about chloric acid. The product, which crystallized 15 min. The filtrate was evaporated and the immediately, was collected by filtration and was residue was taken up in warm isopropyl alcohol washed with isopropyl alcohol (4 x 50 ml) and (about 40"). Filtration gave 11.4 g (86%) of a with diisopropyl ether (3 x 50 ml). Yield 128 g white solid m.p. 112". The compound was crystal(75%). An analytical sample was obtained after lized from acetone by addition of an equal volume dissolution of the carbonate in hot ethyl acetate of diisopropyl ether or from acetonitrile. M.p. (2.5 ml per g) and dilution of the solution with .I 14". Anal. calcd. for C8Hl1NO7S (265.24): half its volume of methanol. M.p. 102" NMR C, 36.22; H, 4.18; N, 5.28; S, 12.09. Found: (hexadeutero acetone) and IR-spectra were in C, 36.3; H, 4.1; N, 5.3; S, 12.0. accordance with expected structure. Anal. calcd. for CloHllNO,S (289.26): C, 41.50; H, 3.86; N, Methylsulfonylethyl phthalimido carbonate (Msc4.84; S, 11.08. Found: C, 41.5; H, 3.9; N, ONPht, ZVb). A solution of 2.44 g (13.1 mmoles) 4.8; S, 11.1. of 111 in 5 ml of acetone was added dropwise to a cold solution of 2.14 g (13.1 mmoles) of NMethyIsulfonyIethyIoxycarbonyIchloride (Msc-Cl hydroxy-phthalimide and 1.83 ml (13.1 mmoles) ZIf). 12.4 g (100 mmoles) of I were dissolved in of triethylamine in 10 rnl of acetone. The addition 25 ml of dry tetrahydrofuran, and the solution was terminated as soon as the red color of the was cooled at -15". Liquid phosgene (12 ml) phthalimidoxy anion had disappeared. Water was quickly added. The flask was closed with a was then added and the precipitate was colground joint bearing an open glass valve and the lected by filtration. The product, m.p. 160"cooling bath was removed. An exothermic reac- 163", weighed 3.84 g (93%). Anal. calcd. for tion set in raising the temperature to about 40". C1zHI1NOTS (313.28): C, 46.01; H, 3.54; N, 301
G. I. TESSER AND I. C. BALVERT-GEERS
4.47; S, 10.23. Found: C, 46.2; H, 3.6; N, 4.4; s, 9.9.
in the pH-stat, at pH 10.5, the Msc-group was slowly lost during the course of 24 h.
Methylsulfonylethyloxycarbonyl uzide (Msc-N3, V ) . 1 g (5.36 mmoles) of 111 was dissolved in a mixture of ethyl acetate (2 ml) and ether (5 ml) by addition of 1 g of sodium azide (1 5.4 mmoles) in 3 ml of water. The two-phase system was stirred for 5 h and then separated. The water layer was extracted with ethyl acetate and discarded. The organic phases were washed with water and dried with sodium sulfate. After evaporation of the solvent, the residue solidified: 845 mg (82%). Crystallization from ethyl acetate (4 ml) and ether (5 ml) gave 705 mg of perfectly colorless crystals, m.p. 41.5".Anal. calcd. for C4H7N304S (193.18): C, 24.87; H, 3.65; N, 21.75; S, 16.60. Found: C, 24.8; H, 3.7; N, 21.8; S, 16.7. IR frequencies (KBr disk): 2180 cm-' (N3), 1730 cm-' (CO), 1275 and 1130 cm-' (SO,).
Methylsulfonylethyloxycarbonylamino acids (general procedures). 20 mmoles of an amino acid were suspended or dissolved in 25 ml of an acetonitrile-water mixture(4: l), and 5.78 g (20mmoles) of solid methylsulfonylethyl p-nitrophenyl carbonate (11) were added. The reaction was started by addition of 2.8 ml (20 mmoles) of triethylamine and the mixture was left for a time interval dependent upon the reactivity of the amino component*. Sometimes a small excess of the acylating agent was used. Non-homogeneous systems were stirred. At the end of the acylation period, the yellow reaction mixture was filtered, if necessary, and concentrated in uacuo. For removal of p-nitrophenol, the residue was dissolved in water, and the solution was acidified to pH 5, either with a strongly acidic ion exchange resin in the H cycle (method A) or with mineral acid (method B). The solution was repeatedly extracted with ether and processed further, depending on the solubility of the product formed.
W-methylsulfonylethyloxycarbonyl-L-lysine.1.83 g (10 mmoles) of lysine monohydrochloride were dissolved in about 50 ml of water containing 1.25 g (5 mmoles) of copper sulfate. After cooling at 4",the pH of the blue solution was increased from 2.2 to 10.8 by careful addition of 5 ml of 4 N sodium hydroxyde (20 mmoles). Maintaining the pH in an autotitrator loaded with 4 N NaOH solution, a solution of 2.24 g (12 mmoles) of methylsulfonylethyloxycarbonyl chloride in 7.5 ml of dry tetrahydrofurane was added dropwise to the mixture. NaOH was rapidly consumed and a precipitate formed. The acylation was complete in about 20 min. The suspension was centrifuged and the blue precipitate was washedwith water and alcohol, and dried with ether. The dry complex, obtained in 85.6% yield, was dissolved in 2 N hydrochloric acid (1 1 ml, 22 mmoles) and diluted with water (30 ml). The resulting green solution was saturated with hydrogensulfide. After about 2 h the suspension was freed from H,S by passage of a current of nitrogen and was then filtered. The acidic solution was filtered through a weakly basic ion exchange resin (Merck 11, OH-cycle) to remove hydrochloric acid. Upon concentration of the neutral eluate, the product crystallized. On recrystallization from water, the compound was obtained as glistening blades. The lysine derivative was stable in 1 N hydrochloric acid at room temperature; 302
Method A . Amino acid derivatives showing a high solubility in cold water, were filtered through a column, containing an excess of a strongly acidic ion exchange resin (Dowex 50 x 2, 2OCUOO mesh, H-cycle). The resin was washed with water and the effluent was evaporated to give the pure amino acid derivative. In some cases, the amino acid derivatives crystallized spontaneously and could be recrystallized from water. If crystallization did not occur, the product was converted in a dicyclohexylammonium salt. Method B. Msc-derivatives showing a limited solubility in cold water, were liberated by addition of potassium hydrogen sulfate solution (2N). With Phe, Leu and Val, spontaneous crystalliza-
* The reaction rate depends strongly on the solubility of the amino component. Boc-Lys, Aca and Z-Lys are completely converted within 15 min. With Ala, Asp, Glu, Glu(OBu'), Gly, Pro, Ser, Thr, Tyr(Bu') and Trp the reaction time was about 1 h. Arg, Ile, Leu, Met, Phe and Val required at least 3 h. With Asn and Gln, aqueous DMF was used as the solvent and tetramethylguanidine as the base. Tyr did not react satisfactorily.
THE METHYLSULFONYLETHYLOXYCARBONYLGROUP
tion occurred. In other cases an oil precipitated and the product was extracted into ethyl acetate, after addition of sodium chloride. Boc-Lys(Msc)O H and Z-Lys( Msc)-OH crystallized spontaneously after removal of the solvent. Msc-Trp-OH was isolated as a dicyclohexyl ammonium salt. Deprotection of Msc-protected amines. Optimal conditions for the removal of Msc-groups from an amino compound were carefully determined for solvent composition and reaction time. (a) Solvent composition. The methylsulfonylethyloxycarbonyl group is quickly lost in a dioxanmethanol mixture containing sufficient aqueous sodium hydroxide to give a 0.1 or 0.2 N solution. The amount of water is critical and should be limited to concentrations up to 5 % for maximum reaction rate. Methanol acts as a scavenger for methylsulfonylethylene and the concentration of this constituent appeared to influence the reaction rate profoundly. The best ratio being dioxanmethanoL(2 or 4 N ) sodium hydroxide = 14:5:1, corresponding to a final base (CH,O- and OH-) concentration of 0.1 or 0.2 N. (b) Time dependency. Dioxan and methanol were mixed in the proportion 14:5 (vlv). Part of this mixture was used as solvent for the protected amino compound ; the solution being transferred into a series of small, capped polyethylene tubes. To the remainder, 1 volume of NaOH(2 or 4 N) was added. With a microsyringe, 2 equivalents of the base were added as rapidly as possible, the vials were closed and agitated on a vortex mixer. After time intervals decreasing from 300 to 5 sec the reaction mixtures were neutralized by addition of an acid (acetic or mineral acid). The neutral solutions were diluted as necessary and spotted onto thin layer plates. After development it was invariably found that with the solvent of the specified composition Msc-groups were quantitatively lost within 5 sec. Methylsulfonylethyloxycarbonyl-L-phenylalanine p-nitrophenyl ester ( VZ).Msc-Phe-OH, crystallized from water as a monohydrate (m.p. 759, was dried over phosphorus pentoxide, giving the anhydrous acid with m.p. 113". This product (2.65 g, 8.41 mmoles) and 1.40 g of p-nitrophenol (10 mmoles) was dissolved in 20 ml of acetonitrile. The mixture was cooled to 0" and 1.82 g (8.83 mmoles) of solid dicyclohexylcarbodiimide
were added. After 16 h at 0", the mixture was filtered and the residue was washed with acetonitrile*. The filtrate was evaporated in vacuo and the crystalline residue was purified by dissolution in warm acetic acid (10 ml) and addition of diisopropyl ether (20 ml), after cooling. The pale yellow crystals were dried over solid moist sodium hydroxide. Yield 3.228 (88%), m.p. 140", [a]22 = -20.1" (c = 1.03 in acetonitrile). Anal.: calcd. for ClsH2,N20eS (436.44): C, 52.29; H, 4.62; N, 6.42; S, 7.35. Found: C, 52.1; H, 4.4; N, 6.5; S, 7.3. Methylsurfonylethyloxycarbonyl-L -phenylalanyl L-arginine mono-hydrate (VZZZ). To a solution of 2.53 g (5.8 mmoles) of VI in a mixture of 20 ml of acetonitrile and 4 ml of water, 0.912 g (5.22 mmoles) of L-arginine (free base) were added. The suspension was stirred at room temperature and became homogeneous in about 30 min. The reaction mixture was concentrated in vacuo. On dilution with water, 0.8 g of unaltered VI separated. The aqueous phase was acidified to pH = 5 with Dowex 50 x 2 (H-cycle) and was extracted with ether to remove p-nitrophenol. On evaporation of the water layer a glassy residue was left, from which, on heating with ethanol, 1.65 g (64.6%) of a white solid was obtained. The compound was recrystallized from water. M.p. 181-184", [ a ] g = -2.3" (c = 1.02 in water), = 8.6" (c = 1.01 in 1N HCI). (489.55): Anal. : calcd. for C19H29N507S.H10 C, 46.62; H, 6.38; N, 14.31; S, 6.55. Found: C,46.7;H,6.1;N, 14.4;S,6.7. Themotherliquor of the analytical sample was evaporated and the residue was boiled for 3 h with 6N hydrochloric acid. On cooling, optically pure Msc-Phe-OH precipitated.
-
Methy lsulfbnylethyloxycarbonyl- L -phenylalanyl L-arginyl-L-tryptophyl-glycinemethyl ester hydrochloride (X).The former zwitterionic dipeptide derivative VIn (2.44 g = 5 mmoles) and 1.60 g of H-Trp-Gly-OMe.HC1 were dissolved in 20 ml of pure dimethylformamide, and 0.81 g (6 mmoles) of 1-hydroxybenzotriazole were added. The solution was cooled to 0" and treated with
* The product sometimes crystallized from thereaction mixture. 303
G. I. TESSER AND I. C. BALVERT-GEERS
finely divided, solid dicyclohexylcarbodiimide (1.03 g, 5 mmoles). The clear solution was kept at 0" for 1 h, left overnight at room temperature, and then cooled again for about 4 h at 0" to complete the precipitation of dicyclohexylurea which was removed by filtration (1.15 g). The filtrate was slowly added into ether to give a precipitate which was triturated with ethyl acetate. The crude product (4.1 g) was purified for analysis by countercurrent distribution in the system EtOAc-benzene-methanol-buffer ( = 10: 1 :5 :7) ; buffer: 19.25 g of ammonium acetate and 28.5 mol of acetic acid made up to 1000 ml with water. After 187 transfers, a single peak was obtained (K = 0.52, rmax= 65), comprising 3.2 g (81%) of the acetate. The compound crystallized from acetic acid upon addition of ether, yielding a hygroscopic product containing an additional equivalent of acetic acid (by titration). TS: RF = 0.33 (nBuOH-AcOH-water = 10:1 :3), = 0.57 (2-propanol-HCOOH-water = 20:1:5); [a]V = -15.8" (c = 1 , MeOH); m.p. 136-1 38.5". Anal. : calcd. for C33H44N80,S.2AcOH. +H,O (857-92): C, 51.80; H, 5.76; N, 13.06. Found: C, 51.8; H, 6.1; N, 13.1. The crude compound was soluble in water. A 1 % solution of the tetrapeptide ester in 0.05 M tris-buffer at pH = 8 was readily and completely digested by trypsin (-5 min). Upon standing for 2 h, the amino component set free by the enzyme underwent cyclization to the corresponding diketopiperazine. The same cyclization was found with authentic IX in tris-buffer. Chromatography, (TS): RF = 0.13 (tris); RF = 0.20 (VIII); R F = 0.33 (X); R F = 0.44 (IX) and RF = 0.64 (diketopiperazine from IX), in BuOH-AcOHwater = 10:1:3. MethylsulfonylethyloxycarbonyI-L-IeucyIgIycine tert-butyl ester. Msc-Leu-OH crystallized from water as a hydrate (m.p. 45"). The air-dried compound lost water and became an oil on further drying. The oil (1.1 g, 3.8 mmoles) was dissolved in dry ethyl acetate, evaporated twice to remove residual water and after addition of 0.53 ml (3.8 mmoles) of triethylamine was converted to the mixed anhydride by addition of 0.54 g (4 mmoles) of isobutylchloroformate at -5'C. After 15 min, 0.52 g (4 mmoles) of HGly-OBu' were added. The reaction mixture 304
was kept for 2 h at room temperature, filtered, and the filtrate was washed with water, sodium hydrogencarbonate and potassium hydrogen sulfate solutions. After drying, 1.43 g (95%) of an oil was obtained which refused to crystallize. The oil was purified by chromatography on silica (18) and gave the expected NMR spectrum after dissolution in CD30D. .4n IR spectrum revealed the presence of the sulfonyl group and carbonyl functions [ a ] g = -17.4" (c = 1 , MeOH). Methylsulfonylethyloxycarbonylglycyl-L-leucine tert-butyl ester. The preparation of this compound was carried out in the same way as described for the foregoing isomeric ester (yield 93%). The compound was purified by chromatography (1 8) but resisted crystallization. The NMR spectrum was in accordance with the expected structure; the presence of sulfonyl and carbonyl groups was demonstrated in an IR spectrum. [a]g = - 38.4" (c = 1, MeOH). Methylsuffonylethloxycarbonyl-L-alanyl-glycine tert-butyl ester was prepared in the way indicated. The oily compound, after chromatographic purification, gave [a]% = - 17.8"(c = 1, MeOH). L-Phenylalanyl-L-arginyl-L-tryptophylglycine( XI). Dioxan (dry, peroxide-free) and absolute methanol were mixed in the proportion 14:5 (vlv). Crude X (766 mg, 1 mmol) was dissolved in about 10 ml of this mixture and with vigorous stirring, 1 ml of 4N sodiumhydroxide (4 equiv.) in 10 ml of the same mixture was immediately added. After 45 sec the reaction mixture was acidified to p H < 4 by addition of hydrochloric acid and the solution was concentrated in uacuo. The product was precipitated by careful addition of sodium hydroxide to pH 1 1 . After filtration and drying, 508 mg (90%) of the crude free base were isolated. The monohydrochloride was obtained, after dissolution of the base in a small amount of a water-acetonitrile mixture (1 :1) containing one equivalent of hydrochloric acid, by slow addition of acetonitrile and cooling. Yield: 419 mg (69.7%). Chromatography, TS: RF = 0.20 (nBuOH-AcOH-water = 4: 1 :1); = 0.36 (nBuOHPyr-AcOH-water = 38 :24: 8 :30) ; = 0.48 (2-propanol-HCOOH-water = 20: 1 :5).
T H E METHYLSULFONYLETHYLOXYCARBONYL GROUP
[n]g = -5.8 (c = 1, 0.1N HCI in 95% MeOH). After equilibration with the humidity of the air. Anal.: C28H37N80,C1.2H20(637.14). Calcd. : C, 52.78; H, 6.49; N, 17.58. Found: C, 52.8; H,6.1 ;N, 17.6. After drying for 24 h over P205, C28HS,Na05Cl.$H20 (601.1 1). Calcd.: C, 55.12; H, 6.27; N, 18.37. Found: C, 55.1; H, 6.2; N, 18.3. Amino acid analysis: Arg :Gly :Phe = 0.94 : 1.00:0.99. ACKNOWLEDGMENTS
We are grateful for the kind interest and advice of Prof. Dr. R. Schwyzer (Institut fiir Molekularbiologie und Biophysik, E.T.H. Zurich, Switzerland), in whose laboratory this work was started, and to Prof. Dr. R. J. F. Nivard (Department of Organic Chemistry, University of Nijmegen) for stimulating discussions. The major part of the work was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.). REFERENCES
1. KADER,A. T. & STIRLING, C. J. M. (1964)
J. Chem. SOC.258-266. R. C. 2. HARDY,P. M.. RYDON.H. N. &THOMPSON, (1972) J. Chem. SOC.,Perkin Trans. I, 5-12. R. (1971) Chem. 3. WUNSCH,E. & SPANGENBERG, Ber. 104,2427-2429. L. A. & HAN,G. Y. (1972) J. Org. 4. CARPINO, Chem. 37, 3404-3409. G. I. & ELLENBROEK, B. W. J. (1967) 5. TESSER, in Peptides 1966, Proceedings of the 8th European Peptide Symposium (Beyerman, H. C., Van de Linde, A. and Maassen van den Brink, W., eds.),
p. 124, North Holland Publishing Co., Amsterdam. 6. BUIS,J. TH.(1973) Thesis,Nijmegen, the Netherlands. 7. MARSHALL, G. R. & MERRIFIELD, R. B. (1971) in Biochemical Aspects of Reactions on Solid Supports (Stark, G . R., ed.), pp. 130-131, Academic Press, New Yotk. 8. IUPAC-IUB Commission for Nomenclature (1 972) Biochem. J. 126, 773-780. 9. SCHULTZ,H. S., FREYERMUTH, H. B. & BWC, S. R. (1963) J. Org. Chem. 28, 1140-1142. 10. WOLTERS,E. TH. M. (1973) Thesis, Nijmegen, the Netherlands. 11. SCHNABEL, E., KLOSTERMEYER, H. & BERNDT, H. (1971) Justus Liebigs Ann. Chem. 749. W108. 12. ARBLJZOV, B. A. & BERDNIKOV, E. A. (1966) Dokl. Acad. Nauk SSSR 171, 860, Intern. J. Su&r Chem. (1971) B6 222. 13. BORDWELL,F. G. (1972), Acad. Chem. Res. 5, 374-381. P.,R I ~ LW., & ZUBER,H. 14. RINIKER,B., SIEBER, (1972) Nature New Biol. 235, 114-115. 15. VAN NISPEN,J. W. F. M. (1974) Thesis, Nijmegen, the Netherlands. 16. PERSONAL COMMUNICATION from Dr. R. Geiger (Hoechst AG. Frankfurt). 17. GRIBNAU,T. C. & TESSER,G. I., unpublished results. 18. HUNT,B. J. & RIGBY, W. (1967) Chem. Ind. 18681869.
Address : Dr. G. I. Tesser Department of Organic Chemistry University of Nijmegen Toernooiveld, Nijmegen the Netherlands
305
THE METHYLSULFONYLETHYLOXYCARBONYL G R O U P , A N E W A N D VERSATILE A M I N O PROTECTIVE F U N C T I O N G. I. TESSER and I. C. BALVERT-GEERS
Department of Organic Chemistry, R.C. University, Nijmegen, the Netherlands
Received 7 September 1974
A new amino protecting group, the methylsulfonylethyloxycarbonyl(Msc) group, is described which combines well with other familiar groups (benzyloxycarbonyl, t-butyloxycarbonyl) in peptide syntheses. Its main characteristics are an extreme acid stability, a high base lability and a high polarity which .enhances solubility in polar solvents including water. The group resists catalytic hydrogenolysis but does not poison the catalyst. It has good crystallizing properties. Application in peptide synthesis is exemplified in the synthesis of Msc-Phe-Arg-Trp-Cly-OMe.HC1. Deprotection of the rnasked tetrapeptide was accomplished within 5 sec with a 1.0 N solution of base (OH- and OCH,-). A reaction scheme for the cleavage of the group is suggested.
The application of the principle of 8-elimination in the design of protecting functions is not new. The usefulness in peptide synthesis has already been demonstrated by Kadar & Stirling (1). Rydon (2), Wunsch (3), and Carpino (4). From our department, an application in solid phase synthesis was announced in a brief note ( 5 ) and worked out by Buis (6). Meanwhile, the same concept has been examined by Merrifield (7). In this paper, we report on general procedures for the introduction of the methylsulfonylethyloxycarbonyl group (Msc*) as an amino protective function, removal of which is also based on P-elimination. The group was developed in order to arrive at a protective group which enhances solubility in polar solvents of intermediates and products in peptide syntheses in solution. The Msc-group combines several interesting and remarkable properties which distinguish it from other protecting groups. For that reason the group may be a n important, new expedient in the synthesis of peptides.
* Abbreviation suggested by Prof. Dr. R. Schwyzer, in accordance with the general convention of the IUPAC-IUB commission for nomenclature (8).
METHODS AND RESULTS
Introduction of the Msc-group in amino compounds The parent compound of the Msc-group, 2-methylsulfonylethanol (I), can readily be obtained from the commerically available 2methylmercaptoethanol by catalytic oxidation with hydrogen peroxide and sodium tungstate (9). The product (I) is a crystalline solid, having limited solubility in organic solvents. It decomposes on distillation, even in high uacuo, by elimination of water, but can be crystallized from isopropyl alcohol. Several suitable reagents (11-V, Fig. 1.) for the preparation of Msc-amino derivatives could be derived from I. Treatment of the alcohol with p-nitrophenyl chloroformate in pyridine, yields the well crystallizing methylsulfonylethyl p-nitrophenyl carbonate (Msc-ONp, 11). An excess of phosgene in tetrahydrofuran solution converts I into the crystalline chloroformate (Msc-CI, 111). Treatment of this compound with hydroxy compounds in the presence of a tertiary amine can be used to obtain mixed carbonates (IVa and b) derived from hydroxy compounds which in themselves do not readily give a chloroformate. Methylsulfonylethyloxy295
G. I. TESSER AND I. C. BALVERT-GEERS
+
CH3SOZCHaCHzOH CICOOCbH4N02-p-
CH~SO~CHZCH~OCOOC~,H~NOZ-~
p-NOnCsHdOH COCIZ
/
I 1 CH~SOZCHZCHzOCOCl -HOY,->
/
I1
/Co-cr
CHJSOZCHZCHZOCOOY
1Va: Y
I11
=
-N
\
CO--CH,
/c o*
\
co
V FIGURE 1 Synthesis of reagents (11-V)for the introduction of an Msc-group.
carbonyl azide (Msc-N,, V) can also be prepared in nearly quantitative yield from 111 and sodium azide. Most of the amino acids react rapidly with Msc-ONp, and this reagent was used for the preparation of all but one of the Msc-derivatives given in.Table 1. To that aim, a suspension or solution of an amino acid in an acetonitrile/ water mixture was stirred for some time with equivalent amounts of the mixed carbonate and triethylamine. The reaction products remained in solution and the Msc-derivatives were separated from p-nitrophenol, after evaporation of acetonitrile, by extraction with ether at pH 5. In some instances (Msc-Phe, Msc-Leu, MscVal) the product crystallized from the water phase on further acidification. Msc-derivatives, having a moderate solubility in water, could be isolated by extraction with ethyl acetate. In all other cases where the Msc-derivatives had a high solubility in water, the aqueous solution containing the product as the triethylammonium salt was filtered through a strongly acidic ionexchange resin and the Msc-derivatives were
296
isolated by evaporation. Some Msc-derivatives crystallized spontaneously; if not, they were converted into dicyclohexylammonium salts which crystallize from iso-propyl alcohol. Msc-azide (V) and especially the mixed carbonates IVa and b are valuable reagents for the introduction of the Msc-group into compounds with a low reactivity, e.g., S-tritylcysteine or peptides having this residue in N terminal position (10). Msc-CI (111) has been used for the acylation of the &-aminofunction in the copper chelate of lysine. Stability of Msc-deriuatices The stability of Msc-protected amino compounds was investigated under the conditions required for removal of other protective groups. Samples of a solution of N"-tert-butyloxycarbonyl N"-methylsulfonylethyloxycarbonyl-L iysine (Boc-Lys(Msc)-OH) in 90% trifluoroacetic acid, kept at room temperature during timeintervals from 5 min to 2 days, were evaporated and analyzed by thin layer chromatography. In all cases, only one spot, coinciding with that
-
-
a
The symbol Aca is used to designate &-aminocaproic acid.
85 126 155 113 171 156 105-110 150 130 128
Acid Acid DCA salt Acid DCA salt DCA salt Acid DCA salt Acid Acid
95% 78% 74% 84% 80% 75% 69% 95% 75% 66%
-
171 176 151 150 172 167 94 175 2 17-224
83 180
M.p.
Acid DCA salt oil DCA salt DCA salt DCA salt DCA salt DCA salt DCA salt Acid DCA salt Zwitterion
Isolated as
75% 71% 95% 77% 85% 73% 82% 100% 96% 76% 82% 78%
-
Proce- Yield dure
Msc-AM-OH" A Msc-Ala-OH A Msc-Arg-OH A Msc-Asn-OH A MSC-ASP-OH A Msc-Gln-OH A MSC-Glu-OH A Msc-Glu(OBut)-OH B M~c-Gly-0H A Msc-Ile-OH A Msc-Leu-OH B H-Lys(Msc)-OH see text B~c-LYs( Msc)-OH B Z-Lys(MsC)-OH B Msc-Met-OH A B Msc-Phe-OH Msc-Ro-OH A Msc-!%-OH A Msc-Thr-OH A MSC-TWOH B B Msc-Tyr(Bu')-OH Msc-Val-OH A
Mcs-derivative
1% in water
_
~
45.44 50.22 52.47 49.52 56.48 52.27 35.68 60.42 52.70 40.44
45.2 50.2 52.3 49.5 56.8 52.7 35.5 60.5 52.9 40.6
51.3 58.6 52.4 60.0 56.5 53.0 42.6 57.1 40.5
51.81 59.50 52.80 60.06 56.16 53.18 42.69 57.1 1 40.53
-
42.9 54.2
-
Analysis N
_
7.12 6.09 8.39 5.43 8.58 8.31 5.61 7.89 6.50 6.41
8.05 9.20 8.23 9.32 8.67 8.43 6.81 9.15 6.80
6.81 8.62
_
7.1 6.1 8.3 5.4 8.6 8.3 5.5 7.7 6.5 6.5
7.07 6.51 5.83 4.44 6.27 6.42 5.20 7.83 3.60 5.24
9.06 6.51 8.80 6.37 5.25 6.89 4.98 6.06 9.45
7.9 9.2 8.2 9.4 8.7 8.4 6.8 9.2 6.5
-
4.98 6.66
7.0 8.7
7.0 6.4 5.8 4.4 6.3 6.3 5.0 7.8 3.6 5.1
8.8 6.2 8.6 6.3 5.3 6.8 5.0 6.0 9.4
_
5.0 6.5
Found Calc. Found Calc. Found
42.49 54.26
Calc.
H
1% in 1N HCI 2% in EtOAc 1% in AcOH.
_
+4.3 +4.2; -7.Zb -2.6 -29.6 -8.7 + 1.6 -5.4 +4.0; +10.3' -0.07; -2.2'
+ 3.9 + 2.5 +7.8 + 3.0 -1.1 + 8.0 + 5.8 -6.0; -9.6b + 13.5' + 3.6d
-
c = 1% in MeOH
C
TABLE 1 Synthetic, physical and analytic data of Msc-amino acids
8.09 7.45 13.34 10.17 7.18 7.34 11.91 5.97 8.28 12.00
11.40 7.62 6.92 4.96 6.71 4.86 6.00 7.89 11.40 6.93 10.82
Calc.
S
8.2 7.3 13.2 10.2 7.3 7.1 11.9 6.0 8.3 12.0
6.9 5.0 6.7 4.7 6.0 7.7 11.1 6.8 10.8
-
11.4 7.8
Found
G. I. TESSER AND 1. C. BALVERT-GEERS
of H-Lys(Msc)-OH, could be detected with ninhydrin as well as with chlorineitolidine. Even after 2 weeks, no trace of free iysine could be found in the solution, demonstrating that the Mscamino group is completely resistant towards 90% TFA [cf. (1 I)]. Very high acid-stability was also observed during cleavage of the benzyloxycarbonyl group from Z-Lys(Msc)-OH in aqueous 12 N hydrochloric acid for 1 h at 40". The residue obtained after evaporation of the solvent over moist sodium hydroxide pellets was dissolved in water and subjected to thin layer chromatography. It appeared that the starting compound had completely disappeared, and that only about 2% free lysine was present in the product, H-Lys(Msc)-OH. Cleavage of the Z-group from ZLys(Msc)-OH by hydrogenation in acetic acid with palladiumlcharcoal (10%) as the catalyst also proceeded with complete preservation of the Msc-group, giving quantitatively H-Lys (Msc)-OH. Alkaline reagents decompose Msc-amino compounds. On dissolution of Msc-Phe-OH in piperidine, a precipitate gradually forms. Thin layer chromatography of the residue, obtained by evaporation of the base after 6 h, revealed that extensive decomposition had occurred and phenylalanine had been formed. In the procedure of Carpino (4) L-phenylalanine was set free in 30 min. The sensitivity towards strong bases is very high. When a solution of Msc-Phe-OH in I N NaOH was immediately neutralized with hydrochloric acid, L-phenylalanine appeared to be the sole amino compound left in the solution. Deprotection of Msc-amino compounds The most suited reagent for the complete and rapid removal of Msc-groups from amino acid or peptide derivatives was found to be the mixture dioxan-methanol-4N NaOH (7.5: 2.25: 0.25) being 0.1N in base (OH- and OCH,-). From a rather large number of experiments the following results may be mentioned. From the pentapeptide derivative, Msc-Val-Lys(Boc)-Val-TyrPro-OBu', the highly selective cleavage of the Msc-group was complete within a few seconds; no by-products could be detected in the reaction mixture. The same result was obtained with MscGlu(0Bu')-OH. tert-Butyl esters of dipeptides
298
containing the Msc-group (e.g., Msc-Gly-LeuOBu') show, however, simultaneous hydrolysis of the ester function during Msc-cleavage. The hydrolysis is much more pronounced with dipeptide esters having a C-terminal glycine residue (Msc-Leu-Gly-OBu', Msc-Ala-Gly-OBu', MscTrp-Gly-OBu'). However, in neither of these cases was formation of a diketopiperazine observed. Cleavage of the Msc-group from the tetrapeptide amide derivative, Msc-Val-Gly-AlaPro-NH,, occurred again without any lysis of the amide bond. An interesting observation was made in the removal of the Msc-group from Msc-Phe-ArgTrp-Gly-OMe. Although the expected product, H-Phe-Arg-Trp-Gly-OH is insoluble in alkali, no precipitate was formed on treatment with the basic cleavage reagent. It arose only after acidification, followed by removal of carbon dioxide and addition of a base. The remainder of the Msc-group on cleavage from Msc-amines with the dioxan-methanolsodium hydroxide mixture is always methylsulfonylethyl methyl ether [cf. (1211. In some experiments the ether was isolated from the reaction mixture and identified by mass spectro=oPY. The results of the cleavage experiments may be explained by the reaction scheme, given in Fig. 2. The elimination of the protecting group is ascribed to the EIcB-mechanism(13), resulting in the loss of methylsulfonylethylene and leaving a negatively charged, soluble carbamate which is stable under the reaction conditions. It is converted into the amino hydrochloride by acidification and elimination of carbon dioxide. The free amine then precipitates on further addition of a base. Application of the Msc-group in peptide synthesis The use of the new protecting group is exemplified by the synthesis of H-Phe-Arg-Trp-Gly-OH (Fig. 3). The tetrapeptide constitutes an intermediate in the synthesis of ACTH (14). Application of the Msc-group is attractive in this case, because removal of other current protective groups (Z, Boc) from tryptophyl peptides always leads to traces of colored by-products as a consequence of oxidative deterioration of the indole nucleus in acidic medium. Moreover the occurrence of carbonium ions in strongly acidic media may
-
THE METHYLSULFONYLETHYLOXYCARBONYL GROUP
Dioxan/MeOH CH~SOzCH~CHzOCO-NH-R
CHJSOZCH- CHZOCONH-R
I
B-
I
CH3S02CH = CH2 + -0OFNH-R (soluble)
I
CHsSOZCH2CHa0CH3
I H+
HOOCNH-R
FIGURE 2 Removal of Msc-groups from Msc-aminocompounds.
+
Msc-Phe-OH
H O W N O 2
C/
/
Msc-Phe-ONp +- H-Arg-OH
Msc-Phe-Arg-OH (VI11)
i
Z-Tr p-G l y-0Me
H-Trp-Gly-OMe.HC1
(1x1
DCC/HOBt 100%
Msc-Phe-Arg-Trp-Gly-0Me.HCI
1
(X) Deprotection go:/,
H-Phe-Arg-Trp-Gly-OH
(XI)
FIGURE 3 Synthesis of a tetrapeptide using the Msc-group for the N-terminal protection.
299
0.I. TESSER AND 1. C. BALVERT-GEERS
lead to side-products by alkylation of the indole group. The tetrapeptide was prepared by fragment condensation because removal of the Msc-group at an earlier stage is precluded by the hydrolysis of the C-terminal ester function. The requisite acetylating dipeptide derivative Msc-Phe-ArgOH (VIII) was readily obtained from MscPhe-ONp (VI) and arginine. The compound crystallized from water. The residue after evaporation of the mother liquor was hydrolyzed with 6 N hydrochloric acid and on cooling deposited optically pure Msc-L-Phe-OH, thus excluding any racemization up to this stage. Condensation of VIlI and tryptophylglycine methyl ester hydrochloride (IX) using dicyclohexylcarbodiimide and N-hydroxybenzotriazole yielded quantitatively the Msc-tetrapeptide ester mono-hydrochloride X as a water soluble compound. Enzymatic hydrolysis of the crude compound with trypsin gave a digest containing VIII, IX and the diketopiperazine from IX, but no trace of X. The end-product XI was obtained in 90% yield by treatment of X with the alkaline reagent specified in the previous section. The reaction time for the removal of the Msc function was less than 5 sec. DISCUSSION
The characteristic chemical properties of Mscamino compounds provide several interesting applications in peptide synthesis as well as in other fields of synthetic chemistry. The excellent acid stability of Msc-derivatives as in N'-protected-W-methylsulfonylethyloxycarbonyllysines (e.g., Boc-Lys(Msc)-OH or ZLys(Msc)-OH) has been explored during the synthesis of a-MSH analogues (15). The high polarity of the Msc-group results in an enhanced solubility of protected peptides in polar solvents, which facilitates purification and processing of such derivatives in polar or even aqueous media. The introduction of Msc-groups into insulin has already been carried out and led to products of increased solubility, as compared with other protective groups (16). The group can be removed in the homogeneous phase, giving a volatile and highly soluble ether in the presence of methanol. These properties meet the criteria of a group
300
that can be applied to the semi synthesis of larger, protein resembling compounds; this is now under investigation. From the high acid stability it may be concluded, that the group will serve well in solid phase peptide synthesis (cf. 11). In combination with the 2-hydroxyethylsulfonylmethyl substituted styrene-divinylbenzene copolymer (5,6,7), unmasked lysyl peptides can be obtained in one operation. In other fields, in which amines or peptides have to be attached uniformly to large carrier molecules (e.g., gels in affinity chromatography or in the preparation of antigenic compounds) the Msc-group is being investigated. Apart from protein chemistry, protection of amino groups with the Msc-function has been applied to acid-sensitive but base-stable compounds such as amino acetals. The easily crystallizable Msc-amino acetals undergo acid catalyzed transacetalizations in alcoholic solution, yielding new amino acetals on deprotection (17). EXPERIMENTAL PROCEDURES
2-Methylsulfonylethanol (I).92.1 g (1 mole) of 2 hydroxyethyl-methyl sulfide and 1 0 0 ml of water were placed in a three-necked roundbottomed flask equipped with a stirrer, thermometer, and a dropping funnel. A solution of 1 g of sodium tungstate dihydrate in 50 ml of water was slowly added with stirring. Then, 100 ml (110 g, 1 mole) of aqueous hydrogen peroxide containing 9.05 mole per kg (by titration) was added dropwise to the well stirred clear solution at a rate which maintained the ensuing exothermic reaction at about 60". A second 100 ml portion was added in the same way but the reaction temperature was now allowed to rise to about 80".After completion of the addition, the reaction mixture was left for 1 h at ambient temperature, then heated again to 50" and treated with 250 mg of palladium on barium sulfate to decompose the remaining hydrogen peroxide. After the evolution of oxygen had ceased, the solution was filtered to remove the palladium catalyst. For removal of sodium tungstate the filtrate was passed through two columns containing 20 ml of Dowex 1 x 2 (OH cycle; 200400 mesh) and 20 ml of Dowex 50 x 2 (H-cycle, 200400 mesh), respectively. The neutral filtrate was concentrated in uacuo. After two evaporations with ethanol and one with isopropyl alcohol a
THE METHYLSULFONYLETHYLOXYCARBONYL GROUP
colorless oil remained that solidified overnight in the refrigerator or directly upon seeding. Yield 125 g (quantitative). The product cannot be distilled without decomposition. Even through a "short-way" distillation in high vacuo the distillate contained methylsulfonylethylene and water (NMR in hexadeutero acetone). The compound can be crystallized, however, from isopropyl alcohol (m.p. 29"). The 3 3 dinitrobenzoate was purified by recrystallization from acetic acid (m.p. 139"). Anal. calcd. for CIOHIONIOIS(318.26): C, 37.74; H, 3.17; N, 8.80; S, 10.07. Found: C, 37.7; H, 3.1; N, 8.7; S, 10.1. The alcohol is very soluble in water, methanol, ethanol, tetrahydrofuran and pyridine. Intermediate solubility is found in isopropyl alcohol and ethyl acetate. Ether, methylene chloride chloroform and benzene do not dissolve the cort.lpound.
Afier about 2 h the outlet valve was connected to a water aspirator and the solution was evaporated to dryness. A pale yellow-colored oil remained, which was dissolved in tetrahydrofuran (15 ml) and cooled in ice. The product crystallized upon addition of 25 ml of dry ethyl ether giving 14.9 g (80%)of white, nearly odorless, and analytically pure crystals. M.p. 4949.5". Anal. calcd. for C4HX104S (186.62): C, 25.97; H, 3.74; S, 17.18; C1, 19.00. Found: C, 25.8; H, 3.8; S, 17.3; CI, 19.1. The chloroformate dissolves quickly in tetrahydrofuran, dioxan and acetone, but has limited solubility in ethyl acetate and methylene chloride, and is insoluble in ether, toluene and petroleum ether. On exposure to moist air, I11 is converted into methylsulfonylethyl carbonate melting at 116-1 17" (from methanol). Anal. calcd. for C7HI4SZO7:C, 30.65; H, 5.14; S, 23.37. Found: C, 30.8; H, 5.1; S, 23.3.
Methylsulfonylethyl succinimido carbonate (MscMethylsulfonylethylp-nitrophenyl carbonate (Msc- ONSu W a ) .To a solution of 5.75 g (50 mmoles) ONp, 11).80.5 g of I (648 mmoles)were dissolved of N-hydroxysuccinimide in 50 ml of dry, freshly in absolute pyridine (200 ml). The solution was distilled acetonitrile, 7 ml (50 mmoles) of tricooled at 0" and 118.9 g (590 mmoles) of p - ethylamine were added, and the mixture was nitrophenyl chloroformate were added with stirr- cooled to 0". A solution of 9.35 g (50 mmoles) ing. The mixture was left for 5 h a t room tem- of I11 in 50 ml of the same solvent was added perature and then concentrated in vacuo. The dropwise with stirring. A precipitate (Et,N.HCI) thick syrup was poured into 1 liter of 1.5 N hydro- that gradually formed, was filtered after about chloric acid. The product, which crystallized 15 min. The filtrate was evaporated and the immediately, was collected by filtration and was residue was taken up in warm isopropyl alcohol washed with isopropyl alcohol (4 x 50 ml) and (about 40"). Filtration gave 11.4 g (86%) of a with diisopropyl ether (3 x 50 ml). Yield 128 g white solid m.p. 112". The compound was crystal(75%). An analytical sample was obtained after lized from acetone by addition of an equal volume dissolution of the carbonate in hot ethyl acetate of diisopropyl ether or from acetonitrile. M.p. (2.5 ml per g) and dilution of the solution with .I 14". Anal. calcd. for C8Hl1NO7S (265.24): half its volume of methanol. M.p. 102" NMR C, 36.22; H, 4.18; N, 5.28; S, 12.09. Found: (hexadeutero acetone) and IR-spectra were in C, 36.3; H, 4.1; N, 5.3; S, 12.0. accordance with expected structure. Anal. calcd. for CloHllNO,S (289.26): C, 41.50; H, 3.86; N, Methylsulfonylethyl phthalimido carbonate (Msc4.84; S, 11.08. Found: C, 41.5; H, 3.9; N, ONPht, ZVb). A solution of 2.44 g (13.1 mmoles) 4.8; S, 11.1. of 111 in 5 ml of acetone was added dropwise to a cold solution of 2.14 g (13.1 mmoles) of NMethyIsulfonyIethyIoxycarbonyIchloride (Msc-Cl hydroxy-phthalimide and 1.83 ml (13.1 mmoles) ZIf). 12.4 g (100 mmoles) of I were dissolved in of triethylamine in 10 rnl of acetone. The addition 25 ml of dry tetrahydrofuran, and the solution was terminated as soon as the red color of the was cooled at -15". Liquid phosgene (12 ml) phthalimidoxy anion had disappeared. Water was quickly added. The flask was closed with a was then added and the precipitate was colground joint bearing an open glass valve and the lected by filtration. The product, m.p. 160"cooling bath was removed. An exothermic reac- 163", weighed 3.84 g (93%). Anal. calcd. for tion set in raising the temperature to about 40". C1zHI1NOTS (313.28): C, 46.01; H, 3.54; N, 301
G. I. TESSER AND I. C. BALVERT-GEERS
4.47; S, 10.23. Found: C, 46.2; H, 3.6; N, 4.4; s, 9.9.
in the pH-stat, at pH 10.5, the Msc-group was slowly lost during the course of 24 h.
Methylsulfonylethyloxycarbonyl uzide (Msc-N3, V ) . 1 g (5.36 mmoles) of 111 was dissolved in a mixture of ethyl acetate (2 ml) and ether (5 ml) by addition of 1 g of sodium azide (1 5.4 mmoles) in 3 ml of water. The two-phase system was stirred for 5 h and then separated. The water layer was extracted with ethyl acetate and discarded. The organic phases were washed with water and dried with sodium sulfate. After evaporation of the solvent, the residue solidified: 845 mg (82%). Crystallization from ethyl acetate (4 ml) and ether (5 ml) gave 705 mg of perfectly colorless crystals, m.p. 41.5".Anal. calcd. for C4H7N304S (193.18): C, 24.87; H, 3.65; N, 21.75; S, 16.60. Found: C, 24.8; H, 3.7; N, 21.8; S, 16.7. IR frequencies (KBr disk): 2180 cm-' (N3), 1730 cm-' (CO), 1275 and 1130 cm-' (SO,).
Methylsulfonylethyloxycarbonylamino acids (general procedures). 20 mmoles of an amino acid were suspended or dissolved in 25 ml of an acetonitrile-water mixture(4: l), and 5.78 g (20mmoles) of solid methylsulfonylethyl p-nitrophenyl carbonate (11) were added. The reaction was started by addition of 2.8 ml (20 mmoles) of triethylamine and the mixture was left for a time interval dependent upon the reactivity of the amino component*. Sometimes a small excess of the acylating agent was used. Non-homogeneous systems were stirred. At the end of the acylation period, the yellow reaction mixture was filtered, if necessary, and concentrated in uacuo. For removal of p-nitrophenol, the residue was dissolved in water, and the solution was acidified to pH 5, either with a strongly acidic ion exchange resin in the H cycle (method A) or with mineral acid (method B). The solution was repeatedly extracted with ether and processed further, depending on the solubility of the product formed.
W-methylsulfonylethyloxycarbonyl-L-lysine.1.83 g (10 mmoles) of lysine monohydrochloride were dissolved in about 50 ml of water containing 1.25 g (5 mmoles) of copper sulfate. After cooling at 4",the pH of the blue solution was increased from 2.2 to 10.8 by careful addition of 5 ml of 4 N sodium hydroxyde (20 mmoles). Maintaining the pH in an autotitrator loaded with 4 N NaOH solution, a solution of 2.24 g (12 mmoles) of methylsulfonylethyloxycarbonyl chloride in 7.5 ml of dry tetrahydrofurane was added dropwise to the mixture. NaOH was rapidly consumed and a precipitate formed. The acylation was complete in about 20 min. The suspension was centrifuged and the blue precipitate was washedwith water and alcohol, and dried with ether. The dry complex, obtained in 85.6% yield, was dissolved in 2 N hydrochloric acid (1 1 ml, 22 mmoles) and diluted with water (30 ml). The resulting green solution was saturated with hydrogensulfide. After about 2 h the suspension was freed from H,S by passage of a current of nitrogen and was then filtered. The acidic solution was filtered through a weakly basic ion exchange resin (Merck 11, OH-cycle) to remove hydrochloric acid. Upon concentration of the neutral eluate, the product crystallized. On recrystallization from water, the compound was obtained as glistening blades. The lysine derivative was stable in 1 N hydrochloric acid at room temperature; 302
Method A . Amino acid derivatives showing a high solubility in cold water, were filtered through a column, containing an excess of a strongly acidic ion exchange resin (Dowex 50 x 2, 2OCUOO mesh, H-cycle). The resin was washed with water and the effluent was evaporated to give the pure amino acid derivative. In some cases, the amino acid derivatives crystallized spontaneously and could be recrystallized from water. If crystallization did not occur, the product was converted in a dicyclohexylammonium salt. Method B. Msc-derivatives showing a limited solubility in cold water, were liberated by addition of potassium hydrogen sulfate solution (2N). With Phe, Leu and Val, spontaneous crystalliza-
* The reaction rate depends strongly on the solubility of the amino component. Boc-Lys, Aca and Z-Lys are completely converted within 15 min. With Ala, Asp, Glu, Glu(OBu'), Gly, Pro, Ser, Thr, Tyr(Bu') and Trp the reaction time was about 1 h. Arg, Ile, Leu, Met, Phe and Val required at least 3 h. With Asn and Gln, aqueous DMF was used as the solvent and tetramethylguanidine as the base. Tyr did not react satisfactorily.
THE METHYLSULFONYLETHYLOXYCARBONYLGROUP
tion occurred. In other cases an oil precipitated and the product was extracted into ethyl acetate, after addition of sodium chloride. Boc-Lys(Msc)O H and Z-Lys( Msc)-OH crystallized spontaneously after removal of the solvent. Msc-Trp-OH was isolated as a dicyclohexyl ammonium salt. Deprotection of Msc-protected amines. Optimal conditions for the removal of Msc-groups from an amino compound were carefully determined for solvent composition and reaction time. (a) Solvent composition. The methylsulfonylethyloxycarbonyl group is quickly lost in a dioxanmethanol mixture containing sufficient aqueous sodium hydroxide to give a 0.1 or 0.2 N solution. The amount of water is critical and should be limited to concentrations up to 5 % for maximum reaction rate. Methanol acts as a scavenger for methylsulfonylethylene and the concentration of this constituent appeared to influence the reaction rate profoundly. The best ratio being dioxanmethanoL(2 or 4 N ) sodium hydroxide = 14:5:1, corresponding to a final base (CH,O- and OH-) concentration of 0.1 or 0.2 N. (b) Time dependency. Dioxan and methanol were mixed in the proportion 14:5 (vlv). Part of this mixture was used as solvent for the protected amino compound ; the solution being transferred into a series of small, capped polyethylene tubes. To the remainder, 1 volume of NaOH(2 or 4 N) was added. With a microsyringe, 2 equivalents of the base were added as rapidly as possible, the vials were closed and agitated on a vortex mixer. After time intervals decreasing from 300 to 5 sec the reaction mixtures were neutralized by addition of an acid (acetic or mineral acid). The neutral solutions were diluted as necessary and spotted onto thin layer plates. After development it was invariably found that with the solvent of the specified composition Msc-groups were quantitatively lost within 5 sec. Methylsulfonylethyloxycarbonyl-L-phenylalanine p-nitrophenyl ester ( VZ).Msc-Phe-OH, crystallized from water as a monohydrate (m.p. 759, was dried over phosphorus pentoxide, giving the anhydrous acid with m.p. 113". This product (2.65 g, 8.41 mmoles) and 1.40 g of p-nitrophenol (10 mmoles) was dissolved in 20 ml of acetonitrile. The mixture was cooled to 0" and 1.82 g (8.83 mmoles) of solid dicyclohexylcarbodiimide
were added. After 16 h at 0", the mixture was filtered and the residue was washed with acetonitrile*. The filtrate was evaporated in vacuo and the crystalline residue was purified by dissolution in warm acetic acid (10 ml) and addition of diisopropyl ether (20 ml), after cooling. The pale yellow crystals were dried over solid moist sodium hydroxide. Yield 3.228 (88%), m.p. 140", [a]22 = -20.1" (c = 1.03 in acetonitrile). Anal.: calcd. for ClsH2,N20eS (436.44): C, 52.29; H, 4.62; N, 6.42; S, 7.35. Found: C, 52.1; H, 4.4; N, 6.5; S, 7.3. Methylsurfonylethyloxycarbonyl-L -phenylalanyl L-arginine mono-hydrate (VZZZ). To a solution of 2.53 g (5.8 mmoles) of VI in a mixture of 20 ml of acetonitrile and 4 ml of water, 0.912 g (5.22 mmoles) of L-arginine (free base) were added. The suspension was stirred at room temperature and became homogeneous in about 30 min. The reaction mixture was concentrated in vacuo. On dilution with water, 0.8 g of unaltered VI separated. The aqueous phase was acidified to pH = 5 with Dowex 50 x 2 (H-cycle) and was extracted with ether to remove p-nitrophenol. On evaporation of the water layer a glassy residue was left, from which, on heating with ethanol, 1.65 g (64.6%) of a white solid was obtained. The compound was recrystallized from water. M.p. 181-184", [ a ] g = -2.3" (c = 1.02 in water), = 8.6" (c = 1.01 in 1N HCI). (489.55): Anal. : calcd. for C19H29N507S.H10 C, 46.62; H, 6.38; N, 14.31; S, 6.55. Found: C,46.7;H,6.1;N, 14.4;S,6.7. Themotherliquor of the analytical sample was evaporated and the residue was boiled for 3 h with 6N hydrochloric acid. On cooling, optically pure Msc-Phe-OH precipitated.
-
Methy lsulfbnylethyloxycarbonyl- L -phenylalanyl L-arginyl-L-tryptophyl-glycinemethyl ester hydrochloride (X).The former zwitterionic dipeptide derivative VIn (2.44 g = 5 mmoles) and 1.60 g of H-Trp-Gly-OMe.HC1 were dissolved in 20 ml of pure dimethylformamide, and 0.81 g (6 mmoles) of 1-hydroxybenzotriazole were added. The solution was cooled to 0" and treated with
* The product sometimes crystallized from thereaction mixture. 303
G. I. TESSER AND I. C. BALVERT-GEERS
finely divided, solid dicyclohexylcarbodiimide (1.03 g, 5 mmoles). The clear solution was kept at 0" for 1 h, left overnight at room temperature, and then cooled again for about 4 h at 0" to complete the precipitation of dicyclohexylurea which was removed by filtration (1.15 g). The filtrate was slowly added into ether to give a precipitate which was triturated with ethyl acetate. The crude product (4.1 g) was purified for analysis by countercurrent distribution in the system EtOAc-benzene-methanol-buffer ( = 10: 1 :5 :7) ; buffer: 19.25 g of ammonium acetate and 28.5 mol of acetic acid made up to 1000 ml with water. After 187 transfers, a single peak was obtained (K = 0.52, rmax= 65), comprising 3.2 g (81%) of the acetate. The compound crystallized from acetic acid upon addition of ether, yielding a hygroscopic product containing an additional equivalent of acetic acid (by titration). TS: RF = 0.33 (nBuOH-AcOH-water = 10:1 :3), = 0.57 (2-propanol-HCOOH-water = 20:1:5); [a]V = -15.8" (c = 1 , MeOH); m.p. 136-1 38.5". Anal. : calcd. for C33H44N80,S.2AcOH. +H,O (857-92): C, 51.80; H, 5.76; N, 13.06. Found: C, 51.8; H, 6.1; N, 13.1. The crude compound was soluble in water. A 1 % solution of the tetrapeptide ester in 0.05 M tris-buffer at pH = 8 was readily and completely digested by trypsin (-5 min). Upon standing for 2 h, the amino component set free by the enzyme underwent cyclization to the corresponding diketopiperazine. The same cyclization was found with authentic IX in tris-buffer. Chromatography, (TS): RF = 0.13 (tris); RF = 0.20 (VIII); R F = 0.33 (X); R F = 0.44 (IX) and RF = 0.64 (diketopiperazine from IX), in BuOH-AcOHwater = 10:1:3. MethylsulfonylethyloxycarbonyI-L-IeucyIgIycine tert-butyl ester. Msc-Leu-OH crystallized from water as a hydrate (m.p. 45"). The air-dried compound lost water and became an oil on further drying. The oil (1.1 g, 3.8 mmoles) was dissolved in dry ethyl acetate, evaporated twice to remove residual water and after addition of 0.53 ml (3.8 mmoles) of triethylamine was converted to the mixed anhydride by addition of 0.54 g (4 mmoles) of isobutylchloroformate at -5'C. After 15 min, 0.52 g (4 mmoles) of HGly-OBu' were added. The reaction mixture 304
was kept for 2 h at room temperature, filtered, and the filtrate was washed with water, sodium hydrogencarbonate and potassium hydrogen sulfate solutions. After drying, 1.43 g (95%) of an oil was obtained which refused to crystallize. The oil was purified by chromatography on silica (18) and gave the expected NMR spectrum after dissolution in CD30D. .4n IR spectrum revealed the presence of the sulfonyl group and carbonyl functions [ a ] g = -17.4" (c = 1 , MeOH). Methylsulfonylethyloxycarbonylglycyl-L-leucine tert-butyl ester. The preparation of this compound was carried out in the same way as described for the foregoing isomeric ester (yield 93%). The compound was purified by chromatography (1 8) but resisted crystallization. The NMR spectrum was in accordance with the expected structure; the presence of sulfonyl and carbonyl groups was demonstrated in an IR spectrum. [a]g = - 38.4" (c = 1, MeOH). Methylsuffonylethloxycarbonyl-L-alanyl-glycine tert-butyl ester was prepared in the way indicated. The oily compound, after chromatographic purification, gave [a]% = - 17.8"(c = 1, MeOH). L-Phenylalanyl-L-arginyl-L-tryptophylglycine( XI). Dioxan (dry, peroxide-free) and absolute methanol were mixed in the proportion 14:5 (vlv). Crude X (766 mg, 1 mmol) was dissolved in about 10 ml of this mixture and with vigorous stirring, 1 ml of 4N sodiumhydroxide (4 equiv.) in 10 ml of the same mixture was immediately added. After 45 sec the reaction mixture was acidified to p H < 4 by addition of hydrochloric acid and the solution was concentrated in uacuo. The product was precipitated by careful addition of sodium hydroxide to pH 1 1 . After filtration and drying, 508 mg (90%) of the crude free base were isolated. The monohydrochloride was obtained, after dissolution of the base in a small amount of a water-acetonitrile mixture (1 :1) containing one equivalent of hydrochloric acid, by slow addition of acetonitrile and cooling. Yield: 419 mg (69.7%). Chromatography, TS: RF = 0.20 (nBuOH-AcOH-water = 4: 1 :1); = 0.36 (nBuOHPyr-AcOH-water = 38 :24: 8 :30) ; = 0.48 (2-propanol-HCOOH-water = 20: 1 :5).
T H E METHYLSULFONYLETHYLOXYCARBONYL GROUP
[n]g = -5.8 (c = 1, 0.1N HCI in 95% MeOH). After equilibration with the humidity of the air. Anal.: C28H37N80,C1.2H20(637.14). Calcd. : C, 52.78; H, 6.49; N, 17.58. Found: C, 52.8; H,6.1 ;N, 17.6. After drying for 24 h over P205, C28HS,Na05Cl.$H20 (601.1 1). Calcd.: C, 55.12; H, 6.27; N, 18.37. Found: C, 55.1; H, 6.2; N, 18.3. Amino acid analysis: Arg :Gly :Phe = 0.94 : 1.00:0.99. ACKNOWLEDGMENTS
We are grateful for the kind interest and advice of Prof. Dr. R. Schwyzer (Institut fiir Molekularbiologie und Biophysik, E.T.H. Zurich, Switzerland), in whose laboratory this work was started, and to Prof. Dr. R. J. F. Nivard (Department of Organic Chemistry, University of Nijmegen) for stimulating discussions. The major part of the work was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.). REFERENCES
1. KADER,A. T. & STIRLING, C. J. M. (1964)
J. Chem. SOC.258-266. R. C. 2. HARDY,P. M.. RYDON.H. N. &THOMPSON, (1972) J. Chem. SOC.,Perkin Trans. I, 5-12. R. (1971) Chem. 3. WUNSCH,E. & SPANGENBERG, Ber. 104,2427-2429. L. A. & HAN,G. Y. (1972) J. Org. 4. CARPINO, Chem. 37, 3404-3409. G. I. & ELLENBROEK, B. W. J. (1967) 5. TESSER, in Peptides 1966, Proceedings of the 8th European Peptide Symposium (Beyerman, H. C., Van de Linde, A. and Maassen van den Brink, W., eds.),
p. 124, North Holland Publishing Co., Amsterdam. 6. BUIS,J. TH.(1973) Thesis,Nijmegen, the Netherlands. 7. MARSHALL, G. R. & MERRIFIELD, R. B. (1971) in Biochemical Aspects of Reactions on Solid Supports (Stark, G . R., ed.), pp. 130-131, Academic Press, New Yotk. 8. IUPAC-IUB Commission for Nomenclature (1 972) Biochem. J. 126, 773-780. 9. SCHULTZ,H. S., FREYERMUTH, H. B. & BWC, S. R. (1963) J. Org. Chem. 28, 1140-1142. 10. WOLTERS,E. TH. M. (1973) Thesis, Nijmegen, the Netherlands. 11. SCHNABEL, E., KLOSTERMEYER, H. & BERNDT, H. (1971) Justus Liebigs Ann. Chem. 749. W108. 12. ARBLJZOV, B. A. & BERDNIKOV, E. A. (1966) Dokl. Acad. Nauk SSSR 171, 860, Intern. J. Su&r Chem. (1971) B6 222. 13. BORDWELL,F. G. (1972), Acad. Chem. Res. 5, 374-381. P.,R I ~ LW., & ZUBER,H. 14. RINIKER,B., SIEBER, (1972) Nature New Biol. 235, 114-115. 15. VAN NISPEN,J. W. F. M. (1974) Thesis, Nijmegen, the Netherlands. 16. PERSONAL COMMUNICATION from Dr. R. Geiger (Hoechst AG. Frankfurt). 17. GRIBNAU,T. C. & TESSER,G. I., unpublished results. 18. HUNT,B. J. & RIGBY, W. (1967) Chem. Ind. 18681869.
Address : Dr. G. I. Tesser Department of Organic Chemistry University of Nijmegen Toernooiveld, Nijmegen the Netherlands
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