Int. J. PeprideProtein Res. 16. 1980, 412-425

P H O T O L A B I L E MULTI-DETACHABLE p - A L K O X Y BENZY L A L C O H O L R E S I N S U P P O R T S F O R P E P T I D E F R A G M E N T O R

S E M I - S Y N T H E S I S

JAMES P. TAM, RlCHARD D. DIMARCHI and R.B. MERRIFIELD

The Rockefeller University, New York, N Y , U.S.A.

Received 15 June, accepted for publication 18 June 1980

Dedicated to the memory of Roderich Walter

Two photolabile multi-detachable alkoxybenzyl alcohol resins, 2-[4- (oxymethyI)phenoxy]propionyl-resin 4 and 4-(4-(ox~vmethyl)pheno.rymethyl/- 3-nitro benzaniidomethyl-resin 5 have been synthesized. Bpoc-peptide attached to resin 4 or 5 when treated with 50% trifluoroacetic acid provided the free, unprotected peptide, but on photolysis gave Bpoc-peptide p-hydroxybenzyl ester. Removal of the p-hydroxybenzyl ester in aqueous base or by ox!dative work u p gave a protected Bpoc-peptide suitable for fragment synthesis at its C-terminus. However, methylation of the ester to Bpoc-peptide p-methoxybenzyl ester followed by removal o f the Bpoc-group gave a protected peptide p-methoxybenzyl ester suitable for fragment coupling a t its N-terminus. The efficacies of these resins were evaluated in the syntheses o f a model tetrapeptide and an octapeptide by using N"-Bpoc-, Fmoc- and Npsanino acids. The use o f 2-thiopyridine with pyridinium hydrochloride as a new and efficient thiolyric reagent for the deprotection o f the Nps-group was studied.

Key words: multidetachable resins; pertide fragments; photolysis in peptide synthesis; solid phase peptide synthesis; thiolytic deprotecting resins; alkoxybenzyl alcohol resins for semi-synthesis.

The commonly used strategy of peptide synthesis on a solid support has been the stepwise addition of amino acids, with

~ ~~ ~~

Abbreviations follow the IUPAC-IUB Tentative Rules on Biochemical Nomenclature Biochem. J. 126,773- 780 (1972). Optically active amino acids are of the Lconfiguration. In addition, the following abbrevi- ations are used: Boc, rert.-butoxycarbonyl; Bpoc, 2 - ( p -biphenylyl)isopropy1(2)oxycarbonyl; DCC, dicyclohexylcarbodiimide; DMAP, N,Ndimethyi- aminopyridine; DMF, dhethylformamide; Fmoc, 9-fluorenylrnethyloxycarbonyl; HOBt, l-hydroxy- benzotriazole; Nps, o-nitrophenylsulfenyl; 0-Bzl(OH), p-hydroxybenzyl ester; TFA, trifluoroacetic acid.

sequential acidolytic removal of the temporary a-amino protecting group, and fmal strong acid cleavage and deprotection of all permanent protecting groups (Merrifield, 1963, 1969). With the better designed protecting groups (Ohno et al., 1972; Yamashiro & Li, 1973a, 6; Erickson & Merrifield, 1973; Engelhard & Merrifield, 1978; Tam et al., 1979a), improved resin supports (Mitchell et al., 1976a; Atherton et d., 1975; Kent & Merrifield, 1978; Smith et aZ., 1979; Tam et al., 19796, 1980) and special handling of final strong acid cleavage (Sakakibara, 1971; Feinberg & Merrifield, 1975 Yajima et al., 1975; Matsuura et al., 1970

412 0367-8377/80/100412-14 %02.00/0 0 1980 Munksgaard, Copenhagen

MULTI-DETACHABLE RESINS

hgano et of., 1978), this general protocol ;hould remain popular in the future. Recently, ilternative tactics that utilize mild or non- icidolytic cleavage of a-amino protecting Zroups in conjunction with trifluoroacetic acid- labile resins have been successfully applied to the stepwise synthesis of a number of peptides including dihydrosoniatostatin (Chang & Meien- hofer, 1978), Pendorphin (Atherton et ~ l . , 1978) and glucagon (Merrifield ef QZ., 1977).

Following the introduction of solid phase- fragment synthesis (Wang, 1975, 1976), it has become clear that this is a valuable alternative route to larger peptides (e.g. Yajima et al., 1974). Our recent efforts in the approach have led to the development of a new and more flexible class of resin, termed multi-detachable resins 1 and 2 (Tam et al., 19793, c , 1980). Each contains two orthogonally removable ester linkages to the resin and can be cleaved by more than one reagent. Both resins can be used for the synthesis of free peptides, protected peptides containing a free carboxyl group, or fully protected peptides containing a handle a t the carboxyl terminus. A suitable protecting group tactic for such resins is to use t-butoxycarbonyl and benzyl derivatives as the a-amino and side chain protecting groups.

0 0

(Wang, 1973) for peptide synthesis. However, protected peptide fragments are not generally obtainable directly from 3, and, furthermore, we have envisioned that protected peptide fragments containing Bpoc and t-butyl groups would also be useful for semi-synthesis in which all the protecting groups could be removed under mild conditions by trifluoroacetic acid. In this paper we wish to report on the design and synthesis of two photolabile multi- detachable alkoxybenzyl alcohol resins 4 and 5 which meet these requirements. Their use in the syntheses of model tetrapeptides with Bpoc, Fmoc and Nps as the temporary a-amino protecting groups, and the use of 2-thiopyridine/ pyridinium hydrochloride as a new and efficient deprotecting reagent for the Nps group, have been studied.

Design of p h o tola bile m ulti-detacha ble p-alkoxybenzyl alcohol resin The design of resins 4 and 5 makes use of two properties of the p-hydroxybenzyl ester (0-Bzl(0H)) of an acid-labile protected amino acid, such as Bpoc-amino acid p-hydroxybenzyl ester 6 : (1) the facile 1 , 6elimination of the ester under basic or oxidative conditions to

CH. 0

I Poo-resin

2 Pon-resin

The development of a-amino protecting groups that are very acid sensitive, e.g. 2-~p.biphenylyl)isopropyl(2.)oxycarbonyl (Bpoc ; Sieber & Iselin, 1968), thiolytic sensitive, e.g. 0-nitrophenylsulfenyl (Nps; Zervas ef al., 1963) and base labile, e.g. 9-fluorenylmethyloxy- carbonyl (Fmoc; Carpino & Han, 1970, 1972) has allowed chemoselective synthetic tactics to be used in combination with the trifluoroacetic acid-labile p-alkoxybenzyl alcohol resin 3

give the quinone methide 7 and Bpoc-amino acid 8 (Scheme l) , and (2) the ease of 6 to be alkylated to form a stable, totally protected peptide p-methoxybenzyl ester 9, wluch could serve, after mild acidic deprotection, as the C-terminal component 10 of a fragment synthesis (Scheme 2).

Derivatives of p-hydroxybenzylic ester 6 have been employed in the design of amino-protecting groups (Wakselman & Guibe-Jampel, 1973:

413

J.P. TAM ET .4L.

LeCorre er al., 1978; Kemp & Hoyng, 1975; Kemp & Roberts, 1975). Its rapid transform- ation 'via 1,6elimination can be effected by many reagents (Turner, 1964; Wagner & Gompper, 1974; Whiting, 1979). However, once the phenolic moiety of 6 is alkylated to the ether 9 , the resulting p-methoxybenzylic ester is stabilized. The amino protecting group can then be removed and the product 10 is suitable for peptide fragment coupling at its N-terminus. Furthermore, a Bpoc-peptide- 0-Bzl(0H) would be useful in semi-synthesis without its conversion to the p-methoxybenzyl ester. Since most semi-synthetic studies require short synthetic fragments and are carried out in buffered solution, selective removal of the Bpoc-group would allow the relatively stable p-hydroxybenzyl ester to serve as the temporary protection at the C-terminus. Both

p-hydroxybenzyl and p-methoxybenzyl esters are removable by trifluoroacetic acid. T& duality of the p-hydroxybenzylic ester greatly facilitates its strategic use in fragment synthesis.

Thus as summarized in the generalized Scheme 3, photolabile Bpoc-peptide alkoxy- benzyl ester resin will retain all the acidic lability of p-alkoxybenzyl alcohol resin 3 and provide a free, unprotected peptide. Whereas, on photolytic cleavage, Bpoc-peptide p-hydro- xybenzyl ester 6 wiU be obtained and can be of further use for either N- or C-terminal fragment condensation.

Synthesis of photolabile alkoxybenzyl ester resin Resin 4 (2- [4(oxymethyl)phenoxyl] propionyl- resin) and 5 (4- [4-(oxymethyl)phenoxymethy~] - 3-nitrobenzamidomethyl-resin) were prepared

6 7 8

SCHEME 1 1,6-Elimination of p-hydroxybenzylic ester.

O O C H .

R O I II

9 10

SCHEME 2 C-terminal fragments from p-hydroxybenzylic ester 6.

414

MULTI-DETACHABLE RESINS

Acidolytic Cleavage

Photolytic Cleavage at bond b 0 5% TFA 50% TFA

h-pptide -OH~-EPOC-PEPTIDE-O-CH, OH----$

0 . 5 % TFA 1 H-PEPTIDE-0-CH, O O C H 3

SCHEME 3 Design of multidetachable photolabile alkoxybenzyl resin 4.

by reacting 4-hydroxymethylphenol with either 2-bromopropionyl-resin 1 1 (Wang, 1976) or 3-nitro-4-bromomethyl- benzamidomethy1-resh 12 (Rich & Gurawa, 1975) together with potassium fluoride and KHC03 in N-methyl- pyrrolidone at 50". 2.Bromopropionyl-resin 11 (Wang, 1976) was conveniently synthesized by Friedel-Crafts acylation of 2-bromopropionyl chloride and NCl3 with unsubstituted 1% cross- linked polystyrene resin. Improvement in the Preparation of resin 11 has also been recently reported (Tam er al., 1980). Resin 12 (Rich tk Gurawa, 1975) was prepared from dicyclohexyl- carhodiimide mediated coupling of 3 -n i t ro4 bromomethylbenzoic acid with aminomethyl resin (Mitchell et al., 19766).

Esterification of Bpoc-amino acid to resin 4 or 5 by dicyclohexylcarbodiimide (DCC) with 4-dimethylamino-pyridine (DMAP) as catalyst in a me thylene chloride and dimethylformamide mixed solvent (Steghch & Hofle, 1969) provided Bpooaminoacy~2[4~oxymethyl)phenoxyl] pro- pionyl-resin 13 or Bpoc-aminoacyld-~4-(oxy- methy1)phenoxymethyl ] - 3 - nitrobenzamido - methyl-resin 14.

Potassium fluoride is both a weak and a poor nucleophile. It has been used in our laboratory to effect esterification of Boc-amino acids to halomethyl or haloacyl resins (Tarn c[ a/.. manuscript in preparation). We have observed that KF was suitable for this reaction with a very reactive resin such as 11 or 12 in order to

N 02 \

I I 12

31 5

J.P. TAM ET AL.

avoid side reactions such as C-alkylation due to the decomposition of 4hydroxymethylphenol to quinone methide, or tautomerization of phenoxide anion (Turner, 1964; Hayashi & Oka, 1974). Thus, the choice of KF, an essentially neutral reagent, allows hydrogen bond-directed ether formation between the weakly acidic phenolic moiety of 4-hydroxy- methylphenol 20 and the bromide resin 11 or 12 to be accomplished selectively in the presence of the less acidic benzyl alcohol. The esterification with KF alone often required 3 days for completion, while addition of KHC03 accelerated the reaction to completion within 2 4 h . Cesium fluoride and cesium bicarbonate were also used to synthesize resin 4, but led to undesirable results with resin 5. ' To minimize a cross-linking reaction of resin 11 or 12 with the dihydroxyl compound, a large excess of 4-hydroxymethylphenol (3-5 equiv.) was used. Although it was difficult experimentally to completely verify the efficiency to this reaction, we obtained evidence that this reaction proceeded largely as anticipated based on the acidic and nucleophilic properties of the product. For example resin 4 (0.6 mmol bromide/g) could be esterified by 4-hydroxymethylphenol to give a bromide- free resin. Subsequent esterification with Bpoc-Ala-OH gave Bpoc-Ala-resin in 92% yield (0.55 mmol/g). On treatment with 50% TFA for 2 h , 0.53mmol/g Ala could be obtained c> 95% yield), but on treatment with glycine methyl ester for 8 h in methylene chloride, less than 3% of Bpoc-Ma-OH was

released from the iesin. These reactions con- firmed that most of the linkage to the resin 11 was through a phenyl ether and the linkagc to the Bpoc-Ala-OH was through a benzy ester.

Attachment of Bpoc-amino acids to th, photolabile multidetachable resins proceede, without difficulties with dicyclohexylcarbo diimide and 4-dimethylaminopyridine. Usuall! greater than 90% of the hydroxymethyl site on the resins were esterified to provid, 0.3-0.8 mmol/g substitution of Bpoc-aminc acid on the resins depending upon the origina substitution. Attachment of Fmoc-amino acid or Npsamino acids could be accomplishec satisfactorily with DCC and DMAP, bu required 0" in a DMF-CH2Clz mixture (1:4 (Meienhofer et al., 1979). Because of tht possibility of low level contamination due tc slow cleavage of Fmoc-amino acid by DMAl during the esterification step, substitution b) Bpoc-amino acid has been suggested (Athertor et al., 1979).

Cleavage of amino acids or peptides from resins 13 and 14 Cleavage of amino acids or peptides from resins 13 and 14 was effected by either acidolysis or photolysis c> 350nm). The acidolytic cleavage gave free peptides while photolytic cleavage provided protected amino acids or peptides.

Trifluoroacetic acid. Treatment of resins 13 ant 14 with TFA-CHzC12 (1 : 1 ,v/v)or TFA-anisolc (9 : 1, v/v) provided free amino acids or fret

I I 4 13

HOCH, O O H Bpoc-amino acid

KF DCCIDMAP NMP

HOCH, O O H Bpoc-amino acid

KF DCCIDMAP NMP

12 5 14

SCHEME 4 Preparation of resin 13 or 14.

416

MULTI-DETACHABLE RESINS

1-4 Bpoc-Val-resin 13c + H-ValOBzl(0Me)

H-Leu-Ala-ValQH

H-Leu-AlaCly -ValQH

Y 7

15

5 J Bpoc-Leu-Ala-resin 13h - Bpoc-Leu-AlaaH

16

1-4 BpocCly-Val-resin 13i -t HGly-Val-O-Bzl(0Me)

17

SCHEME 5 Fragment synthesis using multidetachable alkoxybenzyl alcohol resin 4. (1) Photolysis (> 350 nmt DMF-phenol. 3 days. (2) pH 11.6, Na,SO,, 0", 0.5 h. (3) K,CO,, Met, DMF 1 h. (4) 0.5% CF,CO,H-CH,CI,, 0.5 h. ( 5 ) Photo- lysis (> 350 nm), DMF, 3 days. (6) DCC-HOBt in CH,Cl,-DMF (4: l), O", 2 h. (7) 90% TF A-anisole, 1 h.

peptides with 79-95% yields (Table 1). Re- liminary kinetic data of Bpoc-Val-resin 13c in TFA-CH2C12 (1 : 1) showed that 50% of valine was cleaved in 13min, 80% in 30min and > 95% in 90min, while in TFA-anisole (9 : I), 80% of valine was cleaved in 20min and > 9S% in 60min (Table 2). The presence of 2-3 equiv. thiophenol or mercaptoethanol acceler- ated the cleavage rate only slightly Wha-Serettas & Serettas, 1977). The ad-

dition of a thiol scavenger is also useful in this cleavage reaction as a nucleophilic trapping agent for the p-alkoxybenzyl carbenium ion, particularly when cysteine or tryptophan is present. The acidolytic loss of amino acid due to repetitive and sequential treatment with 0.5% or 1% TFA to remove the Bpoc-group is shown in Table 2. It is calculated to be 0.67% for a 20-min 1% TFA treatment cycle and 0.1% for the 0.5% TFA treatment, but is about 3

TABLE I Cleavage of amino acids or peptides attached t o resins 4 and 5

Amino acid or peptide Esterified to resin Substitution mmol/g Yield (5%)

13a 13b 13, 13d 13e 13f

14a 14b 13h 13i 13j 14c

13s

4 4 4 4 4 4 4 5 5 4 4 4 5

0.30 0.80 0.55 0.5 1 0.52 0.49 0.48 0.20 0.2 1 0.80 0.55 0.5s 0.20

90 92 84 92'

87 91 96

91

us 92

9 F

94=

x 7

81 87 7s

-

80

75 78 71 79

8' 0.21 0.21 0.48

5 79= 14e 5 8 Y rrnoc-(LAGV),d 14k 4

14d

J.P. TAM ET AL.

TABLE 2 Trifluoroacetic acid stability of Bpoc- Val-2-/4-(oxymethyl)phenoxy]propionyl-resin I3c -

70 TFA' k(s-' X lo-') t 112 (h) t > 90% (min) 7% loss per 20 min cycle

0.13 0.5 0.01 177.70 1 .o 0.06 34.40 - 0.67

50.0 6.80 0.28 I 5 -

- -

90.0b 10.70 0.18 40 -

?FA in CH,CI,. b90% TFA in anisole.

times lower than the corresponding p-benzoxy- Senzyl alcohol resin 3 (Wang, 1975; S. Mojsov, personal communication). The better acidolytic stability of resin 4 can probably be attributed to the electron withdrawing properties of the phenacylether linkage. This would be useful in minimizing loss of peptide during a long repetitive synthesis.

Photolysis. The photolytic cleavage of resins 13 or 14 provided protected amino acids or peptides in 75-87% yield (Table 1) . The reaction was conducted in DMF for 3 days. Since the Nps-group is sensitive to light and the Fmoc-group is sensitive to base which may be present in DMF (Atherton ef al., 1979), only amino acids or peptides on resin 13 or 14 with Bpoc as the a-amino protecting group were investigated. Photolytic cleavage of Boc-Ala- resin 13 or Bpoc-Ala-resin 13 in DMF led to 80% recovery of Boc-Ala-OH or Bpoc-Ala-OH, with 20% of the corresponding product as its p-hydroxybenzyl ester. Model studies with Boc- Ala p-hydroxybenzyl ester showed that the decomposition of the ester was due to the basicity of the DMF solvent. Little decom- position (< 3%) of Boc-Ma p-hydroxybenzyl ester was observed in solvents such as dioxane DMF with 0.1% phenol present. Thus, when the photolysis was conducted in the above solvents, particularly in DMF-phenol mixture, the desired Boc-Ala p-hydroxybenzyl esters were obtained in about 80% yield.

Conversion of the p-hydroxybenzyl ester to the free carboxylic acid was achieved by two reagents: (1) pH 11.5 (Na2C03) in the presence of sodium bisulfite or (2) pH 9.5 with 1.2 equiv. K3 Fe(CN), . Both transform-

418

ations were extremely facile and required only Smin for completion in model studies of p-hydroxybenzyl ester of Bpoc-Ala (Bpoc-Ala- 0-Bzl(0H)) and Bpoc-Leu-Ala-Gly -Val (Bpoc- LAGV-0-Bzl(0H)). As expected p-hydroxy- benzyl esters were also cleaved by TFA, under the conditions required to remove the Boc group. Bpoc-Ala-0-Bzl(0H) and Bpoc-Leu- Ala-Gly-Val-OBzl(OH) were observed to be completely deprotected in 30min in 5% TFA to Ma and Leu-Ala-Gly-Val. This manipu- lation would allow a two-stage deprotection from the resin supports and might be of value to obtain protected C-terminal fragment for semi-synthesis.

Busc and nucleophiles. Unlike the multi- detachable Pop-resin 1, the multidetachable alkoxybenzyl resin 4 was fairly resistant to basic or nucleophilic reagents. Treatment of Bpoc-\/al-resin 13c with glycine methvl ester, triethylamine or diisopropylethylamine for 8-24h showed no significant loss of Bpoc-Val- OH from the resin (detection limit 5%). The phenyl ether bond was also resistant to NaOH/ H202 (PH 10.6), a condition found to result in extremely facile cleavage of the phenyl ester linkage (Kenner & Seely, 1972; Hudson et al., 1979). Stro-ng nucleophiles such as tetrabutyl- ammonium cyanide or triethylamine/thiophenol (Tam et d., 1980) slowly yielded Bpoc-Val-OH from 13c (10-20% in 2 days).

Stepwise synthesis of model tetrapepride and octapeptide using Bpoc- and Fmocarnino acids To test resins 4 and 5 in stepwise synthesis, Bpoc-Leu-Ala-Gly-Val-OH and Fmoc-Leu-Ala- Gly-Val-OH were prepared. Bpoc-amino acid

MULTI-DETACHABLE RESINS

DCHA salts were conveniently converted to free carboxylic acid using 0.5M KHS04 in CHzClz to avoid the excessive emulsion formation found when dilute HCI or 1 M citric acid was used. The Bpoc-Val-OH was esterified to resins 4 and 5 and Fmoc-Val-OH was also similarly esterified at 0" for 4 h to resin 5 . The Bpoc protecting group was deprotected in 0.5% TFA in CH2C12 and the Fmoc-amino acids were deprotected in 50% piperidine/CH2C12 for 30min. To avoid the loss of peptide at the dipeptide stage due to base catalyzed diketopiperazine formation, Bpoc-Gly was used instead of Fmoc-Gly in tlus LAGV synthesis. Double coupling was mediated by DCC for 30min each. Bpoc- LAGV-resins 4 and 5 were cleaved by TFA and photolysis while Fmoc-LAGV-resin 5 was cleaved by TFA alone. Satisfactory cleavage yields were obtained as shown in Table 1. The efficacies of these syntheses from resins 4 and 5 and the homogeneity of their crude products (LAGV) were examined by ion-exchange chromatography. Crude LAGV from these syntheses was shown to be 98.4-98.8% homogeneous, with 1-1 .S% of deletion peptides detected (Mitchell et al., 1978). Acidolytic loss of chains during the synthesis with Bpoc-amino acids on resin 4 was found to be 0.14% per step based on the release of LAGV in 0.5% TFA at the fmal step of the synthesis. The amounts of deletion peptides were comparable with those using chloromethyl- resins obtained from the commerical source.

Since the phenacylether bond of resin 4 might be labile to the base condition of Fmoc deprotection, Fmoc-(Leu-Ala-Gly-Va1)3 -OH was synthesized. H-(Le~-Ala-Gly-Val)~ -OH has been well characterized in our laboratory. Thus the efficiency of this synthesis could be evaluated from the well-defined chromatographic separation of the H-(LAGV),-OH and its side Products. The crude octapeptide was found to be 90% pure, with little (< 5%) peptide loss due to the base treatment. These syntheses showed the feasibility of using Bpoc and Fmoc strategy on the rnultidetachable alkoxybenzyl alcohol resins 4 and 5. TO optimize the syntheses, improvements in removal of protecting groups and in coupling efficiency are currently under investigation in our laboratory.

Stepwise synthesis of Leu-Ala-C!,~- Val using Npsarnino acids An alternative to the removal of Bpoc by mild acid and Fmoc by mild base is to use an es- sentidy neutral thiolytic cleavage of the Nps-protecting group. Such strategy will eliminate acidolytic or basic loss of peptides. The Nps group has been reported to be re- moved by 2-thiopyridine in acetic acid (Tun- Kyi, 1978) or in trifluoroacetic acid (Fries et al., 1979). Our synthesis of LAGV using Nps-amino acids with 2-thiopyridine in acetic acid was very poor, giving rise to > 20% of deletion peptides. Model studies with Nps- Val-resin 13g showed incomplete deprotection after 30min. Since the thiolytic reaction is general acid and general base catalyzed, pyridinium hydrochloride should be suitable reagent. Furthermore, the Nps-group is also known to be unstable to treatment with pyridinium hydrochloride (Dorman, 1969; Klostermeyer & Schwertner, 1973). When soluble Nps-amino acids were treated in CH2 C12 with a mixture of 0.05 M 2-thiopyridine and 0.05 M pyridinium hydrochloride, they were > 90% deprotected in 2 min and 100% in 20min. Nps-Val-resin 13g was found to be completely deprotected in 30min by the same reagent. Thus, using the new 2-thiopyridine and pyridinium hydrochloride deprotecting reagent, with reverse addition of DCC during the coupling reaction to prevent premature acidolytic cleavage of the Nps group, crude unpurified LAGV was obtained from resin 4 in 97.5% homogeneity.

A further objective of the thiolytic cleavage of Nps-group is to produce conditions where the by-product will not alkylate amino acids with nucleopiulic side chains such as tryptophan, me th iode or tyrosine. The study carried out by Tun-Kyi with 2-thiopyridine in HOAC showed that the by-products were disulfides which did not alkylate the indole moiety of tryptophan. Our model studies using Nps-lle with 2-thiopyridine in pyridinium hydrochloride with Boc-tryptophan, Boc-methionine and Boc- tyrosine revealed no alkylated product after 24h. The by-product of the Nps-group cleavage was, as reported, the mixed disulfide. In the course of using Nps-amino acids, we found that the formtion of symmetrical anhydride was

419

J . P . TAM ET AL.

difficult, particularly with Nps-Gly. Attempts to form the symmetrical anhydride of NPs-GlY led to a disproportionation giving Nps-GlY-GlY as the major side product, Thus, conventional coupling with inverse addition of DCC and NPs- anin0 acid would be more suitable.

Prel iminar~~ jril.estigarions offragi?ienf synthesis using t?iulri-tletachahle a lko .~)~her izy l alcohol resin 4 As indicated in Scheme 3, methylation of the p-hydroxybenzyl ester of a Bpoc-peptide would allow its use as the C-terminal component in a fragment synthesis. Our preliminary experi- ments using Boc-Ala p-hydroxybenzyl ester indicated that methylation could be accomplished using potassium carbonate with methyl iodide in DMF to obtain Boc-Ala p-me thoxybenzyl ester. However, some decom- position of p-hydroxybenzyl ester was observed, which thus led to a small amount of Boc-Ala- OCH3 as side product. Methylation of the photolyzed Bpoc-Ala-resin 13b also led to similar results. To illustrate the general principle, a tripeptide (H-Leu-Ala-Val-OH) and a tetrapeptide (H-Leu-Ala-Gly-Val-OH) were synthesized. Bpoc-Leu-Ala-OH 16, H-Val p-methoxybenzyl ester 15 and H-Gly-Val p-methoxybenzyl ester 17 were obtained as shown in Scheme 5. Without purification, these fragments were coupled by DCC/HOBt in solution at 0" for 2 h to obtain the corresponding Bpoc-Leu-Ala-Val p-methoxy- benzyl ester and Bpoc-Leu-Ala-Gly-Val p-methoxybenzyl ester. After deprotection with TFA, the resulting LAV and LAGV was analyzed by ionexchange chromatography and satisfactory overall yields (60-6470) were obtained in both cases (Scheme 5). The crude and unpurified material was 91-9676 homogeneous \?rith the methyl ester as the major side product.

EXPERIMENTAL PROCEDURES

Materials and me thods All Bpoc-, Fmoc, or Nps-amino acids were of the L-configuration and were purchased from Chemical Dynamics. p-Hydroxybenzyl alcohol (4hydroxymethylphenol) and 2-thiopyridine were obtajned from Aldrich Chemical Co. Potassium fluoride, cesium fluoride, potassium

420

bicarbonate and cesium bicarbonate (all anhydrous, extra pure grade) were purchased from Alfa Chemical CO. Dimethylformamide (DMF) was obtained as spectrophotometric grade and stored over Linde type 4A molecular sieve and passed through anhydrous neutral alumina before use. Dichloromethane was distilled over anhydrous Na2 C03. Rperidine was fractionally distilled from KOH. N,N- Diisopropylethylamine was fractionally distilled from CaH2. Copoly-(styrene-1% divinylbenzene) beads, Bio Beads SX-1 (200-400 mesh) were purchased from Bio- h d laboratories. 2-Bromopropionyl-resin and 3-Ntro-4-bromomethyl-benzamidomethyl- resin were prepared according to Tam et al. (1980).

Amino acid analyses were performed on the Beckman 121 Amino Acid Analyzer. Peptides or peptide-resins were hydrolyzed in 12 N HC1: acetic acid: 88% phenol (2 : 1 : 1, by vol.) in sealed, evacuated tubes for 24 h at 110". The tripeptide, H-Leu-Ala-Val-OH, the tetrapeptide, H-Leu-Ala-Gly-Val-OH and the octapeptide, H-(Leu-Ala-Gly-Val)z -OH were analyzed on the Beckman 120B Amino Acid Analyzer (Merrifield et al., 1974; Mitchell et al., 1978;0.9 x 54cm, AA-15 resin, eluted with 0.2M sodium citrate buffer, pH 3.49 at 56", 66ml/h; elution times of the peptides were: Leu-Ala-Gly-Val-Leu-Ala- Gly-Val, 133 min; Leu-Ala-Gly-Leu-Ala-Gly-Val, 155 min; Leu-Ala-Gly-Gly-Val, 189 min; Ala- Gly-Val, 201 min; Leu-Ala-Gly, 233 min; Leu- Ala-Gly-Val, 238 min; Gly-Val, 288 min; Leu- Ala-Val, 320 min; Leu-Ala, 332 min; Leu-Gly- Val, 373 min, Leu-Val, 437 min;NH3, 5 10 min). Bpoc-amino acid dicyclohexylamine salts were converted to their corresponding free acids by 0.5 M KHSOI (titrated to pH 2.5 by 1 N NaOH) in CH2C12, dried with MgS04 prior to use. Nps-amino acid dicyclohexylamine salts were converted to their corresponding free acids by 0.5 M citric acid (titrated to pH 3.0 by 1 N NaOH) in CH2C12 which was dried prior to use. Bpoc-Gly - DCHA salt and Nps-Gly - DCHA salt were not soluble in CH,C12 and ethylacetatelDMF was used for the conversion to their free acids. Photolysis was performed in a water-cooled photochemical reaction vessel with a photochemical immersion lamp (Ace-Hanovia, 450 watts, 3-7 Amps,

MULTI-DETACHABLE RESINS

236-254 nm) surrounded by a uranium glass filter.

Preparation of 2-/4-(oxymethyl)phenoxy] - propionyl-resin 4. 2-Bromopropionyl-resin 11 (20 g, 12.0 mmol, prepared according to modified procedure of Tam er al., 1980) suspended in N-methylpyrrolidone (NMP, 200ml) was stirred by a mechanical overhead stirrer with anhydrous potassium fluoride (6.96 g, 120mm01, finely pulverized under nitrogen) and 4-hydroxybenzyl alcohol (7.44 g, 60.0mmol) a t 50". After 2h , anhydrous potassium carbonate (4.14g, 30mmol, or an equivalent amount of potassium bicarbonate), finely pulverized under nitrogen, was added to the reaction mixture. The reaction was allowed to proceed for another 22h. The resulting resin was washed thrice (200ml each) with DMF, dioxane, dioxane-H,O (1 : 1, v/v), dioxane-acetic acid (3 : 1, v/v), dioxane, and acetonitrile. After drying, 20.4g resin was obtained, IR(KBr), 1680-' (ketone), 1220 cm-' (ether), Br 0.08% (0.01 mmol/g). The volume of 1 g of dry and closely packed resin beads was 1.67ml and swelled to 5.2ml in DMF and 7.1 ml in CH2C12.

Preparation of the 4-[4-(oxymethyl)phen- oxymethyI/-3-nitrobenzamidomethyI-resin 5. 3-~itr0-4-bromomethyl-benzamidomethyl resin 12 (2.0g, 0.48mm01, prepared according to modified procedure of Tam et al., 1980) SUS- pended in dimethylformamide (DMF, 20ml) was stirred by a mechanical overhead stirrer with anhydrous potassium fluoride (0.29 g, 5 m o l , finely pulverized under nitrogen) and 4-hydroxybenzyl alcohol (0.1 8 g, 1.44mmol) at 50" for 48h . The resulting resin was washed thrice (20ml each) with DMF, DMF-H20 (1 : 1, Vb), dioxane, dioxane:acetic acid (3 : 1, vh), dioxane, dioxane-H20 (1 : 1, v/v) and aceto- nitrile. After drying, 2.01 g of resin 5 was

Esterification of hydroxymethjd resins 4 and j with Bpoc-, Fmoc- and Nps-amino acids TO a suspension of resin 4 ( 1 g, 0.60 mmol) in CH, c12 (10 ml), N,A'dimethylaminopyridine (DMAP, 0.22 g, 1.8 mmol), Bpoc-Val-OH (0.64 g, 1.8 mmol) and dicyclohexylcarbodi- imide (0.37g, 1.8mmoI) were added successively. The reaction was allowed to proceed for 4h. The resin was then washed thrice (15 ml each) with

and CH3CN. After drying, 1.12 g of esterified resin was obtained, nitrogen analysis (0.77%, 0.55 mmol/g), modified picric acid analysis (0.52 mmol/g), amino acid hydrolysis (Val, 0.54mmol), IR(1680cm-' ketone, broad carbonyl absorption at 1720 cm-' ). To ensure that all hydroxymethyl sites were esterified, the resin was further suspended in CH2C12 (10ml) and treated with pyridine (0.5 ml) and benzoyl chloride (0.89 ml) at 0" for 30 min.

Similar conditions were used for the esterification of Boc-Ma, Bpoc-Gly-(CH2 C12 - DMF solvent, 4 : 1, v/v), Bpoc-Ma, and Bpoc- Val to resins 4 and 5. For Fmoc-Ala and Fmoc-Val, CH2 C12 -DMF (4 : 1 , v/v) was used as the solvent at 0" with DMAP (0.2mol%) as the catalyst. For Nps-Ala and Nps-Val, the CH2 C12 -acetic acid washes after the completion of the esterification procedure were substituted by DMF. Acetic anhydride (1 ml) plus pyridine (1 ml) was used for the termination of the remaining hydroxymethyl sites in lieu of benzoyl chloride and pyridine.

CH2Cl2, CH2Clz-HOAc (3 : 1, v/v), CH2Cl2

Kinetic experiments of Bpoc- VaI-2[4-(oxymethyl)phenyo-ry]- propionyl-resin 13c in trijluoroacetic acid-dichlorome thane Bpoc-Va10CH2C6H40CH(CH3)CO-GH4 -resin 13c (lOOmg, 55pmol) was shaken with tri- fluoroacetic acid-dichloromethane (1 : 1, v/v, 1OOml) and alanine (IOpmol, as internal standard) at 20". At time intervals, from 5 min to 12Omin after mixing, an aliquot of this solution (1 ml) was withdrawn and freed of

Obtained, IR(KEh) 1665cm-I (amide), CH2C12 and TFA by evaporation under a 1515cm-' (nitro), 1240cm-l (ether), Br gentle stream of nitrogen. The residue was analysis 0%. The volume of 1 g of closely dissolved in pH 3.2 buffer for amino acid Packed dry resin beads was 1.67 g. The swollen analysis. Similarly, the rates of acidolytic loss volume was 5.2 in DMF and 7.1 ml in of resin 13c in 1% TFA-CH2C12 (v/v) and 0.5% CH2 C1,. TFA-CH2C12 (v/v) were determined. The data

42 1

J.P. TAM ET AL.

was obtained over the time range from 1 h to 6611. The apparent first order rate constants were determined from plots of 1 n(a/a -x) VS. time where a is the amino acid content of the starting material and x is the amount of amino acid released at a given time. The results are summarized in Table 3.

Clearage af'artlino acids and peprides frortt resins 4 and 5

Cl'irh trifliraroaceric acids. Bpoc-Ala-OCH2 c6 H4 - OCH(CH3 )-COC6H4-resin 13b (IOOmg, 80pmol) was shaken with TFA-CH2C12 (1 : 1, v/v, 10ml) a t 20" for 1 h. Anisole (0.1 ml)and thiophenol (0.01 ml) were added as scavengers. The resin was then filtered and washed with TFA-CH2 Clz (1 : 4, v/v). The combined filtrate was evaporated in vaczio and the residue extracted with anhydrous ether. The ether layer was discarded and the resulting residue was dissolved in water for amino acid analysis. Yield was 70pmol (87%). Similarly, resin 13b (1 OOmg, 80pmol) was treated withTFA-anisole (1 0 ml, 9 : 1, v/v) with thiophenol(O.01 ml) for 1 h to give Ala (74pmo1, 92%). The results of other TFAcleavages are summarized in Table 2.

B y phorolj'sis. Bpoc-Ala-resin 13b (lOOmg, 80pmol) was suspended in DMF (lOml) and phenol (l00mg) in a screw-capped pyrex test tube. The sample was deoxygenated for 1 h in a steady stream of nitrogen and then photolyzed by the Hanovia light source with a uranium glass filter for 72 h. The resin was filtered and washed thrice with DMF ( 5 m l each). The remaining resin was dried and hydrolyzed in HCl:HOAc:phenol (2 : 1 : 1 , by vol.) for 24h at 110". Amino acid analysis indicated that this material contained 20pmol Ala; a 75% photo- lytic cleavage. The product identified from the DMF filtrate was mainly Bpoc-Ma-OW2- C 6 H 4 0 H with 20% of Bpoc-Ala. Bpoc-Ala- OCHz -c6 H4 OH was converted to Bpoc-Ala by dissolution in 85% EtOH at pH 11.6-12 (Na2 CO, -NaHC03) with either K3 Fe(CN)6 (3 equiv.) or sodium bisulfite (10 equiv.) at 0" for 0.5 h.

422

Sjjn thesis of leu cy lalany klY CY h h e

With ,7-(p-bipheny1yl}isopropy~(~)~xycarbonyl- (Bpoc)-umino acids. Bpoc-Val-OCH2 -c6 H, -

0.55 mmol) was placed in a reaction vessel on a shaker and treated as follows for the incorpor- ation of each residue: (1) CHzCl2 (3 x 1 min), (2) 0.5% trifluoroacetic acid (TFA)-CH2C12 (3 x 2min), (3) 0.5% TFA (1 x 25min), (4) CH2Clz (3 x 1 min), ( 5 ) 5% diisopropylethyl. amine (DIEA)-CHZ C l z (2 x 2 min), (6) CH2 C12 ( 5 x 1 min), (7) 4 equiv. DCC in CH2C12 (5ml, 1 x 1 min), 4 equiv. Bpoc-Gly in CH2C12 (5ml,

(3 x 1 min), (10) repeat steps (4) to (8) for recoupling of Bpoc-Gly. The cycle was repeated with Bpoc-Ala and Bpoc-Leu but 3 equiv. preformed symmetrical anhydride was used for step (7). The Bpoc-Leu-Ala-Gly-Val-resin was washed thrice with DMF, CH2C12 and CH3CN and vacuum dried. Amino acid analysis indicated that this material contained 0.37mmol peptide/g of substituted resin and had an amino acid composition of Leu o6 Ala ,, Gly0.% ~

Val,. oz. A portion of the peptide resin (100 mg, 30pmol) was shaken with a mixture of 50% TFA-CH2C12 and thiophenol (0.01 ml) at 20" for 1 h. The liquid phase was filtered and the resin was washed thrice with 50% TFA-CH2 C12. After the combined filtrate was freed of solvent, the residue was dissolved in water (5ml) and part (1 ml) was applied on the long column (0.9 x 54 cm; AA-15 sulfonated polystyrene) of a Beckman 120B Amino Acid Analyzer. The presence of 30.5 pmol Leu-Ala-Gly-Val indicated a cleavage yield of 85%. The desired product Leu-Ma-Gly-Val constituted 98.8 mol% of the total unpurified peptide product. Similar pre- paration and treatments of Bpoc-Val-OCH, - C6H40CHz-(m-N0,-)C6H4-CONHCH2-CgHq- resin 14a provided 92% cleavage yield and 98.7 mol% of Leu-Ala-Gly-Val in the unpurified crude product.

OCH(CH3)-CO-C6 H4-resin 1 3 ~ (1 .o g,

1 x 60min), (8) CH2Cl2 (3 X 1 d), (9) DMF

With 9-fluorenylmethyloxycarbonyl (Fmocj- amino acids. Fmoc-Val-OCH2C6H4-OCH2- (m-N02)-C6 €L, -CONHCH2 -resin 14b (0.5 g, 0.1 mmol) was placed in a reaction vessel on a shaker and treated as follows (1) CH2C12 (3 x 1 min), (2) 5% piperidhe (1 x 5min,

MULTI-DETACHABLE RESINS

1 x 25 min), (3) DMF (3 x 1 min), (4) CH2Cl2 ( 5 x 1 min), (5) 4 equiv. DCC in CH,Cl2 (3 ml, 1 x 1 min), 4 equiv. Bpoc-Gly in CH2 C12 (3 ml, 1 x 60 min), (6) CH2 Cl, (3 x 1 min), (7) DMF (3 x 1 min), (8) repeat steps (3) t o (7) for recoupling of Bpoc-Gly. The next cycle was repeated with steps (1) t o (5) as described above with Bpoc-amino acids to deprotect Bpoc-Gly to minimize diketopiperazine for- mation at the dipeptide stage. Thus, for the third and fourth cycles step (5) was 4 equiv. Fmoc-Ala or Fmoc-Leu in CHzC12 and 4 equiv. DCC in CH2CI2. Fmoc-Leu-Ala- Gly-Val-resin was washed with thrice CH2 Clz - acetic acid (1 : 1, v/v), CH2Clz, CH3CN and vacuum dried. Amino acid analysis indicated that this material contained 0.14mmol of peptide/g of substituted resin and had an amino acid composition of (after removal of Fmoc group). A portion of peptide-resin (100mg, 1 4 ~ 0 1 ) was treated with 50% TFA-CH,Cl, as described with Bpoc-amino acids. The cleavage yield was 79% (12 pmol). The desired Leu-Ala-Gly-Val constituted 98.4% of the unpurified peptide product as analyzed on a Beckman 120B Amino Acid Analyzer after the appropriate treatments to remove the Fmoc group.

Similarly, Fmoc-(Le~-Ala-Gly-Va1)~ -resin was also prepared from Fmoc-Val-resin 13e (300mg, 15.6prnol). The cleavage yield was 82% and the desired H-(Leu-Ala-Gly-Va1)OH constituted 90 mol% of the unpurified peptide product. Amino acid analysis after hydro- lysis: .05 M a l . o o G l ~ 0 . 9 ~ V ~ o . 9 ~ .

With a-nitrophenylsulfenyl (Nps)-amino acids.

13g (0.5 g, 0.24mmol) was placed in a reaction vessel on a shaker and treated as follows for the incorporation of each residue: (1) CH2CL (3 x 1 min), (2) 2-thiopyridine : pyridinium hydrochloride (1 : I)-CH2 Cl, (0.05 M I 2 x 5 min and 1 x 20min), (3) CH2C12 (3 x 1 min), (4) 5% DIEA-CH2C12 (2 x 2min), (5) CH2Cl2 (5 x 1 min), (6) 4equiv. DCC in CH2Clz (1 x 1 min, 3 ml), 4 equiv. Nps-Gly in CHzClz (1 X 60 min, 3 ml), (7) CH2C12 (3 x 1 f in) , (8) DMF (3 x 1 min), (9) repeat steps (3) to (8) for

Nps-Val-OCH, - C,&-OCH(CH3 )-CO-C6& -resin

recoupling of Nps-Gly. The cycle was repeated with Nps-Ala and Nps-Leu. The Nps-Leu-Ala- Gly-Val-resin was washed thrice with CH, Cl, - acetic acid (1 : 1, v/v), CH2 CI2 and CH3 CN and vacuuni dried. Amino acid analysis indicated that t h s material contained 0.20 mmol peptide/g of substituted resin and had an amino acid composition of Leu,.osAla,.,Gly,.loValo.9s . A portion of the peptide-resin (100 mg, 20prnol) was shaken with 50% trifluoroacetic acid- CH2C12 (0.01 ml thiophenol as scavenger) at 20" for 1 h. The liquid phase was treated and analyzed in a Beckman 120B Amino Acid Analyzer described in the synthesis with Bpoc-amino acids. The presence of 16.4 pmol Leu-Ala-Gly-Val indicated a cleavage yield of 82%. The desired Leu-Ala-Gly-Val constituted 97.5 mol% of the unpurified peptide product.

Fragment synthesis of leucylalanylvalinc and leucylalanylglycylvaline Bpoc-Leu-Ala-resin was obtained as described from Bpoc-Ala-resin 13b (2.0 g, 1.6 rnmol). Bpoc-Leu-Ala-OH 16 was obtained in 75% yield after photolysis in DMF and basic work up. T1.c. and ionexchange chromatography analysis (retention time 332min, pH 3.49 buffer on AA-15 column) indicated it was 98% pure. Thus, it was used without purification. Bpoc- Val-p-hydroxybenzyl ester and Bpoc-Gly-Val-p- hydroxybenzyl ester were obtained from the photolytic cleavage of Bpoc-Val-resin 13c (1 g, 0.55 mmol/g) and Bpoc-Cly-Val-resin 13i (1 g, 0.55mmol/g) in DMF-phenol (lord, 100 : 1, v/w). To the DMF solvent filtrate from these photolyses were immediately added K, C 0 3 (finely ground 1 g, 7mmol) and methyl iodide (0.27 g, 2 mmol) and the mixture was allowed t o react for 1 h. The reaction was freed of DMF, and the residue redissolved in ethyl- acetate, washed with aqueous base (pH 11, contained 0.01 % of sodium bisulfite), aqueous acid (pH3, 0.5 M , KHS04), water, saturated sodium chloride and dried in MgSO, . The yield of Bpoc-Val-0-Bzl(0Me) was 60% and Bpoc- Gly-Val-0-Bzl(0Me) was 56% based on ion- exchange chromatography analysis. The Bpoc- group was then removed by 0.5% TFA-CH2C12 for 0 .5h to obtain 15 and 17 and these were coupled separately to Bpoc-Leu-Na-OH 16 by DCC-HOBt in CHZCl2-DMF (4: l ) at 0" for 2 h.

4 2 3

J.P. TAM ET AL.

The final product after the usual work up, deprotection by 90% TFA-anisole and analysis on ion-exchange chromatography gave 64% overall yield of H-Leu-Ala-Val-OH and 60% of H-Leu-Ala-Gly-Val-OH based on Bpoc- Leu-Ala-OH as the starting material.

ACKNOWLEDGMENTS

We wish to thank Ms. A. McNichol for technical assistance and Ms. M. LeDoux for the amino acid analyses. This work was supported by Grant Am 01260 from the U.S. Public Health Service and by a grant from the Hoffmann-La Roche Foundation.

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