Building units for N-backbone cyclic peptides. Part 4.1Synthesis of protected Nalpha;-functionalized alkyl aminoacids by reductive alkylation of natural amino acids

摘要

J. Chem. Soc. Perkin Trans. 1 1997 1501 Building units for N-backbone cyclic peptides. Part 4.1 Synthesis of protected Nmiddot;-functionalized alkyl amino acids by reductive alkylation of natural amino acids Gal Bitan Dan Muller Ron Kasher Evgenia V. Gluhov and Chaim Gilon * Department of Organic Chemistry The Hebrew University of Jerusalem Givat-Ram Jerusalem 91904 Israel A new method for the synthesis of protected Na-(cent;-Y-alkyl) amino acids (Y is a thio amino or carboxy group) and related compounds by reductive alkylation of natural amino acids is reported. These new amino acids serve as building units for the synthesis of backbone-cyclic peptides. They are orthogonally protected at the middot;-amino position by butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) using trimethylsilyl temporary protection to allow for their incorporation into peptides by solid phase peptide synthesis.Introduction Backbone-Cyclization is a method developed in our laboratory for imposing long-range conformational constraint by cyclization of linear bioactive peptides in order to enhance activity stability to metabolic degradation selectivity and bioavailability. 2,3 In the classical peptide cyclization methods the carboxyl or amino termini are often used to cyclize peptides. Alternatively side-chain cyclization can be achieved by closing a lactam ring between the side-chains of lysine and aspartic or glutamic acid residues or a disulfide ring between two cysteine residues. Unfortunately these four natural amino acids offer quite a limited scope of cyclization possibilities. In order to overcome this circumscription several analogous amino acids such as ornithine penicillamine etc.are quite often used. Nevertheless the utilization of such natural or unnatural amino acids often requires their artificial insertion or substitution into the sequence. Consequently crucial functional groups are replaced or altered or the peptide is subjected to conformational changes which frequently lead to loss in or reduction of the biological activity.4 Thus the development of new amino acids which will broaden the scope of cyclization possibilities and will enable minimal alteration of the native sequence is of considerable importance. In Backbone-Cyclization ring closure is effected by bond formation between two functional groups which are linked to the backbone nitrogens by alkyl spacers.Cyclization can thus be accomplished without changing the original sequence or the chemical character of any amino acid residue required for bioactivity. This method also provides a convenient way to stabilize the ubiquitous turn motifs found in peptides by replacing intramolecular hydrogen bonds with suitable covalent chains. If a particular NH group in the peptide is required for biological activity its replacement by an alkylated amide bond might reduce potency. However this problem may usually be easily fixed by shifting the site of cyclization to the next amide bond. In order to perform Backbone-Cyclization of peptides unique unnatural amino acids should be incorporated at the cyclization sites (Fig. 1). These lsquo;building unitsrsquo; are protected Na-(w-Y-alkyl) amino acidsmdash;where Y is an orthogonally protected amino carboxy or thiol groupmdash;which are analogous to the original amino acids at the sites chosen for cyclization.When the synthesis of the peptide is completed the protecting groups of the cyclizing units are removed and cyclization is performed to give a lactam a disulfide a sulfide or a combination of these groups5 (Fig. 2). Although other modes of cyclization are essentially possible we decided to limit our studies to the same groups which are used for cyclization in nature. Building units for Backbone-Cyclization were previously synthesized by us and by others using several methods which were all based on a nucleophilic substitution reaction as a key step.1,6ndash;8 Glycine derivatives were the easiest and hence the first to be prepared by a nucleophilic attack of w-substituted amines on bromoacetic acid or its esters.Other building units based on chiral amino acids could be prepared by the same method albeit in low yield due to extensive side-reactions using a large excess of the attacking amine with appropriate a-chloro acids as substrates.7 Fig. 1 Building units for N-Backbone-Cyclization; (a) w-carboxy (b) w-amine (c) w-thiol. X = Boc Fmoc; R = side-chain of a-amino acid. O N O OH O R Fmoc NH N O OH R Fmoc Boc S N O OH R X Bzl n n n (a) (b) (c) Fig. 2 Incorporation of building units into a peptide and its cyclization P R1 N CH2n1 R2 R3 N CH2n2 R4 N CH2n3 R5 R6 N CH2n4 R7 CO2H SH SH NH2 P R1 N CH2n1 R2 R3 N CH2n2 R4 N CH2n3 R5 R6 N CH2n4 R7 CO S S NH cyclization 1502 J. Chem. Soc. Perkin Trans.1 1997 Reissmann et al. used the amino group of phenylalanine as a nucleophile to attack tert-butyl bromoacetate in the presence of Ag2O and obtained a building unit based on phenylalanine with a carboxy group as the w-functional group see Fig. 1(a) and a tether of one methylene group.6 Unfortunately this method could not be generalized to longer alkyl tethers because the tert-butyl esters of w-halogeno carboxylic acids are not commercially available and their preparation is cumbersome. We have recently shown that several chiral building units with various tether lengths and functional groups could be obtained from a-triflate derivatives of D-hydroxy acids.1,8 However this approach suffered from several drawbacks (1) the synthesis of one chiral building unit required 9.12 stages beginning from costly D-amino acids; (2) triflates based on amino acids with sensitive side-chains are unstable (e.g.tryptophan 9) and the synthesis of building units based on such amino acids is accordingly impractical; (3) in the case of w-thiol building units there was a particular problem in order to prevent a second substitution of the secondary amine formed we had to employ semi-protection of the nucleophile with a benzyl group which was removed after the substitution by catalytic hydrogenation.1 This method was successful for the synthesis of w-amino and wcarbonyl building units yet double substitution was unavoidable in the case of w-thio building units. The semi-protection method is not suitable for the latter type of building units because catalytic hydrogenation fails due to poisoning of the catalyst by the sulfur.These problems inspired us to look for a new method for preparation of building units which would be as general as possible and preferably reduce the cost the number of synthetic steps and be easy to execute. The other most common way for N-alkylation of amines and amino acids is reductive alkylation with aldehydes in the presence of a reducing agent.10 This method was hence chosen to be examined for the preparation of building units for Backbone-Cyclization. Lately one example of the preparation of a building unit based on phenylalanine by reductive alkylation with Boc-glycinal was presented.6 However this was actually the preparation of a pseudo-dipeptide and until the present work no systematic investigation of this method for the preparation of building units was made nor has any unit with a functionalized sidechain been prepared.The current research focused on w-thiol building units since this subgroup was the most problematic to prepare by the nucleophilic substitution method. The reductive alkylation method was however extended also for building units with w-amino or w-carboxy functional groups. Results and discussion Preparation of cent;�-functionalized aldehydes Screening of the relevant literature revealed that essentially all of the previous work which employed reductive alkylation for derivatization of the a-amino group of amino acids used simple and usually commercial aldehydes. An exception to this generalization is found in syntheses of reduced pseudo-peptides in which a Boc- or Fmoc-protected amino aldehyde is condensed with the amino group of an amino acid ester or a growing peptide.In our study of the synthesis of building units by reductive alkylation we adopted the procedure developed by Fehrentz and Caro11 as a general method for the preparation of w-functionalized aldehydes with various chain lengths. In accordance with this method our synthetic root included the preparation of an w-functionalized carboxylic acid. maintaining the w-functional group suitably protected conversion of the acid to the appropriate N,O-dimethyl hydroxamate and reduction to the aldehyde with LiAlH4. In some cases however commercially available acetals could serve as more convenient starting materials for the preparation of the w-functionalized aldehydes. w-(Benzylthio) aldehydes were prepared either from whalogeno acetals or from w-halogeno carboxylic acids.In both cases the first step was substitution of the halogen with toluenea- thiol. w-(Benzylthio) acetals 1 were hydrolysed in 0.5 M H2SO4 to give the desired aldehydes 2 (Scheme 1-I). When w- (benzylthio) carboxylic acids 3 were used then they were first converted to their N,O-dimethyl hydroxamates 4 and then reduced to the aldehydes with LiAlH4 (Scheme 1-II). w- Benzylthio-acet- -propion- -butyr- and -capro-aldehyde were obtained as liquids which could be preserved for long periods under argon at 25 8C. By the same method w-Boc-amino aldehydes 6 were prepared from w-Boc-amino acids through reduction of the appropriate N,O-dimethyl hydroxamates 5 (the preparation of these aldehydes has been previously described 12,13).The preparation of w-(tert-butoxycarbonyl) aldehydes was the most laborious of the three sub-groups prepared due to the necessity to obtain mono-tert-butyl esters of dicarboxylic acids. For the preparation of the mono-protected diacids we found that the opening of cyclic anhydrides with tert-butyl alcohol was most suitable (see also ref. 14). This method was demonstrated in the preparation of mono-tert-butyl glutarate 7 which was then converted to the N,O-dimethyl hydroxamate 8 and then reduced to the aldehyde 9 as described above (Scheme 2). This aldehyde is not known in the literature yet similar aldehydes have been previously prepared.15,16 Scheme 1 Reagents i Bzl-S2 dry NMP; ii 0.5 M H2SO4; iii MeONHMe? HCl BOP TEA; iv LiAlH4 dry Et2O X OEt OEt S OEt OEt Bzl S O H Bzl n-1 X O OH 1iexcl;Euml;a X = Br n = 2 1iexcl;Euml;b X = Cl n = 3 S O OH I II Bzl n-1 n-1 n-1 S O N n-1 Bzl n-1 1iexcl;Euml;c X = Cl n = 4 1iexcl;Euml;b X = Br n = 6 1 OMe Me 2 2 3 4 i ii iii i iv HN Boc n-1 OMe Me HN Boc n-1 H O N 5a n = 2 5b n = 3 6a n = 2 6b n = 3 O Scheme 2 Reagents i But OH ZnCl2; ii MeONHMe?HCl BOP TEA; iii LiAlH4 dry Et2O O O O O OH O O But O N O O But OMe Me O H O O But 3 3 3 7 9 8 i ii iii J.Chem. Soc. Perkin Trans. 1 1997 1503 Table 1 Physical data of zwitterionic building units Compound Starting material Y n Yield () Mp (8C) 10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k 10l 10m 10n 10o 10p 10q 10r 10s 10t 10u 10v 10w g-o-benzylglutamic acid isoleucine isoleucine isoleucine isoleucine leucine leucine leucine e-Boc-lysine methionine methionine phenylalanine phenylalanine phenylalanine O-benzylserine NIn-formyltryptophan O-tert-butyltyrosine valine glycine glycine glycine glycine glycine Bzl-S Bzl-S Bzl-S Boc-NH But-O2C Bzl-S Bzl-S Boc-NH Bzl-S Bzl-S Boc-NH Bzl-S Bzl-S Boc-NH Boc-NH Bzl-S Bzl-S Boc-NH Bzl-S Bzl-S Bzl-S Boc-NH Boc-NH 62334233643233242323434 56 63 56 50 19 55 59 46 30 43 54 61 58 69 29 19 34 59 30 42 41 57 a 150ndash;151 241ndash;243 215ndash;217 204ndash;206 229ndash;231 210ndash;211 191ndash;192 212ndash;214 189ndash;190 204ndash;206 199ndash;201 220ndash;223 206ndash;208 206ndash;209 65ndash;68 167ndash;170 199ndash;200 202ndash;204 181ndash;182 173ndash;175 214ndash;216 a a Not isolated.Preparation of chiral building units Preparation of Na-(w-Y-alkyl) amino acids (other than glycine) by reductive alkylation was found to be a successful method and proved to be simpler and more economical in both time and money in comparison with the former method.1,7 The reaction of various chiral side-chain-protected (where appropriate) L-amino acids with aldehydes 2 6 or 9 was performed with slight modifications according to the procedure of Ohfune et al.17 Thus g-benzylglutamic acid isoleucine leucine e-Boclysine methionine phenylalanine O-benzylserine Ninformyltryptophan O-tert-butyltyrosine and valine were Nalkylated with w-Y-aldehydes to give the Na-(w-Y-alkyl) amino acids 10 with different functional groups and alkyl chains in 20ndash; 60 yield (Scheme 3).The yield depended mainly on the solubility of the product under the reaction conditions. In accord with the results observed by Ohfune et al.17 the N-alkylated amino acids were formed as partially insoluble products which were collected and washed with methanol. In most cases no further purifiction was required.If a product contained any unreduced imine or other impurities it was recrystallized from boiling ethanol. No attempt was made to purify any dissolved product which may have remained in the reaction mixture. Scheme 3 Reagents i NaBH3CN MeOH; ii BTSA; iii Fmoc-Cl or (Boc)2O R(G) CO2H H2N H R(G) CO2H NH H Y D R(G) CO2H N H 2 or 6 or 9 Y D n X n R = side-chains of natural amino acids G = side-chain orthogonal protecting groups BTSA = N,O-bis(trimethylsilyl)acetamide Y D n X NH Boc 2,3 Fmoc CO2 But 4 Fmoc S Bzl 2,3,4,6 Boc Fmoc 10 11 X = Fmoc 12 X = Boc ii iii i Physical data for the zwitterionic units are summarized in Table 1. Toward the end of this work an attempt to develop a method for multiple simultaneous reductive alkylation reactions was made. Since the N-alkylated amino acids produced by this reaction were partly immiscible in the reaction medium it seemed worthwhile to try simultaneously to prepare several products in spatially separated vessels and to purify them together by filtration.Multi-well blocks could offer a simple arrangement for this purpose; however the volume available in each well of commercial multi-well blocks used for solid-phase syntheses is relatively small. Instead we carried out a preliminary exploratory experiment in which the reactions were performed in simple polypropylene vessels arranged in an array and shaken together on a shaker or a vortex. Amino acids were chosen randomly off the shelf and reacted with three aldehydes in the following manner alanine arginine hydrochloride asparagine homophenylalanine isoleucine methionine norvaline phenylglycine and valine with aldehyde 2b; methionine and valine with aldehyde 6a; alanine isoleucine and methionine with aldehyde 9.As no internal stirrer was used in this arrangement it was found to be extremely important to apply vigorous shaking throughout the reaction otherwise the parent amino acids precipitate in the bottom of the vessel and little or no reaction takes place. We used a strong vortex suitable for multiple vessels or arranged the vessels horizontally on an adhesive stripes-type shaker. However evidently neither of these methods was suf- ficient and in all of the cases some unchanged material was detected by TLC. The yields were accordingly low ranging between 7 and 35. Two of the amino acids alanine (with both aldehydes 2b and 6a) and arginine hydrochloride gave soluble products which were qualitatively observed by TLC but which were not isolated.The asparagine product was identified as the dialkylated amino acid Na,Na-bis3-(benzylthio)propylasparagine. All of the other products were the desired Na-(w-Y-alkyl) amino acids. Although no obvious pattern could be deduced for the dependence of the yield upon hydrophobicity it is likely that the method is not suitable for hydrophilic amino acids such as alanine or unprotected arginine and asparagine because the products are soluble in methanol. However appropriate side-chain protection may provide the desired low solubility of the produced N-alkylated amino acid. 1504 J. Chem. Soc. Perkin Trans. 1 1997 Table 2 Physical data of protected building unit Compound Yield () Mp (8C) aT D (c 1 CH2Cl2) Elemental analysis () 11b 11c 11d 11e 11f 11g 11h 11i 11j 11l 11m 11n 11o 11p 11q 11r 11s 12a 12f 12s 12t 12u 12v 12w 42 62 48 15 61 70 20 53 72 51 48 54 30 a 53 72 44 42 a 41 42 88 85 91 78 61 115ndash;117 62ndash;64 56ndash;58 oil 47ndash;48 oil 45ndash;47 semi-solid oil 47ndash;48 53ndash;54 108ndash;110 65ndash;67 60ndash;61 51ndash;52 (decomp.) 58ndash;60 85ndash;86 43ndash;44 oil 71ndash;72 oil oil 124ndash;125 150ndash;152 27.8 18 29.3 30 214.6 25 16.0 25 217.0 24 221.6 23 n.d.b 218.6 24 233.2 25 286.9 25 281.2 23 287,0 25,c 223.5 25 263.5 23 290.2 23 214.7 25 236.3 23 226.2 25 Calc C 71.54; H 6.60; N 2.78 Found C 71.88; H 6.54; N 2.82 Calc C 71.92; H 6.81; N 2.71 Found C 71.83; H 6.82; N 2.69 Calc C 68.21; H 7.50; N 5.49 Found C 67.95; H 7.48; N 5.47 Calc C 70.70; H 7.71; N 2.75 Found C 70.22; H 7.76; N 2.78 Calc C 71.54; H 6.60; N 2.78 Found C 71.45; H 6.71; N 2.71 Calc C 71.92; H 6.81; N 2.71 Found C 71.73; H 6.82; N 2.72 Calc C 68.21; H 7.50; N 5.49 Found C 68.10; H 7.50; N 5.48 Calc C 69.41; H 7.47; N 4.15 Found C 69.26; H 7.41; N 4.08 Calc C 67.73; H 6.42; N 2.55 Found C 67.53; H 6.51; N 2.46 Calc C 73.72; H 5.81; N 2.61 Found C 73.54; H 5.92; N 2.60 Calc C 74.02; H 6.03; N 2.54 Found C 74.10; H 6.12; N 2.58 Calc C 70.57; H 6.66; N 5.14 Found C 70.44; H 6.60; N 5.18 Calc C 68.55; H 6.47; N 5.00 Found C 65.73; H 6.39; N 4.96 Calc C 69.78; H 5.63; N 3.13 Found C 69.65; H 5.69; N 3.24 Calc C 72.88; H 6.45; N 2.30 Found C 72.62; H 6.67; N 2.19 Calc C 67.72; H 7.31; N 5.64 Found C 67.80; H 7.29; N 5.50 Calc C 69.78; H 5.63; N 3.13 Found C 69.61; H 5.74; N 3.23 Calc C 66.27; H 7.60; N 2.58 Found C 66.55; H 7.52; N 2.58 Calc C 62.96; H 8.19; N 3.67 Found C 62.90; H 8.18; N 3.62 Calc C 58.69; H 7.70; N 4.28 Found C 58.55; H 7.72; N 4.28 Calc C 60.15; H 7.42; N 4.13 Found C 59.98; H 7.44; N 4.11 Calc C 61.16; H 7.70; N 3.96 Found C 60.84; H 7.68; N 3.91 bb a Prepared by Method P.b Not determined. c c 1 MeOH. This experiment demonstrated that although simultaneous synthesis of Na-(w-Y-alkyl) amino acids may be feasible and useful some further development is still required to overcome the technical problems which remain. Most of the new building units were protected by tertbutoxycarbonyl (Boc) or fluoren-9-ylmethoxycarbonyl (Fmoc). Protection of the secondary a-amino group of Na-(w-Yalkyl) amino acids was not possible by the common Boc- or Fmoc-introduction procedures since these substances were all insoluble under the reaction conditions required for the introduction of both protecting groups.We have therefore adopted a method of temporary protection by the trimethylsilyl group,6,18 using N,O-bis(trimethylsilyl)acetamide (BTSA) as the silylating agent to introduce either of these protecting groups (Scheme 4). For w-Boc-amino- or w-tert-Butoxycarbonylcontaining units only the Fmoc group was used to protect the a-amine whereas both Boc and Fmoc provided orthogonal protection in the case of w-benzylthiol-containing units. The method proved to be very useful for introduction of Fmoc through Fmoc-Cl yet the yield of Boc-Na-w- (benzylthio)alkyl amino acids was quite poor probably because (Boc)2O is not reactive enough for this reaction. Prolonged reaction times did not increase the yield but probably a more reactive agent such as Boc-Cl or Boc-N3 would be preferable.Physical data for units protected by Fmoc (11) or Boc (12) are summarized in Table 2. The integrity of the final products was verified by RP-HPLC and they were identified by their 1H NMR spectra. Since all of the protected units existed as mixtures of isomers in solution 2D NMR spectra were routinely employed for unambiguous peak assignment. Scheme 4 Reagents i NaBH3CN MeOH; ii Fmoc-Su; iii (Boc)2O Bzl S NH2 O OH O H Bzl S N H CO2H Bzl S N CO2H Bzl S N n + n CO2H n n Fmoc Boc 11 R = H 12 R = H 10 n = 2ndash;4 i ii iii J. Chem. Soc. Perkin Trans. 1 1997 1505 Preparation of glycine-based building units An earlier attempt to force reaction between glycine and protected w-amino aldehydes led to a mixture of products from which the desired product could not be isolated.19 In the current study it was found that Na-w-Y-alkylglycine derivatives could be prepared by a variation of the method of Simon et al.20 The first attempt to react w-(Boc-amino)alkyl amines with glyoxylic acid and then to reduce the imine formed by catalytic hydrogenation as in the original procedure failed to yield the desired glycine building units.However utilizing in situ reductive alkylation of w-substituted primary amines of various lengths with glyoxylic acid in the presence of sodium cyanoborohydride gave the desired products as precipitates when Y was a benzyl-protected thiol. Since glycine building units bearing an w-amino or wcarboxy group were readily prepared by the nucleophilic substitution method,1,7 no special effort was required for their preparation by the reductive alkylation method.Only in one case was a comparison of the two methods in the preparation of Fmoc-Na-3-(Boc-amino)propylglycine and Na-Fmoc-4-(Bocamino) butylglycine made. The overall yield of the first unit was 45 in both methods. In this particular case the zwitterionic Na-3-(Boc-amino)propylglycine did not precipitate from the reaction medium during the reductive alkylation and it was hence necessary to protect the crude product by Fmoc and to further purify it by column chromatography. On the other hand the second unit Na-4-(Boc-amino)butylglycine did precipitate under the same conditions and consequently the overall yield of the Fmoc-protected unit was elevated to 61 when prepared by the reductive alkylation method.The overall yield of this unit was 47 when prepared by the nucleophilic substitution method. It was therefore concluded that the reductive alkylation of w-protected amines with glyoxylic acid was equal to or better than the nucleophilic substitution of benzyl bromoacetate with the same amines for the preparation of building units based on glycine. The reaction between w-functionalized alkylamines and glyoxylic acid was particularly important for the preparation of Na-w-(benzylthio)alkylglycine derivatives. As mentioned above preparation of these compounds by the nucleophilic substitution method suffered from a high amount of double alkylation of the attacking amine. In the current study however w-(benzylthio)alkylamines prepared by previously reported procedures,8 reacted with a slight excess of glyoxylic acid to yield the corresponding Na-w-(benzylthio)alkylglycine with various alkyl chain lengths.In all cases the products were partly immiscible under the reaction conditions and could be filtered off and isolated in 30ndash;40 yield before the next step. Although about the same amount of product still remained in solution its purification was troublesome and we therefore preferred to use larger quantities of the cheap starting materials and did not try to increase the yield above that of the precipitated product. The crude products contained only small amounts of NaCN as the only impurity and could be unambiguously identified by their 1H NMR spectra. The glycine derivatives were soluble in water at pH 7 and the secondary a-amino group could therefore be protected by either Fmoc or Boc protecting groups to give the protected products without any difficulty.The rapid development of efficient screening methods combined with combinatorial chemistry gives new powerful tools to chemists and sets new targets which could not have been achieved less than a decade ago.21 Yet with the ability to prepare large numbers of molecules in a short time and to lsquo;fish outrsquo; only those which are biologically relevant comes the need for novel sophisticated building blocks which will augment the level of diversity and impart through their unique chemistry desirable pharmacological features to the molecules which are being produced. Several laboratories around the world offer a variety of substitutes for the natural building blocks of peptides and proteins which are aimed at maintaining the basic structural and hence the biological properties of amino acids.Many of the makers of such building blocks from N-alkylamino acids which form peptoids to the recently presented betides,22 try to keep the side-chains of the amino acids unchanged while altering the construction which holds them togethermdash;the peptide backbone. In contrast to most of these building blocks which usually focus on one aspect of chemical modification the units described in this work offer threedimensional diversity. The first dimension is the side-chains of 19 natural a-amino acids (excluding proline) to which many unnatural a-aminol acids with a primary amino group can be added. The second dimension is the w-functional groups which may be used for their original functionmdash;cyclizationmdash;but also to connect other useful moieties like chelating agents and affinity crosslinking or radioactive labels.We have limited ourselves in this work to three kinds of w-functional groups but other groups may be preferred for different particular cases. The third dimension is provided by the control of the spacer length which we have been using to explore the conformational space available for bioactive peptides through backbone-cyclic analogue libraries. Although cyclization of short bioactive peptides is a very popular manipulation designated to bestow desirable pharmacological features most laboratories are limited to classical cyclization methods. Backbone-Cyclization offers a smooth way to avoid sequence alteration and side-chain and/or termini modifications usually required for cyclization.The lack of a general rigorous method for the preparation of building units was until recently one of the main obstacles which prevented Backbone-Cyclization from becoming more widely and commonly used. All the previous methods for the preparation of building units were limited to certain amino acids and/or certain tether lengths. This work however offers a simple synthetic pathway for the preparation of building units based on most (side-chain-protected) amino acids with various alkyl chain lengths in 4ndash;5 steps altogether. The first 2ndash;3 steps (depending on the availability of starting compounds) are the preparation of w-substituted aldehydes and the following two steps are reductive alkylation and protection of the a-amine.Experimental Materials and methods Starting materials were purchased from either Merck Darmstadt Germany or Aldrich Milwaukee WI USA and used without further purification. Analytical HPLC was performed on a Merck Hitachi 655A equipped with an L-6200A gradient pump and a UVndash;VIS detector with tunable wavelength set at 220 nm. The flow was fixed at 1 ml min21 and the eluents were triply distilled water (TDW) and MeCN containing 0.1 tri- fluoroacetic acid (TFA) or MeOH. The column was Lichroprep RP-18 250 times; 4.2 mm i.d. from Merck. Mps were measured on a Mel-Temp II capillary equipment. Optical rotations were recorded on Perkin-Elmer 141 or 241 polarimeters in a 10-cm length cell and aD values are given in units of 1021 deg cm2 g21. Elemental analysis was carried out at the microanalytical laboratory of the Hebrew University Jerusalem.1H NMR spectra were recorded on Brucker WP-200 AMX-300 AMX- 400 or DRX-400 spectrometers. 2D Chemical shift correlation (COSY) spectra of final products were routinely recorded and in some cases phase-sensitive 2-D total correlation spectrometry (TOCSY) nuclear Overhauser enhancement spectroscopy (NOESY) NOESY in a rotating frame (ROESY) and Cndash;H correlation spectra were also used to assist with the proton assignment of highly overlapping 1D spectra. The numbering of methylene groups in the N-alkyl chain is always from the Na of the amino acid to the w-functional group. J Values are given in Hz. 1506 J. Chem. Soc. Perkin Trans. 1 1997 Method A. Preparation of cent;-(benzylthio) aldehyde diethyl acetals 1 0.51 Mol of toluene-a-thiol were added dropwise under dry N2 to a stirred suspension of 0.51 mol of NaH in 300 ml of dry NMP at 0 8C.The resulting dark solution was stirred for an additional 30 min and then 0.5 mol of an w-halogeno aldehyde diethyl acetal were slowly added. The mixture was stirred for 18 h at room temperature. Completion of the reaction was monitored by TLC. The mixture was then poured into 1 l of icendash;water and the product was extracted with light petroleum (LP) (6 times; 500 ml). The yellow organic solution was dried over MgSO4 filtered and the solvent was evaporated off in vacuo. The remaining crude products were distilled in vacuo to give the pure title compounds as liquids. Compound 1a was prepared from 2-bromoacetaldehyde diethyl acetal 19a in 85 yield bp 93 8C (0.04 mmHg) (Found C 65.31; H 8.66.C13H20O2S requires C 64.96; H 8.39); dH(CDCl3; 298 K) 7.33ndash;7.22 (5 H m ArH) 4.54 (t J 5.6 1CH) 3.79 (2 H s PhCH2) 3.72ndash;3.43 (4 H m MeCH2) 2.59 (2 H d J 5.6 2-H2) and 1.21 (6 H t J 7.1 CH3CH2). Compound 1b was prepared from 3-chloropropionaldehyde diethyl acetal 19b in 61 yield bp 105 8C (0.02 mmHg); dH- (CDCl3; 298 K) 7.33ndash;7.23 (5 H m ArH) 4.56 (1 H t J 5.6 1CH) 3.71 (2 H s PhCH2) 3.70ndash;3.38 (4 H m MeCH2) 2.47 (2 H t 7.4 3CH2) 1.86 (2 H dt J21 5.7 J23 7.4 2-CH2) and 1.18 (6 H t J 7.0 CH3CH2). Method B. Acid hydrolysis of cent;-(benzylthio) aldehyde diethyl acetals 1 to aldehydes 2a,2b 0.4 Mol of an acetal 1 were stirred for 24 h with 300 ml of 1 M H2SO4 at 60 8C. The progress of the reaction was followed by TLC. The crude product was extracted with LP (6 times; 300 ml) and the solution was dried over MgSO4 filtered on active carbon and evaporated in vacuo.Aldehydes 2 were further distilled in vacuo and were collected as liquids. Aldehyde 2a (87) bp 73 8C (0.06 mmHg); dH(CDCl3; 298 K) 9.40 (1 H t J 2.5 1CH) 7.34ndash;7.24 (5 H m ArH) 3.63 (2 H 5 PhCH2) and 3.07 (2 H d J 1.4 2CH2). Aldehyde 2b (80) bp 101 8C (0.06 mmHg); dH(CDCl3; 298 K) 9.70 (1 H t J 1.1 1-H) 7.34ndash;7.24 (5 H m ArH) 3.73 (2 H s PhCH2) and 2.72ndash;2.65 (4 H m 2- and 3-H2). Method C. Preparation of cent;-(benzylthio) carboxylic acids 3 0.51 Mol of toluene-a-thiol were added dropwise under dry N2 to a mechanically stirred suspension of 1.01 mol of NaH in 1 l of dry NMP at 0 8C. The resulting solution was stirred for an additional 30 min and then 0.5 mol of an w-halogeno carboxylic acid were slowly added.The mixture was stirred for 18 h at room temperature. Completion of the reaction was monitored by TLC. The mixture was diluted with 1 l of icendash;water. The aqueous solution was washed three times with diethyl ether (300 ml) acidified with 0.5 M H2SO4 and extracted with 3 times; 200 ml of diethyl ether. The yellow organic solution which contained some residual toluenethiol was dried over MgSO4 filtered and the solvent was evaporated off in vacuo. The remaining crude product was distilled in vacuo to give the pure title compounds as liquids. Product 3d crystallized on storage. Compound 3c (n = 4) was prepared from 4-chlorobutanoic acid 19c (81) bp 141 8C (0.02 mmHg) lit.,23 170 8C (0.01 mmHg); dH(CDCl3; 298 K) 7.31ndash;7.23 (5 H m ArH) 3.69 (2 H s PhCH2) 2.454 (2 H t J 7.1 2-H2) 2.447 (2 H t J 7.3 4-H2) and 1.95ndash;1.80 (2 H m 3-H2).Compound 3d (n = 6) was prepared from 6-bromohexanoic acid 19d (76) bp 165 8C (0.08 mmHg); dH(CDCl3; 298 K) 7.32ndash;7.23 (5 H m ArH) 3.70 (2 H s PhCH2) 2.42 (2 H t J 7.1 2-H2) 2.33 (2 H t J 7.4 6-H2) 1.69ndash;1.50 (4 H m 3- and 5-H2) and 1.47ndash;1.35 (2 H m 4-H2). Method D. Preparation of cent;-(benzylthio) carboxylic acid N,Odimethyl hydroxamates 4 0.1 Mol of diisopropylethylamine (DIEA) and 0.1 mol of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa- fluorophosphate (BOP) were added to a suspension of 0.1 mol of an w-(benzylthio) carboxylic acid in dichloromethane (DCM). The mixture was stirred for 5 min at room temperature by which time a clear solution was obtained. 0.11 Mol of N,O-dimethylhydroxylamine hydrochloride and 0.11 mol of DIEA were then added and the solution was stirred for 4.5 h at room temperature.The reaction was diluted with 200 ml of DCM washed successively with 3 times; 50 ml of 1 M H2SO4 3 times; 50 ml of saturated aq. KHCO3 and 3 times; 50 ml of saturated NaCl. The organic phase was dried over MgSO4 and the solvent was evaporated off in vacuo. The crude product was further purified by column chromatography (silica; ethyl acetatendash;LP 40 60). Compound 4c (81) (Found C 61.40; H 7.60; N 5.51. C13H19NO2S requires C 61.63; H 7.56; N 5.53); dH(CDCl3; 298 K) 7.32ndash;7.22 (5 H m ArH) 3.71 (2 H s PhCH2) 3.67 (3 H s OCH3) 3.17 (3 H s NCH3) 2.52 (2 H t J 6.7 2-H2) 2.48 (2 H t J 6.9 4-H2) and 1.98ndash;1.84 (2 H m 3-H2). Compound 4d (81) dH(CDCl3; 298 K) 7.32ndash;7.22 (5 H m ArH) 3.70 (2 H s PhCH2) 3.67 (3 H s OCH3) 3.17 (3 H s NCH3) 2.46ndash;2.36 (4 H m 2- and 6-H2) 1.70ndash;1.52 (4 H m 3- and 5-H2) and 1.47ndash;1.35 (2 H m 4-H2).Method E. Reduction of N,O-dimethyl hydroxamates 4 to cent;-(benzylthio) aldehydes 2c and 2d 5 Mol equiv. of LiAlH4 were added in small portions to a solution of 80 mmol of an w-(benzylthio) carboxylic acid N,Odimethyl hydroxamate in 200 ml of dry diethyl ether stirred under N2. The mixture was stirred for 60 min at room temperature then was cooled with an icendash;water-bath and hydrolysed with a solution of 19 g of KHSO4 in 100 ml of water. Diethyl ether (50 ml) was added the layers were separated and the aqueous layer was extracted with 3 times; 50 ml diethyl ether. The combined ethereal layers were washed successively with 3 times; 50 ml of 3 M HCl 3 times; 50 ml of saturated aq.KHCO3 and 3 times; 50 ml of saturated aq. NaCl. The organic phase was dried over MgSO4 and the solvent was evaporated off in vacuo. The aldehydes were collected as colourless or pale yellow oils. Compound 2c (56) dH(CDCl3; 298 K) 9.75 (1 H t J 1.2 1-H) 7.34ndash;7.22 (5 H m ArH) 3.70 (2 H s PhCH2) 2.53 (2 H dt J21 1.2 J23 7.2 2-H2) 2.46 (2 H t J 7.0 4-H2) and 1.91ndash;1.84 (2 H m 3-H2) in agreement with the literature.24 Compound 2d (67) dH(CDCl3; 298 K) 9.72 (1 H t J 1.8 1-H) 7.36ndash;7.20 (5 H m ArH) 3.69 (2 H s PhCH2) 2.40 (2 H t 7.2 6-H2) 2.38 (2 H dt J21 1.7 J23 7.4 2-H2) 1.60ndash;1.52 (4 H m 3- and 5-H2) and 1.39ndash;1.35 (2 H m 4-H2). Method F. Preparation of cent;-(Boc-amino) carboxylic acid N,Odimethyl hydroxamates 5 The title compounds were prepared from Boc-glycine and Bocb- alanine according to Method D.Compound 5a (n = 2) (62) dH(CDCl3; 298 K) 5.27 (1 H br s NH) 4.09 (2 H d J 6.5 2-H2) 3.71 (3 H s OCH3) 3.22 (3 H s NCH3) and 1.39 (9 H s But). Compound 5b (n = 3) (93) dH(CDCl3; 298 K) 5.23 (1 H br s NH) 3.63 (3 H s OCH3) 3.37 (2 H t J 6.3 3-H2) 3.13 (3 H s NCH3) 2.51 (2 H t J 6.7 2-H2) and 1.39 (9 H s But). Method G. Reduction of N,O-dimethyl hydroxamates 5 to cent;-(Boc-amino) aldehydes 6 The title compounds were prepared from hydroxamates 5 according to Method E. Compound 6a (36) dH(CDCl3; 298 K) 9.64 (1 H t J 4.0 1-H) 5.32 (1 H br s NH) 4.06 (2 H d J 4.1 2-H2) and 1.46 (9 H s But). Compound 6b (61) dH(CDCl3; 298 K) 9.73 (1 H t J 3.9 1-H) 5.15 (1 H br s NH) 3.35 (2 H m 3-H2) 2.64 (2 H t J 6.0 2-H2) and 1.36 (9 H s But). J. Chem. Soc. Perkin Trans. 1 1997 1507 Method H.Preparation of mono-tert-butyl glutarate 7 Glutaric anhydride (5.7 g 0.05 mol) was added to a mixture of 28.4 ml (0.3 mol) of dry tert-butyl alcohol and 0.1 g of zinc chloride. The mixture was stirred at 60 8C with exclusion of water for 3 days. 0.5 M Sodium hydroxide (40 ml) was added and after 20 min the product was extracted with diethyl ether (4 times; 40 ml) washed with water (3 times; 50 ml) and dried over MgSO4. The solvent and excess of tert-butyl alcohol were removed in vacuo. The product was obtained as an oil (50) dH(CDCl3; 298 K) 10.70 (1 H br s CO2H) 2.42 (2 H t J 8.0 2-H2) 2.32 (2 H t J 8.0 4-H2) 1.92 (2 H m 3-H2) and 1.45 (9 H s But) (for comparison see ref. 25). Method I. Preparation of glutaric acid tert-butyl ester N,Odimethyl hydroxamate 8 The title compound was prepared from semi-ester 7 according to Method D in 66 yield; dH(CDCl3; 298 K) 3.70 (3 H s OCH3) 3.20 (3 H s NCH3) 2.48 (2 H t J 8.2 2-H2) 2.30 (2 H t J 8.2 4-H2) 1.92 (2 H m 3-H2) and 1.45 (9 H s But).Method J. Reduction of N,O-dimethyl hydroxamate 8 to ldquor;-(tertbutoxycarbonyl) glutaraldehyde 9 The title compound was prepared from hydroxamate 8 according to Method E in 81 yield; dH(CDCl3; 298 K) 9.75 (1 H s 1-H) 2.50 (2 H t J 7.0 4-H2) 2.25 (2 H t J 7.0 2-H2) 1.9 (2 H m 3-H2) and 1.4 (9 H s But). Method K. Preparation of chiral Nmiddot;-cent;-Y-alkyl amino acids 10 A zwitterionic (suitably protected on the side-chain) amino acid (5ndash;10 mmol) was dissolved or suspended in methanol (1.5 ml mmol21). Then 1.5 mol equiv. of aldehyde 2 6 or 9 were added followed by 1.1 mol equiv. of sodium cyanoborohydride and the mixture was stirred at room temperature for 18 h.The precipitated product was collected by filtration on a glass sinter washed with methanol and dried in vacuo. Physical data for compounds 10 are summarized in Table 1. Their 1H NMR data and assignments follow here. Nmiddot;-6-(Benzylthio)hexylglutamic acid ldquor;-benzyl ester 10a. (CD3)2SO; 310 K 7.63ndash;7.29 (8 H m ArH) 7.24ndash;7.20 (2 H m ArH) 4.50 (2 H s OCH2Ph) 4.16ndash;4.13 (1 H m a-H) 3.71 (2 H s SCH2Ph) 3.46 (1 H m 1-H2) 2.82 (1 H m 1-H2) 2.38 (2 H t J 7.3 6-H2) 2.27 (1 H m b-H2) 2.24 (2 H m g-H2) 1.94 (1 H m b-H2) 1.48 (2 H m 5-H2) 1.29 (2 H m 4-H2) and 1.18 (2 H m 3-H2). Nmiddot;-2-(Benzylthio)ethylisoleucine 10b. (D2O; 350 K; as sodium salt) 7.86 (5 H m PhCH2) 4.26 (2 H s CH2Ph) 3.33 (1 H d J 5.7 a-H) 3.21ndash;3.02 (4 H m 1- and 2-H2) 2.03ndash;1.85 (2 H m g-H2) 1.67ndash;1.50 (1 H m b-H) and 1.37ndash;1.30 (6 H m g- and d-H3).Nmiddot;-3-(Benzylthio)propylisoleucine 10c. (D2O; 350 K; as sodium salt) 7.83 (5 H m PhCH2) 4.23 (2 H s CH2Ph) 3.29 (1 H d J 5.8 a-H) 3.01ndash;2.85 (4 H m 1- and 3-H2) 2.22ndash;2.06 (2 H m 2-H2) 2.05ndash;1.85 (2 H m g-H2) 1.60ndash;1.47 (1 H m b-H) and 1.34ndash;1.27 (6 H m g- and d-H3). Nmiddot;-3-(Boc-amino)propylisoleucine 10-d. (D2O; 298 K; as sodium salt) 3.18ndash;3.11 (2 H m 3-H2) 3.01 (1 H d J 5.9 a-H) 2.68ndash;2.49 (2 H m 1-H2) 1.81ndash;1.54 (3 H m 2-H2 and b-H) 1.50 (9 H s But) 1.29ndash;1.16 (2 H m g-H2) 1.00ndash;0.97 (3 H m g-H3) and 0.96ndash;0.93 (3 H m d-H3). Nmiddot;-4-(tert-Butoxycarbonyl)butylisoleucine 10e. (D2O; 298 K; as potassium salt) 2.95 (1 H d J 5.9 a-H) 2.50 (2 H m 1-H2) 2.30 (2 H t J 7.1 4-H2) 1.51 (5 H m 2- and 3-H2 and b-H) 1.45 (9 H s But) 1.22ndash;1.09 (2 H m g-H2) and 0.92ndash;0.85 (6 H m g-and d-H3).Nmiddot;-2-(Benzylthio)ethylleucine 10f. (D2O; 310 K; as sodium salt) 7.45ndash;7.33 (5 H m PhCH2) 3.79 (2 H s CH2Ph) 2.32 (1 H dd J1 6.2 J2 8.3 a-H) 2.71ndash;2.55 (4 H m 1- and 2-H2) 1.59ndash;1.56 (1 H m g-H) 1.44ndash;1.33 (2 H m b-H2) 0.91 (3 H d J 6.8 d-H3) and 0.89 (3 H d J 6.8 d-H3). Nmiddot;-3-(Benzylthio)propylleucine 10g. (D2O; 350 K; as sodium salt) 7.91 (5 H m PhCH2) 4.32 (2 H s CH2Ph) 3.54 (1 H t J 7.2 a-H) 3.07ndash;2.92 (4 H m 1- and 3-H2) 2.30ndash;2.14 (2 H m 2-H2) 2.14ndash;2.01 (1 H m g-H) 1.89 (2 H dd J1 3.4 J2 6.3 b- H2) and 1.40 (6 H m d-H3). Nmiddot;-3-(Boc-amino)propylleucine 10h. (D2O; 298 K; as sodium salt) 3.38 (1 H dd J1 6.0 J2 8.2 a-H) 3.18ndash;3.13 (2 H m 3-H2) 2.63ndash;2.48 (2 H m 1-H2) 1.75ndash;1.38 (5 H m 2- and b- H2 and g-H) 1.50 (9 H s But) and 1.00ndash;0.95 (6 H m d-H3).Nmiddot;-6-(Benzylthio)hexyl-Acirc;-Boc-lysine 10i. Solubility was too low in all attempted systems. Nmiddot;-4-(Benzylthio)butylmethionine 10j. (D2O; 298 K; as sodium salt) 7.37 (5 H m PhCH2) 3.77 (2 H s PhCH2) 3.10 (1 H t J 6.5 a-H) 2.53ndash;2.41 (6 H m 1- 4- and g-H2) 2.09 (3 H s e-H3) 1.86ndash;1.78 (2 H m b-H2) and 1.53 (4 H m 2- and 3-H2). Nmiddot;-3-(Boc-amino)propylmethionine 10k. (D2O; 298 K; as sodium salt) 3.41 (1 H dd J1 5.5 J2 7.4 a-H) 3.17ndash;3.13 (2 H m 3-H2) 2.65ndash;2.48 (4 H m g- and 1-H2) 2.17 (3 H s e-H3) 1.96ndash;1.86 (2 H m b-H2) 1.76ndash;1.61 (2 H m 2-H2) and 1.49 (9 H s But). Nmiddot;-2-(Benzylthio)ethylphenylalanine 10l. (D2O; 298 K; as sodium salt) 7.55ndash;7.36 (10 H m ArH) 3.84 (2 H s CH2Ph) 3.41 (1 H t J 6.7 a-H) 2.99 (2 H d J 6.6 1-H2) and 2.81ndash;2.65 (4 H m b- and 2-H2).Nmiddot;-3-(Benzylthio)propylphenylalanine 10m. (CD3)2SO; 350 K 7.30ndash;7.17 (10 H m ArH) 3.67 (2 H s CH2Ph) 3.35 (1 H t J 6.7 a-H) 2.92 (1 H dd J1 8.0 J2 7.1 b-H2) 2.77 (1 H dd J1 9.6 J2 8.4 b-H2) 2.66ndash;2.51 (2 H m 1-H2) 2.40 (2 H t J 7.2 3-H2) and 1.70ndash;1.55 (2 H m 2-H2). Nmiddot;-3-(Boc-amino)propylphenylalanine 10n. (D2O; 298 K; as calcium salt) 7.34ndash;7.22 (5 H m ArH) 3.29 (1 H t X of ABX J 7.3 a-H) 3.08ndash;2.97 (2 H m 3-H2) 2.90 (2 H dd AB of ABX Jab 6.7 Jbb 13.3 b-H2) 2.59ndash;2.48 (0.9 H m 1-H2-E) 2.46ndash;2.39 (1.1 H m 1-H2-Z) 1.70ndash;1.49 (2 H m 2-H2) and 1.39 (9 H s But). Nmiddot;-2-(Boc-amino)ethyl-O-benzylserine 10o. (D2O; 298 K; as potassium salt) 7.77 (5 H m ArH) 4.92 (2 H s CH2Ph) 4.05 (2 H t J 5.8 2-H2) 3.52 (1 H t J 4.7 a-H) 3.52 (2 H t J 6.0 1-H2) 3.12ndash;2.87 (2 H m b-H2) and 1.77 (9 H s But).Nmiddot;-4-(Benzylthio)butyl-NIn-formyltryptophan 10p. (D2O; 298 K; as sodium salt isomer ratio due to formyl protection E:Z = 1 1.78) 7.76 (2 H t J 7.4 Fmoc 4- and 5-H) 7.66 0.35 H J 6.6 Fmoc 1-H (E) 7.63ndash;7.51 1.65 H m Fmoc 1-H (Z) 7.43ndash;7.36 (2 H m Fmoc 3- and 6-H) 7.32 (2 H t J 8.1 Fmoc 2- and 7-H) 7.28ndash;7.26 (2 H m SBzl o-H) 7.24ndash;7.21 (2 H m SBzl m-H) 7.16ndash;7.14 (1 H m SBzl p-H) 6.89ndash;6.84 2.6 H m Ph (Z) 6.78 0.7 H d J 8.3 Ph o-H (E) 6.56 0.7 H d J 8.2 Ph m-H (E) 4.84 0.35 H dd J12 4.6 J22 10.7 Fmoc CH2 (E) 4.70 0.65 H dd J12 5.6 J22 10.7 Fmoc CH2 (Z) 4.58 0.35 H dd J12 4.8 J2210.7 Fmoc CH2 (E); 4.45 0.65 H dd J12 5.9 J22 10.7 Fmoc CH2 (Z) 4.23ndash;4.19 (1 H m Fmoc CH) 3.78 0.65 H m a-H (Z) 3.67 0.35 H m a-H (E) 3.59 0.7 H s SCH2Ph (E) 3.47ndash;3.39 (1.3 H m SCH2Ph (Z) 3.18ndash;3.15 1.3 H m b-H2 (Z) 3.11 0.35 H m 1-H2 (E) 2.99ndash;2.89 0.65 H m 1-H2 (Z) 2.83ndash;2.79 0.35 H m 1-H2 (E) 2.58 0.35 H m 2-H2 (E) 2.52ndash;2.46 0.65 H m 1-H2 (Z) 2.35ndash;2.29 0.35 H m 2-H2 (E) 2.23ndash;2.19 0.7 H m b-H2 (E) 2.07ndash;2.00 0.65 H m 2-H2 (Z) 1.97ndash;1.94 0.65 H m 2-H2 (Z) 1.33 5.85 H s But (Z) and 1.30 3.15 H s But (E).Nmiddot;-2-(Benzylthio)ethyl-O-tert-butyltyrosine 10q. (D2O; 350 K; as sodium salt) 7.25ndash;7.17 (5 H m SCH2Ph) 7.09 (2 H d J 8.3 Ph m-H) 6.87 (2 H d J 8.0 Ph o-H) 3.55 (2 H s CH2Ph) 3.1 (1 H dd a-H) 2.83 (1 H dd b-H2) 2.62 (1 H dd Jab 7.4 Jbb 13.4 b-H2) 2.56ndash;2.54 (1 H m 1-H2) 2.39 (3 H m 1-H and 2-H2) and 1.18 (9 H s But). Nmiddot;-3-(Boc-amino)propylvaline 10r. (D2O; 298 K; as sodium salt) 3.22ndash;3.12 (2 H m 3-H2) 2.92 (1 H d J 6.2 a-H) 2.69ndash; 2.49 (2 H m 1-H2) 1.69ndash;1.87 (1 H m b-H) 1.85ndash;1.61 (2 H m 2-H2) 1.51 (9 H s But) 1.02 (3 H d J 6.8 g-H3) and 0.98 (3 H d J 6.9 g-H3).1508 J. Chem. Soc. Perkin Trans. 1 1997 Method L. Simultaneous preparation of chiral Nmiddot;-cent;-Yalkyl amino acids 10 These compounds were prepared as in the above procedure (Method K); however the reactions were performed in polypropylene vessels arranged in an array and shaken either by a Genie Vortex 2 from Scientific Industries Bohemia USA or by an adhesive stripes Labotron shaker from INFORS AG Bottmingen Germany. Since this experiment was carried out only once the results are given here separately and the products are not denoted by numbers and letters. N,N-Bis-3-(benzylthio)propylasparagine (35) mp 167ndash; 168 8C; dH(D2O; 298 K; as sodium salt) 7.20ndash;7.12 (8 H m Ar 2- 3- 5- 6-H) 7.07ndash;7.04 (2 H m Ar 4-H) 3.57 (4 H m PhCH2) 3.49 (1 H t J 7.4 a-H) 2.48ndash;2.43 (5 H m 1- and b- H2) 2.37ndash;2.28 (5 H m 1- and b-H2) and 1.57ndash;1.39 (4 H m 2- H2).N-3-(Benzylthio)propylhomophenylalanine (29) mp 212ndash; 213 8C; dH(CD3)2SO; 340 K 7.31ndash;7.13 (10 H m ArH) 3.73 (2 H s PhCH2) 3.07 (1 H t J 6.4 a-H) 2.76ndash;2.59 (4 H m 1- and b-H2) 2.51ndash;2.47 (2 H m 3-H2) 1.92ndash;1.81 (2 H m g-H2) and 1.76ndash;1.71 (2 H m 2-H2). N-3-(Benzylthio)propylisoleucine (23) mp 218 8C NMR spectrum given above. N-4-(tert-Butoxycarbonyl)butylisoleucine (33) mp 230ndash; 231 8C; dH(D2O; 298 K; as potassium salt) 2.95 (1 H d J 5.9 a-H) 2.50 (2 H m 1-H2) 2.30 (2 H t J 7.1 4-H2) 1.51 (5 H s 2- and 3-H2 and b-H); 1.45 (9 H s But) 1.09ndash;1.22 (2 H m g-H2) and 0.85ndash;0.92 (6 H m g-H3 1 d-H3). N-3-(Benzylthio)propylmethionine (34) mp 219ndash;220 8C; dH(D2O; 298 K; as sodium salt) 7.42ndash;7.46 (5 H m PhH) 3.84 (2 H s PhCH2) 3.15 (1 H t J 6.7 H and aCH) 2.48ndash;2.63 (6 H m 1- 3- and g-H2) 2.15 (3 H s g-H3) and 1.74ndash;1.95 (4 H m 2- and b-H2).N-2-(Boc-amino)ethylmethionine (25) mp 240ndash;242 8C; dH(D2O; 298 K; as sodium salt) 3.39 (1 H t J 6.4 a-H) 3.19 (2 H m 2-H2) 2.71ndash;2.51 (4 H m g- and 1-H2) 2.13 (3 H s e-H3) 2.00ndash;1.82 (2 H m b-H2) and 1.46 (9 H s But). N-4-(tert-Butoxycarbonyl)butylmethionine (19) mp 227ndash;228 8C; dH(D2O; 298 K; as potassium salt) 3.13 (1 H t J 6.0 a-H) 2.48ndash;2.58 (4 H m 1- and g-H2) 2.30 (2 H t J 7.1 4-H2) 2.08 (3 H s SCH3) 1.78ndash;1.85 (2 H m b-H2) 1.58 (4 H s g-H2) 0.85ndash;0.92 (6 H m But). N-3-(Benzylthio)propylnorvaline (7) mp 224ndash;225 8C; dH(D2O; 298 K; as sodium salt) 7.43ndash;7.22 (5 H m ArH) 3.81 (2 H s PhCH2) 3.03 (1 H dd J1 5.4 J2 8.2 a-H) 2.56ndash;2.48 (4 H m 1- and 3-H2) 1.79ndash;1.66 (2 H m 2-H2) 1.64ndash;1.43 (2 H m b-H2) 1.355ndash;1.24 (2 H m g-H2) and 0.91 (t J 7.3 d-H3).N-3-(Benzylthio)propylphenylglycine (25) mp 199ndash; 200 8C; dH(CD3)2SO; 298 K 7.40ndash;7.22 (10 H m ArH) 4.21 (1 H s a-H) 3.67 (2 H s PhCH2) 2.77ndash;2.74 (1 H m 1-H2) 2.63ndash;2.60 (1 H m 1-H2) 2.37 (2 H t J 7.1 3-H2) and 1.83ndash;1.79 (2 H m 2-H2). N-3-(Benzylthio)propylvaline (25) mp 218ndash;219 8C; dH(D2O; 298 K; as sodium salt) 7.43ndash;7.22 (5 H m ArH) 3.81 (2 H s PhCH2) 2.82 (1 H t J 6.0 a-H) 2.56ndash;2.48 (4 H m 1- and 3-H2) 1.83ndash;1.80 (1 H m b-H) 1.77ndash;1.74 (2 H m 2-H2) and 0.96ndash;0.86 (6 H m g-H3). N-2-(Boc-amino)ethylvaline (26) mp 256ndash;258 8C; dH(D2O; 298 K; as sodium salt) 3.19 (2 H t J 6.2 2-H2) 2.84 (1 H d J 6.1 a-H) 2.64ndash;2.51 (2 H m 1-H2) 1.83 (1 H m b- H) 1.43 (9 H s But) and 0.94ndash;0.84 (6 H m g-H3).Method M. Protection of the secondary middot;-amino unit of 10 with an Fmoc group by temporary trimethylsilyl (TMS) protection to give 11 Bis(trimethysilyl)acetamide (BTSA) (4.33 ml 1.75 mol equiv.) and 1.74 ml (1 mol equiv.) of DIEA were added to 10 mmol of a substrate 10 suspended in 20 ml of DCM with exclusion of water by a CaCl2 drying tube. When the solution was nearly clear (5ndash;10 min were usually required) 2.72 g (1.05 mol equiv.) of fluoren-9-ylmethoxycarbonyl chloride (Fmoc- Cl) were added and the mixture was stirred for 2 h. Methanol (2 ml) was carefully added and the mixture was stirred for an additional 15 min diluted with 80 ml of DCM washed successively with 1 M HCl (3 times; 50 ml) and saturated aq.NaCl (2 times; 50 ml) and dried over MgSO4 and the solvent was evaporated off in vacuo. The crude product was crystallized from diethyl etherndash; LP. If the product was not sufficiently pure it was further puri- fied by chromatography. Physical data for compounds 11 are summarized in Table 2. Their NMR data and interpretation follow here. Nmiddot;-2-(benzylthio)ethyl-Nmiddot;-Fmoc-isoleucine 11b. dH(CDCl3; 298 K; isomer ratio E:Z = 1 6.14) 7.76 (2 H d J 7.5 Fmoc 4- and 5-H) 7.48 (2 H d J 7.5 Fmoc 1- and 8-H) 7.53 (2 H dd J1 7.4 J2 7.4 Fmoc 3- and 6-H) 7.33ndash;7.20 (7 H m 2- and 7-H and PhCH2) 4.65ndash;4.55 (2 H m Fmoc CH2) 4.19 (1 H t J 5.1 Fmoc CH) 3.96 0.14 H d J 10.0 a-H (E) 3.79ndash;3.73 0.86 H m a-H (Z) 3.52 (2 H s PhCH2) 3.37 0.27 H m 1-H2 (E) 3.25ndash;3.18 0.86 H m 1-H2 (Z) 2.96ndash;2.88 0.86 H m 1-H2 (Z) 2.62 0.28 H m 3-H2 (E) 2.25 1.73 H t J 8.0 3-H2 (Z) 1.95 0.86 H m b-H (Z) 1.68 0.14 H m b-H (E) 1.23ndash;1.18 (2 H m g-H2) 0.91ndash;0.86 2.59 H m g-H3 (Z) 0.83 0.41 H m g-H3 (E) 0.79ndash;0.76 2.59 H m d-H3 (Z) and 0.72 0.41 H m d-H3 (E).Nmiddot;-3-(benzylthio)propyl-Nmiddot;-Fmoc-isoleucine 11c. dH- (CD3)2SO; 298 K; isomer ratio E:Z = 1 1.94 7.88 0.68 H d J 7.2 Fmoc 4- and 5-H (E) 7.81 1.32 H d J 7.3 Fmoc 4- and 5- H (Z) 7.73 0.68 H m Fmoc 1- and 8-H (E) 7.62 1.32 H m Fmoc 1- and 8-H (Z) 7.38ndash;7.23 (9 H m Fmoc 2- 3- 6- and 7- H and PhCH2) 4.60ndash;4.56 (0.5 H m Fmoc CH) 4.44ndash;4.40 (0.5 H m Fmoc CH) 4.35ndash;4.31 0.68 H m Fmoc CH2 (E) 4.24ndash; 4.21 1.32 H m Fmoc CH2 (Z) 3.93 0.34 H d J 10.4 a-H (E) 3.85 0.66 H d J 10.4 a-H (Z) 3.70 0.68 H s PhCH2 (E) 3.61 1.32 H s PhCH2 (Z) 3.30 0.34 H m 1-H2 (E) 3.16 0.34 H m 1-H2 (E) 2.96 0.66 H m 1-H2 (Z) 2.81 0.66 H m 1-H2 (Z) 2.28 0.68 H m 3-H2 (E) 1.96 1.32 H m 3- H2 (Z) 1.62 (1 H m b-H) 1.42 0.68 H m 2-H2 (E) 1.15 3.32 m 2-H2 (Z) and g-H2 0.79 (3 H m g-H3) and 0.74 (3 H m d-H3).Nmiddot;-3-(Boc-amino)propyl-Nmiddot;-Fmoc-isoleucine 11d. dH(CDCl3; 298 K) 7.75 (2 H d J 7.4 Fmoc 4- and 5-H) 7.61ndash;7.56 (2 H m Fmoc 1- and 8-H) 7.41ndash;7.38 (2 H m Fmoc 3- and 6-H) 7.34ndash; 7.26 (2 H m Fmoc 2- and 7-H) 4.78 (0.75 H dd J1 4.7 J2 10.7 Fmoc CH2) 4.69 (0.75 H m Fmoc CH2) 4.56 (0.25 H m Fmoc CH2) 4.40 (0.25 H m Fmoc CH2) 4.21 (1 H m Fmoc CH) 4.10 (0.5 H m a-H) 3.80 (0.5 H m a-H) 3.27 (1 H m 1- H) 3.07 (2 H m 1- and 3-H) 2.76ndash;2.71 (2 H m 3- and b-H) 2.17ndash;1.71 (2 H m 2-H2) 1.44 (9 H s But) 1.21 (2 H m g-H2) 0.89 (3 H m g-H3) and 0.80 (3 H t J 7.0 d-H3).Nmiddot;-4-(tert-Butoxycarbonyl)butyl-Nmiddot;-Fmoc-isoleucine 11e. dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.86) 7.66 (2 H d J 7.2 4- and 5-H) 7.46 (2 H d J 7.2 Fmoc 1- and 8-H) 7.27ndash;7.31 (2 H m Fmoc 3- and 6-H) 7.17ndash;7.23 (2 H m Fmoc 2- and 7-H) 4.60 0.65 H s Fmoc CH2 (Z) 4.54 0.65 H s Fmoc CH2 (Z) 4.46 0.35 H s Fmoc CH2 (E) 4.33 0.35 H s Fmoc CH2 (E) 4.11 (1 H s Fmoc CH) 4.01 0.31 H d J 10.5 a-H (E) 3.87 0.69 H s a-H (Z) 3.14 0.48 H s 1-H2 (E) 2.89 0.73 H s 1-H2 (Z) 2.74 0.79 H s 1-H2 (Z) 2.12 0.7 H s 4-H2 (E) 1.93 2.2 H s 4-H2 (E) and b-H (Z) 1.74 0.25 H s b-H (E) 1.47 1.2 H s 2-H2 (E) and 3-H2 (E) 1.36 (9 H s But) 1.17 0.7 H s 3-H2 (Z) 1.13ndash;1.15 3.4 H m 2-H2 (Z) and g-H2 0.82ndash;0.83 (3 H m g-H3) and 0.71ndash;0.75 (3 H m d-H3).Nmiddot;-2-(Benzylthio)ethyl-Nmiddot;-Fmoc-leucine 11f. dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.86) 7.76 (2 H m Fmoc 4- and 5-H) 7.55 (2 H m Fmoc 1- and 8-H) 7.31 (2 H m Fmoc 3- and 6-H) 7.29 (2 H m Fmoc 2- and 7-H) 7.27ndash;7.21 (5 H m PhCH2) 4.66ndash;4.59 (2 H m Fmoc CH2) 4.54ndash;4.50 0.65 H m a-H (Z) 4.21 (1 H t J 5.7 Fmoc CH) 4.10 0.35 H m a-H (E) 3.73 0.7 H s PhCH2 (E) 3.54 1.3 H s and 0.35 H m PhCH2 (Z) and 1-H2 (E) 3.28ndash;3.20 0.65 H m 1-H2 (Z) 3.09 J. Chem. Soc. Perkin Trans. 1 1997 1509 0.35 H m 1-H2 (E) 2.95ndash;2.87 0.65 H m 1-H2 (Z) 2.69 0.35 H m 2-H2 (E) 2.53 0.35 H m 2-H2 (E) 2.36 0.65 H m 2- H2 (Z) 2.29ndash;2.33 0.65 m 2-H2 (Z) 1.73ndash;1.66 0.65 H m b- H2 (Z) 1.57ndash;1.48 0.65 H m b-H2 (Z) 1.45ndash;1.36 (1 H m g- H) 1.27ndash;1.26 0.7 H m b-H2 (E) 0.90ndash;0.84 3.9 H m d-H3 (Z) 0.75 1.05 H d J 6.1 d-H3 (E) and 0.71 1.05 H d J 6.2 d-H3 (E).Nmiddot;-3-(Benzylthio)propyl-Nmiddot;-Fmoc-leucine 11g. dH(CDCl3; 298 K; isomer ratio E:Z = 1 2.33) 9.79 (1 H br s CO2H) 7.60 (2 H d J 7.4 Fmoc 4- and 5-H) 7.44 (2 H d J 3.6 Fmoc 1- and 8-H) 7.26ndash;7.11 (9 H m PhCH2 and Fmoc 2- 3- 6- and 7-H) 4.53ndash;4.49 (2 H m Fmoc CH2) 4.36 (1 H dd J1 5.0 J2 7.5 a-H) 4.10ndash;4.06 (1 H m Fmoc CH) 3.56 0.6 H s PhCH2 (E) 3.49 (1.4 H s PhCH2 (Z) 3.33 0.6 H m 1-H2 (E) 2.98 0.7 H m 1-H2 (Z) 2.73 0.7 H m 1-H2 (Z) 2.26 0.6 H m 3- H2 (E) 2.05ndash;1.98 1.4 H m 3-H2 (Z) 1.63 0.6 H m 2-H2 (E) 1.55 (2 H m b-H2) 1.41ndash;1.35 2.4 H m 2-H2 (Z) and g-H 0.81 2.1 H d J 6.1 d-H3 (Z) 0.80 2.1 H d J 6.4 d-H3 (Z) 0.71 0.9 H d J 5.6 d-H3 (E) and 0.66 0.9 H d J 5.6 d-H3 (E). Nmiddot;-3-(Boc-amino)propyl-Nmiddot;-Fmoc-leucine 11h.dH(CDCl3; 298 K) 7.76 (2 H m Fmoc 4- and 5-H) 7.57 (2 H d J 7.4 Fmoc 1- and 8-H) 7.39 (2 H m Fmoc 3- and 6-H) 7.32 (2 H m Fmoc 2- and 7-H) 4.66 (2 H m Fmoc CH2) 4.52 (0.5 H m Fmoc CH) 4.38 (0.5 H m Fmoc CH) 4.22 (1 H m a-H) 3.41 (1 H m 1-H2) 3.03ndash;2.89 (3 H m 1-H and 3-H2) 2.77 (2 H m b-H2) 1.62 (2 H m 2-H2) 1.43 (9 H s But) 1.25 (1 H m g- H) and 0.88ndash;0.77 (6 H m d-H3). Nmiddot;-6-(Benzylthio)hexyl-Acirc;-Boc-Nmiddot;-Fmoc-lysine 11i. dH(CDCl3; 298 K; isomer ratio E:Z = 1 2.70) 7.67 (2 H d J 7.5 Fmoc 4- and 5-H) 7.49 (2 H d J 7.5 Fmoc 1- and 8-H) 7.33ndash;7.29 (2 H m Fmoc 3- and 6-H) 7.24ndash;7.19 (5 H m PhCH2) 7.18ndash;7.16 (2 H m Fmoc 2- and 7-H) 4.59ndash;4.53 (2 H m Fmoc CH2) 4.15 (1 H t J 5.4 Fmoc CH) 4.11ndash;4.05 0.73 H m a-H (Z) 3.98 0.27 H m a-H (E) 3.63 (2 H s PhCH2) 3.25 0.27 H m 1-H2 (E) 3.00 0.73 H m 1-H2 (Z) 2.99 (2 H t J 6.8 e-H2) 2.89 0.27 H m 1-H2 (E) 2.71 0.73 H m 1-H2 (Z) 2.31 (2 H t J 7.3 6-H2) 1.90 1.46 H m b-H2 (Z) 1.62 0.54 H m b-H2 (E) 1.41 (2 H m 2-H2) 1.39 0.54 H m d-H2 (E) 1.38 1.46 H m d-H2 (Z) 1.37 (9 H s But) 1.24 (2 H m 5-H2) 1.22 1.46 H m 4-H2 (Z) 1.20 1.46 H m g-H2 (Z) 1.19 0.54 m g-H2 (E) 1.13 0.54 H m 4-H2 (E) and 0.92 (2 H m 3-H2).Nmiddot;-4-(Benzylthio)butyl-Nmiddot;-Fmoc-methionine 11j. dH(CDCl3; 298 K; isomer ratio E:Z = 1 2.33) 9.54 (1 H br s CO2H) 7.73 (2 H d J 7.4 Fmoc 4- and 5-H) 7.56 (2 H d J 7.2 Fmoc 1- and 8-H) 7.40ndash;7.23 (9 H m ArH) 4.66ndash;4.57 (2 H m Fmoc CH2) 4.25 (1 H dd J1 5.0 J2 9.1 a-H) 4.21 0.7 H t J 5.2 Fmoc CH (Z) 4.17 0.3 H m Fmoc CH (E) 3.68 (2 H s PhCH2) 3.47 0.3 H m 1-H2 (E) 3.09ndash;3.04 1 H m 1-H2 (E) and 1-H2 (Z) 2.84ndash;2.80 0.7 H m 1-H2 (Z) 2.53ndash;2.48 0.7 H m g-H2 (Z) 2.41 0.6 H t J 6.9 4-H2 (E) 2.38ndash;2.30 2 H m g-H2 (Z) and b-H2 (E) 2.27 1.4 H t J 6.8 4-H2 (Z) 2.12ndash;2.05 1.4 H m b-H2 (Z) 2.07 2.1 H s e-H3 (Z) 2.02 0.9 H s e- H3 (E) 1.54 1.4 H m 3-H2 (Z) and 1.34ndash;1.27 2.6 H m 3-H2 (E) and 2-H2.Nmiddot;-2-(Benzylthio)ethyl-Nmiddot;-Fmoc-phenylalanine 11l. dH(CDCl3; 298 K; isomer ratio E:Z = 1 4.00) 8.98 (1 H br s CO2H) 7.64ndash;7.61 (2 H m Fmoc 4- and 5-H) 7.40ndash;7.38 (2 H m Fmoc 1- and 8-H) 7.27ndash;7.03 (12.6 H m ArH) 6.92ndash;6.90 (1 H m ArH) 6.58ndash;6.58 (0.4 H m ArH) 4.72 0.2 H dd J12 4.4 J22 10.6 Fmoc CH2 (E) 4.51 0.8 H dd J12 5.9 J22 10.7 Fmoc CH2 (Z) 4.40 0.2 H dd J12 4.4 J22 10.6 Fmoc CH2 (E) 4.30 0.8 H dd J12 6.1 J22 10.7 Fmoc CH2 (Z) 4.09ndash;4.04 (1 H m Fmoc CH) 3.90 0.8 H dd J1 4.6 J2 10.5 a-H (Z) 3.76 0.21 H m a-H (E) 3.470 0.4 H s PhCH2 (E) 3.31 1.6 H s PhCH2 (Z) 3.20ndash;3.06 1.6 H m b-H2 (Z) 2.91 0.8 H m 1-H2 (Z) 2.78 0.2 H m 1-H2 (E) 2.62 0.4 H m b-H2 (E) 2.50 0.8 H m 1-H2 (Z) 2.27 0.2 H m 1-H2 (E) 2.16 0.4 H m 2-H2 (E) and 2.00ndash;1.90 1.6 H m 2-H2 (Z).Nmiddot;-3-(Benzylthio)propyl-Nmiddot;-Fmoc-phenylalanine 11m. (CDCl3; 298 K; isomer ratio E:Z = 1 4.00) 9.00 (1 H br s CO2H) 7.65ndash;7.62 (2 H m Fmoc 4- and 5-H) 7.54ndash;7.42 (2 H m Fmoc 1- and 8-H) 7.31ndash;7.08 (12.6 H m ArH) 7.11ndash;6.99 (1 H m ArH) 6.65ndash;6.64 (0.4 H m ArH) 4.78 0.2 H dd J12 4.3 J22 10.6 Fmoc CH2 (E) 4.56 0.8 H dd J12 5.7 J22 10.7 Fmoc CH2 (Z) 4.47 0.2 H dd J12 4.3 J22 10.6 Fmoc CH2 (E) 4.36 0.8 H dd J12 5.8 J22 10.7 Fmoc CH2 (Z) 4.14ndash;4.08 (1 H m Fmoc CH) 3.93 0.8 H d J1 5.4 J2 9.9 a-H (Z) 3.77 0.2 H m a-H (E) 3.47 0.4 H s PhCH2 (E) 3.44 1.6 H s PhCH2 (Z) 3.25ndash;3.14 1.6 H m b-H2 (Z) 3.03ndash;3.00 0.2 H m 1-H2 (E) 2.90ndash;2.79 0.8 H m 1-H2 (Z) 2.53ndash;2.50 0.2 H m 1-H2 (E) 2.46ndash;2.39 0.8 H m 1-H2 (Z) 2.31 0.4 H m b-H2 (E) 2.17ndash;2.12 0.4 H m 3-H2 (E) 2.09ndash;1.89 1.6 H m 3-H2 (Z) 1.37ndash;1.34 0.4 H m 2-H2 (E) and 1.24ndash;1.12 1.6 H m 2-H2 (Z).Nmiddot;-3-(Boc-amino)propyl-Nmiddot;-Fmoc-phenylalanine 11n. The 1H NMR spectrum was identical with that of the compound which was previously prepared by the nucleophilic substitution method.26 O-benzyl-Nmiddot;-3-(Boc-amino)propyl-Nmiddot;-Fmoc-serine 11o. dH(CDCl3; 298 K; not enough resolution to determine the isomer ratio) 7.76ndash;7.72 (2 H m Fmoc 4- and 5-H) 7.56ndash;7.50 (2 H m Fmoc 1- and 8-H) 7.41ndash;7.36 (2 H m Fmoc 3- and 6-H) 7.35ndash;7.20 (7 H m Fmoc 2- and 7-H and PhCH2) 4.54ndash;4.44 (~1 H m Fmoc CH2) 4.51 (2 H s PhCH2) 4.37ndash;4.34 (~1.25 H m Fmoc CH2 and Fmoc CH) 4.28 (~1 H m a-H) 4.25ndash;4.22 (~0.25 H m Fmoc CH) 3.97ndash;3.95 (~0.5 H m Fmoc CH) 3.77 (~0.5 H m 1-H2) 3.64 (~1 H m b-H2) 3.51 (~0.5 H m 1-H2) 3.42 (~1 H m b-H2) 3.37ndash;3.27 (~2 H m 1-H and 2-H) and 3.06 (~1 H m 2-H2).Nmiddot;-4-(Benzylthio)butyl-Nmiddot;-Fmoc-NIn-formyltryptophan 11p. dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.86) 9.64 0.35 H s CHO (E) 9.24 0.65 H s CHO (Z) 8.24 0.65 H s indole (Z) 7.995 0.35 H s indole (E) 7.65 2 H m Fmoc 4- and 5- H 7.58 (1 H m indole) 7.44 (2 H m Fmoc 1- and 8-H) 7.44 (1 H m indole) 7.30 (2 H m PhCH2 o-H) 7.29 (2 H m Fmoc 3- and 6-H) 7.24 (2 H m Fmoc 2- and 7-H) 7.21 (1 H m PhCH2 p-H) 7.19ndash;7.15 (2 H m PhCH2 m-H) 7.14 (1 H m indole) 6.92 0.65 H indole 8-H (Z) 6.11 0.35 H m indole 8-H (E) 3.68 (2 H s PhCH2) 3.50 (1 H m a-H) 3.19ndash;3.09 (2 H m b-H2) 2.77ndash;2.69 (2 H m 1-H2) 2.40ndash;2.31 (2 H m 4- H2) and 1.55ndash;1.47 (4 H m 2- and 3-H2).Nmiddot;-2-(Benzylthio)ethyl-O-tert-butyl-Nmiddot;-Fmoc-tyrosine 11q. dH(CDCl3; 298 K; resolution too low to determine the isomer ratio) 7.76ndash;7.71 (2 H m Fmoc 4- and 5-H) 7.56ndash;7.50 (2 H m Fmoc 1-and 8-H) 7.41ndash;7.63 (2 H m Fmoc 3-and 6-H) 7.34ndash; 7.20 (7 H m Fmoc 2- and 7-H and PhCH2) 4.54ndash;4.44 (~1 H m Fmoc CH2) 4.51 (2 H s PhCH2) 4.37ndash;4.33 (~1.25 H m Fmoc CH2 and CH) 4.27 (1 H m aCH) 4.25ndash;4.22 (~0.25 H m Fmoc CH) 3.97ndash;3.95 (~0.5 H m Fmoc CH) 3.76 (0.5 H m 1CH2) 3.64 (1 H m bCH2) 3.51 (0.5 H m 1CH2) 3.42 (1 H m bCH2) 3.36ndash;3.26 (2 H 1CH2 and 2CH2) 3.06 (1 H m 2CH2) and 1.39 (9 H s But).Nmiddot;-3-(Boc-amino)propyl-Nmiddot;-Fmoc-valine 11r. dH(CDCl3; 298 K) 7.76 (2 H d J 7.4 Fmoc 4- and 5-H) 7.76ndash;7.52 (2 H m Fmoc 1- and 8-H) 7.42ndash;7.38 (2 H m Fmoc 3- and 6-H) 7.35ndash; 7.31 (2 H m Fmoc 2- and 7-H) 4.74 (1 H m Fmoc CH2) 4.62 (0.5 H m Fmoc CH) 4.46 (0.5 H m Fmoc CH) 4.22 (1 H m Fmoc CH2) 3.87 (0.35 H m a-H) 3.575 (0.65 H m a-H) 3.28 (0.7 H m 1-H2) 3.06 (1.3 H m 1-H2) 2.80ndash;2.74 (2 H m 3-H2) 2.32 (0.65 H m b-H) 2.055 (0.35 H m b-H) 1.73 (0.7 H m 2-H2) 1.44 (9 H s But) 1.25 (1.3 H m 2CH2) 0.95 (1.95 H d J 6.4 g-CH3) 0.903 (1.05 H m g-H3) 0.724 (1.95 H d J 6.4 g-H3) 0.655 (1.05 H m g-H3). Method N. Protection of the secondary middot;-amino group unit of 10 with the Boc group by temporary TMS protection to give 12 This procedure was identical with the latter (Method M) except for the addition of di-tert-butyl dicarbonate instead of Fmoc- 1510 J.Chem. Soc. Perkin Trans. 1 1997 Cl and washing with saturated aq. KHSO4 instead of HCl. Physical data for compounds 12 are summarized in Table 2. Their NMR data and interpretation follow here. Nmiddot;-6-(Benzylthio)hexyl-Nmiddot;-Boc-glutamic acid ldquor;-benzyl ester 12a. dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.08) 7.29ndash;7.19 (9 H m ArH) 7.19ndash;7.16 (1 H m ArH) 5.06 (2 H s OCH2Ph) 4.03 0.52 H m a-H (Z) 3.90 0.48 H m a-H (E) 3.63 (2 H s SCH2Ph) 3.37 0.52 H m 1-H2 (Z) 3.23 0.52 H m 1-H2 (Z) 2.86 0.96 H m 1-H2 (E) 2.40 (2 H t J 6.4 g-H2) 2.32 (2H t J 7.0 6-H2) 2.32 0.96 H m b-H2 (E) 2.08 1.04 H m b-H2 (Z) 1.56ndash;1.43 (2 H m 5-H2) 1.43 1.04 H m 2-H2 (Z) 1.39 0.96 H m 2-H2 (E) 1.38 (9 H s But) 1.30ndash;1.22 (2 H m 4-H2) 1.17ndash;1.13 (2 H m 3-H2).Nmiddot;-2-(Benzylthio)ethyl-Nmiddot;-Boc-leucine 12f. dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.04) 7.97 (1 H br s CO2H) 7.32ndash; 7.23 (5 H m PhCH2) 4.46 0.51 H m a-H (Z) 4.16 0.49 H m a-H (E) 3.75 0.98 H s PhCH2 (E) 3.74 1.02 H s PhCH2 (Z) 3.61 0.49 H m 1-H2 (E) 3.40 0.49 H m 1-H2 (E) 3.09 1.02 H m 1-H2 (Z) 2.72 1.02 H m 2-H2 (Z) 1.78ndash;1.71 (1 H m b-H) 1.64ndash;1.47 (2 H m b- and g-H) 1.44 (9 H s But) 0.91 1.47 H d J 4.7 d-H3 (E) and 0.90 1.53 H d J 4.7 d-H3 (Z). Method O. Preparation of Nmiddot;-cent;-(benzylthio)alkylglycines 10sndash;10u Glyoxylic acid (0.78 g 10.5 mmol) was added to a stirred solution of 10 mmol of an w-(benzylthio)alkylamine 26 and 63 mg (3.3 mmol 1 mol equiv.) of sodium cyanoborohydride in 20 ml of methanol. The mixture was stirred overnight.The precipitated product was filtered off on a glass sinter washed with methanol and dried in vacuo. Nmiddot;-2-(Benzylthio)ethylglycine 10s. dH(D2O; 298 K) 7.413 (5 H m PhCH2) 3.835 (2 H s a-H3) 3.521 (2 H s PhCH2) 3.151 (2 H t J 6.7 1-H2) and 2.781 (2 H t J 6.8 2-H2). Nmiddot;-3-(Benzylthio)propylglycine 10t. dH(D2O; 298 K) 7.392 (5 H m PhCH2) 3.800 (2 H s a-H2) 3.540 (2 H s PhCH2) 3.043 (2 H t J 7.7 1-H2) 2.564 (2 H t J 7.1 3-H2) and 1.986ndash;1.840 (2 H m 2-H2). Nmiddot;-4-(Benzylthio)butylglycine 10u. dH(D2O; 298 K) 7.384 (5 H m PhCH2 3.783 (2 H s a-H2) 3.557 (2 H s PhCH2) 2.980 (2 H t J 7.4 1-H2) 2.515 (2 H t J 6.8 4-H2) and 1.724ndash;1.613 (4 H m 2- and 3-H2). Method P. Preparation of Nmiddot;-cent;-(benzylthio)alkyl-Nmiddot;-Fmocglycines 11sndash;11u Triethylamine (TEA) (5.6 ml 40 mmol) and 6.42 g (20 mmol) of N-(fluoren-9-ylmethoxy)succinimide (Fmoc-OSu) in 120 ml of acetonitrile were added to a solution of 20 mmol of a substrate of 10sndash;10u in 60 ml of water.The reaction mixture was stirred at room temperature for 4 h. Then 180 ml of water were added and the solution was washed successively with LP (3 times; 100 ml) and with a mixture of 3 7 diethyl etherndash;LP (3 times; 100 ml). The aqueous solution was acidified with 40 ml of 1 M HCl and extracted with ethyl acetate (4 times; 100 ml). The combined organic solution was washed with 50 ml of saturated aq. NaCl dried over MgSO4 and evaporated in vacuo. The products were crystallized from diethyl etherndash;LP. The spectrum of compound 11s prepared by the nucleophilic substitution method has been published.26 Nmiddot;-3-(Benzylthio)propyl-Nmiddot;-Boc-glycine 11t.dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.50) 7.31ndash;7.23 (5 H m PhCH2) 3.92 1.2 H s a-H2 (Z) 3.84 0.8 H s a-H2 (E) 3.70 2 H s PhCH2 3.32 (2 H t J 6.7 1-CH2) 2.44ndash;2.41 (2 H m 3-H3) 1.82ndash;1.68 (2 H m 2-H2) 1.45 5.4 H s But (Z) and 1.42 3.6 H s But (E). Method Q. Preparation of Nmiddot;-cent;-(benzylthio)alkyl-Nmiddot;- Boc-glycines 12sndash;12u These products were prepared according to the procedure of Bodanszky and Bodanszky.26 The spectra of compounds 12s and 12t prepared by the nucleophlic substitution method have been published. Nmiddot;-4-(Benzylthio)butyl-Nmiddot;-Boc-glycine 12u. dH(CDCl3; 298 K; isomer ratio E:Z = 1 1.50) 9.39 (1 H br s CO2H) 7.35ndash; 7.20 (5 H m PhCH2) 3.95 1.1 H s a-H2 (Z) 3.84 0.9 H s a-H2 (E) 3.67 (2 H s PhCH2) 3.23ndash;3.20 (2 H m 1-H2) 2.40 (2 H t J 6.5 4-H2) 1.53 (4 H m 2-and 3-H2) 1.43 4.1 H s But (E) and 1.40 4.9 H s But (Z).References 1 Part 3 D. Muller I. Zeltser G. Bitan and C. Gilon J. Org. Chem. 1997 62 411. 2 C. Gilon D. Halle M. Chorev Z. Selinger and G. Byk Biopolymers 1991 31 745. 3 C. Gilon I. Zeltser V. Rashti-Behar D. Muller G. Bitan D. Halle G. Bar-Akiva Z. Selinger and G. Byk in Peptide Chemistry 1992 ed. N. Yanaihara ESCOM Leiden 1993 pp. 482ndash;485. 4 Y. Ovchinnikov G. Chipens and V. Ivanov in Peptides 1982 Walter de Gruyter Berlin 1983 pp. 1ndash;18. 5 C. Gilon D. Halle M. Chorev Z. Selinger and G. Byk in Peptides Chemistry and Biology ESCOM Leiden 1992 pp. 476ndash;477. 6 S. Reissmann G. Greiner J. Jezek C. Amberg B. Muuml;ller L. Seyfarth L. F. P. De Castro and I. Paegelow Biomed. Pept. Proteins Nucleic Acids 1995 1 51. 7 G. Byk and C. Gilon J.Org. Chem. 1992 57 5687. 8 G. Bitan and C. Gilon Tetrahedron 1995 51 10 513. 9 R. W. Feenstra E. H. M. Stokkingreef R. J. F. Nivard and H. C. J. Ottenheijm Tetrahedron 1988 44 5583. 10 H. Wurziger Kontakte (Darmstadt) 1987 8. 11 J. A. Fehrentz and B. Castro Synthesis 1983 676. 12 D. Julie M. Mayer P. Schmitt G. Drapeau D. Regoli and R. Mickelot Eur. J. Med. Chem. 1991 26 921. 13 M. Rodriguez A. Heitz and J. Matinez Int. J. Pept. Protein Res. 1992 39 273. 14 T. Shimizu R. Kobayashi H. Ohmori and T. Nakata Synlett 1995 650. 15 M. Kolb J. Barth J. G. Heydt and M. J. Jung J. Med. Chem. 1987 30 267. 16 J. L. Herrmann G. R. Kieczykowski R. F. Romanet P. J. Wepplo and R. H. Schlessinger Tetrahedron Lett. 1973 4711. 17 Y. Ohfune N. Kurokawa N. Higuchi M. Saito M. Hashimoto and T. Tanaka Chem.Lett. 1984 87 441. 18 D. R. Bolin I. Sytwu F. Humiec and J. Meienhofer Int. J. Pept. Protein Res. 1989 33 353. 19 G. Byk PhD. thesis submitted to the senate of The Hebrew University of Jerusalem 1990. 20 R. J. Simon R. S. Kania R. N. Zuckermann V. D. Huebner D. A. Jewel