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ISSN 1070-3632, Russian Journal of General Chemistry, 2010, Vol. 80, No. 12, pp. 2590­2606. © Pleiades Publishing, Ltd., 2010. Original Russian Text © E. V. Babaev, 2009, published in Rossiiskii Khimicheskii Zhurnal, 2009, Vol. 53, No. 4, pp. 57­72.

Solid-Phase Synthesis for Beginners: Choice of Tools and Techniques for Implementation of Multistage Transformations
E. V. Babaev
Lomonosov Moscow State University, Vorob'evy Gory 1, Moscow, 119991 Russia e-mail: babaev@org.chem.msu.ru
Received July 10, 2009

AbstractSeveral mini-libraries were prepared by solid-phase synthesis: C,N-substituted glycines (1) and 2,5diaminobenzoic acids (2) using Wang resin as well as diarylthioureas (3) and diaryl-guanidines (4) using Rink resin. Comparative analysis of the instruments and technique for these reactions was provided by applying glass vials with filters (BillBoard kit) to prepare the class (1), the tea-bag technique for the class (2), and plastic rods (Lanterns) for the classes (3) and (4). All reactions (supplied with detailed protocols) were optimized for use as a tool of practical student education.

DOI: 10.1134/S1070363210120261 In [1] we remembered the ABC of solid-phase synthesis and dwelt in detail on the experimental problems associated with this technique. The principal inconvenience in the practical work with polystyrene resins is their tendency to swell in solvents. Solvents (and reagents) penetrate into the polymer globules, thus sharply increasing their volume. The gelatin-like nature of the swollen polymer makes it difficult to transfer from one vial to another or to a filter. In the present paper we analyze approaches to solving this problem. Three training tasks tested at the special student laboratory of combinatorial chemistry at the Moscow State University (MSU) were used as an example. In this work, we acquired a certain experience which can be useful for teachers and synthetic organic chemists. Below we consider three examples of tools facilitating manipulations with polymer supports. In the first example, some problems are approached by the use of a container for resins and the BillBoard kit in which reaction vials are combined with filters. The chemical transformation is represented by double modification of a resin-immobilized protected glycine (consecutive -alkylation and N-acylation). In the second example (the tea-bag technique), a resin is contained in a porous plastic bag, and the chemistry is presented by a multistage sequence of transformations. In the third example we consider the methodology of operation with specially modified polypropylene pins (lanterns), a new "synthesis philosophy" which radically changes all traditional views on modern organic synthesis. This technique was employed in a multistage (5­6 stages) synthesis of thioureas and guanidines. The three examples all represent versions of training tasks. BillBoard Technique Let us consider a four-stage modification of glycine (containing benzophenone protection) immobilized on the Wang resin. The main feature of the task is the use of a simple technical tool, the BillBoard kit, which is a specially designed set of filter tubes and convenient plastic facilities for stirring and filtering in several reaction vials. This is an inexpensive kit (if desired, its analog can be readily made) allows one to facilitate and speed-up manipulations when performing concurrent solid-phase reactions. The suggested task was tested by a group of MSU students in the special laboratory course on combinatorial chemistry in 2005. To fulfill the task, three laboratory lessons and a final seminar for purity analysis of the synthesized products (lesson 4) are required. Chemical aspect of the task. The possibility of alkylation of N-substituted amino acids immobilized

2590


SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS Scheme 1.

2591

H2N O

Y

Ph2C=NH, AcOH NMP

Ph Ph N O Y

RX, BEMP NMP

Ph Ph N

R Y O

H3O+, THF

R H2N O Y

= Merrifield, Wang, or Rink resin Y =O, N or polypeptide chain N PN N

N BEMP =

, NMP = N-methylpyrrolidin-2-one

on a support has first been demonstrated 10 years ago [2] (Scheme 1). The amino group in the resin-immobilized glycine is protected with the benzophenone fragment. The 2 group of the resulting imine acquires sufficient acidity characteristic of azaallyl systems, and under the action of a strong base, for example, a substituted phosphazene (), it can be deprotonated and then alkylated. Since the first publications this line of research has substantially extended: Scott, O'Donnell, and coworkers [3­7] have published a big series of papers on the methodology of solid-phase synthesis by this reaction. Analogous reaction could be performed with low-active alkyl halides, as well as with alkenes (Michael reaction). Depending on the nature of the resin, different classes of compounds can be prepared: amides with the Rink resin and aminoaldehydes and aminoketones with the Weinreb resin. It was found that using strong bases allows preparation of dialkylated amino acids. The resulting immobilized intermediates can be reacted with nucleophiles or converted into more complex acyclic and cyclic systems, for example, proline derivatives, lactams, and hydantoins. Specific features of the BillBoard kit. The facility and reproducibility of chemical transformations involving immobilized benzophenone imine derivatives of amino acids promted Prof. Scott to adapt these sequences for the student laboratory course in solidphase synthesis at the Indiana University (USA). The BillBoard training kit was constructed to facilitate concurrent manipulations with resins. BillBoard is a specially designed set of filter tubes (Fig. 1) with double-sided plastic screw caps with

Teflon septa (Fig. 1b). As a result, each vial plays the role of either a reaction vessel (when the caps are screwed) or a filter (when the caps are removed). The filter is soldered unsymmetrically, and the solid support is placed in a larger vial compartment. The tubes are fixed in a special plastic holder (BillBoard as such, Figs. 1b and 1c) which can be used both as a stand (on adding reagents, washing tube contents, and filtering) and as a block for simultaneously rotating and agitating six vials. Two convenient plastic accessories (Fig. 1c) serve as receivers for washings or stands for collection of final cleavage products, respectively. For agitating several BillBoards one can use simple accessories (Fig.1d, see also Figs. 2e and 2f). A complete synthetic kit costs 175 USD, it is designed for multiple use and allows 6 concurrent combinatorial solid-phase reactions. The simplicity and low cost of the kit allowed successful solid-phase combinatorial syntheses to be performed by students and PhD students not only at the Indiana University, but also at the universities of Barselona (Spain) and Lublin (Poland). In 2005, the BillBoard was acquired by the special student laboratory of combinatorial chemistry at the MSU, and in the framework of Prof. Scott's visit to Moscow we practically implemented the task with a group of students and PhD students of the Chemical Department. Brief description of the task. The task involves four stages: (1) alkylation of the H2 group of an amino acid immobilized on the Wang resin (W); (2) removal of the protective group; (3) acylation of the NH2 group of the amino acid; and (4) removal of the doubly modified amino acid from the support (Scheme 2). Each student performed simultaneously six syntheses, using three benzylating and two acylating agents,

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2592

BABAEV Scheme 2.
Ph N Ph
R1X (2 equiv) BEMP, NMP 24 h, 25°C

Ph N O W II

Ph R1
HCl/THF 25°C, 20 min

R2 H3N+ O W III R1
R2COCl DIEA, NMP 24 h, 25°C

O R1
TFA 25°C, 30 min

R2 HN O

O R1

HN O W IV

O W I

OH V

in a single BillBoard labeled according to Scheme 3 (, and I, II, III). Reagents: resin I (protected glycine on the Wang resin) 2 g, phosphazene base (Aldrich-79432, see the formula in Scheme 1) 5 ml, diisopropylethylamine (DIEA) 100 ml, TFA (99%) 100 g, and aqueous l (1 N) 50 ml. Solvents: 1l of N-methylpyrrolidin-2-one (NMP) and by 100 ml of dichloromethane (DCM), THF, and DMF (the latter is needed for rinsing caps of reaction vials). The alkylating agents were three benzyl bromides (from 1 to 5 g): unsubstituted benzyl bromide and its

m-bromo and p-trifluoromethyl derivatives; at the MSU we used additionally a pyridine analog, specifically 2-chloro-5-(chloromethyl)pyridine. Compounds with a high molecular weights, 9-fluorenyl chloroformate (Fmoc-Cl) and 2-naphthoyl chloride (by 5 g), were recommended as acylating agents. Lesson 1. Alkylation (1) Preparation of an isopycnic solution of glycine imine I immobilized on the Wang resin, based on 65 mg (50 mmol) per one experiment (resin capacity 0.77 mmol g­1). Two solvents with different densities, for example, THF and DCM, are added to a dry resin. By varying their ratio one can obtain a homogeneous (as much as practical) gel, when resin particles neither float nor precipitate. At the beginning of the first laboratory lesson students get 6 reaction vials placed in a BillBoard and a solution of resin I (Fig. 2). (2) The required volume of the isopycnic solution distributed between vials, after which the solvent allowed to drain. To expel residual solvent, a simp pump can be used. The resin in each reaction vessel is is le is

(a)

(b)

(c)

(d) Scheme 3.

Stage 1 (alkylation) three benzylating agents
1

R1Br 2R1Br 3R1Br

Stage 3 (acylation) Two acylating agents
1

Fig. 1. BillBoard kit: () double-ended filter vials; (b) fixing of reaction vials; and (c) additional plastic collectors: (left) for washings, (right) for final products, (bottom) BillBoard ready for fixing; and (d) option for simultaneous agitation of six reaction vials. A simple facility allows to fix eight BillBoards and simultaneously agitate 48 vials.

A B

A1 B1 I

A2 B2 II

A3 B3 III

R2Cl R2Cl

2

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SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS

2593

washed with NMP (3â3 ml) by means of 3.5-ml plastic Beral pipette. At all stages of the work, one should meet the following instructions. I. General washing procedure. Washings should be always performed in a strictly specified order. The washing liquid, ~3 ml (~ 80% the total volume of the reaction vial), is added with a 3.5-ml pipette. Wait for 30 s to let the solvent to drain through the filter under the gravitation force. Then the resin should be dried completely (pump purging) (Fig. 2b). A glass should be put beneath the drain pan plug. Finally, the liquid in the glass should be poured into a waste container. II. Use of air pump. The "pump" is a piece of a rubber tube with one end of each plugged with a plastic Beral pipette and the second end attached to a septa with a through slot. When the solvent has drained through the filter (within 30 s), attach the pump (from the septa side) to the vial neck and press the extended part of the pipette. To avoid back soaking of the solvent from the vial to tube, raise slightly the septa over the neck and only then unfold fingers. Repeat the procedure until all solvent drains through the filter. III. Manipulations with caps of reaction vials. Before screwing the cap remove solvent residues from the space between the cap and vial by means of a soft cloth. Before unscrewing turn the side of the BillBoard to be opened up. Unscrew the cap, turn the BillBoard
(a) (b)

around and put it into the pan. Shake BillBoard several times to make sure that all gel residues are removed from inner side of the cap. Open the top caps of all reaction vials and put them into a glass for further use. (3) Mount the BillBoard to a special holder. Screw the bottom caps (closest to soldered-in filters) of each reaction vial (Fig. 2c). (4) Prepare three calibration Beral pipettes for adding benzylating agents 1R1­Br, 2R1­Br, and 3R1­Br (separate pipette for each reagent). Safety measures: glasses and gloves! (5) Add by 0.5 ml of 0.2 solutions of alkylating agents in NMP (100 mmol, 2 equiv). Respectively, 1 R1­Br is added to the reaction vials in the first vertical column (1 and 1), 2R1­Br, to the vials in the second vertical column (2 and 2), and 3R1­Br, to 3 and 3 (Scheme 4). Add by 0.5 ml of a 0.2 solution of B in NMP (100 mmol, 2 equiv) in each of the six reaction vials. Screw the top caps of the BillBoard and mount it to a rotating apparatus (Figs. 2e and 2f). Record reaction initiation time and the BillBoard number. Reaction time 24 h. This reaction (and all subsequent reactions) are performed at room temperature. Before leaving the laboratory, wash the drain tray with acetone over the waste container.
(c)

(d)

(e)

(f)

Fig. 2. Features of practical implementation of the task: () addition of reagents at the first stage (lower caps are closed); (b) pumpdrying of resins on filters; (c) view of a closed BillBoard during reaction, (d) removal of products from resins (under hood!), (e, f) different variants of mixing in 6 reaction vials. RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010


2594 Scheme 4.
Ph Ph N O I
R1X (2 equiv) BEMP (2 equiv.) NMP, 24 h, 25°C

BABAEV

W

(150 mmol, 3 equiv) to each of the six reaction vials. Screw the top caps. The reaction is performed for 24 h in the rotating apparatus (Scheme 6).
Scheme 6.
R1 W O III
O II
R2COCl (2 equiv) DIEA (2 equiv) NMP, 24 h

Ph Ph N

R1

W

H3N+

O R2 N H

R1 O IV

W

Lesson 2. Protection Removal and N-Acylation (6) Record reaction completion time and take the BillBoard out of the rotating apparatus. Unscrew the caps as recommended in Instruction III. (7) Wash out excess reagents from the resin. Wash the alkylated resin (product II) one time with 3 ml of THF and pump-dry (Instruction II). (8) Mount the BillBoard on a holder. Take 12 clean caps, screw the bottom caps of each reaction vial and add about 2.5 ml of a 1 N aqueous solution of a 1:2 HCl­THF mixture to each vial. Screw the top caps and place in the rotating apparatus for 20 min (Scheme 5).
Scheme 5.
Ph Ph N O II R1
1 N HCl/THF (1:2) 25°C, 20 min

Lesson 3. Removal of N,C-Substituted Amino Acid from Support (12) Take the BillBoard out of the rotating apparatus and unscrew caps (Fig. 2d). (13) Filter off and wash product IV with NMP (2â3 ml), THF (2â3 ml), and CH2Cl2 (3â3 ml). (14) Mount the BillBoard on a holder. Screw the bottom end with clean caps. Add 2 ml of CF3COOH/ H2O (95:5) to each vial. Take caution! Screw the top caps and place the BillBoard in a rotating apparatus for 30 min (Scheme 7).
Scheme 7.
O R2 N H IV
CF3COOH/H2O (95:5) 30 min

R1

W

R1 O O R2 N H

W

W

H3N

+

O III

R1 OH O V

(9) Unscrew all caps. (10) Filter off and wash product III in succession with 3 ml of THF and 3 ml of NMP (one time). (11) Mount the BillBoard on a holder. Take 12 clean caps. Screw the bottom caps of each reaction vial and add 0.5 ml of a 0.2 solution of the first acylating agent 1R2­COCl in NMP (100 mmol, 2 equiv) to resin III in the three reaction vials in the first horisontal row (1, 2, and 3). Then add 0.5 ml of a 0.2 M solution of the second acylating agent 2R2­COCl in NMP (100 mmol, 2 equiv) to resin III in the three reaction vials in the second horizontal row (1, 2, and 3). Then add 0.5 ml of a 0.3 solution of DIEA in NMP

(15). As the cleavage reaction is in progress, prepare six weighed receiver vials, label them (11, 12, etc.), and place in a 6-hole rack. (16) Products V which are now in a solution (therefore, one should preserve the filtrate) transfer to the vials. To this end, turn the Billboard over (the bottom caps up), unscrew these caps, and mount receivers on the reaction vials. Make sure that the labels on the reaction and receiver vials are the same. Having accommodated all vials, mount the rack on them, and turn over the whole construction. Unscrew

RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010


SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS Scheme 8.
R1a

2595

HN

O O F VIII

O N+

O-

R1b IX THF

O O aR1

O N+

O-

N R1b

X

O

SnCl2·2H2O DMF R2

O O aR1

O N

R2 R2 O

Cl

XII

O O aR1 NH2 N R1b XI

Py DCM

N R1b XIII
NH3 aq. DMF

O O aR1

R2

O NH
10% TFA/DCM

OH O aR1

R2

O NH +

N R1b XIV

N R1b XV

OH

the upper caps and collect the filtrate into vials, using a pump. (17) Wash the resins one CF3COOH­H2O (95:5) and one CH2Cl2, collecting washings into time pump the solvent thoroughly time with 2 ml of time with 2 ml of receiver vials. Every down.

Lesson 4. Determination of Product Yields and TLC Analysis (20) Weight all final products. (Taking into account that the reactions are performed on a 50 µmol scale, and the molecular weights of products vary in the range 300­400 g mol­1, the theoretical yield in each reaction should be 15­20 mg). The real yield is 10­15 mg. (21) For TLC dissolve each product 0.1 ml of THF per 1 mg of product) and CHCl3/THF/CH3COOH (85/15/2). chromatograms first under UV light iodine. Record Rf in both cases. As earlier synthesized samples. in THF (about use the system Develop the and then with references use

(18) Take the BillBoard off from the vials. Transfer by 0.1 ml of each sample of product V into vials for subsequent LC/MS analysis (Fig. 3). The vials with final products transfer to a vacuum drying oven for evaporation. (Evaporation can also be performed on a rotary evaporator in weighed flasks). (19) Wash the drain pan and BillBoard with acetone.

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2596

BABAEV

Fig. 3. Example LC­MS data for sample 1 (purity 95%, calculated for C25H20F3NO4: 455).

The results obtained in the same combinatorial task in several universities all over the world formed the basis for quite an unusual concept named "Distributed Drug Discovery." Its essence consists in that students of different universities fulfilling the same training task obtain eventually a fairly representative combinatorial library of potential drugs. Synthesis and subsequent testing are planned from a united center

[8]. The contribution of the Moscow team into this project is described in a separate paper [9]. Tea-Bag Technique This technique is a result of a long-term collaboration between the Chemical Diversity Research Institute (CDRI, Khimki) and Higher Educational Institutions of Moscow. The laboratory work was

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SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS (a) (b)

2597

(c)

(d)

Fig. 4. Scheme of the tea-bag technique.

developed based on the practical experience in solidphase synthesis, accumulated at the CDRI. Students of the MSU, involved in fulfilling the tasks of the special laboratory course in combinatorial chemistry could get familiar with the CDRI's procedures during excursions to this institute or Open Days (held since 2004). Below we describe the procedure of a student training task for such special laboratory courses. The laboratory work is based on a technique widely used in solid-phase synthesis and called the tea-bag technique. The container for resin here is a bag of a porous plastic film sealed over the entire perimeter, for example, using a simple hot cutter (Fig. 4a). Such bags (Fig. 4b) can be easily made, put in a great number of vials to add similar reagents (Fig. 4c) or combined and put in one vial (Fig. 4d) to add the same reagent or at repeated resin washing stages. Naturally, the resin swelling effect is unavoidable, but here one can fully avoid resin sticking to glass vial walls. Morever, bags can function as a vessel and a filter simultaneously. Reaction sequence. As a model chemical reaction we chose a sequence shown in Scheme 8. 2-Fluoro-5nitrobenzoic acid, a compound containing a halogen activated for nucleophilic substitution and a nitro group which can be readily reduced to form an amino function and then acylated by the latter, is immobilized on the Wang resin. Theoretically, the starting acid a three-point template, since the third function

(carboxyl), too, can be easily converted into, for example, an amid one. However, in the task under consideration, a shortened sequence is used, and the target products are substituted 2,5-diaminobenzoic acids XV. Required reagents and equipment. The Wang resin, film for bags, and instrumentation for fabrication of the latter are required, as well as a large volume of different solvents, a series of secondary aliphatic amines, chlorides of aromatic (including heteroaromatic) carboxylic acids, SnCl2, pyridine, acetic anhydride, and trifluoroacetic acid. The work is performed on a shaker (mostly at room temperature); varied-size capped vials are used as reservoirs for bags with resins. Stage 1. Immobilization of 2-fluoro-5-nitrobenzoic acid on the Wang resin. Wang resin VII (50 g, capacity 2.0 0.77 mmol g­1) is suspended in a solution of 0.2 mol of 2-fluoro-5nitrobenzoic acid VI (37.41 g) and 25 mg of 4-(dimethylamino)pyridine in 400 ml of absolute dichloromethane (DCM), after which 31.5 ml (0.2 mol) of diisopropylcarbodiimide was added with caution. The reaction mixture is shaken for 2 days at room temperature. The resin is filtered off and washed with solvents in the following order: 2âDCM, DMF, MeOH, 2âDCM, 2âMeOH, 2âDCM, and 2âhexane, and vacuum-dried. Resin VIII is ready for further use.

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2598

BABAEV

OH O F VI

O N

O

+

OH VII

DIC DMAP DCM

O O F VIII

O N

OH

partially destroy the ester bond which links the substituted benzoic acid to polymer. As a result, part of hydroxymethyl groups of the resin get free and can function as a competitive center at the subsequent acylation stage. Since the acylating agent at stage 4 are derivatives of aromatic acids, the introduced acyl residues will persist on the resin until the last stage and contaminate the final product at the linker cleavage stage. Therefore, the hydroxymethyl groups should be protected by a "harmless" residue of a water-soluble acid, say, acetic. In practice, the resins are placed in a pyridine solution and then acetic anhydride is added. The quantities of reagents are estimated as follows: 5 equiv (1.9 mmol, 0.204 ml) of pyridine in abs. DCM per one bag and 5 equiv (1.9 mmol) of acetic anhydride. The vials are agitated in a shaker for 18 h at room temperature. The resins are washed in succession with 2âTHF, DMF, 2âMeOH, 2âDCM, and 2âhexane and vacuum-dried to obtain products X. Stage 3. Reduction of nitro group.
O O R1a O N+

Stage 2. Substitution of fluorine by dialkylamino group.
O O F VIII O
THF

O N

OH

R 1a + R1b IX O N

NH

O-

O R1a

OH

N R1b X O

N R1b X

Resin VIII is distributed over plastic bags (Figs. 4a and 4b) whose number is equal to the number of final products. The resin weigh is a bag is 250 mg (capacity 1.5 0.77 mmol g­1). According to the statements of work (which specify what amine to take and how many experiments to perform), the bags are distributed over vials equal in number to the number of amines (Fig. 4c). The bags are labeled. Each vial is charged with a solution of a required amine dissolved in THF (5 equiv or 1.9 mmol per one portion of resin). The vials are agitated on a shaker for 18 h. The bags are washed successively with 2âTHF, DMF, 2âMeOH, 2âDCM, and 2âhexane. As a result, fluorine in resin VIII is substituted with the corresponding dialkylamino group to form resin X. This stage involves one important operation (capping). The case in point is that secondary amines

SnCl2 H2O DMF

O R1a

NH2 N R1b XI

Since we used the same reaction for all resins X, the labeled bags are put in one reaction vessel (Fig. 1d), after which a 2 solution of SnCl2â2H2O in DMF (50 ml per 1 bag) and shaken at room temperature for 48 h. The bags are successively washed with 2âDMF, 2âDMF/H2O (1:1 v/v), 2âH2O, 2âDMF, 2âMeOH, 2âDCM, 2âMeOH, 2âDCM, and 2âhexane and vacuum dried to obtain resins XI with a free amino group. Stage 4. Acylation of amino group. The operation is performed in two stages. To prevent incomplete reaction, conditions to ensure

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SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS

2599

double acylation of the aniline amino group are chosen. At the following stage, mild removal of one of the amino groups is accomplished, whereas the second amino group remains intact. Double Acylation
O O R 1a NH2 N R1b XI O
Py DCM

The bags with resins XIII are placed into a 25% solution of aqueous ammonia in DMF, agitated in a shaker at room temperature for 18 h, and washed with 2âDMF, 2âMeOH, 2âDCM, 2âMeOH, 2âDCM, and 2âhexane to obtain monoacyl derivatives XIV. Stage 5. Removal of the final product from support.

O + Cl XII O N R2 R O
- OH
2

O
R
2

O R1a

H N O

R2

N R1b XIV

O R1a

10% TFA/DCM

N R1b XIII

O R 1a

H N N R1b XV O

R2

According to the statements of work (which specify how many experiments to perform and what acylating agent to take), the bags with resins VI are again distributed over vials whose number is equal to the number of acyl chlorides (Fig. 4c). The bags are again labeled. To each vial, 5 equiv (1.9 mmol) of pyridine in abs. DCM and acyl chloride VII (5 equiv or 1.9 mmol per one portion of resin) are added. The vials are agitated in a shaker at room temperature for 6­18 h. The bags are washed in succession with 2âDCM, 2âDMF, 2âMeOH, 2âDCM, 2âMeOH, 2âDCM, and 2âhexane and dried to obtain diacyl derivatives XIII. Removal of One of the Acyl Groups
O N N R1b O
NH3 (aq.) DMF

Each bag is placed into an individual vial, treated with 10% TFA/DCM, and left to stand at room temperature for 2 h. The solution is decanted and the solvent was removed at reduced pressure to obtain final products XV. Yield and purity of the obtained products. The isolable yields of products by 100 mg of the initial resin is, on average, 150 mg. The purity of final samples XV, determined by LC-MS (UV-254 and ELSD detectors) was > 90%. Syntheses with Lantern Rods The problem of polymer swelling (which complicated the transfer of resins from one vessel to
Grafted polymer (polystyrene)

O O R1a

R2 R O
2

XIII H N O XIV R2
Base polymer (polypropylene)
Fig. 5. Lantern structure.

O R1a

N R 1b

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2600

BABAEV

another, or to a filter) is solved to success by using a filter tube "container" or a "tea bag." At the same time, one more inconvenience takes place: A fairly large volume of solvents is required to thoroughly wash out excess reagents from pores and internal volume of polymer particles. An ideal solution would be to use a nonswelling polymer like polypropylene. Unfortunately, the desired functional groups (and in the required quantity) are difficult to graft on the polypropylene surface. (With polystyrene, this is readily accomplishable by, for example, chloromethylation). An original solution is to use a polypropylene support surface-coated with a thin polystyrene film. Just this principle forms the basis of the Mimotopes's lantern technology. Lantern is a plastic polypropylene cylinder coated with chemically modified polystyrene. To increase the total volume, an internal cavity and slots are made in the cylinder surface (thus making it similar to a Chinese lantern, Fig. 5). The dimensions of lanterns are chosen so that they fit the cavity of standard plastic
(a) (b)

billets (8â12). By immersing lantern into various reagents (Fig. 2b) and washing it every time with solvents (Fig. 2c), one can perform a multistage synthesis. As a rule, lanterns are used once and thrown away after the last stage ("cutting off" a complex final product from the support). Lanterns (Fig. 6) facilitate the technique of multistage syntheses of large combinatorial libraries, and the quantities of substances, isolted at the last stages (5­50 mg), are more than sufficient for biological tests. The main problem of the lantern technique is logistic in nature, since one should learn how to record correctly the "fate" of each cylinder (not to disorder cylinders in the course of multistage syntheses). To this end, each lantern should be labeled in its specific way. Three approaches are known. The most reliable (and costly) is the so-called Irory technology, when each lantern is provided with a microchip (Fig. 6d), and a special electronic device is required to record and read off from microchips what reactions each lantern should be introduced in (or has
(c) (d)

(e)
C1 B1 A1 C2 B2 A2 2â2â2 = 8 C1 C2 B1 B2 A1 A2

(f)

(g)

(h) Fmoc-NH

NH2
O

VIII

NH

O
2N

NH

H2N
Fig. 6. Technique of the solid-phase synthesis on lanterns (see text). RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010


SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS

2601

(a) Existing protocol with lanterns: ureas from amines

O

NH

O

NH

O

NH

O

NH2 O NHR'

NO2

NH2

N H

O NHR' N H

NH2

O

NH

O

NH

O

NH2 NR NR'

HN

fmoc

N H

S NR'

N H

NR NR'

2

N H

2

(b) Suggested protocol: extension to thioureas and guanidines
Fig. 7. (a) Procedure of diarylurea synthesis, recommended by the producer and (b) synthetic protocol for preparing thioureas and guanidines, optimized as a training task for students.

already been introduced in). A simpler approach is to fix lanterns in the cap of a billet with numbered holes (Fig. 6g) and keep a record by lantern numbers. And, finally, one can fix colored plastic pins on lanterns and mark the reagents already used for synthesis with colored labels or rings (Figs. 6d, 6e, and 6f). This approach is convenient in the case of small libraries. Choice of model reaction. The Mimotopes's website (www.mimotopes.com) reports a great number of examples of efficient usage of lantern in the synthesis of diverse libraries. Out attention was arrested by a simple synthetic sequence leading to N,N'-diarylureas [10] (Fig. 7a). The key stages of this process are quite simple: a m-(or p-)nitrobenzoic acid residue is fixed on a lanern, and then the nitro group is reduced, and the aniline fragment is reacted with isocyanates. Attempting to correlate the structure of expected products with their potential biological activity, we noticed that close structural relatives of diarylureas, viz. diaryl- and triarylguanidines, are well-known medicines (Fig. 8). (Note that thiomorphine and its structural analogs in the libraries, too, contain the guanidine motif).

It was not too difficult to associate the possible synthetic route to guanidines with the aniline intermediate in Fig. 7a: A thiourea residue can be obtained in a high yield by simply replacing isocyanates with isothiocyanates (Fig. 7b). This procedure is welldeveloped in a usual liquid-phase version [11]. Thoureas, in their turn, can be easily converted into guanidines by well-developed (again, in solutions) published procedures [12]. Just this sequence (probably, never realized in the solid-phase version) formed the basis of a task at the special student laboratory at the MSU. Required reagents and equipment. Lanterns VII­ XI, 1.5-ml Eppendorf-type plastic tubes (50), and great number of serum vials are required. For removing the protective group from the resin, a 20% (v/v) solution of piperidine in DMF is prepared. The consumption of reagents is not very high, mostly 1­5 g. Triethylamine and isomeric meta- and para-nitrobenzoyl chlorides (the labels on the bottles are and ) are required for the acylation stage. Reduction is performed using SnCl2â2H2O. The isothiocyanates used are metachlorophenyl isothiocyanate (I) [13] and furfuryl

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2602

BABAEV

O S N N NH N H HN HN O
Phenodianisyl local anesthetics Aptiganel neuroprotectives

NH HN N O

N
Timegadine antiinflammatories

Fig. 8. Drugs of the di(tri)arylguanidine series in the Merck Index.

isothiocyanate (II) [14], which were prepared by known procedures. To convert thioureas into guanidines, NaIO4 and three amines are required: morpholine (), m-toluidine (), and benzylamine (). For the cleavage state we take trifluoroacetic acid; lanterns are washed with methanol and DCM. Transformation sequence and methodical notes. In the work with lanterns, one of the main disadvantages of solid-phase synthesis, viz. difficult control of the degree of progress of multistage reactions, becomes the most evident. Spectral methods are unsuitable. (Actually, plastic pins are difficult to place into spectrometer ampules or cells). At the same time, accumulation of incompletely reacted functional groups (at five or six planned stages) may dramatically affect the purity of the final product. A stage-to-stage purity control is possible: by "cutting-off" intermediates from the support after every further stage. This method is complicated by another reason: In is only with a final product (with a high molecular weight) that one deals with weighable quantities of substance, whereas the quantities of low-molecular intermediates will be negligibly small. Reaction progress is optimally (and what is more, visually) controlled as follows. Let us note (see Fig. 7) that at the first four stages the amino group is either present on the resin surface (the first time after protection removal and the second time after NO2 reduction) or absent (on the initial lantern, in the nitrobenzamide fragment, and in aryl thiourea). Such an alternating appearance and disappearance of the amino group can be detected by means of the Kaiser test.1 It was found (see Fig. 6h) that this test is quite
1

effective, i.e. it makes sense to use a control lantern at each stage (develop it with ninhydrin and then throw away). In practice, lanterns are sprayed with a ninhydrin solution and heat for a short time with a hot air (heat gut or hair drier). In the case of diaryl thioureas, control removal of an intermediate from the support and assessment of its purity by NMR were performed. The purity of final guanidines was controlled by TLC, and their composition was determined by mass spectrometry (direct inlet). The overall sequence and logistics of transformations on 7 lanterns (plus 4 control lanterns) is shwn in Scheme 9. Let us consider the stages in more detail. (1) Protection removal. Lanterns containing the Rink resin (R­CH2­NH­Fmoc) were trated with a piperidine solution to remove the protective group. The ninhydrin test on a control lantern is positive. (2) Acylation of the free amino group of lantern LX (see the denotation in Scheme 9) with para- and metanitrobenzoyl chlorides and B gives resins LX and LX. The ninhydrin test on a control lantern is negative. (3) Reduction of the nitoraryl group to aniline with SnCl2. (The codes of the resulting substances is unchanged.) The ninhydrin test on a control lantern is positive. (4) Formation of diaryl thioureas LXI, LXII, and LXII by the reaction of the amino group with isothiocyanates I and II. The ninhydrin test on a control lantern is negative. (5a) Cutting-off of diaryl thioureas XI, XII, and XII with trifluoroacetic acid (TFA), determination of

See the paper of Babaev and Ermolant'ev in the present issue.

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SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS Scheme 9. Stage 1. Removal of Fmoc protection Stage 2. Reaction with benzoyl chlorides A and B

2603

LX

LX

LX

LX

LX

LX

LX

Denotations L Linker on a polystyrene support X Amino group Lantern Reagent Product

Benzoyl chlorides: A SnCl2

B

Stage 3. Reduction of nitro group Stage 4. Reaction with isothiocyanates I and II Stage 5A. Removal of thioureas from lanterns

LXA

LXA

LXA

LXA

LXA

LXA

LXB

Isothiocyanates: I LXAI

II LXAI LXAI LXAII LXBII TFA

LXAI

LXAI

­L

Thioureas

Stage 5B. Synthesis of guanidines by the reaction with amines , Amines: I , Stage 6. Removal of guanidines from LXAI1 lanterns

II

III

I

XAI XAII XBII

LXAI2 LXAI3 LXAII1 TFA

Guanidines
­L

XAI XAI XAI XAII

the yield (about 10 mg), and measurement of the NMR spectrum. (5b) reatment of the lanterns from experiment (4) with amines , , and . Preparation of guanidines LXI1, LXI2, LXI3, and LXII1. (6) Cutting-off of guanidines XI, XI, XI, and XII with TFA (about 10 mg) and mass spectral ananlysis of the isolated products. Scheme 9 shows consecutive steps which were undertook to optimize the task. Theoretically, 2â2â3 = 12 substances would be expected. The absence of certain combinations is explained by the fact that p-nitrobenzoyl chloride B forms an unsatisfactorily unclear intermediate, which was found out at an early purity control stage (analysis of thioureas). Thiourea II (from furfuryl isothiocyanate II) gave a satisfactory 1H NMR spectrum (Fig. 9a), but analysis of the guanidine obtained from this thiourea revealed a great number of admixtures (probably, formed by side

reactions involving the furan ring). In this connection we suggested to students for practical implementation only a part of Scheme 9, specifically, to use one benzoyl chloride (), one isothiocyanate (I), and three amines, to obtain one thiourea (XI) and three guanidines (XI, XI, and XI). Preparation of N,N',N''-trisubstituted guanidines on lanterns. Before lesson, the teacher demonstrates a negative Kaiser test for a standard lantern. Each student obtains 8 lanterns. Stage 1. Removal of Fmoc protection.
NHFmoc
20% piperidine in DMF 30 min

NH

2

Eight lanterns are placed in 1.5-ml plastic tubes (Eppendorf-type), and 1 ml of 20% piperidine in DMF

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O

NH2

(a)
HN HN S Cl

O

NH2

(b)

HN HN S O
H2N O N HN M = 358.83 N O Cl

O H2N N HN NH Cl

(c)

M = 378.86 O H2N N HN NH Cl

M = 378.86
Fig. 9. 1H NMR spectra of () thiourea II and (b) thioureas I. (c) Mass spectra of final guanidines XI, XI, and XI. RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010


SOLID-PHASE SYNTHESIS FOR BEGINNERS: CHOICE OF TOOLS

2605

is added to each tube. After successively washed with DMF (3â1 ml), and DCM (3â1 ml) and tubes. The control lantern shows a

1 h the sticks are (3â1 ml), methanol transferred into new positive Kaiser test.
NO2

Stage 2. Acylation of amino group.
NH2
Cl O Et3N, 2.5 h

NH O
2

Five lanterns are placed into plastic tubes, and 1 ml of a 1 solution of meta-chlorophenyl isothiocyanate (0.001 mol, 0.169 g, 0.14 ml) in methanol is added to each tube. They are left overnight and then successively washed with methanol (3â1 ml), DCM (3â1 ml), and DMF (3â1 ml). The Kaiser test is negative. Stage 5. Cleavage of amide and separation of final thiourea I.
NH O H2N O HN HN S Cl S N H N H Cl

NO

Seven lanterns are placed into plastic tubes, and 1 ml of a 1 solution of meta-nitrobenzoyl chloride (0.001 mol, 0.185 g, ~28 equiv) and triethylamine (0.001 mol, 0.1 g, 0.14 ml, ~28 equiv) in DCM is added to each tube. The reaction mixture is left overnight, after which the lanterns are successively washed with DCM (3â1 ml), methanol (3â1 ml), and DMF (3â1 ml). The Kaiser test was negative. Stage 3. Reduction of nitro group.
NH O NO2
SnCl2 2H2O 50% DMF/DCM 2h

20% TFA/DCM

NH O NH2

One of the lanterns is placed into 1 ml of a 20% (v/v) solution of TFA in and the mixture is left to stand for 1 h. transferred to a serum vial, and additionally washed with DCM (3â1 bined fractions is first concentrated in in tube, and further concentrated in a oven.

a plastic tube, DCM is added, The solution is the lantern is ml). The comair, transferred vacuum drying

Six lanterns are placed into plastic tubes, and 1 ml of 1 SnCl2â2H2O in DMF (i.e. 0.001 mol of reducer). After a day, lanterns are successively washed with DMF (3â1 ml), DCM (3â1 ml), methanol (3â 1 ml). The Kaiser test is positive. Stage 4. Coupling with isothiocyanate.

N-(m-Chlorophenyl)-N´-(m-phenylcarbamide)thiourea (I), 10 mg, Rf = 0.7 (Silufol, CHCl3/MeOH = 8/2), is obtained. According to TLC, the product contains no admixtures. The 1H NMR spectrum (Fig. 9) contains no extra signals and completely fits the structure. Stage 5B. Guanidation.

NH O NH2
m-Cl-C6H4-N=C=S
NaIO4, R'R''NH

NH O S N H NH O N HN N H

Cl

NH O S N H N H

Cl

DMF, 60°C, 6 h

NR'R''

Cl

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BABAEV

The three remaining lanterns are placed into plastic tubes labeled 1, 2, and 3, and 1 ml of a freshly prepared solution of NaIO4 (0.1 g, 0.00046 mol) and then a solution of corresponding amine I­III (0.0007 mol) in DMF are added to each tube. The reaction mixtures are heated for 6 h at 60°C and left overnight at room temperature. Precipitate formation is observed in all the cases. The laterns are washed with water (5â1 ml), DMF (3â1 ml), methanol (3â1 ml), and DCM (3â1 ml). Stage 6. Cleavage of amide and separation of the final guanidine.

ACKNOWLEDGMENTS The authors are grateful to Prof. V. Scott (USA, Indiana) for consultations and procedures, as well as to student A. Nevskaya for help in their translation. Bags with resins, spectral data for products, and initial procedure (for adaptation to a training task) was provided by PhD Yu. Sandulenko (CDRI). The set of lanterns was provided by Mimotopes, and trial experiments with them were performed by PhD student A. Leont'ev. REFERENCES
1. Babaev, E.V. and Ermolat'ev, D.S., Ross. Khim. Zh. (Zh. Ross. Khim. O­va im. D.I. Mendeleeva), 2009, vol. 53, no. 5, p. 42 2. O'Donnell, M.J., Zhou, C., and Scott, W.L., J. Am. Chem. Soc., 1996, vol. 118, p. 6070. 3. Scott, W.L., Zhou, C., Fang, Z., and O'Donnell, M.J., Tetrahedron Lett., 1997, vol. 38, p. 3695. 4. O'Donnell, M.J., Delgado, F., Dominguez, E., et al., Tetrahedron: Asymmetry, 2001, vol. 12, p. 821. 5. Scott, W.L., O'Donnell, M.J., and Alsina, J., J. Org. Chem., 2002, vol. 67, p. 2960. 6. Scott, W.L., Alsina, J., and O'Donnell, M.J., J. Comb. Chem., 2003, vol. 5, p. 684. 7. Alsina, J., Scott, W.L., and O'Donnell, M.J., Tetrahedron Lett., 2005, vol. 46, p. 3131. 8. Scott, W.L. and O'Donnel, M.J., J. Comb. Chem., 2009, vol. 11, p. 3. 9. Scott, W.L., Alsina, J., Audu, C.O., et al., Ibid., 2009, vol. 11, no. 1, p. 14. 10. http://www.mimotopes.com/files/pdf/ChemNote2.pdf. 11. Mozolis, V.V. and Iokubaitite, S.P., Usp. Khim., 1973, vol. 42, no. 7, p. 1310. 12. Ramadas, K., Janarthanan, N., and Pritha, R., Synlett, 1997, no. 9, p. 1053. 13. Radl, S., Coll. Czech. Chem. Commun., 1992, vol. 57, no. 3, p. 656. 14. Spurlock, L.A. and Fayter, R.G., J. Org. Chem., 1969, vol. 34, no. 12, p. 4035.

NH O N HN
20% TFA/DCM 1h

NR'R''

Cl

H2N O N

NR'R'' N H

Cl

The tubes with lanterns are charged with 1 ml of 20% TFA in DCM and left to stand for 1 h. The solutions are transferred to serum vials with numbers, and the lanterns are additionally washed with DCM (3â1 ml). The combined fractions are concentrated first in air, transferred into new weighed tubes, and further concentrated in a vacuum drying oven to isolate 8­12 mg of the final guanidine. All the three products have close Rf values (~0.4) on Silufol (CHCl3/MeOH = 8/2). By TLC, a minor admixture (5­10%) with Rf = 0.8 was detected. No further purification was performed. The mass spectra show molecular ion peaks with expected m/z (Fig. 9b).

RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010