Research: Science and Education
Chronology of a Difficult Synthesis Fredric M. Menger* and Jennifer L. Sorrells Department of Chemistry, Emory University, Atlanta, GA 30322; *
[email protected] One cannot help but marvel at the planning and execution evident throughout the vast literature in synthetic organic chemistry sometimes encompassing twenty or more steps. If problems are encountered on the way, they are often side-stepped, giving the impression of a smooth and almost inevitable progression toward the ultimate goal. Of course, any practitioner of synthetic organic chemistry knows that these published accounts largely understate the problems experienced during a synthesis. For one thing, space limitations may not permit full disclosure of experiments that failed to work. For another, difficulties may, rightly or wrongly, imply poor planning or lack of laboratory skill, so why advertise them when they would only detract from the main theme? In the present account, we describe the synthesis of compound 1, O
O
O
NH HN 1
O O
O
O S
O∙ Na∙
a project that required almost two years of devoted effort by a graduate student. We could follow the usual route and describe only those few steps that ultimately led to the target. In fact, this is exactly what we did in the original report of our studies where the properties of the compound, not the synthesis per se, were our main focus (1). The synthesis was only of casual interest because it is relatively brief and involves no new chemistry. An alternative approach would be to fully describe all the struggles and reversals and change-of-plans involved in this seemingly “simple” synthesis. We have chosen the latter approach in the hope that this will appeal to younger readers who can thereby acquaint themselves with various problems encountered in synthetic organic chemistry. In summary, this is a description of organic research, but one that traces reality rather than results where failures have been expurgated. It must be stated from the outset that, despite the problems confronted during the synthesis, the exercise had great merit. The challenge was invigorating, the research was educational, the acquired compounds had unique properties (1), and the overall experience was applicable to future chemical aspirations of the student. On balance, we accepted with good cheer the truism that there is no highway for effortless travel, and chemistry is not an exception. Examining the structure of 1, one complication is immediately obvious; there are four functional groups in close quarters: an ester, two amides, and a sulfonate. In addition, there is a long hydrocarbon chain that will likely affect the crystallinity of the compound. Moreover, the diketopiperazine ring system is known to participate in polymeric hydrogen-bonded assemblies and, thereby, adversely affect solubility (2). Purification (chromatography, etc.) might be complicated by the amphiphilic na-
ture of 1 (i.e., it contains both hydrophobic and ionic moieties). There also exist two identical oxygens that must be derivatized unsymmetrically. Finally, conditions must be found that do not endanger the two chiral centers adjacent to carbonyl groups. We wanted to make such a compound to study amphiphilic systems, particularly their properties in water. In general, these systems self-assemble in water because their long chains hydrophobically associate to form micelles or vesicles (3). Ionic head groups typically lie at the surface of these assemblies with the chains directed inwardly and adjacent to each other. Since the ionic head groups electrostatically repel each other, there is a balance between attractive hydrophobic and repulsive electrostatic forces that leads to assemblies with discrete morphologies (3). Our plan was to use 1 to determine how attractive hydrogenbonding forces, acting in concert with hydrophobic forces, perturb the assembly structure. This explains the presence of the strongly self-assembling diketopiperazine ring (i.e., the ring with two amides in it). Note that our desire to study the colloidal nature of 1 demanded synthesizing 0.5–1.0 g of compound rather than merely a few milligrams as is common with many total syntheses. A need for “considerable” quantities of material elevated, of course, the reaction scale and hence the difficulty of the entire synthetic enterprise. The initial plan was to prepare 1 from its cyclic dipeptide core (the l,l-diketopiperazine 2): O
HO
NH OH
HN O 2
Since 2 is commercially available, one may inquire as to why it was not simply purchased. There are four good reasons for this decision: (a) The compound is expensive and, as stated above, we needed a substantial quantity for our colloid studies. (b) The compound can be synthesized via a relatively simple literature route (Scheme I). (c) Carrying out the synthesis was reckoned to be an educational experience (a valuable component of any graduate research). (d) Most importantly, we also wanted to study the diastereomer of 1, namely the d,l compound, that, being commercially unavailable, had to be synthesized. Thus, we might as well synthesize both stereoisomers, but will only describe the l,l in this article. Protecting the serine methyl ester with the Cbz group (i.e., PhCH2OCO– ) in Scheme I and subsequent conversion of the product into the hydrazide (i.e., RCONHNH2), worked well (88% and 78% yield, respectively) (4). Several attempts to couple the hydrazide with the serine methyl ester were, however, unsuccessful despite a reported 32.3% yield for the reaction. An alternate literature-based attempt to couple the two reactants also failed to give the dipeptide owing to a Curtius rearrangement and subsequent ring closure (reactions not mentioned in
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Research: Science and Education
OH OMe ∙ Cl
∙
Cl∙H3N
OH O
O
NaHCO3
O
OMe O
H2O
O
NH O
88% yield NH2NH2·H2O MeOH
OH
OH O O
N H
NH
N
O
OMe
4.5 M HCl/EtOAc Et3N, DMF 0 °C, 2 days
O
O
O
NH
N H
NH2
∙
OMe
∙
Cl∙H3N O
O 78% yield
H2, Pd/C MeOH 4.5 M HCl/EtOAc
OH
OH
OH O
O
OH
O
OH O 4.6 M NH3
OMe
N H ∙ NH3 Cl∙
NH HN
in MeOH
O
OH O
Scheme I. Unsuccessful synthesis of compound 2 from a literature route (4).
OH O O
N H
NH
NH2
1. NaNO HCl/AcOH
O
2. L-serine methylester
O
OH
OH O
1. NaNO HCl/AcOH
OMe
N H
NH
O
O
2. L-serine methylester
OH O
N
N3 O
NH O
O
Curtius rearrangement
O
O
OH C
NH
NH
O
O
NH O
O
Scheme II. An alternate literature-based attempt to couple the two reactants failed to give the dipeptide 2 owing to a Curtius rearrangement and subsequent ring closure (5).
OH O OH O
NH
OH
∙
OH
∙
Cl∙H3N
O
OH
OH O
DCC/HOBt CH2Cl2, Et3N
O
NH
N H
OH O
O
O
5% yield N N
C
N
DCC N,N ′-dicyclohexylcarbodiimide
N N OH HOBt N-hydroxybenzotriazole
Scheme III. Direct coupling of the amino acids using traditional coupling reagents such as DCC.
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Research: Science and Education
OH
C11 H23
O OMe
O
NH
Cl
O
THF
O
∙
C11 H23
O
O OMe
reflux
O
O
NH Pd/C, H2 MeOH HCl/EtOAc
O 60% yield
O O O
N H
C11 H23
DCC/HOBt
O
O NH
C11 H23 O O OMe
EtOAc Et3N
O
∙
O
O
OMe NH3 Cl∙
OH O
O
∙
NH O
O Scheme IV. Unsuccessful attempt to make the fully protected dipeptide.
protected serine in 75% yield (Scheme VI). Deprotection of the Cbz and benzyl groups with Pd/C and H2 gave the dipeptide methyl ester in quantitative yield (!) but only if 45 psi H2 pressure was used. Ambient pressure never worked despite repeated and time-consuming attempts. We then proceeded to cyclize the dipeptide using literature conditions of NH3/MeOH, but we discovered, via X-ray analysis of the diketopiperazine product, that the undesired racemic material had been obtained. Somewhere along the line racemization had occurred.
100
Percent Yield
the publication) as was demonstrated by NMR (Scheme II) (5). This is probably a good point to consider why it is difficult, much more often than one might imagine, to reproduce synthetic reactions in the literature. Two reasons come to mind, but they strike us as highly improbable: (a) yields reported by the original authors might have been exaggerated and (b) laboratory skills of the person attempting to reproduce the reaction might be lacking. A more reasonable explanation invokes a graph in which reaction yield is plotted against a fictitious “environmental function” (encompassing temperature, solvent, reaction time, catalyst, concentration, pH, etc.). Two imaginary plots are shown in Figure 1. A reaction whose plot resembles that of A will be insensitive to the particular choice of reaction conditions. On the other hand, a reaction whose plot resembles that of B will experience a dramatic loss of yield if the correct conditions are not precisely met. A given type-B reaction may not be easily reproduced because it is very difficult to re-create the exact conditions that had been successful at some time previously. Of course, the sensitivity of a reaction to the environmental function is, in general, unknown. But reactions that become generally useful are, one would assume, mainly type-A. With type-B reactions, only laboratories with plentiful resources can afford the multi-dimensional search of the entire environmental field needed to locate the sharp maximum. We next attempted direct coupling of the amino acids using traditional coupling reagents such as DCC (Scheme III). Several attempts at this reaction produced only a 5% yield, suggesting that the free hydroxyls were interfering with the desired amide formation. To alleviate this problem, the serine hydroxyl was protected in 60% yield with a dodecanoyl group (a group that was desired in the final product) (Scheme IV). After removing the Cbz group with Pd/C, we reacted the O-acylated serine ester with the double-protected serine acid in hopes of making the fully protected dipeptide (Scheme IV). Unfortunately, this last reaction failed because, we suspect, a 1,4-O-to-N acyl-transfer occurred in the acylated serine under the basic conditions; acyltransfer was apparently faster than coupling (Scheme V) (6). Successful synthesis of the dipeptide derivative was achieved by reacting the serine methyl ester with commercial doubly-
A
50
B
0
Environmental Function Figure 1. A plot of percent yield vs an environmental function showing a reaction that is insensitive (A) and sensitive (B) to conditions.
C11 H23
C11 H23 O
O
O
∙
4 OH
5
Et3N
OMe
NH3 Cl∙
O
O
O
4 3
3
2
OH
O 2
HN 1
NH2
1
OMe O
5
C11 H23
Scheme V. A 1,4-O-to-N acyl-transfer occurred in the acylated serine under the basic conditions.
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Research: Science and Education O
O O
HN O
O
1. DCC, HOBt EtOAc
HO O
MeO
2. EtOAc 1 equiv Et3N O
∙
O O
∙
O
45 psi H2, Pd/C
O
HO
NH3 Cl∙
H N
MeO
MeOH HCl/EtOAc
O
HO
OH
quantitative yield
75% yield
NH3 Cl∙
MeO
HN
H N
HO
NH3/MeOH
O HO
NH HN O
Scheme VI. Synthesis of racemic compound 2.
racemic
O
O O
HN
O
1. DCC, HOBt DMF, EtOAc
HO O
OH
O
MeO
HN O
HO
2. DMF, EtOAc 1 equiv Et3N O
H N
O O
1. 45 psi H2, Pd/C MeOH
O HO
NH HN
2. 65 °C
O 61% yield
∙
NH3 Cl∙
MeO
OH
88% yield
HO
Scheme VII. Successful synthesis of optically pure l,l-diketopiperazine 2. O
O HO
DMA, DMAP
NH HN
OH
O NH
O HN
O Cl
O
C11 H23
60% yield
OH O
Scheme VIII. Successful acylation of compound 2: DMA is dimethylacetamide (a solvent) and DMAP is dimethylaminopyridine (a strong organic base). SO3·Et3N Et3N, DMA
O
O NH
O HN
OH
O
O
SO3·Pyr
O
DMA
NH HN
SO3·Pyr
O
O O
O S
O
O∙ Na∙
THF , Et3N
Scheme IX. Sulfonation led to a series of unsuccessful attempts, predominately in the purification step.
O
O
1. SO3·Et3N pyridine
NH
O HN
OH
O
O O
O
NH HN
2. NaHCO3 27% yield
O O
O
O S
O∙ Na∙
Scheme X. Successful sulfonation and purification was carried out in basic conditions.
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Research: Science and Education
A change of protocol finally produced optically pure material, and it is instructive to describe the difference. Thus, in the initial racemic route (Scheme VI), double-protected serine was mixed with DCC and HOBT in ethyl acetate to produce a heterogeneous mixture. At this point the serine methyl ester and one equivalent of triethylamine were added. The product was treated with H2, Pd/C in methanol, EtOAc, and HCl. The deprotected material in the ammonium form was added to methanolic ammonia to free the protonated amine and thus effect cyclization. On the other hand, in the route to optically pure material (Scheme VII), the doubly-protected serine was mixed with HOBT and DMF/EtOAc to create a homogeneous solution before the DCC was added. Coupling to the dipeptide was carried out as done previously with the addition of DMF as a cosolvent with EtOAc. When the dipeptide was deprotected with H2 and Pd/C, spontaneous cyclization occurred upon heating in 61% yield with no need to use HCl or NH3 in the sequence, a fact that presumably precluded racemization. At long last we had the optically pure l,l-diketopiperazine 2 in hand. The 61% yield in the spontaneous cyclization brings up the question as to why yields are seldom quantitative. There are three obvious explanations: (a) There is “holdup”—small quantities of material that remain in the flask, on the column, and so forth —that serve to reduce the overall yield. Generally, with care and good technique, this is not an important factor. (b) The reaction may not have gone to completion. Often reactions are run at two different reaction times, and if the yields do not change, the reaction can be regarded as finished. In most cases it is unlikely that low yields are the result of incomplete reactions because this problem is so easy to avoid. (c) There may be a side-product that is usually not isolated and identified. But there is the peculiar aspect to this common enemy of yield. Consider, for example, a reaction with a respectable 75% yield. This means that a sideproduct is formed with a 25% yield. But a rate ratio of 75:25 represents only a small difference in the energy of activation, less than a kilocalorie. In other words, the departure from a quantitative yield is caused by a side reaction that has an activation energy almost identical to that of the desired reaction—a remarkably coincidence. Only two tasks remained once we possessed diketopiperazine 2, namely, to acylate one hydroxyl and to sulfonate the other. It was logical to acylate first, and this was accomplished in 60% yield using dodecanoyl chloride and standard conditions (Scheme VIII). Sulfonation, on the other hand, was more challenging and led to a series of unsuccessful attempts (Scheme IX). The main problem centered around the purification of the final product. Chromatography on silica, crystallization, dialysis, precipitation, and reverse-phase chromatography all failed to give pure product. The continual presence of starting alcohol plagued purification. It was ultimately realized that one major problem stemmed not from an incomplete sulfonation but from an instability of the product under acidic conditions. Excess SO3·Et3N was necessary to ensure complete sulfonation. When the solvent was removed under reduced pressure and water was added, sulfuric acid was formed. Final product was then de-sulfonating back to the alcohol from which it was prepared. We speculate that the neighboring amide carbonyl was assisting the hydrolysis of the normally stable sulfonate group by a type of intramolecular catalysis (7). Careful attention to the pH of the workup solution was required to minimize this reaction.
Thus, sulfonation was carried out as seen in Scheme X. Once a mild base (sodium bicarbonate) was added the compound immediately precipitated from solution in the form of the sodium salt. Compound 1 (purified by a methanol trituration) turned out, unexpectedly, to be too water-insoluble for colloid studies in aqueous solutions. To obtain a more water-soluble compound, we had to circle back and synthesize the decanoyl homolog with two fewer carbons in the chain. Purification of this latter derivative presented a new challenge. Addition of sodium bicarbonate to neutralize the acid led to de-acylation presumably aided by the neighboring nitrogen five atoms away (8). Deacylation had not been observed for the dodecyl salt owing to its instant precipitation from the aqueous solution. To preclude the decomposition problem, a small quantity of pyridine must be present at all times to act as a buffering agent. The sodium counterion was installed through the use of Dowex, an ion exchange resin. Purification was then accomplished through the use of size-exclusion chromatography on columns of Sephadex LH20 with water as the eluent. Sephadex LH20 has been used in the past to separate small molecules such as lipids, vitamins, and steroids according to their size. Thus, a reaction mixture from 500 mg starting alcohol was chromatographed on 100 g Sephadex LH20 while collecting 45 cuts of 22 mL each. According to mass spectrometric analysis of each cut, only 12 cuts contained the desired compound. These cuts were combined and lyophilized, whereupon the residue was chromatographed one or two additional times. In this manner, a pure final product was, after more than 18 months, finally obtained. Characterization of its colloidal behavior could now begin, but that is a whole other story (1). Acknowledgment This work was supported by the National Institutes of Health. Literature Cited 1. Sorrells, J. L.; Menger, F. M. J. Am. Chem. Soc. 2008, 130, 10072– 10073. 2. Hanabusa, K.; Matsumoto, M.; Kimura, M.; Kakehi, A.; Shirai, H. J. Colloid. Interf. Sci. 2000, 224, 231–244. 3. Jonsson, B.; Lindman, B.; Holmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; John Wiley & Sons: Chichester, U.K., 1998. 4. Mori, A.; Imanishi, Y.; Ito, T.; Sakaoku, K. Biomaterials 1985, 6, 325–337. 5. Fruton, J. S. J. Biol. Chem. 1942, 146, 463–470. 6. Kusumoto, S.; Sakai, K.; Shiba, T. Bull Chem. Soc. Jpn. 1986, 59, 1296–1298. 7. Thea, S.; Guanti, G.; Hopkins, A. R.; Williams, A. J. Org. Chem. 1985, 50, 3336–3341. 8. Bergman, J.; Bergman, S. J. Org. Chem. 1985, 50, 1246–1255.
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