Dr Louis A. Carpino (91) passed away at home in Amherst, MA on Friday, January 4, 2019. He was a Professor Emeritus of Organic Chemistry at the University of Massachusetts Amherst.
Dr L.A. Carpino was a pioneer in the development of amino‐protecting groups and coupling reagents for use in the synthesis of biologically active materials such as pharmaceuticals, polynucleotides, PNAs, peptides, and small proteins. His first significant contribution was the development of the tert‐butyloxycarbonyl (Boc) group as a protecting group for amino acids.1 With its much greater acid lability than the benzyloxycarbonyl‐(Z) protecting group, the Boc group facilitated the synthesis of peptides. The discovery of the Boc group enabled a new strategy in the synthesis of peptides. The Boc strategy combined the use of a Boc group as temporary Nα‐amino‐protecting group with the much more acid stable, permanent side‐chain benzyl‐based protecting groups. Boc amino acids are mostly crystalline, easily prepared using a number of synthetic methods, and stable even at room temperature. In general, activation and coupling of Boc amino acids to the N‐terminus of growing peptides is carried out with no side reaction. Because of the rapid removal of Boc groups by TFA/DCM, HCl/dioxane or other acids, Boc amino acids were found to be particularly suited for the development of and the use in solid‐phase peptide synthesis (SPPS) created by RB Merrifield in 1963.2 Boc protection of amino acids has been highly successful in the syntheses of a limited number of proteins and is compatible with automated machines and for parallel synthesis of a large number of short peptides (on pins or in “tea bags”).
Dr Carpino’s next major accomplishment addressed a weak point in the use of Boc‐protecting groups, especially in the presence of other acid labile‐protecting groups. While the temporary Boc group can be removed under much milder conditions than the permanent groups, the latter groups are not completely stable following repeated Boc removals. Thus, partial side‐chain deprotection during the synthesis of long peptides in SPPS may yield undesired side products and complex crude products. Moreover, the final deprotection of side‐chain functionalities as well as cleavage of peptide from resin in the Boc strategy necessitates the use of harsh conditions, such as liquid hydrogen fluoride, which may lead to modification/degradation of the product by side reactions including aspartimide formation, N/O‐shift, etc. Dr Carpino developed a completely orthogonal‐protecting system using the 9‐fluorenylmethoxycarbonyl (Fmoc) group.3 The Fmoc‐protecting group is removed rapidly and efficiently by proton abstraction via secondary amines, conditions that do not affect acid‐labile tert‐butyl–type‐protecting groups placed on the side chains. Moreover, the Fmoc group is completely stable under the conditions used for removal of the resin linkers. Fmoc amino acids are generally easy to prepare in crystalline form and stable during storage. Fmoc amino acids can be highly activated and efficiently coupled without side reactions, and their high ultraviolet (UV) absorption makes it possible to monitor their incorporation during the assembly of the peptide. By using the secondary amines such as piperidine to remove the Fmoc group, the dibenzofulvene formed is trapped on the spot, minimizing possible side reactions. The Fmoc‐based SPPS is nowadays the method of choice for the chemical synthesis of peptides.
Dr Carpino developed a number of other protecting groups that have also found widespread application. The benzothiophenesulfone‐2‐methyloxycarbonyl (Bsmoc)‐protecting group is an example of a large new class of protecting groups that can be removed under the very mild conditions of Michael addition.4 The 2,2,4,6,7‐pentamethyldihydrobenzofuran‐5‐sulfonyl group (Pbf) was developed for side‐chain protection of arginine and is easily removed by trifluoroacetic acid (TFA). Pbf can be used efficiently in the synthesis of arginine‐containing peptides.5 The icyclopropylmethyl (Dcpm) group is used in polypeptide syntheses when it is necessary to minimize peptide insolubility and aggregation.6 In case of threonine/serine‐containing peptides, an alternative route to avoid aggregation problems involves the synthesis of the corresponding O‐acyl‐peptide isomers and subsequent rearrangement back to regular peptides after synthesis and purification.7
In addition to his seminal contributions around amino protecting groups, Dr Carpino developed several new types of coupling reagents based on acid halides and 1‐hydroxy‐7‐azabenzotriazole that are far more efficient than classical reagents because of their speed, generality, and control of epimerization. Fmoc‐amino acid fluorides have facilitated efficient SPPS of peptaibols, such as alamethicin, the synthesis of which was previously reported via a laborious solution‐phase route.8 The more reactive Fmoc‐amino acid chlorides, although being less stable than the corresponding fluorides, enable the coupling of extremely hindered systems such as N‐methyl‐amino‐iso‐butyric acid to amino‐iso‐butyric acid.9 Finally, uronium/guanidinium compounds of 1‐hydroxy‐7‐azabenzotriazole (HOAT) are very efficient activation reagents,10 although their recent use, especially in large‐scale syntheses, has been limited for safety reasons.
Dr Carpino’s contributions over a long and distinguished career have enabled synthetic peptide chemistry as it is widely practiced today. Dr Carpino never stopped thinking about the design of better coupling reagents. In fact, he was working on a new coupling reagent to address some of the safety issues with HOAT over the past year when he passed away.
Dr Carpino was born in Des Moines, Iowa, on December 13, 1927. He was a loving husband, father, and grandfather and is survived by his wife, Barbara, and six children. I got to know him as a great teacher, creative director who shared his ideas, attentive listener who showed respect for anyone, a quiet person who shunned the great arena but fostered intense scientific collaboration over many years. He touched the lives of many people—he will be missed.
Narzissenweg 1, 04416 Markkleeberg, Germany
1. Carpino LA. Oxidative reactions of hydrazines. IV. Elimination of nitrogen from 1,1‐disubstituted‐2‐arenesulfonhydrazides1‐4. J Am Chem Soc. 1957;79(16):4427‐4431.
2. Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc. 1963;85:2149‐2154.
3. Carpino LA, Han GY. 9‐Fluorenylmethoxycarbonyl function, a new base‐sensitive amino‐protecting group. J Am Chem Soc. 1970;92(19):5748‐5749.
4. Carpino LA, Ismail M, Truran GA, et al. The 1,1‐dioxobenzo[b]thiophene‐2‐ylmethyloxycarbonyl (Bsmoc)amino‐protecting group. J Org Chem. 1999;64(12):4324‐4338.
5. Carpino LA, Shroff H, Triolo SA, Mansour EME. The 2,2,4,6,7‐pentamethyldihydrobenzofuran‐5‐sulfonyl group (Pbf) as arginine side chain protectant. Tetrahedron Lett. 1993;34(49):7829‐7832.
6. Carpino LA, Nasr K, Abdel‐Maksoud AA, et al. Dicyclopropylmethyl peptide backbone protectant. Org Lett. 2009;11:3718‐3721.
7. Carpino LA, Krause E, Sferdean CD, et al. Synthesis of ‘difficult’ peptide sequences: application of a depsipeptide technique to the Jung–Redemann 10‐ and 26‐mers and the amyloid peptide Aβ(1–42). Tetrahedron Lett. 2004;45(40):7519‐7523.
8. Carpino LA, Beyermann M, Wenschuh H, Bienert M. Peptide synthesis via amino acid halides. Acc Chem Res. 1996;29(6):268‐274.
9. Carpino LA, Ionescu D, El‐Faham A, et al. Protected amino acid chlorides vs protected amino acid fluorides: Reactivity comparisons. Tetrahedron Lett. 1998;39(3‐4):241‐244.
10. Carpino LA, Imazumi H, El‐Faham A, et al. The uronium/guanidinium peptide coupling reagents: Finally the true uronium salts. Angew Chem Int Ed. 2002;41:441‐445.