CA2266527A1 - Liquid detergents containing proteolytic enzyme, peptide aldehyde and calcium ions - Google Patents

Abstract

Aqueous liquid detergent compositions are described which comprise a proteolytic enzyme wherein the proteolytic activity is reversibly inhibited by a peptide aldehyde and calcium. More specifically, liquid detergent compositions are disclosed which contain a detersive surfactant, a proteolytic enzyme, a peptide aldehyde, and calcium ions. The combination of peptide aldehyde and calcium ions acts to provide synergistic protease inhibitor benefits.

Description

CA 02266~27 1999-03-23 . 1 LIQUID DETERGENTS CONTAINING PROTEOLYTIC
ENZYME, PEPTIDE ALDEHYDE AND CALCIUM IONS
-TECHNICAL FIELD
This invention relates to liquid detergent compositions cont~ining en_ymes.
10 More specifically, this invention pertains to liquid d~ r~elll compositions cont~ining a detersive ~ulr~ l, a proteolytic enzyme, a peptide aldehyde, and calcium ions. The combination of peptide aldehyde and calcium ions act to provide synergistic protease inhibitor benefits.
BACKGROUND OF THE INVENTION
Protease-co~ g Iiquid aqueous detergents are well-known, especially in the context of laundry washing. A commonly encountered problem in such protease-cont~ining liquid aqueous detergents is the degradation phenomenon by the proteolytic enzyme of second enzymes in the composition, such as amylase, lipase, and cellulase, or on the protease itself. As a result, the stability of the second 20 enzyme or the protease itself in the detergent composition is affected and the detergent composition consequently performs less well.
In response to this problem, it has been proposed to use various protease inhibitors or stabilizers. For inct?nre, various references have proposed the use of the following compounds to aid in the stabilization of enzymes: bçn7~midine 25 hydrochloride, lower aliphatic alcohols or carboxylic acids, mixtures of a polyol and a boron compound, aromatic borate esters, and calcium, particularly calcium formate. Recently, it was discovered that certain peptide aldehydes act to stabilize protease enzyme.
Although these compounds have been used to varying success in liquid 30 detergents, they are not free of problems. For example peptide aldehydes are rather ex~ si~/e and create complexities for the fonnnl~tors, especially for liquid d~enl~. Other inhibitors such as calcium and boric acids are less expensive but do not stabilize enzymes ~ well as peptide aldehydes. It is thus an object of the present invention to provide a protease inhibitor system which is economical, 35 effective and suitable for use in a liquid detergent composition.
~ n ~ onse to this object, the present invention proposes to use a combination of calcium ions and peptide aldehydes as reversible protease inhibitors in aqueous ~ ... .. . .. .

CA 02266~27 1999-03-23 W O 98/13459 PCTrUS97/16622 liquid detergent compositions. The presence of both calcium and peptide aldehydeprovides a synergistic stabilization of the protease. This novel combination provides the formulator added flexibility in cle~igning a stabilization system. The levels of peptide aldehyde and calcium can be adjusted to deliver the most cost effective formula and to minimi7e product stability problems that o~en arise from the presence of divalent ions in a liquid detergent matrix.
In particular, the present invention allows for the use of very low levels of peptide aldehydes in the liquid detergent compositions herein. This is particularly critical in the formulation of relatively inexpensive, concentrated liquid detergent 10 compositions which are encompassed by the present invention.
Because the combination of calcium and peptide aldehydes are so efficient in inhibiting proteases, another advantage of the present invention is that even enzymes which are highly sensitive to proteolytic degradation can now be incorporated inliquid detergent compositions comprising a protease. Moreover, it has also been 15 discovered that the increased stability of the protease enzyme allows for improved skincare benefits. These benefits include softening of the skin and hands and less drying from exposure of the hands to the dishwashing liquor.
BACKGROUND ART
It has been proposed to use various protease inhibitors or stabilizers. For 20 instance, US 4,566,985 proposes to use benzamidine hydrochloride; EP 376 705 proposes to use lower aliphatic alcohols or carboxylic acids; EP 381 262 proposes to use a mixture of a polyol and a boron compound; and EP91870072.5 proposes to usearomatic borate esters. See also U.S. Pat. No. 5,030,378 issued July 9, 1991. Also see US4,261,868; US4,404,115; US4,318,818; and EP130,756.
The use of peptide derivatives for the inhibition of proteins appears to have been disclosed in therapeutic applications. EP 293 881 discloses the use of peptide boronic acids as inhibitors of trypsin-like serine proteases. EP 185 390 and US
4,399,065 disclose the use of certain peptide aldehydes derivatives for the inhibition of blood coagulation. J 90029670 discloses the use of optically active alpha amino 30 aldehydes for the inhibition of enzymes in general. See also "Inhibition of Thrombin and Trypsin by Tripeptide Aldehydes", Int. J. Peptide Protein Res., Vol12 (1978), pp. 217-221; Gaal, Bacsy & Rappay, and "Tripeptide Aldehyde Protease Inhibitors May Depress in Vitro Prolactin and Growth Hormone Release"
Endocrinolo~y, Vol. 116, No. 4 (1985), pp. 1426-1432; Rappay, Makara, Bajusz &
35 Nagy. Certain peptide aldehydes have also been disclosed in EP-A-473 502 for inhibiting protease-me~ tecl skin irritation.

CA 02266~27 1999-03-23 W O98/134S9 PCT~US97/16622 In particular see EP185,390, WO94/04651, published 3 March 1994, W094/04652, published 3 March 1994, EP 583,536, published February 23, 1994, EP 583,535, published February 3, 1994, EP 583,534, published February 23, 1994,WO 93/13125, published July 8, 1993, US4,529,525, US4,537,706, US4,537,707, 5 and US5,527,487.
SUMMARY OF THE INVENTION
The invention herein is a liquid detergent composition comprising:
a) an effective amount of a detersive surfactant;
b) an active proteolytic enzyme;
10 c) a source of calcium ions; and d) a peptide aldehyde having the formula:
Z-B-NH-CH(R)-C(O)H
wherein B is a peptide chain comprising from 1 to 5 amino acid moieties; Z is an N-capping moiety selected from the group consisting of phosphoramidate [(R"O)2(O)P-3, sulfenamide [(SR")2-], sulfonamide [(R"(O)2S-], sulfonic acid [SO3H], phosphinamide [(R")2(O)P-], sulfamoyl derivative [R"O(O)2S-], thiourea [(R")2N(O)C-], thiocarbamate [R"O(S)C-], phosphonate [R"-P(O)OH], amidophosphate [R"O(OH)(O)P-], carbamate (R"O(O)C-), and urea (R"NH(O)C-), wherein each R" is independently selected from the group consisting of straight or branched Cl-C6 llncubstituted alkyl, phenyl, C7-Cg alkylaryl, and cycloalkyl moieties, wherein the cycloalkyl ring may span C4-Cg and may contain one or moreheteroatoms selected from the group consisting of O,N,and S (preferred R" is selected from the group con.~icting of methyl, ethyl, and benzyl); and R is selected from the group con.cicting of straight or branched C1 - C6 unsubstituted alkyl, phenyl, and C7 - Cg alkylaryl moieties.
Without being limited by theory, it is believed that the combined source of calcium ion and peptide aldehyde provides more than additive stability to the proteolytic enzyme.
Preferably, the liquid detergent compositions herein comprise, by weight of composition:
a) from about I to about 9S%, preferably from about 8% to about 70%, of said detersive surfactant;
b) from about 0.0001% to about 5%, preferably from about 0.0003% to about 0.1 %, of an active proteolytic enzyme;
c) from about 0.00001% to about 5%, preferably from about 0.0001% to about 1 %, more preferably from about 0.0006% to about 0.5%, of a peptide aldehyde as described hereinbefore; and .

CA 02266~27 1999-03-23 d) from about 0.01% to about 1%, preferably from about 0.05% to about 0.5%, of calcium ion.
The proteolytic enzyme useful herein is preferably a subtilisin-type protease and may be selected from the group consisting of Alcalase~), Subtilisin BPN', Protease A, Protease B, and mixtures thereof.
The source of calcium ion for use herein is preferably selected from calcium formate, calcium xylene sulfonate, calcium chloride, calcium acetate, calcium sulfate. and mixtures thereof.
The dishcare compositions herein may contain further detersive adjuncts, including but not limited to, one or more of the following: suds boosters, chelants, polyacrylate polymers, dispersing agents, dyes, perfumes, processing aids, and mixtures thereof. Moreover for dishcare compositions, the liquid detergent compositions may further comprise an effective amount of amylase enzyme.
Additionally, the dishcare compositions may optionally comprise an effective amount of a source of boric acid and a diol. Typically dishcare compositions will optionally, but preferably, comprise from about 0.25% to about 10%, preferably from about 0.5% to about 5%, more preferably from about 0.75% to about 3%, by weight of boric acid or a compound capable of forming boric acid and a diol, e.g.
1 ,2-propaneidiol .
In a preferred embodiment for heavy duty detergent compositions useful in laundry care, the liquid detergent composition further comprises an effective amount one or more of the following enzymes: lipase, amylase, cellulase, and mixtures thereof. Preferably for laundry compositions, the second enzyme is lipase and isobtained by cloning the gene from Humicola Lanuginos~ and ~x~lc;sslllg the gene in Aspergillus Oryzae. Lipase is utilized in an amount of from about 10 to about 18000 lipase units per gram, preferably from about from about 60 to about 6000 units per gram.
In another pl~rell~d composition useful for laundry care, the second enzyme is a cellulase derived from Humicola Insolens and is utilized in an amount of from about 0.0001% to about 0.1% by weight of the total composition of said cellulase.
The compositions herein may contain further detersive adjuncts, including but not limited to, one or more of the following: suds boosters, builders, soil release polymers, polyacrylate polymers, dispersing agents, dye transfer inhibitors, dyes, perfumes, processing aids, brighteners, and mixtures thereof. Additionally, for laundrycare compositions, the detersive surfactant is typically present in an amount of from about 10% to about 70%, by weight of total composition. Moreover, the laundry compositions may optionally comprise an effective amount of a source of CA 02266~27 1999-03-23 boric acid and a diol. Typically laundry compositions will optionally, but preferably, comprise from about 0.25% to about 10%, preferably from about 0.5% to about 5%, more preferably from about 0.75% to about 3%, by weight of boric acid or a compound capable of forming boric acid and a diol, e.g. 1,2-propaneidiol.
All percentages and proportions herein are by weight, and all references cited are hereby incorporated by reference, unless otherwise specifically indicated.
DETAILED DESCRIPTION OF THE INVENTION
Definitions - The present detergent compositions comprise an "effective amount" or a "stain removal-improving amount" of individual components defined herein. An "effective arnount" or "stain removal-improving amount" is any amountcapable of measurably improving soil cleaning or stain removal from a substrate,i.e., soiled fabric or soiled dishware, when it is washed by the consumer. In general, this amount may vary quite widely.
By "synergy" or "more than additive" as used herein is meant that the enzyme stability benefit when the calcium and peptide aldehydes are combined is greaterthan the sum of the individual benefits obtained when only one of the components is present in a detergent composition.
The liquid aqueous detergent compositions according to the present invention comprise four essential ingredients: (A) a peptide aldehyde or a mixture thereof, (B) a proteolytic enzyme or a mixture thereof, (C) a detersive surfactant, and (D) calcium ion. The compositions according to the present invention preferably further comprise (E) a detergent-compatible second enzyme or a mixture thereof, and may further comprise (F) other optional ingredients.
PeDtide aldeh~des - The detergent compositions according to the present invention comprise, as a first essential ingredient, a peptide aldehyde having the forrnula:
Z-B-NH-CH(R)-C(O)H
wherein B is a peptide chain colllpllsing from 1 to 5 amino acid moieties; Z
is an N-capping moiety selected from the group concicting of phosphorarnidate [(R"0)2(0)P-], sulf~on~mi~le [(SR")2-], sulfonamide [(R"(0)2S-], sulfonic acid [S03H], phosphin~mide [(R")2(0)P-], s--lf~mc yl derivative [R"0(0)2S-], thiourea[(R")2N(O)C-], thioc~banlate [R"O(S)C-], phosphonate [R"-P(O)OH], arnidophosphate [R"O(OH)(O)P-], carbamate (R"O(O)C-), and urea (R"NH(O)C-), wherein each R" is indep~nflently selected from the group consisting of straight or branched C I -C6 unsubstituted alkyl, phenyl, C7-Cg alkylaryl, and cycloalkyl moieties, wherein the cycloalkyl ring may span C4-Cg and may contain one or moreheteroatoms selected from the group concicting of O,N,and S (preferred R" is CA 02266~27 1999-03-23 selected from the group con~icting of methyl, ethyl, and benzyl); and R is selected from the group consisting of straight or branched C I - C6 unsubstituted alkyl, phenyl, _nd C7 - Cg alkylaryl moieties.
Preferred R moieties are selected from the group consisting of methyl, iso-propyl, sec-butyl, iso-butyl, -C6Hs, -CH2-C6Hs, and -CH2CH2-C6Hs, which respectively may be derived from the _mino acids Ala, Val, Ile, Leu, PGly (phenylglycine), Phe, and HPhe (homophenylAIAnine) by converting the carboxylic acid group to an aldehyde group. While such moieties are therefore not amino acids (_nd they may or may not have been synthPci7Pcl from an amino acid precursor), for purposes of simplification of the exemplification of inhibitors useful here, thealdehyde portion of the inhibitors are indicated as derived from amino acids by the addition of "H" after the A~alogous amino acid [e.g., "-Ala~" represents the chemical moiety "-NHCH(CH3)C(O)H"].
Preferred B peptide chains are selected from the group consisting of peptide chains having the amino acid sequences according to the general formula:
Z-A5-A4-A3-A2-A I -NH-CH(R)-C(O)H
such that the following amino acids, when present, are:
Al is selected from Ala, Gly;
A2, if present, is selected from Val, Ala, Gly, Ile;
A3, if present, is selected from Phe, Leu, Val, Ile;
A4, if present, is any amino acid, but preferably is selected from Gly, Ala;
A5, if present, is any amino acid, but preferably is Gly, Ala, Lys.
The present invention aldehydes may be prepared from the corresponding amino acid whereby the C-tf rrninAI end of said amino acid is converted from a carboxylic group to ~ aldehyde group. Such aldehydes may be prepared by ~nown processes, for instance as described in US 5015627, EP 185 930, EP 583,534, and DE3200812.
While not wanting to be bound by theory it is believed that the peptide aldehydes according to the present invention bind to the proteolytic enzyme in the liquid detergent composition, thereby inhibiting said proteolytic enzyme. Upon dilution in water, the proteolytic activity is restored by dissociation of the proteolytic enzyme/peptide aldehyde complex.
The N-tenninAl end of said protease inhibitors according to the present invention is protected by one of the N-capping moiety protecting groups selectedfrom the group con~i~ting of c~bA.nAtPs, ureas, sulfon~mi-lPs, phosphonamides,thioureas, sulfenamides, sulfonic acids, phosphinAmides, thiocarbAm~tes, amidophosphates, and phosphonamides. However, in a highly CA 02266~27 1999-03-23 WO g8/13459 PCI/US97/16622 plefelled embodiment of the present invention, the N-terminal end of said protease inhibitor is protected by a methyl, ethyl or benzyl carbamate [CH30-(O)C-;
CH3CH20-(O)C-; or C6HsCH20-(O)C-], methyl, ethyl or benzyl urea [CH3NH-(O)C-; CH3CH2NH-(O)C-; or C6HsCH2NH-(O)C-], methyl, ethyl or benzyl sulfonamide [CH3S02-; CH3CH2S02-; or C6HsCH2S02-], and methyl, ethyl or benzyl amidophosphate ICH30(0H)(O)P-; CH3CH20(0H)(O)P-; or C6HsCH20(0H)(O)P-] groups.
Synthesis of N-capping groups can be found in the following references:
Protective Groups in Or~anic Chemistry. Greene, T., Wuts, P., John Wiley & Sons,New York, 1991, pp 309-405; March, J, Advanced Or~anic ChemistrY, Wiley Interscience, 1985, pp. 445, 469, Carey, F. Sundberg, R., Advanced Or~anic Chemistr~, Part B, Plenum Press, New York, 1990, pp. 686-89; Atherton, E., Sheppard, R., Solid Phase Peptide Svnthesis, Pierce Chemical, 1989, pp. 3-4; Grant, G., Svnthetic Peptides, W. H. Freeman & Co. 1992, pp. 77-103; Stewart, J., Young, 1 s J., Solid Phase Peptide SYnthesis. 2nd Edition, IRL Press, 1984, pp. 3,5,11,14- 18, 28-29. Bodansky, M., Principles of Peptide Synthesis, Springer-Verlag, 1988, pp.62, 203, 59-69; Bodansky, M., Peptide Chemistry, Springer-Verlag, 1988, pp. 74-81, Bodansky, M., Bodansky, A., The Practice of Peptide Synthesis, Springer-Verlag, 1984, pp. 9-32.
Examples of peptide aldehydes for use herein are: CH3S02Phe-Gly-Ala-Leu-H, CH3S02Val-Ala-Leu-H, C6HsCH20(0H)(O)P-Val-Ala-Leu-H, CH3CH2S02-Phe-Gly-Ala-Leu-H, C6HsCH2S02-Val-Ala-Leu-H, C6HsCH20(0H)(O)P-Leu-Ala-Leu-H, C6HsCH20(0H)(O)P-Phe-Ala-Leu-H, and CH30(0H)(O)P-Leu-Gly-Ala-Leu-H.
In the Synthesis Examples hereinafter methods are disclosed to synthesi7~o certain of these peptide aldehydes.
Synthesis Example 1 Synthesis of the te~lal)el)tide aldehyde Moc-Ala-Phe-Gly-Ala-LeuH
(a) Ala-Leu-OMe.HCL: To a solution of 3.0 g (14.83 mrnol) Ala-Leu-OH, which is dissolved in 50 ml of MeOH and cooled to 0~C, is added 2.43 ml (33.36 mmol) thionyl chloride dropwise. This solution is stirred overnight at room temperature and evaporated to dryness providing quantitative recovery of the desired product.

(b) Cbz-Gly-Ala-Leucine methyl ester: To a solution of 0.414 g (1.98 mmol) Cbz-Gly-OH and 0.500 g (1.98 mmol) Ala-Leu-OMe.HCl in CH2C12 is add 0.607 ml TEA followed immediately by 0.355 ml DEPC. The solution is stirred overnight, evaporated, and the residue partitioned between EtOAc and lN HCI. The organic CA 02266~27 1999-03-23 WO 98/13459 rcT/uss7/16622 phase is washed successively with saturated NaE~C03 and saturated NaCl, dried (MgSO4)and evaporated to afford 0.650 g of pure product.

(c) Moc-Ala-Phe-OH: To a solution of 1.0 g (4.23 mrnol) Ala-Phe which is dissolved in 4.23 ml IN NaOH and cooled to 0~C, 0.419 g (4.44 mmol) is aded methyl chloroformate dropwise. At the same time, in a separate addition funnel, an additional 4.23 ml IN NaOH is added such that the pH is m~int~ined between 9.0-9.5. After addition is complete the reaction is stirred 30 minutes at 0~C and 2 h at room temperature. At this point the solution is cooled to 0~ and the pH adjusted to 0 9.5. This basic solution is washed with EtOAc (lX, 100 ml). The aqueous (0~C) is then adjusted to pH = 2.5 (2N HCl) and extracted with EtOAc (3X, 50 ml), dried (MgSO4) and evaporated to provide 1.07 g pure product.

(d) Moc-Ala-Phe-Gly-Ala-Leu-OMe: To a solution of 0.500 g (1.22 mrnol) Cbz-Gly-Ala-Leucine methyl ester in 10 ml MeOH is added 0.100 g 10% Pd/C. This solution is hydrogenated in the presence of 0.600 ml 4.0M HCI/Dioxane (under balloon pressure) for 1 h, filtered through celite and evaporated. This residue is suspended in CH2C12, 0.342 ml (2.45 mmol) TEA is added followed by 0.359 g (1.22 mmol) Moc-Ala-Phe-OH and 0.219 ml (1.34 mmol) DEPC. After stirring overnight the solvent is evaporated, the residue partitioned between EtOAc and IN
HCI and washed successively with saturated NaHCO3 and NaCI. Drying, evaporation and column chromatography yield 0.450 g of the pure product.

(e) Moc-Ala-Phe-Gly-Ala-Leucinol: A solution is prepared by dissolving 0.182 g (1.64 munol) CaC12 in a mixture of 4 ml ethanol and 2 ml THF. This mixture is cooled to -15~C and 0.450 g (0.820 mrnol) Moc-Ala-Phe-Gly-Ala-Leu-OMe is added followed by 0.124 g (3.28 mrnol) NaBH4. The reaction is stirred for 2 h and q~en~he~l with 10 ml lN HCI. The solvents are evaporated and the rçm~ining aqueous layer partitioned with EtOAc. The organic phase is then washed with saturated NaHCO3 and saturated NaCI. Drying (MgSO4), evaporation and chromatography affords 0.256 g of pure product.

(f) Moc-Ala-Phe-Gly-Ala-LeuH: A solution is prepared by adding 0.623 g (1.47 mmol) Dess-Martin periodinane to 1.8 L CH2Cl2 followed by stirring for 10 minlltes This solution is then cooled to 0~C and 0.256 g (0.490 mmol) Etoc-Phe-Gly-Ala-Leucinol is added in one portion. The reaction is continued for 2 h and poured into a solution con.ci~ting of 2.55 g (10.47 mmol) Na2S2O3 in 30 ml CA 02266~27 1999-03-23 saturated NaHCO3. After stirring for 10 minutes the mixture is extracted with EtOAc (2X, 50 ml). The combined extracts are dried (MgSO4), evaporated. and chromatographed on silica to provide 0.125 g of pure product.

SYnthesis Example 2:
SYnthesis of the tripeptide aldehyde Etoc-Phe-Gly-Ala-LeuH
(a) Ala-Leu-OMe.HCL: To a solution of 450 g (2.20 mol) Ala-Leu-OH, which is dissolved in 4.5 L of MeOH and cooled to 0~C, is added 178.6 ml (4.95 mol) of thionyl chloride dropwise. The solution is stirred overnight at room tenl~e,d~ lre and evaporated to dryness providing 543 g (97.1 % yield) of the desired product to be used as is.

(b) Etoc-Phe-Gly-OH: To a solution of 450 g (2.03 mol) Phe-Gly which is dissolved in 2026 ml IN NaOH and cooled to 0~C, is added methyl chloroformate (3. l ml, 40.0 mmol) dropwise. At the same time, in a separate addition funnel, an additional 2026 ml IN NaOH is added such that the pH is m~int~ine-l between 9.0-9.5. After addition is complete the reaction is stirred 30 minutes at 0~C and 2 h at room temperature. At this point the solution is cooled to 0~ and the pH adjusted to 9.5. This basic solution is washed with EtOAc (IX, 4 L). The aqueous (0~C) is then adjusted to pH = 2.5 (2N HCI) and extracted with EtOAc (3X, 8L), dried (MgSO4), filtered, and the solvent removed to afford 546 g (91.3% yield) pure product.

(c) Etoc-Phe-Gly-Ala-Leu-OMe: To a solution of 470 g (1.86 mol) Etoc-Phe-Gly-OH and 546 g (1.86 mol) Ala-Leu-OMe.HCl in 8 liters CH2CI2 570 ml (4.09 mol) TEA is added followed by 310.4 ml (2.046 mol) DEPC. After stirring overnight thesolvent is evaporated and replaced with EtOAc (4 L). This solution is washed con~ec~l~ively with 2 liters each of 2N HCI, sat'd NaHCO3 and sat'd NaCI. The organic phase is then dried (MgSO4), filtered and evaporated to yield 916 g (93%yield) of the desired m~t~
(d) Etoc-Phe-Gly-Ala-Leucinol: To a solution of 45.10 g (0.406 mol) CaC12 in l Lethanol and I L THF 100 g (0.203 mol) of Etoc-Phe-Gly-Ala-Leu-OMe is added and the mixture cooled to -15~C. To this solution 30.7 g (0.812 mmol) NaBH4 is carefully added followed by stirring for 2 h. Subsequently the reaction is quenched with 100ml 0.1N HCI. This solution is transfered to 4 L of IN HCI and extracted with EtOAc (3X, 2.75 L). The combined EtOAc layers are washed with 4 L

CA 02266~27 1999-03-23 W O98/13459 PCTrUS97/16622 saturated NaHC03, dried (MgSO4) and evaporated. Trituration (twice) with ether (4 L) provides 69.2 g (73.4% yield) of the product.

(e) Etoc-Phe-Gly-Ala-LeuH: A solution is prepared by adding 165.4 g (0.39 mol) Dess-Martin periodinane to 1.8 L CH2C12 followed by stirring for 10 minutes. This solution is then cooled to 0~C and 60 g (0.13 mol) Etoc-Phe-Gly-Ala-Leucinol added in one portion. The reaction is continued for 105 minutes and poured into a solution consisting of 6 L H2O, 393 g NaHCO3 and 431.7 g (1.74 mol) Na2S2O3.
After stirring for 10 minutes the phases are separated and 2 additional extractions 10 (1.5 L each) with CH2C12 are ~c~rulllled. The combined extracts are dried (MgSO4), evaporated, and triturated with (2X, I L) ether to provide 51.7 g (86.2% yield) of the product.

Synthesis Exarnple 3:
Svnthesis of the dipeptide aldehyde Moc-GlY-Ala-LeuH
(a) Ala-Leu-OMe.HCL: To a solution of 3.0 g (14.83 mmol) Ala-Leu-OH, which is dissolved in 50 ml of MeOH and cooled to 0~C, is added 2.43 ml (33.36 mrnol) of thionyl chloride dropwise. The solution is stirred overnight at room temperature and evaporated to dryness providing a quantitative yield of the desired product.
(b) Cbz-Gly-Ala-Leucine methyl ester: To a solution of 0.414 g (1.98 rnrnol) Cbz-Gly-OH and 0.500 g (1.98 mmol) Ala-Leu-OMe.HCI in CH2C12 0.607 ml TEA is added followed im mer~i~tely by 0.355 mi DEPC. The solution is stirred overnightand then evaporated. The residue is partitioned between EtOAc and lN HCI, the 25 organic phase is washed with saturated NaHCO3 and saturated NaCI, dried (MgS04) and evaporated providing 650 mg of pure product.

(c) Moc-Gly-Ala-Leucine methyl ester: To a solution of 2.0 g (4.90 mmol) Cbz-Gly-Ala-Leucine methyl ester which is dissolved in 20 ml MeOH is added 0.200 g 30 10% Pd/C. This is hydrogenated in the presence of 2.45 ml (9.81 mmol) 4.0M
HCI/Dioxane for 2 h after which the reaction is thoroughly ou~g~c~ed and filtered through Celite to remove the catalyst. Evaporation of the MeOH affords 1.45 g ofpure product which is suspended in 45 ml CH2C12 and cooled to 0~C. To this solution 1.45 ml (3.25 mmol) TEA is added followed by 0.362 ml methyl 35 chloroformate. After stirring overnight the CH2C12 is evaporated and the residue partitioned between EtOAc and 1 N HCI. The organic phase is separated and washed CA 02266~27 1999-03-23 sequentially with NaHCO3 and NaCl. Drying, (MgS04), evaporation and chromatographic purification affords 0.~20 g of desired product.

(d) Moc-Gly-Ala-Leucinol: To a solution of 0.168 g (1.51 mmol) CaC12 in 25 ml ethanol and 15 ml THF is added 0.250 g Moc-Gly-Ala-Leucine methyl ester. This solution is cooled to -15~C and 0.114 g (3.02 mmol) NaBH4 is added in one portion.
After stirring 2 h the reaction is quenched with 20 ml lN HCI, concentrated on rotovape and extracted with EtOAc (2x 50ml). The combined extracts are washed with saturated NaHCO3 and NaCI, dried (MgSO4) and evaporated. Purification on 0 silica provides 0.167 g of the pure product.

(e) Moc-Gly-Ala-LeuH- A solution is prepared by adding 0.418 g (0.989 rnmol) Dess-Martin periodinane to 5 ml CH2C12 followed by stirring for 10 minutes. Next0.100 g (0.330 mmol) Moc-Gly-Ala-Leucinol is added in one portion and the 15 reaction stirred for 2 h and poured into a 25 ml solution of saturated NaHCO3cont~ining 1.72 g (6.93 mmol) Na2S2O3. After stirring an additional 10 minutes the solution is extracted with EtOAc (3X, 50 ml), dried (MgSO4) and evaporated.
Chromatography on silica affords 0.016 g of the desired product.

Svnthesis Example 4:
Synthesis of N-(methylsulfonyl)-Phe-Gly-Ala-LeuH
(a) N-Ms-Phe-Gly-OH: To a solution of 2.0 g (9.0 mmol) Phe-Gly-OH, which is dissolved in 9 ml lN NaOH and cooled to 0~C, is added simultaneously 0.766 ml ( 9.9 mmol) of methane sulfonyl chloride and 9 ml lN NaOH, in separate addition 25 funnels. After addition is complete the reaction is stirred 15 minlltes at 0~C and 1 h at room temperature. At this point the solution is cooled to 0~C, the pH adjusted to 9.5 and is washed with EtOAc (lX, 50 m!). The aqueous phase (0~C) is then adjusted to pH = 2.5 (2N HCI) and extracted with EtOAc (3X, 50 ml), dried (MgSO4), filtered, and the solvent removed to afford 2.0 g pure product.
(b) N-Ms-Phe-Gly-Ala-Leucinol: A solution of is prepared by dissolving 0.500 g (1.67 mmol) N-Ms-Phe-Gly-OH in 15 ml THF, cooling to -15~C, and adding 0.366 ml (3.33 mmol) NMM followed by 0.216 ml ( 1.67 mmol) isobutyl chloroformate.
This solution is stirred 5 minutes and 0.374 g (1.67 mmol) Ala-Leucinol.HCI, in a 35 mixture of 10 ml THF and minim~l DMF, are added. Stirring is continued at 0~C for 15 minutes and 2 h at room teln~ldlllre. The solution is quenched with S ml lN
HCl, extracted with EtOAc (3X, 50 ml), the combined extracts are washed with sat'd CA 02266~27 1999-03-23 NaHCO3 and sat'd NaCI. The resulting organic phase is then dried (MgSO4), filtered, evaporated and chromatographed on silica to yield 0.260 g of the desired material.

(c) N-Ms-Phe-Gly-Ala-LeuH: A solution is prepared by adding 0.337 g (0.798 mmol) Dess-Martin periodinane to S ml CH2C12 and stirring for 10 minutes. To this solution 0.125 g (0.266 mmol) N-Ms-Phe-Gly-Ala-Leucinol is added in one portion.The reaction is continued until TLC showed complete conversion at which time thesolution is poured into 25 ml sat'd NaHCO3 cont~inin~ 1.8 g (5.586 mmol) Na2S2O3. After stirring for 10 minutes the mixture is extracted with EtOAc (3X, 50 ml). The combined extracts are dried (MgSO4), evaporated, and chromatographed on silica to afford 0.048 g of the product.

Synthesis Example 5:
SYnthesis of an aldeh~lde protease inhibitor Moc-Leu-OH-L-Leucine (5.0 g, 38.2 mmol) is dissolved in 38 ml lN NaOH and cooled to 0~C. Methyl chloroformate (3.1 ml, 40.0 mmol) is added dropwise while in a separate addition funnel lN NaOH is added as to m~int~in pH at 9.0-9.5. After addition is complete and the pH stabilized at 9.0-9.5 the solution is washed with 200 ml EtOAc, the aqueous phase is then acidified to pH = 2. This mixture is extracted with EtOAc (2X 100 ml), dried (MgSO4), filtered, and the solvent removed to afford 7.15 g pure product.

Moc-Leu-Leucinol- To a solution of 3.5 g (18.52 mmol) Moc-Leu-OH in 100 ml THF, cooled to -15~C, 2.04 ml (18.52 mmol) of N-methyl morpholine is added followed imme~ te~ly by 2.4 ml (18.52 mmol) isobutyl chloroformate. After stirring for 10 minutes 2.37 ml (18.52 mmol) of leucinol in 25 ml of THF is added and the reaction stirred 0.5 h at -15~C and 1 h at room tc~ ,e.~l lre. The mixture is then diluted with 100 ml of H20 and the THF evaporated. The re.n~ining a~ueous phase is partitioned between EtOAc and lN HCl, the organic phase washed with NaHCO3, dried (MgSO4) and evaporated to afford 5.33 g pure product.

Moc-Leu-LeuH-A solution cont~ining 4.4 g (10.41 mmol) Dess-Martin periodinane suspended in 100 ml CH2C12 is pre~ ed and stirred for 10 minutes. To this solution 1.0 g ~3.47 mmol) Moc-Leu-Leucinol is added and the solution stirred 2 h at roomt~ pc.dlllre followed by pouring into 100 ml of saturated NaHCO3 cont~ining 18 g(72.87 mmol) Na2S2O3. This solution is stirred 10 minnt~c and then extracted with CA 02266~27 1999-03-23 ..
EtOAc (2X, 125ml), dried (MgSO4) and the solvent evaporated. Chromatography on silica affords 0.550 g of pure product.

Synthesis Exarnple 6:
Additional peptide aldehydes are synth~i7e~1 according to the following procedures. Some of the interme.li~tçc are purchased from suppliers and in theseinct~nf~es it is noted within the procedure. Dess-Martin periodinane is synthesized according to the procedure of Martin, J.Org. Chem., 1983, 48, 4155.

1 O I. Z-Gly-Ala-Leu-OMe - To a solution of Z-Gly-Ala-OH (20.0 g, 0.071 M) and Leu-OMe.HCl (12.9 g, 0.071 M) in 250 ml dichloromethane is added 21.9 ml (0.157 M) triethylamine (TEA) dropwise over a period of 10 min. This addition is followed by the addition of 11.9 ml (0.078 M) of diethylcyanophosphonate (DECP). The mixture is stirred overnight and the solvent removed. The residue is dissolved in ethyl acetate and washed with lN HCl, saturated NaHCO3, and brine. The solution is dried with MgSO4, filtered and the solvent removed. Recovered will be 29.0 g of product that is homogeneous by TLC. 13C NMR (CDC13) 15.93, 18.60, 21.77, 22.69, 24.72, 40.80, 44.20, 48.70, 50.87, 52.13, 65.28, 66.84, 127.92, 128.00, 128.41, 136.36, 156.76, 169.31, 172.58, 173.24.
II. Moc-Phe-Gly-Ala-Leu-OMe - Z-Gly-Ala-Leu-OMe (29.0 g, 0.071 M) is dissolved in 300 ml MeOH and 35 ml 4.0 M HCI in dioxane. To this solvent mixture is added 5.8 g of 10% Pd/C portionwise. The slurry is ~eg~csed with an aspirator and H2 introduced via balloon. The slurry is m~int~in~d under a positive pressure of H2 and stirred overnight. The slurry is filtered through Celite and a sintered glass funnel and washed thoroughly with MeOH. The solvent is removed and the residue is triturated with ether. The slurry is filtered and the filter cake dried under vacuurn. Recovered 20.2 g of an off-white powder. The crude product and Moc-Phe-OH (15.3 g, 0.068 M) are dissolved in 500 ml CH2Cl2 and 29.9 ml TEA
(0.143 M) added dropwise followed by the dropwise addition of 11.7 ml (0.072 M) of DECP. The mixture iss stirred overnight and the solvent is removed. The residue is dissolved in EtOAc and washed with lN HCI, saturated NaHCO3, and brine. The organic phase is dried (MgS04), filtered and the solvent removed to afford 21.3 g product. 13C NMR (CDC13) 16.66, 16.83, 20,01, 22.46, 23.41, 25.40, 40.11, 41.72,43.75, 49.39, 51.37, 52.87, 56.42, 65.92, 77.39, 77.55, 77.81, 78.24, 127.42, 128.96, 129.19, 130.09, 137.41, 157.62, 169.00, 172.63, 173.24, 174.00.

CA 02266~27 1999-03-23 III. Moc-Phe-Gly-Ala-Leucinol - Moc-Phe-Gly-Ala-Leu-OMe (21.3 g, 44.5 mmol) is dissolved in a mixture of 400 ml EtOH and 250 ml THF. The solution is cooled to 0~C and 9.88 g (89.0 mmol) CaC12 is added. In 5 min the slurry will be homogenized and 6.73 g (178.0 mmol) NaBH4 added portionwise over a period of 5 5 min. The solution is stirred at 0~C for 2 hours and the reaction carefully quenched with lN HCI. The EtOH and THF are removed under vacuum and the rem~ining aqueous mixture extracted with 500 ml EtOAc. This organic phase is washed with saturated NaHCO3, brine, and the organic phase dried with MgSO4. Filtration and removal of solvent affords 20.0 g of an off-white crystalline material.
l o Chromatography on silica (3.5% MeOH/CH2C12) gives 13.0 g pure product. Rf =
0.3 (10% MeOH/CH2Cl2), 13C NMR (CDC13) 17.50, 22.23, 23.12, 24.84, 37.22, 39.76, 43.96, 49.88, 50.93, 52.48, 58.22, 65.27, 98.46, 98.54, 127.04, 128.68, 129.10, }36.62, 157.85, 170.71, 173.85, 174.45 1 5 IV. Moc-Phe-Gly-Ala-Leu-H - 29.9 g (70.7 mmol) of Dess-Martin periodinane is suspended in 500 ml CH2C12 and stirred for 10 min. Moc-Phe-Gly-Ala-Leucinol (10.6 g, 23.5 mmol) iss dissolved in 100 ml C1~2C12 and added at a moderate rate to the periodinane slurry. The mixture is stirred for Ih and poured into 150 ml NaHCO3 cont~ining 123 g Na2S2O3. The mixture is allowed to stir for 15 min and 20 extracted with EtOAc. The organic phase is dried and filtered followed by removal of solvent. Chromatography (3.5% MeOH/CH2C12) on silica gives 5.1 g of pure white solid that is a mixture of the methoxy hemi~ret~l and aldehyde. 13C NMR
(CDC13,CD30D) 17.62, 17.94, 21.53, 21.71, 22.99, 23.30, 23.39, 24.54, 37.05, 37.70, 37.92, 38.24, 42.87, 49.83, 51.79, 52.14, 52.40, 56.75, 57.19, 98.40, 99.18, 25 127.00, 128.60, 129.06, 136.44, 157.27, 169.19, 169.67, 172.73, 173.40, 200.43.

V. Moc-Phe-OH - E-Phenylalanine (5.0 g, 30.2 rnmol) is dissolved in 30 ml lN
NaOH and cooled to 0~C. Methyl chloroformate (2.53 ml, 31.8 mmol) is added dropwise while in a sep~ate addition funnel 30 ml of lN NaOH is added 30 simultaneously. After addition is complete, the solution is washed with 200 ml EtOAc and the aqueous phase acidified to pH = 2. The mixture is extracted with EtOAc (2X 100 ml), dried (MgSO4), filtered, and the solvent removed to afford 6.0 g product. 13C NMR (CDC13) 37.75, 52.57, 54.64, 128.63, 129.35, 135.74, 156.77, 175.76.
VI. Mac-Phe-OH - To a solution of 1.00 g (2.34 mmol) of Phe-OBn.PTSA in Et2O
at room ten,p~.dl~Ire is added 0.36 ml (2.57 mmol) of TEA. This is followed by the CA 02266~27 1999-03-23 WO 98/13459 ~CT/US97/16622 addition of 10 ml MeOH and then 0.14 ml (2.34 mmol) of methyl isocyanate in 4 mlEt2O is added dropwise. The reaction mixture is poured into 50 ml water and the phases separate. The organic phase is dried with MgSO4, filtered and the solventremoved to give 0.66 g of product (96% yield). 13C NMR (CDC13) 27.05, 38.47, 53.45, 54.64, 65.90, 127.43, 127.85, 128.48, 129.28, 130.21, 135.23, 136.22, 158.17, 173.08. To a solution of the crude product (2.11 mmol) in 25 ml MeOH is added 0.120 g Pd/C and the slurry deg~cse(l The slurry is stirred under a positive pressure of H2 via balloon for 1.5 h. The slurry is filtered through Celite and the filter cake washed with MeOH. The solvent is removed to afford 0.430 g product. 13C NMR
0 26.50, 37.92, 54.28, 126.69, 128.28, 129.28, 136.65, 159.36, 175.33.

VII. Mac-Phe-Gly-Ala-Leucinol - To a solution of 0.200 g Mac-Phe-OH (0.900 mmol) and 0.253 g Gly-Ala-Leu-OMe.HCI (0.818 mmol, generated by hydrogenation of I., above, according to the procedure outlined for compound II.) in 15 ml DMF is added 0.250 ml TEA (1.80 mmol) followed by the addition of 0.147 ml DECP (0.900 mmol). The mixture is stirred overnight and the solvent removed.
The residue is redissolved in EtOAc and washed successively with 0.3 N HCI, saturated NaHCO3, and brine. The solution is dried, filtered and the solvent removed to give 0.300 g product. The crude product (0.628 mmol) is dissolved in 17 ml EtOH and cooled to 0~C. To this solution is added 0.140 g CaCl2 (1.25 mmol) in 4 ml THF. To the resulting slurry is added 0.095 g NaBH4 in one portion.
After 45 min. the solution is quenched with water and extracted with EtOAc. The organic phase is dried with MgSO4, filtered and the solvent removed.
Chromatography with 4% MeOH/CH2C12 gave 0.200 g pure product. 13C NMR
(CD30D) 16.84, 21.05, 22.60, 24.51, 25.66, 37.41, 39.73, 42.67, 49.65, 56.63, 64.33, 126.63, 128.32, 128.96, 137.12, 160.01, 170.45, 173.60, 175.03.

VIII. Mac-Phe-Gly-Ala-Leu-H - To a slurry of Dess-Martin periodinane (0.565 g, 1.33 mmol) in 15 ml CH2C12 is added a suspension of Mac-Phe-Gly-Ala-Leucinol (0.200 g, 0.445 mmol) in CH2Cl2 and the resulting slurry stirred for 0.5 h. The mixture is poured into saturated NaHC03 col-t~ i..g 2.32 g Na2S203 and the - solution stirred for 10 min., followed by extraction ~vith EtOAc. The organic phase is dried with MgS04, filtered and the solvent removed. The residue is chromatographed on silica to give 0.081 g product. 13C NMR (10% CD30D in CDC13) 17.18, 17.43, 21.35, 21.55, 23.26, 23.34, 24.40, 24.47, 26.36, 26.60, 37.25, 37.38, 38.60, 42.86, 42.97, 51.77, 51.93, 54.94, 56.75, 57.00, 98.7, 99.32, 126.87, 128.49, 128.91, 136.51, 159.53, 159.55, 169.93, 170.39, 173.63, 173.85, 174.70.

CA 02266~27 1999-03-23 Cbz= carbobenzyloxy Gly = glycine Ala= alanine 5 Leu = leucine Phe = phenylalanine OMe = methyl ester TEA = triethylamine DECP = diethylcyanophosphonate 10 TLC = thin layer chromatography MeOH = methanol Pd/C = palladium on activated carbon EtOH = ethanol THF = tetrahydrofuran Mac = methylaminocarbonyl Moc = methoxycarbonyl Etoc = ethoxycarbonyl Ms = meth~nesulfonyl Proteolytic ~nz,vme - Another essential ingredient in the present liquid detergent compositions is active proteolytic enzyme. Mixtures of proteolytic enzyme are also included. The proteolytic enzyme can be of animal, vegetable or microorganism (preferred) origin. The proteases for use in the detergent compositions herein include (but are not limited to) trypsin, subtilisin, chymotrypsin and elastase-type proteases. Preferred for use herein are subtilisin-type proteolytic enzymes. Particularly p~cr~ . d is bacterial serine proteolytic enzyme obtained from Bacillus subtilis and/or Bacillus licheniforrnis. Protease enzymes are usually present in such liquid detergent compositions at levels sufficient to provide from 0.005 to 0.1 Anson units (AU) of activity per grarn of composition.
Suitable proteolytic enzymes include Novo Industri A/S Alcalase~) (preferred), Esperase~), Savinase~ (Copenhagen, Del~n~k), Gist-brocades' Maxatase~, Maxacal~ and Maxapem 1 5~g~ (protein engineered Maxacal(~)) (Delft, Netherlands), and subtilisin BPN and BPN'(prcfe.lcd), which are commercially available.
Preferred proteolytic enzymes are also modified bacterial serine proteases, such as 3~ those made by Genencor Int~rn~tional, Inc.(San Francisco, California) which are described in European Patent 251,446, filed April 28, 1987 (particularly pages 17, 24 and 98), and which is called herein "Protease B", and U.S. Patent 5,030,378, CA 02266~27 1999-03-23 Venegas, issued July 9, 1991, which refers to a modified bacterial serine proteolytic enzyme (Genencor International) which is called "Protease A" herein (same as BPN'). In particular see columns 2 and 3 of U.S. Patent 5,030,378 for a completedescription, including amino sequence, of Protease A and its variants. Preferredproteolytic enzymes, then, are selected from the group consisting of Alcalase (~) (Novo Industri A/S), BPN', Protease A and Protease B (Genencor), and mixtures thereof. Protease B is most preferred.
Another preferred protease, referred to as "Protease D" is a carbonyl hydrolase variant having an amino acid sequence not found in nature, which is 10 derived from a precursor carbonyl hydrolase by substituting a dirr~.elll amino acid for a plurality of amino acid residues at a position in said carbonyl hydrolase equivalent to position +76, preferably also in combination with one or more amino acid residue positions equivalent to those selected from the group con~ictine of +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, 15 +195, +197, +204, +206, +210, +216, +217, +21~, +222, +260, +265, and/or +274according to the numbering of Bacillus amyloliquefaciens subtilisin, as described in WO 95/10615 published April 20, 1995 by Genencor International.
Useful proteases are also described in PCT publications: WO 95/30010 published Novenber 9, 1995 by The Procter & Gamble Company; WO 95/30011 20 published Novenber 9, 1995 by The Procter & Gamble Company; WO 95/29979 published Novenber 9, 1995 by The Procter & Gamble Company.
Calcium - Any water-soluble calcium salt can be used as a source of calcium ions, including calcium acetate, calcium formate, calcium xylene sulfonate, and calcium propionate. Divalent ions, such as zinc and magnesium ions, can replace 25 the calcium ion completely or in part. Thus in the liquid detergent compositions herein, the source of calcium ions can be partially substituted with a source ofanother divalent ion.
The calcium useful herein is enzyme-~ccessihle. Therefore, the cl~imçd compositions are substantially free of sequestrants, for example, polyacids capable 30 of forming calcium complexes which are soluble in the composition. However, minor amounts of sequestrants such as polyacids or mixtures of polyacids can be used. The enzyme-accessible calcium is defined as the amount of calcium-ions effectively available to the enzyme component. From a practical standpoint the enzyme-accessible calcium is therefore the soluble calcium in the composition in the 35 absence of any storage sequestrants, e.g., having an equilibrium constant of complexation with calcium equal to or greater than 1.5 at 20~C.

CA 02266~27 1999-03-23 W O 98/13459 PCTrUS97/16622 Boric Acid - The compositions herein optionally contain from about 0.25% to about 10%, preferably from about 0.5% to about 5%, more preferably from about 0.75% to about 3%, by weight of boric acid or a compound capable of forming boric acid in the composition (calculated on the basis of the boric acid). Boric acid is preferred, although other compounds such as boric oxide, borax and other alkali metal borates (e.g., sodium ortho-, meta-, pyroborate, an sodium pentaborate) are suitable. Substituted boric acids (e.g., phenylboronic acid, butane boronic acid, and p-bromo phenylboronic acid) can also be used in place of boric acid.
The compositions of the present invention can also contain polyols, especially 10 diols, cont~ining only carbon, hydrogen and oxygen atoms. They preferably contain from about 2 to about 6 hydroxy groups. Examples include propylene glycol (especially 1,2 propanediol, which is preferred), ethylene glycol, glycerol, sorbitol, mannitol, glucose, and mixtures thereof. The polyol generally represents from about 1% to about 15%, preferably from about 1.5% to about 10%, more preferably from about 2% to about 7%, by weight of the composition.
Detersive Surfactant - An effective amount, typically from about I to 95, preferably about 8 to 70, weight %, of detersive surfactant is yet another escenti~l ingredient in the present invention. The detersive surfactant can be selected from the group concicting of anionics, nonionics, cationics, ampholytics, zwitterionics, and 20 mixtures thereof. By selecting the type and amount of detersive surfactant, along with other adjunct ingredients disclosed herein, the present detergent compositions can be formulated to be used in the context of laundry cleaning or in other different cleaning applications, particularly including dishwashing. The particular surfactants used can therefore vary widely depending upon the particular end-use envisioned.The benefits of the present invention are especially pronounced in compositions cont~ining ingredients that are harsh to enzymes such as certain detergency builders and surf~rt~ntc These include (but are not limited to) anionic surfactants such as alkyl ether sulfate linear alkyl benzene sulfonate, alkyl sulfate, etc. Suitable surfactants are described below.
Anionic Surfactants - One type of anionic surfactant which can be utilized encomp~ccec alkyl ester sulfonates. These are desirable because they can be madewith renewable, non-petroleum resources. Preparation of the alkyl ester sulfonate surfactant component can be effected according to known methods disclosed in thetechnical literature. For inct~n~e, linear esters of Cg-C20 carboxylic acids can be 35 sulfonated with gaseous S03 according to "The Journal of the American Oil Chemists Society," 52 (1975), pp. 323-329. Suitable starting materials would include natural fatty substances as derived from tallow, palm, and coconut oils, etc.

CA 02266~27 1999-03-23 W O 98/134S9 PCTrUS97/16622 The preferred alkyl ester sulfonate surfactant, especially for laundry applications, comprises alkyl ester sulfonate surfactants of the structural forrnula:
o R3 -CH-C-oR4 wherein R3 is a Cg-C20 hydrocarbyl, preferably an alkyl, or combination thereof,R4 is a C 1 -C6 hydrocarbyl, preferably an alkyl, or combination thereof, and M is a soluble salt-forming cation. Suitable salts include metal salts such as sodium, potassium, and lithium salts, and substituted or unsubstituted ammoniurn salts, such 0 as methyl-, dimethyl, -trimethyl, and quaternary ammonium cations, e.g.
tetramethyl-ammonium and dimethyl piperdinium, and cations derived from alkanolAmines, e.g. monoethanol-amine, diethanolamine, and triethanolamine.
Preferably, R3 is Clo-Cl6 alkyl, and R4 is methyl, ethyl or isopropyl. Especially preferred are the methyl ester sulfonates wherein R3 is C 1 4-C 16 alkyl.
Alkyl sulfate surfactants are another type of anionic surfactant of importance for use herein. In addition to providing excellent overall cleaning ability when used in combination with polyhydroxy fatty acid amides (see below), including good grease/oil cleaning over a wide range of tel,lpeldlures, wash concentrations~ and wash times, dissolution of alkyl sulfates can be obtained, as well as improved formulability in liquid detergent formulations are water soluble salts or acids of the formula ROSO3M wherein R preferably is a C l o-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C l o-c20 alkyl component, more preferably a C 12-C 1 8 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g., sodium, potassium, lithium), sub~liluled or unsubstituted ammonium cations such as methyl-, dimethyl-, and trimethyl Ammonium and qUAtenlAry ammonium cations, e.g., t~llalll~;l}lyl-ammonium and dimethyl piperdinium, and cations derived from alkanolAmines such as ethanolamine, diethanolamine, triethanolamine, and mixtures thereof, and the like. Typically, alkyl chains of C 12-16 are preferred for lower wash t~ pcldlules (e.g., below about 50~C) and C16 18 alkyl chains are p~ ed for higher wash telll~ alules (e.g., above about 50~C).
Alkyl alkoxylated sulfate surfAct~nt~ are another category of useful anionic surfactant. These surfAct~nt~ are water soluble salts or acids typically of the formula RO(A)mSO3M wherein R is an unsubstituted Clo-C24 alkyl or hydroxyalkyl group having a C1o-C24 alkyl component, preferably a C12-C20 alkyl or hydroxyalkyl, more preferably C12-C18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, mis greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a CA 02266~27 1999-03-23 metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammoniurn or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as - alkyl propoxylated sulfates are contemplated herein. Specific exarnples of substituted ammonium cations include methyl-, dimethyl-, trimethyl-amrnonium and5 quaternary ammonium cations, such as tetramethyl-ammonium, dimethyl piperidinium and cations derived from alkanol~min.-s, e.g. monoethanolamine, diethanolamine, and triethanolamine, and mixtures thereof. Exemplary surfactantsare C12-CIg alkyl polyethoxylate (1.0) sulfate, C12-Clg alkyl polyethoxylate (2.25) sulfate, C 12-c 18 alkyl polyethoxylate (3.0) sulfate, and C 12-c 18 alkyl 1 o polyethoxylate (4.0) sulfate wherein M is conveniently selected from sodium and potassium.
Other Anionic Surfactants - Other anionic surfactants useful for detersive purposes can also be included in the compositions hereof. These can include salts (including, for example, sodium, potassium, ammonium, and substituted amrnonium 15 salts such as mono-, di- and triethanolamine salts) of soap, Cg-C20 linear alkylben7erlesnll honates, Cg-C22 primary or secondary alkanesulphonates, Cg-C24olefinsulphonates, sulphonated polycarboxylic acids prepared by sulphonation of the pyrolyzed product of ~Ik~lin~ earth metal citrates, e.g., as described in British patent specification No. 1,082,179, alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, 20 fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isothionates such as the acyl isothionates, N-acyltaurates, fatty acid amides of methyl tauride, alkyl succin~m~tes and sulfosuccinates, monoesters of sulfosuccinate (especially saturated and unsaturated C 12-c 18 monoesters) diesters of sulfosuccinate (especially saturated and unsaturated C6-C 14 25 diesters), N-acyl sarcosin~es, sulfates of alkylpolysaccharides such as the sulfates of alkylpolyglucoside (the nonionic non~lllf~te~l compounds being described below),blanched primary alkyl snlf~t~s, alkyl polyethoxy carboxylates such as those of the formula ~O(CH2CH20)kCH2COO-M+ wherein R is a Cg-C22 alkyl, k is an integer from O to 10, and M is a soluble salt-forming cation, and fatty acids 30 esterified with isethionic acid and neutralized with sodium hydroxide. Resin acids and hydrogen~te~ resin acids are also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated resin acids present in or derived from tall oil.
Further exarnples are given in "Surface Active Agents and Detergents" (Vol. I and II
by Schwartz, Perry and Berch). A variety of such surfactants are also generally 3~ disclosed in U.S. Patent 3,929,678, issued December 30, 1975 to ~,~ughlin, et al. at Column 23, line 58 through Column 29, line 23 (herein incorporated by reference).

CA 02266~27 1999-03-23 Nonionic Deter~ent Surfactants - Suitable nonionic detergent surfactants are generally disclosed in U.S. Patent 3,929,678, I.~llghlin et al., issued December 30, 1975, at column 13, line 14 through column 16, line 6, incorporated herein by reference. Exemplary, non-limiting classes of useful nonionic surfactants are listed below The polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols. In general, the polyethylene oxide con~lenc~tes are preferred. These compounds include the con-lenc~tion products of alkyl phenols having an alkyl group cont~ining from about 6 to about 12 carbon atoms in either a straight chain or branched chain configuration with the alkylene oxide. In a preferred embodiment,the ethylene oxide is present in an amount equal to from about 5 to about 25 moles of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include Igepal~ C0-630, marketed by the GAF Corporation;
and Triton~) X-45, X-l 14, X-100, and X-102, all marketed by the Rohm & Haas Company. These compounds are commonly referred to as alkyl phenol alkoxylates, (e.g., alkyl phenol ethoxylates).
The conrienc~tion products of aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms. Particularly p~er~,~cd are the condensation products of alcohols having an alkyl group cont~ining from about 10 to about 20 carbon atomswith from about 2 to about 18 moles of ethylene oxide per mole of alcohol.
Examples of co,.,.,lelcially available nonionic surf~ct~ntc of this type includeTergitol~) 1 5-S-9 (the conden.c~tion product of C 1 1 -C 15 linear secondary alcohol with 9 moles ethylene oxide), Tergitol(g) 24-L-6 NMW (the con~en.c~tion product of C12-C14 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; Neodol(~ 45-9 (the con~len~tion product of C 1 4-C 15 linear alcohol with 9 moles of ethylene oxide), Neodol~ 23-6.5 (the con~ien~tion product of C 1 2-C 13 linear alcohol with 6.5 moles of ethylene oxide), Neodol(~ 45-7 (the con~en~tion product of C 1 4-C 15 linear alcohol with 7 moles of ethylene oxide), Neodol~) 45-4 (the con-len.c~tion product of C 1 4-C 15 linear alcohol with 4 moles of ethylene oxide), marketed by Shell Chemical Col"pal1y, and Kyro~) EOB (the condPnc~tion product of C 1 3-C 15alcohol with 9 moles ethylene oxide), marketed by The Procter & Gamble Company.
This category of nonionic surfactant is referred to generally as "alkyl ethoxylates."
The con-len~tion products of ethylene oxide with a hydrophobic base fonned by the conden~tion of propylene oxide with propylene glycol. The hydrophobic , ... _ ... .. . . ....

CA 02266~27 1999-03-23 W O 98tl3459 PCT~US97/16622 portion of these compounds preferably has a molecular weight of from about 1500 to about 1800 and exhibits water insolubility. The addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the con~c~tion product, which corresponds to con.1enc~tion with up to about 40 molesof ethylene oxide. Examples of compounds of this type include certain of the commercially-available Pluronic(~) surf~.~t~nt.~, marketed by BASF.
The con-len~tion products of ethylene oxide with the product resulting from 0 the reaction of propylene oxide and ethylene.1i~mine. The hydrophobic moiety of these products consists of the reaction product of ethylene~ rnin~- and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000.
Examples of this type of nonionic surfactant include certain of the cornmercially available Tetronic~ compounds, marketed by BASF.
Semi-polar nonionic surf~ct~nt.c are a special category of nonionic surfactants which include water-soluble amine oxides cont~ining one alkyl moiety of from 20 about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups cont~ining from about 1 to about 3 carbon atoms; water-soluble phosphine oxides cont~ining one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups cont~inin~ from about I to about 3 carbon atoms;
25 and water-soluble sulfoxides CO~ ing one alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms.
Semi-polar nonionic detergent surfactants include the arnine oxide surfactants having the forrnula O
R3(oR4)XN(R5)2 wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof cont~inin~ from about 8 to about 22 carbon atoms; R4 is an alkylene or hydroxyalkylene group cont~ining from about 2 to about 3 carbon atoms or mixtures 35 thereof; x is from 0 to about 3; and each R5 is an alkyl or hydroxyalkyl group cont~ining from about I to about 3 carbon atoms or a polyethylene oxide group cont~ining from about 1 to about 3 ethylene oxide groups. The R5 groups can be CA 02266~27 1999-03-23 rhP~l to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure.
These amine oxide surfactants in particular include C 1 o-C 18 alkyl dimethyl amine oxides and Cg-C12 alkoxy ethyl dihydroxy ethyl amine oxides.
Alkylpolysaccharides disclosed in U.S. Patent 4,565,647, Llenado, issued January 21, 1986, having a hydrophobic group cont~ining from about 6 to about 30carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g., a polyglycoside, hydrophilic group cont~ining from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide cont~ining 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties. (Optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside.) The intersaccharide bonds can be, e.g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding saccharide units.
Optionally, and less desirably, there can be a polyalkylene-oxide chain joining the hydrophobic moiety and the polysaccharide moiety. The preferred alkyleneoxide is ethylene oxide. Typical hydrophobic groups include alkyl groups, either saturated or unsaturated, branched or unbranched cont~ining from about 8 to about 18, preferably from about 10 to about 16, carbon atoms. Preferably, the alkyl group is a straight chain saturated alkyl group. The alkyl group can contain up to about 3 hydroxy groups and/or the polyalkyleneoxide chain can contain up to about 10, preferably less than 5, alkyleneoxide moieties. Suitable alkyl polysaccharides are octyl, nonyl, decyl, undecyldodecyl, tridecyl, tetradecyl, pent~r~Pcyl, hexadecyl, heptadecyl, and octadecyl, di-, tri-, tetra-, penta-, and hexaglucosides, galactosides, lactosides, glucoses, fructosides, fructoses and/or galactoses. Suitable mixtures include coconut alkyl, di-, tri-, tetra-, and pentaglucosides and tallow alkyl tetra-, penta-, and hexa-glucosides.
The pr~felled alkylpolyglycosides have the formula R20(CnH2nO)t(glYC~sYl)x wherein R2 is selected from the group consisting of alkyl, alkyl-phenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groupscontain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare , ~ . . . . . . . ...

CA 02266~27 1999-03-23 these compounds, the alcohol or alkylpolyethoxy alcohol is forrned first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the l-position). The additional glycosyl units can then be attached between their 1-position and the prece~ling glycosyl units 2-, 3-, 4- and/or 6-position, preferably 5 predominantly the 2-position.
Fatty acid amide surf~ct~nt~ having the formula:
o R6 -C-N (R7 ) 2 wherein R6 is an alkyl group cont~inine from about 7 to about 21 (preferably from 10 about 9 to about 17) carbon atoms and each R7 is selected from the group consisting of hydrogen, C 1 -C4 alkyl, C I -C4 hydroxyalkyl, and -(C2H40)XH where x varies from about 1 to about 3.
Preferred amides are Cg-C20 ammonia amides, monoethanolamides, diethanolamides, and isopropanol~mi~les Cationic Surf~ct~nt.~ - Cationic detersive surfactants can also be included in detergent compositions of the present invention. Cationic surfactants include the ammonium surfactants such as alkyldimethylammonium halogenides, and those surfactants having the formula:
[R2(oR3 )y] [R4(oR3 )y]2R5N+X-wherein R2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R3 is selected from the group consisting of-CH2CH2--CH2CH(CH3)-, -CH2CH(CH2OH)-, -CH2CH2CH2-, and mixtures thereof; each R4 is selected from the group con~i~ting of Cl-C4 alkyl, Cl-C4 hydroxyalkyl, benzyl, ring structures formed by joining the two R4 groups, -CH2CHOHCHOHCOR6CHOH-CH20H wherein R6 is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not O; R5 is the same as R4 or is an alkyl chain wherein the total number of carbon atoms of R2 plus RS is not more than about 18; each y is from 0 to about 10 and the sum of the y values is from 0 to about 15; and X is any compatible anion.
Other cationic surfA~-tAnt~ useful herein are also described in U.S. Patent 4,228,044, Cambre, issued October 14, 1980, incorporated herein by reference.
Other Surfactants - Ampholytic surf~ct~nt~ can be incorporated into the detergent compositions hereof. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can bestraight chain or branched. One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one CA 02266~27 1999-03-23 W O 98/134S9 PCTrUS97/16622 contains an anionic water-solubilizing group, e.g., carboxy, sulfonate, sulfate. See U.S. Patent No. 3,929,678 to L ~ghlin et al., issued December 30, 1975 at column19, lines 18-35 forexamples of ampholytic surfactants.
Zwitterionic surfactants can also be incorporated into the detergent compositions hereof. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyc}ic secondary and tertiary amines, or derivatives of quat~ arnmonium, quaternary phosphonium or tertiary sulfonium compounds. See U.S. Patent No. 3,929,678 to T ~llghlin et al., issued December 30, 1975 at column 19, line 38 through column 22, line 48 for examples 10 of zwitterionic surf~ct~ntc Arnpholytic and zwitterionic surf~ct~nts are generally used in combination with one or more anionic and/or nonionic surfactants.
PolYhydroxy Fatty Acid Amide Surfactant - The liquid detergent compositions hereof may also contain an enzyme-enhancing amount of polyhydroxy fatty acid amide surfactant. By "enzyme-enhancing" is meant that the formulator of the 15 composition can select an amount of polyhydroxy fatty acid amide to be incorporated into the compositions that will improve enzyme cleaning performanceof the detergent composition. In general, for conventional levels of enzyme, theincorporation of about 1 %, by weight, polyhydroxy fatty acid amide will enhanceenzyme performance.
The detergent compositions herein will typically comprise about 1% weight basis, polyhydroxy fatty acid amide surfactant, preferably from about 3% to about 30%, of the polyhydroxy fatty acid arnide. The polyhydroxy fatty acid amide surfactant component comprises compounds of the structural formula:
O Rl R2 - C - N - z wherein: Rl is H, Cl-C4 hyd~oc~l,yl, 2-hydroxy ethyl, 2-hydroxy propyl, or a mixture thereof, p~fcl~bly C1-C4 alkyl, more preferably C1 or C2 alkyl, most preferably C I alkyl (i.e., methyl); and R2 is a Cs-C31 hydrocarbyl, preferably straight chain C7-C 19 alkyl or alkenyl, more preferably straight chain Cg-C 17 alkyl 30 or alkenyl, most preferably straight chain C 11 -C 15 alkyl or alkenyl, or mixtures thereof; and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly co~nectecl to the chain, or an alkoxylated derivative (preferably ethoxylated or propoxylated) thereof. Z preferably will be derived from a reducing sugar in a reductive amination reaction; more preferably Z will be a 35 glycityl. Suitable reducing sugars include glucose, fructose, maltose, lactose, galactose, mannose, and xylose. As raw materials, high dextrose corn syrup, highfructose corn syrup, and high maltose corn syrup can be utilized as well as the ... ., . . . .. .... , .. ~ . . ~ .

CA 02266~27 1999-03-23 W O 98/134S9 PCTnUS97/16622 individual sugars listed above. These corn syrups may yield a mix of sugar components for Z. It should be understood that it is by no means intended to exclude other suitable raw materials. Z preferably will be selected from the group consisting of-CH2-(CHOH)n-CH2OH, -CH(CH2OH)-(CHOH)n l-CH2OH, -CH2-5 (CHOH)2(CHOR')(CHOH)-CH2OH, and alkoxylated derivatives thereof, where n is an integer from 3 to 5, inclusive, and R' is H or a cyclic or aliphatic monosaccharide.
Most preferred are glycityls wherein n is 4, particularly -CH2-(CHOH)4-CH2OH.
R can be, for exarnple, N-methyl, N-ethyl, N-propyl, N-isopropyl, N-butyl, N-2-hydroxy ethyl, or N-2-hydroxy propyl.
1 o R2-CO-NC can be, for example, cocamide, stearamide, oleamide, lauramide, myristamide, capricamide, palmitamide, tallowamide, etc.
Z can be l-deoxyglucityl, 2-deoxyfructityl, I-deoxymaltityl, I-deoxylactityl, 1-deoxygalactityl, 1-deoxymannityl, l-deoxymaltotriotityl, etc.
Methods for m~ing polyhydroxy fatty acid amides are known in the art. In general, they can be made by reacting an alkyl amine with a reducing sugar in a reductive amination reaction to form a corresponding N-alkyl polyhydroxyamine, and then reacting the N-alkyl polyhydroxyamine with a fatty aliphatic ester or triglyceride in a conflen~tion/arnidation step to form the N-alkyl, N-polyhydroxy fatty acid amide product. Processes for making compositions cont~ining polyhydroxy fatty acid amides are disclosed, for exarnple, in G.B. Patent Specification 809,060, published February 18, 1959, by Thomas Hedley & Co., Ltd., U.S. Patent 2,965,576, issued December 20, 1960 to E. R. Wilson, and U.S. Patent2,703,798, Anthony M. Schwartz, issued March 8, 1955, and U.S. Patent 1,985,424,issued December 25, 1934 to Piggott, each of which is incorporated herein by reference.
Second Enzvme - ~efe~ d compositions herein further comprise a performance-enhancing amount of a detergent-compatible second enzyme. By "d~lelgelll-compatible" is meant compatibility with the other ingredients of a liquid detergent composition, such as detersive surfactant and detergency builder. These second enzymes are preferably selected from the group con~i~ting of lipase, amylase, cellulase, and mixtures thereof. The term "second enzyme" excludes the proteolytic enzymes discussed above, so each composition which has a second ert7yme containsat least two kinds of enzyme, including at least one proteolytic enzyme. The amount of second enzyme used in the composition varies according to the type of enzyme.In general, from about 0.0001 to 0.3, more preferably 0.001 to 0.1, weight % of these second enzymes are preferably used. Mixtures of the same class of enzymes .. .... ... . . . ... . . .

CA 02266~27 1999-03-23 (e.g. Iipase) or two or more classes (e.g. cellulase and lipase) may be used. Purified or non-purified forms of the enzyme may be used.
Any lipolytic enzyme suitable for use in a liquid detergent composition can be used in these compositions. Suitable lipase enzymes for use herein include those of bacterial and fungal origin.
Suitable bacterial lipases include those produced by microorg~nicm~ of the Pseudomonas groups, such as Pseudomonas stutzeri ATCC 19.154, as disclosed in British Patent 1,372,034, incorporated herein by reference. Suitable lipases include those which show a positive immunological cross-reaction with the antibody of the lipase produced by the microorganism Pseudomonas fluorescens IAM 1057. This lipase and a method for its purification have been described in J~p~nPse Patent Application 53-20487, laid open on February 24, 1978. This lipase is available from Arnano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P
"Amano," hereinafter referred to as "Amano-P." Such lipases should show a positive immunological cross-reaction with the Amano-P antibody, using the standard and well-known immunodiffusion procedure according to Ouchterlony (Acta. Med. Scan., 133, pages 76-79 (1950)). These lipases, and a method for their immunological cross-reaction with Amano-P, are also described in U.S. Patent 4,707,291, Thom et al., issued November 17, 1987, incorporated herein by reference.
Typical examples thereof are the Amano-P lipase, the lipase ex Pseudomonas fra iFERM P 1339 (available under the trade name Amano-B), lipase ex Pseudomonas nitroreducens var. Iipolyticum FERM P 1338 (available under the trade name Amano-CES), lipases ex Chromobacter viscosum, e.g. Chromobacter viscosum var.
Iipolvticum NRRLB 3673, commercially available from Toyo Jozo Co., Tagata, Japan; and further Chromobacter viscosum lipases from U.S. Biochemical Corp., U.S.A. and Disoynth Co., The Netherlands, and lipases ex Pseudomonas ~ladioli.
Suitable fungal lipases include those producible by Hu~nicola lanuginosa and Thermomyces lanu~inosus. Most preferred is lipase obtained by cloning the gene from Humicola lanu~inosa and ~ressing the gene in Asper~illus orvzae as described in European Patent Application 0 258 068 (Novo Industri A/S), commercially available from Novo Nordisk A/S under the trade name Lipolase(~.
From about 10 to 18,000, preferably about 60 to 6,000, lipase units per grarn (LU/g) of lipase can be used in these compositions. A lipase unit is that amount of Iipase which produces I mmol of titratable fatty acid per minute in a pH stat, where pH is 9.0, tt;lllp~ldlu~e is 30~C, substrate is an emulsion of 3.3wt % of olive oil and 3.3% gurn arabic, in the presence of 13 mmol/l Ca and 20 rnmol/l NaCI in 5 mmol/l Tris-buffer.

, . . , . ~ ,, CA 02266~27 1999-03-23 WO 98/13459 PCTtUS97/16622 Any cellulase suitable for use in a liquid detergent composition can be used in these compositions. Suitable cellulase enzymes for use herein include those frombacterial and fungal origins. Preferably, they will have a pH optimum of between 5 and 9.5. From about 0.0001 to 0.1 weight % cellulase can be used.
Suitable cellulases are disclosed in U.S. Patent 4,435,307, Barbesgaard et al., issued March 6, 1984, incorporated herein by reference, which discloses fungal cellulase produced from Humicola insolens. Suitable cellulases are also disclosed in GB-A-2.075.028, GB-A-2.095.275 and DE-OS-2.247.832.
Examples of such cellulases are cellulases produced by a strain of Humicola 1 o insolens (Humicola ~risea var. therrnoidea), particularly the Humicola strain DSM
1800, and cellulases produced by a fungus of Bacillus N or a cellulase 212-producing fungus belonging to the genus Aeromonas, and cellulase extracted from the hepatopancreas of a marine mollusc (Dolabella Auricula Solander).
Any arnylase suitable for use in a liquid detergent composition can be used in 15 these compositions. Amylases include, for exarnple, amylases obtained from a special strain of B.licheniformis, described in more detail in British Patent Specification No. 1,296,839 (Novo). Amylolytic proteins include, for exarnple, RapidaseR, International Bio-Synthetics, Inc. and TermamylR Novo Industries.
From about 0.0001% to 0.55, preferably 0.0005 to 0.1, w~. % amylase can be 20 used.
Optional In~redients - Detergent builders can optionally be included in the compositions herein, especially for laundry compositions. Inorganic as well as organic builders can be used. When present, the compositions will typically comprise at least about 1 % builder and can be either an inorganic or organic builder.
25 Liquid laundry formulations preferably comprise from about 3% to 30%, more preferably about 5 to 20%, by weight, of detergent builder.
Inorganic detergent builders include, but are not limited to, the alkali metal, ammonium and alkanolarnmonium salts of polyphosphates (exemplified by the tripolyphosph~es, pyrophosphates, and glassy polymeric meta-phosphates), 30 phosphonates, phytic acid, silicates, carbonates (including bicarbonates and sesquicarbonates), sulphates, and alnminosilicates. Borate builders, as well as builders cont~ining borate-forrning materials that can produce borate under detergent storage or wash conditions (hereinafter, collectively "borate builders"), can also be used. Preferably, non-borate builders are used in the compositions of the invention 35 intPndecl for use at wash conditions less than about 50~C, especially less than about 40~C.

, .. ...

CA 02266~27 1999-03-23 Examples of silicate builders are the alkali metal silicates, particularly thosehaving a SiO2:Na20 ratio in the range 1.6:1 to 3.2:1 and layered silicates, such as the layered sodium silicates described in U.S. Patent 4,664,839, issued May 12, 1987 to H. P. Rieck, incorporated herein by reference. However, other silicates may 5 also be useful such as for example m~gnesiurn silicate, which can serve as a cricpening agent in granular formulations, as a stabilizing agent for oxygen bleaches, and as a component of suds control systems.
Examples of carbonate builders are the Alk~line earth and alkali metal carbonates, including sodium carbonate and sesquicarbonate and mixtures thereof 10 with ultra-fine calcium carbonate as disclosed in German Patent Application No.
2,321,001 published on November l S, 1973, the disclosure of which is incorporated herein by reference.
Aluminosilicate builders are useful in the present invention. Aluminosilicate builders are of great importance in most currently marketed heavy duty granular 5 detergent compositions, and can also be a significant builder ingredient in liquid detergent formulations. Aluminosilicate builders include those having the empirical formula:
MZ(zAlo2 YSiO2) wherein M is sodiurn, potassium, ammonium or substituted ammonium, z is from 20 about 0.5 to about 2; and y is 1; this material having a m~gn.-sium ion exchange capacity of at least about SO milligram equivalents of CaC03 hardness per gram of anhydrous aluminosilicate. Preferred alumino-silicates are zeolite builders which have the formula:
Naz[(A102)z (sio2)yJ-xH2o 25 wherein z and y are integers of at least 6, the molar ratio of z to y is in the range from 1.0 to about 0.5, and x is an integer from about lS to about 264.
Useful aluminosilicate ion exchange materials are commercially available.
These aluminosilicates can be crystalline or amorphous in structure and can be naturally-occ~lrring aluminosilicates or synthetically derived. A method for 30 producing aluminosilicate ion exchange materials is disclosed in U.S. Patent 3,985,669, Krummel, et al., issued October 12, 1976, incorporated herein by reference. Preferred synthetic crystalline aluminosilicate ion exchange materials useful herein are available under the desi~n~tions Zeolite A, Zeolite P ~B), andZeolite X. In an especially preferred embodiment, the crystalline aluminosilicate ion 35 exchange material has the formula:
Nal2[(Alo2)l2(sio2)l2] XH20 ~ . . .. . . . .

CA 02266~27 1999-03-23 W O98/134S9 PCT~US97/~6622 wherein x is from about 20 to about 30, especially about 27. This material is known as Zeolite A. Preferably, the aluminosilicate has a particle size of about 0.1 - 10 microns in diameter.
Specific exarnples of polyphosphates are the alkali metal tripolyphosphates, sodium, potassium and ammonium pyrophosphate, sodium and potassium and ammonium pyrophosphate, sodium and potassium orthophosphate, sodium polymeta phosphate in which the degree of polymerization ranges from about 6 to about 21,and salts of phytic acid.
Examples of phosphonate builder salts are the water-soluble salts of ethane I -10 hydroxy- 1, 1 -diphosphonate particularly the sodium and potassium salts, the water-soluble salts of methylene diphosphonic acid e.g. the trisodium and tripotassiumsalts and the water-soluble salts of substituted methylene diphosphonic acids, such as the trisodium and tripotassium ethylidene, isopyropylidene benzylmethylidene and halo methylidene phosphonates. Phosphonate builder salts of the aforementioned types are disclosed in U.S. Patent Nos. 3,159,581 and 3,213,030 issued December 1, 1964 and October 19, 1965, to Diehl; U.S. Patent No. 3,422,021 issued January 14, 1969, to Roy; and U.S. Patent Nos. 3,400,148 and 3,422,137 issued September 3, 1968, and January 14, 1969 to Quimby, said disclosures beingincorporated herein by reference.
Organic detergent builders preferred for the purposes of the present invention include a wide variety of polycarboxylate compounds. As used herein, "polycarboxylate" refers to compounds having a plurality of carboxylate groups, preferably at least 3 carboxylates.
Polycarboxylate builder can generally be added to the composition in acid 25 form, but can also be added in the form of a neutralized salt. When utilized in salt form, alkali metals, such as sodiurn, potassium, and lithiurn, or alkanolarnmonium salts are plefe.lcd.
Included arnong the polycarboxylate builders are a variety of categories of useful materials. One important category of polycarboxylate builders encomp~cses30 the ether polycarboxylates. A number of ether polycarboxylates have been disclosed for use as detergent builders. Examples of useful ether polycarboxylates includeoxydisuccinate, as disclosed in Berg, U.S. Patent 3,128,287, issued April 7, 1964, and Lamberti et al., U.S. Patent 3,635,830, issued January 18, 1972, both of which are incol~oldled herein by reference.
A specific type of ether polycarboxylates useful as builders in the present invention also include those having the general formula:
CH(A)(COOX)-CH(COOX)-O-CH(COOX)-CH(COOX)(B) CA 02266~27 1999-03-23 wherein A is H or OH; B is H or -O-CH(COOX)-CH2(COOX); and X is H or a salt-forming cation. For exarnple, if in the above general formula A and B are both H, then the compound is oxydissuccinic acid and its water-soluble salts. If A is OH and B is H, then the compound is tartrate monosuccinic acid (TMS) and its water-soluble salts. If A is H and B is -O-CH(COOX)-CH2(COOX), then the compound is tartrate disuccinic acid (TDS) and its water-soluble salts. Mixtures of these builders are especially preferred for use herein. Particularly preferred are mixtures of TMS and TDS in a weight ratio of TMS to TDS of from about 97:3 to about 20:80. These builders are disclosed in U.S. Patent 4,663,071, issued to Bush et al., on May 5, O 1987.
Suitable ether polycarboxylates also include cyclic compounds, particularly alicyclic compounds, such as those described in U.S. Patents 3,923,679; 3,835,163;

4,158,635; 4,120,874 and 4,102,903, all of which are incorporated herein by reference.
Other useful detergency builders include the ether hydroxypolycarboxylates represented by the structure:
HO-[C(R)(COOM)-C(R)(COOM)-O]n-H
wherein M is hydrogen or a cation wherein the resultant salt is water-soluble, preferably an alkali metal, ammonium or substituted ammonium cation, n is from about 2 to about 15 (preferably n is from about 2 to about 10, more preferably naverages from about 2 to about 4) and each R is the same or different and selected from hydrogen, C I 4 alkyl or C I 4 substituted alkyl (preferably R is hydrogen).
Still other ether polycarboxylates include copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4, 6-trisulphonic acid, and carboxymethyloxysuccinic acid.
Organic polycarboxylate builders also include the various alkali metal, ammoniurn and substituted ammonium salts of polyacetic acids. Examples include the sodium, potassium, lithium, ammonium and substituted arnmoniurn salts of ethylen~ mine tetraacetic acid, and nitrilotriacetic acid.
Also included are polycarboxylates such as mellitic acid, succinic acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, and carboxymethyloxysuccinic acid, and soluble salts thereof.
Citrate builders, e.g., citric acid and soluble salts thereof (particularly sodium salt), are polycarboxylate builders of particular importance for heavy duty liquid detergent formulations, but can also be used in granular compositions.

. ~._.. .. .. . .

CA 02266~27 1999-03-23 W O 98/13459 PCTrUS97116622 Other carboxylate builders include the carboxylated carbohydrates disclosed in U.S. Patent 3,723,322, Diehl, issued March 28, 1973, incorporated herein by reference.
Also suitable in the detergent compositions of the present invention are the 3,3-dicarboxy-4-oxa-1,6-hexanedioates and the related compounds disclosed in U.S.
Patent 4,566,984, Bush, issued January 28, 1986, incorporated herein by reference.
Useful succinic acid builders include the Cs-C20 alkyl succinic acids and salts thereof. A particularly preferred compound of this type is dodecenylsuccinic acid.
Alkyl succinic acids typically are of the general formula o R-CH(COOH)CH2(COOH) i.e., derivatives of succinic acid, wherein R is hydrocarbon, e.g., Clo-C20 alkyl or alkenyl, preferably C12-C16 or wherein R maybe substituted with hydroxyl, sulfo, sulfoxy or sulfone substituents, all as described in the above-mentioned patents.
The succinate builders are preferably used in the form of their water-soluble 15 salts, including the sodium, potassium, ammonium and alkanolammonium salts.
Specific examples of succinate builders include: laurylsuccinate, myristylsuccinate, palmitylsuccinate, 2-dodecenylsuccinate (plere.led), 2-pentadecenylsuccinate, and the like. Laurylsuccinates are the preferred builders of this group, and are described in European Patent Application 86200690.5/0,200,263, 20 published November 5, 1986.
Examples of useful builders also include sodium and potassiurn carboxymethyloxymalonate, carboxymethyloxysuccinate, cis-cyclo-hexane-hexacarboxylate, cis-cyclope~ e-tetracarboxylate, water-soluble polyacrylates (these polyacrylates having molecular weights to above about 2,000 can also be 25 effectively utilized as disl)e~s~.l~), and the copolymers of maleic anhydride with vinyl methyl ether or ethylene.
Other suitable polycarboxylates are the polyacetal carboxylates disclosed in U.S. Patent 4,144,226, Crutchfield et al., issued March 13, 1979, incolyoldled herein by reference. These polyacetal carboxylates can be ylepa~ed by bringing together, 30 under polymerization conditions, an ester of glyoxylic acid and a polymerization initiator. The resulting polyacetal carboxylate ester is then attached to chemically stable end groups to stabilize the polyacetal carboxylate against rapid depolymerization in ~ lin-o solution, converted to the coll~;syo~lding salt, and added to a surfactant.
Polycarboxylate builders are also disclosed in U.S. Patent 3,30~,067, Diehl, issued March 7, 1967, incorporated herein by reference. Such materials include the CA 02266~27 1999-03-23 water-soluble salts of homo- and copolymers of aliphatic carboxylic acids such as maleic acid, itaconic acid and methylenemalonic acid.
Other organic builders known in the art can also be used. For example, monocarboxylic acids, and soluble salts thereof, having long chain hydrocarbyls can 5 be lltili7~d These would include materials generally referred to as "soaps." Chain lengths of C 1 0-C20 are typically utili7ec~ The hydrocarbyls can be saturated or unsaturated.
Other optional ingredients include soil release agents, chelating agents, clay soil removal/anti redeposition agents, polymeric dispersing agents, bleaches, 10 brl~htençrs, suds suppresors, solvents and aesthetic agents.
The detergent composition herein can be formulated as a variety of compositions, for instance as laundry detergents as well as hard surface cleaners or dishwashing compositions.
The compositions according to the present invention are further illustrated by the following exarnples.
EXAMPLE I
The following compositions are made by combining the listed ingredients in the listed plol,ol~ions. In this example, one or more of the following peptide aldehydes are used:
20 Peptidealdehyde l: CH3O-(O)C-Phe-Gly-Ala-LeuH
Peptide aldehyde 2: CH3N-(O)C-Phe-Gly-Ala-LeuH
Peptide aldehyde 3: CH3O-(O)C-Phe-Gly-Ala-PheH
Peptide aldehyde 4: CH3N-(O)C-Phe-Gly-Ala-PheH
Peptidealdehyde5: CH3SO2Phe-Gly-Ala-Leu-H
25 Peptide aldehyde 6: CH3SO2Val-Ala-Leu-H
Peptide aldehyde 7: C6HsCH2O(OH)(O)P-Val-Ala-Leu-H
Peptidealdehyde8: CH3CH2SO2-Phe-Gly-Ala-Leu-H
Peptide aldehyde 9: C6HsCH2SO2-Val-Ala-Leu-H
Peptide aldehyde 10: C6HsCH2O(OH)(O)P-Leu-Ala-Leu-H
30 Peptidealdehyde 11: C6HsCH2O(OH)(O)P-Phe-Ala-Leu-H
Peptide aldehyde 12: CH3O(OH)(O)P-Leu-Gly-Ala-Leu-H.

Compositions A B C D E F
Linearalkyl benzene 8.5 15 6.5 10 12.5 4 sulfonic acid Sodium C 12-15 1 2 l 2 -- --alkyl sulfate CA 02266~27 1999-03-23 W O 98/13459 PCT~US97116622 C14 1salkyl2.5times10 5 105 11 9 ethoxylated sulfate C12 glucose amide -- -- 9 -- 5 C12 1salcohol7times 3 10 4 7 2.5 ethoxylated Fatty acid 2 5 5 4 2 2 Citricacid 6 7 4 6 4 5 C12 14alkenyl -- 6 -- 5 6 substituted, succinic acid Sodium hydroxide 2 6 2 4 1 1.5 Ethanol 2 1.5 2 4 2 1.5 Monoethanolamine 6 5 4 --1,2-Propanediol 12 10 5 5 4 6 Amylase (143 KNU/g) -- -- 0.1 0.2 Lipolase~) (lOOKLU/g 0.5 0.2 0.5 0.5 0.4 --commercial solution) ProteaseB (34g/L 0.9 -- 0.5 -- 1.2 --commerical solution) Savinase(~ -- 0.3 -- 0.4 0.2 0.3 (commercial solution) Carezymeg) 0.5 1 0.8 -- 0.2 0.8 Peptide aldehydes 1-12 0.009 0.005 0.001 0.0005 0.0011 0.1 Calcium Ions 0.01 0.5 0.1 0.05 0.9 0.25 Water and minors Balance to 100%

EXAMPLE II
The following for nula is tested for % of protease activity rem~ining.
Combinations of 0%, 0.1%, 0.2%, and 0.3% Ca++ (from CaC12) and 0%, 0.0006%, 0.00125%, and 0.0025% peptide aldehyde (Synthesis Example 6) are used. Products are held at 90~F and assayed at weekly intervals for 42 days.
Component Wt (%) Alkyl, 1.4 ethoxylated, sulfate 30 Amine oxide 6 Polyhydroxy fatty acid amide 4 Nonionic surfactant (C 1 1 E9) 5 Mg ion from MgC12 Ca ion from CaC12 see chart below Peptide aldehyde* see chart below Sodium xylene sulfonate 4 Solvent 6 Water to 100%
pH to 8 *Peptide Aldehydes of Synthesis Exarnple 6.

EXAMPLE III
Results showing the petcent protease activity re~n~ining after 42 days at 90~F.
0.01% Protease B enzyme is used.
Calcium Ion Peptide AldehYde -- 0% 0.0006% 0.00125% 0.0025%
0% 48 71 75 86 0.1% 52 84 92 87 0.2% 53 85 98 100 0.3% 60 80 98 92 EXAMPLE IV
The following compositions are made by combining the listed ingredients in the listed ~.opol lions.
In~redients A (wt %) B (wt %) C (wt %) D (wt %) AExS I 22.1 24.7 33.5 3 Polyhydroxyfatty 4.6 1.2 4.2 0 acid amide Amine Oxide 4.6 1.2 4.8 0 Betaine 0 1.2 0 0 Nonionic 6.7 4.1 0 0 Surfactant Mg(OH)2 0.5 0.5 0.7 0 Ca ion from CaC12 0.1 0.3 0.4 0.1 Calciurn xylene 4.5 0 4 0 sulfonate Polyethylene 3 ~ ~ ~
glycol .. .. ~ .. ~

CA 02266~27 1999-03-23 Polypropylene 1.5 0 0 0 glycol 2000 Balance, water to 100% to 100% to 100% to 100%
Protease A or 0.001 -0.01 0.001 -0.01 0.005-0.01 0.0003-0.01 Protease B
Peptide 0.00025- 0.00025- 0.00025- 0.00125-Aldehydes2 0.0025 0.0025 0.0025 0.0025 1 x= the degree of ethoxylation. The average degree of ethoxylation for the compositions are: A=2.2, B=0.6, C=1.4, D=2.2.
2 The peptide aldehydes of Synthesis Example 6 are used herein.

Claims (10)

WHAT IS CLAIMED IS:

1. A liquid detergent composition comprising:
a) from 1% to 95%, by weight of composition, of a detersive surfactant;
b) an active proteolytic enzyme;
c) a source of calcium ions; and d) a peptide aldehyde having the formula Z-B-NH-CH(R)-C(O)H
wherein B is a peptide chain comprising from 1 to 5 amino acid moieties; Z is anN-capping moiety selected from the group consisting of phosphoramidate [(R"O)2(O)P-], sulfenamide [(SR")2-], sulfonamide [(R"(O)2S-], sulfonic acid [SO3H], phosphinamide [(R")2(O)P-], sulfamoyl derivative [R"O(O)2S-], thiourea [(R")2N(O)C-], thiocarbamate [R"O(S)C-], phosphonate [R"-P(O)OH], amidophosphate [R"O(OH)(O)P-], cabamate (R"O(O)C-), and urea (R"NH(O)C-), wherein each R" is independently selected from the group consisting of straight or branched C1 -C6 unsubstituted alkyl, phenyl, C7-C9 alkylaryl, and cycloalkyl moieties, wherein the cycloalkyl ring may span C4-C8 and may contain one or more heteroatoms selected from the group consisting of O,N,and S; and R is selected from the group consisting of straight or branched C1 - C6 unsubstituted alkyl, phenyl, and C7 - C9 alkylaryl moieties.

2. A liquid detergent composition according to Claim 1 wherein the combined source of calcium ion and peptide aldehyde provide more than additive stability to the proteolytic enzyme.

3. A liquid detergent composition according to Claim 1 comprising:
a) from 8 to 70% of said detersive surfactant;
b) from 0.0001% to 5% of an active proteolytic enzyme;
c) from 0.01% to 1% of calcium ion; and d) from 0.00001% to 5% of said peptide aldehyde.

4. A liquid detergent composition according to Claim 1 wherein said R moieties are selected from the group consisting of methyl, iso-propyl, sec-butyl, iso-butyl, -C6H5, -CH2-C6H5. and -CH2CH2-C6H5.

5. A liquid detergent composition according to Claim 4 wherein said B peptide chains are selected from the group consisting of peptide chains having the aminoacid sequences according to the general formula:
Z-A 5-A 4-A 3-A 2-A 1-NH-CH(R)-C(O)H
such that the following amino acids, when present, are:
A1 is selected from Ala, Gly;
A2, if present, is selected from Val, Ala, Gly, Ile;
A3, if present, is selected from Phe, Leu, Val, Ile;
A4, if present, is any amino acid;
A5, if present, is any amino acid.

6. A liquid detergent composition according to Claim 5 wherein the N-terminal end is protected by one of the N-capping moiety protecting groups selected from the group consisting of carbamates, ureas, sulfonamides, phosphonamides, thioureas, sulfenamides, sulfonic acids, phosphinamides, thiocarbamates, amidophosphates, and phosphonamides.

7. A liquid detergent composition according to Claim 6 wherein the N-terminal end of said protease inhibitor is protected by a methyl, ethyl or benzyl carbamate [CH3O-(O)C-; CH3CH2O-(O)C-; or C6H5CH2O-(O)C-], methyl, ethyl or benzyl urea [CH3NH-(O)C-; CH3CH2NH-(O)C-; or C6H5CH2NH-(O)C-], methyl, ethyl or benzyl sulfonamide [CH3SO2-; CH3CH2SO2-; or C6H5CH2SO2-], and methyl, ethyl or benzyl amidophosphate [CH3O(OH)(O)P-; CH3CH2O(OH)(O)P-; or C6H5CH2O(OH)(O)P-] groups.

8. A liquid detergent composition according to Claim 7 wherein said peptide aldehyde is selected from the group consisting of: CH3SO2Phe-Gly-Ala-Leu-H, CH3SO2Val-Ala-Leu-H, C6H5 CH2O(OH)(O)P-Val-Ala-Leu-H, CH3 CH2 SO2-Phe-Gly-Ala-Leu-H, C6H5CH2SO2-Val-Ala-Leu-H, C6H5CH2O(OH)(O)P-Leu-Ala-Leu-H, C6H5CH2O(OH)(O)P-Phe-Ala-Leu-H, and CH3O(OH)(O)P-Leu-Gly-Ala-Leu-H.

9. A liquid detergent composition according to Claim 1 wherein the source of calcium ion is selected from calcium formate, calcium chloride, calcium acetate,calcium xylene sulfonate, calcium sulfate, and mixtures thereof.

10. A liquid detergent composition according to Claim 9 wherein said source of calcium ions is partially substituted with a source of another divalent ion.